Yeasts

Antimicrob Agents Chemother, 2000 Oct, 44(10), 2604 - 8
Comparison of a new triazole, posaconazole, with itraconazole and amphotericin B for treatment of histoplasmosis following pulmonary challenge in immunocompromised mice; Connolly P et al.; A murine model of intratracheally induced histoplasmosis in immunocompromised B6C3F(1) mice was used to evaluate a new triazole antifungal agent, posaconazole . This compound was previously shown to be comparable to amphotericin B and superior to itraconazole for the treatment of histoplasmosis in immunocompetent mice . The current study used mice that were depleted of T lymphocytes by intraperitoneal injection of anti-CD4 and anti-CD8 monoclonal antibodies beginning 2 days before infection and continuing at 5-day intervals until completion of the study . Groups of B6C3F(1) mice that were depleted of CD4 and CD8 T cells were infected with an inoculum of 10(4) Histoplasma capsulatum yeasts . All mice receiving posaconazole at 1 or 0.1 mg/kg of body weight/day, amphotericin B at 2 mg/kg every other day (qod), or itraconazole at 75 mg/kg/day survived to day 29 . Only 60% of mice receiving itraconazole at 10 mg/kg/day and none receiving amphotericin B at 0.2 mg/kg qod survived to that date . Fungal burdens were determined at day 14 of infection, 1 day after discontinuation of therapy . Quantitative colony counts and Histoplasma antigen levels in lung and spleen tissues declined following treatment with amphotericin B at 2 mg/kg qod, posaconazole at 5 and 1 mg/kg/day, and itraconazole at 75 mg/kg/day but not in mice treated with amphotericin B at 0.2 mg/kg qod or itraconazole at 10 mg/kg/day . Posaconazole at 0.1 mg/kg/day reduced fungal colony counts and antigen levels in spleens but not in lungs . This study shows posaconazole activity for the treatment of histoplasmosis in immunosuppressed animals.

J Toxicol Environ Health A, 2000 Sep 15, 61(1), 55 - 67
The fungal cell wall component beta-1,3-glucan has an adjuvant effect on the allergic response to ovalbumin in mice; Ormstad H et al.; The polyglucose beta-1,3-D-glucan is a major structural component of the cell wall of yeasts and fungi . In the present study, the adjuvant activity of beta-1,3-glucan from the fungus Sclerotinia sclerotiorum (SSG) on the response to the model allergen ovalbumin (OA) was studied, using the popliteal lymph node assay (PLNA) in BALB/c mice . The adjuvant activity on the local cellular response was determined by measuring the weight, cell number, and proliferation of the extracted PLNs . The levels of OA-specific immunoglobulin (Ig)E, IgG1, and IgG2a in serum were measured by enzyme-linked immunosorbent assay (ELISA) . Groups of 8 mice were given either SSG + OA, SSG alone, or OA alone on d 0 . Thereafter they were exsanguinated on d 20, or reinjected with OA on d 21, before exsanguination on d 26 or 33 . Only on d 26 was SSG + OA found to significantly increase the PLN weight and cell numbers, but not cell proliferation (thymidine incorporation), compared with OA or SSG alone . SSG + OA was also found to significantly increase both the anti-OA IgE and IgG1 levels on d 20, 26, and 33 compared to OA alone . Compared to SSG alone, SSG + OA increased the OA-specific IgE and IgG 1 levels significantly on d 26 and 33, but not on d 20 . A similar increase was not found for IgG2a . Our results show that beta-1,3-D-glucan provides a clear Th2-dependent (allergic) immune response to OA, indicated by elevated levels of IgE and IgG1 and not IgG2a, in the mouse model used.

J Mol Biol, 2000 Sep 22, 302(3), 593 - 606
Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments; Lehman W et al.; Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure . In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state . On activation, tropomyosin moves away from these sites in two steps, one induced by Ca(2+) binding to troponin and a second by the binding of myosin to actin . In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood . Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells . Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and non-muscle sources, was investigated . Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined . Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small . Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively . In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility . Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins . Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling .

J Zoo Wildl Med, 2000 Jun, 31(2), 211 - 4
Disseminated blastomycosis in two California sea lions (Zalophus californianus); Zwick LS et al.; Two captive California sea lions (Zalophus californianus) from different facilities were diagnosed with disseminated blastomycosis . The first, a 12-yr-old male, died after a 3-wk history of progressive anorexia and lethargy . Gross examination revealed acute jejunitis with focal perforation and associated peritonitis, along with severe purulent bronchopneumonia . The second, a 15-yr-old female, was euthanized after a 2-wk history of severe cutaneous ulceration and declining clinical condition . Gross examination revealed severe pyogranulomatous bronchopneumonia and ulcerative dermatitis . Histopathologic examination in both individuals revealed severe multifocal subacute to chronic pyogranulomatous pneumonia associated with massive numbers of fungal organisms morphologically compatible with Blastomyces sp . Fungal organisms were 8-20-microm-diameter broad-based budding yeasts with thick, refractile, double-contoured walls . The male sea lion had multifocal transmural Blastomyces-induced enteritis with subsequent rupture and peritonitis . The organism was also present in the liver, with minimal associated inflammation . The female had severe multifocal pyogranulomatous ulcerative dermatitis associated with large numbers of intralesional fungal organisms . Dissemination to the spleen had occurred in both animals . A serologic immunodiffusion test for Blastomyces dermatitidis was positive in the male . The presumptive primary pathogen in both cases was Blastomyces dermatitidis.

Bioorg Med Chem, 2000 Jul, 8(7), 1677 - 96
Total synthesis and antifungal evaluation of cyclic aminohexapeptides; Klein LL et al.; The need for new therapies to treat systemic fungal infections continues to rise . Naturally occurring hexapeptide echinocandin B (1) has shown potent antifungal activity via its inhibition of the synthesis of beta-1,3 glucan, a key fungal cell wall component . Although this series of agents has been limited thus far based on their physicochemical characteristics, we have found that the synthesis of analogues bearing an aminoproline residue in the 'northwest' position imparts greatly improved water solubility (> 5 mg/mL) . The synthesis and structure-activity relationships (SAR) based on whole cell and upon in vivo activity of the series of compounds are reported.

Med Mycol, 2000 Aug, 38(4), 289 - 300
Genetic divergence at the SODA locus of six different formae speciales of Pneumocystis carinii; Denis CM et al.; Genetic divergence at the SODA (manganese-dependent superoxide dismutase, MnSOD) locus were compared in six Pneumocystis carinii formae speciales isolated from mouse, rabbit, human, macaque and pig . A degenerate oligonucleotide primer strategy was designed to amplify 85-90% of the full-length SODA gene from P . carinii genomic DNA isolates . DNA sequence analysis revealed an A/T bias in the nucleotide composition (71-77.2%) and the presence of seven small introns (41-142 bp), interrupting each P . carinii open reading frame (ORF) at the same position . The MnSOD deduced amino acid sequences from all P . carinii isolates shared residues which were conserved within the MnSOD family and which are required for enzymatic activity and binding of the cofactor metal . Phylogenetic analysis including MnSOD sequences from representatives of the fungal phyla Basidiomycota and Ascomycota indicated that the P . carinii formae speciales form a monophyletic group that is related to the budding yeasts (subphylum Saccharomycotina, previously called class Hemiascomycetes) in the Ascomycota . In the whole Pneumocystis group, P . carinii f . sp . hominis, P . carinii f . sp . macacae and P . carinii f . sp . oryctolagi MnSOD sequences clustered together, as did the rat-derived P . carinii and P . carinii f . sp . muris sequences.

Microbes Infect, 2000 Jul, 2(9), 997 - 1001
Experimental histoplasmosis in mice treated with anti-murine interferon-gamma antibody and in interferon-gamma gene knockout mice; Clemons KV et al.; Histoplasma capsulatum is an important fungal pathogen in immunocompromised hosts, including AIDS patients . Experimental evidence suggests interferon-gamma (IFN) plays a role in host defense against H . capsulatum . In these studies we sought to demonstrate the importance of IFN in innate resistance to systemic histoplasmosis . The possible exacerbation of infection in BALB/c mice was assessed by administering 200 microg of hamster anti-IFN antibody prior to infection with H . capsulatum (2 x 10(6) yeasts, i.v.) and by comparing the severity of infection between BALB/c IFN gene knockout mice (GKO) and congenic control animals . In two separate studies, we found that anti-IFN treatment caused a dramatic loss of resistance to lethal infection and resulted in earlier mortality of IFN-depleted animals compared with normal IgG or no treatment (P<0.001) . GKO mice were significantly (P<0.001) more susceptible to lethal infection than were control animals, and histological studies corroborated this . These studies clearly demonstrate that IFN is a vital part of the host's innate resistance to systemic infection with H . capsulatum and provide an additional rationale for studying IFN as an immunomodulatory therapeutic for the treatment of this disease.

Biochem Soc Trans, 2000, 28(4), 499 - 504
Biochemical and molecular approaches to understanding protein import into peroxisomes; Baker A et al.; Peroxisomes are eukaryotic organelles that perform diverse and variable functions . Although genetic studies in yeasts and mammals have identified approximately 20 genes (PEX genes) required for the biogenesis of this important organelle, biochemical studies of protein targeting and import have lagged behind and in many cases we have no idea of the function of the PEX gene products (peroxins) . Using an import assay in vitro derived from sunflower cotyledon cells and recombinant proteins, we have obtained translocation intermediates on the peroxisome import pathway and are using cross-linking to identify interacting partners . We have also used antibodies raised against human PEX14 to inhibit the import of matrix proteins in this system . To obtain homologous antibodies for inhibition experiments, to immunoprecipitate cross-linked products and to enable us to study the import pathways of peroxins we have cloned and characterized plant orthologues of three PEX genes, PEX6, PEX10 and PEX14.

Anal Chem, 2000 Aug 1, 72(15), 3590 - 5
A method for determination of particle magnetic susceptibility with analytical magnetapheresis
Fuh CB, Lai MH, Lin LY, Yeh SY.
We recently developed a new method for simple determination of particle magnetic susceptibility using analytical magnetapheresis . This new method does not require laborious calibration plots and trial susceptibility values as do previous analytical magnetapheresis methods . The new method is based on balancing channel flow rates and magnetically induced flow rates for particle deposition in analytical magnetapheresis . The maximal flow rate for complete particle deposition was determined experimentally and set to equal the magnetically induced flow rate for determining particle magnetic susceptibility . This magnetic susceptibility determination generally takes less than 20 min . Several magnetically susceptible and ion-labeled particles were tested using this new method . The carrier magnetic susceptibilities were varied, and erbium ion-labeled particles were studied experimentally, resulting in successful susceptibility determinations of erbium ion-labeled particles and yeasts . The precision of each measurement was generally approximately 10% . Experimental determination of particle magnetic susceptibilities differed by less than 10% from reference measurements taken using a superconducting quantum interference device magnetometer . This method can determine minimal susceptibilities on the order of 10(-9) cgs . The minimum number of erbium labeling ions per particle required for complete deposition of silicas and yeasts was found to be 6.7 x 10(9) . Analytical magnetapheresis shows good potential for use in simple determination of particle magnetic susceptibilities and should become a useful technique.

Genomics, 2000 Jul 1, 67(1), 40 - 7
cDNA cloning and expression analysis of new members of the mammalian F-box protein family; Ilyin GP et al.; F-box proteins are critical components of the SCF ubiquitin-protein ligase complex and are involved in substrate recognition and recruitment for ubiquitination and consequent degradation by the proteasome . We have isolated cDNAs encoding a further 10 mammalian F-box proteins . Five of them (FBL3 to FBL7) share structural similarities with Skp2 and contain C-terminal leucine-rich repeats . The other 5 proteins have different putative protein-protein interaction motifs . Specifically, FBS and FBWD4 proteins contain Sec7 and WD40-repeat domains, respectively . The C-terminal region of FBA shares similarity with bacterial protein ApaG while FBG2 shows homology with the F-box protein NFB42 . The marked differences in F-box gene expression in human tissues suggest their distinct role in ubiquitin-dependent protein degradation.

Pharmazie, 2000 Jul, 55(7), 483 - 9
Antidermatophytic action of new 1-naphthylmethyl and benzo{b}thiophen-7-ylmethyl hydrazones related to inhibitors of squalene epoxidase; Auzzas L et al.; Two series of hydrazonic compounds related to main classes of inhibitors of fungal squalene epoxidase (SE) were designed and prepared on the hypothesis of a pharmacophoric model . The antifungal activity of the new compounds was evaluated in vitro against dermatophytes, moulds and yeasts . Antidermatophytic activity resulted for several hydrazones, particularly for those containing a tett-butylacetylenic group, supporting the hypothesis that the introduction of a hydrazonic function in the model could retain the antimycotic activity.

Chem Pharm Bull (Tokyo), 2000 Jul, 48(7), 982 - 90
New antifungal 1,2,4-triazoles with difluoro(heteroaryl)methyl moiety; Eto H et al.; New 1,2,4-triazoles (1) having a difluoro(heteroaryl)methyl moiety were designed and synthesized via 1-aryl-2,2-difluoro-2-(heteroaryl)ethanones (2), which were prepared by two routes starting from the reaction of ethyl 2,2-difluoro(heteroaryl)acetate with phenyllithiums (Route A) and from the reaction of chlorodifluoro(heteroaryl)methane with benzaldehydes (Route B) . The compounds 1 except for 1g show antifungal activities against yeasts and filamentous fungi in vitro, especially (+)-1f have equal or superior activities compared to those of itraconazole.

J Infect Dis, 2000 Aug, 182(2), 545 - 50 Epub 2000 Jul 28.
Amphotericin B combined with itraconazole or fluconazole for treatment of histoplasmosis; LeMonte AM et al.; To investigate the efficacy of combined treatment with fluconazole (Flu) and amphotericin B (AmB) for Histoplasma capsulatum meningitis, MICs were determined for 10 clinical isolates, following National Committee for Clinical Laboratory Standards guidelines . Weak synergy was observed for 6 of the 10 isolates . For the in vivo models, mice either were sham treated or were given Flu (75 mg/kg/day), AmB (2 mg/kg every other day), itraconazole (Itra; 75 mg/kg/day), AmB+Flu, or AmB+Itra . Following infection with 5x105 yeasts, Flu antagonized AmB's reduction of fungal burden without reducing its effect on survival . When in vivo antagonism was reproduced following infection with 1x104 yeasts, a higher fungal burden was observed in the lungs . Itra had no effect on AmB's activity and was more effective than Flu for clearance of fungal burden . These findings caution against use of AmB+Flu for treatment of histoplasmosis, but studies of the effect of treatment on the fungal burden in the brain are needed to assess combination therapy for meningitis.

J Biol Chem, 2000 Oct 13, 275(41), 31682 - 8
Heat shock protein 90 mediates protein-protein interactions between human aminoacyl-tRNA synthetases; Kang J et al.; Heat shock protein 90 (hsp90) is a molecular chaperone responsible for protein folding and maturation in vivo . Interaction of hsp90 with human glutamyl-prolyl-tRNA synthetase (EPRS) was found by genetic screening, co-immunoprecipitation, and in vitro binding experiments . This interaction was sensitive to the hsp90 inhibitor, geldanamycin, and also ATP, suggesting that the chaperone activity of hsp90 is required for interaction with EPRS . Interaction of EPRS with hsp90 was targeted to the region of three tandem repeats linking the two catalytic domains of EPRS that is also responsible for the interaction with isoleucyl-tRNA synthetase (IRS) . Interaction of EPRS and IRS also depended on the activity of hsp90, implying that their association was mediated by hsp90 . EPRS and IRS form a macromolecular protein complex with at least six other tRNA synthetases and three cofactors . hsp90 preferentially binds to most of the complex-forming enzymes rather than those that are not found in the complex . In addition, inactivation of hsp90 interfered with the in vivo incorporation of the nascent aminoacyl-tRNA synthetases into the multi-ARS complex . Thus, hsp90 appears to mediate protein-protein interactions of mammalian tRNA synthetases.

DNA Res, 2000 Jun 30, 7(3), 223 - 7
Generation of 10,154 expressed sequence tags from a leafy gametophyte of a marine red alga, Porphyra yezoensis; Nikaido I et al.; A total of 10,154 5'-end expressed sequence tags (EST) were established from the normalized and size-selected cDNA libraries of a marine red alga, Porphyra yezoensis . Among the ESTs, 2140 were unique species, and the remaining 8014 were grouped into 1127 species . Database search of the 3267 non-redundant ESTs by BLAST algorithm showed that the sequences of 1080 species (33.1%) have similarity to those of registered genes from various organisms including higher plants, mammals, yeasts, and cyanobacteria, while 2187 (66.9%) are novel . Codon usage analysis in the coding regions of 101 non-redundant EST groups showing significant similarity to known genes indicated the higher GC contents at the third position of codons (79.4%) than the first (62.2%) and the second position (45.0%), suggesting that the genome has been exposed to high GC pressure during evolution . The sequence data of individual ESTs are available at the web site http://www.kazusa.or.jp/en/plant/porphyra/EST/.

J Ethnopharmacol, 2000 Jul, 71(1-2), 179 - 86
Studies on the anti-inflammatory, antipyretic and analgesic properties of Alstonia boonei stem bark; Olajide OA et al.; The methanol extract of the stem bark of Alstonia boonei was investigated for anti-inflammatory property . The analgesic and antipyretic properties of the extract was also evaluated . The extract caused a significant (P<0.05) inhibition of the carrageenan-induced paw oedema, cotton pellet granuloma, and exhibited an anti-arthritic activity in rats . Vascular permeability induced by acetic acid in the peritoneum of mice was also inhibited . The extract also produced marked analgesic activity by reduction of writhings induced by acetic acid, as well as the early and late phases of paw licking in mice . A significant (P<0.05) reduction in hyperpyrexia in mice was also produced by the extract . This study has established anti-inflammatory, analgesic and antipyretic activities of the stem bark of A . boonei.

Mol Cell, 2000 Apr, 5(4), 629 - 38
Membrane protein degradation by AAA proteases in mitochondria: extraction of substrates from either membrane surface; Leonhard K et al.; Two AAA proteases, each with its catalytic site at the opposite membrane surface, mediate the ATP-dependent degradation of mitochondrial inner membrane proteins . We demonstrate here that a model substrate polypeptide containing hydrophilic domains at both sides of the membrane can be completely degraded by either of the AAA proteases, if solvent-exposed domains are in an unfolded state . A short protein tail protruding from the membrane surface is sufficient to allow the proteolytic attack of an AAA protease that facilitates domain unfolding at the opposite side . Our results provide a rationale for the membrane arrangement of AAA proteases in mitochondria and demonstrate that degradation of membrane proteins by AAA proteases involves an active extraction of transmembrane segments and transport of solvent-exposed domains across the membrane.

Virology, 2000 Jun 20, 272(1), 183 - 90
A cellular protein with an RNA-binding activity co-purifies with viral dsRNA from mycovirus-infected Helminthosporium victoriae; Soldevila AI et al.; A cellular protein that co-purifies with mycoviral dsRNA was isolated from the plant pathogenic fungus Helminthosporium victoriae (telomorph: Cochliobolus victoriae) infected with two viruses, the totivirus Helminthosporium victoriae 190S virus and the chrysovirus-like Helminthosporium victoriae 145S virus (Hv145SV) . The cellular protein, which was, designated Hv-p68, accumulated to higher levels in virus-infected isolates compared to virus-free ones . The majority of the Hv145S dsRNAs were found in association with Hv-p68 and not packaged in virions . Hv-p68 could also be detected as a minor component of the virus capsid . Evidence is presented that Hv-p68 occurs in vivo as an octamer and that it possesses RNA-binding activities . Based on partial amino acid sequence analysis, Hv-p68 was shown to share significant sequence identity with alcohol oxidases from methylotrophic yeasts . Hv-p68 is proposed to play a role in viral RNA packaging/replication and in regulating viral pathogenesis .

Biochem Biophys Res Commun, 2000 May 27, 272(1), 29 - 35
Zinc-histidine as nucleation centers for growth of ZnS nanocrystals; Kho R et al.; Histidine is a chelator of zinc, most notably in zinc-finger proteins (zinc coordinated by cysteine and histidine) and in hyperaccumulator plants . Sulfide incorporation into molecules containing metal-cysteinyl complexes has been shown to occur in vivo in certain yeasts, leading to enhanced metal tolerance . Demonstrated here for the first time is incorporation of sulfide into zinc-histidine, resulting in histidine-ZnS nanocrystals (NCs) having unique optical properties . Sulfide complexation occurred optimally at alkaline pH into zinc-(histidine)2 species, and UV/Vis absorption maxima were red-shifted as increasing sulfide addition occurred . Intermediate sulfide concentrations led to multiple, thermodynamically preferred NC species within a sample . Fluorescence of histidine-ZnS NCs was greater than ZnS prepared previously with cysteinyl peptides . Transmission electron microscopy and selected-area electron diffraction indicated hexagonal ZnS crystals having an average size of 4.2 nm . A photocatalytic application of histidine-ZnS NCs was shown by efficient degradation of p-nitrophenol and paraquat in the presence of UV irradiation.

Parassitologia, 1999 Dec, 41(4), 587 - 90
Antifungal activity of Apulia region propolis; Cafarchia C et al.; A study was carried out to assess the in vitro antifungal activity of some natural Apulian propolis extracts of different origin . Their antifungal activity was compared to the antifungal activity of conifers and commercial propolis extracts . All extracts revealed antifungal activity against dermatophytes and Candida species . The antifungal activity differences found depended on the origin of the propolis and the solvent used for extraction . The best antifungal activity was given by the 'Orimini' propolis . The antifungal activity may have been influenced by the presence of different cinnamic and flavonoid components and their different concentration in the extracts . Further investigations are needed to validate this hypothesis.

Am J Hum Genet, 2000 Aug, 67(2), 333 - 44 Epub 2000 Jun 26.
Meiotic recombination and flanking marker exchange at the highly unstable human minisatellite CEB1 (D2S90); Buard J et al.; Unequal crossover has long been suspected to play a role in the germline-specific instability of tandem-repeat DNA, but little information exists on the dynamics and processes of unequal exchange . We have therefore characterized new length alleles associated with flanking-marker exchange at the highly unstable human minisatellite CEB1, which mutates in the male germline by a complex process often resulting in the gene conversion-like transfer of repeats between alleles . DNA flanking CEB1 is rich in single-nucleotide polymorphisms (SNPs) and shows extensive haplotype diversity, consistent with elevated recombinational activity near the minisatellite . These SNPs were used to recover mutant CEB1 molecules associated with flanking-marker exchange, directly from sperm DNA . Mutants with both proximal and distal flanking-marker exchange were shown to contribute significantly to CEB1 turnover and suggest that the 5' end of the array is very active in meiotic unequal crossover . Coconversions involving the interallelic transfer of repeats plus immediate flanking DNA were also common, were also polarized at the 5' end of CEB1, and appeared to define a conversion gradient extending from the repeat array into adjacent DNA . Whereas many mutants associated with complete exchange resulted in simple recombinant-repeat arrays that show reciprocity, coconversions were highly gain-biased and were, on average, more complex, with allele rearrangements similar to those seen in the bulk of sperm mutants . This suggests distinct recombination-processing pathways producing, on the one hand, simple crossovers in CEB1 and, on the other hand, complex conversions that sometimes extend into flanking DNA.

J Biol Chem, 2000 Aug 25, 275(34), 26591 - 8
Interactions between the tetratricopeptide repeat-containing transcription factor TFIIIC131 and its ligand, TFIIIB70 . Evidence for a conformational change in the complex; Moir RD et al.; In the transcription of tRNA and 5 S genes by RNA polymerase III, recruitment of the transcription factor (TF)IIIB is mediated by the promoter-bound assembly factor TFIIIC . A critical limiting step in this process is the interaction between the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC (TFIIIC131) and the TFIIB-related factor Brf1p/TFIIIB70 . To facilitate biochemical studies of this interaction, we expressed a fragment of TFIIIC131, TFIIIC131-(1-580), that includes the minimal TFIIIB70 interaction domain defined by two-hybrid studies together with adjacent sequences, up to the end of TPR9, implicated in the assembly reaction . TFIIIC131-(1-580) interacts with TFIIIB70 in solution and inhibits the formation of TFIIIB70.TFIIIC.DNA complexes . In a coupled equilibrium binding assay, the formation of TFIIIC131-(1-580).TFIIIB70 complexes was adequately described by a single-site binding model and yielded an apparent equilibrium dissociation constant of 334 +/- 23 nm . CD spectroscopy and limited proteolysis experiments defined a well structured and largely protease-resistant core in TFIIIC131-(1-580) comprising part of the hydrophilic amino terminus, TPR1-5, the intervening non-TPR region, and TPR6-8 . CD spectra showed that trifluoroethanol induced significant alpha-helical structure in TFIIIC131-(1-580) . A more modest monovalent ion-dependent CD difference was observed in mixtures of TFIIIC131-(1-580) and TFIIIB70, suggesting that formation of the binary complex may proceed with the acquisition of alpha-helicity.

Antimicrob Agents Chemother, 2000 Jul, 44(7), 1850 - 4
Comparison of the echinocandin caspofungin with amphotericin B for treatment of histoplasmosis following pulmonary challenge in a murine model; Kohler S et al.; Twenty clinical isolates of Histoplasma capsulatum were tested for their in vitro susceptibilities to caspofungin in comparison to those to amphotericin B by following National Committee for Clinical Laboratory Standards guidelines for yeasts . The mean MICs were 16.6 microgram/ml (range, 8 to 32 microgram/ml) for caspofungin and 0.56 microgram/ml (range, 0.5 to 1.0 microgram/ml) for amphotericin B . Survival experiments used a 10(5) dose in a pulmonary challenge model with B6C3F(1) mice . All mice that received amphotericin B at 2 mg/kg of body weight every other day (q.o.d.), 30% of mice that received caspofungin at 8 mg/kg/day, and 20% of mice that received caspofungin at 4 mg/kg/day survived to day 15, while mice that received caspofungin at 2 mg/kg/day and all control mice that received the vehicle died by day 14 . Amphotericin B at 2 mg/kg q.o.d . markedly reduced the fungal burden in the lungs and spleens, as measured by Histoplasma antigen detection techniques and quantitative cultures, for each comparison . Caspofungin at 10 mg/kg twice a day (b.i.d.) did not reduce the fungal burden, as measured by antigen detection techniques, but slightly reduced the levels of fungi in both the lungs and spleens, as determined by quantitative cultures . Caspofungin at 5 mg/kg b.i.d . did not affect fungal burden . Overall, caspofungin had only a slight effect on survival or fungal burden.

J Biol Chem, 2000 Aug 25, 275(34), 26343 - 8
Retention of the human Rad9 checkpoint complex in extraction-resistant nuclear complexes after DNA damage; Burtelow MA et al.; Studies in yeasts and mammals have identified many genes important for DNA damage-induced checkpoint activation, including Rad9, Hus1, and Rad1; however, the functions of these gene products are unknown . In this study we show by immunolocalization that human Rad9 (hRad9) is localized exclusively in the nucleus . However, hRad9 was readily released from the nucleus into the soluble extract upon biochemical fractionation of un-irradiated cells . In contrast, DNA damage promptly converted hRad9 to an extraction-resistant form that was retained at discrete sites within the nucleus . Conversion of hRad9 to the extraction-resistant nuclear form occurred in response to diverse DNA-damaging agents and the replication inhibitor hydroxyurea but not other cytotoxic stimuli . Additionally, extraction-resistant hRad9 interacted with its binding partners, hHus1 and an inducibly phosphorylated form of hRad1 . Thus, these studies demonstrate that hRad9 is a nuclear protein that becomes more firmly anchored to nuclear components after DNA damage, consistent with a proximal function in DNA damage-activated checkpoint signaling pathways.

J Mol Biol, 2000 Jun 16, 299(4), 965 - 77
Signals for TBP/TATA box recognition; Bareket-Samish A et al.; The TATA box-binding protein (TBP) recognizes its target sites (TATA boxes) by indirectly reading the DNA sequence through its conformation effects (indirect readout) . Here, we explore the molecular mechanisms underlying indirect readout of TATA boxes by TBP by studying the binding of TBP to adenovirus major late promoter (AdMLP) sequence variants, including alterations inside as well as in the sequences flanking the TATA box . We measure here the dissociation kinetics of complexes of TBP with AdMLP targets and, by phase-sensitive assay, the intrinsic bending in the TATA box sequences as well as the bending of the same sequence induced by TBP binding . In these experiments we observe a correlation of the kinetic stability to sequence changes within the TATA recognition elements . Comparison of the kinetic data with structural properties of TATA boxes in known crystalline TBP/TATA box complexes reveals several "signals" for TATA box recognition, which are both on the single base-pair level, as well as larger DNA tracts within the TATA recognition element . The DNA bending induced by TBP on its binding sites is not correlated to the stability of TBP/TATA box complexes . Moreover, we observe a significant influence on the kinetic stability of alteration in the region flanking the TATA box . This effect is limited however to target sites with alternating TA sequences, whereas the AdMLP target, containing an A tract, is not influenced by these changes .

Mol Cells, 2000 Apr 30, 10(2), 127 - 34
Isolation of TC/AG repeat microsatellite sequences for fingerprinting rice blast fungus and their possible horizontal transfer to plant species; Kim NS et al.; Genome fingerprinting has been a major role in characterization of population structure and analysis of the variability in phytopathogenic fungi . In order to characterize Korean rice blast fungal isolates, the genomic DNAs were digested with AluI endonuclease and subsequent PCR amplifications using random decamer primers with combinations of microsatellite primers had been carried out . This Alu-Inter SSR technique revealed high polymorphism among the Korean blast fungal isolates . Then, fragments from the Alu-Inter SSR analysis were isolated to be used as probes in Southern hybridization, which also revealed high polymorphism between isolates to distinguish individuals . The sequences of the isolated fragments contained TC/AG tandem repeats interspersed with a 30 bp direct repeat . In gel blot analysis, the isolated TC/AG repeat microsatellite sequences were proved to be useful for characterizing the isolates in blast fungi in addition to the conventional MGR (Magnaporthe grisea repeat) probes . One interesting point was that the rice blast fungus derived TC/AG repeat microsatellite sequences were abundant in non-rice blast fungi and plant species, but not in other fungi and yeasts . A discussion on the possible horizontal gene transfer between phytopathogenic fungi and host plants is presented.

J Med Microbiol, 2000 Jun, 49(6), 575 - 81
Molecular genotyping of Candida species with special respect to Candida (Torulopsis) glabrata strains by arbitrarily primed PCR; Becker K et al.; A set of 46 epidemiologically related or unrelated Candida (Torulopsis) glabrata isolates from four different medical centres in Germany and Hungary, and the type strain of this species, were genetically typed by arbitrarily primed PCR (AP-PCR) . The resulting band patterns of C . glabrata strains were compared with those of other species of the genus Candida including C . albicans, C . guilliermondii, C . kefyr, C . parapsilosis, C . tropicalis and C . krusei . After preliminary trials of various reaction parameters and control experiments to test the reproducibility of this method, it was found that consistently reproducible amplification patterns were obtained only when rigorously optimised and standardised reaction conditions were employed . Discriminatory abilities were studied with 29 generated 10-mer oligonucleotides of different G+C content . Typing of clinical isolates with the optimised AP-PCR protocol was then performed with the primer 50-1, with a G+C content of 50% . Sufficiently discriminatory polymorphisms were observed among the band patterns of the Candida species included . The gel electrophoresis patterns of each species showed an adequate similarity . Variations in minor bands were characteristic for comparison at the isolate level . Only three AP-PCR genotypes were identified among the clinical isolates of C . glabrata tested . Two of these genotypes were closely related and appeared to be widespread within German and Hungarian isolates . The third genotype of C . glabrata showed a distinct band pattern . With optimised, validated and standardised assay conditions, the feasibility, sensitivity and rapidity of AP-PCR may offer a discriminatory method for genotyping of yeasts in epidemiological studies, as well as in the control of nosocomial infections.

J Biol Chem, 2000 Aug 25, 275(34), 26436 - 40
Paxillin localizes to the lymphocyte microtubule organizing center and associates with the microtubule cytoskeleton; Herreros L et al.; Paxillin is a focal adhesion-associated protein that functions as a multi-domain adapter protein, binding several structural and signaling molecules . alpha-Tubulin was identified as an interacting protein in a two-hybrid screen using the paxillin C-terminal LIM domain as a bait . In vitro binding assays with glutathione S-transferase-paxillin demonstrated an interaction of alpha-tubulin with the C terminus of paxillin . Another member of the tubulin family, gamma-tubulin, bound to both the N and the C terminus of paxillin . The interaction between paxillin and both alpha- and gamma-tubulin in vivo was confirmed by co-immunoprecipitation from human T lymphoblasts . Immunofluorescence studies revealed that, in adherent T cells, paxillin localized to sites of cell-matrix interaction as well as to a large perinuclear region . Confocal microscopy revealed that this region corresponds to the lymphocyte microtubule organizing center, where paxillin colocalizes with alpha- and gamma-tubulin . The localization of paxillin to this area was observed in cells in suspension as well as during adhesion to integrin ligands . These data constitute the first characterization of the interaction of paxillin with the microtubule cytoskeleton, and suggest that paxillin, in addition to its well established role at focal adhesions, could also be associated with the lymphocyte microtubule network.

Neuron, 2000 May, 26(2), 443 - 55
Regulation of neuregulin signaling by PSD-95 interacting with ErbB4 at CNS synapses; Huang YZ et al.; Neuregulins (NRGs) and their receptors, the ErbB protein tyrosine kinases, are essential for neuronal development, but their functions in the adult CNS are unknown . We report that ErbB4 is enriched in the postsynaptic density (PSD) and associates with PSD-95 . Heterologous expression of PSD-95 enhanced NRG activation of ErbB4 and MAP kinase . Conversely, inhibiting expression of PSD-95 in neurons attenuated NRG-mediated activation of MAP kinase . PSD-95 formed a ternary complex with two molecules of ErbB4, suggesting that PSD-95 facilitates ErbB4 dimerization . Finally, NRG suppressed induction of long-term potentiation in the hippocampal CA1 region without affecting basal synaptic transmission . Thus, NRG signaling may be synaptic and regulated by PSD-95 . A role of NRG signaling in the adult CNS may be modulation of synaptic plasticity.

Trends Biochem Sci, 2000 Jun, 25(6), 294 - 9
ARID proteins come in from the desert; Kortschak RD et al.; Members of the recently discovered ARID (AT-rich interaction domain) family of DNA-binding proteins are found in fungi and invertebrate and vertebrate metazoans . ARID-encoding genes are involved in a variety of biological processes including embryonic development, cell lineage gene regulation and cell cycle control . Although the specific roles of this domain and of ARID-containing proteins in transcriptional regulation are yet to be elucidated, they include both positive and negative transcriptional regulation and a likely involvement in the modification of chromatin structure.

Trends Biochem Sci, 2000 Jun, 25(6), 290 - 3
Connecting transcription to messenger RNA processing; Proudfoot N; The production of messenger RNA by gene transcription requires at least three RNA-processing mechanisms: capping, splicing and polyadenylation . All three reactions occur in intimate association with the elongating polymerase complex through the C terminus of the largest subunit of RNA polymerase II . The processing of mRNA is therefore orchestrated to act on the nascent RNA as soon as it emerges from the polymerase complex.

Gene, 2000 May 16, 249(1-2), 67 - 74
Molecular and cellular characterizations of a cDNA clone encoding a novel isozyme of aldehyde dehydrogenase from rice; Li Y et al.; Aldehyde dehydrogenases (ALDHs) are a group of enzymes catalyzing the conversion of aldehydes to the corresponding acids . In mammals and yeasts, at least two isozymes of ALDH are known to be involved in ethanol metabolism (cytosolic ALDH1 and mitochondrial ALDH2) . Although mitochondrial ALDH isozymes have previously been identified in several plants, such as maize and tobacco, it is unclear whether cytosolic ALDH isozymes also exist in plants . In this study, we identified and characterized a cDNA clone encoding aldehyde dehydrogenase (ALDH1a) from rice (Oryza sativa L . cv . Nipponbare) . The open reading frame of this clone did not contain a typical mitochondrial targeting signal . Analysis of the subcellular localization of ALDH1a using green fluorescent protein (GFP) suggested that ALDH1a is a cytosolic enzyme rather than a mitochondrial enzyme . A genomic Southern hybridization indicated that sequences homologous to the ALDH1a gene are present in at least two regions of the rice genome . Amplification by RT-PCR showed that ALDH1a is expressed strongly in roots, but not in leaves, of rice seedlings, suggesting that ALDH1a functions in roots.

Mycopathologia, 1999, 146(3), 147 - 54
Evaluation of the mycological status of luncheon meat with special reference to aflatoxigenic moulds and aflatoxin residues; Ismail MA et al.; The luncheon meat samples analyzed, which were produced locally by the two main luncheon meat producing companies in Egypt were relatively highly contaminated either by moulds and yeasts in general, aflatoxigenic species and aflatoxin residues in particular . The most frequently encountered fungi from the samples were yeasts, Aspergillus niger, A . flavus, Penicillium chrysogenum, Rhizopus stolonifer, Mucor circinelloides . Less common were Cladosporium sphaerospermum, Alternaria alternata, Mycosphaerella tassiana, P . aurantiogriseum and P . oxalicum . The most important aflatoxigenic species, A . flavus, was isolated frequently . It was 10% of the total fungal isolates from both samples of the two companies . Seven luncheon meat samples out of 50 analyzed were positive for aflatoxin B1 or B1 and G1, while all samples were negative for aflatoxins B2, G2, M1 and M2 . Aflatoxin B1 was detected only in 4 and 3 samples out of 25 analyzed from each of company A and B, respectively . The highest detectable level, 11.1 ppb, was recorded in a sample from company B and the least, 0.5 ppb, in a sample from company A . Aflatoxin G1, at concentration of 3.2 ppb, was detected in only one sample of the aflatoxin B1--contaminated 3 samples of company B: this sample also had the highest level of aflatoxin B1 . Some luncheon meat samples had higher numbers of aflatoxigenic A . flavus than others, however these samples were negative for aflatoxins . The hazardous potential of such contamination will be discussed.

Am Fam Physician, 2000 May 1, 61(9), 2703 - 10, 2713-4
Treatment of seborrheic dermatitis; Johnson BA et al.; Seborrheic dermatitis is a chronic inflammatory disorder affecting areas of the head and trunk where sebaceous glands are most prominent . Lipophilic yeasts of the Malassezia genus, as well as genetic, environmental and general health factors, contribute to this disorder . Scalp seborrhea varies from mild dandruff to dense, diffuse, adherent scale . Facial and trunk seborrhea is characterized by powdery or greasy scale in skin folds and along hair margins . Treatment options include application of selenium sulfide, pyrithione zinc or ketoconazole-containing shampoos, topical ketoconazole cream or terbinafine solution, topical sodium sulfacetamide and topical corticosteroids.

Biochemistry, 2000 May 9, 39(18), 5355 - 65
Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data; Battiste JL et al.; To test whether distances derived from paramagnetic broadening of (15)N heteronuclear single quantum coherence (HSQC) resonances could be used to determine the global fold of a large, perdeuterated protein, we used site-directed spin-labeling of 5 amino acids on the surface of (15)N-labeled eukaryotic translation initiation factor 4E (eIF4E) . eIF4E is a 25 kDa translation initiation protein, whose solution structure was previously solved in a 3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonate hydrate (CHAPS) micelle of total molecular mass approximately 45-50 kDa . Distance-dependent line broadening consistent with the three-dimensional structure of eIF4E was observed for all spin-label substitutions . The paramagnetic broadening effects (PBEs) were converted into distances for modeling by a simple method comparing peak heights in (15)N-HSQC spectra before and after reduction of the nitroxide spin label with ascorbic acid . The PBEs, in combination with HN-HN nuclear Overhauser effects (NOEs) and chemical shift index (CSI) angle restraints, correctly determined the global fold of eIF4E with a backbone precision of 2.3 A (1.7 A for secondary structure elements) . The global fold was not correctly determined with the HN-HN NOEs and CSI angles alone . The combination of PBEs with simulated restraints from another nuclear magnetic resonance (NMR) method for global fold determination of large proteins (methyl-protonated, highly deuterated samples) improved the quality of calculated structures . In addition, the combination of the two methods simulated from a crystal structure of an all alpha-helical protein (40 kDa farnesyl diphoshphate synthase) correctly determined the global fold where neither method individually was successful . These results show the potential feasibility of obtaining medium-resolution structures for proteins in the 40-100 kDa range via NMR.

Biochem Biophys Res Commun, 2000 May 19, 271(3), 626 - 9
Rho small G-protein-dependent binding of mDia to an Src homology 3 domain-containing IRSp53/BAIAP2; Fujiwara T et al.; mDia1 is a downstream effector of Rho small G protein that is implicated in stress fiber formation and cytokinesis . We isolated an mDia1-binding protein and identified it to be IRSp53/BAIAP2 . IRSp53 and BAIAP2 have independently been isolated as a 58/53-kDa protein tyrosine phosphorylated in response to insulin and a BAI1-binding protein, respectively . BAI1 is a brain-specific seven-span transmembrane protein capable of inhibiting angiogenesis . The proline-rich formin homology 1 domain of mDia1 bound the Src homology 3 domain of IRSp53/BAIAP2 in a GTP-Rho-dependent manner . The results suggest that IRSp53/BAIAP2 is a downstream effector of mDia1 .

Oncogene, 2000 Apr 13, 19(16), 2052 - 9
Evidence for an interaction between the insulin receptor and Grb7 . A role for two of its binding domains, PIR and SH2; Kasus-Jacobi A et al.; The molecular adapter Grb7 is likely to be implicated in the development of certain cancer types . In this study we show that Grb7 binds the insulin receptors, when they are activated and tyrosine phosphorylated . This interaction is documented by two-hybrid experiments, GST pull-down assays and in vivo coimmunoprecipitations . In addition, our results argue in favor of a preferential association between Grb7 and the insulin receptors when compared to other tyrosine kinase receptors like the EGF receptor, the FGF receptor and Ret . Interestingly, Grb7 is not a substrate of the insulin receptor tyrosine kinase activity . Grb7 binds the activated tyrosine kinase loop of the insulin receptors . Two domains of Grb7 are implicated in the insulin receptor binding: the SH2 domain and the PIR (phosphotyrosine interacting region) . The role of these two domains in the interaction with the insulin receptor was already reported for Grb10 and Grb14, the other members of the Grb7 family of proteins . However, the relative importance of these domains varies, considering the receptor and the Grb protein . These differences should be a determinant of the specificity of the receptor tyrosine kinase-Grbs binding, and thus of the implication of Grb7/10/14 in signal transduction.

Nat Struct Biol, 2000 May, 7(5), 362 - 6
A collapsed non-native RNA folding state; Buchmueller KL et al.; At physiological Mg2+ concentrations, the catalytic core of the bI5 group I intron does not fold into its native structure . In contrast, as judged by the global size, this RNA undergoes structural collapse at Mg 2+ concentrations much lower than required to drive folding of the RNA completely to the native state . The bI5 RNA therefore exists in equilibrium between expanded and collapsed non-native states . The activation energy of RNA folding from the collapsed state to the native state is negligible and the reaction is not accelerated by the addition of urea . This collapsed state is thus distinct from the kinetic traps observed during folding of other large RNAs . The collapsed non-native state forms readily in the case of bI5 RNA and may exist generically prior to assembly of other ribonucleoprotein holoenzymes, such as the ribosome.

Eur J Endocrinol, 2000 May, 142(5), 537 - 44
Comparative analysis of follistatin-, activin beta A- and activin beta B-mRNA steady-state levels in diverse porcine tissues by multiplex S1 nuclease analysis; Schneider O et al.; OBJECTIVE: The relation of activins (dimers of the beta-subunits of inhibin) and follistatin (FS) (their binding protein) affect the growth and differentiation of many cell types . Activin- and FS-mRNAs show a widespread co-expression throughout the organism, indicating an essential role for the FS/activin system in diverse physiological processes . The present study was performed to investigate FS-, activin betaA-, and activin beta B-mRNA expression in porcine tissues and to compare the relative mRNA tissue distribution by a newly developed multiplex S1 nuclease protection assay . METHODS: Twenty micrograms total RNA from different porcine tissues were subjected to multiplex S1 analysis . Specific mRNA expression was determined by measurements of optical densities on autoradiographs . RESULTS: Activin beta A-mRNA expression was abundant in the ovary, adrenal gland, fat, vein, artery and uterus, activin beta B-mRNA was highly expressed in the ovary, pituitary, uterus, placenta, aorta and cerebellum . FS-mRNA showed a widespread expression with high levels in ovary, uterus, cerebellum, placenta and fat . The comparison of relative activin beta A-, activin beta B- and FS-mRNA expression within a certain tissue showed a predominance of activin beta A-mRNA in the adrenal gland, fat, artery, spinal cord, cerebrum and colon and of activin beta B-m RNA in pituitary, testis and placenta, while FS-mRNA levels exceeded those of activin subunits in epididymis, liver, lymphoid tissue, muscle, intestine, cerebellum, ovary and uterus . CONCLUSIONS: The presented data provide an overview of FS-, activin beta A-, and activin beta B-mRNA steady state levels in porcine tissues.

J Nutr, 2000 May, 130(5S Suppl), 1350S - 4S
Assessment of marginal zinc status in humans; Wood RJ; The assessment of marginal zinc status is problematic . Currently, there is no universally accepted single measure to assess zinc status in humans . The development of a reliable measure of marginal or moderate zinc status would be useful for a variety of purposes . For example, a simple, yet sensitive and accurate measure of zinc nutritional status is critically needed to further our limited understanding of the possible associations between zinc status and the risk of developing various chronic diseases and in predicting favorable health outcomes in patient populations . A convenient and reliable zinc assessment tool is needed to identify subpopulations who are at a risk of zinc deficiency and as an objective guidepost to determine the need for initiation of zinc supplementation or zinc fortification of the food supply, as well in the refinement of recommendations of dietary zinc allowances . In frank zinc deficiency, clinical signs and static measures of zinc concentrations in a variety of readily available tissues, such as plasma, various blood cell types and hair, may uniformly confirm the presence of depleted body zinc stores . However, in general, the relative insensitivity or imprecision of these measurements has resulted in general disappointment in their use to assess marginal zinc status . Therefore, the search continues to find a useful and reliable marker of marginal zinc deficiency . In an attempt to speculate on possible future developments in the zinc status assessment field, a number of new and potentially promising approaches to this problem are highlighted.

Tissue Cell, 2000 Feb, 32(1), 40 - 8
Different types of response to foreign antigens by leech leukocytes; de Eguileor M et al.; We used morphological and immunocytochemical approaches to characterize and to show the behavior of cells involved in leech inflammatory responses . Leeches were injected with bacterial lipopolysaccharide, fluoresceinated yeasts, sulfate spheres and ciliates (Protozoa) . Shortly after injection, migrating cells appeared in the area of injection . The response of the cells occurred in relation to the injected micro or macro antigens . Each injection first provoked a migration of cells towards the non-self material . Afterwards, different responses (degranulation, phagocytosis, encapsulation, melanization) occurred . The migrating cells involved in these series of processes have a similar behavior and are characterized by CD markers of macrophages, NK cells and granulocytes, which are typical of many invertebrates and vertebrates.

J Biol Chem, 2000 Jul 7, 275(27), 20239 - 42
Serine/Threonine kinases 3pK and MAPK-activated protein kinase 2 interact with the basic helix-loop-helix transcription factor E47 and repress its transcriptional activity; Neufeld B et al.; In the search for physiological substrates of MAPK-activated protein (MAPKAP) kinases, we identified the basic helix-loop-helix (bHLH) transcription factor E47 as an interaction partner of chromosome 3p kinase (3pK) and MAPKAP-K2 (MK2) . The E2A protein E47 is known to be involved in the regulation of tissue-specific gene expression and cell differentiation . E47 is a phosphoprotein, and we identified 3pK and MK2 as E47 kinases in vitro . Furthermore, the expression of either kinase results in a repression of the transcriptional activity of E47 on an E-box containing promoter . In summary, the MAPK-activated protein kinases 3pK and MK2 were identified to form an assembly with the bHLH protein E47 suggesting that these kinases are regulators of E47 activity and E47-dependent gene expression.

IUBMB Life, 2000 Feb, 49(2), 143 - 7
Two systems for phosphate uptake in Yarrowia lipolytica cells grown at acidic conditions; Zvyagilskaya R et al.; Inorganic phosphate (Pi) is accumulated by Yarrowia lipolytica cells grown at acidic pH conditions by two kinetically discrete H+/Pi-cotransport systems with apparent K(m) values for Pi of 12-18 microM and 2-3 mM Pi at pH 5.5, respectively . One of these is derepressible and operates at low external Pi concentrations; the other is most likely constitutively expressed and comes into play at high Pi concentrations . The derepression of the high-affinity Pi transport system is under the control of available extracellular Pi as well as the amount of intracellular polyphosphates stores . Characteristics of the Pi transport behavior in Yarrowia lipolytica are discussed.

J Chromatogr A, 2000 Mar 31, 874(1), 131 - 42
Analytical magnetapheresis of magnetically susceptible particles; Fuh CB et al.; Analytical magnetapheresis is a newly developed technique for analyzing magnetic particles . The magnetically susceptible particles form deposition patterns after flowing through a separation channel in a magnetic field . The separation channel requirements for analytical magnetapheresis are an excellent seal for the carrier flow and ease of disassembly after magnetapheresis . Previously used separation channels often exhibit variable channel leakage and unstable flow velocities . We improved the separation channel assembly to ensure stable, high flow velocities and characterized the system with various magnetically susceptible and labeled particles . Our new separation channel featured silicone sealant with embedded nylon wires and met analytical magnetapheresis requirements . Characterization of this system was performed using several magnetically susceptible particles, and we studied a variety of diamagnetic sample labels with paramagnetic ions and magnetically susceptible particles at different flow-rates and solution pH values . The minimal labeling concentration for complete deposition was determined to be approximately 2.50 x 10(10) ions per particle for test samples at a flow velocity of 0.67 mm s(-1) and a magnetic field gradient of 2.8 T mm(-1) . Silicas, yeasts and blood cells were used for these studies . We determined that the minimal difference in magnetic susceptibility (delta(chi)) for successful separation was approximately 2.00 x 10(-6) {SI} . The magnetic susceptibilities of Dynabeads M-450 at several separation distances and flow-rates were determined to be 0.25 {SI}, within 2% of values published by other workers . The magnetic susceptibilities of various ion-labeled yeasts and cells were determined and most varied by less than 5% at different flow-rates . The results of this study provide very important references for analytical magnetapheresis applications.

Plant Physiol, 2000 Apr, 122(4), 1231 - 8
Genetic enhancement of fatty acid synthesis by targeting rat liver ATP:citrate lyase into plastids of tobacco; Rangasamy D et al.; ATP:citrate lyase (ACL) catalyzes the conversion of citrate to acetyl-coenzyme A (CoA) and oxaloacetate and is a key enzyme for lipid accumulation in mammals and oleaginous yeasts and fungi . To investigate whether heterologous ACL genes can be targeted and imported into the plastids of plants, a gene encoding a fusion protein of the rat liver ACL with the transit peptide for the small subunit of ribulose bisphosphate carboxylase was constructed and introduced into the genome of tobacco . This was sufficient to provide import of the heterologous protein into the plastids . In vitro assays of ACL in isolated plastids showed that the enzyme was active and synthesized acetyl-CoA . Overexpression of the rat ACL gene led to up to a 4-fold increase in the total ACL activity; this increased the amount of fatty acids by 16% but did not cause any major change in the fatty acid profile . Therefore, increasing the availability of acetyl-CoA as a substrate for acetyl-CoA carboxylase and subsequent reactions of fatty acid synthetase has a slightly beneficial effect on the overall rate of lipid synthesis in plants.

Mol Biol Cell, 2000 Apr, 11(4), 1241 - 55
A family of ADP-ribosylation factor effectors that can alter membrane transport through the trans-Golgi; Boman AL et al.; A family of three structurally related proteins were cloned from human cDNA libraries by their ability to interact preferentially with the activated form of human ADP-ribosylation factor 3 (ARF3) in two-hybrid assays . The specific and GTP-dependent binding was later confirmed through direct protein binding of recombinant proteins . The three proteins share large ( approximately 300 residues) domains at their N termini that are 60-70% identical to each other and a shorter (73 residues) domain at their C termini with 70% homology to the C-terminal "ear" domain of gamma-adaptin . Although GGA1 is found predominantly as a soluble protein by cell fractionation, all three proteins were found to localize to the trans-Golgi network (TGN) by indirect immunofluorescence . The binding of GGAs to TGN was sensitive to brefeldin A, consistent with this being an ARF-dependent event . Thus, these proteins have been named Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding proteins, or GGAs . The finding that overexpression of GGAs was sufficient to alter the distribution of markers of the TGN (TGN38 and mannose 6-phosphate receptors) led us to propose that GGAs are effectors for ARFs that function in the regulation of membrane traffic through the TGN.

J Biol Chem, 2000 May 26, 275(21), 16030 - 6
Arginine methylation inhibits the binding of proline-rich ligands to Src homology 3, but not WW, domains; Bedford MT et al.; Src homology 3 (SH3) and WW domains are known to associate with proline-rich motifs within their respective ligands . Here we demonstrate that the proposed adapter protein for Src kinases, Sam68, is a ligand whose proline-rich motifs interact with the SH3 domains of p59(fyn) and phospholipase Cgamma-1 as well as with the WW domains of FBP30 and FBP21 . These proline-rich motifs, in turn, are flanked by RG repeats that represent targets for the type I protein arginine N-methyltransferase . The asymmetrical dimethylation of arginine residues within these RG repeats dramatically reduces the binding of the SH3 domains of p59(fyn) and phospholipase Cgamma-1, but has no effect on their binding to the WW domain of FBP30 . These results suggest that protein arginine methylation can selectively modulate certain protein-protein interactions and that mechanisms exist for the irreversible regulation of SH3 domain-mediated interactions.

Hum Exp Toxicol, 2000 Jan, 19(1), 2 - 31
Chemical hormesis: its historical foundations as a biological hypothesis; Calabrese EJ et al.; Despite the long history of hormesis-related experimental research no systematic effort to describe its early history has been undertaken . The present paper attempts to reconstruct and assess the early history of such research and to evaluate how advances in related scientific fields affected the course of hormesis-related research . The purpose of this paper is not only to satisfy this gap in current knowledge, but also to provide a foundation for the assessment of how the concept of hormetic dose-response relationships may have affected the nature of the bioassay especially with respect to hazard assessment practices within a modern risk assessment framework.

APMIS, 2000 Feb, 108(2), 153 - 60
Characterization of the genetic diversity in superficial and systemic human isolates of Candida parapsilosis by randomly amplified polymorphic DNA (RAPD); Dassanayake RS et al.; The application of randomly amplified polymorphic DNA (RAPD) technology for strain delineation of medically important yeasts has proved to be a valuable tool in clinico-epidemiological studies of Candida species . Candida parapsilosis, a form species of the fungi imperfecti, is an emerging pathogen gaining recognition as an opportunistic agent, especially in the immunocompromised . Therefore, 15 clinical isolates of C . parapsilosis obtained from oral, cutaneous and systemic Candida infections were typed by RAPD analysis using four different primers . The primers RSD6 and RSD9 elicited 7 genotypes each, whereas primers RSD7 and RSD12 revealed 6 and 10 genotypes, respectively . When the data were correlated, a higher degree of genomic heterogeneity in systemic isolates was noted compared with the oral and cutaneous isolates, which shared somewhat similar RAPD profiles . However, a single oral isolate (P5) and two systemic isolates (P13 and P15) elicited radically divergent profiles, dissimilar to their counterparts . RAPD study of the latter two isolates with three additional primers (RSD8, RSD10 and RSD11) confirmed the observed genomic disparity . These data substantiate the previous observations on the genomic heterogeneity in C . parapsilosis and point to genetic shifts which may be associated with ecodiversity, as well as the possible existence of distinct genetic groups within this form species.

J Vet Diagn Invest, 2000 Mar, 12(2), 180 - 3
Diagnosis of sporotrichosis in a donkey using direct fluorescein-labeled antibody testing; Irizarry-Rovira AR et al.; A 4-year-old female donkey residing in an open field in Indiana was admitted for evaluation of facial lesions of 2 years duration . Cytologic and histologic examination of exudate and tissue from the lesions revealed a pyogranulomatous inflammatory reaction with numerous yeasts . Sporothrix schenckii was suspected to be the infectious agent; however, multiple culture attempts did not provide positive identification of the organism . Serologic examination supported infection with S . schenckii . A specific direct immunofluorescent antibody test performed on paraffin-embedded tissue sections confirmed the organism as S . schenckii . Clinical signs resolved after appropriate iodide therapy.

Eur J Dermatol, 2000 Mar, 10(2), 155 - 60
The use of systemic antimycotics in dermatotherapy; Niewerth M et al.; Fungal infections of the skin as well as of the nails and hair due to dermatophytes or due to yeasts or moulds still form a major portion of skin diseases overall . Effective therapy of mycoses is not always simple to achieve . In less severe cases topical therapy can be sufficient, but in extensive cutaneous infections, previous resistance to treatment and especially hyperkeratotic tinea and onychomycosis, systemic therapy can be mandatory . For systemic therapy, in particular azoles, i.e . itraconazole and fluconazole as well as the allylamine terbinafine are worth considering . The older antimycotics, i.e . griseofulvin and also ketoconazole are more and more replaced by other, newer drugs . For optimal treatment of a given mycosis, therapy can and should correspond to the individual situation . This applies both to the type of drug and its mode of application . The treatment of choice is the one with the best benefit to risk ratio and the best benefit to cost ratio . Unfortunately, as yet, a cure cannot be expected in every single case.

J Biol Chem, 2000 Mar 3, 275(9), 6515 - 22
Tissue specificity of E subunit isoforms of plant vacuolar H(+)-ATPase and existence of isotype enzymes; Kawamura Y et al.; Immunoblot analyses and partial amino acid sequencings revealed that both the 40- (E1) and 37-kDa (E2) subunits of V-ATPase in the pea epicotyl were E subunit isoforms . Similarly, both the 35- (D1) and 29-kDa (D2) subunits were D subunit isoforms, although the similarity of the amino acid sequences is still unknown . In immunoblot analyses, two or three E subunit isoforms with molecular masses ranging from 29 to 40 kDa were detected in other plants . Two isotypes of V-ATPase from the pea epicotyl were separated by ion exchange chromatography and had subunit compositions differing only in the ratio of E1 and E2 . There was a difference in the V(max) and K(m) of ATP hydrolysis between the two isotypes . E1 was scarcely detected in crude membrane fractions from the leaf and cotyledon, while E2 was detected in fractions from all of the tissues examined . The compositions of D subunit isoforms in the leaf and epicotyl were different, and the vacuolar membrane in the leaf did not contain D2 . The efficiency of H(+) pumping activity in the vacuolar membrane of the leaf was higher than that of the epicotyl . The results suggest that the presence of the isoforms of D and E subunits is characteristic to plants and that the isoforms are closely related to the enzymatic properties.

J Biol Chem, 2000 Mar 3, 275(9), 6114 - 22
The multifunctional character of a geminivirus replication protein is reflected by its complex oligomerization properties; Orozco BM et al.; Tomato golden mosaic virus (TGMV), a member of the geminivirus family, encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its plant host . TGMV AL1 is an oligomeric protein that binds double-stranded DNA and catalyzes cleavage and ligation of single-stranded DNA . The oligomerization domain, which is required for DNA binding, maps to a region that displays strong sequence and structural homology to other geminivirus Rep proteins . To assess the importance of conserved residues, we generated a series of site-directed mutations and analyzed their impact on AL1 function in vitro and in vivo . Two-hybrid experiments revealed that mutation of amino acids 157-159 inhibited AL1-AL1 interactions, whereas mutations at nearby residues reduced complex stability . Changes at positions 157-159 also disrupted interaction between the full-length mutant protein and a glutathione S-transferase-AL1 oligomerization domain fusion in insect cells . The mutations had no detectable effect on oligomerization when both proteins contained full-length AL1 sequences, indicating that AL1 complexes can be stabilized by amino acids outside of the oligomerization domain . Nearly all of the oligomerization domain mutants were inhibited or severely attenuated in their ability to support AL1-directed viral DNA replication . In contrast, the same mutants were enhanced for AL1-mediated transcriptional repression . The replication-defective AL1 mutants also interfered with replication of a TGMV A DNA encoding wild type AL1 . Full-length mutant AL1 was more effective in the interference assays than truncated proteins containing the oligomerization domain . Together, these results suggested that different AL1 complexes mediate viral replication and transcriptional regulation and that replication interference involves multiple domains of the AL1 protein.

Biochem Biophys Res Commun, 2000 Feb 16, 268(2), 395 - 7
Interactions of cytochrome c peroxidase with lysine peptides; Hirota S et al.; Structural change of Cytochrome c peroxidase (CcP) due to interaction with lysine peptides (Lysptds) has been studied by absorption spectra and measurements on electron transfer between cytochrome c (cyt c) and CcP in the presence of Lysptd . Peaks were observed in the difference absorption spectrum of CcP between in the presence and absence of Lysptds, demonstrating a structural perturbation of CcP, at least at its heme site, on interaction with Lysptd . The interaction between CcP and Lysptd was electrostatic, since no significant peak was detected in the difference absorption spectrum when 100 mM of NaCl was added to the solution . Lysptds competitively inhibited electron transfer from cyt c to CcP, which indicated that they interacted with CcP at the same site as cyt c and would be models of the CcP interacting site of cyt c .

Curr Opin Genet Dev, 2000 Feb, 10(1), 17 - 25
Sensing and responding to DNA damage; Lowndes NF et al.; DNA damage or stalled DNA replication can activate specific signal transduction pathways, termed checkpoints . Checkpoint activation can result in increased repair, induction of a transcriptional programme and inhibition of cell-cycle progression . Recent results have suggested possible mechanisms for the detection of specific DNA structures, provided further information on the organisation of the signal transduction cascade and demonstrated involvement of the checkpoint pathway in DNA repair.

Curr Opin Genet Dev, 2000 Feb, 10(1), 54 - 64
Proteolysis and the cell cycle: with this RING I do thee destroy; Tyers M et al.; The ubiquitin system drives the cell division cycle by the timely destruction of numerous regulatory proteins . Remarkably, the two main activities that catalyze substrate ubiquitination in the cell cycle, the Skp1-Cdc53/cullin-F-box protein (SCF) complexes and the anaphase-promoting complex/cyclosome (APC/C), define a new superfamily of E3 ubiquitin ligases, all based on related cullin and RING-H2 finger protein subunits . The circuits that interconnect the SCF, APC/C and cyclin-dependent kinase activities form a master oscillator that coordinates the replication and segregation of the genome.

Curr Opin Genet Dev, 2000 Feb, 10(1), 65 - 9
Controlling the end of the cell cycle; Cerutti L et al.; The past year has seen significant advances in our understanding of how the events which occur at the end of mitosis, such as cytokinesis and the inactivation of mitotic cyclin dependent kinases are triggered, and also how they are prevented from occurring prematurely or inappropriately . This control is achieved through a combination of temporally ordered proteolytic events and changes in the subcellular localisation of proteins . These studies have also revealed that the nucleolus and spindle pole bodies play a key role in this regulation.

Clin Infect Dis, 2000 Feb, 30(2), 328 - 35
Molecular epidemiology of Blastomyces dermatitidis; McCullough MJ et al.; The inhalation of conidia of Blastomyces dermatitidis, a fungus found in soil, causes disease in humans and animals . We studied the genetic diversity of this pathogen by extracting DNA yeasts and analyzing them with a polymerase chain reaction (PCR)-based typing system we developed, which used restriction fragment analysis of amplicons from the regions between the rDNA repeats and allowed us to class isolates into 3 major groups . Strains were further differentiated by use of PCR fingerprinting with 3 different primers . Fifty-nine isolates collected over 35 years from 15 regions (United States, India, Africa, Canada) were analyzed . Genotypic groups A, B, and C contained 17, 23, and 19 isolates, which were divided into 5, 15, and 12 types, respectively . All 16 isolates from North America in group A were from the upper midwestern United States or Canada, whereas 0 of 20 isolates from the southeastern United States were in group A . Studies of the largest collection from 1 locale (Eagle River, WI), revealed that the soil isolates studied were not responsible for the majority of cases in this outbreak, as previously proposed, and that >1 strain was present in the environment and in patients . Overall, these results provide a tool for the epidemiological study of blastomycosis and illuminate the genetic and geographic diversity of this important pathogen.

Fungal Genet Biol, 1999 Dec, 28(3), 190 - 200
Invasive hyphal growth in Wangiella dermatitidis is induced by stab inoculation and shows dependence upon melanin biosynthesis; Brush L et al.; Stab inoculation of agar medium with yeasts of the human pathogen Wangiella dermatitidis resulted in induction of invasive hyphae . Mechanical penetration of agar was indicated by the observation that an increase in medium gel strength slowed the rate of substrate invasion . A melanized wild-type strain (8656) exhibited much faster invasive growth through 2-8% agar than three melanin-deficient mutants . Inhibition of melanin synthesis in strain 8656 using tricyclazole resulted in a decrease in its rate of invasive growth, while scytalone restored melanin synthesis in the albino mel3 strain and boosted its rate of invasive growth . Earlier research established that cellular melanization is also associated with invasive hyphal growth in the mouse brain, and infections with strain 8656 are invariably lethal . Together, these in vitro and in vivo data indicate that biomechanical characteristics of fungi may be important determinants of virulence and disease progression in human and animal mycoses .

RNA, 2000 Jan, 6(1), 103 - 10
In vivo misreading by tRNA overdose; Navarro F et al.; Rpb5-H147R is an AT-GC transition replacing CAC(His) by CGC(Arg) at a conserved and critical position of ABC27 (Rpb5p), one of the five common and essential subunits shared by all three eukaryotic RNA polymerases . This mutation is viable at 25 degrees C, but has a lethal phenotype at 34 degrees C . A search for dosage-dependent suppressors identified five distinct clones that all bear a copy of the tRNA(His)GUG gene . Suppression was also observed with a small genomic insert bearing this tRNA gene and no other coding sequences, under conditions where there is a sevenfold increase in the cellular concentration of tRNA(His)GUG . Overexpressing tRNA(Arg)ICG, which normally decodes the suppressed CGC codon, counteracted suppression . Suppression is codon specific because it was abolished when replacing CGC by its synonymous codons CGA, CGU, or AGA, but was not detectably affected by several nucleotide substitutions modifying the surrounding sequence and is thus largely insensitive to the nucleotide context . It is proposed that overexpressing tRNA(His)GUG extends its decoding properties from CAC(His) to the noncognate CGC(Arg) codon through an illegitimate U x G pairing at the middle base of the anticodon . Accordingly, tRNA(His)GUG would compete with tRNA(Arg)ICG for chain elongation and generate a significant level of misreading errors under normal growth conditions.

Exp Cell Res, 2000 Feb 25, 255(1), 86 - 94
Human topoisomerase IIalpha and IIbeta interact with the C-terminal region of p53; Cowell IG et al.; The p53 tumor suppressor protein is a critical regulator of cell cycle progression and apoptosis following exposure of cells to DNA damaging agents such as ionizing radiation or anticancer drugs . An important group of anticancer drugs, including compounds such as etoposide and doxorubicin (Adriamycin), interacts with DNA topoisomerase II (topo II), causing the accumulation of enzyme-DNA adducts that ultimately lead to double-strand breaks and cell death via apoptosis . Human topo IIbeta has previously been shown to interact with p53, and we have extended this analysis to show that both topo IIalpha and IIbeta interact with p53 in vivo and in vitro . Furthermore, we show that the regulatory C-terminal basic region of p53 (residues 364-393) is necessary and sufficient for interaction with DNA topo II .

Cell, 2000 Jan 21, 100(2), 229 - 40
OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways; Hata A et al.; We have identified the 30-zinc finger protein OAZ as a DNA-binding factor that associates with Smads in response to BMP2, forming a complex that transcriptionally activates the homeobox regulator of Xenopus mesoderm and neural development, Xvent-2 . OAZ contains a BMP signaling module formed by two clusters of fingers that bind Smads and the Xvent-2 BMP response element, respectively . Previously implicated as a transcriptional partner of Olf-1/EBF in olfactory epithelium and lymphocyte development in the rat, OAZ fulfills this role through clusters of fingers that are separate from the BMP signaling module . The mutually exclusive use of OAZ by the BMP-Smad and Olf pathways illustrates the dual role of a multi-zinc finger protein in signal transduction during development.

EMBO J, 2000 Feb 1, 19(3), 359 - 69
FHL2, a novel tissue-specific coactivator of the androgen receptor; Muller JM et al.; The control of target gene expression by nuclear receptors requires the recruitment of multiple cofactors . However, the exact mechanisms by which nuclear receptor-cofactor interactions result in tissue-specific gene regulation are unclear . Here we characterize a novel tissue-specific coactivator for the androgen receptor (AR), which is identical to a previously reported protein FHL2/DRAL with unknown function . In the adult, FHL2 is expressed in the myocardium of the heart and in the epithelial cells of the prostate, where it colocalizes with the AR in the nucleus . FHL2 contains a strong, autonomous transactivation function and binds specifically to the AR in vitro and in vivo . In an agonist- and AF-2-dependent manner FHL2 selectively increases the transcriptional activity of the AR, but not that of any other nuclear receptor . In addition, the transcription of the prostate-specific AR target gene probasin is coactivated by FHL2 . Taken together, our data demonstrate that FHL2 is the first LIM-only coactivator of the AR with a unique tissue-specific expression pattern.

J Cell Sci, 2000 Feb, 113 ( Pt 4), 729 - 39
TrioGEF1 controls Rac- and Cdc42-dependent cell structures through the direct activation of rhoG; Blangy A et al.; Rho GTPases regulate the morphology of cells stimulated by extracellular ligands . Their activation is controlled by guanine exchange factors (GEF) that catalyze their binding to GTP . The multidomain Trio protein represents an emerging class of &Rgr; regulators that contain two GEF domains of distinct specificities . We report here the characterization of Rho signaling pathways activated by the N-terminal GEF domain of Trio (TrioD1) . In fibroblasts, TrioD1 triggers the formation of particular cell structures, similar to those elicited by RhoG, a GTPase known to activate both Rac1 and Cdc42Hs . In addition, the activity of TrioD1 requires the microtubule network and relocalizes RhoG at the active sites of the plasma membrane . Using a classical in vitro exchange assay, TrioD1 displays a higher GEF activity on RhoG than on Rac1 . In fibroblasts, expression of dominant negative RhoG mutants totally abolished TrioD1 signaling, whereas dominant negative Rac1 and Cdc42Hs only led to partial and complementary inhibitions . Finally, expression of a Rho Binding Domain that specifically binds RhoG(GTP) led to the complete abolition of TrioD1 signaling, which strongly supports Rac1 not being activated by TrioD1 in vivo . These data demonstrate that Trio controls a signaling cascade that activates RhoG, which in turn activates Rac1 and Cdc42Hs.

J Bacteriol, 2000 Feb, 182(4), 874 - 81
WdCHS3, a gene that encodes a class III chitin synthase in Wangiella (Exophiala) dermatitidis, is expressed differentially under stress conditions; Wang Z et al.; Class III chitin synthases are important for hyphal growth in some filamentous fungi but are not found in yeasts . Using a specific PCR product that encodes a portion of the class III chitin synthase of W . dermatitidis as a probe, we isolated the chitin synthase gene, WdCHS3, from this polymorphic melanized pathogen of humans . Northern blotting showed that WdCHS3 was highly expressed under stress conditions, such as the shift of cells to temperatures commensurate with infection, or to conditions that induce cellular morphogenesis in this fungus . Analysis of the 5' upstream sequence of WdCHS3 provided evidence for a negative regulatory element at between -780 and -1600 bp . Western blotting indicated that the production of the WdChs3p was temperature dependent and temporally regulated . Disruption of WdCHS3 in a wild-type strain and in two temperature-sensitive morphological mutants resulted in significantly reduced chitin synthase activities but did not obviously affect their morphologies, growth rates, chitin contents, or virulence . This paradox suggested that the contributions of the high levels of WdCHS3 gene expression and WdChs3p production in strains subjected to stress reside in unknown or unexamined parts of the life cycle of this ecologically poorly known member of the Fungi Imperfecti . Nonetheless, this report presents the first evidence that transcription of a chitin synthase gene is regulated by a negative regulatory element in its 5' upstream sequence.

Arzneimittelforschung, 1999 Dec, 49(12), 1039 - 43
In vitro antifungal evaluation and studies on the mode of action of xanthoxyline derivatives; Pinheiro TR et al.; This study describes the fungistatic effect of xanthoxyline (CAS 90-24-4) and its derivatives against a panel of yeasts, filamentous fungi and dermatophytes, by using the agar dilution method . Results indicated that simple structural modifications led to more potent derivatives, especially in relation with dermatophytes . The most active compound tested (10), which is a benzenesulphonyl derivative, was 12-fold more potent than xanthoxyline itself against Trichophyton rubrum . The evaluation of the mode of action with the whole cell Neurospora crassa assay, suggested that some selected compounds may be acting by the inhibition of fungal cell-wall polymers synthesis or assembly.

J Neurosci, 2000 Dec 1, 20(23), 8551 - 65
Neurobeachin: A protein kinase A-anchoring, beige/Chediak-higashi protein homolog implicated in neuronal membrane traffic; Wang X et al.; We describe the identification and initial characterization of neurobeachin, a neuron-specific multidomain protein of 327 kDa with a high-affinity binding site (K(d), 10 nm) for the type II regulatory subunit of protein kinase A (PKA RII) . Neurobeachin is peripherally associated with pleomorphic tubulovesicular endomembranes near the trans sides of Golgi stacks and throughout the cell body and cell processes . It is also found in a subpopulation of synapses, where it is concentrated at the postsynaptic plasma membrane . In live cells, perinuclear neurobeachin is dispersed by brefeldin A (BFA) within 1 min, and in permeabilized cells a recruitment of neurobeachin from cytosol to Golgi-near membranes is stimulated by GTPgammaS and prevented by brefeldin A . Spots of neurobeachin recruitment are close to but distinct from recruitment sites of COP-I, AP-1, and AP-3 coat proteins involved in vesicle budding . These observations indicate that neurobeachin binding to membranes close to the trans-Golgi requires an ADP-ribosylation factor-like GTPase, possibly in association with a novel type of protein coat . A neurobeachin isoform that does not bind RII, beige-like protein (BGL), is expressed in many tissues . Neurobeachin, BGL, and approximately 10 other mammalian gene products share a characteristic C-terminal BEACH-WD40 sequence module, which is also present in gene products of invertebrates, plants, protozoans, and yeasts, thus defining a new protein family . The prototype member of this family of BEACH domain proteins, lysosomal trafficking regulator (LYST), is deficient in genetic defects of protein sorting in lysosome biogenesis (the beige mouse and Chediak-Higashi syndrome) . Neurobeachin's subcellular localization, its coat protein-like membrane recruitment, and its sequence similarity to LYST suggest an involvement in neuronal post-Golgi membrane traffic, one of its functions being to recruit protein kinase A to the membranes with which it associates.

Int J Food Sci Nutr, 1999 Mar, 50(2), 145 - 8
Study of chemical characteristics and nutritional quality of two food-subproduct flours--multimixture; de Azeredo VB et al.; The objective of this study was to analyze the chemical and nutritional composition of two flours obtained from food subproducts (multimixtures): MM1 based on rice bran and MM2 based on wheat bran . This was done by identifying their macronutrients and performing analyses to determine characteristics such as rancidity, alcohol-soluble acidity, pH and presence of fungi (molds and yeasts) that could affect their quality . Our results show that MM1 has a greater content of nutrients such as lipids, insoluble fibers, calcium, and iron, while MM2 has a higher content of glycids and phosphorus . However, protein contents were similar in both samples . A high index of alcohol-soluble acidity was observed in MM1, and rancidity and high acidity were detected in both samples . No sign of dirtiness was found and the level of molds and yeasts encountered in both flours complies with the legal standards in effect in the country . We conclude that although the multimixtures present good levels of nutrients, they are highly susceptible to decomposition.

Development, 2000 Jan, 127(2), 425 - 35
Regulation of neurogenesis by interactions between HEN1 and neuronal LMO proteins; Bao J et al.; Basic-helix-loop-helix transcription factors regulate neurogenesis and neuronal differentiation by as yet unknown mechanisms . We show that an embryonic neuronal-specific basic-helix-loop-helix protein, HEN1 (also known as NSCL1 or NHLH), interacts with 'LIM only' proteins . Examination of the expression patterns of XHEN1 and XLMO-3, the Xenopus homologues of these human genes, reveals extensive overlap during early neurogenesis: at the onset of gastrulation on the dorsal side of the blastopore lip and, subsequently, in the prospective neural plate . Binding of XLMO-3 increases the transcriptional activity of XHEN1 in vivo . Co-expression of these two genes in Xenopus embryos induces a cascade of expression of neuronal-specific basic-helix-loop-helix proteins that leads to neuronal differentiation . We propose that XHEN1, in concert with XLMO-3, is a critical regulator of neurogenesis.

Chemotherapy, 2000 Jan-Feb, 46(1), 28 - 35
Overview of SPA-S-843 in vitro activity against filamentous fungi; Rimaroli C et al.; In this study, we investigated the in vitro antifungal activity of a new water-soluble partricin A derivative, N-dimethylaminoacetyl-partricin A 2-dimethylaminoethylamide diascorbate, coded SPA-843, currently developed by Societa Prodotti Antibiotici . The activity of SPA-S-843 was compared to that of amphotericin B against 13 strains of Aspergillus spp., 4 strains of Mucor sp., 4 strains of Rhizopus oryzae, 2 strains Paecilomyces variotii, 5 strains of Penicillium spp., 1 strain of Sporothrix schenkii, 7 strains of Trichophyton spp . and 2 strains of Microsporum spp.; the minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) were measured for all the organisms . The in vitro susceptibility testing method employed was an adaptation of the macrodilution reference method for yeasts as described in the National Committee for Clinical Laboratory Standards document M27-A . The in vitro inhibitory activities of SPA-S-843 and amphotericin B against the fungi were evaluated in RPMI-1640 supplemented with L-glutamine and buffered with morpholinepropanesulfonic acid, while the in vitro fungicidal activities were determined by subculturing 0.1 ml from all tubes with no visible growth onto drug-free Sabouraud dextrose agar plates . Comparison with amphotericin B showed that the in vitro inhibitory activity of SPA-S-843 against Aspergillus spp . was better than that of amphotericin B and similar against R . oryzae, P . variotii, Penicillium spp . and S . schenkii . Amphotericin B presented geometric means (GM) of the MICs lower than those of SPA-S-843 against Mucor sp., Microsporum spp . and Trichophyton spp . SPA-S-843 was most fungicidal against Mucor sp . and P . variotii; SPA-S-843 and amphotericin B showed the same fungicidal activity against Aspergillus spp . (GM of the MFCs 12.53 microg/ml), Penicillium spp . (about 12 microg/ml) and S . schenkii (MFC 19.2 microg/ml) . Amphotericin B presented GM of the MFC values lower than those of SPA-S-843 against R . oryzae, Microsporum spp . and Trichophyton spp .

Semin Cell Dev Biol, 1999 Oct, 10(5), 507 - 13
ER protein quality control and proteasome-mediated protein degradation; Brodsky JL et al.; A variety of mutant polypeptides that are associated with human disease are targeted for degradation by an endoplasmic reticulum (ER) quality control system . In addition, physiological signals and viral gene products can target the degradation of several ER resident proteins and secreted proteins passing through the ER . Although the mechanism of protein quality control and the site of degradation were obscure, recent data indicate that degradation requires the cytosolic proteasome . Biochemical and genetic analyses have indicated that both lumenal and integral membrane proteins are selected for proteolysis and exported to the cytosol by a process that in several cases requires ER associated molecular chaperones.

Mycoses, 1999, 42(9-10), 515 - 20
A polymerase chain reaction 'PCR' for a quick diagnosis of aspergillosis; Gabal MA et al.; A polymerase chain reaction (PCR) was developed from sequencing data generated from a specific target band that is unique for Aspergillus fumigatus DNA digested with EcoR1 . The target band was detected through Southern blot hybridization of a non-radioactive probe labelled with DIG-dUTP and DNAs of different aspergilli . The DNA of the target band was purified, concentrated and subjected to sequencing . The size of the sequenced band was approximately 445 bp . One pair of primers was designed and synthesized from the sequencing data of the band . The oligonucleotide primers were specific in amplifying an identical band of A . fumigatus in a population mix containing DNAs of different Aspergillus spp.; Pencillium spp.; yeasts; bacterial and viral organisms that are commonly encountered in clinical specimens of respiratory origin . The reaction proved highly sensitive and as little as 0.0001 microgram of A . fumigatus DNA was detected in the reaction.

Am J Ophthalmol, 1999 Oct, 128(4), 512 - 4
Exophiala jeanselmei causing late endophthalmitis after cataract surgery; Hofling-Lima AL et al.; PURPOSE: To report two cases of late endophthalmitis caused by Exophiala jeanselmei after cataract surgery . METHODS: Case reports, including clinical evaluation, direct examination, and culture of the aqueous humor . RESULTS: In each case, samples from the anterior chamber had positive growth of yeasts with toruloid hyphae and pseudohyphae . Intravitreal and anterior chamber amphotericin B were used in both cases . Apparent clinical resolution was achieved, but after 3 months in one case and 6 months in the other the infection recurred more aggressively, with severe endophthalmitis leading to ocular atrophy . CONCLUSION: E . jeanselmei causes a severe intraocular infection and isolation, and identification of the agent ensures proper diagnosis and treatment . After clinical resolution of the infection, careful and long-term follow-up is recommended to promptly detect relapse and immediately reintroduce treatment.

Biochemistry, 1999 Oct 26, 38(43), 14264 - 70
Diclofenac and its derivatives as tools for studying human cytochromes P450 active sites: particular efficiency and regioselectivity of P450 2Cs; Mancy A et al.; A comparison of the oxidations of diclofenac with microsomes of yeasts expressing various human liver cytochromes P450 showed that P450 2C9 regioselectively led to 4'-hydroxy diclofenac (4'-OHD) whereas P450 3A4 only led to 5-hydroxy diclofenac (5-OHD) . P450 2C19, 2C18, and 2C8 led to the simultaneous formation of 4'-OHD and 5-OHD (respective molar ratios of 1.3, 0.37, and 0.17), and P450 1A1, 1A2, 2D6, and 2E1 failed to give any detectable hydroxylated metabolite under identical conditions . P450 2C9 was found to be much more efficient for diclofenac hydroxylation than all the other P450s tested (k(cat)/K(M) of 1.6 min(-1) microM(-1) instead of 0.025 for the second more active P450), mainly because of markedly lower K(M) values (15 +/- 8 instead of values between 170 and 630 microM) . Oxidation of diclofenac with chemical model systems of cytochrome P450 based on iron porphyrin catalysts exclusively led to the quinone imine derived from two-electron oxidation of 5-OHD, in an almost quantitative yield . Two derivatives of diclofenac lacking its COO(-) function were then synthesized; their oxidation by recombinant human P450 2Cs always led to a major product coming from their 5-hydroxylation . Substrate 2, which derives from reduction of the COO(-) function of diclofenac to the CH(2)OH function, was studied in more detail . All the P450s tested (1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6, and 3A4) almost exclusively led to its 5-hydroxylation . P450s of the 2C subfamily were found to be the most efficient catalysts for this reaction, with k(cat)/K(M) values between 0.2 and 1.6 min(-1) microM(-1) . Oxidation of 2 with an iron porphyrin-based chemical model of cytochrome P450 also led to a product derived from the oxidation of 2 at position 5 . These results show that oxidation of diclofenac and its derivative 2, either with chemical model systems of cytochrome P450 or with recombinant human P450s, generally occurs at position 5 . This position, para to the NH group on the more electron-rich aromatic ring of diclofenac derivatives, is thus, as expected, the privileged site of reaction of electrophilic, oxidant species . The most spectacular exception to this chemoselective 5-oxidation of diclofenac derivatives was found for oxidation of diclofenac itself with P450 2C9 (and P450 2C19 and 2C18 to a lesser extent), which only led to 4'-OHD . A likely explanation for this result is a strict positioning of diclofenac in the P450 2C9 active site, via its COO(-) function, to completely orientate its hydroxylation toward position 4', which is not chemically preferred . P450 2C19, 2C18, and 2C8 would not lead to such a strict positioning as they give mixtures of 4'-OHD and 5-OHD . The above results show that diclofenac derivatives are interesting tools to compare the active site topologies of human P450 2Cs.

Pediatr Infect Dis J, 1999 Nov, 18(11), 1021 - 2
Fungal susceptibility testing; Gianinni MA et al.; The utility of antifungal susceptibility testing has not been broadly determined . Thus, susceptibility testing of fungal isolates is not recommended on a routine basis . For instance, susceptibilty testing may be considered for some Candida species and for patients with Pseudallescheria boydii infections . Testing of yeasts for susceptiblity to azoles is of particular value due to their variability in response to these agents . It may also be important to test the susceptibility of new fungal organisms not previously identified or known to cause human disease because in these situations there are no clinical reports of efficacy to guide the choice of antifungal therapy.

Mol Biol Cell, 1999 Nov, 10(11), 3909 - 26
The interaction and colocalization of Sam68 with the splicing-associated factor YT521-B in nuclear dots is regulated by the Src family kinase p59(fyn); Hartmann AM et al.; Alternative pre-mRNA splicing patterns can change an extracellular stimulus, but the signaling pathways leading to these changes are still poorly characterized . Here, we describe a tyrosine-phosphorylated nuclear protein, YT521-B, and show that it interacts with the nuclear transcriptosomal component scaffold attachment factor B, and the 68-kDa Src substrate associated during mitosis, Sam68 . Northern blot analysis demonstrated ubiquitous expression, but detailed RNA in situ analysis revealed cell type specificity in the brain . YT521-B protein is localized in the nucleoplasm and concentrated in 5-20 large nuclear dots . Deletion analysis demonstrated that the formation of these dots depends on the presence of the amino-terminal glutamic acid-rich domain and the carboxyl-terminal glutamic acid/arginine-rich region . We show that the latter comprises an important protein-protein interaction domain . The Src family kinase p59(fyn)-mediated tyrosine phosphorylation of Sam68 negatively regulates its association with YT521-B, and overexpression of p59(fyn) dissolves nuclear dots containing YT521-B . In vivo splicing assays demonstrated that YT521-B modulates alternative splice site selection in a concentration-dependent manner . Together, our data indicate that YT521-B and Sam68 may be part of a signal transduction pathway that influences splice site selection.

Mol Cell, 1999 Oct, 4(4), 529 - 40
Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins; Murzina N et al.; Mechanisms contributing to the maintenance of heterochromatin in proliferating cells are poorly understood . We demonstrate that chromatin assembly factor 1 (CAF-1) binds to mouse HP1 proteins via an N-terminal domain of its p150 subunit, a domain dispensable for nucleosome assembly during DNA replication . Mutations in p150 prevent association with HP1 in heterochromatin in cells that are not in S phase and the formation of CAF-1-HP1 complexes in nascent chromatin during DNA replication in vitro . We suggest that CAF-1 p150 has a heterochromatin-specific function distinct from its nucleosome assembly function during S phase . Just before mitosis, CAF-1 p150 and some HP1 progressively dissociate from heterochromatin concomitant with histone H3 phosphorylation . The HP1 proteins reassociate with chromatin at the end of mitosis, as histone H3 is dephosphorylated.

Mycoses, 1999, 42(7-8), 443 - 51
Breakthrough invasive fungal infections in neutropenic patients after prophylaxis with itraconazole; Glasmacher A et al.; This study analyses invasive fungal infections in neutropenic patients with haematological malignancies during antifungal prophylaxis with itraconazole . From September 1994 to December 1998 20 patients developed fungal infections . Two patients suffered from disseminated infections by yeasts and 18 patients suffered from pulmonary infections by moulds (eight proven, 10 highly probable in high-resolution CT scans) . In these patients the itraconazole trough concentrations exceeded 500 ng ml-1 (measured by high performance liquid chromatography) significantly less often (median 48%, interquartile range 0-100%) than in another group of 150 leukaemia patients without invasive fungal infections who received 287 courses of prophylaxis with itraconazole at our institution (median 100%, interquartile range 38-100%, P = 0.039) . Twelve patients died, six of these had refractory disease . Patients with fatal invasive fungal infections had lower median itraconazole concentrations immediately before occurrence of the infection than patients with non-fatal infections: 120 (0-478) ng ml-1 versus 690 (305-1908) ng ml-1 (P = 0.039) . In conclusion, this analysis of breakthrough invasive fungal infections during prophylaxis with itraconazole demonstrates that patients with itraconazole trough concentrations below 500 ng ml-1 were significantly more likely to develop fungal infections and that the last itraconazole trough concentration before occurrence of the infection was significantly lower in patients with fatal invasive fungal infections.

EMBO J, 1999 Nov 1, 18(21), 6037 - 49
Human Daxx regulates Fas-induced apoptosis from nuclear PML oncogenic domains (PODs); Torii S et al.; Daxx was first identified as a protein that binds the cytosolic domain of Fas and links this receptor to an apoptosis pathway involving activation of Jun N-terminal kinase (JNK) . We show here that cells overexpressing the human homolog of Daxx (hDaxx) display enhanced sensitivity to apoptosis induced by Fas but not by several other cell death stimuli . hDaxx-mediated enhancement of Fas-induced apoptosis was correlated with accelerated activation of caspases but not with JNK induction . Although specifically enhancing Fas function, hDaxx does not bind Fas and instead is found in the nucleus where it localizes to PML oncogenic domains (PODs) . Moreover, the hDaxx protein also exhibits the ability to repress transcription . Mutagenesis studies demonstrated a correlation between the localization of hDaxx to PODs and its ability to enhance Fas-induced cell death . Arsenic trioxide (As(2)O(3)), an agent that accentuates POD formation, collaborated synergistically with overexpression of hDaxx to increase cellular sensitivity to Fas-induced apoptosis . Taken together, these findings argue that hDaxx promotes sensitivity to Fas from a nuclear location, probably by modulating the transcription of genes involved in Fas-induced caspase activation and apoptosis.

J Chromatogr A, 1999 Oct 1, 857(1-2), 193 - 204
Magnetic split-flow thin fractionation of magnetically susceptible particles; Fuh CB et al.; We recently built a magnetic separation system to extend the applications of split-flow thin (SPLITT) fractionation to magnetically susceptible particles . Here, we characterize the magnetic SPLITT system using magnetically susceptible particles and ion-labeled particles . The flow axis of separation channel was orientated parallel and perpendicular to gravitational forces to exclude and include, respectively, gravitational effects on separation . Both operating modes were used to test the theory experimentally, with emphasis on the parallel mode . The magnetic susceptibilities of carrier and ion-labeled particles were varied, and various ion-labeled and unlabeled particles were studied experimentally, resulting in successful separation of labeled particles, yeasts, and cells from unlabeled ones . The minimal difference in magnetic susceptibility (delta(chi)) required for complete particle separation was about 1.75 x 10(-5) {cgs}, corresponding to about 10(9) labeling ions per particle in this study . The throughput was around 7.2 x 10(8) particles/h using the present setup . Magnetic SPLITT fractionation shows good potential for use in obtaining particles magnetic susceptibilities from a simple theoretical treatment.

Development, 1999 Nov, 126(21), 4807 - 16
TONDU (TDU), a novel human protein related to the product of vestigial (vg) gene of Drosophila melanogaster interacts with vertebrate TEF factors and substitutes for Vg function in wing formation; Vaudin P et al.; The mammalian TEF and the Drosophila scalloped genes belong to a conserved family of transcriptional factors that possesses a TEA/ATTS DNA-binding domain . Transcriptional activation by these proteins likely requires interactions with specific coactivators . In Drosophila, Scalloped (Sd) interacts with Vestigial (Vg) to form a complex, which binds DNA through the Sd TEA/ATTS domain . The Sd-Vg heterodimer is a key regulator of wing development, which directly controls several target genes and is able to induce wing outgrowth when ectopically expressed . Here we show that Vg contains two distinct transcriptional activation domains, suggesting that the function of Vg is to mediate transcriptional activation by Sd . By expressing a chimeric GAL4-Sd protein in Drosophila, we found that the transcriptional activity of the Vg-Sd heterodimer is negatively regulated at the AP and DV boundary of the wing disc . We also identify a novel human protein, TONDU, which contains a short domain homologous to the domain of Vg required for interaction with Sd . We show that TONDU specifically interacts with a domain conserved in all the mammalian TEF factors . Expression of TDU in Drosophila by means of the UAS-GAL4 system shows that this human protein can substitute for Vg in wing formation . We propose that TDU is a specific coactivator for the mammalian TEFs.

Free Radic Res, 1999 Oct, 31(4), 341 - 9
The role of protein phosphatases in the regulation of mitogen and stress-activated protein kinases; Keyse SM; It is now established that a family of dual-specificity protein phosphatases are able to interact with mitogen and stress-activated protein kinases in a highly specific manner to differentially regulate these enzymes in mammalian cells . A role for these proteins in negative feedback regulation of MAP kinase activity is also supported by genetic and biochemical studies in yeasts and Drosophila . More recently it has become clear that other classes of protein phosphatase also play key roles in the regulated dephosphorylation of MAP kinases, including tyrosine-specific protein phosphatases and serine/threonine protein phosphatases . It is likely that a complex balance between upstream activators and these different classes of MAP kinase specific phosphatase are responsible for determining, at least in part, the magnitude and duration of MAP kinase activation and hence the physiological outcome of signalling.

Trends Cell Biol, 1999 Nov, 9(11), 447 - 53
Peroxisomes: simple in function but complex in maintenance; Tabak HF et al.; Peroxisomes compartmentalize part of the anabolic and catabolic pathways and reactions of the cell . Dysfunction of a single peroxisomal enzyme or loss of the whole peroxisomal compartment causes sporadic, but serious, human diseases . Genetic studies in various yeasts have identified PEX genes, which are required for the maintenance of complete peroxisomes . Mutations in PEX genes have proved to be the molecular cause of several human diseases, particularly those involving loss of organelles . Peroxisomes have several properties that distinguish them from other organelles, including the import of folded proteins from the cytosol by an unknown mechanism . By discussing recent highlights from the field of peroxisome research, we aim to share with the general readership our excitement as well as the many mysteries still surrounding peroxisome function and maintenance.

Prog Nucleic Acid Res Mol Biol, 1999, 63, 189 - 221
Genetic disorders associated with cancer predisposition and genomic instability; Vessey CJ et al.; Genomic instability in its broadest sense is a feature of virtually all neoplastic cells . In addition to the mutations and/or gene amplifications that appear to be a prerequisite for the acquisition of a neoplastic phenotype, human cancers exhibit other "markers" of genomic instability--in particular, a high degree of aneuploidy . Indeed, many studies have shown that aneuploidy is an almost invariant feature of cancer cells, and it has been argued by some that the emergence of aneuploid cells is a necessary step during tumorigenesis . The functional link between genomic instability and cancer is strengthened by the existence of several "genetic instability" disorders of humans that are associated with a moderate to severe increase in the incidence of cancers . These disorders include ataxia telangiectasia, Bloom's syndrome, Fanconi anemia, xeroderma pigmentosum, and Nijmegen breakage syndrome, all of which are very rare and are inherited in a recessive manner . Analysis of the cells from such cancer-prone individuals is clearly a potentially fruitful approach for delineating the genetic basis for instability in the genome . It is assumed that by identifying the underlying cause of genetic instability in these disorders, one can derive valuable information not only about the basis of particular genetic diseases, but also about the underlying causes of genomic instability in sporadic cancers in the general population . In this article, we review the clinical and cellular properties of genetic instability disorders associated with cancer predisposition . In particular, we focus on the rapid advances made in our understanding of these disorders that have derived from the cloning of the genes mutated in each case . Because in many instances the affected genes have analogs in lower eukaryotic species, we shall discuss how studies in yeasts in particular have proved valuable in our understanding of human diseases and predisposition to cancer.

J Biol Chem, 1999 Oct 8, 274(41), 28857 - 60
The Rab5 effector EEA1 interacts directly with syntaxin-6; Simonsen A et al.; The fusion of transport vesicles with their cognate target membranes, an essential event in intracellular membrane trafficking, is regulated by SNARE proteins and Rab GTPases . Rab GTPases are thought to act prior to SNAREs in vesicle docking, but the exact biochemical relationship between the two classes of molecules is not known . We recently identified the early endosomal autoantigen EEA1 as an effector of Rab5 in endocytic membrane fusion . Here we demonstrate that EEA1 interacts directly and specifically with syntaxin-6, a SNARE implicated in trans-Golgi network to early endosome trafficking . The binding site for syntaxin-6 overlaps with that of Rab5-GTP at the C terminus of EEA1 . Syntaxin-6 and EEA1 were found to colocalize extensively on early endosomes, although syntaxin-6 is present in the trans-Golgi network as well . Our results indicate that SNAREs can interact directly with Rab effectors, and suggest that EEA1 may participate in trans-Golgi network to endosome as well as in endocytic membrane traffic.

Cell Biochem Biophys, 1999, 31(1), 19 - 48
Structure and function of metal chelators produced by plants: the case for organic acids, amino acids, phytin, and metallothioneins; Rauser WE; Plants produce a range of ligands for cadmium (Cd), copper (Cu), nickel (Ni), and zinc (Zn) . Cd- and Zn-citrate complexes are prevalent in leaves, even though malate is more abundant . In the xylem sap moving from roots to leaves, citrate and histidine are the principal ligands for Cu, Ni, and Zn . Phosphorus-rich globular bodies in young roots are probably Zn-phytate . Metallothioneins (MTs) are cysteine (Cys)-rich ligands . Plants produce class II MTs (MT-IIs) which differ from the archetypal mammalian MT-I in the location and number of Cys . The Ec protein from wheat embryos has Cys in three domains, binds Zn, and disappears with seedling development . The first 59 amino acids have been sequenced for the protein . Fifty-eight genes for MT-IIs, from a range of plants and tissues, predict proteins with Cys in two domains . Most of the predicted proteins have not been isolated, and their metal binding is poorly documented . Three protein bands, corresponding to six MT genes, have been isolated from Arabidopsis, and the amino acids sequenced for nine fragments . The MT-IIIs are atypical, nontranslationally synthesized polypeptides with variously repeating gamma-glutamylcysteine units . Of the five families known, those with carboxy-terminal glycine are the most widespread among plants, algae, and certain yeasts . A heterogeneous grouping of these molecules form Cd-binding complexes with tetrahedral coordination and a Cd-sulfur interatomic distance of 2.52 A . One complex is cytosolic, the dominant one is vacuolar . Together, they can bind a large proportion of cellular Cd; other ligands may also function . Little is known about the counterpart situation for Cu and Zn.

Plant J, 1999 Aug, 19(4), 473 - 80
Some thaumatin-like proteins hydrolyse polymeric beta-1,3-glucans; Grenier J et al.; Thaumatin and 12 purified thaumatin-like (TL) proteins were surveyed for their capacity to hydrolyse beta-1,3-glucans by using an in-gel glucanase assay . Six TL proteins identified by N-terminal amino acid microsequencing were found to be active on carboxymethyl(CM)-pachyman: a barley leaf stress-related permatin, two tomato fruit osmotins, a cherry fruit and two tobacco stigma proteins . TL enzymes ranged in specific activity from 0.07 to 89 nkat mg-1 with CM-pachyman as substrate . Hydrolytic activities were not restricted to TL proteins strongly binding to water-insoluble beta-1,3-glucans since the two osmotins were active without tight binding to pachyman . Some TL proteins hydrolysed crude fungal walls and one barley TL enzyme even lysed fungal spores . No activity was observed on laminarin in the in-gel hydrolase assay . Thin-layer chromatography revealed that the six enzymes acted as endo-beta-1, 3-glucanases leading to the formation of various oligoglucosides . Thus far, the TL enzymes (EC 3.2.1.x) appeared different from the well-known beta-1,3-glucanases (EC 3.2.1.39) . No activity was found with thaumatin, zeamatin, tobacco leaf PR-R protein and four stress-related TL proteins from barley and pea . This is the first demonstration that diverse TL proteins are enzymatically active . The functions of some TL proteins must be reassessed because they display endo-beta-1,3-glucanase activity on polymeric beta-1, 3-glucans.

Plant J, 1999 Aug, 19(4), 441 - 51
Isolation and characterization of a flavonoid 3'-hydroxylase cDNA clone corresponding to the Ht1 locus of Petunia hybrida; Brugliera F et al.; We have isolated a cDNA clone that corresponds to the Ht1 locus of petunia which controls the hydroxylation of dihydrokaempferol to dihydroquercetin and of naringenin to eriodictyol by the action of flavonoid 3'-hydroxylase (F3'H) . The cDNA encodes a 512 amino acid polypeptide with regions of similarity to petunia flavonoid 3',5'-hydroxylases (F3'5'H) . Both F3'H and F3'5'H are cytochromes P450 and are key enzymes in the flavonoid pathway leading to the production of the coloured anthocyanins . The F3 'H transcript is most abundant in petals from flowers at an early stage of development and levels decline as the flower matures . Transcripts are also detected in the ovaries, sepals, peduncles, stems and anthers of the petunia plant . No or very reduced levels of transcripts are detected in ht1/ht1 lines . This is the first report of isolation of a F3 'H cDNA clone from any species.

J Mol Biol, 1999 Sep 10, 292(1), 137 - 49
Structural and functional study of a conserved region in the uncoupling protein UCP1: the three matrix loops are involved in the control of transport; Gonzalez-Barroso MM et al.; It has been reported that the region 261-269 of the uncoupling protein from brown adipose tissue mitochondria, UCP1, has an important role in the control of its proton translocating activity . Thus the deletion of residues Phe267-Lys268-Gly269 leads to the loss of the nucleotide regulation of the protein, while the complete deletion of the segment leads to the formation of a pore . The region displays sequence homology with the DNA-binding domain of the estrogen receptor . The present report analyzes the structure, by NMR and circular dichroism, of a 20 amino acid residue peptide containing the residues of interest . We demonstrate that residues 263-268 adopt an alpha-helical structure . The helix is at the N-terminal end of the sixth transmembrane domain . The functional significance of this helix has been examined by site-directed mutagenesis of the protein expressed recombinantly in yeasts . Alterations in the structure or orientation of the region leads to an impairment of the regulation, by nucleotides and fatty acids, of the transport activity . UCP1 is one member of the family formed by the carriers of the mitochondrial inner membrane . The family is characterized by a tripartite structure with three repeated segments of about 100 amino acid residues . Two of the mutations have also been performed in the first and second matrix loops and the effect on UCP1 function is very similar . We conclude that the three matrix loops contribute to the formation of the gating domain in UCP1 and propose that they form a hydrophobic pocket that accommodates the purine moiety of the bound nucleotide .

Int J Dermatol, 1999 Aug, 38(8), 591 - 5
Onychomycosis in Lahore, Pakistan; Bokhari MA et al.; BACKGROUND: Onychomycosis, a common nail disorder, is caused by yeasts, dermatophytes, and nondermatophyte molds . These fungi give rise to diverse clinical presentations . The present study aimed to isolate the causative pathogens and to determine the various clinical patterns of onychomycosis in the population in Lahore, Pakistan . PATIENTS: In 100 clinically suspected cases, the diagnosis was confirmed by mycologic culture . Different clinical patterns were noted and correlated with causative pathogens . RESULTS: Seventy-two women (mean age, 32.6 +/- 14.8 years) and 28 men (mean age, 40.6 +/- 15.8 years) were studied . Fingernails were involved in 50%, toenails in 23%, and both fingernails and toenails in 27% of patients . The various clinical types noted were distolateral subungual onychomycosis (47%), candidal onychomycosis (36%), total dystrophic onychomycosis (12%), superficial white onychomycosis (3%), and proximal subungual onychomycosis (2%) . Candida was the most common pathogen (46%), followed by dermatophytes (43%) (Trichophyton rubrum (31%), T . violaceum (5%), T . mentagrophytes (4%), T . tonsurans (2%), and Epidermophyton floccosum (1%) and nondermatophyte molds (11%) (Fusarium (4%), Scopulariopsis brevicaulis (2%), Aspergillus (2%), Acremonium (1%), Scytalidium dimidiatum (1%), and Alternaria (1%) . CONCLUSIONS: Onychomycosis is more common in women of 20-40 years of age . Distolateral subungual onychomycosis and candidal onychomycosis are the most common clinical presentations, and Candida and T . rubrum are the major pathogens in Pakistan.

Plant Physiol, 1999 Sep, 121(1), 123 - 34
Antisense repression of hexokinase 1 leads to an overaccumulation of starch in leaves of transgenic potato plants but not to significant changes in tuber carbohydrate metabolism; Veramendi J et al.; Potato (Solanum tuberosum L.) plants transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 1 (StHK1) exhibited altered enzyme activities and expression of StHK1 mRNA . Measurements of the maximum catalytic activity of hexokinase revealed a 22-fold variation in leaves (from 22% of the wild-type activity in antisense transformants to 485% activity in sense transformants) and a 7-fold variation in developing tubers (from 32% of the wild-type activity in antisense transformants to 222% activity in sense transformants) . Despite the wide range of hexokinase activities, no change was found in the fresh weight yield, starch, sugar, or metabolite levels of transgenic tubers . However, there was a 3-fold increase in the starch content of leaves from the antisense transformants after the dark period . Starch accumulation at the end of the night period was correlated with a 2-fold increase of glucose and a decrease of sucrose content . These results provide strong support for the hypothesis that glucose is a primary product of transitory starch degradation and is the sugar that is exported to the cytosol at night to support sucrose biosynthesis.

FEBS Lett, 1999 Sep 3, 457(3), 385 - 8
A novel Jun N-terminal kinase (JNK)-binding protein that enhances the activation of JNK by MEK kinase 1 and TGF-beta-activated kinase 1; Koyano S et al.; We have identified a novel Jun N-terminal kinase (JNK)-binding protein, termed JNKBP1, and examined its binding affinity for JNK1, JNK2, JNK3, and extracellular signal-regulated kinase 2 (ERK2) in COS-7 cells . JNKBP1 preferentially interacted with the JNKs, but not with ERK2 . Furthermore, we investigated the effect of overexpressing JNKBP1 on the JNK and ERK signaling pathways in COS-7 cells . JNKBP1 alone had only a marginal effect on JNK activity . However, the activation of JNK by MEK kinase 1 and TGF-beta-activated kinase 1 was significantly enhanced in the presence of JNKBP1 . In contrast, JNKBP1 had no or very little effect on the ERK signaling pathway . These results suggest that JNKBP1 functions to facilitate the specific and efficient activation of the JNK signaling pathways.

Drug Metab Dispos, 1999 Sep, 27(9), 1068 - 73
Cytochrome P-450 3A4 and 2C8 are involved in zopiclone metabolism; Becquemont L et al.; Zopiclone is a widely prescribed, nonbenzodiazepine hypnotic that is extensively metabolized by the liver in humans . The aim of the present study was to identify the human cytochrome P-450 (CYP) isoforms involved in zopiclone metabolism in vitro . Zopiclone metabolism was studied with different human liver microsomes and a panel of heterologously expressed human CYPs (CYP1A2, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) . In human liver microsomes, zopiclone was metabolized into N-desmethyl-zopiclone (ND-Z) and N-oxide-zopiclone (NO-Z) with the following K(m) and V(m) of 78 +/- 5 and 84 +/- 19 microM, 45 +/- 1 and 54 +/- 5 pmol/min/mg for ND-Z and NO-Z generation, respectively . Ketoconazole (CYP3A inhibitor) inhibited approximately 40% of the generation of both metabolites, sulfaphenazole (CYP2C inhibitor) inhibited the formation of ND-Z, whereas alpha-naphtoflavone (CYP1A), quinidine (CYP2D6), and chlorzoxazone (CYP2E1) did not affect zopiclone metabolism . The generation of ND-Z and NO-Z were highly correlated to testosterone 6beta-hydroxylation (CYP3A activity, r = 0.95 and 0.92, respectively; p =.0001), and ND-Z was highly correlated to CYP2C8 activity (paclitaxel 6alpha-hydroxylase; r = 0.76, p =.004) . Recombinant CYP2C8 had the highest enzymatic activity toward zopiclone metabolism into both its metabolites, followed by CYP2C9 and 3A4 . CYP3A4 is the major enzyme involved in zopiclone metabolism in vitro, and CYP2C8 contributes significantly to ND-Z formation.

J Biol Chem, 1999 Aug 27, 274(35), 25051 - 60
Tetratricopeptide repeat (TPR) motifs of p67(phox) participate in interaction with the small GTPase Rac and activation of the phagocyte NADPH oxidase; Koga H et al.; The small GTPase Rac functions as a molecular switch in several important cellular events including cytoskeletal reorganization and activation of the phagocyte NADPH oxidase, the latter of which leads to production of superoxide, a precursor of microbicidal oxidants . During formation of the active oxidase complex at the membrane, the GTP-bound Rac appears to interact with the N-terminal region of p67(phox), another indispensable activator that translocates from the cytosol upon phagocyte stimulation . Here we show that the p67(phox) N terminus lacks the CRIB motif, a well known Rac target, but contains four tetratricopeptide repeat (TPR) motifs with highly alpha-helical structure . Disruption of any of the N-terminal three TPRs, but the last one, results in defective interaction with Rac, while all the four are required for the NADPH oxidase activation . We also find that Arg-102 in the third repeat is likely involved in binding to Rac via an ionic interaction, and that replacement of this residue with Glu completely abrogates the capability of activating the oxidase both in vivo and in vitro . Thus the TPR motifs of p67(phox) are packed to function as a Rac target, thereby playing a crucial role in the active oxidase complex formation.

J Biol Chem, 1999 Aug 27, 274(35), 24453 - 6
Association of neuronal calcium channels with modular adaptor proteins; Maximov A et al.; Presynaptic voltage-gated calcium (Ca(2+)) channels mediate Ca(2+) influx into the presynaptic terminal that triggers synaptic vesicle fusion and neurotransmitter release . The immediate proximity of Ca(2+) channels to the synaptic vesicle release apparatus is critical for rapid and efficient synaptic transmission . In a series of biochemical experiments, we demonstrate a specific association of the cytosolic carboxyl terminus of the N-type Ca(2+) channel pore-forming alpha(1B) subunit with the modular adaptor proteins Mint1 and CASK . The carboxyl termini of alpha(1B) bind to the first PDZ domain of Mint1 (Mint1-1) . The proline-rich region present in the carboxyl termini of alpha(1B) binds to the SH3 domain of CASK . Mint1-1 is specific for the E/D-X-W-C/S-COOH consensus, which defines a novel class of PDZ domains (class III) . The Mint1-1 PDZ domain-binding motif is present only in the "long" carboxyl-terminal splice variants of N-type (alpha(1B)) and P/Q-type (alpha(1A)) Ca(2+) channels, but not in R-type (alpha(1E)) or L-type (alpha(1C)) Ca(2+) channels . Our results directly link presynaptic Ca(2+) channels to a macromolecular complex formed by modular adaptor proteins at synaptic junction and advance our understanding of coupling between cell adhesion and synaptic vesicle exocytosis.

J Clin Microbiol, 1999 Sep, 37(9), 2927 - 30
Onychomycosis caused by Blastoschizomyces capitatus; D'Antonio D et al.; Blastoschizomyces capitatus was cultured from the nail of a healthy patient with onychomycosis . The identity of the isolate was initially established by standard methods and ultrastructural analysis and was verified by molecular probing . Strains ATCC 200929, ATCC 62963, and ATCC 62964 served as reference strains for these analyses . To our knowledge, this is the first case of nail infection secondary to paronychia caused by this organism reported in the English literature.

Biochem J, 1999 Aug 15, 342 ( Pt 1), 13 - 9
Mapping of the RNA-binding and endoribonuclease domains of NIPP1, a nuclear targeting subunit of protein phosphatase 1; Jin Q et al.; NIPP1 (351 residues) is a major regulatory and RNA-anchoring subunit of protein phosphatase 1 in the nucleus . Using recombinant and synthetic fragments of NIPP1, the RNA-binding domain was mapped to the C-terminal residues 330-351 . A synthetic peptide encompassing this sequence equalled intact NIPP1 in RNA-binding affinity and could be used to dissociate NIPP1 from the nuclear particulate fraction . An NIPP1 fragment consisting of residues 225-351 (Ard1/NIPP1gamma), that may be encoded by an alternatively spliced transcript in transformed B-lymphocytes, displayed a single-strand Mg(2+)-dependent endoribonuclease activity . However, full-length NIPP1 and NIPP1(143-351) were not able to cleave RNA, indicating that the endoribonuclease activity of NIPP1 is restrained by its central domain . The endoribonuclease activity was also recovered in the RNA-binding domain, NIPP1(330-351), but with a 30-fold lower specific activity . Thus, the endoribonuclease catalytic site and the RNA-binding site both reside in the C-terminal 22 residues of NIPP1 . The latter domain does not conform to any known nucleic-acid binding motif.

FEMS Microbiol Lett, 1999 Jul 15, 176(2), 367 - 72
Cryptosporidium parvum possesses a short-type replication protein A large subunit that differs from its host; Zhu G et al.; Replication protein A (RPA) consisting of three subunits is a eukaryotic single-stranded DNA (ssDNA)-binding protein involved in DNA replication, repair and recombination . We report here the identification and characterization of a RPA large subunit (CpRPA1) gene from the apicomplexan Cryptosporidium parvum . The CpRPA1 gene encodes a 53.9-kDa peptide that is remarkably smaller than that from other eukaryotes (i.e . approximately 70 kDa) and is actively expressed in both free sporozoites and parasite intracellular stages . This short-type RPA large subunit has also been characterized from one other protist, Crithidia fasciculata . Three distinct domains have been identified in the RPA large subunit of humans and yeasts: an N-terminal protein interaction domain, a central ssDNA-binding area, and a C-terminal subunit-interacting region . Sequence analysis reveals that the short-type RPA large subunit differs from that of other eukaryotes in that only the domains required for ssDNA binding and heterotrimer formation are present . It lacks the N-terminal domain necessary for the binding of proteins mainly involved in DNA repair and recombination . This major structural difference suggests that the mechanism for DNA repair and recombination in some protists differs from that of other eukaryotes . Since replication proteins play an essential role in the cell cycle, the fact that RPA proteins of C . parvum differ from those of its host suggests that RPA be explored as a potential chemotherapeutic target for controlling cryptosporidiosis and/or diseases caused by other apicomplexans.

J Cell Biol, 1999 Jul 26, 146(2), 465 - 75
ZASP: a new Z-band alternatively spliced PDZ-motif protein; Faulkner G et al.; PDZ motifs are modular protein-protein interaction domains, consisting of 80-120 amino acid residues, whose function appears to be the direction of intracellular proteins to multiprotein complexes . In skeletal muscle, there are a few known PDZ-domain proteins, which include neuronal nitric oxide synthase and syntrophin, both of which are components of the dystrophin complex, and actinin-associated LIM protein, which binds to the spectrin-like repeats of alpha-actinin-2 . Here, we report the identification and characterization of a new skeletal muscle protein containing a PDZ domain that binds to the COOH-terminal region of alpha-actinin-2 . This novel 31-kD protein is specifically expressed in heart and skeletal muscle . Using antibodies produced to a fragment of the protein, we can show its location in the sarcomere at the level of the Z-band by immunoelectron microscopy . At least two proteins, 32 kD and 78 kD, can be detected by Western blot analysis of both heart and skeletal muscle, suggesting the existence of alternative forms of the protein . In fact, several forms were found that appear to be the result of alternative splicing . The transcript coding for this Z-band alternatively spliced PDZ motif (ZASP) protein maps on chromosome 10q22.3-10q23.2, near the locus for infantile-onset spinocerebellar ataxia.

J Cell Biol, 1999 Jul 26, 146(2), 301 - 11
ERG30, a VAP-33-related protein, functions in protein transport mediated by COPI vesicles; Soussan L et al.; Intracellular transport of newly synthesized and mature proteins via vesicles is controlled by a large group of proteins . Here we describe a ubiquitous rat protein-endoplasmic reticulum (ER) and Golgi 30-kD protein (ERG30)-which shares structural characteristics with VAP-33, a 33-kD protein from Aplysia californica which was shown to interact with the synaptic protein VAMP . The transmembrane topology of the 30-kD ERG30 corresponds to a type II integral membrane protein, whose cytoplasmic NH(2) terminus contains a predicted coiled-coil motif . We localized ERG30 to the ER and to pre-Golgi intermediates by biochemical and immunocytochemical methods . Consistent with a role in vesicular transport, anti-ERG30 antibodies specifically inhibit intra-Golgi transport in vitro, leading to significant accumulation of COPI-coated vesicles . It appears that ERG30 functions early in the secretory pathway, probably within the Golgi and between the Golgi and the ER.

Mycoses, 1999, 42(4), 273 - 9
Effects of Pycnoporellus fulgens (Fr.) Donk crude extract on Candida glabrata ultrastructure; Moulin-Traffort J et al.; Among antifungal products of natural origin, Pycnoporellus fulgens (Fr) Donk, is active against pathogenic yeasts . Our study was carried out to observe the ultrastructural modifications produced by P . fulgens crude extract on Candida glabrata . Yeasts were submitted to different concentrations (50, 100, 200, 400 micrograms ml-1) for 72 h under constant stirring at 35 degrees C . Transmission electron microscopy revealed that the extract acted on the cell envelope (cell wall and plasmalemma) . Cell divisions were also affected by thickening of the septum (50 micrograms ml-1) and a deficiency in the daughter cell wall texture . The extent of the antifungal effect clearly depended on the extract concentration.

Mycoses, 1999, 42(4), 265 - 7
A new, semi-automated technique for quantification of Candida adherence to denture acrylic surfaces; Panagoda GJ et al.; Adherence of Candida species to various host surfaces is an important prerequisite for colonization and pathogenesis . Researchers have problems in quantifying candidal adherence to plastic surfaces (used for fabrication of prostheses) due to coaggregation of adherent yeasts and to the laborious, time-consuming method of microscopic quantification . We describe here, a semi-automated image analysis system which is superior to conventional visual light microscopic techniques of quantifying yeasts attached to solid substrates such as acrylic . The results demonstrated a significant positive correlation between the number of cells quantified by traditional light microscopy and the corresponding area obtained by the semi-automated image analysis system (r = 0.83, P < 0.02) . The new system is simple, less laborious and is particularly useful when the adherent attributes of a large battery of yeasts are characterized.

J Comput Biol, 1999 Summer, 6(2), 253 - 9
Positional statistical significance in sequence alignment; Yu L et al.; Beginning with the concept of near-optimal sequence alignments, we can assign a probability that each element in one sequence is paired in an alignment with each element in another sequence . This involves a sum over the set of all possible pairwise alignments . The method employs a designed hidden Markov model (HMM) and the rigorous forward and forward-backward algorithms of Rabiner . The approach can use any standard sequence-element-to-element probabilistic similarity measures and affine gap penalty functions . This allows the positional alignment statistical significance to be obtained as a function of such variables . A measure of the probabilistic relationship between any single sequence and a set of sequences can be directly obtained . In addition, the employed HMM with the Viterbi algorithm provides a simple link to the standard dynamic programming optimal alignment algorithms.

Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8705 - 10
Negative cross-talk between hematopoietic regulators: GATA proteins repress PU.1; Zhang P et al.; The process through which multipotential hematopoietic cells commit to distinct lineages involves the induction of specific transcription factors . PU.1 (also known as Spi-1) and GATA-1 are transcription factors essential for the development of myeloid and erythroid lineages, respectively . Overexpression of PU.1 and GATA-1 can block differentiation in lineages in which they normally are down-regulated, indicating that not only positive but negative regulation of these factors plays a role in normal hematopoietic lineage development . Here we demonstrate that a region of the PU.1 Ets domain (the winged helix-turn-helix wing) interacts with the conserved carboxyl-terminal zinc finger of GATA-1 and GATA-2 and that GATA proteins inhibit PU.1 transactivation of critical myeloid target genes . We demonstrate further that GATA inhibits binding of PU.1 to c-Jun, a critical coactivator of PU.1 transactivation of myeloid promoters . Finally, PU.1 protein can inhibit both GATA-1 and GATA-2 transactivation function . Our results suggest that interactions between PU.1 and GATA proteins play a critical role in the decision of stem cells to commit to erythroid vs . myeloid lineages.

EMBO J, 1999 Jul 15, 18(14), 4004 - 12
Transcriptional regulation by the gamma5 subunit of a heterotrimeric G protein during adipogenesis; Park JG et al.; The adipocyte enhancer-binding protein (AEBP1) is a novel transcriptional repressor with carboxypeptidase activity . A two-hybrid screen was conducted to identify components of AEBP1 that might be important in regulating its activity . The gamma5 subunit of a heterotrimeric G protein was shown to bind specifically to AEBP1 and to attenuate its transcriptional repression activity . Adipogenic stimulation selectively decreased the Ggamma5 level and enhanced the transcriptional repression activity of AEBP1 during mitotic clonal expansion at the onset of adipogenesis . Thus, the actions of Ggamma5 and AEBP1 are directly linked, which could provide the basis for the regulation of transcription at the onset of differentiation . This report shows that a signal-transducing molecule is involved, by direct protein-protein interaction, in the regulation of transcription during adipogenesis.

Diagn Microbiol Infect Dis, 1999 Jul, 34(3), 213 - 20
Nosocomial fungal infections: candidemia; Verduyn Lunel FM et al.; Candida species are frequently encountered as part of the human commensal flora . Colonization mostly precedes candidemia and is an independent risk factor for the development of candidemia . Genotyping methods showed the similarity between colonizing and infecting strains, thus making endogenous origin likely, though exogenous sources like total parenteral nutrition also have been described . Health care workers (HCWs) play an important role in the transmission of yeasts . Candida species are frequently isolated from the hands of HCWs and can be transmitted from hands to patients . Granulocytopenia and damage of the mucosal lining resulting from intensive chemotherapy due to cancer, the increasing use of broad spectrum antibiotics, and the use of intravenous catheters are other important risk factors for the development of candidemia . Candidemia is associated with a high mortality and prolonged hospitalization . Therefore, and because of the high frequency of dissemination, all candidemias should be treated . Amphotericin B was considered the standard drug for the systemic treatment of candidemia . Fluconazole has been shown to be an effective and safe alternative in non-neutropenic patients . 5-Fluorocytosine has been used in combination with amphotericin B in the treatment of deep-seated infections . Liposomal formulations of amphotericin B and other new antifungal drugs currently are under investigation . C . albicans is the most frequently isolated Candida species, although the proportion of infections caused by non-C . albicans species is increasing . Also, there are reports of development of resistance to amphotericin B . C . lusitaniae is known for primary resistance and the development of resistance to amphotericin B . Development of resistance to fluconazole is mainly seen in AIDS patients with recurrent oropharyngeal candidiasis who receive longer courses of therapy.

Mol Cell, 1999 Jun, 3(6), 751 - 60
Synip: a novel insulin-regulated syntaxin 4-binding protein mediating GLUT4 translocation in adipocytes; Min J et al.; Insulin-stimulated glucose transport and GLUT4 translocation require regulated interactions between the v-SNARE, VAMP2, and the t-SNARE, syntaxin 4 . We have isolated a novel syntaxin 4-binding protein, Synip, which specifically interacts with syntaxin 4 . Insulin induces a dissociation of the Synip:syntaxin 4 complex due to an apparent decrease in the binding affinity of Synip for syntaxin 4 . In contrast, the carboxyterminal domain of Synip does not dissociate from syntaxin 4 in response to insulin stimulation but inhibits glucose transport and GLUT4 translocation . These data implicate Synip as an insulin-regulated syntaxin 4-binding protein directly involved in the control of glucose transport and GLUT4 vesicle translocation.

EMBO J, 1999 Jul 1, 18(13), 3834 - 44
Post-replicative base excision repair in replication foci; Otterlei M et al.; Base excision repair (BER) is initiated by a DNA glycosylase and is completed by alternative routes, one of which requires proliferating cell nuclear antigen (PCNA) and other proteins also involved in DNA replication . We report that the major nuclear uracil-DNA glycosylase (UNG2) increases in S phase, during which it co-localizes with incorporated BrdUrd in replication foci . Uracil is rapidly removed from replicatively incorporated dUMP residues in isolated nuclei . Neutralizing antibodies to UNG2 inhibit this removal, indicating that UNG2 is the major uracil-DNA glycosylase responsible . PCNA and replication protein A (RPA) co-localize with UNG2 in replication foci, and a direct molecular interaction of UNG2 with PCNA (one binding site) and RPA (two binding sites) was demonstrated using two-hybrid assays, a peptide SPOT assay and enzyme-linked immunosorbent assays . These results demonstrate rapid post-replicative removal of incorporated uracil by UNG2 and indicate the formation of a BER complex that contains UNG2, RPA and PCNA close to the replication fork.

EMBO J, 1999 Jul 1, 18(13), 3702 - 11
The Pax3-FKHR oncoprotein is unresponsive to the Pax3-associated repressor hDaxx; Hollenbach AD et al.; The Pax3-FKHR fusion protein is present in alveolar rhabdomyosarcoma and results from the t(2;13) (q35;q14) chromosomal translocation . Its oncogenic activity is dependent on a combination of protein-DNA and protein-protein interactions mediated by the Pax3 homeodomain recognition helix . In this report we demonstrate that human Daxx (hDaxx) interacts with Pax3 in vivo and with DNA-bound Pax3 in vitro . This interaction is mediated primarily through the homeodomain recognition helix with the additional involvement of the octapeptide domain and its N-terminal flanking amino acids . Through this interaction hDaxx represses the transcriptional activity of Pax3 by approximately 80% . The Pax3-FKHR fusion is unresponsive to this repressive effect despite an observed endogenous interaction with hDaxx in a rhabdomyosarcoma tumor cell line . Therefore, these data support the model that fusion of FKHR to Pax3 not only adds a strong transactivation domain, but also deregulates transcriptional control of Pax3 by overriding the natural repressive effect of hDaxx.

Front Biosci, 1999 Jul 01, 4, D557 - 70
Activating oligomerization as intermediate level of signal transduction: analysis of protein-protein contacts and active sites in several glycolytic enzymes; Torshin I; A number of enzymes have inactive monomeric and active oligomeric forms . This suggests presence of definite interglobular contact -active site interaction in the enzymes . Although the phenomenon is widely studied in vitro as part of folding process the biological roles of the phenomenon, termed here as "activating oligomerization" are not clearly understood . In this work a procedure for analysis of protein-protein interactions was elaborated . Using spatial structures of several glycolytic enzymes potential role of kinase phosphorylation in regulation of oligomerization of the proteins as well as association of domains in a two-domain protein was assessed . In the enzymes 15-75% of kinase sites (mainly protein kinase C and casein kinase 2 sites) are placed in interglobular contact region(s) . Upon being phosphorylated these sites may prevent oligomer formation . In structures of all the enzymes definite evidences of connection between active site and interglobular contact were found . Two structural mechanisms of interglobular contact influence on the active site were proposed . In addition to known mechanism of oligomerization initiated by allosteric metabolites the influence may be also exerted through functional sequence overlap and/or interdomain contact stabilization mechanisms . Implications for regulation of enzyme cellular function(s), signal transduction and metabolic analysis are considered . It is concluded that activating oligomerization may represent an intermediate level of enzyme cellular regulation.

J Biol Chem, 1999 Jul 9, 274(28), 19949 - 56
Molecular cloning and characterization of a novel dual specificity phosphatase, MKP-5; Tanoue T et al.; A group of dual specificity protein phosphatases negatively regulates members of the mitogen-activated protein kinase (MAPK) superfamily, which consists of three major subfamilies, MAPK/extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p38 . Nine members of this group of dual specificity phosphatases have previously been cloned . They show distinct substrate specificities for MAPKs, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli . Here we have cloned and characterized a novel dual specificity phosphatase, which we have designated MKP-5 . MKP-5 is a protein of 482 amino acids with a calculated molecular mass of 52.6 kDa and consists of 150 N-terminal amino acids of unknown function, two Cdc25 homology 2 regions in the middle, and a C-terminal catalytic domain . MKP-5 binds to p38 and SAPK/JNK, but not to MAPK/ERK, and inactivates p38 and SAPK/JNK, but not MAPK/ERK . p38 is a preferred substrate . The subcellular localization of MKP-5 is unique; it is present evenly in both the cytoplasm and the nucleus . MKP-5 mRNA is widely expressed in various tissues and organs, and its expression in cultured cells is elevated by stress stimuli . These results suggest that MKP-5 is a novel type of dual specificity phosphatase specific for p38 and SAPK/JNK.

J Biol Chem, 1999 Jul 9, 274(28), 19799 - 806
The membrane association domain of RGS16 contains unique amphipathic features that are conserved in RGS4 and RGS5; Chen C et al.; Regulators of G protein signaling (RGS proteins) modulate G protein-mediated signaling pathways by acting as GTPase-activating proteins for Gi, Gq, and G12 alpha-subunits of heterotrimeric G proteins . Although it is known that membrane association is critical for the biological activities of many RGS proteins, the mechanism underlying this requirement remains unclear . We reported recently that the NH2 terminus of RGS16 is required for its function in vivo . In this study, we show that RGS16 lacking the NH2 terminus is no longer localized to the plasma membrane as is the wild type protein, suggesting that membrane association is important for biological function . The region of amino acids 7-32 is sufficient to confer the membrane-targeting activity, of which amino acids 12-30 are predicted to adopt an amphipathic alpha-helix . Site-directed mutagenesis experiments showed that the hydrophobic residues of the nonpolar face of the helix and the strips of positively charged side chains positioned along the polar/nonpolar interface of the helix are crucial for membrane association . Subcellular fractionation by differential centrifugation followed by conditions that distinguish peripheral membrane proteins from integral ones indicate that RGS16 is a peripheral membrane protein . We show further that RGS16 membrane association does not require palmitoylation . Our results, together with other recent findings, have defined a unique membrane association domain with amphipathic features . We believe that these structural features and the mechanism of membrane association of RGS16 are likely to apply to the homologous domains in RGS4 and RGS5.

Syst Appl Microbiol, 1999 May, 22(2), 229 - 36
Four new species in Lipomyces; van der Walt JP et al.; For new species in Lipomyces are described . In terms of nuclear genome comparison the genus Lipomyces comprises three species-clusters . A key to the nine species now assigned to the genus, is given . The delimitation of species by (i) rRNA nucleotide sequence analyses of the 18S and 25S subunits and (II) nDNA homology determinations, do not invariably lead to the recognition of congruent taxa . The family of the Lipomycetaceae, nevertheless provides an illuminative model for phylogenetic studies in terms of more appropriate ribosomal nucleotide sequence analyses of both its teleomorphic and anamorphic members.

Genes Dev, 1999 Jun 15, 13(12), 1540 - 52
A protein phosphatase functions to recycle RNA polymerase II; Cho H et al.; Transcription is regulated by the state of phosphorylation of a heptapeptide repeat known as the carboxy-terminal domain (CTD) present in the largest subunit of RNA polymerase II (RNAPII) . RNAPII that associates with transcription initiation complexes contains an unphosphorylated CTD, whereas the elongating polymerase has a phosphorylated CTD . Transcription factor IIH has a kinase activity specific for the CTD that is stimulated by the formation of a transcription initiation complex . Here, we report the isolation of a cDNA clone encoding a 150-kD polypeptide, which, together with RNAPII, reconstitutes a highly specific CTD phosphatase activity . Functional analysis demonstrates that the CTD phosphatase allows recycling of RNAPII . The phosphatase dephosphorylates the CTD allowing efficient incorporation of RNAPII into transcription initiation complexes, which results in increased transcription . The CTD phosphatase was found to be active in ternary elongation complexes . Moreover, the phosphatase stimulates elongation by RNAPII; however, this function is independent of its catalytic activity.

J Biol Chem, 1999 Jul 2, 274(27), 19441 - 6
Ubc9 interacts with the androgen receptor and activates receptor-dependent transcription; Poukka H et al.; Ubc9, a homologue of the class E2 ubiquitin-conjugating enzymes, has recently been shown to catalyze conjugation of a small ubiquitin-like molecule-1 (SUMO-1) to a variety of target proteins . SUMO-1 modifications have been implicated in the targeting of proteins to the nuclear envelope and certain intranuclear structures and in converting proteins resistant to ubiquitin-mediated degradation . In the present work, we find that Ubc9 interacts with the androgen receptor (AR), a member of the steroid receptor family of ligand-activated transcription factors . In transiently transfected COS-1 cells, AR-dependent but not basal transcription is enhanced by the coexpression of Ubc9 . The N-terminal half of the AR hinge region containing the C-terminal part of the bipartite nuclear localization signal is essential for the interaction with Ubc9 . Deletion of this part of the nuclear localization signal, which does not completely prevent the transfer of AR to the nucleus, abolishes the AR-Ubc9 interaction and attenuates the transcriptional response to cotransfected Ubc9 . The C93S substitution of Ubc9, which prevents SUMO-1 conjugation by abrogating the formation of a thiolester bond between SUMO-1 and Ubc9, does not influence the capability of Ubc9 to stimulate AR-dependent transactivation, implying that Ubc9 is able to act as an AR coregulator in a fashion independent of its ability to catalyze SUMO-1 conjugation.

Cell Mol Life Sci, 1999 May, 55(5), 707 - 34
SNAREs and SNARE regulators in membrane fusion and exocytosis; Gerst JE; Eukaryotes have a remarkably well-conserved apparatus for the trafficking of proteins between intracellular compartments and delivery to their target organelles . This apparatus comprises the secretory (or 'protein export') pathway, which is responsible for the proper processing and delivery of proteins and lipids, and is essential for the derivation and maintenance of those organelles . Protein transport between intracellular compartments is mediated by carrier vesicles that bud from one organelle and fuse selectively with another . Therefore, organelle-specific trafficking of vesicles requires specialized proteins that regulate vesicle transport, docking and fusion . These proteins are generically termed SNAREs and comprise evolutionarily conserved families of membrane-associated proteins (i.e . the synaptobrevin/VAMP, syntaxin and SNAP-25 families) which mediate membrane fusion . SNAREs act at all levels of the secretory pathway, but individual family members tend to be compartment-specific and, thus, are thought to contribute to the specificity of docking and fusion events . In this review, we describe the different SNARE families which function in exocytosis, as well as discuss the role of possible negative regulators (e.g . 'SNARE-masters') in mediating events leading to membrane fusion . A model to illustrate the dynamic cycling of SNAREs between fusion-incompetent and fusion-competent states, called the SNARE cycle, is presented.

Development, 1999 Jun, 126(14), 3159 - 70
XCtBP is a XTcf-3 co-repressor with roles throughout Xenopus development; Brannon M et al.; XTcf-3 is an HMG box transcription factor that mediates Xenopus dorsal-ventral axis formation . As a Wnt pathway effector, XTcf-3 interacts with beta-catenin and activates the expression of the dorsal organizing gene siamois, while in the absence of beta-catenin, XTcf-3 functions as a transcriptional repressor . We show that XTcf-3 contains amino- and carboxy-terminal repressor domains and have identified a Xenopus member of the C-terminal Binding Protein family of transcriptional co-repressors (XCtBP) as the C-terminal co-repressor . We show that two XCtBP binding sites near the XTcf-3 carboxy-terminus are required for the interaction of XTcf-3 and XCtBP and for the transcriptional repression mediated by the XTcf-3 carboxy-terminal domain . By fusing the GAL4 activation domain to XCtBP we have generated an antimorphic protein, XCtBP/G4A, that activates siamois transcription through an interaction with endogenous XTcf-3 . Ectopic expression of XCtBP/G4A demonstrates that XCtBP functions in the regulation of head and notochord development . Our data support a role for XCtBP as a co-repressor throughout Xenopus development and indicate that XCtBP/G4A will be a useful tool in determining how XCtBP functions in various developmental processes.

Differentiation, 1999 Jun, 64(5), 247 - 54
Dictyostelium ribosomal protein genes and the elongation factor 1B gene show coordinate developmental regulation which is under post-transcriptional control; Agarwal AK et al.; Starvation for amino acids initiates the developmental program in the cellular slime mold, Dictyostelium discoideum {19, 20} . One of the earliest developmental events is the decline in ribosomal protein synthesis {2, 17, 29, 30} . The ribosomal protein mRNAs are excluded from polysomes with 20 min to 1 h following the removal of nutrients, and their mRNA levels decline sharply at about 9 h into the 24-h developmental cycle {28, 31, 35, 36} . It has been generally assumed that the decline in r-protein mRNA levels during late development reflected a decline in the transcription rate {12, 32, 43} . Here we demonstrate that this is not the case . The transcription rates of three ribosomal protein genes, rpL11, rpL23 and rpS9 as well as an elongation factor 1B gene have been determined during growth and development in Dictyostelium . Throughout growth and development the transcription rate of the ribosomal protein genes remains relatively constant at 0.2%-0.5% of the rate of rRNA transcription while the elongation factor 1B gene is transcribed at 0.4%-0.6% of the rRNA rate . This low but constant transcription rate is in contrast to a spore coat protein gene Psp D, which is transcribed at 6% of the rRNA rate in late developing cells . The elongation factor 1B gene appears to be co-regulated with the ribosomal protein genes both in terms of its transcription rate and mRNA accumulation . Dictyostelium has been a popular model for understanding signal transduction and the growth to differentiation transition, thus it is of significance that the regulation of ribosome biosynthesis in Dictyostelium resembles that of higher eukaryotes in being regulated largely at the post-transcriptional level in response to starvation as opposed to yeasts where the regulation is largely transcriptional {27}.

Dev Biol, 1999 Jul 1, 211(1), 109 - 23
Spag4, a novel sperm protein, binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme; Shao X et al.; Outer dense fibers are structures unique to the sperm tail . No definite function for these fibers has been found, but they may play a role in motility and provide elastic recoil . Their composition had been described before, but only two of the fiber proteins, Odf1 and Odf2, are cloned . We cloned Odf2 by virtue of its functional and specific interaction with Odf1, which, we show, is mediated by a leucine zipper . Further work demonstrated that the 84-kDa Odf2 protein localizes to both the cortex and the medulla of the fibers, whereas the 27-kDa Odf1 protein is present only in the medulla . Here we report the cloning and characterization of a new Odf1-interacting protein, Spag4 . Spag4 mRNA is spermatid specific, and the 49-kDa Spag4 protein complexes specifically with Odf1, but not Odf2, mediated by a leucine zipper . It also self-associates . In contrast to Odf1 and Odf2, Spag4 protein localizes to two microtubule-containing spermatid structures . Spag4 is detectable in the transient manchette and it is associated with the axoneme in elongating spermatids and epididymal sperm . Our data suggest a role for Spag4 in protein localization to two major sperm tail structures .

Ann N Y Acad Sci, 1999 Apr 20, 873, 262 - 8
On the limits of ellipsoidal models when analyzing dielectric behavior of living cells . Emphasis on red blood cells; Gheorghiu E; The dielectric behavior of red blood cells is simulated by taking into account the real shape (consistent with microscopic observations) and the ellipsoids (prolate and oblate spheroids) having the same surface and volume . We have pointed out that the spectra of the imaginary versus the real part of polarizability, which can be directly derived from the measured data, provide quantitative insight to cell morphology . We emphasize that ellipsoidal approximation is fairly good for random oriented cells, but rather poor whenever oriented cells are measured . This fact is assumed to be the reason for the differences between the reported parameters derived from measurements on single cells and those from observations on (random oriented) cells in suspension.

EMBO J, 1999 Jun 15, 18(12), 3419 - 30
Aiolos transcription factor controls cell death in T cells by regulating Bcl-2 expression and its cellular localization; Romero F et al.; We searched for proteins that interact with Ras in interleukin (IL)-2-stimulated or IL-2-deprived cells, and found that the transcription factor Aiolos interacts with Ras . The Ras-Aiolos interaction was confirmed in vitro and in vivo by co-immunoprecipitation . Indirect immunofluorescence shows that IL-2 controls the cellular distribution of Aiolos and induces its tyrosine phosphorylation, required for dissociation from Ras . We also identified functional Aiolos-binding sites in the Bcl-2 promoter, which are able to activate the luciferase reporter gene . Mutation of Aiolos-binding sites within the Bcl-2 promoter inhibits transactivation of the reporter gene luciferase, suggesting direct control of Bcl-2 expression by Aiolos . Co-transfection experiments confirm that Aiolos induces Bcl-2 expression and prevents apoptosis in IL-2-deprived cells . We propose a model for the regulation of Bcl-2 expression via Aiolos.

EMBO J, 1999 Jun 15, 18(12), 3404 - 18
RYBP, a new repressor protein that interacts with components of the mammalian Polycomb complex, and with the transcription factor YY1; Garcia E et al.; The products of the Polycomb group (PcG) of genes are necessary for the maintenance of transcriptional repression of a number of important developmental genes, including the homeotic genes . A two-hybrid screen was used to search for putative new members of the PcG of genes in mammals . We have identified a new Zn finger protein, RYBP, which interacts directly with both Ring1 proteins (Ring1A and Ring1B) and with M33, two mutually interacting sets of proteins of the mammalian Polycomb complex . Ring1 binds RYBP and M33 through the same C-terminal domain, whereas the RYBP-M33 interaction takes place through an M33 domain not involved in Ring1 binding . RYBP also interacts directly with YY1, a transcription factor partially related to the product of the Drosophila pleiohomeotic gene . In addition, we show here that RYBP acts as a transcriptional repressor in transiently transfected cells . Finally, RYBP shows a dynamic expression pattern during embryogenesis which initially overlaps partially that of Ring1A in the central nervous system, and later becomes ubiquitous . Taken together, these data suggest that RYBP may play a relevant role in PcG function in mammals.

J Am Acad Dermatol, 1999 Jun, 40(6 Pt 2), S21 - 6
Onychomycosis: therapeutic update; Scher RK; Onychomycosis is a common disease of the nail unit caused by dermatophytes, yeasts, and molds . In more than 80% of cases, onychomycosis is caused by the dermatophytes Trichophyton rubrum and Trichophyton mentagrophytes . The prevalence of onychomycosis in the world's population is 2% to 18% or higher and accounts for approximately 50% of all nail disorders . Until recently, available therapies were inadequate because of low cure rates, high relapse rates, and often dangerous side effects . An increased understanding of nail pharmacokinetics has led to the development of safer, more effective systemic therapies for onychomycosis, such as itraconazole, fluconazole, and terbinafine . These new oral antifungal agents allow shorter periods of treatment, provide rapid efficacy, and may improve patient compliance and attitudes regarding therapy . Treatment selection will depend on several factors, including appropriate spectrum of activity, adverse effects, and potential drug interactions plus patient preferences for specific dosing regimens.

J Am Acad Dermatol, 1999 Jun, 40(6 Pt 2), S3 - 8
Diagnosis of onychomycosis made simple; Ellis DH; Because onychomycosis requires long-term systemic therapy, it is essential to diagnose the infection accurately . Although in theory this diagnosis should be simple, results achieved in practice are uneven . Clinicians should be aware of the need to collect an adequate specimen and of the possible need for repeat collections . Direct microscopic techniques for examination of nail scrapings include 10% potassium hydroxide (KOH) with Parker ink, 10% KOH with dimethyl sulfoxide, and 10% KOH with Calcofluor white . When interpreting fungal culture results, it should be noted that negative results are frequent and that contaminant yeasts and molds are common, but that nondermatophytes such as Candida, Scopulariopsis, and Scytalidium can infrequently cause onychomycosis.

Biochim Biophys Acta, 1999 Jun 28, 1428(1), 99 - 105
Single micro electrode dielectrophoretic tweezers for manipulation of suspended cells and particles; Schnelle T et al.; Cells or particles in aqueous suspension close to a single capacitively coupled micro electrode (CCME) driven with high frequency electric fields experience dielectrophoretic forces . The effects near the CCME can be used for trapping and manipulation of single cells using externally metallised glass pipettes and might be used to develop a microscope based on force or capacitance measurements in conductive media.

Microbiol Mol Biol Rev, 1999 Jun, 63(2), 405 - 45
Formation of mRNA 3' ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis; Zhao J et al.; Formation of mRNA 3' ends in eukaryotes requires the interaction of transacting factors with cis-acting signal elements on the RNA precursor by two distinct mechanisms, one for the cleavage of most replication-dependent histone transcripts and the other for cleavage and polyadenylation of the majority of eukaryotic mRNAs . Most of the basic factors have now been identified, as well as some of the key protein-protein and RNA-protein interactions . This processing can be regulated by changing the levels or activity of basic factors or by using activators and repressors, many of which are components of the splicing machinery . These regulatory mechanisms act during differentiation, progression through the cell cycle, or viral infections . Recent findings suggest that the association of cleavage/polyadenylation factors with the transcriptional complex via the carboxyl-terminal domain of the RNA polymerase II (Pol II) large subunit is the means by which the cell restricts polyadenylation to Pol II transcripts . The processing of 3' ends is also important for transcription termination downstream of cleavage sites and for assembly of an export-competent mRNA . The progress of the last few years points to a remarkable coordination and cooperativity in the steps leading to the appearance of translatable mRNA in the cytoplasm.

EMBO J, 1999 Jun 1, 18(11), 2991 - 3006
Recruitment of an alternatively spliced form of synaptojanin 2 to mitochondria by the interaction with the PDZ domain of a mitochondrial outer membrane protein; Nemoto Y et al.; Synaptojanin 1 is an inositol 5'-phosphatase highly enriched in nerve terminals with a putative role in recycling of synaptic vesicles . We have previously described synaptojanin 2, which is more broadly expressed as multiple alternatively spliced forms . Here we have identified and characterized a novel mitochondrial outer membrane protein, OMP25, with a single PDZ domain that specifically binds to a unique motif in the C-terminus of synaptojanin 2A . This motif is encoded by the exon sequence specific to synaptojanin 2A . OMP25 mRNA is widely expressed in rat tissues . OMP25 is localized to the mitochondrial outer membrane via the C-terminal transmembrane region, with the PDZ domain facing the cytoplasm . Overexpression of OMP25 results in perinuclear clustering of mitochondria in transfected cells . This effect is mimicked by enforced expression of synaptojanin 2A on the mitochondrial outer membrane, but not by the synaptojanin 2A mutants lacking the inositol 5'-phosphatase domain . Our findings provide evidence that OMP25 mediates recruitment of synaptojanin 2A to mitochondria and that modulation of inositol phospholipids by synaptojanin 2A may play a role in maintenance of the intracellular distribution of mitochondria.

Am J Health Syst Pharm, 1999 May 1, 56(9), 865 - 71
Management of toenail onychomycosis; Tom CM et al.; The treatment of toenail onychomycosis is reviewed . Onychomycosis contributes to 40% of all nail disorders and appears to be increasing in frequency . Mycotic nail infections are usually caused by dermatophytes, yeasts, and nondermatophyte molds . Most cases of toenail onychomycosis are caused by dermatophytes . Mycotic nail infections do not always resolve spontaneously and may have a substantial impact on the patient's quality of life . Current treatment modalities for onychomycosis include surgery, topical antifungals, and oral antifungals . Surgery is generally not recommended as first-line therapy . Broad-spectrum topical and oral antifungal agents are the most frequently used treatments . Topical treatment is well tolerated but is usually not effective because of poor patient compliance and inadequate penetration of the nail . Oral antifungals are more successful but carry greater risks . Griseofulvin and ketoconazole have been oral antifungals traditionally used for onychomycosis, but these agents are associated with relatively low cure rates . Itraconazole and terbinafine are both safe and effective first-line agents, with reported overall cure rates of 50-90% for dermatophyte-related onychomycosis . Intermittent oral antifungal therapy may reduce the risk of systemic adverse effects and the cost of therapy; more study of this approach is needed . Oral antifungal agents offer patients with toenail onychomycosis greater likelihood of a cure than topical antifungals, but oral therapy carries greater risks and requires closer monitoring.

Nature, 1999 May 13, 399(6732), 155 - 9
Ca2+/calmodulin binds to and modulates P/Q-type calcium channels; Lee A et al.; Neurotransmitter release at many central synapses is initiated by an influx of calcium ions through P/Q-type calcium channels, which are densely localized in nerve terminals . Because neurotransmitter release is proportional to the fourth power of calcium concentration, regulation of its entry can profoundly influence neurotransmission . N- and P/Q-type calcium channels are inhibited by G proteins, and recent evidence indicates feedback regulation of P/Q-type channels by calcium . Although calcium-dependent inactivation of L-type channels is well documented, little is known about how calcium modulates P/Q-type channels . Here we report a calcium-dependent interaction between calmodulin and a novel site in the carboxy-terminal domain of the alpha1A subunit of P/Q-type channels . In the presence of low concentrations of intracellular calcium chelators, calcium influx through P/Q-type channels enhances channel inactivation, increases recovery from inactivation and produces a long-lasting facilitation of the calcium current . These effects are prevented by overexpression of a calmodulin-binding inhibitor peptide and by deletion of the calmodulin-binding domain . Our results reveal an unexpected association of Ca2+/calmodulin with P/Q-type calcium channels that may contribute to calcium-dependent synaptic plasticity.

J Biol Chem, 1999 May 28, 274(22), 15901 - 7
Identification of nuclear receptor corepressor as a peroxisome proliferator-activated receptor alpha interacting protein; Dowell P et al.; Nuclear receptor corepressor (NCoR) was demonstrated to interact strongly with peroxisome proliferator-activated receptor alpha (PPARalpha), and PPARalpha ligands suppressed this interaction . In contrast to the interaction of PPARalpha with the coactivator protein, p300, association of the receptor with NCoR did not require any part of the PPARalpha ligand binding domain . NCoR was found to suppress PPARalpha-dependent transcriptional activation in the context of a PPARalpha.retinoid X receptor alpha (RXRalpha) heterodimeric complex bound to a peroxisome proliferator-responsive element in human embryonic kidney 293 cells . This repression was reversed agonists of either receptor demonstrating a functional interaction between NCoR and PPARalpha.RXRalpha heterodimeric complexes in mammalian cells . NCoR appears to influence PPARalpha signaling pathways and, therefore, may modulate tissue responsiveness to peroxisome proliferators.

Biotechnol Appl Biochem, 1999 Jun, 29 ( Pt 3), 207 - 12
Cofactor recycling in a coupled enzyme oxidation-reduction reaction: conversion of omega-oxo-fatty acids into omega-hydroxy and dicarboxylic acids; Nunez A et al.; Aldehydes are reduced to alcohols by the enzyme alcohol dehydrogenase (ADH), whereas the enzyme aldehyde dehydrogenase (AldDH) oxidizes aldehydes to carboxylic acids . ADH and AldDH require, respectively, the reduced and oxidized forms of the cofactor NAD (NAD+/NADH) . By combining both oxidation and reduction reactions into one process, it is possible to produce alcohols and carboxylic acids simultaneously from aldehydes by continuous recycling of the NAD+/NADH cofactor . However, both enzymes need to be active within the same pH region and buffer system . To test this hypothesis, the pH profile (Vmax and Vmax/Km) as well as the pKa of the prototropic groups involved in catalysis for both dehydrogenases were determined using (Z,Z)-nona-2,4-dienal as a model substrate . The pH profile (Vmax and Vmax/Km) of both enzymes overlapped in the pH range of 6-8 in potassium phosphate buffer . When the coupled enzyme system was used at pH 7 with 10% NAD+ cofactor, over 90% of the starting aldehyde was converted to its corresponding acid and alcohol derivatives in a 1:1 ratio . The sequential action of the enzymes lipoxygenase and hydroperoxide lyase converts polyunsaturated fatty acids to aldehydic fatty acids . The products arising from the oxidation or reduction of the aldehydic functionality are of industrial interest . It was found that 13-oxo-9-(Z),11-(E)-tridecadienoic acid, the product of the sequential reaction of soya bean lipoxygenase and hydroperoxide lyase from Chlorella pyrenoidosa on linoleic acid, is also a substrate in this coupled enzyme system.

J Biol Chem, 1999 May 21, 274(21), 14942 - 7
A model for dynamin self-assembly based on binding between three different protein domains; Smirnova E et al.; Dynamin is a 100-kDa GTPase that assembles into multimeric spirals at the necks of budding clathrin-coated vesicles . We describe three different intramolecular binding interactions that may account for the process of dynamin self-assembly . The first binding interaction is the dimerization of a 100-amino acid segment in the C-terminal half of dynamin . We call this segment the assembly domain, because it appears to be critical for multimerization . The second binding interaction occurs between the assembly domain and the N-terminal GTPase domain . The strength of this interaction is controlled by the nucleotide-bound state of the GTPase domain, as shown with mutations in GTP binding motifs and in vitro binding experiments . The third binding interaction occurs between the assembly domain and a segment that we call the middle domain . This is the segment between the N-terminal GTPase domain and the pleckstrin homology domain . The three different binding interactions suggest a model in which dynamin molecules first dimerize . The dimers are then linked into a chain by a second binding reaction . The third binding interaction might connect adjacent rungs of the spiral.

EMBO J, 1999 May 17, 18(10), 2812 - 22
Transcriptional cofactors of the FOG family interact with GATA proteins by means of multiple zinc fingers; Fox AH et al.; Friend of GATA-1 (FOG-1) is a zinc finger protein that has been shown to interact physically with the erythroid DNA-binding protein GATA-1 and modulate its transcriptional activity . Recently, two new members of the FOG family have been identified: a mammalian protein, FOG-2, that also associates with GATA-1 and other mammalian GATA factors; and U-shaped, a Drosophila protein that interacts with the Drosophila GATA protein Pannier . FOG proteins contain multiple zinc fingers and it has been shown previously that the sixth finger of FOG-1 interacts specifically with the N-finger but not the C-finger of GATA-1 . Here we show that fingers 1, 5 and 9 of FOG-1 also interact with the N-finger of GATA-1 and that FOG-2 and U-shaped also contain multiple GATA-interacting fingers . We define the key contact residues and show that these residues are highly conserved in GATA-interacting fingers . We examine the effect of selectively mutating the four interacting fingers of FOG-1 and show that each contributes to FOG-1's ability to modulate GATA-1 activity . Finally, we show that FOG-1 can repress GATA-1-mediated activation and present evidence that this ability involves the recently described CtBP co-repressor proteins that recognize all known FOG proteins.

J Clin Microbiol, 1999 Jun, 37(6), 1846 - 51
Rapid identification of fungi by using the ITS2 genetic region and an automated fluorescent capillary electrophoresis system; Turenne CY et al.; Invasive fungal disease often plays an important role in the morbidity and mortality of immunocompromised patients . The poor sensitivity of current fungal blood culture and histological practices has led to the development of highly sensitive and specific molecular techniques, such as the PCR . Sequence variability of the internal transcribed spacer 2 (ITS2) region of fungi is potentially useful in rapid and accurate diagnosis of clinical fungal isolates . PCR with fungus-specific primers targeted toward conserved sequences of the 5.8S and 28S ribosomal DNA (rDNA) results in amplification of the species-specific ITS2 regions, which are variable in amplicon length . We have made use of the ABI PRISM 310 genetic analyzer and the ABI PRISM 310 GeneScan analysis software for the determination of variable size differences of the ITS2 region of clinically important fungi, including Candida and non-Candida yeasts, Aspergillus species, and a variety of dermatophytes . No cross-reaction occurred when samples were tested against human and bacterial genomic DNA . We have found that most clinically significant fungal isolates can be differentiated by this method, and it therefore serves to be a promising tool for the rapid (<7 h) diagnosis of fungemia and other invasive fungal infections.

Mol Microbiol, 1999 May, 32(3), 471 - 83
Neutral trehalases catalyse intracellular trehalose breakdown in the filamentous fungi Aspergillus nidulans and Neurospora crassa; d'Enfert C et al.; A cAMP-activatable Ca2+-dependent neutral trehalase was identified in germinating conidia of Aspergillus nidulans and Neurospora crassa . Using a PCR approach, A . nidulans and N . crassa genes encoding homologues of the neutral trehalases found in several yeasts were cloned and sequenced . Disruption of the AntreB gene encoding A . nidulans neutral trehalase revealed that it is responsible for intracellular trehalose mobilization at the onset of conidial germination, and that this phenomenon is partially involved in the transient accumulation of glycerol in the germinating conidia . Although trehalose mobilization is not essential for the completion of spore germination and filamentous growth in A . nidulans, it is required to achieve wild-type germination rates under carbon limitation, suggesting that intracellular trehalose can partially contribute the energy requirements of spore germination . Furthermore, it was shown that trehalose accumulation in A . nidulans can protect germinating conidia against an otherwise lethal heat shock . Because transcription of the treB genes is not increased after a heat shock but induced upon heat shock recovery, it is proposed that, in filamentous fungi, mobilization of trehalose during the return to appropriate growth is promoted by transcriptional and post-translational regulatory mechanisms, in particular cAMP-dependent protein kinase-mediated phosphorylation.

Genes Cells, 1999 Feb, 4(2), 111 - 22
Kinase-independent activity of Cdc2/cyclin A prevents the S phase in the Drosophila cell cycle; Hayashi S et al.; BACKGROUND: The Cdc2-dependent inhibition of S phase is required in G2 for the correct ordering of the S and M phases in yeasts and Drosophila . This function of Cdc2 has been ascribed to its ability to phosphorylate replication factors to prevent the assembly of a preinitiation complex at the origin of replication . Whether this is the sole mechanism of S phase inhibition by Cdc2 in higher metazoans is not known because the pleiotropic functions of this essential cell cycle regulator make genetic analysis difficult . RESULTS: We show that Cdc2 co-expressed with Cyclin A inhibits the S phase in Drosophila salivary glands and diploid abdominal histoblasts . A kinase defective mutant of Cdc2 failed to promote mitosis, but was still able to inhibit the S phase with the same efficiency as the wild-type protein . In addition, Cdc2 and Cyclin A cooperatively inhibit transcriptional activation by the essential S phase regulator E2F . Cdc2 binds to E2F in vitro, and post-transcriptionally promotes its accumulation in vivo . Furthermore, the inhibitory effect of Cdc2 on S phase is overridden by E2F . CONCLUSION: The inhibition of S phase by Cdc2 is achieved in part by a kinase-independent mechanism, which is likely to be mediated by the inhibition of E2F.

J Biol Chem, 1999 May 14, 274(20), 14474 - 81
Biosynthesis of osteogenic growth peptide via alternative translational initiation at AUG85 of histone H4 mRNA; Bab I et al.; The osteogenic growth peptide (OGP) is an extracellular mitogen identical to the histone H4 (H4) COOH-terminal residues 90-103, which regulates osteogenesis and hematopoiesis . By Northern analysis, OGP mRNA is indistinguishable from H4 mRNA . Indeed, cells transfected with a construct encoding {His102}H4 secreted the corresponding {His13}OGP . These results suggest production of OGP from H4 genes . Cells transfected with H4-chloramphenicol acetyltransferase (CAT) fusion genes expressed both "long" and "short" CAT proteins . The short CAT was retained following an ATG --> TTG mutation of the H4 ATG initiation codon, but not following mutation of the in-frame internal ATG85 codon, which, unlike ATG1, resides within a perfect context for translational initiation . These results suggest that a PreOGP is translated starting at AUG85 . The translational initiation at AUG85 could be inhibited by optimizing the nucleotide sequence surrounding ATG1 to maximally support upstream translational initiation, thus implicating leaky ribosomal scanning in usage of the internal AUG . Conversion of the predicted PreOGP to OGP was shown in a cell lysate system using synthetic {His102}H4-(85-103) as substrate . Together, our results demonstrate that H4 gene expression diverges at the translational level into the simultaneous parallel production of both H4, a nuclear structural protein, and OGP, an extracellular regulatory peptide.

Nippon Ishinkin Gakkai Zasshi, 1999, 40(2), 59 - 62
{Taxonomy of genus Malassezia: progress and problems}; Makimura K et al.; The genus Malassezia is composed of lipophilic basidiomycetous yeasts which were recently shown to consist of seven species, one lipid-independent species, M . pachydermatis and six lipid-dependent species, M . furfur, M . sympodialis, M . globosa, M . obtusa, M . restricta and M . slooffiae . Based on this classification, we will be able to analyze pathogenicity or relationship between Malassezia-related diseases and each species.

Chemosphere, 1999 Jun, 38(13), 3041 - 50
Degradation of three phenylurea herbicides (chlortoluron, isoproturon and diuron) by micromycetes isolated from soil; Khadrani A et al.; As part of a study conducted on the fate of xenobiotics in the environment, a selection of 100 strains of micromycetes (Ascomycetes, Basidiomycetes and Yeasts) have been cultivated in liquid synthetic medium with 3 phenylurea herbicides: chlortoluron and isoproturon (100mg L-1) and diuron (20mg L-1) . While 17 strains depleted isoproturon over 50% only 4 depleted diuron and 2 chlortoluron at the same level . The best results were obtained with Bjerkandera adusta and Oxysporus sp which were the most efficient towards the 3 substrates . After 2 weeks Bjerkandera adusta depleted chlortoluron 98%, diuron 92% and isoproturon 88%.

J Exp Med, 1999 Apr 19, 189(8), 1207 - 16
Targeted gene disruption reveals an adhesin indispensable for pathogenicity of Blastomyces dermatitidis; Brandhorst TT et al.; Systemic fungal infections are becoming more common and difficult to treat, yet the pathogenesis of these infectious diseases remains poorly understood . In many cases, pathogenicity can be attributed to the ability of the fungi to adhere to target tissues, but the lack of tractable genetic systems has limited progress in understanding and interfering with the offending fungal products . In Blastomyces dermatitidis, the agent of blastomycosis, a respiratory and disseminated mycosis of people and animals worldwide, expression of the putative adhesin encoded by the WI-1 gene was investigated as a possible virulence factor . DNA-mediated gene transfer was used to disrupt the WI-1 locus by allelic replacement, resulting in impaired binding and entry of yeasts into macrophages, loss of adherence to lung tissue, and abolishment of virulence in mice; each of these properties was fully restored after reconstitution of WI-1 by means of gene transfer . These findings establish the pivotal role of WI-1 in adherence and virulence of B . dermatitidis yeasts . To our knowledge, they offer the first example of a genetically proven virulence determinant among systemic dimorphic fungi, and underscore the value of reverse genetics for studies of pathogenesis in these organisms.

Minerva Stomatol, 1998 Dec, 47(12), 665 - 71
Antifungal activity of chlorhexidine containing mouthrinses . An in vitro study; Pizzo G et al.; BACKGROUND: Oral candidosis has emerged as an increasing problem in immunocompromised patients because oral cavity represents an important port of entry for systemic fungal infections . METHODS: The aim of the present study was to investigate the in vitro antifungal activity of 5 commercial mouthrinses containing chlorhexidine (CHX) . The Minimum Fungicidal Concentration (MFC) against six species of yeasts was determined by a broth macrodilution method . The kill-time of mouthrinses at half the concentration of the commercial formulations was also determined . RESULTS: MFCs were achieved with each mouthrinses against all the organisms under test . However, significant differences in MFC values were found for Ebur Os in comparison with Dentosan, Corsodyl and Plak out (p < 0.001) . Kill-times of Corsodyl and Dentosan were less than or equal to 120 sec with all the species of yeasts, except Torulopsis glabrata . Significant differences were found in kill-time values between Dentosan and Broxo Din only (p < 0.001) . CONCLUSIONS: These results suggest that CHX-containing mouthrinses may represent an appropriate alternative to conventional antifungal drugs in the management of oral candidosis . Effectiveness in preventing systemic fungal infections in immunocompromised patients requires further in vivo studies.

Nat Struct Biol, 1999 Apr, 6(4), 388 - 97
Autoinhibition by an internal nuclear localization signal revealed by the crystal structure of mammalian importin alpha; Kobe B; Importin alpha is the nuclear import receptor that recognizes classical monopartite and bipartite nuclear localization signals (NLSs) . The structure of mouse importin alpha has been determined at 2.5 A resolution . The structure shows a large C-terminal domain containing armadillo repeats, and a less structured N-terminal importin beta-binding domain containing an internal NLS bound to the NLS-binding site . The structure explains the regulatory switch between the cytoplasmic, high-affinity form, and the nuclear, low-affinity form for NLS binding of the nuclear import receptor predicted by the current models of nuclear import . Importin beta conceivably converts the low- to high-affinity form by binding to a site overlapping the autoinhibitory sequence . The structure also has implications for understanding NLS recognition, and the structures of armadillo and HEAT repeats.

Oral Dis, 1998 Dec, 4(4), 260 - 7
The postantifungal effect (PAFE) of antimycotics on oral C . albicans isolates and its impact on candidal adhesion; Ellepola AN et al.; OBJECTIVE: Postantifungal effect (PAFE) is defined as the suppression of growth that persists following limited exposure of yeasts to antimycotics and subsequent removal of the drug . As there are no data on the PAFE of oral C . albicans isolates the main aim of this investigation was to measure the PAFE of 10 oral isolates of C . albicans following limited exposure (1 h) to five antifungal drugs, including nystatin which has not been previously used in PAFE assays . A secondary aim of the study was to evaluate the biological significance of PAFE, using a nystatin pre-exposed isolate of C . albicans and observing its adherence to denture acrylic surfaces, during the PAFE period . DESIGN: A total of 10 oral isolates of C . albicans were examined for the presence of the PAFE after 1 h exposure to five antifungal drugs, nystatin, amphotericin B, 5-fluorocytosine, ketoconazole and fluconazole . PAFE was automatically assessed with the help of a Spectramax machine which utilizes the principle of periodic turbidometric assessment of growth rates at a given temperature over a given period . The data thus collected are automatically processed in a graphic format as a computer printout . The PAFE was determined as the difference in time (h) required for growth of the drug-free control and the drug-exposed test cultures to increase to 0.05 absorbance level following removal of the antifungal agent (by repeated washing) . The adhesion of the single isolate to denture acrylic following limited exposure to nystatin was assessed by a previously described in vitro adhesion assay . RESULTS: Significant PAFE were observed for nystatin, amphotericin-B and 5-fluorocytosine . A marginal PAFE was observed for ketoconazole and little or none for fluconazole . The mean duration of the PAFE of nystatin, amphotericin-B, 5-fluorocytosine, ketoconazole and fluconazole were 2.89 (+/- 0.27) h, 2.83 (+/- 0.23) h, 3.18 (+/- 0.31) h, 0.65 (+/- 0.11) h and 0.16 (+/- 0.06) h, respectively . The mean percentage reduction of adhesion of oral C . albicans BU47204 to denture acrylic during the PAFE period following exposure to nystatin for 10, 30, 50, 70 and 90 min was 9.12%, 61.73%, 65.99%, 82.16% and 83.14%, respectively . CONCLUSIONS: These in vitro findings imply that even a short period of exposure to antifungals may result in modulation of the growth and the virulent attributes of C . albicans, which however is largely dictated by the antimycotic agent in question . Whether such mechanisms operate in vivo needs to be clarified by further studies.

J Neurochem, 1999 Apr, 72(4), 1725 - 34
Subunit assembly and domain analysis of electrically silent K+ channel alpha-subunits of the rat Kv9 subfamily; Stocker M et al.; Alpha-subunits of the voltage-gated potassium channel (Kv) subfamily Kv9 show no channel activity after homomultimeric expression in heterologous expression systems . This report shows that heteromultimeric expression of rKv9.1 and rKv9.3 specifically suppresses the currents mediated by alpha-subunits of the Kv2 and Kv3 subfamilies but does not affect currents mediated by alpha-subunits of the Kv1 and Kv4 subfamilies . To understand the molecular basis of the electrical silence of Kv9 homomultimeric channels, crucial functional domains (amino and carboxy terminus, S4 segment, and pore region) were exchanged between Kv9 alpha-subunits and rKv1.3 . Electrophysiological studies of these chimeras revealed that the pore region is involved in determining the nonconductive behavior of homomultimeric Kv9 channels . This analysis was extended by protein interaction assays, aiming to identify the region of Kv9 subunits responsible for the specific suppression of rKv2.1- and rKv3.4-mediated currents . We could show that the amino-terminal domain of Kv9 alpha-subunits does not support homomultimeric assembly but interacts specifically with the rKv2.1 amino-terminal region . Conversely, the specific intersubfamily assembly of rKv3.4 with rKv9.1 or rKv9.3 is governed by the hydrophobic core and not the amino-terminal domain.

J Biol Chem, 1999 Apr 2, 274(14), 9771 - 7
Definition of a consensus transportin-specific nucleocytoplasmic transport signal; Bogerd HP et al.; The low cytoplasmic and high nuclear concentration of the GTP-bound form of Ran provides directionality for both nuclear protein import and export . Both import and export factors bind RanGTP directly, yet this interaction produces opposite effects; in the former case, RanGTP binding induces nuclear cargo release, whereas in the latter, RanGTP binding induces nuclear cargo assembly . Therefore, nuclear import and export receptors and their protein recognition sites are predicted to be distinct . Nevertheless, the approximately 38-amino acid M9 sequence present in heterogeneous nuclear ribonucleoprotein A1 has been reported to serve as both a nuclear localization signal and a nuclear export signal, even though only one protein, the nuclear import factor transportin, has been shown to bind M9 directly . We have used a combination of mutational randomization followed by selection for transportin binding to exhaustively define amino acids in M9 that are critical for transportin binding in vivo . As expected, the resultant approximately 12-amino acid transportin-binding consensus sequence is also predictive of nuclear localization signal activity . Surprisingly, however, this extensive mutational analysis failed to dissect M9 nuclear localization signal and nuclear export signal function . Nevertheless, transportin appears unlikely to be the M9 export receptor, as RanGTP can be shown to block M9 binding by transportin not only in vitro, but also in the nucleus in vivo . This analysis therefore predicts the existence of a nuclear export receptor distinct from transportin that nevertheless shares a common protein-binding site on heterogeneous nuclear ribonucleoprotein A1.

Mycoses, 1998, 41 Suppl 2, 74 - 7
{Synthesis of fluorochromes and pigments in Malassezia furfur by using tryptophan as the single source of nitrogen}; Mayser P et al.; A new minimal medium consisting only of L-tryptophan (L-trp) and a lipid source induced formation of brown pigmentation only in the species Malassezia furfur . Strains of the species M . sympodialis and M . pachydermatis failed to grow on this medium . Pigmentogenesis was already induced in M . furfur by 0.01 g% tryptophan, the pH optimum was pH = 5 . Alternative amino nitrogen sources given concurrently with trp suppressed pigmentogenesis . The extract of the culture exhibited remarkable fluorescence, and several indole derivatives with a broad spectrum of colors were detected by means of mass spectroscopy and NMR . This finding may have an impact on the clinical appearance of pityriasis versicolor, a very common skin disease caused by lipophilic yeasts of the genus Malassezia . We hypothesize that in pityriasis versicolor metabolic adaptation of Malassezia yeasts to altered nitrogen conditions on superficial skin might be of pathophysiological importance . Tryptophan as inductor of pigmentogenesis probably cumulates during excessive sweating, a well known manifestation factor of pityriasis versicolor.

Antonie Van Leeuwenhoek, 1998 Nov, 74(4), 271 - 81
Temperature and NaCl-tolerance of rock-inhabiting meristematic fungi; Sterflinger K; Black meristematic fungi together with lichens and cyanobacteria dominate the micro-flora of rock surfaces in arid and semi-arid environments of hot and cold deserts . This study shows that rock inhabiting meristematic fungi are extremely tolerant against high temperatures, desiccation and osmotic stress . Their temperature tolerance increases with increasing dehydration of the fungal thallus . Air dried mycelia of black yeasts stand temperatures up to 120 degrees C for at least 0.5 hours . As response to high temperatures multilayered cell walls are developed and trehalose is accumulated whereas the intracellular glycerol regulates the osmotic potential under NaCl stress . Strains from rock in moderate climate (North Germany) show the same tolerance than those isolated from the Mediterranean area . Hortaea werneckii--hitherto only described as agent of human Tinea nigra--is shown to be the most tolerant rock inhabiting species tested . Meristematic fungi cannot be pre-adapted to higher growth temperatures by increased incubation temperatures . Considering the results of this study the justification of the term 'stress' is discussed with regard to rock inhabiting fungi and their natural environment . Consequences for conservation treatments of monuments decayed by meristematic fungi are discussed on the basis of the ecophysiological properties of the fungi.

Trends Microbiol, 1999 Feb, 7(2), 67 - 71
Macrophages in host defense against Histoplasma capsulatum; Newman SL; Macrophages function in both innate and cell-mediated immunity in host defense against pathogenic fungi . They initially serve as a protected environment in which the primary fungal pathogen Histoplasma capsulatum multiplies and disseminates from the lung to other organs . Upon induction of cell-mediated immunity, cytokines activate macrophages to destroy the yeasts and thus remove them from the host.

Biochem Biophys Res Commun, 1999 Mar 24, 256(3), 456 - 61
Interaction of S-SCAM with neural plakophilin-related Armadillo-repeat protein/delta-catenin; Ide N et al.; Synaptic scaffolding molecule (S-SCAM) is a multiple PDZ domain-containing protein, which interacts with neuroligin, a cell adhesion molecule, and the NMDA receptor . In this study, we searched for S-SCAM-interacting proteins and obtained a neuralplakophilin-related armadillo-repeat protein (NPRAP)/delta-catenin . NPRAP/delta-catenin bound to the last PDZ domain of S-SCAM via its carboxyl-terminus in three different cell-free assay systems, was coimmunoprecipitated with S-SCAM from rat crude synaptosomes, and was localized at the excitatory synapses in rat hippocampal neurons . NPRAP/delta-catenin may be implicated in the molecular organization of synaptic junctions through the interaction with S-SCAM .

Curr Opin Genet Dev, 1999 Feb, 9(1), 62 - 8
The regulation of replication origin activation; Donaldson AD et al.; At the start of the cell-division programme, proteins must be assembled onto replication origins to establish competence for initiation of DNA synthesis . At the correct moment, other effectors must then coordinate appropriate firing of the various origins to control entry into and progress through S phase . These processes are key targets of cell-cycle control, and understanding their regulation will provide a deeper knowledge of the mechanisms controlling cell proliferation.

Curr Opin Genet Dev, 1999 Feb, 9(1), 76 - 80
Coordination of cell growth with cell division; Polymenis M et al.; Proliferating cells must increase their mass coordinately with cell division . Recent evidence suggests that coupling of cell growth with cell division might be achieved by making synthesis of activators of cell division particularly sensitive to the capacity of the cell's protein synthesis machinery.

Curr Opin Genet Dev, 1999 Feb, 9(1), 69 - 75
The spindle checkpoint; Amon A; Prior to sister-chromatid separation, the spindle checkpoint inhibits cell-cycle progression in response to a signal generated by mitotic spindle damage or by chromosomes that have not attached to microtubules . Recent work has shown that the spindle checkpoint inhibits cell-cycle progression by direct binding of components of the spindle checkpoint pathway to components of a specialized ubiquitin-conjugating system that is responsible for triggering sister-chromatid separation.

Bioorg Med Chem Lett, 1999 Jan 18, 9(2), 187 - 92
Catalytic activity of carboxypeptidase B and of carboxypeptidase Y with anisylazoformyl substrates; Mock WL et al.; Anisylazoformyllysine (CH3OC6H4-N = N-CO-Lys-OH) is rapidly hydrolyzed at the acyl-lysine linkage by the zinc-enzyme porcine carboxypeptidase B . The catalytic reaction is readily monitored spectrophotometrically by disappearance of the intense absorption (348.5 nm, epsilon 18400) of the azo chromophore, which chemically fragments after substrate cleavage . Carboxypeptidase Y has no activity toward this type of substrate.

Biochim Biophys Acta, 1999 Jan 11, 1429(2), 351 - 64
X-ray absorption spectroscopy of cadmium phytochelatin and model systems; Pickering IJ et al.; Higher plants, algae and some yeasts respond to potentially toxic heavy metals such as cadmium by synthesizing phytochelatins and related cysteine-rich polypeptides . We have used X-ray absorption spectroscopy to study the nature of cadmium binding in such peptides isolated from maize (Zea mays) exposed to low levels of cadmium, and in two synthetic cadmium-peptide complexes, Cd-(gamma-Glu-Cys)3Gly and Cd-(alpha-Glu-Cys)3Gly . We have used the synthetic ions {Cd(SPh)4}2-, {Cd4(SPh)10}2- and {S4Cd10(SPh)16}4-as crystallographically defined models for the cadmium site . The Cd K-edge extended X-ray absorption fine structure (EXAFS) data, together with the Cd K, LI, LII and LIII near-edge spectra, reveal a predominantly tetrahedral coordination of cadmium by sulfur in both the phytochelatin and synthetic peptide complexes . In particular, the Cd LIII-edge lacks a peak at 3534.9 e V which was found to be prominent for oxygen- or nitrogen-coordinated species . The Cd-S distance in the phytochelatin complex is 2.54 A . The Cd K-edge EXAFS does not show any isolated, well-defined Cd-Cd interactions; however, contrary to the conclusion of previous work, their absence is not necessarily indicative of isolated cadmium-thiolate ligation . Evidence from other studies suggests that high static disorder, combined with a large vibrational component, serve to effectively wash out this contribution to the EXAFS . The sulfur K-edge, moreover, shows a low-energy feature both in the phytochelatin and in the synthetic cadmium-peptide complexes which is consistent with sulfide bound in a cluster with cadmium as found for {S4Cd10(SPh)16}4- . This feature strongly suggests the presence of a polynuclear cadmium cluster in maize phytochelatin.

Med Mycol, 1998, 36 Suppl 1, 220 - 9
The role of Malassezia species in the ecology of human skin and as pathogens; Gueho E et al.; The new taxonomic structure of the lipophilic genus Malassezia was presented with key characteristics for the seven described species . Among techniques used for epidemiological surveys, the pulsed field gel electrophoresis (PFGE) was found to be of little value in contrast to randomly amplified polymorphic DNA (RAPD) . Immunological studies still yielded conflicting results but at least the immunomodulatory capacity of Malassezia yeasts appeared to be related to the cell wall lipids . A review of Malassezia infections together with the present consensus for their prevention and treatment was also made.

Med Mycol, 1998, 36 Suppl 1, 52 - 6
Molecular phylogeny and taxonomy of medically important fungi; De Hoog GS et al.; Some recent advances in study of molecular evolution and taxonomy of human pathogens are discussed . In systemic Onygenales as well as in Chaetothyriales, pathogenic species are phylogenetically intermingled with non-pathogenic taxa . When a teleomorph of Coccidioides immitis is eventually found, it is predicted to resemble Uncinocarpus, a genus otherwise comprising environmental species . In the dermatophytes, Trichophyton and Microsporum are paraphyletic, whereas Epidermophyton is polyphyletic . On the basis of 18S and ITS rDNA sequencing data, Exophiala anamorphs (black yeasts) are confirmed to be closely related to the ascomycete genus Capronia . The related neurotropic species Cladophialophora bantiana is remarkable in consistently having introns in its 18S rDNA gene.

Adv Biochem Eng Biotechnol, 1999, 64, 155 - 201
Protein glycosylation: implications for in vivo functions and therapeutic applications; Bhatia PK et al.; The glycosylation machinery in eukaryotic cells is available to all proteins that enter the secretory pathway . There is a growing interest in diseases caused by defective glycosylation, and in therapeutic glycoproteins produced through recombinant DNA technology route . The choice of a bioprocess for commercial production of recombinant glycoprotein is determined by a variety of factors, such as intrinsic biological properties of the protein being expressed and the purpose for which it is intended, and also the economic target . This review summarizes recent development and understanding related to synthesis of glycans, their functions, diseases, and various expression systems and characterization of glycans . The second section covers processing of N- and O-glycans and the factors that regulate protein glycosylation . The third section deals with in vivo functions of protein glycosylation, which includes protein folding and stability, receptor functioning, cell adhesion and signal transduction . Malfunctioning of glycosylation machinery and the resultant diseases are the subject of the fourth section . The next section covers the various expression systems exploited for the glycoproteins: it includes yeasts, mammalian cells, insect cells, plants and an amoeboid organism . Biopharmaceutical properties of therapeutic proteins are discussed in the sixth section . In vitro protein glycosylation and the characterization of glycan structures are the subject matters for the last two sections, respectively.

Protein Eng, 1998 Dec, 11(12), 1155 - 61
Sequence properties of GPI-anchored proteins near the omega-site: constraints for the polypeptide binding site of the putative transamidase; Eisenhaber B et al.; Glycosylphosphatidylinositol (GPI) anchoring is a common post-translational modification of extracellular eukaryotic proteins . Attachment of the GPI moiety to the carboxyl terminus (omega-site) of the polypeptide occurs after proteolytic cleavage of a C-terminal propeptide . In this work, the sequence pattern for GPI-modification was analyzed in terms of physical amino acid properties based on a database analysis of annotated proprotein sequences . In addition to a refinement of previously described sequence signals, we report conserved sequence properties in the regions omega - 11...omega - 1 and omega + 4...omega + 5 . We present statistical evidence for volume-compensating residue exchanges with respect to the positions omega - 1...omega + 2 . Differences between protozoan and metazoan GPI-modification motifs consist mainly in variations of preferences to amino acid types at the positions near the omega-site and in the overall motif length . The variations of polypeptide substrates are exploited to suggest a model of the polypeptide binding site of the putative transamidase, the enzyme catalyzing the GPI-modification . The volume of the active site cleft accommodating the four residues omega - 1...omega + 2 appears to be approximately 540 A3.

J Mol Biol, 1999 Feb 5, 285(5), 2105 - 17
NMR structure of the (52-96) C-terminal domain of the HIV-1 regulatory protein Vpr: molecular insights into its biological functions; Schuler W et al.; The HIV-1 regulatory protein Vpr (96 amino acid residues) is incorporated into the virus particle through a mechanism involving its interaction with the C-terminal portion of Gag . Vpr potentiates virus replication by interrupting cell division in the G2 phase and participates in the nuclear transport of proviral DNA . The domain encompassing the 40 C-terminal residues of Vpr was shown to be involved in cell cycle arrest and binding of nucleocapsid protein NCp7, and suggested to promote nuclear provirus transfer . Accordingly, we show here that the synthetic 52-96 but not 1-51 sequences of Vpr interact with HIV-1 RNA . Based on these results, the structure of (52-96)Vpr was analysed by two-dimensional 1H-NMR in aqueous TFE (30%) solution and refined by restrained molecular dynamics . The structure is characterized by a long (53-78) amphipathic alpha-helix, followed by a less defined (79-96) C-terminal domain . The Leu60 and Leu67 side-chains are located on the hydrophobic side of the helix, suggesting their involvement in Vpr dimerization through a leucine zipper-type mechanism . Accordingly, their replacement by Ala eliminates Vpr dimerization in the two hybrid systems, while mutations of Ile74 and Ile81 have no effect . This was confirmed by gel filtration measurements and circular dichroism, which also showed that the alpha-helix still exists in (52-96)Vpr and its Ala60, Ala67 mutant in the presence and absence of TFE . Based on these results, a model of the coiled-coil Vpr dimer has been described, and its biological relevance as well as that of the structural characteristics of the 52-96 domain for the different functions of Vpr, including HIV-1 RNA binding, are discussed .

Mycoses, 1998 Dec, 41(11-12), 493 - 500
Demonstration of Malassezia furfur and M . sympodialis together with M . pachydermatis in veterinary specimens; Raabe P et al.; In the present study, 47 wild-type isolates of the genus Malassezia were isolated from dog and cat specimens by means of a simple differentiating system recently published . The purpose was to determine whether any of the other seven Malassezia spp . apart from M . pachydermatis occur in carnivores . Of the 47 isolates, three had been obtained from cats (ear 2, skin 1) and 44 from dogs (ear 37, skin 3, faeces 2, claw and paw 2) . After primary isolation, they were subcultured on mDixon agar and then purified and differentiated by means of assimilation of Cremophor EL, splitting of esculin, growth on lipid-free medium and formation of tryptophan-dependent pigments and fluorochromes . Thus, a total of 100 strains could be obtained from the 47 primary isolates . Referring to the source material, M . pachydermatis was found in 83%, M . furfur in 45% and M . sympodialis in 75% . More than 80% of cultures were mixed, comprising two or all three species; a single species was isolated in only nine cases . This shows that animals are not colonized by M . pachydermatis alone, as has been thought until now, but in nearly all cases by mixed cultures . Thus, (domestic) animals could well be a reservoir for other Malassezia species such as M . furfur and M . sympodialis . Surprisingly, Malassezia yeasts were also isolated from dog faeces, indicating that they apparently pass through the gastrointestinal tract in unchanged form after having been taken up by licking colonized areas . The survivability of Malassezia yeasts in highly acid milieu was also demonstrated in vitro . The study confirms the usefulness of the new test procedures and allows new statements concerning the epidemiology of Malassezia yeasts.

Curr Opin Cell Biol, 1998 Dec, 10(6), 769 - 75
Sister chromatid cohesion in mitosis; Biggins S et al.; Sister chromatid cohesion is essential for accurate chromosome segregation during the cell cycle . Newly identified structural proteins are required for sister chromatid cohesion and there may be a link in some organisms between the processes of cohesion and condensation . Proteins that induce and regulate the separation of sister chromatids have also been recently identified.

J Exp Biol, 1992 Nov 1, 172(Pt 1), 431 - 441
A PLANT BIOCHEMIST'S VIEW OF H+-ATPases AND ATP SYNTHASES; Mccarty RE; My twenty-five year fascination with membrane ATPases grew out of my experiences in the laboratories of Andre Jagendorf and Efraim Racker . Andre introduced me to photosynthetic phosphorylation and Ef, to whose memory this article is dedicated, convinced me that ATPases had much to do with ATP synthesis . Astounding progress has been made in the H+-ATPase field in just two decades . By the early 1970s, it was generally recognized that oxidative and photosynthetic ATP synthesis were catalyzed by membrane enzymes that could act as H+-ATPases and that the common intermediate between electron transport and phosphorylation is the electrochemical proton gradient . At that time, it had been shown that a cation-stimulated ATPase activity was associated with plasma membrane preparations from plant roots . The endomembrane or vacuolar ATPases were unknown . The application of improved biochemical methods for membrane isolation and purification, as well as membrane protein reconstitutions, led rapidly to the conclusion that there are three major classes of membrane H+-ATPases, P, V and F . P-ATPases, which will not be considered further in this article, are phosphorylated during their catalytic cycle and have a much simpler polypeptide composition than V- or F-ATPases . The plasma membrane H+-ATPase of plant, yeasts and fungal cells is one example of this class of enzymes (see Pedersen and Carafoli, 1987, for a comparison of plasma membrane ATPases) . Biochemical and gene sequencing analysis have revealed that V- and F-ATPases resemble each other structurally, but are distinct in function and origin . The 'V' stands for vacuolar and the 'F' for F1Fo . F1 was the first factor isolated from bovine heart mitochondria shown to be required for oxidative phosphorylation . Fo was so named because it is a factor that conferred oligomycin sensitivity to soluble F1 . Other F-ATPases are often named to indicate their sources . For example, chloroplast F1 is denoted CF1 (see Racker, 1965, for early work on F1) . Recent successes in reconstitution of vacuolar ATPase have led to a V1Vo nomenclature for this enzyme as well . The term 'ATP synthase' is now in general use to describe F-ATPases . This term emphasizes the facts that although F-ATPases function to synthesize ATP, they do not catalyze, normally, ATP hydrolysis linked to proton flux . In contrast, V-ATPases are very unlikely to operate as ATP synthases . Thus, F-ATPases are proton gradient consumers, whereas V-ATPases generate proton gradients at the expense of hydrolysis . In this brief review, I will compare the structures of F- and V-ATPases . Also, I give some insight into the mechanisms that help prevent wasteful ATP hydrolysis by the chloroplast ATP synthase (CF1Fo).

J Basic Microbiol, 1998, 38(5-6), 415 - 9
N'-methylniphimycin, a novel minor congener of niphimycin from Streptomyces spec . 57-13; Ivanova V et al.; A novel natural niphimycin analog, N'-methylniphimycin was isolated from the culture broth of the Streptomyces spec . 57-13 . The chemical constitution was elucidated from the physico-chemical properties, NMR techniques and mass spectrometry to be a 36-membered macrolide related to azalomycin F5a, shurimycin B and RS-22C . N'-methylniphimycin displayed moderate activity against some yeasts and filamentous fungi.

FEBS Lett, 1998 Dec 4, 440(3), 258 - 63
Structure, function and regulation of the vacuolar (H+)-ATPases; Forgac M; The vacuolar (H+)-ATPases (or V-ATPases) function to acidify intracellular compartments in eukaryotic cells, playing an important role in such processes as receptor-mediated endocytosis, intracellular membrane traffic, protein degradation and coupled transport . V-ATPases in the plasma membrane of specialized cells also function in renal acidification, bone resorption and cytosolic pH maintenance . The V-ATPases are composed of two domains . The V1 domain is a 570-kDa peripheral complex composed of 8 subunits (subunits A-H) of molecular weight 70-13 kDa which is responsible for ATP hydrolysis . The V0 domain is a 260-kDa integral complex composed of 5 subunits (subunits a-d) which is responsible for proton translocation . The V-ATPases are structurally related to the F-ATPases which function in ATP synthesis . Biochemical and mutational studies have begun to reveal the function of individual subunits and residues in V-ATPase activity . A central question in this field is the mechanism of regulation of vacuolar acidification in vivo . Evidence has been obtained suggesting a number of possible mechanisms of regulating V-ATPase activity, including reversible dissociation of V1 and V0 domains, disulfide bond formation at the catalytic site and differential targeting of V-ATPases . Control of anion conductance may also function to regulate vacuolar pH . Because of the diversity of functions of V-ATPases, cells most likely employ multiple mechanisms for controlling their activity.

Genes Dev, 1998 Dec 15, 12(24), 3815 - 20
Molecular interactions between Vestigial and Scalloped promote wing formation in Drosophila; Simmonds AJ et al.; Scalloped (Sd) and Vestigial (Vg) are each needed for Drosophila wing development . We show that Sd is required for Vg function and that altering their relative cellular levels inhibits wing formation . In vitro, Vg binds directly to both Sd and its human homolog, Transcription Enhancer Factor-1 . The interaction domains map to a small region of Vg that is essential for Vg-mediated gene activation and to the carboxy-terminal half of Sd . Our observations indicate that Vg and Sd function coordinately to control the expression of genes required for wing development, which implies that Vg is a tissue-specific transcriptional intermediary factor of Sd.

J Biotechnol, 1998 Dec 11, 66(2-3), 101 - 7
Mycotechnology: the role of fungi in biotechnology; Bennett JW; Fungi have been important in both ancient and modern biotechnological processes . Processes and products that utilize fungi include baking, brewing, and the production of antibiotics, alcohols, enzymes, organic acids, and numerous pharmaceuticals . The advent of recombinant DNA technology and large scale genomics analysis has placed yeasts and filamentous fungi in the forefront of contemporary commercial applications . The term 'mycotechnology' is introduced here to describe the enormous impact of fungi on biotechnology.

Mycoses, 1998 Sep-Oct, 41(7-8), 265 - 71
Synthesis of fluorochromes and pigments in Malassezia furfur by use of tryptophan as the single nitrogen source; Mayser P et al.; A new minimal medium consisting only of L-tryptophan (L-Trp) and a lipid source induced formation of brown pigmentation only in the species Malassezia furfur, which diffuses into the agar . Strains of the species M . sympodialis and M . pachydermatis failed to grow on this medium . On mDixon medium, however, after replacement of peptone by L-Trp, growth of all three Malassezia species was achieved . Under these conditions pigment production was observed with all M . furfur strains tested, although the results for M . pachydermatis strains were inconsistent . M . sympodialis strains showed no pigment production . On the minimal medium pigmentogenesis was induced in M . furfur by only 0.01 g% tryptophan; the pH optimum was pH 5 . In all M . furfur strains, alternative amino nitrogen sources given concurrently with Trp suppressed pigmentogenesis . Furthermore, there were differences in the optimal temperature among the individual M . furfur strains . CBS 7019, CBS 6000 and CBS 6001 failed to produce pigment at 37 degrees C . The extract of the culture exhibited remarkable fluorescence, and several indole derivatives with a broad spectrum of colours were detected . This finding may have an impact on the clinical appearance of pityriasis versicolor, a very common skin disease caused by lipophilic yeasts of the genus Malassezia . We hypothesize that in pityriasis versicolor metabolic adaptation of Malassezia yeasts to altered nitrogen conditions on superficial skin might be of patho-physiological importance . Tryptophan as an inducer of pigmentogenesis probably accumulates during excessive sweating, a well-known manifestation of pityriasis versicolor.

Mol Cell Biol, 1999 Jan, 19(1), 567 - 76
A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 3' end; Mitchell JR et al.; Simple sequence repeat telomeric DNA is maintained by a specialized reverse transcriptase, telomerase . The integral RNA subunit of telomerase contains a template region that determines the sequence added to chromosome ends . Aside from providing the template, little is known about the role of the telomerase RNA . In addition, no hypotheses have been suggested to account for the striking evolutionary divergence in size and sequence between telomerase RNAs of ciliates, yeasts, and mammals . We show that the two- to threefold increase in size of the mammalian telomerase RNAs relative to ciliate telomerase RNAs is due to the presence of an extra domain resembling a box H/ACA small nucleolar RNA (snoRNA) . The human telomerase RNA (hTR) H/ACA domain is essential in vivo for hTR accumulation, hTR 3' end processing, and telomerase activity . By substituting the U64 box H/ACA snoRNA for the hTR H/ACA domain, we demonstrate that a heterologous snoRNA can function to promote chimeric RNA accumulation and 3' end processing but not telomerase activity . In addition, we show that maturation of full-length hTR and its assembly into active telomerase occur from an mRNA promoter-driven RNA polymerase II transcript but not from a U6 snRNA promoter-driven RNA polymerase III transcript . Finally, we show that a small percentage of hTR is associated with nucleoli . These results have implications for the biogenesis and structure of hTR and the human telomerase ribonucleoprotein complex . They also expand the structural and functional diversity of the box H/ACA snoRNA motif.

Gene, 1998 Oct 9, 221(1), 93 - 106
Generation of expressed sequence tags as physical landmarks in the genome of Trypanosoma brucei; Djikeng A et al.; Previous molecular genetic studies on the African trypanosome have focused on only a few genes and gene products, the majority of which are concerned with surface antigenic variation; consequently, an insignificant number of the genes of this organism have been characterized to date . In order to: (1) identify new genes and analyze their expression profile, (2) generate expressed sequence tags (ESTs) for derivation of a physical map of the trypanosome genome, and (3) make available the partial sequence information and the corresponding clones for general biomedical research on the parasite, we have performed single-pass sequencing of random, directionally cloned cDNAs from a bloodstream form Trypanosoma brucei rhodesiense library . Analysis of 2128 such ESTs sequenced so far in this study showed significant similarities {BLASTX P(n)-value < 10(-4), and a match > 10 amino acid residues} with proteins whose genes have been described in diverse organisms including man, rodents, kinetoplastids, yeasts and plants . A number of the ESTs encode homologues of proteins involved in various functions including signal reception and transduction, cell division, gene regulation, DNA repair and replication, general metabolism, and structural integrity . Although some of these genes may have been expected to be present in the African trypanosomes, the majority of them had not previously been described in these organisms . A large proportion, 768 individual ESTs (36%, representing 385 different transcripts), had a significant homology with genes described in organisms other than the African trypanosomes; however, 15% of the ESTs were from genes already described in trypanosomes . Among the ESTs analysed were 462 distinct known genes, only 77 of which have been described in T . brucei . Approximately 52% of the ESTs did not show any significant homology with the sequences in any of the public domain databases.

Eur J Biochem, 1998 Nov 15, 258(1), 223 - 32
Cloning and biochemical characterisation of Aspergillus niger hexokinase--the enzyme is strongly inhibited by physiological concentrations of trehalose 6-phosphate; Panneman H et al.; The Aspergillus niger hexokinase gene hxkA has been cloned by heterologous hybridisation using the Aspergillus nidulans hexokinase gene as a probe . The DNA sequence of the gene was determined, and the deduced amino acid sequence showed significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to those of the budding yeasts . The encoded protein was purified from a multicopy hxkA transformant, and extensively characterised . The hexokinase protein has a molecular mass of 54090, a pI of 4.9 and is a homodimer . D-Glucose, the glucose analogue 2-deoxy-D-glucose, D-fructose, D-mannose and D-glucosamine are phosphorylated by hexokinase, whereas the hexoses D-galactose, L-sorbose, methyl alpha-D-glucoside and the pentoses L-arabinose and D-xylose are not . The enzyme has high affinity for glucose (Km = 0.35 mM at pH 7.5) and for fructose (Km = 2.0 mM at pH 7.5) and is inhibited by ADP . The enzyme is strongly inhibited by physiological concentrations (0.1-0.2 mM) of trehalose 6-phosphate, which may be of importance for in vivo regulation of the enzyme . Inhibition of A . niger hexokinase by trehalose 6-phosphate is competitive towards the sugar substrate (Ki = 0.01 mM) . Based on the kinetic constants of hexokinase and glucokinase their relative contribution to in vivo glucose phosphorylation was calculated and found to be strongly dependent on intracellular pH and glucose concentration . At pH 7.5 glucokinase is predominant, whereas at pH 6.5 hexokinase is predominant at glucose concentrations higher than 0.5 mM . Expression of the hexokinase and the glucokinase gene requires active carbon metabolism . Also on carbon sources which are not substrates for hexokinase or glucokinase, clear expression is observed . The hexokinase and glucokinase enzymes are quite stable in vivo . Even in the absence of transcription, active glucokinase and hexokinase remain present in the cells at almost the same level for at least 3-4 h after depletion of the carbon source.

Nucleic Acids Res, 1999 Jan 1, 27(1), 18 - 24
The EMBL Nucleotide Sequence Database; Stoesser G et al.; The EMBL Nucleotide Sequence Database constitutes Europe's primary nucleotide sequence resource . Main sources for DNA and RNA sequences are direct submissions from individual researchers, genome sequencing projects and patent applications . While automatic procedures allow incorporation of sequence data from large-scale genome sequencing centres and from the European Patent Office (EPO), the preferred submission tool for individual submitters is Webin (WWW) . Through all stages, dataflow is monitored by EBI biologists communicating with the sequencing groups . In collaboration with DDBJ and GenBank the database is produced, maintained and distributed at the European Bioinformatics Institute (EBI) . Database releases are produced quarterly and are distributed on CD-ROM . Network services allow access to the most up-to-date data collection via Internet and World Wide Web interface . EBI's Sequence Retrieval System (SRS) is a Network Browser for Databanks in Molecular Biology, integrating and linking the main nucleotide and protein databases, plus many specialised databases . For sequence similarity searching a variety of tools (e.g . Blitz, Fasta, Blast etc) are available for external users to compare their own sequences against the most currently available data in the EMBL Nucleotide Sequence Database and SWISS-PROT.

Vestn Ross Akad Med Nauk, 1998, (10), 45 - 55
{Molecular bases of prion diseases}; Pokrovskii VI et al.; The paper briefly analyzes the origin of priones and their association with the cellular gene and homologous protein of diseases in man and animals . There is evidence for a direct relationship of the agents that cause spongious encephalitis in the cattle and a new type of Creutzfeldt-Jacob disease in man . The molecular organization of priones and the conformational cellular protein changes underlying the infectious activation of the cell homologue of priones . Emphasis is first laid on the capacity of the cell homologue of priones and their infectiously active derivative to bind to DNA or RNA . In the context of concepts of the priones yeasts an attempt was made to explain the reproduction through the altered control of translation of mRNA that encodes the cellular homologue of priones, which accounts for the duration of the incubation period of the disease . The infections caused by priones are referred to as the so-called slow infections . But in the context of the proposed hypothesis, an infective process in the tissues did not really have some typical signs of infection and resembles accumulation diseases more without the replicative burst typical of infectious processes . The paper gives data on the vital cycle of priones in infected animals and changes in the accumulation of an infective agent . This assesses the currently available diagnostic methods and gives preference to the methods which will be based on the use of monoclonal antibodies that specifically recognize the conformationally altered form of an infectious prione or on the identification of primary oligomeric forms which manifest the onset of amyloidization of the damaged tissues . The main conclusion of the paper is that protein prionization is a common biological phenomenon and the diseases caused by these processes will increase in number in the near future, which makes it necessary to develop diagnostic methods and universal treatments of diseases, such as bacterial infections by using antibiotics.

J Biochem (Tokyo), 1998 Dec 1, 124(6), 1060 - 4
Chromatin remodeling and transcription; Hirose S; The chromatin structure is essential not only for the compact packaging of the eukaryotic genome but also for regulation of transcription . This article provides an overview of chromatin modification upon transcriptional activation or repression, and chromatin remodeling . Interestingly, recent data demonstrate that chromatin remodeling in the promoter region is necessary for transcription.

Lett Appl Microbiol, 1998 Nov, 27(5), 283 - 6
Trichoderma/pathogen interactions: measurement of antagonistic chemicals produced at the antagonist/pathogen interface using a tubular bioassay; Cooney JM et al.; A tubular bioassay was used to measure analytically the local production and concentration of the antifungal Trichoderma secondary metabolite 6-n-pentyl-2H-pyran-2-one (6PAP) at the Trichoderma antagonist/pathogen interface . 6PAP levels significantly increased in the presence of the pathogen Botrytis cinerea, typically 300-700%, and were highest near the pathogen source . The level of response for a particular Trichoderma isolate was found to vary with the test organism used . Two products produced by biotransformation of 6PAP by B . cinerea in response to the interaction were also detected.

J Biotechnol, 1998 Oct 27, 65(2-3), 163 - 71
Overproduction of recombinant Trichoderma reesei cellulases by Aspergillus oryzae and their enzymatic properties; Takashima S et al.; We have established an expression system of Trichoderma reesei cellulase genes using Aspergillus oryzae as a host . In this system, the expression of T . reesei cellulase genes were regulated under the control of A . oryzae Taka-amylase promoter and the cellulase genes were highly expressed when maltose was used as a main carbon source for inducer . The production of recombinant cellulases by A . oryzae transformants reached a maximum after 3-4 days of cultivation . In some cases, proteolysis of recombinant cellulases was observed in the late stage of cultivation . The recombinant cellulases were purified and characterized . The apparent molecular weights of recombinant cellulases were more or less larger than those of native enzymes . The optimal temperatures and pHs of recombinant cellulases were 50-70 degrees C and 4-5, respectively . Among the recombinant cellulases, endoglucanase I showed broad substrate specificities and high activity when compared with the other cellulases investigated here.

Arch Fam Med, 1998 Nov-Dec, 7(6), 587 - 92
Onychomycosis: recognition, diagnosis, and management; Jaffe R; About 20% of the US population between the ages of 40 and 60 years have fungal nail disease, or onychomycosis . The incidence of this infection is increasing worldwide . Most cases of onychomycosis in the United States are caused by dermatophytes, but nondermatophyte fungi (molds or yeasts) may also be causative agents . To confirm the diagnosis of onychomycosis, a potassium hydroxide examination should be performed . A culture is necessary to determine the fungal pathogen and to aid in selecting appropriate therapy . Worldwide, fluconazole (not yet approved in the United States for onychomycosis), itraconazole, and oral terbinafine have superseded griseofulvin and ketoconazole as the agents of choice in treating onychomycosis . These newer systemic compounds have higher cure rates and cause fewer side effects than traditional agents . Intermittent dosing with itraconazole (3 or 4 one-week pulses of 200 or 400 mg daily) is the latest advance in the treatment of onychomycosis . This regimen has been found to be at least as safe and effective as short-term continuous therapy, yet more flexible, convenient, and economical.

Biol Chem, 1998 Oct, 379(10), 1235 - 41
Histone H4 underacetylation in plant facultative heterochromatin; Buzek J et al.; It has been recently shown that facultative heterochromatin in some phyla is H4 and H3 histone underacetylated . Here we present H4 acetylation analyses in a monocotyledonous plant species, Gagea lutea, whose pentaploid endosperm nuclei possess prominent facultative heterochromatin regions . This heterochromatin is attributed to three chromosome sets originated from the chalazal polar nucleus of the embryo sac . We have previously shown that some parts of this heterochromatin contain heavily methylated DNA, but not all the heterochromatin is hypermethylated . In this report we demonstrate that this facultative heterochromatin is characterised by a conspicuous depletion of histone H4 acetylation at N-terminal lysine residues 5, 8, and 12, but not 16 . Endosperm metaphases stained with antiserum against H4Ac5 indicated some heavily labelled chromosomes, while the others displayed no signal (presumably those coming from the three heterochromatinised chromosome sets) . Western blotting analyses have shown that the antisera used, designed to detect human H4 histones, are suitable to recognise specific isoforms of acetylated H4 histones in plants and that the most abundant H4 in G . lutea leaves occurs in its diacetylated isoform . We conclude that flowering plants, similarly to protozoa, yeasts and animals, evolved core histone acetylation/deacetylation as a long-term transcriptional control mechanism to establish and/or transmit epigenetic information on gene expression.

Bull Soc Belge Ophtalmol, 1998, 268, 121 - 6
Infectious crystalline keratopathy: a case report; Roodhooft J et al.; We report a case of crystalline keratopathy . Corneal smears showed yeasts . Cultures showed growth of Pityrosporon ovale . We summarise some causes of crystalline keratopathy and mention some hints for the treatment of infectious crystalline keratopathy.

Mol Cell, 1998 Oct, 2(4), 427 - 36
SH3GL3 associates with the Huntingtin exon 1 protein and promotes the formation of polygln-containing protein aggregates; Sittler A et al.; The mechanism by which aggregated polygins cause the selective neurodegeneration in Huntington's disease (HD) is unknown . Here, we show that the SH3GL3 protein, which is preferentially expressed in brain and testis, selectively interacts with the HD exon 1 protein (HDex1p) containing a glutamine repeat in the pathological range and promotes the formation of insoluble polyglutamine-containing aggregates in vivo . The C-terminal SH3 domain in SH3GL3 and the proline-rich region in HDex1p are essential for the interaction . Coimmunoprecipitations and immunofluorescence studies revealed that SH3GL3 and HDex1p colocalize in transfected COS cells . Additionally, an anti-SH3GL3 antibody was also able to coimmunoprecipitate the full-length huntingtin from an HD human brain extract . The characteristics of the interaction between SH3GL3 and huntingtin and the colocalization of the two proteins suggest that SH3GL3 could be involved in the selective neuronal cell death in HD.

J Med Chem, 1998 Nov 5, 41(23), 4439 - 52
Highly potent bisphosphonate ligands for phosphoglycerate kinase; Jakeman DL et al.; We have synthesized a series of novel analogs of 1, 3-bisphospho-D-glyceric acid, 1,3-BPG,3 and evaluated their binding to phosphoglycerate kinase, PGK (EC 2.7.2.3) . Nonscissile methanephosphonic acids replace the two phosphate monoesters of 1, 3-BPG and lead to several stable, tight-binding mimics of this intermediate species in glycolysis . Multiple fluorine substitution for hydrogen in the alpha-methylene groups of the phosphonic acid 1, 3-BPG analogs markedly improves their binding to PGK as determined by NMR analysis . The best ligands bind some 50-100 times more strongly than does the substrate 3-phospho-D-glyceric acid and show a requirement for pKa3 to be generally below 6.0, while the presence of a beta-carbonyl group seems to be of secondary importance.

Int J Dermatol, 1998 Oct, 37(10), 759 - 65
Tinea pedis et unguium in the Muslim community of Durban, South Africa; Raboobee N et al.; BACKGROUND: In a pilot study performed in eight mosques in the Durban area, it was found that the prevalence of tinea pedis et unguium in the adult Muslim male population regularly attending mosques was higher than in the nonMuslim male population . The aims of the present study were: (i) to determine the prevalence of tinea pedis et unguium in the adult Muslim male population regularly attending mosques; (ii) to investigate the role of mosque carpets and ablution areas in the spread of infection; and (iii) to develop strategies to combat the infection . METHOD: Seventy-eight regular worshippers comprising adult Muslim males, chosen at random from five mosques in the Durban area, were examined for clinical evidence of tinea pedis et unguium . Skin scrapings and nail clippings were taken from clinically infected individuals and submitted for microscopy and culture for fungal organisms . A control group, comprising 72 nonMuslim adult male office workers from the administration departments of King Edward VIII Hospital, was similarly examined . In addition, scrapings from high traffic areas of the mosque carpets and swabs from the ablution areas were cultured for fungi . RESULTS: In the mosque group, it was found that the prevalence of tinea pedis et unguium was 85%, taking either microscopy or culture positivity as indicative of infection . In the control group, the prevalence was 41% . Thus a statistical difference of 44% (P < 0.0001) between the two groups was demonstrated . Dermatophytes and yeasts were isolated from the carpets and/or floors of the ablution areas in all the mosques under investigation . CONCLUSIONS: The high prevalence of tinea pedis et unguium among regular male worshippers in the Muslim community can be attributed to the spread of fungal organisms in the communal ablution areas and prayer carpets of the mosques . Strategies to combat this spread of infection are being developed . These strategies are expected to find important practical applications in other communal environments, such as gymnasia, health spas, swimming pools, changing rooms of sports clubs, public showers, and even hotels.

Biol Psychiatry, 1998 Oct 15, 44(8), 659 - 66
Human gamma-aminobutyric acid B receptor gene: complementary DNA cloning, expression, chromosomal location, and genomic organization; Goei VL et al.; BACKGROUND: The 6p21.3 region of human chromosome 6 is a genetic locus for schizophrenia, juvenile myoclonic epilepsy, and dyslexia . METHODS: Due to our interest in these disorders we performed complementary DNA (cDNA) hybridization selection on genomic DNA clones spanning this region to identify potential positional-candidate genes . RESULTS: We identified a full-length cDNA with an open reading frame of 2883 bp corresponding to a predicted protein of 961 amino acids that shares greater than 95% homology with the rat gamma-aminobutyric acid B (GABAB) receptor . Northern blot hybridization identified a 4.4-kb transcript in human brain . The human gene mapped to two sites on 6p21.3 separated by 2 Mb . Sequence analysis of both sites showed that the centromeric gene is transcribed, whereas the telomeric site is likely a pseudogene . The transcribed gene is distributed over 22 exons spanning 18 kb of genomic DNA . CONCLUSIONS: The genomic location, tissue expression, and function of the human GABAB receptor gene suggest that it is an important positional-candidate for the neurobehavioral disorders with a genetic locus on 6p21.3 . In addition, delineation of the genomic organization will now permit it to be integrated as part of pharmacogenetic studies in trials of anxiolytic, narcotic, antiepileptic, and fluoxetine therapies.

Rev Esp Quimioter, 1998 Mar, 11(1), 70 - 4
{Effect of fluconazole and itraconazole on human polymorphonuclear leukocyte oxidative metabolism and phagocytosis.}; Velert MM et al.; The effect of pretreatment of human polymorphonuclear leukocites (PMNs), for 30 min with fluconazole (0.1, 1, 5 and 50 microgram/ml) and itraconazole (0.05, 0.5 and 5 microgram/ml) on phagocytosis and generation of free radicals (superoxide anion and hydrogen peroxide) was studied in vitro . Phorbol miristate acetate (200 nM) was used as a stimulant . The mean amount of superoxide anion generated by 2.5 x 10(5) PMNs per hour was 4.39 +/- 1.13 nmol for fluconazole-treated PMNs and 4.56 +/- 1.2 nmol for itraconazole, and that of hydrogen peroxide was 11.19 +/- 2.18 and 11.28 +/- 3.61 nmol, respectively . The phagocytosis percentages were 83.8% for the control group and 88 . 7% in antifungal agent- treated PMNs; the phagocytosis index was 3.0 yeasts per PMN for both groups . The differences between the control and treated PMNs were not statistically significant at any of the tested concentrations.

Nippon Ishinkin Gakkai Zasshi, 1998, 39(4), 213 - 8
{Chronic and recurrent vulvovaginal candidiasis}; Kubota T; Approximately 15 % of non-pregnant women and 30 % of pregnant women yield positive cultures of Candida species in the vaginal specimens . A diagnosis of vulvovaginal candidiasis (VVC) should only be made when Candida spp . are isolated from the vulvovaginal area, together with the presence of signs and symptoms . In Japan several topical drugs for vaginal candidiasis are available, but oral azoles has not been approved . According to our studies, treatment with topical drugs resulted in symptomatic cure and negative culture conversion in 80 %-90 % of patients at the end of initial treatment (6-14 days or high-dose one day treatment according to the drug used) . However Candida spp . reappeared in the vagina after several weeks in 7-34 % (according to the drug used) of initially cured cases, and some of them were again symptomatic . A small proportion of women experienced three or more episodes of symptomatic VVC annually . Repeated reinfection from a gastrointestinal reservoir or sexual transmission, subclinical presence of yeasts in the vagina, impaired host defense mechanisms, enhanced Candida virulence have been discussed as factors relating to recurrent vulvovaginal candidiasis (RVVC) . Oral ketoconazole give the best result in treating RVVC, however it is not used because of possible side effects . Several studies have evaluated oral therapy for RVVC with fluconazole and itraconazole that have less side effects . However, several reports have documented the emergence of fluconazole-resistant candidiasis in long-term treatment of mycotic diseases other than VVC . The optimal treatment for RVVC remains difficult to establish.

Cell, 1998 Oct 16, 95(2), 237 - 48
Structural basis for activation of ARF GTPase: mechanisms of guanine nucleotide exchange and GTP-myristoyl switching; Goldberg J; Ras-related GTPases are positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP . The crystal structure of the Sec7 domain GEF bound to nucleotide-free ARF1 GTPase has been determined at 2.8 A resolution and the structure of ARF1 in the GTP-analog form determined at 1.6 A resolution . The Sec7 domain binds to the switch regions of ARF1 and inserts residues directly into the GTPase active site . The interaction leaves the purine-binding site intact but perturbs the Mg2+ and phosphate groups to promote the dissociation of guanine nucleotides . The structure of ARF1 in the GTP-analog form closely resembles Ras, revealing a substantial rearrangement from the GDP conformation . The transition controls the exposure of the myristoylated N terminus, explaining how ARF GTPases couple the GDP-GTP conformational switch to membrane binding.

Trends Cell Biol, 1998 Oct, 8(10), 397 - 403
Proteasome inhibitors: valuable new tools for cell biologists; Lee DH et al.; Proteasomes are major sites for protein degradation in eukaryotic cells . The recent identification of selective proteasome inhibitors has allowed a definition of the roles of the ubiquitin-proteasome pathway in various cellular processes, such as antigen presentation and the degradation of regulatory or membrane proteins . This review describes the actions of these inhibitors, how they can be used to investigate cellular responses, the functions of the proteasome demonstrated by such studies and their potential applications in the future.

J Cell Biol, 1998 Oct 19, 143(2), 429 - 42
Layilin, a novel talin-binding transmembrane protein homologous with C-type lectins, is localized in membrane ruffles; Borowsky ML et al.; Changes in cell morphology and motility are mediated by the actin cytoskeleton . Recent advances in our understanding of the regulators of microfilament structure and dynamics have shed light on how these changes are controlled, and efforts continue to define all the structural and signaling components involved in these processes . The actin cytoskeleton-associated protein talin binds to integrins, vinculin, and actin . We report a new binding partner for talin that we have named layilin, which contains homology with C-type lectins, is present in numerous cell lines and tissue extracts, and is expressed on the cell surface . Layilin colocalizes with talin in membrane ruffles, and is recruited to membrane ruffles in cells induced to migrate in in vitro wounding experiments and in peripheral ruffles in spreading cells . A ten-amino acid motif in the layilin cytoplasmic domain is sufficient for talin binding . We have identified a short region within talin's amino-terminal 435 amino acids capable of binding to layilin in vitro . This region overlaps a binding site for focal adhesion kinase.

J Cell Biol, 1998 Oct 19, 143(2), 309 - 18
Determination of the functional domain organization of the importin alpha nuclear import factor; Herold A et al.; Although importin alpha (Imp alpha) has been shown to act as the receptor for basic nuclear localization signals (NLSs) and to mediate their recruitment to the importin beta nuclear import factor, little is known about the functional domains present in Imp alpha, with the exception that importin beta binding is known to map close to the Imp alpha NH2 terminus . Here, we demonstrate that sequences essential for binding to the CAS nuclear export factor are located near the Imp alpha COOH terminus and include a critical acidic motif . Although point mutations introduced into this acidic motif inactivated both CAS binding and Imp alpha nuclear export, a putative leucine-rich nuclear export signal proved to be neither necessary nor sufficient for Imp alpha nuclear export . Analysis of sequences within Imp alpha that bind to the SV-40 T antigen NLS or to the similar LEF-1 NLS revealed that both NLSs interact with a subset of the eight degenerate armadillo (Arm) repeats that form the central part of Imp alpha . However, these two NLS-binding sites showed only minimal overlap, thus suggesting that the degeneracy of the Arm repeat region of Imp alpha may serve to facilitate binding to similar but nonidentical basic NLSs . Importantly, the SV-40 T NLS proved able to specifically inhibit the interaction of Imp alpha with CAS in vitro, thus explaining why the SV-40 T NLS is unable to also function as a nuclear export signal.

Med Mycol, 1998 Jun, 36(3), 143 - 51
Exophiala dermatitidis and Sarcinomyces phaeomuriformis: ITS1-sequencing and nutritional physiology; Uijthof JM et al.; The internal transcribed spacer 1 (ITS1) sequences of the nuclear rRNA gene (approximately 200 bp) of 33 strains of the Exophiala dermatitidis complex were determined; two similar species were added for comparison . A core group (I), including the type strain CBS 207 . 35, contained 20 identical strains which had previously been found to have introns in their small sub-unit (SSU) rDNA . Eleven remaining strains identified as E . dermatitidis (groups II-V) differed from the core group in 1-4 nucleotide positions (plus a deletion in one strain); most of them lacked introns in their SSU ribosomal genes . The type strain of the meristematic species Sarcinomyces phaeomuriformis CBS 131.88 was found to differ significantly from E . dermatitidis . One strain had the annellidic morphology of E . dermatitidis, but the ITS1 sequence of S . phaeomuriformis . Strain CBS 709.95, an E . dermatitidis reported to have a meristematic synanamorph, was found to have ITS1 identity to the type strain of E . dermatitidis, although SSU sequences established previously suggested a close relationship with S . phaeomuriformis . Slight physiological differences were found between E . dermatitidis and S . phaeomuriformis . An oligonucleotide probe specific for E . dermatitidis was designed, thus able to discriminate this species from closely related black yeasts.

Pathol Biol (Paris), 1998 May, 46(5), 335 - 40
{Fungal and parasitic nosocomial infections: importance and limitations of disinfection methods}; Lebeau B et al.; Due to the increase of opportunistic mycosis and parasitosis for several years, the management of fungal and parasitic risk in hospital has become a necessity and an obligation . The authors remind the main rules and knowledges essential to an optimal management of the fungal and parasitic disinfection in hospital . They summarize the efficiency of different disinfection processes in relation to yeasts, filamentous fungi, Pneumocystis carinii, Cryptosporidia and Microsporidia involved in hospital pathology.

Biosci Biotechnol Biochem, 1998 Aug, 62(8), 1604 - 8
Characterization of ribonucleases from culture medium of Lentinus edodes; Kobayasi H et al.; Lentinus edodes (shiitake) cultivated in potato dextrose medium produced five RNases in the culture filtrate . The two major RNases (RNase Le37 and RNase Le45) were highly purified and their molecular masses, base specificities, N-terminal amino acid sequences, and amino acid compositions were analyzed and compared to RNase Le2 isolated from the fruit bodies of the same mushroom . RNase Le37 and RNase Le45 are base non-specific and adenylic acid preferential RNases like RNase Le2 and their N-terminal sequences are very similar to RNase Le2, but they are glycoproteins and their amino acid compositions are significantly different from that of RNase Le2 . In addition to these enzymes, a guanylic acid-specific RNase with a molecular mass 13 kDa was partially purified . Since RNase Le2, which has very similar N-terminal sequence to RNase Le37 and RNase Le45, was not excreted from the mycelia, the analysis of the structures of these two excreted RNase may shade a light on the mechanism of excretion of RNases in this organism.

Methods, 1998 Aug, 15(4), 355 - 64
Interactions of transcriptional regulators with histones; Edmondson DG et al.; Tremendous advances in the study of chromatin have revealed new classes of transcriptional regulators distinct from classical DNA-binding proteins . Many previously described transcription factors, coactivators, and adaptors are regulators of chromatin structure, interacting directly with the core histone proteins or with nucleosomes . This review describes a method used by our laboratory to examine the interactions of regulatory proteins with the core histone proteins . Far-Western analysis uses a protein probe to detect interactions with histones immobilized on membranes . Variations of this technique can detect the acetylation state of the interacting histones and whether the interaction occurs through the globular domain or the amino-terminal "tail" domain . In addition, we discuss complementary techniques for confirming histone-regulatory protein interactions.

EMBO J, 1998 Sep 1, 17(17), 4909 - 19
Active site mutants in the six regulatory particle ATPases reveal multiple roles for ATP in the proteasome; Rubin DM et al.; A family of ATPases resides within the regulatory particle of the proteasome . These proteins (Rpt1-Rpt6) have been proposed to mediate substrate unfolding, which may be required for translocation of substrates through the channel that leads from the regulatory particle into the proteolytic core particle . To analyze the role of ATP hydrolysis in protein breakdown at the level of the individual ATPase, we have introduced equivalent site-directed mutations into the ATPbinding motif of each RPT gene . Non-conservative substitutions of the active-site lysine were lethal in four of six cases, and conferred a strong growth defect in two cases . Thus, the ATPases are not functionally redundant, despite their multiplicity and sequence similarity . Degradation of a specific substrate can be inhibited by ATP-binding-site substitutions in many of the Rpt proteins, indicating that they co-operate in the degradation of individual substrates . The phenotypic defects of the different rpt mutants were strikingly varied . The most divergent phenotype was that of the rpt1 mutant, which was strongly growth defective despite showing no general defect in protein turnover . In addition, rpt1 was unique among the rpt mutants in displaying a G1 cell-cycle defect . Proteasomes purified from an rpt2 mutant showed a dramatic inhibition of peptidase activity, suggesting a defect in gating of the proteasome channel . In summary, ATP promotes protein breakdown by the proteasome through multiple mechanisms, as reflected by the diverse phenotypes of the rpt mutants.

Insect Mol Biol, 1998 Nov, 7(4), 301 - 6
Ribosomal protein L5 of Bombyx mori: cDNA sequence and tissue differences in the L5 mRNA content; Yang C et al.; A gene encoding the ribosomal protein L5, which assembles with the 5S rRNA into one of the components of the 60S ribosomal subunit, was identified in insects for the first time . We report on the isolation of Bombyx mori cDNA, whose putative translation product is 60-76% identical with the L5 sequences known from other animals and about 50% identical with the L5 of rice and yeasts . Bombyx contains a single copy of the L5 gene, which is constitutively expressed as a 1.1 kb mRNA in all tissues . The ratio of L5 mRNA to total RNA appears to reflect the proteosynthetic tissue capacity . A very high level of L5mRNA is maintained in functional silk glands . A rapid decline of the ratio of L5mRNA to the rRNA, which occurs in the silk glands after cocoon spinning, indicates differences in the stability of these two RNA classes.

Hosp Med, 1998 Apr, 59(4), 309 - 11
Itraconazole pulse: an overview of current use; Warnock DW; Traditionally, fungal infections of the fingernails and toenails have been difficult to treat . Itraconazole is an orally administered triazole antifungal agent with a wide spectrum of activity against dermatophytes, yeasts and some moulds . Pulse therapy with itraconazole is an effective new treatment which offers the shortest treatment course with the least total number of treatment days and lowest total dose.

Microbiol Immunol, 1998, 42(7), 475 - 8
First report of Trichosporon ovoides isolated from the home of a summer-type hypersensitivity pneumonitis patient; Sugita T et al.; Trichosporon species have been known to cause summer-type hypersensitivity pneumonitis (SHP) . During the isolation of yeasts from an SHP patient's house, we recovered a strain belonging to the genus Trichosporon . Morphologically, the isolate produced rectangular arthroconidia when grown on corn meal agar . DNA-DNA hybridization experiments identified the isolate as T . ovoides . A slide agglutination test using specific factor sera demonstrated that the serotype of the strain was type II . Previously, T . asahii, a serotype II species, was considered to be the major antigen of SHP, but it is possible that T . ovoides may also be responsible for SHP . This is the first report of T . ovoides isolated from an SHP patient's home environment.

Ophthalmology, 1998 Aug, 105(8), 1466 - 70
Unilateral Blastomyces dermatitidis endophthalmitis and orbital cellulitis . A case report and literature review; Li S et al.; PURPOSE: The authors report the clinical, cytologic, and histopathologic findings of a unique presentation of concomitant unilateral endophthalmitis and orbital cellulitis secondary to Blastomyces dermatitidis . DESIGN: Case report . METHODS: A 29-year-old healthy woman with a history of pulmonary tuberculosis presented with a painful right eye and rapidly decreasing vision . Fundus examination showed a diffuse elevated choroidal lesion at the posterior pole . With an otherwise unremarkable systemic work-up, the patient was treated with systemic antibiotics and corticosteroids for a presumed diagnosis of choroidal tuberculous granuloma . After an initial response to the treatment, the patient's condition deteriorated rapidly with visual acuity decreasing from 20/25 to no light perception in 3 months . Ipsilateral proptosis developed with magnetic resonance imaging showing a poorly defined orbital mass . Surgical enucleation and an orbital biopsy were performed . RESULTS: Histopathologic examination of the orbital specimen and an intact enucleated globe showed a diffuse necrotizing granulomatous process with the presence of numerous yeasts consistent with B . dermatitidis . This subsequently was confirmed by positive culture of B . dermatitidis from the orbital specimen . CONCLUSIONS: This is a unique case of concurrent unilateral endophthalmitis and orbital cellulitis secondary to B . dermatitidis . Intraocular dissemination of blastomycosis should be suspected in the differential diagnosis of endophthalmitis in patients with previous or active pulmonary lesions of equivocal nature . Early diagnosis and prompt treatment with antifungal medications are essential.

Clin Chim Acta, 1998 Jul 6, 275(1), 71 - 80
A new method for plasma volume measurements with unlabeled dextran-70 instead of 125I-labeled albumin as an indicator; van Kreel BK et al.; A method has been developed to determine plasma volume with dextran-70 without the use of a fluorescent label . The results obtained are compared to those found using the 125I-labeled albumin method, which is taken as the gold standard . The CV of the method is about 5%, compared to 3% with the gold standard . It is shown to be of use for the determination of an increase in plasma volume during pregnancy.

J Biol Chem, 1998 Aug 21, 273(34), 21578 - 84
Identification of residues in the cysteine-rich domain of Raf-1 that control Ras binding and Raf-1 activity; Winkler DG et al.; We have identified mutations in Raf-1 that increase binding to Ras . The mutations were identified making use of three mutant forms of Ras that have reduced Raf-1 binding (Winkler, D . G., Johnson, J . C., Cooper, J . A., and Vojtek, A . B . (1997) J . Biol . Chem . 272, 24402-24409) . One mutation in Raf-1, N64L, suppresses the Ras mutant R41Q but not other Ras mutants, suggesting that this mutation structurally complements the Ras R41Q mutation . Missense substitutions of residues 143 and 144 in the Raf-1 cysteine-rich domain were isolated multiple times . These Raf-1 mutants, R143Q, R143W, and K144E, were general suppressors of three different Ras mutants and had increased interaction with non-mutant Ras . Each was slightly activated relative to wild-type Raf-1 in a transformation assay . In addition, two mutants, R143W and K144E, were active when tested for induction of germinal vesicle breakdown in Xenopus oocytes . Interestingly, all three cysteine-rich domain mutations reduced the ability of the Raf-1 N-terminal regulatory region to inhibit Xenopus oocyte germinal vesicle breakdown induced by the C-terminal catalytic region of Raf-1 . We propose that a direct or indirect regulatory interaction between the N- and C-terminal regions of Raf-1 is reduced by the R143W, R143Q, and K144E mutations, thereby increasing access to the Ras-binding regions of Raf-1 and increasing Raf-1 activity.

J Biol Chem, 1998 Aug 21, 273(34), 21642 - 7
Genomic structure of MUNC18-1 protein, which is involved in docking and fusion of synaptic vesicles in brain; Gotoh K et al.; MUNC18-1 (n-Sec1) is a brain-specific protein and is known to play a role in neurotransmitter release by mediating docking and fusion of synaptic vesicles to presynaptic membranes . The protein is also implicated in the cellular excretion process of hormones and other biological substances in other mammalian tissues and yeasts . We have studied the structure of mouse munc18-1 gene by sequencing the genomic munc18-1 gene and its 5'-flanking region . munc18-1 gene comprises 19 exons whose size ranges from 50 base pairs (2nd exon) to 1676 base pairs (19th exon) with a total gene size of approximately 56 kilobases . In the 5'-flanking region, there are several transcription factor binding sites such as for HSF2, Lyf-1, and Sp1 but no TATA or CAAT sequences . munc18-1 gene was mapped on mouse chromosome 2 between two anchor markers D2Mit152 and D2Mit242 . Transfection experiments employing these and upstream sequences suggest the presence of a sequence(s) that negatively regulates the expression of munc18-1 gene.

Biochem Biophys Res Commun, 1998 Jul 30, 248(3), 464 - 8
Cloning and structural analysis of the gene encoding the ribosomal protein S6 from the parasite Leishmania infantum; Gonzalez-Aseguinolaza G et al.; We have cloned the S6 ribosomal protein encoding gene from a Leishmania infantum cDNA library . This parasite protozoon, responsible for leishmaniasis in Europe, is able to undergo developmental changes in vitro and results a good model to study cell differentiation processes . The LiS6 protein sequence indicates its pertinence to the S6 protein family, related to the early mechanisms of cell division, differentiation and activation, and shows an intermediate position between the yeasts and higher eukaryotes . Thus, LiS6 protein has the same amino acid length as that of the higher eukaryotes and certain common features such nucleus entrance sequences and several kinase phosphorylation sites . However, the key functional protein kinase C phosphorylation sites are at different locations and present several threonine instead of the usual serine residues . The gene structural analysis suggest the presence of three different encoding genes that do not present remarkable changes along the different phases of the parasite.

Biochem Pharmacol, 1998 Jul 1, 56(1), 131 - 9
Molecular cloning and functional analysis of cynomolgus monkey CYP1A2; Sakuma T et al.; Complementary DNA fragments encoding cynomolgus monkey CYP1A2 were amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) method from the liver total RNA of a 3-methylcholanthrene (3-MC)-treated cynomolgus monkey . The nucleotide sequence determined was 1630 bp long and contained an open reading frame for a polypeptide of 516 residues . The nucleotide and the deduced amino acid sequences of cynomolgus monkey CYP1A2 showed 95.1 and 92.8% identities to those of human CYP1A2, respectively . The level of CYP1A2 mRNA in the liver of untreated cynomolgus monkey was very low . Treatment with 3-MC increased it . Still, it was one-fortieth that of CYP1A1 . Cynomolgus monkey CYP1A2 expressed in recombinant yeasts activated 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) and 2-amino-3,8dimethylimidazo{4,5-flquinoxaline (MeIQx) at efficient rates in the umu mutagenicity test . This cytochrome P450 (CYP) also activated 2-amino-l-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP), but less efficiently . These results indicate that cynomolgus monkeys have a functionally active CYPIA2 gene, but its expression level is very low in the liver of untreated cynomolgus monkeys.

Neuron, 1998 Jul, 21(1), 99 - 110
The AMPA receptor GluR2 C terminus can mediate a reversible, ATP-dependent interaction with NSF and alpha- and beta-SNAPs; Osten P et al.; In this study, we demonstrate specific interaction of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins . The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus . We provide evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex . NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis . We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor.

Trends Cell Biol, 1998 Jun, 8(6), 238 - 44
Proteolytic ratchets that control progression through mitosis; Townsley FM et al.; A key player in mitotic progression is a cell-cycle-regulated ubiquitin-protein ligase complex known as the anaphase-promoting complex or cyclosome (APC/C) . The APC/C is part of the machinery that promotes the metaphase-anaphase transition by mediating the ubiquitin-dependent destruction of anaphase inhibitors and initiates exit from mitosis by degrading mitotic cyclins . This review describes the known components and substrates of the mitotic ubiquitination machinery and discusses how a new subfamily of proteins that contain the WD40 repeat (the Fizzy/Cdc20p family) might activate the APC/C to allow temporal differences in substrate ubiquitination during progression through mitosis.

Trends Cell Biol, 1998 Jun, 8(6), 215 - 8
A new beat for the SNARE drum; Gotte M et al.; Eukaryotic cells contain membrane-bound compartments that are connected by trafficking of vesicular intermediates . To maintain compartmental organization, proper targeting of transport vesicles is achieved by specific evolutionarily conserved transmembrane proteins that reside on vesicles and target membranes . According to the original SNARE hypothesis, the formation of a complex of an NEM-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs) and membrane-bound SNAP receptor proteins (SNAREs) ensures docking specificity and leads to membrane fusion driven by the ATPase activity of NSF . Recent results have challenged some aspects of this hypothesis and led to a reassessment of models of SNARE interactions and the events leading to vesicle docking and fusion.

J Nat Prod, 1998 Jul, 61(7), 901 - 6
Three new ellagic acid derivatives from the bark of Eschweilera coriacea from the Suriname rainforest; Yang SW et al.; Bioassay-guided fractionation of Eschweilera coriacea collected in the lowland wet forest of Suriname yielded the new but only weakly active ellagic acid derivative eschweilenol A (1) and the two new but inactive ellagic acid derivatives eschweilenol B (2) and eschweilenol C (3) . The four known compounds, sucrose, ellagic acid, 3-O-galloylepigallocatechin, and epigallocatechin, were also isolated . The structures of the three new compounds were determined by spectrometric methods, primarily from the HMQC, HMBC, NOESY, and ROESY NMR techniques, and chemical methods, including methylation and triethylsilylation . The location of a hydroxyl group in one ellagic acid derivative was determined by a new technique involving an NOE correlation of the protons of a triethylsilyl derivative with a proton on a neighboring aromatic ring.

Bioessays, 1998 May, 20(5), 391 - 9
"Isogaba Maware": quality control of genome DNA by checkpoints; Kitazono A et al.; Checkpoints maintain the interdependency of cell cycle events by permitting the onset of an event only after the completion of the preceding event . The DNA replication checkpoint induces a cell cycle arrest until the completion of the DNA replication . Similarly, the DNA damage checkpoint arrests cell cycle progression if DNA repair is incomplete . A number of genes that play a role in the two checkpoints have been identified through genetic studies in yeasts, and their homologues have been found in fly, mouse, and human . They form signaling cascades activated by a DNA replication block or DNA damage and subsequently generate the negative constraints on cell cycle regulators . The failure of these signaling cascades results in producing offspring that carry mutations or that lack a portion of the genome . In humans, defects in the checkpoints are often associated with cancer-prone diseases . Focusing mainly on the studies in budding and fission yeasts, we summarize the recent progress.

Mikrobiologiia, 1998 Mar-Apr, 67(2), 225 - 30
{Mycocinogeny in Bullera genus: killer activity of Bullera unica and intragenus killer-sensitive relationships}; Golubev VI et al.; The killer toxin secreted by the type strain of Bullera unica is sensitive to proteases and high temperature and displays fungicidal activities in a pH range of 3.5-6 . Ascomycetous yeasts are resistant to this toxin except for Lipomycetaceae and two species of debaryomycetes . This mycocin displays activity against basidiomycetous yeasts, primarily those of tremellaceous affinity . The killer phenotype of B . unica is retained after treatment with agents eliminating extrachromosomal genetic elements . Intrageneric killer-sensitive relationships among within the genus Bullera were studied using all known in this genus; the results are discussed in taxonomic terms.

Curr Opin Cell Biol, 1998 Jun, 10(3), 339 - 45
Life with nucleosomes: chromatin remodelling in gene regulation; Gregory PD et al.; In the past year, the role of chromatin has emerged at the forefront of transcription research . Discovery and characterisation of the chromatin modifying machinery have significantly advanced our understanding of the molecular activities that establish a transcriptionally competent substrate in vivo, and have underscored the importance of the part played by chromatin in the regulation of transcription.

Structure, 1998 May 15, 6(5), 605 - 17
The crystal structure of phenol hydroxylase in complex with FAD and phenol provides evidence for a concerted conformational change in the enzyme and its cofactor during catalysis; Enroth C et al.; BACKGROUND: The synthesis of phenolic compounds as by-products of industrial reactions poses a serious threat to the environment . Understanding the enzymatic reactions involved in the degradation and detoxification of these compounds is therefore of much interest . Soil-living yeasts use flavin adenine dinucleotide (FAD)-containing enzymes to hydroxylate phenols . This reaction initiates a metabolic sequence permitting utilisation of the aromatic compound as a source of carbon and energy . The phenol hydroxylase from Trichosporon cutaneum hydroxylates phenol to catechol . Phenol is the best substrate, but the enzyme also accepts simple hydroxyl-, amino-, halogen- or methyl-substituted phenols . RESULTS: The crystal structure of phenol hydroxylase in complex with FAD and phenol has been determined at 2.4 A resolution . The structure was solved by the MIRAS method . The protein model consists of two homodimers . The subunit consists of three domains, the first of which contains a beta sheet that binds FAD with a typical beta alpha beta nucleotide-binding motif and also a fingerprint motif for NADPH binding . The active site is located at the interface between the first and second domains; the second domain also binds the phenolic substrate . The third domain contains a thioredoxin-like fold and is involved in dimer contacts . The subunits within the dimer show substantial differences in structure and in FAD conformation . This conformational flexibility allows the substrate to gain access to the active site and excludes solvent during the hydroxylation reaction . CONCLUSIONS: Two of the domains of phenol hydroxylase are similar in structure to p-hydroxybenzoate hydroxylase . Thus, phenol hydroxylase is a member of a family of flavin-containing aromatic hydroxylases that share the same overall fold, in spite of large differences in amino acid sequences and chain length . The structure of phenol hydroxylase is consistent with a hydroxyl transfer mechanism via a peroxo-FAD intermediate . We propose that a movement of FAD takes place in concert with a large conformational change of residues 170-210 during catalysis.

Nucleic Acids Res, 1998 May 1, 26(9), 2143 - 9
DNA binding properties of a chemically synthesized DNA binding domain of hRFX1; Cornille F et al.; The RFX DNA binding domain (DBD) is a novel highly conserved motif belonging to a large number of dimeric DNA binding proteins which have diverse regulatory functions in eukaryotic organisms, ranging from yeasts to human . To characterize this novel motif, solid phase synthesis of a 76mer polypeptide corresponding to the DBD of human hRFX1 (hRFX1/DBD), a prototypical member of the RFX family, has been optimized to yield large quantities (approximately 90 mg) of pure compound . Preliminary two-dimensional1H NMR experiments suggested the presence of helical regions in this sequence in agreement with previously reported secondary structure predictions . In gel mobility shift assays, this synthetic peptide was shown to bind in a cooperative manner the 23mer duplex oligodeoxynucleotide corresponding to the binding site of hRFX1, with a 2:1 stoichoimetry due to an inverse repeat present in the 23mer . The stoichiometry of this complex was reduced to 1:1 by decreasing the length of the DNA sequence to a 13mer oligonucleotide containing a single half-site . Surface plasmon resonance measurements were achieved using this 5'-biotylinated 13mer oligonucleotide immobilized on an avidin-coated sensor chip . Using this method an association constant (K a = 4 x 10(5)/M/s), a dissociation constant (K d = 6 x 10(-2)/s) and an equilibrium dissociation constant (K D = 153 nM) were determined for binding of hRFX1/DBD to the double-stranded 13mer oligonucleotide . In the presence of hRFX1/DBD the melting temperature of the 13mer DNA was increased by 16 degreesC, illustrating stabilization of the double-stranded conformation induced by the peptide.

Oncogene, 1998 May, 16(20), 2629 - 37
Lack of transcriptional repression by max homodimers; Yin X et al.; Max, a basic-helix-loop-helix-leucine zipper (bHLH-ZIP) protein, plays a central role in the transcriptional regulation of myc oncoprotein-responsive genes . Myc-max heterodimers bind to consensus E-box motifs near or within the promoters of these genes and activate gene expression, whereas heterodimers between max and members of the mad family of bHLH-ZIP proteins promote transcriptional repression . In contrast to all other members of the myc network, max readily homodimerizes and binds to identical E-box sites in vitro . However, the role for max homodimers in transcriptional repression in vivo is unclear . Upstream stimulatory factor (USF) is a bHLH-ZIP protein which does not interact with members of the myc-max-mad family . By replacing the HLH-ZIP domain of max with that from USF, we created a chimeric protein, max(USF), which was indistinguishable from max with respect to its ability to homodimerize and bind DNA . As expected, however, max(USF) was unable to heterodimerize with any of the tested max partner proteins and was incapable of suppressing c-myc target genes . Thus, transcriptional repression is an exclusive property of max-mad heterodimers and cannot be achieved by max homodimers alone.

EMBO J, 1998 Jun 15, 17(12), 3398 - 412
Two distinct nuclear receptor interaction domains in NSD1, a novel SET protein that exhibits characteristics of both corepressors and coactivators; Huang N et al.; NSD1, a novel 2588 amino acid mouse nuclear protein that interacts directly with the ligand-binding domain (LBD) of several nuclear receptors (NRs), has been identified and characterized . NSD1 contains a SET domain and multiple PHD fingers . In addition to these conserved domains found in both positive and negative Drosophila chromosomal regulators, NSD1 contains two distinct NR interaction domains, NID-L and NID+L, that exhibit binding properties of NIDs found in NR corepressors and coactivators, respectively . NID-L, but not NID+L, interacts with the unliganded LBDs of retinoic acid receptors (RAR) and thyroid hormone receptors (TR), and this interaction is severely impaired by mutations in the LBD alpha-helix 1 that prevent binding of corepressors and transcriptional silencing by apo-NRs . NID+L, but not NID-L, interacts with the liganded LBDs of RAR, TR, retinoid X receptor (RXR), and estrogen receptor (ER), and this interaction is abrogated by mutations in the LBD alpha-helix 12 that prevent binding of coactivators of the ligand-induced transcriptional activation function AF-2 . A novel variant (FxxLL) of the NR box motif (LxxLL) is present in NID+L and is required for the binding of NSD1 to holo-LBDs . Interestingly, NSD1 contains separate repression and activation domains . Thus, NSD1 may define a novel class of bifunctional transcriptional intermediary factors playing distinct roles in both the presence and absence of ligand.

Biochim Biophys Acta, 1998 Apr 17, 1377(2), M61 - 70
The role of protein stability in the cell cycle and cancer; Elledge SJ et al.; In addition to the examples mentioned above, other important regulators of cell proliferation such as cyclin D, cyclin E, p21, p27 are all potentially controlled by ubiquitin-mediated proteolysis . In several of these, phosphorylation has been shown to play a role in targeting the proteins for degradation . It remains to be seen how important the SCF pathway and ubiquitin-mediated proteolysis, in general, will become in cancer research . However, it appears that we have only just scratched the surface on the role of these pathways in the control of important regulatory genes . We suspect there will be much more to come from analysis of these fascinating pathways.

Semin Cell Dev Biol, 1998 Apr, 9(2), 143 - 52
Protein phosphatases and the regulation of MAP kinase activity; Keyse SM; A family of dual specificity (Thr/Tyr) MAP kinase phosphatases (MKPs) have been identified in mammalian cells . These enzymes are implicated in negative feedback control of MAP kinase activity . This idea is supported by genetic and biochemical evidence which implicates homologous enzymes in the regulation of MAP kinases in yeasts and Drosophila . However, recent work in yeasts has shown that, in addition to these dual specificity MKPs, 'classical' tyrosine-specific phosphatases are also involved in the regulated dephosphorylation of MAP kinases . A picture is emerging in which a complex interplay between upstream activators and multiple protein phosphatases is responsible for the regulation of MAP kinase activity . The activities, substrate specificities and subcellular localisation of these protein phosphatases are likely to be key determinants of the biological outcome of signalling through different MAP kinase pathways in mammalian cells and tissues.

Semin Cell Dev Biol, 1998 Apr, 9(2), 111 - 8
Extracellular degradation of agonists as an adaptive mechanism; Ladds G et al.; Many cellular responses are initiated by the binding of an extracellular ligand to receptors at the cell surface . The removal of these ligands, or agonists, would therefore be expected to contribute to the process by which cells recover from stimulation . While dilution in the extracellular medium reduces the concentration of most agonists, many cells have developed specific mechanisms for removing particular ligands . One of the more effective mechanisms is to degrade the extracellular agonist and here we review the production and action of the enzymes responsible for the degradation of a number of these agonists.

Mol Mar Biol Biotechnol, 1998 Mar, 7(1), 62 - 71
Identification and preliminary characterization of PCNA gene in the marine phytoplankton Dunaliella tertiolecta and Isochrysis galbana; Lin S et al.; The gene coding for proliferating cell nuclear antigen (PCNA) was identified in Dunaliella tertiolecta (Chlorophyceae) and Isochrysis galbana (Prymnesiophyceae) . Southern blot hybridization using a PstI fragment of rat PCNA gene (pCR-1) as a probe showed that there is apparently a single copy of this gene per haploid genome in both species . On the Northern blot pCR-1 probed a single messenger RNA for each species of a molecular size close to rat PCNA mRNA (1.1 kilobases {kb}) . Polymerase chain reaction (PCR) with a set of degenerated primers produced a fragment of about 610 base pairs {bp} from genomic DNA of both species; the PCR products appeared close in size to the amplified from rat PCNA and hybridized to the pCR-1 probe . Further analysis with reverse transcription-PCR (RT-PCR), cloning, and sequencing revealed a complementary DNA of a similar size (616 and 576 bp) that possesses an open reading frame encoding 205 and 192 amino acids, respectively, for Dun (D . tertiolecta) and Iso (I . galbana) . Surprisingly, the polypeptides deduced from the two cDNA shared no higher homology to each other (71%) than to animals such as Xenopus (Dun 72%; Iso 73%), rat (Dun 73%; Iso 74%), and human (Dun 73%; Iso 74%), and to higher plants such as soybean (Dun 78%; Iso 72%), Zea mays (Dun 77%; Iso 73%), and rice (Dunn 77%; Iso 72%), although D . tertiolecta has a higher homology (77%) to the Prasinophyceae alga Tetraselmis chiu than does I . galbana (71%) . The homology to PCNA in budding and fision yeasts (63% and 53%, respectively) is also lower than to animals and higher plants . It is thus suggested that with regard to PCNA genes, the three algae are as different from each other as they are from higher plants and animals . In a partially synchronized exponential culture of D . tertiolecta grown with a photocycle of 12 h light and 12 h dark, the abundance of the transcript appeared to be low at hours 3 and 9 (hour 0 = the onset of light period), and increased about 2- to 3-fold at hours 15 and 21 (i.e., during the dark period) . Western blotting and immunofluorescence analysis on concurrent diel samples showed an over 2-fold increase in PCNA protein abundance (in proportion to total cellular protein) and the percentage of cells labeled by PCNA antibody . A similar trend was found for I . galbana grown under the same conditions . The results suggest that the gene transcription was in pace with PCNA synthesis, which was lower in the light period when G1 phase was dominant and higher in the dark period when S (and probably G2 and early M) phase was dominant, and that the expression of this gene may be regulated at the transcriptional level in these two algae.

Drugs, 1998 May, 55(5), 645 - 74
An overview of topical antifungal therapy in dermatomycoses . A North American perspective; Gupta AK et al.; Dermatophytes cause fungal infections of keratinised tissues, e.g . skin, hair and nails . The organisms belong to 3 genera, Trichophyton, Epidermophyton and Microsporum . Dermatophytes may be grouped into 3 categories based on host preference and natural habitat . Anthropophilic species predominantly infect humans, geophilic species are soil based and may infect both humans and animals, zoophilic species generally infect non-human mammals . It is important to confirm mycologically the clinical diagnosis of onychomycosis and other tinea infections prior to commencing therapy . The identity of the fungal organism may provide guidance about the appropriateness of a given topical antifungal agent . Special techniques may be required to obtain the best yield of fungal organisms from a given site, especially the scalp and nails . It is also important to realise the limitations of certain diagnostic aids e.g., Wood's light examination is positive in tinea capitis due to M . canis and M . audouinii (ectothrix organisms); however, Wood's light examination is negative in T . tonsurans (endothrix organism) . Similarly, it is important to be aware that cicloheximide in culture medium will inhibit growth of non-dermatophytes . Appropriate media are therefore required to evaluate the growth of some significant non-dermatophyte moulds . For tinea infections other than tinea capitis and tinea unguium, topical antifungals may be considered . For effective therapy of tinea capitis an oral antifungal is generally necessary . Similarly, oral antifungals are the therapy of choice, especially if onychomycosis is moderate to severe . Furthermore, where the tinea infection involves a large area, in an immunocompromised host or if infection is recurrent with poor response to topical agents, then oral antifungal therapy may be necessary . Topical antifungal agents may be broadly divided into specific and nonspecific agents . The former group includes the polyenes, azoles, allylamines, amorolfine, ciclopirox and butenafine . Generally the topical agent is available as a cream, sometimes for use intravaginally . Less commonly, the formulation may be in the form of a powder, lacquer, spray, gel or solution . Many of these agents have a broad spectrum of activity, being effective against dermatophytes, yeasts and Malassezia furfur . For the treatment of tinea corporis, tinea cruris tinea versicolor and cutaneous candidosis, once or twice daily application may be required, the most common duration of therapy being 2 to 4 weeks . For tinea pedis the most common treatment duration is 4 to 6 weeks.

Appl Environ Microbiol, 1998 May, 64(5), 1947 - 9
Cloning of the ribosomal protein L41 gene of Phaffia rhodozyma and its use a drug resistance marker for transformation; Kim IG et al.; The ribosomal protein L41 gene of Phaffia rhodozyma was cloned and used as a dominant selectable marker for cycloheximide resistance in the transformation of P . rhodozyma . Electrotransformation with a plasmid containing a ribosomal DNA fragment as a targeting signal typically yielded 800 to 1,200 transformants/microgram of DNA with an integrated copy number of about seven per haploid genome.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3603 - 8
Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells; Yan Z et al.; Cdc6 has a critical regulatory role in the initiation of DNA replication in yeasts, but its function in mammalian cells has not been characterized . We show here that Cdc6 is expressed selectively in proliferating but not quiescent mammalian cells, both in culture and within tissues of intact animals . During the transition from a growth-arrested to a proliferative state, transcription of mammalian Cdc6 is regulated by E2F proteins, as revealed by a functional analysis of the human Cdc6 promoter and by the ability of exogenously expressed E2F proteins to stimulate the endogenous Cdc6 gene . Immunodepletion of Cdc6 by microinjection of anti-Cdc6 antibody blocks initiation of DNA replication in a human tumor cell line . We conclude that expression of human Cdc6 is regulated in response to mitogenic signals though transcriptional control mechanisms involving E2F proteins, and that Cdc6 is required for initiation of DNA replication in mammalian cells.

Dev Growth Differ, 1998 Feb, 40(1), 85 - 95
Centrosome-attracting body: a novel structure closely related to unequal cleavages in the ascidian embryo; Hibino T et al.; The mechanism of unequal cleavage is one of the most intriguing subjects in cell biology . Previous studies of unequal cleavage have focused on a limited number of organisms such as yeasts, nematodes, sea urchins and annelids . The cleavage pattern of the ascidian embryo is invariant . In the ascidian embryo, the posterior-most blastomeres divide unequally in three successive cleavages . In the present study, it was shown that the ascidian embryo provides another good experimental system with which to analyze the mechanism of unequal cleavage . A novel structure, designated as CAB (centrosome-attracting body), which was found specifically in the unequally cleaving blastomeres was described . In the course of unequal cleavages, first, a thick microtubule bundle appeared between CAB and one of the centrosomes . Then with the shortening of the microtubule bundle, the nucleus with the centrosome was drawn toward CAB, situated at the posterior cortex of the blastomere . Finally, a cleavage furrow formed in the middle of the asymmetrically located mitotic apparatus and produced two blastomeres of different size, generating a smaller cell that inherits CAB . The CAB seemed to play an essential role in the unequal cleavages in the ascidian embryo.

Antimicrob Agents Chemother, 1998 Apr, 42(4), 762 - 6
Amphotericin B in lipid emulsion: stability, compatibility, and in vitro antifungal activity; Walker S et al.; Newer formulations of amphotericin B (AmB) complexed with liposomes or lipid suspensions have been developed . Preliminary studies have suggested that AmB in Intralipid (IL) may be as effective as, but less toxic than, conventional formulations of AmB, but few data are available regarding its stability, compatibility, or in vitro antifungal activity . A compatibility study was done to evaluate the effects of AmB concentrations in IL containing either 10 or 20% soybean oil . The effects of temperature, shaking, and AmB and IL concentrations on the stability of AmB-IL suspensions were analyzed by visual inspection and liquid chromatography . The in vitro antifungal activity of AmB-IL, compared to that of AmB alone against reference strains of Candida species was determined by using a broth macrodilution method in accordance with National Committee for Clinical Laboratory Standards guidelines (M27-T) . Samples of AmB-IL which were lightly shaken retained more than 90% of the AmB concentration over 21 days when stored at either 4 or 23 degrees C . Varying the AmB concentration did not appear to affect the stability of AmB-IL . However, a precipitate was formed when mixtures with more than 30% lipid as a proportion of the total volume were centrifuged . AmB-IL and AmB alone had similar in vitro antifungal activities against reference strains of yeasts . Further pharmacologic and clinical studies with AmB-IL are warranted, although AmB should not be combined with IL in concentrations capable of producing a precipitate.

Cent Eur J Public Health, 1998 Feb, 6(1), 61 - 6
Growing importance of non-Candida species as opportunistic pathogens; Tomsikova A; A 40-year study of C . albicans, non-C . albicans species and non-Candida species in the excretions of patients with different diseases is described . An increase of non-C . albicans, and non-Candida species since 1980 has been determined . The study is completed by a review of new and emerging yeasts.

Virology, 1998 Mar 15, 242(2), 319 - 26
Group I introns found in Chlorella viruses: biological implications; Nishida K et al.; More than 80 group I introns were detected and characterized in Chlorella viruses isolated from various locations in Japan; the overall average frequency of viruses containing the group I intron was 8.0% . Although most of these introns were inserted in the gene for either transcriptional elongation factor TFIIS (approximately 60%) or URF 14.2 (unidentified open reading frame coding for a 14.2-kDa polypeptide) (approximately 40%), in a few cases, the gene for the major capsid protein Vp52 contained an intron . These introns were biologically active (self-splicing) both in vivo and in vitro . Viruses that contained introns almost usually contained only one, but more than two introns coexisted in several virus isolates . Nucleotide sequence analysis showed that the intron sequences have diverged under strong constraint of the exon genes: introns in the same gene showed more than 99% sequence identity, whereas introns in different genes were only 72-78% identical . Phylogenetic analysis suggested relatedness of these introns to those found in the rRNA genes of a variety of organisms including green algae, red algae, red algae, yeasts, fungi, and protozoa.

Anal Biochem, 1998 Mar 15, 257(2), 218 - 23
Enzymatic synthesis of a neoglycoconjugate by transglycosylation with Arthrobacter endo-beta-N-acetylglucosaminidase: a substrate for colorimetric detection of endo-beta-N-acetylglucosaminidase activity; Takegawa K et al.; The transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae was used for the enzymatic synthesis of a novel oligosaccharide, Man6GlcNAc-p-nitrophenyl-alpha-D-glucose (Man6GlcNAc-Glc-pNP) . The reaction was efficiently induced in aqueous solution containing dimethyl sulfoxide . In the medium containing 20% (v/v) dimethyl sulfoxide with 0.1 M Glc-pNP as an acceptor, the transglycosylation attained yields of 75% by high-performance anion-exchange chromatography . The structure of Man6GlcNAc-Glc-pNP was confirmed by ion mass spectrometry and 400 MHz 1H NMR spectrometry . Various endo-beta-N-acetylglucosaminidases hydrolyzed this oligosaccharide and Man6GlcNAc and Glc-pNP were released from the oligosaccharide by endo-beta-N-acetylglucosaminidase digestion . We have established a new procedure for the colorimetric detection of endo-beta-N-acetylglucosaminidase activity using Man6Glc-NAc-Glc-pNP, which is simple as that for other exoglycosidase assays with pNP-glycosides as substrates.

Ann Pharmacother, 1998 Feb, 32(2), 204 - 14
Current issues in onychomycosis; Trepanier EF et al.; OBJECTIVE: To review the epidemiology, mycology, clinical features and diagnosis, current pharmacotherapy, and pharmacoeconomics of onychomycosis . DATA SOURCES: We conducted a MEDLINE search from 1966 to May 1997 . References from these articles, manufacturers of the discussed antimycotics, and relevant abstracts from recent dermatology conferences were used to collect pertinent data . DATA EXTRACTION: Data were obtained from published controlled studies and case reports . In the pharmacotherapy section, the most weight was placed on fully reported, randomized, controlled comparative trials, but abstracts and case series were included when well-controlled studies were unavailable . DATA SYNTHESIS: Onychomycosis is a common nail disorder that has a substantial impact on patients' quality of life . It is most commonly caused by dermatophytes, but yeasts and molds can also be involved . Diagnosis is made through clinical presentation, potassium hydroxide preparations, and culture of tissue/nail samples . Griseofulvin was the drug of choice for many years, but its low cure rates and the development of newer, more effective drugs made it fall out of favor . Current therapeutic alternatives include fluconazole, itraconazole, and terbinafine . Data on the use of fluconazole are limited to case series and reports . Continuous dosing of itraconazole and terbinafine are well-proven therapies . New data are becoming available on the use of pulse itraconazole dosing, which has recently been approved by the Food and Drug Administration for fingernail infections . These drugs are well tolerated, but attention to drug interactions is necessary with the azoles . CONCLUSIONS: Currently, continuous terbinafine appears to be the most cost-effective drug for dermatophyte onychomycosis.

Mycoses, 1997 Nov, 40(7-8), 243 - 7
The hydroxypyridones: a class of antimycotics of its own; Korting HC et al.; The hydroxypyridones chemically form a class of antimycotics not related to others . For a long time ciclopirox olamine has been the only compound to be used clinically . Recently, ciclopirox as a free acid and rilopirox have also been considered . The congeners are active against a broad spectrum of medically relevant fungi including dermatophytes, yeasts and moulds in vitro . According to investigations based on animal and in vivo models for fungal disease, this also applies in vivo . Low toxicity in the experimental animal has made topical applications in man possible . Clinical experience relates, in particular, to dermatophytosis and vaginal candidosis, but rarer conditions have also been investigated . High tolerability corresponds to efficacy in major dermatomycological conditions . Today, interest mainly focuses on ciclopirox lacquer for onychomycosis . Here as in general, the relative role of hydroxypyridones will have to be finally established in comparative trials.

Mycoses, 1997 Oct, 40(5-6), 193 - 6
Fungaemia caused by Hansenula anomala--an outbreak in a cancer hospital; Thuler LC et al.; Yeasts belonging to the genus Hansenula are rarely encountered as the cause of infection in clinical practice . A wide spectrum of infections caused by these fungi can be seen, ranging from asymptomatic fungaemia to severe disease . We describe an outbreak of 24 cases of infection due to H . anomala in an oncological hospital in Rio de Janeiro, Brazil . The median age of the patients was 11 years, of whom 54.2% were female; 91.7% of the Hansenula fungaemia occurred in the haematology unit . The most frequent primary disease diagnosis was leukaemia (62.5%), and all of those infected had had a central venous catheter or peripheral venous catheter and had been treated previously with broad-spectrum antibiotics . Numerous other risk factors were observed in our cases: previous use of steroids, chemotherapy, radiation therapy and neutropenia (data not shown) . No deaths could be attributed to Hansenula.

Biochemistry (Mosc), 1997 Nov, 62(11), 1242 - 53
Telomere functions . A review; Kurenova EV et al.; Telomeres are structurally and functionally complex . They consist of an array of simple DNA repeats at the extreme end of the chromosome with a more complex array of repeats adjacent to it . A large number of proteins have been identified that bind to the telomeric DNA repeats or to the protein complexes that are built at the chromosome end . Telomeres tend to form associations with each other . These associations have been implicated in the formation of nuclear domains that may be important for transcriptional regulation, for sister chromatid pairing at mitosis, and for homologous meiotic synapsis . Telomeric chromosome ends do not cause delays in cell cycle progression, nor are they subject to DNA repair as are broken chromosome ends . Telomeres also provide a separate mechanism for adding additional copies of the telomeric DNA to chromosome ends . This is needed to counterbalance the loss of DNA sequences from chromosome ends due to incomplete DNA replication . The components that participate in the latter mechanism and this process have been characterized in detail; the other functions of telomeres are less well understood but are the subjects of active investigation.

J Neurosci, 1998 Feb 1, 18(3), 914 - 22
Interaction of presenilins with the filamin family of actin-binding proteins; Zhang W et al.; Mutations in presenilin genes PS1 and PS2 account for approximately 50% of early-onset familial Alzheimer's disease (FAD) . The PS1 and PS2 genes encode highly homologous transmembrane proteins related to the Caenorhabditis elegans sel-12 and spe-4 gene products . A hydrophilic loop region facing the cytoplasmic compartment is likely to be functionally important because at least 14 mutations in FAD patients have been identified in this region . We report here that the loop regions of PS1 and PS2 interact with nonmuscle filamin (actin-binding protein 280, ABP280) and a structurally related protein (filamin homolog 1, Fh1) . Overexpression of PS1 appears to modify the distribution of ABP280 and Fh1 proteins in cultured cells . A monoclonal antibody recognizing ABP280 and Fh1 binds to blood vessels, astrocytes, neurofibrillary tangles, neuropil threads, and dystrophic neurites in the AD brain . Detection of ABP280/Fh1 proteins in these structures suggests that these presenilin-interacting proteins may be involved in the development of AD and that interactions between presenilins and ABP280/Fh1 may be functionally significant . The ABP280 gene is located on the human X chromosome, whereas the newly identified Fh1 gene maps to human chromosome 3 . These results provide a new basis for understanding the function of presenilin proteins and further implicate cytoskeletal elements in AD pathogenesis.

Br J Dermatol, 1997 Nov, 137(5), 764 - 7
Susceptibility of Malassezia furfur subgroups to terbinafine; Leeming JP et al.; Malassezia furfur, the fungus causing pityriasis versicolor, has been reported to be sensitive to terbinafine in vitro but although topical therapy is effective in the treatment of pityriasis versicolor, oral therapy is not . This phenomenon was investigated by determining the susceptibility to terbinafine of different M . furfur subgroups in vivo (during topical or oral application) and in vitro . All M . furfur subgroups were suppressed (approximately 10-fold) by topical terbinafine . Oral treatment resulted in no significant suppression of cutaneous M . furfur populations with the exception of a single subgroup (A), which was reduced to undetectable levels on the skin of eight of 10 patients receiving oral terbinafine . Isolates of subgroup A were also markedly more susceptible to terbinafine in laboratory tests . The importance of the recognition of distinct subgroups within the cutaneous lipophilic yeasts when evaluating their antifungal susceptibility and their role in disease is discussed.

Mol Cell Biol, 1998 Feb, 18(2), 710 - 20
The Elk-1 ETS-domain transcription factor contains a mitogen-activated protein kinase targeting motif; Yang SH et al.; The phosphorylation of transcription factors by mitogen-activated protein kinases (MAP) is a pivotal event in the cellular response to the activation of MAP kinase signal transduction pathways . Mitogenic and stress stimuli activate different pathways and lead to the activation of distinct groups of target proteins . Elk-1 is targeted by three distinct MAP kinase pathways . In this study, we demonstrate that the MAP kinase ERK2 is targeted to Elk-1 by a domain which is distinct from, and located N-terminally to, its phosphoacceptor motifs . Targeting via this domain is essential for the efficient and rapid phosphorylation of Elk-1 in vitro and full and rapid activation in vivo . Specific residues involved in ERK targeting have been identified . Our data indicate that the targeting of different classes of MAP kinases to their nuclear substrates may be a common mechanism to increase the specificity and efficiency of this signal transduction pathway.

Trends Genet, 1997 Dec, 13(12), 489 - 96
The case for epigenetic effects on centromere identity and function; Karpen GH et al.; The centromere is required to ensure the equal distribution of replicated chromosomes to daughter nuclei . Centromeres are frequently associated with heterochromatin, an enigmatic nuclear component that causes the epigenetic transcriptional repression of nearby marker genes (position-effect variegation or silencing) . The process of chromosome segregation by movement along microtubules to spindle poles is highly conserved, yet the putative cis-acting centromeric DNA sequences bear little or no similarity across species . Recently, studies in several systems have revealed that the centromere itself might be epigenetically regulated and that the higher-order structure of the underlying heterochromatin contributes to centromere function and kinetochore assembly.

Mol Cell Biol, 1998 Jan, 18(1), 576 - 89
Role of UEV-1, an inactive variant of the E2 ubiquitin-conjugating enzymes, in in vitro differentiation and cell cycle behavior of HT-29-M6 intestinal mucosecretory cells; Sancho E et al.; By means of differential RNA display, we have isolated a cDNA corresponding to transcripts that are down-regulated upon differentiation of the goblet cell-like HT-29-M6 human colon carcinoma cell line . These transcripts encode proteins originally identified as CROC-1 on the basis of their capacity to activate transcription of c-fos . We show that these proteins are similar in sequence, and in predicted secondary and tertiary structure, to the ubiquitin-conjugating enzymes, also known as E2 . Despite the similarities, these proteins lack a critical cysteine residue essential for the catalytic activity of E2 enzymes and, in vitro, they do not conjugate or transfer ubiquitin to protein substrates . These proteins constitute a distinct subfamily within the E2 protein family and are highly conserved in phylogeny from yeasts to mammals . Therefore, we have designated them UEV (ubiquitin-conjugating E2 enzyme variant) proteins, defined as proteins similar in sequence and structure to the E2 ubiquitin-conjugating enzymes but lacking their enzymatic activity (HW/GDB-approved gene symbol, UBE2V) . At least two human genes code for UEV proteins, and one of them, located on chromosome 20q13.2, is expressed as at least four isoforms, generated by alternative splicing . All human cell types analyzed expressed at least one of these isoforms . Constitutive expression of exogenous human UEV in HT-29-M6 cells inhibited their capacity to differentiate upon confluence and caused both the entry of a larger proportion of cells in the division cycle and an accumulation in G2-M . This was accompanied with a profound inhibition of the mitotic kinase, cdk1 . These results suggest that UEV proteins are involved in the control of differentiation and could exert their effects by altering cell cycle distribution.

Curr Opin Hematol, 1996 Jan, 3(1), 27 - 34
Stress- and mitogen-activated signal transduction in hematopoietic cells; Kieran MW et al.; The ability of an organism to respond to its environment is critical to survival . The mechanisms by which cells recognize and interpret different stimuli vary enormously and can be manifested by proliferation, differentiation, apoptosis, or altered metabolic activity . Recently, a series of signaling cascades was identified that links actions on the cell surface with activation or suppression of transcription . These signals are transmitted via phosphorylation and include the stress-activated protein kinases and the mitogen-activated protein kinases . This review describes these cascades in reference to the hematopoietic cell lineages.

Math Biosci, 1997 Dec, 146(2), 115 - 30
Mathematical description of synergistic interaction of hyperthermia and ionizing radiation; Petin VG et al.; A simple mathematical model of simultaneous combined action of ionizing radiation and hyperthermia has been proposed . The model suggests that the synergistic interaction of ionizing radiation and hyperthermia is expected to result from the additional lethal damage arising from the interaction of sublesions induced by both agents . These sublesions are considered nonlethal after each agent taken alone . The model was applied to the quantitative analysis of the simultaneous action of hyperthermia and ionizing radiation . It predicts the dependence of synergistic interaction of the ratio of lethal events produced by every agent used, as well as the maximal value of the synergistic effect, conditions at which the maximal interactive effect can be achieved, and the dependence of synergistic effect on dose rate . The predictions of the model have been tested with four experimental data sets reported in the literature . The theory appears to be appropriate and the conclusions valid.

Diagn Microbiol Infect Dis, 1997 Nov, 29(3), 147 - 53
An outbreak of Candida parapsilosis prosthetic valve endocarditis; Diekema DJ et al.; Candida parapsilosis, an important nosocomial pathogen and the most common species of Candida found on the hands of health care workers, is a rare cause of prosthetic valve endocarditis (PVE) . From March through June 1994, four cases of C . parapsilosis PVE were diagnosed at a 400-bed community hospital . The mean time to presentation after valve replacement surgery was 148 days (range, 20 to 345) . Three of the four patients died of complications of PVE . Multiple environmental cultures were performed, and only one was positive for C . parapsilosis . Cultures from the bypass pump, cell saver, cardioplegia solution, and subsequent valves were all negative . All valve replacements were performed by the same operating room team . Interviews with the surgeon and physician assistant, the only personnel involved in all cases, revealed that their hypoallergenic gloves were subject to frequent tears during valve replacement procedures, often requiring several glove changes per procedure . Hand cultures of personnel were obtained, and cultures from 20 individuals (26%) were positive for C . parapsilosis . Hand cultures of the surgeon and physician assistant obtained 8 months after the last case had surgery were negative for yeasts . Molecular typing of the 3 available case isolates, 14 epidemiologically unrelated patient isolates, 1 environmental isolate, and 20 hand isolates was performed by electrophoretic karyotyping and restriction endonuclease analysis of genomic DNA using restriction enzymes BssHII and EagI followed by pulsed field gel electrophoresis . The three case isolates were identical by restriction endonuclease analysis of genomic DNA, and two of the three shared the same electrophoretic karyotyping profile . The remaining patient, environmental, and hand isolates represented 29 different DNA types and were distinctly different from the case isolates . All of the isolates tested were susceptible to amphotericin B, 5FC, fluconazole, and itraconazole . The circumstantial evidence suggests the probability of glove tears during valve replacement surgery and subsequent transmission of C . parapsilosis to patients.

Plant Cell, 1997 Nov, 9(11), 1973 - 83
The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development; Choi W et al.; Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection structure called an appressorium that is crucial for host plant penetration . Previously, it was found that cAMP regulates appressorium formation . To further understand the cellular mechanisms involved in appressorium formation, we have cloned a gene (MAC1) encoding adenylate cyclase, a membrane-bound enzyme that catalyzes the production of cAMP from ATP, by using a polymerase chain reaction-based strategy . The entire gene was isolated and subcloned from a large insert bacterial artificial chromosome library . Sequence characterization showed that MAC1 has a high degree of identity with other adenylate cyclase genes from several filamentous fungi as well as yeasts . Gene deletion resulted in reduced vegetative growth, conidiation, and conidial germination . Transformants lacking MAC1 were unable to form appressoria on an inductive surface and were unable to penetrate susceptible rice leaves . mac1- transformants were also sterile and produced no perithecia . Appressorium formation was restored in the presence of exogenous cAMP derivatives . These results confirm that cell signaling involving cAMP plays a central role in the development and pathogenicity of M . grisea.

Biochemistry, 1997 Dec 2, 36(48), 14661 - 75
NMR studies of the role of hydrogen bonding in the mechanism of triosephosphate isomerase; Harris TK et al.; Triosephosphate isomerase (TIM) catalyzes the reversible interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP), with Glu-165 removing the pro-R proton from C1 of DHAP and neutral His-95 polarizing the carbonyl group of the substrate . TIM and its complexes with the reactive intermediate analogs, phosphoglycolic acid (PGA) and phosphoglycolohydroxamic acid (PGH), were studied by 1H NMR at 600 MHz and at low temperature (-4.8 degrees C) . His-95 shows an N epsilon H resonance at 13.1 ppm which shifts to 13.3 ppm in the TIM-PGA complex and to 13.5 ppm in the TIM-PGH complex . In the TIM-PGH complex, His-95 N epsilon H shows a slow, pH-independent exchange rate with water (kex = 80 s-1 at 30 degrees C, Eact = 19 kcal/mol), which is 44-fold slower than that of an exposed histidine suggesting partial shielding from bulk solvent, and a fractionation factor phi = 0.71 +/- 0.02 consistent with its donation of a normal hydrogen bond . The formation of the TIM-PGH complex results in the appearance of several deshielded proton resonances, including one at 14.9 ppm and one at 10.9 ppm which overlaps with another resonance . The resonance at 14.9 ppm is absent and the resonance at 10.9 ppm is much weaker in the TIM complex of PGA, which lacks the hydroxamic acid (-NHOH) moiety . 15N-labeled PGH was synthesized and the NH proton of free {15N}PGH shows a single 1H-15N HMQC cross peak with delta (1H) = 10.3 ppm and delta (15N) = 168 ppm which shifts to delta (1H) = 10.9 ppm and delta (15N) = 174 ppm in the TIM complex of {15N}PGH . The 15N-1H coupling in the complex indicates covalent N-H bonding, and the deshielded delta (15N) indicates a significant contribution of the imidate resonance form of PGH . The 14.9 ppm resonance is assigned to the NOH proton of bound PGH . This resonance shows a pH-independent exchange rate with water (kex = 3900 s-1 at 30 degrees C, Eact = 8.9 kcal/mol) which may reflect the dissociation of the TIM-PGH complex, and meets the criteria for a low-barrier hydrogen bond on the basis of the significant downfield shift of 6.2 ppm from the NOH proton of the model compound acetohydroxamic acid, and a very low fractionation factor phi = 0.38 +/- 0.06 . In the X-ray structure of the TIM-PGH complex {Davenport, R . C., Bash, P . A., Seaton, B . A., Karplus, M., Petsko, G . A., and Ringe, D . (1991) Biochemistry 30, 5821}, the NOH proton of bound PGH is hydrogen bonded to Glu-165 . A low-barrier hydrogen bond from PGH NOH to Glu-165 suggests a dual role for Glu-165 in catalysis of proton transfer not only between the C1 and C2 carbons but also between the O1 and O2 oxygens in the interconversion of DHAP and GAP in wild type TIM . Such a mechanism, together with the measured exchange rate of the His-95 N epsilon H proton with solvent protons can accommodate the classical measurements of tritium incorporation from DHAP into GAP.

FEBS Lett, 1997 Nov 3, 417(1), 109 - 13
Proteins interacting with the molecular chaperone hsp70/hsc70: physical associations and effects on refolding activity; Gebauer M et al.; We investigated several hsp70/hsc70 interacting proteins and established by two independent techniques that hsp40 and Hop/p60 specifically interact with the 257 residue carboxy-terminal domain of hsp70 while Hap-46 and Hip/p48 bind the 383 residue amino-terminal ATP binding domain . Hap-46 and Hip/p48 competed for binding to hsc70, while Hap-46 had no effect on the binding of either Hop/p60 or hsp40 to hsc70 . Hap-46 inhibited the refolding of thermally denatured firefly luciferase in an hsc70 and hsp40 dependent assay, and this effect was largely compensated by Hop/p60 . These interacting proteins thus appear to cooperate in affecting the chaperoning activity of hsp70/hsc70.

Yeast, 1997 Oct, 13(13), 1231 - 42
Molecular characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Phaffia rhodozyma; Verdoes JC et al.; The glyceraldehyde-3-phosphate dehydrogenase (GPD; EC1.2.1.12)-encoding gene (gpd) was isolated from a genomic library of Phaffia rhodozyma CBS 6938 . Unlike some other eukaryotic organisms the gpd gene is represented by a single copy in P . rhodozyma . The complete nucleotide sequence of the coding, as well as the flanking non-coding regions was determined . The nucleotide sequence of gpd predicted six introns and a polypeptide chain of 339 amino acids . The codon usage in the gpd gene of P . rhodozyma was highly biased and was significantly different from the codon usage in other yeasts . Phylogenetic analysis of different yeasts and filamentous asco- and basidiomycetes gpd sequences indicated that the gpd gene of P . rhodozyma forms a cluster with the corresponding genes of filamentous basidiomycetes.

J Nat Prod, 1997 Oct, 60(10), 986 - 90
Three new pseudodistomins, piperidine alkaloids from the ascidian Pseudodistoma megalarva; Freyer AJ et al.; Bioassay-guided fractionation of the MeOH-CH2Cl2 extract of the Micronesian ascidian Pseudodistoma megalarva yielded three new piperidine alkaloids, pseudodistomins D-F (3-5) and the previously reported pseudodistomins B and C (1 and 2) . The structure and stereochemistry of these compounds were established by interpretation of spectral data . Pseudodistomins B-F were found to be active in a cell-based assay for DNA damage induction, but the activity was due to an alternative mechanism.

Curr Med Res Opin, 1997, 13(10), 627 - 34
HIV antibodies: further questions and a plea for clarification; Papadopulos-Eleopulos E et al.; The existence of specific antibody/protein reactions is the crucial assumption underlying proof of HIV isolation, proof of HIV infection and the causative role of HIV in AIDS . However, since 1 . antibodies which react with the 'HIV' proteins arise following allogenic stimuli in non-HIV-infected animals and humans, as well as in mice and humans with autoimmune disorders; antibodies to antigens from both mycobacteria and yeasts cross-react with HIV env and gag proteins; 2 . individuals belonging to the AIDS risk groups are subjected to allogenic stimuli and have high levels of autoimmune antibodies, while the vast majority of patients in the AIDS risk groups are infected with either or both mycobacteria or yeasts; the evidence for the existence of HIV and its putative role in AIDS must be reappraised.

Microbiol Immunol, 1997, 41(9), 747 - 50
Fc receptor-mediated phagocytosis, superoxide production and calcium signaling of beta 2 integrin-deficient bovine neutrophils; Nagahata H et al.; Fc receptor for immunoglobulin G-mediated phagocytosis, superoxide production and intracellular calcium ({Ca2+}i) signaling of complement receptor type 3 (CR3)-deficient neutrophils from a heifer with leukocyte adhesion deficiency (BLAD) were compared to those of control heifers . The mean phagocytic activity of IgG-coated yeasts and aggregated bovine IgG (Agg-IgG)-induced superoxide production of CR3-deficient neutrophils were 10% and 77.9%, respectively, of those of control neutrophils . The {Ca2+}i signals in CR3-deficient neutrophils stimulated with Agg-IgG or concanavalin A were different with mean peak {Ca2+}i concentrations of 78% and 41.9%, respectively, of those of control neutrophils . These findings suggest that Fc receptor-mediated neutrophil functions are closely dependent on the presence of CR3 (CD11b/CD18) on the neutrophil cell surfaces.

Rinsho Byori, 1997 Jun, 45(6), 599 - 601
{A case of sporotrichosis with numerous fungal elements}; Furuta T et al.; We report a case of cutaneous sporotrichosis with numerous fungal elements in a 59-year-old Japanese male treated with topical corticosteroid ointment . Histologically, granulomas were found with a few of neutrophils, many lymphocytes and plasmocytes in the upper dermis . Numerous yeasts were found in the granulomas . They were stained with periodic acid Schiff, but not with hematoxylin and eosin . No asteroid body was detected . After treatment of the corticosteroid, histological pattern is different from usual pattern, such as the number of fungal elements is larger, extent of neutrophil infiltration is slighter and very rare appearance of asteroid body . These histological differences are likely to be induced by the local immunosuppression after corticosteroid treatment.

Cancer Surv, 1997, 29, 221 - 61
Functions of the DNA dependent protein kinase; Jin S et al.; The DNA dependent protein kinase (DNA-PK) is a trimeric nuclear complex consisting of a large protein kinase and the Ku heterodimer that regulates kinase activity by its association with DNA . Recent findings have shown structural similarities between DNA-PK and a family of lipid and putative protein kinases (PIK family) . DNA-PK is one of the PIK members known to be a protein kinase with clearly identified effector subunits . A broad range of observations link DNA-PK to dual roles in double strand DNA break (DSB) repair and transcription . Unlike its most closely related PIKs, DNA-PK is not required for activating cell cycle regulated DNA damage signalling mechanisms . Instead, the phenotypes and biochemical properties of DNA-PK are most consistent with functions in DSB repair and joining steps in recombination mechanisms . DNA-PK is associated with RNA polymerase II and RNA polymerase I transcription complexes, where it most frequently has a negative regulatory role.

Cancer Surv, 1997, 29, 133 - 50
The anaphase promoting complex; Page AM et al.; We have proposed a preliminary model of how the anaphase promoting complex functions throughout the cell cycle, but despite the flurry of recent publications characterizing the APC--its components, regulation and substrate specificity--many fundamental questions remain to be answered . Firstly, the remaining components of the APC need to be identified and characterized . We do not know if all cyclosome components are conserved in all eukaryotes, or if higher eukaryotes, having a more complicated cell cycle machinery, maintain additional subunits for more sophisticated functional and regulatory control . In addition, we need to determine the identity of the various kinases and phosphatases that regulate the APC itself . The biochemistry of individual APC components is also a mystery, and a specific biochemical function has not been assigned to any known members of the complex . It is not at all clear which subunit(s) of the complex actually recognizes the E2 enzyme and which subunit(s) recognizes the cyclin destruction box . It is likely that many cyclosome substrates remain to be identified, and it will be interesting to determine whether all cyclosome substrates require a destruction box for their degradation or whether the APC recognizes other determinants of protein instability . Finally, we assume that the APC degrades mitotic cyclins in all proliferating cells, but whether it degrades unique cell cycle related substrates in specific tissues is unclear . Furthermore, nothing is known about APC function during meiosis, or whether the APC degrades other substrates that are not related to the cell cycle . This is an exciting and rapidly developing field in the exciting world of cell cycle biology . We expect that new findings will surely reveal many interesting surprises about this essential protein complex.

Indian J Exp Biol, 1997 Mar, 35(3), 313 - 4
Lipid production by Candida Y-1; Thaker P et al.; Ten oleaginous yeasts were isolated from sources like fruits, flowers, curd whey and soil samples . Lipid production varied from 24 to 71.2% of dry weight of cell biomass . Strain Y-1, which was isolated from curd whey, produced the highest amount of lipid . It was identified belonging to the genus Candida . The optimal cultural conditions for lipid production by Candida Y-1 were found to be glucose (5%) as carbon source, ammonium sulfate (1%) as nitrogen source, pH 4.5, and temperature 28 degrees C under shake-flask conditions (125 rpm).

J Clin Invest, 1997 Sep 15, 100(6), 1465 - 74
Modulation of the effector function of human macrophages for Histoplasma capsulatum by HIV-1 . Role of the envelope glycoprotein gp120; Chaturvedi S et al.; We have demonstrated that monocyte-derived macrophages (Mphi) from HIV+ individuals are deficient in their capacity to phagocytose Histoplasma capsulatum (Hc) yeasts, and are more permissive for the intracellular growth of Hc . To determine whether these defects in Mphi function were caused by HIV infection of the Mphi and/or by pathological events associated with HIV infection, cultured normal human Mphi were infected with the HIV-1BaL strain . Virus production, quantified by reverse transcriptase activity and p24 antigen, was evident on day 8 after infection and peaked on day 16 . On days 12, 16, and 20 after infection, HIV-1-infected Mphi were deficient in their capacity to recognize and bind Hc yeasts compared with control Mphi, and also were more permissive for the intracellular growth of Hc . Culture of normal Mphi with the envelope glycoprotein gp120 inhibited phagocytosis of Hc yeasts by Mphi in a concentration-dependent manner, but did not cause more rapid intracellular growth of Hc . Normal Mphi cultured in the serum of HIV+ individuals with impaired Mphi function subsequently were deficient in their capacity to phagocytose Hc yeasts, and were more permissive for the intracellular growth of yeasts compared with Mphi cultured in normal serum . Conversely, culture of normal Mphi in the serum of HIV+ patients with normal Mphi function did not affect the interaction of Hc yeasts with Mphi . Moreover, when Mphi from HIV+ individuals that were initially defective in host defense against Hc were cultured in normal HIV- serum, normal Mphi function was demonstrated . Adsorption of gp120 from the serum of two HIV+ patients removed the capacity of the serum to cause a Mphi defect in phagocytosis of Hc, but had no effect on the capacity of the serum to cause accelerated intracellular growth . These data demonstrate that observed defects in Mphi interaction with Hc yeasts may be caused by gp120 and other, as yet unknown serum component(s) probably released into serum by HIV-infected cells.

J Biol Chem, 1997 Sep 12, 272(37), 22995 - 9
A conditionally expressed third partner stabilizes or prevents the formation of a transcriptional activator in a three-hybrid system; Tirode F et al.; We describe a three-hybrid system that involves three polypeptides that allow or prevent the formation of the transcriptional activator . Beside the two-hybrid fusion proteins, the third partner is under the control of the Met25 promoter, which is positively regulated in medium lacking methionine . We document a situation where such a third partner promotes interaction between two proteins, one fused to a DNA-binding domain and the other fused to an activator domain . This is demonstrated for cdk7-MAT1 interaction stabilized by the presence of cyclin H; these three polypeptides are found either free or associated with the transcription/DNA repair factor TFIIH . We also document the capacity of our system to conditionally inhibit the interaction between two polypeptides that otherwise elicit a positive two-hybrid response . This is demonstrated for Ras-Raf interaction precluded by an excess of Raf . The presence of a methionine-regulated promoter provides an "on" or "off" switch for the formation of the transcriptional activator, thus also providing an excellent control to evaluate the activation or inhibition properties of the third partner.

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