Radionuclide liver scans in tumor detection: current concepts. Because of the frequency of spread of tumors arising elsewhere to the liver, hepatic evaluation plays a key role in planning therapy. Dynamic radionuclide transit studies depend upon the dual blood supply of the liver (hepatic artery and portal vein) and can be a useful prelude to static imaging, as most intrahepatic tumors have an hepatic arterial blood supply. Since most tumors do not have the ability to accumulate a radioactive colloid, static views are usually obtained after injection of particulates, which are accumulated by the reticuloendothelial system. A gross defect on the radiocolloid scan simply indicates the presence of an intrahepatic abnormality, but does not identify the nature of the lesion. The possible role to be played by use of additional radiopharmaceuticals, with different properties, in identifying the intrahepatic process is discussed. Other uses of liver scans, such as in evaluating the response of known intrahepatic lesions to therapy, are pointed out.


Skeletal scanning in neoplastic disease. Bone imaging techniques are a sensitive and accurate method for the detection of primary and secondary neoplastic disease of the skeleton. Such procedures have been demonstrated to be superior to evaluation via blood chemistry levels or routine skeletal radiographic surveys. These techniques are also helpful in establishing sites for biopsy and for objective evaluation of therapeutic modalities. They suffer the disadvantage of being nonspecific, and as a result, all positive areas identified by scanning techniques should be correlated with radiographic changes.


Ultrasonic imaging of neoplasms.

Ultrasonic imaging of neoplasms. Reflected ultrasound scanning (echography) is rapidly developing into a prime tool for tumor diagnosis, outlining, and detection. The principles of echographic diagnosis of neoplasms, the accepted uses in detection and diagnosis, as well as some of the principles of internal and contour patterns are discussed. Echography is very helpful in determining the size, location, organ origin, internal characteristics, and course of neoplasms in the abdominal area, neck, and extremities. Echography is helpful in radiation therapy planning and is expected to assume an increasing role in the diagnosis and management of tumors.


Interventional radiology.

Interventional radiology. Direct unencumbered visualization of areas of interest by image intensification combined with more invasive techniques of percutaneous puncture and angiography have extended the scope of the radiologist in the diagnosis and management of patients with neoplastic diseases. Percutaneous biopsies can be obtained more safely and with greater reliability from lesions of the lungs, skeleton, kidneys, liver, pancreas, and retroperitoneal lymph nodes. Intravascular therapy by arterial occlusion of the neoplasm has been employed to control hemorrhage, to decrease tumor mass, and preoperatively to facilitate surgical removal. The possibility of initiating an immune response to the ischemic neoplasm is also discussed.


Carcinoembryonic antigen: clinical application. Carcinoembryionic antigen (CEA) a glycoprotein extracted from colonic cancer tissue (beta-globulin electrophoretic mobility, sedimentation coefficient 7 to 8S, and mol wt approximately 200,000) can be detected and measured by radioimmunoassay. Clinical evaluations of CEA determination have given the following results: In health: (1) Serum CEA level is not influenced by sex, age, blood type, time of blood sampling, or family history of cancer; (2) serum CEA level is influenced by a history of smoking or inflammatory disease of the bowel, lung, pancreas, and other organs (occasionally, a CEA level as high as 10 ng/ml is noted); and (3) currently, CEA positivity is defined as greater than 2.5 ng/ml, however, 5 ng/ml may be more realistic. In cancer: (1) CEA level may be increased in primary cancer of the gastrointestinal (GI) tract as well as in non-GI neoplasia; (2) the CEA test is not recommended for screening to detect early cancer; (3) serum CEA level depends on the stage of the neoplasia and usually is not influenced by the grade of differentiation; and (4) markedly increased (greater than 25 ng/ml serum CEA values are highly suggestive of metastatic cancer, particularly hepatic metastasis. In biological fluid: The CEA or CEA-like activity can be measured in gastrointestinal secretions. Quantitative studies of CEA levels in such fluids may yield more information than is obtainable from studies of serum. However, this possibility needs more study at present. Therefore, the currently available CEA tests cannot replace any of the now standard diagnostic methods for cancer detection. This use for assessment of therapy in selected patients or for following those known to be a high risk for cancer appears promising in preliminary studies, but clinical value, if any, remains to be determined.


Validity of screening.

Validity of screening. The technical aspects of evaluation of screening programs to detect early disease are discussed. Screening is differentiated from diagnosis. The practical importance of specificity and sensitivity of screening tests is illustrated. The concept of evaluation of the total program is introduced. The determinants of successful programs are discussed with illustrations.


Cancer of the pancreas: detection and diagnosis. Since cancer of the pancreas is now the fourth most common cause of death from cancer in the United States, and since the signs and symptoms of this disease are such poor indicators of its presence, other more effective diagnostic methods must be utilized in an attempt to lower the mortality of pancreatic cancer. The newer studies which appear to hold some promise include endoscopy alone and in combination with pancreatography, selective and superselective angiography with or without pharmacologic assistance, and various forms of scanning. Resolution of some of the problems associated with the technique of biopsy and immunologic studies may make these studies of greater value. A clearer distinction between cancer arising in the pancreas itself from those lesions arising in the ampullary area will be of great value in clarifying both thinking and therapeutic approaches to the problem. Activation of the National Pancreatic Cancer Project and some other ongoing multi-institutional cooperative studies should do much to stimulate further advances in our knowledge about pancreatic cancer.


The diagnosis of prostatic cancer.

The diagnosis of prostatic cancer. The diagnosis and detection of genitourinary cancer covers a broad range of physical signs and instrumental observations which are not necessarily diagnostic. The primary diagnosis in most entities remains dependent upon histologic confirmation. Adenocarcinoma of the prostate is the most common, and at times, most difficult urogenital cancer to diagnose and detect. Many newer techniques today are designed to evaluate the stage of disease, and to detect heretofore occult metastatic foci. Immunologic assays may be of future prognostic value.


Early detection of malignant melanoma.

Early detection of malignant melanoma. Case examination of the skin is the first and foremost tool in the detection of skin cancer. Early diagnosis of malignant melanoma is possible with appreciation of certain color changes, surface changes, and border changes in pigmented lesions. Emphasis is placed upon these criteria for diagnosis and other associated phenomena that may lead to suspicion of early malignant melanoma.


Advances in detection and diagnosis of head and neck tumors. Cancers of the head and neck are accessible to both inspection and palpation in the majority of patients. This accessibility allows for improvements in detection, through education of the medical profession and also of the allied health personnel. Some of the new advances in detection and diagnosis have been associated with development of new instruments. A new lens system developed by Hopkins allows a brighter endoscopic image and a significantly greater breadth of field. Needle aspiration biopsy techniques have also improved both in facility of performance and in accuracy of diagnosis. Asteriography aids in the detection of carotid body tumors. Selective parathormone studies have considerably improved the preoperative accuracy of evaluating parathyroid disease. Scans, both using ultrasound and a fluorescent iodine scan have also improved the preoperative evaluation of thyroid tumors. A challenge of detection is the realization that there is an increased risk of the development of thyroid cancer in childhood and in adults in those patients who received radiation therapy to the head and neck during infancy or childhood. A national campaign to alert potential individuals at risk should be a prime objective of the American Cancer Society.


Excretion of catecholamines in relatives of patients with familial neuroblastoma. The urinary excretion of catecholamines in 13 close relatives of two distantly related patients with congenital neuroblastoma was investigated. One of these relatives was a child with already known ganglioneuroblastoma. The catecholamines noradrenaline and dopamine were determined. Vanilmandelic acid and homovanillic acid also were determined and numerous other catecholamine metabolites were measured. In contrast to some previous reports in the literature, no pathological excretion patterns were found in the healthy family members.


Tumor angiogenesis activity in cells grown in tissue culture. Normal, viral transformed and tumor-derived cells grown in tissue culture and representing different species were tested for their ability to produce an extracellular tumor angiogenesis factor (TAF). TAF was assayed by measuring the host-mediated vascular response of the chorioallantoic membrane to TAF preparations. All of the viral transformed and tumor-derived cells tested, including SVT2, SVW126, Welker 256 rat carcinoma, B-16 mouse melanoma, human glioblastoma, and human meningioma cells, produced TAF. The potency of the TAF preparations, as measured by the number of cells needed to induce a positive vascular response on the chorioallantoic membrane, varied from cell line to cell line. The most potent cells tested were the glioblastoma and maningioma brain tumor cells. Since these brain tumors are found to be the most highly vascularized tumors in vivo, it was concluded that a correlation exists between the vascularity of a tumor in vivo and the potency of TAF in vitro. There was no detectable angiogenesis activity induced by density-inhibited BALB/c primary mouse embryo or early-passage human skin fibroblasts, even when relatively large numbers of cells were used to make a sample. However, density-inhibited BALB/c 3T3 aan W138 human embryonic lung fibroblasts, two cell lines widely regarded as demonstrating "normal" growth behavior in culture, produced TAF. From these and other observations, it was suggested that BALB/c 3T3 and W138 are not fully "normal" cells. Furthermore, it was suggested that the production of TAF is an early event in the cell transformation process that precedes the loss of density inhibition of growth in vitro.


Comparison of nude mice with the host species for evaluation of the tumorigenicity of guinea pig and hamster cells transformed in vitro by chemical carcinogens. Nude mice (nu/nu) were compared with the species of origin for determination of the tumorigenicity of cells from chemical carcinogen-transformed and noncarcinogenic chemically treated, nontransformed guinea pig and Syrian hamster cultures. The incidence and time of appearance of progressively growing tumors were similar in the host species and in nude mice after injection of 10(7) transformed cells. Inoculation of 10(8) nontransformed cells routinely was nontumorigenic in the host species and in nude mice. The nude mouse has potential as a sensitive and reliable alternative host to the species of origin to elaluate the tumorigenicity of xenogeneic mammalian cells from cell culture model systems of carcinogenesis.


Early effects of a single intrarectal dose of 1,2-dimethylhydrazine in mice. The early morphological and biochemical effects of intrarectally administered 1,2-dimethylhydrazine dihydrochloride on mouse colon were studied. Using [3H]thymidine autoradiography, it was found that 1,2-dimethylhydrazine dihydrochloride, 250 mg/kg decreased the number of prelabeled DNA-synthesizing cells in the distal colon as early as 30 min after instillation. During the interval from 24 hr to 2 weeks, however, the opposite effect was seen; incorporation of [3H]thymidine increased 3- to 5-fold over controls. At lower doses (0.25 to 25 mg/kg), a similar trend was observed. Histological examination showed no dramatic changes in cell structure or in tissue architecture. No changes were seen in labeling indices in the proximal colon. In the liver, cellular alterations were seen at concentrations of 25 to 250 mg/kg, particularly in the centrolobular region. These changes were evident at 2 hr and disappeared by 4 hr. The kidney was unaffected by 1,2-dimethylhydrazine dihydrochloride at any concentration. Our results suggest that enzymes capable of activating 1,2-dimethylhydrazine dihydrochloride are located within the mucosal cells of the distal colon.


Cell cycle synchronization of human lymphoid cells in vitro by 2,3-dihydro-1h-imidazo[1,2-b]pyrazole. 2,3-Dihydro-1H-imidazo[1,2-b]pyrazole (IMPY), a DNA synthesis-inhibitory drug, reversibly arrests growth of human lymphoblasts in vitro. DNA distribution histograms of cultures exposed to 0.5 to 2.0 mM IMPY show accumulation of cells with G1-early S DNA content. On reincubation in fresh medium, cell cycle traverse is resumed by the blocked cells in a synchronized manner. Maximum incorporation of [H3]thymidine into DNA (174 to 220% of control) and labeling indexes (72 to 86%) are seen after 4 hr of incubation, and a major increase in cell number is seen between the 9th and 13th hr. DNA distribution histograms of cells reincubated in fresh medium (after double block), show an initial increase in the number od cells with S-G2-M DNA content and a corresponding decrease in the number of G1-early S cells. After 4 hr of reincubation, a gradual increase in the number of G1-early S cells was seen as the earlier blocked cells completed cell cycle traverse and mitosis. Cells exposed to 2.0 mM IMPY took approximately 2 hr longer to traverse than did cells exposed to 0.5 mM IMPY.


Effect of adriamycin on the cell cycle traverse and kinetics of cultured human lymphoblasts. Exposure of cultured human lymphoblasts to adriamycin (ADM) (0.1 mug/ml for 24 hr or 0.5 mug/ml for 1 hr) leads to an accumulation of cells with the DNA content of late S and G2. Higher concentrations of ADM (0.5 to 10 mug/ml) inhibit cell cycle traverse. Effect of ADM on cell cycle traverse, cell growth, and incorporation of labeled precursors into DNA is dependent on drug concentration and length of exposure to ADM. Synchronized cells in G1 or G2 part of the cell cycle are less sensitive to ADM than cells in S phase. Similarly, plateau-phase cells are less sensitive to ADM than cells from log-phase cultures.


Responsiveness of senescent mice to the antitumor properties of Corynebacterium parvum. The antitumor properties of Corynebacterium parvum have been studied in young (3- to 8-month-old) and aged 18 or more months old) BALB/c mice given s.c., i.m., i.p., or i.v. transplants of the highly malignant, weakly immunogenic line 1 lung carcinoma, and in aged (25- to 33-month-old) BALB/c mice bearing primary mammary tumors. These aged BALB/c mice were shown to be less immunoresponsive than their younger counterparts, and this, in combination with nonimmunological factors, made them more sensitive to the lethal effects of the line 1 carcinoma. Correspondingly, C. parvum proved to have less antitumor activity in aged mice than it did in young mice. In spite of this relatively weaker antitumor activity for C. parvum in aged mice, repeated injections of this agent were able to induce temporary regressions of the primary mammary tumors studied and therby prolong survival time.


Some biological properties and an in vivo evaluation of tyrosine phenol-lyase on growth of B-16 melanoma. Tyrosine phenol-lyase from Erwinia herbicola was purified with the goal of assessing its effect on growth of malignant melanoma. Ammonium sulfate-sodium citrate fractionation and diethylaminoethyl cellulose-hydroxylapatite chromatography were used. The purified enzyme was shown to reduce plasma tyrosine levels when administered to normal C57BL x DBA/2 F1 mice. The plasma half-life value of the enzyme was found to be 6 to 7 hr. Unlike results reported with glutaminase and asparaginase preparations, the lactate dehydrogenase-elevating virus had no significant influence on plasma clearance of tyrosine phenol-lyase. The enzyme significantly inhibited growth of established B-16 melanoma tumors.


Pituitary role in the growth of metastasizing MRMT-1 mammary carcinoma in rats. MRMT-1 is a mammary carcinoma induced in immunologically impaired female Sprague-Dawley rats fed 3-methylcholanthrene. Its biological characteristics include transplantability to syngeneic normal rats and spontaneous metastases to various organs. Hypophysectomy performed 48 hr after tumor inoculation resulted in tumor regression accompanied by the disappearance of the metastasis in lungs of all the animals. The hypophysectomized animals were given replacement treatments, such as transplantation of pituitary homogenates, pituitary homograft underneath the renal capsule, or prolactin administration, and the inhibited tumor growth was markedly reactivated, with the reappearance of lung metastasis. These results indicate that MRMT-1 mammary carcinoma is prolactin dependent.


Intradermal transplantation in mice of small numbers of sarcoma cells followed by tumor growth and regression. Relatively small numbers of Sarcoma 1 cells were transplanted intradermally in A/WySn mice to study the course of growth when the size of the transplant is comparable to an early stage of spontaneous cancer. Transplants of 31 to 32,000 cells took one of three courses: no apparent growth; development of tumor followed by regression; or progressive growth. Transplants of 8,000 cells were the most evenly divided among these three courses and were selected for further study. Bilateral tumors took the same course to regression or to continued growth with significant frequency. There was complete correlation between regression of a tumor and immunity to a challenge made 1 to 5 weeks after the original transplant. Persistent tumors had a similar relationship, but the immunity was less complete. Limited, inapparent growth, followed by regression, was indicated in some mice, simulating immunological surveillance. Serum from such mice, when administered systemically in other mice, apparently induced enhancement of intradermal transplants.


Red blood cell metabolism in AKR mice in the prelymphomatous phase. The purpose of this study was to evaluate erythrokinetics and in vitro red blood cell (RBC) glucose utilization, 2,3-diphosphoglycerate production, and adenosine triphosphate levels following incubation in AKR mice from early in life until the onset of AKR lymphoma. Normal BALB/c mice served as controls. While hemoglobin concentration and RBC survival remained constant and similar in both groups of mice, the half disappearance time of injected radioactive 59Fe was longer and the 48-hr reappearance of 59Fe was less in AKR mice, compared with those of BALB/c mice. In vitro RBC metabolic studies indicated increased glucose utilization and 2,3-diphosphoglycerate production and decreased adenosine triphosphate levels following incubation in AKR RBC, in contrast to those in BALB/c RBC. RBC metabolic studies were also done in a small group of low leukemic C3H mice, and were similar to BALB/c mice. These differences became most marked in RBC from mice aged 15 to 30 weeks. Overt lymphoma began to occur after age 40 weeks. Hence, these erythropoietic changes occurred prior to the onset of lymphoma. The data imply a direct effect of virus infection on RBC or their precursors. The results are similar to changes in RBC metabolism noted in Rauscher-infected BALB/c mice. The broader implication of these findings in reference to viral host interactions and human leukemogenesis is discussed.


Malignant neoplasms in the families of patients with ataxia-telangiectasia. Ataxia-telangiectasia (A-T) is an autosomal recessive syndrome associated with a greatly increased incidence of malignant neoplasms in homozygous affected individuals. Heterozygotes for the gene for A-T are thought to comprise about 1% of the general population and, therefore, it is important to know whether this gene also predisposes the heterozygous carrier to cancers. Heterozygous carriers of this gene are common among the close relatives of patients with A-T, although individual carriers cannot be identified by any clinical criterion or laboratory test. For this reason, we compared the incidence of death from malignant neoplasms in 2 families of patients with A-T to that expected in a random sample of the general population. There were 59 deaths from malignant neoplasms in relatives dying before age 75, compared to 42.6 expected (p less than 0.02). For A-T heterozygotes younger than age 45, the risk of dying from a malignant neoplasm was estimated to be greater than 5 times the risk for the general population. A-T heterozygotes may comprise more than 5% of all persons dying from a cancer before age 45. The incidence of ovarian, gastric, and biliary system carcinomas and of leukemia and lymphoma was increased in these A-T families. Other neoplasms that may be associated with this gene in heterozygotes include pancreatic, basal cell, colonic, breast, and cervical carcinomas.


Immunosensitivity and histocompatibility antigens in drug-altered leukemic cells. New antigenic properties of experimental lymphomas have been reported previously following in vivo treatment with antitumor agents. 5-(3,3-Dimethyl-1-triazeno)imidazole-4-carboxamide (DIC) induced new antigenic characteristics on L1210 and L5178Y lymphomas, that were previously investigated in studies in animals compatible with the original untreated parental tumors. Here the L1210/DIC and L5178Y/DIC susceptibility to the cytotoxic effects of allogeneic and xenogeneic lymphocytes and sera obtained from animals sensitized to DBA/2 histocompatibility antigens were studied. The original and the DIC tumors showed the same sensitivity to anti-DBA/2 cellular and humoral cytotoxicity. The immune response electied in allogeneic mice by the original and DIC sublines was evaluated by in vitro cell-mediated and humoral cytotoxic assay. Beyond the immune response to histocompatibility antigens, a specific, anti-DIC-antigen immunoreaction was not found. Inhibition assay of the cell-mediated cytotoxicity and absorption of the humoral cytotoxicity demonstrated that DIC-induced antigens are not reciprocally related in cell-surface concentration to the natural DBA/2 histocompatibility antigens associated with tumor cells of DIC lines. An experiment was conducted in which specific activity against the DIC-treated L5178Y/DIC cells was observed with anti-L5178Y/DIC rabbit immune serum absorbed with the parental L5178Y lymphoma. This finding provides additional support to previous studies indicating that treatment with DIC induced new antigens on the lymphoma cells.


Suppressive role of indole on 2-acetylaminofluorene hepatotoxicity. Indole is known to suppress the hepatotoxicity and carcinogenicity of 2-acetylaminofluorene (AAF) in rats and hamsters. For elucidation of the mechanism of its protective role, 2 experiments were conducted using young male rats. In the 1st experiment, the 24-hr biliary excretion of N-hydroxy-2-acetylaminofluorene (N-OH-AAF)-glucuronide was measured after 2 and 4 weeks of dietary administration of 0.03% AAF with or without 1.6% indole. The amount of [9-14C]N-OH-AAF that was excreted as the glucuronide following a single i.p. injection of [9-14C]AAF was lower after 2 weeks in animals fed AAF and indole, as compared to those fed AAF alone [1.5 +/- 1.2% versus 19.6 +/- 3.6% S.E. (p less than 0.001)]. After 4 weeks of AAF administration without indole, the biliary excretion fell to 4.8 +/- 2.1%. This was also significantly higher than that of the animals fed both AAF and indole [1.8 +/- 1.2% (p less than 0.025)]. The suppressive role of indole on the conjugate excretion was also reflected in a decreased biliary excretion of all [9-14C]AAF metabolites in animals treated with indole alone. In the 2nd experiment, the protective action of indole was assessed by survival following daily i.p. injections of N-OH-AAF and Na2SO4 solution. Na2SO4 increased the hepatotoxicity of N-OH-AAF. Indole suppressed the toxicity of N-OH-AAF even in the presence of Na2SO4. This protective role of indole was partially overcome only when excess sulfate was coadministered. These results indicate that indole suppresses the biliary excretion of the O-glucuronide of N-OHAAF during the initial exposure of the animal to the carcinogen, possibly reflecting decreased N-OH-AAF formation. Indole also modifies the metabolism of AAF FOLLOWING N-hydroxylation, perhaps activating N-OH-AAF, depending upon the concentration of sulfate available.


The effect of gonadal ablation on transplacentally induced neurogenic tumors in hamsters. The effects of gonadectomy on tumors induced transplancentally by the ethylnitrosourea precursors, ethylurea and sodium nitrite, were investigated in hamsters. The pregnant hamsters were exposed to four daily doses of ethylurea (100 mg/kg) and sodium nitrite (50 mg/kg) administered from Day 12 to 15 of pregnancy. Weaned offspring were gonadectomized when they reached the age of 5 weeks. Orchiectomized male progeny showed a multiplicity and greater frequency of peripheral nervous system tumors and of any other tumor types than did intact males or their ovariectomized and intact female siblings. The possible inhibitory effects of endogenous androgens on the development and growth of neurogenic tumors in the peripheral nervous system and the influence of an induced endocrinal imbalance on prenatally induced neoplasms are discussed.


Non-histone chromosomal proteins of chemically transformed neoplastic cells in tissue culture. Chromatin proteins from control and dimethylnitrosamine-transformed baby hamster kidney cells were comparbd by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results indicate that non-histone chromosomal proteins from transformed cells contained protein components of low and intermediate electrophoretic mobility, which were deficient in normal cells. Comparison of the relative amount of incorporation of labeled amino acids into non-histone chromosomal proteins showed that protein components with a molecular weight of about 60,000 M.W. had a markedly increased labeling activity in the chemically transformed cells. These results suggest that changes in non-histone chromosomal proteins are associated with neoplastic transformation by chemical carcinogens.


Placenta-like alkaline phosphatase in gynecological cancers. In 302 patients with tumors of the cervix, corpus uteri, and ovaries, assessment by clinical staging (tumors-nodules-metastasis system) (4) and histopathology has been related to the presence of serum heat-stable, placenta-lide alkaline phosphatase (PLAP) activity. Early stages of cervical tumors show the highest incidence of this isoenzyme. In advanced stages of this disease, a decrease in frequency was observed that might be interpreted as the result of gradual dedifferentiation of the tumor cells to a point where synthesis of PLAP became undetectable. The same observation was made in adenocarcinomas of the corpus uteri, i.e., patients with advanced disease tended to have the lowest incidence of serum PLAP. Only in cancers of the ovaries did we find a positive correlation between this enzyme marker and the extent of the disease. In more than one-third of the patients examined, PLAP levels were an index of the tumor burden.


Exchangeable intracellular methotrexate levels in the presence and absence of vincristine at extracellular drug concentrations relevant to those achieved in high-dose methotrexate-folinic acid "rescue" protocols. Studies were undertaken to (a) assess intracellular methotrexate (MTX) levels at extracellular drug concentrations comparable to those achieved in high-dose MTX-folinic acid rescue protocols and (b) establish whether there is a rationale for the use of vincristine in these regimens. The data indicate that only low levels of exchangeable MTX (intracellular MTX in excess of the tightly bound fraction) accumulated in Ehrlich ascites tumor cells at high extracellular MTX concentration. For instance, the exchangeable steady-state intracellular MTX level was approximately 6.5 muM when the extracellular drug concentration was 85 muM. Over the interval of these experiments, exchangeable intracellular MTX did not exceed approximately 10 muM even when extracellular MTX was raised to 250 muM. These exchangeable intracellular MTX concentrations are comparable to those levels required experimentally to suppress (a) tetrahydrofolate synthesis from dihydrofolate and (b) tetrahydrofolate-dependent purine, pyrimidine, and amino acid synthesis in these cells in vitro. Vincristine (10 muM) augmented net MTX accumulation when the extracellular MTX level was 10, 100, or 250 muM. The limited capacity of cells to accumulate exchangeable intracellular MTX and the apparent role for this intracellular MTX component in achieving the metabolic effects of this agent may account for the necessity for high MTX blood levels in the treatment of some tumors and may be the basis, in part, for the enhanced chemotherapeutic efficacy of high-dose MTX regimens. These studies provide a rationale for the combined use of vincristine and MTX in high-dose MTX protocols. The addition of vincristine may permit the achievement of the level of exchangeable intracellular MTX that is required to critically inhibit tetrahydrofolate synthesis without an increase in the extracellular MTX concentration. This may permit a reduced MTX dose, diminishing the excretory load on the kidney and minimizing nephrotoxicity due to deposition of MTX in the renal tubule and interstitium. While the data indicate that the ratio of the concentration of exchangeable intracellular MTX to the extracellular drug concentration may be very low under steady-state conditions at high extracellular drug levels, further studies are required to establish that these steady-state gradients for MTX represent nonequilibrium conditions.


Tumor colony formation by Friend virus-infected cells in immunosuppressed mice. We have investigated the role of host immunological factors in the formation of "tumor colonies" in the spleens of unirradiated C57BL/6 X C3Hf/Bi FI mice 9 days after i.v. injection of spleen cells from Friend virus (FV)-infected C3Hf/Bi donors. Pretreatment of hosts with antilymphocyte serum (ATS) increased the number of tumor colonies. Pretreatment with formalinized FV-infected cells had the opposite effect, and ATS diminished the inhibitory effect of preimmunization. Cell suspensions from 11 individual FV-infected donors were examined. The suspensions differed with respect to their behavior on transplantation into untreated and ATS-pretreated F1 hybrid hosts. With several suspensions, the number of tumor colonies produced was approximately proportional to the number of cells injected; in all of these, ATS increased the slope of the line relating colony number to cell number. With most of the suspensions, tumor colony-forming efficiencies in untreated hosts strikingly decreased with increasing number of cells injected; ATS induced an increase in the number of tumor colonies and rendered the colony-forming response more nearly proportional to cell number. With two suspensions, few or no colonies developed; pretreatment with ATS had no significant effect. When the 11 cell suspensions were considered together, a proportional relation was found between the magnitude of the ATS effect (i.e., colony number in the presence of ATS minus colony number in the absence of ATS) and the colony-forming efficiency in ATS-treated mice. The ATS effect on the average was equivalent to a 2-fold increase in tumor colony-forming efficiency. We interpret these findings to indicate that two factors interact to determine the number of tumor colonies produced by spleen cells from FV-infected C3H donors in untreated F1 hybrid hosts. One is a property of the FV-infected cell population and includes its frequency of tumor colony-forming units; this factor varies widely among different cell suspensions. The other is a property of the tumor colony-forming units-host interrelationship and includes the vulnerability of tumor colony-forming units to the host immune response elicited by the injected cells; this factor appears to be constant with different cell suspensions. The present results show that the two factors can be dissociated in immunosuppressed hosts.


Effect of N1-(2'-tetrahydrofuryl)-5-fluorouracil and 5-fluorouracil on nucleic acid and protein biosyntheses in Ehrlich ascites cells. N1-(2'-Tetrahydrofuryl)-5-fluorouracil (FT-207) is a derivative of 5-fluorouracil (5-FU) and has been accepted as a new chemotherapeutic drug. The inhibitory effects of FT-207 and 5-FU on nucleic acid and protein biosyntheses in Ehrlich ascites cells were compared. Both drugs markedly inhibited the incorporation in vivo of the labeled precursors into nucleic acid and protein. The inhibitory effect of FT-207 on DNA and RNA synthesis lasted for a long period of time. Two hr after administration of 5-FU (250 mug/g body weight), the absolute size of uracil pool of liver increased by at least 50%. However, in an in vitro study, FT-207 at a concentration of 60 mug/ml produced no effect on the incorporation of precursors into DNA and RNA of Ehrlich ascites cells 3 hr incubation. If 5-FU was added to Ehrlich ascites cell suspension simultaneously with [5-3H]uridine, the incorporation of the labeled precursor into RNA increased by 30 to 50%.


Clinical and pharmacological evaluation of triazinate in humans. Twenty-four patients with advanced solid tumors and seven with acute leukemia were treated with a triazine folate antagonist, triazinate, to determine the toxicity spectrum, the maximum tolerated dose, and the pharmacological disposition of the drug. Negligible toxicity was seen with single doses of 20 to 225 mg/sq m given as a 0.5-hr infusion. Single doses of 300 to 600 mg/sq m infused over 0.5 to 3 hr caused moderate to severe central neurological impairment with light headedness, somnolence, visual disturbances, weakness, and in one patient, severe respiratory distress and cyanosis. Skin, mucous membrane, and bone marrow toxicity were mild to moderate with single doses. When triazinate was given by a multiple-dose schedule every 12 to 24 hr, there was no neurological toxicity, but mucositis, skin toxicity, and myelotoxicity were increased. Five patients developed an erythematous to desquamative rash at the site of previous or concurrent radiotherapy. Serum disappearance of triazinate was at least bionsiderable variation from patient to patient. Single i.v. doses of 300 mg/sq m resulted in serum levels of 10(-5) M or higher for 8 hr and, with repeated doses, this level could be maintained. Administration p.o. resulted in serum concentrations less than 10% of that achieved after i.v. administration. Cerebrospinal fluid concentrations were 2% or less of the serum levels in five or six patients, 1 to 4 hr after i.v. treatment. Urinary excretion varied from 12 to 71% (median, 43%) of the total dose injected during the first 24 hr. Measurable objective solid tumor responses were not seen in this Phase 1 study, although two patients had stabilization of previously advancing disease. Decreases in peripheral blasts occurred in both types of acute leukemia, but improvement in the bone marrow was not observed.


The conversion of an ovariectomy-nonresponsive to an ovariectomy-responsive mammary tumor strain. MTW9, a transplantable mammary tumor in Wistar Furth rats, shows little growth unless host serum prolactin is increased. This study compares the response to ovariectomy of MTW9-MtT, a tumor developed in rats bearing the mammosomatotropic tumor MtTW10 (serum prolactin 500 to 7000 ng/ml) with MTW9-P developed in rats given chronic perphenazine treatment (4 mg/kg/day). Serum prolactin concentrations were 150 to 600 ng/ml in MTW9-P bearing rats. MTW9-MtT does not regress after ovariectomy but does regress after surgical removal (resection) of MtT. Ovariectomy plus MtT resection leads to greater tumor regression than MtT resection alone. MTW9-P does not regress when perphenazine administration is stopped but does regress after ovariectomy, whether or not rats are given perphenazine. Administration of estradiol (10 mug/day) to rats with complete ovariectomy-induced regression of MTW9-P results in regrowth of tumor. These data suggest that MTW9-P may represent a clone of MTW9 with a lower requirement for prolactin.


The inhibition of DNA synthesis by cannabinoids. Several of the cannabinoids found in marihuana have been shown to inhibit tumor growth and increase the life-span of mice bearing the Lewis lung adenocarcinoma. When trypsin-dispersed isolated Lewis lung cells are incubated in vitro, they maintain their capacity to carry out macromolecular synthesis (RNA, DNA, protein). This process can be inhibited by cytosine arabinoside, actinomycin D, or methyl-1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, whereas cyclophosphamide, an agent that must be bioactivated, was inactive. Inhibition of DNA synthesis as measured by [3H]thymidine uptake into acid-insoluble material was used as an index of cannabinoid activity against isolated Lewis lung cells, L1210 leukemia cells, and bone marrow cells incubated in vitro delta9-, delta8-, 1-hydroxy-3-n pentyl-, and 1-delta8-tetrahydrocannabinol, and cannabinol demonstrated a dose-dependent inhibition of DNA synthesis whereas cannabidiol and 1-hydroxy-3-n-pentylcannabidiol were markedly less inhibitory in our in vitro cell systems. Furthermore, our in vitro observations with these cannabinoids are supported by in vivo tumor inhibition studies. Ring modifications as in cannabichromene or cannabicyclol abolish in vitro activity as does dihydroxylation at the 8beta and 11 positions of 1-delta9-trans-tetrahydrocannabinol. Delta9-trans-tetrahydrocannabinol demonstrated the least toxicity of all inhibitory cannabinoids in vivo; this is supported by its lesser effect on bone marrow DNA synthesis in vitro.


Stabilization of concanavalin A by metal ligands. Metal-free concanavalin A is readily and irreversibly inactivated by temperatures above 60 degrees. Manganese ion completely prevents the thermal aggregation of the protein at 60 and 70 degrees, and partially protects at 80 degrees, but shows no protective properties at 90 degrees. Managanese protection against thrermal aggregation was found to be maximal at pH 4-8. The precipitation between glycogen and Mn2+-stabilized conanavian A is partially inhibited at temperatures greater than 30 degrees, but can be reversed by cooling to room temperature...


Selectively blocked derivatives of muco-inositol and their conversion into derivatives of epi- and cis-inositol. Benzylation, and then hydrolysis, of 1, 2:4, 5-di-O-isopropylidene-muco-inositol (1) gave 3, 6-di-O-benzyl-muco-inositol (3). This was converted into a series of derivatives, including the 1, 5-di-O-benzoyl-3, 6-di-O-benzyl-2, 4-di-p-toluenesulfonate 7. The resistance to displacement of the sulfonate groups in 7 prevented conversion of 7 into an intermediate for the synthesis of aminoglycoside antibiotics. Monobenzylation of 1, followed by an oxidation-reduction cycle, yielded 6-O-benzyl-1, 2:4, 5-di-O-isopropylidene-epi-inositol (10). From this was synthesized a series of epi-inositol derivatives, analogous to the muco series but less complete. For derivatives of 1, 2:5, 6-di-O-isopropylidene-epi- and muco-inositol, the p. m. r. data indicate modified skew conformations. The reaction of the 3, 6-di-p-bromobenzenesulfonate (17) of 1 with anhydrous hydrazine proceeded in part by S-O cleavage to regenerate 1, and in part by displacement of both sulfonate groups by the same nitrogen atom. The resulting, novel 1, 4-epimino-cis-inositol was converted into further derivatives.


Hydrazinolysis and nitrous acid deamination of the carbohydrate moiety of alpha1-acid glycoprotein. Hydrazinolysis followed by nitrous acid deamination of alpha1-acid glycoprotein gave acidic and neutral mono- and oligo-saccharides that contain 2,5-anhydro-D-mannose as reducing residue: alpha-D-Manp-(1 leads to 3)-[alpha-D-Manp-(1 leads to 6)]-beta-D-Manp-(1 leads to 4)-2,5-anhydro D-mannose (1), beta D-Galp-(1 leads to 4)-2,5-anhydro-D-mannose (3), 2,5-anhydro-D-mannose, and two N-acetylneuraminic acid-containing oligosaccharides having the common partial sequence: NeuNAc-(2 leads to ?)-[BETA-D-Galp-(1 leads to 4)-2,5-anhydro-D-mannose] (5). This specific cleavage of 2-amino-s-deoxy-D-glucosyl linkages released almost quantitatively a very limited number of saccharides. Reduction with sodium borotritide of the products of cleavage allowed the precise determination of the molar proportion of 1, 3, and free 2,5-anhydro-D-mannose.


Selective N-desulfation of heparin with dimethyl sulfoxide containing water or methanol. A solvolytic N-desulfation of heparin was developed by treatment of its pyridinium salt with dimethyl sulfoxide containing 5% of water or methanol for 1.5 h at 50 degrees. Chemical and chromatographic studies showed that the solvolytic desulfation is a useful method for N-desulfation of heparin without depolymerization of the heparin molecule. The partially N-desulfated heparins were also obtained by treatment with dimethyl sulfoxide containing 5% of water at 20 degrees, and their anticoagulant activity is related to the degree of N-desulfation.


The cellular organization and nervous supply of the basilar papilla in the lizard, Calotes versicolor. The basilar papilla of the lizard Calotes versicolor contains about 225 sensory cells. These are of two types: the short-haired type A cells in the ventral (apical) part of the organ, and the type B cells with long hair bundles, in the dorsal (basal) part of the organ. The type A cells are unidirectionally oriented and are covered by a tectorial membrane while the type B cells lack a covering structure and their hair bundles are oriented bidirectionally. Apart from those differences, the type A and type B cells are similar. They are columnar, and display the features common to most sensory cells in inner ear epithelia. The sensory cells are separated by supporting cells, which have long slender processes that keep the sensory cells apart. Close to the surface of the basilar papilla a "terminal bar" of specialized junctions interlocks adjacent cells. Below this adjacent supporting cells are linked by an occluding junction. The cochlear nerve enters from the medial (neural) aspect. The fibres of the nerve lose their myelin sheaths as they enter the basilar papilla. Each sensory cell is associated with several nerve endings. All the nerves identified were afferent. Marked variations were seen between nerve endings in the basilar papilla, but no morphological equivalents of any functional differences were observed.


Distribution of the glio-interstitial system in molluscs. II. Electron microscopy of tonic and phasic muscles in the digestive tract of Aplysia and other opisthobranchs. The muscular walls of the buccal mass and the oesophagus of Aplysia rosea and Glossodoris tricolor were studied by electron microscopy. The cytological features of the muscle cells, neuro-muscular junctions and a neuro-glial junction are described. This junction between an axon and a teloglial process, in the oesophagus of Aplysia, possesses all the cytological differentiations of a typical molluscan synapse. Particular attention is drawn to the distribution of the glio-interstitial tissue and the size of the extracellular spaces in these muscular organs. The classification of these muscle into 'tonic' and 'phasic' types is discussed. From this study and other data, it is concluded that the development of the glio-interstitial tissue in the muscular organs of molluscs is correlated with the size of the extracellular spaces rather than with the type of contraction of the muscle.


Intracellular transport of sulfated macromolecules in parotid acinar cells. Intracellular transport of sulfated macromolecules in parotid acinar cells was investigated by electron microscopic radioautography after injection of 35S-sulfate. Ten minutes after injection radiosulfate was concentrated in the Golgi region. By 1 hr, much of the radioactive material had been transported to condensing vacuoles. These vacuoles were subsequently transformed into zymogen granules which contained almost 70% of the radioactivity 4 hrs after injection. These results indicate that, in addition to its packaging function, the Golgi apparatus in parotid acinar cells is capable of utilizing inorganic sulfate for the production of sulfated macromolecules. These molecules, following an intracellular route similar to that taken by digestive enzymes, become an integral component of zymogen granules. The possibility that sulfated macromolecules play a role in exocrine secretion by aiding in the packaging of exportable proteins is discussed.


Comparison of in vitro and in vivo cell cycle parameters of lymphoid cells in pig spleens. Parameters of the cell cycle of lymphoid cells were estimated by analyzing percent labeled mitoses curves after a 3H-thymidine flash. Either anaesthetized pigs were labeled and multiple biopsies taken from the spleen in vivo or isolated perfused pig spleens were labeled in vitro. The data from in vivo and in vitro experiments were very similar. The mean values for cell cycle parameters were: 20.2 to 20.5 hours for the generation time, about 0.5 to 1 hour for G2, about 1.2 to 1.3 hours for M; about 17 to 16.4 hours for S and about 1.5 to 1.7 hours for G1. The mean grain count halving time of labeled mitoses was in accordance with the measured generation time. The isolated perfused spleen seems to give results equal to in vivo data and could, therefore, be employed as a model for studying cell cycle parameters not only in animal but also in human lymphoid tissue.


Circadian changes in prolactin cell activity in the pituitary of the teleost Poecilia latipinna in freshwater. Quantitative morphometric studies with the electron microscope were made on the prolactin cells of wild freshwater sailfin mollies taken in Florida in August at four different times of day. The results indicate a circadian rhythm in the prolactin cell, the period of highest synthetic activity being from midday to evening, as indicated by the condition of nucleus, Golgi apparatus and rough endoplasmic reticulum, and by the incidence of granule release profiles. No circadian changes were detected in the ACTH cells. Several distinct sites of prolactin granule release were recognised. However, there was no conclusive evidence of granule release by any mechanism other than classical exocytosis.


The pineal gland of the mole (Talpa europaea L.) III. A fluorescence histochemical study. The pineal gland of the mole, a mammal which lives in permanent darkness, has been studied using fluorescence histochemistry. An extensive catecholaminergic innervation is demonstrated. A yellow formaldehyde-induced fluorescence, characteristic of indoleamines, was not observed. If formaldehyde vapour treatment was omitted in the procedure, numerous cells containing yellow-orange autofluorescent material could be shown. The nature and possible function of this material is discussed.


Immuno-cytochemical demonstration of the inability of the homozygous Brattleboro rat to synthesize vasopressin and vasopressin-associated neurophysin. Immuno-enzyme cytochemical investigations have shown that, (1) the hypothalamic supraoptic and paraventricular nuclei of the Brattleboro rat, as in the normal rat, contain separate neurons which produce oxytocin + neurophysin; (2) the hereditary inability of the Brattleboro rat to synthesize vasopressin and its associated neurophysin is due to a biochemical defect of separate "neurophysin-vasopressin" neurons in the supraoptic and the paraventricular nuclei. These observations strongly support the hypotheses that (1) vasopressin and its associated neurophysin are formed via a common precursor, and (2) the initial point of intracellular appearance of the hereditary defect in the Brattleboro rat lies in the synthesis of this precursor, which occurs on ribosomes. Moreover, observations have demonstrated that, in the Brattleboro rat, in addition to the hereditary inability of the hypothalamic magnocellular neurosecretory system to synthesize vasopressin, there also exists a similar hereditary defect in the hypothetical parvicellular suprachiasmatic-median eminence neurosecretory system.


Specific arrangements of membrane particles at sites of exo-endocytosis in the freeze-etched neurohypophysis. Images have been obtained from freeze-etch replicas of neurohypophyses which are consistent with the view that orderly arranged aggregates of membrane particles occur in regions where fragments of membrane are being added to and taken away from the plasma membrane during secretion. Aggregates of particles included rosette-like and necklace-like patterns similar to those described by other authors at sites of exocytosis and endocytosis.


The prebifurcation section of the axon of the rat spinal ganglion cell. The long nonmyelinated portion of the unipolar process of spinal ganglion cells resembles in many instances the perikaryon and is characterized by the following structural pecularities: 1. The surface membrane displays numerous invaginations and evaginations, which interdigitate with folds of the investing satellite cells, resulting in a considerable increase of the area of intercellular contact. The intercellular gap frequently widens to intercellular cisternae. 2. The axolemma of the most distal part of the nonmyelinated portion is undercoated by dense material and thus resembles the "initial segment" of multipolar nerve cells. 3. The unipolar process of spinal ganglion cells shows a conspicuously high density of neurotubules. The neurotubules frequently collect into fascicles in the same manner as was described for the initial segment of multipolar nerve cells. 4. The nonmyelinated part as well as the first internodes of the myelinated part of the unipolar cell process contain a highly developed axoplasmic reticulum, many-partly huge-mitochondria, a striking number of dense bodies and clusters of ribosomes. The myelin sheath of the first internodes of the unipolar process is unusually thin in relation to their axon diameters. At successive internodes the thickness of the myelin sheath increases stepwise, the Schwann cell loops of the paranodal zones changing their appearence correspondingly. In the great number of ultrathin sections scanned in this study, not one synapse was found, neither in the unmyelinated initial segment nor in the soma.


Organ culture of adult rat and mouse tracheal epithelium: I. Ultrastructure following various culture periods. Electron microscopic studies of adult rat and mouse tracheal epithelium maintained in organ culture for a period of up to 6 days were performed. In specimens cultured for 60 minutes no conspicuous micromorphological alterations could be observed. Following culture periods from 1-6 days the number of cilia in some of the ciliated cells was reduced while their structure and the other ultrastructural details of the epithelial cells were preserved. In specimens cultured for 5-6 days some additional alterations could be noticed: polymorphism of mitochondria, increased number of lysosomes, appearance of intracellular vacuoles, exhaustion of goblet cells and disappearance of granulated mast-cell like cells in the rat tracheal epithelium.


Electron microscopic evidence for a muscle layer in the wall of the terminal hepatic venule in the rat. Electron microscopically smooth muscle cells could be detected in the walls of the terminal hepatic venules of the rat liver. These muscle cells are double layered in the following venous channels, the intercalated veins. It is supposed that these smooth muscle cells play an important role as outlet sphincters of the liver sinusoids. Furthermore, fenestrations could be found in the endothelial lining of terminal hepatic venules.


Distribution of monoaminergic neurons in the nervous system of non-malacostracan crustaceans. A comparative investigation of the distribution of monoaminergic neurons in non-malacostracan crustaceans was performed with the histochemical fluorescence method of Falck-Hillarp. Two fluorophores were found: the more widespread of the two emits a green fluorescence; and the more sparsely distributed emits a yellow to brown-yellow fluorescence. Specific green fluorescent areas were shown to exist in the protocerebrum. The central body and the optic ganglia of the compound eye (where present) are always fluorescent. Moreover, the centre of the nauplius eye may have a green fluorophore, as in ostracods, and a neuropile area, here called the frontal area. These neuropile centres are known from ordinary histological studies of the nervous system. In addition, there are specific monoaminergic centres, such as the so-called dorsal area of phyllopods and anostracans as well as the copepod specific areas. Specific monoaminergic areas appear in the deutocerebrum and the suboesophageal ganglion where they are particularly well developed. Presumed sensory neurons in the cavity receptor organ of Artemia saliva are shown to be monoaminergic. Monoaminergic sensory neurons have not been described previously in Arthropods. Presumed motor innervation of hind-gut and trunk muscles is also found, and it is concluded that in crustaceans neurons of every type (sensory, internuncial, motor) may be monoaminergic.


Morphological changes of osteoblasts in vitro. The appearance of neonate rat endocranial osteoblasts exposed in situ and fixed immediately was compared with that of similar osteoblasts organ cultured for short periods of up to 48 hours in control medium and serum alone, or with added parathyroid extract (PTE). Normal osteoblasts showed a range of variation in size and shape, degree of elongation and orientation. Culturing the osteoblasts resulted in an overall loss of elongation and ordering of the cells, and the production of dorsal ruffles which were more complex and larger in the longer culture times. PTE added to the culture medium caused an increase in cell elongation and a striking recordering of the osteoblasts into domains of parallel cells. The swirling patterns made by these domains were similar to those of the underlying bone collagen. Ruffles, where present, were small and more often peripheral than dorsal. The results indicate that fully differentiated osteoblasts are able to adapt very rapidly to survival and function in culture conditions, and that the endocytosis necessary for synthetic activity is suppressed by PTE although some cell movement may continue.


Eelctron microscopy of unstained fresh air-dried spreads of mouse pancreas acinar cells and energy dispersive x-ray microanalysis of zymogen granules. Electron microscopy of unstained, fresh air-dried spreads of the mouse pancreas has disclosed various substructures of acinar cells. The nucleus with a nucleolus, rough endoplasmic reticulum with ribosomes, mitochondria with dense granules, lysosomes, intracisternal granules, and zymogen granules with a core surrounded by a halo, and a boundary membrane were observable under a conventional electron microscope. Five zymogen granules of an acinar cell were examined by energy dispersive X-ray microanalysis. Peaks for phosphorus, sulfur, chlorine and potassium were detected in all of the zymogen granules examined, which confirmed a previous report (Takaya, 1975b). By increasing the integrated pulse counting time up to 300 seconds, calcium and magnesium were detected in two of the granules in addition to the elements mentioned above. No peaks for either copper or zinc were observed when nylon grids were used. The electrolyte content of zymogen granules and of acinar initial fluid is discussed in relation to the secretory mechanism of the exocrine pancreas.


Topographical differences of RNA labelling in rat brain after intraventricular administration of labelled RNA precursors. 14C-uridine or 14C-orotic acid was injected into the third or fourth brain ventricle of adult rats. The rate of incorporation of these precursors into the RNA of various brain regions was studied by autoradiography. 0.5 hour, 1 hour, 2 hours and 4 hours after the injection of labelled uridine and/or orotic acid into the third ventricle, a very uneven labelling of different brain regions was observed. The highest grain density was found over the ventricular walls and in the closely adjacent brain tissue; the intensity of labelling decreased sharply with distance from the ventricular lumen. 24 hours after intraventricular injection, a medio-lateral gradient of grain density was no longer observed. An intense labelling of leptomeninx (especially at the base of the brain) and of ependymal cells was observed at all time intervals investigated. At time intervals 0.5-2 hours the grain density of these structures surpassed by a considerable amount the grain density over neurona, glial cells or neuropile. Two hours after the injection of 14C-orotic acid into the fourth ventricle, the grains were mainly localised over leptomeningeal cells and vessels at the base of the brain and in the adjacent narrow strip of brain tissue. The rest of the brain was only very faintly labelled.


Quantitative autoradiographic assessment of 3H-estradiol uptake in immunocytochemically characterized pituitary cells. Dry-mount autoradiography was combined with peroxidase immunocytochemistry to examine estrogen uptake in four pituitary cell types. Quantification by silver grain counts was used to compare 3H-estradiol uptake in nuclei of pituitary cells 60 min after i.v. injection into short-term (control) and long-term ovariectomized and in long-term thyroidectomized rats. Under all three hormonal states, the order of labeling intensity was: gonadotropes greater than somatotropes greater than lactotrops greater than thyrotropes. Long-term ovariectomy caused a significant increase in estrogen uptake of gonadotropes, somatotropes and lactotropes, while uptake in thyrotropes decreased. Long-term thyroidectomy decreased uptake in somatotropes, lactotropes and thyrotropes while gonadotropes remained unchanged.


Freeze-fracture observations on the intercellular junctions of Sertoli cells and of Leydig cells in the human testis. Normal and feminized human testes were examined by means of freeze-fracture. In both cases, the junctional complexes between adjacent Sertoli cells show a unique characteristic feature. Many parallel linear occluding junctions are located circumferentially around the cell surface at the level of the nucleus. They are more than forty in number over one Sertoli cell surface. In the A face, the particles are not prominent on the ridge of the junctions. Instead, they are clearly seen in the center of the groove of the junctions on the B face. Gap junctions consisting of particle aggregation are not found between the Sertoli cells. The gap junctions between the Leydig cells are frequently observed in both normal and feminized testes. They are round or elliptic in contour and about several micrometers in diameter. The particles, about 8 nm in diameter, are closely packed to form a quasi-hexagonal pattern. The pits are found on the B face corresponding to the particle aggregation.


Scanning electron microscopy of bone cells in culture. Embryonic and young rat bone cells have been growing in culture and examined in the scanning electron microscopy (SEM). Compared with cells fixed in situ and taken directly from the animal, the cultured osteoblastic cells were smoother, flatter and more extensive and showed tighter intercellular contacts. Some matrix is formed in culture and undergoes at least partial mineralization as judged by the accumulation of Ca and P measured by energy dispersive x-ray analysis. Findings concerning the morphology of the collagen arrangement were indecisive. Some superficial cells, free of surrounding matrix, resembled osteocytes in normal in vivo bone. This may indicate that a proportion of the extracellular matrix produced by the cultured cells failed to polymerise into recognizable bone matrix, and that osteocytic morphology is not dependent upon the physical characteristics of the bone matrix.


A critical reevaluation of the structure of the rat uriniferous tubule as revealed by scanning electron microscopy. The fine structure of luminal surface of clearly identified portions of uriniferous tubules has been studied by scanning electron microscopy to elucidate some controversies concerning the topography of certain surface formations. The results show a characteristic pattern of the luminal surface in the region of Henle's loop, which was assumed by previous authors, to belong to the collecting tubule. Furthermore it is demonstrated that no cilia are present within the terminal portion of the collecting tubules.


Morphometric classification of neurosecretory granules in the neurohypophysis of the hagfish, Eptatretus burgeri. Neurosecretory axons in the neurohypophysis of the hagfish, Eptatretus burgeri, were statistically classified into six types according to the size of secretory granules. Thes types are comparable with those in higher vertebrates. The concentration of each axon type is different in three regions: anterior dorsal wall, posterior dorsal wall, and ventral wall. The regional differences of the hagfish neurohypophysis are discussed in relation to the regional differentiation of the tetrapod neurohypophysis into the median eminence and the pars nervosa.


Ultrastructural diversity in motor units of crustacean stomach muscles. The physiological and ultrastructural properties of muscle fiber.s comprising three motor units in the gastric mill of blue crabs are described. In their contractile properties muscle fibers in all motor units are similar and resemble the slow type fibers in crustacean limb muscles. The majority of fibers generate large excitatory post-synaptic potentials which do not facilitate strongly. Structurally two types of fibers are found. The one type has long sarcomeres (greater than 6 mum), thin to thick myofilament ratios of 5-6:1 and diads located near the ends of the A-band. The other type has shorter sarcomeres (less than 6 mum), thin to thick myofilament ratios of 3:1 and diads located at mid sarcomere level. Both types of fibers occur within a single motor unit and this differs from the vertebrate situation. Furthermore, the finding of fibers with a low thin to thick myofilament ratio of 3:1 demonstrates that they are not exclusive to fast type crustacean muscle but also occur in slow stomach muscles.


Electron microscopic study on the gill bars of amphioxus (Branchiostoma californiense) with special reference to neurociliary control. Both primary and secondary (tongue) bars of the pharyngeal gill basket are covered by epithelial cells that are continuous with the cells that line the atrium. Anterior and posterior faces of the gill bars are covered with lateral ciliated cells, which possess a single cilium, ringed by microvilli, and an elaborate basal mitochondria-rootlet apparatus. Pharyngeal faces of the gill bars are covered with ciliated pharyngeal cells, atrial faces by mucus secreting atrial cells. The surface epithelium rests on a stromal septum, a flattened tube of basal lamina which dilates to form the visceral blood vessel (along the pharyngeal face) and skeletal blood vessel (along the atrial face). This basal lamina surrounds paired skeletal rods which run through the longitudinal axis of the gill bars near the atrial face. Between the skeletal rods and atrial cells of primary gill bars is a coelomic channel lined by  epithelioid coelomic cells. Neuronal processes, some with neurosecretory granules, are located among the bases of the atrial cells. Some axons may contact lateral ciliated cells where the latter meet atrial cells, but synaptoid endings have not been found here or elsewhere in the gill bars. Nervous tissue has not been identified among lateral ciliated cells even though ciliary activity of these cells is supposedly regulated by atrial nervous tissue.


Retinal alterations induced by continuous light in immature rats. I. Fine structure and electroretinography. Immature albino rats were exposed to continuous illumination for 5-93 days and the light induced ultrastructural and electroretinographic changes were studied. Another group was exposed to continuous light for 7-9 days and then kept in complete darkness, or in cyclic light-dark up to 90 days. By comparison with the results obtained in adult animals, lesions appeared faster in the immature group. Tubular transformation of rods, phagocytosis of altered outer and inner segments with resulting changes in retinal organization, synaptic degeneration in the outer plexiform layer, and cell lysis of some photoreceptor cell perikarya are described. ERG recovery, following the period of darkness or cyclic light-dark was only partial, the amplitude of the "b" wave reached only 50-60% of the control preillumination values. However, the fine structure of the recovered outer segments was similar to that found in normal retinae.


The ultrastructure of the byssal apparatus of Mytilus galloprovincialis. IV. Observations by transmission electron microscopy. The ultrastructure of the byssus of Mytilus galloprovincialis was analysed by transmission electron microscopy in thin sections of either embedded or frozen samples. All parts of the byssus (stem core laminae, stem outer laminae, threads proximal and distal parts) appear to be formed by the same basic filamentous components organized in different ways at the submicroscopic level and embedded in a variable quantity of matrix. The filaments appear to consist of a central electron-lucent zone (3 nm in diameter), surrounded by an electron-dense rim (total diameter 7 nm). The matrix has a granular or microfilamentous structure. The stem and the threads differ greatly in their submicroscopic organization, but their basic constituents (filaments and matrix) are similar. Peculiar filamentous banded elements (FBE) were found mainly in the stem outer laminae. A relation between the ultrastructure and mechanical properties of the different parts of the byssus was established. The presence of collagen is discussed; since no morphological evidence of any of the known forms of collagen organization was revealed by electron microscopy, it is suggested that byssus collagen may be localized in the matrix and in the FBE.


Fine structure of the chromophobe in the pars distalis of the common snapping turtle, Chelydra serpentina. Chromophobes of the pars distalis in young Chelydra serpentina have sparse cytoplasm with no specific granules; however, many cytoplasmic filaments are present. The chromophobes are connected to the other cell types by desmosomes, while different types of junctional specializations occur between adjacent chromophobes. Cytoplasmic filaments traverse the cytoplasm in a random manner and terminate on both the junctional complexes and the nuclear envelope. It is proposed that, in addition to providing a structural framework, the chromophobes may be involved in integrating cellular responses of the parenchyma to changes in the endocrine milieu.


Some observations on the fine structure of the myotendinous junction in myotomal muscle of the tadpole tail. Myotendinous junctions in the myotomal tail muscles of the tadpole of Rana rugosa were examined by electron microscopy. At the site of the myotendinous junction, the sarcolemma is covered on its sarcoplasmic aspect by the connecting filament layer and the attachment layer, and on the extracellular aspect by the intermediary later and the external lamina, with associated collagen fibrils. The intermediary layer consists of filamentous structures which closely resemble "microfibrils" (Hanak and Böck, 1971), "spine-like or thread-like profiles" (Korneliussen, 1973) and "intermediary layer" (Nakao, 1975a, b) in the myotendinous junctions of other vertebrate skeletal muscles. Particularly interesting is the fact that all the coverings and linings of the sarcolemma, including the external lamina, are completely absent in the terminal segment of the finger-like sarcolemmal invagination characteristic of the myotendinous junction. Furthermore, special types of coupling between a sac of sarcoplasmic reticulum and a part of the sarcolemmal invagination are frequently observed. These couplings always occur along the region of the sarcolemma where the external lamina is absent. The couplings show features similar to those to the triad, such as "SR feet", "scalloped SR membranes" and "granular content of the SR sac", suggesting that they are analogous and functionally similar to the triad and other equivalent structures.


Silicon in rat liver organelles: electron probe microanalysis. Electron probe microanalysis of unfixed freeze-substituted rat liver tissue embedded in Spurr's low viscosity epoxy resin demonstrated the occurrence of Si as well as P, S, and Cl in the nucleus, nucleolus, mitochondria, and rough endoplasmic reticulum. Chemical analysis confirmed that the Si in the organelles did not originate from instrumental contaminants. This suggests that Si may be involved in the biochemistry of these subcellular organelles.


Rhabdom changes in the shrimp, Palaemonetes. The fine structure of the principal compound eye of the shrimp, Palaemonetes, was studied under conditions of light and dark adaptation. Ommatidium the situation in other decapod crustaceans. Light and dark adapted eyes differ in that the rhabdom changes its shape; morphological evidence suggests a possible sequence of events involving production, utilization, and degradation of photoreceptor membrane, a discontinuous process occurring only during changes from light to dark and dark to light. A hypothesis of membrane turnover is proposed.


The development and maintenance of smooth muscle in control and aneurogenic amphibians (Ambystoma). Ultrastructural investigations showed that development and maintenance of smooth muscle was similar in control and aneurogenic amphibian larvae. This applies to both multi-unit and unitary smooth muscles. The gut musculature displayed a regional variation in smooth muscle morphology and a variety of intermuscular appositions even under conditions of nervelessness.


A molecular orbital study on the interaction between the cytochrome P-450 and its substrates. Models for the interaction of the cytochrome P-450 with its substrates, namely for the type I interaction are proposed in which the molecular planes of the aromatic substrate and the porphyrins of P-450 are in parallel. Optical values such as transition energies and oscillator strengths for the isolated P-450 and P-450-substrate complex are calculated by means of molecular orbital method (ASMO SCF CI method), and they are compared with the observed values. The fact that no appreciable shift in the absorption peak of Soret band of P-450 was observed upon addition of substrate is reflected well in the calculation. Similarly, the well-known fact that the absorbance of the P-450 was decreased by mixing it with the substrate is also explained well by the calculated oscillator strength for the isolated P-450 and the stacked complexes. From the good agreement between the calculated results and the observed ones, it is suggested that the proposed models might be true reflections of the interactions between the P-450 and its substrates.


Biochemical paramters of BHT-induced cell growth in mouse lung. Male mice of 7 different strains were injected i.p. with 400 mg/kg of butylated hydroxytoluene (BHT). 2 and 4 days later, the incorporation of thymidine into pulmonary DNA was significantly increased in all treated animals and this was accompanied by an increase in lung weight and pulmonary DNA. Thymidine kinase activity and DNA polymerase activity were enhanced in the lungs of BHT-treated animals and maximum activity of these enzymes appeared to precede maximum thymidine incorporation by 24 h. 3 days after BHT a good correlation was found between administered dose and thymidine kinase activity. Measuring the activity of this enzyme might serve as a convenient biochemical marker to follow and to quantitate BHT-produced cell proliferation in lung. The concentrations of cyclic AMP and the activity of adenylate cyclase were not altered by BHT on days 1-9 after administration. BHT produced also some dose-dependent, time-dependent increases in the activities of pulmonary 5'-nucleotidase and glucose-6-phosphate dehydrogenase (G6PDH), but had little effect on isocitric dehydrogenase (ICDH), pyruvate kinase (PK) and lactic dehydrogenase (LDH).


Evidence for caffeine-sensitive damage in methylazoxymethanol acetate-treated L5178Y cells. The effects of methylazoxymethanol (MAM) acetate on colony survival, cell proliferation and DNA synthesis of murine lymphoma L5178Y cells are studied. Decreased sensitivity and immediate depression of cell proliferation and DNA synthesis were found in L5178Y cells in contrast to the reports on HeLa cells. Pre-labelling with 5-bromodeoxyuridine (BUdR) did not enhance significantly the carcinogen-induced cell lethality. Post-treatment with caffeine greatly enhanced cell lethality and depression of cell proliferation. These effects of caffeine were diminished when the cells had passed through two generations following the MAM acetate treatment. Experiments with synchronized cells showed that the action of caffeine was located primarily in S phase following the MAM acetate-treatment. These results strongly suggest that in L5178Y cells, MAM acetate induces damage, which is repaired by a mechanism analogous to post-replication repair of UV light-induced damage.


Respiratory control depression by tetraalkylammonium bromides in rat liver mitochondria. Six different lipophilic (hydrophobic) organic cations, tetraethyl-, tetrapropyl, tetrabutyl-, tetrapentyl-, tetrahexyl-, and tetraheptylammonium bromide, depressed respiratory control in rat liver mitochondria. Evaluation of mitochondrial responses in terms of a quadratic equation in log P (an index of lipophilicity) indicated that the NADH dehydrogenase receptor site for inhibitor (diminution of control of glutamate, alpha-ketoglutarate, and beta-hydroxybutyrate respiration) was more lipophilic than receptor sites for flavin-linked substrates (reduction of control of succinate, choline and alpha-glycerophosphate respiration). The succinate dehydrogenase receptor site for inhibition by the tetraalkylammonium bromides was more hydrophillic (less lipophilic) than the choline or alpha-glycerophosphate dehydrogenase receptor sites. Depression of respiratory control may be a function of charge density and of lipophilicity at specific inner membranal sites and the susceptible site may differ for different respiratory substrates.


The circadian variation of hepatic microsomal drug and steroid metabolism in the golden hamster. Rhythmic variations in a number of parameters of hepatic microsomal drug and steroid metabolism and binding were observed in the adult male golden hamster. With an illumination schedule of a light phase between 06.30 and 18.30 h, maximal activities were encountered between 04.00 and 08.00 h with corresponding minima between 16.00 and 18.00 h. In addition, kinetic changes were observed in both the metabolism and spectral interaction of aniline and biphenyl at times of maximal and minimal activities. Possible mechanisms responsible for the circadian variation in hamster hepatic microsomal xenobiotic metabolism are discussed.


Biochemical changes in rat kidney on exposure to elemental mercury vapor: effect on biosynthesis of metallothionein. Evidence is presented that exposure of rats to elemental mercury vapor results in increased amounts of a metallothionein-like protein in kidney tissue but not in liver. After three or more daily exposures, each of 2 h duration, to elemental mercury vapor, more than 50% of the mercury in kidney tissue is bound to a protein having a molecular weight (mol. wt.) of about 10 000 as determined by Sephadex G-75 gel filtration chromatography. Cystine is incorporated into a 10 000 mol. wt. protein fraction from kidneys of rats which were injected with [U-14C] cystine after five daily 2-h exposures to mercury vapor. In contrast, no significant incorporation of [U-14C] cystine into this protein fraction was observed in kidneys of control rats or in livers of both control and mercury vapor-exposed rats. The in vivo incorporation of 109Cd into the fraction followed the same pattern as that of [14C] cystine in rats injected with tracer doses of CdCl2 labeled with radioactive 109Cd isotope. This 10 000 mol. wt. protein, newly synthesized in response to repeated exposures to mercury vapor, exhibited identical properties to metallothionein, namely in its subcellular localization, molecular weight, heat stability and isoelectric points. A significant incorporation of [U-14C]-cystine into this protein in rat kidney alone on exposure to mercury vapor confirms its induced biosynthesis in the kidney.


The binding of benz(a)anthracene derivatives to glyceraldehyde-3-phosphate dehydrogenase and mammary tissue following exposure to laboratory light. 7,12-Dimethylbenz(a)anthracene (DMBA) and 7-methoxymethyl-12-methylbenz(a)anthracene (MeO-DMBA) are converted to a number of products during short exposures in aqueous suspension to laboratory illumination. The mixture of products binds to glyceraldehyde-3-phosphate dehydrogenase (GPDH) while inhibiting its activity but there is no apparent relationship between the binding and inhibition of enzyme activity. There is little, or no, binding or enzyme inhibition when the compounds are protected from light. 7-Bromomethyl-12-methylbenz(a)anthracene (Br-DMBA) binds to GPDH whether photoactivated or not but enzyme inhibition depends upon light exposure. The binding of light-exposed DMBA by surviving rat mammary tissue was five-times greater than with the unchanged hydrocarbon. Binding of MeO-DMBA products also occurred after light exposure but not in the dark.


Studies on the mechanism of inhibition of 2-acetylaminofluorene toxicity by butylated hydroxytoluene. Male rats were placed on a diet containing 0.05% (w/w) of the hepatic carcinogen 2-acetylaminofluorene (AAF). They ceased to gain weight. However, when the carcinogenic diet was supplemented with butylated hydroxytoluene (BHT) (0.5% w/w), an antioxidant, the animals gained weight at approximately one-half of the normal rate. This observation led to a series of experiments aimed at elucidating the mechanism(s) by which BHT reduced the toxicity of AAF. These initial studies were directed towards the effect of BHT on the extent and duration of the covalent binding of AAF with DNA. BHT feeding was shown to reduce the binding of carcinogen to hepatic DNA. Studies employing cells in culture demonstrated that BHT does not influence either excision repair or post-replication repair of DNA. These data indicate that a potential mechanism of action of BHT is at the anti-initiation level of carcinogen-induced DNA damage.


Evidence for a dual innervation affecting local blood flow in the hypothalamus of the conscious rabbit. We have attempted to evaluate the role of adrenergic nerves which arise from the superior cervical ganglia or which are intracerebral throughout their course, in the control of local cerebral blood flow (CBF). Hypothalamic blood flow (HBF) was measured in the conscious rabbit by the 133Xe-clearance technique. Stimulation of the upper brainstem, using 5-Hz, 3-V, 1-msec, square wave pulses, increased by HBF by a mean of 7.6 ml/100 g per min (P less than 0.005). This effect was abolished by the intrahypothalamic injection of the beta-adrenoreceptor blocker, propranolol, and by chemical sympathectomy of the hypothalamus or of the upper brainstem with 6-hydroxydopamine, but was not altered by bilateral cervical ganglionectomy. Intrahypothalamic injection of 0.1 mug of tyramine caused a mean decrease in HBF of 15.6 ml/100 g per min (P less than 0.001). This effect of intrahypothalamic injection of tyramine was abolished by bilateral cervical sympathectomy but not by chemical sympathectomy of the upper brainstem. These results support the idea that local CBF, at least in the hypothalamus, is mediated by two distinct pathways. The first consists of the sympathetic nerves which arise in the cervical ganglia, and which activate intrahypothalamic alpha-receptors to cause constriction. The second is an entirely intracerebral noradrenergic pathway which stimulates beta-receptors to cause vasodilation.


The effects of tension on acetylstrophanthidin-induced transient depolarizations and aftercontractions in canine myocardial and Purkinje tissues. Transmembrane potentials and contractile activity were recorded from isolated canine Purkinje and ventricular muscle preparations exposed to acetylstrophantidin (AS) and subjected to a resting tension equal to 80% of that required to elicit peak developed tension. AS induced transient depolarizations (TD's) accompanied by aftercontractions in Purkinje tissue. AS also induced aftercontractions in 11 muscle preparations, and in seven of these the mechanical events were associated with TD's. Aftercontractions and TD's in both and the coupling intervals were directly related to the preceding basic cycle length (BCL). The amplitudes of aftercontractions and TD's reached a maximum at a coupling interval of 600-700 msec. Tension increased the amplitude of TD's in Purkinje tissue and promoted the appearance of TD's in muscle. TD's in muscle occasionally reached threshold in the presence of tension. The results of this study suggest that stretch or increased resting tension may promote the types of cardiac arrhythmias that are causally related to digitalis-induced TD's. The results are compatible with the hypothesis that TD's are caused either by a transmembrane influx of calcium or by an internal release of calcium ions.


Vascular responses to arachidonic acid in the perfused canine lung. We compared the effects of arachidonic acid (AA), the bisenoic prostaglandin precursor, with those of prostaglandin F2a (PGF2a) and norepinephrine (NE) on pulmonary vascular resistance in the isolated (in situ), perfused canine lung lobe. The isolated lobe was perfused with autologous blood or an artificial perfusate under conditions of constant flow. Lobar artery and venous pressures were constantly monitored after bolus injections of AA, PGF2a, and NE into the inflow cannula. AA (100 mug/kg produced a significant increase in the pressure gradient (93.3 +/- 8.4%, SE) in the lobe. Similarly, PGF2a (1 mug/kg) significantly increased the pressure gradient (41.2 +/- 6.5%), as did NE (1 mug/kg, 41.6 +/- 3.2%). Aspirin (25 mg/kg) completely blocked the pulmonary vascular effect of AA, but did not affect the response to PGF2a, Linoleic acid, a control fatty acid, did not produce pulmonary vasoconstriction. The pressor effect of AA was not blocked by pretreatment with phentolamine, propranolol, cyproheptadine, or atropine. The use of an artificial perfusate free of cellular elements did not prevent the vasoconstrictor action of AA. The times to onset of action of the three agents were similar, and short. These results suggest that AA is converted into vasoactive intermediates or a prostaglandin, and the vasoactive intermediates or the prostaglandin act directly on precapillary pulmonary vascular smooth muscle rather than through platelet, plasma, adrenergic, or cholinergic mechanisms.


Depletion of cardiac norepinephrine during two forms of hemolytic anemia in the rat. Knowledge of the status of cardiac norepinephrine (NE) during anemia could lead to a better understanding of the role the sympathetic nervous system plays in cardiac function during anemia. Rats were made anemic by treatment with phenylhydrazine (PHZ). After the rapid onset of anemia, 60% of the stored NE in the heart was lost within 48 hours after treatment. Associated with the loss of cardiac NE was an increase in the wet weight of the heart, which reached a value 40% above control 48 hours after treatment. PHZ itself probably does not directly mediate this depletion of NE, since the vas deferens, brain and spleen had a normal store of NE at 48 hours. This contention was supported when rats, treated with PHZ, were transfused with normal rat red blood cells. This transfusion resulted in PHZ-treated rats which were not anemic. The hearts of these rats were not depleted of NE, but the hearts of the nontransfused, PHZ-treated controls were. Anemia also was induced by treating rats with anti-rat red blood cell serum. The hearts of these rats also were depleted of NE. These experiments show that during two forms of anemia there is a loss of NE from the sympathetic neurons innervating the heart. The effect of this on regulation of cardiac function remains to be determined.


Renal tubular transport of 3H-digoxin in saline diuresis in rats. We evaluated urinary excretion and tubular transport of 3H-digoxin by three different methods in anesthetized rats made diuretic by infusion of 2.5% saline. In one group small volumes of 3H-digoxin and 14C-inulin were injected simultaneously into surface proximal convolutions, and urine was collected serially from both ureters. Digoxin recovery was lower after early (62.1 +/- 5.3%) than after late (86.9 +/- 7.7%) proximal administration but inulin recovery was complete (99.6 +/- 2.7%) after all injections. Most of the digoxin was excreted simultaneously with inulin. Delayed recovery was low. In another group of rats digoxin and inulin were applied directly to the capsule of the left kidney. Two-thirds of the recovered digoxin appeared from the left ureter and one-third from the right. The difference (41.9 +/- 7.4%) is an estimate of transtubular digoxin influx. Digoxin excretion preceded inulin only on the left. Digoxin to inulin concentration ratios were 6 times higher from the left than the right, whereas inulin recoveries from the two sides were similar. In a third group of rats tubular fluid was collected from surface convolutions of proximal and distal tubule. In the accessible segment of the proximal tubule 35.9% of the filtered digoxin was reabsorbed. In the more distal nephron, drug was added into the lumen; this resulted in a net urinary excretion of 80.2 +/- 18.2%. These findings are compatible with free filtration of digoxin at the glomerulus followed by passive proximal tubular reabsorption and an influx against a concentration gradient in the distal nephron.


Effect of hindlimb isolation procedure on isogravimetric capillary pressure and transcapillary fluid dynamics in dogs. We measured isogravimetric capillary pressure (Pci) and plasma colloid osmotic pressure (pip) in isolated dog hindlimbs. A very rapid isolation technique and a perfusion technique involving no weight change were developed in order to compare the effects of different isolation procedures. Also, a "previous isolation procedure" (PIP) was used to approximate isolation procedures previously reported; this procedure included (A) anesthesia for 1 to 1 1/2 hours before limb isolation, (B) 1/2 hour of denervation before isolation, and (C) perfusion after isolation for 1/2 hour at an arterial pressure of 100 mm Hg and a venous pressure of 6 mm Hg. These different procedures altered average (+/- SE) capillary pressure and fluid dynamics in the hindlimb as shown in the foly pressure in the intact resting dog hindlimb may be 8 mm Hg below plasma colloid osmotic pressure, or about 9 mm Hg, and that many reported values have been heavily influenced by the isolation technique.


The role of angiotensin in the canine renal vascular response to barbiturate anesthesia. The influence of barbiturate anesthesia on renal blood flow was assessed by the xenon washout method in trained dogs with catheters chronically implanted in the renal artery. Anesthesia induced with either thiopental sodium or pentobarbital sodium resulted in a striking reduction in renal blood flow (4.1 +/- 0.1 vs. 2.6 +/- 0.2 ml/g per min; P less than 0.001) without a change in arterial pressure. The reduction in blood flow was prevented by a high salt intake and partially reversed by agents which interrupt the renin-angiotensin system (BPF 9a; 1-Sar,8-Ala-angiotensin II; propranolol) but not by alpha-adrenergic blocking agents (phentolamine and phenoxybenzamine). Anesthesia blunted the renal vascular response to angiotensin II (P less than 0.0005) whereas responsiveness to norepinephrine was increased (P less than 0.05). We conclude that barbiturate anesthesia induces a major, angiotensin-mediated renal vascular response which must be considered in the interpretation of experiments performed under these conditions.


The effect of lidocaine on diastolic transmembrane currents determining pacemaker depolarization in cardiac Purkinje fibers. We studied the effects of lidocaine (1-5 mg/liter) on the diastolic currents of sheep Purkinje fibers by the two-microelectrode voltage clamp technique to obtain additional information on how lidocaine decreases the slope of spontaneous diastolic depolarization of mammalian Purkinje fibers. During voltage clamps we measured both the magnitude and time course of activation and deactivation of the time- and voltage-dependent potassium "pacemaker current" (iK2), and also the steady state current-voltage relationship throughout the pacemaker voltage range. At a concentration of 1 mg/liter lidocaine had no effect on the amplitude of iK2. In contrast, at 5 mg/liter, lidocaine diminished the magnitude of iK2 throughout the voltage range of pacemaker depolarization. Lidocaine (1-5 mg/liter) had no effect on either (1) the transmembrane voltage at which iK2 is half-activated, (2) the reversal voltage for iK2, or (3) the kinetics of iK2. Lidocaine (1-5 mg/liter) increased the steady state outward transmembrane current. This effect of lidocaine can be attributed to a variable contribution from both an increase in time-independent outward potassium current (iK1) and a decrease in background inward current.


Mineralocorticoid secretion in essential hypertension with normal and low plasma renin activity. In 19 control subjects, 33 patients with essential hypertension and normal plasma renin activity (PRA) and 11 patients with low PRA, secretory rates of 18-hydroxy-11-deoxy-corticosterone (18-OH DOC), 11-deoxycorticosterone (DOC) and corticosterone were measured. Patients with low PRA were significantly older and had higher arterial pressure and slightly lower plasma potassium levels than patients with normal PRA. Mean 18-OH DOC secretion rate was higher in patients with normal PRA (603 +/- 112 SEM mug/24 hr) than in control subjects (219 +/- 19) and considerably higher (P less than 0.001) in patients with low PRA (1800 +/- 472). DOC and corticosterone secretion rates were within normal limits in most hypertensive patients. Plasma aldosterone was significantly higher in the hypertensive population than in control subjects whereas no significant difference was observed between the low- and normal-renin groups. A significant (P less than 0.01) mutual positive correlation was found between the secretion rates of 18-OH DOC, DOC and corticosterone in patients with low plasma renin activity. In contrast, there was no correlation between the secretion rates of the three mineralocorticoids in control subjects and patients with normal plasma renin activity. These data suggest a biosynthetic variation of the mineralocorticoid pathways in essential hypertension.


Precordial ST-segment mapping. 2. Effects of oxygen inhalation on ischemic injury in patients with acute myocardial infarction. Precordial ST-segment mapping was serially applied in the Coronary Care Unit for the study of the effect of oxygen inhalation on the ischemic injury in 17 patients with acute anterior transmural myocardial infarction. A 49-lead ECG system was used. The sum of all ST elevations (sigmaST) recorded was taken as an index of magnitude of ischemic injury and the number of recording sites showing ST elevation (NST) was taken as an index of extent of ischemic damage. Stability of the precordial maps was observed over a period of one hour while the patients were on ambient air. Oxygen inhalation for a mean of 66 min resutled in a fourfold increase of PaO2 and a mean of 16% reduction of both sigmaST and NST. When the patients were returned to ambient air breathing, a mean of 13% increase of sigmaST and 19% of NST from the levels recorded during oxygen inhalation were observed. Levels of sigmaST and NST on ambient air following discontinuation of oxygen inhalation were not significantly different from the corresponding values from maps recorded before onset of oxygen breathing. Blood pressure and heart rate remained unchanged throughout the study. Clinical status of the patients was unchanged during the study period save for two patients who showed changes in intensity of their chest pain.


Asymptotic slope of log pressure vs log volume as an approximate index of the diastolic elastic properties of the myocardium in man. The goal of this study was to develop, on a rational basis, an index of the intrinsic diastolic elastic properties of the left ventricle. A relatively simple analytic model employing a thin-walled spherical geometry coupled with an approximate formulation of a two-dimensional constitutive relation, was used to examine the primary determinants of the pressure-volume relationship of the intact heart. The results permit comparison with other indices of compliance or wall stiffness. The slope of the dP/dV vs P curve was found to be sensitive to the non-linear elastic constant K, but is also sensitive to variations in cardiac muscle volume. VdP/dV was found to be sensitive to pressure. m = d(log P)/d(log V) = (V/P)(dP/dV) is proposed as an index sensitive to K and relatively insensitive to both pressure and initial cardiac geometry. The index is compared with published studies. Using the data of Fester and Samet, mean values of the asymptotic log-log P-V slope, m, evaluated at end-diastole for normal, idiopathic hypertrophy, mild, moderate and severe coronary artery disease were 3.95 +/- 0.60 (SEM), 5.05 +/- 1.60, 5.24 +/- 0.96, 8.35 +/- 2.06, and 15.13 +/- 3.0, respectively. At values of LVEDP less than 7 mm Hg the concept of simple distension is questioned. The advantages and limitations of this approximate index are discussed. This index seems to afford a practical measure of the elastic properties of the wall over a rather wide range of pressure, volume, wall mass and wall thickness.


Human ventricular refractoriness. Effects of cycle length, pacing site and atropine. The effective refractory period of the right ventricle (ERP-V) was measured in 27 patients during atrial or ventricular pacing using the ventricular extra stimulus method. Pacing was conducted with impulses of 1.5-2 times diastolic threshold. The ERP-V was directly related to the basic cycle length (BCL) although the ERP-V was always greater for atrial pacing than for ventricular pacing at a given BCL. The ratio ERP-V/BCL was greater at shorter cycle lengths indicating that a larger fraction of the cycle was refractory at faster heart rates. The ratio ERP-V/QT interval did not change over a range of BCLs, but the ratio was larger for atrial pacing (.77 +/- .05 SD) than for ventricular pacing (.60 +/- .05). Atropine (1 mg i.v.) was given to six patients. The drug did not affect the ERP-V in six of eleven determinations, prolonged ERP-V twice and shortened it slightly three times. Measurements were reproducible over an hour but varied at a given BCL when measured on separate days. Asymptomatic repetitive beating occurred in seven of 27 patients when the premature stimulus was within 20 msec of the ERP-V. The effective refractory period of the right ventricle in man can be determined reproducibly and with safety. Changes induced by various perturbations parallel results from in vitro single cell and myocardial studies.


Electrophysiological and clinical observations in patients with alternating bundle branch block. Electrophysiological studies (His bundle recordings and atrial stimulation) were performed in nine patients who manifested periods of both right and left bundle branch block (RBBB and LBBB). In seven of the patients, alternating bundle branch block appeared to reflect intermittent or chronic bundle branch block superimposed on incomplete (but electrocardiographically complete) block of the contralateral bundle branch. In three of these seven, shift from one bundle branch block pattern to the other was associated with reproducible change in H-V (mean change 30 msec), and could be induced by alteration of cardiac rate with carotid massage, coupled atrial stimulation, and rapid atrial pacing. In one of the seven, RBBB with a P-R of 0.20 seconds preceded chronic LBBB with a P-R of 0.24 seconds, implying that RBBB had been incomplete. In three of the seven, although a definite mechanism of alternation could not be demonstrated, transient contralateral bundle branch block occurred superimposed on chronic ipsilateral bundle branch block, implying that the ipsilateral block was incomplete. Two patients manifested periods of narrow QRS, LBBB, RBBB, and paroxysmal A-V block. Based upon pathological data (one case), this pattern appeared to reflect a lesion involving the distal His bundle and proximal bundle branches. In the total group of patients, clinical course was primarily determined by the severity of heart disease and not by occurrence of A-V block. The conduction defect in the majority of patients was surprisingly benign.