Proteomics . 2005 Jan 3; {Epub ahead of print} Lipoproteomics I: Mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry; Karlsson H et al.; The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear . Therefore, detailed information about the protein composition of LDL may contribute to reveal its role in atherogenesis and the mechanisms that lead to coronary disease in humans . Here, we sought to map the proteins in human LDL by a proteomic approach . LDL was isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix assisted laser desorption/ionization-time of flight-mass spectrometry and with amino acid sequencing using electrospray ionization tandem mass spectrometry . These procedures identified apo B-100, apo C-II, apo C-III (three isoforms), apo E (four isoforms), apo A-I (two isoforms), apo A-IV, apo J and apo M (three isoforms not previously described) . In addition, three proteins that have not previously been identified in LDL were found: serum amyloid A-IV (two isoforms), calgranulin A, and lysozyme C . The identities of apo M, calgranulin A, and lysozyme C were confirmed by sequence information obtained after collision-induced dissociation fragmentation of peptides characteristic for these proteins . Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus . These results indicate that in addition to the dominating apo B-100, LDL contains a number of other apolipoproteins, many of which occur in different isoforms . The demonstration, for the first time, that LDL contains calgranulin A and lysozyme C raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall. BMC Infect Dis . 2004 Dec 22;4(1):62 {Epub ahead of print} Catheter-related bacteremia due to Kocuria rosea in a patient undergoing peripheral blood stem cell transplantation; Altuntas F et al.; BACKGROUND: Micrococcus species may cause intracranial abscesses, meningitis, pneumonia, and septic arthritis in immunosuppressed or immunocompetent hosts . In addition, strains identified as Micrococcus spp . have been reported recently in infections associated with indwelling intravenous lines, continuous ambulatory peritoneal dialysis fluids, ventricular shunts and prosthetic valves . CASE PRESENTATION: We report on the first case of a catheter-related bacteremia caused by Kocuria rosea, a gram-positive microorganism belonging to the family Micrococcaceae, in a 39-year-old man undergoing peripheral blood stem cell transplantation due to relapsed Hodgkin disease . This uncommon pathogen may cause opportunistic infections in immunocompromised patients . CONCLUSIONS: This report presents a case of Kocuria rosea catheter related bacteremia after stem cell transplantation successfully treated with vancomycin and by catheter removal. Mol Biochem Parasitol, 2005 Jan, 139(1), 65 - 73 Mosquito innate immunity: involvement of beta 1,3-glucan recognition protein in melanotic encapsulation immune responses in Armigeres subalbatus; Wang X et al.; Beta 1,3-glucan recognition proteins (GRP) have specific affinity for beta 1,3-glucan, a component on the surface of fungi and bacteria . By interacting with beta 1,3-glucan, GRP initiates activation of prophenoloxidase, a key enzyme in the signaling pathway leading to melanotic encapsulation in invertebrates . In this study, we characterize a novel hemocyte-specific GRP from the mosquito, Armigeres subalbatus (AsGRP) . The 1.57kb cDNA clone encodes a 499 deduced amino acid sequence, which contains a region that displays significant similarity to the glucanase-like regions of other GRPs and Gram-negative bacteria binding proteins found in other organisms . AsGRP is constitutively expressed in the hemolymph of adult female mosquitoes, and is upregulated following challenge with Escherichia coli, Micrococcus luteus, and the filarial worm Dirofilaria immitis . AsGRP specifically recognizes curdlan (insoluble beta 1,3-glucan), but not mannose or N-acetyl-d-glucosamine . AsGRP binds a low percentage of E . coli, most M . luteus and D . immitis microfilariae . AsGRP double-stranded RNA interference strongly inhibits melanotic encapsulation of D . immitis in Ar . subalbatus . These results suggest that AsGRP has the capacity to bind to a variety of pathogens, functions as a pattern recognition receptor, and is required for effective melanotic encapsulation immune responses in Ar . subalbatus. Eur J Biochem, 2004 Dec, 271(23-24), 4825 - 33 Antimicrobial activity of histones from hemocytes of the Pacific white shrimp; Patat SA et al.; The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated . In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp, Litopenaeus vannamei . The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching . The L . vannamei histone proteins were found to be highly homologous to histones of other species . Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin . Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibition against Micrococcus luteus . Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 microm while a mixture of histones H2B and H4 was active at 3 microm . In addition, a fraction containing a fragment of histone H1 was also found to be active . The synthetic peptide similar to buforin was active at submicromolar concentrations . These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a defense mechanism previously unreported in an invertebrate . Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought. Protein Expr Purif, 2004 Dec, 38(2), 272 - 8 Molecular cloning, overexpression, and purification of Micrococcus luteus K-3-type glutaminase from Aspergillus oryzae RIB40; Masuo N et al.; We have for the first time found and cloned the cDNA (AoglsA) of Aspergillus oryzae RIB40, which encodes a 49.9-kDa protein sharing 40% homology with the salt-tolerant glutaminase of Micrococcus luteus K-3 (Micrococcus glutaminase) . AoglsA was subcloned into a series of expression vectors and expressed in Saccharomyces cerevisiae and Escherichia coli . The gene product, which we named AoGls, showed glutaminase activity and was produced in a cell wall fraction of S . cerevisiae and a soluble protein in E . coli . The highest expression level of 186 U/mg was obtained when the AoglsA was inserted into six bases downstream of the Shine-Dalgarno (SD) sequence of pKK223-3 and expressed in E . coli Rosetta (DE3) . AoGls was purified by SuperQ-TOYOPEARL, glutamine affinity chromatography, and Butyl-TOYOPEARL . This is the first report on the overexpression and purification of a M . luteus K-3-type glutaminase cloned from an eucaryote. Biofouling, 2004 Jun, 20(3), 167 - 75 Photocatalytic inhibition of microbial adhesion by anodized titanium; Gopal J et al.; Biofouling is one of the concerns in the use of titanium for seawater cooled condensers of power plants . Earlier studies have shown that anodized titanium and its alloys with a thin film of anatase (TiO(2)) on its surface can inhibit attachment of Pseudomonas sp . when illuminated with near-UV light (350 - 380 nm) . In the present study, a comparison of the photocatalytic inhibition of microbial attachment on titanium surfaces anodized at different voltages was carried out . Thin films of anatase of varying thickness were produced on titanium grade-2 by anodizing in dilute orthophosphoric acid solution at 30 V, 50 V and 100 V . The photocatalytic efficiency of these anodized surfaces was measured by the methylene blue degradation method . The anodised surfaces were exposed to liquid cultures of Gram-negative Pseudomonas sp., Gram-positive Micrococcus sp . and to a mixed algal culture . Photocatalytic inhibition of microbial attachment was maximum on the titanium surface anodized at 30 V, followed by the surface anodized at 50 V and then at 100 V . The photocatalytic inhibition of microbial attachment was also found to be dependent on the cell wall characteristics of the organism . The Gram-negative Pseudomonas sp . with a lipoproteinaceous outer membrane was the most susceptible to the photocatalytic effect, while the Gram-positive Micrococcus sp . with peptidoglycan cell wall showed moderate susceptibility and the algae with siliceous cell wall showed no susceptibility at all. Mikrobiologiia, 2004 Jul-Aug, 73(4), 567 - 70 {The ability of saprotrophic bacteria isolated from natural habitats to lyse yeasts} {Ultrastructure of resting cells of some non-spore-forming bacteria} {No authors listed} Using electron microscopy (ultrathin sections and freeze-fractures), we investigated the ultrastructure of the resting cells formed in the cultures of Micrococcus luteus, Arthrobacter globiformis, and Pseudomonas aurantiaca under conditions of prolonged incubation (up to 9 months) . These resting cells included cyst-like forms that were characterized by complex cell structure and the following ultrastructural properties: (i) a thickened or multiprofiled cell wall (CW), typically made up of a layer of the preexisting CW and one to three de novo synthesized murein layers; (ii) a thick, structurally differentiated capsule; (iii) presence of large intramembrane particles (d = 180-270 A), occurring both on the PF and EF sides of the membrane fractures of M . luteus and A . globiformis; (iv) a peculiar structure of the cytoplasm, which was either fine-grained or lumpy (coarse-grained) in different parts of the cell population; and (v) a condensed nucleoid . Intense formation of cyst-like cells occurred in aged (2- to 9-month-old) bacterial cultures grown on diluted complex media or on nitrogen-, carbon-, and phosphorus-limited synthetic media, as well as in suspensions of cells incubated in media with sodium silicate . The general morphological properties, ultrastructural organization, and physiological features of cyst-like cells formed during the developmental cycle suggest that constitutive dormancy is characteristic of non-spore-forming bacteria. J Virol, 2004 Nov, 78(22), 12566 - 75 ORC, MCM, and histone hyperacetylation at the Kaposi's sarcoma-associated herpesvirus latent replication origin; Stedman W et al.; The viral genome of Kaposi's sarcoma-associated herpesvirus (KSHV) persists as an extrachromosomal plasmid in latently infected cells . The KSHV latency-associated nuclear antigen (LANA) stimulates plasmid maintenance and DNA replication by binding to an approximately 150-bp region within the viral terminal repeats (TR) . We have used chromatin immunoprecipitation assays to demonstrate that LANA binds specifically to the replication origin sequence within the KSHV TR in latently infected cells . The latent replication origin within the TR was also bound by LANA-associated proteins CBP, double-bromodomain-containing protein 2 (BRD2), and the origin recognition complex 2 protein (ORC2) and was enriched in hyperacetylated histones H3 and H4 relative to other regions of the latent genome . Cell cycle analysis indicated that the minichromosome maintenance complex protein, MCM3, bound TR in late-G(1)/S-arrested cells, which coincided with the loss of histone H3 K4 methylation . Micrococcal nuclease studies revealed that TRs are embedded in a highly ordered nucleosome array that becomes disorganized in late G(1)/S phase . ORC binding to TR was LANA dependent when reconstituted in transfected plasmids . DNA affinity purification confirmed that LANA, CBP, BRD2, and ORC2 bound TR specifically and identified the histone acetyltransferase HBO1 (histone acetyltransferase binding to ORC1) as a potential TR binding protein . Disruption of ORC2, MCM5, and HBO1 expression by small interfering RNA reduced LANA-dependent DNA replication of TR-containing plasmids . These findings are the first demonstration that cellular replication and origin licensing factors are required for KSHV latent cycle replication . These results also suggest that the KSHV latent origin of replication is a unique chromatin environment containing histone H3 hyperacetylation within heterochromatic tandem repeats. Methods Mol Biol, 2004, 291, 13 - 20 Modification of the 32P-Postlabeling Method to Detect a Single Adduct Species as a Single Spot; Ochiai M et al.; The original 32P-postlabeling method developed by Randerath and his colleagues has been modified to detect a single type of adduct as a single spot in thin-layer chromatography (TLC), because some types of adducts gave multiple adduct spots by the original method . In the remodified methods, DNA is first digested with micrococcal nuclease and phophodiesterase II and then labeled with {gamma-32P}ATP under standard or adduct-intensification conditions . Since the labeled digest includes adducted mono-, di-, and/or oligo-deoxynucleotides, it is further treated with phosphatase and phosphodiesterase prior to TLC . The labeled digest is treated with nuclease P1 (NP1) in method I, and with T4 polynucleotide kinase and NP1 in method II, and then with phosphodiesterase I in both cases, and subjected to TLC . The advantage of these methods is that the number of adduct species formed can be estimated by TLC. Glycobiology . 2004 Oct 13; {Epub ahead of print} Dimannosyldiacylglycerol serves as a lipid anchor precursor in the assembly of the membrane-associated lipomannan in Micrococcus luteus1; Pakkiri LS et al.; Based on recent analytical and enzymological studies, a topological model for the role of alpha-Dmannosyl-(1-->3)-alpha-D-mannosyl-(1-->3)-diacylglycerol (Man2-DAG) as a lipid anchor precursor and mannosylphosphorylundecaprenol (Man-P-Und) as a mannosyl donor in the assembly of a membrane-associated lipomannan (LM) in M . luteus has been proposed . In this study, a {(3)H}mannose-suicide selection procedure has been used to identify temperature-sensitive (ts) mutants defective in LM assembly . Two micrococcal mutants with abnormally low levels of Man2-DAG and LM at the non-permissive temperature (37 degrees C), mms1 and mms2, have been isolated and characterized . In vivo and in vitro biochemical assays indicate that mms1 cells have a defect in the mannosyltransferase catalyzing the conversion of Man-DAG to Man2-DAG, and mms2 has a temperature-sensitive defect in the synthesis of Man-P-Und . Since mms1 cells are depleted of endogenous Man2-DAG, membranes from this mutant efficiently converted purified, exogenous {(3)H}Man2-DAG to {(3)H}LM by a Man-P-Und-dependent process . An obligatory role for Man-P-Und as a mannosyl donor in the elongation process was also demonstrated by showing that the conversion of exogenous {(3)H}Man2-DAG to {(3)H}LM by membranes from mms1 cells in the presence of GDP-Man was inhibited by amphomycin . In addition, consistent with Man2-DAG serving as a lipid anchor precursor for LM assembly, endogenous, pre-labeled {(3)H}Man2-DAG was converted to {(3)H}LM when membranes from mms2 cells were incubated with purified, exogenous Man-P-Und . These studies provide the first direct proof for the role of Man2-DAG as the lipid anchor precursor for LM, and suggest that Man2-DAG may be essential for the normal growth of M . luteus cells. J Biol Chem, 2004 Dec 10, 279(50), 52069 - 74 Epub 2004 Dec 10. The histone chaperone Asf1p mediates global chromatin disassembly in vivo; Adkins MW et al.; The packaging of the eukaryotic genome into chromatin is likely to be mediated by chromatin assembly factors, including histone chaperones . We investigated the function of the histone H3/H4 chaperones anti-silencing function 1 (Asf1p) and chromatin assembly factor 1 (CAF-1) in vivo . Analysis of chromatin structure by accessibility to micrococcal nuclease and DNase I digestion demonstrated that the chromatin from CAF-1 mutant yeast has increased accessibility to these enzymes . In agreement, the supercoiling of the endogenous 2mu plasmid is reduced in yeast lacking CAF-1 . These results indicate that CAF-1 mutant yeast globally under-assemble their genome into chromatin, consistent with a role for CAF-1 in chromatin assembly in vivo . By contrast, asf1 mutants globally over-assemble their genome into chromatin, as suggested by decreased accessibility of their chromatin to micrococcal nuclease and DNase I digestion and increased supercoiling of the endogenous 2mu plasmid . Deletion of ASF1 causes a striking loss of acetylation on histone H3 lysine 9, but this is not responsible for the altered chromatin structure in asf1 mutants . These data indicate that Asf1p may have a global role in chromatin disassembly and an unexpected role in histone acetylation in vivo. Antimicrob Agents Chemother, 2004 Oct, 48(10), 3662 - 9 Multilayer polyelectrolyte films functionalized by insertion of defensin: a new approach to protection of implants from bacterial colonization; Etienne O et al.; Infection of implanted materials by bacteria constitutes one of the most serious complications following prosthetic surgery . In the present study, we developed a new strategy based on the insertion of an antimicrobial peptide (defensin from Anopheles gambiae mosquitoes) into polyelectrolyte multilayer films built by the alternate deposition of polyanions and polycations . Quartz crystal microbalance and streaming potential measurements were used to follow step by step the construction of the multilayer films and embedding of the defensin within the films . Antimicrobial assays were performed with two strains: Micrococcus luteus (a gram-positive bacterium) and Escherichia coli D22 (a gram-negative bacterium) . The inhibition of E . coli D22 growth at the surface of defensin-functionalized films was found to be 98% when 10 antimicrobial peptide layers were inserted in the film architecture . Noticeably, the biofunctionalization could be achieved only when positively charged poly(l-lysine) was the outermost layer of the film . On the basis of the results of bacterial adhesion experiments observed by confocal or electron microscopy, these observations could result from the close interaction of the bacteria with the positively charged ends of the films, which allows defensin to interact with the bacterial membrane structure . These results open new possibilities for the use of such easily built and functionalized architectures onto any type of implantable biomaterial . The modified surfaces are active against microbial infection and represent a novel means of local host protection. Astrobiology, 2004 Fall, 4(3), 345 - 58 The structure of resting bacterial populations in soil and subsoil permafrost; Soina VS et al.; The structure of individual cells in microbial populations in situ of the Arctic and Antarctic permafrost was studied by scanning and transmission electron microscopy methods and compared with that of cyst-like resting forms generated under special conditions by the non-spore-forming bacteria Arthrobacter and Micrococcus isolated from the permafrost . Electron microscopy examination of microorganisms in situ revealed several types of bacterial cells having no signs of damage, including "dwarf" curved forms similar to nanoforms . Intact bacterial cells in situ and frozen cultures of the permafrost isolates differed from vegetative cells by thickened cell walls, the altered structure of cytoplasm, and the compact nucleoid, and were similar in these features to cyst-like resting forms of non-spore-forming "permafrost" bacterial strains of Arthrobacter and Micrococcus spp . Cyst-like cells, being resistant to adverse external factors, are regarded as being responsible for survival of the non-spore-formers under prolonged exposure to subzero temperatures and can be a target to search for living microorganisms in natural environments both on the Earth and on extraterrestrial bodies. Mol Endocrinol, 2005 Jan, 19(1), 138 - 47 Epub 2004 Sep 16. The pituitary-specific transcription factor, Pit-1, can direct changes in the chromatin structure of the prolactin promoter; Kievit P et al.; The chromatin structure of a promoter is an important determinant of its transcriptional activity . Many promoters are assembled into repressive polynucleosomal arrays that are subsequently remodeled to allow for the activation of gene expression . This study addresses the contribution of a single transcription factor, Pit-1, in orchestrating the chromatin structure of the prolactin gene . Utilizing an in vivo reconstitution system, we found that Pit-1 can bind to multiple sites in the chromatin-assembled 5'-flanking region of the prolactin gene and activate transcription from the chromatin-assembled template . Interestingly, Pit-1 was able to substantially alter micrococcal nuclease digestion of the prolactin 5'-flanking region, and the results are consistent with presence of a translationally positioned nucleosome on the prolactin promoter . Changes in micrococcal nuclease digestion were also observed with a truncated Pit-1 mutant containing only the DNA-binding domain . As the truncation mutant was unable to activate transcription from the chromatin-assembled template, the ability of Pit-1 to alter chromatin structure of the prolactin gene is not dependent on transcriptional activation . We propose that Pit-1 likely plays a role in altering chromatin to facilitate recruitment and subsequent transcriptional activation by additional factors. Cancer Res, 2004 Sep 15, 64(18), 6416 - 23 Potential role of a novel transcriptional coactivator PELP1 in histone H1 displacement in cancer cells; Nair SS et al.; The estrogen receptor plays an important role in breast cancer progression . Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), also called modulator of nongenomic activity of estrogen receptor (MNAR), a novel coactivator of estrogen receptor, modulates estrogen receptor transactivation functions . The mechanisms by which PELP1 modulates estrogen receptor genomic functions is not known . Here, using biochemical and scanning confocal microscopic analysis, we have demonstrated nuclear localization and functional implications of PELP1 . Subnuclear fractionation showed PELP1 association with chromatin and nuclear matrix fractions . Ligand stimulation promoted recruitment of PELP1 to 17beta-estradiol responsive promoters, its colocalization with acetylated H3, and increased PELP1-associated histone acetyltransferase enzymatic activity . Far Western analysis revealed that PELP1 interacts with histone 1 and 3, with more preference toward histone 1 . Using deletion analysis, we have identified the PELP1 COOH-terminal region as the histone 1 binding site . The PELP1 mutant lacking histone 1-binding domain acts as a dominant-negative and blocks estrogen receptor alpha-mediated transcription . Chromatin immunoprecipitation analysis showed a cyclic association and dissociation of PELP1 with the promoter, with recruitment of histone 1 and PELP1 occurring in opposite phases . PELP1 overexpression increased the micrococcal nuclease sensitivity of estrogen response element-containing nucleosomes . Our results provide novel insights about the transcription regulation of PELP1 and suggest that PELP1 participates in chromatin remodeling activity via displacement of histone 1 in cancer cells. Arch Gerontol Geriatr, 1999 Apr, 28(2), 149 - 58 Effect of 17beta estradiol and progesterone on the conformation of the chromatin of the liver of female Japanese quail during aging; Mahendra G et al.; Plasma levels of 17beta estradiol and progesterone were measured by radioimmunoassay in immature, adult and old female Japanese quails . The levels of progesterone and the progesterone/estradiol ratio were maximum in adult, egg laying birds . Conformation of the chromatin of the liver of birds of various ages before and after administration of steroid hormones was studied by digesting the nuclei with micrococcal nuclease (MNase) and pancreatic deoxyribonuclease I (DNase I) followed by electrophoretic resolution of the DNA fragments . The pattern of bands shows that the chromatin of the adult is more sensitive to DNase I and MNase than that of young and old birds . Administration of 17beta estradiol and progesterone enhances the digestion of the chromatin by DNase I and MNase in old birds . Such enhancement does not occur in adult birds as the chromatin is already in relaxed and open conformation due to the high levels of the hormones already present at this age. Mol Cells, 2004 Aug 31, 18(1), 100 - 6 Chromatin remodeling facilitates DNA incision in UV-damaged nucleosomes; Lee K et al.; The DNA repair machinery must locate and repair DNA damage all over the genome . As nucleosomes inhibit DNA repair in vitro, it has been suggested that chromatin remodeling might be required for efficient repair in vivo . To investigate a possible contribution of nucleosome dynamics and chromatin remodeling to the repair of UV-photoproducts in nucleosomes, we examined the effect of a chromatin remodeling complex on the repair of UV-lesions by Micrococcus luteus UV endonuclease (ML-UV endo) and T4-endonuclease V (T4-endoV) in reconstituted mononucleosomes positioned at one end of a 175-bp long DNA fragment . Repair by ML-UV endo and T4-endoV was inefficient in mononucleosomes compared with naked DNA . However, the human nucleosome remodeling complex, hSWI/SNF, promoted more homogeneous repair by ML-UV endo and T4-endo V in reconstituted nucleosomes . This result suggests that recognition of DNA damage could be facilitated by a fluid state of the chromatin resulting from chromatin remodeling activities. J Biol Chem, 2004 Dec 10, 279(50), 52376 - 81 Epub 2004 Dec 10. Catalase reaction by myoglobin mutants and native catalase: mechanistic investigation by kinetic isotope effect; Kato S et al.; The catalase reaction has been studied in detail by using myoglobin (Mb) mutants . Compound I of Mb mutants (Mb-I), a ferryl species (Fe(IV)=O) paired with a porphyrin radical cation, is readily prepared by the reaction with a nearly stoichiometric amount of m-chloroperbenzoic acid . Upon the addition of H2O2 to an Mb-I solution, Mb-I is reduced back to the ferric state without forming any intermediates . This indicates that Mb-I is capable of performing two-electron oxidation of H2O2 (catalatic reaction) . Gas chromatography-mass spectroscopy analysis of the evolved O2 from a 50:50 mixture of H2(18)O2/H2(16)O2 solution containing H64D or F43H/H64L Mb showed the formation of 18O2 (m/e = 36) and 16O2 (m/e = 32) but not 16O18O (m/e = 34) . This implies that O2 is formed by two-electron oxidation of H2O2 without breaking the O-O bond . Deuterium isotope effects on the catalatic reactions of Mb mutants and catalase suggest that the catalatic reactions of Micrococcus lysodeikticus catalase and F43H/H64L Mb proceed via an ionic mechanism with a small isotope effect of less than 4.0, since the distal histidine residue is located at a proper position to act as a general acid-base catalyst for the ionic reaction . In contrast, other Mb mutants such as H64X (X is Ala, Ser, and Asp) and L29H/H64L Mb oxidize H2O2 via a radical mechanism in which a hydrogen atom is abstracted by Mb-I with a large isotope effect in a range of 10-29, due to a lack of the general acid-base catalyst. Genome Biol . 2004;5(9):R62 . Epub 2004 Aug 20. Global nucleosome occupancy in yeast; Bernstein BE et al.; BACKGROUND: Although eukaryotic genomes are generally thought to be entirely chromatin-associated, the activated PHO5 promoter in yeast is largely devoid of nucleosomes . We systematically evaluated nucleosome occupancy in yeast promoters by immunoprecipitating nucleosomal DNA and quantifying enrichment by microarrays . RESULTS: Nucleosome depletion is observed in promoters that regulate active genes and/or contain multiple evolutionarily conserved motifs that recruit transcription factors . The Rap1 consensus was the only binding motif identified in a completely unbiased search of nucleosome-depleted promoters . Nucleosome depletion in the vicinity of Rap1 consensus sites in ribosomal protein gene promoters was also observed by real-time PCR and micrococcal nuclease digestion . Nucleosome occupancy in these regions was increased by the small molecule rapamycin or, in the case of the RPS11B promoter, by removing the Rap1 consensus sites . CONCLUSIONS: The presence of transcription factor-binding motifs is an important determinant of nucleosome depletion . Most motifs are associated with marked depletion only when they appear in combination, consistent with a model in which transcription factors act collaboratively to exclude nucleosomes and gain access to target sites in the DNA . In contrast, Rap1-binding sites cause marked depletion under steady-state conditions . We speculate that nucleosome depletion enables Rap1 to define chromatin domains and alter them in response to environmental cues. Methods Mol Biol, 2005, 288, 319 - 42 DNase I footprinting of small molecule binding sites on DNA; Bailly C et al.; Nuclease footprinting techniques were initially developed to investigate protein-deoxyribonucleic acid (DNA) interactions but these tools of molecular biology have also become instrumental for probing sequence-selective binding of small molecules to DNA . Here, the method is described and technical details are given for performing deoxyribonuclease (DNase) I footprinting with DNA-binding drugs . An example is presented where DNase I is used (as well as DNase II and micrococcal nuclease) to probe the patterns of sequence-selective recognition of DNA by the anticancer antibiotic actinomycin D . DNase I is a convenient endonuclease for detecting and locating the position of actinomycin-binding sites within GC-rich sequences. J Virol, 2004 Sep, 78(18), 10178 - 86 During lytic infection herpes simplex virus type 1 is associated with histones bearing modifications that correlate with active transcription; Kent JR et al.; Herpes simplex virus type 1 (HSV-1) is a large (150-kb) double-stranded DNA virus that forms latent infections in neuronal cells of the human peripheral nervous system . Previous work determined that the HSV-1 genome is found in an ordered nucleosomal structure during latent infection . However, during lytic infection, it was unclear whether viral DNA was in a chromatin state . We examined HSV-1 during lytic infection using micrococcal nuclease digestion and chromatin immunoprecipitation . The HSV-1 genome is at least partially nucleosomal, although apparently not in a regular repeating structure . Analysis of histones associated with HSV-1, within both the promoter and the transcribed regions, revealed covalent amino tail modifications similar to those associated with active host mammalian genes . Certain of the modifications were detected in the temporal order expected of the immediate-early, early, and late gene classes . These data suggest that productive infection may be accompanied by acquisition of a permissive chromatin state . Cytogenet Genome Res, 2004, 107(1-2), 132 - 8 An evolutionary change in telomere sequence motif within the plant section Asparagales had significance for telomere nucleoprotein complexes; Rotkova G et al.; In association with a phylogenetic tree of Asparagales, our previous results showed that a distinct clade included plant species where the ancestral, Arabidopsis-type of telomeric repeats (TTTAGGG)n had been partially, or fully, replaced by the human-type telomeric sequence (TTAGGG)n . Telomerases of these species synthesize human repeats with a high error rate in vitro . Here we further characterize the structure of telomeres in these plants by analyzing the overall arrangement of major and minor variants of telomeric repeats using fluorescence in situ hybridization on extended DNA strand(s) . Whilst the telomeric array is predominantly composed of the human variant of the repeat, the ancestral, Arabidopsis-type of telomeric repeats was ubiquitously observed at one of the ends and/or at intercalary positions of extended telomeric DNAs . Another variant of the repeat typical of Tetrahymena was observed interspersed in about 20% of telomerics . Micrococcal nuclease digestions indicated that Asparagales plants with a human-type of telomere have telomeric DNA organised into nucleosomes . However, unexpectedly, the periodicity of the nucleosomes is not significantly shorter than bulk chromatin as is typical of telomeric chromatin . Using electrophoretic mobility shift assays we detected in Asparagales plants with a human type of telomere a 40-kDa protein that forms complexes with both Arabidopsis- and human-type G-rich telomeric strands . However, the protein shows a higher affinity to the ancestral Arabidopsis-type sequence . Two further proteins were found, a 25-kDa protein that binds specifically to the ancestral sequence and a 15-kDa protein that binds to the human-type telomeric repeat . We discuss how the organisation of the telomere repeats in Asparagales may have arisen and stabilised the new telomere at the point of mutation . J Cell Biol, 2004 Aug 16, 166(4), 493 - 505 Epub 2004 Aug 09. Mouse centric and pericentric satellite repeats form distinct functional heterochromatin; Guenatri M et al.; Heterochromatin is thought to play a critical role for centromeric function . However, the respective contributions of the distinct repetitive sequences found in these regions, such as minor and major satellites in the mouse, have remained largely unsolved . We show that these centric and pericentric repeats on the chromosomes have distinct heterochromatic characteristics in the nucleus . Major satellites from different chromosomes form clusters associated with heterochromatin protein 1alpha, whereas minor satellites are individual entities associated with centromeric proteins . Both regions contain methylated histone H3 (Me-K9 H3) but show different micrococcal nuclease sensitivities . A dinucleosome repeating unit is found specifically associated with major satellites . These domains replicate asynchronously, and chromatid cohesion is sustained for a longer time in major satellites compared with minor satellites . Such prolonged cohesion in major satellites is lost in the absence of Suv39h histone methyltransferases . Thus, we define functionally independent centromeric subdomains, which spatio-temporal isolation is proposed to be important for centromeric cohesion and dissociation during chromosome segregation. J Biol Chem, 2004 Oct 8, 279(41), 42677 - 86 Epub 2004 Aug 04. On the mechanism of constitutive Pdr1 activator-mediated PDR5 transcription in Saccharomyces cerevisiae: evidence for enhanced recruitment of coactivators and altered nucleosome structures; Gao C et al.; Drug resistance as a result of overexpression of drug transporter genes presents a major obstacle in the treatment of cancers and infections . The molecular mechanisms underlying transcriptional up-regulation of drug transporter genes remains elusive . Employing Saccharomyces cerevisiae as a model, we analyzed here transcriptional regulation of the drug transporter gene PDR5 in a drug-resistant pdr1-3 strain . This mutant bears a gain-of-function mutation in PDR1, which encodes a transcriptional activator for PDR5 . Similar to the well studied model gene GAL1, we provide evidence showing that PDR5 belongs to a group of genes whose transcription requires the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex . We also show that the drugindependent PDR5 transcription is associated with enhanced promoter occupancy of coactivator complexes, including SAGA, Mediator, chromatin remodeling SWI/SNF complex, and TATA-binding protein . Analyzed by chromatin immunoprecipitations, loss of contacts between histones and DNA occurs at both promoter and coding sequences of PDR5 . Consistently, micrococcal nuclease susceptibility analysis revealed altered chromatin structure at the promoter and coding sequences of PDR5 . Our data provide molecular description of the changes associated with constitutive PDR5 transcription, and reveal the molecular mechanism underlying drug-independent transcriptional up-regulation of PDR5. Extremophiles, 2004 Dec, 8(6), 441 - 6 Epub 2004 Jul 30. Digestion by serine proteases enhances salt tolerance of glutaminase in the marine bacterium Micrococcus luteus K-3; Yoshimune K et al.; Salt-tolerant glutaminase (Micrococcus glutaminase, with an apparent molecular mass of 48.3 kDa, intact glutaminase) from the marine bacterium Micrococcus luteus K-3 was digested using protease derived from M . luteus K-3 . The digestion products were a large fragment (apparent molecular mass of 38.5 kDa, the glutaminase fragment) and small fragments (apparent molecular mass of 8 kDa) . The digestion was inhibited by phenylmethanesulfonyl fluoride (PMSF) . Digestion of intact glutaminase by serine proteases including trypsin, elastase, lysyl endopeptidase, and arginylendopeptidase also produced the glutaminase fragment . The N-terminus of the glutaminase fragment was the same as that of intact glutaminase . The N-termini of two small fragments were Ala394 and Ala396, respectively . The enzymological and kinetic properties of the glutaminase fragment were almost the same as those of intact glutaminase except for salt-tolerant behavior . The glutaminase fragment was a higher salt-tolerant enzyme than the intact glutaminase, suggesting that Micrococcus glutaminase is digested in the C-terminal region by serine protease from M . luteus K-3 to confer salt tolerance on glutaminase. Rapid Commun Mass Spectrom, 2004, 18(14), 1541 - 7 Selective digestion and novel cleanup techniques for detection of benzo{a}pyrene diol epoxide-DNA adducts by capillary electrophoresis/mass spectrometry; Gennaro LA et al.; Benzo{a}pyrene (BP) is a ubiquitous environmental polycyclic aromatic hydrocarbon (PAH) which, upon metabolic conversion to reactive benzo{a}pyrene-7,8-diol-9,10-epoxide (BPDE), has been found to attach covalently to DNA . Given the low level of DNA adducts typically present in vivo or in vitro, an essential first step prior to capillary electrophoresis/mass spectrometry (CE/MS) (or liquid chromatography/mass spectrometry (LC/MS)) analysis of the DNA digests is the removal of the bulk non-adducted nucleotides, enzymes or salts, and isolation of enriched adducts . This report focuses on the development of novel sample handling methods aimed at facilitating the analysis of BPDE-DNA adducts by CE/MS . This approach involves a simple variation on the digestion procedure, in combination with the use of metal affinity ZipTips for the more efficient cleanup of BPDE-DNA adducts formed in vitro for subsequent CE/MS analysis . The previously described digestion procedure, consisting of micrococcal nuclease, spleen phosphodiesterase and nuclease P1, allows for selective dephosphorylation of normal nucleotides, while leaving adducted nucleotides intact . Metal affinity ZipTips, typically used for selective extraction of phosphopeptides, were used here for extraction of adducted nucleotides . The utility of metal affinity SPE was tested on mixtures of dG and dGp, wherein nucleotide extracts contained no detectable nucleosides by CE/UV analysis . An in vitro BPDE-DNA incubation was then digested using the above procedure . Metal affinity solid-phase extraction (SPE) was subsequently used for the selective isolation of phosphorylated components, i.e., adducted nucleotides, from the mixture of enzymes and non-adducted nucleosides . SPE extracts were enriched in nucleotide adducts and analyzed using sample stacking and CE/MS . This method has several advantages over previously described cleanup procedures for dGp-BPDE adducts: fast, simple, uses commercially available materials, no need for excessive dilution (small scale), the suitability for use with automation, and possible applicability to other bulky hydrophobic adducts . Nucleic Acids Res . 2004 Jul 28;32(13):e111. Transcriptionally competent chromatin assembled with exogenous histones in a yeast whole cell extract; Rodriguez-Campos A et al.; We describe a cell-free chromatin assembly system derived from the yeast Saccharomyces cerevisiae, which efficiently packages DNA into minichromosomes in a reaction dependent on exogenous core histones and an ATP-regenerating system . Both supercoiled and relaxed plasmid DNA serve as templates for nucleosomal loading in a gradual process that takes at least 6 h for completion at 30 degrees C . Micrococcal nuclease digestion of the assembled minichromosomes displays an extended nucleosomal ladder with a repeat length of 165 bp . The purified minichromosomes contain the four core histones in stoichiometric proportion and exhibit phased nucleosomes over the mouse mammary tumour virus (MMTV) promoter . The progesterone receptor and NF1 synergize on these minichromosomes resulting in efficient cell-free transcription . The ease of manipulation and the potential use of yeast strains carrying mutations in the chromatin handling machinery make this system suitable for detailed mechanistic studies. Methods Mol Biol, 2004, 287, 65 - 75 Measuring changes in chromatin using micrococcal nuclease; Steward N et al.; This chapter documents a simple protocol to identify the nucleosome positioning of any given genes . The procedure includes partitioning 200-bp DNA fragments constituting the nucleosomal core region by micrococcal nuclease digestion and semiquantitative polymerase chain reaction amplification using multiple sets of primers covering arbitrary regions of approximately 150 bp in the gene . If the nuclease-digested 200-bp DNA is efficiently amplified, the region is inside the core . If the amplification is poor, the region spans the linker region . By a combination of this method with direct methylation mapping, the core region of a maize gene, ZmMI1, was shown to be less methylated than the linker region . The potential usefulness of the technique is discussed. Methods Mol Biol, 2004, 287, 21 - 44 Native chromatin immunoprecipitation; Thorne AW et al.; Chromatin immunoprecipitation (ChIP) is a technique widely used for determining the genomic location of modified histones and other chromatin-associated factors . Here we describe the methodology we have used in our laboratory for the immunoprecipitation of chromatin isolated from cells in the absence of crosslinking . Chromatin released from nuclei by micrococcal nuclease digestion is centrifuged through sucrose gradients to allow selection of mono- or dinucleosomes . This allows a protein or modification at a particular gene or locus to be mapped at higher resolution than in a crosslinked ChIP experiment . Two methods for the immunoprecipitation of chromatin are described: a large-scale fractionation by which it is possible to visualize the proteins of the immunoprecipitate by polyacrylamide gel electrophoresis, PAGE and a small-scale method that is more appropriate when the quantity of chromatin is limited . The sequence content of DNA extracted from the immunoprecipitated chromatin is analyzed by hybridization of Southern or slot blots, or by quantitative polymerase chain reaction . Enrichment of particular sequences in the immunoprecipitated fraction reveals the presence and extent of the modification at this location. EMBO J, 2004 Aug 18, 23(16), 3314 - 24 Epub 2004 Jul 15. Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA; Bao Y et al.; H2A.Bbd is an unusual histone variant whose sequence is only 48% conserved compared to major H2A . The major sequence differences are in the docking domain that tethers the H2A-H2B dimer to the (H3-H4)(2) tetramer; in addition, the C-terminal tail is absent in H2A.Bbd . We assembled nucleosomes in which H2A is replaced by H2A.Bbd (Bbd-NCP), and found that Bbd-NCP had a more relaxed structure in which only 118+/-2 bp of DNA is protected against digestion with micrococcal nuclease . The absence of fluorescence resonance energy transfer between the ends of the DNA in Bbd-NCP indicates that the distance between the DNA ends is increased significantly . The Bbd docking domain is largely responsible for this behavior, as shown by domain-swap experiments . Bbd-containing nucleosomal arrays repress transcription from a natural promoter, and this repression can be alleviated by transcriptional activators Tax and CREB . The structural properties of Bbd-NCP described here have important implications for the in vivo function of this histone variant and are consistent with its proposed role in transcriptionally active chromatin. Mol Cell, 2004 Jul 2, 15(1), 43 - 56 Cyclin A repression in quiescent cells is associated with chromatin remodeling of its promoter and requires Brahma/SNF2alpha; Coisy M et al.; Cell cycle-dependent expression of cyclin A is controlled by transcriptional repression in early phase of the cell cycle . In this study, we directly examine the chromatin structure of the mouse cyclin A promoter through in vivo micrococcal nuclease footprinting . We describe here that cyclin A repression is associated with two positioned nucleosomes and that histones progressively lose DNA contact synchronously with gene activation . This particular nucleosomal organization is disrupted by mutations of the cyclin A bipartite repressor sequence . Moreover, the same sequence recruits the chromatin remodeling factor Brahma/SNF2alpha (Brm) onto the cyclin A promoter . Accordingly, cyclin A proximal promoter is not wrapped around nucleosomes and not repressed in quiescent cells lacking Brm . These results provide molecular explanations for the transcriptional repression state of cyclin A, as well as insights into the action of Brm chromatin remodeling factor as cell cycle regulator. J Food Prot, 2004 Jun, 67(6), 1190 - 4 Controlled release of antimicrobial compounds from highly swellable polymers; Buonocore GG et al.; The suitability of antimicrobial release films made from highly swellable polymers for use in food packaging was evaluated . The possibility of modulating the release kinetics of active compounds either by regulating the degree of cross-link of the polymer matrix or by using multilayer structures was addressed . The release kinetics of lysozyme, nisin, and sodium benzoate (active compounds with different molecular weights) were determined at ambient temperature (25 degrees C) . The effectiveness of the proposed active films in inhibiting microbial growth was addressed by determining the antimicrobial efficiency of the released active compounds . Micrococcus lysodeikticus, Alicyclobacillus acidoterrestris, and Saccharomyces cerevisiae were used to test the antimicrobial efficiency of released lysozyme, nisin, and sodium benzoate, respectively . Results indicate that the release kinetics of both lysozyme and nisin can be modulated through the degree of cross-link of the polymer matrix, whereas multilayer structures need to be used to control the release kinetics of sodium benzoate . All the active compounds released from the investigated active films were effective in inhibiting microbial growth. Tuberculosis (Edinb), 2004, 84(3-4), 167 - 79 Global expression profiling of strains harbouring null mutations reveals that the five rpf-like genes of Mycobacterium tuberculosis show functional redundancy; Downing KJ et al.; SETTING: Aged, dormant cultures of Mycobacterium tuberculosis can be resuscitated by a secreted, proteinaceous growth factor from Micrococcus luteus, known as resuscitation-promoting factor (Rpf) . M . tuberculosis contains five rpf-like genes, rpfA (Rv0867c), rpfB (Rv1009), rpfC (Rv1884c), rpfD (Rv2389c) and rpfE (Rv2450c), that bear significant similarity to Mi . luteus rpf, suggesting that these too may play a role in growth and/or reactivation from a quiescent state . OBJECTIVE AND DESIGN: Unmarked deletion mutants of each of the five rpf-like genes of M . tuberculosis H37Rv were constructed and their phenotypes and global gene expression profiles were assessed . RESULTS AND CONCLUSIONS: Deletion of any one of the rpf-like genes did not affect growth or survival of M . tuberculosis in liquid culture, but some alterations in colony-forming ability and colonial morphology were observed . Global gene expression profiling suggested that loss of rpfC affected the expression of the largest number of genes and there was a significant overlap in the differential gene expression profile of the rpfC mutant with those of the rpfB, rpfD and rpfE mutants . The expression profile of the rpfA mutant was notably less similar, but inverse associations with genes affected in the other mutants were observed . These results, together with those obtained by real-time, quantitative RT-PCR, suggest that the rpf-like genes serve wholly or partially overlapping functions in M . tuberculosis. J Biol Chem, 2004 Aug 13, 279(33), 34101 - 6 Epub 2004 Jun 09. A pattern recognition serine proteinase triggers the prophenoloxidase activation cascade in the tobacco hornworm, Manduca sexta; Ji C et al.; A serine proteinase cascade in insect hemolymph mediates prophenoloxidase activation, a defense mechanism against pathogen or parasite infection . Little is known regarding its initiating proteinase or how this enzyme is activated in response to invading microorganisms . We have isolated from the tobacco hornworm, Manduca sexta, a cDNA encoding a modular protein designated hemolymph proteinase 14 (HP14) . It contains five low density lipoprotein receptor class A repeats, a Sushi domain, a unique Cys-rich region, and a proteinase-catalytic domain . The HP14 mRNA exists in fat body and hemocytes of the naive larvae, and its level increases significantly at 24 h after a bacterial challenge . We expressed proHP14 with a carboxyl-terminal hexahistidine tag in a baculovirus/insect cell system and detected the recombinant protein in two forms . The 87-kDa protein was primarily intracellular, whereas the 75-kDa form was present in the medium . Interaction with peptidoglycan resulted in proteolytic processing of the purified zymogen and generation of an amidase activity . Supplementation of hemolymph with proHP14 greatly enhanced prophenoloxidase activation in response to Micrococcus luteus . These data suggest that proHP14 is a pattern recognition protein that binds to bacteria and autoactivates and triggers the prophenoloxidase activation system in the hemolymph of M . sexta. Microbiology, 2004 Jun, 150(Pt 6), 1687 - 97 Formation of 'non-culturable' cells of Mycobacterium smegmatis in stationary phase in response to growth under suboptimal conditions and their Rpf-mediated resuscitation; Shleeva M et al.; Conditions were investigated that promote the formation of 'non-culturable' (NC) cells of Mycobacterium (Myc.) smegmatis in stationary phase . After cultivation in a rich medium, or under conditions that may be considered optimal for bacterial growth, or starvation for carbon, nitrogen or phosphorus, bacteria failed to enter a NC state . However, when grown under suboptimal conditions, resulting in a reduced growth rate or maximal cell concentration (e.g . in modified Hartman's-de Bont medium), bacteria adopted a stable NC state after 3-4 days incubation in stationary phase . Such conditions are not specific as purF and devR mutants of Myc . smegmatis also showed (transient) loss of culturability following growth to stationary phase in an optimized medium, but under oxygen-limited conditions . The behaviour of the same mutants in oxygen-sufficient but nutrient-inappropriate medium (modified Hartman's-de Bont medium) was similar to that of the wild-type (adoption of a stable NC state) . It is hypothesized that adoption of a NC state may represent an adaptive response of the bacteria, grown under conditions when their metabolism is significantly compromised due to the simultaneous action of several factors, such as usage of inappropriate nutrients or low oxygen availability or impairment of a particular metabolic pathway . NC cells of wild-type Myc . smegmatis resume growth when transferred to a suitable resuscitation medium . Significantly, resuscitation was observed when either recombinant Rpf protein or supernatant derived from a growing bacterial culture was incorporated into the resuscitation medium . Moreover, co-culture with Micrococcus (Mcc.) luteus cells (producing and secreting Rpf) also permitted resuscitation . Isogenic strains of Myc . smegmatis harbouring plasmids containing the Mcc . luteus rpf gene also adopt a similar NC state after growth to stationary phase in modified Hartman's-de Bont medium . However, in contrast to the behaviour noted above, these strains resuscitated spontaneously when transferred to the resuscitation medium, presumably because they are able to resume endogenous synthesis of Mcc . luteus Rpf . Resuscitation was not observed in the control strain harbouring a plasmid lacking Mcc . luteus rpf . In contrast to wild-type, the NC cells of purF and devR mutants obtained under oxygen-limited conditions resuscitate spontaneously, presumably because the heterogeneous population contains some residual viable cells that continue to make Rpf-like proteins. Dtsch Tierarztl Wochenschr, 2004 Apr, 111(4), 162 - 5 Pharmacokinetics and tissue residue profiles of erythromycin in broiler chickens after different routes of administration; Goudah A et al.; This study investigated the disposition kinetics and plasma availability of erythromycin in broiler chickens after single intravenous (i.v.), intramuscular (i.m.), subcutaneous (s.c.) and oral administrations (p.o.) of 30 mg kg(-1) b . wt . Tissue residue profiles were also studied after multiple intramuscular, subcutaneous, and oral administration of 30 mg kg(-1) b . wt., twice daily for three consecutive days . Plasma and tissue concentrations of erythromycin were determined using microbiological assay methods with Micrococcus luteus as the test organism . Following intravenous injection, plasma concentration-vs-time curves were best described by a two compartment open model . The decline in plasma drug concentration was bi-exponential with half-lives of (t(1/2alpha)) 0.19 h and (t(1/2beta)) 5.3 h for distribution and elimination phases, respectively . After intramuscular, subcutaneous and oral administration erythromycin at the same dose was detected in plasma at 10 min and reached its minimum level 8 h post-administration . The peak plasma concentration (Cmax) were 5.0, 5.3, and 6.9 microg x ml(-1) and were attained at 1.7, 1.4, and 1.3 h (Tmax), respectively . The elimination half-lives (T(1/2el)) were 3.9, 2.6, and 4.1 h and the mean residence times (MRT) were 3.5, 3.2, and 3.6 h, respectively . The systemic bioavailabilities were 92.5, 68.8, and 109.3%, respectively . In vitro protein binding percent of erythromycin in broiler plasma was ranged from 21 to 31% . The limit of quantification (LOQ) for the assay was 0.03 microg x ml(-1) in plasma and tissues . The tissue level concentrations were highest in the liver, and decreased in the following order: plasma > kidney > lung > muscle and heart . No erythromycin residues were detected in tissues and plasma after 24 h except in liver and kidney where it persisted during 48 h following intramuscular and oral administrations. J Gen Virol, 2004 Jun, 85(Pt 6), 1763 - 76 Significance of the 3'-terminal region in minus-strand RNA synthesis of Hibiscus chlorotic ringspot virus; Wang HH et al.; RNA-dependent RNA polymerase (RdRp) was solubilized from crude extracts of Hibiscus cannabinus infected by Hibiscus chlorotic ringspot virus (HCRSV), a member of the Carmoviridae . After treatment of the extracts with micrococcal nuclease to remove the endogenous templates, the full-length genomic RNA and the two subgenomic RNAs were efficiently synthesized by the partially purified RdRp complex in vitro . When the full-length RNAs of Potato virus X, Tobacco mosaic virus, Odontoglossum ringspot virus and Cucumber mosaic virus were used as templates, no detectable RNA was synthesized . Synthesis of HCRSV minus-strand RNA was shown to initiate opposite the 3'-terminal two C residues at the 3' end in vitro and in vivo . The CCC-3' terminal nucleotide sequence was optimal and nucleotide variations from CCC-3' diminished minus-strand synthesis . In addition, two putative stem-loops (SLs) located within the 3'-terminal 87 nt of HCRSV plus-strand RNA were also essential for minus-strand RNA synthesis . Deletion or disruption of the structure of these two SLs severely reduced or abolished RNA synthesis . HCRSV RNA in which the two SLs were replaced with the SLs of Turnip crinkle virus could replicate in kenaf protoplasts, indicating that functionally conserved structure, rather than nucleotide sequence, plays an important role in the minus-strand synthesis of HCRSV . Taken together, the specific sequence CCC at the 3' terminus and the two SLs structures located in the 3'UTR are essential for efficient minus-strand synthesis of HCRSV. Microb Ecol, 2004 Jul, 48(1), 120 - 7 Epub 2004 May 28. Micrococcus luteus -- survival in amber; Greenblatt CL et al.; A growing body of evidence now supports the isolation of microorganisms from ancient materials . However, questions about the stringency of extraction methods and the genetic relatedness of isolated organisms to their closest living relatives continue to challenge the authenticity of these ancient life forms . Previous studies have successfully isolated a number of spore-forming bacteria from organic and inorganic deposits of considerable age whose survival is explained by their ability to enter suspended animation for extended periods of time . However, despite a number of putative reports, the isolation of non-spore-forming bacteria and an explanation for their survival have remained enigmatic . Here we describe the isolation of non-spore-forming cocci from a 120-million-year-old block of amber, which by genetic, morphological, and biochemical analyses are identified as belonging to the bacterial species Micrococcus luteus . Although comparison of 16S rRNA sequences from the ancient isolates with their modern counterparts is unable to confirm the precise age of these bacteria, we demonstrate, using complementary molecular and cell biological techniques, evidence supporting the view that these (and related modern members of the genus) have numerous adaptations for survival in extreme, nutrient-poor environments, traits that will assist in this bacteria's persistence and dispersal in the environment . The bacteria's ability to utilize succinic acid and process terpine-related compounds, both major components of natural amber, support its survival in this oligotrophic environment. Insect Mol Biol, 2004 Jun, 13(3), 273 - 82 A novel lectin with a fibrinogen-like domain and its potential involvement in the innate immune response of Armigeres subalbatus against bacteria; Wang X et al.; Mosquitoes have an efficient cellular innate immune response that includes phagocytosis of microbial pathogens and encapsulation of metozoan parasites . In this study, we describe a novel lectin in the mosquito, Armigeres subalbatus (aslectin or AL-1) . The 1.27 kb cDNA clone for the AL-1 gene (AL-1) encodes a 279 deduced amino acid sequence that contains a C-terminal fibrinogen-like domain . AL-1 is transcribed in all life stages . AL-1 mainly exists in the haemolymph of adult female mosquitoes, and is upregulated following both Escherichia coli and Micrococcus luteus challenge . AL-1 specifically recognizes N-acetyl-d-glucosamine and is able to bind both E . coli and M . luteus . These results suggest that AL-1 might function as a pattern recognition receptor in the immune response in Ar . subalbatus. J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Jun 15, 805(2), 315 - 23 Affinity membrane chromatography: relationship of dye-ligand type to surface polarity and their effect on lysozyme separation and purification; Arica MY et al.; Two different dye-ligands, i.e . Procion Brown MX-5BR (RB-10) and Procion Green H-4G (RG-5) were immobilised onto poly(2-hydroxyethylmethacrylate) (pHEMA) membranes . The polarities of the affinity membranes were determined by contact angle measurements . Separation and purification of lysozyme from solution and egg white were investigated . The adsorption data was analysed using two adsorption kinetic models the first order and the second order to determine the best-fit equation for the separation of lysozyme using affinity membranes . The second-order equation for the adsorption of lysozyme on the RB-10 and RG-5 immobilised membranes systems is the most appropriate equation to predict the adsorption capacity for the affinity membranes . The reversible lysozyme adsorption on the RB-10 and RG-5 did not follow the Langmuir model, but obeyed the Temkin and Freundlich isotherm model . Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate . The purities of the eluted lysozyme, as determined by HPLC, were 76 and 92% with recovery 63 and 77% for RB-10 and RG-5 membranes, respectively . For the separation and purification of lysozyme the RG-5 immobilised membrane provided the best results . The affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated adsorption-elution cycles. J Virol, 2004 May, 78(10), 5056 - 67 Interaction between human immunodeficiency virus type 1 reverse transcriptase and integrase proteins; Hehl EA et al.; Reverse transcriptase (RT) and integrase (IN) are two key catalytic enzymes encoded by all retroviruses . It has been shown that a specific interaction occurs between the human immunodeficiency virus type 1 (HIV-1) RT and IN proteins (X . Wu, H . Liu, H . Xiao, J . A . Conway, E . Hehl, G . V . Kalpana, V . R . Prasad, and J . C . Kappes, J . Virol . 73:2126-2135, 1999) . We have now further examined this interaction to map the binding domains and to determine the effects of interaction on enzyme function . Using recombinant purified proteins, we have found that both a HIV-1 RT heterodimer (p66/p51) and its individual subunits, p51 and p66, are able to bind to HIV-1 IN . An oligomerization-defective mutant of IN, V260E, retained the ability to bind to RT, showing that IN oligomerization may not be required for interaction . Furthermore, we report that the C-terminal domain of IN, but not the N-terminal zinc-binding domain or the catalytic core domain, was able to bind to heterodimeric RT . Deletion analysis to map the IN-binding domain on RT revealed two separate IN-interacting domains: the fingers-palm domain and the carboxy-terminal half of the connection subdomain . The carboxy-terminal domain of IN alone retained its interaction with both the fingers-palm and the connection-RNase H fragments of RT, but not with the half connection-RNase H fragment . This interaction was not bridged by nucleic acids, as shown by micrococcal nuclease treatment of the proteins prior to the binding reaction . The influences of IN and RT on each other's activities were investigated by performing RT processivity and IN-mediated 3' processing and joining reactions in the presence of both proteins . Our results suggest that, while IN had no influence on RT processivity, RT stimulated the IN-mediated strand transfer reaction in a dose-dependent manner up to 155-fold . Thus, a functional interaction between these two viral enzymes may occur during viral replication. J Antibiot (Tokyo), 2004 Feb, 57(2), 125 - 35 Isolation and structural characterization of siderophores, madurastatins, produced by a pathogenic Actinomadura madurae; Harada K et al.; Madurastatins Al (1), A2 (2) and A3 (3), novel pentapeptides that were acylated with salicylic acid at the N-terminus, were isolated from the culture broth of a pathogenic Actinomadura madurae IFM 0745 strain . These structures were mainly determined by 2D NMR and MS/MS spectral techniques . The strain produced simultaneously madurastatins B1 (4) and B2 (5) consisting of Ser and salicylic acid moieties . Compounds 1 and 4 had an antibacterial activity against Micrococcus luteus, indicating that the presence of the aziridine ring is essential for such activity . Because 1 has a strong affinity with ferric ion due to the presence of two hydroxamic acids and a salicylic acid, it is considered to be a siderophore that is a low molecular weight iron chelater . The production of siderophores may be one of the characteristics of pathogenic microorganisms. Methods Mol Biol, 2004, 265, 59 - 72 Induction and biochemical purification of RNA-induced silencing complex from Drosophila S2 cells; Caudy AA et al.; The discovery of RNA interference (RNAi) has greatly simplified the process of suppressing genes in many experimental systems, including Caenorhabditis elegans, Drosophila, and mammalian cells . A sequence-specific nuclease complex, called the RNA-induced silencing complex (RISC), can be purified from cells undergoing RNAi . RISC shows RNase activity when exposed to RNAs homologous to the input double-stranded RNA (dsRNAs) but lacks activity in the presence of nontargeted RNAs . We describe the induction of RNAi by dsRNA in cultured Drosophila Schneider-2 (S2) cells and detail procedures for RISC purification from these cells . This purification approach has allowed us to identify several RISC components, including siRNAs, Argo naute 2 (Ago-2), Drosophila Fragile X related protein (dFXR), Vasa intronic gene (VIG), and the micrococcal nuclease family member Tudor-SN (Drosophila CG7008) . RNAi is carried out by an endogenous pathway important for normal development in many organisms . In fact, organisms express hundreds of different microRNAs (miRNAs), small hairpin RNAs that function through the RNAi pathway to suppress expression of endogenous genes . The function of miRNAs is poorly understood, and most of their targets are unknown . Purified RISC complexes contain short interfering RNAs and endogenously expressed miRNAs and will be useful for studying many aspects of the RNAi machinery. Biochem Biophys Res Commun, 2004 May 14, 317(4), 1052 - 60 The instantly released Drosophila immune proteome is infection-specific; Vierstraete E et al.; In this study, we analyzed the hemolymph proteome of Drosophila third instar larvae, which were induced with a suspension of Gram-positive bacteria or yeast . Profiling of the hemolymph proteins of infected versus non-infected larvae was performed by two-dimensional difference gel electrophoresis . Infection with Micrococcus luteus or Saccharomyces cerevisiae induced, respectively, 20 and 19 differential protein spots . The majority of the spots are specifically regulated by one pathogen, whereas only a few spots correspond to proteins altered in all cases of challenging (including after challenge with lipopolysaccharides) . All of the upregulated proteins can be assigned to specific aspects of the immune system, as they did not increase in the hemolymph of sterile pricked larvae . Next to known immune proteins, unannotated proteins were identified such as CG4306 protein, which has homologues with unknown function in all metazoan genome databases available today. Mol Cell Biol, 2004 Apr, 24(7), 3036 - 47 Chromatin-mediated restriction of nuclear factor 1/CTF binding in a repressed and hormone-activated promoter in vivo; Belikov S et al.; Mouse mammary tumor virus (MMTV) promoter-driven transcription is induced by glucocorticoid hormone via binding of the glucocorticoid receptor (GR) . The MMTV promoter also harbors a binding site for nuclear factor 1 (NF1) . NF1 and GR were expressed in Xenopus oocytes; this revealed GR-NF1 cooperativity both in terms of DNA binding and chromatin remodeling but not transcription . A fraction of NF1 sites were occupied in a hormone-dependent fashion, but a significant and NF1 concentration-dependent fraction were constitutively bound . Activation of the MMTV promoter resulted in an approximately 50-fold increase in the NF1 accessibility for its DNA site . The hormone-dependent component of NF1 binding was dissociated by addition of a GR antagonist; however, the antagonist RU486, which supports partial GR-DNA binding, also maintained partial NF1 binding . Hence GR-NF1 cooperativity is independent of agonist-driven chromatin remodeling . NF1 induced the formation of a micrococcal-nuclease-resistant protein-DNA complex containing the DNA segment from -185 to -55, the MMTV enhanceosome . Coexpression of NF1 and Oct1 resulted in a significant stimulation of hormone-induced MMTV transcription and also in increased basal transcription . We propose that hormone-independent NF1 binding may be involved in maintaining transcriptional competence and establishment of tissue-specific gene networks. Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 605 - 8 Arsenicicoccus bolidensis gen . nov., sp . nov., a novel actinomycete isolated from contaminated lake sediment; Collins MD et al.; An unknown Gram-positive, catalase-positive, facultatively anaerobic, non-spore-forming, coccus-shaped bacterium originating from sediment was characterized using phenotypic, molecular chemical and molecular phylogenetic methods . Chemical studies revealed the presence of a cell-wall murein based on LL-diaminopimelic acid (type LL-Dpm-glycine(1)), a complex mixture of saturated, monounsaturated and iso- and anteiso-methyl-branched, non-hydroxylated, long-chain cellular fatty acids and tetrahydrogenated menaquinones with eight isoprene units {MK-8(H(4))} as the major respiratory lipoquinone . This combination of characteristics somewhat resembled members of the suborder Micrococcineae, but did not correspond to any currently described species . Comparative 16S rRNA gene sequencing confirmed that the unidentified coccus-shaped organism is a member of the Actinobacteria and represents a hitherto-unknown subline related to, albeit different from, a number of taxa including Intrasporangium, Janibacter, Terrabacter, Terracoccus and Ornithinicoccus . Based on phenotypic and phylogenetic considerations, it is proposed that the unknown bacterium originating from lake sediment be classified as a new genus and species, Arsenicicoccus bolidensis gen . nov., sp . nov . (type strain CCUG 47306(T)=DSM 15745(T)). Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 557 - 61 Xylanibacterium ulmi gen . nov., sp . nov., a novel xylanolytic member of the family Promicromonosporaceae; Rivas R et al.; A bacterial strain designated XIL08(T) was isolated from an elm tree affected by Dutch elm disease . Strain XIL08(T) is Gram-positive, aerobic, rod-shaped and non-motile . The complete 16S rDNA sequence of this micro-organism was obtained and phylogenetic analysis based on the neighbour-joining method indicated that the closest related organism belongs to the genus Xylanimonas of the family Promicromonosporaceae, suborder Micrococcineae . Cell-wall analyses revealed the presence of type A4alpha, L-lys-L-ala-D-Glu peptidoglycan . The cell-wall sugars found were rhamnose in large amounts, fucose, mannose and galactose and traces of arabinose and glucose . HPLC analysis of menaquinones revealed two peaks, the main peak corresponding to MK-9(H(4)) and the smaller one to MK-8(H(4)) . The major fatty acid found was anteiso-C(15 : 0) . Mycolic acids were absent . The polar lipids detected were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides . The G+C content of the DNA was 72 mol% . Isolate XIL08(T) hydrolysed xylan but not cellulose . Growth was observed with many carbohydrates including acetate and xylan as the only carbon source . Catalase activity was not detected . The data from this polyphasic study suggest that this bacterium belongs to a novel genus of the family Promicromonosporaceae . It is proposed that isolate XIL08(T) (=LMG 21721(T)=CECT 5731(T)) be classified in a new genus, Xylanibacterium gen . nov., as the type strain of Xylanibacterium ulmi sp . nov. Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 525 - 31 Yania halotolerans gen . nov., sp . nov., a novel member of the suborder Micrococcineae from saline soil in China; Li WJ et al.; A novel coccoid, halotolerant actinobacterium, designated strain YIM 70085(T), was isolated from a soil sample that was collected in Xinjiang Province, China, and characterized by using a polyphasic approach . Optimum growth temperature was 28 degrees C and growth occurred optimally in culture media that contained 10 % KCl . The peptidoglycan type was A4alpha, L-lys-gly-L-Glu . Whole-cell sugars consisted of xylose, mannose and galactose . Phospholipids were diphosphatidylglycerol, phosphatidylglycerol, one unknown phospholipid, one unknown glycolipid and traces of phosphatidylinositol . Menaquinones were MK-8 (83 %), MK-7 (12 %) and MK-9 (15 %) . Predominant fatty acids were i-C(15 : 0) (44.29 %), ai-C(15 : 0) (35.60 %) and ai-C(17 : 0) (9.74 %) . The DNA G+C content was 53.5 mol% . Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 70085(T) occupies a branch that is distinct from, although very close to, the family Micrococcaceae in the suborder Micrococcineae . Based on its phenotypic characteristics, phylogenetic position (as determined by 16S rRNA gene sequence analysis) and 16S rDNA signature nucleotide data, it is concluded that the isolate represents a novel member of the suborder Micrococcineae, for which the name Yania halotolerans gen . nov., sp . nov . is proposed . The type strain is YIM 70085(T) (=CCTCC AA001023(T)=DSM 15476(T)). Nucleic Acids Res, 2004 Feb 19, 32(4), 1251 - 60 Print 2004. In vivo interactions of the Acanthamoeba TBP gene promoter; Chen L et al.; Transcription of the TATA box binding protein (TBP) gene in Acanthamoeba castellanii is regulated by TATA box binding protein promoter binding factor (TPBF), which binds to an upstream TBP promoter element to stimulate transcription, and to a TATA proximal element, where it represses transcription . In order to extend these observations to the in vivo chromatin context, the TBP gene was examined by in situ footprinting and chromatin immunoprecipitation (ChIP) . Acanthamoeba DNA is nucleosomal with a repeat of approximately 160 bp, and an intranucleosomal DNA periodicity of 10.5 bp . The TBP gene comprises a 220 bp micrococcal nuclease hypersensitive site corresponding to the promoter regulatory elements previously identified, flanked by protected regions of a size consistent with the presence of nucleosomes . ChIP data indicated that TPBF is associated with the TBP, TPBF and MIL gene promoters, but not to the CSP21, MIIHC, 5SrRNA or 39SrRNA promoters, or to the MIL gene C-terminal region . Binding by TPBF to the TPBF and MIL gene promoters was confirmed by in vitro assays . These results validate the in vitro model for TBP gene regulation and further suggest that TPBF may be autoregulated and may participate in the regulation of the MIL gene. J Chromatogr A, 2004 Feb 27, 1028(1), 149 - 53 Protein separation with surfactant-coated polystyrene involving Cibacron Blue 3GA-conjugated triton X-100; Saitoh T et al.; Through mixing of porous polystyrene particles (Amberlite XAD-4), non-ionic surfactants, and surfactant-conjugated substrates (affinity ligand) in an aqueous solution led to the formation of a novel medium (affinity admicelle) for protein separation . The ligand (CB-Triton) was synthesized by mixing a triazine dye (Cibacron Blue 3GA (CB)) and a polyoxyethylene-type non-ionic surfactant (Triton X-100) in weakly alkaline solutions . Triton X-100 and CB-Triton were competitively sorbed onto XAD-4 . Albumin (bovine serum), alcohol dehydrogenase (yeast), and lysozyme (chicken egg) having specific interaction to CB were collected onto the affinity admicelle . On the other hand, the collection of ovalubmin (chicken egg white), having no binding ability to CB, was negligibly small . Lysozyme in 100 microl of chicken egg white, diluted with 900 microl of 10 mM Tris-HCl (pH 7.4), was successfully collected on 18 mg of CB-Triton admicelles and, then, it was eluted with 1 ml of aqueous solution of 100 mM phosphate (pH 7.4) . The recovery based on the activity for the lysis of micrococcus and the concentration factor were 60% and 40 (n = 3), respectively. Clin Chim Acta, 2004 Mar, 341(1-2), 165 - 72 Blood lactoferrin release induced by running exercise in normal volunteers: antibacterial activity; Inoue H et al.; BACKGROUND: The aim of this study was to determine serum lactoferrin concentrations and serum antibacterial activity before and after running exercise . METHODS: Twenty-four healthy young men were randomly assigned to high, middle, or low intensity of exercise groups (5000 steps running at 180, 130, and 80 steps/min, respectively) . Blood samples were collected at baseline and immediately, 1 and 4 h after exercise . Concentrations of circulating neutrophils, serum lactoferrin, iron in whole blood, and serum iron were measured . Antibacterial activity of serum was evaluated using live Micrococcus luteus . RESULTS: The numbers of circulating neutrophils were increased by 20.0% and 15.5% 1 h after exercise in high and middle groups (both P<0.01), respectively . Serum lactoferrin concentrations were significantly increased immediately after exercise by 48.3% and 33.0% in the high and middle groups (both P<0.01), respectively . No significant changes in total iron or serum iron concentrations were observed during the study . Antibacterial activities of serum collected immediately after exercise in the high and middle groups were significantly stronger than those before exercise, by 31.2% and 25.4% (both P<0.05), respectively . CONCLUSIONS: Serum lactoferrin concentrations are increased immediately after running exercise and may play an antibacterial role in host defenses before mobilization of neutrophils into the circulating pool. J Virol, 2004 Mar, 78(5), 2179 - 86 Underrepresentation of the 3' region of the capsid pregenomic RNA of duck hepatitis B virus; Ostrow KM et al.; The pregenomic RNA (pgRNA) of hepadnaviruses is packaged into capsids where it is reverse transcribed to yield mature DNA genomes . This report describes differences between the 3' region and other regions of the pgRNA isolated from capsids . Analysis of capsid pgRNA isolated by using an established method involving micrococcal nuclease treatment demonstrated reduced levels of the 3' region of the pgRNA compared to the 5' region . This underrepresentation of the 3' region was partly a result of microccocal nuclease digestion of the 3' region because isolation of capsid pgRNA by an alternative method that did not involve nuclease treatment led to a greater, but not complete, recovery of the 3' region . These results indicate that the 3' region of the capsid pgRNA is susceptible to micrococcal nuclease digestion during its isolation and that the 3' region can still be underrepresented when capsid pgRNA is isolated without nuclease digestion . Additional experiments show that the 3' ends of capsid pgRNA isolated by micrococcal nuclease treatment are heterogeneously dispersed from nucleotide 2577 to the poly(A) tail . These data provide evidence that the 3' region of the capsid pgRNA has biochemical properties different from those of its 5' region . Possibly, the 3' region of the pgRNA is not packaged into the interior of the capsid but rather is associated with a part of the capsid where it is susceptible to microccocal nuclease digestion. J Biochem (Tokyo), 2003 Dec, 134(6), 819 - 26 Significance of highly conserved aromatic residues in Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase; Kharel Y et al.; Undecaprenyl diphosphate synthase catalyzes the sequential condensation of eight molecules of isopentenyl diphosphate (IPP) in the cis-configuration into farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of the bacterial cell wall . This cis-type prenyltransferase exhibits a quite different mode of binding of homoallylic substrate IPP from that of trans-type prenyltransferase {Kharel Y . et al . (2001) J . Biol . Chem . 276, 28459-28464} . In order to know the IPP binding mode in more detail, we selected six highly conserved residues in Regions III, IV, and V among nine conserved aromatic residues in Micrococcus luteus B-P 26 UPP synthase for substitution by site-directed mutagenesis . The mutant enzymes were expressed and purified to homogeneity, and then their effects on substrate binding and the catalytic function were examined . All of the mutant enzymes showed moderately similar far-UV CD spectra to that of the wild-type, indicating that none of the replacement of conserved aromatic residues affected the secondary structure of the enzyme . Kinetic analysis showed that the replacement of Tyr-71 with Ser in Region III, Tyr-148 with Phe in Region IV, and Trp-210 with Ala in Region V brought about 10-1,600-fold decreases in the kcat/Km values compared to that of the wild-type but the Km values for both substrates IPP and FPP resulted in only moderate changes . Substitution of Phe-207 with Ser in Region V resulted in a 13-fold increase in the Km value for IPP and a 1,000-2,000-fold lower kcat/Km value than those of the wild-type, although the Km values for FPP showed about no significant changes . In addition, the W224A mutant as to Region V showed 6-fold and 14-fold increased Km values for IPP and FPP, respectively, and 100-250-fold decreased kcat/Km values as compared to those of the wild-type . These results suggested that these conserved aromatic residues play important roles in the binding with both substrates, IPP and FPP, as well as the catalytic function of undecaprenyl diphosphate synthase. Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku, 2003, (121), 76 - 7 {Lysozyme Reference Standard (Control 031) of National Institute of Health Sciences}; Kaihara A et al.; The "Lysozyme Reference Standard (Control 951031)" of the National Institute of Health Sciences was prepared . The lysozyme potency of the standard material was assayed against the Lysozyme Reference Standard (Control 951) by turbidimetric method two turbidimetric methods using the dried y-cells of Micrococcus luteus as a substrate . The potency of the standard material was in satisfactory agreement with that of Lysozyme Reference Standard (Control 951) and was defined as 1 mg {potency} per mg. J Mol Biol, 2004 Jan 30, 335(5), 1199 - 211 Redefinition of the cleavage sites of DNase I on the nucleosome core particle; Cousins DJ et al.; DNase I has been widely used for the footprinting of DNA-protein interactions including analyses of nucleosome core particle (NCP) structure . Our understanding of the relationship between the footprint and the structure of the nucleosome complex comes mainly from digestion studies of NCPs, since they have a well-defined quasi-symmetrical structure and have been widely investigated . However, several recent results suggest that the established consensus of opinion regarding the mode of digestion of NCPs by DNase I may be based on erroneous interpretation of results concerning the relationship between the NCP ends and the dyad axis . Here, we have used reconstituted NCPs with defined ends, bulk NCPs prepared with micrococcal nuclease and molecular modelling to reassess the mode of DNase I digestion . Our results indicate that DNase I cuts the two strands of the nucleosomal DNA independently with an average stagger of 4 nt with the 3'-ends protruding . The previously accepted value of 2 nt stagger is explained by the finding that micrococcal nuclease produces NCPs not with flush ends, but with approximately 1 nt 5'-recessed ends . Furthermore we explain why the DNA stagger is an even and not an odd number of nucleotides . These results are important for studies using DNase I to probe nucleosome structure in complex with other proteins or any DNA-protein complex containing B-form DNA . We also determine the origin of the 10n +/- 5 nt periodicity found in the internucleosomal ladder of DNase I digests of chromatin from various species . The explanation of the 10n +/- 5 nt ladder may have implications for the structure of the 30 nm fibre. Curr Eye Res, 2004 Jan, 28(1), 25 - 36 Quantitative and conformational characterization of lysozyme deposited on balafilcon and etafilcon contact lens materials; Senchyna M et al.; PURPOSE: To determine whether differences in lysozyme deposition and/or activity exist on worn etafilcon and balafilcon contact lenses following care with a polyquaternium-based system (PQ) or a polyhexanide-based system (PHMB) . METHODS: Following acid-based deposit extraction, lysozyme concentration was determined via Western blotting and lysozyme activity was determined by a micrococcyl assay . RESULTS: Lysozyme deposition on etafilcon lenses was greater following disinfection with the PHMB-based system (1551 +/- 371 micro g/lens vs 935 +/- 271 micro g/lens; p < 0.001) . Deposition on balafilcon lenses was not influenced by the care regimen (10 +/- 3.5 micro g/lens vs 10 +/- 5 micro g/lens; p = 0.89) . For both materials, the percentage of denatured lysozyme was greater when they were exposed to the PHMB-based system (28 vs 21%; p = 0.05 (etafilcon) and 57 vs 40%; p = 0.04 (balafilcon)) . CONCLUSIONS: The quantity and conformation of lysozyme deposited on hydrogel contact lens materials is significantly influenced by both lens material and care regimen. Eur J Ophthalmol, 2003 Nov-Dec, 13(9-10), 770 - 2 The effect of anterior chamber maintainer on anterior chamber contamination; Baykara M et al.; PURPOSE: To evaluate the effect of anterior chamber continuous infusion maintainer system on the contamination of anterior chamber in phacoemulsification surgery . METHODS: Clear corneal phacoemulsification surgery was performed in 132 eyes of 132 randomly selected patients with cataract who were divided into two groups of 66 eyes according to the use of an anterior chamber maintainer (ACM) system . The fluid specimens were taken from anterior chamber in the beginning and at the end of the surgery . They were transferred under anaerobic conditions and investigated by culturing onto blood agar and thiogluconate broth media . Differences between the two groups with respect to contamination of the specimens were investigated . RESULTS: The mean age of the group undergoing surgery without a maintainer system (Group A) was 63 +/- 10 years (min = 41, max = 80) versus 59 +/- 10 years (min = 33, max = 80) in the other group (Group B) in which the maintainer was used during surgery . In the postoperative specimen, Micrococcus species were isolated from one eye (1.5%) in Group A and S . pyogenes in one eye (1.5%) from Group B . Mean follow-up interval was 12 +/- 6 (min = 4, max = 28) months . CONCLUSIONS: The use of ACM system in clear corneal phacoemulsification surgery carries no additional risks as far as contamination is concerned. J Infect Chemother, 2003 Dec, 9(4), 358 - 60 Penetration of oral telithromycin into female genital tissues; Mikamo H et al.; To estimate the potential efficacy of telithromycin in the treatment of gynecological infections, a pharmacokinetic study was conducted in 13 Japanese subjects . Telithromycin was administered orally, at a dose of 600 mg, to patients undergoing hysterectomy, 3.0 to 7.5 h prior to the hysterectomy . At surgical operation, cubital venous blood, uterine arterial blood, vaginal cervix uteri (portio vaginalis), supravaginal uterine cervix, uterine endometrium, uterine myometrium, oviduct, and ovary specimens were collected separately . The blood and tissue concentrations of telithromycin were measured with a bioassay, using Micrococcus luteus ATCC 9361 as the test organism . The concentrations of telithromycin in these tissues and their proportions in relation to that in cubital venous blood (in parentheses) were as follows: cubital venous blood, 0.119 to 1.270 mg/l; uterine arterial blood, 0.111 to 1.230 mg/l; vaginal cervix uteri (portio vaginalis), 0.356 to 1.850 mg/kg (1.324 to 5.640), supravaginal uterine cervix, 0.376 to 4.520 mg/kg (1.108 to 16.807), uterine endometrium, 0.234 to 5.300 mg/kg (0.975 to 12.185), uterine myometrium, 0.309 to 5.050 mg/kg (1.288 to 19.832), oviduct, 0.375 to 5.550 mg/kg (1.563 to 10.000); and ovary, 0.495 to 5.250 mg/kg (1.835 to 6.851) . From these results, it was concluded that the concentrations of telithromycin in female genital tissues are generally higher than those in blood . Taking the antimicrobial spectrum of telithromycin into consideration, it was suggested that telithromycin could potentially be a good candidate for the treatment of gynecological infections, including cases associated with sexually transmitted diseases. Infect Immun, 2004 Jan, 72(1), 515 - 26 Individual Mycobacterium tuberculosis resuscitation-promoting factor homologues are dispensable for growth in vitro and in vivo; Tufariello JM et al.; Mycobacterium tuberculosis possesses five genes with significant homology to the resuscitation-promoting factor (Rpf) of Micrococcus luteus . The M . luteus Rpf is a secreted approximately 16-kDa protein which restores active growth to cultures of M . luteus rendered dormant by prolonged incubation in stationary phase . More recently, the Rpf-like proteins of M . tuberculosis have been shown to stimulate the growth of extended-stationary-phase cultures of Mycobacterium bovis BCG . These data suggest that the Rpf proteins can influence the growth of mycobacteria; however, the studies do not demonstrate specific functions for the various members of this protein family, nor do they assess the function of M . tuberculosis Rpf homologues in vivo . To address these questions, we have disrupted each of the five rpf-like genes in M . tuberculosis Erdman, and analyzed the mutants for their growth in vitro and in vivo . In contrast to M . luteus, for which rpf is an essential gene, we find that all of the M . tuberculosis rpf deletion mutant strains are viable; in addition, all show growth kinetics similar to Erdman wild type both in vitro and in mouse organs following aerosol infection . Analysis of rpf expression in M . tuberculosis cultures from early log phase through late stationary phase indicates that expression of the rpf-like genes is growth phase-dependent, and that the expression patterns of the five M . tuberculosis rpf genes, while overlapping to various degrees, are not uniform . We also provide evidence that mycobacterial rpf genes are expressed in vivo in the lungs of mice acutely infected with virulent M . tuberculosis. RNA, 2004 Jan, 10(1), 75 - 89 Saccharomyces SRP RNA secondary structures: a conserved S-domain and extended Alu-domain; Van Nues RW et al.; The contribution made by the RNA component of signal recognition particle (SRP) to its function in protein targeting is poorly understood . We have generated a complete secondary structure for Saccharomyces cerevisiae SRP RNA, scR1 . The structure conforms to that of other eukaryotic SRP RNAs . It is rod-shaped with, at opposite ends, binding sites for proteins required for the SRP functions of signal sequence recognition (S-domain) and translational elongation arrest (Alu-domain) . Micrococcal nuclease digestion of purified S . cerevisiae SRP separated the S-domain of the RNA from the Alu-domain as a discrete fragment . The Alu-domain resolved into several stable fragments indicating a compact structure . Comparison of scR1 with SRP RNAs of five yeast species related to S . cerevisiae revealed the S-domain to be the most conserved region of the RNA . Extending data from nuclease digestion with phylogenetic comparison, we built the secondary structure model for scR1 . The Alu-domain contains large extensions, including a sequence with hallmarks of an expansion segment . Evolutionarily conserved bases are placed in the Alu- and S-domains as in other SRP RNAs, the exception being an unusual GU(4)A loop closing the helix onto which the signal sequence binding Srp54p assembles (domain IV) . Surprisingly, several mutations within the predicted Srp54p binding site failed to disrupt SRP function in vivo . However, the strength of the Srp54p-scR1 and, to a lesser extent, Sec65p-scR1 interaction was decreased in these mutant particles . The availability of a secondary structure for scR1 will facilitate interpretation of data from genetic analysis of the RNA. Mikrobiologiia, 2003 Sep-Oct, 72(5), 594 - 9 {Free radicals in mercury-resistant bacteria indicate a novel metabolic pathway}; Ostrovskii DN et al.; A mercury resistant-soil bacterium P.10.15, identified as a close relative of Pseudomonas veronii, was shown to accumulate a specific compound in the stationary phase of growth . This compound is converted to a long-lived free radical under oxidizing conditions, as registered by its EPR signal at room temperature . The compound was purified by ion-exchange and gel-filtration chromatography and identified by mass spectroscopy, 2D NMR, and EPR as a trisaccharide beta-D-GlcpNOH,CH3-(1-->6)-alpha-D-Glcp-(1-->1)-alpha-D-Glcp, or, in other words, as 6-O-(2-deoxy-2-{N-methyl}hydroxylamino-beta-D- glucopyranosyl)-alpha-alpha-trehalose, previously discovered in Micrococcus luteus (lysodeikticus) and named lysodektose . The compound is suggested to be a novel intermediate of a previously unknown basic metabolic pathway of trehalose transformation in bacteria, a potential target for antibacterial drug development. J Pediatr Hematol Oncol, 2003 Dec, 25(12), 969 - 74 Fatal pulmonary hemorrhage associated with micrococcal infection in two children with acute lymphoblastic leukemia; Payne JH et al.; Pulmonary hemorrhage is a rare cause of death in patients with acute leukemia . Within a 2-month period the authors observed two fatal pediatric cases, which were associated with opportunistic organisms of the genus Micrococcus . Both patients were receiving consolidation treatment for acute lymphoblastic leukemia . The authors discuss the causes of pulmonary hemorrhage in patients with leukemia and review the relevant literature . Micrococci have previously been considered as non-pathogenic, but there is considerable evidence for morbidity and mortality occurring, particularly in immunocompromised patients . The authors propose that micrococcal infection may have been a major predisposing factor for pulmonary hemorrhage in these thrombocytopenic patients. J Photochem Photobiol B, 2003 Dec 5, 72(1-3), 69 - 78 Antitumor activity of 4-amino and 8-methyl-4-(3diethylamino propylamino)pyrimido{4',5':4,5}thieno (2,3-b) quinolines; Gopal M et al.; The interaction of 4-aminopyrimido {4',5':4,5} thieno (2,3-b) quinoline and 8-methyl-4-(3-diethylaminopropylamino) pyrimido {4',5':4,5} thieno (2,3-b) quinoline with DNA was studied by UV-Vis and fluorescence spectrophotometry as well as by hydrodynamic methods . On binding to DNA, the absorption spectra underwent bathochromic and hypochromic shifts and the fluorescence was quenched . These compounds are able to bind to DNA with an affinity of about 10(6) M(-1) for calf thymus DNA at ionic strength 0.01 M and their intercalating characteristic (lengthening of the DNA) depends upon the length of the chain . Binding to the GC-rich DNA of Micrococcus lysodeikticus was stronger than the binding to calf thymus DNA at ionic strength 0.01 M . The cytotoxicities of these compounds on leukemia HL-60, melanoma B16F10 and neuro 2a cells are quite similar and inhibition (IC50) is in the range of 0.992-3.968 microM . The anticancer efficacy against B16 melanoma, has provided evidence of major antitumor activity for 8-methyl-4-(3diethylaminopropylamino) pyrimido {4',5':4,5} thieno(2,3-b)quinoline . Single or multiple intraperitonial (i.p) doses of drug proved high level activity against the subcutaneous (s.c) grafted B16 melanoma, significantly increasing survival (p<0.001) and inhibiting tumor growth (T/C of 4%) . This study offers a new intercalation functional group to DNA-targeted drug design. Cancer Res, 2003 Nov 15, 63(22), 7968 - 74 RNA-directed actions of 8-chloro-adenosine in multiple myeloma cells; Stellrecht CM et al.; The purine analogue, 8-chloro-adenosine (8-Cl-Ado), induces apoptosis in a number of multiple myeloma (MM) cell lines . This ribonucleoside analogue accumulates as a triphosphate and selectively inhibits RNA synthesis without perturbing DNA synthesis . Cellular RNA is synthesized by one of three polymerases (Pol I, II, or III); thus, the inhibition of one or more RNA polymerases may be mediating 8-Cl-Ado cytotoxicity . Here, we have addressed this question by dissecting the RNA-directed actions of 8-Cl-Ado in MM cells . Differential alterations in {(3)H}uridine incorporation were found in the three major classes of RNA after a 20-h exposure with 10 microM 8-Cl-Ado . The synthesis rate of Pol III transcripts, 5 S and tRNA, remained unchanged, whereas Pol I-mediated rRNA synthesis decreased by approximately 20% . In contrast, mRNA synthesis, which is transcribed by Pol II, rapidly declined within 4 h and reached a 50% decrease, which was maintained for 20 h . Parallel to RNA synthesis inhibition, 8-Cl-Ado was maximally incorporated in the mRNA (>13 nmol/mg RNA), which was 5-fold higher than the tRNA and rRNA incorporation . Electrophoretic and radiographic analysis of newly synthesized and processed {(14)C}uridine-labeled transcripts indicated that the analogue blocks transcription elongation . Consistent with that result, high-performance liquid chromatography analysis of micrococcal nuclease and spleen phosphodiesterase-digested RNA demonstrated that the analogue incorporation is at the 3' terminus . In conclusion, our data demonstrate that in MM cells, 8-Cl-Ado is preferentially incorporated into mRNA, suggesting a propensity toward Pol II, and inhibits RNA synthesis by premature transcriptional chain termination. EMBO J, 2003 Nov 3, 22(21), 5851 - 62 A chromosomal SIR2 homologue with both histone NAD-dependent ADP-ribosyltransferase and deacetylase activities is involved in DNA repair in Trypanosoma brucei; Garcia-Salcedo JA et al.; SIR2-like proteins have been implicated in a wide range of cellular events including chromosome silencing, chromosome segregation, DNA recombination and the determination of life span . We report here the molecular and functional characterization of a SIR2-related protein from the protozoan parasite Trypanosoma brucei, which we termed TbSIR2RP1 . This protein is a chromosome-associated NAD-dependent enzyme which, in contrast to other known proteins of this family, catalyses both ADP-ribosylation and deacetylation of histones, particulary H2A and H2B . Under- or overexpression of TbSIR2RP1 decreased or increased, respectively, cellular resistance to DNA damage . Treatment of trypanosomal nuclei with a DNA alkylating agent resulted in a significant increase in the level of histone ADP-ribosylation and a concomitant increase in chromatin sensitivity to micrococcal nuclease . Both of these responses correlated with the level of TbSIR2RP1 expression . We propose that histone modification by TbSIR2RP1 is involved in DNA repair. Mol Cell Biol, 2003 Nov, 23(22), 7937 - 46 Replication-independent assembly of nucleosome arrays in a novel yeast chromatin reconstitution system involves antisilencing factor Asf1p and chromodomain protein Chd1p; Robinson KM et al.; Chromatin assembly in a crude DEAE (CD) fraction from budding yeast is ATP dependent and generates arrays of physiologically spaced nucleosomes which significantly protect constituent DNA from restriction endonuclease digestion . The CD fractions from mutants harboring deletions of the genes encoding histone-binding factors (NAP1, ASF1, and a subunit of CAF-I) and SNF2-like DEAD/H ATPases (SNF2, ISW1, ISW2, CHD1, SWR1, YFR038w, and SPT20) were screened for activity in this replication-independent system . ASF1 deletion substantially inhibits assembly, a finding consistent with published evidence that Asf1p is a chromatin assembly factor . Surprisingly, a strong assembly defect is also associated with deletion of CHD1, suggesting that like other SNF2-related groups of nucleic acid-stimulated ATPases, the chromodomain (CHD) group may contain a member involved in chromatin reconstitution . In contrast to the effects of disrupting ASF1 and CHD1, deletion of SNF2 is associated with increased resistance of chromatin to digestion by micrococcal nuclease . We discuss the possible implications of these findings for current understanding of the diversity of mechanisms by which chromatin reconstitution and remodeling can be achieved in vivo. Mol Cell Biol, 1982 Dec, 2(12), 1608 - 18 Linear DNA does not form chromatin containing regularly spaced nucleosomes; Mertz JE; The topological state of DNA may play a role in regulating chromatin structure and gene expression in eucaryotes . To test this hypothesis, the arrangements of nucleosomes on circular and unit-length linear simian virus 40 (SV40) DNAs incubated in nuclei of Xenopus oocytes were determined by (i) analyzing changes in the electrophoretic properties of the DNAs and (ii) examining the patterns of DNA fragments resulting from digestions with micrococcal nuclease . Whereas circular DNA became associated with nucleosomes that were arranged along the DNA at regular intervals of approximately 195 base pairs, linear DNA failed to reconstitute into chromatin containing regularly spaced nucleosomes . DNA that failed to form proper chromatin was gradually degraded, indicating that histone proteins in proper association with DNA may be the cellular component that normally protects chromosomal DNA from endonucleolytic attack . When either circular or linear DNA was incubated in an in vitro transcription system made from a whole-cell extract of HeLa cells, most of the molecules did not associate with histone proteins to form regularly spaced nucleosomes . Furthermore, linearization of mRNA-encoding DNAs, including SV40, reduces their transcriptional activity in Xenopus oocytes to a level comparable to that obtained with the in vitro transcription system employed here . Therefore, proper association of DNA with appropriate cellular chromosomal factors may be a prerequisite for proper transcription by RNA polymerase II. J Agric Food Chem, 2003 Oct 22, 51(22), 6468 - 74 Physicochemical properties and bioactivity of nisin-containing cross-linked hydroxypropylmethylcellulose films; Sebti I et al.; Cross-linked hydroxypropylmethylcellulose (HPMC) cast films with citric acid as polycarboxylic cross-linker were elaborated to study the effect of cross-linking level on various properties . Increased amounts of cross-linking agent were not connected to statistically different tensile strength and Young's modulus . Whatever the cross-linking level of the film was, the ultimate elongation parameter decreased by approximately 60% compared to the HMPC control film . Moisture sorption isotherms and water contact angle meter showed that the effect of cross-linking degree tends to reduce the hygroscopic and hydrophilic characteristics of films . In addition, to control bacteria growth on food surfaces, the antimicrobial activity of both 98% cross-linked HPMC-nisin and control HPMC-nisin films was tested on Micrococcus luteus . Despite the incorporation of a significant content of nisin, cross-linked HPMC-nisin films were completely inactive on the microbial strain compared to the HPMC-nisin control films . Cross-linking conditions likely either denatured the nisin or irreversibly bound nisin to the cross-linked HPMC . However, nisin adsorbed into films made from previously cross-linked HPMC maintained its activity. J Virol, 2003 Nov, 77(21), 11425 - 35 Chromatin remodeling of the Kaposi's sarcoma-associated herpesvirus ORF50 promoter correlates with reactivation from latency; Lu F et al.; The switch from latent to lytic infection of Kaposi's sarcoma-associated herpesvirus is initiated by the immediate early transcriptional activator protein Rta/open reading frame 50 (ORF50) . We examined the transcriptional regulation of the ORF50 core promoter in response to lytic cycle stimulation . We show that the ORF50 promoter is highly responsive to sodium butyrate (NaB) and trichostatin A (TSA), two chemicals known to inhibit histone deacetylases . The NaB and TSA responsive element was mapped to a 70-bp minimal promoter containing an essential GC box that binds Sp1/Sp3 in vitro and in vivo . Micrococcal nuclease mapping studies revealed that a nucleosome is positioned over the transcriptional initiation and the Sp1/3 binding sites . Stimulation with NaB or TSA increased histone acetylation and restriction enzyme accessibility of the ORF50 promoter transcription initiation site . Chromatin immunoprecipitation assay was used to demonstrate that the ORF50 promoter is associated with several different histone deacetylase proteins (including HDAC1, 5, and 7) in latently infected cells . NaB treatment led to the rapid association of Ini1/Snf5, a component of the Swi/Snf family of chromatin remodeling proteins, with the ORF50 promoter . Ectopic expression of the CREB-binding protein (CBP) histone acetyltransferase (HAT) stimulated plasmid-based ORF50 transcription in a HAT-dependent manner, suggesting that CBP recruitment to the ORF50 promoter can be an initiating event for transcription and viral reactivation . Together, these results suggest that remodeling of a stably positioned nucleosome at the transcriptional initiation site of ORF50 is a regulatory step in the transition from latent to lytic infection. Eukaryot Cell, 2003 Oct, 2(5), 876 - 85 Nucleosome position-dependent and -independent activation of HIS7 epression in Saccharomyces cerevisiae by different transcriptional activators; Valerius O et al.; ARO4 and HIS7 are two tandemly orientated genes of Saccharomyces cerevisiae that are transcribed into the same direction . The ARO4 terminator and the HIS7 promoter regions are sensitive to Micrococcus nuclease (Mnase) and separated by a positioned nucleosome . The HIS7 promoter is target for the transcription factors Gcn4p and Bas1p/Bas2p that activate its transcription upon amino acid starvation and purine limitation, respectively . Activation of the HIS7 gene by Gcn4p overexpression but not by Bas1p/Bas2p releases an ordered nucleosome distribution to yield increased Mnase sensitivity throughout the intergenic region . This remodeling is SNF2 dependent but mostly GCN5 independent . Accordingly, SNF2 is necessary for the Gcn4p-mediated transcriptional activation of the HIS7 gene . GCN5 is required for activation upon adenine limitation by Bas1p/Bas2p . Our data suggest that activation of HIS7 transcription by Gcn4p and Bas1p/Bas2p is supported by a nucleosome position-dependent and -independent mechanism, respectively . Whereas Gcn4p activation causes Swi/Snf-mediated remodeling of the nucleosomal architecture at the HIS7 promoter, the Bas1p/Bas2p complex presumably activates in combination with Gcn5p-dependent histone acetylation. Glycobiology, 2004 Jan, 14(1), 73 - 81 Epub 2003 Oct 09. Structural and topological studies on the lipid-mediated assembly of a membrane-associated lipomannan in Micrococcus luteus; Pakkiri LS et al.; The biosynthesis of three mannolipids and the presence of a membrane-associated lipomannan in Micrococcus luteus (formerly Micrococcus lysodeikticus) were documented over 30 years ago . Structural and topological studies have been conducted to learn more about the possible role of the mannolipids in the assembly of the lipomannan . The major mannolipid has been purified and characterized as alpha-D-mannosyl-(1 --> 3)-alpha-D-mannosyl-(1 --> 3)-diacylglycerol (Man2-DAG) by negative-ion electrospray-ionization multistage mass spectrometry (ESI-MSn) . Analysis of the fragmentation patterns indicates that the sn-1 position is predominantly acylated with a 12-methyltetradecanoyl group and the sn-2 position is acylated with a myristoyl group . The lipomannan is shown to be located on the exterior face of the cytoplasmic membrane, and not exposed on the surface of intact cells, by staining of intact protoplasts with fluorescein isothiocyanate (FITC)-linked concanavalin A (Con A) . When cell homogenates of M . luteus are incubated with GDP-{3H}mannose (GDP-Man), {3H}mannosyl units are incorporated into Man1-2-DAG, mannosylphosphorylundecaprenol (Man-P-Undec) and the membrane-associated lipomannan . The addition of amphomycin, an inhibitor of Man-P-Undec synthesis, had no effect on the synthesis of Man1-2-DAG, but blocked the incorporation of {3H}mannose into Man-P-Undec and consequently the lipomannan . These results strongly indicate that GDP-Man is the direct mannosyl donor for the synthesis of Man1-2-DAG, and that the majority of the 50 mannosyl units in the lipomannan are derived from Man-P-Undec . Protease-sensitivity studies with intact and lysed protoplasts indicate that the active sites of the mannosyltransferases catalyzing the formation of Man1-2-DAG and Man-P-Undec are exposed on the inner face, and the Man-P-Undec-mediated reactions occur on the outer surface of the cytoplasmic membrane . Based on all of these results, a topological model is proposed for the lipid-mediated assembly of the membrane-bound lipomannan. Nucleic Acids Res, 2003 Oct 15, 31(20), 5897 - 906 Effects of genomic context and chromatin structure on transcription-coupled and global genomic repair in mammalian cells; Feng Z et al.; It has been long recognized that in mammalian cells, DNA damage is preferentially repaired in the transcribed strand of transcriptionally active genes . However, recently, we found that in Chinese hamster ovary (CHO) cells, UV-induced cyclobutane pyrimidine dimers (CPDs) are preferentially repaired in both the transcribed and the non-transcribed strand of exon 1 of the dihydrofolate reductase (DHFR) gene . We mapped CPD repair at the nucleotide level in the transcriptionally active DHFR gene and the adjacent upstream OST gene, both of which have been translocated to two chromosomal positions that differ from their normal endogeneous positions . This allowed us to study the role of transcription, genomic context and chromatin structure on repair . We found that CPD repair in the transcribed strand is the same for endogenous and translocated DHFR genes, and the order of repair efficiency is exon 1 > exon 2 > exon 5 . However, unlike the endogenous DHFR gene, efficient repair of CPDs in the non-transcribed strand of exon 1 is not observed in the translocated DHFR gene . CPDs are efficiently repaired in the transcribed strand in endogenous and translocated OST genes, which indicates that efficient repair in exon 1 of the non-transcribed strand of the endogenous DHFR gene is not due to the extension of transcription-coupled repair of the OST gene . Using micrococcal nuclease digestion, we probed the chromatin structure in the DHFR gene and found that chromatin structure in the exon 1 region of endogenous DHFR is much more open than at translocated loci . These results suggest that while transcription-coupled repair is transcription dependent, global genomic repair is greatly affected by chromatin structure. Chem Res Toxicol, 2003 Sep, 16(9), 1130 - 7 Identification of DNA adducts derived from riddelliine, a carcinogenic pyrrolizidine alkaloid; Chou MW et al.; Riddelliine is a naturally occurring carcinogenic pyrrolizidine alkaloid that produces liver tumors in experimental animals . Riddelliine requires metabolic activation to dehydroriddelliine and 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) to exert its toxicity . Previously, (32)P-postlabeling HPLC was used to detect a set of eight DHP-derived adduct peaks from DNA modified both in vitro and in vivo . Among these DHP-derived DNA adducts, two were identified as epimers of DHP-2'-deoxyguanosine 3'-monophosphate . In this study, the remaining adducts have been characterized as DHP-modified dinucleotides . A series of dinucleotides, TpGp, ApGp, TpCp, ApCp, TpAp, ApAp, TpTp, and ApTp, were obtained by enzymatic digestion of calf thymus DNA with micrococcal nuclease (MN) and spleen phosphodiesterase (SPD) followed by HPLC separation and structural identification by negative ion electrospray tandem mass spectrometry (ES/MS/MS) . Incubation of individual dinucleotides with DHP produced DHP-modified dinucleotide adducts that were also characterized using LC-ES/MS/MS . A parallel analysis of the isolated DHP-modified dinucleotides using (32)P-postlabeling recapitulated the series of unidentified adduct peaks that we previously reported from DHP-modified calf thymus DNA in vitro and rat liver DNA in vivo . Intact calf thymus DNA was also reacted with DHP and then digested by MN/SPD under the same conditions . The adduct profile obtained from LC-ES/MS/MS analysis was similar to that observed from the isolated dinucleotides . Structural analysis using LC-ES/MS/MS showed that DHP bound covalently to both 3'- and 5'-guanine, -adenine, and -thymine bases (but not cytosine) of dinucleotides to produce two or more isomers of each DHP-dinucleotide adduct . By comparing adduct formation at dissimilar bases within individual dinucleotides, the relative reactivity of DHP with individual bases was determined to be guanine > adenine approximately thymine . Identification of the entire set of DHP-derived DNA adducts further validates the conclusion that riddelliine is a genotoxic carcinogen and enhances the applicability of these biomarkers for assessing carcinogenic risks from exposure to pyrrolizidine alkaloids. Fish Shellfish Immunol, 2003 Oct, 15(4), 325 - 31 cDNA cloning of the lysozyme of the white shrimp Penaeus vannamei; Sotelo-Mundo RR et al.; Lysozyme, an antibacterial protein, has been implicated in innate immunity in invertebrates, but its activity in shrimp remained to be determined . We cloned the white shrimp lysozyme cDNA using a PCR strategy and detected its activity in haemocytes using a lytic-zone assay against <