In vivo infectivity of the fibrotropic C-type viral isolates from C57BL/Ka mice. Of the three fibrotropic C-type viral isolates from C57BL/Ka mice, only the BL/Ka(B) virus is capable of infecting normal hematopoietic and lymphoid cell populations of C57BL/Ka mice in vivo, and none are tumorigenic. Inoculation of this virus alone into neonates resulted in transient replication in the bone marrow, spleen, and occasionally the thymus. Thymocytes could, however, be permanently infected in such animals if BL/Ka(B) were coinoculated with the xenotropic BL/Ka(X) virus. Neonatal injection of BL/Ka(B) prior to fractionated wholebody irradiation yielded an increase in the percentage of virus-productive radiogenic lymphomas and a decrease in incidence of such tumors. Injection of BL/Ka(B) into normal adult C57BL/Ka mice did not yield overt expression of virus replication in any of the tissues tested; latent infection could, however, be detected in the marrow and in the reticuloepithelium of the thymus. Whole-body X-irradiation of adults with 400 rads partially restored the neonatal susceptibility of bone marrow cells to infection by the isolate. BL/Ka(B) injection after fractionated whole-body irradiation of weanling C57BL/Ka mice increased the percentage of virus-positive lymphomas and revealed that a bone marrow cell subpopulation permissive for infection by the virus increases greatly in abundance soon after irradiation.


Prolactin receptors in human breast cancer cells in long-term tissue culture. Prolactin receptors have been identified for the first time in a number of human breast cancer cell lines and a normal human breast cell line maintained in long-term tissue culture. Optimal conditions for determining the binding of 125I-labeled human prolactin to these cells were established. Five different tumor cell lines have different content of prolactin receptors ranging from 2,300 to 26,000 sites/cell. All tumor cell lines contained more prolactin receptors than does one normal breast cell line (1700 sites/cell). The prolactin receptors in these human mammary tumor cells not only bind human prolactin but also recognize other lactogenic hormones such as human growth hormone, human placental lactogen, and sheep prolactin, but not animal growth hormone, which are not lactogenic. The affinity (Ka) of binding of human prolactin to these cells is 4 x 10(9) M-1 (Kd = 2.5 x 10(-10)M). The hormone specificity and affinity for hormone of these human mammary tumor cells are very similar to that found for the rabbit mammary gland. These human mammary tumor cell lines in long-term culture should prove very useful to study the biology of prolactin receptors in living human cells and the role of prolactin in the tumorigenesis of the human breast.


Hormone-dependent growth of a rat chondrosarcoma in vivo. The importance of various hormonal factors in the growth of a transplantable chondrosarcoma has been studied in vivo. Tumor growth was reduced by 95% in adrenalectomized or hypophysectomized rats as compared to normal animals. The number of tumors developing in either adrenalectomized or hypophysectomized rats was reduced more in male than in female rats. However, ovariectomy or orchiectomy did not alter the growth of the tumor. The inhibition of tumor growth in adrenalectomized and hypophysectomized animals was only observed after the first 10 days following inoculation. Cortisone (4-pregnen-17 alpha,21-diol-3,11,20-trione) administration fully restored tumor growth in adrenalectomized animals while adrenocorticotropic hormone or growth hormone were only partially effective in supporting tumor growth in hypophysectomized animals. High-affinity glucocorticoid receptors (7 to 10S) were present in the cytosls prepared from the tumor cells and were found to be increased in tumors from adrenalectomized animals. These results indicate that the growth of this chondrosarcoma is strongly dependent upon endocrine factors of adrenal and pituitary origin.


Relationship between inhibition of protein methylase I and inhibition of Rous sarcoma virus-induced cell transformation. A correlation was found between inhibition of protein methylase I and inhibition of virus-induced cell transformation by structural analogs of S-adenosylhomocysteine; all good inhibitors of this enzyme are also good inhibitors of Rous sarcoma virus-induced chicke embryo fibroblast transformation. The inhibitory effect of these analogs was similar on enzymes from normal and transformed cells; no significant variation of the inhibition constants was observed after purification of protein methylase I. From the kinetic constants obtained, a structure-activity relationship can be established for protein methylase I.


Metabolism of three radiolabeled pancreatic carcinogenic nitrosamines in hamsters and rats. The in vivo metabolism and disposition of three radiolabeled N-nitrosamines which are carcinogenic for the pancreas of the hamster but not the rat have been examined. N-[1-14C]Nitrosobis(2-oxopropyl)amine (BOP), N-[1-14C]nitrosobis(2-hydroxypropyl)amine (BHP), and their suggested proximate pancreatic carcinogenic metabolite N-[1-14C]nitroso-(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) were metabolized and exhaled as 14CO2 to various extents somewhat proportional to their carcinogenic potency. More than 50% of the dose of BOP and HPOP was exhaled as 14CO2, whereas 26% of BHP was excreted this way, and 40% of BHP was excreted unchanged in the urine. Administered BOP was excreted to a small extent in the urine of both species as HPOP and BHP. No other nitrosamine metabolites were detected in urine. HPOP and BHP were detected in the pancreatic juice and bile of both species after administration of BOP and BHP. The results suggest that pancreatic ductular carcinogenesis in the hamster as a result of exposure to BOP is not due to secretion of carcinogenic metabolities in the pancreatic juice or reflux of bile containing nitrosamine metabolites into the ducts. Carcinogen metabolic activation appears to be by an oxidative pathway.


Enhanced collagenase production by fibroblasts derived from human basal cell carcinomas. Fibroblast cultures derived from human basal cell carcinomas demonstrated an increased capacity to synthesize and secrete collagenase. Although the levels of collagenase were up to 8-fold greater than those of normal control cell lines, this phenotypic trait was not permanent and was expressed only for a few passages following primary explanation. The basal cell carcinoma fibroblast collagenase was secreted as a proenzyme. The kinetics of activation and the catalytic efficiency of the basal cell carcinoma fibroblast enzyme were equal to control collagenase, indicating that increased activity was due to increased synthesis of enzyme protein. Increased synthesis of collagenase was not due either to altered cell growth or to an overall increase in protein synthesis. Furthermore, synthesis of another major protein, of another major protein, collagen, was not enhanced. The data suggest that the tumors may have stimulated adjacent fibroblasts to produce more collagenase which is of importance in tumor invasion.


Relationship between Rous sarcoma virus-induced expression of membrane antigen and phenotypic transformation. Rous sarcoma virus-transformed hamster BHK fibroblasts express a virus-induced cell surface antigen undetectable in cells either transformed by unrelated viruses or infected by transformation-defective strains of Rous sarcoma virus. To clarify whether this antigen plays any role in the process of malignant transformation or is expressed at the cell surface only as a consequence of the acquisition of the transformed phenotype, we have investigated its expression at the cell surface of Rous sarcoma virus-transformed BHK cells treated with dibutyryl cyclic adenosine 3':5'-monophosphate and at the surface of parental BHK cells transiently transformed by the tumor promoter phorbol myristate acetate. In the dibutyryl cyclic adenosine 3':5'-monophosphate-treated cells, in which most of the parameters of the transformed phenotype are reverted to normality, but the product of the transforming gene is still present, virus-induced cell surface antigen is expressed. In the mirror experiment, this antigen is not expressed by phenotypically transformed but genetically normal phorbol myristate acetate-treated cells. It is concluded that the tumor membrane antigen studied is intimately associated with the expression of the function(s) controlled by the transforming gene.


Effects of 12-O-tetradecanoylphorbol-13-acetate and mezerein on epidermal ornithine decarboxylase activity, isoproterenol-stimulated levels of cyclic adenosine 3':5'-monophosphate, and induction of mouse skin tumors in vivo. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the antileukemic agent mezerein are diterpene esters of plant origin with certain structural similarities. Both compounds, when applied topically to mouse skin, were equipotent on a molar basis in inducing hyperplasia, inflammation, and ornithine decarboxylase activity, as well as in reducing cyclic adenosine 3':5'-monophosphate accumulation in response to beta-adrenergic stimulation. In contrast, mezerein was much less effective as a tumor promoter; the phorbol ester at 8.5 nmol/application yielded 78-fold more tumors than did 8.5 nmol mezerein per application to similarly initiated SENCAR mice. The superiority of the phorbol ester was nearly as great in CD-1 mice.


Putrescine as a biochemical marker of malignant brain tumors. Putrescine, spermidine, and spermine levels were determined in normal brain and central nervous system-related tumor tissues obtained at operation from 50 patients. The biochemical data were correlated with morphological histopathological descriptions of the same tissues. There was little variation in putrescine levels in normal cerebral cortical tissue. Subcortical white matter had lower putrescine but higher spermidine content than those of the overlying cortex. Putrescine levels were elevated in all astrocytomas assayed, and the magnitude of this elevation was proportional to the malignancy of the tumor as determined by histopathological criteria. In contradistinction, putrescine content of "benign" tumors was generally equal to or lower than that of the normal cerebral cortex. Spermidine and spermine levels varied widely in the tumors assayed and did not correlate with criteria of malignancy. It is concluded that putrescine may be a good biochemical marker of malignancy in central nervous system-related tumors.


Sequence homology of nucleic acids from human breast cancer cells and complementary DNA's from murine mammary tumor virus and Mason-Pfizer monkey virus. Simultaneous presence of murine mammary tumor virus- and Mason-Pfizer monkey virus-specific sequences has been detected in nucleic acids isolated from some human breast tumors and from MCF-7 cells, a well-characterized human breast cancer cell line. Carefully characterized long complementary DNA transcripts were used in the molecular hybridization experiments. From the data that are presently available, it would appear that when homology is detected with one of the mammary tumor probes the other also generally shows shows homology. Among all the complementary DNA-RNA hybrids only three, all murine mammary tumor virus hybrids, show Tm values close to 80 degrees. The rest of the hybrids are low melting with shallow slopes for their Crt curves, indicating partial and imperfect hybrids in the majority of cases. Low levels of weak hybrid formation are also detectable with the tumor DNA's. The present experiments cannot ascertain whether the hybridizing sequences from Mason-Pfizer monkey virus and murine mammary tumor virus code for specific viral functions in their natural hosts. Annealing experiments using gene specific cDNA's would be required for fully characterizing these sequences.


Evaluation of impaired triglyceride fatty acid transport and oxidation for the detection of cancer in mice. We have tested the hypothesis proposed by Costa, Lyles, and Ullrich. (Effect of Human and Experimental Cancer on the Conversion of 14C Tripalmitin to 14CO2. Cancer (Phila.), 38:1259-1265, 1976) that the transport and/or oxidation of triglyceride fatty acids is markedly impaired in rodents bearing a growing s.c. carcinoma. Specifically, we have tested whether oxidation of triglyceride fatty acids is depressed in cancer-bearing animals. Mice inoculated s.c. with Ehrlich carcinoma cells were given injections (i.v. and i.p.) of 14C-labeled triglyceride fatty acids prepared as very-low-density lipoproteins by physiological methods or (i.p.) with [-14C]tripalmitin dissolved in peanut oil during both early (3 to 4 days) and advanced (7 to 8 weeks) stages of tumor growth. Specific activity of the expired 14CO2 was measured for periods ranging from 1 to 7 hr following injections. Because cancer-bearing mice can become severely hypertriglyceridemic, plasma triglyceride pool sizes were also measured during each experiment to account for the effects of possible differential dilution of the tracers. At no instance did we find any significant differences between specific activities of expired 14CO2 or plasma triglyceride pool sizes of the cancer-bearing animals and controls. Thus, a cancer-induced impairment of triglyceride fatty acid transport and metabolism to CO2, such as reported by Costa et al., does not seem to be a universal phenomenon in rodents.


cis-Dichlorodiammineplatinum(II) and VP-16-213: an active induction regimen for small cell carcinoma of the lung. Thirty-eight patients with small cell carcinoma and no prior therapy were treated with a combination chemotherapy program including 60 mg/m2 of cis-dichlorodiammineplatinum(II) (cis-platinum) iv on Days 1 and 22 and 120 mg/m2 of VP-16-213 iv on Days 4, 6, 8, 25, 27, and 29. This was followed by 1000 mg/m2 of cyclophosphamide, 40 mg/m2 of Adriamycin, and 1.4 mg/m2 of vincristine, all given iv on Days 42, 63, 84, and 105. The program was then recycled, with cis-platinum and VP-16-213 beginning on Day 126 and the regimen repeated as above. All patients received prophylactic whole-brain radiation (usually at a dose of 3000 rads in ten fractions) between Days 42 and 63. The projected duration of treatment is 18 months in the absence of relapse. In 21 patients with limited disease, the complete response rate was 52% (5.75+--15.25+ months) and the partial remission rate was 48% (2.25--10.5 months). The extensive-disease group showed a complete remission rate of 41% (4.75--11+ months) and a partial remission rate of 47% (3.75--11.5+ months). Response to therapy with cis-platinum and VP-16-213 was very rapid and invariably maximal by the end of the 6-week induction period. Survival for the limited-disease group appears encouraging but followup time is insufficient. Toxicity included nausea and vomiting, myelosuppression, alopecia, and renal insufficiency which was dose-limiting in two patients. The cis-platinum and VP-16-213 combination is clearly an active induction regimen in small cell carcinoma of the lung, but whether it will play a role in increasing long-term survival rates remains to be seen.


Antigenic variation in visna virus.

Antigenic variation in visna virus. Two antigenic variants of visna virus were isolated sequentially from a single sheep inoculated with a plaque-purified strain of virus designated 1514. The genetically stable variants, LV1-1 and LV1-4, are of two classes: LV1-1 is partially neutralized by antibody to the inoculum strain 1514, while LV1-4 is not neutralized by antibody to 1514. The genetic mechanism responsible for generating the antigenic variants was investigated by comparing the chymotryptic and tryptic maps of the envelope glycoprotein gp135 and core polypeptides (p30, p16, p14), and by comparing the pattern of large oligonucleotides produced by digestion of the RNAs by T1 ribonuclease. We show that only the peptide maps of gp135 differ among strains, that the number of peptide fragments altered is small and that gp135 is the polypeptide that elicits neutralizing antibody. The maps of the RNAs are identical. We conclude that mutation in the glycoprotein gene rather than recombination is more probably responsible for antigenic variation, and speculate on the special aspects of visna virus replication relevant to this phenomenon.


Germ cell tumors of the testis.

Germ cell tumors of the testis. The histologic patterns of the various germ cell tumors of the testis are reviewed and these tumors have been defined and classified according to the World Health Organization criteria. The importance of obtaining adequate sections of the primary and metastatic tumors is stressed and more extensive use of the World Health Organization classification of testicular tumors is encouraged so that meaningful comparisons may be made between different centers treating these patients.


Phase II trial of cis-dichlorodiammineplatinum(II) in advanced malignant lymphoma and small cell lung cancer: preliminary results. In an ongoing cooperative study of the Cancer and Acute Leukemia Group B, 21 evaluable patients with advanced malignant lymphomas were treated with 70 mg/m2 of cis-dichlorodiammineplatinum(II) (cis-platinum) once every 3 weeks. All patients had received extensive prior therapy. Partial remission was obtained in two of seven patients with Hodgkin's disease, for 1+ and 7 months, and in three of 14 patients with non-Hodgkin's lymphoma, for 2, 2+, and 2.5 months. In another ongoing trial, 11 patients with advanced, pretreated small cell cancer of the lung received 80 mg/m2 of cis-platinum once every 3 weeks. Four patients achieved partial remissions. These lasted 2+ and 2.5 months in the two patients evaluable for duration of response. Two further clear-cut tumor regressions were noted. The major toxic effects were myelosuppression and vomiting. In the second trial, one case of probable drug-related fatal nephrotoxicity was encountered despite optimal forced diuresis with mannitol and furosemide. cis-Platinum definitely warrants further evaluation in these diseases because of significant effectiveness even after extensive prior treatment.


Characterization of different tumor antigens present in cells transformed by simian virus 40. In addition to large T and small t antigens, cells transformed by simian virus 40 (SV40) commonly contain other proteins which specifically immunoprecipitate with SV40 anti-T serum and which are  not detected in untransformed cells. The additional tumor antigens (T-Ags) fall into two groups: those having a close structural relationship with normal SV40 T-Ags, and those unrelated to large T and small t. The latter are probably nonviral T-Ags (NVT-Ags). The NVT-Ags comprise a family of proteins of molecular weight 50,000-55,000. Fingerprint analysis shows that NVT-Ags have few if any peptides in common with large T or small t, and that they lack the amino terminal tryptic peptide and the peptides unique to small t. NVT-Ags from different species have different fingerprints, but those isolated from different transformants of the same cell line are identical. The size of NVT is unaltered in cells transformed by mutants of SV40 with deletions in the region 0.60-0.55 map units. The mRNA for NVT does not hybridize to SV40 DNA. The other forms of T-Ag isolated from transformed cells fall into three classes: shortened forms of large T (truncated large T); multiple species of T-Ag with molecular weights very similar to, but distinct from, those of normal large T (large T doublets and triplets); and elongated forms of large T (super T). These proteins all contain the normal amino terminus of SV40 T-Ags, and the truncated forms of large T lack peptides from the carboxy terminal half of large T. One species of super T (molecular weight 130,000) contains only those methionine tryptic peptides present in normal large T, although it may contain some peptides in more than one copy.


Heterogeneity of genetic loci in chickens: analysis of endogenous viral and nonviral genes by cleavage of DNA with restriction endonucleases. Restriction endonucleases can be used to define the structure and position of genetic loci for which specific molecular hybridization reagents are available. We have used this approach to compare 18 chicken embryos with respect to several cellular genes; endogenous viral DNA related to the replicative genes of avian sarcoma virus (ASV) or to RAV-O, an endogenous virus of chickens; and sequences related to the transforming (src) gene of ASV. Each cellular gene eas remarkably homogeneous within our test population. We found little or no variation in globin and ovomucoid genes; ovalbumin and transferrin (with one exception) showed variation which is probably allelic in nature. The endogenous viral DNA which has homology with RAV-O was found at several different positions in host DNA and its structure resembled that of proviruses acquired by experimental infection, with sequences from both ends of viral RNA repeated near both ends of viral DNA. Within the population of 18 chickens, one endogenous provirus was always present, whereas the several other proviruses were each found in only a few members of this group. However, screening of additional chickens identified individuals lacking the provirus common to the initial 18 animals surveyed; in at least one embryo no RAV-O-related DNA was detected. These findings suggest that the endogenous RAV-O-related sequences have entered the germ line by relatively recent infection and are still segregating in several contemporary chicken flocks. The sequences in the chicken genome which have homology with the src gene of ASV are invariant from bird to bird and in this sense resemble a cellular gene rather than a viral sequence.


Chicken hematopoietic cells transformed by seven strains of defective avian leukemia viruses display three distinct phenotypes of differentiation. Chicken hematopoietic cells transformed in vitro and in vivo by seven strains of replication-defective avian leukemia viruses were assayed for the expression of six erythroid and five myeloid differentiation parameters, including differentiation-specific surface antigens as detected by newly developed antisera. The transformed cells were found to display three distinct phenotypes of differentiation. First, cells transformed by AEV resemble erythroblasts. They express heme, globin, carbonic anhydrase and erythrocyte cell surface antigen at low levels, and histone H5 and erythroblast cell surface antigen at high levels. Second, cells transformed by MC29, CMII, OK10 and MH2 viruses have macrophage-like properties. They strongly express Fc receptors, phagocytic capacity and macrophage cell surface antigen, but only weakly express myeloblast cell surface antigen and are negative for ATPase activity. Third, cells transformed by AMV and E26 viruses resemble myeloblasts in that they weakly express Fc receptors, phagocytic capacity and macrophage cell surface antigen but strongly express myeloblast cell surface antigen and ATPase activity. No difference was found between in vitro- and in vivo-transformed cells in the parameters tested. In light of recent genetic and biochemical evidence, we believe that these phenotypes reflect the action of three new types of viral-transforming genes, designated erb (erythroblast), mac (macrophage) and myb (myeloblast).


The asymmetric segregation of parental nucleosomes during chrosome replication. SV40 DNA replicated in the presence of cycloheximide was more sensitive to staphylococcal nuclease digestion and had a lower superhelical density than viral DNA replicated in the absence of this drug. These data indicate that fewer nucleosomes are associated with progeny SV40 DNA molecules after DNA replication in the absence of protein synthesis and that these nucleosomes are derived from the parental histones. We designed an experiment to determine whether these parental SV40 nucleosomes segregate to the leading side of the replication form where DNA synthesis is continuous, the lagging side of the fork where DNA synthesis is discontinuous or randomly to both sides of the fork. The results indicate that the parental histones distributed themselves asymmetrically, preferentially (80-90%) segregating with the leading side of both SV40 DNA replication forks during bidirectional replication in the absence of protein synthesis. In the case of SV40, the same parental DNA strands are the templates for the leading side of DNA replication at both forks as well as the templates for the informational or coding strand of early and late viral mRNA synthesis. Based on this correspondence, we designed an experiment to test whether chicken cells growing in culture and replicating their DNA in the absence of protein synthesis segregated their parental histones asymmetrically to the progeny DNA strand that also coded for stable nuclear RNA transcripts. The results of these experiments indicate that, like SV40, parental cellular histones segregate asymmetrically and are preferentially associated with those DNA template strands that code for stable nuclear RNA species detected by hybridization to single-copy DNA.


Transcription maps of mtDNAs of two strains of saccharomyces: transcription of strain-specific insertions; Complex RNA maturation and splicing. We have developed a two-dimensional method for simultaneously mapping on the yeast mtDNA genome all the transcripts representing more than 0.01% of mtRNA. In two yeast strains, Saccharomyces carlsbergensis NCYC-74 and Saccharomyces cerevisiae KL14-4A, about 25 discrete transcripts were found apart from tRNAs. The mtDNAs of these strains differ by the absence (NCYC-74) or presence (KL 14-4A) of various large insertions located within genetically active regions. The transcripts can all be related to known loci on the genetic map. In nearly all cases the RNAs are much longer than required to specify the known protein product of the locus concerned. The organization of the transcripts is similar in the two strains except at the positions of the large insertions (500-3300 bp) in the oxi-3 and cob loci. The sequences of these insertions are present in RNA species larger than 25S, but are absent from smaller transcripts of the same regions. This is probably due to splicing, since the coding sequences for most of these smaller transcripts are noncontiguous. The smaller transcripts of other loci also seem to arise from processing of larger RNA species. The oxi-3 locus, containing the structural gene for cytochrome c oxidase subunit l, is transcribed in a very complex fashion that suggests differential splicing into partially overlapping transcripts. This may indicate that oxi-3 has additional genetic functions, including possible control of the biosynthesis of cytochrome c oxidase holoenzyme or its assembly into the mitochondrial inner membrane. As in the case of the eucaryote nucleus, the regulation of mitochondrial gene expression seems to occur more at the level of RNA processing than has been recognized thus far.


Messenger RNA for the Ad2 DNA binding protein: DNA sequences encoding the first leader and heterogenity at the mRNA 5' end. During the early stage of Ad2 infection of human cells, RNA is transcribed from five separate transcription units. Early region II encodes the mRNA for a 72K single-stranded DNA binding protein (DBP) which functions in DNA replication. This report describes the structure of the first leader of the DBP mRNA and the flanking sequences in the DNA. The leader, labeled in vivo with 32P, was isolated by DNA filter hybridization to the viral restriction fragment Eco RI F, and its RNAase T1 and RNAase A oligonucleotides were analyzed by RNA fingerprinting techniques. Comparison of this RNA sequence information with the DNA sequence of Eco RI F has located a 68 nucleotide region of the Hae III C subfragment at coordinate 75.1 that encodes the leader. This position is near the coordinate to which nascent chain analysis and ultraviolet transcription mapping have mapped an RNA initiation site, or promoter, for the DBP mRNA. The DNA sequence that overlaps the leader on the 3' side contains a donor sequence for splicing this leader to a second downstream leader. The splicing sequence shows a seven base homology with the comparable structure of the Ad2 major late leader, and a mouse globin mRNA splicing sequence. The DNA sequence upstream from the cap, the region oof the potential promoter site does not, however, contain a "TA-TAAA"-type homology of the sort noted by D. Hogness, M. Goldberg and R. Lifton (personal communication) for many cellular transcription units, and by other investigations for the Ad2 major late transcription unit. Also, the leader is found with two distinct capped 5' termini, 7meGpppA and 7meGpppG, which are encoded at adjacent positions in the DNA and thus are from mRNAs which are staggered by one nucleotide in length at the 5' end. The staggering at the 5' terminus and the lack of the upstream homolgy distinguish the DBP mRNA from many viral and cellular messenger. In both these respects, however, the DBP mRNA resembles the late messengers of SV40 and polyoma viruses. In this paper, we discuss the implications of these findings for the mechanism of specifying mRNA 5' ends.


["Giant" oncocytic renal adenomas].

["Giant" oncocytic renal adenomas]. Postmortem and bioptic material from the 1953-1976 period included 7 cases of oncocytic renal adenoma larger than 5 cm including 3 larger than 10 cm. Macroscopic characteristics included red-brownish coloration and extensive foci of central hyalinosis. Microscopy showed them as being made up of cells resembling oncocytes with relatively active nuclei with solid, occasionally focus-like tubular structure. In spite of the large size and, in some rarer cases, focal nuclear atypia all of the tumours appeared to behave in a benign way. The causes of their increased frequency in the past decade remain to be clarified yet.


Mechanisms of inhibition of active transport ATPases by mercurials. Inhibition by methylmercury and mercuric chloride of Mg,Ca ATPase and Na,K ATPase activities in human erythrocyte ghosts was correlated with the binding capacity of ghosts for the mercurial. Full inhibition was always reached below saturation of binding capacity, and half-inhibition at levels as low as 10% saturation. Under such conditions, concentrations of free inhibitor were negligibly low, and existing mathematical models of inhibition were not applicable. New inhibitor partition equations were introduced to model the mechanisms of action of mercurials. Up to 7 methylmercury groups were calculated to bind to one Na,K ATPase molecule at non-inhibitory sites, while only one reacted with the inhibitory site. Mg,Ca ATPase showed simple one-hit inhibition (one mercurial per enzyme); further washing of ghosts, however, unmasked a second binding site (cooperative two-hit inhibition). Affinities of mercurials to sites of inhibition were calculated relative to other ligands in erythrocyte membranes: the ratios ranged from 3 : 1 to 50 : 1. The results demonstrated the use of binding capacity assays and inhibitor partition equations to measure and compare the susceptibilities of membrane-bound enzymes to poisoning by mercurials.


Benzo[a]pyrene--serum : albumin/cysteine interactions: fluorescence and electron spin resonance studies. The complex between bovine serum albumin (BSA) and benzo[a]pyrene (BaP) exhibits three different types of fluorescence. The visible fluorescence at 407, 431 and 458 nm is modified by the formation of both hydroxy-BaP derivatives and BaP products strongly bound to the protein. The ultraviolet fluorescence I is characterized by a triple-peaked structural fluorescence with maxima at 340, 357 and 378 nm. Upon addition of mercaptoethanol, this ultraviolet fluorescence decreases. The ultraviolet fluorescence II appears at 380 nm and corresponds to that of pyrene-like products. When irradiated with ultraviolet (365 nm) light, both the ultraviolet fluorescence I and the visible fluorescence decreases. In the interactionsof BaP with BSA, a new radical has also been found in addition to the known 6-oxo-BaP radical. The lyophilized BSA-BaP complex exhibits two broad ESR bands, one of which increases in lipid-free BSA. In the concentrated aqueous solution of the BSA-BaP complex, the ESR signal is converted to a six-line spectrum. The benzene extract, which removes non-covalently bound BaP products, shows an ESR signal similar to that in aqueous solution except for the absence of two lower g-valued lines. When irradiated with ultraviolet (365 nm) light the signal intensity of the new radical species decreases, while that of 6-oxo-BaP increases. Upon addition of of mercaptoethanol, the signal of the new radical also diminishes and is replaced by a single narrow signal. Ths mixture of BaP and cysteine kept at room temperature for one day in the dark produces both a six-line ESR spectrum and a broad ultraviolet fluorescence at 330 nm, which gradually decay in about one week. When BaP and cysteine are mixed at 65 degrees C for several hours, little ultraviolet fluorescence and ESR signal are detected. The results indicate the formation of a BaP radical as an intermediate in the interaction of BaP with BSA and cysteine in the presence of oxygen and suggest the involvement of SH-groups in this interaction.


Binding of nitrosamines to cytochrome P-450 of liver microsomes. The interactions of 5 carcinogenic and 1 non-carcinogenic nitrosamines with hepatic microsomal cytochrome (cyt.) P-450 were investigated, using both optical difference and electron paramagnetic resonance (EPR) spectroscopic methods. Liver microsomes from phenobarbital (PB)-pretreated mice and 3-methylcholanthrene (3-MC)-pretreated rats were used, in order to have an increased specific content of cyt. P-450 and cyt. P-448 respectively. The optical and EPR spectral data obtained in the oxidised state suggest that nitrosamines are able to bind both as substrates and as ligands to the hemoprotein cyt. P-450, depending on the concentration of nitrosamine, its chemical identity and the cytochrome species present. After reduction with dithionite or NADPH in the optical difference spectrum a Soret band developed between 444 and 453 nm to an extent, which is dependent on the particular nitrosamine present. This initial nitrosamine-induced spectrum might represent a ferrous nitric oxide (NO)-cyt. P-450 complex. It appears unstable and is converted kinetically into a spectrum lacking a Soret band, but with a predominant absorbance minimum at about 425 nm. A visible band is located at 585 nm. In the EPR spectrum a sharp 3-line signal around g = 2.01 appears concomitantly. Both spectral parameters are typical of a NO-cyt. P-420 complex. These results, in conjunction with metabolic studies, indicate that nitrosamines are denitrosated by a reductive process in which cyt. P-450 appears to be involved. The resulting NO-cyt. P-450 complex denatures to a NO-cyt. P-420 complex when the dioxygen level is not sufficiently high to complete successfully.


Effect of moderate physical exercise on serum lipoproteins. A controlled clinical trial with special reference to serum high-density lipoproteins. A controlled trial is reported on the effects of mild-to-moderate physical activity on serum lipoproteins. After two baseline examinations 100 asymptomatic middle-aged men were randomly assigned to exercise and control groups. The exercise group participated in a 4-month exercise program that consisted of 3-4 weekly sessions. The control group was advised to maintain their previous exercise habits. The success of the program was corroborated by the increase in VO2 in the training group, but not in the control group. Serum triglycerides decreased from 1.54 +/- 0.10 to 1.27 +/- 0.08 mmol/1 (p less than 0.001) and high-density lipoprotein (HDL) cholesterol increased from 1.27 +/- 0.04 to 1.41 +/- 0.04 mmol/1 (p less than 0.01) in the exercise group during the trial. No change was seen in the control group. As the concentration of apolipoprotein AI stayed constant in both groups, the ratio HDL cholesterol/apolipoprotein AI increased only in the exercise group. The level of low-density lipoprotein (LDL) cholesterol and apolipoprotein AII decreased in both groups during the trial. The alterations in serum triglycerides and HDL cholesterol in the exercise group were not dependent on weight reduction; similar changes were also seen in subjects with constant body weight during the intervention.


Survival of the ischemic brain: a progress report. The number of patients with cerebral infarctions increases as the population ages, despite campaigns against hypertension, the greatest risk factor. Cerebral ischemia initiates events that are presumed to defer the stage of irreversible injury. These events cause an increase of perfusion around the central ischemic zone and trigger the Bohr effect, both of which preserve tissue viability. Almost simultaneously, mitochondrial function fails, resulting in insufficient energy for the enzyme systems to control Na and K ion equilibrium. At the same time, protein synthesis slows and cellular respiratory enzymes decrease their activity, initiating an irreversible state of tissue change. Tissue fatty acids increase as a result of dissolution of cell membrane lipoprotein structure. Barbiturates reduce the extent of experimental infarction. Resperine and aminophylline are also effective, but there are no corroborative clinical trials. That ischemic brain damage may be the result of toxic substances in the ischemic tissue represents a new concept.


Electrophoretic separation of high-density lipoprotein choelsterol evaluated and compared with the modified lipid research clinic procedure. We evaluated a new agarose-gel-electrophoretic procedure (Corning) (I) for separating and quantitating of high-density lipoprotein cholesterol (HDLC), comparing it with the modified Lipid Research Clinics (LRC) procedure (heparin 183 kilounits/L, MnCl2 92 mmol/L) (II). Method I was insensitive to an HDLC concentration of 50 mg/L, but gave a linear dose-response curve between 130 and 1200 mg/L. Method II is sensitive to 50 mg/L and linear from 50--1200 mg/L. The within-plate CV for the Corning method varied from 26.2% for an HDLC of 168 mg/L to 6.8% for 580 mg/L. Within-day between-plate CV for the Corning method ranged from 22.1% at 155 mg/L to 8.0% at 651 mg/L, compared to 3.0 and 0.8% for the modified LRC procedure. Between-day CV for method I was 20, 12.6, 4.3, and 3.5% for HDLC concentrations of 175, 435, 542, and 678 mg/L, respectively; for method II it was 14, 5, 3.5, and 2.6%, respectively. Analysis of HDLC in 100 patients by both procedures showed mean HDLC values to be significantly lower (mean + SD, 27.8 +/- 1.7 mg/L; p less than 0.001) by method I. In 46 patients with HDLC less than 450 mg/L, this difference was accentuated (mean + SD = 40.5 +/- 2.6 mg/L) and clinically significant. Electrophoretic methods offer a promising further alternative method for HDLC separation and quantitation, but the negative bias, present limited sensitivity, and lack of precision at less than 450 mg/L indicate that they are not yet optimal for routine clinical use for patients with values less than 450 mg/L.


Quantitative determination of high-density lipoprotein cholesterol by agarose gel electrophoresis. We have developed a procedure for the determination of high-density lipoprotein cholesterol by agarose gel electrophoresis. Only 2 micro L of sample was applied to the gel. After electrophoresis at 90 V for 35 min, an enzymatic cholesterol reagent was applied. After a 30-min incubation, the high-density lipoprotein cholesterol was quantified by densitometry. Precision for this measurment approaches that reported for the heparin-manganese/Abell-Kendall method (Clin. Chem, 25: 596--609, 1979). We evaluated accuracy by comparing high-density lipoprotein cholesterol concentration measured by electrophoresis to that determined in the Framingham Heart Study procedure (J. Biol. Chem. 195: 357, 1952). The resulting correlation was excellent. By the paired Student's t-test, there was no significant difference between the two methods. The proposed method gives a linear standard curve when the concentration of total cholesterol is between 1.0 and 3.5 g/L. By accurate quantitation of high-density lipoprotein cholesterol, agarose gel electrophoresis can aid in assessment of coronary heart disease risk for a large segment of the population.


High concentrations of free kinins and kinin system components in abdominal transudate of a patient with nephrotic syndrome. High levels of bradykinin (60--80 ng/ml) were found in abdominal transudate from a patient with nephrotic syndrome caused by chronic glomerulonephritis. The abdominal transudate contained neutral kininogenase and its precursor, identified with plasma kallikrein and prekallikrein, respectively, as well as both forms of kininogen, the low-molecular-weight form predominating. The abdominal transudate was characterized also by very low kininase activity and low levels of alpha 1-antitrypsin (0.46 g/l) and alpha 2-macroglobulin. Large amounts of very low density lipoproteins were present in the transudate. Despite the difference in total protein content between the abdominal transudate and the patient's serum (4.3 g/l and 48 g/l, respectively) their protein fraction composition was similar. The data obtained suggest that bradykinin is important in maintenance of long-lasting blood vessel hyperpermeability, which, in turn, is a driving force in the pathogenesis of refractory nephrotic edema.


[Spectrophotometric assay of angiotensin I-converting enzyme (author's transl)]. The method of chemical assay of angiotensin I-converting enzyme described is a modification of the previously published spectrophotometric assay based on quantitation of hippuric acid released from hippuryl-L-histidyl-L-leucine. The new procedure involves extraction of hippuric acid with ethylacetate, evaporation to dryness of the extract, solubilization of the residue with 1 mol/l NaCl and purification with petroleum ether before measurements of the absorbance at 228 nm of the aqueous phase. Under these conditions, hippuric acid insoluble in petroleum ether remains in the aqueous phase, whereas other A228-absorbing materials, readily soluble in the ether and able to interfere with the assay, are eliminated.


[Quantitative analysis of lipoproteins in hyperlipoproteinemias of type IIa, IIb and IV (author's transl)]. Cholesterol and triglyceride content of serum lipoprotein fractions isolated by ultracentrifugation have been studied in 33 healthy subjects and in 56 subjects affected by hyperlipoproteinemia of type IIa, IIb and IV. Patients with atherosclerotic disease were characterized by a general decrease of HDL cholesterol and by a negative correlation between HDL cholesterol and VLDL triglycerides; patients with type IIb and IV showed an increase of LDL lipoproteins. The increase of triglycerides in type IIb and IV was caused by elevation of VLDL triglycerides or LDL triglycerides, and the increase of cholesterol in type IIb is sometimes caused by elevation of VLDL cholesterol. It is evident that several subtypes exist within Fredrickson's classification. Patients with cerebral arterial disease when compared with patients affected by non-ischaemic disease, showed a negative and significant correlation between HDL cholesterol and total cholesterol.


Influence of urine on "in vitro" crystallization rate of calcium oxalate: determination of inhibitory activity by a [14C]oxalate technique. A simple radiochemical method is proposed for the in vitro assay of the inhibitory activity of urine with respect to calcium oxalate crystal growth using [14C]oxalate as a tracer. The method shows an improved sensitivity over existing methods and indicates that citrate, pyrophosphate and chondroitin sulphate are active inhibitors of calcium oxalate crystal growth down to concentrations of 10(-5), 10(-7) and 10(-10) mol/l respectively. The inhibitory activity in the urines of 12 recurrent calcium stone-formers was significantly lower than in the urines of matched control subjects (P less than 0.01), confirming the clinical usefulness of the test.


An improved method for the determination of acetaldehyde in human blood with minimal ethanol interference. A sensitive method for the determination of acetaldehyde in human blood is described which involves the derivatization of acetaldehyde with semicarbazide followed by head-space gas chromatographic analysis of acetaldehyde in plasma samples after acid hydrolysis of the semicarbazone derivative. Quantitative recovery of acetaldehyde from human blood was achieved and interference from ethanol, a major problem associated with earlier methods, was negligible.


The specific release of sialytransferase activity by human hepatoma cell lines. Sialyltransferase (EC 2.4.99.1) is released in large amounts by two hepatoma cell lines (SK-H-MA and CLH) established from patients with hepatocellular carcinoma (hepatoma). This release requires protein synthesis and glycoprotein synthesis, but not cell division. In contrast, sialyltransferase is released in minimal amounts by a cell line derived from normal human liver (Chang). The hepatoma cells also contain more surface and cellular sialyltransferase activity than Change cells. Hepatoma sialyltransferase has properties similar to other sialyltransferases. Using a calibrated Sephadex G-200 column, it is resolved into two forms with molecular weights of 65 000 and 80 000.


Response of plasma lipoproteins and acute phase proteins to myocardial infarction. Plasma concentrations of lipoprotein-lipids, apolipoprotein B (apoB) and of seven other proteins have been estimated serially in 27 patients up to three months following myocardial infarction. Results were compared with those from age- and sex-matched control subjects. At three months the mean total, low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol concentrations were higher than those of the control subjects, whereas very low density lipoprotein (VLDL) cholesterol, total and VLDL triglyceride, and total and LDL apolipoprotein B concentrations were not significantly different. Relative to concentrations at three months total and LDL cholesterol and apolipoprotein B concentrations fell markedly, and a slight fall occurred in HDL cholesterol following infarction. VLDL cholesterol and total and VLDL triglyceride were decreased only on day one. Albumin and transferrin concentrations were higher and alpha 1-acid glycoprotein was lower at three months than in the control subjects; alpha 2-macroglobulin, caeruloplasmin, haptoglobin and immunoglobulin IgM were not significantly different. Following infarction albumin and transferrin fell, alpha 2-macroglobulin did not change, and alpha 1-acid glycoprotein, caeruloplasmin, haptoglobin and IgM rose. The changes in both lipids and protein are probably part of the general metabolic response to trauma.


Increased 99mTc-pyrophosphate uptake with radiation pneumonitis. A case of radiation pneumonitis confirmed by tissue biopsy is presented with demonstration of increased uptake of 99mTc-pyrophosphate.


The early clinical presentation of synovial sarcoma. While synovial sarcoma most commonly presents as a painless mass, occasionally the cancer emerges in a misleading manner resulting in an unfavorable delay or error in diagnosis. A review of the litrature reveals 4 such occult patterns: pretumor phase characterized only by pain or tenderness; the acute inflammatory lesion presenting as a "hot" arthritis or bursitis; the chronic contracture; the post traumatic tumor. These conditions, especially when otherwise unaccounted for, are indications for biopsy. Four avoidable pitfalls in biopsy management also emerged from the review.


Acyclovir kinetics after intravenous infusion. The disposition and safety of the antiviral drug acyclovir were studied in 14 subjects with advanced malignancies. Acyclovir was administered by a 1-hr intravenous infusion at doses of 0.5, 1.0, 2.5, and 5.0 mg/kg. At the end of infusion, mean peak plasma levels (+/- SEM), determined by radioimmunoassay, were 6.4 +/- 0.7, 12.1 +/- 2.3, 14.9 +/- 2.7, and 33.7 +/- 7.1 microM. The plasma concentration-time profiles could be described by a biexponential equation. The half-life of acyclovir in the slow disposition phase ranged from 2.2 to 5 hr and the drug was detected in the plasma for at least 18 hr after infusion. The total body clearance ranged from 117 to 396 ml/min/1.73 m2. A proportionality between area under the curve and dose suggests that acyclovir exhibits dose-independent kinetics in the dose range studied. There was wide variation in cumulative urinary excretion of unchanged drug, ranging from 30 to 69% of the dose. From renal clearances of acyclovir, which were higher than creatinine clearances, it appears that both glomerular filtration and tubular secretion contribute to its renal excretion. Analysis of the urine by reverse-phase high-performance liquid chromatography revealed the presence of the metabolite 9-carboxymethoxymethylguanine. There was no indication of toxicity either clinically or from laboratory findings in any of the study subjects. This study demonstrates that in addition to selectivity and low toxicity, the kinetic profile and metabolic disposition of acyclovir make it an attractive candidate for therapy in a variety of herpes infections.


The physical and genetic organization of a viral genome. Mutants of SV40 with deletions of a few to several thousand base pairs have been constructed in vitro and cloned in cultured monkey cells. The location and size of these deletions has been determined by restriction endonuclease mapping and electron microscopic and enzymatic analysis of DNA heteroduplex molecules. Analysis of the phenotype of these deletion mutants permits us to specify the locations of the known SV40 genes, in particular, the novel organization of SV40s two early genes that are required for oncogenesis.


Acute respiratory failure following severe arsenic poisoning. A 47-year-old man had an episode of severe respiratory failure after acute intoxication with arsenic. Features of the initial clinical presentation included nausea, vomiting, and diarrhea, acute psychosis, diffuse skin rash, and marked pancytopenia. A peripheral neuropathy then developed which resulted in severe weakness of all muscles of the limbs, the shoulder and pelvis girdles, and the trunk. The neuropathy continued to progress despite treatment with dimercaprol (BAL in oil). Five weeks after the initial exposure, the patient was no longer able to maintain adquate ventilation and required mechanical ventilatory support. Improvement in the patient's neuromuscular status permitted successful weaning from the ventilator after one month of mechanical ventilation. Long-term follow-up revealed no further respiratory difficulty and slow improvement in the strength of the peripheral muscles.


Enzyme replacement and other biochemical approaches to the therapy of adenosine deaminase deficiency. Addition of adenosine deaminase (ADA) restored in vitro responses of lymphocytes from a patient with ADA deficiency and severe combined immunodeficiency (SCID). Enzyme replacement therapy, using red blood cells as a source of encapsulated human ADA, restored both T and B cell function in this patient. Ten other ADA--SCID patients have been treated with this form of enzyme replacement and five have responded to therapy. Lymphocytes from ADA--SCID patients treated with enzyme replacement become immunocompetent but remain enzyme deficient. Studies of these cells provide evidence supporting both cyclic AMP- and dATP-mediated immunosuppressive mechanisms in ADA--SCID. These observations suggest that inhibition of cyclic AMP synthesis and/or deoxycytidine (and possibly thymidine) supplementation may be useful new biochemical approaches to the therapy of ADA--SCID.


[Importance of follow-up investigation in colonic carcinoma (author's transl)]. Extensive follow-up investigations were done in 1978 in 135 patients, most of whom had been operated for colo-rectal carcinoma of all stages in 1977. In 71% of the cases no or no sufficient postoperative control had been performed in the patients' own area. At the follow-up investigations 11 recurrences in the anastomosis, 10 of which were operable, 20 distant metastases, 1 simultaneous second carcinoma, 15 adenomas, partially with numerous atypical cells, 1 polyposis coli and 23 nonspecific granulomas of the anastomosis were detected. The Haemoccult test was positive in 3 of the 11 anastomotic recurrences. Only in cases of distant metastases were values of carcinoembryonic antigen clearly increased (greater than 5 ng/ml). These findings underline the necessity of intensifying the regular follow-up investigation in colo-rectal carcinoma.


Radioiodinated nondegradable gonadotropin-releasing hormone analogs: new probes for the investigation of pituitary gonadotropin-releasing hormone receptors. Studies of pituitary plasma membrane gonadotropin-releasing hormone (GnRH) receptors using [125I]-iodo-GnRH suffer major disadvantages. Only a small (less than 25%) proportion of specific tracer binding is to high affinity sites, with more than 70% bound to low affinity sites (Ka = 1 x 10(6) M-1). [125I]Iodo-GnRH is also inactivated during incubation with pituitary plasma membrane preparations. Two superactive analongs of GnRH, substituted in positions 6 and 10, were used as the labeled ligand to overcome these problems. Both analogs bound to the same high affinity sites as GnRH on bovine pituitary plasma membranes, though the affinity of the analogs was higher than that of the natural decapeptide (Ka = 2.0 x 10(9), 6.0 x 10(9), and 3.0 x 10(8) M-1 for [D-Ser(TBu)6]des-Gly10-GnRH ethylamide, [D-Ala6]des-Gly10-GnRH ethylamide, and GnRH, respectively. The labeled analogs bound to a single class of high affinity sites with less than 15% of the specific binding being to low affinity sites (Ka approximately equal to 1 x 10(6) M-1). The labeled analogs were not inactivated during incubation with the pituitary membrane preparations. Using the analogs as tracer, a single class of high affinity sites (K1 = 4.0 x 10(9) M-1) was also demonstrated on crude 10,800 x g rat pituitary membrane preparations. Use of these analogs as both the labeled and unlabeled ligand offers substantial advantages over GnRH for investigation of GnRH receptors, allowing accurate determination of changes in their numbers and affinities under various physiological conditions.


Effect of tetradecapeptide renin substrate on blood pressure, plasma renin activity, and vasopressin and adrenocorticotropin concentrations. The effects of third ventricular injection of tetradecapeptide renin substrate (TDP) and natural renin substrate prepared from dog cerebrospinal fluid were compared in anesthetized dogs. Central injection of 350 pmol TDP caused a long lasting increase in arterial blood pressure, a reduction in PRA, and increases in plasma levels of vasopressin, and ACTH. In marked contrast, central administration of equimolar doses of natural renin substrate had no effect on these variables. Intracranial administration of the converting enzyme inhibitor SQ 20881 prevented the effects of central injection of TDP. Thus, TDP exerts its effects via conversion to angiotensin II and does not necessitate the postulation of the action of an enzyme with renin-like activity in the brain.


Follicle-stimulating hormone-induced, adenosine 3',5'-monophosphate-mediated movement of immature rat sertoli cells in primary culture. Sertoli cells dissociated from 10-day-old rat testes form colonies in primary culture in response to FSH. FSH and dibutyryl cAMP stimulated the attachment of Sertoli cells to an equal extent; however, FSH-treated cultures contained a small number of large colonies while dibutyryl cAMP-treated cultures contained a large number of small colonies. This relationship was not altered by the addition of a number of other peptide or steroid hormones. Extracellular cAMP levels and colony density were negatively correlated. Colony size at 24 h of culture was diminished in FSH-treated cultures by the addition of a cAMP antibody at 6 or 12 h of incubation. The addition of cAMP at 24 h to FSH-treated cultures caused a dose-dependent stimulation of colony size but not colony density at 48 h of culture. A point source of cAMP (4 x 10(-3) M in agar) inhibited migration of cells toward the agar spot. An agar spot on the dish substratum containing Sepharose-bound FSH exhibited a halo of cells next to the spot, with a zone lacking cells distal to it. Radioautographs of [125I]iodo-FSH-treated cultures exhibited a nonhomogeneous distribution of silver grains; colony size increased faster than the number of labeled cells. Taken together, the results suggest that 1) FSH is stimulating the attachment of Sertoli cells through an increase in intracellular cAMP, 2) FSH is promoting active aggregation of Sertoli cells in culture through a modulation of extracellular cAMP, and 3) cells with a large amount of bound FSH are acting as centers for aggregation.


Study of oxytocin receptor in human myometrium using highly specific 3H-labeled oxytocin. A highly specific tritium labeled oxytocin (3H-OT) was synthesized utilizing the method of catalytic substitution of halogen for hydrogen. The specific activity of 3H-OT was 19 Ci/mM and the biologic activity was 350 U/mg, which was sufficient for the OT radioreceptor assay. The maximum % uptake of 3H-OT in the human myometrium was observed in 20,000 X g pellets under the optimal conditions of pH 7.4, at 20 degrees C and the incubation time of 90 min and it was augmented in the presence of Mn++. It was observed from the Scatchard plot, that the binding site of OT in the human myometrial specimens was a single type within the range of OT concentration of 0.4 nM to 1.6 nM. The dissociation constants (Kd) and the number of binding sites (NBS) showed a relative increase as gestation advance. The apparent Kd of term pregnancies was 1.25 X 10(-9) M regardless of the presence or absence of labor pains, while the NBS of term pregnancies with and without labor pain was 1.2 X 10(-12) and 4.7 X 10(-12) moles/mg, protein, respectively.


Induction of specific human growth hormone binding sites in rat liver by human growth hormone. 125I-Labeled hGH was bound to liver plasma membranes which were obtained from female rats. The binding was displaced by hGH, hPRL, bPRL, rPRL and bGH but not by rGH. This result indicated that hGH was bound to lactogenic binding sites in rat livers. After hypophysectomy, the binding was markedly decreased. Treatment of hypophysectomized rats with hGH (80 micrograms/day) for 10 days increased the binding sites for hGH. These binding sites were different from those found in normal female rat livers because of their high affinity and specificity for hGH. These results indicate that hGH induces specific binding sites for hGH in rat livers.


Effects of ischemia on the removal of extracellular potassium in cat cortex during pentylenetetrazol seizures. Changes in cortical extracellular potassium activity ([K+]0), NADH fluorescence, and oxygen consumption were studied in anesthetized cats during pentylenetetrazol seizures. The effects of partial ischemia induced by either hypotension or intermittent carotid artery occlusion on these parameters were investigated. Nonischemic seizures were characterized by gradual generalized decreases in cortical NADH fluorescence and increases in O2 consumption, along with rapid increases in [K+]0, which then usually fell slightly as the ictal discharge continued. Ischemic seizures, on the other hand, were accompanied by complex changes in NADH fluorescence, by smaller delayed maximal increases in O2 consumption that lasted beyond the end of ictal activity, and by more sustained increases in [K+]0. The decay of [K+]0 after the termination of seizures in both nonischemic and moderately ischemic animals was not a monoexponential function: plots of ln delta [K+]0 versus time showed an initial linear decline (of slope M1) that rather abruptly slowed (to slope M2) after 2 to 5 sec and then often increased to the original rate. Both M1 and M2 were proportionately decreased by ischemia. In addition, the rate of [K+]0 removal could be slowed by acute ischemia induced either during or after the end of ictal activity. The initial rate of postictal [K+]0 removal (M1) was found to be linearly and inversely related to the level of cortical NADH fluorescence at the time of seizure termination. The results of this study suggest that an O2-dependent transport mechanism plays a major role in the removal of [K+]0 during and following the termination of generalized pentylenetetrazol seizures in the cat.


Carcinogenicity of chloroform.

Carcinogenicity of chloroform. Chloroform is carcinogenic in rats, mice, and probably in dogs. Chloroform induced carcinomas of the liver and kidney and malignant tumors in other organs in rats and mice. Liver neoplasms have been described in three strains of mice. Carcinomas of the kidney were found in a first study in mice and in the repeat of that study. Dogs given chloroform developed neoplasms of the liver as well as in other organs. Rats given chloroform also developed toxic changes, particularly male rats, as a result of treatment. These lesions included interstitial fibrosis of the kidney; polyarteritis of the mesenteric, pancreatic, and other arterioles and arteries; and atrophy of the testes. These toxic changes may have interfered with the development of neoplasms in male rats.


Effect of sciatic nerve crush on enzyme activities of skeletal muscles of the rat. Weight loss and reduction in specific activity of cytochrome oxidase of both red (soleus) and white muscles (gastrocnemius and plantaris) of the rat was greatest 5 days after sciatic nerve crush (p less than 0.001) and then became minimal. In neither was there a significant, concomitant loss of protein. By 19 days, the specific activity of cytochrome oxidase was the same in both muscle types. The specific activity of lactate dehydrogenase was reduced significantly (p less than 0.001) in the white muscles, to a value approaching that of the red soleus by 19 days postoperatively, but remained unaltered in the soleus. Nerve crush is proposed as a model experimental system for studying neural regulation of skeletal muscle metabolism.


Effect of air ionization on heart rate and perceived exertion during a bicycle exercise test. A double-blind cross-over study. The influence of ionization of air on heart rate (HR) and ratings of perceived exertion (RPE) during bicycle exercise was studied in nine healthy medical students selected according to a randomized schedule from the class of 90 students. The exercise tests were performed both under negative and positive ionization. The study was made with a double-blind, cross-over design. The body surface exposed to ionic current was made large by reducing the clothing of the subject. A significant overall tendency to lower HR and RPE values under negative ionization was observed (p less than 0.01, sign tests). The RPE values were significantly lower (p less than 0.01, paired t-test and the Wilcoxon test) under negative than under positive ionization at the maximal work load level but not at other relative load levels. However, when separately tested at each relative load level HR values did not differ significantly in negative and positive ionization. The results of this pilot study indicate that ionic composition of the air can modify the RPE and possibly also HR during exercise; negative air ionization seems to be beneficial compared with positive ionization. The mechanisms involved are obscure, but we suggest that negative ionization of air may increase oxidative metabolism through generation of a superoxide radical (O2-) that is reduced to H2O2 by superoxide dismutases.


The control of phosphoprotein phosphatase by the second-site phosphorylation of a substrate. Studies with H2B histone as model substrate. The phosphorylation of Ser-32, in addition to Ser-36 of H2B histone, stimulated the rate of Pi release from Ser-36 by the small form (Mr 31 000) of pig heart phosphoprotein phosphatase both in the absence and presence of 50 mM magnesium acetate. By phosphorylation at Ser-32, the Km value for Ser-36 phosphate in H2B histone was increased from 0.38 microM to 1.16 microM in the absence of magnesium acetate, but not significantly changed (from 37.4 microM to 26.2 microM) in the presence of magnesium acetate. With the large form (Mr 224000) of the phosphoprotein phosphatase, however, the phosphorylation at Ser-32 suppressed the rate of Pi release from Ser-36 both in the absence and presence of magnesium acetate. The Km value of the large form for Ser-36 phosphatase in H2B histone was nevertheless increased by phosphorylation at Ser-32, from 1.2 microM to 5.3 microM in the presence of magnesium acetate, but not changed (from 0.26 microM to 0.23 microM) in the absence of magnesium acetate.


Evidence for a second desensitized state of beta-adrenergic receptor with low affinity for beta-antagonists and normal reactivity towards beta-agonists in adipocyte membranes previously exposed to beta-antagonists. When adipocyte membranes are successively exposed to (-)-propranolol or (+/- alprenolol at 25 or 4 degrees C, repeatedly washed and then assayed for (-)-[3H]dihydroalprenolol binding, the apparent number of beta-adrenergic binding sites is markedly decreased. Induction of this peculiar type of receptor desensitization does not require prolonged exposure of the membranes to the beta-adrenergic antagonists (half-time: 1 min), is stereospecific, concentration-dependent and almost complete with high concentrations of antagonists. p[NH]ppG, which reduces the affinity of fat cell beta-adrenergic receptors for agonists, does not prevent the antagonist-induced decrease in the receptor number. The magnitude of the desensitizating effect induced separately by (-)-isoproterenol and (-)-propranolol is not additive in membranes exposed to both drugs, suggesting that the receptors lost after exposure to agonists are the same sites as part of those lost after exposure to antagonists. However, contrary to the results found in membranes desensitized by agonists, adenylate cyclase activity remained fully responsive to catecholamines in membranes exposed to beta-antagonists. As shown by kinetic studies on (-)-[3H]dihydroalprenolol binding, this beta-antagonist-induced receptor desensitization is reversible after prolonged incubation. These data which have never yet been described in the other reported desensitizable beta-adrenergic systems, suggest that, when exposed to beta-antagonists, the fat cell beta-adrenergic receptors undergo a conformational change leading to a peculiar state which has low affinity for antagonists but behaves towards agonists as does the receptor in its resting state.


Conversion of a mitochondrial precursor polypeptide into subunit 1 of cytochrome oxidase in the mi-3 mutant of Neurospora crassa. 1. The cytochrome-alpha alpha 3-deficient mi-3 cytoplasmic mutant of Neurospora crassa synthesizes a mitochondrial translation product which crossreacts with antibodies specific to subunit 1 of cytochrome oxidase. The immunoprecipitated polypeptide migrates more slowly during gel electrophoresis than the authentic 41 000-Mr subunit 1 of the wild-type enzyme. An apparent molecular weight of about 45 000 was estimated for the mutant product. 2. Radioactive labelling experiments in vivo show that the crossreacting material found in the mutant is relatively stable and does not form complexes with other subunits of the oxidase. 3. After induction of a functional cytochrome oxidase in the mutant cells with antimycin A, the 45 000-Mr polypeptide is converted to a 41 000-Mr component, which exhibits the same electrophoretic mobility as subunit 1 of the oxidase. Pulse-chase labelling kinetics reveal a typical precursor product relationship. 4. The converted polypeptide becomes assembled with other enzyme subunits to form a protein complex which has the immunological characteristics of cytochrome oxidase. A possible physiological role of the post-translational processing of the mitochondrially synthesized component is discussed.


Regulation of transcription by DNA-bound non-histone nuclear proteins. Purified non-histone proteins from mouse mammary cells bind specifically to homologous DNA or chromatin. Complexes of non-histone protein with DNA or chromatin, isolated on agarose columns, were transcribed with both Escherichia coli RNA polymerase and RNA polymerase B from calf thymus. The fact that complexing of DNA with non-histone proteins increases transcription by E. coli RNA polymerase but not by RNA polymerase B suggests different mechanisms of transcription by these two enzymes. Similar experiments with mouse and Drosophila chromatin indicate that non-histone proteins specifically stimulate the transcription of mouse chromatin by RNA polymerase B. Non-histone proteins stimulate the transcription of mouse mammary tumor virus sequences in chromatin by RNA polymerase B but not by E. coli RNA polymerase. We conclude that those non-histone proteins bound specifically to chromatin are able to activate the transcription of specific genes by eukaryotic RNA polymerase.


The calcium-dependent phosphatidylinositol-phosphodiesterase of rat brain. Mechanisms of suppression and stimulation. 1. The activity of the soluble, calcium-dependent phosphatidylinositol-specific phosphodiesterase (EC 3.1.4.10) against [32P]phosphatidylinositol has been investigated. 2. KC1 (only at neutral pH), Mg2+, positively-charged proteins such as histone, and phospholipids containing a choline headgroup are all inhibitory to the enzyme. Choline-phospholipids cause a 90% inhibition at an equimolar ratio to phosphatidylinositol. 3. Other phospholipids (phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine and phosphatidic acid) are all potent stimulators of the enzyme: maximum stimulation being observed at a ratio of 1 mol activator/5--10 mol phosphatidylinositol. 4. Unsaturated amphiphiles such as oleic and oleoyl alcohol also stimulate the activity, maximum stimulation being observed at about an equimolar ratio to phosphatidylinositol. Saturated amphiphiles (such as stearic acid and stearoyl alcohol) are less effective. 5. The activation by acidic phospholipids and unsaturated amphiphiles appear to be independent as they are additive and, under certain conditions, synergistic. 6. Both types of stimulator (independently or together) can reverse the inhibition caused by histone or phosphatidylcholine. 7. Possible mechanisms of the suppression of the phosphatidylinositol phosphodiesterase in vivo, of its activation, and of the amplification of phosphatidylinositol breakdown are discussed.


Kinetic mechanism of NADH-dependent glutamate synthase from lupin nodules. From initial-rate studies, a partially random kinetic mechanism has been deduced for NADH-dependent glutamate synthase from lupin nodules. The mechanism involves compulsory binding of NADH as first substrate, followed by random-order binding of glutamine and 2-oxoglutarate. Patterns of inhibition by glutamate substantiate the mechanism. Dithionite was incapable of acting as an alternative reducing substrate although it is known to reduce the flavine groups of the enzyme. The implications of these results are discussed. Published rate equations for this type of mechanism were found to be unsatisfactory for this enzyme and suitable new equations are produced. These equations should have general application where the obligatory first substrate binds very tightly.


NAD-dependent formate dehydrogenase from methylotrophic bacterium, strain 1. Purification and characterization. 1. NAD-dependent formate dehydrogenase was isolated from gram-negative methylotrophic bacteria, strain 1, grown on methanol. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography and preparative isotachophoresis or gel filtration; it resulted in a yield of 40%. 2. The final enzyme preparations were homogeneous as judged by sedimentation in an ultracentrifuge. Formate dehydrogenase purified in the presence of EDTA reveals two bands on electrophoresis in polyacrylamide gel both after protein and activity staining. Two components are transformed into a single one after prolonged storage in the presence of 2-mercaptoethanol. 3. Formate dehydrogenase is a dimer composed of identical or very similar subunits. The molecular weight of the enzyme is about 80 000. 4. Amino acid composition and some other physico-chemical properties of the enzyme were studied. 5. Formate dehydrogenase is specific for formate and NAD as electron acceptor. The Michaelis constant was 0.11 mM for NAD and 15 mM for formate (pH 7.0, 37 degrees C). 6. Formate dehydrogenase was rapidly inactivated in the absence of -SH compounds. The enzyme retained full activity upon storage at ambient temperature in solution for half a year in the presence of 2-mercaptoethanol or EDTA.


Use of protein-mediated lipid exchange in the study of membrane-bound enzymes. The lipid dependence of glucose-6-phosphatase. The ability of liver lipid-exchange proteins to introduce foreign phospholipids into microsomes was used in a study of the lipid dependence of glucose-6-phosphatase. Supplementation of intact rat liver and hepatoma microsomes with exogeneous aminophospholipids prevents the decline of glucose-6-phosphatase activity during incubation, whereas the introduction of exogeneous phosphatidylcholine has no protective effect. On the contrary with deoxycholate-disrupted hepatoma microsomes, introduction of additional phosphatidylcholine causes activation while phosphatidylethanolamine has only little effect. The results are explained by assuming that the transport unit and the catalytic moiety of the glucose-6-phosphatase system have different lipid requirements, the activity of the former protein depending mainly on phosphatidylethanolamine and phosphatidylserine and that of the catalytic protein depending on phosphatidylcholine. In deoxycholate-disrupted liver microsomes (in which both the glucose-6-phosphatase activity and the phosphatidylcholine content are much higher than in hepatoma microsomes) incubation with phosphatidylcholine and lipid-exchange proteins alters neither the phospholipid composition nor the enzyme activity. THis suggests that the diminished activity of glucose-6-phosphatase in hepatomas may be partly due to a low level of phosphatidylcholine.


Tubulin-associated nucleoside diphosphokinase. Microtubule protein, prepared by cycles of polymerisation and dissociation, contained a nucleoside diphosphokinase (NDP kinase) activity (EC 2.7.4.6). This activity was not intrinsic to the tubulin dimer or the so-called microtubule-associated proteins. The NDP kinase had the following properties. (1) The enzyme existed in a low-molecular-weight form and in association with the complex of microtubule-associated proteins and tubulin (i.e. multimeric tubulin). (2) The low-molecular-weight species was also formed by dissociation of multimeric tubulin by salt or by removal of microtubule-associated proteins on phosphocellulose. (3) GDP bound to the exchangeable site of multimeric tubulin and also GDP derived from the E site of the tubulin dimer was a substrate for the NDP kinase. (4) The NDP kinase showed a 7-fold increase in activity during ATP-dependent microtubule assembly. On the basis of these properties, it is proposed that microtubule protein contains an NDP kinase specifically associated with tubulin and its functions.


Uptake of 131I-19-cholesterol by normal and spontaneously hyperfunctioning canine adrenals. In six normal dogs the adrenals could be visualized as separate areas of radioactivity at 7--10 days after injection of 20--40 muCi 131I-iodocholesterol per kg of body weight. Image analysis revealed uptake values of 0.15--0.3% of the injected dose. In five dogs with pituitary-dependent hyperadrenocorticism the adrenals became visible at 3--5 days after injection of the radiopharmaceutical, with uptake values of 0.38--2.2%. In six dogs with hyperadrenocorticism due to adrenocortical tumor the scintigraphy contributed to the diagnosis and the presurgical localization. The uptake values were within the normal range; the tumor could be observed at 3--10 days after injection. Additional findings on adrenal asymmetry and a case of fluctuating pituitary-dependent hyperadrenocorticism are discussed.


The sensitivity of scintigraphic myocardial imaging by the use of 99mTc-labelled pyrophosphate in the diagnosis of cardiomyopathy of various etiology. A total of 10 patients were examined. In five children who suffered from secondary cardiomyopathy accompanying progressive muscular dystrophy scintigraphic examination of the myocardium was made using 99mTc-labelled pyrophosphate. In two cases the scan was distinctly positive, in three cases negative. Five men with poorly defined primary nonobstructive cardiomyopathy were examined too. The scan was positive in three cases and negative in two cases. The positivity of the pyrophosphate scan of the heart is therefore not pathonomonic of ischaemic damage. An increased accumulation of pyrophosphate in the myocardium in cardiomyopathy indicates an acute phase of the disease.


The particular usefulness of radioisotope methods in some benign bone diseases. The authors have performed radioisotope examinations in 271 patients with various non-neoplastic bone diseases. According to their opinion, early diagnosis and follow-up of therapeutic results are the main characteristics which allow radioisotopes to play an important and irreplaceable role. They particularly emphasize the usefulness of radioisotope methods in femoral aseptic necrosis and Paget's disease.


HDL- and LDL-cholesterol in normal weight healthy children 13 years of age. From 108 healthy 13 years old school children who took part on a longitudinal study in regard to serum lipids and lipoproteins 64 with normal body weight were examined. Lipoproteins were estimated by means of preparative ultracentrifuge and polyanionprecipitation (Heparin and Manganese-chloride) according to the Lipid Research Clinics Methods, NIH, Bethesda. Mean values and S.D. were calculated as: Total cholesterol: 155 +/- 29, 150 +/- 35 mg/dl; LDL-cholesterol 98 +/- 21 and 96 +/- 34 mg/dl; HDL-cholesterol 44 +/- 13 and 44 +/- 13 mg/dl for males and females respectively. The few data from the literature showing wide variations in LDL- and HDL-cholesterol concentrations are compared with our results; methodological considerations indicate that for further epidemiological studies concerning the possible risk factors for premature atherosclerosis standaradized laboratory tests should be provided.


Effects of alkanols and halothane on rat brain muscarinic and alpha-adrenergic receptors. Effects of the primary alcohols ethanol, butanol, pentanol and of halothane were measured on the binding functions of muscarinic and alpha-adrenergic receptor preparations in rat brain homogenates, with the use of the antagonists 3H-quinuclidinyl benzilate and 3H-WB-4101. IC50 concentrations of the alkanols for the muscarinic and alpha-receptors respectively were: ethanol, 2.0 M and 1.4 M; butanol, 0.24 M and 0.16 M; heptanol, 3.7 X 10(-3) M and 2.6 X 10(-3) M. The plot of IC50 values versus number of carbon atoms in the alkanol was linear and of the same slope as the plot of membrane fluidity changes, thus indicating the importance of the membrane/water partition coefficient of the alkanol. Halothane at clinical concentrations had no effect on the receptors, although significant inhibition of radioligand binding was produced by 2.5 mM halothane, and inhibition was complete in presence of 17.5 mM anesthetic. From the correlation of receptor binding inhibitions with membrane fluidity changes reported by other workers, it is suggested that the activity of membrane receptors may be modulated by the fluidity of their membranes.


Heterosynaptic postactivation potentiation in hippocampal CA 3 neurons: long-term changes of the postsynaptic potentials. CA 3 neurons were excited synaptically by stimulation in the dentate hilus and the stratum radiatum of CA 1 in guinea pig hippocampal slices. Following repetitive stimulation (10--20 c/s, 10 s) of either stimulation site, the amplitudes of orthodromic population spikes or the probability of unitary discharges increased. Changes of the intracellularly recorded potentials were either (a) increased EPSP amplitudes associated with decreased IPSP amplitudes, or (b) increased IPSP amplitudes. A cell showing enhanced IPSPs after repetitive activation could respond with increased EPSP amplitudes and decreased IPSP amplitudes upon further repetitive activation. The potentiation, which was always preceded by a 5--10 min depression, lasted up to 3 h. This potentiation was heterosynaptic, since the responses to the non-stimulated input also changed and since the inputs were found to excite the pyramidal cells through separate synapses in double shock experiments. The heterosynaptic mode of the potentiation as well as the changes of the IPSPs indicate that not only the excitatory pathway but also the inhibitory pathway must be considered in explaining postactivation potentiation in this hippocampal field.


Biochemical compartmentation of fish tissues. II. Nonspecific phosphomonoesterases in brain. The specific activities of acid and alkaline phosphatases in different regions of the brain of 9 nutritionally important fishes were worked out. The region consisting of pituitary, hypothalamus and thalamus showed the highest acid and alkaline phosphatase activities. Least activities of both the enzymes were found in the cerebellum and medulla oblongata. The piscivorous fishes contain the highest acid and alkaline phosphatase activity followed by cat fishes and major carps. The distributional pattern of these 2 enzymes in 4 regions of the brain of 9 fishes is the same.


The influence of insulin, cAMP and the calcium ionophore X 537 A on the growth of the cartilage analagen of limb buds in vitro. Limb buds of 11-day-old mouse embryos were cultured for 6 days with insulin, dibutyryl cAMP and X 537 A. The cartilage anlage was reduced by insulin and enlarged by dibutyryl cAMP and X 537 A. The effects are due to changes in the amount of intercellular substance.


Globin gene expression in MSV-transformed fibroblasts. The activation of globin gene expression on viral transformation of 3T3 cells was investigated. Globin mRNA was determined using a radioactive complementary DNA probe. No difference was found between 3T3 and transformed 3T3 cells. There does not therefore appear to be a random activation of extensive regions of the cellular genome.


Penicillin-induced formation of ribonuclease in rice (Oryza sativa L.) endosperm and its inhibition by abscisic acid. Penicillin stimulates the formation of ribonuclease in embryoless rice (Oryza sativa L.) endosperm and enhances gibberelin-induced response. Penicillin-induced RNase production is completely inhibited by abscisic acid.


Correlation of immunogenicity with suppression of lymphocyte adenosine 3',5'-monophosphate-dependent protein kinase. Cyclic-AMP-dependent protein kinase activity was depressed in whole spleen as well as in isolated splenic lymphocytes from 3-methylcholanthrene (MCA), R3230 AdCa mammary adenocarcinoma, N-hydroxy-2-acetylaminofluorene, and 4-dimethylaminoazobenzene (DMAAB) tumor-bearing Fischer rats as compared to control animals. The magnitude of depression increased with the immunogenicity of the tumor. The depressed enzyme activity was the result of a reduced Vmax for adenosine 3',5'-monophosphate (cAMP)-stimulated histone phosphorylation.


Adrenergic reinnervation of the denervated rat urinary bladder. In rats undergoing unilateral extirpation of the pelvic ganglion, the adrenergic innervation disappeared on the ipsilateral side of the urinary bladder. It had reappeared after 6--9 weeks.


In vivo inhibition of the tyrosinase of hamster melanoma with sodium diethyldithiocarbamate. In vivo inhibition of the DOPA-oxydase activity of the soluble tyrosinase fraction of melanotic hamster melanoma is found after i.p. administration of diethyldithiocarbamate, with considerable increase in the content of SH-groups which probably play the role of free radical scavengers.


TSH-receptor antibodies, HLA B8 and thyroid autoantibodies in patients with Graves' disease in therapeutically induced euthyroidism. The prevalence of TSH-receptor antibodies and of thyroid autoantibodies was studied in 48 HLA-typed patients with Graves' disease, who were in an euthyroid state after antithyroid therapy with methimazole. TSH-receptor antibodies, which were found in 35% of the patients, did not correlate with the positivity of HLA B8. By contrast the persistence of thyroid microsomal antibodies was significantly associated with HLA B8.


Leydig cell function in streptozotocin-induced diabetic rats. Streptozotocin diabetic rats were infertile as a result of decreased Leydig cell function of the testes. The major changes found were: decreased number of Leydig cells and their spontaneous secretion of testosterone. No change in the receptors to LH on the Leydig cells was observed. LH was found to be obligatory for the regulation of Leydig cell function and fertility.


Role of lipoprotein lipase in plasma triglyceride removal. Intravenous injections of anti-lipoprotein lipase serunis quantitatively block the catabolism of very low density lipoprotein (VLDL) and portomicron triglyceride and specifically inhibit triglyceride transport into ovarian follicles. The immunological studies presented provide information on the site of action of lipoprotein lipase (LPL). In the anti-LPL serum-treated animals initial plasma triglyceride accumulation occurs at the time of antiserum injection. This instantaneous inhibition of triglyceride removal provides direct evidence that the functional LPL responsible for VLDL and portomicron triglyceride hydrolysis is located in sites within the plasma compartment readily accessible to immunoglobulins. In vitro immunological studies show that the adipose, heart, ovarian, and liver LPL share common immunological determinants. Biochemical studies on highly purified heart and adipose LPL suggest that these enzymes have identical protein moieties.


The partial adrenocortical hydroxylase deficiency syndrome in infertile women. Eighteen infertile women desiring conception were selected for additional diagnostic studies on the basis of clinical evidence of hyperandrogenism characterized by acne and/or facial hirsutism. These 18 patients underwent stimulation tests with adrenocorticotropic hormone which demonstrated the presence of partial adrenocortical hydroxylase deficiences. All patients were then placed on glucocorticoid suppression therapy. Ten patients conceived and, of these, six required no therapy other than prednisone. Four conceived using a combination of prednisone and clomiphene therapy. Of othe eight patients who did not conceive, seven had other significant infertility factors. Therefore, of the patients without other significant fertility factors, 91% conceived. This study demonstrates that partial adrenocortical enzyme defects play a significant role in some women with clinical evidence of hyperandrogenism, and, in such women, either glococorticoid therapy alone or in combination with clomiphene is indicated.


The effect of factors causing induction of DNA breaks on transfection of chicken cells by the XC DNA, and the kinetics of appearance of virus-producing cells after transfection. The effect of BUdR and 4NQO treatment of the recipient chicken fibroblast cultures on the efficiency of transfection by the XC DNA was investigated. The efficiency of transfection was 2-fold higher when the recipient cultures were incubated in the presence of 10 micrograms BUdR/ml in medium 199 (48%) than when cultures were not treated (25%). The efficiency was not further increased by exposure of BUdR-treated cultures to visible light for 20 min (31%). Growth of BUdR-treated cultures decreased after light irradiation which indicated that BUdR was incorporated into the host cell DNA. Treatment of the recipient cultures with 4NGO at a concentration of 0.4 micrograms/ml for 2 h before transfection exerted only unfavourable effects on transfection efficiency. In cultures positive in transfection, transformation was first detected 3 days after exposure to DNA by the infectious centre assay, with a frequency of 4.76 +/- 6.46 transformed cells per 10(6) cells.


[Studies on the heterogeneity of urinary HCG molecules by RRA and RIA (author's transl)]. A sensitive bioassay (Radio receptor assay: RRA) for urinary hCG which is independent of plasma half life and metabolism has been developed by utilizing the specific binding of 125I-hCG to 150 approximately 2000g fraction of homogenates of the rat testis. The receptor and immunological activity (RRA/RIA) ratio of urinary hCG in pregnant and trophoblastic diseased women was examined. In addition, the urine of women with normal pregnancies was studied for quantitative and qualitative differences among hCG molecules, by gel filtration on a Sephadex G-100, and each fraction was measured by RRA and radioimmunoassay (RIA). 1) The sensitivity of RRA was 4 ng/ml with a precision (lambda) of 0.03. The intraassay coefficient of variation and that for inter assay were 2 approximately 10% and 12.1% respectively. There was a complete lack of cross reaction with PRL, hCG alpha, hCG beta, and FSH; this indicated a high specificity of the RRA. 2) Receptor reactive hCG in urine throughout pregnancy showed a major peak in the 10th week and declined, but remained fairly constant until the 31st week of pregnancy. The RRA/RIA ratio in urinary hCG of normal pregnancies was highest in the first trimester and lower in the second and third trimester. The average value throughout gestation was 2.04 +/- 0.18 (SE), which was about 2 times greater than that (1.08 +/0 0.11(SE)) of trophoblastic diseases (p less than 0.01). 3) The RRA/RIA ratio in the patients with trophoblastic tumors was highest in the hydatidiform mole and lowest in the chorioepithelioma. 4) When the urine of normal pregnancies was gel filtrated on a Sephadex G-100, it was found that receptor active molecules of hCG were present in urine of the first and second trimester. These results indicated that the RRA/RIA ratio in urinary hCG was distinct in normal pregnancies from trophoblastic tumors and would contribute sufficiently to an early diagnosis and the treatment of successive trophoblastic disease in follow-ups of patients with hydatidiform mole.


Localization of LH receptors on luteal cells with a ferritin--LH conjugate. In order to study the distribution of LH (HCG) receptors on luteal cells ferritin was coupled to ovine LH with glutaraldehyde and purified by gel chromatography. The conjugate (FELH) competed with 125I-hCG for binding to isolated luteal membranes and stimulated a dose-dependent release of progesterone (P) from isolated luteal cells which was inhibited by PGF2 alpha. FELH was distributed as single molecules or in small clusters at intervals on the surfaces of luteal cells labeled at 37 degrees C, 4 degrees C or with formaldehyde prefixation. Capping or preferential labeling at one site was not observed. The general distribution of LH (hCG) binding sites at 37 degrees C was confirmed by light-microscopic autoradiography. The distribution at 4 degrees C or with prefixation was more diffuse than at 37 degrees C suggesting that FELH binding induces small changes in receptor aggregation. Binding of FELH was specific since excess hCG reduced FELH binding to luteal cells. In cells labeled at 4 degrees C, rinsed and warmed to 37 degrees C FELH was observed along cell surfaces and within some coated vesicles and a few lysosomes within minutes suggesting that receptor internalization is a rapid and possibly continual process.


The effect of induced hyperprolactinaemia on Leydig cell function and LH-induced loss of LH-receptors in the rat testis. Anterior pituitary glands were transplanted beneath the kidney capsule of intact, adult male rats to induce hyperprolactinaemia. This resulted in reduced serum levels of LH and FSH and increased adrenal weight. In pituitary-transplanted rats, testicular hCG-receptor binding was increased by 55 to 175%, whilst the capacity of the testis to secrete testosterone in vitro was greatly reduced. Injection of ovine LH into control and pituitary-transplanted rats resulted in similar percentage reductions in hCG-receptor binding in the two groups. This treatment impaired the in vitro steroidogenic responsiveness of testes from control rats at 24 h after injection, but had no major effect on the already-impaired, steroidogenic responsiveness of testes from pituitary-transplanted rats. Although induction of hyperprolactinaemia resulted in marked changes in Leydig cell function, these alterations were possibly due to the chronically reduced serum gonadotrophin levels in hyperprolactinaemic rats as well as a direct effect of prolactin on the Leydig cell.


The effect of insulin and guanosine nucleotides on protein phosphorylations by sarcolemma membranes from skeletal muscle. In a previous report we have shown that insulin increases the phosphorylation of an endogenous protein of mol. wt. 16 000 daltons in sarcolemma membranes. In the present work we have demonstrated that phosphorylations of exogenous histones by the sarcolemma membranes are also increased by insulin. These results indicate that insulin activates a cyclic-AMP-independent protein kinase in sarcolemma membranes. The stimulatory effect of insulin on protein phosphorylations is increased by GTP and its analogue GMP-P(NH)P. The insulin effect was increased 3--4-fold by micromolar concentrations of GTP. The effect by the analogue GMP-P(NH)P was somewhat less. In the absence of insulin guanosine nucleotides had no effect on phosphorylation of the proteins. The results suggest that GTP is a modulator in the activation of a sarcolemma membrane protein kinase by insulin.


Monoamine oxidase and catechol-O-methyltransferase activity in hamster and rat insulinomas. Hamster and rat insulinomas were assayed for norepinephrine, dopamine and serotonin concentration and for monoamine oxidase and catechol-o-ethyltransferase (COMT) activity. The concentration of norepinephrine (mean 0.55 mumol/kg, range less than 0.20 to 2.64 mumol/kg) and serotonin (mean 5.22 mucol/kg, rang less than 0.6 to 26.5 mumol/kg) in hamster insulinomas were comparable to previously reported concentrations. Dopamine conentration (mean 0.34 mumol/kg, range less than 0.20 to 0.95 mumol/kg) was only 2 to 2.5% of that reported previously. Monoamine oxidase activity of the hamster and rat insulinomas were comparable to those of normal hamster islets. In contrast, the COMT activity of both insulinomas was much greater than the COMT activity of normal pancreatic islets of both species and was greater than in several other tissues and tumours. The tumour COMT, which was predominantly in the cytosol, was Mg2+ dependent and had a comparable sensitivity to inhibition by tropolone as purified beef-liver COMT. Hamster insulinoma monoamine oxidase was more sensitive than rat insulinoma monoamine oxidase to inhibition by tranylcypromine and deprenyl, while rat insulinoma monoamine oxidase was more sensitive to inhibition by clorgyline and was more heat labile.


The use of the psychiatric consultation record for residency training. The Psychiatric Consultation Record (PCR) is an instrument developed to aid in the teaching of consultation psychiatry, to evaluate the impact of this teaching, and to provide an overall assessment of the consultation training program at St. Luke's Hospital. The PCR both structures the resident's training experience and provides a tool with which he or she can integrate theoretical and practical knowledge pertaining to consultation work. Its use has enabled supervisors to detect the degree to which didactic material has become integrated into residents' thinking and has provided data through which the scope and quality of each resident's work can be rapidly assessed. Finally, its use has brought to light deficiencies in service delivery which, before the use of this instrument, had not been apparent.


Cloning the argF gene from Escherichia coli K-12 with simian virus 40. We have inserted a gene coding for ornithine transcarbamylase (OTCase) from Escherichia coli K-12 into the late gene region of simian virus 40 (SV40) DNA and propagated the hybrid molecules as free episomes or by co-infection with an SV40 tsA helper virus. In the first case, the E. coli argF gene was inserted via the EcoRI and BamHI termini in the late gene region of SV40 and the recombinant molecules were used to transfect monkey kidney cells. The hybrid DNA, which was too large to be encapsidated, was replicated for a short time (14 days) but was eventually lost from the surviving cells. In order to allow the argF gene to be packaged into virions, we purified two SV40 vectors containing large deletions of late gene region sequences. One was a 3325 base pair segment from a HaeII + BamHI digest. The argF gene was joined to both vectors at the BamHI site and these linear molecules were used to transfect monkey cells in the presence of SV40 tsA58 DNA as helper. These hybrid DNAs were replicated and packaged into virions. Late in the lytic infection of monkey cells, polyadenylated, cytoplasmic argF transcripts were detected, but significant translation of these trancripts was not observed.


Immunogenicity and bioactivity of glucagon, modified at methionine-27. Porcine glucagon was modified at methionine-27 by methylation or oxidation. Antisera against the glucagon derivatives were obtained. One of these antisera showed a high affinity for glucagon, with no cross-reactivity with gut-GLI 1. Biological activities of these derivatives were assessed on rat hepatocytes. Both derivatives had the same maximal glucose-mobilising activity as native glucagon, but a decrease potency, suggesting a crucial role of methionine in the binding of glucagon to its hepatic receptor.