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