Nat Rev Mol Cell Biol, 2002 Dec, 3(12), 919 - 31 Sorting out the cellular functions of sorting nexins; Worby CA et al.; Sorting nexins are a diverse group of cellular trafficking proteins that are unified by the presence of a phospholipid-binding motif - the PX domain . The ability of these proteins to bind specific phospholipids, as well as their propensity to form protein-protein complexes, points to a role for these proteins in membrane trafficking and protein sorting . It will be interesting to determine whether the various sorting nexins have specialized or generalized roles in protein trafficking. Food Addit Contam, 2002 Nov, 19(11), 1081 - 90 Post-harvest storage of corn: effect of beginning moisture content on mycoflora and fumonisin contamination; Ono EY et al.; The effect of storage on mycoflora profile was monitored bimonthly in 36 corn (Zea mays L.) samples, dividing the same sample into groups dried to 11 and 14% moisture content (1008 analysis) . These groups were further subdivided based on the initial total count (moulds and yeasts) up to 10(4) CFU g(-1) (12 samples, range 1.6 x 10(4) to 9.0 x 10(4), mean 3.8 x 10(4) CFU g(-1)) and up to 10(5) CFU g(-1) (24 samples, range 1.0 x 10(5) to 5.0 x 10(5), mean 2.7 x 10(5) CFU g(-1)) . In the corn group dried to 11%, the fumonisin content was analysed at the initial stage (freshly harvested) and at the end of 12-month storage . Fusarium spp . and Penicillium spp . prevailed at the freshly harvested stage (100%), maintaining this profile throughout 12 months, in corn dried to both 11 and 14% . Cladosporium spp., Aspergillus spp . and Phoma spp . were also detected at lower frequencies during the storage . Fusarium spp . and the total fungal colony count during 12-month storage carried out with samples dried to 11 or 14% moisture content were statistically evaluated using ANOVA for randomized complete block design . The correlation between storage time and Fusarium spp . and total fungal colony count data was analysed by Pearson's correlation test . There was no difference in Fusarium spp . and total counts in the 10(4) CFU g(-1) initial total count group throughout the storage time (p < 0.05) . There was a negative correlation between fungal population and storage time (p < 0.05) in the 10(5) CFU g(-1) initial total count group . Fumonisins were detected in all freshly harvested corn, at a mean concentration of 9.9 +/- 6.0 micro g g(-1) (range 0.74-22.6 micro g g(-)1) . These values did not change in the 12-month stored corn (mean of 9.9 +/- 5.8 micro g g(-1), range 0.81-23.7 micro g g(-1)) . These post harvest data indicated the importance of moisture content at the crop harvesting/predrying stage to control fungal growth and further fumonisin production. Eukaryot Cell, 2002 Jun, 1(3), 378 - 90 A G-protein beta subunit required for sexual and vegetative development and maintenance of normal G alpha protein levels in Neurospora crassa; Yang Q et al.; The genome of the filamentous fungus Neurospora crassa contains a single gene encoding a heterotrimeric G-protein beta subunit, gnb-1 . The predicted GNB-1 protein sequence is most identical to G beta proteins from the filamentous fungi Cryphonectria parasitica and Aspergillus nidulans . N . crassa GNB-1 is also 65% identical to the human GNB-1 protein but only 38 and 45% identical to G beta proteins from budding and fission yeasts . Previous studies in animal and fungal systems have elucidated phenotypes of G beta null mutants, but little is known about the effects of G beta loss on G alpha levels . In this study, we analyzed a gnb-1 deletion mutant for cellular phenotypes and levels of the three G alpha proteins . Delta gnb-1 strains are female-sterile, with production of aberrant fertilized reproductive structures . Delta gnb-1 strains conidiate more profusely and have altered mass on solid medium . Loss of gnb-1 leads to inappropriate conidiation and expression of a conidiation-specific gene during growth in submerged culture . Intracellular cyclic AMP levels are reduced by 60% in vegetative plate cultures of delta gnb-1 mutants . Loss of gnb-1 leads to lower levels of the three G alpha proteins under a variety of conditions . Analysis of transcript levels for the gna-1 and gna-2 G alpha genes in submerged cultures indicates that regulation of G alpha protein levels by gnb-1 is posttranscriptional . The results suggest that GNB-1 directly regulates apical extension rate and mass accumulation . In contrast, many other delta gnb-1 phenotypes, including female sterility and defective conidiation, can be explained by altered levels of the three N . crassa G alpha proteins. Mikrobiologiia, 2002 Sep-Oct, 71(5), 667 - 74 {The structure of the micromycete complexes of oligotrophic peat deposits in the southern taiga subzone of West Siberia}; Golovchenko AV et al.; The analysis of the micromycete complexes of oligotrophic peat deposits in the Vasyugan Marsh by direct count and culture methods showed that the micromycete carbon comprises no more than 3% of the total peat carbon and that the microscopic fungal biomass varies from 2 to 13 tons/hectare, depending on the season and the peat deposit thickness . Fungal spores were found in all layers of the peat deposits, whereas the mycelium was found only in the active peat layer . The high abundance of eukaryotic cells in the peats was due to the presence of yeastlike cells rather than fungal spores . Analyses by culture methods showed that micromycetes were present in all peat layers and that their abundance tended to decrease with depth, except for yeasts, which were uniformly distributed in a vertical direction . The micromycete complexes of the peat deposits were similar in the diversity and abundance of dominant species but differed in the composition of minor species . Peat yeasts were dominated by ascomycetes. Trends Genet, 2002 Dec, 18(12), 636 - 42 Intracellular mRNA localization: motors move messages; Tekotte H et al.; Intracellular mRNA localization is a common mechanism of post-transcriptional regulation of gene expression . In a wide range of organisms, mRNA localization coupled with translational regulation target the proteins to their site of function . Here, we describe recent exciting evidence that some mRNAs are transported as particles along the cytoskeleton by the molecular motors dynein, kinesin or myosin . We discuss the key questions of how localized mRNAs might be linked to motors and what determines their cytoplasmic destinations. Trends Cell Biol, 2002 Nov, 12(11), 509 - 16 Checking on the fork: the DNA-replication stress-response pathway; Osborn AJ et al.; To ensure the fidelity of DNA replication, cells activate a stress-response pathway when DNA replication is perturbed . This pathway regulates not only progress through the cell cycle but also transcription, apoptosis, DNA repair/recombination and DNA replication itself . Mounting evidence has suggested that this pathway is important for the maintenance of genomic integrity . Here, we discuss recent findings about how this pathway is activated by replication stress and how it regulates the DNA-replication machinery to alleviate the stress. Curr Biol, 2002 Nov 19, 12(22), 1908 - 18 Caenorhabditis elegans HUS-1 is a DNA damage checkpoint protein required for genome stability and EGL-1-mediated apoptosis; Hofmann ER et al.; BACKGROUND: The inability to efficiently repair DNA damage or remove cells with severely damaged genomes has been linked to several human cancers . Studies in yeasts and mammals have identified several genes that are required for proper activation of cell cycle checkpoints following various types of DNA damage . However, in metazoans, DNA damage can induce apoptosis as well . How DNA damage activates the apoptotic machinery is not fully understood . RESULTS: We demonstrate here that the Caenorhabditis elegans gene hus-1 is required for DNA damage-induced cell cycle arrest and apoptosis . Following DNA damage, HUS-1 relocalizes and forms distinct foci that overlap with chromatin . Relocalization does not require the novel checkpoint protein RAD-5; rather, relocalization appears more frequently in rad-5 mutants, suggesting that RAD-5 plays a role in repair . HUS-1 is required for genome stability, as demonstrated by increased frequency of spontaneous mutations, chromosome nondisjunction, and telomere shortening . Finally, we show that DNA damage increases expression of the proapoptotic gene egl-1, a response that requires hus-1 and the p53 homolog cep-1 . CONCLUSIONS: Our findings suggest that the RAD-5 checkpoint protein is not required for HUS-1 to relocalize following DNA damage . Furthermore, our studies reveal a new function of HUS-1 in the prevention of telomere shortening and mortalization of germ cells . DNA damage-induced germ cell death is abrogated in hus-1 mutants, in part, due to the inability of these mutants to activate egl-1 transcription in a cep-1/p53-dependent manner . Thus, HUS-1 is required for p53-dependent activation of a BH3 domain protein in C . elegans. J Nat Prod, 2002 Nov, 65(11), 1712 - 4 Aldehyde dehydrogenase inhibitors from the mushroom Clitocybe clavipes; Kawagishi H et al.; Five fatty acid derivatives including three novel compounds were isolated from the mushroom Clitocybe clavipe . Their structures were elucidated by spectral analyses . These compounds inhibited aldehyde dehydrogenase in vitro. Nucleosides Nucleotides Nucleic Acids, 2002 Jun-Jul, 21(6-7), 449 - 62 Analysis of relative positions of ribonucleotide bases in a crystal structure of ribosome; Takasu A et al.; Relative positions of bases to bases in a crystal structure of ribosome were analyzed extensively . It was found that there is no clear relation between bases apart more than 15 A and, thus, the relative location of bases can be analyzed within 15 A of the reference bases . As for base pairing, major positioning was found to be due to the Watson-Crick type base pairs . Some other positions corresponding to non-Watson-Crick type base pairs were also found in some extents . As for base-base stacking, it was observed that the bases stacked to adenine base are dispersive . It was found that less non-Watson-Crick base pairs was found close to the protein binding site, suggesting that the protein components have a tendency to bind to the regular stem structures . The database of relative location of bases must be useful for improvement of structural determination and structural modeling systems. Biochem Soc Trans, 2002 Nov, 30(Pt 6), 1047 - 50 Regulation of lipid accumulation in oleaginous micro-organisms; Ratledge C; A small number of eukaryotic micro-organisms, the oleaginous species, can accumulate triacylglycerols as cellular storage lipids, sometimes up to 70% of the biomass . Some of these lipids, particularly those containing high proportions of polyunsaturated fatty acids of nutritional and dietary importance, are now in commercial production; these are known as single-cell oils . The biochemistry of lipid accumulation has been investigated in yeasts and filamentous fungi and can now be described in some detail: lipid accumulation is triggered by cells exhausting nitrogen from the culture medium, but glucose continues to be assimilated . Activity of isocitrate dehydrogenase within the mitochondrion, however, now slows or even stops due to the diminution of AMP within the cells . This leads to the accumulation of citrate, which is transported into the cytosol and cleaved to acetyl-CoA by ATP:citrate lyase, an enzyme that does not occur in non-oleaginous species . This enzyme is therefore essential for lipid accumulation . The presence of this enzyme does not, however, explain why different species of oleaginous micro-organisms have different capacities for lipid accumulation . The extent of lipid accumulation is considered to be controlled by the activity of malic enzyme (ME), which acts as the sole source of NADPH for fatty acid synthase (FAS) . If ME is inhibited, or genetically disabled, then lipid accumulation is very low . There is no general pool of NADPH which can otherwise be used by FAS . The stability of ME is therefore crucial and it is proposed that ME is physically attached to FAS as part of the lipogenic metabolon . ME activity correlates closely with lipid accumulation in two filamentous fungi, Mucor circinelloides and Mortierella alpina . When ME ceases to be active, lipid accumulation also stops . No other enzyme activity shows such a correlation. Curr Opin Investig Drugs, 2002 Oct, 3(10), 1437 - 45 DNA binding compounds targeting fungal pathogens: an emerging concept in the discovery of novel antifungal agents; Lou L et al.; With the urgent need for novel agents to combat emerging fungal resistance to existing drugs, activity in the exploration of small molecule DNA binders has increased . Recently, selected cationic heterocyclic compounds belonging to a broad class of molecules known to bind to the minor groove of DNA were revealed to have potent antifungal activity . These molecules are different from the conventional DNA-interacting drugs such as topoisomerase inhibitors, DNA alkylators or intercalators . Selected compounds are fungicidal towards a variety of pathogenic yeasts and molds, and a selected lead compound is efficacious in a mouse model of systemic candidosis. Mutat Res, 2002 Nov 30, 509(1-2), 35 - 47 RecQ helicases and cellular responses to DNA damage; Wu L et al.; The faithful replication of the genome is essential for the survival of all organisms . It is not surprising therefore that numerous mechanisms have evolved to ensure that duplication of the genome occurs with only minimal risk of mutation induction . One mechanism of genome destabilization is replication fork demise, which can occur when a translocating fork meets a lesion or adduct in the template . Indeed, the collapse of replication forks has been suggested to occur in every replicative cell cycle making this a potentially significant problem for all proliferating cells . The RecQ helicases, which are essential for the maintenance of genome stability, are thought to function during DNA replication . In particular, RecQ helicase mutants display replication defects and have phenotypes consistent with an inability to efficiently reinitiate replication following replication fork demise . Here, we review some current models for how replication fork repair might be effected, and discuss potential roles for RecQ helicases in this process. EMBO J, 2002 Nov 15, 21(22), 6162 - 73 Nutrient control of gene expression in Drosophila: microarray analysis of starvation and sugar-dependent response; Zinke I et al.; We have identified genes regulated by starvation and sugar signals in Drosophila larvae using whole-genome microarrays . Based on expression profiles in the two nutrient conditions, they were organized into different categories that reflect distinct physiological pathways mediating sugar and fat metabolism, and cell growth . In the category of genes regulated in sugar-fed, but not in starved, animals, there is an upregulation of genes encoding key enzymes of the fat biosynthesis pathway and a downregulation of genes encoding lipases . The highest and earliest activated gene upon sugar ingestion is sugarbabe, a zinc finger protein that is induced in the gut and the fat body . Identification of potential targets using microarrays suggests that sugarbabe functions to repress genes involved in dietary fat breakdown and absorption . The current analysis provides a basis for studying the genetic mechanisms underlying nutrient signalling. Microsc Res Tech, 2002 Nov 15, 59(4), 325 - 30 Lifespan extension by caloric restriction: an aspect of energy metabolism; Yamaza H et al.; Caloric restriction (CR) may retard aging processes and extend lifespan in organisms by altering energy-metabolic pathways . In CR rodents, glucose influx into tissues is not reduced, as compared with control animals fed ad libitum (AL), although plasma concentrations of glucose and insulin are lower . Gene expression profiles in rodents have suggested that CR promotes gluconeogenesis and fatty acid biosynthesis in skeletal muscle . In the liver, CR promotes gluconeogenesis but decreases fatty acid synthesis and glycolysis . In lower organisms such as yeasts and nematodes, incomplete blocks in steps of insulin/insulin-like growth factor-1 (IGF-1) signal pathway extend lifespan . The life-prolonging effect of CR in yeasts requires NPT1 and SIR2 genes, both of which relate to sensing energy status and silencing genes . These findings stress the substantial role of energy metabolism on CR . Future studies on metabolic adaptation and gene silencing with regard to lower caloric intake will be warranted to understand the mechanisms of the anti-aging and life-prolonging effects of CR . Exp Cell Res, 2002 Nov 1, 280(2), 212 - 21 Ubc9 is essential for viability of higher eukaryotic cells; Hayashi T et al.; Ubc9 is an enzyme involved in the conjugation of SUMO-1 (small ubiquitin related modifier 1) to target proteins . The SUMO-1 conjugation system is well conserved from yeasts to higher eukaryotes, but many SUMO-1 target proteins reported recently in higher eukaryotic cells, including IkappaBalpha, MDM2, p53, and PML, are not present in yeasts . To determine the physiological roles of SUMO-1 conjugation in higher eukaryotic cells, we constructed a conditional UBC9 mutant of chicken DT40 cells containing the UBC9 transgene under control of a tetracycline-repressible promoter and characterized their loss of function phenotypes . Ubc9 disappeared 3 days after the addition of tetracycline and the increase in viable cell number stopped 4 days after the addition of drug . In contrast to the cases of ubc9 mutants of budding and fission yeasts, which show defects in progression of G2 or early M phase and in chromosome segregation, respectively, we did not observe accumulation of cells in G2/M phase or a considerable increase in the frequency of chromosome missegregation upon depletion of Ubc9 but we did observe an increase in the number of cells containing multiple nuclei, indicating defects in cytokinesis . A considerable portion of the Ubc9-depleted cell population was committed to apoptosis without accumulating in a specific phase of the cell cycle, suggesting that chromosome damages are accumulated in Ubc9-depleted cells, and apoptosis is triggered without activating checkpoint mechanisms under conditions of SUMO-1 conjugation system impairment. Genetics, 2002 Oct, 162(2), 977 - 85 Incorporation of large heterologies into heteroduplex DNA during double-strand-break repair in mouse cells; Raynard SJ et al.; In this study, the formation and repair of large (>1 kb) insertion/deletion (I/D) heterologies during double-strand-break repair (DSBR) was investigated using a gene-targeting assay that permits efficient recovery of sequence insertion events at the haploid chromosomal immunoglobulin (Ig) mu-locus in mouse hybridoma cells . The results revealed that (i) large I/D heterologies were generated on one or both sides of the DSB and, in some cases, formed symmetrically in both homology regions; (ii) large I/D heterologies did not negatively affect the gene targeting frequency; and (iii) prior to DNA replication, the large I/D heterologies were rectified. J Cell Biochem, 2002, 87(3), 258 - 65 Roles for cytoplasmic polyadenylation in cell cycle regulation; Read RL et al.; Polyadenylation of eukaryotic mRNAs in the nucleus promotes their translation following export to the cytoplasm and is an important determinant of mRNA stability . An additional level of control of gene expression is provided by cytoplasmic polyadenylation, which activates translation of a number of mRNAs important in orchestrating cell cycle events in oocytes . Recent studies indicate that cytoplasmic polyadenylation may be a mechanism of translational activation that is more widespread in eukaryotic cells . Here we discuss the roles of a recently identified family of nucleotidyl transferases (encoded by the cid1 gene family) in cell cycle regulation . To date, this family has been characterised mainly in yeasts, but it is conserved throughout the eukaryotes . Biochemical studies have indicated that a subset of members of this family function as cytoplasmic poly(A) polymerases targeting specific mRNAs for translation . This form of translational control appears to be particularly important for cell cycle regulation following inhibition of DNA synthesis . Biol Pharm Bull, 2002 Oct, 25(10), 1307 - 10 Antifungal evaluation of bis Mannich bases derived from acetophenones and their corresponding piperidinols and stability studies; Gul HI et al.; The development of resistance to current antifungal therapeutics drives the search for effective new agents . The fact that some acetophenone-derived Mannich bases had shown antifungal activities in our previous studies led us to design and synthesize acetophenone-derived bis Mannich bases, B1-B5, bis(beta-aroylethyl)methylamine hydrochlorides, to evaluate their antifungal activity . These bis Mannich bases were then converted to the corresponding piperidinols, C1-C5, which are structural isomers of bis derivatives, 3-aroyl-4-aryl-1-methyl-4-piperidinol hydrochlorides, to see alterations in biological activity . A stability study of B1 and Cl was also carried out to estimate whether they alkylate the thiols . All compounds studied have shown antifungal activity, especially against dermatophytes (Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, and Microsporum canis), in the concentration range studied (2-128 microng/ml) . The activity was especially apparent against T . tonsurans . All compounds had at least equal antifungal activity compared with the reference compound amphotericin-B against T . tonsurans . Bis Mannich bases were generally found to be more potent compounds than their structural isomer piperidinols . The results of our stability studies suggest that thiol alkylation may contribute to the antifungal activity of the Mannich bases synthesized . Even though all compounds showed antifungal activity against dermatophytes, bis Mannich bases B1, B2, B4, and B5 appear to have potential for developing novel antifungal agents against dermatophytes. Int J Dermatol, 2002 Oct, 41(10), 647 - 51 Epidemiology of onychomycosis in patients with diabetes mellitus in India; Dogra S et al.; BACKGROUND: The number of individuals diagnosed with diabetes mellitus is increasing worldwide . Although onychomycosis is often observed in diabetics, there have been no large studies of its epidemiology in this patient group in India . METHODS: We studied the prevalence of onychomycosis in diabetics attending the Diabetes Clinic at the Postgraduate Institute of Medical Education and Research, Chandigarh, India, and compared it with that in a nondiabetic control group . A total of 400 diabetic subjects (237 males, 163 females), aged 48.8 +/- 0.5 years (mean +/- SD), were evaluated . RESULTS: The prevalence of onychomycosis in the diabetic and control groups was 17% and 6.8%, respectively, the difference being statistically significant (P < 0.001) . The presence of onychomycosis was found to correlate significantly with increasing age (P < 0.01) and male gender (P < 0.05) in both diabetic and control groups . From diabetics, yeasts were the most common isolate (48.1%), followed by dermatophytes and nondermatophyte molds in 37% and 14.8%, respectively . In the control group, the distribution of yeasts, dermatophytes, and nondermatophyte molds was 25%, 62.5%, and 12.5%, respectively . After controlling for age and sex, a stepwise logistic regression demonstrated that significant predictors for onychomycosis included the duration of diabetes (P < 0.01), absent or feeble peripheral pulses (P < 0.15), peripheral neuropathy (P < 0.05), and retinopathy (P < 0.001) . CONCLUSIONS: Diabetics were found to be 2.5 times more likely to have onychomycosis than the controls . Predisposing factors included increasing age, male gender, duration of diabetes, impaired peripheral circulation, peripheral neuropathy and retinopathy. Mol Biochem Parasitol, 2002 Sep-Oct, 124(1-2), 51 - 62 Peroxisomal targeting protein 14 (PEX14) from Leishmania donovani . Molecular, biochemical, and immunocytochemical characterization; Jardim A et al.; Pathogens of the Leishmania and Trypanosoma genera compartmentalize glycolytic and other nutritional pathways in glycosomes, unique subcellular organelles related to the peroxisomes of mammals and yeasts . Most glycosomal proteins are targeted to the glycosomes by a COOH-terminal tripeptide signal similar to the peroxisomal targeting signal-1 (PTS-1) . It has been proposed that PTS-1 forms a complex with the PEX5 receptor protein which then docks to the glycosomal membrane through interactions with the membrane associated PEX14 protein . To analyze the role of PEX14 in glycosomal protein import, the gene encoding the L . donovani PEX14 (LdPEX14) was isolated and shown to encode a 464 amino acid protein that exhibited very limited sequence homology with peroxisomal PEX14 proteins . In vitro binding experiments with purified recombinant LdPEX14 and LdPEX5 confirmed that LdPEX14-LdPEX5 interacted with a K(d) of 2.75 microM . When LdPEX5 was preloaded with a PTS-1 peptide, the affinity of the LdPEX14-LdPEX5 interaction affinity increased . Furthermore, binding experiments with truncated forms of LdPEX5 and LdPEX14 showed that the interaction domains localized to the amino terminal region of both proteins . Finally, confocal microscopy, subcellular fractionation, and differential extraction experiments indicated that LdPEX14 is a soluble protein that associates tightly with the glycosomal membrane and further support the role of LdPEX14 in forming a docking complex involved in glycosome biogenesis. Curr Opin Cell Biol, 2002 Aug, 14(4), 434 - 47 Phosphoinositides and the golgi complex; De Matteis M et al.; Phosphoinositides act as precursors of second messengers and membrane ligands for protein modules . Specific lipid kinases and phosphatases are located and differentially regulated in cell organelles, generating a non-uniform distribution of phosphoinositides . Although it is not clear whether and how the phosphoinositide pools are integrated, it is certain that they locally control fundamental processes, including membrane trafficking . This applies to the Golgi complex, where a direct, central role of the phosphatidylinositol 4,5-bisphosphate precursor phosphatidylinositol 4-phosphate has recently been reported. Plant Physiol, 2002 Oct, 130(2), 688 - 97 Arabidopsis ABI5 subfamily members have distinct DNA-binding and transcriptional activities; Kim SY et al.; A small family of novel basic leucine zipper proteins that includes abscisic acid (ABA)-INSENSITIVE 5 (ABI5) binds to the promoter region of the lea class gene Dc3 . The factors, referred to as AtDPBFs (Arabidopsis Dc3 promoter-binding factors), were isolated from an immature seed cDNA library . AtDPBFs bind to the embryo specification and ABA-responsive elements in the Dc3 promoter and are unique in that they can interact with cis-elements that do not contain the ACGT core sequence required for the binding of most other plant basic leucine zipper proteins . Analysis of full-length cDNAs showed that at least five different Dc3 promoter-binding factors are present in Arabidopsis seeds; one of these, AtDPBF-1, is identical to ABI5 . As expected, AtDPBF-1/ABI5 mRNA is inducible by exogenous ABA in seedlings . Despite the near identity in their basic domains, AtDPBFs are distinct in their DNA-binding, dimerization, and transcriptional activity. Genome Biol . 2002 Sep 24;3(10):REPORTS4032 . Epub 2002 Sep 24. Developmental biologists cast a net over sequenced genomes; Gerberding M et al.; A report on the annual meeting of the Society of Developmental Biology, Madison, Wisconsin, USA, 21-24 July 2002. J Biol Chem, 2002 Dec 20, 277(51), 49230 - 7 Epub 2002 Oct 04. Characterization of the two coactivator-interacting surfaces of the androgen receptor and their relative role in transcriptional control; Christiaens V et al.; The androgen receptor interacts with the p160 coactivators via two surfaces, one in the ligand binding domain and one in the amino-terminal domain . The ligand binding domain interacts with the nuclear receptor signature motifs, whereas the amino-terminal domain has a high affinity for a specific glutamine-rich region in the p160s . We here describe the implication of two conserved motifs in the latter interaction . The amino-terminal domain of the androgen receptor is a very strong activation domain constituent of Tau5, which is mainly active in the absence of the ligand binding domain, and Tau1, which is only active in the presence of the ligand binding domain . Both domains are, however, implicated in the recruitment of the p160s . Mutation analysis of the p160s has shown that the relative contribution of the two recruitment mechanisms via the signature motifs or via the glutamine-rich region depend on the nature of the enhancers tested . We propose, therefore, that the androgen receptor-coactivator complex has several alternative conformations, depending partially on the context of the enhancer. J Basic Microbiol, 2002, 42(5), 295 - 301 Laccase production by some Phlebia species; Arora DS et al.; The present study was carried out to examine the ability of four species of the genus Phlebia, viz . P . floridensis, P . brevispora, P . radiata and P . fascicularia, to produce the ligninolytic enzyme laccase in liquid culture . P . floridensis was the most active species that even surpassed laccase production by Trametes versicolor, and was chosen for further study . Among several carbon sources tested, malt extract turned out to be the best medium for subsequent laccase concentration by ammonium sulfate precipitation . Specific enzyme activity increased 12-fold during this procedure and a K(m) value of 0.33 mM was calculated for the resulting laccase preparation using guaiacol as the substrate . Concentrated laccase from P . floridensis was relatively thermostable and retained 70% and 15% of its activity after 1 h preincubation at 50 degrees C and 60 degrees C, respectively. Swiss Med Wkly, 2002 Jun 15, 132(23-24), 303 - 11 Antifungal chemotherapy: advances and perspectives; Groll AH et al.; Invasive fungal infections have emerged as important causes of morbidity and mortality in immunocompromised patients . In response to this challenge, the field of antifungal chemotherapy has considerably expanded . Fluconazole and itraconazole, introduced in the late 1980s, were the first durably useful alternatives to amphotericin B deoxycholate . The clinical development of the lipid formulations of amphotericin B, and, more recently, that of novel echinocandin derivatives and improved antifungal triazoles each represent milestones in antifungal drug research that have further amplified our therapeutic options . Major progress has been made in harmonising disease definitions, in defining the paradigms of antifungal intervention, and in designing and implementing clinical trials . Standardised methods for in vitro susceptibility testing of yeasts and filamentous fungi have become available, and pharmacodynamic concepts have entered preclinical and clinical drug development . This article reviews the evolution of therapeutic options over the past decade, advances in chemoprevention and empirical antifungal therapy, progress in early diagnosis and pre-emptive therapy, the promise of the new echinocandins and second generation triazoles, as well as perspectives for combination therapies and adjuvant immunoreconstitution . Invasive fungal infections will remain a frequent and important complication of modern medicine; the current momentum in the field of laboratory and clinical antifungal drug research provides hope for substantial progress in prevention and management of these life-threatening infections in the near future. Curr Biol, 2002 Oct 1, 12(19), 1670 - 4 Poleward microtubule flux is a major component of spindle dynamics and anaphase a in mitotic Drosophila embryos; Maddox P et al.; During cell division, eukaryotic cells assemble dynamic microtubule-based spindles to segregate replicated chromosomes . Rapid spindle microtubule turnover, likely derived from dynamic instability, has been documented in yeasts, plants and vertebrates . Less studied is concerted spindle microtubule poleward translocation (flux) coupled to depolymerization at spindle poles . Microtubule flux has been observed only in vertebrates, although there is indirect evidence for it in insect spermatocytes and higher plants . Here we use fluorescent speckle microscopy (FSM) to demonstrate that mitotic spindles of syncytial Drosophila embryos exhibit poleward microtubule flux, indicating that flux is a widely conserved property of spindles . By simultaneously imaging chromosomes (or kinetochores) and flux, we provide evidence that flux is the dominant mechanism driving chromosome-to-pole movement (anaphase A) in these spindles . At 18 degrees C and 24 degrees C, separated sister chromatids moved poleward at average rates (3.6 and 6.6 microm/min, respectively) slightly greater than the mean rates of poleward flux (3.2 and 5.2 microm/min, respectively) . However, at 24 degrees C the rate of kinetochore-to-pole movement varied from slower than to twice the mean rate of flux, suggesting that although flux is the dominant mechanism, kinetochore-associated microtubule depolymerization contributes to anaphase A. Proteins, 2002 Nov 15, 49(3), 302 - 20 Rebuilt 3D structure of the chloroplast f1 ATPase-tentoxin complex; Minoletti C et al.; The F1 part of the chloroplast H+ adenosine triphosphate (ATP)-synthase (CF1) strongly interacts with tentoxin, a natural fungous cyclic tetrapeptide known to inhibit the chloroplast enzyme and not the mammalian mitochondrial enzyme . Whereas the synthesis or the hydrolysis of ATP requires the stepwise rotation of the protein rotor gamma within the (alphabeta)3 crown, only one molecule of tentoxin is needed to fully inhibit the complex . With the help of an original homology modeling technique, based on robust distance geometry protocols, we built a tridimensional model of the alpha3beta3gamma CF1) subcomplex (3200 esidues), in which we introduced three different nucleotide occupancies to check their possible influence on the tentoxin binding site . Simultaneous comparison of three available high-resolution X-ray structures of F1, performed with a local structural alignment search tool, led to characterizing common structural blocks and the distorsions experienced by the complex during the catalytic turnover . The common structural blocks were used as a starting point of the spinach CF1 structure rebuilding . Finally, tentoxin was docked into its putative binding site of the reconstructed structure . The docking method was initially validated in the mitochondrial enzyme by its ability to relocate nucleotides into their original position in the crystal . Tentoxin binding was found possible to the two alpha/beta interfaces associated with the empty and adenosine diphosphate (ADP)-loaded catalytic sites, but not to the one associated with the ATP-loaded site . These results suggest a mechanism of CF1 inhibition by one molecule of tentoxin, by the impossibility of the alpha/beta interface bearing tentoxin to pass through the ATP-loaded state . Hum Mol Genet, 2002 Oct 1, 11(20), 2479 - 88 Cancer epigenomics; Plass C; Research in cancer epigenomics is driven by the development of novel technologies and the utilization of model organisms ranging from yeasts to plants to vertebrates . For decades, the search for cancer genes has focused on genetic defects that were used as tags for identification of these genes . With the realization that epigenetic modifications, most importantly DNA methylation events, are frequently involved in transcriptional changes in both tumor suppressor genes and oncogenes, techniques have been developed that support the identification of novel cancer genes altered by DNA methylation alone or in combination with genetic events . Recent data demonstrate that, in addition to DNA methylation, chromatin modifications are also involved in gene regulation . We are now beginning to understand this interesting interplay between chromatin modifications, DNA methylation and gene regulation . This review will summarize our current knowledge of DNA methylation and histone modification in normal cells, introduce emerging concepts that show the intimate link between DNA methylation and chromatin modifications, and highlight recent advancements in our understanding of aberrant DNA methylation, with special emphasis on genome-wide hypermethylation. Cell, 2002 Sep 6, 110(5), 545 - 50 RNA logic in time and space; Darnell RB; An EMBO-sponsored workshop entitled "Translational Control in Development and Neurobiology" was held recently in Mallorca, Spain . The talks covered mechanisms of translational regulation, particularly in terms of temporal and spatial regulation, over a wide range of biological systems. Med Mycol, 2002 Aug, 40(4), 383 - 6 High rate of vaginal infections caused by non-C . albicans Candida species among asymptomatic women; Dan M et al.; A prospective observational study of patients attending a gynecological clinic and those referred to a clinic for genitourinary infections was undertaken with the purpose of evaluating the relative prevalence of non-C . albicans Candida species among Candida isolates from the vagina in different clinical settings in an area with high occurrence of vulvovaginal candidiasis . The rate of non-C . albicans Candida species was 44.5% among asymptomatic women, 19.4% among those with sporadic vaginitis and 21% among patients with chronic vaginal symptoms (p < 0.001 for asymptomatic vs . pooled symptomatic women) . No increase in the rate of non-C . albicans Candida was observed during a period of 4 years (1995-1998) despite a 1.57-fold increase in the sales of azole antifungal agents . Unlike some previous reports we could not document an association of non-C . albicans Candida species with chronic vaginal symptoms or increased use of azole antifungal agents . The significantly higher rate of these yeasts in asymptomatic women is in accord with the known tendency of non-C . albicans Candida species to cause mild symptoms. Plant Physiol, 2002 Sep, 130(1), 179 - 89 Engineering herbicide metabolism in tobacco and Arabidopsis with CYP76B1, a cytochrome P450 enzyme from Jerusalem artichoke; Didierjean L et al.; The Jerusalem artichoke (Helianthus tuberosus) xenobiotic inducible cytochrome P450, CYP76B1, catalyzes rapid oxidative dealkylation of various phenylurea herbicides to yield nonphytotoxic metabolites . We have found that increased herbicide metabolism and tolerance can be achieved by ectopic constitutive expression of CYP76B1 in tobacco (Nicotiana tabacum) and Arabidopsis . Transformation with CYP76B1 conferred on tobacco and Arabidopsis a 20-fold increase in tolerance to linuron, a compound detoxified by a single dealkylation, and a 10-fold increase in tolerance to isoproturon or chlortoluron, which need successive catalytic steps for detoxification . Two constructs for expression of translational fusions of CYP76B1 with P450 reductase were prepared to test if they would yield even greater herbicide tolerance . Plants expressing these constructs had lower herbicide tolerance than CYP76B1 alone, which is apparently a consequence of reduced stability of the fusion proteins . In all cases, increased herbicide tolerance results from more extensive metabolism, as demonstrated with exogenously fed phenylurea . Beside increased herbicide tolerance, expression of CYP76B1 has no other visible phenotype in the transgenic plants . Our data indicate that CYP76B1 can function as a selectable marker for plant transformation, allowing efficient selection in vitro and in soil-grown plants . Plants expressing CYP76B1 may also be a potential tool for phytoremediation of contaminated sites. Plant Physiol, 2002 Sep, 130(1), 22 - 46 Inositol phospholipid metabolism in Arabidopsis . Characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C; Mueller-Roeber B et al.; Phosphoinositides (PIs) constitute a minor fraction of total cellular lipids in all eukaryotic cells . They fulfill many important functions through interaction with a wide range of cellular proteins . Members of distinct inositol lipid kinase families catalyze the synthesis of these phospholipids from phosphatidylinositol . The hydrolysis of PIs involves phosphatases and isoforms of PI-specific phospholipase C . Although our knowledge of the roles played by plant PIs is clearly limited at present, there is no doubt that they are involved in many physiological processes during plant growth and development . In this review, we concentrate on inositol lipid-metabolizing enzymes from the model plant Arabidopsis for which biochemical characterization data are available, namely the inositol lipid kinases and PI-specific phospholipase Cs . The biochemical properties and structure of characterized and genome-predicted isoforms are presented and compared with those of the animal enzymes to show that the plant enzymes have some features clearly unique to this kingdom. Mem Inst Oswaldo Cruz, 2002 Jul, 97(5), 747 - 50 Clinical and mycological study of scalp white piedra in the State of Paraíba, Brazil; Pontes ZB et al.; White piedra is a superficial mycoses characterized by nodules on the hair shaft, caused by the basidiomycetous yeasts . In the present study, clinical and mycological findings of scalp white piedra caused by Trichosporon spp . are related . Twenty three cases of scalp white piedra were observed with a high incidence in women (87%) and preschool children from 2 to 6 (74%) years old . These groups presented a relationship of dependence with this infection . Despite the low socio-economic status, poor standards of hygiene, (48% of the patients) as well as the fact that 30.4% of the children shared the same nursery, these factors were not significant for the transmission of the mycosis . These were the first reports of scalp white piedra in Joao Pessoa city, Paraiba, Brazil. J Mol Biol, 2002 Sep 6, 322(1), 123 - 35 Hydrophobic interactions at the Ccap position of the C-capping motif of alpha-helices; Ermolenko DN et al.; We investigated the possible role of residues at the Ccap position in an alpha-helix on protein stability . A set of 431 protein alpha-helices containing a C'-Gly from the Protein Data Bank (PDB) was analyzed, and the normalized frequencies for finding particular residues at the Ccap position, the average fraction of buried surface area, and the hydrogen bonding patterns of the Ccap residue side-chain were calculated . We found that on average the Ccap position is 70% buried and noted a significant correlation (R=0.8) between the relative burial of this residue and its hydrophobicity as defined by the Gibbs energy of transfer from octanol or cyclohexane to water . Ccap residues with polar side-chains are commonly involved in hydrogen bonding . The hydrogen bonding pattern is such that, the longer side-chains of Glu, Gln, Arg, Lys, His form hydrogen bonds with residues distal (>+/-4) in sequence, while the shorter side-chains of Asp, Asn, Ser, Thr exhibit hydrogen bonds with residues close in sequence (<+/-4), mainly involving backbone atoms . Experimentally we determined the thermodynamic propensities of residues at the Ccap position using the protein ubiquitin as a model system . We observed a large variation in the stability of the ubiquitin variants depending on the nature of the Ccap residue . Furthermore, the measured changes in stability of the ubiquitin variants correlate with the hydrophobicity of the Ccap residue . The experimental results, together with the statistical analysis of protein structures from the PDB, indicate that the key hydrophobic capping interactions between a helical residue (C3 or C4) and a residue outside the helix (C", C3' or C4') are frequently enhanced by the hydrophobic interactions with Ccap residues. Ultramicroscopy, 2002 Aug, 92(3-4), 243 - 50 Optical interference artifacts in contact atomic force microscopy images; Mendez-Vilas A et al.; Atomic force microscopy images are usually affected by different kinds of artifacts due to either the microscope design and operation mode or external environmental factors . Optical interferences between the laser light reflected off the top of the cantilever and the light scattered by the surface in the same direction is one of the most frequent sources of height artifact in contact (and occasionally non-contact) images . They are present when imaging highly reflective surfaces, or even when imaging non-reflective materials deposited onto reflective ones . In this study interference patterns have been obtained with a highly polished stainless steel planchet . The influence of these artifacts in surface roughness measurements is discussed, and a semi-quantitative method based on the fast Fourier transform technique is proposed to remove the artifacts from the images . This method improves the results obtained by applying the usual flattening routines. Nat Rev Genet, 2002 Sep, 3(9), 683 - 97 The art and design of genetic screens: filamentous fungi; Casselton L et al.; In the 1940s, screens for metabolic mutants of the filamentous fungus Neurospora crassa established the fundamental, one-to-one relationship between a gene and a specific protein, and also established fungi as important genetic organisms . Today, a wide range of filamentous species, which represents a billion years of evolutionary divergence, is used for experimental studies . The developmental complexity of these fungi sets them apart from unicellular yeasts, and allows the development of new screens that enable us to address biological questions that are relevant to all eukaryotes. Nat Rev Mol Cell Biol, 2002 Sep, 3(9), 639 - 50 Seven-transmembrane receptors; Pierce KL et al.; Seven-transmembrane receptors, which constitute the largest, most ubiquitous and most versatile family of membrane receptors, are also the most common target of therapeutic drugs . Recent findings indicate that the classical models of G-protein coupling and activation of second-messenger-generating enzymes do not fully explain their remarkably diverse biological actions. Plant J, 2002 Sep, 31(5), 663 - 73 The VERNALIZATION INDEPENDENCE 4 gene encodes a novel regulator of FLOWERING LOCUS C; Zhang H et al.; The late-flowering, vernalization-responsive habit of many Arabidopsis ecotypes is mediated predominantly through repression of the floral programme by the FLOWERING LOCUS C (FLC) gene . To better understand this repressive mechanism, we have taken a genetic approach to identify novel genes that positively regulate FLC expression . We identified recessive mutations in a gene designated VERNALIZATION INDEPENDENCE 4 (VIP4), that confer early flowering and loss of FLC expression in the absence of cold . We cloned the VIP4 gene and found that it encodes a highly hydrophilic protein with similarity to proteins from yeasts, Drosophila, and Caenorhabditis elegans . Consistent with a proposed role as a direct activator of FLC, VIP4 is expressed throughout the plant in a pattern similar to that of FLC . However, unlike FLC, VIP4 RNA expression is not down-regulated in vernalized plants, suggesting that VIP4 is probably not sufficient to activate FLC, and that VIP4 is probably not directly involved in a vernalization mechanism . Epistasis analysis suggests that VIP4 could act in a separate pathway from previously identified FLC regulators, including FRIGIDA and the autonomous flowering promotion pathway gene LUMINIDEPENDENS . Mutants lacking detectable VIP4 expression flower earlier than FLC null mutants, suggesting that VIP4 regulates flowering-time genes in addition to FLC . Floral morphology is also disrupted in vip4 mutants; thus, VIP4 has multiple roles in development. Plant J, 2002 Sep, 31(5), 565 - 76 Regulation of ADL6 activity by its associated molecular network; Lam BC et al.; Plant dynamin-like proteins consist of a group of high molecular weight GTPase with diverse structural arrangements and cellular localizations . In addition, unlike animal dynamins, there was no evidence for the involvement of any plant dynamin-like protein in clathrin-mediated vesicle trafficking . In this study we demonstrate that ADL6 (Arabidopsis dynamin-like protein 6), due to its domain arrangement, behaves similarly to the animal dynamins . The association of ADL6 with clathrin-coated vesicles was demonstrated by co-fractionation and immunocytochemical studies . ADL6 also interacted via its C-terminus with gamma-adaptin, an adaptor protein of clathrin-coated vesicles . Our results suggest that ADL6 participates in clathrin-mediated vesicle trafficking originating from the Golgi . In addition, our studies demonstrate that ADL6 intrinsic GTPase activity is regulated by its association with acidic phospholipids and an SH3 (Src homology 3)-containing protein. Angew Chem Int Ed Engl, 2002 Jul 2, 41(13), 2259 - 64 Chemical strategies for iron acquisition in plants; Staiger D; Iron is an essential element for plant nutrition . Although iron is the fourth most abundant element (3 %) of the earth's crust, it is not readily available because of its low solubility . Therefore, plants need an active mechanism to extract iron from the soil . They have evolved several chemical strategies to acquire iron ions and the physiology of these mechanisms has been known for a long time . Only recently, the use of molecular genetic approaches has led to a biochemical and molecular characterization of the players involved, thus providing an entry to the manipulation of iron uptake in plants. Anal Chem, 2002 Aug 15, 74(16), 4235 - 49 High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics; Shen Y et al.; We describe high-efficiency (peak capacities of approximately 10(3)) nanoscale (using column inner diameters down to 15 microm) liquid chromatography (nanoLC)/low flow rate electrospray (nanoESI) mass spectrometry (MS) for the sensitive analysis of complex global cellular protein enzymatic digests (i.e., proteomics) . Using a liquid slurry packing method with carefully selected packing solvents, 87-cm-length capillaries having inner diameters of 14.9-74.5 microm were successfully packed with 3-microm C18-bonded porous (300-A pores) silica particles at a pressure of 18,000 psi . With a mobile-phase delivery pressure of 10,000 psi, these packed capillaries provided mobile-phase flow rates as low as approximately 20 nL/min at LC linear velocities of approximately 0.2 cm/s, which is near optimal for separation efficiency . To maintain chromatographic efficiency, unions with internal channel diameters as small as 10 microm were specially produced for connecting packed capillaries to replaceable nanoESI emitters having orifice diameters of 2-10 microm (depending on the packed capillary dimensions) . Coupled on-line with a hybrid-quadrupole time-of-flight MS through the nanoESI interface, the nanoLC separations provided peak capacities of approximately 10(3) for proteome proteolytic polypeptide mixtures when a positive feedback switching valve was used for quantitatively introducing samples . Over a relatively large range of sample loadings (e.g., 5-100 ng, and 50-500 ng of cellular proteolytic peptides for 14.9- and 29.7-microm-i.d . packed capillaries, respectively), the nanoLC/nanoESI MS response for low-abundance components of the complex mixtures was found to increase linearly with sample loading . The nanoLC/nanoESI-MS sensitivity also increased linearly with decreasing flow rate (or approximately inversely proportional to the square of the capillary inner diameter) in the flow range of 20-400 nL/min . Thus, except at the lower loadings, decreasing the separation capillary inner diameter has an effect equivalent to increasing sample loading, which is important for sample-limited proteomic applications . No significant effects on recovery of eluting polypeptides were observed using porous C18 particles with surface pores of 300-A versus nonporous particles . Tandem MS analyses were also demonstrated using the high-efficiency nanoLC separations . Chromatographic elution time, MS response intensity, and mass measurement accuracy was examined between runs with a single column (with a single nanoESI emitter), between different columns (same and different inner diameters with different nanoESI emitters), and for different samples (various concentrations of cellular proteolytic peptides) and demonstrated robust and reproducible sensitive analyses for complex proteomic samples. Mol Cell, 2002 Aug, 10(2), 347 - 58 The three-dimensional structure of the autoproteolytic, nuclear pore-targeting domain of the human nucleoporin Nup98; Hodel AE et al.; Nup98 is a component of the nuclear pore that plays its primary role in the export of RNAs . Nup98 is expressed in two forms, derived from alternate mRNA splicing . Both forms are processed into two peptides through autoproteolysis mediated by the C-terminal domain of hNup98 . The three-dimensional structure of the C-terminal domain reveals a novel protein fold, and thus a new class of autocatalytic proteases . The structure further reveals that the suggested nucleoporin RNA binding motif is unlikely to bind to RNA . The C terminus also contains sequences that target hNup98 to the nuclear pore complex . Noncovalent interactions between the C-terminal domain and the cleaved peptide tail are visible and suggest a model for cleavage-dependent targeting of hNup98 to the nuclear pore. Curr Genet, 2002 Aug, 41(5), 311 - 22 Epub 2002 Jul 11. Maintenance of mitochondrial DNA integrity: repair and degradation; Kang D et al.; Mitochondria have their own genome, which is essential for proper oxidative phosphorylation and hence for a large part of ATP production in a cell . Although mitochondrial DNA-less (rho(0)) cells can survive under certain conditions, the integrity of the mitochondrial genome is critical for the survival of multicellular organisms . Mitochondrial DNA (mtDNA) is damaged more than nuclear DNA because mitochondria produce a large amount of reactive oxygen species and tend to accumulate toxic xenobiotics . Therefore, there is keen interest in mechanisms that maintain the integrity of mtDNA . DNA repair may play an important role . The repair of mtDNA has been investigated less intensely than nuclear DNA repair because, for a long time, it was thought that mitochondria lacked DNA repair systems . In fact, DNA damage can be repaired in mitochondria . Base-excision repair in mitochondria is well established . The enzymes responsible for mtDNA repair have been identified and are encoded by the same genes as their nuclear counterparts . Mitochondrion-targeting sequences are generated through alternative splicing of mRNAs, alternative use of transcription initiation sites, or alternative use of translation initiation sites . In addition to DNA repair, the degradation of damaged mtDNA may be tolerated because there are multiple copies of mtDNA molecules in a cell. Epidemiol Mikrobiol Imunol, 2002 Aug, 51(3), 131 - 4 {Resistance of microscopic fungi to selected antimycotics}; Kunova Z; The author attended to enquiries of developing resistance of microscopic fungi (yeasts, micromycetes) to antimycotics used in therapy and their mechanisms of action. Physiol Genomics, 2002 Aug 14, 10(2), 79 - 91 RNA interference of peroxisome-related genes in C . elegans: a new model for human peroxisomal disorders; Petriv OI et al.; RNA-mediated interference (RNAi) for the posttranscriptional silencing of genes was used to evaluate the importance of various peroxisomal enzymes and peroxins for the development of Caenorhabditis elegans and to compare the roles of these proteins in the nematode to their roles in yeasts and humans . The nematode counterparts of the human ATP-binding cassette half-transporters, the enzymes alkyldihydroxyacetonephosphate synthase and Delta(3,5)-Delta (2,4)-dienoyl-CoA isomerase, the receptors for peroxisomal membrane and matrix proteins (Pex19p and Pex5p), and components of the docking and translocation machineries for matrix proteins (Pex13p and Pex12p) are essential for the development of C . elegans . Unexpectedly, RNAi silencing of the acyl-CoA synthetase-mediated activation of fatty acids, the alpha- and beta-oxidation of fatty acids, the intraperoxisomal decomposition of hydrogen peroxide, and the peroxins Pex1p, Pex2p, and Pex6p had no apparent effect on C . elegans development . The described analysis of functional gene knockouts through RNAi provides a basis for the use of C . elegans as a valuable model system with which to study the molecular and physiological defects underlying the human peroxisomal disorders. Rev Argent Microbiol, 2002 Apr-Jun, 34(2), 95 - 9 Onychomycosis in João Pessoa City, Brazil; Pontes ZB et al.; Onychomycosis epidemiology is a combination of various factors which include, among others, clinical presentation, etiologic agents of the infection and the patient's history background . Out of a total of 672 nail samples examined, 460 (68.4%) were microscopy positive for fungi and 306 (66.5%) of these were culture positive, including Candida (82%), dermatophytes (13.4%), Trichosporon spp (3.6%) and nondermatophyte molds (1%) . Onychomycosis was more frequent in females (79.7%) than in males (20.3%) . These were more common in fingernails (96.1%) than in toenails (60%) and yeasts were the most isolated etiologic agents . Among the clinical presentations, paronychia (CP) (57.2%) and onycholysis (CO) (24.8%) were the most common, caused frequently by C . albicans in 52.6% and 60.5% of the cases, respectively . T . rubrum (44.4%) and Trichosporon spp (22.2%) were the most frequent species in the case of distal lateral subungual onychomycosis (DLSO) . Fusarium spp was the agent responsible for 33.3% of the cases of proximal subungual onychomycosis (PSO) and for 14.3% of white superficial onychomycosis (WSO), whereas Acremonium spp was responsible for 14.3% of the cases of WSO. Science, 2002 Sep 6, 297(5587), 1692 - 6 Epub 2002 Aug 08. Structure, mechanism, and regulation of the Neurospora plasma membrane H+-ATPase; Kuhlbrandt W et al.; Proton pumps in the plasma membrane of plants and yeasts maintain the intracellular pH and membrane potential . To gain insight into the molecular mechanisms of proton pumping, we built an atomic homology model of the proton pump based on the 2.6 angstrom x-ray structure of the related Ca2+ pump from rabbit sarcoplasmic reticulum . The model, when fitted to an 8 angstrom map of the Neurospora proton pump determined by electron microscopy, reveals the likely path of the proton through the membrane and shows that the nucleotide-binding domain rotates by approximately 70 degrees to deliver adenosine triphosphate (ATP) to the phosphorylation site . A synthetic peptide corresponding to the carboxyl-terminal regulatory domain stimulates ATPase activity, suggesting a mechanism for proton transport regulation. Am J Clin Pathol, 2002 Aug, 118(2), 278 - 86 Evaluation of the status of laboratory practices and the need for continuing education in medical mycology; Rosner ER et al.; A survey to determine the need for training in medical mycology was sent to 605 US laboratories . Training needs were determined by comparing actual laboratory mycology practices with recommended practices, documenting the extent of mycology training reported by employees, and asking respondents to specify the fungi they considered most difficult to identify . The response rate was 56.7% (with only 316 laboratories providing sufficient information) . Results showed a large degree of interlaboratory variation in practices and suggested that more judicious practices could lower costs and improve clinical relevance . Only 55.6% of laboratories reported that at least 1 employee attended a formal mycology continuing education program in the 4 years before the survey . Species of dermatophytes, dematiaceous fungi, and non-Candida yeasts were the most difficult to identify . Training may be needed in basic isolation procedures and in advanced topics such as identification of problematic molds and yeasts and antifungal susceptibility testing . Educators should consider clinical relevance and cost-containment without sacrificing quality when designing courses . Support for additional mycology training may improve if hospital and laboratory administrators are alerted to potential dangers and costs involved in treating patients with invasive fungal infections. Mol Cell Biochem, 2002 May-Jun, 234-235(1-2), 3 - 9 Cyclic oxidation and reduction of protein methionine residues is an important antioxidant mechanism; Stadtman ER et al.; Almost all forms of reactive oxygen species (ROS) oxidize methionine residues of proteins to a mixture of the R- and S-isomers of methionine sulfoxide . Because organisms contain methionine sulfoxide reductases (Msr's) that can catalyze the thioredoxin-dependent reduction of the sulfoxides back to methionine, it was proposed that the cyclic oxidation/reduction of methionine residues might serve as antioxidants to scavenge ROS, and also to facilitate the regulation of critical enzyme activities . We summarize here results of studies showing that organisms possess two different forms of Msr--namely, MsrA that catalyzes reduction of the S-isomer and MsrB that catalyzes the reduction of the R-isomer . Deletion of the msrA gene in mice leads to increased sensitivity to oxidative stress and to a decrease (40%) in the maximum lifespan . This suggests that elimination of both Msr's would have more serious consequences. Cell, 2002 Jul 12, 110(1), 13 - 8 Shmoos, rafts, and uropods- the many facets of cell polarity; Dustin ML; The recent Juan March Foundation meeting on "Regulation and functional insights in cellular polarity" focused on cellular polarity in yeasts, Dictyostelium, epithelial cells, fibroblasts, and immune cells . The molecular systems covered included membrane rafts, actin and tubulin cytoskeleton, polarized transcription, signaling, and cell-cell adhesion . Across these diverse biological and molecular systems, important general concepts emerged, including new ideas for establishing and maintaining polarity that are likely to be applicable across models and experimental systems. Mol Cell, 2002 Jul, 10(1), 105 - 15 90S pre-ribosomes include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors; Grandi P et al.; We report the characterization of early pre-ribosomal particles . Twelve TAP-tagged components each showed nucleolar localization, sedimented at approximately 90S on sucrose gradients, and coprecipitated both the 35S pre-rRNA and the U3 snoRNA . Thirty-five non-ribosomal proteins were coprecipitated, including proteins associated with U3 (Nop56p, Nop58p, Sof1p, Rrp9, Dhr1p, Imp3p, Imp4p, and Mpp10p) and other factors required for 18S rRNA synthesis (Nop14p, Bms1p, and Krr1p) . Mutations in components of the 90S pre-ribosomes impaired 40S subunit assembly and export . Strikingly, few components of recently characterized pre-60S ribosomes were identified in the 90S pre-ribosomes . We conclude that the 40S synthesis machinery predominately associates with the 35S pre-rRNA factors, whereas factors required for 60S subunit synthesis largely bind later, showing an unexpected dichotomy in binding. J Clin Microbiol, 2002 Aug, 40(8), 2953 - 8 Multicenter comparative evaluation of six commercial systems and the national committee for clinical laboratory standards m27-a broth microdilution method for fluconazole susceptibility testing of Candida species; Morace G et al.; Fluconazole susceptibility among 800 clinical Candida isolates (60% C . albicans) and two control strains (C . krusei ATCC 6258 and C . parapsilosis ATCC 22019) was tested with the NCCLS M27-A method (gold standard) and six commercial products (Candifast, disk, Etest, Fungitest, Integral System Yeasts, and Sensititre YeastOne) . Results were classified as susceptible, susceptible-dose dependent, or resistant using M27-A breakpoints or, for Fungitest, Integral System Yeasts, and Candifast, as susceptible, intermediate, or resistant, according to the manufacturers' instructions . Concordance with NCCLS M27-A results was analyzed with the chi(2) test . Intra- and interlaboratory reproducibility was also evaluated . NCCLS M27-A (90.1%), Etest (93.1%), Sensititre YeastOne (93.1%), disk (96.7%), Fungitest (92.6%), Integral System Yeasts (40.6%), and Candifast (6.0%) classified the indicated percentages of C . albicans isolates as susceptible . Among non-C . albicans strains, the percentages of susceptible isolates were as follows: NCCLS M27-A, 74.0%; Etest, 83.8%; Sensititre YeastOne, 64.1%; disk, 60.6%; Fungitest, 76.6%; Integral System Yeasts, 28.3%; and Candifast, 27.4% . All methods except Candifast and Integral System Yeasts showed good agreement with NCCLS M27-A results for both C albicans and non-C . albicans isolates . Intralaboratory reproducibility was excellent for NCCLS M27-A, Etest, Sensititre YeastOne, disk, and Fungitest (88 to 91%) . Similar results emerged from the interlaboratory reproducibility evaluation . Our findings indicate that some commercial methods can be useful for fluconazole susceptibility testing of clinical Candida isolates . Those characterized by a lack of medium standardization and/or objective interpretative criteria should be avoided . Particular caution is necessary when testing is being done for clinical and epidemiological purposes. Mol Pathol, 2002 Aug, 55(4), 250 - 61 A role for CCN3 (NOV) in calcium signalling; Li CL et al.; AIMS: In animals and humans increased expression of CCN3 (NOV) is detected in tissues where calcium is a key regulator, such as the adrenal gland, central nervous system, bone and cartilage, heart muscle, and kidney . Because the multimodular structure of the CCN proteins strongly suggests that these cell growth regulators are metalloproteins, this study investigated the possible role of CCN3 in ion flux and transport during development, control of cell proliferation, differentiation, and pathobiology . METHODS: The isolation of CCN3 partners was performed by means of the two hybrid system . Yeasts were cotransfected with an HL60 cDNA library fused to the transactivation domain of the GAL4 transcription factor, and with a plasmid expressing CCN3 fused to the DNA binding domain of GAL4 . Screening of the recombinant clones selected on the basis of leucine, histidine, and tryptophan prototrophy was performed with a beta-galactosidase assay . After the interaction between CCN3 and its putative partners was checked with a GST (glutathione S-transferase) pull down assay, the positive clones were identified by cloning . To establish whether the CCN3 protein affected calcium ion flux, a dynamic imaging microscopy system was used, which allowed the fluorometric measurement of the intracellular calcium concentration . The proteins used in the assays were GST fused with either CCN3 or CCN2 (CTGF) and GST alone as a control . RESULTS: The two hybrid system identified the S100A4 (mts1) calcium binding protein as a partner of CCN3 and the use of the GST fusion proteins showed that the addition of CCN3 and CCN2 to G59 glioblastoma and SK-N-SH neuroblastoma cells caused a pronounced but transient increase of intracellular calcium, originating from both the entry of extracellular calcium and the mobilisation of intracellular stores . CONCLUSIONS: The interaction of CCN3 with S100A4 may account, in part, for the association of CCN3 with carcinogenesis and its pattern of expression in normal conditions . The increased intracellular calcium concentrations induced by CCN3 and CCN2 both involve different processes, among which voltage independent calcium channels might be of considerable importance in regulating the calcium flux associated with cell growth control, motility, and spreading . These observations assign for the first time a biological function to the CCN3 protein and point out a broader role for the CCN proteins in calcium ion signalling. Acta Biochim Pol, 2002, 49(1), 1 - 10 Aminoacyl-tRNA synthetases and aminoacylation of tRNA in the nucleus; Mucha P; This review is focused on findings concerning the presence of translation apparatus components (aminoacyl-tRNA synthetases, aminoacyl-tRNA, elongation factors) as well as translation itself in the nucleus . A nuclear role of these molecules is unknown . New findings suggest that well-accepted model of spatial segregation of transcription and translation in eukaryotic cell may be oversimplifcation . Nuclear coupling of both these processes show us how exciting and surprising may be the world of the living cell. Genetics, 2002 Jul, 161(3), 1089 - 99 Identification of six loci in which mutations partially restore peroxisome biogenesis and/or alleviate the metabolic defect of pex2 mutants in podospora; Ruprich-Robert G et al.; Peroxins (PEX) are proteins required for peroxisome biogenesis . Mutations in PEX genes cause lethal diseases in humans, metabolic defects in yeasts, and developmental disfunctions in plants and filamentous fungi . Here we describe the first large-scale screening for suppressors of a pex mutation . In Podospora anserina, pex2 mutants exhibit a metabolic defect {inability to grow on medium containing oleic acid (OA medium) as sole carbon source} and a developmental defect (inability to differentiate asci in homozygous crosses) . Sixty-three mutations able to restore growth of pex2 mutants on OA medium have been analyzed . They fall in six loci (suo1 to suo6) and act as dominant, allele-nonspecific suppressors . Most suo mutations have pleiotropic effects in a pex2(+) background: formation of unripe ascospores (all loci except suo5 and suo6), impaired growth on OA medium (all loci except suo4 and suo6), or sexual defects (suo4) . Using immunofluorescence and GFP staining, we show that peroxisome biogenesis is partially restored along with a low level of ascus differentiation in pex2 mutant strains carrying either the suo5 or the suo6 mutations . The data are discussed with respect to beta-oxidation of fatty acids, peroxisome biogenesis, and cell differentiation. Peptides, 2002 Jul, 23(7), 1351 - 9 Transmissible spongiform encephalopathies: the story of a pathogenic protein; Van Everbroeck B et al.; An overview is provided from the first description of the transmissible spongiform encephalopathies (TSE) to recent major discoveries in this research field . The TSE are a group of diseases in animal and in man caused by a unique pathogen: the prion protein . The exact nature of the etiological agent or the prion protein is thought to be a misfolded protein . Although current research has provided a wealth of data indicating that a structural isoform of the prion protein is the responsible pathogen, this hypothesis is not yet experimentally proven. Drug Resist Updat, 2002 Feb, 5(1), 3 - 10 Recent progress in the diagnosis of fungal infections in the immunocompromised host; Erjavec Z et al.; The management of invasive fungal infections has been hampered by the inability to diagnose the infection at an early stage of disease . Although proving the presence of infection by histology and culture remains the cornerstone of the diagnosis, non-culture based methods are becoming available that enable early detection . Molecular diagnosis by PCR appears very promising since fungal DNA can be detected in the blood of infected patients before conventional methods . Furthermore, a broad range of yeasts and molds can be identified to species level . Automation of sample preparation and use of real-time PCR systems will help standardize the procedure and reduce false positive results due to contamination . Promising assays for the detection of fungal antigens in serum have been commercialized, including detection systems for mannan (Candida) and galactomannan (Aspergillus) . Circulating antigens can be detected at an early stage of infection, often before the onset of clinical symptoms . Antigen detection is limited to detecting only one genus and not enabling speciation . Furthermore, both PCR and antigen detection can be used to monitor the response of patients to treatment with anti-fungal agents . Although prospective screening of high-risk patients for the presence of circulating markers of fungal infection appears to be an appropriate strategy, studies are needed to help to establish the optimal approach to managing invasive fungal infections that incorporates the benefits of non-culture based methods. Curr Opin Struct Biol, 2002 Jun, 12(3), 368 - 73 Computational methods for the prediction of protein interactions; Valencia A et al.; Establishing protein interaction networks is crucial for understanding cellular operations . Detailed knowledge of the 'interactome', the full network of protein-protein interactions, in model cellular systems should provide new insights into the structure and properties of these systems . Parallel to the first massive application of experimental techniques to the determination of protein interaction networks and protein complexes, the first computational methods, based on sequence and genomic information, have emerged. Trends Plant Sci, 2002 Jul, 7(7), 301 - 8 Mitogen-activated protein kinase cascades in plants: a new nomenclature; MAPK Group; Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes, including yeasts, animals and plants . These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses . In plants, MAPK cascades are involved in responses to various biotic and abiotic stresses, hormones, cell division and developmental processes . Completion of the Arabidopsis genome-sequencing project has revealed the existence of 20 MAPKs, 10 MAPK kinases and 60 MAPK kinase kinases . Here, we propose a simplified nomenclature for Arabidopsis MAPKs and MAPK kinases that might also serve as a basis for standard annotation of these gene families in all plants. J Biol Chem, 2002 Jul 19, 277(29), 26098 - 102 Epub 2002 May 06. A point mutation of the AF2 transactivation domain of the glucocorticoid receptor disrupts its interaction with steroid receptor coactivator 1; Kucera T et al.; Glucocorticoids cause a 10-fold increase in hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene transcription through two low affinity glucocorticoid receptor (GR) binding sites and a complex array of accessory factor DNA elements and associated proteins . To analyze how co-activators interact with the GR in this context, we took advantage of the C656G GR mutant that binds ligand with very high affinity . This GR activates PEPCK gene transcription at a 500-fold lower dexamethasone concentration than does wild type GR . Transfected C656G GR containing additional mutations or deletions was tested on PEPCK gene expression in H4IIE hepatoma cells . We found that the AF2 domain is the only one of the three defined transactivation domains in GR that is required for PEPCK gene expression and that mutation of this domain disrupts the direct interaction of GR with steroid receptor coactivator 1 (SRC-1) . These data help define the functional interaction between GR and SRC-1 and further define the role of the GR in glucocorticoid-mediated expression of the PEPCK gene. Kidney Int, 2002 Aug, 62(2), 679 - 87 Efficient in vitro lowering of carbonyl stress by the glyoxalase system in conventional glucose peritoneal dialysis fluid; Inagi R et al.; BACKGROUND: Reactive carbonyl compounds (RCOs) present in heat-sterilized peritoneal dialysis (PD) fluid have been incriminated in the progressive deterioration of the peritoneal membrane observed in long-term PD patients . The present study utilized the glyoxalase I (GLO I) system as a new approach to lower in vitro the peritoneal fluid content of RCOs such as methylglyoxal (MGO), glyoxal (GO) and 3-deoxyglucosone (3-DG) . METHODS: GO, MGO, and 3-DG solutions or conventional glucose PD fluids were incubated in vitro with various RCO lowering compounds . The evolution of GO, MGO, and 3-DG levels was monitored by high-performance liquid chromatography . The tested compounds included aminoguanidine and glutathione (GSH), alone or together with GLO I . The human GLO I gene was overexpressed in Chinese hamster ovary (CHO) cells, or ubiquitously in transgenic mice . Cell supernatant of the CHO transfectant and protein extracts of various organs of the transgenic mice were also tested . RESULTS: Aminoguanidine incubated with MGO/GO/3-DG mixtures, promptly reduced RCO levels . GSH alone had a similar but milder and slower effect . Together with GLO I, it promptly decreased GO and MGO levels but was less efficient toward 3-DG . After incubation with glucose PD fluid, GSH together with GLO I had the same effect on MGO, GO, and 3-DG levels . Addition of transfected cell supernatant or tissue extracts overexpressing GLO I, together with GSH to either GO, MGO, or 3-DG solutions, promptly and markedly reduced GO and MGO but not 3-DG levels . CONCLUSIONS: GLO I together with GSH efficiently lowers glucose-derived RCOs, especially GO and MGO, both in conventional glucose PD fluids and in RCO solutions . The fact that genetically manipulated cells overexpressing GLO I activity have a similar effect suggests that maneuvers raising GLO I activity in peritoneal cells or in the peritoneal cavity might help prevent the deleterious effects of the peritoneal carbonyl stress in PD patients . The clinical relevance of this approach is yet to be documented. Int Immunopharmacol, 2002 May, 2(6), 797 - 806 Modulation of rat macrophage function by the Mangifera indica L . extracts Vimang and mangiferin; Garcia D et al.; Vimang is an aqueous extract of Mangiferia indica L., traditionally used in Cuba as an anti-inflammatory, analgesic and antioxidant . In the present study, we investigated the effects of Vimang and of mangiferin (a C-glucosylxanthone present in the extract) on rat macrophage functions including phagocytic activity and the respiratory burst . Both Vimang and mangiferin showed inhibitory effects on macrophage activity: (a) intraperitoneal doses of only 50-250 mg/kg markedly reduced the number of macrophages in peritoneal exudate following intraperitoneal injection of thioglycollate 5 days previously (though there was no significant effect on the proportion of macrophages in the peritoneal-exudate cell population); (b) in vitro concentrations of 0.1-100 microg/ml reduced the phagocytosis of yeasts cells by resident peritoneal and thioglycollate-elicited macrophages; (c) in vitro concentrations of 1-50 microg/ml reduced nitric oxide (NO) production by thioglycollate-elicited macrophages stimulated in vitro with lipopolysaccharide (LPS) and IFNgamma; and (d) in vitro concentrations of 1-50 microg/ml reduced the extracellular production of reactive oxygen species (ROS) by resident and thioglycollate-elicited macrophages stimulated in vitro with phorbol myristate acetate (PMA) . These results suggest that components of Vimang, including the polyphenol mangiferin, have depressor effects on the phagocytic and ROS production activities of rat macrophages and, thus, that they may be of value in the treatment of diseases of immunopathological origin characterized by the hyperactivation of phagocytic cells such as certain autoimmune disorders. Mol Endocrinol, 2002 Jul, 16(7), 1538 - 51 Evolution of glycoprotein hormone subunit genes in bilateral metazoa: identification of two novel human glycoprotein hormone subunit family genes, GPA2 and GPB5; Hsu SY et al.; The canonical members of the human glycoprotein hormone subunit family of cystine knot-forming polypeptides include the common alpha-subunit, and four beta-subunit genes, FSHbeta, LHbeta, TSHbeta, and hCGbeta . Using pairwise sequence analysis of the complete human genome, we have identified two novel glycoprotein hormone subunit-related genes . Based on unique sequence similarity to the alpha- and beta-subunits of glycoprotein hormones, they were named glycoprotein-alpha2 (GPA2) and glycoprotein-beta5 (GPB5), respectively . PCR analysis using a panel of human cDNAs from 14 different tissues demonstrated that GPB5 is similar to other beta-subunits showing restricted tissue expression, mainly in pituitary and brain . In contrast, the GPA2 transcript is found in diverse tissues . Furthermore, immunoreactive GPA2 and GPB5 were detected in the anterior pituitary of mouse and frog, whereas the expression of GPA2 and GPB5 in transfected cells resulted in the secretion of recombinant polypeptides in conditioned medium . After GenBank searches in lower organisms, glycoprotein hormone beta-subunit-related genes were identified from the genome of nematode Caenorhabditis elegans, hookworm Ancylostoma caninum, and Drosophila melanogaster . The evolutionary conservation of these invertebrate homologs can be seen in several key sequence characteristics, and the data suggest that the glycoprotein hormone beta-subunit gene ancestor evolved before the emergence of bilateral metazoa, thus providing a better understanding of the evolution of this group of classic polypeptide hormones and their receptors . Studies of the complete inventory of genes homologous to glycoprotein hormone subunits in the human genome and lower organisms will allow future functional characterization and identification of their respective receptors. Philos Trans R Soc Lond B Biol Sci, 2002 Jun 29, 357(1422), 749 - 60 Plant D-type cyclins and the control of G1 progression; Oakenfull EA et al.; The basic pattern of controls that operate during the G1 phase of the plant cell cycle shows much closer similarity to animals than to the yeasts and other fungi . The activity of D-type cyclin (CycD) kinases is induced in response to stimulatory signals, and these phosphorylate the plant homologue of the retinoblastoma tumour susceptibility (Rb) protein . It is likely that Rb phosphorylation results in the activation of genes under the control of E2F transcription factors, including those required for S phase entry . As the initial triggers of the cascade, attention has focused on the CycDs, and a family of 10 genes is present in Arabidopsis, divided into three major and three minor groups . Analysis to date suggests that these groups are functionally distinct. J Cell Sci, 2002 Jul 1, 115(Pt 13), 2651 - 8 Compartmentation of enzymes in a microbody, the glycosome, is essential in Trypanosoma brucei; Guerra-Giraldez C et al.; All kinetoplastids contain membrane-bound microbodies known as glycosomes, in which several metabolic pathways including part of glycolysis are compartmentalized . Peroxin 2 is essential for the import of many proteins into the microbodies of yeasts and mammals . The PEX2 gene of Trypanosoma brucei was identified and its expression was silenced by means of tetracycline-inducible RNA interference . Bloodstream-form trypanosomes, which rely exclusively on glycolysis for ATP generation, died rapidly upon PEX2 depletion . Insect-form (procyclic) trypanosomes do not rely solely on glycolysis for ATP synthesis . PEX2 depletion in procyclic forms resulted in relocation of most tested matrix proteins to the cytosol, and these mutants also died . Compartmentation of microbody enzymes is therefore essential for survival of bloodstream and procyclic T . brucei . In contrast, yeasts and cultured mammalian cells grow normally in the absence of peroxisomal membranes unless placed on selective media. Mycoses, 2002, 45 Suppl 1, 47 - 52 {Histologic studies on otomycosis}; Vennewald I et al.; Fungal infections of the ear are mostly described as mycoses of the auditory canal . The aim of our investigations was to find out how fungi colonize the ear in immunocompetent patients . In the years from 1993 to 2000, 128 patients suspected of having otomycosis were examined . Of these 115 patients suffered from chronic otitis media with persisting tympanum perforation and otorrhea . A further 13 patients had clinical signs of otitis externa only . In 54 out of 139 samples, fungi were found in the auditory canal, in five on the tympanic membrane, and in five in the middle ear . Two-thirds were isolated as moulds and one-third as yeasts . Dominating species were Aspergillus niger and Candida parapsilosis . Samples of 15 patients suspected of having mastoiditis or cholesteatoma were examined histologically . Fungal hyphae were observed in the middle ear cavity and/or between horny lamellae of cholesteatoma in 4 patients . In the middle ear of immunocompetent patients chronic-hyperplastic (polypous) inflammation was detected with increased production of mucus, which probably promotes the colonization with pathogenic fungi as in the middle ear just like in the auditory canal. Virchows Arch, 2002 Jun, 440(6), 573 - 82 Epub 2002 Apr 09. Telomere lengthening in telomerase-negative cells: the ends are coming together; Scheel C et al.; Telomeres are crucial for chromosomal stability and cell viability . Activation of telomerase, a specialized reverse transcriptase, is the predominant mechanism for maintaining telomere length and function in yeasts and human cells . Telomere maintenance is regarded as a key element in the immortalization of cells and hence in oncogenesis . Although more than 90% of all malignant tumors display telomerase activity, there appear to be alternatives to this mechanism . Alternative lengthening of telomeres (ALT) in the absence of telomerase has been described in various organisms and recently also in immortalized and transformed human cells . This article will discuss how ALT is detected and how it affects telomere morphology . It will review the frequency and relevance of ALT in in vitro immortalized cell lines, tumor-derived cell lines, and primary tumors . We have only begun to link mechanisms by means of which ALT may act in immortalized human cells to the growing knowledge about telomeres, and we can look forward to further fascinating insights into telomere biology . Our review will also emphasize recent advances in our understanding of the induction and repression of ALT and the demonstration of ALT in cancer cells in the light of new treatment strategies targeted against telomere maintenance mechanisms. Curr Opin Cell Biol, 2002 Jun, 14(3), 377 - 83 Replication timing and transcriptional control: beyond cause and effect; Gilbert DM; In general, transcriptionally active euchromatin replicates during the first half of S phase, whereas silent heterochromatin replicates during the second half . Moreover, changes in replication timing accompany key stages of development . Although there is not a strict correlation between replication timing and transcription per se, recent results reveal a strong relationship between heritably repressed chromatin and late replication that is conserved in all eukaryotes . A long-standing question is whether replication timing dictates the structure of chromatin or vice versa . Mounting evidence supports a model in which replication timing is both cause and consequence of chromatin structure by providing a means to inherit chromatin states that, in turn, regulate replication timing in the subsequent cell cycle . Moreover, new findings relating aberrations in replication timing to defects in centromere function, chromosome cohesion and genome instability suggest that the role of replication timing extends beyond its relationship to transcription . Novel systems in both yeasts and mammals are finally beginning to reveal some of the determinants that regulate replication timing, which should pave the way for a long-anticipated molecular dissection of this complex liaison. J Cell Biol, 2002 Jun 10, 157(6), 941 - 51 Epub 2002 Jun 10. Rlp7p is associated with 60S preribosomes, restricted to the granular component of the nucleolus, and required for pre-rRNA processing; Gadal O et al.; Many analyses have examined subnucleolar structures in eukaryotic cells, but the relationship between morphological structures, pre-rRNA processing, and ribosomal particle assembly has remained unclear . Using a visual assay for export of the 60S ribosomal subunit, we isolated a ts-lethal mutation, rix9-1, which causes nucleolar accumulation of an Rpl25p-eGFP reporter construct . The mutation results in a single amino acid substitution (F176S) in Rlp7p, an essential nucleolar protein related to ribosomal protein Rpl7p . The rix9-1 (rlp7-1) mutation blocks the late pre-RNA cleavage at site C2 in ITS2, which separates the precursors to the 5.8S and 25S rRNAs . Consistent with this, synthesis of the mature 5.8S and 25S rRNAs was blocked in the rlp7-1 strain at nonpermissive temperature, whereas 18S rRNA synthesis continued . Moreover, pre-rRNA containing ITS2 accumulates in the nucleolus of rix9-1 cells as revealed by in situ hybridization . Finally, tagged Rlp7p was shown to associate with a pre-60S particle, and fluorescence microscopy and immuno-EM localized Rlp7p to a subregion of the nucleolus, which could be the granular component (GC) . All together, these data suggest that pre-rRNA cleavage at site C2 specifically requires Rlp7p and occurs within pre-60S particles located in the GC region of the nucleolus. Bioorg Med Chem, 2002 Aug, 10(8), 2657 - 62 Non-thiol farnesyltransferase inhibitors: utilization of an aryl binding site by 5-arylacryloylaminobenzophenones; Mitsch A et al.; We recently described a novel aryl binding site of farnesyltransferase . The 2-naphthylacryloyl residue was developed as an appropriate substituent for our benzophenone-based AAX-peptidomimetic capable of occupying this binding site, resulting in a non-thiol farnesyltransferase inhibitor with nanomolar activity . The activity of this inhibitor is readily explained on the basis of docking studies which show the 2-naphthyl residue fitting into the aryl binding site. Structure (Camb), 2002 Jun, 10(6), 763 - 72 Intra- and intermolecular interactions in sucrose transporters at the plasma membrane detected by the split-ubiquitin system and functional assays; Reinders A et al.; Interaction of two separately expressed halves of sucrose transporter SUT1 was detected by an optimized split-ubiquitin system . The halves reconstitute sucrose transport activity at the plasma membrane with affinities similar to the intact protein . The halves do not function independently, and an intact central loop is not required for membrane insertion, plasma membrane targeting, and transport . Under native conditions, the halves associate into higher molecular mass complexes . Furthermore, the N-terminal half of the low-affinity SUT2 interacts functionally with the C-terminal half of SUT1 . Since the N terminus of SUT2 determines affinity for sucrose, the reconstituted chimera has lower affinity than SUT1 . The split-ubiquitin system efficiently detects intramolecular interactions in membrane proteins, and can be used to dissect transporter structure. Biochem Biophys Res Commun, 2002 May 31, 294(1), 145 - 8 Native transfer RNA catalyzes Diels-Alder reaction; Mielcarek M et al.; In this paper we show that transfer ribonucleic acids (tRNAs) catalyze the Diels-Alder cycloaddition reaction . A new DNA oxidative damage product, 6-furfuryladenine (kinetin) or its riboside (diene), was transformed with dimethyl acetylenedicarboxylate or maleic anhydride (dienophile) . The reaction proceeds in the presence of tRNA at high pressure but not at ambient condition . If so tRNA in prebiotic conditions (RNA world) had at least two functions: catalytic and a carrier of genetic information . It means that tRNA at high pressure shows catalytic properties and is a true Diels-Alderase. Int J Infect Dis, 2002 Mar, 6 Suppl 1, S47 - 53 Antifungal drug resistance: does it matter? Rogers TR. The objectives of this review are: to review the modes of action of currently available antifungal drugs; to define drug resistance and discuss the mechanisms by which fungi can develop resistance to antifungal drugs; to consider the epidemiological and host factors that contribute to the outcome of antifungal therapy and whether the available in vitro susceptibility test methods can reliably predict clinical response; and to assess the overall relevance of drug resistance to the outcome of fungal infections.The incidence of antifungal drug resistance among pathogens causing invasive fungal infections appears to be increasing . In the case of Candida spp., this may in part be a consequence of selective pressure brought about by more intensive antifungal use leading to a 'pathogen shift' . Non-albicans Candida spp . are more likely to demonstrate reduced susceptibility to fluconazole compared to C . albicans . Susceptibility breakpoints developed by the National Committee for Clinical Laboratory Standards to test azoles and flucytosine against Candida spp . are helpful in guiding therapy . Antifungal drug resistance in yeasts is of clinical importance . Increasingly reliable methods of in vitro susceptibility testing can help predict clinical response to therapy, but other considerations, including host- and drug-related factors, can also have an important bearing on the ultimate outcome of treatment. Mycopathologia, 2002, 154(1), 25 - 8 Edible dates (Phoenix dactylifera), a potential source of Cladosporium cladosporioides and Sporobolomyces roseus: implications for public health; Moore JE et al.; Edible dates (Phoenix dactylifera) were examined for the presence of endogenous yeasts and filimentous fungi . Mean counts of fungi were 530 colony forming units (cfu) per gram of fruit, representing a mixture of two phenotypic colony types . Subsequent DNA extraction and PCR amplification of these two morphotypes yielded an amplicon of approximately 350 bp with the 5.8S-28S rRNA ITS region . Sequence analysis identified these to be Cladosporium cladosporioides (230 cfu/g) and Sporobolomyces roseus . Both organisms have been previously reported in opportunistic infections involving skin or in immunocompromised patients . This is the first report of edible dates being a source of these organisms and we emphasize the importance of the common practice of washing hands following the consumption of these fruits by hand. J Biol Chem, 2002 Jul 19, 277(29), 26281 - 5 Epub 2002 May 14. Mediation of the DCC apoptotic signal by DIP13 alpha; Liu J et al.; DCC (deleted in colorectal cancer) is a candidate tumor suppressor gene . However the function of DCC remains elusive . Previously, we demonstrated that forced expression of DCC induces apoptosis or cell cycle arrest (Chen, Y . Q., Hsieh, J . T., Yao, F., Fang, B., Pong, R . C., Cipriano, S . C . & Krepulat, F . (1999) Oncogene 18, 2747-2754) . To delineate the DCC-induced apoptotic pathway, we have identified a protein, DIP13 alpha, which interacts with DCC . The DIP13 alpha protein has a pleckstrin homology domain and a phosphotyrosine binding domain . It interacts with a region on the DCC cytoplasmic domain that is required for the induction of apoptosis . Although ectopic expression of DIP13 alpha alone causes only a slight increase in apoptosis, co-expression of DCC and DIP13 alpha results in an approximately 5-fold increase in apoptosis . Removal of the DCC-interacting domain on DIP13 alpha abolishes its ability to enhance DCC-induced apoptosis . Inhibition of endogenous DIP13 alpha expression by small interfering RNA blocks DCC-induced apoptosis . Our data suggest that DIP13 alpha is a mediator of the DCC apoptotic pathway. Clin Microbiol Infect, 2002 Mar, 8(3), 162 - 73 Identification of Malassezia species from patient skin scales by PCR-RFLP; Gaitanis G et al.; OBJECTIVE: This study was aimed at the development of a DNA-based procedure directly applicable to pathological skin scales and at the assessment of its value in rapid laboratory confirmation and identification of each of the seven Malassezia species . These lipophilic basidiomycetous yeasts in predisposed individuals are involved in pityriasis versicolor, seborrheic dermatitis, blepharitis, folliculitis, atopic dermatitis and fungemia . Standard identification procedures to species level are available, but so far no system for direct detection and characterization of Malassezia species in clinical specimens is available . METHODS: Malassezia DNA was extracted from pathological skin scales by a modified hexadecyltrimethylammonium bromide (CTAB) method and amplified by single and nested polymerase chain reaction (PCR), assays using the general fungal ITS 1/4 and 3/4 primers for amplification of sequences from the Malassezia major ribosomal DNA complex . Restriction fragment length polymorphism (RFLP) analysis of PCR products was used in subsequent species identification . DNA extracted from culture-positive skin scales was also tested by PCR and the RFLP patterns obtained were analyzed . RESULTS: A total of 36 isolates were tested . Distinct pure culture and skin-scale ITS 3/4 HinfI and AluI restriction patterns differentially identified M . furfur, M . globosa, M . restricta, M . sympodialis, M . pachydermatis, M . obtusa and M . slooffiae . Malassezia DNA was extracted from pathological skin scales and RFLP identified solitary and multiple Malassezia species in the same specimen . Molecular identification was confirmed by cultures and biochemical tests . Concurrent detection and identification of Candida and Yarrowia species was also feasible from skin scales . CONCLUSION: The proposed method, described for the first time, could provide a sensitive and rapid detection and identification system for Malassezia species, which may be applied to epidemiological surveys and routine practice. Biochim Biophys Acta, 2002 May 20, 1597(1), 36 - 41 The distinctiveness of ATP:citrate lyase from Aspergillus nidulans; Adams IP et al.; ATP:citrate lyase (ACL), an important enzyme in lipid synthesis, has been purified from Aspergillus nidulans to a specific activity of 19.6 micromol min(-1) mg(-1), almost twice that of any other purified ACL and shown to be distinct from any previously purified ACL . The enzyme is a 371+/-31 kDa hexamer of 3 alpha, 3 beta proteins, unlike the 4 alpha tetramer found in rats or yeasts . The molecular weights of the alpha and beta protein subunits were determined by SDS-PAGE to be 70 and 55 kDa.ACL in A . nidulans (unlike Aspergillus niger) appears to be regulated by the carbon source present in the media . In crude extracts, it was found at high activity (88 micromol min(-1) mg protein(-1)) in glucose-grown cells but only at low activity (10 micromol min(-1) mg protein(-1)) in acetate-grown cells. Biochemistry, 2002 May 7, 41(18), 5765 - 75 Mapping the G-actin binding surface of cofilin using synchrotron protein footprinting; Guan JQ et al.; Cofilin is an actin regulatory protein that binds to both monomeric and filamentous actin, and has filament severing activity . Although crystal structures for the monomeric forms of both G-actin and cofilin have been described, the structure of the binary cofilin-G-actin complex is not available . Synchrotron protein footprinting is used to identify specific side chain residues on the cofilin surface that are buried in the formation of the cofilin-G-actin binary complex . Exposure to synchrotron X-rays results in stable oxidative modifications of aromatic, aliphatic, and sulfur-containing side chains, with the rate of modification for a particular residue being dependent on its intrinsic reactivity and solvent accessibility . The rates of modification were monitored for a number of peptides generated by digestion of oxidized cofilin, both in isolation and in its binary complex with G-actin . After binding to G-actin takes place, a significant decrease in modification rates, indicating protection of side chain groups, is seen for cofilin peptides corresponding to residues 4-20, 10-17, 83-96, 91-105, and 106-117 . A number of other peptides show no change in reactivity, and are presumed to represent regions distal to the binding site . Tandem mass spectrometry demonstrates that residues Leu 13, Pro 94, Met 99, and Leu 108 and 112 directly participate in the binding interface . These results are generally consistent with, and complementary to, the results of previous site-directed mutagenesis studies and extend our understanding of the G-actin binding surface of cofilin. Brain Res Mol Brain Res, 2002 Mar 28, 99(2), 145 - 9 Protein inhibitor of neuronal nitric oxide synthase interacts with protein kinase A inhibitors; Yu J et al.; Protein kinase A (PKA) and neuronal nitric oxide synthase (nNOS) are important signaling molecules . It is well known that PKA can specifically phosphorylate nNOS . But the underlying molecular mechanism is still obscure . Our data indicate that the protein inhibitor of nNOS (PIN) binds to protein kinase A inhibitors (PKIs), which suggests that PKIs, together with PIN, might mediate the phosphorylation of nNOS by PKA. Curr Opin Infect Dis, 2002 Apr, 15(2), 133 - 42 Malassezia species in skin diseases; Crespo Erchiga V et al.; Since the taxonomic revision carried out in 1996, enlarging the genus Malassezia to comprise seven different species, a number of studies have investigated from different points of view -- mycological, molecular and immunological -- the relationships of these species with the pathologies associated with lipophilic yeasts, as well as its presence in healthy skin . From these studies, it now appears clear that Malassezia globosa is the main species associated with pityriasis versicolor, which is the only cutaneous disease in which the involvement of Malassezia is undisputed . Nevertheless, this species can also be found in normal skin, in which the predominant species is Malassezia sympodialis . In the remaining dermatological disorders related to Malassezia, the role of these yeasts is controversial . In seborrhoeic dermatitis, atopic dermatitis and folliculitis, several studies have focused on the immunological aspects that could explain the pathogenic mechanism . In other diseases, such as confluent and reticulate papillomatosis, neonatal pustulosis, otitis and onychomycosis, the presence or significance of Malassezia is still a matter of dispute. Food Addit Contam, 2002 Apr, 19(4), 408 - 14 Potential ochratoxin A producers from wine grapes in Argentina and Brazil; Da RR et al.; The aim was to identify the normal mycoflora in wine grapes from Argentina and Brazil . We collected 50 grapes samples from Malbec and Chardonnay varieties in each country during the 1997-98 harvest . Yeasts were a major component of the fungal population, and the most frequent genera of filamentous fungi isolated were: Aspergillus, Penicillium and Botrytis . Other genera identified (in decreasing order) were: Phythophthora, Moniliella, Alternaria and Cladosporium . From grapes, the mean frequency of filamentous fungi ranged from 1.3 x 10(4) to 5.4 x 10(6) CFU g(-1) . We isolated 48 Aspergillus niger strains from Argentinian grape, of which eight could produce ochratoxin A . Sixteen of 53 A . niger strains from Brazilian grapes produced ochratoxin A . The results indicate that similar mycobiota were isolated from Argentinian and Brazilian wine grapes and there could be ochratoxin A production in this substrate. Proc Natl Acad Sci U S A, 2002 Apr 16, 99(8), 5331 - 6 The frameshift signal of HIV-1 involves a potential intramolecular triplex RNA structure; Dinman JD et al.; The cis-acting mRNA elements that promote programmed -1 ribosomal frameshifting present a natural target for the rational design of antiretroviral chemotherapies . It has been commonly accepted that the HIV-1 frameshifting signal is special, because its downstream enhancer element consists of a simple mRNA stem loop rather than a more complex secondary structure such as a pseudoknot . Here we present three lines of evidence, bioinformatic, structural, and genetic, showing that the biologically relevant HIV-1 frameshift signal contains a complex RNA structure that likely includes an extended RNA triple-helix region . We suggest that the potential intramolecular triplex structure is essential for viral propagation and viability, and that small molecules targeted to this RNA structure may possess antiretroviral activities. Mol Biol Cell, 2002 Apr, 13(4), 1390 - 407 Morphology and dynamics of the endocytic pathway in Dictyostelium discoideum; Neuhaus EM et al.; Dictyostelium discoideum is a genetically and biochemically tractable social amoeba belonging to the crown group of eukaryotes . It performs some of the tasks characteristic of a leukocyte such as chemotactic motility, macropinocytosis, and phagocytosis that are not performed by other model organisms or are difficult to study . D . discoideum is becoming a popular system to study molecular mechanisms of endocytosis, but the morphological characterization of the organelles along this pathway and the comparison with equivalent and/or different organelles in animal cells and yeasts were lagging . Herein, we used a combination of evanescent wave microscopy and electron microscopy of rapidly frozen samples to visualize primary endocytic vesicles, vesicular-tubular structures of the early and late endo-lysosomal system, such as multivesicular bodies, and the specialized secretory lysosomes . In addition, we present biochemical and morphological evidence for the existence of a micropinocytic pathway, which contributes to the uptake of membrane along side macropinocytosis, which is the major fluid phase uptake process . This complex endosomal compartment underwent continuous cycles of tubulation/vesiculation as well as homo- and heterotypic fusions, in a way reminiscent of mechanisms and structures documented in leukocytes . Finally, egestion of fluid phase from the secretory lysosomes was directly observed. Nat Cell Biol, 2002 May, 4(5), 329 - 36 The TRPM7 channel is inactivated by PIP(2) hydrolysis; Runnels LW et al.; TRPM7 (ChaK1, TRP-PLIK, LTRPC7) is a ubiquitous, calcium-permeant ion channel that is unique in being both an ion channel and a serine/threonine kinase . The kinase domain of TRPM7 directly associates with the C2 domain of phospholipase C (PLC) . Here, we show that in native cardiac cells and heterologous expression systems, G alpha q-linked receptors or tyrosine kinase receptors that activate PLC potently inhibit channel activity . Numerous experimental approaches demonstrated that phosphatidylinositol 4,5-bisphosphate (PIP(2)), the substrate of PLC, is a key regulator of TRPM7 . We conclude that receptor-mediated activation of PLC results in the hydrolysis of localized PIP(2), leading to inactivation of the TRPM7 channel. Mol Cell, 2002 Mar, 9(3), 505 - 14 A positive-strand RNA virus replication complex parallels form and function of retrovirus capsids; Schwartz M et al.; We show that brome mosaic virus (BMV) RNA replication protein 1a, 2a polymerase, and a cis-acting replication signal recapitulate the functions of Gag, Pol, and RNA packaging signals in conventional retrovirus and foamy virus cores . Prior to RNA replication, 1a forms spherules budding into the endoplasmic reticulum membrane, sequestering viral positive-strand RNA templates in a nuclease-resistant, detergent-susceptible state . When expressed, 2a polymerase colocalizes in these spherules, which become the sites of viral RNA synthesis and retain negative-strand templates for positive-strand RNA synthesis . These results explain many features of replication by numerous positive strand RNA viruses and reveal that these viruses, reverse transcribing viruses, and dsRNA viruses share fundamental similarities in replication and may have common evolutionary origins. Mol Endocrinol, 2002 Apr, 16(4), 757 - 73 The nuclear receptor coactivators p300/CBP/cointegrator-associated protein (p/CIP) and transcription intermediary factor 2 (TIF2) differentially regulate PKA-stimulated transcriptional activity of steroidogenic factor 1; Borud B et al.; Steroidogenic factor-1 (SF-1) is a member of the nuclear receptor superfamily that plays essential roles in the development of endocrine organs . Steroid receptor coactivator 1 and transcription intermediary factor 2 (TIF2) belong to the p160 coactivator family that mediates transcriptional activation by several nuclear receptors, including SF-1 . Here, it is reported that another of the p160 coactivators, p/CIP, interacts with SF-1 through the activation function-2 domain . Both p300/CBP/cointegrator-associated protein (p/CIP) and TIF2 potentiated SF-1-mediated transcription from two reporter gene constructs in transfected nonsteroidogenic COS-1 cells and in adrenocortical Y1 cells . PKA was shown to stimulate SF-1 transcriptional activity, and coexpression of p/CIP together with the PKA catalytic subunit stimulated SF-1-mediated transactivation even further . In contrast, PKA catalytic subunit overexpression impaired the ability of TIF2 to potentiate SF-1-dependent transcription . Activation of PKA also inhibited the TIF2-mediated coactivation of other nuclear receptors such as PPAR alpha/-gamma and liver X receptor-alpha . The TIF2 mRNA levels were not affected by PKA, but instead we found that PKA activation led to a decrease in the levels of TIF2 protein . Moreover, the C-terminal activation domain 2 of TIF2 was required for the inhibitory effect of PKA, suggesting that this region is the target for the PKA-mediated down-regulation . Thus, in contrast to the regulation of p/CIP and steroid receptor coactivator 1, we suggest that activation of PKA leads to selective down-regulation of TIF2 and subsequently repression of TIF2 coactivator function. Biochem Biophys Res Commun, 2002 Mar 29, 292(2), 300 - 7 DOC-2/DAB2 is the binding partner of myosin VI; Inoue A et al.; Myosin VI is a molecular motor that moves processively along actin filaments and is believed to play a role in cargo movement in cells . Here we found that DOC-2/DAB2, a signaling molecule inhibiting the Ras cascade, binds to myosin VI at the globular tail domain . DOC-2/DAB2 binds stoichiometrically to myosin VI with one molecule per one myosin VI heavy chain . The C-terminal 122 amino acid residues of DOC-2/DAB2, containing the Grb2 binding site, is identified to be critical for the binding to myosin VI . Actin gliding assay revealed that the binding of DOC-2/DAB2 to myosin VI can support the actin filament gliding by myosin VI, suggesting that it can function as a myosin VI anchoring molecule . The C-terminal domain but not the N-terminal domain of DOC-2/DAB2 functions as a myosin VI anchoring site . The present findings suggest that myosin VI plays a role in transporting DOC-2/DAB2, a Ras cascade signaling molecule, thus involved in Ras signaling pathways . (c)2002 Elsevier Science (USA). Life Sci Space Res, 1972, 10, 211 - 25 An integrated multi-purpose biology instrument utilizing a single detector, the mass spectrometer; Radmer R et al.; A mass spectrometer is used to analyze the gas phase in a number of reaction vessels filled with Martian soil . By choosing appropriate incubation conditions this instrument can be used to perform a wide spectrum of experiments ranging from the observation of general indices of life, i.e . processes and patterns unexplainable by physico-chemical mechanisms, to assays utilizing isotopes which probe for specific metabolic processes . Of particular interest is the in situ incubation in which a Martian soil sample is maintained at a constant temperature and its gas phase composition analyzed with time . Properly interpreted, this is a very general life-detection probe which makes minimal assumption as to the nature of Martian biology . Other assays and measurements concerning the soil and the atmosphere compatible with this method are also described. Am J Clin Dermatol, 2002, 3(2), 71 - 81 Six novel antimycotics; Rubin AI et al.; We have reviewed six new antimycotic agents which have potential applications for human cutaneous and mucosal diseases . Information on these six drugs was obtained via an English language search of PubMed through the US National Library of Medicine . The antimycotic agents reviewed include rilopirox, lanoconazole, NND-502, butenafine, eberconazole and voriconazole . Rilopirox is a synthetic pyridone derivative, related to ciclopirox, with a fungicidal action . Rilopirox is a hydrophobic, topical agent with potential application in mucosal candida infections, tinea versicolor and seborrheic dermatitis . Lanoconazole, an imidazole, is a topical agent with potential application in tinea infections and cutaneous candidiasis . The drug has been available for clinical use in Japan since 1994 and once-daily application to affected areas is recommended . In addition to its antifungal effect, animal data suggest that application of lanoconazole 0.5 or 1% cream is associated with accelerated wound healing . NND-502, a stereoselective analog of lanoconazole, is a topical agent with potential application in tinea pedis infection . NND-502 appears to be more effective in inhibiting ergosterol biosynthesis than lanoconazole or bifonazole and clinical trials comparing these agents are awaited . Butenafine is the first member of a new class of antifungals, the benzylamine derivatives, and has been approved for topical use in Japan (since 1992) and the US . Butenafine has a potent fungicidal action and the drug has been shown to be effective in multiple clinical trials in patients with tinea pedis, tinea corporis and tinea cruris . Butenafine has also been reported to exert an anti-inflammatory action after topical application and this may offer potential benefit over other topical antifungal agents . Eberconazole, an imidazole derivative, is a topical antifungal agent that has been shown to be effective in clinical trials in patients with tinea infections . Preliminary data indicate that the eberconazole is effective against some triazole-resistant yeasts such as Candida krusei and Candida glabrata . Voriconazole is an azole antifungal derivative of fluconazole . The drug is available in both oral and parenteral formulations . Oral voriconazole 200mg twice daily has been effective in treating oropharyngeal candidiasis and apergillosis in immunocompromised patients . After 12 weeks' treatment, a similar dosage of the drug elicited a positive response in 69% of nonimmunocompromised patients with invasive aspergillosis. Nat Struct Biol, 2002 Apr, 9(4), 263 - 7 Phosphorylation of linker histones regulates ATP-dependent chromatin remodeling enzymes; Horn PJ et al.; Members of the ATP-dependent family of chromatin remodeling enzymes play key roles in the regulation of transcription, development, DNA repair and cell cycle control . We find that the remodeling activities of the ySWI/SNF, hSWI/SNF, xMi-2 and xACF complexes are nearly abolished by incorporation of linker histones into nucleosomal array substrates . Much of this inhibition is independent of linker histone-induced folding of the arrays . We also find that phosphorylation of the linker histone by Cdc2/Cyclin B kinase can rescue remodeling by ySWI/SNF . These results suggest that linker histones exert a global, genome-wide control over remodeling activities, implicating a new, obligatory coupling between linker histone kinases and ATP-dependent remodeling enzymes. J Invest Dermatol, 2001 Dec, 117(6), 1405 - 11 Two different mutations in the cytoplasmic domain of the integrin beta 4 subunit in nonlethal forms of epidermolysis bullosa prevent interaction of beta 4 with plectin; Koster J et al.; The integrin alpha 6 beta 4 plays a crucial role in the assembly and maintenance of hemidesmosomes . Previous work has shown that the recruitment of plectin into hemidesmosomes is dependent on beta 4 and involves a region of the beta 4 cytoplasmic domain, which contains the first two fibronectin (FNIII) repeats and a short region of the connecting segment . Two missense mutations (R1225H and R1281W) in beta 4 that are responsible for nonlethal forms of epidermolysis bullosa are located in the second FNIII repeat . One of them is confined to a loop region that connects two beta strands (EC') whereas the other is located at the N-terminal end of the second FNIII repeat . We here report that these mutations render beta 4 unable to interact with plectin and prevent the localization of plectin in hemidesmosomes . Substitution of a lysine residue (K1279W) that forms part of the same loop as R1281 had no effect on the ability of beta 4 to recruit plectin . Furthermore, we show that an extended loop structure in beta 4, composed of the amino acids DDN (1262--1264), which resembles the RGD integrin-binding loop in fibronectin, is not involved in the binding to plectin . These results further demonstrate that binding of beta 4 to plectin is essential for the proper formation and function of hemidesmosomes and that loss of the interaction between beta 4 and plectin is associated with a mild form of epidermolysis bullosa. Vet Res, 2002 Jan-Feb, 33(1), 71 - 82 Effect of 1-24ACTH administration on sheep blood granulocyte functions; Paltrinieri S et al.; The objective of the present study was to determine the efficiency of blood neutrophils (PMN) taken from sheep during acute stress . Ten healthy Charolle sheep were sampled before treatment (T0) and 1 (T1), 2 (T2), 24 (T24) and 48 (T48) hours after 1-24ACTH administration . Ten sheep serving as the controls were sampled at the same time intervals, using saline solution instead of 1-24ACTH . At each time sampling, rectal temperature, heart rate, cortisol, glucose, non-esterified fatty acids (NEFA), total and differential leukocyte counts were evaluated . PMN were isolated after centrifugation of whole blood and hypotonic lysis of RBC . Chemotaxis was evaluated on a modified Boyden chamber using a nitrate cellulose filter and both Zymosan activated serum (ZAS) and interleukin-8 (IL-8) as chemoattractants . Phagocytosis was measured using both non-opsonized latex beads and fluoresceinated yeasts opsonized with homologous serum . Superoxide (O(-)2) production was evaluated by measuring superoxide dismutase-inhibitable reduction of ferricytochrome C, and adherence by a colorimetric assay of acid phosphatase activity of adherent cells . The administration of 1-24ACTH induced an acute stress reaction, indicated by the presence of clinical, biochemical and hematological changes . Adherence significantly increased from T0 to T2 in treated sheep . This might be responsible for the depression of non-specific immunity in stressed animals . Studies using stressors other than 1-24 ACTH are needed to verify the influence of other components of the stress reaction on PMN functions. EMBO J, 2002 Mar 1, 21(5), 1063 - 73 A Caenorhabditis elegans TGF-beta, DBL-1, controls the expression of LON-1, a PR-related protein, that regulates polyploidization and body length; Morita K et al.; Using cDNA-based array analysis combined with double-stranded RNA interference (dsRNAi), we have identified yk298h6 as a target gene of Caenorhabditis elegans TGF-beta signaling . Worms overexpressing dbl-1, a TGF-beta ligand, are 16% longer than wild type . Array analysis shows yk298h6 to be one of several genes suppressed in such worms . Disruption of yk298h6 function by dsRNAi also resulted in long worms, suggesting that it is a negative regulator of body length . yk298h6 was then mapped to, and shown to be identical to, lon-1, a known gene that affects body length . lon-1 encodes a 312 amino acid protein with a motif sequence that is conserved from plants to humans . Expression studies confirm that LON-1 is repressed by DBL-1, suggesting that LON-1 is a novel downstream component of the C.elegans TGF-beta growth regulation pathway . Consistent with this, LON-1 is expressed mainly in the larval and adult hypodermis and has dose-dependent effects on body length associated with changes in hypodermal ploidy, but not hypodermal cell proliferation. J Nat Prod, 2002 Feb, 65(2), 198 - 202 New sugar-mimic alkaloids from the pods of Angylocalyx pynaertii; Yasuda K et al.; Chromatographic separation of the pod extract of Angylocalyx pynaertii resulted in the isolation of 13 sugar-mimic alkaloids (1-13) . The structures of the new alkaloids were elucidated by spectroscopic methods as the 6-O-beta-D-glucoside (10) and N-hydroxyethyl derivative (11) of 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) (1), 1,6-dideoxynojirimycin (12), and 1,3,4-trideoxynojirimycin (13) . 2,5-Imino-1,2,5-trideoxy-L-glucitol (7), 2,5-dideoxy-2,5-imino-D-fucitol (8), and beta-homofuconojirimycin (9), isolated from the pods as well as the bark, were very specific inhibitors of alpha-L-fucosidase with no significant inhibitory activity toward other glycosidases . In this work, 1,4-dideoxy-1,4-imino-D-ribitol (6) was found to be a better inhibitor of lysosomal beta-mannosidase than 2,5-imino-1,2,5-trideoxy-D-mannitol (2) . N-Hydroxyethyl-1-deoxynojirimycin (miglitol), which is commercially available for the treatment of diabetes, retained its inhibitory potential toward rat intestinal maltase and sucrase, whereas 11 and the synthetic N-hydroxyethyl derivative of 2,5-dideoxy-2,5-imino-D-mannitol markedly lowered or abolished their inhibition toward all enzymes tested. Mycoses, 2002 Feb, 45(1-2), 29 - 37 Onychomycosis in Northern Greece during 1994-1998; Koussidou T et al.; In the 5-year period 1994-1998, 13957 patients were examined in the Mycological Laboratory of the State Hospital for Skin and Venereal Diseases, in Thessaloniki, Greece . Of the 2766 patients presenting with onychomycoses (20%), 67% were women and 33% were men . In the toenail infections, dermatophytes were most often isolated (72.3%), especially in women, followed by moulds (9.6%) and yeasts (2%); 16.1% of the infections were mixed . In the fingernail infections mostly yeasts were isolated (72%), especially in women, followed by dermatophytes (10%) and moulds (5.6%); 12.4% of the infections were mixed. Mycoses, 2002 Feb, 45(1-2), 15 - 8 Recovery of Candida dubliniensis from sputum of cystic fibrosis patients; Peltroche-Llacsahuanga H et al.; Yeasts have been cultivated in a high percentage of sputum samples from patients with cystic fibrosis (CF) . In cases of Candida dubliniensis a high recovery rate (15-25%) has so far only been reported from the oral cavity of HIV-infected persons . When studying sputum samples of patients suffering from cystic fibrosis (n = 54) we could repeatedly observe C . dubliniensis in 6 patients (11.1%) by using a complex isolation procedure involving the use of Staib agar . This revealed that the prevalence rate was significantly higher than the estimated overall prevalence for all non-HIV infected patients (0.8%) currently reported. J Agric Food Chem, 2002 Feb 27, 50(5), 1306 - 11 Fungal flora and ochratoxin a production in grapes and musts from france; Sage L et al.; Eleven samples of grapes and musts used in red table wines were investigated for the occurrence of potential ochratoxin A (OTA)-producing molds . From these samples, 59 filamentous fungi and 2 yeasts were isolated . Among the 30 genera isolated, Deuteromycetes were the most frequent (70%) followed by Ascomycetes (10%) . Six of the eleven grapes samples were contaminated by potentially ochratoxinogenic strains (Penicillium chrysogenum and Aspergillus carbonarius) . When cultivated in vitro on solid complex media, the 14 strains of A . carbonarius produced OTA . No other species produced OTA under the same conditions . Among must samples, eight of eleven were found to be contaminated by OTA (concentrations from <10 to 461 ng/L) . There is a strong correlation between the presence of ochratoxin-producing strains on grapes and OTA in musts . These findings should be connected with the OTA contamination of human blood in these areas and in France. Arch Dermatol, 2002 Feb, 138(2), 215 - 8 Skin colonization by Malassezia species in neonates: a prospective study and relationship with neonatal cephalic pustulosis; Bernier V et al.; OBJECTIVES: To assess skin colonization by Malassezia species in full-term healthy newborns, to investigate factors associated with colonization, and to look at acnelike cephalic pustulosis associated with this carriage . DESIGN: Samples were obtained from neonates and their mothers 0 to 5 days after birth and again 3 weeks later . Clinical patterns of common acnelike pustulosis were reported as mild (<10 papulopustules), moderate (> or =10 papulopustules), or absent . Direct examination and culture of sample . Identification of yeasts was based on microscopic and physiologic criteria . SETTING: A maternity hospital and the pediatric dermatology unit of a university hospital . PARTICIPANTS: Consecutive series of 102 neonates and their mothers . MAIN OUTCOME MEASURES: Incidence of skin colonization and type of Malassezia species found in neonates and correlation with neonatal cephalic pustulosis (neonatal acne) . RESULTS: At the first visit, 11 neonates and 36 mothers had cultures positive for Malassezia . Malassezia sympodialis and Malassezia globosa were preferentially cultured . At 3 weeks, 29 (52%) of 56 neonates and 18 (32%) of 56 mothers had cultures positive for only M sympodialis and M globosa . Breastfeeding was not associated with a higher prevalence of Malassezia carriage in neonates . Malassezia colonization was higher when pustulosis was more severe and M sympodialis was found in pustules . CONCLUSIONS: Malassezia colonization begins at birth and increases in the first weeks of life . A high prevalence of M sympodialis in neonates is noted from birth . Its association with neonatal acne is confirmed . Further investigation is needed to study the role of sebum secretion rate and quality in the neonatal period. J Mol Biol, 2002 Feb 1, 315(5), 951 - 63 Non-cognate template usage and alternative priming by a group II intron-encoded reverse transcriptase; Morozova T et al.; Group II introns are retroelements that site-specifically insert into DNA through homing . They are also implicated in related phenomena such as ectopic site insertions and precise intron deletions, but little is known about how group II intron reverse transcriptases (RTs) interact with non-cognate substrates . Here we show that wild-type aI2 RT readily reverse transcribes non-cognate RNAs in mitochondrial RNP particles when the aI2 intron structure is misfolded . In two closely related priming mutants, 1degree2(DeltaD5) and 1(+)2(DeltaD5), which contain wild-type RT but a disrupted intron structure, the RT has substantially lost specificity for aI2 RNA and copies multiple RNAs present in the RNP particles, using an alternative priming mechanism . The RT in 1degree2(DeltaD5) RNP particles can also copy exogenous RNAs but unlike the endogenous templates, a complementary primer is required, suggesting that the alternative priming event is specific to RT-RNA interactions formed in vivo . Alternatively primed cDNAs from strains 1degree2(DeltaD5), 1(+)2(DeltaD5) and 1degree2(P714T) (containing the mutation P714T in the RT) do not use homing site DNA as a primer, but appear to utilize a non-complementary DNA primer of approximately ten nucleotides . The alternative priming mechanism and reverse transcription of non-cognate templates has implications for in vivo reverse transcription of non-intronic RNAs, which is expected to occur during intron deletions and other retroprocessing events . Antonie Van Leeuwenhoek, 2001 Dec, 80(3-4), 275 - 86 Molecular taxonomy and biodiversity of rock fungal communities in an urban environment (Vienna, Austria); Sterflinger K et al.; The diversity of fungal communities on three different historical monuments in the city of Vienna (Austria) was analyzed and compared to the fungal diversity of microfungi on rock in the original quarry located in a rural area (Zogelsdorf, Austria) . The fungal strains isolated were characterized by morphology and the complete rock fungal community was identified based on molecular data, that is, by sequencing parts of the small ribosomal subunit (18S) and internal transcribed spacer region 1 (ITS1) . The genera Coniothyrium, Epicoccum and Phoma were found to be dominant on monument and rock surfaces . Additionally, black yeasts such as Exophiala species and microcolonial fungi like Sarcinomyces and Coniosporium which hitherto were regarded as typical rock inhabitants in semi-arid environments are frequently found on all rock surfaces in Vienna . The biodiversity of the fungi in the urban environment was much higher than on the same rock type in a rural environment, this difference can be attributed to the elevated organic pollution in the city. Nat Rev Mol Cell Biol, 2002 Jan, 3(1), 21 - 9 Mechanisms of transcription-coupled DNA repair; Svejstrup JQ; Several types of helix-distorting DNA lesions block the passage of elongating RNA polymerase II . Surprisingly, such transcription-blocking lesions are usually repaired considerably faster than non-obstructive lesions in the non-transcribed strand or in the genome overall . In this review, our knowledge of eukaryotic transcription-coupled repair (TCR) will be considered from the point of view of transcription, and current models for the mechanism of TCR will be discussed. Bioorg Med Chem, 2002 Mar, 10(3), 615 - 20 Non-thiol farnesyltransferase inhibitors: structure-activity relationships of benzophenone-based bisubstrate analogue farnesyltransferase inhibitors; Schlitzer M et al.; Investigations on the structure-activity relationships of benzophenone-based bisubstrate analogue farnesyltransferase inhibitors yielded a bisubstrate analogue farnesyltransferase inhibitor lacking any prenylic or peptidic substructures with nanomolar activity . This represents a considerable progress in comparison to those non-prenylic, non-peptidic bisubstrate analogue farnesyltransferase inhibitors we have described before which utilized AAX-peptidomimetic substructures different from the benzophenone since those inhibitors displayed activity only in the micromolar range. Cesk Patol, 2001 Nov, 37(4), 172 - 6 {Mycotic diseases of the heart}; Tomsova M; There has been an increasing frequency of deep mycotic infections over the last years complicating mostly disease states with immunosuppression . During the last 30 years, 54 cases of mycotic infection of the heart were identified at the Fingerland Department of Pathology in Hradec Kralove, with 32 cases during the last 10 years alone (prevalence 0.2% of 28 199 autopsies) . A review of the sex, age, etiology, source of infection, principal disease and morphological cardiac findings is presented . There was male predominance (74%) . The mean age of the patients was 48 years (ranging from 15 days to 87 years) . Yeasts of genus Candida dominated, followed in incidence by genus Aspergillus . The postmortem haemocultivation was positive in only 25% of all cases, while cultivation from a vegetation or a myocardial abscess was positive in 61% . The probable source of infection was detected in 28 patients; very often it was bronchopneumonia or an infected central venous catheter . Endocarditis was found in 28 cases, including six cases on a prosthetic valve and four cases on mural endocardium . Isolated myocarditis was found in 25 patients . There was also one case of isolated mycotic pericarditis . In more than half of all patients there was a state of immunosuppression, usually caused by a haematologic malignancy. IUBMB Life, 2001 Sep-Nov, 52(3-5), 165 - 73 The mitochondrial uncoupling protein UCP1: a gated pore; Arechaga I et al.; The uncoupling protein UCP1 is a member of a superfamily of homologous proteins formed by the mitochondrial metabolite transporters . Although they act in vivo as carriers, under specific experimental conditions some of these transporters have been shown to behave as channels . This dual transport operation suggests that these carriers are likely to be formed by two differentiated functional and structural domains . The kinetic model termed "single binding center gated pore" is well suited to understand the behaviour of these carriers . It proposes that in the protein core there must exist a hydrophilic translocation pore whose access is controlled by gates . It is highly likely that the hydrophilic channel is formed by the transmembrane alpha-helices and that loops contribute to the formation of the gates . UCP1 is regulated physiologically by fatty acids and purine nucleotides . Nucleotides maintain the proton conductance inhibited while fatty acids act as cytosolic second messengers of noradrenaline to active UCP1 . Based on photoaffinity labeling and mutagenesis data, we propose a structural model for the localization of the binding site . The nucleotide enters through a gate in the cytosolic side and binds deep inside the protein . The three matrix loops contribute to the formation of a hydrophobic binding pocket that would accommodate the purine moiety . Three arginine residues (in helices II, IV, and VI) would interact with the phosphate groups . His214 and Glu190 have been involved in the pH regulation of the nucleotide binding but because they are on the cytosolic side of the protein, we propose that their state of protonation will determine the access of the nucleotide to the binding center. Acta Microbiol Immunol Hung, 2001, 48(3-4), 533 - 43 Searching for new-type antifungal drugs (an outline for possible new strategies); Pocsi I et al.; New approaches for treatment of invasive fungal infections are necessary to cope with emerging resistant fungal pathogens of humans . In this paper, three different strategies are presented and evaluated to find new-type antifungal drugs and their targets . While experimental data obtained with potent chitinase inhibitors, e.g . allosamidin, and small-size antifungal proteins of fungal origin are encouraging more efforts are needed to verify and exploit the possible involvement of intracellular thiols, e.g . glutathione, and their metabolic enzymes in the pathogenesis of mycoses caused by dimorphic fungi . Chitinase inhibitors seem to hinder the cell separation of yeasts and the fragmentation of filamentous fungi quite effectively and, hence, they may be implicated in future therapies of systemic mycoses . In addition, small-size antifungal proteins possessing a broad inhibition spectrum may also provide us with promising new agents for the treatment of different kinds of (e.g . cutaneous) fungal infections. Curr Biol, 2002 Jan 8, 12(1), R32 - 40 Signaling complexes: junctions on the intracellular information super highway; Smith FD et al.; An enigmatic yet fundamental principle of signal transduction is that parallel signaling pathways assembled from a common repertoire of enzymes are able to propagate diverse physiological responses . A key feature of such a mechanism is that separate signaling pathways are organized into localized transduction units, each tailored to respond optimally to a particular signal . Protein-protein interactions maintained by anchoring, adapter and scaffolding proteins provide the molecular glue that holds these signal transduction units together . A major objective of the signaling community is to ascertain how signals flow through compartmentalized transduction units that contain transmembrane receptors, protein kinases, phosphatases and their substrates. Biotechnol Bioeng, 2002 Mar 5, 77(5), 489 - 94 Oxygen transfer rate and sophorose lipid production by Candida bombicola; Guilmanov V et al.; Sophorose lipids (SLs) have applications as surfactants and are produced at high levels by several yeasts . We developed a fed-batch shake-flask method for the production of SLs by Candida bombicola ATCC 22214 . Optimal aeration, expressed in terms of oxygen transfer rate, was between 50 and 80 mM O(2)/L h(-1) and resulted in maximum values for both volumetric product formation (1-1.5 g/L h(-1)) and SL yield (350 g/L) . The lowest aeration levels resulted in the enrichment in saturated fatty acid SLs at the expense of unsaturated fatty acid SLs . J Mol Biol, 2002 Jan 18, 315(3), 297 - 310 Productive folding to the native state by a group II intron ribozyme; Swisher JF et al.; Group II introns are large catalytic RNA molecules that fold into compact structures essential for the catalysis of splicing and intron mobility reactions . Despite a growing body of information on the folded state of group II introns at equilibrium, there is currently no information on the folding pathway and little information on the ionic requirements for folding . Folding isotherms were determined by hydroxyl radical footprinting for the 32 individual protections that are distributed throughout a group II intron ribozyme derived from intron ai5gamma . The isotherms span a similar range of Mg(2+) concentrations and share a similar index of cooperativity . Time-resolved hydroxyl radical footprinting studies show that all regions of the ribozyme fold slowly and with remarkable synchrony into a single catalytically active structure at a rate comparable to those of other ribozymes studied thus far . The rate constants for the formation of tertiary contacts and recovery of catalytic activity are identical within experimental error . Catalytic activity analyses in the presence of urea provide no evidence that the slow folding of the ai5gamma intron is attributable to the presence of unproductive kinetic traps along the folding pathway . Taken together, the data suggest that the rate-limiting step for folding of group II intron ai5gamma occurs early along the reaction pathway . We propose that this behavior resembles protein folding that is limited in rate by high contact order, or the need to form key tertiary interactions from partners that are located far apart in the primary or secondary structure . Eur J Biochem, 2002 Jan, 269(1), 283 - 90 Cloning and expression of sterol Delta 14-reductase from bovine liver; Roberti R et al.; Biosynthesis of cholesterol represents one of the fundamental cellular metabolic processes . Sterol Delta 14-reductase (Delta 14-SR) is a microsomal enzyme involved in the conversion of lanosterol to cholesterol in mammals . Amino-acid sequence analysis of a 38-kDa protein purified from bovine liver in our laboratory revealed > 90% similarity with a human sterol reductase, SR-1, encoded by the TM7SF2 gene, and with the C-terminal domain of human lamin B receptor . A cDNA encoding the 38-kDa protein, similar to human TM7SF2, was identified by analysis of a bovine expressed sequence tag (EST) database . The cDNA was synthesized by RT-PCR, cloned, and sequenced . The cDNA encodes a 418 amino-acid polypeptide with nine predicted transmembrane domains . The deduced amino-acid sequence exhibits high similarity with Delta 14-SR from yeasts, fungi, and plants (55-59%), suggesting that the bovine cDNA encodes Delta 14-SR . Northern blot analysis of bovine tissues showed high expression of mRNA in liver and brain . The polypeptide encoded by the cloned cDNA was expressed in COS-7 cells . Immunofluorescence analysis of transfected cells revealed a distribution of the protein throughout the ER . COS-7 cells expressing the protein exhibited Delta 14-SR activity about sevenfold higher than control cells . These results demonstrate that the cloned bovine cDNA encodes Delta 14-SR and provide evidence that the human TM7SF2 gene encodes Delta 14-SR. Science, 2002 Jan 4, 295(5552), 147 - 50 Sitosterol-beta-glucoside as primer for cellulose synthesis in plants; Peng L et al.; Cellulose synthesis in plants requires beta-1,4-glucan chain initiation, elongation, and termination . The process of chain elongation is likely to be distinct from the process of chain initiation . We demonstrate that a CesA glucosyltransferase initiates glucan polymerization by using sitosterol-beta-glucoside (SG) as primer . Cotton fiber membranes synthesize sitosterol-cellodextrins (SCDs) from SG and uridine 5'-diphosphate-glucose (UDP-Glc) under conditions that also favor cellulose synthesis . The cellulase encoded by the Korrigan (Kor) gene, required for cellulose synthesis in plants, may function to cleave SG from the growing polymer chain. Mycoses, 2001 Nov, 44(9-10), 390 - 4 Mites in fungal cultures; Duek L et al.; Recently all existing Trichophyton mentagrophytes colonies within our laboratory, which had originally appeared normal, rapidly and at an early stage became perforated . Therefore the aims of this study were to expose the cause of these major morphological changes and to find out if this phenomenon may occur in other fungal cultures . Microscopic examination of specimens taken from the damaged colonies showed many mites at different developmental stages, which were subsequently identified as the acarus, Tyrophagus putrescentiae . The laboratory experiments demonstrated that mites feed on the spores and hyphae of all the dermatophytes, moulds and yeasts tested . For the time being Tyrophagus putrescentiae is an unpleasant pest which damages fungal cultures but future use of the acari in biological control may be considered. Xenobiotica, 2001 Nov, 31(11), 757 - 67 Differential inhibition of human CYP1A1 and CYP1A2 by quinidine and quinine; Ching MS et al.; 1 . The inhibition of recombinant CYP1A1 and CYP1A2 activity by quinidine and quinine was evluated using ethoxyresorutin O-deethylation, phenacetin O-deethylation and propranolol desisopropylation as probe catalytic pathways . 2 . With substrate concentrations near the Km of catalysis, both quinidine and quinine potently inhibited CYP1A1 activity with {I}(0.5) approximately 1-3 microM, whereas in contrast, there was little inhibition of CYP1A2 activity . The Lineweaver-Burk plots with varying inhibitor concentrations suggested that inhibition by quinidine and quinine was competitive . 3 . There was only trace metabolism of quinidine by recombinant CYP1A1, whereas rat liver microsomes as a control showed extensive consumption of quinidine and metabolite production . 4 . This work suggests that quinidine is a non-classical inhibitor of CYP1A1 and that it is not as highly specific at inhibiting CYP2D6 as previously thought. IUBMB Life, 2001 Jun, 51(6), 345 - 50 Translocation of proteins into mitochondria; Hoogenraad NJ et al.; The translocase of the outer mitochondrial membrane (TOM) is composed of receptors, a channel protein, and its modulators that function together to import proteins into mitochondria . Although the import pathway of proteins directed to the mitochondrial matrix has been well characterized, recent studies into the import pathway taken by proteins into the other submitochondrial compartments have broadened our understanding into the way the TOM machinery recognizes, interacts, and translocates proteins. J Biochem (Tokyo), 2002 Jan, 131(1), 107 - 11 Tyrosine residues near the FAD binding site are critical for FAD binding and for the maintenance of the stable and active conformation of rat monoamine oxidase A; Ma J et al.; Monoamine oxidase is a flavin-containing enzyme located at the mitochondrial outer membrane that catalyzes the oxidative deamination of amines . To investigate the role of tyrosine residues near the FAD-binding site, Cys-406, of monoamine oxidase A, the tyrosine residues at posiyions 402, 407, and 410 were indurdually replaced with alanine or phenylalanine and the effects of the mutations on catalytic activity, FAD binding, and enzyme structure were examined . Half or fewer of the mutant proteins incorporated FAD . The mutation of Tyr-407 to alanine led to an almost completely loss of catalytic activity for serotonin, PEA, tyramine, and tryptamine . A substantial decrease in the catalytic activity was also observed with the enzymes mutated at Tyr-402 and Tyr-410 to alanine, although the effect of the latter mutation was much less . All these mutants were sensitive to trypsin treatment of the purified enzyme, while the wild type enzyme was resistant to treatment . On the other hand, substitution of Tyr-402 or Tyr-407 with phenylalanine had little effect on these properties . Taken together, we conclude that tyrosine residues near Cys-406 may be form a pocket to facilitates FAD incorporation, the catalytic center, and a stable conformation, probably through interactions among the aromatic rings of the tyrosine residues and FAD. FEBS Lett, 2001 Dec 14, 509(3), 395 - 8 Transferrin binds insulin-like growth factors and affects binding properties of insulin-like growth factor binding protein-3; Storch S et al.; In the circulation, most of the insulin-like growth factors (IGFs) are bound to a ternary 150 kDa complex with IGF-binding protein (IGFBP)-3 and the acid labile subunit . In the current study, we identify transferrin (Tf) by mass spectrometry, and immunoprecipitation as a component of a major IGF-binding fraction separated from human plasma . IGF ligand blotting, cross-linkage experiments and surface plasmon resonance spectrometry have been used to demonstrate the capability of Tf to bind IGFs specifically . In combination with Tf, IGFBP-3 showed a 5-fold higher affinity for IGF-II than IGFBP-3 alone . The data suggest that Tf may play an important role in regulating IGF/IGFBP-3 functions. EMBO Rep, 2001 Dec, 2(12), 1089 - 94 Epub 2001 Nov 21. Tramtrack69 interacts with the dMi-2 subunit of the Drosophila NuRD chromatin remodelling complex; Murawsky CM et al.; dMi-2, the ATPase subunit of the Drosophila nucleosome remodelling and histone deacetylation (dNuRD) complex, was identified in a two-hybrid screen as an interacting partner of the transcriptional repressor, Tramtrack69 (Ttk69) . A short region of Ttk69 is sufficient to mediate this interaction . Ttk69, but not the Ttk88 isoform, co-purifies with the dNuRD complex isolated from embryo extracts . dMi-2 and Ttk69 co-immunoprecipitate from embryonic extracts, indicating that they can associate in vivo . Both dMi-2 and Ttk69 co-localize at a number of discrete sites on polytene chromosomes, showing that they bind common target loci . We also demonstrate that dMi-2 and Ttk interact genetically, indicating a functional interaction in vivo . We propose that Ttk69 represses some target genes by remodelling chromatin structure through the recruitment of the dNuRD complex. J Cell Biol, 2001 Dec 10, 155(6), 911 - 21 Epub 2001 Dec 10. BRCA1-induced large-scale chromatin unfolding and allele-specific effects of cancer-predisposing mutations; Ye Q et al.; The breast cancer susceptibility gene BRCA1 encodes a protein that has been implicated in multiple nuclear functions, including transcription and DNA repair . The multifunctional nature of BRCA1 has raised the possibility that the polypeptide may regulate various nuclear processes via a common underlying mechanism such as chromatin remodeling . However, to date, no direct evidence exists in mammalian cells for BRCA1-mediated changes in either local or large-scale chromatin structure . Here we show that targeting BRCA1 to an amplified, lac operator-containing chromosome region in the mammalian genome results in large-scale chromatin decondensation . This unfolding activity is independently conferred by three subdomains within the transactivation domain of BRCA1, namely activation domain 1, and the two BRCA1 COOH terminus (BRCT) repeats . In addition, we demonstrate a similar chromatin unfolding activity associated with the transactivation domains of E2F1 and tumor suppressor p53 . However, unlike E2F1 and p53, BRCT-mediated chromatin unfolding is not accompanied by histone hyperacetylation . Cancer-predisposing mutations of BRCA1 display an allele-specific effect on chromatin unfolding: 5' mutations that result in gross truncation of the protein abolish the chromatin unfolding activity, whereas those in the 3' region of the gene markedly enhance this activity . A novel cofactor of BRCA1 (COBRA1) is recruited to the chromosome site by the first BRCT repeat of BRCA1, and is itself sufficient to induce chromatin unfolding . BRCA1 mutations that enhance chromatin unfolding also increase its affinity for, and recruitment of, COBRA1 . These results indicate that reorganization of higher levels of chromatin structure is an important regulated step in BRCA1-mediated nuclear functions. J Cell Biol, 2001 Dec 10, 155(6), 885 - 91 Epub 2001 Dec 10. Golgi matrix proteins interact with p24 cargo receptors and aid their efficient retention in the Golgi apparatus; Barr FA et al.; The Golgi apparatus is a highly complex organelle comprised of a stack of cisternal membranes on the secretory pathway from the ER to the cell surface . This structure is maintained by an exoskeleton or Golgi matrix constructed from a family of coiled-coil proteins, the golgins, and other peripheral membrane components such as GRASP55 and GRASP65 . Here we find that TMP21, p24a, and gp25L, members of the p24 cargo receptor family, are present in complexes with GRASP55 and GRASP65 in vivo . GRASPs interact directly with the cytoplasmic domains of specific p24 cargo receptors depending on their oligomeric state, and mutation of the GRASP binding site in the cytoplasmic tail of one of these, p24a, results in it being transported to the cell surface . These results suggest that one function of the Golgi matrix is to aid efficient retention or sequestration of p24 cargo receptors and other membrane proteins in the Golgi apparatus. J Cell Biol, 2001 Dec 10, 155(6), 877 - 83 Epub 2001 Dec 10. A GRASP55-rab2 effector complex linking Golgi structure to membrane traffic; Short B et al.; Membrane traffic between the endoplasmic reticulum (ER) and Golgi apparatus and through the Golgi apparatus is a highly regulated process controlled by members of the rab GTPase family . The GTP form of rab1 regulates ER to Golgi transport by interaction with the vesicle tethering factor p115 and the cis-Golgi matrix protein GM130, also part of a complex with GRASP65 important for the organization of cis-Golgi cisternae . Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins . Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport . These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure. Protoplasma, 2001, 216(3-4), 119 - 42 Cyclin/Cdk complexes: their involvement in cell cycle progression and mitotic division; John PC et al.; DNA replication and mitosis are dependent on the activity of cyclin-dependent protein kinase (CDK) enzymes, which are heterodimers of a catalytic subunit with a cyclin subunit . Cyclin binding to specific individual proteins is thought to provide potential substrates to Cdk . Protein binding by cyclins is assessed in terms of its mechanisms and biological significance, using evidence from diverse organisms including substrate specificity in animal Cdk enzymes containing D-, A-, and B-type cyclins and extensive cyclin gene manipulations in yeasts . Assembly of protein complexes with cyclin/Cdk is noted and the capacity of the cyclin-dependent kinase subunit Cks, in such complex, to extend the range of Cdk substrates is documented and discussed in terms of cell cycle regulation . Cell cycle progression involves changing abundance of individual cyclins, due to changing rates of their transcription or proteolysis, with consequent changes in the substrates of CDK through the cell cycle . Some overlap of the functions of individual cyclins in vivo has been identified by cyclin deletions and is suggested to follow a pattern in which cyclins can commonly complete functions initiated by the preceding cyclins well enough to preserve viability as groups of cyclins are removed by proteolysis . Cyclin accumulation is particularly important in terminating the G1 phase, when it raises CDK activity and starts events leading to DNA replication . It is suggested that plants share this mechanism . The distribution of cyclins and Cdk in maize root tip cells during mitosis and cytokinesis indicates the presence of Cdk1 (Cdc2a) and cyclin CycB1zm;2 at the mature and disassembling preprophase band and the presence of CycB1zm;2 at condensing and condensed chromosomes . Both observations correlate with the earlier-reported capacity of injected metaphase cyclin/CDK to accelerate preprophase band disassembly and chromosome condensation and with observations of the location of Cdk and cyclins in other laboratories . Additionally CycB1zm;2 is seen at the nuclear envelope during its breakdown, which correlates with an acceleration of the process by injected metaphase cyclin B/CDK . A phenomenon possibly unique to the plant kingdom is the persistence of mitotic cyclins after anaphase . Participation of cyclins in cytokinesis is indicated by the concentration of the mitotic cyclin CycA1;zm;1 at the phragmoplast . It is suggested that cyclins have a general function of spatially focusing Cdk activity and that in the plant cell the concentrations of cyclins are important mediators of CDK activity at the cytoskeleton, chromosomes, spindle, nuclear envelope, and phragmoplast. Curr Biol, 2001 Nov 27, 11(23), 1870 - 3 Apoptotic mimicry by an obligate intracellular parasite downregulates macrophage microbicidal activity; de Freitas Balanco JM et al.; Programmed cell death by apoptosis of unnecessary or potentially harmful cells is clearly beneficial to multicellular organisms . Proper functioning of such a program demands that the removal of dying cells proceed without an inflammatory reaction . Phosphatidylserine (PS) is one of the ligands displayed by apoptotic cells that participates in their noninflammatory removal when recognized by neighboring phagocytes . PS ligation induces the release of transforming growth factor-beta (TGF-beta), an antiinflammatory cytokine that mediates the suppression of macrophage-mediated inflammation . In Hydra vulgaris, an organism that stands at the base of metazoan evolution, the selective advantage provided by apoptosis lies in the fact that Hydra can survive recycling apoptotic cells by phagocytosis . In unicellular organisms, it has been proposed that altruistic death benefits clonal populations of yeasts and trypanosomatids . Now we show that advantageous features of the apoptotic process can operate without death as the necessary outcome . Leishmania spp are able to evade the killing activity of phagocytes and establish themselves as obligate intracellular parasites . Amastigotes, responsible for disease propagation, similar to apoptotic cells, inhibit macrophage activity by exposing PS . Exposed PS participates in amastigote internalization . Recognition of this moiety by macrophages induces TGF-beta secretion and IL-10 synthesis, inhibits NO production, and increases susceptibility to intracellular leishmanial growth. Biochem Biophys Res Commun, 2001 Dec 7, 289(3), 738 - 43 Identification of structural domains affecting transactivation potential of Nm23; Cho SJ et al.; The strong transactivation activity of the C-terminal half (amino acids 76-152) of Nm23 was reported previously . Here we examined a structural domain preventing or necessary to its transactivation activity . The C-terminal 1/4 (amino acids 109-152) was sufficient for transactivation, but the C-terminal half with a longer N-terminal extension (amino acids 58-152) caused the loss of the transactivation ability . Furthermore, coexpression of the N-terminal half with the C-terminus of Nm23-H1 blocked the transactivation activity of the C-terminal half, where direct interaction of both truncated proteins was demonstrated in vitro . Transactivation activities in the C-terminal halves of the known mutants (P96S, H118F, S120G, and S120A) exhibiting differential antimetastasis effects were also tested . Significant reduction of transactivation activity was observed only in H118F, indicating that NPD kinase active-site histidine is required . This suggests that transactivation potential of Nm23 is related to NDP kinase activity but not to metastasis suppressor activity . (c) 2001 Elsevier Science. J Cell Sci, 2001 Nov, 114(Pt 21), 3805 - 12 Dis1/TOG universal microtubule adaptors - one MAP for all? Ohkura H, Garcia MA, Toda T. Microtubules play central roles in various cellular processes in eukaryotes . The dynamics and organisation of interphase microtubules and mitotic spindles are dramatically altered during the cell cycle and development . However, the molecular mechanisms underlying this dynamic behaviour remain to be understood . In recent years, a novel family of microtubule-associated proteins (MAPs), the Dis1/TOG family, has emerged as a versatile regulator of microtubule function . These MAPs are highly conserved in eukaryotes from yeasts and plants to humans . The localisation and function of these MAPs are not determined simply by their intrinsic microtubule-binding activity . Instead this family executes its diverse roles by interacting with other regulatory molecules, including microtubule motors and centrosomal proteins . The modular structure of these MAPs may allow them to interact with multiple proteins and thereby be involved in a wide variety of microtubule and spindle functions. Trends Cell Biol, 2001 Dec, 11(12), 519 - 25 From snapshots to moving pictures: new perspectives on nuclear organization; Heun P et al.; The positioning of chromosomal domains in the interphase nucleus is proposed to facilitate gene regulation in simple cells such as yeasts and to coordinate patterns of gene expression and activation of origins of replication during cell differentiation in complex organisms . Over the past 10-12 years, detailed information on the organization of interphase chromosomes has accumulated from three-dimensional microscopy of fixed cells labeled by in situ hybridization and immunofluorescence techniques . Recently, time-lapse fluorescence microscopy of GFP-tagged domains has shown that interphase chromatin can be highly dynamic, moving distances >0.5 microm within seconds . Novel fluorescence techniques show that most nuclear proteins are also highly mobile . Both the rapid oscillations of chromatin and long-range movements of chromosomes suggest new mechanisms for spatial and temporal control of transcription and other nuclear events. New Microbiol, 2001 Oct, 24(4), 397 - 404 Fluconazole susceptibility of Italian Candida dubliniensis clinical isolates determined by reference and simplified tests; Giammanco GM et al.; Candida dubliniensis ia an opportunistic pathogen mainly associated with oral candidiasis in human immunodeficiency virus (HIV)-infected individuals . We recently recovered the first Italian clinical isolates of C . dubliniensis from the oral cavities of seven HIV-seropositive subjects . The in vitro susceptibility to fluconazole (FLCZ) of these isolates was determined according to the National Committee for Clinical Laboratory Standards (NCCLS) M27-A broth microdilution method for yeasts . All seven isolates of C . dubliniensis were susceptible to FLCZ (MICs < or =0.5 microg/ml) . Results of this reference method were compared to those obtained with simplified tests, more adapted to routine evaluation in hospital laboratories . Fungitest and Sensititre YeastOne colorimetric microplate-based methods have been evaluated . The agar disk diffusion method has also been tested on two different media: RPMI 1640-2% glucose and High Resolution-2% glucose-0.5 microg/ml methylene blue . All of the simplified methods tested were able to correctly identify FLCZ-susceptibility of this group of Italian C . dubliniensis isolates. Dev Cell, 2001 Jul, 1(1), 6 - 8 Self-destruction in the line of duty; Subramani S; Three cellular processes, microautophagy, macroautophagy, and the cytoplasm-to-vacuole (Cvt) pathway, are involved in the cargo delivery from the cytosol to the vacuole or lysosome . Recent findings have identified Cvt19 at the receptor for specific cargo binding in the Cvt pathway. Genome Inform Ser Workshop Genome Inform, 2000, 11, 196 - 204 A gene network inference method from continuous-value gene expression data of wild-type and mutants; Kyoda KM et al.; In this paper we introduce a new inference method of a gene regulatory network from steady-state gene expression data . Our method determines a regulatory structure consistent with an observed set of steady-state expression profiles, each generated from wild-type and single deletion mutant of the target network . Our method derives the regulatory relationships in the network using a graph theoretic approach . The advantage of our method is to be able to deal with continuous values of steady-state data, while most of the methods proposed in past use a Boolean network model with binary data . Performance of our method is evaluated on simulated networks with varying the size of networks, indegree of each gene, and the data characteristics (continuous-value/binary), and is compared with that of predictor method proposed by Ideker et al . As a result, we show the superiority of using continuous values to binary values, and the performance of our method is much better than that of the predictor method. J Toxicol Environ Health A, 2001 Oct 26, 64(4), 311 - 25 Acute inflammation and recovery in rats after intratracheal instillation of a 1-->3-beta-glucan (zymosan A); Young SH et al.; Although endotoxin is a known potent stimulant of inflammatory responses, the magnitude of pulmonary response following exposure to various organic dusts does not always correlate with endotoxin content of the dusts alone . Other components, such as 1-->3-beta-glucans, derived from the inner cell wall of yeasts and fungi, have been implicated in organic dust toxic syndrome . However, animal studies report conflicting results concerning the inflammatory potency of 1-->3-beta-glucan . In this experiment, the pulmonary reaction of rats to 1-->3-beta-glucan (zymosan A) exposure was assessed . Male Sprague-Dawley rats were exposed via intratracheal instillation (IT) to zymosan A (dose range 0-5 mg/kg body weight) . Rats were sacrificed 1-7 d postexposure and the following pulmonary responses were monitored: (1) breathing frequency, (2) differential cell counts of hronchoalveolar lavage (BAL) cells, (3) chemiluminescence (CL) as a measure of alveolar macrophage activation, (4) nitric oxide production by alveolar macrophages, (5) albumin levels, and (6) lactate dehydrogenase (LDH) activity in the first acellular lavage fluid . Upon challenge with zymosan A, rats exhibited a dose-dependent pulmonary response at 1 d post IT that was significantly higher than the control level at a dose of 1-2.5 mg/kg body weight for each of these pulmonary parameters . Post-IT enhancement of breathing frequencies and polymorphonuclear leukocytes (PMN) obtained by BAL both correlated very well with zymosan A concentration (r = .95 and .99, respectively) . Elevation of albumin levels and LDH activity of the acellular BAL fluid also correlated (r = .80) with the dose of zymosan . The recovery from a single intratracheal administration of zymosan A (2.5 mg/kg body weight) was monitored over 7 d . PMN and CL showed significant recovery from d 1 level by 3 d postexposure . Breathing frequencies and nitric oxide production showed significant recovery from d 1 level by 4 d postexposure . A good correlation (r2= .8) between recovery of PMN in BAL, CL, or nitric oxide production and the days postexposure was observed. Diagn Microbiol Infect Dis, 2001 Sep-Oct, 41(1-2), 29 - 35 Multicenter prospective surveillance of oral Candida dubliniensis among adult Brazilian human immunodeficiency virus-positive and AIDS patients; Milan EP et al.; The incidence of C . dubliniensis in South America has not yet been determined . In the present study, oral swab samples were taken from 108 HIV-infected/AIDS individuals attending 6 separate Brazilian HIV-treatment centers to determine the incidence of C . dubliniensis in this population . Swabs were plated onto CHROMagar Candida medium and 155 isolates, presumptively identified as C . albicans or C . dubliniensis were further investigated . In a preliminary screen for C . dubliniensis, 13 of the 155 isolates showed no or poor growth at 42 degrees C, and all them were subjected to randomly amplified polymorphic DNA (RAPD) and polymerase chain reaction (PCR) analysis using C . dubliniensis-specific primers . We confirmed that 4 out of 13 isolates were C . dubliniensis, representing an incidence rate of 2.8% for the Brazilian HIV-infected population infected with yeasts exhibiting green colonies on CHROMagar Candida . This value is significantly lower than those reported in Ireland and the United States. J Biochem (Tokyo), 2001 Nov, 130(5), 657 - 64 Three-dimensional structural model analysis of the binding site of lithocholic acid, an inhibitor of DNA polymerase beta and DNA topoisomerase II; Mizushina Y et al.; The molecular action of lithocholic acid (LCA), a selective inhibitor of mammalian DNA polymerase beta (pol beta), was investigated . We found that LCA could also strongly inhibit the activity of human DNA topoisomerase II (topo II) . No other DNA metabolic enzymes tested were affected by LCA . Therefore, LCA should be classified as an inhibitor of both pol beta and topo II . Here, we report the molecular interaction of LCA with pol beta and topo II . By three-dimensional structural model analysis and by comparison with the spatial positioning of specific amino acids binding to LCA on pol beta (Lys60, Leu77, and Thr79), we obtained supplementary information that allowed us to build a structural model of topo II . Modeling analysis revealed that the LCA-interaction interface in both enzymes has a pocket comprised of three amino acids in common, which binds to the LCA molecule . In topo II, the three amino acid residues were Lys720, Leu760, and Thr791 . These results suggested that the LCA binding domains of pol beta and topo II are three-dimensionally very similar. Mol Cell, 2001 Oct, 8(4), 732 - 3 New twists in understanding the fate of antisense oligodeoxynucleotide mRNA targets; Steiger MA et al.; In this issue of Molecular Cell, Thoma et al . show that after a target mRNA is cleaved, upon treatment with an antisense oligodeoxynucleotide, the 3' cleavage product persists and is translated to produce an N-terminally truncated version of the protein encoded by the target mRNA. Genes Cells, 2001 Oct, 6(10), 857 - 68 Two domains of Nrf2 cooperatively bind CBP, a CREB binding protein, and synergistically activate transcription; Katoh Y et al.; BACKGROUND: Nrf2 belongs to the Cap-N-Collar (CNC) transcription factor family and is essential for the antioxidant responsive element (ARE)-mediated expression of a group of detoxifying and antioxidant genes . The forced expression of Nrf2 in mammalian cells activates the expression of target genes through the ARE, with Nrf2 showing the highest transactivation activity among the CNC family of transcription factors . To elucidate the molecular mechanisms generating this potent transactivation activity, we examined the functions of the domains within Nrf2 . RESULT: We found that Nrf2 contains two transcription activation domains, Neh4 and Neh5, which act synergistically to attain maximum a activation of reporter gene expression . Neh4 and Neh5 both individually and cooperatively bind to CBP (CREB (cAMP Responsive Element Binding protein) Binding Protein) . In fact, the specific inhibitor of CBP, adenovirus E1A protein, significantly reduced Nrf2 activity . Importantly, the CBP-binding activity of Nrf2 deletion mutants positively correlated with their transactivation activity . Neh5 contains a motif which is commonly conserved among the CNC factors, whereas Neh4 contains the novel CBP-interacting motif recently identified in p53 and E2F . CONCLUSIONS: Our results indicate that Nrf2 exploits the cooperative binding of two independent transactivation domains to CBP in the acquisition of a potent transactivation activity. Curr Biol, 2001 Oct 16, 11(20), R838 - 41 Pre-mRNA processing: insights from nonsense; Brogna S; In eukaryotic cells, translation is thought to be confined to cytoplasm, but two recent studies have challenged this notion, one showing that an mRNA's open reading frame influences nuclear events as early as release from the site of transcription, and the other by providing evidence for protein synthesis within the nucleus. Curr Biol, 2001 Oct 16, 11(20), R834 - 7 Cell cycle: Waiters serving the Destruction machinery; Vodermaier HC; Targeting regulatory proteins for destruction, and thereby controlling progression through mitosis, is the crucial task for the anaphase-promoting complex (APC) . Recent evidence suggests that essential APC activators, WD40 repeat proteins of the Cdc20 family, act as long-suspected receptors for APC substrates. Anal Biochem, 2001 Oct 15, 297(2), 111 - 6 Fluorescence polarization: analysis of carbohydrate-protein interaction; Kakehi K et al.; Fluorescence polarization has been widely used for the studies on the molecular motion in solution and has been applied to immunoassays for proteins, therapeutic drug monitoring in clinical pharmacy, and assays for environmentally toxic compounds . Because fluorescence polarization is most readily applicable to the kinetic analysis of the binding reaction between a substance having small molecular mass and a receptor molecule, this method is easily applied to the analysis of carbohydrate-lectin binding . In this tutorial Thematic Review, we briefly introduce the principles of fluorescence polarization and some applications of fluorescence polarization technique to glycobiology . Altern Lab Anim, 2001 Sep-Oct, 29(5), 547 - 56 Evaluation of the toxicity of substituted benzthioanilides by using in vitro tests; Kowalska-Pylka H et al.; The cytotoxicity of 12 benzthioanilides substituted in the N-aromatic ring, and of two commercial preparations (imaverol and thiuram) for comparison, was studied with clone 81 cat cells, by determining the highest tolerated dose, and by using the neutral red uptake assay and the kenacid blue assay for total protein . The concentrations that induced 20%, 50% and 80% (IC20, IC50 and IC80) inhibition relative to controls were calculated from dose-response curves . For some compounds, rat LD50 values were also determined . All the benzthioanilide preparations showed in vitro toxicities lower than those of the fungicides imaverol and thiuram . It was confirmed that the cytotoxicities of the compounds depend on the type of substituent . The least toxic compound contained a CONHCH(2)CO(2)H substituent in the para position of the N-aromatic ring, and the most toxic compounds contained chloro and fluoro, or three chloro substituents in the anilide moiety . All the benzthioanilides tested showed fungistatic activity for dermatophytes; two of the compounds (compound 5 and compound 12) also inhibited the development of yeasts at concentrations lower than those which caused toxicity in vitro . The LD50 values and the cytotoxic concentrations in vitro were linearly related. Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 589 - 601 Mating-type genes for classical strain improvements of ascomycetes; Poggeler S; The ability to mate fungi in the laboratory is a valuable tool for genetic analysis and for classical strain improvement . In ascomycetous fungi, mating typically occurs between morphologically identical partners that are distinguished by their mating type . In most cases, the single mating-type locus conferring mating behavior consists of dissimilar DNA sequences (idiomorphs) in the mating partners . All ascomycete mating-type idiomorphs encode proteins with confirmed or putative DNA-binding motifs . These proteins control, as master regulatory transcription factors, pathways of cell speciation and sexual morphogenesis . Mating-type organization of four of the six classes of ascomycetes has been studied at the molecular level over the past 20 years . This review gives a short overview of the structural organization of the mating-type loci of yeasts and filamentous ascomycetes . In addition, this review describes how the availability of mating-type sequences allows the investigation of key issues concerning genetic and phylogenetic analyses of fungal species. Arch Biochem Biophys, 2001 Oct 15, 394(2), 189 - 200 Interaction of new sulfaphenazole derivatives with human liver cytochrome p450 2Cs: structural determinants required for selective recognition by CYP 2C9 and for inhibition of human CYP 2Cs; Ha-Duong NT et al.; A series of new derivatives of sulfaphenazole (SPA), in which the NH(2) and phenyl substituents of SPA are replaced by various groups or in which the sulfonamide function of SPA is N-alkylated, were synthesized in order to further explore CYP 2C9 active site and to determine the structural factors explaining the selectivity of SPA for CYP 2C9 within the human P450 2C subfamily . Compounds in which the NH(2) group of SPA was replaced with R(1) = CH(3), Br, CH = CH(2), CH(2)CH = CH(2), and CH(2)CH(2)OH exhibited a high affinity for CYP 2C9, as shown by the dissociation constant of their CYP 2C9 complexes, K(s), which was determined by difference visible spectroscopy (K(s) between 0.1 and 0.4 microM) and their constant of CYP 2C9 inhibition (K(i) between 0.3 and 0.6 microM) . This indicates that the CYP 2C9-iron(III)-NH(2)R bond previously described to exist in the CYP 2C9-SPA complex does not play a key role in the high affinity of SPA for CYP 2C9 . Compounds in which the phenyl group of SPA was replaced with various aryl or alkyl R(2) substituents only exhibited a high affinity for CYP 2C9 if R(2) is a freely rotating and sufficiently electron-rich aryl substituent . Finally, compounds resulting from a N-alkylation of the SPA sulfonamide function (R(3) = CH(3), C(2)H(5), or C(3)H(7)) did not retain the selective inhibitory properties of SPA toward CYP 2C9 . However, they are reasonably good inhibitors of CYP 2C8 and CYP 2C18 (IC(50) approximately 20 microM) . These data allow one to better understand the structural factors that are important for selective binding in the CYP 2C9 active site . They also provide us with clues towards new selective inhibitors of CYP 2C8 and CYP 2C18 . Microbiology, 2001 Oct, 147(Pt 10), 2857 - 64 Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina; Wynn JP et al.; The biochemical events associated with the onset of lipid accumulation in Mucor circinelloides and Mortierella alpina, under conditions of nitrogen-limited growth, have been elucidated; they differ in key aspects from those described in oleaginous yeasts . The NAD+:isocitrate dehydrogenases of Mc . circinelloides and Mort . alpina were not absolutely dependent on AMP for activity . Furthermore, changes in the cellular adenine nucleotide pools and energy charge were different from those reported for oleaginous yeasts . In Mc . circinelloides ATP, ADP and AMP concentrations all decreased by 50% after nitrogen limitation, leading to a constant energy charge at the expense of the size of the total adenylate pool . Pyruvate carboxylase in Mc . circinelloides was cytosolic, having implications for the organization of lipid synthesis in filamentous fungi . As a result of the data obtained, a revised and more concerted mechanism for the initiation of storage lipid accumulation is put forward for filamentous fungi. Trends Cell Biol, 2001 Oct, 11(10), 426 - 33 Integrating stress-response and cell-cycle checkpoint pathways; Pearce AK et al.; The DNA integrity checkpoint and stress kinase (SAPK/JNK and p38) pathways function to modulate cell-cycle, apoptotic and transcriptional responses to stress . Although initially considered to function independently, recent advances indicate a number of links between the stress-response and checkpoint pathways . Here, we consider the relationship between the stress-response and checkpoint pathways and how they interact to modulate cell-cycle control. Biochemistry (Mosc), 2001 Aug, 66(8), 932 - 6 Cleaving of ketosubstrates by transketolase and the nature of the products formed; Solov'eva ON et al.; The interaction of transketolase ketosubstrates with the holoenzyme has been studied . On addition of ketosubstrates cleaving both irreversibly (hydroxypyruvate) and reversibly (xylulose 5-phosphate), identical changes in the CD spectrum at 300-360 nm are observed . The changes in this spectral region, as previously shown, are due to the formation of the catalytically active holoenzyme from the apoenzyme and the coenzyme, and the cleavage of ketosubstrates by transketolase . The identity of the changes in transketolase CD spectrum caused by the addition of reversibly or irreversibly cleaving substrates indicates that in the both cases the changes are due to the formation of an intermediate product of the transketolase reaction--a glycolaldehyde residue covalently bound to the coenzyme within the holoenzyme molecule . Usually, in the course of the transferase reaction, the glycolaldehyde residue is transferred to an aldose (acceptor substrate), resulting in the recycling of the holoenzyme free of the glycolaldehyde residue . The removal of the glycolaldehyde residue from the holoenzyme appears to proceed even in the absence of an aldose . However, the glycolaldehyde cannot be found the free state because it condenses with another glycolaldehyde residue formed in the course of the cleavage of another ketosubstrate molecule yielding erythrulose. Bioorg Med Chem, 2001 Sep, 9(9), 2485 - 91 The contribution of the methyl groups on thymine bases to binding specificity and affinity by alanine-rich mutants of the bZIP motif; Kise KJ Jr et al.; We have used fluorescence anisotropy to measure in situ the thermodynamics of binding of alanine-rich mutants of the GCN4 basic region/leucine zipper (bZIP) to short DNA duplexes, in which thymines were replaced with uracils, in order to quantify the contributions of the C5 methyl group on thymines with alanine methyl side chains . We simplified the alpha-helical GCN4 bZIP by alanine substitution: 4A, 11A, and 18A contain four, 11, and 18 alanine mutations in their DNA-binding basic regions, respectively . Titration of fluorescein-labeled duplexes with increasing amounts of protein yielded dissociation constants in the low-to-mid nanomolar range for all bZIP mutants in complex with the AP-1 target site (5'-TGACTCA-3'); binding to the nonspecific control duplex was >1000-fold weaker . Small changes of <1 kcal/mol in binding free energies were observed for wild-type bZIP and 4A mutant to uracil-containing AP-1, whereas 11A and 18A bound almost equally well to native AP-1 and uracil-containing AP-1 . These modest changes in binding affinities may reflect the multivalent nature of protein-DNA interactions, as our highly mutated proteins still exhibit native-like behavior . These protein mutations may compensate for changes in enthalpic and entropic contributions toward DNA-binding in order to maintain binding free energies similar to that of the native protein-DNA complex. Cell, 2001 Sep 7, 106(5), 575 - 84 TATA binding protein-associated CK2 transduces DNA damage signals to the RNA polymerase III transcriptional machinery; Ghavidel A et al.; Here we report that RNA polymerase (pol) III transcription is repressed in response to DNA damage by downregulation of TFIIIB, the core component of the pol III transcriptional machinery . Protein kinase CK2 transduces this stress signal to TFIIIB . CK2 associates with and normally activates the TATA binding protein (TBP) subunit of TFIIIB . The beta regulatory subunit of CK2 binds to TBP and is required for high TBP-associated CK2 activity and pol III transcription in unstressed cells . Transcriptional repression induced by DNA damage requires CK2 and coincides with downregulation of TBP-associated CK2 and dissociation of catalytic subunits from TBP-CK2 complexes . Therefore, CK2 is the terminal effector in a signaling pathway that represses pol III transcription when genome integrity is compromised. Adv Space Res, 1983, 3(9), 107 - 11 Changes in the amino acid code; Jukes TH; The genetic code is characterized by a pattern arising from "wobble-pairing" between codons and anticodons, so that one nucleotide in the first anticodon position can pair with more than one nucleotide in the third position of a codon . Earlier codes may have existed in which there were fewer anticodons than at present, so that these earlier codes contained fewer amino acids . The universal code was formerly thought to be the only currently existing code used by terrestrial species . It is now known that differences exist from the universal code in mitochondrial coding systems, and also that mitochondrial systems differ from each other . These findings lend support to the proposal that archetypal codes preceded the present universal code . Such archetypal codes may have had some resemblances to mitochondrial codes. Adv Space Res, 1994 Oct, 14(10), 235 - 48 Repair of DNA double-strand breaks and its effect on RBE; Frankenberg D; DNA double-strand breaks (DSB) are induced linearly with absorbed dose both for sparsely and densely ionizing radiations . By enzymatic repair the linear relationship between the number of DSB and absorbed dose is converted into a non linear one . Furthermore, the RBE-values of high LET radiations for residual DSB increase with increasing amount of DSB repair especially in the low dose range . Unrepaired and/or misrepaired DSB are supposed to be responsible for chromosomal aberrations, cell killing, oncogenic cell transformation and gene mutation . At low doses, for these endpoints much higher RBE-values than those for initial DSB are observed . However, with increasing doses the RBE-values for these endpoints approach those for initial DSB . These observations are likely to be interpreted using the following two parameters of the energy deposition structure: 1 . The distribution of clusters with respect to their size at the nm-scale and to the number of ionizations per cluster (cluster distribution) . 2 . The distribution of distances between clusters of definite size and with definite number of ionizations (distance distribution of clusters) . For the induction of DSB solely the ionization density in clusters of nm-dimensions (i.e . the cluster distribution) is important . For unrepaired or misrepaired DSB (responsible for chromosome aberrations, cell killing, oncogenic cell transformation and gene mutation) both the cluster distribution and the distance distribution of clusters are relevant . At low doses the distance distribution of clusters along a single particle track determines the RBE-value . However, with increasing dose the distribution of clusters produced by all particles traversing the cell nucleus becomes increasingly determinant . Here, solely the cluster distribution is important as it is the case for the induction of DSB. Adv Space Res, 1984, 4(10), 199 - 204 On the quantitative interpretation of cellular heavy ion action; Kiefer J et al.; Current analyses of heavy ion action assume that the survival probability of a cell hit by a heavy ion depends only on the energy absorbed in its critical site . It is known, however, that the efficiency to produce a biological effect depends also on the spatial pattern of energy deposition . This has to be included in the quantitative evaluation of heavy ion action . Based on recent models of lesion formation by ionizing radiation (Goodhead and Brenner, Phys . Med . Biol . 28, 485, 1983) data with lighter ions (LET < 500 keV/micrometer) were re-analysed . It is shown that the behaviour of various cell systems can be described by a common curve which can be used to estimate the contribution of "non-linear" components (i.e . where the distribution of energy deposition plays a role) with heavy ions . It is concluded that even with Uranium ions the regions of non-linear effects does not extend beyond 50 nm from the trade core . These data will be used to assess quantitatively survival curves obtained with very heavy ion exposure. Adv Space Res, 1989, 9(10), 59 - 72 Cellular and subcellular effect of heavy ions: a comparison of the induction of strand breaks and chromosomal aberration with the incidence of inactivation and mutation; Kraft G et al.; Radiobiological effects of heavy charged particles are compared for a large variety of ions from Helium to Uranium and energies between 1 and 1000 MeV/u which correspond to LET values between 10 and 16000 keV/micrometers . The different cross section for the induction of strand breaks and chromosomal aberrations as well as for inactivation and mutation induction exhibit striking similarities when compared as function of the linear energy transfer (LET) . At LET values below 100 keV/micrometers all data points of one specific effect form one single curve as a function of LET, independent of the atomic number of the ion . In this LET range, the biological effects are independ from the particle energy or track structure and depend only on the energy transfer . Therefore, LET is a good parameter in this regime . For LET values greater than 100 keV/micrometers, the curves for the different ions separate from the common curve in order of increasing atomic numbers . In this regime LET is no longer a good parameter and the physical parameters of the formation of particle tracks are important . The similarity of the sigma-LET curves for different endpoints indicates that the 'hook-structure' is produced by physical and chemical effects which occur before the biologically relevant lesions are formed . However, from the existing data of biological effects, it can be concluded that the efficiencies for cell killing are always smaller than those extrapolated from X-ray data on the basis of the energy deposition only . Therefore, cells which are directly hit by an HZE particle are not killed and undergo a finite risk of mutation and transformation. Adv Space Res, 1989, 9(10), 21 - 9 Quantitative interpretation of heavy ions effects: models for the biological effects of heavy ions; Kiefer J et al.; Heavy ions are an important part of space radiation . Although they contribute only about 1 percent in number the fraction in terms of energy deposited is much higher . Also the quality of radiation is different from the other components since the LET is generally quite high . This poses the problem of Relative Biological Effectiveness (RBE) . It is considerably more important in space than on earth because shielding measures are costly and sometimes not even feasible . Radiation hazards appear to be the limiting factor In long term space flights and their evaluation constitutes a major task . There is still no general agreement about RBE of earthbound radiation, and even less concerning the biological weighting of very heavy and very energetic ions in space . Because of the lack of experimental data--particularly for risk estimates in humans-- theoretical approaches may be very helpful in this respect and provide the only means to judge the radiation protection situation in outer space . In order to be useful careful checks of their consistency are necessary . This paper summarizes some of the more common approaches in a critical manner . The unhappy conclusion at the end will be that at present it is not possible to understand even heavy ion action on survival quantitatively with an acceptable precision. J Cell Biol, 2001 Sep 3, 154(5), 909 - 11 A new view of the spindle checkpoint; Hoyt MA; Previous studies of the spindle checkpoint suggested that its ability to prevent entry into anaphase was mediated by the inhibition of the anaphase-promoting complex (APC) ubiquitin ligase by Mad2 . Two new studies challenge that view by demonstrating that another checkpoint protein, BubR1, is a far more potent inhibitor of APC function. Clin Microbiol Infect, 2001, 7 Suppl 2, 46 - 53 Present status of the detection of antifungal resistance: the perspective from both sides of the ocean; Cuenca-Estrella M et al.; The NCCLS reference methodology for antifungal susceptibility testing is a new milestone of the evolution of medical mycology . The use of this methodology however, is not problem-free . At present, major limitations are a trailing phenomenon with azoles, unreliable detection of resistance to amphotericin B, poor growth of some organisms and unpractical procedures for the clinical laboratory . Herein a overview of NCCLS guidelines for yeasts and filamentous fungi is presented . Likewise, a review of studies conducted trying to overcome the limitations of reference procedures is also included . Several alternative approaches are reviewed as alternative media, inoculum size and incubation time . Modifications of reading procedure and endpoint determination are also evaluated . Agar diffusion methods and other methods for susceptibility testing are cited . Finally, we discuss the data on correlation of the in vitro results with the in vivo activity. Genome Biol . 2001;2(7):REVIEWS3009 . Epub 2001 Jul 05. Inhibitor of apoptosis proteins and their relatives: IAPs and other BIRPs; Verhagen AM et al.; SUMMARY: Apoptosis is a physiological cell death process important for development, homeostasis and the immune defence of multicellular animals . The key effectors of apoptosis are caspases, cysteine proteases that cleave after aspartate residues . The inhibitor of apoptosis (IAP) family of proteins prevent cell death by binding to and inhibiting active caspases and are negatively regulated by IAP-binding proteins, such as the mammalian protein DIABLO/Smac . IAPs are characterized by the presence of one to three domains known as baculoviral IAP repeat (BIR) domains and many also have a RING-finger domain at their carboxyl terminus . More recently, a second group of BIR-domain-containing proteins (BIRPs) have been identified that includes the mammalian proteins Bruce and Survivin as well as BIR-containing proteins in yeasts and Caenorhabditis elegans . These Survivin-like BIRPs regulate cytokinesis and mitotic spindle formation . In this review, we describe the IAPs and other BIRPs, their evolutionary relationships and their subcellular and tissue localizations. Dtsch Med Wochenschr, 2001 Aug 17, 126(33), 905 - 8 {Retrospective analysis of fluconazole efficacy in Candida-colonized, non-neutropenic, surgical patients in long-term intensive care}; Kappe R et al.; BACKGROUND AND OBJECTIVE: The early clinical diagnosis of invasive candidiasis is difficult . Fluconazole, which has been available since the early 1990s, is a relatively atoxic intravenously applicable antimycotic agent . For this reason it has been widely used - possibly too much . The aim of this study was the retrospective critical evaluation of the efficacy of systemic antifungal chemotherapy in non-neutropenic, Candida-colonized, surgical patients in long-term intensive care . PATIENTS AND METHODS: 69 patients (54 men and 15 women, aged 55.8 {range 18-87} years) of 364 patients of the anaesthesiological intensive care unit (ICU) of the University Hospital of Heidelberg in 1991 and 1992 were selected for the study . None of the 69 patients was suffering from proven invasive candidiasis according to the gold-standard criteria of positive histology, blood culture, or isolation from a sterile compartment . However, 35 of the 69 patients were systemically treated with fluconazole (on average 295 mg per day for 10.2 days intravenously) . 34 patients did not receive any antifungal therapy . Retrospectively we analysed the course of the disease in both groups of patients . Furthermore, 173 serum samples of these patients were available for investigations by Western blot for anti-Candida antibodies of the immune globulin classes M and G . RESULTS: Both groups, antimycotically treated and untreated patients, had similar characteristics at base-line: age, sex, underlying disease, severity of the disease (APACHE II Score), and also mortality (approximately 20 % in both groups) . Only times in the ICU and on mechanical ventilation were significantly enhanced in fluconazole treated patients (p values 0.0004 each) . Before therapy, the fluconazole patients had significantly more often yeasts in primarily non-sterile compartments (chi (2) test 0.05) . The yeasts were partly eradicated by fluconazole (32/54, 59.3 %) . Anti-Candida antibodies significantly correlated with higher age (anti 47 kDa antigen, p = 0.02), but not with other, clinically, diagnostically or prognostically relevant parameters . CONCLUSION: Fluconazole in non-neutropenic, Candida-colonized, surgical patients in long-term ICU care neither improved the clinical course nor the mortality rate among these patients . These observations indicate that there was a trend of overestimating the clinical significance of Candida in this group of patients . Fluconazole therapy may be significantly reduced in such patients. Prep Biochem Biotechnol, 2001 Aug, 31(3), 305 - 16 Purification and partial characterization of glyoxalase I from bovine brain; Lupidi G et al.; Glyoxalase I was purified to homogeneity from bovine brain using affinity chromatography on S-hexylglutathione-Sepharose 6B with a yield of 22% . The enzyme was a dimer (44,000 Daltons) composed of, apparently, identical subunits (22,000 Daltons), as shown by SDS electrophoresis, and contained one mole of Zn2+/monomer . The active site metal ion, Zn2+, was removed by dialysis against EDTA, but the activity of the apoenzyme obtained was not completely restored after addition of Co2+ and Zn2+ (<25%), while a recovery of 50% was obtained after addition of Mg2+ . The enzyme was inhibited by S-bromobenzylglutathione and S-p-nitrobenzylglutathione with a Ki value of 21 microM and 32 microM, respectively . The highest dissociation constant observed for the brain enzyme with respect to that reported for human erythrocytes, or other mammalian forms of enzyme could be related to a tissue-specific dependence of the glyoxalase I activity. J Am Soc Mass Spectrom, 2001 Aug, 12(8), 885 - 8 Prediction of peptide ion mobilities via a priori calculations from intrinsic size parameters of amino acid residues; Shvartsburg AA et al.; Ion mobility spectrometry (IMS) has recently been established as a powerful tool to separate the protease digest mixtures and identify their peptide components . As accurate calculation of mobilities is critical for this technique, a new rapid method based on intrinsic size parameters (ISPs) of amino acid residues has been devised . However, those parameters had to be obtained by tedious statistical analysis of a large body of experimental data . Here we demonstrate that they can instead be derived a priori, based on the stoichiometry of a residue . Our main finding is that the ISP of a residue is essentially determined by its density, that is, the average mass/size ratio of its constituent atoms . This is in accordance with an interpretation in which peptides assume compact conformations in the gas phase dominated by the solvation of ionic charge. Nat Rev Genet, 2001 Aug, 2(8), 584 - 96 Determining centromere identity: cyclical stories and forking paths; Sullivan BA et al.; The centromere is the genetic locus required for chromosome segregation . It is the site of spindle attachment to the chromosomes and is crucial for the transfer of genetic information between cell and organismal generations . Although the centromere was first recognized more than 120 years ago, little is known about what determines its site(s) of activity, and how it contributes to kinetochore formation and spindle attachment . Recent work in this field has supported the hypothesis that most eukaryotic centromeres are determined epigenetically rather than by primary DNA sequence . Here, we review recent studies that have elucidated the organization and functions of centromeric chromatin, and evaluate present-day models for how centromere identity and propagation are determined. Nat Cell Biol, 2001 Aug, 3(8), 708 - 14 TGF-beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation; Perlman R et al.; Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has a principal role in growth control through both its cytostatic effect on many different epithelial cell types and its ability to induce programmed cell death in a variety of other cell types . Here we have used a screen for proteins that interact physically with the cytoplasmic domain of the type II TGF-beta receptor to isolate the gene encoding Daxx - a protein associated with the Fas receptor that mediates activation of Jun amino-terminal kinase (JNK) and programmed cell death induced by Fas . The carboxy-terminal portion of Daxx functions as a dominant-negative inhibitor of TGF-beta-induced apoptosis in B-cell lymphomas, and antisense oligonucleotides to Daxx inhibit TGF-beta-induced apoptosis in mouse hepatocytes . Furthermore, Daxx is involved in mediating JNK activation by TGF-beta . Our findings associate Daxx directly with the TGF-beta apoptotic-signalling pathway, and make a biochemical connection between the receptors for TGF-beta and the apoptotic machinery. Nippon Ishinkin Gakkai Zasshi, 2001, 42(3), 123 - 6 {Assimilation test of Malassezia furfur isolated from the environment}; Tanaka R et al.; The lipophilic yeasts Malassezia species are the causative agents of tinea versicolor and known also to be a member of normal skin flora . They are commonly isolated from the skin of humans and animals, but not from the environment . This is the first report of the isolation of Malassezia sp . from the environment (a hospital floor) . The results of assimilation tests of lipids and karyotyping showed that these isolates were M . furfur . They assimilated not only lipids including floor wax and car wax but also some ointments (except antifungal agents) used clinically . The results suggest that we need to take care when using such ointments to treat skin diseases. Cytogenet Cell Genet, 2001, 93(1-2), 19 - 22 C18orf2, a novel, highly conserved intronless gene within intron 5 of the GNAL gene on chromosome 18p11; Vuoristo JT et al.; We have characterized a novel intronless human gene (C18orf2) which is embedded in intron 5 of the G-protein gene (GNAL) on chromosome 18p11 . This gene codes for a 199 amino acid polypeptide with a predicted molecular weight of 22.1 kDa . It is highly homologous to a number of predicted developmental proteins in organisms ranging from yeasts to Drosophila . C18orf2 mRNA was found to be expressed in various tissues . J Clin Microbiol, 2001 Aug, 39(8), 2873 - 9 Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections; Ferrer C et al.; The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis) . Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA . Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR . PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method . It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method . All negative controls (human and bacterial DNA) were PCR negative . The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR . Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001 . PCRs were subsequently performed with 11 ocular samples . The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health . The results were PCR positive for fungal primers for three corneal scrapings, one aqueous sample, and one vitreous sample; one of them was negative by culture . Molecular fungal identification was successful in all cases . Bacterial detection by PCR was positive for three aqueous samples and one vitreous sample; one of these was negative by culture . Amplification of ITS2/5.8S rDNA and molecular typing shows potential as a rapid technique for identifying fungi in ocular samples. Biochim Biophys Acta, 2001 Aug 6, 1513(2), 95 - 121 Slippage and uncoupling in P-type cation pumps; implications for energy transduction mechanisms and regulation of metabolism; Berman MC; P-type ATPases couple scalar and vectorial events under optimized states . A number of procedures and conditions lead to uncoupling or slippage . A key branching point in the catalytic cycle is at the cation-bound form of E(1)-P, where isomerization to E(2)-P leads to coupled transport, and hydrolysis leads to uncoupled release of cations to the cis membrane surface . The phenomenon of slippage supports a channel model for active transport . Ability to occlude cations within the channel is essential for coupling . Uncoupling and slippage appear to be inherent properties of P-type cation pumps, and are significant contributors to standard metabolic rate . Heat production is favored in the uncoupled state . A number of disease conditions, include ageing, ischemia and cardiac failure, result in uncoupling of either the Ca(2+)-ATPase or Na(+)/K(+)-ATPase. Protein Sci, 2001 Aug, 10(8), 1677 - 84 Haspin-like proteins: a new family of evolutionarily conserved putative eukaryotic protein kinases; Higgins JM; Haspin (haploid germ cell-specific nuclear protein kinase) is reported to be a serine/threonine kinase that may play a role in cell-cycle cessation and differentiation of haploid germ cells . In addition, Haspin mRNA can be detected in diploid cell lines and tissues . Here, Haspin-like proteins are identified in several major eukaryotic phyla-including yeasts, plants, flies, fish, and mammals-and an extended group in Caenorhabditis elegans . The Haspin-like proteins have a complete but divergent eukaryotic protein kinase domain sequence . Although clearly related to one another and to other eukaryotic protein kinases, the Haspin-related proteins lack conservation of a subset of residues that are almost invariant in known kinases and possess distinctive inserted regions . In fact, phylogenetic analysis indicates that the Haspin-like proteins form a novel eukaryotic protein kinase family distinct from those previously defined . The identification of related proteins in model organisms provides some initial insight into their functional properties and will provide new experimental avenues by which to determine the function of the Haspin proteins in mammalian cells. Protein Sci, 2001 Aug, 10(8), 1490 - 7 The N-terminal portion of mature aldehyde dehydrogenase affects protein folding and assembly; Zhou J et al.; Human liver cytosolic (ALDH1) and mitochondrial (ALDH2) aldehyde dehydrogenases are both encoded in the nucleus and synthesized in the cytosol . ALDH1 must fold in the cytosol, but ALDH2 is first synthesized as a precursor and must remain unfolded during import into mitochondria . The two mature forms share high identity (68%) at the protein sequence level except for the first 21 residues (14%); their tertiary structures were found to be essentially identical . ALDH1 folded faster in vitro than ALDH2 and could assemble to tetramers while ALDH2 remained as monomers . Import assay was used as a tool to study the folding status of ALDH1 and ALDH2 . pALDH1 was made by fusing the presequence of precursor ALDH2 to the N-terminal end of ALDH1 . Its import was reduced about 10-fold compared to the precursor ALDH2 . The exchange of the N-terminal 21 residues from the mature portion altered import, folding, and assembly of precursor ALDH1 and precursor ALDH2 . More of chimeric ALDH1 precursor was imported into mitochondria compared to its parent precursor ALDH1 . The import of chimeric ALDH2 precursor, the counterpart of chimeric ALDH1 precursor, was reduced compared to its parent precursor ALDH2 . Mature ALDH1 proved to be more stable against urea denaturation than ALDH2 . Urea unfolding improved the import of precursor ALDH1 and the chimeric precursors but not precursor ALDH2, consistent with ALDH1 and the chimeric ALDHs being more stable than ALDH2 . The N-terminal segment of the mature protein, and not the presequence, makes a major contribution to the folding, assembly, and stability of the precursor and may play a role in folding and hence the translocation of the precursor into mitochondria. Drugs, 2001, 61(8), 1121 - 9; discussion 1130-1 Caspofungin; Keating GM et al.; Caspofungin is the first in a new class of antifungal agents, the glucan synthesis inhibitors, that interfere with fungal cell wall synthesis . Caspofungin exhibited in vitro and in vivo efficacy against a wide range of fungi and yeasts including Aspergillus and Candida species . A complete or partial response to caspofungin therapy was seen in 40.7% of immunocompromised adults with invasive aspergillosis who did not respond to, or did not tolerate, other antifungal agents in a noncomparative multicentre study . Caspofungin was effective in patients with oropharyngeal or oesophageal candidiasis, according to the preliminary results of 2 randomised double-blind trials . Caspofungin was generally well tolerated in a multicentre noncomparative trial involving patients with invasive aspergillosis . One or more drug-related clinical adverse effects were experienced by 13.8% of caspofungin recipients (the most common were fever, nausea, vomiting and complications associated with the vein into which caspofungin was infused) . The tolerability of caspofungin appeared to be better than that of amphotericin B and similar to that of fluconazole in double-blind, randomised trials involving patients with mucosal candidiasis. J Oral Pathol Med, 2001 Jul, 30(6), 347 - 54 Oral Candida in HIV-infected heterosexuals and intravenous drug users in Thailand; Nittayananta W et al.; The objectives of this study were to determine levels of oral yeasts in Thai people at different stages of HIV infection compared with HIV-negative controls, to identify factors associated with the levels of oral Candida, and to determine whether the levels of the organism can be used as a predictive clinical marker in HIV-infected individuals . One hundred and eighty HIV-infected heterosexual persons and intravenous drug users (IVDUs) were enrolled (152 men, 28 women) . Eighty-three HIV-free subjects from the same population were included as controls (48 men, 35 women) . Oral yeasts were isolated in 103 HIV-infected subjects (57.2%) and 36 HIV-negative controls (43.3%) . The mean number of colony forming units (CFU) of oral Candida in the first group was 1.9x10(4) CFU/ml (range 2.2x10(2)-4.0x10(6) CFU/ml), which was significantly different statistically when compared with 1.7x10(3) CFU/ml (range 4.0x10(2)-1.2x10(5) CFU/ml) in the control group (P=0.0000) . The following factors are significantly associated statistically with the levels of oral Candida among the subjects (P<0.05): age, stage of HIV infection, total number of lymphocyte cell count, risk group, nutritional status, general health status, weight loss, type of oral lesions and number of oral lesions and number of sites affected . The study revealed that HIV serostatus, stage of HIV infection, and the occurrence of oral lesions among HIV-infected subjects may be predicted by the levels of oral Candida (P<0.05) . By using a cut-off point of 2.0x10(3) CFU/ml, the sensitivity and predicitve values of the level of oral Candida on the HIV serostatus was higher than that based on the culture positivity results alone, although the specificity was similar . These findings suggest that, in conjunction with some other clinical signs and laboratory findings, the levels of oral Candida may be used as a predictive marker of disease in HIV infection. Br J Clin Pharmacol, 2001 Jul, 52(1), 53 - 63 In vitro metabolism and drug interaction potential of a new highly potent anti-cytomegalovirus molecule, CMV423 (2-chloro 3-pyridine 3-yl 5,6,7,8-tetrahydroindolizine I-carboxamide); Bournique B et al.; AIMS: To identify the enzymes involved in the metabolism of CMV423, a new anticytomegalovirus molecule, to evaluate its in vitro clearance and to investigate its potential involvement in drug/drug interactions that might occur in the clinic . METHODS: The enzymes involved in and the kinetics of CMV423 biotransformation were determined using pools of human liver subcellular fractions and heterologously expressed human cytochromes P450 (CYP) and FMO . The effect of CMV423 on CYP probe activities as well as on indinavir and AZT metabolism was determined, and 26 drugs were tested for their potential to inhibit or activate CMV423 metabolism . RESULTS: CMV423 was oxidized by CYP and not by FMO or cytosolic enzymes . The Km values for 8-hydroxylation to rac-RPR 127025, an active metabolite, and subsequent ketone formation by human liver microsomes were 44 +/- 13 microM and 47 +/- 11 microM, respectively, with corresponding Vmax/Km ratios of 14 and 4 microl min(-1) nmol(-1) P450 . Inhibition with selective CYP inhibitors indicated that CYP1A2 was the main isoform involved, with some participation from CYP3A . Expressed human CYP1A1, 1A2, 2C9, 3A4 and 2C8 catalysed rac-RPR 127025 formation with Km values of < 10 microM, 50 +/- 21 microM, 55 +/- 19 microM, circa 282 +/- 61 microM and circa 1450 microM, respectively . CYP1B1, 2A6, 2B6, 2C19, 2D6, 2E1 or 3A5 did not catalyse the reaction to any detectable extent . CYP1A1 and 3A4 also catalysed ketone formation from rac-RPR 127025 . In human liver microsomes, CMV423 at 1 and 10 microM inhibited CYP1A2 activity up to 31% and 63%, respectively, CYP3A4 activity up to 40% (10 microM) and CYP2C9 activity by 35% (1 and 10 microM) . No effect was observed on CYP2A6, 2D6 and 2E1 activities . CMV423 had no effect on indinavir and AZT metabolism . Amongst 26 drugs tested, none inhibited CMV423 metabolism in vitro at therapeutic concentrations . CONCLUSIONS: CMV423 is mainly metabolized by CYP1A2 and 3A4 . Its metabolism should not be saturable at the targeted therapeutic concentrations range (Cmax < 1 microM) . CMV423 will probably affect CYP1A2 and 1A1 activities in vivo to some extent, but no other drug-drug interactions are expected. Dtsch Tierarztl Wochenschr, 2001 Jun, 108(6), 246 - 8 Effects of oligosaccharides on functional parameters of the intestinal tract of growing pigs; Breves G et al.; Feeding experiments were carried out in growing pigs using topinambur powder or inactivated yeasts as prebiotic additives with an application period of at least 3 weeks . At the end of the experimental periods the animals were killed and segments of the intestinal tract were used for measuring transport physiological parameters and for mucin histochemistry . Jejunal epithelia were mounted in Ussing chambers for measuring electrical tissue parameters, paracellular permeability and short circuit current response to mucosal glucose . Both prebiotics had no effects on basal or forskolin stimulated short circuit currents . Total tissue conductances tended to be higher in response to both prebiotics . Topinambur increased mucosal-to-serosal fluxes of mannitol in the proximal and distal jejunum, which could only be demonstrated for the distal jejunum when inactivated yeasts were fed . Mucosal application of glucose induced higher current responses in jejunal tissues . From histochemistry it could be demonstrated that both prebiotics increased the number of ileal goblet cells and the thickness of the colonic mucosa. Med Mycol, 2001 Jun, 39(3), 243 - 51 Quantitative culture of Malassezia species from different body sites of individuals with or without dermatoses; Gupta AK et al.; Quantitative cultures were obtained using contact plates to determine whether the quantity and composition of Malassezia species at a given anatomic site in normal individuals differs from that of patients with various cutaneous dermatoses . The sample included 20 clinically healthy individuals (without any dermatosis) and 110 patients with dermatoses (including 31 with atopic dermatitis {AD}, 28 with psoriasis {PS}, 28 with seborrheic dermatitis {SD} and 23 with pityriasis versicolor {PV}) . Contact plates filled with special culture medium were used to obtain a quantitative culture from five body sites (scalp, forehead, arm, trunk and leg) of every individual . The number of cfu were recorded for every plate that grew Malassezia yeasts, and 3-5 colonies were isolated for identification to species level using microscopic, physiological and molecular characteristics . The mean cfu counts observed among patients with AD, PS and SD was significantly lower than normal control subjects (P < 0.05) . The mean cfu counts from PV patients was not different from that of healthy control subjects . Overall, for all conditions considered together, the mean cfu counts in lesional sites were significantly lower than in non-lesional sites (P <0.05) . Furthermore, the mean cfu counts from lesional sites in patients with AD and PS were significantly lower than the corresponding value in patients with PV (P <0.05) . Six Malassezia species were recovered from the different dermatoses . Malassezia sympodialis was the most common species associated with AD and PV patients and healthy control subjects, while M . globosa was most frequently isolated from PS and SD patients . More than one Malassezia species was recovered at any given anatomic site from both controls as well as individuals with dermatoses . M . globosa was equally likely to be recovered from scalp, forehead and trunk, but less likely to derive from arms and legs . M . restricta and M . slooffiae were recovered more frequently from the upper body (scalp and forehead) than from the lower body . Among normal individuals and for patients with AD and PV, M . sympodialis was significantly more likely to affect the forehead than the legs. Scand J Infect Dis, 2001, 33(5), 367 - 74 Prospective study of fungaemia in a single cancer institution over a 10-y period: aetiology, risk factors, consumption of antifungals and outcome in 140 patients; Kovacicova G et al.; Over a 10-y period (1989-99) we prospectively evaluated all patients with fungaemia among 16,555 admissions (21,004 blood cultures) at a national cancer referral institution in the Slovak Republic . A prospective protocol was completed on 140 patients with fungaemia, which was then analysed in terms of aetiology, clinical characteristics, potential risk factors and outcome . The most frequently isolated organism was C . albicans, in 75 patients (52.9%), followed by non-albicans Candida spp . in 45 patients (32.1%) . Non-Candida spp . yeasts represented 16 episodes in 16 patients (11.4%) . Moulds caused 4 episodes in 4 patients (3.6% of all fungaemias) and all were caused by Fusarium spp . Mucositis (p = 0.025), > or = 3 positive blood cultures (p = 0.02), acute leukaemia (p = 0.00001), neutropenia (p = 0.0015), quinolone prophylaxis (p < 0.000005) and breakthrough fungaemia (p = 0.004) during prophylaxis with fluconazole (p = 0.03) and itraconazole (p = 0.005) were significantly more associated with non-Candida than C . albicans spp . Furthermore, attributable mortality was higher in the subgroup of non-Candida than C . albicans spp . (50.0 vs . 18.7%, p < 0.02) . The only independent risk factor for inferior outcome was antifungal therapy of < 10 d duration (odds ratio 2.1, 95% confidence interval, p < 0.001) . Aetiology, neutropenia and mucositis were not independent risk factors for higher mortality in multivariate analysis; however, they were risk factors for inferior outcome in univariate analysis (p < 0.05-0.005). J Hosp Infect, 2001 Jul, 48(3), 193 - 7 Low rate of Candida parapsilosis-related colonization and infection in hospitalized preterm infants: a one-year prospective study; Gagneur A et al.; We determined the rate of Candida parapsilosis colonization in preterm neonates (NN) and the relationship between colonization and systemic infection through a prospective study in the Neonatal Intensive Care Unit of a university hospital . All NN born at a gestational age of 32 weeks or less were included . Specimens from rectum, mouth and retro-auricular skin were obtained at admission and weekly thereafter . All samples were inoculated on to Sabouraud agar, CHROMagar and Dixon media . Candida species were identified using API Candida and API 20C . DNA analysis was performed using pulse field gel electrophoresis.Fifty-four patients were included (mean age: 30 +/- 1.5 weeks; mean birthweight: 1347 +/- 301 g; male: 40%) . Fungal colonization was detected in 43 (79.6%) . Causative agents were C . parapsilosis (N= 7);Malassezia furfur (N= 30);C . albicans (N= 21), C . guillermondii (N= 1) . No sample was positive for two different yeasts at the same time . C . parapsilosis colonization included anal (N= 6), buccal (N= 1), and skin (N= 2) . The average age at time of colonization was 17.8+/-9.8 days . Neither fungal septicaemia nor death were observed in colonized infants . Two central venous catheters were found to be colonized, one with C . parapsilosis and one with M . furfur . Logistic regression showed a link between colonization and gestational age alone . Three different DNA profiles were observed.This study suggests that in our units, the occurrence of C . parapsilosis colonization is low and bears no relation to systemic infection . The systematic identification of C . parapsilosis carriers for the purposes of isolation and preventive treatment does not appear to be warranted . Nucleic Acids Res, 2001 Jul 1, 29(13), E67 - 7 Novel display of knotted DNA molecules by two-dimensional gel electrophoresis; Trigueros S et al.; We describe a two-dimensional agarose gel electrophoresis procedure that improves the resolution of knotted DNA molecules . The first gel dimension is run at low voltage, and DNA knots migrate according to their compactness . The second gel dimension is run at high voltage, and DNA knots migrate according to other physical parameters such as shape and flexibility . In comparison with one-dimensional gel electrophoresis, this procedure segregates the knotted DNA molecules from other unknotted forms of DNA, and partially resolves populations of knots that have the same number of crossings . The two-dimensional display may allow quantitative and qualitative characterization of different types of DNA knots simply by gel velocity. J Biol Chem, 2001 Sep 7, 276(36), 33540 - 6 Epub 2001 Jun 29. Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts) . Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide; Xu X et al.; Ordered lipid domains enriched in sphingolipids and cholesterol (lipid rafts) have been implicated in numerous functions in biological membranes . We recently found that lipid domain/raft formation is dependent on the sterol component having a structure that allows tight packing with lipids having saturated acyl chains (Xu, X., and London, E . (2000) Biochemistry 39, 844-849) . In this study, the domain-promoting activities of various natural sterols were compared with that of cholesterol using both fluorescence quenching and detergent insolubility methods . Using model membranes, it was shown that, like cholesterol, both plant and fungal sterols promote the formation of tightly packed, ordered lipid domains by lipids with saturated acyl chains . Surprisingly ergosterol, a fungal sterol, and 7-dehydrocholesterol, a sterol present in elevated levels in Smith-Lemli-Opitz syndrome, were both significantly more strongly domain-promoting than cholesterol . Domain formation was also affected by the structure of the sphingolipid (or that of an equivalent "saturated" phospholipid) component . Sterols had pronounced effects on domain formation by sphingomyelin and dipalmitoylphosphatidylcholine but only a weak influence on the ability of cerebrosides to form domains . Strikingly it was found that a small amount of ceramide (3 mol %) significantly stabilized domain/raft formation . The molecular basis for, and the implications of, the effects of different sterols and sphingolipids (especially ceramide) on the behavior and biological function of rafts are discussed. Acta Biol Hung, 2001, 52(2-3), 299 - 306 Effects of double-stranded RNA viruses on the reproduction of Phaffia rhodozyma; Pfeiffer I et al.; DsRNA viruses were transferred from a virus-containing strain to a virus-free strain of Phaffia rhodozyma by protoplast fusion . The resulting new strain carried all three types of dsRNA of the virus-containing strain and had the electrophoretic karyotype of the virus-free strain . The effects of the dsRNA viruses on the host fitness were checked by following the asexual and the sexual reproductivity . The results demonstrated that viruses have no effect on the growth rate during the lag and log phases of the vegetative reproduction, but the maximum cell numbers in the stationary phase differ significantly . Inconclusive results were obtained as concerns the effects of viruses on the sexual reproduction. Glycobiology, 2001 May, 11(5), 71R - 9R Lectin-like proteins in model organisms: implications for evolution of carbohydrate-binding activity; Dodd RB et al.; Classes of intracellular lectins that recognize core-type structures and mediate intracellular glycoprotein trafficking are present in vertebrates, model invertebrates such as Caenorhabditis elegans and Drosophila melanogaster, plants, and yeasts . Lectins that recognize more complex structures at the cell surface, such as C-type lectins and galectins, are also found in invertebrate organisms as well as vertebrates, but the functions of these proteins have evolved differently in different animal lineages. Res Microbiol, 2001 Apr-May, 152(3-4), 391 - 9 Translational regulation by ABC systems; Chakraburtty K; Elongation factor 3 is a cytosolic protein required by the fungal ribosomes for in vitro protein synthesis and for in vivo growth . EF-3 stimulates binding of EF-1:GTP:aa-tRNA ternary complex to the ribosomal A site by facilitated release of the deacylated tRNA from the E site . The reaction requires ATP hydrolysis . EF-3 contains two ATP binding sequence (NBS) motifs . NBSI is sufficient for the intrinsic ATPase activity . NBSII is essential for the ribosome-stimulated functions. Protein Sci, 2001 Jul, 10(7), 1343 - 52 Heat capacity changes upon burial of polar and nonpolar groups in proteins; Loladze VV et al.; In this paper we address the question of whether the burial of polar and nonpolar groups in the protein locale is indeed accompanied by the heat capacity changes, DeltaC(p), that have an opposite sign, negative for nonpolar groups and positive for polar groups . To accomplish this, we introduced amino acid substitutions at four fully buried positions of the ubiquitin molecule (Val5, Val17, Leu67, and Gln41) . We substituted Val at positions 5 and 17 and Leu at position 67 with a polar residue, Asn . As a control, Ala was introduced at the same three positions . We also replaced the buried polar Gln41 with Val and Leu, nonpolar residues that have similar size and shape as Gln . As a control, Asn was introduced at Gln41 as well . The effects of these amino acid substitutions on the stability, and in particular, on the heat capacity change upon unfolding were measured using differential scanning calorimetry . The effect of the amino acid substitutions on the structure was also evaluated by comparing the (1)H-(15)N HSQC spectra of the ubiquitin variants . It was found that the Ala substitutions did not have a considerable effect on the heat capacity change upon unfolding . However, the substitutions of aliphatic side chains (Val or Leu) with a polar residue (Asn) lead to a significant (> 30%) decrease in the heat capacity change upon unfolding . The decrease in heat capacity changes does not appear to be the result of significant structural perturbations as seen from the HSQC spectra of the variants . The substitution of a buried polar residue (Gln41) to a nonpolar residue (Leu or Val) leads to a significant (> 25%) increase in heat capacity change upon unfolding . These results indicate that indeed the heat capacity change of burial of polar and nonpolar groups has an opposite sign . However, the observed changes in DeltaC(p) are several times larger than those predicted, based on the changes in water accessible surface area upon substitution. J Antimicrob Chemother, 2001 Jul, 48(1), 83 - 7 In vitro comparison of the antimycotic activity of a miconazole-HP-beta-cyclodextrin solution with a miconazole surfactant solution; Piel G et al.; The antimycotic activity of a new parenteral solution containing miconazole was compared with that of a marketed solution (Daktarin IV solution) . This solution has been withdrawn from the Belgian market, probably because of toxic effects related to the presence of polyoxyl 35 castor oil . We propose a new formulation containing miconazole (10 mg/mL) (like the marketed solution), in combination with hydroxypropyl-beta-cyclodextrin and lactic acid . The MICs of these two solutions were determined by a broth microdilution method (based on NCCLS guidelines) for 67 yeasts and 50 filamentous fungi isolates . This study shows that the MICs obtained with these two solutions are not significantly different. Hautarzt, 2001 May, 52(5), 418 - 22 {Incidence of fungal involvement in nail psoriasis}; Stander H et al.; BACKGROUND AND OBJECTIVE: Because it is clinically so similar, onychomycosis is one of the most important differential diagnostic considerations in psoriatic nail changes . We determined the frequency of fungal involvement in nails of psoriatic patients . PATIENTS/METHODS: Fungal colonization of the nails of psoriatic patients with and without clinically obvious nail changes was investigated in a collective of 250 patients compared to a group of 102 non-psoriatic persons . RESULTS: There was no significant difference in the frequency of nail colonization with dermatophytes and moulds between healthy persons and psoriatics . Yeasts were more commonly found in psoriatic nails . CONCLUSIONS: Nail changes in psoriatic patients are mostly caused by psoriasis . However secondary colonization by yeasts is more common in psoriatic nails than in the general population . It remains to be clarified why dermatophytes are not more common in psoriatic nails. Plant Physiol, 2001 Jun, 126(2), 759 - 69 Rapid accumulation of phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate correlates with calcium mobilization in salt-stressed arabidopsis; DeWald DB et al.; The phosphoinositide phosphatidylinositol 4,5-bisphosphate {PtdIns(4,5)P(2)} is a key signaling molecule in animal cells . It can be hydrolyzed to release 1,2-diacyglycerol and inositol 1,4,5-trisphosphate (IP(3)), which in animal cells lead to protein kinase C activation and cellular calcium mobilization, respectively . In addition to its critical roles in constitutive and regulated secretion of proteins, PtdIns(4,5)P(2) binds to proteins that modify cytoskeletal architecture and phospholipid constituents . Herein, we report that Arabidopsis plants grown in liquid media rapidly increase PtdIns(4,5)P(2) synthesis in response to treatment with sodium chloride, potassium chloride, and sorbitol . These results demonstrate that when challenged with salinity and osmotic stress, terrestrial plants respond differently than algae, yeasts, and animal cells that accumulate different species of phosphoinositides . We also show data demonstrating that whole-plant IP(3) levels increase significantly within 1 min of stress initiation, and that IP(3) levels continue to increase for more than 30 min during stress application . Furthermore, using the calcium indicators Fura-2 and Fluo-3 we show that root intracellular calcium concentrations increase in response to stress treatments . Taken together, these results suggest that in response to salt and osmotic stress, Arabidopsis uses a signaling pathway in which a small but significant portion of PtdIns(4,5)P(2) is hydrolyzed to IP(3) . The accumulation of IP(3) occurs during a time frame similar to that observed for stress-induced calcium mobilization . These data also suggest that the majority of the PtdIns(4,5)P(2) synthesized in response to salt and osmotic stress may be utilized for cellular signaling events distinct from the canonical IP(3) signaling pathway. Am J Physiol Cell Physiol, 2001 Jul, 281(1), C257 - 69 Regulation by GDI of RhoA/Rho-kinase-induced Ca2+ sensitization of smooth muscle myosin II; Gong MC et al.; We characterized the role of guanine nucleotide dissociation inhibitor (GDI) in RhoA/Rho-kinase-mediated Ca2+ sensitization of smooth muscle . Endogenous contents (approximately 2-4 microM) of RhoA and RhoGDI were near stoichiometric, whereas a supraphysiological GDI concentration was required to relax Ca2+ sensitization of force by GTP and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) . GDI also inhibited Ca2+ sensitization by GTP . G14V RhoA, by alpha-adrenergic and muscarinic agonists, and extracted RhoA from membranes . GTPgammaS translocated Rho-kinase to a Triton X-114-extractable membrane fraction . GTP . G14V RhoA complexed with GDI also induced Ca2+ sensitization, probably through in vivo dissociation of GTP . RhoA from the complex, because it was reversed by addition of excess GDI . GDI did not inhibit Ca2+ sensitization by phorbol ester . Constitutively active Cdc42 and Rac1 inhibited Ca2+ sensitization by GTP . G14V RhoA . We conclude that 1) the most likely in vivo function of GDI is to prevent perpetual "recycling" of GDP . RhoA to GTP . RhoA; 2) nucleotide exchange (GTP for GDP) on complexed GDP . RhoA/GDI can precede translocation of RhoA to the membrane; 3) activation of Rho-kinase exposes a hydrophobic domain; and 4) Cdc42 and Rac1 can inhibit Ca2+ sensitization by activated GTP . RhoA. Mol Cell Biol, 2001 Jul, 21(13), 4162 - 8 TFIIS enhances transcriptional elongation through an artificial arrest site in vivo; Kulish D et al.; Transcriptional elongation by RNA polymerase II has been well studied in vitro, but understanding of this process in vivo has been limited by the lack of a direct and specific assay . Here, we designed a specific assay for transcriptional elongation in vivo that involves an artificial arrest (ARTAR) site designed from a thermodynamic theory of DNA-dependent transcriptional arrest in vitro . Transcriptional analysis and chromatin immunoprecipitation experiments indicate that the ARTAR site can arrest Pol II in vivo at a position far from the promoter . TFIIS can counteract this arrest, thereby demonstrating that it possesses transcriptional antiarrest activity in vivo . Unexpectedly, the ARTAR site does not function under conditions of high transcriptional activation unless cells are exposed to conditions (6-azauracil or reduced temperature) that are presumed to affect elongation in vivo . Conversely, TFIIS affects gene expression under conditions of high, but not low, transcriptional activation . Our results provide physical evidence for the discontinuity of transcription elongation in vivo, and they suggest that the functional importance of transcriptional arrest sites and TFIIS is strongly influenced by the level of transcriptional activation. Science, 2001 Jun 1,