J Mol Biol, 2000 Nov 17, 304(1), 21 - 33 Two DNA-binding sites on the globular domain of histone H5 are required for binding to both bulk and 5 S reconstituted nucleosomes; Duggan MM et al.; We have previously shown the existence of two DNA-binding sites on the globular domain of H5 (termed GH5), both of which are required for nucleosome organisation, as judged by the protection of a 166 bp chromatosome intermediate during micrococcal nuclease digestion of chromatin . This supports a model in which GH5 contacts two duplexes on the nucleosome . However, studies of a nucleosome assembled on the 5 S rRNA gene have argued against the requirement for two DNA-binding sites for chromatosome protection, which has implications for the role of linker histones . We have used this proposed difference in the requirement for a second site on the globular domain in the two models as a means of investigating whether bulk and reconstituted 5 S nucleosomes are indeed fundamentally different . GH5 protects a 166 bp chromatosome in both "bulk" and 5 S systems, and in both cases protection is abolished when all four basic residues in site II are replaced by alanine . Binding to four-way DNA junctions, which present a pair of juxtaposed duplexes, is also abolished . Single mutations of the basic residues did not abolish chromatosome protection in either system, or binding to four-way junctions, suggesting that the residues function as a cluster . Both bulk and 5 S nucleosomes thus require a functional second DNA-binding site on GH5 in order to bind properly to the nucleosome . This is likely to reflect a similar mode of binding in each case, in which two DNA duplexes are contacted in the nucleosome . There is no indication from these experiments that linker histones bind fundamentally differently to 5 S and bulk nucleosomes . J Biol Chem, 2001 Feb 2, 276(5), 3635 - 40 Epub 2000 Nov 02. Core histone acetylation is regulated by linker histone stoichiometry in vivo; Gunjan A et al.; We investigated the relationship between linker histone stoichiometry and the acetylation of core histones in vivo . Exponentially growing cell lines induced to overproduce either of two H1 variants, H1(0) or H1c, displayed significantly reduced rates of incorporation of {(3)H}acetate into all four core histones . Pulse-chase experiments indicated that the rates of histone deacetylation were similar in all cell lines . These effects were also observed in nuclei isolated from these cells upon labeling with {(3)H}acetyl-CoA . Nuclear extracts prepared from control and H1-overexpressing cell lines displayed similar levels of histone acetylation activity on chromatin templates prepared from control cells . In contrast, extracts prepared from control cells were significantly less active on chromatin templates prepared from H1-overexpressing cells than on templates prepared from control cells . Reduced levels of acetylation in H1-overproducing cell lines do not appear to depend on higher order chromatin structure, because it persists even after digestion of the chromatin with micrococcal nuclease . The results suggest that alterations in chromatin structure, resulting from changes in linker histone stoichiometry may modulate the levels or rates of core histone acetylation in vivo. Am J Vet Res, 2000 Oct, 61(10), 1294 - 7 Detection of lysozyme in llama, sheep, and cattle tears; Gionfriddo JR et al.; OBJECTIVE: To determine whether the tears of llamas, sheep, and cattle contain lysozyme and compare lysozyme concentrations in tears among these species . ANIMALS: 40 llamas, 5 sheep, and 36 cattle . PROCEDURE: Electrophoresis, western blot immunoassay for lysozyme, a spectrophotometric assay to detect tear lysozyme by its ability to lyse a suspension of Micrococcus lysodeiticus, and a microtiter plate colorometric assay were performed . RESULTS: A 13.6-kd protein band was detected by use of electrophoresis and western blot immunoassay in llama and sheep tears but not cattle tears . Results of spectrophotometric assay suggested that llama and sheep tears had high concentrations of lysozyme, whereas cattle tears had low concentrations . Results of the microtiter plate colorometric assay suggested that llama tears had high concentrations of lysozyme, whereas concentrations in sheep and cattle tears were lower . CONCLUSIONS AND CLINICAL RELEVANCE: Lysozyme concentrations in tears may vary among species and this variability may contribute to differing susceptibilities to ocular diseases such as infectious keratoconjunctivitis. FEBS Lett, 2000 Sep 1, 480(2-3), 208 - 12 In vitro nuclear reconstitution could be induced in a plant cell-free system; Zhao Y et al.; A cell-free system derived from carrot cell cytosol extract has been developed for reassembling nuclear structure around the added demembranated sperm chromatin of Xenopus . Morphological evidence suggests that reassembled nuclei display the typical characteristics of normal eukaryotic nuclei, such as double-layered nuclear membrane and nuclear pores . Micrococcal nuclease treatment indicates that remodeling of the demembranated sperm chromatin has occurred and the structure of nucleosome is formed during nuclear reconstitution . These data indicate that the nuclear reconstitution can be induced in cell-free systems from plants, and the self-assembly of the nucleus is ubiquitous in both animal and plant cells. Chromosome Res, 2000, 8(6), 527 - 41 Comparative analysis of DNA methylation in tobacco heterochromatic sequences; Kovarik A et al.; Cytosine methylation levels and susceptibility to drug-induced hypomethylation have been studied in several Nicotiana tabacum (tobacco) DNA repetitive sequences . It has been shown using HapII, MspI, BamHI and Sau3AI methylation-sensitive restriction enzymes that the degree of 5'-mCmCG-3' methylation varied significantly between different repeats . There were almost saturation levels of 5-methylcytosine at the inner (3') cytosine position and variable degrees of methylation at the outer (5') cytosine at the enzyme recognition sites . The non-transcribed high copy satellite sequences (HRS60, GRS) displayed significant heterogeneity in methylation of their basic units while middle repetitive sequences (R8.1, GRD5, 5S rDNA) were more uniformly modified at both cytosine residues . Dihydroxypropyladenine (DHPA) treatment, which is thought to reduce DNA methyltransferase activity by increasing S-adenosylhomocysteine levels, resulted in extensive demethylation of the outer cytosine in all repeats, and the partial hypomethylation of cytosines at the inner positions in less densely methylated repeats such as HRS60 and GRS . The results suggest that hypomethylation of 5'-mCmCG-3' sites with DHPA is a gradual non-random process proceeding in the direction mCmCG-->CmCG-->CCG . The 18S-5.8S-25S rDNA was remarkably hypomethylated relative to the 5S rDNA at all restriction sites studied . Fluorescence in-situ hybridization showed that DNA decondensation within and between the 18S-5.8S-25S and 5S rDNA loci was variable in different nuclei . All nuclei had condensed and decondensed sequence . The chromatin of 18S-5.8S-25S rDNA was more readily digested with micrococcal nuclease than the 5S rDNA suggesting that the overall levels of decondensation were higher for 18S-5.8S-25S rDNA . Variable decondensation patterns within and between loci were also observed for GRS and HRS60 . Cytosine methylation of the tobacco repeats is discussed with respect to transcription, overall levels of condensation and overall structure. J Microbiol Methods, 2000 Oct, 42(2), 159 - 65 A sensitive standardised micro-gel well diffusion assay for the determination of antimicrobial activity; du Toit EA et al.; We have developed a highly sensitive micro-gel well diffusion assay for the determination of antimicrobial activity . In essence, the normal radial diffusion type assay was adapted to perform it in a microtiter plate . We compared our micro-gel well diffusion assay to a radial diffusion assay and a microtiter broth dilution method, using gramicidin S as model antibiotic, and Micrococcus luteus as the indicator organism . The micro-gel well diffusion assay was as sensitive as the microtiter broth dilution method, and approximately twice as sensitive as the radial diffusion method . Data analysis to calculate minimum inhibitory concentration, 50% microbial growth inhibition and maximum inhibitory concentration was refined by generating dose-response curves with the software package Prism 3.0 (Graphpad Software Inc.) . The minimum inhibitory concentrations, determined by the three methods, were significantly different (P<0 . 001), highlighting the limitations involved in comparing data obtained from different methods. Int J Food Microbiol, 2000 Sep 25, 60(2-3), 147 - 52 Effects of combined exposure of micrococcus luteus to nisin and pulsed electric fields; Dutreux N et al.; Death and injury following exposure of Micrococcus luteus to nisin and pulsed electric field (PEF) treatment were investigated in phosphate buffer (pH 6.8, sigma = 4.8 ms/cm at 20 degrees C) . Four types of experiment were carried out, a single treatment with nisin (100 IU/ml at 20 degrees C for 2 h), a single PEF treatment, a PEF treatment followed by incubation with nisin (as before) and addition of nisin to the bacterial suspension prior to the PEF treatment . The application of nisin clearly enhanced the lethal effect of PEF treatment . The bactericidal effect of nisin reduced viable counts by 1.4 log10 units . Treatment with PEF (50 pulses at 33 kV/cm) resulted in a reduction of 2.4 log10 units . PEF treatment followed by nisin caused a reduction of 5.2 log10 units in comparison with a 4.9 log10 units reduction obtained with nisin followed by PEF . Injury of surviving cells was investigated using media with different concentrations of salt . Sublethally damaged cells of M . luteus could not be detected by this means, following PEF treatment. J Biochem (Tokyo), 2000 Oct, 128(4), 575 - 84 Genetic characterization of rbt mutants that enhance basal transcription from core promoters in Saccharomyces cerevisiae; Kunoh T et al.; While this Saccharomyces cerevisiae SIN4 gene product is a component of a mediator complex associated with RNA polymerase II, various studies suggest the involvement of Sin4 in the alteration of higher-order chromatin structure . Our previous analysis of a sin4 mutant suggested that the mechanisms of transcriptional repression by Sin4 (mediator) and the Tup1-Ssn6 complex (general repressor) are different . To elucidate the way in which these two repression systems are interrelated, we isolated mutants that exhibit enhanced transcription of a reporter gene harboring the upstream activation sequence (UAS), but still are subject to Tup1-Ssn6-mediated repression . Besides sin4, rgr1, tup1, and ssn6 mutants, we also obtained new mutants that enhance basal transcription even from a core promoter without UAS . Such mutants, designated rbt for regulator of basal transcription, can be classified into at least six complementation groups, i.e., four single (rbt1 to rbt4) and two apparently double (rbt5 rbt6 and rbt7 rbt8) mutations . The phenotype of rbt mutants is dependent on the TATA box and not specific to the integration site or kind of core promoter . No significant difference in micrococcal nuclease (MNase) accessibility to the core promoter of test genes was observed between rbt mutants and the wild-type strain, indicating that the higher-order chromatin structure of the core promoter region is not significantly altered in these mutants . The rbt1 to rbt4 mutations are suppressed by the Dgal11 mutation as in the case of the sin4 mutation, but give rise to a different profile from the sin4 mutation with regard to the activity of some of the promoters . From these observations, we suggest that RBT gene product(s) could be novel mediators that act with or in close association with Sin4 but have a function distinct from that of Sin4 . Moreover, the fact that rbt mutations nullify Tup1-Ssn6 general repressor-mediated repression is consistent with the idea that the mechanisms of Rbt (mediator)- and Tup1-Ssn6 (general repressor)-mediated repression are interconnected but substantially different. Biochem Biophys Res Commun, 2000 Sep 16, 276(1), 57 - 63 Inorganic Polyphosphate/ATP-NAD kinase of Micrococcus flavus and Mycobacterium tuberculosis H37Rv; Kawai S et al.; An enzyme with both inorganic polyphosphate {poly(P)}- and ATP-dependent NAD kinase activities was isolated from Micrococcus flavus . The enzyme was a dimer consisting of 34 kDa subunits, and was named poly(P)/ATP-NAD kinase . Internal amino acid sequences of the enzyme showed homologies with some function-unknown proteins released on the GenBank database . Among such proteins, hypothetical Rv1695 protein (Accession No . Z98268-16), which was encoded by a gene named "Rv1695" on genomic DNA of Mycobacterium tuberculosis H37Rv, was proposed to be poly(P)-dependent NAD kinase . By cloning and expression in Escherichia coli, Rv1695 was shown to encode poly(P)/ATP-NAD kinase and named ppnk . The ppnk product, recombinant-poly(P)/ATP-NAD kinase (Ppnk) was purified and characterized . The enzyme was a tetramaer consisting of 35 kDa subunits when expressed in E . coli . Poly(P)/ATP-NAD kinases of M . flavus and Ppnk of M . tuberculosis H37Rv specifically and completely phosphorylated NAD by utilizing commercially available poly(P)s and nucleoside triphosphates as phosphoryl donors . Arch Androl, 2000 Jul-Aug, 45(1), 61 - 71 Participation of DNA structure on sperm chromatin organization; Fuentes-Mascorro G et al.; The in vitro interaction between purified bovine liver and sperm DNA with somatic histones, to form nucleosomes, and with bovine and salmon protamines were studied . DNAse or microccocal nuclease digestion of liver DNA-histone reassociated chromatin produced the expected polynucleosome type of fragments . Electrophoretic patterns of digested sperm-DNA nucleosomes were different . Micrococcal nuclease digestion produced mainly fragments smaller than 100 bp and some nucleosome-type particles . Under DNAse activity most of the products were smaller than 100 bp, indicating an increased susceptibility of the sperm DNA-histone complexes to the hydrolytic activity of both nucleases, particularly toward DNAse I . This differential susceptibility was confirmed by sucrose gradient spectrophotometric analysis . Acridine orange (AO) staining of histone-DNA reassociated nucleosomes showed significant differences in fluorescence intensity, sperm DNA-histone complexes being almost twice as fluorescent as liver DNA-histone complexes . On the contrary, liver DNA/protamine complexes stained with AO were consistently more fluorescent than sperm DNA-protamine complexes . Finally, no differences in either fluorescence intensity or spectra were observed when liver and sperm DNA were stained with AO after interaction with salmon protamines . The data suggest that sperm DNA has important structural characteristics that differentiates it from somatic DNA . These differences seem to be species specific and must surely play an important role on the determination of the dramatic sequence of that participates sperm chromatin organization. Nucleic Acids Res, 2000 Sep 1, 28(17), 3346 - 53 Levels of free PABP are limited by newly polyadenylated mRNA in early Spisula embryogenesis; de Melo Neto OP et al.; The poly(A) tail of eukaryotic mRNAs regulates translation and RNA stability through an association with the poly(A)-binding protein (PABP) . The role of PABP in selective polyadenylation/deadenylation and translational recruitment/repression of maternal mRNAs that occurs in early development is not fully understood . Here, we report studies including UV-crosslinking and immunoblotting assays to characterise PABP in the early developmental stages of the clam Spisula solidissima . A single, 70 kDa PABP, whose sequence is highly homologous to vertebrate, yeast and plant PABPs, is detected in oocytes . The levels of clam PABP are constant in early embryogenesis, although its ability to crosslink labelled poly(A) is 'masked' shortly after fertilisation and remains so until the larval stage . Full RNA-binding potential of PABP in embryo lysates was achieved by brief denaturation with guanidinium hydrochloride followed by dilution for binding and crosslinking or by controlled treatment of lysates with Ca(2+)-dependent micrococcal nuclease . Masking of PABP, which accompanies cytoplasmic polyadenylation in maturing oocytes and in in vitro activated oocyte lysates, is very likely due to an association with mRNAs that bear new PABP target binding sites and thus prevent protein binding to the labelled A-rich probe . Functional implications of these findings as well as the potential application of this unmasking method to other RNA-binding proteins is discussed. Mol Gen Genet, 2000 Jul, 263(6), 966 - 77 The role of ABC transporters from Aspergillus nidulans in protection against cytotoxic agents and in antibiotic production; Andrade AC et al.; This paper describes the characterization of atrC and atrD (ABC transporters C and D), two novel ABC transporter-encoding genes from the filamentous fungus Aspergillus nidulans, and provides evidence for the involvement of atrD in multidrug transport and antibiotic production . BLAST analysis of the deduced amino acid sequences of AtrCp and AtrDp reveals high homology to ABC transporter proteins of the P-glycoprotein cluster . AtrDp shows a particularly high degree of identity to the amino acid sequence of Afu Mdr1p, a previously characterized ABC transporter from the human pathogen A . fumigatus . Northern analysis demonstrates an increase in transcript levels of atrC and atrD in fungal germlings upon treatment with natural toxic compounds and xenobiotics . The atrC gene has a high constitutive level of expression relative to attrD, which suggests its involvement in a metabolic function . Single knock-out mutants for atrC and atrD were generated by gene replacement using pyrG from A . oryzae as a selectable marker . DeltatrD mutants display a hypersensitive phenotype to compounds such as cycloheximide, the cyclosporin derivative PSC 833, nigericin and valinomycin, indicating that AtrDp is involved in protection against cytotoxic compounds . Energy-dependent efflux of the azole-related fungicide fenarimol is inhibited by substrates of AtrDp (e.g . PSC 833, nigericin and valinomycin), suggesting that AtrDp plays a role in efflux of this fungicide . Most interestingly, (delta)atrD mutants display a decrease in penicillin production, measured indirectly as antimicrobial activity against Micrococcus luteus . These results suggest that ABC transporters may be involved in secretion of penicillin from fungal cells. J Biol Chem, 2000 Oct 27, 275(43), 33336 - 45 Regulation of c-myc mRNA decay in vitro by a phorbol ester-inducible, ribosome-associated component in differentiating megakaryoblasts; Brewer G; The K562 leukemia cell line is bipotential for erythroid and megakaryoblastic differentiation . The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) activates a genetic program of gene expression in these cells leading to their differentiation into megakaryoblasts, a platelet precursor . Thus, K562 cells offer a means to examine early changes in gene expression necessary for megakaryoblastic commitment and differentiation . An essential requirement for differentiation of many hematopoietic cell types is the down-regulation of c-myc expression, because its constitutive expression blocks differentiation . TPA-induced differentiation of K562 cells causes rapid down-regulation of c-myc expression, due in part to an mRNA decay rate that is 4-fold faster compared with dividing cells . A cell-free mRNA decay system reconstitutes TPA-induced destabilization of c-myc mRNA, but it requires at least two components for reconstitution . One component fractionates to the post-ribosomal supernatant from either untreated or treated cells . This component is sensitive to cycloheximide and micrococcal nuclease . The other component is polysome-associated and is induced or activated by TPA . Although in dividing cells c-myc mRNA decays via a sequential pathway involving removal of the poly(A) tract followed by degradation of the mRNA body, TPA activates a deadenylation-independent pathway . The cell-free mRNA decay system reconstitutes this alternate decay pathway as well. Ophthalmic Surg Lasers, 1999 Jul-Aug, 30(7), 540 - 6 Open drops in ophthalmology offices: expiration and contamination; Wessels IF et al.; OBJECTIVE: To determine the relationship between eye drop use and contamination rate in ophthalmology offices . DESIGN: Following permission request, open bottles were examined and the nozzle tip and one drop of content was cultured on solid media . OUTCOME MEASURES: Drug category, volume, weight compared to full, clean legible label, expiration date; 2 or more bacterial colonies along the inoculation site . RESULTS: In 18 offices, of 1,485 open bottles (mean 12.2, range 4 to 23 per lane) on average 19.8% (range 0% to 88%) were expired (16.2 of 82.5 bottles per office) . The frequency of occurrence (%) and expiration (%E) were 40.3% cycloplegics (19.4%E); 16.4% glaucoma (33.7%E); 10.8% anesthetics (8.8%E); and 4% steroids (8.8%E; or 42.2%E including one outlier) . Most likely expired were glaucoma (P < 0.001); small 2-3 ml (P < 0.02), nearly empty (P < 0.05), or dirty (P < 0.001) bottles . Only one (5 ml cyclopentolate, not expired) grew a Micrococcus (0.07%) . CONCLUSIONS: Drops in ophthalmology offices may be expired but are not contaminated. Artif Organs, 2000 Jul, 24(7), 577 - 80 Adsorption of microorganism components by lixelle beads; Tsuchida K et al.; We reported previously that Lixelle, which was used for beta-2 microglobulin (BMG) adsorption columns, could adsorb not only BMG but also inflammatory cytokines . We then were interested in the application of Lixelle to patients with systemic inflammatory response syndrome (SIRS) and tried to find out its ability to adsorb microorganism components in vitro using lipopolysaccharide (LPS) (E . coli: B8), endotoxin (ET) containing water, and peptidoglycan (PG: Micrococcus luteus) . The initial concentrations of each solution were LPS (ET: 29,135 EU/L), contaminated water (ET: 3,523 EU/L), and PG (67.1 ng/ml) and 2.5 ml of each of the stock solutions and adjusted diluted solutions contained 0.5 ml of Lixelle beads . After shaking at 37 degrees C for 2 h, ET in the solutions was determined by the ET specific-limulus amebocyte lysate (ES-LAL) method and PG by the silkworm larbae plasma (SLP) method . The results revealed that even when ET concentrations in LPS and contaminated water were high, the samples containing Lixelle beads showed significant decreases . There was some adsorption of PG but no significant differences . Thus, Lixelle beads can adsorb not only BMG but also microorganism components such as ET and PG . These findings, together with the ability to adsorb inflammatory cytokines by Lixelle, show the possibility of application for the treatment of infectious SIRS. Mayo Clin Proc, 2000 Jul, 75(7), 705 - 8 Effects of 4 hand-drying methods for removing bacteria from washed hands: a randomized trial; Gustafson DR et al.; OBJECTIVE: To evaluate the effects of 4 different drying methods to remove bacteria from washed hands . SUBJECTS AND METHODS: One hundred adult volunteers participated in this randomized prospective study . All bacterial counts were determined using a modified glove-juice sampling procedure . The difference was determined between the amounts of bacteria on hands artificially contaminated with the bacterium Micrococcus luteus before washing with a nonantibacterial soap and after drying by 4 different methods (cloth towels accessed by a rotary dispenser, paper towels from a stack on the hand-washing sink, warm forced air from a mechanical hand-activated dryer, and spontaneous room air evaporation) . The results were analyzed using a nonparametric analysis (the Friedman test) . By this method, changes in bacterial colony-forming unit values for each drying method were ranked for each subject . RESULTS: The results for 99 subjects were evaluable . No statistically significant differences were noted in the numbers of colony-forming units for each drying method (P = .72) . CONCLUSION: These data demonstrate no statistically significant differences in the efficiency of 4 different hand-drying methods for removing bacteria from washed hands. Klin Lab Diagn, 2000 Jun, (6), 13 - 5 {Measurements of salivary lysozyme activity}; Storozhuk PG et al.; A new method for measuring salivary lysozyme is proposed, which is based on lysozyme capacity to lyze glycosaminoglycanes and proteoglycanes in plasma membranes of Micrococcus lysodeikticus . Specific activity of the enzyme is identified from the amount of protein in glycosaminoglycanes and proteoglycanes . The quantity of glycosaminoglycanes and proteoglycanes containing 0.001 mg protein/ml/min is suggested to be takes as a unit of lysozyme. J Biol Chem, 2000 Oct 6, 275(40), 31347 - 52 Multiple histone acetyltransferases are associated with a chicken erythrocyte chromatin fraction enriched in active genes; Hebbes TR et al.; We have examined salt-soluble chromatin released by micrococcal nuclease from a 15-day-old chicken embryo erythrocyte nuclei for histone acetyltransferase (HAT) activities . This chromatin is enriched in transcriptionally active sequences from within the active beta-globin locus and contains elevated levels of acetylated core histones . HAT activities present in this fraction target histones H4, H3, and H2A when the chromatin itself is used as the substrate . In gel HAT activity assay demonstrates that the salt-soluble chromatin fraction contains four acetyltransferase molecules distinguished by their different molecular masses (47, 33, 32, and 28 kDa) . Further separation of the chromatin by centrifugation through sucrose gradients shows that the acetyltransferases segregate into chromatin-bound and chromatin-free populations . The 32- and 28-kDa HATs are associated with chromatin, whereas the 47- and 33-kDa HAT molecules are not . The chromatin-bound HAT activities predominantly target H4 to give the diacetyl and triacetyl species; some acetylation of H2A can also be seen . Our results suggest that the chromatin-associated acetyltransferases have a role in gene regulation. Inflamm Res, 2000 May, 49(5), 214 - 23 Nutritional supplementation with copper in the rat . I . Effects on adjuvant arthritis development and on some in vivo- and ex vivo-markers of blood neutrophils; Milanino R et al.; OBJECTIVE AND DESIGN: The aims of the work were: 1) to confirm the preliminarily observed anti-arthritic potential of a 200 ppm copper-supplemented diet in the rat: 2) to study the impact of the nutritional treatment and of the experimental pathology on neutrophil activity . ANIMALS AND CELLS: Two hundred female Sprague-Dawley rats were used . Polymorphonuclear leukocytes were isolated from these animals for the ex vivo studies . TREATMENT: Control-rats were maintained on a standard diet containing 5 ppm of copper . Supplemented-rats were kept on a diet containing 200 ppm of the metal . METHODS: Mycobacterium butyricum-induced arthritis was studied . Flame atomic absorption spectroscopy was used to assess copper and zinc levels . The "microplate-assay" technique was used to determine serum lysozyme concentration (lysis of Micrococcus lysodeikticus cell walls), as well as neutrophil O2- generation (superoxide dismutase-inhibitable reduction of ferricytochrome-c), and adhesion (activity of the membrane enzyme acid phosphatase) . The results were statistically evaluated by the Student's t test . RESULTS: The nutritional copper-supplementation: 1) significantly inhibited the adjuvant-arthritis development (33% +/- 5, P<0.01); 2) did not modify lysozyme secretion or superoxide production; 3) significantly decreased the percentage of cell adhesion by an average of 41% +/- 19 (P<0.01) . CONCLUSIONS: The copper-supplemented diet has an anti-arthritic effect which may be also primed by the effect of copper on the expression of the neutrophil cell-adhesion molecules. Genome Res, 2000 Jun, 10(6), 874 - 86 A human genomic library enriched in transcriptionally active sequences (aDNA library); Pelling AL et al.; Core histone hyperacetylation, in particular of H4, is concentrated in the promoter-upstream regions of active genes and in certain cases is locuswide . Antibodies to hyperacetylated H4 were used to immunoprecipitate dinucleosomal chromatin derived from K562 human erythroleukemic cells by micrococcal nuclease digestion . The extracted DNA was made into a genomic library and was expected to contain sequences from genes active in K562 cells (an active, 'aDNA' library) . Clones (180) were randomly selected from the library; 24 of 103 tested (23%) contained highly repeated sequences, as determined by their hybridization to total genomic DNA, and were not analyzed further . An additional 10 clones (6%) were shown to contain no insert DNA . The remaining 146 were sequenced and compared with the nucleic acid databases and in all six frames to the protein databases: Sixeen clones could be assigned to known genes, the majority of which (12) were tissue specific . All but 2 of these 16 corresponded to segments 5' of the coding sequences, as expected if H4 acetylation is concentrated at promoter regions . Thirty-three clones (23%) displayed high sequence identity to cDNAs in the expressed sequence tag database (dbEST) . Northern blots and reverse transcription (RT)-PCR were used to determine the proportion of clones representing sequences expressed in K562 cells: Although only 1 of 34 tested clones showed a band in Northern hybridization, RT-PCR demonstrated that at least 12 of 40 tested clones (30%) were present in the mRNA population . Because a further 8 of these 40 clones were identified as gene fragments by database sequence comparisons, it follows that about half of this subset of 40 clones is derived from genes . The aDNA library is thus very gene rich and not skewed toward the most highly expressed sequences, as in mRNA libraries . The aDNA library is also rich in promoters and could be a valuable source of such sequences, particularly those that lack CpG islands or other features that allow their specific selection. Microbiology, 2000 Jun, 146 ( Pt 6), 1295 - 310 Phylogenetic and physiological diversity of Arthrobacter strains isolated from unconsolidated subsurface sediments; Crocker FH et al.; Forty strains of Gram-positive, aerobic, heterotrophic bacteria isolated from saturated subsurface lacustrine, paleosol and fluvial sediments at the US Department of Energy's Hanford Site in south central Washington State were characterized by phylogenetic analysis of 16S rRNA gene sequences and by determination of selected morphological, physiological and biochemical traits . Phylogenetic analyses of 16S rDNA sequences from subsurface isolates in the context of similar sequences from previously described bacterial species indicated that 38 of the subsurface strains were most closely related to Arthrobacter: The other two strains appeared to be most closely related to Kocuria . The subsurface isolates fell into seven phylogenetically coherent and distinct clusters, indicating that there was a significant degree of diversity among them . Additional diversity was detected by analysis of cellular fatty acids and physiological traits . The general morphological, physiological and biochemical traits of the subsurface strains were consistent with those of Arthrobacter, Micrococcus and genera recently separated from Micrococcus, such as Kocuria . Some of the subsurface strains were phylogenetically closely related to certain species of Arthrobacter . (16S rDNA sequence similarities >99%) . However, most of the subsurface isolates did not cluster with previously established species in phylogenetic analyses of 16S rRNA gene sequences or with hierarchical cluster analysis of cellular fatty acid profiles . Moreover, many of the subsurface isolates that were most closely related to Arthrobacter . also differed from all established species of that genus in several of their specific physiological characteristics . Most of the subsurface isolates, then, are likely to be novel strains or species of Arthrobacter. Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 1247 - 51 Characterization of a Rothia-like organism from a mouse: description of Rothia nasimurium sp . nov . and reclassification of Stomatococcus mucilaginosus as Rothia mucilaginosa comb . nov; Collins MD et al.; An unknown, Gram-positive, ovoid-shaped bacterium isolated from the nose of a mouse was subjected to a polyphasic taxonomic analysis . Comparative 16S rRNA gene sequencing demonstrated that the unknown organism was a member of the family Micrococcaceae and possessed a specific phylogenetic association with Rothia dentocariosa and Stomatococcus mucilaginosus . Phenotypically, the bacterium closely resembled R . dentocariosa and S . mucilaginosus but could be distinguished from these species by biochemical tests and electrophoretic analysis of whole-cell proteins . Based on both phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified in the genus Rothia, as Rothia nasimurium sp . nov . In addition, it is proposed that S . mucilaginosus be reclassified in the genus Rothia, as Rothia mucilaginosa comb . nov. Org Lett, 1999 Dec 2, 1(11), 1843 - 6 Synthesis of the Bycroft-Gowland structure of micrococcin P1; Ciufolini MA et al.; {formula: see text} We describe the chemical synthesis of the accepted structure of micrococcin P1, a member of the thiostrepton group of antibiotics, and we show that this architecture does not correspond to that of the natural product . Methods developed during the present study should greatly facilitate ongoing efforts centering on the determination of the actual structure of microccin P1, in addition to being applicable to the synthesis of more complex thiostrepton congeners. Biosci Biotechnol Biochem, 2000 Apr, 64(4), 767 - 74 Some properties of a macromolecular conjugate of lysozyme prepared by modification with a monomethoxypolyethylene glycol derivative; Nodake Y et al.; Hen egg-white lysozyme was modified with a succinyl ester derivative of monomethoxypolyethylene glycol (mPEG-COONSu), and some properties of the resulting conjugate (mPEG-lysozyme) were studied . The conjugate was prepared by modification of lysozyme with mPEG-COONSu and purified with use of columns of CM-Toyopearl 650M and Sephadex G-75 . Analytical data indicated that in the conjugate, 1.05 moles of mPEG with an average molecular weight of 5,000 were covalently attached to the lysozyme molecule . Tryptic peptide analysis of the conjugate showed that Lys 33 in lysozyme is the residue mainly modified with mPEG-COONSu . Covalent attachment of the mPEG-derivative to amino groups greatly increased the thermostability of lysozyme without any conformational change of the protein molecule . mPEG-lysozyme retained full enzyme activity for glycol chitin, but lytic activity for Micrococcus luteus cells in neutral media was 75% of that of native lysozyme and its optimal pH was at pH 5.0 . It was also found that the reactivity of lysozyme with anti-lysozyme antibody from BALB/c mice or human lymphocytes was decreased by modification with mPEG-COONSu . From these findings, it was suggested that mPEG-COONSu can be advantageously used for protein tailoring of lysozyme. Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 355 - 63 Frigoribacterium faeni gen . nov., sp . nov., a novel psychrophilic genus of the family Microbacteriaceae; Kampfer P et al.; The taxonomic position of five actinobacterial strains isolated from dust, an animal shed, the air inside a museum and soil was investigated using a polyphasic approach . The growth characteristics were unusual for actinomycetes . Optimal growth was at temperatures ranging from 2 to 10 degrees C . After small-step adaptation (5 degrees C steps) to higher temperatures, the strains were also able to grow at 20 degrees C . Cell wall analyses revealed that the organisms showed a hitherto undescribed, new group B-type peptidoglycan {type B2beta according to Schleifer & Kandler (1972), but with lysine instead of ornithine} . All strains contained menaquinone MK-9 . Mycolic acids were not detected . Diphosphatidylglycerol, phosphatidylglycerol and an unknown glycolipid were detected in the polar lipid extracts . The main fatty acids were 12-methyl-tetradecanoic acid (15:0 anteiso), 12-methyl-tetradecenoic acid (15:1 anteiso), 14-methyl-pentadecanoic acid (16:0 iso) and 14-methyl-hexadecanoic acid (17:0 iso), as well as an unusual compound identified as 1,1-dimethoxy-anteiso-pentadecane (15:0 anteiso-DMA) . The G+C content of DNA was approximately 71 mol% . The results of 16S rRNA gene sequence comparisons revealed that the strains represent a new lineage in the suborder Micrococcineae and the family Microbacteriaceae of the order Actinomycetales . On the basis of these results the new genus Frigoribacterium gen . nov . is proposed, harbouring the new species Frigoribacterium faeni sp . nov . (type strain = 801T = DSM 10309T). Extremophiles, 2000 Apr, 4(2), 123 - 30 Structural analysis of the elongation factor G protein from the low-temperature-adapted bacterium Arthrobacter globiformis SI55; Berchet V et al.; The first structural analysis of elongation factor G (EF-G) from a cold-adapted bacterium is presented . EF-G is an essential protein involved in the elongation process during protein synthesis and is therefore thought to play a crucial role in the low-temperature adaptation of cold-adapted microorganisms . To define its importance, the EF-G gene (fus) from the psychrotolerant bacterium Arthrobacter globiformis SI55 was cloned and sequenced . The deduced primary structure of the elongation factor is composed of 700 amino acids with a predicted molecular mass of 77.4 kDa . A three-dimensional model of the protein was constructed based on the known crystal structures of structurally homologous proteins . Structural features that might potentially be important for activity and flexibility at low temperature were deduced by comparisons with models of the EF-G proteins from the closely related mesophiles Micrococcus luteus and Mycobacterium tuberculosis . These features include a loss in the number of salt bridges in intradomain and interdomain positions, increased solvent interactions mediated by greater charge and polarity on domain surfaces, loop insertions, loss of proline residues in loop structures, and an increase of hydrophobicity in core regions . Specific changes have also been identified in the catalytic domain (G domain) and sites of potential ribosome interaction, which may directly affect guanosine triphosphate (GTP) hydrolysis and elongation rates at low temperature. Virology, 2000 Apr 25, 270(1), 215 - 28 Mutational analysis of the bovine respiratory syncytial virus nucleocapsid protein using a minigenome system: mutations that affect encapsidation, RNA synthesis, and interaction with the phosphoprotein; Khattar SK et al.; The nucleocapsid (N) protein of bovine respiratory syncytial virus (BRSV) is a multifunctional protein that plays a central role in transcription and replication of viral genomic RNA . To investigate the domains and specific residues involved in different N activities, we generated a total of 27 deletion and 12 point mutants of the N protein . These mutants were characterized using an intracellular BRSV-CAT minigenome replication system for the ability to (1) direct minigenome RNA synthesis, (2) direct minigenome encapsidation, and (3) form a complex with the phosphoprotein (P) . The mutations tested were defective in synthesis of RNA from the BRSV-CAT minigenome template with the exception of the following: a deletion involving the first N-terminal amino acid and mutations involving conservative substitution at the second amino acid and at certain internal cysteine residues . Micrococcal nuclease enzyme protection assays showed that mutations involving amino acids 1-364 of the 391-amino-acid N protein prevented minigenome encapsidation . Thus the BRSV N protein has a C-terminal, 27-amino-acid tail that is not required for encapsidation . Interestingly, two of the mutations that ablated encapsidation did not greatly affect RNA synthesis; the mutant involving deletion of the N-terminal amino acid and the mutant involving a substitution at position 2 . This finding indicates that the formation of a nucleocapsid sufficient to protect the RNA from nuclease is not required for template function . Coimmunoprecipitation of N and P using N- or P-specific antiserum revealed two regions of the N protein that are important for association with the P protein: a central portion of 244-290 amino acids and a C-terminal portion of 338-364 amino acids . J Biol Chem, 2000 Jun 16, 275(24), 18482 - 8 Molecular cloning, expression, and functional analysis of a cis-prenyltransferase from Arabidopsis thaliana . Implications in rubber biosynthesis; Oh SK et al.; cis-Prenyltransferase catalyzes the sequential condensation of isopentenyl diphosphate with allylic diphosphate to synthesize polyprenyl diphosphates that play vital roles in cellular activity . Despite potential significance of cis-prenyltransferase in plant growth and development, no gene of the enzyme has been cloned from higher plants . Using sequence information of the conserved region of cis-prenyltransferase cloned recently from Escherichia coli, Micrococcus luteus, and yeast, we have isolated and characterized the first plant cis-prenyltransferase from Arabidopsis thaliana . Sequence analysis revealed that the protein is highly homologous in several conserved regions to cis-prenyltransferases from M . luteus, E . coli, and yeast . In vitro analyses using the recombinant protein overexpressed in E . coli revealed that the enzyme catalyzed the formation of polyprenyl diphosphates ranging in carbon number from 100 to 130 with a predominance of C(120) . The enzyme exhibited a higher affinity for farnesyl diphosphate than for geranylgeranyl diphosphate, with the K(m) values being 0.13 and 3.62 micrometer, respectively, but a lower affinity for isopentenyl diphosphate, with a K(m) value of 23 micrometer . In vitro rubber biosynthesis analysis indicated that the Arabidopsis cis-prenyltransferase itself could not catalyze the formation of higher molecular weight polyprenyl diphosphates similar to natural rubber . A reverse transcriptase-polymerase chain reaction analysis showed that the gene was expressed at low levels in Arabidopsis plant, in which expression of the cis-prenyltransferase in leaf and root was higher than that in stem, flower, and silique . These results indicate the tissue-specific expression of cis-prenyltransferase and suggest a potential role and significance of the enzyme in the polyisoprenoid biosynthesis in plants. Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 715 - 9 Characterization of Micrococcus antarcticus sp . nov., a psychrophilic bacterium from Antarctica; Liu H et al.; A Gram-positive, cold-adapted, aerobic, spherical actinobacterium (strain T2T) with a quite low cardinal growth temperature was isolated from Chinese Great-Wall station in Antarctica . Sequence comparisons of the 16S rDNA indicated the isolate to be a phylogenetic member of the genus Micrococcus, family Micrococcaceae, in which it represents a novel lineage . The phylogenetic distinctness of the isolate with respect to the type strains Micrococcus luteus and Micrococcus lylae was supported by DNA-DNA similarity values of less than 40% . Chemotaxonomic properties supported the placement of the isolate in the genus Micrococcus . The diagnostic diamino acid of the cell-wall peptidoglycan is lysine . The predominant menaquinones are MK-8 and MK-8(H2) . The G + C content of the DNA of the isolate is 66.4 mol% . Genotypic, morphological and physiological characteristics were used to describe a new species of Micrococcus, for which the name Micrococcus antarcticus is proposed . The type strain is T2T (= AS 1.2372T). J Biol Chem, 2000 Jun 23, 275(25), 18676 - 81 In vivo structure of the cell cycle-regulated human cdc25C promoter; Korner K et al.; The cdc25C promoter is regulated during the cell cycle by the transcriptional repressor CDF-1 that inhibits the activation function of upstream transcriptional activators, most notably the nuclear factor Y/CAAT box binding factor (NF-Y/CBF) . In this report a detailed analysis of the in vivo structure of the cdc25C promoter was made . Micrococcus nuclease and methidiumpropyl-EDTA footprinting strongly suggest that the proximal promoter encompassing the cell cycle-dependent element/cell cycle genes homology region and the upstream NF-Y sites is organized in a positioned nucleosome throughout the cell cycle . Furthermore, structural perturbations were detected by DNase I, phenanthroline copper, and KMnO(4) footprinting at the NF-Y binding sites in vivo, which is in agreement with the reported property of NF-Y to bend DNA in vitro . Similar results were obtained with the structurally and functionally related cyclin A promoter . The structural perturbations seen in DNase I and phenanthroline copper footprints were less pronounced in G(0) cells when compared with cycling cells . This presumably reflects a weakened in vivo interaction of NF-Y with its cognate DNA element in G(0) . It is likely that these structural perturbations, together with the reported ability of NF-Y to recruit histone acetyl transferase activity, contribute to an opened chromatin structure as a prerequisite for optimal regulation through activation and repression. Microbiology, 2000 Mar, 146 ( Pt 3), 611 - 9 Identification of a gene cluster for antibacterial polyketide-derived antibiotic biosynthesis in the nystatin producer Streptomyces noursei ATCC 11455; Zotchev S et al.; Streptomyces noursei ATCC 11455 produces the antifungal polyene antibiotic nystatin containing the deoxysugar moiety mycosamine . Part of the deoxythymidyl diphosphate (TDP)-glucose dehydratase gene (gdhA) known to be involved in deoxysugar biosynthesis was amplified by PCR from genomic DNA of S . noursei ATCC 11455 . A gene library for S . noursei was made and screened with the gdhA probe . Several overlapping phage clones covering about 30 kb of the S . noursei genome were physically mapped . A partial DNA sequencing analysis of this region resulted in the identification of several putative genes typical of macrolide antibiotic biosynthetic gene clusters . A gene-transfer system for 5 . noursei has been established, and gene deletion or disruption experiments within the putative biosynthetic gene cluster were performed . All of the knock-out mutants retained the ability to produce nystatin, suggesting that the identified gene cluster is not involved in biosynthesis of this antibiotic . Culture extracts from the wild-type strain and three knock-out mutants were analysed by TLC followed by a bioassay against Micrococcus luteus . Two antibacterial compounds were found to be synthesized by the wild-type strain while only one was produced by the mutants . This provided evidence for the involvement of the identified gene cluster in the biosynthesis of a presumably novel antibacterial macrolide antibiotic in S . noursei. Hum Reprod, 2000 Apr, 15(4), 778 - 84 Antimicrobial activity of human cervical mucus; Eggert-Kruse W et al.; The antibacterial activity of human cervical mucus (CM) was examined on standardized microbial colonized agar plates (agar diffusion test) . In parallel, the lysozyme content of CM was determined by means of a turbidimetric test system in aliquots of the same CM specimens . Suspensions of living lyophilized Micrococcus lysedeikticus were used as bacterial substrate . Testing was performed in a total of 133 CM samples, obtained at mid-cycle from sexually active women from unselected infertile couples with a median age of 30 (range 21-42) years . All mucus specimens showed considerable antibacterial activity with clearly visible circular inhibition zones around the CM-filled holes in the colonized agar plates . Related to the effect of hen's egg white (HEW)-lysozyme on the same plates, the median activity of the CM specimens in the agar diffusion test was equivalent to 33.0 (range 6.4-391.4) microg/ml HEW-lysozyme . However, there was a wide inter-individual range of antibacterial effects of cervical secretions . The cervical index did not significantly influence the outcome of either test . The pH of the endocervical CM also was not correlated with the antibacterial effect . Sexual activity leading to the presence of spermatozoa in CM considerably increased its antibacterial effect . The activity was markedly higher in samples obtained within hours after intercourse compared with those taken after sexual abstinence of >/=5 days (P < 0.05) . In microbially colonized CM specimens compared to sterile CM, all obtained under hormonally standardized conditions, the antibacterial activity in the agar plate test was significantly lower (P < 0.05) . The results of this pilot study demonstrate the considerable antibacterial activity of human CM. Bioseparation, 1999, 8(1-5), 247 - 54 Use of a micro-expanded bed containing immobilised lysozyme for cell disruption in flow injection analysis; Nandakumar MP et al.; A method for cell disruption in Flow Injection Analysis (FIA) systems has been developed . The principle involves on-line cell disruption by means of immobilised lysozyme followed by an ultrasonic treatment . In order to avoid flow problems in the analytical system, the lysozyme was immobilised to Streamline that was used in an expanded bed in the flow system . Samples of suspensions of Micrococcus lysodeikticus were treated and the success of the treatment was evaluated in terms of released protein and as a decrease in the optical density at 450 nm . The new technology offers a powerful tool in flow injection analyses for quantification of intracellular compounds . The concept of integration, i.e . combining cell disruption with handling of cell debris and assay procedure in one continuous flow process facilitates its use and increases the probability of reaching reproducible and reliable results. Mol Gen Genet, 2000 Feb, 263(1), 38 - 47 Histone hyperacetylation facilitates chromatin remodelling in a Drosophila embryo cell-free system; Krajewski WA; We found that Drosophila embryo extract contains a protein activity (or activities) that can destabilize nucleosomes, resulting in increased sensitivity to DNase I, release of nucleosomal supercoiling, high levels of conformational flexibility of DNA and more diffuse micrococcal nuclease digestion patterns . Incorporation of histone H1 did not significantly affect this nucleosome remodelling . Remodelling occurs more efficiently in hyperacetylated chromatin . It was shown previously that hyperacetylated chromatin, reconstituted in a Drosophila embryo cell-free system, exhibits increased DNase I sensitivity and a high degree of conformational flexibility of DNA . The present data suggest that the more diffuse structure of acetylated chromatin is a result of chromatin remodelling by protein activities in the Drosophila embryo extract. Theriogenology, 1998 Sep, 50(4), 559 - 73 Bacteriology of preserved stallion semen and antibiotics in semen extenders; Varner DD et al.; Three experiments were conducted to evaluate the effects of different antibiotics in a milk-glucose semen extender on motility of equine sperm and elimination of bacteria following storage of extended semen in vitro . In Experiment 1, 7 antibiotics were compared: amikacin, gentamicin, streptomycin, potassium penicillin, sodium penicillin, ticarcillin, and polymixin B . In Experiment 2, 3 antibiotic treatments were compared: potassium penicillin G, amikacin, or a combination of potassium penicillin G and amikacin . In Experiment 3, 3 antibiotic treatments were compared: potassium penicillin G-amikacin, ceptiofur, and a combination of ticarcillin and clavulanic acid (Timentin) . Control treatments (antibiotic-free extender) were included in each experiment . Six motility variables were evaluated: percentage of motile sperm; percentage of progressively-motile sperm; percentage of rapidly-motile sperm; mean curvilinear velocity; mean average path velocity; and mean straight-line velocity . In Experiment 1, mean percentages of motile, progressively motile and rapidly motile sperm were lower (P < 0.05) in semen exposed to polymixin B then in other treatments . Mean average-path velocity of sperm in extender containing polymixin B was lower (P < 0.05) than that of all other treatments, with exception of control or ticarcillin . Mean straight-line velocity of sperm in extender containing polymixin B was lower (P < 0.05) than that of all other treatments, with exception of control, streptomycin or ticarcillin . Semen samples containing gentamicin, amikacin, streptomycin, or potassium penicillin were more effective (P < 0.05) at eliminating bacterial growth than those samples containing polymixin B . Semen samples containing gentamicin were also more effective (P < 0.05) at eliminating bacterial growth than those samples containing ticarcillin or sodium penicillin . In Experiment 2, mean percentage of rapidly-motile sperm, and mean curvilinear, average-path, and straight-line velocities were greater (P < 0.05) for potassium penicillin-amikacin than values for all other treatments . In 2 of 3 stallions, an effect of treatment on percentage of motile sperm was detected (P < 0.05) . For one stallion, mean motility of potassium penicillin-amikacin was greater (P < 0.05) than that of all other treatment groups . For another stallion, mean motility of the control was lower (P < 0.05) than that of the other treatments . Following storage, potassium penicillin (16/18 {89%}) or potassium penicillin-amikacin (17/19 {94%}) were more effective (P < 0.05) at controlling aerobic and anaerobic bacterial isolates in semen specimens than was amikacin (10/18 {56%}) . In Experiment 3, a difference among treatment groups for motility variables was not detected (P < 0.05) . No bacterial growth was recovered in antibiotic-treated semen, with exception of Micrococcus sp . (2 colonies) which were isolated from one semen specimen treated with ceptiofur. J Biol Chem, 2000 Mar 17, 275(11), 8226 - 32 Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on naked DNA and chromatin substrates; Widlak P et al.; Here we report the co-factor requirements for DNA fragmentation factor (DFF) endonuclease and characterize its cleavage sites on naked DNA and chromatin substrates . The endonuclease exhibits a pH optimum of 7.5, requires Mg(2+), not Ca(2+), and is inhibited by Zn(2+) . The enzyme generates blunt ends or ends with 1-base 5'-overhangs possessing 5'-phosphate and 3'-hydroxyl groups and is specific for double- and not single-stranded DNA or RNA . DFF endonuclease has a moderately greater sequence preference than micrococcal nuclease or DNase I, and the sites attacked possess a dyad axis of symmetry with respect to purine and pyrimidine content . Using HeLa cell nuclei or chromatin reconstituted on a 5 S rRNA gene tandem array, we prove that the enzyme attacks chromatin in the internucleosomal linker, generating oligonucleosomal DNA ladders sharper than those created by micrococcal nuclease . Histone H1, high mobility group-1, and topoisomerase II activate DFF endonuclease activity on naked DNA substrates but much less so on chromatin substrates . We conclude that DFF is a useful reagent for chromatin research. Eur J Dermatol, 2000 Jan-Feb, 10(1), 41 - 2 Correlation between bacterial population and axillary and plantar bromidrosis: study of 30 patients; Guillet G et al.; Although studies on the chemistry of odors are expanding to identify the chemical structures of odorous substances, there are no universal standards as yet to measure odor and intensity of bromidrosis . Clinical evaluation can be made on a subjective scoring from 0 to 3 prior to prescription of an antiseptic soap . In order to appreciate the correlation between the intensity of bromidrosis (BI) and bacterial activity, a study was carried out with both clinical and bacterial assessment in thirty patients with axillary or plantar BI . Odor intensity was evaluated by two physicians using a score from 0 to 3 (i.e . absent, minor, moderate, major), meanwhile bacterial composition and density were assessed before and after 10 days of hygiene using an antiseptic detergent (trichlocarbanilide) provided on the first visit . Baseline count of diphtheroids/cm2 was 35.104 and baseline micrococci average was 32.104/cm2 . At the end of the study, the reduction of odor intensity was observed in 20 patients (67%) without any change in sweat production . The clinical improvement correlated with a reduction of both micrococci (70%) or diphtheroids (73%) as compared with initial data . In patients presenting persistant bromidrosis, the bacterial count/cm2 did not significantly decrease and remained above 104 diphtheroids/cm2 . Thus, this study suggests that body odor may be at least indirectly correlated to microbia counts with a bacteria threshold of BI ranging around and above 104. J Appl Microbiol, 1999 Dec, 87(6), 794 - 803 Effects of environmental conditions on microbial proteolysis in a pork myofibril model system; Kenneally PM et al.; A number of bacterial strains used for meat fermentations were screened for proteolytic activity . A strain of Micrococcus which was found to be proteolytic was evaluated for the effects of environmental conditions on its proteolytic activity against pork myofibrillar proteins using response surface methodology . Three strains of micrococci were also tested for the ability to produce free amino acids from pork myofibrils . Analysis of the effects of environmental conditions showed that proteolytic activity would be minimal under conditions normally found in fermented sausages, thereby suggesting that proteolysis in these products is largely due to endogenous meat enzymes . The three strains of micrococci were shown to produce free amino acids from pork myofibrils, thereby demonstrating the presence of peptidase activity in these strains. Biochem Biophys Res Commun, 2000 Feb 16, 268(2), 293 - 7 Purification, cloning, and three-dimensional structure prediction of Micrococcus luteus FAD-containing tyramine oxidase; Roh JH et al.; The FAD-containing tyramine oxidase enzyme and gene from the Gram (+) bacterium Micrococcus luteus were isolated, and computer prediction was used to propose a preliminary 3D model of the protein . A 2.8-kb Sau3AI fragment containing the structural gene of tyramine oxidase was cloned from a M . luteus genomic DNA library . The 1332 bp gene encodes a protein of 443 amino acids, with a calculated molecular mass of 49.1 kDa . The enzyme was found to be a homodimer with a molecular weight of 49,000 . It oxidizes tyramine, adrenaline, 3-hydroxytyramine, dopamine, and noradrenaline, and was reversibly inhibited by FAD-containing monoamine oxidase A and B specific inhibitors . Sequence comparison show that tyramine oxidase is smaller than other FAD-amine oxidases but that it contains well-conserved amino acid residues reported in all other FAD-amine oxidases . A hypothetical three-dimensional structure of tyramine oxidase has also been proposed based on secondary structure predictions, threading, and comparative modeling . Nucleic Acids Res, 2000 Mar 1, 28(5), 1126 - 32 Tissue-specific chromatin structure of the phenobarbital-responsive unit and proximal promoter of CYP2B1/2 and modulation by phenobarbital; Kim J et al.; Phenobarbital induction of transcription of CYP2B genes is mediated by an enhancer, termed a phenobarbital responsive unit (PBRU), approximately 2000 bp 5' of the transcription start site . To further delineate the mechanism of phenobarbital induction, protein binding in native chromatin and the nucleosomal structure of the PBRU and proximal promoter were examined in liver and kidney, in which the CYP2B1/2 genes are expressed and not expressed, respectively . Protein binding to the PBRU in kidney chromatin was not detected even though in vitro DNase I footprints were not detectably different with nuclear extracts from liver and kidney . Likewise, protein binding to regulatory motifs was not detected in the proximal promoter region in kidney chromatin . In liver chromatin, however, DNase I hypersensitivity and partial protection of the regulatory motifs from DNase I digestion or reaction with dimethyl sulfate was observed and phenobarbital treatment increased the hypersensitivity but only modestly affected protection . Low resolution Southern analysis of micrococcal nuclease-digested chromatin from untreated rats revealed micrococcal nuclease hypersensitive regions in the proximal promoter and PBRU regions in liver, but not in kidney . Phenobarbital treatment increased hyper-sensitivity in liver in both regions . Micrococcal nuclease hypersensitivity in the PBRU was largely restricted to a linker region between phased nucleosomes while in the proximal promoter hypersensitivity extended over approximately 200 bp suggesting disruption of a nucleosome in this region . These data indicate that in liver phenobarbital treatment substantially alters protein binding to regulatory motifs in the PBRU, while not greatly affecting such binding in the proximal promoter, and substantially alters chromatin structure in both regions, presumably as a result of chromatin modifying factors recruited to the PBRU . In the kidney, chromatin is probably in a closed conformation that prevents binding of regulatory factors. Comp Biochem Physiol B Biochem Mol Biol, 1999 Dec, 124(4), 475 - 81 Purification and characterization of chitin-binding proteins from the hemolymph of sweet potato hornworm, Agrius convolvuli; Chae KS et al.; Three chitin-binding proteins (CBPs: CBP9, CBP15, CBP66) were identified from the larval hemolymph of sweet potato hornworm, Agrius convolvuli . Two (CBP9 and CBP15) of them have been isolated and purified by gel filtration (Superdex HR 75), cation-exchange chromatography (Mono S), and reverse-phase chromatography (muRPC PC 2.1/3) . In experiments to detect CBPs in hemolymph, we examined whether ionic strength and existence of bovine serum albumin in the incubation solution influenced binding affinity of CBPs to chitin . The N-terminal sequences of three CBPs were determined by the automated Edman degradation and showed the sequence homology in basic local alignment search tool search CBP15 and CBP66 were quite similar to lysozymes and bovine serum albumins, respectively . In contrast, CBP9 is not similar to any other known protein, as judged from databank comparisons . Therefore, we concluded that CBP9 is a novel protein with binding capacity to chitin that is a component of the fungal cell wall . CBP9 has no antibacterial activity against Escherichia coli and Micrococcus luteus, and also showed negative response in hemagglutination assay . CBP9 is confirmed as a monomer with a molecular mass of 9.14 kDa by electron spray ionization and matrix-assisted laser desorption ionization mass spectrometry. Protein Expr Purif, 2000 Feb, 18(1), 46 - 55 Purification and characterization of a unique alkaline elastase from Micrococcus luteus; Clark DJ et al.; Micrococcus luteus isolated from human skin secretes an alkaline protease which degrades elastin . M . luteus protease (MLP) was produced in the late logarithmic and stationary phases of growth . MLP, purified to homogeneity by a three-step process, had a molecular mass of 32,812 Da and an isoelectric point of 9.3 . MLP was active and highly stable in solution for 24 h from pH 6.0 to 10.5; it had maximal activity at temperatures between 57 and 59 degrees C . The presence of calcium in the solution was essential for enzyme activity and to prevent autolysis . Optimal activity occurred between pH 9.0 and 9.5, with 60% maximal activity from pH 6.5 to 11.0 . The enzyme was inhibited by the serine enzyme inhibitors phenylmethylsulfonyl fluoride and chymostatin but not by the metalloenzyme inhibitor 1,10-phenanthroline or sulfhydryl enzyme inhibitors . Casein, bovine serum albumin, ovalbumin, beta-lactoglobulin, and elastin were digested by the protease while collagen and keratin were resistant to digestion . MLP demonstrated both esterase and amidase activity on synthetic peptide substrates . MLP preferentially cleaved the Leu(15)-Tyr(16) and Phe(24)-Phe(25) bonds of the oxidized beta-chain of insulin . Longer digests of insulin and the pattern of activity against synthetic substrates suggest that MLP has a cleavage specificity for bulky, hydrophobic, or aromatic amino acids in the P(1) or P(1)' positions . Amino acid sequences from the N-terminus and internal peptides of MLP were unique . Mech Dev, 2000 Feb, 90(2), 217 - 26 Embryonic inheritance of the chromatin organisation of the imprinted H19 domain in mouse spermatozoa; Banerjee S et al.; Insulin-like growth factor 2 (Igf 2) and H19 genes are oppositely imprinted and as such have been most extensively studied imprinted genes both genetically and at the molecular level . Imprints of the H19 gene, being established during spermatogenesis, are epigenetically transmitted to the somatic cells of the embryo . Current hypotheses attempting to explain the allele-specific silence of the H19 gene include DNA methylation and chromatin condensation . In order to understand the molecular basis of H19 epigenesis, it is crucial to identify the markings in the chromatin organising the imprinted domain in spermatozoa . Using Micrococcal nuclease (MNase), DNase I and Methidiumpropyl-EDTA . iron II (MPE.Fe(II)) as chromatin probes, we demonstrate that in mouse epididymal spermatozoa, at least 4kb DNA upstream of the H19 'cap' site, containing the imprinted and differentially methylated domain (DMD), is heterochromatic . The cleavage sites in this domain (-2 to -4kb) exhibit approximately 425bp periodicity . This structure is maintained in the paternal allele of normal embryos and is disrupted at -2.2, -2.65 and at -3.5kb in embryos maternally disomic for the distal end of chromosome 7 (MatDp 7) . The hypersensitive sites in chromatin precisely register the MPE.Fe(II) cleavage sites in chromosomal DNA . Therefore, the DNA sequences in the imprinted domain constrain the chromatin structure in a way similar to that of 1.688g/cm(3) Drosophila satellite chromatin . In addition, we find that condensation of the paternal allele correlates with methylation-dependent alteration in the structure of DNA sequences in DMD . These results suggest that CpG-methylation induces localised changes in DNA conformation and these facilitate consequent remodelling of chromatin thereby allowing the paternal and maternal H19 alleles to be distinguished. J Clin Lab Anal, 1999, 13(6), 301 - 7 Development of a microparticle-enhanced nephelometric immunoassay for quantitation of human lysozyme in pleural effusion and plasma; Caballero M et al.; A microparticle-enhanced nephelometric immunoassay, based on polystyrene beads coated with antihuman lysozyme antibody, has been developed for lysozyme quantification in sera and pleural effusions . The standard curve extends from 0.58 mg/l to 18.75 mg/l and no antigen effect was observed . The results showed a good serial precision . The intra-assay precision (n = 20) expressed as CV was between 2.2 and 4.2 in three different concentrations . The inter-assay precision, with different calibration curves (n = 12) was between 6.4 and 7.1 . The analytical assay showed a sufficient linearity (r > 0.999) . There were no interferences either with haemoglobin (up to 4 g/l), lipids (up to 0.5%, expressed as 1% Lipofundina content), or bilirubin (up to 5 mg/dl) . The analytical sensitivity was lower than 0.6 mg/l . The correlation with a Micrococcus lysodeikticus turbidimetric assay showed a correlation coefficient of 0.915 . We have studied 92 patients with pleural effusion . In each case, pleural fluid adenosine deaminase activity and pleural fluid to plasma lysozyme ratio were determined . The lysozyme ratio showed similar clinical sensitivity and specificity as to adenosine deaminase. Mol Microbiol, 2000 Jan, 35(1), 223 - 33 Deletion of the unique gene encoding a typical histone H1 has no apparent phenotype in Aspergillus nidulans; Ram inverted question markon A et al.; We have cloned the H1 histone gene (hhoA) of Aspergillus nidulans . This single-copy gene codes for a typical linker histone with one central globular domain . The open reading frame is interrupted by six introns . The position of the first intron is identical to that of introns found in some plant histones . An H1-GFP fusion shows exclusive nuclear localization, whereas chromosomal localization can be observed during condensation at mitosis . Surprisingly, the deletion of hhoA results in no obvious phenotype . The nucleosomal repeat length and susceptibility to micrococcal nuclease digestion of A . nidulans chromatin are unchanged in the deleted strain . The nucleosomal organization of a number of promoters, including in particular the strictly regulated niiA-niaD bidirectional promoter is not affected. Immunity, 1999 Dec, 11(6), 665 - 75 Rapid and selective remodeling of a positioned nucleosome during the induction of IL-12 p40 transcription; Weinmann AS et al.; Nucleosomes are important for gene regulation, but comprehensive studies of nucleosome positioning, remodeling, and transcription factor binding at inducible mammalian promoters have not been reported . We have analyzed the IL-12 p40 promoter, which is induced in macrophages by bacterial products . High-resolution micrococcal nuclease analyses revealed that a positioned nucleosome, nucleosome 1, spans the promoter, with three positioned nucleosomes further upstream . Upon activation, nucleosome 1 was rapidly and selectively remodeled in a protein synthesis-dependent manner . In primary macrophages, IFNgamma synergistically enhanced p40 expression, but little effect on remodeling or promoter occupancy was observed . These results suggest that remodeling complexes are selectively targeted to a single, promoter-encompassing nucleosome and that IFNgamma influences an event that is independent or downstream of remodeling. DNA Cell Biol, 1999 Dec, 18(12), 895 - 901 A method for efficient extraction of bovine papilloma virus-based minichromosomes that preserves native chromatin structure; Karpova E et al.; Aiming to create an adequate model for investigation of the molecular mechanisms involved in transcriptional regulation by steroid hormones, a number of cell lines carrying bovine papilloma virus (BPV) based constructs containing the mouse mammary tumor virus long terminal repeat (LTR) were established (Ostrowski et al., Mol . Cell . Biol . 3, 2945-2957, 1983) . However, all our attempts to extract from the cells such minichromosomes as nucleoprotein complexes using a method previously described (Ostrowski, Nucleic Acids Res . 15, 6957-6971, 1987) failed . Here, we show that this failure was attributable to DNA rearrangements in most of the cell lines, resulting in the integration of the BPV-based constructs into the host cell genome . We have identified two cell lines where the constructs are episomal . Micrococcal nuclease digestion of the nuclei demonstrated the presence of nucleosomes positioned over the episomal MMTV LTR . We managed to optimize conditions for preparation of nuclei and minichromosomes, which allowed extraction of approximately 40% of the minichromosomes, most of them being in circular superhelical form . Our data show clearly that the main factor preventing the release of minichromosomes from the nuclei is the presence of polyamines in the cell lysis buffer . The organization of MMTV promoter chromatin was unaffected by the extraction procedure, suggesting that these minichromosomes could be valuable templates for in vitro transcription studies and to identify proteins involved in chromatin remodeling during transcription. FEBS Lett, 1999 Dec 31, 464(3), 153 - 8 Protein purification and gene isolation of chlamysin, a cold-active lysozyme-like enzyme with antibacterial activity; Nilsen IW et al.; An antibacterial approximately 11 kDa protein designated chlamysin was isolated from viscera of the marine bivalve Chlamys islandica . Chlamysin inhibited the growth of all Gram-positive and Gram-negative bacteria tested . The isolated protein was highly efficient in hydrolyzing Micrococcus luteus cells only at low pH (4.5-6.2) and at low temperature (4-35 degrees C) . No significant loss of enzyme activity was observed after 30 days storage at room temperature or after heating to 70 degrees C for 15 min, suggesting relatively high protein structure stability . Sequence-analyzed fragments of the protein revealed data which guided the isolation of the cDNA gene, encoding a 137 amino acid chlamysin precursor in scallops . The deduced protein contains a high portion of cysteine, serine and histidine residues and has a predicted isoelectric point below 7 . The chlamysin protein was found to have sequence homology to an isopeptidase and to a recently published bivalve lysozyme. Appl Environ Microbiol, 2000 Jan, 66(1), 431 - 4 Coaggregation between aquatic bacteria is mediated by specific-growth-phase-dependent lectin-saccharide interactions; Rickard AH et al.; Coaggregating strains of aquatic bacteria were identified by partial 16S rRNA gene sequencing . The coaggregation abilities of four strains of Blastomonas natatoria and one strain of Micrococcus luteus varied with culture age but were always maximum in the stationary phase of growth . Each member of a coaggregating pair carried either a heat- and protease-sensitive protein (lectin) adhesin or a saccharide receptor, as coaggregation was reversed by sugars. J Biol Chem, 1999 Dec 31, 274(53), 37950 - 6 Effects of H1 histone variant overexpression on chromatin structure; Gunjan A et al.; The importance of histone H1 heterogeneity and total H1 stoichiometry in chromatin has been enigmatic . Here we report a detailed characterization of the chromatin structure of cells overexpressing either H1(0) or H1c . Nucleosome spacing was found to change during cell cycle progression, and overexpression of either variant in exponentially growing cells results in a 15-base pair increase in nucleosome repeat length . H1 histones can also assemble on chromatin and influence nucleosome spacing in the absence of DNA replication . Overexpression of H1(0) and, to a lesser extent, H1c results in a decreased rate of digestion of chromatin by micrococcal nuclease . Using green fluorescent protein-tagged H1 variants, we show that micrococcal nuclease-resistant chromatin is specifically enriched in the H1(0) variant . Overexpression of H1(0) results in the appearance of a unique mononucleosome species of higher mobility on nucleoprotein gels . Domain switch mutagenesis revealed that either the N-terminal tail or the central globular domain of the H1(0) protein could independently give rise to this unique mononucleosome species . These results in part explain the differential effects of H1(0) and H1c in regulating chromatin structure and function. Mol Cell Biol, 2000 Jan, 20(1), 61 - 9 Histone H1 is dispensable for methylation-associated gene silencing in Ascobolus immersus and essential for long life span; Barra JL et al.; A gene encoding a protein that shows sequence similarity with the histone H1 family only was cloned in Ascobolus immersus . The deduced peptide sequence presents the characteristic three-domain structure of metazoan linker histones, with a central globular region, an N-terminal tail, and a long positively charged C-terminal tail . By constructing an artificial duplication of this gene, named H1, it was possible to methylate and silence it by the MIP (methylation induced premeiotically) process . This resulted in the complete loss of the Ascobolus H1 histone . Mutant strains lacking H1 displayed normal methylation-associated gene silencing, underwent MIP, and showed the same methylation-associated chromatin modifications as did wild-type strains . However, they displayed an increased accessibility of micrococcal nuclease to chromatin, whether DNA was methylated or not, and exhibited a hypermethylation of the methylated genome compartment . These features are taken to imply that Ascobolus H1 histone is a ubiquitous component of chromatin which plays no role in methylation-associated gene silencing . Mutant strains lacking histone H1 reproduced normally through sexual crosses and displayed normal early vegetative growth . However, between 6 and 13 days after germination, they abruptly and consistently stopped growing, indicating that Ascobolus H1 histone is necessary for long life span . This constitutes the first observation of a physiologically important phenotype associated with the loss of H1. Mol Biol Cell, 1999 Dec, 10(12), 4217 - 30 Differentiation of chromatin during DNA elimination in Euplotes crassus; Jahn CL; In Euplotes crassus, most of the micronuclear genome is eliminated during formation of a transcriptionally active macronucleus . To understand how this is mediated throughout the genome, we have examined the chromatin structure of the macronucleus-destined sequences and Tec transposons, which are dispersed in 15,000 copies in the micronuclear genome and completely eliminated during formation of the macronuclear genome . Whereas the macronucleus-destined sequences show a typical pattern of nucleosomal repeats in micrococcal nuclease digests, the Tec element chromatin structure digests to a nucleosome-like repeat pattern that is not typical: the minimum digestion products are approximately 300-600 base pairs, or "subnucleosomal," in size . In addition, the excised, circular forms of the Tec elements are exceedingly resistant to nucleases . Nevertheless, an underlying nucleosomal structure of the Tec elements can be demonstrated from the size differences between repeats in partial micrococcal nuclease digests and by trypsin treatment of nuclei, which results in mononucleosome-sized products . Characterization of the most micrococcal nuclease-resistant DNA indicates that micronuclear telomeres are organized into a chromatin structure with digestion properties identical to those of the Tec elements in the developing macronucleus . Thus, these major repetitive sequence components of the micronuclear genome differ in their chromatin structure from the macronuclear-destined sequences during DNA elimination . The potential role of developmental stage-specific histone variants in this chromatin differentiation is discussed. Biosci Biotechnol Biochem, 1999 Oct, 63(10), 1671 - 6 Molecular analysis of prenyl chain elongating enzymes; Koyama T; Multiple alignments of primary structures of many kinds of prenyltransferases that participate in the most fundamental prenyl-chain backbone synthesizing process in isoprenoid biosynthesis showed seven conserved regions in the primary structures of (E)-prenyl diphosphate synthases . However, no information has been available about the structures of (Z)-prenyl diphosphate synthases until our recent isolation of the gene for the undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26 . The amino acid sequence of the (Z)-prenyl diphosphate synthase is totally different from those of (E)-prenyl chain elongating enzymes . Protein data base searches for sequences similar to that of the undecaprenyl diphosphate synthase yielded many unknown proteins which have not yet been characterized . Two of the proteins have recently been identified as the undecaprenyl diphosphate synthase of Escherichia coli and the dehydrodolichyl diphosphate synthase of Saccharomyces cerevisiae, indicating that there are three highly conserved regions in the primary structure of (Z)-prenyl chain elongating enzymes. Dev Comp Immunol, 1999 Oct-Dec, 23(7-8), 563 - 70 Apolipophorin-III in Galleria mellonella potentiates hemolymph lytic activity; Halwani AE et al.; Heat-inactivated serum of non-immune Galleria mellonella larvae enhanced the lytic activity of larval cell-free hemolymph against Micrococcus lysodeikticus . The increase in bacterial lysis was due to a 17.2 kDa protein known previously to bind to bacterial lipopolysaccharides . The protein enhanced the lytic activity of insect cell-free hemolymph and hen lysozyme in vitro and insect hemolymph in vivo . The hydrophobic protein, which adhered to M . lysodeikticus, was identified by its amino acid sequence homology as apolipophorin-III . The titer of apolipophorin-III in 200-250 mg last instar larvae was 8.7 mg/ml of hemolymph . Apolipophorin-III did not bind to lysozyme . A possible mode of action of apolipophorin-III with lysozyme in the insect is proposed. Mol Cell Biol, 1999 Dec, 19(12), 8591 - 603 NF-Y associates with H3-H4 tetramers and octamers by multiple mechanisms; Caretti G et al.; NF-Y is a CCAAT-binding trimer with two histonic subunits, NF-YB and NF-YC, resembling H2A-H2B . We previously showed that the short conserved domains of NF-Y efficiently bind to the major histocompatibility complex class II Ea Y box in DNA nucleosomized with purified chicken histones . Using wild-type NF-Y and recombinant histones, we find that NF-Y associates with H3-H4 early during nucleosome assembly, under conditions in which binding to naked DNA is not observed . In such assays, the NF-YB-NF-YC dimer forms complexes with H3-H4, for whose formation the CCAAT box is not required . We investigated whether they represent octamer-like structures, using DNase I, micrococcal nuclease, and exonuclease III, and found a highly positioned nucleosome on Ea, whose boundaries were mapped; addition of NF-YB-NF-YC does not lead to the formation of octameric structures, but changes in the digestion patterns are observed . NF-YA can bind to such preformed DNA complexes in a CCAAT-dependent way . In the absence of DNA, NF-YB-NF-YC subunits bind to H3-H4, but not to H2A-H2B, through the NF-YB histone fold . These results indicate that (i) the NF-Y histone fold dimer can efficiently associate DNA during nucleosome formation; (ii) it has an intrinsic affinity for H3-H4 but does not form octamers; and (iii) the interactions between NF-YA, NF-YB-NF-YC, and H3-H4 or nucleosomes are not mutually exclusive . Thus, NF-Y can intervene at different steps during nucleosome formation, and this scenario might be paradigmatic for other histone fold proteins involved in gene regulation. Mol Cell Biol, 1999 Dec, 19(12), 7944 - 50 High-resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating-type locus HMRa; Ravindra A et al.; Genetic and biochemical evidence implicates chromatin structure in the silencing of the two quiescent mating-type loci near the telomeres of chromosome III in yeast . With high-resolution micrococcal nuclease mapping, we show that the HMRa locus has 12 precisely positioned nucleosomes spanning the distance between the E and I silencer elements . The nucleosomes are arranged in pairs with very short linkers; the pairs are separated from one another by longer linkers of approximately 20 bp . Both the basic amino-terminal region of histone H4 and the silent information regulator protein Sir3p are necessary for the organized repressive chromatin structure of the silent locus . Compared to HMRa, only small differences in the availability of the TATA box are present for the promoter in the cassette at the active MATa locus . Features of the chromatin structure of this silent locus compared to the previously studied HMLalpha locus suggest differences in the mechanisms of silencing and may relate to donor selection during mating-type interconversion. Insect Biochem Mol Biol, 1999 Nov, 29(11), 989 - 97 Purification and characterization of the lysozyme from the gut of the soft tick Ornithodoros moubata; Kopacek P et al.; The gut of the adult soft ticks Ornithodoros moubata displays high lytic activity against the bacteria Micrococcus luteus . The activity differed in the range of two orders of magnitude among individual animals and increased on average 4 fold during the first week following ingestion . In homogenates of first instar nymphs the activity was much lower increasing exponentially as nymphs neared the first molt . The protein responsible for this activity was purified out of gut contents of adult ticks by means of affinity adsorption on magnetic-chitin followed by two chromatography steps on cation exchange FPLC column MonoS . The homogeneous active protein has a mass of 14006 +/- 20 Daltons as determined by MALDI-TOF mass spectrometry . The N-terminal amino-acid sequence of this protein is K-V-Y-D-R-C-S-L-A-S-E-L-R with the highest similarity to the lysozyme from liver of rainbow trout and to lysozymes from digestive tracts of several mammals . The motif DRCSLA is specific for the digestive lysozymes of several dipteran insects . Based on this evidence, we have identified the protein as the tick gut lysozyme . The tick gut lysozyme has a pI near 9.7 and retains its full activity after treatment at 60 degrees C for 30 minutes . The pH optimum of the tick lysozyme was in the range from pH 5-7 . Only marginal activity could be detected at pH > 8 which raises the question about the function of lysozyme in anti-bacterial defense in the environment of the tick gut. Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1733 - 40 Beutenbergia cavernae gen . nov., sp . nov., an L-lysine-containing actinomycete isolated from a cave; Groth I et al.; Two aerobic, Gram-positive bacteria, strains HKI 0122T and HKI 0132, were isolated from a cave . Cells are not acid-fast, non-motile, non-spore-forming and exhibit a rod-coccus growth cycle . The cell wall peptidoglycan contains lysine in position 3 of the peptide subunit and an interpeptide bridge of L-Lys<--L-Glu . The major menaquinone is MK-8(H4), 13-methyl and 12-methyl tetradecanoic acids are the predominating fatty acids . The polar lipids consist of phosphatidylinositol, diphosphatidylglycerol and three unknown phospholipids . Mycolic acids are absent . The DNA base composition is 71 mol% G + C . Phylogenetic analysis revealed that strain HKI 0122T forms a novel taxon among the families and unassigned genera of the suborder Micrococcineae, within the order Actinomycetales . On the basis of the genotypic, chemotaxonomic, morphological and physiological characteristics of these two isolates it is proposed to assign strains HKI 0122T and HKI 0132 to a new genus and species for which the name Beutenbergia cavernae gen . nov., sp . nov . is proposed . The type strain is HKI 0122T (= DSM 12333T). Arch Virol, 1999, 144(10), 1977 - 90 Rescue of a bovine respiratory syncytial virus genomic RNA analog by bovine, human and ovine respiratory syncytial viruses confirms the "functional integrity" and "cross-recognition" of BRSV cis-acting elements by HRSV and ORSV; Yunus AS et al.; The nucleotide sequences of the 3' leader and 5' trailer regions were determined for genomic RNA of bovine respiratory syncytial virus (BRSV) strain A-51908 . The leader and trailer sequences are '45' and '161' nucleotides in length, respectively . The functionality of BRSV leader and trailer sequences and their recognition by HRSV and ovine respiratory syncytial virus (ORSV) proteins were examined with a in vitro transcribed BRSV genomic RNA analog carrying the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of BRSV transcription signals . Upon transfection into BRSV, HRSV or ORSV infected cells, the BRSV minireplicons were 'rescued' such that the reporter gene was expressed, the minigenome was replicated and packaged into micrococcal nuclease resistant-infectious minireplicons . The passage of infectious minireplicons could be blocked by a polyclonal BRSV neutralizing antiserum . Bovine parainfluenza virus-3, a heterologous paramyxovirus was inactive in rescuing BRSV genomic RNA analog . Mutational substitution of the G residue at position 4 of leader sequence in the BRSV genomic RNA analog, with an A or U residue inhibited its transcription and replication, while replacement with a C residue had no significant effect on rescue . These results show that the cis-acting elements of BRSV are functional and are also recognized by the proteins of HRSV and ORSV . The helper virus complemented rescue system developed here will be useful for characterizing the cis-acting elements of BRSV. J Virol, 1999 Nov, 73(11), 8919 - 25 Characterization of intracellular reverse transcription complexes of Moloney murine leukemia virus; Fassati A et al.; To examine the early events in the life cycle of Moloney murine leukemia virus (MoMLV), we analyzed the intracellular complexes mediating reverse transcription . Partial purification of the reverse transcription complexes (RTCs) by equilibrium density fractionation and velocity sedimentation indicated that three distinct species of intracellular complexes are formed shortly after cell infection . Only one of these species is able to start and complete reverse transcription in the cell cytoplasm . This RTC is composed of at least the viral genome, capsid, integrase, and reverse transcriptase proteins . The RTC becomes permeable to micrococcal nuclease but not to antibodies . Shortly after initiation of reverse transcription, the viral strong stop DNA within the RTC is protected from nuclease digestion . The sedimentation velocity of the RTC decreases during reverse transcription . After entry into the nucleus, most capsid proteins are lost from the RTC and its sedimentation velocity decreases further. J Periodontol, 1999 Sep, 70(9), 960 - 6 Periodontal tissue disposition of azithromycin in patients affected by chronic inflammatory periodontal diseases; Blandizzi C et al.; BACKGROUND: The recognition that periodontal diseases are associated with specific pathogens has led to interest in the use of antibacterial drugs for inhibition of these microorganisms . On these bases, the present study was aimed at evaluating the tissue distribution of the new macrolide antibiotic azithromycin in patients subjected to oral surgery for chronic inflammatory diseases of both marginal and periapical periodontium . METHODS: Thirty-two patients were treated with azithromycin 500 mg/day orally for 3 consecutive days, and drug concentrations in plasma, saliva, normal gingiva, and pathological periodontal tissues were evaluated . For this purpose, samples of blood, saliva, normal gingiva, granulation tissue, and radicular granuloma or cyst wall (from dentigerous cyst) were collected during oral surgery or 0.5, 2.5, 4.5, and 6.5 days after the end of pharmacological treatment; then, azithromycin levels were measured by a microbiological plate assay, using Micrococcus luteus NCTC 8440 as the indicator organism . RESULTS: The concentrations of azithromycin in plasma, saliva, normal gingiva, and pathological tissues reached the highest values 12 hours after the last dose (0.37+/-0.05 mg/l, 2.12+/-0.30 mg/l, 6.30+/-0.68 mg/kg, and 11.60+/-1.50 mg/kg, respectively) and then declined gradually . Consistent levels of the drug in normal gingiva and pathological tissues could be detected, however, up to 6.5 days, indicating that azithromycin was retained in target tissues for a long time after the end of treatment . Moreover, azithromycin levels in both normal gingiva and pathological tissues exceeded the minimum inhibitory concentrations of most pathogens involved in the pathophysiology of chronic inflammatory periodontal diseases . Notably, azithromycin levels in pathological tissues were significantly higher than those in normal gingiva 0.5, 2.5, and 4.5 days after the last dose . CONCLUSIONS: The present results indicate a marked penetration of azithromycin into both normal and pathological periodontal tissues, suggesting that azithromycin represents a promising option in both adjunctive and prophylactic treatments of chronic inflammatory periodontal diseases. Biochimie, 1999 Jul, 81(7), 727 - 32 The site of binding of linker histone to the nucleosome does not depend upon the amino termini of core histones; An W et al.; Using nucleosomes reconstituted on a defined sequence of DNA, we have investigated the question as to whether the N-terminal tails of core histones play a role in determining the site of binding of a linker histone . Reconstitutes used histone cores of three types: intact, lacking the N-terminal H3 tails, or lacking all tails . In each case the same, single defined position for the histone core was observed, using high-resolution mapping . The affinity for binding of linker histone H1(o) was highest for the intact cores, lowest for the tailless cores . However, the location of the linker histone, as judged by micrococcal nuclease protection, was exactly the same in each case, an asymmetric site of about 17 bp to one side of the core particle DNA. Mol Cell Biol, 1999 Oct, 19(10), 6963 - 71 Altered telomere nuclear matrix interactions and nucleosomal periodicity in ataxia telangiectasia cells before and after ionizing radiation treatment; Smilenov LB et al.; Cells derived from ataxia telangiectasia (A-T) patients show a prominent defect at chromosome ends in the form of chromosome end-to-end associations, also known as telomeric associations, seen at G(1), G(2), and metaphase . Recently, we have shown that the ATM gene product, which is defective in the cancer-prone disorder A-T, influences chromosome end associations and telomere length . A possible hypothesis explaining these results is that the defective telomere metabolism in A-T cells are due to altered interactions between the telomeres and the nuclear matrix . We examined these interactions in nuclear matrix halos before and after radiation treatment . A difference was observed in the ratio of soluble versus matrix-associated telomeric DNA between cells derived from A-T and normal individuals . Ionizing radiation treatment affected the ratio of soluble versus matrix-associated telomeric DNA only in the A-T cells . To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, we examined such interactions in human cells expressing either a dominant-negative effect or complementation of the ATM gene . The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper motif mimics the altered interactions of telomere and nuclear matrix similar to that of A-T cells . A-T fibroblasts transfected with wild-type ATM gene had corrected telomere-nuclear matrix interactions . Further, we found that A-T cells had different micrococcal nuclease digestion patterns compared to normal cells before and after irradiation, indicating differences in nucleosomal periodicity in telomeres . These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix, and alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene. Chem Res Toxicol, 1999 Sep, 12(9), 796 - 801 A novel method for the isolation and identification of stable DNA adducts formed by Dibenzo{a,l}pyrene and Dibenzo{a,l}pyrene 11, 12-dihydrodiol 13,14-epoxides in vitro; Devanesan P et al.; Our laboratory previously reported the identification and quantification of depurinating DNA adducts of dibenzo{a,l}pyrene (DB{a,l}P) in vitro, which comprise about 84% of all the DNA adducts that are formed {Li, K.-M., et al . (1995) Biochemistry 34, 8043-8049} . To determine a complete adduct profile and identify both stable and depurinating DNA adducts, we have developed a relatively simple, nonradioactive method for the identification of stable DNA adducts by combining enzymatic digestion, HPLC, and fluorescence line-narrowing spectroscopy (FLNS) techniques . Calf thymus DNA, bound to either (+/-)-anti- or (+/-)-syn-DB{a,l}PDE or rat liver microsome-activated DB{a,l}P, was first digested to 3'-mononucleotides with micrococcal nuclease and spleen phosphodiesterase . The adducts were then separated by HPLC with an ion-pair column and identified by FLNS by using the spectra of standards for comparison . In reactions with (+/-)-anti-DB{a,l}PDE, three adducts, an anti-cis-DB{a,l}PDE-dGMP, an anti-trans-DB{a, l}PDE-dAMP, and an anti-cis-DB{a,l}PDE-dAMP, were identified by HPLC and FLNS . In reactions with (+/-)-syn-DB{a,l}PDE, a pair of syn-trans-DB{a,l}PDE-dGMP adducts as well as a syn-cis-DB{a, l}PDE-dGMP, a syn-cis-DB{a,l}PDE-dAMP, and a pair of syn-trans-DB{a, l}PDE-dAMP adducts were identified . From the digest of microsome-activated DB{a,l}P-bound DNA, a syn-trans-DB{a,l}PDE-dGMP, an anti-cis-DB{a,l}PDE-dGMP, a syn-trans-DB{a,l}PDE-dAMP, and a syn-cis-DB{a,l}PDE-dAMP adduct were identified . An anti-cis-DB{a, l}PDE-dAMP adduct was identified only by (32)P-postlabeling . A total of five of the stable adducts formed by DB{a,l}P and nine of the stable adducts formed by DB{a,l}PDE in vitro have been identified . These adducts were also correlated to adduct spots in the (32)P-postlabeling method by cochromatography with standards . Approximately 93% of the stable adducts formed in reactions with (+/-)-anti-DB{a,l}PDE, 90% of adducts with (+/-)-syn-DB{a,l}PDE, and 85% of adducts formed with microsome-activated DB{a,l}P have been identified as Gua or Ade adducts . Equal amounts of stable Gua and Ade adducts were observed in the microsome-catalyzed binding of DB{a, l}P to calf thymus DNA, while 1.4 times more Gua adducts than Ade adducts were obtained in reactions with (+/-)-anti- or (+/-)-syn-DB{a,l}PDE. Acta Crystallogr D Biol Crystallogr, 1999 Sep, 55 ( Pt 9), 1606 - 7 Crystallization and preliminary X-ray diffraction studies of undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26; Fujihashi M et al.; Undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26, one of the Z-prenyl chain-elongating enzymes, was crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate and lithium sulfate as precipitants . The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 127.2, b = 60.2, c = 75.7 A, beta = 105.6 degrees . The crystals diffract X-rays to at least 2.2 A resolution using synchrotron radiation and are suitable for high-resolution crystal structure analysis. J Biol Chem, 1999 Sep 17, 274(38), 27128 - 38 Selective nucleosome disruption by drugs that bind in the minor groove of DNA; Fitzgerald DJ et al.; Previous studies have shown that drugs which bind in the DNA minor groove reduce the curvature of bent DNA . In this article, we examined the effects of these drugs on the nucleosome assembly of DNA molecules that display different degrees of intrinsic curvature . DAPI (4,6-diamidino-2-phenylindole) inhibited the assembly of a histone octamer onto a 192-base pair circular DNA fragment from Caenorhabditis elegans and destabilized a nucleosome that was previously assembled on this segment . The inhibitory effect was highly selective since it was not seen with nonbent molecules, bent molecules with noncircular shapes, or total genomic DNA . This marked template specificity was attributed to the binding of the ligand to multiple oligo A-tracts distributed over the length of the fragment . A likely mechanism for the effect is that the bound ligand prevents the further compression of the DNA into the minor groove which is required for assembly of DNA into nucleosomes . To further characterize the effects of the drug on chromatin formation, a nucleosome was assembled onto a 322-base pair DNA fragment that contained the circular element and a flanking nonbent segment of DNA . The position of the nucleosome along the fragment was then determined using a variety of nuclease probes including exonuclease III, micrococcal nuclease, DNase I, and restriction enzymes . The results of these studies revealed that the nucleosome was preferentially positioned along the circular element in the absence of DAPI but assembled onto the nonbent flanking sequence in the presence of the drug . DAPI also induced the directional movement of the nucleosome from the circular element onto the nonbent flanking sequence when a nucleosome preassembled onto this template was exposed to the drug under physiologically relevant conditions. Nucleic Acids Res, 1999 Aug 15, 27(16), 3364 - 70 Heterochromatic silencing of Drosophila heat shock genes acts at the level of promoter potentiation; Cryderman DE et al.; In a variety of organisms, genes placed near heterochromatin are transcriptionally silenced . In order to understand the molecular mechanisms responsible for this block in transcription, high resolution in vivo chromatin structure analysis was performed on two heat shock genes, hsp26 and hsp70 . These genes normally reside in euchromatin where GAGA factor and RNA Pol II are present on the promoter prior to heat shock induction . P-element transformation experiments led to the identification of stocks in which these two genes were inserted within heterochromatin of the chromosome 4 telomeric region . These transgenes exhibit silencing that is partially suppressed by mutations in the gene encoding HP1 . Micrococcal nuclease analysis revealed that the heterochromatic transgenes were packaged in a more regular nucleosome array than when located in euchromatin . High resolution DNase I analysis demonstrated that GAGA factor and TFIID were not associated with these promoters in heterochromatin; potassium permanganate experiments showed a loss of Pol II association . Taken together, these data suggest that occlusion of trans-acting factors from their cis- acting regulatory elements leading to a block in promoter potentiation is a mechanism for heterochromatin gene silencing. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1997 Jun, 19(3), 222 - 6 {Effects of the glucoprotein component of musk on the functions of rat polymorphonuclear leukocytes activated by fMLP in vitro}; Wang W et al.; To investigate the effects of musk-1, a glucoprotein component isolated from the water extract of musk, on the release of superoxide anion as well as beta-glucuronidase and lysozyme of rat polymorphonuclear leukocytes activated by fMLP . An in vitro incubation system with rat polymorphonuclear leukocytes was used . Superoxide anion production was determined by cytochrome C reduction . beta-glucuronidase and lysozyme release was quantitated by enzyme reactions in which phenolphthaleinglucuronic acid and micrococcus lysodeikticus were as the substrates, respectively . In comparison with control, musk-1 at final concentrations of 1-100 micrograms/ml can increase superoxide anion production by 23.0%-83.6% and decrease beta-glucuronidase and lysozyme release by 7%-47% and 9%-22%, respectively, in rat polymorphonuclear leukocytes . It is concluded that Musk-1 can significantly affect the functions of rat polymorphonuclear leukocytes . Therefore, inhibition of lysosomal enzyme release might be considered as one of the mechanisms of anti-inflammatory role of musk. J Mol Biol, 1999 Aug 20, 291(3), 561 - 73 Demethylation of thymine residues affects DNA cleavage by endonucleases but not sequence recognition by drugs; Bailly C et al.; The 5-methyl group of thymidine residues protrudes into the major groove of double helical DNA . The structural influence of this exocyclic substituent has been examined using a PCR-made 160 bp fragment in which thymidine residues were replaced with uridine residues . We show that the dT-->dU substitution and the consequent deletion of the methyl group affects the cleavage of DNA by deoxyribonuclease I and micrococcal nuclease . Analysis of the DNase I cleavage sites, in terms of di and trinucleotides, indicates that homopolymeric tracts of d(AT) become significantly more susceptible to DNase I cleavage when uridine is substituted for thymidine residues . The results indicate that removal of the thymidine methyl groups from the major groove at AT tracts induces structural perturbations that transmit into the opposite minor groove, where they can be detected by endonuclease probing . In contrast, DNase I footprinting experiments with different mono and bis-intercalating drugs reveal that dT-->dU substitution does not markedly affect sequence-specific drug-DNA recognition in the minor or major groove of the double helix . The consequences of demethylation of thymidine residues are discussed in terms of changes in the minor groove width connected to variations in the flexibility of DNA and the intrinsic curvature associated with AT tracts . The study identifies the methyl group of thymine as an important molecular determinant controlling the width of the minor groove and/or the flexibility of the DNA . J Photochem Photobiol B, 1999 May, 50(1), 66 - 73 The degradation of L-histidine and trans- and cis-urocanic acid by bacteria from skin and the role of bacterial cis-urocanic acid isomerase; Hug DH et al.; UV-B radiation suppresses cell-mediated immunity . Histidine forms trans-urocanic acid (trans-UCA) enzymatically in the stratum corneum . Photoisomerization of trans-UCA to cis-urocanic acid (cis-UCA) has been proposed for the initiation of an immunosuppressive process . Many microorganisms described in the literature metabolize histidine and/or trans-UCA . Our enrichment cultures of soil and sewage contain organisms that can degrade cis-UCA . We have tested microorganisms for degradation of cis-UCA, trans-UCA, or L-histidine when they are incorporated at 0.2% in nutrient broth . Six out of 10 selected genera isolated by our clinical microbiology laboratory degrade one or more of the imidazole substrates . We have cultured over 60 aerobic isolates from human skin . Of these, 33 degrade one or more of the three imidazole substrates and 12 degrade cis-UCA . Isolates from BALB/c mice are also active on cis-UCA . We have identified a cis-UCA-degrading bacterium as Micrococcus luteus . Four ATCC strains of M . luteus have been tested and three are active on histidine or trans-UCA; two are active on cis-UCA . Micrococci that degrade cis-UCA contain a new enzyme, cis-UCA isomerase, which converts the substrate to the trans-isomer . This enzyme provides access to the classical L-histidine degradation pathway . We hypothesize that an epidermal microflora that degrades L-histidine, trans-UCA, or cis-UCA influences the concentration of urocanic acids on the skin and, thus, affects immune suppression. Ocul Immunol Inflamm, 1999 Mar, 7(1), 7 - 15 Clinical application of a homogeneous colorimetric assay for tear lysozyme; Klaeger AJ et al.; PURPOSE: Tear lysozyme and tear lactoferrin are enzymes synthesized by the lacrimal gland . Their concentration in human tears reflects tear gland function . Tear gland dysfunction can lead to ocular surface disease . We developed a colorimetric lysozyme assay . The objective of this study was to determine the diagnostic power and the clinical application of this assay that allows rapid and precise quantification of tear lysozyme . METHODS: Tear specimens of 120 eyes (30 Sjogren's patients and 30 controls) were collected using standardized filter paper discs . Tear lysozyme concentration was determined using p-nitrophenyl penta-N-acetyl-beta-chitopentaoside as substrate in the colorimetric assay . The results were compared to clinical findings and to two commonly used tests, the Micrococcus agar diffusion assay for tear lysozyme and the tear lactoferrin immunodiffusion assay . RESULTS: The colorimetric assay showed a good dose-response relationship . The use of the assay as a method of diagnosing aqueous tear deficiency, using the clinical findings and the medical history as gold standard, demonstrated 85% sensitivity and 92% specificity . The results of the colorimetric assay when compared with the Micrococcus agar diffusion assay showed a linear relationship of r=0.77; when compared with the lactoferrin immunoassay r=0.73 . CONCLUSIONS: The colorimetric assay is simple to perform and does not require sophisticated laboratory equipment and personnel . Results can be precisely quantified within one hour after tear collection . The diagnostic power of the test is comparable to previously reported assays for lysozyme and lactoferrin and will be useful in the diagnosis of ocular surface disease. Indian J Biochem Biophys, 1998 Oct, 35(5), 266 - 72 Chemical modification of 3-HBA-6-hydroxylase by phenylglyoxal: kinetic and physicochemical studies on the modified enzyme; Sumathi S et al.; The inactivation of 3-HBA-6-hydroxylase isolated from Micrococcus species by phenylglyoxal and protection offered by 3-HBA against inactivation indicate the presence of arginine residue at or near the substrate binding site . The loss of enzyme activity was time and concentration dependent and displayed pseudo-first order kinetics . A 'n' value of 0.9 was obtained thus suggesting the modification of a single arginine residue per active site which led to the loss of enzyme activity . The enzyme activity could be restored by extensive dialysis at neutral pH . Quenching of the intrinsic fluorescence and reduction in the ellipticity value at 280 nm in the near-UV CD spectrum of the enzyme was noticed after its treatment with phenylglyoxal . These observations probably imply distinct perturbations in the environment of adjacent aromatic amino acid residues such as tryptophan as a consequence of arginine modification. Biol Pharm Bull, 1999 Jun, 22(6), 654 - 6 Purification, properties and reactivity of the esterase from Micrococcus sp; Imura A et al.; The esterase from Micrococcus sp., which hydrolyzes n-propyl-2-fluorocyclopropanecarboxylate (3) enantioselectively, was highly purified by three types of chromatography . The purified enzyme was inactivated by Hg and diisopropyl fluorophosphate (DFP) . It was a monomer with a molecular weight of about 35000 . The enzyme exhibits esterase activity towards many aliphatic propyl esters . The enantioselectivity for substrate (3) using purified enzyme did not differ from that of crude enzyme. Arch Microbiol, 1999 Jul, 172(1), 9 - 14 Stimulation of the multiplication of Micrococcus luteus by an autocrine growth factor; Mukamolova GV et al.; Viable cells of Micrococcus luteus secrete a proteineous growth factor (Rpf) which promotes the resuscitation of dormant, nongrowing cells to yield normal, colony-forming bacteria . When washed M . luteus cells were used as an inoculum, there was a pronounced influence of Rpf on the true lag phase and cell growth on lactate minimal medium . In the absence of Rpf, there was no increase in colony-forming units for up to 10 days . When the inoculum contained less than 10(5) cells ml-1, macroscopically observable M . luteus growth was not obtained in succinate minimal medium unless Rpf was added . Incubation of M . luteus in the stationary phase for 100 h resulted in a failure of the cells to grow in lactate minimal medium from inocula of small size although the viability of these cells was close to 100% as estimated using agar plates made from lactate minimal medium or rich medium . The underestimation of viable cells by the most-probable-number (MPN) method in comparison with colony-forming units was equivalent to the requirement that at least 10(5) cells grown on succinate medium, 10(3) cells from old stationary phase, or approximately 10-500 washed cells are required per millilitre of inoculum for growth to lead to visible turbidity . The addition of Rpf in the MPN dilutions led to an increase of the viable cell numbers estimated to approximately the same levels as those determined by colony-forming units . Thus, a basic principle of microbiology - "one cell-one culture" - may not be applicable in some circumstances in which the metabolic activity of "starter" cells is not sufficient to produce enough autocrine growth factor to support cell multiplication. Jpn J Cancer Res, 1999 May, 90(5), 523 - 9 Differentiation of a cell line of human cervical argyrophil small cell carcinoma to macrophage lineage cells; Ichimura H et al.; To investigate the origin of argyrophil small cell carcinoma (ASCC) of the uterine cervix, we examined the influence of dibutyryl cyclic adenosine 3',5'-monophosphate (dB-cAMP), a known differentiation inducer, on the characteristics of an ASCC cell line, TC-YIK, which has been shown to be a useful in vitro experimental model of ASCC . In TC-YIK cells after treatment with dB-cAMP, two specific antigenic markers of macrophages, CD14 and human leukocyte antigen-DR, were detected by flow cytometric analysis . In addition, interferon-gamma mRNA was detected by reverse transcription-polymerase chain reaction and interferon-gamma protein was detected by ELISA . More than 90% of the cells stained positive for alpha-naphthyl butyrate esterase, 1% of the cells showed phagocytotic activity against Micrococcus lysodeikticus, and 22% of the cells had M . lysodeikticus adsorbed on their surface . Furthermore, granulocyte-macrophage colony stimulating factor accelerated the proliferation of TC-YIK cells . These results indicate that dB-cAMP promotes differentiation of ASCC cells to macrophages . In contrast, less than 10% of the cells showed stellate morphology, suggesting differentiation to neuronal cells after treatment with dB-cAMP, as reported previously . Thus, TC-YIK cells have been shown to differentiate both into macrophage lineage cells and neuronal cells, suggesting that ASCC originates from undifferentiated stem cells. J Mol Biol, 1999 Jun 18, 289(4), 675 - 81 Archaeal nucleosome positioning sequence from Methanothermus fervidus; Pereira SL et al.; DNA in Methanothermus fervidus, a hyperthermophilic archaeon, is constrained into archaeal nucleosomes in vivo by the archaeal histones HMfA and HMfB . Here, w