Yeasts

J Protein Chem, 1991 Feb, 10(1), 25 - 9
Eukaryotic glucose-6-phosphate dehydrogenases: structural screening of related proteins; Bergman T et al.; Rapid assessment of structural relationships between yeast glucose-6-phosphate dehydrogenases and other eukaryotic types of this enzyme is described . Separation and size estimation of large fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis, electroblotting onto disks, and sequencer analysis provide data that permit alignment of the segments thus characterized with the related proteins, and utilize existing structural knowledge to assess new enzyme structures . Affinity labeling allows further correlations . The results establish the overall structural arrangements of the new proteins, including the location of the active-site lysine residue, even though the yeast enzyme structures are found to differ markedly from the few previously characterized glucose-6-phosphate dehydrogenases.

Bone Marrow Transplant, 1991 Feb, 7(2), 127 - 31
Does ketoconazole prevent fungal infection in children treated with high dose chemotherapy and bone marrow transplantation? Results of a randomized placebo-controlled trial; Benhamou E et al.; Between August 1985 and October 1988 125 children who were candidates for a bone marrow transplantation entered a randomized double-blind placebo-controlled trial (63 children in ketoconazole (K) group and 62 children in placebo (P) group) to evaluate the efficacy of ketoconazole in decreasing the incidence of digestive yeast colonization . Among the 38 children who were initially colonized, the proportion of children who became decolonized was higher (p less than 0.05) in group K (47%) than in group P (16%) . Among the 87 children without initial colonization, the proportion of children in whom digestive colonization occurred was lower (p less than 0.01) in group K (20%) than in group P (49%) . Three children in group K (5%) and six children in group P (9%) developed a documented fungal infection (NS) . Thirty-nine children in each group developed a fever of unknown origin which did not respond to a 7-day course of antibiotherapy . One hundred and three children received amphotericin B (Ampho B) (83% in group K and 82% in group P) . The mean duration (+/- SD) of Ampho B was similar in group K (28 +/- 26 days) and in group P (28 +/- 25 days) . The mean total dose of Ampho B was similar in group K (475.5 +/- 1060 mg) and in group P (540.4 +/- 979 mg) . The actuarial survival from the day of randomization until bone marrow recovery was not different in the two groups . We conclude that ketoconazole is efficient in preventing gut yeast colonization but does not reduce the incidence of fungal documented infections and fevers of unknown origin unresponsive to antibiotherapy which necessitate empirical coverage and treatment with Ampho B.

Anal Biochem, 1991 Feb 1, 192(2), 358 - 61
The use of pH indicators to identify suitable environments for freezing samples in aqueous and mixed aqueous/nonaqueous solutions; Hill JP et al.; The colours of frozen solutions containing pH indicators are shown to provide a test for changes in pH in the solvent environment which occur on freezing . Yeast alcohol dehydrogenase loses activity on freezing in phosphate buffer (a buffer in which pH indicator colour changes shows a marked decrease in pH on freezing) but when frozen in bis-tris, Hepes, or N-glycylglycine buffers (all of which show little change in the colour of universal pH indicator and hence of pH on freezing) is stable on freezing . The effects of freezing in different buffer systems on the rate of decomposition of NADPH, and on the rate hydrolysis of 4-nitrophenyl acetate, are rationalised in terms of the pH shifts in these buffers which were determined using universal pH indicator . It is proposed that a major reason for the instability of samples on freezing is the pH changes which occur when some systems are frozen . From the results a general scheme for selecting the best environment for safely freezing samples is proposed which is based on the use of pH indicators.

Mycopathologia, 1991 Feb, 113(2), 109 - 15
Macrocyclic trichothecenes produced by Stachybotrys isolated from Egypt and eastern Europe; el-Maghraby OM et al.; Twenty seven isolates of Stachybotrys chartarum, S . albipes, S . kampalensis and S . microspora from Egypt and Eastern Europe were tested for production of macrocyclic trichothecenes . Twenty of the 27 isolates, grown on rice seeds, were toxic to brine shrimp larvae . Based on TLC and HPLC analyses, 5 macrocyclic trichothecenes (verrucarin J, roridin E, satratoxins F, G & H) as well as trichoverrols were identified . When grown in liquid culture on rice extract medium, only 3 isolates were toxic and produced verrucarin J, roridin E and satratoxins G & H . Extracts from mycelial mats were more toxic than culture filterates of two isolates grown on rice extract and both contained the same macrocyclic trichothecenes (285.5 mg/4 L), in addition to trichoverrols A & B (31 mg/4 L) found in mycelial mats only . When grown on 3% sucrose Czapek's medium supplemented with peptone and yeast extract (still cultures), all isolates were non-toxic to brine shrimp and no trichothecenes could be detected in the extracts.

Biochem Cell Biol, 1991 Feb-Mar, 69(2-3), 211 - 5
Sequence of an acidic ribosomal protein from the jellyfish Polyorchis penicillatus; Gallin W; We have isolated a cDNA clone from the jellyfish Polyorchis penicillatus that encodes the homologue of the A1 acidic ribosomal protein previously characterized in human, brine shrimp, fruit fly, and yeast . The sequence of this protein is strongly conserved among the five eukaryotic species for which it has been determined . Conservation is greatest in the amino-terminal 51 amino acids and the carboxyl-terminal 25 amino acids . This suggests that these regions are necessary for interactions with other components of the protein synthetic machinery, while the central part of the protein has a less specific role to play . Comparison of the sequences obtained from the different species indicate that the metazoan lineages all appear to have arisen at approximately the same time and significantly later than the time of divergence of yeast from the common ancestor of the Metazoa.

J Cutan Pathol, 1991 Feb, 18(1), 28 - 35
Majocchi's granuloma; Smith KJ et al.; Majocchi's granuloma (nodular granulomatous perifolliculitis) is a well recognized but uncommon infection of dermal and subcutaneous tissue by fungal organisms (dermatophytes) usually limited to the superficial epidermis . The organism usually associated with Majocchi's granuloma is Trichophyton rubrum; however, other dermatophytes including T . mentagrophytes (variety granulosum), T . epilans, T . violaceum, M . audouinii, M . gypseum, M . ferrugineum, and M . canis may be the causative agent . A review of 17 cases revealed not only the variety of possible organisms but also a marked variation from the usual hyphal forms . The morphologic variations including yeast forms, bizarre hyphae, mucinous coatings, and the Splendore-Hoeppeli phenomenon may be factors which allow the dermatophytes to persist and grow in an abnormal location . Also, there is evidence that Majocchi's granuloma may occur in two distinct groups of patients.

J Mol Endocrinol, 1991 Feb, 6(1), 63 - 70
Cloning and characterization of a gene encoding pig epidermal growth factor; Pascall JC et al.; A portion of the pig epidermal growth factor (EGF) gene has been isolated and characterized . The nucleotide sequencies of exons 20 and 21, which encode the EGF region of the precursor protein, show 85% similarity with the human EGF gene sequence . In addition, conservation of the intron-exon boundaries between the two species was generally observed . Although the pig exon 21 appeared to lack a single nucleotide at its 5' end relative to the human gene, sequences obtained by direct amplification of the genomic DNA around the 5' end of this exon using the polymerase chain reaction, and from a pig EGF cDNA recombinant isolated from a kidney library, indicated that the deletion was probably a cloning artifact . Comparison of the predicted amino acid sequence of pig EGF with that of EGF from other species, as well as with several other polypeptides which bind to the EGF receptor, indicated conservation of Gly18, Tyr37, Gly39 and Arg41 in addition to all six cysteine residues and Leu47, which are known to be critical for biological activity . A synthetic gene encoding the predicted amino acid sequence of pig EGF was expressed in yeast . The recombinant polypeptide was shown to compete with 125I-labelled mouse EGF for binding to cells and to stimulate DNA synthesis in quiescent monolayers of Swiss 3T3 cells.

Mutat Res, 1991 Feb, 262(2), 93 - 9
Effects of selenium deficiency on sperm morphology and spermatocyte chromosomes in mice; Watanabe T et al.; Sperm morphology and spermatocyte chromosomes were examined in mice maintained on a Torula yeast diet for 5 weeks . In the selenium-deficient group, the proportion of abnormal sperm was high, ranging from 6.8% to 49.6%, while in the control group it ranged from only 4.0% to 15.0% . The most frequently occurring abnormalities in sperm shape were in the sperm head . There was also a tendency for abnormalities in other regions (neck, midpiece and tail) to be increased . However, in metaphase-I spermatocytes, the frequencies of various types of abnormal chromosomes (univalent chromosomes, translocations and structural anomalies) did not differ between the selenium-deficient and control groups . These findings indicate that selenium day be an essential constituent for spermatogenesis in mice.

Hum Genet, 1991 Feb, 86(4), 350 - 4
Chromosome assignment of four RAS-related RAB genes; Rousseau-Merck MF et al.; The human RAB genes share structural and biochemical properties with the RAS gene superfamily . The encoded RAB proteins show 38 to 75% amino acid identity with the yeast YPT1 and SEC4 gene products . We used four human RAB-cDNAs, RAB3B, RAB4, RAB5 and RAB6, to map the corresponding genes on human chromosomes . These genes were assigned to 1p32-p31, 1q42-q43, 3p24-p22 and 2q14-q21, respectively, by in situ hybridization.

Genes Dev, 1991 Feb, 5(2), 298 - 309
Identification of functional domains in the maize transcriptional activator C1: comparison of wild-type and dominant inhibitor proteins; Goff SA et al.; Genes encoding fusions between the maize regulatory protein C1 and the yeast transcriptional activator GAL4 and mutant C1 proteins were assayed for their ability to trans-activate anthocyanin biosynthetic genes in intact maize tissues . The putative DNA-binding region of C1 fused to the transcriptional activation domain of GAL4 activated transcription of anthocyanin structural gene promoters in c1 aleurones, c1 Rscm2 embryos, and c1 r embryogenic callus . Cells receiving these constructs accumulated purple anthocyanin pigments . The C1 acidic region fused to the GAL4 DNA-binding domain activated transcription of a GAL4-regulated promoter . An internal deletion of C1 also induced pigmentation; however, frameshifts in either the amino-terminal basic or carboxy-terminal acidic region blocked trans-activation, and the latter generated a dominant inhibitory protein . Fusion constructs between the wild-type C1 cDNA and the dominant inhibitor allele C1-I cDNA were used to identify the amino acid changes in C1 responsible for the C1-I inhibitory phenotype . Results from these studies establish that amino acids within the myb-homologous domain are critical for transcriptional activation.

J Infect Dis, 1991 Feb, 163(2), 219 - 25
Safety and immunogenicity of a genetically engineered human immunodeficiency virus vaccine; Wintsch J et al.; A phase 1 trial of a candidate human immunodeficiency virus type 1 (HIV-1) vaccine was done in 25 healthy seronegative subjects . The antigen, env2-3 (SF2), was a nonglycosylated polypeptide representing the gp120 region of the env gene of the HIV-1(SF2) isolate . It was produced in genetically engineered yeast as a denatured molecule incapable of binding CD4 . A synthetic lipophilic muramyl tripeptide (MTP-PE) was used as an adjuvant . Ten subjects received adjuvant alone and 15 received 50- or 250-micrograms doses of env2-3 (SF2) administered intramuscularly in two immunization regimens . In general, adjuvant and vaccine were well tolerated . Antibody responses to both the homologous antigen, env2-3 (SF2), and antigens from other highly divergent HIV isolates were elicited in the majority of vaccine recipients . However, antibody titers were low, without neutralizing activity . In 9 of 11 subjects who received the complete vaccine immunization series, a significant specific T lymphocyte response was observed.

Mol Biol Med, 1991 Feb, 8(1), 95 - 100
Additional mutations in argininosuccinate synthetase causing citrullinemia; Kobayashi K et al.; Deficiency of argininosuccinate synthetase causes arginine auxotrophy in lower organisms and causes citrullinemia in humans and cattle . Previously, seven missense mutations, four mutations associated with an absence of an exon in mRNA, and one splicing mutation have been identified in human neonatal citrullinemia . Reverse transcription of mRNA, amplification of cDNA and sequencing of cDNA clones were used to identify two additional missense mutations causing citrullinemia . One mutation involves substitution of leucine for serine at position 18 (S18L) and the other a substitution of cysteine for arginine at position 86 (R86C) . Both of these mutations represent C----T transitions in CpG dinucleotides, and eight of nine missense mutations causing human citrullinemia involve similar transitions in CpG dinucleotides . The nucleotide coding sequence and deduced amino acid analysis are available for four mammalian species, yeast and three bacterial species . Six of nine missense mutations in humans occur in amino acid positions that are completely conserved in these organisms . Mutations causing human citrullinemia are extremely heterogeneous, and all non-consanguineous individuals studied to date are compound heterozygotes.

Mycopathologia, 1991 Feb, 113(2), 121 - 5
Different media and methodologies for the detection of aflatoxin production by Aspergillus flavus strains isolated from trout feed; Cutuli MT et al.; We have studied the aflatoxin producing capacity of 41 Aspergillus flavus strains isolated from the mycoflora present of natural media (wheat, rice and mixed feed) synthetic medium (Aflatoxin Producing Ability Medium) and semisynthetic media (Coconut Agar Medium and Glucose Yeast Extract Agar) were compared . Aflatoxins were analysed on days 4 and 8 post-inoculation under an incubation temperature of 28 degrees C . A total of 30 strains (75.7%) were producers on natural media as detected by Thin Layer Chromatography: 23 strains on wheat, 27 on rice and 12 on mixed feed . The results by qualitative fluorescence tests on synthetic and semisynthetic media were: 3 strains positive on Coconut Agar Medium (CAM) 1 on Glucose Yeast Extract Agar (GY + Agar) and none on Aflatoxin Producing Ability Medium (APA).

Mutat Res, 1991 Feb, 259(2), 165 - 76
Aneuploidy in Drosophila, IV . Inhalation studies on the induction of aneuploidy by nitriles; Osgood C et al.; The Drosophila ZESTE system was used to monitor the induction of sex chromosome aneuploidy following inhalation exposure of adult females to four nitriles: acetonitrile, propionitrile, acrylonitrile and fumaronitrile . Acetonitrile and propionitrile were highly effective aneuploidogens, inducing both chromosome loss and chromosome gain following brief exposures to low concentrations of these chemicals, and these nitriles also induced rapid paralysis . Acrylonitrile-induced chromosome loss only but did not induce paralysis . Fumaronitrile, in contrast with the results reported in yeast, was ineffective in inducing chromosome loss or gain . Virtually all exceptional offspring induced by acetonitrile and propionitrile were recovered in the first sampled eggs, corresponding to treated mature oocytes . Additionally, the time interval between treatment and sampling was shown to be important, suggesting rapid loss or detoxification of the nitriles . Genetic analysis demonstrated that most aneuploids resulted from induced segregation errors during the first division of meiosis . Cold treatments were found to be ineffective in enhancing the effects of acetonitrile, suggesting important differences between the Drosophila and yeast aneuploidy detection systems . Possible mechanisms by which nitriles may disrupt chromosome segregation in Drosophila oocytes are considered.

Antonie Van Leeuwenhoek, 1991 Feb, 59(2), 81 - 93
Bulleromyces genus novum (Tremellales), a teleomorph for Bullera alba, and the occurrence of mating in Bullera variabilis; Boekhout T et al.; Mating is observed in Bullera alba and B . variabilis, resulting in the formation of dikaryotic mycelium with clamps, haustorial branches, and lateral and terminal dikaryotic, clavate, lageniform or subglobose cells . These cells develop in B . alba into tremellaceous phragmobasidia . Karyogamy has been observed in young non-divided basidia . Germination of the phragmobasidia occurred by acropetal chains of yeast cells, ballistospores or hyphae . Septal pores are dolipores with parenthesomes made up of U-shaped vesicles (Tremellales type) . For the teleomorph of B . alba a new genus, Bulleromyces, is proposed, with only one species, viz . Bulleromyces albus.

Plant Cell, 1991 Feb, 3(2), 105 - 13
Monocot regulatory protein Opaque-2 is localized in the nucleus of maize endosperm and transformed tobacco plants; Varagona MJ et al.; Protein targeting to the nucleus has been studied extensively in animal and yeast systems; however, nothing is known about nuclear targeting in plants . The Opaque-2 (O2) gene produces a regulatory protein that is responsible for inducing transcription of the alpha-zein class of storage proteins in maize kernels . The cloned O2 gene encodes a protein that contains a leucine zipper DNA binding domain that can interact with zein gene promoters . We have used immunolocalization to show that the O2 protein is present in nuclei in the maize endosperm tissues known to produce alpha-zeins . In addition, neither embryo tissue from wild-type kernels nor endosperm from kernels harboring a null o2 allele contain the O2 protein . Analysis of a transposable, element-induced o2 allele, o2-m20, revealed that sectors of endosperm cells contained the nuclear-localized O2 protein, indicating excision of the transposable element . To study further the nuclear transport of the O2 protein, we have transformed this gene, under the control of a constitutive promoter, into tobacco . Plants were shown to have detectable levels of steady-state O2 mRNA and O2 protein . Immunolocalization of O2 protein in transformed tobacco plants indicated that the O2 protein was transported into tobacco nuclei . Therefore, we have developed a system to study nuclear targeting in plants and have established that the nuclear transport machinery is similar in monocots and dicots.

Chromosoma, 1991 Feb, 100(2), 118 - 24
He-T family DNA sequences in the Y chromosome of Drosophila melanogaster share homology with the X-linked stellate genes; Danilevskaya ON et al.; The genome of Drosophila melanogaster contains a class of repetitive DNA sequences called the He-T family, which is unusual in being confined to telomeric and heterochromatic regions . The specific He-T fragment designated Dm665 was cloned in yeast by selection for an autonomously replicating sequence (ARS) . Dm665 contains a restriction fragment length polymorphism (RFLP) that is specific to males and thus derives from the Y chromosome . Deletion mapping using X-Y translocations indicates that sequences homologous to Dm665 occur in at least one major cluster in each arm of the Y chromosome . Among 20 yeast artificial chromosome (YAC) clones containing Drosophila sequences homologous with Dm665, four clones derive from defined regions of the long arm of the Y and two from the short arm . The sequence of Dm665 is 2443 bp long, consists of 59% A + T, and contains no significant open reading frames or direct or inverted repeats . However, Dm665 contains a region of 650 bp that shares homology with portions of the X-linked locus Stellate.

Int J Hematol, 1991 Feb, 54(1), 49 - 55
Aclacinomycin, an anti-leukemic anthracycline, impairs human neutrophil functions; Katoh M et al.; The effects of aclacinomycin, an anti-leukemic anthracycline, on human neutrophil functions were investigated . The release of superoxide (O2-) in neutrophils stimulated by opsonized zymosan, myristate, or phorbol myristate acetate was inhibited by aclacinomycin in a dose- and time-dependent manner . The phagocytosis of yeast particles and oil droplets, and membrane potential changes stimulated by phorbol myristate acetate were also inhibited by aclacinomycin . On the other hand, the O2(-)-producing enzyme (NADPH oxidase) in the particulate fraction prepared from myristate-stimulated neutrophils was not affected by aclacinomycin . When high concentrations of aclacinomycin (10-100 micrograms/ml) were employed, significant inhibition of O2- release, phagocytosis, and membrane potential changes was observed within 5 min . Phagocytic activity was also inhibited when neutrophils were preincubated for 13 h at 37 degrees C with a low concentration (40 ng/ml) of aclacinomycin, which could be obtained by intravenous administration of 20 mg aclacinomycin . Myristate-induced O2- release was not impaired by cytosine arabinoside (2-800 micrograms/ml), vincristine (0.1-100 micrograms/ml), adriamycin (25-100 micrograms/ml), or daunomycin (5-75 micrograms/ml) when the cells were preincubated with these drugs for 5 min at 37 degrees C . These findings suggest that aclacinomycin inhibits the respiratory burst by impairing the activating system of NADPH oxidase and phagocytic activity.

Plant Mol Biol, 1991 Feb, 16(2), 283 - 92
Specificity of leaf mitochondrial and chloroplast processing systems for nuclear-encoded precursor proteins; Whelan J et al.; The specificity of the mitochondrial and chloroplast processing enzymes for the nuclear-encoded precursor proteins was investigated . Mitochondrial precursor proteins of the Nicotiana plumbaginifolia and the Neurospora crassa beta subunits of F1-ATPase and the Neurospora Rieske FeS precursor protein were processed to the correct mature size by matrix extracts isolated from spinach leaves, yeast, rat liver and beef heart . The mitochondrial extracts failed to process chloroplast precursor proteins of the stromal small subunit of ribulose 1,5-bisphosphate carboxylase and the thylakoid 33 kDa protein of the oxygen-evolving complex . Both mitochondrial F1 beta precursors were specifically processed by a soluble stromal extract from chloroplasts . However, no processing of the Rieske FeS precursor protein was observed under the same conditions with the chloroplast extract . The cleavage of the mitochondrial F1 beta precursors by the chloroplast extract was shown to be sensitive to the metal chelators EDTA and ortho-phenanthroline . The cleavage site of the mitochondrial F1 beta precursor by the chloroplast soluble extract appears to be located at the N-terminus.

Trends Biochem Sci, 1991 Feb, 16(2), 63 - 7
Mitochondrial import receptors for precursor proteins; Pfanner N et al.; The specific targeting of precursor proteins synthesized in the cytosol to various cell organelles is a central aspect of intracellular protein traffic . Several hundred different proteins are imported from the cytosol into the mitochondria . Recent studies have identified the mitochondrial outer membrane proteins MOM19, MOM72, MOM38 (approximately ISP42) and p32 which have a role in initial steps of protein import . The first three components are present in a multi-subunit complex that catalyses recognition and membrane insertion of precursor proteins.

Biochemistry, 1991 Jan 29, 30(4), 1010 - 6
Rat liver polysome N alpha-acetyltransferase: isolation and characterization; Yamada R et al.; Rat liver polysome N alpha-acetyltransferase has been purified to homogeneity by a four-step procedure that utilizes ammonium sulfate precipitation, gel filtration, hydroxylapatite chromatography, and Mono Q ion exchange chromatography . The enzyme is greatly stabilized by the inclusion of EDTA and 0.01% deoxycholate in the isolation buffers . The purified enzyme has a native molecular weight of 190,000 and a subunit molecular weight of 95,000, suggesting that it is a homodimer . The enzyme shows a pH optimum of 8.0 and is strongly inhibited by Cl-, I-, SCN-, and ClO4- and to a lesser degree by sulfate and acetate . It is unaffected by phosphate, citrate, and F- and by Na+ and K+; NH4+ is partially inhibitory . The enzyme is also sensitive to iodoacetic acid . It is generally more similar to yeast N alpha-acetyltransferase {Lee, F.-J . S., Lin, L.-W., & Smith, J . A . (1988) J . Biol . Chem . 263, 14948-14955} than to the hen oviduct enzyme, which contains a 7S RNA subunit {Kamitani, K., & Sakiyama, F . (1989) J . Biol . Chem . 264, 13194-13198}, although the amino acid compositions are quite different.

Biochemistry, 1991 Jan 29, 30(4), 997 - 1004
Secondary structure of a complement control protein module by two-dimensional 1H NMR; Barlow PN et al.; The complement control protein (CCP) module (also known as the short consensus repeat) is a consensus sequence of about 60 amino acid residues which is thought to fold independently . It occurs over 140 times in more than 20 extracellular mosaic proteins including 12 proteins of the complement cascade . An isolated CCP module, the 16th repeat from human complement factor H, has been expressed in a yeast vector and shown to fold with the same pattern of disulfide bond formation as is seen in the native protein . Two-dimensional 600-MHz 1H NMR spectra of this module have been recorded at pH 3.3 and 6.0 and analyzed to permit determination of secondary structure in solution . The CCP module comprises two predominantly extended segments (Glu1-His13 and Ala17-Glu27), two segments of double-stranded antiparallel beta-sheet (Gly14-Val16 paired with Tyr31-Cys33 and Gly38-Asp40 paired with Ser57-Ile59), and a short piece of triple-stranded beta-sheet (Glu27-Thr30, Ile44-Leu48, and Lys51-Ser53) . Turns occur at Asp22, Gly36, and Glu50, while Gly41-Ala43 appear to form a looped-out segment or bulge . This structure is compared with a secondary structure prediction made on the basis of an alignment scheme of 101 sequences for CCP modules {Perkins, S . J., Haris, P . I., Sim, R . B., & Chapman, D . (1988) Biochemistry 27, 4004-4012}--the experimentally determined secondary structure bears an overall resemblance to the predicted one but differs in the number and position of turns . Some of those amino acid residues which are highly conserved throughout the range of CCP modules appear to play a role in stabilizing the global fold.

J Biol Chem, 1991 Jan 25, 266(3), 1448 - 55
The primary structure of human glutaminyl-tRNA synthetase . A highly conserved core, amino acid repeat regions, and homologies with translation elongation factors; Fett R et al.; We describe the nucleotide sequences of several overlapping cDNA clones specific for human glutaminyl-tRNA synthetase . The identified open reading frame indicates that the enzyme is composed of 1440 amino acids . A stretch of about 360 amino acids of the human enzyme is highly conserved in bacterial and yeast glutaminyl-tRNA synthetases . However, the human enzyme is three times larger than the bacterial and twice as large as the yeast enzyme suggesting that a considerable part of human glutaminyl-tRNA synthetase has evolved to perform functions other than the charging of tRNA . The sequence outside of the conserved core region includes three 57-amino acid repeats followed by a consecutive stretch of 11 charged amino acids . A computer assisted search of two protein data banks reveals that the human glutaminyl-tRNA synthetase shares small blocks of amino acid similarities with several other synthetases of different amino acid specificities . Interestingly, the enzyme also possesses some regions of similarities with eukaryotic translation elongation factor EF-1 but not with any other sequence stored in the protein data banks . The coding regions of human and mouse glutaminyl-tRNA synthetase cDNAs are identical at 94% of the codons . However, the 3'-noncoding regions of mouse and human mRNAs are more divergent (approximately 68%) but both possess the potential to form stable secondary structures of similar general architecture.

Nucleic Acids Res, 1991 Jan 25, 19(2), 287 - 96
A novel 40S multi-snRNP complex isolated from rat liver nuclei; Guialis A et al.; Two structurally distinct RNP complexes (MI and MII), each with a sedimentation value of approx . 40S, were isolated from rat liver nuclear extracts by sucrose gradient centrifugation and subsequent native gel electrophoresis of the 40S hnRNP-containing fractions . MII RNP contained the bulk of hnRNA and hnRNP proteins (i.e . the 32-45KD core proteins and polypeptides of 60-80 and 110-130KD) . MI RNP was characterized by the exclusive presence of U-snRNAs (U1, U2, U4, U5 and U6), their well known snRNP polypeptides and a number of Sm-associated proteins in the range of 50-210KD . Immunoselection experiments employing a monoclonal antibody with an established specificity for the U2-snRNP-specific B" polypeptide proved that the RNA and protein components characteristic of MI were part of a single multi-snRNP unit . The prominent 200/210KD protein doublet of MI was identified immunochemically as the rat homologue of the yeast PRP8 protein, a known U5-associated splicing component . Based on the major biochemical and immunochemical features of MI and MII RNP complexes, we conclude that MII represents the monomeric 40S hnRNP structure, whereas MI defines a novel multi-snRNP entity.

Science, 1991 Jan 25, 251(4992), 426 - 30
A genetic model for interaction of the homeodomain recognition helix with DNA; Hanes SD et al.; The Bicoid homeodomain protein controls anterior development in the Drosophila embryo by binding to DNA and regulating gene expression . With the use of genetic assays in yeast, the interaction between the Bicoid homeodomain and a series of mutated DNA sites was studied . These experiments defined important features of homeodomain binding sites, identified specific amino acid-base pair contacts, and suggested a model for interaction of the recognition alpha-helices of Bicoid and Antennapedia-class homeodomain proteins with DNA . The model is in general agreement with results of crystallographic and magnetic resonance studies, but differs in important details . It is likely that genetic studies of protein-DNA interaction will continue to complement conventional structural approaches.

Int J Cancer, 1991 Jan 21, 47(2), 281 - 4
Treatment of leukemia L1210 and P388 by arabinosylcytosine-polysaccharide conjugates; Novotny L et al.; Conjugates of 1-beta-D-arabinofuranosylcytosine (araC) with two polysaccharides such as polygalacturonic acid (PGA) and carboxymethylated yeast beta-D-glucan (CMG) were tested for their antileukemic activity in vitro on a L1210 cell line in suspension culture, in soft agar assay and in vivo on L1210, L1210/araC- and P388-leukemia-bearing mice . Both conjugates showed high activity in vitro in soft agar assay, compared with araC . Single administration of PGA-araC or CMG-araC increased the survival time 1.5 x or 1.7 x, respectively, compared with araC in vivo in L1210-leukemia-bearing mice . The conjugates were not active against araC-resistant leukemia line L1210/araC . The marked effect of both PGA-araC and CMG-araC against leukemia L1210 and P388 is probably due to the prolonged release of free araC from conjugates caused by hydrolysis.

Gene, 1991 Jan 15, 97(2), 191 - 8
Cloning of several lignin peroxidase (LIP)-encoding genes: sequence analysis of the LIP6 gene from the white-rot basidiomycete, Phanerochaete chrysosporium; Zhang YZ et al.; A Phanerochaete chrysosporium BKMF1767 genomic library, constructed in the BamHI site of vector YRp12, was screened with the lignin peroxidase(LIP)-encoding cDNAs, CLG4 and CLG5, that have been shown to encode LIP2 (previously H2) and LIP6 (previously H10), respectively . Six distinct LIP genomic clones, designated pGLG1, pGLG2, pGLG3, pGLG4, pGLG5, and pGLG6, were isolated in this study . Probe CLG4 hybridized only to pGLG1 . Probe CLG5 gave intense hybridization to pGLG2 and weaker hybridization to clones pGLG3 through pGLG6, but showed little or no hybridization to pGLG1 . These results, in agreement with previous biochemical results, indicate the existence of LIP gene subfamilies . The limits and transcriptional orientation of the LIP gene in each clone were determined . The sequence data showed that pGLG2 contains the LIP6 gene, which encodes a protein identical in amino acid (aa) composition to that encoded by CLG5 . It contains a leader sequence of 27 aa and a mature protein of 344 aa (Mr 36,607) . Archetypal TATA-box-like and CAAT-box-like sequences in the 5'-noncoding region are located 51 and 97 nt upstream from the cDNA start point, respectively . S1 nuclease analysis of the 5' region of LIP6 revealed two transcription start points 8 nt apart downstream from the TATA box . Comparison of the sequence of LIP6 with its corresponding cDNA CLG5 showed that the gene contains nine small introns which range in size from 50 to 62 bp . These introns contained consensus splice junction sequences similar to those reported in other fungal and yeast introns.

Proc Natl Acad Sci U S A, 1991 Jan 15, 88(2), 561 - 4
X-ray scattering indicates that the leucine zipper is a coiled coil; Rasmussen R et al.; Dimerization of the bZIP class of eukaryotic transcriptional control proteins requires a sequence motif called the leucine zipper . We have grown two distinct crystal forms of a 33-amino acid peptide corresponding to the leucine zipper of the yeast transcriptional activator GCN4 . This peptide is known to form a dimer of parallel helices in solution . X-ray scattering from both crystal forms shows reflections that are diagnostic of coiled coils . The most notable reflections occur at approximately 5.2 A resolution and correspond to the pitch of helices in coiled coils . There is no diffraction maximum near 5.4 A, the characteristic pitch of straight helices . Our results provide direct evidence that the leucine zipper of GCN4 is a coiled coil.

Ned Tijdschr Geneeskd, 1991 Jan 12, 135(2), 68 - 73
{Utilization of food supplements in The Netherlands}; Dorant E et al.; In 1987 and 1988 a dietary survey was carried out in a representative sample of the Dutch population, under the authority of the Ministries of Welfare, Health and Cultural Affairs, and Agriculture and Fisheries . By means of a two day dietary record data were collected on food consumption and the use of dietary supplements . More than seventeen percent of the Dutch population has been using at least one dietary supplement on at least one day of the survey . Age, sex, season, social class, alternative food habits, smoking and diet are related to the use of supplements . In young persons mainly fluoride and vitamin AD preparations are used, while as age progresses a shift towards other supplements, like garlic and brewer's yeast preparations, is observed . The use of single vitamin C supplements is not related to the level of mean daily vitamin C intake from foods.

Nucleic Acids Res, 1991 Jan 11, 19(1), 149 - 54
Structural and transcriptional analysis of a human subtelomeric repeat; Cheng JF et al.; A human subtelomeric repeat (designated as the HST repeat) has been isolated and characterized from a yeast artificial chromosome containing one human telomere . This repeat is located immediately adjacent to the telomeric T2AG3 repeats at the extreme termini of the human chromosomes . The DNA sequence of 3.6 kb of the HST repeat has been determined . The HST repeat spans over 3.6 kb in length, and contains one evolutionarily conserved CpG-rich region . The copy number of the HST repeat varies among telomeres . Genomic hybridization experiments suggest that the HST repeat consists of two distinct segments, and the distal portions of the HST repeat are also distributed elsewhere in the genome . In HeLa cells, the HST repeat sequence appears to be transcribed into a 6 kb polyadenylated RNA and a variety of non-polyadenylated RNA species.

Cell, 1991 Jan 11, 64(1), 149 - 57
S . pombe gene sds22+ essential for a midmitotic transition encodes a leucine-rich repeat protein that positively modulates protein phosphatase-1; Ohkura H et al.; The fission yeast dis2+ gene encodes one of the two type 1 protein phosphatases (PP1) in this organism . Its semidominant mutant dis2-11 is defective in mitosis . Here we report the characterization of a high dosage suppressor, sds22+, that complements dis2-11 . Sequencing of the cloned sds22+ gene predicts a novel 30 kd protein, which consists almost entirely of leucine-rich 22 amino acid repeats and is enriched in the insoluble nuclear fraction . sds22+ is an essential gene required for the mitotic metaphase/anaphase transition; gene disruption causes cell cycle arrest at midmitosis . Unexpectedly, the sds22+ gene becomes dispensable upon high dosage of the PP1 genes . The sds22+ product appears to facilitate PP1-dependent dephosphorylation, but does not substitute PP1 . We propose that the sds22+ protein forms a repeating helical rod that is capable of enhancing a PP1-dependent dephosphorylation activity that is essential in midmitosis.

Nature, 1991 Jan 3, 349(6304), 79 - 81
A small GTP-binding protein dissociates from synaptic vesicles during exocytosis; Fischer von Mollard G et al.; Low-molecular-weight GTP-binding proteins are strong candidates for regulators of membrane traffic . In yeast, mutations in the sec4 or ypt1 genes encoding small GTP-binding proteins inhibit constitutive membrane flow at the plasma membrane or Golgi complex, respectively . It has been suggested that membrane fusion-fission events are regulated by cycling of small GTP-binding proteins between a membrane-bound and free state, but although most of these small proteins are found in both soluble and tightly membrane-bound forms, there is no direct evidence to support such cycling . In rat brain a small GTP-binding protein, rab3A, is exclusively associated with synaptic vesicles, the secretory organelles of nerve terminals . Here we use isolated nerve terminals to study the fate of rab3A during synaptic vesicle exocytosis . We find that rab3A dissociates quantitatively from the vesicle membrane after Ca2(+)-dependent exocytosis and that this dissociation is partially reversible during recovery after stimulation . These results are direct evidence for an association-dissociation cycle of a small GTP-binding protein during traffic of its host membrane.

J Chromatogr, 1991 Jan 2, 562(1-2), 421 - 34
Liquid secondary ion mass spectrometry applied to structural confirmation of enzymically prepared C-terminal-truncated derivatives of recombinant hirudin; Maftouh M et al.; The thrombin-specific inhibitor, hirudin variant rHV2-Lys 47 (rHirudin), is a 65-amino acid polypeptide produced by recombinant DNA technology in yeast . Previous studies have shown that the acidic C-terminal segment of hirudin is susceptible to enzymic degradation . To address the question of C-terminal-truncated forms of the protein in terms of by-products or metabolites, well-defined reference compounds are needed . We prepared nine derivatives by carboxypeptidase Y digestion of rHirudin followed by a two-step chromatographic purification . Liquid secondary ion mass spectrometric measurements performed on peptides collected after reversed-phase high-performance liquid chromatography showed three pure forms (1-64, 1-63 and 1-56) and three mixtures of two forms each (1-62 + 1-61, 1-58 + 1-57 and 1-55 + 1-54), which were readily distinguished from one another by their mass spectra . Further purification of these co-eluted samples was achieved by ion-exchange chromatography and their structures were confirmed by liquid secondary ion mass spectrometry . Preliminary studies conducted on intact rHirudin indicated that this is an excellent analytical tool for mass measurements of hirudin-related proteins . Indeed, it allowed rapid (within 10-15 min), precise (0.50 a.m.u . relative to expected value), reproducible (mean MH+ = 6907.64 +/- 0.42 a.m.u.), sensitive (up to 500 ng, i.e . 72 pmol) and specific measurement of the quasi-molecular ion (MH+) of the protein, and was thus readily applicable to the analysis of several derivatives.

Antibiot Khimioter, 1991 Jan, 36(1), 35 - 7
{Effect of proper-myl on humoral interactions between activated lymphocytes and neutrophils in patients with myeloproliferative diseases}; Guseva SA; The lymphokine synthesizing function of peripheral blood lymphocytes (PBL) was studied in 22 patients with chronic myeloid leukemia (CML), 28 patients with subleukemic myelosis (SLM) and 15 healthy persons . The index of the neutrophil stimulation (INS) in CML (1.73 +/- 0.068) and SLM (1.458 +/- 0.004) was statistically significantly lower than that in the healthy persons (2.6 +/- 0.07) . The use of proper-myl in the complex therapy markedly increased the ability of PBL to produce the factor stimulating phagocytic activity of neutrophils (INS 1.94 +/- 0.04 and 1.832 +/- 0.092) . On the basis of the findings it was recommended to use proper-myl for prevention and treatment of infectious complications.

Am J Clin Oncol, 1991, 14 Suppl 1, S51 - 63
Clinical effects of recombinant human interleukin-3; Ganser A et al.; Interleukin-3 (IL-3) is a glycoprotein belonging to the hematopoietic growth factor family that in preclinical in vitro and in vivo studies has exhibited a multilineage activity . Phase I/II trials with recombinant human IL-3 (rhIL-3) expressed in yeast are being done in patients with advanced malignancies as well as in patients with bone marrow failure states . Subcutaneous administration of rhIL-3 at dosages between 30 and 500 micrograms/m2 for 15 consecutive days has resulted in a dose-dependent increase in platelet counts as well as in a substantial increase in the number of circulating neutrophils, eosinophils, monocytes, and lymphocytes in patients with advanced malignancies but normal hematopoiesis . Erythropoiesis is less stimulated with an increase in hemoglobin concentration only in a minority of patients . In patients with secondary hematopoietic failure due to prolonged chemo-/radiotherapy or bone marrow infiltration by tumor cells, treatment with rhIL-3 leads to a clinically significant restoration of hematopoiesis, especially of thrombopoiesis and granulopoiesis . rhIL-3 has also been shown to improve neutrophil and platelet counts in patients with myelodysplastic syndromes, while improvement of hematopoiesis is rarely observed in patients with severe aplastic anemia with the presently used treatment schedules . Adverse effects of rhIL-3 are minor at the clinically used dosages and include fever, bone pain, headache, and stiffness of the neck . Transient thrombocytopenia has been observed in a few patients with myelodysplastic syndrome or aplastic anemia treated at dosages of 250-500 micrograms/m2 . rhIL-3 is a multilineage hematopoietic cytokine with promising effects on platelet and neutrophil counts and special usefulness in patients with secondary hematopoietic failure.

Intervirology, 1991, 32(3), 160 - 72
Antibody reactivity to deletion mutants of the HIV-1 SF2 envelope; Back NK et al.; In human immunodeficiency virus type 1 (HIV-1) infected individuals, the antibody response to the external envelope (gp120) is associated with in vitro neutralization . To further characterize the anti-gp120 response, we examined the IgG reactivity of 75 HIV-1-seroconverted and 200 HIV-1-seropositive individuals to deletion mutants of gp120 in an enzyme immunoassay . We used yeast-derived, non-glycosylated recombinant HIV-1 SF2 gp120 equivalent and-variants deleted in variable regions . We observed two distinctive response patterns: IgG non-responders (SF2-V3-restricted responders) and IgG responders to conserved regions of gp120 . This divergence in response pattern occurred soon after gag/env HIV-1 antibody seroconversion and persisted in time within an individual . In addition, the SF2-V3-restricted responders had a higher frequency of HIV-1 core antigen positivity and HIV-1 core antibody negativity than the non-restricted responders . These results suggest that specific and persistent host antibody response patterns to gp120 develop early in HIV-1 infection and that these patterns are associated with differences in HIV-1 expression.

J Clin Lab Anal, 1991, 5(2), 121 - 6
Correlation of Histoplasma capsulatum polysaccharide antigen with the severity of infection in murine histoplasmosis; Williams BJ et al.; We sought to determine if Histoplasma capsulatum polysaccharide antigen (HPA) levels correlate with the extent of infection in murine of histoplasmosis . Separate groups of mice were inoculated intratracheally with varying numbers of H . capsulatum yeast cells . After 1 week, HPA levels and fungal burden (quantitative culture of lung and spleen and histopathologic stain of lung) were determined in lung and spleen, and HPA levels in serum . HPA levels, cultures and histopathological stain results of lung and spleen tissue showed a direct correlation with increasing inoculum size . HPA levels in serum also correlated with the size of inoculum . H . capsulatum antigen in lung correlated with silver stain scores of lung tissue, (R = 0.948, P less than 0.001) and with quantitative culture scores of lung, (R = 0.929, P less than 0.001) . HPA levels in spleen tissue also correlated with spleen culture scores, (R = 0.724, P less than 0.001) . These results indicate that determination of HPA level in serum and tissue may be a useful test in evaluating the severity of diseases as well as efficacy of antifungal therapy in histoplasmosis.

Can J Microbiol, 1991 Jan, 37(1), 86 - 95
Isolation and sequencing of a genomic DNA clone containing the 3' terminus of the 6-methylsalicylic acid polyketide synthetase gene of Penicillium urticae; Wang IK et al.; A 7.7-kilobase (kb) Penicillium urticae genomic DNA fragment containing the 3' terminus of the 6-methylsalicylic acid polyketide synthetase gene was cloned using a 41-mer mixed oligodeoxynucleotide probe which was based on a cyanogen bromide cleavage peptide of 35 amino acids obtained from pure synthetase . Nucleotide sequence analysis of a 2.2-kb region of the cloned fragment revealed a large open reading frame of 1866 bases which was devoid of introns and which corresponded to amino acids of the carboxyl terminus of the enzyme . This was followed by a putative transcription termination--polyadenylation signal . A putative acyl carrier protein domain at the 3' terminus was preceded by a beta-ketoreductase domain . These functionalities were identified by amino acid sequences known to be characteristic of the active sites of fatty acid synthetase functional domains . Their relative positions contrast with those in yeast and P . urticae fatty acid synthetase genes where the two functional domains are located at the 5' terminus and in reverse order . Furthermore, amino acid sequence identities indicated a striking homology with vertebrate rather than either yeast or P . urticae fatty acid synthetases.

Bull Cancer, 1991 Jan, 78(1), 1 - 21
{Different regulation systems of cell cycle events (dysregulation of these events in the tumoral cell)}; Colomb E et al.; Preservation of the shape and the integrity of multicellular eukaryotes needs rigorous cell proliferation monitoring . During the prereplicative G1 phase, a finely adjusted and specific control supervises the proliferant/non proliferant states of the cells . Some molecular mechanisms of growth regulation have been identified in recent years . Changes in normal cell attachment on extracellular matrix and intercellular chemical signalling (secretion of informative molecules) activate intracellular signals for division . The transduction mechanisms of the extracellular signalling to the nucleus have been partially elucidated for steroid hormones and growth factors . Molecular biology research and proto-oncogene discoveries have led to considerable progress in understanding the role of these normal genes in the control of cellular proliferation . The initiation of the response to extracellular factors requires: i), direct transducers (specific binding of the steroid hormone on its cytoplasmic or nuclear receptor and high affinity binding of this activated complex to specific DNA sequences); and ii) indirect transducers (binding of growth factors on extracellular domains of specific receptor proteins which convert this extracellular event into several intracellular signals, secondary messengers, protein kinases and specific nuclear regulatory factors) . Whatever the transduction system, nuclear events control transcription of growth regulatory genes . The series of enzymatic reactions set in motion by indirect transduction systems require strict regulation systems, the diversity and the complexity of which has been perceived in studies on jun and fos gene families . Each proliferation step is governed by growth stimulators and growth inhibitors, the transformation of normal cells to cancer cells resulting from alterations of these regulatory process . Independent of extracellular stimuli and of their transfer to the nucleus, intracellular controls coordinate cell cycle phases (G1, S, G2 and M) to produce daughter cells identical to the original cell . Two control points are particularly critical: one in G1 (the "start" point) and the other in G2 just before mitosis . Although intermediate steps between extracellular and intracellular controls are still unknown, yeast gene analyses have allowed determination of molecular regulatory mechanisms implicated in the passage of these critical points . A considerable advance was made by the discovery that some of the involved components presented strong sequence and function homologies in organisms from yeast to man, suggesting a phyllogenetically conserved mechanism . It seems likely that the phosphorylation state of protein p34, its association with a G1-phase specific cyclin or a M-phase specific cyclin, and its protein kinase activity regulate the proliferation state of higher eukaryotic cells . In spite of significant advances, much research is still necessary to elucidate all the mechanisms involved in cell cycle control.

Pharmacol Biochem Behav, 1991 Jan, 38(1), 21 - 7
Analgesic and acute central nervous system side effects of the intravenously administered enkephalinase inhibitor SCH 32615; Chipkin RE et al.; The analgesic and acute central nervous system (CNS) side effect potential of the enkephalinase inhibitor SCH 32615 (N-{L-(1-carboxy-2-phenyl)ethyl}-L-phenyl-alanine-beta-alanine) were evaluated after IV administration to mice, rats and squirrel monkeys . In mice, SCH 32615 caused dose-related suppression of acetic acid-induced writhing (minimal effective dose, MED = 3 mg/kg IV) . In rats, SCH 32615 produced dose-related increases in the response latencies in the yeast inflamed-paw test (MED = 10 mg/kg IV) . In squirrel monkeys, using a new hot-water bath tail-flick test, SCH 32615 significantly prolonged the escape latencies (MED = 100 mg/kg IV) . These results in primates are the first data showing an analgesic action of an enkephalinase inhibitor in a reflex model of pain . When measured for its CNS side effect potential, SCH 32615 had no significant effects in rats (up to 100 times its analgesically active doses) or in monkeys (up to three times) . In the mouse, at doses 100 times its minimal effective dose, SCH 32615 produced brief convulsions; these lasted only a minute, resolved quickly, and did not cause lethality . In contrast, in rats and squirrel monkeys, the standard opioid analgesic morphine produced profound CNS side effects; this was particularly notable in monkeys, in which morphine's maximal analgesic effects were associated with near lethal respiratory depression . These data demonstrate that SCH 32615 produces selective analgesic actions and that its acute side effect liability is less than that seen with a clinically used standard.

Am J Trop Med Hyg, 1991 Jan, 44(1), 28 - 33
Plasmodium vivax sporozoite antibodies in individuals exposed during a single malaria outbreak in a non-endemic area; Fontes CJ et al.; We studied seroreactivity against Plasmodium vivax antigens in 62 individuals living in a small community near Mantena, Minas Gerais, Brazil, an area outside the endemic malaria zone Brazil . Eight months earlier, there had been transmission of P . vivax for a period of 50 days, which was then totally controlled by chemotherapy and insecticides . An anti-sporozoite response, measured by ELISA using a recombinant protein expressed in yeast, was detected in 45% (14 of 31) of individuals eight months after infection and persisted for 20 months in 12% . Eighteen individuals were treated prophylactically for malaria because they lived in houses in which an overt infection had occurred . Seven of these individuals were ELISA positive; of these, 5 had antibodies against the blood stage parasites . Among 13 other individuals in the endemic area who did not have positive smears, had not been ill, and had not received prophylaxis, five were anti-circumsporozoite positive up to a 40-fold serum dilution . They did not develop asexual blood stage antibodies and remained parasite-free for the following 20 months.

J Nutr, 1991 Jan, 121(1), 50 - 6
Comparative antioxidant effectiveness of dietary beta-carotene, vitamin E, selenium and coenzyme Q10 in rat erythrocytes and plasma; Zamora R et al.; Five groups of five weanling rats were each fed a Torula yeast-based diet either unsupplemented or supplemented with 30 mg beta-carotene/kg, 30 IU vitamin E/kg, 1 mg selenium/kg or 30 mg coenzyme Q10/kg . Elevated levels of plasma aspartate aminotransferase and alanine aminotransferase are sensitive indicators of liver damage . The former enzyme was lower (P less than 0.01) in the vitamin E-, selenium- and beta-carotene-supplemented groups than in the unsupplemented control group, and the latter enzyme was lower in the vitamin E- and selenium-supplemented groups, suggesting a relatively equal effectiveness of these three antioxidants against liver damage . Erythrocytes were tested for protection against uninduced oxidative damage or that induced by 1 mmol/L bromotrichloromethane (BrCl3C) by measuring thiobarbituric acid-reactive substances (TBARS), hemoglobin, hemolysis, protein precipitation, alanine release and several enzyme activities . In untreated erythrocytes, selenium, beta-carotene and coenzyme Q10 exhibited protection by lowering (P less than 0.05) TBARS and alanine release, but only vitamin E protected against hemolysis . In BrCl3C-treated erythrocytes, vitamin E, selenium and beta-carotene protected by decreasing (P less than 0.05) protein precipitation, whereas selenium and beta-carotene decreased alanine release . The results of this study suggested that, in a manner analogous to vitamin E and selenium, beta-carotene and coenzyme Q10 function as antioxygenic nutrients.

J Allergy Clin Immunol, 1991 Jan, 87(1 Pt 1), 107 - 10
Caffeine, a naturally occurring acaricide; Russell DW et al.; Since caffeine is a plant alkaloid that has been described as a naturally occurring insecticide, its acaricidal effect on Dermatophagoides pteronyssinus (Dp) was investigated . Twelve cultures were established by adding 30 Dp to 200 mg of Tetramin fish food and brewer's yeast (8:2 ratio); six cultures were treated with 20 mg of finely ground caffeine . All 12 cultures were incubated at 75% relative humidity, 25 degrees C, and observed during 8 weeks . Live mites were then counted under a stereoscope, cultures were extracted, and supernatants were analyzed for Der p I and Der f I allergen content with a two-site monoclonal RIA . Live mite counts in untreated cultures varied from 146 to 274 (215 +/- 47.1), and in caffeine-treated cultures from 0 to 3 (1 +/- 1.2; p less than or equal to 0.0001) . Der p I concentrations in untreated cultures varied from 588 to 9000 ng/gm (3138.3 +/- 2990.8 ng/gm), and in caffeine-treated cultures from 52 to 117 ng/gm (78 +/- 23.8 ng/gm; p less than or equal to 0.01) . Der p I was not detected in the food media or caffeine; Der f I was not detected in any of the cultures . Results demonstrate that caffeine inhibits mite growth and allergen production.

Cancer Res, 1991 Jan 1, 51(1), 105 - 9
Variant human breast tumor estrogen receptor with constitutive transcriptional activity; Fuqua SA et al.; Since progesterone receptor (PgR) is normally induced by estrogen, breast cancer lacking estrogen receptor (ER) would also be expected to lack PgR . However, a small percentage of breast cancers are ER- yet PgR+ . These tumors might possess an ER which is defective in estrogen binding but is still functional in stimulating estrogen-responsive genes such as PgR . We have now detected such a variant, lacking exon 5 of the hormone-binding domain, using complementary DNA amplified by the polymerase chain reaction . This variant was the predominate ER RNA expressed in three ER-/PgR+ tumors . Furthermore, the variant ER constitutively activates transcription of a normally estrogen-dependent gene construct in yeast cells . The variant ER could explain the expression of PgR in certain tumors and have therapeutic implications.

Infect Immun, 1991 Jan, 59(1), 428 - 32
Genetic control of natural resistance in mouse macrophages regulating intracellular Legionella pneumophila multiplication in vitro; Yoshida S et al.; It is known that Legionella pneumophila proliferates in peritoneal macrophage cultures derived from A/J mice but not in macrophage cultures derived from many other strains, including C57BL/6 mice . To analyze the genetic control of this trait and the location of the Legionella resistance-susceptibility gene, we prepared segregating progeny of A/J and C57BL/6 mice and determined the levels of susceptibility of individual mice . Peritoneal macrophages were collected by injecting thioglycolate medium, and macrophage monolayers were infected in vitro with L . pneumophila Philadelphia-1 . Counting of colonies on buffered charcoal yeast extract agar plates and Gimenez staining of macrophage monolayers were carried out daily . There was a 10-fold increase in bacterial burden 1 day after infection and a 100-fold increase after 2 days in A/J (susceptible) macrophages . The increase in bacterial burden was always less than 10-fold in macrophages from C57BL/6 (resistant) progenitors, A/J x C57BL/6 F1 hybrids, and C57BL/6 x F1 backcross progeny . The ratios of resistant individuals to susceptible individuals were 22:6 for F2 progeny and 20:22 for A/J x F1 backcross progeny . The fact that the organism did not proliferate in macrophages from B10.A mice demonstrated that major histocompatibility antigens did not regulate the macrophage resistance of C57BL/6-derived mice . The sex and coat color genes of mice were not linked to the resistance-susceptibility gene . We suggest that resistance and susceptibility are controlled by a single gene or closely linked genes which are autosomal and that the resistance allele is dominant . The results of a comparison of the strain distribution pattern of this trait with the distribution pattern of 185 allelic markers in A/J x C57BL/6 and C57BL/6 x A/J recombinant inbred strains suggest that this susceptibility-resistance gene is located in the proximal part of chromosome 15.

Proc Natl Acad Sci U S A, 1991 Jan 1, 88(1), 144 - 8
Molecular cloning and characterization of interferon alpha/beta response element binding factors of the murine (2'-5')oligoadenylate synthetase ME-12 gene; Yan C et al.; Seven clones encoding interferon response element binding factors have been isolated from a mouse fibroblast lambda gt11 cDNA library by using a 32P end-labeled tandem trimer of the mouse (2'-5')oligoadenylate synthetase gene interferon response element as a probe . Clone 16 shares strong similarity (95%) at both DNA and amino acid level with YB-1, a human major histocompatibility complex class II Y-box DNA-binding protein, and with dbpB, a human epidermal growth factor receptor gene enhancer region binding protein . The product of the gene represented by clone 16 may represent a factor that regulates multiple genes by binding to a variety of 5' regulatory elements . Clone 25 is a 2407-base-pair-long cDNA and contains a putative 311-amino acid open reading frame corresponding to an estimated mass of 35.5 kDa . This putative protein, designated as interferon response element binding factor 1 (IREBF-1), contains an acidic domain, three heptad repeat leucine arrays, and a region that shares similarity with the yeast transcriptional factor GAL4 DNA-binding domain . Furthermore, the C terminus of IREBF-1 shows an unusual amphipathic property: within a 79-amino acid range, one side of the alpha-helical region contains a preponderance of hydrophobic amino acids and the other side contains hydrophilic amino acids . This type of structure provides a strong hydrophobic force for protein-protein interaction.

Mol Cell Biol, 1991 Jan, 11(1), 240 - 9
Structure and regulation of histone H2B mRNAs from Leishmania enriettii; Genske JE et al.; We have studied the structure and expression of histone H2B mRNA and genes in the parasitic protozoan Leishmania enrietti . A genomic clone containing three tandemly repeated genes has been sequenced and shown to encode three identical histone proteins and two types of closely related mRNA sequence . We have also sequenced three independent cDNA clones and demonstrated that the Leishmania H2B mRNAs are polyadenylated, similar to the basal histone mRNAs of higher eucaryotes and the histone mRNAs of yeast . In addition, the Leishmania mRNAs contain inverted repeats near the poly(A) tail which could form stem-loops similar in secondary structure, but not in sequence, to the 3' stem-loops of nonpolyadenylated replication-dependent histones of higher eucaryotes . Unlike the replication-dependent histones, the Leishmania histone H2B mRNAs do not decrease in abundance following treatment with inhibitors of DNA synthesis . The histone mRNAs are differentially expressed during the parasite life cycle and accumulate to a higher level in the extracellular promastigotes (the form which in nature lives within the gut of the insect vector) than in the intracellular amastigotes (the form that lives within the mammalian host macrophages).

J Immunol, 1991 Jan 1, 146(1), 244 - 9
Moderate zinc deficiency in rhesus monkeys . An intrinsic defect of neutrophil chemotaxis corrected by zinc repletion; Vruwink KG et al.; Experimental animals fed zinc-deficient diets are well known for susceptibility to infections and impaired mitogen response and Ig production . However, the levels of zinc deficiency used have generally been severe, not comparable to human populations, and have not addressed neutrophil function . To address this issue we have studied the effect in rhesus monkeys of a well defined moderately zinc-deficient (MZ) diet on polymorphonuclear leukocyte (PMN) function . Female adult rhesus monkeys were fed either a control (100 micrograms Zn/g) or MZ (2 micrograms Zn/g) diet for 9 mo with quantitation of PMN chemotaxis, and phagocytosis of opsonized yeast . In addition, membrane potential and secretion responses (changes in 90 degrees light scatter) and changes in PMN shape (forward light scatter shifts) were also measured . When compared to the PMN of animals fed control diets, there was a significant reduction in chemotaxis to FMLP of MZ-fed monkey PMN . Although shape change, cell membrane depolarization, as well as phagocytosis were not significantly different among the two groups, the PMN of MZ animals had significantly lower relative loss of orthogonal light scatter (degranulation) due primarily to a lower resting orthogonal light scatter and also a smaller loss when stimulated with FMLP . In vitro addition of zinc to the cells (25 microM) did not improve chemotaxis, and in fact, was inhibitory for most control and zinc-deficient cells . However, after 2 wk of dietary zinc repletion (100 micrograms Zn/g), chemotaxis in the low zinc group was higher and comparable to the control response . These data indicate that zinc deficiency is associated with an intrinsic PMN defect that specifically affects chemotaxis and is corrected with dietary zinc repletion.

Ciba Found Symp, 1991, 159, 174 - 83; discussion 183-7
Antibody catalysis of carbon-carbon bond formation; Hilvert D; We have used rationally designed transition state analogues to generate antibodies that catalyse two important carbon-carbon bond forming reactions: a bimolecular Diels-Alder cycloaddition and a unimolecular Claisen rearrangement . Our tailored immunoglobulin catalysts (abzymes) exhibit all the properties of naturally occurring enzymes, including substantial rate accelerations, substrate specificity, and high regio- and stereoselectivity . As first generation abzymes are generally inferior to naturally occurring enzymes, we are also employing classical genetic selection strategies to augment their chemical efficiency . We have expressed the antibody that catalyses the Claisen rearrangement of chorismate in yeast cells that lack natural chorismate mutase activity . Improved versions of the abzyme will be identified, following random mutagenesis, by their ability to repair this metabolic defect . The development and study of highly efficient catalytic antibodies promises to advance our understanding of how enzymes work and evolve, how protein function correlates with structure, and how entirely new enzymic activities can be created for use in research, industry and medicine.

J Med Vet Mycol, 1991, 29(5), 335 - 8
Candida haemulonii from clinical specimens in the USA; Gargeya IB et al.; Classical yeast identification procedures and DNA relatedness studies confirmed the occurrence of Candida haemulonii among clinical specimens in the USA, particularly isolations from the foot . None of the six clinical isolates studied produced identical API 20C profile codes.

Mycoses, 1991 Jan-Feb, 34(1-2), 47 - 52
Pathogenesis of postoperative candidosis: no detectable fungemia during reoperations after abdominal surgery; Rantala A et al.; Pathogenesis of systemic candidosis in surgical patients is unsettled . Results from animal models suggest that invasion from colonized intestine is the major portal of entry into circulation . To study the mechanisms of systemic candidosis in surgical patients after intestinal surgery, multiple blood cultures were taken during reoperations with manipulation of the intestinum to detect possible perioperative fungemia . The lysis centrifugation method (Isolator) was used for blood cultures . Of the 30 subjects in the material, 12 were demonstrated to be colonized with yeast before the reoperation . Three patients had positive blood cultures during reoperation, but no yeasts were found in the 146 perioperative blood cultures . Three patients had severe nonsuperficial yeast infection after reoperation, but none had disseminated candidosis . This finding in high risk patients supports the view that immediate perioperative fungemia and persorption from colonized intestinum is rare . However, persorption may occur later via healing wounds of the colonized intestinum . Both persorption and the exogenous route (e.g . vascular catheters) are possible in the pathogenesis of postoperative candidosis.

Mycoses, 1991 Jan-Feb, 34(1-2), 1 - 18
The medically important dematiaceous fungi and their identification; Dixon DM et al.; Dematiaceous fungi include a large group of organisms that are darkly pigmented (dark brown, olivaceous, or black) . In most cases the pigment is melanin, and specifically, dihydroxynaphthalene melanin . The diseases produced include chromoblastomycosis, eumycotic mycetoma, and phaeohyphomycosis . Phaeohyphomycosis is a new classification for a diverse group of previously known entities grouped together on the basis of finding dematiaceous hyphal and/or yeast-like forms in tissue; tissue involvement may be superficial, cutaneous and corneal, subcutaneous, or systemic . Identification of these fungi is based mostly upon morphology . Important structures include annellides (Phaeoannellomyces, Exophiala), phialides (Phialophora, Wangiella), adelophialides (Phialemonium without collarettes, Lecythophora with collarettes), differentiation of conidiophores (Xylohypha versus Cladosporium) and conidial hilum, septation and germination (Bipolaris, Drechslera, Exserohilum) . Useful laboratory tests include the 12% gelatin test (controversial), nitrate assimilation (W . dermatitidis is negative, most other species are positive), and determination of temperature maxima (especially 37 degrees C for E . jeanselmei, 40 degrees C for W . dermatitidis and B . spicifera, 42 degrees C for X . bantiana, and 45 degrees C for Dactylaria constricta var . gallopava and Scedosporium inflatum).

Int J Vitam Nutr Res, 1991, 61(2), 130 - 4
Bioavailability of folate following ingestion of cholestyramine in the rat; Hoppner K et al.; The effect of cholestyramine ingestion on the intestinal deconjugation and absorption of folic acid (PGA) and brewers yeast folate was investigated using a rat bioassay and liver folate uptake as the response parameter . Male weanling Sprague Dawley rats were depleted on a low AIN-76A formulated basal diet for 21 days . During a 14 day repletion period folic acid (PGA) and brewers yeast were added to provide 0.25, 0.5 and 1.0 mg of folate per kg of diet . Cholestyramine was administered directly as part of the diet at 1.1% . All diets were made isonitrogenous and isocaloric . Based on a parallel line assay, the relative biological value of folate for PGA + cholestyramine (79) was significantly different from the standard diet (PGA = 100), while those for brewers yeast (88) and for brewers yeast + cholestyramine (88) did not differ from the standard diet . Ingestion of cholestyramine significantly reduced the bioavailability of PGA versus brewers yeast folate in rats.

Biochem Int, 1991 Jan, 23(1), 151 - 6
Diadenosine polyphosphates inhibit the action of ribonucleotide reductase on ADP; Wasternack C et al.; Using permeabilized cells prepared according to Lammers, M . and Follmann, H . (Arch . Biochem . Biophys . 244, 430-438, 1986), the ribonucleotide reductase (EC 1.17.4.1) of yeast was assayed in situ . In our experimental conditions, 40-200 pmoles of ADP were reduced in 10 min per 5 x 10(7) cells . Concentrations of 0.01 mM of diadenosine tri, tetra, penta and hexaphosphates elicited inhibitions of 37, 40, 63 and 58%, respectively . The enzyme was almost completely inhibited in the presence of 0.05 mM diadenosine pentaphosphate . As some of these dinucleotides are present in yeast cells at these concentrations, the reported inhibition may have physiological meaning.

Int J Vitam Nutr Res, 1991, 61(1), 72 - 6
Chromatography of selenoproteins in human serum using matrix-bound heparin; Akesson B et al.; Since previous experiments indicated that a major selenoprotein in human serum interacts with heparin, chromatography of serum on matrix-bound heparin was studied . When human serum was applied to heparin-agarose columns, approximately half of the applied selenium was not retained on the columns . Approx . 40% of the selenium could then be eluted either with increasing concentrations of heparin or ammonium acetate . Using a scaled-down version of this procedure, selenoproteins from 0.5 ml serum were separated into the heparin-binding and non-heparin-binding fractions . In an experiment where healthy subjects were given supplements of yeast selenium (200 micrograms/d) for eight weeks, the concentration of selenium in serum was almost doubled and then approached the original concentration 16 weeks after the end of the supplementation . During supplementation, no change in the concentration of heparin-binding selenoproteins was observed, and instead the increase in serum selenium occurred in non-heparin-binding proteins . This suggests that the need for selenium by the heparin-binding proteins was saturated already at the starting serum selenium level (1.0 mumol/l) . Since interaction with heparin has been observed also for selenoprotein P isolated from rat plasma, the protein in the heparin-binding fraction, demonstrated in this paper, may be a human analogue to selenoprotein P.

J Rheumatol, 1991 Jan, 18(1), 66 - 71
Effects of pyrophosphatase on dissolution of calcium pyrophosphate dihydrate crystals; Xu Y et al.; Understanding the dissolution mechanisms involved in calcium pyrophosphate dihydrate (CPPD) crystals may prove important for the development of therapy for CPPD arthropathy . We demonstrate that yeast pyrophosphatase effectively dissolved CPPD crystals in solutions . Maximum enzymatic dissolution of CPPD crystals was achieved at neutral pH and when the enzyme had access to the crystal surface . The enzymatic dissolution of CPPD crystals was highly dependent on ambient {Mg++} and {Ca++} . The stimulating effects of Mg++ on crystal dissolution in the presence of the enzyme is due to stimulation of pyrophosphatase activity and to enhanced direct release of pyrophosphate ions from the crystal surface . The inhibiting effect of Ca++ on crystal dissolution by the enzyme is mainly due to the suppression of pyrophosphatase activity.

Int J Syst Bacteriol, 1991 Jan, 41(1), 6 - 14
Characterization of mitochondrial DNA in various Candida species: isolation, restriction endonuclease analysis, size, and base composition; Su CS et al.; A practical and effective method for the extraction of mitochondrial DNA from Candida species was developed . Zymolyase was used to induce yeast protoplasts, and mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations . Restriction endonuclease analyses of mitochondrial DNAs from 19 isolates representing seven species of Candida (C . albicans, C . kefyr, C . lusitaniae, C . maltosa, C . parapsilosis, C . shehatae, and C . tropicalis) and Lodderomyces elongisporus revealed different cleavage patterns that appeared to be specific for the species . Few common restriction fragments were evident . The genome sizes of the mitochondrial DNAs ranged from 26.4 to 51.4 kilobase pairs, and the guanine-plus-cytosine contents ranged from 20.7 to 36.8 mol% . There was no correlation between the base compositions of nuclear and mitochondrial DNAs . Eight isolates of C . parapsilosis, including the type culture, and an ascosporogenous strain of L . elongisporus, which was once proposed as the teleomorph of C . parapsilosis, had similar mitochondrial DNA molecular sizes (30.2 and 28.8 kilobase pairs); however, restriction endonuclease patterns of these organisms were distinct . These data provide additional support for discrimination of these two species . The results of our experiments demonstrate that mitochondrial DNA analyses may provide useful criteria for the differentiation of yeast species.

Am J Respir Cell Mol Biol, 1991 Jan, 4(1), 72 - 81
Exposure of humans to ambient levels of ozone for 6.6 hours causes cellular and biochemical changes in the lung; Devlin RB et al.; An acute (2 h) exposure of humans to 0.4 ppm ozone initiates biochemical changes in the lung that result in the production of components mediating inflammation and acute lung damage as well as components having the potential to lead to long-term effects such as fibrosis . However, many people are exposed to lower levels of ozone than this, but for periods of several hours . Therefore, it is important to determine if a prolonged exposure to low levels of ozone is also capable of causing cellular and biochemical changes in the lung . Nonsmoking males were randomly exposed to filtered air and either 0.10 ppm ozone or 0.08 ppm ozone for 6.6 h with moderate exercise (40 liters/min) . Bronchoalveolar lavage (BAL) was performed 18 h after each exposure, and cells and fluid were analyzed . The BAL fluid of volunteers exposed to 0.10 ppm ozone had significant increases in neutrophils (PMNs), protein, prostaglandin E2 (PGE2), fibronectin, interleukin-6 (IL-6), and lactate dehydrogenase (LDH) compared with BAL fluid from the same volunteers exposed to filtered air . In addition, there was a decrease in the ability of alveolar macrophages to phagocytize yeast via the complement receptor . Exposure to 0.08 ppm ozone resulted in significant increases in PMNs, PGE2, LDH, IL-6, alpha 1-antitrypsin, and decreased phagocytosis via the complement receptor . However, BAL fluid protein and fibronectin were no longer significantly elevated . We conclude that exposure of humans to as low a level as 0.08 ppm for 6.6 h is sufficient to initiate an inflammatory reaction in the lung.

Princess Takamatsu Symp, 1991, 22, 3 - 19
Molecular basis of multistage carcinogenesis; Harris CC; Revealing the molecular basis of human disease including cancer will be viewed as one of the triumphs of biomedical research in the 20th Century . One successful strategy has been to analyze abnormalities in cancer-related genes occurring in preneoplastic and neoplastic lesions in humans and animal models . These gene abnormalities, e.g., mutations, can be specifically linked in some cases to environmental carcinogens in molecular epidemiological studies of human populations and in more controlled experimental conditions using animal or in vitro models of carcinogenesis . A second successful strategy has been to capitalize on advances from basic research such as defining signal transduction pathways in mammalian cells and the genetic control of the cell cycle in yeast . Mutations in yeast cell division control genes can lead to genomic instability and aneuploidy which are hallmarks of cancer . Therefore, the role of these genes in human carcinogenesis is being intensely investigated . The involvement of the p53 gene in the majority of human cancers has focused attention on the molecular and biochemical mechanisms of this tumor suppressor gene . The analysis of the p53 mutational spectrum in human cancers has provided evidence that both exogenous and endogenous causes of mutation contribute to human carcinogenesis . The increased understanding of the molecular basis of carcinogenesis has important implications in the prevention, diagnosis and treatment of human cancer.

Princess Takamatsu Symp, 1991, 22, 145 - 52
Spi1 GTPase interacts with RCC1 to maintain interdependency of cell cycle events; Matsumoto T et al.; A mutant which can enter mitosis at any cell cycle stage has been isolated and characterized in fission yeast . The pim1 (premature initiation of mitosis) mutant prearrested at G1/S can develop a mitotic spindle and has tightly condensed chromosomes upon shift to the restrictive temperature . pim1-induced mitosis requires maturation promoting factor (MPF) activity, but not the essential mitotic inducer, cdc25 . The pim1+ gene encodes a homolog of regulator of chromosome condensation 1 (RCC1), a regulator of onset of mitosis in mammalian cells . A multicopy suppressor of pim1, spi1, was isolated, and found to encode a 25 kDa GTPase . The primary sequence of the spi1 GTPase shows extensive identity (80%) to human TC4, whose function is unknown . The spi1/TC4 GTPase defines a novel class in the "ras-like" GTPase family, which is distinct from ras, rho, or ypt . Disruption of the spi1+ gene causes genomic instability in a heterozygous diploid . These genetic data suggest that pim1+ and spi1+ interact to coordinate correct entry into mitosis . Immunological experiments demonstrate that the pim1+ and spi1+ products are physically associated . Mutation in the pim1 gene results in lowered affinity of the protein for the spi1 protein in vitro, which may explain why high dosages of the spi1 protein can rescue the pim1 mutant in vivo . The pim1/spi1 complex dissociates in the presence of Mg2+ and GTP . The current data suggests that pim1+ acts as a GTP exchanger for the spi1 GTPase.

Rev Inst Med Trop Sao Paulo, 1991 Jan-Feb, 33(1), 74 - 9
Subcutaneous phaeohyphomycosis caused by Bipolaris hawaiiensis . A case report; Costa AR et al.; A case of phaeohyphomycosis caused by Bipolaris hawaiiensis is reported . The patient, an immunocompetent host, presented a verrucous lesion on the first finger of the left foot . Dematiaceous septate hyphae and yeast-like elements were seen in direct and histological examination . The isolated strain was identified on the basis of micro and macromorphological aspects . Treated with electrocoagulation, the lesions healed and presented no relapse after two years follow-up.

Nucleic Acids Symp Ser, 1991, (25), 149 - 50
Sequence specific purification of a particular tRNA by solid phase DNA probe; Tsurui H et al.; A novel method for the purification of a specific tRNA using solid phase DNA probe is developed . With this method, the probe DNA immobilized on HPLC gel hybridized with target tRNA within a minute at room temperature . The hybridizing capacity of the solid phase probe was about 20 O.D . per gram dry gel when yeast phenylalanine tRNA was used . The specificity of this method was extremely high and the recovery rate was about 90%.

DNA Seq, 1991, 1(3), 197 - 206
An H3-H4 histone gene pair in the marine copepod Tigriopus californicus, contains an intergenic dyad symmetry element; Porter D et al.; Histone genes are one of the most widely studied multigene families in eucaryotes . Over 200 histone genes have been sequenced, primarily in vertebrates, echinoderms, fungi and plants . We present here the structure and genomic orientation of an H3-H4 histone gene pair from the marine copepod, Tigriopus californicus . These histone gene sequences are the first to be determined for the class Crustacea and among the first to be determined for protostomes . The H4 and H3 genes in Tigriopus are shown to be adjacent, to have opposite polarity, and to contain a 26 bp region of dyad symmetry centrally located within the spacer region between the two genes . A similarly located dyad element has been found in yeast which contributes to the coordinated cell cycle control of the adjacent histone genes . The Tigriopus H3-H4 histone gene pair is clustered with one H2A and two H2B histone genes on a 15 kb genomic Bam H1 fragment . The H4 gene sequence predicts an H4 protein with an unusual serine to threonine substitution at the amino terminal residue . The H3 gene sequence predicts an H3 protein which is identical to the vertebrate H3.2 histone.

Acta Derm Venereol Suppl (Stockh), 1991, 167, 1 - 36
Seborrhoeic dermatitis and Pityrosporum ovale: cultural, immunological and clinical studies; Bergbrant IM; Seborrhoeic dermatitis is a common skin disease mainly affecting the scalp and face . The etiology of seborrhoeic dermatitis is unknown but a connection with the lipophilic yeast Pityrosporum ovale has been found in a number of treatment studies . P . ovale belongs to the normal cutaneous flora but is also an opportunistic pathogen . The purpose of these studies was to examine how the density of P . ovale changes with age, to determine the number of P . ovale in seborrhoeic dermatitis compared to controls, to study the immunological functions in patients with seborrhoeic dermatitis, to evaluate different methods of detecting antibodies against P . ovale and to describe how the patients experience their disease . The number of P . ovale on clinically normal skin decreases with increasing age . In patients with seborrhoeic dermatitis, the number of P . ovale in lesional skin was not increased compared to healthy skin in the patients or in healthy controls . A reduction of the skin surface lipids was seen in elderly healthy individuals . The lipid content on the skin in patients with seborrhoeic dermatitis was higher than in controls (p = 0.0001) . Serum IgG antibodies against P . ovale measured with indirect immunofluorescence decreased parallel to increasing age in healthy individuals and no difference was found between patients with seborrhoeic dermatitis and healthy controls . ELISA with a P . ovale protein extract was the only method that demonstrated a difference in immune response between patients and controls when this method was compared with four other assays (p = 0.03) . Immunological screening was done in 30 patients with seborrhoeic dermatitis . No major abnormalities in the humoral and local immune system were found but T-cell and NK-cell aberrations were found in several patients with seborrhoeic dermatitis . One-third of the patients had low lymphocyte stimulations with Concanavalin-A and phytohaemagglutinin and almost half of the patients had high frequencies of circulating natural killer-cells . In a questionnaire answered by 431 patients with seborrhoeic dermatitis, we found indications that hereditary, the season, mental stress and the work environment influence the disease . The investigations suggest that the number of P . ovale in seborrhoeic dermatitis is of minor importance . How each individual reacts to P . ovale and the amount of skin surface lipids are probably of greater importance in the development of seborrhoeic dermatitis.

Int J Technol Assess Health Care, 1991, 7(3), 379 - 402
Cost-benefit analysis of hepatitis-B vaccination . A computerized decision model for Spain; Jonsson B et al.; The availability and efficacy of recombinant deoxyribonucleic acid yeast-derived hepatitis-B vaccine, at a price much lower than the previously available plasma-derived hepatitis-B vaccines against hepatitis-B virus infections, motivate a new cost-benefit analysis of hepatitis-B vaccination . Spanish data were used to calculate direct and indirect costs of hepatitis-B infection and the costs and benefits of different vaccination strategies in defined risk groups of the Spanish population . A vaccination program will reduce direct expenditures for hepatitis B if the attack rate in the target population is higher than 4.9% . If indirect costs are included, the threshold for cost saving is reduced to 0.9% . The results are sensitive to the price of the vaccine, the duration of protection, assumptions about consequences for quality of life, and to indirect costs.

J Cell Sci Suppl, 1991, 14, 143 - 5
Dynamin: a microtubule-associated GTP-binding protein; Obar RA et al.; We recently identified dynamin as a third nucleotide-sensitive microtubule-associated protein in brain tissue, in addition to kinesin and cytoplasmic dynein . Molecular cloning analysis has revealed that dynamin contains the three consensus elements characteristic of GTP-binding proteins, and biochemical results support a role for GTP in dynamin function . Dynamin is also homologous to the Mx proteins, involved in interferon-induced viral resistance, and the product of the yeast VPS1 gene, involved in vacuolar protein sorting . These results identify a novel class of GTP-utilizing proteins, with apparently diverse functions.

EXS, 1991, 58, 50 - 69
Oligonucleotide fingerprinting using simple repeat motifs: a convenient, ubiquitously applicable method to detect hypervariability for multiple purposes; Epplen JT et al.; A panel of simple repetitive oligonucleotide probes has been designed and tested for multilocus DNA fingerprinting in some 200 fungal, plant and animal species as well as man . To date at least one of the probes has been found to be informative in each species . The human genome, however, has been the major target of many fingerprinting studies . Using the probe (CAC)5 or (GTG)5, individualization of all humans is possible except for monozygotic twins . Paternity analyses are now performed on a routine basis by the use of multilocus fingerprints, including also cases of deficiency, i.e . where one of the parents is not available for analysis . In forensic science stain analysis is feasible in all tissue remains containing nucleated cells . Depending on the degree of DNA degradation a variety of oligonucleotides are informative, and they have been proven useful in actual case work . Advantages in comparison to other methods including enzymatic DNA amplification techniques (PCR) are evident . Fingerprint patterns of tumors may be changed due to the gain or loss of chromosomes and/or intrachromosomal deletion and amplification events . Locus-specific probes were isolated from the human (CAC)5/(GTG)5 fingerprint with a varying degree of informativeness (monomorphic versus truly hypervariable markers) . The feasibility of three different approaches for the isolation of hypervariable mono-locus probes was evaluated . Finally, one particular mixed simple (gt)n(ga)m repeat locus in the second intron of the HLA-DRB genes has been scrutinized to allow comparison of the extent of exon-encoded (protein-) polymorphisms versus intronic hypervariability of simple repeats: adjacent to a single gene sequence (e.g . HLA-DRB1*0401) many different length alleles were found . Group-specific structures of basic repeats were identified within the evolutionarily related DRB alleles . As a further application it is suggested here that due to the ubiquitous interspersion of their targets, short probes for simple repeat sequences are especially useful tools for ordering genomic cosmid, yeast artificial chromosome and phage banks.

Folia Microbiol (Praha), 1991, 36(2), 198 - 204
The immunoadjuvant effect of soluble glucan derivatives in mice; Wagnerova J et al.; We examined the effect of soluble derivatives of yeast glucan on the humoral immune response of various strains of inbred mice after administration of different doses according to various schedules . Glucan was injected i.v . or s.c . in a single dose or repeatedly . The immune response was examined by determining the titres of serum hemagglutinins against sheep erythrocytes (SRBC-Ab) . The immunoadjuvant effect of glucan derivatives depends on the inbred strain used, on the dose of glucan, mode and time of administration with respect to antigen injection . The results have shown that the stimulatory effect of glucan derivatives occurred already after a single injection, the optimum dose being 10-20 mg/kg . Intravenous injection was more efficient than the subcutaneous one . In some cases, a slight increase of the spleen mass was observed.

Folia Microbiol (Praha), 1991, 36(2), 148 - 52
Sterol composition of nystatin-resistant Candida maltosa mutants; Mikhailova NP et al.; Composition of sterol fractions of nystatin-resistant Candida maltosa strains was determined . Using UV-spectrometry, TLC and GLC-MS it was demonstrated that resistance to nystatin is connected with the composition alterations of yeast cell sterols . Block of different stages of ergosterol biosynthesis was revealed in some mutants, viz . C-24-transmethylation, delta 8----delta 7-isomerization, 14 alpha-demethylation, C-5(6)-dehydrogenation, reduction of C-14(15) and C-24(28) double bonds.

Folia Microbiol (Praha), 1991, 36(4), 406 - 7
A rapid and simple method for DNA preparation from Candida utilis; Krizkova L et al.; A method for the extraction of genomic DNA from the industrial yeast Candida utilis is described . The method is rapid, simple and produces DNA that is sufficiently pure for restriction analysis and should be suitable for Southern blotting and the construction of gene libraries.

Folia Microbiol (Praha), 1991, 36(4), 367 - 74
Effect of pH and organic matter on the toxicity of heavy metals to growth of some fungi; Bagy MM et al.; Increasing the pH from 5 to 9 decreased the toxicity of mercuric chloride, zinc sulfate, lead nitrate, copper sulfate and nickel chloride toward the growth of Aspergillus flavus, Penicillium chrysogenum, Cunninghamella echinulata, Myrothecium verrucaria and Phoma humicola . On the other hand, the toxicity of cadmium chloride was increased by the increasing pH . Also increasing the concentration of organic matter (peptone and yeast extract) from 0.5 to 1.5% induced a significant reduction in the toxicity of all heavy metals toward the growth of all test fungi.

Folia Microbiol (Praha), 1991, 36(4), 343 - 6
Production of laccase by Curvularia sp; Banerjee UC et al.; A Curvularia sp . isolated from soil was found to contain laccase activity toward guaiacol as substrate . The organism produced an extracellular laccase in a medium containing yeast extract, peptone and dextrose . Initial medium pH 4.0 and cultivation temperature 30 degrees C were found to be most suitable for maximum enzyme production . The optimum pH and temperature for laccase activity were found to be 5.2 and 50 degrees C, respectively . Under optimum conditions, the enzyme had a Km (guaiacol) of 0.75 mmol/L and a V of 1.50 CU min-1 ml-1 . Some divalent metal ions inhibited laccase activity at very low concentrations.

IARC Sci Publ, 1991, (115), 113 - 7
Comparative acute nephrotoxicity of Penicillium aurantiogriseum in rats and hamsters; Hard GC et al.; Air-dried mycelium of Penicillium aurantiogriseum2, grown as a surface culture on yeast extract-sucrose medium, was incorporated into powdered diet and fed to rats and hamsters for different periods up to 28 days . At intervals, animals were anaesthetized and the kidneys fixed in situ by perfusion . In rats, the fungus produced scattered exfoliation of pyknotic cells and an increased frequency of mitotic figures involving the pars recta segment of proximal tubular epithelium . This lesion was detectable as early as three days after beginning of treatment and was well developed by 14 days . No degenerative tubular change or mitogenic effect was observed in hamsters, even after feeding for 35 days; and there was no apparent renal pelvic or interstitial lesion in either species.

Arch Invest Med (Mex), 1991 Jan-Mar, 22(1), 35 - 40
Human leukocyte migration inhibition factor (LIF) increases polymorphonuclear cell endocytosis; Cortes-Castillo MA et al.; We prepared supernatants of Concanavalin-A activated human lymphocytes containing high titers of leukocyte migration inhibition factor (LIF) . A pool of these supernatants was filtered thorough sephadex 6-100 as well as a pool of supernatants from parallel non activated cultures . A migration assay was carried out for each activated fraction, using as control migration the same fraction from non activated supernatants . In this way we found a fraction from activated supernatants with high LIF activity . We assayed the effect of this LIF containing fraction on a yeast endocytosis assay by polymorphonuclear (PMN) cells . We found that the LIF containing fraction increased the number of endocytic PMN in about 80% . This effect was absent from control supernatant and from other fractions from activated supernatant but without LIF activity . The LIF containing fraction did not increase the average number of endocytosed yeast per cell nor the ability to reduce NBT . The endocytosis enhancing effect was blocked by the specific LIF blocker N-acetyl-D-glucosamine . We conclude that LIF can increase the endocytic activity of PMN cells.

Cytobios, 1991, 68(273), 77 - 83
Effect of parvalbumin and S-100 protein on protein synthesis in rabbit reticulocyte lysate; Cheema IR et al.; The effect of two high affinity Ca+ binding acidic proteins, parvalbumin and S-100 protein on protein synthesis in rabbit reticulocyte lysates (RRL), was investigated . Nuclease-treated RRL, supplemented with yeast mRNA, and 3H-leucine were incubated at 37 degrees C, and incorporation of 3H-leucine into protein was determined for 24 min . At 20 micrograms/100 microliters lysate concentration, both parvalbumin and S-100 protein caused a marked inhibition of protein synthesis compared with the control lysate . At a lower concentration parvalbumin was less inhibitory than histone H1; the effect of S-100 protein was not significant . The combined inhibitory effect of parvalbumin and H1 was not additive probably due to strong interaction between them as was evidenced by the enhanced absorbance of parvalbumin-H1 mixture . Spectrophotometric profiles of parvalbumin-tRNA mixture indicated that, unlike H1, parvalbumin did not inhibit protein synthesis by binding with nucleic acids . These results suggest an important role for parvalbumin in translational regulation.

C R Seances Soc Biol Fil, 1991, 185(5), 290 - 305
{Protein farnesyl and geranylgeranyl transferases}; de Gunzburg J; Posttranslational prenylation of proteins synthesized as soluble precursors enhances their hydrophobicity and enables them to bind biological membranes . These modifications consist in the attachment of a C15 farnesyl or a C20 geranylgeranyl moiety to the cysteine residue(s) of proteins bearing CAAX, CC or CXC C-terminal sequences (where C = cysteine, A = aliphatic residue and X = any amino-acid), such as proteins of the ras superfamily, gamma subunits of heterotrimetric G proteins, lamin B as well as yeast mating factor a . A farnesyl transferase (FTase) and two distinct geranylgeranyl transferases (GGTases I and II) have been recently identified . FTase and GGTase I modify proteins containing a C-terminal CAAX motif; such a sequence is necessary and sufficient for recognition by the enzymes . The nature of the fourth residue determines the nature of the modification: when X is a serine, a methionine or a phenylalanine, the protein is farnesylated, whereas the presence of a leucine residue results in the attachment of a geranylgeranyl group . Both these enzymes are alpha beta heterodimers; their purification, molecular cloning of their coding sequences as well as mutational studies in yeast have shown that they share a common alpha subunit, and that their beta subunits exhibit a significant level of sequence similarity . GGTase II modifies ras-related proteins exhibiting CC and CXC C-terminal sequences; the enzyme as well as its recognition motif are yet largely uncharacterized.

Histol Histopathol, 1991 Jan, 6(1), 107 - 13
Ultrastructural changes induced by alpha-sarcin in a human pulmonary tumor grown in naked mice; Mohamed-Ali H et al.; alpha-Sarcin is a cytotoxic polypeptide produced by Aspergillus giganteus . It suppresses protein synthesis in yeast and wheat germ extracts and has a purine-specific RNase activity . The substance has been tested for its antitumor properties in a series of induced tumor systems in mice such as sarcoma and carcinoma among others . Although some of the in vitro effects of alpha-Sarcin on certain cellular components have been elucidated, the biological effects leading to cellular damage are still obscure . In this work we analysed the morphological changes in tumor cells derived from human pulmonary adenocarcinoma heterotransplanted and grown in naked mice, induced shortly (24 hours) after a single intratumoral injection of alpha-Sarcin (0.4 mg/tumor) . The results obtained were: 1) swelling of mitochondria; 2) cell necrosis with partial removal of necrotic cells by phagocytosis; 3) thickening of interlobular connective tissue; 4) hyperplasia of goblet-cell-like clear cells . The mode of action concerning these cellular changes is presently uncertain . In view of the severity of these structural alterations it seems conceivable that alpha-Sarcin may enter the cell undergoing interactions with different intracellular structures . This would require a selective membrane permeabilization, perhaps induced upon formation of complexes with negatively-charged membrane phospholipids.

Rocz Panstw Zakl Hig, 1991, 42(1), 1 - 7
{Histamine and tyramine levels in selected food products}; Gajewska R et al.; Histamine and tyramine contents were determined in parallel in fish and fish products ripening and processed cheese, yeast, wine, cabbage and sauerkraut, and tomato paste . Histamine was assayed by the colorimetric method of Hardy and Smith, and by TLC . Tyramine was determined by TLC . Levels of histamine and tyramine were found to be low in all products tested . For histamine and tyramine, respectively, they amounted: in raw fish to 0.0-8.0 and 0.0-2.6 mg/100 g, in fish products to 0.0-16.0 and 0.0-10.0 mg/100 g, and in cheeses to 0.0-0.8 and 1.3-20.0 mg/100 g . In the remaining food products (tomato paste, yeast, wine, cabbage and sauerkraut), histamine content was between 0.0-16.6 mg/100 g (highest in tomato paste), and tyramine content fluctuated between 0.0-8.0 mg/100 g (highest in sauerkraut).

Int J Immunopharmacol, 1991, 13(5), 501 - 8
Effect of saikosaponin on the immune responses in mice; Ushio Y et al.; The in vivo effects of saikosaponin, isolated from Bupleurum radix, on the immune responses are still poorly understood . We have already shown that saikosaponin-d increases phagocytic activities of murine peritoneal macrophages such as spreading activity, phagocytosis, lysosomal enzyme activity and intracellular killing activity of living yeast . This work extends these observations by showing that treatment with saikosaponin also increased the antibody response in plaque-forming cell numbers after in vivo immunization with sheep red blood cells (SRBC) and an augmentation of spleen cell proliferation responses to stimulation with T- or B-cell mitogens both before and after immunization . Furthermore, after SRBC immunization, the macrophages from mice treated with saikosaponin-d revealed significant increases in spreading activity and lysosomal enzyme activity . The chemiluminescences of the macrophages from mice treated with saikosaponin-d stimulated by opsonized zymosan and PMA were enhanced and interleukin-1 production by the cells was increased in a dose-dependent manner . These results demonstrate that saikosaponin-d may stimulate in vivo immunological lymphocyte functions, partly by activating some macrophage functions.

DNA Seq, 1991, 2(2), 133 - 5
Nucleotide sequence of a human heart cDNA encoding the mitochondrial phosphate carrier; Dolce V et al.; We have isolated and characterized a full length cDNA clone encoding the precursor of the human heart mitochondrial phosphate carrier protein . The entire clone is 1330 bp in length with 5'- and 3'-untranslated regions of 48 and 184 bp, respectively . The open reading frame encodes the mature protein consisting of 312 amino acids, preceded by a presequence of 49 amino acids . The amino acid sequence of the mature human phosphate carrier is 93.6, 94.2 and 33.6% identical to that of the phosphate carrier from beef, rat and yeast, respectively . Like other mitochondrial transport proteins, the human phosphate carrier has a tripartite structure . Each of the three repeats contains two hydrophobic regions which presumably span the membrane in the form of alpha-helices.

Nephrol Dial Transplant, 1991, 6 Suppl 3, 35 - 40
Phagocytosis activity of polymorphonuclear cells of normal persons and dialysis patients is influenced by different dialysis membranes; Schauer S et al.; Leukocyte (PMN) functional capacity has been investigated through evaluation of phagocytosis of opsonised yeast cells in a radiometric test system . The PMN of dialysis patients (DP) had a slightly lower ability to ingest opsonised yeast cells in comparison with normal persons (NP), suggesting that an intrinsic cellular defect may exist . Under the influence of six membranes (cellulose acetate, regenerated cellulose, modified cellulose, cuprophane, polysulphone, and polymethylmethacrylate) the phagocytosis index decreased significantly between 10 and 17% in NP and between 13 and 23% in DP . There is a clear correlation with the membrane surface area . These results are not explained by the number of dead leukocytes (4-6.5% in DP and also in NP independently of membrane contact) . The direct membrane effect could be responsible for the diminished phagocytic activity of leukocytes in NP and DP . Aqueous extracts of membranes alone resulted in no change of the phagocytic ability of PMN . Extracellular or 'uraemic factors' were excluded by the test procedure . The killing rate of yeast cells by PMN in NP and DP was not influenced in any of the membranes tested.

Doc Ophthalmol, 1991, 77(3), 185 - 92
Longitudinal measures in children receiving ENCAD for hereditary retinal degeneration; Birch DG et al.; A hydrolysate of yeast RNA (ENCAD) is used in the Soviet Union for the treatment of hereditary retinal degenerations . We report longitudinal data from three young patients who have made at least two visits to the Soviet Union over a five-year period to receive treatment with ENCAD . Two children were diagnosed with cone-rod degeneration and the third has an isolated (simplex) form of retinitis pigmentosa . Visual function measurements were obtained before and after each visit to Moscow . In the comparison of previsit and postvisit visual acuity, 30 Hz flicker amplitude, and visual fields, ENCAD treatment had no significant short-term effect . Despite treatment with ENCAD, each patient has shown a significant decrease in visual function over the 5-year period . The rate of progression in these patients appears similar to previously published data on the natural history of their retinal degenerative disorders.

Acta Otolaryngol, 1991, 111(5), 943 - 5
Is Pityrosporum ovale a pathogen of the external auditory meatus?
Stenfors LE, Raisanen S.
Fifteen dry wax samples, 15 wet wax samples and 7 dandruffy skin samples obtained from the external auditory meatus (EAM) of 26 individuals (age range from 1 to 76 years) were examined for the identification of Pityrosporum ovale . After staining the samples with Gram's stain, cultures were made on Sabouraud's medium with olive oil added . Thirteen of the dry wax samples, 1 of the wet wax samples and 6 of the dandruffy skin samples harboured P . ovale . Lipophilic yeast is frequently present in dry wax in adults and must be considered an aetiological agent of dandruff in the EAM.

Biotechnol Ther, 1991, 2(1-2), 63 - 89
Importance of conformation on the neutralizing antibody response to HIV-1 gp120; Steimer KS et al.; We have investigated the role of conformation of HIV-1 gp120 on its potential efficacy as a subunit vaccine . The questions that we set out to answer were: 1) Are there neutralizing antibodies directed to conformational epitopes in gp120? 2) If so, what is the spectrum of virus isolates neutralized by these antibodies? 3) Is a conformationally correct gp120 subunit more effective in the induction of neutralizing antibodies than a denatured subunit? 4) Does native gp120 subunit vaccination induce a broader neutralizing response than a gp120 antigen that cannot display conformational epitopes? To address these questions, we characterized the gp120-specific antibody response of HIV-1-infected humans and of experimental animals immunized with recombinant native and nonnative gp120 subunits . Two versions of recombinant gp120 produced from the HIV-SF2 isolate of HIV-1 were employed in these studies: 1) a nonglycosylated, denatured version produced in genetically engineered yeast, which we presume is capable of presenting only linear determinants, and 2) a fully glycosylated, native version, produced in genetically engineered mammalian cells, that is capable of displaying linear as well as conformational epitopes . Antibodies directed exclusively to conformational epitopes in gp120 were purified from pooled HIV antibody-positive human sera using these two versions of HIV-SF2 gp120 . These antibodies exhibited neutralizing activity, and this activity was effective in the neutralization of a different, broader spectrum of HIV-1 isolates than that of antibodies to linear determinants in gp120 purified from the same serum pool . When these two versions of HIV-SF2 gp120 were used as subunit immunogens in baboons, clear differences in their abilities to elicit neutralizing antibodies were observed . The native version was more effective in the induction of neutralizing antibodies effective against HIV-SF2, the homologous virus isolate . The isolate specificity of the neutralizing response to these two versions of HIV-SF2 gp120 also differed . The nonglycosylated version induced neutralizing antibodies that were effective against only the isolate, or closely related isolates, from which the antigen was derived . In contrast, the native version induced a neutralizing response that was effective against a broad panel of HIV-1 isolates, including at least one isolate that one would not expect to be neutralized by antibodies to the PND of HIV-SF2 gp120.

Symp Soc Exp Biol, 1991, 45, 57 - 62
Development of a system for efficient chromosome walking in Arabidopsis; Grill E et al.; The small genome size of Arabidopsis and the low level of repetitive DNA sequences make this crucifer an attractive system for chromosome walking to isolate genes . Mapping of a mutant locus relative to restriction fragment length polymorphism (RFLP) markers provides the first step towards isolating the corresponding gene . The RFLP marker closest to the target gene serves as a starting point . The distance between gene and marker is generally in the range of 50-200 kb . In order to facilitate chromosome walking of this magnitude, we constructed a yeast artificial chromosome (YAC) library of Arabidopsis . Large fragments of Arabidopsis DNA were cloned into a YAC vector and transformed into yeast . The library contains more than 10 equivalents of the Arabidopsis genome . YACs containing sequences of RFLP markers of Arabidopsis revealed an average insert size of 150 kb . Thus, 1-3 (contiguous) YACs should be sufficient to clone genes from Arabidopsis by chromosome walking . In order to use the system to isolate genes involved in the signal transduction of abscisic acid, we fine-mapped the mutant loci abi-1 and abi-2, which confer abscisic acid insensitivity, relative to RFLPs and isolated the corresponding YACs.

Comp Biochem Physiol C, 1991, 100(3), 389 - 96
Isolation, partial purification, and characterization of the cytochrome P-450-dependent monooxygenase system from the midgut of the earthworm Lumbricus terrestris; Berghout AG et al.; 1 . Cytochrome P-450 was purified from microsomes of the midgut of the earthworm Lumbricus terrestris up to a maximal specific content of 5.5 nmol P-450/mg protein . 2 . At least 3 different cytochromes P-450 with apparent molecular weights of 48,000, 51,000 and 53,000 were identified by SDS-PAGE . 3 . Western blot analysis with various polyclonal antibodies did not show structural epitopes common to the cytochromes P-450 of rodents or yeast and L . terrestris . 4 . The microsomes contained about 43 pmol P-450/mg protein corresponding to 0.51 nmol P-450/g midgut and 64 pmol P-450/g body weight, respectively, and converted benzyloxyresorufin into resorufin with a Vmax of 2.12 pmol resorufin/min.mg protein and a Km of 770 nM benzyloxyresorufin at 25 degrees C, pH 8.0 . 5 . The microsomes exhibited a NADPH-cytochrome P-450 reductase activity of 9.4 nmol cytochrome c/min.mg protein . 6 . The apparent molecular weight of the threefold-purified reductase was 63,000.

Prog Clin Biol Res, 1991, 362, 33 - 66
The molecular genetics of retinal photoreceptor proteins involved in cGMP metabolism; Pittler SJ et al.; Metabolism of cGMP is critically important for the functioning of phototransduction in the mammalian retina . In rod and cone photoreceptors, two types of antagonistic enzymes, guanylate cyclases and cGMP phosphodiesterases, carefully balance the available amount of the intracellular messenger . Guanylate cyclase produces cGMP and phosphodiesterase rapidly hydrolyzes cGMP upon bleaching of the photopigment . Regulation of their activity in light and dark, influence of Ca++, and feed-back mechanisms are currently under intense investigation . A molecular analysis on both the gene and protein levels will contribute significantly to our understanding of their respective roles in phototransduction . The two types of enzymes have been characterized molecularly to a very different extent . Rod phosphodiesterase was purified to homogeneity almost fifteen years ago, but photoreceptor guanylate cyclase has evaded all attempts for molecular characterization . Characterization of retinal guanylate cyclase cDNA(s), however, will most likely be achieved in the near future . Cone PDE was shown to be a distinct enzyme, different from, but related to, the rod enzyme . Molecular cloning has provided sequence information of two of the three subunits of rod PDE; the small inhibitory subunit has been expressed in bacterial expression vectors, giving us an elegant tool for exploring mechanisms of activation and inhibition . The gene encoding the alpha subunit was shown to be a member of a large gene family of cyclic nucleotide phosphodiesterases, present in many eucaryotes ranging from unicellular organisms (yeast) to mammals . While much has been achieved, many questions remain to be answered . The beta subunit of rod phosphodiesterase has evaded complete molecular characterization, and its origin (one gene and posttranslational modification of the gene product generating alpha and beta, alternative splicing, or two separate genes with distinct gene products) has not been elucidated . Mechanisms of interaction of subunits, activation and inhibition, the active site(s) of the enzyme are undefined . Virtually nothing is known about the molecular organization of the photoreceptor guanylate cyclase(s) . Recent cloning of two apparently unrelated mammalian guanylate cyclases, however, containing a common homologous domain signals increasingly rapid progress in this field.

Acta Microbiol Hung, 1991, 38(2), 133 - 40
Temperature induced changes in microsomal (Na+,K+)-ATPase and lipid composition of Candida kefyr; Singh B et al.; Growth temperature affected both the membrane lipid composition and microsoma (Na+, K+)-ATPase activity of Candida kefyr . Higher growth, temperature (37 degrees C) increased the amount of total lipids, phospholipids and free sterol . Ratios of phosphatidylcholine to phosphatidylethanolamine as well saturated to unsaturated fatty acids increased with a rise in growth temperature . Km of the ATPase isolated from the yeast grown at 27 degrees C was minimum, suggesting that the membranes of C . kefyr grown at optimal growth temperature provide the most suitable environment for the activity of ATPase.

Biochem Int, 1991 Jan, 23(1), 83 - 92
Superoxide anion production by lipoamide dehydrogenase redox-cycling: effect of enzyme modifiers; Grinblat L et al.; Redox-cycling of porcine heart lipoamide dehydrogenase in the presence of NADH and oxygen produced O2- . (NADH-oxidase activity) as demonstrated by (a) reduction of cytochrome c; (b) reduction of the Fe(III)-ADP complex; (c) lucigenin luminescence and (d) the inhibitory effect of superoxide dismutase . NAD+ and p-chloromercuribenzoate inhibited O2- . generation whereas arsenite enhanced it . Comparison of heart and yeast enzyme preparations revealed a close correlation between lipoamide reductase and NADH-oxidase activities . It is concluded that O2- . production is a molecular property of lipoamide dehydrogenase.

Life Sci, 1991, 49(1), 23 - 8
Role of glutathione reductase system in disulfiram conversion to diethyldithiocarbamate; Nagendra SN et al.; Experiments were carried out to establish the role of glutathione reductase (GR), if any, in the metabolic conversion of disulfiram (DS) to diethyldithiocarbamate (DDC) . It was observed that, under standard assay conditions, whereas DS was incorporated as a substrate instead of oxidised glutathione (GSSG), the enzymes from both human liver extract and yeast sources failed to reduce the parent compound, implying that glutathione reductase perse do not reduce disulfiram . However, the incorporation of disulfiram into an assay system comprising of GSSG, NADPH and reductase resulted in DS reduction to DDC . Further, the observation, that the GR assay system devoid of either GSSG or NADPH was found to lack DS reducing ability, implies that GSH as a reaction product of GR system is responsible for the reduction of DS to DDC . The results of in-vitro experiments indicated that GSH perse could reduce DS to DDC nonenzymatically, with a stoichiometric relationship of 2:1 . Thus it is inferred that GR perse do not reduce DS, whereas GSH, as an intermediary metabolite of GR system, brings about non-enzymatic reduction of DS via a sulfhydral group exchange reaction.

Bioseparation, 1991, 2(1), 23 - 9
Effects of periodic backflushing on ultrafiltration performance; Kim BS et al.; Periodic backflushing was introduced to a membrane separation process to improve the performance . Hemoglobin (M.W . = 62,500) and dextran (M.W . = 10,000) were used as model compounds . Filtration performance of an ultrafiltration membrane system (Amicon hollow fiber membrane, H1P30-43, molecular weight cutoff = 30,000) was measured in terms of apparent permeability and retention coefficient of dextran to determine the effects of backflushing frequency and duration of one cycle . An optimum frequency around 0.2 min-1 existed to give a maximum permeability while the retention of dextran decreased with increasing frequencies . The improvement in permeability by periodic backflush was more than doubled . The retention of dextran decreased as backflushing duration was increased in one cycle . With the duration of 33.75 s, the retention of dextran was less than 50% and dextran output was 1.14 g/h, which was 1.3 times the value without backflushing . Also, periodic backflush made possible the long-term filtration of yeast cells for more than 20 h.

J Biotechnol, 1991 Jan, 17(1), 51 - 66
Proteolytic events in the processing of secreted proteins in fungi; Calmels TP et al.; Secreted heterologous proteins have been found to be produced much less efficiently by fungi than secreted homologous ones . This could be due, at least in part, to proteolytic cleavage by site-specific endoproteases of the secretory pathway, similar to the yeast KEX2 protease and the mammalian dibasic endoproteinases found in secretory pathways . Mature secreted fungal proteins may be protected from such cleavage due to the absence of cleavable sites in exposed regions . A comparison of the dipeptide distributions of 33 secreted and 34 cytoplasmic proteins from fungal producers of extracellular enzymes indicated a significant bias for some doublets, including the basic dipeptides Lys-Arg, Arg-Arg and Arg-Lys which have also been demonstrated to be KEX2 substrates . Other combinations were also found to be rare in secreted proteins, which could indicate either a broader specificity of the considered endopeptidase, or the presence either in the secretory organelles or among the secreted proteins of additional proteases with different specificities . Experimental evidence that the Lys-Arg site is processed in Tolypocladium geodes was provided by cloning a synthetic prosequence upstream of a phleomycin resistance (Sh ble) gene and analyzing the N-terminus of the corresponding protein purified from the culture supernatant . This system also provides a tool for further studies of specific proteases of fungi.

Nucleic Acids Res, 1990 Dec 25, 18(24), 7299 - 303
A gene from the VSG expression site of Trypanosoma brucei encodes a protein with both leucine-rich repeats and a putative zinc finger; Revelard P et al.; The transcription unit of the gene for the variant specific glycoprotein (VSG) AnTat 1.3A of Trypanosoma brucei contains several associated genes (ESAGs, for Expression Site-Associated Genes), 7 of which have already been described . We report here the characterization of a further ESAG, which we term ESAG 8, present 1 kb downstream from the putative adenylate cyclase gene ESAG 4 . ESAG 8 encodes a 70 kd protein whose sequence indicates that it is probably not exposed at the cell surface . With the exception of the N-terminal domain which contains a presumptive DNA-binding zinc finger, the ESAG 8 protein consists exclusively of leucine-rich repeats of 23 amino acids, typical of protein-interacting domains such as the RAS-interacting region of the yeast adenylate cyclase . ESAG 8 transcripts are only found in bloodstream forms, and their level is particularly low, suggesting a high rate of degradation . The ESAG 8 protein may be involved in stage-specific regulatory processes, such as gene expression control or adenylate cyclase activation.

Curr Opin Biotechnol, 1990 Dec, 1(2), 180 - 7
The molecular basis of genetic disease; Boehm CD et al.; The pace of localization and characterization of genes affected in human genetic disorders is quickening . Many important genes were localized or characterized recently: genes for in cystic fibrosis, NF-2, Marfan's syndrome and xeroderma pigmentosum, to name a few . Also, in the past 15 months, the CFTR gene affected in cystic fibrosis has been isolated, the first disease gene to be isolated without use of previous cytogenetic clues, such as deletions or translocations in sporadic cases . Other examples should follow, although we have been disappointed to date by the difficulties encountered in the isolation of Huntington's disease gene which was localized a number of years ago to distal chromosome 4p . It is still very difficult to isolate a disease gene without critical cytogenetic information . New improved techniques for finding the desired expressed sequences in a large cloned segment of human DNA are needed . Our ability to find mutant alleles of a given sequence has expanded greatly with the recent technical advances in denaturing gradient gel electrophoresis, chemical cleavage, and single-stranded conformational electrophoresis . One would predict that information derived from the human genome project will have a major impact upon the isolation of further disease genes . As whole regions of human chromosomes or indeed entire chromosomes are physically mapped and cloned as continuous, overlapping YACs (yeast artificial chromosomes), isolation of disease genes will become easier and easier.(ABSTRACT TRUNCATED AT 250 WORDS)

Genes Dev, 1990 Dec, 4(12A), 2132 - 45
Lethal and temperature-sensitive mutations and their suppressors identify an essential structural element in U2 small nuclear RNA; Ares M Jr et al.; U2 snRNA is an essential component of the splicing apparatus in eukaryotic cells . Three possible secondary structures for the highly conserved 5' half of U2 snRNA are consistent with U2 phylogenetic sequence variation . To distinguish among these models and to test the function of U2 structural elements, we made greater than 35 mutations in the yeast U2 snRNA gene . Some of the mutations were designed in pairs so that combinations could be made that would restore base-pairing to differentiate helix requirements from primary sequence requirements . The mutations identify an essential stem-and-loop structure adjacent to the branchpoint interaction region . A conserved complementarity to the loop just upstream of the Sm site and an additional conserved stem-loop are dispensable for U2 function, even in the background of a previously identified large internal deletion . Non-Watson-Crick base appositions at the 53-62 base pair in the essential stem lead to a variety of temperature and KCl-sensitive phenotypes, as well as an accumulation of unspliced precursors in vivo . Chemical structure probing of U2 RNA in vivo reveals that the bulk of U2 in a yeast cell adopts a structure in good agreement with that deduced from genetic results . We suggest that this stem-loop is not a binding site for an intrinsic U2 snRNP protein but may interact with other factors during spliceosome assembly or splicing.

Mol Reprod Dev, 1990 Dec, 27(4), 366 - 75
Cell-cycle aspects of growth and maturation of mammalian oocytes; Motlik J et al.; In this review, recent data concerning growth and maturation of nonmammalian and mammalian female germ cells are compiled with regard to the increased understanding of somatic cells mitotic cycles, from yeast to human tissues . These data allow us to conclude that growing oocytes of nonvertebrates, lower vertebrates, and mammals resemble somatic cells in the G1 phase of the mitotic cycle in their metabolic and cell cycle behavior . Transcriptional and translational activity of growing oocytes and G1 somatic cells is not compatible with the activation of maturation promoting factor (MPF), with chromatin condensation or with nuclear membrane disintegration . Growing oocytes, even when they are in the dictyate stage of the first meiotic division, promptly inactivate MPF introduced into their cytoplasm by fusion or microinjection, just as do somatic interphase cells . In mammals, the LH surge induces "de novo" RNA and protein synthesis in granulosa cells . This metabolic change in granulosa cells abolishes their inhibitory activity, and meiosis in fully grown oocytes in preovulatory follicles is then resumed . Resumption of meiosis requires an activation of pre MPF molecules within oocytes . This can be achieved either without (mouse, rat, and rabbit) or with (pig, sheep, and cow) an active protein synthesis by the oocytes . The species specificity is probably dependent on the presence or absence of cyclin-like and/or mos-like molecules in fully grown oocytes . Both major events during GVBD, chromatin condensation, and nuclear envelope disintegration require protein phosphorylation . Experimentally, these two phosphorylation activities can be separated one from another . The active MPF molecules are amplified autocatalytically in amphibian and starfish oocytes . However, an increase of MPF activity in mouse and pig oocytes, similarly as in Rana pipiens and sturgeon oocytes, requires an active protein synthesis.

Proc Natl Acad Sci U S A, 1990 Dec, 87(24), 9625 - 9
Molecular analysis of human argininosuccinate lyase: mutant characterization and alternative splicing of the coding region; Walker DC et al.; Argininosuccinic acid lyase (ASAL) deficiency is a clinically heterogeneous autosomal recessive urea cycle disorder . We previously established by complementation analysis that 28 ASAL-deficient patients have heterogeneous mutations in a single gene . To prove that the ASAL structural gene is the affected locus, we sequenced polymerase chain reaction-amplified ASAL cDNA of a representative mutant from the single complementation group . Fibroblast strain 944 (approximately 1% of residual ASAL activity), from a late-onset patient who was the product of a consanguineous mating, had only a single base-pair change in the coding region, a C-283----T transition at a CpG dinucleotide in exon 3 . This substitution converts Arg-95 to Cys (R95C), occurs in a stretch of 13 residues that is identical in yeast and human ASAL, and was present in both of the patient's alleles but not in 14 other mutant or 10 normal alleles . Expression in COS cells demonstrated that the R95C mutation produces normal amounts of ASAL mRNA but little protein and less than 1% ASAL activity . We observed that amplified cDNA from mutant 944 and normal cells (liver, keratinocytes, lymphoblasts, and fibroblasts) contained, in addition to the expected 5' 513-base-pair band, a prominent 318-base-pair ASAL band formed by the splicing of exon 2 from the transcript . The short transcript maintains the ASAL reading frame but removes Lys-51, a residue that may be essential for catalysis, since it binds the argininosuccinate substrate . We conclude (i) that the identification of the R95C mutation in strain 944 demonstrates that virtually all ASAL deficiency results from defects in the ASAL structural gene and (ii) that minor alternative splicing of the coding region occurs at the ASAL locus.

Proc Natl Acad Sci U S A, 1990 Dec, 87(23), 9378 - 82
Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site; Wise RJ et al.; Intracellular proteolytic processing of precursor polypeptides is an essential step in the maturation of many proteins, including plasma proteins, hormones, neuropeptides, and growth factors . Most frequently, propeptide cleavage occurs after paired basic amino acid residues . To date, no mammalian propeptide processing enzyme with such specificity has been purified or cloned and functionally characterized . We report the isolation and functional expression of a cDNA encoding a propeptide-cleaving enzyme from a human liver cell line . The encoded protein, called PACE (paired basic amino acid cleaving enzyme), has structural homology to the well-characterized subtilisin-like protease Kex2 from yeast . The functional specificity of PACE for mediating propeptide cleavage at paired basic amino acid residues was demonstrated by the enhancement of propeptide processing of human von Willebrand factor when coexpressed with PACE in COS-1 cells.

Mol Cell Biol, 1990 Dec, 10(12), 6335 - 47
Factors involved in specific transcription by mammalian RNA polymerase II: role of transcription factors IIA, IID, and IIB during formation of a transcription-competent complex; Maldonado E et al.; Human transcription factor TFIID, the TATA-binding protein, was partially purified to a form capable of associating stably with the TATA motif of the adenovirus major late promoter . Binding of the human and yeast TFIID to the TATA motif was stimulated by TFIIA . TFIIA is an integral part of a complex capable of binding other transcription factors . A complex formed with human TFIID and TFIIA (DA complex) was specifically recognized by TFIIB . We found that TFIIB activity was contained in a single polypeptide of 32 kDa and that this polypeptide participated in transcription and was capable of binding to the DA complex to form the DAB complex . Formation of the DAB complex required TFIIA, TFIID, and sequences downstream of the transcriptional start site; however, the DA complex could be formed on an oligonucleotide containing only the adenovirus major late promoter TATA motif . Using anti-TFIIB antibodies and reagents that affect the stability of a transcription-competent complex, we found that yeast and human TFIID yielded DAB complexes with different stabilities.

EMBO J, 1990 Dec, 9(13), 4455 - 65
Cyclic-AMP-responsive transcriptional activation of CREB-327 involves interdependent phosphorylated subdomains; Lee CQ et al.; Cyclic AMP-regulated gene expression is mediated by specific phosphoproteins (CREBs) which bind to cAMP-responsive elements of gene promoters . By analyzing the transactivation activities and phosphorylations in vivo of deletion and point mutated chimeric fusion proteins of the placental CREB-327, in which the DNA-binding domain is replaced by the heterologous binding-domain of the yeast transcription factor GAL4, we localized the cAMP-responsive and phosphorylated domain to a minimal-essential sequence module of 46 amino acids (residues 92-137) . This serine-rich, multiply-phosphorylated sequence consists of at least three interdependent subdomains required for transcriptional activation . Although phosphorylation of serine-119 by cyclic AMP-dependent protein kinase A is necessary for transcriptional activation, such activation requires both a phosphorylated heptadecapeptide domain located ten residues amino terminal to the serine-119 and an eleven-residue domain carboxyl terminal to the serine-119 . Deletion of these two domains does not impair phosphorylation of serine-119 . Further, deletion of the carboxyl-terminal domain does not alter phosphorylation of the heptadecapeptide domain . We propose that akin to the phosphorylation-dependent activation of enzymes, the transcriptional transactivation functions of CREB-327 involve a phosphorylation-dependent allosteric conformational mechanism.

J Nutr, 1990 Dec, 120(12), 1692 - 9
Respiratory burst and candidacidal activity of peritoneal macrophages are impaired in copper-deficient rats; Babu U et al.; To investigate the effect of dietary copper deficiency on the function of peritoneal macrophages, weaned male Lewis rats were pair-fed diets containing either adequate (7 mg/kg diet; +Cu) or deficient (0.7 mg/kg diet; -Cu) levels of copper for 5 wk . Cellular copper content and the activity of Cu, Zn superoxide dismutase were significantly lower in both resident and thioglycollate-elicited macrophages from -Cu rats than in cells from +Cu controls . Reduced cellular Cu status was associated with impaired respiratory burst as assessed by zymosan-induced chemiluminescent activity and superoxide anion (O2-) generation . Candidacidal activity of macrophages from -Cu rats was also reduced and was highly correlated with chemiluminescent activity and O2- generation . In contrast, phagocytosis of opsonized erythrocytes by peritoneal macrophages from -Cu rats was normal . Elicited peritoneal macrophages from marginally Cu-deficient rats also killed significantly fewer yeast cells than macrophages from +Cu rats . These results demonstrate that macrophage function is impaired by dietary Cu deficiency and that the candidacidal activity of these cells may provide a sensitive indicator of Cu status.

Mol Cell Biol, 1990 Dec, 10(12), 6225 - 35
Purification of RIP60 and RIP100, mammalian proteins with origin-specific DNA-binding and ATP-dependent DNA helicase activities; Dailey L et al.; Replication of the Chinese hamster dihydrofolate reductase gene (dhfr) initiates near a fragment of stably bent DNA that binds multiple cellular factors . Investigation of protein interactions with the dhfr bent DNA sequences revealed a novel nuclear protein that also binds to domain B of the yeast origin of replication, the autonomously replicating sequence ARS1 . The origin-specific DNA-binding activity was purified 9,000-fold from HeLa cell nuclear extract in five chromatographic steps . Protein-DNA cross-linking experiments showed that a 60-kDa polypeptide, which we call RIP60, contained the origin-specific DNA-binding activity . Oligonucleotide displacement assays showed that highly purified fractions of RIP60 also contained an ATP-dependent DNA helicase activity . Covalent radiolabeling with ATP indicated that the DNA helicase activity resided in a 100-kDa polypeptide, RIP100 . The cofractionation of an ATP-dependent DNA helicase with an origin-specific DNA-binding activity suggests that RIP60 and RIP100 may be involved in initiation of chromosomal DNA synthesis in mammalian cells.

J Antibiot (Tokyo), 1990 Dec, 43(12), 1579 - 85
Biological activities of cyclophellitol; Atsumi S et al.; Cyclophellitol {1S,2R,3S,4R,5R,6R)-5-hydroxymethyl-7-oxabicyclo{4,1,0} heptane-2,3,4-triol) was tested against 9 glycosidases and found to be a specific inhibitor of almond beta-glucosidase . Cyclophellitol inhibited almond beta-glucosidase activity by 50% at 0.8 micrograms/ml and was a competitive inhibitor of almond beta-glucosidase as revealed by Lineweaver-Burk plot . Cyclophellitol was inactive against yeast alpha-glucosidase, beta-galactosidase, beta-glucuronidase, alpha-L-fucosidase, end-beta-N-acetyl glucosaminidase, alpha-mannosidase, and cellulase . It was weakly active toward fungal beta-xylosidase . Cyclophellitol-treated almond beta-glucosidase was equally suppressed after dialysis; thus cyclophellitol is likely to bind to almond beta-glucosidase irreversibly . The inhibitor was found by fluorimetric assay to be active against beta-glucosidase but inactive toward alpha-glucosidase in Molt-4 microsomal fraction . It also inhibited Molt-4 beta-glucocerebrosidase completely at 2 micrograms/ml when the enzyme was assayed with a synthetic labeled substrate, and the inhibitory activity was more than one hundred times higher than that of nojirimycin, castanospermine, or of deoxynojirimycin . Mice administered 1 mg of cyclophellitol daily for 5 days began to exhibit severe abnormalities of nervous system similar to those found in Gaucher's mouse.

Appl Environ Microbiol, 1990 Dec, 56(12), 3718 - 22
Differentiation of Penicillium griseofulvum Dierckx isolates by enzyme assays and by patulin and griseofulvin analyses; Jimenez M et al.; The production of patulin and griseofulvin by 49 different isolates of Penicillium griseofulvum Dierckx was analyzed by high-performance liquid chromatography . Eleven isolates were obtained from pistachio nuts, 37 were obtained from wheat seeds, and 1 was obtained from the American Type Culture Collection . Activities of 19 enzymes were also assayed by the API ZYM system . From these results it may be deduced that there are two different groups among the strains tested which cannot be distinguished by morphological and cultural characteristics . One group of isolates did not produce detectable amounts of patulin and griseofulvin when grown in sucrose-yeast extract and Wickerham media, while enzymatic activities were quantitatively distinct from the other group, which produced patulin and griseofulvin in variable proportions . Leucine arylamidase, phosphoamidase, and beta-D-glucosidase are the main enzymes with differing activities between the two groups . Differences in physiological characteristics among isolates of a single species reveal shortcomings in the classification of the penicillia based only on morphological criteria . Thus, determination of the ability to yield mycotoxins and antibiotics as well as determination of enzymatic activities appear to be very valuable tools in the taxonomy of these fungi and for food toxicology.

Semin Cancer Biol, 1990 Dec, 1(6), 371 - 82
The myb genes; Weston KM; The v-myb oncogene and its cellular progenitor c-myb are both DNA binding proteins capable of transcriptional activation, and are implicated in the regulation of the switch between growth and differentiation in hematopoietic cells . Studies attempting to define the oncogenic determinants of v-myb and activated c-myb genes implicate N- and/or C-terminal truncation as important; both these events appear to increase the affinity of the myb protein for DNA . Myb-like genes have been found in organisms ranging from yeast, through plants, to humans; in the more distantly related cases, only the myb DNA binding domain, situated at the N-terminus of the protein, has been conserved.

J Bioenerg Biomembr, 1990 Dec, 22(6), 725 - 51
Mitochondrial protein import; Geli V et al.; Most polypeptides of mitochondria are imported from the cytosol . Precursor proteins contain targeting and sorting information, often in the form of amino-terminal presequences . Precursors first bind to receptors in the outer membrane . Two putative import receptors have been identified: a 19-kilodalton protein (MOM19) in Neurospora mitochondria, and a 70-kilodalton protein (MAS70) in yeast . Some precursors integrate directly into the outer membrane, but the majority are translocated through one or both membranes . This process requires an electrochemical potential across the inner membrane . Import appears to occur through a hydrophilic pore, although the inner and outer membranes may contain functionally separate translocation machineries . In yeast, a 42-kilodalton protein (ISP42) probably forms part of the outer membrane channel . After import, precursors interact with "chaperonin" ATPases in the matrix . Presequences then are removed by the matrix protease . Finally, some proteins are retranslocated across the inner membrane to the intermembrane space.

Biochem Genet, 1990 Dec, 28(11-12), 601 - 13
Dosage compensation and dietary glucose repression of larval amylase activity in Drosophila miranda; Norman RA et al.; The functional locus for alpha-amylase (Amy) in Drosophila miranda is in the evolutionarily new X2 chromosome . X2 evolved from an autosome in response to an ancestral autosome-Y translocation that gave rise to the "neo-Y" chromosome of this species . Y-linked Amy, if still present in the ancestrally translocated element, is unexpressed . Dosage compensation for amylase activity was examined in larvae of the S 204 strain . Since dietary glucose is known to repress Amy expression in Drosophila melanogaster, dosage compensation of amylase activity in male larvae of D . miranda was tested by rearing larvae of both sexes on yeast diets with or without a glucose supplement . The WT 10 strain of Drosophila persimilis, a sibling species in which Amy is autosomally linked, was used as a reference for tests of amylase activity differences between the sexes . On the diet with glucose, Amy expression was repressed in both WT 10 and S 204 larvae and male larvae of S 204 displayed dosage compensation for amylase activity . On the nonrepressing diet consisting of yeast alone, S 204 continued to display dosage compensation.

Trends Biochem Sci, 1990 Dec, 15(12), 473 - 7
Small GTP-binding proteins in vesicular transport; Balch WE; Recent recognition of the abundance of small GTP-binding proteins in eukaryotic cells has sparked off a search for the possible function of these proteins . Evidence is accumulating that SAR1, ARF, SEC4 and YPT1 in yeast and the rab and arf family in mammalian cells play a central role in the regulation of vesicle transport and organelle function.

Eur J Clin Microbiol Infect Dis, 1990 Dec, 9(12), 886 - 91
Evaluation of a gold-silver staining method for detection and identification of Candida species by light microscopy; Cailliez JC et al.; A gold-silver staining procedure was evaluated for detection of Candida species of medical importance . Probes were prepared by coupling lectins or antibodies (polyclonal and monoclonal) directly or indirectly to colloidal gold particles . Structures reacting to these probes were specifically revealed by light microscopy in cells present in infected kidney tissue sections or in isolated yeast cells on glass slides . Definition, contrast and sensitivity were of a high order . Preliminary data showed that it was possible, using discriminating dilutions, to identify cells from different species of the genus Candida, grown in vitro, according to their ability to stain with polyclonal monospecific antisera . The advantages of gold-silver staining compared with other staining procedures currently used in routine mycological laboratories are its sensitivity, good definition, ease and rapidity, and long conservation of reaction . It is suggested that the procedure has applications for research and identification of yeasts in clinical samples.

Plant Cell, 1990 Dec, 2(12), 1261 - 72
Upstream sequences other than AAUAAA are required for efficient messenger RNA 3'-end formation in plants; Mogen BD et al.; We have characterized the upstream nucleotide sequences involved in mRNA 3'-end formation in the 3' regions of the cauliflower mosaic virus (CaMV) 19S/35S transcription unit and a pea gene encoding ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) . Sequences between 57 bases and 181 bases upstream from the CaMV polyadenylation site were required for efficient polyadenylation at this site . In addition, an AAUAAA sequence located 13 bases to 18 bases upstream from this site was also important for efficient mRNA 3'-end formation . An element located between 60 bases and 137 bases upstream from the poly(A) addition sites in a pea rbcS gene was needed for functioning of these sites . The CaMV -181/-57 and rbcS -137/-60 elements were different in location and sequence composition from upstream sequences needed for polyadenylation in mammalian genes, but resembled the signals that direct mRNA 3'-end formation in yeast . However, the role of the AAUAAA motif in 3'-end formation in the CaMV 3' region was reminiscent of mRNA polyadenylation in animals . We suggest that multiple elements are involved in mRNA 3'-end formation in plants, and that interactions of different components of the plant polyadenylation apparatus with their respective sequence elements and with each other are needed for efficient mRNA 3'-end formation.

Mol Gen Genet, 1990 Dec, 224(3), 469 - 76
Elicitor-specific induction of one member of the chitinase gene family in Arachis hypogaea; Herget T et al.; Chitinases are believed to play an important role in plant defence against bacterial and fungal attack . In peanut (Arachis hypogaea) chitinase genes form a small multigene family . Four chitinase cDNAs (chit 1-4) were isolated from cultured peanut cells . Expression of individual chit genes was assayed by the polymerase chain reaction (PCR) followed by analysis of restriction fragment length polymorphisms (RFLP) . UV irradiation, dilution of cell cultures and treatment with Phytophthora megasperma (Pmg) elicitor or yeast extract were used to induce expression of chit genes . The chit 3 gene is constitutively expressed at a low level in untreated as well as in treated cultures; the expression of chit 4 gene is induced by each of the stimuli tested, whereas the chit 1 gene is activated by cell culture dilution and by yeast extract treatment . The chit 2 gene is strongly activated by treatment with cell wall components from the fungus Phytophthora megasperma but not by the other stimuli . These results indicate that chit 2 gene expression may be controlled by pathogen-specific regulatory elements.

J Med Virol, 1990 Dec, 32(4), 219 - 24
Kinetics of antibody response to hepatitis B virus determinants and to recombinant vaccines in Italy; Zanetti AR et al.; The kinetics of antibody response to the group determinant a and subdeterminants d and y of hepatitis B virus were studied after infection and following immunisation with two recombinant DNA yeast-derived hepatitis B vaccines . The initial antibody response was to the subdeterminant epitopes, whereas anti-a antibody, which provides protection against different subtypes of the virus, was not detected for some weeks or months . The delay in the development of anti-a antibody after active immunisation raises important issues of early protection against infection.

Mol Cell Biol, 1990 Dec, 10(12), 6654 - 63
Expression of a gene family in the dimorphic fungus Mucor racemosus which exhibits striking similarity to human ras genes; Casale WL et al.; Sporulation, spore germination, and yeast-hypha dimorphism in the filamentous fungus Mucor racemosus provide useful model systems to study cell development in eucaryotic cells . Three RAS genes (MRAS1, MRAS2, and MRAS3) from M . racemosus have been cloned, and their nucleotide sequences have been determined . The predicted amino acid sequences and the sizes of the three MRAS proteins exhibit a high degree of similarity with other ras proteins, including that encoded by H-ras, which have been implicated in regulation of proliferation and development in eucaryotic cells by mediating signal transduction pathways . The MRAS proteins show conservation of functional domains proposed for ras proteins, including guanine nucleotide interaction domains, an effector domain, a binding epitope for neutralizing antibody Y13-259, and the COOH-terminal CAAX box, which is a site of thiocylation and membrane attachment . Amino acid sequences unique to each MRAS protein occur adjacent to the CAAX box, consistent with the location of the hypervariable region in other ras proteins . Northern (RNA) analysis was used to study expression of the three MRAS genes in relation to cell development . Gene-specific probes for two of these genes, MRAS1 and MRAS3, hybridized to different 1.3-kb mRNA transcripts . The accumulation of these transcripts depended on the developmental stage, and this pattern was different between the two MRAS genes . No transcript for MRAS2 was detected in the developmental stages examined . The unique patterns of MRAS transcript accumulation suggest that individual MRAS genes and proteins may play distinct roles in cell growth or development.

Biochemistry, 1990 Nov 27, 29(47), 10585 - 9
A novel minimum ribozyme with oxidoreduction activity; Yanagawa H et al.; A nucleoside catalyzing the oxidoreduction of NADH and K3Fe(CN)6 was isolated from Torula yeast RNA and also obtained in 0.05% yield by a series of steps: SDS-phenol extraction, nuclease P1 digestion, alkaline phosphatase digestion, anion exchange chromatography, and HPLC on an ODS column . Its chemical structure was clearly determined at 5-hydroxycytidine, from the results of FAB-MS and 1H and 13C NMR spectroscopies . The mass spectra, chromatographic behavior, UV spectra, and NMR spectra of this nucleoside from natural and synthetic sources were identical . This is the first report of an RNA catalyst having catalytic activity except for the cleavage and ligation of phosphodiester bonds of RNA . That an RNA has oxidoreduction activity indicates new possibilities for RNAs as "living molecules" . 5-Hydroxycytidine may be a vestige of RNAs that formerly possessed metabolizing ability.

FEBS Lett, 1990 Nov 26, 275(1-2), 135 - 8
Site-directed mutagenesis of the putative catalytic residues of Trichoderma reesei cellobiohydrolase I and endoglucanase I; Mitsuishi Y et al.; Site directed mutagenesis has been performed to test hypotheses concerning the putative active sites of Trichoderma reesei cellobiohydrolase I and endoglucanase I . It is shown that mutagenesis of the residue E126, previously proposed to be the proton donor in CBHI, did not totally inactivate the enzyme while mutagenesis of the residue E127 in the homologous enzyme EGI resulted in complete loss of activity . These results are compared with those obtained in similar studies of other glucanases and the effects on enzymatic activity of hyperglycosylation of the yeast produced cellulases are discussed.

Eur J Biochem, 1990 Nov 26, 194(1), 167 - 76
Physical and immunological characterization of human transcription factor IIIA; Waldschmidt R et al.; Human transcription factor IIIA (htFIIIA), specifically required for transcription of the gene for 5S ribosomal RNA has been characterized with respect to some of its physical, immunological and functional properties . TFIIIA from HeLa cells, which selectively binds 5S RNA, is a monomer of approximately 35 kDa with a Stokes' radius of approximately 2.65 nm and a sedimentation coefficient of approximately 2.8 S . These values indicate that the human protein is of rather globular shape and hence diverges not only in molecular mass but also in most of the molecular properties from its highly asymmetric counterpart in Xenopus laevis oocytes . By raising specific polyclonal antibodies against hTFIIIA it was shown in Western immunoblots that there was no cross-reaction between anti-hTFIIIA antibodies and the amphibian protein . Conversely, monoclonal antibodies against three domains of X . laevis TFIIIA antibodies and the amphibian protein . Conversely, monoclonal antibodies against three domains of X . laevis TFIIIA did not cross-react with the human transcription factor . The polyclonal antisera raised against hTFIIIA specifically neutralized binding of the human transcription factor to 5S DNA and abolished in vitro transcription of 5S RNA but these antibodies were unable to inhibit 5S RNA synthesis in cellular extracts from Xenopus, Drosophila or yeast cells . Finally, the species variation of TFIIIA could be substantiated by electrophoretic mobility shift assays revealing preferential binding of hTFIIIA to the homologous 5S RNA gene.

Nucleic Acids Res, 1990 Nov 25, 18(22), 6649 - 57
The structure of a subterminal repeated sequence present on many human chromosomes; Cross S et al.; All telomeres which have been studied consist of an array of simple G/C rich repeats . Human telomeres were shown to share sequence similarity with those of lower eukaryotes by cross-hybridization and human telomeric sequences have been cloned by complementation of telomere function in yeast . Analysis of human telomeric sequences cloned in this way is described here . The terminal part of the cloned human telomeric DNA consists of an array of simple repeats, principally of the sequence TTAGGG and derivatives . The very terminal part consists of yeast-type telomeric repeats which suggests that the human telomeric sequences have acted as a primer for the addition of additional telomeric repeats in the yeast . Subterminal sequences are shared between a number of clones and in situ data shows that these subterminal sequences are present at several different chromosomal ends . Related sequences are present at internal as well as telomeric positions . Differences in the hybridization patterns of subterminal sequences in somatic compared to germ-line tissues are described which indicate differential modification of these sequences during development.

Nucleic Acids Res, 1990 Nov 25, 18(22), 6545 - 51
Group II intron RNA-catalyzed recombination of RNA in vitro; Morl M et al.; We report the first evidence for a novel reaction mediated by the self-splicing yeast mitochondrial group II intron bl1; the site-specific recombination of RNA molecules in vitro . Upon incubation of the intron lariat with two different RNAs, each harbouring a short sequence complementary to exon binding site 1 (EBS1) of the intron, novel recombined RNAs are formed . As a result of this intron-mediated shuffling of gene segments, the 5' part of RNA1 is ligated to the 3' part of RNA2 and, reciprocally, the 5' part of RNA2 to the 3' part of RNA1 . Sequence analysis of the recombinant junction shows that the site of recombination is precisely located 3' to intron binding site 1 (IBS1) . The hypothesized mechanism of recombination involves exchange of RNA 5' parts after the first step of a reverse splicing reaction . The possible role of this mechanism in vivo and during prebiotic evolution is discussed.

J Biol Chem, 1990 Nov 25, 265(33), 20621 - 6
Sequence of a cDNA that specifies the uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetylglucosamine-1-phosphate transferase from Chinese hamster ovary cells; Scocca JR et al.; We have isolated a portion of the uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetyl-glucosamine-1-phosphate transferase gene (GTR2) from the genome of a tunicamycin-resistant clonal Chinese hamster ovary cell line, 3E11 . The genomic fragment was selected by its hybridization to the yeast ALG-7 gene at low stringency . A 2.46-kilobase cDNA was isolated from a library prepared from 3E11 mRNA and probed with GTR2 . The cDNA contained an open reading frame that encodes a protein of 408 amino acids with a molecular mass of 44.9 kDa . This protein was 43% identical in amino acid sequence to the protein of 448 amino acids encoded by the ALG-7 gene . The GTR2 gene fragment contained sequences for four exons coding for the carboxyl-terminal half of the protein . Transferase DNA sequences in 3E11 cells were 12-fold elevated over wild-type cells and 25-fold elevated when 3E11 cells were grown in the presence of tunicamycin . Transferase RNA levels in 3E11 cells were also elevated over wild-type levels but appeared unchanged by the presence of tunicamycin in the medium.

Schweiz Rundsch Med Prax, 1990 Nov 20, 79(47), 1457 - 62
{The efficacy of drug therapy in structural lesions of the hair and in diffuse effluvium--comparative double blind study}; Petri H et al.; Growth and quality of hair was studied after treatment with Pantogar, another prescription (Verum-2) and placebo for four months in 60 patients with diffuse effluvium capillorum and agnogenic structural alternations of hair . Efficacy was assessed by measurements of swelling, dye-binding and thickness for hair-quality and evaluation of hair-density and trichograms for hair-growth . Statistical analysis of swelling properties and trichogram data indicated that Pantogar was effective, the second preparation improved quality of hair and retarded hair loss . Placebo was ineffective judged by the used parameters . Tolerance of the treatment was good and adverse effects could not be substantiated.

Science, 1990 Nov 16, 250(4983), 994 - 7
Wilms tumor locus on 11p13 defined by multiple CpG island-associated transcripts; Bonetta L et al.; Wilms tumor is an embryonal kidney tumor involving complex pathology and genetics . The Wilms tumor locus on chromosome 11p13 is defined by the region of overlap of constitutional and tumor-associated deletions . Chromosome walking and yeast artificial chromosome (YAC) cloning were used to clone and map 850 kilobases of DNA . Nine CpG islands, constituting a "CpG island archipelago," were identified, including three islands that were not apparent by conventional pulsed-field mapping, and thus were at least partially methylated . Three distinct transcriptional units were found closely associated with a CpG island within the boundaries of a homozygous DNA deletion in a Wilms tumor.

J Biol Chem, 1990 Nov 15, 265(32), 19479 - 85
Studies on the 4-carbon precursor in the biosynthesis of riboflavin . Purification and properties of L-3,4-dihydroxy-2-butanone-4-phosphate synthase; Volk R et al.; The formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione requires a phosphorylated 4-carbon intermediate which has been designated as Compound X (Neuberger, G., and Bacher, A . (1985) Biochem . Biophys . Res . Commun . 127, 175-181) . The enzyme catalyzing the formation of Compound X has been purified about 600-fold from the cell extract of the flavinogenic yeast Candida guilliermondii by chromatographic procedures . The purified protein appeared homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of a single polypeptide of 24 kDa . The committed substrate of the enzyme was identified as D-ribulose 5-phosphate . The enzyme yields two products which were identified as L-3,4-dihydroxy-2-butanone 4-phosphate and formate by NMR and CD spectroscopy . Mg2+ is required for activity.

N Z Med J, 1990 Nov 14, 103(901), 539 - 41
Hepatitis B vaccine boosters: further studies in children; Milne A et al.; Two groups of children were given reduced dose boosters with Merck Sharp and Dohme (MSD) recombinant DNa, yeast derived hepatitis B vaccine (YDV), 30 and 34 months respectively after primary immunisation with MSD plasma derived vaccine (PDV) . In the first group of unselected children the geometric mean titre (GMT) rose from 387 to 8346 IU/L, with all children protected . The second group were selected as the poorest responders to their primary course of vaccine . The booster raised the GMT from 14.6 to 325 IU/L, with 95% having protective levels of anti-HBs (greater than 10 IU/L) . This study confirms that the vaccine used by the New Zealand Department of Health for the hepatitis B immunisation programme in preschoolers, will provide protection for the great majority of children for at least three years.

Nature, 1990 Nov 8, 348(6297), 137 - 43
Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins; Kang PJ et al.; By analysis of a temperature-sensitive yeast mutant, a heat-shock protein in the matrix of mitochondria, mitochondrial hsp70 (Ssc1p), is found to be involved both in translocation of nuclear-encoded precursor proteins across the mitochondrial membranes and in (re)folding of imported proteins in the matrix.

J Biol Chem, 1990 Nov 5, 265(31), 18961 - 7
Structure, exon pattern, and chromosome mapping of the gene for cytosolic copper-zinc superoxide dismutase (sod-1) from Neurospora crassa; Chary P et al.; A 4.8-kilobase BamHI-HindIII fragment encoding the entire Neurospora crassa CuZn superoxide dismutase gene (herein designated sod-1) was isolated from a genomic library using two 60-base deoxyoligonucleotide probes corresponding to the published N . crassa amino acid sequence . The nucleotide sequence of the gene encodes an amino acid sequence matching the published protein sequence at 152 of 153 positions . Codon preference shows an unusually strong bias such that only 32 of the possible 61 codons are used, with no codons ending in A . Codon usage is that of highly expressed N . crassa genes . The gene contains three introns, none of which corresponds to any of the introns previously identified in the human gene . Analysis of the intron positions provides support for the hypothesis that CuZn superoxide dismutases evolved by gene duplication and fusion followed by the addition of exons encoding an N-terminal beta-hairpin and a zinc-binding subdomain . The N . crassa gene has an intron mapping to amino acid residue 114 in a sequence-conserved region of the protein whereas the human gene has an intron mapping to a similar but not identical position at residue 118 . The discordant position of these introns suggests that one of them was inserted relatively recently . The first N . crassa intron contains a sequence that is similar to the transcriptional regulatory site, UAS1, of the yeast CYC1 (iso-1-cytochrome c) gene and to a putative UAS from the yeast manganese superoxide dismutase gene . A 10-nucleotide portion of this region also matches exactly a sequence in intron 2 of the con-10 gene of N . crassa . sod-1 was mapped to the left arm of chromosome I by following the segregation of a restriction fragment length polymorphism in a sexual cross . Although results indicate that there is a single gene for cytosolic CuZn superoxide dismutase, two additional, perhaps distantly related, sequences were identified that hybridized weakly to both oligonucleotide probes.

J Reprod Med, 1990 Nov, 35(11), 992 - 4
Terconazole for the treatment of vulvovaginal candidiasis; Thomason JL et al.; A double-blind, randomized trial was conducted to evaluate the efficacy and safety of terconazole for vulvovaginal candidiasis . Treatment consisted of daily intravaginal application of one of the following regimens: 80-mg terconazole suppositories for 3 days, miconazole nitrate suppositories for 7 days or placebo suppositories for 7 days . The terconazole and miconazole nitrate groups had significantly higher therapeutic cure rates than did the placebo group . Evaluation of vaginal secretions with microscopic examination showed no evidence of leukocyte proliferation . Proline aminopeptidase activity, present in patients who have bacterial vaginosis, could not be detected in the vaginal secretions from patients with yeast vulvovaginitis.

J Pharmacol Exp Ther, 1990 Nov, 255(2), 511 - 22
Pharmacology of pravadoline: a new analgesic agent; Haubrich DR et al.; Pravadoline is a new chemical entity with analgesic activity in humans . This report describes the pharmacology of pravadoline and compares the activity of pravadoline with that of two major classes of analgesics, the opioids and the nonsteroidal anti-inflammatory drugs (NSAIDs) . Like the NSAIDs, pravadoline inhibited the synthesis of prostaglandins (PGs) in mouse brain both in vitro (IC50, 4.9 microM) and ex vivo (ED50, 20 mg/kg p.o.) and displayed antinociceptive activity in rodents subjected to a variety of chemical, thermal and mechanical nociceptive stimuli . Administration of pravadoline prevented the writhing response induced by i.p . administration of acetylcholine (ED50, 41 mg/kg p.o.) or PGE2 (ED50, 24 mg/kg p.o.) and prolonged the response latency induced by tail immersion in hot water at a temperature of 55 degrees C (minimum effective dose, 100 mg/kg s.c.) . In the rat, treatment with pravadoline prevented acetic acid-induced writhing (ED50, 15 mg/kg p.o.), brewer's yeast-induced hyperalgesia (Randall-Selitto test) (minimum effective dose, 1 mg/kg p.o.), the nociceptive response induced by paw flexion in the adjuvant-arthritic rat (ED50, 41 mg/kg p.o.) and bradykinin-induced head and forepaw flexion (ED50, 78 mg/kg, p.o.) . The antinociceptive activity of pravadoline cannot be explained by an opioid mechanism, because pravadoline-induced antinociception was not antagonized by naloxone (1 mg/kg s.c.) and pravadoline did not bind to opioid receptors at concentrations up to 10 microM . However, like the opioid analgesics, pravadoline diminished the electrically induced twitch response of mouse vas deferens preparations, but, in contrast to opioids, this action of pravadoline was not attenuated by naloxone . The possibility is discussed that this effect of pravadoline upon isolated tissues may contribute to its antinociceptive activity . In contrast to NSAIDs, pravadoline was more potent ex vivo as an inhibitor of the formation of PGs in brain vs . stomach . In addition, pravadoline failed to produce gastrointestinal lesions when administered p.o . to rats or mice, and did not possess significant anti-inflammatory activity at antinociceptive doses . Also unlike NSAIDs, pravadoline inhibited rat gastrointestinal transit when administered at doses similar to those which were antinociceptive . The overall pharmacologic profile of pravadoline suggests that the compound may be capable of managing more diverse or more severe pain than is achieved by anti-inflammatory analgesics, without producing side effects commonly associated with either the opioid or the nonopioid analgesics.

Am Surg, 1990 Nov, 56(11), 688 - 90
An objective appraisal of the role of computed tomographic (CT) guided drainage of intra-abdominal abscesses; Lent WM et al.; Computed tomographic (CT) guided drainage is an important tool in the treatment of intra-abdominal abscess . Its most important role is in the treatment of small, unilocular, well-placed abscesses . Success rates in our experience diminish considerably in abscesses involving necrotic tumors or those infected with yeast . As is frequently characteristic of new technologic procedures, the initial evaluation of the success rate of the procedure is overly optimistic . The procedure carries a considerable complication rate (13%) and mortality rate (15%) . Most importantly, success is usually evident early; within the first 24 to 48 hours . After this length of time, careful evaluation to consider further treatment should be contemplated.

Nature, 1990 Nov 1, 348(6296), 76 - 80
Cloning of a mitogen-inducible gene encoding a kappa B DNA-binding protein with homology to the rel oncogene and to cell-cycle motifs; Bours V et al.; We have cloned and characterized a mitogen-inducible gene isolated from human T cells that predicts a protein of 968 amino acids . The amino-terminal domain has regions homologous to the oncogene rel and to the developmentally important gene dorsal of Drosophila . The carboxy-terminal domain contains repeat structures found in a variety of proteins that are involved in cell-cycle control of yeast and in tissue differentiation in Drosophila and Ceanorhabditis elegans, as well as in the putative human oncogene bcl-3 and in the ankyrin protein . A truncated form of the product of this gene translated in vitro is a DNA-binding protein which interacts specifically with the kappa B binding site found in many inducible genes, including the enhancer in human immunodeficiency virus . This gene is yet another in a growing list of important regulatory molecules whose expression is transcriptionally induced upon cellular activation.

Mol Cell Biol, 1990 Nov, 10(11), 5914 - 20
An amino-terminal c-myc domain required for neoplastic transformation activates transcription; Kato GJ et al.; The product of the c-myc proto-oncogene is a nuclear phosphoprotein whose normal cellular function has not yet been defined . c-Myc has a number of biochemical properties, however, that suggest that it may function as a potential regulator of gene transcription . Specifically, it is a nuclear DNA-binding protein with a short half-life, a high proline content, segments that are rich in glutamine and acidic residues, and a carboxyl-terminal oligomerization domain containing the leucine zipper and helix-loop-helix motifs that serve as oligomerization domains in known regulators of transcription, such as C/EBP, Jun, Fos, GCN4, MyoD, E12, and E47 . In an effort to establish that c-Myc might regulate transcription in vivo, we sought to determine whether regions of the c-Myc protein could activate transcription in an in vitro system . We report here that fusion proteins in which segments of human c-Myc are linked to the DNA-binding domain of the yeast transcriptional activator GAL4 can activate transcription from a reporter gene linked to GAL4-binding sites . Three independent activation regions are located between amino acids 1 and 143, a region that has been shown to be required for neoplastic transformation of primary rat embryo cells in cooperation with a mutated ras gene . These results demonstrate that domains of the c-Myc protein can function to regulate transcription in a model system and suggest that alterations of Myc transcriptional regulatory function may lead to neoplastic transformation.

J Photochem Photobiol B, 1990 Nov, 7(2-4), 231 - 50
Phototumorigenesis studies of 5-methoxypsoralen in bergamot oil: evaluation and modification of risk of human use in an albino mouse skin model; Young AR et al.; The skin of the female hairless albino mouse (Skh 1) was used to study the enhancement of solar simulated radiation (SSR) tumorigenesis by 5-methoxypsoralen (5-MOP) in model perfumes that contain bergamot oil . This work was done in association with yeast mutagenicity studies and human skin phototoxicity studies . Analyses of time-to-onset of tumour observation with 5-MOP at 0, 5, 15 and 50 ppm show a highly significant 5-MOP dose effect and the data indicate that 5-MOP has phototumorigenic potential even at 5 ppm . The addition of 0.5% UVB and 0.5% UVA sunscreens significantly reduces the tumorigenicity associated with the vehicle (i.e . 5-MOP at 0 ppm) and 5-MOP at all concentrations . Pairwise comparisons of 5-MOP (at 5 or 15 ppm) plus sunscreens with vehicle plus sunscreens show that the sunscreens afford total protection at the lower 5-MOP concentrations . Additional studies show that a 5-6 h delay between 5-MOP application and SSR exposure defers the time-to-onset of tumours as does intermittent 5-MOP and SSR treatment . A comparison of 5-MOP at 50 ppm in bergamot oil with 5-MOP at 50 ppm prepared from pure 5-MOP crystals shows identical results, indicating that the active phototumorigenic agent in bergamot oil is 5-MOP and not other related compounds, which may be present at greater concentrations . Analyses of tumour histology at death show, in general, similar patterns of malignancy for all groups . Thus although it is possible to delay tumorigenesis by various strategies, the tumours that eventually develop are just as likely to be malignant, if not more so, when compared with non-delayed groups.

Eur J Clin Microbiol Infect Dis, 1990 Nov, 9(11), 832 - 5
Inactivity of terbinafine in a rat model of pulmonary aspergillosis; Schmitt HJ et al.; In a model of bronchopulmonary aspergillosis terbinafine did not improve survival of experimental animals in doses up to 80 mg/kg/day despite adequate lung concentrations . Pretreatment and aerosolization of the compound were also ineffective . Terbinafine was markedly less active in vitro when serum was used instead of Yeast-Nitrogen-Glucose-broth . It is concluded that a lack of bioavailability in the presence of serum may explain the lack of activity of terbinafine in experimental aspergillosis.

EMBO J, 1990 Nov, 9(11), 3527 - 32
Cotranslational glycosylation of proteins in systems depleted of protein disulphide isomerase; Bulleid NJ et al.; The role of protein disulphide isomerase (PDI) and other resident proteins of the endoplasmic reticulum (ER) lumen in co- and post-translational modification of secretory proteins has been studied in experiments on translation in vitro . We have devised procedures for extracting the lumenal content proteins of dog pancreas microsomal vesicles by alkaline buffer, or detergent washing, and for reconstitution of the depleted membrane fraction . When microsomal membranes are depleted of content by washing at pH 9.1, they are able to co-translationally glycosylate human interferon-gamma (IFN-gamma) and yeast pro-alpha-factor and the products appear to be identical to those produced by control microsomes . However, when microsomal membranes are depleted of content by washing with saponin they are still able to co-translationally translocate and glycosylate human IFN-gamma, but the products were of higher apparent Mr than those generated by control microsomes . When saponin-washed microsomal membranes were reconstituted with homogeneous protein disulphide isomerase (PDI), the generated vesicles gave the same pattern of co-translationally glycosylated IFN-gamma as saponin-washed microsomal membranes lacking PDI . These results are discussed in relation to the roles of resident ER proteins in co-translational modification; they suggest that PDI is not an essential component of the machinery of co-translational N-glycosylation, but that detergent washing may inactivate or remove some ER glycosidases.

Mycoses, 1990 Nov-Dec, 33(11-12), 513 - 7
Cutaneous sporotrichosis in Thailand: first reported case; Kwangsukstith C et al.; A case of cutaneous sporotrichosis is reported for the first time in Thailand . The infection occurred in a 33-year-old Thai female who has been in good health and had no history of previous trauma or contact with any animals . Histopathology revealed pseudoepitheliomatous hyperplasia of the epidermis and a combination of granulomatous and pyogenic reactions in the dermis and subcutaneous tissue . Typical asteroid bodies (Splendore-Hoeppli phenomenon) with central yeast cells were seen . Sporothrix schenckii was recovered from skin biopsy specimens . The patient responded well to the treatment with saturated solutions of potassium iodide within three months . No recurrence was seen after more than six months follow-up.

Mol Biol (Mosk), 1990 Nov-Dec, 24(6), 1675 - 8
{Organization of genes of ribosomal RNA from the mushroom Verticillium dahliae}; Mukhamedov RS et al.; Using yeast probe, a complete ribosomal DNA unit from a plant pathogenic fungus, Verticillium dahliae, was cloned into a plasmid vector pTZ19R . Partial DNA sequence of the clones, when compared to the yeast ribosomal DNA sequence, allowed to establish the physical map of the fungal rDNA . The overall organization was shown to be similar to other fungal rDNAs previously known.

Infection, 1990 Nov-Dec, 18(6), 342 - 6
Legionellosis in patients with HIV infection; Bangsborg JM et al.; During the five-year period 1984-1988 we received 192 specimens from 180 patients infected with the human immunodeficiency virus (HIV) for investigation of Legionella infection . The majority of specimens were bronchoalveolar lavage (BAL) fluids (84%), but tracheal suctions and lung tissue from autopsies were also examined . The diagnostic methods used were a direct immunofluorescence assay (DFA) for the detection of Legionella antigen, and culture on buffered charcoal yeast extract (BCYE-alpha) media . All specimens were also examined for the presence of other bacterial lung pathogens, and all BAL specimens additionally for Pneumocystis carinii and mycobacteria . Legionellosis was not found to be common among HIV-infected patients, as only six specimens (3%) from six patients were found positive by DFA, and no specimens were culture-positive for Legionella species . Dual infection with Legionella and P . carinii occurred in two patients . Clinical data of the six patients are presented, and currently used methods for diagnosing legionellosis are discussed.

Biochem Biophys Res Commun, 1990 Oct 30, 172(2), 371 - 6
The difference in murine CDC2 kinase activity between cytoplasmic and nuclear fractions during the cell cycle; Yasuda H et al.; The mouse analog of yeast CDC2+ kinase was detected in the cytoplasmic and nuclear fractions of cultured mouse FM3A cells . Its activity in the nuclear fraction increased in the G2/M phase became seven times higher than that in the G1/S phase, while the activity in the cytoplasmic fraction remained was almost constant from the G1/S to G1 phases . The activity in the cytoplasmic fraction was similar to that in the nuclear fraction in the G2/M phase . The amount of the enzyme remained almost constant during the cell cycle in both the nuclear and cytoplasmic fractions . These findings suggest that the cytoplasmic enzyme might play an independent role in the cell cycle.

J Biol Chem, 1990 Oct 25, 265(30), 18621 - 7
The calcium-binding site of clathrin light chains; Nathke I et al.; Clathrin light chains are calcium-binding proteins (Mooibroek, M . J., Michiel, D . F., and Wang, J . H . (1987) J . Biol . Chem . 262, 25-28) and clathrin assembly can be modulated by calcium in vitro . Thus, intracellular calcium may play a regulatory role in the function of clathrin-coated vesicles . The structural basis for calcium's influence on clathrin-mediated processes has been defined using recombinant deletion mutants and isolated fragments of the light chains . A single calcium-binding site, formed by residues 85-96, is present in both mammalian light chains (LCa and LCb) and in the single yeast light chain . This sequence has structural similarity to the calcium-binding EF-hand loops of calmodulin and related proteins . In mammalian light chains, the calcium-binding sequence is flanked by domains that regulate clathrin assembly and disassembly.

Biochim Biophys Acta, 1990 Oct 23, 1087(2), 226 - 34
Glutaminyl-tRNA synthetase as a component of the high-molecular weight complex of human aminoacyl-tRNA synthetases . An immunological study; Schray B et al.; The human glutaminyl-tRNA synthetase is three times larger than the corresponding bacterial and twice as large as the yeast enzyme . It is possible that the additional sequences of the human glutaminyl-tRNA synthetase are required for the formation of the multienzyme complex which is known to include several of aminoacyl-tRNA synthetases in mammalian cells . To address this point we prepared antibodies against three regions of the human glutaminyl-tRNA synthetase, namely against its enzymatically important core region, and against two sections in its large C-terminal extension . In intact multienzyme complexes the core region was accessible to specific antibody binding . However, the C-terminal sections became available to specific antibody binding only when certain components of the multienzyme complex were either absent or degraded . These findings allow first conclusions as to the relative position of some components in the mammalian aminoacyl-tRNA synthetase complex.

Blood, 1990 Oct 15, 76(8), 1514 - 20
The pharmacokinetics of plasminogen activator inhibitor-1 in the rabbit; Mayer EJ et al.; The pharmacokinetics of the activated and latent forms of plasminogen activator inhibitor-1 (PAI-1) isolated from HT1080 fibrosarcoma cells (HT1080 PAI-1) and a nonglycosylated form of human PAI-1 isolated from a yeast expression system (rPAI-1) were followed in the rabbit . As assessed by an immunologic assay specific for human PAI-1, guanidine HCI activated HT1080 PAI-1 and rPAI-1 entered the total plasma volume following intravenous bolus administration and exhibited a biphasic clearance pattern . The t1/2s of HT1080 PAI-1 for the initial and beta phases equalled 6.0 and 24.8 minutes, respectively . The t1/2s of rPAI-1 for the initial and beta phases equalled 8.8 and 34.0 minutes, respectively . Similar results were obtained by measuring PAI-1 activity in plasma and with trace amounts of 125I-rPAI-1, suggesting that the above pharmacokinetic behavior could also apply to endogenous PAI-1 . The liver was the main site of rPAI-1 clearance . Unactivated, latent PAI-1 exhibited a very different pharmacokinetic profile . Over 80% of latent rPAI-1 cleared from the circulation within 10 minutes (t1/2 = 1.7 minutes) . The difference in clearance behavior between activated and latent PAI-1 may be related to the ability of activated PAI-1, but not latent PAI-1, to rapidly form high-molecular-weight complexes with plasma binding factors which were observed in vitro and in vivo . Because PAI-1 could potentially tilt the fibrinolytic balance toward a prothrombotic state, its rapid clearance may represent an important control mechanism governing the circulating levels of this key component of the fibrinolytic pathway.

Gene, 1990 Oct 15, 94(2), 165 - 71
Two ubiquitin-long-tail fusion genes arranged as closely spaced direct repeats in barley; Gausing K et al.; Ubiquitin (Ubi) genes encode two types of fusion proteins: polyUbi with a varying number of direct repeats of Ubi, and Ubi-tail fusions with long or short basic C-terminal extensions . A barley (Hordeum vulgare) genomic clone has been isolated with two very similar, intronless genes encoding monoUbi-long-tail fusion peptides . The genes are arranged as direct repeats separated by 3 kb of DNA and account for two of the probable three long-tail genes in the haploid barley genome . Both genes are active and give rise to messengers about 800 nt long . The sequence of the encoded Ubi moieties is identical to the sequence of Ubi repeats of polyUbi precursors from barley and other plants . The basic tails of the peptides are 79 aa long and 71-72% homologous to corresponding sequences from yeast and man . Recently, it was found that the long and short tails are ribosomal proteins in yeast {Finley et al., Nature 338 (1989) 394-401} and the evolutionary conservation of the structure of the Ubi-tail fusion genes suggests that they serve the same function in plants . The similarity between yeast and barley Ubi-long-tail fusion genes may extend to the regulatory regions, since upstream activating sites characteristic of ribosomal protein-encoding genes in yeast (UASrpg) were found in the barley genes.

Science, 1990 Oct 12, 250(4978), 267 - 71
A plant leucine zipper protein that recognizes an abscisic acid response element; Guiltinan MJ et al.; The mechanism by which phytohormones, like abscisic acid (ABA), regulate gene expression is unknown . An activity in nuclear extracts that interacts with the ABA response element (ABRE) from the 5' regulatory region of the wheat Em gene was identified . A complementary DNA clone was isolated whose product is a DNA binding protein (EmBP-1) that interacts specifically with an 8-base pair (bp) sequence (CACGTGGC) in the ABRE . A 2-bp mutation in this sequence prevented binding of EmBP-1 . The same mutation reduced the ability of the ABRE to confer ABA responsiveness on a viral promoter in a transient assay . The 8-bp EmBP-1 target sequence was found to be conserved in several other ABA-responsive promoters and in promoters from plants that respond to signals other than ABA . Similar sequences are found in promoters from mammals, yeast, and in the major late promoter of adenovirus . The deduced amino acid sequence of EmBP-1 contains conserved basic and leucine zipper domains found in transcription factors in plants, yeast, and mammals . EmBP-1 may be a member of a highly conserved family of proteins that recognize a core sequence found in the regulatory regions of various genes that are integrated into a number of different response pathways.

J Biol Chem, 1990 Oct 5, 265(28), 16940 - 7
Purification and characterization of growth-associated H1 histone kinase from Novikoff hepatoma cells; Chambers TC et al.; Growth-associated H1 histone kinase, a homolog of the yeast cdc2+/CDC28 protein kinases that control entry into mitosis, is a chromatin-bound cyclic nucleotide-independent enzyme found only in growing cells . In a procedure involving salt extraction of chromatin, ammonium sulfate precipitation, and three chromatographic steps, the enzyme has been purified greater than 10,000-fold from Novikoff hepatoma cells . Enzyme purified by this procedure catalyzes the transfer to H1 histone of 2.7 mumol of phosphate/min/mg, a specific activity within the range of those reported for a number of homogeneous or nearly homogeneous protein kinases . Further purification to near homogeneity was achieved by an additional step of sucrose density gradient fractionation . Enzyme activity in the sucrose gradient is associated with two polypeptides of apparent Mr 60,000 and 33,000 on sodium dodecyl sulfate-gel electrophoresis . Substrate specificity studies show that in addition to H1, proteins with H1-like structure and function including histone H1(0), the erythrocyte-specific H5 histone, and the testis-specific H1t histone are phosphorylated . Nucleosome core histone H3, high mobility group proteins 1, 2, 14, and 17, protamine, casein, and ribosomal protein S6 are not substrates.

Biologicals, 1990 Oct, 18(4), 345 - 50
Report of a collaborative study for assessing the potency of hepatitis B vaccines; Ferguson M et al.; A collaborative study was carried out to examine the suitability of a hepatitis B vaccine derived from plasma as an immunogenicity reference for vaccines produced by recombinant DNA technology in yeast . The use of a plasma derived vaccine as reference appeared satisfactory, although the use of homologous reference improved agreement in potency estimates . The use of a recombinant standard did not however improve agreement for a recombinant vaccine produced by a different manufacturer . The variation in the dilution of vaccine required to induce antibodies in 50% of test animals and in potency estimates varied widely between laboratories (25-fold and 10-fold respectively) . However this was similar to the variation found in a previous collaborative study.

Appl Environ Microbiol, 1990 Oct, 56(10), 2951 - 6
Influence of water activity and temperature on survival of and colony formation by heat-stressed Chrysosporium farinicola aleuriospores; Beuchat LR et al.; The ability of sublethally heat-stressed aleuriospores of Chrysosporium farinicola to form colonies on yeast extract-glucose agar (YGA) supplemented with sufficient glucose, sorbitol, glycerol, and NaCl to achieve reduced water activity (aw) in the range of 0.88 to 0.95 was determined . The effects of the aw of diluent and incubation temperature during recovery and colony formation were also investigated . Aleuriospores harvested from 14-day-old cultures grown at 25 degrees C were less resistant to heat inactivation compared with aleuriospores from 20-day-cultures . Increased populations of heat-stressed aleuriospores were recovered as the aw of YGA was decreased from 0.95 (glucose and glycerol) and 0.94 (sorbitol) to 0.89 and 0.88, respectively . In NaCl-supplemented YGA, populations recovered at an aw of 0.94 were greatly reduced compared with populations detected at an aw of 0.92; no colonies were formed on NaCl-supplemented YGA at an aw of 0.88 . Tolerance to aw values above 0.88 to 0.89 as influenced by solute type was in the order of glucose greater than sorbitol greater than glycerol greater than NaCl . Incubation at 20 degrees generally resulted in an increase in recoverable aleuriospores compared with incubation at 25 degrees C or at 30 degrees C for 14 days followed by 20 degrees C for 10 days . The lethal effect of NaCl on heat-stressed aleuriospores was enhanced at 30 degrees C . The retention of viability of aleuriospores held in sucrose-peptone water diluent (aw, 0.936) for 20 min was essentially the same as that observed when aleuriospores were held in peptone water (aw, 0.997).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Cell Biol, 1990 Oct, 68(10), 1151 - 65
Boehringer Mannheim Award lecture . Phosphatidylcholine metabolism: masochistic enzymology, metabolic regulation, and lipoprotein assembly; Vance DE; Phosphatidylcholine is apparently essential for mammalian life, since there are no known inherited diseases in the biosynthesis of this lipid . One of its critical roles appears to be in the structure of the eucaryotic membranes . Why phosphatidylcholine is required and why other phospholipids will not substitute are unknown . The major pathway for the biosynthesis of phosphatidylcholine occurs via the CDP-choline pathway . Choline kinase, the initial enzyme in the sequence, has been purified to homogeneity from kidney and liver and also catalyzes the phosphorylation of ethanolamine . Most evidence suggests that the next enzyme in the pathway, CTP:phosphocholine cytidylyltransferase, catalyzes the rate-limiting and regulated step in phosphatidylcholine biosynthesis . This enzyme has also been completely purified from liver . Cytidylyltransferase appears to exist in the cytosol as an inactive reservoir of enzyme and as a membrane-bound form (largely associated with the endoplasmic reticulum), which is activated by the phospholipid environment . There is evidence that the activity of this enzyme and the rate of phosphatidylcholine biosynthesis are regulated by the reversible translocation of the cytidylyltransferase between membranes and cytosol . Three major mechanisms appear to govern the distribution and cellular activity of this enzyme . (i) The enzyme is phosphorylated by cAMP-dependent protein kinase, which results in release of the enzyme into the cytosol . Reactivation of cytidylyltransferase by binding to membranes can occur by the action of protein phosphatase 1 or 2A . (ii) Fatty acids added to cells in culture or in vitro causes the enzyme to bind to membranes, where it is activated . Removal of the fatty acids dissociates the enzyme from the membrane . (iii) Perhaps most importantly, the concentration of phosphatidylcholine in the endoplasmic reticulum feedback regulates the distribution of cytidylyltransferase . A decrease in the level of phosphatidylcholine causes the enzyme to be activated by binding to the membrane, whereas an increase in phosphatidylcholine mediates the release of enzyme into the cytosol . The third enzyme in the CDP-choline pathway, CDP-choline:1,2-diacylglycerol choline-phosphotransferase, has been cloned from yeast but never purified from any source . In liver an alternative pathway for phosphatidylcholine biosynthesis is the methylation of phosphatidylethanolamine by phosphatidylethanolamine N-methyltransferase . This enzyme is membrane bound and has been purified to homogeneity . It catalyzes all three methylation reactions involved in the conversion of phosphatidylethanolamine to phosphatidylcholine.(ABSTRACT TRUNCATED AT 400 WORDS)

Trends Biochem Sci, 1990 Oct, 15(10), 374 - 6
Histone deletion mutants challenge the molecular clock hypothesis; Behe MJ; A basic tenet of the molecular clock hypothesis is that the rate of sequence drift for a protein depends on the number of amino acid residues that are critical for its function . However, recent experiments have determined that, although core histone sequences are highly conserved among eukaryotes, large regions of the proteins are dispensable for growth in yeast.

Inflammation, 1990 Oct, 14(5), 471 - 83
Antiinflammatory reactivity of copper(I)-thionein; Miesel R et al.; In unseparated human blood the reactivity of yeast copper (I)-thionein on TPA-activated polymorphonuclear leukocytes was evaluated and compared with low Mr copper chelates exerting Cu2Zn2 superoxide dismutase mimetic activity . Cu, 18 microM, in the form of Cu-thionein was sufficient to inhibit the superoxide production of activated human blood phagocytes by 50% . Furthermore, the scavenging of hydroxyl radicals and singlet oxygen by Cu(I)-thionein was determined, using the 2-deoxyribose fragmentation assay induced by decaying K3CrO8 and the NADPH oxidation caused by UVA illuminated psoralen, respectively . The inhibitory reactivity of Cu-thionein in both assays was compared with that of serum proteins including albumin, ceruloplasmin, transferrin, and ferritin . The galactosamine/endotoxin-induced hepatitis in male NMRI mice was used to evaluate the antiinflammatory reactivity of Cu-thionein in vivo . The serum copper, superoxide dismutase, and sorbitol dehydrogenase concentrations, as well as the activity of polymorphonuclear leukocytes in unseparated blood seemed most appropriate to quantify the protective capacity of Cu-thionein in the course of an oxidative stress-dependent liver injury . The intraperitoneal application of 32.5 mumols/kg thionein-Cu limited this damage to 45%.

Ethiop Med J, 1990 Oct, 28(4), 155 - 61
Preliminary studies on antipyretic and analgesic properties of Taverniera abyssinica; Dagne E et al.; In an attempt to ascertain the pharmacological basis of the use of the marketed traditional drug Taverniera abyssinica A . Rich . (Amharic name Dingetegna), crude extracts as well as purified substances of this plant were tested for their antipyretic and analgesic properties . Antipyretic activity was determined on rats made hyperthermic by yeast injection and analgesic activity was determined by the hot plate, as well as the acetic acid induced writhing, methods . The study showed that the plant possesses significant antipyretic and analgesic activities.

J Allergy Clin Immunol, 1990 Oct, 86(4 Pt 1), 503 - 11
Characteristics of patients with food-related complaints; Parker SL et al.; Forty-five patients with classic food-allergic symptoms and/or subjective food-related complaints not traditionally associated with food allergy underwent evaluation . On the basis of a comprehensive clinical history, skin testing, and placebo-controlled, double-blind food challenges, patients were assigned to one of two groups: patients with reactions highly suggestive of IgE-mediated food hypersensitivity (group A, N = 22) and patients with atypical adverse food reactions that could not be confirmed by double-blind food challenge (group B, N = 23) . Most patients in both groups were female, 77.3% and 91.3% of patients in group A and B, respectively . In group B, onset of symptoms occurred at an older age than in group A, 28.9 years +/- 17.2 versus 17.1 +/- 12.1 (p = 0.0015), respectively, and involved more foods, 25.6 +/- 22.1 versus 5.2 +/- 5.5 (p = 0.0002) . Foods causing most prominent symptoms among patients in group A included legumes, tree nuts, crustaceans, and fish . In group B, milk, white sugar, wheat, egg, smoked/cured meat, and yeast were among the most troublesome foods . All but one patient in group A gave a positive skin test response to food; only four patients in group B had a positive response . We conclude that a subset of patients with food-related complaints can be accurately predicted to have a negative double-blind challenge with suspected foods on the basis of information obtained by history and skin testing.

Proc Natl Acad Sci U S A, 1990 Oct, 87(19), 7673 - 7
The gamma subunit of transducin is farnesylated; Lai RK et al.; Protein prenylation with farnesyl or geranylgeranyl moieties is an important posttranslational modification that affects the activity of such diverse proteins as the nuclear lamins, the yeast mating factor mata, and the ras oncogene products . In this article, we show that whole retinal cultures incorporate radioactive mevalonic acid into proteins of 23-26 kDa and one of 8 kDa . The former proteins are probably the "small" guanine nucleotide-binding regulatory proteins (G proteins) and the 8-kDa protein is the gamma subunit of the well-studied retinal heterotrimeric G protein (transducin) . After deprenylating purified transducin and its subunits with Raney nickel or methyl iodide/base, the adducted prenyl group can be identified as an all-trans-farnesyl moiety covalently linked to a cysteine residue . Thus far, prenylation reactions have been found to occur at cysteine in a carboxyl-terminal consensus CAAX sequence, where C is the cysteine, A is an aliphatic amino acid, and X is undefined . Both the alpha and gamma subunits of transducin have this consensus sequence, but only the gamma subunit is prenylated . Therefore, the CAAX motif is not necessary and sufficient to direct prenylation . Finally, since transducin is the best understood G protein, both structurally and mechanistically, the discovery that it is farnesylated should allow for a quantitative understanding of this post-translational modification.

J Cell Biol, 1990 Oct, 111(4), 1419 - 26
Predominance of clathrin light chain LCb correlates with the presence of a regulated secretory pathway; Acton SL et al.; Two forms of clathrin light chains, LCa and LCb, are expressed in all mammalian and avian tissues that have been examined, whereas only one type is found in yeast . Regions of structural dissimilarity between LCa and LCb indicate possible functional diversity . To determine how LCa and LCb might differentially influence clathrin function, light chain expression patterns and turnover were investigated . Relative expression levels of the two light chains were determined in cells and tissues with and without a regulated secretory pathway . LCa/LCb ratios ranged from 5:1 to 0.33:1 . A higher proportion of LCb was observed in cells and tissues that maintain a regulated pathway of secretion, suggesting a specialized role for the LCb light chain in this process . The ratio of light chains in assembled clathrin was found to reflect the levels of total light chains expressed in the cell, indicating no preferential incorporation into triskelions or coated vesicles . The half-lives of LCa, LCb, and clathrin heavy chain were determined to be 24, 45, and 50 h, respectively . Thus, LCa is turned over independently of the other subunits . However, the half-lives of all three subunits are sufficiently long to allow triskelions to undergo many rounds of endocytosis, minimizing the possibility that turnover contributes to regulation of clathrin function . Rather, differential levels of LCa and LCb expression may influence tissue specific clathrin regulation, as suggested by the predominance of LCb in cells maintaining a regulated secretory pathway.

EMBO J, 1990 Oct, 9(10), 3201 - 8
Purified presequence binding factor (PBF) forms an import-competent complex with a purified mitochondrial precursor protein; Murakami K et al.; In vitro mitochondrial import of the purified precursor form (pOTC) of rat ornithine carbamoyltransferase (OTC) is stimulated by a cytosolic factor(s) contained in rabbit reticulocyte lysate . A protein factor that binds to pOTC but not to mature OTC and was named presequence binding factor or PBF, was purified 91,000-fold from the lysate by affinity chromatography using pOTC-bound Sepharose, DEAE-5PW HPLC and sucrose gradient centrifugation . The purified PBF migrated as a single polypeptide of 50,000 daltons on SDS-PAGE . On sucrose gradients, urea-denatured pOTC sedimented to the bottom, whereas PBF sedimented with an S20,w value of 5.5S . When pOTC and PBF were centrifuged together, both polypeptides sedimented as a complex of 7.1S . Formation of the pOTC-PBF complex was inhibited by micromolar concentrations of the synthetic presequence of pOTC and those of other mitochondrial precursor proteins . The purified PBF markedly stimulated the import of purified or in vitro synthesized pOTC into the mitochondria . PBF-stimulated pOTC import was further enhanced by a 70 kd heat shock protein (hsp 70) purified from yeast; the hsp70 alone had little effect . Thus, PBF binds to the presequence portion of the precursors and may hold them in a transport-competent form in cooperation with hsp70.

Mol Cell Biol, 1990 Oct, 10(10), 5473 - 85
The mouse c-rel protein has an N-terminal regulatory domain and a C-terminal transcriptional transactivation domain; Bull P et al.; We have shown that the murine c-rel protein can act as a transcriptional transactivator in both yeast and mammalian cells . Fusion proteins generated by linking rel sequences to the DNA-binding domain of the yeast transcriptional activator GAL4 activate transcription from a reporter gene linked in cis to a GAL4 binding site . The full-length mouse c-rel protein (588 amino acids long) is a poor transactivator; however, the C-terminal portion of the protein between amino acid residues 403 to 568 is a potent transcriptional transactivator . Deletion of the N-terminal half of the c-rel protein augments its transactivation function . We propose that c-rel protein has an N-terminal regulatory domain and a C-terminal transactivation domain which together modulate its function as a transcriptional transactivator.

FEBS Lett, 1990 Oct 1, 271(1-2), 215 - 8
Firefly luciferase synthesizes P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) and other dinucleoside polyphosphates; Guranowski A et al.; The synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) has been considered, for a long time, to be catalyzed mainly by some aminoacyl-tRNA synthetases {Brevet et al . (1989) Proc . Natl . Acad . Sci . USA 86, 8275-8279} . Recently, yeast Ap4A phosphorylase, acting in reverse (Guranowski et al . (1988) Biochemistry 27, 2959-2964), was shown to synthesize Ap4A, too . In the case of the synthetases, the intermediate complex E-aminoacyl-AMP may serve as donor of AMP to ATP, yielding Ap4A . Here we demonstrate that firefly luciferase (EC 1.13.12.7) which forms the E-luciferin-AMP intermediate also synthesizes Ap4A as well as other dinucleoside polyphosphates . We suggest moreover that: other enzymes (mainly synthetases and some transferases), which catalyze the transfer of a nucleotidyl moiety, via nucleotidyl-containing intermediates and releasing PPi may produce dinucleoside polyphosphates.

Proc Natl Acad Sci U S A, 1990 Oct, 87(19), 7414 - 8
Tumorigenic 3T3 cells maintain an alkaline intracellular pH under physiological conditions; Gillies RJ et al.; One of the earliest events in the response of mammalian cells to mitogens is activation of Na+/H+ exchange, which increases intracellular pH (pHin) in the absence of HCO3- or at external pH values below 7.2 . The proliferative response can be blocked by preventing the pHin increase; yet, the proliferative response cannot be stimulated by artificially raising pHin with weak bases or high medium pH . These observations support the hypothesis that optimal pHin is a necessary, but not sufficient, component of the proliferative-response sequence . This hypothesis has recently been challenged by the observation that transfection of NIH 3T3 cells with yeast H(+)-ATPase renders them tumorigenic . Although previous measurements indicated that these transfected cells maintain a higher pHin in the absence of HCO3-, whether H(+)-ATPase transfection raised the pHin under physiologically relevant conditions was not known . The current report shows that these transfected cells do maintain a higher pHin than control cells in the presence of HCO3-, supporting the possibility that elevated pHin is a proliferative trigger in situ . We also show that these cells are serum-independent for growth and that they glycolyze much more rapidly than phenotypically normal cells.

AIDS, 1990 Oct, 4(10), 967 - 73
Performance characteristics of a novel immunoassay based on hybrid Ty virus-like particles (Ty-VLPs): rapid differentiation between HIV-1 and HIV-2 infection; Gilmour JE et al.; Recombinant antigens containing all or parts of the HIV-1 proteins p24, Nef and gp41 and HIV-2 gp36 have been purified and used to develop a rapid immunoassay to detect and differentiate between HIV-1 and HIV-2 antibodies in a single test . The antigens were produced as particulate fusion proteins by exploiting the ability of a protein encoded by the yeast retrotransposon Ty to assemble into virus-like particles (Ty-VLPs) . Hybrid HIV: Ty-VLPs carrying each of the antigens were applied to nitrocellulose strips at specified locations in a slot-blot format and then used to detect antibodies present in human serum and plasma samples of diverse geographical origin . Previously confirmed HIV-1- and HIV-2-positive samples were readily and reliably identified . The assay was used to identify a case of HIV-2 infection in an African woman who had been resident in the Oxford region for the last 3 years and to analyse the prevalence of anti-HIV antibodies in a longitudinal study of seroconverting patients . We also demonstrate that the assay works efficiently with whole blood.

Microbiologica, 1990 Oct, 13(4), 343 - 6
"Giant cell" production by C . albicans cultured in xylitol; Ameglio F et al.; "Giant cell" production by C . albicans cultured in the presence of substances belonging to the pentitol family is described in this study . After two days of incubation, at pentitol concentrations ranging between 0.1 and 20 mg%, the production of very large, frequently multibudding cells became evident . Their number increased with time, in some cases reaching 20% of the culture cells and their size could exceed 25 mcm . Comparisons with other sugar/alcohols and with other Candida spp . are presented . A possible explanation of this observation is consistent with an accumulation of pentitol catabolites in the yeast cytoplasm followed by an increased osmotic strength and cell swelling.

Electrophoresis, 1990 Oct, 11(10), 893 - 4
A general-purpose pulsed field controller; Abbal P et al.; A pulsed field controller compatible with various types of electrophoresis tanks and power supplies is described . The system is based on a high-precision timing circuit, with an output isolated from the main high voltage supply, and four high voltage transistors . The switching time is programmed manually between 5-160 s . With a transverse alternating field electrophoresis system, intact yeast chromosomes could be completely separated after 24 h.

J Biomol Struct Dyn, 1990 Oct, 8(2), 413 - 30
Structural analysis of a group II intron by chemical modifications and minimal energy calculations; Kwakman JH et al.; Folding of the yeast mitochondrial group II intron aI5c has been analysed by chemical modification of the in vitro synthesised RNA with dimethylsulfate and diethylpyrocarbonate . Computer calculations of the intron secondary structure through minimization of free energy were also performed in order to study thermodynamic properties of the intron and to relate these to data obtained from chemical modification . Comparison of the two sets of data with the current phylogenetic model structure of the intron aI5 reveals close agreement, thus lending strong support for the existence of a typical group II intron core structure comprising six neighbouring stem-loop domains . Local discrepancies between the experimental data and the model structures have been analyzed by reference to thermodynamic properties of the structure . This shows that use of the latest refined set of free energy values improves the structure calculation significantly.

Eur J Biochem, 1990 Sep 24, 192(3), 777 - 81
Primary structure of profilins from two species of Echinoidea and Physarum polycephalum; Takagi T et al.; Profilin is a small G-actin-binding protein, the amino acid sequence of which was previously reported for calf, human, Acanthamoeba and yeast . Here the amino acid sequences of three profilins obtained from eggs of two species of Echinoidea, Clypeaster japonicus (order, Clypeasteroida) and Anthocidaris crassispina (order, Echinoida), and plasmodium of Physarum polycephalum were determined . Two echinoid profilins were composed of 139 amino acid residues, N-termini were acylated and the molecular mass was calculated to be 14.6 kDa, slightly larger than that of 13 kDa estimated by SDS/PAGE {Mabuchi, I . & Hosoya, H . (1982) Biomed . Res . 3, 465-476} . On the other hand, Physarum profilin was composed of 124 amino acid residues, the N-terminus was acylated, and the calculated molecular mass was 13132 Da . The sequences of C . japonicus and A . crassispina profilins were homologous (84% identical) . However, the similarity of these profilins with those form other organisms was low . The sequence of Physarum profilin was homologous with Acanthamoeba profilin isoforms (51% identical) and with yeast profilin (42% identical), but not with other profilins . The relatively conservative sequence of profilins from yeast, Physarum, Acanthamoeba, echinoid eggs and mammalian cells was found in the N-terminal region, which was suggested to be a common actin-binding region . The C-terminal region was also conserved, although to a lesser extent than the N-terminal region.

Mol Cell Biochem, 1990 Sep 21, 97(2), 145 - 51
Preparation and characterization of monoclonal antibodies to human hexokinase type I; Chiarantini L et al.; 1 . Three different immunization protocols and several screening procedures were used to prepare seven mouse monoclonal antibodies to human placenta hexokinase type I . None of these monoclonals were able to recognize the native enzyme but all detected hexokinase when adsorbed onto polystyrene plates or on immunoblots after SDS/polyacrylamide-gel electrophoresis . 2 . All seven monoclonals recognize the two different subtypes of human hexokinase I equally well . Limited tryptic digestion of hexokinase followed by Western blotting and immunodetection show that these monoclonals recognize epitopes that lie in different tryptic peptides . 3 . Comparative ELISA studies showed that human hexokinase types I and II have great immunological similarities while hexokinase I from different mammalian species and yeast hexokinase are recognized with different affinities.

Biochemistry, 1990 Sep 18, 29(37), 8696 - 701
Structurally unique plant cytochrome c oxidase isolated from wheat germ, a rich source of plant mitochondrial enzymes; Peiffer WE et al.; Purification and characterization of plant cytochrome c oxidases have been impeded by the difficulty of obtaining enough plant mitochondria . We have found commercial wheat germ to be a rich and convenient source of mitochondrial membranes containing respiratory chain complexes in ratios and amounts similar to mitochondria prepared from etiolated seedlings . Cytochrome c oxidase was purified from these membranes by anion-exchange (MonoQ) fast protein liquid chromatography . The enzyme is highly active (turnover number up to 1000 s-1) and exhibits biphasic cytochrome c reaction kinetics similar to those of beef heart oxidase . As with other plant oxidases, the visible spectrum of wheat germ oxidase in the reduced form is blue-shifted compared to other eukaryotic cytochrome oxidases, with peaks at 441 and 602 nm . The electron paramagnetic resonance spectrum of CuA of the wheat germ enzyme is very similar to that of the maize and beef heart enzymes, suggesting that the copper environment is not altered . Sodium dodecyl sulfate-polyacrylamide gels show a subunit composition in which subunits I-IV resemble those of the yeast enzyme in size and antigenicity, while three to four smaller peptides are dissimilar to yeast and other eukaryotic oxidases . A difference between the subunit composition of the wheat germ and wheat seedling enzymes suggests the existence of a developmental or tissue-specific form of cytochrome oxidase in plants.

Biochem Pharmacol, 1990 Sep 15, 40(6), 1299 - 305
Metabolism of oltipraz and glutathione reductase inhibition; Moreau N et al.; A decrease in glutathione reductase (GR) activity was observed in Schistosoma mansoni isolated from oltipraz(OPZ)-treated mice . Yeast and Schistosoma mansoni GR-activity was inhibited by OPZ derivatives only . These OPZ-derivatives showed in vitro schistosomicidal activity . Using yeast GR and dithiolium salts of OPZ, time-dependent inactivation and gel chromatography experiments revealed irreversible inhibition dependent on the redox state of the enzyme . Binding of radiolabelled ({3H}7-methyl-8-methylthio-pyrrolo{1,2-a}pyrazine disulphide 1b) obtained from OPZ was observed using exclusion chromatography and equilibrium dialysis . These results indicate that GR can be considered as the target of schistosomicidal activity of OPZ . The lack of inhibitory activity of OPZ and dithiole-thione analogues, and the potent activity of the corresponding pyrrolo-pyrazine derivatives, is consistent with the hypothesis that OPZ is a pro-drug.

Experientia, 1990 Sep 15, 46(9), 975 - 7
Isolation and characterization of a new hemagglutinin from the red alga Gracilaria bursa-pastoris; Okamoto R et al.; A new agglutinin has been isolated from the red alga Gracilaria bursa-pastoris by affinity chromatography on a yeast mannan-Cellulofine column . This agglutinin was isolated as a monomeric glycoprotein with a relatively low molecular weight . It had an isoelectric point of 4.7 and contained large amounts of Gly, Asx and Glx . It agglutinated trypsin-treated rabbit erythrocytes at the low concentration of 30 ng/ml . The activity was inhibited only by glycoproteins bearing N-glycans . This agglutinin also showed mitogenic activity for mouse splenic lymphocytes.

Biochem Biophys Res Commun, 1990 Sep 14, 171(2), 676 - 83
cDNA cloning and sequencing of component C8 of proteasomes from rat hepatoma cells; Tanaka K et al.; The primary structure of component C8 of rat proteasomes (multicatalytic proteinase complexes) has been determined by sequencing on isolated cDNA clone . C8 consists of 255 amino acid residues with a calculated molecular weight of 28,417 . These values are consistent with those obtained by protein chemical analyses . Computer-assisted homology comparison showed that C8 is a new protein, differing from all proteins reported so far . The overall amino acid sequence of C8 resembles those of most other components of proteasomes reported, such as components C2, C3 and C9 of rat proteasomes and certain components of other eukaryotic proteasomes, such as those of Drosophila and yeast, but shows little similarity with component C5 of rat proteasomes . C8 showed particularly close structural similarity to component YC1 of yeast proteasomes, suggesting that C8 has been highly conserved during evolution and functions ubiquitously in all eukaryotes.

Nucleic Acids Res, 1990 Sep 11, 18(17), 5077 - 81
Use of repetitive DNA probes as physical mapping strategy in Caenorhabditis elegans; Cangiano G et al.; A method for linking genomic sequences cloned in yeast artificial chromosomes (YACs) has been tested using Caenorhabditis elegans as a model system . Yeast clones carrying YACs with repeated sequences were selected from a C . elegans genomic library, total DNA was digested with restriction enzymes, transferred to nylon membranes and probed with a variety of repetitive DNA probes . YAC clones that overlap share common bands with one or more repetitive DNA probes . In 159 YAC clones tested with one restriction enzyme and six probes 28 overlapping clones were detected . The advantages and limitations of this method for construction of YAC physical maps is discussed.

Biochim Biophys Acta, 1990 Sep 10, 1087(1), 31 - 8
Class II ribonuclease H comigrates with, but is distinct from, the third largest subunit of calf thymus RNA polymerase I; Vonwirth H et al.; It has been reported (Iborra et al . (1979) J . Biol . Chem . 254, 10920-10924) that the third and the fifth largest subunit of yeast RNA polymerase I exhibit ribonuclease H activity . The authors suggested that the third largest subunit is identical with the chromatin-associated ribonuclease H49, the putative yeast equivalent of bovine ribonuclease H IIb . Although the third largest subunit of calf thymus RNA polymerase I and ribonuclease H IIb display nearly identical molecular masses under denaturing conditions, serological analysis reveals that, in contrast to their counterparts in yeast, these mammalian proteins are distinct entities . Interestingly, sera from some patients with mixed connective tissue disease which contain antibodies directed against RNA polymerase I, also react with ribonuclease H IIb epitopes . This observation suggests that a protein displaying ribonuclease H IIb antigenicity could be associated with RNA polymerase I . Additional indications supporting this conclusion are discussed.

J Biol Chem, 1990 Sep 5, 265(25), 14922 - 31
Genomic organization and chromosomal localization of the human nucleolin gene; Srivastava M et al.; Nucleolin, a eukaryotic nucleolar phosphoprotein, is involved in the synthesis and maturation of ribosomes . To characterize the genomic organization and regulatory sequences of this gene, two overlapping lambda clones containing the human nucleolin gene plus flanking regions were isolated from a genomic library using human nucleolin cDNA . Southern blots of genomic DNA from human, several mammals, chicken, and yeast revealed that the nucleolin gene is well conserved across these species . The gene consists of 14 exons with 13 intervening sequences and spans approximately 11 kilobases of DNA . Analysis of the splice junctions indicated that the amino-terminal domain and the four RNA binding domains plus the nuclear localization signal are split into adjacent exons . Sequences from the 5'-flanking and the first intron contain a high content of GC residues which is consistent with nucleolin being a "housekeeping" gene . Promoter elements include an atypical TATA box (GTTA), one CCAAT box much further from the initiation site, three reverse compliments of CCAAT (ATTGG), and two pyrimidine-rich nucleotide stretches . In addition, this region and the first intron contain numerous potential Sp1, GCF, CRE-fos, GCN, AP-1, AP-2, UCE, and sequences similar to the glucocorticoid receptor binding site . The transcription start site was determined by primer extension and S1 nuclease mapping of RNA from human liver . One Kpn and three Alu repeats were found within two of the middle introns . The 3'-untranslated portion of the gene contains five homology blocks in a 100-base pair region that are highly conserved among human, mouse, and hamster genomes . Finally, we have determined that the human nucleolin gene is located on chromosome 2q12-qter and is present at one copy per haploid genome . A restriction fragment length polymorphism with EcoRI has been detected in the gene.

EMBO J, 1990 Sep, 9(9), 2923 - 9
Molecular cloning, primary structure and expression of the human X linked A1S9 gene cDNA which complements the ts A1S9 mouse L cell defect in DNA replication; Zacksenhaus E et al.; The temperature-sensitive ts A1S9 mutation of mouse L cells was previously shown to affect nuclear DNA replication and to be complemented by active and inactive human X chromosomes in human-ts A1S9 somatic cell hybrids . We report the isolation of cDNA clones which correct the ts A1S9 lesion, using as a probe a genomic fragment derived from the human A1S9 locus . The nucleotide sequence of the A1S9 cDNA encompasses a single open reading frame of 2409 bp which could encode a heretofore unreported protein of 90 393 daltons . Southern blot hybridization of the A1S9 cDNA probe with DNA from various species revealed homologous sequences in vertebrates but not in yeast . Northern blot analysis of serum-starved, synchronized cells demonstrated that the A1S9 gene was expressed at a relatively low level in quiescent cells and at a higher and constant level throughout the cell cycle . Human cell lines harbouring increasing numbers of inactive X chromosomes (47, XXX, 49, XXXXX) were found to express the A1S9 gene at the same level as control cells (45, X), suggesting that the gene does not escape X chromosome inactivation.

Zhonghua Zhong Liu Za Zhi, 1990 Sep, 12(5), 328 - 31
{Effects of selenium deficiency and supplementation on tumor immune response in mice}; Hu XZ; DBA/2 mice were fed for 16 weeks with Torula yeast-based synthetic diet containing various concentrations of selenium (Se) . At 13th week, the mice were immunized with syngenetic L5178 Y lymphoma cells and their specific and non-specific tumor immune responses were examined 3 weeks after immunization . The results indicated that in mice fed with a diet containing 0.007 ppm Se, the serum Se level was extremely low (0.02 micrograms/ml) . These Se-deficient mice were unable to elicit normal tumor-specific immune responses . Both the specific proliferation of T cells in MLTC and tumoricidal activity of CTL were very much depressed . In addition, these mice also showed impaired NK and LAK cell activity . The effects of Se supplementation varied depending on the amount of Se given . When 0.170 ppm Se was added to the low Se diet, all the immune parameters examined were restored to the normal level . When 0.567 ppm Se was added, however, the tumor immune responses remained as low as those in Se-deficient mice . This study implies that the prevalence of primary hepatocellular carcinoma in areas where Se is deficient has a profound immunological basis . Se supplementation is obviously indicated for cancer prevention in these areas but the amount of Se supplied is crucial.

Eur J Clin Nutr, 1990 Sep, 44(9), 629 - 35
Absorption and retention in acute diarrhoea; Mann MD et al.; 15N-yeast protein absorption, nitrogen and fat retention and stool reducing substances and lactate were measured in 6 infants who had acute diarrhoea and 15 who had had severe diarrhoea for 4 d . The results were compared with those of previously reported infants, who had had diarrhoea for 8 d . The infants were fed a full cream cows' milk, soy based or low lactose formula . In all cases the losses of nitrogen and energy in stool rose as stool weight increased . In severe diarrhoea, the losses of nutrients in stool were so great that oral feeds did not provide adequate nitrogen and energy . The smallest loss of nitrogen and fat were found in infants who had had diarrhoea for 4 d and who were fed a soy based formula.

J Cell Sci, 1990 Sep, 97 ( Pt 1), 193 - 204
Peroxisomes induced in Candida boidinii by methanol, oleic acid and D-alanine vary in metabolic function but share common integral membrane proteins; Goodman JM et al.; Peroxisomes massively proliferate in the methylotrophic yeast Candida boidinii when cultured on methanol as the only carbon and energy source . These organelles contain enzymes that catalyze the initial reactions of methanol utilization . The membranes contain abundant proteins of unknown function; their apparent molecular masses are 20, 31, 32 and 47 x 10(3) Mr and are termed PMP20, PMPs31-32 and PMP47 . Recently, we reported that peroxisomes in this yeast are also induced by oleic acid and D-alanine as carbon sources, and that these peroxisomes contain increased concentrations of the enzymes of fatty acid beta-oxidation or D-amino acid oxidase, respectively . This report extends these findings and further compares the enzyme composition from peroxisomes induced by methanol, oleic acid and D-alanine . the patterns of matrix proteins represented on SDS-polyacrylamide gels from peroxisomes induced by oleic acid or D-alanine were found to be very different from those of peroxisomes induced by methanol . In order to differentiate between membrane proteins that have specific functions in pathways of substrate utilization from those with more generalized functions, peroxisomal membranes from cultures grown on methanol, oleic acid or D-alanine were purified . Analysis of these fractions demonstrated that while PMP20 is found only in peroxisomes induced by methanol, the PMPs31-32 and PMP47 were the abundant peroxisomal membrane proteins (PMP) regardless of inducing substrate . The data strongly suggest that the function of PMP20 is related to methanol metabolism . In contrast, the functions of PMPs31-32 and PMP47 are 'substrate-nonspecific' . We speculate that they may relate to the structure, assembly or general function of the organelle.

Ultrastruct Pathol, 1990 Sep-Oct, 14(5), 439 - 52
Ultrastructural observations on Penicillium marneffei in natural human infection; Chan YF et al.; The ultrastructure of Penicillium marneffei and the host response to the infection were studied in two patients . One was immunocompetent and the other an immunosuppressed renal graft recipient . In the immunocompetent patient it was observed that all the yeast cells were phagocytosed and were found either within membrane-bound vacuoles or lying freely within the cytoplasm of the macrophages . It was postulated that continuous lysosomal fusion with the phagolysosomes and multiplication of the fungi within the phagocytic vacuoles might eventually lead to the rupture of the vacuoles with release of the organisms into the cytoplasm of the macrophages . In the second patient, the immunosuppressive effects of corticosteroids might account for the large number of nonphagocytosed fungi in the tissue space, and the failure to form large phagocytic vacuoles.

Somat Cell Mol Genet, 1990 Sep, 16(5), 437 - 41
Targeted gene replacement at the endogenous APRT locus in CHO cells; Adair GM et al.; We demonstrate the feasibility of targeted gene replacement at an endogenous, chromosomal gene locus in cultured mammalian cells, employing a two-step strategy similar to an approach routinely used for genetic manipulation in yeast . Utilizing an APRT+ recombinant generated by targeted integration of plasmid sequences (including a functional copy of the gpt gene) at the CHO APRT locus, we have been able to select gpt- "pop-out" recombinants that have arisen by intrachromosomal recombination between APRT direct repeats at the targeted integration site . Reciprocal exchanges leading to "pop-out" of integrated plasmid/gpt gene sequences occur at a rate of approximately 6.3 x 10(-6) per cell generation . Depending on the site of crossover, such "pop-out" events result in either replacement or restoration of the original APRT target gene sequence.

Exp Lung Res, 1990 Sep-Oct, 16(5), 451 - 79
Migratory behaviors of alveolar macrophages during the alveolar clearance of light to heavy burdens of particles; Lehnert BE et al.; We investigated the unstimulated and stimulated migratory activities of lavaged alveolar macrophages (AMs) in vitro over the course of alveolar clearance of three different mass lung burdens of microspheres . Our intent was to uncover potentially important relationships between the migratory behaviors of the AM and the retention kinetics of particles . Groups of adult, male Fischer-344 rats were intratracheally instilled with approximately 86 micrograms (low burden, LB), approximately 1 mg (medium burden, MB), or approximately 3.7 mg (high burden, HB) of polystyrene microspheres (2.13 microns diameter), or with carrier vehicle (phosphate buffered saline, PBS) alone . The lung retention kinetics of the particles were determined over an approximately 170 day period . On days 14, approximately 57, and approximately 85, lavaged AMs were assessed for their abilities to migrate through 5-microns pore membranes in response to inactivated rat serum (unstimulated condition) and yeast-activated rat serum (stimulated condition) . The retention characteristics of the three burdens could be satisfactorily described by two-component, negative exponential equations . The kinetics of retention of the LB and MB were similar, although some evidence indicated the MB slightly retarded the lung clearance process . Deposition of the HB resulted in more marked prolongations of both the early, more rapid, and the slower, longer term components of alveolar clearance . The unstimulated migration indices of AMs from the particle-instilled lungs were generally not significantly different from those of AMs from PBS-instilled lungs except for a significant increase in the migration indices of LB AMs at the last assay time . The stimulated migration indices of MB and HB AMs were significantly decreased on assay days 14 and approximately 57 . On day approximately 85, however, the migration indices of LB, MB, and HB AMs were all increased above the PBS AMs . Comparisons of the frequency distributions of particles in the unstimulated and stimulated AM that migrated to those in corresponding parent AM populations consistently indicated a preferential migration of particle-free AMs and of AMs with lesser loads of microspheres . The overall results of this study suggest that the unstimulated and stimulated migratory activities of particle-laden AMs are depressed in vitro . Our results also suggest that the migratory activities of generally particle-free AMs may be enhanced over a prolonged period of time following the deposition of particles in the lung.

Am J Clin Pathol, 1990 Sep, 94(3), 323 - 5
A new slide latex agglutination test for the diagnosis of acute Candida vaginitis; Sobel JD et al.; Two hundred two slide latex agglutination (SLA) tests were performed on 137 women attending a vaginitis clinic to evaluate the efficacy of this new test in diagnosing acute symptomatic Candida vaginitis . In 77 patients with acute Candida vaginitis, the SLA test revealed a positive reaction in 56 patients, reflecting a sensitivity of 72.7%, lower than that observed with the 10% potassium hydroxide microscopic examination (sensitivity 90%) . False negative SLA reactions could not be accounted for by lower numbers of yeast cultured from the vagina or by the presence of nonalbicans strains of Candida . Following successful antimycotic therapy, the SLA test promptly became negative in all mycologically negative patients . Application of the SLA test in asymptomatic healthy control women revealed extremely few false positive reactions for Candida (5.6%) . This easily performed and rapid slide latex agglutination test should provide a useful adjunct to the diagnosis of Candida vaginitis, but only for physicians who do not routinely perform microscopy on vaginal secretions.

J Clin Microbiol, 1990 Sep, 28(9), 2087 - 93
Diagnosis of Mycoplasma pneumoniae pneumonia: sensitivities and specificities of serology with lipid antigen and isolation of the organism on soy peptone medium for identification of infections; Kenny GE et al.; The sensitivities and specificities of isolation and serology for detection of Mycoplasma pneumoniae infections were determined for 3,546 pneumonia patients for whom both isolation and serological data were available . Soy peptone, fresh yeast extract, horse serum-supplemented agar, and diphasic medium were employed for isolation, and the lipid antigen of M . pneumoniae was used for serodiagnosis by complement fixation . The number of M . pneumoniae colonies most frequently detected was 200 to 600 per throat swab, with a range of less than or equal to 60 to greater than or equal to 2,000 . The use of diphasic medium increased the number of isolates by 26% compared with direct isolation on agar plates . The organism was isolated from 360 of 525 patients who showed fourfold or greater antibody increases in their paired sera, resulting in a sensitivity of culture of 68% . When persons with titers of greater than or equal to 32 but no fourfold increase were used as the reference, the sensitivity of culture was 58% . The combined sensitivity of the culture method for persons with serological evidence of infection (fourfold increase and titer of greater than or equal to 32) was 64% . The specificity of the culture method was 97% for the 2,527 serologically negative persons . Fourfold antibody increases were found in 360 of 674 persons with isolates of the organism, resulting in a sensitivity of 53% . An additional 247 persons showed titers of greater than or equal to 32 (without a fourfold increase), resulting in a combined sensitivity of 90% for serology with the lipid antigen for the detection of antibodies in culture-positive persons . Fourfold antibody increases were found in 6% of culture-negative persons, resulting in a specificity of 94% . The quantitative culture results provide important base-line data for the development of rapid diagnostic tests for M . pneumoniae infection.

Genetics, 1990 Sep, 126(1), 157 - 66
Induced rates of mitotic crossing over and possible mitotic gene conversion per wing anlage cell in Drosophila melanogaster by X rays and fission neutrons; Ayaki T et al.; As a model for chromosome aberrations, radiation-induced mitotic recombination of mwh and flr genes in Drosophila melanogaster strain (mwh +/+ flr) was quantitatively studied . Fission neutrons were five to six times more effective than X rays per unit dose in producing either crossover-mwh/flr twins and mwh singles-or flr singles, indicating that common processes are involved in the production of crossover and flr singles . The X-ray-induced rate/wing anlage cell/Gy for flr singles was 1 X 10(-5), whereas that of crossover was 2 x 10(-4); the former and the latter rate are of the same order of magnitude as those of gene conversion and crossover in yeast, respectively . Thus, we conclude that proximal-marker "flr" singles induced in the transheterozygote are gene convertants . Using the model based on yeast that recombination events result from repair of double-strand breaks or gaps, we propose that mitotic recombination in the fly is a secondary result of recombinational DNA repair . Evidence for recombinational misrepair in the fly is given . The relative ratio of radiation-induced mitotic crossover to spontaneous meiotic crossover is one order of magnitude higher in the fly than in yeast and humans.

Plant Mol Biol, 1990 Sep, 15(3), 383 - 97
A low molecular weight DNA polymerase from wheat embryos; Castroviejo M et al.; The study of plant DNA polymerases lags far behind that concerning their animal or yeast counterpart . In this work we describe the first extensive purification to apparent homogeneity, as well as a detailed biochemical and immunological characterization, of a low molecular weight DNA polymerase (DNA polymerase CI) purified from wheat embryos . The monomeric enzyme is a basic protein having a molecular weight of 52 kDa . Polyclonal antibodies raised in rabbits against DNA polymerase CI did not inhibit animal DNA polymerases alpha and beta or wheat DNA polymerase A, whereas wheat DNA polymerases CII and B were much less affected than the CI enzyme . Several properties of enzyme CI were studied . Some known inhibitors of DNA polymerase activity including aphidicolin, phosphonoacetic acid and heparin, did not affect DNA polymerase CI while the activity of this enzyme was strongly inhibited by ddTTP and N-ethylmaleimide . The polyamine spermine decreased markedly the enzyme activity, while spermidine produced a strong stimulation at the same concentrations that spermine inhibited the enzyme . The best template for this enzyme is poly dA-oligo dT, although polymerase CI can recognize significantly some synthetic polyribonucleotide templates (poly rC-oligo dG, poly rA-oligo dT) but only at a given protein/template primer ratio . The enzyme is blocked at the amino terminus, thus preventing the automatic sequencing of the protein . The amino acid analysis showed a striking similarity with the animal low molecular weight DNA polymerase beta . The latter observation, as well as the effect of inhibitors (except N-ethylmaleimide which does not inhibit the animal polymerase) indicate that the DNA polymerase described in this work is a plant DNA polymerase very similar to the low molecular weight animal DNA polymerase beta, an enzyme believed to be involved in nuclear DNA repair.

Mycoses, 1990 Sep-Oct, 33(9-10), 491 - 7
White piedra: ultrastructure and a new microecological aspect; de Almeida Junior HL et al.; White piedra or trichosporosis is a superficial mycosis of the hair shaft, caused by the yeast Trichosporon beigelii; it has been found in all continents and may involve the hair of any part of the body . We report a case of white piedra on the hairs of the inguinal fold with ultrastructural studies . Transmission electron microscopy showed that the nodules have the same morphological aspects as the fungus in culture (hyphae and arthrospores) except for the presence of a cementant substance . By scanning electron microscopy the elimination of spores was seen on the nodule surface . Interestingly similar nodules were found on cotton fibres of the patient's underwear, which were also studied by scanning electron microscopy . This finding can explain therapeutic failure and demands special hygienic conditions related to clothes.

Mycoses, 1990 Sep-Oct, 33(9-10), 483 - 9
Enzyme immunoassay detection of antibodies in canine blastomycosis using Blastomyces dermatitidis lysate antigens; Seawell BW et al.; A Blastomyces dermatitidis yeast phase lysate antigen (T-58) prepared in our laboratory was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in 201 serum specimens from dogs with blastomycosis . In addition 36 sera from non-infected dogs from an endemic area for blastomycosis and 36 sera from non-infected dogs residing in a blastomycosis non-endemic area were assayed . Sensitivity values ranged from 96.2% (53 pre-treatment sera) to 88.5% (148 post-treatment sera) with the specimens from dogs with confirmed blastomycosis . Minimal reactivity was experienced with the sera from non-infected dogs . Positive reactions were obtained with 8.3% of the endemic area sera and with 5.6% of the sera from the non-endemic area . Mean ELISA index values ranged from 7.66 with the pretreatment sera to 1.11 and 1.03 with the endemic area sera and non-endemic area specimens respectively.

Mycoses, 1990 Sep-Oct, 33(9-10), 469 - 75
Experimental phaeohyphomycosis; Pospisil L et al.; The authors performed an experimental infection of the rabbit eye with Wangiella dermatitidis which had been isolated from the corneal ulcer of a patient . The fungus was inoculated into the front chamber and the vitreous body . The disease showed a trend to spontaneous recovery . The individual phases of the experimental infection were followed by histology and electronmicroscopy, both TEM and SEM . Different stages of development of the polymorphous fungus, as sclerotic bodies, mycelial filaments and yeast-like cells, could be demonstrated.

Br J Clin Pract Suppl, 1990 Sep, 71, 70 - 2
Diagnostic laboratory techniques in vaginal candidosis; Evans EG; The laboratory diagnosis of vulvovaginal candidosis is complicated by the fact that the Candida spp., mainly C . albicans, are found as commensals in the normal vagina . Therefore, the demonstration of Candida in material from the vagina by microscopy (KOH mounts or Gram smears) and/or culture on Sabouraud's agar does not necessarily confirm infection . For this reason, attention is frequently paid to the quantity of yeast isolated in culture and to the morphological form of the organism (yeast/mycelium) seen in the clinical material . However, there is controversy as to whether either of these two criteria, which may be influenced by sampling methods, sample type, sample age, etc, are reliable indicators of Candida infection and correlate with the signs and symptoms attributed to this condition . A more accurate measure of the quantity of Candida present in the vagina may be obtained by measurement of yeast antigen levels in material such as vaginal washings . Correlation of antigen levels with signs and symptoms of infection is required, as is an appraisal of the role of these antigens in the pathogenesis of vulvovaginal candidosis . A new and more rapid method for diagnosis of vulvovaginal candidosis, based on the detection of Candida antigens with a slide latex agglutination test, has been recently introduced.

Genet Anal Tech Appl, 1990 Sep, 7(5), 114 - 8
YAC cloning of DNA embedded in an agarose matrix; McCormick MK et al.; Yeast artificial chromosome (YAC) cloning of DNA in agarose is an alternative method to cloning from aqueous solutions . It minimizes any shearing that may result from handling of high molecular weight DNA and can be done with nanogram to microgram amounts of material, which facilitates construction of YACs from sources of DNA other than genomic DNA isolated from cells . The average size of the YACs recovered (200-1000 kb) and efficiency of transformation of ligation products (200-1000 cfu/micrograms) are similar to those reported using aqueous protocols . This method has been used to construct chromosome specific YACs, and it should be possible to apply the technique to the construction of chromosome specific libraries using flow sorted chromosomes as source material, and the cloning of restriction fragments isolated by preparative pulsed field gel electrophoresis.

Genet Anal Tech Appl, 1990 Sep, 7(5), 100 - 6
Current methods for YAC clone characterization; Nelson DL; Yeast artificial chromosome (YAC) cloning has allowed isolation of much longer DNA fragments than was previously possible . While this technology makes feasible the isolation of large regions of complex genomes in overlapping cloned segments, it has also required altered methods for manipulation of cloned DNAs . In particular, the isolation of insert sequences from YAC clones has been especially difficult . Methods for characterization of YAC inserts are described and compared.

Genomics, 1990 Sep, 8(1), 168 - 70
Characterization of polymorphic loci on a telomeric fragment of DNA from the long arm of human chromosome 7; Dietz-Band J et al.; The 240-kb yeast artificial chromosome (YAC) HTY146 (D7S427) containing the telomere from the q arm of human chromosome 7 was subcloned into the cosmid vector sCOS-1 . Cosmid subclones were screened for DNA polymorphisms by Southern blot analysis of restriction digests of DNA from random individuals . Four distinct polymorphisms were characterized . These markers provide a resource for defining the end of the genetic map for the long arm of human chromosome 7.

Proc Natl Acad Sci U S A, 1990 Sep, 87(17), 6664 - 8
DNA polymerases alpha, delta, and epsilon: three distinct enzymes from HeLa cells; Syvaoja J et al.; DNA polymerases alpha, delta, and epsilon have been purified and characterized from the same HeLa cell extract in order to determine their relationship by comparing them from the same cell type . The catalytic properties and the primary structures of the large subunits of the DNA polymerases as compared by partial peptide mapping with N-chlorosuccinimide are different . Likewise, the small subunit of DNA polymerase epsilon appears to be distinct from the large subunit of the same polymerase and from the smaller subunits of DNA polymerase alpha . HeLa DNA polymerase delta is processive only when HeLa proliferating cell nuclear antigen is present, whereas DNA polymerase epsilon is quite processive in its absence . Inhibitor and activator spectra of DNA polymerases alpha, delta, and epsilon also distinguish the three enzymes . These results and immunologic comparisons published elsewhere support the premise that HeLa DNA polymerases alpha, delta, and epsilon are distinct enzymes that have common properties with yeast DNA polymerases I, III, and II, respectively.

Brain Pathol, 1990 Sep, 1(1), 33 - 40
Progress toward the isolation and characterization of the genes causing neurofibromatosis; Menon AG et al.; Neurofibromatosis 1 and neurofibromatosis 2 are clinically distinct autosomal dominant disorders that affect an estimated 1.5 million individuals throughout the world . The genetic defect in each disorder has been mapped to different chromosomes, NF1 to chromosome 17 and NF2 to chromosome 22 . Progress towards the cloning of the NF1 gene has proceeded rapidly . The NF1 locus was bracketed using genetic linkage analysis on NF1 affected pedigrees . Physical mapping methods were then used to precisely map the translocation breakpoints in each of two NF1 affected individuals who harbored constitutional chromosomal translocations in the putative NF1 region of chromosome 17 . The region of DNA located between the two translocations has been cloned in cosmids and yeast artificial chromosomes and a number of RNA coding sequences have been identified . The identification of the NF1 gene will depend on finding mutations in the DNA of affected individuals . In the case of NF2, progress seems to have been less rapid, in part due to the lower availability of NF2 affected pedigrees . The genetic defect has been mapped to the long arm of chromosome 22 by studies of chromosomal loss in the tumours associated with this disease . Subsequent genetic mapping has confirmed this location . Flanking DNA markers for the NF2 locus have been identified . The region of DNA between these markers is in the order of 5-10 Mb . The identification of chromosomal aberrations in patients with NF2 that involve chromosome 22 will play an important role in the identification of the NF2 gene in much the same way as they have in NF1.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 69 - 73
The primary structure of rat ribosomal proteins: the amino acid sequences of L27a and L28 and corrections in the sequences of S4 and S12; Wool IG et al.; The amino acid sequences of rat ribosomal proteins L27a and L28 were deduced from the sequences of nucleotides in recombinant cDNAs and confirmed from the NH2-terminal amino acid sequences of the proteins . L27a contains 147 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 16 476 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 18-22 copies of the L27a gene . The mRNA for the protein is about 600 nucleotides in length . L27a is homologous to mouse L27a (there are 3 amino acid changes) and to yeast L29 . Rat ribosomal protein L28 has 136 amino acids (its NH2-terminal methionine is also processed after translation) and has a molecular weight of 15 707 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 9 or 10 copies of the L28 gene . The mRNA for the protein is about 640 nucleotides in length . L28 contains a possible internal duplication of 9 residues . Corrections are recorded in the sequences reported before for rat ribosomal proteins S4 and S12.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 186 - 92
Aminoacylation of 3' terminal tRNA-like fragments of turnip yellow mosaic virus RNA: the influence of 5' nonviral sequences; Mans RM et al.; The present model of the L-shaped tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA encompasses 82 nucleotides . A previous kinetic study on 3' terminal TYMV RNA fragments that contain the tRNA-like structure and a 5' nonviral GGGAGA sequence, suggested that viral sequences upstream of the tRNA-like domain, i.e., upstream of nucleotide 82, increase the rate of aminoacylation (Dreher et al . (1988) Biochimie 70, 1719-1727) . Here we report an increase in the aminoacylation rate when the number of nonviral nucleotides at the 5' end of TYMV RNA transcripts was reduced . The influence of these 5' proximal nonviral sequences on the conformation of the RNA molecule was investigated by structure mapping experiments . A structure that deviates from the tRNA-like structure was found in some of the transcripts . The formation of this alternative structure is dependent upon: (1) the nature and number of the nonviral nucleotides; (2) the number and secondary structure of viral nucleotides between the nonviral nucleotides and the tRNA-like domain . Footprinting experiments with valyl-tRNA synthetase from yeast suggest that the enzyme does not recognize the alternative structure.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 160 - 2
The role of mammalian initiation factor eIF-4D and its hypusine modification in translation; Hershey JW et al.; Initiation factor eIF-4D functions late in the initiation pathway, apparently during formation of the first peptide bond . The factor is post-translationally modified at a specific lysine residue by reaction with spermidine and subsequent hydroxylation to form hypusine . A precursor form lacking hypusine is inactive in the assay for methionyl-puromycin synthesis, but activity is restored following in vitro modification to deoxyhypusine, thereby suggesting that the modification is essential for function . Since formylated methionyl-tRNA is less dependent on eIF-4D in the puromycin assay, we postulate that eIF-4D and its hypusine modification may stabilize charged Met-tRNA binding to the peptidyl transferase center of the 60S ribosomal subunit . Analysis of eIF-4D genes in yeast indicate that eIF-4D and its hypusine modification are essential for cell growth.

J Biol Chem, 1990 Aug 25, 265(24), 14234 - 41
Changes in crystallographic structure and thermostability of a Cu,Zn superoxide dismutase mutant resulting from the removal of a buried cysteine; McRee DE et al.; In principle, protein thermostability depends on efficient interior packing of apolar residues and on avoidance of irreversible denaturation in the unfolded state . To study these effects, the single free cysteine in the highly stable enzyme bovine Cu,Zn superoxide dismutase was mutated to alanine (Cys6----Ala), and the recombinant protein was expressed in yeast, purified, characterized for reversible and irreversible denaturation, crystallized isomorphously to the wild-type enzyme, and used to determine the atomic structure . Removal of the chemically reactive thiol significantly decreased the rate of irreversible denaturation (as monitored by thermal inactivation at 70 degrees C), but the observed energetic cost (delta delta G of 0.7-1.3 kcal/mol as determined by differential scanning calorimetry) was much less than predicted from either the change in hydrophobicity or packing due to removal of the interior sulfur atom . X-ray diffraction data were collected to 2.1-A resolution using an area detector, and the atomic model for the mutant enzyme was determined by fitting to electron density difference maps, followed by reciprocal space refinement both with stereochemical restraints using PROLSQ and with molecular dynamics using X-PLOR . The refined 2.1-A resolution crystallographic structure suggests that small concerted and compensating shifts (less than 0.5 A) of the surrounding side chains and of the adjacent N- and C-terminal beta-strands significantly reduced the energetic cost of the interior mutation by improving packing and stereochemistry in the mutant enzyme . Taken together, these results differentiate between the effects of reversible and irreversible processes as they impact the design of thermostable proteins and suggest that relatively subtle concerted shifts can significantly reduce the energetic cost of evolutionary variation in internal residues of proteins with Greek key beta-barrel folds.

Nucleic Acids Res, 1990 Aug 25, 18(16), 4901 - 3
repa, a repetitive and dispersed DNA sequence of the filamentous fungus Podospora anserina; Deleu C et al.; The sequences of homologous DNA regions of two wild-type strains of the fungus Podospora anserina, revealed in one strain the presence of a 349bp insertion leading to a RFLP . This DNA sequence is repeated in the genome and some of its locations are different in various wild-type strains . This DNA element exhibits structural similarities with the yeast solo delta, sigma or tau elements.

Biochim Biophys Acta, 1990 Aug 24, 1027(2), 141 - 8
Import into mitochondria of precursors containing hydrophobic passenger proteins: pretreatment of precursors with urea inhibits import; Law RH et al.; We have studied the import into isolated yeast mitochondria of three hydrophobic passenger proteins attached to the N-terminal cleavable presequence of mitochondrial ATPase subunit 9 from Neurospora crassa . One natural precursor (pN9) contained N . crassa subunit 9; two chimaeric precursors, N9L/Y8-1 and N9L/Y9-2, respectively contained yeast mitochondrial ATPase subunits 8 and 9 . In the absence of urea, pN9 and N9L/Y8-1 are imported efficiently but N9L/Y9-2 is not imported . After pretreatment of precursors in 4 M urea, binding of pN9 to mitochondria is marginally affected while its import is substantially inhibited; the binding to mitochondria of chimaeric proteins, N9L/Y8-1 and N9L/Y9-2, is greatly enhanced but no import is observed . This behaviour of import precursors containing hydrophobic passenger proteins is contrasted with that of a hydrophilic chimaeric precursor pCOXIV-DHFR, whose binding and import are enhanced by pretreatment with a high concentration of urea (8 M) . The import of N9L/Y8-1 is very sensitive to the presence of low concentrations of urea in the import reaction mixture, and is abolished above 0.5 M urea although precursor binding to mitochondria is increased . By contrast, neither the import nor binding of pCOXIV-DHFR is affected directly by urea up to 0.8 M . These deleterious effects of urea on import of the chimaeric precursors N9L/Y8-1 and N9L/Y9-2 are interpreted in terms of a non-productive binding of these precursors to mitochondria, brought about by exposure of their hydrophobic domains resulting from urea unfolding . The generalization that membrane translocation of mitochondrial import precursors is enhanced by their prior unfolding in urea thus does not apply in the case of these precursors containing hydrophobic passenger proteins.

FEBS Lett, 1990 Aug 20, 269(1), 89 - 92
Inhibition of oxygen toxicity by targeting superoxide dismutase to endothelial cell surface; Inoue M et al.; Since enzymes that degrade reactive oxygens, such as superoxide dismutase (SOD), are significantly lower in plasma than in intracellular compartments, cell surface membranes should be protected against hazardous oxygens particularly when animals are challenged with oxidative stress . To minimize oxygen toxicity on endothelial cell surface, a fusion gene consisting of cDNA coding human Cu2+/Zn2(+)-SOD and heparin-binding peptide was constructed and expressed in yeast . The resulting enzyme (HB-SOD) bound to a heparin-Sepharose column and cultured endothelial cells; binding was inhibited either by high NaCl concentrations or heparin . When injected intravenously, HB-SOD predominantly bound to vascular endothelial cell surface . Carrageenin-induced paw edema and cold-induced brain edema of the rat were markedly inhibited by a single dose of HB-SOD . These results suggest that superoxide radical and/or its metabolite(s) occurring at or near the outer surface of vascular endothelial cells might play a critical role in the pathogenesis of vasogenic edema.

FEBS Lett, 1990 Aug 20, 269(1), 194 - 6
Fast atom bombardment mass spectrometry and chemical analysis in determinations of acyl-blocked protein structures; Egestad B et al.; Peptide generation and fast atom bombardment mass spectrometry in combination with conventional chemical analysis was used to identify the blocking group and establish the N-terminal structure of six different proteins at the nanomole level . In this manner, the first terminal structures of three non-mammalian alcohol dehydrogenases were determined, demonstrating the presence of N-terminal acetylation in these piscine, amphibian, and avian enzymes . Similarly, two different yeast glucose-6-phosphate dehydrogenases and a minor variant of a human alcohol dehydrogenase were found to be acetylated . The exact end location of C-terminal structures was also established . Together, the analyses permit the definition of terminal regions and blocking groups, thus facilitating the delineation of remaining structures.

Biochem Biophys Res Commun, 1990 Aug 16, 170(3), 1352 - 8
F0F1-ATPase of plant mitochondria: isolation and polypeptide composition; Hamasur B et al.; A simple and high yield purification procedure for the isolation of F0F1-ATPase from spinach leaf mitochondria has been developed . This is the first report concerning purification and composition of the plant mitochondrial F0F1-ATPase . The enzyme is selectively extracted from inner membrane vesicles with the zwitterionic detergent, 3-{(3-cholamidopropyl) dimethyl ammonio}-1- propane sulfonate (CHAPS) . The purified enzyme exhibits a high oligomycin-sensitive ATPase activity (3,6 mumol.min-1.mg-1) . SDS-PAGE of the purified F0F1-ATPase complex reveals protein bands of molecular masses of 54 kDa (F1 alpha,beta), 33 kDa (F1 gamma), 28 kDa, 23 kDa, 21 kDa (F1 delta), 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa (F1 epsilon) and 8.5 kDa . All polypeptides migrate as one complex in a polyacrylamide gradient gel under non-denaturing conditions in the presence of 0.1% Triton X-100 . Five polypeptides could be identified as subunits of F1 . Polypeptides of molecular masses 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa constitute the F0 part of the complex . Our results show that polypeptide composition of the plant mitochondrial F0 differs from other eukaryotic F0 of yeast, mammals and chloroplasts.

Biochem J, 1990 Aug 15, 270(1), 205 - 11
Intracellular transport of endocytosed proteins in rat liver endothelial cells; Kindberg GM et al.; 1 . Receptor-mediated endocytosis of mannose-terminated glycoproteins in rat liver endothelial cells has been followed by means of subcellular fractionation and by immunocytochemical labelling of ultrathin cryosections after intravenous injection of ovalbumin . For subcellular-fractionation studies the ligand was labelled with 125-tyramine-cellobiose adduct, which leads to labelled degradation products being trapped intracellularly in the organelle where the degradation takes place . 2 . Isopycnic centrifugation in sucrose gradients of a whole liver homogenate showed that the ligand is sequentially associated with three organelles with increasing buoyant densities . The ligand was, 1 min after injection, recovered in a light, slowly sedimenting vesicle and subsequently (6 min) in larger endosomes . After 24 min the ligand was recovered in dense organelles, where also acid-soluble degradation products accumulated . 3 . Immunocytochemical labelling of ultrathin cryosections showed that the ligand appeared rapidly after internalization in coated vesicles and subsequently in two larger types of endosomes . In the 'early' endosomes (1 min after injection) the labelling was seen closely associated with the membrane of the vesicle; after 6 min the ligand was evenly distributed in the lumen . At 24 min after injection the ligand was found in the lysosomes . 4 . A bimodal distribution of endothelial cell lysosomes with different buoyant densities was revealed by centrifugation in iso-osmotic Nycodenz gradients, suggesting that two types of lysosomes are involved in the degradation of mannose-terminated glycoproteins in liver endothelial cells . Two populations of lysosomes were also revealed by sucrose-density-gradient centrifugation after injection of large amounts of yeast invertase . 5 . In conclusion, ovalbumin is transferred rapidly through three endosomal compartments before delivering to the lysosomes . The degradation seems to take place in two populations of lysosomes.

Science, 1990 Aug 10, 249(4969), 635 - 40
The cellular functions of small GTP-binding proteins; Hall A; A substantial number of novel guanine nucleotide binding regulatory proteins have been identified over the last few years but the function of many of them is largely unknown . This article will discuss a particular family of these proteins, structurally related to the Ras oncoprotein . Approximately 30 Ras-related small guanosine triphosphate (GTP)-binding proteins are known, and from yeast to man they appear to be involved in controlling a diverse set of essential cellular functions including growth, differentiation, cytoskeletal organization, and intracellular vesicle transport and secretion.

Cell, 1990 Aug 10, 62(3), 599 - 608
The neurofibromatosis type 1 gene encodes a protein related to GAP; Xu GF et al.; cDNA walking and sequencing have extended the open reading frame for the neurofibromatosis type 1 gene (NF1) . The new sequence now predicts 2485 amino acids of the NF1 peptide . A 360 residue region of the new peptide shows significant similarity to the known catalytic domains of both human and bovine GAP (GTPase activating protein) . A much broader region, centered around this same 360 amino acid sequence, is strikingly similar to the yeast IRA1 product, which has a similar amino acid sequence and functional homology to mammalian GAP . This evidence suggests that NF1 encodes a cytoplasmic GAP-like protein that may be involved in the control of cell growth by interacting with proteins such as the RAS gene product . Mapping of the cDNA clones has confirmed that NF1 spans a t(1;17) translocation mutation and that three active genes lie within an intron of NF1, but in opposite orientation.

Am J Med Sci, 1990 Aug, 300(2), 98 - 101
Histoplasmosis as a cause of pleural effusion in the acquired immunodeficiency syndrome; Marshall BC et al.; Disseminated histoplasmosis is an increasingly important opportunistic infection in patients with the acquired immunodeficiency syndrome (AIDS) . We report the first case of histoplasmosis as a cause of pleural effusion in a patient with AIDS . Recognition of the typical intracellular yeast on a Wright-Giemsa stained smear of the pleural fluid cells allowed prompt initiation of amphotericin B.

Agents Actions, 1990 Aug, 31(1-2), 117 - 26
AHR-10037, a non-steroidal anti-inflammatory compound of low gastric toxicity; Sancilio LF et al.; AHR-10037 is an anti-inflammatory compound possessing analgesic and antipyretic properties and a high therapeutic index . AHR-10037 was comparable to indomethacin in suppressing acute (Evans blue-carrageenan pleural effusion) and chronic (adjuvant-induced arthritis) inflammation . There was a delayed onset of antipyresis (yeast-induced hyperthermia in rats), analgesia (acetylcholine-induced abdominal constriction in mice) and inhibition of caster oil-induced diarrhea in rats . Antipyresis occurred 3 hours after administration of AHR-10037, 4 mg/kg, PO . vs 1 hour after administration of acetylsalicylic acid, 100 mg/kg, PO; maximum analgesic activity (ED50 = 4.1 mg/kg) occurred at 4 hours . AHR-10037 was inferior to indomethacin in suppressing castor oil-induced diarrhea in rats . The therapeutic index of AHR-10037 (relating acute anti-inflammatory potency to gastric toxicity potency relative to indomethacin) ranged from 56-91 . The pharmacological profile suggests that AHR-10037 is a prodrug converted in vivo to a cyclooxygenase inhibitor.

Mol Gen Genet, 1990 Aug, 223(1), 65 - 75
Homologous domains of the largest subunit of eucaryotic RNA polymerase II are conserved in plants; Nawrath C et al.; Genomic and cDNA clones homologous to the RpII215 gene of Drosophila were isolated from Arabidopsis thaliana and assigned to a single copy gene encoding a transcript of 6.8 kb . Nucleotide sequence analysis of Arabidopsis genomic and cDNAs revealed a striking homology to yeast, Caenorhabditis, Drosophila and mouse genes encoding the largest subunit of RNA polymerase II . The Arabidopsis gene rpII215 contains 13 introns, 12 of which interrupt the coding sequence of a protein of 205 kDa . The position of the first intron is conserved between plant and animal genes, while an intron located in the 3' untranslated region of the rpII215 gene is unique to Arabidopsis . Common domains present in all known largest subunits of eucaryotic RNA polymerase II were identified in the predicted sequence of the Arabidopsis RpII215 protein . Both the order and the position of N-terminal Zn2+ finger and of DNA and alpha-amanitin binding motifs are conserved in Arabidopsis . The C-terminal region of the Arabidopsis protein contains 15 consensus and 26 variant YSPTSPS repeats (CTDs) . Highly conserved structure among the various C-terminal domains suggests that the largest subunit of RNA polymerase II in plants may also interact with transcription factors and with protein kinases that control the cell cycle as in other organisms.

Psychiatr Neurol Med Psychol (Leipz), 1990 Aug, 42(8), 457 - 66
{Effect of pulsating electromagnetic field therapy on cell volume and phagocytosis activity in multiple sclerosis and migraine}; Mix E et al.; PEMF treatment was studied in 10 patients with multiple sclerosis and 10 patients with migraine . In both patients' groups a single treatment induced a significant rise of yeast particle uptake by blood granulocytes . The percentage of phagocytizing cells was increased in migraine patients only . In both patients' groups 20 PEMF treatments caused a reduction of particle uptake, whereas the percentage of phagocytizing cells remained unchanged . In migraine patients the opsonic capacity of serum and the mean cell volume of erythrocytes, lymphocytes and granulocytes were initially reduced, but increased during the course of 20 PEMF treatments . The biphasic changes of cell volume and phagocytic activity are interpreted as a result of counter-regulation of the organism in response to the primary PEMF effect.

Pathol Res Pract, 1990 Aug, 186(4), 514 - 7; discussion 518
Histoplasmosis duboisii (African histoplasmosis) . An African case reported from Chile with ultrastructural study; Oddo D et al.; A case of histoplasmosis duboisii in a 30 year-old engineer is presented . The diagnosis was made with the help of light microscopy, electron microscopy and cultures . Although diagnosed in Chile, the patient probably acquired the disease in the endemic African area, more precisely in the Ivory Coast . Differential diagnosis between Histoplasma capsulatum var . capsulatum and Histoplasma capsulatum var . duboisii is based primarily on the larger in-vivo yeast form size of the latter . Electron microscopy study, the first done on the duboisii variety of Histoplasma capsulatum in human material to our knowledge, was not essential for this purpose . Differential diagnosis between Histoplasma capsulatum var . duboisii and Blastomyces dermatitidis is based on morphological tissue changes and mycologic characteristics . Once more, a case of an "exotic" or geographically restricted disease is detected far from its endemic area, thanks to easier means of transportation . Earth is a shrinking planet.

Poult Sci, 1990 Aug, 69(8), 1285 - 91
Dietary and monensin effects on activity of hepatic microsomal mixed function oxidase system in chickens; Brenes A et al.; Four experiments were conducted to investigate if the degree of activation of the microsomal mixed-function oxidase (MFO) system was related to the degree of growth depression associated with the addition of monensin to the diet . The experiments were conducted with broiler chicks in battery brooders in which the chicks were fed diets of various composition and containing monensin at 0 to 160 ppm . In all experiments, the activity of the MFO system was estimated by the change in the content of cytochrome P-450 in the hepatic microsomes . Activities of some microsomal enzymes were also measured in some of the experiments . Feeding a diet with 24% protein containing fish meal, alfalfa meal, and torula yeast significantly increased the activity of the MFO system in comparison with feeding an isonitrogenous and isoenergetic corn and soybean diet, but there was no difference between the diets in the toxicity of monensin as measured by growth rate . Supplementing a 16% protein but not a 24% protein diet with monensin significantly reduced growth rate . In none of the four experiments was there a statistically significant change in the hepatic content of cytochrome P-450 as a result of feeding monensin . Thus, variation in the magnitude of growth depression caused by monensin in diets of different protein concentration or ingredient composition does not appear to be explained on the relative degree of the activation of the MFO system.

Mol Biochem Parasitol, 1990 Aug, 42(1), 55 - 62
Analysis of the sequences flanking the translational start sites of Plasmodium falciparum; Saul A et al.; The 5' and 3' regions adjacent to the initiation codon in 22 Plasmodium falciparum sequences were examined . A 5' consensus sequence (AAAA/ATG) was found . Although P . falciparum non-translated DNA is A-rich, A occurred significantly more frequently in the 4 positions preceding the initiation ATG than in adjacent non-translated DNA, suggesting that this consensus sequence has functional significance in the initiation of translation . This region has similarities with the equivalent sequences in yeast and Drosophila but differs markedly from that in vertebrates . No significant bias in nucleotide frequencies was found 3' to the initiation codon.

J Biochem (Tokyo), 1990 Aug, 108(2), 166 - 8
Characterization of cyanide-resistant respiration and appearance of a 36 kDa protein in mitochondria isolated from antimycin A-treated Hansenula anomala; Sakajo S et al.; Mitochondria exhibiting cyanide-resistant respiration were isolated from Hansenula anomala which had been incubated in the presence of antimycin A to induce cyanide-resistant respiration . The cyanide-resistant respiration in isolated mitochondria was not inhibited by antimycin A or myxothiazol, suggesting that the branching of the pathway from the normal cyanide-sensitive pathway takes place at the coenzyme Q level . Analysis of mitochondrial proteins by sodium dodecyl sulfate gel electrophoresis indicated that a 36 kDa protein was induced by antimycin A treatment of the yeast . It is suggested that this protein is a component of the cyanide-resistant respiratory pathway.

Antimicrob Agents Chemother, 1990 Aug, 34(8), 1619 - 21
Comparison of cilofungin and amphotericin B for therapy of murine candidiasis; Smith KR et al.; We compared the efficacies of cilofungin and amphotericin B treatment in a murine model of disseminated candidiasis . Three different dosages of each drug plus controls were evaluated . Statistically improved survival was noted only among mice treated with 1 mg of amphotericin B per kg of body weight (P less than 0.05) . While all amphotericin B regimens and the two lower-dosage cilofungin regimens significantly reduced yeast cell counts in kidneys compared with the controls, the amphotericin B-treated mice had a significantly higher percentage of sterile kidneys following therapy compared with those treated with cilofungin (P = 0.0001).

Protein Eng, 1990 Aug, 3(8), 733 - 7
Construction of a novel artificial-ribozyme-releasing plasmid; Taira K et al.; A novel 'active-ribozyme-releasing system' was constructed, taking advantage of the consensus sequence of a new class of ribozyme . An active ribozyme sequence, targeted for the SFL1 gene (a yeast suppressor gene for flocculation) was fused just downstream of the T7 promoter . The 3' terminus of the first ribozyme was designed to be trimmed by the second ribozyme connected to the downstream of the first active ribozyme . In vitro experiments revealed that the active ribozyme targeted to SFL1 was successfully released by the action of the second ribozyme, subsequently cleaving the SFL1 mRNA at the predetermined site . Since the first active ribozyme with a defined 3'-terminus can be produced even when a circular DNA is used as a template, this kind of construct has a potential to release an 'active ribozyme' tailored to destroy a target gene (RNA) in vivo . Moreover, the second ribozyme in this construct can be utilized as a universal pseudo-terminator for generation of any RNA transcripts inserted in place of the cassette portion of the first ribozyme.

Biophys J, 1990 Aug, 58(2), 379 - 89
Orientation and lateral mobility of cytochrome c on the surface of ultrathin lipid multilayer films; Pachence JM et al.; We have previously shown that cytochrome c can be electrostatically bound to an ultrathin multilayer film having a negatively charged hydrophilic surface; furthermore, x-ray diffraction and absorption spectroscopy techniques indicated that the cytochrome c was bound to the surface of these ultrathin multilayer films as a molecular monolayer . The ultrathin fatty acid multilayers were formed on alkylated glass, using the Langmuir-Blodgett method . In this study, optical linear dichroism was used to determine the average orientation of the heme group within cytochrome c relative to the multilayer surface plane . The cytochrome c was either electrostatically or covalently bound to the surface of an ultrathin multilayer film . Horse heart cytochrome c was electrostatically bound to the hydrophilic surface of fatty acid multilayer films having an odd number of monolayers . Ultrathin multilayer films having an even number of monolayers would not bind cytochrome c, as expected for such hydrophobic surfaces . Yeast cytochrome c was covalently bound to the surface of a multilayer film having an even number of fatty acid monolayers plus a surface monolayer of thioethyl stearate . After washing extensively with buffer, the multilayer films with either electrostatically or covalently bound cytochrome c were analyzed for bound protein by optical absorption spectroscopy; the orientation of the cytochrome c heme was then investigated via optical linear dichroism . Polarized optical absorption spectra were measured from 450 to 600 nm at angles of 0 degrees, 30 degrees, and 45 degrees between the incident light beam and the normal to the surface plane of the multilayer . The dichroic ratio for the heme alpha-band at 550 nm as a function of incidence angle indicated that the heme of the electrostatically-bound monolayer of cytochrome c lies, on average, nearly parallel to the surface plane of the ultrathin multilayer . Similar results were obtained for the covalently-bound yeast cytochrome c . Furthermore, fluorescence recovery after photobleaching (FRAP) was used to characterize the lateral mobility of the electrostatically bound cytochrome c over the monolayer plane . The optical linear dichroism and these initial FRAP studies have indicated that cytochrome c electrostatically bound to a lipid surface maintains a well-defined orientation relative to the membrane surface while exhibiting measurable, but highly restricted, lateral motion in the plane of the surface.

Proc Natl Acad Sci U S A, 1990 Aug, 87(16), 6029 - 33
Cloning and expression of rat liver CTP: phosphocholine cytidylyltransferase: an amphipathic protein that controls phosphatidylcholine synthesis; Kalmar GB et al.; CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) is a key regulatory enzyme in the synthesis of phosphatidylcholine in higher eukaryotes . This enzyme can interconvert between an inactive cytosolic form and an active membrane-bound form . To unravel the structure of the transferase and the mechanism of its interaction with membranes, we have cloned a cytidylyltransferase cDNA from rat liver by the oligonucleotide-directed polymerase chain reaction . Transfection of the rat clone into COS cells resulted in a 10-fold increase in cytidylyltransferase activity and content . The activity of the transfected transferase was lipid-dependent . The central portion of the derived protein sequence of the rat clone is highly homologous to the previously determined yeast cytidylyltransferase sequence {Tsukagoshi, Y., Nikawa, J . & Yamashita, S . (1987) Eur . J . Biochem . 169, 477-486} . The rat protein sequence lacks any signals for covalent lipid attachment and lacks a hydrophobic domain long enough to span a bilayer . However, it does contain a potential 58-residue amphipathic alpha-helix, encompassing three homologous 11-residue repeats . We propose that the interaction of cytidylyltransferase with membranes is mediated by this amphipathic helix lying on the surface with its axis parallel to the plane of the membrane such that its hydrophobic residues intercalate the phospholipids.

Singapore Med J, 1990 Aug, 31(4), 314 - 6
The evaluation of Engerix-B in healthy Malaysian medical students; Isahak I et al.; Fifty medical students were screened for hepatitis B serological markers of whom 42 students entered the study . Those who were found to be negative for all markers were vaccinated with 1.0 ml (20 mcg HBsAg) Engerix-B vaccine intramuscularly in the deltoid region according to the 0, 1, 6 month schedule . Blood samples were taken at 1, 2, 3, 6, 9 months . One month following the first dose, 7% showed detectable AntiHBs with a GMT of 11 IU/I . By the sixth month, just before the third dose was given, 79% seroconverted with a GMT of 2952 IU/I . Three months following the third dose all had seroconverted with a GMT of 18,381 IU/I . No serious adverse reactions were noted and none of the subjects showed evidence of hepatitis B infection during the study . This study thus confirms the high immunogenicity and safety of recombinant yeast-extract hepatitis B vaccine.

J Gen Microbiol, 1990 Aug, 136 ( Pt 8), 1483 - 90
Purification and properties of two lactose hydrolases from Trichosporon cutaneum; West M et al.; Two enzymes that hydrolysed lactose were purified essentially to homogeneity from cell extracts of the oleaginous yeast Trichosporon cutaneum . One enzyme of Mr 120,000 had properties typical of a beta-galactosidase (EC 3.2.1.23) . It hydrolysed lactose, lactulose and nitrophenyl-beta-D-galactosides . The enzyme required K+ or Rb+ for activity, and other monovalent cations tested were not effective . Enzyme activity was abolished by EDTA and stimulated by Mg2+, Mn2+ and Ca2+ . The beta-galactosidase was induced by lactose, galactose, lactulose and lactobionic acid . The other enzyme, a beta-glycosidase (EC 3.2.1.21) of Mr 52,000 showed no ionic requirements and it hydrolysed lactose, nitrophenyl-beta-D-galactosides, 4-nitrophenyl-beta-D-glucoside, cellobiose, laminaribiose, laminaritriose and sophorose, but not gentiobiose, 4-nitrophenyl-beta-D-mannoside or sucrose . This enzyme was induced by lactose, galactose and lactulose, and also by cellobiose.

Biochim Biophys Acta, 1990 Jul 30, 1049(3), 255 - 60
Eukaryotic tRNAs(Pro): primary structure of the anticodon loop; presence of 5-carbamoylmethyluridine or inosine as the first nucleoside of the anticodon; Keith G et al.; The modified nucleoside U*, located in the first position of the anticodon of yeast, chicken liver and bovine liver tRNA(Pro) (anticodon U*GG), has been determined by means of TLC, HPLC, ultraviolet spectrum and gas chromatography-mass spectrometry . The structure was established as 5-carbamoylmethyluridine (ncm5U) . In addition, we report on the primary structures of the above-mentioned tRNAs as well as those which have the IGG anticodon . In yeast, the two tRNA(Pro) (anticodons U*GG and IGG) differ by eight nucleotides, whereas in chicken and in bovine liver, both anticodons are carried by the same 'body tRNA' with one posttranscriptional exception at position 32, where pseudouridine is associated with ncm5U (position 34) in tRNA(Pro) (U*GG) and 2'-O-methylpseudouridine is associated with inosine (position 34) in tRNA(Pro) (IGG).

Cell, 1990 Jul 27, 62(2), 317 - 29
Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments; Chavrier P et al.; A set of 11 clones encoding putative GTP binding proteins highly homologous to the yeast YPT1/SEC4 gene products have been isolated from an MDCK cell cDNA library . We localized three of the corresponding proteins in mammalian cells by using affinity-purified antibodies in immunofluorescence and immunoelectron microscopy studies . One, the MDCK homolog of rab2, is associated with a structure having the characteristics of an intermediate compartment between the endoplasmic reticulum and the Golgi apparatus . The second, rab5, is located at the cytoplasmic surface of the plasma membrane and on early endosomes, while the third, rab7, is found on late endosomes . These findings provide evidence that members of the YPT1/SEC4 subfamily of GTP binding proteins are localized to specific exocytic and endocytic subcompartments in mammalian cells.

Nature, 1990 Jul 26, 346(6282), 387 - 90
Highly conserved core domain and unique N terminus with presumptive regulatory motifs in a human TATA factor (TFIID); Hoffman A et al.; The factor TFIID is one of several general factors that are necessary and sufficient for transcription initiation by mammalian RNA polymerase II . Stable interactions with the common TATA element lead both to template commitment and to the assembly of the other general factors into a functional preinitiation complex . Consistent with its key role in the promoter activation pathway, human TFIID also seems to be a target for some regulatory factors, as evidenced both by physical and functional studies of interactions between these components . The evolutionary conservation of functional properties led to the purification and cloning of yeast TFIID, the identification of presumptive structural motifs, and direct structure-function studies . Here we report the cloning of a complementary DNA encoding a functional human TFIID . This reveals an evolutionarily conserved core which corresponds precisely to the 180-residue DNA binding/activation domain determined for yeast TFIID, a near absolute conservation of component structural motifs (direct repeats, central basic core/lysine repeat, and sigma homology), providing further support for their functional importance, and a unique N-terminal structure that suggests involvement in species-specific regulatory factor interactions.

Nature, 1990 Jul 26, 346(6282), 379 - 82
Triggering of cyclin degradation in interphase extracts of amphibian eggs by cdc2 kinase; Felix MA et al.; The cell cycles of early Xenopus embryos consist of a rapid succession of alternating S and M phases . These cycles are controlled by the activity of a protein kinase complex (cdc2 kinase) which contains two subunits . One subunit is encoded by the frog homologue of the fission yeast cdc2+ gene, p34cdc2 and the other is a cyclin . The concentration of cyclins follows a sawtooth oscillation because they accumulate in interphase and are destroyed abruptly during mitosis . The association of cyclin and p34cdc2 is not sufficient for activation of cdc2 kinase, however; dephosphorylation of key tyrosine and threonine residues of p34cdc2 is necessary to turn on its kinase activity . The activity of cdc2 kinase is thus regulated by a combination of translational and post-translational mechanisms . The loss of cdc2 kinase activity at the end of mitosis depends on the destruction of the cyclin subunits . It has been suggested that this destruction is induced by cdc2 kinase itself, thereby providing a negative feedback loop to terminate mitosis . Here we report direct experimental evidence for this idea by showing that cyclin proteolysis can be triggered by adding cdc2 kinase to a cell-free extract of interphase Xenopus eggs.

J Biol Chem, 1990 Jul 25, 265(21), 12486 - 93
Ubiquitin extension proteins of Arabidopsis thaliana . Structure, localization, and expression of their promoters in transgenic tobacco; Callis J et al.; The highly conserved protein ubiquitin is synthesized in eukaryotes as two types of protein fusions from which active ubiquitin is derived by proteolytic processing . We report here the isolation and characterization of multiple genes from one type that encode ubiquitin extension proteins from the higher plant, Arabidopsis thaliana (L.) . Two genes with 90% nucleotide identity in their exons encode ubiquitin and identical 52-amino acid (aa) extension proteins with 85 and 79% aa identity to 52-aa extension proteins from humans and yeast, respectively . Two other genes with 90% nucleotide identity encode ubiquitin and 81-aa extension proteins that differ by 4 amino acids from each other and are approximately 70% identical to the 76- and the 80-aa extension proteins from yeast and humans, respectively . Antibodies recognizing the 52- and 81-aa Arabidopsis extension proteins identify them as constituents of ribosomes . By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 52- and 81-aa extension proteins migrate at 6.8 and 11.5 kDa, respectively, and neither cross-reacts with anti-ubiquitin antibodies, indicating that extension proteins are cleaved from ubiquitin following translation . Ubiquitin extension protein genes encode the smallest transcript size class of ubiquitin mRNAs in Arabidopsis . The 5'-flanking regions of both UBQ1 and UBQ6, genes representative of the both extension proteins, direct the expression of readily detectable levels of the marker enzyme beta-glucuronidase in transgenic tobacco, suggesting the utility of these promoters for expression of foreign genes in higher plants.

J Biol Chem, 1990 Jul 25, 265(21), 12611 - 7
Mammalian DNA ligases . Catalytic domain and size of DNA ligase I; Tomkinson AE et al.; DNA ligase I is the major DNA ligase activity in proliferating mammalian cells . The protein has been purified to apparent homogeneity from calf thymus . It has a monomeric structure and a blocked N-terminal residue . DNA ligase I is a 125-kDa polypeptide as estimated by sodium dodecyl sulfate-gel electrophoresis and by gel chromatography under denaturing conditions, whereas hydrodynamic measurements indicate that the enzyme is an asymmetric 98-kDa protein . Immunoblotting with rabbit polyclonal antibodies to the enzyme revealed a single polypeptide of 125 kDa in freshly prepared crude cell extracts of calf thymus . Limited digestion of the purified DNA ligase I with several reagent proteolytic enzymes generated a relatively protease-resistant 85-kDa fragment . This domain retained full catalytic activity . Similar results were obtained with partially purified human DNA ligase I . The active large fragment represents the C-terminal part of the intact protein, and contains an epitope conserved between mammalian DNA ligase I and yeast and vaccinia virus DNA ligases . The function of the N-terminal region of DNA ligase I is unknown.

J Mol Biol, 1990 Jul 20, 214(2), 585 - 95
High-resolution three-dimensional structure of horse heart cytochrome c; Bushnell GW et al.; The 1.94 A resolution three-dimensional structure of oxidized horse heart cytochrome c has been elucidated and refined to a final R-factor of 0.17 . This has allowed for a detailed assessment of the structural features of this protein, including the presence of secondary structure, hydrogen-bonding patterns and heme geometry . A comprehensive analysis of the structural differences between horse heart cytochrome c and those other eukaryotic cytochromes c for which high-resolution structures are available (yeast iso-1, tuna, rice) has also been completed . Significant conformational differences between these proteins occur in three regions and primarily involve residues 22 to 27, 41 to 43 and 56 to 57 . The first of these variable regions is part of a surface beta-loop, whilst the latter two are located together adjacent to the heme group . This study also demonstrates that, in horse cytochrome c, the side-chain of Phe82 is positioned in a co-planar fashion next to the heme in a conformation comparable to that found in other cytochromes c . The positioning of this residue does not therefore appear to be oxidation-state-dependent . In total, five water molecules occupy conserved positions in the structures of horse heart, yeast iso-1, tuna and rice cytochromes c . Three of these are on the surface of the protein, serving to stabilize local polypeptide chain conformations . The remaining two are internally located . One of these mediates a charged interaction between the invariant residue Arg38 and a nearby heme propionate . The other is more centrally buried near the heme iron atom and is hydrogen bonded to the conserved residues Asn52, Tyr67 and Thr78 . It is shown that this latter water molecule shifts in a consistent manner upon change in oxidation state if cytochrome c structures from various sources are compared . The conservation of this structural feature and its close proximity to the heme iron atom strongly implicate this internal water molecule as having a functional role in the mechanism of action of cytochrome c.

Biochem J, 1990 Jul 15, 269(2), 451 - 8
Nucleotide sequence of cDNA coding for rat liver pI 6.1 esterase (ES-10), a carboxylesterase located in the lumen of the endoplasmic reticulum; Robbi M et al.; A commercial rat liver cDNA library in lambda gt11 was screened with a rabbit antiserum to native pI 6.1 esterase (ES-10) . The inserts of the immunoreactive clones were short (0.9-1.1 kbp) . One of these was used as a probe to rescreen the library, yielding 30 clones, two of which contained relatively long (approx . 1.9 kbp) and widely overlapping cDNA inserts . They did not contain the first two nucleotide residues of the initiator codon, nor the 5'-end untranslated portion of the mRNA . These were derived from a home-made rat liver cDNA library in lambda gt11, screened with an oligonucleotide corresponding to the 5'-end of the already known cDNA sequence . The nucleotide sequence consists of 48 bp of 5'-end non-coding region, 1695 bp of coding region and 212 bp of 3'-end non-coding region including a 20 bp poly(A) tail . The signal peptide and the mature protein subunit are 18 and 547 residues long respectively . Tyr is confirmed as N-terminal residue . The predicted amino acid sequence is highly similar to those of rabbit liver esterase forms 1 (77% identity) and 2 (56% identity), determined by protein sequencing {Korza & Ozols (1988) J . Biol . Chem . 263, 3486-3495; Ozols (1989) J . Biol . Chem . 264, 12533-12545} . The three enzymes share the Ser and His residues presumed to be part of the active site, four Cys residues and a high proportion of charged side chains at their C-terminus . The C-terminal tetrapeptides of the three esterases (-HVEL, -HIEL and -HTEL for pI 6.1 and forms 1 and 2 esterases respectively) are reminiscent of, but not identical with, the localization signal identified in other proteins of the endoplasmic-reticulum lumen (-KDEL in animal cells {Munro & Pelham (1987) Cell 48, 899-907}; -HDEL in yeast {Pelham, Hardwick & Lewis (1988) EMBO J . 7, 1757-1762}) . We still lack direct evidence to decide whether or not these C-terminal tetrapeptides commit esterases to reside in the endoplasmic reticulum . In that case the antepenultimate residue (D, V, I or T) would be only weakly stringent, and some sequences primed by H instead of K would be recognized in animal as well as in yeast cells.

J Biol Chem, 1990 Jul 15, 265(20), 11692 - 9
The stereospecificities of seven dehydrogenases from Acholeplasma laidlawii . The simplest historical model that explains dehydrogenase stereospecificity; Glasfeld A et al.; Stereospecificities are reported for seven dehydrogenases from Acholeplasma laidlawii, an organism from an evolutionarily distinct branch of life which has not previously been studied from a stereochemical point of view . Three of the activities examined (alcohol dehydrogenase, lactate dehydrogenase, and alanine dehydrogenase) catalyze the transfer of the pro-R (A) hydrogen from NADH . Four other activities (3-hydroxy-3-methylglutaryl-CoA reductase, glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, and NADH oxidase) catalyze the transfer of the pro-S (B) hydrogen from NAD(P)H . The stereospecificity of hydroxymethylglutaryl-CoA reductase is notable because it is the opposite of that of hydroxymethylglutaryl-CoA reductases from yeast and rat . These data are used to derive the simplest historical model capable of explaining available experimental facts.

Experientia, 1990 Jul 15, 46(7), 731 - 3
Chromate reduction in Streptomyces; Das S et al.; Streptomyces species 3M grew in peptone yeast extract medium with 1000 micrograms/ml K2Cr2O7 . Incubation of the chromate with different cell fractions in the presence of NADH and NADPH resulted in a decrease of Cr6+ in the reaction mixture . The level of Cr6+ was reduced by 82.7% by a particulate cell fraction obtained by centrifugation at 105,000 x g for 1 h, in the presence of NADH . The reducing enzyme was associated with this cell fraction . The enzyme was constitutive and reduced Cr6+ to Cr3+.

Science, 1990 Jul 13, 249(4965), 181 - 6
Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NF1 patients; Wallace MR et al.; Von Recklinghausen neurofibromatosis (NF1) is a common autosomal dominant disorder characterized by abnormalities in multiple tissues derived from the neural crest . No reliable cellular phenotypic marker has been identified, which has hampered direct efforts to identify the gene . The chromosome location of the NF1 gene has been previously mapped genetically to 17q11.2, and data from two NF1 patients with balanced translocations in this region have further narrowed the candidate interval . The use of chromosome jumping and yeast artificial chromosome technology has now led to the identification of a large (approximately 13 kilobases) ubiquitously expressed transcript (denoted NF1LT) from this region that is definitely interrupted by one and most likely by both translocations . Previously identified candidate genes, which failed to show abnormalities in NF1 patients, are apparently located within introns of NF1LT, on the antisense strand . A new mutation patient with NF1 has been identified with a de novo 0.5-kilobase insertion in the NF1LT gene . These observations, together with the high spontaneous mutation rate of NF1 (which is consistent with a large locus), suggest that NF1LT represents the elusive NF1 gene.

Nucleic Acids Res, 1990 Jul 11, 18(13), 3871 - 9
Characterization of a human cDNA encoding a widely expressed and highly conserved cysteine-rich protein with an unusual zinc-finger motif; Liebhaber SA et al.; A human term placental cDNA library was screened at low stringency with a human prolactin cDNA probe . One of the cDNAs isolated hybridizes to a 1.8 kb mRNA present in all four tissues of the placenta as well as to every nucleated tissue and cell line tested . The sequence of the full-length cDNA was determined . An extended open reading frame predicted an encoded protein product of 20.5 kDa . This was directly confirmed by the in vitro translation of a synthetic mRNA transcript . Based upon the characteristic placement of cysteine (C) and histidine (H) residues in the predicted protein structure, this molecule contains four putative zinc fingers . The first and third fingers are of the C4 class while the second and fourth are of the C2HC class . Based upon sequence similarities between the first two and last two zinc fingers and sequence similarities to a related rodent protein, cysteine-rich intestinal protein (CRIP), these four finger domains appear to have evolved by duplication of a preexisting two finger unit . Southern blot analyses indicate that this human cysteine-rich protein (hCRP) gene has been highly conserved over the span of evolution from yeast to man . The characteristics of this protein suggest that it serves a fundamental role in cellular function.

Nature, 1990 Jul 5, 346(6279), 35 - 9
The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors; Yanofsky MF et al.; Mutations in the homeotic gene agamous of the plant Arabidopsis cause the transformation of the floral sex organs . Cloning and sequence analysis of agamous suggest that it encodes a protein with a high degree of sequence similarity to the DNA-binding region of transcription factors from yeast and humans and to the product of a homeotic gene from Antirrhinum . The agamous gene therefore probably encodes a transcription factor that regulates genes determining stamen and carpel development in wild-type flowers.

J Biol Chem, 1990 Jul 5, 265(19), 11265 - 72
A peptidase in human platelets that deamidates tachykinins . Probable identity with the lysosomal "protective protein"; Jackman HL et al.; We discovered an enzyme in human platelets that deamidates substance P and other tachykinins . Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase . The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94 . The purified enzyme exhibits esterase, peptidase, and deamidase activities . The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0 . With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5 . Enkephalins, released upon deamidation of enkephalinamides, were not cleaved . Gly9-NH2 of oxytocin was released without deamidation . Peptides with a penultimate Arg residue were not hydrolyzed . Some properties of the deamidase are similar to those reported for cathepsin A . The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective . In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer . After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . {3H}diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain . The first 25 amino acids of both chains were sequenced . They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast . This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability . A defect in this protein is the cause of galactosialidosis, a severe genetic disorder . The ability of physiological stimuli (e.g . thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.

FEBS Lett, 1990 Jul 2, 267(1), 117 - 20
Formation and decay of cytochrome c peroxidase compound ES during aerobic reduction with dithionite; Orii Y et al.; Stopped-flow and rapid scanning studies have clearly demonstrated that mixing of an oxygen-saturated solution of yeast cytochrome c peroxidase with sodium dithionite yields compound ES, indicating generation of H2O2 . The formation of compound ES was most pronounced when {Na2S2O4}/{O2} approximately 1, and it reverted to the ferric form while standing . Even in the presence of an excess of dithionite ({Na2S2O4}/{O2} = 3.4) compound ES was formed immediately, but was soon replaced by the ferric form, followed by its final reduction to the ferrous state . The apparent first order rate constant for the decay of compound ES to the ferric form increased linearly with the square root of the dithionite concentration, thus involvement of SO2- in that process being suggested.

Virus Genes, 1990 Jul, 4(2), 121 - 36
RNA pseudoknots: translational frameshifting and readthrough on viral RNAs; ten Dam EB et al.; Ribosomal frameshifting on retroviral RNAs has been proposed to be mediated by slippage of two adjacent tRNAs into the -1 direction at a specific heptanucleotide sequence . Here we report a computer-aided analysis of the structure around the established or putative frameshift sites in a number of retroviral, coronaviral, toroviral, and luteoviral RNAs and two dsRNA yeast viruses . In almost all cases a stable hairpin was predicted four to nine nucleotides downstream of the shifty heptanucleotide . More than half of the resulting hairpin loops give rise to potential pseudoknotting with sequences downstream of this hairpin . Especially in the case of the shifty heptanucleotides U UUA AAC and G GGA AAC, stable downstream pseudoknots are present . Indications were also found for the presence of pseudoknots downstream of amber stop condons at readthrough sites in some retroviral RNAs.

J Submicrosc Cytol Pathol, 1990 Jul, 22(3), 367 - 78
Freeze fracture and scanning electron microscope studies on the nuclear envelope and perinuclear cytomembranes (parabasal apparatus) in the protozoan, Lophomonas blattarum; Kessel RG et al.; Phase contrast, scanning electron microscope (SEM), and freeze fracture studies on the parasitic flagellate, Lophomonas blattarum, have demonstrated that the endomembrane system (parabasal apparatus) is highly ordered, restricted in position to a perinuclear zone at the anterior end of the organism, and is supported in this localized cytoplasmic region by overlapping sheets or plates of microtubules, previously called the calyx and axial filament, which may participate in supporting the nucleus-endomembrane system in a restricted region of the cell . Light microscope observations, SEM and freeze fracture data provide support to previous views that the rough- and smooth-surfaced endoplasmic reticulum are interconnected and attached to the outer layer of the nuclear envelope . The continuity of these membrane systems provides an orderly and restricted packing in the perinuclear cytoplasm since other areas of the cell may become filled with yeast . These flagellates are especially adept at phagocytosis of entire yeast . In yeast-laden cells, the flagella-nucleus-parabasal body-calyx-axial filament complex may separate from the remainder of the cell and assume a motile existence for a time . The significance of the described relationships, in addition to providing efficiency in endomembrane localization, may also reflect synthesis of enzymes and proteins by the RER and packaging in the SER, both of which are continuous . Granules characteristic of glycogen are concentrated around the SER which may be involved in glycogen metabolism . Although critical information is lacking, the endomembrane system may also be involved in the synthesis and morphogenesis of lysosomes, and perhaps peroxisomes . Lophomonas thus amplifies a highly ordered spatial relationship between the nuclear envelope and the ER.

AIDS Res Hum Retroviruses, 1990 Jul, 6(7), 855 - 69
Importance of hypervariable regions of HIV-1 gp120 in the generation of virus neutralizing antibodies; Haigwood NL et al.; Variants of the envelope gene of the HIV-SF2 isolate of HIV-1 with deletions of one or more of the hypervariable domains of gp120 were produced in genetically engineered yeast as nonglycosylated denatured polypeptide analogs of gp120 . Purified antigens were used to immunize experimental animals to determine whether the removal of hypervariable regions from this type of gp120 immunogen had any effect on (1) the ability of the antigen to elicit virus neutralizing antibodies; and (2) the isolate specificity of the neutralizing antibodies that were elicited . The results of these studies demonstrate that, in addition to the previously identified V3 domain, at least two other hypervariable regions in gp120 are capable of eliciting neutralizing antibodies in experimental animals . However, when all five of the hypervariable regions were deleted, the resulting antigen was no longer capable of eliciting neutralizing antibodies . Finally, the neutralizing antibodies elicited by all of these nonglycosylated antigens were effective against HIV-SF2, the isolate from which the antigens were derived, but were not able to neutralize two divergent isolates, HIV-BRU or HIV-Zr6.

Appl Environ Microbiol, 1990 Jul, 56(7), 2228 - 33
Oxidation of benzaldehydes to benzoic acid derivatives by three Desulfovibrio strains; Zellner G et al.; Desulfovibrio vulgaris Marburg, "Desulfovibrio simplex" XVI, and Desulfovibrio sp . strain MP47 used benzaldehydes such as vanillin, 3,4,5-trimethoxybenzaldehyde, protocatechualdehyde, syringaldehyde, p-anisaldehyde, p-hydroxybenzaldehyde, and 2-methoxybenzaldehyde as electron donors for sulfate reduction and carbon dioxide and/or components of yeast extract as carbon sources for cell synthesis . The aldehydes were oxidized to their corresponding benzoic acids . The three sulfate reducers oxidized up to 7 mM vanillin and up to 4 mM p-anisaldehyde . Higher concentrations of vanillin or p-anisaldehyde were toxic . In addition, pyridoxal hydrochloride and o-vanillin served as electron donors for sulfate reduction . Salicylaldehyde, pyridine-2-aldehyde, pyridine-4-aldehyde, and 4-hydroxy-3-methoxybenzylalcohol were not oxidized . No molecular hydrogen was detected in the gas phase . The oxidized aldehydes were not further degraded.

Mol Cell Biol, 1990 Jul, 10(7), 3415 - 20
DNA-binding and transcriptional properties of human transcription factor TFIID after mild proteolysis; Van Dyke MW et al.; The existence of separable functions within the human class II general transcription factor TFIID was probed for differential sensitivity to mild proteolytic treatment . Independent of whether TFIID was bound to DNA or free in solution, partial digestion with either one of a variety of nonspecific endoproteases generated a protease-resistant protein product that retained specific DNA recognition, as revealed by DNase I footprinting . However, in contrast to native TFIID, which interacts with the adenovirus major late (ML) promoter over a very broad DNA region, partially proteolyzed TFIID interacted with only a small region of the ML promoter immediately surrounding the TATA sequence . This novel footprint was very similar to that observed with the TATA factor purified from yeast cells . Partially proteolyzed human TFIID could form stable complexes that were resistant to challenge by exogenous templates . It could also nucleate the assembly of transcription complexes on the ML promoter with an efficiency comparable to that of native TFIID, yielding similar levels of transcription initiation . These results suggest a model in which the human TFIID protein is composed of at least two different regions or polypeptides: a protease-resistant "core," which by itself is sufficient for promoter recognition and basal transcriptional levels, and a protease-sensitive "tail," which interacts with downstream promoter regions and may be involved in regulatory processes.

Proc Natl Acad Sci U S A, 1990 Jul, 87(14), 5397 - 401
Cell division in higher plants: a cdc2 gene, its 34-kDa product, and histone H1 kinase activity in pea; Feiler HS et al.; The mitotic cell cycle of yeast and animal cells is regulated by the cdc2 gene and its product, the p34 protein kinase, and by other components of the MPF or histone H1 kinase complex . We present evidence that cdc2, p34, and a histone H1 kinase also exist in higher plants . Protein extracts from 10 plant species surveyed display a 34-kDa component recognized by a monoclonal antibody directed against an evolutionarily conserved epitope of fission yeast p34 . Nondenatured protein extracts of mitotic Pisum sativum (garden pea) tissues were fractionated by gel filtration, electrophoretically separated under denaturing conditions, and immunoblotted . p34 crossreactive material was apparent in both low and high molecular mass fractions, indicating that pea p34 occurs as both a monomer and as part of a high molecular mass complex . Histone H1 kinase activity was found predominantly in the higher molecular mass fractions, those to which the least phosphorylated form of pea p34 was confined . We also report the cloning of the pea homologue of cdc2 by polymerase chain reaction . DNA sequence analysis reveals perfect conservation of the hallmark "PSTAIR" sequence motif found in all cdc2 gene products analyzed to date.

EMBO J, 1990 Jul, 9(7), 2289 - 98
Self-splicing of the mobile group II intron of the filamentous fungus Podospora anserina (COI I1) in vitro; Schmidt U et al.; The first intron of the mitochondrial gene coding for cytochrome oxidase subunit I (COI I1) of Podospora anserina can undergo self-splicing in vitro at high concentrations of NH4Cl or KCl . Under these conditions cleavage at the 5' splice junction takes place without branch formation probably via hydrolysis by water or OH- and the intron is released in a linear form . In vitro transcripts that contain mutated introns with large deletions in nonconserved domain IV comprising greater than 50% of the intronic sequence display a more efficient splicing reaction and, surprisingly, 5' cleavage via transesterification and lariat formation is re-established to a low degree under NH4Cl . In contrast to the self-splicing group II introns aI5 gamma and bI1 from yeast mitochondria cleavage at the 3' splice site of the Podospora intron is reduced and cleavage by hydrolysis in trans (i.e . exon reopening) is almost completely suppressed . Both observations could be interpreted as a result of unfavourable spatial conformations of the intron that (i) lead to a steric hindrance of the 5' exon to attack the 3' splice site in cis and (ii) block intron-dependent cleavage reaction of the ligated exons in trans . Alternatively, the possibility that a weak overall interaction of the postulated exon- with the corresponding intron-binding sites (EBS-IBS pairings) is responsible for the remarkable differences to the self-splicing reaction of other group II introns is discussed.

Chem Res Toxicol, 1990 Jul-Aug, 3(4), 325 - 32
Heterocyclic derivatives of 3-substituted-1,1,1-trifluoro-2-propanones as inhibitors of esterolytic enzymes; Szekacs A et al.; A series of (alkylthio)trifluoropropanones containing a heterocyclic moiety was synthesized . The compounds were tested for in vitro inhibition of four hydrolytic enzymes including insect juvenile hormone esterase (JHE), eel acetylcholinesterase (AChE), yeast lipase (LP), and bovine alpha-chymotrypsin . The I50 values ranged from 10(-3) to 10(-7) M . 3-(2-Pyridylthio)-1,1,1-trifluoro-2-propanone was found to be the most potent inhibitor as compared to the other tested heterocyclic analogues with an I50 value of 98 nM against JHE from the fifth-instar larvae of Trichoplusia ni . Results from X-ray crystallography showed that the compound exists in a tetrahedral gem-diol form stabilized by an intramolecular hydrogen bond in the solid state . X-ray crystallography of a less potent inhibitor, 3-(4-pyridylthio)-1,1,1-trifluoro-2- propanone, showed that it also exists in the hydrated form, but it lacks an intramolecular hydrogen bond . These results provide indirect support that trifluoromethyl ketones are transition-state mimic inhibitors of esterases, and the bearing of the results on the transition-state mimic theory is discussed . The I50 values against AChE were in the micromolar range . Compounds containing a imidazolyl, triazolyl, and pyrimidyl moiety showed the highest inhibition of this enzyme . Differential selectivity of inhibition was associated with the bond distances between the nitrogen and the carbonyl group as in the natural substrate, when measured in the molecules in their minimal energy conformations . Inhibition of LP was moderate to weak, when compared to JHE and AChE . None of the tested compounds showed significant inhibition of alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Mycoses, 1990 Jul-Aug, 33(7-8), 375 - 81
Comparison of enzyme immunoassay and immunodiffusion for the detection of canine blastomycosis; Seawell BW et al.; Two Blastomyces dermatitidis commercial immunodiffusion antigens, Meridian Diagnostics and Nolan-Scott Laboratories, and two B . dermatitidis yeast phase lysate antigens, T-58 and K-Le, prepared in our laboratory were utilized in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in 61 serum specimens from dogs with blastomycosis . All antigens used in the ELISA were diluted to 50 ng of protein ml-1 . Immunodiffusion (ID) tests were also performed on the sera using a Nolan-Scott ID antigen . Greater reactivity, based on the ELISA index value, was evidenced with the two yeast phase lysate antigens over the two commercial reagents in the ELISA . In addition the sensitivity of the ELISA with the T-58, K-Le and Meridian antigens was considerably greater than that of the ID test . The Nolan-Scott antigen, however, was able to detect more positive specimens when used in the ID procedure than in the ELISA . Therefore encouraging results were obtained with respect to the continued development and optimization of the ELISA using B . dermatitidis cell lysate antigens for the diagnosis of blastomycosis.

Diagn Microbiol Infect Dis, 1990 Jul-Aug, 13(4), 297 - 302
Candidal infection of bone . Assessment of serologic tests in diagnosis and management; Quindos G et al.; In this case report, 30 sera from a 25-year-old heroin abuser with intervertebral candidosis were treated for the presence of anti-Candida albicans antibodies by agglutination, counterimmunoelectrophoresis, and indirect immunofluorescent assay . Sera were also adsorbed with heat-killed blastospores to remove antibodies against yeast-phase cells and tested by indirect immunofluorescent assay for anti-C . albicans germ tube antibodies (CAGTAs) . Humoral responses to candidal 47-kD antigen were studied by immunoblotting in 23 unadsorbed sera . Anti-C . albicans antibodies were found in high titers by the three procedures but correlated poorly with the clinical evolution of the disease . CAGTAs were present from the beginning of the infection: Titers decreased in association with antifungal treatment and the patient's improvement, eventually becoming negative . Only class IgG antibodies to the 47-kD antigen were detected . These were present during the full course of the infection, failing to disappear at the end of the study . In this case, detection of CAGTAs appeared to be an important aid to diagnosis of the bony candidal infection, as they are detected early during the illness and seemed to have a prognostic significance.

J Am Acad Dermatol, 1990 Jul, 23(1), 82 - 6
Correlation of Pityosporum ovale density with clinical severity of seborrheic dermatitis as assessed by a simplified technique; Heng MC et al.; One hundred patients with facial seborrheic dermatitis and 42 control subjects were studied . The number of periodic acid-Schiff-positive Pityosporum ovale yeast cells in skin scrapings per high-power field were counted and designated 1 + to 4 + . Our data indicate a correlation between the density of P . ovale and the clinical severity of seborrheic dermatitis, both before and after therapy with a precipitated sulfur/salicyclic acid shampoo . The data support the concept that yeast contributes to the pathogenesis of seborrheic dermatitis.

Thromb Haemost, 1990 Jun 28, 63(3), 499 - 504
Physico-chemical properties of recombinant desulphatohirudin; Electricwala A et al.; Physico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No . CGP 39393) were reinvestigated . As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure . However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation . Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al . for a natural hirudin-variant . Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies.

Nature, 1990 Jun 28, 345(6278), 829 - 32
Regulation of the pituitary-specific homeobox gene GHF1 by cell-autonomous and environmental cues; McCormick A et al.; Homeodomain proteins function in determination of mating type in yeast, segmentation in fruit flies and cell-type specific gene expression in mammals . In Drosophila, expression of homeobox genes is controlled by cell-autonomous interactions between regulatory proteins and environmental clues . Similar controls may operate during mammalian limb development and frog embryogenesis . But, the exact way in which expression of homeodomain proteins is regulated in these systems is not clear and requires biochemical analysis of homeobox gene transcription . We now describe such an analysis of the GHF1 gene, which encodes a mammalian homeodomain protein specifying expression of the growth hormone (GH) gene in anterior pituitary somatotrophs . GHF1 is transcribed in a highly restricted manner and the presence of GHF1 protein is correlated both temporally and spatially with activation of the GH gene during pituitary development . Analysis of the GHF1 promoter indicates that transcription is also controlled by cell-autonomous interactions involving positive autoregulation by GHF1, and environmental cues that modulate the intracellular level of cyclic AMP and thereby the activity of cAMP response element binding protein (CREB), a ubiquitous transactivator that binds to the GHF1 promoter.

J Biol Chem, 1990 Jun 25, 265(18), 10589 - 96
Mapping the binding sites of human erythrocyte ankyrin for the anion exchanger and spectrin; Davis LH et al.; This report describes initial characterization of the binding sites of ankyrin for spectrin and the anion exchanger using defined subfragments isolated from purified ankyrin domains . The spectrin-binding domain of ankyrin is comprised of two subdomains: an acidic, proline-rich region (pI = 4) involving the amino-terminal 80 residues from 828 to 908 and a basic region (pI = 8.8) that extends from 898 to 1386 . The amino-terminal 70 amino acids of the spectrin-binding domain are critical for association with spectrin, since a subfragment missing this region is only 5% as active as the intact domain in displacing binding of spectrin to inside-out membrane vesicles, while deletion of the first 38 residues of the acidic domain results in a 10-fold reduction in activity . The anion exchanger-binding site is confined to an 89-kDa domain that was isolated and characterized as a globular molecule with approximately 30% alpha-helical configuration . A subfragment of the 89-kDa domain extending from residues 403 to 779 (or possibly 740) retains ability to associate with the anion exchanger . The 89-kDa domain is comprised of a series of tandem repeats of 33 amino acids that extend from residues 35 to 778 (Lux, S., John, K., and Bennett, V . (1990) Nature 344, 36-42) . The activity of residues 403-779 demonstrates that the 33-amino acid repeats of the 89-kDa domain are responsible for association between ankyrin and the anion exchanger . The 33-amino acid repeating sequence of ankyrin represents an ancient motif also found in proteins of Drosophila, yeast, and Caenor habditis elegans . The finding that the 33-amino acid repeating sequence is involved in interaction with the anion exchanger implies that this motif may perform a role in molecular recognition in diverse proteins.

J Biol Chem, 1990 Jun 25, 265(18), 10582 - 8
Isolation of distinct small ribonucleoprotein particles containing the spliced leader and U2 RNAs of Trypanosoma brucei; Michaeli S et al.; Messenger RNA maturation in trypanosomes involves an RNA trans-splicing reaction in which a 39 nucleotide 5'-spliced leader (SL), derived from an independently transcribed 139 nucleotide SL RNA, is joined to pre-mRNAs . Trans-splicing intermediates are structurally consistent with a mechanism of SL addition which is similar to that of cis-splicing of nuclear pre-mRNAs; homologous components (e.g . the U small nuclear RNAs) exist in both cis- and trans-splicing systems, suggesting that these also participate in the two types of splicing reactions . In this study, ribonucleoprotein (RNP) complexes containing the trypanosome SL and U2 RNAs were purified and characterized . Although present at low levels in cellular extracts, the SL and U2 RNPs are the two most abundant of the several non-ribosomal small RNP complexes in these cells . The purification scheme utilizes ion-exchange chromatography, equilibrium density centrifugation, and gel filtration chromatography and reveals that the SL RNP shares biophysical properties with U RNPs of trypanosomes and other eukaryotes; its sedimentation coefficient in sucrose gradients is approximately 10 S, and it is resistant to dissociation during Cs2SO4 equilibrium density centrifugation . Complete separation of the SL and U2 RNPs was achieved by non-denaturing polyacrylamide gel electrophoresis . Proteins purifying with the SL and U2 RNPs were identified by 125I-labeling of tyrosine residues . Four SL RNP proteins with approximate molecular masses of 36, 32, 30, and 27 kDa and one U2 RNP protein of 31 kDa were identified, suggesting that different polypeptides are associated with these two RNAs . These particles are not immunoprecipitated by anti-Sm sera which recognizes U snRNP proteins of other eukaryotes including humans plants and yeast.

Biochim Biophys Acta, 1990 Jun 21, 1049(2), 219 - 22
Characterization of a cDNA encoding cottonseed catalase; Ni W et al.; A 1.7 kb cDNA clone was isolated from our lambda gt11 library constructed from poly(A) RNA of 24-h-old cotyledons . The cDNA encodes a full-length catalase peptide (492 amino acid residues) . The calculated molecular mass is 56,800, similar to that determined for purified enzyme (57,000 SDS-PAGE) . Among higher plant catalases, this cotton catalase shows the highest amino acid sequence identity (85%) to the subunit of homotetrameric maize CAT 1, a developmental counterpart to the homotetrameric CAT A isoform of cotton seeds . Comparison of sequences from cotton, sweet potato, maize CAT 1, and yeast with bovine catalase revealed that the amino acid residues and regions that are involved in catalytic activity and/or required to maintain basic catalase structure, are highly conserved . The C-terminus region, which has the lowest nucleotide sequence identity between plant and mammalian catalases, does not terminate with a tripeptide, S-K/R/H-L, a putative targeting signal for peroxisomal proteins.

J Mol Biol, 1990 Jun 20, 213(4), 583 - 91
Superfamily of UvrA-related NTP-binding proteins . Implications for rational classification of recombination/repair systems; Gorbalenya AE et al.; A superfamily of proteins encoded by bacterial, phage and eukaryotic genomes and performing a wide range of NTP-dependent functions was delineated by amino acid sequence comparison . The new superfamily brought together bacterial proteins UvrA, RecF, RecN, MutH and HexA, T4 phage gp46, T5 phage D13 protein, lambda phage EA59 protein and yeast Rad50 protein, all involved in recombination, repair and, in some cases, also in replication of respective genomes, and a family of bacterial and eukaryotic proteins implicated in active transport of various compounds, cell division and nodulation whose relationship to UvrA had been recognized previously . For some of the members of the new superfamily, NTPase activity or NTP-binding capacity have been demonstrated . All these proteins encompassed four distinct conserved sequence motifs, of which two constituted the NTP-binding pattern typical of a vast class of ATP and GTP-binding proteins, whereas the other two were unique for the new superfamily . The new superfamily was characterized by an unusually large span of length variation of polypeptide segments separating the two conserved motifs of the NTP-binding pattern . Sequence similarity was revealed, on the one hand, between the N-terminal NTP-binding domain of UvrA, recN, gp46 and D13, and on the other hand, between the C-terminal NTP-binding domain of UvrA, recF and EA59 . Possible relationships between different pathways of DNA repair and recombination are briefly analyzed from the viewpoint of involvement of NTPases of different groups.

Cancer Res, 1990 Jun 15, 50(12), 3761 - 6
Inhibition of p34cdc2 kinase activity by etoposide or irradiation as a mechanism of G2 arrest in Chinese hamster ovary cells; Lock RB et al.; The mammalian homologue of the yeast cdc2 gene product, p34cdc2, is a cell cycle-regulated protein essential for mitosis . We have used polyclonal antisera raised against a peptide corresponding to the carboxyl terminus of the sequence of human cdc2 to study p34cdc2 in Chinese hamster ovary (CHO) cells . Major bands are immunoprecipitated at a molecular weight of 34,000, although not in the presence of competing antigenic peptide . p34cdc2 is coimmunoprecipitated with proteins of molecular weights of 52,000 and 57,000 . Immunoprecipitates express histone H1 kinase activity which varies throughout the cell cycle, maximal activity being observed in G2-M . The activity of the p34cdc2 kinase varies according to its association with the Mr 52,000 and 57,000 proteins and according to their phosphorylation state . Treatment of either asynchronous CHO cells or an enriched G2 population with the antitumor agent, etoposide, results in rapid inhibition of immunoprecipitated p34cdc2 kinase activity, which is not due to a direct effect of drug upon the enzyme . p34cdc2 kinase activity recovers as cells arrest in G2 and a second etoposide treatment further inhibits p34cdc2 kinase activity and prolongs G2 arrest . Exposure of asynchronous CHO cells to gamma-irradiation also inhibits p34cdc2 kinase activity within 1 h . Again this activity recovers as cells accumulate in G2 . These results suggest that DNA damage in CHO cells elicits a response which results in inhibition of p34cdc2 kinase activity and, consequently, G2 arrest.

Biochem Biophys Res Commun, 1990 Jun 15, 169(2), 325 - 31
Tissue and species distribution of liver type and tumor type nuclear poly(A) polymerases; Hengst-Zhang JA et al.; Previous studies in this laboratory have identified two distinct nuclear poly(A) polymerases, a 48 kDA tumor type enzyme and a 36-38 kDA liver type enzyme . To investigate the tissue and species specificity of these enzymes, nuclear extracts were prepared from various rat tissues, pig brain and two human cell lines . These as well as whole cell extract from yeast were probed for the two enzymes by immunoblot analysis using polyclonal anti-tumor poly(A) polymerase antibodies or autoimmune sera which contain antibodies specific for the liver type enzyme . Results indicate that both tumor and liver type enzymes are conserved across species ranging from rat to human . The yeast enzyme does not appear to be immunologically related to the liver or the tumor type poly(A) polymerase . The liver type enzyme appears to be specific for normal tissues whereas the tumor type enzyme is detected only in tissues in a "tumorigenic" state or cell lines originating from tumor tissues.

J Biol Chem, 1990 Jun 15, 265(17), 9881 - 7
Matrix processing peptidase of mitochondria . Structure-function relationships; Schneider H et al.; The mitochondrial processing peptidase (MPP) and the processing enhancing protein (PEP) cooperate in the proteolytic cleavage of matrix targeting sequences from nuclear-encoded mitochondrial precursor proteins . We have determined the cDNA sequence of Neurospora MPP after expression cloning . MPP appears to contain two domains of approximately equal size which are separated by a loop-like sequence . Considerable structural similarity exists to the recently sequenced yeast MPP as well as to Neurospora and yeast PEP . Four cysteine residues are conserved in Neurospora and yeast MPP . Inactivation of MPP can be achieved by using sulfhydryl reagents . MPP (but not PEP) depends on the presence of divalent metal ions for activity . Both MPP and PEP are synthesized as precursors containing matrix targeting signals which are processed during import into mitochondria by the mature forms of MPP and PEP.

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