Polyacrylamide gel electrophoresis of visna virus polypeptides isolated by agarose gel chromatography. The proteins of visna are separated into nine major peaks by agarose gel chromatography in 6 M guanidine hydrochloride (GuHCl). The polypeptides in eack peak were isolated by acid precipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The patterns of SDS-PAGE show that the excluded material from the GuHCl column contains an aggregate of 10 non-glycosylated polypeptides. It is shown that this aggregate represents virus substructures that are not completely solubilized by GuHCl. Two glycoproteins, gp175 and gp115, were isolated from the column eluate. The major glycoprotein gp115 was coeluted with P90, P68, and P61 in GuHCl 4. Each of the four major peaks (GuHCl 5 to 8) contains more than one nonglycosylated polypeptide. However, a small polypeptide, P12, can be isolated in a homogeneous form in the last peak, GuHCl 9. Analysis of the virus proteins (100 microgram) by SDS-PAGE shows that 20 radioactive bands can be recognized. During fractionation of the protein on agarose gel columns followed by analysis with SDS-PAGE, a number of minor polypeptides that were not detected before became clearly recognizable. Thus, the combined use of column chromatography and SDS-PAGE shows that visna virus is composed of 25 proteins.


Physical map of the Kirsten sarcoma virus genome as determined by fingerprinting RNase T1-resistant oligonucleotides. From analysis of the large RNase T1-resistant oligonucleotides of Kirsten sarcoma virus (Ki-SV), a physical map of the virus genome was deduced. Kirsten murine leukemia virus (Ki-MuLV) sequences were detected in T1 oligonucleotides closest to the 3' end of the viral RNA and extended approximately 1,000 nucleotides into the genome. The rat genetic sequences started at this point and extended all the way to the very 5' end of the RNA molecules, where a small stretch of Ki-MuLV sequence was detected. By comparison of the fingerprints of Ki-SV RNA and the RNA of the endogenous rat src genetic sequences, it was found that more than 50% of the T1 oligonucleotides were similar between Ki-SV and the endogenous rat src RNA, suggesting an identical primary nucleotide sequence in over 50% of the viral genomes. The results indicate that Ki-SV arose by recombination between the 5' and 3' ends of Ki-MuLV and a large portion of the homologous sequences of the endogenous rat src RNA.


DNA-binding proteins in the cytoplasm of vaccinia virus-infected mouse L-cells. Mouse L cell fibroblasts were infected with vaccinia virus and labeled 2 to 3 h postinfection with [35S]methionine. Labeled proteins were fractionated on native and denatured DNA-cellulose columns and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Twenty-four 90,000 to 12,500, were detected. VDP-12A (molecular weight, 29,750) had affinity for denatured but not native DNA, and its synthesis was dependent on viral DNA replication. VDP-20 (molecular weight, 41,000) bound very tightly to native and denatured DNA and was displaced only after boiling the protein-DNA-cellulose matrix in 1% sodium dodecyl sulfate. VDP-8,-11,-12,-13, -and-14 behaved electrophoretically like the polypeptide species previously shown to be present in DNA-protein complexes prepared from infected cells. The molecular weights of VDP-10 (50,000), VDP-11 (36,000), and VDP-8 (67,000) were similar to the polypeptide subunits of polyadenylate polymerase and phosphohydrolase I, enzymes purified from virions which have also been shown to have affinity for DNA.


Endoribonuclease activity associated with animal RNA viruses. A specific endoribonucleolytic activity was detected when detergent-lysed vesicular stomatitis of Sendai virus was incubated with the precursor to Escherichia coli tRNA Tyr. The cleavage products produced and the characteristics of the reaction were similar to those previously reported for human KB cell RNase NU. Like RNase NU, the virus-associated reaction generates 5'-hydroxyl and 3'-phosphate groups at the cleavage sites. At protein concentrations similar to those used to test vesicular stomatitis and Sendai viruses, virions of Sindbis virus and poliovirus also exhibited endoribonucleolytic activity, but reovirus, simian virus 40, and minute virus of mice did not. This endoribonuclease may be of physiological relevance to some of the viruses we tested.


Transcription of host-substituted simian virus 40 DNA in whole cells and extracts. Viral transcriptional complexes were extracted from the nuclei of monkey kidney cells infected with wild-type simian virus 40 (SV40) or a variant strain containing a high proportion of host-substituted DNA molecules. The RNAs synthesized by these complexes in an in vitro system were analyzed for their content of SV40 and host sequences by a technique of sequential hybridization to plaque-purified and substituted viral DNAs. The relative labeling of the two types of sequences was commensurate with their proportion in the viral DNA (about 20% host). The substituted virus contains both reiterated and unique types of cellular sequences, and both kinds appeared to be transcribed. Transcripts of the substituted sequences formed a much smaller proportion of the virus related RNA recovered from intact infected cells, suggesting that host sequence transcripts are synthesized but rapidly degraded in the whole cell. The alternative, that transcription of these sequences is artificially enhanced in the in vitro system, cannot be rigorously excluded. We compared the self-annealing of viral RNAs from nuclear extracts of cells infected with wild-type and substituted viruses; transcripts labeled both in vivo and in vitro showed a two- to threefold-higher level of self-annealing in the case of the variant than in the case of wild type SV40.


Binding and transcription of simian virus 40 DNA by DNA-dependent RNA polymerase from Escherichia coli. Supercoiled simian virus 40 was transcribed more efficiently than nonsupercoiled DNA. The effect was increased from two- to fivefold by the addition of rifampin with triphosphates. The number and locations of polymerase binding sites with respect to Hin II-III restriction fragments were determined. The total number of binding sites was nine, as determined by UV difference spectroscopy. The locations of these binding sites were on the A, B, D, E, F, and G fragments, as determined by gel electrophoresis. The number of sites was the same for both supercoiled and relaxed or Hin II-III-digested DNA, and the point of saturation of supercoiled DNA by polymerase remained the same with increasing concentrations of rifampin from 0 to 8 microgram/ml.


Effect of chemical modification of supercoiled simian virus 40 DNA on the rate of in vitro transcription. Superhelical simian virus 40 FI DNA could be modified with the single-strand-specific reagent N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide (CMC). A limited reaction, of less than 2% of the base pairs, resulted in almost total inhibition of in vitro transcription by DNA-dependent RNA polymerase from Escherichia coli. This effect was shown to be due to DNA modification and not to inhibition of polymerase activity by the reagent. Inhibition of enzyme activity occurred if the contaminating reagent was not absorbed with another protein before polymerase addition. No inhibition was observed when DNA and polymerase were incubated together to allow the formation of pre-initiation complexes before CMC was added. Studies of template saturation with polymerase showed that the inhibition of transcription by DNA modification was due to a loss of binding ability of the enzyme to the reacted, supercoiled DNA when reaction times of less than 2 h were used.


Presence of human chromosome 21 alone is sufficient for hybrid cell sensitivity to human interferon. Human/mouse somatic cell hybrids with chromosome 21 as the only detectable human genetic material were sensitive to both human leukocyte and fibroblast interferons. The presence of additional human chromosomes decreased the amount of interferon needed to attain a given level of virus resistance. Decreased cytopathic effects, decreased virus yields, and the appearance of a specific phosphorylated protein associated with interferon treatment were all observed in hybrids maintaining only human chromosome 21. The phosphorylated protein found in extracts of these human interferon-treated hybrid cells was of mouse origin.


Polyoma virus-human cell interactions: persistence of T-antigen in two cell lines with and without transformation. The interaction of polyoma virus and human cells was investigated. Abortive infection as evidenced by the synthesis of T-antigen was observed in normal fibroblast and abnormal (transformed) cells but not in normal epithelial cells. A high percentage of simian virus 40-transformed WI-18 Va2 and spontaneously transformed BE skin cells produced T-antigen after high-multiplicity infection, but most of the cells rapidly lost antigen-producing capacity upon cell passage, and the cultures became negative by passage 3. All fibroblast cells displayed varying degrees of susceptibility to infection, but most of the cell lines became negative for T-antigen except for two. In one, T-antigen persisted in a small percentage of the cells throughout the lifetime of the culture, without cellular transformation occurring. In the other, the entire culture became morphologically transformed and eventually consisted of 100% T-antigen-positive cells. This is the first time that normal diploid human fibroblast cells have been transformed by polyoma virus.


Comparison of two viable variants of simian virus 40. The DNAs of two viable strains of simian virus 40, 776 and 777, have been compared by using restriction endonucleases. Differences between the two strains were detected at five separate points on the simian virus 40 genome. One of these differences, in the region of DNA coding for the major viral coat protein, was confirmed by tryptic peptide analysis of coat proteins from the two strains. Some physiological differences between the two strains were examined and can, in general, be explained by differences observed between the DNAs of the two strains. In addition, defective variants derived from strain 777 interfere more efficiently with the replication of strain 777 than with the replication of strain 776.


Herpesvirus ateles DNA and its homology with Herpesvirus saimiri nucleic acid. Analysis of the structural organization of Herpesvirus ateles DNA shows that two types of viral DNA molecules are encapsidated in virions: (i) M-genomes, which contain 74% light sequences (L-DNA, 38% guanine plus cytosine) and 26% highly repetitive heavy sequences (H-DNA, 75% guanine plus cytosine), and (ii) defective H-genomes, which consist exclusively of repetitive H-DNA. The structure of M-genomes from H. ateles consists of an L-DNA region of about 70 x 10(6) daltons inserted between H-DNA termini of variable length. M-genomes with a shorter H-DNA region at one end of the molecule have a long stretch of H-DNA at the other end, resulting in a total molecular weight of 89.8 +/- 8.5 x 10(6). Thus it resembles the structure of M-genomes of H. saimiri. H-DNA of the two independent H. ateles isolates, strains 810 and 73, reveals different patterns after cleavage with restriction endonuclease Sma I. H-DNA of H. ateles 810 appears to consist of identical tandem repeat units with a molecular weight of 1,035,000; the H-DNA repeat unit of strain 73 is shorter (930,000 molecular weight). Corresponding DNA sequences of the two H. ateles strains (810 and 73) are completely homologous in cross-hybridizations. However, a discrete nucleotide sequence divergence between these virus strains is detected by measuring melting temperatures (T(m)) of DNA hybrid molecules. Some homology exists between H. ateles and H. saimiri DNA. Hybridization of L-DNA from H. ateles with L-DNA from H. saimiri shows about a 35% homology between the respective L-DNA sequences; the resulting heteroduplex molecules show a decrease of T(m) by 13.5 degrees C, corresponding to about a 9% mismatching in cross-hybridizing parts of L-regions. Very little homology is found between H-DNA of H. ateles and H. saimiri.


Synthesis and processing of precursor polypeptides to murine mammary tumor virus structural proteins. Biosynthesis of murine mammary tumor virus (MuMTV) proteins was studied in the chronically MuMTV-infected epithelial cell line MuMT-73 by using monospecific antisera to the major MuMTV core protein p27 and the major envelope glycoprotein gp47. In pulse-labeling experiments using [35S]methionine, monospecific antisera to p27 precipitated a 75,000-molecular-weight protein as the major intracellular component. Analysis of the same cellular extracts with monospecific antisera to gp47 revealed that the gp47 precursor was a 70,000-dalton protein. After chase periods, there was a loss of label from the precursors and a concomitant increase of labeled extracellular mature viral proteins. The glycoprotein precursor incorporated labeled glucosamine and seemed to be processed more rapidly than the p27 precursor. Considerable amounts of apparently nonvirion-associated gp47 and glycoprotein precursor could be detected in the extracellular culture fluid.


Structure of 50 to 70S RNA from Moloney sarcoma viruses. The 50 to 70S RNAs of two clonal isolates of defective Moloney sarcoma-leukemia helper virus complex were analyzed by gel electrophoresis and electron microscopy. The RNAs extracted from both clone 3 and clone 124-5R of Moloney sarcoma-leukemia virus complex contained some large monomer subunits ca. 10,000 nucleotides in length (10 kilobases), which are believed to be the Moloney leukemia virus subunits. Both RNAs had an excess of a smaller, sarcoma-specific subunit, 5 kilobases (clone 3) or 6 kilobases (clone 124-5R) in length. Electron microscopy of intact 50 to 70S dimer RNA molecules showed for both clones many dimers of two small subunits, some dimers of two large subunits, but few if any heterodimers with one large and one small subunit. This result was unexpected because the sequences near the 5'end of the RNA subunits, which are believed to be involved in the dimer linkage, are probably homologous between the large and small subunits. We also observed that some small-small dimers migrated anomalously slowly on nondenaturing gels. The nature of this slow-migrating complex is unkown; it could be a higher aggregate of the small-small dimer with additional small or large subunits, or it could be an extended conformation of the small-small dimer.


Anatomy of bovine mammillitis DNA. I Restriction endonuclease maps of four populations of molecules that differ in the relative orientation of their long and short components. In this paper, we report that the DNA of bovine mammillitis virus (BMV) consists of two covalently linked components that are 71.5 x 10(6) and 15.7 x 10(6) in molecular weight and designated L and S, respectively. We further report that the BMV DNA consists of four equimolar populations differing only in the orientation of the L and S components relative to each other. This conclusion is based on the following: (i) The sum molecular weight of fragments generated by digestion of BMV DNA with Hsu I, Hpa I, Bgl II, or Xba I significantly exceeds the established molecular weight of the intact DNA. (ii) In each digest, the fragments form three groups differing in molar concentration. In reference to the molar concentration of intact DNA, each enzyme digest contained a set of four fragments 0.25 M in concentration, a set of four fragments 0.5 M in concentration, and a variable size set, unique for each enzyme digest, 1.0 M in concentration. (iii) Experiments involving digestion of intact DNA by lambda exonuclease followed by restriction endonuclease digestion established that each of four 0.5 M fragments were positioned at the termini of the BMV DNA. (iv) Complete maps for the fragments generated by each enzyme established that the 0.25 M fragments arise by fusion of the sequences of the terminal fragments when these are juxtaposed as a consequence of the inversion of L and S components. The maps also established the dimensions of the L and S components. We conclude that the structure of BMV DNA is similar to that of HSV DNA previously shown to consist of two unequal size components that invert relative to each other.


Encephalomyocarditis virus RNA. III. Presence of a genome-associated protein. A low-molecular-weight protein was found to be associated with intact 35S RNA isolated from purified encephalomyocarditis virus. This protein was positively charged at pH 3.5, sensitive to proteinase K treatment, and labeled with either 3H-amino acids or 32Pi.


Inhibition of herpes simplex virus type 1 replication in temperature-sensitive cell cycle mutants. Herpes simplex virus type 1 DNA synthesis and infections progeny production were studied in five different conditional hamster (BHK-21) cell cycle mutants. At the nonpermissive temperature (39.5 degrees C), both events were strongly inhibited in four of these cell lines. The degree of inhibition was a reproducible characteristic of each cell mutant and in two cases was dependent upon the multiplicity of infection. Experiments involving shifts to the nonpermissive temperature at least 3 h postinfection at 33.5 degrees C suggested that the defects in viral replication were not due to faulty adsorption, penetration, or uncoating, whereas experiments involving shifts of infected cells from the nonpermissive temperature to 33.5 degrees C revealed the reversible nature of the inhibition.


Alterations in the protein synthetic apparatus of Friend erythroleukemia cells infected with vesicular stomatitis virus or herpes simplex virus. We have compared the effects of infection with herpes simplex virus (HSV) and vesicular stomatitis virus (VSV) on the protein synthetic apparatus of Friend erythroleukemia cells. Previous studies demonstrated that infection with HSV rapidly shuts off the synthesis of globin and other cellular polypeptides (Y. Nischioka and S. Silverstein, 1977, Proc. Natl. Acad. Sci. U.S.A. 74: 2370-2374). In contrast to these findings, globin synthesis persists in Friend erythroleukemia cells infected with VSV. Physical measurements of the size of bulk-infected cell mRNA, using hybridization with polyuridylic acid, demonstrated that there was no detectable change in the size of mRNA's after infection with VSV. A comparison of the kinetics of hybridization of cytoplasmic RNA extracted from cells infected with either HSV or VSV with globin complementary DNA revealed that by 4 h postinfection with HSV only about 15% of the globin mRNA sequences remained, whereas there was no discernible change in the sequence abundance of globin mRNA in VSV-infected cells.


5' Terminus of defective and nondefective Sendai viral genomes is ppp Ap. Using RNase T2 digestion of 32P-labeled Sendai virus nondefective and defective-interfering RNAs, the 5' termini were isolated and demonstrated to be pppAp for both the plus and minus strands.


Initiation points for DNA replication in nontransformed and simian virus 40-transformed BALB/c 3T3 cells. The number of initiation points for DNA synthesis per unit length of DNA in rapidly growing cells is greater for simian virus 40-transformed than for nontransformed BALB/c 3T3 cells.


Adenovirus DNA synthesis in vitro in an isolated complex. DNA-protein complexes isolated from adenovirus-infected cells by a modification of the M-band technique were used as an in vitro system for the study of adenovirus DNA replication. The synthesis in vitro was semiconservative, inhibited by N-ethylmaleimide, and stimulated by ATP. Studies on DNA-negative mutants of adenovirus showed that the DNA synthesis in vitro represents a continuation of adenovirus DNA replication in vivo. DNA synthesis in vitro was inhibited 38% by 20 microgram of phosphonoacetic acid per ml, which is several-fold higher than the inhibition obtained with purified DNA polymerase beta or gamma, but was similar to the degree of inhibition of DNA polymerase alpha. DNA synthesis in complexes from uninfected cells was much less sensitive to inhibition by phosphonoacetic acid. In addition, complexes from infected cells contained a greater proportion of the alpha-polymerase than complexes from uninfected cells, suggesting that an association of alpha-polymerase with the replication complex may be occurring during adenovirus infection, with subsequent utilization of the alpha-polymerase for viral DNA synthesis.


Radiobiological inactivation of Epstein-Barr virus. Lymphocyte transforming properties of B95-8 strain Epstein-Barr virus (EBV) are very sensitive to inactivation by either UV or X irradiation. No dose of irradiation increases the transforming capacity of EBV. The X-ray dose needed for inactivation of EBV transformation (dose that results in 37% survival, 60,000 rads) is similar to the dose required for inactivation of plaque formation by herpes simplex virus type 1 (Fischer strain). Although herpes simplex virus is more sensitive than EBV to UV irradiation, this difference is most likely due to differences in the kinetics or mechanisms of repair of UV damage to the two viruses. The results lead to the hypothesis that a large part, or perhaps all, of the EBV genome is in some way needed to initiate transformation. The abilities of EBV to stimulate host cell DNA synthesis, to induce nuclear antigen, and to immortalize are inactivated in parallel. All clones of marmoset cells transformed by irradiated virus produce extracellular transforming virus. These findings suggest that the abilities of the virus to transform and to replicate complete progeny are inactivated together. The amounts of UV and X irradiation that inactivate transformation by B95-8 virus are less than the dose needed to inactivate early antigen induction by the nontransforming P(3)HR-1 strain of EBV. Based on radiobiological inactivation, 10 to 50% of the genome is needed for early antigen induction. Inactivation of early antigen induction is influenced by the cells in which the assay is performed. Inactivation proceeds more rapidly in EBV genome-free cells than in genome carrier Raji or in P(3)HR-1 converted EBV genome-free cells clone B(1). These results indicate that the resident EBV genome participates in the early antigen induction process. Variation in radio-biological killing of B95-8 and P(3)HR-1 EBV is not attributable to variations in the repair capacities of the cells in which the viruses were assayed, since inactivation of HSV was the same in primary lymphocytes and in all lymphoid cell lines tested.


Glycoproteins of avian tumor virus recombinants: evidence for intragenic crossing-over. The envelope glycoproteins of several avian tumor virus recombinants selected for the host range of a leukosis virus and the transforming function of a sarcoma virus were compared with each other and with those of their parents. It was found that the glycoproteins of different recombinant viruses, derived from the same parents, differed in their electrophoretic mobilities measured in polyacrylmide gels. The glycoproteins that had lower electrophoretic mobilities had higher precentages of carbohydrate. The carbohydrate of viral glycoproteins was estimated to range between 8 and 18% from their buoyant densities in CsCl, using known glycoproteins as standards. After exhaustive Pronase digestion, the carbohydrate was recovered from viral glycoproteins as a mixture of glycopeptides with molecular weights ranging from 2,500 to 5,000. It was estimated that distinct viral glycoproteins contained between two and five such oligosaccharide chains and that the glycoproteins of different recombinants expressing the same host range marker may differ in the number of oligosaccharide chains and consequently also in their polypeptide structure. Those with lower electrophoretic mobility contain more oligosaccharide chains per molecule than those with higher electrophoretic mobilities. It is suggested that not oligosaccharide chains define the viral host range.


Adenocarcinoma of the urachus.

Adenocarcinoma of the urachus. During the last 50 years 8 patients with adenocarcinoma of the urachus were managed at our hospital. The various modalities of therapy included radiation therapy, transurethral resection, segmental resection of the bladder and total cystectomy. A retrospective analysis of these cases is presented, including the histologic patterns, sites of spread and survival according to therapy.


[2 models of indomethacin-induced arterial hypertension in rats in chronic experiments]. Chronic administration of small doses of prostaglandin-synthesis inhibitor indometacin against the background of a salt load or unilateral nephrectomy induces the development of arterial hypertension in rats. Arterial pressure increases (two fold on the average) on the 6th week of the experiment in 60-80% of the animals. Arterial hypertension developing against the background of a salt load is marked by retention of sodium in the organism and increase the intravascular volume, while that developing in unilateral nephrectomy--by increased sodium excretion and reduced intravascular volume. Depressed activity of the renin-angiotensin-aldosterone system and conspicious changes in the renal vascular channel are noted in both forms of arterial hypertension. It is assumed that disorders in the metabolism of cyclic nucleotides underlie the changes occurring in the renal vessels due to the effect of indometacin. Similar generalized changes in the peripheral vascular channel on the whole may be the cause of the increased vascular resistance and one of the causative factors of the hypertension development.


[Typology of the hyperlipoproteinemias in ischemic heart disease]. A total of 227 patients with ischaemic heart disease were examined. Hypercholesterolemia was revealed in 72 percent, hypertriglyceridemia--in 48 percent, and various types of hyperlipoproteinemia--in 82 percent. Type II hyperlipoproteinemia was seen most often (59 percent), type IV in 21 percent. Hyperlipoproteinemia of Type IV was found in men twice as often as in women (29 and 13 percent respectively). Women had normal levels of blood serum lipoproteins 1.5 times more often. No spontaneous normalization of hyperlopoproteinemia was noted.


[Crystalline corneal dystrophy (Schnyder) in the presence of familial type IIa hyperlipoproteinaemia (author's transl)]. A family with dominant autosomal hyperlipoproteinaemia typ IIa is reported. One sibling, a 19-year-old man, showed in addition to the hyperlipoproteinaemia typical corneal signs of crystalline corneal dystrophy of Schnyder. From 1964 to 1976 these corneal deposits increased. The problems of hyperlipoproteinaemia combined with corneal crystalline dystrophy are discussed.


A naturally occurring epizootic caused by Sendai virus in breeding and aging rodent colonies. I. Infection in the mouse. An acute Sendai virus epizootic occurred simultaneously in a breeding colony, in experimental and stock animal rooms, and in a colony of aging mice. During the 2-month period that the infection was at its maximum, death rates were approximately doubled. In some strains, the preweanling death rate reached 100%. RFM and BALB/c mice were most susceptible and NZB mice least susceptible. The mortality during the period of Sendai virus infection was increased for most strains and age groups except for the oldest female RFM and NZB mice. Death rates during the epizootic were lowest in young adult mice (greater than 10 weeks of age) and highest in the very young mice (less than 10 weeks of age) and in the oldest male and the moderately aged female mice. Although a substantial number of older mice died during the epizootic, examination of the age-specific death rates indicated that the increase in deaths remained relatively constant for all ages over 10 weeks. This showed that the older mice were not more susceptible to Sendai virus infection. As a sequela of the epizootic, focal chronic pneumonia was found in 10-40% of the mice coming to necropsy even 1 year later.


A naturally occurring epizootic caused by Sendai virus in breeding and aging rodent colonies. II. Infection in the rat. Sendai virus infected a hysterectomy derived, barrier maintained breeding colony and a conventional aging rat colony. The virus produced seroconversion in the colonies followed by a 7-month period of decreasing titers. Clinical signs were absent during the months when titers were highest, and there was no increase in mortality, but multifocal interstitial pneumonia with perivascular and peribronchial cuffing by lymphocytes and plasma cells was present in rat lungs examined histologically. Such lesions were absent before the period of seroconversion. During the months of declining titers, the interstitial and perivascular lesions decreased in frequency and severity. The peribronchial lesions did not decrease, however, and were still present in many rats 7 months after the acute infection. Attempts to isolate the virus from weanling rats were unsuccessful.


Development of a guinea pig colony free of complement-fixing antibodies to parainfluenza virus. Complement-fixing antibodies to parainfluenza 3 virus were found in Hartley strain [Cds: (HA)] guinea pigs from the authors' production colony. The prevalence and distribution of these antibodies were determined by testing guinea pigs of five age categories: 4 weeks, 8 weeks, 12 weeks, 6 months to 1 year, and over 1 year of age. Forty-seven percent (28 of 60) were positive to parainfluenza 3 antigen. Positive reactors were found in all age groups except those 8 weeks old. The 12-week-old group had the highest titers; the group over 1 year of age had the highest percentage of positives (92%). When 8-week-old guinea pigs were isolated, 55% were positive at some time between 8 and 34 weeks of age. The titers characteristically rose rapidly and then dropped slowly to low or undetectable levels. Four pairs of breeders over 6 months of age (most of whom were positive for parainfluenza 3 virus antibodies and, therefore, presumed to be immune to the virus) were isolated and allowed to breed. Their offspring  were found to be free of complement-fixing antibodies to parainfluenza 3 virus.


Cytochemical distinction between azurophils and catalase-containing granules in leukocytes. I. Studies in developing neutrophils and monocytes from patients with myeloperoxidase deficiency: comparison with peroxidase-deficient chicken heterophils. The neutrophils and monocytes of two patients with hereditary myeloperoxidase (MPO) deficiency lacked MPO activity as determined by light and electron microscopic cytochemical staining. With a technique employing neutral 3,3'-diaminobenzidine, azurophils of precursor and mature neutrophils were devoid of MPO whereas eosinophil, basophil, and platelet peroxidases exhibited normal activity. After incubation in alkaline DAB medium, which stains catalase, some small granules were strongly reactive in both immature and mature neutrophils and monocytes. These catalase-containing granules were distinct from all other categories of granules. Their number decreased with maturation. In the presence of cyanide or aminotriazole, peroxidatic activity could also be detected in ellipsoid azurophils, although large spherical granules remained unreactive. This peroxidatic activity is apparently not due to MPO inasmuch as it has been demonstrated that this protein is not synthesized in these patients. Thus, the significance of the last finding is unclear but suggests a heterogeneity of azurophil content. In contrast to human MPO-deficient cells, chicken heterophils naturally devoid of peroxidase are unable to produce hydrogen peroxide upon phagocytosis and were also devoid of catalase-containing particles. This observation suggests that catalase is involved in the control of the intracellular level of hydrogen peroxide in human cells.


Glycosylation of VSV glycoprotein is similar in cystic fibrosis, heterozygous carrier, and normal human fibroblasts. The single envelope glycoprotein of vesicular stomatitis virus was used as a specific probe of glycosyltransferase activities in fibroblasts from two cystic fibrosis patients, an obligate heterozygous carrier and a normal individual. Gel filtration of pronase-digested glycopeptides from both purified virions and infected cell-associated VSV glycoprotein which had been labeled with[3H] glucosamine did not reveal any significant differences in the glycosylation patterns between the different cell cultures. All 4 cell lines were apparently able to synthesize the mannose- and glucosamine- containing core structure and branch chains terminating in sialic acid which are characteristic of asparagine-linked carbohydrate side chains in cellular glycoproteins. Analysis of tryptic glycopeptides by anion-exchange chromotography indicated that the same 2 major sites on the virus polypeptide were recognized and glycosylated in all 4 VSV-infected cell cultures. These studies suggest that the basic biochemical defect(s) in cystic fibrosis is not an absence or deficiency in enzymes responsible for the biosynthesis of complex carbohydrate side chains.


Coenzyme models. 10. Rapid oxidation of NADH by a flavin immobilized in cationic polyelectrolytes. The title flavin rapidly oxidizes NADH, the observed enhancement being of 460--4650-fold compared with 3-methyltetra-O-acetylriboflavin. Analysis of the kinetic data at 30 degrees C established that the reaction proceeds according to the Michaelis-Menten kinetics at low ionic strength (mu = 0.02: Km = ca. 10(-5) M, kact. = 0.06-0.37 s-1) and according to the simple second-order kinetics at high ionic strength (mu = 0.3), the largest rate difference being 342-fold. On the other hand, the oxidation of 1-benzyl-1,4-dihydronicotinamide, a NADH model compound, was not accelerated by the use of the title flavin. It is concluded, therefore, that the formation of the polymeric flavin-NADH complexes due to the electrostatic interaction is responsible for the marked rate enhancement. This is the first example that the oxidation of NADH is facilitated in the nonenzymatic system.


Plasma membrane associated metabolic parameters and the aging of human diploid fibroblasts. Substrate uptake, portions of the cyclic AMP system, membrane fluidity and cellular phospholipid content are some of the parameters which are structurally associated with the plasma membrane and which have been linked to the control of cell proliferation. These parameters were studied with respect to cellular aging of human embryo lung fibroblasts (HELF) in culture. We observed in late passage an increase in the rate of uridine transport and in cellular cyclic AMP levels. These results were examined in relation to the increase in cell volume which occurs in senescing HELF. We also observed an increase in Vmax of uridine transport, and a decrease in the Km of cyclic AMP phosphodiesterase (PDE) as quiescent, passage 18-25, HELF were stimulated to divide with fresh serum. A similar effect of serum occurred in late passage (p. 43) cells despite the inability of these late passage cultures to undergo further proliferation. There was no change in cAMP-PDE activity with increasing passage number suggesting that the observed alterations of the cAMP levels, basal and in response to extracellular effectors, were due to alterations in the adenyl cyclase system. We observed no change in senescent HELF in membrane fluidity or phospholipid and neutral fat content.


Retrolabyrinthine approach: technique and newer indications. Excellent exposure of the cerebellopontine angle is obtained by an approach through the mastoid posterior to the labyrinth. Since the major portion of the dissection is extradural, this approach is associated with a very low morbidity. The retrolabyrinthine approach has been used for several years for selective partial section of the posterior root of the trigeminal nerve in cases of trigeminal neuralgia. Complete relief of pain has been accomplished in 25 of 28 cases, and the other 3 patients had partial relief of pain. The only complications in these patients were partial hearing impairment in 2, and 1 partial abducens nerve paralysis which subsequently recovered completely. Two patients required secondary closure of cerebrospinal fluid leaks. This approach has also been used for exploration and biopsy of cerebellopontine angle tumors and for treatment of other cranial nerve problems. We conclude that the retrolabyrinthine approach is the preferred route to the cerebellopontine angle in a variety of clinical conditions.


Melanotic neuroectodermal tumor of infancy. The lack of knowledge regarding the histogenesis of the melanotic neuroectodermal tumor of infancy may account for the numerous names given to this neoplasm. The most appropriate nomenclature is melanotic neuroectodermal tumor of infancy as this term is both clinically descriptive and reflects its most likely histogenesis. The histological diagnosis of this neoplasm and its known benign biological behavior should be appreciated so that radical surgery or radiotherapy is not undertaken. Our case report includes an electron microscopic analysis which is in keeping with other reported cases. We have not confirmed high urinary VMA levels prior to resection of the lesion.


Therapeutic innovation: a model for mental health planning. Enlightened and progressive care of the mentally ill is closer to reality with the creation of the psychiatric halfway house. The halfway house is not an institutional edifice but a residential facility located in the community and indistinguishable from the other neighboring dwellings. The feature which sets apart the halfway house programmatically is its provision of a housing unit with some services for ex-hospital patients. Human incarceration continues to be a major enforcement instrument for the control of deviant behavior. Mental illness, as one type of poorly understood behavior, is for the most part controlled by institutionalizing the mentally ill person in a large dehumanized total institution such as the state mental hospital. Although major reforms are underway in many total institutions to humanize treatment procedures, innovative alternatives to custodial care are gaining impetus in the community. One such fascinating and rapidly expanding treatment innovation is the psychiatric halfway house. This presentation represents the results and implications of a comparative study of twenty such programs in the United States.


Selective effects of thiazide on intestinal absorption of calcium and adsorptive and renal hypercalciurias. The effect of long-term thiazide therapy on the intestinal Ca absorption was measured in 10 well-defined cases of absorptive hypercalciuria with intestinal hyperabsorption of Ca and 8 with renal hypercalciuria ("renal leak" of Ca), many of whom had hyperabsorption of Ca. In most cases of absorptive hypercalciuria, the intestinal hyperabsorption of Ca persisted during treatment, despite restoration of normal urinary Ca. In contrast, the intestinal Ca absorption decreased significantly during thiazide therapy in 7 of 8 patients with renal hypercalciuria commensurate with the "correction" of the renal leak of Ca and secondary hyperparathyroidism. The results support the hypothesis that the intestinal hyperabsorption of Ca in absorptive hypercalciuria may be primary, whereas that in renal hypercalciuria may be associated with the hyperparathyroid state.


Early detection of breast cancer.

Early detection of breast cancer. One thousand referred patients with breast symptoms, a history of breast disease, or a family history of breast carcinoma have been examined at a suburban breast clinic. This is a preliminary survey of the data and results so far obtained from the initial examination of these patients. The examination consisted of history-taking by means of a questionnaire, physical examination of the breasts, thermography and xeromammography. A report correlating the findings was forwarded to the referring doctor in each case and any necessary action was indicated in the report. Seventy-eight patients were thought to require biopsy of a suspicious lump or mammographic lesion (7.8% of the total number); 76 patients actually had biopsies, and 18 patients were found to have carcinoma of the breast (23.8% of the total number of patients who had biopsies). The overall incidence of carcinoma in the 1000 women attending the clinic was 1.8% at the initial visit. While general population screening for breast carcinoma may be hard to justify, from both the detection rate and an economic point of view, it would appear from our experience to date that screening of selected high-risk referred patients is justified and, in fact, desirable.


[Effect of oxygen concentration and the physiological state of cells on the respiration chain of Candida mycoderma]. The effect of oxygen concentration and limitation of the cultural growth by the substrate to be oxidized on the functionation of the respiration chain and the ratio of cytochromes a : c : b was studied in intact cells of Candida mycoderma. As the concentration of oxygen in the medium decreases and the growth of the culture slows down, the cyanide-resistant pathway of electron transport (CrPET), which is inhibited by benzhydroxamic acid, is found in the yeast cells. At the same time, the content of cytochrome a in the cells decreases, probably because cytochrome oxidase is now less involved in electron transport via CrPET, whereas the content of cytochromes c and b increases. The latter fact seems to be due to the activation of the cytochrome-c-peroxidase pathway of electron transport whose functioning in the studied strain of C. mycoderma has been shown earlier. CrPET was found to function also at a high concentration of oxygen, but under conditions of the transition of the culture to the stationary phase, as well as during the incubation of resting cells taken at the exponential phase of growth. The manifestation of CrPET in conditions of a low concentration of oxygen can be caused not only by the direct effect of its deficiency on the respiration chain, but also by a change in the physiological state of yeast cells at their growth decelerates or ceases.


Deletion mutants of temperate Bacillus subtilis bacteriophage phi105. Six deletion mutants of temperate Bacillus subtilis phage phi105 have been isolated on the basis of their increased resistance to chelating agents. The size and position of the deletions was determined by electronmicroscopy of DNA heteroduplexes. All deletions are located in a region about 55-70% from one end of the DNA molecule, in the right half of the known genetic map of the phage. The segment 55-65% does not contain any genes essential for lytic growth or lysogenization. A gene(s) for immunity is located in a segment 65-70% from the left end. By electronmicroscopy by partially denatured phi105 DNA two A-T rich regions have been localized in the right half of the molecule. One of these regions falls within the non-essential 55-65% DNA segment.


Restoration of mutability in non-mutable Escherichia coli carrying different plasmids. N and I group plasmids, which increase methylmethane sulfonate (MMS) mutagenesis in lexA+ strains of E. coli WP2 may be divided into two classes: those restoring part of the mutability of lexA- stains (class I) and those leaving lexA- strains non-mutable (class II). Almost complete restoration of MMS mutability is obtained by class I plasmids in a partially suppressed lexA rnm strain, while clase II plasmids cause far fewer MMS revertants in this strain than in lexA+. A pair of class I and II plasmids in lexA- shows a synergistic effect on mutability. These two classes do not coincide with plasmid division into incompatibility groups.


An evaluation of D-glucosamine as a gratuitous catabolite repressor of Saccharomyces carlsbergensis. Glucose represses mitochondrial biogenesis and the fermentation of maltose, galactose and sucrose in yeast. We have analyzed the effect of D-glucosamine on these functions in order to determine if it can produce a similar repression. It was found that glucosamine represses the respiration rate (QO2) but more rapidly than glucose and to a final level slightly higher than in glucose-treated cells. Derepression of the respiration rate following either glucose or glucosamine repression was similar. A two hour lag was followed by a linear increase in QO2 to the derepressed level. Both glucose and glucosamine repressed the level of cytochrome oxidase to the same level. Glucosamine was also found to repress maltose and galactose fermentation but not sucrose fermentation. The derepression of maltase synthesis was inhibited by glucosamine. The constitutive synthesis of maltase was repressed by the addition of glucosamine. Glucosamine was judged to produce a repressed state similar to glucose repression in many respects.


Effect of "fasting" and different concentrations of glucose on alpha-linolenic acid metabolism in HTC cells. Correlation with the ultrastructural study. HTC cells are able to convert alpha-linolenic acid into higher homologs by desaturating and elongating reactions. When the cells were cultured in a Krebs Ringer bicarbonate solution ("fasted cells") a decrease in both biosynthetic reactions took place. "Refeeding" the cells with Swim's 77 medium without glucose enhanced the biosynthesis of polyunsaturated fatty acids from alpa-linolenic acid family, but when glucose was added to the medium, alpha-linolenic acid was preferably elongated rather than converted into eicose-pentaenoic acid. The ultrastructural study revealed HTC cells with a simple cytoplasmic organization, showing little evidence of their origin from hepatocytes. The cells cultured in a complete medium appeared well preserved and were similar to those "fasted" for 12 hours and "refed" for another 12 hours using Swim's 77 medium without serum. The amount of glucose in the medium plays a role in preserving the cell structure. This effect does not occur if glucose is added in the absence of aminoacids and vitamins.


Studies of poly(A+)-RNA in mouse hepatoma and cortisone-stimulated rat liver. The content of poly(A)-containing RNA in subcellar fractions has been investigated both in cortisone-treated rat liver and experimental hepatoma cells. The fractions included nuclei, cytoplasm, mitochondria, free and membrane-bound polyribosomes. 1) In both cases of genome activation (cortisone induction and hepatoma cells) an increase in poly(A) content of all subcellular fractions except free polyribosomes was observed. 2) Cortisone was found to induce elongation of poly(A) segments detected in both nuclei and cytoplasm. 3) An increase in the poly(A) block size also was found to be stimulated in nuclei and cytoplasm of hepatoma cells. 4) The observed elongation in poly(A) length occurred against the background of an increase of the population of of poly(A)-RNA's.


[Domiciliary assisted respiration by tracheotomy for chronic respiratory insufficiency (author's transl)]. Prerequisites for success in domiciliary assisted respiration for chronic respiratory insufficiency are the understanding of the patient and his active participation in the treatment as well as adjustment and attitude of his environment. The authors report their experience in 89 patients. Spirographic data were obtained in 72 patients which permitted subdivision into a group with restrictive disturbances and one with obstructive changes. The differences in prognosis in these two groups of patients were surprising. The survival rates in the obstructive diseases after 3, 5 and 10 years were 54%, 38% and 33% respectively; the corresponding values for the restrictive diseases were 84%, 84% and 69%. These differences are statistically significant. The restrictive changes are therefore considerably more favorable to assess, also with respect to a better quality of life.


[Domiciliary assisted respiration in the Languedoc-Roussillon region (author's transl)]. The activities of the Society for Domiciliary Assisted Respiration founded in the Languedoc-Roussillon region in 1973 are reported. Up to September 30, 1976, 281 patients were treated, 189 are still alive. Obstructive chronic bronchopneumopathies were predominant. In the second place were restrictive respiratory insufficiences and respiratory muscle failure in the third. Obviously, careful attention by a specially trained doctor and nurse must be guaranteed, as well as technical support. Blood gas analyses and spirographic investigations control the course of treatment. Domiciliary assisted respiration not only prolongs the life of numerous patients, but also improves its quality.


Molecular dosimetry of the mutagen ethyl methanesulfonate in Drosophila melanogaster spermatozoa: linear relation of DNA alkylation per sperm cell (dose) to sex-linked recessive lethals. The dosage-response curve for EMS was determined with dose measured as ethylations of DNA per sperm cell, and response measured as the relative frequency of sex-linked recessive lethals induced in sperm cells of Drosophila melanogaster. Dose can be converted to ethylations per nucleotide of DNA by dividing ethylations of DNA per sperm cell by 3 X 10(8) nucleotides per sperm cell. Adult males were exposed to equal amounts of either [3H]EMS for determining dose or nonlabeled EMS for determining mutational response. By feeding EMS for 24 h in a concentration of 25 mM, a high dose of 1.4 X 10(-2) ethylations per nucleotide was observed. With 1.4% of the nucleotides ethylated, 57% of the X-chromosomes were hemizygously viable; therefore, ethylation per se is not very efficient in inducing mutations. The relative frequency of mutations increased linearly with the dose from a dose of 2.1 X 10(-4) to 1.4 X 10(-2) ethylations per nucleotide. No threshold was apparent, and the statistical limits of the exponent, 1.0 +/- 0.1, excluded an exponent as high as 1.2. This linear relation suggests no change in mechanism of mutagenesis occurs from low to high dose in Drosophila. A nonlinear relation was found between exposure and dose; when exposure was increased by a factor of 250 (from 0.1 to 25 mM EMS in the feeding medium) dose was increased by a factor of only 68. By extrapolating down from our lowest dose of 2.1 X 10(-4) ethylations per nucleotide with an observed frequency of 0.55% +/- 0.08% sex-linked recessive lethals, we estimate the doubling dose for sex-linked recessive lethals to be 4 X 10(-5) ethylations per nucleotide.


Tumour-specific phenylalanine tRNA contains two supernumerary methylated bases. Every malignant tumour examined contains aberrant tRNA methyltransferases and a few tRNAs which are absent from the normal tissue of origin. To determine whether tumour-specific tRNAs have different modifications from those in normal tissue, we purified the most frequently occurring tumour-specific isoaccepting tRNA from two malignant tissues. The isoaccepting phenylalanine tRNA from Novikoff hepatoma and Ehrlich ascites cells both contain two supernumerary methylated bases. One of these l-methylguanine, is absent from the phenylalanine tRNA of normal rat, mouse, rabbit and calf liver. An increase in the levels of 5-methylcytidine and dihydrouridine was also detected.


Rotational behaviour and cGMP responses following manipulation of nigral mechanisms with chlordiazepoxide. Evidence for enhancement of GABA transmission by benzodiazepines. Unilateral stereotaxic injections of 1 microgram of the soluble benzodiazepine chlordiazepoxide hydrochloride into the predominantly GABA-containing zona reticulata of the substantia nigra of amphetamine-pretreated rats induced rotational behaviour similar to that seen following unilateral elevation of nigral GABA levels and amphetamine treatment; this effect was not seen following injections into the vicinity of the predominantly dopamine-containing zona compacta. Chlordiazepoxide-induced rotations were abolished by the GABA-antagonist picrotoxin. Both chlordiazepoxide and GABA depressed production of cyclic 3',5'-guanosine monophosphate in samples of nigral tissue in vitro as estimated by radioimmunoassay. It is concluded that chlordiazepoxide may enhance GABA transmission within the substantia nigra, by some as yet unidentified mechanism, to create asymmetric activity in GABA-modulated neurones and hence induce rotation.


Monoamine oxidase in rat reticulocytes: subcellular localization and identification of isoenzymes. After "chemically induced reticulocytosis" in rats by treatment with acetyl-phenylhydrazide, monoamine oxidase (MAO) activities were determined in erythrocyte preparations of these animals. Studies on subcellular fractions obtained by differential centrifugation showed that the enzyme activity of rat reticulocytes is a classical mitochondrial MAO. The patterns of inhibition produced by clorgyline (A-type MAO), deprenil (B-type MAO) and pargyline or tranylcypromine (both types of MAO) in reticulocytes were determined in vitro using tryptamine as a substrate for both types of MAO and phenylethylamine as a substrate for the B-type. The results indicate that both A-type (approximately 75%) and B-type (approximately 25%) MAO are present in rat reticulocytes; while tryptamine was mainly deaminated by the A-type enzyme, both types of MAO were shown to contribute to the deamination of phenylethylamine. These findings were confirmed in investigations on the thermostabilities of the tryptamine and phenylethylamine deaminating activities of rat reticulocyte MAO.


Correlation between drug-induced supersensitivity of dopamine dependent striatal mechanisms and the increase in striatal content of the Ca2+ regulated protein activator of cAMP phosphodiesterase. Rats were injected daily with 1.3 mumol/kg of haloperidol s.c. for 10 days. From the second to the ninth day after haloperidol withdrawal the rats developed supersensitivity to the behavioral affects of apomorphine. Concomitantly, the Ka of dopamine for the activation of striatal adenylate cyclase was lowered and the striatal content of the Ca2+ dependent protein that activates cAMP phosphodiesterase was increased. This activator protein is stored in striatal membranes and can be released by membrane phosphorylation in cytosol. This protein increases the activity of the high Km phosphodiesterase (Uzunov et al., 1976) but when it is bound to striatal membranes it facilitates the activation of striatal adenylate cyclase by dopamine (Gnegy et al., 1976b). The increase in protein activator content of striatal membranes caused by haloperidol could be a primary factor in causing supersensitivity to the biochemical and behavioral effects of dopamine receptor agonists.


Effects of guanidine on synaptic transmission in the spinal cord of the frog. The effects of guanidine on motoneurons of the isolated frog spinal cord were studied by adding the drug to the solution bathing the cord during intracellular recording. Guanidine (5.10(-4) M) did not alter the membrane potential of motoneurons. The main effect was a marked increase of the amplitudes and frequencies of small spontaneously occurring inhibitory postsynaptic potentials. The hyperpolarizing component of postsynaptic potentials evoked by stimulation of dorsal roots was also enhanced by guanidine. Higher concentrations of guanidine (5.10(-3) M) resulted in a very large and irreversible increase of the small spontaneously occurring inhibitory potentials, which now appeared in a regular, rhythmic pattern. The effects of guanidine could easily be blocked by increasing the magnesium ions (15 mM) in the bath solution. These results indicate that guanidine facilitates the release of an inhibitory transmitter in afferent terminals of the frog spinal cord either by a direct action on these terminals or indirectly by an action on nerve endings impinging on inhibitory interneurons.


Disturbances in kidney functions and calcium and phosphate metabolism in cadmium-poisoned rats. In an attempt to elucidate the etiology and pathogenesis of 'Itai-Itai' disease, the relationship between bone and kidney lesions in cadmium-poisoned rats was determined using the standard renal clearance technique. Calcium and cadmium contents in the femur and kidney were also estimated. The severity of renal lesions was histologically evident both in the tubule and glomerulus, and paralleled the decrease in GFR and the increase in FF. There was no significant change in fractional excretion of phosphate, but fractional excretion of calcium increased in the cadmium-poisoned rats. The secondary hyperparathyroidism followed by uremic renal osteodystrophy did not result in an osteomalacia. Thus, it is apparent that disturbance in calcium reabsorption in the tubules plays a role in the loss of calcium, and cadmium associated with low levels of calcium has a direct effect on the bone-causing osteomalacia.


Influence of lysine vasopressin on the EEG in man. Preliminary results. The aim of the present study was to determine the influence of lysine vasopressin on the electroencephalogram in man. Our results suggest a dose-dependent activity of lysine vasopressin on the EEG, which might be similar to that observed in animal and man after administration of nicotine. This effect seems to be independent of the action of the corticotropic hormone simultaneously released.


Cell-mediated immunity to measles virus in multiple sclerosis: correlation with disability. The blastogenic transformation of lymphocytes on exposure to antigen was used to investigate cell-mediated immune responses to viruses implicated in the pathogenesis of multiple sclerosis. In 12 patients with "early" multiple sclerosis, lymphocyte reactivity to measles virus, parainfluenza virus, and vaccinia virus did not differ significantly from reactivity in a healthy control group. However, there was a significant inverse correlation between the lymphocyte blastogenic response to measles and vaccinia viruses and the predetermined degree of disability from multiple sclerosis. A deficient cellular response to measles virus, and possibly to other common viruses, is therefore probably a consequence of the disease itself, and not a causal factor.


[Value of electromyography for the detection of peripheral neuropathies in the course of chronic hepatopathies]. It has recently been noted that peripheral nerve fibres are involved in the course of chronic liver disease. The existence of hepatic polyneuropathy is still a matter of debate. A series of 29 subjects (not including diabetics or alcoholics) were examined clinically and electromyographically. Signs of distal lower extremity polyneuritis (paresthesia, hypoesthesia and osteotendinous hypo-areflexia) were noted in 31%. Latent neuropathy was observed in the form of slow maximum and minimum motor conduction rates in clinically unimpaired nerves. Electromyographical alterations were more evident in subjects with frankly chronic as opposed to barely chronic liver disease.


[Cephalic zoster with involvement of the 5th, 7th, 8th, 9th and 10th cranial nerves]. A case of cephalic zoster with involvement of the 5th, 7th, 8th, 9th and 10th left cranial nerves is described. The anatomopathological findings are surveyed. These show that lesions are often found in several areas of the nervous system. The pathogenesis of these forms is examined, with particular reference to the mechanism of involvement of several cranial nerves. It is felt that this is primarily due to reactivation of the virus in several ganglia.


Mercury levels in residents of a geothermal area in the Central North Island. As a result of previous high trace metal findings in the soil, cattle, vegetation and water of the Broadlands area of New Zealand a study was made of the levels of mercury, selenium, arsenic and zinc in a group of children from this region. Arsenic and zinc levels were found to be normal but significantly raised urinary spot mercury concentrations were found in some of the children when studied in December 1975 and 24 hour urinary mercury concentrations recorded six months later were also raised, but to a lesser degree. Samples of household tap water showed increased mercury concentrations (median 0.001 micromol/L). In agreement with other New Zealand studies the blood selenium concentrations were low (76.7 mu/L) and they did not vary in relation to urinary mercury levels. The children were also examined for signs of peripheral neuropathy but only minor abnormalities were found. The source of the elevated mercury is uncertain but it may come from drinking water or possibly from locally produced meat or vegetables.


Genital herpes and the FTA-ABS.

Genital herpes and the FTA-ABS. The fluorescent treponemal antibody-absorption test (FTA-ABS) was obtained on a series of 55 healthy men and women with documented recurrent genital herpes. In contrast to a previous report indicating that a significant number of these patients demonstrated a false positive FTA-ABS, the present series failed to confirm these findings. Of interest, however, was the high incidence of borderline FTA-ABS tests seen in both the control group and the group with herpes. The significance and use of the FTA-ABS are briefly discussed.


Origin of estrogen in isosexual precocious pseudopuberty due to a granulosa-theca cell tumor. The purpose of the present study was to quantify the rates of production and to define the mechanism(s) or origin of sex steroid hormones in a 3 1/2-year-old girl with isosexual precocious pseudopuberty caused by a granulosa-theca cell tumor of the ovary. The results suggest that the hyperestrogenism occurs principally due to the secretion of estradiol-17beta synthesized de novo by the tumor, but that the tumor in vivo was also capable of aromatizing C19 plasma precursor hormones to estrogen.


Basement membrane changes under neoplastic oral mucous membrane. Ultrastructural observations, review of the literature, and a unifying concept. This study explores the morphologic nature of the basement membrane under neoplastic oral mucous membrane. Of particular interest is the progression of changes associated with dysplasia, carcinoma in situ, and invasive carcinoma. On the basis of a critical review of the literature and our own ultrastructural observations, we present a unifying concept for the development of these basement membrane changes. This concept proposes that neoplastic epithelial cells may produce a collagenolytic enzyme which is released into the epithelial intercellular spaces. This enzyme permeates to the basement membrane, causes breaks in the basement membrane, and focal loss of stromal area contiguous with intercellular spaces. The neoplastic basal cells develop pseudopodia that eventually extend through the breaks in the basement membrane. This concept suggests that the basement membrane changes herald the progression of carcinoma in situ to invasive carcinoma.


In vitro comparison of sialographic agents. An experimental model is presented for in vitro comparative evaluation of radiopaque media. Experimental results parallel the clinical picture of fractionated filling films and comparative radiodensity of oil- and water-soluble media.


[Therapy of inoperable bronchial carcinoma]. The results of a prospective randomized study on carcinoma of the bronchus are reported. In a total of 56 patients polychemotherapy, high-dosage vitamin-A treatment, Endoxan and immunotherapy, as well as the application of proteolytic enzymes in altogether 5 combinations of therapy were tested.


[Action of calcitonin on osteoclasts in Paget's bone disease during long term treatment (author's transl)]. Comparison of biopsies from patients with Paget's bone disease before and during long-term treatment with calcitonin reveals that the ration of bone-adherent osteoclasts to free osteoclasts is not modified by the hormone. Nor does treatment alter the average number of nuclei in osteoclasts. Under electron microscopy, all the cytological anomalies of osteoclasts in Paget's bone disease and, in particular, the characteristic nuclear inclusions, persist through treatment. Thus, although such osteoclasts do react to calcitonin as demonstrated by several authors, the specific morphological anomalies remain unaffected by the treatment. It is likely that the osteoclast in Paget's bone disease is an abnormal cell with peculiarities which may be related to the yet unknown etiology of the disease.


Pituitary adenomas.

Pituitary adenomas. Pituitary adenomas arise from and consist of adenohypophyseal cells. Based on the tinctorial characteristics of the cell cytoplasm, they were divided previously into chromophobic, acidophilic, and basophilic adenoma types. This classification is only of limited value, since it fails to consider the endocrine function of the adenoma cells and the cell type from which the tumor originates. Advanced morphologic techniques, including electron microscopy and immunocytology, led to a new pituitary adenoma classification reflecting current knowledge and attributing greater significance to clinical features, structure-function relationships, and cytogenesis. The morphologic study of pituitary adenomas is still in a relatively early stage and much more work is required to understand the basic principles of pituitary cytopathology. Thus, the classification used in this review may change as new facts accumulate. We feel justified to say that attempts to correlate structural features of pituitary adenomas with secretory activity and their separation into distinct entities cannot be regarded as examples of curiosity-oriented research. This type of investigation represents not only an intellectually rewarding experience, but is also of practical value and provides important information for the endocrinologists.


Application of a live varicella vaccine in children with acute leukemia or other malignant diseases. A live varicella vaccine was used in 11 susceptible children in remission from acute leukemia, ten of whom had been in remission for six months or less, and in 6 children with neuroblastoma and retinoblastoma. In the immunological checkup before vaccination, most of them showed a positive reaction in the skin tests with dinitrochlorobenzene, phytohemagglutinin, purified protein derivative, and viral antigens. Leukopenia (three cases, less than 3,000/cu mm) and decreased IgG level (two cases, 380 mg/dl and 445 mg/dl) were observed in the children with leukemia. Anticancer medication was suspended from one week before vaccination to one week after vaccination. The only clinical reaction was a minute rash that appeared three weeks after vaccination in two children with leukemia and that disappeared within three days. Serological responses by complement fixing and neutralizing (NT) tests were detected in all the vaccinated children four weeks after vaccination, and NT antibody was still detected 28 months after vaccination in the two patients tested. Three of the vaccines were exposed to natural varicella at home and in the classroom 2 to 18 months after vaccination, but they were free from any varicella symptoms.


Clinical and serologic testing of a live varicella vaccine and two-year follow-up for immunity of the vaccinated children. A live varicella vaccine derived from the Oka strain was given on 181 children who had no history of varicella and were seronegative by complement fixation (CF) and neutralization (NT) tests; 125 children were hospitalized and 54 were receiving steroid therapy. Overall, seroconversion was achieved in 85.1% of the children by the CF test and in 97.8% by the NT test. Clinical reaction consisting of mild fever and rash appeared in only two children. One hundred seventy-nine of the vaccinated children were followed up by questionnaire and 51 were followed up serologically approximately two years later, at which time 10 of 51 (19.6%) were seropositive by the CF test and 50 of 51 (98.0%) by the NT test. Only one out of 80 children who had postvaccinal contact with varicella contracted mild varicella 16 months after vaccination. None of the vaccinees developed herpes zoster. These results suggest that this live varicella vaccine may safely and effectively be used for children with or without underlying diseases, including those receiving steroid therapy, and that immunity of at least two years' duration is conferred upon the vaccinated subjects.


Leigh's encephalomyelopathy in a patient with cytochrome c oxidase deficiency in muscle tissue. A patient is described with subacute necrotizing encephalomyelopathy proven by autopsy. A slight increase of blood pyruvate and lactate levels with an increased lactate/pyruvate ratio and frequently increased beta-hydroxybutyrate/acetoacetate ratio suggested a disorder of mitochondrial oxidation. A cytochrome c oxidase deficiency was shown in peripheral muscle tissue with some residual cytochrome c oxidase activity in heart muscle. Normal cytochrome c oxidase activity was present in liver tissue. Because of the markedly higher levels of pyruvate and lactate in CSF compared with blood and an increased lactate/pyruvate ratio in CSF, there may also have been defective activity of cytochrome c oxidase in brain tissue. After a period of apparently normal development, the child's clinical condition gradually deteriorated and she died at age 6 years due to respiratory insufficiency. This study illustrates the fact that Leigh's disease is not linked to a single inherited molecular defect.


Sodium pump stimulation by oxytocin and cyclic AMP in the isolated epithelium of the frog skin. Activity of the Na pump was judged by Na extrusion in epithelial cells loaded with Na by a previous incubation in K-free solutions in the cold. Oxytocin significantly stimulated Na extrusion either at normal (3.5 mM) or low (0.25 mM) K in the medium. It was stimulated as well by cyclic AMP. Maximal concentrations of either agent caused about the same degree of stimulation. Addition of ouabain or removal of K prevented the action of both agents, but amiloride showed no effect at all. These results strongly suggest that, a) neurohypophyseal hormones not only increase Na entry across the mucosal barrier of the epithelium but they also stimulate the serosal Na pump, b) cyclic AMP not only mediates the action of neurohypophyseal hormones on Na and water permeability of the mucosal barrier, but it also mediates the action of the hormones on the Na pump of the serosal barrier.


[Moschowitz's disease: efficacy of anti-platelet aggregation agents]. In November 1976, a 52 year old woman presented with a Moschowitz syndrome with clinical manifestations of continuous fever at 39 degrees and a transient Wernicke type aphasia. Laboratory findings included schizocytosis, a peripheral thrombocytopaenia and functional renal insufficiency. The ethanol tests was positive but there was no frank defibrination syndrome. After corticosteroid therapy failed, the patient was treated with Dipyridamole 400 mg/24 hours IV and acetylsalicylic acid 4 g/24 hours IV. Fever disappeared on the same day and the thrombocytopaenia was corrected in 48 hours. The patient was considered to be cured 15 days later. No precise aetiology to explain the Moschowitz syndrome was discovered apart from the recent ingestion of oestrogens. The authors emphasise the considerable progress which this use of a combination of Dipyridamole and aspirin represents, resulting in the cure of Moschowitz syndrome, a condition considered to be fatal up until a few years ago.


[The identification of hepatitis A virus in faeces. Diagnosis and epidemiological value (author's transl)]. During 1976, two hepatitis A epidemics in institutions enabled the authors to study the presence of the virus in the faeces during the course of the disease. After concentration of feacal extracts in polyethyleneglycol 6000, virions were observed by immune electron microscopy. Among 13 stool extracts examined, 11 proved positive. The two negative extracts were samples collected one month after the onset of clinical symptoms and signs. The samples richest in particles were those collected during the preicteric phase of the disease. In those cases in which samples were collected during jaundice, the viral concentration was lower. The limit of detection was a period of five days after the onset of jaundice. Thus, examination for hepatitis A virus in the faeces by electron microscopy may be used to obtain an aetiological diagnosis at the beginning of the disease. In one of the patients, a study of the kinetics of the appearance of antibodies was possible. From the time of onset of jaundice, the presence of specific antibodies was noted, reaching a maximum after two months. In the children's institution, no cases of hepatitis A were seen among those entrants who had received an injection of standard polyvalent gammaglobulin at the time of admission.


The use of nuclease P1 in sequence analysis of end group labeled RNA. A method is described for the direct sequence analysis of 20-25 nucleotides from the termini of 5'- or 3'-end-group [32P] labeled RNA. The method involves partial endonucleolytic digestion of the labeled RNA with nuclease P1 (from Penicillium citrinum) followed by separation of the partial digestion products by two-dimensional homochromatography, the nucleotide sequence being determined by mobility shift analysis. This procedure has been applied to the sequence analysis of the terminal regions of tRNAs and of high molecular weight RNA, such as messenger RNA or viral RNA. A further application involves its use in conjunction with snake venom phosphodiesterase to determine the sequence of 5'-end group labeled oligonucleotides, containing modified bases, derived from T1 or pancreatic RNase digestion of tRNA.


End labeling of enzymatically decapped mRNA. A method is presented for rapid and efficient 5' end labeling with 32P of capped mRNAs, by a series of three enzymatic reactions: the blocking nucleotide of the cap structure is removed by tobacco acid pyrophosphatase, and after dephosphorylation with alkaline phosphatase the 5' end is labeled with gamma-32-P-ATP and T4 polynucleotide kinase.


DNA-relaxing enzyme from Micrococcus luteus. A DNA-relaxing enzyme which catalyzes the conversion of superhelical DNA to a non-superhelical covalently closed form has been purified from Micrococcus luteus to near homogeneity by two chromatographic steps. The enzyme is a single polypeptide chain. As determined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and gel filtration on Sephadex G 150, the molecular weight is 115,000. The DNA-relaxing activity determined as a function of enzyme concentration follows a sigmoidal curve. The enzyme requires Mg++ for activity. In the presence of 4.5 mM Mg++ addition of 50-250 mM KCl yields incompletely relaxed DNA molecules (intermediates); intermediates are also observed in the absence of KCl, when the reaction is carried out at 0 degree C or at Mg++ concentrations exceeding 10 mM.


The sv40 transcription complex. I. Effect of viral chromatin proteins on endogenous RNA polymerase activity. SV40 chromatin obtained from infected monkey cells was used to study the effect of the viral chromatin proteins on endogenous RNA polymerase II. Ammonium sulfate activated the rate of transcription by endogenous RNA polymerase in two ways: 1) by direct action on the enzyme; and 2) by causing a reversible conformational change in the viral chromatin. Under optimal reaction conditions, the viral chromatin proteins did not limit the rate of RNA chain elongation, and high molecular weight RNA (1.6 X 10(6) d) was synthesized by the SV40 chromatin.


The sv40 transcription complex. II. Non-dissociation of protein from SV40 chromatin during transcription. A small fraction of the SV40 chromatin isolated from infected monkey cell cultures by the Triton method contains active RNA polymerase which had initiated transcription in vivo. This viral transcription complex (VTC) was utilized to answer the question of whether proteins dissociate from chromatin during transcription in vitro. 3H-RNA was synthesized by the VTC under conditions such that over half the label was in transcripts which were longer than half the length of the SV40 genome. Virtually all of the 3H-RNA remained associated with the SV40 chromatin, causing an increase in sedimentation rate from 55S to 78S. The density of the VTC-3H-RNA complex indicated that less than 5% of the original protein dissociated from the SV40 DNA which served as a template for transcription. We conclude that SV40 chromatin can be transcribed while the proteins remain associated with the DNA.


[Some aspects of protein methylation].

[Some aspects of protein methylation]. The paper gives a review of the literature publications on protein methylation and probable donors of methyl groups. It presents experimental data demonstrating the capability of 14CH3-B12 to methylate in vitro proteins of tRNA methylases from Zajdela ascite hepatoma and rat liver as well as commercial RNase and albumin preparations.


[Biological method of quantitating vitamin D in different preparations and diets]. A biological method for quantitative assay of vitamin D3 in various preparations has been developed. The method is based on a close correlation between the vitamin D3 dose and the content of calcium-binding protein in the duodenal mucosa of rachitic chickens 72 hrs after vitamin D3 administration. Calcium-binding protein is assayed by radial immunodiffusion in agar-agar. The calibration curve of the dose-protein dependence allows measurements of I to 500 IU of vitamin D3 in the sample tested. The method shows good accuracy and reproducibility. The paper presents the results of quantitation of vitamin D in various preparations.


[Effect of ACTH on protein phosphorylation in rat adrenal ribosomes]. The authors studied the influence of ACTH on the steroidogenesis and 32P-labeled KH2PO4 incorporation into the ribosome proteins under conditions of incubation of the rat adrenal glands in vitro. 30 minutes before the incubation ACTH added into the medium stimulated corticosteroid biosynthesis by 116 +/- 26% and increased the radioactive phosphorus incorporation into the sum total ribosome proteins by 38 +/- 7%. Ribosome proteins were separated by electrophoresis in polyacrylamide gel at pH 4.5 into 30 fractions. 5--6 protein fractions proved to be phosphorylated during the incubation. ACTH altered the phosphorylation of individual protein fractions differently: there was stimulation in 2 fractions and inhibition of phosphorylation--in 3. It was revealed that, along with steroidogenesis, ACTH altered the phosphorylation of ribosome proteins. The change of the rate of ribosome phosphorylation was possibly one of the mechanisms with the aid of which the steroidogenic action of ACTH is realized.


Nephrogenous adenosine 3'5' monophosphate as an index of parathyroid function in chronic renal disease. The purpose of the study was the evaluation of nephrogenous adenosine 3'5' monophosphate (ncAMP) determinations in the diagnosis of hyperparathyroidism (HPT) in chronic renal disease (CRD). The ratio of cAMP clearance to creatinine clearance (CcAMP/Ccr) was applied as the index of the participation of ncAMP in 24 hr urinary cAMP excretion. In seventy CRD patients, the mean CcAMP/Ccr value was higher than in healthy individuals. A good correlation between plasma iPTH and CcAMP/Ccr ratio was noted. In patients with a ratio higher than 2.5, the signs of HPT were found. The determination of CcAMP/Ccr ratio in CRD may be of value in the assessment of parathyroid hyperactivity.


Structure of cytochrome c555 of Chlorobium thiosulfatophilum: primitive low-potential cytochrome c. Cytochrome c555 is an 86-residue type c cytochrome derived from Chlorobium thiosulfatophilum, an obligately anaerobic green sulfur bacterium which is among the most primitive of living organisms. Here is presented a preliminary structural description of the cytochrome c555 molecule based on its crystallographic structure determination at 2.7-A resolution by multiple isomorphous replacement methods. This structure is of interest not only because of its evolutionary significance but also because the cytochrome c555 molecule possesses an unusually low physiologic oxidoreduction potential (Em,7 = +145 mV) compared with related members of the cytochrome c family. Consequently, determination of its structure may permit a direct assessment of the structural factors responsible for prosthetic group redox potential regulation in type c cytochromes.


Characterization of a common precursor to corticotropin and beta-lipotropin: identification of beta-lipotropin peptides and their arrangement relative to corticotropin in the precursor synthesized in a cell-free system. Radioactive proteins synthesized in an mRNA-dependent reticulocyte cell-free system under the direction of mRNA from AtT-20/D-16v mouse cells were isolated by specific immunoprecipitation using antiserum to either alpha(1-24) corticotropin or beta-endorphin [beta(61-91) lipotropin]. Each immunoprecipitate was fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and shown to contain only one labeled protein with an apparent molecular weight of 28,500. Tryptic peptide analysis of the Mr 28,500 corticotropin and beta-lipotropin molecules isolated from the gels demonstrated that the two proteins had the same lysine, methionine, and tryptophan peptides. Four tryptic peptides from the cell-free product exhibited the same electrophoretic and chromatographic mobilities as marker tryptic peptides from bovine beta-melanotropin and porcine beta-endorphin. The identification of these peptides was confirmed by amino acid composition studies with a variety of labeled amino acids. The beta-lipotropin tryptic peptides were also shown to be located carboxy terminal to the corticotropin tryptic peptides.


2'-Deoxy-2'-azidocytidine inhibits the initiation of polyoma DNA synthesis. 2'-Deoxy-2'-azidocytidine, a nucleoside analogue, inhibits polyoma DNA synthesis. The inhibition results in a 5.5-fold decrease in the amount of replicating intermediates, as detected by electron microscopy. The results indicate that replication is inhibited at an early step, possibly in the initiation of new rounds of replication. In contrast, the effect on the elongation of DNA chains appears to be less pronounced. As a result of the inhibition, polyoma DNA molecules at an early stage of replication accumulate in the nuclei. Upon incubation of such nuclei, DNA synthesis is induced in these molecules. The replication starts from the normal origin and elongation proceeds bidirectionally at a rate close to that observed during in vivo conditions. Analysis of polyoma DNA replication in nuclei isolated from control cells indicates that both initiation and termination of replication are impaired under in vitro conditions.


5'-Terminal sequences and coding region of late simian virus 40 mRNAs are derived from noncontiguous segments of the viral genome. The region of the simian virus 40 genome complementary to the 5' end of the most abundant poly(A)-containing 19S and 16S mRNAs was mapped by hybridization of double-labeled RNA ([3H]methyl group and [14C]uridine) to specific DNA fragments. Chemical identification of methylated residues indicated that a common "leader" sequence adjacent to the 5' terminus of both 19S and 16S mRNA is transcribed from DNA sequences located between 0.67 and 0.76 map units. The estimated size of this "leader" RNA, which does not code for any known viral protein, is 170-200 nucleotides. Our results indicate that sequences complementary to the "leader" region and coding portion of 16S mRNA are located in separate parts of the simian virus 40 genome.


Renaturation of complementary single-stranded DNA circles: complete rewinding facilitated by the DNA untwisting enzyme. Renaturation of two complementary single-stranded circles should be limited by topological constraints against the rewinding of the DNA helix. If a mixture of complementary single-stranded rings is annealed and then treated with the DNA untwisting enzyme, the DNA circles completely renature as judged by (i) the presence of interlocked rings that sediment at 53 S in alkali, (ii) the buoyant density of the renatured DNA in CsCl gradients containing ethidium bromide, and (iii) the resistance of the product to the single-strand-specific S1 nuclease. Therefore, the DNA untwisting enzyme is able to provide a transient single-strand break that is sufficient to allow the two strands to completely rewind. The possibility that the untwisting enzyme might facilitate the initiation of the process of genetic recombination is discussed.


The 5'-terminal structures of poliovirion RNA and poliovirus mRNA differ only in the genome-linked protein VPg. The 5'-terminal, RNase T1-resistant oligonucleotide of poliovirus mRNA has been isolated. Its sequence is pU-U-A-A-A-A-C-A-Gp, which is identical to that of virion RNA except that the genome-linked protein VPg is absent [Nomoto, A., Detjen, B., Pozzatti, R. & Wimmer, E. (1977) Nature 268, 208-213]. Because all newly synthesized viral RNAs are VPg-linked, we propose that VPg is cleaved from progeny RNA at the linkage between protein and nucleic acid prior to polyribosome formation. This may represent a new mode of processing of viral macromolecules. Virion RNA from which VPg has been cleaved proteolytically retains its specific infectivity, an observation suggesting that VPg is not involved in early steps (penetration and translation) of the infectious cycle initiated by RNA.


Asymmetric binding of the inhibitor di(adenosine-5') pentaphosphate (Ap5A) to adenylate kinase. The effect of binding diadenosine pentaphosphate (Ap(5)A) to adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3) has been investigated by (31)P nuclear magnetic resonance. The symmetric molecule, Ap(5)A, is a potent inhibitor of the adenylate kinase reaction, 2 ADP right arrow over left arrow ATP + AMP. Free Ap(5)A has two groups of signals in its (31)P nuclear magnetic resonance spectrum centered at 11.1 and 22.8 parts/million (ppm) upfield from 85% H(3)PO(4) that are assigned to the end (1-P and 5-P) and middle (2-, 3-, and 4-P) phosphates, respectively. Addition of Mg(2+) shifts the centers of these resonances to 11.7 and 22.3 ppm. The spectrum of Ap(5)A bound to porcine adenylate kinase shows five groups of signals centered at 10.9, 11.9, 20.5, 22.7, and 24.0 ppm; the resonances at 11.1 ppm (1-P and 5-P) and at 22.8 ppm (2-P and 4-P) are now clearly split, indicating asymmetric binding of Ap(5)A to the enzyme. The asymmetry is strikingly enhanced in enzyme-bound MgAp(5)A, which has resonances at 10.5, 12.5, 18.6, 22.7, and 25.6 ppm. By the addition of Mn(2+) to the enzyme.MgAp(5)A complex, the observed signals in increasing order of shifts were tentatively assigned to 1-P, 5-P, 4-P, 3-P, and 2-P, where the 3-, 4-, and 5-P positions correspond to the ATP-binding site on the enzyme. The asymmetry introduced in the phosphate chain of enzyme.MgAp(5)A is indicated by the (31)P chemical shift of 7 ppm between 2- and 4-P, which is one of the largest thus far observed for phosphate substrates bound noncovalently to enzymes.


Guanosinetriphosphatase activity of tubulin associated with microtubule assembly. Tubulin, purified by cycles of assembly followed by phosphocellulose chromatography, exhibits a characteristic GTPase activity that is polymerization dependent and can be attributed to the tubulin itself. This activity has been observed, in a standard reassembly buffer containing low Mg2+, under three conditions that induce microtubule assembly: in the presence of microtubule-associated proteins, in the presence of DEAE-dextran, or after addition of high Mg2+ and glycerol. The phosphocellulose-purified tubulin showed no GTPase activity under the following nonpolymerizing conditions: in buffer with low Mg2+ in the absence of microtubule-associated proteins or DEAE-dextran, in buffer with high Mg2+ and glycerol at tubulin concentrations below the critical concentration, or when microtubule assembly was inhibited by vinblastine. Colchicine, on the other hand, while blocking microtubule assembly, induced a significant GTPase activity in the phosphocellulose-purified tubulin. During the process of assembly, GTP appears to be hydrolyzed as a free tubulin dimer polymerizes into a microtubule. A constant GTPase activity when polymerization equilibrium is reached apparently reflects the cyclic polymerization-depolymerization of tubulin dimers at the ends of the microtubules.


Deuterium magnetic resonance studies of the interaction of lipids with membrane proteins. The deuterium magnetic resonance spectra of lipid-protein particles containing cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) isolated from beef heart mitochondria and the specifically deuterated lipid 1-(16,16,16-trideuteropalmitoyl)-2-palmitoleoyl phosphatidylcholine are presented. These reconstituted particles are of uniform lipid and protein content; however, the spectra clearly show two environments characterized by distinctly different residual quadrupolar splittings or order parameters. The less-ordered environment shows a splitting similar to but slightly less than that of the pure lipid alone at a given temperature. The more restricted environment appears to be induced by the presence of the protein. The amount of the restricted lipid is clearly temperature dependent with a 2- to 3-fold decrease in relative amount from 2 to 22 degrees. The rate of exchange of lipid between the free and restricted environments is slower than 10(3)/sec. The significance of these phenomena is discussed.


Transfer of ADP-ribose from NAD to choleragen: a subunit acts as catalyst and acceptor protein. Choleragen selectively incorporates 3H from [3H]NAD labeled on the adenosine moiety and not 14C from [14C]NAD labeled on the nicotinamide moiety. This reaction does not require protein in addition to choleragen. Incorporation of isotope does not proceed at 4 degrees, requires dithiothreitol, is stable after extensive washing with cold trichloroacetic acid, and is decreased 80% by boiling in trichloroacetic acid. Studies with the A and B subunits of choleragen show that the A subunit catalyzes ADP-ribosylation and serves as an acceptor protein. The B subunit does not show catalytic or acceptor activity. We conclude that choleragen and its A subunit catalyze the hydrolysis of NAD and the enzymatic transfer of ADP-ribose to the A subunit.


Characterization of early simian virus 40 transcriptional complexes: late transcription in the absence of detectable DNA replication. Isolation of early viral transcriptional complexes and incorporation in vitro of radiolabeled precursors into nascent RNA has permitted an analysis of early simian virus 40 (SV40) transcription. Under conditions such that viral DNA replication was undetectable, both early and late SV40 RNA were synthesized. This finding provides evidence that viral DNA replication is not an absolute requirement for late transcription and supports earlier observations that late viral RNA is synthesized in SV40-infected nonpermissive mouse cells. The majority of the early viral transcriptional activity can be solubilized, indicating that a substantial portion of this RNA is transcribed from free rather than integrated templates. Sedimentation analysis of the transcriptional complexes resulted in the detection of two separate peaks of activity, suggesting the possibility of two distinct types of early SV40 templates.


Stimulation of sugar uptake and thymidine incorporation in mouse 3T3 cells by calcium phosphate and other extracellular particles. Evidence is presented that the marked stimulation of sugar uptake and thymidine incorporation by addition of extra Ca2+ to stationary phase mouse 3T3 cells in culture is phosphate dependent and due to the action of the calcium phosphate precipitate formed in the medium. The cells are similarly stimulated by a variety of particulate materials, including calcium pyrophosphate, barium sulfate, kaolin, and polystrene beads. The precipitate effects on sugar uptake are of the same magnitude as those seen with certain hormones (insulin, epidermal growth factor) or with fresh 10% calf serum. The effect of barium sulfate on thymidine incorporation is also of the same magnitude as seen with these hormones, but much less than half that found with fresh calf serum. The stimulation by barium sulfate or hormones of thymidine incorporation is not phosphate dependent.


Feline oncornavirus-associated cell membrane antigen: evidence for an immunologically crossreactive feline sarcoma virus-coded protein. The feline oncornavirus-associated cell membrane antigen (FOCMA) acts as a target for natural immuno-surveillance against tumor development in the cat. In the present study, mink and rat cells nonproductively transformed by feline sarcoma virus (FeSV) were shown to express FOCMA as well as 5'-terminal feline leukemia virus (FeLV) gag gene proteins, p15 and p12. In contrast, such cells lack detectable levels of other FeLV gag gene-coded proteins or the env gene product, gp70. FOCMA, p15, and p12 antigen expression is initially in the form of an 80,000-100,000 molecular weight precursor which, upon post-translational cleavage, gives rise to a 65,000 molecular weight component that contains FOCMA and a 25,000 molecular weight component containing p15 and p12. Feline lymphoma cells, including those from several tumors that lacked detectable levels of FeLV structural protein expression, were shown to be FOCMA-positive. These findings strongly suggest that FOCMA represents an FeSV-coded transformation specific protein and provide preliminary information regarding the position within the FeSV genome coding for its synthesis.


Lymphospecific toxicity in adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency: possible role of nucleoside kinase(s). Inherited deficiencies of the enzymes adenosine deaminase (adenosine aminohydrolase; EC 3.5.4.4) and purine nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase; EC 2.4.2.1) preferentially interfere with lymphocyte development while sparing most other organ systems. Previous experiments have shown that through the action of specific kinases, nucleosides can be "trapped" intracellularly in the form of 5'-phosphates. We therefore measured the ability of newborn human tissues to phosphorylate adenosine and deoxyadenosine, the substrate of adenosine deaminase, and also inosine, deoxyinosine, guanosine, and deoxyguanosine, the substrates of purine nucleoside phosphorylase. Substantial activities of adenosine kinase were found in all tissues studied, while guanosine and inosine kinases were detected in none. However, the ability to phosphorylate deoxyadenosine, deoxyinosine, and deoxyguanosine was largely confined to lymphocytes. Adenosine deaminase, but not purine nucleoside phosphorylase, showed a similar lymphoid predominance. Other experiments showed that deoxyadenosine, deoxyinosine, and deoxyguanosine were toxic to human lymphoid cells. The toxicity of deoxyadenosine was reversed by the addition of deoxycytidine, but not uridine, to the culture medium. Based upon these and other experiments, we propose that in adenosine deaminase and purine nucleoside phosphorylase deficiency, toxic deoxyribonucleosides produced by many tissues are selectively trapped in lymphocytes by phosphorylating enzyme(s).


Selectivity of action of an antiherpetic agent, 9-(2-hydroxyethoxymethyl) guanine. A guanine derivative with an acyclic side chain, 2-hydroxyethoxymethyl, at position 9 has potent antiviral activity [dose for 50% inhibition (ED(50)) = 0.1 muM] against herpes simplex virus type 1. This acyclic nucleoside analog, termed acycloguanosine, is converted to a monophosphate by a virus-specified pyrimidine deoxynucleoside (thymidine) kinase and is subsequently converted to acycloguanosine di- and triphosphates. In the uninfected host cell (Vero) these phosphorylations of acycloguanosine occur to a very limited extent. Acycloguanosine triphosphate inhibits herpes simplex virus DNA polymerase (DNA nucleotidyltransferase) 10-30 times more effectively than cellular (HeLa S3) DNA polymerase. These factors contribute to the drug's selectivity; inhibition of growth of the host cell requires a 3000-fold greater concentration of drug than does the inhibition of viral multiplication. There is, moreover, the strong possibility of chain termination of the viral DNA by incorporation of acycloguanosine. The identity of the kinase that phosphorylates acycloguanosine was determined after separation of the cellular and virus-specified thymidine kinase activities by affinity chromatography, by reversal studies with thymidine, and by the lack of monophosphate formation in a temperature-sensitive, thymidine kinase-deficient mutant of the KOS strain of herpes simplex virus type 1 (tsA1).


Interspecies radioimmunoassay for the major structural proteins of primate type-D retroviruses. A competition radioimmunoassay has been developed in which type-D retroviruses from three primate species compete. The assay utilizes the major structural protein (36,000 daltons) of the endogenous squirrel monkey retrovirus and antisera directed against the major structural protein (27,000 daltons) of the Mason-Pfizer monkey virus isolated from rhesus monkeys. Purified preparations of both viruses grown in heterologous cells, as well as extracts of heterologous cells infected with squirrel monkey retrovirus or Mason-Pfizer monkey virus, compete completely in the assay. Addition of an endogenous virus of the langur monkey also results in complete blocking. No blocking in the assay is observed with type-C baboon viruses, woolly monkey virus, and gibbon virus. Various other type-C and type-B viruses also showed no reactivity. An interspecies assay has thus been developed that recognizes the type-D retroviruses from both Old World monkey (rhesus and langur) and New World monkey (squirrel) species.


Opiate receptor: cooperativity of binding observed in brain slices. Kinetic constants for binding of 3H-labeled morphine and naloxone were determined from Hill plot analyses and from experiments in which the concentration of tritiated drug was constant and that of nonlabeled drug varied. With brain slices, the binding of either drug exhibited strong positive cooperativity (Hill slope greater than 3); this was not observed in brain homogenates. Thus, in slices the relationship between opiate binding site and ligand may be more relevant physiologically and pharmacologically.