Yeasts

EMBO J, 1991 Nov, 10(11), 3321 - 9
Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates; Norbury C et al.; The p34cdc2 protein kinase is a conserved regulator of the eukaryotic cell cycle . Here we show that residues Thr14 and Tyr15 of mouse p34cdc2 become phosphorylated as mouse fibroblasts proceed through the cell cycle . We have mutated these residues and measured protein kinase activity of the p34cdc2 variants in a Xenopus egg extract . Phosphorylation of residues 14 and 15, which lie within the presumptive ATP-binding region of p34cdc2, normally restrains the protein kinase until it is specifically dephosphorylated and activated at the G2/M transition . Regulation by dephosphorylation of Tyr15 is conserved from fission yeast to mammals, while an extra level of regulation of mammalian p34cdc2 involves Thr14 dephosphorylation . In the absence of phosphorylation on these two residues, the kinase still requires cyclin B protein for its activation . Inhibition of DNA synthesis inhibits activation of wild-type p34cdc2 in the Xenopus system, but a mutant which cannot be phosphorylated at residues 14 and 15 escapes this inhibition, suggesting that these phosphorylation events form part of the pathway linking completion of DNA replication to initiation of mitosis.

Lakartidningen, 1991 Oct 30, 88(44), 3665 - 8
{Lysozyme--an enzyme of both historical and current interest as a therapeutical agent}; Burman LG et al.; Lysozyme, a bacteriolytic protein discovered by Fleming in 1922 and found to be phylogenetically ancient and almost ubiquitous among living organisms, is probably the most studied enzyme in biology and medicine . Evidence of its involvement in resistance to bacterial infection is compelling but remains indirect . Muramyl peptides (fragments of bacterial cell wall peptidoglycan) exert many effects on the immune system and the CNS, and appear to contribute to non-specific resistance to infection, fever, fatigue, and the pathogenesis of bacterial infection . Synthetic muramyl peptide analogues are currently used as adjuvants in vaccine trials in humans . Several pathological conditions are associated with changes in lysozyme concentrations, and egg-white lysozyme treatment has been tried on a small scale . With the cloning of the human lysozyme gene in yeast cells the enzyme can now be produced on a large scale, which will enable its therapeutic applications to be evaluated.

J Immunol, 1991 Oct 15, 147(8), 2565 - 73
Leukocyte adhesion molecule-1 (LAM-1, L-selectin) interacts with an inducible endothelial cell ligand to support leukocyte adhesion; Spertini O et al.; The human lymphocyte homing receptor, LAM-1, mediates the adhesion of lymphocytes to specialized high endothelial venules (HEV) of peripheral lymph nodes . We now report that LAM-1 is also a major mediator of leukocyte attachment to activated human endothelium . In a novel adhesion assay, LAM-1 was shown to mediate approximately 50% of the adhesion of both lymphocytes and neutrophils to TNF-activated human umbilical vein endothelial cells at 4 degrees C . The contribution of LAM-1 to leukocyte adhesion was only detectable when the assays were carried out under rotating (nonstatic) conditions, suggesting that LAM-1 is involved in the initial attachment of leukocytes to endothelium . In this assay at 37 degrees C, essentially all lymphocyte attachment to endothelium was mediated by LAM-1, VLA-4/VCAM-1, and the CD11/CD18 complex, whereas neutrophil attachment was mediated by LAM-1, endothelial-leukocyte adhesion molecule-1, and CD11/CD18 . Thus, multiple receptors are necessary to promote optimal leukocyte adhesion to endothelium . LAM-1 also appeared to be involved in optimal neutrophil transendothelial migration using a videomicroscopic in vitro transmigration model system . LAM-1-dependent leukocyte adhesion required the induction and surface expression of a neuraminidase-sensitive molecule that was expressed for at least 24 h on activated endothelium . Expression of the LAM-1 ligand by endothelium was optimally induced by LPS and the proinflammatory cytokines TNF-alpha and IL-1 beta, whereas IFN-gamma and IL-4 induced lower levels of expression . The LAM-1 ligand on HEV and cytokine treated endothelium may be similar carbohydrate-containing molecules, because phosphomannan monoester core complex from yeast Hansenula hostii cell wall blocked binding of lymphocytes to both cell types, and identical epitopes on LAM-1-mediated lymphocyte attachment to HEV and activated endothelium . Thus, LAM-1 and its inducible endothelial ligand constitute a new pair of adhesion molecules that may regulate initial leukocyte/endothelial interactions at sites of inflammation.

Nature, 1991 Oct 24, 353(6346), 769 - 72
Hypervariable C-terminal domain of rab proteins acts as a targeting signal; Chavrier P et al.; Mammalian cells express many ras-like low molecular mass GTP-binding proteins (rab proteins) that are highly homologous to the Ypt1 and Sec4 proteins involved in controlling secretion in yeast . Owing to their structural similarity and to their variety, rab proteins have been postulated to act as specific regulators of membrane traffic in exocytosis and endocytosis, and rab5 has been shown to be involved in early endosome fusion in vitro . In agreement with their postulated functions, all rab proteins studied so far have been found in distinct subcompartments along the exocytic or endocytic pathways . To define the region mediating their specific localization, we transiently expressed rab2, rab5 and rab7 hybrid proteins in BHK cells, and determined their intracellular localization by immunofluorescence confocal microscopy and subcellular fractionation . Here we present evidence that the highly variable C-terminal domain contains structural elements necessary for the association of rab proteins with their specific target membranes in the endocytic pathway.

Nature, 1991 Oct 24, 353(6346), 726 - 30
Peptide-binding specificity of the molecular chaperone BiP; Flynn GC et al.; Members of the heat-shock protein family (hsp70s) can distinguish folded from unfolded proteins . This property is crucial to the role of hsp70s as molecular chaperones and is attributable to the amino-acid specificity of the peptide-binding site . The specificity for peptide ligands is investigated using a set of peptides of random sequence but defined chain length . The peptide-binding site selects for aliphatic residues and accommodates them in an environment energetically equivalent to the interior of a folded protein.

FEBS Lett, 1991 Oct 21, 291(2), 192 - 4
Isolation of mitotic p34cdc2 apoenzyme from human cells; Meikrantz W et al.; A simple procedure was devised for isolating from homogenates of mitotic cells the human homolog to the fission yeast cdc2 gene product . The identity of the purified protein was established with anti-p34cdc2 antibodies and p13suc 1, both specific ligands for p34cdc2 . Active-site labeling with oxidized {alpha 32P}ATP showed the purified molecule to be an ATP-binding protein . Its ability to phosphorylate casein but not histone, and its phosphorylation on tyrosine, detected by anti-phosphotyrosine antibodies, indicates the form of p34cdc2 purified is the inactive or apoenzyme form . Purified quantities of human p34cdc2 should be of considerable value in establishing the mechanism of its activation at mitosis by phosphatases.

Cell, 1991 Oct 18, 67(2), 283 - 91
Parallel activation of the NIMA and p34cdc2 cell cycle-regulated protein kinases is required to initiate mitosis in A . nidulans; Osmani AH et al.; We show that in Aspergillus nidulans, p34cdc2 tyrosine dephosphorylation accompanies activation of p34cdc2 as an H1 kinase at mitosis . However, the nimA5 mutation arrests cells in G2 with p34cdc2 tyrosine dephosphorylated and fully active as an H1 kinase . Activation of NIMA is therefore not required for p34cdc2 activation . Furthermore, mutation of nimT, which encodes a protein with 50% similarity to fission yeast cdc25, causes a G2 arrest and prevents tyrosine dephosphorylation of p34cdc2 but does not prevent full activation of the NIMA protein kinase . Mitotic activation of p34cdc2 by tyrosine dephosphorylation is therefore not required for activation of NIMA . These data suggest that activation of either the p34cdc2 protein kinase or the NIMA protein kinase alone is not sufficient to initiate mitosis . Parallel activation of both cell cycle-regulated protein kinases is required to trigger mitosis.

Biochim Biophys Acta, 1991 Oct 16, 1095(1), 1 - 4
Binding of cathepsin D to the mannose receptor on rat peritoneal macrophages; Young PR et al.; Adherent cultures of rat peritoneal macrophages secrete lysozyme and the lysosomal marker enzymes beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase; the levels of secreted lysosomal cathepsin D, however, were found to be insignificant . Incubation of the cells at 4 degrees C for 15 min with yeast mannan or with 50 mM mannose, methyl alpha-glucopyranoside, or N-acetylglucosamine caused the concentration of cathepsin D in the culture medium to increase 30-40-fold; mannose-6-phosphate had no effect . 125I-labeled cathepsin D was prepared and the binding constant to the macrophage cell surface was determined to be KD = 27 nM . The data suggest that cathepsin D binds to the mannose receptor of macrophages and that binding to this receptor is not in equilibrium with the bulk medium.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9051 - 5
Origin of human chromosome 2: an ancestral telomere-telomere fusion; IJdo JW et al.; We have identified two allelic genomic cosmids from human chromosome 2, c8.1 and c29B, each containing two inverted arrays of the vertebrate telomeric repeat in a head-to-head arrangement, 5'(TTAGGG)n-(CCCTAA)m3' . Sequences flanking this telomeric repeat are characteristic of present-day human pretelomeres . BAL-31 nuclease experiments with yeast artificial chromosome clones of human telomeres and fluorescence in situ hybridization reveal that sequences flanking these inverted repeats hybridize both to band 2q13 and to different, but overlapping, subsets of human chromosome ends . We conclude that the locus cloned in cosmids c8.1 and c29B is the relic of an ancient telomere-telomere fusion and marks the point at which two ancestral ape chromosomes fused to give rise to human chromosome 2.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 8977 - 81
Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase; Chu E et al.; Thymidylate synthase (TS; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) is essential for the de novo synthesis of thymidylate, a precursor of DNA . Previous studies have shown that the cellular level of this protein is regulated at both the transcriptional and posttranscriptional levels . The regulation of human TS mRNA translation was studied in vitro with a rabbit reticulocyte lysate system . The addition of purified human recombinant TS protein to in vitro translation reactions inhibited translation of TS mRNA . This inhibition was specific in that recombinant TS protein had no effect on the in vitro translation of mRNA for human chromogranin A, human folate receptor, preplacental lactogen, or total yeast RNA . The inclusion of dUMP, 5-fluoro-dUMP, or 5,10-methylene-tetrahydrofolate in in vitro translation reactions completely relieved the inhibition of TS mRNA translation by TS protein . Gel retardation assays confirmed a specific interaction between TS protein and its corresponding mRNA but not with unrelated mRNAs, including human placenta, human beta-actin, and yeast tRNA . These studies suggest that translation of TS mRNA is controlled by its own protein end product, TS, in an autoregulatory manner.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 8895 - 9
Inhibitors of DNA topoisomerase II prevent chromatid separation in mammalian cells but do not prevent exit from mitosis; Downes CS et al.; DNA topoisomerase II (EC 5.99.1.3) is necessary for chromosome condensation and disjunction in yeast but not for other functions . In mammalian cells, it has been reported to be necessary for progression toward mitosis but not for transit through mitosis . We have found, on the contrary, that specific inhibition of topoisomerase II (but not of topoisomerase I) interferes with mammalian mitotic progression . Metaphase is prolonged, and anaphase separation of chromatids is completely inhibited, in cells given high concentrations of topoisomerase II inhibitors; nevertheless these cells attempt cleavage, sometimes generating nucleate and anucleate daughters . Lower concentrations of inhibitors interfere with anaphase and produce abnormalities of segregation . DNA topoisomerase II activity is therefore necessary for mammalian chromatid separation, but it is not tightly coupled to the control of other mitotic events.

Biochemistry, 1991 Oct 15, 30(41), 9873 - 81
Proton linkage of complex formation between cytochrome c and cytochrome b5: electrostatic consequences of protein-protein interactions; Mauk MR et al.; Two potentiometric methods have been used to study the pH-dependent changes in proton binding that accompany complex formation between cytochrome c and cytochrome b5 . With one method, the number of protons bound or released upon addition of one cytochrome to the other has been measured as a function of pH . The results from these studies are correlated with the complexation-induced difference titration curve calculated from the titration curves of the preformed complex and of the individual proteins . Both methods demonstrate that complex formation at acid pH is accompanied by proton release, that complex formation at basic pH is accompanied by proton uptake, and that the change in proton binding at neutral pH, where stability of complex formation is maximal, is relatively small . Under all conditions studied, the stoichiometry of cytochrome c-cytochrome b5 complex formation is 1:1 with no evidence of higher order complex formation . Although the dependence of complex formation on pH for interaction between different species of cytochrome c and cytochrome b5 are qualitatively similar, they are quantitatively different . In particular, complex formation between yeast iso-1-cytochrome c and lipase-solubilized bovine cytochrome b5 occurs with a stability constant that is 10-fold greater than observed for the other two pairs of proteins under all conditions studied . Interaction between these two proteins is also significantly less dependent on ionic strength than observed for complexes formed by horse heart cytochrome c with either form of cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1991 Oct 11, 19(19), 5307 - 12
Binding of human glutaminyl-tRNA synthetase to a specific site of its mRNA; Schray B et al.; The human glutaminyl-tRNA synthetase is able to bind to its own mRNA . The enzyme contains two binding regions . One is located in the central section of the enzyme which includes its most hydrophilic portion with ten lysine residues in a block of 20 amino acids . This part of the enzyme binds unspecifically to all RNA sequences tested . A second binding region is located in that part of the enzyme which shows high degrees of sequence similarities with the bacterial and yeast glutaminyl-tRNA synthetases, and which is most likely responsible for the charging of tRNA with glutamine . This second RNA binding region specifically interacts with a site in the 3' noncoding region of the synthetase's mRNA . The binding site in the mRNA is characterized by an extended secondary structure that includes elements of the 'identity set' of nucleotides recognized by the enzyme when interacting with tRNA . We discuss possible physiological implications of the interaction between glutaminyl-tRNA synthetase and its mRNA.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5269 - 74
A rat brain mRNA encoding a transcriptional activator homologous to the DNA binding domain of retroviral integrases; Duilio A et al.; We have isolated a rat cDNA, named FE65, hybridizing to an mRNA of about 2,300 nucleotides present in rat brain, undetectable in rat liver and very poorly represented in other tissues . An mRNA of the same size is present in human neuroblastoma cells and is absent from other human cell lines . The FE65 cDNA contains an open reading frame (ORF) coding for a polypeptide of 499 amino acids in which 143 residues can be aligned with the DNA binding domain of the integrases encoded by mammalian immunodeficiency viruses . The remaining part of the FE65 ORF is not homologous with the correspondent regions of the integrases; the first 206 residues of the FE65 ORF show numerous negative charges and a short sequence not dispensable for the function of the transactivating acidic domain of the jun family transcriptional factors . A plasmid which expresses FE65 amino acids 1-232 fused to the yeast GAL4 DNA binding domain was co-transfected with a plasmid containing five GAL4 binding sites upstream of a minimal Adenovirus promoter controlling the expression of the CAT gene . This experiment showed that the fused protein GAL4-FE65 is able to obtain a 30-40 fold increase of the CAT gene expression compared to the expression observed in the presence of the GAL4 DNA binding domain alone . Two types of FE65 mRNA are present in rat brain, differing only for six nucleotides . We demonstrate that this is the consequence of a neuron-specific alternative splicing of a six-nucleotide miniexon, which is also present in the human genome, in an intron/exon context very similar to that of the rat FE65 gene.

Biochim Biophys Acta, 1991 Oct 10, 1075(2), 181 - 6
Cooperative stacking and hydrogen bond pairing interactions of fragment peptide in cap binding protein with mRNA cap structure; Ueda H et al.; The stacking and hydrogen bonding abilities of Trp-(Gly)n-Glu (n = 0 approximately 3) for the interaction with 7-methylguanine (m7G) base were examined by fluorescence and 1H-NMR methods, and it was shown that they correlate with the distance between the Trp and Glu residues, and become most significant when both residues are separated from each other by two Gly residues (n = 2) . Based on this insight, the sequence conserved between the human and yeast cap binding proteins (CBPs) was surveyed, and the sequence of Trp-Glu-Asp-Glu (No . 102-105 in human CBP) was selected as a probable site for the binding with mRNA cap structure . Thus, the stacking and hydrogen bonding abilities of Trp-Glu-Asp-Glu with m7G cap structure were examined by comparative experiments using its analogous peptides . The results showed that the fourth Glu residue is important not only for the construction of hydrogen bond pairing with m7G base but also for strengthening the stacking interaction between the Trp indole ring and m7G base . Taking account of the recognition analysis using the mutant CBP proteins by site-directed mutagenesis (Ueda, H., Iyo, H., Doi, M., Inoue, M., Ishida, T., Morioka, H., Tanaka, T., Nishikawa, S . and Uesugi, S . (1991) FEBS Lett . 280, 207-210), this cooperative interaction could be important for the recognition of mRNA cap structure.

Biochim Biophys Acta, 1991 Oct 8, 1090(2), 277 - 80
Isolation and characterisation of a bovine cDNA encoding eukaryotic initiation factor 2 alpha; Green SR et al.; Two cDNA clones have been isolated, from a bovine lymphosarcoma library, that encode the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha) . The predicted 315 amino acid sequence showed more than 99% amino acid identity with rat and human eIF-2 alpha . Galactose-regulated expression of a full length bovine eIF-2 alpha cDNA in yeast resulted in the synthesis of a polypeptide of the predicted molecular mass (36 kDa) . Furthermore, the expressed polypeptide cross-reacted with an antibody raised against rabbit eIF-2 alpha confirming the identity of the cDNA.

Cell, 1991 Oct 4, 67(1), 197 - 211
cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2; Gautier J et al.; cdc25 controls the activity of the cyclin-p34cdc2 complex by regulating the state of tyrosine phosphorylation of p34cdc2 . Drosophila cdc25 protein from two different expression systems activates inactive cyclin-p34cdc2 and induces M phase in Xenopus oocytes and egg extracts . We find that the cdc25 sequence shows weak but significant homology to a phylogenetically diverse group of protein tyrosine phosphatases . cdc25 itself is a very specific protein tyrosine phosphatase . Bacterially expressed cdc25 directly dephosphorylates bacterially expressed p34cdc2 on Tyr-15 in a minimal system devoid of eukaryotic cell components, but does not dephosphorylate other tyrosine-phosphorylated proteins at appreciable rates . In addition, mutations in the putative catalytic site abolish the in vivo activity of cdc25 and its phosphatase activity in vitro . Therefore, cdc25 is a specific protein phosphatase that dephosphorylates tyrosine and possibly threonine residues on p34cdc2 and regulates MPF activation.

Cell, 1991 Oct 4, 67(1), 189 - 96
The cdc25 protein contains an intrinsic phosphatase activity; Dunphy WG et al.; Genetic and biochemical studies have indicated that the cdc25 protein controls the entry into mitosis by triggering tyrosine dephosphorylation of the cdc2 protein kinase . We show that the isolated cdc25 protein can catalyze dephosphorylation of several model phosphatase substrates, including p-nitrophenyl phosphate and two distinct tyrosine-phosphorylated peptides . The cdc25-dependent cleavage reaction closely resembles dephosphorylation by known tyrosine phosphatases: the reaction requires a reducing agent, shows high sensitivity to sodium vanadate, and proceeds efficiently in the presence of metal chelators . Moreover, the phosphatase activity of the cdc25 protein is eliminated by treatment with N-ethylmaleimide or by alteration of a single conserved cysteine residue by site-directed mutagenesis . These observations indicate that the cdc25 protein can function as a tyrosine phosphatase in the absence of any other protein.

Nature, 1991 Oct 3, 353(6343), 437 - 40
A product of the prune locus of Drosophila is similar to mammalian GTPase-activating protein; Teng DH et al.; The X-linked prune (pn) eye-colour mutation of Drosophila melanogaster has a highly specific, complementary lethal interaction with the conditional dominant Killer of prune (awdK-pn) mutation . Although awdK-pn flies have no apparent phenotype on their own, pn awdK-pn double mutants die as second or third larval instars . The awd locus encodes a nucleoside diphosphate kinase, an enzyme that catalyses the transfer of high-energy phosphate bonds between nucleoside diphosphates and nucleoside triphosphates, which is essential for the normal development of Drosophila . Analysis of the pn locus has suggested that the complementary DNA, TcD37, encodes a putative pn+ product . Here we report the nucleotide sequence of TcD37 and the similarity of its deduced protein product to the catalytic domain of mammalian GTPase-activating proteins (GAPs); GAPs stimulate the GTPase activity of Ras (ref . 6), which are plasma membrane-bound proteins involved in the regulation of cell proliferation and differentiation . These results suggest that the Drosophila TcD37 protein participates in a biochemical pathway similar to that of Ras and GAPs in mammals and yeast . We propose that the interaction between pn and awd is due to a neomorphic mutation that enhances the ability of AwdK-pn nucleoside diphosphate kinase to induce a regulatory GTPase into a GTP-bound 'on' state, whereas Pn modulates the activity of this GTPase either by switching it to a GDP-bound 'off' state or by interfering with its effector function.

Mutat Res, 1991 Oct, 261(2), 85 - 91
Dichloroacetonitrile, a by-product of water chlorination, induces aneuploidy in Drosophila; Osgood C et al.; Nitriles have been shown to be potent inducers of aneuploidy in yeast and Drosophila test systems . Haloacetonitriles are by-products of water chlorination that have been shown to be mutagenic and carcinogenic following topical application . In this report we show that dichloroacetonitrile, but not dibromoacetonitrile, is an effective inducer of aneuploidy in oocytes of Drosophila melanogaster . Following inhalation exposure of ZESTE adult females, dichloroacetonitrile (8.6 ppm) induced highly significant increments in the frequencies of sex chromosome loss and gain . Sodium cyanide was also found to be a highly effective inducer of germline aneuploidy, suggesting that cyanide toxicity may contribute to potency of nitriles as inducers of aneuploidy.

Mol Cell Biol, 1991 Oct, 11(10), 4822 - 9
Dominant inhibitory mutations in the Mg(2+)-binding site of RasH prevent its activation by GTP; Farnsworth CL et al.; We have previously demonstrated that substitution of Asn for Ser at position 17 of RasH yields a dominant inhibitory protein whose expression in cells interferes with endogenous Ras function (L . A . Feig, and G . M . Cooper, Mol . Cell . Biol . 8:3235-3243, 1988) . Subsequent structural studies have shown that the hydroxyl group of Ser-17 contributes to the binding of Mg2+ associated with bound nucleotide . In this report, we show that more subtle amino acid substitutions at this site that would be expected to interfere with complexing Mg2+, such as Cys or Ala, also generated dominant inhibitory mutants . In contrast, a Thr substitution that conserves a reactive hydroxyl group maintained normal Ras function . These results argue that the defect responsible for the inhibitory activity is improper coordination of Mg2+ . Preferential affinity for GDP, observed in the original Asn-17 mutant, was found exclusively in inhibitory mutants . However, this binding specificity did not completely block the mutant proteins from binding GTP in vivo since introduction of the autophosphorylation site, Thr-59, in 17N Ras resulted in the phosphorylation of the double mutant in cells . Furthermore, inhibitory mutants failed to activate a model downstream target, yeast adenylate cyclase, even when bound to GTP . Thus, the consequence of improper complexing of Mg2+ was to lock the protein in a constitutively inactive state . A model is presented to explain how these properties could cause the mutant protein to inhibit the activation of endogenous Ras by competing for a guanine nucleotide-releasing factor.

Exp Parasitol, 1991 Oct, 73(3), 354 - 61
Angiostrongylus costaricensis: culture of third-stage larvae to young adults in a defined medium; Hata H et al.; The third-stage larvae of Angiostrongylus costaricensis were successfully cultured to young adults in a chemically defined medium . The most suitable medium for the development was Waymouth's medium among eight defined media examined . Twenty-eight days after cultivation in this medium, 77% of the larvae developed to young adults, although these worms gradually died thereafter . When Waymouth's medium was supplemented with mouse red blood cells, these young adult worms continued their development . The mean body lengths of the worms cultivated in Waymouth's medium supplemented with RBCs were significantly larger than those of the worms in the medium without RBCs on Days 14 and 21 after cultivation . Addition of RBCs was essential for their further development . At 28 days after cultivation, the maximum body length of the worms was 2.1 mm for males and 3.3 mm for females . Additions of serum, yeast extract lactalbumin hydrolysate, and growth factors to Waymouth's medium did not provide any additional benefits for worm development.

EMBO J, 1991 Oct, 10(10), 2965 - 73
Transcriptional activation by heterodimers of the achaete-scute and daughterless gene products of Drosophila; Cabrera CV et al.; The achaete-scute complex (AS-C) and the daughterless (da) genes encode helix-loop-helix proteins which have been shown to interact in vivo and to be required for neurogenesis . We show in vitro that heterodimers of three AS-C products with DA bind DNA strongly, whereas DA homodimers bind weakly and homo or heterocombinations of AS-C products not at all . Proteins unable to dimerize did not bind DNA . Target sequences for the heterodimers were found in the promoters of the hunchback and the achaete genes . Using sequences of the former we show that the DNA binding results obtained in vitro fully correlate with the ability of different combinations to activate the expression of a reporter gene in yeast . Embryos deficient for the lethal of scute gene fail to activate hunchback in some neural lineages in a pattern consistent with the lack of a member of a multigene family.

Plant Mol Biol, 1991 Oct, 17(4), 773 - 85
Substances in nuclear wheat germ extracts which interfere with polymerase III transcriptional activity in vitro; Furter R et al.; Wheat germ nuclear extracts inhibited an active yeast polymerase III (pol III) transcription extract . We isolated two chromatin-associated fractions which harbored biochemically distinguishable inhibitory activities, each contributing about 40-50% to the total inhibitory activity . One fraction, which was released from the chromatin upon treatment with 350 to 900 mM NaCl, was purified to homogeneity and identified as histone H1 . It inhibited the yeast extract by excluding the transcription machinery from the template DNA . It can be partially antagonized by additional nontemplate DNA together with templates that have strong pol III promoters . The other fraction, which was released from the chromatin between 0 and 350 mM NaCl, inhibited transcription by affecting transcription complex formation partially through transcription factor-inhibitor interactions . Furthermore, it affected the rate of transcription reinitiation but not the elongation rate . Ways to move towards an active DNA-dependent pol III plant extract are discussed.

Mol Gen Genet, 1991 Oct, 229(2), 273 - 7
The initiation site for recombination cog is at the 3' end of the his-3 gene in Neurospora crassa; Bowring FJ et al.; Recombination at his-3 in Neurospora crassa is thought to be initiated through a site designated cog which lies in the his-3 to ad-3 interval of linkage group I . Fragments of the his-3 gene were used to transform various his-3 mutant alleles to prototrophy in order to link the genetic map to the nucleotide sequence . It was established that cog is at the 3' end of his-3 and is therefore not the his-3 promoter . This suggests that cog may be dissimilar to a number of yeast recombinators which are associated with promoters of transcription.

Vrach Delo, 1991 Oct, (10), 57 - 60
{The characteristics of the humoral interaction of lymphocytes with neutrophilic granulocytes of the peripheral blood in patients with chronic lympholeukemia under the influence of Proper-Myl}; Tishchenko LM et al.; A study of 32 patients with chronic lympholeucosis (CL) revealed a reduction of the number of phagocytosing neutrophil granulocytes and a reduction of production of humoral mediators by lymphocytes that stimulated the phagocytic function of granulocytes . It is shown that treatment by usually used cytostatic agents did not essentially effect the investigated values . Inclusion of proper-myl in the treatment course of patients with chronic lympholeucosis furthered increase of the number of phagocytosing neutrophil granulocytes and an increased production of humoral mediator stimulating the phagocytic activity of granulocytes.

Protein Eng, 1991 Oct, 4(7), 821 - 9
Mutations in the FAD-binding fold of alcohol oxidase from Hansenula polymorpha; de Hoop M et al.; Alcohol oxidase of methylotrophic yeast is an FAD-containing enzyme . When in its active form, the enzyme is an octamer and located in the peroxisomes . To study the importance of FAD-binding on the activity, octamerization and intracellular localization of the enzyme, alcohol oxidase of Hansenula polymorpha was mutated in its presumed nucleotide-binding domain, which is formed by the N-terminal sequence . Whereas mutations of a glutamic acid residue (E42) reduced the stability of the octamer, it hardly affected enzyme activity and expression . However, replacements of three conserved glycines (G13, G15 and G18) and a conserved glutamic acid (E39) within the fold had severe effects . The mutations not only resulted in loss of enzyme activity but in reduced protein levels as well, probably due to decreased stability of the mutant alcohol oxidase . However, octamerization of the protein still occurred . The existence of inactive octameric proteins provides information about the formation pathway of this octameric flavoprotein.

Biologicals, 1991 Oct, 19(4), 347 - 53
Immunogenicity of hepatitis B surface antigen derived from the baculovirus expression vector system: a mouse potency study; Attanasio R et al.; A standard mouse potency test was performed to evaluate the immunogenicity of recombinant hepatitis B surface antigen (HBsAg) produced in the baculovirus/insect cell expression system . Groups of NIH Swiss mice were immunized with serial four-fold amounts of either baculovirus-derived HBsAg adsorbed to aluminum sulfate or a commercially available yeast-derived recombinant HBsAg vaccine preparation . Results from these experiments showed that the effective dose of baculovirus- and yeast-derived HBsAg vaccine preparations necessary to seroconvert 50% of the animals were similar . The duration of the antibody response to HBsAg was studied in mice immunized with the highest doses of the two recombinant vaccine preparations 3 and 6 months after injection . No decrease in the anti-HBs response was observed 6 months after injection . No decrease in the anti-HBs response was observed 6 months after immunization with either of the two vaccine preparations . These results indicate that the baculovirus-derived recombinant HBsAg could serve as an alternative vaccine candidate for hepatitis B virus.

J Pharm Sci, 1991 Oct, 80(10), 928 - 30
Kinetics of drug action in disease states . XXXVIII: Effect of body temperature on the convulsant activity of pentylenetetrazol in rats; Walker JS et al.; The purpose of this investigation was to determine the effect of body temperature on the pharmacodynamics (convulsant activity) of pentylenetetrazol (PTZ) . Rats received an iv infusion of PTZ until the onset of maximal seizures, at which time samples of cerebrospinal fluid (CSF), brain, and blood (for serum) were obtained for subsequent determination of PTZ concentrations by HPLC . The PTZ infusion caused a decrease in body temperature of approximately 4 degrees C within 20 min and onset of seizures in approximately 40 min . Compared with animals whose temperature was maintained in the normal range by heating pads, the hypothermic rats required significantly larger doses and higher serum, brain, and CSF concentrations of PTZ to produce seizures . Other rats received an injection of brewer's yeast to produce fever . Then, PTZ was infused 6, 12, or 24 h later when body temperature was elevated by an average of 1.3, 1.1, or 0.4 degrees C, respectively . Compared with control rats, whose temperature was maintained in the normal range by heating pads, moderate hyperthermia had no significant effect on the dose and concentrations of PTZ required to produce maximum seizures . Pentylenetetrazol exemplifies a drug that can produce hypothermia which, in turn, reduces the sensitivity of rats to its pharmacologic action . Unlike the central nervous system (CNS) depressants phenobarbital and ethanol, whose pharmacologic activity in rats is enhanced at elevated body temperature, the activity of the CNS stimulant PTZ is apparently not altered by fever.

J Gen Microbiol, 1991 Oct, 137 ( Pt 10), 2469 - 75
A metabolic grid among versiconal hemiacetal acetate, versiconol acetate, versiconol and versiconal during aflatoxin biosynthesis; Yabe K et al.; Dichlorvos treatment of aflatoxigenic Aspergillus parasiticus SYS-4 (NRRL 2999) or a verscolorin A-accumulating mutant, NIAH-9, resulted in accumulation of versiconol acetate (VOAc) and versiconal hemiacetal acetate (VHA), whereas the production of aflatoxins, versicolorin A (VA), and versiconol (VOH) decreased . In feeding experiments using another non-aflatoxigenic mutant, NIAH-26, aflatoxins were newly produced from each of VHA, VOAc, VOH, versicolorin B (VB) and versicolorin C (VC) . In these experiments, aflatoxin production from VHA or VOAc was inhibited by dichlorvos, whereas that from each of VOH, VB and VC was insensitive to dichlorvos . In cell-free experiments using the cytosol fraction of NIAH-26, VHA was converted to VC (or VB) and a substance tentatively identified as versiconal (VHOH) . By further addition of NADH or NADPH to the same reaction mixture, VOAc and VOH were also formed together with VC (VB) and VHOH . VOH was produced from VOAc irrespective of nicotinamide adenine nucleotide . Also, the incubation of VOH in the presence of NAD or NADP led to the formation of VC (VB) . The production of VC (VB) and VHOH from VHA, and that of VOH from VOAc was inhibited by dichlorvos, whereas the production of VOAc from VHA, and that of VC (VB) from VOH, was insensitive to dichlorvos . These results indicate that a metabolic grid catalysed by dehydrogenase and esterase among VHA, VOAc, VOH and VHOH, and a reaction from VHOH to VC (VB) are involved in aflatoxin biosynthesis . These enzyme activities were also detected when yeast extract peptone medium was used, or when A . oryzae SYS-2 was examined.

J Rheumatol, 1991 Oct, 18(10), 1606 - 10
Alkaline phosphatase dissolves calcium pyrophosphate dihydrate crystals; Xu Y et al.; We have shown that yeast pyrophosphatase dissolves calcium pyrophosphate dihydrate (CPPD) crystals in solutions . In this investigation we demonstrate that alkaline phosphatase (ALP) effectively dissolves CPPD crystals in vitro . CPPD dissolution by ALP had a pH optimum of 7.4, which is the optimum pH for its pyrophosphatase (PPiase) activity . The CPPD dissolution and PPiase activity by ALP are magnesium dependent, whereas its phosphoester hydrolytic activity is not . Calcium, which inhibited the enzymatic CPPD dissolution and PPiase activity of ALP had no effect on its phosphoester hydrolytic activity . These data indicate that PPiase activity of ALP is responsible for CPPD dissolution and not its phosphoester hydrolytic activity . Matrix molecules such as proteoglycans and chondroitin sulfate had no effect on the enzymatic and nonenzymatic dissolution of CPPD crystals . ALP acted more effectively on CPPD crystals than on soluble pyrophosphate relative to yeast PPiase . Our data suggest that chondrocyte ALP may play an important role in the dissolution of CPPD crystals in cartilage.

Antimicrob Agents Chemother, 1991 Oct, 35(10), 2128 - 30
Treatment of murine invasive candidiasis with amphotericin B and cilofungin: evidence for enhanced activity with combination therapy; Sugar AM et al.; The in vivo interactions of cilofungin, an echinocandin antifungal agent, and amphotericin B, a polyene derivative, in a murine model of disseminated candidiasis have been investigated . While single therapy with either drug alone prolonged survival of infected mice, kidney colony counts were not appreciably reduced . In contrast, combination therapy, especially at higher doses of both drugs, resulted in significant prolongation of survival and suppression of growth of yeast cells in the kidneys . Combination therapy of experimental candidiasis with cilofungin and amphotericin B did not result in antagonism; rather, additive or synergistic effects were seen . Future preclinical work with other echinocandin and polyene derivatives should include studies evaluating the in vivo interactions of both classes of compounds.

Immunol Lett, 1991 Oct, 30(2), 241 - 8
Genetic regulation of macrophage priming/activation: the Lsh gene story; Blackwell JM et al.; This paper describes functional and genetic studies on the macrophage resistance gene Lsh/Ity/Bcg first described almost two decades ago . Working in vitro with resident peritoneal, liver (Kupffer cells) and bone marrow derived macrophages from congenic B10 (LshS) and B10.L-LshR mice it has been possible to demonstrate that the final effector mechanism for the gene in regulating antileishmanial activity involves production of reactive nitrogen rather than reactive oxygen intermediates . This in turn is dependent upon priming/activation of macrophages for enhanced TNF-alpha release which acts back on the macrophage in an autocrine manner to increase nitric oxide production . The precise point at which Lsh acts to control macrophage priming/activation has not been identified, but studies of early response gene expression show differences in KC mRNA levels at 2 h after LPS stimulation, and in c-fos mRNA as early as 20 min after stimulation with PMA plus ionophore, in peritoneal macrophages from congenic LshS and LshR mice . Data available suggest that both negative and positive signals may be involved in macrophage priming/activation, with LshS macrophages down-regulating their capacity for continued response to the autocrine loop . Work in progress will examine the role of TPA and cAMP response element-binding proteins in regulating gene expression in Lsh congenic mice . A major new initiative has also commenced to clone the Lsh gene by reverse genetics using yeast artificial chromosomes to walk towards Lsh from the closet proximal and distal markers on mouse chromosome 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Zentralbl Bakteriol, 1991 Oct, 275(4), 436 - 50
Minimal requirements for growth of Brucella suis and other Brucella species; Plommet M; Minimal nutritional requirements and temperature limits of growth were studied in Brucella suis and, comparatively, in a few other Brucella species . In a saline basic medium including thiosulphate, ammonium sulphate and glucose with addition of 2 or 4 vitamins (nicotinic acid, thiamin and panthotenic acid, biotin), 24 out of 25 B . suis, 4/6 B . melitensis and 1/6 B . abortus strains were able to grow . Some strains, however, needed to be initially induced to grow by other ingredients, CO2, other vitamins, or amino acids, or by a prolonged incubation . In the saline basic medium without ammonium, glutamic acid and/or alanine and arginine, with or without glucose, supported the growth of all the B . suis and B . melitensis strains, except 2 which required a sulphur amino acid . Five out of 6 B . abortus strains did not grow in either medium without addition of one or several aromatic amino acids or, for one strain, aspartic acid, or valine . One strain could also be induced to grow in ammonium medium by other amino acids . In a rich medium with yeast extract, all Brucella species grew at 18 degrees C and 42.5 degrees (except one) while most B . suis (14/17) grew also at 15 degrees C and 44 degrees C, in contrast to other brucellae of which a few strains only grew at these temperatures . In saline ammonium glucose medium, yeast extract at 0.1 g/l provided all the required vitamins and amino acids for all brucellae and at 1 g/l, it even provided enough nitrogen to support growth without ammonium . Such basic saline medium with yeast extract may be advantageously used in routine Brucella culture, instead of the classic undefined peptone mediums . B . suis biovar 1 strains did not differ significantly in their minimal nutritional requirements, precluding the use of these requirements to differentiate the strains, in particular the Chinese vaccine strain S2 from the reference strain 1330 or from other strains from different parts of the world . Finally, B . suis which is endowed with a nearly complete synthetic potential may represent the parental Brucella species from which the melitensis and abortus species may have evolved.

Alcohol Clin Exp Res, 1991 Oct, 15(5), 804 - 7
Multi-enzyme catalyzed rapid ethanol lowering in vitro; Whitmire DR et al.; Ethanol was oxidized to acetate by an enzyme system using yeast alcohol dehydrogenase (YADH), yeast aldehyde dehydrogenase (YALDH), and lactic dehydrogenase (LDH) recycling NAD in two model duodenal fluids and in canine duodenal aspirate in vitro . Sufficient enzyme activities were maintained to convert as much as 34% of the original ethanol to acetate with negligible acetaldehyde accumulation.

Clin Microbiol Rev, 1991 Oct, 4(4), 411 - 21
Histoplasma variation and adaptive strategies for parasitism: new perspectives on histoplasmosis; Eissenberg LG et al.; This review summarizes the biology of Histoplasma capsulatum in relation to a wide variety of corresponding pathologies in histoplasmosis . Features of these disease syndromes can be explained in part by natural variations within the fungal population and adaptations made by individual organisms to specific environments . H . capsulatum grows as mycelia and conidia in the soil; once inhaled, the organism undergoes a dramatic morphological and physiological conversion to a yeast form . The yeasts proliferate within the phagolysosomes of macrophages, using a variety of specific strategies for intracellular survival . Even avirulent strains or variants are able to avoid being killed by macrophages and instead establish inapparent or persistent infections . The ingested avirulent organisms assume enlarged shapes similar in appearance to those seen in histological sections of tissues from patients with histoplasmosis . Respiratory tract epithelial cells also appear to play a role in persistence: within them yeasts undergo phenotypic switching akin to the phase variation observed in other pathogens . This particular change involves the loss or modification of cell wall alpha-(1,3)-glucan, which is also correlated with the spontaneous appearance of avirulent variants . The repertoire of adaptive responses and natural variations within this species probably evolved from the need to adjust to a wide range of dynamic environments . In combination with the immune status of the host, these characteristics of H . capsulatum appear to influence the epidemiology, extent, and persistence of histoplasmosis.

Virus Res, 1991 Oct, 21(2), 141 - 54
Structural relationships between hepatitis B surface antigen in human plasma and dimers from recombinant vaccine: a monoclonal antibody study; Thanh LT et al.; Ten monoclonal antibodies were obtained from mice immunized with a yeast recombinant hepatitis B vaccine . They were selected at an early stage for their ability to bind to native surface antigen particles (HBsAg) in human plasma . All antibodies recognized conformational epitopes which were destroyed completely or almost completely by reduction of disulphide bridges . They were divided into five epitope groups by their competition for binding to recombinant S protein, though epitopes within each group are not identical . Recombinant S protein migrated on SDS-PAGE in the absence of reducing agents as a mixture of monomers and dimers/oligomers . Sucrose gradient analysis suggests that all these forms are co-aggregated into HBsAg-like particles . On Western blots, all ten antibodies either bound only to dimers/oligomers or strongly preferred them over monomers . The results suggest that, of the antibodies produced in response to recombinant vaccine in mice, most of those which bind strongly to 'native' HBsAg particles in human plasma recognize surface structures created by interaction between two subunits.

Clin Exp Immunol, 1991 Oct, 86(1), 66 - 70
Analysis of antibodies to RNA in patients with systemic lupus erythematosus and other autoimmune rheumatic diseases; Blanco F et al.; The frequency and clinical associations of anti-RNA antibodies measured by ELISA were assessed in 138 patients with systemic lupus erythematosus (SLE) . Of the sera from these patients 9.4% had anti-RNA antibodies but no distinguishing features, clinical, serological or immunogenetic, between those with or without these antibodies could be identified . However, investigations of patients with other autoimmune rheumatic diseases did not reveal any anti-RNA positivity, which indicates a marked disease specificity for anti-RNA antibodies in SLE . The initial anti-RNA antibody screen used a soluble yeast extract as test antigen . The positive sera were further tested against a range of RNAs from 10 different types of rat tissue . In essence few differences were observed, suggesting that the anti-RNA response is directed against common, highly conserved epitopes.

Lab Anim Sci, 1991 Oct, 41(5), 407 - 10
An epizootic of histoplasmosis duboisii (African histoplasmosis) in an American baboon colony; Butler TM et al.; Histoplasmosis duboisii, a chronic granulomatous disease caused by Histoplasma capsulatum var . duboisii, was diagnosed in 21 baboons at a large primate colony in San Antonio, Texas . Diagnosis was based on finding 8 to 15 microns-diameter yeast cells in histologic sections . Therapy with drugs was unsuccessful . Surgical removal of lesions was the primary treatment . Epidemiologic data suggest the incubation period to be at least 9 months . The most likely route of infection was oral and happened during grooming by the baboons.

J Mol Evol, 1991 Oct, 33(4), 367 - 78
The evolution of rhodopsins and neurotransmitter receptors; Fryxell KJ et al.; Rhodopsins share a limited number of amino acid identities with a variety of other integral membrane proteins . Most of these proteins have seven putative transmembrane segments and are likely to play a role in transmembrane signaling . We have undertaken a systematic series of comparisons of primary and secondary structure in order to clarify the functional and evolutionary significance of these sequence similarities . On the basis of consistently high similarity scores, we find that the most internally consistent definition of the rhodopsin gene family would include vertebrate rhodopsins, alpha- and beta-adrenergic receptors, M1 and M2 muscarinic acetylcholine receptors, substance K receptors, and insect rhodopsins, while excluding bacteriorhodopsin, the mass human oncogene, vertebrate and insect nicotinic acetylcholine receptors, and the yeast STE2 and STE3 peptide receptors . The rhodopsin gene family is highly diverged at the primary sequence level but has maintained a conserved secondary structure, including a previously unidentified hierarchy of transmembrane segment hydrophobicity . We have developed new computer algorithms for progressive multiple sequence alignment and the analysis of local conservation of protein domains, and we have used these algorithms to examine the phylogeny of the rhodopsin gene family and the changing domains of sequence conservation . The results show striking differences and similarities in the conserved domains in each of the three main branches of the rhodopsin gene family, and indicate that color vision arose independently in the lines of descent leading to modern humans and fruit flies.

J Virol Methods, 1991 Oct, 34(3), 333 - 41
Detection of immobilised Murray Valley encephalitis virus RNA using oligonucleotide probes with varying degrees of mismatch; Howard MJ et al.; The design of oligonucleotides used for hybridisation studies often utilises available sequence information of the type strain of a particular virus . If hybridisation studies, using such oligonucleotides, are carried out with field isolates of the same virus, the problem of base pair mismatches and consequent difficulties in detection may arise . This study examined the effect of base pair mismatches on the hybridisation between membrane-bound Murray Valley encephalitis virus (MVE) RNA derived from various strains and deliberately mismatched oligonucleotide probes . Under conditions of very low stringency, probes containing up to 5 mismatches were able to detect MVE RNA, but not yeast RNA . Under washing conditions of increased stringency, hybridisation could be detected between MVE virus RNA and probes with only 3 to 4 mismatches . However, the extent of this interaction was dependent on the number and type of mismatches and their relative sequence position.

Exp Mol Pathol, 1991 Oct, 55(2), 119 - 34
The effect of mild hyperthermia on the morphology and function of murine resident peritoneal macrophages; van Bruggen I et al.; During short term culture of murine resident peritoneal macrophages, increasing the temperature from 37 to 39 degrees C resulted in an increased activity of several surface receptors (FcR and receptor for gluteraldehyde-fixed sheep red blood cells), enhanced phagocytosis of yeast particles, improved spreading, and an accelerated reduction of nitroblue tetrazolium . At 41 degrees C, however, significant reduction of several functional properties (endocytosis of colloidal gold and horseradish peroxidase, phagocytosis of yeast particles) and a decrease in the reduction of nitroblue tetrazolium, the incorporation of tritiated uridine, and Fc and C3b surface receptor activity were observed . In addition morphological evidence of apoptosis, observed in a small number of cells cultured at 39 degrees C and in the majority of macrophages maintained at 41 degrees C, was confirmed by DNA electrophoresis . The data indicates that a reduction of several functional activities of macrophages occurs at 41 degrees C and apoptosis may largely account for these effects.

Curr Opin Biotechnol, 1991 Oct, 2(5), 735 - 41
Expression cloning systems; Aruffo A; This review will cover the use of expression cloning in Xenopus oocytes, fission yeast, and mammalian cells . Of the systems covered herein, transient expression cloning systems in Xenopus oocytes and mammalian cells have proven to be the most effective and versatile, as demonstrated by the large number of cDNA clones isolated by these two methods in the past year . Of particular interest, are recent advances in the screening methodologies used in conjunction with transient expression in mammalian cells which have permitted the application of this system in the isolation of cDNAs encoding intracellular proteins.

Biochem Biophys Res Commun, 1991 Sep 30, 179(3), 1181 - 6
Sequence requirements for proteolytic cleavage of precursors with paired basic amino acids; Oda K et al.; When expressed in COS cells, human prorenin was secreted into the medium without being processed to an active renin . Co-expression of furin, a mammalian homologue of the yeast KEX2 gene product, did not affect proteolytic processing of prorenin . A mutant proreninR-4 constructed by site-directed mutagenesis of Pro (-4) to Arg was not cleaved by an endoprotease in the COS cell . However, proreninR-4 was detectably cleaved to yield the active renin upon co-transfection with furin DNA, indicating that Arg at position -4 is important for recognition and processing by furin in addition to the absolute requirement for paired basic amino acids . Another mutant precursor in which Leu (+1) of proreninR-4 was replaced with Ser was found to be much more efficiently processed than proreninR-4, regardless of co-expression of furin . The results suggest that not only a basic amino acid at position -4 but also Leu at position +1 significantly affect the processing of prorenin catalyzed by the COS cell endoprotease or furin.

Cell, 1991 Sep 6, 66(5), 981 - 93
Activation of class II gene transcription by regulatory factors is potentiated by a novel activity; Meisterernst M et al.; A novel activity (USA) stimulated activator-dependent transcription in a reconstituted system in conjunction with natural TFIID, resulting in 10- to 50-fold levels of induction by regulatory factors . USA mediated a modest induction by USF in conjunction with either recombinant human TFIID, intact yeast TFIID, or the evolutionarily conserved C-terminal portion of yeast TFIID . Upon further purification, USA was resolved into two components that had opposite effects on core promoter activity and that in combination potentiated activator function . Gel mobility shift experiments indicated physical interactions between the inhibitory activity and TFIID, suggesting that the additional components (cofactors) associate with the preinitiation complex both to reduce promoter activity in the absence and to increase promoter activity in the presence of transcriptional activators.

J Biol Chem, 1991 Sep 5, 266(25), 16954 - 9
Functional expression of furin demonstrating its intracellular localization and endoprotease activity for processing of proalbumin and complement pro-C3; Misumi Y et al.; We have cloned a rat cDNA encoding furin which is structurally related to yeast Kex2 protease . Products of 88 and 94 kDa were obtained by in vitro transcription/translation of the cDNA in the absence and presence of microsomes . When the cDNA was transfected into COS-1 cells, furin was expressed as a major glycosylated form of 94 kDa, accompanied by a minor proteolytic form of 86 kDa, and found to be localized in the Golgi complex . Proalbumin and complement pro-C3 are intracellularly processed into their mature forms by cleavage at the dibasic residues Arg-Arg, a common cleavage signal found in many pro-type precursors . In cells transfected with the cDNA of C3 or albumin alone, only about half of each proform expressed was processed by an endogenous activity of the cells . When furin was coexpressed, the proforms of both C3 and albumin were completely processed into their mature forms . In addition, co-expression of rat alpha 1-protease inhibitor mutant (Met352----Arg) resulted in inhibition of the endogenous and exogenous processing activities, as observed for the naturally occurring mutant Pittsburgh which has been identified as a specific inhibitor for the processing enzyme . Taken together, these results indicate that furin is an endoprotease localized to the Golgi complex and capable of processing proalbumin and pro-C3 into the mature forms.

Biochemistry, 1991 Sep 3, 30(35), 8661 - 5
Subsite interactions of ribonuclease T1: viscosity effects indicate that the rate-limiting step of GpN transesterification depends on the nature of N; Steyaert J et al.; We report on the effect of the viscogenic agents glycerol and ficoll on the RNase T1 catalyzed turnover of GpA, GpC, GpU, and Torula yeast RNA . For wild-type enzyme, we find that the kcat/Km values for the transesterification of GpC and GpA as well as for the cleavage of RNA are inversely proportional to the relative viscosity of glycerol-containing buffers; no such effect is observed for the conversion of GpU to cGMP and U . The second-order rate constants for His40Ala and Glu46Ala RNase T1, two mutants with a drastically reduced kcat/km ratio, are independent of the microviscosity, indicating that glycerol does not affect the intrinsic kinetic parameters . Consistent with the notion that molecular diffusion rates are unaffected by polymeric viscogens, addition of ficoll has no effect on the kcat/Km for GpC transesterification by wild-type enzyme . The data indicate that the second-order rate constants for GpC, GpA, and Torula yeast RNA are at least partly limited by the diffusion-controlled association rate of substrate and active site; RNase T1 obeys Briggs-Haldane kinetics for these substrates (Km greater than Ks) . Calculations suggest that the equilibrium dissociation constants (Ks) for the various GpN-wild-type enzyme complexes are virtually independent of N whereas the measured kcat values follow the order GpC greater than GpA greater than GpU . This is also revealed by the steady-state kinetic parameters of Tyr38Phe and His40Ala RNase T1, two mutants that follow simple Michaelis-Menten kinetics because of a dramatically reduced kcat value (i.e., Km = Ks).(ABSTRACT TRUNCATED AT 250 WORDS)

Ophthalmology, 1991 Sep, 98(9), 1356 - 9
Ocular histoplasmosis with retinitis in a patient with acquired immune deficiency syndrome; Specht CS et al.; Disseminated histoplasmosis is one of the life-threatening opportunistic infections associated with acquired immune deficiency syndrome (AIDS) . A 29-year-old man with AIDS and disseminated histoplasmosis complained of a hazy spot in the vision of his left eye . Results of examination showed distinct creamy white intraretinal and subretinal infiltrates in both eyes . The patient died within a month from pulmonary infection with Histoplasma capsulatum and cytomegalovirus . Examination with light microscopy showed that the right and left eyes contained histoplasma yeast cells in lesions of retinitis, optic neuritis, and uveitis . These lesions contained variable numbers of lymphocytes and histiocytes . Electron microscopy of the histoplasma in tissue showed characteristic features . This case illustrates the funduscopic appearance and histopathology of histoplasmic retinitis, an uncommon although important ophthalmologic complication of AIDS.

Acta Cytol, 1991 Sep-Oct, 35(5), 557 - 9
Fine needle aspiration diagnosis of Penicillium marneffei infection; Ma KF et al.; A disseminated infection with Penicillium marneffei, a rare human pathogen that may infect both healthy and immunocompromised patients, was diagnosed by fine needle aspiration cytology in a patient infected with the human immunodeficiency virus . The presence of yeast-form organisms with an eccentric or central dot and occasional septate and elongated forms highly suggested the diagnosis, which was confirmed on culture . Establishment of the diagnosis is important because this infection is potentially curable.

Oncogene, 1991 Sep, 6(9), 1555 - 9
Differential expression of two types of the neurofibromatosis type 1 (NF1) gene transcripts related to neuronal differentiation; Nishi T et al.; A 360 residue region encoded by the neurofibromatosis type 1 (NF1) gene shows significant homology to the catalytic domains of both mammalian GTPase-activating proteins (GAP) and yeast IRA proteins . This GAP-related domain of the NF1 gene (NF1-GRD), like the GAP and IRA protein, has been reported to mediate hydrolysis of Ras-bound GTP to GDP, resulting in inactivation of Ras protein . In the present study, we identified two different types of NF1-GRD cDNA . One (type I) is identical to the previously reported sequence, and the other (type II) contained an additional 63 bp insertion that encodes for a region of 21 amino acids in the center of the NF1-GRD molecule . Alternative splicing is the most likely mechanism by which these two types of transcripts arise . Our observations reveal that the type I transcript is predominantly expressed in undifferentiated cells, whereas the type II transcript predominates in differentiated cells . Furthermore, the expression pattern of type I and type II NF1-GRD mRNA immediately changed in SH-SY5Y neuroblastoma cells when neuronal differentiation programs were induced by retinoic acid treatment . We propose that the differential expression of type I and type II NF1-GRD transcripts might be an 'on/off' switch that regulates the catalytic activity of the NF1 gene product, which plays an important role in the regulation of neuronal differentiation.

Mol Gen Genet, 1991 Sep, 228(3), 424 - 32
Identification of the genes coding for the second-largest subunits of RNA polymerases I and III of Drosophila melanogaster; Seifarth W et al.; We have isolated cDNA and genomic clones of Drosophila melanogaster by cross-hybridization with a 658 bp fragment of the yeast gene coding for the second-largest subunit of RNA polymerase III (RET1) . Determination of the sequence by comparison of genomic and cDNA regions reveals an ORF of 3405 nucleotides which is interrupted in the genomic sequence by an intron of 48 bp . The deduced polypeptide consists of 1135 amino acids with a calculated molecular weight of 128 kDa . The protein sequence shows the same conserved regions of homology as those observed for all the second-largest subunits of RNA polymerases cloned so far . The gene (DmRP128) obviously codes for a second-largest subunit of an RNA polymerase which is different from DmRP140 and DmRP135 . We have purified three distinct RNA polymerase activities from D . melanogaster . By using specific RNA polymerase inhibitors in enzyme assays and by comparing their subunit composition we were able to distinguish between RNA polymerase I, II, and III . RNA polymerase preparations of D . melanogaster were blotted and the second-largest subunits were identified with antibodies raised against polypeptides expressed from DmRP128 and DmRP135 . Anti-DmRP135 antibodies react strongly with the second-largest subunit of RNA polymerase I but do not react with the respective subunits of RNA polymerase II and III . The second-largest subunit of RNA polymerase III is only recognized by anti-DmRP128 . Previously, we have claimed that DmRP135 codes for the second-largest subunit of RNA polymerase III.(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Hum Genet, 1991 Sep, 49(3), 656 - 61
Isolation of a human DNA sequence which spans the fragile X; Kremer EJ et al.; To identify the sequences involved in the expression of the fragile X and to characterize the molecular basis of the genetic lesion, we have constructed yeast artificial chromosomes (YACs) containing human DNA and have screened them with cloned DNA probes which map close to the fragile site at Xq27.3 . We have isolated and partly characterized a YAC containing approximately 270 kb of human DNA from an X chromosome which expresses the fragile X . This sequence in a yeast artificial ring chromosome, XTY26, hybridizes to the two closest DNA markers, VK16 and Do33, which flank the fragile site . The human DNA sequence in XTY26 also spans the fragile site on chromosome in situ hybridization . When a restriction map of XTY26, derived by using infrequently cutting restriction enzymes, is compared with similar YAC maps derived from non-fragile-X patients, no large-scale differences are observed . This YAC, XTY26, may enable (a) the fragile site to be fully characterized at the molecular level and (b) the pathogenetic basis of the fragile-X syndrome to be determined.

Proc Natl Acad Sci U S A, 1991 Sep 1, 88(17), 7674 - 8
Evidence for a factor required for transcriptional stimulation by the chimeric acidic activator GAL-VP16 in HeLa cell extracts; White JH et al.; We provide biochemical evidence for the existence of a transcriptional intermediary factor (TIF) in HeLa whole-cell extracts (WCE) that is distinct from the basic transcription factors and that is required for transcriptional stimulation by the chimeric acidic activator GAL-VP16 . We have fractionated HeLa WCE by heparin-agarose chromatography . Of transcriptionally active fractions eluting in a step between 0.24 and 0.6 M KCl, the initial fractions are refractory to GAL-VP16 stimulation, whereas subsequent fractions are strongly stimulated by the activator . Aliquots of GAL-VP16-responsive fractions efficiently complement refractory fractions for transcriptional stimulation . Aliquots of responsive fractions are also far more efficient than those of refractory fractions in overcoming transcriptional inhibition that is brought about by high concentrations of GAL-VP16 . Experiments performed with heat-treated WCE support the idea that HeLa cells contain a TIF that is essential for GAL-VP16 stimulation, but that is not required for basal transcription . Addition of recombinant yeast or human transcription factor TFIID (rTFIIDY and rTFIIDH, respectively) to a WCE heated at 48 degrees C for 15 min restores basal transcription, but in neither case is the reconstituted system activated by GAL-VP16 . However, a 45 degrees C heat-treated WCE reconstituted with either rTFIIDH or rTFIIDY is stimulated by GAL-VP16, suggesting that a HeLa TIF can be selectively inactivated by heating at 48 degrees C, but not at 45 degrees C . Interestingly, a TFIID fraction partially purified from HeLa cell extracts, but not rTFIIDH, efficiently relieves transcriptional inhibition by GAL-VP16, suggesting that there may be an association between TIF(s) and TFIID and, moreover, that TIF(s) may be the direct target of the acidic domain of GAL-VP16 . In summary, our results support the existence of a TIF that is not essential for basal transcription but that is required to mediate the stimulatory activity of the acidic activator GAL-VP16.

Mol Cell Biol, 1991 Sep, 11(9), 4389 - 97
Double-strand gap repair in a mammalian gene targeting reaction; Valancius V et al.; To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells . A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium . The structures of the recombinant loci were then determined by genomic Southern blot hybridizations . We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction . Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms . Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost . These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments.

EMBO J, 1991 Sep, 10(9), 2635 - 44
Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants; Goodall GJ et al.; We have found previously that the sequences important for recognition of pre-mRNA introns in dicot plants differ from those in the introns of vertebrates and yeast . Neither a conserved branch point nor a polypyrimidine tract, found in yeast and vertebrate introns respectively, are required . Instead, AU-rich sequences, a characteristic feature of dicot plant introns, are essential . Here we show that splicing in protoplasts of maize, a monocot, differs significantly from splicing in a dicot, Nicotiana plumbaginifolia . As in the case of dicots, a conserved branch point and a polypyrimidine tract are not required for intron processing in maize . However, unlike in dicots, AU-rich sequences are not essential, although their presence facilitates splicing if the splice site sequences are not optimal . The lack of an absolute requirement for AU-rich stretches in monocot introns in reflected in the occurrence of GC-rich introns in monocots but not in dicots . We also show that maize protoplasts are able to process a mammalian intron and short introns containing stem--loops, neither of which are spliced in N.plumbaginifolia protoplasts . The ability of maize, but not of N.plumbaginifolia to process stem--loop-containing or GC-rich introns suggests that one of the functions of AU-rich sequences during splicing of dicot plant pre-mRNAs may be to minimize secondary structure within the intron.

Plant J, 1991 Sep, 1(2), 141 - 8
Identification of a novel S-phase-specific gene during the cell cycle in synchronous cultures of Catharanthus roseus cells; Ito M et al.; The cell-cycle specific cDNAs were isolated from a cDNA library prepared from cells in the S phase in the synchronous cultures of Catharanthus roseus . One of the isolated genes, which we refer to as cyc07, was analyzed in detail . The full-length cDNA of cyc07 contains an open reading frame of 735 nucleotides, encoding a protein of 245 amino acids with a molecular weight of 28,356 Da . The protein predicted from the nucleotide sequence is highly basic, as are mammalian histones . cyc07 mRNA was detected specifically in cells at the S phase in synchronous cultures . The induction and accumulation of mRNA in the S phase were suppressed when DNA synthesis was inhibited by aphidicolin . In the intact plant, cyc07 mRNA was found preferentially in root tips that contained meristematic tissue . A databank search revealed that a sequence homologous to the nucleotide sequence of cyc07 cDNA is present in the downstream region of the SIR3 gene in the yeast genome . The amino acid sequence predicted from the corresponding region of the yeast genome exhibited significant homology with that of cyc07 protein . These similarities between cyc07 and the corresponding region in yeast suggest that the homologous sequence in yeast is a novel gene that is functionally homologous to cyc07 . Our results presented here suggest the possibility that cyc07 may play a role in the proliferation of higher plant cells, in particular in the entry into or progression of the S phase of the cell cycle.

Clin Exp Dermatol, 1991 Sep, 16(5), 331 - 8
An immunological study in patients with seborrhoeic dermatitis; Bergbrant IM et al.; The humoral and cellular immune-status was studied in 30 patients with seborrhoeic dermatitis . Increased frequencies of natural killer cells were found in 46% of patients . Furthermore, subnormal mitogen stimulation responses were demonstrated in 13 patients, whereas two individuals were found to have very high numbers of activated T lymphocytes in peripheral blood . Higher-than-normal total serum IgG and IgA was observed in 14 and 11 patients, respectively . For nine of 12 patients with skin lesions, dermal perivascular cell infiltrates were seen . The majority of the infiltrating cells reacted with anti-CD4 antibodies . HLA-DR-expressing keratinocytes were found in two biopsies . The study suggests that patients with seborrhoeic dermatitis may have depressed T-cell function . This could have a bearing on their susceptibility to the Pityrosporum ovale-associated dermatitis . The very high frequencies of activated T cells observed in the peripheral blood of two otherwise healthy seborrhoeic individuals suggests that intermittent systemic immune activation may occur . Seborrhoeic dermatitis is a common skin disease . It can be diagnosed by its characteristic red to yellow-brown lesions covered with greasy scales distributed in areas with a high number of sebaceous glands, such as the scalp, face and upper trunk . There is an association between seborrhoeic dermatitis and the lipophilic yeast Pityrosporum ovale but its exact aetiological role is not known . The yeast is a member of the normal cutaneous flora but also the aetiological agent of pityriasis versicolor and Pityrosporum folliculitis . P . ovale can activate complement via the direct and alternative pathways . This may play some part in the induction of inflammation.(ABSTRACT TRUNCATED AT 250 WORDS)

Arh Hig Rada Toksikol, 1991 Sep, 42(3), 267 - 79
{Immunologic changes and pulmonary ventilatory function in animal feed processing workers}; Zuskin E et al.; Respiratory symptoms and immunological reactions were examined in 35 animal food workers . The most frequent positive skin prick reactions occurred to fish flour (82.9%), followed by carotene (77.1%), cornflour (65.7%), four-leaf clover (62.9%), sunflower (54.3%), chicken meat (31.4%), soy (28.6%) and yeast (22.7%) . The IgE serum level was increased in 40% of the animal food workers and in 2.6% of the controls . A significantly higher prevalence of chronic respiratory symptoms was found in animal food workers than in controls . However, there was no significant difference in prevalence of chronic respiratory symptoms between workers with positive and those with negative skin tests to house dust and fish flour or between those with increased and those with normal IgE levels (except for dyspnoea) . There were significant acute across-shift reductions in ventilatory capacity, particularly for FEF25 . The workers with positive skin tests to fish flour demonstrated significantly larger acute FEF25 reductions than those with negative skin tests . An extract of animal food caused constriction of isolated guinea pig tracheal smooth muscle in vitro . It appears that animal food dust in addition to immunological response may produce a direct irritative effect on the airways of exposed workers.

Chem Pharm Bull (Tokyo), 1991 Sep, 39(9), 2301 - 7
Synthesis and antifungal activities of a series of (1,2-disubstituted vinyl)imidazoles; Ogawa M et al.; A series of vinylimidazoles containing a hetero atom such as sulfur or oxygen at a beta-position of the vinyl group was prepared and the antifungal activities were tested . It was found that sulfur-substituted derivatives such as (E)-1-{2-(methylthio)-1-{2-(pentyloxy)phenyl}ethenyl}-1H-imidazole (5a-5) and (E)-1-{1-{2-(hexyloxy)phenyl}-2-(methylthio)ethenyl}-1H-imidazole (5a-6) showed excellent antifungal activities against dermatophytes and yeast cells . The stereochemistry of the hydrochloride salt of 5a-5 was determined by X-ray crystallography . The structure-activity relationships were discussed.

Indian J Exp Biol, 1991 Sep, 29(9), 875 - 6
Effect of selected chemicals on mosquito larval orientation behaviour using a new apparatus; Vartak PH et al.; An apparatus was designed and fabricated for studying the effects of aromatic chemicals on the IV instar larvae of Aedes aegypti (L.) . The test chemicals (essential oils and monoterpenoids) were incorporated in agar along with yeast and dog biscuits which formed an attractant food source (bait) . A fairly rapid decrease in the migration of larvae towards the bait cum chemical source was observed during the first hour after which a steady state was observed . Minimum migration (9.67%) was seen with terpeneol anhydrous and maximum (15.0%) with beta-citronellal as compared to control (65.67%), at the end of a 4 hr exposure period . Food particles were also detected on microscopic examination.

Mol Biochem Parasitol, 1991 Sep, 48(1), 47 - 58
Induction and localization of Plasmodium falciparum stress proteins related to the heat shock protein 70 family; Kumar N et al.; Induction of heat shock-related stress proteins Pfhsp and Pfgrp, similar in sequence to hsp70 (heat shock protein) and grp78 (glucose-regulated protein), respectively, was studied in culture-derived parasite Plasmodium falciparum . Elevation in temperature from 26 degrees C to 37 degrees C and higher caused significant induction of Pfhsp with a moderate effect on the synthesis of Pfgrp also . Synthesis of Pfgrp, however, was not induced by partial glucose deprivation . On the contrary, lack of glucose in the medium resulted in cessation of protein synthesis in the parasites . Other known inducers of grp synthesis in mammalian cells, i.e., calcium ionophore A23187 and inhibitors of glycosylation (tunicamycin, 2-deoxy glucose) were also without any apparent effect on the synthesis of Pfgrp . Heat shock-induced responses were transient in nature: removal of stress caused repression of these responses . The effect of glucose deprivation was only partially reversible with better recovery if parasites were subjected to glucose starvation at 26 degrees C than at 37 degrees C . Northern blot analysis and in vitro translation of mRNA revealed a parallel increase in the levels of mRNA for Pfhsp upon heat shock . Immuno-gold electron microscopy with cultured parasites revealed nuclear location of Pfhsp and primarily cytoplasmic (probably endoplasmic reticulum) location of Pfgrp . These findings suggest that SDEL (carboxy terminal sequence of Pfgrp) might play a similar role in the cellular localization of Pfgrp as does the sequence KDEL in mammalian cells and HDEL in yeast.

Blood Rev, 1991 Sep, 5(3), 177 - 92
Cell cycle regulation; Bybee A et al.; Over the past few years there has been a resurgance of interest in the cell cycle . The excitement has mainly been due to the fact that researchers all over the world who had been working on seemingly different processes in yeast, fruit flies, frogs and man have found that many of the processes and individual proteins have been highly conserved . In essence, what regulates the cell cycle in yeast also works in man . This review is biased towards cell cycle regulation in mammalian cells but we have also covered what is known about similar mechanisms in other organisms to provide a broader perspective to the field . We outline what is currently known about the cell cycle and the key points at which cell proliferation is controlled . We summarise recent work on cell cycle control genes and antioncogenes and the post-transcriptional regulation of the proteins for which they code . Finally we cover the relationship between the cell cycle and differentiation.

J Clin Microbiol, 1991 Sep, 29(9), 2007 - 12
Application of an indirect immunofluorescence test for detection of Mycoplasma pneumoniae in respiratory exudates; Hirai Y et al.; We prepared polyclonal antibody specific to Mycoplasma pneumoniae and examined the conditions influencing the ability of an indirect immunofluorescence test to detect the specific antigen in respiratory exudates . The antibody did not cross-react with normal human serum or with respiratory exudates from 10 healthy persons . Cross-reactivity of the antibody with species of mycoplasmas other than M . genitalium was fully diminished when absorbed with horse serum and yeast extract, components of the culture medium . Though the absorbed antibody cross-reacted with M . genitalium, the titer was significantly lower than when tested against M . pneumoniae . Two types of antigen-specific fluorescence were observed in clinical specimens: one is large or small fluorescent granular aggregates found in mucus, and the other is fine fluorescent particles diffused on the entire surface of small epithelial cells . Throat smears from 49 patients with serologically confirmed M . pneumoniae infections were examined by our indirect immunofluorescence method . Positive results were obtained in 42 cases, many of which were positive before a rise in serum antibody titer could be demonstrated, indicating that the method is useful for a preliminary diagnosis at an early stage of the infection.

Vopr Med Khim, 1991 Sep-Oct, 37(5), 73 - 6
{The effect of selenium on enzymes metabolizing xenobiotics in rat liver}; Kravchenko LV et al.; Activity of enzymes involved in metabolism of xenobiotics was not altered in liver tissue of rats kept on a ration enriched with selenium at a dose of 2.5 mg/kg . Both organic form of selenium (yeast meal, selenomethionine) and inorganic derivatives (sodium selenite) at a dose of 5 mg/kg of ration caused distinct activation of epoxide hydrolase, UDP-glucuronosyl transferase and glutathione transferase within 6 weeks after the experiment beginning, while content of cytochrome P-450, glutathione-SH and glutathione peroxidase activity were not significantly altered . Within 9 weeks the enzymatic activity remained at the higher rate only in rats kept on the ration with sodium selenite . Relationship between toxic effects of selenium high doses and alterations in activity of enzymes involved in metabolism of xenobiotics is discussed.

J Dairy Sci, 1991 Sep, 74(9), 2965 - 75
Isolation and phagocytic properties of neutrophils and other phagocytes from nonmastitic bovine milk; Sandgren CH et al.; A technique for the separation of neutrophils from macrophages-epithelial cells in samples of nonmastitic bovine milk with low cell counts has been developed . The procedure is based on centrifugation in a discontinuous metrizamide gradient and is rapid, taking less than 40 min . The recovery of the neutrophils is about 30% and their viability about 90% . The isolated neutrophils showed an appreciable unstimulated luminol- and lucigenin-dependent chemiluminescence, which was due to NADPH oxidase rather than to xanthine oxidase . The neutrophils had a higher rate of ingestion of C3-opsonized particles than macrophages-epithelial cells, whereas no significant differences in phagocytosis of IgG-opsonized yeast or unopsonized yeast were detected between the two cell populations . The macrophages-epithelial cells produced no luminol-dependent chemiluminescence and induced considerably lower activity in the lucigenin-dependent system than neutrophils, indicating that these cells contain no myeloperoxidase . Analyses of the activity of the neutrophils in response to C3-opsonized yeast particles showed that the luminol-dependent chemiluminescence of cells isolated from residual milk increased significantly over the lactation period . Moreover, a tendency to a higher phagocytosis and chemiluminescence of neutrophils isolated from residual milk than from stripping milk was indicated.

Curr Genet, 1991 Sep, 20(4), 319 - 29
The first analysed archegoniate mitochondrial gene (COX3) exhibits extraordinary features; Marienfeld JR et al.; The first mitochondrial-encoded gene of an archegoniate has been identified, cloned and sequenced . The cytochrome oxidase III gene (cox3) of the moss Physcomitrella patens consists of a 618 bp open reading frame with high homology (around 72%) to known cox3 sequences of higher plants . Nevertheless, it is a quarter shorter than these . The cox3 gene of P . patens contains no introns and reveals a G + C-content of 41.3% . The region containing the cox3 gene exists as a single copy in the mitochondrial genome as shown by restriction mapping . In the 5' flanking sequence a putative ribosome binding site and a putative secondary structure were found . Two main transcripts of 2.4 kb and 2.6 kb were detected indicating a complex mitochondrial transcription pattern possibly due to co-transcription . Additional open reading frames were found downstream from, as well as upstream of, the cox3 gene . In Western blots a polyclonal cox3 antibody from yeast detected one single band with an apparent molecular weight of 22 kDa.

Biochem Cell Biol, 1991 Sep, 69(9), 586 - 607
Cytochrome c oxidase: structure, function, and membrane topology of the polypeptide subunits; Cooper CE et al.; Mitochondrial cytochrome c oxidase and its bacterial homologs catalyze electron transfer and proton translocation reactions across membranes . The eukaryotic enzyme complex consists of a large number of polypeptide subunits . Three of the subunits (I, II, and III) are mitochondrially encoded while the remaining 6 (yeast) to 10 (bovine) are nuclear encoded . Antibody and chemical-labelling experiments suggest that subunits I-III and most (but not all) of the nuclear-encoded subunits span the inner mitochondrial membrane . Subunits I and II are the catalytic core of the enzyme . Subunit I contains haem a, haem a3 and CuB, while subunit II contains CuA and the cytochrome c binding site . Subunit III and most of the nuclear subunits are essential for the assembly of a functional catalytic enzyme . Some nuclear subunits are present as isozymes, although little functional difference has yet been detected between enzyme complexes composed of different isozymes . Therefore, any additional role attributed to the nuclear-encoded subunits beyond that of enzyme assembly must be tentative . We suggest that enough evidence exists to support the idea that modification of the larger nuclear subunits (IV, V, and possibly VI) can effect enzyme turnover in vitro . Whether this is a physiological control mechanism remains to be seen.

Nature, 1991 Aug 29, 352(6338), 821 - 4
Genetic evidence for base pairing between U2 and U6 snRNA in mammalian mRNA splicing; Datta B et al.; Removal of introns from eukaryotic nuclear messenger RNA precursors is catalysed by a large ribonucleoprotein complex called the spliceosome, which consists of four small nuclear ribonucleoprotein particles (U1, U2, U5, and U4/U6 snRNPs) and auxiliary protein factors . We have begun a genetic analysis of mammalian U2 snRNA by making second-site mutations in a suppressor U2 snRNA . Here we find that several mutations in the 5' end of U2 (nucleotides 3-8) are deleterious and that one of these can be rescued by compensatory base changes in the 3' end of U6 (nucleotides 92-95) . The results demonstrate genetically that the base-pairing interaction between U2 (nucleotides 3-11) and U6 snRNA (nucleotides 87-95), originally proposed on the basis of psoralen photocrosslinking experiments, can influence the efficiency of mRNA splicing in mammals . The U2/U6 interaction in yeast, however, is fairly tolerant to mutation (D.J . Field and J.D . Friesen, personal communication), emphasizing the potential for facultative RNA interactions within the spliceosome.

Nature, 1991 Aug 29, 352(6338), 803 - 7
Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A; Flanagan WM et al.; Cyclosporin A and FK506 inhibit T- and B-cell activation and other processes essential to an effective immune response . In T lymphocytes these drugs disrupt an unknown step in the transmission of signals from the T-cell antigen receptor to cytokine genes that coordinate the immune response . The putative intracellular receptors for FK506 and cyclosporin are cis-trans prolyl isomerases . Binding of the drug inhibits isomerase activity, but studies with other prolyl isomerase inhibitors and analysis of cyclosporin-resistant mutants in yeast suggest that the effects of the drug result from the formation of an inhibitory complex between the drug and isomerase, and not from inhibition of isomerase activity . A transcription factor, NF-AT, which is essential for early T-cell gene activation, seems to be a specific target of cyclosporin A and FK506 action because transcription directed by this protein is blocked in T cells treated with these drugs, with little or no effect on other transcription factors such as AP-1 and NF-kappa B . Here we demonstrate that NF-AT is formed when a signal from the antigen receptor induces a pre-existing cytoplasmic subunit to translocate to the nucleus and combine with a newly synthesized nuclear subunit of NF-AT . FK506 and cyclosporin A block translocation of the cytoplasmic component without affecting synthesis of the nuclear subunit.

Biochim Biophys Acta, 1991 Aug 27, 1090(1), 115 - 8
An adult male specific gene in Drosophila containing the repetitive element opa; Grabowski DT et al.; A cDNA has been isolated for an adult male specific gene in Drosophila that contains the repetitive element opa . We have named this gene Dromsopa for Drosophila male specific opa containing gene . The 0.6 kb mRNA for this gene is only found in the abdominal region of adult male Drosophila and in no other tissue or at other developmental stages . The Dromsopa opa repeat codes for the usual stretch of poly(glutamine) interrupted by histidine residues . The opa repetitive element was originally found in the Drosophila Notch gene (Kidd, S . et al . (1983) Cell 34, 431-433 and Wharton, K.A . (1985) Cell 40, 55-62) and has, more recently, been found in genes under developmental or tissue specific control from yeast to humans . The gene was cloned using a genomic fragment during a chromosomal walk upstream of the AP3 gene located at chromosomal location 79CD on the left arm of the third chromosome (Kelley, M.R . et al . (1989) Mol . Cell . Biol . 9, 965-973) . The Dromsopa gene has no other identity with genes currently in the databases, once the opa repeat is excluded . The possibility that the Dromsopa gene is a male specific regulatory factor is under investigation as is its precise location within the abdomen, such as in germ line tissue.

Biochim Biophys Acta, 1991 Aug 27, 1090(1), 102 - 8
Molecular cloning of cDNA for antimycin A-inducible mRNA and its role in cyanide-resistant respiration in Hansenula anomala; Sakajo S et al.; A cDNA for mRNA induced by antimycin A in Hansenula anomala was cloned . The mRNA for the cDNA was expressed in the yeast under the conditions expressing the cyanide-resistant respiration activity . The nucleotide sequence revealed a long open reading frame of 342 codons encoding a protein with a molecular weight of 40,282 in the cDNA . An antibody recognizing the protein encoded by the open reading frame was produced . Immunoblotting of H . anomala proteins with this antibody showed that a 36 kDa protein localized in mitochondria was a mature form of the protein encoded by the cDNA . It is suggested that the cloned cDNA encodes a protein involved in the cyanide-resistant respiratory pathway.

Biochim Biophys Acta, 1991 Aug 27, 1090(1), 125 - 8
Nucleotide sequence of a cDNA coding for the mitochondrial precursor protein of cytochrome c oxidase subunit IV from the slime mold Dictyostelium discoideum; Rizzuto R et al.; Subunit-specific polyclonal antibodies were used to isolate cDNA clones encoding subunit IV of Dictyostelium discoideum cytochrome c oxidase . DNA sequence analysis reveals an open reading frame of 149 amino acids . As shown by sequencing of the protein N-terminus, the subunit is synthesized with a 24 residue cleavable presequence which leads to a mature polypeptide of 14305 Da . The slime mold subunit exhibits a low but significant degree of similarity with subunit Va of human and subunit VI of yeast cytochrome c oxidase.

J Biol Chem, 1991 Aug 25, 266(24), 15992 - 8
Missense mutation serine106----proline causes 17 alpha-hydroxylase deficiency; Lin D et al.; Steroid 17 alpha-hydroxylase deficiency is caused by defects in cytochrome P450c17, the single enzyme that has 17-alpha hydroxylase and 17,20-lyase activities . We describe a rapid and efficient polymerase chain reaction tactic for identifying these genetic lesions and identify Ser106----Pro as the cause of 17 alpha-hydroxylase deficiency in two unrelated homozygous patients from Guam . We used site-directed mutagenesis of the normal P450c17 cDNA to construct the Pro106 mutant, and expressed both the normal and mutant sequences in monkey COS-1 cells and in yeast . Expression of the normal sequence permitted the cells to convert pregnenolone to 17-OH pregnenolone, progesterone to 17-OH progesterone, and 17-OH pregnenolone to dehydroepiandrosterone, showing the normal sequence conferred both 17 alpha-hydroxylase and 17,20-lyase activities . Expression of the mutant sequence generated P450c17 mRNA, but conferred none of these activities, proving that the Ser106----Pro mutation abolished the 17 alpha-hydroxylase and 17,20-lyase activities . An HhaI restriction site created by the mutation should permit screening of large populations.

J Mol Biol, 1991 Aug 20, 220(4), 903 - 14
Use of high coverage reference libraries of Drosophila melanogaster for relational data analysis . A step towards mapping and sequencing of the genome; Hoheisel JD et al.; Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated . Also, a jumping library has been created by a new method that takes advantage of methylation differences between genomic DNA and vector . Thirdly, two cDNA libraries have been picked . All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones . As a reference system, such filters are distributed and identified clones are provided . Single-copy probes have identified on average 1.4 cosmids per genome equivalent . Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations . cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization.

J Mol Biol, 1991 Aug 20, 220(4), 843 - 53
Unidirectional replication as visualized by two-dimensional agarose gel electrophoresis; Martin-Parras L et al.; Two-dimensional (2D) agarose gel electrophoresis is progressively replacing electron microscopy as the technique of choice to map the initiation and termination sites for DNA replication . Two different versions were originally developed to analyze the replication of the yeast 2 microns plasmid . Neutral/Neutral (N/N) 2D agarose gel electrophoresis has subsequently been used to study the replication of other eukaryotic plasmids, viruses and chromosomal DNAs . In some cases, however, the results do not conform to the expected 2D gel patterns . In order to better understand this technique, we employed it to study the replication of the colE1-like plasmid, pBR322 . This was the first time replicative intermediates from a unidirectionally replicated plasmid have been analyzed by means of N/N 2D agarose gel electrophoresis . The patterns obtained were significantly different from those obtained in the case of bidirectional replication . We showed that identification of a complete are corresponding to molecules containing an internal bubble is not sufficient to distinguish a symmetrically located bidirectional origin from an asymmetrically located unidirectional origin . We also showed that unidirectionally replicated fragments containing a stalled fork can produce a pattern with an inflection point . Finally, replication appeared to initiate at only some of the potential origins in each multimer of pBR322 DNA.

Biochim Biophys Acta, 1991 Aug 20, 1085(1), 126 - 30
Lipid hydrogenation induces elevated 18:1-CoA desaturase activity in Candida lipolytica microsomes; Horvath I et al.; Microsomal membranes prepared from the mesophilic yeast Candida lipolytica grown at 10 degrees C were hydrogenated by the homogeneous Pd-catalyst, palladium di (sodium alizarine sulfonate) (Pd(QS)2) . After hydrogenation to various levels, the microsomes were washed free of the Pd-complex and transferred to a reaction mixture (containing NADH, MgCl2, ATP, CoA and {14C}18:1-CoA) for assay of 18:1-CoA desaturase activity . Microviscosity alterations were also followed by measuring changes in DPH fluorescence polarization . Rapid catalytic hydrogenation of unsaturated fatty acids of the lipids occurred within 20-120 s, resulting in large increases in 16:0, 18:0 and 18:1 acids and decreases in 18:2 acid . In the range 7-20% 18:0 content, a pronounced increase in desaturase activity was observed, with a maximum of greater than 2-fold at a 18:0 content of 12%, followed by a decrease to the initial activity at 33% 18:0 content . These changes were well-correlated with changes in microviscosity, maximal desaturase activity occurring in the DPH fluorescence anisotropy range of 0.23-0.24; above and below this range, desaturase activities were close to the initial control values . It is suggested that the hydrogenation-induced increase in the formation of 18:2 from 18:1-CoA (proceeding partly through direct desaturation of PC) may be due to changes in conformation of the membrane-bound desaturase enzyme complex as a result of controlled rigidification of the surrounding lipids . The operation of such a self-regulating control mechanism would be consistent with a previously proposed model for microsomal desaturase action.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 7006 - 10
Functional reintroduction of human telomeres into mammalian cells; Farr C et al.; Telomeric sequences of eukaryotes consist of short tandem repeats organized in arrays of variable length in which the guanine-rich strand runs 5'----3' toward the chromosomal end . The terminal repeats in yeast are the only elements necessary for telomere function in this organism . To test whether mammalian terminal repeats can function after reintroduction into a mammalian cell, a repeat-containing terminal fragment from a human chromosome was electroporated into a hamster-human hybrid cell line . In 6 of 27 independent transformants analyzed, the introduced sequences were found at the ends of chromosomes, based on all available criteria . Terminal restriction-fragment heterogeneity and the survival of these chromosomes demonstrate that these telomeres are functional . Cytogenetic evidence from one of these cell lines suggests that chromosome breakage with healing at the integration site is the mechanism responsible for the terminal location.

J Biol Chem, 1991 Aug 15, 266(23), 15202 - 12
Structural requirements for transformation of substrates by the (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase; Nes WD et al.; The membrane-bound enzyme of microsomes obtained from sunflower embryos that catalyzes the bi-substrate transfer reaction whereby the methyl group of (S)-adenosyl-L-methionine is transferred to C-24 of the sterol side chain has been investigated . Optimal incubation conditions for assay of the microsomal (S)-adenosyl-L-methionine:sterol delta 24-methyl transferase (SMT) have been established for the first time . The microsomal preparation was found to catalyze the formation of a delta 24(28)-sterol and to be free of contaminating methyl transferase enzymes, e.g . those which form delta 23-24 methyl sterols (cyclosadol) and delta 25-24 beta-methyl sterols (cyclolaudenol) and other sterolic enzymes which might transform the acceptor molecule to metabolites which could compete in the assay with the test substrate . From a series of incubations with 27 sterol and sterol-like (triterpenoids) substrates of which 23 compounds possessed a 24,25-double bond, we observed a marked dependence on precise structural features and three-dimensional shape of the acceptor molecule in its ability to be transformed by the SMT . In contrast to the yeast SMT where cycloartenol fails to bind to the SMT and zymosterol is the best substrate for methylation, the sunflower SMT studied here utilizes cycloartenol preferentially to zymosterol and the other substrates . Of the chemical groups which distinguishes cycloartenol, a free 3 beta-OH,9 beta,19-cyclopropyl group, trimethylated saturated nucleus, and delta 24-double bond, only the nucleophilic centers at C-3 and C-24 were obligatory for substrate binding and methylation . Of the bent or flat conformations which cycloartenol may orient in the enzyme-substrate complex, our results indicate a selection for acceptor molecules which possess the shape that closely resembles the crystal state and solution orientation of cycloartenol which is now known to be flat rather than bent (Nes, W . D., Benson, M., Lundin, R . E., and Le, P . H . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 5759-5763).

J Biol Chem, 1991 Aug 15, 266(23), 15060 - 7
Isolation and structural characterization of the Chlamydomonas reinhardtii gene for cytochrome c6 . Analysis of the kinetics and metal specificity of its copper-responsive expression; Hill KL et al.; We have isolated a 5-kilobase pair fragment of genomic DNA containing the entire coding region for the Chlamydomonas reinhardtii gene encoding the copper-repressible Cyt c6 . A region comprising 2.6 kilobase pairs contains the entire transcribed region plus 852 nucleotides upstream of the Cyt c6 transcription start site and 495 nucleotides downstream of the conserved C . reinhardtii polyadenylation signal . Comparison of the genomic sequence with the cDNA sequence (Merchant, S., and Bogorad, L . (1987) J . Biol . Chem . 262, 9062-9067) revealed that the coding region is interrupted by two introns, each of which is flanked by C . reinhardtii consensus intron/exon boundaries . Primer extension and S1 nuclease protection analyses identified the 5' border of the Cyt c6 mRNA at approximately 79 base pairs upstream from the initiator methionine . Analysis of the 5' upstream region reveals no significant similarity to sequences found in upstream regions of other copper-regulated genes . Time-course studies indicate that 1) the mature Cyt c6 mRNA has a half-life of approximately 45-60 min and is completely lost within 4 h, and 2) the primary, unspliced transcript has a half-life of approximately 10 min and is completely lost within 30 min after the addition of copper ions to copper-depleted cells . These results indicate that the response to copper occurs very rapidly upon elevation of extracellular copper levels . Although this gene is unresponsive to silver ions in vivo, in contrast to the yeast copper-responsive CUP1 gene (Furst, P., Hu, S., Hackett, R., and Hamer, D . (1988) Cell 55, 705-717), it does respond to mercury ions, albeit with less sensitivity . Mercury ions cannot, however, substitute for copper in allowing the accumulation of plastocyanin in vivo.

Genomics, 1991 Aug, 10(4), 921 - 6
Linkage mapping and fluorescence in situ hybridization of TCTE1 on human chromosome 6p: analysis of dinucleotide polymorphisms on native gels; Kwiatkowski TJ Jr et al.; Highly informative dinucleotide repeat polymorphisms were identified at the T-complex-associated-testes-expressed-1 (TCTE1) locus on human chromosome 6p . Electrophoresis of single-stranded DNA on native gels facilitated the analysis of the dinucleotide polymorphisms . Linkage mapping positions this marker midway between the centromere and HLA with recombination fractions as follows: D6Z1-0.21-TCTE1-0.24-HLA . Two-color fluorescence in situ hybridization places TCTE1 proximal to CRIL171 (D6S19) . Together, linkage and in situ hybridization indicate that the order of the loci is D6Z1-D6S4-D6S90-TCTE1-D6S19-D6S29-HL A-telomere . A sequence tagged site (STS) was established, and three yeast artificial chromosome (YAC) clones were identified for the TCTE1 locus.

Nucleic Acids Res, 1991 Aug 11, 19(15), 4075 - 81
Transcript mapping reveals different expression strategies for the bicistronic RNAs of the geminivirus wheat dwarf virus; Dekker EL et al.; We have characterised the major transcripts of the Czech isolate of wheat dwarf virus (WDV-CJI) which show that WDV uses two different mechanisms for expressing overlapping open reading frames (ORFs) . Mapping of the virion sense RNAs identified a single polyadenylated transcript of 1.1kb spanning the overlapping ORFs V1 and V2 which encode cell-cell spread functions and the coat protein respectively . This finding distinguishes WDV from other monocot-infecting geminiviruses studied so far which were shown to encode two 3' co-terminal transcripts capable of expressing either the V1 or V2 ORF . A survey of codon usage at the junction between the V1 and V2 ORF has led us to propose that translational frame shifting analogous to that in the yeast Ty element may occur . Analysis of polymerase chain reaction (PCR) amplified complementary sense cDNA clones has revealed the presence of mature spliced and unspliced RNAs which could encode products of an intron mediated C1:C2 ORF fusion or the C1 ORF product alone . Mapping of the 5' and 3' extremities of the major WDV encoded transcripts has allowed us to identify putative transcription regulatory sequences and the presence of multiple overlapping transcripts may suggest temporal regulation of transcription.

Cell, 1991 Aug 9, 66(3), 451 - 64
The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions; Woods DF et al.; Mutations of the lethal(1)discs large-1 (dlg) tumor suppressor gene of Drosophila cause neoplastic overgrowth of the imaginal discs . Sequencing of a near full-length cDNA predicts a protein containing a domain homologous to yeast guanylate kinase and a region homologous to SH3, a putative regulatory motif in nonreceptor protein tyrosine kinases and other signal transduction proteins . Immunofluorescence analysis using antibodies directed against fusion peptides shows that the dlg gene product is localized in an apical belt of the lateral cell membrane, at the position of the septate junction . The results suggest that a signal transduction process involving guanine nucleotides occurs at the septate junction and is necessary for cell proliferation control in Drosophila epithelia.

J Biol Chem, 1991 Aug 5, 266(22), 14754 - 62
Human lysosomal protective protein has cathepsin A-like activity distinct from its protective function; Galjart NJ et al.; The protective protein was first discovered because of its deficiency in the metabolic storage disorder galactosialidosis . It associates with lysosomal beta-galactosidase and neuraminidase, toward which it exerts a protective function necessary for their stability and activity . Human and mouse protective proteins are homologous to yeast and plant serine carboxypeptidases . Here, we provide evidence that this protein has enzymatic activity similar to that of lysosomal cathepsin A: 1) overexpression of human and mouse protective proteins in COS-1 cells induces a 3-4-fold increase of cathepsin A-like activity; 2) this activity is reduced to approximately 1% in three galactosialidosis patients with different clinical phenotypes; 3) monospecific antibodies raised against human protective protein precipitate virtually all cathepsin A-like activity in normal human fibroblast extracts . Mutagenesis of the serine and histidine active site residues abolishes the enzymatic activity of the respective mutant protective proteins . These mutants, however, behave as the wild-type protein with regard to intracellular routing, processing, and secretion . In contrast, modification of the very conserved Cys60 residue interferes with the correct folding of the precursor polypeptide and, hence, its intracellular transport and processing . The secreted active site mutant precursors, endocytosed by galactosialidosis fibroblasts, restore beta-galactosidase and neuraminidase activities as effectively as wild-type protective protein . These findings indicate that the catalytic activity and protective function of the protective protein are distinct.

Mol Cell Biol, 1991 Aug, 11(8), 4053 - 64
Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras; Stacey DW et al.; Two dominant inhibitory Ras mutant proteins were analyzed by microinjection . One, {Asn-17}Ras, had a substitution in the putative Mg(2+)-binding site of Ha-Ras . The other, RAST, had a mutation in a yeast RAS protein that impaired its GTPase activity and increased its affinity for GAP . RAST also had a mutation that blocked its localization to the plasma membrane . In NIH 3T3 cells {Asn-17}Ras inhibited the function of normal Ras much more efficiently than that of oncogenic Ras . In contrast, RAST interfered with the transforming activity of oncogenic Ras more efficiently than that of normal Ras . These conclusions were based on two separate types of analysis . The inhibitory Ras mutant proteins were first microinjected into cells stably transformed either by oncogenic Ras or by high levels of expression of cellular Ras . Results obtained in stably transformed cells were then verified by coinjection of the inhibitory Ras mutant proteins together with transforming concentrations of either oncogenic or normal Ras protein . Whereas RAST was active in soluble form . {Asn-17}Ras required membrane localization for activity . Furthermore, mutations in the GAP/effector-binding domain reduced or eliminated the inhibitory activity of RAST but had no detectable effect on {Asn-17}Ras . These results are consistent with the possibility that {Asn-17}Ras functions by blocking the activation of endogenous Ras proteins, while RAST functions by blocking the ability of activated Ras to stimulate a downstream target within the cells . The properties of RAST suggest that interference with the GAP/effector-binding function of RAS represents a strategy for the preferential inactivation of oncogenic Ras in cells.

Mol Cell Biol, 1991 Aug, 11(8), 3850 - 9
Mapping of replication initiation sites in mammalian genomes by two-dimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix; Dijkwel PA et al.; Two complementary two-dimensional gel electrophoretic techniques have recently been developed that allow initiation sites to be mapped with relative precision in eukaryotic genomes at least as complex as those of yeast and Drosophila melanogaster . We reported the first application of these mapping methods to a mammalian genome in a study on the amplified dihydrofolate reductase (DHFR) domain of the methotrexate-resistant CHO cell line CHOC 400 (J.P . Vaughn, P.A . Dijkwel, and J.L . Hamlin, Cell 61:1075-1087, 1990) . Our results suggested that in this 240-kb domain, initiation of nascent DNA strands occurs at many sites within a 30- to 35-kb zone mapping immediately downstream from the DHFR gene . In the course of these studies, it was necessary to develop methods to stabilize replication intermediates against branch migration and shear . This report describes these stabilization methods in detail and presents a new enrichment protocol that extends the neutral/neutral two-dimensional gel mapping method to single-copy loci in mammalian cells . Preliminary analysis of replication intermediates purified from CHO cells by this method suggests that DNA synthesis may initiate at many sites within a broad zone in the single-copy DHFR locus as well.

J Virol, 1991 Aug, 65(8), 4211 - 5
Genomic structure and RNA polymerase activity in Leishmania virus; Widmer G et al.; Viral particles infecting some stocks of the protozoan parasite Leishmania braziliensis subsp . guyanensis contain a double-stranded RNA genome of ca . 5 kbp and are associated with an RNA-dependent RNA polymerase which synthesizes in vitro double-stranded and single-stranded, genome-length transcripts . The majority of viral transcripts are single-stranded and templated from one genomic strand . The putative replicase generates double-stranded RNA by synthesizing the opposite strand on a preexisting RNA template . These data are compatible with a replicative cycle proposed for the yeast viruses . Purification of the Leishmania virus on CsCl yields virus without double-strand synthesis activity, while this activity is consistently present in unpurified virus and in particles from sucrose gradients . The deficiency in double-strand synthesis in CsCl-derived virions correlates with the accessibility of the viral polymerase and genomic RNA to exogenously added enzymes, indicative of a structural modification of the viral capsid.

EMBO J, 1991 Aug, 10(8), 2069 - 75
A cdc2-like kinase phosphorylates histone H1 in the amitotic macronucleus of Tetrahymena; Roth SY et al.; Genetic and biochemical studies have shown that cdc2 protein kinase plays a pivotal role in a highly conserved mechanism controlling the entry of cells into mitosis . It is generally believed that one function of cdc2 kinase is to phosphorylate histone H1 which in turn promotes mitotic chromosome condensation . However, direct evidence linking H1 phosphorylation to mitotic chromatin condensation is limited and the exact cellular function(s) of H1 phosphorylation remains unclear . In this study, we show that mammalian cdc2 kinase phosphorylates H1 from the amitotic macronucleus of Tetrahymena with remarkable fidelity . Furthermore, we demonstrate that macronuclei from Tetrahymena contain a growth-associated H1 kinase activity which closely resembles cdc2 kinase from other eukaryotes . Using polyclonal antibodies raised against yeast p34cdc2, we have detected a 36 kd immunoactive polypeptide in macronuclei which binds to Suc1 (p13)-coated beads and closely follows H1 kinase activity . Since macronuclei divide without mitotic chromosome condensation, these data demonstrate that H1 phosphorylation by cdc2 kinase may be necessary, but is not sufficient to promote mitotic chromatin condensation . The fact that an activity which strongly resembles mammalian cdc2 kinase is active during cell growth in a nucleus which does not undergo mitosis and chromosome condensation suggests that other factors are needed for a true mitotic division to occur . These data also reinforce the notion that H1 phosphorylation has important functions outside mitosis both in Tetrahymena and in mammalian cells.

J Reprod Med, 1991 Aug, 36(8), 593 - 7
Antifungal agents vs . boric acid for treating chronic mycotic vulvovaginitis; Jovanovic R et al.; Ninety-two women with chronic mycotic vaginal infections were followed with microscopic examination of the vaginal discharge during prolonged therapy with antifungal agents and boric acid . A microscopic picture unique to chronic mycotic vaginitis was observed, representing the cytologic reaction of the mucous membrane to chronic yeast infection . This diagnostic tool proved extremely effective in detecting both symptomatic and residual, subclinical mycotic infection and provided a highly predictive measure of the probability of relapse . The ineffectiveness of conventional antifungal agents appeared to be the main reason for chronic mycotic infections . In contrast, boric acid was effective in curing 98% of the patients who had previously failed to respond to the most commonly used antifungal agents and was clearly indicated as the treatment of choice for prophylaxis.

Mol Cell Biol, 1991 Aug, 11(8), 3997 - 4004
Identification of amino acid residues required for Ras p21 target activation; Marshall MS et al.; The Krev-1 gene has been shown to suppress ras-mediated transformation in vitro . Both ras and Krev-1 proteins have identical effector domains (ras residues 32 to 40), which are required for biological activity and for the interaction of Ras p21 with Ras GTPase-activating protein (GAP) . In this study, five amino acid residues flanking the ras effector domain, which are not conserved with the Krev-1 protein, were shown to be required for normal protein-protein interactions and biological activity . The substitution of Krev-1 p21 residues 26, 27, 30, 31, and 45 with the corresponding amino acid residues from Ras p21 resulted in a Krev-1 protein which had ras function in both mammalian and yeast biological assays . Replacement of these residues in Ras p21 with the corresponding Krev-1 p21 amino acids resulted in ras proteins which were impaired biologically or reduced in their affinity for in vitro GAP binding . Evaluation of these mutant ras proteins have implications for Ras p21-GAP interactions in vivo.

Neuron, 1991 Aug, 7(2), 183 - 96
Mechanisms of complex transcriptional regulation: implications for brain development; He X et al.; The large number of transcription factors, their diverse sequence-specific interactions with DNA sites and with other transcription factors, and their ability to be modified in response to a variety of environmental cues and intracellular signals provide combinatorial codes for highly complex and yet highly organized patterns of gene expression likely to underlie the determination of diversity of neuronal phenotypes . Subtle differences in the combinations of transcription factors are likely to have profound consequences for cell phenotype, similar to the mechanism involved in the specification of cell types in yeast (reviewed in Herskowitz, 1989) . Although our current understanding of transcriptional regulation in the brain comes largely from phenomenological studies, recent technical progress on two fronts promises a bright future . Homologous recombination technology in embryonic stem cells (reviewed in Capecchi, 1989; Rossant, 1990) allows the disruption of particular genes in transgenic mice and definition of the roles of identified transcription factors in mammalian neurogenesis . A second technological advance, targeted tumorigenesis, has provided neuronal model cell lines (Mellon et al., 1990; reviewed in Cepko, 1988; McKay et al., 1988) that mimic certain neuronal differentiation pathways . These combined genetic, cell biological, and biochemical approaches will greatly facilitate the study of neural development and function.

Pediatr Clin North Am, 1991 Aug, 38(4), 991 - 1017
Disorders of hypopigmentation in children; Pinto FJ et al.; The most common disorders of hypopigmentation in children are pityriasis alba, vitiligo, nevus depigmentosus, and tinea versicolor . Pityriasis alba usually presents as ill defined, scaly patches of hypomelanosis on the cheeks of children with an atopic diathesis . The face is also a favored site for vitiligo, but the distribution is periorificial, and the pigment loss is complete because of a destruction of melanocytes . Vitiligo is an acquired, progressive disorder in contrast to nevus depigmentosus, which is a stable, congenital leukoderma . The localized form of nevus depigmentosus must be distinguished from an ash leaf spot, the earliest cutaneous manifestation of tuberous sclerosis, whereas the systematized form may be confused with hypomelanosis of Ito, another neurocutaneous disorder . The lesions of tinea versicolor favor the upper trunk of adolescents, and potassium hydroxide examination of the associated scale reveals hyphal and yeast forms of P . orbiculare . Any inflammatory process in the skin such as dermatitis or psoriasis can resolve with areas of hypopigmentation.

J Clin Invest, 1991 Aug, 88(2), 519 - 23
Mutations of P450c21 (steroid 21-hydroxylase) at Cys428, Val281, and Ser268 result in complete, partial, or no loss of enzymatic activity, respectively; Wu DA et al.; Steroid 21-hydroxylase deficiency is the major cause of congenital adrenal hyperplasia (CAH), a common genetic disease . To define the relationship between gene mutations and enzyme deficiency, we generated missense mutations of the 21-hydroxylase cDNA at three different sites and characterized the mutant proteins after expressing them in cultured mammalian and yeast cells . Among them, Ser268 and Val281 have been found to be mutated in CAH patients, whereas Cys428 has been implicated as the heme ligand . Our results show mutations at these sites result in complete, partial, or no loss of the enzymatic activity . All the Cys428 mutants had neither enzymatic activity nor P450 absorption, thus supporting the notion that Cys428 is the heme ligand . All the 268-mutants exhibited the same activity as normal 21-hydroxylase, demonstrating that the clinically observed Ser268----Thr change represents a polymorphism rather than the cause of the enzyme deficiency . The 281-mutants had normal Km but greatly reduced Vmax values that also paralleled the reduction in the heme content, in the order Val281 (normal, 100%) greater than Ile281 (50%) greater than Leu281 (20%) greater than Thr281 (10%) . Our findings suggest that the methyl group at the beta-carbon of Val281 is required for heme incorporation and consequently enzymatic activity.

J Bacteriol, 1991 Aug, 173(16), 4959 - 69
Methylamine metabolism in Hansenula polymorpha: an in vivo 13C and 31P nuclear magnetic resonance study; Jones JG et al.; Methylamine uptake, oxidation, and assimilation were studied in Hansenula polymorpha, a methylotrophic yeast . The constitutive ammonia transport system was shown to be effective at accumulating methylamine within cells cultured with methylamine or ammonia as a nitrogen source . {13C}methylamine oxidation rates were measured in vivo in methylamine-adapted cells by 13C nuclear magnetic resonance and were found to be lower than its uptake rate into the cells . The 13C label of methylamine was found exclusively in trehalose and glycerol, and {13C}formaldehyde was also extensively assimilated, indicating the presence of an assimilation pathway for the methylamine carbon . In vivo 31P nuclear magnetic resonance analysis showed major differences in the endogenous polyphosphate levels and mean chain length during adaptation of the cells from ammonia to methylamine, indicating that methylamine accumulated in the vacuole in the same manner as basic amino acids and purines . {13C}glucose metabolism was drastically altered during adaptation of the cells from ammonia to methylamine as a nitrogen source . The total rate of glucose utilization and the rate of ethanol production fell . Direct trehalose synthesis from glucose increased, indicating a switch from carbon utilization for growth to that for storage . The rate of methylamine oxidation was sufficient to support a much higher flow of carbon into central biosynthetic pathways . These results suggest that this reduction in biosynthetic carbon flow, rather than nitrogen availability, was the main factor responsible for reducing the growth rate of the yeast when ammonia was replaced by methylamine as the nitrogen source.

J Econ Entomol, 1991 Aug, 84(4), 1257 - 61
Effect of larval diet on cat flea (Siphonaptera: Pulicidae) developmental times and adult emergence; Moser BA et al.; The natural diet of cat flea, Ctenocephalides felis (Bouche), larvae is primarily adult flea feces, but dried bovine blood may be substituted in the laboratory . Percentage adult emergence (79.4% on feces; 78.9% on blood) and developmental times (20.6 d on feces; 17.1 d on blood) did not significantly differ for the two diets . The drying temperature of blood determined its quality; blood dried at 120 degrees C was unsatisfactory for larval development . The dietary value of dried bovine blood was not enhanced when supplemented with brewer's yeast, rodent chow, or a combination of those constituents . Blood particle size ranging from less than 180 to greater than 500u did not affect the value of blood as a diet . Rodent chow, yeast, albumen, hemoglobin, and mixtures of these constituents were unsuitable as larval diets.

Semin Cell Biol, 1991 Aug, 2(4), 261 - 70
The substrates of the cdc2 kinase; Nigg EA; The eukaryotic cell cycle is characterized by two major events, DNA replication (S phase) and mitosis (M phase) . According to the current paradigm of the cell cycle as a cdc2 cycle, both of these events are driven by serine-threonine specific protein kinases encoded by functional homologs of the fission yeast cdc2 gene . To understand how cdc2 kinases function, it is necessary to identify their physiological substrates and to determine how phosphorylation of these substrates promotes cell cycle progression . Definitive information about substrates relevant to early stages of the cell cycle (G1 and S phases) remains scarce, but several likely physiological targets of the mitotic cdc2 kinase have recently been identified . Current evidence indicates that cdc2 kinase may trigger entry of cells into mitosis not only by initiating important regulatory pathways but also by direct phosphorylation of abundant structural proteins.

FEMS Microbiol Lett, 1991 Aug 1, 66(2), 157 - 61
Proteolytic activation of alpha,alpha-trehalose 6-phosphate synthase in Candida utilis; Vicente-Soler J et al.; Total trehalose 6-phosphate synthase activity increased in cell-free extracts from Candida utilis following short-term preincubation of the enzyme samples at 37 degrees C . This endogenous activation was prevented by the inhibitors of serine-type proteases, phenylmethylsulfonyl fluoride, antipain or chymostatin, but not by other protease inhibitors such as pepstatin . Fractionation of the cell extracts by Sephadex G-200 gel filtration revealed that the activity of one of the two synthase enzymes present in these cells was enhanced after the activation treatment . These observations indicate the existence of a proteolytically activatable enzyme form in the trehalose 6-phosphate synthase complex of this yeast in addition to the previously characterized enzyme, whose activity appears to be inactivated by reversible phosphorylation.

EMBO J, 1991 Aug, 10(8), 2291 - 6
Both Oct-1 and Oct-2A contain domains which can activate the ubiquitously expressed U2 snRNA genes; Yang J et al.; The U2 snRNA genes, which are transcribed by RNA polymerase II at high levels in all tissues examined, require both a distal and a proximal sequence element for efficient expression . The distal sequence element which has many properties in common with transcriptional enhancers contains, in addition to Sp1 binding sites, an octamer binding site which mediates activation through interactions with the ubiquitous transcription factor Oct-1 . In the present study we have attempted to answer the question whether Oct-1 contains a unique activating domain which is required for activation of snRNA genes or whether ubiquitously expressed and lymphoid specific octamer binding factors both have the capacity to activate snRNA transcription . Our results show that in the presence of Oct-1, overexpression of Oct-2A in HeLa or COS1 cells neither inhibits nor stimulates transcription of U2 constructions which contain octamer binding sites with or without an adjacent Sp1 binding site . Moreover, an Oct-2A--GAL4 fusion protein in which the DNA binding domain of Oct-2A was substituted for by the one of the yeast transcription activator GAL4 activates transcription of a human U2 snRNA gene in which the octamer binding site was replaced by a GAL4 binding site . From the results it is concluded that both Oct-1 and Oct-2A contain domains which can activate the ubiquitously expressed U2 snRNA genes.

J Clin Microbiol, 1991 Aug, 29(8), 1723 - 4
Variability in commercial histoplasma complement fixation antigens; Leland DS et al.; Using Immuno-Mycologics (IMMY; Norman, Okla.) histoplasmal yeast (HY) and mycelial (HM) antibody complement fixation test antigens, we retested 1,386 samples that were initially tested with Meridian Diagnostics, Inc . (Cincinnati, Ohio), antigens . Histoplasma antibody was identified (greater than or equal to 1:16) in 20% of HY and 5% of HM samples reported to have titers of less than 1:8 with Meridian reagents . IMMY titers were at least fourfold higher than Meridian titers in 39% of HY and 54% of HM samples that initially had titers of greater than or equal to 1:8 with Meridian antigens . Because 30 of 58 (52%) samples from confirmed cases of histoplasmosis yielded negative results with Meridian antigens and positive results upon retesting with IMMY antigens, we concluded that the Meridian antigens had less reactivity with human histoplasmal antibody.

J Immunol, 1991 Aug 1, 147(3), 942 - 9
Function and evolutionary conservation of distinct epitopes on the leukocyte adhesion molecule-1 (TQ-1, Leu-8) that regulate leukocyte migration; Spertini O et al.; The leukocyte adhesion molecule-1 (LAM-1, TQ=1, Leu-8) in humans, like its murine homologue, MEL-14, is the principal receptor that mediates the binding of leukocytes to high endothelial venules (HEV) of peripheral lymph nodes . In this study, several regions of the protein which mediate receptor function were identified by using a large panel of murine mAb reactive with LAM-1 . Individual mAb reacted with LAM-1+ cells with characteristic intensities of immunofluorescence staining, and each bound both lymphocytes and neutrophils . Lymphocyte attachment to HEV was significantly inhibited by the binding of five mAb . In contrast, only two of these mAb were able to completely block the binding of phosphomannan monoester core complex from the yeast Hansenula holstii cell wall (PPME), a phosphomannan monoester core polysaccharide that serves as a soluble model of the natural ligand of LAM-1 . Interestingly, the binding of two anti-LAM-1 mAb to cells induced a significant increase in PPME binding, reminiscent of the increase in receptor affinity observed after leukocyte activation . Antibody cross-blocking studies indicated that many of the functionally important epitopes were spatially distinct, and domain mapping indicated that they recognized distinct domains of LAM-1 . The expression and function of these epitopes were further assessed by using a variety of animal species to further characterize the functionally relevant epitopes defined in these studies . At least some anti-LAM-1 mAb reacted with leukocytes from monkey, cow, rabbit, sheep, dog, cat, pig, and goat, but not from chicken, rat, or mouse . The reactivity of anti-LAM-1 mAb in several animal species correlated with the ability of leukocytes to bind PPME, and mAb that inhibited lymphocyte binding to HEV in man could also inhibit this function in rhesus monkey and dog . Thus, several LAM-1 epitopes are structurally and functionally well conserved throughout recent mammalian evolution, emphasizing an important role for LAM-1 in the regulation of leukocyte traffic.

Semin Cell Biol, 1991 Aug, 2(4), 243 - 50
Cell cycle control of DNA replication by p34cdc2; Blow JJ; Commitment to DNA replication is one of the major control points of the eukaryotic cell cycle, and one that has been curiously hard to analyse . However, homologous components of this process are now being identified by genetic analysis of yeast and by biochemical analysis of cell-free systems from higher eukaryotes . This homology suggests that these components are part of a universal mechanism for controlling the eukaryotic cell cycle . The most important component of this mechanism is the cdc2 protein, which controls the initiation of both DNA replication and mitosis . At present, however, its precise role in DNA replication is unclear.

Curr Opin Biotechnol, 1991 Aug, 2(4), 561 - 7
Alteration of enzyme specificity and catalysis by protein engineering; Wilks HM et al.; New substrate specificities can be introduced into existing enzymes for the purpose of making them more suitable for the chemoenzymic synthesis of single compound drugs and other chiral compounds . The most productive route used in the past year has involved the utilization of the catalytic and substrate-binding properties from homologous enzymes found in nature, one example being the broadening of the substrate specificity of yeast alcohol dehydrogenase . Other highlights include the creation of thermostable dehydrogenases that will interconvert NADPH and NADH, and the design of mutant enzymes with improved catalytic rates compared with their wild-type counterparts.

J Immunol Methods, 1991 Jul 26, 141(1), 63 - 72
A new technique, requiring small amounts of cells, for the parallel study of chemiluminescence and phagocytosis via different receptors in the same cell population; Sandgren CH et al.; An assay permitting the parallel assessment of phagocytosis and chemiluminescence in the same cell population has been developed . The method is based on the phagocytosis of fluorescein isothiocyanate-conjugated yeast particles, either unopsonized or opsonized with complement factor C3 or IgG, by purified cells in suspension in a luminometer . Only a small number of cells (2 x 10(4)-1 x 10(5)) is required, and the reproducibility is high . Moreover, the technique permits phagocytosis to be related to oxygen-dependent killing activity in the same cell population . Since phagocytosis, degranulation and oxygen radical formation as a consequence of well-defined receptor recognition mechanisms can be characterized in very small cell populations, the method is suitable for monitoring the phagocytic function of cells from extravascular sites.

J Biol Chem, 1991 Jul 25, 266(21), 13964 - 70
Resolution of a low molecular weight G protein in neutrophil cytosol required for NADPH oxidase activation and reconstitution by recombinant Krev-1 protein; Eklund EA et al.; Activation of the membrane-associated NADPH oxidase in intact human neutrophils requires a receptor-associated heterotrimeric GTP-binding protein that is sensitive to pertussis toxin . Activation of this NADPH oxidase by arachidonate in a cell-free system requires an additional downstream pertussis toxin-insensitive G protein (Gabig, T . G., English, D., Akard, L . P., and Schell, M . J . (1987) (J . Biol . Chem . 262, 1685-1690) that is located in the cytosolic fraction of unstimulated cells (Gabig, T . G., Eklund, E . A., Potter, G . B., and Dykes, J . R . (1990) J . Immunol . 145, 945-951) . In the present study, immunodepletion of G proteins from the cytosolic fraction of unstimulated neutrophils resulted in a loss of the ability to activate NADPH oxidase in the membrane fraction . The activity in immunodepleted cytosol was fully reconstituted by a partially purified fraction from neutrophil cytosol that contained a 21-kDa GTP-binding protein . Purified human recombinant Krev-1 p21 also completely reconstituted immunodepleted cytosol whereas recombinant human H-ras p21 or yeast RAS GTP-binding proteins had no reconstitutive activity . Rabbit antisera raised against a synthetic peptide corresponding to the effector region of Krev-1 (amino acids 31-43) completely inhibited cell-free NADPH oxidase activation, and this inhibition was blocked by the synthetic 31-43 peptide . An inhibitory monoclonal antibody specific for ras p21 amino acids 60-77 (Y13-259) had no effect on cell-free NADPH oxidase activation . Activation of the NADPH oxidase in intact neutrophils by stimulation with phorbol myristate acetate caused a marked increase in the amount of membrane-associated antigen recognized by 151 antiserum on Western blot . Thus a G protein in the cytosol of unstimulated neutrophils antigenically and functionally related to Krev-1 may be the downstream effector G protein for NADPH oxidase activation . This system represents a unique model to study molecular interactions of a ras-like G protein.

J Biol Chem, 1991 Jul 25, 266(21), 13560 - 3
Determination of disulfide bond pairs and stability in recombinant tick anticoagulant peptide; Sardana M et al.; Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of blood coagulation factor Xa (Waxman, L., Smith, D.E., Arcuri, K.E., and Vlasuk, G.P . (1990) Science 248, 593-596) . The 60-amino acid sequence of TAP shows limited homology to Kunitz-type inhibitors, including cysteines at positions 5, 15, 33, 39, 55, and 59 . For detailed biochemical and pharmacological studies, a recombinant version of TAP (rTAP) has been produced in yeast . To determine the arrangement of the disulfide bonds, rTAP was cleaved with trypsin and chymotrypsin and the purified peptides sequenced using a gas-phase sequenator . The positions of the disulfide bonds were assigned by identifying the cycle(s) at which di-phenylthiohydan-toin-cystine was released . The specific disulfide bridges, Cys-5 to Cys-59, Cys-15 to Cys-39, and Cys-33 to Cys-55, are analogous to those in the prototype Kunitz-type inhibitor, bovine pancreatic trypsin inhibitor (BPTI) . While treatment of BPTI with dithiothreitol rapidly and specifically reduced one disulfide bond, the reduction of disulfide bonds in rTAP proceeded at a slower rate and appeared to be nonspecific, reaching a maximum of two disulfides reduced . Reduced rTAP derivatized with either iodoacetic acid or iodoacetamide lost 59% of its inhibitory activity . In contrast, BPTI alkylated with iodoacetic acid inhibited trypsin half as well as the iodoacetamide derivative . Although the arrangement of disulfides in the two inhibitors is the same, their susceptibility to reduction is markedly different.

Biochim Biophys Acta, 1991 Jul 23, 1089(3), 386 - 8
Characterization of a cDNA encoding subunit VI of cytochrome c oxidase from the slime mold Dictyostelium discoideum; Rizzuto R et al.; The primary structure of subunit VI of cytochrome c oxidase from the slime mold Dictyostelium discoideum has been determined by sequencing cDNA and N-terminus of the protein . The 92 amino acid residues long polypeptide (Mr = 10,535) shows homology with subunit IV of mammalian and subunit V of yeast cytochrome c oxidase . Though smaller and synthesized without a cleavable presequence, the slime mold oxidase subunit maintains the presence of a putative membrane spanning region.

Eur J Biochem, 1991 Jul 15, 199(2), 441 - 50
Conserved amino acid sequences among plant proteins sorted to protein bodies and plant vacuoles . Can they play a role in protein sorting?
Sebastiani FL, Farrell LB, Vasquez M, Beachy RN.
Amino acid sequence comparisons were made between the soybean alpha subunit of beta-conglycinin and 34 members of different plant protein families targeted to seed protein bodies or vacuoles . A number of short conserved amino acid sequences were identified in seed storage proteins, plant protease inhibitors and lectins, and the probable functions of these sequences are discussed . For proteins of known tertiary structure, these sequences map to the surface of the respective molecules . It is postulated that these regions produce a common secondary structure which could interact with other molecules involved in the sorting process . One of these regions, region A, is similar to the yeast carboxypeptidase Y (CPY) vacuolar targeting signal, and is present in both storage proteins and lectins . Computer modeling based upon the tertiary structure of concanavalin A (ConA) was used to generate models representing the structure of two highly related lectins from Dolichos biflorus, one of which is targeted to protein bodies and the other secreted . A different glycosylation pattern together with amino acid sequences upstream of the identified conserved amino acid sequences are predicted to modulate the presentation of the sorting domains in the lectins and be the determinant in the sorting of these lectins.

Biochem J, 1991 Jul 15, 277 ( Pt 2), 483 - 92
Properties and structural requirements for substrate specificity of cytochrome P-450-dependent obtusifoliol 14 alpha-demethylase from maize (Zea mays) seedlings; Taton M et al.; The biochemical properties of cytochrome P-450-dependent obtusifoliol 14 alpha-demthylase (P-450OBT.14DM) from maize (Zea mays) seedlings were defined . In particular, the enzyme was shown by differential centrifugation to be localized in the endoplasmic reticulum . P-450OBT.14DM had an apparent Km of 160 +/- 5 microM and an apparent Vmax of 65 +/- 5 pmol/min per mg of protein for its best substrate, obtusifoliol . The substrate specificity of P-450OBT.14DM was thoroughly investigated by comparing the demethylation of obtusifoliol with that of a series of 15 natural or novel synthetic analogues of obtusifoliol . The results obtained clearly indicate that three distinct domains of the sterol substrate are governing obtusifoliol demethylation by P-450OBT.14DM . They revealed that (i) P-450OBT.14DM has probably a specific apolar binding site for the side chain, (ii) the delta 8-double bond is an absolute requirement for substrate demethylation and (iii) the 3-hydroxy group plays a critical role in the enzyme-substrate interaction . Interestingly the binding site, beyond the C-3 position, contains a cleft which cannot accommodate a 4 beta-methyl substituent present in lanosterol or eburicol, the precursors of 14-desmethylsterols respectively in mammals and yeast . This result indicates that P-450OBT.14DM is a novel constitutive cytochrome P-450 with a high degree of substrate and product specificity.

Biochem J, 1991 Jul 15, 277 ( Pt 2), 451 - 6
Down-regulation of mannose receptors on macrophages after infection with Leishmania donovani; Basu N et al.; Macrophages express a mannose-specific endocytosis receptor that binds and internalizes mannose-terminated glycoproteins . Infection of mouse peritoneal macrophages with Leishmania donovani resulted in a decrease in mannose-receptor activity . With 125I-labelled beta-glucuronidase as ligand, a 2-fold decrease in uptake rate was observed in infected cells, with no change in Kuptake . Cell-surface binding of 125I-mannose-BSA was diminished 2.5-fold after infection . The decrease in ligand binding appeared to be due to a decrease in the number of sites, with no change in affinity . Elimination of parasites from infected cells by treatment with neoglycoprotein-conjugated methotrexate resulted in an increase in receptor number . Cycloheximide suppressed the drug-treatment-mediated rise in receptor number in infected macrophages . A decrease in receptor activity was also observed in liver Kupffer cells isolated from parasite-infected mice . Binding of ligand by another carbohydrate receptor, the mannose 6-phosphate receptor, was not altered by infection . Phagocytosis of yeast cells was also not altered . These results suggest that mannose receptor synthesis in macrophages is specifically suppressed after infection with Leishmania parasites.

J Immunol, 1991 Jul 15, 147(2), 621 - 6
Arachidonic acid is essential for IgG Fc receptor-mediated phagocytosis by human monocytes; Lennartz MR et al.; Phagocytosis is a specialized function of neutrophils and macrophages that requires coordination of multiple biochemical and biophysical events . Considerable progress has been made in identifying the membrane receptors involved in phagocytosis, but the intracellular signaling pathways that are necessary for particle ingestion are poorly understood . In an effort to address this complex question, we investigated the role of arachidonic acid (AA) in the uptake of yeast and IgG-coated E (EIgG) or C-coated E . Human monocytes, labeled with 3H AA, released this label during phagocytosis of yeast and EIgG, but not in response to EC3b . The PL inhibitors bromophenacyl bromide and manoalide abolished the release of 3H and inhibited phagocytosis of EIgG in parallel . Both drugs caused a similar inhibition of yeast-mediated 3H release but had little effect on yeast ingestion . Similar results were obtained with the inhibitor quinacrine (mepacrine) . Exogenously added AA and dihomo-gamma-linolenic acid restored bromophenacyl bromide-inhibited EIgG ingestion; arachidonate analogs eicosatrienoic acid and eicosapentanoic acid did not . Inhibition of the cyclooxygenase and lipoxygenase pathways for AA metabolism by indomethacin or BW755C did not affect EIgG phagocytosis, demonstrating that these major AA metabolic pathways are not involved in phagocytic signaling . These experiments suggest that release of AA is essential for EIgG ingestion and that phagocytosis in monocytes proceeds by at least two mechanisms, one dependent on AA (EIgG) and one independent of it (yeast).

J Chromatogr, 1991 Jul 12, 548(1-2), 329 - 34
Separation of acidic peptides by reversed-phase ion-pair chromatography . Analytical application to a series of acidic substrates of casein kinases; Calderan A et al.; A series of small peptides including clusters of glutamyl residues, synthesized to study the site specificity of rat liver (L-CK2) and yeast (Y-CK2) casein kinase-2, are analytically characterized by ion-pair high-performance liquid chromatography using tetrabutylammonium as counter-ion and acetonitrile as modifier of the aqueous phase . Under these conditions peptides of slightly different acidity can be separated and the elution order parallels the hydrophobicity of the ion-pair-peptide complexes, which increases with the number of the acidic functions present in the sequence.

J Biol Chem, 1991 Jul 5, 266(19), 12127 - 30
Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway; Hosaka M et al.; Many peptide hormones are produced from larger precursors by endoproteolysis at pairs of basic amino acids (e.g . Lys-Arg and Arg-Arg) within the regulated secretory pathway in endocrine cells . However, many other secretory and membrane proteins appear to be produced from precursors through cleavage at multiple, rather than paired, basic residues within the constitutive secretory pathway in non-endocrine cells . By surveying various precursors processed constitutively, we noticed that most of them have the consensus sequence, Arg-X-Lys/Arg-Arg (RXK/RR), at the cleavage site . When expressed in endocrine and non-endocrine cells, a precursor with the RXKR sequence was cleaved in both types of cells, whereas that with the Lys-Arg pair was cleaved only in the endocrine cells . When the RXKR precursor was coexpressed with furin and PC3, both of which are mammalian homologues of the yeast precursor-processing endoprotease Kex2, in non-endocrine cells, enhancement of the precursor cleavage by furin but not by PC3 was observed . By contrast, when the Lys-Arg precursor was coexpressed with the two mammalian proteases in endocrine cells with no endogenous processing activity at dibasic sites, it was cleaved only by PC3 . These results indicate that the basic pair and the RXK/RR sequence are the signals for precursor cleavages catalyzed by PC3 within the regulated secretory pathway and by furin within the constitutive pathway, respectively.

Eur J Biochem, 1991 Jul 1, 199(1), 231 - 8
Probing the catalytic sites of triosephosphate isomerase by 31P-NMR with reversibly and irreversibly binding substrate analogues; Schnackerz KD et al.; We have explored the degree of independence of the two catalytic centers, interactions between the catalytic centers and the subunit-subunit contact sites, and different conformations of triosephosphate isomerase (TPI), by simultaneously employing irreversibly (covalent) and reversibly binding substrate analogues and monitoring their 31P-NMR resonances . 3-Chloroacetol phosphate (CAP) was bound to the active site by reaction with Glu165 . The resulting, inactive (CAP-TPI)2 complex exhibited two distinct 31P-NMR resonances which were independent of pH and represent two conformational forms of the enzyme . Dissociation in guanidine hydrochloride followed by redimerization resulted in a single conformation . This was observed with the enzyme from chicken, rabbit and yeast . The inactive (CAP-TPI)2 dimer was mixed with native TPI, and dissociated/reassociated to form heterodimers (CAP-TPI)(TPI) in which one subunit contained the CAP label and the other subunit was unmodified . This hybrid migrated intermediate between the native and CAP-modified enzyme on nondenaturing PAGE . The heterodimer exhibited 50% the activity of the native dimer, but kinetic properties were otherwise indistinguishable . The reversibly binding transition state analogue, 2-phosphoglycolate (PGA), was used to probe the remaining vacant active site of the heterodimer . Bound PGA exhibited a pH-independent 31P-NMR resonance which was readily distinguishable from resonances of CAP-TPI and free PGA . No differences were observed in the binding of PGA to the vacant subunit of the heterodimer or the native dimer, further pointing to the independent nature of the two catalytic centers . However, the (CAP-TPI)(TPI) heterodimer was more susceptible to subunit dissociation in guanidine hydrochloride than the native dimer . Thus, it appears that the two active sites function completely independently of each other, but that the binding of CAP at the active center loosens the subunit-subunit contact . In addition, the two forms of the enzyme-inhibitor complex trapped by reaction with CAP may represent conformations with the hinged lid or flexible loop (residues 166-176) in the open and closed positions.

Eur J Biochem, 1991 Jul 1, 199(1), 169 - 79
Heterogeneity of the complex N-linked oligosaccharides at specific glycosylation sites of two secreted carrot glycoproteins; Sturm A; The N-linked glycans from the 52/54-kDa medium protein and cell wall beta-fructosidase, two glycoproteins secreted by carrot suspension culture cells, were characterized . Carrot cells were labelled with {3H}glucosamine or {3H}fucose . The 52/54-kDa medium protein was isolated from the culture medium and beta-fructosidase from cell walls . The purified proteins were digested with trypsin and glycopeptides were isolated and sequenced . Glycans obtained from individual glycopeptides were separated by gel filtration chromatography and characterized by concanavalin A chromatography, by treatments with exoglycosidases and by sugar composition analysis . The 52/54-kDa medium protein and cell wall beta-fructosidase have one high-mannose-type glycan similar to those from yeast and animal glycoproteins . In addition, the 52/54-kDa medium protein has three complex-type and cell wall beta-fructosidase two complex-type glycans per polypeptide . The complex-type glycans isolated from individual glycosylation sites are fairly large and very heterogeneous . The smallest of these glycans has the structure {Xyl}(Man)3{Fuc}(GlcNAc}2Asn (square brackets indicating branching) whereas the larger ones carry additional sugars like terminal N-acetylglucosamine and possibly rhamnose and arabinose in the case of the 52/54-kDa medium protein and only arabinose in the case of cell wall beta-fructosidase . These terminal sugars are linked to the alpha-mannose residues of the glycan cores . The 52/54-kDa medium protein is secreted with large and homogeneous complex glycans, their heterogeneity originates from slow processing after secretion . The complex glycans from cell wall beta-fructosidase are processed before the enzyme is integrated into the cell wall.

Dev Biol, 1991 Jul, 146(1), 246 - 9
Maturation-promoting factor and p34cdc2 kinase during oocyte maturation of the Japanese quail; Mori M et al.; Maturation-promoting factor and a homolog of fission yeast cdc2+ gene product (p34cdc2) were investigated during the final 24 hr of maturation of quail oocytes . Kinase activity of p34cdc2 in the oocyte germinal disk (GD) increased 15 times at maturation . Two bands, at 32 and 34 kDa, were detected in immature oocytes by immunoblotting of SDS-PAGE with anti-p34cdc2 monoclonal antibody . A new band, which is close to the 32-kDa band but with a slightly faster mobility, appeared during maturation . No p34cdc2 could be detected outside the GD . Microinjection of GD extract from mature oocytes caused maturation of Xenopus oocytes.

Behring Inst Mitt, 1991 Jul, (89), 35 - 45
Paramyxovirus tropism dependent on host proteases activating the viral fusion glycoprotein; Nagai Y et al.; An essential step in paramyxovirus fusion (F) glycoprotein biosynthesis is the posttranslational endoproteolytic cleavage of the inactive precursor glycoprotein Fo by host cell proteases . When the Fo possesses a pair or a cluster of basic residues at the cleavage site, cleavage is catalyzed by a ubiquitous protease(s) and the infection is consequently pantropic . When the site is monobasic with a single arginine, cleavage is allowed to occur only by the enzyme(s) expressed in limited tissue types and the infection is localized there . We have isolated from chick embryo an example of the latter type of endoprotease specific for the single arginine motif and demonstrate its identity with the clotting factor Xa . The ectopic expression of the FXa appeared to be the sole determinant for the viral tropism in chick embryo . The latter type of protease specific for a paired or multiple basic cleavage motif have neither been identified nor characterized extensively . We show here that this cleavage can be induced by the yeast KEX2 protease, a unique subtilisin-like serine protease, responsible for pro factor processing at the paired basic sites.

Plant Foods Hum Nutr, 1991 Jul, 41(3), 253 - 9
Utilization of expeller pressed partially defatted peanut cake meal in the preparation of bakery products; Chavan JK et al.; Expeller pressed partially defatted peanut cake obtained from skin-free kernels was used as graded supplements in the preparation of breads, sweet buns, cupcakes and yeast-raised doughnuts . Incorporation of cake meal lowered the specific volume and sensory properties, but improved the fresh weight, water holding capacity and protein content of the products . The products containing 10% peanut cake meal were found to be acceptable.

Somat Cell Mol Genet, 1991 Jul, 17(4), 411 - 20
Expression of a human cDNA encoding a protein containing GAR synthetase, AIR synthetase, and GAR transformylase corrects the defects in mutant Chinese hamster ovary cells lacking these activities; Chang FH et al.; The isolation of a human cDNA encoding the multifunctional protein containing GAR synthetase, AIR synthetase, and GAR transformylase by functional complementation of purine auxotrophy in yeast has been reported . Chinese hamster ovary (CHO) cell mutant purine auxotrophs deficient in GAR synthetase (Ade-C) or AIR synthetase plus GAR transformylase (Ade-G) activities were transfected with this human GART cDNA subcloned into a mammalian expression vector . This restored 49-140% of the activities of GAR synthetase, AIR synthetase, and GAR transformylase in transfected cells when compared to wild-type CHO K1 parental cells . Study of one stably expressing transfectant, AdeC2, revealed that the human GART cDNA was incorporated into the CHO genome . The enzyme activities appear to be associated with an expressed protein of 110 kDa, very similar to that of purified human GART trifunctional enzyme . The Ade-C mutant shows reduced amounts of GART mRNA compared to CHO K1 and a protein of apparently reduced size, results consistent with the purine requirement and enzyme deficiency observed in the mutant . These experiments provide definitive evidence that the human GART cDNA encodes and can direct the production of active human GART trifunctional protein in mammalian cells . They also provide important evidence that the Ade-C and Ade-G mutants of CHO cells are defective in this gene.

J Anim Sci, 1991 Jul, 69(7), 2904 - 17
Assessment of the influence of dietary vitamin E on sows and offspring in three parities: reproductive performance, tissue tocopherol, and effects on progeny; Mahan DC; Sixty crossbred (Yorkshire-Hampshire X Duroc) gilts were fed one of four corn-soybean meal diets fortified with .3 ppm Se and 0, 16, 33, or 66 IU of DL-alpha-tocopheryl acetate/kg . The study was conducted over a three-parity period to evaluate sow reproductive performance and the vitamin E tissue status of both sows and progeny at various time periods postcoitum and(or) postpartum . The basal diet averaged 8.4 mg of alpha-tocopherol/kg and .38 ppm of Se . Although litter size at birth was lowest (P less than .15) when sows were fed the basal diet, a higher incidence of agalactia when sows were fed the lower dietary vitamin E levels resulted in an increased (P less than .05) litter size at 7 d postpartum as dietary vitamin E increased . Sow serum alpha-tocopherol increased (P less than .01) at each measurement period as dietary vitamin E level increased . Colostrum and milk alpha-tocopherol concentrations increased (P less than .01) as dietary vitamin E level increased, and colostrum values were three to five times higher than at later milks . Colostrum alpha-tocopherol declined by parity from sows fed less than or equal to 16 IU/kg but was similar at each parity for sows fed greater than or equal to 33 IU/kg, resulting in a dietary vitamin E x parity interaction (P less than .01) . The Se content of sow milk declined with parity but was not affected by dietary vitamin E level . Sow liver tocopherol at weaning (28 d postpartum) increased (P less than .01) as dietary vitamin E increased and increased with parity (P less than .05) . Pig serum and liver alpha-tocopherol concentrations were elevated at birth and 7 and 28 d of age as sow dietary level of vitamin E increased . Upon weaning, pigs were fed a torula yeast-dextrose diet that contained 3.0 mg of alpha-tocopherol/kg and .32 ppm Se for a 28-d postweaning period . Liver and serum alpha-tocopherol concentrations declined during the postweaning period . Evidence of the vitamin E deficiency occurred at 28 d postweaning in the progeny from sows fed the basal diet or 16 IU of vitamin E; the incidence was more prevalent in the pigs from Parities II and III . These results suggest that a supplemental level of 16 IU of vitamin E/kg of diet was inadequate for the reproducing sow; higher levels are justified, particularly when females are retained in the herd for several parities.

Int J Syst Bacteriol, 1991 Jul, 41(3), 410 - 6
Isolation and characterization of a dimethyl sulfide-degrading methanogen, Methanolobus siciliae HI350, from an oil well, characterization of M . siciliae T4/MT, and emendation of M . siciliae; Ni SS et al.; We isolated strain HI350 from a gas and oil well in the Gulf of Mexico, characterized it, and found that it is closely related to Methanolobus siciliae T4/MT (T = type strain), which we also characterized . The previously published characterization of the type strain of M . siciliae was limited to the optimum temperature for growth, and our characterization suggested the species description given below . Cells are irregular, nonmotile, coccoid, and 1.5 to 3 microns in diameter . The catabolic substrates used include methanol, trimethylamine, and dimethyl sulfide, but not H2-CO2, formate, or acetate . Growth is fastest in the presence of 0.4 to 0.6 M Na+, in the presence of 60 to 200 mM Mg2+, at pH 6.5 to 6.8, and at 40 degrees C . Growth on trimethylamine is stimulated by yeast extract . An electrophoretic analysis confirmed that strain HI350 is closely related to strain T4/MT and indicated that major changes in the intracellular proteins of M . siciliae HI350 occur when the growth substrate is switched between dimethyl sulfide and trimethylamine.

Plant Cell, 1991 Jul, 3(7), 667 - 75
The basic domain of plant B-ZIP proteins facilitates import of a reporter protein into plant nuclei; van der Krol AR et al.; The import of large molecules into the nucleus is an active process that requires the presence in cis of a nuclear localization signal (NLS) . Although these signals have been well characterized in mammalian, yeast, and amphibian nuclear proteins, no plant NLS has yet been described . The NLSs identified so far generally contain clusters of basic amino acids . This characteristic feature prompted us to test several basic domains from the plant DNA-binding proteins TGA-1A and TGA-1B and the TATA box-binding protein TFIID for nuclear targeting function . When tested as N-terminal fusions to the beta-glucuronidase protein, only those constructs containing the DNA binding (basic) domain of the basic-zipper (B-ZIP) region of TGA-1A or TGA-1B conferred nuclear import . These results suggest a close association or overlap of the DNA binding and nuclear targeting domains of B-ZIP proteins . We also demonstrated that a wild-type but not a mutant simian virus 40 large T-antigen NLS facilitates import into plant nuclei, indicating a strong conservation between nuclear import mechanisms in animals and plants.

Plant Cell, 1991 Jul, 3(7), 657 - 65
Cell-specific expression of plant histone H2A genes; Koning AJ et al.; Histone H2A is a component of eukaryotic chromatin whose expression has not been studied in plants . We isolated and characterized a tomato and a pea cDNA encoding histone H2A . We found that in tomato H2A is encoded by a small gene family and that both the pea and the tomato mRNAs are polyadenylated . Tomato H2A has 82% amino acid residue identity to pea H2A, 83% to wheat, and 65% to human and yeast H2A . Plant H2As differ from fungal and animal H2As in their amino-terminal and carboxy-terminal regions . Carboxy-terminal plant H2A regions contain the motif SPKK, a peptide implicated in binding of A/T-rich DNA regions . By using RNA gel blot analysis, we determined that the steady-state mRNA level of these genes was abundant in apices and early developing fruit and very low in mature tissues . In situ RNA hybridization showed strong spatial regulation because the mRNA was abundant in some cells and not detectable in others . In tomato shoot tips, H2A-expressing cells were distributed irregularly in or near meristems . In tomato or pea root tips, expressing cells were concentrated near the apex, and their distribution was consistent with that expected of cycling cells . Other H2A transcripts were found in nondividing cortical cells that are known to undergo endoduplication during the late maturation phase of primary development.

Proc Natl Acad Sci U S A, 1991 Jul 1, 88(13), 5809 - 13
Enhanced hematopoietic activity of a human granulocyte/macrophage colony-stimulating factor-interleukin 3 fusion protein; Curtis BM et al.; Granulocyte/macrophage colony-stimulating factor-interleukin 3 (GM-CSF-IL-3) fusion proteins were generated by construction of a plasmid in which the coding regions of human GM-CSF and IL-3 cDNAs were connected by a synthetic linker sequence followed by subsequent expression in yeast . Both GM-CSF-IL-3 and IL-3-GM-CSF fusion proteins were purified to homogeneity and shown to bind to cell-surface receptors through either their GM-CSF or IL-3 domains . The fusion proteins exhibited enhanced receptor affinity, proliferative activity, and hematopoietic colony-stimulating activity compared with either IL-3 and/or GM-CSF alone . This suggests that GM-CSF-IL-3 fusion proteins may hold future promise as therapeutic agents.

Proc Natl Sci Counc Repub China B, 1991 Jul, 15(3), 153 - 9
Potentiation effect of corn extract on the production of eremofortin C, EC oxidase, and PR toxin by Penicillium roqueforti; Chang SC et al.; Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti . Their structures are similar and differ only by an alcohol and an aldehyde group at the C-12 position . EC has been demonstrated to be the precursor of PR toxin, and EC is transformed to PR toxin by EC oxidase . These two compounds and EC oxidase are secreted by P . roqueforti in the culture medium, which is usually composed of 15% sucrose and 2% yeast extract . Recently, we discovered that the addition of corn extract to this medium increased the production of EC and PR toxin and the activity of EC oxidase in a coordinative manner . In a time-course study, we found that the peak yield of EC and PR toxin and the maximum activity of EC oxidase in the culture medium containing 7.5% sucrose, 1% yeast extract, and 20% corn extract were increased 6.2, 4.6, and 4.7-fold, respectively, as compared with those obtained in the medium without corn extract . Moreover, corn extract increased the production of EC and PR toxin and the activity of EC oxidase by P . roqueforti in a dose-dependent manner . On the other hand, when the concentrations of sucrose and yeast extract were increased while fixing the ratio of corn extract, we found that the levels of EC and PR toxin and the enzyme activity were decreased concomitantly . We thus conclude that corn extract can enhance the production of EC, PR toxin and EC oxidase by P . roqueforti when grown in a minimal medium and that the potentiation effect of corn extract is suppressed when the fungi are grown in a rich medium.

Antonie Van Leeuwenhoek, 1991 Jul, 60(1), 55 - 9
On the false positive urease activity of Yarrowia lipolytica; Peter G et al.; The ability of Yarrowia lipolytica to produce ammonia from urea was found variable on some media . The colour change of the indicator in Christensen's urea agar was not due to the urease activity of this species but was a non-specific alkalization reaction . Rapid urea broth was reliable giving no false positive results . It was found that Y . lipolytica is a urease negative yeast species.

Izv Akad Nauk SSSR Biol, 1991 Jul-Aug, (4), 611 - 4
{The possible effect of amino acids forming a loop in the surface layer of subunits on the electrophoretic mobility of enzymes in the alcohol/polyol dehydrogenase family}; Sudovtsov VE; Relative electrophoretic mobility (REM) of alcohol dehydrogenases from equine hepatocyte cytoplasm was low probably due to the presence of a loop which consisted of 21 amino acid residues in the surface layer of the enzyme subunits . The REM of multiple molecular forms of alcohol dehydrogenases from yeast cell cytoplasm was higher as consistent with the absence of this loop in the surface layer of the enzyme subunits . Possible role of amino acid residues comprising the loop, in the formation of total charge and their effect on REM values of enzymes from the alcohol/polyol dehydrogenase family are discussed.

Mol Cell Endocrinol, 1991 Jul, 78(3), 171 - 8
Primary structure and tissue distribution of anglerfish carboxypeptidase H; Roth WW et al.; Most peptide hormones are synthesized as part of larger precursor proteins which must be processed after translation to generate bioactive peptides . This usually involves cleavage of the precursor by an endopeptidase at sites marked by basic amino acids, followed by removal of N- or C-terminal basic residues by the action of an aminopeptidase or carboxypeptidase . These processing events have been observed in a variety of species, from yeast to mammals . As part of an effort to characterize prohormone processing enzymes in the anglerfish, Lophius americanus, we have cloned and sequenced a cDNA for the fish prohormone processing carboxypeptidase H (CPH) . Polyadenylated RNA from anglerfish (AF) islet organs was used to construct a cDNA library in phage lambda gt11 . The library was screened with a probe derived from the cDNA for rat CPH . A 2400 base pair AF cDNA clone was isolated . This cDNA encodes a polypeptide which is similar in size and composition to mammalian CPH . The sequence data indicate that the AF CPH precursor is a 454 amino acid polypeptide . The derived amino acid sequence of the putative fish CPH is 81% homologous to the rat and bovine CPH enzymes . Significantly, all of the amino acid residues thought to be important for metal ion and substrate binding, glycosylation, and catalytic activity of mammalian CPH are conserved in the fish enzyme . Northern hybridization using RNA from AF tissues indicates that a 2.5 kb fish CPH mRNA is expressed in brain, pituitary and islet organs, but not in other tissues which do not secrete peptide hormones.

Med Vet Entomol, 1991 Jul, 5(3), 283 - 92
A simple larval diet for population studies on the blowfly Lucilia sericata (Diptera: Calliphoridae); Daniels S et al.; 1 . A simple artificial diet was devised for larvae of Lucilia sericata Meigen . 2 . A basic diet of 20 g/l agar with 20% horse blood and added yeast sustained normal growth and development, as compared with a lamb meat control . 3 . When yeast was added to the blood agar diet at 50 g/l, both peak and final larval weights were increased by 25-50% at higher larval densities (2-8 larvae/g diet) and the hatchling-adult development period was reduced by about 3 days . 4 . No adult insects emerged on an agar-yeast diet without blood . Increasing the concentration of blood from 10% to 20% increased adult weight at emergence by 50%, though developmental period was not significantly affected . 5 . Increasing larval density significantly reduced the weights of pupae and emergent adults (by up to 50%), both on the blood-agar-yeast diet and the lamb meat control . Percentage survival fell from 80-90% to below 10% on the blood-agar-yeast diet at the highest densities of 4 or 8 larvae/g diet, though survival on lamb was not significantly affected by density.

Cell Growth Differ, 1991 Jul, 2(7), 343 - 9
p34cdc2 is physically associated with and phosphorylated by a cdc2-specific tyrosine kinase; Ferris DK et al.; The mammalian homologue of the yeast cdc2 gene encodes a 34-kilodalton serine/threonine kinase that is a subunit of M phase-promoting factor . Recent studies have shown that p34cdc2 is also a major tyrosine-phosphorylated protein in HeLa cells and that its phosphotyrosine content is cell cycle regulated and related to its kinase activity . Here, we show that cdc2 is physically associated with and phosphorylated in vitro by a highly specific tyrosine kinase . Tyrosine phosphorylation of cdc2 in vitro occurs at tyrosine 15, the same site that is phosphorylated in vivo . The association between the two kinases takes place in the cytosolic compartment and involves cyclin B-associated cdc2 . Evidence is presented that a substantial fraction of cytosolic cdc2 is hypophosphorylated, whereas nuclear cdc2 is hyperphosphorylated . Finally, we show that the tyrosine kinase associated with cdc2 may be a 67-kilodalton protein and is distinct from src, abl, fms, and other previously reported tyrosine kinases.

Hum Antibodies Hybridomas, 1991 Jul, 2(3), 142 - 7
Serodiagnosis of cancer by using Candida cytochrome c recognized by human monoclonal antibody HB4C5; Hashizume S et al.; Cytochrome c from various sources, such as Candida krusei, yeast, horse, and cattle, was found to be recognized by human monoclonal antibody HB4C5 specific to lung cancer . Therefore, the cytochrome c was applied to the measurement of antibody amount in patient sera with a similar reactivity to the antibody HB4C5 for serodiagnosis of cancer . The cytochrome c from Candida krusei was most valuable for the serodiagnosis of various cancers, and the yeast cytochrome c was also useful . However, horse and bovine cytochrome c did not react with antibody of the cancer patients . By using Candida cytochrome c lung, bile duct, esophagus, and liver cancers were detected at high rates of more than 50% . In the case of lung cancer, the detection rates of small-cell, squamous, large-cell and adenocarcinoma were 78%, 63%, 100%, and 34%, respectively . The rate for small-cell carcinoma was higher than that with the currently used NSE assay system, and the rate for squamous carcinoma was comparable to that with the SCC assay system, although the system using cytochrome c did not show similar reactivity to that with the SCC system . Furthermore, lung cancer was detected at early stages by using cytochrome c, and even in the case of adenocarcinoma, the rate at early stages with the cytochrome c system was higher than that with the CEA assay system . On the other hand, false positive rates of benign diseases and normal were low--8% and 2%, respectively.

Mol Gen Genet, 1991 Jul, 227(3), 356 - 60
Reciprocal homologous junctions generated in mouse cells; Desautels L et al.; We analysed pairs of reciprocal homologous junctions resulting from intermolecular conservative homologous recombination in mouse cells . The assay used did not rely on the reconstitution of a selectable gene . This permitted the introduction of multiple markers in the parental homologous sequences which in turn enabled us to compare the contribution of each parent to the reciprocal products of a given recombination event . In all recombinants analysed we found, when comparing the reciprocal junctions, a middle segment originating from only one parent . This segment of uniparental origin occurred randomly throughout the region of homology and could extend over a thousand base pairs . These results are consistent with a gap repair process like the one proposed for homologous recombination in yeast . However, introducing a double-strand break in the region of homology did not enhance but rather decreased the proportion of recombinants with reciprocal homologous junctions relative to other types of recombinants.

J Med Microbiol, 1991 Jul, 35(1), 29 - 34
Killing of Histoplasma capsulatum by gamma-interferon-activated human monocyte-derived macrophages: evidence for a superoxide anion-dependent mechanism; Brummer E et al.; The interaction of human macrophages with the yeast form of the thermally dimorphic fungal pathogen, Histoplasma capsulatum, was studied . Macrophages derived from monocytes by culture in vitro for 3 days ingested H . capsulatum, but were neither fungicidal or fungistatic . In contrast, when monocytes were exposed to human recombinant gamma-interferon (gamma-IFN) during their differentiation into macrophages, those macrophages were able to reduce the number of ingested or adherent cfu of H . capsulatum by 44-75% in 2 h . Activation of macrophages for fungicidal activity by gamma-IFN was dose dependent and 500-1000 units ml were optimal . Antibody to gamma-IFN abrogated the gamma-IFN activation process . Killing of H . capsulatum by activated macrophages in 2-h assays could be inhibited by superoxide dismutase but not by sodium azide.

Endocrinology, 1991 Jul, 129(1), 503 - 10
An inducible functional peripheral benzodiazepine receptor in mitochondria of steroidogenic granulosa cells; Amsterdam A et al.; Granulosa cell lines, transformed by SV40 T-antigen and Ha-ras oncogene, have recently been established that can produce progesterone at levels comparable to those of highly differentiated cultures of primary granulosa cells (1-4) . Here, the hypothesis that these cells contain a mitochondrial benzodiazepine receptor, and that stimulation of the receptor can trigger progesterone production in these cells, was tested . The agonist of the peripheral benzodiazepine receptor, Ro5-4864, produced a 3- to 5-fold stimulation (P less than 0.005) of progesterone production both in differentiated granulosa primary cultures and in the oncogene-transformed cell lines . Ro5-2807 (diazepam, Valium) exerts a similar effect on granulosa cell steroidogenesis while the specific agonist of central benzodiazepine receptor Ro15-4513 was without effect . The effects of Ro5-4864 or Ro5-2807 were not additive to those of gonadotropins and cAMP . Intact isolated mitochondria possessed high-affinity binding sites to {3H}-Ro5-4864 (Kd = 3.03 +/- 0.70 nM), which were enriched by 1 order of magnitude in these organelles compared to total cell homogenate . Bound Ro5-4864 could be competitively displaced with 1 microM unlabeled Ro5-4864 and Ro5-2807, but not with specific ligands of central benzodiazepine receptors Ro15-4513 and Ro15-1788 . Prolonged elevation of cAMP in these cells caused a 30% (P less than 0.01) rise in the number of receptors . Mitochondria of NIH 3T3 cells contained only 30-40% (P less than 0.001) of the Ro5-4864 binding sites of mitochondria from steroidogenic cells, whereas yeast mitochondria lacked them completely . The existence of functional peripheral benzodiazepine receptors in mitochondria suggests that they may have a physiological role in the mobilization of cholesterol into mitochondria, and in elevating progesterone production in ovarian cells . The modulation of the interaction between benzodiazepine compounds and the gamma-aminobutyric acid receptor by progesterone metabolites suggests new interrelationships between peripheral and central nervous system receptors sensitive to benzodiazepines.

Agric Biol Chem, 1991 Jul, 55(7), 1757 - 64
Evidence that more than one gene encodes n-alkane-inducible cytochrome P-450s in Candida maltosa, found by two-step gene disruption; Ohkuma M et al.; An n-alkane-assimilating yeast, Candida maltosa, has a diploid genome . Probed with the previously isolated gene of n-alkane-inducible cytochrome P-450 (P-450alk), its allelic gene had been isolated, its nucleotides sequenced, and the interallelic divergence examined . Using one of the allelic genes, we disrupted the two alleles of the cytochrome P-450alk gene by a two-step gene disruption system . Surprisingly, the disruptant still assimilated n-alkane and contained n-alkane-inducible cytochrome P-450 . This result indicates that, other than the disrupted two alleles, there is at least one other gene that encodes an n-alkane-inducible cytochrome P-450.

J Biotechnol, 1991 Jul, 19(2-3), 259 - 70
Use of ars18 based vectors to increase protein production in Yarrowia lipolytica; Nicaud JM et al.; The isolation of ars sequence from the yeast Yarrowia lipolytica has recently been reported (Fournier et al., 1991) . Vectors containing ars18 have been used to increase homologous and heterologous protein production . Examples presented are the Yarrowia lipolytica alkaline extracellular protease (AEP), the porcine alpha 1-interferon and the bovine prochymosin . A 2- to 6-fold increase in the corresponding protein production was observed and in several cases it was established that it corresponded to the copy number of plasmid in the cell.

Philos Trans R Soc Lond B Biol Sci, 1991 Jun 29, 332(1264), 271 - 6
The Florey Lecture, 1990 . How is the cell division cycle regulated?
Nurse P.
It is argued in this lecture that in most eukaryotic cells onset of mitosis is coupled to attainment of a critical cell mass and to completion of the previous S-phase . In fission yeast these controls operate through a regulatory gene network that activates the p34cdc2 protein kinase at mitosis . This is brought about by dephosphorylation of a tyrosine residue located in the ATP binding site of the kinase . The p34cdc2 protein kinase is also important for regulating the onset of mitosis in vertebrate cells suggesting that there is a universal control regulating mitosis in all eukaryotic cells.

Science, 1991 Jun 21, 252(5013), 1651 - 6
Complementary DNA sequencing: expressed sequence tags and human genome project; Adams MD et al.; Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs) . ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences . Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor . Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction . This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.

J Biol Chem, 1991 Jun 15, 266(17), 10989 - 94
Interaction of CAP sequence site binding factor and transcription factor IID preceding and following binding to the adenovirus 2 major late promoter; Safer B et al.; Interaction of cloned yeast, drosophila, and human transcription factor IID (yTFIID, dTFIID, and hTFIID, respectively) with the adenovirus 2 major late promoter (Ad2 MLP) confers a more limited pattern of DNase I protection than that obtained using highly purified native hTFIID (Hahn, S., Buratowski, S., Sharp, P . A . and Guarente, L . (1989) EMBO J . 8, 3379-3382; Van Dyke, M . W., and Sawadogo, M . (1990) Mol . Cell . Biol . 10, 3415-3420; Horikoshi, M., Wang, C.K., Fujii, H., Cromlish, J.A., Weil, P.A., and Roeder, R.G . (1989) Nature 341, 299-303; Peterson, M . G., Tanese, N., Pugh, B.F., and Tjian, R . (1990) Science 248, 1625-1630; Hoey, T., Dynlacht, B . D., Peterson, M.G., Pugh, B.F., and Tjian, R . (1990) Cell 61, 1179-1186) . Since the mass of the cloned TFIIDs is considerably less than that of native hTFIID (27-38 kDa versus 120-140 kDa), it is considered likely that native hTFIID exists as a mixed heterodimer . We have recently identified, purified, and characterized a novel transcription factor that binds to the CAP site region (+1 to +23) of the Ad2 MLP . This CAP site binding factor, designated CBF, is required for optimal transcriptional activity . We now show that when bound to the Ad2 MLP, yTFIID and CBF interact to generate the extended pattern of DNase I protection conferred by native hTFIID . In addition, bound yTFIID and CBF interact such that the stability of the complex exceeds that of each factor bound alone . We also demonstrate the existence in nuclear extracts of a hTFIID and CBF heterodimer by the electrophoretic mobility shift analysis . CBF, therefore, may represent the first identified member of a large family of gene-specific TFIID-associated factors that are required for the regulated gene-specific expression of TFIID activity.

Proc Natl Acad Sci U S A, 1991 Jun 15, 88(12), 5297 - 301
Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes; Thomas L et al.; Two mammalian gene products, PC2 and PC3, have been proposed as candidate neuroendocrine-precursor processing enzymes based on the structural similarity of their catalytic domains to that of the yeast precursor-processing endoprotease Kex2 . In this report we demonstrate that these two proteases can cleave proopiomelanocortin (POMC) in the secretory pathway of mammalian cells . Similarly to pituitary corticotrophs, PC3 expressed in processing-deficient BSC-40 cells cleaved native mouse POMC at the -Lys-Arg- sites flanking corticotropin . The -Lys-Arg- within beta-lipotropin was less efficiently cleaved to release beta-endorphin . Expression of PC2 together with PC3 resulted in efficient conversion of beta-lipotropin, as occurs in pituitary melanotrophs . Furthermore, coexpression of PC2 together with mouse POMC in bovine adrenomedullary chromaffin cells resulted in conversion of beta-lipotropin to gamma-lipotropin and beta-endorphin in the regulated secretory pathway . Finally, the processing selectivities of PC3 and PC2 expressed together in BSC-40 cells were determined by using a series of mutant mouse POMCs containing all possible pairs of basic residues at certain sites . The observed pattern of cleavage site selectivities mimicked that of the endogenous endoproteases of the insulinoma and bovine adrenomedullary chromaffin cells, suggesting that PC2 and PC3 may represent important core endoproteases in the catalysis of prohormone processing in many neuroendocrine cell types.

Nature, 1991 Jun 13, 351(6327), 588 - 90
Reduced binding of TFIID to transcriptionally compromised mutants of VP16; Ingles CJ et al.; Activator proteins that control transcription initiation by RNA polymerase II usually have two domains: one binds to DNA, and the other activates transcription . A particularly potent acidic activation domain at the C terminus of the herpes simplex virus protein VP16 binds directly and selectively to the human and yeast TATA box-binding factor TFIID . We have now investigated the biological significance of this in vitro interaction by using mutant forms of VP16 . For changes at the critical phenylalanine residue at position 442 of VP16 there was a good correlation between transactivation activity in vivo and the binding of VP16 to TFIID in vitro . In contrast, mutants with reduced negative charge were more defective for binding than for activation.

J Biol Chem, 1991 Jun 5, 266(16), 10632 - 7
Nucleotide sequence of the unique nitrate/nitrite-inducible cytochrome P-450 cDNA from Fusarium oxysporum; Kizawa H et al.; A cDNA clone for the nitrate/nitrite-inducible cytochrome P-450 (P-450) of the fungus Fusarium oxysporum (tentatively termed P-450dNIR) was isolated by an immunoscreening method . Sequence determination revealed a polypeptide of 403 amimo acid residues (Mr = 44,371), which was shown to contain the full-length sequence of the fungal P-450 . The amino terminus region of the predicted sequence contained neither the signal-like, hydrophobic domain that is commonly observed in microsomal P-450s nor the tagging prosequence that is essential for localization of mitochondrial P-450s . Further, the sequence exhibited higher homologies against those of soluble bacterial P-450s, in particular P-450s of Streptomyces, rather than those of eukaryotic P-450s including yeast and fungal P-450s . These results are highly indicative that P-450dNIR is the first soluble P-450 derived from eukaryotic organisms . The unique features might be related to the novel function of P-450dNIR, which is involved in a dissimilatory reduction of nitrite by the fungus . P-450dNIR was classified into a new family, P-450LV, and the corresponding gene of the fungus was named CYP55.

J Med Chem, 1991 Jun, 34(6), 1838 - 44
Oxidation of dihydropyridine calcium channel blockers and analogues by human liver cytochrome P-450 IIIA4; Guengerich FP et al.; A series of 21 different 4-substituted 2,6-dimethyl-3-(alkoxycarbonyl)-1,4-dihydropyridines was considered with regard to oxidation to pyridine derivatives by human liver microsomal cytochrome P-450 (P-450) . Antibodies raised against P-450 IIIA4 inhibited the microsomal oxidation of nifedipine and felodipine to the same extent, as did cimetidine and the mechanism-based inactivator gestodene . Gestodene was approximately 10(3) times more effective an inhibitor than cimetidine, on a molar basis . When rates of oxidation of the 1,4-dihydropyridines were compared to each other in different human liver microsomal preparations, all were highly correlated with each other with the exceptions of a derivative devoid of a substituent at the 4-position and an N1-CH3 derivative . A P-450 IIIA4 cDNA clone was expressed in yeast and the partially purified protein was used in reconstituted systems containing NADPH-cytochrome P-450 reductase and cytochrome b5 . This system catalyzed the oxidation of all of the 1,4-dihydropyridines except the two for which poor correlation was seen in the liver microsomes . Principal component analysis supported the view that most of these reactions were catalyzed by the same enzyme in the yeast P-450 IIIA4 preparation and in the different human liver microsomal preparations, or by a closely related enzyme showing nearly identical properties of catalytic specificity and regulation . The results indicate that the enzyme P-450 IIIA4 is probably the major human catalyst involved in the formal dehydrogenation of most but not all 1,4-dihydropyridine drugs.

J Parasitol, 1991 Jun, 77(3), 441 - 8
Excystation of Giardia muris induced by a phosphate-bicarbonate medium: localization of acid phosphatase; Feely DE et al.; Giardia muris cysts were incubated briefly in an aqueous induction medium of 0.1 M potassium phosphate with 0.1, 0.2, or 0.3 M sodium bicarbonate . High rates of excystation (91.1-96.7%) were recorded within 5 min after the cysts were placed in trypticase-yeast extract-iron-serum (TYI-S) medium . Substitution of phosphate-buffered saline for TYI-S as the excystation medium resulted in high rates (95.9%) of excystation but required an incubation of 15 min . Excystation was inhibited by the presence of 4-4'-diisothiocyanatostilbene-2-2'-disulfonic acid (DIDS), a specific inhibitor of vacuolar and lysosomal acidification . Microscopic observation showed the loss of the peritrophic space and a change in the refractile nature of the cyst wall prior to excystation . Histochemical studies demonstrated a reaction product of acid phosphatase activity in the lysosomelike peripheral vacuoles in induced cysts and in the peritrophic space of cysts placed in excystation medium . Staining with acridine orange suggested that the peripheral vacuoles become acidified during induction . This staining was inhibited also by DIDS . These studies show that in vitro excystation can be produced at high rates by easily prepared media without exogenous enzymes, low pH, reducing agents, or complex components . The data also suggest that excystation may be stimulated by the bicarbonate-phosphate medium accompanied by acidification of the peripheral vacuoles and the release of their contents into the peritrophic space.

J Nutr, 1991 Jun, 121(6), 908 - 16
Dietary plant materials and development of diabetes in the BB rat; Hoorfar J et al.; The present study was designed to examine further the impact of individual plant protein sources found in a diabetogenic, cereal-based, rodent laboratory diet, NIH-07 {open formula, nonpurified rat and mouse diet (positive control)}, on the development of diabetes . Diabetes-prone BB rats that were pan-T(OX19+)-lymphopenic were fed a low diabetogenic diet during gestation and lactation . Progeny of these rats were fed a normal or autoclaved NIH-07 diet, or one of eight other diets based on the AIN-76A formulation, with modified protein sources as follows: hydrolyzed casein (HC), soybean meal, HC+ trypsin inhibitor (TI) in water (2 mg/mL, wheat germ, alfalfa seeds, Brewer's yeast, red lentils and a plant protein mixture . Feeding soybean meal increased the incidence of diabetes compared with the negative control, HC diet (47% vs . 12% incidence, P = 0.02) . Wheat germ, alfalfa seeds and plant protein mixture resulted in an intermediate incidence of diabetes of 33%; the incidence was lower for Brewer's yeast and lentils (20% and 13%) . Autoclaving (121 degrees C, 10 min) the NIH-07 diet or the presence of TI in drinking water had a minimal effect on diabetes frequency, suggesting heat-labile plant toxicants were not directly involved . Thus, certain dietary plant protein sources or associated agents may influence the development of spontaneous diabetes in the BB rat.

J Leukoc Biol, 1991 Jun, 49(6), 592 - 8
Effect of dietary fish oil on development and selected functions of murine inflammatory macrophages; Hubbard NE et al.; Inflammatory macrophages from mice fed diets containing menhaden fish oil (MFO) have a reduced capacity for cytotoxicity of mastocytoma cells upon activation with interferon-gamma (IFN gamma) and lipopolysaccharide due to an altered responsiveness to IFN gamma . In an effort to elucidate further how dietary MFO effects macrophage function, we have studied the maturation of inflammatory macrophages from mice fed MFO compared with mice fed safflower oil (SFO) using several processes that serve as markers of the activational state . No significant differences in the recruitment or percentage of peritoneal exudate cells as macrophages after thioglycollate injection and no differences in spreading, binding, or phagocytosis of sheep erythrocytes or phagocytosis of yeast by inflammatory macrophages were observed when the dietary groups were compared . However, MFO macrophages had an altered capacity for peroxide release when stimulated with unopsonized zymosan (10-200 micrograms/ml) . Furthermore, to elucidate how MFO feeding could alter IFN gamma-induced responses of inflammatory macrophages, we assessed phorbol-12-myristate-13-acetate-induced hydrogen peroxide production and expression of class II MHC determinants (Ia) . There were no differences between macrophages from mice fed the two diets with respect to the production of peroxide when they were preincubated with 0.1-10 U/ml of IFN gamma . However, MFO macrophages had greater peroxide production after enhancement with 100 U/ml of IFN gamma . With respect to Ia induction, the percentage of macrophages responding to IFN gamma was not altered by diet, and there were no differences in expression of Ia induced by 24 hr exposure to IFN gamma . Thus the differential effect of MFO compared with SFO is probably mediated not by an alteration in the maturation of inflammatory macrophages but rather through the alteration of IFN gamma-induced functions such as peroxide production.

Z Gesamte Inn Med, 1991 Jun, 46(8), 263 - 70
{Therapy of HIV infection (AIDS)}; Haustein UF; The stages of human immunodeficiency viruses (HIV) life cycle are described as guide to therapeutic intervention . Practical therapeutic recommendations are given . They should be directed to viruses as the causal agent and to the features of opportunistic infections as well as of associated malignant tumors . Recently 3 progresses could be reached: (1) the application of azidothymidine in the latency phase, when the number of CD4 positive cells decreases below 500/mm3, whereby the progression of the disease can be delayed and side-effects can be reduced; (2) the prophylaxis of pneumocystis carinii pneumonia by inhalation of pentamidin; and (3) the introduction of fluconazole acting against yeast fungus infection, whereby development of resistant yeast strains is still missing and side-effects are smaller than with other antimycotics . In addition, the application of HIV-vaccine in already HIV-infected persons seems to be effective . By combining several drugs their toxicity is to be reduced . Interdisciplinary research and good cooperation among clinicians are conditions for an effective therapy . Last but not least psychosocial aspects and a good psychological guidance and counseling of the affected persons should be considered.

Planta Med, 1991 Jun, 57(3), 225 - 31
Analgesic, antipyretic and anti-inflammatory properties of Euphorbia hirta; Lanhers MC et al.; Lyophilised aqueous extract of Euphorbia hirta L . (Euphorbiaceae) has been evaluated for analgesic, antipyretic and anti-inflammatory properties in mice and rats, in order to complete its activity profile, after the confirmation of the existence of a central depressant activity particularly expressed by a strong sedative effect, associated with anxiolytic effects . This study leads us to the conclusion that this plant extract exerts central analgesic properties . Such a dose-dependent action was obtained against chemical (writhing test) and thermic (hot plate test) stimuli, respectively, from the doses of 20 and 25 mg/kg and it was inhibited by a naloxone pretreatment, a specific morphinic antagonist compound . An antipyretic activity was obtained at the sedative doses of 100 and 400 mg/kg, on the yeast-induced hyperthermia . Finally, significant and dose-dependent anti-inflammatory effects were observed on an acute inflammatory process (carrageenan-induced edema test in rats) from the dose of 100 mg/kg . On the other hand, plant extract remained inactive on chronic processes such as Freund's adjuvant-induced rheumatoid arthritis, after a chronic treatment during fourteen days at the daily dose of 200 or 400 mg/kg; however, if inefficacy was observed on rat backpaws edema and on loss of weight, the aqueous extract reduced the inflammatory hyperalgia.

Curr Opin Cell Biol, 1991 Jun, 3(3), 444 - 51
Telomeres; Greider CW; Telomeres are essential for chromosome stability and replication . Maintaining a balance between telomere shortening and lengthening is essential for cell viability . Recent work on telomeres from yeast, Drosophila and mammals, and on telomerase has provided insight into the mechanisms of both the shortening and lengthening processes.

Curr Opin Cell Biol, 1991 Jun, 3(3), 407 - 13
Replication origins, factors and attachment sites; Gasser SM; The initiation of eukaryotic DNA synthesis occurs at specific sites determined by both cis- and trans-acting elements . Here I review advances in the characterization of yeast origins, origin-binding proteins and the relationship of DNA replication to nuclear substructure in yeast.

J Clin Microbiol, 1991 Jun, 29(6), 1106 - 13
Isolation and characterization of Sporothrix schenckii from clinical and environmental sources associated with the largest U.S . epidemic of sporotrichosis; Dixon DM et al.; The largest recorded epidemic of sporotrichosis in the United States occurred in 1988 and involved a total of 84 cases in 15 states . All cases were associated with Wisconsin-grown sphagnum moss . Twenty-one clinical isolates of Sporothrix schenckii and 69 environmental isolates of Sporothrix spp . from the epidemic were characterized and compared . The environmental isolates were recovered from 102 samples of sphagnum moss and other material by using direct plating techniques . Characteristics examined included macroscopic and microscopic morphology, conversion to a yeast phase, exoantigen reactions, and virulence in mice . On the basis of these studies, eight environmental isolates were identified as S . schenckii, five were identified as Ophiostoma stenoceras, and the remainder were identified as Sporothrix species . The environmental isolates of S . schenckii were recovered from moss samples from one Pennsylvania nursery and from three New York State Soil and Water Conservation districts, but none were recovered from moss directly from the bogs in Wisconsin.

Chromosoma, 1991 Jun, 100(5), 289 - 92
Coming to grips with a complex matter . A multidisciplinary approach to the synaptonemal complex; Loidl J; Research into the synaptonemal complex (SC) is currently finding renewed interest . This is because the primarily cytological knowledge of the SC has been increasingly supplemented by immunochemical characterization and genetical studies over recent years . Moreover, yeast, offering one of the most handy and rewarding experimental systems, has joined the group of organisms in which the SC can be studied cytologically . In this essay I shall present aspects concerning the SC and its role in meiotic pairing that have emerged over the past few years, parts of which were discussed at a recent meeting in Obertraun, Austria.

J Exp Med, 1991 Jun 1, 173(6), 1511 - 20
Isolation and characterization of beta-glucan receptors on human mononuclear phagocytes; Czop JK et al.; beta-glucan receptors, with ligand specificity for yeast and fungal carbohydrate polymers, have been studied as phagocytic receptors of human monocytes . To characterize their structure, binding studies were carried out with human U937 cells and a rabbit IgG anti-Id that recognizes epitopes on monocyte beta-glucan receptors . Unstimulated U937 cells specifically bound large amounts of the anti-Id, but almost none of the control anti-isotype . At saturation, the number of anti-Id molecules bound per U937 cell was 2.6 x 10(6) with an apparent Ka of 1.9 x 10(7) M-1 . Immunoprecipitates from detergent lysates of surface-radioiodinated U937 cells contained only two membrane proteins with antigenic specificity for the anti-Id, one having a mol wt of 180 kD and the other 160 kD . Both proteins were disulfide-linked and presented, after reduction, as five polypeptides of 95, 88, 60, 27, and 20 kD . Detergent lysates of unlabeled U937 cells, purified by affinity chromatography on anti-Id-Sepharose, yielded the same two nonreduced proteins and five reduction products in slab gels stained with Coomassie blue . In Western blots probed with the anti-Id, the most immunoreactive nonreduced and reduced affinity-purified products were the 160 and 20 kD molecules, respectively . Immunoblots of two-dimensional gels showed the 180 and 160 kD proteins to express a common epitope through disulfide linkage to the 20 kD polypeptide . By immunoblot analysis, U937 cell glucan-binding proteins from detergent lysates contained two cell proteins antigenic for the anti-Id that were indistinguishable from affinity-purified molecules in size and subunit composition . Studies of affinity-purified proteins from detergent lysed human monocytes were characterized by immunoblot analysis and found to be identical to U937 cell beta-glucan receptors . They consisted of two disulfide-linked proteins, with mol wt of 180 and 160 kD, and had in common a 20 kD polypeptide with the anti-Id epitope.

Hum Cell, 1991 Jun, 4(2), 100 - 8
{Cell division and the microtubular cytoskeleton}; Izutsu K; Kinetochore microtubules result from an interaction between astral microtubules and the kinetochore of the chromosomes after breakdown of the nuclear envelope at the end of prophase . In this process, the end of a microtubule projecting from one of the polar regions contacts the primary constriction of a chromosome . The latter then undergoes rapid poleward movement . Concerning the mechanism of anaphase chromosome movement, the motive force for the chromosome-to-pole movement appears to be generated at the kinetochore or in the region very close to it . It has not been determined whether chromosomes propel themselves along stationary kinetochore microtubules by a motor at the kinetochore, or they are pulled poleward by a traction fiber consisting of kinetochore microtubules and associated motors . As chromosomes move poleward coordinate disassembly of kinetochore microtubules might occur from their kinetochore ends . In diatom and yeast spindles, elongation of the spindle in anaphase (anaphase B) may be explained by microtubule assembly at polar microtubule ends in the spindle mid-zone and sliding of the antiparallel microtubules from the opposite poles . The sliding force appears to be generated through an ATP-dependent microtubule motor . In isolated sea urchin spindles, the microtubule assembly at the equator alone might provide the force for spindle elongation, although, in addition, involvement of microtubule sliding by a GTP-requiring mechanochemical enzyme cannot be excluded . Discussions were made on possible participation in anaphase chromosome movement of such microtubule motors as dynein, kinesin, dynamin and the claret segregation protein.

Biol Trace Elem Res, 1991 Jun, 29(3), 289 - 94
A preliminary report on the intervention trials of primary liver cancer in high-risk populations with nutritional supplementation of selenium in China; Yu SY et al.; The purpose of this study was to evaluate the effect of selenium (Se) in the prevention of human primary liver cancer . Three intervention trials were conducted among the residents at high risk to primary liver cancer (PLC) in Qidong county, Jiang-su province, the People's Republic of China . This area has the second highest rate of PLC in China . One trial was undertaken among the general population in a township with supplement of table salt fortified with 15 ppm anhydrous sodium selenite (Se-salt) for 5 y and the other four townships with similar PLC incidence rate served as the controls using normal table salt . The second trial was undertaken among hepatitis B virus surface antigen carriers (HBVsAg+) receiving supplement of 200 micrograms Se in form of selenized yeast (Se-yeast) daily vs placebo for 4 y . The third trial was carried out in members of families with high PLC incidence using Se-yeast (200 micrograms of Se daily) vs placebo for 2 y . The results showed that nutritional supplement of Se could reduce the PLC incidence significantly.

Proc Natl Acad Sci U S A, 1991 Jun 1, 88(11), 4671 - 4
Steel-Dickie mutation encodes a c-kit ligand lacking transmembrane and cytoplasmic domains; Brannan CI et al.; Mice homozygous for the viable Sl allele steel-Dickie (Sld) are sterile, severely anemic, and black-eyed white . The nature of the Sld mutation was investigated at the molecular level and was found to be due to a 4.0-kilobase intragenic deletion in mast cell growth factor (MGF) genomic sequences, providing conclusive evidence that Sl encodes MGF . As a consequence of this deletion, Sld is only capable of encoding a soluble truncated growth factor that lacks both transmembrane and cytoplasmic domains . Northern analysis indicates that Sld mRNA is expressed at approximately wild-type levels in adult tissues, and yeast expression studies suggest that the Sld protein is as biologically active as wild-type soluble MGF . These studies provide a molecular basis for explaining the Sld phenotype, a description of a germ-line mutation in the transmembrane and cytoplasmic domains of a membrane-bound growth factor, and in vivo evidence for the importance of membrane-bound forms of growth factors in mammalian development.

Zhonghua Nei Ke Za Zhi, 1991 Jun, 30(6), 357 - 9, 383
{Kinetic observation on BRC immune function of patients with epidemic hemorrhagic fever}; Kang Y et al.; The RBC immune function of 88 patients with epidemic haemorrhagic fever (EHF) was examined kinetically with complement-labeled yeast method established by Guo Feng . The results showed that the percentage of RBC c3b receptor rossette, activity of RCIA-enhancing factor and c3 in sera of patients during the 3 different phases of EHF were markedly lower than those of the normal controls, while the percentage of RBC IC rossete, activity of RCIA-inhibiting factor and CIC in sera of the patients were manifestly higher (P less than 0.01) . The change of all the indices observed above was most apparent in oliguric phase . This suggested that RBC immune function is lowered in the process of EHF.

EMBO J, 1991 Jun, 10(6), 1427 - 33
Active repression of transcription by the engrailed homeodomain protein; Jaynes JB et al.; The Drosophila engrailed gene product (En) is a homeodomain-containing protein that contributes to segmental patterning . In transfection assays it acts as a transcriptional repressor . We show that En is an active repressor, blocking activation by mammalian and yeast activators that bind to sites some distance away from those bound by En . Active repression is distinct from the effects of passive homeodomain-containing proteins, which repress when competing with activators for binding sites and activate when competing with En . Active repression activity maps outside the En homeodomain, and this activity can be transferred to a heterologous DNA binding domain.

J Biochem (Tokyo), 1991 Jun, 109(6), 803 - 6
Cloning and functional expression of a novel endoprotease involved in prohormone processing at dibasic sites; Nakayama K et al.; We cloned and sequenced a cDNA from a library of mouse pituitary AtT-20 cells which are known to cleave an endogenous and various foreign prohormones at dibasic sites . This cDNA encodes a novel 753-residue protein, named PC3, which is structurally related to the yeast Kex2 protease involved in precursor cleavage at dibasic sites and to recently identified mammalian Kex2-like proteins, furin and PC2 . Among examined cell lines and tissues, PC3 mRNA was only detected in AtT-20 cells . The substrate specificity of PC3 expressed in mammalian cells was similar to that observed in AtT-20 cells . We conclude that PC3 is a resident prohormone processing endoprotease in AtT-20 cells.

Mol Biochem Parasitol, 1991 Jun, 46(2), 229 - 39
Characterization of the gene encoding the largest subunit of Plasmodium falciparum RNA polymerase III; Li WB et al.; We report here the isolation, sequence analysis, structure, and expression of the gene encoding the largest subunit of RNA polymerase III (RPIII) from Plasmodium falciparum . The P . falciparum RPIII gene consists of 5 exons and 4 introns, is expressed in all of the asexual erythrocytic stages of the parasite as a 8.5-kb mRNA, and is present in a single copy on chromosome 13 . The predicted 2339 amino acid residue RPIII subunit contained 5 regions that were conserved between different eukaryotic RPIII subunits, and 4 variable regions that separated the conserved regions . Three of the variable regions were greatly enlarged in comparison to the corresponding variable regions in other RPIII subunits . Variable region C' represented nearly one-third of the P . falciparum RPIII subunit (750 amino acid residues), included a unique repeated decapeptide sequence, and had some homology with yeast DNA topoisomerase II . Noteworthy amino acid sequences and structures were identified in both the conserved regions and in the enlarged variable regions, and their possible role(s) as domains that regulate RPIII enzyme activity is discussed.

New Biol, 1991 Jun, 3(6), 581 - 91
Alteration of specific amino acid residues in the acidic domain I of VSV phosphoprotein (P) converts a GAL4-P(I) hybrid into a transcriptional activator; Takacs AM et al.; As part of a study of transcriptional regulation by viral proteins, we examined whether an acidic region from a regulatory protein of an RNA virus could function as a trans-activator . The NH2-terminal highly acidic domain I of the phosphoprotein (P) of vesicular stomatitis virus (VSV) was fused to the DNA-binding domain of the yeast trans-activator, GAL4 . In transient transfection assays, the resulting chimeric protein failed to activate transcription of a reporter CAT gene . However, mutation of basic amino acid residues located at positions 6 and 8 or the alteration of eight amino acids within the acidic domain to eight different amino acids converted the chimeric protein into a transcriptional activator comparable to wild-type GAL4 . When subjected to SDS-polyacrylamide gel electrophoresis, the P proteins containing trans-activation-positive mutations in domain I showed an altered mobility, suggesting that these mutations may have caused a conformational change that is critical for trans-activation . Since the acidity of P domain I is not sufficient to activate transcription, additional features of this region must play an important role in GAL4-mediated trans-activation . None of the trans-activation-positive mutants supported VSV RNA transcription in vitro . These results suggest that the amino acid residues within P domain I that can be made to function in the trans-activation of DNA-dependent RNA transcription are distinct from those involved in VSV RNA-dependent RNA transcription.

J Gen Virol, 1991 Jun, 72 ( Pt 6), 1393 - 9
Development of immunogenic recombinant Oka varicella vaccine expressing hepatitis B virus surface antigen; Shiraki K et al.; Recombinant Oka varicella vaccine expressing hepatitis B virus (HBV) surface antigen (HBs) was constructed by inserting the HBs gene into the viral thymidine kinase (TK) gene and was examined for its immunogenicity in guinea-pigs . The HBs gene encoding 25 amino acids of preS2 and the whole of the S region was inserted into the TK gene of the cloned plasmid . The chimeric plasmid DNA and Oka varicella vaccine DNA were cotransfected and recombinant virus was isolated after immunofluorescence screening using a monoclonal antibody to HBs and a fluorescein-conjugated anti-mouse antibody . Expression of viral HBs was detected in the cytoplasm of infected cells and was stable over several repeated passages in vitro . The recombinant virus expressed 26K and 30K HBs molecules in infected cells and the culture supernatant contained 30K and 35K HBs molecules . HBs was purified at a density of 1.20 g/ml from the culture supernatants . The recombinant virus induced an antibody response to HBs as well as to varicella-zoster virus (VZV) in guinea-pigs, and the antibody titre to HBs was comparable to that induced by a recombinant HBs subunit vaccine produced in yeast . Thus a single dose of live recombinant Oka varicella vaccine could induce good immunity to VZV and HBs . The recombinant Oka varicella vaccine expressing HBs may be a good candidate for a combined HBV and VZV vaccine.

Biochem Biophys Res Commun, 1991 May 31, 177(1), 497 - 503
Identification of lanosterol 14 alpha-methyl demethylase in human tissues; Raucy JL et al.; Lanosterol 14 alpha-methyl demethylase was investigated in human tissues using a radio-HPLC assay to detect the 4,4-dimethyl-5 alpha-cholesta-8, 14-dien-3 beta-ol (diene) metabolite . The sequence of events leading to the demethylated product in human liver microsomes involves the conversion of the diol to the aldehyde followed by diene formation . Enzyme activity displayed a greater than 10 fold variation among the 9 liver samples studied . Kinetic parameters were determined and shown to differ between two separate liver samples . Addition of inhibitors of yeast lanosterol 14 alpha demethylase, ketoconazole and miconazole, resulted in extensive inhibition of formation of the demethylated metabolite . The enzyme, detected in microsomes isolated from human kidney and lymphocytes, also catalyzed the conversion of dihydrolanosterol to oxylanosterol intermediates and the diene . The presence of this enzyme in microsomes from various human tissues suggests that it may play a role in cellular regulation of cholesterol synthesis.

Biochem Biophys Res Commun, 1991 May 31, 177(1), 279 - 85
Recognition of human immunodeficiency virus glycoproteins by natural anti-carbohydrate antibodies in human serum; Tomiyama T et al.; Anti-carbohydrate antibodies were isolated from Human immunodeficiency virus (HIV) negative human serum by affinity chromatography using yeast mannan followed by protein A . The purified mannan-binding IgG (MBIgG) bound to HIV glycoproteins gp 160, gp 120 and gp 41 in Western blot . Immunofluorescence revealed that MBIgG bound to HIV/IIIB-infected H9 cells but not to uninfected H9 cells, suggesting that carbohydrate structures recognized by MBIgG are specifically expressed on HIV-infected cells . MBIgG did not neutralize infectivity of HIV . These results show that normal human serum contains natural antibodies reactive to carbohydrate structures of HIV glycoproteins propagated in human cells.

Cell, 1991 May 31, 65(5), 825 - 36
Gamma-tubulin is a highly conserved component of the centrosome; Stearns T et al.; We have cloned and characterized gamma-tubulin genes from both X . laevis and S . pombe, and partial genes from maize, diatom, and a budding yeast . The proteins encoded by these genes are very similar to each other and to the original Aspergillus protein, indicating that gamma-tubulins are an ubiquitous and highly conserved subfamily of the tubulin family . A null mutation of the S . pombe gene is lethal . gamma-tubulin is a minor protein, present at less than 1% the level of alpha- and beta-tubulin, and is limited to the centrosome . In particular, gamma-tubulin is associated with the pericentriolar material, the microtubule-nucleating material of the centrosome . gamma-Tubulin remains associated with the centrosome when microtubules are depolymerized, suggesting that it is an integral component that might play a role in microtubule organization.

Nature, 1991 May 30, 351(6325), 411 - 4
Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic; van der Bliek AM et al.; Temperature-sensitive paralysis is the most striking defect of adult Drosophila carrying the shibire mutation . This is believed to be due to a reversible block of endocytosis, which prevents membrane cycling and thus depletes synaptic vesicles . The shibire mutation also affects many tissues outside the nervous system . We have now mapped and characterized the shibire gene . A 275-kilobase yeast artificial chromosome was subcloned into cosmids, among which the gene was then located by analysing with restriction-fragment length polymorphisms . A 15-kilobase fragment of wild-type DNA rescues the mutant phenotype and the sequence of two mutant alleles show differences with wild type, demonstrating that we have isolated the shibire gene . The gene encodes a protein that is highly similar to rat dynamin, 69% of the amino-acid sequence is identical . Dynamin is a GTP-driven mechanochemical enzyme related to mammalian mx-proteins and to the yeast vps 1 gene product . Because the shibire gene product and dynamin have extensive similarity, we propose that they are cognate homologues . Dynamin causes microtubules to slide along each other in vitro and in extracts it is associated with a distinct, but so far uncharacterized, membrane fraction . In light of the shibire phenotype, we suggest that these proteins provide the motor for vesicular transport during endocytosis.

J Biol Chem, 1991 May 25, 266(15), 9610 - 6
Structure of the human type I DNA topoisomerase gene; Kunze N et al.; We describe the molecular organization of the human gene coding for type I DNA topoisomerase . The coding sequence is split into 21 exons distributed over at least 85 kilobase pairs (kb) of human genomic DNA . The sizes of the 20 introns vary widely between 0.2 and at least 30 kb and all contain the sequence elements known to be required for pre-mRNA splicing . Several of the intron sequences separate exons encoding parts of the enzyme that are highly conserved between human and yeast suggesting that at least some of the exons may code for individual, structurally, or functionally important domains of the enzyme . We also describe the promoter sequence of the human topoisomerase I gene and show that it is composed of distinct functional elements.

Biochemistry, 1991 May 21, 30(20), 4978 - 84
Phosphorylated aminosugars: synthesis, properties, and reactivity in enzymatic reactions; Sem DS et al.; A number of phosphorylated aminosugars have been prepared and tested as substrates for metabolic reactions . 6-Aminoglucose is a slow substrate for yeast hexokinase with a Vmax that is only 0.012% that for glucose . While Vmax is pH independent, V/K decreases below the pK of 9.0 of the amino group . 6-Aminoglucose is a competitive inhibitor vs glucose with a Ki value increasing below the pK of 9 but leveling off at 33 mM below pH 7.16 . Thus, protonation decreases binding affinity by 2.4 kcal/mol and only the neutral amine is catalytically competent . 6-Aminoglucose-6-P was synthesized enzymatically with hexokinase . Its pK's determined by 31P NMR were 2.46 and 8.02 (alpha anomer) and 2.34 and 7.85 (beta anomer), with a beta:alpha ratio of 3.0 . It is most stable at pH 12 (half-life 228 h at 22 degrees C), while as a monoanion its half-life is 3 h . The free energy of hydrolysis at 25 degrees C and pH 9.25 is -10.3 kcal/mol . The phosphorylated amino analogues of 6-P-gluconate, ribulose-5-P, fructose-6-P, fructose-1,6-bis-P (amino group at C-6 only), and glyceraldehyde-3-P were synthesized enzymatically . The 31P NMR chemical shifts of these analogues are 8-8.5 ppm at pH 9.5 . Their relative stability is 6-aminogluconate-6-P greater than 3-aminoglyceraldehyde-3-P greater than 6-aminoglucose-6-P greater than 6-aminofructose-1,6-bis-P congruent to 6-aminofructose-6-P greater than 5-aminoribulose-5-P . These analogues were tested as substrates for their respective enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Science, 1991 May 17, 252(5008), 958 - 61
Site-specific recombination between homologous chromosomes in Drosophila; Golic KG; The ability to mark a cell and its descendants genetically so that the resulting cell clone can be distinguished from neighboring cells facilitates studies in animal biology and development . A method of generating clones by inducing homologous mitotic recombination in Drosophila with a site-specific yeast recombinase is described . This method allows for frequent mosaicism after mitotic exchange is induced at predefined sites in the genome.

Nature, 1991 May 16, 351(6323), 242 - 5
Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein; Strausfeld U et al.; Oocytes arrested in the G2 phase of the cell cycle contain a p34cdc2/cyclin B complex which is kept in an inactive form by phosphorylation of its p34cdc2 subunit on tyrosine, threonine and perhaps serine residues . The phosphatase(s) involved in p34cdc2 dephosphorylation is unknown, but the product of the fission yeast cdc25+ gene, and its homologues in budding yeast and Drosophila are probably positive regulators of the transition from G2 to M phase . We have purified the inactive p34cdc2/cyclin B complex from G2-arrested starfish oocytes . Addition of the purified bacterially expressed product of the human homologue of the fission yeast cdc25+ gene (p54CDC25H) triggers p34cdc2 dephosphorylation and activates H1 histone kinase activity in this preparation . We propose that the cdc25+ gene product directly activates the p34cdc2-cyclin B complex.

Cancer, 1991 May 15, 67(10 Suppl), 2712 - 7
Interleukin-3 . Biologic effects and clinical impact; Oster W et al.; Human interleukin-3 (IL-3) is expressed in yeast and has a specific activity of 5 x 10(7) U/mg of protein . It exerts functional and proliferative effects on multiple hematopoietic cell lineages including the neutrophil, eosinophil, basophil, monocytic, and thrombopoietic cell lines . IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) share common binding capacities on hematopoietic cells . Each of these agents has entered clinical trials . The clinical experiences with IL-3 alone and in combination with GM-CSF in a Phase I/II trial are summarized in this report.

Cancer, 1991 May 15, 67(10 Suppl), 2705 - 7
Hematopoietic effects of a granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein; Williams DE et al.; The common functional characteristics of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) may be explained by the presence of a subpopulation of cell surface receptors capable of binding both growth hormones . A GM-CSF/IL-3 fusion protein (pIXY 321) was produced in a yeast expression host . Receptor binding studies with HL-60, JM-1, AML-193, and KG-1 cell lines suggested that the GM-CSF and IL-3 regions had adopted a native conformation within the fusion protein . The fusion protein also exhibited enhanced biologic activity compared with GM-CSF or IL-3 in assays of normal, primary human hematopoietic progenitor cells . pIXY 321 may offer significant clinical advantages over the individual cytokines.

Anal Biochem, 1991 May 15, 195(1), 57 - 62
Preparation and characterization of the NAD vinylogue, 3-pyridylacryloamide adenine dinucleotide; Anderson BM et al.; The vinylogue of NAD, 3-pyridylacryloamide adenine dinucleotide, was prepared from NAD and 3-pyridylacryloamide through the snake venom NADase-catalyzed transglycosidation reaction . The analog, purified by ion-exchange chromatography, was obtained in a 55% yield . The cyanide adduct and reduced form of the analog exhibited absorbance maxima at 358 nm and 378 nm, respectively, with extinction coefficients in each case being 2.3-times higher than those reported for the corresponding NAD derivatives . 3-Pyridylacryloamide adenine dinucleotide served as a coenzyme with bovine liver glutamic dehydrogenase and to a lesser extent with malate and lactate dehydrogenases . The analog was not reduced in reactions catalyzed by yeast and horse liver alcohol dehydrogenases, sheep liver sorbitol dehydrogenase, and rabbit muscle glycerophosphate dehydrogenase . Substitution of the pyridylacryloamide analogs for NAD and NADH in the assay of substrates for glutamic dehydrogenase was demonstrated.

J Immunol, 1991 May 15, 146(10), 3557 - 62
Staurosporine inhibits neutrophil phagocytosis but not iC3b binding mediated by CR3 (CD11b/CD18); Roubey RA et al.; C receptor CR3 (iC3b-receptor, CD11b/CD18) plays an essential role in several phagocytic and adhesive neutrophil functions . Recent evidence suggests that stimulus-induced phosphorylation of the CR3 beta-chain, CD18, may mediate certain neutrophil functions by transiently converting the molecule to an activated state . Staurosporine, a protein kinase C inhibitor that blocks PMA-induced CD18 phosphorylation, was used to study the functional relevance of this event . Neutrophils adhered to glass were assayed for binding and phagocytosis of iC3b-opsonized sheep E (EC3bi) in the presence or absence of PMA and/or staurosporine . Binding of EC3bi was markedly increased, not only by PMA, but also by staurosporine and by a combination of both agents (three- to sevenfold) . The enhancement of rosetting by staurosporine was likely caused by increased surface expression of CR3 via exocytosis of specific granular contents . In contrast, staurosporine alone did not stimulate phagocytosis of EC3bi and markedly inhibited PMA-induced phagocytosis . Staurosporine also inhibited phagocytosis of yeast beta glucan particles, a CR3 ligand that, in contrast to EC3bi, is bound and ingested without additional prior treatment with PMA . beta glucan phagocytosis was associated with a low level of CD18 phosphorylation . Staurosporine did not block phagocytosis in general, because this agent had relatively little effect on FcR-mediated phagocytosis . These data demonstrate that phagocytosis mediated by CR3 requires activation of CR3 via a staurosporine-sensitive pathway . Increased binding of EC3bi, a function of increased surface expression of CR3, does not require activation of CR3 by such a pathway, confirming previous evidence for the independence of these two phenomena . A direct role for CD18 phosphorylation in the activation of CR3 for phagocytosis is consistent with these data.

Arch Biochem Biophys, 1991 May 15, 287(1), 141 - 50
DNA polymerase B from wheat embryos: a plant delta-like DNA polymerase; Richard MC et al.; Studies in eucaryotic cells (mainly animals and yeast) indicate that at least two DNA polymerases are involved in DNA replication at the level of the replication fork: DNA polymerase alpha, which is associated with DNA primase, is involved in the replication of the lagging strand; DNA polymerase delta, associated with an exonuclease activity, synthesizes the forward continuous DNA strand . Much less information exists concerning plant systems . Previous work from this laboratory provided preliminary evidence of an association between DNA polymerase B from wheat embryo and an exonucleolytic activity . In this paper, we present additional data on the biochemical properties of DNA polymerase B . An improved purification procedure described in this article has been developed . During all the purification steps the nuclease activity was associated with DNA polymerase activity . A biochemical study of this enzyme activity shows that it is an exonuclease which hydrolyses DNA in the 3' to 5' direction . Moreover, this exonuclease confers a proofreading function to DNA polymerase B . Comparison of DNA polymerase B properties (template specificity, sensitivity to DNA replication inhibitors like aphidicolin and butyl-phenyl dGTP, copurification of DNA polymerase and exonuclease activities) with those of animal DNA polymerase delta indicates that these enzymes share many common features . To our knowledge, this is the first report of DNA polymerase delta in higher plants.

Eur J Biochem, 1991 May 8, 197(3), 631 - 41
Proton-NMR studies of the effects of ionic strength and pH on the hyperfine-shifted resonances and phenylalanine-82 environment of three species of mitochondrial ferricytochrome c; Moench SJ et al.; Ferricytochromes c from three species (horse, tuna, yeast) display sensitivity to variations in solution ionic strength or pH that is manifested in significant changes in the proton NMR spectra of these proteins . Irradiation of the heme 3-CH3 resonances in the proton NMR spectra of tuna, horse and yeast iso-1 ferricytochromes c is shown to give NOE connectivities to the phenyl ring protons of Phe82 as well as to the beta-CH2 protons of this residue . This method was used to probe selectively the Phe82 spin systems of the three cytochromes c under a variety of solution conditions . This phenylalanine residue has previously been shown to be invariant in all mitochondrial cytochromes c, located near the exposed heme edge in proximity to the heme 3-CH3, and may function as a mediator in electron transfer reactions {Louie, G . V., Pielak, G . J., Smith, M . & Brayer, G . D . (1988) Biochemistry 27, 7870-7876} . Ferricytochromes c from all three species undergo a small but specific structural rearrangement in the environment around the heme 3-CH3 group upon changing the solution conditions from low to high ionic strength . This structural change involves a decrease in the distance between the Phe82 beta-CH2 group and the heme 3-CH3 substituent . In addition, studies of the effect of pH on the 1H-NMR spectrum of yeast iso-1 ferricytochrome c show that the heme 3-CH3 proton resonance exhibits a pH-dependent shift with an apparent pK in the range of 6.0-7.0 . The chemical shift change of the yeast iso-1 ferricytochrome c heme 3-CH3 resonance is not accompanied by an increase in the linewidth as previously described for horse ferricytochrome c {Burns, P . D . & La Mar, G . N . (1981) J . Biol . Chem . 256, 4934-4939} . These spectral changes are interpreted as arising from an ionization of His33 near the C-terminus . In general, the larger spectral changes observed for the resonances in the vicinity of the heme 3-CH3 group in yeast iso-1 ferricytochrome c with changes in solution conditions, relative to the tuna and horse proteins, suggest that the region around Phe82 is more open and that movement of the Phe82 residue is less constrained in yeast ferricytochrome c . Finally, it is demonstrated here that both the heme 8-CH3 and the 7 alpha-CH resonances of yeast ferricytochrome c titrate with p2H and exhibit apparent pK values of approximately 7.0 . The titrating group responsible for these spectral changes is proposed to be His39.

Biochemistry, 1991 May 7, 30(18), 4398 - 405
Proton NMR assignments of heme contacts and catalytically implicated amino acids in cyanide-ligated cytochrome c peroxidase determined from one- and two-dimensional nuclear Overhauser effects; Satterlee JD et al.; Proton NMR assignments of the heme pocket and catalytically relevant amino acid protons have been accomplished for cyanide-ligated yeast cytochrome c peroxidase . This form of the protein, while not enzymatically active itself, is the best model available (that displays a resolvable proton NMR spectrum) for the six-coordinate low-spin active intermediates, compounds I and II . The assignments were made with a combination of one- and two-dimensional nuclear Overhauser effect methods and demonstrate the utility of NOESY experiments for paramagnetic proteins of relatively large size (Mr 34,000) . Assignments of both isotope exchangeable and nonexchangeable proton resonances were obtained by using enzyme preparations in both 90% H2O/10% D2O and, separately, in 99.9% D2O solvent systems . Complete resonance assignments have been achieved for the proximal histidine, His-175, and His-52, which is a member of the catalytic triad on the distal side of the heme . In addition, partial assignments are reported for Trp-51 and Arg-48, catalytically important residues, both on the distal side . Aside from His-175, partial assignments for amino acids on the proximal side of the heme are proposed for the alanines at primary sequence positions 174 and 176 and for Thr-180 and Leu-232.

Mol Biol Evol, 1991 May, 8(3), 345 - 55
Evolutionary relationships of avian Eimeria species among other Apicomplexan protozoa: monophyly of the apicomplexa is supported; Barta JR et al.; Direct, reverse transcriptase-mediated, partial sequencing of the small-subunit (16S-like) ribosomal RNA (srRNA) of Eimeria tenella and E . acervulina was performed . Sequences were aligned by eye with six previously published, partial or complete srRNA sequences of apicomplexan protists (Plasmodium berghei, Theileria annulata, Cryptosporidium sp., Toxoplasma gondii, Sarcocystis muris, and S . gigantea) . Six eukaryotic protists (a slime mold, a yeast, two dinoflagellates, and two ciliates) acted as an outgroup for a parsimony-based phylogenetic analysis (PAUP Ver . 3.0) . The 188 phylogenetically informative sites (i.e., those positions that neither were unvaried nor had only autapomorphic substitutions) supported a single tree topology 481 steps in length with a consistency index of 0.65 in which the monophyly of the Apicomplexa was supported . The two Eimeria species and S . muris, S . gigantea, and T . gondii formed a pair of monophyletic groups that were sister groups . The two Sarcocystis species were not hypothesized to be sister taxa . The genera Plasmodium and Cryptosporidium were hypothesized to form the sister group to these five coccidia and T . annulata . A priori data-editing techniques that deleted "variable" positions prior to analysis failed to recognize the monophyly of the Apicomplexa when the same parsimony-based tree-building algorithm was used . Inability of the outgroup taxa to root the well-supported ingroup tree (Apicomplexa) at a unique site when these taxa were used individually for this purpose reinforces the need for an appropriate, multiple-taxon outgroup in such analyses.

Immunology, 1991 May, 73(1), 88 - 94
Differential presentation of hepatitis B S-preS(2) particles and peptides by macrophages and B-cell like antigen-presenting cells; Scheerlinck JP et al.; Different cell types, including dendritic cells, macrophages and Ia+ B cells, have been described to present soluble antigen (Ag) to T-cell hybridomas . However, it is still not clear whether these different cell types can act as antigen-presenting cells (APC) for complex and insoluble Ag such as viral particles . Using yeast recombinant hepatitis B S-preS(2)-containing particles, T-cell hybridomas were generated and used as a tool to study processing and presentation of antigen . Different types of APC were compared in regard to their capacity to process and present the protein-lipid composed S-preS(2) particles and the thereof derived T-cell epitope containing peptides by different types of APC . While a S-preS(2)-derived T-cell epitope containing peptide, which does not require processing, could be presented both by macrophage and B-cell like APC, the presentation of S-preS(2) particles required the presence of macrophages . The fact that B-cell like APC and macrophages behave differently with regard to the presentation of S-preS(2) particles suggest that the uptake and/or processing of this type of Ag by B-cell like APC and macrophages is different.

Br J Cancer, 1991 May, 63(5), 758 - 62
Collagen production by macrophages in tumour encapsulation and dormancy; Vaage J et al.; Dormant and regressing implants of C3H mammary carcinoma MC2 were always found to be surrounded by a cellular fibrous capsule where macrophages and T cells predominated as the cellular elements . Macrophages were always closely associated with the collagen deposition, and stained with anti-collagen type I immuno-peroxidase in tissue sections . The capacities of macrophages and T-lymphocytes to function in collagen formation was investigated with the use of Nucleopore chambers implanted i.p . in normal mice . The procollagen that entered the chambers via the pores, was assumed to have been produced by the packed layer of peritoneal macrophages that adhered firmly to the outside of washed chambers . The adherent cells all stained with Mac-1 immuno-peroxidase, and phagocytosed yeast in short-term culture . The formation of collagen fibres in the chambers was enhanced if the chambers contained T lymphocytes . It appears that macrophages have the capacity to function as collagen producing cells in tumour encapsulation.

EMBO J, 1991 May, 10(5), 1225 - 36
The maternally expressed Drosophila gene encoding the chromatin-binding protein BJ1 is a homolog of the vertebrate gene Regulator of Chromatin Condensation, RCC1; Frasch M; Using monoclonal antibodies I have identified a nuclear protein of Drosophila, BJ1 (Mr approximately 68 kd), and isolated its gene . Biochemical analysis demonstrates that the BJ1 protein is associated with nucleosomes and is released from chromatin by agents which intercalate into DNA, as previously shown for the high mobility group proteins (HMGs) . On polytene chromosomes the protein is localized in all bands, with no preference for particular loci . Both the BJ1 protein and in particular the BJ1 mRNA are strongly expressed maternally . In early embryos all nuclei contain equal amounts of BJ1 . During neuroblast formation, BJ1 mRNA becomes restricted to cells of the central nervous system, and higher protein levels are found in the nuclei of this tissue . In late embryonic stages, the mRNA almost completely disappears, but significant amounts of BJ1 protein persist until morphogenesis . The BJ1 gene encodes a 547 amino acid polypeptide featuring two different types of internal repeats . The sequence from amino acids 46 to 417 containing seven repeats of the first type has been highly conserved in evolution . 45% of the amino acids in this region are conserved in seven similar tandem repeats of the human gene Regulator of Chromatin Condensation, RCC1 . The phenotype of a cell line carrying a mutation of RCC1 suggested a main function for this gene in cell cycle control . A yeast gene, SRM1/PRP20, also contains these repeats and shows 30% amino acid identity to BJ1 in this region . Mutations in this gene perturb mRNA metabolism, disrupt nuclear structure and alter the signal transduction pathway for the mating pheromone . Complementation experiments argue for a common function of these genes in the different species . I propose that their gene products bind to the chromatin to establish or maintain a proper higher order structure as a prerequisite for a regulated gene expression . Disruption of this structure could cause both mis-expression and default repression of genes, which might explain the pleiotropic phenotypes of the mutants.

Indian J Exp Biol, 1991 May, 29(5), 468 - 73
Effects of protein restriction on functional properties of rat peritoneal macrophages; Machaiah JP; Rats were maintained on 20% and 4% protein diets for 3 weeks . The functional properties of thioglycollate (TG) elicited macrophages from these groups were compared with the non elicited resident cells from the protein fed group . Elicitation of macrophages in response to TG was low in the protein deficient group . These cells also exhibited low adherence in overnight cultures compared to those isolated from the protein fed group; however their viability and total protein content remained unaltered . Normal resident and TG elicited cells from 4% protein fed group exhibited an initial lag period in H2O2 production in response to zymosan stimulation . The lag period could be correlated to the high endogeneous catalase activity in these cells . Incubation with zymosan resulted in rapid decline in catalase levels, facilitating evolution of H2O2 . On prolonged incubation, the elicited cells from the protein fasted rats evolved about 87% H2O2 compared to the protein fed samples . In the absence of zymosan all the samples possessed comparable NADPH oxidase activity . Zymosan induced activation of this enzyme was higher in TG cells from the protein fed groups, compared to the protein fasted and the resident samples . The cellular enzyme activity, however was not altered in the TG cells of both the groups though it declined rapidly in the corresponding resident cells . Significant reduction (congruent to 50%) in both serum iron and transferrin in the low protein fed samples did not correspondingly affect the oxidative burst process . However the engulfment of yeast cells was greatly impaired due to protein restriction . Adherence and phagocytic properties of macrophages are regulated by the activity of their membrane constituents.(ABSTRACT TRUNCATED AT 250 WORDS)

Trends Genet, 1991 May, 7(5), 155 - 61
Integrated maps of the Drosophila genome: progress and prospects; Kafatos FC et al.; A physical map of the Drosophila melanogaster genome is being assembled, consisting of ordered overlapping cosmid clones . The map is constructed in steps, separately for each chromosomal division . Gaps in this map are to be bridged with yeast artificial chromosome clones . Hybridization to previously cloned genes and extensive use of in situ hybridization to polytene chromosomes ensure that the cosmid map is firmly anchored to the wealth of available genetic and cytogenetic information . The intention is to make the physical map widely available as part of an overall, integrated genetic resource for the Drosophila research community.

J Protozool, 1991 May-Jun, 38(3), 211 - 6
A serum-free, partly defined medium, PDM-805, for axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and other Entamoeba; Diamond LS et al.; We describe the first serum-free, partly defined medium (PDM-805) for cultivating the human enteric pathogen, Entamoeba histolytica, and the reptilian amebae E . barreti, E . invadens, and E . terrapinae . PDM-805 was developed by the stepwise replacement of yeast extract, bovine serum, and a casein peptone digest in TYI-S-33, a medium widely used for the axenic cultivation of these parasites . The defined components include amino acids, carbohydrates, B vitamins, ascorbic acid, tocopherol, thioctic acid, nucleic acid precursors, trace metals, and phosphate buffers . The undefined components include a highly purified bovine serum albumin, a lipoprotein-cholesterol solution from bovine serum, and a dialyzable, autoclavable, water-soluble growth factor(s) having a molecular weight of less than 3,500 prepared from casein peptone . To date, studies on the growth requirements of E . histolytica, strain 200:NIH, show the following are essential for sustained multiplication of this ameba: iron, glucose, biotin, folic acid, niacinamide, pantothenate, pyridoxal, riboflavin, thiamine, cysteine, an ammonium moiety (in addition to that present in cysteine), bovine serum albumin, lipoprotein-cholesterol, and casein peptone dialysate.

Mycopathologia, 1991 May, 114(2), 77 - 81
Deficiency in the incorporation of labeled thymidine and inhibition in the biosynthesis of interleukin-2 in lymphocytes obtained from Histoplasma capsulatum infected mice; Islas-Rodriguez AE et al.; The incorporation of (3H) thymidine and the biosynthesis of interleukin-2(IL-2) were investigated in Concanavalin A (ConA) and histoplasmin stimulated lymphocytes from spleen of infected Balb/c mice with the yeast phases of Histoplasma capsulatum . The ability to incorporate (3H) thymidine of Con A stimulated lymphocytes in culture from spleen of Histoplasma capsulatum infected mice, as well as the IL-2 content present in the supernatants of that cultures, were depressed along the first three weeks of the experiments, but starting week five, normal values were restored or even discretly increased . Incorporation of (3H) thymidine in histoplasmin stimulated lymphocytes remained inhibited along the seven weeks the experiment lasted . Results showed that inoculation of H . capsulatum yeast in mice provoked a temporary immunosuppression on cell mediated immunity, that can be explained by means of the inability of T cells to produce enough IL-2 necessary for the proliferation of T cells in culture.

Rev Infect Dis, 1991 May-Jun, 13(3), 379 - 82
Human infection caused by Exophiala pisciphila: case report and review; Sughayer M et al.; One year after receiving a liver transplant and 2 months after treatment with high doses of steroids and monoclonal anti-CD3 for an episode of rejection, a 38-year-old woman developed a skin papule above the left medial malleolus . The papule, which at first had an annular shape, evolved into a pustule, ulcerated, drained, and assumed a crusted verrucous appearance . Multiple satellite papules appeared around the lesion, which was incompletely excised and thought to represent squamous cell carcinoma . Review of the histologic slides revealed pseudoepitheliomatous hyperplasia with multiple epidermal and dermal abscesses, pigmented hyphae, and yeast-like forms . Culture of material obtained at reexcision yielded a dematiaceous fungus that was identified as Exophiala pisciphila . No evidence of dissemination was found . This represents a unique report of human infection with this fungus, a well-recognized pathogen of fish . Except for the absence of sclerotic bodies, the clinicopathologic features resembled those of chromoblastomycosis rather than those of the subcutaneous cystic form of phaeohyphomycosis often associated with species of Exophiala.

Appl Environ Microbiol, 1991 May, 57(5), 1340 - 5
Enzymatic conversion of norsolorinic acid to averufin in aflatoxin biosynthesis; Yabe K et al.; 5'-Hydroxyaverantin (HAVN) was isolated from a mold, Emericella heterothallica IFO 30842 . Aspergillus parasiticus NIAH-26, a UV-irradiated mutant of A . parasiticus SYS-4, produced neither aflatoxins nor precursors in yeast extract-sucrose (YES) medium . When the postmicrosome (cytosol) fraction of NIAH-26, which had been prepared from the culture in YES medium, was incubated with norsolorinic acid (NA) in the presence of NADH or NADPH, averantin (AVN) was produced . The reverse reaction from AVN to NA was promoted by the addition of NAD or NADP (dehydrogenase reaction) . When the microsome fraction of NIAH-26 was incubated with AVN, HAVN was produced in the presence of NADPH (monooxygenase reaction) . HAVN was, furthermore, oxidized to averufin (AVR) by the cytosol fraction of NIAH-26 in the presence of NAD or NADP (dehydrogenase reaction) . In the feeding experiments with A . parasiticus NIAH-26, aflatoxins were produced from AVN, HAVN, NA, and AVR but not from averufanin or averythrin . These results indicate that the reaction sequence NA in equilibrium AVN----HAVN----AVR is involved in the biosynthetic pathway of aflatoxins . The enzyme activities described here were dependent on the culture medium, and no enzyme activities were observed in the nonaflatoxigenic strain A . oryzae SYS-2 (IFO 4251).

Am J Clin Nutr, 1991 May, 53(5), 1222 - 9
Effects of antioxidant supplementation on platelet function: a randomized pair-matched, placebo-controlled, double-blind trial in men with low antioxidant status; Salonen JT et al.; We investigated the effect on platelet function of supplementing men with low antioxidant status with 600 mg ascorbic acid, 300 mg alpha-tocopherol, 27 mg beta-carotene, and 75 micrograms selenium in yeast daily . Eighty men were randomly assigned in pairs (matched for smoking, baseline antioxidant status, and time and day of entry) by use of a double-blind design to receive supplement or placebo for 5 mo . Compared with 39 control subjects, 39 antioxidant-supplemented men experienced the following net reductions during the double-blind period: 20% (P = 0.012) in serum lipid peroxides, 24% (P = 0.035) in ADP-induced platelet aggregation, 42% (P = 0.040) in the rate of ATP release during aggregation, 51% (P = 0.018) in serum (platelet-produced) thromboxane B2, and 29% (P = 0.024) in plasma beta-thromboglobulin concentration . The data support our hypothesis that antioxidant supplementation of men with low antioxidant status and high fat intake reduces lipid peroxidation, the capacity of platelets to aggregate and to produce thromboxane A2, and in vivo platelet activation.

Zhonghua Zhong Liu Za Zhi, 1991 May, 13(3), 162 - 4
{A preliminary study on the anti-mutagenic effect of seleno-malt}; Luo HZ; Seleno-malt is an organic selenium product recently developed in China . Seleno-malt itself is not genotoxic . It antagonized the mutagenic effect of aflatoxin B1 . Its antagonistic activity was 10-15 times higher then seleno-yeast . Seleno-malt may be used as a food additive in areas with low selenium level for the prevention of Ke-shan disease and cancer.

Am J Reprod Immunol, 1991 May, 25(4), 143 - 5
Failure to detect integrins of the beta-2 subclass on the human sperm surface; Fusi F et al.; Several cell-to-cell recognition systems utilize the tripeptide Asp-Gly-Arg (RGD) as a cell surface signaling sequence . Mac-1, a member of the integrin subfamily defined by the beta-2 subunit, recognizes C3bi by means of an RGD sequence . Because C3 complement components have been detected on the gamete surfaces and RGD has been demonstrated to be involved in sperm-egg interaction, we explored the possibility that a member of the b2 integrin subfamily was present on the sperm surface and involved in egg recognition . Two methods were utilized to detect integrins on the sperm surface . Sperm were exposed to Mabs directed against different epitopes of each member of this integrin subfamily; and spermatozoa were tested for their ability to bind zymosan or yeast, since Mac-1 bearing cells possess this property . Both methods failed to demonstrate such integrins on the sperm surface, suggesting that another RGD receptor is involved in gamete recognition.

Biochem Int, 1991 May, 24(2), 391 - 6
Hexose metabolism in pancreatic islets . Insignificance of D-glucose futile cycling in rat islets; Liemans V et al.; When rat pancreatic islets were incubated in the presence of unlabelled D-glucose (16.7 mM) and 3HOH, the production of 3H-labelled material susceptible to be phosphorylated by yeast hexokinase and then detritiated by yeast phosphoglucoisomerase did not exceed 2.66 +/- 0.21 pmol/islet per 180 min, i.e . about 1% of the rate of exogenous D-{5-3H}glucose utilization . Such a material accounted for 43 +/- 4% of the total radioactivity, associated with tritiated hexose(s) . It is proposed, therefore, that the futile cycling of D-glucose in the reactions catalyzed in the islet cells by the hexokinase isoenzymes and glucose-6-phosphatase represents a negligible fraction of the total rate of D-glucose phosphorylation.

Science, 1991 Apr 26, 252(5005), 576 - 9
Regulation of Ras-GAP and the neurofibromatosis-1 gene product by eicosanoids; Han JW et al.; Ras-GAP (GTPase activating protein) is a regulatory protein that stimulates the intrinsic guanosine triphosphatase (GTPase) activity of the proto-oncogene product p21ras . A domain of the neurofibromatosis gene product (NF1) that has sequence similarity to the catalytic domain of Ras-GAP and to yeast IRA gene products also has a specific stimulatory activity toward p21ras GTPase . Arachidonic acid and phosphatidic acid inactivate GAP, but no agents have been identified that stimulate GAP and thereby switch p21ras off . With the use of recombinant Ha-c-Ras and Ras-GAP, NF1, and GAP catalytic domains, it was found that prostaglandins PGF2 alpha and PGA2 stimulated Ras-GAP and that prostacyclin PGI2 inhibited Ras-GAP . The stimulatory effect of PGF2 alpha was saturable and structure-specific and competed with the inhibitory effect of arachidonic acid . Arachidonic acid also inhibited the catalytic activity of NF1, but prostaglandins were not stimulatory . These results suggest a mechanism for the allosteric control of Ras function through the modulation of arachidonate metabolism.

J Biol Chem, 1991 Apr 25, 266(12), 7804 - 11
Transcription initiated by RNA polymerase II and transcription factors from liver . Structure and action of transcription factors epsilon and tau; Conaway JW et al.; We have fractionated rat liver and identified a set of transcription factors that are essential for accurate initiation by RNA polymerase II . These factors were resolved into five distinct enzyme fractions designated alpha, beta gamma, delta, epsilon, and tau . Four of these fractions can now be replaced with purified proteins . alpha and beta gamma were previously purified to apparent homogeneity (Conaway, J . W., and Conaway, R . C . (1989) J . Biol . Chem . 264, 2357-2362) . Here, we report purification to near homogeneity of transcription factor epsilon . Epsilon has a native molecular mass of approximately 90 kDa and is composed of 34- and 58-kDa polypeptides . Both the 34- and 58-kDa polypeptides are required for runoff transcription . In addition, we show that transcription factor tau is a rat liver homologue of the TATA factor (TFIID or BTF1) that can be efficiently replaced in transcription in vitro by recombinant yeast TFIID . Comparison of the two factors reveals, however, that they differ significantly in their abilities to direct the transcription system to discriminate between promoters of different sequences.

Nature, 1991 Apr 25, 350(6320), 715 - 8
Phosphorylation of two small GTP-binding proteins of the Rab family by p34cdc2; Bailly E et al.; Entry of a cell into mitosis induces a series of structural and functional changes including arrest of intracellular transport . Knowledge of how the mitotic cycle is driven progressed substantially with the identification of the p34cdc2 protein kinase as a subunit of maturation-promoting factor, the universal regulating component of the mitotic cycle . Activation of the kinase at the onset of mitosis is thought to trigger the important mitotic events by phosphorylating key proteins . Small guanine nucleotide-binding proteins have been implicated in regulating transport pathways . For instance, two small Ras-related GTP-binding proteins, Sec4p and Ypt1p, control distinct stages of the secretory pathway in budding yeast . The GTP-binding proteins of the Rab family in rats and humans display strong homologies with Sec4p and Ypt1p, and might therefore also be involved in regulating intracellular transport . Indeed, distinct Rab proteins are located in the exocytotic and endocytotic compartments . Interruption of vesicular transport during mitosis might involve modification of these proteins . We now present biochemical evidence for a mitosis-specific p34cdc2 phosphorylation of Rab1Ap and Rab4p . By contrast, Rab2p and Rab6p are not phosphorylated . We also show that the distribution of Rab1Ap and Rab4p between cytosolic and membrane-bound forms is different in interphase and mitotic cells . This may provide a clue to the mechanism by which phosphorylation could affect membrane traffic during mitosis.

Biochemistry, 1991 Apr 16, 30(15), 3634 - 9
Secondary 18O isotope effects for hexokinase-catalyzed phosphoryl transfer from ATP; Jones JP et al.; Secondary 18O isotope effects in the gamma-position of ATP have been measured on phosphoryl transfer catalyzed by yeast hexokinase in an effort to deduce the structure of the transition state . The isotope effects were measured by the remote-label method with the exocyclic amino group of adenine as the remote label . With glucose as substrate, the secondary 18O isotope effect per 18O was 0.9987 at pH 8.2 and 0.9965 at pH 5.3, which is below the pK of 6.15 seen in the V/K profile for MgATP . With the slow substrate 1,5-anhydro-D-glucitol, the value was 0.9976 at pH 8.2 . While part of the inverse nature of the isotope effect may result from an isotope effect on binding, the more inverse values when catalysis is made more rate limiting by decreasing the pH or switching to a slower substrate suggest a dissociative transition state for phosphoryl transfer, in agreement with predictions from model chemistry . The 18O equilibrium isotope effect for deprotonation of HATP3- is 1.0156, while Mg2+ coordination to ATP4- does not appear to be accompanied by an 18O isotope effect larger than 1.001.

Proc Natl Acad Sci U S A, 1991 Apr 15, 88(8), 3120 - 4
The arflike gene encodes an essential GTP-binding protein in Drosophila; Tamkun JW et al.; We have identified a Drosophila gene (arflike, arl) encoding a protein that is structurally related (approximately 55% identity) to the ADP-ribosylation factors (ARFs) of yeast and mammals . Biochemical analyses of purified recombinant arl-encoded protein revealed properties similar to the ARF proteins, including the ability to bind and hydrolyze GTP . Clear functional differences between arl and ARF proteins, including a complete lack of ARF activity, suggest that arl is not a functional homolog of ARF . A recessive lethal arl mutation was recovered, demonstrating that the arl locus is an essential gene . We conclude that the arl locus encodes an essential member of the ARF subfamily of small GTP-binding proteins in Drosophila.

Proc Natl Acad Sci U S A, 1991 Apr 15, 88(8), 3446 - 50
Three-dimensional structure of human basic fibroblast growth factor, a structural homolog of interleukin 1 beta; Zhang JD et al.; The three-dimensional structure of the 146-residue form of human basic fibroblast growth factor (bFGF), expressed as a recombinant protein in yeast, has been determined by x-ray crystallography to a resolution of 1.8 A . bFGF is composed entirely of beta-sheet structure, comprising a three-fold repeat of a four-stranded antiparallel beta-meander . The topology of bFGF is identical to that of interleukin 1 beta, showing that although the two proteins share only 10% sequence identity, bFGF, interleukin 1, and their homologs comprise a family of structurally related mitogenic factors . Analysis of the three-dimensional structure in light of functional studies of bFGF suggests that the receptor binding site and the positively charged heparin binding site correspond to adjacent but separate loci on the beta-barrel.

Biochem J, 1991 Apr 15, 275 ( Pt 2), 427 - 33
Molecular structure of the human muscle-specific enolase gene (ENO3); Peshavaria M et al.; The single human gene for muscle-specific enolase was isolated and its structure was characterized, from which the mature mRNA transcript and encoded protein were also deduced . The gene contains 12 exons, spans approx . 6 kb and encodes a protein of 433 residues . The gene structure is similar to that found for the rat neuron-specific enolase gene, and the deduced protein aligns precisely with other enolase sequences, including the sequence of the only published crystallized enolase, yeast eno-1 . The 5' boundary of the gene includes a 5' non-coding exon and is characterized by an upstream TATA-like box and CpG-rich region . This region contains potential recognition motifs for general transcriptional regulation involving Sp1, activator protein 1 and 2, CCAAT box transcription factor/nuclear factor I and cyclic AMP, and for muscle-specific transcriptional regulation involving a CC(A + T-rich)6GG box, M-CAT-box CAATCCT and two myocyte-specific enhancer-binding factor 1 boxes.

Nature, 1991 Apr 11, 350(6318), 512 - 5
A novel cyclin encoded by a bcl1-linked candidate oncogene; Motokura T et al.; We have previously identified a candidate oncogene (PRAD1 or D11S287E) on chromosome 11q13 which is clonally rearranged with the parathyroid hormone locus in a subset of benign parathyroid tumours . We now report that a cloned human placental PRAD1 complementary DNA encodes a protein of 295 amino acids with sequence similarities to the cyclins . Cyclins can form a complex with and activate p34cdc2 protein kinase, thereby regulating progress through the cell cycle . PRAD 1 messenger RNA levels vary dramatically across the cell cycle in HeLa cells . Addition of the PRAD1 protein to interphase clam embryo lysates containing inactive p34cdc2 kinase and lacking endogenous cyclins allows it to be isolated using beads bearing p13suc1, a yeast protein that binds cdc2 and related kinases with high affinity and coprecipitates kinase-associated proteins . Addition of PRAD1 also induces phosphorylation of histone H1, a preferred substrate of cdc2 . These data suggest that PRAD1 encodes a novel cyclin whose overexpression may play an important part in the development of various tumours with abnormalities in 11q13.

J Biol Chem, 1991 Apr 5, 266(10), 6467 - 71
Structural analysis of the gene encoding rat cholesterol alpha-hydroxylase, the key enzyme for bile acid biosynthesis; Nishimoto M et al.; The gene encoding cholesterol 7 alpha-hydroxylase (P450VIIA) was isolated from rat genomic DNA . The gene spanned about 11 kilobases and contained six exons . Blotting analysis of genomic DNA and complete matching of restriction maps of several isolated genomic clones indicated that there appeared to be only one gene in the rat genome . The putative transcription initiation site was present 61 base pairs upstream from the ATG codon . The typical TATA sequence and CCAAT promoter element were found at 24 and 47 base pairs upstream from the transcription initiation site, respectively . Alignment of several P450 proteins showed that the cholesterol 7 alpha-hydroxylase gene shared location of introns with none of the other P450 genes except for intron 5, which was in the same position as intron 10 of the gene encoding P450IVA1 . The alignment also indicated that the distal helix of cholesterol 7 alpha-hydroxylase contained an asparagine in place of the well conserved threonine that is postulated to be involved in the O2 binding site . Unusual residues, Asn-126 and Thr-442, were also found at the sites where all other P450s have positively charged amino acids, which are considered to be involved in interaction with heme propionate . These replacements may be related to the unique function and unusual lability of the hydroxylase . Analysis of evolutionary distance between the cholesterol 7 alpha-hydroxylase gene and other known P450 genes indicated that yeast P450LIA is most closely related to P450VIIA . This finding suggests that the cholesterol 7 alpha-hydroxylase gene is an evolutionarily old P450 gene.

J Prosthet Dent, 1991 Apr, 65(4), 528 - 32
In vivo fungal presence and growth on two resilient denture liners; Graham BS et al.; To determine whether two intraoral-setting resilient denture liners supported the in vivo presence or growth of oral commensal fungi, the liners were randomly placed in the mandibular complete dentures of 14 patients . Cytologic smears were made from the liner surfaces at 1 hour and 1, 2, 7, 14 and 30 days after intraoral placement . Yeast forms were observed in six patient trials of material A and in two trials of material B . Hyphae were observed in only one patient trial of material A and in two trials of material B . When yeast forms and hyphae findings were combined and were considered as indicative of fungal presence or growth, the prevalence up to 30 days was seven for material A and four for material B . Statistical analysis revealed no significant difference in the prevalence of fungal presence or growth between the two resilient denture liners tested.

Gene, 1991 Apr, 100, 123 - 9
Glycolytic pathway of the human malaria parasite Plasmodium falciparum: primary sequence analysis of the gene encoding 3-phosphoglycerate kinase and chromosomal mapping studies; Hicks KE et al.; We have isolated and characterised the gene (PGK) encoding the glycolytic enzyme 3-phosphoglycerate kinase (PGK) from the human malaria parasite Plasmodium falciparum . This was achieved using the polymerase chain reaction (PCR) to amplify genomic DNA with primers constructed on the basis of conserved regions identified within PGK molecules of other organisms, and using the PCR product to isolate genomic clones . The gene is present in a single copy, encoding a protein of 416 amino acids (aa) . The predicted aa sequence (45.5 kDa) displays approx . 60% identity to both human and yeast PGK molecules, and of the three P . falciparum glycolytic enzymes reported to date, has the greatest sequence identity to the host homologue . All aa residues implicated in substrate and cofactor binding and catalysis are conserved in the malarial PGK molecule, but there are major differences in overall composition, with implications for enzyme stability . In asexual blood-stage parasites, a single mRNA transcript of approx . 2.1 kb is observed . We have mapped the PGK gene to chromosome 9 of the parasite, and a further gene encoding a glycolytic enzyme, aldolase, to chromosome 14.

Tex Med, 1991 Apr, 87(4), 88 - 90
Disseminated histoplasmosis with bilateral adrenal enlargement: diagnosis by computed tomography-directed needle biopsy; Schonfeld AD et al.; Disseminated histoplasmosis is a rare and potentially fatal disease caused by the dimorphic soil fungus Histoplasma capsulatum . We describe a 67-year-old man with diabetes who presented with a 6-month history of fever, weight loss, and mental deterioration; using computed tomography (CT), we found hepatosplenomegaly and bilateral adrenal masses . CT-directed adrenal biopsy showed yeast forms consistent with H capsulatum . Therapy with ketoconazole was accompanied by adrenal insufficiency (treated successfully with prednisone and fludrocortisone) and produced an excellent therapeutic response . We address the differential diagnosis of bilateral adrenal enlargement, and we discuss the clinical features, diagnosis, and treatment of disseminated histoplasmosis, a disease that includes parts of the Southwestern United States in its endemic region.

Genitourin Med, 1991 Apr, 67(2), 137 - 42
A study of candidosis: the role of fomites; Rashid S et al.; This study investigates treatment failure and recurrences of vulvo-vaginal candidosis . It reviews the factors possibly associated with both . Patients attending a department of genitourinary medicine with recurrent candidosis (N = 186) were entered in the trial . The patients were investigated for evidence of candidosis from vagina, rectal wall and buccal mucosa and were given antifungal therapy . Prevention and reinfection via fomites was studied by means of a single blind parallel study comparing the effect of soaking undergarments in the amphoteric biocide Tego 103G with the effect of a placebo soak . General and possible contributory factors influencing treatment and failure and recurrences were considered . The success rate of miconazole therapy was typical of any imidazole therapy: 85.4% . There was no evidence that modern oral contraceptives played a role in candidosis . Oral nystan reduced the rectal wall carriage from 39.2% to 23.2% . Oral yeast carriage rate in women was 37.6% . The recurrence rate over a six month period was 47.4% . The laboratory results of Tego soaking reduced the yeast carriage on panties from 85.2% to 23.4% . However, no evidence was found in the trial results that panties were a significant source of reinfection.

Exp Cell Res, 1991 Apr, 193(2), 411 - 9
A novel protein related to cell cycle-dependent alterations of chromatin structure; Pfeffer U et al.; We have detected a novel nuclear antigen, AF-2, which appears to be involved in cell cycle-dependent alterations of chromatin structure . Specific monoclonal antibodies detect the antigen spread over the whole cell during mitosis and in islet-like structures in the nuclei of a subpopulation of cells in interphase . Upon nucleolytic digestion of fixed cells, the antigen becomes available to the antibodies in all cells, indicating that AF-2 antigen is present during the whole cell cycle but differentially accessible . Digestion with the single strand specific S1 nuclease reveals that the alteration of chromatin structure induced by the introduction of nicks into the DNA rather than the digestion of DNA bound to the immunogenic epitope accounts for the change in accessibility of AF-2 antigen in interphase nuclei . The epitope recognized by the antibody in human cells is present in two polypeptides of 65 and 36 kDa, respectively, which are tightly bound to chromatin and cross-linkable to the nuclear matrix . The proteins also occur in the midbody during cytokinesis . The immunogenic epitope is conserved between man and fission yeast.

J Leukoc Biol, 1991 Apr, 49(4), 342 - 51
Effect of soluble aminated beta-1,3-D-polyglucose on human monocytes: stimulation of cytokine and prostaglandin E2 production but not antigen-presenting function; Doita M et al.; Glucans are insoluble polymers of beta-1,3-linked glucose derived from yeast cell walls that effectively activate macrophages . Recently, aminated derivatives of beta-1,3-D-polyglucose have been developed that are soluble but also activate murine macrophages . The current studies were undertaken to determine whether soluble aminated beta-1,3-D-polyglucose (AG) would also stimulate human monocytes . The AG employed contained less than 2 ng endotoxin/mg . AG induced the production of intracellular, membrane-associated, and secreted forms of interleukin 1 (IL1) in a dose-dependent manner, with 50 micrograms/ml yielding maximal responses . AG also induced tumor necrosis factor-alpha (TNF alpha) secretion by human monocytes . Prostaglandin E2 (PGE2) production was also stimulated in a concentration-dependent manner . Quantitatively, optimal stimulatory concentrations of AG were comparable to endotoxin in the capacity to induce production of these various mediators . In contrast to its capacity to induce production of IL1, TNF alpha, and PGE2, AG did not stimulate monocytes to become more effective antigen presenting cells . These results indicate that AG is potent inducer of proinflammatory mediators from human monocytes but does not enhance their capacity to initiate immune responses.

Indian J Biochem Biophys, 1991 Apr, 28(2), 83 - 92
Influence of cellular differentiation on ultraviolet induced DNA damage and its repair mechanisms in B . cereus; Kamat AS et al.; Susceptibility to UV irradiation of B . cereus BIS-59 spores undergoing germination at various stages-dormant spores to vegetative cell stage and their ability to recover from radiation damage were studied . For a given dose of radiation, the number of spore photoproducts (SPP) formed in the DNA of dormant spores was about 5-times greater than that of thymine dimers (TT) formed in the DNA of vegetative cells . At intermediate stages of the germination cycle, there was a rapid decline in the UV radiation-induced SPP formed in DNA with a concomitant increase in the UV radiation-induced TT formed in DNA . Bacterial spores undergoing germination (up to 3 hr) in the low nutrient medium (0.3% yeast extract) displayed much higher resistance to UV radiation than those germinating in the rich nutrient medium, even though there was no discernible difference under the two incubation conditions in respect of the extent of germination and the time at which the outgrowth stage appeared (3 hr) . This was due to the formation TT in the DNA of spores germinating in the low nutrient as compared to that of spores germinating in the rich-nutrient medium . In UV-irradiated dormant spores, SPP formed in the spore DNA did not disappear even after prolonged incubation in the non-germinating medium . However, when the UV-irradiated dormant spores were germinated in low or rich nutrient medium, a significant proportion of SPP in DNA was eliminated . The dormant spores incubated in either of the germinating media for 15 min and then UV-irradiated were capable of eliminating SPP (presumably by monomerization) even by incubation in a non-germinating medium and in the complete absence of protein synthesis (buffer holding recovery), thereby implying that spore-repair enzymes were activated in response to initial's germination . The acquisition of photo-reactivation ability appeared in spores subjected to germination only in the rich-nutrient medium at the outgrowth stage and required de novo synthesis of the required enzymes.

Curr Opin Cell Biol, 1991 Apr, 3(2), 269 - 75
Mitotic control; Maller JL; 1990 has been a year of continued exciting developments in cell cycle control . Progress has occurred in delineating the mechanism of activation of maturation-promoting factor during entry into mitosis and the mechanism of cyclin degradation responsible for exit from mitosis . Notable advances have also occurred in our understanding of the dependence of mitotic entry on completion of DNA synthesis . Both genetic and biochemical data link this crucial checkpoint to the function of the cdc25 gene product and the extent of phosphorylation of Tyr15 in cdc2 kinase.

Arzneimittelforschung, 1991 Apr, 41(4), 423 - 6
Anti-inflammatory and immunomodulating effects of the novel agent gamma-(2-aminoethylamino)-2-butyrothienone . 1st communication: inhibitory effects on mouse paw edema; Gavalas A et al.; A series of 4-hydroxy-(or-amino)-ethyl-amino butyrophenones or butyrothienones were synthesized . For detail studies on antiinflammatory effects, gamma-(2-aminoethylamino)-2-butyrothienone (gamma-ABT) was chosen as representative of these new non-steroidal anti-inflammatories (NSAID) . The effect on mouse paw edema induced by various phlogistic agents was first investigated . The inhibitory effect of gamma-ABT on carrageenin-induced edema was remarkable and nearly equal to that of indometacin . Similarly to indometacin, gamma-ABT inhibited the early and late stage of yeast-induced edema in contrast to concanavalin A (Con A) induced edema which was only inhibited by gamma-ABT . Both the above induced edema are supposed to be unrelated to prostaglandins in the rat system . gamma-ABT displayed an inhibitory effect on nystatin-induced edema similar to indometacin suggesting that gamma-ABT has significant membrane stabilizing action and a strong blocking action on synthesis of prostaglandins . gamma-ABT inhibited as well the sustained edema induced by mustard . In conclusion gamma-ABT is an effective agent not only on acute but also on subacute and chronic inflammation and its mode of action appears similar to other NSAID . gamma-ABT posses in addition the advantage of antioxidant activity which is not shared by selective cyclooxygenase inhibitors and thus should be potentially effective in autoimmune diseases.

Proc Natl Acad Sci U S A, 1991 Apr 1, 88(7), 2613 - 7
Drosophila P-element transposase is a transcriptional repressor in vitro; Kaufman PD et al.; Mobility of P transposable elements in Drosophila melanogaster depends on the 87-kDa transposase protein encoded by the P element . Transposase recognizes a 10-base-pair DNA sequence that overlaps an A + T-rich region essential for transcription from the P-element promoter . We report here that transposase represses transcription from the P-element promoter in vitro . This transcriptional repression is blocked by prior formation of an RNA polymerase II transcription complex on the template DNA . Binding of transposase on the P-element promoter is blocked by prior binding of either the Drosophila RNA polymerase II complex or the yeast transcription factor TFIID . These data suggest that transposase represses transcription by preventing assembly of an RNA polymerase II complex at the P-element promoter.

Mol Cell Biol, 1991 Apr, 11(4), 2035 - 9
Accurate transcription of a plant mitochondrial gene in vitro; Hanic-Joyce PJ et al.; To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII) . Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon . We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo . Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA . This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.

G E N, 1991 Apr-Jun, 45(2), 105 - 10
{Giardia lamblia: comparison of two diagnostic methods and evaluation of response to treatment with metronidazole}; Guerreiro NM et al.; During the period from March through November 1989, 70 children who were attended at the Pediatric Department at Central Hospital in Valencia, were enrolled in the study, it was thought that Giardia lamblia infection might be present . Giardia L . were identified using two different diagnostic procedures: from stool samples and duodenal aspirates for cysts or trophozoites examination . These children were treated with Metronidazole three dosage of 15, 30 and 50 mg/kg per day for a ten day period . Our study showed predominant giardiasis in children with ages ranging from 2 to 6 years old (60%) with a relationship between female and male sex 1.05:1 . In this series, 72.8% of patients presented normal nutrition, and 55.7% of them were from the suburban area . The most frequent symptoms were abdominal pain, diarrhea, vomiting, abdominal distention, constipation and flatulence . The infants prevalent symptom was diarrhea (83.3%) and the older children and school children prevalent symptom was abdominal pain with 78.5 and 100% respectively . In this study, stool examination was positive in 97.1% of the children and duodenal aspirate was positive in all 70 children (100%); the first procedure showed predominant Giardia cysts (88.2%) and the second one showed predominant trophozoites (47.1%) . All 70 patients (100%) were cured with Metronidazole to different dosage . Side effects were seen with only the maxim dose, such as nausea 40%, headache 10% and appearance of yeast into 50% of duodenal aspirate.

Plant Mol Biol, 1991 Apr, 16(4), 567 - 77
Characterization of cDNA and genomic clones encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Hevea brasiliensis; Chye ML et al.; Hevea brasiliensis is the major producer of natural rubber which is cis-1,4-polyisoprene . The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is involved in the biosynthesis of rubber and other plant products . We have used a hamster HMGR cDNA clone as a heterologous hybridization probe to isolate and characterize cDNA and genomic clones of HMGR from H . brasiliensis . Sequence analysis revealed that these clones fall into two different classes, HMGR1 and HMGR2 . Comparison of the two classes shows 86% nucleotide sequence homology and 95% amino acid homology . The carboxy-termini of Hevea HMGRs are highly homologous to those of hamster, yeast and Arabidopsis HMGR . The amino-terminus of Hevea HMGR contains two potential membrane-spanning domains as in Arabidopsis HMGR while seven such domains are found in the HMGRs of other organisms . The apparent molecular mass of Hevea HMGR was estimated in western blot analysis to be 59 kDa . Northern blot analysis indicated that the HMGR1 transcript of 2.4 kb is more highly-expressed in laticifer than in leaf . Genomic Southern analysis using 3'-end cDNA probes indicates the presence of at least two HMGR genes in Hevea.

Leukemia, 1991 Apr, 5(4), 300 - 8
Regulation of leukocyte adhesion molecule-1 (TQ1, Leu-8) expression and shedding by normal and malignant cells; Spertini O et al.; The human leukocyte adhesion molecule-1 (LAM-1, TQ1, Leu-8) is involved in the binding of human leukocytes to high endothelial venules (HEV) of peripheral lymph nodes (LN) . The regulation of LAM-1 expression is unique in that leukocyte stimulation induces a rapid down-modulation of LAM-1 from the cell surface . In this study, the regulation and function of LAM-1 was studied in detail in normal lymphocytes and compared with the LAM-1 of malignant leukocytes . Modulation of LAM-1 from the cell surface occurred concomitantly with the appearance of LAM-1 in the culture medium indicating that LAM-1 is cleaved from the cell surface . Shedding of LAM-1 was decreased in the presence of protein kinase C (PKC) inhibitors . As with normal lymphocytes, cells transfected with the LAM-1 cDNA and chronic lymphocytic leukemia (CLL) cells also shed LAM-1 following phorbol myristate acetate (PMA) exposure . CLL cells expressed the same Mr LAM-1 protein as normal lymphocytes and LAM-1+ CLL cells were able to specifically bind to HEV . In addition, normal lymphocytes and LAM-1+ CLL cells were capable of binding polyphosphomonester core polysaccharide (PPME) derived from yeast cell wall, a carbohydrate which mimics an essential component of the natural ligand for LAM-1, and PPME and HEV binding was specifically blocked by a new monoclonal antibody (mAb) reactive with LAM-1 . The expression of LAM-1 and other adhesion molecules was examined on cells of 118 hematopoietic malignancies . LAM-1 was most frequently expressed on CLL and follicular or diffuse small cleaved cell lymphomas, whereas most other malignancies were LAM-1- . Thus, most CLL cells and some non-Hodgkin's lymphoma cells express a functionally active LAM-1 molecule which may correlate with their capacity to migrate through the circulation and disseminate into peripheral LN.

J Cell Biol, 1991 Apr, 113(1), 65 - 76
Cleavage of precursors by the mitochondrial processing peptidase requires a compatible mature protein or an intermediate octapeptide; Isaya G et al.; Many precursors of mitochondrial proteins are processed in two successive steps by independent matrix peptidases (MPP and MIP), whereas others are cleaved in a single step by MPP alone . To explain this dichotomy, we have constructed deletions of all or part of the octapeptide characteristic of a twice cleaved precursor (human ornithine transcarbamylase {pOTC}), have exchanged leader peptide sequences between once-cleaved (human methylmalonyl-CoA mutase {pMUT}; yeast F1ATPase beta-subunit {pF1 beta}) and twice-cleaved (pOTC; rat malate dehydrogenase (pMDH); Neurospora ubiquinol-cytochrome c reductase iron-sulfur subunit {pFe/S}) precursors, and have incubated these proteins with purified MPP and MIP . When the octapeptide of pOTC was deleted, or when the entire leader peptide of a once-cleaved precursor (pMUT or pF1 beta) was joined to the mature amino terminus of a twice-cleaved precursor (pOTC or pFe/S), no cleavage was produced by either protease . Cleavage of these constructs by MPP was restored by re-inserting as few as two amino-terminal residues of the octapeptide or of the mature amino terminus of a once-cleaved precursor . We conclude that the mature amino terminus of a twice-cleaved precursor is structurally incompatible with cleavage by MPP; such proteins have evolved octapeptides cleaved by MIP to overcome this incompatibility.

Biochemistry, 1991 Mar 26, 30(12), 3011 - 9
Electrophilic catalysis in triosephosphate isomerase: the role of histidine-95; Komives EA et al.; Electrophilic catalysis by histidine-95 in triosephosphate isomerase has been probed by using Fourier transform infrared spectroscopy and X-ray crystallography . The carbonyl stretching frequency of dihydroxyacetone phosphate bound to the wild-type enzyme is known to be 19 cm-1 lower (at 1713 cm-1) than that of dihydroxyacetone phosphate free in solution (at 1732 cm-1), and this decrease in stretching frequency has been ascribed to an enzymic electrophile that polarizes the substrate carbonyl group toward the transition state for the enolization . Infrared spectra of substrate bound to two site-directed mutants of yeast triosephosphate isomerase in which histidine-95 has been changed to glutamine or to asparagine show unperturbed carbonyl stretching frequencies between 1732 and 1742 cm-1 . The lack of carbonyl polarization when histidine-95 is removed suggests that histidine-95 is indeed the catalytic electrophile, at least for dihydroxyacetone phosphate . Kinetic studies of the glutamine mutant (H95Q) have shown that the enzyme follows a subtly different mechanism of proton transfers involving only a single acid-base catalytic group . These findings suggest an additional role for histidine-95 as a general acid-base catalyst in the wild-type enzyme . The X-ray crystal structure of the H95Q mutant with an intermediate analogue, phosphoglycolohydroxamate, bound at the active site has been solved to 2.8-A resolution, and this structure clearly implicates glutamate-165, the catalytic base in the wild-type isomerase, as the sole acid-base catalyst for the mutant enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1991 Mar 25, 19(6), 1273 - 8
A differentially expressed murine RNA encoding a protein with similarities to two types of nucleic acid binding motifs; Ayane M et al.; Using differential screening, a murine cDNA, termed X16, was isolated corresponding to an mRNA which is more strongly expressed in pre-B cell lines relative to mature B-cell lines . The complete coding sequence of the mRNA predicts a 19kD protein with two domains connected by a proline-rich spacer . The N-terminal domain of about 90 amino acids encodes an RNA binding motif including the ribonucleoprotein consensus octapeptide found in one class of RNA-binding proteins and highly conserved from yeast to man . Within the very basic C-terminal domain of about 60 amino acids, several copies of two different peptides are found which are also present in several proteins which bind DNA or RNA . The expression of X16 is not limited to the lymphoid lineage . In adult mice, although the strongest expression was seen in thymus, mRNA was also found in testis, brain, spleen, and very low in heart . X16 mRNA was not detected in liver and kidney . In tissue culture, the expression of X16 mRNA can be induced by serum . The conserved protein motifs and expression pattern suggest that X16 could be involved in RNA processing correlating with cellular proliferation.

Proc Natl Acad Sci U S A, 1991 Mar 15, 88(6), 2118 - 21
Stable integrative transformation of Trypanosoma brucei that occurs exclusively by homologous recombination; Eid J et al.; A calmodulin-neomycin-resistance fusion gene was introduced into Trypanosoma brucei by electroporation, and stably transformed cell lines were obtained . In all of the transformants, the fusion gene had integrated into the host genome at the cognate locus, evidently by homologous recombination within flanking calmodulin DNA . This unusual observation distinguishes trypanosomes as the only eukaryote other than yeast known to undergo gene targeting in essentially 100% of the stable transformants . It should now be possible to systematically manipulate the trypanosome genome, directing predetermined mutations to virtually any chromosomal locus.

J Biol Chem, 1991 Mar 15, 266(8), 4810 - 5
Ligand-binding characteristics of rat serum-type mannose-binding protein (MBP-A) . Homology of binding site architecture with mammalian and chicken hepatic lectins; Lee RT et al.; Sugar-binding characteristics of rat serum mannose-binding protein (MBP) were studied using the carbohydrate-recognition domain of this protein expressed from a cloned cDNA . To assess the binding affinity of various test compounds, they were added as inhibitors in a binding assay in which 125I-MBP was incubated with yeast cells and the extent of binding was estimated from the radioactivity associated with the pelleted cells . The results of such inhibition assays suggest that MBP has a small binding site which is probably of the trough-type . The 3- and 4-OH of the target sugar are indispensable, while the 6-OH is not required . These characteristics are shared by the rat hepatic lectin and chicken hepatic lectin, both of which are C-type lectins containing carbohydrate-recognition domains highly homologous to that of MBP . Apparently, the related primary structures of these lectins give rise to similar gross architecture of their binding sites, despite the fact that each exhibits different sugar binding specificities.

Science, 1991 Mar 15, 251(4999), 1351 - 5
Recombinase-mediated gene activation and site-specific integration in mammalian cells; O'Gorman S et al.; A binary system for gene activation and site-specific integration, based on the conditional recombination of transfected sequences mediated by the FLP recombinase from yeast, was implemented in mammalian cells . In several cell lines, FLP rapidly and precisely recombined copies of its specific target sequence to activate an otherwise silent beta-galactosidase reporter gene . Clones of marked cells were generated by excisional recombination within a chromosomally integrated copy of the silent reporter . By the reverse reaction, integration of transfected DNA was targeted to a specific chromosomal site . The results suggest that FLP could be used to mosaically activate or inactivate transgenes for analysis of vertebrate development, and to efficiently integrate transfected DNA at predetermined chromosomal locations.

Biochem Biophys Res Commun, 1991 Mar 15, 175(2), 500 - 6
Characterization of a cAMP-independent Ca2(+)-inhibited protamine kinase from Candida lipolytica; Rahmatullah M et al.; A cAMP-independent protamine kinase has been purified from extracts of the yeast Candida lipolytica by ion-exchange and affinity chromatography . Two subunits with apparent Mr's of 52,000 and 36,000 were resolved by SDS-PAGE . The purified kinase exhibited about 20% activity with casein and histone Type VII-S as substrates relative to protamine . The enzyme was inactive against other protein substrates tested, and was essentially insensitive to AMP, cAMP, cGMP up to 0.2 mM, the polyamines spermine and spermidine up to 1 mM, N-ethylmaleimide (5 mM), 2-mercaptoethanol (20 mM), or dithiothreitol (2 mM), and several cations like Zn2+, N1+, or Co2+ at 0.1 mM each . Ca2+ at 3 mM inhibited protamine kinase activity by 50%, which was reversed by EGTA.

J Biol Chem, 1991 Mar 5, 266(7), 4387 - 91
Carboxyl methylation of platelet rap1 proteins is stimulated by guanosine 5'-(3-O-thio)triphosphate; Huzoor-Akbar et al.; Carboxyl methylation of platelet ras-related proteins, known as rap proteins, was investigated in this study . Platelet membrane proteins of Mr 23,000 incorporated radioactivity in the presence of S-{methyl-3H}adenosylmethionine and platelet cytosol . About 97% of the radioactivity present in the Mr 23,000 proteins was liberated as volatile methanol under basic (1 M sodium hydroxide) conditions . Cycloheximide, an inhibitor of protein synthesis, inhibited incorporation of S-{methyl-3H}adenosylmethionine by 25% . These results suggest that at least 75% of the radioactivity present in the Mr 23,000 proteins is due to carboxyl methylation and not due to the incorporation of S-{methyl-3H}adenosylmethionine into proteins or due to the incorporation of base-stable methyl groups into side chains of arginine, histidine, or lysine residues . Protein methylation did not occur if membranes or cytosol alone was incubated with S-{methyl-3H}adenosylmethionine . Guanosine 5'(3-O-thio)triphosphate increased methylation of the Mr 23,000 proteins in a time- and concentration-dependent manner . Acetyl-farnesylcysteine, a synthetic substrate for carboxyl methyltransferases, completely blocked methylation of the Mr 23,000 membrane proteins . On the basis of one- and two-dimensional Western blots using rap-specific antisera, the Mr 23,000 methylated proteins were identified as rap1 proteins . The existence of the carboxyl-terminal CAAX motif in rap1 proteins, similar to the CAAX motif present in p21ras as well as in the yeast mating factors, leads us to suggest that methylation of rap1 proteins possibly occurs at the alpha-carboxyl-terminal cysteine.

Alcohol, 1991 Mar-Apr, 8(2), 91 - 6
Effect of chronic ethanol consumption on selenium status and utilization in rats; Cho HK et al.; Effects of chronic ingestion of 2 levels of alcohol on selenium (Se) utilization were determined in initially Se-depleted rats . Male weanling rats were fed ad lib a Se deficient (0.012 mg/kg) basal diet for 4 weeks and then were meal-fed low or marginally adequate Se in the form of high Se yeast for 4 weeks . During Se repletion, ethanol, which replaced medium-chain triglycerides in the diet, provided 10 or 20 percent of food energy . The basal diet provided 80% of food energy as well as adequate protein, vitamins and minerals . In rats given adequate Se moderate chronic ethanol consumption did not influence Se absorption or retention, but increasing ethanol level raised Se in liver and whole blood in a linear fashion and in kidney in a quadratic manner . In this rat model measures of Se status were reduced by low Se intake, not chronic moderate ethanol ingestion.

J Clin Microbiol, 1991 Mar, 29(3), 475 - 8
Legionella fairfieldensis sp . nov . isolated from cooling tower waters in Australia; Thacker WL et al.; Three Legionella-like organisms were isolated from water from the cooling towers of two Australian institutions . The strains grew on buffered charcoal-yeast extract (BCYE) agar but not on BCYE agar in the absence of L-cysteine . Gas-liquid chromatography profiles of the isolates were consistent with those for Legionella spp . They were serologically distinct from other legionellae in a slide agglutination test . DNA hybridization studies showed that the three isolates belong to a new species of Legionella, Legionella fairfieldensis (ATCC 49588).

J Intraven Nurs, 1991 Mar-Apr, 14(2), 130 - 5
What the practicing nurse should know about thiamine; Baumgartner TG; Thiamine (vitamin B1) is an essential nutritional component that acts as a coenzyme in the oxidative decarboxylation of alpha-keto acids . It also serves as the connection between the glycolytic cycle and the high energy-producing Krebs (or citric acid) cycle . Unlike other B vitamins, it activates the guanylate cyclase/cyclic guanosine monophosphate (GMP) system but not the adenylate cyclase system . The active coenzyme, thiamine pyrophosphate (TPP) is an antiberiberi substance . Thiamine itself is a pharmacologic antagonist of acetylcholine, which may explain the nerve lesions caused by thiamine deficiency . Liver, pork, yeast, and rice-polishings are rich in thiamine; however, several antithiamine factors are also found in common foods . For example, a thermal labile factor in the viscera of fresh water fish and tea leaves antagonizes thiamine.

Am J Clin Nutr, 1991 Mar, 53(3), 748 - 54
Selenium distribution in blood fractions of New Zealand women taking organic or inorganic selenium; Butler JA et al.; Three groups of 11 New Zealand women each received, for 32 wk, yeast tablets with no added selenium (placebo) or 200 micrograms Se/d in tablets either as selenate or as selenium-enriched yeast (SeMet) in a double-blind selenium trial . Plasma and erythrocyte (RBC) samples were collected bimonthly . Gel filtration of plasma from women taking SeMet revealed two major selenium-containing peaks with most of the selenium in the second peak . In contrast, the first peak contained most of the selenium in plasma from women taking selenate . Chromatography of RBC lysates indicated that the majority of the selenium was with hemoglobin (Hb) in women taking SeMet but was about equally distributed between glutathione peroxidase (GSH-Px) and Hb in women taking selenate . The percentage of selenium associated with GSH-Px was found to be greater in RBCs and plasma of women taking selenate than of those taking SeMet.

Proc Natl Acad Sci U S A, 1991 Mar 1, 88(5), 1844 - 8
Chromosome microdissection and cloning in human genome and genetic disease analysis; Kao FT et al.; A procedure has been described for microdissection and microcloning of human chromosomal DNA sequences in which universal amplification of the dissected fragments by Mbo I linker adaptor and polymerase chain reaction is used . A very large library comprising 700,000 recombinant plasmid microclones from 30 dissected chromosomes of human chromosome 21 was constructed . Colony hybridization showed that 42% of the clones contained repetitive sequences and 58% contained single or low-copy sequences . The insert sizes generated by complete Mbo I cleavage ranged from 50 to 1100 base pairs with a mean of 416 base pairs . Southern blot analysis of microclones from the library confirmed their human origin and chromosome 21 specificity . Some of these clones have also been regionally mapped to specific sites of chromosome 21 by using a regional mapping panel of cell hybrids . This chromosome microtechnology can generate large numbers of microclones with unique sequences from defined chromosomal regions and can be used for processes such as (i) isolating corresponding yeast artificial chromosome clones with large inserts, (ii) screening various cDNA libraries for isolating expressed sequences, and (iii) constructing region-specific libraries of the entire human genome . The studies described here demonstrate the power of this technology for high-resolution genome analysis and explicate their use in an efficient search for disease-associated genes localized to specific chromosomal regions.

Proc Natl Acad Sci U S A, 1991 Mar 1, 88(5), 1701 - 5
Lymphocyte activation induces rapid changes in nuclear and cytoplasmic glycoproteins; Kearse KP et al.; A unique form of nucleoplasmic and cytoplasmic protein glycosylation, O-linked GlcNAc, (O-GlcNAc) is present on proteins ranging from those of yeast to man, including many chromatin proteins, transcription factors, nuclear pore proteins, and certain types of cytoskeletal proteins . In this report we have studied the effects of cellular activation on O-GlcNAc-modified proteins, using T lymphocytes as a model system . Results indicate that the apparent levels of O-GlcNAc on many nuclear proteins increases rapidly after lymphocyte activation, returning to control levels after a few hours . In contrast, the apparent levels of O-GlcNAc on a distinct population of cytosolic proteins decreases rapidly after cellular activation and also returns to control levels after a few hours . These data are consistent with the hypothesis that O-GlcNAc is a regulatory modification and suggest that O-GlcNAc modification may play an important role in the early stages of T-lymphocyte activation.

Ukr Biokhim Zh, 1991 Mar-Apr, 63(2), 59 - 65
{Features of thiamine diphosphate-dependent enzyme inhibition by oxodihydrothiochrome and its phosphorylated derivatives}; Zimatkina TI et al.; Anticoenzyme action of new derivatives of thiamine: oxodihydrothiochrome and its mono- and diphosphoric esters has been studied in the experiments on mice . It is shown that the given compounds exert an inhibiting action on transketolase and pyruvate dehydrogenase and do not change activity of 2-oxoglutarate dehydrogenase in the animal organism . Antivitamin effect of the studied inhibitors is observed with the lower doses and in the earlier terms as compared with the other known inhibitors of thiamine-diphosphate-dependent enzymes . The preparations inhibit activity of the yeast pyruvate-decarboxylase by the mixed (with respect to thiamine-diphosphate) type (Ki for oxodihydrothiochrome and its mono- and diphosphoric esters: 2.3 x 10(-3), 7.2 x 10(-4), 5.6 x 10(-5) M, respectively) . Possible mechanisms of the action of the mentioned compounds as thiamine antimetabolites are discussed.

Jpn J Med, 1991 Mar-Apr, 30(2), 135 - 7
A case of Trichosporon pullulans infection of the lung with adult T-cell leukemia; Shigehara K et al.; Fungal infections are often reported, but Trichosporon infection is very rare . A 78-year-old man with adult T-cell leukemia complicated with pulmonary infections is presented . Bronchial exudate culture revealed many yeast-like colonies, which were morphologically and biochemically identified as Trichosporon pullulans.

Zentralbl Veterinarmed B, 1991 Mar, 38(2), 90 - 4
Alterations of cellular immune response during intensive training of event horses; Buschmann H et al.; During strenuous exercise of horses that are prepared for international Three-Day-Events a significant decrease in the in vitro killing rate of phagocytosed yeast cells by the blood granulocytes has been observed . Other immunological parameters, such as the phorbolmyristate dependent chemiluminescence in granulocytes and the mitogenic stimulation of blood lymphocytes, remained unchanged.

Arch Intern Med, 1991 Mar, 151(3), 574 - 8
Suboptimal response to hepatitis B vaccine in drug users; Rumi M et al.; Fifty-five drug users institutionalized in a community for drug detoxification were vaccinated against hepatitis B virus (HBV) with a recombinant yeast-derived vaccine (Engerix B, Smith Kline Biologicals, Belgium) . At 0, 1, and 6 months, 20 micrograms of vaccine was given in the deltoid to these patients, of whom 17 had no serum HBV markers, 15 had only the antibody to core antigen (anti-HBc), and 23 had anti-HBc and antibody to the surface antigen (anti-HBs) values lower than the allegedly "protective" value of 10 mIU/mL . Forty-one healthy controls were vaccinated in parallel . At month 7 (ie, 1 month after the third dose of vaccine), in 76% (13/17) of drug users with no HBV markers, 6% (1/15) of those with isolated anti-HBc, and 69% (16/23) of those with anti-HBc and nonprotective anti-HBs, protective anti-HBs titers (greater than or equal to 10 mIU/mL) developed, compared with 97% (40/41) of controls . At month 24, these rates fell to 43% for drug users with no HBV marker, 0% for those with anti-HBc, 31% for those with anti-HBc and anti-HBs, and 86% for controls . The drug users who did not respond to vaccination were more likely to be those with evidence of prior HBV infection and anergy to skin tests . This indicates that unresponsiveness to hepatitis B vaccine in drug users may be due to altered immunity.

J Clin Invest, 1991 Mar, 87(3), 901 - 7
Increase in the expression of a family of small guanosine triphosphate-binding proteins, rab proteins, during induced phagocyte differentiation; Maridonneau-Parini I et al.; Rab is a newly identified family of small G-proteins that share 35-70% homology with the yeast Sec4p and Ypt1p involved in the regulation of the secretory pathway . Mature phagocytes display functions requiring organized intracellular traffic and, for this reason, we questioned whether phagocyte differentiation could correlate with the increased expression of rab proteins . Rabbit antisera raised against the recombinant proteins rab1Ap, 2p, 4p, and 6p were able to detect the corresponding proteins in the human monoblast leukemic cell line U937 . When these cells were induced to differentiate into monocyte/macrophage-like cells displaying functional characteristics of a normal phagocyte, rab1Ap, 2p, 4p, and 6p were increased and this correlated with an increase in the rab transcripts . Using a rab5 probe, we also observed an increased expression of the rab5 gene in differentiated cells . Similarly, differentiation of the human leukemic myeloblast HL60 cell line along either monocyte or granulocyte pathways induced an increased expression of the rab proteins . Rab proteins were also detected in human neutrophils and in guinea pig alveolar macrophages . As degranulation is one of the phagocyte functions acquired in the late stage of differentiation, we investigated whether rab proteins would be involved in this process . Although rab proteins were tightly membrane bound, none of them was detected in the specific or azurophil granules purified from human neutrophils . The increased expression of rab proteins in mature phagocytes suggests that they may promote functions highly developed in these cells.

Southeast Asian J Trop Med Public Health, 1991 Mar, 22(1), 39 - 40
Immune response to hepatitis B vaccine in premature neonates; Chawareewong S et al.; The immunogenecity of a recombinant yeast-derived hepatitis B vaccine in 25 premature infants who were born to HBsAg negative mothers was studied . The gestational ages were 28-37 weeks and birth weights were 1300-2000 grams . Seroconversion occurred in 22 infants, the anti HBs titers varied between 50 and 13,470 IU/L (Geometric mean titer = 542 IU/L) . Seroconversion rate = 88% . The response did not vary with gestational age, birth weight or illness status.

Vaccine, 1991 Mar, 9(3), 163 - 9
Induction of protection level of anti-pre-S2 antibodies in humans immunized with a novel hepatitis B vaccine consisting of M (pre-S2 + S) protein particles (a third generation vaccine); Kuroda S et al.; An enzyme-linked immunosorbent assay (ELISA) for anti-pre-S2 antibodies was developed by the use of both recombinant yeast-derived S and M (pre-S2 + S) protein particles as antigens . By this ELISA was determined the amount of both human and chimpanzee anti-pre-S2 antibodies produced by a new type of yeast-derived hepatitis B (HB) vaccine (TGP-943, subtype adr), which consists of modified M protein particles . In seven randomly selected human individuals who were vaccinated intramuscularly with 10 micrograms (as a protein) TGP-943 three times (0, 4th and 24th week), a detectable level of anti-pre-S2 antibodies was found to be rapidly elicited at 4th or 8th week after the first vaccination . The protective level of anti-pre-S2 antibodies in humans was tentatively assessed by comparing the anti-pre-S2 antibody titres in the vaccinated human individuals with that in chimpanzees which produced only anti-pre-S2 antibodies to tolerate well against the challenge by 1000 chimpanzee infectious units of HBV (subtype ayw) . From this assessment, it was speculated that all human individuals tested had already acquired the protective level of anti-pre-S2 antibodies at 4th or 8th week after the first vaccination with TGP-943.

J Leukoc Biol, 1991 Mar, 49(3), 266 - 76
Role of endogenously derived leukotrienes in the regulation of lysosomal enzyme expression in macrophages exposed to beta 1,3-glucan; Lew DB et al.; The expression of the lysosomal enzyme hexosaminidase has been shown to be stimulated by the exposure of mouse macrophages to beta 1,3-glucan, a particulate component of yeast cell walls and zymosan particles . Exposure of mouse peritoneal macrophages to particulate beta 1,3-glucan (100 micrograms/ml) was also found to stimulate the production of eicosanoids from both the cyclooxygenase (prostaglandin E2) and 5'-lipoxygenase (leukotriene C4) pathways . The objective of this study was to determine the relationship, if any, between the production of arachidonic acid metabolites and the increased expression of lysosomal enzymes . To determine if products of the cyclooxygenase or 5'-lipoxygenase pathway were involved in the regulation of hexosaminidase expression, macrophages were exposed to beta 1,3-glucan in the presence of indomethacin, an inhibitor of cyclooxygenase; 4,7,10,13 ETYA, an inhibitor of both cyclooxygenase and 5'-lipoxygenase; or AA861, a selective inhibitor of 5'-lipoxygenase . While the increased expression of hexosaminidase was not affected in macrophages stimulated with beta 1,3-glucan in the presence of indomethacin, both 4,7,10,13 ETYA and AA861 completely blocked the response, suggesting a role for products of the 5'-lipoxygenase pathway in the regulation of hexosaminidase expression . To further explore the relationship between arachidonate release and the increase expression of hexosaminidase, macrophages were exposed to phospholipase A2 in an attempt to circumvent the interaction between beta 1,3-glucan and the macrophage membrane . Incubation with phospholipase A2 was found both to induce the accumulation of LTC4 in the culture supernatant and to stimulate the increased expression of hexosaminidase . The mechanism of regulation of hexosaminidase expression by products of the 5'-lipoxygenase pathway was investigated by incubating macrophages with purified luekotrienes in either the presence or absence of beta 1,3-glucan . Incubation of macrophages with purified LTC4 or LTB4 in the absence of beta 1,3-glucan failed to stimulate the expression of hexosaminidase . However, challenging macrophage monolayers with LTC4 or LTB4 in the presence of a suboptimal concentration of beta 1,3-glucan (1 microgram/ml) led to a synergistic increase in the expression of hexosaminidase . Collectively these data suggest that the leukotriene products of the 5'-lipoxygenase pathway, LTC4 and LTB4, regulate the expression of lysosomal enzymes by apparently priming macrophages, thereby increasing their sensitivity to triggering agents such as beta 1,3-glucan . Since macrophages produce LTC4, and the increased expression of hexosaminidase is prevented by inhibitors of the 5'-lipoxygenase pathway, the data further suggest that LTC4 may prime macrophages in an autocrine or paracrine fashion.

Mycoses, 1991 Mar-Apr, 34(3-4), 173 - 6
The preservation of fungal cultures by lyophilization; Bunse T et al.; A method for the preservation of fungal strains is presented . The cultures are grown on Sabouraud glucose agar in glass ampoules and lyophilized without further processing . By this method the macroscopical morphology of the cultures is preserved, so that these can be used immediately without recultivation as reference cultures . All tested mould and yeast strains remained viable over the six months duration of the experiment, whereas the dermatophyte strains could only partially be recultivated.

Clin Exp Immunol, 1991 Mar, 83(3), 452 - 9
Characterization and large production of human monoclonal antibodies against the HIV-1 envelope; Boyer V et al.; Peripheral blood lymphocytes from a volunteer immunized with a recombinant vaccinia virus VSC-25 expressing the gp160 env protein of HTLV-IIIB strain and from an asymptomatic HIV-infected individual were immortalized by Epstein-Barr (EBV) . Clones which secrete human monoclonal antibodies from the two individuals (DZ, IgG1, lambda and C31, IgG1, kappa) were obtained and were stable for more than 2 years . The two monoclonals were directed against the gp160 env protein of HIV, DZ directed against the gp41 and C31 directed against the gp120 . C31 was group-specific, whereas DZ was directed against the HTLV-IIIB and HTLV-RF strains . The epitope recognized by DZ was mapped to the carboxy terminus of the gp41, by expression of HIV DNA fragments in a yeast system and peptide analysis . The C31 epitope was not expressed by the yeast library and not present among the peptides which were tested . Monoclonal antibodies had no inhibitory effect in an HIV-induced cell fusion assay, but DZ showed a weak neutralizing activity against the HTLV-IIIB strain . Cloned EBV-transformed cell lines were fused to a murine myeloma, which allowed the heteromyeloma to be cultivated in serum-free medium . The monoclonal antibodies were produced in large quantity in a hollow-fibre reactor at defined culture conditions and purification procedures.

Genes Dev, 1991 Mar, 5(3), 484 - 95
AGL1-AGL6, an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes; Ma H et al.; The predicted products of floral homeotic genes, AGAMOUS (AG) from Arabidopsis thaliana and DEFICIENS A (DEF A) from Antirrhinum majus, have been shown previously to share strong sequence similarity with transcription factors from humans (SRF) and yeast (MCM1) . The conserved sequence between these proteins is localized within a domain known to be necessary for the DNA binding and for the dimerization of SRF . We have isolated six new genes from A . thaliana, AGL1-AGL6, which also have this conserved sequence motif . On the basis of the sequence comparison between the AG and AGL genes, they can be assigned to two subfamilies of a large gene family . RNA dot blot analysis indicates that five of these genes (AGL1, AGL2, AGL4, AGL5, and AGL6) are preferentially expressed in flowers . In addition, in situ RNA hybridization experiments with AGL1 and AGL2 show that their mRNAs are detected in some floral organs but not in others . Our results suggest that these genes may act to control many steps of Arabidopsis floral morphogenesis . In contrast, the AGL3 gene is expressed in vegetative tissues as well as in flowers, suggesting that it functions in a broader range of tissues . We discuss possible roles of this gene family during the evolution of flowers.

Int J Epidemiol, 1991 Mar, 20(1), 60 - 7
Primary hepatocellular carcinoma in an area of low incidence: evidence for a viral aetiology from routinely collected data; Lamont DW et al.; In recent years a strong case has been made in support of a viral aetiology for at least some primary hepatocellular carcinoma (PHC) in areas of low incidence . By pooling routinely collected cancer registration and infection data, study of the relationship between hepatitis B virus (HBV) infection and the incidence of PHC in Scotland over the period 1972-1985 has confirmed this view . Over this period the incidence of PHC in men increased, there was a relationship between the incidence of notification of HBV infection and that of hepatocellular carcinoma in different parts of the country, and an increased risk attached to those chronically infected with the virus . Given the recent introduction of lower cost yeast derived vaccines, there may now be more scope for prevention both of primary liver cancer and of other liver disease.

Eur J Biochem, 1991 Feb 26, 196(1), 11 - 7
H(+)-translocating inorganic pyrophosphatase of plant vacuoles . Inhibition by Ca2+, stabilization by Mg2+ and immunological comparison with other inorganic pyrophosphatases; Maeshima M; The effects of divalent cations, especially Ca2+ and Mg2+, on the proton-translocating inorganic pyrophosphatase purified from mung bean vacuoles were investigated to compare the enzyme with other pyrophosphatases . The pyrophosphatase was irreversibly inactivated by incubation in the absence of Mg2+ . The removal of Mg2+ from the enzyme increased susceptibility to proteolysis by trypsin . Vacuolar pyrophosphatase required free Mg2+ as an essential cofactor (K0.5 = 42 microM) . Binding of Mg2+ stabilizes and activates the enzyme . The formation of MgPPi is also an important role of magnesium ion . Apparent Km of the enzyme for MgPPi was about 130 microM . CaCl2 decreased the enzyme activity to less than 60% at 40 microM, and the inhibition was reversed by EGTA . Pyrophosphatase activity was measured under different conditions of Mg2+ and Ca2+ concentrations at pH 7.2 . The rate of inhibition depended on the concentration of CaPPi, and the approximate Ki for CaPPi was 17 microM . A high concentration of free Ca2+ did not inhibit the enzyme at a low concentration of CaPPi . It appears that for Ca2+, at least, the inhibitory form is the Ca2(+)-PPi complex . Cd2+, Co2+ and Cu2+ also inhibited the enzyme . The antibody against the vacuolar pyrophosphatase did not react with rat liver mitochondrial or yeast cytosolic pyrophosphatases . Also, the antibody to the yeast enzyme did not react with the vacuolar enzyme . Thus, the catalytic properties of the vacuolar pyrophosphatase, such as Mg2+ requirement and sensitivity to Ca2+, are common to the other pyrophosphatases, but the vacuolar enzyme differs from them in subunit mass and immunoreactivity.

Biochemistry, 1991 Feb 26, 30(8), 2204 - 16
Protein-induced inactivation and phosphorylation of rabbit muscle phosphofructokinase; Zhao ZZ et al.; Several previously untested proteins promote the reversible inactivation of rabbit skeletal muscle phosphofructokinase . Grouped in decreasing order of effectiveness, they include the following: skeletal muscle troponin C greater than troponin, the two smooth muscle myosin light chains, alpha-actinin, and S-100 much greater than parvalbumin and soybean trypsin inhibitor . The efficiency of troponin C in this process may even exceed that previously reported for calmodulin . Sequences near calcium binding site III are apparently involved in the troponin C-phosphofructokinase interaction . Troponin C and calmodulin exert calcium-dependent effects on the physical and chemical properties of muscle phosphofructokinase . When calcium is present, comigration with either protein allows the enzyme to enter the stacking gel during urea-polyacrylamide gel electrophoresis . Both enhance the phosphorylation of phosphofructokinase catalyzed by the cAMP-dependent protein kinase, with phosphate incorporations approaching 2 mol of P/mol of protomer . Reaction occurs at Ser774 and at Ser376--a novel site whose phosphorylation is highly sensitive to troponin C and less so to calmodulin . Maximum phosphorylation has slight effect on the catalytic activity of the enzyme under standard assay conditions . The troponin C induced or calmodulin-induced phosphorylation of phosphofructokinase requires calcium and is strongly inhibited by either fructose 2,6-bisphosphate or fructose 1,6-bisphosphate . Inactivation occurs in the presence or absence of calcium, with generally higher concentrations of effectors required for protection in the latter case . Liver and yeast phosphofructokinases shows little activity loss in the presence of either calmodulin or troponin C . We have developed and tested a general mathematical model for the protein-induced inactivation of phosphofructokinase which may find application to other systems.

Nucleic Acids Res, 1991 Feb 25, 19(4), 707 - 12
Plant nonsense suppressor tRNA(Tyr) genes are expressed at very low levels in vitro due to inefficient splicing of the intron-containing pre-tRNAs; Szweykowska-Kulinska Z et al.; Oligonucleotide-directed mutagenesis was used to generate amber, ochre and opal suppressors from cloned Arabidopsis and Nicotiana tRNA(Tyr) genes . The nonsense suppressor tRNA(Tyr) genes were efficiently transcribed in HeLa and yeast nuclear extracts, however, intron excision from all mutant pre-tRNAs(Tyr) was severely impaired in the homologous wheat germ extract as well as in the yeast in vitro splicing system . The change of one nucleotide in the anticodon of suppressor pre-tRNAs leads to a distortion of the potential intron-anticodon interaction . In order to demonstrate that this caused the reduced splicing efficiency, we created a point mutation in the intron of Arabidopsis tRNA(Tyr) which affected the interaction with the wild-type anticodon . As expected, the resulting pre-tRNA was also inefficiently spliced . Another mutation in the intron, which restored the base-pairing between the amber anticodon and the intron of pre-tRNA(Tyr), resulted in an excellent substrate for wheat germ splicing endonuclease . This type of amber suppressor tRNA(Tyr) gene which yields high levels of mature tRNA(Tyr) should be useful for studying suppression in higher plants.

J Biol Chem, 1991 Feb 25, 266(6), 3782 - 90
Role of the proregion in the production and secretion of the Yarrowia lipolytica alkaline extracellular protease; Fabre E et al.; The yeast Yarrowia lipolytica secretes an alkaline extracellular protease (AEP) . It is first synthesized as a precursor comprising a putative signal peptide, a stretch of 10 X-Ala or X-Pro sequences that are substrates for a dipeptidyl aminopeptidase, a large pro-region that contains a glycosylation site and two Lys-Arg sites that can be cleaved by a KEX2-like endoprotease and finally the mature protease itself . A defect in the XPR6 (KEX2-like) gene results in the secretion of an inactive proenzyme (Matoba, S., and Ogrydziak, D . M . (1989) J . Biol . Chem . 264, 6037-6043), showing that the proregion inhibits protease activity . To determine whether the proregion plays an additional role in protease secretion, we have generated deletions and point mutations in the corresponding region of the structural gene . In this paper we examine the effects of these mutations on AEP secretion and maturation and show that the proregion is essential for its secretion . All deletions affecting the proregion resulted in the intracellular accumulation of unprocessed precursors . Deletion of the glycosylation site in the proregion resulted in the production of an unglycosylated precursor that was secreted and matured correctly at 18 degrees C but accumulated in the cells at 28 degrees C . From these results, we propose that the AEP prosequence plays an additional essential role in guiding the proper folding of the protein into a conformation compatible with secretion.

J Biol Chem, 1991 Feb 25, 266(6), 3402 - 7
Molecular cloning of a novel human cdc2/CDC28-like protein kinase; Johnson KW et al.; A homology probing approach was utilized to isolate a new human protein kinase . Deoxyoligonucleotide probes recognizing a conserved subdomain in the COOH-terminal portion of protein kinases identified a cDNA clone encoding a putative kinase with predicted serine/threonine phosphorylation specificity . The full-length, 1.7-kilobase pair cDNA hybridizes to 1.7- and 3.4-kilobase mRNA transcripts in a number of tissues . The size of the encoded protein is 454 amino acids and consists of an NH2-terminal 130-residue segment, which may represent a regulatory region, followed by a 324-residue catalytic domain . Comparisons and alignments of the primary sequence and predicted secondary structure of the catalytic region to other known kinases reveal that the new kinase, denoted "CLK" (for CDC-like kinase), represents a prototype for a new family of human protein kinases bearing significant homology to the yeast cdc2/CDC28 kinases that regulate the cell cycle.

J Mol Biol, 1991 Feb 20, 217(4), 629 - 35
A widely distributed "CAT" family of repetitive DNA sequences; Martinez-Soriano JP et al.; The yeast genome contains a family of repetitive sequences consisting primarily of a tandemly arranged trinucleotide, CAT, or a closely related CGT sequence . To characterize similar sequences in divergent organisms, a previously isolated "CAT" sequence was used to isolate homologous genomic clones from a human cell line, an insect and a higher plant . Sequence analyses show that comparable repetitive sequences are widely distributed and may be present in all eukaryotic genomes . In situ hybridization analyses indicate that in yeast, the CAT elements are dispersed among all the chromosomes, and a more detailed analysis in Drosophila indicates that at least one of these sequences maps on the X chromosome between known genetic loci which are actively expressed . Repeated searches of yeast cDNA libraries indicate that these CAT clusters are not expressed but substantial effects on the expression of a cloned gene strongly suggest that they play an important role in gene regulation.

Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1115 - 9
Identification of a protein kinase multigene family of Dictyostelium discoideum: molecular cloning and expression of a cDNA encoding a developmentally regulated protein kinase; Haribabu B et al.; We have identified protein kinase genes of Dictyostelium by using highly conserved amino acid sequence motifs to design the synthesis and amplification of DNA fragments by polymerase chain reactions (PCRs) . Cloning and sequencing the PCR products have revealed five different members of the protein kinase multigene family . These five putative kinases showed varying degrees of amino acid sequence similarity (40-70%) to protein kinases in data bases and contained invariant amino acid residues characteristic of protein kinases . DNA from PCR was labeled and used to isolate several lambda gt11 cDNA clones, including one full-length one (Dd kinase-2) . The nucleotide sequence of Dd kinase-2 contained a region identical to one of the cloned kinase fragments amplified by PCR, and based on the deduced amino acid sequence Dd kinase-2 encodes a protein of 479 amino acids . A 350-amino acid kinase domain at the C-terminal end shows high homology to the catalytic domains of protein kinase A, protein kinase C, S-6 kinase of Xenopus, and the suppressor of cdc25 of yeast . The N-terminal domain is highly basic and also contains alternating threonine/proline residues . The cDNA hybridized to a single copy gene but to two differentially regulated mRNAs--a 2.0-kilobase mRNA that is expressed in vegetative cells and a 2.2-kilobase mRNA that is expressed during development . The larger mRNA is induced by cAMP by using a cell-surface receptor-mediated signal transduction pathway.

Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1138 - 42
Carbohydrate ligands for endothelial-leukocyte adhesion molecule 1; Tiemeyer M et al.; The acute inflammatory response requires that circulating leukocytes bind to and penetrate the vascular wall to access the site of injury . Several receptors have been implicated in this interaction, including a family of putative carbohydrate-binding proteins . We report here the identification of an endogenous carbohydrate ligand for one of these receptors, endothelial-leukocyte adhesion molecule 1 (ELAM-1) . Radiolabeled COS cells transfected with a plasmid containing the cDNA for ELAM-1 were used as probes to screen glycolipids extracted from human leukocytes . COS cells transfected with this plasmid adhered to a subset of sialylated glycolipids resolved on TLC plates or adsorbed on polyvinyl chloride microtiter wells . Adhesion to these glycolipids required calcium but was not inhibited by heparin, chondroitin sulfate, keratan sulfate, or yeast phosphomannan . Monosaccharide composition, linkage analysis, and fast atom bombardment mass spectrometry of the glycolipids indicate that the ligands for ELAM-1 are terminally sialylated lactosylceramides with a variable number of N-acetyllactosamine repeats and at least one fucosylated N-acetylglucosamine residue.

Nucleic Acids Res, 1991 Feb 11, 19(3), 505 - 10
A test case for physical mapping of human genome by repetitive sequence fingerprints: construction of a physical map of a 420 kb YAC subcloned into cosmids; Bellanne-Chantelot C et al.; A rapid and safe method of Yeast Artificial Chromosome (YAC) physical mapping by cosmid 'fingerprinting' is presented . YACs are subcloned into cosmids which are prepared without previous separation of cloned DNA from host DNA . Groups of overlapping clones are detected according to their restriction fragments size and intensity after hybridization with total human DNA . To test this approach, a cosmid library was constructed from total DNA of a yeast strain containing a 420 kb YAC . A single contig of 84 clones was obtained with a minimal detectable overlap of 60% i.e . a 9.2 fold representative library . Large scale physical mapping of YACs would take full advantage of the DNA preparation procedure employed in this work and allows to take into account restriction fragment intensities.

FEBS Lett, 1991 Feb 11, 279(1), 105 - 9
A 28 kDa mitochondrial protein is radiolabelled by crosslinking with a 125I-labelled presequence; Font B et al.; A 13-residue peptide containing the first 12 amino acids of the N-terminal part of the signal sequence of yeast cytochrome c oxidase subunit IV is shown by chemical crosslinking to interact with a mitochondrial protein . This result is obtained with mitochondria from four different origins . Submitochondrial localization experiments suggest that the 28 kDa labelled component is present on the outer face of the inner membrane . Since such addressing peptides are imported into mitochondria through the same machinery as protein precursors, the 28 kDa protein might be a component of the translocation apparatus.

Cell, 1991 Feb 8, 64(3), 625 - 33
Preferential inhibition of the oncogenic form of RasH by mutations in the GAP binding/"effector" domain; Farnsworth CL et al.; The double mutation, D33H/P34S, reduced the transforming activity of oncogenic RasH proteins, G12V and Q61L, 400- and 20-fold, respectively . Remarkably, this same mutation did not reduce the transforming activity of normal RasH, nor did it impair the ability of the protein to restore a functional Ras pathway in cells whose endogenous Ras proteins were inhibited . Another mutation in this region, D38N, had similar effects . The mutations reduced downstream coupling efficiency of normal Ras as assessed by yeast adenylyl cyclase stimulation . However, this was offset by decreased GTPase activating protein (GAP) binding, since the latter resulted in elevated GTP-bound mutant Ras in cells . The mutations produced a similar decrease in downstream coupling efficiency of oncogenic Ras, but decreased GAP binding did not compensate because the GTPase activity of oncogenic Ras is not stimulated by GAP . These results imply that preferential inactivation of oncogenic Ras in human tumors may be achieved by reagents designed to inhibit the GAP-binding/"effector" domain of Ras proteins.

J Biol Chem, 1991 Feb 5, 266(4), 2675 - 80
A family of ras-like GTP-binding proteins expressed in electromotor neurons; Ngsee JK et al.; The cDNAs encoding seven low molecular weight (LMW) GTP-binding proteins were isolated from an electrode lobe library of the marine ray Discopyge ommata . Four were assigned as the ray homologues of previously identified LMW GTP-binding proteins rab1, ral, Krev, and rho . Three others showed unique sequences, including two exhibiting significant similarity to the yeast SEC4 protein . Northern analysis indicated that several of the transcripts are enriched in neural tissues with a moderate level of expression in cardiac muscle . This tissue distribution was corroborated with affinity purified antibodies against the LMW GTP-binding proteins . Subcellular fractionation revealed that the proteins co-purify with cholinergic synaptic vesicles . Immunohistochemical analysis confirms this localization . At least two of the proteins, oral and o-rho, are localized to the pre-synaptic terminals.

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