Yeasts

Biochemistry, 1992 Dec 8, 31(48), 12147 - 54
Identification of topaquinone and its consensus sequence in copper amine oxidases; Janes SM et al.; The nature of the active site cofactor and the amino acid sequence flanking this structure have been determined in a range of copper amine oxidases . For enzymes from porcine plasma, porcine kidney, and pea seedlings, proteolytic digestion was performed on phenylhydrazone or p-nitrophenylhydrazone derivatives . Thermolysin treatment leads to relatively small active site peptides, which have been characterized by Edman degradation and by resonance Raman spectroscopy . Resonance Raman spectra of peptides show identical peak positions and intensities relative to each other and to a model p-nitrophenylhydrazone derivative of topaquinone hydantoin, establishing topaquinone as the cofactor in each instance . Edman degradation of peptides provides active site sequences for comparison to previous determinations with bovine serum and yeast amine oxidases . The available data establish a consensus sequence of Asn, Topa, Asp/Glu . Trypsin leads to significantly longer peptides, which reveal a high degree of sequence identity between plasma proteins from bovine and porcine sources (89%), with significantly decreased identity between the porcine serum and intracellular amine oxidases (56%) . A lower degree of identity (45%) is observed between the pea seedling and mammalian enzymes . As an alternative to the isolation of active site peptides for topaquinone identification, visible spectra of intact proteins have been investigated . It is shown that p-nitrophenylhydrazone derivatives of native enzymes, active site-derived peptides, and a topaquinone model exhibit identical behavior, absorbing at 457-463 nm at neutral pH (pH 7.2) and at 575-587 nm in basic solution (1-2 M KOH) . These spectral properties, which appear unique to topaquinone, provide a rapid and simple test for the presence of this cofactor in intact enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Pigment Cell Res, 1992 Dec, 5(6), 400 - 3
Effect of amphotericin B on dopachrome tautomerase activity and other melanogenic parameters in cultured B16/F10 melanoma cells; Peinado P et al.; The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 micrograms/ml) used for preventing molds and yeast growth in cultures of animal cells . However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities . At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased . The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone . After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase . The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res . 3, 173-175) for tyrosine hydroxylase.

Nippon Rinsho, 1992 Dec, 50(12), 3086 - 92
{Recent progress in molecular biology of inherited tubular transport abnormalities}; Indo Y et al.; Recent progress in the molecular biological approach to analysis of inherited tubular transport abnormalities is reviewed . 1) cDNAs of several mammalian proteins, related to amino acid transport in renal tubular cell, have been cloned using an expression cloning in Xenopus oocytes . One of them stimulates the transport of cystine, dibasic amino acids and neutral amino acids and will accelerate the analysis of cystinuria . 2) Isolation of cDNAs, encoding human and rat vasopressin V2 receptors, has been reported . The deduced amino acid sequence seems to be a member of receptors with seven putative transmembrane regions . Analysis of this gene from patients with nephrogenic diabetes insipidus is in progress . 3) Analysis of carbonic anhydrase II (CA II) gene in a Belgian family with renal tubular acidosis associated with osteoporosis and cerebral calcification has shown a point mutation replacing an invariant histidine residue of CA II protein with tyrosine . 4) Oculocerebrorenal syndrome of Lowe (OCRL) is a X-linked disorder affecting the lens, brain and kidneys . The OCRL locus has been mapped to Xq24-26 by linkage analysis and by finding de novo X-autosome translocations at Xq24-26 in two unrelated females with OCRL . A cDNA has been isolated using yeast artificial chromosome and DNA inserts that span the X chromosome breakpoint from a female patient . Transcript for this cDNA is absent in unrelated male patients . The open reading frame encodes a new protein similar to human inositol-polyphosphate-5-phosphatase, raising a possibility that OCRL is an inborn error of inositol phosphate metabolism.

Genomics, 1992 Dec, 14(4), 857 - 62
YAC-assisted cloning of a putative G-protein mapping to the MHC class I region; Denizot F et al.; We report the successful use of whole yeast artificial chromosomes (YACs) as probes for direct positional cloning of novel expressed sequences in a given genomic fragment . The class I region of the human major histocompatibility complex, in particular the chromosomal fragment spanning the HLA-E locus, was investigated . The screening of a cDNA library with a 210-kb-long YAC clone led to the identification of a new gene, positionally conserved in the major histocompatibility complex of the mouse genome and encoding a putative GTP binding protein . Although its precise function remains unknown, the interspecies conservation of both sequence and map position suggests a regulatory or functional link with the histocompatibility cluster.

Genomics, 1992 Dec, 14(4), 1010 - 8
Structure and linkage of the D2 dopamine receptor and neural cell adhesion molecule genes on human chromosome 11q23; Eubanks JH et al.; The gene encoding the D2 dopamine receptor (DRD2) is located on human chromosome 11q23 and has been circumstantially associated with a number of human disorders including Parkinson's disease, schizophrenia, and susceptibility to alcoholism . To determine the physical structure of the DRD2 gene, we utilized cosmid cloning, isolation of yeast artificial chromosomes (YACs), and pulsed-field gel electrophoresis to construct a long-range physical map of human chromosome 11q23 linking the genes for the DRD2 and neural cell adhesion molecule (NCAM) . The D2 dopamine receptor gene extends over 270 kb and includes an intron of approximately 250 kb separating the putative first exon from the exons encoding the receptor protein . The resulting physical map spans more than 1.5 mb of chromosome band 11q23 and links the DRD2 gene with the gene encoding the NCAM located 150 kb 3' of the DRD2 gene and transcribed from the same DNA strand . We additionally located the sites of at least four hypomethylated HTF islands within the physical map, which potentially indicate the sites of additional genes . High-resolution fluorescent in situ suppression hybridization using cosmid and YAC clones localized this gene cluster between the ApoAI and STMY loci at the interface of bands 11q22.3 and 11q23.1.

Curr Opin Genet Dev, 1992 Dec, 2(6), 907 - 12
Position effect and related phenomena; Henikoff S; Position-effect variegation in Drosophila, the mosaic expression of genes juxtaposed to heterochromatin, remains an enigmatic long-range phenomenon . While the chromatin-conformation model has been challenged, compelling contrary evidence is lacking . Nevertheless, progress has been made in the genetic and molecular analysis of genes involved in the process of heterochromatin formation and in the characterization of genetic elements normally located in pericentric heterochromatin . In addition, telomeric position effect in yeast provides a new model system for the study of the quasi-stable inheritance of an inactivated state.

Cell Growth Differ, 1992 Dec, 3(12), 889 - 97
Heterodimerization with c-Fos is not required for cell transformation of chicken embryo fibroblasts by Jun; Hughes M et al.; c-Jun belongs to a family of proteins that require dimerization for activity . Dimerization occurs through a leucine-rich region near the carboxy terminus called the leucine zipper . Jun can form dimeric complexes with other Jun family as well as Fos family members . The relative proportion of these different dimeric complexes is determined by the relative abundance of each family member at a particular time . Overexpression of v-Jun or c-Jun alone will lead to cell transformation of chicken embryo fibroblasts, albeit with varying efficiencies . Upon overexpression, v-Jun or c-Jun presumably becomes the predominant AP-1 component in the cell . Theoretically, this should lead to a larger proportion of homodimers than heterodimers . It is not clear what role, if any, the other Jun and Fos family proteins play during cell transformation . We have examined the ability of Jun to induce cell transformation in chicken embryo fibroblasts in the absence of interaction with other Jun or Fos family proteins . To this end, we have constructed a chicken v-Jun mutant that is incapable of heterodimerization . This was accomplished by replacing the leucine zipper region of Jun with that of the yeast transcription factor GCN4 . This chimeric protein, VJ-GLZ, retains all of the DNA binding and transcriptional activation domains of v-Jun . As expected, in vitro translated VJ-GLZ was found to be incapable of forming heterodimers with c-Fos, FosB, and JunD.(ABSTRACT TRUNCATED AT 250 WORDS)

Br J Ind Med, 1992 Dec, 49(12), 850 - 4
Reductions in lymphocyte subpopulations after repeated exposure to 1.5 ppm nitrogen dioxide; Sandstrom T et al.; In this investigation the effects of repeated exposure to 1.5 ppm NO2 on immune competent cells in bronchoalveolar lavage (BAL) fluid was studied . Special attention was focused on effects on lymphocyte subpopulations . Eight healthy subjects were exposed to 1.5 ppm NO2 every second day on six occasions . Bronchoalveolar lavage fluid was collected at least three weeks before the exposure series as reference and 24 hours after the last exposure . The results obtained were analysed using a non-parametric test for paired observations, with each subject as his own control . Significant reductions were found in the total number and percentage of T cytotoxic-suppressor cells in BAL fluid; this caused an increase in the ratio of T helper-inducer: cytotoxic-suppressor cells . The total number of natural killer cells in the BAL fluid was also reduced . The numbers of all other cell types were unchanged after exposure . No reduction of phagocytosis of opsonised yeast particles by alveolar macrophages in vitro was detected . It is concluded that repeated short term exposures to 1.5 ppm NO2, a moderate occupational concentration, induces significant effects on immune competent bronchoalveolar lymphocytes . This indicates that previous findings of changes in the lymphoid immune system induced by NO2 in animals may well be applicable to humans.

Scand J Immunol, 1992 Dec, 36(6), 885 - 91
Phagocytosis following translocation of the neutrophil b-cytochrome from the specific granule to the plasma membrane is associated with an increased leakage of reactive oxygen species; Lundqvist H et al.; The effect of neutrophil b-cytochrome translocation on the respiratory burst activation generated during phagocytosis of yeast particles was investigated . Secretion of neutrophil specific granules was induced by the calcium ionophore ionomycin prior to phagocytosis . The secretory process is associated with a translocation from the specific granules to the plasma membrane of the respiratory burst b-cytochrome . Respiratory burst activity was measured as release of hydrogen peroxide in the absence of azide (extracellular leakage) and in the presence of azide (total production) . The subcellular localization of the b-cytochrome was found to affect the extracellular release of hydrogen peroxide in that a plasma membrane localization was associated with a significantly increased release during phagocytosis . It should be pointed out, however, that most of the hydrogen peroxide, both in control and in ionomycin-treated cells, is produced intracellularly, probably in the phagosomes.

J Exp Med, 1992 Dec 1, 176(6), 1673 - 80
Phagocytic chimeric receptors require both transmembrane and cytoplasmic domains from the mannose receptor; Kruskal BA et al.; Phagocytosis has traditionally been viewed as a specialized function of myeloid and monocytic cells . The mannose receptor (MR) is an opsonin-independent phagocytic receptor expressed on tissue macrophages . When human MR cDNA is transfected into Cos cells, these usually non-phagocytic cells express cell surface MR and bind and ingest MR ligands such as zymosan, yeast, and Pneumocystis carinii . Expression of cDNA for Fc gamma RI (CD64), the high-affinity Fc receptor, in Cos cells confers binding but barely detectable phagocytosis of antibody-opsonized erythrocytes (EA) . We report here that chimeric receptors containing the ligand-binding ectodomain of the Fc receptor and the transmembrane and cytoplasmic domains of the MR ingest bound EA very efficiently, whereas chimeras with the Fc receptor ecto- and transmembrane domains and the MR tail, or the Fc receptor ecto- and cytoplasmic domains and the MR transmembrane region, are significantly less phagocytic . All of the chimeric receptors bind ligand with equal avidity, but gain of functional phagocytosis is only conferred by the MR transmembrane and cytoplasmic domains . Endocytosis of monomeric immunoglobulin G by chimeric receptors demonstrates a similar pattern, with optimal uptake by the chimera containing both tail and transmembrane regions from the MR . The chimeric receptors with only the transmembrane or the cytoplasmic domain contributed by the MR were less efficient . Site-directed mutagenesis of the single tyrosine residue in the cytoplasmic tail (which is present in a motif homologous to an endocytosis consensus motif in the LDL receptor cytoplasmic tail {Chen, W.-J., J . L . Goldstein, and M . S . Brown . 1990 . J . Biol . Chem . 265:3116}) reduces the efficiency of phagocytosis and endocytosis to a similar extent.

DNA Cell Biol, 1992 Dec, 11(10), 727 - 34
Complete human NF1 cDNA sequence: two alternatively spliced mRNAs and absence of expression in a neuroblastoma line; Bernards A et al.; Neurofibromatosis type 1 (NF1) is caused by mutations in a large gene on chromosome 17q11.2 . Previously described partial cDNAs for this gene predicted a protein related to yeast IRA1/IRA2 and the mammalian RAS GTPase activator protein GAP . To initiate a detailed study of the role of this gene in NF1, we have characterized a set of overlapping cDNAs that represent its complete coding sequence . Our results show that two differentially expressed human NF1 mRNAs differ by a 63-bp insertion in the GAP-related domain . These mRNAs predict two 2,818- and 2,839-amino acid proteins with calculated molecular masses of approximately 317 and 319 kD . Extensive similarity to IRA proteins is evident in a 1,450-amino-acid central segment, roughly between amino acids 900 and 2,350 . However, the remainder of the NF1 protein is not significantly similar to other proteins . Interestingly, the SK-N-SH human neuroblastoma line expresses no detectable NF1 mRNA, indicating that expression of NF1 is not essential for viability of this neural crest-derived tumor cell line.

Mol Cell Biol, 1992 Dec, 12(12), 5563 - 70
Isolation and structural analysis of a 1.2-megabase N-myc amplicon from a human neuroblastoma; Schneider SS et al.; Oncogene amplification is observed frequently in human cancers, but little is known about the mechanism of gene amplification or the structure of amplified DNA in tumor cells . We have studied the N-myc amplified domain from a representative neuroblastoma cell line, SMS-KAN, and compared the map of the amplicon in this cell line with that seen in normal DNA . The SMS-KAN cell line DNA was cloned into yeast artificial chromosomes (YACs), and clones were identified by screening the YAC library with amplified DNA probes that were obtained previously (B . Zehnbauer, D . Small, G . M . Brodeur, R . Seeger, and B . Vogelstein, Mol . Cell . Biol . 8:522-530, 1988) . In addition, YAC clones corresponding to the normal N-myc locus on chromosome 2 were obtained by screening two normal human YAC libraries with these probes, and the restriction maps of the two sets of overlapping YACs were compared . Our results suggest that the amplified domain in this cell line is a approximately 1.2-Mb circular molecule with a head-to-tail configuration, and the physical map of the normal N-myc locus generally is conserved in the amplicon . These results provide a physical map of the amplified domain of a neuroblastoma cell line that has de novo amplification of an oncogene . The head-to-tail organization, the general conservation of the normal physical map in the amplicon, and the extrachromosomal location of the amplified DNA are most consistent with the episome formation-plus-segregation mechanism of gene amplification in these tumors.

J Cell Biol, 1992 Dec, 119(5), 1117 - 28
Evidence for a novel route of wheat storage proteins to vacuoles; Levanony H et al.; Wheat seed storage proteins are deposited in protein bodies (PB) inside vacuoles, but their subcellular site of aggregation and their route to vacuoles are still controversial . In the present work, an ultra structural analysis of developing wheat endosperm at early to mid maturation was performed to address these issues . Golgi complexes were rarely detected, indicating that their role in wheat storage protein transport is limited . In contrast, a considerable amount of PB was detected in the cytoplasm . Many of these PB were surrounded by RER membranes and were enlarged by fusion of smaller PB . Small, electron lucent vesicles were detected around the surfaces of the PB in the cytoplasm, or attached to them, suggesting that such attachments and subsequent fusion of the vesicles with each other lead to the formation of small vacuoles containing PB inclusions . Immunogold labeling with serum raised against yeast-BiP, an ER-localized protein, demonstrated that the wheat BiP homolog was present within the PB in the cytoplasm as well as inside vacuoles . This confirmed that the PB were formed within the RER and that the Golgi complex was not involved in their transport to vacuoles . It is concluded that a considerable part of the wheat storage proteins aggregate into PB within the RER and are then transported as intact PB to the vacuoles by a novel route that does not utilize the Golgi complex.

Proteins, 1992 Dec, 14(4), 475 - 82
Investigation of the function of mutated cellulose-binding domains of Trichoderma reesei cellobiohydrolase I; Reinikainen T et al.; The function of the cellulose-binding domain (CBD) of the cellobiohydrolase I of Trichoderma reesei was studied by site-directed mutagenesis of two amino acid residues identified by analyzing the 3D structure of this domain . The mutant enzymes were produced in yeast and tested for binding and activity on crystalline cellulose . Mutagenesis of the tyrosine residue (Y492) located at the tip of the wedge-shaped domain to alanine or aspartate reduced the binding and activity on crystalline cellulose to the level of the core protein lacking the CBD . However, there was no effect on the activity toward small oligosaccharide (4-methylumbelliferyl beta-D-lactoside) . The mutation tyrosine to histidine (Y492H) lowered but did not destroy the cellulose binding, suggesting that the interaction of the pyranose ring of the substrate with an aromatic side chain is important . However, the catalytic activity of this mutant on crystalline cellulose was identical to the other two mutants . The mutation P477R on the edge of the other face of the domain reduces both binding and activity of CBHI . These results support the hypothesis that both surfaces of the CBD are involved in the interaction of the binding domain with crystalline cellulose.

Cancer Res, 1992 Dec 1, 52(23), 6682 - 9
Fibroblasts transformed by different ras oncogenes show dissimilar patterns of protease gene expression and regulation; Zhang JY et al.; NIH 3T3 cells transformed by different activated ras genes showed different patterns of protease gene expression, indicating the existence of least two pathways for NIH 3T3 transformation from mutated ras genes . In cells transformed by activated mammalian EJ-ras and chimeric EJ/vHa-ras, high constitutive levels of urokinase plasminogen activator (uPA) mRNA and/or phorbol-12-myristate-13-acetate (PMA) inducibility of the uPA mRNA was observed . However, PMA did not induce cathepsin L (CL) mRNA levels in these same cell lines . In contrast, NIH 3T3 cells transformed by homologous yeast RAS1Leu sequences showed low levels of uPA mRNA and a lack of PMA inducibility of uPA mRNA, but did show high constitutive levels of the mRNA for CL and/or PMA inducibility of CL mRNA expression . Based on their differences in PMA inducibility these two phenotypes are designated rasuPA+/CL- and rasCL+/uPA-, respectively . Run-on assays indicated the differences in the levels of CL and uPA mRNA with ras transformation and phorbol ester induction are due to changes in transcription rates . Based on the observation of the two ras-transformed phenotypes for protease expressions, we asked whether uPA and CL can substitute for each other in the promotion of experimental metastasis . Injection of in vitro antisense inhibited cells in nude mice showed an inhibition of lung colonization by anti-uPA only in the rasuPA+/CL- phenotype and by anti-CL only in the rasCL+/uPA- phenotype . The data thus show the existence of two distinct activated ras-transformed metastatic phenotypes induced in the same parental cell line and that uPA or CL protease expressions alternatively facilitate the metastasis of cells with one ras phenotype and not with the other.

Biochem Cell Biol, 1992 Dec, 70(12), 1356 - 67
hsp80 of Neurospora crassa: cDNA cloning, gene mapping, and studies of mRNA accumulation under stress; Roychowdhury HS et al.; Using mRNA isolated from Neurospora crassa mycelium, grown for 14 h at normal growth temperature of 28 degrees C, and heat shocked for 1 h at 48 degrees C, a cDNA library was prepared in the expression vector lambda gt11 . Following immunoscreening of this library with a polyclonal antiserum raised against a 80-kilodalton heat-shock protein (HSP80), cDNA clones containing 1.1- and 1.4-kilobase inserts were selected . Analysis of the partial nucleotide sequence and the deduced amino acid sequence of the cDNA clones revealed a remarkable extent of homology with other eukaryotic stress-90 family proteins; 85% identity of the amino acid sequence with that of yeast HSP90(82) was seen . The C-terminal end of the sequence contained the MEEVD motif, characteristic of eukaryotic stress proteins with a predominantly cytosolic localization . The gene for N . crassa HSP80 was mapped to the right arm of linkage group V, using restriction fragment length polymorphism mapping . Its expression during heat shock and recovery was monitored by probing Northern blots of RNA isolated from mycelium grown under various stress conditions.

J Cell Biol, 1992 Dec, 119(5), 1069 - 76
Long-term sensitization training in Aplysia leads to an increase in the expression of BiP, the major protein chaperon of the ER; Kuhl D et al.; Long-term memory for sensitization of the gill- and siphon-withdrawal reflexes in Aplysia californica requires RNA and protein synthesis . These long-term behavioral changes are accompanied by long-term facilitation of the synaptic connections between the gill and siphon sensory and motor neurons, which are similarly dependent on transcription and translation . In addition to showing an increase in over-all protein synthesis, long-term facilitation is associated with changes in the expression of specific early, intermediate, and late proteins, and with the growth of new synaptic connections between the sensory and motor neurons of the reflex . We previously focused on early proteins and have identified four proteins as members of the immunoglobulin family of cell adhesion molecules related to NCAM and fasciclin II . We have now cloned the cDNA corresponding to one of the late proteins, and identified it as the Aplysia homolog of BiP, an ER resident protein involved in the folding and assembly of secretory and membrane proteins . Behavioral training increases the steady-state level of BiP mRNA in the sensory neurons . The increase in the synthesis of BiP protein is first detected 3 h after the onset of facilitation, when the increase in overall protein synthesis reaches its peak and the formation of new synaptic terminals becomes apparent . These findings suggest that the chaperon function of BiP might serve to fold proteins and assemble protein complexes necessary for the structural changes characteristic of long-term memory.

Farmaco, 1992 Dec, 47(12), 1495 - 511
4H-thieno{3,4-c}pyrazole derivatives with antiinflammatory, analgesic, antipyretic and platelet antiaggregating activities; Menozzi G et al.; The synthesis of a series of 1-aryl-1,6-dihydro-4H-thieno{3,4-c}pyrazol-4-ones by cyclization of 3-{(2-arylhydrazino)methylene}thiophene-2,4(3H,5H)-diones, prepared by reacting 3-dimethylaminomethylenethiophene-2,4(3H,5H)-dione with arylhydrazines, is described . The 4-fluorophenyl derivative showed remarkable analgesic, antiinflammatory and antipyretic activities in mice or rats, as well as a platelet antiaggregating activity in vitro comparable to that of acetylsalicylic acid.

Mech Dev, 1992 Dec, 39(3), 129 - 42
The ubiquitous transactivator Zfp-38 is upregulated during spermatogenesis with differential transcription; Chowdhury K et al.; We describe the complete nucleotide sequence of a full length cDNA clone encoding a new mouse zinc finger protein gene, Zfp-38 and localize it on chromosome 5 by the interspecific backcross analysis . The N-terminal domain of the Zfp-38 protein (64 kDa) contains 358 amino acids and the C-terminal domain of 197 residues encodes 7 zinc fingers . We also present evidence that Zfp-38 is a strong transcriptional activator . The transactivation domain was localized in the non finger region and a fusion protein containing 112 amino acid residues from this region of the Zfp-38 and the DNA binding domain of the yeast Gal 4 protein, very efficiently transactivated the expression of a reporter CAT plasmid, harboring the Gal4 target site . By in situ hybridization and northern blotting technique, the Zfp-38 transcript can be detected at a highly elevated level during spermatogenesis . Its expression accompanies the progression from pachytene spermatocytes to round spermatids . The undifferentiated spermatogonia or the haploid elongated spermatid and the spermatozoa do not show any detectable level of the transcript . Interestingly, other tissues express low levels of a slightly shorter transcript with a different 5' end as determined by RNase protection . The presence of both a transcriptional activating domain and 7 DNA binding zinc fingers, coupled with the cell type(s) specific expression pattern, suggests that Zfp-38 has the potential to regulate transcription during spermatogenesis.

Plant Cell, 1992 Dec, 4(12), 1575 - 88
Expression of antisense or sense RNA of an ankyrin repeat-containing gene blocks chloroplast differentiation in arabidopsis; Zhang H et al.; The Arabidopsis AKR gene that encodes a protein with four ankyrin repeats (a 33-amino acid motif that appears in the 89K domain of the human protein ankyrin) was isolated and characterized . A short sequence outside the ankyrin repeats is similar to that of the protein of the Drosophila muscle segment homeobox (msh) gene . The expression of the AKR gene is light dependent, and transgenic Arabidopsis plants with two or more copies of an antisense or sense AKR construct became chlorotic in a developmentally regulated manner . The chlorotic phenotype was genetically transmitted to the next generation, although most chlorotic plants produced much less seed . Reduced presence of thylakoid membranes and loss of grana are found in the plastids of chlorotic leaves, indicating that antisense or sense AKR has blocked chloroplast differentiation . This study indicates the importance of ankyrin repeat-containing proteins, not only in yeast and animals, but in plants as well.

Biochemistry, 1992 Nov 24, 31(46), 11524 - 35
Effect of Asp-235-->Asn substitution on the absorption spectrum and hydrogen peroxide reactivity of cytochrome c peroxidase; Vitello LB et al.; The spectroscopic properties of a mutant cytochrome c peroxidase, in which Asp-235 has been replaced by an asparagine residue, were examined in both nitrate and phosphate buffers between pH 4 and 10.5 . The spin state of the enzyme is pH dependent, and four distinct spectroscopic species are observed in each buffer system: a predominantly high-spin Fe(III) species at pH 4, two distinct low-spin forms between pH 5 and 9, and the denatured enzyme above pH 9.3 . The spectrum of the mutant enzyme at pH 4 is dependent upon specific ion effects . Increasing the pH above 5 converts the mutant enzyme to a predominantly low-spin hydroxy complex . Subsequent conversion to a second low-spin form is essentially complete at pH 7.5 . The second low-spin form has the distal histidine, His-52, coordinated to the heme iron . To evaluate the effect of the changes in coordination state upon the reactivity of the enzyme, the reaction between hydrogen peroxide and the mutant enzyme was also examined as a function of pH . The reaction of CcP(MI,D235N) with peroxide is biphasic . At pH 6, the rapid phase of the reaction can be attributed to the bimolecular reaction between hydrogen peroxide and the hydroxy-ligated form of the mutant enzyme . Despite the hexacoordination of the heme iron in this form, the bimolecular rate constant is approximately 22% that of pentacoordinate wild-type yeast cytochrome c peroxidase . The bimolecular reaction of the mutant enzyme with peroxide exhibits the same pH dependence in nitrate-containing buffers that has been described for the wild-type enzyme, indicating a loss of reactivity with the protonation of a group with an apparent pKa of 5.4 . This observation eliminates Asp-235 as the source for this heme-linked ionization and strengthens the hypothesis that the pKa of 5.4 is associated with His-52 . The slower phase of the reaction between peroxide and the mutant enzyme saturates at high peroxide concentration and is attributed to conversion of unreactive to reactive forms of the enzyme . The fraction of enzyme which reacts via the slow phase is dependent upon both pH and specific ion effects.

Science, 1992 Nov 20, 258(5086), 1353 - 5
Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis; Arondel V et al.; A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition . Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library . A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation . Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase.

J Mol Biol, 1992 Nov 20, 228(2), 421 - 32
Structure of the pericentric long arm region of the human Y chromosome; Cooper KF et al.; We have analysed the sequence organization of the DNA in the pericentric region of the long arm of the human Y chromosome . The structures of one cosmid and three yeast artificial chromosome clones were determined . The region consists of a mosaic of the known 5, 48 and 68 base-pair tandemly repeated sequences and at least five novel repeated sequence families . A long range-map of approximately 3.5 x 10(6) base-pairs of genomic DNA was constructed that placed the clones between about 500 x 10(3) and 850 x 10(3) base-pairs from the long arm edge of the centromeric alphoid DNA array.

J Mol Biol, 1992 Nov 20, 228(2), 327 - 37
Chromatin reconstitution on small DNA rings . V . DNA thermal flexibility of single nucleosomes; Hamiche A et al.; The thermal flexibility of DNA minicircles reconstituted with single nucleosomes was measured relative to the naked minicircles . The measurement used a new method based on the electrophoretic properties of these molecules, whose mobility strongly depended on the DNA writhe, either of the whole minicircle, when naked, or of the extranucleosomal loop, when reconstituted . The experiment was as follows . The DNA length was first increased by one base-pair (bp), and the correlative shift in mobility resulting from the altered DNA writhe was recorded . Second, the gel temperature was increased so that the former mobility was restored . Under these conditions, the untwisting of the thermally flexible DNA due to the temperature shift exactly compensates for the increase in the DNA mean twist number resulting from the one bp addition . The relative thermal flexibility was then calculated as the ratio between the increases in temperature measured for the naked and the reconstituted DNAs, respectively . The figure, 0.69 (+/- 0.07), was used to derive the length of DNA in interaction with the histones, 109 (+/- 25) bp . Such length was in good agreement with the mean value of 115 bp we have previously obtained from the distribution of the angles between DNAs at the entrance and exit of similar nucleosomes measured from high resolution electron microscopy . This consistency further reinforces our previous conclusion that minicircle-reconstituted nucleosomes, with 1.3(109/83) to 1.4(115/83) turns of superhelical DNA, show no crossing of entering and exiting DNAs when the loop is in its most probable configuration, and therefore, that these nucleosomes behave topologically as "single-turn" particles . The present data are also within the range of values, 50 to 100 bp of thermally rigid DNA per nucleosome, obtained by others for yeast plasmid chromatin, suggesting that the "single-turn" particle notion may be extended to this particular case of naturally-occurring H1-free chromatin . However, these data are quite different from the 230 bp figure derived from thermal measurements of reconstituted H1-free minichromosomes . It is proposed that nucleosome interactions occurring in this chromatin, but not in yeast chromatin, may be partly responsible for the discrepancy.

Biochim Biophys Acta, 1992 Nov 20, 1160(2), 213 - 20
Plant cytosolic pyruvate kinase: a kinetic study; Podesta FE et al.; The kinetic properties of cytosolic pyruvate kinase (PKc) from germinating castor oil seeds (COS) have been investigated . From experiments in which the free Mg2+ concentration was varied at constant levels of either the complexed or free forms of the substrates it was determined that the true substrates are the free forms of both phosphoenolpyruvate (PEP) and ADP . This conclusion is corroborated by the quenching of intrinsic PKC tryptophan fluorescence by free PEP and ADP . Mg2+ is bound as the free bivalent cation but is likely released as MgATP . The fluorescence data, substrate interaction kinetics, and pattern of inhibition by products and substrate analogues (adenosine 5'-O-(2-thiodiphosphate) for ADP and phenyl phosphate for PEP) are compatible with a sequential, compulsory-ordered, Tri-Bi type kinetic reaction mechanism . PEP is the leading substrate, and pyruvate the last product to abandon the enzyme . The dissociation constant and limiting Km for free PEP (8.2 to 22 and 38 microM, respectively) and the limiting Km for free ADP (2.9 microM) are considerably lower than those reported for the non-plant enzyme . The results indicate that COS PKc exists naturally in an activated state, similar to the fructose 1,6-bisphosphate-activated yeast enzyme . This deduction is consistent with a previous study (F.E . Podesta and W.C . Plaxton (1991) Biochem . J . 279, 495-501) that failed to identify any allosteric activators for the COS PKc, but which proposed a regulatory mechanism based upon ATP levels and pH-dependent alterations in the enzyme's response to various metabolite inhibitors . As plant phosphofructokinases display potent inhibition by PEP, the overall rate of glycolytic flux from hexose 6-phosphate to pyruvate in the plant cytosol will ultimately depend upon variations in PEP levels brought about by the regulation of PKc.

Nature, 1992 Nov 19, 360(6401), 270 - 3
Exocytotic fusion is activated by Rab3a peptides; Oberhauser AF et al.; Studies of intracellular traffic in yeast and mammalian systems have implicated members of the Rab family of small GTP-binding proteins as regulators of membrane fusion . We have used the patch clamp technique to measure exocytotic fusion events directly and investigate the role of GTP-binding proteins in regulating exocytosis in mast cells . Intracellular perfusion of mast cells with GTP-gamma S is sufficient to trigger complete exocytotic degranulation in the absence of other intracellular messengers . Here we show that GTP is a potent inhibitor of GTP-gamma S-induced degranulation, indicating that sustained activation of a GTP-binding protein is sufficient for membrane fusion . We have found that synthetic oligopeptides, corresponding to part of the effector domain of Rab3a, stimulate complete exocytotic degranulation, similar to that induced by GTP-gamma S . The response is selective for Rab3a sequence and is strictly dependent on Mg2+ and ATP . This suggests that sustained activation of a Rab3 protein causes exocytotic fusion . The peptide response can be accelerated by GDP-beta S, suggesting that Rab3a peptides compete with endogenous Rab3 proteins for a binding site on a target effector protein, which causes fusion on activation.

Biochemistry, 1992 Nov 17, 31(45), 10969 - 75
Evidence that a minor groove-binding peptide and a major groove-binding protein can simultaneously occupy a common site on DNA; Oakley MG et al.; Affinity cleaving proteins have been synthesized based on the DNA-binding domain of the yeast transcriptional activator GCN4 with the DNA cleaving moiety Fe.EDTA attached at the NH2 terminus {Oakley, M . G., & Dervan, P . B . (1990) Science 248, 847} . Cleavage patterns generated by Fe-EDTA-GCN4(226-281) bound to the DNA sites 5'-CTGACTAAT-3' and 5'-ATGACTCTT-3' reveal that the NH2 termini of the GCN4 DNA-binding domain are located in the major groove of DNA, 9-10 base pairs apart, consistent with a Y-shaped dimeric structure . 1-Methylimidazole-2-carboxamide netropsin (2-ImN) is a designed synthetic peptide which binds in the minor groove of DNA at 5'-TGACT-3' sites as an antiparallel, side-by-side dimer {Mrksich, M., Wade, W . S., Dwyer, T . J., Geierstanger, B . H., Wemmer, D.E., & Dervan, P . B . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 7586} . Through the use of Fe.EDTA-GCN4(226-281) as a sequence-specific footprinting agent, it is shown that the dimeric protein GCN4-(226-281) and the dimeric peptide 2-ImN can simultaneously occupy their common binding site in the major and minor grooves of DNA, respectively . The association constants for 2-ImN in the presence and in the absence of Fe.EDTA-GCN4(226-281) are found to be similar, suggesting that the binding of the two dimers is not cooperative.

Biochem Biophys Res Commun, 1992 Nov 16, 188(3), 982 - 91
Analysis of endogenous and exogenous nuclear translocation of fibroblast growth factor-1 in NIH 3T3 cells; Zhan X et al.; Nuclear localization of fibroblast growth factors (FGF) have been reported by many laboratories . We demonstrate here that FGF-1, the precursor for acidic FGF contains a putative nuclear translocation sequence (NTS) NYKKPKL, which is able to direct the expression of the bacterial beta galactosidase (beta gal) gene to the nucleus of transfected NIH 3T3 cells . However, this NTS is unable to target either FGF-1 itself or a FGF-1-beta gal fusion protein into the nucleus, suggesting that FGF-1 may contain an additional sequence which prevents endogenously expressed FGF-1 from being translocated into the nucleus . Indeed, when FGF-1 was fused to the NTS derived from the yeast histone 2B gene, the chimeric construct also failed to be transported into the nucleus either by itself or as a beta gal fusion protein . Interestingly, when 125I-FGF-1 was used to stimulate quiescent NIH 3T3 cells, a significant amount of internalized 125I-FGF-1 (approximately 10%) was found within the nucleus and the nuclear localization of FGF-1 through the exogenous pathway could be significantly reduced by suramin, an inhibitor of the interaction of FGF-1 with its receptor . These data suggest that while FGF-1 contains a NTS, nuclear translocation requires an exogenous and not an endogenous pathway.

J Biol Chem, 1992 Nov 15, 267(32), 23165 - 9
Sequence of the fifth largest subunit of RNA polymerase II from plants; Ulmasov T et al.; An affinity-purified antibody raised against the fifth largest subunit of cauliflower (Brassica oleracea) RNA polymerase II was used to screen an expression library and isolate an Arabidopsis thaliana cDNA clone . This cDNA clone was used to isolate a soybean (Glycine max) cDNA clone, and both clones were sequenced . The open reading frames contain 176 amino acids and predict polypeptides of 19.5 and 19.6 kDa for Arabidopsis and soybean, respectively . The amino acid sequences of the Arabidopsis and soybean polypeptides are 91.5% identical . The fifth largest subunit in plant RNA polymerase II is present at unit stoichiometry in purified enzyme and does not dissociate from the holoenzyme during nondenaturing polyacrylamide gel electrophoresis . The gene encoding the 19.5-kDa subunit has been isolated and sequenced from Arabidopsis . The gene is single copy and contains five introns . The size of the mRNA encoding this RNA polymerase II subunit in Arabidopsis and soybean is approximately 1 kilobase . None of the published yeast or animal RNA polymerase subunit sequences show similarity to the fifth largest subunit in plants.

Biochim Biophys Acta, 1992 Nov 15, 1171(1), 88 - 92
Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana; Reddy AS et al.; We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein . The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein . Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology . The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein . The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids . Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested . Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

Proc Natl Acad Sci U S A, 1992 Nov 15, 89(22), 10867 - 71
Neuronal cdc2-like kinase: a cdc2-related protein kinase with predominantly neuronal expression; Hellmich MR et al.; Recent studies have shown that there exists a family of protein kinases structurally and functionally related to the yeast cell cycle regulatory kinase cdc2 {Meyerson, M., Faha, B., Su, L.-K., Harlow, E . & Tsai, L.-H . (1991) Cold Spring Harbor Symp . Quant . Biol . 56, 177-186 and Meyerson, M., Enders, G . H., Wu, C.-L., Su, L.-K., Gorka, C., Nelson, C., Harlow, E . & Tsai, L.-H . (1992) EMBO J . 11, 2909-2917} . Two members of cdc2 family, p34cdc2 (also named cdk1) and cdk2, have been identified in mammalian cells . cdk1 kinase regulates the progression from G2 to M phase, and cdk2 kinase has been proposed to regulate the progression from G1 to S phase . In this work, we have cloned and structurally characterized a third member of the cdc2 kinase family with 58% amino acid sequence identity to mouse cdk1 and 61% identity to human cdk2 . We call this kinase neuronal cdc2-like kinase (nclk) because, in contrast to either cdk1 or cdk2, nclk is expressed at high levels in terminally differentiated neurons no longer in the cell cycle . Previous studies have shown {Hisanaga, S., Kusubata, M., Okumura, E . & Kishimoto, T . (1991) J . Biol . Chem . 266, 21798-21803 and Guan, R . J., Hall, F . L . & Cohlberg, J . A . (1992) J . Neurochem . 58, 1365-1371} that cdk1 kinase, but not other structurally defined protein kinases, could phosphorylate the repeated Lys-Ser-Pro (KSP) motifs found in mammalian high and middle molecular mass neurofilament subunits in vitro, but the precise molecular nature of the endogenous neuronal KSP kinase has remained undefined . The structural similarity of nclk to cdk1 kinase and its high level of expression in terminally differentiated neurons suggest that nclk may play a role in the phosphorylation of the neurofilament KSP repeats in vivo, a function distinct from cell cycle regulation.

Cell, 1992 Nov 13, 71(4), 691 - 700
Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias; Tkachuk DC et al.; We have identified a human homolog of the Drosophila trithorax protein that is structurally altered by 11q23 translocations in acute leukemias . Human trithorax (HRX) is a predicted 431 kd protein containing two potential DNA-binding motifs consisting of zinc fingers conserved with the fly protein and nonconserved amino-terminal "AT hook" motifs related to the DNA-binding motifs in HMG proteins . 11q23 translocations disrupt the HRX gene between these two motifs, and in a t(11;19)-carrying cell line fusion transcripts are expressed from both derivative chromosomes . The more abundant derivative 11 transcript codes for a chimeric protein containing the AT hook motifs fused to a previously undescribed protein (ENL) from chromosome 19 . These data suggest a novel role for a trithorax-homologous protein in multilineage human leukemias that may be mediated by DNA binding within the minor groove at AT-rich sites, implicated to play an important role in bacterial IHF-, yeast datin-, and mammalian HMG-mediated gene activation.

J Pharmacol Exp Ther, 1992 Nov, 263(2), 533 - 9
Effects of in vivo and in vitro administration of morphine sulfate upon rhesus macaque polymorphonuclear cell phagocytosis and chemotaxis; Liu Y et al.; The effects of morphine administration were evaluated upon the polymorphonuclear (PMN) cell activities of rhesus macaques . Sixteen animals were used in the study . Seven macaques were treated with saline and nine animals were administered morphine in the form of morphine sulfate, following a control period of saline injections . The dosage regime was initiated at 1 mg of morphine sulfate per kg of b.wt., 3 times daily, with incremental steps to a stable dose of 5 mg/kg, 3 times daily . PMN cells prepared from morphine-treated animals showed a marked, transient reduction in their ability to kill ingested yeast blastospores, although the ingestive capacity of the PMN cells was not impaired . PMN cells prepared from three of the nine morphine-treated animals showed a transient reduction in chemotaxis toward a complement-derived chemotactic factor . In vitro studies on PMN cells prepared from morphine-naive animals suggested that the killing activity and the chemotaxis of the cells were reduced by treatment with 50 pM morphine sulfate, but not with 50 fmol of morphine sulfate.

J Biol Chem, 1992 Nov 5, 267(31), 22054 - 9
Identification of actin and HSP 70 as cyclosporin A binding proteins by photoaffinity labeling and fluorescence displacement assays; Moss ML et al.; A novel family of cyclosporin A (CsA) binding proteins was identified by using the biologically active, radioiodinated photoaffinity probe {D-Lys-N epsilon-(4-azido-3-{125I}iodophenyl)propionyl)}8-CsA . In addition to cyclophilin, proteins with molecular masses of 43 kDa and approximately 50-55 kDa were labeled in Jurkat extracts and bovine calf thymus . Sequence analysis of the 43-kDa protein purified from calf thymus and subsequent Western analysis of CsA affinity-purified material from Jurkat extracts identified the 43-kDa component as actin . {D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)}8-CsA, a fluorescent analogue of CsA, was prepared and used to measure the binding constants of cyclosporin derivatives to actin by means of a new fluorescence displacement assay . {D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)}8-CsA and {N delta-t-butoxycarbonyl diaminobutyryl)}8-CsA bind to bovine actin at physiologically relevant concentrations, with dissociation constants of 60 +/- 33 and 570 +/- 380 nM, respectively . Because the ATPase fragment of heat shock cognate 70 (HSC 70) is structurally related to actin, the yeast homologue SSA1 was tested and found to be radiolabeled by the cyclosporin A photoaffinity reagent . The binding constant for {D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)}8-CsA to SSA1 was determined and is 53 +/- 48 nM . These results indicate that actin and the 70-kDa heat shock protein family contain a structurally related domain for binding of cyclosporin A-related peptides.

Prikl Biokhim Mikrobiol, 1992 Nov-Dec, 28(6), 889 - 93
{Penicillium-species fungi--producers of ochratoxin A in grain}; L'vova LS et al.; The occurrence of ochratoxin A and ochratoxigenic fungi in the commercial batches of various domestic grains (wheat, rye, barley, corn and rice) has been studied . Penicillium cyclopium, P . viridicatum and P . chrysogenum isolated from grain synthesized on a sucrose-yeast medium predominantly patulin, penicillic and kojic acids . Only 4.4% of the fungal isolates were able to synthesize ochratoxin A . The concentration of the mycotoxin accumulated by the fungi was less than 500 micrograms/kg . 230 samples of wheat and 502 samples of corn were examined . The analysis showed that ochratoxin A was present in 0.9% and 0.1% of samples tested, respectively . The mycotoxin accumulated in grain mainly during its spontaneous heating and was concentrated in mold-damaged kernels.

Prenat Diagn, 1992 Nov, 12(11), 931 - 43
Analysis of chromosome 21 copy number in uncultured amniocytes by fluorescence in situ hybridization using a cosmid contig; Zheng YL et al.; A comparison of the use of chromosome 21-specific libraries, DOP-PCR 21 paints, yeast artificial chromosome (YAC) clones, single cosmids, and a 21q cosmid contig as probes for the detection of the copy number of chromosome 21 in interphase cells by fluorescence in situ hybridization shows that the cosmid contig is a satisfactory probe for interphase analysis of chromosome 21 . The contig cCMP21.a, which is 55 kb in length, is highly chromosome 21-specific and produces intense, compact signals in a high proportion of interphase cells . A retrospective blind analysis of coded uncultured amniotic fluid samples correctly detected four trisomy 21 cases out of 49 samples.

Mol Microbiol, 1992 Nov, 6(22), 3365 - 73
The beta-tubulin gene from rat and human isolates of Pneumocystis carinii; Edlind TD et al.; The development of new drugs for treating Pneumocystis carinii infections in AIDS patients is hampered by the lack of long-term culture systems, and by our generally limited knowledge of this organism . Recently, however, we observed significant activity of various benzimidazoles against growth of this organism in short-term cultures . Benzimidazoles inhibit microtubule polymerization; there is strong evidence that the primary target is the beta-tubulin subunit . To understand the basis for benzimidazole activity against P . carinii, and to examine the apparent relatedness of this organism to fungi, we have cloned and sequenced the single beta-tubulin gene from a rat P . carinii isolate . There was 89-91% identity at the amino acid level to beta-tubulins from filamentous fungi, but only 79-82% identity to yeast and protozoal beta-tubulins . Also, eight introns were distributed throughout the P . carinii beta-tubulin gene in a pattern characteristic of filamentous fungi . Specific residues previously implicated in benzimidazole sensitivity were conserved in P . carinii beta-tubulin . The polymerase chain reaction was used to amplify a segment of P . carinii beta-tubulin DNA from bronchoalveolar lavages obtained from two patients with AIDS . There was considerable divergence at the DNA level between the human and rat sequences, but 100% identity at the amino-acid level.

Chem Pharm Bull (Tokyo), 1992 Nov, 40(11), 2954 - 7
Syntheses of cerulenin and its analogs . II . Synthesis and biological activity of dl-carbacerulenin, a carbocyclic analog of cerulenin; Shimazawa R et al.; 2,3-Epoxy-4-hydroxy-4-((E,E)-3,6-octadienyl)cyclopentanone (dl-carbacerulenin 5) was synthesized via the epoxyketones 15a and 15b as a mimic of the active form of the antibiotics cerulenin 1, a potent inhibitor of fatty acid synthetase (FAS) . The monobenzyl ethers (12 and 13), synthetic intermediates of 15, were prepared by direct benzylation of the epoxycyclopentene (7) . Inhibitory activity of synthesized 5 toward yeast FAS was less than that of cerulenin by a factor of 1000.

Comp Biochem Physiol B, 1992 Nov, 103(3), 749 - 53
Purification and characterization of a humoral opsonin from the solitary urochordate Styela clava; Kelly KL et al.; 1 . We have previously identified opsonic activity in the plasma of the solitary urochordate, Styela clava . 2 . Here, we report the purification and further characterization of the opsonic molecule . 3 . Two purification methods were employed . 4 . Gel filtration yielded one strongly opsonic fraction that contained a single, electrophoretically-resolved protein . 5 . Opsonic activity was dose-dependent and sensitive to tryptic digestion and heat denaturation . 6 . SDS-PAGE and calibrated gel filtration indicated the opsonic protein was a 17.5 kDa monomer while isoelectrofocusing indicated a single pI of 7.0 . 7 . In an alternative procedure, a similar opsonic activity and protein were isolated by affinity purification using whole yeast cells.

Vis Neurosci, 1992 Nov, 9(5), 461 - 9
Electrophysiological sensitivity of carotenoid deficient and replaced Drosophila; Chen DM et al.; R1-6 dominated electroretinographic (ERG) spectral sensitivities were determined as a function of days posteclosion from carotenoid deprived and replaced white-eyed Drosophila . The sensitivity of flies deprived from egg to adult waxed (about 1.5 log units by day 3), and then waned gradually from 3-11 days (over 2 log units by day 11) . Carotenoid replacement (feeding nothing but carrot juice) effected recovery to near the replete controls' level in about 1 day throughout (tested at 0, 4, and 11 days) . The normal yellow cornmeal-agar-molasses-brewers yeast fly food (in our laboratory, supplemented with beta-carotene) renders a slower recovery (requiring 7-9 days) since it is a medium designed largely for larval growth . Placing replete adults on deprivational medium did not create a deprivational syndrome in over 11 days . At 3-7 days, deprived flies reared and maintained in constant darkness had substantially enhanced sensitivity, beyond the 1.5 log unit increment already described for cyclic light rearing conditions . All spectral analyses are consistent with the ultraviolet (UV) sensitization of the blue (480 nm) rhodopsin by a replacement-dependent retinoid including two unexpected findings: (1) sensitivity recovery with carrot juice was so fast that the UV peak was already high at 6 h; and (2) the waxing of the deprived fly's sensitivity in dark rearing was so great that the UV peak was present at 4-7 days.

J Am Vet Med Assoc, 1992 Nov 1, 201(9), 1375 - 7
In vitro susceptibility to antimycotics of Microsporum canis isolates from cats; Puccini S et al.; One hundred thirty-four isolates of Microsporum canis, obtained from cats, were tested for in vitro susceptibility to various antifungal agents . The fungi were classified as susceptible, resistant, and intermediate by measuring the size of the zone of inhibited growth on yeast nitrogen base agar medium . Clotrimazole had the highest activity (99.2%), followed by tioconazole (89.6%), griseofulvin (88.8%), econazole (73.1%), ketoconazole (50.7%), miconazole (15.7%), and isoconazole (12.7%) . We found 14.9% of the isolates to be susceptible to all the assayed drugs, whereas the highest resistance frequency (41.8%) was against 2 antimycotics . A simultaneous resistance to all the tested antimycotics was not found . The differences among the antifungal drugs activity were examined, and administration of drugs for which a simultaneous resistance was minimal is suggested.

Genomics, 1992 Nov, 14(3), 769 - 74
Construction and characterization of a region-specific microdissection library from human chromosome 2q35-q37; Yu J et al.; A region-specific genomic library for human chromosome 2q35-q37 has been constructed using the microdissection and polymerase chain reaction-mediated linker-adaptor microcloning method . Twenty fragments from the chromosome region 2q35-q37 were dissected and a library consisting of 20,000 recombinant microclones was obtained . The insert size ranged between 50 and 800 bp, with a mean of approximately 270 bp . About 50-60% of the microclones contained unique sequences . The microdissection library has been demonstrated to derive from the dissected region 2q35-q37 by chromosome painting using the fluorescence in situ hybridization (FISH) technique . Southern blot analysis of the unique sequence microclones from the library showed that 54% (26/48) of the clones are of human origin and chromosome 2 specific . Four of these microclones have been further mapped to the 2q37 region by using a cell hybrid containing only 2q37 . The unique sequence microclones have also been characterized for their insert size and the hybridizing genomic fragments cleaved with HindIII . As shown previously, these microclones will be useful in isolating corresponding yeast artificial chromosome (YAC) clones with large inserts for high-resolution physical mapping and also in screening cDNA libraries to isolate expressed gene sequences as candidate genes to facilitate search for the crucial genes underlying genetic diseases and specific forms of cancer assigned to the region.

Blood, 1992 Nov 1, 80(9), 2172 - 5
Breakpoints at 11q23 in infant leukemias with the t(11;19)(q23;p13) are clustered; Morgan GJ et al.; We have analyzed a series of nine infant leukemias that carry a t(11;19)(q23;p13) . They had the morphologic features of acute lymphoblastic leukemia (ALL) and expressed markers typical of B-cell progenitor ALL or pre-B ALL; one coexpressed myeloid markers in addition to lymphoid markers (biphenotypic) . Two probes (P/S4 and 98.40) subcloned from a yeast artificial chromosome (YAC) known to span the breakpoint in the t(4;11) were used to investigate DNA isolated from the leukemic cells of these patients . A total of approximately 15 kb of genomic DNA in the vicinity of the probes was examined by conventional Southern blot analysis using a series of restriction enzymes . In eight of the nine cases, the breakpoint could be mapped to an approximately 10-kb BamHI fragment disclosed by hybridization to the P/S4 probe.

Virology, 1992 Nov, 191(1), 98 - 105
The nucleotide sequence of apple stem grooving capillovirus genome; Yoshikawa N et al.; The complete nucleotide sequence of apple stem grooving virus (ASGV) genome has been determined . The genome is 6496 nucleotides in length excluding a 3'-terminal poly(A) tail and contains two overlapping open reading frames (ORFs) . ORF1 begins at nucleotide position 37 and is terminated at position 6341, encoding a protein with a molecular weight of 241 kDa . ORF2, which is in a different reading frame within ORF1, begins at position 4788 and can encode a 36-kDa protein . The 241-kDa protein contains two consensus sequences associated with the RNA-dependent RNA polymerase and the NTP-binding helicase . Comparisons of amino acid sequences around these conserved motifs with other RNA viruses revealed that ASGV has extensive similarities with apple chlorotic leaf spot, tymo-, carla-, and potexviruses, and is a member of the sindbis-like supergroup . ASGV coat protein is found to be located in the C-terminal region of the 241-kDa polyprotein . The 36-kDa protein encoded by ORF2 contains the consensus sequence Gly-Asp-Ser-Gly found in the active site of several cellular and viral serine proteases.

Virology, 1992 Nov, 191(1), 506 - 10
Nucleotide sequence changes in the polymerase basic protein 2 gene of temperature-sensitive mutants of influenza A virus; Lawson CM et al.; Influenza A viruses bearing temperature-sensitive (ts) mutations are restricted in replication in the respiratory tract of animals and humans and are therefore attenuated . Nucleotide sequences were determined for the RNA segment coding for the polymerase basic protein 2 (PB2) from a panel of 12 influenza A/Udorn/307/72 (H3N2) ts viruses, previously characterized to have a ts mutation in the PB2 gene . Each of the viruses with a ts mutation in the PB2 gene had a single amino acid change located at position 65, 100, 112, 174, 298, 310, 386, 391, 556, or 658 of the PB2 protein . The sites of the single mutations were scattered throughout the length of the protein and occurred in regions that are highly conserved among the influenza A virus PB2 predicted amino acid sequences . Interestingly, the substitution of aspartic acid for asparagine at position 556 was found to lie within a region that has homology with cap-binding motifs of human and yeast proteins . Taken together, the findings of lesion sites in the A/Udorn/307/72 PB2 gene and the three reported amino acid changes at positions 265, 417, and 512 for A/AA/6/60, A/WSN/33, and A/FPV/Ros/34 ts PB2 genes, respectively, indicate that the PB2 gene can sustain a viable ts mutation at different sites . This information will allow us to construct cloned cDNA copies of the A/Udorn/307/72 PB2 gene mutagenized at specific sites . Different configurations of two or more ts mutations may be incorporated into the cDNA PB2 gene constructs . We have a host-range reassortant virus that should permit rescue of in vitro-produced transcripts of the PB2 gene into infectious virus . The rescue of these mutated PB2 RNA segments into an infectious influenza A virus may lead to the development of live attenuated reassortant virus vaccines that are satisfactorily attenuated, genetically stable, and immunogenic in humans.

EMBO J, 1992 Nov, 11(11), 3995 - 4005
Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15; Gu Y et al.; We have examined the role of phosphorylation in the regulation of human cyclin-dependent kinase-2 (CDK2), a protein closely related to the cell cycle regulatory kinase CDC2 . We find that CDK2 from HeLa cells contains three major tryptic phosphopeptides . Analysis of site-directed mutant proteins, expressed by transient transfection of COS cells, demonstrates that the two major phosphorylation sites are Tyr15 (Y15) and Thr160 (T160) . Additional phosphorylation probably occurs on Thr14 (T14) . Replacement of T160 with alanine abolishes the kinase activity of CDK2, indicating that phosphorylation at this site (as in CDC2) is required for kinase activity . Mutation of Y15 and T14 stimulates kinase activity, demonstrating that phosphorylation at these sites (as in CDC2) is inhibitory . Similarly, CDK2 is activated in vitro by dephosphorylation of Y15 and T14 by the phosphatase CDC25 . Analysis of HeLa cells synchronized at various cell cycle stages indicates that CDK2 phosphorylation on T160 increases during S phase and G2, when CDK2 is most active . Phosphorylation on the inhibitory sites T14 and Y15 is also maximal during S phase and G2 . Thus, the activity of a subpopulation of CDK2 molecules is inhibited at a time in the cell cycle when overall CDK2 activity is increased.

Cancer Res, 1992 Nov 1, 52(21), 6083 - 7
The RCK gene associated with t(11;14) translocation is distinct from the MLL/ALL-1 gene with t(4;11) and t(11;19) translocations; Akao Y et al.; We previously demonstrated that the 11q23 breakpoint region, designated the RCK locus, of the RC-K8 B-lymphoma cell line with t(11;14)(q23;q32) is centromeric to PBGD, while breakpoints of infantile leukemia cell lines with t(11;19)(q23;p13) are detectable by pulsed-field gel electrophoresis with the CD3D probe . In the present study, using a probe within 1.0 kilobase of the t(11;14) breakpoint, we isolated a partial complementary DNA clone for the putative RCK gene, which detects a 7.5-kilobase mRNA . Sequence analysis predicted a novel protein of 472 amino acids which demonstrated sequence homology to a translation initiation factor/helicase family . We also isolated a phage clone from the CD3D/G yeast artificial chromosome clone (yB22B2) which detects 11- and 12-kilobase mRNAs, most likely for the MLL/ALL-1 gene associated t(4;11)(q21;q23) and t(11;19)(q23;p13) translocations . By pulsed-field gel electrophoresis after NotI digestion, this recombinant clone is on a 96-kilobase fragment, while RCK and PBGD probes are on a more telomeric 690-kilobase NotI fragment . These results, altogether, suggested that two different genes, RCK and MLL/ALL-1, are associated with 11q23 translocation of hematopoietic tumors.

J Biotechnol, 1992 Nov, 26(2-3), 183 - 201
Studies on the enzymatic reduction of N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester; Nassenstein A et al.; The enzymatic reduction of N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester, the key intermediate in the stereoselective synthesis of a statinanalogue, was studied with Hansenula anomala and Hansenula silvicola . Using whole cells of H . anomala gives complete conversion and a diastereomeric excess of 88% of the desired 3S, 4S statinanalogue . The strain contains two NADPH-dependent oxidoreductases, that can be separated by ion exchange chromatography or gelfiltration, yielding the 3S, 4S or 3R, 4S stereoisomers, respectively, with > 99% diastereomeric excess (DE) . In the crude extract the 3S, 4S oxidoreductase is very unstable and could be purified with << 1% yield only . In contrast, H . silvicola, which gave poor conversions using whole cells, exhibited about 80-fold higher specific activity in the crude extract than H . anomala . The NADPH-dependent oxidoreductase was purified 317-fold in 12% yield . A single enzyme of 54 kDa reduces the substrate with 97.4% DE . Besides the statinanalogue a wide range of other compounds could be reduced, most notably diones and chinones such as isatin or campherchinone . It was demonstrated that the enzymes often discussed for the reduction of beta-ketoesters with yeast e.g . L-3-hydroxyacyl CoA dehydrogenase (EC 1.1.1.35), the beta-ketoreductase of the fatty acid synthase complex and also the 3-hydroxy-3-methyl glutaryl-CoA dehydrogenase (EC 1.1.1.34) are separated during the purification steps from the oxidoreductase acting on N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester . The physiological role of the new enzyme is still unknown.

Biotechnol Prog, 1992 Nov-Dec, 8(6), 469 - 78
Recombinant human insulin; Ladisch MR et al.; Insulin is a well-characterized peptide that can be produced by recombinant DNA technology for human therapeutic use . A brief overview of insulin production from both traditional mammalian pancreatic extraction and recombinant bacterial and yeast systems is presented, and detection techniques, including electrophoresis, are reviewed . Analytical systems for insulin separation are principally based on reversed-phase chromatography, which resolves the deamidation product(s) (desamido insulin) of insulin, proinsulin, and insulin . Process-scale separation is a multistep process and includes ion exchange, reversed-phase, and size exclusion chromatography . Advantages and/or disadvantages of various separation approaches, as described by the numerous literature references on insulin purification, are presented.

Int J Immunopharmacol, 1992 Nov, 14(8), 1363 - 73
Stimulation of human monocyte beta-glucan receptors by glucan particles induces production of TNF-alpha and IL-1 beta; Abel G et al.; beta-glucans are pharmacologic agents that rapidly enhance host resistance to a variety of biologic insults through mechanisms involving macrophage activation . To determine whether stimulation of the beta-glucan receptors on human monocytes resulted in cytokine production, monolayers of monocytes were incubated with purified yeast glucan particles and measured for tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) mRNA and protein . By Northern blot analysis, TNF-alpha mRNA was detected within 30 min of incubation with glucan particles, peaked at 2 h, and remained elevated for at least 8 h . Glucan induction of IL-1 beta mRNA followed a similar time-course of initiation and accumulation . By enzyme-linked immunosorbent assays (ELISAs), significant levels of TNF-alpha and IL-1 beta were present in supernatants of glucan-treated cells within 1 h and plateau levels of both cytokines were approached within 4 h . At particle-to-cell ratios of from 0.4 to 18, glucan particles induced dose-dependent increases in TNF-alpha and IL-1 beta mRNA and corresponding increases in TNF-alpha and IL-1 beta proteins . Exposure of monocytes to glucan particles for 0-30 min and washing before continued incubation for 4 h in particle-free buffer induced production and secretion of TNF-alpha and IL-1 beta in a time-dependent fashion compatible with phagocytosis . The pretreatment of monocyte monolayers with trypsin reduced glucan-induced production of TNF-alpha and IL-1 beta in a dose-dependent manner with 5 micrograms/ml of trypsin effecting reductions of greater than 50% . Thus, glucan particles induce human monocyte production of TNF-alpha and IL-1 beta by a mechanism that is dependent on trypsin-sensitive beta-glucan receptors.

Free Radic Biol Med, 1992 Nov, 13(5), 557 - 80
Metabolism of oxygen radicals in peroxisomes and cellular implications; del Rio LA et al.; Peroxisomes are subcellular respiratory organelles which contain catalase and H2O2-producing flavin oxidases as basic enzymatic constituents . These organelles have an essentially oxidative type of metabolism and have the potential to carry out different important metabolic pathways . In recent years the presence of different types of superoxide dismutase (SOD) have been demonstrated in peroxisomes from several plant species, and more recently the occurrence of SOD has been extended to peroxisomes from human and transformed yeast cells . A copper,zinc-containing SOD from plant peroxisomes has been purified and partially characterized . The production of hydroxyl and superoxide radicals has been studied in peroxisomes . There are two sites of O2- production in peroxisomes: (1) in the matrix, the generating system being xanthine oxidase; and (2) in peroxisomal membranes, dependent on reduced nicotinamide adenine dinucleotide (NADH), and the electron transport components of the peroxisomal membrane are possibly responsible . The generation of oxygen radicals in peroxisomes could have important effects on cellular metabolism . Diverse cellular implications of oxyradical metabolism in peroxisomes are discussed in relation to phenomena such as cell injury, peroxisomal genetic diseases, peroxisome proliferation and oxidative stress, metal and salt stress, catabolism of nucleic acids, senescence, and plant pathogenic processes.

Mol Reprod Dev, 1992 Nov, 33(3), 287 - 96
Okadaic acid and p13suc1 modulate the reinitiation of meiosis in mouse oocytes; Gavin AC et al.; Short-term exposure to okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, induced resumption of meiosis, including metaphase spindle formation, in mouse oocytes treated with a phosphodiesterase inhibitor, while long incubations with OA arrested oocyte maturation at a step prior to spindle formation . To explore the basis for this difference, the overall patterns of protein synthesis and phosphorylation and the production of tissue-type plasminogen activator (tPA), the synthesis of which is induced after germinal vesicle breakdown (GVBD), were analyzed under various OA treatments . Short-term exposure to OA led to tPA production and did not greatly affect the maturation-associated changes in protein phosphorylation . By contrast, a long application of OA did not result in tPA production and induced more marked changes in protein phosphorylation . Microinjection into prophase oocytes of the product of the fission yeast gene p13suc1, known to inhibit p34cdc2 kinase activation and/or activity, prevented meiotic reinitiation . This effect was overcome by microinjection of OA, at concentrations higher than those required for induction of maturation in the absence of p13suc1 . These observations suggest that inhibition of phosphatase 1 or 2A or both triggers meiotic resumption by acting at the same site or at a site proximal to the p13suc1-sensitive step of cdc2 kinase activation.

Anal Biochem, 1992 Nov 1, 206(2), 328 - 33
An anion-exchange radioassay for glucose 6-phosphate phosphatase: use in topological studies with endoplasmic reticulum vesicles; Rush JS et al.; A simple procedure is presented for the enzymatic preparation of {2-3H}mannose 6-phosphate (Man 6-P) with purified yeast hexokinase and unlabeled ATP . The enzymatically synthesized {2-3H}Man 6-P is utilized as the radiolabeled substrate in a new rapid assay for glucose 6-phosphate (Glc 6-P) phosphatase . The principle of the assay procedure is that the unreacted substrate, {2-3H}Man 6-P, is retained by the anion-exchange resin, AG 1-X8 (acetate), while the enzymatic product, {2-3H}-mannose, is eluted directly into a scintillation counting vial . When Glc 6-P phosphatase activity associated with mouse liver endoplasmic reticulum (ER) vesicles is assayed by the new chromatographic assay, the same characteristic latency and properties are observed, as determined by the commonly used colorimetric assay of inorganic phosphate produced . The anion-exchange radioassay described should be useful for a variety of topological studies on enzymes associated with membrane vesicles derived from liver and kidney ER.

Plant Mol Biol, 1992 Nov, 20(3), 367 - 75
Molecular characterization of type 1 serine/threonine phosphatases from Brassica oleracea; Rundle SJ et al.; We describe the isolation of cDNA clones encoding type 1 serine/threonine protein phosphatase (PP1) from Brassica oleracea stigmas . We demonstrate that PP1 form a multigene family in Brassica . Within their most conserved domain, these phosphatases are 80-90% identical at the amino acid level . One cDNA (BoPP1) was found to encode a protein that shows 78-80% sequence identity to maize, rabbit, and yeast PP1 . The accumulation of BoPP1 mRNA is developmentally regulated . Varying levels of BoPP1-homologous transcripts were detected in leaves, cotyledons, pistils, anthers and roots . In addition, distinct species of BoPP1 transcripts accumulated at different stages of Brassica microspore development, and mature trinucleate microspores contained a unique BoPP1 mRNA species not found at other stages of the plant's life cycle . Lastly, we show by genomic Southern blots that the Brassica genome might contain homologues of the mammalian PP1 inhibitor-1.

Mycoses, 1992 Nov-Dec, 35(11-12), 317 - 20
Shorter treatment for vaginal candidosis: comparison between single-dose oral fluconazole and three-day treatment with local miconazole; Timonen H; Fluconazole is an effective, simple and safe, although slightly expensive, agent for the treatment of vaginal candidosis . Single-dose fluconazole (150 mg) administered orally in capsule form was compared with three-day local treatment with miconazole pessaries in the treatment of vaginal candidosis in a randomized study in Finland . Cure rates were good (> 80%) in randomized patient groups assessed both clinically and by the results of yeast cultures . Oral administration was preferred to local therapy by patients in both the miconazole and fluconazole groups . For the time being, fluconazole is not recommended for use during pregnancy or lactation.

Hum Mol Genet, 1992 Nov, 1(8), 579 - 85
A YAC contig in Xp21 containing the adrenal hypoplasia congenita and glycerol kinase deficiency genes; Walker AP et al.; The gene loci for adrenal hypoplasia congenita (AHC) and glycerol kinase deficiency (GK) map in Xp21 distal to Duchenne muscular dystrophy (DMD), and proximal to DXS28 (C7), by analysis of patient deletions . We have constructed a yeast artificial chromosome (YAC) contig encompassing a 1.2 Mb region extending distally from DMD, and containing DXS708 (JC-1), the distal junction clone of a patient with GK and DMD . A pulsed-field gel electrophoresis map of the YAC contig identified 3 potential CpG islands . Whole YAC hybridization identified cosmids both for construction of cosmid contigs, and isolation of single copy probes . Thirteen new single copy probes and DXS28 and DXS708 were hybridized on a panel of patients; the deletion mapping indicates that the YAC contig contains both GK and at least part of AHC, and together with the physical map defines a GK critical region of 50-250 kb . In one AHC patient with a cytogenetically detectable deletion we used the new probes to characterize a complex double deletion . Non-overlapping deletions observed in other unrelated AHC patients indicate that the AHC gene is large, extending over at least 200-500 kb . This mapping provides the basis for the identification of the AHC and GK genes.

Cytokine, 1992 Nov, 4(6), 418 - 28
Cloning, expression and characterization of ovine interleukins 1 alpha and beta; Fiskerstrand CE et al.; Ovine interleukin 1 alpha (IL-1 alpha) c-DNA, obtained by polymerase chain reaction, has been cloned into pTZ18R and pTZ19R . The resulting DNA sequence shows close homology with the bovine sequence . The derived amino-acid sequence shows conserved motifs similar to those observed in all species studied so far . No signal peptide is seen . Northern blots of RNA from lipopolysaccharide (LPS)-stimulated ovine alveolar macrophages show IL-1 beta m-RNA to be produced earlier than and to be more transient than IL-1 alpha m-RNA . c-DNAs coding for the IL-1 alpha proprotein and IL-1 alpha and IL-1 beta mature proteins have been cloned and expressed in the yeast Ty-VLP system as fusion proteins . The resultant IL-1 protein preparations, cleaved from their fusion partners by the action of activated coagulation Factor Xa, are 80-95% pure and show biological activity in standard thymocyte co-mitogen and cartilage degradation assays for IL-1 . Some species specificity is observed in that sheep thymocytes are more responsive to ovine rIL-1 than are mouse thymocytes . The presence of a Factor Xa cleavage site in the IL-1 alpha proprotein suggests that Factor Xa may be involved in the processing of ovine IL-1 alpha to its mature form.

Antonie Van Leeuwenhoek, 1992 Nov, 62(4), 285 - 90
Characterization of a novel enzyme, N6-acetyl-L-lysine: 2-oxoglutarate aminotransferase, which catalyses the second step of lysine catabolism in Candida maltosa; Schmidt H et al.; A novel aminotransferase catalyzing the second step of lysine catabolism, the oxidative transamination of the alpha-group of N6-acetyllysine, was identified and characterized in the yeast Candida maltosa . The enzyme was strongly induced in cells grown on L-lysine as sole carbon source . Its activity was specific for both N6-acetyllysine and 2-oxoglutarate . The Km values were 14 mM for the donor, 4 mM for the acceptor and 1.7 microM for pyridoxal-5-phosphate . The enzyme had a maximum activity at pH 8.1 and 32 degrees C . Its molecular mass estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis was 55 kDa . Since the native molecular mass determined by gel filtration was 120 kDa, the enzyme is probably a homodimer.

Antonie Van Leeuwenhoek, 1992 Nov, 62(4), 321 - 9
Nutritional pattern and eco-physiology of Hortaea werneckii, agent of human tinea nigra; de Hoog GS et al.; The life cycle of Hortaea werneckii includes yeast-like, hyphal and meristematic growth . The preponderance of each form of propagation can be influenced by environmental conditions . The clinical entity 'tinea nigra' is explained by ecological similarities between supposed natural niches and human hyperhydrotic skin . The species is recognizable by assimilation of lactose, nitrate and nitrite, no or little growth with L-lysine, cadaverine, creatine and creatinine, and tolerance of 10% NaCl . It generally does not grow at 36 degrees C.

Ann N Y Acad Sci, 1992 Oct 28, 660, 209 - 18
Cell cycle effects of microinjected antisense oligodeoxynucleotides to p34cdc2 kinase; Mercer WE et al.; In this study the effect of antisense oligomers targeted against the mRNA transcripts of p34cdc2 kinase on G1 progression into S-phase was examined . For this purpose, antisense, sense, or nonsense oligomers were introduced directly into the cytoplasm of T98G cells grown in monolayer cultures by glass-capillary microinjection . The microinjection of antisense oligomers (but not sense or nonsense oligomers) into growth-arrested cells before serum stimulation inhibited G1 progression into S-phase . This inhibition was correlated with a reduction in the steady-state levels of nuclear p34cdc2 protein . Microinjection of antisense oligomers into cells at 2 and 6 hours after serum stimulation also resulted in a marked inhibition in the ability of cells to enter S-phase . The inhibitory effect decreased when cells were microinjected at 12 hours after serum stimulation . When cells were microinjected at 18 and 24 hours after serum stimulation, only a slight inhibition was observed . As the antisense oligomers were introduced directly into the cytoplasm of cells at each of the time points examined, the observed differences in the inhibitory effects of the antisense oligomers at later times after serum stimulation cannot be explained by differences in uptake . An alternative explanation is that after a certain threshold level of nuclear p34cdc2 protein is reached in late G1 phase; no further increase is necessary, because the cells become committed to enter S-phase . In yeast, p34cdc2 appears to play an important role in the G1/S-phase transition at a control point in late G1 phase called START (reviewed by Lewin) . In mammalian cells a control point that could be equivalent to START is the "restriction point" which is defined as the time after which inhibition of protein synthesis fails to block entry into S-phase (reviewed by Pardee) . The effects observed with antisense oligomers to p34cdc2 kinase are strikingly similar to what is observed when low concentrations of the drug cycloheximide are added to these cells at different times after serum stimulation; entry into S-phase is significantly inhibited when cycloheximide is added up to 12 hours postimulation . Thus, the results reported in this study are in agreement with the idea that p34cdc2 kinase plays a role in the G1/S phase transition in mammalian cells . Finally, introduction of antisense oligomers directly into the cytoplasm of cells grown in monolayer cultures by glass-capillary microinjection appears to be a viable alternative to simply adding the oligomers to the culture medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1992 Oct 27, 31(42), 10322 - 30
Left-sided substrate binding of lysozyme: evidence for the involvement of asparagine-46 in the initial binding of substrate to chicken lysozyme; Inoue M et al.; The "right-sided" and "left-sided" substrate binding modes at the lower saccharide binding subsites (D-F sites) of chicken lysozyme were investigated by utilizing mutant lysozymes secreted from yeast . We constructed the following mutant lysozymes; "left-sided" substitution of Asn46 to Asp, deletion of Thr47, and insertion of Gly between Thr47 and Asp48 and "right-sided" substitution of Asn37 to Gly . Analyses of their activities and substrate binding abilities showed that Asn46 and Thr47 are involved in the initial enzyme-substrate complex and Asn37 is involved in the transition state . These results support an earlier proposal that interactions between substrate and residues at the left side of lysozyme stabilize a catalytically inactive enzyme-substrate complex, while interactions between substrate and residues at the right side stabilize the catalytically active complex {Pincus, M . R., & Scheraga, H . A . (1979) Macromolecules 12, 633-644} . These results are also consistent with the proposed kinetic mechanism for lysozyme reaction that the rearrangement of an initial enzyme-substrate complex (beta-complex) to another complex (gamma-complex) is required for catalytic hydrolysis {Banerjee S . K., Holler, E., Hess, G . P., & Rupley, J . A . (1975) J . Biol . Chem . 250, 4355-4367}.

FEBS Lett, 1992 Oct 26, 311(3), 226 - 30
Conformation and length of the signal sequence affect processing of secretory protein; Kohara A et al.; Processing of human lysozyme with artificially designed signal sequences was examined in an in vitro translation-translocation system and compared with their secretory capabilities in yeast . It has been shown that the conformation of the C-terminal region of the signal sequence and the length of the hydrophobic segment are important factors for efficient cleavage of the signal sequence.

J Biol Chem, 1992 Oct 25, 267(30), 21738 - 45
The role of the cathepsin D propeptide in sorting to the lysosome; Conner GE; The propeptides of lysosomal enzymes have been implicated in membrane association and mannose 6-phosphate-independent sorting to the lysosome (Rijnboutt, S., Aerts, H., Geuze, H . J., Tager, J . M., and Strous, G . J . (1991) J . Biol . Chem . 266, 4862-4868; McIntyre, G . F., and Erickson, A . H . (1991) J . Biol . Chem . 266, 15438-15445) . In this report, the function of the propeptide of procathepsin D in sorting to the lysosome was directly assessed using a cathepsin D deletion mutant lacking the propeptide, and using a chimeric cDNA encoding the cathepsin D propeptide fused to the secretory protein alpha-lactalbumin . Proteins encoded by these cDNAs were expressed in mouse Ltk- cells and in human hepatoma Hep G2 cells, and then immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis . The deletion mutant was glycosylated but was rapidly degraded in a chloroquine-independent fashion and did not assume an active conformation . Thus the propeptide appeared to be necessary for correct folding . The chimeric protein was glycosylated and secreted . The coincidence of complex oligosaccharide modification and secretion of the chimeric protein suggested that it was slowly released from the endoplasmic reticulum and rapidly passed through the cell to the extracellular compartment . This was confirmed by immunofluorescent localization of the proteins . The data indicated that the propeptide appeared to be necessary for folding of cathepsin D but, unlike the yeast vacuolar propeptides, was not sufficient to direct a secretory protein to the lysosome in fibroblasts or in epithelial cells.

Proc R Soc Lond B Biol Sci, 1992 Oct 22, 250(1327), 1 - 10
The Florey Lecture, 1992 . The secretion of proteins by cells; Pelham HR; In eukaryotic cells, protein secretion provides a complex organizational problem . Secretory proteins are first transported, in an unfolded state, across the membrane of the endoplasmic reticulum (ER), and are then carried in small vesicles to the Golgi apparatus and finally to the cell membrane . The ER contains soluble proteins which catalyse the folding of newly synthesized polypeptides . These proteins are sorted from secretory proteins in the Golgi complex: they carry a sorting signal (the tetrapeptide KDEL or a related sequence) that allows them to be selectively retrieved and returned to the ER . This retrieval process also appears to be used by some bacterial toxins to aid their invasion of the cell: these toxins contain KDEL-like sequences and may, in effect, follow the secretory pathway in reverse . The membrane-bound receptor responsible for sorting luminal ER proteins has been identified in yeast by genetic means, and related receptors are found in mammalian cells . Unexpectedly, this receptor has a second role: in yeast it is required to maintain the normal size and function of the Golgi apparatus . By helping to maintain the composition of both ER and Golgi compartments, the KDEL receptor has an important role in the organization of the secretory pathway.

Gene, 1992 Oct 21, 120(2), 325 - 6
Sequence of a canine cDNA clone encoding a Ran/TC4 GTP-binding protein; Dupree P et al.; We report the isolation and characterization of a canine cDNA encoding a 216-amino acid GTP-binding protein of the Ras superfamily . The protein is almost identical to the human TC4 {Drivas et al., Mol . Cell . Biol . 10 (1990) 1793-1798} and Ran {Bischoff and Ponstingl, Proc . Natl . Acad . Sci . USA 88 (1991) 10830-10834; Nature 354 (1991) 80-82} proteins, the latter of which has been found to be involved in cell cycle control . Furthermore, the protein is highly similar to the fission yeast spi1 gene product {Matsumoto and Beach, Cell 66 (1991) 347-360} . The high degree of evolutionary conservation in this protein suggests that it plays a vital role in the eukaryotic cell.

Gene, 1992 Oct 21, 120(2), 249 - 54
Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase; Le Guen L et al.; The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized . The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region . The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1) . SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase . Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A . thaliana . Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves . AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.

FEBS Lett, 1992 Oct 12, 311(1), 55 - 9
Processing of mutated proinsulin with tetrabasic cleavage sites to bioactive insulin in the non-endocrine cell line, COS-7; Yanagita M et al.; The amino acid sequence, Arg-4-X-3-Lys/Arg-2-Arg-1 decreases X+1, is thought to be a consensus processing site for a constitutive secretory pathway in non-endocrine cells . We created a mutant proinsulin DNA with a peptide structure of B chain-Arg-Arg-Lys-Arg-C peptide-Arg-Arg-Lys-Arg-A chain, which compares to the native proinsulin structure of B chain-Arg-Arg-C peptide-Lys-Arg-A chain . When the mutant insulin was expressed in a monkey kidney-derived cell line, COS-7, approximately 60% of the total immunoreactive insulin appeared as mature insulin in the culture medium . This conversion to the mature form was strikingly facilitated by co-expressing the mutant proinsulin with furin, a homologue of the yeast endoprotease, Kex2.

Mol Cell Biochem, 1992 Oct 7, 115(2), 137 - 42
Dual anomeric specificity of phosphomannoisomerase assessed by 2D phase sensitive 13C EXSY NMR; Malaisse-Lagae F et al.; The reversible conversion between D-mannose 6-phosphate and D-fructose 6-phosphate catalyzed by yeast phosphomannoisomerase was studied by phase sensitive 2D 13C-(1H) EXSY NMR spectroscopy at 100.623 MHz, using 13C enriched substrates in the C2 position of the D-hexose 6-phosphates . The unique pair of isomerization cross-peaks observed in the 2D EXSY map correlates the 13C2 resonances of the beta-anomers of both D-{2-13C}-mannose 6-phosphate and D-{213C}-fructose 6-phosphate . This indicates that phosphomannoisomerase specifically catalyzes the reversible conversion between beta-D-mannose 6-phosphate and beta-D-fructose 6-phosphate . Since phosphoglucoisomerase was recently found to catalyze specifically the interconversion of alpha-D-glucose 6-phosphate and beta-D-fructose 6-phosphate, the beta-anomer of the ketohexose ester could be directly channeled in a multi-enzyme system involving phosphoglucoisomerase, phosphomannoisomerase and phosphofructokinase.

Science, 1992 Oct 2, 258(5079), 103 - 9
Genome analysis and the human X chromosome; Mandel JL et al.; A unified genetic, physical, and functional map of the human X chromosome is being built through a concerted, international effort . About 40 percent of the 160 million base pairs of the X chromosome DNA have been cloned in overlapping, ordered contigs derived from yeast artificial chromosomes . This rapid progress toward a physical map is accelerating the identification of inherited disease genes, 26 of which are already cloned and more than 50 others regionally localized by linkage analysis . This article summarizes the mapping strategies now used and the impact of genome research on the understanding of X chromosome inactivation and X-linked diseases.

J Med Chem, 1992 Oct 2, 35(20), 3648 - 52
Prodrugs of nitroxyl as inhibitors of aldehyde dehydrogenase; Lee MJ et al.; In the preceding paper, analogs of chlorpropamide with an OMe substituent on the sulfonamide nitrogen were shown to inhibit aldehyde dehydrogenase (AlDH), and it was postulated that these compounds were bioactivated by O-demethylation to release nitroxyl (HN = O, nitrosyl hydride), which is an inhibitor of AlDH . Further evidence for the production of nitroxyl from compounds with O-acyl instead of OMe on the sulfonamide nitrogen is now presented . Thus, nitrous oxide (N2O), the end product of nitroxyl dimerization and disproportionation, was found to be generated on alkaline or enzymatic hydrolysis of N,O-diacylated N-hydroxyarylsulfonamides . Since the latter compounds strongly inhibit yeast AlDH in vitro after bioactivation by an esterase intrinsic to this enzyme, nitroxyl generated from these compounds must be the common intermediate that inhibits AlDH.

Science, 1992 Oct 2, 258(5079), 60 - 6
The human Y chromosome: overlapping DNA clones spanning the euchromatic region; Foote S et al.; The human Y chromosome was physically mapped by assembling 196 recombinant DNA clones, each containing a segment of the chromosome, into a single overlapping array . This array included more than 98 percent of the euchromatic portion of the Y chromosome . First, a library of yeast artificial chromosome (YAC) clones was prepared from the genomic DNA of a human XYYYY male . The library was screened to identify clones containing 160 sequence-tagged sites and the map was then constructed from this information . In all, 207 Y-chromosomal DNA loci were assigned to 127 ordered intervals on the basis of their presence or absence in the YAC's, yielding ordered landmarks at an average spacing of 220 kilobases across the euchromatic region . The map reveals that Y-chromosomal genes are scattered among a patchwork of X-homologous, Y-specific repetitive, and single-copy DNA sequences . This map of overlapping clones and ordered, densely spaced markers should accelerate studies of the chromosome.

Arch Biochem Biophys, 1992 Oct, 298(1), 49 - 55
Ligand recognition by purified human mannose receptor; Kery V et al.; In this work we examine the carbohydrate binding properties of human placental mannose receptor (HMR) using a rapid and sensitive enzyme-linked immunosorbent microplate assay . The assay is based on the inhibition of binding of highly purified receptor to yeast mannan-coated 96-well plates . The specificity of ligand binding was inferred from the potency of different saccharides in blocking HMR binding to the mannan-coated wells . The relative inhibitory potency of monosaccharides was L-Fuc greater than D-Man greater than D-Glc greater than D-GlcNAc greater than Man-6-P much greater than D-Gal much greater than L-Rha much greater than GalNAc . The inhibitory potency of mannose increased by two orders of magnitude when linear oligomers were used . Oligomers containing alpha-1-3- and alpha-1-6-linked mannose residues were more inhibitory than those containing alpha-1-2- and alpha-1-4-linked mannoses . Linear or branched oligomannosides larger than three units did not have a significant influence on their inhibitory potency; rather, potency was found to decrease in comparison with oligomannosides with three units . Compared to linear oligomers, inhibition of binding was the best using branched mannose oligosaccharides, alpha-D-Man-bovine serum albumin conjugates, or mannan . A model is discussed in which branched ligand is bound to spatially distinct sites on the HMR.

Arch Biochem Biophys, 1992 Oct, 298(1), 271 - 8
Functional consequences of mutation of highly conserved serine residues, found at equivalent positions in the N- and C-terminal domains of mammalian hexokinases; Baijal M et al.; Despite the extensive sequence similarity between the N- and C-terminal halves of the 100-kDa molecular weight mammalian hexokinases (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), reflecting their evolutionary origin by duplication and fusion of a gene coding for a smaller ancestral hexokinase, there is evidence for a functional division, with the C-terminal domain retaining a catalytic role while the N-terminal domain serves a regulatory function {binding of the product inhibitor, glucose 6-phosphate) (Glc-G-P)} . Conversion of Ser603 to Ala in the C-terminal domain of rat Type I hexokinase, expressed in COS-1 cells, resulted in drastic reduction of catalytic activity; Ser603 is analogous to Ser158, a residue of critical catalytic importance in the homologous yeast hexokinase . In contrast, conversion of Ser155 to Ala in the N-terminal domain (analogous to Ser603 in the C-terminal domain) of rat Type I hexokinase had no effect on catalytic activity or on inhibition of the enzyme by the Glc-6-P analog, 1,5-anhydroglucitol-6-P . Immunoreactivity with monoclonal antibodies recognizing conformationally sensitive epitopes was not affected, indicating that neither mutation resulted in gross structural perturbation . These results are consistent with the assignment of catalytic function, involving Ser603, to the C-terminal domain, and demonstrate that the analogous Ser155 is not critical for either catalytic or regulatory function . The Type I isozyme, expressed in COS-1 cells, retained the ability to bind to mitochondria in a Glc-6-P-sensitive manner, as previously found with the enzyme isolated from mammalian tissues.

J Cell Sci, 1992 Oct, 103 ( Pt 2), 381 - 8
Cytostellin: a novel, highly conserved protein that undergoes continuous redistribution during the cell cycle; Warren SL et al.; Cytostellin, a 240 kDa protein, has been purified from mammalian cells by immunoaffinity chromatography using monoclonal antibody H5 . Immunofluorescence microscopy shows diffuse and punctate cytostellin immunoreactivity in interphase nuclei . Nuclease digestion and salt extraction are not required to expose the epitope . The onset of prophase is marked by the appearance of multiple intensely immunofluorescent cytostellin-containing 'bodies' within the nucleus . Nuclear disassembly is heralded by the movement of cytostellin bodies from the nucleus to multiple positions throughout the cell . Cytostellin bodies in metaphase, anaphase and telophase cells are widely dispersed, including some in cell processes far removed from the mitotic spindle apparatus . However, a distinct subset of larger, more intensely staining bodies surrounds the mitotic spindle apparatus . Cytostellin bodies remain in the cytoplasm of the daughter cells and disappear after the appearance of nascent nuclei . Cytostellin is immunologically distinct from other nuclear and cytoplasmic proteins, and it has been detected by immunoblot analysis in all species tested from yeast to humans . Based upon these findings, we postulate that cytostellin has a cell cycle-dependent function which is conserved in higher and lower eukaryotic cells.

Genet Res, 1992 Oct, 60(2), 87 - 101
Phenotypic plasticity for life-history traits in Drosophila melanogaster . III . Effect of the environment on genetic parameters; Gebhardt MD et al.; We estimated genetic and environmental variance components for developmental time and dry weight at eclosion in Drosophila melanogaster raised in ten different environments (all combinations of 22, 25 and 28 degrees C and 0.5, 1 and 4% yeast concentration, and 0.25% yeast at 25 degrees C) . We used six homozygous lines derived from a natural population for complete diallel crosses in each environment . Additive genetic variances were consistently low for both traits (h2 around 10%) . The additive genetic variance of developmental time was larger at lower yeast concentrations, but the heritability did not increase because other components were also larger . The additive genetic effects of the six parental lines changed ranks across environments, suggesting a mechanism for the maintenance of genetic variation in heterogenous environments . The variance due to non-directional dominance was small in most environments . However, there was directional dominance in the form of inbreeding depression for both traits . It was pronounced at high yeast levels and temperatures but disappeared when yeast or temperature were decreased . This meant that the heterozygous flies were more sensitive to environmental differences than homozygous flies . Because dominance effects are not heritable, this suggests that the evolution of plasticity can be constrained when dominance effects are important as a mechanism for plasticity.

Biotechnol Appl Biochem, 1992 Oct, 16(2), 188 - 94
Red blood cells as an antigen-delivery system; Magnani M et al.; The use of adjuvants is usually required to induce strong immunological responses to protein antigens . However, in many cases these adjuvants cannot be extensively applied in human and veterinary vaccinations because of associated inflammatory reactions or granuloma formation . We show here that protein antigens (bovine serum albumin, hog liver uricase, and yeast hexokinase), coupled to autologous red blood cells by way of a biotin-avidin-biotin bridge, elicit an immunological response in mice similar to or higher than that obtained by the use of Freund's adjuvant . Quantities as low as 0.5 micrograms/mouse are high enough to generate these immunological responses . Furthermore, splenocytes of mice immunized by red blood cell-coupled antigens can be used to generate hybridomas secreting monoclonal antibodies . Thus, the delivery of antigens by autologous red blood cells is an effective way to avoid the use of adjuvants for producing anti-peptide antibodies and possibly to generate peptide vaccines.

Plant Cell, 1992 Oct, 4(10), 1295 - 307
A maize protein associated with the G-box binding complex has homology to brain regulatory proteins; de Vetten NC et al.; The G-box element is a moderately conserved component of the promoter of many inducible genes, including the alcohol dehydrogenase genes of Arabidopsis and maize . We used monoclonal antibodies generated against partially purified G-box binding factor (GBF) activity to characterize maize proteins that are part of the DNA binding complex . Antibodies interacted with partially purified maize GBF complexes to produce a slower migrating complex in the gel retardation assay . Immunoprecipitation experiments suggested that the protein recognized by the antibody is not a DNA binding protein in and of itself, but rather is associated with the DNA binding complex . These monoclonal antibodies were used to isolate cDNA clones encoding a protein that we have designated GF14 . Maize GF14 contains a region resembling a leucine zipper and acidic carboxy and amino termini, of which the latter can form an amphipathic alpha-helix similar to known transcriptional activators such as VP16 and GAL4 . Protein gel blot analysis of cell culture extract showed that a single, major protein of approximately 30 kD is recognized by anti-GF14; the protein is also present predominantly in the kernel and root . The deduced amino acid sequence of maize GF14 is more than 80% identical to Arabidopsis GF14 and Oenothera PHP-O, and is more than 60% identical to a class of mammalian brain proteins described as both protein kinase C inhibitors and activators of tyrosine and tryptophan hydroxylases . GF14 is found in a variety of monocotyledons and dicotyledons, gymnosperms, and yeast . This suggests a deep evolutionary conservation of a potential regulatory protein associated with a core sequence found in the promoter region of many genes.

Am J Trop Med Hyg, 1992 Oct, 47(4), 429 - 39
Antibodies to the major merozoite surface coat protein of Plasmodium falciparum (gp195) in a human population living in a malaria-endemic area of the Philippines; Kramer KJ et al.; The seroprevalence of naturally acquired antibodies against Plasmodium falciparum merozoite surface protein gp195 was assessed in 726 individuals living in the Napsan region of Palawan in The Philippines . Antibodies against gp195 were detected using parasite-derived antigens in an enzyme-linked immunosorbent assay . The lowest seroprevalence of anti-gp195 antibodies (45%) was found in the 0-4-year-old age group . By 10-19 years of age, the seroprevalence of anti-gp195 antibodies had leveled off at approximately 90% . Anti-gp195 antibody titers were determined for 59 randomly selected individuals using parasite-derived gp195 and two yeast recombinant polypeptides corresponding to the N-terminal (195A) and C-terminal (p42) processing fragments of gp195 . For each antigen, the lowest antibody titers were found in the 0-4-year-old age group . The 5-9-year-old age group had anti-gp195 antibody titers comparable with the older age groups . Immunoblotting experiments with parasite-derived gp195 revealed that all serum samples tested had detectable antibodies to the 195-kD gp195 precursor molecule and the 83-kD N-terminal processing fragment . Individuals with anti-gp195 titers greater than 1:400 had antibodies against both the N-terminal and C-terminal processing fragments of gp195 . These results suggest that the gp195 C-terminal region may be less immunogenic than the N-terminal region when presented on the parasite surface during natural malaria infections.

Genomics, 1992 Oct, 14(2), 256 - 62
A random STS strategy for construction of YAC contigs spanning defined chromosomal regions; Cole CG et al.; Sequence tagged sites (STSs) that were generated via Alu-element-mediated polymerase chain reaction (Alu-PCR) and mapped to human Xq26 were used to isolate and overlap yeast artificial chromosomes (YACs) . By collating the results of primary pool screening, the order of STSs and YACs was postulated directly . Subsequent isolation of 11 key YACs from 75 positive pools confirmed the proposed contig . Although only a small subset of the available Alu-PCR fragments was used, the STSs were generated at sufficient density to isolate all the YACs required and to identify all except one overlap directly . The results confirmed physical linkage of HPRT to DXS86 and DXS144E . Long-range continuity was determined purely by analysis of the 11 YAC colonies and required no end-rescue . This strategy is therefore an effective approach for the construction of YAC contigs spanning discrete chromosomal regions contained within somatic cell hybrids, with minimal prior knowledge of the region.

Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 9247 - 51
"Enzymogenesis": classical liver alcohol dehydrogenase origin from the glutathione-dependent formaldehyde dehydrogenase line; Danielsson O et al.; Analysis of the activity and structure of lower vertebrate alcohol dehydrogenases reveals that relationships between the classical liver and yeast enzymes need not be continuous . Both the ethanol activity of class I-type alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and the glutathione-dependent formaldehyde activity of the class III-type enzyme {formaldehyde:NAD+ oxidoreductase (glutathione-formylating), EC 1.2.1.1} are present in liver down to at least the stage of bony fishes (cod liver: ethanol activity, 3.4 units/mg of protein in one enzyme; formaldehyde activity, 4.5 units/mg in the major form of another enzyme) . Structural analysis of the latter protein reveals it to be a typical class III enzyme, with limited variation from the mammalian form and therefore with stable activity and structure throughout much of the vertebrate lineage . In contrast, the classical alcohol dehydrogenase (the class I enzyme) appears to be the emerging form, first in activity and later also in structure . The class I activity is present already in the piscine line, whereas the overall structural-type enzyme is not observed until amphibians and still more recent vertebrates . Consequently, the class I/III duplicatory origin appears to have arisen from a functional class III form, not a class I form . Therefore, ethanol dehydrogenases from organisms existing before this duplication have origins separate from those leading to the "classical" liver alcohol dehydrogenases . The latter now often occur in isozyme forms from further gene duplications and have a high rate of evolutionary change . The pattern is, however, not simple and we presently find in cod the first evidence for isozymes also within a class III alcohol dehydrogenase . Overall, the results indicate that both of these classes of vertebrate alcohol dehydrogenase are important and suggest a protective metabolic function for the whole enzyme system.

Nature, 1992 Oct 1, 359(6394), 380 - 7
Continuum of overlapping clones spanning the entire human chromosome 21q; Chumakov I et al.; A continuous array of overlapping clones covering the entire human chromosome 21q was constructed from human yeast artificial chromosome libraries using sequence-tagged sites as landmarks specifically detected by polymerase chain reaction . The yeast artificial chromosome contiguous unit starts with pericentromeric and ends with subtelomeric loci of 21q . The resulting order of sequence-tagged sites is consistent with other physical and genetic mapping data . This set of overlapping clones will promote our knowledge of the structure of this chromosome and the function of its genes.

Infect Immun, 1992 Oct, 60(10), 4230 - 8
Early activation of splenic macrophages by tumor necrosis factor alpha is important in determining the outcome of experimental histoplasmosis in mice; Wu-Hsieh BA et al.; Experimental infection of animals with Histoplasma capsulatum caused a massive macrophage infiltration into the spleen and induced the production of tumor necrosis factor alpha (TNF-alpha) locally . The cytokine was also produced in vitro by peritoneal exudate macrophages exposed to a large inoculum of yeast cells . Depletion of the cytokine by injection of polyclonal sheep anti-TNF-alpha antibody was detrimental to sublethally infected mice . Fungous burdens in the spleens of TNF-alpha-depleted mice were higher than they were in the infected control mice at days 2, 7, and 9 after infection, and the antibody-treated animals succumbed to the infection . Histopathological study of spleen sections revealed that splenic macrophages were not able to control proliferation of intracellular yeasts as a result of TNF-alpha depletion . It seems that TNF-alpha plays a role in early activation of splenic macrophages which is important in controlling the outcome of an infection.

Blood, 1992 Oct 1, 80(7), 1825 - 31
Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt; Erickson P et al.; We have developed a restriction map of the chromosome 21 breakpoint region involved in t(8;21)(q22;q22.3) acute myelogenous leukemia (AML) and have isolated a genomic junction clone containing chromosome 8 and 21 material . Using probes from these regions, rearrangements have been identified in each of nine cases of t(8;21) AML examined . In addition, we have isolated cDNA clones from a t(8;21) AML cDNA library that contain fused sequences from chromosome 8 and 21 . The chromosome 8 component, referred to as ETO (for eight twenty-one), is encoded over a large genomic region, as suggested by the analysis of corresponding yeast artificial chromosomes (YACs) . The DNA sequence of the chromosome 21 portion of the fusion transcript is derived from the normal AML1 gene . A striking similarity (67% identity over 387 bp, with a corresponding 69% amino acid identity) was detected between AML1 and the Drosophila segmentation gene, runt . The critical consequence of the translocation is the juxtaposition of 5' sequences of AML1 to 3' sequences of ETO, oriented telomere to centromere on the der(8) chromosome.

Plant Mol Biol, 1992 Oct, 20(1), 71 - 9
Sorghum mitochondrial atp6: divergent amino extensions to a conserved core polypeptide; Mullen JA et al.; Sorghum mitochondrial atp6 occurs as one copy in the line Tx398 and as two copies in IS1112C . In IS1112C a repeated sequence diverged within the atp6 open reading frames . The two open reading frames (1137 bp, atp6-1; 1002 bp, atp6-2) share an identical conserved region of 756 bp but are flanked 5' by divergent extensions of 246 (atp6-1) or 381 bp (atp6-2) . Tx398 carried only atp6-2 . The breakpoint of the repeated sequence of the conserved core region corresponds to the amino acid sequence Ser-Pro-Leu-Asp, which is the amino terminus of the proteolytically processed yeast ATP6 . The 5' extensions of atp6-1 and atp6-2 were similar to those of rice and maize, respectively . Each open reading is transcribed, however nuclear background influenced transcriptional patterns of atp6-2 in IS1112C.

Genes Chromosomes Cancer, 1992 Oct, 5(3), 244 - 51
Molecular cloning and analysis of chromosome band 11q23 involved in leukaemia-associated translocations; Das S et al.; Three overlapping yeast artificial chromosomes (YACs) spanning a 780 kb region of DNA around the CD3 locus on chromosome 11 have been isolated and characterised . The individual cloned regions have been mapped by in situ hybridisation to chromosome band 11q23, and a restriction enzyme map of this region has been constructed . The positions of these clones in relation to a series of leukaemia-associated chromosomal translocations has also been determined . It was concluded that, although two clones lay entirely proximal to the breakpoints examined, the third clone (13HH4) encompassed the breakpoints for the translocations t(4;11), t(6;11), and t(9;11) . The t(9;11) was observed in an acute myeloid leukaemia in a patient previously treated for an unrelated malignancy . It would thus appear that the breakpoints at chromosome band 11q23 occurring in therapy-related leukaemias are in the same region as those found in adult and childhood acute leukaemias and may result from a common underlying mechanism.

Brain Pathol, 1992 Oct, 2(4), 287 - 95
The X-linked dystonia-parkinsonism syndrome (XDP): clinical and molecular genetic analysis; Graeber MB et al.; Dystonia and parkinsonism are two major representatives of movement disorders . The X-linked dystonia-parkinsonism syndrome (XDP) serves as a model system for the study of both dystonia and parkinsonism since both symptom complexes occur together and are inherited as Mendelian traits with very high penetrance . XDP, which is endemic to the Philippine island of Panay, originated by a single mutation ("genetic founder effect"), thus assuring homogeneity of the disorder at the molecular level . The disease locus, DYT3, has been assigned to the proximal long arm (Xq12-21.1) of the human X chromosome . A strategy is described to isolate this gene by positional cloning . The rationale of this strategy, the major methods involved and technical terms are explained.

J Psychiatr Res, 1992 Oct, 26(4), 309 - 26
Mapping psychiatric disease genes: impact of new molecular strategies; Gilliam TC; Genetic mapping of genes which predispose to psychiatric illness is discussed in relation to recent developments in molecular genetic technology . Among the psychiatric disorders, the mechanism by which genetic factors contribute to illness is poorly understood, and the classification of phenotype (ill-status) is extremely complicated . These uncertainties, together with other complicating factors, tend to undermine the effectiveness of genetic linkage analysis . Two very powerful new molecular strategies have the potential to improve the overall gene mapping effort . First, new applications of polymerase chain reaction (PCR) technology will allow laboratories to generate much more genetic data than has been previously possible . Some of the factors which confound psychiatric linkage analysis should be mitigated by the larger data sets that will be generated with this technology . Second, the cloning of large segments of human chromosomes into yeast artificial chromosomes (YACs) has given rise to strategies to clone and catalog the entire human genome . The goal of constructing overlapping YAC clones (contigs) end-to-end across each human chromosome now appears imminent . This development will have immense effect upon our ability to identify disease genes.

Genomics, 1992 Oct, 14(2), 369 - 76
The gene for a novel epidermal antigen maps near the neurofibromatosis 1 gene; Kayes LM et al.; Recently the M17S1 gene, encoding an epidermal antigen thought to play a role in cell adhesion, was mapped to chromosome bands 17q11-q12, placing it in the vicinity of the gene for the genetic disorder neurofibromatosis 1 (NF1) . The pleomorphic cutaneous lesions of NF1 and the precedent for other genes being embedded within the NF1 gene prompted us to investigate whether the M17S1 gene mapped near, or within, the NF1 gene . Genetic linkage analyses revealed that M17S1 was tightly linked to NF1 and mapped within the interval bounded by D17S58 and D17S54 . Physical mapping of an M17S1 cDNA on somatic cell hybrids, yeast artificial chromosomes, and an NF1 patient with a deletion involving an entire NF1 allele demonstrated that M17S1 is located at least 180 kb centromeric to the NF1 gene . The distance between the genes suggests that M17S1 is unlikely to contribute to the NF1 phenotype since a gross chromosomal rearrangement would be required to disrupt expression of both genes.

Mol Cell Biol, 1992 Oct, 12(10), 4334 - 46
The MRF4 activation domain is required to induce muscle-specific gene expression; Mak KL et al.; MRF4 is a member of the basic helix-loop-helix muscle regulatory factor family that also includes MyoD, myogenin, and Myf-5 . Overexpression of MRF4 or the other muscle regulatory factors in fibroblasts converts the cells to differentiated muscle fibers and transcriptionally activates expression of endogenous and cotransfected muscle genes . Although these factors induce a similar phenotype, they also exhibit some distinct biological activities . For example, MyoD trans activates alpha-actin and troponin I reporter genes to very high levels, whereas MRF4 efficiently activates only alpha-actin expression . Since these proteins have a common basic helix-loop-helix domain, it is likely that portions of the proteins outside of this region impart some specificity to the activity of each muscle regulatory factor . As an initial step in determining the mechanism by which MRF4 and MyoD activate gene transcription, the transcriptional activation domain of MRF4 has been characterized . Experiments utilizing chimeric proteins containing the yeast GAL4 DNA-binding domain and portions of the MRF4 protein indicate that the MRF4 activation domain is located within amino acids 10 to 30 . This amino terminus is both necessary and sufficient to elicit a transcriptional response in transfected cells . The MRF4 activation domain and the related amino-terminal MyoD activation domain are capable of substituting for one another in converting fibroblasts to a myogenic phenotype and in activating expression of an alpha-actin reporter gene, although the MRF4 and MyoD activation domains on these chimeric proteins also dictate the specificity of transcriptional activation . The different primary amino acid sequences of these regions leave open the possibility that different coregulator proteins interact with the muscle regulatory factors to elicit their correct transcriptional activity during skeletal muscle development.

Pharmacogenetics, 1992 Oct, 2(5), 207 - 16
Guinea pig or rabbit lung flavin-containing monooxygenases with distinct mobilities in SDS-PAGE are allelic variants that differ at only two positions; Nikbakht KN et al.; Both guinea pig and rabbit express two variants of the 'lung' flavin-containing monooxygenase (FMO), observed as three distinct phenotypes based on mobility differences in SDS-PAGE . Samples of messenger RNA prepared from lungs of the two homozygous phenotypes of the guinea pig were used for the construction of two cDNA libraries . The libraries were screened with a cDNA encoding the rabbit lung FMO, and positive clones for each guinea pig lung FMO variant were isolated and sequenced . A full length clone from each library was found to encode a protein of 535 amino acids containing two pyrophosphate binding sites . Comparison of the sequences of the guinea pig and rabbit lung FMOs shows that their primary structures are 86% identical . The coding region sequences of the guinea pig variants differ at only two positions, and both differences result in amino acid substitutions . Sequence analysis has also been completed on a partially characterized variant of the rabbit lung FMO . As with the guinea pig, the nucleotide and amino acid sequences of the rabbit variants differ at only two positions . The cDNAs encoding the guinea pig variants were expressed in yeast . The activities of the enzymes are characteristic of the lung FMO, and the mobilities of the expressed enzymes are the same as those observed for the variants present in guinea pig pulmonary microsomal preparations . Similar to findings for the rabbit, analysis of genomic DNA indicates that the guinea pig lung FMO is associated with a single gene . The results of cDNA sequence analysis, expression in yeast, and analysis of genomic DNA indicate that the multiple lung FMOs in guinea pig and rabbit are allelic variants whose mobilities in SDS-PAGE are markedly altered by minimal changes in primary structure.

Biochem Cell Biol, 1992 Oct-Nov, 70(10-11), 920 - 45
Towards understanding the control of the division cycle in animal cells; Masui Y; The author reviewed the historical process by which classical knowledge of cell division accumulated, to give rise to the molecular biology of the cell cycle, and discussed the perspective of this field of research . The study of the control of cell division began at the turn of the century . It was hypothesized that cell division was a physiological regulation necessary for growing cells to maintain a proper nucleocytoplasmic ratio to survive, which was later substantiated by the finding that amoeba cells could be prevented from dividing by repeated excision of the cytoplasm . However, the observation in Tetrahymena that heat-shocked cells grow exceedingly, but fail to divide, suggested that the cell required the accumulation of a labile "division protein" to initiate division . Mechanisms that control the cell cycle were studied in oocytes by nuclear transplantation and cytoplasmic transfer, and in cultured mammalian cells, protozoa, and Physarum plasmodia by cell fusion . These experiments demonstrated the existence of cytoplasmic factors that control the cell cycle . Maturation promoting factor (MPF) thus discovered in frog oocytes became known to be an ubiquitous cytoplasmic factor that causes the transition from interphase to metaphase in all organisms . The insight into the molecular control of cell growth and division was gained from yeast cell genetics . For biochemical analysis of the cell cycle control, the method to observe the cell cycle in vitro was developed using frog egg extracts . Thus, MPF was identified as a cdc2--cyclin protein complex . Its activity was found to depend on synthesis and phosphorylation of these proteins . However, recently it was found that there were cell cycle phenomena that were difficult to explain in these terms . Various other cellular factors, including nucleocytoplasmic ratio and microtubule assembly, were also found to control MPF, as well as the cell cycle . It remained open to future how these factors control MPF to alter the pattern of the cell cycle.

Tidsskr Nor Laegeforen, 1992 Sep 30, 112(23), 2970 - 1
{Skin tests with Pityrosporum ovale extract in atopic dermatitis}; Steinkjer B; The etiology of atopic dermatitis is multifactorial . Immediate-type sensitivity to the lipophilic yeast, Pityrosporum ovale, may be important, especially in a subgroup of patients with dermatitis predominantly in the head and neck region . 100 patients with atopic dermatitis were investigated by means of skin prick tests, including Pityrosporum ovale extract . Positive tests were found more often in atopic patients with "head and neck"-dermatitis, but the correlation between a positive prick test to Pityrosporum and the severity of atopic dermatitis, estimated by means of clinical criteria and total IgE, was even higher.

Biochim Biophys Acta, 1992 Sep 23, 1159(2), 203 - 8
Caution: the glycylmethyl and glycylethyl esters of glutathione are substrates for glyoxalase I; Hamilton DS et al.; The glycylmethyl and glycylethyl esters of glutathione have been synthesized and carefully characterized by both 1H-NMR and tandem FAB mass spectrometry . Contrary to previously published studies, these compounds (as their methylglyoxal-thiohemiacetals) do indeed serve as moderately efficient substrates for yeast glyoxalase I, with kcat values that are approx . 3-fold smaller and Km values that are approx . 3-fold larger than those of the thiohemiacetal formed from glutathione . Product inhibition studies show that the glycylmethyl and glycylethyl esters of (S)-D-lactoylglutathione bind approx . 1.4-fold less tightly to the active site than (S)-D-lactoylglutathione . These observations exclude an essential role for the glycyl-CO2- of substrate in active site binding and catalysis.

Biochemistry, 1992 Sep 22, 31(37), 8747 - 54
Functional signal peptide reduces bilayer thickness of phosphatidylcholine liposomes; Tahara Y et al.; To investigate the interaction between a signal peptide and the lipid bilayer, two kinds of peptides, L8-M5 (L8 = MRL8PLAALG, M5 = KVFER) and L14-M5 (L14 = MRL14PLAALG), were examined in membranes composed of dioleoylphosphatidylcholine (DOPC) . Peptides L8 and L14 are artificially designed signal sequences, and M5 is the N-terminal five residues of human lysozyme; L8 mediated effective secretion of human lysozyme in yeast, while L14 did not {Yamamoto, Y., et al . (1987) Biochem . Biophys . Res . Commun . 149, 431-436} . DOPC liposomes incorporating L8-M5 or L14-M5 were observed by electron cryomicroscopy as pairs of concentric circles, and the separation of the bilayer was measured along the membrane . Peptide L8-M5 was found to reduce the bilayer thickness, but L14-M5 did not . CD measurements revealed that L8-M5 adopted an alpha-helical conformation with random coil in the liposome membranes and that L14-M5 adopted a more helical and less random conformation than L8-M5 . Fluorescence spectroscopy using both aqueous and membranous probes revealed that L8-M5 destabilized the lipid bilayer more strongly than L14-M5 . These results suggest that functional L8-M5 reduces the bilayer thickness and destabilizes the lipid bilayer and that these activities are important for signal peptide function.

Science, 1992 Sep 18, 257(5077), 1689 - 94
Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle; Koff A et al.; Human cyclin E, originally identified on the basis of its ability to function as a G1 cyclin in budding yeast, associated with a cell cycle-regulated protein kinase in human cells . The cyclin E-associated kinase activity peaked during G1, before the appearance of cyclin A, and was diminished during exit from the cell cycle after differentiation or serum withdrawal . The major cyclin E-associated kinase in human cells was Cdk2 (cyclin-dependent kinase 2) . The abundance of the cyclin E protein and the cyclin E-Cdk2 complex was maximal in G1 cells . These results provide further evidence that in all eukaryotes assembly of a cyclin-Cdk complex is an important step in the biochemical pathway that controls cell proliferation during G1.

Cell, 1992 Sep 18, 70(6), 887 - 900
CLIP-170 links endocytic vesicles to microtubules; Pierre P et al.; Binding of endocytic carrier vesicles to microtubules depends on the microtubule-binding protein CLIP-170 in vitro . In vivo, CLIP-170 colocalizes with a subset of transferrin receptor-positive endocytic structures and, more extensively, with endosomal tubules induced by brefeldin A . The structure of CLIP-170 has been analyzed by cloning its cDNA . The predicted non-helical C- and N-terminal domains of the homodimeric protein are connected by a long coiled-coil domain . We have identified a novel motif present in a tandem repeat in the N-terminal domain of CLIP-170 that is involved in binding to microtubules . This motif is also found in the Drosophila Glued and yeast BIK1 proteins . These features, together with its very elongated structure, suggest that CLIP-170 belongs to a novel class of proteins, cytoplasmic linker proteins (CLIPs), mediating interactions of organelles with microtubules.

Nature, 1992 Sep 17, 359(6392), 246 - 50
Centractin is an actin homologue associated with the centrosome; Clark SW et al.; Actin is one of the most ubiquitous, abundant and well-conserved proteins of eukaryotes, participating in many crucial cellular processes including the maintenance of cell shape, motility and cell division . Actins from the most divergent sources still share amino-acid identities in excess of 70% (ref . 3) . This may well explain why low-abundance homologues of actin have been difficult to isolate . Genes encoding distant relatives of actin in budding and fisson yeast have now been cloned . We report here the discovery of a vertebrate actin-like protein, which we name centractin . A full-length complementary DNA clone was isolated whose sequence reveals amino-acid identities with actin of over 50%, increasing to more than 70% when conservative amino-acid changes are considered . Northern analysis and western blotting indicate a ubiquitous tissue and species distribution . Morphological and biochemical criteria show that centractin is associated with centrosomes.

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8822 - 6
Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3; Smeekens SP et al.; Experiments using recombinant vaccinia viruses expressing rat proinsulin I coinfected into COS-7 cells with recombinant vaccinia virus expressing human furin, human PC2, mouse PC3 (subtilisin-related proprotein convertases 1-3, respectively), or yeast Kex2 indicate that in this system both Kex2 and furin produce mature insulin, whereas PC2 selectively cleaves proinsulin at the C-peptide-A-chain junction . This is a property consistent with its probable identity with the rat insulinoma granule type II proinsulin processing activity as described by Davidson et al . {Davidson, H . W., Rhodes, C . J . & Hutton, J . C . (1988) Nature (London) 333, 93-96} . PC3 generates mature insulin but cleaves preferentially at the proinsulin B-chain-C-peptide junction . This pattern of cleavage by PC3 is similar, but not identical, to that of the highly B-chain-C-peptide junction-selective type I activity as described by Davidson et al., perhaps due to the presence of a P4 arginine residue near the C-peptide-A-chain junction unique to the rat proinsulins . These results along with data presented on the expression of both PC2 and PC3 in islet beta cells strongly support the conclusion that these proteases are involved in the conversion of proinsulin to insulin in vivo.

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8596 - 600
Molecular organization of Leishmania RNA virus 1; Stuart KD et al.; The complete 5284-nucleotide sequence of the double-stranded RNA genome of Leishmania RNA virus 1 (LRV1) was determined and contains three open reading frames (ORFs) on the plus (+) (mRNA) strand . The predicted amino acid sequence of ORF3 has motifs characteristic of viral RNA-dependent RNA polymerases . ORF2, which may encode the major viral coat protein, overlaps ORF3 by 71 nucleotides, suggesting a +1 translational frameshift to produce a gag-pol type of fusion protein . Two alternative models for the frameshift are presented . The 5' splice leader sequence of kinetoplastid mRNAs is not in LRV1 RNA . This suggests that the 450-base region at the 5' end of the LRV1 (+)-strand, which contains ORF1 and is highly conserved among viral strains, does not encode protein but has a role in initiation of translation and/or RNA stability . The similarity of LRV1 genomic organization, replication cycle, and RNA-dependent RNA polymerase sequence to those of the yeast virus ScV L-A suggests a common ancestral origin . The possibility that LRV1 affects pathogenesis in leishmaniasis is intriguing.

Gene, 1992 Sep 10, 118(2), 273 - 8
The Drosophila melanogaster ribosomal protein L17A-encoding gene; Noselli S et al.; The structure and sequence of the gene encoding the Drosophila melanogaster homolog of the human and yeast large-subunit ribosomal protein L17A (rpL17A) is presented . The deduced amino acid (aa) sequence of 140 residues exhibits 87% and 77% identity to that of the human (140 aa) and yeast (137 aa) rpL17As, respectively . The D . melanogaster rpL17A gene is single copy and maps at 58F6-59A3, a chromosome region encompassing a previously characterized Minute locus, M(2)I . Despite this extensive homology in their protein products, the D . melanogaster and yeast rpL17A genes display different exon-intron structures, with the first D . melanogaster intron mapping within the 5'-untranslated mRNA leader . The rpL17A gene gives rise to a single 600-nucleotide transcript present throughout development, and is located close to another similarly expressed gene . The 5' end of the D . melanogaster rpL17A mRNA contains a polypyrimidine tract displayed by several mammalian rp genes and involved in translational control of their expression.

J Biol Chem, 1992 Sep 5, 267(25), 18047 - 54
Identification of an estrogen response element in the 3'-flanking region of the murine c-fos protooncogene; Hyder SM et al.; We have used transient transfection assays with reporter plasmids expressing chloramphenicol acetyltransferase, linked to regions of mouse c-fos, to identify a specific estrogen response element (ERE) in this protooncogene . This element is located in the untranslated 3'-flanking region of the c-fos gene, 5 kilobases (kb) downstream from the c-fos promoter and 1.5 kb downstream of the poly(A) signal . This element confers estrogen responsiveness to chloramphenicol acetyltransferase reporters linked to both the herpes simplex virus thymidine kinase promoter and the homologous c-fos promoter . Deletion analysis localized the response element to a 200-base pair fragment which contains the element GGTCACCACAGCC that resembles the consensus ERE sequence GGTCACAGTGACC originally identified in Xenopus vitellogenin A2 gene . A synthetic 36-base pair oligodeoxynucleotide containing this c-fos sequence conferred estrogen inducibility to the thymidine kinase promoter . The corresponding sequence also induced reporter activity when present in the c-fos gene fragment 3 kb from the thymidine kinase promoter . Gel-shift experiments demonstrated that synthetic oligonucleotides containing either the consensus ERE or the c-fos element bind human estrogen receptor obtained from a yeast expression system . However, the mobility of the shifted band is faster for the fos-ERE-complex than the consensus ERE complex suggesting that the three-dimensional structure of the protein-DNA complexes is different or that other factors are differentially involved in the two reactions . When the 5'-GGTCA sequence present in the c-fos ERE is mutated to 5'-TTTCA, transcriptional activation and receptor binding activities are both lost . Mutation of the CAGCC-3' element corresponding to the second half-site of the c-fos sequence also led to the loss of receptor binding activity, suggesting that both half-sites of this element are involved in this function . The estrogen induction mediated by either the c-fos or the consensus ERE was blunted by the antiestrogen tamoxifen . Based on these studies, we believe the 3'-fos ERE sequence we have identified may be a major cis-acting element involved in the physiological regulation of the gene by estrogens in vivo.

J Biol Chem, 1992 Sep 5, 267(25), 17498 - 501
Analysis of the oligomerization of myogenin and E2A products in vivo using a two-hybrid assay system; Chakraborty T et al.; Members of the helix-loop-helix (HLH) family of proteins bind DNA and activate transcription as homo- and heterodimers . Myogenin is a muscle-specific HLH protein that binds DNA in vitro as a heterodimer with several widely expressed HLH proteins, such as the E2A gene products E12 and E47 . We describe a method for detection of protein-protein interactions among HLH proteins in vivo in which dimerization through the HLH motif reconstructs a hybrid transcription factor containing the DNA-binding domain of yeast GAL4 linked to one HLH motif and the activation domain of VP-16 linked to another . We have used this assay to investiagate whether myogenin forms homomeric or heteromeric complexes in vivo and to determine whether growth factors and oncogenes that inhibit myogenesis influence myogenin's ability to dimerize . The results show that myogenin heterodimerizes with E12 and E47 in vivo, but it does not homodimerize to a measurable extent . Peptide growth factors, as well as the immediate early gene products c-Jun, v-Fos, and c-Myc, inhibit the activity of myogenin through a mechanism independent of its association with E2A products.

Biochim Biophys Acta, 1992 Sep 4, 1159(1), 81 - 93
Conformational analysis of a mitochondrial presequence derived from the F1-ATPase beta-subunit by CD and NMR spectroscopy; Bruch MD et al.; Previous studies on mitochondrial targeting presequences have indicated that formation of an amphiphillic helix may be required for efficient targeting of the precursor protein into mitochondria, but the structural details are not well understood . We have used CD and NMR spectroscopy to characterize in detail the structure of a synthetic peptide corresponding to the presequence for the beta-subunit of F1-ATPase, a mitochondrial matrix protein . Although this peptide is essentially unstructured in water, alpha-helix formation is induced when the peptide is placed in structure-promoting environments, such as SDS micelles or aqueous trifluoroethanol (TFE) . In 50% TFE (by volume), the peptide is in dynamic equilibrium between random coil and alpha-helical conformations, with a significant population of alpha-helix throughout the entire peptide . The helix is somewhat more stable in the N-terminal part of the presequence (residues 4-10), and this result is consistent with the structure proposed previously for the presequence of another mitochondrial matrix protein, yeast cytochrome oxidase subunit IV . Addition of increasing amounts of TFE causes the alpha-helical content to increase even further, and the TFE titration data for the presequence peptide of the F1-ATPase beta-subunit are not consistent with a single, cooperative transition from random coil to alpha-helix . There is evidence that helix formation is initiated in two different regions of the peptide . This result helps to explain the redundancy of the targeting information contained in the presequence for the F1-ATPase beta-subunit.

Cell, 1992 Sep 4, 70(5), 803 - 10
New reactions catalyzed by a group II intron ribozyme with RNA and DNA substrates; Morl M et al.; Here we describe three novel reactions of the self-splicing group II intron bI1 (the first intron of the COB gene of yeast mitochondria) demonstrating its catalytic versatility: reversal of the first step of the self-splicing reaction catalyzed by a linear form of the intron utilizing the energy of a phosphoanhydride bond for transesterification, ligation of a single-stranded DNA to an RNA, and cleavage of a single-stranded DNA substrate . These results have the following evolutionary implications: use of the alpha-beta bond of a terminal triphosphate for transesterification suggests that an RNA RNA replicase could use mononucleotide triphosphates as precursors, and cleavage of single-stranded DNA and DNA-RNA ligation suggests that excised group II introns might integrate directly into DNA without prior reverse transcription.

Nature, 1992 Sep 3, 359(6390), 70 - 3
Expression cloning of a human DNA repair gene involved in xeroderma pigmentosum group C; Legerski R et al.; Xeroderma pigmentosum (XP) is a rare human autosomal recessive disease characterized by solar sensitivity, high predisposition for developing cancers on areas exposed to sunlight, and, in some cases, neurological abnormalities . XP cells are defective in DNA repair, and complementation of this defect has been used to identify eight genetic groups (A-G and variant) . We have developed a simple, highly efficient complementary DNA expression system for use in human cells . Here we use this system to isolate a cDNA clone that restores the ultraviolet sensitivity and unscheduled DNA synthesis of XP-C cells to normal levels . The XP-C complementing clone XPCC encodes a highly hydrophilic protein which is composed of a predicted 823 amino acids and shares limited homology with the product of the yeast DNA repair gene RAD4 . The XPCC transcript is undetectable by northern blotting in most XP-C cell lines examined.

J Cell Biol, 1992 Sep, 118(6), 1347 - 58
Pathways of internalization of the hCG/LH receptor: immunoelectron microscopic studies in Leydig cells and transfected L-cells; Ghinea N et al.; Monoclonal anti-receptor antibodies were used to study the cellular traffic of the hCG/LH receptor by immunoelectron microscopy . The LHR38 antibody was shown to bind to the extracellular domain of the receptor but not to interfere with hormone binding, adenylate cyclase activation or with the rate of internalization of the receptor . Pig Leydig cells and a permanent L-cell line expressing the LH receptor were used for the study . Incubation with LHR38-gold complexes showed the LH receptors to be randomly distributed over the cell surface including the clathrin coated pits . The LH receptors were internalized via a route including coated pits, coated vesicles and multivesicular bodies to lysosomes . This route is different from that observed for beta-adrenergic, muscarinic, and yeast mating factor receptors and considered previously as possibly general for G-protein-coupled receptors . The use of {125I}LHR38 allowed precise measurement of the rate of internalization, showing the existence of a constitutive pathway which was increased 11-fold by hormone administration . Double labeling experiments suggested that the hormone (hCG-Au15nm) and the receptor (labeled with LHR38-Au5nm) have similar routes of endocytosis, both of them being degraded in lysosomes . Studies of the reappearance of LHR38-Au5nm on the surface of the cells and the use of monensin indicated that only a very small proportion of the receptor molecules were recycled to the cell surface . The distribution and the intracellular pathways of LH receptors are very similar in Leydig cells and transfected L-cells . This opens the possibility of using the latter to study, by in vitro mutagenesis, the molecular mechanisms involved in the cellular traffic of LH receptors.

Anesthesiology, 1992 Sep, 77(3), 467 - 74
Identification of the pharmacogenetic determinants of alfentanil metabolism: cytochrome P-450 3A4 . An explanation of the variable elimination clearance; Yun CH et al.; There is considerable variability in the elimination clearance of the opioid analgesic alfentanil . It has been shown previously that alfentanil clearance is independent of the polymorphic debrisoquine hydroxylase (P-450 2D6), and it is therefore of interest to identify the human cytochrome P-450 enzymes involved in noralfentanil formation, the primary reaction involved in the oxidative N-dealkylation at the piperidine nitrogen . Purified human P-450 3A4 showed appreciable catalytic activity, and yeast recombinant P-450 3A4 also showed alfentanil oxidation activity . When microsomes prepared from different human liver samples were compared, noralfentanil formation activity was well correlated (r = 0.95,P less than 0.005) with nifedipine oxidation (a P-450 3A4 marker) but not with markers of other P-450s, including phenacetin O-deethylation (P-450 1A2), chlorzoxazone 6-hydroxylation (P-450 2E1), and (S)-mephenytoin 4'-hydroxylation (a P-450 2C enzyme) . Using antibodies that recognize specific human P-450 enzymes (immunoinhibition techniques), it was possible to demonstrate that anti-P-450 3A4 nearly completely inhibited alfentanil oxidation activity in the human liver microsomes, but no other antibodies showed a measurable inhibitory effect . Selective chemical inhibitors of P-450 3A4, gestodene and troleandomycin, inhibited as much as 90% of the microsomal noralfentanil formation activity, but other chemical inhibitors did not show a detectable inhibitory effect . 7,8-Benzoflavone inhibited as much as 90% of the alfentanil oxidation activity of the microsomal or reconstituted P-450 3A4 system . This work indicates that P-450 3A4 contributes significantly to human liver microsomal alfentanil oxidation, whereas P-450 2D6 does not contribute.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 8245 - 8
Delineation of the dystonia-parkinsonism syndrome locus in Xq13; Graeber MB et al.; The X chromosome-linked dystonia-parkinsonism syndrome (XDP) is a severe movement disorder, characterized by both dystonia and parkinsonism . XDP is a genetically homogeneous disorder . Known ancestry of all patients has been traced back to Panay, Philippines, where the disease probably originated from a single mutation (founder effect) . The gene locus, DYT3, has been previously assigned to the proximal long arm of the X chromosome (Xq12-q21.1) . Using four dinucleotide tandem repeat (DNTR) sequences from Xq13-derived yeast artificial chromosomes (YACs), we further delineate DYT3 within Xq13 . Observation of a recombination event between DYT3 and DNTR locus 4548-7, derived from a YAC encompassing locus DXS56, establishes 4548-7 as a distal flanking marker . Assignment of DYT3 to a region in Xq13, flanked by loci 4548-7 and DXS159, is further supported by highly significant allelic association between DYT3 and a total of four DNTR loci--PY2-31, PY5-10, 4548-1, and 4548-7--located in a region defined by PGK1 and DXS56 . /phi/ and /delta/ values were 0.82/0.35, 0.78/0.42, 0.65/0.34, and 0.88/0.58 for PY2-31, PY5-10, 4548-1, and 4548-7 at P less than 10(-2), P less than 10(-4), P less than 10(-3), and P less than 10(-6).

Am J Clin Nutr, 1992 Sep, 56(3), 565 - 72
Estimated mineral intakes of toddlers: predicted prevalence of inadequacy in village populations in Egypt, Kenya, and Mexico; Murphy SP et al.; Intakes of minerals and factors that might affect their bioavailability were estimated for 255 toddlers aged 18-30 mo living in villages in Egypt, Kenya, and Mexico . Mean intakes over 1 y were compared with international-requirement estimates by using a probability approach . The prevalence of iron intakes likely to be inadequate to prevent anemia was estimated as 35% in Egypt, 13% in Kenya, and 43% in Mexico . The prevalence of zinc intakes likely to be inadequate to meet basal requirements was estimated as 57% and 25% in Kenya and Mexico, respectively, but only 10% in Egypt, where the use of yeast-leavened breads was judged to have improved zinc availability . There was no suggestion that estimated copper or magnesium intakes were inadequate, but calcium intakes in Kenya and Egypt were well below recommended amounts . Studies of factors affecting mineral bioavailability in the diets of these countries' populations could suggest dietary changes that might improve effective mineral intake with minimal cost.

Res Microbiol, 1992 Sep, 143(7), 731 - 5
An improved method for quantitative culture of Malassezia furfur; Bergbrant IM et al.; Quantitative culture of Malassezia furfur from clinically healthy skin in 25 individuals was performed with two different methods using contact plates . The best results were obtained when a glucose peptone yeast extract medium, with the addition of milk, Tween-60, glycerol and glycerol monostearate was used . Different techniques for incubation and the reproducibility of this method were evaluated . Incubation can be done in a plastic bag at 32 or 37 degrees C . This new method is simple, the colonies are easy to identify and the counts are high and reliable.

Biochem Cell Biol, 1992 Sep, 70(9), 792 - 9
The largest subunit of RNA polymerase II in Dictyostelium: conservation of the unique tail domain and gene expression; Lam TY et al.; cDNAs encoding the largest subunit of RNA polymerase II were isolated from a Dictyostelium cDNA library . A total of 2.9 kilobases (kb) of cDNA was sequenced and the amino acid sequence of the carboxyl-terminal half of the protein was deduced . Similar to other eukaryotic RNA polymerases II, the largest subunit of Dictyostelium RNA polymerase II contains a unique repetitive tail domain at its carboxyl-terminal region . It consists of 24 highly conserved heptapeptide repeats, with a consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser . In addition to the tail domain, five segments of the deduced primary structure show > 50% sequence identity with either yeast or mouse protein . RNA blots show that cDNA probes hybridized with a single mRNA species of approximately 6 kb and immunoblots using a monoclonal antibody raised against the tail domain lighted up a single protein band of 200 kilodaltons . Interestingly, expression of the largest subunit of RNA polymerase II appears to be under developmental regulation . The accumulation of its mRNA showed a 60% increase during the first 3 h of development, followed by a steady decrease during the next 6 h . Cells began to accumulate a higher level of the RNA polymerase II mRNA after 9 h of development . When cells were treated with low concentrations of cAMP pulses to stimulate the developmental process, the pattern of mRNA accumulation moved 3 h ahead, but otherwise remained similar to that of control cells.

Pediatr Infect Dis J, 1992 Sep, 11(9), 713 - 6
Prophylactic miconazole oral gel for the prevention of neonatal fungal rectal colonization and systemic infection; Wainer S et al.; Prior rectal colonization with fungi may be an important risk factor for development of systemic fungal infection in the neonate . This placebo-controlled study evaluated the benefits of miconazole oral gel in the prevention of fungal rectal colonization and systemic infection in high risk neonates admitted to the Neonatal Intensive Care Unit . Repeated oral application of miconazole gel reduced the overall prevalence of postnatally acquired rectal colonization; a yeast was grown in 19.5% of the weekly rectal swabs in the miconazole-treated group compared with 36.2% in the control group (69 of 354 vs . 146 of 403, P < 0.0001) . There was no reduction in the incidence of systemic fungal infection in the two groups although the overall incidence of the infection was low in both groups, at 2.0% vs . 2.6% (6 of 298 vs . 8 of 302, P not significant) . No relationship was shown between prior rectal colonization and subsequent systemic fungal infections in either of the two groups . This study does not support the use of prophylactic miconazole oral gel for the prevention of neonatal systemic fungal infections.

Xenobiotica, 1992 Sep-Oct, 22(9-10), 1083 - 92
Polymorphism in stereoselective hydroxylations of mephenytoin and hexobarbital by Japanese liver samples in relation to cytochrome P-450 human-2 (IIC9); Kato R et al.; 1 . Stereoselective 4'-hydroxylations of R-(--)-mephenytoin and S-(+)-mephenytoin were determined in liver microsomes of 19 Japanese subjects . 2 . The content of P-450 human-2 assessed by Western-blots correlated with microsomal S-(+)-mephenytoin 4'-hydroxylation . Antibody raised against P-450 human-2 effectively inhibited microsomal S-(+)-mephenytoin 4'-hydroxylation, but was less efficient for inhibition of R-(--)-mephenytoin 4'-hydroxylation in extensive metabolizers, and 4'-hydroxylation of both mephenytoin enantiomers in poor metabolizers . 3 . Similar results were observed on the stereoselective hydroxylations of R-(--)- and S-(+)-hexobarbital . Clear correlations were observed for the content of P-450 human-2 and microsomal R-(--)-hexobarbital 3'alpha-hydroxylation and S-(+)-hexobarbital 3'beta-hydroxylation . 4 . Moreover, yeast microsomes expressing P-450 human-2 cDNA showed high stereoselectivities for hydroxylations of mephenytoin and hexobarbital similar to those observed in human liver . 5 . Two other cytochromes P-450(IIC 9/10) expressed in yeast, whose cDNA were synthesized by site-directed mutagenesis from human-2 cDNA, showed no stereoselectivity for the hydroxylations of mephenytoin and hexobarbital, in spite of the modification of only two amino acid substitutions or deletions in the whole sequence . 6 . Only a cytochrome derived from P-450 human cDNA corresponding to P-450 human-2 was expressed in human livers, the two cytochromes of the three related IIC9/10 forms were not expressed . 7 . These findings indicate that P-450 human-2 is the major cytochrome P-450 responsible for the polymorphisms in stereoselective hydroxylations of mephenytoin and hexobarbital.

Parasite Immunol, 1992 Sep, 14(5), 471 - 9
Transmission blocking antibody of the Plasmodium falciparum zygote/ookinete surface protein Pfs25 also influences sporozoite development; Lensen AH et al.; The Plasmodium falciparum zygote/ookinete surface protein, Pfs25, persists in the oocyst wall throughout its development . Anti-25 kD transmission blocking antibody, given to infected Anopheles stephensi or A . gambiae mosquitoes in an additional bloodmeal, 3-6 days after being fed gametocyte infected blood, penetrated the oocyst and reacted with the 25 kD protein within it . This reaction caused a significant reduction in the number of developing sporozoites . Mouse serum containing antibodies raised by immunization with a recombinant 25 kD yeast product showed a similar effect.

J Infect, 1992 Sep, 25(2), 201 - 4
Fatal disseminated Scedosporium inflatum infection in a neutropenic immunocompromised patient; Farag SS et al.; Disseminated infection with the fungus Scedosporium inflatum in a neutropenic patient with non-Hodgkins lymphoma presented with the triad of muscle tenderness, papular skin lesions and fever, and progressed rapidly to a fatal outcome . This represents the first reported instance of fatal widely disseminated infection with this organism, and demonstrates that the triad of presenting clinical features, formerly reported to be pathognomic of systemic candidiasis, can no longer be regarded as specific for infection with a particular species of yeast or fungus.

Genomics, 1992 Sep, 14(1), 75 - 82
Organization of the human monoamine oxidase genes and long-range physical mapping around them; Chen ZY et al.; A 265-kb yeast artificial chromosome containing sequences for human monoamine oxidase A and B (MAO-A and MAO-B) genes has been characterized . These two genes are localized within a region of about 240 kb and are arranged in a tail-to-tail configuration, with the 3' coding sequences separated by about 50 kb . A region about 2.5 Mb around the MAO loci was mapped by pulsed-field gel electrophoresis (PFGE) . Comparisons between the restriction maps derived from the YAC and the long-range map derived from genomic digestions were in general agreement . The important features identified include a CpG island at the 5' end of the MAO-A and MAO-B genes, respectively . The combined information supports the order of markers within this region to be DXS77-DXS7-MAOA-MAOB.

Am J Physiol, 1992 Sep, 263(3 Pt 1), G353 - 9
Effects of nerve stimulation and zymosan on glycogenolysis in perfused livers from cold-exposed rats; Shiota M et al.; The effects of sympathetic nerve stimulation and zymosan (cell wall particles from yeast) on glycogenolysis were studied in perfused livers from rats kept for 5 and 20 days at 4 degrees C . The rate of glycogenolysis induced by nerve stimulation decreased significantly without any decrease in norepinephrine outflow during cold exposure, and the rate induced by norepinephrine did not change . By contrast, the rate of zymosan-induced glycogenolysis increased markedly during cold exposure . The rats with denervated hepatic nerves did not show the increased response to zymosan . In cold-exposed rats, both mepacrine and ibuprofen inhibited the effects of zymosan and of nerve stimulation without any inhibition of the outflow of norepinephrine . Neither inhibitor had any effect on the effects of norepinephrine . The metabolic effects of nerve stimulation and zymosan were not additive in cold-exposed rats . These results suggest that cold exposure may modulate the metabolism of arachidonic acid in Kupffer cells via hepatic nerve and decrease the eicosanoid-dependent glycogenolysis by nerve stimulation.

Mutat Res, 1992 Sep, 277(3), 187 - 200
Review of the mutagenicity/genotoxicity of butylated hydroxytoluene; Bomhard EM et al.; Butylated hydroxytoluene (BHT) is an effective, widely used, low cost antioxidant . A host of studies examining the potential of BHT to cause point mutations have been published . They include in vitro studies on various bacterial species and strains and on various types of mammalian cell lines as well as in vivo studies on Drosophila melanogaster, silk worms and also the mouse specific locus test (involving long-term exposure) . Together these studies convincingly show the absence of a potential for BHT to cause point mutations . A great number of studies on many cell types and species have also been carried out to examine the potential of BHT to cause chromosome aberrations . In vitro studies have been published using plant cells and the WI-38, CHL, CHO, and V79 mammalian cell lines . In vivo studies have been carried out on somatic and/or germ cells of Drosophila melanogaster, rats and mice . Nearly all studies, especially those using validated test systems, indicate that BHT lacks clastogenic potential . In vitro studies on bacterial, yeast and various mammalian cell lines including DON, CHO, CHL cells and primary hepatocytes demonstrate the absence of interactions with or damage to DNA . Taking all the existing data into account, the weight of evidence suggests that BHT does not represent a relevant mutagenic/genotoxic risk to man.

Rev Inst Med Trop Sao Paulo, 1992 Sep-Oct, 34(5), 441 - 6
{The sources of histoplasmosis infection on the Isla de la Juventud, Cuba}; Fernandez Andreu CM et al.; The purpose of this work is to report the isolation of Histoplasma capsulatum, etiologic agent of histoplasmosis, from soil in sites inhabited by bats and chicken in the Island of Youth, Cuba . The fungus was cultured from four species of cave dwelling bats too . The identification of H . capsulatum was done by mycelial to yeast conversion and exoantigen test . It is pointed out the epidemiological value of some of these isolations in caves of great importance from the archaeological, speleological or tourist point of view, and the potential risk that they represent to human health . The authors conclude with some recommendation to prevent the infection with H . capsulatum in people who have to keep in contact with those environments.

Rinsho Byori, 1992 Sep, 40(9), 911 - 7
{Fundamental studies of hepatitis C virus: a review}; Fujiyama S et al.; In 1974, Prince et al . reported the existence of posttransfusion hepatitis with a long incubation period which was not related to hepatitis B virus (HBV) . These cases were named "non-A, non-B" (NANB) hepatitis . The genome of NANB hepatitis virus was discovered recently using a recombinant complementary DNA (cDNA) approach . It was termed the hepatitis C virus (HCV), and a specific diagnostic tool for the circulating HCV antibody (anti-HCV) was developed using a purified viral polypeptide derived from recombinant yeast expressing a small part of the HCV genome . HCV is believed to be the main cause of blood-borne non-A, non-B hepatitis worldwide, which frequently evolves to chronic hepatitis and cirrhosis, and which may also be involved in the development of hepatocellular carcinoma . HCV is classified as part of the flaviviridae family and contains a positive-stranded RNA molecule by approximately 10 kb nucleotides . The HCV genome encodes a large polyprotein precursor, which is processed in structural nucleocapsid and envelope proteins and in non-structural proteins (NS1-NS5) . Nucleotide sequence comparisons of distinct HCV isolates have shown a significant genetic variability between the different HCV strains . At present the diagnosis of HCV infection depends on various anti-HCV tests including second generation HCV Ab . Antigenic markers for HCV are being developed but the concentrations of HCV antigens in serum are at the lower limit of detectability by existing immunoassay technology . A polymerase chain reaction has been used to detect HCV RNA in the serum and liver . Serum HCV RNA disappears from serum after effective IFN treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

J Virol, 1992 Sep, 66(9), 5500 - 8
The Epstein-Barr virus R transactivator (Rta) contains a complex, potent activation domain with properties different from those of VP16; Hardwick JM et al.; Rta, encoded by Epstein-Barr virus (EBV), is a potent activator of transcription via enhancer sequences located upstream of several viral genes . To identify the domains of Rta that facilitate transcription by interacting with cellular transcription factors, different segments of Rta were linked to the DNA binding domain of yeast transactivator GAL4 (residues 1 to 147) . These GAL4-Rta fusion proteins were tested in transfected cells for their ability to activate the adeno E1b promoter with an upstream GAL4 DNA binding site . The acidic C-terminal domain of Rta (amino acids 520 to 605) was a potent activator but behaved differently from VP16 in dose-response and competition experiments . A subterminal domain of Rta (amino acids 416 to 519) linked to GAL4 had weak activation activity . Deletion of these domains from native Rta showed that the C-terminal domain was required for transactivation, but the subterminal domain was required only in B cells . The C-terminal activation domain of Rta contains a pattern of positionally conserved hydrophobic residues shared with VP16 and other transactivators . Substitution of several conserved hydrophobic amino acids in Rta severely impaired transactivation . The improtance of hydrophobic residues was further substantiated by comparing EBV Rta with that of herpesvirus saimiri, which revealed little sequence similarity except for a few acidic residues and the positionally conserved hydrophobic amino acids . The C-terminal domain of EBV Rta contains three partially overlapping copies of this hydrophobic motif . Mutational analysis indicated that all three copies were required for full activity . However, two of the three copies appeared to be sufficient to produce full activity on a target promoter with multiple binding sites, suggesting that these motifs are functional subdomains that can synergize.

J Theor Biol, 1992 Aug 21, 157(4), 487 - 503
A parallel computation revealing the role of the in vivo environment in shaping the catalytic structure of a mitochondrial RNA transcript; Fernandez A; We study the search for folded structures performed by an RNA molecule as it is being assembled by sequential incorporation of nucleotides . The specific process we shall focus on is the transcription of intron 4 of the yeast apocytochrome b gene . We prove that the structure generated by sequential folding is endowed with catalytic potential . More specifically, the formation of all conserved interactions in the catalytic core and intramolecular splicing substrate may be achieved by sequential folding in vivo, concurrent with transcription itself . We base our analysis on a parallel Monte Carlo simulation, assigning an individual processor to each competing folding pathway . In this way, we show that the group I mitochondrial intron requires an in vivo environment to aid the folding into a structure presenting the catalytically-active helix P7 and the splicing substrate upon which the catalytic core exerts its function . It is inferred that a base pair disruption caused by interaction with a ribosome is required to bias the folding pathway so that helix P7 is formed . Furthermore, it is shown that a disruption in the early stages of transcription, followed by the perturbation described is essential to shape the 3' splicing site . Our study suggests that pre-mRNAs of group I might achieve catalytic potential in a fundamentally different way from ribosomal RNAs of the same group, where thermodynamics appear to control the formation of the catalytic structural motif . The role of the trans-acting factor, on the other hand, cannot be recovered in vitro by recombination with the transcript.

Mol Cell Biochem, 1992 Aug 18, 113(2), 105 - 21
Oscillations in glycolysis: multifactorial quantitative analysis in muscle extract; Marynissen G et al.; A multifactorial quantitative analysis of oscillations in glycolysis was conducted in the postmicrosomal supernatant of rat muscle homogenates incubated in the presence of yeast hexokinase . Oscillations in adenine nucleotides, D-fructose 1,6-bisphosphate, triose phosphates, L-glycerol 3-phosphate, 3HOH generation from D-{5-3H}glucose, NADH and L-lactate production were documented . The occurrence of such oscillations were found to depend mainly on the balance between the consumption of ATP associated with the phosphorylation of D-glucose, as catalyzed by both yeast and muscle hexokinase, and the net production of ATP resulting from the further catabolism of D-fructose 6-phosphate, as initiated by activation of phosphofructokinase . The oscillatory pattern was suppressed in the presence of D-fructose 2,6-bisphosphate . It is proposed that the quantitative information gathered in this study may set the scene for further studies in extracts of cells other than myocytes, e.g . hepatocytes and pancreatic islet cells, in which no oscillation of glycolysis was so far observed.

FEBS Lett, 1992 Aug 17, 308(2), 207 - 10
Rat liver BiP/GRP78 is down-regulated by a peroxisome-proliferator, clofibrate; Motojima K et al.; Administration of clofibrate in rat results in down-regulation of several liver proteins and a vast induction of peroxisomal proteins . One protein was identified as BiP/GRP78 using antibodies and cDNA cloning . The level of mRNA was reduced by the drug.

Arch Biochem Biophys, 1992 Aug 15, 297(1), 123 - 31
Plant sterol biosynthesis: novel potent and selective inhibitors of cytochrome P450-dependent obtusifoliol 14 alpha-methyl demethylase; Salmon F et al.; The R-(-) isomer of methyl 1-(2,2-dimethylindan-1-yl)imidazole-5-carboxylate (CGA 214372; 2) strongly inhibited P450-dependent obtusifoliol 14 alpha-demethylase (P450OBT.14DM) (I50 = 8 x 10(-9) M, I50/Km = 5 x 10(-5) in a maize (Zea mays) microsomal preparation . Kinetic studies indicated uncompetitive inhibition with respect to obtusifoliol . The corresponding S-(+) isomer was a 20-fold weaker inhibitor for P450OBT.14DM . The molecular features of a variety of analogues of 2 were related to their potency as inhibitors of P450OBT.14DM in vitro, allowing delineation of the key structural requirements governing inhibition of the demethylase . CGA 214372 proved to have a high degree of selectivity for P450OBT.14DM . This allowed easy distinction of this activity from other P450-dependent activities present in the maize microsomal preparation and gave strong evidence that P450OBT.14DM is a herbicidal target . Microsomal maize P450OBT.14DM and yeast P450LAN.14DM, the only known examples of P450-dependent enzymes carrying out an identical metabolic function in different eukaryotes, showed distinct inhibition patterns with CGA 214372 and ketoconazole, a substituted imidazole anti-mycotic.

Eur J Biochem, 1992 Aug 15, 208(1), 83 - 90
The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes; Bussink HJ et al.; Aspergillus niger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin . One of the two previously characterized genes coding for the abundant polygalacturonases I and II (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage lambda EMBL4 using conditions of moderate stringency . The products of these genes were detected in the culture medium of Aspergillus nidulans transformants on the basis of activity measurements and Western-blot analysis using a polyclonal antibody raised against PGI . These transformants were, with one exception, constructed using phage DNA . A . nidulans transformants secreted high amounts of PGI and PGII in comparison to the previously characterized A . niger transformants and a novel polygalacturonase (PGC) was produced at high levels by A . nidulans transformed with the subcloned pgaC gene . This gene was sequenced and the protein-coding region was found to be interrupted by three introns; the different intron/exon organization of the three sequenced A . niger polygalacturonase genes can be explained by the gain or loss of two single introns . The pgaC gene encodes a putative 383-amino-acid prepro-protein that is cleaved after a pair of basic amino acids and shows approximately 60% amino acid sequence similarity to the other polygalacturonases in the mature protein . The N-terminal amino acid sequences of the A . niger polygalacturonases display characteristic amino acid insertions or deletions that are also observed in polygalacturonases of phytopathogenic fungi . In the upstream regions of the A . niger polygalacturonase genes, a sequence of ten conserved nucleotides comprising a CCAAT sequence was found, which is likely to represent a binding site for a regulatory protein as it shows a high similarity to the yeast CYC1 upstream activation site recognized by the HAP2/3/4 activation complex.

Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7698 - 702
Human glucokinase gene: isolation, characterization, and identification of two missense mutations linked to early-onset non-insulin-dependent (type 2) diabetes mellitus; Stoffel M et al.; DNA polymorphisms in the glucokinase gene have recently been shown to be tightly linked to early-onset non-insulin-dependent diabetes mellitus in approximately 80% of French families with this form of diabetes . We previously identified a nonsense mutation in exon 7 in one of these families and showed that it was the likely cause of glucose intolerance in this dominantly inherited disorder . Here we report the isolation and partial sequence of the human glucokinase gene and the identification of two missense mutations in exon 7, Thr-228----Met and Gly-261----Arg, that cosegregate with early-onset non-insulin-dependent diabetes mellitus . To assess the molecular mechanism by which mutations at these two sites may affect glucokinase activity, the crystal structure of the related yeast hexokinase B was used as a simple model for human beta-cell glucokinase . Computer-assisted modeling suggests that mutation of Thr-228 affects affinity for ATP and mutation of Gly-261 may alter glucose binding . The identification of mutations in glucokinase, a protein that plays an important role in hepatic and beta-cell glucose metabolism, indicates that early-onset non-insulin-dependent diabetes mellitus may be primarily a disorder of carbohydrate metabolism.

Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7408 - 11
Serine tRNA complementary to the nonuniversal serine codon CUG in Candida cylindracea: evolutionary implications; Yokogawa T et al.; In the asporogenic yeast Candida cylindracea, the codon CUG is read as serine instead of leucine . This is an unusual instance in which the amino acid assignment of a codon deviates from the universal code . To infer the evolutionary process of this change, the tRNA with the anticodon sequence CAG, which is complementary to and thus responsible for translation of the codon CUG, has been identified . Indeed, this tRNA translates an in-frame CUG codon in a synthetic mRNA as serine in an in vitro translation system . The gene for the tRNA is interrupted by an intron in the anticodon loop . Sequence comparisons of the tRNA and its gene suggest that a single cytidine was inserted into the anticodon loop of the gene for tRNA(Ser)IGA during evolution to produce tRNA(Ser)CAG . The tRNA(Ser)CAG may be produced from its precursor molecule containing the cytidine insertion by splicing.

Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7476 - 80
A carboxyl-terminal-domain kinase associated with RNA polymerase II transcription factor delta from rat liver; Serizawa H et al.; We previously purified RNA polymerase II transcription factor delta from rat liver and found that it has an associated DNA-dependent ATPase (dATPase) activity . In this report, we show that delta is also closely associated with a protein kinase activity that catalyzes phosphorylation of the largest subunit of RNA polymerase II . Kinase activity copurifies with transcription and DNA-dependent ATPase (dATPase) activities when delta is analyzed by anion- and cation-exchange HPLC as well as by sucrose gradient sedimentation, arguing that delta possesses all three activities . Phosphorylation of the largest subunits of both rat and yeast RNA polymerase II is stimulated by DNA, whereas phosphorylation of a synthetic peptide containing multiple copies of the carboxyl-terminal heptapeptide repeat is not . Although both ATP and GTP appear to function as phosphate donors, GTP is utilized less than 10% as well as ATP . These findings suggest that delta may exert its action in transcription at least in part through a mechanism involving phosphorylation of the largest subunit of RNA polymerase II.

Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7491 - 5
Wild-type p53 is a cell cycle checkpoint determinant following irradiation; Kuerbitz SJ et al.; Cell cycle checkpoints appear to contribute to an increase in cell survival and a decrease in abnormal heritable genetic changes following exposure to DNA damaging agents . Though several radiation-sensitive yeast mutants have been identified, little is known about the genes that control these responses in mammalian cells . Recent studies from our laboratory have demonstrated a close correlation between expression of wild-type p53 genes in human hematopoietic cells and their ability to arrest in G1 phase after certain types of DNA damage . In the present study, this correlation was first generalized to nonhematopoietic mammalian cells as well . A cause and effect relationship between expression of wild-type p53 and the G1 arrest that occurs after gamma irradiation was then established by demonstrating (i) acquisition of the G1 arrest after gamma irradiation following transfection of wild-type p53 genes into cells lacking endogenous p53 genes and (ii) loss of the G1 arrest after irradiation following transfection of mutant p53 genes into cells with wild-type endogenous p53 genes . A defined role for p53 (the most commonly mutated gene in human cancers) in a physiologic pathway has, to our knowledge, not been reported previously . Furthermore, these experiments illustrate one way in which a mutant p53 gene product can function in a "dominant negative" manner . Participation of p53 in this pathway suggests a mechanism for the contribution of abnormalities in p53 to tumorigenesis and genetic instability and provides a useful model for studies of the molecular mechanisms of p53 involvement in controlling the cell cycle.

Biochem Biophys Res Commun, 1992 Aug 14, 186(3), 1688 - 93
The primary structure of rat ribosomal protein S25; Chan YL et al.; The amino acid sequence of the rat 40S ribosomal subunit protein S25 was deduced from the sequence of nucleotides in a recombinant cDNA . Ribosomal protein S25 has 125 amino acids and has a molecular weight of 13,733 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 19 to 22 copies of the S25 gene . The mRNA for the protein is about 550 nucleotides in length . Rat S25 is homologous to ribosomal proteins from other eukaryotes (human and yeast).

Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 6678 - 82
Conservation of the prohormone convertase gene family in metazoa: analysis of cDNAs encoding a PC3-like protein from hydra; Chan SJ et al.; A subclass of proteolytic enzymes that correctly cleave precursor proteins at paired basic residues and are structurally related to the bacterial subtilisins has recently been identified . In yeast, a single membrane-bound proteolytic processing enzyme encoded by the kex2 gene has been found, whereas in higher vertebrates cDNAs encoding four distinct enzymes (PC2, PC3, furin, and PACE 4) have been identified . Like kex2, furin (also known as PACE) contains a hydrophobic transmembrane domain, but PC2, PC3, and PACE 4 lack this feature . All five enzymes exhibit striking similarities in their catalytic domains, and this suggests that they have arisen from a common ancestral subtilisin-like gene . We report here the identification of cDNAs encoding a protein that is similar in structure to PC3 from a simple metazoan, Hydra vulgaris (formerly Hydra attenuata) . cDNAs encoding two isoforms of this PC3-like enzyme were obtained that differ only in their carboxyl-terminal sequences, probably due to alternative splicing of a common pre-mRNA . Neither form contains a transmembrane domain . Predicted amino acid sequence comparisons revealed that the hydra PC3-like enzyme is 55.4% and 56.7% identical in the catalytic domain to mouse PC3 and human furin, respectively . RNA blot analyses revealed that the PC3-like RNA is expressed predominantly in the hydra body column and not in the head region, although the hydra head contains a high density of nerve cells, which synthesize a variety of neuropeptides . For this reason, we suspect that another proprotein cleavage enzyme isoform may be expressed in head nerve cells . The isolation of a PC3-like cDNA from hydra is consistent with the presence of neuroendocrine cells and indicates that the PC/furin gene family has been well conserved in all metazoa . A simplified nomenclature for the group of mammalian processing proteases is proposed.

Nucleic Acids Res, 1992 Aug 11, 20(15), 3905 - 10
Purification and characterization of DNA ligase I from the trypanosomatid Crithidia fasciculata; Brown GW et al.; A DNA ligase has been purified approximately 5000-fold, to near homogeneity, from the trypanosomatid Crithidia fasciculata . The purified enzyme contains polypeptides with molecular masses of 84 and 80 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Both polypeptides formed enzyme-adenylate complexes in the absence of DNA, contained an epitope that is highly conserved between human and bovine DNA ligase I and yeast and vaccinia virus DNA ligases, and were identified in fresh lysates of C . fasciculata by antibodies raised against the purified protein . Hydrodynamic measurements indicate that the enzyme is an asymmetric protein of approximately 80 kDa . The purified DNA ligase can join oligo(dT) annealed to poly(dA), but not oligo(dT) annealed to poly(rA), and can ligate blunt-ended DNA fragments . The enzyme has a low Km for ATP of 0.3 microM . The DNA ligase absolutely requires ATP and Mg2+, and is inhibited by N-ethylmaleimide and by KCI . Substrate specificity, Km for ATP, and the conserved epitope all suggest that the purified enzyme is the trypanosome homologue of DNA ligase I.

J Biol Chem, 1992 Aug 5, 267(22), 15737 - 43
The bimB3 mutation of Aspergillus nidulans uncouples DNA replication from the completion of mitosis; May GS et al.; A conditionally lethal mutation in the bimB gene of Aspergillus nidulans disrupts the normal regulatory patterns associated with mitotic events . This results in DNA replication in the absence of the completion of mitosis in the mutant at restrictive temperature . This defect yields large polyploid nuclei after several hours at restrictive temperature . The bimB gene has been cloned by genetic mapping and chromosome walking from the previously cloned amdS gene . The cloned DNA complements the temperature-sensitive recessive bimB3 mutation . Sequence analysis of overlapping complementary DNA clones for bimB predicts a polypeptide of 2,068 amino acids . The predicted polypeptide of 227,958 Da is shown to have a carboxyl-terminal region similar to those of the budding yeast ESP1 and fission yeast cut1+ genes . In contrast these genes exhibit no other regions of similarity to one another . The conserved domain in these three proteins and the similarity of the terminal mutant phenotypes for these genes are suggestive of a conserved function for this domain in each of the predicted polypeptides . We also present evidence for a second gene in the genome of A . nidulans which also has this conserved carboxyl-terminal region, suggesting that bimB, ESP1, and cut1+ may be members of a small gene family.

Plant Mol Biol, 1992 Aug, 19(5), 793 - 801
Identification and characterisation of cDNA clones encoding cinnamyl alcohol dehydrogenase from tobacco; Knight ME et al.; Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) is an enzyme involved in lignin biosynthesis . We have previously isolated pure CAD enzyme as two closely related polypeptides of 44 and 42.5 kDa from tobacco stems . In this paper, we report partial amino acid sequences of these two polypeptides . Based on the peptide sequences mixed oligonucleotides were used to screen a tobacco stem cDNA library and CAD cDNA clones encoding the two polypeptides were identified . DNA sequence comparisons indicate very high sequence identity between these clones both in the coding and in the 5' and 3' untranslated sequences . The close similarity between the two CAD genes leads us to suggest that they do not represent different isoforms but are the same gene from each of the two parental lines of Nicotiana tabacum cv . Samsun . Sequence comparisons with alcohol dehydrogenase 1 (ADH1) from yeast shows sequence similarities of ca . 30%, while comparisons with maize, barley and potato ADH1 sequences show similarities of not more than 23%.

Am J Hum Genet, 1992 Aug, 51(2), 263 - 72
Isolation, characterization, and regional mapping of microclones from a human chromosome 21 microdissection library; Yu J et al.; Thirty-four unique-sequence microclones were isolated from a previously described microdissection library of human chromosome 21 and were regionally mapped using a cell hybrid mapping panel which consists of six cell hybrids and divides chromosome 21 into eight regions . The mapping results showed that the microclones were unevenly distributed along chromosome 21, with the majority of microclones located in the distal half portion of the long arm, between 21q21.3 and 21qter . The number of unique-sequence clones began to decrease significantly from 21q21.2 to centromere and extending to the short arm . This finding is consistent with those reported in other chromosome 21 libraries . Thus, it may be inferred that the proximal portion of the long arm of chromosome 21 contains higher proportions of repetitive sequences, rather than unique sequences or genes . The microclones were also characterized for insert size and were used to identify the corresponding genomic fragments generated by HindIII . In addition, we demonstrated that the microclones with short inserts can be efficiently used to identify YAC (yeast artificial chromosome) clones with large inserts, for increased genomic coverage for high-resolution physical mapping . We also used 200 unique-sequence microclones to screen a human liver cDNA library and identified two cDNA clones which were regionally assigned to the 21q21.3-q22.1 region . Thus, generation of unique-sequence microclones from chromosome 21 appears to be useful to isolate and regionally map many cDNA clones, among which will be candidate genes for important diseases on chromosome 21, including Down syndrome, Alzheimer disease, amyotrophic lateral sclerosis, and one form of epilepsy.

J Nutr, 1992 Aug, 122(8), 1620 - 6
Dietary selenium stabilizes glutathione peroxidase mRNA in rat liver; Christensen MJ et al.; The objective of these studies was to determine the step at which dietary selenium (Se) regulates the pre-translational expression of the gene for cytosolic Se-glutathione peroxidase (Se-GPX) in rat liver . Weanling male Sprague-Dawley rats were fed a Torula yeast-based Se-deficient diet, or the same diet supplemented with 0.5 mg/kg as Na2SeO3, for at least 40 d . Livers were excised and divided into three portions for isolation of nuclei, for purification of total RNA and for assay of Se-GPX activity . Nuclei were used in run-on transcription assays and for isolation of total nuclear RNA . Nuclear RNA and total liver RNA were probed with segments from a rat Se-GPX cDNA in Northern blot and slot blot assays . Despite marked differences in Se-GPX activity, there were no significant differences between dietary groups in the transcription rates of the Se-GPX gene . Species hybridizing to Se-GPX were not detected in nuclear RNA . However, steady state levels of Se-GPX mRNA were markedly reduced by Se deficiency . These results suggest that dietary Se exerts its effect on pretranslational Se-GPX gene expression at the level of cytosolic mRNA stabilization.

EMBO J, 1992 Aug, 11(8), 3011 - 9
Ternary complex formation over the c-fos serum response element: p62TCF exhibits dual component specificity with contacts to DNA and an extended structure in the DNA-binding domain of p67SRF; Shaw PE; The serum response element (SRE) plays an essential role in the transcriptional regulation of proto-oncogene c-fos . A ternary complex, consisting of transcription factors p67SRF and p62TCF bound to the SRE is present in several cell types and its formation has been correlated with inducibility of the gene in different cells by serum, epidermal growth factor and phorbol esters . Interaction of p62TCF with the SRE in vitro exhibits both a degree of sequence specificity and a strict dependence on the presence of bound p67SRF . A 90 amino acid DNA-binding domain of p67SRF (coreSRF) suffices for ternary complex formation . DNase I footprinting and UV-mediated DNA-protein crosslinking experiments presented here show that direct DNA contacts are made by p62TCF with the 5' sequence of the SRE and thus explain the sequence dependence of ternary complex formation . Additionally, analysis of ternary complex formation by chimaeras of coreSRF and the related yeast protein ArgRI as well as comprehensive mutagenesis of non-conserved residues between the two proteins has yielded a coreSRF mutant specifically unable to interact with p62TCF and demonstrates that an extended structure in coreSRF is required for this interaction . Thus p62TCF exhibits dual component specificity in ternary complex formation over the c-fos SRE.

Genomics, 1992 Aug, 13(4), 957 - 61
Identification of new markers in Xp21 between DXS28 (C7) and DMD; Worley KC et al.; Characterization of Xp21 distal to Duchenne muscular dystrophy (DMD) in the region containing the genes for adrenal hypoplasia congenita (AHC) and glycerol kinase deficiency (GKD) has been limited due to a paucity of probes . Two probes were localized between DXS28 (C7) and AHC, the yeast artificial chromosome insert YHX39 (DXS727) and the polymorphic phage clone QST59 (DXS319) . A genomic clone, FT1 (DXS726), 3' to DMD, was also characterized . Portions of the three probes were sequenced and primer pairs were generated to amplify a sequence-tagged site within each probe . Amplification of DNA from patients confirmed the deletion results obtained by Southern blot analysis, and these three sequence-tagged sites were successfully combined for triplex PCR . In addition to facilitating molecular genetic diagnosis in Xp21, these probes can be used to identify additional YACs and other probes to further increase the genomic information and diagnostic capabilities in this region.

Genomics, 1992 Aug, 13(4), 942 - 50
Determination of the exon structure of the distal portion of the dystrophin gene by vectorette PCR; Roberts RG et al.; The structure of the 3' one-third of the dystrophin gene has not previously been established . We have used vectorette PCR on a yeast artificial chromosome containing part of the human dystrophin gene to determine that there are 20 exons in this region and to characterize adjacent intron sequences of each one . Combined with previous information on the remainder of the gene, this study shows that the coding sequence is distributed between 79 exons . We have used PCR between exons to measure the distances that separate the more closely clustered exons . Vectorette PCR products were used as probes on Southern blots to assign all the 3' exons to genomic HindIII fragments that are commonly detected in the analysis of dystrophin gene deletions . The results will be useful for determining the effect of genomic deletions on the translational reading frame, for setting up genomic PCR assays to confirm point mutations, for analyzing splice site mutations, and for investigating potential cis-acting elements involved in tissue-specific alternative splicing . Vectorette PCR using primers derived from cDNA sequence represents an efficient and widely applicable method for establishing gene structure and obtaining intron sequence flanking exons, starting from a genomic clone and a cDNA sequence.

Clin Geriatr Med, 1992 Aug, 8(3), 513 - 27
Oral candidiasis; Peterson DE; Candida spp . can frequently cause oral infections in the elderly . A number of factors, including yeast virulence factors and compromised host defenses, contribute to outcomes of clinical disease . Precise mechanisms that determine the varied clinical appearances of oral candidiasis have not been delineated fully . Oral candidiasis should be suspected at the clinical level when oral mucosal lesions consistent with the various presentations of candidiasis are observed in patients at risk . Culture remains the gold standard for assessment, although results may be equivocal . Topical or systemic antifungal therapy may result in resolution of symptoms and lesions, but lesions may recur if underlying risk factors remain.

J Cell Biol, 1992 Aug, 118(4), 785 - 94
cdc25 is a nuclear protein expressed constitutively throughout the cell cycle in nontransformed mammalian cells; Girard F et al.; A family of proteins homologous to the cdc25 gene product of the fission yeast bear specific protein tyrosine phosphatase activity involved in the activation of the p34cdc2-cyclin B kinase . Using affinity-purified antibodies raised against a synthetic peptide corresponding to the catalytic site of the cdc25 phosphatase, we show that cdc25 protein is constitutively expressed throughout the cell cycle of nontransformed mammalian fibroblasts and does not undergo major changes in protein level . By indirect immunofluorescence, cdc25 protein is found essentially localized in the nucleus throughout interphase and during early prophase . Just before the complete nuclear envelope breakdown at the prophase-prometaphase boundary, cdc25 proteins are redistributed throughout the cytoplasm . During metaphase and anaphase, cdc25 staining remains distributed throughout the cell and excludes the condensed chromosomes . The nuclear locale reappears during telophase . In light of the recent data describing the cytoplasmic localization of cyclin B protein (Pines, J., and T . Hunter . 1991 . J . Cell Biol . 115:1-17), the data presented here suggest that separation in two distinct cellular compartments of the cdc25 phosphatase and its substrate p34cdc2-cyclin B may be of importance in the regulation of the cdc2 kinase activity.

Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 7247 - 51
Phorbol ester-induced amino-terminal phosphorylation of human JUN but not JUNB regulates transcriptional activation; Franklin CC et al.; Phorbol ester tumor promoters activate gene transcription by regulating both the synthesis and posttranslational modification of the activator protein 1 (AP-1) transcription factor, c-Jun and JunB are components of the mammalian AP-1 complex . Here we demonstrate that in U-937 human leukemic cells, phorbol esters stimulate the phosphorylation of the amino terminus of human c-Jun (JUN) but not human JunB (JUNB) . Mutational analysis indicates that serine-63 and -73, which reside within the putative regulatory domain of JUN, are required for both constitutive and phorbol 12-myristate 13-acetate-inducible N-terminal JUN phosphorylation . To determine the functional role of this N-terminal phosphorylation, we prepared several chimeric proteins containing the N-terminal 84 amino acids (positions 5-89) of human JUN or murine JUNB fused to the yeast GAL4 DNA-binding domain . This region was found to be sufficient for the phorbol ester-inducible transcriptional activity of JUN, but not JUNB . This induction was abolished by the mutation of serine-63 and -73 to leucine residues . Thus, we propose that phorbol esters enhance the trans-activation potential of JUN, but not JUNB, by the phosphorylation of the N-terminal regulatory domain of JUN.

Vet Med (Praha), 1992 Aug, 37(8), 427 - 33
{Use of parasitic wasps (Hymenoptera: Pteromalidae) in the biological control of domestic flies in pig housing}; Muska M; Adaptability of two parasitoid species S . nigroaenea and M . zaraptor to conditions of stable microclimate was investigated in a farrowing house . The colony was reared in an insectary at a temperature of 24-26 degrees C and relative humidity of 60-70% in cages of the size 0.3 x 0.3 x 0.2 m . The development of the species M . zaraptor from egg to adult lasted 19 to 23 days, in S . nigroaenea it was 23 to 25 days . Rates of parasitism of house fly pupae were followed in plastic pots (8 x 4 x 9 cm) with larval medium for fly rearing . The larval medium consisted of milk powder, wheat bran and dried yeast . Both species were demonstrated to be able to penetrate to pupae in deeper layers of the medium (Tab . 1) and in this way to control the amounts of fly adults after their eclosion . The results were evaluated by Student's t-test . 98% of flies in the stable were house flies (Musca domestica L.), the remaining flies were stable flies (Stomoxys calcitrans L.) . No parasitoids were observed in the stable . Two pots containing 2-3,000 pupae parasitized by S . nigroaenea and M . zaraptor were placed in the stable on 30th January 1990 . Parasitoids were monitored at three locations of the stable according to parasitism rates of lab-reared house fly pupae exposed in plastic pots with larval medium . Twenty-two checks were made in 2 to 4 week intervals from February to November 1990, and one final check after a six-month interruption (Tab . II) . Both species persisted in the stable for the whole period of observation (Fig . 1) . The species S . nigroaenea, the population levels of which were much higher, showed the greater migration activity after its individuals had been released to the stable . Sprayings with the Alfacron insecticide were performed in the stable in March and September 1990 in the course of this experiment.

Genet Anal Tech Appl, 1992 Aug, 9(4), 103 - 6
A simple method for randomly mutating cloned DNA fragments by using chemical mutagens and the polymerase chain reaction; Deshler JO; Random mutagenesis of cloned DNA fragments can be an important tool for understanding genes and/or gene products . I report here a simple procedure for generating random mutations in any cloned DNA fragment in vitro . This method utilizes the polymerase chain reaction (PCR) to amplify chemically damaged single-stranded DNA containing the DNA fragment of interest . A mutagenized pool of plasmid DNA is produced by cloning the PCR product (containing chemically induced sequence heterogeneity) into a new vector . Therefore, extreme chemical treatments can be used that biologically inactivate the original vector . In this report, 10% of the plasmids contained a mutation in a 97-nucleotide DNA insert, giving a mutagenesis frequency of 10(-3)/base pair . Of 3000 yeast transformants screened, seven intron mutations were isolated that activate a cryptic 3' splice site, suggesting that 2% of the mutations could give rise to the desired phenotype . The seven mutations included transitions, transversions and single nucleotide deletions, demonstrating the well-balanced repertoire of nucleotide changes derived from this procedure.

J Ethnopharmacol, 1992 Aug, 37(1), 71 - 6
Preliminary pharmacologic evaluation of crude whole plant extracts of Elephantopus scaber . Part I: In vivo studies; Poli A et al.; Elephantopus scaber has been used in Brazil as a traditional remedy to cause diuresis, antipyresis and to eliminate bladder stones . In the current study, aqueous and hydroalcoholic extracts of whole plants were tested for acute toxicity, analgesic, antipyretic, antiinflammatory, cardiovascular, diuretic and constipating activities . Both extracts (0.3-6 g/kg i.p.) induced writhing, loss of muscle tone, ataxia, prostration and death in mice . No analgesic effects of these extracts were detected using mouse hot-plate and acetic acid-induced writhing tests . Both extracts failed to modify diuresis or carrageenan-induced rat paw edema . In contrast, given intraperitoneally, both reduced brewer's yeast-induced hyperthermia in rats, but when given orally did not affect it . Moreover, the aqueous extract decreased the intestinal transit time in mice while the hydroalcoholic extract increased it . Finally, these extracts, given intravenously, reduced blood pressure and heart rate in rats; these effects could be blocked by atropine but not by co-administration of pyrilamine and cimetidine.

Klin Monatsbl Augenheilkd, 1992 Aug, 201(2), 122 - 4
{Endogenous candida endophthalmitis in a drug dependent patient: intravenous therapy with liposome encapsulated amphotericin B}; Neppert B et al.; We report this exemplary case of a drug addict with candida endophthalmitis where we used the new form of amphotericin B, encapsulated in liposomes . We were able to reconfirm the reduced number of side effects and the minimized nephrotoxicity reported by authors of other specialties . In our patient, a reduction or elimination of the yeast was probably achieved, nevertheless, he developed a traction retinal detachment . In future cases of fungal endophthalmitis, we recommend liposomal amphotericin B in higher doses.

Eur J Cell Biol, 1992 Aug, 58(2), 429 - 44
The lethal prune/Killer-of-prune interaction of Drosophila causes a syndrome resembling human neurofibromatosis (NF1); Hackstein JH; The eye color mutant prune (pn) of Drosophila melanogaster shows a lethal interaction with the Killer-of-prune (K-pn) allele of the abnormal wing disc (awd) locus . The awd gene is the Drosophila homologue of the mammalian tumor metastasis gene nm23, and it has been postulated that pn encodes a protein with similarity to a GAP, a GTPase-activating protein . Such GAPs potentially control Ras-like proteins, which are important molecular switches . However, there is only a low sequence homology with the genes for human GAP and neurofibromatosis (NF1), and with yeast IRA1 and IRA2, and there is no evidence for the functional significance of this homologization . I now show that pn mutations lower the concentrations of larval pteridines, and that this phenomenon is enhanced by two orders of magnitude by the lethal interaction between pn and awdK-pn . These gradual effects on the pteridin concentrations indicate a corresponding drop of the pools of free GTP, and favor the involvement of GTP-binding proteins . In addition, cytology reveals a considerable hypertrophy of the neuroglia and the perineurium of the larval brain . Furthermore, the lymph glands of the larvae are highly abnormal and form melanotic (pseudo)tumors upon ageing of the larvae . These pseudotumors consist predominantly of lamellocytes which are part of the cellular defence system of Drosophila . These observations most likely indicate hyperactivity of a Ras-like protein which becomes manifest in cell types equivalent to the cell types affected by human neurofibromatosis (NF1) . Thus, it is very suggestive to regard the synthetic lethal system prune/Killer-of-prune as the Drosophila model for human neurofibromatosis.

Curr Opin Cell Biol, 1992 Aug, 4(4), 593 - 9
Regulation of intracellular membrane transport; Gruenberg J et al.; A number of proteins that are necessary for membrane transport have been identified using cell-free assays and yeast genetics . Although our knowledge of transport mechanisms remains limited, common themes are clearly emerging . In particular, specific GTP-binding proteins appear to be involved, not only at all steps of membrane traffic but also at more than one check-point within each step . The ordered sequence of events occurring during vesicle formation, targeting and fusion may be regulated in a stepwise manner by specific GTP-dependent switches, which act as modular elements of the transport mechanism.

J Bioenerg Biomembr, 1992 Aug, 24(4), 407 - 14
Structural conservation and functional diversity of V-ATPases; Nelson N; The vacuolar system of eukaryotic cells contains a large number of organelles that are primary energized by an H(+)-ATPase that was named V-ATPase . The structure and function of V-ATPases from various sources was extensively studied in the last few years . Several genes encoding subunits of the enzyme were cloned and sequenced . The sequence information revealed the relations between V-ATPases and F-ATPases that evolved from common ancestral genes . The two families of proton pumps share structural and functional similarity . They contain distinct peripheral catalytic sectors and hydrophobic membrane sectors . Genes encoding subunits of V-ATPase in yeast cells were interrupted to yield mutants that are devoid of the enzyme and are sensitive to pH and calcium concentrations in the medium . The mutants were used to study structure, function, molecular biology, and biogenesis of the V-ATPase . They also shed light on the functional assembly of the enzyme in the vacuolar system.

Bioessays, 1992 Aug, 14(8), 565 - 71
Genes, genes and more genes in the human major histocompatibility complex; Milner CM et al.; The human major histocompatibility complex (MHC), on the short arm of chromosome 6, represents one of the most extensively characterised regions of the human genome . This approximately 4 Mb segment of DNA contains genes encoding the polymorphic MHC class I and class II molecules which are involved in antigen presentation during an immune response . Recently the whole of the MHC has been cloned in cosmids and/or yeast artificial chromosomes (YACs) and large portions have been characterised for the presence of novel genes . Many unrelated genes, both housekeeping and tissue specific, have been identified and the gene density in some regions is now approaching one gene every few kilobases . Some of the novel genes encode proteins involved in the intracellular processing and transport of antigens that are presented by MHC class I molecules . Others, however, have no obvious role in the immune response . The MHC is located in the chromosome band 6p21.3 which is a Giemsa (G)-light band . The detection of such a large number of functional genes (at least 70) in this region is compatible with the idea that both housekeeping and tissue-specific genes are localised predominantly in G-light bands.

Curr Genet, 1992 Aug, 22(2), 135 - 40
Transmission of mitochondrial DNA in Ustilago violacea; Wilch G et al.; Mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) were used as genetic markers for following mitochondrial transmission in the basidiomycete Ustilago violacea . Yeast-like cells of opposite mating types (a1 and a2) were mated on 2% water agar and were treated with alpha-tocopherol to induce formation of dikaryotic hyphae . Upon depletion of the alpha-tocopherol, the hyphae budded off haploid cells with parental nuclear genotypes . These cells were examined for mitochondrial RFLP phenotype . In progeny expressing the a1 mating type, mitochondria from either parent were observed equally frequently . In progeny with the a2 mating type, mitochondria were almost exclusively (94%) from the a2 parent.

Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 7169 - 73
The A alpha mating locus of Schizophyllum commune encodes two dissimilar multiallelic homeodomain proteins; Stankis MM et al.; The A alpha mating locus is one of four multiallelic loci that govern sexual development in the basidiomycete fungus Schizophyllum commune . We have determined the nucleotide sequence encoding three A alpha mating types, A alpha 1, A alpha 3, and A alpha 4 . We have found that the locus for A alpha 3 and A alpha 4 consists of two genes: Y and Z . The locus for A alpha 1 encodes only one gene, Y . The Z polypeptides encoded by different alleles exhibit 42% identity . The Y polypeptides exhibit 49-54% identity . The finding that the deduced Z and Y polypeptides have homeodomain motifs suggests that these polypeptides may be DNA-binding regulatory proteins that control the expression of developmental genes . The deduced Z polypeptide also has acidic regions that might be functionally analogous to the acidic regions in yeast GAL4 and GCN4 that activate transcription . The Y polypeptide has a serine-rich region and a basic region that shows some identity to the lysine-rich region of H1 histones.

J Biochem (Tokyo), 1992 Aug, 112(2), 243 - 8
Mitochondrial porin can be translocated across both endoplasmic reticulum and mitochondrial membranes; Sakaguchi M et al.; Mitochondrial porin is a major integral membrane protein of the outer membrane . To assess the stop-transfer sequence in the yeast porin molecule (P), we constructed the following chimeric proteins . (i) The signal sequence of interleukin 2, a secretory protein, was fused to the amino-terminus of porin (SP) . (ii) The matrix targeting presequence of cytochrome c oxidase subunit IV was fused to the amino-terminus of porin (CP) . (iii) The amino-terminal segment consisting of 42 amino acid residues of "70 kDa protein" of yeast mitochondria, a major membrane protein of the outer membrane, was introduced into the middle portion of interleukin 2 (IL70) . These chimeric proteins were expressed with an in vitro transcription-translation system and their integration into microsomal membrane or mitochondrial membranes was examined . When the proteins were synthesized in vitro with wheat germ cell-free system in the presence of rough microsomal membrane (RM), SP was completely translocated across the membrane, processed by the signal peptidase, and glycosylated . The translocation of IL70 molecule across RM was stopped at the introduced amino-terminal segment of 70 kDa protein . The authentic porin did not interact with the microsomal membrane . To assess the interaction with mitochondria, porin and CP were synthesized with the reticulocyte lysate system and subjected to posttranslational import reaction with isolated rat liver mitochondria . The authentic porin was integrated into the outer membrane in an alkali-resistant fashion . CP was imported into the mitochondria and its presequence was cleaved by the processing protease in the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

J Antimicrob Chemother, 1992 Aug, 30(2), 181 - 8
Effects of itraconazole on phagocytosis and killing of Candida glabrata by polymorphonuclear leucocytes from guinea pigs; Xhonneux B et al.; Itraconazole, a systemically active antifungal, was tested for its effects on microscopically assessed phagocytosis and killing of Candida glabrata 233 in vitro . Yeast cells were exposed to itraconazole in culture and guinea-pig peritoneal polymorphonuclear leucocytes were exposed to the drug injected intraperitoneally in vivo . At a concentration of 10(-7) M and with exposure times of 1 h, itraconazole pre-treatment of the leucocytes had no effect on the ability of PMNL to ingest or kill C . glabrata . However, pre-treatment of the growing C . glabrata cells under the same conditions significantly increased their vulnerability to both phagocytosis and intracellular killing . Longer exposures of the yeasts to itraconazole further increased their susceptibility to leucocyte phagocytosis, and it also rendered the cells vulnerable to killing merely by immersion in sodium deoxycholate solution . These findings indicate that short exposures of C . glabrata to low itraconazole concentrations damages the cells sublethally and renders them highly susceptible to leucocyte killing . Itraconazole had no direct effects on leucocyte function itself.

Biochem J, 1992 Aug 1, 285 ( Pt 3), 985 - 92
Mouse UDP-GlcNAc: dolichyl-phosphate N-acetylglucosaminephosphotransferase . Molecular cloning of the cDNA, generation of anti-peptide antibodies and chromosomal localization; Rajput B et al.; A cDNA encoding UDP-GlcNAc-dolichyl-phosphate N-acetylglucosaminephosphotransferase (GPT; EC 2.7.8.15), an enzyme that catalyses the first step in the synthesis of dolichol-linked oligosaccharides, was isolated from mRNA prepared from mouse mammary glands . The cDNA contains an open reading frame that codes for a protein of 410 amino acids with a predicted molecular mass of 46.472 kDa . Mouse GPT has two copies of a putative dolichol-recognition sequence that has so far been identified in all eukaryotic enzymes which interact with dolichol, and four consensus sites for asparagine-linked glycosylation . It shows a high degree of conservation with yeast and hamster GPTs at the amino acid level . The mouse GPT cDNA recognized a single mRNA species of about 2 kb in mouse mammary glands when used as a probe in Northern blot analysis . An antiserum raised against a 15-residue peptide, derived from the predicted amino acid sequence of the cloned mouse cDNA, specifically precipitated the activity of GPT from solubilized mouse mammary gland microsomes, and detected a protein of about 48 kDa on Western blot . This size is in good agreement with that predicted from the cDNA sequence, and also with that (46 and 50 kDa) of purified bovine GPT . With the use of a panel of mouse/hamster somatic-cell hybrids and a specific probe derived from the 3'-non-coding region of the mouse cDNA, the GPT gene was mapped to mouse chromosome 17.

EMBO J, 1992 Aug, 11(8), 2903 - 8
Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation; Kosako H et al.; Xenopus MAP kinase activator, a 45 kDa protein, has been shown to function as a direct upstream factor sufficient for full activation and both tyrosine and serine/threonine phosphorylation of inactive MAP kinase . We have now shown by using an anti-MAP kinase activator antiserum that MAP kinase activator is ubiquitous in tissues and is regulated post-translationally . Activation of MAP kinase activator is correlated precisely with its threonine phosphorylation during the oocyte maturation process . It is a key question whether MAP kinase activator is a kinase or not . We have shown that Xenopus MAP kinase activator purified from mature oocytes is capable of undergoing autophosphorylation on serine, threonine and tyrosine residues . Dephosphorylation of purified activator by protein phosphatase 2A treatment inactivates its autophosphorylation activity as well as its activator activity . Thus, Xenopus MAP kinase activator is a protein kinase with specificity for both serine/threonine and tyrosine . Partial protein sequencing of purified activator indicates that it contains a sequence homologous to kinase subdomains VI and VII of two yeast protein kinases, STE7 and byrl.

FEBS Lett, 1992 Jul 28, 307(2), 131 - 4
The WD-40 repeat; van der Voorn L et al.; An amino acid sequence motif, called the WD-40 repeat, has been found as a repeat in a large variety of proteins that do not share any obvious functional properties . At present, the function of the repeated motif is not known for any of these proteins . Interestingly, recent experiments in yeast indicate that several proteins containing the WD-40 repeat are genetically associated with members of the TPR-family, a protein family that is characterized by the presence of another repeated motif of unknown function: the tetratricopeptide repeat . It is conceivable that proteins containing the WD-40 repeat interact physically with members of the TPR-family via their respective repeated motifs.

FEBS Lett, 1992 Jul 27, 307(1), 34 - 9
Crystallographic binding studies with triosephosphate isomerases: conformational changes induced by substrate and substrate-analogues; Wierenga RK et al.; TIM catalyses the interconversion of a triosephosphate aldehyde into a triosephosphate ketone . This is a simple chemical reaction in which only protons are transferred . The crystallographic studies of TIM from chicken, yeast and trypanosome complexed with substrate and substrate analogues are discussed . The substrate binds in a deep pocket . On substrate binding, large conformational changes are induced in three loops . As a result of these conformational changes in the liganded structure, the active site pocket is sealed off from bulk solvent and the sidechain of the catalytic glutamate becomes optimally positioned for catalysis.

Nucleic Acids Res, 1992 Jul 25, 20(14), 3763 - 72
Sequence and variability of the 5.8S and 26S rRNA genes of Pneumocystis carinii; Liu Y et al.; The sequence of the coding region of the rRNA operon of rat-derived Pneumocystis carinii has been completed, including the genes for 5.8S and 26S rRNA . These genes show homology to the rRNA genes of yeast, and an apparent group I self-splicing intron is present in the 26S rRNA gene . Like a similar intron in the 16S rRNA gene, this intron is in a phylogenetically conserved region . Variation in the 26S rRNA sequence was noted between P . carinii organisms isolated from two different sources.

J Biol Chem, 1992 Jul 25, 267(21), 15229 - 36
Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family; Bennett DL et al.; Enzymological studies have implicated two Ca(2+)-dependent endopeptidases in the conversion of proinsulin to insulin; a type 1 activity which cleaves on the C-terminal side of Arg31-Arg32 and a type 2 activity which cleaves C-terminally to Lys64-Arg65 in the proinsulin sequence . The possibility that these enzymes are related to the recently discovered family of mammalian subtilisin-like gene products (furin, PC2, and PC3) and the yeast propheromone-converting enzyme (KEX-2), was investigated . Degenerate oligonucleotide primers flanking the putative catalytic domain within this gene family were used in a polymerase chain reaction to amplify related sequences from rat insulinoma cDNA . One major product of 700 base pairs was obtained which was greater than 99% identical to the corresponding rat PC2 sequence . This cDNA was subcloned into the bacterial expression vector pGEX-3X to generate a recombinant protein for antibody production . Western blot analysis showed the immunoreactivity was prominent in neuroendocrine tissues as a 65-kDa protein . It was concentrated in secretory granule-enriched fractions of insulinoma tissue, where it was present as a readily solubilized monomeric protein . Deglycosylation studies using endoglycosidase H and N-glycanase showed that the 65-kDa protein was comprised of approximately 9% carbohydrate, consistent with the presence of three consensus sequences for N-linked glycosylation in rat PC2 . The immunoreactivity co-eluted with the type 2 proinsulin endopeptidase on gel filtration and ion-exchange chromatography and the antisera specifically immunoprecipitated type 2 activity from insulin granule extracts . N-terminal sequence analysis of the immunoreactive protein gave two sequences which corresponded to residues 109-112 and 112-119 of rat PC2 . This indicated that posttranslational processing of PC2 itself occurs C-terminally to basic amino acids to produce the mature enzyme . It is concluded that PC2 is the type 2 endopeptidase involved in proinsulin conversion . Localization of PC2 immunoreactivity to other tissues of the diffuse neuroendocrine system suggests that the type 2 endopeptidase also functions in the processing of precursor forms of other prohormones and polypeptide neurotransmitters.

J Mol Biol, 1992 Jul 20, 226(2), 349 - 66
Sequence-specific DNA binding by a two zinc-finger peptide from the Drosophila melanogaster Tramtrack protein; Fairall L et al.; We show that the DNA-binding domain of the Drosophila melanogaster regulatory protein Tramtrack consists of a 66 amino acid sequence containing two zinc-finger motifs and a short sequence N-terminal to the first finger motif . This short N-terminal sequence is essential for DNA binding and we suggest it is involved in maintaining the three-dimensional structure of the first finger domain, as has been seen in the nuclear magnetic resonance structure of one of the zinc-finger domains of the yeast transcription factor SW15 . The characterization of the DNA-binding activity of this 66 residue peptide (delta 911zf) shows that it binds in a sequence-specific manner, as a monomer, to a natural target site with an apparent KD approximately 4 x 10(-7) M . The shortest delta 911zf binding site, which retains full affinity, consists of an 11 base-pair sequence with a one nucleotide overhang at each 5' end . DNase I, hydroxyl radical and methylation protection footprinting studies show that, in common with other zinc-finger proteins, delta 911zf binds in the major groove of DNA . The data presented are consistent with the zinc-fingers of Tramtrack contacting both strands of the DNA, and thus the binding differs in detail to that observed in the crystal structure of the three zinc-fingers of Zif268 complexed to their target DNA.

FEBS Lett, 1992 Jul 20, 306(2-3), 181 - 4
Molecular cloning of a novel ras-like protein from chicken; Trueb J et al.; We have isolated a cDNA clone from a chicken DNA expression library which codes for a ras-like polypeptide of 216 amino acid residues . This polypeptide is closely related to the human protein TC4 and to the yeast protein Spil, two novel proteins that may be involved in the coordination of the cell cycle . In the amino-terminal region, the three polypeptides possess a P-loop motif characteristic of GTP-binding proteins . At the carboxy-terminal end, however, they lack the typical CAAX-box which is usually responsible for membrane anchorage of ras-like proteins . It is therefore likely that the three polypeptides define a new subclass of GTP-binding proteins within the ras-like superfamily.

Nature, 1992 Jul 16, 358(6383), 249 - 52
T-complex polypeptide-1 is a subunit of a heteromeric particle in the eukaryotic cytosol; Lewis VA et al.; The murine t-complex encodes t-complex polypeptide-1 (TCP1), which is constitutively expressed in almost all cells, and upregulated during spermatogenesis . Mammalian sequences have greater than 96% identity with each other, and greater than 60% identity with Drosophila melanogaster and yeast orthologues . TCP1 is essential in yeast, and is postulated to be the cytosolic mammalian equivalent of groEL . We report here that, in the native state, murine and human TCP1 is distributed throughout the cytosol as an 800K-950K hetero-oligomeric particle in association with four to six unidentified proteins and two Hsp70 heat-shock proteins . Negative-stain electron microscopy indicates that the structure is two stacked rings, 12-16 nm in diameter . Therefore, despite similarities with the chaperonin 60 proteins, these data indicate that TCP1 is biochemically and structurally unique . We suggest that TCP1 may represent one of a family of molecules in the eukaryotic cytosol involved in protein folding and regulated in part by their heteromeric associations.

Nature, 1992 Jul 16, 358(6383), 239 - 42
The Lowe's oculocerebrorenal syndrome gene encodes a protein highly homologous to inositol polyphosphate-5-phosphatase; Attree O et al.; Lowe's oculocerebrorenal syndrome (OCRL) is a human X-linked developmental disorder of unknown pathogenesis and has a pleiotropic phenotype affecting the lens, brain and kidneys . The OCRL locus has been mapped to Xq25-q26 by linkage and by finding de novo X; autosome translocations at Xq25-q26 in two unrelated females with OCRL . Here we use yeast artificial chromosomes with inserts that span the X chromosomal breakpoint from a female OCRL patient in order to isolate complementary DNAs for a gene that is interrupted by the translocation . We show that the transcript is absent in both female OCRL patients with X; autosome translocations and that it is absent or abnormally sized in 9 of 13 unrelated male OCRL patients with no detectable genomic rearrangement . The open reading frame encodes a new protein with 71% similarity to human inositol polyphosphate-5-phosphatase . Our results suggest that OCRL may be an inborn error of inositol phosphate metabolism.

Biochem J, 1992 Jul 15, 285 ( Pt 2), 391 - 4
A member of the eukaryotic subtilisin family (PC3) has the enzymic properties of the type 1 proinsulin-converting endopeptidase; Bailyes EM et al.; PC3, a mammalian homologue of the yeast subtilisin-like proteinase Kex2, was expressed in Xenopus oocytes and its activity was characterized . PC3 cleaved human proinsulin at one of the two dibasic sites (KTRR32 but not LQKR65) . The specificity, inhibitor profile, pH optimum (5.5) and Ca(2+)-dependence (K0.5 = 2.5-3 mM) paralleled those of the insulin-granule type 1 endopeptidase activity, suggesting a role for PC3 in the conversion of prohormones.

Eur J Biochem, 1992 Jul 15, 207(2), 615 - 20
A rat cerebellar protein containing the cdc10/SWI6 motif; Taoka M et al.; Systematic analysis of soluble proteins in developing rat cerebellum by an automated two-dimensional liquid-chromatography system detected a number of proteins which increased transiently during the initial stage of postnatal development . One of the proteins, V-1, was isolated using a liquid-chromatography system, and its amino acid sequence was determined by analysis of the purified protein . The sequence showed that the V-1 protein consists of 117 amino acids with an acetylated N-terminus, and has 2.5 internal sequence repeats of 33 amino acids . Computer retrieval of the sequence indicated that the repeated sequences have a structural characteristics of the cdc10/SWI6 motif, which is found in a series of proteins, including those involved in cell-cycle control and cell-fate determination in yeast, Drosophila melanogaster and Caenorhabditis elegans . The structure of V-1, coupled with its controlled expression in early postnatal development, implies a potential role for V-1 in cerebellar morphogenesis.

Proc Natl Acad Sci U S A, 1992 Jul 15, 89(14), 6403 - 7
Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity; Kato K et al.; We have introduced a variety of amino acid substitutions into carboxyl-terminal CA1A2X sequence (C = cysteine; A = aliphatic; X = any amino acid) of the oncogenic {Val12}Ki-Ras4B protein to identify the amino acids that permit Ras processing (isoprenylation, proteolysis, and carboxyl methylation), membrane association, and transformation in cultured mammalian cells . While all substitutions were tolerated at the A1 position, substitutions at A2 and X reduced transforming activity . The A2 residue was important for both isoprenylation and AAX proteolysis, whereas the X residue dictated the extent and specificity of isoprenoid modification only . Differences were observed between Ras processing in living cells and farnesylation efficiency in a cell-free system . Finally, one farnesylated mutant did not undergo either proteolysis or carboxyl methylation but still displayed efficient membrane association (approximately 50%) and transforming activity, indicating that farnesylation alone can support Ras transforming activity . Since both farnesylation and carboxyl methylation are critical for yeast a-factor biological activity, the three CAAX-signaled modifications may have different contributions to the function of different CAAX-containing proteins.

J Immunol, 1992 Jul 15, 149(2), 548 - 55
A carboxyl-terminal fragment of Plasmodium falciparum gp195 expressed by a recombinant baculovirus induces antibodies that completely inhibit parasite growth; Chang SP et al.; The major merozoite surface Ag (gp195) of Plasmodium falciparum has been shown to protect monkeys against parasite infection, and gp195-based synthetic peptides and recombinant polypeptides have been evaluated as potential malaria vaccines . A major problem in developing a gp195-based recombinant vaccine has been the difficulty in obtaining a recombinant polypeptide that is immunologically equivalent to the native protein . In this study, the carboxyl-terminal processing fragment (p42) of gp195 was produced in yeast and in a baculovirus recombinant system . Immunologic analyses indicated that the secreted baculovirus p42 (BVp42) expressed native, disulfide-dependent conformational epitopes, whereas these epitopes were poorly represented in the intracellular yeast p42 . BVp42, but not yeast p42, was also recognized by the majority of gp195-specific antibodies of animals immunized with purified native gp195, indicating that the anti-gp195 response of these animals was focused on conformational determinants of the p42 processing fragment . Sera against native gp195 of congenic mice of diverse H-2 haplotypes recognized the BVp42 polypeptide, demonstrating that a genetically heterogeneous population is capable of responding to p42 epitopes . BVp42 was highly immunogenic and induced high titers of antibodies that were cross-reactive with purified native gp195 in an ELISA and also reacted with schizonts and merozoites by immunofluorescence . Anti-BVp42 antibodies completely inhibited the in vitro growth of the malaria parasite, whereas anti-yeast p42 antibodies had no effect . These results indicate that native, conformational epitopes of p42 are critical for the induction of gp195-specific, parasite growth-inhibitory antibodies and that the BVp42 polypeptide efficiently induces antibodies specific for these native determinants.

Infect Immun, 1992 Jul, 60(7), 2565 - 71
An 80-kilodalton antigen from Histoplasma capsulatum that has homology to heat shock protein 70 induces cell-mediated immune responses and protection in mice; Gomez FJ et al.; An extract of the cell wall and cell membrane from Histoplasma capsulatum yeast cells was assayed by Western blot (immunoblot) for reactivity with two monoclonal antibodies to heat shock protein 70 . Four bands with molecular masses of 80, 66, 54, and 32 kDa bound both antibodies . The 80-kDa protein was isolated, analyzed for homology to heat shock protein 70, and tested for antigenicity and immunogenicity in C57BL/6 mice . The 80-kDa protein reacted with monoclonal antibody to heat shock protein 70 . Sera from mice immunized with the antigen recognized H . capsulatum heat shock protein 70 . Moreover, the amino-terminal sequence of the 80-kDa protein revealed substantial homology with heat shock protein 70 from several species . The 80-kDa protein induced delayed-type hypersensitivity responses in mice immunized with either viable yeast cells or antigen . Splenocytes from mice immunized with yeast cells or with antigen responded in vitro to the 80-kDa antigen . Immunization of mice with the antigen enhanced host resistance against a sublethal inoculum of H . capsulatum yeast cells, but it did not reduce the mortality of mice given a lethal challenge of yeast cells . Thus, this antigen manifests homology with members of the heat shock protein 70 family . Furthermore, the 80-kDa protein elicits cellular immune responses to H . capsulatum, and it mediates protective immunity.

Nucleic Acids Res, 1992 Jul 11, 20(13), 3419 - 25
Construction of a human chromosome 4 YAC pool and analysis of artificial chromosome stability; Sleister HM et al.; In order to construct a human chromosome 4-specific YAC library, we have utilized pYAC4 and a mouse/human hybrid cell line HA(4)A in which the only human chromosome present is chromosome 4 . From this cell line, approximately 8Mb of chromosome 4 have been cloned . The library includes 65 human-specific clones that range in size from 30kb to 290kb, the average size being 108kb . In order to optimize the manipulation of YAC libraries, we have begun to investigate the stability of YACs containing human DNA in yeast cells; these studies will also determine if there are intrinsic differences in the properties of chromosomes containing higher eukaryotic DNAs . We are examining two kinds of stability: 1} mitotic stability, the ability of the YAC to replicate and segregate properly during mitosis, and 2} structural stability, the tendency of the YAC to rearrange . We have found that the majority of YACs examined are one to two orders of magnitude less stable than authentic yeast chromosomes . Interestingly, the largest YAC analyzed displayed a loss rate typical for natural yeast chromosomes . Our results also suggest that increasing the length of an artificial chromosome improves its mitotic stability . One YAC that showed a very high frequency of rearrangement by mitotic recombination proved to be a mouse/human chimera . In contrast to studies using total human DNA, the frequency of chimeras (i.e., mouse/human) in the YAC pool appeared to be low.

Nucleic Acids Res, 1992 Jul 11, 20(13), 3297 - 303
SRF and MCM1 have related but distinct DNA binding specificities; Wynne J et al.; The mammalian transcription factor SRF and the yeast regulatory protein MCM1 contain DNA binding domains that are 70% identical; moreover, both proteins can bind the serum response element in the human c-fos promoter . Here we present an analysis of MCM1 sequence specificity by selection of sites from random sequence oligonucleotides . In this assay the MCM1 DNA binding domain selects binding sites containing the consensus (NotC)CCY(A/T)(A/T)(T/A)NN(A/G)G, distinct from the SRF binding consensus CC(A/T)6GG . Carboxylethylation interference analysis of a set of selected sites suggests that MCM1 contacts DNA in its major groove throughout one helical turn . These differences in specificity are largely due to sequence differences between the N terminal basic parts of the SRF and MCM1 DNA binding domains . Comparison of the relative binding affinities of MCM1 and SRF for a panel of representative binding sites showed that many high affinity MCM1 sites have negligible affinity for SRF and vice versa . Thus MCM1 and SRF have significantly different sequence specificities.

Nucleic Acids Res, 1992 Jul 11, 20(13), 3333 - 9
The AT-rich tract of the SV40 ori core: negative synergism and specific recognition by single stranded and duplex DNA binding proteins; Galli I et al.; The SV40 origin of replication comprises a run of thymine and adenine residues . Integrity of this AT-rich sequence is known to be essential for replication . We set out to study whether or not these elements can work synergistically to sustain replication . Quite surprisingly, additional copies of the AT stretch linked to a functional SV40 ori core dramatically reduce its replication in Cosl cells, probably by creating some physical block . Interestingly, the same inhibiting effect can be observed with the addition in cis of the yeast ARS consensus, which is homologous to the SV40 AT stretch . This modulation is possibly due to the action of cellular factors that recognize either of the two sequences . In fact, we demonstrate the existence of factor(s) in Cosl crude nuclear extracts that in vitro can specifically bind to either of them . Moreover, we show that these sequence-specific factor(s) (MW about 50 kDa), named SOAP, recognize both single (T-rich strand) and double stranded forms of the AT tracts . Binding to single stranded AT stretches can be specifically inhibited by the corresponding duplex form, but not vice versa.

J Mol Biol, 1992 Jul 5, 226(1), 227 - 38
Crystal structure solution and refinement of the semisynthetic cobalt-substituted bovine erythrocyte superoxide dismutase at 2.0 A resolution; Djinovic K et al.; The semisynthetic Co-substituted bovine erythrocyte superoxide dismutase (SOD) has been crystallized in a new crystalline form and the structure determined at 2.0 A (1 A = 0.1 nm) resolution . The crystals belong to space group P2(1)2(1)2(1) with cell constants: a = 51.0, b = 147.6, c = 47.5 A, and contain one dimeric molecule of 32,000 M(r) per asymmetric unit . The structure has been solved by molecular replacement techniques using the Cu,Zn bovine enzyme as a search model, and refined by molecular dynamics with the crystallographic pseudo-energy term, followed by conventional crystallographic refinement . The R-factor for the 18,964 unique reflections in the resolution range from 10.0 to 2.0 A is 0.176 for a model comprising 2188 protein atoms and 200 solvent molecules; the root-mean-square deviation from the ideal bond lengths is 0.010 A, and the average atomic temperature factor is 26.5 A2 . The dimeric molecule of the enzyme is composed of two identical subunits related by a non-crystallographic 2-fold axis . The subunit has as its structural scaffolding the conventional SOD-flattened antiparallel eight-stranded beta-barrel, with three external loops . The co-ordination geometry of the metal center in the active site is fairly well preserved when compared with the native Cu,Zn bovine enzyme . Co2+ is in tetrahedral co-ordination, while the Cu2+ ligands show an uneven distortion from the square planar geometry . The least-squares superposition of the metals ligands and the catalytically important Arg141 of the native and Co-substituted enzyme yields a root-mean-square value of 0.401 A, the largest deviation occurring at the Co2+ ligand Asp81 . An additional copper ligand, compatible with a water molecule, is observed at 2.38 A from Cu2+ in the active-site channel, at the supposed binding site of the O2- anion substrate . Several ordered water molecules have been observed on the protein surface and in the active-site channel; their structural locations coincide remarkably with those of related water molecules found in the crystal structure of the phylogenetically distant superoxide dismutase from yeast.

J Biol Chem, 1992 Jul 5, 267(19), 13772 - 7
Cap structure of U3 small nucleolar RNA in animal and plant cells is different . gamma-Monomethyl phosphate cap structure in plant RNA; Shimba S et al.; U3 small nucleolar RNA (snoRNA) is an abundant small RNA involved in the processing of pre-ribosomal RNA of eukaryotic cells . U3 snoRNA has been previously characterized from several sources, including human, rat, mouse, frog, fruit fly, dinoflagellates, slime mold, and yeast; in all these organisms, U3 snoRNA contains trimethylguanosine cap structure . In all instances where investigated, the trimethylguanosine-capped snRNAs including U3 snoRNA, are synthesized by RNA polymerase II . However, in higher plants, the U3 snoRNA is synthesized by RNA polymerase III and contains a cap structure different from trimethylguanosine (Kiss, T., and Solymosy, F . (1990) Nucleic Acids Res . 18, 1941-1949; Marshallsay, C., Kiss, T., and Filipowicz, W . (1990) Nucleic Acids Res . 18, 3451-3458; Kiss, T., Marshallsay, C., and Filipowicz, W . (1991) Cell 65, 517-526) . In this study, we present evidence that cowpea and, most likely, tomato plant U3 snoRNA contains a methyl-pppA cap structure . These data show that the same U3 snoRNA contains different cap structures in different species and suggest that the kind of cap structure that an uridylic acid-rich small nuclear RNA contains is dependent on the RNA polymerase responsible for its synthesis . In vitro synthesized plant U3 snoRNA, with pppA or pppG as its 5' end, was converted to methyl-pppA/G cap structure in vitro when incubated with extracts prepared from wheat germ or HeLa cells . These data show that the capping machinery is conserved in organisms as evolutionarily distant as plants and mammals . Nucleotides 1-45 of tomato U3 snoRNA, which are capable of forming a stem-loop structure, are sufficient to direct the methyl cap formation in vitro.

J Biol Chem, 1992 Jul 5, 267(19), 13229 - 38
Molecular cloning of human mevalonate kinase and identification of a missense mutation in the genetic disease mevalonic aciduria; Schafer BL et al.; Mevalonic aciduria is the first proposed inherited disorder of the cholesterol/isoprene biosynthetic pathway in humans, and it is presumed to be caused by a mutation in the gene coding for mevalonate kinase . To elucidate the molecular basis of this inherited disorder, a 2.0-kilobase human mevalonate kinase cDNA clone was isolated and sequenced . The 1188-base pair open reading frame coded for a 396-amino acid polypeptide with a deduced M(r) of 42,450 . The predicted protein sequence displayed similarity to those of galactokinase and the yeast RAR1 protein, indicating that they may belong to a common gene family . Southern hybridization studies demonstrated that the mevalonate kinase gene is located on human chromosome 12 and is a single copy gene . No major rearrangements were detected in the mevalonic aciduria subject . The relative size (2 kilobases) and amounts of human mevalonate kinase mRNA were not changed in mevalonic aciduria fibroblasts . Approximately half of the mevalonic aciduria cDNA clones encoding mevalonate kinase contained a single base substitution (A to C) in the coding region at nucleotide 902 that changed an asparagine residue to a threonine residue . The presence of this missense mutation was confirmed by polymerase chain reaction amplification and allele-specific hybridization of the genomic DNAs from the proband and the proband's father and brother . Similar analysis failed to detect this mutation in the proband's mother, seven normal subjects, or four additional mevalonic aciduria subjects, indicating that the mutation does not represent a common gene polymorphism . Functional analysis of the defect by transient expression confirmed that the mutation produced an enzyme with diminished activity . Our data suggest that the index case is a compound heterozygote for a mutation in the mevalonate kinase gene.

Nature, 1992 Jul 2, 358(6381), 70 - 3
Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase; Kuo CJ et al.; The macrolide rapamycin induces cell cycle G1 arrest in yeast and in mammalian cells, which suggests that an evolutionarily conserved, rapamycin-sensitive pathway may regulate entry into S phase . In mammals, rapamycin inhibits interleukin-2 receptor-induced S phase entry and subsequent T-cell proliferation, resulting in immunosuppression . Here we show that interleukin-2 selectively stimulates the phosphorylation and activation of p70 S6 kinase but not the erk-encoded MAP kinases and rsk-encoded S6 kinases . Rapamycin completely and rapidly inhibits interleukin-2-induced phosphorylation and activation of p70 S6 kinase at concentrations comparable to those blocking S phase entry of T cells (0.05-0.2 nM) . The structurally related macrolide FK506 competitively antagonizes the actions of rapamycin, indicating that these effects are mediated by FKBP, which binds the transition-state mimic structure common to both rapamycin and FK506 (refs 4, 6, 9-11) . The selective blockade of the p70 S6 kinase activation cascade by the rapamycin-FKBP complex implicates this signalling pathway in the regulation of T cell entry into S phase.

J Cell Biochem, 1992 Jul, 49(3), 266 - 71
Extremely divergent histone H4 sequence from Trypanosoma cruzi: evolutionary implications; Toro GC et al.; Trypanosoma cruzi presents six histones electrophoretically resolved in three gel systems . Indirect evidence shows that one of these histones, named e, corresponds to H4 in other species . We present evidence that histone e is H4 by sequencing its amino terminal end . The amino terminal of T . cruzi histone H4, unlike that of other H4s examined thus far is not blocked . Moreover, this protein presents two variants . This partial amino acid sequence of T . cruzi histone H4 differs greatly from homologous sequences of human, yeast, or Tetrahymena . Since the conservatism of the core histones (H2A, H2B, H3, and H4) is clearly illustrated by comparative sequence analyses, the data shown here demonstrates that T . cruzi histone H4 is the most divergent reported . Quantitative analysis of the data suggests that the rate of substitutions in the histone H4 amino terminal sequence varies among different lineages . We postulate a slow-down in the evolutionary rate of histone H4 amino terminal domain in the metazoa branch related perhaps to the appearance of a novel function for this domain.

APMIS, 1992 Jul, 100(7), 586 - 92
Disseminated histoplasmosis in a badger (Meles meles) in Denmark; Jensen HE et al.; We report the first case of disseminated histoplasmosis in an animal in Scandinavia . Yeast cells compatible with those of Histoplasma capsulatum var . capsulatum were found in the skin, liver, spleen, a kidney, and a lymph node of a wild badger (Meles meles) . The diagnosis was confirmed by electron microscopy and immunofluorescence staining of the yeast cells in tissue sections.

Genomics, 1992 Jul, 13(3), 826 - 8
Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification; Lengauer C et al.; Alu-PCR protocols were optimized for the generation of human DNA probes from yeast strains containing yeast artificial chromosomes (YACs) with human inserts between 100 and 800 kb in size . The resulting DNA probes were used in chromosome in situ suppression (CISS) hybridization experiments . Strong fluorescent signals on both chromatids indicated the localization of specific YAC clones, while two clearly distinguishable signals were observed in greater than or equal to 90% of diploid nuclei . Signal intensities were generally comparable to those observed using chromosome-specific alphoid DNA probes . This approach will facilitate the rapid mapping of YAC clones and their use in chromosome analysis at all stages of the cell cycle.

Genomics, 1992 Jul, 13(3), 726 - 30
YAC mapping by FISH using Alu-PCR-generated probes; Breen M et al.; Human genomic mapping has been greatly advanced by the independent development of three new methods: large DNA fragment cloning in yeast artificial chromosomes, amplification from complex DNAs of human specific segments by Alu-PCR, and high-resolution localization of complex DNA probes by fluorescent in situ hybridization . We describe here the combination of these three analytical tools for efficient and accurate localization of randomly screened or especially selected human YAC recombinants to chromosome 11 . We map a YAC clone encompassing the pepsinogen A (PGA) locus to 11q13.1-11q13.3.

Acta Cytol, 1992 Jul-Aug, 36(4), 527 - 8
Disseminated histoplasmosis diagnosed by percutaneous needle biopsy of a retroperitoneal lymph node . A case report; Neumann MP et al.; A case of disseminated histoplasmosis diagnosed by percutaneous needle biopsy cytology is reported . The patient presented with fever and pancytopenia . Computed tomography (CT) revealed retroperitoneal lymphadenopathy . Cytology smears prepared from a CT-guided screw needle biopsy of one of the lymph nodes showed numerous histiocytes with intracytoplasmic yeast forms consistent with Histoplasma capsulatum . Fungal cultures prepared from additional needle biopsy material confirmed the diagnosis . This case illustrates the utility of needle biopsy in the evaluation of radiographically detected retroperitoneal lymphadenopathy and in the rapid diagnosis of infectious disease in certain clinical settings.

J Cell Biol, 1992 Jul, 118(2), 347 - 58
Association of calmodulin and an unconventional myosin with the contractile vacuole complex of Dictyostelium discoideum; Zhu Q et al.; mAbs specific for calmodulin were used to examine the distribution of calmodulin in vegetative Dictyostelium cells . Indirect immunofluorescence indicated that calmodulin was greatly enriched at the periphery of phase lucent vacuoles . The presence of these vacuoles in newly germinated (non-feeding) as well as growing cells, and the response of the vacuoles to changes in the osmotic environment, identified them as contractile vacuoles, osmoregulatory organelles . No evidence was found for an association of calmodulin with endosomes or lysosomes, nor was calmodulin enriched along cytoskeletal filaments . When membranes from Dictyostelium cells were fractionated on equilibrium sucrose density gradients, calmodulin cofractionated with alkaline phosphatase, a cytochemical marker for contractile vacuole membranes, at a density of 1.156 g/ml . Several high molecular weight calmodulin-binding proteins were enriched in the same region of the gradient . One of the calmodulin-binding polypeptides (molecular mass approximately 150 kD) cross-reacted with an antiserum specific for Acanthamoeba myosin IC . By indirect immunofluorescence, this protein was also enriched on contractile vacuole membranes . These results suggest that a calmodulin-binding unconventional myosin is associated with contractile vacuoles in Dictyostelium; similar proteins in yeast and mammalian cells have been implicated in vesicle movement.

Mol Cell Biol, 1992 Jul, 12(7), 3130 - 7
Activation domains of L-Myc and c-Myc determine their transforming potencies in rat embryo cells; Barrett J et al.; Members of the Myc family of proteins share a number of protein motifs that are found in regulators of gene transcription . Conserved stretches of amino acids found in the N-terminal transcriptional activation domain of c-Myc are required for cotransforming activity . Most of the Myc proteins contain the basic helix-loop-helix zipper (bHLH-Zip) DNA-binding motif which is also required for the cotransforming activity of c-Myc . L-Myc, the product of a myc family gene that is highly amplified in many human lung carcinomas, was found to cotransform primary rat embryo cells with an activated ras gene . However, L-Myc cotransforming activity was only 1 to 10% of that of c-Myc (M . J . Birrer, S . Segal, J . S . DeGreve, F . Kaye, E . A . Sausville, and J . D . Minna, Mol . Cell . Biol . 8:2668-2673, 1988) . We sought to determine whether functional differences between c-Myc and L-Myc in either the N-terminal or the C-terminal domain could account for the relatively diminished L-Myc cotransforming activity . Although the N-terminal domain of L-Myc could activate transcription when fused to the yeast GAL4 DNA-binding domain, the activity was only 5% of that of a comparable c-Myc domain . We next determined that the interaction of the C-terminal bHLH-Zip region of L-Myc or c-Myc with that of a Myc partner protein, Max, was equivalent in transfected cells . A Max expression vector was found to augment the cotransforming activity of L-Myc as well as that of c-Myc . In addition, a bacterially synthesized DNA-binding domain of L-Myc, like that o c-Myc, heterodimerizes with purified Max protein to bind the core DNA sequence CACGTG . To determine the region of L-Myc responsible for its relatively diminished cotransforming activity, we constructed chimeras containing exons 2 (constituting activation domains) and 3 (constituting DNA-binding domains) of c-Myc fused to those of L-Myc . The cotransforming potencies of these chimeras were compared with those of full-length L-Myc of c-Myc in rat embryo cells . The relative cotransforming activities suggest that the potencies of the activation domains determine the cotransforming efficiencies for c-Myc and L-Myc . This correlation supports the hypothesis that the Myc proteins function in neoplastic cotransformation as transcription factors.

Curr Genet, 1992 Jul, 22(1), 21 - 7
Isolation and characterization of the Aspergillus niger pyruvate kinase gene; de Graaff L et al.; The Aspergillus niger gene encoding pyruvate kinase was cloned by heterologous hybridization using a fragment from the corresponding yeast gene as a probe . The primary structure of the gene, including 5' and 3' flanking sequences, was determined . The structural part of the A . niger pkiA gene is 2054 bp long and is interrupted by seven putative introns . Splicing of the intron sequences results in an open reading frame of 1578 bp, encoding a protein of 526 amino-acid residues and a molecular weight of 58,130 Da . Extensive homology is found with pyruvate kinase from A . nidulans; only 33 amino acids are different between both proteins . Transformation experiments using the pyrA gene as a selection marker and the subcloned pkiA gene as a co-transforming marker led to increased levels of pyruvate kinase . Analysis of the transformants showed that in none of the transformants integration had occurred at the pkiA locus . Predominantly co-integration of the pyrA- and the pkiA-containing plasmids was found in the cases examined.

Mol Biol Cell, 1992 Jul, 3(7), 749 - 59
In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal; Sommer JM et al.; The compartmentalization of glycolytic enzymes into specialized organelles, the glycosomes, allows the bloodstream form of Trypanosoma brucei to rely solely on glycolysis for its energy production . The biogenesis of glycosomes in these parasites has been studied intensively as a potential target for chemotherapy . We have adapted the recently developed methods for stable transformation of T . brucei to the in vivo analysis of glycosomal protein import . Firefly luciferase, a peroxisomal protein in the lantern of the insect, was expressed in stable transformants of the procyclic form of T . brucei, where it was found to accumulate inside the glycosomes . Mutational analysis of the peroxisomal targeting signal serine-lysine-leucine (SKL) located at the C-terminus of luciferase showed that replacement of the serine residue (Serine548) with a small neutral amino acid (A, C, G, H, N, P, T) still resulted in an import efficiency of 50-100% of the wild-type luciferase . Lysine549 could be substituted with an amino acid capable of hydrogen bonding (H, M, N, Q, R, S), whereas the C-terminal leucine550 could be replaced with a subset of hydrophobic amino acids (I, M, Y) . Thus, a peroxisome-like C-terminal SKL-dependent targeting mechanism may function in T . brucei to import luciferase into the glycosomes . However, a few significant differences exist between the glycosomal targeting signals identified here and the tripeptide sequences that direct proteins to mammalian or yeast peroxisomes.

Biol Chem Hoppe Seyler, 1992 Jul, 373(7), 427 - 32
Antibodies to rat procathepsin B recognize the active mature enzyme; Rowan AD et al.; Use of mature cathepsin B for immunization invariably yields antisera that react with the denatured protein but not with the native enzyme . This is thought to be due to spontaneous denaturation of the immunizing antigen on introduction into the animal . Recombinant rat procathepsin B has been expressed in yeast as a secreted product . A procathepsin B mutant (Cys29Ser), where autoprocessing is prevented, has been purified and used to raise a rabbit polyclonal antiserum . Both immunodiffusion analysis and an activity depletion assay demonstrated that this antibody recognized native mature cathepsin B . It appears that conformational epitopes existing on the active enzyme are lost on denaturation . The stability of the proenzyme however permits their presentation for antibody generation.

Biol Chem Hoppe Seyler, 1992 Jul, 373(7), 413 - 8
Investigation of structure function relationships in cathepsin B; Hasnain S et al.; Previous suggestions from sequence alignment studies and examination of the recently determined X-ray crystal structures of cathepsin B point to roles for several specific residues in substrate binding and catalysis . The role of these groups is being examined by studying cathepsin B mutants produced using a yeast expression system . The substitutions Gly198Asp, Arg202Ala, His111Gln and Glu245Gln provide a mechanistic basis for the exopeptidase activity of cathepsin B and the ability of this cysteine proteinase to accept an arginine residue in the S2 subsite.

Int J Syst Bacteriol, 1992 Jul, 42(3), 404 - 7
Legionella shakespearei sp . nov., isolated from cooling tower water; Verma UK et al.; A Legionella-like organism (strain 214T {T = type strain}) was isolated from a cooling tower in Stratford-upon-Avon, England . This strain required L-cysteine and contained cellular branched-chain fatty acids that are typical of the genus Legionella . Strain 214T produced pink colonies on buffered charcoal-yeast extract agar . Ubiquinone Q-12 was the major quinone . Strain 214T was serologically distinct from other legionellae as determined by a slide agglutination test . The results of DNA hybridization studies showed that strain 214T (= ATCC 49655T) is a member of a new Legionella species, Legionella shakespearei.

J Am Board Fam Pract, 1992 Jul-Aug, 5(4), 375 - 80
Evaluation of a latex agglutination test for the identification of Candida species in vaginal discharge; Reed BD et al.; BACKGROUND: The diagnosis of Candida vulvovaginitis using historical symptoms, pelvic examination findings, and results of traditional in-office laboratory tests is often inaccurate . Although Candida cultures can verify the diagnosis, they are not routinely used . METHODS: We prospectively compared the accuracy of a commercially available, in-office, rapid, latex agglutination test (CandidaSure) for the identification of Candida species with results from Candida culture . RESULTS: The sensitivity of the latex agglutination test was 66 percent and the specificity was 63 percent when compared with Candida culture results in patients with vaginal symptoms . When compared with a microscopic evaluation using potassium hydroxide (KOH), the CandidaSure test had greater sensitivity but a lower specificity . In 74 percent of cases with a positive KOH preparation, yeast forms were present on culture, and little was gained by adding the CandidaSure test in this situation . The addition of the CandidaSure test in those cases with a negative KOH preparation resulted in increased sensitivity but also increased the number of false-positive diagnoses . CONCLUSIONS: The CandidaSure test has greater sensitivity than the KOH preparation, but it is less predictive of a positive culture . Because of this limitation, Candida should be documented by culture for any patient who has recurrent or persistent disease and a negative KOH slide.

Mikrobiologiia, 1992 Jul-Aug, 61(4), 585 - 90
{Inhibition of Candida valida growth by copper ions}; Chistiakova TI et al.; The high toxicity of copper ions for Candida valida growth was established at pH-auxostat regime . The value of mu max decreased even at the residual Cu2+ concentration 1.0 mg/l . The inhibition constant (Ki) that characterized a copper ion concentration at which yeast specific growth rate was halved was equal to 7.7 mg/l . A linear dependence of 1/mu max on a residual concentration of copper ions indicates that yeast growth inhibition is due to inhibition of one enzymic reaction which is the most sensitive to copper . Yeast growth inhibition by copper was accompanied by accumulation of Cu2+ ions in biomass, a decrease in nucleic acid and true protein contents, and changes in amino acid composition of protein . The amounts of cystine and cysteine in protein increased and tryptophane content decreased with inhibition of yeast growth . Yeast growth inhibition by copper did not affect the lipid content but significantly reduced the degree of unsaturation due to a decrease in the amounts of polyunsaturated linoleic and alpha-linolenic acids.

J Biochem (Tokyo), 1992 Jul, 112(1), 155 - 62
Further studies on chimeric P450 2C2/2C14 having testosterone 16 beta-hydroxylase activity which is absent in the parental P450s; Uno T et al.; cDNAs for various chimeras between P450 2C2, P450 2C14, P450 2B5, and P450 2E1 were constructed, the chimeric P450s were expressed in yeast cells, and their catalytic activities were compared in the reconstituted system containing partially purified P450 preparations . The chimera P450(2Hc3), consisting of the 462 amino-terminal residues of P450 2C2 and the remaining 28 residues of P450 2C14, had testosterone 16 beta-hydroxylase activity, which is not seen in either of the parental P450s, in addition to higher activities of laurate (omega-1)-hydroxylation and benzphetamine N-demethylation than the parental P450s {Uno, T . et al . (1990) Biochem . Biophys . Res . Commun . 167, 498-503} . When either of the segments from P450 2C2 and P450 2C14 in this chimera was replaced with the corresponding sequences of P450 2E1 or when the 35 carboxy-terminal residues of P450(2Hc3) were replaced with those of P450 2B5, the 16 beta-hydroxylase activity disappeared . When the 262 amino-terminal residues, except for residues 90-125 (region 90-125), of P450(2Hc3) were replaced with those of P450 2C14, the resulting chimera retained both testosterone 16 beta- and laurate (omega-1)-hydroxylase activities . Further replacing the region 90-125 with that of P450 2C14 resulted in disappearance of the 16 beta-hydroxylase activity and profound decrease in the (omega-1)-hydroxylase activity . Testosterone 16 beta-hydroxylation was inhibited by laurate and laurate (omega-1)-hydroxylation by testosterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Biofizika, 1992 Jul-Aug, 37(4), 643 - 6
{Biological detector of weak cosmic fields}; Kopvillem UKh et al.; Possibility of use of biological object as a detector of faint cosmophysical fields is discussed . The signals of free induction and electric echo caused by a short-term effect of light with an intensity of approximately 0.2 mWt/sm2 on biological systems were observed . The experiments with Blatella germanica, Muska domestica were performed in vivo and with red and green seaweeds and yeast cells in vitro . The signals were detected by means of the dielectric permeability measurement in a dynamic regime . Main features of the observed responses are described and assumption on mechanisms of their origin are given . An example of registration of the Lunar eclipse on a detector with in active element of Blatella germanica is given.

Arzneimittelforschung, 1992 Jul, 42(7), 954 - 8
Anti-inflammatory, analgesic, and antipyretic effects and gastrointestinal toxicity of the new anti-inflammatory drug N-(3-{3-(piperidinylmethyl)phenoxy}propyl)-carbamoylmethylthio }ethyl 1-(p-chlorobenzoyl) 5-methoxy-2-methyl-3-indolylacetate; Segawa Y et al.; The anti-inflammatory, analgesic, and antipyretic effects and gastrointestinal toxicity of N-(3-{3-(piperidinylmethyl) phenoxy} propyl)- carbamoylmethylthio} ethyl 1-(p-chlorobenzoyl) 5-methoxy-2-methyl-3-indolylacetate (CP-331, CAS 127966-70-5), a new anti-inflammatory drug, were evaluated using indomethacin as a control . CP-331 exerted anti-inflammatory, analgesic and antipyretic effects on the models of carrageenin-induced paw edema, increased vascular permeability, ultraviolet light-induced erythema, granuloma proliferation, adjuvant arthritis, inflammatory pain, and yeast-induced fever . However, these effects were observed at a molar level similar to or higher than that of indomethacin . In addition, CP-331 influenced more markedly than indomethacin the delayed type hypersensitivity to sheep red blood cells . On the other hand, CP-331 did not damage the gastric mucosa even at a high dose of 1,000 mg/kg and also induced slighter damage to the intestinal mucosa than indomethacin . Thus, CP-331 exerted anti-inflammatory, analgesic, and antipyretic effects but without showing gastric toxicity, which is a common side effect of anti-inflammatory drugs . These results suggest the clinical applicability of this drug in the long-term therapy of inflammatory diseases such as rheumatoid arthritis.

Arzneimittelforschung, 1992 Jul, 42(7), 935 - 44
Pharmacological studies of the new antiinflammatory agent 3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-o ne . 1st communication: antiinflammatory, analgesic and other related properties; Tanaka K et al.; The antiinflammatory, analgesic and antipyretic activities of 3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-on e (T-614, CAS 123663-49-0) were investigated in various animal models and compared with those of nimesulide, indomethacin and ibuprofen . The antiinflammatory potency of T-614 on carrageenin-induced paw edema, paper disk granuloma and established adjuvant arthritis was greater than that of ibuprofen, but slightly lower than those of nimesulide and indomethacin . In acute inflammatory models, unlike indomethacin, T-614 suppressed the edemas provoked by dextran and bromelain in rats, but its inhibitory action on ultraviolet erythema in guinea-pigs was weak . Although the analgesic activity of T-614 was hardly demonstrated in writhing tests in mice, its potency against the inflammatory pain such as Randall-Selitto test, adjuvant-induced hyperalgesia and antigen-induced arthritic pain in rats was superior to that of ibuprofen . Moreover, it had a potent analgesic effect on urate-induced synovitis in dogs . T-614 exerted a prompt and strong antipyretic effect in both yeast-induced febrile rats and lipopolysaccharide-induced febrile rabbits . T-614 had virtually no gastrointestinal ulcerogenic action and did not affect water and sodium excretion in rats . T-614 is a novel antiinflammatory compound with different pharmacological properties from that of the reference drugs.

Am J Physiol, 1992 Jul, 263(1 Pt 2), F171 - 4
cDNA sequence and tissue expression of bovine vacuolar H(+)-ATPase M(r) 70,000 subunit; Marushack MM et al.; cDNA clones encoding the 70,000 relative molecular weight (M(r)) subunit of the bovine vacuolar proton-adenosine triphosphatase (H(+)-ATPase) were isolated, and a total length of 3.1 kb in overlapping clones from bovine kidney and brain libraries was sequenced . The cDNA contains a 1.9-kb coding region and yields a deduced protein sequence of 618 amino acids . The subunit sequence has 50% amino acid identity with the corresponding subunit from yeast, carrot, and Neurospora vacuolar H(+)-ATPases . The internal regions of the protein are most highly conserved, whereas the NH2- and COOH-terminals exhibit variability . mRNA levels of the M(r) 70,000 subunit were examined in multiple bovine tissues and were found to be expressed at highest levels in kidney medulla and cortex, at moderate levels in brain and adrenal gland, and at low levels in liver, muscle, and heart.

Genes Chromosomes Cancer, 1992 Jul, 5(1), 50 - 6
Distinct breakpoints in band 11q23 of the t(4;11) and t(11;14) associated with leukocyte malignancy; Radice P et al.; Several non-random translocation breakpoints associated with leukemia or lymphoma have been shown to occur in chromosome band 11q23 between the genes CD3G and PBGD, a distance of approximately 750 kb . A combination of yeast artificial chromosome (YAC) cloning, in situ hybridization, and pulsed field gel electrophoresis (PFGE) experiments has further refined the interval containing one of these breakpoints, t(4;11)(q21;q23), to within 200 kb of CD3G . We have extended the PFGE analysis to show that the t(4;11) breakpoint lies in a region of approximately 100 kb, situated 100 kb distal to CD3G . Furthermore, we show that a second 11q23 breakpoint, t(11;14)(q23;q32), which was also previously mapped between CD3G and PBGD, is distinct from that of the t(4;11) chromosome . The 11q23 sequences that are involved at the t(11;14) breakpoint are not present in a YAC containing the t(4;11) breakpoint . The t(11;14) breakpoint has been localized on the PFGE map of the CD3G-PBGD interval and is at least 110 kb distal to the t(4;11) breakpoint, thus demonstrating heterogeneity among 11q23 breakpoints.

Curr Microbiol, 1992 Jul, 25(1), 19 - 23
Comparison of culture methods and an immunofluorescence assay for the detection of Legionella pneumophila in domestic hot water devices; Alary M et al.; The objective of this study was to compare an indirect immunofluorescence assay with culture methods for the identification of Legionella pneumophila serogroups 1 to 6 in hot water samples taken from domestic environments . Hot water samples were obtained from the water heater, the shower heads, and the most frequently used faucet of 211 private houses . Concentrated water samples were inoculated on buffered charcoal yeast extract agar (BCYE) and on a semi-selective culture medium (GPV) . Colonies with a morphology similar to that of Legionellaceae were subcultured on BCYE and on blood agar plates; those that grew on the former but not the latter were further characterized and identified by direct immunofluorescence techniques . The concentrated samples were also smeared on multiple-well microscope slides and tested by indirect immunofluorescence with monoclonal antibodies against L . pneumophila, serogroups 1 to 6 . Of the houses studied, 30% were found to contain culturable L . pneumophila in at least one water sample, whereas 63% were positive by indirect immunofluorescence . The sensitivity of this assay compared with culture varied from 16.7-21.1%, and its specificity was between 76.7% and 88.3% depending on the sample source (water heater, shower heads, or faucet) . In the 38 houses with at least one positive sample found by both immunofluorescence and culture, total or partial agreement between serogroups identified by both techniques was only 34% . The results obtained in this study strongly suggest that indirect immunofluorescence is not an adequate alternative for the identification of L . pneumophila in hot water systems.

Hum Genet, 1992 Jul, 89(5), 531 - 8
A 530kb YAC contig tightly linked to the Friedreich ataxia locus contains five CpG clusters and a new highly polymorphic microsatellite; Fujita R et al.; Friedreich ataxia (FA) is a severe autosomal recessive neurodegenerative disease . The defective gene has been previously assigned to chromosome 9q13-q21 by demonstration of tight linkage to the two independent loci D9S15 and D9S5 . Linkage data indicate that FRDA is at less than 1 cM from both markers . Previous physical mapping has shown that probes defining D9S15 (MCT112) and D9S5 (26P) are less than 260 kb apart and are surrounded by at least six CpG clusters within 450 kb, which might indicate the presence of "candidate" genes for FA . We isolated and characterized a 530 kb YAC (yeast artificial chromosome) contig that contains five of the CpG clusters . The YACs were used to search for new polymorphic markers needed to map FRDA precisely with respect to the cloned segment . In particular, we found a (CA)n microsatellite polymorphism, GS4, that detects 13 alleles with a PIC value of 0.83 and allows the definition of haplotypes extending over 310 kb when used in combination with polymorphic markers at D9S5 and D9S15.

Plant J, 1992 Jul, 2(4), 443 - 55
Bean homologs of the mammalian glucose-regulated proteins: induction by tunicamycin and interaction with newly synthesized seed storage proteins in the endoplasmic reticulum; D'Amico L et al.; Treatment of developing bean cotyledons with the inhibitor of N-glycosylation tunicamycin enhanced the synthesis of at least two polypeptides with molecular mass 78 kDa and 97 kDa . Pulse-chase experiments and subcellular fractionation indicated that these are endoplasmic reticulum (ER) residents . The 78 kDa protein is a major component of the ER protein fraction and, by N-terminal sequencing, was identified as a bean homolog of the mammalian 78 kDa glucose-regulated protein (GRP78) . This is a molecular chaperone that is probably involved in the folding and oligomerization of several animal and yeast proteins in the ER . When newly synthesized storage glycoproteins phaseolin, phytohemagglutinin or alpha-amylase inhibitor were immunoprecipitated from an ER preparation of tunicamycin-treated tissue, the GRP78 homolog was always co-precipitated . Bound GRP78 homolog could be released by ATP treatment . These results suggest that, at least when glycosylation is inhibited, this protein plays a role in the early stages of the synthesis of vacuolar storage proteins.

Dev Biol, 1992 Jul, 152(1), 113 - 20
Isolation and characterization of goldfish cdk2, a cognate variant of the cell cycle regulator cdc2; Hirai T et al.; This paper reports the nucleotide and predicted amino acid sequences of the goldfish cdk2, a cognate variant of the cell cycle regulator cdc2 . The predicted protein sequence shows strong homology to the other known cdk2 (88% for Xenopus and 90% for human) . A monoclonal antibody against the C-terminal sequence of goldfish cdk2 recognized a 34-kDa protein in extracts from various goldfish tissues . The protein level was high in such tissues as testis and ovary containing actively dividing cells . Protein cdk2 binds to p13sucl, the fission yeast suc1+ gene product, but not to cyclin B, with which cdc2 forms a complex . The kinase activity of cdk2 increased 30-fold when oocytes matured, although its protein level did not remarkably change . Anti-cdk2 immunoprecipitates from 32P-labeled mature oocyte extracts contained a 47-kDa protein, which was not recognized by either anti-cyclin A or anti-cyclin B antibody, indicating complex formation of cdk2 with a protein other than cyclins A or B.

Immunopharmacology, 1992 Jul-Aug, 24(1), 37 - 45
Acetaminophen inhibits the human polymorphonuclear leukocyte function in vitro; Shalabi EA; The aim of the study is to investigate the effect of Acetaminophen (Am) on the oxidative respiratory burst of isolated human polymorphonuclear leukocytes (PMNs) . Acetaminophen inhibited the luminolchemiluminescence (CL) peak response of PMNs stimulated with phorbol myristate acetate (PMA) or opsonized zymosan in a concentration dependent manner . The inhibitory effect of Am on PMA-stimulated PMNs-CL response was partially reversible . The level of CL inhibition with Am plus the hydroxyl radical scavengers allopurinol, dimethyl sulfoxide (DMSO) or superoxide dismutase (SOD) is greater than that with Am alone . Generation of superoxide (O2-) by stimulated PMNs, as assayed by superoxide dismutase inhibitable reduction of Ferricytochrome c, was markedly inhibited by Am . Furthermore, the phagocytic activity of PMNs as tested for by the ingestion of opsonized dead yeast was significantly reduced in Am-treated cells . These results indicate clearly that Am causes significant inhibition of the human PMNs function in vitro.

Rev Fr Transfus Hemobiol, 1992 Jul, 35(3), 155 - 62
{Hepatitis C virus}; Janot C et al.; Hepatitis C Virus, major causative agent of parenterally transmitted non-A non-B hepatitis was identified by Choo et al . in 1988 using molecular biology technologies . This virus contains a positive stranded RNA genome, it has been shown to have a small diameter . These structure and properties suggest that HCV shares many features in common with Pestiviruses and Flaviviruses . After many years of research, two major objectives were reached: -the identification of viral genome and the main purified viral polypeptide derived from recombinant yeast . Major information were recently appearing about the nucleotide sequences of different isolates coming from US, Europe and Japan; -the preparation of specific tool (ELISA anti-HCV) for detection of circulating HCV antibodies.

Environ Health Perspect, 1992 Jul, 97, 149 - 52
Phagolysosomal pH in alveolar macrophages; Nyberg K et al.; We studied phagolysosomal pH in alveolar macrophages (AM) using fluorescein-labeled yeast (FYP) and silica particles (FSP) as probes . Fluorescence intensities from the ingested test particles were measured on populations of AM using fluorescence spectrometry and on individual phagolysosomes using fluorescence microscopy . Measurements were performed on rabbit AM, which had been incubated with FYP or FSP (in vitro procedure) . We also instilled FYP or FSP via the trachea into rabbit lungs and after 1 day, 1 week, 1 month, and 3 months lavaged the lungs and measured the pH in AM (in vivo procedure) . Phagolysosomal pH was independent of the number and size of the fluorescent particles . Measurements of populations of AM with fluorescence spectrometry and of individual phagolysosomes with fluorescence microscopy gave similar average pH . For the FYP, pH decreased during the first day after lavage both in the in vitro and the in vivo procedures . For the FSP, pH was unchanged during the same period . After 1 day pH was similar for both particles . Electron microscopy showed a larger number of lysosomes in contact with phagosomes and a higher percentage of vacuolated phagosomes for FYP than for FSP . In the in vivo procedure, pH was unchanged at least up to 1 month, and this pH was lower than that in the in vitro procedure . The difference was probably due to conditions at the time of phagocytosis . Particles retained in the lung parenchyma were within AM, and their location within the AM appeared unchanged from 1 week up to 3 months.

J Vet Diagn Invest, 1992 Jul, 4(3), 231 - 7
Competitive ELISA for serodiagnosis of bluetongue: evaluation of group-specific monoclonal antibodies and expressed VP7 antigen; Afshar A et al.; The performance of 2 competitive enzyme-linked immunosorbent assays (C-ELISA) was compared with the reference C-ELISA I for the detection of antibodies to bluetongue virus (BTV) . One of the assays (C-ELISA II) used a group-specific monoclonal antibody (MAb) to BTV, obtained from the American Type Culture Collection (8A3B-6) and tissue culture (TC)-derived BTV antigen (Ag), and the other assay (C-ELISA III) used BTV core protein VP7 (expressed in yeast) and the reference MAb (Pirbright Laboratory, 3-17-A3) . Test sera were obtained by sequential blood samples from 22 calves, each inoculated with a different serotype (T) of BTV (South African {SA} T-1-T-16 and T-18-T-20 and USA T-11, T-13, and T-17) . Sera were also obtained from 4 calves and 4 sheep inoculated with USA BTV T-10 and from several groups of calves exposed to single or multiple doses of epizootic hemorrhagic disease virus (EHDV) T-1-T-4 grown in TC (BHK-21) or suckling mouse brain (SMB) . A total of 618 bovine and ovine field sera collected from BT-free and BT-endemic areas were also tested . The C-ELISA III was more sensitive than the C-ELISA II in the detection of anti-BTV antibody in sera from cattle and sheep early after infection with BTV . Seroconversion was demonstrated by the 3 C-ELISAs in all animals inoculated with BTV by 20 days postinfection (DPI), except in calves that received SA T-3 or USA T-13, which became positive at 40 DPI.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 5857 - 61
Human chromosomal localization of genes encoding the gamma 1 and gamma 2 subunits of the gamma-aminobutyric acid receptor indicates that members of this gene family are often clustered in the genome; Wilcox AS et al.; The gamma-aminobutyric acid (GABA) receptors are the major inhibitory neurotransmitter receptors in the brain and the site of action of a number of important pharmacological agents including barbiturates, benzodiazepines, and ethanol . The gamma 1 and gamma 2 subunits have been shown to be important in mediating responses to benzodiazepines, and a splicing variant of the gamma 2 subunit, gamma 2L, has been shown to be necessary for ethanol actions on the receptor, raising the possibility that the gamma 2 gene may be involved in human genetic predisposition to the development of alcoholism . We have assigned the human genes encoding the gamma 1 and gamma 2 subunits of the GABAA receptor to chromosomes 4 and 5, respectively, by PCR amplification of human-specific products from human-hamster somatic cell hybrid DNAs . Using panels of chromosome-specific natural deletion hybrids, we have further localized the gamma 1 gene (GABRG1) to 4p14-q21.1 and the gamma 2 gene (GABRG2) to 5q31.1-q33.2 . These data indicate that the gamma 1 gene may be clustered together with the previously mapped alpha 2 and beta 1 genes on chromosome 4 and that the gamma 2 gene may be close to the previously localized alpha 1 gene on chromosome 5 . To further examine the latter possibility the alpha 1 gene was mapped using the chromosome 5 deletion hybrids and shown to be within the same region as the gamma 2 gene, 5q31.1-q33.2 . A PCR-based screening strategy was used to isolate a 450-kilobase human genomic yeast artificial chromosome clone containing both the alpha 1 and gamma 2 genes . Pulsed-field gel restriction mapping of the yeast artificial chromosome indicates that the two genes are within 200 kilobases of each other . The data presented here provide further evidence for the nonrandom organization of the human genome by demonstrating that members of the GABAA receptor gene family often occur in small gene clusters widely distributed in the genome.

Infect Immun, 1992 Jul, 60(7), 2683 - 7
Inhibition of murine macrophage protein kinase C activity by nonviable Histoplasma capsulatum; Wolf JE et al.; Ingestion of Histoplasma capsulatum yeast cells inhibits the oxidative burst response of murine macrophages (M phi's) . Since protein kinase C (PKC) is believed to be an integral part of the signal transduction pathway involved in the production of reactive oxygen intermediates, we investigated the relationship between PKC activity and oxidative burst inhibition in H . capsulatum-containing murine peritoneal M phi's . An inhibitory effect on both basal and phorbol myristate acetate-mobilized membrane PKC activities was observed in M phi's which had ingested H . capsulatum but not latex spheres . These results suggest that one way in which H . capsulatum may disrupt the oxidative burst is through a PCK-dependent mechanism.

Infect Immun, 1992 Jul, 60(7), 2559 - 64
Alterations in murine macrophage arachidonic acid metabolism following ingestion of nonviable Histoplasma capsulatum; Wolf JE et al.; The effect of ingestion of heat-killed Histoplasma capsulatum yeast cells on the metabolism of arachidonic acid (20:4) to prostenoids and leukotrienes was examined in murine peritoneal macrophages (M phi s) . H . capsulatum-containing M phi s exhibited a metabolite profile similar to that of zymosan-challenged phagocytes; however, there were differences with respect to the relative and total amounts of products produced . While proteose peptone-elicited M phi s exposed to H . capsulatum released quantitatively less prostaglandin E2 (PGE2) and leukotriene C4 than zymosan-treated M phi s, they metabolized a greater percentage of total product to prostenoids . In addition, whereas in vitro priming with gamma interferon increased both the PGE2 and leukotriene C4 contents of zymosan-stimulated M phi supernatants, similarly primed M phi s challenged with H . capsulatum selectively increased only PGE2 production . The immunosuppressive effect of a relative excess of prostenoids in H . capsulatum-containing M phi s may contribute to the overall disturbance in cell-mediated immunity characteristic of disseminated histoplasmosis.

Science, 1992 Jun 26, 256(5065), 1815 - 7
Reversal of the orientation of an integral protein of the mitochondrial outer membrane; Li JM et al.; The NH2-terminus of the signal-anchor sequence of an integral, bitopic protein of the outer mitochondrial membrane was extended both in amino acid length (from 11 to 38 amino acids) and net charge (from +4 to +8)--changes that confer on the NH2-terminus characteristics of a strong matrix-targeting signal . The protein was inserted into the outer membrane but in an inverted orientation (Ncyto-Cin) . These findings suggest that, in common with other membrane systems, the orientation of a protein in the outer mitochondrial membrane can be determined by a signal that causes retention of the NH2-terminus on the cytosolic side of the membrane.

Cell, 1992 Jun 26, 69(7), 1227 - 36
Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases; Chung J et al.; The macrolide rapamycin blocks cell cycle progression in yeast and various animal cells by an unknown mechanism . We demonstrate that rapamycin blocks the phosphorylation and activation of the 70 kd S6 protein kinases (pp70S6K) in a variety of animal cells . The structurally related drug FK506 had no effect on pp70S6K activation but at high concentrations reversed the rapamycin-induced block, confirming the requirement for the rapamycin and FK506 receptor, FKBP . Rapamycin also interfered with signaling by these S6 kinases, blocking serum-stimulated S6 phosphorylation and delaying entry of Swiss 3T3 cells into S phase . Neither rapamycin nor FK506 blocked activation of a distinct family of S6 kinases (RSKs) or the MAP kinases . These studies identify a rapamycin-sensitive signaling pathway, argue for a ubiquitous role for FKBPs in signal transduction, indicate that FK506-FKBP-calcineurin complexes do not interfere with pp70S6K signaling, and show that in fibroblasts pp70S6K, not RSK, is the physiological S6 kinase.

J Biol Chem, 1992 Jun 25, 267(18), 12592 - 9
Alpha-isoenzyme of alcohol dehydrogenase from monkey liver . Cloning, expression, mechanism, coenzyme, and substrate specificity; Light DR et al.; The cDNA for the alpha-isoenzyme from rhesus monkey (Macaca mulatta) liver was cloned and expressed in yeast . The alpha-isoenzymes of human and monkey liver alcohol dehydrogenase differ from the other human and horse liver enzymes in having Met57, Ala93, and Val116 instead of Leu57, Phe93, and Leu116 in the substrate binding pocket and Gly47 instead of Arg47 near the pyrophosphate moiety of the coenzyme . The effects of these differences on the kinetic mechanism, substrate specificity, and coenzyme binding were studied with the purified, recombinant monkey alpha-isoenzyme (MmADH alpha) and mutated enzymes with Gly47 substituted with His or Arg . The mechanism appears to be random for the binding of NAD+ and ethanol and ordered for NADH and acetaldehyde, with formation of a dead-end enzyme-NADH-ethanol complex . MmADH alpha reacts 130-fold slower (V/K) with ethanol and 3-25-fold slower with 2-methyl alcohols but 20-fold faster with cyclohexanol, as compared with horse (Equus caballus) liver EE isoenzyme (EqADH) . MmADH alpha is stereoselective for the R isomer of 2-butanol, whereas EqADH favors the S isomer . Both enzymes have comparable reactivity with larger primary alcohols . MmADH alpha is more reactive with secondary alcohols and has highest activity with cyclohexanol . However, it does not react with steroids such as 5 beta-androstane-17 beta-ol-3-one . Molecular modeling suggests that the differences between MmADH alpha and EqADH are a result of the substitution of Ala for Phe93 and Thr for Ser48 . MmADH alpha binds NAD+ most rapidly when a group with a pK of 7.4 is unprotonated, implicating His51 in this reaction . The G47R substitution decreased the dissociation constants for NAD+ and NADH and turnover numbers only about 2-fold, whereas the G47H substitution increased dissociation constants 7-14-fold and turnover numbers 4-fold . A basic residue at position 47 is not crucial for activity, as multiple interactions determine coenzyme affinity.

Biochemistry, 1992 Jun 23, 31(24), 5545 - 53
Multiple role of hydrophobicity of tryptophan-108 in chicken lysozyme: structural stability, saccharide binding ability, and abnormal pKa of glutamic acid-35; Inoue M et al.; Trp108 of chicken lysozyme is in van der Waals contact with Glu35, one of two catalytic carboxyl groups . The role of Trp108 in lysozyme function and stability was investigated by using mutant lysozymes secreted from yeast . By the replacement of Trp108 with less hydrophobic residues, Tyr (W108Y lysozyme) and Gln (W108Q lysozyme), the activity, saccharide binding ability, stability, and pKa of Glu35 were all decreased with a decrease in the hydrophobicity of residue 108 . Namely, at pH 5.5 and 40 degrees C, the activities of W108Y and W108Q lysozymes against glycol chitin were 17.3 and 1.6% of that of wild-type lysozyme, and their dissociation constants for the binding of a trimer of N-acetyl-D-glucosamine were 7.4 and 309 times larger than that of wild-type lysozyme, respectively . For the reversible unfolding at pH 3.5 and 30 degrees C, W108Y and W108Q lysozymes were less stable than wild-type lysozyme by 1.4 and 3.6 kcal/mol, respectively . As for the pKa of Glu35, the values for W108Y and W108Q lysozymes were found to be lower than that for wild-type lysozyme by 0.2 and by 0.6 pKa unit, respectively . The pKa of Glu35 in lysozyme was also decreased from 6.1 to 5.4 by the presence of 1-3 M guanidine hydrochloride, or to 5.5 by the substitution of Asn for Asp52, another catalytic carboxyl group . Thus, both the hydrophobicity of Trp108 and the electrostatic interaction with Asp52 are equally responsible for the abnormally high pKa (6.1) of Glu35, compared with that (4.4) of a normal glutamic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1992 Jun 22, 305(1), 51 - 4
Formation and quantification of protein complexes between peroxisomal alcohol oxidase and GroEL; Evers ME et al.; We have studied the use of yeast peroxisomal alcohol oxidase (AO) as a model protein for in vitro binding by GroEL . Dilution of denatured AO in neutral buffer leads to aggregation of the protein, which is prevented by the addition of GroEL . Formation of complexes between GroEL and denatured AO was demonstrated by a gel-shift assay using non-denaturing polyacrylamide gel electrophoresis, and quantified by laser-densitometry of the gels . In the presence of MgAMP-PNP or MgADP the affinity of GroEL for AO was enhanced . Under these conditions up to 70% of the purified GroEL formed a complex with this protein . Release was stimulated at room temperature by MgATP, and was further enhanced by addition of GroES.

Biochim Biophys Acta, 1992 Jun 19, 1100(3), 217 - 34
Malate dehydrogenase isoenzymes: cellular locations and role in the flow of metabolites between the cytoplasm and cell organelles; Gietl C; Malate dehydrogenases belong to the most active enzymes in glyoxysomes, mitochondria, peroxisomes, chloroplasts and the cytosol . In this review, the properties and the role of the isoenzymes in different compartments of the cell are compared, with emphasis on molecular biological aspects . Structure and function of malate dehydrogenase isoenzymes from plants, mammalian cells and ascomycetes (yeast, Neurospora) are considered . Significant information on evolutionary aspects and characterisation of functional domains of the enzymes emanates from bacterial malate and lactate dehydrogenases modified by protein engineering . The review endeavours to give up-to-date information on the biogenesis and intracellular targeting of malate dehydrogenase isoenzymes as well as enzymes cooperating with them in the flow of metabolites of a given pathway and organelle.

Biochim Biophys Acta, 1992 Jun 15, 1131(2), 227 - 32
Cloning and nucleotide sequences of two lipase genes from Candida cylindracea; Longhi S et al.; Two lipase-encoding genes (LIP1 and LIP2) have been isolated from a SacI genomic library of the yeast Candida cylindracea and their nucleotide sequences have been determined . Comparison with the sequence of a cDNA ruled out the presence of introns in the two genes . Both ORFs encode for mature proteins of 534 residues with putative signal peptides of 15 and 14 amino acids, respectively . When compared with other lipase sequences, the two C . cylindracea lipases showed homology only with the Geotrichum candidum lipase, whereas they shared a significant similarity with several esterases.

BMJ, 1992 Jun 13, 304(6841), 1536 - 9
New rapid test for prenatal detection of trisomy 21 (Down's syndrome): preliminary report; Bryndorf T et al.; OBJECTIVE--To devise and evaluate a rapid screening method for detecting trisomy 21 (Down's syndrome) in samples of uncultured amniotic fluid cells . DESIGN--Non-radioactive in situ hybridisation with HY128, a 500,000 base pair yeast artificial chromosome probe specific for chromosome 21 . Blinded study of 12 karyotypically normal amniotic fluid samples and eight samples trisomic for chromosome 21 . SETTING--Cytogenetic and obstetric services at a tertiary referral centre, Copenhagen . MAIN OUTCOME MEASURES--Time necessary to complete the test . Proportion of cell nuclei containing two and three hybridisation signals in karyotypically normal and abnormal amniotic fluid samples . RESULTS--The test could be completed within three to four days after amniocentesis . In the normal samples a mean of 73% (range 61-82%) of the amniotic cell nuclei showed two hybridisation signals and 6% (0-18%) showed three signals . By contrast, among the trisomic samples 29% (19-38%) of the nuclei exhibited two signals and 48% (31-60%) showed three signals . CONCLUSION--The technique clearly distinguished between normal and trisomic samples . Prenatal diagnosis with in situ hybridisation with chromosome specific probes was fast and may make it possible to screen for selected, aneuploidies . However, the technique is still at a preliminary stage and needs further evaluation and refinement.

Cell, 1992 Jun 12, 69(6), 977 - 88
twine, a cdc25 homolog that functions in the male and female germline of Drosophila; Alphey L et al.; twine is the second homolog of the fission yeast gene cdc25 to be found in Drosophila . Both string and twine cDNAs can rescue a temperature-sensitive cdc25 mutation in fission yeast, but not a deletion . We detect the expression of string but not twine transcripts in the proliferating cells of newly cellularized embryos, in third instar larval brains, and in imaginal discs . Both genes are abundantly expressed in nurse cells during oogenesis, the maternal transcripts persisting throughout the syncytial stage of embryonic development . In the testis, twine transcripts are seen in the growing stage of premeiotic cysts . Analysis of a twine mutant suggests a requirement for the gene during oogenesis, during syncytial embryonic development, and for male meiosis . Meiosis does not occur in homozygous twine males, which produce cysts containing 16 rather than 64 spermatids.

Lancet, 1992 Jun 6, 339(8806), 1371 - 5
Use of nonoxynol-9 and reduction in rate of gonococcal and chlamydial cervical infections; Niruthisard S et al.; The spermicide nonoxynol-9 (N-9) has been used as a contraceptive for over 30 years, but the use of a vaginal spermicide and condoms for the prevention of sexually transmitted infections has not been examined in randomised studies . We report a single-blind randomised field trial to assess the effect of N-9 film on the rate of gonococcal and chlamydial cervical infection in women at high risk of these diseases . 343 women were randomly assigned to use either condoms and N-9 (186 women) or condoms and a placebo (157) . Compliance with condom use was much the same in the two groups . Overall, N-9 reduced the rate of cervical infection by 25% (rate ratio {RR} 0.75, 95% confidence interval {Cl} 0.5-1.1); in women who used N-9 for more than 75% of their coital acts the infection rate was reduced by 40% (RR 95% Cl 0.3-1.0) . The rate of yeast vulvovaginitis or genital ulcers was not higher in N-9 users than in placebo users, but the rate of symptomatic irritation was increased by 70% (RR 95% Cl 1.1-2.6) among N-9 users . Condom use was more protective against cervical infection than N-9 use . The rate of infection was 50% (RR 95% Cl 0.3-0.7) lower with 75% than with 0-50% condom compliance . The use of a vaginal N-9 spermicide with condoms whenever possible seems to be a better strategy than the use of condoms only for prevention of gonococcal and chlamydial cervical infection.

Biochemistry, 1992 Jun 2, 31(21), 5005 - 9
Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain; Yang JH et al.; Several mixed DNA/RNA and 2'-O-methylribonucleotide/RNA analogues derived from the "hammerhead" domain of RNA catalysis have been prepared to study the minimum ribonucleotide requirement for catalytic activity . Oligodeoxyribonucleotides containing from seven to as few as four ribonucleotides are active in cleaving a substrate RNA . Predominantly deoxyribonucleotide-containing analogues have kcat values 20-300 and kcat/KM values approximately 100-2000 times lower than those of all-RNA ribozyme . In the case of predominantly 2'-O-methyl analogues, at least five ribonucleotides are needed to assure catalytic activity . In addition, both predominantly deoxyribonucleotide and 2'-O-methyl oligomers are at least 3 orders of magnitude more stable than an all-RNA ribozyme in incubations with RNase A and a yeast extract . These results suggest that the ribophosphate backbone is not a strict requirement for ribozyme-type catalysis . The identification of the four required ribonucleotides in the hammerhead catalytic domain provides valuable information for the rational design of chemical species having ribonuclease activities.

Arch Dermatol, 1992 Jun, 128(6), 811 - 4
Reiter's syndrome of the vulva . The psoriasis spectrum; Edwards L et al.; BACKGROUND--Reiter's syndrome is a disease characterized by crusted, scaling, acral and genital plaques; urethritis or cervicitis; and arthritis, which occur in genetically susceptible patients in response to any of many infections . This disease rarely occurs in women, and specific characterizations of vulvar and cervical lesions are rare . OBSERVATIONS--We describe a 39-year-old woman with a history of mucocutaneous candidiasis that was refractory to oral ketoconazole therapy . She presented with well-demarcated, erythematous, crusted plaques over the vulva, hands, and feet, as well as with cervical lesions and a history of conjunctivitis and iritis . Following the biopsy of characteristic skin lesions, recognition of systemic signs, and cultures that were negative for yeast, her condition was diagnosed as Reiter's syndrome . CONCLUSIONS--Reiter's syndrome of the vulva, vagina, and cervix may not be recognized because of its uncommon occurrence in women and the physician's consequent unfamiliarity with its clinical appearance in the genital area . This disease and pustular psoriasis share many common features and exist on a spectrum . A high index of suspicion and correlation of the many facets of the disease will better enable the clinician to make this diagnosis.

J Bacteriol, 1992 Jun, 174(11), 3723 - 8
Significance of NADP/NAD glutamate dehydrogenase ratio in the dimorphic behavior of Benjaminiella poitrasii and its morphological mutants; Khale A et al.; Studies on the levels of glutamate dehydrogenase (GDH), glutamine synthetase, and glutamate synthase were carried out as a function of temperature, nutritional conditions, and the morphological (yeast or mycelium) form of Benjaminiella poitrasii . Since both NAD- and NADP-dependent GDH activities were found in B . poitrasii, the quantitative relation between these two enzymes expressed as the NADP-GDH/NAD-GDH activity ratio (GDH ratio) was studied to evaluate its possible role in the morphogenesis . In the yeast-to-mycelium transition, a decrease in the GDH ratio occurred (between 1 and 2 h) and germ tube formation could be observed only at 3 h . Under similar sets of experimental conditions, exogenous addition 1.0 mM of alpha-ketoglutarate delayed germ tube emergence (4 h) compared with the control . On the other hand, in the presence of 1.0 mM glutamate an earlier onset of the germ tube formation was noted . The morphological (monomorphic) mutants, Y-2 and Y-5, showed a high GDH ratio and maintained the yeast morphology.

Virology, 1992 Jun, 188(2), 869 - 74
Retroviral envelope protein fusions to secreted and membrane markers; Mace MC et al.; We have analyzed a series of Moloney murine leukemia (M-MuLV) envelope (env) protein fusions to the marker proteins invertase and placental alkaline phosphatase (PLAP), expressed in Psi2 retrovirus packaging cells . The yeast invertase protein, fused at its third amino acid residue to the amino-terminal signal sequence and 17 residues of the mature M-MuLV env protein, retained its enzymatic activity and was secreted from mammalian cells . However, env protein fusions to the C-terminal portion of invertase were inactive . In contrast, some, but not all, env protein fusions at the C-terminal region of PLAP were enzymatically active: PLAP fusions containing long C-terminal portions of env localized to the rough endoplasmic reticulum (RER) and possessed low enzyme activity levels, while fusion constructs containing relatively short portions of the M-MuLV env gene localized to the Golgi and had higher activity levels . Those proteins that localized to the Golgi also were processed, in part, to forms of 67 to 68 kDa, the size of the mature PLAP protein . Since PLAP ordinarily is transferred to a phosphatidyl-inositol glycan tail (PIG-tail) in the Golgi and then transported to the plasma membrane, it appears that Golgi-localized PLAP-env fusions are processed imperfectly . PLAP itself, when expressed in Psi2 cells, accumulated at the plasma membrane and, unlike the PIG-tailed Thy-1 protein, was not incorporated into virus particles . Thus, the reported incorporation of the Thy-1 protein into M-MuLV virions does not appear to be a consequence of its glycoprotein tail.

EMBO J, 1992 Jun, 11(6), 2139 - 49
An extra copy of nimEcyclinB elevates pre-MPF levels and partially suppresses mutation of nimTcdc25 in Aspergillus nidulans; O'Connell MJ et al.; Previous work has shown that nimA encodes a cell cycle regulated protein kinase that is required along with the p34cdc2 histone H1 kinase (MPF) for mitosis in Aspergillus nidulans . We have now identified two other gene products required for mitosis in A.nidulans . nimT encodes a protein similar to the fission yeast cdc25 tyrosine phosphatase and is required for the conversion of pre-MPF to MPF and nimE encodes a B-type cyclin which is a subunit of MPF . A new genetic interaction between nimEcyclinB and nimTcdc25 type genes is reported . Increased copy number of nimEcyclinB can suppress mutation of nimTcdc25 and also lead to increased accumulation of tyrosine phosphorylated p34cdc2 (pre-MPF) . This biochemical observation suggests an explanation for the genetic complementation . If nimEcyclinB recruits p34cdc2 for tyrosine phosphorylation to form pre-MPF it follows that increased expression of nimEcyclinB would increase the level of pre-MPF . The increased level of pre-MPF generated may then allow the mutant nimTcdc25 protein to convert enough pre-MPF to MPF and thus permit some mitotic progression . We also demonstrate that correct cell cycle regulation by the p34cdc2 protein kinase pathway is essential for correct developmental progression in A.nidulans.

J Cell Biol, 1992 Jun, 117(5), 1041 - 53
Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210; Hamaguchi JR et al.; The mouse FT210 cell line is a temperature-sensitive cdc2 mutant . FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle . FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases . H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1 . The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo . A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2 . The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase . We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.

Protein Eng, 1992 Jun, 5(4), 295 - 303
Main structural and functional features of the basic cytosolic bovine 21 kDa protein delineated through hydrophobic cluster analysis and molecular modelling; Schoentgen F et al.; A 21 kDa protein purified from bovine brain cytosol was previously described as a hydrophobic ligand binding protein; however, its accurate biological function remained still uncertain . In order to get further information about its potential biological role, an extended prediction of its secondary and three dimensional structures was undertaken . We describe here a process which permitted us to discover a structural homology between the 21 kDa protein and the N-domain of yeast phosphoglycerate kinase (PGK) . This process is based on comparing the 21 kDa protein with all the proteins presenting a slight homology, by using the Hydrophobic Cluster Analysis (HCA) method . According to the observed similarity between the N-domain of yeast PGK and the 21 kDa protein, we built a model which was shown to possess a potential binding site for nucleotides . Moreover, the model obtained presents three-dimensional (3D) structure similarity with adenylate kinase . These results suggest two main hypotheses: (i) the 21 kDa protein may belong to the kinase family; (ii) the binding of a nucleotide could imply a modification of the 3D structure of the 21 kDa protein that can promote the transfer of hydrophobic ligands to the plasma membrane . Meanwhile, verification of these hypotheses has been in part performed experimentally: the 21 kDa protein binds MgATP as well as, to a lesser extent, phosphoglycerate.

Biochimie, 1992 Jun, 74(6), 519 - 26
Structural modeling of the human erythrocyte bisphosphoglycerate mutase; Craescu CT et al.; Using the crystallographic structure of yeast monophosphoglycerate mutase (MPGM) as a framework we constructed a three-dimensional model of the homologous human erythrocyte bisphosphoglycerate mutase (BPGM) . The modeling procedure consisted of substituting 117 amino acid residues and positioning 19 C-terminal residues (unresolved in the X-ray structure) by empirical methods, followed by energy minimization . Among several differences in the active site region the most significant appears to be the replacement of Ser11 in MPGM by Gly in BPGM . The C-terminal segment, which contains mainly basic amino acids, lines the cavity of the active site . The seven amino acid residues, which have been shown to be essential for the three catalytic functions of the human BPGM, interact with the amino acids in the protein core, near the active site . In addition, a cluster of several positively charged residues, particularly arginines, has been identified at the entrance of the active site; this cluster may serve as a secondary binding site for polyanionic substrates or cofactors, as required by a two-binding-site model of the catalytic activities . This model is in agreement with recent studies of an inactive BPGM variant substituent at an Arg position situated in this positively charged cluster . The position of Cys20 in the model constructed suggests that this residue is responsible for inactivation of the enzyme by sulfhydryl reagents . Subunit interfaces have also been constructed for BPGM by analogy with MPGM and suggest that, in addition to the known dimerization of BPGM, tetramerization may occur under certain conditions.

Biol Chem Hoppe Seyler, 1992 Jun, 373(6), 315 - 21
Induction and detection of anti-peptide antibody specificity is critically affected by the mode of hapten presentation; Moroder L et al.; C-Terminal cholecystokinin (CCK)-peptides of increasing chain lengths were all linked at their N-termini to the single surface-exposed cysteine residue 107 of yeast iso-1-cytochrome c by the maleimide/thiol reaction . The resulting CCK/cytochrome 1:1 conjugates with the haptenic peptides in the identical protein environment were used to immunize outbred guinea pigs in order to assess the critical size of CCK peptides required for the expression of a CCK-specific epitope and the induction of antibodies not crossreacting with the homologous gastrin sequence . By using standard ELISA techniques with polystyrene-adsorbed antigen to evaluate the specificity of the antisera, none of the conjugates were found to induce anti-CCK antisera not crossreacting with gastrin . However, when the biotinyl-CCK-antigen was immobilized by polystyrene-adsorbed avidin, i.e . via a procedure which assures maximum accessibility of the bound antigen, we were able to demonstrate that with CCK-12 and particularly CCK-13, linked through their N-termini to the carrier, the critical length for the expression and recognition of a CCK-specific epitope was reached . The related polyclonal antisera did not crossreact with the homologous gastrin in the modified ELISA.

AIDS Res Hum Retroviruses, 1992 Jun, 8(6), 1165 - 70
Identification of a neutralizing domain in the external envelope glycoprotein of simian immunodeficiency virus; Benichou S et al.; Two murine monoclonal antibodies (MAbs), designated MATG2014 and MATG2033, were generated . They are reactive with the external envelope glycoprotein gp130 of the simian immunodeficiency virus of macaque monkey (SIVmac251), and display a cell-free virus neutralizing activity in vitro . In addition, MATG2014 cross-reacts with HIV-2Rod gp140 . Epitope mapping of these MAbs was performed by screening and SIVmac peptide library expressed in yeast and confirmed using synthetic peptides . MATG2014 and MATG2033 recognize two overlapping epitopes localized in an 18 residue domain between amino acid 171 and 188 of the SIVmac251 gp130 . Sera from experimentally SIV-infected macaques are immunoreactive with this neutralizing domain . Sequence comparison with related SIV and HIV-2 viral strains indicates a low variability of this region, consistent with the cross-reactivity of MATG2014 with HIV-2Rod gp140 . This domain should then be considered in designing experimental vaccines.

J Formos Med Assoc . 1992 Jun;91 Suppl 2:S92.
{Study on P450c21 gene}; Chung BC; Yeast cells harboring mutant plasmids were incubated with 0.1, 0.15, 0.2, 0.3, 0.4, 0.45, 0.6, or 1.0 mM (14C)-17-hydroxyprogesterone for 1h . The steroids in the media were then separated by thin layer chromatography and counted to calculate enzymatic activities . Wt = wild type enzyme . Pt: patient enzyme.

J Bacteriol, 1992 Jun, 174(12), 4057 - 63
Development of multipurpose peroxisomes in Candida boidinii grown in oleic acid-methanol limited continuous cultures; Waterham HR et al.; We have studied the development and metabolic significance of peroxisomes in the yeast Candida boidinii following adaptation of the organism to cultivation conditions which require the simultaneous presence and activity of two independent peroxisome-mediated pathways for growth . After the addition of methanol to oleic acid-grown cells at late exponentional growth, a number of new small peroxisomes developed which, apart from the presence of beta-oxidation enzymes, were characterized by the presence of enzymes involved in methanol metabolism (alcohol oxidase and dihydroxyacetone synthase) . The latter proteins, however, were absent in the larger organelles which were originally present in the oleic acid-grown cells prior to the addition of methanol and which contained only enzymes of the beta-oxidation pathway . Subsequent experiments on cells from continuous cultures grown on a mixture of oleic acid and methanol at steady-state conditions revealed that both the enzymes of the beta-oxidation pathway and those involved in methanol metabolism were found in one and the same compartment . Thus, under these conditions the cells contained peroxisomes which were concurrently involved in the metabolism of two different carbon sources simultaneously used for growth . Our results indicated that the heterogeneity in the peroxisomal population of a single cell, observed in the transient state following the addition of methanol, is only temporary and due to heterogeneity among these organelles with respect to their capacity to incorporate newly synthesized matrix proteins.

Eur J Med, 1992 Jun, 1(3), 132 - 8
Production and characterization of monoclonal antibodies to human Pneumocystis carinii for the diagnosis of P . carinii pneumonia; Nato F et al.; OBJECTIVES: Monoclonal antibodies against human Pneumocystis carinii were produced by fusion of myeloma cells (X63-AG8.653) with splenocytes from Biozzi mice that had been immunized against P . carinii cysts isolated from infected human lung . The aim of this study was to evaluate the usefulness of these monoclonal antibodies for the diagnosis of P . carinii pneumonia by indirect immunofluorescence in comparison with a modified silver stain method and commercial kits . METHODS: One hundred fifty-seven specimens from 87 patients, infected or non-infected with human immunodeficiency virus, were examined for the presence of P . carinii . Specimens were either induced sputum samples or bronchoalveolar lavage fluids . Indirect immunofluorescence was performed with six stable clones obtained by limiting dilution . Four of the monoclonal antibodies were IgG1 isotypes, one was an IgG3 and one was an IgM . Their isoelectric points varied from 6.5 to 8.3 . Tests were also performed with silver methenamine staining and with two commercially available monoclonal antibodies (Monofluo kit from Diagnostics Pasteur and MAb from Dako) . RESULTS: The 6 antibodies all recognized P . carinii cysts in indirect immunofluorescence . No cross reactivity was observed with yeast or host cells . P . carinii antigens could not be identified with western immunoblotting suggesting that the monoclonal antibodies recognized native antigens . This result was confirmed by dot blot analysis . Spots were observed with native but not with denatured antigens . Inhibition studies showed that these 6 antibodies recognized the same or overlapping sites . The sensitivities of detection of P . carinii in sputum were 87% by silver stain and from 93.5 to 96.7% by immunofluorescence . The sensitivities of detection in bronchoalveolar lavage were 67.3% by silver stain and from 75.7% to 76.8% by immunofluorescence . CONCLUSION: Immunofluorescence was more sensitive than silver staining and the best results were obtained with E5-8 and A8-13 monoclonal antibodies and with Monofluo kit.

J Cell Sci, 1992 Jun, 102 ( Pt 2), 285 - 97
Association of cyclin-bound p34cdc2 with subcellular structures in xenopus eggs; Leiss D et al.; Cell cycle progression is controlled by changes in kinase activity of homologs of the fission yeast protein p34cdc2 . The p34cdc2 kinase is activated by its association with a cyclin subunit, followed by post-translational modifications . Here, we show that in Xenopus eggs stimulated to enter the early embryonic cell cycle by an electric shock, part of the p34cdc2 becomes associated with subcellular fractions as the eggs progress towards mitosis . This occurs as a result of cyclin accumulation because most of the B-type cyclins and some of the A-type cyclins are found in the particulate fraction . Moreover, as soon as cyclins are degraded, p34cdc2 is released in the soluble fraction . The p34cdc2-cyclin complex can be solubilised by 80 mM beta-glycerophosphate (in the standard MPF extraction buffer) or by high salt concentrations . The post-translational modifications leading to cdc2 kinase activation by cyclin occur in the insoluble form . Following fractionation of egg extracts by sucrose gradient centrifugation, the p34cdc2-cyclin B complex is found in several fractions, but especially in two discrete peaks . We present evidence that in the slow-sedimenting peak the p34cdc2-cyclin B complex is associated with the 60 S subunit of monoribosomes . It could be targeted in this fashion to substrates such as ribosomal proteins and maybe to cytoskeletal proteins, since ribosomes bind to microtubules and are present in the spindle . The p34cdc2-cyclin B complex is also found in a faster-migrating fraction containing various membranous structures, including Golgi stacks . Therefore, as observed by immunofluorescence in other systems, it seems that cyclin subunits target p34cdc2 to specific cellular sites and this is certainly important for its function . In addition, we present preliminary evidence suggesting that some component present in the ribosome-containing fraction is required for activation of the p34cdc2-cyclin B complex.

EMBO J, 1992 Jun, 11(6), 2189 - 99
cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication; Dutta A et al.; RPA is a single-stranded DNA binding protein complex purified from human cells and is essential for the initiation and elongation stages of SV40 DNA replication in vitro . In both human and yeast cells, the 34 kDa polypeptide subunit of RPA is phosphorylated in the S and G2 phases of the cell cycle and not in G1 . One of the major RPA kinases present in extracts of human cells was purified and shown to be the cyclin B-cdc2 complex . This purified kinase, and a closely related cyclin A associated cdc2-like kinase, phosphorylated RPA p34 on a subset of the chymotryptic peptides that were phosphorylated in vivo at the G1-S transition . Two serines near the N-terminus of RPA p34 were identified as possible sites of phosphorylation by cdc2 kinase . These same serines were necessary for RPA phosphorylation in vivo . The purified cdc2 kinase stimulated SV40 DNA replication in vitro when added to G1 cell extracts . The kinase also stimulated unwinding at the origin of replication, one of the earliest steps in DNA replication requiring RPA, but only in the presence of an additional factor present in G1 cell extracts . Thus, one or more members of the cyclin-cdc2 kinase family may be required for the initiation and maintenance of S phase, in part due to their ability to phosphorylate and activate a cellular DNA replication factor, RPA.

Mol Cell Biol, 1992 Jun, 12(6), 2514 - 24
Specific transcription factors stimulate simian virus 40 and polyomavirus origins of DNA replication; Guo ZS et al.; The origins of DNA replication (ori) in simian virus 40 (SV40) and polyomavirus (Py) contain an auxiliary component (aux-2) composed of multiple transcription factor binding sites . To determine whether this component stimulated replication by binding specific transcription factors, aux-2 was replaced by synthetic oligonucleotides that bound a single transcription factor . Sp1 and T-antigen (T-ag) sites, which exist in the natural SV40 aux-2 sequence, provided approximately 75 and approximately 20%, respectively, of aux-2 activity when transfected into monkey cells . In cell extracts, only T-ag sites were active . AP1 binding sites could replace completely either SV40 or Py aux-2 . Mutations that eliminated AP1 binding also eliminated AP1 stimulation of replication . Yeast GAL4 binding sites that strongly stimulated transcription in the presence of GAL4 proteins failed to stimulate SV40 DNA replication, although they did partially replace Py aux-2 . Stimulation required the presence of proteins consisting of the GAL4 DNA binding domain fused to specific activation domains such as VP16 or c-Jun . These data demonstrate a clear role for transcription factors with specific activation domains in activating both SV40 and Py ori . However, no correlation was observed between the ability of specific proteins to stimulate promoter activity and their ability to stimulate origin activity . We propose that only transcription factors whose specific activation domains can interact with the T-ag initiation complex can stimulate SV40 and Py ori-core activity.

Nat Genet, 1992 Jun, 1(3), 204 - 8
Isolation and characterization of a candidate gene for Norrie disease; Chen ZY et al.; Previous analysis has refined the location of the gene for Norrie disease, a severe, X-linked, recessive neurodevelopmental disorder, to a yeast artificial chromosome subfragment of 160 kilobases (kb) . This fragment was used to screen cDNA libraries from human fetal and adult retina . As a result, we have identified an evolutionarily conserved cDNA, which is expressed in fetal and adult brain and encodes a predicted protein of 133 amino acids . The cDNA detects genomic sequences which span a maximum of 50 kb, and which are partly deleted in several typical Norrie disease patients . An EcoRI polymorphism with a calculated heterozygosity value of 43% was observed . The locus identified is a strong candidate for the Norrie disease gene.

Nat Genet, 1992 Jun, 1(3), 176 - 9
Peripheral myelin protein-22 gene maps in the duplication in chromosome 17p11.2 associated with Charcot-Marie-Tooth 1A; Matsunami N et al.; Charcot-Marie-Tooth disease 1A (CMT1A) is a hereditary demyelinating peripheral neuropathy, associated with a DNA duplication on chromosome 17p11.2 . A related disorder in the mouse, trembler (Tr), maps to mouse chromosome 11 which has syntenic homology to human chromosome 17p . Recently, the peripheral myelin protein-22 (pmp-22) gene was identified as the likely Tr locus . We have constructed a partial yeast artificial chromosome contig spanning the CMT1A gene region and mapped the PMP-22 gene to the duplicated region . These observations further implicate PMP-22 as a candidate gene for CMT1A, and suggest that over-expression of this gene may be one mechanism that produces the CMT1A phenotype.

Cell, 1992 May 29, 69(5), 871 - 81
Specific interaction between the nonphosphorylated form of RNA polymerase II and the TATA-binding protein; Usheva A et al.; Fractionation of a transcription-competent HeLa cell extract on a column containing one copy of the heptamer repeat (YSPTSPS) present in the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II resulted in the loss of transcriptional activity . Fractionation of the extracts on columns containing mutations of the heptamer repeat was without effect . Such transcriptionally inactive extracts regained their ability to specifically transcribe different class II promoters upon the addition of human TFIID, recombinant yeast TATA-binding protein (TBP), or proteins bound to the column . Fractionation of RNA polymerase II on columns containing human or yeast TBP resulted in the specific retention of the nonphosphorylated form of RNA polymerase II . The phosphorylated form of the enzyme was unable to interact with TBP . The specific interaction of RNA polymerase II with TBP was mediated by the CTD of RNA polymerase II.

FEBS Lett, 1992 May 18, 302(3), 217 - 9
Possible involvement of superoxide anion in the induction of cyanide-resistant respiration in Hansenula anomala; Minagawa N et al.; A chemiluminescence study showed that Qi site inhibitors such as antimycin A induce O2- generation in respiring cyanide-sensitive mitochondria from the yeast, Hansenula anomala . The O2- generation was suppressed by radical scavengers such as flavone, butylated hydroxyanisole, and Co0 . Induction of cyanide-resistant respiration in H . anomala cells by Qi site inhibitors was also inhibited by these radical scavengers . Furthermore, antimycin A-induced synthesis of the mitochondrial 36-kDa protein, which is thought to be the alternative oxidase functional in the cyanide-resistant respiratory pathway, was abolished by the addition of flavone . These observations suggest that O2- is somehow involved in the induction of cyanide-resistant respiration.

Nucleic Acids Res, 1992 May 11, 20(9), 2223 - 32
Effect of the higher-order structure of tRNAs on the stability of hybrids with oligodeoxyribonucleotides: separation of tRNA by an efficient solution hybridization; Kumazawa Y et al.; In the course of developing a method to purify a single tRNA species efficiently, we have examined hybridization efficiencies between some tRNAs and short oligodeoxyribonucleotide probes both by the filter and solution hybridization methods without denaturants . The hybridization efficiencies varied considerably among probes which are complementary to different regions of the tRNAs, although there was little efficiency variation in the probes toward DNA substrates including the same nucleotide sequence . This efficiency variation was shown to be due to tRNA-specific higher-order structures as well as a hypermodified nucleotide in the anticodon loop . Characterization of the tRNA-probe hybrids by both nondenaturing gel electrophoresis and chemical modification showed the existence of two stable hybridizing states as a function of ionic strength . Our results indicate that RNA molecules with a number of intramolecular base pairings are able to form stable hybrids with complementary sequences under nondenaturing conditions . On the basis of these data, an appropriate probe was designed to successfully purify yeast tRNA(Phe) by making a tRNA(Phe)-probe hybrid, which has a longer retention time in hydroxyapatite high performance liquid chromatography than the tRNA(Phe) itself.

Science, 1992 May 8, 256(5058), 825 - 7
Implication of GAP in Ras-dependent transactivation of a polyoma enhancer sequence; Schweighoffer F et al.; Controversy exists as to whether the interaction of a guanosine triphosphatase activating protein (GAP) with Ras proteins functions both to initiate and to terminate Ras-dependent signaling events or only to terminate them . GAP-C, a carboxyl-terminal fragment of GAP that is sufficient to stimulate GTPase activity, inhibited the stimulation of transcription produced by some oncoproteins (v-Src, polyoma middle T, wild-type Ras, and oncogenic Ras) but not that produced by v-Mos . Wild-type GAP did not affect transcription induced by oncogenic Ras but reversed the inhibitory effect of GAP-C on transcription induced by oncogenic Ras . These results indicate that GAP is a negative regulator of wild-type Ras and elicits a downstream signal by interacting with Ras-GTP (guanosine triphosphate).

Biochim Biophys Acta, 1992 May 7, 1131(1), 103 - 7
Molecular cloning and expression of hsp82 gene of the dimorphic pathogenic fungus Histoplasma capsulatum; Minchiotti G et al.; We have cloned a nucleotide sequence from Histoplasma capsulatum G222B corresponding to a heat inducible hsp82 gene, and determined its entire sequence and the flanking regions . During the temperature-controlled mycelium-to-yeast phase transition the gene is more actively transcribed at 37 degrees C in the temperature tolerant and mouse-virulent G222B strain, while 34 degrees C is the optimum for transcription in the temperature sensitive and mouse-avirulent Downs strain.

Biochemistry, 1992 May 5, 31(17), 4334 - 46
1H, 15N, 13C, and 13CO assignments of human interleukin-4 using three-dimensional double- and triple-resonance heteronuclear magnetic resonance spectroscopy; Powers R et al.; The assignment of the 1H, 15N, 13CO, and 13C resonances of recombinant human interleukin-4 (IL-4), a protein of 133 residues and molecular mass of 15.4 kDa, is presented based on a series of 11 three-dimensional (3D) double- and triple-resonance heteronuclear NMR experiments . These studies employ uniformly labeled 15N- and 15N/13C-labeled IL-4 with an isotope incorporation of greater than 95% for the protein expressed in yeast . Five independent sequential connectivity pathways via one-, two-, and three-bond heteronuclear J couplings are exploited to obtain unambiguous sequential assignments . Specifically, CO(i)-N(i + 1),NH(i + 1) correlations are observed in the HNCO experiment, the C alpha H(i), C alpha (i)-N(i + 1) correlations in the HCA(CO)N experiment, the C alpha(i)-N(i + 1),NH(i + 1) correlations in the HNCA and HN(CO)CA experiments, the C alpha H(i)-N(i + 1),NH(i + 1) correlations in the H(CA)NH and HN(CO)HB experiments, and the C beta H(i)-N(i + 1),NH(i + 1) correlations in the HN(CO)HB experiments . The backbone intraresidue C alpha H(i)-15N(i)-NH(i) correlations are provided by the 15N-edited Hartmann-Hahn (HOHAHA) and H(CA)NH experiments, the C beta H(i)-15N(i)-NH(i) correlations by the 15N-edited HOHAHA and HNHB experiments, the 13C alpha(i)-15N(i)-NH(i) correlations by the HNCA experiment, and the C alpha H(i)-13C alpha(i)-13CO(i) correlations by the HCACO experiment . Aliphatic side-chain spin systems are assigned by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and total correlated (HCCH-TOCSY) spectroscopy . Because of the high resolution afforded by these experiments, as well as the availability of multiple sequential connectivity pathways, ambiguities associated with the limited chemical shift dispersion associated with helical proteins are readily resolved . Further, in the majority of cases (88%), four or more sequential correlations are observed between successive residues . Consequently, the interpretation of these experiments readily lends itself to semiautomated analysis which significantly simplifies and speeds up the assignment process . The assignments presented in this paper provide the essential basis for studies aimed at determining the high-resolution three-dimensional structure of IL-4 in solution.

Biochemistry, 1992 May 5, 31(17), 4205 - 10
Conserved residues flanking the thiol/disulfide centers of protein disulfide isomerase are not essential for catalysis of thiol/disulfide exchange; Lu X et al.; Protein disulfide isomerase (PDI) catalyzes the oxidative folding of proteins containing disulfide bonds by increasing the rate of disulfide bond rearrangements which normally occur during the folding process . The amino acid sequences of the N- and C-terminal redox active sites (PWCGHCK) in PDI are completely conserved from yeast to man and display considerable identity with the redox-active center of thioredoxin (EWCGPCK) . Available data indicate that the two thiol/disulfide centers of PDI can function independently in the isomerase reaction and that the cysteine residues in each active site are essential for catalysis . To evaluate the role of residues flanking the active-site cysteines of PDI in function, a variety of mutations were introduced into the N-terminal active site of PDI within the context of both a functional C-terminal active site and an inactive C-terminal active site in which serine residues replaced C379 and C382 . Replacement of non-cysteine residues (W34 to Ser, G36 to Ala, and K39 to Arg) resulted in only a modest reduction in catalytic activity in both the oxidative refolding of RNase A and the reduction of insulin (10-27%), independent of the status of the C-terminal active site . A somewhat larger effect was observed with the H37P mutation where approximately 80% of the activity attributable to the N-terminal domain (approximately 40%) was lost . However, the H37P mutant N-terminal site expressed within the context of an inactive C-terminal domain exhibits 30% activity, approximately 70% of the activity of the N-terminal site alone.(ABSTRACT TRUNCATED AT 250 WORDS)

FASEB J, 1992 May, 6(8), 2537 - 44
Endogenous retroviruses: potential etiologic agents in autoimmunity; Krieg AM et al.; The genomes of all organisms, from yeast to humans, contain thousands of endogenous retroviruses (ERV) . In most species all or almost all ERV are noninfectious, but some ERV retain open reading frames capable of encoding proteins . RNA and proteins derived from ERV are expressed in humans and other species . Until recently, there was little evidence that this ERV expression resulted in any immunologic effects . Recent studies make it increasingly clear that some ERV have important immunologic effects . The immune effects of ERV expression raise the question of a possible pathogenic role in idiopathic autoimmune diseases . Interest in this question has been heightened by the observation that some infectious retroviruses cause manifestations of autoimmunity . Nonetheless, attempts to isolate infectious retroviruses from patients with idiopathic autoimmune diseases have generally failed . The possible role of ERV in idiopathic autoimmune diseases has not yet been fully explored . This review focuses on the known and the potential immune effects of ERV, especially as they may relate to autoimmune diseases.

Mol Pharmacol, 1992 May, 41(5), 975 - 80
Human liver microsomal N-hydroxylation of dapsone by cytochrome P-4503A4; Fleming CM et al.; One of the major routes of elimination of dapsone (4,4'-diaminodiphenylsulfone) is by N-oxidation, to produce a hydroxylamine metabolite . The specific form of cytochrome P-450 (P-450) involved in this oxidation reaction was examined in human liver microsomal preparations previously characterized with respect to their content of several known P-450 enzymes . Among five preparations, the rank order of activity for dapsone hydroxylamine formation was most well correlated with the immunochemically determined level of P-4503A4 (r = 0.94, p less than 0.03) . Moreover, inhibition of microsomal oxidation was observed with antibodies specific to P-4503A, with a maximum reduction of greater than 90%, but was not produced by antibodies specific to P-4501A2, P-4502CMP, or P-4502E1 . Prior incubation of microsomes with gestodene (100 microM) or troleandomycin (20 microM), known selective mechanism-based inhibitors of P-4503A enzymes (in the presence of NADPH), led to 75% and 40% reductions in catalytic activity, respectively . In contrast, preincubation with increasing concentrations of alpha-naphthoflavone, a known activator of P-4503A4, increased dapsone N-hydroxylation in a concentration-dependent manner, with 5-fold activation being observed at 50 microM alpha-naphthoflavone . Finally, P-4503A4 isolated from human liver microsomes and cDNA-expressed P-4503A4 (in yeast) were both able to catalyze dapsone N-hydroxylation, with the latter preparation exhibiting a 3-fold activation in the presence of 100 microM alpha-naphthoflavone . Collectively, these findings demonstrate that N-oxidation of dapsone in human liver is predominantly mediated by P-4503A4, and they suggest that quantitative measurement of this metabolic pathway in vivo might serve as an index of the activity of this enzyme.

Toxicol Appl Pharmacol, 1992 May, 114(1), 147 - 55
Metabolism of trans,trans-muconaldehyde by aldehyde and alcohol dehydrogenases: identification of a novel metabolite; Goon D et al.; The metabolism of trans,trans-muconaldehyde (MA), a highly reactive alpha,beta-unsaturated dialdehyde, was examined in vitro using purified yeast alcohol and aldehyde dehydrogenases (ADH and ALDH, respectively) . In the presence of NAD(+)-fortified ALDH, the mono-oxidation product (acid/aldehyde) was the primary metabolite formed with trace amounts of the dioxidation product (trans,trans-muconic acid) . In NADH-fortified reactions with ADH, both the mono- and direduction products (hydroxy/aldehyde and dihydroxy, respectively) were readily detected . Oxidation and reduction products of MA were formed in incubates containing both dehydrogenases together with either NAD+ or NADH . Unexpectedly, an additional metabolite was detected, which was a major product in both NAD(+)- and NADH-fortified systems containing ALDH and ADH in combination and whose formation could be inhibited by pyrazole (an ADH inhibitor) . ALDH-mediated oxidation of a synthetic standard of the hydroxy/aldehyde derivative of MA resulted in formation of this new metabolite, which was also a major product formed by rat hepatocytes incubated with MA . Using HPLC/photodiode array detection, the new metabolite was found to cochromatograph and have a uv spectrum identical to that of a synthetic standard of the hydroxy/acid derivative of MA . The metabolite was confirmed as the hydroxy/acid derivative of MA after preparative HPLC, TMS derivatization, and GC/MS analysis . The hydroxy/acid metabolite was not formed during ADH-mediated reduction of the mono-oxidation product of MA, suggesting that this metabolite was formed by yeast dehydrogenases via a primary reduction of MA and subsequent oxidation of the hydroxy/aldehyde to the hydroxy/acid . These data show that the hydroxy/acid derivative is a novel metabolite of MA, which arises from the interaction of both oxidative and reductive routes of metabolism.

Genomics, 1992 May, 13(1), 49 - 61
Physical mapping of genes to specific chromosomes in Dictyostelium discoideum; Kuspa A et al.; Cloned genes were used to probe a highly redundant library of large cloned fragments of the Dictyostelium discoideum genome carried in yeast artificial chromosomes (YACs) . Each gene recognized several independent YAC clones, thereby grouping them into a contig . Individual YACs were arranged within the contig by positioning genes relative to rare restriction sites and the YAC ends . Genes that had been previously assigned to one of the six linkage groups by parasexual genetics were used to establish physically mapped regions on specific chromosomes . Previously unmapped genes were assigned to specific chromosomes when they recognized members of a mapped contig . Linkage was confirmed by congruence of large-scale restriction maps centered on either the previously mapped or the newly mapped genes . At present, the chromosome-assigned map segments comprise approximately 50% of the genome . About half of each map segment is covered by overlapping YACs.

Genes Dev, 1992 May, 6(5), 864 - 75
Functional analysis of the transcriptional activator encoded by the maize B gene: evidence for a direct functional interaction between two classes of regulatory proteins; Goff SA et al.; The B, R, C1, and Pl genes regulating the maize anthocyanin pigment biosynthetic pathway encode tissue-specific transcriptional activators . B and R are functionally duplicate genes that encode proteins with the basic-helix-loop-helix (b-HLH) motif found in Myc proteins . C1 and Pl encode functionally duplicate proteins with homology to the DNA-binding domain of Myb proteins . A member of the b-HLH family (B or R) and a member of the myb family (C1 or Pl) are both required for anthocyanin pigmentation . Transient assays in maize and yeast were used to analyze the functional domains of the B protein and its interaction with C1 . The results of these studies demonstrate that the b-HLH domain of B and most of its carboxyl terminus can be deleted with only a partial loss of B-protein function . In contrast, relatively small deletions within the B amino-terminal-coding sequence resulted in no trans-activation . Analysis of fusion constructs encoding the DNA-binding domain of yeast GAL4 and portions of B failed to reveal a transcriptional activation domain in the B protein . However, an amino-terminal domain of B was found to recruit a transcriptional activation domain by an interaction with C1 . Formation of this complex resulted in the activation of a synthetic promoter containing GAL4 recognition sites, demonstrating that this interaction does not require the normal target promoters for B and C1 . B and C1 fusions with yeast GAL4 DNA-binding and transcriptional activation domains were also found to interact when synthesized and assayed in yeast . The domains responsible for this interaction map to a region that contains the Myb homologous repeats of the C1 protein and to the amino terminus of the B protein, which does not contain the b-HLH motif . These studies suggest that the regulation of the maize anthocyanin pigmentation pathway involves a direct interaction between members of two distinct classes of transcriptional activators.

Blood, 1992 May 1, 79(9), 2349 - 55
Preferred sequence requirements for cleavage of pro-von Willebrand factor by propeptide-processing enzymes; Rehemtulla A et al.; Maturation of pro-von Willebrand factor (vWF) to its active form requires proteolytic processing after a pair of dibasic amino acids (-LysArg-) at residue 763 . By coexpression of vWF and various propeptide processing enzymes in COS-1 cells, we here demonstrate that vWF is preferentially processed by the paired dibasic amino acid-cleaving enzyme PACE (furin) . Processing of vWF by the yeast homologue of PACE, Kex2, was inefficient and not specific for the authentic site . Two additional recently identified mammalian propeptide-processing enzymes PC2 and PC3 had no detectable vWF-processing activity . The inability of PC2 and PC3 to cleave vWF was apparently not due to the absence of a transmembrane domain, since deletion of the transmembrane domain from PACE resulted in a secreted form which retained its propeptide processing activity within the secretory apparatus . The inability of PC2 and PC3 to process wild-type vWF or any of the vWF mutants described suggests different members of subtilisin-related propeptide-processing enzyme family have evolved to selectively recognize and cleave specific sets of substrates . In addition to paired dibasic residues at the propeptide cleavage site, many proteins, including vWF, also contain an arginine at the P4 position . We have generated mutant vWFs with substitutions at the P2 lysine and/or the P4 arginine to investigate their significance in substrate specificity . A conservative substitution of the P4 arginine by lysine resulted in a decrease in vWF processing by PACE, as did a nonconservative substitution to alanine . Substitution of the P2 lysine to aspartic acid decreased processing and little or no processing was detected when both the P4 and P2 were mutated to lysine and aspartic acid, respectively . These data indicate that both the P4 arginine and the P2 lysine play an important role in substrate recognition by PACE.

J Infect Dis, 1992 May, 165(5), 891 - 7
Prophylactic intravenous amphotericin B in neutropenic autologous bone marrow transplant recipients; Perfect JR et al.; This study assessed the efficacy, toxicity, and pharmacology of low-dose amphotericin B given prophylactically to patients (serum concentrations of 0.2-0.4 microgram/ml) undergoing bone marrow transplantation . Yeast isolates from patients' oropharyngeal areas had MICs of 0.1-0.2 microgram/ml, and none were amphotericin B resistant . The effect of low-dose amphotericin B on reducing the numbers of yeast colonizing the oropharyngeal area was significant (P less than .01) . The average delay in switching to high-dose prophylactic amphotericin B was only 1 day; the decision to do so because of a perceived fungal infection occurred more frequently for the placebo group (P = .06) . Fewer patients from the low-dose amphotericin B group (8.8%) than from the placebo group (14.3%) had fungi isolated from normally sterile body sites (P = .35) . Infusion-related side effects but not systemic toxicities were significantly greater (P less than .001) in the amphotericin B group . The 6-week survival was greater in those receiving amphotericin B (P less than .03), but the improved survival could not be attributed to the prevention of fungal infections.

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