Cell, 1989 Sep 8, 58(5), 833 - 46 Isolation of a human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2; Pines J et al.; This paper reports the nucleotide and predicted amino acid sequence of a human B-type cyclin . The predicted protein sequence shows strong homology to the other known cyclins in the central third of the protein . We show that the level of cyclin mRNA is regulated during the cell cycle, increasing during G2 phase to four time that present in G1 . The protein accumulates steadily during G2 to at least 20 times its level in G1 and is abruptly destroyed at mitosis . In G2/M phase, cyclin is associated with p34cdc2, the human homolog of the fission yeast gene cdc2+, and this complex has histone H1 kinase activity. Nature, 1989 Sep 7, 341(6237), 74 - 6 Changing fos oncoprotein to a jun-independent DNA binding protein with GCN4 dimerization specificity by swapping "leucine zippers"; Sellers JW et al.; A structural motif for DNA-binding proteins, the 'leucine zipper', has been proposed for the jun, fos and myc gene products, the yeast transcriptional activator GCN4, and the C/EBP enhancer-binding protein . These proteins all contain a region with four or five leucine residues spaced exactly seven amino acid residues apart whose sequence is consistent with the formation of an amphipathic alpha-helix . It has been proposed that the leucine zipper consists of two interdigitated alpha-helices, one from each monomer, that constitute the dimerization function necessary for high-affinity binding to DNA; an adjacent region of basic residues is thought to be responsible for specific protein-DNA contacts . In support of this model, substitution of the leucine residues within the motif can abolish dimerization and DNA-binding, and a synthetic peptide corresponding to the GCN4 leucine zipper forms alpha-helical dimers . Despite the conserved leucine residues, however, each protein has a distinct dimerization specificity . Specifically, GCN4 homodimer, Jun homodimer and Fos-Jun heterodimer proteins bind to the same DNA site, whereas Fos is unable to form homodimers, bind DNA, or interact with GCN4 (refs 8-14) . Here, we alter the dimerization specificity of Fos by precisely replacing its leucine zipper with that from GCN4 . This Fos-GCN4 chimaeric protein is able to bind to the target site in the absence of Jun, and can form DNA-binding heterodimers with GCN4 but not with Jun . These results indicate that the leucine zipper is sufficient to confer dimerization specificity and strongly suggest that Fos contacts DNA directly. Am J Med, 1989 Sep 4, 87(3A), 26S - 29S Protective efficacy of a recombinant deoxyribonucleic acid hepatitis B vaccine in institutionalized mentally handicapped clients; Van Damme P et al.; Mentally handicapped clients in institutions are at high risk for hepatitis B virus (HBV) infection . In 1985, 770 mentally handicapped residents from four institutions in the Antwerp area were screened for HBV markers . The prevalence of hepatitis B surface antigen was 10.3 percent (range, 6.1 to 15.2 percent); 42.3 percent (range, 11.5 to 60.1 percent) had antibodies to hepatitis B surface antigen and the hepatitis B core antigen . In 1986, 275 seronegative mentally handicapped residents were vaccinated intramuscularly in the deltoid region with 20 micrograms (1.0 ml) of a recombinant deoxyribonucleic acid yeast-derived hepatitis B vaccine (Engerix-B, SmithKline Biologicals, Rixensart, Belgium) on a zero-, one-, six-month schedule . Serum samples were collected at Months 1, 2, 7, 12, and 24 and were tested for HBV markers by radioimmunoassay . The seroconversion rates for hepatitis B surface antigen antibodies were 39 percent at Month 1 (geometric mean concentration, 6.4 IU/liter), 82 percent at Month 2 (geometric mean concentration, 23.4 IU/liter), 97 percent at Month 7 (geometric mean concentration, 1,034 IU/liter), and 96 percent at Month 12 (geometric mean concentration, 269 IU/liter) . Among the 214 residents evaluated at Month 12, 69 percent had hepatitis B surface antigen antibody levels greater than 100 IU/liter (geometric mean concentration, 603 IU/liter) . No significant adverse reactions were observed . Within the first seven months of observation, HBV infection was detected in eight of 271 subjects (estimated annual incidence of 5 percent) . During this period, none of the clients developed clinical hepatitis or showed biochemical evidence of liver damage . Between eight and 24 months, no additional HBV infections were detected . These data can be compared with an annual incidence of HBV infection of 8.7 percent in a historical cohort of mentally handicapped residents in one of the four institutions. Am J Vet Res, 1989 Sep, 50(9), 1481 - 5 Characterization of the attachment of Treponema hyodysenteriae to Henle intestinal epithelial cells in vitro; Bowden CA et al.; Properties of the attachment of Treponema hyodysenteriae to Henle intestinal epithelial (HIE 407) cells were examined . The frequency of attachment depended on the motility and viability of the spirochetes . Rabbit hyperimmune and swine convalescent antisera inhibited attachment . Treatment of HIE cells with neuraminidase had no effect on attachment; however, treatment of spirochetes with the enzyme decreased adherence significantly (P = 0.01) . Attachment was inhibited by N-acetylneuraminic acid, D-glucuronic acid, and fetuin . Adherence was increased following coincubation with N-acetylglucosamine or yeast mannan . Surface antigens of T hyodysenteriae, isolated by chemical extraction, competitively inhibited adherence . Concentrated T hyodysenteriae culture supernatant fractions inhibited adherence, but concentrated phosphate buffered-saline washings of the spirochete and concentrated uninoculated media did not inhibit adherence . Sialic acid was detected in unwashed T hyodysenteriae and spent culture supernatant fractions in higher concentrations than from washed spirochetes and uninoculated media . It was concluded that the binding adhesins on T hyodysenteriae for cultured HIE cells may contain sialic acid residues. Mol Cell Biol, 1989 Sep, 9(9), 3614 - 20 Sequence identification of cytochrome b in Plasmodium gallinaceum; Aldritt SM et al.; We have identified a gene that encodes the polypeptide cytochrome b in the avian malarial parasite Plasmodium gallinaceum . The gene containing the open reading frame was found to be located on a 6.2-kilobase multimeric extrachromosomal element . The amino acid translation from this gene demonstrated significant similarities to cytochrome b sequences from yeast, mammal, and fungus genomes . We present evidence that the P . gallinaceum cytochrome b transcript is part of a larger primary transcript from the element that is subsequently processed . The message for P . gallinaceum cytochrome b was found to be 1.2 kilobases in size . This is the first report identifying a mitochondrial nucleic acid sequence in malaria-causing organisms and suggests that a functional cytochrome system may exist in these parasites. J Clin Microbiol, 1989 Sep, 27(9), 2003 - 7 New fluorescence assay for the quantitation of fungi; Coleman T et al.; Quantitative determination of fungal mass is easily achieved with a new procedure that detects particle epifluorescence . Fungi are detected after exposure to a fluorescent stain (Fungiqual; CIBA-GEIGY Corp., Summit, N.J.) by using a fluorescence particle concentration analyzer . This report describes a simple fluorescence method for quantitation of either yeast or mycelial forms of fungi . The nature of the staining reaction was studied, and a practical application of this procedure for determination of fungal susceptibility to an antifungal agent is presented. Proc Natl Acad Sci U S A, 1989 Sep, 86(17), 6773 - 7 Human platelet glycoprotein IX: an adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures; Hickey MJ et al.; The glycoprotein (GP) Ib-IX complex on the surface of human platelets functions as the von Willebrand factor receptor and mediates von Willebrand factor-dependent platelet adhesion to blood vessels . GPIX is a relatively small (Mr, 17,000) protein that may provide for membrane insertion and orientation of the larger component of the complex, GPIb (Mr, 165,000) . Using antibody screening, we cloned a cDNA encoding GPIX from a human erythroleukemia cell cDNA library constructed in phage lambda gt11 . Lacking a 5' untranslated region and start codon, the cDNA sequence includes 604 nucleotides, beginning with 495 bases at the 5' end coding for 165 amino acids, followed by a stop codon and 106 noncoding bases at the 3' end . By Northern blot analysis, the GPIX cDNA hybridizes with a single 1.0-kilobase species of platelet poly(A)+ RNA . Translation of the cDNA sequence gives a predicted protein sequence beginning with a truncated putative signal sequence of 5 amino acid followed by a sequence of 17 amino acids matching that determined directly by Edman degradation of intact GPIX . The predicted amino acid sequence of mature GPIX includes an NH2-terminal extracytoplasmic domain of 134 residues, a transmembrane domain of 20 residues, 6 intracytoplasmic residues, and 1 N-linked glycosylation site . GPIX contains a leucine-rich glycoprotein (LRG) sequence of 24 amino acids similar to conserved LRG sequences in GPIb and other proteins from humans, Drosophila, and yeast . "Flanking" sequences of approximately 22 amino acids are present at the NH2 and/or COOH sides of the "central" LRG sequence(s) in GPIX, GPIb, and the other human and Drosophila members of the LRG family . The role of the flank-LRG center-flank structure in the evolution and function of the LRG proteins remains to be defined. Proc Natl Acad Sci U S A, 1989 Sep, 86(17), 6686 - 90 Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources; Nelson DL et al.; Current efforts to map the human genome are focused on individual chromosomes or smaller regions and frequently rely on the use of somatic cell hybrids . We report the application of the polymerase chain reaction to direct amplification of human DNA from hybrid cells containing regions of the human genome in rodent cell backgrounds using primers directed to the human Alu repeat element . We demonstrate Alu-directed amplification of a fragment of the human HPRT gene from both hybrid cell and cloned DNA and identify through sequence analysis the Alu repeats involved in this amplification . We also demonstrate the application of this technique to identify the chromosomal locations of large fragments of the human X chromosome cloned in a yeast artificial chromosome and the general applicability of the method to the preparation of DNA probes from cloned human sequences . The technique allows rapid gene mapping and provides a simple method for the isolation and analysis of specific chromosomal regions. Indian J Exp Biol, 1989 Sep, 27(9), 785 - 91 Development of improved media for axenic cultivation of Acanthamoeba culbertsoni, Singh and Das 1970; Shukla OP et al.; Several varieties of peptone supported growth of A . culbertsoni to different extents reaching a maximum cell density of 1-2 X 10(6)/ml . Proteose peptone and tryptone also yielded good growth when combined with thiamine and vitamin B12 . A combination of proteose peptone with glucose, yeast extract and salts promoted excellent growth of A . culbertsoni with cell density reaching 1-2 X 10(7) cells/ml; tryptone and one of the indigenous peptones also yielded comparable growth when substituted for proteose peptone in this medium . Casamino acids also supported good growth of amoebae and requirement of yeast extract could be met by a combination of thiamine, vitamin B12 and biotin . Bacto peptone did not support good growth of this amoeba but supplementation of peptone with casamino acids or amino acid mixture improved the growth supporting capacity of the medium . Development of several media with or without glucose will aid in cultivation of A . culbertsoni, studies on its metabolism as well as screening of potential drugs. Mycoses, 1989 Sep, 32(9), 463 - 8 Candida guilliermondii var . guilliermondii infection in infertile women; Nagy B et al.; The sera of 263 women--217 infertile and 46 pregnant--were examined by various serological methods (precipitation test, agglutination, indirect immunofluorescence) to detect Candida guilliermondii var . guilliermondii (C.g.) infection . The precipitation reaction was performed with extracellular C . guilliermondii antigen, the agglutination reaction was employed parallel with C . albicans . In the infertile group 122 (56.2%) proved to be C.g . positive, while in the fertile 11 women (23.9%) proved to be so, the level of significance being p less than 0.0001 between the two groups . A one-month ketoconazole treatment (one tablet, 200 mg/day) was adequate for eliminating the C.g . infection . In a few cases hystological examinations were also performed according to Gomori-Grocott and yeast cells could be detected in the stroma of the ovary . IgA, IgG, IgM, Gc-globulin, transferrin and ferritin determinations were carried out before and after the ketoconazole treatment, and there were significant differences in the IgM and transferrin levels between the infected and non-infected groups . The authors achieved 5 pregnancies of 56 treated women in 6 months. Trends Biochem Sci, 1989 Sep, 14(9), 368 - 73 Quantum mechanical effects in enzyme-catalysed hydrogen transfer reactions; Klinman JP; Hydrogen tunneling at room temperature has recently been demonstrated in the reactions catalysed by yeast alcohol dehydrogenase and bovine serum amine oxidase . These results suggest that quantum mechanical effects may be a fundamental and pervasive feature of enzyme-catalysed hydrogen transfer reactions . Our ability to detect such behavior introduces a new probe for the investigation of reaction barrier shape and protein dynamics in enzyme catalysis. G Ital Dermatol Venereol, 1989 Sep, 124(9), XLVII - IL {Efficacy of fenticonazole in patients with pityriasis versicolor}; Di Silverio A et al.; Thirty patients suffering from Pityriasis versicolor were treated with fenticonazole in cream or lotion form with two applications a day . At microscopy, Malassezia furfur was encountered in 25 cases and Pityrosporum orbiculare in 5 . Disappearance of the yeast was obtained on average in 2 weeks of treatment . Measurement of sebum content with a Sebumeter apparatus did not reveal significant difference (p greater than 0.05) between patients suffering from Pityriasis versicolor and controls and in treated patients, in the course of topical therapy. Arch Pharm (Weinheim), 1989 Sep, 322(9), 531 - 4 Antifungal activity of novel 5-carbonyl derivatives of 3-phenyl-3-(1H-imidazol-1-ylmethyl)-2-methylisoxazolidines; Bennett GA et al.; The synthesis and antifungal activity of a series of novel 5-carbonyl derivatives of 3-phenyl-3-(1H-imidazol-1-ylmethyl)-2-methylisoxazolidines (4) are discussed . The preparation of the title compounds involved a 1,3-dipolar cycloaddition reaction of alpha-substituted ketonitrones with either acrylic esters, acrylamide or methyl vinyl ketone to furnish cis/trans-diastereomeric mixtures of the desired 5-carbonyl isoxazolidines 4 . The anifungal activity was evaluated in vitro in solid agar cultures . Some of the compounds tested exerted moderate to potent activity against a wide variety of dermatophytes and yeast and systemic fungi. Am J Gastroenterol, 1989 Sep, 84(9), 1079 - 83 Fungal colonization of the esophagus; Vermeersch B et al.; A prospective study was conducted in 224 patients to determine the clinical significance of esophageal colonization with yeasts under different conditions . In accordance with the results of direct smear microscopic examination and culture of esophageal brushings, patients were divided into three groups: positive, negative, and the patients, in whom saprophytic forms were detected . A higher prevalence of positive findings was noted in patients with predisposing factors for yeast invasion than in patients free of underlying disease . Eleven percent of patients with an endoscopically normal appearing esophagus were positive . We have the impression that this situation may represent a preclinical condition of fungal esophagitis . Patients treated with H2-blocking agents showed a significantly higher incidence of positive findings, than did those without such treatment . Whether patients suffering from a refluxesophagitis resistant to long-term treatment with H2 blockers, but with a significant colonization by yeasts, could benefit by an additional treatment with antimycotics remains a controversial issue and should be studied in a controlled way. J Biochem (Tokyo), 1989 Sep, 106(3), 396 - 400 N-terminal half of a mitochondrial presequence peptide takes a helical conformation when bound to dodecylphosphocholine micelles: a proton nuclear magnetic resonance study; Endo T et al.; Two-dimensional proton nuclear magnetic resonance (NMR) spectra of a synthetic peptide (p25) corresponding to the amino-terminus of the yeast mitochondrial cytochrome oxidase subunit IV precursor protein have been analyzed . Sequence-specific resonance assignments of the peptide have been made in the presence of micelles of a phospholipid analog, perdeuterated dodecylphosphocholine (DPC), with the aid of such techniques as HOHAHA, DQF-COSY, and NOESY . The interresidue nuclear Overhauser effects (NOEs) indicate that the N-terminal half of p25 (S3-F11) takes a helical structure while the C-terminal half does not take a regular secondary structure . Addition of DPC to the solution of p25 induced chemical shift changes only of the resonances from the residues in the N-terminal half, suggesting that the N-terminal half of p25 is directly involved in binding to DPC . The induced helical structure in the N-terminal half at a lipid-water interface may be important in the ability of this presequence to direct a "passenger" protein into mitochondria. J Med Entomol, 1989 Sep, 26(5), 487 - 8 Larval diet and the vector competence of Culex annulirostris (Diptera: Culicidae) for Murray Valley encephalitis virus; Kay BH et al.; Culex annulirostris Skuse larvae were reared on two different amounts of powdered dog chow and yeast, low intake (2.8 micrograms/larva) and high intake (8.4 micrograms/larva), to produce adults with mean wing lengths of 3.25 and 3.7 mm, respectively . The resulting adults were fed different dosages of Murray Valley encephalitis virus and after 10 d extrinsic incubation at 28 degrees C, the infection rate, transmission rate, salivary gland, and body titers were determined . There were no significant differences in any of these parameters. Anal Biochem, 1989 Sep, 181(2), 227 - 33 Computer-controlled discontinuous rotating gel electrophoresis for separation of very large DNA molecules; Gekeler V et al.; The newly designed equipment for alternating field gel electrophoresis which permits the separation of very large DNA molecules and the simultaneous analysis of up to 35 samples is described . The field alternation is effected by intermittently rotating the submerged agarose gel by optitional angles . The time intervals between changes of position are controlled by a computer program driving a simple switching device which was designed to suit any technique using periodic switching or inversion of the electrical field . Because the electrophoresis unit provides an absolutely homogeneous electrical field, no distortion of migration lanes occurs and the resolution is very good . Moreover, by using a switching time interval gradient an almost perfect linear relationship between migration distances and molecule sizes in the range of about 100-1250 kilobase pairs is observed . In two-dimensional separations, different switching time programs for the first and second dimension allow maximum resolution of selected size ranges . Field inversion gel electrophoresis is possible as well . The performance of the method is demonstrated by comparing the chromosome sizes of different yeast strains. J Clin Invest, 1989 Sep, 84(3), 886 - 91 Receptor-mediated phagocytosis in human neutrophils is associated with increased formation of inositol phosphates and diacylglycerol . Elevation in cytosolic free calcium and formation of inositol phosphates can be dissociated from accumulation of diacylglycerol; Fallman M et al.; Phagocytosis of C3bi- or IgG-opsonized yeast particles in human neutrophils was found to be associated with an increased formation of inositol phosphates and diacylglycerol . Pertussis toxin only marginally affected phagocytosis of IgG- and C3bi-opsonized particles and the associated formation of second messengers . Forskolin, which induced a threefold rise of cellular cAMP, however, markedly inhibited both C3bi- and IgG-mediated phagocytosis as well as the particle-induced formation of inositol phosphates and diacylglycerol . These observations are in contrast to what was found to occur with chemotactic factors and indicate that chemotactic and phagocytic signaling can be regulated independently in human neutrophils . Since C3bi-mediated phagocytosis has been shown to occur at vanishingly low cytosolic free calcium levels, calcium-depleted cells were used to study the importance of the inositol cycle for the engulfment of C3bi-opsonized particles . Despite a total lack of receptor-induced formation of inositol phosphates, a significantly increased accumulation of diacylglycerol accompanied the ingestion of C3bi-opsonized particles . These data show that the engulfment of C3bi-opsonized particles can occur independently of both a calcium transient and an increased inositol phosphate production . However, the observed accumulation of diacylglycerol, not derived from phosphoinositides, suggests that this second messenger play a role in the control of the engulfment process. Br Poult Sci, 1989 Sep, 30(3), 633 - 9 Utilisation of free and protein dietary lysine in chicks estimated with isotopes; Otto M et al.; 1 . Utilisation of supplementary free L-lysine hydrochloride was estimated in growing chicks and compared to that of protein-bound lysine . The technique used is based on the oxidative catabolism of either free (U-14C)-L-lysine or the labelled lysine incorporated into yeast proteins . 2 . The animals received a wheat-wheat gluten diet which was L-lysine-supplemented with either unlabelled yeast proteins or a mixture of synthetic amino acids simulating the yeast proteins or L-lysine hydrochloride alone . 3 . At 13 and 15 days after hatching, expiry of (14C)--carbon dioxide was followed 8 h after dosing with the appropriate radiolabelled diet . After 4 h, 0.66% of the protein-bound and 5.3 to 5.7% of the free lysine radioactivity appeared as 14C--carbon dioxide . 4 . It is concluded that under these conditions lysine from both sources was utilised more efficiently than had been assumed hitherto, protein-bound lysine being slightly better utilised than free lysine. J Mol Evol, 1989 Sep, 29(3), 202 - 7 Evolution of the mitochondrial genetic code . I . Origin of AGR serine and stop codons in metazoan mitochondria; Osawa S et al.; AGA and AGG (AGR) are arginine codons in the universal genetic code . These codons are read as serine or are used as stop codons in metazoan mitochondria . The arginine residues coded by AGR in yeast or Trypanosoma are coded by arginine CGN throughout metazoan mitochondria . AGR serine sites in metazoan mitochondria are occupied mainly in corresponding sites in yeast or Trypanosoma mitochondria by UCN serine, AGY serine, or codons for amino acids other than serine or arginine . Based on these observations, we propose the following evolutionary events . AGR codons became unassigned because of deletion of tRNA Arg (UCU) and elimination of AGR codons by conversion to CGN arginine codons . Upon acquisition by serine tRNA of pairing ability with AGR codons, some codons for amino acids other than arginine mutated to AGR, and were captured by anticodon GCU in serine tRNA . During vertebrate mitochondrial evolution, AGR stop codons presumably were created from UAG stop by deletion of the first nucleotide U and by use of R as the third nucleotide that had existed next to the ancestral UAG stop. J Neurogenet, 1989 Sep, 6(1), 11 - 26 The expression of the neurogenic locus Notch during the postembryonic development of Drosophila melanogaster and its relationship to mitotic activity; Markopoulou K et al.; The molecular analysis of the Notch locus of Drosophila melanogaster demonstrated that it codes for a protein which shows homology to the epidermal growth factor as well as to the products of certain yeast genes involved in the control of the cell cycle (Wharton et al., 1985a; Breeden and Nasmyth, 1987) . The structure of the protein suggests that Notch is involved in a cell interaction mechanism which controls the differentiation of several different tissues during development . Here we examine Notch expression during imaginal development using in situ hybridization to tissue sections and demonstrate that Notch is not expressed ubiquitously during the postembryonic stages, but rather is confined to specific tissues . During the larval and early pupal period Notch transcripts are predominantly localized in the imaginal discs and the central nervous system . In the middle and late pupal period the signal levels in these tissues drop dramatically and in the adult animal Notch transcripts are essentially detected only in the ovaries . In the larval stages the pattern of Notch expression appears to be closely correlated with mitotically active tissues, while in later stages this correlation appears less strict . The findings reported here indicate that there is a requirement for normal Notch function in a number of tissues at several developmental stages and that the pleiotropic phenotypic manifestation of Notch mutations is a context dependent developmental result . The observed association of Notch expression with mitotically active cell populations raises the possibility that Notch may play a role in the cell cycle. Jpn J Cancer Res, 1989 Sep, 80(9), 879 - 86 The tumor rejection antigen separated from Rous sarcoma virus-induced murine fibrosarcoma exhibits a molecular weight of approximately 60 kD but differs from functional pp60src; Suda T et al.; The tumor antigen capable of inducing tumor resistance (tumor rejection antigen; TRA) was obtained in a solubilized form by sodium dodecyl sulfate (SDS) extraction of plasma membrane fraction from Rous sarcoma virus (RSV)-induced CSA1M fibrosarcoma cells (BALB/c origin) . Analyses by Sephacryl S-300 gel filtration and SDS-polyacrylamide gel electrophoresis revealed that TRA activity was recovered in the fraction with a molecular weight of approximately 60 kD . Unfractionated crude SDS-solubilized preparation contained gp70 as detected by rabbit anti-gp70 antiserum, whereas such reactivity was lost in the fraction exhibiting the molecular weight of about 60 kD . Since this fraction retained pp60src activity, the relation of TRA to pp60src was further investigated . pp60v-src was also obtained from the lysate of v-src-expressing yeast transformant . Immunization of BALB/c mice with such pp60v-src-containing lysate failed to induce any significant tumor protection . The above 60 kD fraction of CSA1M solubilized antigens was allowed to bind to Sepharose beads coupled with anti-pp60src monoclonal antibody and separated into the bead-bound and bead-unbound fractions . The bead-bound fraction that was recovered from pp60src-binding beads (pp60src-positive fraction) did not exhibit the TRA activity . In contrast, immunization with the fraction depleted of pp60src activity (bead-unbound fraction) resulted in potent tumor protection . These results indicate that the solubilized membranous component(s) of CSA1M with a molecular weight of approximately 60 kD, which is distinct from functional pp60src, functions as the TRA against RSV-induced CSA1M tumor cells. Biochim Biophys Acta, 1989 Aug 31, 997(3), 236 - 41 Nonpolar interactions in the modification of an essential sulfhydryl of sorbitol dehydrogenase by N-alkylmaleimides; Beier KH et al.; A series of N-alkylmaleimides varying in chainlength from N-methyl- to N-octylmaleimide inclusive was shown to effectively inactivate sheep liver sorbitol dehydrogenase at pH 7.5 and 25 degrees C . The apparent second-order rate constants for inactivation increased with increasing chainlength of the N-alkylmaleimide used . Positive chainlength effects were also indicated by the Kd values for the N-ethyl and N-heptyl derivatives obtained from studies of the saturation kinetics observed for inactivation of the enzyme at high concentrations of these maleimides . The complete inactivation of sorbitol dehydrogenase was demonstrated to occur through the selective covalent modification of one cysteine residue per subunit of enzyme . The stoichiometry of enzyme inactivation was supported on the one hand by fluorescence titration with fluorescein mercuric acetate of the native and the inactivated enzyme, and, on the other hand, by the simultaneous inactivation of the enzyme with selective modification of one sulfhydryl per subunit by N-{p-(2-benzoxazolyl)phenyl}maleimide . Protection of the enzyme from N-alkylmaleimide inactivation was observed with the binding of NADH, whereas both NAD and sorbitol were ineffective as protecting ligands . Diazotized 3-aminopyridine adenine dinucleotide, in contrast to previous studies of this reagent with yeast alcohol dehydrogenase and rabbit muscle glycerophosphate dehydrogenase, did not function as a site-labeling reagent for sorbitol dehydrogenase. Wien Med Wochenschr, 1989 Aug 31, 139(15-16), 381 - 3 {Prevention of candidiasis in at-risk patients}; Hain K; Colonisation by yeasts and invasive yeast-growth (candidosis, yeast-mycosis) increase whenever a patient's power of resistance is lowered . Early recognition of colonizing yeasts in a patient can be as important for his vital functions, as the therapy of the underlying disease will be . This seems to be quite complicated in many cases and often it is omitted by several reasons . An "easy to handle method" is a "rapid yeast culture" . Blood-cultures and serodiagnosis can be added if held necessary . It is more important to find out, if there are few or many yeast-cells in a patient, than to count them . Hidden "yeast-nests" can in short time recruit eliminated yeasts . Too much sugar means too many yeasts as well . Anti-yeast-therapeutics have to be selected and dosed according to their characteristic qualities. Wien Med Wochenschr, 1989 Aug 31, 139(15-16), 379 - 80 {Dermatomycoses and an antifungal diet}; Putzier E; Treatment of dermatomycoses and some other dermatoses, e.g . seborrheic eczema, is more effective if the intestinal tract is free of pathogenic fungi . In order to reduce the yeast colonies between the intestinal villi and to avoid the persorption of living yeast cells into the lymph-tract and the blood circulation it is recommendable to perform the so-called antifungal diet: less sugar of all kind and much more vegetable fibres in the food. J Biol Chem, 1989 Aug 25, 264(24), 14240 - 5 Proalbumin to albumin conversion by a proinsulin processing endopeptidase of insulin secretory granules; Rhodes CJ et al.; A lysate of purified insulin secretory granules, which contains two types of proinsulin processing activity (type 1, Arg-Arg-directed and type II, Lys-Arg-directed (Davidson, H.W., Rhodes, C.J., and Hutton, J . C . (1988) Nature 333, 93-96), was found to process proalbumin by specific proteolytic cleavage of the COOH-terminal side of the Arg-2-Arg-1 sequence . The subcellular distribution of proalbumin processing activity in insulinoma tissue paralleled that for proinsulin conversion and occurred principally in a secretory granule fraction . Cleavage appeared to result from the Arg-Arg-directed type 1 proinsulin processing endo-peptidase . It was Ca2+-dependent (K0.5 activation = 1.0-1.5 mM Ca2+), unaffected by group-specific inhibitors of serine, cysteinyl, or aspartyl proteinases, and had an acidic pH optimum (5.5) . Active-site inhibitor studies showed this activity had a preference for dibasic over monobasic amino acid sequences and indicated that the sequence of the dibasic site was an important determinant of the susceptibility of the substrate to cleavage . The activity did not process the proalbumin Christchurch mutant (Arg-2-Arg-1 to Arg-2-Gln-1) . It was inhibited by the variant alpha 1-antitrypsin Pittsburgh (Met358 to Arg358; K0.5 = 100 nM) but not by other related proteins normally co-secreted with albumin from hepatocytes, namely alpha 1-antitrypsin M, alpha 2-macroglobulin, or antithrombin III . The insulin secretory granule proalbumin processing activity was indistinguishable from a proalbumin endopeptidase reported in rat liver membranes and similar to the yeast KEX-2 protease . These findings suggest that a highly conserved set of proprotein endopeptidases exists, which are specific for a dibasic sequence but broadly specific for proprotein substrates . Such enzymic activities appear to be active within both the constitutive and regulated pathways of secretion . Intraorganellar Ca2+ and pH appear to play a key role in regulating their activities. J Mol Biol, 1989 Aug 20, 208(4), 587 - 99 Processing of mitochondrial RNA in Aspergillus nidulans; Dyson NJ et al.; Genes for cytochrome oxidase subunit I (oxiA), ATPase subunit 9, NADH dehydrogenase subunit 3 (ndhC) and cytochrome oxidase subunit II (oxiB) are located within a 7.2 kb (1 kb = 10(3) bases or base-pairs) segment of the Aspergillus nidulans mitochondrial genome . Northern hybridization shows that abundant RNA molecules of 4.0, 2.5 and 1.5 kb, each containing copies of two or more genes, are transcribed from this region . The 4.0 kb molecule, which contains copies of each of the four genes but lacks the three oxiA introns, is cleaved at a point just upstream from ndhC to give rise to the 2.5 kb RNA, which contains copies of oxiA and the ATPase subunit 9 gene, and the 1.5 kb RNA, which carries ndhC and oxiB . The ATPase subunit 9 gene, which has no identified function, is therefore transcribed into an abundant RNA . S1 nuclease analysis indicates that there are no additional introns in the amino-terminal region of oxiA and that the 4.0 and 2.5 kb transcripts of this gene have staggered 5' termini, the most upstream of which is adjacent to the 3' end of the histidinyl-tRNA gene . The results suggest that transcription of this genome proceeds via a very limited number of primary transcripts with mature RNAs produced by extensive processing events including tRNA excision . RNA synthesis and processing in A . nidulans mitochondria therefore resembles the events occurring in metazoa rather than yeast. Nature, 1989 Aug 17, 340(6234), 568 - 71 Leucine zippers of fos, jun and GCN4 dictate dimerization specificity and thereby control DNA binding; Kouzarides T et al.; The products of the fos and jun protooncogenes form a stable heterodimer which binds to the TPA-responsive element (TRE) TGACTCA with high affinity . These two proteins, together with the yeast GCN4 protein, belong to a growing family of transcription factors, including FosB, Fra1, JunB and JunD, whose members share a highly conserved DNA-binding domain . This domain is composed of two structures: a basic motif, which is thought to bind directly to DNA; and a leucine zipper, which provides a dimerization interface . Although this domain is highly conserved in Fos, Jun and GCN4, each of these three proteins has very different relative affinities for the TRE . To understand these differences, we used 'domain-swapping' experiments designed to test the relative contributions of the basic motif and the leucine zipper to TRE-binding affinity . Here we show that fos, jun and GCN4 have different affinities for the TRE due to differences in the hetero- or homo-dimerization capacity of their leucine zipper domains; the basic motifs of these three proteins have comparable DNA binding potential . These results indicate that leucine zippers control the types of protein complexes which can associate with a TRE and regulate gene expression. Mol Cell Biochem, 1989 Aug 15, 89(1), 57 - 67 Phosphoglucoisomerase-catalyzed interconversion of hexose-phosphates . Isotopic discrimination between hydrogen and tritium; Malaisse-Lagae F et al.; The rate of conversion of D-glucose 6-phosphate to D-fructose 6-phosphate as catalyzed by yeast phosphoglucoisomerase is about fourfold lower when 3H, rather than 1H, is present on the C2 of D-glucose 6-phosphate . This difference appears to be due mainly to a change in maximal velocity, rather than affinity . Phosphoglucoisomerase also distinguishes between 1H and 3H in terms of either their intramolecular transfer from C2 to C1 or their incorporation from water on the C1 of D-fructose 6-phosphate. J Biol Chem, 1989 Aug 15, 264(23), 13929 - 37 Two genes encode the major membrane-associated protein of methanol-induced peroxisomes from Candida boidinii; Garrard LJ et al.; A massive proliferation of peroxisomes occurs in the yeast Candida boidinii when methanol is utilized as the sole carbon source; these peroxisomes contain the enzymes which catalyze the initial steps of methanol utilization . The most abundant peroxisomal membrane-associated protein has an apparent molecular mass of 20 kDa and is termed PMP20 . We report the isolation of two genes that encode very similar forms of PMP20; this is the first report of genes that encode proteins associated with peroxisomal membranes . Southern analysis demonstrates that the two genes are on different loci, although there are several homologous regions of both 5'- and 3'-untranslated sequence . One of the areas of 5' homology is within the untranslated region of the mRNA . Within the coding region there are 35 base differences between the two genes that are reflected in only five amino acid differences . The mRNAs representing both genes of PMP20 are induced in cells grown in methanol-containing medium and are below detection in cells grown in glucose . S1 nuclease protection analysis indicates that there is a 2.5-fold difference in mRNA expression between the two genes when induced . The predicted sequences of both PMP20 genes show the absence of a cleaved amino-terminal leader sequence and the presence of only 1 cysteine residue . In agreement with previous biochemical data suggesting a peripheral association of this protein with the membrane (Goodman, J . M., Maher, J., Silver, P . A., Pacifico, A., and Sanders, D . (1986) J . Biol . Chem . 261, 3464-3468), there are no obvious membrane spanning regions predicted in the sequences . Both PMP20 gene products contain the carboxyl-terminal sequence AKL, similar to the putative SKL peroxisomal sorting sequence (Gould, S . J., Keller, G.-A., and Subramani, S . (1988) J . Cell Biol . 107, 897-905). Eur J Biochem, 1989 Aug 15, 183(3), 661 - 9 Purification and characterization of trimming glucosidase I from pig liver; Bause E et al.; Trimming glucosidase I has been purified about 400-fold from pig liver crude microsomes by fractional salt/detergent extraction, affinity chromatography and poly(ethylene glycol) precipitation . The purified enzyme has an apparent molecular mass of 85 kDa, and is an N-glycoprotein as shown by its binding to concanavalin A-Sepharose and its susceptibility to endo-beta-N-acetylglucosaminidase (endo H) . The native form of glucosidase I is unusually resistant to non-specific proteolysis . The enzyme can, however, be cleaved at high, that is equimolar, concentrations of trypsin into a defined and enzymatically active mixture of protein fragments with molecular mass of 69 kDa, 45 kDa and 29 kDa, indicating that it is composed of distinct protein domains . The two larger tryptic fragments can be converted by endo H to 66 kDa and 42 kDa polypeptides, suggesting that glucosidase I contains one N-linked high-mannose sugar chain . Purified pig liver glucosidase I hydrolyzes specifically the terminal alpha 1-2-linked glucose residue from natural Glc3-Man9-GlcNAc2, but is inactive towards Glc2-Man9-GlcNAc2 or nitrophenyl-/methyl-umbelliferyl-alpha-glucosides . The enzyme displays a pH optimum close to 6.4, does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (Ki approximately 2.1 microM), N,N-dimethyl-1-deoxynojirimycin (Ki approximately 0.5 microM) and N-(5-carboxypentyl)-1-deoxynojirimycin (Ki approximately 0.45 microM), thus closely resembling calf liver and yeast glucosidase I . Polyclonal antibodies raised against denatured pig liver glucosidase I, were found to recognize specifically the 85 kDa enzyme protein in Western blots of crude pig liver microsomes . This antibody also detected proteins of similar size in crude microsomal preparations from calf and human liver, calf kidney and intestine, indicating that the enzymes from these cells have in common one or more antigenic determinants . The antibody failed to cross-react with the enzyme from chicken liver, yeast and Volvox carteri under similar experimental conditions, pointing to a lack of sufficient similarity to convey cross-reactivity. J Biol Chem, 1989 Aug 15, 264(23), 13697 - 700 Regulation of the product of a possible human cell cycle control gene CDC2Hs in B-cells by alpha-interferon and phorbol ester; Thomas NS; The product of the cell cycle control gene cdc2 is required in yeast for transition through both G1 and G2 control points of the cell cycle . The homologous protein in higher eukaryotes has been shown to be a component of the mitosis promoting factor complex and may thus regulate entry through the G2 control point into mitosis . It is suggested from the work presented here that, as in yeast, the human CDC2Hs gene product (p34CDC2Hs) may also play a role in cell cycle control in the G1(G0) phase of the cell cycle . Interferon-alpha inhibits the growth of the human B-cell line Daudi in the G1(G0) phase of the cell cycle and prevents cells from entering S-phase . Culturing the cells with interferon-alpha inhibits the phosphorylation of p34CDC2Hs and causes the down-regulation of CDC2Hs mRNA . Phorbol ester also inhibits the Daudi cell cycle in G1(G0) and causes the inhibition of p34CDC2Hs phosphorylation and a reduction of CDC2Hs mRNA . These studies provide insights into the process of growth control and the cytostatic mechanism of interferon-alpha. Cell, 1989 Aug 11, 58(3), 553 - 63 glp-1 and lin-12, genes implicated in distinct cell-cell interactions in C . elegans, encode similar transmembrane proteins; Yochem J et al.; Genomic DNA closely related in sequence to lin-12, a gene that specifies certain cell fates during C . elegans development, was isolated from a C . elegans library by low stringency hybridization . DNA sequencing of genomic and cDNA clones predicts the new sequence to encode an integral membrane protein that shares three repeated amino acid sequence motifs with the lin-12 product and the Drosophila Notch product: an epidermal growth factor-like motif, the "lin-12/Notch Repeat," and a motif present in two yeast gene products that have cell cycle dependent functions . Austin and Kimble (see accompanying paper) present evidence that this sequence corresponds to glp-1, a gene implicated in cell-cell interactions distinct from those involving lin-12 . Possible implications of the predicted structure of the glp-1 product with respect to these cell-cell interactions are discussed. Cell, 1989 Aug 11, 58(3), 473 - 83 The AU-rich sequences present in the introns of plant nuclear pre-mRNAs are required for splicing; Goodall GJ et al.; Plant cells do not in general process the introns of transcripts expressed from introduced vertebrate genes . By studying the processing of model introns in transfected plant protoplasts, we have investigated the special requirements for intron recognition by plant cells . Our results indicate that the requirements for intron recognition in plants are different from those of both metazoa and yeast . A synthetic intron of arbitrary sequence but incorporating splice site consensus sequences and a high proportion of U and A nucleotides, a characteristic feature of plant introns, was efficiently spliced in protoplasts . We have studied the effects of various sequence alterations and conclude that AU-rich sequences are necessary for intron recognition . In addition, we find that the criteria for branch site selection are relaxed, as they are in vertebrates, but a polypyrimidine tract is not necessary. Nature, 1989 Aug 10, 340(6233), 487 - 8 Transcription factor IIIA induced bending of the Xenopus somatic 5S gene promoter; Schroth GP et al.; Transcription factor IIIA (TFIIIA), the canonical zinc-finger protein, is a protein of relative molecular mass 39,000 (39K) that is required for transcription of 5S-ribosomal subunit genes in Xenopus . It binds in a sequence-specific manner to the internal control region of the 5S gene (see Fig . 1) and facilitates transcription of the gene by RNA polymerase III . It also binds to the 5S gene product to form a 7S ribonucleoprotein particle . In oocytes the 7S particle acts as a storage form of the RNA to be utilized later in development . TFIIIA binds to DNA through its 30 K N-terminal domain, which contains nine zinc-fingers . TFIIIA was the first protein described to have this type of DNA binding motif, but numerous other proteins have now been shown to have zinc-finger domains . A structure for a single zinc-finger from the yeast protein ADR1, was recently proposed based on two-dimensional NMR data (ref . 8), and a similar structure was proposed based on comparison with crystal structures of other metalloproteins . Although models for the interaction of TFIIIA with the 5S-ribosomal gene DNA have been proposed, based on nuclease digestion and methylation interference data, little precise structural information is available for TFIIIA and the physical basis for the interaction of zinc-fingers with DNA is not understood . Using both circular permutation and circularization assays we provide convincing biochemical evidence that TFIIIA bends the DNA at the internal promoter of the 5S gene. J Chromatogr, 1989 Aug 4, 476, 205 - 25 High-performance liquid chromatography of amino acids, peptides and proteins . XCII Thermodynamic and kinetic investigations on rigid and soft affinity gels with varying particle and pore sizes; Anspach FB et al.; In these investigations Cibacron Blue F3GA was immobilized on soft gels, porous silicas, and non-porous glass beads . Hen egg white lysozyme, human serum albumin and yeast alcohol dehydrogenase were used as adsorbates with the dye-affinity sorbents . Batch experiments with continuous monitoring of protein concentration were employed to evaluate thermodynamic and kinetic behaviour of these proteins in finite bath systems . The observed adsorption kinetic rates of interaction of the above proteins with each of the dye-affinity sorbents were found to decrease with increasing protein molecular weight . Equilibration times, in the batch experimental mode, of the adsorption of lysozyme on the dye-affinity sorbents varied from 20 s for the non-porous glass beads with a size range of 20-40 microns to more than 60 min in the case of a porous sorbent with a particle diameter of 100-300 microns and 60 nm pore size . Furthermore, equilibration times, which represent the overall adsorption rates incorporating all the non-equilibrium effects, increased with all affinity systems when adsorption took place in the non-linear portion of the isotherm . The most dramatic increase was observed when sorbents with relatively high protein size to pore size ratios, lambda, were employed. Biochim Biophys Acta, 1989 Aug 3, 975(3), 377 - 83 Inhibition of the bovine-heart mitochondrial F1-ATPase by cationic dyes and amphipathic peptides; Bullough DA et al.; The bovine heart mitochondrial F1-ATPase is inhibited by a number of amphiphilic cations . The order of effectiveness of non-peptidyl inhibitors examined as assessed by the concentration estimated to produce 50% inhibition (I0.5) of the enzyme at pH 8.0 is: dequalinium (8 microM), rhodamine 6G (10 microM), malachite green (14 microM), rosaniline (15 microM) greater than acridine orange (180 microM) greater than rhodamine 123 (270 microM) greater than rhodamine B (475 microM), coriphosphine (480 microM) greater than safranin O (1140 microM) greater than pyronin Y (1650 microM) greater than Nile blue A (greater than 2000 microM) . The ATPase activity was also inhibited by the following cationic, amphiphilic peptides: the bee venom peptide, melittin; a synthetic peptide corresponding to the presence of yeast cytochrome oxidase subunit IV (WT), and amphiphilic, synthetic peptides which have been shown (Roise, D., Franziska, T., Horvath, S.J., Tomich, J.M., Richards, J.H., Allison, D.S . and Schatz, G . (1988) EMBO J . 7, 649-653) to function in mitochondrial import when attached to dihydrofolate reductase (delta 11.12, Syn-A2, and Syn-C) . The order of effectiveness of the peptide inhibitors as assessed by I0.5 values is: Syn-A2 (40 nM), Syn-C (54 nM) greater than melittin (5 microM) greater than WT (16 microM) greater than delta 11,12 (29 microM) . Rhodamines B and 123, dequalinium, melittin, and Syn-A2 showed noncompetitive inhibition, whereas each of the other inhibitors examined (rhodamine 6G, rosaniline, malachite green, coriphosphine, acridine orange, and-Syn-C) showed mixed inhibition . Replots of slopes and intercepts from Lineweaver-Burk plots obtained for dequalinium were hyperbolic indicating partial inhibition . With the exception of Syn-C, for which the slope replot was hyperbolic and the intercept replot was parabolic, steady-state kinetic analyses indicated that inhibition by the other inhibitors was complete . The inhibition constants obtained by steady-state kinetic analyses were in agreement with the I0.5 values estimated for each inhibitor examined . Rhodamine 6G, rosaniline, dequalinium, melittin, Syn-A2, and Syn-C were observed to protect F1 against inactivation by the aziridinium of quinacrine mustard in accord with their experimentally determined I0.5 values.(ABSTRACT TRUNCATED AT 400 WORDS) Blut, 1989 Aug, 59(2), 165 - 70 Lineage- and differentiation-dependent alterations in the expression of receptors for glycoconjugates (lectins) in different human hematopoietic cell lines and low grade lymphomas; Gabius S et al.; Important biological functions and cellular recognition phenomena are supposedly governed by specific sugar-protein interactions . Human hematopoietic cell lines offer an excellent model for the study of the expression of endogenous receptors for the carbohydrate part of glycoconjugates with respect to cell lineage and modulation by differentiation . Initially, a panel of fluorescent (neo)glycoproteins was successfully employed to demonstrate cytologically the actual presence of such receptors on different cell lines: the B lymphoblast line, Daudi; the T cell lymphoblastic leukemia line, P12; the multipotent leukemic line, K562 and the promyelocytic line, HL060 . Biochemical analyses were performed using affinity chromatography on supports with immobilized lactose and asialofetuin (simple or complex beta-galactosides), melibiose (alpha-galactoside), fucose, N-acetyl-D-galactosamine, maltose (alpha-glucoside), the mannose-rich yeast glycoprotein, mannan, glycopeptides containing sialic acid residues and heparin . Subsequently, sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis was used to detect cell lineage-dependent changes in theses parameters . Differentiation-dependent changes in the expression of receptors with specificity to galactose, N-acetylgalactosamine, maltose and heparin were similarly uncovered upon dimethyl sulfoxide-induced differentiation of HL60 cells . Differences in this type of cellular characteristic were also apparent for lymphoma cells from patients with various histological subtypes of lowgrade lymphomas . This initial description of lineage- and differentiation-dependent differences in various human hematopoietic cell lines and in cells from patients with lowgrade lymphomas suggests that advances in the knowledge of the composition of endogenous sugar receptors (lectins) may aid in understanding aspects of the biological behavior of hematopoietic cells and their related malignancies via participation of sugar-protein (lectin) interactions. Am J Clin Nutr, 1989 Aug, 50(2), 346 - 52 Qinghaosu, dietary vitamin E, selenium, and cod-liver oil: effect on the susceptibility of mice to the malarial parasite Plasmodium yoelii; Levander OA et al.; Young female mice were fed torula-yeast-based diets deficient in vitamin E or selenium or supplemented with cod-liver oil to determine the effect of host antioxidant status on the therapeutic efficacy of the Chinese traditional antimalarial drug qinghaosu (QHS), a sesquiterpene endoperoxide . Vitamin E deficiency enhanced the antimalarial action of QHS against Plasmodium yoelii, both in terms of decreased parasitemia and improved survival but Se deficiency did not . A vitamin E-deficient diet containing 5% cod-liver oil had such strong antimalarial activity in itself that no additional therapeutic benefit of QHS could be demonstrated . Hematocrit values in parasitized mice treated with QHS or fed the cod-liver-oil-supplemented, vitamin E-deficient diet were normal . Nutritional manipulation of host antioxidant status may provide a promising prophylactic and/or therapeutic tool for the control of malaria. Res Commun Chem Pathol Pharmacol, 1989 Aug, 65(2), 249 - 52 Effect of selenium supplementation after acute myocardial infarction; Korpela H et al.; The effect of selenium supplementation was evaluated in 81 patients with acute myocardial infarction in a double-blind, placebo-controlled study . Patients were randomised into two treatment groups receiving either selenium-rich yeast (100 micrograms/day) or placebo in addition to conventional drug therapy for a 6-month period . During treatment the mean serum selenium concentration increased from 82 micrograms/l to 122 micrograms/l (p less than 0.001) in the selenium supplemented group and remained unaltered in the placebo group (83 micrograms/l) . During the 6-month follow-up period there were 4 cardiac deaths in the placebo group whereas no patients in the selenium group died during the follow-up period . Furthermore, there were 2 nonfatal reinfarctions in the placebo group and one nonfatal reinfarction in the selenium group . The results encourage further studies to evaluate the efficacy of antioxidants in the prevention and therapy of acute myocardial infarction. FASEB J, 1989 Aug, 3(10), 2151 - 63 Structural and functional properties of ras proteins; Santos E et al.; The ras proteins belong to a family of related polypeptides that are present in all eukaryotic organisms from yeast to human . Their extraordinary evolutionary conservation suggests that they have essential cellular functions, although their exact role remains unknown . Mutations in specific amino acids and overexpression of normal proteins have been linked to altered proliferation and/or differentiation and, particularly, to neoplastic processes . Mature ras proteins are located on the inner side of the plasma membrane, and their biochemical properties include binding and exchange of guanine nucleotides and GTPase activity . The favored hypothesis for ras function is that these proteins exist in an equilibrium between an inactive conformation (p21.GDP) and an active conformation (p21 . GTP) in which they are able to interact with their as yet unknown cellular target or targets . Similarities in cellular location, structure, and biochemistry with other known regulatory (G) proteins suggest that they play a role in transduction of signals from the cell surface . The elucidation of the crystal structure of normal and transforming ras proteins and the identification of cellular proteins that interact directly with them (GAP, CDC25) or suppress some of their biological effects (Krev-1) have opened new avenues in the search for their elusive cellular targets and in the elucidation of the functional role of ras gene products. J Dent Que, 1989 Aug, 26, 425 - 8 {Vaccination as a means of prevention}; Montplaisir S et al.; While recognizing the fact that the use of strict hygienic techniques, disposable materials and quality control are essential for sterilizing instruments, vaccination still remains the most efficient way to combat infection . In this article, the authors discuss the principle problems associated with the use of vaccines and especially as they relate to the dental office . Hepatitis B is certainly of interest to all dentists and their personnel, especially since its primary mode of transmission is by contact of blood with the skin and the mucosa . The two forms of vaccines presently available, are derived from the human plasma of carriers or from yeast and cause a genetic reaction which produces hepatitis B virus antigens . These vaccines protect at least over 90% of all healthy individuals and do not generate any secondary infection or unfavorable reactions . Faced with the reality of hepatitis B, it is very wise to remember that it is BETTER TO BE VACCINATED THAN TO HAVE TO LIVE WITH THE EFFECTS OF HEPATITIS. Proc Natl Acad Sci U S A, 1989 Aug, 86(15), 5698 - 702 Association of charge clusters with functional domains of cellular transcription factors; Brendel V et al.; Using rigorous statistical methods, we have identified and evaluated unusual properties of the distribution of charged residues within the sequences of eukaryotic cellular transcription factors . Virtually all transcription factors, including GAL4, c-Jun, C/EBP, CREB, Oct-1, Oct-2, Sp1, Egr-1, CTF-1, steroid and thyroid hormone receptors, and others, carry one or more highly significant charge clusters . For the most part these clusters (conserved within families of homologous proteins) are of positive net charge but contain also substantial numbers of acidic residues . Predominantly basic charge clusters are often, but not exclusively, associated with DNA-binding domains, and vice versa . Negative charge clusters of note occur only in the yeast protein PHO4 and in the proteins encoded at the Drosophila loci zeste (zeta) and knrl . This dearth of statistically significant negative charge clusters raises questions with respect to the generality of acidic activation domains . A number of sequences (Oct-1, Oct-2, zeste, Dhr23, E75, and knrl) contain multiple charge clusters together with one or more significantly long uncharged regions . The occurrence of multiple charge clusters is a rare phenomenon (found in less than 3% of all proteins, mainly in Drosophila developmental control proteins and in transactivators of eukaryotic DNA viruses) . Most of the proteins with zinc-binding "fingers" carry a mixed charge cluster centered at the zinc-finger motif preceded by a long uncharged stretch, suggestive of a modular structure for these proteins. EMBO J, 1989 Aug, 8(8), 2275 - 82 Cyclin is a component of the sea urchin egg M-phase specific histone H1 kinase; Meijer L et al.; A so-called 'growth-associated' or 'M-phase specific' histone H1 kinase (H1K) has been described in a wide variety of eukaryotic cell types; p34cdc2 has previously been shown to be a catalytic subunit of this protein kinase . In fertilized sea urchin eggs the activity of H1K oscillates during the cell division cycle and there is a striking temporal correlation between H1K activation and the accumulation of a phosphorylated form of cyclin . H1K activity declines in parallel with proteolytic cyclin destruction of the end of the first cell cycle . By virtue of the high affinity of the fission yeast p13suc1 for the p34cdc2 protein, H1K strongly binds to p13-Sepharose beads . Cyclin, p34cdc2 and H1K co-purify on this affinity reagent as well as through several conventional chromatographic procedures . Anticyclin antibodies immunoprecipitate the M-phase specific H1K in crude extracts or in purified fractions . Sea urchin eggs appear to contain much less cyclin than p34cdc2, suggesting that p34cdc2 may interact with other proteins . These results demonstrate that cyclin and p34cdc2 are major components of the M-phase specific H1K. Biokhimiia, 1989 Aug, 54(8), 1294 - 9 {Molecular characteristics of beta-galactosidase secreted by Penicillium canescens}; Nikolaev IV et al.; Extracellular beta-galactosidase from P . canescens culture medium was purified by ion-exchange chromatography on DEAE and CM-Sepharose CL-6B and gel filtration . The enzyme active form was shown to be a monomer with a molecular weight of about 120 kDa; the isoelectric point is 6.7 and the sedimentation coefficient is 6.5 . In terms of physico-chemical and catalytic properties, the purified enzyme is similar to beta-galactosidases of other fungi of genus Penicillium . The amino acid composition and the NH2-terminal sequence of 24 residues non-homologous to the corresponding sequences of bacterial and yeast beta-galactosidases were determined. Clin Immunol Immunopathol, 1989 Aug, 52(2), 271 - 8 Monocyte function in systemic lupus erythematosus; Boswell J et al.; Systemic lupus erythematosus (SLE) is characterized by multiple defects affecting B and T lymphocyte cell function . In view of the influence that monocytes may have on these functions, we have examined monocyte function in patients with SLE and normals . Monocytes were examined as to number, their ability to modulate IgG synthesis, phagocytose yeast particles, and acid phosphatase activity . Patients had more monocytes and mononuclear cells made more IgG, but these factors appear to be independent of each other . Monocytes from lupus patients are less phagocytic but show similar levels of acid phosphatase activity to normal controls . Normal, but not lupus monocytes, show enhanced phagocytosis following treatment with LPS . The effect of monocyte stimulation on acid phosphatase activity is the same in lupus and normal monocytes . Taken together, these observations suggest that the increased numbers of monocytes in lupus patients and some subtle functional differences in these cells may be important in the progression of this disease. J Bioenerg Biomembr, 1989 Aug, 21(4), 461 - 70 Hexokinase-binding properties of the mitochondrial VDAC protein: inhibition by DCCD and location of putative DCCD-binding sites; Nakashima RA; The outer mitochondrial membrane receptor for hexokinase binding has been identified as the VDAC protein, also known as mitochondrial porin . The ability of the receptor to bind hexokinase is inhibited by pretreatment with dicyclohexylcarbodiimide (DCCD) . At low concentrations, DCCD inhibits hexokinase binding by covalently labeling the VDAC protein, with no apparent effect on VDAC channel-forming activity . The stoichiometry of {14C}-DCCD labeling is consistent with one to two high-affinity DCCD-binding sites per VDAC monomer . A comparison between the sequence of yeast VDAC and a conserved sequence found at DCCD-binding sites of several membrane proteins showed two sites where the yeast VDAC amino acid sequence appears to be very similar to the conserved DCCD-binding sequence . Both of these sites are located near the C-terminal end of yeast VDAC (residues 257-265 and 275-283) . These results are consistent with a model in which the C-terminal end of VDAC is involved in binding to the N-terminal end of hexokinase. Biochimie, 1989 Aug, 71(8), 963 - 8 Ionic mitochondrial channels: characteristics and possible role in protein translocation; Henry JP et al.; Most of the mitochondrial proteins are synthesized in the cytoplasm as precursors which are then translocated into the organelle . These precursors have a NH2-terminal extension which functions as a mitochondrial targeting signal . The import process through mitochondrial membranes is voltage-dependent; its mechanism is still unknown . Translocation has been proposed to occur through specific channels, thus, indicating the interest of the study of mitochondrial ionic channels . Two anion channels with different electrical characteristics have been described in the outer and the inner membranes . Using the technique of "Tip-Dip", we have shown the existence of a cation channel of large conductance in mitochondria . The characteristics of this channel differ from that of the other mitochondrial anion channels . A positively charged 13-residue synthetic peptide, with the sequence of the amino terminal extremity of the nuclear-coded subunit IV of yeast cytochrome C oxidase, induces a blockade of the cationic channel . From the characteristics of the blockade, it is likely that the channel could be permeable to the peptide . The specificity of this effect suggests that this channel might be involved in protein translocation. Exp Cell Res, 1989 Aug, 183(2), 361 - 75 M-phase-specific protein kinase from mitotic sea urchin eggs: cyclic activation depends on protein synthesis and phosphorylation but does not require DNA or RNA synthesis; Arion D et al.; Histone H1 kinase (H1K) undergoes a transient activation at each early M phase of both meiotic and mitotic cell cycles . The mechanisms underlying the transient activation of this protein kinase were investigated in mitotic sea urchin eggs . Translocation of active H1K from particulate to soluble fraction does not seem to be responsible for this activation . H1K activation cannot be accounted for by the transient disappearance of a putative H1K inhibitor present in soluble fractions of homogenates . Aphidicolin, an inhibitor of DNA synthesis, and actinomycin D, an inhibitor of RNA synthesis, do not impede the transient appearance of H1K activity . H1K activation therefore does not require DNA or RNA synthesis . Fertilization triggers a rise in intracellular pH responsible for the increase of protein synthesis . H1K activation is highly dependent on the intracellular pH . Ammonia triggers an increase of intracellular pH and stimulates protein synthesis and H1K activation . Acetate lowers the intracellular pH, decreases protein synthesis, and blocks H1K activation . Protein synthesis is an absolute requirement for H1K activation as demonstrated by their identical sensitivities to emetine concentration and to time of emetine addition . About 60 min after fertilization, H1K activation and cleavage become independent of protein synthesis . The concentration of p34, a homolog of the yeast cdc2 gene product which has been recently shown to be a subunit of H1K, does not vary during the cell cycle and remains constant in emetine-treated cells . H1K activation thus requires the synthesis of either a p34 postranslational modifying enzyme or another subunit . Finally, phosphatase inhibitors and ATP slow down in the in vitro inactivation rate of H1K . These results suggest that a subunit or an activator of H1K is stored as an mRNA in the egg before mitosis and that full activation of H1K requires a phosphorylation. Cell, 1989 Jul 28, 58(2), 361 - 72 Regulation of p34cdc2 protein kinase during mitosis; Moreno S et al.; The cell-cycle timing of mitosis in fission yeast is determined by the cdc25+ gene product activating the p34cdc2 protein kinase leading to mitotic initiation . Protein kinase activity remains high in metaphase and then declines during anaphase . Activation of the protein kinase also requires the cyclin homolog p56cdc13, which also functions post activation at a later stage of mitosis . The continuing function of p56cdc13 during mitosis is consistent with its high level until the metaphase/anaphase transition . At anaphase the p56cdc13 level falls dramatically just before the decline in p34cdc2 protein kinase activity . The behavior of p56cdc13 is similar to that observed for cyclins in oocytes . p13suc1 interacts closely with p34cdc2; it is required during the process of mitosis and may play a role in the inactivation of the p34cdc2 protein kinase . Therefore, the cdc25+, cdc13+, and suc1+ gene products are important for regulating p34cdc2 protein kinase activity during entry into, progress through, and exit from mitosis. Gene, 1989 Jul 15, 79(2), 207 - 17 Structure and expression of a gene encoding heat-shock protein Hsp70 from the Oomycete fungus Bremia lactucae; Judelson HS et al.; A gene encoding a protein homologous to a 70-kDa heat-shock protein (Hsp70) was isolated from Bremia lactucae and its structure and pattern of expression were determined . This is the first report on the structure of a protein-coding gene from an Oomycete fungus . The cloned gene is a member of a small multigene family . The level of hsp70 mRNA in germlings increases from a low constitutive level in response to heat or cold treatment . A high level of the mRNA is also detected in spores . The hsp70 gene is expressed as a primary transcript of 2241 nucleotides (nt) and contains a continuous open reading frame of 2025 nt . Near the C terminus of the coding sequence is an unusual region that contains repeated enhancer-like sequences . This insert has not been described in other hsp70 genes and is not an intron . Upstream from the 5' terminus of the mRNA are multiple CCAAT motifs, a sequence similar to a consensus heat-shock regulatory element, and an A + T-rich putative 'TATA' box . A canonical polyadenylation recognition sequence is present downstream from the coding sequence . The deduced amino acid sequence is equally similar to yeast and maize Hsp70, providing further evidence of the dissimilarity between Oomycetes and true fungi . The cloning of this gene is part of our strategy to develop a transformation system for B . lactucae. J Biol Chem, 1989 Jul 15, 264(20), 11928 - 33 Synthesis and properties of 5-azido-UDP-glucose . Development of photoaffinity probes for nucleotide diphosphate sugar binding sites; Drake RR Jr et al.; A new active site directed photoaffinity probe, which is a model compound for studying nucleotide diphosphate sugar binding proteins, has been synthesized by coupling 5-azido-UTP and {32P}Glc-1-P using yeast UDP-glucose pyrophosphorylase to produce {beta-32P}5-azidouridine 5'-diphosphoglucose (5N3UDP-Glc) . This probe has photochemical properties similar to that of 5-azidoUTP (Evans, R . K., and Haley, B . E . (1987) Biochemistry 26, 269-276) . The efficacy of 5N3UDP-Glc as an active site directed probe was demonstrated using yeast UDP-Glc pyrophosphorylase . Saturation effects of photoinsertion were observed with an apparent Kd of 51 microM and the natural substrate, UDP-Glc, prevented photoinsertion of {beta-32P}5N3UDP-Glc with an apparent Kd of 87 microM . Prevention of photoinsertion was also seen with UTP and pyrophosphate with apparent Kd values less than 200 microM . UMP, UDP, ATP, and GTP were much less effective competitors . Selective photoinsertion was observed with several partially purified enzymes including UDP-Glc dehydrogenase, UDP-Gal-4-epimerase, Gal-1-P uridyltransferase, and phosphorylase a . The absence of nonselective photoinsertion into bulk proteins was demonstrated with crude homogenates of rabbit liver as well as with several UDP-Glc binding proteins . Of the six purified enzymes tested, only phosphoglucomutase has been shown to incorporate radiolabel from the photoprobe in the absence of UV irradiation . These results and a discussion of the utility of 5N3UDP-Glc for detecting UDP-Glc binding proteins and isolating active site peptides are presented. Cell, 1989 Jul 14, 58(1), 69 - 76 Evolution of plant mitochondrial genomes via substoichiometric intermediates; Small I et al.; Comparison of the modern fertile maize mitochondrial genome (N) with an ancestral maize mitochondrial genome (RU) reveals a 12 kb duplication (containing the atpA gene) in the modern genome that is absent from the ancestor . Cloning, mapping, and sequencing of the relevant portions of the ancestral genome shows that this duplication probably arose via a three-stage recombination process involving substoichiometric intermediates . Comparison with analogous observations on yeast mitochondrial genomes suggests that this three-stage model of genome reorganization can be generally applied to plant mitochondrial genomes to explain both deletions and the creation of novel repeats, common features of plant mitochondrial genome evolution. Cell, 1989 Jul 14, 58(1), 37 - 45 Site selection by Xenopus laevis RNAase P; Carrara G et al.; Investigation of the mechanism of cleavage site selection by Xenopus RNAase P reveals that the acceptor stem, a 7 bp helix common to all tRNA precursors, is required for cleavage . We propose that Xenopus RNAase P recognizes conserved features of the mature tRNA and that the cleavage site is selected by measuring the length of the acceptor stem . In support of this, we demonstrate that insertion of 2 bp in the acceptor stem of yeast pre-tRNA(3Leu) relocates the cleavage site 2 bases 3' to the original one . In addition, insertion of 1 bp in the acceptor stem of the end-matured yeast pre-tRNA(Phe) generates an RNAase P cleavage site: the enzyme produces a mature tRNA with the characteristic 7 bp stem and releases one 5' flanking nucleotide . Since it has previously been shown that cleavage sites of the splicing endonuclease are determined by the length of the anticodon stem, RNAase P and the splicing endonuclease apparently use different stems to determine their cutting sites. Cell, 1989 Jul 14, 58(1), 85 - 93 Transcriptional activation by the v-myb oncogene and its cellular progenitor, c-myb; Weston K et al.; The v-myb oncogene, like its cellular progenitor c-myb, encodes a short-lived nuclear protein involved in processes affecting growth and differentiation in a number of cell types . Fusion proteins, in which v-myb sequences are linked to the DNA binding domain of the yeast transcriptional activator GAL4, can activate transcription from a reporter gene linked in cis to a GAL4 binding site . The domain of v-myb responsible for transcriptional activation is located between residues 204 and 254, and is both necessary and sufficient for activation . Intact v-myb and c-myb proteins can also activate transcription, via a myb binding site linked in cis to a reporter gene . A v-myb protein bearing a deletion in the activator domain is no longer capable of stimulating transcription. Biochemistry, 1989 Jul 11, 28(14), 5794 - 801 Structure of an unmodified tRNA molecule; Hall KB et al.; We have used NMR to study the structure of the yeast tRNA(Phe) sequence which was synthesized by using T7 RNA polymerase . Many resonances in the imino 1H- spectrum of the transcript have been assigned, including those of several tertiary interactions . When the Mg2+ concentration is high, the transcript appears to fold normally, and the spectral features of the transcript resemble those of tRNA(Phe) . The transcript has been shown to be aminoacylated with kinetics similar to the modified tRNA(Phe) {Sampson, J . R., & Uhlenbeck, O . C . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 1033-1037}, suggesting that the structure of the two molecules must be similar . In the absence of Mg2+ or at {tRNA}:{Mg2+} ratios less than 0.2, the transcript does not adopt the native structure, as shown by both chemical shifts and NOE patterns . In these low Mg2+ conditions, a second GU base pair is found, suggesting a structural rearrangement of the transcript . NMR data indicate that the structure of a mutant having G20 changed to U20 is nearly identical with that of the normal sequence, suggesting that the low aminoacylation activity of this variant is not due to a substantially different conformation. Biochemistry, 1989 Jul 11, 28(14), 6035 - 41 A novel, arsenite-sensitive E2 of the ubiquitin pathway: purification and properties; Klemperer NS et al.; In the multienzyme ubiquitin-dependent proteolytic pathway, conjugation of ubiquitin to target proteins serves as a signal for protein degradation . Rabbit reticulocytes possess a family of proteins, known as E2's, that form labile ubiquitin adducts by undergoing transthiolation with the ubiquitin thiol ester form of ubiquitin activating enzyme (E1) . Only one E2 appears to function in ubiquitin-dependent protein degradation . The others have been postulated to function in regulatory ubiquitin conjugation . We have purified and characterized a previously undescribed E2 from rabbit reticulocytes . E2(230K) is an apparent monomer with a molecular mass of 230 kDa . The enzyme forms a labile ubiquitin adduct in the presence of E1, ubiquitin, and MgATP and catalyzes conjugation of ubiquitin to protein substrates . Exogenous protein substrates included yeast cytochrome c(Km = 125 mu M; kcat approximately 0.37 min-1) and histone H3 (Km less than 1.3 mu M; kcat approximately 0.18 min-1) as well as lysozyme, alpha-lactalbumin, and alpha-casein . E2(230K) did not efficiently reconstitute Ub-dependent degradation of substrates that it conjugated, either in the absence or in the presence of the ubiquitin-protein ligase that is involved in degradation . E2(230K) may thus be an enzyme that functions in regulatory Ub conjugation . Relative to other E2's, which are very iodoacetamide sensitive, E2(230K) was more slowly inactivated by iodoacetamide (k(obs) = 0.037 min-1 at 1.5 mM iodoacetamide; pH 7.0, 37 degrees C) . E2(230K) was also unique among E2's in being subject to inactivation by inorganic arsenite (k(i)max = 0.12 min-1; K(0.5) = 3.3 mM; pH 7.0, 37 degrees C) . Arsenite is considered to be a reagent specific for vicinal sulfhydryl sites in proteins, and inhibition is usually rapidly reversed upon addition of competitive dithiol compounds . Inactivation of E2(230K) by arsenite was not reversed within 10 min after addition of dithiothreitol at a concentration that blocked inactivation if it was premixed with arsenite; inactivation is therefore irreversible or very slowly reversible . We postulate that a conformation change of E2(230K) may be rate-limiting for interaction of enzyme thiol groups with arsenite. Biochim Biophys Acta, 1989 Jul 7, 1008(2), 243 - 6 Transformation of Neurospora crassa by an integrative transforming plasmid is not enhanced by ribosomal DNA sequences; Russell PJ et al.; Two integrative transforming plasmids of Neurospora crassa that differed only by the presence of almost all of a ribosomal DNA repeat unit on one plasmid were constructed . The plasmids were used to test the target concentration hypothesis which states that the transformation frequency is proportional to the number of genomic copies of a homologous sequence located on the transforming plasmid . Since there are approx . 200 copies of the rDNA sequences in the genome, the target concentration hypothesis would have been proved if the transformation frequency was 200-fold higher for the rDNA-containing plasmid compared with the plasmid without rDNA . The results indicated no difference in the transformation for the two plasmids, thereby providing no support for the hypothesis . The target concentration hypothesis has been proved for yeast, and thus mechanisms different from that responsible for integrative transformation in yeast must operate in N . crassa, perhaps including non-homologous recombination events. Vopr Med Khim, 1989 Jul-Aug, 35(4), 132 - 4 {Determination of glutathione peroxidase activity}; Model' MA; A simplified procedure is developed for estimation of glutathione peroxidase activity involving glutathione reductase in the conjugated reaction . The procedure developed enabled to use commonly employed spectrophotometers and to decrease distinctly the glutathione reductase consumption . Isolation of glutathione reductase from yeast is described. Mycopathologia, 1989 Jul, 107(1), 51 - 5 Ketoconazole resistance in Torulopsis glabrata; Nobre G et al.; The majority of clinical isolates of T . glabrata has been shown highly sensitive to ketoconazole, when tested with an agar dilution method, and resistant, when using a broth dilution method . The fact was accounted for the high degree of mutation associated with the haploid state present in this species . Experimental T . glabrata infection in immunocompetent and immunocompromised mice appears not respond to the oral administration of the drug . The results agree with that of broth dilution tests and not with those produced by the agar methods . It seems that agar dilution sensitivity tests are inadequate for yeast species with an high rate of mutation, as T . glabrata. Nippon Shokakibyo Gakkai Zasshi, 1989 Jul, 86(7), 1519 - 24 {The effects of biotin on the metabolism of ammonia and amino acids in urease-induced hyperammonemic rats}; Nagamine T et al.; The effects of oral and intraperitoneal administration of biotin in urease-induced hyperammonemic rats, as well as the influence of biotin deficiency, have been studied . Biotin deficiency was produced by feeding standard diet MF (Oriental Yeast Co.) supplemented with dry egg-white (egg-white group) . Egg-white + biotin group had free access to 0.0014% of biotin solution at all time . Following an intraperitoneal injection of urease, 25 U/kg (B.W.), plasma ammonia levels in egg-white + biotin group were lower than in egg-white group, especially there was significance (p less than 0.05) at 8 hours after the urease injection . Similarly, plasma ammonia levels in biotin-injected rats, in which 1 mg of biotin had been injected intraperitoneally prior to the experiment, were significantly low compared with saline-injected controls at 4 and 6 hours after urease administration . Results of plasma amino acid analysis, 9 hours after the urease injection indicated that Fischer's molar ratio (Leu + Ileu + Val/Tyr + Phe) was significantly higher in the biotin-injected rats than the saline-injected control . It suggests that biotin might decrease blood ammonia by facilitating the detoxification mechanism as follow: L-glutamate + NH3----L-glutamine. J Biolumin Chemilumin, 1989 Jul, 4(1), 159 - 63 Enhanced chemiluminescence ELISA for the detection of antibody to hepatitis B virus surface antigen; Ireland D et al.; A chemiluminescent enzyme linked immunosorbent assay (ELISA) for the detection of antibody to hepatitis B virus surface antigen (anti-HBs) in human serum has been developed . Polystyrene microtitre plates were coated with recombinant, yeast-derived hepatitis B surface antigen (rec-HBsAg) . Patient serum samples and appropriate controls were added to the rec-HBsAg-coated wells and incubated to bind anti-HBs . The wells were then washed and a fluorescein isothiocyanate (FITC) conjugate of a human plasma-derived hepatitis B surface antigen (HBsAg) was added . Following incubation and further washing, the bound FITC-labelled HBsAg was detected after addition of a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody and assaying for the enzyme . The activity of the HRP was measured using luminol and hydrogen peroxide as substrates and iodophenol as a chemiluminescence enhancer . The luminescence was recorded using a camera luminometer . Preliminary tests have shown the assay to be suitable for the detection of antibody in sera from both vaccinees and also from individuals with a past hepatitis B virus infection . The use of the FITC-anti-FITC system together with the measurement of a chemiluminescence signal makes possible the completion of this assay in a few hours . The assay has been shown to be both specific and sensitive and provides a permanent photographic record. Res Commun Chem Pathol Pharmacol, 1989 Jul, 65(1), 35 - 56 Research on heterocyclic compounds . XXV . Phenyl derivatives of fused imidazole systems: antiinflammatory activity; Abignente E et al.; A group of seven acidic phenyl derivatives of some fused imidazole systems were subjected to a series of tests in vivo in order to evaluate their pharmacological activity . Antiinflammatory activity was studied by means of the carrageenin-induced rat paw edema . Hot plate test and writhing induced by acetic acid in mice were used to evaluate analgesic activity . Yeast-induced hyperthermia in rats was employed to study antipyretic action . Irritative and ulcerogenic action on the rat gastric mucosa was examined at higher doses . The antiaggregating activity and the inhibition of platelet malondialdehyde production were studied in vitro . All experimental results are discussed from the point of view of structure-activity relationships and mode of action. S Afr Med J, 1989 Jul 1, 76(1), 13 - 6 Blastomyces dermatitidis infections in the RSA; Frean JA et al.; Twenty cases of blastomycosis have been confirmed in the RSA, 9 of which are presented for the first time . Patients came from all four provinces and the mean age was 40 years . Six cases were diagnosed between 1985 and 1987 . Differences between strains of Blastomyces dermatitidis isolated in the RSA and in North America include morphological and cultural characteristics, mycelial-yeast conversion, antigenic structure, and compatibility in cross-mating experiments . The diagnosis of this disease can be made by direct examination of unstained specimens, by histological examination or by culture of the organism . Culture should be attempted in all cases for confirmation of microscopic findings. Res Microbiol, 1989 Jul-Aug, 140(6), 373 - 8 Differentiation of Candida guilliermondii varieties by lectin-like substances from marine algae; Fabregas J et al.; Lectin-like substances of 54 marine algae, corresponding to 30 red algae and 24 brown algae, were studied to estimate the agglutination activity against Candida guilliermondii . Extracts of Dictyopteris membranacea, Bifurcaria bifurcata, Halidrys siliquosa, Ascophyllum nodosum, Fucus serratus and F . spiralis algae showed selective agglutinating activities for C . guilliermondii "sensu stricto" and some of its varieties . According to their response to agglutination (positive or negative), certain algal extracts enabled the differentiation of varieties of C . guilliermondii independently of the titre values . Three extracts of brown algae were sufficient to identify 4 varieties of C . guilliermondii . The results suggest that lectin-like substances from marine algae may be valuable reagents as assistant tools for the identification of yeast strains. Vestn Khir Im I I Grek, 1989 Jul, 143(7), 39 - 43 {Preventive use of immunostimulants in patients with infectious endocarditis}; Kostiuchenko AL et al.; Leakadine++, levamisole++, proper-myl were used in the postoperative period in 36 patients operated upon for infectious endocarditis with regard for the character of the immunopathology found . The directed immunostimulation results in resolution of immunodeficiency and normalization of the immune status of the patients. Mol Biol (Mosk), 1989 Jul-Aug, 23(4), 1171 - 6 {Characteristics of DNA isolated from a complex form of DNA-polymerase alpha from the rat liver}; Timchenko NA et al.; A complex from of DNA polymerase alpha was isolated from the nuclear membrane of hepatocytes . DNA fragments were shown to be among components of the complex under study . In this paper we present evidence that DNA from the alpha-polymerase complex from quiescent hepatocytes (DNA-G) differs in its nucleotide composition from its counterpart (DNA-S) isolated from hepatocytes synthesizing DNA . As judged by dot hybridization, DNA-G0 does not contain nucleotide sequences which are complementary to ribosomal or messenger RNA, whereas the abovementioned sequences are present in DNA-S . At the same time DNA-G0 is found to contain sequences which are homologous to both SV40 DNA and yeast TRPI-ARS1 DNA . The difference in nucleotide sequences between DNA-G0 and DNA-S indicates that in the process of replication DNA is being stretched across the multienzyme complex located on the nuclear membrane. Prostaglandins, 1989 Jul, 38(1), 79 - 89 Effects of inadequate vitamin E and/or selenium nutrition on the release of arachidonic acid metabolites in rat alveolar macrophages; Eskew ML et al.; Effects of vitamin E and/or selenium (Se) deficiency on the secretion of arachidonic acid metabolites by zymosan-stimulated pulmonary alveolar macrophages (AM) were examined using cells from male Long-Evans hooded rats fed torula-yeast based diets with or without the supplementation of vitamin E (150 IU/kg) or Se (0.5 mg/kg) . Alveolar macrophages obtained by lavage were purified by adherence and cultured for 4 h in Hank's balanced salt solution containing bovine serum albumin (0.1%) and zymosan (300 micrograms/ml) . The arachidonic acid metabolites present in the culture supernatant were measured by radioimmunoassay . Altered vitamin E and Se nutrition had no effect on the number of cells or cell types recovered from the pulmonary airways . Alveolar macrophages derived from animals fed on diets deficient in vitamin E or Se or both nutrients secreted higher levels of prostaglandin E2 and thromboxane B2 . Levels of both 5-hydroxyeicosatetraenoic acid and leukotriene B4 were significantly increased only in the group fed the diet adequate in Se but deficient in vitamin E . Our data suggest that vitamin E and Se might play an important role to control the levels of several physiologically and pathologically important arachidonic acid metabolites. Proc Natl Acad Sci U S A, 1989 Jul, 86(14), 5325 - 9 Molecular cloning of rat homologues of the Drosophila melanogaster dunce cAMP phosphodiesterase: evidence for a family of genes; Swinnen JV et al.; To study the structure and function of cyclic nucleotide phosphodiesterases (PDEs) involved in mammalian gametogenesis, a rat testis cDNA library was screened at low stringency with a cDNA clone coding for the Drosophila melanogaster dunce-encoded PDE as a probe . This screening resulted in the isolation of two groups of cDNA clones, differing in their nucleotide sequences (ratPDE1 and ratPDE2) . In the rat testis, RNA transcripts corresponding to both groups of clones were expressed predominantly in germ cells . Additional screenings of a Sertoli cell cDNA library with a ratPDE2 clone as a probe led to the isolation of two more groups of clones (rat-PDE3 and ratPDE4) . Unlike ratPDE1 and ratPDE2, these clones hybridized to transcripts present predominantly in the Sertoli cell . In the middle of the coding region, all four groups of clones were homologous to each other . The deduced amino acid sequences of part of this region were also homologous to the D . melanogaster dunce PDE and to PDEs from bovine and yeast . These data indicate that a family of genes homologous to the D . melanogaster dunce-encoded PDE is present in the rat and that these genes are differentially expressed in somatic and germ cells of the seminiferous tubule . These findings provide a molecular basis for the observed heterogeneity of cAMP PDEs. Allergol Immunopathol (Madr), 1989 Jul-Aug, 17(4), 205 - 7 Polymorphonuclear functions in patients with mixed connective tissue disease; Bodolay E et al.; Polymorphonuclear leukocytes (PMNs) from 29 patients with mixed connective tissue disease (MCTD) were studied "in vitro" for their phagocytic and chemotactic function as well as for granulocyte alkaline phosphatase (GAP) activity . Fc-receptor expression detected by EA-rosette formation was comparable to the control . Yeast-phagocytosis, C3b-receptor mediated phagocytosis and chemotaxis of PMNs, however, significantly decreased in MCTD . At the same time, photometric measure of alkaline phosphatase activity indicated a nearly two fold increase in PMNs from patients with MCTD . Although no correlation was found between PMN functions and the activity of the disease, PMN disorders may play a role in pathogenesis of these connective tissue disorders. Vet Med (Praha), 1989 Jul, 34(7), 421 - 30 {Detection of cyclopiazonic acid and its producers in food}; Ostry V et al.; In the course of six months, 60 samples of foods were examined for their contents of cyclopiazonic acid . These samples were subjected to a basal mycological screening aimed at Aspergillus flavus and Penicillium sp . strains . Cyclopiazonic acid contents in samples of Hermelin cheese, peanuts, rice, peeled barley grains, Folican salami, and packaged meat did not exceed the value of 0.5 mg.kg-1 . When using a modification of the method of cyclopiazonic acid isolation described by Dorner et al . (1983), 521 mg of this mycotoxin were isolated from a culture of Penicillium griseofulvum CCM 8006 strain grown in liquid medium containing 2% yeast autolysate and 2.5% sucrose . About 47% of the isolated Aspergillus flavus strains were bitoxicogenic (produced both cyclopiazonic acid and aflatoxin) . Cyclopiazonic acid was produced by 23.5% of the isolated Penicillium sp . strains . No cyclopiazonic acid was produced in vitro by Penicillium nalgoviensis strains from the Czechoslovak collection on sweet wort agar containing peptone from soybean . Penicillium commune F-426 and Penicillium aurantiogriseum F-708 strains are efficient producers of this acid. J Mol Evol, 1989 Jul, 29(1), 40 - 51 Dinoflagellates in evolution . A molecular phylogenetic analysis of large subunit ribosomal RNA; Lenaers G et al.; The sequence of the large subunit ribosomal RNA (LsuRNA) gene of the dinoflagellate Prorocentrum micans has been determined . The inferred rRNA sequence {3408 nucleotides (nt)} is presented in its most probable secondary structure based on compensatory mutations, energy, and conservation criteria . No introns have been found but a hidden break is present in the second variable domain, 690 nt from the 5' end, as judged by agarose gel electrophoresis and primer extension experiments . Prorocentrum micans LsuRNA length and G+C content are close to those of ciliates and yeast . The conserved portions of the molecule (1900 nt) have been aligned with corresponding sequences from various eukaryotes, including five protista, one metaphyta, and three metazoa . An extensive phylogenetic study was performed, comparing two phenetic methods (neighbor joining on difference matrix, and Fitch and Margoliash on Knuc values matrix) and one cladistic (parsimony) . The three methods led to similar tree topologies, except for the emergence of yeast that groups with ciliates and dinoflagellates when phenetic methods are used, but emerges later in the most parsimonious tree . This discrepancy was checked by statistical analyses on reduced trees (limited to four species) inferred using parsimony and evolutionary parsimony methods . The data support the phenetic tree topologies and a close relationship between dinoflagellates, ciliates, and yeast. Proc Natl Acad Sci U S A, 1989 Jul, 86(13), 4892 - 6 Multiple alpha subunits of guanine nucleotide-binding proteins in Dictyostelium; Pupillo M et al.; Previous results have shown that chemotaxis and the expression of several classes of genes in Dictyostelium discoideum are regulated through a cell surface cAMP receptor interacting with guanine nucleotide-binding proteins (G proteins) . We now describe cloning and sequencing of cDNAs encoding two G alpha protein subunits from Dictyostelium . The derived amino acid sequences show that they are 45% identical to each other and to G alpha protein subunits from mammals and yeast . Both cDNAs are complementary to multiple mRNAs that are differentially expressed during development . This evidence and analysis of mutants presented elsewhere suggest that they have distinct physiological functions. Arch Biochem Biophys, 1989 Jul, 272(1), 97 - 102 Phytoalexin synthesis in soybean cells: elicitor induction of reductase involved in biosynthesis of 6'-deoxychalcone; Welle R et al.; Chromatofocusing on Mono P proved to be an efficient purification procedure for the NADPH-dependent reductase from soybean (Glycine max L.) cell cultures which acts together with chalcone synthase in the biosynthesis of 2',4',4-trihydroxychalcone (6'-deoxychalcone) . By isoelectric focusing the pI of reductase was determined to be 6.3 . Addition of pure soybean reductase to cell-free extracts from stimulated cell cultures of parsley and bean (Phaseolus vulgaris) and from young flowers of Dahlia variabilis caused in each case synthesis of 6'-deoxychalcone . When 4-coumaroyl-CoA was replaced by caffeoyl-CoA in the reductase assay, formation of 2',4',3,4-tetrahydrochalcone (butein) was observed . A polyclonal antireductase antiserum was raised in rabbits and proved to be specific in Ouchterlony diffusion experiments, Western blots and immunotitration . The reductase antiserum showed no cross-reactivity with soybean chalcone synthase (CHS) . A biotin/{125I}streptavidin system provided a quantitative Western blot for the reductase . Changes in the activities, amounts of protein, and mRNA activities of reductase and CHS were determined after challenge of soybean cell cultures by elicitor (from Phytophthora megasperma f.sp . glycinea or yeast) . For both enzymes a pronounced and parallel increase in activity and amounts of protein was observed after elicitor addition with a maximum at about 16 h after challenge . Parallel increases in mRNA activities occurred earlier . The results indicate a parallel induction of de novo synthesis of reductase and CHS which coact in synthesis of 6'-deoxychalcone. Mol Plant Microbe Interact, 1989 Jul-Aug, 2(4), 165 - 8 Expression of reverse transcriptase genes in Fulvia fulva; McHale MT et al.; Antibodies raised against intercellular fluid antigens isolated from diseased tomato leaves have revealed that the fungal pathogen Fulvia fulva expresses genes for a fungal reverse transcriptase (RNA-dependent DNA polymerase) . This enzyme is required for the replication of retroviruses and retroviral-like transposable elements and could provide a mechanism for increasing the mutation rate of fungal pathogens, perhaps explaining their ability to evolve new races rapidly . We report here the DNA sequence of a 225-bp clone from a lambda gt11 genomic library of F . fulva . This clone, designated P5, exhibits a high degree of sequence homology with the reverse transcriptase (pol) gene of the Drosophila melanogaster copia-like retrotransposon 17.6 . Southern blot analysis of genomic DNA of F . fulva showed that P5-related sequences are moderately reiterated with 30-100 copies, some of which exhibit restriction fragment length polymorphism in different races of the pathogen . Western blot analysis of extracts from F . fulva with antibodies raised to purified reverse transcriptase (from human immunodeficiency virus-1) revealed immunoreactive proteins . Reverse transcriptase previously has been detected in a variety of organisms including yeast, insects, protozoa, and mammals, but to our knowledge, this is the first report of its occurrence in filamentous fungi. Cell, 1989 Jun 30, 57(7), 1167 - 77 All ras proteins are polyisoprenylated but only some are palmitoylated; Hancock JF et al.; The C-terminal CAAX motif of the yeast mating factors is modified by proteolysis to remove the three terminal amino acids (-AAX) leaving a C-terminal cysteine residue that is polyisoprenylated and carboxyl-methylated . Here we show that all ras proteins are polyisoprenylated on their C-terminal cysteine (Cys186) . Mutational analysis shows palmitoylation does not take place on Cys186 as previously thought but on cysteine residues contained in the hypervariable domain of some ras proteins . The major expressed form of c-K-ras (exon 4B) does not have a cysteine residue immediately upstream of Cys186 and is not palmitoylated . Polyisoprenylated but nonpalmitoylated H-ras proteins are biologically active and associate weakly with cell membranes . Palmitoylation increases the avidity of this binding and enhances their transforming activity . Polyisoprenylation is essential for biological activity as inhibiting the biosynthesis of polyisoprenoids abolishes membrane association of p21ras. Cell, 1989 Jun 30, 57(7), 1259 - 73 The gradient morphogen bicoid is a concentration-dependent transcriptional activator; Struhl G et al.; The bicoid (bcd) protein is expressed in an anteroposterior gradient in early Drosophila embryos and controls the zygotic activation of the segmentation gene hunchback (hb) in a broad but precisely bounded anterior domain . Here we show that the hb gene contains multiple regulatory elements that mediate transcriptional activation in response to bcd protein . Further, we demonstrate that the resulting patterns of expression in vivo depend critically on both the bcd gradient profile and the number and quality of these hb elements . Finally, we show that these same elements mediate bcd-dependent transcriptional activation in yeast and that this interaction requires distinct DNA binding and activating regions in the bcd protein . Our results argue that bcd protein normally binds and activates the hb gene in a concentration-dependent fashion, thereby allowing the gradient of bcd protein to dictate where the hb gene is initially turned on in early embryos . They also suggest that the bcd gradient has the instructive capacity to activate other subordinate control genes by the same mechanism, each in a distinct spatial domain according to its affinity for bcd protein. J Biol Chem, 1989 Jun 25, 264(18), 10649 - 53 The two Xenopus laevis SRC genes are co-expressed and each produces functional pp60src; Steele RE et al.; The haploid genome of Xenopus laevis contains two src genes, and transcripts from both genes are found in the maternal RNA pool of the oocyte (Steele, R . E . (1985) Nucleic Acids Res . 13, 1747-1761) . We have now isolated cDNA clones which contain complete coding sequences from both src mRNAs . In vitro translation of RNAs transcribed in vitro from these clones produces in each case a protein with an apparent molecular mass of 57 kDa . The in vitro-synthesized proteins show identical protease cleavage patterns . Sequence analysis of the coding regions of the two cDNAs revealed that they both produce 532-amino acid polypeptides which differ from each other at only eight sites . Analysis of silent site changes between the two coding sequences suggests that the two genes began diverging about 25 million years ago . Hybridization with probes specific for each of the two src RNAs indicates that the two genes are co-expressed in embryos and in at least some adult tissues as well as during oogenesis . Finally, expression of each of the cDNA clones in yeast causes the appearance of proteins which are recognized by an antibody which binds to phosphotryosine. Science, 1989 Jun 23, 244(4911), 1475 - 7 Regulatory role for GTP-binding proteins in endocytosis; Mayorga LS et al.; Guanosine 5'-triphosphate (GTP)-binding proteins have been implicated in the transport of newly synthesized proteins along the secretory pathway of yeast and mammalian cells . Early vesicle fusion events that follow receptor-mediated endocytosis as measured by three in vitro assays were blocked by guanosine 5'-O-(3-thiotriphosphate) and aluminum fluoride . The effect was specific for guanosine nucleotides and depended on the presence of cytosolic factors . Thus, GTP-binding proteins may also have a role in the transport of molecules along the endocytic pathway. Carbohydr Res, 1989 Jun 15, 189, 289 - 99 Purification of endo-N-acetyl-beta-D-glucosaminidase H by substrate-affinity chromatography; Greber UF et al.; Endo-N-acetyl-beta-D-glucosaminidase H (Endo H) was purified to homogeneity (3000-fold) from a culture filtrate to Streptomyces plicatus . The key step was substrate-affinity chromatography, which afforded a 1000-fold purification and yielded a protease- and exoglycosidase-free preparation of Endo H . Proteins from the crude sample were applied to the substrate-affinity column, consisting of yeast-invertase glycopeptides bound to Sepharose-immobilized concanavalin A . After washing off the unbound proteins, Endo H was quantitatively eluted by methyl alpha-D-mannopyranoside . Various conditions were tested to achieve an optimal binding of Endo H to this substrate-affinity gel . After substrate-affinity chromatography, Endo H was separated from the coeluted gylcopeptide substrate and some protein impurities by gel filtration and hydrophobic chromatography. Biochemistry, 1989 Jun 13, 28(12), 5226 - 31 Molecular organization of developmentally regulated Dictyostelium discoideum ubiquitin cDNAs; Ohmachi T et al.; Dictyostelium discoideum ubiquitin mRNAs are regulated in a complex fashion during spore germination and multicellular development . Species of mRNA of 1900, 1400, 1100, 840, 580, and 500 nucleotides (nt) are found which are expressed differentially during different stages of development . DNA blot analysis indicates that ubiquitin genes constitute a multigene family of at least six genes . cDNAs representing all the ubiquitin mRNA transcripts were isolated and sequenced . The Dictyostelium mRNAs are organized as tandem repeats of the 76 amino acid ubiquitin unit (228 nt) . We isolated one cDNA containing seven of these tandem repeats, and two different five- and three-repeat cDNAs . In addition, 2 cDNAs containing a single ubiquitin repeat fused at its 3' end to an unrelated 52 and 78 amino acid extension were identified . There is a remarkable similarity in the sequences of the non-ubiquitin extensions among yeast and mammalian counterparts . The extensions are very basic, containing approximately 30% lysine/arginine . Another common feature of these proteins is the presence of a common structural motif containing cysteine residues at conserved positions, suggesting a metal binding domain that matches a consensus sequence of Xenopus transcription factor TFIIIA and other nucleic acid binding proteins . The characterization of ubiquitin cDNAs and genomic sequences in D . discoideum now makes the understanding of its developmental regulation feasible. J Biol Chem, 1989 Jun 5, 264(16), 9141 - 4 A synthetic peptide-directed antibody as a probe of the phosphorylation site of pyruvate dehydrogenase; Miernyk JA et al.; A synthetic peptide, corresponding to the 14-amino acid tryptic fragment containing phosphorylation sites one and two of bovine mitochondrial pyruvate dehydrogenase, was coupled to Limulus polyphemus hemocyanin and used to produce rabbit polyclonal antibodies . A positive signal was observed when Western blots of bovine, porcine, or yeast mitochondrial pyruvate dehydrogenase complexes were probed with the antibodies but not with blots of bacterial, cellular slime mold, plant mitochondrial, or plant plastid pyruvate dehydrogenase complexes . The antibodies also gave a positive signal when used to probe blots of the bovine kidney branched chain 2-oxo acid dehydrogenase complex . The ATP-dependent phosphorylation/inactivation of rat liver mitochondrial pyruvate dehydrogenase complex could be inhibited by prior incubation with the anti-peptide antibodies. J Biol Chem, 1989 Jun 5, 264(16), 9103 - 6 Guanylate cyclase, a cell surface receptor; Garbers DL; Guanylate cyclase appears to represent a central member of a diverse family of proteins involved in cell signaling mechanisms including the protein kinases, a low Mr ANP receptor, and possibly adenylate cyclase (based on limited sequence identity with the yeast enzyme) . A membrane form of guanylate cyclase represents a new model for cell surface receptors, although such a model was once envisioned for adenylate cyclase (79) . In original models for adenylate cyclase, hormone was thought to bind with either the enzyme or with an unknown protein to enhance cyclic AMP production (79) . Guanylate cyclase appears to fall into the first adenylate cyclase model where binding of a ligand to an extracellular site on the enzyme transmits a signal to an intracellular catalytic site . The production of cyclic GMP, a second messenger, and of pyrophosphate are then increased . The protein tyrosine kinase family of receptors (80) and possibly another forthcoming family of cell surface receptors containing protein tyrosine phosphatase activity (81-83) contain a single transmembrane domain like guanylate cyclase . Furthermore, the protein tyrosine kinases are activated by ligand binding to the extracellular domain . However, the activation of guanylate cyclase, unlike these cell surface receptors, results in the formation of a low molecular weight second messenger. Cell, 1989 Jun 2, 57(5), 835 - 45 The genes and transcripts of an antigen gene expression site from T . brucei; Pays E et al.; The AnTat 1.3A antigen gene expression site of T . brucei was cloned from genomic libraries of the 200 kb expressor chromosome . In addition to the antigen gene, it contains seven putative coding regions (ESAGs, for expression site-associated genes), as well as a RIME retroposon . The polypeptide encoded by ESAG 4 shows homology to yeast adenylate cyclase, and possesses structural features of a transmembrane protein . The expression site is transcribed by a pol l-like polymerase in the parasite bloodstream form only, but sequences similar to ESAGs 5, 4, and 2 are also transcribed constitutively elsewhere, by a polymerase sensitive to alpha-amanitin . Ultraviolet irradiation, which seems to block RNA processing, allows the tentative mapping of a transcription promoter about 45 kb upstream of the antigen gene. Vrach Delo, 1989 Jun, (6), 10 - 2 {Optimization of chemotherapy of stomach cancer}; Chernyi VA et al.; Results are analyzed of the treatment of 520 patients with diffuse gastric cancer using different schemes that included chemotherapy with fluorouracil, intraabdominal polychemotherapy with fluorouracil and adriablastin, immunotherapy with yeast polysaccharides and thymus hormone--taktivin . Chemotherapy supplemented by immunotherapy essentially increases survival . Intraabdominal administration of antitumour drugs in gastric cancer is anatomically more adequate than the intravenous route. Akush Ginekol (Mosk), 1989 Jun, (6), 20 - 3 {Urogenital chlamydia infection in women with habitual abortion}; Chuchupalov PD et al.; The Chlamydozoan population was identified in 43.6 per cent of 110 females with a history of habitual abortion . It was found that the infection was more frequent in females aged under 30 years, in those who was born preterm, in patients with early (premarital) sexual contacts or those with spontaneously stopped pregnancy during the first trimester, as well as in those females who suffered from cervicitis, yeast colpitis, pyelonephritis or had post-inflammatory changes of placental tissue . Higher incidence of cervicitis, cervical erosion, yeast colpitis, salpingo--oophoritis and pyelonephritis was documented in females infected with Chlamydozoa. Lipids, 1989 Jun, 24(6), 518 - 25 A novel spectrophotometric assay for lipase activity utilizing cis-parinaric acid; Rogel AM et al.; A new spectrophotometric assay for determining the activity of acylglycerol hydrolases (lipases, E.C . 3.1.1.3) was developed and optimized for yeast lipase (Candida cylindracea) . Studies with porcine pancreatic lipase were also conducted and the influence of various detergents and divalent cations on the assay was evaluated . The assay uses cis-parinaric acid (PnA), a naturally occurring fatty acid that has unique spectroscopic properties, and takes advantage of the reversible binding of fatty acids to bovine serum albumin (BSA) . Free PnA has an ultraviolet absorption peak at 321.2 nm . When PnA is bound to BSA, however, the peak shifts to 324.2 nm . The assay mixture contains 6 microM PnA, 1 microM BSA, 75 microM triolein, and 0.3 mM taurocholate in a 50 mM tris-HCl buffer with 1 microM EDTA . The release of oleic acid from triolein is monitored over time by measuring the ratio of optical densities (OD) at 319.0 and 329.0 nm . Initially, there is maximum binding of PnA to BSA, and the OD ratio is approximately 1.0 . Upon addition of lipase, PnA is displaced from the BSA by oleic a