Arch Biochem Biophys, 1997 Aug 15, 344(2), 295 - 300 Characterization of a pollination-related cDNA from Phalaenopsis encoding a protein which is homologous to human peroxisomal acyl-CoA oxidase; Do YY et al.; The first putative plant acyl-CoA oxidase cDNA has been isolated from a Phalaenopsis cDNA library constructed by poly(A)+ RNA extracted from petals 1 day after pollination . This cDNA, pOACO31, contains a 2100-bp open reading frame which encodes a polypeptide named PACO1 of 699 amino acids . The predicted isoelectric point of PACO1 is 8.74 and the molecular weight is 78,032 Da, similar to that of a monomer of predicted plant acyl-CoA oxidase . Southern blot analysis indicated that this gene occurs in one copy or a low number of copies per haploid genome . When compared with sequences in databases, PACO1 revealed significant similarity only to peroxisomal acyl-CoA oxidase particularly within 13 conserved regions and a putative FMN-binding site. Antimicrob Agents Chemother, 1997 Aug, 41(8), 1835 - 6 In vitro antifungal activity of pneumocandin L-743,872 against a variety of clinically important molds; Del Poeta M et al.; The in vitro activity of the new antifungal drug pneumocandin L-743,872 against 55 isolates of clinically important molds was examined by an adapted macrobroth dilution method for yeasts . Pneumocandin L-743,872 exhibited in vitro antifungal activity against Alternaria sp., Aspergillus flavus, Aspergillus fumigatus, Curvularia lunata, Exophiala jeanselmei, Fonsecaea pedrosoi, Paecilomyces variotii, and Scedosporium apiospermum . The drug appeared to lack significant in vitro inhibitory activity against Fusarium oxysporum, Fusarium solani, Rhizopus arrhizus, Paecilomyces lilacinus, and Scedosporium prolificans. Antimicrob Agents Chemother, 1997 Aug, 41(8), 1832 - 4 In vitro evaluation of voriconazole against some clinically important fungi; McGinnis MR et al.; Voriconazole was compared to amphotericin B, fluconazole, and itraconazole by using an in vitro macrobroth dilution test based upon current National Committee for Clinical Laboratory Standards tentative standards against the dimorphic fungi and several opportunistic molds and yeasts . In all instances, the voriconazole MICs were lower than those of fluconazole . In most instances, the MICs were lower than the recorded MICs of amphotericin B and itraconazole. J Clin Microbiol, 1997 Aug, 35(8), 2031 - 9 Identification of Candida species by randomly amplified polymorphic DNA fingerprinting of colony lysates; Steffan P et al.; We have characterized a method that produces simple yet diagnostic fingerprints that are unique to isolates of Candida species . DNA from individual colonies can be amplified from crude single-colony lysates . Randomly amplified polymorphic DNA (RAPD) fingerprints generated from a single primer correctly identified the species of most (>98%) of the isolates identified with CHROMagar Candida plates as non-Candida albicans Candida species . RAPD fingerprints were much more informative than the plates, since they distinguished between all tested species and required less time . Most (91%) of these identifications agreed with those assigned by API 20C tests . In almost every incident of species identity mismatch, electrophoretic karyotyping showed that the RAPD fingerprint was correct . This underscores the improved objectivity and reliability of this method over those of conventional diagnostic tools . The identities of approximately 30% of C . albicans isolates identified in clinical laboratories by positive germ tube tests are not verified by either testing on CHROMagar Candida plates or RAPD fingerprinting . Data suggest that clinical isolates conventionally identified as C . albicans in clinical settings are heterogeneous, consisting of both misidentified and atypical yeasts . RAPD fingerprints obtained from primary culture plate colonies allows for rapid, highly accurate determinations of Candida species, hence permitting earlier selection of appropriate antifungal agents in the clinical setting. Can J Microbiol, 1997 Jul, 43(7), 649 - 57 Murine macrophage elastolytic activity induced by Aspergillus fumigatusstrains in vitro: evidence of the expression of two macrophage-induced protease genes; Rodriguez E et al.; The interaction between Aspergillus fumigatus conidia and murine macrophages of various origins was investigated . Cocultures were carried out between A . fumigatus strains and freshly isolated murine pulmonary alveolar macrophages or two murine macrophage cell-lines: murine alveolar cell-line MALU and murine astrocytoma cell-line J774 . By measuring the variation of elastolytic activity in the coculture supernatants with two elastin substrates, we demonstrated that either viable or fixed A . fumigatus or C . albicans yeasts or nonspecific particles induced significant macrophage elastolytic activity . The effect of A . fumigatus supernatant or the purified A . fumigatus galactomannan suggested also the possible involvement of this polysaccharide in macrophage-protease gene expression, release, and activity in invasive aspergillosis . The effect of inhibitory compounds demonstrated the potential implication of a macrophagic metalloprotease and a macrophagic cysteine protease . RNA analysis allowed us to demonstrate the induction of expression of two macrophagic protease genes in stimulated macrophages . Two distinctive mechanisms appeared to be implicated in macrophage protease induction: nonspecific phagocytosis in the earliest times of the coculture and (or) specific galactomannan recognition after its gradual release by the mycelium. Infect Immun, 1997 Jul, 65(7), 2564 - 9 Intrapulmonary response to Histoplasma capsulatum in gamma interferon knockout mice; Allendoerfer R et al.; We examined the immunobiological responses to Histoplasma capsulatum in lungs of gamma interferon (IFN-gamma) knockout mice (GKO mice) . Naive GKO mice succumbed by day 9 to intranasal challenge with 2.5 x 10(6) yeasts, whereas all wild-type (WT) mice survived for 45 days . Compared to lungs of WT mice, the lungs of acutely infected GKO mice exhibited dramatically elevated numbers of CFU in lungs and significantly higher levels of tumor necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not interleukin-12 (IL-12) or IL-4 . To determine if IFN-gamma is necessary in reexposure histoplasmosis, GKO and WT mice were inoculated with 10(4) yeasts intranasally and given amphotericin B for 3 weeks . Six weeks later, mice were rechallenged with 2.5 x 10(6) yeasts . All GKO mice died by day 6, whereas all WT mice survived for 45 days . Lungs of GKO mice contained substantially elevated numbers of CFU and higher TNF-alpha and GM-CSF levels but not IL-12 or IL-4 . Thus, IFN-gamma is requisite for control of pulmonary histoplasmosis in naive and reexposed mice. Cell, 1997 Jun 27, 89(7), 1155 - 64 Coassembly of TRP and TRPL produces a distinct store-operated conductance; Xu XZ et al.; The Drosophila retinal-specific protein, TRP (transient receptor potential), is the founding member of a family of store-operated channels (SOCs) conserved from C . elegans to humans . In vitro studies indicate that TRP is a SOC, but that the related retinal protein, TRPL, is constitutively active . In the current work, we report that coexpression of TRP and TRPL leads to a store-operated, outwardly rectifying current distinct from that owing to either TRP or TRPL alone . TRP and TRPL interact directly, indicating that the TRP-TRPL-dependent current is mediated by heteromultimeric association between the two subunits . We propose that the light-activated current in photoreceptor cells is produced by a combination of TRP homo- and TRP-TRPL heteromultimers. Cell, 1997 Jun 27, 89(7), 1067 - 76 Daxx, a novel Fas-binding protein that activates JNK and apoptosis; Yang X et al.; The Fas cell surface receptor induces apoptosis upon receptor oligomerization . We have identified a novel signaling protein, termed Daxx, that binds specifically to the Fas death domain . Overexpression of Daxx enhances Fas-mediated apoptosis and activates the Jun N-terminal kinase (JNK) pathway . A C-terminal portion of Daxx interacts with the Fas death domain, while a different region activates both JNK and apoptosis . The Fas-binding domain of Daxx is a dominant-negative inhibitor of both Fas-induced apoptosis and JNK activation, while the FADD death domain partially inhibits death but not JNK activation . The Daxx apoptotic pathway is sensitive to both Bcl-2 and dominant-negative JNK pathway components and acts cooperatively with the FADD pathway . Thus, Daxx and FADD define two distinct apoptotic pathways downstream of Fas. J Biol Chem, 1997 Jun 20, 272(25), 15834 - 40 Expression cloning of PIG-L, a candidate N-acetylglucosaminyl-phosphatidylinositol deacetylase; Nakamura N et al.; Many eukaryotic cell surface proteins are bound to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor . Several genes involved in GPI anchor biosynthesis have been cloned using complementation of mutant mammalian cell lines and yeasts that are defective in its biosynthesis pathway . However, the gene involved in the second step of this pathway, in which N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI) is N-deacetylated to form glucosaminyl (GlcN)-PI, has not been cloned . In this study, we established a GPI anchor-deficient mutant of Chinese hamster ovary (CHO) cells defective in the second step . Complementation analysis with the known GPI anchor mutant cells demonstrated that it belonged to the same complementation group as the CHO cell mutant G9PLAP.85 . Using the new mutant, we cloned a rat gene termed PIG-L (for phosphatidylinositol glycan class L) that is involved in this step . PIG-L encodes a 252-amino acid, endoplasmic reticulum membrane protein, most of which is in the cytoplasmic side . This orientation of PIG-L protein is consistent with the notion that the second step of GPI anchor biosynthesis occurs on the cytoplasmic side of the endoplasmic reticulum. Cell, 1997 Jun 13, 89(6), 859 - 66 The eucaryal tRNA splicing endonuclease recognizes a tripartite set of RNA elements; Di Nicola Negri E et al.; The tRNA splicing endonuclease cleaves intron-containing tRNA precursors on both sides of the intron . The prevailing belief has been that the enzyme binds only to the mature domain through the invariant bases . We show instead that, for recognition, the endonuclease utilizes distinct sets of structural elements, several of which are within the intron . One subset of recognition elements, localized in the mature domain, is needed for recognition of both cleavage sites, while two other subsets, localized at the exon-intron boundaries, are used for recognition of either one or the other cleavage site . The two cleavage sites are essentially independent: neither is required by the other for cleavage to take place . These results support a two-active-site model for the eucaryal endonuclease. J Am Osteopath Assoc, 1997 Jun, 97(6), 339 - 46 Advances in the treatment of superficial fungal infections: focus on onychomycosis and dry tinea pedis; Del Rosso JQ; Onychomycosis is one of the most stubborn superficial mycoses . With few exceptions, oral antifungal therapy is needed to achieve resolution . Before oral itraconazole, fluconazole, and terbinafine hydrochloride became available, physicians had to rely on prolonged therapy with griseofulvin or oral ketoconazole . Of the newer oral agents, itraconazole appears to have the broadest spectrum of action, with therapeutic activity against dermatophytes, yeasts, and some nondermatophyte molds . Tissue pharmacokinetics accounts for significantly greater efficacy and much shorter treatment courses for fungal infections of the skin and nails . In general, oral itraconazole, fluconazole, and terbinafine are very well tolerated . The newer oral agents offer improved efficacy over griseofulvin and ketoconazole for onychomycosis and dry tinea pedis. Chem Biol, 1997 Jun, 4(6), 461 - 71 Methionine aminopeptidase (type 2) is the common target for angiogenesis inhibitors AGM-1470 and ovalicin; Griffith EC et al.; BACKGROUND: Angiogenesis, the formation of new blood vessels, is essential for tumor growth . The inhibition of angiogenesis is therefore emerging as a promising therapy for cancer . Two natural products, fumagillin and ovalicin, were discovered to be potent inhibitors of angiogenesis due to their inhibition of endothelial cell proliferation . An analog of fumagillin, AGM-1470, is currently undergoing clinical trials for the treatment of a variety of cancers . The underlying molecular mechanism of the inhibition of angiogenesis by these natural drugs has remained unknown . RESULTS: Both AGM-1470 and ovalicin bind to a common bifunctional protein, identified by mass spectrometry as the type 2 methionine aminopeptidase (MetAP2) . This protein also acts as an inhibitor of eukaryotic initiation factor 2alpha (elF-2alpha) phosphorylation . Both drugs potently inhibit the methionine aminopeptidase activity of MetAP2 without affecting its ability to block elF-2alpha phosphorylation . There are two types of methionine aminopeptidase found in eukaryotes, but only the type 2 enzyme is inhibited by the drugs . A series of analogs of fumagillin and ovalicin were synthesized and their potency for inhibition of endothelial cell proliferation and inhibition of methionine aminopeptidase activity was determined . A significant correlation was found between the two activities . CONCLUSIONS: The protein MetAP2 is a common molecular target for both AGM-1470 and ovalicin . This finding suggests that MetAP2 may play a critical role in the proliferation of endothelial cells and may serve as a promising target for the development of new anti-angiogenic drugs. Lipids, 1997 Jun, 32(6), 605 - 10 Sesamol as an inhibitor of growth and lipid metabolism in Mucor circinelloides via its action on malic enzyme; Wynn JP et al.; Sesamol, a nonoil component of sesame seed oil, inhibited growth, fatty acid synthesis, and desaturation by Mucor circinelloides in vivo . Although sesamol also inhibited the growth of other fungi and yeasts, its effect on the lipid metabolism of M . circinelloides was exceptional . An enzymological study demonstrated that sesamol affected lipid synthesis primarily by the inhibition of malic enzyme activity, thereby limiting the NADPH supply for fatty acid synthesis and desaturation . Sesamol itself had no inhibitory effect on malic enzyme activity in vitro . A metabolite of sesamol is therefore probably responsible for the in vivo effects of sesamol on lipid metabolism. J Clin Microbiol, 1997 Jun, 35(6), 1353 - 60 Detection and identification of fungal pathogens in blood by using molecular probes; Einsele H et al.; A PCR assay was developed for the detection and identification of Candida and Aspergillus species . The design of the oligonucleotide primer pair as well as the species-specific probes used for species identification was derived from a comparison of the sequences of the 18S rRNA genes of various fungal pathogens . The primers targeted a consensus sequence for a variety of fungal pathogens . The assay was tested for sensitivity and specificity with 134 fungal and 85 nonfungal isolates . To assess clinical applicability, 601 blood samples from four defined groups were tested: group A (n = 35), controls; groups B to D (n = 86), patients with febrile neutropenia, without fungal colonization (group B; n = 29) and with fungal colonization (group C; n = 36); and patients with documented invasive fungal infection (IFI) (group D; n = 21) . The assay detected and, by species-specific hybridization, identified most of the clinically relevant Candida and Aspergillus species at 1 CFU/ml of blood . Amplification was 100% sensitive for all molds and yeasts tested, with Histoplasma capsulatum being the only non-Aspergillus species hybridizing with the Aspergillus spp . probe . None of 35 group A patients and only 3 of 65 group B and C patients were PCR positive . The sensitivity of the assay for specimens from patients with IFI (21 patients in group D) was 100% if two specimens were tested . For specificity, 3 of 189 specimens from patients at risk but with negative cultures were positive by the assay, for a specificity of 98% . PCR preceded radiological signs by a median of 4 days (range, 4 to 7 days) for 12 of 17 patients with hepatosplenic candidiasis or pulmonary aspergillosis . For the 10 patients with IFI responding to antifungal therapy, PCR assays became persistently negative after 14 days of treatment, in contrast to the case for 11 patients, who remained PCR positive while not responding to antifungal therapy . Thus, the described PCR assay allows for the highly sensitive and specific detection and identification of fungal pathogens in vitro and in vivo . Preliminary data from the screening of a selected group of patients revealed some value in the early diagnosis and monitoring of antifungal therapy. Curr Opin Genet Dev, 1997 Apr, 7(2), 220 - 32 Life and death in the cytoplasm: messages from the 3' end; Wickens M et al.; The cytoplasmic life of an mRNA revolves around the regulation of its localization, translation and stability . Interactions between the two ends of the mRNA may integrate translation and mRNA turnover . Regulatory elements in the region between the termination codon and poly(A) tail - the 3' untranslated region - have been identified in a wide variety of systems, as have been some of the key players with which these elements interact. Genes Dev, 1997 Mar 1, 11(5), 640 - 53 ALY, a context-dependent coactivator of LEF-1 and AML-1, is required for TCRalpha enhancer function; Bruhn L et al.; LEF-1 is a transcription factor that participates in the regulation of the T-cell receptor alpha (TCR alpha) enhancer by facilitating the assembly of multiple proteins into a higher order nucleoprotein complex . The function of LEF-1 is dependent, in part, on the HMG domain that induces a sharp bend in the DNA helix, and on an activation domain that stimulates transcription only in a specific context of other enhancer-binding proteins . With the aim of gaining insight into the function of context-dependent activation domains, we cloned ALY, a novel LEF-1-interacting protein . ALY is a ubiquitously expressed, nuclear protein that specifically associates with the activation domains of LEF-1 and AML-1 (CBF alpha2, PEBP2 alpha(B), which is another protein component of the TCR alpha enhancer complex . In addition, ALY can increase DNA binding by both LEF-1 and AML proteins . Overexpression of ALY stimulates the activity of the TCR alpha enhancer complex reconstituted in transfected nonlymphoid HeLa cells, whereas down-regulation of ALY by anti-sense oligonucleotides virtually eliminates TCR alpha enhancer activity in T cells . Similar to LEF-1, ALY can stimulate transcription in the context of the TCR alpha enhancer but apparently not when tethered to DNA through an heterologous DNA-binding domain . We propose that ALY mediates context-dependent transcriptional activation by facilitating the functional collaboration of multiple proteins in the TCR alpha enhancer complex. Plant Physiol, 1997 Mar, 113(3), 943 - 9 Binding of the peroxisomal targeting sequence SKL is specified by a low-affinity site in castor bean glyoxysomal membranes . A domain next to the SKL binds to a high-affinity site; Wolins NE et al.; The carboxyl-terminal amino acid sequence serine-lysine-leucine (SKL) is the consensus peroxisomal targeting sequence 1 (PTS1) and is sufficient to direct a polypeptide to peroxisomes in vivo in plants, animals, and yeasts . However, there are also two sites on alkali-stripped glyoxysomal membranes from castor bean (Ricinus communis) endosperm that bind the peptide YHKHLKPLQSKL (SKLp), the sequence of the last 12 amino acids of acyl-coenzyme A oxidase (N.E . Wollins, R.P . Donaldson {1994} J Biol Chem 289: 1149-1153) . It was hypothesized that one of these sites interacts with information other than the PTS1 . To explore the sequence requirements for each SKLp binding site, we tested the peptides YHKHLKPQSKG and YHKHLKPLQS and found that they bound to the high-affinity site, but not to the low-affinity site . When the high-affinity site was blocked with YHKHLKPQSKG, SKLp bound to the low-affinity site with a dissociation constant (Kd) of 8.5 microM . In an attempt to disrupt high-affinity binding, two the upstream, positively charged residues were replaced with negatively charged residues to make the peptide YHKETEPLQSKL . YHKETEPLQSKL did not bind to either site on the glyoxysomal membranes . These results indicate that the PTS1 binds to the low-affinity site and that the adjacent, positively charged domain binds to the high-affinity site. J Immunol, 1997 Feb 15, 158(4), 1779 - 86 Activation of human macrophage fungistatic activity against Histoplasma capsulatum upon adherence to type 1 collagen matrices; Newman SL et al.; Human monocyte/macrophages (Mphi) were adhered to extracellular matrix proteins, and the intracellular growth of Histoplasma capsulatum (Hc) yeasts were quantified and compared with their growth in Mphi adhered to plastic . Freshly isolated monocytes and cultured monocyte/derived Mphi adhered to type 1 collagen gels, but not to nongelled collagen-, fibronectin-, laminin-, or vitronectin-coated surfaces, demonstrated significant fungistatic activity against Hc yeasts . Activation of Mphi developed immediately upon adherence to the collagen matrices (1 h) and did not require additional time in culture . In addition, many of the yeasts were digested by 24 h postinfection . Mphi adhered to collagen maintained their fungistatic activity for up to 4 days, during which time monolayers cultured on plastic were destroyed . Culture of Mphi in the presence of IFN-gamma or TNF-alpha for 24 h before infection did not augment the fungistatic activity of collagen-adherent Mphi . Likewise, culture of monocytes on collagen gels with IL-3, granulocyte-Mphi CSF (GM-CSF) or Mphi CSF (M-CSF) for 7 days did not enhance Mphi fungistatic activity above that obtained by monocytes cultured on collagen alone . The mechanism(s) of Mphi-mediated fungistasis was not associated with production of toxic oxygen radicals, nitric oxide, or the restriction of intracellular iron . However, experiments with horseradish peroxidase-labeled gold colloids and immunoelectron microscopy demonstrated that phagolysosomal fusion, which is minimal in Hc-infected Mphi adhered to plastic, is enhanced significantly at both 1 h and 24 h postinfection in Mphi adhered to collagen matrices . These data suggest that in vivo, matrix-bound Mphi may express a previously unrecognized antifungal activity that proceeds in the absence of exogenous cytokines and is mediated, in part, by overcoming the capacity of Hc yeasts to inhibit Mphi phagolysosomal fusion. Eur J Epidemiol, 1997 Feb, 13(2), 235 - 8 Transmission of nocardiosis and molecular typing of Nocardia species: a short review; Provost F et al.; Nocardia species are ubiquitous in the environment and may be found in the soil . They are generally responsible for sporadic pulmonary diseases acquired by inhalation of spores, with secondary localizations in the central nervous system and subcutaneous tissues . There is no absolute evidence for person to person transmission . Presumptive outbreaks of nocardiosis were observed in immunocompromised patients, more frequently in kidney transplant patients than in cardiac transplant patients . Nocardia spp., being present in dust particles, closure and disinfection of the transplantation unit with formaldehyde arrested the sequence of cases of nocardiosis . The original sources of the Nocardia sp . remain doubtful . Other possible sources of contamination are other patients, medical staff and the hospital environment . The first studies of Nocardia spp . typing were based on the detection of extracellular antigens, on the susceptibility of actinomycete strains to killer yeasts, and on the biochemical profiles with fluorogenic substrate . The use of molecular typing techniques have given very promising results . Analysis of plasmid profiles is an interesting way to compare the identity of isolates, although the reliability of this method depends of the presence of plasmids in the isolates . Other typing methods, including analysis of restriction length fragment polymorphism of total DNA, ribosomal DNA fingerprinting, require further investigations to evaluate their discriminating power or to be easily interpretable, whereas a random amplified polymorphic DNA (RAPD) assay was successful for epidemiological purposes . Progress in epidemiological analysis of cases of nocardiosis will be consistent when an improved diagnosis of this infection (molecular and serological diagnosis) will be available, when the genetic diversity of Nocardia spp . isolates will be better known, and when molecular typing, that hold promise in complementing investigations of outbreak of these infections, will be systematically performed when an abnormal increase of cases of nocardiosis in a population with risk factors is observed. Plant Cell, 1997 Feb, 9(2), 223 - 35 Cell cycle phase specificity of putative cyclin-dependent kinase variants in synchronized alfalfa cells; Magyar Z et al.; The eukaryotic cell division cycle is coordinated by cyclin-dependent kinases (CDKs), represented by a single major serine/threonine kinase in yeasts (Cdc2/CDC28) and a family of kinases (CDK1 to CDK8) in human cells . Previously, two cdc2 homologs, cdc2MsA and cdc2MsB, have been identified in alfalfa (Medicago sativa) . By isolating cDNAs using a cdc2MsA probe, we demonstrate here that at least four additional cdc2 homologous genes are expressed in the tetraploid alfalfa . Proteins encoded by the new cdc2MsC to cdc2MsF cDNAs share the characteristic functional domains of CDKs with the conserved and plant-specific sequence elements . Transcripts from cdc2MsA, cdc2MsB, cdc2MsC, and cdc2MsE genes are synthesized throughout the cell cycle, whereas the amounts of cdc2MsD and cdc2MsF mRNAs peak during G2-to-M phases . The translation of Cdc2MsA/B, Cdc2MsD, and Cdc2MsF proteins follows the pattern of transcript accumulation . The multiplicity of kinase complexes with cell cycle phase-dependent activities was revealed by in vitro phosphorylation experiments . Proteins bound to p13suc1-Sepharose or immunoprecipitated with Cdc2MsA/B antibodies from cells at G1-to-S and G2-to-M phase boundaries showed elevated kinase activities . the Cdc2MsF antibodies separated a G2-to-M phase-related kinase complex . Detection of histone H1 phosphorylation activities in fractions immunoprecipitated with antimitotic cyclin (CyclinMs2) antibodies from G2-to-M phase cells indicates the complex formation between this cyclin and a kinase partner in alfalfa . The observed fluctuation of transcript levels, amounts, and activities of kinases in different cell cycle phases reflects a multilevel regulatory system during cell cycle progression in plants. FEBS Lett, 1997 Jan 3, 400(2), 206 - 10 Identification of Asp804 and Asp808 as Na+ and K+ coordinating residues in alpha-subunit of renal Na,K-ATPase; Pedersen PA et al.; Mutations to Asp804 and Asp808 in the alpha-subunit almost abolish Na,K-ATPase activity, but high-affinity binding of {3H}ATP or {3H}ouabain at equilibrium and E1-E2 transitions are preserved . Titration of K+-ion displacement of {3H}ATP or {3H}ouabain shows that the mutations interfere with occlusion of K+ in the E2{2K} conformation . Reduced phosphorylation levels or affinities for Na+ in presence of oligomycin indicate that Asp804 and Asp808 also contribute to coordination of Na+ in the E1P{3Na} form . Demonstration of alternate interactions of Na+ or K+ with Asp804 and Asp808 support the notion of cation binding in a ping-pong sequence in catalytic models of Na,K-pumping. Mycoses, 1997 Jan-Feb, 40(1-2), 3 - 24 Fungal infections of the cardiovascular system--a review; Kuttin ES; Various yeasts and filamentous fungal infections of human and animal heart and blood vessels, as well as the experimental investigations, are reviewed . Clinical, diagnostic, therapeutic and pathological aspects are discussed . The prevalence of mycotic infection of the heart, once considered to be an uncommon disease, has been reported frequently during recent years . The rate will certainly be higher if cases are more thoroughly investigated. Biochem Cell Biol, 1997, 75(2), 95 - 102 Influence of the adenovirus 5 E1A oncogene on chromatin remodelling; Mymryk JS et al.; In the eukaryotic nucleus, compaction of DNA into chromatin can limit the access of trans-acting factors, providing an additional level of regulation to processes such as transcription, replication, and repair . Recent studies have suggested that the protein products of the adenovirus 5 E1A oncogene can influence SWI-SNF and histone acetylase activities, two cellular processes that facilitate transcription in the context of chromatin . This review focuses on the unexpected effects of E1A on cellular processes that remodel chromatin in relation to its transcriptional and transforming activities. Adv Biochem Eng Biotechnol, 1997, 58, 185 - 230 Fluidized bed adsorption as a primary recovery step in protein purification; Thommes J; Fluidized bed adsorption has been introduced as an integrative technology combining clarification, concentration, and initial purification in a single step . In the paper presented here, the use of fluidized adsorbents in the primary recovery of proteins starting from unclarified broths is reviewed . First the principle of fluidizing adsorbent particles is discussed, subsequently possible experimental procedures for whole broth adsorption are demonstrated . The system parameters governing the performance of the sorption process in a fluidized bed are discussed in the second part of the paper and considerations on how operating parameters and process design influence the limiting steps are provided . Finally, examples for the successful operation of whole broth adsorption employing fluidized adsorbents are shown and conditions are defined under which this technology may be an alternative to traditional protein purification methods. DNA Cell Biol, 1997 Jan, 16(1), 59 - 72 The transcriptional and translational control of diazepam binding inhibitor expression in rat male germ-line cells; Kolmer M et al.; The diazepam binding inhibitor {DBI, also known as acyl-CoA-binding protein, (ACBP), or endozepine} is a 10-kD protein that has been suggested to be involved in the regulation of several biological processes such as acyl-CoA metabolism, steroidogenesis, insulin secretion, and gamma-aminobutyric acid type A (GABA(A))/benzodiazepine receptor modulation . DBI has been cloned from vertebrates, insects, plants, and yeasts . In mammals, DBI is expressed in almost all the tissues studied . Nevertheless, DBI expression is restricted to specific cell types . Here we have studied DBI gene expression in the germ-line cells of rat testis . The DBI gene was intensively transcribed in postmeiotic round spermatids from stages VI to VIII of the seminiferous epithelial cycle . A prominent, spermatid-specific upstream transcription initiation site was identified in addition to the multiple common transcriptional initiation sites found in the somatic tissues . However, no DBI protein was detected in round spermatids, suggesting that the DBI transcripts were translationally arrested . The DBI protein was detected in the late spermatogenic stages starting from elongating spermatids from step 18 (stage VI) onward . The DBI protein was also detected in mature spermatozoa and in ejaculated human sperms . The majority of DBI was located at the middle piece of the spermatozoons tail enriched with mitochondria . On the basis of this observation and the well-established role of DBI in acyl-CoA metabolism, we propose that DBI expression in spermatozoa reflects the usage of fatty acids as a primary energy source by spermatozoa . The biological function of DBI in spermatozoa could thus be related to the motility function of sperm. Bioessays, 1997 Jan, 19(1), 57 - 66 Peroxisome biogenesis; Waterham HR et al.; Peroxisomes are eukaryotic organelles that are the subcellular location of important metabolic reactions . In humans, defects in the organelle's function are often lethal . Yet, relative to other organelles, little is known about how cells maintain and propagate peroxisomes or how they direct specific sets of newly synthesized proteins to these organelles (peroxisome biogenesis/assembly) . In recent years, substantial progress has been made in elucidating aspects of peroxisome biogenesis and in identifying PEX genes whose products, peroxins, are essential for one or more of these processes . The most progress has been made in understanding the mechanism by which peroxisome matrix proteins are imported into the organelles . Signal sequences responsible for targeting proteins to the organelle have been defined . Potential signal receptor proteins, a receptor docking protein and other components of the import machinery have been identified, along with insights into how they operate . These studies indicate that multiple peroxisomal protein-import mechanisms exist and that these mechanisms are novel, not simply variations of those described for other organelles. Leukemia, 1997 Jan, 11(1), 86 - 96 Elf-2, a rhombotin-2 binding ets transcription factor: discovery and potential role in T cell leukemia; Wilkinson DA et al.; Rhombotin-2 (RBTN-2) is a proto-oncogene only in the context of T lymphocytes . We postulated that the oncogenic effect of RBTN-2 in T cells is likely mediated by binding protein(s) with T cell-specific expression . By screening a T cell cDNA library, we identified a novel ets transcription factor that binds RBTN-2 . This protein was named elf-2 because its DNA-binding domain is virtually identical to that of ets family member elf-1 . Northern analyses showed similar levels of two elf-2 transcripts (3.5 kb and 3.8 kb) in all tissues except thymus . Thymocytes expressed four- to 10-fold greater amounts of the 3.5 kb transcript than other tissues . Sequence analyses of cDNA clones indicated that these transcripts encode proteins differing only at their amino termini, and likely represent alternatively spliced isoforms . These isoforms (elf-2a and elf-2b) contain identical RBTN-2 binding regions and DNA-binding domains . Elf-2b lacks a putative transactivation domain . The expression patterns suggest that RBTN-2 normally interacts equally with elf-2a and elf-2b . In contrast, when RBTN-2 is inappropriately expressed in T cells, RBTN-2 would interact predominantly with elf-2b; this interaction may lead to T cell proliferation. Plant Cell, 1996 Dec, 8(12), 2223 - 34 PK12, a plant dual-specificity protein kinase of the LAMMER family, is regulated by the hormone ethylene; Sessa G et al.; The ethylene signal is transduced in plant cells via phosphorylation events . To identify protein kinases whose levels of expression are modulated by the plant hormone ethylene, we utilized a differential reverse transcriptase-polymerase chain reaction approach using mRNA extracted from ethylene-treated and untreated tobacco leaves . An ethylene-induced cDNA clone, PK12, encoding a protein kinase, was isolated . PK12 is a new member of the recently defined LAMMER family of protein kinases, which has been identified in mammals, flies, yeasts, and plants . The LAMMER kinases are related to the cell cycle-dependent CDC2-type kinases and are characterized by their similarity at kinase subdomain X . The recombinant PK12 protein autophosphorylates in vitro on serine, threonine, and tyrosine residues, thereby making it a member of the dual-specificity protein kinases . Immunoprecipitation of PK12 from plant extracts and kinase assay revealed that the apparent PK12 activity is rapidly and transiently increased when plants are treated with ethylene . By using in situ hybridization, we detected accumulation of the PK12 transcript in leaves after ethylene treatment and in the untreated flower abscission zone . The tissue in this zone is known to constitutively express ethylene-regulated genes. FEMS Microbiol Lett, 1996 Dec 1, 145(2), 261 - 5 A method for the large scale isolation of high transformation efficiency fungal genomic DNA; Zhang D et al.; A procedure for isolation of genomic DNA from the zygomycete Cunninghamella elegans and other filamentous fungi and yeasts is reported . This procedure involves disruption of cells by grinding using dry ice, removal of polysaccharides using cetyltrimethylammonium bromide and by phenol extractions, and precipitation of DNA with isopropanol at room temperature . The isolation method produced large scale (approximate 1 mg DNA/5 g wet cells) and highly purified high molecular mass DNA . Sau3AI partially digested DNA showed high transformation efficiency (> 10(6)/100 ng DNA) when ligated to ZAP-express lambda vector. Biol Signals, 1996 Nov-Dec, 5(6), 301 - 8 Protozoan cell cycle control; Wong JT; Many genes belonging to the cyclin-dependent kinase (cdk) family have been isolated from protozoans . While their role in cell cycle has yet to be proven unequivocally, at least one cdk can complement the cdc2ts/cdc28ts mutants in yeasts . Among the interesting questions relating to cdks in protozoa are: whether one cdk acts throughout the whole cell cycle and whether cyclin partnership is absolutely required . In protozoa, cell cycle control is closely associated with developmental control . Many life cycle differentiation phases can only occur during a specific window in the cell cycle . Because of different DNA biosynthetic pathways, some protozoa among the earliest eukaryotic lineages are unresponsive to common inhibitors of DNA synthesis like hydroxyurea . However, many protozoa do have different checkpoint controls in relation to their response to cell cycle inhibitors. J Med Vet Mycol, 1996 Nov-Dec, 34(6), 371 - 8 Cell-mediated immunity in host resistance against infection caused by Penicillium marneffei; Kudeken N et al.; Penicillium marneffei is one of the most important opportunistic infectious pathogens in AIDS patients in Thailand and Southeast Asia . However, very little is known about the host defence mechanisms against P . marneffei infection . In the present study, we established the first experimental murine model of chronic pulmonary and disseminated infection using P . marneffei, and examined the immunological response to such infection in euthymic and athymic mice . In this model, micro-organisms inoculated intratracheally multiplied progressively in the lungs and disseminated to the liver and spleen . However, the number of organisms decreased gradually in these organs . In contrast, congenitally athymic mice developed severe pulmonary and disseminated systemic mycosis . Pulmonary penicilliosis marneffei was associated with a marked cellular inflammatory response as evident by histological abnormalities and increased intraparenchymal leucocyte count . To confirm the importance of cell-mediated immunity in host resistance to P . marneffei infection, we transferred nylon wool non-adherent spleen cells into the athymic mice . Such treatment significantly reduced the number of yeasts in the organs of athymic mice . Taken together, our results demonstrate that the cell-mediated immunity play a central role in a host defence mechanism against infection with P . marneffei, and suggest that our new model may be a useful approach for studying the pathogenesis of this fungal disease. Cell, 1996 Nov 1, 87(3), 577 - 88 Crystal structure at 2.4 angstroms resolution of the complex of transducin betagamma and its regulator, phosducin; Gaudet R et al.; The crystal structure of transducin's betagamma subunits complexed with phosducin, which regulates Gtbetagamma activity, has been solved to 2.4 angstroms resolution . Phosducin has two domains that wrap around Gtbetagamma to form an extensive interface . The N-terminal domain binds loops on the "top" Gtbeta surface, overlapping the Gtalpha binding surface, explaining how phosducin blocks Gtbetagamma's interaction with Gtalpha . The C-terminal domain shows structural homology to thioredoxin and binds the outer strands of Gtbeta's seventh and first blades in a manner likely to disrupt Gtbetagamma's normal orientation relative to the membrane and receptor . Phosducin's Ser-73, which when phosphorylated inhibits phosducin's function, points away from Gtbetagamma, toward a large flexible loop . Thus phosphorylation is not likely to affect the interface directly, but rather indirectly through an induced conformational change. J Biol Chem, 1996 Oct 25, 271(43), 26868 - 75 PIG-A and PIG-H, which participate in glycosylphosphatidylinositol anchor biosynthesis, form a protein complex in the endoplasmic reticulum; Watanabe R et al.; Many eukaryotic cell surface proteins are bound to the membrane via a glycosylphosphatidylinositol (GPI) anchor . Assembly of the GPI anchor precursor is a sequential addition of components to phosphatidylinositol (PI) in the endoplasmic reticulum (ER) . The first step is the transfer of N-acetylglucosamine (GlcNAc) to PI from UDP-GlcNAc to generate GlcNAc-PI . This simple step, however, is regulated by at least three genes because in both mammals and yeasts, there are three mutants of different complementation classes . To clarify this complexity, we analyzed the products of two cloned human genes, PIG-A and PIG-H . Here we demonstrate 1) that PIG-A is an ER transmembrane protein with a large cytoplasmic domain that has homology to a bacterial GlcNAc transferase and a small lumenal domain; 2) that PIG-H is a cytoplasmically oriented, ER-associated protein; and 3) that they form a protein complex . We also show that part of the small lumenal domain of PIG-A plays an essential functional role in targeting itself to the rough ER . Taken together with the cytoplasmic orientation of GlcNAc-PI, these results indicated that PIG-A and PIG-H are subunits of the GPI GlcNAc transferase that transfers GlcNAc to PI on the cytoplasmic side of the ER. Biochim Biophys Acta, 1996 Oct 17, 1297(2), 235 - 43 Further studies on the bioaffinity chromatography of NAD(+)-dependent dehydrogenases using the locking-on effect; O'Carra P et al.; Previous studies have capitalized on ordered kinetic mechanisms in the design of biospecific affinity chromatographic methods for highly efficient purifications and mechanistic studies of enzymes . The most direct tactic has been the use of immobilised analogues of the following, usually enzyme-specific substrates, e.g., lactate/pyruvate in the case of lactate dehydrogenase for which NAD+ is the leading substrate . Such immobilised specific substrates are, however, often difficult or impossible to synthesise . The locking-on strategy reverses the tactic by using the more accessible immobilised leading substrate, immobilised NAD+, as adsorbent with soluble analogues of the enzyme-specific ligands (e.g., lactate in the case of lactate dehydrogenase) providing a substantial reinforcement of biospecific adsorption sufficient to effect adsorptive selection of an enzyme from a group of enzymes such as the NAD(+)-specific enzymes . The value of this approach is demonstrated using model studies with lactate dehydrogenase (LDH, EC 1.1.1.27), alcohol dehydrogenase (ADH, EC 1.1.1.1), glutamate dehydrogenase (GDH, EC 1.4.1.3) and malate dehydrogenase (MDH, EC 1.1.1.37) . Purification of bovine liver GDH in high yield from crude extracts is described using the tactic. J Hosp Infect, 1996 Oct, 34(2), 123 - 9 Colonization and infection associated with Malassezia and Candida species in a neonatal unit; Shattuck KE et al.; The objectives of this study were to determine, in neonates of < 1250 g birthweight (N = 57), the initial time of skin colonization by Malassezia furfur, rate of colonization by Candida spp., and whether skin colonization by these yeasts was predictive of central line colonization or fungaemia . By age two weeks, 51% of neonates were culture-positive for M . furfur on umbilical or groin skin . During hospitalization, positive skin cultures for M . furfur or Candida spp . were obtained in 70% and 37% of neonates, respectively . Risk factors associated with positive skin cultures were mechanical ventilation and three or more episodes of suspected sepsis . Eight of the 52 infants with central venous catheters, had positive blood cultures withdrawn from the lines; five (62%) of these had positive skin surveillance cultures . Although positive skin cultures for M . furfur, Candida spp., or both were commonly observed in this population, they were not predictive of positive central line cultures or systemic illness. J Biol Chem, 1996 Sep 6, 271(36), 22189 - 95 Regulation of the RNA polymerase I and III transcription systems in response to growth conditions; Clarke EM et al.; To better understand the mechanisms that regulate stable RNA synthesis, we have analyzed the RNA polymerase I and III transcriptional activities of extracts isolated from cells propagated under a variety of conditions . Under balanced growth conditions the levels of both RNA polymerase I- and III-specific transcription increased proportionally with growth rate . Upon nutritional starvation, RNA polymerase I transcription rapidly declined, followed by 5 S rDNA and eventually tDNA transcription . Transcriptional activities in extracts were restored when the nongrowing cultures were resuspended in fresh medium, although growth did not resume . The differential expression of 5 S rDNA and tDNA genes in extracts prepared from cells subjected to partial starvation was traced to a 5 S rDNA-specific inhibitor and not to a defect in any RNA polymerase III transcription factor . Characterization of this inhibitor indicated that it was not 5 S rRNA . It was sensitive to phenol extraction and resistant to RNase, and its target did not appear to be transcription factor IIIA . Not all treatments that slowed or stopped growth down-regulated the stable RNA transcription apparatus . Cells that have been subjected to either energy starvation or cycloheximide treatment still retain the ability to synthesize stable RNA in vitro, suggesting the presence of alternative regulatory mechanisms. Biochim Biophys Acta, 1996 Sep 5, 1296(2), 145 - 51 Molecular characterisation of a thermoactive beta-1,3-glucanase from Oerskovia xanthineolytica; Parrado J et al.; Molecular characterisation of a lytic thermoactive beta-1,3-glucanase from Oerskovia xanthineolytica LL-G109 has been performed . A molecular mass of 27 195.6 +/- 1.3 Da and an isoelectric point of 4.85 were determined by electrospray mass spectrometry and from its titration curve, respectively . Its thermoactivity profile shows it to be a heat-stable enzyme with a temperature optimum of 65 degrees C . The secondary structure content of the protein was estimated by circular dichroism to be approx . 25% alpha-helix, 7% random coil, and 68% beta-sheet and beta-turn structure . Nuclear magnetic resonance spectra confirm the high content of beta-structure . Furthermore, the presence of a compact hydrophobic core is indicated by the presence of slowly exchanging amide hydrogens and the enzyme's relatively high resistance to proteolysis . The N-terminal sequences of the intact protein and of a tryptic peptide each exhibit significant similarity to family 16 of glycosyl hydrolases whose overall fold is known to contain almost exclusively beta-sheets and surface loops . Moreover, the sequenced tryptic peptide appears to encompass residues of the Oerskovia xanthineolytica glucanase active site, since it contains a portion of the family 16 active-site motif E-{L/I/V}-D-{L/I/V}-E. Ann Pathol, 1996 Sep, 16(4), 266 - 70 {Role of guided fine needle punction in the diagnosis of deep-seated Aids-related infections . Report of a case of hepato-nodal histoplasmosis}; Longchampt E et al.; A case of disseminated histoplasmosis diagnosed by fine needle aspiration biopsy is reported . The patient suffering from acquired immune deficiency syndrome (AIDS) had enlarged liver, spleen and mesenteric lymph nodes . Cytological smears prepared from a CT scan guided fine needle aspiration biopsy of one of the lymph node and the liver, showed numerous free or intrahistiocytic yeasts consistent with Histoplasma capsulatum . Yeasts and protozoars morphologically close to Histo-plasma capsulatum are reviewed . The indications of fine needle aspiration biopsy for the diagnosis of infections in AIDS patients are emphasized . This method enables to send rapidly material for cultures and to start immediately an appropriate treatment. J Enzyme Inhib, 1996 Aug, 11(1), 51 - 66 Piperastatin B: a new selective serine carboxypeptidase inhibitor from Streptomyces lavendofoliae MJ908-WF13; Murakami S et al.; Piperastatin B, a new inhibitor of serine carboxypeptidase was purified from a culture broth of Streptomyces lavendofoliae MJ908-WF13 as a minor component by monitoring its inhibitory activity against carboxypeptidase Y (CP-Y) . Its structure was determined to be N-formyl-Val-Thr-Leu-Val-Pip-Leu-Pip (pip: piperazic acid, hexahydropyridadine-3-carboxylic acid) . Piperastatin B is a highly specific competitive inhibitor of CP-Y (Ki = 55 nM) with little effect on related enzymes and resembles the major component, piperastatin A, in these respects. Pediatr Allergy Immunol, 1996 Aug, 7(3), 151 - 5 Domestic fungal viable propagules and sensitization in children with IgE mediated allergic diseases; Dill I et al.; In order to study the possible relationship between domestic fungal exposure and sensitization to fungi, the homes of 20 children with allergic diseases (mainly bronchial asthma) were inspected for visible fungal growth and airborne viable fungal propagules were sampled . Inclusion criterion was sensitization to at least one of the tested fungi as shown by proof of specific IgE in serum . Twelve of 19 homes (63%) revealed a considerable amount of viable fungal propagules and/or visible fungal growth . Penicillium, Cladosporium, Aspergillus, Mycelia sterilia and yeasts were most prevalent . There was no clear-cut relationship to serologically proven sensitization. Clin Infect Dis, 1996 Aug, 23(2), 305 - 13 Update on the management of onychomycosis: highlights of the Third Annual International Summit on Cutaneous Antifungal Therapy; Elewski BE et al.; Onychomycosis is an increasingly common fungal infection of the nail, which has traditionally been difficult to diagnose and treat and has physical and psychological consequences for the patient . Onychomycosis can be caused by dermatophytes, nondermatophytic filamentous fungi, and yeasts . The relative percentages of cases due to these etiologic agents vary with geographic location; however, in the United States, dermatophytes are the most common pathogens . Toenails are affected four times as often as fingernails . Microscopy and culture are the diagnostic "gold standards" for onychomycosis, although biopsy of the nail may be required to obtain a definitive diagnosis when conditions that mimic onychomycosis, such as psoriasis, are suspected . The treatment of onychomycosis includes a combination of topical therapy, surgical or chemical nail avulsion, and systemic therapy . The new generation of systemic agents (itraconazole, fluconazole, and terbinafine) is associated with a higher cure rate and shorter courses of treatment than are the older systemic antifungal drugs (i.e., griseofulvin and ketoconazole); these characteristics have sparked new interest in onychomycosis . Of these newer antifungals, itraconazole and terbinafine are the only agents currently approved by the U.S . Food and Drug Administration for the treatment of onychomycosis. Genes Dev, 1996 Aug 1, 10(15), 1858 - 69 The EVES motif mediates both intermolecular and intramolecular regulation of c-Myb; Dash AB et al.; The c-Myb transcription factor is a proto-oncoprotein whose latent transforming activity can be unmasked by truncation of either terminus . Because both ends of Myb are involved in negative regulation, we tested whether they could associate in a two-hybrid assay and identified a carboxy-terminal motif that interacts with the amino-terminal DNA-binding domain . The EVES motif is highly conserved in vertebrate c-Myb proteins and contains a known site of phosphorylation previously implicated in the negative regulation of c-Myb . Interestingly, a related EVES motif is present in p100, a ubiquitously expressed transcriptional coactivator found in diverse species . We show that p100 interacts with and influences the activity of c-Myb, implicating it in the regulation of c-Myb, differentiation, and cell growth . Our results suggest that Myb is regulated by a novel mechanism in which intramolecular interactions and conformational changes control the intermolecular associations among Myb, p100, and the transcriptional apparatus. Mol Cell Biol, 1996 Aug, 16(8), 4486 - 94 A consensus motif in the RFX DNA binding domain and binding domain mutants with altered specificity; Emery P et al.; The RFX DNA binding domain is a novel motif that has been conserved in a growing number of dimeric DNA-binding proteins, having diverse regulatory functions, in eukaryotic organisms ranging from yeasts to humans . To characterize this novel motif, we have performed a detailed dissection of the site-specific DNA binding activity of RFX1, a prototypical member of the RFX family . First, we have performed a site selection procedure to define the consensus binding site of RFX1 . Second, we have developed a new mutagenesis-selection procedure to derive a precise consensus motif, and to test the accuracy of a secondary structure prediction, for the RFX domain . Third, a modification of this procedure has allowed us to isolate altered-specificity RFX1 mutants . These results should facilitate the identification both of additional candidate genes controlled by RFX1 and of new members of the RFX family . Moreover, the altered-specificity RFX1 mutants represent valuable tools that will permit the function of RFX1 to be analyzed in vivo without interference from the ubiquitously expressed endogenous protein . Finally, the simplicity, efficiency, and versatility of the selection procedure we have developed make it of general value for the determination of consensus motifs, and for the isolation of mutants exhibiting altered functional properties, for large protein domains involved in protein-DNA as well as protein-protein interactions. Chemotherapy, 1996 Jul-Aug, 42(4), 294 - 307 Antiproliferative effects of delta 24(25) sterol methyl transferase inhibitors on Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies; Urbina JA et al.; We have studied the antiproliferative effects of two sterol analogs previously reported as potent inhibitors of delta 24(25) sterol methyl transferase (E.C . 2.1.1.43) of yeasts and fungi on epimastigotes and amastigotes on Trypanosoma (Schizotrypanum) cruzi, the causative agents of Chagas disease, as well as its chemotherapeutic effects in a murine model of the disease . On the epimastigote form proliferating in liver infusion tryptose medium at 28 degrees C 22,26-azasterol (AZA), a cholestanol analog with a 6-membered aza ring as a side chain produced a dose-dependent reduction of the growth rate up to 3 microM, but at 10 microM complete growth arest and cell lysis took place after 120-144 h . For 24(R,S),25-epiminolanosterol (EIL), complete growth arrest and lysis took place with 6 microM . In both cases the antiproliferative effects were potentiated by the simultaneous incubation of the epimastigotes with inhibitors of sterol C-14 alpha-demethylase such as ketoconazole or SDZ 89,485, as indicated by concave isobolograms and fractional inhibitory concentrations ranging from 0.11 to 0.46 . Analysis of the sterol composition in control and treated cells by thin-layer and capillary gas-liquid chromatography coupled to mass spectrometry showed that growth inhibition correlated with the complete disappearance of the native endogenous sterols of the parasite (ergosterol and 24-ethyl analogs) and the accumulation of 24-desalkyl sterols . Against the clinically relevant amastigote form proliferating inside cultured Vero cells at 37 degrees C, AZA eradicated the parasite of 100 nM, while the corresponding concentration for EIL was 300 nM . Synergic effects of both inhibitors when combined with ketoconazole against this form of the parasite was demonstrated using a three-dimensional analytic method which allowed the identification of optimal drug concentrations . Finally, it was found that daily oral administration of AZA at 50 mg/kg/day for a total of 43 doses to mice infected with a lethal inoculum of T . cruzi allowed survival of all treated animals 25 days after infection, while all control (untreated) animals were dead at this point of time . Increased survival correlated with a 90% reduction in parasitemia in the treated animals . The antiparasitic effects of the azasterol were potentiated in combined treatments with ketoconazole . This is the first report of a successful application of a sterol methyl transferase inhibitor as a chemotherapeutic agent in a protozoal infection. Oncogene, 1996 Jun 6, 12(11), 2291 - 300 A 15 amino acid stretch close to the Grb2-binding domain defines two differentially expressed hSos1 isoforms with markedly different Grb2 binding affinity and biological activity; Rojas JM et al.; We compared structure, expression and functional properties of two hSos1 cDNA isoforms (IsfI and Isf II) isolated, respectively, from human fetal brain and adult skeletal muscle libraries . IsfI and IsfII nucleotide sequences differ only by the presence in IsfII of an inframe 45 hp insertion located near the first proline-rich motif required for Grb2 binding . Some human tissues express only one isoform whereas others express different proportions of both in fetal and adult stages . In vitro binding assays and in vivo functional studies showed that MI exhibits significantly higher Grb2 binding affinity and biological activity than IsfI . These results suggest that functionally different hSos1 isoforms, with differential tissue expression and distribution, play important regulatory roles in the mechanisms controlling Ras activation in different tissues and/or developmental stages. Mech Dev, 1996 Jun, 57(1), 3 - 20 Five years on the wings of fork head; Kaufmann E et al.; Since its discovery five years ago the conserved family of fork head/HNF-3-related transcription factors has gained increasing importance for the analysis of gene regulatory mechanisms during embryonic development and in differentiated cells . Different members of this family, which is defined by a conserved 110 amino acid residues encompassing DNA binding domain of winged helix structure, serve as regulatory keys in embryogenesis, in tumorigenesis or in the maintenance of differentiated cell states . The purpose of this review is to summarize the accumulating amount of data on structure, expression and function of fork head/HNF-3-related transcription factors. Mol Endocrinol, 1996 Jun, 10(6), 694 - 704 Estrogen receptor affinity and location of consensus and imperfect estrogen response elements influence transcription activation of simplified promoters; Nardulli AM et al.; We have examined the ability of estrogen receptor (ER) to bind and bend DNA fragments containing the Xenopus laevis vitellogenin A2 estrogen response element (ERE), which contains a palindromic, consensus ERE sequence, the X . laevis vitellogenin B1 ERE2, which contains a 1-bp mismatch in the 5'-end of the half-palindrome, and the human pS2 ERE, which contains a 1-bp mismatch in the 3'-end of the half-palindrome . ER binding induced a 65 degrees bend in DNA fragments containing the consensus ERE, the vitellogenin B1 ERE2, or the pS2 ERE . However, ER affinity for the consensus ERE was 2-fold greater than for either the vitellogenin B1 ERE2 or the pS2 ERE . When Chinese hamster ovary (CHO) cells were transfected with reporter plasmids containing either the consensus ERE, the vitellogenin B1 ERE2, or the pS2 ERE separated from the TATA sequence by 26 helical turns, exposure to 10 nm 17 beta-estradiol increased transcription 12.7-, 2.4-, and 3.8-fold, respectively . Increasing the spacing between the ERE and TATA sequence to three helical turns decreased the ability of the consensus ERE to activate transcription by 55% and increased the ability of the pS2 ERE to activate transcription by 35% but had no significant effect on vitellogenin B1 ERE2 activity . Further increasing the distance between the ERE and TATA sequence to 3.6 helical turns restored the activity of promoters containing the consensus ERE and pS2 ERE but decreased the activity of the promoter containing the relatively weak vitellogenin B1 ERE2 . These data support the idea that 1) the affinity of ER for the ERE, 2) the location of an ERE within the promoter, and 3) the magnitude and orientation of DNA bends induced by binding of ER or other proteins are important in transcription activation of estrogen-responsive genes. Rinsho Byori, 1996 Jun, 44(6), 512 - 7 {The antigen (CANDTEC antigen) detected by CAND TEC test for diagnosis of candidiasis}; Mitsuya M et al.; Most guinea pigs inoculated with 5.4 x 10(9) of C . albicans intraperitoneally, produce CANDTEC antigen (GPCANDTECAG) in sera . The antigen is heat-labile (at 56 degrees C for 30 min) as is that in humans . According to gel filtration, the molecule size of the antigen from guinea pigs was 4000KDa or more . ELISA revealed the antigen-positive gel fractions to contain a small amount of mannan from the yeasts and C3 . ELISA using rabbit anti-GPCANDTECAG serum indicated that the two CANDTEC antigens from guinea pigs and humans shared determinants . Gel filtration indicated that the CANDTEC antigen from patients was from 4000KDa to 3000KDa . In the antigen-positive gel fractions, IgM was detected by ELISA, but mannan and C3 were not detected . However, immunoblotting analysis on the antigen-positive fraction revealed a unique band of 200KDa, stained with concanavalin A-ALP . These findings indicate that CANDTEC antigens in guinea pigs and humans are immune complexes formed after infection of Candida, although the antigens have different components. J Clin Microbiol, 1996 Jun, 34(6), 1583 - 5 Duration of incubation of fungal cultures; Morris AJ et al.; To determine the optimum duration of incubation for recovery of fungi, the results of 2,173 consecutive clinical cultures were reviewed . Overall, 94% of fungal isolates were detected by day 7 and 98% were detected by day 14 . Yeasts were usually (98%) detected within the first week of incubation . Recovery of molds required more time, but 81% were detected by day 7 and more than 96% were detected by day 14. Cas Lek Cesk, 1996 May 29, 135(11), 335 - 9 {Oncogenes and the malignancy process}; Mares J et al.; An important group of genes for the development of neoplastic diseases are, in addition to tumour suppressor genes, protooncogenes . The latter are highly preserved genes present in a similar sequence in the cell genomes of different species (yeasts - man) . They encode components of biochemical signalling pathways by which external mitotic signals stimulate cell proliferation and products which inhibit cell differentiation . The result of activation of protooncogenes into oncogenes (mutations, chromosomal rearrangements, amplifications, viral insertions, insertion mutagenesis) is in particular hyperstimulation of cells resulting in uncontrolled proliferation . Mutations are of the dominant type, elimination of one allele leads to the transformation of a protooncogene into an oncogene . Oncogenes are classified with regard to the transmission level of the mitogenic signal on which they act . Originally they were detected in the genome of oncogenic viruses . However, they do not form their constant and specific constituent, the virus acts as a vector which transmits cellular protooncogenes (or oncogenes) during the reproductive cycle from one cell to another . The activity of various types of oncogenes is the necessary prerequisite for the genesis and development of various neoplastic diseases . Detection of oncogene alterations provides in some instances important diagnostic, prognostic and therapeutic findings. Biochem Biophys Res Commun, 1996 May 24, 222(3), 802 - 8 The polyether bistratene A activates protein kinase C-delta and induces growth arrest in HL60 cells; Griffiths G et al.; Bistratene A (BisA) induced growth arrest in G2/M in HL60 cells . In addition, BisA-treated cells (50 nM for 48 h) became adherent and expressed the adhesion molecule CD11c, but did not express the monocyte enzyme alpha-napthyl acetate esterase or phagocytose complement coated yeasts . BisA activated protein kinase C (PKC)-delta and induced translocation of PKC-delta to the nucleus . This suggests that activation of PKC-delta can induce growth arrest and cell adhesion, but is insufficient to mediate full differentiation of HL60 cells . BisA has potential as a new probe for determining the function of PKC isoenzymes, specifically PKC-delta. FEBS Lett, 1996 May 20, 386(2-3), 255 - 9 ADP delivery from adenylate kinase in the mitochondrial intermembrane space to oxidative phosphorylation increases in the presence of macromolecules; Laterveer FD et al.; Macromolecules were added to isolated rat liver mitochondria to mimic cytosolic macromolecules and tested for their effects on the ADP delivery from adenylate kinase in the intermembrane space to oxidative phosphorylation . In the presence of 10% (w/v) dextran M20 or bovine serum albumin, approximately 60% of the maximal ADP flux from adenylate kinase to oxidative phosphorylation was not accessible to an extramitochondrial ADP scavenger . In the absence of macromolecules this was 34% . ADP determinations from incubations with macromolecules demonstrated the existence of flux-dependent ADP concentration gradients across the outer membrane which can be as high as 12 microM. Mutat Res, 1996 May 15, 363(1), 67 - 75 Multiple nuclear localization signals in XPG nuclease; Knauf JA et al.; We report here evidence for the mechanism of nuclear localization of XPG nuclease in human cells . Several candidate nuclear localization signal (NLS) peptides have been proposed for XPG protein . We have identified XPG peptides containing functional NLS and a potential nuclear retention signal (NRS) using in situ immunofluorescene localization of transiently expressed beta-galactosidase fusion proteins . Two XPG regions with putative NLS {amino acid (AA) coordinates: NLS-B (AA 1057-1074) and NLS-C (AA 1171-1185)} were each shown to independently localize the beta-gal extensively (> 80%) to the nucleus of HeLa cells . The C-terminus peptide containing NLS-C, an NLS conserved evolutionarily between yeasts and humans, also directed sub-localization of beta-galactosidase to intranuclear foci reminiscent of native XPG protein, as well as to peri-nucleolar regions . Peptides in the putative XPG 'NLS domain' (AA approximately 1051-1185) apparently function in concert for nuclear localization and also for retention of XPG in nuclear matrix-associated foci . Evidence presented elsewhere (Park et al., 1995) indicates that the peptide containing NLS-C (AA 1146-1185) also regulates the dynamic localization of XPG in the nucleus following UV-irradiation. Mycoses, 1996 May-Jun, 39(5-6), 195 - 9 Disseminated infection with Trichosporon asahii; Itoh T et al.; Trichosporon fungaemia and disseminated, purpuric, papular skin lesions developed on the head, trunk and extremities of a 5-year-old female with acute lymphocytic leukaemia . Histopathologically, the skin lesions demonstrated dermal budding yeasts . She died despite treatment with antifungal drugs . The isolate from the blood was further identified morphologically and physiologically as Trichosporon asahii, based on the revision of the genus Trichosporon by Gueho et al . (1992) . According to the new revision, T . asahii is the only taxon regularly involved in systemic mycoses, so that most of the isolates previously reported as T . beigelii (formerly, T . cutaneum) in human deep mycoses are now thought to belong to T . asahii. Hepatogastroenterology, 1996 May-Jun, 43(9), 771 - 5 Focal lesion of African histoplasmosis presenting as a malignant gastric ulcer; Sanguino JC et al.; We describe the case of a localized lesion of African Histoplasmosis, presenting as a gastric ulcer, with radiological and endoscopic features suggesting malignancy, that was submitted to surgery . Histologic examination of the resected specimen revealed typical yeasts of Histoplasma duboisii . There where no clinical, radiological or endoscopic signs of disseminated disease and conventional antifungal therapy was not prescribed . The patient has been followed for 11 years now, without evidence of relapse . There are few reports of gastrointestinal Histoplasmosis, and even fewer specifically caused by H . Duboissii Previous descriptions of gastric ulcer in immunocompetent hosts are related to disseminated forms of American Histoplasmosis . Although focal digestive lesions have been found in African patients, there is no available data on the incidence of gastric ulcer as a presenting sign of the disease. Horm Metab Res, 1996 May, 28(5), 223 - 6 Influence of zinc and selenium deficiency on parameters relating to thyroid hormone metabolism; Kralik A et al.; 48 weaned male Sprague-Dawley rats with an initial average body weight of 41 g were divided into 4 groups of 12 animals (zinc-deficient; zinc-adequate, pair-fed with zinc-deficient group; selenium-deficient; selenium-adequate) for 40 days . All groups were fed a semisynthetic diet with casein being the source of protein . In the selenium-deficient diet, there was a selenium concentration of 0.038 mg/kg . The other diets were supplemented with Na-selenite in order to adjust the selenium concentration to 0.3 mg/kg . In the zinc-deficient diet, there was a zinc concentration of 4.1 mg/kg . The zinc concentrations in the other diets were adjusted to 45 mg/kg by the addition of zinc-sulfate heptahydrate . Zinc-deficient rats were characterized by a markedly reduced alkaline phosphatase activity in their serum, whilst selenium-deficient rats showed a markedly reduced glutathione peroxidase in serum proving their respective zinc-deficient and selenium-deficient states . Zinc deficiency decreased concentrations of triiodothyronine (T3) and free thyroxine (fT4) in serum by approximately 30% when compared with zinc-adequate controls . The concentration of thyroxine (T4) in serum was not affected by zinc deficiency . Selenium-deficient animals had lower concentrations of T3 and T4 than selenium-adequate animals . The concentration of fT4 in serum was not affected by selenium deficiency . The activity of hepatic type I 5'deiodinase was decreased by 67% by zinc deficiency and by 47% by selenium deficiency compared to adequate controls . The study data show that both zinc and selenium deficiency affect the metabolism of thyroid hormones. Antonie Van Leeuwenhoek, 1996 Apr, 69(3), 217 - 22 Stationary phase development of Trimmatostroma abietis; Figueras MJ et al.; Processes of anamorph cell replication in Trimmatostroma abietis are described . Growth and conidiation are delimited on the basis of morphological, ultrastructural and ecological criteria . Cellular expansion shifts from bidirectional intercalary in exponential phase cells to isodiametric in late stationary phase cells, in the latter case with endogenous asexual reproduction . Ultrastructural similarities to dothideaceous black yeasts are discussed. Drugs, 1996 Apr, 51(4), 585 - 620 Itraconazole . A reappraisal of its pharmacological properties and therapeutic use in the management of superficial fungal infections; Haria M et al.; Itraconazole is an orally administered triazole antifungal agent . Its spectrum of activity includes dermatophyte, dimorphic and dematiaceous fungi, yeasts, and some moulds . In clinical trials, mycological cure was attained in approximately 70 to 80, > or = 70 and > or = 80% of patients with, respectively, fingernail and toenail onychomycosis (200 mg/day for 3 months), dermatophytosis (100 mg/day for 2 to 4 weeks) and vaginal candidiasis (400 mg/day for 1 day or 200 mg/day for 3 days) . Approximately 20 to 30% of patients with onychomycosis may relapse after completion of therapy; relapse rate data are limited for the other indications . Recently developed intermittent regimens of itraconazole (400 mg/day for 1 week per month for 3 to 4 months) appear to have similar efficacy to standard regimens in the treatment of onychomycosis . Shorter, higher dosage itraconazole treatment regimens (200 or 400 mg/day for 1 week) are also beneficial in dermatomycoses . Discrepancies and limitations of study design hamper conclusions about efficacy relative to other antifungal drugs . Newer intermittent and short course higher dosage itraconazole regimens have also not been evaluated in comparative studies . Available studies show that the efficacy of itraconazole appears to be greater than that of griseofulvin, but similar to or lower than that of terbinafine in patients with dermatophyte onychomycosis or cutaneous fungal infections . Moreover, the efficacy of itraconazole may be similar to or lower than that of fluconazole in the treatment of cutaneous mycoses . Comparative data from patients with acute vaginal candidiasis suggest that itraconazole is at least as effective as intravaginal clotrimazole and oral fluconazole, and superior to intravaginal econazole . These results require confirmation . Prescription-event monitoring data indicate that itraconazole is generally well tolerated . Gastrointestinal disturbances, dizziness and headache occur most commonly; liver toxicity has been rarely described . Its usefulness in some clinical situations may be limited because of its ability to interact with various therapeutic agents . In conclusion, itraconazole along with other established agents should be considered a first-line treatment for patients with extensive or recalcitrant cutaneous fungal infections, mixed dermatophyte and Candida onychomycosis or vaginal candidiasis . It is currently considered a second-line drug for dermatophyte onychomycosis; the use of newer intermittent itraconazole treatment regimens may, however, extend its role in the management of this condition . Although itraconazole offers greater benefit than conventional therapies (griseofulvin and ketoconazole) in terms of efficacy and tolerability, wider clinical experience is required to determine its merits relative to the newer agents, terbinafine and fluconazole. Am J Hum Genet, 1996 Apr, 58(4), 694 - 702 Thiopurine S-methyltransferase deficiency: two nucleotide transitions define the most prevalent mutant allele associated with loss of catalytic activity in Caucasians; Tai HL et al.; The autosomal recessive trait of thiopurine S-methytransferase (TPMT) deficiency is associated with severe hematopoietic toxicity when patients are treated with standard doses of mercaptopurine, azathioprine, or thioguanine . To define the molecular mechanism of this genetic polymorphism, we cloned and characterized the cDNA of a TPMT-deficient patient, which revealed a novel mutant allele (TPMT*3) containing two nucleotide transitions (G460-->A and A719-->G) producing amino acid changes at codons 154 (Ala-->Thr) and 240 (Tyr--> Cys), differing from the rare mutant TPMT allele we previously identified (i.e., TPMT*2 with only G238-->C) . Site-directed mutagenesis and heterologous expression established that either TPMT*3 mutation alone leads to a reduction in catalytic activity (G460-->A, ninefold reduction; A719-->G, 1.4-fold reduction), while the presence of both mutations leads to complete loss of activity . Using mutation specific PCR-RFLP analysis, the TPMT*3 allele was detected in genomic DNA from approximately 75 percent of unrelated white subjects with heterozygous phenotypes, indicating that TPMT*3 is the most prevalent mutant allele associated with TPMT-deficiency in Caucasians. Cell, 1996 Mar 22, 84(6), 853 - 62 Site-specific phosphorylation of IkappaBalpha by a novel ubiquitination-dependent protein kinase activity; Chen ZJ et al.; Signal-induced activation of the transcription factor NF-kappaB requires specific phosphorylation of the inhibitor IkappaBalpha and its subsequent proteolytic degradation . Phosphorylation of serine residues 32 and 36 targets IkappaBalpha to the ubiquitin (Ub)-proteasome pathway . Here we report the identification of a large, multisubunit kinase (molecular mass approximately 700 kDa) that phosphorylates IkappaBalpha at S32 and S36 . Remarkably, the activity of this kinase requires the Ub-activating enzyme (E1), a specific Ub carrier protein (E2) of the Ubc4/Ubc5 family, and Ub . We also show that a ubiquitination event in the kinase complex is a prerequisite for specific phosphorylation of IkappaBalpha . Thus, ubiquitination serves a novel regulatory function that does not involve proteolysis. Science, 1996 Mar 15, 271(5255), 1533 - 9 Phosphoinositides as regulators in membrane traffic; De Camilli P et al.; Phosphorylated products of phosphatidylinositol play critical roles in the regulation of membrane traffic, in addition to their classical roles as second messengers in signal transduction at the cell surface . Growing evidence suggests that phosphorylation-dephosphorylation of the polar heads of phosphoinositides (polyphosphorylated inositol lipids) in specific intracellular locations signals either the recruitment or the activation of proteins essential for vesicular transport . Cross talk between phosphatidylinositol metabolites and guanosine triphosphatases is an important feature of these regulatory mechanisms. Science, 1996 Mar 15, 271(5255), 1526 - 33 Coat proteins and vesicle budding; Schekman R et al.; The trafficking of proteins within eukaryotic cells is achieved by the capture of cargo and targeting molecules into vesicles that bud from a donor membrane and deliver their contents to a receiving department . This process is bidirectional and may involve multiple organelles within a cell . Distinct coat proteins mediate each budding event, serving both to shape the transport vesicle and to select by direct or indirect interaction the desired set of cargo molecules . Secretion, which has been viewed as a default pathway, may require sorting and packaging signals on transported molecules to ensure their rapid delivery to the cell surface. Biochemistry, 1996 Mar 12, 35(10), 3141 - 6 Secondary structure and topology of a mitochondrial presequence peptide associated with negatively charged micelles . A 2D H-NMR study; Chupin V et al.; In this study the secondary structure and topology of the peptide, corresponding to the presequence of cytochrome oxidase subunit IV (p25) in a negatively charged membrane-mimetic environment, were assessed by circular dichroism and two-dimensional nuclear magnetic resonance . The micelles used consisted of dodecylphosphoglycol (DPG), a mild anionic detergent with a headgroup resembling that of phosphatidylglycerol . The secondary structure was analyzed by interresidue nuclear Overhauser enhancement measurements and chemical shifts of backbone protons . The data revealed alpha-helix formation of the peptide upon interaction with the micelles, both in the N- and in the C-terminal halves, which are separated from each other by the proline residue at position 13 . The topology of the peptide was studied by determining the effect of spin-labeled 12-doxylstearate on the line widths of the peptide proton resonances . This method revealed the insertion of hydrophobic residues of both the N- and the C-terminal halves of p25 into the hydrophobic environment of the micelles, demonstrating the orientation of the amphiphilic helix. FEBS Lett, 1996 Mar 11, 382(1-2), 18 - 20 Rabbit translation elongation factor 1 alpha stimulates the activity of homologous aminoacyl-tRNA synthetase; Negrutskii BS et al.; Functional and structural sequestration of aminoacyl-tRNA has been recently found in eukaryotic cells and the aminoacyl-tRNA channeling has been suggested {B.S . Negrutskii et al., Proc . Natl . Acad . Sci . 91 (1994) 964-968}, but molecular details and mechanism of the process remained unclear . In this paper we have verified a possible interaction between rabbit aminoacyl-tRNA synthetase and homologous translation elongation factor 1 alpha (EF-1 alpha), the proteins which may play a role of sequential components involved in the transfer of the aminoacyl-tRNA along the protein synthetic metabolic chain . The stimulation of the phenylalanyl-tRNA synthetase activity by EF-1 alpha is found . The effect is shown to be specific towards the origin of tRNA and elongation factor molecules . The data obtained favor the direct transfer mechanism of the aminoacyl-tRNA channeling process during eukaryotic protein synthesis. Lipids, 1996 Mar, 31(3), 253 - 9 Study of the delta 12-desaturase system of Lipomyces starkeyi; Lomascolo A et al.; The specific activity of the microsomal delta 12-desaturase system, which transforms oleic acid into linoleic acid, was about 16 pmol/min/mg protein . However, most of the total activity was nonsedimentable even after a 200000 x g centrifugation for 100 min . The study of various physicochemical parameters showed that this enzymatic complex, functioning optimally between pH 7 and 8, had low thermal stability . Ca2+ which may cause an aggregation of the microsomes, and Hg2+ completely inhibited the activity, whereas Mg2+, Mn2+, and Zn2+ were activators . The delta 12-desaturase system was relatively specific toward oleic acid, though isomers of this fatty acid also had an action, either as substrates or as competitive inhibitors, on the activity of the system . The study of the effect of the exogenous oleoyl-CoA and elaidoyl-CoA on the specific activity of the delta 12-desaturase system showed a preference toward oleoyl-CoA. Vet Pathol, 1996 Mar, 33(2), 238 - 41 Visceral mycosis in Chinook salmon (Oncorhynchus tschawytscha) due to Sporobolomyces salmonicolor; Muench TM et al.; One-month-old Chinook salmon fry from a cold-water hatchery were presented live for euthanasia and necropsy . Gross lesions were emaciation in 90% of the fry and ascites and increased cutaneous pigmentation in the remaining 10% . A cause for the emaciation was not determined . Histologically, the fry with ascites and increased pigmentation had visceral mycosis with aerocystitis, myositis, peritonitis, and dermatitis . Sporobolomyces salmonicolor, a rare human pathogen, was isolated and identified in tissue sections from affected fry. J Med Vet Mycol, 1996 Mar-Apr, 34(2), 145 - 7 Isolation of Malassezia sympodialis from feline skin; Bond R et al.; Carriage of Malassezia yeasts was investigated in 17 cats in two colonies using a lipid-supplemented culture medium . Malassezia pachydermatis was isolated from one cat . Lipid-dependent Malassezia yeasts with electrophoretic karyotypes consistent with M . sympodialis were isolated from all six cats in one group and from one of 11 in the second group . To our knowledge, this is the first report of the isolation of lipid-dependent yeasts from cats. J Mol Evol, 1996 Feb, 42(2), 321 - 2 CUG codons in Candida spp.; Jukes TH et al.; Codon CUG is used for serine instead of for leucine, its usual assignment, in several yeasts of the genus Candida . We propose a series of steps for the reassignment, including disappearance of leucine CUG and its anticodon CAG, formation of a new serine tRNA, with anticodon CAG, from a duplication of the gene for serine tRNA (IGA), and then production of CUG codons by mutation at sites that are mostly "nonessential." J Antimicrob Chemother, 1996 Feb, 37(2), 275 - 83 Relative growth measurement of Candida species in a single concentration of fluconazole predicts the clinical response to fluconazole in HIV infected patients with oral candidosis; Cartledge JD et al.; The growth of 113 Candida spp . isolated prospectively from 104 HIV-positive patients treated for thrush was determined using a single concentration of 3 mg/L (10(-5) M) fluconazole relative to growth in drug free medium . Using a receiver operator characteristic curve, a relative growth in fluconazole of > or = 88% best discriminated between isolates from 56 patients who responded to treatment with fluconazole and 48 who failed to respond to 7 days, treatment with at least 100 mg/d drug with a sensitivity of 98% and a specificity of 96% . When the isolates from the eight patients with mixed infections due to both resistant and susceptible yeasts were excluded, the sensitivity and specificity both reached 100% . Relative growth of Candida spp . in a single concentration of fluconazole can therefore be used to accurately predict the clinical response to standard fluconazole treatment of thrush in patients infected with HIV. Br J Oral Maxillofac Surg, 1996 Feb, 34(1), 23 - 5 A comparative study of the efficacy of fluconazole and amphotericin B in the treatment of oropharyngeal candidosis in patients undergoing radiotherapy for head and neck tumours; Finlay PM et al.; Radiotherapy given during treatment of oral and pharyngeal malignancy is frequently associated with colonization of the oral mucosa by Candida species . Treatment of these infections has included topical and systemic agents . In the present study 73 patients with oropharyngeal candidosis were treated with either amphotericin B (10 mg lozenges, four times daily for 14 days, 36 patients) or fluconazole (50 mg daily for 7 days, 37 patients) . The yeasts most frequently isolated were C albicans and C glabrata . Clinical signs and symptoms showed improvement at end of treatment in 72% of patients who received amphotericin B compared with 92% of patients who received fluconazole . Mycological cure at end of treatment was achieved in 31% of the amphotericin B group and 46% of patients who received fluconazole . For both treatments the cure rate was less in denture wearers than in non denture wearers. J Am Acad Dermatol, 1996 Feb, 34(2 Pt 1), 273 - 7 Present and potential diagnostic techniques in onychomycosis; Pierard GE et al.; The problem of onychomycosis has been frequently addressed during recent years . To make the diagnosis of onychomycosis dermatologists have relied on clinical presentation, culture, and microscopy . These approaches are hampered by false-negative and false-positive results that have confused treatment outcomes . Two new diagnostic techniques, immunohistochemistry and flow cytometry, provide an effective means of identifying different dermatophytes, yeasts, and nondermatophytic molds . Immunohistochemistry employs antibodies to certain fungi to enable positive identification in situ, whereas flow cytometry differentiates fungi on the basis of molecular differences . These techniques provide new evidence that nondermatophytic molds and yeasts can actively invade nail tissue and that mixed infections occur . These findings could have important implications for the treatment of onychomycosis. Arch Dermatol, 1996 Feb, 132(2), 190 - 3 Neonatal Malassezia furfur pustulosis; Rapelanoro R et al.; BACKGROUND: Papulopustular eruptions of the face in neonates are frequently referred to as neonatal acne or sebaceous miliaria . Our findings suggest that there is an association between this type of eruption and Malassezia furfur infection . OBSERVATIONS: Direct examination of pustule smears showed M furfur yeasts in eight of 13 cases involving neonates with erythema and papulopustules of the face, neck, and scalp (mean age at onset, 22 days {range, 7 to 30 days}) . The pustules were predominantly neutrophilic . Treatment with 2% ketoconazole cream applied topically twice daily was effective in 1 week . CONCLUSION: Malassezia furfur is frequently associated with a common nonfollicular pustulosis of the newborn, probably improperly termed neonatal acne. J Inorg Biochem, 1996 Feb, 61(2), 125 - 42 Ag(I)-binding to phytochelatins; Mehra RK et al.; Phytochelatins (PCs) are glutathione-derived peptides with the general structure (gamma-Glu-Cys)nGly, where n varies from 2 to 11 . A variety of metal ions such as Cu(II), Cd(II), Pb(II), Zn(II), and Ag(I) induce PC synthesis in plants and some yeasts . It has generally been assumed that the inducer metals also bind PCs . However, very little information is available on the binding of metals other than Cu(I) and Cd(II) to PCs . In this paper, we describe the Ag(I)-binding characteristics of PCs with the structure (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly, and (gamma-Glu-Cys)4Gly . The Ag(I)-binding stoichiometries of these three peptides were determined by (i) UV/VIS spectrophotometry, (ii) luminescence spectroscopy at 77 K, and (iii) reverse-phase HPLC . The three techniques yielded similar results . ApoPCs exhibit featureless absorption in the 220-340 nm range . The binding of Ag(I) to PCs induced the appearance of specific absorption shoulders . The titration end point was indicated by the flattening of the characteristic absorption shoulders . Similarly, luminescence at 77 K due to Ag(I)-thiolate clusters increased with the addition of graded Ag(I) equivalents . The luminescence declined when Ag(I) equivalents in excess of the saturating amounts were added to the peptides . At neutral pH, (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly, and (gamma-Glu-Cys)4Gly bind 1.0, 1.5, and 4.0 equivalents of Ag(I), respectively . The Ag(I)-binding capacity of (gamma-Glu-Cys)2Gly and (gamma-Glu-Cys)3Gly was increased at pH 5.0 and below so that Ag(I)/-SH ratio approached 1.0 . A similar pH-dependent binding of Ag(I) to glutathione was also observed . The increased Ag(I)-binding to PCs at lower pH is of physiological significance as these peptides accumulate in acidic vacuoles . We also report lifetime data on Ag(I)-PCs . The relatively long decay-times (approximately 0.1-0.3 msec) accompanied with a large Stokes shift in the emission band are indicative of spin-forbidden phosphorescence. Tsitologiia, 1996, 38(9), 889 - 913 {The structural and functional characteristics of the dolichol cycle enzymes}; Shpakov AO et al.; The literature and the authors' our data on the biosynthesis of a dolichol derivative Dol-PP-GlcNAc2Man9Glc3, involved in protein N-glycosylation in the endoplasmic reticulum (ER) of the eukaryotic cells, have been summarized and analysed . The structural and functional characteristics of dolichol-coupled enzymes, catalyzing biosynthesis of Dol-PP-GlcNAc2Man9Glc3, are considered . It is shown that the dolichol cycle enzymes, in conformity with their structural peculiarities and ER membrane topology, can be divided into three groups having the common evolutionary origin . Possible mechanisms of the dolichol derivative translocation through membrane is discussed . A conclusion is made about formation of multicomponent complexes by dolichol-coupled enzymes . These complexes secure functional co-ordination of the enzymes. Environ Mol Mutagen, 1996, 28(3), 176 - 81 Aneuploidy in germ cells: disruption of chromosome mover components; Preston RJ; The task of the Workgroup on "Disruption of Chromosome Mover Components" was to establish what cellular structures are involved in chromosome segregation and how disruption of these could occur . Recent research on the mechanism of action of the cellular components that segregate chromosomes accurately during mitosis or meiosis has served to highlight the number of potential targets for disruption . The process of chromosome segregation represents an orchestrated chain of events centered on the activities of cellular motors, kinesins and dyneins . These motors are involved in arranging chromosomes at the metaphase plate, providing the spindle tension necessary for progression, and the actual segregation of the chromosomes to the poles . The Workgroup determined that there is a lack of information on the effects of chemical exposure to cell motors and other chromosome mover components, and that there is a clear need for further research . This article describes the discussions of the Workgroup and highlights areas of future research into chromosome movement, particularly in human meiotic and mitotic cells . The Workgroup emphasized that obtaining mechanistic data on the induction of aneuploidy will allow for extrapolation of the dose response curves for chemical exposures below the level of observation and for using aneuploidy data for quantitative risk assessment for adverse health effects. Adv Enzyme Regul, 1996, 36, 3 - 15 Differences in the properties of mammalian ribonucleotide reductase toward its substrates; Cory JG et al.; These studies, using three different reagents, show that the substrate properties of ribonucleotide reductase are specific but can be variable depending upon the nature of the interaction of the reagent with the holoenzyme or the individual subunit . Etheno-CDP, which acts as a competitive inhibitor with respect to CDP, interacts with the active site of the holoenzyme . This interaction was the result of rather tight structural requirements as epsilon-ADP did not result in a similar level of inhibition of either CDP or ADP reductase activities . The YL 1/2 antibody which binds very tightly to the NHI subunit has a much greater effect on CDP reductase activity than ADP reductase activity . The nonapeptide that corresponds to the C-terminus amino acid sequence of the NHI subunit and which binds to the EB subunit and aborts the formation of the NHI-EB active complex has a greater effect on ADP reductase activity than on CDP reductase activity . The use of reagents such as these can be helpful in dissecting the subtle but important differences in the substrate properties of mammalian ribonucleotide reductase. EXS, 1996, 77, 307 - 20 Toxic metal-responsive gene transcription; Zhu Z et al.; Metals play a dual role in biological systems, serving as essential co-factors for a wide range of biochemical reactions yet these same metals may be extremely toxic to cells . To cope with the stress of increases in environmental metal concentrations, eukaryotic cells have developed sophisticated toxic metal sensing proteins which respond to elevations in metal concentrations . This signal is transmitted to stimulate the cellular transcriptional machinery to activate expression of metal detoxification and homeostasis genes . This review summarizes our current understanding of the biochemical and genetic mechanisms which underlie cellular responses to toxic metals via metalloregulatory transcription factors. Rapid Commun Mass Spectrom, 1996, 10(11), 1371 - 8 Delayed extraction improves specificity in database searches by matrix-assisted laser desorption/ionization peptide maps; Jensen ON et al.; Peptide mass maps obtained by matrix-assisted laser desorption ionization (MALDI) are an attractive means to identify proteins by searches in sequence databases . Here we demonstrate that the recently introduced delayed ion-extraction technique, when coupled to reflectron MALDI time-of-flight mass spectrometry, leads to dramatically improved search specificity . Routine resolution in the range of 6,000 to 12,000 allows assignment of monoisotopic masses throughout the peptide mass range . Database searches can be performed with high precision by use of a mass accuracy which is currently better than 30 ppm over a wide mass range and better than 5 ppm for a narrow mass range . This high performance makes it possible to identify proteins with fewer peptide masses than before . Additional low intensity peaks can be assigned after a search because of the improved signal-to-noise ratio of delayed-extraction peptide mass spectra, increasing sequence coverage of matched proteins . The improvements in database search specificity can be used to identify the components of simple protein mixtures . In combination with advanced sample preparation and automation techniques, delayed-extraction MALDI time-of-flight mass spectrometry is now an extremely powerful tool for the database identification of proteins. Ann Dermatol Venereol, 1996, 123(3), 157 - 64 {Allergologic survey in 251 patients with moderate or severe dermatitis . Incidence and value of the detection of contact eczema, food allergy or sensitization to air-borne allergens}; Guillet MH et al.; INTRODUCTION: Because of the increased recruitment of uncontrolled atopic dermatitis (AD) necessitating chronic use of dermocorticosteroids, we developed a prospective allergologic survey in a serie of 251 patients presenting with moderate or severe AD . PATIENTS AND METHOD: 251 patients were refered for allergologic assessment and followup . The clinical severity was assessed by use of standardized scores . Patients were grouped by age: group 1 (70 children younger than 2 years), group 2 (93 children between 2 and 7 years), group 3 (23 children between 7 and 15 years), group 4 (65 children over 15 years and adults) . All the patients were systematically screened for contact dermatitis and IgE mediated sensitization (inhallant and food allergens) with blood tests for IgE, prior to evaluation of clinical relevance . RESULTS: Aero-allergen sensitization was demonstrated in 51 p . 100 of children and 89 p . 100 of adults . It was present earlier in severe AD with main clinical involvement for nose and throat and respiratory symptoms . Clinical responsibility for dermatitis was documented in only 6 p . 100 of AD . Food allergy was early incriminated as flare factors in most of severe AD (96 p . 100 of children and 81 p . 100 of adults) with major and persistant improvement under eviction diet . Main allergens were eggs (46 p . 100), pea-nuts (29 p . 100), shellfish (24 p . 100), milk (20 p . 100), flour (14 p . 100), fish (14 p . 100), soybeans (8.9 p . 100) . Food allergy to yeasts (7.2 p . 100) was important in adults . Food allergy is the earliest allergy in the course of severe AD and the number of involved trophallergens increases in older patients . Patch tests were positive in 40 p . 100 of patients (i . e . 31 p . 100 of children and 66 p . 100 of adults) with a greater incidence in moderate AD . Main allergens were metals (54 p . 100), fragrances (19 p . 100), balsam of Peru (10 p . 100), parabens (8 p . 100) and lanoline (4 p . 100) . CONCLUSION: When AD is not efficiently controlled by dermocorticosteroids, allergologic screening and treatment of children and adults proves to be very interesting . Specific measures regarding food allergy and contact dermatitis reduce or vanish cutaneous flares . As for inhallant sensitizations, Dermatologists should be awared that they may play a role regarding assessment of sensitization and prevention of respiratory symptoms in moderate and severe AD since the risk of complications is important in both groups. J Ethnopharmacol, 1995 Dec 15, 49(3), 163 - 9 Antifungal activity of turmeric oil extracted from Curcuma longa (Zingiberaceae); Apisariyakul A et al.; Turmeric oil and curcumin, isolated from Curcuma longa L., were studied against fifteen isolates of dermatophytes, four isolates of pathogenic molds and six isolates of yeasts . The inhibitory activity of turmeric oil was tested in Trichophyton-induced dermatophytosis in guinea pigs . The results showed that all 15 isolates of dermatophytes could be inhibited by turmeric oil at dilutions of 1:40-1:320 . None of the isolates of dermatophytes were inhibited by curcumin . The other four isolates of pathogenic fungi were inhibited by turmeric oil at dilutions of 1:40-1:80 but none were inhibited by curcumin . All six isolates of yeasts tested proved to be insensitive to both turmeric oil and curcumin . In the experimental animals, turmeric oil (dilution 1:80) was applied by dermal application on the 7th day following dermatophytosis induction with Trichophyton rubrum . An improvement in lesions was observed in 2-5 days and the lesions disappeared 6-7 days after the application of turmeric oil. Curr Opin Cell Biol, 1995 Dec, 7(6), 781 - 9 The self-destructive personality of a cell cycle in transition; Deshaies RJ; The transition from G1 to S phase, sister chromatid separation in anaphase, and the exit from mitosis are driven by the destruction of cell cycle regulatory proteins by distinct ubiquitin-dependent proteolytic pathways . The components and targets of these key degradation pathways are now becoming clear . Genetic and biochemical dissections of these extremely specific and well regulated destruction pathways are providing fundamental insights into the mechanisms of control of the cell division cycle. Cell, 1995 Dec 1, 83(5), 683 - 92 Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins; Rexach M et al.; The molecular dynamics of nuclear protein import were examined in a solution binding assay by testing for interactions between a protein containing a nuclear localization signal (NLS), the transport factors karyopherin alpha, karyopherin beta, and Ran, and FXFG or GLFG repeat regions of nucleoporins . We found that karyopherins alpha and beta cooperate to bind FXFG but not GLFG repeat regions . Binding of the NLS protein to karyopherin alpha was enhanced by karyopherin beta . Two novel reactions were discovered . First, incubation of a karyopherin heterodimer-NLS protein complex with an FXFG repeat region stimulated the dissociation of the NLS protein from the karyopherin heterodimer . Second, incubation of the karyopherin heterodimer with RanGTP (or with a Ran mutant that cannot hydrolyze GTP) led to the dissociation of karyopherin alpha from beta and to an association of Ran with karyopherin beta; RanGDP had no effect . We propose that movement of NLS proteins across the nuclear pore complex is a stochastic process that operates via repeated association-dissociation reactions. FEBS Lett, 1995 Nov 27, 376(1-2), 91 - 4 Automation of micro-preparation and enzymatic cleavage of gel electrophoretically separated proteins; Houthaeve T et al.; To achieve high throughput, protein microcharacterization sample preparation must be automated . We describe a cartesian robot capable of processing 32 protein samples in parallel . The system is based on specially designed flow-through reactors for contamination-free reagent delivery and removal . Washing of excised gel pieces, reduction and alkylation, proteolytic cleavage and peptide extraction are performed in these reactors . Compatibility of the system with HPLC peptide separation and Edman degradation as well as with laser desorption mass spectrometry of the unseparated mixture is demonstrated . This is the first report describing automated preparation and processing of multiple protein samples. Mol Gen Genet, 1995 Nov 15, 249(2), 185 - 90 Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii; Remacle C et al.; The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies . In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent . In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur . Six mitochondrial mutants unable to grow in the dark (dk- mutants) were crossed in various combinations and the percentages of wild-type dk+ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome . In crosses between strains mutated in the COB (apocytochrome b) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (+/- 3.2%) . The corresponding physical distance between the mutation sites was 4.3 kb . In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (+/- 0.02%) was found . Two other mutants not yet characterized at the molecular level were also used for recombinational studies . From these data, a linear genetic map of the mitochondrial genome could be drawn . This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map . The frequency of recombination per physical distance unit (3.2% +/- 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas. Cell, 1995 Nov 3, 83(3), 423 - 32 Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion; Stenmark H et al.; We have identified a novel 100 kDa coiled-coil protein, rabaptin-5, that specifically interacts with the GTP form of the small GTPase Rab5, a potent regulator of endocytic transport . It is mainly cytosolic, but a fraction colocalizes with Rab5 to early endosomes . Expression of a GTPase-deficient Rab5 mutant enhances the binding of rabaptin-5 to enlarged endosomes . Overexpression of rabaptin-5 alone is sufficient to promote expansion of early endosomes . Rab5 recruits rabaptin-5 to purified early endosomes in a GTP-dependent manner, demonstrating functional similarities with other members of the Ras superfamily . Immunodepletion of rabaptin-5 from cytosol strongly inhibits Rab5-dependent early endosome fusion . Rabaptin-5 is thus a Rab effector required for membrane docking and fusion. Pediatr Allergy Immunol, 1995 Nov, 6(4), 181 - 6 Viable fungi in indoor air in homes and schools in the Sør-Varanger community during winter; Dotterud LK et al.; The present study investigated the content of fungal aerospores in homes and schools of house-dust-mite (HDM)-sensitized and control children in a subarctic area . During winter, airborne microfungi were collected from the homes and schools of 19 HDM-sensitized children and 19 nonatopic controls, all living in the community of Sor-Varanger, northern Norway . The samples were cultivated and microfungal growth was identified microscopically . Indoor humidity, temperature, and carbon dioxide (CO2) concentrations were measured . Housing conditions and sociodemographic and symptom data were obtained by a questionnaire . Penicillium was the most common microfungus in both homes and schools, followed by various yeasts, Aspergillus, Cladosporium, and Mucor . The number of infected homes was equal in the HDM-sensitized group and the control group, but aerospore counts were higher in the HDM-sensitized group than in the control group . The lowest aerospore counts were found in the schools . High aerospore counts also appeared to be related to high indoor humidity . The keeping of pets and damp indoor conditions were more frequent in homes of HDM-sensitized children than in the control group, whereas parental smoking and carpeting occurred with equal frequency in both groups . This indicates that no allergy sanitation measures had been undertaken, especially in the homes of the HDM-sensitized children. J Antibiot (Tokyo), 1995 Nov, 48(11), 1262 - 6 Chondramides A approximately D, new antifungal and cytostatic depsipeptides from Chondromyces crocatus (myxobacteria) . Production, physico-chemical and biological properties; Kunze B et al.; Novel depsipeptides, named chondramides were produced at levels up to 4.3 mg/liter by several myxobacteria of the genus Chondromyces . The compounds are structurally closely related to jaspamide/jasplakinolide from marine sponges of the genus Jaspis . Initially the chondramides were detected in acetone extracts of the biomass of Chondromyces crocatus, strain Cm c2 . So far, four structural variants could be characterized, the chondramides A approximately D . They inhibited the growth of a few yeasts and showed high cytostatic activity against cultivated human and animal cells. J Med Microbiol, 1995 Nov, 43(5), 360 - 7 An alpha 5 beta 1-like integrin receptor mediates the binding of less pathogenic Candida species to fibronectin; Santoni G et al.; The present study was undertaken to investigate whether less pathogenic Candida species (C . tropicalis, C . stellatoidea, C . krusei and C . glabrata) express a fibronectin receptor (FNr) antigenically related to alpha 5 beta 1 integrin, which mediates their binding to fibronectin (FN) . By flow cytometric analysis, a monoclonal antibody (MAb) directed against human alpha 5 integrin subunit (clone SAM-1) and two different antisera to FNr positively stained C . tropicalis, C . stellatoidea and C . glabrata, with the greatest expression observed for C . tropicalis . No or only marginal immunoreactivity was found on C . krusei . C . tropicalis, C . stellatoidea, C . glabrata, but not C . krusei yeasts specifically adhered to FN; higher levels of adhesion were found for C . tropicalis and C . stellatoidea with respect to C . glabrata . Less pathogenic Candida spp . bound to the Arg-Gly-Asp (RGD) containing 120-kDa fragment of FN and adhesion to intact FN was markedly inhibited by Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), but not by Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) peptides . In addition, anti-alpha 5 SAM-1 MAb and both anti-FNr antisera strongly blocked binding of less pathogenic Candida spp . to FN . Overall, these results indicate that less pathogenic Candida spp., including C . tropicalis, C . stellatoidea and C . glabrata, express a receptor antigenically related to alpha 5 beta 1 integrin which mediates their adhesion to FN. Biochemistry, 1995 Oct 24, 34(42), 13934 - 42 Probing intradomain and interdomain conformational changes during equilibrium unfolding of phosphoglycerate kinase: fluorescence and circular dichroism study of tryptophan mutants; Sherman MA et al.; Phosphoglycerate kinase is a monomeric protein composed of two globular domains of the alpha/beta type . Extensive domain-domain interactions involve three segments of the polypeptide chain that are distant from one another in the primary sequence: the N-terminus, the C-terminus, and a centrally located alpha-helix . In order to monitor spectroscopically the conformational changes that occur in the individual domains and at the interdomain interface during the unfolding process, we have constructed a series of single-tryptophan mutants . In addition to two previously described mutants, each with single tryptophans in the C-terminal domain (W308 and W333) {Szpikowska, B . K., Beechem, J . M., Sherman, M . A., & Mas, M . T . (1994) Biochemistry 33, 2217-2225}, four new single-tryptophan mutants have been constructed: two with tryptophans located in the interdomain region (W194 and W399) and two with tryptophans in the N-terminal domain (W48 and W122) . The equilibrium unfolding transitions induced by guanidine hydrochloride were monitored using far-UV CD, near-UV CD, steady-state, and time-resolved fluorescence . These studies reveal two unfolding transitions and suggest a sequential unfolding process for the mutants described in this paper . During the first transition (Cm approximately 0.5 M) the interdomain region and C-terminal domain unfold; the N-terminal domain remains relatively compact but lacks much of the tertiary structure that characterizes the native state . A hyperfluorescent intermediate is detected during this transition by tryptophan probes placed within the N-terminal domain . Complete unfolding of the N-terminal domain occurs during the second transition (Cm approximately 0.9 M). Trends Biochem Sci, 1995 Oct, 20(10), 405 - 11 Post-translational modification of poly(ADP-ribose) polymerase induced by DNA strand breaks; Lindahl T et al.; There are one million molecules of poly(ADP-ribose) polymerase (PARP) in mammalian cell nuclei and the enzyme is found in most eukaryotes, with the notable exception of yeasts . In response to DNA damage caused by ionizing radiation or alkylating agents, PARP binds to strand interruptions in DNA and undergoes rapid automodification with synthesis of long branched polymers of highly negatively charged poly(ADP-ribose) . DNA repair occurs after dissociation of modified PARP from DNA strand breaks . Biochemical data with enzyme-depleted extracts and studies of enzyme-deficient mice show that PARP does not participate directly in DNA repair . Possible roles for poly(ADP-ribose) synthesis are discussed. J Mol Evol, 1995 Oct, 41(4), 407 - 13 Molecular and phylogenetic analysis of PCR-amplified cyclin-dependent kinase (CDK) family sequences from representatives of the earliest available lineages of eukaryotes; Riley DE et al.; Cyclin-dependent kinase (CDK) and cell division control (CDC2) sequences are strongly conserved among eukaryotes and may complement the use of other sequence families in eukaryotic phylogenetic inference . We synthesized degenerate PCR primers to amplify the catalytic region of CDK homologs in representatives of the earliest available lineages of eukaryotes . CDK family sequence-based, maximum-likelihood distance measurements with neighbor-joining, and Fitch-Margoliash least-squares analyses produced unrooted dendrograms that included protists, yeasts, and higher eukaryotes . Bootstrap confidence estimates supported CDK-based phylogenetic groupings among the protists, fungi, and vertebrates although resolution within these groups was often insignificant . However, Trichomonas vaginalis and Giardia lamblia exhibited two of the most div