The immediate-early growth response in regenerating liver and insulin-stimulated H-35 cells: comparison with serum-stimulated 3T3 cells and identification of 41 novel immediate-early genes.

Liver regeneration provides a unique system for analysis of mitogenesis in intact, fully developed animals. Cellular immediate-early genes likely play an important role in cell cycle regulation and have been extensively studied in mitogen-stimulated fibroblasts lymphocytes but not in liver. We have begun to characterize the immediate-early growth response genes of mitogen-stimulated liver cells, specifically, regenerating liver and insulin-stimulated Reuber H-35 hepatoma cells, and to address differences in growth response between different cell types. Through subtraction and differential screening of cDNA libraries from regenerating liver and insulin-treated H-35 cells, we have extensively characterized 341 differentially expressed clones and identified 52 immediate-early genes. These genes have been partially sequenced and subjected to Northern (RNA) blot analysis, and 41 appear to be novel. Surprisingly, two-thirds of these genes are also expressed in BALB/c 3T3 cells, but only 10 were identified in previous studies of 3T3 cells, and of these, 6 include well-known genes like jun and fos, and only 4 are novel. Approximately one-third of the immediate-early genes identified in mitogen-stimulated liver cells or serum-stimulated NIH 3T3 cells are expressed in a tissue-specific fashion, indicating that cell type-specific regulation of the proliferative response occurs during the immediate-early period. Our findings indicate that the immediate-early response is unusually complex for the first step in a regulatory cascade, suggesting that multiple pathways must be activated. The abundance of immediate-early genes and the highly varied pattern of their expression in different cell types suggest that the tissue specificity of the proliferative response arises from the particular set of these genes expressed in a given tissue.     

Quantification of Clostridium botulinum type A toxin and organisms in the feces of a case of infant botulism and examination of other related specimens

Bacteriological examinations were performed on the first case of infant botulism in Japan (an infant boy aged 79 days at onset of illness). Clostridium botulinum type A toxin and organisms were detected continually in the stools of the infant for at least 31 days and 39 days, respectively. The highest levels of the toxin and of the population of the organisms, 7.8 X 10(4) LD50/g and 1.3 X 10(6) colony forming units (cfu)/g, were detected in the stool specimen taken on the 20th day of illness. Type A organisms were detected also in the honey fed to the infant before onset of illness, teats of his feeding bottle, soil specimens taken at the house entry and the vacuum-cleaner dust. Fecal excretion of the toxin and organisms was no longer detected from the 68th day of illness and he recovered.     

Amino acid substitutions in mitochondrial ATPase subunit 9 of Saccharomyces cerevisiae leading to oligomycin or venturicidin resistance

A series of isonuclear oligomycin-resistant mutants of Saccharomyces cerevisiae carrying mutations in the mitochondrial oli1 gene has been studied. DNA sequence analysis of this gene has been used to define the amino acid substitutions in subunit 9 of the mitochondrial ATPase complex. A domain of amino acids involved in oligomycin resistance can be recognized which encompasses residues in each of the two hydrophobic portions of the subunit 9 polypeptide that are thought to span the inner mitochondrial membrane. Certain amino acid substitutions also confer cross-resistance to venturicidin: these residues define an inner domain for venturicidin resistance. The expression of venturicidin resistance resulting from one particular substitution is modulated by nuclear genetic factors.     

Examination of protein sequence homologies: III. Ribosomal protein YS25 fromSaccharomyces cerevisiae and its counterparts fromSchizosaccharomyces pombe,rat liver,andEscherichia coli

Summary The sequences of the ribosomal proteins YS25, SP-S28, RL-S21, and Ec-S6, fromSaccharomyces cerevisiae, Schizosaccharomyces pombe, rat liver, andEscherichia coli, respectively, have been examined using a computer program that searches for homologous tertiary structures. Matrices of comparisons among the eukaryotic sequences show that they match each other sequentially without any internal gaps. The average values of the correlation coefficients obtained from the comparison matrices are higher for the first halves of the sequences than for the latter halves. This result suggests that the first halves of the sequences may represent a more important domain than the latter halves. The comparison matrices between the eukaryotic and bacterial sequences of ribosomal proteins, however, do not show sequentially arranged homology, though there are six well-matching segments arranged in different orders in the two types of sequences. This implies that the eukaryotic sequences of the ribosomal protein were reconstituted by two internal transpositions and six deletions of 4–12 residues each from the ancestral sequence during the divergence between bacterial and eukaryotic genes. These findings may give insight into structural and quantitative studies of evolutionary divergence between eukaryotes and prokaryotes.     

The bacterial flora of the intestine of Ascaris suum and 5-hydroxytryptamine production

Representative facultative anaerobes of the bacterial flora from the intestine of female Ascaris suum were isolated and identified. The number of bacteria in the intestine was approximately 4 X 10(9) per g wet weight of intestine. Seventeen of 19 of the isolated colonies were found to secrete 5-hydroxytryptamine in culture. Holding A. suum in an antibiotic-containing medium did not affect the levels of 5-hydroxytryptamine in the worm, which were 231 +/- 14 ng/g in antibiotic-media as compared to 250 +/- 16 ng/g in control media. This implied that the bacteria may not be contributing to the level of 5-hydroxytryptamine in the tissues of A. suum.     

Mitotic inhibition and aneuploidy induction by naturally occurring and synthetic estrogens in Chinese hamster cells in vitro

We used a predominantly diploid Chinese hamster cell line to test a number of naturally occurring and synthetic estrogens for their ability to arrest cells at metaphase, their potential for allowing anaphase recovery, and their capability of inducing aneuploid progeny. The chemicals employed included diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol and estriol. We also tested progesterone, estrone and testosterone in this regard. Only estrogens and their synthetic analogs caused mitotic arrest and aneuploidy, while progesterone, estrone and testosterone did not cause mitotic disturbances. Among the estrogens, DES was the most effective arrestant on a comparative molar basis, whereas dienestrol was most potent over a wide range of concentrations. Estriol was the least potent as an arrestant but was an effective inducer of aneuploidy. The addition of a metabolic activator (S9) did not alter the ability of DES to arrest mitosis. Following the removal of the drugs, cells were able to quickly reorganize a spindle apparatus and enter anaphase. Diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol and estriol caused significant increase in aneuploidy within a narrow range of high concentrations in recovering cell populations. Aneuploidy was induced in a non-random manner. Immunofluorescence studies with anti-tubulin antibody indicate that estrogens may have a mechanism of mitotic arrest similar to that of colchicine and colcemid, viz inhibiting the polymerization of tubulin to form microtubules. These data suggest that the interaction between estrogens and microtubules may mediate the induction of aneuploidy in somatic cells. Aneuploidy induction by DES and similar compounds may be related to their carcinogenic potential.     

Histopathological evidence for protective immunity induced by sonicated Salmonella vaccine

The highly susceptible inbred C3H/HeNMTV mice were vaccinated with fragments derived from sonicated Salmonella typhimurium and then infected with the pathogen. All of the vaccinated mice survived an otherwise rapidly fatal challenge of 10(5) organisms, i.e., greater than 10(3) x mean lethal dose (LD50). The vaccine also protected two-thirds of the mice infected with 10(6) bacteria and extended the survival time of the remainder in their fatal disease. Histopathological findings showed that, like the control mice, the vaccinated and infected mice developed abscesses with infiltration of polymorphonuclear leukocytes in the organs of the reticuloendothelial system during the early stage of the infection. However, unlike the primary lesions in the control mice, the lesions of the vaccinated mice tended to be discrete and self-limiting. They began to transform into granulomas after the first week of infection. Recovery and regeneration of tissues were evident 3 weeks after the infection.     

Anti-kinetochore antibodies: use as probes for inactive centromeres.

Application of a modified immunofluorescence technique using an anti-kinetochore serum enables cytogeneticists to obtain quality metaphase spreads and to localize kinetochores. In a patient with a 45, XX, -9, -11, tdic (9p;11p) constitution, we found that the dicentric marker chromosome has an intensely fluorescent kinetochore (no. 11), the functional centromere, and a less intensely fluorescent kinetochore (no. 9), the inactive centromere. The data suggest that in the process of tandem fusion (telomere-telomere between 11p and 9p), the centromere of chromosome 9 was not deleted, but, rather, inactivated.     

Purification and characterization of human lipoprotein lipase and hepatic triglyceride lipase. Reactivity with monoclonal antibodies to hepatic triglyceride lipase

Human lipoprotein lipase and hepatic triglyceride lipase were purified to homogeneity from post-heparin plasma. These enzymes were purified 250,000- and 100,000-fold with yields of 27 +/- 15 and 19 +/- 6%, respectively. Molecular weight determination by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents yielded Mr of 60,500 +/- 1,800 and 65,200 +/- 400, respectively, for lipoprotein lipase and hepatic triglyceride lipase. These lipase preparations were shown to be free of detectable antithrombin by measuring its activity and by probing of Western blots of lipases with a monospecific antibody against antithrombin. In additions, probing of Western blots with concanavalin A revealed no glycoproteins corresponding to the molecular weight of antithrombin. Four stable hybridoma-producing distinct monoclonal antibodies (mAb) to hepatic triglyceride lipase were isolated. The specificity of one mAb, HL3-5, was established by its ability to immunoprecipitate hepatic triglyceride lipase catalytic activity. Interaction of HL3-5 with this lipase did not inhibit catalytic activity. The three other mAb interacted with hepatic triglyceride lipase only after denaturation of the enzyme with detergents. The relatedness of these two enzymes was examined by comparing under the same conditions the thermal inactivation, the sensitivity to sulfhydryl and reducing agents, amino acid composition, and the mobility of peptide fragments generated by cyanogen bromide cleavage. The results of these studies strongly support the view that the two enzymes are different proteins. Immunological studies confirm this conclusion. Four mAb to hepatic triglyceride lipase did not interact with lipoprotein lipase in Western blots, enzyme-linked immunosorbent assay, and immunoprecipitation experiments. These immunological studies demonstrate that several epitopes of the hepatic triglyceride lipase protein moiety are not present in the lipoprotein lipase molecule.