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