Bacterial growth in seafoood on restaurant premises.

Operational guidelines for a chain of the newer type of seafood restaurants and take-out stores were derived from plots of bacteriological and trimethylamine changes during the preparation and storage of cod fillets, shrimp, and clams. If fish is to be defrosted at room temperature it should be washed and portioned within 14 h. Subsequent storage at 5 degrees C should not exceed 72 h and once removed from the refrigerator it should be fried within 3 h. New stores should consider improved methods of defrosting. Trimethylamine content was a more sensitive measure of quality loss than bacterial growth. Shrimp quality can be improved by emphasizing the way shells are removed because most of the contamination was external. Shrimp shelled while still frozen had the best potential keeping quality. Clam meat is handled less and is always held at 5 degrees C; but bacterial growth indicated that it should not be held on the premises for more than 3 days including the time necessary for defrosting. Batter prepared fresh daily can be feft at room temperature. Frying can obliterate poor handling procedures, but adequate cooking is essential under any conditions. Clams are a gourmet item but the practice of cooking them lightly at customer request proved inadvisable.


Kindling, unit discharge patterns and neural plasticity.

Two approaches to the study of the kindling phenomenon were discussed: 1) an attempt to identify the pattern of neural activity required to produce the changes underlying kindling and 2) an investigation into the nature of those changes. Three experiments were reported that used the neocortical transcallosal system as a monosynaptic model system in which to study possible synaptic mechanisms of the kindling effect. Experiment I showed an increase in the transcallosal evoked potential following neocortical kindling. Experiment II showed an increase in the strength of the transcallosal evoked cell discharge following neocortical kindling. Experiment III reported the results of an histological examination of neocortical tissue in kindled and non-kindled animals using the Golgi-Cox technique. Spine density, spine dimension and branching were measured for pyramidal cell apical dendrites. No differences were found between primary and secondary (contralateral) foci or between kindled and non-kindled animals.


Behavioral and epileptic determinants of predatory attack behavior in the cat.

This report presents studies which relate limbic epileptic excitability to behavioral measures of defensive suppression of predatory attack in cats. Correlated with heightened defensiveness to environmental stimuli among non-killer cats is a heightened amygdaloid epileptic excitability, as well as a heightened conduction of amygdaloid epileptic activity to thalamic and hypothalamic substrates of predatory response in the amygdala to the complex visual stimuli presented by rat prey. These neurosensory responses correlate well with measures of epileptic excitability. Brain and behavior measures appear related since enhancement of excitability in the amygdala and of projection of epileptic activity by repeated electrical stimulation of predatory attacks. Furthermore, the ventral hippocampus seems capable of antagonizing the behaviorally suppressive effects of heightened amygdaloid excitability perhaps at points of convergence of amygdaloid and hippocampal output.


Frontal cortical kindling in cats.

Our observaiton of amygdoloid seizure development in cats indicated early afterdischarge propagation into basal cortical areas prior to the development of bifrontal sharp theta discharge. In view of the implied participation of the frontal lobe in amygdaloid kindling, both fractional lesioning and kindling of selective areas were performed. This paper summarizes out stimulation study involving premotor, prefrontal, mesial frontal and orbital cortices. Except for the orbital series which showed a rather strikingly similar pattern of seizure development to that of amygdaloid kindling, all areas showed significantly different features in terms of the speed of seizure development, afterdischarge propagation, fragility of developing seizure, final stage 5 seizure and post-ictal behavior pattern, interictal discharge morphology and propagation, and generalized seizure triggering threshold intensity. All these findings suggest that the frontal lobe participates in, but is not essential for, the amygdaloid seizure development. The results of ongoing fractional lesion series support such a conclusion.


Frontal lobe and kindling in the rat.

To test the hypothesis that the cortex participates in amygdaloid kindling in rats, bilateral aspiration lesions were made in various cortical areas in rats prior to kindling. Lesions in orbital cortex (on the dorsal lip of the rhinal sulcus) or prefrontal cortex (area 10) significantly retarded the rate of amygdaloid kindling; lesions in motor cortex, anterior cingulate cortex, or visual cortex were without effect. Detailed analysis indicated that the orbital lesioned and frontal-lesioned rats kindled relatively normally up to the second-last stage of amygdaloid kindling, in which stage they perseverated significantly longer than the controls and the other lesioned rats. These results suggest that areas of the frontal lobe participate in the elaboration and generalization of amygdaloid seizures in rats. Although retarded in rate, kindling nonetheless occurred in the lesioned rats, indicating that these cortical areas are not essential for the development of amygdaloid seizures.


Cardiomyopathy after widely separated courses of adriamycin exacerbated by actinomycin-D and mithramycin.

A 22-year-old man with a synovial cell sarcoma attained an excellent response to therapy with adriamycin (NSC-123127) and dimethyltriazeno imidazole carboxamide (NSC-45388). Therapy was discontinued at a cumulative dose of adriamycin of 600 mg/m2. Relapse occurred 13 1/2 months later, and therapy with adriamycin was restarted. Because of tumor progression, therapy was discontinued after a cumulative dose of adriamycin of 120 mg/m2. Ten weeks later, severe congestive heart failure developed which ultimately caused the patient's death. Exacerbations of the heart failure were temporally related to the administration of the antitumor antibiotics actinomycin-D (NSC-3053) and mithramycin (NSC-24559). Electron microscopic examination of the heart revealed changes characteristic of adriamycin cardiomyopathy. Thus, even after a long hiatus, it may not be safe to exceed the recommended maximum cumulative dose level of adriamycin. The pathogenic mechanisms involved in the development of adriamycin cardiomyopathy are reviewed, and the possible synergistic effect of other antitumor antibiotics is discussed.


A laboratory model for the study of the immunobiology of osteosarcoma.

Inoculation of Moloney sarcoma virus into the marrow cavity of the tibia of newborn Wistar-Lewis rats resulted in the appearance of an initially localized osteosarcoma in 97.7% of these animals. At least 77.9% of the rats developed lung metastases and died, usually within 6 weeks of inoculation. The remaining 22.1% showed regression of disease after initial growth of the tumor. Tumor cells were maintained in tissue culture and used as target cells for a visual and isotopic (3H-thymidine or 125IUdR) microcytotoxicity assay. Cell-mediated immunity could be measured by these methods throughout the course of the illness in animals with progressive disease as well as in those whose tumors eventually regressed. The presence of serum factors capable of modifying the  level of CMI was documented. This Moloney-sarcoma-virus-induced rat osteosarcoma and human osteosarcoma thus appear to have several basic pathologic and immunologic similarities. The model may be useful for studying the effects of a variety of treatment protocols upon the clinical course and immune response to osteosarcoma.


Antibodies to Epstein-Barr virus in patients with Hodgkin's disease and leukemia.

Sera from 67 Hodgkin's disease patients, 71 leukemia patients, and 186 healthy subjects were tested for antibodies to Epstein-Barr (EB) viral antigens by immunofluorescence methods. In both disease categories, in particular Hodgkin's disease patients, levels of antibodies to the viral capsid antigen (EBV-VCA) and MGT were higher than in the healthy controls. Significantly higher titers were found in Jewish patients of Asian-African origin, as compared to Jews of European origin, with Arab patients as intermediates. The effect of ethnic origin was independant of age and histopathologic type. Sex had no effect on titer. Inconsistent differences in titer were found between age groups in the various ethnic-histopathologic type groups. Some of the leukemia patients had no detectable antibodies to EBV, while all Hodgkin's disease patients showed previous contact with EB virus. Antibodies to the early antigen (EBV-EA) were found in 27% of Hodgkin's and 37% of leukemia patients, and in none of the healthy controls tested.


Megavoltage irradiation in the treatment of gliomas of the brain and spinal cord.

Curative radiotherapy was attempted in treating 256 patients with unresected or partially excised gliomas of the brain and spinal cord. Survival decreased with increasing age, reflecting the different incidences of tumor types in various age groups. Actuarial 5-year survival ranged from 85% for cerebellar astrocytomas, to 47% for medulloblastomas, to 41% for cerebral hemispheric astrocytomas, and 0% for glioblastoma multiforme. Further improvements in survival utilizing radiotherapy are unlikely until new adjuncts are developed, for higher radiation doses may lead to a disproportionate increase in radiation complications.


Carcinoma of the cervix with cylindromatous features believed to arise in mesonerphric duct.

A detailed description of the clinical and morphological aspects of a case of carcinoma of the cervix arising from mesonephric duct remnants is presented, and the literature pertaining to these rare tumors is discussed. At the time of initial surgical treatment the tumor was confined to the cervix; however, eventual metastatic dissemination to the bony and soft tissues of the pelvis, the lungs, and the lower vertebral column resulted in the patient's death 8 years later. The tumor appeared refractory to further attempts at treatment with radiation, progestational therapy, and variety of chemotherapeutic agents. The observation of a focal cylindromatous growth pattern, not previously described in tumors of this type, is presented.


Pleomorphic adenoma of the salivary gland. With special reference to histochemical and electron microscopic studies and biochemical analysis of glycosaminoglycans in vivo and in vitro.

Pleomorphic adenoma of the salivary gland is classified into two types based on the morphology of the cells and biochemical analysis of glycosaminoglycans. Type 1: the tumors consisted mainly of spindle cells, producing a large amount of mucinous material, and contained not only chondroitin 4- and 6-sulfate and hyaluronic acid but also heparan sulfate. When the tumors of this type were dissociated and cultivated, spindle cells proliferated vigorously as mulitlayer and secreted mucinous substance into the intercellular space. The cells looked like non-epithelial cells in a conventional culture bottle, but showed an epithelial-like arrangement when they were cultivated in sponge matrix or inoculated subcutaneously into mice. Fine fibres stained with orceine or resorcin-fuchsin were observed in the intercellular material both in resected tissue and in culture. Type 2: the tumors consisted mainly of ductal cells, having no mucinous area, and contained little heparan sulfate. When the tumors were cultivated, the growth of epithelial-like polygonal cells was observed in monotonous pattern, producing scanty intercellular substance, and proliferating, as monolayer, much less rapidly than the cells of Type 1.


Childhood sarcomas and lymphomas. Characterization of new cell lines and search for type-C virus.

Four cell lines were derived from childhood malignancies: rhabdomyosarcoma, sarcoma, lymphosarcoma and an American Burkitt's lymphoma. Cells of the four lines formed tumors in immunosuppressed newborn hamsters. The tumors had a microscopic appearance like that of the tumors from which the  cell lines were derived. No virus-like particles were detected by electron microscopy in the cell lines after treatment with bromodeoxyuridine; no hamster type-C virus expression was present in cell lines derived from the hamster tumors. Chromosome constitution and in vitro growth properties of the cell lines were studied as was the fibrinolytic function of the sarcoma lines.


Estrogen receptors in hormone-dependent breast cancers.

The determination of estrogen receptor (estrophilin) in human breast cancers, both primary and metastatic, can furnish information useful to the clinician in his choice of the optimal therapy for the individual patient with advanced disease. Of patients with significant tumor estrophilin levels, most, but not all, will respond favorably to endocrine therapy. Women whose cancers lack sufficient amounts of estrophilin have little or no chance of benefit from endocrine ablation or hormone administration and probably should be treated directly by alternative types of therapy.


Potential carcinogenicity of food additives and contaminants.

The potential role in carcinogenesis of food additives and contaminants presents a complex problem in terms of assessing the risk to the general public. Long-term testing in laboratory animals is still the most feasible method for determining potential carcinogenicity of various chemicals. The disadvantages encountered in the present methods of animal testing are discussed and a review is made of the current status of particular food additives and contaminants under scrutiny as possible carcinogens. It is suggested that, since it may not be possible to remove all carcinogenic materials from the environment, methods to mitigate or neutralize their harmful effects should be sought. Greater cooperation is called for among food technologists, toxicologists, laboratory researchers, and epidemiologists in the decision-making process regarding the role of possibly carcinogenic additives and contaminants.


Inhibition of replication of virus-transformed fibroblasts by antibodies to RNA.

The activity of 7S immunoglobulins (Ig) antibody to RNA, obtained in rabbits after a prolonged immunization with RNA-methylated bovine serum albumin complex was evaluated in vitro on normal (3T3) and simian virus 40-transformed (SV 3T3) mouse fibroblasts. The presence of anti-RNA antibody in the culture medium inhibited both the SV 3T3 cell proliferation and the [3H]thymidine incorporation. In contrast, an increased [3H]uridine incorporation was evident within 48 and 96 hr of culture. No significant modification in these 3 parameters was observed in 3T3 cultures treated in the same manner. Both 3T3 and SV 3T3 showed cytoplasmic fluorescence when cultured in the presence of fluoresceinated anti-RNA Ig. However, with the indirect fluorescence technique anti-RNA Ig were detected in SV 3T3 cytoplasm only. These data suggest that anti-RNA Ig were taken up by both 3T3 and SV 3T3, but only in SV 3T3 did the anti-RNA Ig retain their antigenic properties and block cellular proliferation.


Cultivation of medulloblastoma cells derived from simian adenovirus SA7-induced hamster brain tumor.

In vitro cultivation of medulloblastoma cells was successfully established from hamster brain tumors induced by simian adenovirus SA7. These tumor cells possess morphological features in cultures that are very similar to those of the original tumor. Both tubular and rosette growth patterns were evident. At the ultrastructural level only small numbers of cytoplasmic organelles could be detected in the tumor cells, typical of generally immature and/or undifferentiated cells. The proliferation of these tumor cells depended upon properly dense initial platings. In addition, it was found that single medulloblastoma cells could be stimulated to produce colonies if treated for 10 days with conditioned medium. Inoculation of these cells into synergenic animals resulted in 100% uptake. Survival of the hamsters was directly correlated with the number of cells injected. This new medulloblastoma cell line may prove to be a useful model for experimental brain tumor studies.


Inhibition by methioninyl adenylate of focus formation by Rous sarcoma virus.

Methioninyl adenylate is a specific and potent inhibitor of the enzyme methionyl-tRNA synthetase and, consequently, of protein biosynthesis. In cultures of chick embryo fibroblasts infected with Rous sarcoma virus, incubation for a 2-day period with 1 to 3 mM concentrations of this inhibitor, as late as 4 days after infection, irreversibly prevented subsequent formation of foci of transformed cells. Later addition could also elicit the irreversible disappearance of already existing foci, by phenotypic reversion and/or cell killing. Virus production in transformed cells and replication in newly infected cells were also inhibited but to a lesser degree. Under the same conditions, the same concentrations of methioninyl adenylate caused only a reversible growth arrest of normal cells. The selective toxicity of the inhibitor for transformed cells is not due to a greater affinity for the target enzyme, but it may be due to the fact that inhibition of protein biosynthesis is only partially reversible in these cells, whereas it is fully reversible in normal cells.


Focal infection and transformation in situ of thymus cell subclasses by a thymotropic murine leukemia virus.

Studies on the maturational lineages of thymic lymphocytes have revealed several subclasses which are distinguishable on the basis of cell size, topographic distribution within the thymus, DNA synthetic and mitotic activity, migratory behavior, and other properties. Strain C57BL/Ka mice were inoculated with radiation leukemia virus at different concentrations, and tissues were removed at defined intervals. Sequential sections were analyzed for virus-specific cytoplasmic antigen expression, for morphological evidence of neoplastic transformation, and for alkaline phosphatase activity. The first detectable sign of MuLV infection was the focal appearance of cytoplasmic viral antigens in cells of the outer thymic cortex, followed by coalescence of such foci and, several weeks later, by the appearance of morphologically transformed and alkaline phosphatase-positive cells, again often focally distributed in the outer thymic cortex. These observations strongly suggest that the large, mitotically active cells of the outer thymic cortex are the principal source of target cells for both productive infection and subsequent lymphoma induction by the virus.


RNA sulfurtransferase activity in rat liver and chemically induced hepatomas.

RNA sulfurtransferase activity has been detected in rat liver and in hepatomas from rats fed a diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene for 14 to 18 weeks. The reaction measured was the transfer of sulfur from cysteine to acceptor sites in Escherichia coli B transfer RNA (tRNA). Specific activities of the enzymes in liver and hepatoma supernatant fractions were similar, as were the rates and extents of sulfur transfer to tRNA. DEAE-cellulose chromatography of digests of the [35S]tRNA reaction products revealed 3 peaks associated with nucleotide material, the amounts of these peaks differing in tRNA from liver and hepatoma systems. This may suggest differences in specific sulfurtransferases in these tissues.


Polyamine metabolism in normal and in virus-transformed chick embryo fibroblasts.

The effect of transformation by Rous sarcoma virus on the ornithine decarboxylase levels of chick embryo fibroblasts was studied. Infection with Rous sarcoma virus resulted in increased ornithine decarboxylase activity which preceded morphological alterations by at least 24 hr. The ornithine decarboxylase levels of normal controls, unlike those in Rous sarcoma virus-infected cells, declined with age, so that the enzyme activity of transformed cells was increased twentyfold 5 days postinfection. Infection with a temperature-sensitive mutant of Rous sarcoma virus, T5, stimulated ornithine decarboxylase activity of chick embryo fibroblasts at the permissive (37 degrees) but not at the nonpermissive (42 degrees) temperature. The ornithine decarboxylase activity of T5-infected cells shifted from 42 degrees to 37 degrees increased within 2 hr and reached the levels of wild-type, Schmidt-Ruppin-17A-transformed cells in 11 hr. When T5-transformed fibroblasts were shifted to the nonpermissive temperature, ornithine decarboxylase activity decreased fivefold within 4 hr and paralleled the levels of normal controls in 11 hr. Shifting of temperatures of incubation caused comparable alterations in cellular putrescine but not in spermine or spermidine content.


Functional properties of mitochondria isolated from murine L5178 lymphoblasts grown in cell culture.

Mitochondria were isolated from lymphoblasts grown for 5 days in cell culture. Measurement of mitochondrial respiratory activity revealed poor response to adenosine 5'-diphosphate with reduced nicotinamide adenine dinucleotide-linked substrates but well-coupled active respiration with succinate as substrate. These mitochondria also exhibited rapid initial rates of respiration-supported calcium uptake as measured by dual-beam spectrophotometry. H+/2e- and Ca2+/2e- ratios were in normal limits for the lymphoblast mitochondria during calcium uptake in the presence of phosphate. In the absence of phosphate no calcium uptake, H+ ejection, or stimulation of oxygen consumption was observed. However, the lack of discharge of the accumulated calcium from the lymphoblast mitochondria upon inhibition of respiration suggests possible different mechanisms of cation transport compared to mitochondria from normal, mammalian cell types. Electron microscopy of freshly prepared mitochondrial suspensions revealed preparations with intact outer membranes and  abundant cristae and that were relatively free of other cellular structures. These studies demonstrate the feasibility of obtaining intact respiring mitochondria from cultured lymphoid cells and indicate that active ion transport in these mitochondria may be significantly different from "normal" cell mitochondria.


Influence of formycin B on polyadenosine diphosphoribose synthesis in vitro and in vivo.

Formycin B inhibited growth of L5178Y mouse leukemia cells in concentrations of less than twice the concentration that inhibits cell proliferation at 50% by cytostasis; at higher concentrations (more than twice the 50% concentration mentioned), the cells were killed. In cells treated with the concentration that inhibits cell proliferation at 50%, the average cell volume did not change. The formycin B inhibitory effect on cell proliferation was reduced by coincubation with nicotinamide adenine diphosphate or adenosine. The biosyntheses of DNA,RNA, and protein in whole cells were sensitively inhibited by formycin B as checked by incorporation studies with radioactive precursors. In addition, the formation of polyadenosine diphosphoribose was reduced even with higher sensitivity; in particular the extent of adenosine diphosphate ribosylation of histone subfraction H1 was reduced. Formycin B has been shown to be an inhibitor for the polyadenosine diphosphoribose polymerase, isolated from oviduct nuclei of quails. Both the chromatin-bound and the soluble enzyme are inhibited competitively; the relative affinity (Ki/Km) of formycin B to the polyadenosine diphosphoribose polymerase is 1.5.


Inhibition of formation of microtubular paracrystals in HeLa-S3 cells by neocarzinostatin.

The effect of an antitumor antibiotic, neocarzinostatin (NCS), on the formation of microtubular paracrystals (PC) induced by vinblastine sulfate, 10 mug/ml, in HeLa-S3 cells was examined by phase-contrast microscopy. The pretreatment of HeLa-S3 cells with NCS, 5 to 50 mug/ml, for 4 hr prevented the PC formation, and there was a dose response of NCS to the degree of inhibition. When the same inhibitory effect on PC formation was examined with other antitumor agents at high doses (50 mug/ml), colchicine was found to be one of the most effective agents, like NCS. Puromycin, antimycin, adriamycin, cytochalasin B, and cycloheximide revealed moderate activity, and the other antibiotics, such as mitomycin C, bleomycin, and rifampicin, did not show any effect at all. NCS was a unique antibiotic that inhibited PC formation among inhibitors of DNA synthesis. It was suggested that NCS affects the microfibrillar-microtubular proteins system in vivo, resulting in the inhibitions of organization of spindle fibers from microtubules at the G2 phase in HeLa cells.


The enzymatic basis of the selective action of cyclophosphamide.

The initial metabolic products of cyclophosphamide (4-hydroxy-cyclophosphamide and aldophosphamide) were prepared biologically in unpurified form. Their toxicity to tumor cells were tested by bioassay techniques and in cell culture, and the deactivation abilities of various tissue-soluble fractions were quantitated. Liver and kidney cytosol effectively deactivated the primary metabolites, whereas cytosols from gastrointestinal tract mucosa, Walker ascites tumor, and spleen were less efficient. When [14C]cyclophosphamide was activated and incubated with liver cytosol, 34% of all radioactivity was identified as carboxyphosphamide, by mass spectrometry of the methyl ester. Measurement of alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (EC 1.2.1.3) activities by reduced nicotinamide adenine dinucleotide production revealed a qualitative correspondence between aldehyde dehydrogenase activity and deactivation ability. Unpurified aldophosphamide and the analogs prepared from 6-methyl- and 5,5-dimethylcyclophosphamides were substrates for nicotinamide adenine dinucleotide-requiring enzymes, whereas incubation of 4-hydroxy-4-methylcyclophosphamide in an unfractionated incubation mixture with liver soluble enzymes did not cause reduced nicotinamide adenine dinucleotide production.


RNA type C virus antigens in hamster cells transformed by carcinogenic DNA viruses and chemicals.

The passive hemagglutination inhibition technique was used to test serologically for the presence of Syrian hamster type C virus antigen(s) (SHCVA) in a wide variety of normal and transformed hamster cells and tissues. SHCVA could not be detected in normal tissues or nonneoplastic tissues of tumor-bearing Syrian hamsters. Normal hamster embryo cells or cells transformed in vitro by simian adenovirus, by chemical alone, or doubly transformed by simian adenovirus and chemical did not contain SHCVA; however, SHCVA was found in a majority of tumors resulting from transplantation of these in vitro-transformed cells. No consistent pattern was observed in the capacity of individual transformed cell lines to produce SHCVA-positive or -negative tumors. When cells of a given transformed line were inoculated at 4 sites on each of 8 hamsters, SHCVA-positive tumors were found not to be randomly distributed but rather to be clustered on a few animals. SHCVA could be detected in only a few primary tumors induced by inoculation of carcinogenic DNA viruses; however, both the incidence and titer of SHCVA were significantly increased in a variety of transplanted tumors. These data suggest that SHCVA may be introduced into transplanted, transformed hamster cell tumors during passage in the host animal. Alternatively, in vivo conditions may allow expression of viral antigens not found under in vitro conditions; however, if this is true, only certain animals appear to be capable of activating SHCVA.


Binding of the azocarcinogen 3'-methyl-p-dimethylaminoazobenzene to cellular components of normal rat liver and azocarcinogen-induced hepatomas.

The location of binding sites for 3'-methyl-p-dimethylaminoazobenzene (3'-Me-DAB) or metabolites on components of rat liver cells and hepatoma cells in tumors induced by this carcinogen was determined at 2 stages during the induction of tumors in rats: (a) in normal liver immediately following the application of a massive dose of the azocarcinogen by intragastric feeding, and (b) in liver and tumor after hepatomas had developed following repeated exposures to the carcinogen by s.c. injections. Bound 3'-Me-DAB or metabolites were detected by the use of rabbit antisera directed against either p-azoazobenzene or p'-azo-p-dimethylaminoazobenzene in an indirect fluorescent antibody technique. Soon after massive intragastric doses of 3'-Me-DAB, the staining observed when the anti-p-azoazobenzene antiserum was used was principally on cytoplasmic components of liver cells with some staining of the intranuclear components. When the second antiserum, anti-p'-azo-p-dimethylaminoazobenzene antiserum, was used, the most intense fluorescent staining was on the nuclear membranes, although there was some cytoplasmic and intranuclear staining as well.


Short-term effects of ACTH on protein synthesis in adrenal cortex cells of young rats.

Two units of ACTH were administered intraperitoneally to young 20 gm-rats which received an intravenous injection of L-leucine-3H thirteen min later. ACTH-injected rats, and control rats which received the isotope alone, were killed at 2-, 10-, 30- and 60-min intervals. Electron microscope autoradiographs in control animals showed strong amino-acid uptake at pulse time (2-min) in the cytoplasm of adrenal zona fasciculata cells. Label was shared between the endoplasmic reticulum (ER) and mitochondria, and a lower but still considerable uptake was seen in nucleoli. At first chase time interval (10-min) cytoplasmic labelling declined, while nuclear and nucleolar labelling increased, both changing little thereafter, and there was a 10-30 min Golgi peak. ACTH administration provoked an overall increase in amino-acid incorporation into cytoplasm, nucleus and nucleolus at pulse time, with no changes in the distribution of the reactions among organelles. Intensification of labelling was most evident over nucleoli, the grain density of which was four-times as high as in controls. The short-term increase in ER and mitochondrial protein synthesis observed after ACTH injections was considered to be consistent with the hypothesis that most newly-formed proteins in these cells may be involved in the regulation of steroidogenesis. The marked increase in nucleolar labelling suggested the presence of proteins involved in RNA synthesis.


Alteration of cytochrome c oxidase activity during spermatogenesis in Carcinus maenas.

During spermatogenesis in the crab, Carcinus maenas, mitochondria in the developing spermatids degenerate. In close association with mitochondrial fragments in late spermatids, an elaborate lattice-like complex of fused membranes is formed and lies in a position adjacent to the acrosome. Mature sperm possess no mitochondria, but a whorl of membranes is present alongside the acrosome in the diffuse nucleus. To ascertain whether or not cytochrome c oxidase activity is lost as the mitochondria disintegrate, differentiating Carcinus maenas spermatids and mature sperm have been studied cytochemically. Cytochfome c oxidase activity was localised by means of the osmophilic compound 3,3'-diaminobenzidine (DAB). Cytochrome c oxidase activity is confined to the cristae of mitochondria in the testis. As spermatids mature most mitochondria are lost. A few mitochondrial fragments may be caught up within the lamellar complex. While they are recognisalbe as mitochondria they retain cytochrome c oxidase activity. The lamellar complex does not show this enzyme activity. These results therefore suggest that the mature sperm of Carcinus maenas do not contain the enzymes normally incorporated in the mitochondrial membrane, capable of oxidative phosphorylation.


Effect of methyl methanesulphonate (MMS) and methylene dimethanesulphonate (MDMS) on the template activity of DNA isolated from MDMS-sensitive and -resistant Yoshida cells.

Both methylene dimethanesulphonate (MDMS) and methyl methanesulphonate (MMS) cause the template activity of Yoshida cell DNA to decrease in a dose-dependent manner. The MDMS-resistant subline of Yoshida tumour is less sensitive in terms of DNA template activity than the MDMS-sensitive subline towards both agents. Although the difference in sensitivity is not reflected in the survival data after each agent, it does suggest that the DNA from each cell line differs in its capacity to function as an efficient template.


The inactivation of bacteriophage R17 by ethylating agents: the lethal lesions.

The biological inactivation of bacteriophage R17 by ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENUA) has been studied. At the mean lethal dose for the first compound 8 moles ethyl are bound/mole RNA and with the nitroso compound 3.5 moles ethyl are bound. Analysis of the amounts of the different ethylated derivatives formed shows that the toxicity of the sulphonate can be accounted for by the formation of 3-ethylcytosine, O6-ethylguanine, 1-ethyladenine and chain breaks produced on the hydrolysis of ethyl phosphotriesters. With the nitroso derivative on the other hand, the sum of chain breaks and of bases alkylated on a position involved in specific hydrogen bonding between base pairs only accounts for 65% of the observed toxicity. The possibility that 3-ethyladenine may constitute a lethal lesion is discussed.


Assays for phosphotriester formation in the reaction of bacteriophage R17 with a group of alkylating agents.

The interaction of bacteriophage R17 with 8 compounds has been studied, comparing the contribution of degradation of ribonucleic acid to the total toxicity. Breaks in the RNA chain result from the hydrolysis of phosphotriesters and thus are a measure of the extent of O-alkylation and of the SN1-type mechanism of the reaction. With many alkylating agents mutagenicity and carcinogenicity increase with increasing SN1 character of the reaction. In experiments with methyl methanesulphonate no evidence of degradation was observed at up to 19 times the mean lethal dose (620 methylations/RNA molecule). Breaks in the RNA chain accounted for 1 in 10 of the lethal lesions with beta-hydroxyethyl methanesulphonate, 1 in 60 with bis-(2-chloromethyl)methylamine (nitrogen mustard, HN2), less than 1 in 125 with 2,2-dichlorvinyl dimethyl phosphate (dichlorovos, DDVP), and 1 in 200 with propylene oxide. The hydrolysis rate of bis-(2 chloroethyl)ether was too slow for any reaction to be detected. In reactions with the carcinogen bis-(2-chloromethyl)ether the toxicity observed could be accounted for by the formaldehyde produced on hydrolysis. Cross-linking of the bacteriophage components by formaldehyde reduced the survival range over which the physical state of the RNA could be studied. No evidence of RNA degradation was observed. Reaction of the formaldehyde led to a progressive loss of biological activity over 24 h, a loss which was partially reversed by dialysis.


[Immunofluorencence localization of secretions related to gonadotropic, corticotropic, melanotropic and somatotropic hormones in hypophysis of Nectophrynoides occidentalis].

In Nectophrynoïdes occidentalis, ovine anti-LH anti beta (1-24) corticotropin, both anti alpha and anti beta MSH and bovine anti-STH antisera allowed us to identify by immunofluorescence gonadotropic, corticotropic, melanotropic and somatotropic hypophysial cells of a viviparous toad. The identification of these different cell types was confirmed by applying a classical cytologic technique to the same sections.


[Identification of cAMP dependant protein kinases in human lymphocytes].

Chromatographic purification by "DEAE" cellulose resolves the cAMP binding proteins in human lymphocytes into three parts. In presence of Mg++ each one possesses cAMP dependent protein-kinase activity, one of them showing allosteric characteristics.


[Demonstration of antigen-antibody complex of TSV5C12 cell membranes by means of guinea pig fluorescent anti-complement antibodies].

The application of rabbit anti-guinea pig complement fluorescent antibody to SV40 transformed cells demonstrated the presence of immune complexes on the cell membrane surface. The presence of such complexes was not detectable by using fluorescent rabbit anti-hamster immunoglobulins antisera.


[Resistance to association of antibiotics (polymyxin and penicillin) in relation to cell concentration of two bacterial species in the same inoculum. Complementary inoculum effect].

Dense suspensions of Sa. typhi (Polys Penr) and of St. aureus (Polyr Pens) proliferate on gelose medium added with the two antibiotics only if the inoculum is mixed. The growth is probably due to the partial neutralization of the antibiotics following the formation of complexes between these antibiotics and some constituents of the two strains.


[RNA fragments rich in G and A nucleotides obligatory for in vitro DNA replication of phages phi-X 174 and lambda].

Evidence was presented that in vitro conversion of single-stranded DNA of phage phi X 174 to the double-stranded replicative form by partially purified DNA-dependent DNA polymerase I requires a specific RNA fragment acting as primer (25-50 nucleotides). RNA fragments highly rich in nucleotides A and G were obtained by partial degradation of E. coli M 500 Sho-R ribosomal RNA with pancreatic ribonuclease. They become covalently bound to the newly synthesized DNA chain of the replicative form of phage phi X 174. These RNA fragments are also required for in vitro replication of lambda phage DNA.


[Correlation between agglutinability of concanavalin A and nucleic acid synthesis induced by polyoma virus].

We have previously shown the induction of agglutinability by concanavalin A by abortice infection of BHK 21 cells with polyoma virus. In this system, the maximal agglutination occurred simultaneously with the first mitosis. Using inhibitors of macromolecule synthesis in an amitotic system, we showed that this agglutinability was correlated with cell DNA synthesis.


[Electron microscopic study of the penetration and distribution of somitic cells in the mesoblast of the limb buds of reptiles (Anguis fragilis and Lacerta viridis)].

Based on characteristics of mitochondria and on the amount of lipid inclusions, a distinction between somitic cells and mesoblastic somatopleural cells is possible, at the early stages of the development of the limb bud in Reptiles (Anguis fragilis and Lacerta viridis). The dislocation of the ventral processes of the somites and the localisation of the somitic cells in the mesoblast of the anterior limb buds could be studied.


[Effect of antioxidant substances on the level of free organic radicals naturally present in the rat diaphragm].

Using the electron spin resonance method, the effect of antioxydants on the number of natural organic free radicals is studied in the case of the diaphragm of the Rat. The effect observed shows an important decrease in the free radical concentration, which may, in the case of ascorbic acid and BHT, lead to their complete disappearance. These results are discussed in the light of the hypothesis according to which antioxydants play a role in the lengthening of the life span because of their fundamental character of inhibitors of free radicals.


[Activity of specific collagenases in developing granulomatous tissues].

Specific collagenases are activated rapidly in inflamed tissues. This activity reaches a higher level in acute inflammation than in chronic inflammation. It appears to be related to an increased metabolic activity of the local fibroblasts.


[Confirmation of the localization on human chromosome F19 of a structural gene of poliovirus receptors].

The study of permissivity for poliovirus and the study of enzymatic markers in man-mouse somatic cell hybrids presents a very positive correlation between the presence of the poliovirus receptor and the presence of a well known enzyme marker, the phosphoglucoseisomerase (GPI), localized on the human chromosome F 19.


[Transfer of the tetracycline-chloramphenicol plasmid in Clostridium perfringens].

Two plasmids which confer resistance to Tetracycline-Chloramphenicol and Erythromycine-Clindamycine were found in a strain of Clostridium perfringens. The plasmid (Tet-Chl) was shown to be transferable to sensitive strains of C. perfringens. The transfer experiments were made with the wild type strain or strains cured by one plasmid or the other as donor strains.


Facilitation of adrenergic transmission by locally generated angiotensin II in rat mesenteric arteries.

When studied on isolated rat mesenteric arteries perfused with Tyrode's solution, angiotensin I and angiotensin II (1 ng/ml), a synthetic tetradecapeptide renin substrate, and a purified hog renin substance (50-100 ng/ml) potentiated vasoconstrictor responses to sympathetic nerve stimulation and to injected norepinephrine without altering basal pressure. These agents produced a greater augmentation of the vasoconstrictor responses to nerve stimulation than to injected norepinephrine. The potentiation of vasoconstrictor responses to sympathetic nerve stimulation and injected norepinephrine which was elicited by renin substrate and angiotensin I was abolished by an inhibitor of angiotensin I-converting enzyme, SQ 20,881, and by an angiotensin II receptor antagonist, [Sar1-Ile8]angiotensin II. In contrast, the potentiating effect of angiotensin II was blocked only by the latter compound. We conclude that utilization of renin substrate within the vascular wall by renin or renin-like enzymes results in the formation of angiotensin I, which is converted to angiotensin II. Angiotensin in turn potentiates the vasoconstrictor responses to adrenergic stimuli presumably by augmenting release of the adrenergic transmitter and inhibiting its neuronal reuptake as well as by increasing vascular reactivity to norepinephrine.


Diagnosis of type III hyperlipoproteinemia by chromatography of plasma lipoproteins on columns containing agarose.

Agarose column chromatography has been used to separate plasma lipoproteins into very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). Applied to the diagnosis of primary type III hyperlipoproteinemia, the procedure is capable of demonstrating three characteristic and specific changes from normality in the elution pattern of lipoproteins from patients with this condition. In the type III profile there is (a) incomplete separation of VLDL from putative LDL material, (b) early elution of the type III LDL with respect to a normal LDL marker, and (c) relative deficiency of type III LDL with elution characteristics of normal LDL. We advocate the use of this method in the diagnosis of type III hyperlipoproteinemia.


Cholesterol metabolism in hypertriglyceridaemia and the effects of treatment.

The faecal output of bile acids and endogenous neutral steroids was increased in three hypertriglyceridaemic patients. One patient had familail type IIb, one had type IV and the third had type V hyperlipoproteinaemia. The hyperlipidaemia in the type IV and type V patients was associated with diabetes and a high alcohol intake. The plasma cholesterol and triglyceride concentrations and the faecal output of bile acids decreased significantly when the type IIb patient was given D-thyroxine plus propranolol, and when the type IV and type V patients were treated by withdrawal of alcohol, a low=carbohydrate diet and insulin or glibenclamide. The findings are discussed in relation to the possibility that hypertriglyceridaemia and increased bile acid synthesis in these patients have a common metabolic origin.


The fluorimetric determination of ammonia in protein-free filtrates of human blood plasma.

Ammonia has been determined in filtrates of human plasma after precipitation of the proteins by perchloric acid. After restoration of the pH to around 7.5, addition of 2-oxoglutarate, NADH and glutamate dehydrogenase (GDH) convers the ammonia to L-glutamate with oxidation of the NADH to NAD. This latter reaction was utilised in two ways. In the first, reduction of native NADH fluorescence under the conditions of the GDH reaction provided a measure of ammonia concentration. In the second, residual NADH was destroyed by acid treatment, and the fluorescent product generated from NAD under strongly alkaline conditions was assayed. The optimal requirements for both methods were defined, their linearity and precision ascertained, and their relative merits compared. The first method was convenient for "one-off" estimations, and the second for larger batches. Ammonia concentration increased in plasma and in acid protein-free filtrates of plasma irrespective of the conditions of storage; however when the latter were neutralised, storage at -20 degrees C was effective. The distribution of plasma ammonia concentration in healthy subjects was log-normal. The range for males was 21-58 mumol/1 and for females 17-51 mumol/1; this difference was statistically significant (P less than 0.01).


The radiology of excavated Saxon and medieval human remains from Winchester.

Radiography of many of the human bones discovered during the archeological excavations at Winchester has been an integral part of the study of the Saxon and medieval populations found there. Thus, a radiological study of bone pathology in successive populations from a single site spanning a period of 600 years is presented.


Ganglia of the nerve. Presentation of two unusual cases, a review of the literature, and a discussion of pathogenesis.

Two cases of intraneural ganglia are reported: the first reported involvement of the sciatic nerve and the first reported involvement of the tibial nerve in the tarsal tunnel. Both were successfully treated by excision with sparing the neural elements leading to neurological recovery. A review of the literature fails to reveal any consensus that lesions arise from normal synovial cavities. Incision, evacuation, and complete excision under magnification sparing neural elements gives satisfactory results. Excision of the nerve is not indicated. Pathologic examination suggests that the lesion arises by multicentric metaplasia of connective tissue elements of the nerve rather than by invasion.


The angiographic evaluation of the internal carotid artery.

The importance of adequate evaluation of the internal carotid artery is stressed since pathology within it may mimic many intracranial diseases. Some of the many appearances seen on carotid angiography are described.


The effects of a diphosphonate and dietary calcium on the metabolism of vitamin D3 (cholecalciferol) in the chick.

1. Vitamin D-deficient chicks, maintained on a diet adequate in calcium and treated with ethane-1-hydroxy-1,1-diphosphonate for 2 days before a single oral dose of cholecalciferol (vitamin D3), converted the vitamin into 24,25-dihydroxycholecalciferol instead of into the normal metabolite 1,25-dihydroxycholecalciferol. 2. This inhibition of the renal 1-hydroxylase disappeared on withdrawal of the diphosphonate. 3. Kidneys from chicks given diphosphonate for 12 days converted 25-hydroxycholecalciferol into 24,25-dihydroxycholecalciferol on incubation in vitro. 4. The inhibition of the 1-hydroxylase was markedly accelerated by treating the birds with cholecalciferol. 5. No inhibition of renal 1-hydroxylation was observed in birds maintained on a diet low in calcium. 6. A possible mechanism producing this effect is discussed.


Observations on the passage of apoproteins from plasma lipoproteins into peripheral lymph in two men.

1. The passage of radioactive apolipoproteins into lymph draining the foot was investigated in two men, each given a single intravenous injection of low-density lipoprotein containing 131I-labelled apoprotein B and of very-low-density lipoprotein containing 125I-labelled apoprotein A and apoprotein C. 2. Protein-bound 125I and 131I appeared in the lymph of both subjects. Immunoelectrophoresis of lymph lipoproteins against anti-(high-density lipoprotein) and anti-(low-density lipoprotein) showed the presence of apo-high-density lipoprotein and apo-low-density lipoprotein with faster mobilities than plasma high-density and low-density lipoprotein respectively. Most of the protein-bound 131I in lymph was recovered in the precipitin line formed by the apoprotein B-containing lipoprotein after immunoelectrophoresis. Polyacrylamide gel electrophoresis of the lymph lipoprotein fraction showed the presence of 125I-containing bands with mobilities similar to those of the apoprotein A of high-density lipoprotein and of three of the fast-moving C apoproteins. 3. These results suggest that most, if not all, of the apoproteins of plasma lipoproteins reach the interstitial fluids and that some lipoproteins undergo modification during their passage into peripheral lymph.


Serological diagnosis of infection with the putative bovine leukemia virus.

We report on an accurate, rapid and inexpensive test for the identification of animals infected with the Bovine C-type virus (BLV). The test involves the detection of serum antibodies to BLV using the immunofluorescent (IF) technique on acetone-fixed, infected cells. The specificity of the test was demonstrated by the fact that virus was found by electron microscopy in 90% of cattle showing positive reactions. In contrast, virus was not found despite extensive examination in antibody negative animals. Thus, the presence of IF antibody is an accurate indicator of current rather than past BLV infection. In order for the IF test to be specific it is of critical importance that the target cells used are infected only with BLV. BLV antibodies can also be detected by the immunoprecipitation (Ouchterlony) technique. However, a significant proportion of BLV infected animals showing positive reactions in the IF test failed to show precipitin antibodies to the virus. Likewise, BLV infection was demonstrated by both the IF test and electron microscopy in many animals with persistently normal levels of blood lymphocytes. Thus, neither the precipitin test nor the blood lymphocyte count (Bendixen's key) can be used to rule out BLV infection.


Interaction of ethacrynic acid with control sites of renal glucose metabolism.

Ethacrynic acid stimulates in vitro concentration dependent renal gluconeogenesis from substrates which enter the gluconeogenic pathway at the level of the triosephosphates like glycerol or fructose or from substrates which have to pass the oxaloacetate shuttle like pyruvate or from intermediary products of fatty acid oxydation or citrate cycle. Our results suggest that a site of action of ethacrynic acid in this metabolic aspect is the enzyme system fructose diphosphatase/frutose-6-phosphate kinase and eventually additionally pyruvate carboxylase.


Metabolic patterns in various structures of the rat nephron. The distribution of enzymes of carbohydrate metabolism.

By means of the microdissection technique applied on kidney tissue, the following results were obtained: Hexokinase, an enzyme of glycolysis, revealed a low activity in the proximal and a high activity in the distal tubule. This distribution pattern is consistent with the finding that glucose is the main fuel for the distal tubule. Glucose-6-phosphatase, an enzyme of gluconeogenesis, demonstrates a significant activity in the distal tubule and in the glomerulus. Both structures are, however, no glucose producers. Phosphoenolpyruvate carboxykinase, the key enzyme of gluconeogenesis, is found only in the segments of the proximal tubule. The distal tubule lacks any activity. This is also the case during starvation and metabolic acidosis when gluconeogenesis is stimulated. Glutamic dehydrogenase, -an enzyme possibly connected with ammoniagenesis-, malate- and lactate dehydrogenase-, enzymes involved with hydrogen transfer through the mitochondrial membrane-, showed a close parallelism to phosphoenolpyruvate carboxykinase in their distribution along the proximal tubule. The bidirectional function of glyceraldehyde-P dehydrogenase is well documented by the close correlation to phosphoenolpyruvate carboxykinase (gluconeogenesis) in the proximal tubule and to pyruvic kinase (glycolysis) in the distal tubule.


Assignment of the gene for human uridine monophosphate kinase to chromosome 1 using somatic cell hybrid clone panels.

Evidence from mouse-human somatic cell hybrids is presented for the assignment of the gene for uridine monophosphate kinase (ATP: nucleoside monophosphate phosphotransferase, E.C. 2.7.4.4) to human chromosome 1. The use of the "clone panel" in this determination and its value as a systematic method for gene mapping is discussed.


A new method of in situ hybridization.

A new method for gene mapping at the chromosome level using in situ hybridization and scanning electron microscopy is described and has been applied to mapping the rRNA genes of Drosophila melanogaster. Biotin is covalently attached to Drosophila rRNA via a cytochrome c bridge at a ratio of one cytochrome-biotin per 130 nucleotides by a chemical procedure. Polymethacrylate spheres with a diameter of ca. 60 nm are prepared by emulsion polymerization and are covalently attached to the protein avidin at a ratio of 5-20 avidins per sphere. The biotin-labeled rRNA is hybridized to denatured DNA in a chromosome squash. Upon incubation with a sphere solution, some of the biotin sites become labeled with spheres because of the strong non-covalent interaction between biotin and avidin. The chromosome squash is examined in the scanning electron microscope (SEM). Polymer spheres, which are visible in the SEM, are observed to label the nucleolus, where the rRNA genes are located.


Circadian rhythm of urinary 17-hydroxycorticosteroids during metyrapone-induced ACTH release in normal subjects.

The circadian rhythm of urinary 17-OHCS was studied in nine normal subjects before and after oral metyrapone administration (750 mg every 4 hrs over a period of 24 hrs). The test was carried out twice in each subject with a shift of 12 hrs in the time of first administration (0800-test and 2000-test). For data from serial measurements of steroid metabolites on urine collected at 2-h intervals, the least squares fit of a 24-h cosine curve allowed the demonstration of a rhythm both in control conditions and during the 48-h span following metyrapone administration. Circadian acrophases were substantially similar. Total urinary 17-OHCS output in the first 24-h day following metyrapone was higher in the 2000-test as compared with the 0800-test (P less than .001). Conversely, in the second 24-h day, higher values were obtained in the 0800-test (P less than .025). The combined 48-h exretion, however, was identical in the two groups. The results suggest a time-limited action of metyrapone upon the pituitary-adrenal axis, since the increased excretion of 17-OHCS seems essentially accounted for by an exaggerated impulsive phase of ACTH secretion during the early morning hours.


[Occult nasopharyngeal carcinoma (author's transl)].

Clinical and histological examination of a cervical lymph node, removed from a 15-year-old boy, revealed nasopharyngeal carcinoma as the probable diagnosis. But the decisive diagnostic pointer was a markedly raised Epstein-Barr virus antibody titre, which gives the diagnosis at a time when there may not as yet be clinical evidence of carcinoma. Serial serum titre determinations are of prognostic value: with increasing spread of the tumour there is a rise in antibody titre. Autopsy confirmed that the nasopharynx had been the site of the primary tumour.


Beclomethasone dipropionate inhaler: a review of its pharmacology, therapeutic value and adverse effects. I: Asthma.

Beclomethasone dipropionate is a topically active corticosteroid used as an adjuvant in the control of chronic asthma when given by inhalation as an aerosol. It is not intended for treatment of acute attacks. It appears that the main difference between beclomethasone dipropionate and other corticosteroids previously used by inhalation is its high topical activity together with a lower systemic activity due to metabolic inactivation of the swallowed portion of the dose. Clinical experience has shown that at doses of 200 to 600mug daily, beclomethasone dipropionate inhaler is preferable to oral corticosteroids, because of lack of side-effects, when adult patients and children who are inadequately controlled by full doses of sodium cromoglycate and bronchodilators, are first considered to need maintenance corticosteroids. Inhaled beclomethasone dipropionate can allow a worthwhile reduction in maintenance doses of systemic corticosteroids in many patients already receiving these drugs and can replace systemic steroids entirely in some patients, particularly when their initial dose of steroids is less than 10mg daily of prednisone or its equivalent. Substitution should be attempted when the patient's asthma is well controlled on their usual doses of systemic steroids and full doses of other adjuvant therapy. Withdrawal of systemic corticosteroids should be performed slowly and carefully. Because recovery from impaired adrenocortical function caused by prolonged systemic steroid therapy is usually slow, special care is necessary for 9 to 12 months after transfer to beclomethasone dipropionate aerosol until the hypothalamo-pituitary-adrenal axis has sufficiently recovered to cope with emergencies such as trauma, surgery, severe infections or an acute attack of asthma. It is essential that additional therapy including high doses of systemic corticosteroids be used immediately to control any acute exacerbation of asthma which occurs during maintenance therapy with beclomethasone dipropionate aerosol. Tests of adrenal function suggest that beclomethasone dipropionate at dosages of 400 to 800 mug daily has little or no adverse effect. The most common side-effect associated with the continuous use of beclomethasone dipropionate inhaler has been oropharyngeal candidiasis, which appears to be dose-related and more common in women than in men. Systemic steroid withdrawal effects, like being generally unwell, and exacerbation of underlying allergic diseases such as allergic rhinitis, have been reported after substitution of beclomethasone dipropionate inhaler for systemic steroids. However, systemic withdrawal effects seldom occur if systemic steroids are withdrawn slowly.


Cyclic AMP response of isolated Snell adrenocortical carcinoma 494 cells to trophic hormones and other substances.

Suspensions of viable cells were prepared from solid tumor of the Snell adrenocortical carcinoma 494 without the use of proteolytic enzymes. Cyclic AMP formation in these cells was stimulated by ACTH, LH, FSH and TSH but not by prostaglandins (E1, E2, F1alpha and F2alpha), insulin and secretin. Glucagon tested at a single dose level of 50 mug increased cyclic AMP to about 65% of the maximum amounts obtained with ACTH. When Ca++ was omitted from the incubation medium, the response to ACTH was considerably reduced while that to LH was essentially unchanged. Low concentrations of EGTA (0.3 MM) abolished the ACTH response almost completely but caused only a partial reduction in the response to LH; as much as 10 mM EGTA was required to obtain complete inhibition of the latter.


Cyclic AMP in the thyroid of the rat fed prophylthiouracil: in vitro unresponsiveness to thyrotropin.

Fragments of thyroid from rats fed Purina +0.1% propylthiouracil were incubated in vitro and the concentration of cyclic AMP measured. The normal gland showed a 3-fold increase in cyclic AMP with 50 mU thyrotropin per ml or 10(-4) M prostaglandin E1; tissue from rats fed propylthiouracil for 10 days to 6 months responded to prostaglandin E1 but not to thyrotropin. Feeding Purina without propylthiouracil for 1 month after 5 months of goitrogen restored thyrotropin-responsiveness, as did, to a lesser extent, injection of triiodothyronine, 50 mug twice daily for 5 days.


Inhibition of a parathyroid hormone-stimulated renal adenylate cyclase by cyclic AMP.

Plasma membranes were isolated from bovine renal cortex. This particulate, adenylate cyclase-containing fraction was stimulated to produce cyclic AMP by parathyroid hormone and fluoride. When the time-course of adenylate cyclase activity was investigated, it was found that while PTH-stimulated cyclic AMP production comes to a halt in about 15 minutes after the initiation of the reaction, fluoride-stimulated activity continues unabated for at least an hour. Experiments to determine the cause of this showed that the cyclase enzyme is not degraded under our experimental conditions, but is inhibited by a soluble, unbound product of the reaction which requires ATP for its synthesis. In our experiments degradation of parathyroid hormone was relatively slow and could not account for the rapid inhibition of PTH-stimulated cyclase activity. Of the various agents tested, cyclic AMP was found capable of inhibiting PTH-stimulated cyclic AMP production by our purified membrane preparation. Half-maximal inhibition was observed at around 10(-6) M concentrations of the nucleotide. Pyrophosphate, adenosine, 5'-AMP and ADP had no effects. The significance of these results in relation to the regulation of adenylate cyclase activity is discussed.


Regulation of enzymes of serine and one-carbon metabolism by testosterone in rat prostate, liver, and kidney.

A significant decrease in the specific activity of 3 enzymes of serine and one-carbon metabolism (3-phosphoglycerate dehydrgenase, phosphoserine phosphatase, and serine hydroxy-methyltransferase) was found in the rat prostate gland with castration. A single injection of testosterone propionate to rats 3 days after castration resulted in a significant increase in the 3 enzyme activities within 24 h. This increase in specific activity was maximal 72 h after injection of testosterone in the case of 3-phosphoglycerate dehydrogenase and serine hydroxymethyltransferase. When cycloheximide was administered in conjunction with testosterone, the increase in 3-phosphoglycerate dehydrogenase and serine hydroxy-methyltransferase activity was significantly reduced compared to injection of testosterone alone. N6, O2'-dibutyryl adenosine-3',5'-cyclic monophosphate [(Bu)2 cAMP] and theophylline injected at 8 h intervals to rats 3 days after castration failed to mimic the action of testosterone on these 3 enzymes 24 and 72 h after beginning injections. Castration had no effect on the specific activity of these enzymes in the kidney; however, 3-phosphoglycerate dehydrogenase was significantly diminished in the liver 6 days after castration. A single injection of testosterone to rats 3 days after castration restored the activity to sham-operated levels. Serine hydroxy-methyltransferase and phosphoserine phosphatase activity in the liver were unaffected 6 days after castration. Thus testosterone exerts a regulatory role on serine and one-carbon metabolism in the prostate and liver which (Bu), cAMP is unable to minic.


A direct effect of testosterone on muscle cells in tissue culture.

A direct effect of physiological levels of testosterone on skeletal muscle cells in tissue culture is reported. The effect is a 25% stimulation of labeling index by radioautographic analysis of cultures following incubation with [3H]thymidine at the end of a 48-h exposure to 10(-8)M testosterone in 2% gelding serum. This stimulation was observed in primary myoblasts, an established myogenic cell line (Yaffe's L6 cells), and muscle fibroblasts. The effect was specific for testosterone: the labeling index did not change significantly from control values when estradiol, 5beta-pregnanediol, androstenedione, or dihydrotestosterone were added, although small increases were noted in the latter two cases. The effect on the labeling index was localized as a decrease in time spent in the G1 phase of the cell cycle, which decreased about 30% in muscle cells exposed to testosterone. This first report of an effect of testosterone on isolated muscle cells, coupled with the recent description of a receptor for anabolic steroids in muscle cytoplasm, indicates that the effects of male sex hormones result from a direct interaction with muscle rather than from a primary interaction with some other tissue.


The role of ACTH and adrenal glucocorticoids in the salt appetite of wild rabbits (Oryctolagus cuniculus (L)).

The selective appetites of wild rabbits for 500 mEq/1 solutions of NaC1, KC1, MgC1(2), and CaC1(2) were studied in intact and adrenalectomized rabbits during daily treatment with either 4 IU long acting ACTH, 1.0 or 2.5 mg cortisol acetate, or 2.5 mg corticosterone. The animals were individually caged and external sodium balances performed. In intact rabbits, cortisol or corticosterone produced a significant stimulation of NaC1 appetite. The response to concurrent dosage of cortisol and corticosterone was less than half of that obtained with ACTH which produced a comparable alteration of blood glucocorticoid levels but a 10-fold increase in NaC1 intake. CaC1(2) intake was increased in intact rabbits by cortisol treatment but not by corticosterone or ACTH. Adrenalectomized rabbits maintained on daily steroid replacement therapy of 0.1 mg deoxycorticosterone acetate and 0.75 mg cortisone acetate showed a normal pattern of electolyte, food, and water intake. Under these conditions ACTH produced a 4-fold increase in NaC1 intake. Further addition of cortisol and corticosterone to steroid replacement therapy produced an increase in NaC1 intake comparable to their effect on normal rabbits. Thereupon supplementation with ACTH resulted in an increase to a level at least as great as that found in ACTH treated, normal rabbits. The effects of ACTH and glucocorticoids on NaC1 appetite were synergistic. Sodium balance showed that increases in NaC1 intake were not the result of the treatment initially producing a body sodium deficit, which was then corrected by increased intake. The results provide further evidence for the hypothesis that NaC1 appetite may be hormonally regulated, and demonstrate that ACTH is capable of stimulating NaC1 intake by a previously unsuspected non-adrenal pathway.


Renal medullary adenylate cyclase in rats with hypothalamic diabetes insipidus.

The activities of enzymes related to the cellular action of vasopressin as well as the activities of other enzymes were studied in an animal model of hypothalamic diabetes insipidus. Rats with hereditary hypothalamic diabetes insipidus (homozygotes of Bratteboro strain) were found to have significantly lower renal medullary adenylate cyclase activity, either basal activity or activity stimulated by vasopressin, as compared with controls (heterozygotes of the same strain). There were no differences between the two strains in the activities of cyclic AMP phosphodiesterase, other hormone-sensitive adenylate cyclases, or the other renal medullary enzymes studied, which are apparently unrelated to the vasopressin action. The treatment of rats with hereditary hypothalamic diabetes insipidus with exogenous vasopressin increased the activity of renal medullary adenylate cyclase stimulated in vitro by maximal doses of vasopressin, but had no effect on the basal activity of adenylate cyclase or on the activity of cyclic AMP phosphodiesterase. This suggests that low adenylate cyclase activity in the renal medulla of rats with diabetes insipidus may be related to the subnormal concentrating ability observed in these animals.


Induction of human endometrial estradiol dehydrogenase by progestins.

Estradiol-17beta dehydrogenase activity in proliferative human endometrium (average of 1.5 nmole of estrone formed from estradiol/mg protein/h) was stimulated as much as as 6-fold during incubations of tissue slices in culture medium containing progesterone. Stimulation was already detectable at 7 h and the highest activity values were reached at 48-72 h of incubation in the presence of excess progesterone. Maximal stimulation was achieved with concentrations of the hormone of 0.25 mug/ml or higher. At concentrations approximately equal to midluteal plasma levels (20 ng/ml) more than 50% of the maximal response was observed. Norgestrel (17alpha-ethynyl-18-methyl-19-nortestosterone) was also effective in inducing enzymatic activity. The similarity of the effects obtained with progesterone (a possible substrate for estradiol dehydrogenase) and the synthetic progestin indicates that the stimulation of enzymatic activity was not due to substrate induction. Addition of estradiol to the culture medium had no influence on the activity of the enzyme. The induction of estradiol dehydrogenase by progesterone was inhibited by puromycin or actinomycin D. These observations indicate that progestational agents increase the rate of de novo synthesis of the enzyme. Stimulation of endometrial estradiol dehydrogenase was also observed after 2-3 day oral administration of medroxyprogesterone acetate to women in the follicular phase. In contrast, the enzymatic activity in endometrium obtained from women taking estrogens was found to be as low as in normal proliferative tissue. These in vitro and in vivo results point to progesterone as the agent responsible for the 10-fold increase in endometrial estradiol dehydrogenase activity observed during the luteal phase in menstruating women. Data obtained from superfusion studies of estrogen dynamics in endometrium indicate that changes in enzyme concentrations may play a physiologic role in the regulation of tissue levels of estradiol.


Binding of insulin to thymocytes from suckling and hypophysectomized rats: evidence for two mechanisms regulating insulin sensitivity.

Insulin more effectively increased amino acid influx into thymocytes isolated from insulin-sensitive suckling and hypophysectomized rats than into cells obtained from normal adult animals. Thymocytes from hypophysectomized adult rats bound more insulin than did cells from sham-operated controls, suggesting that insulin sensitivity in this condition was due to an increase in the number of insulin receptors. In contrast, thymocytes from suckling rats bound the same amount of insulin as did cells from adult animals, suggesting that in this instance insulin sensitivity was the result of changes in events subsequent to the binding of insulin.


The role of the pentose phosphate shunt in thyrotropin-induced thyroid hormone secretion: in vivo and vitro studies with 6-aminonicotinamide in mouse thyroids.

The possible role of the pentose phosphate shunt in thyroid hormone secretion was investigated in vivo and in vitro with mouse thyroid glands. Thyroidal endocytosis in response to TSH, a step of thyroid hormone secretion, was evaluated for its dependency upon the pentose phosphate shunt by using 6-aminonicotinamide (6-AN), an antimetabolite in the synthesis of pyridine nucleotides. Formation of 14CO2 from glucose labeled either in the C-1 or C-6 position was studied to estimate the pentose phosphate shunt activity. A dose of 6-AN markedly reduced oxidation of [1-14C]glucose but did not affect that of [6-14C]glucose induced by TSH. Concomitantly there was a marked decrease in thyroidal endocytotic response to TSH. These inhibitions by 6-AN were completely abolished by the pretreatment with nicotinamide. Methylene blue, which oxidizes NADPH and thus stimulates activity of the pentose shunt, significantly depressed thyroidal endocytosis in response to TSH in vitro. These inhibitions of colloid droplet formation by 6-AN or methylene blue were not manifested against dibutyryl cyclic AMP stimulation. Furthermore, a dose of 6-AN, which seems to inhibit only the pentose phosphate shunt, markedly depressed TSH-induced formation of cyclic amp. These findings suggest that the pentose phosphate shunt might play an important role in triggering TSH stimulation of thyroid hormone secretion by supplying NADPH, and further, that NADPH dependency in thyroid hormone secretion is at a site prior to the generation of cyclic AMP.


Diabetes insipidus and galactorrhea caused by histiocytosis X.

A 44-year-old woman with diabetes insipidus of 3 years duration was found to have histiocytosis X. This was based on clinical, radiological and pathological findings consistent with the diagnosis. Furthermore, she developed spontaneous galactorrhea during the course. Endocrine studies of hypothalamic-pituitary function revealed completely impaired secretion of gonadotropin, growth hormone and anti-diuretic hormone, and possible partial impairment of adrenocorticotropic hormone secretion, while thyroid stimulating hormone secretion remained intact. Persistently elevated plasma levels of human prolactin were also demonstrated, which were unaffected by administration of either thyrotropin releasing hormone, l-DOPA or water loading, but suppressed significantly by CB-154, an ergot alkaloid. These results suggest that abnormalities of the patient's endocrine function may be mainly accounted for by a single hypothalamic lesion.


ACTH secretion during sleep in a patient with Nelson's syndrome.

Plasma ACTH determinations were made by radioimmunoassay, every 10 min for 8 hours during sleep in a patient with Nelson's syndrome. The plasma ACTH concentrations were relatively stable, fluctuating in a narrow range. It is strongly suggested that the pituitary tumor itself was secreting ACTH in an autonomous manner and it is concluded that a neuronally initiated program of ACTH secretion, was absent in the present patient with Nelson's syndrome.


Alteration of thyroidal responsiveness to TSH under the influence of circulating thyroid hormone: short feed-back regulatory effect.

In order to examine the hypothesis that the thyroidal responsiveness to TSH is under the influence of thyroid hormone, following the T3 injection to the mice, serum T3 concentrations and the response of thyroid tissue to a fixed dose of TSH in terms of intracellular colloid droplet formation was studied. The colloid droplets induced by TSH was significantly reduced when serum T3 was decreasing, while it was significantly increased when serum T3 was increasing. This results demonstrate for the first time the existence of short feed-back loop regulating intra-thyroidal function by circulating T3. To delineate the possible mechanism of action of T3, the thyroid gland of mouse whose serum T3 concentration was elevated by injecting 50 mug T3, was incubated with TSH in vitro. TSH-induced cyclic AMP generation was not inhibited at all but colloid droplet formation was significantly inhibited in the thyroid tissue of the animal whose serum T3 concentration was enormously high. Thus, it was demonstrated that the site at which T3 affects is beyond cylcic AMP generation but prior to endocytosis, being consistent with our previous results.


Incorporation rate of glucose carbon, palmitate carbon and leucine carbon into metabolites in relation to enzyme activities and RNA levels in human skeletal muscles.

The activities (Vmax) of hexokinase, glycogen phosphorylase, glucose-6-phosphate dehydrogenase, phosphofructokinase, lactate dehydrogenase, citrate synthase, cytochrome c oxidase, and 3-OH-acyl-CoA dehydrogenase in human skeletal muscles were compared with the in vitro utilization of glucose and palmitic acid assessed under optimal conditions. Statistically significant correlations between substrate fluxes and enzyme activities were found suggesting that the substrate incorporation rate in vitro in some way reflects the capacity of metabolic pathways. The incorporation rate of leucine into muscle proteins was also statistically significantly correlated to the RNA concentration in the muscle tissue. Glycolytic and glycogenolytic enzymes correlated significantly to each other and correlations were also found between aerobic enzymes supporting the validity of constant proportions between certain key enzymes in human skeletal muscles.


Adenyl cyclase and cyclic nucleotide phosphodiesterase activities in murine muscular dystrophy.

Adenyl cyclase and cyclic nucleotide phosphodiesterase activities were assayed in homogenates of hind leg skeletal muscle from dystrophic and normal mice. Adenyl cyclase activity was stimulated 2.5 times by epinephrine and 6 times by fluoride over the basal activity in both dystrophic and normal mice. The activity of adenyl cyclase from dystrophic muscle of mice was significantly higher than that of normal mice under all the conditions tested (i.e. basal, epinephrine and fluoride). Cyclic nucleotide phosphodiesterase from skeletal muscle of mice has two Km's (2.1 and 11 mumol/l) which suggests the existence of either two forms of enzyme or a single enzyme with negative cooperativity. The activity of this enzyme was significantly elevated in the skeletal muscle of dystrophic mice compared to the normal controls. The available evidence suggests that the same cyclic nucleotide phosphodiesterase is responsible for the hydrolysis of both cyclic AMP and cyclic GMP.


Enzyme activities in muscle and connective tissue of M. Vastus lateralis in habitually training and sedentary 33 to 70-year-old men.

A cross-sectional study was carried out to examine the activities of certain enzymes representing aerobic and anaerobic energy metabolism as well as the biosynthesis of collagen of M. vastus lateralis in 23 male endurance athletes in habitual training, aged 33 to 70 years. 23 sedentary healthy men of corresponding ages were selected for the control group. The mean maximal oxygen uptake of the trained subjects was 53.6 ml-kg--1. min--1 and that of the control subjects 36.3 ml-kg--1. min--1. As compared to the control group the trained subjects had significantly higher values in the muscle malate dehydrogenase, succinate dehydrogenase and prolyl hydroxylase activities, whereas the opposite was true in the activity of lactate dehydrogenase. In hexokinase and creatine phosphokinase no marked differences between the groups were observed. The results showed that endurance training leads to increased activities of oxidative enzymes in the skeletal muscle. The adaptation changes were also observed in old men. The increased activity of prolyl hydroxylase may reflect the general enzymatic adaptation to physical training. A possibility exists that the turnover of muscle collagen in endurance athelets is continuously faster than that in sedentary men of corresponding ages.


The 3'-amido and 5'-amido analogues of adenosine 3':5'-monophosphate; interaction with cAMP-specific proteins.

The sensitivity for recognition of adenosine 3:5'-monophosphate (cAMP) by its coordinate proteins towards chemical changes in the six-membered cyclic phosphate ring has been investigated. A comparison of the interaction parameters of the 3' and 5'-amido analogues (I, II) and of unsubstituted cAMP has been made using two different protein kinases and the phosphodiesterase from bovine heart. Binding affinity and the capacity of the amido analogues to stimulate the phosphotransferase activity of the kinases is greatly reeuced relative to cAMP, the 3'-position being more sensitive towards the modification than the 5'-position. The coordinate noncyclic derivatives, 3'-deoxy-3'-amino-5'-AMP (IV) and 5'-deoxy-5'-amino-3'-amp (iii), were also tested. Surprisingly activity towards protein kinases was found to be considerable for the 5'-deoxy-5'-amino-3'-AMP (III), while the 3'-deoxy-3'-amino-5'-AMP (IV) is practically inactive. A possible reason for this is that the noncylic 5'-analogue (III) may be able to assume a cyclic structure maintained by internal salt formation. The phosphodiesterase splits both cyclic amido analogues but with reduced rates compared to that of natural cAMP. Kinetic data obtained from different methods reveal a stronger affinity for the 5'-analogue (I) than the 3'-analogue (II) for the active site, although the reaction rate at saturated substrate concentration is significantly higher with II than with I. The properties of the amido and the noncyclic amino analogues are discussed with available data from chemotaxis of the cellular slime moulds. Furthermore data of the respective methylene cyclic derivatives are used for a more comprehensive comparison. The above is interpreted in terms of the electronic features of the substitutions and of the changes in bond distances or angles upon replacement of O by NH or CH2 in the cyclic phosphate ring (obtained from X-ray work).


Heterogeneity of enterotoxin-like protein extracted from spores fo Clostridium perfringens type A.

Enterotoxin-like protein was extracted from spores of three enterotoxin-positive and three enterotoxin-negative strains of Clostridium perfringens type A by urea/mercaptoethanol, alkaline mercaptoethanol and alkaline dithiothreitol. Disc immunoelectrophoresis demonstrated that three distinct enterotoxin-like proteins could be extracted. In 7% acrylamide gels, type I, type II, and type III enterotoxinlike proteins had relative mobilities of 0.52, 0.63, and 0.73 respectively. In contrast to disc immunoelectrophoresis, immunoelectrophoresis in agar gel demonstrated identical electrophoretic properties for the various entertoxin-like proteins. Immunoelectrofocusing experiments gave isoelectric points of 4.43, 4.43, 4.36, and 4.52 for purified entertoxin and type I, type II, and type III enterotoxin-like proteins respectively. Ferguson plots (i.e., log relative mobility versus acrylamide concentration) yielded nonparallel lines which intersected at a nonsieving concentration of acrylamide indicating that the various species of enterotoxin-like protein differed in size. Estimation of the molecular weight of purified enterotoxin and the three species of enterotoxin-like protein was done by comparing the slopes obtained in Ferguson plots with those obtained using proteins of a known molecular weight. Molecular weights of 38000, 36500, 23000, and 15400 were obtained for purified enterotoxin, type I, type II, and type III enterotoxin-like protein respectively. Collectively, the evidence indicates that fractionation of the different species of enterotoxin-like protein was due primarily to differences in their size, and that different forms of enterotoxin-like protein can be extracted from spores of different strains of C. perfringens type A.


On the mechanism of inhibition of enzyme induction in Escherichia coli by distamycin A.

The mechanism of the interference of the antiviral antibiotic distamycin A with the bacterial cell has been investigated. Labelled distamycin A is firmly bound by E. coli cells and the binding process does not require metabolic energy as indicated by the use of inhibitors. The antibiotic does not induce gross alteration in the cell membrane but inhibits cyclic AMP accumulation in the cells exposed to a glucose-free medium. This inhibition is concomitant with that exerted on the synthesis of an inducible enzyme such as beta-galactosidase. By the method of pulse induction it appears that distamycin A exterts its inhibiting effect on inducible synthesis at the level of transcription. This effect is probably related to an interference with the positive control of enzyme synthesis performed via the system represented by cyclic AMP and the CRP protein.


[Synthesis and properties of semisynthetic penicillins containing the furoxan nucleus].

Synthesis, antibacterial activity and stability in acid solution of potassium salts of 6-(3-methyl-4-furoxancarboxyamido)penicillanic acid and 6-(4-methyl-3-furoxancarboxyamido)penicillanic acid are reported.


Quantification of triglyceride transport in blood plasma: a critical analysis.

Reliable and precise quantification of endogenous triglyceride transport in man has not been possible with simple means to date. Direct measurement of net splanchnic secretion of triglyceride fatty acids (TGFA) in very low density lipoproteins (VLDL) provides the must unambiguous information, but precision is low. Coupling infusion of labeled fatty acid with sampling of arterial and hepatic venous blood increases precision; however, the contribution of precursors other than plasma free fatty acids (FFA) must be assessed. Measurement of the rate of hydrolysis of plasma triglycerides after displacing lipases into the blood with heparin holds promise as a simple, nonisotopic method, but it has not been carefully validated and heparin itself alters FFA and triglyceride transport. Multicompartmental analysis following pulse injection of labeled fatty acid offers a practical approach, but uncertainties about the number  and location of interacting compartments have made it impossible to determine an absolute value for transport. Reinjection of biologically labeled plasma VLDL is impractical for large scale use, and validity of this approach remains uncertain because of heterogeneity of VLDL-triglycerides and their complex metabolic behavior. Methods to label VLDL-triglycerides in vitro deserve more study as does labeling of other components, such as the B-apoprotein. Such approaches will require rigorous comparison with biologically labeled material as well as careful assessment of alterations in kinetic behavior that may occur when VLDL are separated from blood plasma.


Triglyceride turnover: a comparison of simultaneous determinations using the radioglyceride and the lipolytic rate procedures.

Two different methods of examining triglyceride turnover are described. One measures the clearance of endogenously labeled triglyceride glycerol, the radioglyceride method. The other assesses the hydrolysis of endogenous very low density lipoproteins after a primed infusion of maximal doses of heparin, the lipolytic rate. Both depend on widely different assumptions. A comparison of data obtained by both methods at the same time in single individuals with widely differing triglyceride concentrations revealed them to agree quite closely. This correspondence indirectly suggests the validity of the procedures. The procedures have been applied to show that fat-free high-carbohydrate diets accelerate triglyceride production in some humans.


Preliminary model for human lipoprotein metabolism in hyperlipoproteinemia.

A model is proposed for the metabolism of plasma lipoprotein apoproteins based on studies of a hyperlipoproteinemic subject who received 2.5 mCi[3H]leucine intravenously. Measurements included apoprotein specific activities (apo-B and apo-C) of very low density lipoprotein (VLDL) and of three low density lipoprotein (LDL) subspecies, Sf 17 LDL, Sf 10 LDL, and Sf 4 LDL. Activities of plasma albumin were also determined. The data were analyzed using a compartmental model and the SAAM computer program. A chain-like series of compartments were necessary to simulate plasma VLDL kinetics, suggesting a multistep delipidation process. The data are consistent with the notion that VLDL is the dominant LDL precursor. Two modes of conversion from VLDL to LDL are required. After partial delipidation some VLDL is converted to the Sf 17 LDL, while the remainder undergoes further delipidation before being converted to Sf 4 LDL, the major plasma LDL component. Some direct release of LDL into plasma had to be introduced to fit the data, about 24% of total LDL production. The three LDL subspecies follow a precursor-product relationship (Sf 17 leads to Sf 10 leads to Sf 4). The analysis also indicates that in using labeled leucine as a tracer, the slow exchange of leucine with the total body protein pool must be considered in trying to resolve the LDL subsystem and in the estimation of steady-state apoprotein levels. In view of the fact that the proposed model is based predominantly on the data from a single patient, no generalizations can be made about parameter values. The study is most valuable, however, in pointing out metabolic pathways not considered before and in calling attention to variables that must be considered in the design of experiments to study lipoprotein kinetics.


Erythropoietin and the differentiation of red blood cells.

Erythropoietin is the primary factor regulating red blood cell formation in mammals and some other animals. It has been purified from plasma derived from anemic sheep and from the urine of anemic patients and is a glycoprotein. The sheep hormone has a molecular weight of 46,000 and appears to consist of a single chain. The carbohydrate and amino acid compositions where known, are summarized, as are the known structural requirements for biological activity. Its mode of action as the inducer of red cell differentiation has been studied in marrow cell and fetal liver cell cultures. Erythropoietin interacts first with a protein receptor on its target cell causing the appearance of a cytoplasmic protein thought to be a mediator that, in turn, causes an increased rate of transcription in the target cell nuclei. There are several different species of RNA synthesized before the cells initiate hemoglobin synthesis. The effect on transcription is only indirectly dependent on DNA synthesis. Some properties of the primitive erythropoietin-responsive cell are discussed and a cellular model for blood cell differentiation is presented.


The expression of avian gs antigen in mammalian Rous sarcoma virus-transformed cells after treatment with 5-iododeoxyuridine and dexamethasone.

The content of avian gs antigen in RSV-transformed rat cells LW13-RsK1 and LW13-RsK4 showed a transient increase after treatment with 5-iododeoxyuridine. Higher levels of gs antigen were found when treated cells were cultivated in the presence of dexamethasone. In no case was the production of RSV found in treated RSV-transformed rat cell lines when, in addition, XC cells were tested.


Effect of prophylactic and therapeutic administration of BCG vaccine on growth of experimental sarcoma.

The effect of prophylactic and therapeutic administration of BCG vaccine on growth of transplantable sarcomas induced by MSV-Harvey (MSVT1) and 3-methylcholanthrene (MC 11 and MC 12) in C57BL/10Sn mice was studied. Growth of MSVT1 sarcomas was not inhibited by oral or intratumoral BCG therapy. Nor was prophylactic oral administration of BCG followed by therapeutic oral BCG treatment effective. On the other hand, intradermal administration of an adequate dose of BCG vaccine preceding the tumour transplantation inhibited tumour growth. The low (0.05-0.2 mg.) doses of BCG induced resistance comparable to the effect of subcutaneous immunization with tumour cells, whereas the higher (0.5-1.0 mg) doses had no effect. Growth of MC 11 and MC 12 tumours was not affected by prophylactic intradermal BCG administration.


Long-term preservation of transfecting activity of DNA isolated from rat virogenic XC cells transformed by Prague strain of Rous sarcoma virus.

The DNA isolated from rat virogenic XC cells transformed by Rous sarcoma virus retains standard transfecting activity for 7 months when the DNA solution in 0.1 X PBS, containing 10% glycerol is stored at -70 degrees C. The value of the sedimentation constant S20w does not significantly change during storage.


Mode of dermal-epidermal adhesion.

The mode of dermal-epidermal adhesion was investigated by a systematic enzyme digestion of the dermo-epidermal junction. The anchoring fibrils and filaments were found to consist of elastin and collagen-like filaments which intertwine to form the adepidermal membrane. A model on the possible in situ conditions is proposed.


Mechanism of nucleotide inhibition of gonadotropin binding to cell membranes of bovine corpus luteum.

ATP, CTP, ADP, AMP, cyclic 3',5'-AMP (cAMP) and cyclic 3',5'-CMP (cCMP) effectively inhibited the specific binding of 125I-labelled human chorionic gonadotropin ([125I]HCG) to bovine corpus luteum cell membranes. This inhibition was observed with 2.5 X 10(-4) M to 1.0 X 10(-3) M nucleotide concentrations, regardless of the presence of a nucleotide regenerating system. Submaximal concentrations of combinations of the nucleotides were additive in inhibiting binding. The inhibition of [125I]HCG binding was observed when the nucleotides were added at the beginning of or during incubation or preincubation of the membranes with nucleotides. Preincubation of membranes with CTP and cAMP, subsequent washing and reincubation with hormone, showed time-dependent inhibition of [125I]HCG binding when the preincubation temperature was 38 degrees C but not at 4 degrees C. The concentrated supernates from nucleotides preincubated with membranes had no inhibitory effect on [125I]HCG binding to fresh membranes. In the absence of added nucleotides, [125I]HCG-membrane interaction had the following apparent binding constants: a Kd of 1.5 X 10(-10) M, 46.3 fmoles of binding sites per mg membrane protein, and rate constants for association and dissociation 4.0 X 10(6) M-1 sec-1 and 1.0 X 10(-3) sec-1, respectively. At steady state conditions of [125I]HCG binding, CTP inhibited [125I]HCG at lower concentrations of added hormone (less than 3 X 10(-9) M) whereas at higher concentrations, this nucleotide enhanced [125I]HCG binding. Scatchard analysis of the data revealed that inhibition and enhancement of [125I]HCG binding in the presence of CTP were due to lowered affinity of gonadotropin receptors (32-37) fold) and to exposure of new low-affinity binding sites for [125I]HCG, respectively. At non-steady-state conditions, nucleotides increased dissociation rates (80 to 100%) and decreased association rates (30 to 38%). The data appear to be compatible with the suggestion that the nucleotides may bind to sites in the membranes and subsequently induce conformational changes in membrane components, resulting in a decreased affinity of gonadotropin receptors. The physiological significance of these findings needs to be determined.


Cyclic nucleotide phosphodiesterases in uterine development.

Activities of cyclic nucleotide phosphodiesterase were studied in rat uterus as a function of age, DNA and protein content. Linear kinetics were observed for uterine homogenate cyclic GMP (cGMP) phosphodiesterase activity, but anomalous double-reciprocal plots, suggestive of multiple enzyme forms, were observed for cyclic AMP (cAMP) hydrolysis, cAMP phosphodiesterase was therefore measured at high and low substrate concentrations, 200 muM and 0.25 muM cAMP, respectively, to approximate multiple enzyme activities. Based upon total organ content, the total cAMP and cGMP phosphodiesterase activities increased throughout uterine development, from 5-50 days of age. On the same basis, the apparent low KM cAMP phosphodiesterase increased only between days 5 and 15 and showed no significant increase between days 15 and 50. On the other hand, specific activities of an apparent low KM cAMP phosphodiesterase, expressed per mg of protein or per mug of DNA, showed a marked reduction in activity between 30 and 50 days of age. Chronic administration of 17beta-estradiol to immature rats increased their uterine protein content and decreased the specific activity of the apparent low KM cAMP phosphodiesterase. In another estrogen target tissue, the anterior pituitary, protein and DNA content also increased during development but no changes in specific activities of cyclic nucleotide phosphodiesterase were noted. These results suggest the possible participation of cyclic nucleotide phosphodiesterases in the induction of uterine growth and development by ovarian hormones.


Effect of partially purified NSILA on adenylate cyclase, phosphodiesterase and 3',5'-cyclic AMP in fat cells.

Partially purified, non-suppressible, insulin-like material (NSILA-S)  was studied with respect to its effect on the levels of 3',5'-cyclic adenosine monophosphate (cAMP) and its mechanism of action in the control of this nucleotide in rat fat cells. NSILA-S prevents the rise of cAMP in fat cells under the influence of isoproterenol with similar kinetics to insulin. A maximal effect is observed at about 70 ng/ml with a biological activity equivalent to 200 muU/ml of insulin. NSILA-S inhibits norepinephrine-stimulated adenylate cyclase activity in fat cell ghosts and partially purified plasma membrane preparations. At 10 mM Mg2+, the inhibition is characterized by an effect of Vmax without change in affinity towards ATP (apparent KM 30 muM). Similarly there is no observed change in affinity towards Mg2+. With respect to inhibition of norepinephrine-stimulated adenylate cyclase, the dose-response curve of NSILA-S is similar to that already found with intact cells. The effect of norepinephrine is inhibited throughout the dose-response range between 5 X 10(-7) and 5 X 10(-4) M. In contrast to previous observations with insulin in ghosts, NSILA-S inhibits the basal adenylate cyclase activity. Cyclic nucleotide phosphodiesterase activity in homogenates as measured at 1.0 muM substrate is increased by 90% after previous incubation of fat cells with NSILA-S. The study suggests that the anti-lipolytic effect of NSILA-S is mediated by a lowering of cAMP through inhibition of the adenylate cyclase and/or stimulation of the phosphodiesterase system.


Studies on isolated rat adrenal cells metabolism of hydroxylated sterols.

Corticosterone production by isolated rat adrenal cells in the absence of ACTH is stimulated by 20alpha-hydroxycholesterol, 22R-hydroxycholesterol and 25-hydroxycholesterol. This effect is also seen at sub-maximal ACTH concentrations. Aminoglutethimide (AGI) inhibits the stimulation caused by the three hydroxylated cholesterols. In the presence of ACTH, AGI also inhibits steroid production both under control conditions and in the presence of the sterols. The stimulation by 25-hydroxycholesterol is dose-dependent, both in the presence and absence of a sub-maximal ACTH concentration. At maximal ACTH concentrations 25-hydroxycholesterol does not give additional stimulation. Cycloheximide has no effect on corticosterone production from 25-hydroxycholesterol whether ACTH is present or not. Our results indicate that 25-hydroxycholesterol is a good substrate for the study of the cholesterol side-chain cleaving system and the mechanism of action of ACTH at the levels of the intact cell.