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Null mutations in three genes encoding cyclin-like proteins (CLN1, CLN2, and CLN3) in Saccharomyces cerevisiae cause cell cycle arrest in G1 (cln arrest). In cln1 cln2 cln3 strains bearing plasmids containing the CLN3 (also called WHI1 or DAF1) coding sequence under the transcriptional control of a galactose-regulated promoter, shift from galactose to glucose medium (shutting off synthesis of CLN3 mRNA) allowed completion of cell cycles in progress but caused arrest in the ensuing unbudded G1 phase. Cell growth was not inhibited in arrested cells. Cell division occurred in glucose medium even if cells were arrested in S phase during the initial 2 h of glucose treatment, suggesting that CLN function may not be required in the cell cycle after S phase. However, when the coding sequence of the hyperactive C-terminal truncation allele CLN3-2 (formerly DAF1-1) was placed under GAL control, cells went through multiple cycles before arresting after a shift from galactose to glucose. These results suggest that the C terminus of the wild-type protein confers functional instability. cln-arrested cells are mating competent. However, cln arrest is distinct from constitutive activation of the mating-factor signalling pathway because cln-arrested cells were dependent on the addition of pheromone both for mating and for induction of an alpha-factor-induced transcript, FUS1, and because MATa/MAT alpha (pheromone-nonresponsive) strains were capable of cln arrest in G1 (although a residual capacity for cell division before arrest was observed in MATa/MAT alpha strains). These results are consistent with a specific CLN requirement for START transit. Show less

The ABA-induced MA12 cDNA from maize, which encodes a set of highly phosphorylated embryo proteins, was used to isolate the corresponding genomic clone. This gene, called RAB-17 (responsive to ABA), encodes a basic, glycine-rich protein (mol. wt. 17,164) containing a cluster of 8 serine residues, seven of them contiguous. It is a homologue of the rice RAB-21 gene (Mundy J, Chua NH, EMBO J 7; 2279-2286, 1988). Phosphoamino acid analysis of the isolated protein indicates that only the serine residues are phosphorylated and a putative casein-type kinase phosphorylatable sequence was identified in the protein. The pattern of expression and in vivo phosphorylation of the RAB-17 protein was studied during maize embryo germination and in calli of both meristematic or embryonic origin. ABA treatment induced the synthesis of RAB-17 mRNA and protein in calli, however, the RAB-17 proteins were found to be highly phosphorylated only in embryos. Show less

We studied expression of laminin, fibronectin, and Type IV collagen in the testis by means of immunofluorescence and immunoblot analysis and also examined gene expression of fibronectin using the ribonuclease protection assay. By immunofluorescence on sections from 20-day-old rats, laminin, fibronectin, and Type IV collagen were found in the basement membrane of the seminiferous tubules and in the interstitial regions of the testis. No localization of any extracellular matrix components was found inside the sectioned cells. However, when Sertoli cells were cultured on glass coverslips, laminin and Type IV collagen were both found inside the cells, suggesting new synthesis. In cultured peritubular cells, Type IV collagen, laminin, and fibronectin were found within the cells. When examined by immunoblot analysis, freshly isolated Sertoli and peritubular cells from 20-day-old rats did not demonstrate production of laminin or fibronectin. After 5 days in culture, peritubular cells produced both laminin and fibronectin, whereas cultured Sertoli cells produced only laminin. In contrast, freshly isolated and cultured Sertoli and peritubular cells all produced Type IV collagen. Moreover, the ribonuclease protection assay indicated that the bulk of fibronectin gene expression occurs within the first 10 days of postnatal development, with lower maintenance levels occurring thereafter. These results indicate that in the testis the highest levels of expression of laminin and fibronectin occur during development and in primary cell culture, whereas expression of Type IV collagen is higher at later stages. Show less

Limited proteolysis of carboxypeptidase A from bovine pancreas with subtilisin Carlsberg generates a stable intermediate, carboxypeptidase S, whose esterase and peptidase activities are increased and decreased, respectively, under standard assay conditions. Carboxypeptidase S was isolated by affinity chromatography. Sequence analysis shows that it is cleaved solely at the Ala154-Gly155 bond. Its enzymatic properties were determined under stopped-flow conditions with Dns-Gly-Ala-Phe and its ester analogue Dns-Gly-Ala-OPhe. For both substrates, the Km values are increased 30-40-fold. The kcat value for peptide hydrolysis is virtually unaffected whereas that for ester hydrolysis is increased 10-fold. The magnitude of the Km effect is equivalent to a loss of 9 kJ/mol of binding energy and likely reflects a disruption of the network of hydrogen bonds that links Tyr-248 and Arg-145 to the backbone carbonyls of Ala-154 and Gly-155. The difference in kcat effects for the two substrate classes is related to differences in the chemical nature of the rate-determining step. Product release is rate determining for catalytic hydrolysis of ester substrates, and hence, the increase in kcat indicates that dissociation of products is facilitated as a result of the Ala154-Gly155 bond scission. The changes in enzymatic activity accompanying limited proteolysis are due to conformational alterations in the vicinity of the active center of the molecule. The affinity of a monoclonal antibody, mAb 100, directed toward the antigenic determinant located between residues 209 and 218 in carboxypeptidase A is diminished considerably for carboxypeptidase S.(ABSTRACT TRUNCATED AT 250 WORDS) Show less

The RPC31 gene encoding the C31 subunit of Saccharomyces cerevisiae RNA polymerase C (III) has been isolated, starting from a C-terminal fragment cloned on a lambda gt11 library. It is unique on the yeast genome and lies on the left arm of chromosome XIV, very close to a NotI site. Its coding sequence perfectly matches the amino acid sequence of two oligopeptides prepared from purified C31. It is also identical to the ACP2 gene previously described as encoding an HMG1-like protein (W. Haggren and D. Kolodrubetz, Mol. Cell. Biol. 8:1282-1289, 1988). Thus, ACP2 and RPC31 are allelic and encode a subunit of RNA polymerase C. The c31 protein has a highly acidic C-terminal tail also found in several other chromatin-interacting proteins, including animal HMG1. Outside this domain, however, there is no appreciable homology to any known protein. The growth phenotypes of a gene deletion, of insertions, and of nonsense mutations indicate that the C31 protein is strictly required for cell growth and that most of the acidic domain is essential for its function. Random mutagenesis failed to yield temperature-sensitive mutants, but a slowly growing mutant was constructed by partial suppression of a UAA nonsense allele of RPC31. Its reduced rate of tRNA synthesis in vivo relative to 5.8S rRNA supports the hypothesis that the C31 protein is a functional subunit of RNA polymerase C. Show less

It is well known that cultured Sertoli cells secrete plasminogen activators (Lacroix et al., Mol Cell Endocrinol 1977; 9:227-236; Hettle et al., Biol Reprod 1986; 34:895-904). We now show that testicular cells in culture also secrete gelatinolytic metalloproteinases. Gelatin zymographic analysis of concentrated culture medium proteins reveals that Sertoli cells secrete gelatinases of 185 kDa, 110 kDa, 83 kDa, 76 kDa, and 72 kDa in addition to plasminogen activators (PAs). Gelatinase 185 kDa is induced by FSH. Media from Sertoli (epithelial)/peritubular (mesenchymal) cell cocultures contain the Sertoli cell gelatinases and one FSH-stimulated gelatinase of 50 kDa, indicating that gelatinase 50 kDa is regulated by both FSH and cell-cell interactions. A 50-kDa fibronectinolytic activity is also present in the coculture medium from cells grown in the presence of FSH. Casein zymography demonstrates a prominent 30-kDa protease only in media from cocultures. Peritubular cells secrete urokinase-type plasminogen activator (u-PA) and exhibit slight degrading activity at 86 kDa and 74 kDa. The gelatinases are most active in the pH range 7.3-8.5 and are completely or partially inhibited by metal ion chelators indicating that they are metalloproteinases. Our data demonstrate that testicular cells in culture secrete several gelatinases in addition to PAs, and that FSH and coculture conditions regulate some of these secreted proteases. We suggest that the highly regulated secretion of these proteases may well be of physiological importance during testicular development and spermatogenesis. Show less

The genetics and linkage relationships of several isozymatic and morphological markers have been investigated in different cultivars of rye (Secale cereale L.). The inheritance and the variability among cultivars of three new isozymatic zones are described: GOT2 and LAP, each of them under the control of a two-allele single locus, namely Got2 and Lap, respectively; and 6PGD1 controlled by two loci, 6Pgd1a and 6Pgd1b, which have alleles in common. Four linkage groups have been found: Acp2-Acp3, Got3-Mdh2-Lper4, Mdh1-6Pgd2-Pgi2, and Pgm-Eper2-[Eper1-Eper3]. The assignment of these four groups to the chromosomes 7R, 3R, 1R, and 4R is discussed. Show less

Deletion analysis offers a powerful alternative to linkage and karyotypic approaches for human chromosome mapping. A panel of deletion hybrids has been derived by mutagenizing J1, a hamster cell line that stably retains chromosome 11 as its only human DNA, and selecting for loss of MIC1, a surface antigen encoded by a gene in band 11p13. A unique, self-consistent map was constructed by analyzing the pattern of marker segregation in 22 derivative cells lines; these carry overlapping deletions of 11p13, but selectively retain a segment near the 11p telomere. The map orders 35 breakpoints and 36 genetic markers, including 3 antigens, 2 isozymes, 12 cloned genes, and 19 anonymous DNA probes. The deletions span the entire short arm, dividing it into more than 20 segments and define a set of reagents that can be used to rapidly locate any newly identified marker on 11p, with greatest resolution in the region surrounding MIC1. The approach we demonstrate can be applied to map any mammalian chromosome. To test the gene order, we examined somatic cell hybrids from five patients, whose reciprocal translocations bisect band 11p13; these include two translocations associated with familial aniridia and two with acute T-cell leukemia. In each patient, the markers segregate in telomeric and centromeric groups as predicted by the deletion map. These data locate the aniridia gene (AN2) and a recurrent T-cell leukemia breakpoint (TCL2) in the marker sequence, on opposite sides of MIC1. To provide additional support, we have characterized the dosage of DNA markers in a patient with Beckwith-Wiedemann syndrome and an 11p15-11pter duplication. Our findings suggest the following gene order: TEL - (HRAS1, MER2, CTSD, TH/INS/IGF2, H19, D11S32) - (RRM1, D11S1, D11S25, D11S26) - D11S12 - (HBBC, D11S30) - D11S20 - (PTH, CALC) - (LDHA, SAA, TRPH, D11S18, D11S21) - D11S31 - D11S17 - HBVS1 - (FSHB, D11S16) - AN2 - MIC1 - TCL2 - delta J - CAT - MIC4 - D11S9 - D11S14 - ACP2 - (D11S33, 14L) - CEN. We have used the deletion map to show the distribution on 11p of two centromeric repetitive elements and the low-order interspersed repeat A36Fc. Finally, we provide evidence for an allelic segregation event in the hamster genome that underlies the stability of chromosome 11 in J1. The deletion map provides a basis to position hereditary disease loci on 11p, to distinguish the pattern of recessive mutations in different forms of cancer and, since many of these genes have been mapped in other mammalian species, to study the evolution of a conserved syntenic group. Show less

Epidermal growth factors receptor (EGFR) was localized immunocytochemically in the testes of mature and immature rats and immature monkeys. One polyclonal antibody, recognizing the intracellular domain (RK2) of the receptor, was used to carry out the EGFR immunodetection. The RK2 antibody revealed the presence of the EGFR predominantly in Sertoli cells of mature and immature rats and of immature monkeys, although limited interstitial localization of the EGFR was also discerned in the mature rat. In cultured Sertoli cells of immature rats, grown in the absence of epidermal growth factor (EGF), the EGFR was randomly distributed at the cell surface, whereas after the addition of EGF the receptor became aggregated into distinct focal regions. In addition, EGFR of cultured Sertoli cells exhibited autophosphorylation activity upon stimulation with EGF, but failed to transcytose iodinated EGF across a permeability barrier formed by the cultured cells. Instead, all of the added iodinated EGF was internalized and degraded. Show less

Carbamyl phosphate synthetase I (CPS1) is an initial enzyme of urea synthetase system. It exists exclusively in liver cells and epithelial cells of the small intestine. By immunocytochemistry, 70.5% of 88 surgically resected gastric carcinomas (42 advanced and 46 early gastric carcinomas) was found to be CPS1 immunoreaction positive, whereas all other carcinomas (of the esophagus, colon, pancreas, lung, breast, ovary, kidney, prostate and urinary bladder) tested were negative. CPS1 expression in gastric carcinoma was closely related to the types of mucin secreted by the carcinoma cells. Most carcinomas secreting sialomucin were CPS1 positive, yet those secreting sulfomucin or neutral mucin did not express CPS1. The types of intestinal metaplasia adjacent to the carcinoma correlated neither with CPS1 expression nor with the histological types of carcinoma. Owing to the fact that gastric carcinomas with CPS1 expression possess the characteristics of small intestinal epithelium, it is very likely that CPS1 can be used as a novel marker for gastric carcinoma originating from complete type intestinal metaplasia. Show less

The cDNAs encoding human prostatic acid phosphatase were cloned and characterized. The mRNAs contain 3' noncoding regions of heterogeneous sizes 646, 1887 or 1913 nucleotides. A dimer and a monomer of the conserved Alu-repeats are present in the longer 3' noncoding sequences. The complete sequence of 354 amino acids for the mature enzyme was determined by sequencing both cDNA and protein. Human prostatic and lysosomal acid phosphatases exhibit 50% sequence homology, including five Cys residues and two putative N-linked glycosylation sites. The Acp-3 gene coding for human prostatic acid phosphatase was mapped onto chromosome 3 in this investigation. The Acp-2 gene coding for lysosomal acid phosphatase has previously been located on chromosome 11, while the Acp-1 gene coding for red blood cell acid phosphatase is on chromosome 2. Show less

The effects of cold on protein and RNA metabolism in leaves of rice (Oryza sativa L.) seedlings were investigated. Treatment with a diurnal cycle of 15/10 degrees C or 11/6 degrees C for up to 1 week resulted in progressive changes in the protein synthesis pattern after in vivo labeling of intact rice leaves with [(35)S]methionine. These changes were reversed when the seedlings were returned to normal growth temperatures. While de novo accumulation of several abundant proteins was suppressed, some polypeptides were consistently found to be cold-induced. Synthesis of ribulose 1,5-bisphosphate carboxylase (Rubisco) was drastically reduced after 7 days of cold. Using immunoprecipitation of Rubisco, evidence was obtained that the suppression was greater for the small subunit (over 90%) than for the large subunit (80%), indicating a partial loss of coordination in their synthesis. Preformed Rubisco as well as other cold-suppressed proteins were stable for up to 7 days at 11/6 degrees C. Cold-sensitive rice cultivars responded with similar but more drastic changes in the protein synthesis pattern when compared to cold-tolerant varieties. The suppression of Rubisco synthesis by cold was shown to result from reduced levels of the mRNAs encoding both subunits; their decrease paralleled the lower protein synthesis of each. The levels of other chloroplast-encoded mRNAs, especially psaB, and of the nuclear encoded chlorophyll a/b binding protein, also strongly decreased in the cold, whereas the transcripts of the mitochondrial genes apt9, coxIII, and most nuclear genes analyzed were unaffected or only slightly reduced. These data indicate that some chloroplast functions are disturbed during cold stress. One nuclear gene known to be induced by water stress and ABA (Rab21) was also found to be induced by cold treatment. Show less

alpha-Melanotropin (alpha-melanocyte-stimulating hormone, alpha-MSH) is a tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2. The minimal sequence of alpha-MSH required for agonism in the lizard (Anolis carolinensis) skin bioassay was determined to be Ac-His-Phe-Arg-Trp-NH2 (Ac-alpha-MSH6-9-NH2). Smaller fragments of this sequence (Ac-alpha-MSH6-8-NH2, Ac-alpha-MSH6-7-NH2, Ac-alpha-MSH7-9-NH2, and Ac-alpha-MSH7-8-NH2) were devoid of melanotropic activity. The tetrapeptide, Ac-alpha-MSH7-10-NH2, was also inactive, thus again demonstrating the importance of His at position 6 for minimal activity. The important potentiating amino acids were found to be Met-4, Lys-11, and Pro-12, since Ac-alpha-MSH4-10-NH2 was about 100 times more potent than Ac-alpha-MSH5-10-NH2, and Ac-[Nle4]-alpha-MSH4-11-NH2 was about 40 times more potent than Ac-alpha-MSH4-10-NH2 or Ac-[Nle4]-alpha-MSH4-10-NH2. Ac-alpha-MSH4-12-NH2 and Ac-[Nle4]-alpha-MSH4-12-NH2 were equipotent and about six times more potent than alpha-MSH. Since [Nle4]-alpha-MSH and Ac-[Nle4]-alpha-MSH4-13-NH2 were both equipotent but about sixfold less active than Ac-[Nle4]-alpha-MSH4-12-NH2, it is clear that valine at position 13 does not contribute to the potency of alpha-MSH, except possibly in a negative way. The minimal message sequence for equipotency to alpha-MSH appears to be Ac-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-NH2, since the analog, Ac-[Nle4]-alpha-MSH4-11-NH2, was as active as the native hormone. Ser-1, Tyr-2, Ser-3, Glu-5, and Val-13 are not important for melanotropic potency since Ac-alpha-MSH4-12-NH2 was more potent than alpha-MSH, and Ac-alpha-MSH5-10-NH2 and Ac-alpha-MSH6-10-NH2 were equipotent, being about 4,000 times less active than alpha-MSH. Show less

A monoclonal antibody with high affinity to acid phosphatase isoenzyme 2 (Ab-AcP2) was selected to examine its binding to different normal and tumor tissues using the indirect immunohistochemical method. Both mature prostatic epithelial cells in the prostate and the highly dedifferentiated prostatic cancer cells in the bone marrow showed strong binding to the antibody. Among nonprostatic tissues, only bone marrow, breast, and kidney showed trace staining in some specimens. The specificity of Ab-AcP2 was much better than that of the polyclonal antibody to acid phospatase previously reported. When the antibody to the prostate-specific antigen (Ab-PSA) was used, weak background staining was often encountered, and weak to moderate stains were seen in the prostatic stroma, bone marrow, lung, skin, and melanoma. Show less

Cyclins were discovered in marine invertebrates based on their dramatic cell cycle periodicity. Recently, the products of three genes associated with cell cycle progression in S. cerevisiae were found to share limited homology with cyclins. Mutational elimination of the CLN1, CLN2, and DAF1/WHI1 products leads to cell cycle arrest independent of cell type, while expression of any one of the genes allows cell proliferation. Using strains where CLN1 was expressed conditionally, the essential function of Cln proteins was found to be limited to the G1 phase. Furthermore, the ability of the Cln proteins to carry out this function was found to decay rapidly upon cessation of Cln biosynthesis. The data are consistent with the hypothesis that Cln proteins activate the Cdc28 protein kinase, shown to be essential for the G1 to S phase transition in S. cerevisiae. Because of the apparent functional redundancy of these genes, DAF1/WHI1 has been renamed CLN3. Show less

The H-2 region of mouse chromosome 17 is known to include one or more genes that affect susceptibility to cortisone-induced cleft palate. We have now studied congenic strains that possess crossovers in the interval between H-2S and H-2D and have observed significant differences in susceptibility among recombinants that had been believed to possess the same H-2 haplotypes. Pregnant mice were injected on days 11 through 14 of gestation with 100 mg of cortisone per kg of body weight. The frequency of cleft palate in B10.A(2R) was significantly greater than in B10.A(1R), despite the fact that both have H-2a/H-2b crossovers in the interval between the S and D loci and have the same alleles at all loci that have been previously characterized. Both B10.BAR5 and B10.BAR12 were significantly more susceptible than B10.A(18R), although these strains also share the same alleles at all loci that have been previously characterized. All three of these strains have H-2b/H-2a recombinant chromosomes, with crossovers in the S/D interval. Genetic linkage between H-2 and the high-susceptibility gene of B10.BAR5 was confirmed by testing H-2 homozygotes derived by intercrossing backcross animals. These data therefore suggest that a gene coding for susceptibility, which we designate Cps-1, maps in the 350-kb interval between H-2S and H-2D, and the congenic strains that we have found to be different have different crossover points within this interval. Alleles at the Cps-1 locus have embryonic effects, but no demonstrable effects on the maternal environment. Show less

With cDNA fragments of CPS1, OCT and ACT as probes, dot and Northern blot analyses of poly(A)+-RNA from rat liver with different pathological lesions during carcinogenesis induced by diethylnitrosamine were conducted. It was shown that the level of mRNA of tissue-specific enzymes, CPSI and OCT decreased while that of the proliferating enzyme ACT mRNA increased, and the alteration was correlated with the degree of pathological changes in each case. The relative changes in the mRNA level of these enzymes during hepatocarcinogenesis were correlated with that of enzyme activities. Implication of these findings in the mechanism of carcinogenesis in terms of cell proliferation and differentiation was discussed. Show less

cDNA coding for carbamyl phosphate synthetase I was cloned from recombinant plasmid with insert complementary to the mRNA for CPS1 followed by hybrid-selected translation screening. The length of the insert CPS1 cDNA was approximately 800 base pairs. Using this cDNA as a probe, it was found by dot-blot analysis of the total RNAs and poly(A)+-RNAs isolated from rat livers with different pathological lesions induced by diethylnitrosamine that the levels of CPS1 mRNA were decreased, the decrease being correlated with the malignancy of hepatocytes during carcinogenesis. Show less

The legionellae are facultative intracellular bacterial pathogens which multiply in host phagocytes. Legionella micdadei cells contain an acid phosphatase (ACP2) which blocks superoxide anion production by human neutrophils stimulated with formyl-Met-Leu-Phe (fMLP) [A. K. Saha, et al. (1985) Arch. Biochem. Biophys. 243, 150-160]. In the present study, we have purified the Legionella phosphatase to homogeneity as indicated by the finding of a single 68,000-Da band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We explored the possibility that ACP2 acts by interfering with polyphosphoinositide hydrolysis and the production of the intracellular second messengers, inositol trisphosphate (IP3) and diacylglycerol, following neutrophil stimulation. Phosphatidylinositol 4,5-bisphosphate (PIP2) was hydrolyzed rapidly by ACP2 in vitro. The rate of hydrolysis of PIP2 was higher at pH 7.0 (Km 2.0 microM; 4 X 10(3) units/mg protein; 1 unit equals 1 nmol of Pi released/h) than at lower pH. IP3 was also a good substrate for ACP2 in vitro. When human neutrophil phosphoinositides were prelabeled with 32Pi, subsequent incubation with ACP2 resulted in an 85% loss of the labeled PIP2 over 2 h. Following fMLP stimulation of [3H]inositol-labeled neutrophils, the quantity of IP3 produced by ACP2-treated cells was reduced by 44%. Prior treatment of neutrophils with ACP2 also reduced by 45% the amount of diacylglycerol they produced when stimulated by fMLP. These results indicate that the Legionella phosphatase may compromise the neutrophils' microbicidal response to the organism by hydrolyzing PIP2, the progenitor of IP3 and diacylglycerol, and by hydrolyzing IP3 itself. Show less

The high-mobility-group (HMG) proteins, a group of nonhistone chromatin-associated proteins, have been extensively characterized in higher eucaryotic cells. To test the biological function of an HMG protein, we have cloned and mutagenized a gene encoding an HMG-like protein from the yeast Saccharomyces cerevisiae. A yeast genomic DNA library was screened with an oligonucleotide designed to hybridize to any yeast gene containing an amino acid sequence conserved in several higher eucaryotic HMG proteins. DNA sequencing and Northern (RNA) blot analysis revealed that one gene, called ACP2 (acidic protein 2), synthesizes a poly(A)+ RNA in S. cerevisiae which encodes a 27,000-molecular-weight protein whose amino acid sequence is homologous to those of calf HMG1 and HMG2 and trout HMGT proteins. Standard procedures were used to construct a diploid yeast strain in which one copy of the ACP2 gene was mutated by replacement with the URA3 gene. When this diploid was sporulated and dissected, only half of the spores were viable. About half of the nonviable spores proceeded through two or three cell divisions and then stopped dividing; the rest did not germinate at all. None of the viable spores contained the mutant ACP2 gene, thus proving that the protein encoded by ACP2 is required for cell viability. The results presented here demonstrate that an HMG-like protein has an essential physiological function. Show less

Primary cultures of Sertoli cells maintained in conventional cultures on plastic culture vessels do not retain many of the structural and functional properties of their in vivo counterparts. Sertoli cell phenotype is better maintained by incorporating certain environmental parameters, intrinsic to the testis, into the Sertoli cell culture system. These environmental parameters include a) high cell density, b) a unique extracellular matrix, c) a semipermeable support between the basal plasma membrane of the cells and blood-derived nutrients in the interstitium, d) chemically distinct microenvironments at the apical and basal surfaces of the cells, and e) cell-to-cell interactions among Sertoli cells and other testicular cell types. Using three variations of Sertoli cell culture we have demonstrated the importance of each of these environmental parameters in obtaining a better Sertoli cell culture model. Show less

Contrabithorax (Cbx) alleles are dominant mutations in the Ultrabithorax (Ubx) gene of Drosophila that cause its ectopic expression in the mesothorax. We have studied the role of the homologous chromosome in the mesothoracic phenotype in several Cbx heterozygotes. None of the Cbx alleles studied shows variations in phenotype with extra doses of the Ubx gene. Only Cbx2 and CbxIRM (a revertant of Cbx1) show synapsis-dependent gene expression ("transvection"). The mesothoracic phenotypes of CbxIRM and Cbx2 heterozygotes are strongly modified when the homologous chromosome carries breakpoints proximal to or at the Ubx locus or null alleles of this gene. These lesions in the homologous chromosome enhance the Cbx2 mutant phenotype and reduce that of CbxIRM one. The genetic analysis of these transvection effects suggests that the transcription of the CbxIRM and Cbx2 alleles depends on RNAs of short radius of action from the homologous Ubx gene. Show less

Isolated Sertoli cells were cultured on MatrigelTM-coated Millipore filters in bicameral chambers. The Sertoli cells form confluent epithelial sheets that, by virtue of the Sertoli cell tight junctions, form transepithelial permeability barriers between the apical and basal domains of the cells. These Sertoli cells secrete metabolically labeled proteins in a polarized manner. Three peptides, P1 (pI = 4.5-5.0, MW = 70,000), P2 (pI = 4.5-5.0, MW = 50,000), and P3 (pI = 4.0-4.7, MW = 34,000) are secreted apically from the epithelial sheets of Sertoli cells and are not found in basal secretions from the same Sertoli cells. Pachytene spermatocyte-conditioned medium contains proteins released from the germ cells that are uniquely different from the Sertoli cell-secreted proteins. Addition of the pachytene spermatocyte-conditioned medium to the apical reservoir of the bicameral chambers over an epithelial sheet of Sertoli cells stimulated the synthesis and secretion of total protein, transferrin, and specifically induced peptides S1 and S2 from Sertoli cells. As controls, conditioned medium from 3T3 fibroblasts and round spermatids did not stimulate the Sertoli cells. Hence, the ability of pachytene spermatocyte proteins to induce specific Sertoli cell secretion indicates that the pachytene spermatocytes are able to influence their surrounding milieu, and provides further support to the concept of a paracrine interaction between germ cells and Sertoli cells during spermatogenesis. Show less

The seasonal variation in respiratory syncytial virus (RSV) infections in children was examined in Benin City. Nasopharyngeal washes were obtained from children under 3 years hospitalised for acute lower respiratory infections during two seasons - rainy season (June-August) and dry season (December-February). RSV surface antigen was identified by ELISA. 54% of patients in the rainy season were ELISA positive for RSV compared to 8.8% during the dry season. Clinical features in the RSV infected patients were not different between the two seasons and were similar to that reported from temperate climates. It is concluded that RSV infections occur all year round with a peak during the rainy season. Show less

The ADP-ribosylation factor (ARF) is a 21-kDa GTP-binding protein that serves as the cofactor in the cholera toxin-catalyzed activation of the stimulatory guanine nucleotide-binding protein of adenylate cyclase (Gs). An oligonucleotide probe based on the partial amino acid sequence was used to clone ARF from a bovine adrenal chromaffin cDNA library. The yeast (Saccharomyces cerevisiae) ARF gene was then cloned from a YCp50 genomic library by cross-species hybridization by using the coding region of the bovine gene. RNA gel blots of poly(A)+ RNA indicate that only one ARF message size (900 and 2000 base pairs) is present in yeast and cows, respectively. Comparison of the cDNA-derived amino acid sequences of ARF to other GTP-binding proteins reveals a structural relationship between ARF and the ras family of proteins. A slightly better structural relationship is detected when ARF is compared to the alpha subunits of the trimeric GTP-binding proteins, including Gs alpha. All of the biochemical characteristics of the purified ARF, including the lack of GTPase activity and the posttranslational myristoylation, are consistent with the derived sequences. Comparison of the ARF sequences to that of the chicken processed pseudogene (CPS-1), previously reported as a ras homologue, reveals that CPS-1 is actually an ARF-derived gene. These results demonstrate that ARF is a GTP-binding protein with structural features of both the ras and the trimeric GTP-binding protein families. Show less

The authors studied the wild type strain, N2, and three mutant strains of the nematode, Caenorhabditis elegans, in order to measure genetically produced changes in responses to nine volatile anesthetics. They determined the anesthetic ED50s of N2 for thiomethoxyflurane, methoxyflurane, chloroform, halothane, enflurane, isoflurane, fluroxene, flurothyl, and diethylether. The log-log relationship of the oil-gas partition coefficients (O/G) and the ED50s of these agents for N2 yields a straight line with a slope of -.997 with a R2 of .98 over a range of O/G (at 37 degrees C) from 48 to 7230. When the O/Gs are corrected to 22 degrees C, the slope is -.964 with an R2 of .98. This relationship is similar to that found in other animals. Two mutant strains, unc-79 and unc-80, show altered responses to these anesthetics. These strains are two to three times more sensitive than N2 to anesthetics with an O/G greater than that of halothane (220 at 37 degrees C), yet they differ little from N2 in response to anesthetics with lower O/Gs. unc-79 and unc-80 are about 30% more sensitive than N2 to diethylether. The double mutant unc-79; unc-80 is more sensitive to halothane, isoflurane, and fluroxene than is either mutant alone. The authors believe these data indicate an alteration at the site of action of volatile anesthetics in unc-79 and unc-80. They also postulate that the interaction of unc-79 and unc-80 indicate these genes code for enzymes in a common pathway, and that unc-79 precedes unc-80 in this pathway. Show less

Nuclei from the normal mouse liver were partially digested with micrococcal nuclease, followed by DNA extraction, agarose gel electrophoresis and dot blot hybridization with 32P-labeled cDNA probes of CPS1 and ACT complex. It was clearly shown that the CPS1 genes were distributed on the monomer, dimer. and trimer of nucleosomes, while the genes coding for ACT complex were distributed on the condensed oligonucleosomes. An opposite manner of distribution of CPS1 and ACT complex genes was, however, noted in the case of ascites hepatoma cells, in which the specific activity of ACT was 13 times higher than that in the normal liver, while that of CPS1 was remarkably reduced. Similar patterns of change in mRNA level of CPS1 and ACT complex were observed in the normal mouse liver and ascites hepatoma cells, indicating a close relationship between chromatin structure and gene expression of these enzymes. Show less

Chromosomal locations of 10 isozyme loci in rice (Oryza sativa L.) were determined through trisomic analysis. All 10 genes produced altered allozyme banding patterns in specific F1 trisomics. This served as the primary source of evidence for chromosome locations of Est-5, Icd-1, Acp-1, and Pgd-1. The locations of Amp-1, Amp-2, Amp-4, Pox-5, Got-1, and Cat-1 were further confirmed from segregation data in BC1 generations, as the ratios deviated significantly from 1:1 in the critical trisomics but agreed with the expected trisomic ratios. Triallelic heterozygotes were recovered for Amp-1 and Amp-2. On the basis of these data Got-1, Est-5, and Icd-1 were located to chromosome 1, Amp-1 to chromosome 2, Cat-1 and Pox-5 to chromosome 3, Acp-1 to chromosome 6, Amp-2 and Amp-4 to chromosome 8, and Pgd-1 to chromosome 11. Because Acp-2 and Pox-2 are known to be linked with Acp-1, they must also be on chromosome 6. The gene order and recombination values between isozyme loci on chromosomes 3, 6, 8, and 11 are presented. Show less

The identification of microfilaments contained within Sertoli cell ectoplasmic specializations (ES) in intact rat testes is reported. In order to determine the presence and configuration of ES during the cycle of the seminiferous epithelium, frozen sections of testes were prepared and stained with NBD-phallicidin (NBDP). Results revealed that Sertoli cell ES become most prominent immediately adjacent to acrosomal caps of spermatids, once these begin their elongation phase of maturation. Significant association of ES with spermatogenic cells earlier than round spermatids was not detected with NBDP. Intense staining of the ES continued up to the final stages preceding sperm release, and was followed by dissipation of the fluorescence. These results indicate that the disappearance of ES, as detected with NBDP, does not correlate precisely with sperm release. Show less

The distribution of laminin, type IV collagen, heparan sulfate proteoglycan, and fibronectin was investigated in the rat testicular lamina propria by electron microscopic immunocytochemistry. Distinct patterns were observed for each antigen within the extracellular matrix (ECM) layers of the lamina propria. Laminin, type IV collagen, and heparan sulfate proteoglycan all localized to the seminiferous tubule basement membrane. Type IV collagen and heparan sulfate proteoglycan, but not laminin, localized to the seminiferous tubule side of the peritubular myoid cells. All four of the antigens were localized between the peritubular and lymphatic endothelial cells. Failure to localize fibronectin in the ECM layer between the Sertoli and peritubular myoid cells tends to support the concept that adult Sertoli cells do not produce this protein in vivo. Intracellular immunostaining was insufficient to allow unambiguous identification of the cellular source of any of the ECM molecules. Show less