Micrococci

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 Micrococcus luteus . The cloning was based on a reported EST (dbEST BE18831) . The deduced amino acid sequence resulted in 150 amino with 46% identity to hen egg white lysozyme . RT-PCR was used to detect lysozyme mRNA in haemocytes . Analysis of the amino acid sequence of the shrimp lysozyme showed that it belongs to the C-type family of lysozymes . Furthermore, the lysozyme amino acid sequence contained extra residues at its C-terminus, which are characteristic of marine invertebrates . This information will be useful in future studies on the molecular mechanisms of immunity in marine invertebrates.

Lett Appl Microbiol, 2003, 37(4), 314 - 7
Structural analysis and genetic variation of the 16S-23S rDNA internal spacer region from Micrococcus luteus strains; Haga S et al.; AIMS: To clone and sequence the 16S-23S ribosomal DNA (rDNA) internal spacer region (ISR) from Micrococcus luteus . METHODS AND RESULTS: The primer pair for 16S-23S rDNA ISR amplified a fragment of about 850 bp in length for two strains, JCM3347 and JCM3348 and a fragment of about 790 bp for a strain, ATCC9341 . After sequencing the ISRs were identified by the comparison of the ISRs and the flanking regions of ISR . CONCLUSIONS: Although the sequence difference of the ISR occurred at only one position between the two JCM strains, the highly variable length (440 and 370 bp) and sequence similarity (about 40%) were demonstrated between the ISRs of the two JCM strains and a ATCC strain . SIGNIFICANCE AND IMPACT OF THE STUDY: A CCTCCT sequence was first detected at the 3'-end of the 16S rDNA of the three strains . Moreover, highly similar sequence to the 21-bp region containing a putative rRNA processing site was observed in the ISR of the three strains . Interestingly, no intercistronic tRNAs were demonstrated in the ISRs from the three strains.

Mol Ecol, 2003 Oct, 12(10), 2771 - 82
Mitochondrial DNA sequences reveal extensive cryptic diversity within a western American springsnail; Liu HP et al.; We analysed cytochrome c oxidase subunit I and NADH dehydrogenase subunit I sequence variation among 29 populations of a widely ranging southwestern springsnail (Pyrgulopsis micrococcus) and 18 regional congeners . Cladistic analyses of these sequences depict P . micrococcus as a polyphyletic composite of five well-supported clades . Sequence divergences among these clades and subclades imply the possible occurrence of as many as seven or eight cryptic species in addition to P . micrococcus . Our finding that P . micrococcus contains multiple, genetically distinct and geographically restricted lineages suggests that diversification within this highly speciose aquatic genus has been structured in large part by the operation of terrestrial barriers to gene flow . However, these sequence data also indicate that recent dispersal among hydrographically separated areas has occurred within one of these lineages, which we attribute to passive transport on migratory waterbirds.

J Biol Chem, 2003 Nov 21, 278(47), 46556 - 64 Epub 2003 Sep 09.
Manduca sexta serpin-3 regulates prophenoloxidase activation in response to infection by inhibiting prophenoloxidase-activating proteinases; Zhu Y et al.; Many serine proteinase inhibitors of the serpin superfamily have evolved in vertebrates and invertebrates to regulate serine proteinase cascades that mediate the host defense responses . We have isolated an immune-responsive serpin from the tobacco hornworm, Manduca sexta . This inhibitor, M . sexta serpin-3, contains a reactive site loop strikingly similar to the proteolytic activation site in prophenoloxidase (pro-PO) . Molecular cloning and sequence comparison indicate that serpin-3 is orthologous to Drosophila melanogaster serpin 27A, a regulator of melanization . M . sexta serpin-3 is constitutively present in hemolymph at a low concentration of 5-12 microg/ml and increases to 30-75 microg/ml after a microbial challenge . Recombinant serpin-3 efficiently blocks pro-PO activation in the hemolymph, and it forms SDS-stable acyl-enzyme complexes with purified pro-PO-activating proteinases (PAPs) from M . sexta . PAP-serpin-3 complexes were isolated by immunoaffinity chromatography from hemolymph activated by treatment with Micrococcus luteus . Kinetic analysis of PAP-serpin-3 association strongly suggests that serpin-3 is a physiological regulator of the pro-PO activation reaction.

Dev Comp Immunol, 2004 Jan, 28(1), 1 - 7
Purification and cDNA cloning of a novel antibacterial peptide with a cysteine-stabilized alphabeta motif from the longicorn beetle, Acalolepta luxuriosa; Saito A et al.; An antibacterial peptide from the hemolymph of a coleopteran insect, Acalolepta luxuriosa, in the superfamily Cerambyocidea was characterized . The mature antibacterial peptide had 27 amino acid residues with a theoretical molecular weight of 3099.29 and it showed antibacterial activity against Escherichia coli and Micrococcus luteus . The deduced amino acid sequence of the peptide showed that it had a cysteine-stabilized alphabeta motif with a C...CXXXC...C...CXC consensus sequence, like insect defensins . However, the results of a multiple sequence alignment and phylogenetic analysis with CLUSTAL X indicated that this peptide is a novel peptide with a cysteine-stabilized alphabeta motif that is distant from insect defensins.

Biol Chem, 2003 Jul, 384(7), 1019 - 27
Interaction of maize chromatin-associated HMG proteins with mononucleosomes: role of core and linker histones; Lichota J et al.; Two groups of plant chromatin-associated high mobility group (HMG) proteins, namely the HMGA family, typically containing four A/T-hook DNA-binding motifs, and the HMGB family, containing a single HMG-box DNA-binding domain, have been identified . We have examined the interaction of recombinant maize HMGA and five different HMGB proteins with mononucleosomes (containing approx . 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin . The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA in the presence of H1 differed from that observed in the absence of H1 . HMGA and the HMGB proteins bound H1-containing nucleosome particles with similar affinity . The plant HMG proteins could also bind nucleosomes that were briefly treated with trypsin (removing the N-terminal domains of the core histones), suggesting that the histone N-termini are dispensable for HMG protein binding . In the presence of untreated nucleosomes and trypsinised nucleosomes, HMGB1 could be chemically crosslinked with a core histone, which indicates that the trypsin-resistant part of the histones within the nucleosome is the main interaction partner of HMGB1 rather than the histone N-termini . In conclusion, these results indicate that specific nucleosome binding of the plant HMGB proteins requires simultaneous DNA and histone contacts.

Chem Biol, 2003 Aug, 10(8), 769 - 78
Structural basis for contrasting activities of ribosome binding thiazole antibiotics; Lentzen G et al.; Thiostrepton and micrococcin inhibit protein synthesis by binding to the L11 binding domain (L11BD) of 23S ribosomal RNA . The two compounds are structurally related, yet they produce different effects on ribosomal RNA in footprinting experiments and on elongation factor-G (EF-G)-dependent GTP hydrolysis . Using NMR and an assay based on A1067 methylation by thiostrepton-resistance methyltransferase, we show that the related thiazoles, nosiheptide and siomycin, also bind to this region . The effect of all four antibiotics on EF-G-dependent GTP hydrolysis and EF-G-GDP-ribosome complex formation was studied . Our NMR and biochemical data demonstrate that thiostrepton, nosiheptide, and siomycin share a common profile, which differs from that of micrococcin . We have generated a three-dimensional (3D) model for the interaction of thiostrepton with L11BD RNA . The model rationalizes the differences between micrococcin and the thiostrepton-like antibiotics interacting with L11BD.

Biosci Biotechnol Biochem, 2003 Aug, 67(8), 1751 - 60
Primary structure of inorganic polyphosphate/ATP-NAD kinase from Micrococcus flavus, and occurrence of substrate inorganic polyphosphate for the enzyme; Kawai S et al.; The gene encoding an inorganic polyphosphate/ATP-NAD kinase was cloned from Micrococcus flavus, and its primary structure was analyzed . Alignment of the primary structure with those of other characterized NAD kinases revealed candidate amino acid residues, mainly charged ones, that would be related to inorganic polyphosphate use . The alignment also showed that the primary structure found carried a protruding C-terminal polypeptide . Although the C-terminal polypeptide was demonstrated to be dispensable for the kinase activities, and was proposed to be removed in M . flavus, the entire primary structure including the C-terminal polypeptide was homologous with that of the ATP synthase beta chain . The inorganic polyphosphate used by the inorganic polyphosphate/ATP-NAD kinase as a phosphoryl donor was isolated from cells of M . flavus, suggesting that the ability of the enzyme to use inorganic polyphosphate is of physiological significance and is not an evolutionary trait alone.

Eur J Biochem, 2003 Sep, 270(18), 3750 - 9
In vivo cross-linking of nucleosomal histones catalyzed by nuclear transglutaminase in starfish sperm and its induction by egg jelly triggering the acrosome reaction; Nunomura K et al.; A histone heterodimer, designated as p28, which contains an Nepsilon(gamma-glutamyl)lysine cross-link between Gln9 of histone H2B and Lys5 or Lys12 of histone H4, is present in starfish (Asterina pectinifera) sperm . Treatment of sperm nuclei with micrococcal nuclease produced soluble chromatin, which was size-fractionated by sucrose-gradient centrifugation to give p28-containing oligonucleosome and p28-free mononucleosome fractions, indicating that the cross-link is internucleosomal . When sperm nuclei were incubated with monodansylcadaverine, a fluorescent amine, in the presence or absence of Ca(2+), histone H2B was modified only in the presence of Ca(2+) . Gln9, in the N-terminal region, was modified, but the other Gln residues located in the internal region were not, suggesting that the modification takes place on the surface of the nucleosome core by the in situ action of a Ca(2+)-dependent nuclear transglutaminase . Treatment of sperm with the egg jelly, which activates Ca(2+) influx to induce the acrosome reaction, resulted in a significant elevation of the p28 content in the nucleus . This is the first demonstration of an in vivo activation of transglutaminase leading to the formation of a cross-link in intracellular proteins.

J Mol Biol, 2003 Sep 5, 332(1), 111 - 25
Dinucleosome DNA of human K562 cells: experimental and computational characterizations; Kato M et al.; Dinucleosome formation is the first step in the organization of the higher order chromatin structure . With the ultimate aim of elucidating the dinucleosome structure, we constructed a library of human dinucleosome DNA . The library consists of PCR-amplifiable DNA fragments obtained by treatment of nuclei of erythroid K562 cells with micrococcal nuclease followed by extraction of DNA and adaptor ligation to the blunt-ended DNA fragments . The library was then cloned using a plasmid vector and the sequences of the clones were determined . The dominating clones containing the Alu elements were removed . A total of 1002 clones, which comprised a dinucleosome database, contained 84 and 918 clones from the clones before and after removing Alu elements, respectively . Approximately 70% of the clones were between 300 and 400 bp in size and they were distributed to various locations of all chromosomes except the Y chromosome . The clones containing A(2)N(8)A(2)N(8)A(2) or T(2)N(8)T(2)N(8)T(2) sequences were classified into three types, Type I (N shape), Type II (V shape) and Type III (M shape) according to DNA curvature plots . The locations of experimentally determined curved DNA segments matched well with the calculated ones though the clones of Types I and III showed additional curved DNA segments as revealed by the curvature plots . The distributions of complementary dinucleotides in the nucleosome DNA, at the ends of the dinucleosome DNA clones, allowed us to predict the positions of the nucleosome dyad axis, and estimate the size of the nucleosome core DNA, 125nt . The distributions of AA and TT dinucleotides, as well as other RR and YY dinucleotides, showed a periodicity with an average period of 10.4 bases, close to the values observed before . Mapping of nucleosome positions in the dinucleosome database based on the observed periodicity revealed that the nucleosomes were separated by a linker of 7.5+ approximately 10 x n nt . This indicates that the nucleosome-nucleosome orientations are, typically, halfway between parallel and antiparallel . Also an important finding is that the distributions of AA/TT and other RR/YY dinucleotides, apparently, reflect both DNA curvature and DNA bendability, cooperatively contributing to the nucleosome formation.

Science, 2003 Aug 22, 301(5636), 1096 - 9
Transcription elongation factors repress transcription initiation from cryptic sites; Kaplan CD et al.; Previous studies have suggested that transcription elongation results in changes in chromatin structure . Here we present studies of Saccharomyces cerevisiae Spt6, a conserved protein implicated in both transcription elongation and chromatin structure . Our results show that, surprisingly, an spt6 mutant permits aberrant transcription initiation from within coding regions . Furthermore, transcribed chromatin in the spt6 mutant is hypersensitive to micrococcal nuclease, and this hypersensitivity is suppressed by mutational inactivation of RNA polymerase II . These results suggest that Spt6 plays a critical role in maintaining normal chromatin structure during transcription elongation, thereby repressing transcription initiation from cryptic promoters . Other elongation and chromatin factors, including Spt16 and histone H3, appear to contribute to this control.

Proc Natl Acad Sci U S A, 2003 Sep 2, 100(18), 10546 - 51 Epub 2003 Aug 14.
HILS1 is a spermatid-specific linker histone H1-like protein implicated in chromatin remodeling during mammalian spermiogenesis; Yan W et al.; Chromatin remodeling is a major event that occurs during mammalian spermiogenesis, the process of spermatid maturation into spermatozoa . Nuclear condensation during spermiogenesis is accomplished by replacing somatic histones (linker histone H1 and core histones) and the testis-specific linker histone, H1t, with transition proteins and protamines . It has long been thought that H1t is the only testis-specific linker histone, and that all linker histones are replaced by transition proteins, and subsequently by protamines during spermiogenesis . Here, we report the identification and characterization of a spermatid-specific linker histone H1-like protein (termed HILS1) in the mouse and human . Both mouse and human HILS1 genes are located in intron 8 of the alpha-sarcoglycan genes . HILS1 is highly expressed in nuclei of elongating and elongated spermatids (steps 9-15) . HILS1 displays several biochemical properties that are similar to those of linker histones, including the abilities to bind reconstituted mononucleosomes, produce a chromatosome stop during micrococcal nuclease digestion, and aggregate chromatin . Because HILS1 is expressed in late spermatids that do not contain core histones, HILS1 may participate in spermatid nuclear condensation through a mechanism distinct from that of linker histones . Because HILS1 also belongs to the large winged helix/forkhead protein superfamily, HILS1 may also regulate gene transcription, DNA repair, and/or other chromosome processes during mammalian spermiogenesis.

Med Dosw Mikrobiol, 2003, 55(1), 75 - 80
{Susceptibility to antibiotics of bacteria from genera Micrococcus, Kocuria, Nesterenkonia, Kytococcus and Dermacoccus}; Szczerba I; Two hundred and nineteen strains from genera Micrococcus, Kocuria, Nesterenkonia, Kytococcus and Dermacoccus isolated from different sources, such as saliva, skin of palm and forearm and from vestibule of nose were tested . The susceptibility to doxycycline, cetriaxone, cefuroxyme, amikacyne, amoksycillin with clavulanic acid, trimethoprim/sulfamethoxazole, ampicillin, ciprofloxacin, penicillin and erythromycin were estimated . In general, bacteria from these genera are sensitive to most of selected antibiotics . Most of the strains showed resistance to ampicillin and erythromycin . None of these strains produced beta-lactamases . In infectious caused by bacteria from genera Micrococcus, Kocuria, Nesterenkonia, Kytococcus and Dermacoccus in clinical treatment should be used amoksycillin with clavulanic acid, doxycycline, cetriaxone, cefuroxyme, or amikacine because, with one's own range of activity embrace highest percentage of investigated strains.

Med Dosw Mikrobiol, 2003, 55(1), 67 - 74
{Occurrence and number of bacteria from the Micrococcus, Kocuria, Nesterenkonia, Kytococcus and Dermacoccus genera on skin and mucous membranes in humans}; Szczerba I; The aim of work was to evaluate the frequency of occurrence of bacteria from genera of Micrococcus, Kocuria, Nesterenkonia, kytococcus and Dermatococcus on human skin and mucous membranes in healthy population . Among 150 investigated persons these bacteria were found in 122 (81.3%) . The frequency of isolation was similar in both sex (82.4% in female and 79.7% in male) . Most often the strains were isolated from oral cavity (48.7%), and from skin of palm and forearm (40.7% and 37.3%) . Least frequency of occurrence was observed in vestibule of nose (26%) . The predominant isolated strains were: M . luteus (26.2%), and N . halobia (21%) followed by K . varians (16.4%), M . lylae (12.2%), D . sedentarius (9.1%), K . kristinae (7.3%), K . nishinomiyaensis (7.3%), K . rosea (0.3%).

Tuberculosis (Edinb), 2003, 83(4), 261 - 9
Resuscitation factors from mycobacteria: homologs of Micrococcus luteus proteins; Zhu W et al.; SETTING: Resuscitation promoting factors (Rpfs) are proteins, originally identified in Micrococcus luteus, that promote recovery of bacteria from a viable but non-replicating phase (e.g., stationary phase or latency) to a replicating phase . Purified M . luteus Rpf can stimulate growth and increase recovery of M . luteus bacteria as well as Mycobacterium tuberculosis bacteria from prolonged stationary cultures . OBJECTIVE: To clone and characterize Rpfs from mycobacteria . DESIGN: We cloned one M . avium subsp . paratuberculosis rpf gene and one M . tuberculosis rpf gene into the pET19b or pET21a vector for expression in Escherichia coli . The His-tag recombinant proteins were purified and characterized.RESULTS: When the purified recombinant proteins were added to Sauton medium (a relatively minimal medium) at 100-500 pM, lag phase for mycobacteria from non-replicating cultures was shortened and there was a 10- to 100-fold increase in colony-forming units compared with control samples . In most probable number assays, the mycobacterial Rpfs increased recovery of mycobacteria from late stationary culture by about 10-fold . The Rpfs also promoted recovery of extensively washed Mycobacterium smegmatis bacteria inoculated into Sauton medium . Rpfs had only minor effects on growth of M . tuberculosis in BACTEC 12B broth, a rich medium . CONCLUSION: The mycobacterial Rpfs demonstrate resuscitation activities similar to those of the M . luteus Rpf.

Chin Med Sci J, 2000 Jun, 15(2), 124 - 6
Preparation of anti-idiotypic antibodies specific for anti-HEL and analysis of their functional mimicry; Mingyuan L et al.; OBJECTIVE: This study is to investigate the functional mimicry by using anti-idiotypic antibodies of enzymes . METHODS: Monoclonal anti-idiotypic antibodies against anti-HEL (hen egg-white lysozyme, HEL) antibodies were obtained by fusion of Sp2/0 myeloma cells with spleen cells of syngeneic mice immunized with monoclonal anti-HEL antibodies against HEL's different antigenic epitopes . Then bacteriolysis of the anti-idiotypic antibodies were observed . RESULTS: Eight hybridomas strains secreting anti-idiotypic antibodies were selected and characterized . It was shown that two of eight anti-idiotypic antibodies secreted by two hybridomas (1A10C9 and 2A11C1B3) could mimic HEL catalytic activity to lyse Micrococcus lysodeikticus and that the catalytic effect of mixed anti-idiotypic antibodies of 1A10C9 and 2A11C1B3 was stronger than that of one of them, but less than HEL . CONCLUSION: The results demonstrated that the anti-idiotypic antibodies that could mimic enzyme activity existed in the idiotype network during anti-enzymatic immune response.

Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 995 - 7
Reclassification of ATCC 9341 from Micrococcus luteus to Kocuria rhizophila; Tang JS et al.; Strain ATCC 9341, currently known as Micrococcus luteus, has been designated as a quality-control strain in a number of applications . It is also cited as the standard culture in several official methods and manuals, as well as the Code of Federal Regulations . Over the years, it has become apparent that ATCC 9341 does not resemble other M . luteus strains; however, its phenotypic characteristics alone were ambiguous . Recently, a polyphasic study was performed in which molecular data were combined with cytochemical properties and physiological characteristics . The results clearly indicate that ATCC 9341 is a member of the genus Kocuria . Thus, it is proposed to reclassify ATCC 9341 as Kocuria rhizophila and to alert users worldwide of this name change.

Biophys Chem, 2003 Jun 1, 104(2), 381 - 92
Acetylated nucleosome assembly on telomeric DNAs; Cacchione S et al.; The role of histone N-terminal domains on the thermodynamic stability of nucleosomes assembled on several different telomeric DNAs as well as on 'average' sequence DNA and on strong nucleosome positioning sequences, has been studied by competitive reconstitution . We find that histone tails hyperacetylation favors nucleosome formation, in a similar extent for all the examined sequences . On the contrary, removal of histone terminal domains by selective trypsinization causes a decrease of nucleosome stability which is smaller for telomeres compared to the other sequences examined, suggesting that telomeric sequences have only minor interactions with histone tails . Micrococcal nuclease kinetics shows enhanced accessibility of acetylated nucleosomes formed both on telomeric and 'average' sequence DNAs . These results suggest a more complex role for histone acetylation than the decrease of electrostatic interactions between DNA and histones.

Infect Immun, 2003 Aug, 71(8), 4789 - 94
Proteins of the Rpf family: immune cell reactivity and vaccination efficacy against tuberculosis in mice; Yeremeev VV et al.; It was shown recently that Mycobacterium tuberculosis expresses five proteins that are homologous to Rpf (resuscitation promoting factor), which is secreted by growing cells of Micrococcus luteus . Rpf is required to resuscitate the growth of dormant Micrococcus luteus organisms, and its homologues may be involved in mycobacterial reactivation . Mycobacterial Rpf-like products are secreted proteins, which makes them candidates for recognition by the host immune system and anti-Rpf immune responses potentially protective against reactivated tuberculosis . Here we report that the Rpf protein itself and four out of five of its mycobacterial homologues, which were administered as subunit vaccines to C57BL/6 mice, are highly immunogenic . Rpf-like proteins elicit immunoglobulin G1 (IgG1) and IgG2a responses and T-cell proliferation and stimulate production of gamma interferon, interleukin-10 (IL-10), and IL-12 but not IL-4 or IL-5 . Both humoral and T-cell responses against these antigens show a high degree of cross-reactivity . Vaccination of mice with Rpf-like proteins results in a significant level of protection against a subsequent high-dose challenge with virulent M . tuberculosis H37Rv, both in terms of survival times and mycobacterial multiplication in lungs and spleens.

J Basic Microbiol, 2003, 43(4), 337 - 40
Studies on the genomic heterogeneity of Micrococcus luteus strains by macro-restriction analysis using pulsed-field gel electrophoresis; Murayama O et al.; Macro-restriction analysis by means of double digestion using DraI and VspI demonstrated that they cleaved the genomic DNAs from Micrococcus luteus JCM1464(T), JCM3347, and JCM3348 into four to five fragments in a distinguishable manner by pulsed-field gel electrophoresis (PFGE) . Separate digestion with DraI and VspI cleaved the genomic DNA from M . luteus ATCC9341 into a relatively limited number of restriction fragments (six pieces) . SspI and XbaI cleaved the genomic DNA from each of the four strains into restriction fragments in a distinctly different and distinguishable manner.Thus, PFGE profiles after digestion with these four restriction enzymes that recognize six specific base sequences demonstrated the heterogeneity at the whole genomic DNA level among the four strains of M . luteus.The size of the genomic DNA of M . luteus ATCC9341 was estimated to be approximately 2.3 Mb by the summing the lengths of the restriction fragments generated by each three restriction enzymes (DraI, SspI, and VspI) and averaging the results.

Nucleic Acids Res, 2003 Jul 15, 31(14), 4085 - 90
Correlation between premeiotic DNA replication and chromatin transition at yeast recombination initiation sites; Murakami H et al.; The DNA double-strand breaks (DSBs) that initiate meiotic recombination in Saccharomyces cerevisiae are preceded first by DNA replication and then by a chromatin transition at DSB sites . This chromatin transition, detected as a quantitative increase in micrococcal nuclease (MNase) sensitivity, occurs specifically at DSB sites and not at other MNase-sensitive sites . Replication and DSB formation are directly linked: breaks do not form if replication is blocked, and delaying replication of a region also delays DSB formation in that region . We report here experiments that examine the relationship between replication, the DSB-specific chromatin transition and DSB formation . Deleting replication origins (and thus delaying replication) on the left arm of one of the two parental chromosomes III affects DSBs specifically on that replication-delayed arm and not those on the normally replicating arm . Thus, replication timing determines DSB timing in cis . Delaying replication on the left arm of chromosome III also delays the chromatin transition at DSB sites on that arm but not on the normally replicating right arm . Since the chromatin transition precedes DSB formation and requires the function of many genes necessary for DSB formation, these results suggest that initial events for DSB formation in chromatin are coupled with premeiotic DNA replication.

J Biol Chem, 2003 Sep 12, 278(37), 35145 - 51 Epub 2003 Jul 01.
La protein is associated with terminal oligopyrimidine mRNAs in actively translating polysomes; Cardinali B et al.; La is an abundant, mostly nuclear, RNA-binding protein that interacts with regions rich in pyrimidines . In the nucleus it has a role in the metabolism of several small RNAs . A number of studies, however, indicate that La protein is also implicated in cytoplasmic functions such as translation . The association of La in vivo with endogenous mRNAs engaged with polysomes would support this role, but this point has never been addressed yet . Terminal oligopyrimidine (TOP) mRNAs, which code for ribosomal proteins and other components of the translational apparatus, bear a TOP stretch at the 5' end, which is necessary for the regulation of their translation . La protein can bind the TOP sequence in vitro and activates TOP mRNA translation in vivo . Here we have quantified La protein in the cytoplasm of Xenopus oocytes and embryo cells and have shown in embryo cells that it is associated with actively translating polysomes . Disruption of polysomes by EDTA treatment displaces La in messenger ribonucleoprotein complexes sedimenting at 40-60 S . The results of polysome treatment with either low concentrations of micrococcal nuclease or with high concentrations of salt indicate, respectively, that La association with polysomes is mediated by mRNA and that it is not an integral component of ribosomes . Moreover, the analysis of messenger ribonucleoprotein complexes dissociated from translating polysomes shows that La protein associates with TOP mRNAs in vivo when they are translated, in line with a positive role of La in the translation of this class of mRNAs previously observed in cultured cells.

Cell Tissue Res, 2003 Jul, 313(1), 117 - 27 Epub 2003 Jun 28.
Hemocyte-mediated phagocytosis and melanization in the mosquito Armigeres subalbatus following immune challenge by bacteria; Hillyer JF et al.; Mosquitoes are important vectors of disease . These insects respond to invading organisms with strong cellular and humoral immune responses that share many similarities with vertebrate immune systems . The strength and specificity of these responses are directly correlated to a mosquito's ability to transmit disease . In the current study, we characterized the hemocytes (blood cells) of Armigeres subalbatus by morphology (ultrastructure), lectin binding, enzyme activity, immunocytochemistry, and function . We found four hemocyte types: granulocytes, oenocytoids, adipohemocytes, and thrombocytoids . Granulocytes contained acid phosphatase activity and bound the exogenous lectins Helix pomatia agglutinin, Galanthus nivalis lectin, and wheat germ agglutinin . Following bacteria inoculation, granulocytes mounted a strong phagocytic response as early as 5 min postexposure . Bacteria also elicited a hemocyte-mediated melanization response . Phenoloxidase, the rate-limiting enzyme in the melanization pathway, was present exclusively in oenocytoids and in many of the melanotic capsules enveloping bacteria . The immune responses mounted against different bacteria were not identical; gram(-) Escherichia coli were predominantly phagocytosed and gram(+) Micrococcus luteus were melanized . These studies implicate hemocytes as the primary line of defense against bacteria.

Biopolymers, 2003, 72(4), 207 - 16
Fourier transform IR spectroscopic appraisal of radiation damage in Micrococcus luteus; Perromat A et al.; Fourier transform IR spectroscopy (FTIR) is used to analyze cells of Micrococcus luteus, the type species of the highly heterogeneous genus Micrococcus that belongs to the Micrococcaceae family . The cells of M . luteus, which is a Gram-positive and yellow-pigmented bacterium, are submitted to increasing doses of gamma radiation . Irradiation leads to the generation of reactive oxygen species that induce biochemical changes as shown in spectral profiles . Beyond a dose of 0.70 kGy, significant differences between samples are observed, particularly in the 1485-900 cm(-1) region, which contains information about membrane lipids, cell wall polysaccharides, and nucleic acids . After a dose of 16.50 kGy, M . luteus is reincubated for times ranging from 1 to 24 h . Postirradiation reincubated bacteria are found far from the control and irradiated cells (mainly in the 985-900 cm(-1) range), suggesting that a biomolecular rearrangement occurs as soon as reincubation begins in the growth medium . Thus, FTIR spectroscopy appears to be a very useful technique for the rapid visualization of the alterations induced by both the radiation and mutagenic response during reincubation . The use of mathematical methods gives good insight into the biomolecular compounds involved in these two mechanisms . In view of these preliminary results, we hypothesize that it can be successfully applied to any type of tissue and that it may be a future interesting tool for evaluating the effects of radiation in humans .

Int J Biochem Cell Biol, 2003 Nov, 35(11), 1588 - 600
Purification of N-terminally truncated histone H2A-monoubiquitin conjugates from leukemic cell nuclei: probable proteolytic products of ubiquitinated H2A; Okawa Y et al.; To gain insight into the significance of nuclear ubiquitinated proteins, two serial extracts prepared from various leukemic cells were analysed by western blotting with anti-ubiquitin antibody . Two previously unidentified ubiquitinated proteins with molecular masses of 10 and 17 kDa were found in 8 M urea-soluble extracts, obtained from Tris-buffer-insoluble materials, of acute myeloid leukemia OCI/AML 1a cells and the cells from the leukemia patients . Both proteins were successfully purified from the OCI/AML 1a cells and identified as monoubiquitin-truncated H2A conjugates, the 10 kDa ubiquitinated H2A(115-129) and the 17 kDa ubiquitinated H2A(54-129), suggesting that both proteins were produced by limited proteolysis of an intact form (23 kDa) of ubiquitinated H2A(1-129) . The 17 kDa protein as well as the 23 kDa ubiquitinated histone H2A were localised in chromatin fractions of the OCI/AML cells and released by high concentrations of salt in a micrococcal nuclease-sensitive manner, suggesting their association with chromatin . In contrast, the 10 kDa protein remained insoluble even when the nuclei were treated with nuclease under high salt concentrations, presumably due to binding to the nuclear matrix . An antibody recognising H2A(70-81) also detected the 17 kDa protein in anti-ubiquitin immunoprecipitates obtained from the OCI/AML cell nuclei . In addition, the 17 kDa protein levels in THP-1 cells were transiently increased, concomitant with a decrease in the 23 kDa ubiquitinated H2A, by treatment with phorbol 12-myristate 13-acetate or all-trans-retinoic acid, both of which induce differentiation . This is the first report of probable proteolytic products of ubiquitinated H2A, which might have a role in nuclear functions.

Eye Contact Lens, 2003 Jan, 29(1 Suppl), S75 - 9; discussion S83-4, S192-4
Lysozyme and lipid deposition on silicone hydrogel contact lens materials; Jones L et al.; PURPOSE: We sought to determine whether there were differences in lysozyme (quantity and conformation) and lipid deposition on in vivo worn conventional (etafilcon) and silicone hydrogel (balafilcon and lotrafilcon) contact lenses . METHODS: After extraction, lysozyme concentration in each extract was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting . Lysozyme activity was determined by the rate of lysis of Micrococcis lysodeikticus cells . Lipid deposition was determined by high-performance liquid chromatography . RESULTS: Lysozyme deposition on etafilcon lenses was significantly greater than that measured on silicone hydrogel (SH) lenses (985 microg per lens versus 10 and 3 microg per lens for balafilcon and lotrafilcon materials, respectively; P<0.001) . The degree to which lysozyme was denatured was influenced by the lens material, with the lowest degree of denaturation (22%) seen on the conventional lens material, as compared with 50% for balafilcon and 80% for lotrafilcon (P<0.001) . Lipid deposition was greatest on the SH materials, with up to 600 microg per lens of certain lipid classes being deposited on balafilcon, as compared with 20 microg per lens on etafilcon (P<0.001) . CONCLUSION: The quantity and conformation of lysozyme and the quantity of lipid deposited on hydrogel contact lenses is significantly influenced by the composition of the lens material . SH contact lens materials deposit low levels of lysozyme and high levels of lipid deposition compared with ionic contact lens materials . Although SH materials deposit only small amounts of lysozyme, the degree of lysozyme denaturation that occurs is higher relative to that seen on ionic lens materials.

Biochem Biophys Res Commun, 2003 May 16, 304(4), 818 - 24
Lysozyme conjugate immune complex formation and the effects on substrate hydrolysis; Smales CM et al.; The defined estrone glucuronide-lysozyme conjugate E3, that is acylated solely at K33, was used as a probe for the steric requirements of the active site cleft of chicken type lysozymes . When the immune complex was formed with an anti-estrone glucuronide antiserum, the rate of lysis of the E3 conjugate with the large bacterial substrate Micrococcus lysodeikticus was inhibited by over 90% . However, when the small hexamer of N-acetyl glucosamine was used as the substrate, the rate of hydrolysis by the immune complex was accelerated by 350% compared with the control rate . Thus, inhibition by the anti-estrone glucuronide cannot be caused simply by steric occlusion of the active site . Other factor(s) in the immune complex activate the hydrolysis reaction, most likely by favouring the conformations that lead to the transition state.

Biopolymers, 2003 May, 69(1), 42 - 59
The solution structures and activity of caerin 1.1 and caerin 1.4 in aqueous trifluoroethanol and dodecylphosphocholine micelles; Wegener KL et al.; The caerin 1 peptides are among the most powerful of the broad-spectrum antibiotic amphibian peptides . Caerin 1.1 has previously been shown to form an amphipathic helix-bend-helix structure in aqueous trifluoroethanol (H . Wong, J . H . Bowie, and J . A . Carver European Journal Biochemistry, 1997, Vol . 247, pp . 545-557) and structure-activity relationship studies indicate that both helices are required for activity, as well as flexibility in the bend region connecting the two . The structure of caerin 1.1 in dodecylphosphocholine micelles was investigated and shown to be very similar to that determined in aqueous trifluoroethanol . Caerin 1.4, which is identical to caerin 1.1, but with serine residues replacing Val5 and Gly7, is less active than caerin 1.1 against most bacterial species but has improved activity against Escherichia coli and Micrococcus luteus . The solution NMR structure of caerin 1.4 was determined in both aqueous trifluoroethanol and dodecylphosphocholine micelles, and was shown to be similar to caerin 1.1 . It was concluded that differences in the hydrophobicity and hydrophilic angle of the first helix are probably responsible for the different spectra of antibacterial activity . The similarity of the structures calculated in aqueous trifluoroethanol and dodecylphosphocholine micelles suggests that, for caerin 1.1 and 1.4, these solvent systems are equally as good at representing a membrane environment .

Biochemistry, 2003 Apr 15, 42(14), 4035 - 41
Mutational analysis of allylic substrate binding site of Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase; Fujikura K et al.; Undecaprenyl diphosphate (UPP) synthase catalyzes the sequential cis-condensation of isopentenyl diphosphate (IPP) onto (E,E)-farnesyl diphosphate (FPP) . In our previous reports on the Micrococcus luteus B-P 26 UPP synthase, we have shown that the conserved residues in the disordered region from Ser-74 to Val-85 is crucial for the binding of FPP and the catalytic function {Fujikura, K., et al . (2000) J . Biochem . (Tokyo) 128, 917-922} and the existence of a structural P-loop motif for the FPP binding site {Fujihashi, M., et al . (2001) Proc . Natl . Acad . Sci . U.S.A., 98, 4337-4342} . To elucidate the allylic substrate binding site in more detail, we prepared eight mutant enzymes and examined their kinetic behavior . The mutant with respect to the two complementarily conserved Arg residues among the structural P-loop motif, G32R-R42G, retained the activity and showed product distribution pattern exactly similar to that of the wild-type, indicating that the complementarily conserved Arg is important for maintaining the catalytic function . Substitutions of Asp-29, Arg-33, or Arg-80 with Ala resulted in a large loss of enzyme activity, suggesting that these residues are essential for catalytic function . However, the K(m) values of these mutant enzymes for Z-GGPP, which is the first intermediate during the enzymatic cis-condensations of IPP onto FPP, were only moderately different or little changed from those of the wild type . These results suggest that the binding site for the intermediate Z-GGPP having a cis double bond is different to that for the intrinsic allylic substrate, FPP, whose diphosphate moiety is recognized by the structural P-loop.

J Biol Chem, 2003 Jul 4, 278(27), 24388 - 98 Epub 2003 Apr 16.
Architecture of the flaviviral replication complex . Protease, nuclease, and detergents reveal encasement within double-layered membrane compartments; Uchil PD et al.; Flavivirus infection causes extensive proliferation and reorganization of host cell membranes to form specialized structures called convoluted membranes/paracrystalline arrays and vesicle packets (VP), the latter of which is believed to harbor flaviviral replication complexes . Using detergents and trypsin and micrococcal nuclease, we provide for the first time biochemical evidence for a double membrane compartment that encloses the replicative form (RF) RNA of the three pathogenic flaviviruses West Nile, Japanese encephalitis, and dengue viruses . The bounding membrane enclosing the VP was readily solubilized with nonionic detergents, rendering the catalytic amounts of enzymatically active protein component(s) of the replicase machinery partially sensitive to trypsin but allowing limited access for nucleases only to the vRNA and single-stranded tails of the replicative intermediate RNA . The RF co-sedimented at high speed from nonionic detergent extracts of virus-induced heavy membrane fractions along with the released individual inner membrane vesicles whose size of 75-100 nm as well as association with viral NS3 was revealed by immunoelectron microscopy . Viral RF remained nuclease-resistant even after ionic detergents solubilized the more refractory inner VP membrane . All of the viral RNA species became nuclease-sensitive following membrane disruption only upon prior trypsin treatment, suggesting that proteins coat the viral genomic RNA as well as RF within these membranous sites of flaviviral replication . These results collectively demonstrated that the newly formed viral genomic RNA associated with the VP are oriented outwards, while the RF is located inside the nonionic detergent-resistant vesicles.

EMBO J, 2003 Apr 15, 22(8), 1889 - 97
Pseudouridylation (Psi) of U2 snRNA in S . cerevisiae is catalyzed by an RNA-independent mechanism; Ma X et al.; Pseudouridylation of snRNAs in vertebrates is guided by small nucleolar/Cajal body-specific RNAs (sno/scaRNAs) . We developed an in vitro system using cell extracts and single site-radiolabeled U2 snRNAs to study pseudouridylation in Saccharomyces cerevisiae . Micrococcal nuclease-treated cell extracts are fully competent to catalyze U2 pseudouridylation, suggesting an RNA-independent process . A pseudouridylase activity for Psi(35) within yeast U2 is identified via a screen of an S.cerevisiae GST-ORF protein library . This activity is associated with YOR243c ORF, which has not previously been assigned function . When the GST-YOR243c protein is expressed in Escherichia coli, pseudouridylation activity is comparable to that expressed in S.cerevisiae, demonstrating that this protein (designated Pus7) alone can catalyze Psi(35) formation in U2 . Both in vitro and in vivo analyses using wild-type and pus7-Delta strains show that Pus7 is indispensable for Psi(35) formation in U2 . Using site-specific radiolabeled U2 and U2 fragments, we show that Pus7 activity is specific for Psi(35) and that the U2 stem- loop II region is essential for the pseudouridylation reaction . A BLAST search revealed Pus7 homologs in various organisms.

Plant Mol Biol, 2003 Mar, 51(5), 665 - 75
Matrix attachment regions enhance transcription of a downstream transgene and the accessibility of its promoter region to micrococcal nuclease; Fukuda Y et al.; Nuclear matrix attachment regions (MARs) are thought to influence the expression of flanking genes . In this study, we investigated the activation of genes by tobacco MARs that had previously been identified in the 5' region of the basic class I chitinase gene, CHNS0 . In transgenic tobacco cells, a construct consisted of the 35S promoter of cauliflower mosaic virus (CaMV) fused to a beta-glucuronidase gene (uidA) with 5' MAR elements was expressed at a 10-fold higher level than a similar construct without MAR sequences . However, expression of a similar construct with 3' MARs and of a construct with a truncated (-46) 35S minimal promoter and uidA with 5' MARs was not similarly enhanced, suggesting that MARs might act by increasing the activity of downstream enhancers . Deletion analysis of the MAR sequences revealed that the function of the MARs that increased the expression of the transgene was redundant . Moreover, assays of the transient expression of transgenes suggested that MAR elements might be involved in the structure and organization of chromatin . To examine the influence of MARs on chromatin structure, we investigated the effects of micrococcal nuclease (MNase) on the DNA in the reporter gene around the MARs . Analysis of the time-course of digestion of nuclei with MNase revealed that the 35S promoter region with 5' MARs was much more sensitive to MNase than the same region without MARs, suggesting that MARs might mediate the opening of chromatin in the region of a downstream promoter, with consequent enhancement of transcription.

Life Sci Space Res, 1976, 14, 359 - 62
On micro-organisms of the stratosphere; Imshenetsky AA et al.; The lower parts of the biosphere are well studied since various live beings are found in oceans and at the bottom of large hollows . Contrary to this, we have no data about the upper boundaries of the biosphere . Samples were obtained with the help of specially constructed analysers which were installed in meteorological rockets and reached an altitude of 100 km . With the help of methods completely excluding the possibility of contamination of analysers with outside microflora it became possible to prove that earth microbes carried by air currents are present in the stratosphere . At an altitude of 48-77 km Circinella muscae, Asp . niger, Penicillium notatum were found as well as mycobacterium and micrococcus . The correlation of these cultures with external factors is studied and the weight of one conidium or one cell in isolated micro-organisms is estimated . These investigations will continue.

J Invertebr Pathol, 2003 Mar, 82(3), 162 - 6
Detection of lysozyme-like enzymatic activity secreted by an immune-responsive mosquito cell line; Nasr NM et al.; Using molecular approaches, we have recently shown that the C7-10 mosquito cell line from Aedes albopictus, and the Aag-2 line from Aedes aegypti, secrete a variety of immune peptides into the culture medium, including cecropins, defensins, transferrin, and lysozyme . The diversity of these peptides makes it difficult to quantify the relative activities of each molecule, because possible synergistic interactions may occur . Using a microtiter plate assay with live bacteria, we now show that C7-10 cells secrete an activity that is more potent against the Gram-positive bacterium, Micrococcus luteus, than against Gram-negative Escherichia coli . This lysozyme-like activity is accompanied by production of a lytic zone in an agarose plate assay containing commercially available, lyophilized M . luteus . Properties of the lysozyme-like activity from C7-10 cells included a broad pH optimum from 5.5 to 6.5, and heat-sensitivity above 42 degrees C . Amounts of secreted activity increased during the initial 24h of incubation with heat-killed bacteria . During this induction, lysozyme-like activity was found primarily in the cell culture supernatant.

J Parasitol, 2003 Feb, 89(1), 62 - 9
Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti; Hillyer JF et al.; Mosquitoes are vectors of many deadly and debilitating pathogens . In the current study, we used light and electron microscopies to study the immune response of Aedes aegypti hemocytes to bacterial inoculations, Plasmodium gallinaceum natural infections, and latex bead injections . After challenge, mosquitoes mounted strong phagocytic and melanization responses . Granulocytes phagocytosed bacteria singly or pooled them inside large membrane-delimited vesicles . Phagocytosis of bacteria, Plasmodium sporozoites, and latex beads was extensive; we estimated that individual granulocytes have the capacity to phagocytose hundreds of bacteria and thousands of latex particles . Oenocytoids were also seen to internalize bacteria and latex particles, although infrequently and with low capacity . Besides phagocytosis, mosquitoes cleared bacteria and sporozoites by melanization . Interestingly, the immune response toward 2 species of bacteria was different; most Escherichia coli were phagocytosed, but most Micrococcus luteus were melanized . Similar to E . coli, most Plasmodium sporozoites were phagocytosed . The immune response was rapid; phagocytosis and melanization of bacteria began as early as 5 min after inoculation . The magnitude and speed of the cellular response suggest that hemocytes, acting in concert with the humoral immune response, are the main force driving the battle against foreign invaders.

Chemistry, 2003 Apr 4, 9(7), 1556 - 65
Getting closer to the real bacterial cell wall target: biomolecular interactions of water-soluble lipid II with glycopeptide antibiotics; Vollmerhaus PJ et al.; A novel synthesized water-soluble variant of lipid II (LII) was used to evaluate the noncovalent interactions between a number of glycopeptide antibiotics and their receptor by bioaffinity electrospray ionization mass spectrometry (ESI-MS) . The water-soluble variant of lipid II is an improved design, compared to the traditionally used tripeptide N,N'-diacetyl-L-lysyl-D-alanyl-D-alanine (KAA), of the target molecule on the bacterial cell wall . A representative group of glycopeptide antibiotics was selected for this study to evaluate the validity of the novel cell-wall-mimicking target LII . Structure-function relationships of various glycopeptide antibiotics were investigated by means of 1) bioaffinity mass spectrometry to evaluate solution-phase molecular interactions with both LII and KAA, 2) fluorescence leakage experiments to study the interactions with the membrane-embedded lipid II, and 3) minimum inhibitory concentrations against the indicator strain Micrococcus flavus . Our results with the novel LII molecule reveal that some antibiotics interact differently with KAA and LII . Additionally, our data cast doubt on the hypothesis that antibiotic selfdimerization assists in the in-vivo efficacy . Finally, the water-soluble lipid II proved to be a better model of the bacterial cell wall.

Biochem Biophys Res Commun, 2003 Mar 28, 303(1), 8 - 13
Incorporation of DUF/FACT into chromatin enhances the accessibility of nucleosomal DNA; Seo H et al.; DNA unwinding factor (DUF) was discovered as an essential DNA replication factor in Xenopus egg extracts . DUF consists of an HMG protein and a homolog of Cdc68p/Spt16p, and has the capability of unwinding dsDNA . Here we have examined the interaction of DUF with chromatin . DUF was incorporated into chromatin assembled from sperm heads and from plasmid DNA in egg extracts . It was revealed that the chromatin assembled in egg extracts immunodepleted of DUF is less sensitive to micrococcal nuclease (NNase) digestion than that assembled in control extracts, indicating that chromatin containing DUF has more decompact structure than that without DUF . Also we found that DUF has a high affinity for core histones in vitro . We suggest that the function of DUF may be to make the chromatin structure accessible to replication factors.

J Environ Biol, 2002 Jul, 23(3), 289 - 94
Enzymological aspects of bioconversion of m-dinitrobenzene; Dey S; Three main enzymes, responsible for bioconversion of 1,3 dinitrobenzene (m-DNB) by Micrococcus colpogenes MCM B 410, were isolated from the sonicated cell mass, grown in presence of m-DNB in a synthetic medium, for 7 days . The soluble proteins were separated by differential precipitation and also separated by native PAGE . Each fraction obtained from both the protocols, was tested for nitro aryl reductase, aryl mono oxygenase and resorcinol 2,3 dioxygenase . The apparent molecular weight of the proteins were nitro aryl reductase (30 kDa), aryl mono oxygenase (110 kDa) and resorcinol 2,3 di oxygenase (65 kDa).

Life Sci Space Res, 1977, 15, 37 - 9
Resistance of stratospheric and mesospheric micro-organisms to extreme factors; Imshenetsky AA et al.; Studies of the stratosphere and mesosphere, by means of special analysers installed on meteorological rockets, have thrown more light on our knowledge of the upper boundary of the biosphere . The presence of the following micro-organisms was registered at heights of 49-77 km: Aspergillus niger, Penicillium notatum, Circinella muscae, Papulaspora anomala, Mycobacterium luteum and Micrococcus albus . The isolated micro-organisms were subjected to the action of gamma-irradiation, high vacuum and UV radiation in order to evaluate the quality of sterilization by gamma-rays (3.2-3.5 Mrad) prior to sampling and the resistance of these micro-organisms to physical factors of the stratosphere and mesosphere . No species with high radio-resistance were detected among the isolated cultures . The D10 index for fungal spores and bacterial vegetative cells, freeze-dried or suspended in a physiological solution, did not exceed 290 krad . These data confirm that sterilization of the analyser with gamma-rays assured the purity of biological experiments during sampling . The isolated micro-organisms were found to be very resistant to high vacuum (10(-9) mmHg) and UV radiation, with the exception of the pigmentless Micrococcus albus . This evidence shows that pigmented micro-organisms can survive in the earth's atmosphere at high altitudes.

Int Immunopharmacol, 2003 Feb, 3(2), 159 - 67
A novel prodigiosin-like immunosuppressant from an alkalophilic Micrococcus sp; Pandey R et al.; A novel red pigment, 2,2'-{3-methoxy-1'amyl-5'-methyl-4-(1-pyrryl)} dipyrryl-methene (MAMPDM), which has properties similar to those of prodigiosins, has been isolated for the first time from a bacterium putatively identified as Micrococcus sp . Our studies showed that MAMPDM inhibited proliferation of both human T as well as B cells and murine T cells, in response to polyclonal mitogens, in a concentration-dependent manner while murine B cell proliferation induced by lipopolysaccharide was inhibited only at high concentration . The effect of MAMPDM on constitutive cell cycling was ascertained using four mouse and human tumour cell lines . At 100 nM, the concentration that inhibited con A induced proliferation of mouse spleen cells, the viability of these cell lines was not affected . At 10-100-fold higher concentration of MAMPDM, however, there was a decrease in cell viability with T cell-derived cell lines being more sensitive . MAMPDM did not block the secretion of IL-2 or expression of CD25 though it inhibited the proliferation of con A stimulated T cells . The higher amount of IL-2 in the supernatant of the con A stimulated T cells, cultured in the presence of the immunomodulator, indicated accumulation of IL-2 due to its reduced utilisation . At inhibitory concentration, MAMPDM induced apoptosis in con A stimulated cells . Thus, MAMPDM may have considerable and selective T cell immunosuppressive potential and appears to act by a mechanism distinct from that of other known immunosuppressors .

Water Sci Technol, 2003, 47(1), 251 - 6
Bacterial biosorbent for removing and recovering copper from electroplating effluents; Lo W et al.; Investigations were carried out to study the removal and recovery of Cu(II) ions from wastewater by Micrococcus sp . The Langmuir isotherm model described very well the equilibrium behavior of copper biosorption, with maximum biosorption capacity (q(max)) reaching 52.1 mg Cu2+/g dry cell at pH 6 . Biomass prewashed with sulfuric acid (0.05 mol 1(-1)) and sodium sulfate (1 mol l(-1)) solutions were shown to increase the copper removal capabilities up to 27% and 16%, respectively . Copper uptake by cells was negligible at pH 2.0 and then increased quickly with increasing pH until 6.0 . Cells of Micrococcus sp . were immobilized in 2% calcium alginate and 10% polyacrylamide gel beads . A counter-current process comprising a series of immobilized cell reactors was developed for removing and recovering copper from electroplating effluents . This process was capable of producing an effluent at low copper concentration, with only a minimum amount of desorbing agent used . The technique of scanning electron microscopy coupled with X-ray dispersion analysis shows that Cu2+ exchanged with K+ and Ca2+ on the cell wall of Micrococcus sp., thereby suggesting ion exchange as one of the dominant mechanisms of metal biosorption for this bacterial strain.

Blood, 2003 Jun 15, 101(12), 4894 - 902 Epub 2003 Feb 06.
Human IL-12(p35) gene activation involves selective remodeling of a single nucleosome within a region of the promoter containing critical Sp1-binding sites; Goriely S et al.; To get insight into the regulation of human interleukin-12 (IL-12) synthesis, we determined the chromatin organization of the IL-12(p35) promoter region . First, we determined positioning of nucleosomes within the IL-12(p35) promoter using the indirect end-labeling technique in the THP-1 monocytic cell line . On stimulation with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), hypersensitivity to digestion with DNase I, micrococcal nuclease, and specific restriction enzymes was detected in the region encompassing nucleotide (nt) -310 to -160, indicating selective inducible chromatin remodeling involving disruption of a single nucleosome (named nuc-2) . Using p35 promoter deletion mutants and reporter gene assays, we demonstrated that the -396/-241 region contained critical cis-acting elements . Within this latter region, we characterized physically and functionally 2 Sp1-binding sites, which were acting as key regulatory elements for both basal and LPS/IFN-gamma-inducible p35 gene expression: Sp1#1 lies within the remodeled nuc-2 region and Sp1#2 is located in the nucleosome-free region immediately upstream of nuc-2 . Finally, we extended the chromatin structure analysis to dendritic cells (DCs) derived from human monocytes and observed the same nucleosomal organization and remodeling as in the THP-1 cell line . Moreover, we found that in DCs, LPS and IFN-gamma synergized in the induction of nucleosomal remodeling and that chromatin remodeling at the p35 locus immediately preceded IL-12(p35) mRNA synthesis . Taken together, our results demonstrate that IL-12(p35) gene activation in the course of DC maturation involves selective and rapid remodeling of a single positioned nucleosome within a region of the promoter containing critical Sp1-binding sites.

Cancer Res, 2003 Feb 1, 63(3), 689 - 95
A chromosome 3-encoded repressor of the human telomerase reverse transcriptase (hTERT) gene controls the state of hTERT chromatin; Szutorisz H et al.; Telomerase is crucial for human carcinogenesis . The limiting component of telomerase activity is telomerase reverse transcriptase (hTERT), undetectable in differentiated somatic cells but present in most tumor cells . There is evidence that hTERT transcription is shut down by a repressor in normal cells, but the mechanisms that turn on or maintain expression in tumor cells are not understood . To identify cis-acting regulatory elements, we scanned the hTERT gene for nuclease sensitive sites . In tumor cells and in in vitro transformed fibroblasts that contain hTERT mRNA, we detected a pattern of nuclease-sensitive sites in the second intron different from that in normal fibroblasts . To test whether the chromatin state characterized by the increased nuclease sensitivity plays a role in tumor-specific hTERT expression, we used a telomerase-positive breast carcinoma line, 21NT . Introduction of a normal chromosome 3 into these cells is known to down-regulate hTERT expression, probably through transcriptional silencing . 21NT cells displayed a similar pattern of micrococcal nuclease (MNase) sensitivity to other telomerase-positive lines, whereas the hTERT chromatin of the chromosome 3-hybrids resembled that of normal fibroblasts . In segregants that had lost the normal chromosome 3, the MNase sensitivity pattern characteristic of telomerase-positive cells was restored, and some (but not all) re-expressed the hTERT gene . The simplest model compatible with these results, and with data on the mapping of an hTERT repressor on chromosome 3, is that hTERT expression in tumor cells depends on an open state of intron 2-chromatin . We propose that, during the development of the breast carcinoma from which the 21NT cell line was derived, loss of function of this repressor led to chromatin remodeling necessary (but probably not sufficient) for expression of the hTERT gene . An improved understanding of the precise mechanism of hTERT dysregulation in human cancer may well find applications in the development of antitelomerase cancer therapy.

Biochemistry, 2003 Feb 11, 42(5), 1209 - 16
Low-temperature-induced structural changes in human lysozyme elucidated by three-dimensional NMR spectroscopy; Kumeta H et al.; The three-dimensional solution structures of human lysozyme were determined at 35 and 4 degrees C using the heteronuclear multidimensional NMR spectroscopy, which were compared with each other to clarify the structural response of this enzyme to lowering of the temperature . Together with the data of the temperature dependence experiments of the lytic activity against Micrococcus luteus, we consider the implication of the observed structural change for the low-temperature-induced reduction of the activity of human lysozyme . The structures of human lysozyme determined at the two temperatures are found to be similar, both of which comprise four alpha-helices (A- to D-helices) and three antiparallel beta-strands (beta(1)-beta(3)), leading to the constructions of the alpha- and beta-domains as previously identified in the X-ray crystal structure . A significant structural change was observed for the "active site lobe" comprising the loop region connecting C- and D-helices and the following D-helix, which moves toward the active site cleft located between the alpha- and beta-domains so as to obstruct the cleft according to the temperature lowering . It further appeared that the total volume as well as the accessible surface area of human lysozyme decreases with lowering of the temperature, suggesting that the internal cavity of this enzyme shrinks under low temperature environment . Because in human lysozyme the region comprising the active site lobe is responsible for turnover of the enzymatic reaction against the substrate, the low-temperature-induced structural change of the active site lobe presumably controls the efficiency of the lytic activity under low temperatures.

Biochem J, 2003 May 1, 371(Pt 3), 697 - 708
Cleavage of fragments containing DNA mismatches by enzymic and chemical probes; Brown J et al.; We prepared synthetic 50-mer DNA duplexes, each containing four mismatched base-pairs in similar positions . We examined their cleavage by DNases I and II, micrococcal nuclease (MNase), methidiumpropyl-EDTA-Fe(II) {MPE-Fe(II)} and hydroxyl radicals . We find that single mismatches only produce subtle changes in the DNase I-cleavage pattern, the most common of which is attenuated cleavage at locations 2-3 bases on the 3'-side of the mismatch . Subtle changes are also observed in most of the DNase II-cleavage patterns, although GT and GG inhibit the cleavage over longer regions and generate patterns that resemble footprints . MNase cleaves the heteroduplexes at the mismatches themselves (except for CC), and in some cases cleaves CpG and CpC steps . None of the mismatches causes any change in the cleavage patterns produced by hydroxyl radicals or MPE-Fe(II) . We also examined the cleavage patterns of fragments containing tandem GA mismatches in the sequences RGAY/RGAY and YGAR/YGAR (R, purine; Y, pyrimidine) . RGAY causes only subtle changes in the cleavage patterns, which are similar to those seen with single mismatches, except that there are no changes in MNase cleavage . However, YGAR inhibits DNases I and II cleavage over 4-6 bases, and attenuates MPE-Fe(II) and hydroxyl radical cleavage at 2 bases . These changes suggest that this mismatch has a more pronounced effect on the local DNA structure . These changes are discussed in terms of the structural and dynamic effects of each mismatch.

Mol Cell Biol, 2003 Feb, 23(4), 1460 - 9
Maintenance of open chromatin and selective genomic occupancy at the cell cycle-regulated histone H4 promoter during differentiation of HL-60 promyelocytic leukemia cells; Hovhannisyan H et al.; During the shutdown of proliferation and onset of differentiation of HL-60 promyelocytic leukemia cells, expression of the cell cycle-dependent histone genes is downregulated at the level of transcription . To address the mechanism by which this regulation occurs, we examined the chromatin structure of the histone H4/n (FO108, H4FN) gene locus . Micrococcal nuclease, DNase I, and restriction enzymes show similar cleavage sites and levels of sensitivity at the H4/n locus in both proliferating and differentiated HL-60 cells . In contrast, differentiation-related activation of the cyclin-dependent kinase inhibitor p21(cip1/WAF1) gene is accompanied by increased nuclease hypersensitivity . Chromatin immunoprecipitation assays of the H4/n gene reveal that acetylated histones H3 and H4 are maintained at the same levels in proliferating and postproliferative cells . Thus, the chromatin of the H4/n locus remains in an open state even after transcription ceases . Using ligation-mediated PCR to visualize genomic DNase I footprints at single-nucleotide resolution, we find that protein occupancy at the site II cell cycle element is selectively diminished in differentiated cells while the site I element remains occupied . Decreased occupancy of site II is reflected by loss of the site II binding protein HiNF-P . We conclude that H4 gene transcription during differentiation is downregulated by modulating protein interaction at the site II cell cycle element and that retention of an open chromatin conformation may be associated with site I occupancy.

Wei Sheng Wu Xue Bao, 1999 Feb, 39(1), 55 - 9
{Purification and properties of human lysozyme in engineered bacterium E . coli}; Ye J et al.; To obtain the SDS-PAGE-pure human lysozyme, the crude enzyme of engineered bacterium E . coli was purified by chromatography on cation ion exchange of Express-Ion S . The optimum reaction temperature and pH of this lysozyme were 45 degrees C and 6.5, respectively . The isoelectric point is pH 8.91, and Km of the enzyme for Micrococcus lysodeikticus is 0.0311 mg/mL . The thermal stability of the engineered enzyme is more sensible than hen egg white lysozyme and human milk lysozyme . The sequence of 5 amino acids in N-end is same as designed, except an Met at the first . The affects of some metal ion on this enzyme were shown . Cu2+ of 0.01 mmol/L could completely inactivate the enzyme.

Zhongguo Zhong Yao Za Zhi, 2001 Jan, 26(1), 50 - 3
{Effects of musk glucoprotein on the function of rat polymorphonuclear leukocytes activated by IL-8 in vitro}; Wang WJ et al.; OBJECTIVE: To investigate the effects of musk-1, a glucoprotein component isolated from the water extract of musk, on some functions of rat polymorphonuclear leukocytes activated by IL-8 in vitro . METHOD: An in vitro incubation system was used . Superoxide anion production was determined by cytochrome C reduction . beta-glucuronidase and lysozyme release was quantitated by enzyme reactions in which phenolph-thaleinglucuronic acid and Micrococcus Lysodeikticus were as the substrates, respectively . RESULTS: In comparison with control, musk-1 at concentration 1-100 micrograms.ml-1 can increase superoxide anion production by 91.7%-291%, and decrease beta-glucuronidase and lysozyme release by 2.2%-58.1% and 3.9%-39.8%, respectively . CONCLUSION: Inhibition of lysosomel enzyme release might be considered as one of mechanisms of antiinflammatory action of musk.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Sep-Oct, (5), 11 - 5
{Detection of biologically active substances with antagonistic and stimulating activity in Spirulina platensis}; Blinkova LP et al.; The results of studies on the detection of biologically active substances (BAS) in biomass dilutions and culture fluid of Spirulina platensi and algae (Chlorella, Fucus, Laminaria) by the agar diffusion method are presented . After the sterilization of the solutions with chloroform (CF) a substance with lysozyme-like activity and 2 substances with antagonistic activity deep in agar and on its surface were detected with the use of the micrococcal indicator strain . After CF treatment, depending on the concentration of S . platensis strains, a compound stimulating the growth of bacteria and sensitive to heat treatment was detected . BAS were also detected with the use of other indicator cultures.

J Biol Chem, 2003 Mar 7, 278(10), 8163 - 71 Epub 2002 Dec 30.
Interaction of NF-E2 in the human beta-globin locus control region before chromatin remodeling; Onishi Y et al.; When transcription is initiated under repressive conditions, such as when chromatin are packed together, binding followed by the functioning of key components in the transcriptional apparatus should be appropriately facilitated in the chromatin architecture . We provide evidence that the erythroid-specific enhancer- binding protein NF-E2 interacts with the cognate motif at DNase I-hypersensitive site 2 of the human beta-globin locus control region in a repressive state . The nucleosome containing the NF-E2-binding site showed characteristic rotational and translational phases in vitro . The binding site had less affinity to the histone octamers than nearby regions while showing greater accessibility to DNase I and micrococcal nuclease . Furthermore, the motif was recognized by the exogenous NF-E2 protein expressed in HeLa cells, which have a repressive state of chromatin at the beta-globin locus, as shown by ligation-mediated PCR and chromatin immunoprecipitation assay . These lines of evidence indicate that NF-E2 interacts with the cognate motif on the nucleosome before chromatin is remodeled.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2095 - 100
Citricoccus muralis gen . nov., sp . nov., a novel actinobacterium isolated from a medieval wall painting; Altenburger P et al.; A Gram-positive, aerobic, spherical actinobacterium, designated strain 4-0(T), was isolated from a medieval wall painting and characterized to determine its taxonomic position . The peptidoglycan of strain 4-0(T) was of type A4alpha, with lysine as the diagnostic cell wall diamino acid and an interpeptide bridge of Lys-Gly-Glu . Its quinone system contained predominantly MK-9(H2) (64%) and its polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, four unknown glycolipids, two unknown phospholipids and an unknown lipid . The fatty acid profile of strain 4-0(T) was represented by significant amounts of ai-C15:0 and moderate amounts of ai-C17:0, i-C16:0 and i-C15:0 fatty acids . Spermidine was predominant in the polyamine pattern . The G+C content of the genomic DNA was 68 mol% . Comparative 16S rDNA sequence studies revealed the highest similarity values (95.4-96.1%) between strain 4-0(T) and species of the genus Micrococcus and certain species of the genus Arthrobacter, including Arthrobacter pascens DSM 20545(T), Arthrobacter ramosus DSM 20546(T), Arthrobacterprotophormiae DSM 20168(T), Arthrobacternicotianae DSM 20123(T) and Arthrobacter globiformis DSM 20124(T) . Phylogenetic analyses demonstrated that strain 4-0(T) branches deeply on the Micrococcus lineage . Because it is almost equidistant phylogenetically from the genera Micrococcus and Arthrobacter and possesses significant differences in chemotaxonomic characteristics from members of these genera, it is suggested that strain 4-0(T) be classified as a novel species in a new genus; the name Citricoccus muralis gen . nov., sp . nov . is proposed . The type strain is strain 4-0(T) (= DSM 11442(T) CCM 4981(T)).

J Gen Appl Microbiol, 1998 Aug, 44(4), 243 - 249
Exposure to low dose of gamma radiation enhances the excision repair in Saccharomyces cerevisiae; Dutta K; The effect of low doses of ionizing and nonionizing radiation on the radiation response of yeast Saccharomyces cerevisiae toward ionizing and nonionizing radiation was studied . The wild-type strain D273-10B on exposure to 54 Gy gamma radiation (resulting in about 10% cell killing) showed enhanced resistance to subsequent exposure to UV radiation . This induced UV resistance increased with the incubation time between the initial gamma radiation stress and the UV irradiation . Exposure to low doses of UV light on the other hand showed no change in gamma or UV radiation response of this strain . The strains carrying a mutation at rad52 behaved in a way similar to the wild type, but with slightly reduced induced response . In contrast to this, the rad3 mutants, defective in excision repair, showed no induced UV resistance . Removal of UV-induced pyrimidine dimers in wild-type yeast DNA after UV irradiation was examined by analyzing the sites recognized by UV endonuclease from Micrococcus luteus . The samples that were exposed to low doses of gamma radiation before UV irradiation were able to repair the pyrimidine dimers more efficiently than the samples in which low gamma irradiation was omitted . The nature of enhanced repair was studied by scoring the frequency of induced gene conversion and reverse mutation at trp and ilv loci respectively in strain D7, which showed similar enhanced UV resistance induced by low-dose gamma irradiation . The induced repair was found to be essentially error-free . These results suggest that irradiation of strain D273-10B with low doses of gamma radiation enhances its capability for excision repair of UV-induced pyrimidine dimers.

Biochim Biophys Acta, 2003 Jan 2, 1619(1), 59 - 69
Structural changes in DNA mediated by cationic lipids alter in vitro transcriptional activity at low charge ratios; Prasad TK et al.; Lipid/DNA complexes or Lipoplexes have been characterized by various biochemical and biophysical methods to understand the physical basis of transfection . Here we have addressed the effect of cationic liposomes, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), on transcription of DNA templates in vitro . Transcriptional activity of DNA-dependent RNA polymerase at DNA templates complexed with the cationic lipid varied as a function of charge ratio of lipid/DNA . At low charge ratios of 0.3:1 lipid/DNA and up to 1:1, we observed stimulation in transcription, while at higher charge ratios of lipid/DNA 3:1, complete inhibition in the activity occurred . Cetyl tri-methyl ammonium bromide (CTAB), a cationic detergent, and polyethylenimine (PEI), a cationic polymer, also bring about similar changes although to a lesser extent . The stimulation in transcription motivated us to probe into the molecular nature of the lipid/DNA interactions by absorbance spectroscopy and circular dichroism (CD) . Upon interaction with lipids, hyperchromicity and susceptibility to micrococcal nuclease has increased, which suggests that the DNA was partially denatured . On complexation with the cationic lipid (DOTAP), the magnitude of the positive band in CD spectra decreased, accompanied with a red shift, as a function of charge ratio . Results from spectroscopic and enzyme assays suggest that at low charge ratios DNA may be partially unwound.

J Gen Appl Microbiol, 2001 Dec, 47(6), 307 - 312
Isolation of hexavalent chromium-reducing Cr-tolerant facultative anaerobes from tannery effluent; Srinath T et al.; Several facultative anaerobes tolerant to high levels of chromate (>400 mg/ml) were isolated from tannery effluents . These isolates displayed varying degrees of Cr(VI) reduction under aerobic and anaerobic conditions at room temperature (24+/-2 degrees C) . Interestingly, eight isolates were efficient in reducing 70% Cr(VI) anaerobically . This includes 5 isolates of genus Aerococcus, two isolates of Micrococcus and single isolate of genus Aeromonas . These isolates were subjected to further characterization for possible use in Cr(VI) detoxification of industrial wastes . This is the first report of Aerococcus sp . capable of Cr(VI) reduction >70% anaerobically . These bacteria were further checked for tolerance to a variety of other heavy metals . Our study indicates the possible use of these bacteria in environmental clean up.

Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 1972 - 82 Epub 2002 Nov 23.
The structures of Micrococcus lysodeikticus catalase, its ferryl intermediate (compound II) and NADPH complex; Murshudov GN et al.; The crystal structure of the bacterial catalase from Micrococcus lysodeikticus has been refined using the gene-derived sequence both at 0.88 A resolution using data recorded at 110 K and at 1.5 A resolution with room-temperature data . The atomic resolution structure has been refined with individual anisotropic atomic thermal parameters . This has revealed the geometry of the haem and surrounding protein, including many of the H atoms, with unprecedented accuracy and has characterized functionally important hydrogen-bond interactions in the active site . The positions of the H atoms are consistent with the enzymatic mechanism previously suggested for beef liver catalase . The structure reveals that a 25 A long channel leading to the haem is filled by partially occupied water molecules, suggesting an inherent facile access to the active site . In addition, the structures of the ferryl intermediate of the catalase, the so-called compound II, at 1.96 A resolution and the catalase complex with NADPH at 1.83 A resolution have been determined . Comparison of compound II and the resting state of the enzyme shows that the binding of the O atom to the iron (bond length 1.87 A) is associated with increased haem bending and is accompanied by a distal movement of the iron and the side chain of the proximal tyrosine . Finally, the structure of the NADPH complex shows that the cofactor is bound to the molecule in an equivalent position to that found in beef liver catalase, but that only the adenine part of NADPH is visible in the present structure.

J Mol Biol, 2002 Nov 29, 324(3), 501 - 17
Asymmetric binding of histone H1 stabilizes MMTV nucleosomes and the interaction of progesterone receptor with the exposed HRE; Vicent GP et al.; Packaging of mouse mammary tumor virus (MMTV) promoter sequences in nucleosomes modulates access of DNA binding proteins and influences the interaction among DNA bound transcription factors . Here we analyze the binding of histone H1 to MMTV mononucleosomes assembled with recombinant histones and study its influence on nucleosome structure and stability as well as on progesterone receptor (PR) binding to the hormone responsive elements (HREs) . The MMTV nucleosomes can be separated into three main populations, two of which exhibited precise translational positioning . Histone H1 bound preferentially to the 5' distal nucleosomal DNA protecting additional 27-28 nt from digestion by micrococcal nuclease . Binding of histone H1 was unaffected by prior crosslinking of protein and DNA in nucleosomes with formaldehyde . Neither the translational nor the rotational nucleosome positioning was altered by histone H1 binding, but the nucleosomes were stabilized as judged by the kinetics of nuclease cleavage . Unexpectedly, binding of recombinant PR to the exposed distal HRE-I in nucleosomes was enhanced in the presence of histone H1, as demonstrated by band shift and footprinting experiments . This enhanced PR affinity may contribute to the reported positive effect of histone H1 on the hormonal activation of MMTV reporter genes.

Proc Natl Acad Sci U S A, 2002 Nov 26, 99(24), 15393 - 7 Epub 2002 Nov 18.
Mitotic chromosomes are chromatin networks without a mechanically contiguous protein scaffold; Poirier MG et al.; Isolated newt (Notophthalmus viridescens) chromosomes were studied by using micromechanical force measurement during nuclease digestion . Micrococcal nuclease and short-recognition-sequence blunt-cutting restriction enzymes first remove the native elastic response of, and then to go on to completely disintegrate, single metaphase newt chromosomes . These experiments rule out the possibility that the mitotic chromosome is based on a mechanically contiguous internal non-DNA (e.g., protein) "scaffold"; instead, the mechanical integrity of the metaphase chromosome is due to chromatin itself . Blunt-cutting restriction enzymes with longer recognition sequences only partially disassemble mitotic chromosomes and indicate that chromatin in metaphase chromosomes is constrained by isolated chromatin-crosslinking elements spaced by approximately 15 kb.

OMICS, 2002, 6(3), 259 - 72
%(G+C) variation and prediction by a model of bacterial gene transfer and codon adaptation; Buckley CO et al.; The %(G + C) of bacterial genomes ranges from 25% in Mycoplasma to 75% in Micrococcus . Our model for horizontal gene flow enabled a theoretical study of the adaptation of relative codon frequency to match the pattern of the tRNA set of a new host . This study explored the dynamic relationship of %(G + C) to vectors of relative codon frequency (F(gamma)), relative amino acid coding frequency (F(alpha)), and absolute codon frequency (F(|gamma|)) in chromosomes of nine, fully sequenced bacterial genomes that varied widely in %(G + C) . At constant F(alpha), the theoretical maximum average range possible was %(G + C) = 37.4 +/- 0.9% . In simulations of F(gamma) adaptation to a new host following hypothetical gene transfer, we modeled %(G + C) as a function of F(gamma) and F(alpha) . The simulation revealed that %(G + C) is dependent on F(gamma) and F(alpha) in an explicit relationship described in this paper . We conclude that (1) F(gamma) and F(alpha) determine %(G + C), and (2) the degree of adaptation of %(G + C) in a transferred gene depends upon the degree of F(gamma) equilibration and the similarity of F(alpha) of the transferred gene to that of the new host.

J Invertebr Pathol, 2002 Sep, 81(1), 12 - 8
Comparison of parasitism by Cotesia glomerata with bacterial infection and wounding in Pieris brassicae: induction of new haemolymph polypeptides and changes in humoral immune response; Ockroy KS et al.; In Pieris brassicae, parasitism by Cotesia glomerata and bacterial infection are differentiated with respect to haemolymph protein arrays, and production or suppression of antibacterial agents . Bacteriolytic activity in haemolymph from parasitized larvae was slightly, but significantly, higher 24h post-treatment than that of untreated and wounded controls . Micrococcus lysodeikticus- or lipopolysaccharide-(LPS) injected insects exhibited an 11-fold greater response than those parasitized . At 24h post-treatment, antibacterial activity against Escherichia coli was observed in haemolymph from all but untreated larvae . Injection of Grace's medium, M . lysodeikticus or LPS, caused a greater than threefold response than parasitization or wounding . The protein banding patterns of parasitized hosts did not correspond to those of the other treatments . Two parasitoid-induced proteins (38 and 128 kDa) were examined . Both were found in parasitized insects, not in those wounded, injected with Grace's medium, M . lysodeikticus or LPS . Neither protein was bacteriolytic or bacteriostatic in inhibition zone assays.

Blood, 2003 Mar 15, 101(6), 2388 - 92 Epub 2002 Oct 31.
Increased inflammation in lysozyme M-deficient mice in response to Micrococcus luteus and its peptidoglycan; Ganz T et al.; More than 70 years ago, Alexander Fleming discovered lysozyme and proposed that nonpathogenic bacteria fail to cause disease because they are very susceptible to destruction by lysozyme, an enzyme that is one of the principal proteins of phagocytes . Although much has been learned about the effects of lysozyme in vitro, its biological role in vivo has not been determined . We examined transgenic mice deficient in lysozyme M after challenge by the normally nonpathogenic and highly lysozyme-sensitive bacterium Micrococcus luteus . Despite partial compensation by newly expressed lysozyme P in macrophages, lysozyme M-deficient mice developed much more severe lesions than wild-type mice . The tissue injury was due to the failure of lysozyme M-deficient mice to inactivate peptidoglycan, resulting in an intense and prolonged inflammatory response . Our data indicate that tissue injury is normally limited by prompt degradation of bacterial macromolecules that trigger innate immunity and inflammation.

Mol Microbiol, 2002 Nov, 46(3), 623 - 35
A family of autocrine growth factors in Mycobacterium tuberculosis; Mukamolova GV et al.; Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus . Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells . We show here that the five cognate proteins from M . tuberculosis have very similar characteristics and properties to those of Rpf . They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations . Several lines of evidence indicate that they exert their activity from an extra-cytoplasmic location, suggesting that they are also involved in intercellular signalling . The five M . tuberculosis proteins show cross-species activity against M . luteus, Mycobacterium smegmatis and M . bovis (BCG) . Actively growing cells of M . bovis (BCG) do not respond to these proteins, whereas bacteria exposed to a prolonged stationary phase do . Affinity-purified antibodies inhibit bacterial growth in vitro, suggesting that sequestration of these proteins at the cell surface might provide a means to limit or even prevent bacterial multiplication in vivo . The Rpf family of bacterial growth factors may therefore provide novel opportunities for preventing and controlling mycobacterial infections.

Mol Microbiol, 2002 Nov, 46(3), 611 - 21
The rpf gene of Micrococcus luteus encodes an essential secreted growth factor; Mukamolova GV et al.; Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signalling functions and is required for the resuscitation of dormant cells . Originally isolated from the supernatant of actively growing cultures, Rpf was also detected on the surface of actively growing bacteria . Most molecules may be sequestered non-productively at the cell surface, as a truncated form of the protein, encompassing only the 'Rpf domain' is fully active . The C-terminal LysM module, which probably mediates binding to the cell envelope, is not required for biological activity . Rpf was essential for growth of M . luteus . Washed cells, inoculated at low density into a minimal medium, could not grow in its absence . Moreover, the incorporation of anti-Rpf antibodies into the culture medium at the time of inoculation also prevented bacterial growth . We were unable to inactivate rpf using a disrupted form of the gene, in which most of the coding sequence was replaced with a selectable thiostrepton resistance marker . Gene disruption was possible in the presence of a second, functional, plasmid-located copy of rpf, but not in the presence of a rpf derivative whose protein product lacked the secretory signal sequence . As far as we are aware, Rpf is the first example of a truly secreted protein that is essential for bacterial growth . If the Rpf-like proteins elaborated by Mycobacterium tuberculosis and other mycobacteria prove similarly essential, interference with their proper functioning may offer novel opportunities for protecting against, and treating, tuberculosis and other mycobacterial disease.

Parasitology, 2002 Oct, 125(Pt 4), 359 - 66
Humoral immune response of Simulium damnosum s.l . following filarial and bacterial infections; Klager SL et al.; The time-course of the humoral immune response of female blackflies after a challenge with bacteria, different Onchocerca microfilariae species, bacterial endotoxin and microfilarial extract was investigated . Strong bacteriolytic and growth inhibition activities against the Gram-positive bacterium Micrococcus luteus were induced by all agents . Specific differences were found in activity levels and time-course . Notably the endotoxin lipopolysaccharide (LPS) induced a very early, profound bacteriolytic and antibacterial response, which declined within a day after injection . In contrast, the bacteriolytic activities after Escherichia coli D31 and Onchocerca microfilariae infections were lower, but remained elevated over the observation period of 4 days . The bacteriolytic activity was correlated to a haemolymph protein with a molecular weight of around 14 kDa . Anti-Gram-positive activity in the E . coli infected group appeared within the first 6 h . However, it took 4 days in the microfilarial infected blackflies to reach significant levels . The active agent was identified to be a peptide with a molecular weight of around 4-4.5 kDa . Activity against the Gram-negative bacteria E . coli was detected in blackflies injected with E . coli D31, O . dukei microfilariae and microfilarial extract on days 1 and 4 after injection . The immune response in S . damnosum s.l . naturally infected via a bloodmeal on cattle supported the findings of the experimental infections . Similarities of the immune response kinetics between bacterial and filarial infections suggested that intracellular Wolbachia bacteria, released from microfilariae, could be responsible for the antibacterial response . This is supported by the observation that the induction of an immune response in the Drosophila melanogaster mbn-2 cell line by the filarial extract is blocked by polymyxin B, which forms inactive complexes with bacterial LPS.

Food Addit Contam, 2002 Sep, 19(9), 819 - 28
Accelerated solvent extraction of animal feedingstuffs for microbial growth inhibition screening for the presence of antimicrobial feed additives; Higgins HC et al.; Three plate systems (combinations of indicator organism and growth medium) were evaluated for the detection of analytical standards of the banned feed additives avoparcin, bacitracin zinc, spiramycin, tylosin and virginiamycin . When authorized in the EU, the previously recommended minimum inclusion rate (MIR) for each compound was 5 mg kg(-1) . One of the plate systems (Micrococcus luteus ATCC 10240, nutrient agar) detected all five additives . This plate was used in a further study that evaluated the suitability of accelerated solvent extraction (ASE) as a first step in the development of a rapid single-plate screening assay . A drug-free (negative control) feedingstuff was fortified with the compounds (0-50 mg kg(-1)), extracted by ASE and the extracts applied to the plate at each of three pH ranges - unadjusted extract (pH 5.7-5.9), pH 6.5 and 8.0 . At pH 6.5, sub-MIR concentrations of virginiamycin and tylosin were detectable . Avoparcin was detectable at 6.3 mg kg(-1) . The detection of zinc bacitracin was#10; pH-independent (10 mg kg(-1)) . At pH 8.0, spiramycin was detectable at 5.4 mg kg(-1) . Mean +/- SD analytical recoveries from fortified feedingstuffs (n = 10) ranged from 57 +/- 1.5% for avoparcin to 96 +/- 4% for virginiamycin . The five additives were also detectable following ASE extraction from a range of different feedingstuffs fortified with each of the drugs . A further 24 compounds permitted for use in animal feeds were tested . Of these, nine were detectable at their recommended MIR . It is concluded that ASE is a versatile technique suitable for the automated extraction of a range of antimicrobials from animal feedingstuffs . Employing ASE with this single-plate detection system permits the rapid antimicrobial screening of animal feedingstuffs and allows the detection of the banned additives . Whilst the method is applicable as a screening test, more specific postscreening methods would be necessary for subsequent identification (and quantification) of antimicrobials in screening-positive samples.

Life Sci Space Res, 1979, 17, 105 - 10
Microorganisms of the upper layer of the atmosphere and the protective role of their cell pigments; Imshenetsky AA et al.; Of the six species of microorganisms isolated from the mesosphere, five contained pigments and were more resistant to UV radiation compared with their pigment-free mutants . The black pigment isolated from the conidia of Aspergillus niger considerably increased the UV resistance of the unpigmented mutant conidia of Penicillium notatum, the spore Circinella muscae and the vegetative cells of Micrococcus albus . From the data it is possible to conclude that in the upper layers of the Earth's atmosphere the predominant proportion of pigmented microorganisms is the consequence of natural selection by UV radiation.

J Biol Chem, 2002 Nov 8, 277(45), 43474 - 80 Epub 2002 Sep 03.
Human testis/sperm-specific histone H2B (hTSH2B) . Molecular cloning and characterization; Zalensky AO et al.; Human sperm, unlike the sperm of other mammals, contain replacement histones with unknown biological functions . Here, we report the identification of the novel human gene coding for a testis/sperm-specific histone H2B (hTSH2B) . This variant histone is 85% homologous to somatic H2B and has over 93% homology with the testis H2B of rodents . Using genomic PCR, two genetic alleles of hTSH2B were found in the human population . The hTSH2B gene is transcribed exclusively in testis, and the corresponding protein is also present in mature sperm . We expressed recombinant hTSH2B and identified this protein with a particular H2B subtype expressed in vivo . The subnuclear distribution of H2B variants in sperm was determined using biochemical fractionation and immunoblotting . The H2B variant associated with telomere-binding activity () was solubilized by Triton X-100 or micrococcal nuclease extraction, whereas hTSH2B was relatively tightly bound in nuclei . Immunofluorescence showed that hTSH2B was concentrated in spots located at the basal nuclear area of a subpopulation (20% of cells) of mature sperm . This fact may be of particular importance, because the hTSH2B "positive" and "negative" sperm cells may undergo significantly different decondensation processes following fertilization.

Med Dosw Mikrobiol, 2002, 54(1), 29 - 34
{Occurrence of bacteria in the mouth from genera of Micrococcus, Kocuria, Nesterenkonia, Kytococcus and Dermacoccus}; Szczerba I et al.; The aim of the study was to assess the prevalence of different bacteria in the oral cavity . The bacteria were present in the oral cavities of 73 (48.7%) of 150 individuals . Nesterenkonia halobia, the most frequently isolated species, was found in 20 (27%) individuals, Micrococcus luteus in 16 (22%), Kocuria kristinae in 12 (16%), Kocuria varians in 10 (14%), Dermacoccus sedentarius in 9 (12%), Micrococcus lylae in 8 (11%), and Kytococcus nishinomiyaensis in 3 (4%) . Mean counts of these microorganisms were relatively low and amounted in log10 CFU/ml saliva for M . luteus 1.87 +/- 0.52, for M . lylae 2.03 +/- 0.39, for N . halobia 2.14 +/- 0.56, for K . kristinae 2.20 +/- 0.69, for K . varians 2.19 +/- 0.67, for K . nishinomiyaensis 1.72 +/- 0.39, and for D . sedentarius 2.27 +/- 0.55 . The factor limiting the population sizes of these microorganisms was most probably the antagonistic activity of the bacteria living in oral cavity.

Genes Dev, 2002 Aug 15, 16(16), 2108 - 19
The Drosophila heterochromatic gene encoding poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during development; Tulin A et al.; Poly(ADP-ribose) polymerase (PARP) is a major NAD-dependent modifying enzyme that mediates important steps in DNA repair, transcription, and apoptosis, but its role during development is poorly understood . We found that a single Drosophila Parp gene spans more than 150 kb of transposon-rich centromeric heterochromatin and produces several differentially spliced transcripts, including a novel isoform, PARP-e, predicted to encode a protein lacking enzymatic activity . An insertion mutation near the upstream promoter for Parp-e disrupts all Parp expression . Heterochromatic but not euchromatic sequences become hypersensitive to micrococcal nuclease, nucleoli fail to form, and transcript levels of the copia retrotransposon are elevated more than 50-fold; the variegated expression of certain transgenes is dominantly enhanced . Larval lethality can be rescued and PARP activity restored by expressing a cDNA encoding PARP-e . We propose that PARP-e autoregulates Parp transcription by influencing the chromatin structure of its heterochromatic environment . Our results indicate that Parp plays a fundamental role organizing the structure of Drosophila chromatin.

J Biol Chem, 2002 Oct 11, 277(41), 38305 - 10 Epub 2002 Aug 06.
Transcription-coupled and transcription-independent repair of cyclobutane pyrimidine dimers in the dihydrofolate reductase gene; Hu W et al.; Using a ligation-mediated polymerase chain reaction technique, we have mapped the repair of ultraviolet light-induced cyclobutane pyrimidine dimers (CPDs) at the nucleotide level in exons 1, 2, and 5 of the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary cells . We found that CPDs are preferentially repaired in the transcribed strand (T strand) and that the order of repair efficiency is exon 1 > exon 2 > exon 5 . In the cells with a deletion of the DHFR gene encompassing the promoter region and the first four exons, CPDs are not repaired in the T strand of the residual DHFR gene . These results substantiate the idea that the preferential repair of CPDs in the T strand is transcription dependent . However, in the wild type gene we have found that CPDs are efficiently repaired in the nontranscribed strand (NT strand) of exon 1 but not in the NT strand of exons 2 and 5 . Probing the chromatin structure of exons 1, 2, and 5 of the DHFR gene with micrococcal nuclease revealed that the exon 1 region is much more sensitive to micrococcal nuclease digestion than the exon 2 and exon 5 regions, suggesting that the chromatin structure in the exon 1 region is much more open . These results suggest that, although preferential repair of the T strand of the DHFR gene is transcription dependent, repair of the NT strand is greatly affected by chromatin structure.

Eur J Cell Biol, 2002 Jul, 81(7), 413 - 8
Differentiation of immune cells challenged by bacteria in the common European starfish, Asterias rubens (Echinodermata); Coteur G et al.; Amoebocytes are the main effector cells of the echinoderm immune system . In starfishes, a taxon in which bacterial diseases have been rarely reported, amoebocytes are considered to be the only circulating and immune cell type . The present paper addresses the question of amoebocyte differentiation in the starfish Asterias rubens when challenged by bacteria . Starfishes were injected with FITC-coupled bacteria (Micrococcus luteus) . Amoebocytes were collected at regular time intervals for 24 h . The cytometric characteristics and the phagocytic activity were studied by flow cytometry . Three amoebocyte groups of different size were identified . The cell concentrations of the two largest and more numerous of these groups (G2 and G3) were modulated by immune stimulation while the group of smallest, less numerous, cells (G1) was unaffected . All of these cell groups were phagocytic but their kinetics of cell activation and bacteria ingestion differed . G1 cells showed the lowest phagocytic activity while G3 cells had the highest and fastest phagocytic activity . Starfish amoebocytes appear to be segregated in three groups, two of them (G2 and G3) being immunomodulated and one of them presenting a very fast reaction to bacteria . It is suggested that the high efficiency of the immune system in starfishes is related to this fast reaction.

J Biol Chem, 2002 Oct 4, 277(40), 37741 - 6 Epub 2002 Jul 17.
Periodic DNA methylation in maize nucleosomes and demethylation by environmental stress; Steward N et al.; When maize seedlings were exposed to cold stress, a genome-wide demethylation occurred in root tissues . Screening of genomic DNA identified one particular fragment that was demethylated during chilling . This 1.8-kb fragment, designated ZmMI1, contained part of the coding region of a putative protein and part of a retrotransposon-like sequence . ZmMI1 was transcribed only under cold stress . Direct methylation mapping revealed that hypomethylated regions spanning 150 bases alternated with hypermethylated regions spanning 50 bases . Analysis of nuclear DNA digested with micrococcal nuclease indicated that these regions corresponded to nucleosome cores and linkers, respectively . Cold stress induced severe demethylation in core regions but left linker regions relatively intact . Thus, methylation and demethylation were periodic in nucleosomes . The following biological significance is conceivable . First, because DNA methylation in nucleosomes induces alteration of gene expression by changing chromatin structures, vast demethylation may serve as a common switch for many genes that are simultaneously controlled upon environmental cues . Second, because artificial demethylation induces heritable changes in plant phenotype (Sano, H., Kamada, I., Youssefian, S., Katsumi, M., and Wabilko, H . (1990) Mol . Gen . Genet . 220, 441-447), altered DNA methylation may result in epigenetic inheritance, in which gene expression is modified without changing the nucleotide sequence.

Antimicrob Agents Chemother, 2002 Aug, 46(8), 2344 - 8
Hexapeptide derivatives of glycopeptide antibiotics: tools for mechanism of action studies; Allen NE et al.; Hexapeptide (des-N-methylleucyl) derivatives of LY264826 were prepared in order to examine further the role of N-substituted hydrophobic side chains in defining the mechanisms of action of semisynthetic glycopeptide antibiotics . The hexapeptide of LY264826 binds to the cell wall intermediate analog L-Lys-D-Ala-D-Ala with a 100-fold lower affinity than LY264826 and inhibits Micrococcus luteus almost 200-fold more poorly than LY264826 does . Alkylation of the 4-epi-vancosamine moiety of the disaccharide significantly enhanced the antibacterial activity of the hexapeptide . Alkylation did not affect the binding affinity for D-alanyl-D-alanine residues; however, it did enhance dimerization 7,000-fold and enhanced binding to bacterial membrane vesicles noticeably compared with the levels of dimerization and binding for the unsubstituted hexapeptide . The findings from this study complement those presented in an earlier report (N . E . Allen, D . L . LeTourneau, and J . N . Hobbs, Jr., J . Antibiot . 50:677-684, 1997) and are consistent with the conclusion that the enhanced antibacterial activities of semisynthetic glycopeptide antibiotics derive from the ability of the hydrophobic side chain to markedly affect both dimerization and binding to bacterial membranes.

Transgenic Res, 2002 Jun, 11(3), 229 - 39
Expression of functional recombinant human lysozyme in transgenic rice cell culture; Huang J et al.; Using particle bombardment-mediated transformation, a codon-optimized synthetic gene for human lysozyme was introduced into the calli of rice (Oryza sativa) cultivar Taipei 309 . The expression levels of recombinant human lysozyme in the transformed rice suspension cell culture approached approximately 4% of total soluble protein . Recombinant human lysozyme was purified to greater than 95% homogeneity using a two-step chromatography process . Amino acid sequencing verified that the N-terminus of the mature recombinant human lysozyme was identical to native human lysozyme . This indicates that the rice RAmy3D signal peptide was correctly cleaved off from the human lysozyme preprotein by endogenous rice signal peptidase . Recombinant human lysozyme was found to have the same molecular mass, isoelectric point and specific activity as native human lysozyme . The bactericidal activity of recombinant human lysozyme was determined by turbidimetric assay using Micrococcus lysodeikticus in 96-well microtiter plates . The bactericidal activity of lysozyme on gram-negative bacteria was examined by adding purified lysozyme to mid-log phase cultures of E . coli strain JM109 . In this study, significant bactericidal activity was observed after E . coli cells were exposed to recombinant human lysozyme for 60 min . Both native and recombinant human lysozyme displayed the same thermostability and resistance to degradation by low pH . The potential for using rice-derived lysozyme as an antimicrobial food supplement, particularly for infant formula and baby foods, is discussed.

Acta Vet Hung, 2002, 50(2), 143 - 50
A possible relationship between bumblefoot responsive to potassium arsenite and micrococci in the blood of three birds of prey; Tarello W; Pododermatitis (bumblefoot) is a major health problem of falcons world-wide because healing processes in the talons are difficult and lengthy . A peregrine (Falco peregrinus), a merlin (Falco columbarius) and a saker falcon (Falco cherrug) with bumblefoot at different stages ranging from III to V, were all found to be carriers of micrococcus-like organisms in the blood and two of them were successfully treated with 0.5% potassium arsenite in low dosage given intravenously . A number of considerations are made on the immune dysfunction aspects of bumblefoot in birds of prey and on the emerging role of arsenic-based medicaments in the treatment of animal and human immune dysfunction syndromes.

J Pharm Biomed Anal, 2002 Jul 31, 29(5), 957 - 61
Microbiological assay for azithromycin in pharmaceutical formulations; Breier AR et al.; The validation of a microbiological assay, applying the cylinder-plate method, for the determination of the antibiotic azithromycin is described . Using a strain of Micrococcus luteus ATCC 9341 as the test organism, azithromycin at concentrations ranging from 0.1 to 0.4 microgml(-1) could be measured in capsules and suspensions . A prospective validation of the method showed that it was linear (r=0.998), precise (RSD=1.40-capsules; RSD=1.19-powder for suspension and RSD=1.73-oral suspension) and accurate (it measured the added quantities) . We conclude that the microbiological assay is satisfactory for quantitation of in vitro antibacterial activity of azithromycin.

Indian Heart J, 2002 Mar-Apr, 54(2), 181 - 3
Prospective evaluation of the risk of bacteremia induced by transesophageal echocardiography; Dhas KL et al.; BACKGROUND: The incidence of bacteremia induced by transesophageal echocardiography is controversial in the Indian population . This study aimed to find out the occurrence of bacteremia following transesophageal echocardiography . METHODS AND RESULTS: Between February 2000 and January 2001, 47 patients (26 males and 21 females) were enrolled for the study . Their ages ranged from 13 to 61 years (mean: 35 +/- 11.4 years) . Patients with prosthetic valves, suspected infective endocarditis and those on antibiotics were excluded . For each procedure, two sets of blood cultures were obtained immediately before and after the procedure . For each blood culture, 10 ml of blood was evenly inoculated into brain-heart infusion broth and biphasic infusion medium and incubated for 7 days . Transesophageal echocardiography was carried out under oropharyngeal anesthesia (xylocaine gel and spray) . Two blood cultures taken before the procedure were positive and excluded from the final analysis . Of the remaining 45 patients whose preprocedure blood cultures were sterile, 6 samples (13.3%) were positive after the procedure diphtheroids in 3, micrococci in 2 and aerobic spore formers in 1 . CONCLUSIONS: This study demonstrates that the incidence of bacteremia related to transesophageal echocardiography is not insignificant, as reported in previous studies . Though routine antibiotic prophylaxis before transesophageal echocardiography is not advocated, it should be recommended in high-risk patients such as those with prosthetic valves, multivalvular involvement or those with a past history of infective endocarditis.

Biomed Sci Instrum, 2002, 38, 495 - 500
L-glutamic acid production in a continuous stirred tank bioreactor using coimmobilized bio-catalyst using a fluorosensor; Prabhu N et al.; The production of L-Glutamic acid has been studied using coimmobilized whole cells of pseudomonas reptilivora and micrococcus glutamicus in a two litre Tokyo Rikakikai fermentor using glucose as selected production medium . The process was carried out at an optimum temperature of 32 degree Celsius and a pH of 7.2 . The progress of the reaction was recorded using Dr . Ingold fluorosensor . The effect of initial substrate concentration, speed of agitation, volume ofcalcium alginate beads and aeration rate on the yield of glutamic acid has been investigated . It has been found that the acid production increases exponentially with substrate concentration, and mass transfer co-efficient varied linearly with aeration rate . The kinetic parameters also had been estimated.

Biochim Biophys Acta, 2002 Jun 12, 1590(1-3), 140 - 9
2',5'-oligoadenylate synthetase from a lower invertebrate, the marine sponge Geodia cydonium, does not need dsRNA for its enzymatic activity; Lopp A et al.; Recently, the presence of 2',5'-linked oligoadenylates and a high 2',5'-oligoadenylate synthetase activity were discovered in a lower invertebrate, the marine sponge Geodia cydonium . It has been demonstrated that mammalian 2-5A synthetase isozymes require a dsRNA cofactor for their enzymatic activity . Our results show that, unlike mammalian 2-5A synthetases, the 2-5A synthetase from the sponge acts in a dsRNA-independent manner in vitro . A prolonged incubation of the G . cydonium extract with a high concentration of a micrococcal nuclease had no effect on the activity of the 2-5A synthetase . At the same time, the micrococcal nuclease was effective within 30 min in degrading dsRNA needed for the enzymatic activity in IFN-induced PC12 cells . These results indicate that the 2-5A synthetase from G . cydonium may be active per se or is activated by some other mechanism . The sponge enzyme is capable of synthesizing a series of 2-5A oligomers ranging from dimers to octamers . The accumulation of a dimer in the predominant proportion during the first stage of the reaction was observed, followed by a gradual increase in longer oligoadenylates . By its product profile and kinetics of formation, the sponge 2-5A synthetase behaves like a specific isoform of enzymes of the 2-5A synthetase family.

J Mol Biol, 2002 May 24, 319(1), 27 - 35
Initiation factor IF2, thiostrepton and micrococcin prevent the binding of elongation factor G to the Escherichia coli ribosome; Cameron DM et al.; The bacterial translational GTPases (initiation factor IF2, elongation factors EF-G and EF-Tu and release factor RF3) are involved in all stages of translation, and evidence indicates that they bind to overlapping sites on the ribosome, whereupon GTP hydrolysis is triggered . We provide evidence for a common ribosomal binding site for EF-G and IF2 . IF2 prevents the binding of EF-G to the ribosome, as shown by Western blot analysis and fusidic acid-stabilized EF-G.GDP.ribosome complex formation . Additionally, IF2 inhibits EF-G-dependent GTP hydrolysis on 70 S ribosomes . The antibiotics thiostrepton and micrococcin, which bind to part of the EF-G binding site and interfere with the function of the factor, also affect the function of IF2 . While thiostrepton is a strong inhibitor of EF-G-dependent GTP hydrolysis, GTP hydrolysis by IF2 is stimulated by the drug . Micrococcin stimulates GTP hydrolysis by both factors . We show directly that these drugs act by destabilizing the interaction of EF-G with the ribosome, and provide evidence that they have similar effects on IF2.

J Mol Biol, 2002 Apr 26, 318(2), 333 - 49
In vivo topography of Rap1p-DNA complex at Saccharomyces cerevisiae TEF2 UAS(RPG) during transcriptional regulation; De Sanctis V et al.; We have analyzed in detail the structure of RAP1-UAS(RPG) complexes in Saccharomyces cerevisiae cells using multi-hit KMnO(4), UV and micrococcal nuclease high-resolution footprinting . Three copies of the Rap1 protein are bound to the promoter simultaneously in exponentially growing cells, as shown by KMnO(4) multi-hit footprinting analysis, causing extended and diagnostic changes in the DNA structure of the region containing the UAS(RPG) . Amino acid starvation does not cause loss of Rap1p from the complex; however, in vivo UV-footprinting reveals the occurrence of structural modifications of the complex . Moreover, low-resolution micrococcal nuclease digestion shows that the chromatin of the entire region is devoid of positioned nucleosomes but is susceptible to changes in accessibility to the nuclease upon amino acid starvation . The implications of these results for the mechanism of Rap1p action are discussed . (c) 2002 Elsevier Science Ltd.

Yao Xue Xue Bao, 1998 Aug, 33(8), 621 - 5
{Comparison of roxithromycin bioavailability of a conventional and a dispersible tablet formulation}; Zhong D et al.; Following oral administrations of a single dose of 150 mg roxithromycin dispersible tablet (formulation A, a new formulation for clinical trial) and conventional tablet (formulation B, purchased from the market) to each of 10 healthy male volunteers in a randomized crossover design, the plasma levels of active drug at different times were determined by a microbial assay with Micrococcus luteus CMCC (B) 28001 and the plasma concentration-time profiles were obtained . The Tmax values of formulation A and B obtained were 1.7 +/- 0.9 and 3.7 +/- 1.6 h, the Cmax values were 4.97 +/- 1.17 and 2.04 +/- 1.26 micrograms.ml-1 and the AUC0-->infinity values were 62.2 +/- 11.9 and 35.0 +/- 16.9 micrograms.h.ml-1, respectively . Surprisingly, the relative bioavailability of formulation B was found to be only 59.8% +/- 32.6% (P < 0.01) . The low bioavailability of the latter formulation was attributed to poor release of roxithromycin in the gastric site, which was proposed as a key factor to influence drug absorption . Other related factors were also discussed.

J Am Chem Soc, 2002 May 15, 124(19), 5518 - 27
Femtosecond electron-transfer reactions in mono- and polynucleotides and in DNA; Reid GD et al.; Quenching of redox active, intercalating dyes by guanine bases in DNA can occur on a femtosecond time scale both in DNA and in nucleotide complexes . Notwithstanding the ultrafast rate coefficients, we find that a classical, nonadiabatic Marcus model for electron transfer explains the experimental observations, which allows us to estimate the electronic coupling (330 cm(-1)) and reorganization (8070 cm(-1)) energies involved for thionine-{poly(dG-dC)}(2) complexes . Making the simplifying assumption that other charged, pi-stacked DNA intercalators also have approximately these same values, the electron-transfer rate coefficients as a function of the driving force, DeltaG, are derived for similar molecules . The rate of electron transfer is found to be independent of the speed of molecular reorientation . Electron transfer to the thionine singlet excited state from DNA obtained from calf thymus, salmon testes, and the bacterium, micrococcus luteus (lysodeikticus) containing different fractions of G-C pairs, has also been studied . Using a Monte Carlo model for electron transfer in DNA and allowing for reaction of the dye with the nearest 10 bases in the chain, the distance dependence scaling parameter, beta, is found to be 0.8 +/- 0.1 A(-1) . The model also predicts the redox potential for guanine dimers, and we find this to be close to the value for isolated guanine bases . Additionally, we find that the pyrimidine bases are barriers to efficient electron transfer within the superexchange limit, and we also infer from this model that the electrons do not cross between strands on the picosecond time scale; that is, the electronic coupling occurs predominantly through the pi-stack and is not increased substantially by the presence of hydrogen bonding within the duplex . We conclude that long-range electron transfer in DNA is not exceptionally fast as would be expected if DNA behaved as a "molecular wire" but nor is it as slow as is seen in proteins, which do not benefit from pi-stacking.

Eur J Biochem, 2002 May, 269(9), 2383 - 93
Re-oxygenation of hypoxic simian virus 40 (SV40)-infected CV1 cells causes distinct changes of SV40 minichromosome-associated replication proteins; Riedinger HJ et al.; Hypoxia interrupts the initiation of simian virus 40 (SV40) replication in vivo at a stage situated before unwinding of the origin region . After re-oxygenation, unwinding followed by a synchronous round of viral replication takes place . To further characterize the hypoxia-induced inhibition of unwinding, we analysed the binding of several replication proteins to the viral minichromosome before and after re-oxygenation . T antigen, the 34-kDa subunit of replication protein A (RPA), topoisomerase I, the 48-kDa subunit of primase, the 125-kDa subunit of polymerase delta, and the 37-kDa subunit of replication factor C (RFC) were present at the viral chromatin already under hypoxia . The 70-kDa subunit of RPA, the 180-kDa subunit of polymerase alpha, and proliferating cell nuclear antigen (PCNA) were barely detectable at the SV40 chromatin under hypoxia and significantly increased after re-oxygenation . Immunoprecipitation of minichromosomes with T antigen-specific antibody and subsequent digestion with micrococcus nuclease revealed that most of the minichromosome-bound T antigen was associated with the viral origin in hypoxic and in re-oxygenated cells . T antigen-catalysed unwinding of the SV40 origin occurred, however, only after re-oxygenation as indicated by (a) increased sensitivity of re-oxygenated minichromosomes against digestion with single-stranded DNA-specific nuclease P1; (b) stabilization of RPA-34 binding at the SV40 minichromosome; and (c) additional phosphorylations of RPA-34 after re-oxygenation, probably catalysed by DNA-dependent protein kinase . The results presented suggest that the subunits of the proteins necessary for unwinding, primer synthesis and primer elongation first assemble at the SV40 origin in form of stable, active complexes directly before they start to work.

J Biol Chem, 2002 Jun 21, 277(25), 22497 - 508 Epub 2002 Apr 15.
Transcriptional control of the human thromboxane synthase gene in vivo and in vitro; Yaekashiwa M et al.; Thromboxane A(2), a potent mediator of vasoconstriction and platelet aggregation, is synthesized from prostaglandin H(2) by thromboxane synthase (TXAS) . We report here on promoter analyses of human TXAS using in vitro transcription and in vivo transfection methods . The 39-bp core promoter, containing both TATA and initiator elements, accurately initiates transcription in an orientation-dependent manner in a cell-free transcription system . Mutation of either TATA or initiator abolished transcriptional activity, but the upstream sequence had no effect on TXAS promoter activities in vitro, suggesting that the core promoter is sufficient for transcriptional activity from a naked DNA template . In contrast, mutation of both elements drastically decreased the promoter activity in vivo . Furthermore, TXAS proximal promoter containing the nucleotides -90 to -56 relative to the transcriptional start site was necessary for promoter transactivation in vivo . Transcriptional activities from 5'-deletion mutants indicated that the effects of the nucleotides -90/-56 were more pronounced in stably transfected cells (a 150-fold difference) than in the transiently transfected cells (an 8-fold difference), reflecting the effects of TXAS transcriptional activation from replicating and nonreplicating DNA templates . Partial micrococcal nuclease digestion indicated that the sequence -90/-56, where the NF-E2 site is located, is associated with alterations of nucleosomal structure at the regions of promoter and reporter gene but not the region further downstream . Mutagenesis and forced expression studies demonstrated a critical role of p45 NF-E2 in controlling TXAS expression under native chromatin conditions . Band shifting and chromatin immunoprecipitation assays indicated that p45 NF-E2 was bound to the TXAS promoter in vitro and in vivo . Indirect end labeling and ligation-mediated PCR analyses further demonstrated that the occupation of TXAS promoter NF-E2 site was associated with disruption of nucleosomal structure . Collectively, these results indicate that binding of NF-E2 is critical both for alteration of the nucleosomal structure and for activation of the TXAS promoter, whereas the TATA and initiator elements are essential for transcription.

Int J Biochem Cell Biol, 2002 Jun, 34(6), 632 - 44
Chromatin structure analysis of the rat Na, K-ATPase beta2 gene 5'-flanking region; Alvarez de la Rosa D et al.; The Na, K-ATPase is formed by two major subunits (alpha and beta) encoded by a gene family of at least four alpha and three beta isoforms . These genes show distinctive expression patterns involving complex tissue-specific and developmental regulation, although the control mechanisms are not well understood . Here we study the role of chromatin structure in the tissue-specific expression of rat Na, K-ATPase beta2 isoform, which is mainly found in the central nervous system . We have examined the presence and characteristics of nuclease hypersensitive sites and the cytosine methylation patterns in the 5'-flanking region of the beta2 isoform gene from various nuclear preparations . Our results show that in this 5'-flanking region there is only one nuclease hypersensitive site . It is located upstream of the transcription initiation site and shows tissue-specific characteristics . Digestion with deoxyribonuclease I (DNase I), S1 nuclease and micrococcal nuclease yield patterns consistent with a triple-helix structure present only in the active state of the promoter . We also demonstrate that the 5'-flanking region of the beta2 gene co-localizes with a CpG island free of methylation in every tissue tested . The results presented here support a role for specific chromatin remodeling events in the regulation of the Na, K-ATPase beta2 gene expression . They also provide the basis for future studies of the transcription factors involved in the regulation of this gene.

J Biol Inorg Chem, 2002 Apr, 7(4-5), 473 - 82 Epub 2002 Jan 15.
New potent agents binding to a poly(dT) sequence in double-stranded DNA: bis(Zn(2+)-cyclen) and tris(Zn(2+)-cyclen) complexes; Kikuta E et al.; In an effort to search for mechanistically new and more potent agents than conventional drugs that target AT-rich sequences in double-stranded DNA, we have tested multi(Zn(2+)-cyclen) complexes . Indeed, they selectively bound to poly(dT) sequences to melt the A-T hydrogen bonds; only 2.5 microM or 4 microM of the p-tris(Zn(2+)-cyclen) complex were required to completely melt a 50 microM nucleobase of double-stranded poly(dA) x poly(dT) or poly(dA-dT)(2) at 25 degrees C . The region with seven consecutive T's in native DNA (150 bp) was protected from micrococcal nuclease hydrolysis, as revealed by footprinting assays, with IC(50) values of 2 microM for p-bis(Zn(2+)-cyclen) and 0.5 microM for p-tris(Zn(2+)-cyclen) . The high affinity to AT-rich sequences of these Zn(2+)-cyclen complexes matches or surpasses those of the conventional AT-binding drugs distamycin A (IC(50)=2 microM) and DAPI (5 microM) . Moreover, the p-tris(Zn(2+)-cyclen) complex selectively binds to the TATA box sequence of the SV40 early promoter to inhibit the binding of the TATA binding protein as effectively as distamycin A, with an IC(50) value of 0.4 microM . In vitro transcription of poly(dA) x poly(dT) using Escherichia coli RNA polymerase was effectively inhibited by p-tris(Zn(2+)-cyclen) . The {(3)H}-ATP incorporation into RNA was more strongly blocked (IC(50)=0.8 microM) than the {(3)H}-UTP incorporation (IC(50)=40 microM), a fact indicating that the p-tris(Zn(2+)-cyclen) complex interacts only with the poly(dT) strand in the double-stranded DNA template.

Mol Cell Biol, 2002 May, 22(9), 2974 - 83
Dual roles of p300 in chromatin assembly and transcriptional activation in cooperation with nucleosome assembly protein 1 in vitro; Asahara H et al.; In a yeast two-hybrid screen to identify proteins that bind to the KIX domain of the coactivator p300, we obtained cDNAs encoding nucleosome assembly protein 1 (NAP-1), a 60-kDa histone H2A-H2B shuttling protein that promotes histone deposition . p300 associates preferentially with the H2A-H2B-bound form of NAP-1 rather than with the unbound form of NAP-1 . Formation of NAP-1-p300 complexes was found to increase during S phase, suggesting a potential role for p300 in chromatin assembly . In micrococcal nuclease and supercoiling assays, addition of p300 promoted efficient chromatin assembly in vitro in conjunction with NAP-1 and ATP-utilizing chromatin assembly and remodeling factor; this effect was dependent in part on the intrinsic histone acetyltransferase activity of p300 . Surprisingly, NAP-1 potently inhibited acetylation of core histones by p300, suggesting that efficient assembly requires acetylation of either NAP-1 or p300 itself . As p300 acted cooperatively with NAP-1 in stimulating transcription from a chromatin template in vitro, our results suggest a dual role of NAP-1-p300 complexes in promoting chromatin assembly and transcriptional activation.

J Biol Chem, 2002 Jun 14, 277(24), 21440 - 5 Epub 2002 Apr 05.
Multiple factors prevent transcriptional interference at the yeast ARO4-HIS7 locus; Valerius O et al.; Increased transcriptional activity may cause transcriptional interference in organisms with compact genomes such as the yeast Saccharomyces cerevisiae . Replacement of the yeast ARO4 promoter by the stronger ACT1 promoter increases ARO4 transcription and simultaneously reduces the basal transcription of the downstream HIS7 gene . The open reading frames of ARO4 and HIS7 are tandemly transcribed and are separated by 416 bp . In wild-type cells, a nuclease-resistant site suggests that the two genes are separated by a single positioned nucleosome . Transcriptional interference correlates with Micrococcus nuclease accessibility of this otherwise nuclease-resistant site . Deletion analyses of the region between the two open reading frames revealed that transcriptional interference increases upon removal of either parts of the ARO4 3' end or HIS7 promoter sequences . The abolishment of the Abf1p-binding site within the HIS7 promoter significantly enhances transcriptional interference, resulting in a histidine auxotrophic strain . Our data suggest that the yeast cell prevents transcriptional interference by the combined action of efficient ARO4 transcription termination, the positioning of a fixed nucleosome, and transcription factor binding to the HIS7 promoter.

Fish Shellfish Immunol, 2002 Mar, 12(3), 187 - 200
Reactive oxygen species (ROS) production by amoebocytes of Asterias rubens (Echinodermata); Coteur G et al.; An adapted peroxidase, luminol-enhanced chemiluminescence method in an EDTA-free, Ca++-containing medium is described and used to characterise reactive oxygen species (ROS) production by starfish immunocytes using a standard microplate reader luminometer . ROS production was stimulated by direct interaction of immunocytes with bacteria or bacterial wall components, but not by the soluble stimulant PMA nor the lectin concanavalin A . Produced ROS detected by this method are apparently superoxide anions, hydrogen peroxide and peroxynitrite . Comparison with other chemiluminescence methods indicates that the described method is the only one to detect the stimulation of starfish immunocytes by the Gram-positive bacteria, Micrococcus luteus, a fact that questions previous reports indicating a lack of stimulation by pathogens . The adapted method provides a rapid determination of the overall ROS production, which is suitable for both disease control and immunotoxicological studies in echinoderms.

Biol Reprod, 2002 Apr, 66(4), 1061 - 7
Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis; Sakkas D et al.; Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa . The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood . The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters . We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I . We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression . In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa . This DNA protection is poorer in men with abnormal semen parameters . We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process . Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.

J Inorg Biochem, 2002 Feb, 88(3-4), 251 - 3
Gold helps bacteria to oxidize methane; Levchenko LA et al.; With the use of labeled methane-14C and by chromatographic analysis it was shown that gold-containing protein ("Au-protein"), isolated from goldphilic Micrococcus luteus bacteria, catalyzes the oxidation of methane to methanol in the system also containing NADH, air, K(3)Fe(CN)(6) and Tris-HCl buffer . Presumably Au-protein helps bacteria to survive when usual sources of carbon and energy are scarce.

Oncogene, 2002 Mar 7, 21(11), 1649 - 57
Relations between clusters of oxidatively damaged nucleotides and active or open nucleosomes in the rat Nth 1 gene; Nomoto M et al.; The distribution of oxidative damage to bases such as 8-hydroxyguanine (8-OH-Gua), was determined at the nucleotide level of resolution using the ligation-mediated PCR technique . Administration of a renal carcinogen, ferric nitrilotriacetate (Fe-NTA), is known to induce oxidative stress and subsequent formation of 8-OH-Gua in the kidney . Whole genomic DNA was isolated from the rat kidney with or without Fe-NTA treatment and then digested with formamidopyrimidine-DNA glycosylase (Fpg) . As a target, we focused on the gene of a DNA repair enzyme for thymine glycol, Nth 1 . Cleaved signals were found in exon 1 and exon 3, but not exon 5 . Nucleosomes in these regions, enriched in damaged nucleotides, were highly accessible to micrococcal nuclease, especially in the kidney . Taking into account the function of the protein segment encoded by these regions, we discussed the molecular mechanism of the restricted formation of the damaged nucleotides.

Mol Cell Biol, 2002 Apr, 22(7), 2229 - 41
CENP-A, -B, and -C chromatin complex that contains the I-type alpha-satellite array constitutes the prekinetochore in HeLa cells; Ando S et al.; CENP-A is a component of centromeric chromatin and defines active centromere regions by forming centromere-specific nucleosomes . We have isolated centromeric chromatin containing the CENP-A nucleosome, CENP-B, and CENP-C from HeLa cells using anti-CENP-A and/or anti-CENP-C antibodies and shown that the CENP-A/B/C complex is predominantly formed on alpha-satellite DNA that contains the CENP-B box (alphaI-type array) . Mapping of hypersensitive sites for micrococcal nuclease (MNase) digestion indicated that CENP-A nucleosomes were phased on the alphaI-type array as a result of interactions between CENP-B and CENP-B boxes, implying a repetitive configuration for the CENP-B/CENP-A nucleosome complex . Molecular mass analysis by glycerol gradient sedimentation showed that MNase digestion released a CENP-A/B/C chromatin complex of three to four nucleosomes into the soluble fraction, suggesting that CENP-C is a component of the repetitive CENP-B/CENP-A nucleosome complex . Quantitative analysis by immunodepletion of CENP-A nucleosomes showed that most of the CENP-C and approximately half the CENP-B took part in formation of the CENP-A/B/C chromatin complex . A kinetic study of the solubilization of CENPs showed that MNase digestion first released the CENP-A/B/C chromatin complex into the soluble fraction, and later removed CENP-B and CENP-C from the complex . This result suggests that CENP-A nucleosomes form a complex with CENP-B and CENP-C through interaction with DNA . On the basis of these results, we propose that the CENP-A/B/C chromatin complex is selectively formed on the I-type alpha-satellite array and constitutes the prekinetochore in HeLa cells.

Mol Biochem Parasitol, 2002 Mar, 120(1), 23 - 31
Endoribonuclease activities of Trypanosoma brucei mitochondria; Salavati R et al.; RNA editing in kinetoplastids is a type of post-transcriptional processing that changes mitochondrial mRNA sequences by the addition or deletion of uridines . Multiple enzymatic activities, such as endoribonuclease and RNA ligase, are associated with this process and exist in a multienzyme complex . Endonuclease activities from Trypanosoma brucei mitochondrial extracts were fractionated by sequential ion exchange and gel filtration chromatography . The RNA editing specific endonuclease activity co-fractionated with in vitro editing while another endonuclease activity with a different substrate specificity, and the majority of mtRNase P activity fractionated away from the editing activity . The pH, salt, temperature, and Mg(2+) optima of all three endonucleases were determined . All three activities are sensitive to high temperature and protease digestion . In addition, treatment with micrococcal nuclease resulted in partial disruption of the editing complex and decreased pre-cleaved in vitro insertion editing activity, suggesting that both RNA(s) and protein(s) are necessary in the intact functional complex.

Pflugers Arch, 2001, 443 Suppl 1, S45 - 9 Epub 2001 Aug 04.
CFTR and lysozyme secretion in human airway epithelial cells; Duszyk M; Lysozyme is secreted in large quantities in human airways (10-20 mg/day), where it helps to defend against bacterial and fungal infection . Lysozyme expression is restricted to the serous cells of the submucosal glands, which also express high levels of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels . It is often assumed that mucus secretion in human airways is coupled to anion secretion through CFTR Cl(-) channels located in the apical membrane . Therefore, a defect in CFTR function could cause abnormal mucus secretion leading to persistent bacterial infection and inflammation of the airways . In this study we measured simultaneous secretion of lysozyme and Cl(-) from human airway epithelial serous cells . Secretion of lysozyme was measured by a turbidimetric assay that relies on the ability of lysozyme to disrupt the wall of the bacterium Micrococcus lysodeikticus, thus causing a fall in the optical density of the sample . Secretion of Cl(-) was measured as short-circuit current in a modified Ussing chamber . Activation of Cl(-) secretion by stimulation of cAMP- or Ca(2+)-dependent pathways caused comparable increases in lysozyme secretion . Similarly, blockers of Cl(-) secretion, such as diphenylamine-2-carboxylate (DPC), also reduced lysozyme secretion . However, while treatment of airway submucosal gland cells with antisense oligonucleotides directed against CFTR reduced Cl(-) secretion, it had no significant effect on the total amount of lysozyme secretion . These results suggest a role for functional CFTR in regulation of lysozyme secretion in human airways.

J Immunol, 2002 Feb 15, 168(4), 1542 - 6
Cutting edge: the toll pathway is required for resistance to gram-positive bacterial infections in Drosophila; Rutschmann S et al.; In Drosophila, the response against various microorganisms involves different recognition and signaling pathways, as well as distinct antimicrobial effectors . On the one hand, the immune deficiency pathway regulates the expression of antimicrobial peptides that are active against Gram-negative bacteria . On the other hand, the Toll pathway is involved in the defense against filamentous fungi and controls the expression of antifungal peptide genes . The gene coding for the only known peptide with high activity against Gram-positive bacteria, Defensin, is regulated by both pathways . So far, survival experiments to Gram-positive bacteria have been performed with Micrococcus luteus and have failed to reveal the involvement of one or the other pathway in host defense against such infections . In this study, we report that the Toll pathway, but not that of immune deficiency, is required for resistance to other Gram-positive bacteria and that this response does not involve Defensin.

J Biol Chem, 2002 Apr 12, 277(15), 12777 - 83 Epub 2002 Jan 30.
Transcription-coupled DNA repair is genomic context-dependent; Feng Z et al.; DNA damage is preferentially repaired in the transcribed strand of many active genes . Although the concept of DNA repair coupled with transcription has been widely accepted, its mechanisms remain elusive . We recently reported that in Chinese hamster ovary cells while ultraviolet light-induced cyclobutane pyrimidine dimers (CPDs) are preferentially repaired in the transcribed strand of dihydrofolate reductase gene, CPDs are efficiently repaired in both strands of adenine phosphoribosyltransferase (APRT) locus, in either a transcribed or nontranscribed APRT gene (1) . These results suggested that the transcription dependence of repair may depend on genomic context . To test this hypothesis, we constructed transfectant cell lines containing a single, actively transcribed APRT gene, integrated at different genomic sites . Mapping of CPD repair in the integrated APRT genes in three transfectant cell lines revealed two distinct repair patterns, either preferential repair of CPDs in the transcribed strand or very poor repair in both strands . Similar kinetics of micrococcal nuclease digestion were seen for all three transfectant APRT gene domains and endogenous APRT locus . Our results suggest that both the efficiency and strand-specificity of repair of an actively transcribed gene are profoundly affected by genomic context but do not reflect changes in first order nucleosomal structure.

Arch Microbiol, 2002 Feb, 177(2), 173 - 83 Epub 2001 Dec 04.
Enzymes of dimethylsulfone metabolism and the phylogenetic characterization of the facultative methylotrophs Arthrobacter sulfonivorans sp . nov., Arthrobacter methylotrophus sp . nov., and Hyphomicrobium sulfonivorans sp . nov; Borodina E et al.; Novel methylotrophic Arthrobacter and Hyphomicrobium species are described . Constitutive membrane-associated dimethylsulfone- and dimethylsulfoxide-reductases were found in Arthrobacter methylotrophus strain TGA and Hyphomicrobium sulfonivorans strain S1 . Enzyme activities increased during growth with dimethylsulfone or dimethylsulfoxide, respectively, and different ratios of activity with different growth substrates indicated that they are separate enzymes . SDS-PAGE showed some membrane-associated polypeptides to be enhanced during growth with dimethylsulfone (54 kDa in H . sulfonivorans, 21-24 kDa, 54 kDa and 80 kDa in A . methylotrophus) . Western blotting with anti-dimethylsulfoxide-reductase antibody showed cross-reaction with 54- and 21-kDa polypeptides in A . methylotrophus . All strains contained rhodanese and sulfur oxygenase after growth with dimethylsulfone . Sulfite was oxidized in the Arthrobacter species by APS reductase and sulfite dehydrogenase . H . sulfonivorans oxidized sulfite with APS reductase, which is unusual for an alpha-proteobacterium . The Arthrobacter species were distinguished from each other and from other Arthrobacter and Micrococcus species by 16S rRNA gene sequence analysis . The menaquinone and fatty acid profiles of the Arthrobacter species were similar . Their peptidoglycan structures were L-Lys- L-Ser- L-Thr- L-Ala for A . sulfonivorans and L-Lys- L-Ala(2-4) for A . methylotrophus . H . sulfonivorans exhibited gross morphology typical for Hyphomicrobium, but possessed helically twisted prosthecae . 16S rRNA gene sequence analysis showed it to be distinct from all the other Hyphomicrobium, Filomicrobium and Pedomicrobium species sequenced to date . Formal descriptions of the new species are given.

Plasmid, 2002 Jan, 47(1), 1 - 9
A 50-kb plasmid rich in mobile gene sequences isolated from a marine micrococcus; Zhong Z et al.; A 50,709-bp cryptic plasmid isolated from a marine Micrococcus has been sequenced and found to contain a number of putative mobile genetic elements . The coding regions for 11 putative transposases comprise approximately 17% of the total plasmid sequence . The majority of these transposases are located within a 13-kb cluster which includes a 1553-bp direct repeat consisting of a duplicated pair of transposase genes . The remaining putative ORFs showed similarity to a variety of proteins, the most notable being spider silk .

Mikrobiologiia, 2001 Nov-Dec, 70(6), 776 - 87
{Fine structure of mummified cells of microorganisms formed under exposure to a chemical analog of anabiosis autoinducer}; Suzina NE et al.; Under the influence of alkyl hydroxybenzene (C6-AHB) added to cell suspensions at a concentration of (1-5) x 10(-3) M, the cells of Saccharomyces cerevisiae, Micrococcus luteus, and Thioalkalivibrio versutus underwent dramatic changes in the ultrastructural organization of cell membranes, cytoplasm, and inclusions . In yeast suspension, the first changes were observed after 15 min in the structure of slit-like invaginations in the cytoplasmic membrane (CM): they were shortened and thickened . In the subsequent 30 to 60 min, CM ruptures were formed in the regions devoid of intramembrane protein particles and in the slit-like invaginations . After 24 h, complete disintegration of the intracellular membrane structures and conglomeration of the ribosomal part of the cytoplasm occurred . Similar changes were observed on the exposure of gram-positive and gram-negative bacteria to AHB . However, the cell wall in all the microorganisms studied was not destroyed, and in Micrococcus luteus it was even thickened . These mummified forms were preserved as morphologically intact but nonviable cells for more than three years of observations . By their ultrastructural characteristics, these mummified forms of microorganisms were similar to the fossilized microorganisms discovered by us in fibrous kerite . The concept of micromummies was formulated . AHB are supposed to play an important role in the process of fossilization of microorganisms in nature.

J Clin Microbiol, 2002 Jan, 40(1), 311 - 3
Catheter-related bacteremia due to Kocuria kristinae in a patient with ovarian cancer; Basaglia G et al.; We report on the first case of a catheter-related recurrent bacteremia caused by Kocuria kristinae, a gram-positive microorganism belonging to the family Micrococcaceae, in a 51-year-old woman with ovarian cancer . This unusual pathogen may cause opportunistic infections in patients with severe underlying diseases.

Biochemistry, 2002 Jan 8, 41(1), 179 - 84
Reconstitution of nucleosomes with histone macroH2A1.2; Changolkar LN et al.; MacroH2A histones have an unusual hybrid structure, consisting of an N-terminal domain that is approximately 65% identical to a full-length histone H2A and a large C-terminal nonhistone domain . To develop an in vitro approach for investigating the effects of macroH2A proteins on chromatin structure and function, we reconstituted nucleosomes with recombinant macroH2A1.2, substituting for conventional H2A . Recombinant macroH2A1.2 was able to efficiently replace both of the conventional H2As in reconstituted nucleosomes . The substitution of macroH2A1.2 for H2A did not appear to grossly perturb the basic structure of the nucleosome core, as assessed by sedimentation and by digestion with micrococcal nuclease or DNase I . However, two differences were observed . First, the region around the midpoint of the nucleosomal core DNA was more resistant to digestion by DNase I in nucleosome core particles reconstituted with macroH2A1.2 . Second, preparations of core particles reconstituted with macroH2A1.2 had a greater amount of material that sedimented more rapidly than mononucleosomes, suggesting that macroH2A1.2 may promote interactions between nucleosomes . Recombinant macroH2A proteins should be valuable tools for examining the effects of macroH2A on nucleosome and chromatin structure.

Int J Hyg Environ Health, 2001 Nov, 204(2-3), 123 - 6
Hand disinfection according to the European Standard EN 1500 (hygienic handrub): a study with gram-negative and gram-positive test organisms; Goroncy-Bermes P; It was the aim of this study to compare the efficacy of alcohol-based hand disinfectants according to European Standard EN 1500 (hygienic handrub), using the routine test organism Escherichia coli and, additionally, Micrococcus luteus as a surrogate for Gram-positive pathogens . One ethanol-based hand disinfectant (product A) and one propanol-based hand disinfectant (product B) were used in all experiments . Product B (propanol-based) was significantly more effective against both test organisms than product A (ethanol-based) in quantitative suspension tests but not in tests simulating practical conditions . In the experiments according to EN 1500 germ reduction rates obtained with the ethanol-containing formulation A were identical for E . coli and M . luteus . Product B was slightly, but not significantly more effective against M . luteus . To conclude, using E . coli as the test organism for evaluating the antibacterial efficacy of alcoholic hand disinfectants under practical conditions even appears to be sufficient to permit the drawing of conclusions for Gram-positive pathogens . However, more alcohol-based hand disinfectants should be tested in further studies to verify the results obtained.

Insect Biochem Mol Biol, 2001 Dec, 32(1), 75 - 84
Two novel insect defensins from larvae of the cupreous chafer, Anomala cuprea: purification, amino acid sequences and antibacterial activity; Yamauchi H; A humoral immune response in larvae of the coleopteran insect, Anomala cuprea has been examined for exploring the molecular basis of host-pathogen interactions . The antibacterial activity against the Gram-positive strain, Micrococcus luteus was detected at a low level in absence of injection . The activity increased strikingly in the hemolymph of the larvae challenged with Escherichia coli, showing the fluctuating profile through a time course, which consists of the static induction phase, the production phase rising to a maximum level, and the reduction phase extending over a long duration . Two peptides were purified and characterized by reverse-phase HPLC, Edman degradation and mass spectrometry . They were isoforms, composed of similar sequences with two amino acid substitutions in 43 residues, and novel members of the insect defensins, cysteine-rich antibacterial peptides . Anomala defensins A and B showed potent activity against Gram-positive bacteria, with slight differences in activity against a few strains of tested bacteria . Anomala defensin B was active at high concentration of 40 microM against the Gram-negative strain, Xenorhabdus japonicus, a pathogen toward the host, A . cuprea larvae.

Mol Biol Cell, 2001 Nov, 12(11), 3365 - 74
Glucocorticoid receptor activation of the I kappa B alpha promoter within chromatin; Deroo BJ et al.; The glucocorticoid receptor (GR) is a ligand-activated transcription factor that induces expression of many genes . The GR has been useful for understanding how chromatin structure regulates steroid-induced transcription in model systems . However, the effect of glucocorticoids on chromatin structure has been examined on few endogenous mammalian promoters . We investigated the effect of glucocorticoids on the in vivo chromatin structure of the glucocorticoid-responsive I kappa B alpha gene promoter, the inhibitor of the ubiquitous transcription factor, nuclear factor kappa B (NF kappa B) . Glucocorticoids inhibit NF kappa B activity in some tissues by elevating the levels of I kappa B alpha . We found that glucocorticoids activated the I kappa B alpha promoter in human T47D/A1-2 cells containing the GR . We then investigated the chromatin structure of the I kappa B alpha promoter in the absence and presence of glucocorticoids with the use of micrococcal nuclease, restriction enzyme, and deoxyribonuclease (DNaseI) analyses . In untreated cells, the promoter assembles into regularly positioned nucleosomes, and glucocorticoid treatment did not alter nucleosomal position . Restriction enzyme accessiblity studies indicated that the I kappa B alpha promoter is assembled as phased nucleosomes that adopt an "open" chromatin architecture in the absence of hormone . However, glucocorticoids may be required for transcription factor binding, because DNaseI footprinting studies suggested that regulatory factors bind to the promoter upon glucocorticoid treatment.

Dtsch Med Wochenschr, 2001 Nov 2, 126(44), 1229 - 32
{Glomerulonephritis secondary to chronic infection of a ventriculoatrial shunt}; Ozcan F et al.; HISTORY AND ADMISSION FINDINGS: A 39-year-old man was referred for assessment of a nephrotic syndrome . He reported deteriorating health with bouts of fever and microhaematuria and proteinuria in the past year . At the age of 24 years a ventriculoatrial shunt had been inserted for an internal hydrocephalus . At another hospital he was given steroids for a nephrotic syndrome suspected of being associated with membranoproliferative glomerulitis, but the disease progressed . On admission he had severe generalised oedema with a temperature of 38,5;C . His general condition was poor . He had no neck stiffness . INVESTIGATIONS: Parameters of inflammation were raised . Serum creatinine and creatinine clearance were normal . Levels of complements C3 and C4 were reduced . The proteinuria was 9g/24h . Renal biopsy revealed type 1 membranoproliferative glomerulonephritis . Micrococcus roseus/varians was demonstrated several times by aerobic blood cultures . TREATMENT AND COURSE: The findings suggested chronically infected ventriculoatrial shunt as cause of the glomerulonephritis . The shunt was, therefore, removed . The same pathogens were grown from it on aerobic culture medium . Six months after removal and replacement of the shunt and treatment of the infection the proteinuria had fallen to 0.45 mg/h; serum creatinine was 1.0 mg/dl . CONCLUSION: When membranoproliferative glomerulonephritis has been demonstrated, secondary forms should be considered in the differential diagnosis . In most cases specific treatment can prevent progression of the renal disease.

Chem Phys Lipids, 2001 Nov, 113(1-2), 23 - 7
Total synthesis of the novel bacterial fatty acid 16-methyl-8(Z)-heptadecenoic acid; Carballeira NM et al.; The recently discovered bacterial fatty acid 16-methyl-8(Z)-heptadecenoic acid was synthesized for the first time in four steps (22% overall yield) starting from commercially available 8-methylnonanoic acid . The synthetic approach provided enough material to corroborate the structure and stereochemistry of the acid, which was recently identified in a Micrococcus bacterium from Lake Pomorie in Bulgaria . Reference equivalent-chain length values in nonpolar capillary gas chromatography for methyl 16-methyl-8(Z)-heptadecenoate and methyl 16-methyl-8(E)-heptadecenoate are also reported . This information will be helpful in subsequent characterizations of these fatty acids, as well as in the total identification of the fatty acid profile of bacteria producing these compounds.

J Pharm Biomed Anal, 2002 Jan 1, 27(1-2), 91 - 6
Microbiological assay for determination of ofloxacin injection; Ev Lda S et al.; A simple, sensitive and specific agar diffusion bioassay for the antibacterial ofloxacin was developed using a strain of Micrococcus luteus ATCC 9341 as the test organism, ofloxacin at concentration ranging 12-27 microg ml(-1) could be measured in injection . A prospective validation of the method showed that method was linear (r=0.9994) and precise (CV=1.14) . UV spectrophotometric and high performance liquid chromatographic techniques were chosen as a comparison methods for the determination of ofloxacin . The results obtained by three methods were statistically analyzed by analysis of variance (ANOVA) and the results obtained indicate that there is no significant difference among these methods.

Planta, 2001 Sep, 213(5), 770 - 80
Identification of a novel low-temperature-response element in the promoter of the barley (Hordeum vulgare L) gene blt101.1; Brown AP et al.; Two winter barley (Hordeum vulgare L . cv . Igri) genomic clones, lambda gblt101.1 and lambda gblt101.2, encoding the blt101 gene family, were isolated from a genomic library . Deletion analysis of the blt101.1 promoter, using transient beta-glucuronidase (GUS) reporter expression assays, indicated that it contains at least three regulatory regions . A 107-bp region between nucleotides -168 and -275 with respect to the translation initiation codon, confers high-level GUS reporter expression at low temperature and contains a sequence (designated CR1) that is highly conserved in equivalent positions within the promoters of both members of the blt101 gene family . A 10-bp motif contained within CR1 binds proteins present in nuclear extracts from both control and low-temperature-treated barley tissue . Loss-of-function experiments, using transient-expression analysis, confirmed that this motif acts as a previously unreported low-temperature-responsive element . Nuclease sensitivity analysis of intact chromatin indicated that the blt101.1 promoter becomes more susceptible to DNase and micrococcal nuclease at low temperature, consistent with chromatin reorganisation upon transcriptional induction . It is proposed that both the 10-bp motif and chromatin reorganisation are involved in the regulation of blt101.1 at low temperature . This is the first detailed analysis of a low-temperature-specific plant promoter and identifies a novel low-temperature-response element.

Protein Expr Purif, 2001 Nov, 23(2), 328 - 37
Expression and purification of Manduca sexta prophenoloxidase-activating proteinase precursor (proPAP) from baculovirus-infected insect cells; Wang Y et al.; Analogous to human thrombin, prophenoloxidase-activating proteinase (PAP) is a terminal enzyme of a serine proteinase cascade in the tobacco hornworm Manduca sexta . In order to purify and study the activating enzyme for PAP from this insect, we produced the zymogen of PAP (proPAP) in a bacterial expression system . The affinity-purified protein was then used as an antigen to generate a specific rabbit antiserum . Immunoblot analysis indicated that the proPAP was present at a low level in Manduca larval hemolymph, but was induced by six- to eightfold in larvae that had been injected with Escherichia coli or Micrococcus lysodeikticus . To produce the native proenzyme for functional analyses, we constructed a recombinant baculovirus to infect Spodoptera frugiperda Sf21 cells . ProPAP was secreted into the medium at a low concentration of approximately 0.37 mg/liter under the optimal conditions . We then developed a simple, efficient scheme to enrich and purify this protein, which involves two lectin affinity and one HPLC ion-exchange chromatographic steps . Immunoblot analysis following SDS-polyacrylamide gel electrophoresis indicated that the recombinant proPAP is nearly identical in mobility to the zymogen from Manduca hemolymph . After the purified proPAP was added to the larval hemolymph, it was readily activated by an unknown proteinase in the presence of M . lysodeikticus .

Inorg Chem, 1996 Apr 10, 35(8), 2373 - 2377
ESR Studies of A(1u) and A(2u) Oxoiron(IV) Porphyrin pi-Cation Radical Complexes . Spin Coupling between Ferryl Iron and A(1u)/A(2u) Orbitals; Fujii H et al.; This study shows the ESR spectra of oxoiron(IV) porphyrin pi-cation radicals of 1-8 in dichloromethane-methanol (5:1) mixture . We reported in a previous paper that oxoiron(IV) porphyrin pi-cation radicals of 1-4 are in an a(1u) radical state while those of 5-8 are in an a(2u) radical . The ESR spectra (g( perpendicular)(eff) approximately 3.1 and g( parallel)(eff) approximately 2.0) for the a(1u) radical complexes, 1-4, appear quite different from those reported previously for the oxoiron(IV) porphyrin pi-cation radical of 5 (g(y) = 4.5, g(x) = 3.6, and g(z) = 1.99) . The unique ESR spectra of the a(1u) radical complexes rather resemble those of compound I from Micrococcus lysodeikticus catalase (CAT) and ascorbate peroxidase (ASP) . This is the first examples to mimic the ESR spectra of compound I in the enzymes . From spectral analysis based on a spin Hamiltonian containing an exchange interaction, the ESR spectra of 1-4 can be explained as a moderate ferromagnetic state (J/D approximately 0.3) between ferryl S = 1 and the porphyrin pi-cation radical S' = (1)/(2) . The magnitudes of zero-field splitting (D) for ferryl iron and isotropic J value, estimated from the temperature-dependence of the half-saturation power of the ESR signals, are approximately 28 and approximately +8 cm(-1), respectively . A change in the electronegativity of the beta-pyrrole substituent hardly changes the ESR spectral feature while that of the meso-substituent slightly does owing to the change in the E/D value . On the basis of the present ESR results, we propose the a(1u) radical state for compound I of CAT and ASP.

Proc Natl Acad Sci U S A, 1992 Jun 15, 89(12), 5256 - 60
Ribosomal gene clusters are uniquely proportioned between open and closed chromatin structures in both tomato leaf cells and exponentially growing suspension cultures; Conconi A et al.; The accessibility of regulatory molecules to specific DNA sequences and chromatin regions in the nucleus is crucial to gene expression . In this study, we examined the chromatin structure in tomato leaf cells and in exponentially growing tomato cell suspension cultures . The structure of ribosomal chromatin was investigated by micrococcal nuclease and psoralen photocrosslinking . We showed that ribosomal genes in tomato are folded into two distinct types of chromatin: an open chromatin conformation and a closed nucleosomecontaining chromatin . In contrast to previous findings in Friend cells, where half of the ribosomal genes were found to be complexed within an inactive chromatin structure, we demonstrated that the canonical nucleosome-containing chromatin is present in the majority (approximately 80%) of the tomato rRNA-encoding DNA clusters . The minor open chromatin population (approximately 20% of the ribosomal genes) could be detected only after analysis following psoralen crosslinking . The relative amounts of the two ribosomal chromatin structures are similar in stationary and exponentially growing cells . This suggests that the proportions of open and closed chromatin structures present in either stationary or exponentially growing tomato cells are not dependent on the transcriptional process.

Mol Cell Biol, 2001 Nov, 21(22), 7682 - 95
Nucleosomes are translationally positioned on the active allele and rotationally positioned on the inactive allele of the HPRT promoter; Chen C et al.; Differential chromatin structure is one of the hallmarks distinguishing active and inactive genes . For the X-linked human hypoxanthine phosphoribosyltransferase gene (HPRT), this difference in chromatin structure is evident in the differential general DNase I sensitivity and hypersensitivity of the promoter regions on active versus inactive X chromosomes . Here we characterize the nucleosomal organization responsible for the differential chromatin structure of the active and inactive HPRT promoters . The micrococcal nuclease digestion pattern of chromatin from the active allele in permeabilized cells reveals an ordered array of translationally positioned nucleosomes in the promoter region except over a 350-bp region that is either nucleosome free or contains structurally altered nucleosomes . This 350-bp region includes the entire minimal promoter and all of the multiple transcription initiation sites of the HPRT gene . It also encompasses all of the transcription factor binding sites identified by either dimethyl sulfate or DNase I in vivo footprinting of the active allele . In contrast, analysis of the inactive HPRT promoter reveals no hypersensitivity to either DNase I or a micrococcal nuclease and no translational positioning of nucleosomes . Although nucleosomes on the inactive promoter are not translationally positioned, high-resolution DNase I cleavage analysis of permeabilized cells indicates that nucleosomes are rotationally positioned over a region of at least 210 bp on the inactive promoter, which coincides with the 350-bp nuclease-hypersensitive region on the active allele, including the entire minimal promoter . This rotational positioning of nucleosomes is not observed on the active promoter . These results suggest a model in which the silencing of the HPRT promoter during X chromosome inactivation involves remodeling a transcriptionally competent, translationally positioned nucleosomal array into a transcriptionally repressed architecture consisting of rotationally but not translationally positioned nucleosomal arrays.

Exp Cell Res, 2001 Oct 15, 270(1), 96 - 101
Nuclear assembly of demembranated Xenopus sperm in plant cell-free extracts from Nicotiana ovules; Lu P et al.; The cell-free preparation derived from Nicotiana tabaccum ovules induced chromatin decondensation and pronuclear formation from demembranated Xenopus laevis sperm nuclei . Fluorescent microscope and phase-contrast microscope visualization of assembly intermediates indicated that 95.6% of X . leavis sperm changed their tadpole-like shape to circular shape or elliptical shape after over 1.5 h of incubation . Transmission electron microscope visualization showed that nuclear membrane was assembled around the periphery of the dispersed chromatin . Nuclear envelope of most reassembled nuclei was composed of a double membrane inlaid with a little single membrane . Nucleosome assembly was verified by means of micrococcal nuclease digestion . After 2 to 5 h of incubation, digestion of the product of nuclear assembly with micrococcal nuclease produced at least six nucleosome fragments of about 250 bp each .

Zhongguo Zhong Yao Za Zhi, 1998 Apr, 23(4), 238 - 40, inside back cover
{Effects of the glucoprotein component of musk on functions of rat polymorphonuclear leukocytes activated by LTB4 in vitro}; Wang W et al.; To investigate the effects of musk-1, a glucoprotein component isolated from the water extract of musk, on some functions of rat polymorphonuclear leukocytes activated by LTB4, an in vitro incubation system with rat polymorphonuclear leukocytes was used . The superoxide anion production was determined by cytochrome C reduction, and the beta-glucuronidase and lysozyme release was quantitated by enzyme reactions in which phenolphthaleinglucuronic acid and micrococcus lysodeikticus were used as the substrates . In comparison with the control, musk-1 at final concentrations of 1 microgram/ml-100 micrograms/ml can increase the superoxide anion production by 28.7%-202.1% and decrease the beta-glucuronidase and lysozyme release by 3%-46% and 6%-32% respectively in rat polymorphonuclear leukocytes . It is concluded that musk-1 can significantly affect the functions of rat polymorphonuclear leukocytes activated by LTB4 . One of the mechanisms of this anti-inflammatory action of musk may consist in the inhibition of lysosomal enzyme release.

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1653 - 61
Salana multivorans gen . nov., sp . nov., a novel actinobacterium isolated from an anaerobic bioreactor and capable of selenate reduction; von Wintzingerode F et al.; Three facultatively anaerobic, Gram-positive bacteria, strains Se-3111T, Se-13111 and Se-1311A, were isolated from an anaerobic, dechlorinating bioreactor culture enriched from sediment of the River Saale in Germany . All strains were isolated from the dechlorinating mixed culture through their ability to reduce selenate anaerobically to elemental selenium . All three strains shared identical 16S rDNA sequences and phylogenetic analysis revealed that strain Se-3111T forms a novel taxon within the suborder Micrococcineae of the class Actinobacteria, related most closely to Beutenbergia cavernae . On the basis of genotypic, chemotaxonomic and physiological characteristics, it is proposed that the novel strains Se-3111T, Se-13111 and Se-1311A be classified in a new genus as Salana multivorans gen . nov., sp . nov . The type strain of the novel species is Se-3111T (= DSM 13521T = NRRL B-24118T).

J Immunol, 2001 Oct 15, 167(8), 4494 - 503
Chromatin remodeling, measured by a novel real-time polymerase chain reaction assay, across the proximal promoter region of the IL-2 gene; Rao S et al.; The structure of chromatin and its remodeling following activation are important aspects of the control of inducible gene transcription . The IL-2 gene is induced in a cell specific-manner in T cells following an antigenic stimulus . We show, using a novel real-time PCR assay, that significant chromatin remodeling of the IL-2 proximal promoter region occurred upon stimulation of both the murine EL-4 T cell line and primary CD4(+) T cells . Chromatin remodeling appears to be limited to the first 300 bp of the proximal promoter region as measured by micrococcal nuclease and restriction enzyme accessibility . Time course studies indicated that chromatin remodeling was observed at 1.5 h postinduction and was maintained for up to 16 h . The remodeling is reversible upon removal of the stimulus . The region immediately upstream from the transcription start site, however, remains accessible for up to 16 h . Upon restimulation, remodeling occurs much more rapidly, consistent with a more rapid rise in IL-2 mRNA levels . Using a number of pharmacological inhibitors we show that remodeling is dependent on the presence of specific transcription factors, but not on the modification of histones . The development of this novel chromatin accessibility assay based on real-time PCR has allowed rapid, sensitive, and quantitative measurements on the IL-2 gene following cellular activation in both T cell lines and primary cells.

Biosci Biotechnol Biochem, 2001 Jul, 65(7), 1680 - 3
Purification and characterization of an esterase from Micrococcus sp . YGJ1 hydrolyzing phthalate esters; Akita K et al.; An esterase hydrolyzing phthalate esters has been purified from Micrococcus sp . YGJ1 . The enzyme, a monomeric protein (Mr = 56 kDa) with a pI of 4.0, hydrolyzes various aliphatic and aromatic carboxylesters . The medium chain (C3-C4) esters are the most preferred substrates . The enzyme is inhibited by HgCl2 and p-chloromercuribenzoate but not by phenylmethylsulfonyl fluoride.

Folia Microbiol (Praha), 2001, 46(1), 17 - 20
Steroid biotransformation by different strains of Micrococcus sp; Dogra N et al.; A strain of Micrococcus sp . was isolated for its capability of side chain degradation of cholesterol . This strain was characterized and identified as Micrococcus roseus . It was found to be the best strain for the production of androsta-1,4-diene-3,17-dione and androst-4-ene-3,17-dione compared with other Micrococcus strains.

Acta Pharmacol Sin, 2000 Aug, 21(8), 765 - 8
Immunosuppressive effects of rubidatum in mice; Yang SL et al.; AIM: To study the effect of rubidatum (Rub) on immune function in normal mice . METHODS: Serum lysozyme concentration (SLC) was measured using micrococcus lysodiekticus as a substrate . Delayed type hypersensitivity (DTH) was determined by measuring the thickness of the right hind footpad 24 h after the injection of 1 x 10(8) washed sRBC (50 microL 10% sRBC) . Serum hemolysin concentration was determined by OD measuring at A540 after the serum was treated with 2-mercaptoethanol . Phagocytic function of peripheral leukocyte (Leu) were determined by the incorporated radioactivity of {3H}TdR . The hemolytic activity of plaque forming cell (PFC) was determined by measuring the lymphocytemediated hemolysis of sheep red blood cell in vitro . T- and B-lymphocyte transformation (TLT and BLT) were induced by phytohemagglutinin (PHA) and lipopolysacharide (LPS) respectively and measured by the incorporation of {3H}TdR . RESULTS: Rub 125, 500, 2000 mg.kg-1.d-1 p.o . to BALB/c (or NIH) mice decreased the SLC; inhibited the phagocytosing functions of peripheral leukocytes; diminished the hemolytic activity of PFC; decreased the HC50; inhibited the DTH reaction; and showed inhibitory effects on TLT and BLT . CONCLUSION: Rub has immunosuppressive effects on immune system in mice by affecting M phi, T, and B lymphocyte, which suggests that Rub has inhibitory effects on both nonspecific and specific immune function.

J Environ Biol, 2001 Apr, 22(2), 119 - 28
Bioconversion of 1,3 dinitrobenzene by Micrococcus colpogenes strain MCM B410: quantitation and characterization of the intermediates; Dey S; Micrococcus colpogenes MCM B 410, indigenous to soil, collected from nitro aromatic contaminated site, could transform 1,3 dinitrobenzene (m-DNB) initially to m-nitroaniline, m-nitrophenol, m-aminophenol and resorcinol at 30 degrees C under shake culture condition . Carbon mineralization studies with unlabelled and radio labelled 1,3 (U14 C) dinitrobenzene subtrates indicated that the above metabolities appeared within 4 days . After 7 days incubation a significant traction of the source compound was degraded to C Q through aliphatic acids . Presence of nitro aryl reductase, aryl monooxygenase and resorcinol 1,3 di oxygenase was also noted in the sonicated cell mass.

Biochem Cell Biol, 2001, 79(3), 349 - 63
Linker DNA destabilizes condensed chromatin; Green GR et al.; The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei . Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance . Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells . Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei . Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei . Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature . We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA . The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.

Mol Pharmacol, 2001 Aug, 60(2), 394 - 402
Histone H3 phosphorylation is coupled to poly-(ADP-ribosylation) during reactive oxygen species-induced cell death in renal proximal tubular epithelial cells; Tikoo K et al.; Although the cellular response to chemical-induced stress is relatively well characterized, particularly the response to DNA damage, factors that govern the outcome of the stress response (cell survival or cell death) are less clearly defined . In this context, the mitogen-activated protein kinase (MAPK) family responds to a variety of physical and chemical stresses . The activation of MAPKs, especially the extracellular-regulated protein kinase subfamily, seems to play a causal role in death of renal proximal tubular epithelial cells (LLC-PK1) induced by reactive oxygen species (ROS) . In this study, we show that extracellular signal receptor-activated kinase (ERK) activation may be coupled with LLC-PK1 cell death via changes in chromatin structure, which is mediated by increases in the phosphorylation of histone H3 (a post-translational modification required for both chromosome condensation and segregation during mitosis) and premature chromatin/chromosomal condensation, leading to cell death . In support of this view, 2,3,5-tris-(glutathione-S-yl)hydroquinone (TGHQ)-induced phosphorylation of histone H3 is accompanied by increases in chromatin condensation, as observed with the use of 4,6-diamidino-2-phenylindole-fluorescent staining, and by decreases in the sensitivity of chromatin to digestion by micrococcal nuclease . Changes in chromatin structure precede cell death . TGHQ-induced histone H3 phosphorylation and chromatin condensation are inhibited by PD098059, which selectively inhibits MAPK kinase, an upstream regulator of ERKs . Moreover, histone phosphorylation is modulated by poly(ADP-)ribosylation . Thus, the inhibition of poly(ADP-ribose)polymerase with 3-aminobenzamide prevents histone H3 phosphorylation and increases cell survival, suggesting that ADP-ribosylation and histone H3 phosphorylation are coupled in this model of ROS-induced DNA damage and cell death . The coupling of histone phosphorylation with ribosylation has not been previously demonstrated.

Nucleic Acids Res, 2001 Jul 1, 29(13), E64 - 4
The mapping of nucleosomes and regulatory protein binding sites at the Saccharomyces cerevisiae MFA2 gene: a high resolution approach; Teng Y et al.; We have developed an end-labelling approach to map the positions of nucleosomes and protein binding sites at nucleotide resolution by footprinting micrococcal nuclease (MNase)-sensitive sites . Using this approach we determined that the MFA2 gene and its upstream control regions have four positioned nucleosomes when transcription is repressed in mating type alpha cells and that the nucleosomes lose their positioning when the gene became transcriptionally active in mating type a cells . We also detected MNase-hypersensitive sites in the alpha2 operator region of MFA2 in alpha cells but not in a cells . These probably result from the change in the local DNA conformation due to protein(s) binding in this region that governs MFA2 transcription.

Biochemistry, 2001 Jul 3, 40(26), 7860 - 7
Differential chromatin association and nucleosome binding of the maize HMGA, HMGB, and SSRP1 proteins; Lichota J et al.; In plants, chromosomal high mobility group (HMG) proteins have been identified in the HMGA family, containing A/T-hook DNA binding motifs, and in the HMGB family, containing an HMG-box DNA binding domain, that are considered architectural factors in chromatin . We have characterized the association of the HMGA protein, five different HMGB proteins, and the structure-specific recognition protein 1 (SSRP1) with maize chromatin by extraction experiments using NaCl, ethidium bromide, spermine, and distamycin A . The difference in the release of the proteins from chromatin by these reagents indicates that they are differentially associated with chromatin . This was confirmed by treatment of chromatin with micrococcal nuclease, demonstrating that the HMGA, HMGB2/3, and SSRP1 proteins are enriched in the highly nuclease-sensitive fraction of chromatin, which is likely to be transcriptionally competent . As examined by electrophoretic mobility shift analyses, the HMGA protein and the proteins containing an HMG domain (HMGB proteins and SSRP1) bind specifically to purified maize mononucleosomes that contain a histone octamer and approximately 165 bp of DNA . The mode of interaction with the nucleosomes differs for HMGA and HMGB proteins . In the case of the HMGB1 protein, the full-length protein is required for specific nucleosome binding, as the individual HMG-box DNA binding domain (which is sufficient for DNA interactions) interacts nonspecifically with the nucleosomes . Collectively, these findings indicate that HMGA, the various HMGB proteins, and SSPR1 are differentially associated with plant chromatin and may act as architectural factors in different nucleoprotein structures.

Chromosome Res, 2001, 9(4), 309 - 23
Transition between two forms of heterochromatin at plant subtelomeres; Sykorova E et al.; The manner of packing of the terminal DNA loci into nucleosomes and higher order structures may strongly influence their functional interactions . Besides the structural flexibility of telomeric DNA sequences, conserved features of their chromatin including short nucleosome phasing (157 bp) and nucleosome sliding have been described previously . To gain a complementary knowledge of subtelomeres, we have analysed the chromatin structure of two subtelomeric tandem repeats from the plant Silene latifolia: X43.1 and 15Ssp . X43.1 shows two distinct nucleosome periodicities--157 and 188 bp . Preferred positions of its two nucleosomes have been mapped at both low and high resolution and the experimental results correspond to computer-predicted positions . 15Ssp is a newly-discovered sequence showing a telomere-associated position by PCR and a subtelomeric location by pulsed-field gel electrophoresis and fluorescence in situ hybridisation . Its 159 bp sequence unit shows a tandem arrangement and the presence of micrococcal nuclease-hypersensitive sites when either naked DNA or chromatin is digested . Use of a chemical nuclease results in a regular nucleosome ladder of 157 bp periodicity . Moreover, 15Ssp mononucleosomes show instability and absence of specific positioning, features typical for telomeric chromatin.

Mol Cell Biol Res Commun, 2000 Oct, 4(4), 206 - 11
An open chromatin structure in a liver-specific enhancer that confers high level expression to human apolipoprotein b transgenes in mice; Levy-Wilson B et al.; A number of DNaseI-hypersensitive (DH) sites have been mapped within a regulatory region situated upstream of the human apolipoprotein B (apoB) promoter (-5262 to -899) that is required for high level expression of human apoB transgenes in the livers of mice . These DH sites were observed in nuclei from transcriptionally active liver-derived HepG2 cells, but were absent from transcriptionally inactive HeLa cell nuclei . Several nuclear protein binding sites were detected in the DNaseI-hypersensitive region by DNaseI footprinting with HepG2 nuclear extracts, representing putative binding sites for the liver-specific activators . The locations of binding sites for these transcription factors were revealed via computer analysis of the DNA sequence of this region against a transcription factor database . Many micrococcal nuclease hypersensitive (MH) sites were also observed in nuclei from HepG2 cells but not in HeLa cell nuclei, implying that in hepatic cells, nucleosomes are either absent or have been displaced from this region by the liver-specific transcriptional activators, as inferred by the correspondence between the DH sites, the MH sites and the footprints .

Curr Biol, 2001 Apr 3, 11(7), 463 - 73
Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing; Sharp JA et al.; BACKGROUND: Position-dependent gene silencing in yeast involves many factors, including the four HIR genes and nucleosome assembly proteins Asf1p and chromatin assembly factor I (CAF-I, encoded by the CAC1-3 genes) . Both cac Delta asfl Delta and cac Delta hir Delta double mutants display synergistic reductions in heterochromatic gene silencing . However, the relationship between the contributions of HIR genes and ASF1 to silencing has not previously been explored . RESULTS: Our biochemical and genetic studies of yeast Asf1p revealed links to Hir protein function . In vitro, an active histone deposition complex was formed from recombinant yeast Asf1p and histones H3 and H4 that lack a newly synthesized acetylation pattern . This Asf1p/H3/H4 complex generated micrococcal nuclease--resistant DNA in the absence of DNA replication and stimulated nucleosome assembly activity by recombinant yeast CAF-I during DNA synthesis . Also, Asf1p bound to the Hir1p and Hir2p proteins in vitro and in cell extracts . In vivo, the HIR1 and ASF1 genes contributed to silencing the heterochromatic HML locus via the same genetic pathway . Deletion of either HIR1 or ASF1 eliminated telomeric gene silencing in combination with pol30--8, encoding an altered form of the DNA polymerase processivity factor PCNA that prevents CAF-I from contributing to silencing . Conversely, other pol30 alleles prevented Asf1/Hir proteins from contributing to silencing . CONCLUSIONS: Yeast CAF-I and Asf1p cooperate to form nucleosomes in vitro . In vivo, Asf1p and Hir proteins physically interact and together promote heterochromatic gene silencing in a manner requiring PCNA . This Asf1/Hir silencing pathway functionally overlaps with CAF-I activity.

Genetics, 2001 Jun, 158(2), 597 - 611
Yeast spt6-140 mutation, affecting chromatin and transcription, preferentially increases recombination in which Rad51p-mediated strand exchange is dispensable; Malagon F et al.; We have shown that the spt6-140 and spt4-3 mutations, affecting chromatin structure and transcription, stimulate recombination between inverted repeats by a RAD52-dependent mechanism that is very efficient in the absence of RAD51, RAD54, RAD55, and RAD57 . Such a mechanism of recombination is RAD1-RAD59-dependent and yields gene conversions highly associated with the inversion of the repeat . The spt6-140 mutation alters transcription and chromatin in our inverted repeats, as determined by Northern and micrococcal nuclease sensitivity analyses, respectively . Hyper-recombination levels are diminished in the absence of transcription . We believe that the chromatin alteration, together with transcription impairment caused by spt6-140, increases the incidence of spontaneous recombination regardless of whether or not it is mediated by Rad51p-dependent strand exchange . Our results suggest that spt6, as well as spt4, primarily stimulates a mechanism of break-induced replication . We discuss the possibility that the chromatin alteration caused by spt6-140 facilitates a Rad52p-mediated one-ended strand invasion event, possibly inefficient in wild-type chromatin . Our results are consistent with the idea that the major mechanism leading to inversions might not be crossing over but break-induced replication followed by single-strand annealing.

Biochim Biophys Acta, 1974 Apr 27, 349(1), 13 - 22
Isolation of a short, cytosine-rich repeating unit from the DNA of Escherichia coli; Lin HJ; Hybridization of heterologous nucleic acids has provided the means for isolating a repeating sequence which is located next to template regions of DNA . Separated single strands of 32P-labelled DNA from Escherichia coli were to a limited extent able to anneal with DNA of Micrococcus lysodeikticus immobilized on nitrocellulose membrane filters . The resulting hybrid was resistant to enzymes specific for unpaired strands, nuclease S1 (Aspergillus oryzae) and exonuclease I (E . coli) . The E . coli DNA so hybridized was isolated and characterized . It contained all four bases with cytosine predominating; strand length was about 50-60 nucleotides . Since these units occupied about 1-2% of the length of the E . coli chromosome, they would have to be repeated about 2000 times in a single cell . Formation of the unusual hybrid was not diminished by prior saturation of the E . coli DNA with homologous 3H-labelled RNA . In fact both RNA and additional increments of DNA were detected on the filters approximately in a 1:1 ratio, showing that some of the repeating sequences were physically continuous with transcribed regions of DNA.

Bioresour Technol, 2001 Aug, 79(1), 15 - 22
Characterization of de-emulsification capabilities of a Micrococcus species; Das M; Effect of post harvest washing as well as cell concentration on de-emulsification characteristics of an isolated Micrococcus species has been tested with Tween 60 Span 60 stabilised oil in water (o/w) and L-92 pluronic surfactant stabilised water in oil (w/o) model emulsions (kerosene water) . The cells used were 140 h old and grown under submerged conditions at 37 degrees C in a medium containing n-tetradecane (4% v/v) as the carbon source . The harvested bacterial cells when in an unwashed condition (at a cell concentration of 2 mg/ml of emulsion) were found to de-emulsify the o/w system at a much faster rate than the w/o system exhibiting half-life values for the respective system as 10.2 and 127.7 h . Post harvest washing of the cells with any lipid solubilising solvent (n-pentane, n-hexane, kerosene, chloroform-methanol-water (CMW)) yielded a decrease in their de-emulsification power for w/o emulsion . But the decay of o/w emulsion became faster with n-pentane- and kerosene-washed cells as evident from their corresponding half-life values of 3.3 and 4.6 h . Compared to the w/o system, an increase in the concentration of kerosene-washed cell had a direct effect on de-emulsification for the o/w system . For cell concentrations of 2, 3 and 4 mg/ml of the emulsion, the half-life values for the w/o system were 364.8, 442.0 and 454.9 h, respectively . For 2 and 4 mg/ml cell contents, the half-life values for the o/w system were 4.6 and 1.0 h . The decay of both the emulsions was very slow or even incomplete for cell concentrations less than 2 mg/ml . De-emulsifying capacity of the n-tetradecane grown Micrococcus species towards o/w model emulsion improved considerably after washing the cells with n-pentane and kerosene, and use of kerosene-washed cells (4 mg/ml) reduced the half-life to 1 h.

J Virol, 2001 Jul, 75(13), 6235 - 41
Variant chromatin structure of the oriP region of Epstein-Barr virus and regulation of EBER1 expression by upstream sequences and oriP; Wensing B et al.; Most of the Epstein-Barr virus genome in latently infected cells is in a standard nucleosomal structure, but the region encompassing oriP and the Epstein-Barr virus-encoded small RNA (EBER) genes shows a distinctive pattern when digested with micrococcal nuclease . This pattern corresponds to a previously mapped nuclear matrix attachment region . Although the EBER genes are adjacent to oriP, there is only a two- to fourfold effect of oriP on EBER expression . However, sequences containing a consensus ATF site upstream of EBER1 are important for EBER1 expression.

Phytochemistry, 2001 May, 57(2), 273 - 8
Benzopyran derivatives from Mallotus apelta; An TY et al.; From the leaves of Mallotus apelta, seven benzopyran compounds were obtained and their structures were determined using spectroscopic methods . One showed moderate antibiotic activity against Micrococcus lutens.

Curr Microbiol, 2001 Jul, 43(1), 69 - 73
Biodegradation of carbaryl by a Micrococcus species; Doddamani HP et al.; A bacterium capable of utilizing carbaryl as sole source of carbon was isolated from garden soil and identified as a Micrococcus species . The organism also utilized carbofuran, naphthalene, 1-naphthol, and several other aromatic compounds as growth substrates . The organism degraded carbaryl by hydrolysis to yield 1-naphthol and methylamine . 1-Naphthol was further metabolized via salicylate by a gentisate pathway, as evidenced by oxygen uptake and enzymatic studies.

J Bacteriol, 2001 Jun, 183(12), 3729 - 36
Isolation and characterization of IS1409, an insertion element of 4-chlorobenzoate-degrading Arthrobacter sp . strain TM1, and development of a system for transposon mutagenesis; Gartemann KH et al.; A new insertion element of 1,449 bp with 25-bp perfect terminal repeats, designated IS1409, was identified in the chromosome of 4-chlorobenzoate-degrading Arthrobacter sp . strain TM1 NCIB12013 . Upon insertion, IS1409 causes a target duplication of 8 bp . IS1409 carries only a single open reading frame of 435 codons encoding the transposase TnpA . Both TnpA and the overall organization of IS1409 are highly similar to those of some related insertion elements of the ISL3 group (J . Mahillon and M . Chandler, Microbiol . Mol . Biol . Rev . 62:725--774, 1998) . IS1409 was also found in other 4-chlorobenzoate-degrading Arthrobacter strains and Micrococcus luteus . Based on IS1409, a series of transposons carrying resistance genes for chloramphenicol and gentamicin were constructed . These transposons were used to demonstrate transposition events in vivo and to mutagenize Arthrobacter sp . strains.

J Bacteriol, 2001 Jun, 183(12), 3689 - 703
Plasmid-encoded phthalate catabolic pathway in Arthrobacter keyseri 12B; Eaton RW; Several 2-substituted benzoates (including 2-trifluoromethyl-, 2-chloro-, 2-bromo-, 2-iodo-, 2-nitro-, 2-methoxy-, and 2-acetyl-benzoates) were converted by phthalate-grown Arthrobacter keyseri (formerly Micrococcus sp.) 12B to the corresponding 2-substituted 3,4-dihydroxybenzoates (protocatechuates) . Because these products lack a carboxyl group at the 2 position, they were not substrates for the next enzyme of the phthalate catabolic pathway, 3,4-dihydroxyphthalate 2-decarboxylase, and accumulated . When these incubations were carried out in iron-containing minimal medium, the products formed colored chelates . This chromogenic response was subsequently used to identify recombinant Escherichia coli strains carrying genes encoding the responsible enzymes, phthalate 3,4-dioxygenase and 3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase, from the 130-kbp plasmid pRE1 of strain 12B . Beginning with the initially cloned 8.14-kbp PstI fragment of pRE824 as a probe to identify recombinant plasmids carrying overlapping fragments, a DNA segment of 33.5 kbp was cloned from pRE1 on several plasmids and mapped using restriction endonucleases . From these plasmids, the sequence of 26,274 contiguous bp was determined . Sequenced DNA included several genetic units: tnpR, pcm operon, ptr genes, pehA, norA fragment, and pht operon, encoding a transposon resolvase, catabolism of protocatechuate (3,4-dihydroxybenzoate), a putative ATP-binding cassette transporter, a possible phthalate ester hydrolase, a fragment of a norfloxacin resistance-like transporter, and the conversion of phthalate to protocatechuate, respectively . Activities of the eight enzymes involved in the catabolism of phthalate through protocatechuate to pyruvate and oxaloacetate were demonstrated in cells or cell extracts of recombinant E . coli strains.

Biochemistry, 2001 May 29, 40(21), 6398 - 405
Membrane activity of the peptide antibiotic clavanin and the importance of its glycine residues; van Kan EJ et al.; The peptide antibiotic clavanin A (VFQFLGKIIHHVGNFVHGFSHVF-NH(2)) is rich in histidine and glycine residues . In this study the antimicrobial activity and membrane activity of wild-type clavanin A and seven Gly --> Ala mutants thereof were investigated . Clavanin A effectively killed the test microorganism Micrococcus flavus and permeabilized its cytoplasmic membrane in the micromolar concentration range, suggesting that the membrane is the target for this molecule . Consistent with this suggestion, it was observed that clavanin A efficiently inserted into different phospholipid monolayers mainly via hydrophobic interactions . Bilayer permeabilization was observed for both low and high molecular mass fluorophores enclosed in unilamellar vesicles and occurred at the same concentration as the antimicrobial activity . It is therefore suggested that the loss of barrier function does not involve specific receptors in the target membrane . Circular dichroism spectroscopy indicated that under membrane mimicking conditions a random coil --> helical transition was induced for all clavanin derivatives tested . Observed differences in peptide-membrane interaction and biological activity between the various clavanin derivatives demonstrated the functional importance of Gly at the positions 6 and 13 . These two glycines may act as flexible hinges that facilitate the hydrophobic N-terminal end of clavanin to deeply insert into the bilayer . On the contrary, no such role is evident for Gly 18, as its substitution by Ala actually stimulated membrane interaction and biological activity . This study suggests that the combined hydrophobicity, overall state of charge, and conformational flexibility of the peptide determine the (membrane) activity of clavanin A and its Gly --> Ala mutants.

Mol Genet Genomics, 2001 Apr, 265(2), 311 - 5
Polytene chromosome interband DNA is organized into nucleosomes; Schwartz YB et al.; The molecular basis that underlies the maintenance of polytene chromosome banding pattern remains unclear . To test the possibility that the decondensed state of interbands is provoked by the absence of nucleosomes, we have subjected chromatin from the previously defined 61C7/C8 interband to digestion with micrococcal nuclease . We have demonstrated that interband DNA forms nucleosomes both in salivary glands and in the bulk of larval tissues . This finding strongly suggests that the difference in compaction between DNA in polytene chromosome bands and interbands results from differences that appear at the higher levels of chromatin organization.

Biomed Sci Instrum, 2001, 37, 457 - 62
L-glutamic acid production in a novel three phase fluidized bed reactor using coimmobilized bio-catalyst; Baskar R et al.; The production of L-Glutamic acid has been studied using coimmobilized whole cells of pseudomonas reptilivora and micrococcus glutamicus in a three phase fluidized bed bioreactor with selected production medium enriched with glucose . The effect of initial substrate concentration, temperature, pH and aeration rate on the yield of glutamic acid has been investigated . It has been found that the acid production increases exponentially with substrate concentration and mass transfer co-efficient varied linearly with temperature and aeration rate . The optimum temperature and pH are 31 degree Celsius and 7.2 respectively.

J Biol Chem, 2001 Jul 27, 276(30), 28459 - 64 Epub 2001 May 09.
Identification of Significant residues for homoallylic substrate binding of Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase; Kharel Y et al.; The primary structure of cis-prenyltransferase is totally different from those of trans-prenyltransferases (Shimizu, N., Koyama, T., and Ogura, K . (1998) J . Biol . Chem . 272, 19476-19481) . To better understand the molecular mechanism of enzymatic cis-prenyl chain elongation, we selected seven charged residues in the conserved Region V and two of Phe-Ser motif in Region III of undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26 for substitutions by site-directed mutagenesis and examined their effects on substrate binding and catalysis . Kinetic studies indicated that replacements of Arg-197 or Arg-203 with Ser, and Glu-216 with Gln resulted in 7-11-fold increases of Km values for isopentenyl diphosphate and 18-1200-fold decreases of kcat values compared with those of the wild-type enzyme . In addition, two mutants with respect to the Phe-Ser motif in Region III, F73A and S74A, showed 16-32-fold larger Km values for isopentenyl diphosphate and 12-16-fold lower kcat values than those of the wild-type . Furthermore, product analysis indicated that three mutants, F73A, S74A, and E216Q, yielded shorter chain prenyl diphosphates as their main products . These facts together with the protein structural analysis recently carried out (Fujihashi, M., Zhang, Y.-W., Higuchi, Y., Li, X.-Y., Koyama, T., and Miki, K . (2001) Proc . Natl . Acad . Sci . U . S . A . 98, 4337-4342) indicated that the diphosphate moiety of homoallylic substrate is electrostatically recognized by the three charged amino acids, Arg-197, Arg-203, and Glu-216, in Region V and the Phe-Ser motif in Region III, also indispensable for homoallylic substrate binding as well as catalytic function . It was suggested that the undecaprenyl diphosphate synthase takes a different mode for the binding of isopentenyl diphosphate from that of trans-prenyl chain elongating enzymes.

J Pharm Pharmacol, 2001 Apr, 53(4), 549 - 54
Turbidimetric and HPLC assays for the determination of formulated lysozyme activity; Liao YH et al.; In several studies lysozyme has been employed as a model protein to investigate the effects of formulation factors upon biological activity . The aim of this work was to develop and validate an HPLC technique to assay lysozyme and to compare the results with biological activity determined from a validated turbidimetric assay . The turbidimetric assay was based upon the lytic action of lysozyme on Micrococcus lysodeikticus cells, whilst the reverse-phase HPLC assay employed an acetonitrile gradient in 0.1% trifluoroacetic acid . The limits of detection and quantification were 3.84 and 6.24 microg mL(-1) for HPLC assay, whilst the corresponding values for turbidimetric assay were 1.94 and 3.86 microg mL(-1) . The methods were used to monitor the loss of enzyme activity after heating . Lysozyme concentrations determined from HPLC peak height were found to correlate (r2 = 0.9963) with those obtained from turbidimetric assay.

J Biol Chem, 2001 Jul 13, 276(28), 26317 - 23 Epub 2001 May 01.
Subcellular localization of the human proto-oncogene protein DEK; Kappes F et al.; Recent data revealed that DEK associates with splicing complexes through interactions mediated by serine/arginine-repeat proteins . However, the DEK protein has also been shown to change the topology of DNA in chromatin in vitro . This could indicate that the DEK protein resides on cellular chromatin . To investigate the in vivo localization of DEK, we performed cell fractionation studies, immunolabeling, and micrococcal nuclease digestion analysis . Most of the DEK protein was found to be released by DNase treatment of nuclei, and only a small amount by treatment with RNase . Furthermore, micrococcal nuclease digestion of nuclei followed by glycerol gradient sedimentation revealed that DEK co-sedimentates with oligonucleosomes, clearly demonstrating that DEK is associated with chromatin in vivo . Additional chromatin fractionation studies, based on the different accessibilities to micrococcal nuclease, showed that DEK is associated both with extended, genetically active and more densely organized, inactive chromatin . We found no significant change in the amount and localization of DEK in cells that synchronously traversed the cell cycle . In summary these data demonstrate that the major portion of DEK is associated with chromatin in vivo and suggest that it might play a role in chromatin architecture.

Appl Biochem Biotechnol, 2001 Mar, 90(3), 233 - 49
Cooperativity and substrate specificity of an alkaline amylase and neopullulanase complex of Micrococcus halobius OR-1; Rajdevi KP et al.; The saccharifying alkaline amylase and neopullulanase complex of Micrococcus halobius OR-1 hydrolyzes both alpha-(1,4)- and alpha-(1,6)-glycosidic linkages of different linear and branched polysaccharides . The following observations were made concerning the analysis of the coexpressed amylase and neopullulanase enzymes . Even though the enzymes were subjected to a rigorous purification protocol, the activities could not be separated, because both the enzymes were found to migrate in a single peak . By contrast, two independent bands of amylolytic activity at 70 kDa and pullulanolytic activity at 53 kDa were evident by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reducing and nonreducing PAGE, and zymographic analysis on different polysaccharides . Preferential chemical modification of the enzyme and concomitant high-performance thin-layer chromatographic analyses of the saccharides liberated showed that amylase is sensitive to 1-(dimethylamino-propyl)-3-ethyl carbodiimide-HCl and cleaved alpha-(1,4) linkages of starch, amylose, and amylopectin producing predominantly maltotriose . On the other hand, formalin-sensitive neopullulanase acts on both alpha-(1,4) and alpha-(1,6) linkages of pullulan and starch with maltotriose and panose as major products . It is understood that neopullulanase exhibits dual activity and acts in synergy with amylase toward the hydrolysis of alpha-(1,4) linkages, thereby increasing the overall reaction rate; however, such a synergism is not seen in zymograms, in which the enzymes are physically separated during electrophoresis . It is presumed that SDS-protein intercalation dissociated the enzyme complex, without altering the individual active site architecture, with only the synergism lost . The optimum temperature and pH of amylase and neopullulanase were 60 degreesC and 8.0, respectively . The enzymes were found stable in high alkaline pH for 24 h . Therefore, the saccharifying alkaline amylase and neopullulanase of M . halobius OR-1 evolved from tapioca cultivar shows a highly active and unique enzyme complex with several valuable biochemical features.

Eur J Anaesthesiol, 2001 Mar, 18(3), 151 - 8
The influence of intravenous anaesthetics on the activity of enzymes released from polymorphonuclear leucocytes in vitro; Krumholz W et al.; BACKGROUND AND OBJECTIVE: Polymorphonuclear leucocytes make a decisive contribution to defence against bacterial infections . In particular, the effects of anaesthetics on non-oxidative bactericidal mechanisms have previously only been superficially examined . Although the influence of anaesthetic agents on oxidative bactericidal activity has been thoroughly examined, our study concentrated on the effect on non-oxidative processes, which appears to have been a neglected field of research . METHODS: The effects of methohexital, etomidate, ketamine, fentanyl and morphine on the activity of lysozyme and beta-glucuronidase released from polymorphonuclear leucocytes have been studied in vitro . The activity of lysozyme was determined by recording the changes in the turbidity of a suspension of micrococcus lysodeicticus caused by the enzymatic action of lysozyme . beta-glucuronidase activity was photometrically measured by the enzymatic cleavage of phenolphthalein glucuronic acid . RESULTS: High concentrations of methohexital inhibited lysozyme activity; however, etomidate and morphine caused an increase of beta-glucuronidase activity in therapeutic plasma concentrations . While there was no effect of etomidate on lysozyme activity, all concentrations tested significantly stimulated beta-glucuronidase activity . This result was unexpected because intravenous anaesthetics have previously shown a tendency to suppress polymorphonuclear leucocyte functions . Whereas the inhibition of lysozyme activity by the high concentration of methohexital was no surprise, the increase of beta-glucuronidase activity caused by etomidate, ketamine, fentanyl and morphine was quite unexpected . CONCLUSIONS: At present, the underlying mechanism for the increase of beta-glucuronidase activity caused by etomidate, ketamine, fentanyl and morphine is unknown . The fact that there was no influence of these agents on lysozyme activity possibly suggests that the anaesthetic agents have different effects on azurophilic and specific granules . Since in vitro investigations have their limitations, it is too early to draw practical consequences from our study . Moreover, at present it is unclear whether an increase of beta-glucuronidase activity in vivo is an advantage or not . In any case, we think it advisable to perform further investigations on the influence of anaesthetic agents on oxygen-independent bactericidal mechanisms.

Mol Cell Biol, 2001 Apr, 21(8), 2867 - 79
Long-range nucleosome ordering is associated with gene silencing in Drosophila melanogaster pericentric heterochromatin; Sun FL et al.; We have used line HS-2 of Drosophila melanogaster, carrying a silenced transgene in the pericentric heterochromatin, to investigate in detail the chromatin structure imposed by this environment . Digestion of the chromatin with micrococcal nuclease (MNase) shows a nucleosome array with extensive long-range order, indicating regular spacing, and with well-defined MNase cleavage fragments, indicating a smaller MNase target in the linker region . The repeating unit is ca . 10 bp larger than that observed for bulk Drosophila chromatin . The silenced transgene shows both a loss of DNase I-hypersensitive sites and decreased sensitivity to DNase I digestion within an array of nucleosomes lacking such sites; within such an array, sensitivity to digestion by MNase is unchanged . The ordered nucleosome array extends across the regulatory region of the transgene, a shift that could explain the loss of transgene expression in heterochromatin . Highly regular nucleosome arrays are observed over several endogenous heterochromatic sequences, indicating that this is a general feature of heterochromatin . However, genes normally active within heterochromatin (rolled and light) do not show this pattern, suggesting that the altered chromatin structure observed is associated with regions that are silent, rather than being a property of the domain as a whole . The results indicate that long-range nucleosomal ordering is linked with the heterochromatic packaging that imposes gene silencing.

J Biol Chem, 2001 Jun 8, 276(23), 20482 - 90 Epub 2001 Feb 20.
The chromatin structure of the dual c-myc promoter P1/P2 is regulated by separate elements; Albert T et al.; The proto-oncogene c-myc is transcribed from a dual promoter P1/P2, with transcription initiation sites 160 base pairs apart . Here we have studied the transcriptional activation of both promoters on chromatin templates . c-myc chromatin was reconstituted on stably transfected, episomal, Epstein-Barr virus-derived vectors in a B cell line . Episomal P1 and P2 promoters showed only basal activity but were strongly inducible by histone deacetylase inhibitors . The effect of promoter mutations on c-myc activity, chromatin structure, and E2F binding was studied . The ME1a1 binding site between P1 and P2 was required for the maintenance of an open chromatin configuration of the dual c-myc promoters . Mutation of this site strongly reduced the sensitivity of the core promoter region of P1/P2 to micrococcal nuclease and prevented binding of polymerase II (pol II) at the P2 promoter . In contrast, mutation of the P2 TATA box also abolished binding of pol II at the P2 promoter but did not affect the chromatin structure of the P1/P2 core promoter region . The E2F binding site adjacent to ME1a1 is required for repression of the P2 promoter but not the P1 promoter, likely by recruitment of histone deacetylase activity . Chromatin precipitation experiments with E2F-specific antibodies revealed binding of E2F-1, E2F-2, and E2F-4 to the E2F site of the c-myc promoter in vivo if the E2F site was intact . Taken together, the analyses support a model with a functional hierarchy for regulatory elements in the c-myc promoter region; binding of proteins to the ME1a1 site provides a nucleosome-free region of chromatin near the P2 start site, binding of E2F results in transcriptional repression without affecting polymerase recruitment, and the TATA box is required for polymerase recruitment.

Eur J Biochem, 2001 Apr, 268(7), 2134 - 40
Extensive deproteinization of Dictyostelium discoideum RNase P reveals a new catalytic activity; Stathopoulos C et al.; Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures . After treatment with proteinase K followed by phenol extraction of samples containing D . discoideum RNase P activity, a new enzymatic activity was recovered . The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D . discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5'-hydroxyl ends . This activity seems to be associated with one or two RNA molecules copurifying with D . discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.

J Biol Chem, 2001 Jun 22, 276(25), 22772 - 8 Epub 2001 Mar 26.
Two phases of chromatin decondensation during dedifferentiation of plant cells: distinction between competence for cell fate switch and a commitment for S phase; Zhao J et al.; Cellular dedifferentiation is the major process underlying totipotency, regeneration, and formation of new stem cell lineages in multicellular organisms . In animals it is often associated with carcinogenesis . Here, we used tobacco protoplasts (plant cells devoid of cell wall) to study changes in chromatin structure in the course of dedifferentiation of mesophyll cells . Using flow cytometry and micrococcal nuclease analyses, we identified two phases of chromatin decondensation prior to entry of cells into S phase . The first phase takes place in the course of protoplast isolation, following treatment with cell wall degrading enzymes, whereas the second occurs only after protoplasts are induced with phytohormones to re-enter the cell cycle . In the absence of hormonal application, protoplasts undergo cycles of chromatin condensation/decondensation and die . The ubiquitin proteolytic system was found indispensable for protoplast progression into S phase, being required for the second but not the first phase of chromatin decondensation . The emerging model suggests that cellular dedifferentiation proceeds by two functionally distinct phases of chromatin decondensation: the first is a transitory phase that confers competence for cell fate switch, which is followed, under appropriate conditions, by a second proteasome-dependent phase representing a commitment for the mitotic cycle . These findings might have implications for a wide range of dedifferentiation-driven cellular processes in higher eukaryotes.

J Biol Chem, 2001 Jun 8, 276(23), 20197 - 205 Epub 2001 Mar 26.
Targeted and extended acetylation of histones H4 and H3 at active and inactive genes in chicken embryo erythrocytes; Myers FA et al.; Affinity-purified polyclonal antibodies recognizing the most highly acetylated forms of histones H3 and H4 were used in immunoprecipitation assays with chromatin fragments derived from 15-day chicken embryo erythrocytes by micrococcal nuclease digestion . The distribution of hyperacetylated H4 and H3 was mapped at the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the tissue-specific gene, carbonic anhydrase (CA) . H3 and H4 acetylation was found targeted to the CpG island region at the 5' end of both these genes, falling off in the downstream direction . In contrast, at the beta(A)-globin gene, both H3 and H4 are highly acetylated throughout the gene and at the downstream enhancer, with a maximum at the promoter . Low level acetylation was observed at the 5' end of the inactive ovalbumin gene . Run-on assays to measure ongoing transcription showed that the GAPDH and CA genes are transcribed at a much lower rate than the adult beta(A)-globin gene . The extensive high level acetylation at the beta(A)-globin gene correlates most simply with its high rate of transcription . The targeted acetylation of histones H3 and H4 at the GAPDH and CA genes is consistent with a role in transcriptional initiation and implies that transcriptional elongation does not necessarily require hyperacetylation.

Indian J Ophthalmol, 2000 Mar, 48(1), 50 - 2
Micrococcal endophthalmitis following extracapsular cataract extraction with foldable silicone intraocular lens implantation; Fogla R et al.; A case of postoperative endophthalmitis caused by micrococci, after phacoemulsification and foldable silicone intraocular lens (IOL) implantation is reported.

J Cell Biol, 2001 Jan 22, 152(2), 375 - 84
A novel chromatin protein, distantly related to histone H2A, is largely excluded from the inactive X chromosome; Chadwick BP et al.; Chromatin on the mammalian inactive X chromosome differs in a number of ways from that on the active X . One protein, macroH2A, whose amino terminus is closely related to histone H2A, is enriched on the heterochromatic inactive X chromosome in female cells . Here, we report the identification and localization of a novel and more distant histone variant, designated H2A-Bbd, that is only 48% identical to histone H2A . In both interphase and metaphase female cells, using either a myc epitope-tagged or green fluorescent protein-tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout . In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes . Chromatin fractionation demonstrates association of H2A-Bbd with the histone proteins . Separation of micrococcal nuclease-digested chromatin by sucrose gradient ultracentrifugation shows cofractionation of H2A-Bbd with nucleosomes, supporting the idea that H2A-Bbd is incorporated into nucleosomes as a substitute for the core histone H2A . This finding, in combination with the overlap with acetylated forms of H4, raises the possibility that H2A-Bbd is enriched in nucleosomes associated with transcriptionally active regions of the genome . The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.

Infect Immun, 2001 Apr, 69(4), 2270 - 6
Micrococci and peptidoglycan activate TLR2-->MyD88-->IRAK-->TRAF-->NIK-->IKK-->NF-kappaB signal transduction pathway that induces transcription of interleukin-8; Wang Q et al.; This study was done to elucidate the signal transduction pathway of interleukin-8 (IL-8) induction by gram-positive bacteria . Bacteria (micrococci) and peptidoglycan (PGN) induced transcription of IL-8 in HEK293 cells expressing Toll-like receptor 2 (TLR2) and CD14 but not in those expressing TLR1 or TLR4 . A mutation within the NF-kappaB site in the IL-8 promoter abrogated transcriptional induction of IL-8 by the two stimulants . Dominant negative myeloid differentiation protein (MyD88), IL-1 receptor-associated kinase (IRAK), NFkappaB-inducing kinase (NIK), and IkappaB kinase (IKK) mutant forms completely inhibited micrococcus- and PGN-induced activation of NF-kappaB and expression of the gene for IL-8 . Induction of NF-kappaB was partially inhibited by dominant negative tumor necrosis factor receptor-associated kinase 6 (TRAF6) but not TRAF2, whereas induction of IL-8 gene was partially inhibited by both TRAF6 and TRAF2 . These data indicate that micrococci and PGN induce TLR2-dependent activation of the gene for IL-8 and that this activation requires MyD88, IRAK, NIK, IKK, and NF-kappaB and may also utilize TRAF6 and, to a lesser extent, TRAF2.

Infect Immun, 2001 Apr, 69(4), 2025 - 30
Micrococcus luteus teichuronic acids activate human and murine monocytic cells in a CD14- and toll-like receptor 4-dependent manner; Yang S et al.; Teichuronic acid (TUA), a component of the cell walls of the gram-positive organism Micrococcus luteus (formerly Micrococcus lysodeikticus), induced inflammatory cytokines in C3H/HeN mice but not in lipopolysaccharide (LPS)-resistant C3H/HeJ mice that have a defect in the Toll-like receptor 4 (TLR4) gene, both in vivo and in vitro, similarly to LPS (T . Monodane, Y . Kawabata, S . Yang, S . Hase, and H . Takada, J . Med . Microbiol . 50:4-12, 2001) . In this study, we found that purified TUA (p-TUA) induced tumor necrosis factor alpha (TNF-alpha) in murine monocytic J774.1 cells but not in mutant LR-9 cells expressing membrane CD14 at a lower level than the parent J774.1 cells . The TNF-alpha-inducing activity of p-TUA in J774.1 cells was completely inhibited by anti-mouse CD14 monoclonal antibody (MAb) . p-TUA also induced interleukin-8 (IL-8) in human monocytic THP-1 cells differentiated to macrophage-like cells expressing CD14 . Anti-human CD14 MAb, anti-human TLR4 MAb, and synthetic lipid A precursor IV(A), an LPS antagonist, almost completely inhibited the IL-8-inducing ability of p-TUA, as well as LPS, in the differentiated THP-1 cells . Reduced p-TUA did not exhibit any activities in J774.1 or THP-1 cells . These findings strongly suggested that M . luteus TUA activates murine and human monocytic cells in a CD14- and TLR4-dependent manner, similar to LPS.

Plant Cell, 2001 Mar, 13(3), 599 - 612
Targeted histone acetylation and altered nuclease accessibility over short regions of the pea plastocyanin gene; Chua YL et al.; The chromatin structure of the pea plastocyanin gene (PetE) was examined at three different transcriptional states by investigating the acetylation states of histones H3 and H4 and the nuclease accessibility of the gene in pea roots, etiolated shoots, and green shoots . The acetylation states of histones associated with different regions of PetE were analyzed by chromatin immunoprecipitation with antibodies specific for acetylated or nonacetylated histone H3 or H4 tails, followed by polymerase chain reaction quantification . Comparison of pea tissues indicated that histone hyperacetylation was associated with increased PetE transcription in green shoots . Moreover, hyperacetylation of both histones H3 and H4 was targeted to the enhancer/promoter region in green shoots, suggesting that only specific nucleosomes along the gene were modified . Time-course digestions of nuclei with micrococcal nuclease and DNaseI indicated that the enhancer/promoter region was more resistant to digestion in the inactive gene in pea roots than was the same region in the active gene in shoots, whereas the transcribed region of PetE was digested similarly among the tissues . This finding indicates that transcription is accompanied by changes in the nuclease accessibility of the enhancer/promoter region only . Moreover, these results indicate that the changes in nuclease accessibility are organ specific, whereas histone hyperacetylation is light dependent, and they suggest that changes in nuclease accessibility precede histone hyperacetylation during PetE activation.

Anal Biochem, 2001 Mar, 290(2), 292 - 301
Formation and efficacy of vancomycin group glycopeptide antibiotic stereoisomers studied by capillary electrophoresis and bioaffinity mass spectrometry; Bonnici PJ et al.; The conformational stability of vancomycin group antibiotics (i.e., vancomycin and avoparcin) in aqueous solution has been studied . These complex glycopeptide antibiotics contain many chiral centers allowing the potential formation of stereoisomers . Using capillary electrophoresis these stereoisomers could be separated and detected by UV and/or mass spectrometry . Fresh aqueous samples of both vancomycin and avoparcin already contained a plethora of stereoisomers . Thermal degradation of the antibiotics was studied as well . For vancomycin thermal degradation led primarily to the formation of CDP-I and aglycons . In the case of avoparcin thermal degradation led mainly to the interconversion between stereoisomers . These antibiotic stereoisomers may exhibit different antibacterial efficacy . Solution-phase association constants of fresh and heated samples of these antibiotics and their bacterial cell wall mimicking receptors were determined by bioaffinity mass spectrometry and revealed that the heated samples exhibited, in general, a lower affinity . Minimum inhibitory concentrations (Micrococcus flavus) were determined and confirmed the decrease in antibacterial efficacy upon heating .

Microbios, 2001, 104(407), 55 - 61
PCR cloning of the resuscitation-promoting factor (Rpf) gene from Micrococcus luteus, sequencing and expression in Escherichia coli; Matsuda M et al.; A polymerase chain reaction (PCR) cloning procedure was developed for the resuscitation-promoting factor (Rpf) gene of Micrococcus luteus using strains NCIMB 13267, JCM 1464T, JCM 3347, and JCM 3348 . A PCR product of the Rpf gene fragment was ligated into a cloning vector pBluescript II KS (+) with the restriction endonucleases Eco RI and Bam HI . The ligation mixture was used to transform Escherichia coli DH5alpha . The DNA sequence of the Rpf gene cloned from strain JCM 1464T was 84% homologous with that of NCIMB 13267, and from strains JCM 3347 and JCM 3348 it was 100% and 86% homologous, respectively . Recombinant Rpf proteins of M . luteus NCIMB 13267 and JCM 1464T after expression in E . coli BL21 harbouring the pET-19b-Rpf plasmid, and after purification, were approximately 16 kD for both strains.

FEBS Lett, 2001 Feb 23, 491(1-2), 159 - 63
Antibacterial activity of a pepsin-derived bovine hemoglobin fragment; Froidevaux R et al.; Peptic digestion of bovine hemoglobin yields a fragment with antibacterial activity . This peptide was purified to homogeneity by a two-step procedure including anion exchange chromatography and preparative reversed-phase HPLC . Mass determination and fragmentation indicated that this peptide corresponded to the 1-23 fragment of the alpha chain of hemoglobin . The minimum inhibitory concentration and mode of action of this peptide towards Micrococcus luteus strain A270 were determined . Hemolytic assay, interaction with liposomes, and study of its structure in solution were also performed.

Panminerva Med, 2000 Sep, 42(3), 231 - 2
Micrococcus luteus: a putative cause of hepatic abscess?
Andreopoulos T, Papanikolaou G, Politou M, Konstantopoulos K, Stefanou J, Loukopoulos D.
Micrococcus luteus was repeatedly isolated in blood cultures during a prolonged feverish syndrome in a patient who presented with multiple hepatic abscesses as well . In contrast to the literature, this case is not related to prosthetic devices; an untreated limb wound may have been the site of microbial entry.

Int J Syst Evol Microbiol, 2001 Jan, 51(Pt 1), 81 - 7
Ornithinimicrobium humiphilum gen . nov., sp . nov., a novel soil actinomycete with L-ornithine in the peptidoglycan; Groth I et al.; A Gram-positive bacterium originating from garden soil was taxonomically studied . Cells are non-motile, non-sporulating, irregular rods and cocci . The cell wall peptidoglycan contains L-ornithine as diagnostic diamino acid and an interpeptide bridge consisting of L-Orn<--L-Ala<--Gly<--D-Asp . The major menaquinone is MK-8(H4) . 13-Methyl tetradecanoic acid and 14-methyl pentadecanoic acid are the predominant fatty acids . The polar lipids are phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, three unknown glycolipids and three unknown phospholipids . Mycolic acids are absent . The DNA G+C composition is 70 mol% . The acyl type of the glycan chain of peptidoglycan is acetyl . Glucose is the dominating whole cell sugar; arabinose, rhamnose and xylose are present in traces . Results of 16S rDNA sequence comparisons revealed that strain HKI 0124T represents a novel lineage within the suborder Micrococcineae of the order Actinomycetales adjacent to the recently described genus Ornithinicoccus . On the basis of the clearly pronounced morphological, physiological, chemotaxonomic and phylogenetic differences between strain HKI 0124T and all members of the suborder Micrococcineae, it is proposed to assign strain HKI 0124T to a new genus and species, Ornithinimicrobium humiphilum gen . nov., sp . nov . The type and only strain is HKI 0124T (= DSM 12362T = CIP 106634T).

J Med Microbiol, 2001 Jan, 50(1), 4 - 12
Induction of necrosis factor-alpha and interleukin-6 in mice in vivo and in murine peritoneal macrophages and human whole blood cells in vitro by Micrococcus luteus teichuronic acids; Monodane T et al.; Earlier studies showed that Micrococcus luteus cells and cell walls induced anaphylactoid reactions leading to death, in some instances within 1 h, in C3H/HeN mice primed with muramyl dipeptide (MDP) . They also induced serum cytokines in the surviving mice . The present study investigated the structural components responsible for these activities . Teichuronic acids, a component of M . luteus cell walls, induced tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in MDP-primed C3H/HeN mice . Peptidoglycans had little effect on the cytokine-inducing activities . Reducing teichuronic acids, i.e., teichuronic acids whose carboxyl groups had been reduced, lost their cytokine-inducing activities . Neither peptidoglycans nor teichuronic acids induced anaphylactoid reactions in the MDP-primed mice . Purified teichuronic acids also induced TNF-alpha and IL-6 production in C3H/HeN murine peritoneal macrophages and human whole-blood cells in the culture, but reduced teichuronic acids did not . The purified teichuronic acids induced no TNF-alpha and only low levels of IL-6 in MDP-primed C3H/HeJ mice, and neither cytokine in peritoneal macrophage cultures from C3H/HeJ mice with a single point of mutation in Toll-like receptor 4 (TLR4) gene . These findings suggest that induction of cytokines by teichuronic acids is mainly TLR4-dependent.

Glycobiology, 2001 Jan, 11(1), 89 - 98
An alternative cis-isoprenyltransferase activity in yeast that produces polyisoprenols with chain lengths similar to mammalian dolichols; Schenk B et al.; Dolichyl monophosphate (Dol-P) is a polyisoprenoid glycosyl carrier lipid essential for the assembly of a variety of glycoconjugates in the endoplasmic reticulum of eukaryotic cells . In yeast, dolichols with chain lengths of 14--17 isoprene units are predominant, whereas in mammalian cells they contain 19--22 isoprene units . In this biosynthetic pathway, t,t-farnesyl pyrophosphate is elongated to the appropriate long chain polyprenyl pyrophosphate by the sequential addition of cis-isoprene units donated by isopentenyl pyrophosphate with t,t,c-geranylgeranyl pyrophosphate being the initial intermediate formed . The condensation steps are catalyzed by cis-isoprenyltransferase (cis-IPTase) . Genes encoding cis-IPTase activity have been identified in Micrococcus luteus, Escherichia coli, Arabidopsis thaliana, and Saccharomyces cerevisiae (RER2) . Yeast cells deleted for the RER2 locus display a severe growth defect, but are still viable, possibly due to the activity of an homologous locus, SRT1 . The dolichol and Dol-P content of exponentially growing revertants of RER2 deleted cells (Delta rer2) and of cells overexpressing SRT1 have been determined by HPLC analysis . Dolichols and Dol-Ps with 19--22 isoprene units, unusually long for yeast, were found, and shown to be utilized for the biosynthesis of lipid intermediates involved in protein N-glycosylation . In addition, cis-IPTase activity in microsomes from Delta rer2 cells overexpressing SRT1 was 7- to 17-fold higher than in microsomes from Delta rer2 cells . These results establish that yeast contains at least two cis-IPTases, and indicate that the chain length of dolichols is determined primarily by the enzyme catalyzing the chain elongation stage of the biosynthetic process.

Insect Biochem Mol Biol, 2001 Mar 1, 31(3), 241 - 8
The defensin peptide of the malaria vector mosquito Anopheles gambiae: antimicrobial activities and expression in adult mosquitoes; Vizioli J et al.; A recombinant Anopheles gambiae defensin peptide was used to define the antimicrobial activity spectrum against bacteria, filamentous fungi and yeast . Results showed that most of the Gram-positive bacterial species tested were sensitive to the recombinant peptide in a range of concentrations from 0.1 to 0.75 microM . No activity was detected against Gram-negative bacteria, with the exception of some E . coli strains . Growth inhibitory activity was detected against some species of filamentous fungi . Defensin was not active against yeast . The kinetics of bactericidal and fungicidal effects were determined for Micrococcus luteus and Neurospora crassa, respectively . Differential mass spectrometry analysis was used to demonstrate induction of defensin in the hemolymph of bacteria-infected adult female mosquitoes . Native peptide levels were quantitated in both hemolymph and midgut tissues . The polytene chromosome position of the defensin locus was mapped by in situ hybridization.

Biochem Biophys Res Commun, 2001 Feb 2, 280(4), 961 - 3
Columnar packing of telomeric nucleosomes; Fajkus J et al.; Telomeres belong to the key functional elements of eukaryotic chromosomes . Like all the other parts of the genome, they exist and function as complexes of DNA with histones and various nonhistone proteins, including specific telomere-binding proteins . Studies of telomeric chromatin have shown on the one hand a lack of nucleosome positioning and on the other hand a specific nucleosome spacing as revealed by micrococcal nuclease digestion . Based on these properties and on accumulated experimental data, we present a model for a columnar packing of nucleosomes in telomeric chromatin .

Mol Cell Biol, 2001 Feb, 21(4), 1155 - 63
Acetylation of a specific promoter nucleosome accompanies activation of the epsilon-globin gene by beta-globin locus control region HS2; Gui CY et al.; On stably replicating episomes, transcriptional activation of the epsilon-globin promoter by the beta-globin locus control region HS2 enhancer is correlated with an increase in nuclease sensitivity which is limited to the TATA-proximal nucleosome (N1) . To elucidate what underlies this increase in nuclease sensitivity and the link between chromatin modification and gene expression, we examined the nucleoprotein composition and histone acetylation status of transcriptionally active and inactive promoters . Micrococcal nuclease digestion of active promoters in nuclei released few nucleosome-like nucleoprotein complexes containing N1 sequences in comparison to results with inactive promoters . We also observed that N1 DNA fragments from active promoters are of a subnucleosomal length . Nevertheless, chromatin immunoprecipitation experiments indicate that histones H3 and H4 are present on N1 sequences from active promoters, with H3 being dramatically hyperacetylated compared with that from inactive promoters and vector sequences . Strikingly, H3 in the adjacent upstream nucleosome (N2) does not appear to be differentially acetylated in active and inactive promoters, indicating that the nucleosome modification of the promoter that accompanies transactivation by HS2 is highly directed and specific . However, global acetylation of histones in vivo by trichostatin A did not activate transcription in the absence of HS2, suggesting that HS2 contributes additional activities necessary for transactivation . N1 sequences from active promoters also contain reduced levels of linker histone H1 . The detection of a protected subnucleosomal sized N1 DNA fragment and the recovery of N1 DNA sequences in immunoprecipitations using anti-acetylated H3 and H4 antibodies argue that N1 is present, but in an altered conformation, in the active promoters.

Mol Cell Biol, 2001 Jan, 21(2), 548 - 61
The RNase P associated with HeLa cell mitochondria contains an essential RNA component identical in sequence to that of the nuclear RNase P; Puranam RS et al.; The mitochondrion-associated RNase P activity (mtRNase P) was extensively purified from HeLa cells and shown to reside in particles with a sedimentation constant ( approximately 17S) very similar to that of the nuclear enzyme (nuRNase P) . Furthermore, mtRNase P, like nuRNase P, was found to process a mitochondrial tRNA(Ser(UCN)) precursor {ptRNA(Ser(UCN))} at the correct site . Treatment with micrococcal nuclease of highly purified mtRNase P confirmed earlier observations indicating the presence of an essential RNA component . Furthermore, electrophoretic analysis of 3'-end-labeled nucleic acids extracted from the peak of glycerol gradient-fractionated mtRNase P revealed the presence of a 340-nucleotide RNA component, and the full-length cDNA of this RNA was found to be identical in sequence to the H1 RNA of nuRNase P . The proportions of the cellular H1 RNA recovered in the mitochondrial fractions from HeLa cells purified by different treatments were quantified by Northern blots, corrected on the basis of the yield in the same fractions of four mitochondrial nucleic acid markers, and shown to be 2 orders of magnitude higher than the proportions of contaminating nuclear U2 and U3 RNAs . In particular, these experiments revealed that a small fraction of the cell H1 RNA (of the order of 0.1 to 0.5%), calculated to correspond to approximately 33 to approximately 175 intact molecules per cell, is intrinsically associated with mitochondria and can be removed only by treatments which destroy the integrity of the organelles . In the same experiments, the use of a probe specific for the RNA component of RNase MRP showed the presence in mitochondria of 6 to 15 molecules of this RNA per cell . The available evidence indicates that the levels of mtRNase P detected in HeLa cells should be fully adequate to satisfy the mitochondrial tRNA synthesis requirements of these cells.

Comp Immunol Microbiol Infect Dis, 2001 Jan, 24(1), 57 - 70
Chronic fatigue syndrome in horses: diagnosis and treatment of 4 cases; Tarello W; A report from England has suggested that Chronic Fatigue Syndrome exists in equines and constitutes an emerging veterinary problem . Preliminary epidemiological studies seem to confirm the zoonotic implications of CFS . An arsenical drug, sodium thiacetarsamide, was administered to four horses with a diagnosis of Chronic Fatigue Syndrome (CFS), already treated unsuccessfully with different medications . The CFS-like lethargy, with accompanying symptoms and signs, of the four animals obtained a complete remission after intravenous treatment with this drug at low dosage (0.1 mg/kg/day) . No adverse side effects were ever noticed . This clinical response was associated with recovery from anaemia and decrease of muscular enzyme values in two of the four horses . In all patients, micrococci-like bacteria found before treatment adhering to the outer surface of many red blood cells, disappeared at post-treatment controls . Considerations are made on the possible action of an arsenical drug, used in isolation, in the treatment of CFS.

Mol Pharmacol, 2001 Jan, 59(1), 69 - 75
Regulation of bleomycin-induced DNA breakage and chromatin structure in lung endothelial cells by integrins and poly(ADP-ribose) polymerase; Jones CB et al.; Activation of endothelial cell integrins inhibits DNA breakage by diverse agents, including the DNA-damaging agent bleomycin . DNA breaks activate nuclear poly(ADP-ribose) polymerase (PARP), which regulates chromatin structure and DNA repair . We determined the role of PARP in suppression of bleomycin genotoxicity by integrins using wild-type and PARP knockout mouse lung endothelial cells (MLEC), and the PARP inhibitor, 3-aminobenzamide (3AB) . Activation of beta1 integrins by antibody clustering enhanced the sensitivity of wild-type nuclei to digestion with micrococcal nuclease and deoxyribonuclease I, indicating that chromatin structure was altered . 3AB blocked this effect . Knockout and 3AB-treated wild-type MLEC were hypersensitive to deoxyribonuclease I compared with wild-type cells, demonstrating that PARP regulates chromatin structure . Integrin clustering reduced the hypersensitivity of knockout cells, suggesting additional, PARP-independent mechanisms that inhibit nuclease interaction with chromatin . Bleomycin caused DNA breakage in wild-type and knockout MLEC . Breaks were eliminated after 60 min incubation of wild-type cells in drug-free medium, whereas 3AB or PARP knockout inhibited DNA repair . Integrin clustering protected wild-type cells from DNA breakage, and 3AB and PARP knockout inhibited this protection . Bleomycin caused large increases in PARP activity in wild-type but not knockout MLEC, and integrin clustering inhibited the activation of PARP . The results indicate that the antigenotoxic effects of integrin activation require PARP and that integrins alter chromatin structure by PARP-dependent and -independent mechanisms.

FEMS Immunol Med Microbiol, 2000 Dec, 29(4), 233 - 40
Culturability of Mycobacterium tuberculosis cells isolated from murine macrophages: a bacterial growth factor promotes recovery; Biketov S et al.; Very little is known about the culturability and viability of mycobacteria following their phagocytosis by macrophages . We therefore studied populations of the avirulent 'Academia' strain of Mycobacterium tuberculosis isolated from murine peritoneal macrophage lysates several days post-infection in vivo . The resulting bacterial suspensions contained a range of morphological types including rods, ovoid forms and coccoid forms . Bacterial viability measured using the MPN method (dilution to extinction in liquid medium) was often much higher than that measured by CFU (plating on solid medium) . Viability in the MPN assay was further enhanced when the Micrococcus luteus protein, Rpf, was incorporated into the liquid culture medium at picomolar concentrations . Rpf is an example of a family of autocrine growth factors found throughout the high G+C cohort of Gram-positive bacteria including M . tuberculosis . M . tuberculosis cells obtained from macrophages had altered surface properties, as compared with bacteria grown in vitro . This was indicated by loss of the ability to adsorb bacteriophage DS6A, a reduced tendency to form clumps, acquisition of ethidium bromide stainability following heat treatment, and loss of Rpf-mediated resuscitation following freezing and thawing . These results indicate that a proportion of 'unculturable' M . tuberculosis cells obtained from macrophages is either injured or dormant and that these cells may be recovered or resuscitated using Rpf in liquid medium.

EMBO J, 2000 Dec 15, 19(24), 6804 - 13
Chromatin-mediated transcriptional regulation by the yeast architectural factors NHP6A and NHP6B; Moreira JM et al.; The Saccharomyces cerevisiae NHP6A and NHP6B proteins are chromatin architectural factors, functionally and structurally related to the mammalian high mobility group (HMG)-1 and -2 proteins, a family of non-sequence-specific DNA binding proteins . nhp6a nhp6b mutants have various morphological defects and are defective in the induced expression of several RNA polymerase II-transcribed genes . We found that NHP6A/B proteins are also required for full induction of the yeast CHA1 gene . Importantly, CHA1 basal level expression is increased 10-fold in an nhp6a nhp6b double deletion mutant . Micrococcal nuclease and DNase I analysis of the CHA1 gene in this strain showed an open promoter structure, characteristic of the activated state of this promoter, even under non-inducing conditions . To address the possible function of the NHP6A/B proteins in chromatin-mediated gene regulation, we performed whole-genome transcriptional profiling of a Deltanhp6a Deltanhp6b yeast strain . Our results suggest that NHP6A/B proteins play an important regulatory role, repressing as well as potentiating expression of genes involved in several cellular processes, and that NHP6A/B control is exerted at the level of the individual gene.

Biomed Chromatogr, 2000 Nov, 14(7), 489 - 92
Application of capillary electrophoresis to the analysis of soluble chromatin; Asatiani NV et al.; Capillary electrophoresis (CE) has been applied to study DNA-protein complexes using as the test system soluble chromatin from chicken erythrocytes and rapidly proliferated cultured Chinese hamster fibroblast-like cells B11-dii-FAF-28 . Separation was performed with home-made CE apparatus, using a regulated high-voltage power supply, UV-detector and fused silica capillaries with inner diameter 75 microm . The heterogeneity of nucleosomal particles with different DNA lengths after micrococcal nuclease digestion was detected .

Biochem Biophys Res Commun, 2000 Dec 9, 279(1), 213 - 8
Chromatin structure of telomere domain in human sperm; Zalenskaya IA et al.; Telomeres in human sperm nucleus are clustered at the nuclear periphery . Chromosomes in the sperm are highly condensed with protamines, however, a small portion of DNA remains associated with histones; the role of the nucleohistone is unknown . To examine structure of the telomeric chromatin, the sperm nuclei were treated with micrococcal nuclease . Chromatin released by the digestion was free from protamines, but contained histones and revealed nucleosomal organization . It was enriched with telomeric DNA organized into closely spaced nucleosomes with a periodicity of 148 +/- bp . Thus, while the most of the sperm genome is packed into extremely dense nucleoprotamine structure, at least a part of the telomeric DNA is arranged into nucleosomes and can be released by the nuclease . We suggest that telomeres might be among the first structures in the sperm nucleus that respond to oocyte signals for male pronucleus development at fertilization .

J Biol Chem, 2001 Mar 2, 276(9), 6337 - 42 Epub 2000 Dec 01.
The human origin recognition complex protein 1 dissociates from chromatin during S phase in HeLa cells; Kreitz S et al.; We investigated the association of human origin recognition complex (ORC) proteins hOrc1p and hOrc2p with chromatin in HeLa cells . Independent procedures including limited nuclease digestion and differential salt extraction of isolated nuclei showed that a complex containing hOrc1p and hOrc2p occurs in a nuclease-resistant compartment of chromatin and can be eluted with moderate high salt concentrations . A second fraction of hOrc2p that dissociates in vitro at low salt conditions was found to occur in a chromatin compartment characterized by its high accessibility to micrococcal nuclease . Functional differences between these two sites become apparent in HeLa cells that synchronously enter the S phase after a release from a double-thymidine block . The hOrc1p/hOrc2p-containing complexes dissociate from their chromatin sites during S phase and reassociate at the end of mitosis . In contrast, the fraction of hOrc2p in nuclease-accessible, more open chromatin remains bound during all phases of the cell cycle . We propose that the hOrc1p/hOrc2p-containing complexes are components of the human origin recognition complex . Thus, the observed cell cycle-dependent release of the hOrc1p/hOrc2p-containing complexes is in line with previous studies with Xenopus and Drosophila systems, which indicated that a change in ORC stability occurs after prereplication complex formation . This could be a powerful mechanism that prevents the rereplication of already replicated chromatin in the metazoan cell cycle.

J Biochem (Tokyo), 2000 Dec, 128(6), 917 - 22
Significance of Asn-77 and Trp-78 in the catalytic function of undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26; Fujikura K et al.; The primary structures of cis-prenyltransferases are completely different from those of trans-prenyltransferases . To obtain information about amino acid residues relating to catalytic function, random mutation of the undecaprenyl diphosphate synthase gene of Micrococcus luteus B-P 26 was carried out to construct a mutated gene library using an error-prone polymerase chain reaction . From the library, the mutants showing poor enzymatic activity were selected by the colony autoradiography method . Among 31 negative clones selected from 3,000 mutants, two clones were found to contain only one amino acid substitution at either Asn-77 or Trp-78 . To determine the functional roles of these interesting residues, we prepared six mutated enzymes with substitutions at residues Asn-77 or Trp-78 by site-directed mutagenesis . Substitution of Asn-77 with Ala, Asp, or Gln resulted in a dramatic decrease in catalytic activity, but the K(m) values for both allylic and homoallylic substrates of these mutant enzymes were comparable to those of the wild-type . On the other hand, three Trp-78 mutants, W78I, W78R, and W78D, showed 5-20-fold increased K(m) values for farnesyl diphosphate but not for Z-geranylgeranyl diphosphate . However, these mutants showed moderate levels of enzymatic activity and comparable K(m) values for isopentenyl diphosphate to that of the wild-type . These results suggest that the Asn-Trp motif is involved in the binding of farnesyl diphosphate and enzymatic catalysis.

J Biol Chem, 2001 Mar 2, 276(9), 6420 - 8 Epub 2000 Nov 28.
Oct-1 preferentially interacts with androgen receptor in a DNA-dependent manner that facilitates recruitment of SRC-1; Gonzalez MI et al.; Gene regulation by steroid hormone receptors depends on the particular character of the DNA response element, the array of neighboring transcription factors, and recruitment of coactivators that interface with the transcriptional machinery . We are studying these complex interactions for the androgen-dependent enhancer of the mouse sex-limited protein (Slp) gene . This enhancer has, in addition to multiple androgen receptor (AR)-binding sites, a central region (FPIV) with a binding site for the ubiquitous transcription factor Oct-1 that appears crucial for hormonal regulation in vivo . To examine the role of Oct-1 in androgen-specific gene activation, we tested the interaction of Oct-1 with AR versus glucocorticoid receptor (GR) in vivo and in vitro . Oct-1 coimmunoprecipitated from cell lysates with both AR and GR, but significant association with AR required both proteins to be DNA-bound . This was confirmed by sensitivity of the protein association to treatment with ethidium bromide or micrococcal nuclease . Addition of DNA to micrococcal nuclease-treated samples restored interaction, even when binding sites were on separate DNA molecules, suggesting association was due to direct protein-protein interaction and not indirect tethering via the DNA . AR/GR chimeras revealed that interaction of the N and C termini of AR was required to communicate the DNA-bound state that enhances interaction with Oct-1 . Protease digestion assays of hormone-bound receptors revealed further conformational changes in the ligand binding domain of AR, but not GR, upon DNA binding . Furthermore, these conformational changes led to increased interaction with the coactivator SRC-1, via the NID 4 domain, suggesting DNA binding facilitates recruitment of SRC-1 by the AR-Oct-1 complex . Altogether, these results suggest that the precise arrangement of binding sites in the Slp enhancer ensures proper hormonal response by imposing differential interactions between receptors and Oct-1, which in turn contributes to SRC-1 recruitment to the promoter.

J Nat Prod, 2000 Nov, 63(11), 1573 - 5
Two novel iso-branched octadecenoic acids from a Micrococcus species; Carballeira NM et al.; The novel fatty acids 16-methyl-6(Z)-heptadecenoic acid and 16-methyl-8(Z)-heptadecenoic acid were identified for the first time in nature in a species of the bacterium Micrococcus isolated from Lake Pomorie in Bulgaria . The principal fatty acids in this bacterium were a series of iso-anteiso fatty acids with chain lengths between C(14) and C(24), while the most interesting series of monounsaturated fatty acids was a family of Delta(6) fatty acids with chain lengths between C(14) and C(17) . The novel compounds were characterized using a combination of GC-MS and chemical transformations, such as dimethyl disulfide derivatization and catalytic hydrogenation . The results established for the first time a bacterial origin for some of these Delta(6) fatty acids.

Exp Cell Res, 2000 Nov 25, 261(1), 284 - 92
The proliferation-specific human Ki-67 protein is a constituent of compact chromatin; Kreitz S et al.; The human nuclear Ki-67 protein (Ki-67p) is expressed in proliferating, but not in quiescent, cells and is therefore widely used as a proliferation marker in histopathological research and practice . However, information regarding its intranuclear location is scarce and controversial . Here we describe the results of cell fractionation and nuclease digestion experiments using nuclei isolated from human HeLa cells in interphase . Ki-67p dissociates at 0.3-0.4 M NaCl from its nuclear binding sites, and gradient centrifugations indicate that the released Ki-67p is most likely a single molecular entity and not complexed to other proteins . In nuclei, prepared under physiological salt conditions, the binding sites are largely resistant against micrococcal nuclease . However, when prepared at very low ionic strengths, chromatin regions with associated Ki-67p become accessible to micococcal-nuclease-producing chromatin fragments that carry bound Ki-67p . We conclude that Ki-67p is a chromatin protein and resides at densely packed regions, probably heterochromatin . Our data provide a useful basis for further biochemical research on this human nuclear protein .

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