Usefulness of drill biopsy in the diagnosis of breast tumors. A retrospective study was undertaken to analyze the usefulness of 600 drill biopsies taken from 557 breast tumors. Based on the first biopsy, the malignant or benign nature of the tumor was accurately estimated in 93.6% of the cases. A diagnosis of cancer was established microscopically in 87.4% of the tumors. In our opinion, this method proved to be valuable and is recommended for outpatient clinics.


Brain metastasis from prostatic carcinoma. Between 1959 and 1971 there were 91 patients with clinically diagnosed prostatic carcinoma who were autopsied at Roswell Park Memorial Institute. In four of these 91 (4.4%) intracerebral metastasis were found at autopsy, but only in one of these four was the diagnosis arrived at pre-mortem. This report describes the diagnosis and management of intracerebral metastasis from prostate carcinoma. It appears, on the basis of our initial experience, that the clinical diagnosis of this entity deserves more frequent consideration.


Standardized nomenclature for inbred strains of mice: sixth listing. Rules for designating inbred strains of mice are presented, along with a list of strains with their origins and characteristics, a table of biochemical polymorphisms, and standard subline designations.


Testicular relapse in childhood acute lymphocytic leukemia during bone marrow remission. Seven children with acute lymphocytic leukemia developed testicular swelling during bone marrow remission. Needle biopsies and one orchiectomy specimen revealed leukemic infiltration. Another patient encountered concurrent medullary and testicular relapses. The leukemic nature of the testicular infiltration can be readily confirmed on needle biopsies. Orchiectomy should be discouraged. The leukemic infiltration mainly took place in the interstitial spaces. The seminiferous tubules were atrophic and lined by Sertoli cells. Occasional spermatogenic cells could be found in some cases. The leukemic cells were also observed to invade and accumulate beneath the Sertoli cell layer. Destruction of the tubules by the infiltrate was seen in advanced cases. Postradiation biopsies were characterized by fibrosis. The sigeveloped subsequent marrow relapse despite the effective control of the testicular relapse by irradiation. Another important observation is the high incidence of testicular relapse (16.2% so far).


Specific receptors for glucocorticoid in the cytoplasm of the liver of AH 130 tumor-bearing rats. Specific receptors for dexamethasone (11beta, 17alpha, 21-trihydroxy-9alpha-fluoro-16alpha-methyl-1,4-pregnadiene-3,20-dione) in the cytoplasm of the liver from AH 130 (solid type) tumor-bearing rats markedly increased in the advanced stage of tumor growth. The cytoplasmic receptors of the livers of normal and tumor-bearing rats differed in their affinities for dexamethasone, and their apparent equilibrium (dissociation) constants (K) for dexamethasone were 4.0 and 2.6 X 10(-9) M, respectively. The rates of dissociation of dexamethasone-receptor complexes and the heat denaturations of the receptors in the livers of normal and tumor-bearing rats were similar. The glucocorticoid receptors of tumor-bearing rat liver had slightly higher affinities than did those of normal liver for all the steroids tested. Only a trace amount of receptors for dexamethasone could be detected in the cytoplasm of AH 130 ascites cells.


An investigation into the binding of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one to DNA in vitro. After metabolic activation the carcinogen 15,16-dihydro-11-[3H]methylcyclopenta[a]phenanthren-17-one binds to DNA in vitro, and this binding is prevented by 7,8-benzoflavone. Radioactivity cannot be removed from the DNA with organic solvents or by chromatography on Sephadex G-50, even after heat denaturation of the DNA. Enzymatic hydrolysis yields radioactive fractions, which elute from a column of Sephadex LH-20 immediately after the natural nucleosides. At least two species of reactive metabolites are involved in this bending, those with a half-life of a few hr and others with greater stability. After extraction from the aqueous incubation mixture, they could be detected in discrete polar fractions from separations of the complex metabolite mixture by high-pressure liquid chromatography. Their ability to bind to DNA decreased with time at ambient temperature, and they were rapidly deactivated by acid. 7,8-Benzolflavone acted by suppressing the formation of polar metabolites derived from enzymatic oxidation of the aromatic double bonds. The inhibitor had no effect on the enzymes hydroxylating saturated carbon; hence it is unlikely that metabolism of the methyl group is important in conversion of this carcinogen to its proximate form, although the presence of the 11-methyl group is essential for carcinogenic activity in this series.


The interaction of rhodium(II) carboxylates with enzymes. The effect of rhodium(II) acetate, propionate, and methoxyacetate on the activity of 17 enzymes was evaluated. The enzymes were preincubated with the rhodium(II) complexes in order to detect irreversible inhibition. All enzymes that have essential sulfhydryl groups in or near their active site were found to be irreversibly inhibited. Those enzymes without essential sulfhydryl groups were not affected. In each case, the rate of inactivation closely paralleled the observed toxicity and antitumor activity of rhodium(II) carboxylates; that is, rhodium(II) propionate greater than rhodium(II) acetate greater than rhodium(II) methoxyacetate. In addition, those enzymes that have been demonstrated to be most sensitive to established sulfhydryl inhibitors, such as glyceraldehyde-3-phosphate dehydrogenase, were also most sensitive to rhodium(II) carboxylate inactivation. Proton nuclear magnetic resonance measurements made during the titration of rhodium(II) acetate with cysteine showed that breakdown of the carboxylate cage occurred as a result of reaction with this sulfhydryl-containing amino acid.


Decreased liver and lung drug-metabolizing activity in mice treated with Corynebacterium parvum. Injections of killed suspensions of Corynebacterium parvum (i.p.) in young male mice were followed by time- and dose-dependent decreases in the drug-metabolizing activity of liver microsomes and lung homogenates. In vitro assays with model substrates [aminopyrine, aniline, p-nitroanisole, and benzo(a)pyrene] were used to quantitate drug-metabolizing activity. It is likely that such decreases in mixed function oxidases activity will act to significantly alter the pharmacokinetics of concurrently or subsequently administered drugs. The results provide a possible mechanism to explain several previously reported immunochemotherapeutic interactions.


Electronic volume analysis of L1210 chemotherapy. The rapid analysis of in vivo chemotherapy on the L1210 ascites tumor grown in C57BL/6 X DBA/2F1 mice has been shown by means of an electronic volume analysis. The drugs were injected on the 4th day of tumor growth, and the cells in the peritoneal cavity were studied at 24-hr intervals on the 5th through 7th day. Using the electronic cell volume distributions, combined with labeling indices, cell morphology, and cell counts, it was found that the alkylating agents. 1,3-bis(2-chloroethyl)-1-nitrosourea and cyclophosphamide, at the dosages used, were more effective than the S-phase-specific drugs, palmitoyl ester of 1-beta-D-arabinofuranosylcytosine, vincristine, and methotrexate.


The direct spectrophotometric observation of benzo(a)pyrene phenol formation by liver microsomes. Optical spectral repetitive scan analysis during the oxidative metabolism of benzo(a)pyrene by liver microsomal suspensions reveals the time-dependent formation of an intermediate(s) of which the visible spectra resemble those of several benzo(a)pyrene phenols. Liver microsomes from 3-methylcholanthrene-treated rats showed a greater rate of formation of the phenols than did microsomes from control animals; the rate of formation catalyzed by liver microsomes from phenobarbital-pretreated rats was intermediate. When 3-hydroxybenzo(a)pyrene was used as a standard for comparison of activity, the rates of formation of phenols were compared when measured by fluorometric, spectrophotometric, or high-pressure liquid chromatographic analytical techniques. An epoxide hydrase inhibitor, 1,1,1-trichloropropene-2,3-oxide, enhanced phenol formation regardless of the source of liver microsomes, and 7,8-benzoflavone inhibited control and 3-methylcholanthrene-induced microsomal metabolism of benzo(a)pyrene, 7,8-Benzoflavone did not effect benzo(a)pyrene metabolism by liver microsomes from phenobarbital-pretreated rats. The effect of inhibitors on the spectrophotometric assay correlates well with the results obtained from benzo(a)pyrene metabolite analysis using high-pressure liquid chromatography.


The epizootiology and pathogenesis of thyroid hyperplasia in coho salmon (Oncorhynchus kisutch) in Lake Ontario. The thyroid glands of coho salmon collected at different stages of their anadromous migration exhibited progressive and extensive hyperplasia and hypertrophy. The incidence of overt nodule formation rose from 5% in fish collected in August to 24% in fish collected in October. The histological picture of the goiters was similar to that found in thiourea-treated teleosts and thiouracil-treated mammals. There was a concomitant, significant decrease in serum thyroxine and triiodothyronine values between September and October (thyroxine, 1.0+/-0.3 mug/100 ml and 0.4 mug/100 ml in September and October, respectively; triiodothyronine, 400.3+/-51.6 ng/100 ml and 80.2 ng/100 ml in September and October, respectively) and marked hypertrophy and hyperplasia of thyrotrophs. These data indicate a progressive hypothyroid condition which, although it may be linked to iodide deficiency, may well be enhanced by other environmental factors. The evidence for involvement of other factors is discussed.


Differences in benzo(a)pyrene metabolism between rodent liver microsomes and embryonic cells. Differences in benzo(a)pyrene metabolite pattern have been shown by rodent liver microsomes (Sprague-Dawley) and rodent embryo cells from Syrian hamsters and NIH Swiss mice. Rodent liver induced by methylcholanthrene shows marked quantitative variation between species. Additional pattern changes were found in mouse and hamster embryo secondary cultures with a reduction of the K-region metabolites and a marked increase in 9-hydroxybenzo(a)-pyrene. These results are indicative of a region-specific attack on the carcinogen by the cell monooxygenases which is distinct from the liver attack of microsomal enzymes on benzo(a)pyrene. These results suggest that activation and detoxification of benzo(a)pyrene may be species and tissue variable, and susceptibility and resistence to malignant transformation may be predicted on induction of a fortuitous combination of intermediate metabolic steps.


The incorporation of 5-iodo-2'-deoxyuridine into the DNA of HeLa cells and the induction of alkaline phosphatase activity. Inhibition of DNA synthesis during the period of exposure of HeLa cells to 5-iodo-2'-deoxyuridine (IUdR) inhibited the induction of alkaline phosphatase activity. This finding, taken together with previous findings that IUdR did not induce alkaline phosphatase activity in the presence of 2-fold molar excess thymidinemonstrated that IUdR incorporation into DNA is correlated with the increase in alkaline phosphatase activity. With the exception of an interim period described in the text, induction of alkaline phosphatase activity was linearly related to medium concentrations of IUdR of up to at least 3 muM. However, the extent of IUdR substitution in DNA did not appear to be related to the degree of enzyme induction. Alkaline phosphatase activity continued to increase at medium concentrations of IUdR from 1 to 3 muM, while little further substitution of DNA occurred.


Glycopeptides from the surgace of human neuroblastoma cells. Glycopeptides suggesting a complex oligosaccharide composition are present on the surface of cells from human neuroblastoma tumors and several cell lines derived from the tumors. The glycopeptides, labeled with radioactive L-fucose, were removed from the cell surface with trypsin, digested with Pronase, and examined by chromatography on Sephadex G-50. Human skin fibroblasts, brain cells, and a fibroblast line derived from neuroblastoma tumor tissue show less complex glycopeptides. Although some differences exist between the cell lines and the primary tumor cells, the similarities between these human tumors and animal tumors examined previously are striking.


Action of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver. The effects of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver were investigated. The enzyme was isolated from the nuclear fraction essentially according to the method of Baril et al.; it was characterized as the alpha polymerase on the basis of its response to synthetic templates and its inhibition with N-ethylmaleimide. Although polycytidylic acid had no effect on the DNA polymerase alpha either as a template or as an inhibitor, partially thiolated polycytidylic acid (MPC) was found to be a potent inhibitor, its activity being directly related to its extent of thiolation (percentage of 5-mercaptocytidylate units in the polymer). In comparison, the DNA polymerase beta which was purified from normal rat liver nuclear fraction, was much less sensitive to inhibition by MPC. Analysis of the inhibition of the alpha polymerase by the method of Lineweaver and Burk showed that the inhibitory action of MPC was competitively reversible with the DNA template, but the binding of the 7.2%-thiolated MPC to the enzyme was much stronger than that of the template (Ki/Km less than 0.03). Polyuridylic acid as such showed some inhibitory activity which increased on partial thiolation, but the 8.4%-thiolated polyuridylic acid was less active than the 7.2% MPC. When MPC was annealed with polyinosinic acid, it lost 80% of its inhibitory activity in the double-stranded configuration. However, 1 to 2%-thiolated DNA isolates were significantly more potent inhibitors than were comparable (1.2%-thiolated) MPC and showed competitive reversibility with the unmodified (but "activated") DNA template. These results indicate that the inhibitory activities of partially thiolated polynucleotides depend not only on the percentage of 5-mercapto groups but also on the configuration, base composition, and other specific structural properties.


Stimulation of thymidine uptake and cell proliferation in mouse embryo fibroblasts by conditioned medium from mammary cells in culture. Undialyzed conditioned medium from several cell culture sources did not stimulate thymidine incorporation or cell overgrowth in quiescent, density-inhibited mouse embryo fibroblast cells. However, dialyzed conditioned medium (DCM) from clonal mouse mammary cell lines MCG-V14, MCG-T14, MCG-T10; HeLa cells; primary mouse adenocarcinoma cells; and BALB/c normal mouse mammary epithelial cells promoted growth in quiescent fibroblasts. The amount of growth-promoting activity produced per cell varied from 24% (HeLa) to 213% (MCG-V14) of the activity produced by primary tumor cells. The production of growth-promoting activity was not unique to tumor-derived cells or cells of high tumorigenicity. The amount of growth-promoting activity produced per cell in the active cultures was not correlated with any of the following: tumorigenicity, growth rat, cell density achieved at saturation, cell type, or species of cell origin. It is concluded that transformed and non-transformed cells of diverse origin, cell type, and tumorigenicity can produce growth factors in culture. The growth-promoting potential of the active media from primary tumor cultures accumulated with time of contact with cells and was too great to be accounted for entirely by the removal of low-molecular-weight inhibitors by dialysis. The results are consistent with the hypothesis that conditioned medium from the active cultures contained a dialyzable, growth-promoting activity. Different cell lines exhibited differential sensitivity to tumor cell DCM and fetal bovine serum. Furthermore, quiescent fibroblasts were stimulated by primary tumor cell DCM in the presence of saturating concentrations of fetal bovine serum. These observations support the notion that the active growth-promoting principle in primary tumor cell DCM may not be a serum factor(s).


Suppression of Moloney sarcoma virus immunity following sensitization with attenuated virus. Murine sarcoma virus (Moloney strain) (MSV-M)-induced tumors are unusual in that they regularly appear less than 2 weeks after virus inoculation, progress for 1 to 2 weeks, and are rejected by normal adult BALB/c mice. Rejectio leaves the animals immune to tumor induction. In the present study, presensitization of normal adult BALB/c mice with attenuated MSV-M resulted in an altered pattern of tumor immunity. Injection of active MSV-M into the presensitized animals resulted in tumor induction and rejection similar to that observed in normal animals, but rejection failed to produce protection against the secondary inoculation with MSV-M. After the second inoculation with active MSV-M, tumors appeared and progressed but ultimately were rejected. Over 80% of the mice died, 25% after the primary challenge and the remainder after the secondary challenge. At death, all mice had histological evidence of leukemia which was the probable cause of death. The animals that died following the secondary challenge also had evidence of disseminated MSV-M. Solid tumor nodules were found in skeletal muscle distant from the original site of inoculation, and active MSV-M was isolated from spleen and lungs. The possibility that the results were produced by specific suppression of MSV-Moloney leukemia virus immunity is discussed.


Classification of human colorectal adenocarcinoma cell lines. Eleven human colorectal adenocarcinoma cell lines established in this laboratory were classified into three groups based on morphological features (light and electron microscopy), modal chromosome number, and ability to synthesize carcinoembryonic antigen (CEA). Group 1 cell lines contained both dedifferentiated and differentiating cells growing in tight clusters or islands of epithelium-like cells; their modal chromosome number was about 47, and they synthesized small to moderate amounts of CEA. Group 2 cell lines were more dedifferentiated, were hyperdiploid, and synthesized small amounts of CEA. Group 3 cell lines were morphologically similar to those of Group 1 by light microscopy. They differed ultrastructurally by containing microvesicular bodies; the modal chromosome number varied from hyperdiploid to hypertriploid or they had bimodal populations of hypodiploid and hypertriploid cells, and they synthesized relatively large amounts of CEA. No correlation could be found between Broder's grade or Duke's classification of the original tumor and modal chromosome number or ability to synthesize CEA. These findings support Nowell's hypothesis that the stem line is different for each solid tumor, which makes it difficult to relate chromosomal changes to the initiation of the neoplastic state.


Blood thymidine level and iododeoxyuridine incorporation and reutilization in DNA in mice given long-acting thymidine pellets. A long-acting thymidine pellet consisting of 190 mg of cholesterol and 60 mg of thymidine has been developed for the study of thymidine metabolism and reutilization in vivo. Implantation of such a pellet s.c. in adult mice will maintain the blood plasma concentration of thymidine at levels between 40 and 8 X 10(-6) M, which are from 36 to 7 times those of normal mice, for periods up to 48 hr. During this period, in vivo uptake and reutilization of [125I]iododeoxyuridine, a thymidine analog, into intestinal and tumor DNA were almost completely suppressed. While iododeoxyuridine reutilization is not large in normal proliferative tissue even in the absence of pellet implants, reutilization of over 30% was measured in large, rapidly growing ascites tumors. The inhibition of iododeoxyuridine incorporation by elevated thymidine blood levels is directly proportional to serum concentration. This appears to be due to a thymidine pool in rapid equilibrium with blood thymidine. This pool is at least 10 times larger than the 4-nmole pool of extracellular thymidine.


Diphtheria toxin effects on human cells in tissue culture. HeLa cells exposed to a single sublethal concentration of diphtheria toxin were found to have diminished sensitivity when subsequently reexposed to the toxin. Three cells strains exhibiting toxin resistance were developed. In the cells that had previously been exposed to toxin at 0.015 mug/ml, 50% inhibition of protein synthesis required a toxin concentration of 0.3 mug/ml, which is more than 10 times that required in normal HeLa cells. There appears to be a threshold level of diphtheria toxin action. Concentrations of toxin greater than that required for 50% inhibition of protein synthesis (0.01 mug/ml) are associated with cytotoxicity, whereas those below this concentration may not be lethal. Several established human cell lines of both normal and neoplastic origin were tested for their sensitivity to the effects of the toxin. No special sensitivity was observed with the cells of tumor origin. Fifty % inhibition of protein synthesis of HeLa cells was achieved with diphtheria toxin (0.01 mug/ml) as compared to the normal human cell lines tested (0.03 and 0.5 mug/ml) and a cell line derived from a human pancreatic adenocarcinoma (0.2 mug/ml). A human breast carcinoma cell line showed a maximum of 45% inhibition of protein synthesis. This required a diphtheria toxin concentration of 5 mug/ml. These results suggest that different human cell lines show wide variation in their sensitivity to the toxin.


The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture. We have established or characterized six lines of human breast cancer maintained in long-term tissue culture for at least 1 year and have examined these lines for estrogen responsiveness. One of these cell lines, MCF-7, shows marked stimulation of macromolecular synthesis and cell division with physiological concentrations of estradiol. Antiestrogens are strongly inhibitory, and at concentrations greater than 3 X 10(-7) M they kill cells. Antiestrogen effects are prevented by simultaneous treatment with estradiol or reversed by addition of estradiol to cells incubated in antiestrogen. Responsive cell lines contain high-affinity specific estradiol receptors. Antiestrogens compete with estradiol for these receptors but have a lower apparent affinity for the receptor than estrogens. Stimulation of cells by estrogens is biphasic, with inhibition and cell death at concentrations of 17beta-estradiol or diethylstilbestrol exceeding 10(-7) M. Killing by high concentrations of estrogen is probably a nonspecific effect in that we observe this response with 17alpha-estradiol at equivalent concentrations and in the otherwise unresponsive cells that contain no estrogen receptor sites.


The effects of glucocorticoids and progesterone on hormone-responsive human breast cancer in long-term tissue culture. Glucocorticoids, at physiological concentration, inhibit cell division and thymidine incorporation in three lines of human breast cancer maintained in long-term tissue culture. At steroid concentrations sufficient to inhibit thymidine incorporation 50%, little or no effect is seen on protein synthesis 48 hr after hormone addition. All three of these lines are shown to have glucocorticoid receptors demonstrable by competitive protein binding assays. Receptors are extensively characterized in one line by sucrose density gradient analysis and binding specificity studies. Good correlation between receptor-binding specificity and biological activity is found except for progesterone, which binds to glucocorticoid receptor but is noninhibitory. Cross-competition and quantification studies demonstrate a separate receptor for progesterone. This receptor has limited binding specificities restricted largely to progestational agents, whereas the glucocorticoid receptor bound both glucocorticoids and progesterone. Two other human breast cancer lines neither contain glucocorticoid receptor nor are inhibited by glucocorticoids. It is concluded that in some cases glucocorticoids can directly limit growth in human breast cancer in vitro without requiring alterations in other trophic hormones.


The effects of androgens and antiandrogens on hormone-responsive human breast cancer in long-term tissue culture. We have examined five human breast cancer cell lines in continuous tissue culture for androgen responsiveness. One of these cell lines shows a 2- to 4-fold stimulation of thymidine incorporation into DNA, apparent as early as 10 hr following androgen addition to cells incubated in serum-free medium. This stimulation is accompanied by an acceleration in cell replication. Antiandrogens [cyproterone acetate (6-chloro-17alpha-acetate-1,2alpha-methylene-4,6-pregnadiene-3,20-dione) and R2956 (17beta-hydroxy-2,2,17alpha-trimethoxyestra-4,9,11-triene-1-one)] inhibit both protein and DNA synthesis below control levels and block androgen-mediated stimulation. Prolonged incubation (greater than 72 hr) in antiandrogen is lethal. The MCF- cell line contains high-affinity receptors for androgenic steroids demonstrable by sucrose density gradients and competitive protein binding analysis. By cross-competition studies, androgen receptors are distinguishable from estrogen receptors also found in this cell line. Concentrations of steroid that saturate androgen receptor sites in vitro are about 1000 times lower than concentrations that maximally stimulate the cells. Changes in quantity and affinity of androgen binding to intact cells at 37 degrees as compared with usual binding techniques using cytosol preparation at 0 degrees do not explain this difference between dissociation of binding and effect. However, this difference can be explained by conversion of [3H]-5alpha-dihydrotestosterone to 5alpha-androstanediol and more polar metabolites at 37 degrees. An examination of incubation media, cytoplasmic extracts and crude nuclear pellets reveals probable conversion of [3H]testosterone to [3H]-5alpha-dihydrotestosterone. Our data provide compelling evidence that some human breast cancer, at least in vitro, may be androgen dependent.


Effect of hepatocarcinogens on the binding of glucocorticoid-receptor complex in rat liver nuclei. The effects of a number of carcinogens and hepatotoxins on the binding kinetics of the interactions of glucocorticoidcytosol receptor complex with nuclear acceptor sites in rat liver were investigated. Both the apparent sites in rat liver were investigated. Both the apparent concentration of nuclear binding sites and the Kd were significantly diminished following treatment of rats with sublethal doses of the carcinogens aflatoxin B1, diethylnitrosamine, dimethylnitrosamine, thioacetamide, 3'-methyl-4-dimethylaminoazobenzene, 4-dimethylaminoazobenzene, and 3-methylcholanthrene. Treatment with actinomycin D resulted in a slight reduction in the apparent concentration of nuclear acceptor sites but had no effect on the nuclear binding Kd. The hepatotoxic but noncarcinogenic analgesic, acetaminophen, as well as the weakly toxic aflatoxin B1 cognate, aflatoxin B2, were without effect on the kinetics or binding capacity of glucocorticoid-nuclear acceptor site interaction. These experiments suggest that chemically induced alteration of functional glucocorticoid binding sites on chromatin may be involved in the biochemical effects produced in liver by carcinogens of several chemical types. This experimental model may provide a useful approach for further elucidation of early events in carcinogenesis.


Long-term transplantability and morphological stability of three experimentally induced urinary bladder carcinomas in rats. Three transitional cell carcinomas induced in Fischer 344 rats by a methylcholanthrene pellet or a foreign body inserted locally into the bladder have been serially transplanted in the syngeneic strain for up to 6.5 years. There have been no changes in the individual morphological characteristics of the tumors during this time. Cells cultured in vitro for varying numbers of passages reproduce regularly the morphology of each tumor when they are injected back into the animals and results from a microcytotoxicity assay for cellular immunity indicate that they retain a common, bladder tumor-specific antigen. These tumors are useful for research in turmo biology and are offered to other scientists seeking transplantable carcinomas for experimentation.


Effect of sex difference on the in vitro and in vivo metabolism of aflatoxin B1 by the rat. Hepatic microsome-catalyzed metabolism of aflatoxin B1 (AFB1) to aflatoxin M1 and aflatoxin Q1 and the "metabolic activation" of AFB1 to DNA-alylating metabolite(s) were studied in normal male and female Sprague-Dawley rats, in gonadectomized animals, and in castrated males and normal females treated with testosterone. Microsomes from male animals formed 2 to 5 times more aflatoxin M1, aflatoxin Q1, and DNA-alkylating metabolite(s) than those from females. Castration reduced the metabolism of AFB1 by the microsomes from males by about 50%, whereas ovariectomy had no significant effect on AFB1 metabolism by the microsomes from females. Testosterone treatment (4 mg/rat, 3 times/week for about 6 weeks) of castrated immature males and immature females enhanced the metabolism of AFB1 by their microsomes. A sex difference in the metabolism of AFB1 by liver microsomes was also seen in other strains of rats tested: Wistar, Long-Evans, and Fischer. The activity of kidney microsomes for metabolic activation was 1 to 4% that of the liver activity and was generally lower in microsomes from male rats as compared to those from female rats of Sprague-Dawley, Wistar, and Long-Evans strains. The in vitro results obtained with hepatic microsomes correlated well with the in vivo metabolism of AFB1, in that more AFB1 became bound in vivo to hepatic DNA isolated from male rats and from a female rat treated with testosterone than that isolated from control female rats. These data suggest that the differences in hepatic AFB1 metabolism may be the underlying cause of the sex difference in toxicity and carcinogenicity of AFB1 observed in rats.


Tissue pharmacokinetics, inhibition of DNA synthesis and tumor cell kill after high-dose methotrexate in murine tumor models. In Sarcoma 180 and L1210 ascites tumor models, the initial rate of methotrexate accumulation in tumor cells in the peritoneal cavity and in small intestine (intracellularly) after s.c. doses up to 800 mg/kg, showed saturation kinetics. These results and the fact that initial uptake in these tissues within this dosage range was inhibited to the expected relative extent by the simultaneous administration of leucovorin suggest that carrier mediation and not passive diffusion is the major route of drug entry at these extremely high doses. Maximum accumulation of intracellular drug occurred within 2 hr and reached much higher levels in small intestine than in tumor cells at the higher dosages. At a 3-mg/kg dose of methotrexate s.c., intracellular exchangeable drug levels persisted more than four times longer in L1210 cells than in small intestine, but differences in persistence (L1210 cell versus gut) diminished markedly with increasing dosage. At 96 mg/kg, the difference in persistence was less than 2-fold. In small intestine and L1210 cells, theduration of inhibition of DNA synthesis at different dosages correlated with the extent to which exchangeable drug was retained. Toxic deaths occurred when inhibition in small intestine lasted longer than 25 to 30 hr. Recovery of synthesis in small intestine and L1210 cells occurred synchronously and only below dosages of 400 mg/kg. Within 24 hr after dosages of greater than 24 mg/kg, the rate of tumor cell loss increased to a point characterized by a single exponential (t1/2=8.5 hr). The total cell loss, but not the rate of cell loss, was dose dependent.


Follow-up of American Cancer Society Special Postdoctoral Research Fellowship recipients. A follow-up study of the 44 recipients of American Cancer Society, Inc., Special Postdoctoral Research Fellowship from 1962 to 1973 revealed that 11 of 21 M.D. candidates obtained their second (Ph.D.) degree at the end of training. By contrast, all but one among the 23 Ph.D. candidates were awarded the second (M.D.) degree. A great majority of either group remain in active research, regardless of whether or not they obtained the second degree. A very high percentage of their research is cancer related.


Ambiguity of the thymidine index.

Ambiguity of the thymidine index. The observed thymidine indices of seven experimental tumor lines are compared as a function of duration of emulsion exposure. The effects of dose level of tritiated thymidine and background threshold are also evaluated. The results indicate that an arbitrary high background threshold discriminates against "lightly" labeled cells at short periods of exposure but that the chosen threshold becomes less critical with longer exposure. The observed thymidine index increases with increasing duration of emulsion exposure but appears to approach a plateau for all tumor systems. The "thymidine index curves" are significantly different for each tumor. There is an inverse relationship between the dose of tritiated thymidine and the duration of exposure required to recognize the same fraction of cells as labeled in a given tumor. Similar experimental conditions do not necessarily guarantee a valid basis for comparison of observed thymidine indices among tumors.


Effects and specificity of anticancer agents on the respiration and energy metabolism of tumor cells. Using 35 new anticancer agents from the screening program of the National Cancer Institute we have performed a biochemical study of the effects on the respiration and oxidative phosphorylation of rat liver mitochondria and on the respiration of leukocyte, liver, L1210 leukemia, and Ehrlich ascites cell suspensions. Fifteen of the 35 compounds were found to be potent respiratory inhibitors as defined by 50% inhibition of mitochondrial respiration at concentrations of 110 mu mol/liter or less. The mechanism of respiratory inhibition by the drugs was either a rotenone-, antimycin-, or oligomycin-like effect. One triazine derivative showed some specificity for inhibiting tumor cell respiration in comparison with normal cell respiration. Two naphthoquinone derivatives showed inhibition of respiration in in vivo treatments at chemotherapeutic doses. It was concluded that data on respiratory effects may assist in the interpretation of the results of in vivo and in vitro screening tests of the drugs, and that in some cases, as with the naphthoquinone derivatives, the effects on respiration could be related to the mechanism of action or the mechanism of toxicity of the drugs.


Deveopment of resistance to combinations of six antimetabolites in mice with L1210 leukemia. The development of resistance to combinations of 6-mercaptopurine, 6-thioguanine, 6-methylmercaptopurine riboside, methotrexate, 5-fluorouracil, and cytosine arabinoside was studied in L1210 leukemia through 60 transfer generations. The treatment schedules were either simultaneous or offset. In simultaneous administration, one sixth of the LD10 of each of the six drugs was administered within a few minutes, daily for 6 days. In offset administration, the drugs were given either in the above-listed order or in reversed order, with one drug given each day. In the simultaneous combination treatment protocol 31 transfer generations were necessary to reach partial resistance, but in the two offset combination schedules only five and three generations were needed. The relative rate of development of resistance to the individual drugs was slower in the three combination schedules than in single-drug schedules. Resistance to 6-mercaptopurine and 6-thioguanine was completed after four generations on the offset combination schedules, but only after 28 generations on the simultaneous schedule.


5-Fluorouracil (NSC-19893), DTIC (NSC-45388), BCNU (NSC-409962), and vincristine (NSC-67574) in advanced colorectal carcinoma. Thirty-three patients with advanced colorectal carcinoma were treated with 5-day courses of 5-fluorouracil (10 mg/kg/day, iv, Days 1-5), DTIC (3 mg/kg/day, iv, Days 1 and 2), BCNU (1.5 mg/kg/day, iv, Day 1 only), and vincristine (0.025 mg/kg/day, iv, Day 1 only) at 5-week intervals. One patient achieved CR and six patients achieved PRs. Five of ten patients treated within 8 weeks after detection of advanced cancer had CR or PR (P = 0.02). After most courses toxicity was moderate, although it necessitated a change to another regimen for one patient. One patient refused further cancer chemotherapy and one patient died due to severe bone-marrow toxicity.


Comparative evaluation of three combination regimens for advanced malignant melanoma: results of an international cooperative study. The therapeutic results of a controlled study with three multiple-drug regimens (regimen A: DTIC, vincristine, BCNU; regimen B: DTIC, vincristine, hydroxyurea; and regimen C: DTIC, actinomycin D, BCNU) in a total of 274 evaluable patients with advanced malignant melanoma are reported. CRs were significantly more frequent (P less than 0.01) in regimens A (9.3%) and C (16.4%) compared with regimen B (1.1%). No significant difference in terms of CR plus PR was detected among the three regimens. In all regimens a higher number of CRs plus PRs was seen in patients with soft tissue metastases only, compared with those who had visceral involvement. In all three regimens patients achieving CR showed a longer duration of response and survival in comparison with patients achieving PR. The incidence of brain metastases was neither lowered nor delayed by the presence of BCNU in regimens A and C.


Phase II study of cis-dichlorodiammineplatinum(II) (NSC-119875) in advanced adenocarcinoma of the ovary. Cis-dichlorodiammineplatinum(II) was used in the treatment of 34 patients with advanced adenocarcinoma of the ovary who were resistant to conventional chemotherapy. Two dose schedules were explored: a high-dose schedule with 30 mg/m2 given daily on 3 successive days every 4 weeks and a low-dose schedule with 30 mg/m2 given once every 2-3 weeks. Nine patients (26.5%) showed a therapeutic response with a median duration of 6 months (range, 3-15 months). The main toxic effects included myelosuppression, renal function impairment, and nausea and vomiting. The high-dose schedule proved to be more toxic without any therapeutic advantage.


Combination chemotherapy with adriamycin (NSC-123127) and cyclophosphamide (NSC-26271) for solid tumors: a phase II trial. One hundred and nine adult patients with metastatic carcinoma were treated at 3-4-week intervas with a combination of adriamycin (40 mg/m2 given iv on Day 1) and cyclophosphamide (200 mg/m2/day given orally in divided doses on Days 3-6). Ninety-two of 96 patients who had an adequate trial (minumum of two courses or progression of disease after one course) had follow-up observations of tumor sites and were considered evaluable for response. Overall objective response rates by tumor type were as follows: stage III or IV ovarian adenocarcinoma, 61% (14 of 23 patients); endometrial adenocarcinoma, 67% (four of six patients); cervical adenocarcinoma, 33% (one of three patients); prostatic adenocarcinoma, 18% (two of 11 patients); testicular carcinoma, 33% (one of three patients); lung carcinoma, 21% (four of 19 patients); renal adenocarcinoma, 14% (one of seven patients); gastrointestinal adenocarcinoma, 18% (two of 11 patients); melanoma, 25% (one of four patients); and miscellaneous tumors, no responses in five patients. In patients with ovarian adenocarcinoma who had not previously received any cytotoxic chemotherapy the response rate was 80% (12 of 15 patients) with 33% five of 15 patients achieving complete clinical remission. CRs in these patients have now been maintained for periods ranging from 7 to 12 months. The major toxic effects were mild to moderate leukopenia, alopecia, and nausea with vomiting. Hemorrhagic cystitis was observed in three patients. The combination of adriamycin and cyclophosphamide is an effective treatment for carcinoma of the breast (reported elsewhere), ovary, and endometrium and should be considered for initial chemotherapy in patients with these tumors. Further investigations of its use for melanoma and carcinoma of the lung, prostate, kidney, and gastrointestinal tract are also warranted.


Multidisciplinary curative treatment for disseminated carcinoma of the breast. Eighty-four patients with disseminated breast carcinoma were treated with the combination of hormonal therapy and combination chemotherapy from the time of diagnosis. Hormonal treatment included castration for women still menstruating, estrogens for women menopausal for greater than 5 years, and androgens or progesterone for women menopausal for less than 5 years. Combination chemotherapy consisted of monthly 5-day courses of 5-fluorouracil (500 mg/day, Days 1-5), cyclophosphamide (300 mg/day, Days 1 and 4), vincristine (1 mg/day, Days 2 and 5), and methotrexate (100 mg, Day 1) followed by citrovorum factor (12 mg/day, Days 2 and 3). The overall response rate was 83.3%: 43 CRs (51%) and 27 PRs. The median survival time was 17 months: 6 months for the failures, 12 months for the 27 PRs, and 27 months for the 43 CRs. Fifteen patients with CR are still alive: one after 16 months, one after 17 months, two after 19 months, and one each after 23, 24, 25, 31, 35, 37, 38, 43, 48, 68 and 70 months.


Study of the infrared spectra of oligosaccharides in the region 1,000-40 cm-1. The i.r. spectra of disaccharides differing in monosaccharide composition and in the position and configuration of the glycosidic linkage, and also those of raffinose and model saccharides, were studied in the region 1,000-40 cm-1. Two ranges may be of interest for structural analysis. The first, called "the anomeric region=, is suitable for the determination of the configuration of the glycosidic linkage. The spectra of the oligosaccharides in the second region, called "the region of crystallinity", depend upon the packing of the molecules in the solid. The reasons for the present impossibility of using the far-infrared region of the i.r. spectra of lower oligosaccharides for the determination of the position of the glycosidic linkage are considered.


The molecular-weight range of mucosal-heparin preparations. A recently reported method describes the determination of the molecular-weight range of heparins by using an electrofocusing procedure to isolate individual molecular species. Commercially available heparins were fractionated on a column of polyacrylamide-agarose gel to give fractions whose molecular weights were estimated by viscometry. Fractions with mutually exclusive molecllar-weight ranges gave an appreciable number of common bands when subjected to the electrofocusing procedure; therefore, each of these bands cannot be formed from a single molecular species of heparin. Other mucopolysaccharides also gave band sequences indistinguishable from those of heparin; they differed in position and intensity with different ampholyte batches, and probably arose from individual molecular species of the ampholyte rather than the mucopolysaccharide. The molecular-weight range of the heparin was observed to be broader than that usually reported.


Characterization and structure of a sialic acid-containing hexasaccharide isolated from human pregnancy urine. A hexasaccharide containing sialic acid has been isolated from the urine of pregnant women. It contains D-glucose, N-acetyl-D-glucosamine, and sialic acid in the proportions of 2:1:1:2. Its structure, established by methylation analysis, enzymic digestion, and potassium borohydride reduction, is that of lacto-N-tetraose with one of the residues of sialic acid linked alpha-(2 leads to 3) to the terminal nonreducing residue of D-galactose, and the other sialic acid residue linked alpha-(2 leads to 6) to the N-acetyl-D-glucosamine residue. This hexasaccharide is related to human pregnancy, as it has not been found in nonpregnant, normal-woman urine and is present in human milk.


Echocardiography in the diagnosis of pericardial effusion. Echocardiography is perhaps the most useful and accurate method of diagnosis of pericardial effusion. Its noninvasive nature and the ease with which it may be repeated make it doubly attractive. In the hands of a skilled echocardiographer, its accuracy is unequaled by other methods such as carbon dioxide injections, radionuclide angiography, and intra-atrial angiography. In addition, echocardiography as an aid to safe pericardiocentesis may become widely applicable.


Pericardial heart disease: a study of its causes, consequences, and morphologic features. This report reviews morphologic aspects of pericardial heart disease. A morphologic classification for this condition is presented. An ideal classification of pericardial heart disease obviously would take into account clinical, etiologic and morphologic features of this condition but a single classification combining these three components is lacking. Pericardial heart disease is relatively uncommon clinically, and when present at necropsy it usually had not been recognized during life. The term "pericarditis" is inaccurate because most pericardial diseases are noninflammatory in nature. Morphologically chronic pericardial heart disease may present clinically as an acute illness. Even when clinical symptoms are present, however, few patients develop evidence of cardiac dysfunction (constriction). When pericardial constriction occurs, it is the result of increased pericardial fluid or increased pericardial tissue or both. Increased fluid is treated by drainage; increased tissue is treated by excision. In most patients with chronic constrictive pericarditis the etiology is not apparent even after histologic examination of pericardia.


Acute viral pericarditis.

Acute viral pericarditis. Viral infections of the pericardium occur frequently. Unfortunately, the viruses responsible for the infection are rarely isolated or even identified. The clinical manifestations are those of fluid in the pericardium and usually an associated friction rub. There is no specific treatment for a viral agent, but supportive care is important and valuable. The diagnostic and therapeutic principles in the clinical care of patients with viral pericarditis are discussed. Viral pericarditis never occurs without associated subepicardial myocarditis and often occurs in association with diffuse myocarditis. For this reason the disease may be better termed viral myopericarditis.


Nonviral infectious pericarditis.

Nonviral infectious pericarditis. Infections of the pericardium can occur as incidental findings in patients with systemic disease or dominate the clinical situation, representing a major threat to life. These infections appear to arise from the contiguous spread of infected tissue or from hematogenous dissemination. The clinical manifestations can resemble the "textbook description," but more often are subtle, nonspecific, or altered by the patient's basic disease. The diagnosis should never be discarded because dyspnea or chest pain are not elicited, friction rub and pulsus paradoxus are not detected, and chest x-rays and electrocardiograms are not "confirmatory." It is generally conceded that there has been a recent shift in the etiology of infectious pericarditis. This change has been attributed to the increased average age of hospitalized patients, modern diagnostic and therapeutic advances, and the impact of antibiotics. However, three concepts have stood the test of time: accurate diagnosis is the cornerstone of treatment; the availability of antibiotics must not relegate the role of surgery to a secondary position; and therapy must be directed against the pericarditis and the additional sources of infected tissue.


Constrictive pericarditis.

Constrictive pericarditis. Constrictive pericarditis, although still a relatively rare disease, continues to be a clinical problem that most practicing cardiologists may encounter. A major clinical clue to diagnosis is the continued elevation of the central venous pressure after adequate diuresis. The diagnosis is further supported by (1) prominent X and Y descents in the jugular venous pulse, (2) a relatively normal or only slightly enlarged cardiac silhouette in a patient with congestive heart failure, (3) pericardial calcification or significant congestive failure especially when the right sided signs predominate without obvious cause. When the disease is suspected, appropriate investigation should be undertaken using both the noninvasive and the catheterization studies. If the diagnosis is supported, then the choice of therapy at present is based primarily on severity of symptoms with surgical removal of the constricting pericardium being the therapy of choice in patients unable to be managed medically.


The postpericardiotomy and similar syndromes. A syndrome of pericardial and often pleural reaction with effusions and with fever occurs in three different settings. It bears the descriptive term of the condition which it follows: the postpericardiotomy syndrome, the postmyocardial infarction syndrome, and the postpericardial trauma syndrome. The manifestations, incidence, theories of etiology, treatment, and possibility of prevention were discussed. The syndrome is probably due to an immunologic response with an associated viral illness, since in a prospective study of children undergoing cardiac surgery in which the pericardium was entered, we found that the development of heart-reactive antibody in high titer and a significant rise in antiviral antibody occurred in patients with clinically evident syndrome.


Analysis of cell cycle compartments of hepatocytes after partial hepatecomy. An analysis of cell kinetic parameters as a function of intralobular localization of hepatocytes was performed using autoradiographic methods to obtain a basis for a quantitative description of cell cycle compartments after partial hepatectomy. The influx into the S compartment revealed a maximum in a lobular zone not immediately adjacent to the portal tract, with a decrease towards the perivenous and periportal area of the lobule. The maximum influx was found in the intermediate zone at 34 hr and a lower one in the perivenous parts of the lobule at 40 hr. The influx pattern at 56 hr was similar to the situation at 18 hr. The fraction of labelled mitoses in the whole liver and in subunits of the lobule disclosed a constant duration of S whereas the duration of G2 + M was prolonged from the periportal to the perivenous zone. A graphical model of kinetic events after partial hepatectomy is proposed, which describes the sizes of the various cell cycle compartments; the model successfully predicted the results of a continuous labelling experiment.


Regeneration of CFUs in the marrow of mice exposed to 300 rads after having recovered from 950 rads. Exposure to 950 rads 60Co radiation has been reported to cause long-lasting damage to the hematopoietic stroma (HS), although the size of the CFUs population recovers to pre-irradiation levels. In these studies HS damage was detected only after subcutaneously implanting the femurs of the irradiated mice into syngeneic hosts. To exclude the possibility that what was considered to be HS damage was merely caused by artifacts due to the process of implantation in a new host, we compared the rate of regeneration of CFUs in mice which had recovered from 950 rads prior to receiving 300 rads 60Co radiation (950 + 300 rads group) with that of mice which received only 300 rads (0 + 300 rads group). The CFUs population in the 950 + 300 rads group grew exponentially for 2 weeks at a rate which did not differ significantly from that of CFUs in the 0 + 300 rads group. However, the rate of CFUs growth reached a plateau before full recovery was achieved in contrast to that in the 0 + 300 rads mice. We therefore conclude that the incomplete regeneration of CFUs in the marrows of 950 + 300 rads mice was most likely caused by X-irradiation-induced damage to the HS rather than damage to the inherent repopulating potential of the CFUs per se.


Cell kinetics of a chondrosarcoma.

Cell kinetics of a chondrosarcoma. The cell kinetics of the transplantable DC-II mouse chondrosarcoma have been studied by the pulse labelled mitoses method. The analysis gave the following estimates for the phases of the cell cycle: G1, 10-5 hr; S, 9-5 hr; G2, 4 hr with an intermitotic time of 23-5 hr. Consideration of the overall growth of the tumour indicated that the growth fraction and cell loss factor both had values of about 0-5. The results are compared with cell kinetic data from sarcomas and other cartilage tissues.


The perturbed cell proliferation kinetics of the EMT6/M/AC tumour in mice treated with anti-mouse lymphocyte serum. The cell proliferation kinetics of the EMT6/M/AC tumour were determined at two volumes, 6-3 mm3 and 180 mm3, in mice treated with anti-mouse lymphocyte serum, AMLS. Comparison of the growth curve with that obtained in non-AMLS treated animals showed a marked increase in the growth rate at all volumes in the treated group. In contrast, the cell cycle time and the intermitotic phase times were not significantly different in the treated and untreated groups at comparable volumes. The increase in the growth rate in AMLS treated mice was obtained in spite of decreases in both the rate constant for cell production and the growth fraction, and was due to a marked decrease in the rate constant for cell loss.


Regenerative proliferation of mouse epidermal cells following adhesive tape stripping. Micro-flow fluorometry of isolated epidermal basal cells combined with 3H-TdR incorporation and a stathmokinetic method (colcemid). The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) Laerum, 1969) and brought into a monodisperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G1, S and (G1 + M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. 3H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G2 cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G2 pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G2 phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G2 phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the G1 phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.


The proliferative status of intestinal epithelial clonogenic cells: sensitivity to S phase specific cytotoxic agents. High concentrations of tritiated thymidine and cytosine arabinoside (Ara-C) have been used to selectively kill cells in the crypts of Lieberkuhn that are synthesizing DNA. The effect of these agents on the number of regenerating microcolonies seen 3 1/2 days after a range of radiation doses indicates that a majority of the clonogenic cells are proliferating rapidly and that the slowly proliferating cells at the base of the crypt do not represent the whole clonogenic population.


The relation between cell proliferation, differentiation and ultrastructural development in rat intestinal epithelium. The ultrastructural development of the principal cells in rat small intestine was studied by morphometric analyses in relation to the exact cell position along crypt and villus. From the bottom to the tip of the crypt, a gradual increase occurred in absolute size of the total cell, the cytoplasm, the terminal web and of nearly all cell organelles. Also, the relative size of the cytoplasm, mitochondria, microvilli and endoplasmic reticulum increased during crypt cell differentiation. No sudden changes in ultrastructure were observed in the so-called "critical decision zone", normally located halfway up the crypt where the proliferative activity ceases. At the crypt-villous junction a 1.4-3 fold increase in cell size, cytoplasm, terminal web and of most organelles was noted. Expansion of the proliferative cell compartment over the total length of the crypt as occurs during recovery after a low X-irradiation dose (72 h after 400 R) does not affect the normal development of cellular ultrastructure. These findings are discussed in relation to biochemical and cell kinetic data.


An ultrastructural in vitro study on the regulation of neurosecretory activity in the freshwater snail Lymnaea stagnalis (L.) with particular reference to caudo-dorsal cells. The neurosecretory Caudo-Dorsal Cells (CDC) in the cerebral ganglia of the freshwater pulmonate snail Lymnaea stagnalis produce an ovulation stimulating hormone. Previously it has been shown that neuronal and non-neuronal inputs are involved in the regulation of their activity. The degree of autonomy of these cells has been investigated by studying with morphometric methods the ultrastructure of CDC maintained in vitro. CDC of isolated cerebral ganglia which were cultured for 7 days show a considerable rate of synthesis, transport and release of neurohormone. Apparently these processes can proceed in the absence of neuronal and hormonal inputs from outside the cerebral ganglia. Completely isolated CDC, however, do not show neurosecretory activity in vitro; active Golgi zones, indicating the formation of neurosecretory elementary granules, are absent from such cells. Isolation does not seem to affect general cell functions such as protein synthesis and respiration. It is suggested that a neuronal input, originating within the cerebral ganglia, is necessary for the stimulation of CDC neurosecretory activity. Techniques are described for the isolation and culture of neurosecretory cells of L. stagnalis.


Morphometric in vitro analysis of the control of the activity of the neurosecretory dark green cells in the freshwater snail Lymnaea stagnalis (L.).. The neurosecretory Dark Green Cells (DGC) in the pleural and parietal ganglia of the freshwater snail Lymnaea stagnalis seem to be involved in osmoregulation. Previous experiments have indicated that changes of the osmolality of the environment induce activity changes of the DGC. Furthermore, it was shown that information on environmental osmolality reaches the DGC via the blood. In the present study right pleural and parietal ganglion complexes were cultured for 3 days in vitro under different osmotic conditions. Quantitative electron microscopy revealed that, compared with the control osmolality (130mOsm/kg H2O), osmolalities of 160 and 190 mOsm/kg H2O caused a reduced synthesis and an increased storage of neurohormone in the DGC. Apparently, the activity of the DGC depended on the osmotic pressure of the medium. It is proposed that in vivo the osmotic pressure of the blood (which is related to the osmolality of the environment) regulates DGC activity.


Basal lamina of human seminiferous tubule- its role in material transport. I. In presence of tunica vaginal hydrocele. Electron microscopy of testicular biopsies obtained from two adult males with tunica vaginal hydrocele revealed some protrusions from the basal lamina to the germinal epithelium in the seminiferous tubule. The protrusions were of three types: some between the spermatogonia and Sertoli cells, some directly within the Sertoli cell cytoplasm and others inside the spermatogonia. The protrusions inside the spermatogonia were only 0.5 mum deep whereas the other types were from 1-11 mum deep. Occasionally some cut off portions of these protrusions were seen inside the ground cytoplasm of the Sertoli cell without an apparent connection with the original stalk. The matrix of the protrusions contained a homogenous component (composed of a fine filamentous element) and granular and membranous components. These components closely resemble the materials found in the basal lamina of the seminiferous tubule. It has been suggested that under mild pathological conditions, i.e., hydrocele, the junctions between the seminiferous tubule epithelium and the basal lamina become somewhat more flexible. As a result, the protrusions become longer and a passage might be formed to allow the flow of raw materials in or out of the seminiferous tubule.


The morphology and innervation of subepithelial "striated" muscle cells in the male guinea-pig urethra. Light and electron microscopic techniques have been used to determine the distribution, morphology and innervation of subepithelial striated muscle cells in the wall of the proximal urethra of the male guinea-pig. These cells form a continuous layer, immediately beneath the urethral epithelium extending from the bladder neck to the termination of the ejaculatory ducts into the proximal urethra. They differ from "typical" striated muscle fibres (as seen in the external urethral sphincter) by their small size, rich acetylcholinesterase content and the irregular arrangement of intracellular myofilaments and sarcoplasmic reticulum. In addition, motor end plate regions have not been observed on these striated cells when examined using a light microscopic histochemical technique. The cells are related to acetylcholinesterase positive nerves which run between them in a manner compatible with the occurrence of "en passant" synaptic interactions. Using electron microscopy, axonal varicosities containing small (50 nm diameter) agranular vesicles are encountered 50 nm from the striated cells; membrane specialisations characteristic om motor end plates have not been observed on the cells. The findings are discussed, particularly in relation to the distribution, unusual morphology and innervation of these subepithelial muscle cells.


Ultrastructure and differentiation of ascidian muscle. I. Caudal musculature of the larva of Diplosoma macdonaldi. The larval caudal musculature of the compound ascidian Diplosoma macdonaldi consists of two longitudinal bands of somatic striated muscle. Approximately 800 mononucleate cells, lying in rows between the epidermis and the notochord, constitute each muscle band. Unlike the caudal muscle cells of most other ascidian larvae, the myofibrils and apposed sarcoplasmic reticulum occupy both the cortical and the medullary sarcoplasm. The cross-striated myofibrils converge near the tapered ends of the caudal muscle cell and integrate into a field of myofilaments. The field originates and terminates at intermediate junctions at the transverse cellular boundaries. Close junctions and longitudinal and transverse segments of nonjunctional sarcolemmata flank the intermediate junctions, creating a transverse myomuscular (TMM) complex which superficially resembles the intercalated disk of the vertebrate heart. A perforated sheet of sarcoplasmic reticulum (SR) invests each myofibril. The sheet of SR spans between sarcomeres and is locally undifferentiated in relation to the cross-striations. Two to four saccular cisternae of SR near each sarcomeric Z-line establish interior (dyadic) couplings with an axial analogue of the vertebrate transverse tubular system. The axial tubules are invaginations of the sarcolemma within and adjacent to the intermediate junctions of the TMM complex. The caudal muscle cells of larval ascidians and the somatic striated muscle fibers of lower vertebrates bear similar relationships to the skeletal organs and share similar locomotor functions. At the cellular level, however, the larval ascidian caudal musculature more closely resembles the vertebrate myocardium.


Silicon-containing granules of rat liver, kidney and spleen mitochondria. Electron probe X-ray microanalysis. Isolated rat liver mitochondria containing granule aggregates (25-75 nm in diameter) and small (5-10 nm) electron opaque granules were examined by electron probe X-ray microanalysis. The granule aggregates gave an intense Si signal, while the small granules gave both Si and P signals. Isolated mitochondria of rat liver, spleen and kidney, subjected to detergent solubilization and differential centrifugation, produced two granule fractions: (1) a 10,000 g fraction consisting predominantly of granule aggregates (25--75 nm) composed of smaller granules (5--10 nm in diameter), and (2) a 10,000--30,000 g fraction of non-aggregated small granules (5-10 nm). Thin sections of isolated granule aggregates gave Si X-ray signals similar to those obtained from in situ granules. In addition S, Cl, Mg, Cr and Fe X-ray signals were observed. Cr occurred only in the large kidney granules, while Fe occurred in both fractions of the spleen and kidney granules. The presence of Si in the granules was confirmed by chemical analysis of the isolated granules and in vivo radiolabeling of the granules with 31Si and 68Ge. Contamination within the electron microscope was eliminated by a liquid nitrogen anticontamination device.


Autoradiographic study on the localization of estradiol-17beta in neonatal mouse uterus and cervix. The uptake of 3H-estradiol-17beta in the neonatal mouse uterus and cervix has been studied by an autoradiographic method. When the radioactive hormone is administered in vivo and in vitro, grains are found to be concentrated above the nuclei both in the uterine and cervical epithelium and stroma. Grain counts revealed that the nuclear concentration of grains is higher at 4 h than at 2 h after isotope injection. The cervical epithelium has a higher nuclear concentration than the uterine epithelium both in vivo and in vitro. In the stroma, this situation is reversed except after in vitro treatment of the tissues. In the cervix, more of the hormone seems to be located within the nucleus while in the uterus a higher proportion of the grains are found in the vicinity of the nuclear periphery. Although the nuclear concentration of grains is higher at 4 h than at 2 h, the number of grains above the sections is lower at 4 h. Both in vivo and in vitro, the number of grains is higher above the stromal than above the epithelial compartments of the uterus and cervix. Five days old animals showed the same labeling pattern. The differences in uptake and distribution of 3H-estradiol are discussed in relation to other known differences in the hormone responsiveness in these tissues.


On the fine structure of the electrocyte of Electrophorus electricus L. The general ultrastructure of the electrocyte, the basic unit of the electric organs of Electrophorus electricus, is analyzed. Presented herein are detailed observations of the syncytial surface, its fibrillar coat, invaginations of the plasma membrane and synaptic terminals. Using Thiéry's method glycogen granules were identified in the syncytial cytoplasm and inside the synaptic terminals, their size and structure being compatible with the muscular origin of the electric organs, to which the filamentous meshwork found in the cytoplasm may be related. Among the perinuclear-organelles, are dense bodies with crystalline patterns. The mitochondrial matrix contains dense granules, their size and structure varying according to the organ to which they belong and to the fixation method used.


Relationship between the chromatoid body and the acrosomal system in early spermatids of Myxine glutinosa L. A transient close relationship between the chromatoid body and the developing acrosome is demonstrated in early spermatids of Myxine glutinosa.


Subsarcolemmal mitochondria and capillarization of soleus muscle fibers in young rats subjected to an endurance training. A morphometric study of semithin sections. Rats, 6 weeks old, were subjected to a program of endurance running for 3, 6 and 12 weeks. 0.5 to 0.8 mum thick sections of Epon embedded soleus muscles were studied with morphometric methods. In cross-sections the area occupied by subsarcolemmal mitochondria was independent of the age, but was 53% higher after 12 weeks of training. The mean depth of the zones with subsarcolemmal mitochondria increased only 15% to about 0.9 mum. Thus, the subsarcolemmal mitochondria showed a pronounced spreading at the muscle fiber surface in trained muscles.-The number of capillaries per fiber decreased slightly in controls and increased not significantly in trained muscles. It is concluded that the subsarcolemmal mitochondria supply the energy for the active transport of metabolites through the sarcolemma in oxidative muscle fibers, and that they are the limiting factor for endurance performance of the soleus muscle fibers because the changes in the capillarization were only small. It is suggested that the subsarcolemmal and the interfibrillar mitochondria have different functions and may therefore represent different types of mitochondria which can be distinguished by their morphology as well as by their biochemical properties.


Hypothalamic and hormonal control of the photoperiodically induced vernal functions in the white-crowned sparrow, Zonotrichia leucophrys gambelii. 1. The effects of hypothalamic lesions and exogenous hormones. To assess the role of the hypothalamo-hypophysial complex in the control of photoperiodically induced vernal premigratory responses in the White-crowned Sparrow, the effects of hypothalamic lesions and systemic administration of several hormones on these responses were investigated. Lesions that destroyed the posterior median eminence (PME) or the entire median eminence (ME) inhibited photoperiodically induced testicular growth, premigratory fattening and Zugunruhe. Lesions in the basal infundibular nucleus (IN) that resulted in complete inhibition of testicular growth abolished Zugunruhe, but allowed varying degrees of fattening. The systemic administration of prolactin, testosterone propionate (TP) or the combination thereof in the PME-lesioned birds induced fattening similar to that observed in photostimulated controls but did not induce Zugunruhe. It is concluded that testosterone and prolactin are the most important hormones involved in the control of vernal premigratory fattening. The role of these hormones in the induction of vernal Zugunruhe is not positively proven.


The embryonic diapause of Aulocara elliotti (Orthoptera, Acrididiae). Histological and morphometric changes during diapause development and following experimental termination with juvenile hormone analogue. Progressive hypertrophy of fat body, muscles, gonads and pleuropodial glands was observed with lengthening diapause incubation in embryos of Aulocara elliotti. Embryonic hemolymph stained more darkly and lipoid materials accumulated in the fat body and other tissues. Gradual increase in volumes was observed in the corpora cardiaca, corpora allata and ventral head glands (ecdysial glands). Termination of diapause was achieved in embryos treated with juvenile hormone analogue (JHA) (4 ethyl phenyl 6.7 epoxy geranyl ether--87%, Stauffer Chemical) applied directly to the egg undiluted or when eggs were immersed in a 10% solution in methanol. Treated embryos terminating diapause underwent rapid teratological morphogenesis, achieving pigmentation and sclerotization as in definitive embryos without engulfing their yolk. In treated embryos which remained in diapause, unusual paired masses of pigmented cells, resembling the melanotic pseudotumors of Drosophila, were observed ventral and lateral to the stomodeum and are thought to be subesophageal body cells (pericardial cell homologues). In some embryos these cells were enlarged, lysed or their nuclei rhexic and pycnotic. This response suggests these cells are target organs for JHA, or perhaps for ecdysone or its precursor released by JHA treatment. Endocrine glands in treated embryos had significantly larger volumes (.01) than those of controls or untreated embryos. The ventral head glands of treated embryos appeared to be cytoplasmically depleted. It is suggested that JHA triggered the release of stored ecdysone (or its precursor produced in the ventral head gland) at a rate too rapid for normal morphogenetic processes, such as dorsal closure, to accompany epidermal maturation. The response to JHA cannot be interpreted as juvenilization, but rather must be considered to be precocious maturation. These results indicate that: 1. diapause development involves a transfer of nutrient materials from the yolk into the fat body and other embryonic tissues where they are available to meet the demands of post-diapause morphogenesis; 2. that embryonic diapause in A. elliotti is a consequence of endocrine deficiency which probably results ultimately from the lack of neurosecretory stimulus from the brain-corpora cardiaca complex; and 3. that the subesophageal body cells, and perhaps the pericardial cells, play an essential role in the embryonic physiology, perhaps in the production of carrier protein and/or an essential growth regulator.


The fine structure of the drum muscles of the tigerfish, Therapon jarbua, as compared with the trunk musculature. The fibers of drum and trunk muscles of the Tigerfish. Therapon jarbua, differ greatly in diameter. The myofibrils of the trunk muscles are irregularly oriented, while those of the drum muscles are rolled into spiral or concentric bands. Both muscle types possess the sarcomere structure typical of cross-straited musculature. However, the myofibrils of the drum muscles differ greatly in sarcomere length and width from those in the trunk musculature. The trunk muscles contain few mitochondria, whereas in the drum muscles mitochondria are abundant. The sarcoplasmic reticulum (SR) of the drum muscles takes the form of elongated tubes in both the A and the I region; that of the trunk musculature consists of small vesicles. Of the two muscle types, the drum muscle contains more SR. With respect to the form of the T system, the trunk musculature is of the Z type and the drum muscles of the A-I type. The drum muscle displays a considerably greater number of motor endplates; these lack typical junctional folds and have mitochondria with very few cristae. No fat could be demonstrated in either the drum or the trunk muscles. However, the concentration of glycogen is higher in the drum muscles than in the musculature of the trunk.


The effects of prostaglandin F2alpha on the fine structure of the corpus luteum of the pregnant hamster. Corpora lutea (C.L.) from normal and prostaglandin-treated pregnant hamsters were examined by light and electron microscopy. Luteal cells from pregnant control animals were hypertrophied and contained an abundance of agranular endoplasmic reticulum (AER) suggesting steroidogenic activity. Animals which were injected on day 4 of pregnancy had ovulated by the third day following the injection, hence, two generations of corpora lutea were present in the ovary. Luteal cells from the "old" C.L. of pregnancy underwent a progressive degeneration during the four days following the single prostaglandin injection, whereas, cells from "new" C.L. appeared viable in most respects. The presence of aggregated platelets in both "old" and "new" C.L. was noted. It is suggested that the "new" C.L. are responsible for the rebound of plasma progesterone reported in previous investigations three days following prostaglandin treatment in pregnant hamsters.


Histological, histochemical and electron microscopical studies on the nervous apparatus of the pineal organ in the tiger salamander, Ambystoma tigrinum. 150-190 photoreceptor cells form a basic structural component of the pineal organ of Ambystoma tigrinum. Most of the outer and inner segments of these cells project into the lumen horizontally. Only 10% of the total number of photoreceptor cells are located within the pineal roof which is composed of a single cell layer. The photoreceptor cells are connected with nerve cells by synapses displaying characteristic ribbons. Different types of synaptic contacts, i.e. simple, tangential, dyad, triad and invaginated, are found. They are embedded in extended neuropil zones. A particular type of synapse indicates the presence of interneurons. The basal processes of some photoreceptor cells leave the pineal organ and make synaptic contacts with nervous elements located within the area of the subcommissural organ. Employing the method of Karnovsky and Roots (1964) for histochemical demonstration of acetylcholinesterase (AChE) approximately 70 neurons (intrapineal neurons) can be discerned in the pineal organ of Ambystoma tigrinum. In analogy to the distribution of photoreceptor cells only few nerve cells are observed in the roof portion of the pineal organ. Evidently, two different types of AChE-positive intrapineal neurons are present. About 40-50 AChE-positive neurons (extrapineal neurons) are scattered in the area of the subcommissural organ. In this area two types of nerve cells can be distinguished: 1) neurons which send pinealofugal (afferent) axons toward the posterior commissure and 2) neurons which emit pinealopetal (efferent) axons into or toward the pineal organ. The nervous pathways connecting the pineal organ with the diencephalomesencephalic border area are represented by a distinct pineal pedicle and several accessory pineal tracts. Granular nerve fibers run within the posterior commissure and establish synaptic contacts in the commissural region adjacent to the pineal organ. Some of these granular elements enter the pineal organ. The morphology of the nervous apparatus of the pineal organ of Ambystoma tigrinum is discussed in context with evidence from physiological experiments.


Aggregation-induced alterations in fibroblast morphology. An ultrastructural study. The different stages during aggregation of diploid human skin fibroblasts have been examined by transmission and scanning electron microscopy. As a result of aggregation, fibroblasts form a complex tissue configuration. Numerous intercellular junctions can be observed, while the cells remain polygonal and do not develop an organised intracellular cytoskeleton. Cell division occurs only rarely. After aggregation, signs of progressive auto-digestion develop. Adhesion to a substrate results in outgrowth of the cells and monolayer formation, even when extensive cell damage had occurred. The morphology of fibroblasts in aggregates and in the monolayers, from which they were derived is compared and the contribution of the aggregate system to the study of fibroblast behavior is discussed.


The ultrastructural organization of the visual system of the wax moth, Galleria mellonella: the retina. The ultrastructural organization of ommatidial components of the retina of the moth. Galleria mellonella are described from electron microscopic observations. Each ommatidium is composed of 12 common retinula cells and one basal eccentric cell. The retinula cells are connected together by a desmosomal strip along their length. The rhabdom occupies the basal thirty percent of the ommatidium and can be divided into nine segments of parallel microvilli. Several cells may contribute to an individual rhabdomere. The rhabdomeres are arranged in a cross with single cell rhabdomeres lying between the arms of the cross. Thin sections of ommatidium absorb polarized light differentially. The total amount of plane polarized light absorbed varies with angle of rotation for an entire ommatidium but there are also differences between the amount of absorption of adjacent rhabdomeric segments. Galleria appears to be the only lepidopteran in which the possibility of the polarized light reception has been reported.


The ultrastructural organization of the visual system of the wax moth, Galleria mellonella: the optic tract. The ultrastructural organization of the axons of retinula cells of the eye of the wax moth Galleria mellonella are described. The axons traverse an appreciable distance between the basement membrane of the retina and the lamina ganglionaris of the optic lobe of the brain. The optic tract was reconstructed from serial thin sections. Axons emanating from a single ommatidium are closely associated together in the optic tract. Adjacent cartridges fuse together to form large clusters of axons (8 to 10 cartridges). There is further coalescence between these large clusters. Extracellular space within the optic tract is severely limited and axons are sheathed by glial lamellae. Extracellular space between the axons and glia has been measured between 50 and 120 A. Calculations are presented that suggest that the glial interstices between the axons could increase the space constants of the axons significantly. Potentials could be transmitted along the length of the axons with between 59 to 37% decremental decrease, depending upon the number of glial interstices.


Ultrastructural studies on prolactin and growth hormone cells in Anguilla pituitaries in vitro. Eel hemi-pituitaries were cultured in vitro on high or low sodium media which are known to affect differentially prolactin and growth hormone release. Ultrastructural examination of the prolactin cells after 24 h culture showed the Golgi bodies were markedly more abundant and widely distributed in hemi-pituitaries from the low sodium medium. Secretory granule release profiles and dense bodies were also more frequent, but the percentage of the cytoplasmic volume occupied by secretory granules was lower than on the high sodium medium. RER was only slightly modified. Significant differences were noted in the shape and processes of the non-granulated (stellate) cells of the RPD, but there were only slight differences in the the ultrastructure of the somatotropes.


Scanning electron microscopy of photoreceptor cells in the light- and dark-adapted retina of Haplochromis burtoni (Cichlidae, Teleostei). The photoreceptor layer in the retina of Haplochromis burtoni (Cichlidae, Teleostei) was studied by scanning electron microscopy. Three types of receptors were identified: rods, single-cones and double-cones. The three-dimensional arrangement of these photoreceptors is described in the light- and dark-adapted retina. The surface of the inner segment of the photo-receptor cells displays fine vertical fissures which give rise to slender processes. These so called calycal processes which are of different lengths in rods and cones, surround the beginning of the smooth-surfaced outer segment. The myoid, the contractile part of the receptor, which is located beneath the ellipsoid, was examined in the single-cones of the dark-adapted retina. It is a slender structure with surface infoldings. The myoid, studied by transmission electron microscopy, contains bundles of parallel myofilaments, which are thought to be contractile.


The rete testis in man: ultrastructural aspects. The ultrastructure of the normal human rete testis was analyzed. The rete testis cavities are irregularly shaped and contain virtually no spermatozoa. Smooth muscle cells often surround the cavities. In the epithelial lining, two cell types are distinguishable. Flat, dark cells exhibit numerous slender microvilli, and numerous apical and basal micro-vesicles. Prismatic, lighter cells have more cell organelles, mostly polarized towards a supranuclear position. Both cell types contain variable amounts of glycogen and fat, and an occasional cilium. All cells display intricate lateral cell surfaces that possess different cell-to-cell attachment devices. Intermediate cell types are frequently found. On a morphological basis, the epithelial cells seem to be involved in the release of substances into the lumen and probably also in transport towards the base. Connective tissue elements are found subjacent to the epithelium. Scattered among the fibrocytes are typical smooth muscle cells. Expansions of some smooth muscle cells are connected to the epithelial basement membrane by a network of microfibrillar materia. The smooth muscle cells may be involved in changing the shape of the rete testis channels, thus promoting the flux of the rete testis fluid. Different types of nerve fibre bundles are distinguished in the connective tissue of the rete testis which may correspond to autonomic and sensory nerves or sensory receptors.


The occurrence of argyrophilic and argentaffin cells in the gut of Ciona intestinalis L. Argyrophilic and argentaffin cells occur in the stomach and intestinal epithelium of the sea-squirt, Ciona intestinalis L. These cells are characterized by their basal swelling which contains the nucleus surrounded by small secretory granules and by a filamentous cell-apex which reaches the gut lumen. The cells are scattered unevenly within the epithelium. Their number decreases rapidly towards the lower part of the intestine. The localization, size of granules and their shape are features which differentiate these cells from other secretory cells in the gut epithelium such as mucous cells. These cells are thought to possess an endocrine function.


Studies of cardiac ganglia in pre- and postnatal rabbits. This investigation was undertaken to describe the ultrastructure of cardiac ganglia in rabbits from day 18 of gestation to day 35 postpartum. Special attention was directed to the types of synaptic contacts made with the principal neurons and with the small granule-containing cells. The cardiac ganglia in all animals consisted mainly of parasympathetic postganglionic neurons, supporting cells, and small granule-containing (small intensely fluorescent) cells. The neurons received afferent  synaptic terminals of two types. One type contained mainly small clear vesicles typical of most cholinergic terminals. The second type contained mainly small dense-core vesicles (these were most prominent after treatment of the animal with 5-hydroxydopamine), and were considered to be adrenergic terminals. These adrenergic terminals are probably part of an inhibitory system in the ganglia. The small granule-containing cells received typical afferent synaptic terminals of the cholinergic type, and also formed specialized contacts with certain axonal terminals. These latter specializations are considered to be reciprocal synapses which probably have a role in modulating ganglionic transmission.


Indoleamine-accumulating neurons in the retina of rabbit, cat and goldfish. Special neurons accumulating indoleamines have been detected in the retina of rabbit, cat and goldfish. They have their perikarya in the innermost cell row of the inner nuclear layer, among the amacrine cells, and send their processes to various parts of the inner plexiform layer. The distribution of the processes is different in the different animals investigated. The neurons do not correspond to the previously known dopaminergic retinal neurons, which have a different distribution of their terminals and which can be demonstrated with a specially developed technique, simultaneously with the indoleamine-accumulating neurons.


Tritiated thymidine autoradiographic study of cell migration and renewal in the pyloric mucosa of golden hamsters. The kinetics of cell proliferation, migration and renewal in the pyloric mucosa of golden hamsters were studied by flash, cumulative and pulse labelling autoradiography following 3H-thymidine injections. By flash labelling autoradiography, it was shown that the labelled epithelial cells are exclusively confined to a zone several cells wide in the region of the isthmus between the gastric pits and the pyloric glands. In the cumulative and pulse labelling experiments, this cell proliferation in the isthmus region was shown to be for replacement of both the surface epithelial and the glandular cells. The surface epithelial cells of the pyloric mucosa arising in the upper portion of the isthmus come to line the pits and the surface, and are sloughed off into the gastric lumen within a week. The mucin-containing glandular cells, which arise more deeply in the isthmus region, migrate downwards and are apparently lost at the deepest level of the glands. The life span of the mucin-containing glandular cells was estimated at about 14 days. This cell type appears to undergo renewal of the "first produced, first lost" pipe line variety. However, a small number of glandular cells was found to survive for more than 20 days (up to 30 days), suggesting the existence of a sub-population of cells with different kinetics in the pyrloric glands.


Differential sensitivity of rods and cones in Xenopus retina to hemicholinium-3. Hemicholinium-3 (HC-3), a drug which prevents synthesis of acetylcholine in neurons, when injected intraperitoneally in doses as low as 2 X 5 mg/kg produces marked ultrastructural changes and damage in rod but not in cone photoreceptors. In rods it causes a reduction in cytoplasmic background density, swelling of the endoplasmic reticulum, ballooning of the outer membrane of the nucleus, leaching of the nucleoplasm and clumping of the nuclear chromatin. In dark-adapted rods HC-3 produces some loss of cytoplasmic synaptic vesicles but no reduction in numbers of those vesicles which lie adjacent to the synaptic ribbons. In light-adapted rods the drug does not cause such an apparent reduction of synaptic vesicles but does induce a considerable reduction in numbers of vesicles associated with the ribbons. These structural changes are discussed in the light of what is known about the pharmacology of HC-3 and neurotransmitter release from vertebrate photoreceptors.


Ultrastructural changes of pituitary gonadotropic cells in estrogen-treated pregnant rats. An ultrastructural study of gonadotropic pituitary cells was performed in estrogen-treated pregnant rats. Estradiol-treatment on Day 10 of pregnancy led to signs of ovulation or luteinization on Day 12 in 50% of the animals. Degranulation was observed in the FSH and LH cells of estrogen-responsive rats, whereas in the unresponsive group, the same cells were intensely granulated. The FSH cells of the control group showed signs of degranulation which could be correlated with follicular development. LH cells were sometimes degranulated. The role played by FSH and LH cells in the triggering of ovulation and luteinization by estrogen in the pregnant rat is discussed in the light of the ultrastructural observations.


The connectivity of cones and cone horizontal cells in a mosaic-type teleost retina. A total of 20 Golgi-impregnated cone horizontal cells of Nannacara anomala (Cichlidae) were studied in alternating semi- and ultrathin sections in order to examine their connections with the overlying square mosaic of equal double and central single cones. Cone horizontal cells exhibiting three types of processes: (a) the long horizontal "axon", (b) short horizontal dendrites with a terminal swelling, and (c) cone contacting processes ascending towards the outer plexiform layer. As seen in tangential sections, the latter processes are arranged in the form of two concentric circles including a central "spot". The processes of the inner circle contact the eight double cone pedicles of one square unit: processes of the outer circle contact eight more double cone pedicles which are directly adjacent to the square unit. The central "spot" represents a process which contacts the central single cone. Processes of the inner circle most often terminate in a dichotomous branching which represents the lateral elements to one ribbon synapse, whereas in the outer circle only a single terminal swelling is observed. Because of the mosaic of the cones and the constancy of this pattern of connectivity a model can be constructed where the dendritic fields of the cone horizontal cells overlap to a considerable extent. From this model, it follows that each double cone pedicle is contacted by four different horizontal cells. The functional significance of these findings for color vision is discussed in the light of recent work with the microspectrophotometer characterizing the cone system of this species as bichromatic. The mosaic-like arrangement of the horizontal cell dendrites supports the conclusion that the parallels between the patterns of receptor and horizontal cells are no coincidence but play an important role in lateral inhibition and neural adaptation of the retina.


Histological and histochemical observations on the neurosecretory cells in the diencephalon of Chthonerpeton indistinctum and Ichthyophis paucisulcus (Gymnophiona, Amphibia). In the diencephalon of two species of Gymnophiona (Amphibia) two neurosecretory nuclei were examined with histological (Alcian Blue, Aldehyde Fuchsin, Brookes Trichrome stain) and enzyme histochemical techniques (acid phosphatase, alpha-naphthyl acetate esterase, acetylcholinesterase (AChE)). In the preoptic nucleus two categories of secretory neurons were distinguished: large and medium sized neurons. The perikarya of both cell types contain very little neurosecretory material. The Alcian Blue method stained the medium sized neurons faintly but selectively. The tractus praeopticohypophyseus is marked by the presence of Herring bodies, which, however, are relatively scarce. The neurohypophysis, in contrast, contains large amounts of neurosecretory material. Both cell types of the preoptic nucleus are characterized by their very strong AChE and alpha-naphthylacetate esterase activity. The AChE also marks the tractus praeoptico-hypophyseus. In the large neurons acid phosphatase is present around the nucleus; in the medium sized neurons this enzyme is concentrated close to the origin of the axon. In the dorso-caudal hypothalamus a small group of neurons is stained with Alcian-Blue. These neurons, which also contain AChE, are located immediately under the ependyma which seems to be specialized in this region.


The structure of some cephalopod statoliths. The statoliths of Sepia officinalits, Octopus vulgaris, Alloteuthis subulata and Taonius megalops have a smooth outline, but an irregular shape. They have projections and indentations. The statoliths from a pair of statocysts are usually quite similar in size and shape, and the general pattern is probably maintained throughout the size range of the species. Statoliths from large animals are marginally larger than those from smaller ones. The statolith usually occupies only a small part of the cavity of the statocyst, and it is situated in the anterior part of the statocyst. They are joined to the macula by hairs extending from it. These hairs are very delicate and easily broken during preparation of the specimens. The hairs are much longer and narrower than the receptor cilia of the macula. The receptor cilia are enclosed within holes in the tangled hairlike anchoring fibrils. The statolith is made up of crystalline subunits, the statoconia. The crystals vary in size, they are usually elongated, hexagonal with pointed ends. The statolith consists of a closely packed mass of these crystals, sometimes they are irregularly arranged, where in others they are stacked with their long axes parallel. In Sepia officinalis and Taonius megalops, the crystals are arranged in regular shaped packets and these packets of crystals are stacked together. These larger subunits are not always arranged in a regular way, and their major axes can be organised in several different ways. The size and outline of these large subunits do vary in different parts of the statolith. The external surface of the statolith is macroscopically smooth. Over some parts there is a surface layer covering the rod-like crystals that make up the major bulk of the stone. In other regions, the surface is rough at a microscopic level, the roughness is produced by the exposed ends of the filamentous crystals. The crystals are composed of calcium carbonate in the form of aragonite.


Studies on secretory glycoproteins in the rat exocrine pancreas. I. Fine structure of the Golgi complex and release of fucose-labeled proteins after in vivo stimulation with caerulein. Using a double-label technique on isolated rat pancreatic lobules, the rate of synthesis and discharge of regular and fucosylated secretory proteins was studied under control conditions and after in vivo prestimulation with caerulein. Both labeled leucine and fucose were incorporated into pancreatic proteins at a linear rate, which was potentiated by in vivo stimulation. In pulse-chase experiments both regular and fucosylated secretory proteins were discharged into the medium in parallel. The in vivo pretreatment with caerulein caused an earlier discharge and increased the total amount released. Kinetic analysis of unstimulated (baseline) discharge of both classes of secretory proteins indicated in vitro sensitivity by the previous in vivo treatment with caerulein. The biochemical data were compared to the fine structure of the Golgi complex under both control and prestimulated ocnditions. The Golgi stacks were composed of four to six individual cisternae which in some cases were connected by intercisternal pores. Transporting vesicles were observed fusing along the total length of the outermost cisterna on both the cis- and trans-side and with the lateral ends of the intermediate cisternae. Under control conditions only the last trans-cisterna contained some electron opaque material; in vivo prestimulation led to distension and filling of all disternae in an individual Golgi-unit. Numerous stages of transformation of the last transcisterna into condensing vacuoles were observed, lending support to the hypothesis that during packaging of secretory products the membranes of the Golgi complex undergo a continuous turnover.