Microorganism

J Med Microbiol, 2004 Mar, 53(Pt 3), 207 - 12
Characterization of elongated Helicobacter pylori isolated from a patient with gastric-mucosa-associated lymphoid-tissue lymphoma; Toyokawa T et al.; To date, two Helicobacter species, Helicobacter pylori and 'Helicobacter heilmannii' (formerly named 'Gastrospirillum hominis'), have been identified from the human stomach . In this study, we observed non-H . pylori-shaped bacteria in gastric tissue sections and successfully isolated them by cultivation . Elongated bacteria were isolated from a patient with gastric-mucosa-associated lymphoid-tissue lymphoma who had been diagnosed as H . pylori-negative by culture, rapid urease test and histopathology in another hospital . The bacteria were grown only on chocolate agar in a CO2 incubator, appeared more than 10 microm long in histological sections, formed small colonies and showed poor growth in a brain heart infusion broth; these characteristics apparently differed from common clinical isolates of H . pylori . However, the bacteria were identified as H . pylori by PCR of the urease gene, 16S rDNA sequencing, protein profile and antigenicity examined by anti-H . pylori polyclonal antibody . These observations suggest that the H . pylori strain identified in this study may contribute to the development of gastroduodenal diseases in cases judged as H . pylori-negative by ordinary methods.

J Biol Chem, 2004 Apr 23, 279(17), 17834 - 41 Epub 2004 Feb 17.
Solution structure of the pore-forming protein of Entamoeba histolytica; Hecht O et al.; Amoebapore A is a 77-residue protein from the protozoan parasite and human pathogen Entamoeba histolytica . Amoebapores lyse both bacteria and eukaryotic cells by pore formation and play a pivotal role in the destruction of host tissues during amoebiasis, one of the most life-threatening parasitic diseases . Amoebapore A belongs to the superfamily of saposin-like proteins that are characterized by a conserved disulfide bond pattern and a fold consisting of five helices . Membrane-permeabilizing effector molecules of mammalian lymphocytes such as porcine NK-lysin and the human granulysin share these structural attributes . Several mechanisms have been proposed to explain how saposin-like proteins form membrane pores . All mechanisms indicate that the surface charge distribution of these proteins is the basis of their membrane binding capacity and pore formation . Here, we have solved the structure of amoebapore A by NMR spectroscopy . We demonstrate that the specific activation step of amoebapore A depends on a pH-dependent dimerization event and is modulated by a surface-exposed histidine residue . Thus, histidine-mediated dimerization is the molecular switch for pore formation and reveals a novel activation mechanism of pore-forming toxins.

Adv Drug Deliv Rev, 2004 Mar 3, 56(4), 511 - 25
M cell targeting by lectins: a strategy for mucosal vaccination and drug delivery; Jepson MA et al.; Bioadhesins are a recognised method of enhancing the absorption of drugs and vaccines at mucosal surfaces . Additionally, bioadhesins allow for cell specific targeting . Lectin-mediated targeting and delivery exploits unique surface carbohydrates on mucosal epithelial cells . The antigen-sampling M cells offer a portal for absorption of colloidal and particulate delivery vehicles, including bacteria, viruses and inert microparticles . We review work supporting the use of lectins to aid targeting to intestinal M cells . Consideration is also given to lectin-mediated targeting in non-intestinal sites and to the potential application of other bioadhesins to enhance M cell transport . While substantial hurdles must be overcome before mucosal bioadhesins can guarantee consistent, safe, effective mucosal delivery, this strategy offers novel opportunities for drug and vaccine formulation.

Lab Invest, 2004 Apr, 84(4), 425 - 32
Recurrent perivascular inflammation induced by lipopolysaccharide (endotoxin) results in the formation of atheromatous lesions in vivo; Engelmann MG et al.; Bacteria and viruses are suspected to induce arteriosclerosis; however, most investigators have focused on coincidences rather than causal relationships . The aim of this work was to establish a rabbit model in which the vessel reaction to local perivascular injection of defined bacterial products can be analyzed . A total of 23 rabbits were randomized to four groups . Groups A and B were fed a normal diet, groups C and D were fed a cholesterol-enriched diet . Groups A and C were treated with a single perivascular injection of bacterial lipopolysaccharide (LPS, endotoxin) placed next to auricular, carotid and femoral arteries, and sodium chloride placed next to the contralateral arteries (control) . Group B and D animals were treated with repeated perivascular injections over 90 days . Vascular tissues (n=116 treated segments of 23 rabbits) were analyzed using morphometry at histology, and using immunohistochemistry to detect macrophages, lymphocytes and vascular smooth muscle cells . LPS treatment resulted in transient focal intima thickening . After single LPS application, no increase in atheromatous lesion formation was observed in comparison with controls (group C, lesion area index 0.031+/-0.012 vs 0.015+/-0.006, P=1.0) . Repeated LPS application resulted in significant atheromatous lesion formation compared with saline control (group D, lesion area index 0.148+/-0.049 vs 0.008+/-0.006, P=0.003) in hypercholesterolemic rabbits . Repeated LPS inflammation in normocholesterolemic did not lead to atheromatous lesion formation (intima media ratio 0.04+/-0.01 vs 0.04+/-0.007, P=1.0) . Single perivascular administration of low-dose bacterial LPS resulted in transient focal intimal thickening, while significant increase in lesion formation occurred after repeated LPS application in cholesterol-fed animals . In conclusion, this animal model will allow the assessment of the impact of defined dosages of different bacterial pathogens onto the vascular wall in the context of atherogenesis . The atheromatous lesion-promoting effect of repeated perivascular administration of LPS supports the hypothesis that bacterial pathogens may be involved in atherogenesis.

Exp Lung Res, 2004 Jan-Feb, 30(1), 17 - 29
Interaction between mycobacteria and mucus on a human respiratory tissue organ culture model with an air interface; Middleton AM et al.; Mycobacteria adhere specifically to extracellular matrix (ECM) and mucus with a fibrous, but not globular, appearance, in organ cultures of human respiratory mucosa examined by scanning electron microscopy . Previously, light microscopy sections made of tissue infected for 7 days demonstrated mycobacteria associated with mucus on the organ culture surface, and within submucosal glands in areas of damaged epithelium . The authors have now investigated the interactions between Mycobacterium avium complex (MAC), Mycobacterium tuberculosis (MTB), and Mycobacterium smegmatis (MS) and mucus by preincubating bacteria with purified mucins MUC5AC and MUC5B prior to inoculation onto the organ culture mucosal surface . They have also measured mucin production by the organ culture after mycobacterial infection . Mucus did not cause clumping of mycobacteria . There was a significant (P=.03) increase in the amount of fibrous mucus, but not globular mucus, observed on tissue inoculated with mucins compared to controls . The number of bacteria adhering to ECM was markedly reduced after incubation with mucins, which could indicate a protective effect . Mycobacterial infection did not increase mucin production by the organ culture . Mycobacterial adherence to mucins may play a role in the pathogenicity of mycobacteria in diseases such as cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary disease (COPD), in which there are changes in mucus composition and clearance.

Biochem J, 2004 May 15, 380(Pt 1), 75 - 81
Tissue-specific loss of fucosylated glycolipids in mice with targeted deletion of alpha(1,2)fucosyltransferase genes; Iwamori M et al.; Glycolipids in epithelial tissues of the gastrointestinal tract act as receptors for enteric bacteria and are implicated in the activation of the intestinal immune system . To clarify the genes involved in the fucosylation of the major glycolipids, substrate glycolipids and fucosylated products were measured in tissues of wild-type and mutant mice lacking alpha(1,2)fucosyltransferase genes FUT1 or FUT2 . Quantitative determination was performed by TLC-immunostaining for GA1 (Gg4Cer), FGA1 (fucosyl GA1), GM1 (II3NeuAc-Gg4Cer), FGM1 (fucosyl GM1), and Forssman glycolipids . Both FGM1 and FGA1 completely disappeared from the antrum, cecum, and colon of FUT2-null mice, but not those of FUT1-null and wild-type mice . Precursor glycolipids, GM1 and GA1, accumulated in tissues of FUT2-null mice, indicating that the FUT2-encoded enzyme preferentially participates in the fucosylation of GA1 and GM1 in these tissues . Female reproductive organs were similarly found to utilize FUT2 for the fucosylation of glycolipids FGA1 (uterus and cervix), and FGM1 (ovary), due to their absence in FUT2-null mice . In FUT1-null mice FGA1 was lost from the pancreas, but was present in wild-type and FUT2-null mice, indicating that FUT1 is essential for fucosylation of GA1 in the pancreas . Ulex europaeus agglutinin-I lectin histochemistry for alpha(1,2)fucose residues confirmed the absence of alpha(1,2)fucose residues from the apical surface of pancreatic acinar glands of FUT1-null mice . Ileum, epididymis, and testis retained specific fucosylated glycolipids, irrespective of targeted deletion of either gene, indicating either compensation for or redundancy of the alpha(1,2)fucosyltransferase genes in these tissues.

PLoS Biol . 2004 Feb;2(2):E31 . Epub 2004 Feb 17.
Engineered biosynthesis of regioselectively modified aromatic polyketides using bimodular polyketide synthases; Tang Y et al.; Bacterial aromatic polyketides such as tetracycline and doxorubicin are a medicinally important class of natural products produced as secondary metabolites by actinomyces bacteria . Their backbones are derived from malonyl-CoA units by polyketide synthases (PKSs) . The nascent polyketide chain is synthesized by the minimal PKS, a module consisting of four dissociated enzymes . Although the biosynthesis of most aromatic polyketide backbones is initiated through decarboxylation of a malonyl building block (which results in an acetate group), some polyketides, such as the estrogen receptor antagonist R1128, are derived from nonacetate primers . Understanding the mechanism of nonacetate priming can lead to biosynthesis of novel polyketides that have improved pharmacological properties . Recent biochemical analysis has shown that nonacetate priming is the result of stepwise activity of two dissociated PKS modules with orthogonal molecular recognition features . In these PKSs, an initiation module that synthesizes a starter unit is present in addition to the minimal PKS module . Here we describe a general method for the engineered biosynthesis of regioselectively modified aromatic polyketides . When coexpressed with the R1128 initiation module, the actinorhodin minimal PKS produced novel hexaketides with propionyl and isobutyryl primer units . Analogous octaketides could be synthesized by combining the tetracenomycin minimal PKS with the R1128 initiation module . Tailoring enzymes such as ketoreductases and cyclases were able to process the unnatural polyketides efficiently . Based upon these findings, hybrid PKSs were engineered to synthesize new anthraquinone antibiotics with predictable functional group modifications . Our results demonstrate that (i) bimodular aromatic PKSs present a general mechanism for priming aromatic polyketide backbones with nonacetate precursors; (ii) the minimal PKS controls polyketide chain length by counting the number of atoms incorporated into the backbone rather than the number of elongation cycles; and (iii) in contrast, auxiliary PKS enzymes such as ketoreductases, aromatases, and cyclases recognize specific functional groups in the backbone rather than overall chain length . Among the anthracyclines engineered in this study were compounds with (i) more superior activity than R1128 against the breast cancer cell line MCF-7 and (ii) inhibitory activity against glucose-6-phosphate translocase, an attractive target for the treatment of Type II diabetes.

Acta Paediatr Taiwan, 2003 Sep-Oct, 44(5), 297 - 9
An outbreak of scombroid fish poisoning in a kindergarten; Wu SF et al.; We report an outbreak of scombroid poisoning at one kindergarten on September 25, 1997 . There were 94 cases . The onset of symptoms of scombrotoxin after ingestion of fish is rapid (40 to 50 minutes after consumption) . Clinical manifestation consisted of hyperemia, particularly on the face and neck (94.7%), nausea and vomiting (17.0%), abdominal pain (17.0%), pruritus (4.3%), headache and dizziness (4.3%) and diarrhea (3.2%) . The duration of symptoms was 3 hours on average . Most patients responded to antihistamine very well . The poisoning was caused by the ingestion of spoiled scombroid fish . The tissues of scombroid fish had undergone a number of changes provoked by bacteria and the uncooked fish containing 2,104 ppm of histamine whereas cooked fish containing 1,980 ppm (198 mg/100 gm) of histamine was found in this accident.

J Egypt Soc Parasitol, 2003 Aug, 33(2), 373 - 84
Experimental demonstration of hepatitis C virus (HCV) in an Egyptian strain of Culex pipiens complex; Hassan MI et al.; Reverse transcriptase (RT) polymerase chain reaction (PCR) was used to detect hepatitis C Virus (HCV) RNA among heads, guts, larvae and eggs of Culex pipiens complex . The mosquitoes were trapped from homes of hepatitis C patients or among the same organs of symbiotic (normal gut bacteria) and aposymbiotic (without gut bacteria) mosquitoes fed HCV positive blood by an artificial membrane feeder . The eggs and larvae resulted from symbiotic females fed HCV positive blood was tested for HCV-RNA . Hepatitis C virus RNA was detected only in heads of symbiotic mosquitoes collected from homes of HCV positive patients at 3h and 6h after feeding . The virus was detected at 3d and 8d after being fed on HCV-RNA positive blood in guts of the same group . The virus was not detected in the eggs or larvae resulted from female mosquitoes fed on HCV-RNA positive blood . The results raise the possibility of the mechanical and/or biological transmission of HCV by Cx . pipiens, and pave the way to the ongoing study on the effect of gut bacteria of Cx . pipiens in a trial to identify an anti-HCV agent.

New Microbiol, 2004 Jan, 27(1), 75 - 7
Mycobacterium avium sub . paratuberculosis in tissue samples of Crohn's disease patients; Sechi LA et al.; Crohn's disease is a non-specific chronic transmural inflammatory disease . The disease was associated with a frameshit mutation in the NOD2 gene . Nevertheless, other researchers associated the presence of M . paratuberculosis within the intestinal tissues of patients with the disease . An adapted "in situ hybridization" technique was used to detect IS900 M . paratuberculosis DNA in paraffin embedded tissue from Crohns tissue disease samples . We were able to identify M . paratuberculosis DNA in around 69% of the paraffine embedded intestinal samples of Crohn's disease patients analysed . The presence of M . paratuberculosis DNA in the intestinal samples analysed does not necessarily mean that M . paratuberculosis is responsible for Crohn's disease . Our results support the hypothesis that infection may be caused by cell wall defective M . paratuberculosis since no bacteria were detected by Ziehl Neelsen stain.

J Environ Qual, 2004 Jan-Feb, 33(1), 61 - 71
Attenuation of methane and volatile organic compounds in landfill soil covers; Scheutz C et al.; The potential for natural attenuation of volatile organic compounds (VOCs) in landfill covers was investigated in soil microcosms incubated with methane and air, simulating the gas composition in landfill soil covers . Soil was sampled at Skellingsted Landfill at a location emitting methane . In total, 26 VOCs were investigated, including chlorinated methanes, ethanes, ethenes, fluorinated hydrocarbons, and aromatic hydrocarbons . The soil showed a high capacity for methane oxidation resulting in very high oxidation rates of between 24 and 112 microg CH4 g(-1) h(-1) . All lower chlorinated compounds were shown degradable, and the degradation occurred in parallel with the oxidation of methane . In general, the degradation rates of the chlorinated aliphatics were inversely related to the chlorine to carbon ratios . For example, in batch experiments with chlorinated ethylenes, the highest rates were observed for vinyl chloride (VC) and lowest rates for trichloroethylene (TCE), while tetrachloroethylene (PCE) was not degraded . Maximal oxidation rates for the halogenated aliphatic compounds varied between 0.03 and 1.7 microg g(-1) h(-1) . Fully halogenated hydrocarbons (PCE, tetrachloromethane {TeCM}, chlorofluorocarbon {CFC}-11, CFC-12, and CFC-113) were not degraded in the presence of methane and oxygen . Aromatic hydrocarbons were rapidly degraded giving high maximal oxidation rates (0.17-1.4 microg g(-1) h(-1)) . The capacity for methane oxidation was related to the depth of oxygen penetration . The methane oxidizers were very active in oxidizing methane and the selected trace components down to a depth of 50 cm below the surface . Maximal oxidation activity occurred in a zone between 15 and 20 cm below the surface, as this depth allowed sufficient supply of both methane and oxygen . Mass balance calculations using the maximal oxidation rates obtained demonstrated that landfill soil covers have a significant potential for not only methane oxidation but also cometabolic degradation of selected volatile organics, thereby reducing emissions to the atmosphere.

Mol Biol Evol, 2004 May, 21(5), 841 - 50 Epub 2004 Feb 12.
GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity; Shagin DA et al.; Homologs of the green fluorescent protein (GFP), including the recently described GFP-like domains of certain extracellular matrix proteins in Bilaterian organisms, are remarkably similar at the protein structure level, yet they often perform totally unrelated functions, thereby warranting recognition as a superfamily . Here we describe diverse GFP-like proteins from previously undersampled and completely new sources, including hydromedusae and planktonic Copepoda . In hydromedusae, yellow and nonfluorescent purple proteins were found in addition to greens . Notably, the new yellow protein seems to follow exactly the same structural solution to achieving the yellow color of fluorescence as YFP, an engineered yellow-emitting mutant variant of GFP . The addition of these new sequences made it possible to resolve deep-level phylogenetic relationships within the superfamily . Fluorescence (most likely green) must have already existed in the common ancestor of Cnidaria and Bilateria, and therefore GFP-like proteins may be responsible for fluorescence and/or coloration in virtually any animal . At least 15 color diversification events can be inferred following the maximum parsimony principle in Cnidaria . Origination of red fluorescence and nonfluorescent purple-blue colors on several independent occasions provides a remarkable example of convergent evolution of complex features at the molecular level.

J Biol Chem, 2004 Apr 23, 279(17), 17205 - 16 Epub 2004 Feb 12.
Helicobacter pylori CagA induces Ras-independent morphogenetic response through SHP-2 recruitment and activation; Higashi H et al.; The CagA protein of Helicobacter pylori, which is injected from the bacteria into bacteria-attached gastric epithelial cells, is associated with gastric carcinoma . CagA is tyrosine-phosphorylated by Src family kinases, binds the SH2 domain-containing SHP-2 phosphatase in a tyrosine phosphorylation-dependent manner, and deregulates its enzymatic activity . We established AGS human gastric epithelial cells that inducibly express wild-type or a phosphorylation-resistant CagA, in which tyrosine residues constituting the EPIYA motifs were substituted with alanines . Upon induction, wild-type CagA, but not the mutant CagA, elicited strong elongation of cell shape, termed the "hummingbird" phenotype . Time-lapse video microscopic analysis revealed that the CagA-expressing cells exhibited a marked increase in cell motility with successive rounds of elongation-contraction processes . Inhibition of CagA phosphorylation by an Src kinase inhibitor, PP2, or knockdown of SHP-2 expression by small interference RNA (siRNA) abolished the CagA-mediated hummingbird phenotype . The morphogenetic activity of CagA also required Erk MAPK but was independent of Ras or Grb2 . In AGS cells, CagA prolonged duration of Erk activation in response to serum stimulation . Conversely, inhibition of SHP-2 expression by siRNA abolished the sustained Erk activation . Thus, SHP-2 acts as a positive regulator of Erk activity in AGS cells . These results indicate that SHP-2 is involved in the Ras-independent modification of Erk signals that is necessary for the morphogenetic activity of CagA . Our work therefore suggests a key role of SHP-2 in the pathological activity of H . pylori virulence factor CagA.

Bioinformatics, 2004 Jul 10, 20(10), 1557 - 64 Epub 2004 Feb 12.
BioOptimizer: a Bayesian scoring function approach to motif discovery; Jensen ST et al.; MOTIVATION: Transcription factors (TFs) bind directly to short segments on the genome, often within hundreds to thousands of base pairs upstream of gene transcription start sites, to regulate gene expression . The experimental determination of TFs binding sites is expensive and time-consuming . Many motif-finding programs have been developed, but no program is clearly superior in all situations . Practitioners often find it difficult to judge which of the motifs predicted by these algorithms are more likely to be biologically relevant . RESULTS: We derive a comprehensive scoring function based on a full Bayesian model that can handle unknown site abundance, unknown motif width and two-block motifs with variable-length gaps . An algorithm called BioOptimizer is proposed to optimize this scoring function so as to reduce noise in the motif signal found by any motif-finding program . The accuracy of BioOptimizer, which can be used in conjunction with several existing programs, is shown to be superior to using any of these motif-finding programs alone when evaluated by both simulation studies and application to sets of co-regulated genes in bacteria . In addition, this scoring function formulation enables us to compare objectively different predicted motifs and select the optimal ones, effectively combining the strengths of existing programs . AVAILABILITY: BioOptimizer is available for download at www.fas.harvard.edu/~junliu/BioOptimizer/

Eur J Haematol, 2004 Feb, 72(2), 149 - 53
Association of pyoderma gangrenosum and sterile osteomyelitis in a patient having myelodysplastic syndrome with der(1;7)(q10;q10); Yoshida C et al.; Neutrophilic dermatoses such as Sweet's disease and pyoderma gangrenosum (PG) are occasionally associated with myelodysplastic syndrome (MDS) . We present here a 67-yr-old male having PG and sterile osteomyelitis in association with underlying MDS (refractory anemia) and Crohn's disease . To establish the diagnosis of MDS, sternal bone marrow puncture was performed, which showed chromosomal abnormality containing der(1;7)(q10;q10) . After the puncture, he suffered from gradually progressive skin ulceration, flare, and bone pain . Magnetic resonance imaging (MRI) of the sternum showed severe inflammation in the sternum and the overlying subcutaneous tissue . All of the cultures obtained from the wound were negative for both bacteria and fungus . Biopsy was performed from the antero-sternal skin lesion, which showed epidermal ulceration with prominent infiltration of neutrophils . He was thus diagnosed as having PG and sterile osteomyelitis, and was treated with prednisolone, which completely resolved the symptoms . We consider that the bone marrow aspiration in the present patient provoked PG and sterile osteomyelitis . As was previously reported by others, certain chromosomal abnormalities in MDS may be related with the development of neutrophilic dermatoses.

J Appl Microbiol, 2004, 96(3), 546 - 51
A nested-PCR assay for detection of Xylella fastidiosa in citrus plants and sharpshooter leafhoppers; Ciapina LP et al.; AIMS: Detection of Xylella fastidiosa in citrus plants and insect vectors . METHODS AND RESULTS: Chelex 100 resin matrix was successfully standardized allowing a fast DNA extraction of X . fastidiosa . An amplicon of 500 bp was observed in samples of citrus leaf and citrus xylem extract, with and without symptoms of citrus variegated chlorosis, using PCR with a specific primer set indicating the presence of X . fastidiosa . The addition of insoluble acid-washed polyvinylpyrrolidone (PVPP) prior to DNA extraction of insect samples using Chelex 100 resin together with nested-PCR permitted the detection of X . fastidiosa within sharpshooter heads with great sensitivity . It was possible to detect up to two bacteria per reaction . From 250 sharpshooter samples comprising four species (Dilobopterus costalimai, Oncometopia facialis, Bucephalogonia xanthopis and Acrogonia sp.), 87 individuals showed positive results for X . fastidiosa in a nested-PCR assay . CONCLUSIONS: The use of Chelex 100 resin allowed a fast and efficient DNA extraction to be used in the detection of X . fastidiosa in citrus plants and insect vectors by PCR and nested-PCR assays, respectively . SIGNIFICANCE AND IMPACT OF THE STUDY: The employment of efficient and sensitive methods to detect X . fastidiosa in citrus plants and insect vectors will greatly assist epidemiological studies.

Artif Organs, 2004 Feb, 28(2), 210 - 7
Pyrogen transfer across high- and low-flux hemodialysis membranes; Weber V et al.; The extent to which bacterial products from contaminated dialysate enter a patient's blood depends upon the type and permeability of the hemodialysis membrane in use . This study was performed to assess the transfer of pyrogenic substances across both high- and low-flux membranes (DIAPES, Fresenius Polysulfone, Helixone, Polyamide S) . All experiments were carried out in the saline-saline model . The dialysate pool was contaminated either with purified lipopolysaccharide (LPS) (250 and 500 EU/mL) or with sterile bacterial culture filtrates (20 EU/mL), and in vitro dialysis was performed under diffusive and convective conditions . A significant transfer of endotoxin was observed for both low- and high-flux DIAPES challenged with either LPS or with bacterial culture filtrates . Under identical conditions, no transfer of endotoxins was detectable across Fresenius Polysulfone and Helixone upon challenge with purified LPS . With bacterial culture filtrates, endotoxin concentrations for Polyamide S and Fresenius Polysulfone were about 10% and 1%, respectively, of those measured for DIAPES, whereas no transfer of endotoxin was detectable for Helixone . Using an alternative assay (induction of interleukin-1 receptor antagonist, IL-1Ra, in whole blood), only the DIAPES membrane showed the passage of cytokine-inducing substances . Thus, when saline is present in both the blood and dialysate compartments (i.e., the situation during predialysis priming procedures), dialysis membranes differ profoundly with respect to their permeability to endotoxins.

Mar Biotechnol (NY), 2002 Jan, 4(1), 6 - 11
Gene transfer and cloning of flanking chromosomal regions using the medaka fish Tol2 transposable element; Koga A et al.; For the ultimate purpose of developing genetic tools using the medaka fish Tol2 transposable element, we examined whether it can transfer a marker gene into the fish genome and also be applied for cloning of chromosomal regions adjacent to insertion points . An internal region of Tol2 was removed and replaced with the green fluorescent protein (GFP) gene and a bacterial plasmid replication origin . This modified Tol2 clone was microinjected into fertilized eggs together with messenger RNA for the Tol2 transposase . The GFP gene was found to be integrated into chromosomes and transmitted to subsequent generations . Restriction enzyme digestion of genomic DNA of a transformant fish, followed by ligation and introduction into bacteria, produced a plasmid containing the entire element and flanking chromosomal regions . Sequencing analysis of this clone demonstrated transposition of the element in the germline of the first generation . Thus, the basic requirements for a gene transfer vector and gene tagging system were fulfilled.

Mar Biotechnol (NY), 2000 Nov, 2(6), 577 - 86
Molecular cloning and antiserum development of cyclin box in the brown tide alga Aureococcus anophagefferens; Lin S et al.; Cyclins can be useful cell cycle markers for growth rate studies on harmful algal blooms . In this study, a gene fragment corresponding to cyclin box was cloned for the brown tide alga Aureococcus anophagefferens . This algal gene fragment, designated as Btcycl1, was most similar to cyclin B . Oligopeptides based on the deduced amino acid sequence were synthesized and used to raise an antiserum that reacted on Western blots with a protein of about 63 kDa, the same size as cyclin B in other organisms . The cyclin B-like protein recognized by this antiserum, and the messenger RNA amplified using the primers, were more abundant in exponential cultures and decreased markedly in stationary cultures . This protein also appeared to be cell cycle dependent . Immunofluorescence labeling showed that this antiserum specifically stained a protein in Aureococcus cells and had no cross-reaction with bacteria that were present in the algal culture . The Btcycl1 sequence and the antiserum will provide a useful tool for studies on regulation of in situ growth rate for this brown tide alga.

Nature, 2004 Feb 12, 427(6975), 652 - 6
Structural basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase; Fromme JC et al.; The genomes of aerobic organisms suffer chronic oxidation of guanine to the genotoxic product 8-oxoguanine (oxoG) . Replicative DNA polymerases misread oxoG residues and insert adenine instead of cytosine opposite the oxidized base . Both bases in the resulting A*oxoG mispair are mutagenic lesions, and both must undergo base-specific replacement to restore the original C*G pair . Doing so represents a formidable challenge to the DNA repair machinery, because adenine makes up roughly 25% of the bases in most genomes . The evolutionarily conserved enzyme adenine DNA glycosylase (called MutY in bacteria and hMYH in humans) initiates repair of A*oxoG to C*G by removing the inappropriately paired adenine base from the DNA backbone . A central issue concerning MutY function is the mechanism by which A*oxoG mispairs are targeted among the vast excess of A*T pairs . Here we report the use of disulphide crosslinking to obtain high-resolution crystal structures of MutY-DNA lesion-recognition complexes . These structures reveal the basis for recognizing both lesions in the A*oxoG pair and for catalysing removal of the adenine base.

Methods Mol Med, 2004, 94, 333 - 72
Solid supports in enzyme-linked immunosorbent assay and other solid-phase immunoassays; Butler JE; Most modern immunoassays involve the use of synthetic solid phases to immobilize one of the reactants, often by simple adsorption . These solid-phase immunoassays (SPIs) involve ligand-receptor interactions that occur within a reaction volume close to the solution/solid-phase interface . As a consequence, the immunochemistry/biochemistry of these ligand-receptor interactions differ from their counterparts in solution . Nevertheless, mass law equations can be derived for measuring the antigen capture of solid-phase antibodies, for determining the affinity of solid phases for protein adsorption, and for estimating antibody affinity . Many proteins adsorbed on polystyrene or silicone suffer adsorption-induced conformational changes (ACC) and are partially or largely denatured . Alternative methods for immobilizing proteins and virus, while preserving antigenicity, may yield only a modest increase in functional reactant concentration . Peptides and small recombinant proteins appear to benefit especially from nonadsorptive immobilization . Not all solid phases commonly used in SPIs have the same properties, the same capacity for reactant immobilization, cause the same level of denaturation, or experience the same level of nonspecific binding . Empiricism, adherence to a few practical rules of thumb, and avoidance of certain "old wives tales" can be valuable in the successful development of SPIs.

Biochim Biophys Acta, 2004 Feb 12, 1696(2), 193 - 202
Occurrence of proteinaceous endoxylanase inhibitors in cereals; Goesaert H et al.; Cereals contain proteinaceous inhibitors of endoxylanases, which affect the efficiency and functionality of these enzymes in cereal processing . This review relates their first discovery in wheat and the subsequent purification of two distinct classes of endoxylanase inhibitors, namely Triticum aestivum xylanase inhibitor (TAXI)-type and xylanase inhibitor protein (XIP)-type inhibitors in cereals . Both inhibitor classes occur in monocots as multi-isoform families . The reported data provide an overview of the relative quantitative and qualitative variation of these inhibitors in cereals . Wheat and rye are particularly rich in TAXI-type and XIP-type inhibitors with the latter inhibitors being more abundant . Lower inhibitor levels are present in durum wheat and barley, while maize contains solely XIP-type inhibitors . No inhibitors have been isolated from rice, oats and buckwheat.

Insect Biochem Mol Biol, 2004 Mar, 34(3), 273 - 81
An ecdysone-inducible putative "DEAD box" RNA helicase in the spruce budworm (Choristoneura fumiferana); Zhang DY et al.; RNA helicases are a family of enzymes that unwind nucleic acid duplexes, such as RNA/RNA and RNA/DNA, in a 3' to 5' direction into single-stranded polynucleotides . A putative RNA helicase cDNA (CfrHlc64) was isolated from the spruce budworm, Choristoneura fumiferana . CfrHlc64 was 1998 nucleotides in length, and the deduced protein had 565 amino acids with a predicted molecular mass of 64 kDa . It contained eight functional motifs conserved in the "DEAD box" family of RNA helicases . The deduced amino acid sequence showed 10-50% identities to homologues of other species from bacteria to human . In vitro expression of the cDNA resulted in recombinant proteins of 64 kDa as expected from the deduced amino acid sequence . Northern blotting and RT-PCR analyses revealed the presence of CfrHlc64 mRNA in all developmental stages from embryo to adult . Higher levels of CfrHlc64 mRNA were detected in the fat body and midgut than in the epidermis of sixth instar larvae . The CfrHlc64 protein was distributed mainly in the fat body . Female adults expressed CfrHlc64 mRNA at higher levels than male adults . The nonsteroidal ecdysone agonist, tebufenozide, enhanced the expression of CfrHlc64 in a dose-dependent manner.

J Immunol Methods, 2004 Feb 1, 285(1), 111 - 27
Immunosuppressive properties of anti-CD3 single-chain Fv and diabody; Le Gall F et al.; The mouse anti-human CD3 monoclonal antibody OKT3 is a potent immunosuppressive agent used in clinical transplantation . However, OKT3 therapy is associated with unpleasant and often serious side effects which appear to result from cytokine release, complement activation and a human anti-mouse antibody (HAMA) response . To decrease these adverse side effects, we constructed antibody fragments comprising OKT3 variable domains without any constant domains . Single-chain Fv (scFv) monomers, dimers and trimers were generated by changing the linker length between the V(H) and V(L) domains . The linkers used were the natural extensions of the V(H) into the C(H)1 domain . The dimeric molecules (diabodies) demonstrated the best CD3-binding activity . The diabody with the six amino acid linker was produced in bacteria with a tenfold higher yield than other scFvs and possessed CD3-binding affinity approaching that of the parental mAb . In contrast to OKT3 mAb, the anti-CD3 diabody and scFv monomer did not cause any T-cell activation and cytokine release in vitro, while demonstrating CD3 modulation . In mixed lymphocyte cultures, both diabody and scFv, but not the monoclonal antibody OKT3, were able to suppress T-cell activation and secretion of IL-2 and IFN-gamma in a dose-dependent manner . The anti-CD3 diabody may provide a potent immunosuppressive drug with low toxicity and immunogenicity.

J Am Chem Soc, 2004 Feb 18, 126(6), 1858 - 71
Electrostatic study of the proton pumping mechanism in bovine heart cytochrome C oxidase; Popovic DM et al.; Cytochrome c oxidase (CcO) is the terminal enzyme of the cell respiratory chain in mitochondria and aerobic bacteria . It catalyzes the reduction of oxygen to water and utilizes the free energy of the reduction reaction for proton pumping across the inner-mitochondrial membrane, a process that results in a membrane electrochemical proton gradient . Although the structure of the enzyme has been solved for several organisms, the molecular mechanism of proton pumping remains unknown . In the present paper, continuum electrostatic calculations were employed to evaluate the electrostatic potential, energies, and protonation state of bovine heart cytochrome c oxidase for different redox states of the enzyme along its catalytic cycle . Three different computational models of the enzyme were employed to test the stability of the results . The energetics and pH dependence of the P-->F, F-->O, and O-->E steps of the cycle have been investigated . On the basis of electrostatic calculations, two possible schemes of redox-linked proton pumping are discussed . The first scheme involves His291 as a pump element, whereas the second scheme involves a group linked to propionate D of heme a(3) . In both schemes, loading of the pump site is coupled to ET between the two hemes of the enzyme, while transfer of a chemical proton is accompanied by ejection of the pumped H(+) . The two models, as well as the energetics results are compared with recent experimental kinetic data . The proton pumping across the membrane is an endergonic process, which requires a sufficient amount of energy to be provided by the chemical reaction in the active site . In our calculations, the conversion of OH(-) to H(2)O provides 520 meV of energy to displace pump protons from a loading site and overall about 635 meV for each electron passing through the system . Assuming that the two charges are translocated per electron against the membrane potential of 200 meV, the model predicts an overall efficiency of 63%.

Cell Mol Life Sci, 2004 Feb, 61(3), 301 - 25
Human clade B serpins (ov-serpins) belong to a cohort of evolutionarily dispersed intracellular proteinase inhibitor clades that protect cells from promiscuous proteolysis; Silverman GA et al.; Serpins are unique among the various types of active site proteinase inhibitors because they covalently trap their targets by undergoing an irreversible conformational rearrangement . Members of the serpin superfamily are present in the three major domains of life (Bacteria, Archaea and Eukarya) as well as several eukaryotic viruses . The human genome encodes for at least 35 members that segregate evolutionarily into nine (A-I) distinct clades . Most of the human serpins are secreted and circulate in the bloodstream where they reside at critical checkpoints intersecting self-perpetuating proteolytic cascades such as those of the clotting, thrombolytic and complement systems . Unlike these circulating serpins, the clade B serpins (ov-serpins) lack signal peptides and reside primarily within cells . Most of the human clade B serpins inhibit serine and/or papain-like cysteine proteinases and protect cells from exogenous and endogenous proteinase-mediated injury . Moreover, as sequencing projects expand to the genomes of other species, it has become apparent that intracellular serpins belonging to distinct phylogenic clades are also present in the three major domains of life . As some of these serpins also guard cells against the deleterious effects of promiscuous proteolytic activity, we propose that this cytoprotective function, along with similarities in structure are common features of a cohort of intracellular serpin clades from a wide variety of species.

Arch Microbiol, 2004 Apr, 181(4), 299 - 304 Epub 2004 Feb 10.
' Candidatus Hepatincola porcellionum' gen . nov., sp . nov., a new, stalk-forming lineage of Rickettsiales colonizing the midgut glands of a terrestrial isopod; Wang Y et al.; The midgut glands (hepatopancreas) of terrestrial isopods are densely colonized by hitherto uncultivated bacteria . In the case of the Common Woodlouse, Porcellio scaber (Crustacea: Isopoda), the symbionts represent a novel lineage in the alpha-subdivision of Proteobacteria . Based on comparative sequence analysis of their 16S rRNA genes, their closest (albeit distant) relatives were among the Rickettsiales, which are intracellular symbionts or pathogens of many animals . Transmission electron microscopy and in situ hybridization with fluorescently labeled oligonucleotide probes revealed a homogeneous population of symbionts intimately associated with the endothelium of the hepatopancreas, which apparently interact with the microvilli of the brush border by means of a stalk-like cytoplasmic appendage . Based on isolated phylogenetic position and unique cytological properties, the provisional name ' Candidatus Hepatincola porcellionum' is proposed to classify this new taxon of Rickettsiales colonizing the hepatopancreas of P . scaber.

Proc Natl Acad Sci U S A, 2004 Feb 17, 101(7), 1852 - 7 Epub 2004 Feb 09.
Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins; Jonsson K et al.; Helicobacter pylori binds a number of host cell proteins, including the plasma protein plasminogen, which is the proenzyme of the serine protease plasmin . Two H . pylori plasminogen-binding proteins have been described; however, no genes were identified . Here we report the use of a phage display library to clone two genes from the H . pylori CCUG 17874 genome that mediate binding to plasminogen . DNA sequence analysis of one of these genes revealed 96.6% homology with H . pylori 26695 HP0508 . A subsequent database search revealed that the amino acid sequence of a lysine-rich C-terminal segment of HP0508 is identical to the C terminus of HP0863 . Recombinant proteins expressed from HP0508 and HP0863 bound plasminogen specifically and in a lysine-dependent manner . We designate these genes pgbA and pgbB, respectively . These proteins are expressed by a variety of H . pylori strains, have surface-exposed domains, and do not inhibit plasminogen activation . These results indicate that pgbA and pgbB may allow H . pylori to coat its exterior with plasminogen, which subsequently can be activated to plasmin . The surface acquisition of protease activity may enhance the virulence of H . pylori.

Proc Natl Acad Sci U S A, 2004 Feb 17, 101(7), 2203 - 8 Epub 2004 Feb 09.
The outer plastid envelope protein Oep16: role as precursor translocase in import of protochlorophyllide oxidoreductase A; Reinbothe S et al.; A 16-kDa plastid envelope protein was identified by chemical crosslinking that interacts with the precursor of NADPH:protochlorophyllide oxdidoreductase A (pPORA) during its posttranslational import into isolated barley chloroplasts . Protein purification and subsequent protein sequencing showed that the 16-kDa protein is an ortholog of a previously identified outer plastid envelope protein, Oep16 . A protein of identical size was present in barley etioplasts and interacted with pPORA . Similar 16-kDa protein-dependent crosslink products of pPORA were detected in wheat, pea, and Arabidopsis chloroplasts . Database analyses revealed that the 16-kDa protein belongs to a family of preprotein and amino acid transporters found in free-living bacteria and endosymbiotic mitochondria and chloroplasts . Antibodies raised against the 16-kDa protein inhibited import of pPORA, highlighting its role in protein import.

FEMS Microbiol Lett, 2004 Feb 9, 231(1), 131 - 6
The key Sinorhizobium meliloti succinoglycan biosynthesis gene exoY is expressed from two promoters; Cheng HP et al.; Bacterial exopolysaccharide, succinoglycan, plays an important role in eliciting infection thread formation, which is a key step in the establishment of Sinorhizobium meliloti-alfalfa (Medicago sativa) nitrogen fixing symbiosis . To understand the regulatory mechanisms that control production of succinoglycan, the expression of the key succinoglycan biosynthesis gene, exoY, was analyzed by constructing a set of nested deletions of the exoY promoter region . Two exoY promoters were identified based on the promoter activities and confirmed by direct detection of the transcripts . The expression from both promoters was induced in the exoR95 and exoS96 mutant backgrounds suggesting that both promoters are regulated by the ExoR protein and the ExoS/ChvI two-component signal transduction system . The identification of the exoY promoters provides additional avenue for further analysis of the role of succinoglycan in S . meliloti-alfalfa symbiosis.

Water Res, 2004 Feb, 38(4), 983 - 91
Anaerobic stabilisation and conversion of biopolymers in primary sludge--effect of temperature and sludge retention time; Mahmoud N et al.; The effect of sludge retention time (SRT) and process temperature on the hydrolysis, acidification and methanogenesis of primary sludge was investigated in completely stirred tank reactors (CSTRs) . The CSTRs were operated to maintain SRTs of 10, 15, 20 and 30 days at process temperatures of 25 degrees C and 35 degrees C . The rates of hydrolysis and the biodegradability of primary sludge were assessed in batch reactors incubated at 15 degrees C, 25 degrees C and 35 degrees C . The results revealed that the major amount of sludge stabilisation occurred between 0 and 10 days at 35 degrees C and 10 and 15 days at 25 degrees C . Hydrolysis was found to be the rate limiting-step of the overall digestion process, for the reactors operated at 35 degrees C and 25 degrees C, except for the reactor operated at 10 days and 25 degrees C . At the latter conditions, methanogenesis was the rate-limiting step of the overall digestion process . Proteins hydrolysis was limited to a maximum value of 39% at 30 days and 35 degrees C due to proteins availability in the form of biomass . The biodegradability of primary sludge was around 60%, and showed no temperature dependency . The hydrolysis of the main biopolymers and overall particulate COD of the primary sludge digested in CSTRs were well described by first-order kinetics, in case hydrolysis was the rate-limiting step . Similarly, the hydrolysis of the overall particulate COD of the primary sludge digested in batch reactors were described by first-order kinetics and revealed strong temperature dependency, which follows Arrhenius equation.

Environ Toxicol Chem, 2004 Jan, 23(1), 17 - 23
Methylmercury production in High Arctic wetlands; Loseto LL et al.; Mercury is present at elevated levels in the top predators living in High Arctic ecosystems . Because only methylmercury (MeHg) bioaccumulates in food chains, the sources need to be identified . In temperate environments, wetlands are considered to be the principal sources of MeHg, with sulfate-reducing bacteria (SRB) thought to be responsible . The present study investigated whether High Arctic wetlands produced MeHg and whether SRB were involved in MeHg formation . Frozen soil was collected from 18 High Arctic wetlands before ground thaw, and when analyzed for MeHg, values were low, averaging 0.065 ng/g . When soils were incubated for 30 and 60 d at typical summer Arctic soil temperatures (4 degrees C and 8 degrees C), MeHg increased up to 100-fold . These laboratory observations were consistent with field measurements of wetland surface water, where MeHg concentrations increased from near detection limits (0.02 ng/L) at the inflow to an average of 1.21 ng/L at the outflow . Both laboratory and field data showed MeHg production in High Arctic wetlands . The prevalence of SRB in soil was low, however, and DNA analysis of the dissimilatory sulfate-reductase gene specific to SRB was positive at only one site . The present study showed that wetlands in the High Arctic can produce MeHg but that SRB may not the dominant mercury methylators.

Int J Mol Med, 2004 Mar, 13(3), 451 - 4
Nucleic acids from intact epithelial cells as a target for stool-based molecular diagnosis of colorectal cancer; Spethmann S et al.; Stool-based molecular techniques may improve strategies for colorectal cancer screening . Molecular methods have successfully been applied to detect tumour DNA in stool from patients diagnosed for colorectal carcinoma . In these assays human DNA has to be analyzed against a background of excess nucleic acids from bacteria and dietary waste products . More recently a different diagnostic approach has been described characterizing intact cells isolated from stool . In this study we combine both approaches preparing nucleic acids from isolated epithelial cells to evaluate if: a) tumour cell-specific RNA can be analyzed since cellular RNA molecules are prevented from early digestion by an intact cell membrane; and b) specificity or sensitivity of established DNA-based methods can be improved when epithelial cells are separated from other stool components . Comparing different protocols we found cell isolation using epithelium-specific antibodies to be more effective and reproducible than a technique using density gradient centrifugation . A detection limit of 10(4) cells per ml stool was determined when samples from healthy volunteers were spiked with epithelial cells . Amplification of human sequences from total stool DNA was more efficient than a correspondent amplification of DNA extracted from isolated cells, so that an improvement of DNA-based methods cannot be expected by introducing cell isolation procedures . RNA detection was successful in 1 of 5 patients with confirmed diagnosis of colorectal cancer . The authors suggest that low numbers of detectable cells might rather be a biological than an analytical problem limiting a routinely performed method for colorectal cancer diagnosis.

Mol Biol Cell, 2004 Apr, 15(4), 1853 - 61 Epub 2004 Feb 06.
The Oxa2 protein of Neurospora crassa plays a critical role in the biogenesis of cytochrome oxidase and defines a ubiquitous subbranch of the Oxa1/YidC/Alb3 protein family; Funes S et al.; Proteins of the Oxa1/YidC/Alb3 family mediate the insertion of proteins into membranes of mitochondria, bacteria, and chloroplasts . Here we report the identification of a second gene of the Oxa1/YidC/Alb3 family in the genome of Neurospora crassa, which we have named oxa2 . Its gene product, Oxa2, is located in the inner membrane of mitochondria . Deletion of the oxa2 gene caused a specific defect in the biogenesis of cytochrome oxidase and resulted in induction of the alternative oxidase (AOD), which bypasses the need for complex IV of the respiratory chain . The Oxa2 protein of N . crassa complements Cox18-deficient yeast mutants suggesting a common function for both proteins . The oxa2 sequence allowed the identification of a new subfamily of Oxa1/YidC/Alb3 proteins whose members appear to be ubiquitously present in mitochondria of fungi, plants, and animals including humans.

J Clin Microbiol, 2004 Feb, 42(2), 660 - 4
Detection of species-specific helicobacter ribosomal DNA in intestinal biopsy samples from a population-based cohort of patients with ulcerative colitis; Streutker CJ et al.; The inflammatory bowel diseases are considered an abnormal host immune response to an environmental stimulus . Evidence suggests a role for intestinal bacteria in initiating and/or providing an ongoing stimulus for inflammation in inflammatory bowel disease . Helicobacter pylori is the major cause of active chronic gastritis and peptic ulcers in humans and has been linked to gastric carcinoma and lymphoma . Studies in various animal models, particularly mice, have identified enterohepatic Helicobacter species that are capable of causing hepatitis and enterocolitis . We hypothesize that Helicobacter species may have a role in maintaining inflammation in humans with inflammatory bowel disease . In order to investigate this, biopsy specimens were obtained from patients with and without inflammatory bowel disease . DNA was extracted from the tissues and subjected to PCR with primers designed to detect the ribosomal DNA of members of the Helicobacter species . DNA from six biopsy samples from 60 inflammatory bowel disease patients tested positive . This included 5 of 33 ulcerative colitis patients that were positive compared to 0 of 29 age-matched controls (P < 0.04) . Sequencing of the bands produced by PCR amplification revealed >or=99% homology with H . pylori . These results indicate that a member of the Helicobacter species may be involved in some cases of ulcerative colitis.

Appl Environ Microbiol, 2004 Feb, 70(2), 679 - 85
Expression, secretion, and glycosylation of the 45- and 47-kDa glycoprotein of Mycobacterium tuberculosis in Streptomyces lividans; Lara M et al.; The gene encoding the 45/47 kDa glycoprotein (Rv1860) of Mycobacterium tuberculosis was expressed in Streptomyces lividans under its own promoter and under the thiostrepton-inducible Streptomyces promoter PtipA . The recombinant protein was released into the culture medium and, like the native protein, migrated as a double band at 45 and 47 kDa in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels . However, in contrast to the native protein, only the 47-kDa recombinant protein could be labeled with concanavalin A (ConA) . Carbohydrate digestion with jack bean alpha-D-mannosidase resulted in a reduction in the molecular mass of the recombinant protein upper band and completely eliminated ConA binding . Two-dimensional gel electrophoresis revealed only one isoelectric point for the recombinant protein . Comparative fingerprinting analysis of the individually purified upper and lower recombinant protein bands, treated under the same conditions with specific proteases, resulted in similar peptide patterns, and the peptides had the same N-terminal sequence, suggesting that migration of the recombinant protein as two bands in SDS-PAGE gels could be due to differences in glycosylation . Mass spectrometry analysis of the recombinant protein indicated that as in native protein, both the N-terminal and C-terminal domains of the recombinant protein are glycosylated . Furthermore, it was determined that antibodies of human tuberculosis patients reacted mainly against the carbohydrate residues of the glycoprotein . Altogether, these observations show that expression of genes for mycobacterial antigens in S . lividans is very useful for elucidation of the functional role and molecular mechanisms of glycosylation in bacteria.

Front Biosci, 2004 Jan 01, 9, 283 - 9
Wound healing: an overview of acute, fibrotic and delayed healing; Diegelmann RF et al.; Acute wounds normally heal in a very orderly and efficient manner characterized by four distinct, but overlapping phases: hemostasis, inflammation, proliferation and remodeling . Specific biological markers characterize healing of acute wounds . Likewise, unique biologic markers also characterize pathologic responses resulting in fibrosis and chronic non-healing ulcers . This review describes the major biological processes associated with both normal and pathologic healing . The normal healing response begins the moment the tissue is injured . As the blood components spill into the site of injury, the platelets come into contact with exposed collagen and other elements of the extracellular matrix . This contact triggers the platelets to release clotting factors as well as essential growth factors and cytokines such as platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta) . Following hemostasis, the neutrophils then enter the wound site and begin the critical task of phagocytosis to remove foreign materials, bacteria and damaged tissue . As part of this inflammatory phase, the macrophages appear and continue the process of phagocytosis as well as releasing more PDGF and TGF beta . Once the wound site is cleaned out, fibroblasts migrate in to begin the proliferative phase and deposit new extracellular matrix . The new collagen matrix then becomes cross-linked and organized during the final remodeling phase . In order for this efficient and highly controlled repair process to take place, there are numerous cell-signaling events that are required . In pathologic conditions such as non-healing pressure ulcers, this efficient and orderly process is lost and the ulcers are locked into a state of chronic inflammation characterized by abundant neutrophil infiltration with associated reactive oxygen species and destructive enzymes . Healing proceeds only after the inflammation is controlled . On the opposite end of the spectrum, fibrosis is characterized by excessive matrix deposition and reduced remodeling . Often fibrotic lesions are associated with increased densities of mast cells . By understanding the functional relationships of these biological processes of normal compared to abnormal wound healing, hopefully new strategies can be designed to treat the pathological conditions.

Bioresour Technol, 2004 May, 92(3), 321 - 6
On the behavior of the periodic anaerobic baffled reactor (PABR) during the transition from carbohydrate to protein-based feedings; Stamatelatou K et al.; The influence of the organic substrate composition in the feed of an innovative reactor, the periodic anaerobic baffled reactor (PABR) is examined . A laboratory-scale PABR fed on a synthetic medium composed of mixtures of glucose (a carbohydrate) and gelatin (a protein) in various ratios performed well . The PABR seemed to be minimally affected during the gradual substitution of glucose by gelatin . In fact, the reactor performance remained at an optimal level (approximately 98%), while operated under an organic loading rate of 3.125 gCOD/l/d.

Math Biosci, 2004 Mar-Apr, 188, 221 - 33
Rippling of myxobacteria; Igoshin OA et al.; Myxobacteria colonies during their aggregation phase propagate complex waves over their surface . These waves are fundamentally different from the analogous phenomenon in diffusion-reaction systems or in populations of Dictyostelium discoideum where colliding waves annhilate . Myxobacterial waves appear to pass through one another, analogous to solitons . Moreover, individual bacteria oscillate back and forth, exhibiting no net mass transfer . A mathematical model can explain virtually all of the experimentally observed properties of these waves and draw several conclusions about the properties of the intercelular signaling system.

J Am Vet Med Assoc, 2004 Feb 1, 224(3), 419 - 23
Use of somatic cell counts and California mastitis test results from individual quarter milk samples to detect subclinical intramammary infection in dairy cattle from a herd with a high bulk tank somatic cell count; Middleton JR et al.; OBJECTIVE: To determine whether somatic cell counts (SCCs) or California mastitis test (CMT) scores for individual quarter milk samples could be used to detect subclinical intramammary infection among dairy cattle in a herd with a high bulk tank SCC . DESIGN: Prospective clinical trial . ANIMALS: 278 Holstein-Friesian dairy cattle from a single herd . PROCEDURE: Individual quarter milk samples were collected and submitted for bacterial culture, California mastitis testing, and determination of SCC . Additional milk samples were collected 34 days later and submitted for bacterial culture . RESULTS: During the initial visit to the herd, milk samples were collected from all 278 cows . However, because of blind mammary quarters or missing data, results for 1,057 quarter milk samples were included . Bacterial culture did not yield any growth for 622 (58.8%) of these samples . Regardless of the cutoff that was used, sensitivity of the CMT score was < or = 0.50 and sensitivity of the SCC linear score (SCS) was < or = 0.60 . For 497 mammary quarters, results of bacterial culture of samples collected 34 days apart were concordant; bacterial culture did not yield any growth for 342 (68.8%) of these quarters . Regardless of the cutoff that was used, sensitivity of the CMT score was < or = 0.61 and sensitivity of the SCS was < or = 0.76 for mammary quarters with concordant bacterial culture results . CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that neither CMT score nor SCC is sensitive enough to be useful as a screening test for identifying infected mammary quarters among dairy cattle in a herd with high bulk tank SCC.

J Vet Intern Med, 2004 Jan-Feb, 18(1), 56 - 64
A prospective study of canine infective endocarditis in northern California (1999-2001): emergence of Bartonella as a prevalent etiologic agent; MacDonald KA et al.; A prospective study was performed (June 1999 to May 2001) to determine the incidence of infective endocarditis (IE) due to Bartonella in dogs in northern California and to compare these patients with other dogs with IE . IE was diagnosed antemortem based on clinical signs and echocardiography in 18 dogs . The etiologic agent was Bartonella sp . in 5 dogs (28%) and was diagnosed by high seroreactivity to Bartonella (titer > 1:512; range, 1:1,024-1:4,096); and confirmed postmortem by positive polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) from the infected valve and partial DNA sequencing of the citrate synthase gene (glt A) . Conventional bacteria were causative agents in 7 dogs (39%) . An etiologic agent was not identified in 6 dogs (33%) . Bartonella vinsonii berkhoffii (n = 3), B clarridgeiae (n = 1), and a B clarridgeiae-like organism (n = 1) were identified . Blood culture was positive only for the IE case due to B clarridgeiae . All dogs with IE due to Bartonella were also seroreactive to Anaplasma phagocytophilum . All dogs with IE due to Bartonella had lesions only on the aortic valve . Of the cases of IE not due to Bartonella, 31% involved the aortic valve, 61% the mitral valve, and 8% both valves . Dogs with mitral valve IE lived longer than all dogs with aortic valve IE (P = .004) and dogs with IE of the aortic valve due to Bartonella (P = .002) . In conclusion, Bartonella is a common cause of IE in dogs of northern California . A high Bartonella serologic titer (> 1:512) is useful antemortem to diagnose aortic valve IE due to Bartonella.

J Vet Intern Med, 2004 Jan-Feb, 18(1), 47 - 51
Antinuclear antibodies can be detected in dog sera reactive to Bartonella vinsonii subsp . berkhoffii, Ehrlichia canis, or Leishmania infantum antigens; Smith BE et al.; The presence of antinuclear antibodies (ANAs) is used to support a clinical diagnosis of systemic lupus erythematosus (SLE) in dogs . However, clinicians must interpret the detection of ANAs with caution, particularly in light of increasing evidence that dogs with known bacterial and protozoal infections can have high ANA titers . Retrospectively, medical records were reviewed for all dogs that were concurrently tested for antinuclear antigens and Bartonella vinsonii (berkhoffii), Ehrlichia canis, or Rickettsia rickettsii antigens between 1990 and 2000 . When analyzed on the basis of reactivity to a specific infectious agent, 75% of the B vinsonii (berkhoffii) seroreactors, 16.7% of the E canis seroreactors, and 0% of the R rickettsii seroreactors had concurrent ANAs . Subsequent prospective testing did not detect ANAs in convalescent sera from dogs experimentally infected with B vinsonii (berkhoffii), E canis, or R rickettsii . However, 10-20% B vinsonii (berkhoffii), E canis, or Leishmania infantum reactive sera from naturally infected dogs contained ANAs . In addition, 45% of sera from dogs that are reactive to multiple vectorborne organisms were more likely to contain ANAs when compared to sera from dogs reactive to only 1 test antigen . When interpreting the relevance of seroreactivity to nuclear antigens, clinicians should recognize that dogs with seroreactivity to B vinsonii (berkhoffii), E canis, or L infantum antigens (especially those with seroreactivity to more than one of these pathogens) may produce ANAs.

Clin Infect Dis, 2004 Feb 15, 38(4), 547 - 55 Epub 2004 Jan 29.
Infectious disease images on the World Wide Web; Priest DH et al.; Infectious diseases (ID) physicians are often in need of medical images to enhance their teaching, research, and clinical practice . We explored the Internet for Web sites with images that would be useful to ID physicians . A total of 24 sites were included for review . Of these, 10 sites were broad in their scope and included images of bacteria, parasites, viruses, and/or fungi . In addition, 4 sites reviewed were specific for fungi, 4 sites were specific for viruses, and 6 sites focused on parasites . The number and size of images, copyright restrictions, and fees were noted for all sites . The authors gave each site a subjective navigation and layout score . Features of the sites, including microscopy images, laboratory images, clinical images, clinical vignettes, medical illustrations and/or clip art, medical illustrations of life cycles, slides of educational material, radiographs, and animation or video, were also described . A variety of image resources are available to the ID physician.

J Immunol, 2004 Feb 15, 172(4), 2595 - 606
Inflammatory gene profiles in gastric mucosa during Helicobacter pylori infection in humans; Wen S et al.; Helicobacter pylori infection is associated with an inflammatory response in the gastric mucosa, ultimately leading to cellular hyperproliferation and malignant transformation . Hitherto, only expression of a single gene, or a limited number of genes, has been investigated in infected patients . cDNA arrays were therefore used to establish the global pattern of gene expression in gastric tissue of healthy subjects and of H . pylori-infected patients . Two main gene expression profiles were identified based on cluster analysis . The data obtained suggest a strong involvement of selected Toll-like receptors, adhesion molecules, chemokines, and ILs in the mucosal response . This pattern is clearly different from that observed using gastric epithelial cell lines infected in vitro with H . pylori . The presence of a "Helicobacter-infection signature," i.e., a set of genes that are up-regulated in biopsies from H . pylori-infected patients, could be derived from this analysis . The genotype of the bacteria (presence of genes encoding cytotoxin-associated Ag, vacuolating cytotoxin, and blood group Ag-binding adhesin) was analyzed by PCR and shown to be associated with differential expression of a subset of genes, but not the general gene expression pattern . The expression data of the array hybridization was confirmed by quantitative real-time PCR assays . Future studies may help identify gene expression patterns predictive of complications of the infection.

Cell Microbiol, 2004 Mar, 6(3), 201 - 11
The role and regulation of programmed cell death in plant-pathogen interactions; Greenberg JT et al.; It is commonly known that animal pathogens often target and suppress programmed cell death (pcd) pathway components to manipulate their hosts . In contrast, plant pathogens often trigger pcd . In cases in which plant pcd accompanies disease resistance, an event called the hypersensitive response, the plant surveillance system has learned to detect pathogen-secreted molecules in order to mount a defence response . In plants without genetic disease resistance, these secreted molecules serve as virulence factors that act through largely unknown mechanisms . Recent studies suggest that plant bacterial pathogens also secrete antiapoptotic proteins to promote their virulence . In contrast, a number of fungal pathogens secrete pcd-promoting molecules that are critical virulence factors . Here, we review recent progress in determining the role and regulation of plant pcd responses that accompany both resistance and susceptible interactions . We also review progress in discerning the mechanisms by which plant pcd occurs during these different interactions.

Eur J Clin Invest, 2004 Feb, 34(2), 149 - 55
Genetic deficiency of CD16, the low-affinity receptor for immunoglobulin G, has no impact on the functional capacity of polymorphonuclear neutrophils; Wagner C et al.; BACKGROUND: Of the three receptors for immunoglobulin G (IgG), the low-affinity receptor CD16 is constitutively expressed on polymorphonuclear neutrophils (PMNs), monocytes and NK-cells . CD16 participates in various effector functions, notably phagocytosis of opsonized particles or of immune complexes, and in antibody-dependent cellular cytotoxicity (ADCC) . In the present study we report a case of total CD16 deficiency on PMNs and monocytes . DESIGN: Polymorphonuclear neutrophils, monocytes and NK-cells were analyzed for surface-receptor expression by cytofluorometry and laser scan microscopy . Moreover, CD16-specific mRNA was assessed by RT-PCR . As functional parameters, phagocytosis of opsonized bacteria was tested, as was superoxide production . RESULTS: Polymorphonuclear neutrophils and monocytes totally deficient in CD16 were detected by chance in an apparently healthy individual . Further analysis revealed that two more members of his family, his father and sister, were also deficient in CD16 . All were healthy and there was no evidence of an increased frequency, or of exceptionally severe or persistent infections . Despite the lack of CD16, phagocytosis of antibody-coated bacteria was within the normal range, as was the superoxide production . CONCLUSION: Deficiency of CD16 does not compromise the host defence . Apparently, the other receptors for IgG, CD32 and CD64, can compensate for the lack of CD16.

Mol Microbiol, 2004 Feb, 51(4), 1003 - 14
The Mycobacterium tuberculosis ino1 gene is essential for growth and virulence; Movahedzadeh F et al.; Inositol is utilized by Mycobacterium tuberculosis in the production of its major thiol and of essential cell wall lipoglycans . We have constructed a mutant lacking the gene encoding inositol-1-phosphate synthase (ino1), which catalyses the first committed step in inositol synthesis . This mutant is only viable in the presence of extremely high levels of inositol . Mutant bacteria cultured in inositol-free medium for four weeks showed a reduction in levels of mycothiol, but phosphatidylinositol mannoside, lipomannan and lipoarabinomannan levels were not altered . The ino1 mutant was attenuated in resting macrophages and in SCID mice . We used site-directed mutagenesis to alter four putative active site residues; all four alterations resulted in a loss of activity, and we demonstrated that a D310N mutation caused loss of the active site Zn2+ ion and a conformational change in the NAD+ cofactor.

J Oral Sci, 2003 Dec, 45(4), 233 - 5
Is there evidence of a sphincter system in Wharton's duct? Etiological factors related to sialolith formation; Teymoortash A et al.; The exact cause of the formation of sialoliths is unknown . Detailed knowledge of the pathogenesis of sialolithiasis is necessary to define new therapeutic procedures . The possible presence of a sphincter system in Wharton's duct has been described frequently in the context of diagnostic sialendoscopy . Serial histological examination of the entire Wharton's duct in four samples revealed no anatomical correlation for the presence of a sphincter . Secretory disturbances and viscous secretions as well as microlith formation and ductal obstruction cannot fully explain the genesis of sialoliths . The coaction of those factors with participation of bacteria leads to the development of sialoliths.

J Oral Sci, 2003 Dec, 45(4), 201 - 6
The anti-adherence effect of Piper betle and Psidium guajava extracts on the adhesion of early settlers in dental plaque to saliva-coated glass surfaces; Razak FA et al.; The aqueous extracts of Piper betle and Psidium guajava were prepared and tested for their anti-adherence effect on the adhesion of early plaque settlers (Strep . mitis, Strep . sanguinis and Actinomyces sp.) . The saliva-coated glass surfaces were used to simulate the pellicle-coated enamel surface in the oral cavity . Our results showed that the anti-adherence activities of Piper betle and Psidium guajava extracts towards the bacteria were different between the bacterial species . Psidium guajava was shown to have a slightly greater anti-adherence effect on Strep . sanguinis by 5.5% and Actinomyces sp . by 10% and a significantly higher effect on Strep . mitis (70%) compared to Piper betle . The three bacterial species are known to be highly hydrophobic, and that hydrophobic bonding seemed to be an important factor in their adherence activities . It is therefore suggested that the plant extracts, in expressing their anti-adherence activities, could have altered the hydrophobic nature of the bonding between the bacteria and the saliva-coated glass surfaces.

Curr Biol, 2004 Feb 3, 14(3), 236 - 41
Diverse substrate recognition mechanisms for rhomboids; thrombomodulin is cleaved by Mammalian rhomboids; Lohi O et al.; The rhomboids are a recently discovered family of intramembrane proteases that are conserved across evolution . Drosophila was the first organism in which they were characterized, where at least Rhomboids 1-3 activate EGF receptor signaling by releasing the active forms of EGF-like growth factors . Subsequent work has begun to shed light on the role of these proteases in bacteria and yeast, but nothing is known about the function of rhomboids in vertebrates beyond evidence that the subclass of mitochondrial rhomboids is conserved . Here, we report that the anticoagulant cell-surface protein thrombomodulin is the first mammalian protein to be a rhomboid substrate in a cell culture assay . The thrombomodulin transmembrane domain (TMD) is cleaved only by vertebrate RHBDL2-like rhomboids . Thrombomodulin TMD cleavage is directed not by sequences within the TMD, as is the case with Spitz but by its cytoplasmic domain, which, at least in some contexts, is necessary and sufficient to determine cleavage by RHBDL2 . These data suggest that thrombomodulin could be a physiological substrate for rhomboid . Moreover, the discovery of a second mode of substrate recognition by rhomboids implies mechanistic diversity in this family of intramembrane proteases.

Zhonghua Liu Xing Bing Xue Za Zhi, 2003 Dec, 24(12), 1126 - 8
{A new type of spotted fever group Rickttsiaes detected in the area of Changbai mountain, Jilin province}; Hao YJ et al.; OBJECTIVE: In order to find out the current situation of tick-borne spotted fever in the area of Changbai mountain, Jilin province . METHODS: In this study, a polymerase chain reaction (PCR) method was developed with primers R . rOmpA 190.70p and R . rOmpA 190.701n designed on the basis of rOmpA gene, which is specific for examining spotted fever group Rickttsiaes (SFGR) . Six hundred nighty-three ticks were tested and a positive PCR product amplified from D . silvarum specimen (named JL-02) was cloned and sequenced . RESULTS: The SFGR DNA was detected from D . silvarum, Haemaphysalis concinna with the positive rates were 53.81% and 7.41% respectively . Its nucleotide sequence of 587 bp rOmpA and derived amino-acids showed 100.00% similarity with nucleotide sequence of DnS 14 and 99.00% with DnS 28 from the Former Soviet Union according to the result of BLUST and CLUSTAL, which was differential from the DNA sequences of strains previously detected in China . CONCLUSION: The natural focus of tick-borne spotted fever did exist in the area of Changbai mountain . The DNA sequence of SFGR was similar to that of DnS 14, which was first reported in China.

J Clin Periodontol, 2003 Nov, 30(11), 937 - 43
Expression of RNAs encoding for alpha and beta integrin subunits in periodontitis and in cyclosporin A gingival overgrowth; Bolcato-Bellemin AL et al.; BACKGROUND: Variation of integrin expression in healthy and diseased gingiva revealed a potential biological role for these cell matrix receptors during gingival remodeling . AIM: Here we determined the level of RNA and tissue localization of different integrin subunits in periodontitis and cyclosporin A-induced gingival overgrowth . METHODS: The level of expression was determined by Reverse Transcriptase Polymerase Chain Reaction in 12 periodontitis-affected patients, four patients exhibiting severe cyclosporin A-induced gingival overgrowth and seven healthy patients as controls . RESULTS: The RNA encoding for beta1, alpha2 and alpha5 integrin subunits were reduced in periodontitis gingiva . The reduction observed was stronger in cyclosporin A-treated patients as compared to the healthy controls, while RNA encoding for alpha1 subunit was increased . The RNA encoding for alpha6 integrin was only reduced in cyclosporin A-treated gingiva . Immunohistochemistry showed that i) integrin alpha2 expression is restricted to the gingival epithelium of cyclosporin A-treated patients, ii) the reduction of alpha6 integrin expression in cyclosporin A-treated gingiva is due to loss of expression at focal contacts and iii) beta1 integrin is evenly distributed in the three populations with an intensity decrease in periodontitis and cyclosporin A-treated gingiva . CONCLUSION: Taken together these results showed a role for the integrin receptors in periodontal diseases and cyclosporin A-induced gingival overgrowth.

Int J Med Microbiol, 2003 Dec, 293(6), 391 - 401
Housekeeping enzymes as virulence factors for pathogens; Pancholi V et al.; Housekeeping enzymes are ubiquitously present in almost all living beings to perform essential metabolic functions for the purpose of survival . These enzymes have been characterized in detail for many years . In recent years, there has been a number of reports indicating that some of these enzymes perform a variety of other functions . In case of many pathogens, certain enzymes play a role to enhance virulence . To perform such a function, enzymes must be located on the surface of pathogens . Although they do not have the typical signal sequence or membrane anchoring mechanisms, they do get secreted and are displayed on the surface, probably by their reassociation . Once on the surface, these enzymes interact with host components, such as fibronectin and plasminogen, or interact directly with the host cells, to trigger signal transduction and thereby enable the pathogens to colonize, persist and invade the host tissue . Therefore, certain housekeeping enzymes may act as putative virulence factors and targets for the development of new strategies to control the infection by using agents that can block their secretion and/or reassociation.

Arch Microbiol, 2004 Mar, 181(3), 245 - 9 Epub 2004 Feb 03.
Phenyl methyl ethers: novel electron donors for respiratory growth of Desulfitobacterium hafniense and Desulfitobacterium sp . strain PCE-S; Neumann A et al.; Desulfitobacterium hafniense and Desulfitobacterium sp . strain PCE-S grew under anoxic conditions with a variety of phenyl methyl ethers as electron donors in combination with fumarate as electron acceptor . The phenyl methyl ethers were O-demethylated to the corresponding phenol compounds . O-demethylation was strictly dependent on the presence of fumarate; no O-demethylation occurred with CO2 as electron acceptor . One mol phenyl methyl ether R-O-CH3 was O-demethylated to R-OH per 3 mol fumarate reduced to succinate . The growth yields with vanillate or syringate plus fumarate were approximately 15 g cells (dry weight) per mol methyl moiety converted . D . hafniense utilized vanillate or syringate as an electron donor for reductive dehalogenation of 3-Cl-4-hydroxyphenylacetate, whereas strain PCE-S was not able to dechlorinate tetrachloroethene with phenyl methyl ethers . Crude extracts of both organisms showed O-demethylase activity in the O-demethylase assay with vanillate or syringate as substrates when the organism was grown on syringate plus fumarate . Besides the homoacetogenic bacteria, only growing cells of Desulfitobacterium frappieri PCP-1 have thus far been reported to be capable of phenyl methyl ether O-demethylation . This present study is the first report of Desulfitobacteria utilizing phenyl methyl ethers as electron donors for fumarate reduction and for growth.

Crit Care Med, 2004 Feb, 32(2), 533 - 8
Mesenteric lymph from burned rats induces endothelial cell injury and activates neutrophils; Deitch EA et al.; OBJECTIVE: Our previous studies indicated that mesenteric lymph duct ligation prevented burn-induced lung injury . Thus, the goal of the present study was to begin to investigate potential mechanisms of this protective effect . DESIGN: Prospective animal study with concurrent control . SETTING: Small animal laboratory . SUBJECTS: Adult male Sprague-Dawley rats . INTERVENTIONS: Mesenteric lymph and portal vein plasma were collected from male rats subjected to a 40% third-degree scald burn or sham burn . The biological effects of these lymph and plasma samples were tested for their ability to kill human umbilical vein endothelial cells (HUVECs), increase HUVEC monolayer permeability, and activate polymorphonuclear leukocytes (PMNs), as reflected in CD11b adhesion molecule expression and superoxide production . Additionally, ileal specimens were harvested at the end of the experiment (6 hrs postburn) for histologic analysis . MEASUREMENTS AND MAIN RESULTS: Postburn mesenteric lymph produced during the first 2 hrs after burn injury and tested at a 5% concentration, but not sham-burn lymph or portal plasma from burned rats, was toxic for HUVECs resulting in cell death after an 18-hr incubation period . Similarly, only postburn lymph increased HUVEC monolayer permeability . Postburn lymph activated both rat and human PMNs as reflected in increased CD11b expression and augmentation of the phorbol myristate acetate-induced superoxide response . Neither sham-burn lymph nor postburn portal vein plasma activated PMNs . Both the burn and sham-burn lymph samples were sterile, indicating that the effects of burn lymph on the HUVECs or PMNs were not due to translocating bacteria . Last, an association was found between burn-induced gut injury and the production of toxic burn lymph . CONCLUSIONS: Burn-induced gut injury results in the production of biologically active factors that are carried in the mesenteric lymph, but not the portal plasma, which injure endothelial cells and activate PMNs and thus could contribute to distant organ injury.

Glycoconj J, 2004, 19(7-9), 575 - 81
Galectins as inflammatory mediators; Almkvist J et al.; Over the last decade a vast amount of reports have shown that galectin-1 and galectin-3 are important mediators of inflammation . In this review we describe how the galectins may be involved in several parts of the inflammatory process, including the recruitment of neutrophils into an infected tissue and the recognition and killing of bacteria by activation of the tissue destructive phagocytic respiratory burst . During bacterial infection or aseptic inflammatory processes, galectins are produced and released by e.g . infected epithelium, activated tissue-resident macrophages and endothelial cells . These extracellular galectins may facilitate binding of neutrophils to the endothelium by cross-linking carbohydrates on the respective cells . Further the galectins improve binding of the neutrophil to the extracellular matrix proteins laminin and fibronectin, and are potential chemotactic factors, inducing migration through the extracellular matrix towards the inflammatory focus . When the cells encounter bacteria, galectin-3 could function as an opsonin, cross-linking bacterial lipopolysaccharide or other carbohydrate-containing surface structures to phagocyte surface glycoconjugates . Both galectin-1 and galectin-3 have the capacity to induce a respiratory burst in neutrophils, provided that the cells have been primed by degranulation and receptor upregulation . The reactive oxygen species produced may be destructive to the invading micro-organisms as well as to the surrounding host tissue, pointing out the possible role of galectins, not only in defence toward infection, but also in inflammatory-induced tissue destruction.

Eur J Pharm Sci, 2004 Feb, 21(2-3), 179 - 89
The use of formulation technology to assess regional gastrointestinal drug absorption in humans; Basit AW et al.; The aim of this study was to assess the feasibility of using oral modified-release formulations for the purposes of site-specific targeting and regional drug absorption assessment in man . An immediate release pellet formulation containing ranitidine as the model drug of choice for the study was fabricated by extrusion-spheronisation, and then film coated with either the enteric polymer polyvinyl acetate phthalate or the bacteria-degradable polymer amylose, in combination with ethylcellulose, to effect drug release within the small intestine and colon, respectively . Optimised formulations were evaluated in vivo in ten healthy volunteers, who each received, on four separate occasions, the immediate release, small intestinal release and colonic release formulations (each equivalent to 150mg ranitidine), and an intravenous injection of ranitidine (equivalent to 50mg ranitidine) . Blood samples were collected and assessed for ranitidine concentration, and radiolabelled placebo pellets were co-administered with the coated ranitidine pellets to monitor their gastrointestinal transit using a gamma camera . Ranitidine was rapidly released and absorbed from the immediate release formulation, whereas the enteric formulation (10% coat weight gain) delayed drug release until some or all of the pellets had emptied into the small intestine . The amylose-ethylcellulose coated formulation (coat ratio 1:3, coat weight gain 25%) retarded ranitidine release until the pellets had reached the colon . The mean absolute bioavailability of ranitidine from the immediate release, small intestinal release and colonic release formulations were 50.6, 46.1 and 5.5%, respectively . These data are in general agreement to those obtained from a previous regional intubation study . The present study therefore demonstrates the practical potential of utilising a non-invasive, formulation-based approach to assess drug absorption from different regions of the human gastrointestinal tract.

J Neurosci Methods, 2004 Feb 15, 133(1-2), 91 - 8
Transport of a synaptotagmin-YFP fusion protein in sympathetic neurons during early neurite outgrowth in vitro after transfection in vivo; Narayan S et al.; Developing neurons are engaged in neurite outgrowth as well as the synthesis and transport of proteins involved in synaptic transmission . Very little is known about when transport is established in these rudimentary neurites . We used a novel technique to visualize protein transport during the early hours of neurite outgrowth in culture . Recombinant adenoviruses were used to express a synaptotagmin-YFP fusion protein in the superior cervical ganglia of neonatal rats in vivo and protein transport was examined in neuronal cultures established from the superior cervical ganglions (SCGs) . We find that, as early as 4h in culture, synaptotagmin-YFP was present in the cytoplasm, lamellipodia, filopodia and growth cones . Protein expression appeared punctate in neurites at 8h in vitro and is consistent with a vesicular localization . These results indicate that the machinery to transport synapse-specific proteins is functional in rudimentary neurites at this time and indicates that this technique can be used to study early neuronal development.

FEMS Microbiol Lett, 2004 Jan 30, 230(2), 265 - 74
The activator of the Rhodospirillum rubrum PHB depolymerase is a polypeptide that is extremely resistant to high temperature (121 degrees C) and other physical or chemical stresses; Handrick R et al.; Hydrolysis of native (amorphous) polyhydroxybutyrate (nPHB) granules isolated from different sources by soluble PHB depolymerase of Rhodospirillum rubrum in vitro requires the presence of a heat-stable compound (activator) . The activator was purified and was resistant against various physical and chemical stresses such as heat (up to 130 degrees C), pH 1-12, dryness, oxidation by H2O2, reducing and denaturing compounds (2-mercaptoethanol, 5 M guanidinium-HCl) and many solvents including phenol/chloroform . The activator coding gene was identified by N-terminal sequencing of the purified protein, and the deduced protein showed significant homology to magnetosome-associated protein (Mms16) of magnetotactic bacteria . Analysis of the activation process in vitro showed that the activator acts on nPHB granules but not on the depolymerase . The effect of the activator could be mimicked by pretreatment of nPHB granules with trypsin or other proteases but protease activity of the purified activator was not detected . Evidence is shown that different mechanisms were responsible for activation of nPHB by trypsin and activator, respectively . PHB granule-associated protein (PhaP) of Ralstonia eutropha nPHB granules were cleaved by trypsin but no cleavage occurred after activator treatment . Hydrolysis of artificial protein-free PHB granules coated with negatively charged detergents (sodium dodecyl sulfate (SDS), cholate but not cetyltrimethyl-ammonium bromide (CTAB)) did not require activation and confirmed that surface layer proteins of nPHB granules are the targets of the activator rather than lipids . All experimental data are in agreement with the assumption that trypsin and the activator enable the PHB depolymerase to find and to bind to the polymer surface: trypsin by removing a portion of proteins from the polymer surface, the activator by modifying the surface structure in a not yet understood manner presumably by interaction with phasins of the proteinous surface layer of nPHB.

Dermatol Ther, 2004, 17(1), 102 - 10
The diagnosis and treatment of infectious vaginitis; Edwards L; Inflammation of the vagina as a result of infectious agents is very common, both as an overgrowth of normal or common colonizers, or as a frank infection . The most common causes of infectious vaginitis are yeast, bacteria, protozoa, viruses, and parasites . Infections of the vagina produce an increase in vaginal secretion, vulvar symptoms of itching or irritation from contact with irritating vaginal fluid, and sometimes odor . A careful microscopic examination of vaginal secretions generally yields the correct diagnosis, but atypical or recalcitrant disease deserves a confirmatory culture, as noninfectious inflammatory processes can produce similar symptoms.

Dermatol Ther, 2004, 17(1), 47 - 9
Desquamative inflammatory vaginitis; Murphy R; Desquamative inflammatory vaginitis (DIV) is not a diagnosis in itself, and may be the presentation of a range of blistering disorders including pemphigus vulgaris, lichen planus and mucous membrane pemphigoid . The existence of an idiopathic subset of DIV remains controversial and is discussed in the present article . Desquamative inflammatory vaginitis is a rare but disabling condition . It presents in women of any age with a history of discomfort, irritation and painful sexual intercourse . Patients may also report an increased vaginal discharge . Examination of the vulva is normal, but erythematous regions on the vaginal walls are evident with increased vaginal secretion . This secretion is high in polymorphonuclear leukocytes, with an increased number of immature squamous epithelial cells . Repeated cultures are negative for bacteria, viruses and yeast . This is a sterile inflammatory vaginitis that can be difficult to treat, but successful therapy has been reported with topical steroids and clindamycin.

Mol Microbiol, 2004 Jan, 51(2), 299 - 306
Evolution of fungal sex chromosomes; Fraser JA et al.; Sexual reproduction enables organisms to shuffle two parental genomes to produce recombinant progeny, and to purge the genome of deleterious mutations . Sex is conserved in virtually all organisms, from bacteria and fungi to plants and animals, and yet the mechanisms by which sexual identity are established share both conserved general features and are remarkably diverse . In animals, sexual identity is established by dimorphic sex chromosomes, whereas in fungi a specialized region of the genome, known as the mating-type locus, governs the establishment of cell type identity and differs in DNA sequence between cells of different mating-types . Recent studies on the mating-type loci of fungi and algae reveal features shared with the mammalian X and Y chromosomes, suggesting that these represent early steps in the evolution of sex chromosomes.

Biochemistry, 2004 Feb 10, 43(5), 1135 - 44
Structural insights into chloride and proton-mediated gating of CLC chloride channels; Pusch M; CLC Cl(-) channels fulfill numerous physiological functions as demonstrated by their involvement in several human genetic diseases . They have an unusual homodimeric architecture in which each subunit forms an individual pore whose open probability is regulated by various physicochemical factors, including voltage, Cl(-) concentration, and pH . The voltage dependence of Torpedo channel CLC-0 is derived probably indirectly from the translocation of a Cl(-) ion through the pore during the opening step . Recent structure determinations of bacterial CLC homologues marked a breakthrough for the structure-function analysis of CLC channels . The structures revealed a complex fold with 18 alpha-helices and two Cl(-) ions per subunit bound in the center of the protein . The side chain of a highly conserved glutamate residue that resides in the putative permeation pathway appears to be a major component of the channel gate . First studies have begun to exploit the bacterial structures as guides for a rational structure-function analysis . These studies confirm that the overall structure seems to be conserved from bacteria to humans . A full understanding of the mechanisms of gating of eukaryotic CLC channels is, however, still lacking.

J Exp Zoolog B Mol Dev Evol, 2004 Jan 15, 302(1), 69 - 91
Evo-devo aspects of classical and molecular data in a historical perspective; Sander K et al.; We discuss the interplay between evolution and development as reflected in data and concepts since about 1800 . Darwin and his "continental apostle" Haeckel put the striking similarity between early vertebrate embryos in an evolutionary context . Haeckel's partly illicit generalizations discredited evolutionary thinking among early experimental embryologists who moreover noted riddles incompatible with contemporary concepts of homology and evolution . Relevant solutions were suggested by the more recent concept of ontogenetic networks that embody complex regulatory properties and genes with partly overlapping functions . Molecular data on development increasingly reveal evolutionary opportunism, for instance when a widespread signaling chain involved in primitive immune defense was apparently recruited later on for dorso-ventral axis determination in some evolutionarily advanced insect groups . Recently, Rickettsia-related bacteria colonizing many arthropod species were found to "manipulate" the first steps of host development to the advantage of their own propagation, but by ways that could also promote host speciation . Molecular genetics can now document evolutionary steps in ontogenetic networks . In the fruit fly for instance, the novel bicoid gene has superseded a crucial patterning function within a pre-existing network--a case of "molecular caenogenesis." The expression patterns of conserved genes that antagonistically determine dorso-ventral polarity support a literal revolution envisioned almost 200 years ago . This is the dorso-ventral inversion of the body plan in some metazoans--ascribed then to the Articulata, now to the Chordata . The final section posits that the opportunistic character of evolutionary innovations is detrimental to parsimony in development .

Parasitol Res, 2004 Mar, 92(5), 414 - 20 Epub 2004 Feb 04.
Cloning, expression and partial characterization of a gene encoding the S15a ribosomal protein of Taenia solium; Jimenez L et al.; Ribosomes, ribosomal proteins (r-proteins), and messenger and transfer RNAs catalyze the synthesis of proteins in organisms . To understand and define the components involved in this event in Taenia solium, we isolated and characterized a T . solium cDNA encoding the basic ribosomal protein S15a (TsS15a) . The TsS15a cDNA produces a protein with M(r) (relative molecular mass) 14,988, which contains 22.3% of basic amino acids . Analysis comparing TsS15a protein with other S15a r-proteins indicates that this protein is highly conserved . A recombinant TsS15a protein with similar M(r) was produced in bacteria . Antibodies against recombinant TsS15a react with a 15-kDa protein in extracts from all life stages of T . solium and from all helminths tested . Hybridization studies showed the presence of two genes encoding a mRNA of 0.5 kb . Moreover, the gene presents an intron of 30 bp . Our phylogenetic analysis using S15a r-proteins reproduced the topologies reported for 16/18S rRNA.

Ann Thorac Surg, 2004 Feb, 77(2), 704 - 6
Role of Bartonella henselae endocarditis in the nucleation of aortic valvular calcification; Ghidoni JJ; A 6-year-old boy presented with fatigability, shortness of breath, and bulging neck veins . Echocardiography revealed large vegetations, aortic insufficiency, a dilated left ventricle, and bicuspid aortic valve . There was no history of immunocompromise, fevers, or feline exposures . Blood cultures were negative; antibodies against Bartonella henselae were positive . Gentamicin was administered intravenously . Ross procedure was performed and patient was discharged on antibiotics in 5 days . Native valve was thickened by scar and fibrinous vegetations . Warthin-Starry stain demonstrated coccobacilli . Light and ultrastructural morphology, and monoclonal staining implicated B . henselae . Bacterial membranes contain calcium apatite crystals . Antigenic material was present in bacteria and calcified nodules . This case illustrates calcified protobacteria becoming incorporated into scar tissue during endocarditis.

ScientificWorldJournal, 2004 Jan 16, 4, 9 - 34
The use of plants for remediation of metal-contaminated soils; Vassilev A et al.; The use of green plants to remove, contain, inactivate, or degrade harmful environmental contaminants (generally termed phytoremediation) is an emerging technology . In this paper, an overview is given of existing information concerning the use of plants for the remediation of metal-contaminated soils . Both site decontamination (phytoextraction) and stabilization techniques (phytostabilization) are described . In addition to the plant itself, the use of soil amendments for mobilization (in case of phytoextraction) and immobilization (in case of phytostabilization) is discussed . Also, the economical impacts of changed land-use, eventual valorization of biomass, and cost-benefit aspects of phytoremediation are treated . In spite of the growing public and commercial interest and success, more fundamental research is needed still to better exploit the metabolic diversity of the plants themselves, but also to better understand the complex interactions between metals, soil, plant roots, and micro-organisms (bacteria and mycorrhiza) in the rhizosphere . Further, more demonstration experiments are needed to measure the underlying economics, for public acceptance and last but not least, to convince policy makers.

Amino Acids, 2004 Feb, 26(1), 3 - 8 Epub 2003 Jul 29.
Metabolism and function in animal tissues of agmatine, a biogenic amine formed from arginine; Grillo MA et al.; Recently agmatine, decarboxylated arginine, has been shown to be an important biological compound in several animal tissues . This paper summarizes the known information regarding the transport of arginine, its decarboxylation and the effects of the agmatine formed mainly on NO and polyamine synthesis.

J Biomol NMR, 2004 Mar, 28(3), 235 - 47
Protein signal assignments using specific labeling and cell-free synthesis; Shi J et al.; The goal of structural genomics initiatives is to determine complete sets of protein structures that represent recently sequenced genomes . The development of new high throughput methods is an essential aspect of this enterprise . Residue type and sequential assignments obtained from specifically labeled samples, when combined with 3D heteronuclear data, can significantly increase the efficiency and accuracy of the assignment process, the first step in structure determination by NMR . A protocol for the design of specifically labeled samples with high information content is presented along with a description of the experiments used to extract essential information using 2D versions of 3D heteronuclear experiments . In vitro protein synthesis methods were used to produce four specifically labeled samples of the 23.5 kDa protein phosphoserine phosphatase (PSP) from Methanoccous jannaschii (MJ1594) . Each sample contained two (13)C/(15)N-labeled amino acids and one (15)N-labeled amino acid . The 135 type and 14 sequential assignments obtained from these samples were used in conjunction with 3D data obtained from uniformly (13)C/(15)N-labeled and (2)H/(13)C/(15)N-labeled protein to manually assign the backbone (1)H(N), (15)N, (13)CO, (13)C(alpha), and (13)C(beta) signals . Using an automated assignment algorithm, 30% more assignments were obtained when the type and sequential assignments were used in the calculations.

Urology, 2004 Jan, 63(1), 175 - 6
Dorsal urethral fistula: case report and review of literature; Grandhi TM et al.; Urethral fistula is a rare, but recognized, entity . We report the case of a young diabetic patient who developed urethral fistula on the dorsum of penis after debridement for necrotizing fasciitis . This cause and location for urethral fistula is extremely rare, and we were unable to find a similar case in published studies . A brief literature search for various causes of urethral fistula was made, and the likely mechanisms for the cause of the fistula were explored.

Microsc Microanal, 2003 Dec, 9(6), 542 - 55
Extending energy-filtered transmission electron microscopy (EFTEM) into three dimensions using electron tomography; Weyland M et al.; The length scales on which materials microstructures are being formed, grown, and even designed are becoming increasingly small and increasingly three-dimensional . For such complex structures two-dimensional transmission electron microscopy (TEM) analysis is often inadequate and occasionally misleading . One approach to this problem is the modification of electron tomography techniques, developed for structural biology, for use in materials science . Energy-Filtered (EF) TEM elemental distribution images approximate to true projections of structure, and, as such, can be used to reconstruct the three-dimensional distribution of chemical species . A sample holder has been modified to allow the high tilt (+/-60 degrees ) required for tomography and a semiautomatic acquisition script designed to manage energy-loss acquisition . Tilt series data sets have been acquired from two widely different experimental systems, Cr carbides in 316 stainless steel and magnetite nanocrystals in magnetotactic bacteria, demonstrating single- and multiple-element tomography . It is shown that both elemental maps and jump-ratio images are suitable for reconstruction, despite the effects of diffraction contrast in the former and thickness changes in the latter . It is concluded that the image contrast, signal, and signal-to-noise ratio (SNR) are key to the achievable reconstruction quality and, as such, the technique may be of limited value for high energy loss/small inelastic cross section edges.

Clin Rheumatol, 2004 Feb, 23(1), 73 - 5 Epub 2004 Jan 08.
An unusual pattern of arthritis in a child with Kawasaki syndrome; Duzova A et al.; Arthritis is reported in one-third of cases with Kawasaki syndrome . It may have an early or a late onset form . We present a 15-month-old-girl who had been referred with complaints of pain and swelling in her left shoulder . Physical examination revealed bulbar conjunctival injection, erythematous lips and pharynx, strawberry tongue, erythematous rash, edema and erythema of the left shoulder, left knee, right elbow and right wrist, and moderate distress in the left shoulder and left hip . She was diagnosed with Kawasaki syndrome, and intravenous immunoglobulin infusion (IVIG) 2 g/kg and aspirin (100 mg/kg/day) were instituted . The patient had two additional episodes of arthritis involving the hip joint on the 8th day, and the shoulder and metacarpophalangeal (MCP) and interphalangeal (IP) joints of her right hand on the 15th day . Turbid material was aspirated in both instances; Gram and Wright's staining of this material showed many leukocytes but no bacteria . A second dose of IVIG (1 g/kg) was given . At the end of the third week all extremities were painless, with a normal range of motion . Arthritis in our patient was the presenting sign, having a 'septic arthritis mimicking' and 'biphasic' pattern . Although the patient presented with severe and recurrent arthritis, which is significantly correlated with severe multisystem disease and the presence or development of coronary artery aneurysm, the response to IVIG was excellent.

Nature, 2004 Jan 29, 427(6973), 445 - 8
A newly discovered Roseobacter cluster in temperate and polar oceans; Selje N et al.; Bacterioplankton phylotypes of alpha-Proteobacteria have been detected in various marine regions, but systematic biogeographical studies of their global distribution are missing . Alpha-Proteobacteria comprise one of the largest fractions of heterotrophic marine bacteria and include two clades, SAR11 and Roseobacter, which account for 26 and 16% of 16S ribosomal RNA gene clones retrieved from marine bacterioplankton . The SAR11 clade attracted much interest because related 16S rRNA gene clones were among the first groups of marine bacteria to be identified by cultivation-independent approaches and appear to dominate subtropical surface bacterioplankton communities . Here we report on the global distribution of a newly discovered cluster affiliated to the Roseobacter clade, comprising only as-yet-uncultured phylotypes . Bacteria of this cluster occur from temperate to polar regions with highest abundance in the Southern Ocean, but not in tropical and subtropical regions . Between the south Atlantic subtropical front and Antarctica, we detected two distinct phylotypes, one north and one south of the polar front, indicating that two adjacent but different oceanic provinces allow the persistence of distinct but closely related phylotypes . These results suggest that the global distribution of major marine bacterioplankton components is related to oceanic water masses and controlled by their environmental and biogeochemical properties.

J Neurosci, 2004 Jan 28, 24(4), 972 - 81
Dopamine neurons mediate a fast excitatory signal via their glutamatergic synapses; Chuhma N et al.; Dopamine neurons are thought to convey a fast, incentive salience signal, faster than can be mediated by dopamine . A resolution of this paradox may be that midbrain dopamine neurons exert fast excitatory actions . Using transgenic mice with fluorescent dopamine neurons, in which the axonal projections of the neurons are visible, we made horizontal brain slices encompassing the mesoaccumbens dopamine projection . Focal extracellular stimulation of dopamine neurons in the ventral tegmental area evoked dopamine release and early monosynaptic and late polysynaptic excitatory responses in postsynaptic nucleus accumbens neurons . Local superfusion of the ventral tegmental area with glutamate, which should activate dopamine neurons selectively, produced an increase in excitatory synaptic events . Local superfusion of the ventral tegmental area with the D2 agonist quinpirole, which should increase the threshold for dopamine neuron activation, inhibited the early response . So dopamine neurons make glutamatergic synaptic connections to accumbens neurons . We propose that dopamine neuron glutamatergic transmission may be the initial component of the incentive salience signal.

J Neurosurg Spine, 2004 Jan, 100(1), 46 - 51
Rotational and transpositional flaps for the treatment of spinal wound dehiscence and infections in patient populations with degenerative and oncological disease; Vitaz TW et al.; OBJECT: Wound-related complications following complex posterior spine procedures may result in the need for serial debridements and may place the instrumentation at risk . Numerous treatments have been advocated for this problem, but each has limitations . In this article the authors discuss the experience from two large teaching institutions at which rotational and transpositional flaps were used in the management of deep wound infections and dehiscences . METHODS: The authors generated a list of patients treated via posterior or posterolateral approaches for metastatic tumors or complex degenerative disorders in whom wound complications subsequently developed . Data were obtained from the medical records and reviewed retrospectively . Thirty-seven patients were treated with rotational or transpositional flaps at the two institutions during the study period . Patients underwent a mean of 1.3 procedures for the treatment of wound healing problems, and cultures were positive in 70% . In three patients (8%) this treatment failed due to protrusion of hardware through the skin or repeated dehiscence requiring reclosure . Spinal instrumentation was salvaged in 97% of the cases . CONCLUSIONS: The use of local tissue flaps is advantageous for treatment of posterior wound complications due to spine surgery . In this procedure highly vascularized tissue is used to increase healing, accelerate clearance of bacteria, and fill any dead space.

Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 344 - 6 Epub 2004 Jan 23.
Cloning, purification, crystallization and preliminary crystallographic studies of Bradyrhizobium fucosyltransferase NodZ; Brzezinski K et al.; The alpha-1,6-fucosyltransferase NodZ from Bradyrhizobium sp . WM9 (Lupinus), composed of 325 amino acids with a molecular weight of 37 kDa, has been cloned, expressed and purified . Protein crystals suitable for X-ray diffraction were obtained under optimized crystallization conditions using ammonium dihydrogen phosphate as a precipitant . The crystals are hexagonal and belong to space group P6(1)22 or P6(5)22, with unit-cell parameters a = 125.5, c = 95.6 A, and contain 56.8% solvent and a single protein molecule in the asymmetric unit . Native data were collected to 2.85 A using synchrotron radiation and cryogenic conditions . The native crystals were soaked in a mother-liquor solution containing 2.5 mM {Ta(6)Br(12)}(2+) cluster for derivatization and SAD data were collected to 3.4 A at the tantalum L(III) absorption peak.

Biophys J, 2004 Feb, 86(2), 665 - 80
Proton transfer dynamics at the membrane/water interface: dependence on the fixed and mobile pH buffers, on the size and form of membrane particles, and on the interfacial potential barrier; Cherepanov DA et al.; Crossing the membrane/water interface is an indispensable step in the transmembrane proton transfer . Elsewhere we have shown that the low dielectric permittivity of the surface water gives rise to a potential barrier for ions, so that the surface pH can deviate from that in the bulk water at steady operation of proton pumps . Here we addressed the retardation in the pulsed proton transfer across the interface as observed when light-triggered membrane proton pumps ejected or captured protons . By solving the system of diffusion equations we analyzed how the proton relaxation depends on the concentration of mobile pH buffers, on the surface buffer capacity, on the form and size of membrane particles, and on the height of the potential barrier . The fit of experimental data on proton relaxation in chromatophore vesicles from phototropic bacteria and in bacteriorhodopsin-containing membranes yielded estimates for the interfacial potential barrier for H(+)/OH(-) ions of approximately 120 meV . We analyzed published data on the acceleration of proton equilibration by anionic pH buffers and found that the height of the interfacial barrier correlated with their electric charge ranging from 90 to 120 meV for the singly charged species to >360 meV for the tetra-charged pyranine.

Mol Biochem Parasitol, 2004 Mar, 134(1), 105 - 14
A novel form of actin in Leishmania: molecular characterisation, subcellular localisation and association with subpellicular microtubules; Sahasrabuddhe AA et al.; To study the occurrence and subcellular distribution of actin in trypanosomatid parasites, we have cloned and overexpressed Leishmania donovani actin gene in bacteria, purified the protein, and employed the affinity purified rabbit polyclonal anti-recombinant actin antibodies as a probe to study the organisation and subcellular distribution of actin in Leishmania cells . The Leishmania actin did not cross react with antimammalian actin antibodies but was readily recognized by the anti-Leishmania actin antibodies in both the promastigote and amastigote forms of the parasite . About 10(6) copies per cell of this protein (M(r) 42.05 kDa) were present in the Leishmania promastigote . Unlike other eukaryotic actins, the oligomeric forms of Leishmania actin were not stained by phalloidin nor were dissociated by actin filament-disrupting agents, like Latrunculin B and Cytochalasin D . Analysis of the primary structure of this protein revealed that these unusual characteristics may be related to the presence of highly diverged amino acids in the DNase I-binding loop (amino acids 40-50) and the hydrophobic plug (amino acids 262-272) regions of Leishmania actin . The subcellular distribution of actin was studied in the Leishmania promastigotes by employing immunoelectron and immunofluorescence microscopies . This protein was present not only in the flagella, flagellar pocket, nucleus and the kinetoplast but it was also localized on the nuclear, vacuolar and cytoplasmic face of the plasma membranes . Further, the plasma membrane-associated actin was colocalised with subpellicular microtubules, while most of the actin present in the kinetoplast colocalised with the k-DNA network.These results clearly indicate that Leishmania contains a novel form of actin which may structurally and functionally differ from other eukaryotic actins . The functional significance of these observations is discussed.

Spectrochim Acta A Mol Biomol Spectrosc, 2004 Feb, 60(3), 527 - 34
Raman properties of chlorophyll d, the major pigment of Acaryochloris marina: studies using both Raman spectroscopy and density functional theory; Chen M et al.; The Raman spectroscopy of purified chlorophyll (Chl) d extracted from Acaryochloris marina has been measured over the wide region of 250-3200 cm(-1) at 77 K following excitation of its Soret band at 488 nm and analyzed with the aid of hybrid density-functional vibrational analyses . A Raman peak specific to Chl d, which arises from the formyl group 3(1) C=O stretching, was clearly observed at 1659 cm(-1) with medium intensity . Peaks due to other C=O stretching vibrations of the 13(1) keto-, 13(3) ester- and 17(3) groups were also observed . Four very strong peaks were observed in the range of 1000-1600 cm(-1), assigned to the CC stretching and mixtures of the CH3 bend and CN stretching . CCC and NCC bending contribute to medium intensity peaks at 986 and 915 cm(-1) . Out-of-plane CH bending at Chl d methine sites 10, 5 and 20 contribute to observed peaks at 885, 864 and 853 cm(-1), respectively . A few modes involving the MgN stretching and MgNC bending motions were observed in the very low frequency range . Density functional theory (DFT) calculations have been used to make assignments on the observed Raman spectrum and the DFT results have been found to be in good agreement with the experimental results.

Trends Parasitol, 2004 Feb, 20(2), 54 - 7
More plastids in human parasites?
Waller RF, McConville MJ, McFadden GI.
Trypanosomatid parasites are disease agents with an extraordinarily broad host range including humans, livestock and plants . Recent work has revealed that trypanosomatids harbour numerous genes sharing apparent common ancestry with plants and/or bacteria . Although there is no evidence of a plastid (chloroplast-like organelle) in trypanosomatids, the presence of such genes suggests lateral gene transfer from some photosynthetic organism(s) during trypanosomatid evolution . Remarkably, many products of these horizontally acquired genes now function in the glycosome, a highly modified peroxisome unique to trypanosomatids and their near relatives.

Biochim Biophys Acta, 2004 Jan 20, 1676(2), 119 - 26
Identification and characterization of mouse TRMU gene encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase; Yan Q et al.; The nucleotide modification in tRNA plays a pivotal role in the fidelity of translational process . The mutated mitochondrial tRNA (mt tRNA) associated with human diseases often exhibited a defect in nucleotide modification at wobble position of anticodons . Recently, the product of trmU, 5-methylaminomethyl-2-thiouridylate-methyltransferase, has been shown to be one component of enzyme complex for the biosynthesis of mnm5s2U in the wobble position of the bacterial tRNAs . Here we report the identification and characterization of mouse TRMU homolog . A 1532 bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated . The mouse TRMU gene containing 11 exons encodes a 417 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs related to tRNA modification . The mouse TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver and brain . Furthermore, immunofluorescence analysis of NIH3T3 cells expressing TRMU-GFP fusion protein demonstrated that the mouse Trmu localizes in mitochondria . These observations suggest that the mouse TRMU is a structural and functional homolog of bacterial TrmU, thereby playing a role in the mt tRNA modification and protein synthesis.

J Tenn Dent Assoc, 2003 Fall, 83(4), 20 - 4
Curing halitosis: the sweet smell of success; Weesner BW Jr; Halitosis is socially incapacitating, physically damaging, and difficult to discuss . As the dentist is responsible for the oral cavity, it behooves the dental health provider to diagnose and treat halitosis if we are to offer total oral health care to our patients . While oral malodor has been recognized in the literature throughout our history, interest has recently increased in treating the problem and research is now providing the answers to this condition . This article summarizes some of the current research in managing halitosis.

Bioessays, 2004 Feb, 26(2), 151 - 8
Post-replication repair in DT40 cells: translesion polymerases versus recombinases; Hochegger H et al.; Replication forks inevitably stall at damaged DNA in every cell cycle . The ability to overcome DNA lesions is an essential feature of the replication machinery . A variety of specialized polymerases have recently been discovered, which enable cells to replicate past various forms of damage by a process termed translesion synthesis . Alternatively, homologous recombination can be used to restart DNA replication across the lesion . Genetic and biochemical studies have shed light on the impact of these two post-replication repair pathways in bacteria and yeast . In vertebrates, however, a genetic approach to study post-replication repair has been compromised because many of the genes involved appear to be essential for embryonic development . We have taken advantage of the chicken cell line DT40 to perform a genetic analysis of translesion synthesis and homologous recombination and to characterize genetic interactions between these two pathways in vertebrates . In this article, we aim to summarize our current understanding of post-replication repair in DT40 in the perspective of bacterial, yeast and mammalian genetics .

Hum Pathol, 2004 Jan, 35(1), 113 - 21
Age-dependent development of the splenic marginal zone in human infants is associated with different causes of death; Kruschinski C et al.; Infants are more susceptible to infections caused by T cell- independent type 2 (TI-2) polysaccharide antigens of certain encapsulated bacteria . Immune responses against this type of antigen are related to the splenic marginal zone (MZ) . However, only few data exist on the age-dependent developmental stages of the human spleen in early childhood and on their association with different diseases . Therefore, the present study aimed to investigate spleens of a large number of children at very young ages (12 days to 32 months), derived from autopsy cases . Immunohistochemical labeling was performed on paraffin sections of 34 spleens using a panel of monoclonal antibodies . The shape and size of the white pulp compartments were examined and correlated to the cause of death of the children . Results show that the development of the different compartments was statistically age-dependent, but no clear-cut time point for the maturity of each compartment was seen . Furthermore, the MZ was significantly more often missing when sudden infant death (SID) and/or infection were the cause of death, compared with other violent or traumatic reasons that served as controls . This association supports the concept that an immature state of the spleen and especially of the MZ might contribute to the increased susceptibility to bacterial infections in young infants.

J Mol Evol, 2003 Dec, 57(6), 672 - 80
Speciation in Chlamydia: genomewide phylogenetic analyses identified a reliable set of acquired genes; Ortutay C et al.; Horizontal gene transfer (HGT), a process through which genomes acquire sequences from distantly related organisms, is believed to be a major source of genetic diversity in bacteria . A central question concerning the impact of HGT on bacterial genome evolution is the proportion of horizontally transferred sequences within genomes . This issue, however, remains unresolved because the various methods developed to detect potential HGT events identify different sets of genes . The present-day consensus is that phylogenetic analysis of individual genes is still the most objective and accurate approach for determining the occurrence and directionality of HGT . Here we present a genome-scale phylogenetic analysis of protein-encoding genes from five closely related Chlamydia, identifying a reliable set of sequences that have arisen via HGT since the divergence of the Chlamydia lineage . According to our knowledge, this is the first systematic phylogenetic inference-based attempt to establish a reliable set of acquired genes in a bacterial genome . Although Chlamydia are obligate intracellular parasites of higher eukaryotes, and thus suspected to be isolated from HGT more than the free-living species, our results show that their diversification has involved the introduction of foreign sequences into their genome . Furthermore, we also identified a complete set of genes that have undergone deletion, duplication, or rearrangement during this evolutionary period leading to the radiation of Chlamydia species . Our analysis may provide a deeper insight into how these medically important pathogens emerged and evolved from a common ancestor.

J Math Biol, 2004 Feb, 48(2), 218 - 42 Epub 2003 Aug 20.
Macrophage response to Mycobacterium tuberculosis infection; Gammack D et al.; The immune response to Mycobacterium tuberculosis (Mtb) infection is the formation of multicellular lesions, or granolomas, in the lung of the individual . However, the structure of the granulomas and the spatial distribution of the immune cells within is not well understood . In this paper we develop a mathematical model investigating the early and initial immune response to Mtb . The model consists of coupled reaction-diffusion-advection partial differential equations governing the dynamics of the relevant macrophage and bacteria populations and a bacteria-produced chemokine . Our novel application of mathematical concepts of internal states and internal velocity allows us to begin to study this unique immunological structure . Volume changes resulting from proliferation and death terms generate a velocity field by which all cells are transported within the forming granuloma . We present numerical results for two distinct infection outcomes: controlled and uncontrolled granuloma growth . Using a simplified model we are able to analytically determine conditions under which the bacteria population decreases, representing early clearance of infection, or grows, representing the initial stages of granuloma formation.

Cell Mol Life Sci, 2004 Jan, 61(2), 192 - 208
Diversity of structures and properties among catalases; Chelikani P et al.; More than 300 catalase sequences are now available, divided among monofunctional catalases (> 225), bifunctional catalase-peroxidases (> 50) and manganese-containing catalases (> 25) . When combined with the recent appearance of crystal structures from at least two representatives from each of these groups (nine from the monofunctional catalases), valuable insights into the catalatic reaction mechanism in its various forms and into catalase evolution have been gained . The structures have revealed an unusually large number of modifications unique to catalases, a result of interacting with reactive oxygen species . Biochemical and physiological characterization of catalases from many different organisms has revealed a surprisingly wide range of catalatic efficiencies, despite similar sequences . Catalase gene expression in micro-organisms generally is controlled either by sensors of reactive oxygen species or by growth phase regulons, although the detailed mechanisms vary considerably.

J Exp Med, 2004 Feb 2, 199(3), 315 - 21 Epub 2004 Jan 26.
The metallo-beta-lactamase/beta-CASP domain of Artemis constitutes the catalytic core for V(D)J recombination; Poinsignon C et al.; The V(D)J recombination/DNA repair factor Artemis belongs to the metallo-beta-lactamase (beta-Lact) superfamily of enzymes . Three regions can be defined within the Artemis protein sequence: (a) the beta-Lact homology domain, to which is appended (b) the beta-CASP region, specific of members of the beta-Lact superfamily acting on nucleic acids, and (c) the COOH-terminal domain . Using in vitro mutagenesis, here we show that the association of the beta-Lact and the beta-CASP regions suffices for in vivo V(D)J recombination of chromosome-integrated substrates . Single amino acid mutants point to critical catalytic residues for V(D)J recombination activity . The results presented here define the beta-Lact/beta-CASP domain of Artemis as the minimal core catalytic domain needed for V(D)J recombination and suggest that Artemis uses one or two Zn(II) ions to exert its catalytic activity, like bacterial class B beta-Lact enzymes hydrolyzing beta-lactam compounds.

Zhonghua Er Ke Za Zhi, 2003 Aug, 41(8), 578 - 81
{Development of a gap ligase chain reaction for detection of Chlamydia trachomatis in newborn infants}; Wei H et al.; OBJECTIVE: To establish a gap ligase chain reaction (G-LCR) assay for the detection of Chlamydia trachomatis (Ct) in neonates with pneumonia . METHODS: A G-LCR DNA amplification assay that targeted the outer major membrane protein gene (omp1) of Ct was developed to detect Ct . The sensitivity and specificity of the G-LCR test was examined by the use of highly purified elementary bodies (EBs) . Nasopharyngeal swabs taken from 328 neonates with pneumonia were analyzed by Gap-LCR and cell culture . RESULTS: The detection limit of G-LCR was 2 EBs . G-LCR could detect five species of Ct and was not cross-reacted with C psittaci and other bacteria . The prevalence of Ct in 328 neonates with pneumonia, using an expanded gold standard of a positive cell culture or two confirmed positive non-culture tests, was 21% (69/328) . After analysis of discrepant results, the sensitivity, specificity, and positive and negative predictive values for the G-LCR were 98.6%, 100%, 100% and 99.6%, respectively; whereas those for culture were 86.9%, 100%, 100% and 96.6%, respectively . CONCLUSION: This study demonstrated that the G-LCR was a highly sensitive nonculture technique and good alternative test for the detection of chlamydial infections.

Infect Immun, 2004 Feb, 72(2), 1107 - 15
Protection against CD95-induced apoptosis by chlamydial infection at a mitochondrial step; Fischer SF et al.; Chlamydiae are obligate intracellular bacteria that infect human epithelial and myeloid cells . Previous work has established that chlamydiae are able to protect a cell against apoptosis induced by certain experimentally applied stimuli . Here we provide an analysis of this protective activity against the signal transduction during CD95-induced apoptosis . In HeLa cells overexpressing CD95, infection with Chlamydia trachomatis inhibited the appearance of apoptotic morphology, effector caspase activity, the activation of caspase-9 and -3, and the release of cytochrome c from mitochondria . However, caspase-8-processing and activity (measured as cleavage of Bid) were unaffected by the chlamydial infection . Similarly, infection with the species C . pneumoniae did not prevent the activation of caspase-8 but inhibited the appearance of effector caspase activity upon signaling through CD95 . Furthermore, infection with C . trachomatis was able to inhibit CD95-induced apoptosis in Jurkat lymphoid cells, where a mitochondrial contribution is required, but not in SKW6.4 lymphoid cells, where caspase-8 directly activates caspase-3 . Taken together, these data show that chlamydial infection can protect cells against CD95-induced apoptosis but only where a mitochondrial signaling step is necessary for apoptotic signal transduction.

Infect Immun, 2004 Feb, 72(2), 896 - 907
RhuR, an extracytoplasmic function sigma factor activator, is essential for heme-dependent expression of the outer membrane heme and hemoprotein receptor of Bordetella avium; Kirby AE et al.; Genes involved in iron (Fe) acquisition often are regulated in response to the local availability of Fe . In many bacteria, Fe-dependent responsiveness is mediated by Fur, a global Fe-dependent transcriptional repressor . Tighter regulatory control of Fur-responsive genes is afforded by incorporating additional regulators into Fur-dependent regulatory cascades . RhuI, a Fur-dependent extracytoplasmic function sigma factor of Bordetella avium, in response to the dual stimulation of Fe starvation and the presence of heme (or hemoproteins), regulates P(bhuR), a heme-responsive promoter which directs expression of the bhuRSTUV heme utilization operon . While BhuR, the outer membrane heme receptor, and RhuI have been shown to be indispensable for heme-dependent activation of P(bhuR), collateral components of the regulatory cascade have not been described . In this investigation, RhuR, an integral cytoplasmic membrane protein with homology to anti-sigma factors, is shown to be an essential activator of P(bhuR) expression . The functional domain of RhuR required for heme-dependent activation of P(bhuR) expression was mapped to the N-terminal 97 amino acids of the protein by use of a chimeric RhuR-BlaM fusion . Expression of the chimera in a rhuR mutant rendered P(bhuR) constitutive, thereby decoupling the promoter from heme dependency . Growth studies confirmed that B . avium requires RhuR for optimal utilization of hemoglobin, but not hemin, as a sole source of nutrient Fe . These data imply that B . avium expresses, in addition to the BhuR heme/hemoprotein utilization system, an alternative RhuR-independent heme utilization mechanism . A model is proposed in which RhuR is the functional bridge between BhuR and RhuI in a heme-dependent regulatory cascade.

Infect Immun, 2004 Feb, 72(2), 742 - 9
Host-inducible immunogenic sphingomyelinase-like protein, Lk73.5, of Leptospira interrogans; Artiushin S et al.; Leptospira interrogans causes a variety of clinical syndromes in animals and humans . Although much information has accumulated on the importance of leptospiral lipopolysaccharide in protective antibody responses, relatively little is known about proteins that participate in immune responses . Identification of those proteins induced only in the host is particularly difficult . Using a novel double-antibody screen designed to identify clones in a gene library of L . interrogans serovar Pomona expressing host-inducible proteins, we have characterized a gene (lk75.3) encoding a sphingomyelinase-like preprotein of 648 amino acids with cytotoxic activity for equine pulmonary endothelial cells and weak hemolytic activity for equine and rabbit erythrocytes . lk73.5 was found as a single gene copy in all serovars of L . interrogans but not in other Leptospira spp . except L . inadai . The open reading frame (ORF) for Lk73.5 is followed by another partially homologous sequence containing an ORF (sph-like 2) for a 28.7-kDa peptide . Lk73.5 and Sph-like 2 share 95.1 and 97.7% amino acid identity with putative sphingomyelinases Sph2 and Sph1 (N terminus) from L . interrogans serovar Lai (S.-X . Ren, G . Fu, X.-G . Jiangk, R . Zeng, Y.-G . Miao, H . Xu, Y.-X . Zhang, H . Xiong, G . Lu, L.-F . Lu, H.-Q . Jiang, J . Jia, Y.-F . Tu, J.-X . Jiang, W.-Y . Gu, Y.-Q . Zhang, Z . Cai, H.-H . Sheng, H.-F . Yin, Y . Zhang, G.-F . Zhu, M . Wank, H.-L . Huangk, Z . Qian, S.-Y . Wang, Wei Ma, Z.-J . Yao, Y . Shen, B.-Q . Qiang, Q.-C . Xia, X.-K . Guo, A . Danchinq, I . S . Girons, R . L . Somerville, Y.-M . Wen, M.-H . Shik, Z . Chen, J.-G . Xuk, and G.-P . Zhao, Nature 422:88-893, 2003) . Substantial homologies to sphingomyelinases from other leptospiras and other bacteria are also present . Lk73.5 was not detected in leptospiras cultured at 30 or 37 degrees C . The recombinant protein reacted strongly with sera from recently infected mares but not with sera from horses vaccinated with commercial pentavalent bacterin . The host-inducible immunogenic Lk73.5 should have value in distinguishing vaccine from infection immune response.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 215 - 20
Bartonella chomelii sp . nov., isolated from French domestic cattle (Bos taurus); Maillard R et al.; Two strains of bacteria isolated from the blood of French domestic cows were found to be similar to Bartonella species on the basis of phenotypic characteristics . Genotypic analysis based on sequence comparison of the 16S rRNA and citrate synthase (gltA) genes and on DNA-DNA hybridization showed that the two isolates represent a distinct and new species of Bartonella . Moreover, the phylogenetic analysis inferred from comparison of 16S rRNA and gltA sequences demonstrated that the new Bartonella species is related to other ruminant-derived Bartonella species . The name Bartonella chomelii is proposed for the new species . The type strain of Bartonella chomelii sp . nov . is A828T (=CIP 107869T=CCUG47497T).

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 203 - 9
Phylogenetic analysis of Streptomyces spp . isolated from potato scab lesions in Korea on the basis of 16S rRNA gene and 16S-23S rDNA internally transcribed spacer sequences; Song J et al.; The 16S rRNA gene sequences for 34 strains, including 11 isolates, were determined to classify scab-causing Streptomyces spp . and relatives isolated from potato scab lesions collected in Jeju, Korea . The 16S-23S rDNA internally transcribed spacer (ITS) sequences were determined to investigate whether the 16S-23S ITS region is useful for analysing intra- and interspecific relationships in these bacteria . On the basis of phylogenetic analysis of 16S rRNA gene sequences, most of the isolates were classified as Streptomyces scabiei and Streptomyces acidiscabies . Isolate KJO61 was placed in an ambiguous taxonomic position between Streptomyces reticuliscabiei and Streptomyces turgidiscabies . 16S-23S ITS region sequence analysis showed that tRNA genes were not found in this region of Streptomyces spp . The 16S-23S ITS regions of Streptomyces spp . exhibited various lengths and highly variable sequence similarities (35-100%) within strains as well as intra- and interspecies . It was revealed that Streptomyces europaeiscabiei could be clearly differentiated from Streptomyces scabiei . However, it was clarified that ITS regions are not useful in phylogenetic analysis of Streptomyces spp.

Acta Biochim Pol, 2003, 50(4), 1245 - 56
Plant purple acid phosphatases - genes, structures and biological function; Olczak M et al.; The properties of plant purple acid phosphatases (PAPs), metallophosphoesterases present in some bacteria, plants and animals are reviewed . All members of this group contain a characteristic set of seven amino-acid residues involved in metal ligation . Animal PAPs contain a binuclear metallic center composed of two irons, whereas in plant PAPs one iron ion is joined by zinc or manganese ion . Among plant PAPs two groups can be distinguished: small PAPs, monomeric proteins with molecular mass around 35 kDa, structurally close to mammalian PAPs, and large PAPs, homodimeric proteins with a single polypeptide of about 55 kDa . Large plant PAPs exhibit two types of structural organization . One type comprises enzymes with subunits bound by a disulfide bridge formed by cysteines located in the C-terminal region around position 350 . In the second type no cysteines are located in this position and no disulfide bridges are formed between subunits . Differences in structural organisation are reflected in substrate preferences . Recent data reveal in plants the occurrence of metallophosphoesterases structurally different from small or large PAPs but with metal-ligating sequences characteristic for PAPs and expressing pronounced specificity towards phytate or diphosphate nucleosides and inorganic pyrophosphate.

Acta Biochim Pol, 2003, 50(4), 1129 - 46
Melatonin as an antioxidant: biochemical mechanisms and pathophysiological implications in humans; Reiter RJ et al.; This brief resume enumerates the multiple actions of melatonin as an antioxidant . This indoleamine is produced in the vertebrate pineal gland, the retina and possibly some other organs . Additionally, however, it is found in invertebrates, bacteria, unicellular organisms as well as in plants, all of which do not have a pineal gland . Melatonin's functions as an antioxidant include: a), direct free radical scavenging, b), stimulation of antioxidative enzymes, c), increasing the efficiency of mitochondrial oxidative phosphorylation and reducing electron leakage (thereby lowering free radical generation), and 3), augmenting the efficiency of other antioxidants . There may be other functions of melatonin, yet undiscovered, which enhance its ability to protect against molecular damage by oxygen and nitrogen-based toxic reactants . Numerous in vitro and in vivo studies have documented the ability of both physiological and pharmacological concentrations to melatonin to protect against free radical destruction . Furthermore, clinical tests utilizing melatonin have proven highly successful; because of the positive outcomes of these studies, melatonin's use in disease states and processes where free radical damage is involved should be increased.

Acta Biochim Pol, 2003, 50(4), 891 - 908
Non-homologous DNA end joining; Pastwa E et al.; DNA double-strand breaks (DSBs) are a serious threat for the cell and when not repaired or misrepaired can result in mutations or chromosome rearrangements and eventually in cell death . Therefore, cells have evolved a number of pathways to deal with DSB including homologous recombination (HR), single-strand annealing (SSA) and non-homologous end joining (NHEJ) . In mammals DSBs are primarily repaired by NHEJ and HR, while HR repair dominates in yeast, but this depends also on the phase of the cell cycle . NHEJ functions in all kinds of cells, from bacteria to man, and depends on the structure of DSB termini . In this process two DNA ends are joined directly, usually with no sequence homology, although in the case of same polarity of the single stranded overhangs in DSBs, regions of microhomology are utilized . The usage of microhomology is common in DNA end-joining of physiological DSBs, such as at the coding ends in V(D)J (variable(diversity) joining) recombination . The main components of the NHEJ system in eukaryotes are the catalytic subunit of DNA protein kinase (DNA-PK(cs)), which is recruited by DNA Ku protein, a heterodimer of Ku70 and Ku80, as well as XRCC4 protein and DNA ligase IV . A complex of Rad50/Mre11/Xrs2, a family of Sir proteins and probably other yet unidentified proteins can be also involved in this process . NHEJ and HR may play overlapping roles in the repair of DSBs produced in the S phase of the cell cycle or at replication forks . Aside from DNA repair, NHEJ may play a role in many different processes, including the maintenance of telomeres and integration of HIV-1 genome into a host genome, as well as the insertion of pseudogenes and repetitive sequences into the genome of mammalian cells . Inhibition of NHEJ can be exploited in cancer therapy in radio-sensitizing cancer cells . Identification of all key players and fundamental mechanisms underlying NHEJ still requires further research.

Virus Genes, 2004 Jan, 28(1), 41 - 53
Base usage and dinucleotide frequency of infectious bursal disease virus; Yew TD et al.; Base usage and dinucleotide frequency have been extensively studied in many eukaryotic organisms and bacteria, but not for viruses . In this paper, a comprehensive analysis of these aspects for infectious bursal disease virus (IBDV) was presented . The analysis of base usage indicated that all of the IBDV genes possess equivalent overall nucleotide distributions . However when the base usage at each codon positions was analysed by using cluster analysis, the VP5 open reading frame (ORF) formed a different cluster isolated from the other genes . The unusual base usage of VP5 ORF may indicate that the gene was originated by the virus "overprinting strategy", a strategy in which virus may create novel gene by utilizing the unused reading frames of its existing genes . Meanwhile, the GC content of the IBDV genes and the chicken's coding sequences was comparable; suggesting the virus imitation of the host to increase its translational efficiency . The analysis of dinucleotide frequency indicated that IBDV genome had dinucleotide bias: the frequencies of CpG and TpA were lower and the TpG was higher than the expected . Classical methylation pathway, a process where CpG converted to TpG, may explain the significant correlation between the CpG deficiency and TpG abundance . "Principal component analysis of the dinucleotide frequencies" (DF-PCA) was used to analyse the overall dinucleotide frequencies of IBDV genome . DF-PCA on the hypervariable region and polyprotein (VPX-VP4-VP3) gene showed that the very virulent IBDV (vvIBDV) was segregated from other strains; which meant vvIBDV had a unique dinucleotide pattern . In summary, the study of base usage and dinucleotide frequency had unravelled many overlooked genomic properties of the virus.

Plant Physiol, 2004 Feb, 134(2), 801 - 12 Epub 2004 Jan 22.
Adenylate gradients and Ar:O(2) effects on legume nodules: I . Mathematical models; Wei H et al.; Mathematical models were developed to test the likelihood that large cytosolic adenylate concentration gradients exist across the bacteria-infected cells of legume nodules . Previous studies hypothesized that this may be the case to account for the unusually low adenylate energy charge (AEC; 0.65) measured in the plant fraction of metabolically active nodules (M.M . Kuzma, H . Winter, P . Storer, I . Oresnik, C.A . Atkins, D.B . Layzell {1999} Plant Physiol 119: 399-407) . Simulations coupled leghemoglobin-facilitated O(2) diffusion into the infected cell, through bacteroid nitrogenase activity, with the ATP demand for transport and ammonia assimilation in the plant fraction of ureide- and amide-producing nodules . Although large cytosolic adenylate gradients were predicted to exist in both nodule types, amide nodules were predicted to have steeper AEC gradients (0.82-0.52) than ureide nodules (0.82-0.61) . The differences were attributed to an additional ATP demand for Asn synthesis in the amide nodule . Simulations for nodules transferred to an Ar:O(2) atmosphere predicted a major reduction in the magnitude of adenylate gradients and an increase in the AEC of the plant fraction . Results were consistent with a number of experimental studies and were used to propose an experimental test of the models.

Biochim Biophys Acta, 2004 Jan 22, 1670(2), 156 - 64
Expression of human arginine decarboxylase, the biosynthetic enzyme for agmatine; Zhu MY et al.; Agmatine, an amine formed by decarboxylation of L-arginine by arginine decarboxylase (ADC), has been recently discovered in mammalian brain and other tissues . While the cloning and sequencing of ADC from plant and bacteria have been reported extensively, the structure of mammalian enzyme is not known . Using homology screening approach, we have identified a human cDNA clone that exhibits ADC activity when expressed in COS-7 cells . The cDNA and deduced amino acid sequence of this human ADC clone is distinct from ADC of other forms . Human ADC is a 460-amino acid protein that shows about 48% identity to mammalian ornithine decarboxylase (ODC) but has no ODC activity . While naive COS-7 cells do not make agmatine, these cells are able to produce agmatine, as measured by HPLC, when transfected with ADC cDNA . Northern blot analysis using the cDNA probe indicated the expression of ADC message in selective human brain regions and other human tissues.

Microbes Infect, 2004 Jan, 6(1), 113 - 28
Pathogenesis and host responses in human onchocerciasis: impact of Onchocerca filariae and Wolbachia endobacteria; Brattig NW; Onchocerca volvulus is a tissue-invasive parasitic nematode causing skin and eye pathology in human onchocerciasis . The filariae habour abundant intracellular Wolbachia bacteria, now recognised as obligatory symbionts, and therefore emerging as a novel target for chemotherapy . Recent research demonstrates that both the filariae and endobacteria contribute to the pathogenesis of onchocerciasis, and molecules have been identified that promote inflammatory or counter-inflammatory immune mechanisms, divert the host's immune response or procure evasion of the parasite.

Microbes Infect, 2004 Jan, 6(1), 93 - 100
Interaction between Brucella abortus and cellular prion protein in lipid raft microdomains; Watarai M; A wide variety of pathogens employ lipid raft microdomains to infect host cells . Here, we review selected aspects of interaction between Brucella abortus and cellular prion protein, one of the lipid raft-associated molecules on the plasma membrane, when bacteria infect macrophages, and discuss the correlates of proliferation in mice.

Vet Microbiol, 2004 Jan 14, 98(1), 63 - 9
Identification of Bartonella strains isolated from wild and domestic ruminants by a single-step PCR analysis of the 16S-23S intergenic spacer region; Maillard R et al.; Of the 20 species or subspecies of Bartonella currently known, 7 cause various diseases in humans with many being zoonotic . However, some Bartonella species appear only to cause asymptomatic bacteraemia in their hosts . In ruminants, three Bartonella species (B . bovis, B . capreoli and B . schoenbuchensis) have recently been described . However, limited or no information has yet been published concerning their mode of transmission and their possible pathogenicity for domestic cattle . The phylogenetic relationship of these species with other bacteria of the Bartonella genus has only been recently investigated . It is therefore necessary to develop appropriate tools that will easily allow identification of these ruminant strains for epidemiological and clinical studies . A single-step PCR assay, based on the amplification of a fragment of the 16S-23S rRNA intergenic spacer (ITS), was evaluated for identification of Bartonella isolated from domestic cattle and from free-ranging or captive cervids . For each Bartonella species tested, the PCR assay led to a product that was unique either for its length or its sequence . All ruminant isolates tested could be easily differentiated among themselves and from the other Bartonella species . Furthermore, sequence analysis of the PCR products revealed a close relationship between all ruminant Bartonella strains . Therefore, ITS PCR testing appears to be a convenient tool for a quick diagnosis of ruminant Bartonella species.

Curr Biol, 2004 Jan 20, 14(2), R73 - 4
Evolution of cooperation: two for one?
Ackermann M, Chao L.
How can cooperation thrive in a selfish world? Recent evolution experiments show how bacteria themselves can generate conditions that make cooperation a winning strategy . At least in the short term.

Curr Biol, 2004 Jan 20, 14(2), 138 - 44
Abstrakt, a DEAD box protein, regulates Insc levels and asymmetric division of neural and mesodermal progenitors; Irion U et al.; Asymmetric cell division generates cell diversity in bacteria, yeast, and higher eukaryotes . In Drosophila, both neural and muscle progenitors divide asymmetrically . In these cells the Inscuteable (Insc) protein complex coordinates cell polarity and spindle orientation . Abstrakt (Abs) is a DEAD-box protein that regulates aspects of cell polarity in oocytes and embryos . We use a conditional allele of abs to investigate its role in neural and muscle progenitor cell polarity . In neuroblasts we observe loss of apical Insc crescents, failure in basal protein targeting, and defects in spindle orientation . In the GMC4-2a cell we observe loss of apical Insc crescents, defects in basal protein targeting, and equalization of sibling neuron fates; muscle precursors show a similar equalization of sibling cell fates . These phenotypes resemble those of insc mutants; indeed, abs mutants show a striking loss of Insc protein levels but no change of insc RNA levels . Furthermore, we find that the Abs protein physically interacts with insc RNA . Our results demonstrate a novel role for Abs in the posttranscriptional regulation of insc expression, which is essential for proper cell polarity, spindle orientation, and the establishment of distinct sibling cell fates within embryonic neural and muscle progenitors.

Clin Tech Small Anim Pract, 2003 Nov, 18(4), 254 - 61
Maintenance of gastrointestinal endoscopes; Shumway R et al.; Endoscopes are used for visualization and biopsy of gastrointestinal lesions, as well as therapeutic procedures, such as foreign body retrieval . In the past, they were primarily used in large institutional settings where specialized personnel could focus on their maintenance . Today, they are becoming increasingly common in general practice . The maintenance of gastrointestinal endoscopes in the veterinary setting involves many challenges, including safe handling, reprocessing, and storage . Meeting these challenges requires well-trained personnel and strict protocols . Reprocessing, which includes cleaning and disinfection, offers the greatest challenge . The complex structure of flexible endoscopes, particularly the long, narrow channels, makes them difficult to clean . Gastrointestinal endoscopes operate in a contaminated environment, exposing them to high levels of organic matter and bacteria . High-level disinfection is necessary for infection control, but liquid germicides available for endoscope disinfection can be toxic to medical personnel and patients.

J Immunol Methods, 2004 Jan, 284(1-2), 133 - 40
Efficient gene transfer into the human natural killer cell line, NKL, using the Amaxa nucleofection system; Maasho K et al.; Natural killer (NK) cell lines are useful for studying facets of NK cell biology . Such cell lines are notoriously difficult to transfect by traditional methods, a fact that has hampered NK cell biology studies for a long time . To overcome this, we investigated the use of the Amaxa nucleofection system that directly transfers DNA into the nucleus of the cell . This technology has revolutionized transfection studies with heretofore relatively transfection resistant cell types such as T cells, B cells and dendritic cells . Despite these advances, NK cells and NK cell lines have remained relatively resistant to transfection, including nucleofection . In this study we employed cDNA for SHP1 and various Rab proteins cloned in enhanced green/yellow fluorescent protein (EGFP/EYFP) expression plasmids for transient transfections into NKL cells . The expression of EGFP/EYFP fusion proteins was analyzed by flow cytometry, immunoblot and confocal microscopic analyses . We achieved 40-70% transfection efficiency with high levels of expression in this cell line with 85-90% viability . The method used in this report proves to be far superior to existing methods for delivering DNA into this well studied NK cell line and, consequently, provides new experimental opportunities.

Pac Health Dialog, 2002 Sep, 9(2), 225 - 8
The viral diarrhoea epidemic 2002 in the Marshall Islands: its impact on a small island pharmacy; Harding A; This paper describes the increased demands on the pharmacy department of Majuro Hospital in the Marshall Islands during a recent viral diarrhoea epidemic . It discusses the increased number of patients, the increased usage of drugs and also the increased costs during this epidemic . The number of patients only rose 10% . However, the increase in workload of the outpatient department, laboratory and the pharmacy department was much greater . The percentage of children presenting to the outpatient department with diarrhoea increased from 10% to 80% . Oral rehydration solution (ORS) was the most common medication given during the epidemic in line with the World Health Organisation Guidelines.

J Cardiovasc Surg (Torino), 2003 Oct, 44(5), 605 - 9
Helicobacter pylori seropositivity in patients with unstable angina; Pellicano R et al.; AIM: The pathogenesis of ischemic heart diseases has been correlated, on epidemiological and pathogenetic grounds, with infections by viruses and bacteria, including Helicobacter pylori (H . pylori) . The AIM: of this study were to investigate the association of unstable angina (UA) with anti-H . pylori seropositivity in a case-control study and to search for the classic cardiovascular risk factors in both infected and uninfected patients . METHODS: We studied 32 consecutive patients (20 males, 12 females), mean age 65 years (range 42-89), with final diagnosis of UA . A total of 64 subjects (40 males, 24 females, mean age 65 years, range 42-89) admitted to the Emergency Care Unit, age and sex-matched, served as controls . The presence of hypertension, serum levels of cholesterol and glucose, plasma levels of fibrinogen, smoking habit and social class were investigated in all patients . Cases and controls were inhabitants of NorthWestern Italy, and had similar socioeconomic status as based on working place and on instruction level . H . pylori seroprevalence was assessed by the presence of antibodies (IgG) against H . pylori by means of a commercial enzyme immunosorbent assay . RESULTS: Antibodies to H . pylori were found in 26/32 (81%) of the patients and in 34/64 (53%) of the controls (p=0.007); the odds ratio was 3.82 (95% confidence interval 1.27 to 12.04) . Classical cardiovascular risk factors, such as socio-economic status, did not differ among patients with and without antibodies to H . pylori . CONCLUSION: Patients with unstable angina had a significantly higher seroprevalence of anti-H . pylori than the control population . Classical risk factors for ischemic heart disease, such as the indicators of socio-economic status, were equally distributed among infected or uninfected patients with UA.

Exp Biol Med (Maywood), 2004 Feb, 229(2), 170 - 81
Posttraumatic inflammation is a complex response based on the pathological expression of the nervous, immune, and endocrine functional systems; Aller MA et al.; The successive phases that make up both the local and systemic posttraumatic acute inflammatory response could represent the expression of three concatenated pathological or "primitive" functional systems with trophic properties: the nervous, immune, and endocrine ones . The nervous functional system would play an important role in the phenomenon of ischemia-reperfusion, which would be represented by nutrition by diffusion that is either anaerobic (ischemia) or with defective use of oxygen (reperfusion) and, thus, with a limited energy requirement . The immune functional system would be represented by the infiltration of the tissues by inflammatory cells and bacteria, which would become mediators in providing nutrition to the injured tissues . Although the use of oxygen would still be defective, hypermetabolism and fever would occur . In these inflammatory response phases, the lymphatic is the most important circulation . The endocrine functional system would be the most specialized and would have high energy requirements because it would be represented by the blood capillary-mediated nutrition . Highly specialized epithelial cells would already possess a perfected oxidative metabolism . The successive expression of these three functional systems during embryonic development and also during the evolutionary development of our species could explain why the inflammatory response is a ubiquitous mechanism that is common to multiple diseases, because it is an integrator of the ontogeny and phylogeny.

J Biol Chem, 2004 Apr 2, 279(14), 13652 - 8 Epub 2004 Jan 20.
MeaB is a component of the methylmalonyl-CoA mutase complex required for protection of the enzyme from inactivation; Korotkova N et al.; Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA . In humans, deficiencies in the mutase lead to methylmalonic aciduria, a rare disease that is fatal in the first year of life . Such inherited deficiencies can result from mutations in the mutase structural gene or from mutations that impair the acquisition of cobalamins . Recently, a human gene of unknown function, MMAA, has been implicated in methylmalonic aciduria (Dobson, C . M., Wai, T., Leclerc, D., Wilson, A., Wu, X., Dore, C., Hudson, T., Rosenblatt, D . S., and Gravel, R . A . (2002) Proc . Natl . Acad . Sci . U . S . A . 99, 15554-15559) . MMAA orthologs are widespread in bacteria, archaea, and eukaryotes . In Methylobacterium extorquens AM1, a mutant defective in the MMAA homolog meaB was unable to grow on C(1) and C(2) compounds because of the inability to convert methylmalonyl-CoA to succinyl-CoA (Korotkova N., Chistoserdova, L., Kuksa, V., and Lidstrom, M . E . (2002) J . Bacteriol . 184, 1750-1758) . Here we demonstrate that this defect is not due to the absence of adenosylcobalamin but due to an inactive form of methylmalonyl-CoA mutase . The presence of active mutase in double mutants defective in MeaB and in the synthesis of either R-methylmalonyl-CoA or adenosylcobalamin indicates that MeaB is necessary for protection of mutase from inactivation during catalysis . MeaB and methylmalonyl-CoA mutase from M . extorquens were cloned and purified in their active forms . We demonstrated that MeaB forms a complex with methylmalonyl-CoA mutase and stimulates in vitro mutase activity . These results support the hypothesis that MeaB functions to protect methylmalonyl-CoA mutase from irreversible inactivation.

J Biol Chem, 2004 Apr 9, 279(15), 15076 - 83 Epub 2004 Jan 20.
Two distinct crt gene clusters for two different functional classes of carotenoid in Bradyrhizobium; Giraud E et al.; Aerobic photosynthetic bacteria possess the unusual characteristic of producing different classes of carotenoids . In this study, we demonstrate the presence of two distinct crt gene clusters involved in the synthesis of spirilloxanthin and canthaxanthin in a Bradyrhizobium strain . Each cluster contains the genes crtE, crtB, and crtI leading to the common precursor lycopene . We show that spirilloxanthin is associated with the photosynthetic complexes, while canthaxanthin protects the bacteria from oxidative stress . Only the spirilloxanthin crt genes are regulated by light via the control of a bacteriophytochrome . Despite this difference in regulation, the biosyntheses of both carotenoids are strongly interconnected at the level of the common precursors . Phylogenetic analysis suggests that the canthaxanthin crt gene cluster has been acquired by a lateral gene transfer . This acquisition may constitute a major selective advantage for this class of bacteria, which photosynthesize only under conditions where harmful reactive oxygen species are generated.

J Cell Biol, 2004 Jan 19, 164(2), 233 - 41
Ordered proteolysis in anaphase inactivates Plk1 to contribute to proper mitotic exit in human cells; Lindon C et al.; We have found that key mitotic regulators show distinct patterns of degradation during exit from mitosis in human cells . Using a live-cell assay for proteolysis, we show that two of these regulators, polo-like kinase 1 (Plk1) and Aurora A, are degraded at different times after the anaphase-promoting complex/cyclosome (APC/C) switches from binding Cdc20 to Cdh1 . Therefore, events in addition to the switch from Cdc20 to Cdh1 control the proteolysis of APC/C(Cdh1) substrates in vivo . We have identified a putative destruction box in Plk1 that is required for degradation of Plk1 in anaphase, and have examined the effect of nondegradable Plk1 on mitotic exit . Our results show that Plk1 proteolysis contributes to the inactivation of Plk1 in anaphase, and that this is required for the proper control of mitotic exit and cytokinesis . Our experiments reveal a role for APC/C-mediated proteolysis in exit from mitosis in human cells.

Zhonghua Er Ke Za Zhi, 2003 Sep, 41(9), 645 - 7
{Clinical and chest X-ray characteristics of 5 cases with severe acute respiratory syndrome in children in Shenzhen area}; Lu PX et al.; OBJECTIVE: To explore clinical and chest X-ray features of SARS in children to facilitate correct diagnosis . METHODS: Clinical manifestations and chest X-ray findings in five children suffering from SARS admitted for treatment in the hospital between February and May, 2003 in Shenzhen area were analyzed . The diagnosis was confirmed by epidemiological, clinical, laboratory and radiological examinations . Among the 5 cases, 1 was a boy and the others were girls at the age of 4 to 13 years . RESULTS: Of the 5 SARS children, 3 presented a history of close contact with SARS patients . Fever was the initiative symptom, 4 had a body temperature of over 38 degrees C with the highest being 40 degrees C; fever sustained from 4 to 7 days with an average of 5.6 days . All the 5 cases developed nonproductive cough; on auscultation, both moist and dry rales could be heard in 3 out of the 5 cases . Mean total white count of peripheral blood was (2.96 - 6.9) x 10(9)/L, and was < 5.0 x 10(9)/L in 4 cases . SARS associated coronavirus specific RNA fragment was found positive by RT-PCR in 1 case; 1 case was positive for both IgM and IgG antibodies to the virus; 1 case was positive for only IgM antibody and another 2 cases were positive for only IgG antibody . IgG and IgM antibodies to Mycoplasma pneumoniae and Chlamydia pneumoniae as well as blood culture for bacteria were all negative . Findings on chest X-ray examination: 4 cases showed presence of patchy or macular opacities with cord-like shadows in unilateral lung plates while 1 case each showed ground-glass-like opacity and migratory changes; 1 case showed interstitial changes in the lungs in the form of irregular reticular lattice and cord-like shadows . Two cases received CT scanning and macular-patchy or spotty shadows were seen all over the lung . The shortest time for absorption of foci in the lungs was 7 days while the longest was 33 days with a mean of 15 +/- 6 days . None of the cases had any signs of fibrosis in the lungs . All the 5 cases were completely cured and discharged 7 to 40 days (mean 18 +/- 11 days) after admission . CONCLUSION: Compared with adult cases with SARS, children with SARS had milder symptoms and signs . Presence of unilateral patchy shadow in lungs represented the main chest X-ray findings.

Environ Technol, 2003 Nov, 24(11), 1323 - 30
Fate of phosphorus from biological aerobic treatment of pig slurry . By-products characterization and recovery; Daumer ML et al.; The fate of phosphorus distribution in the products obtained from biological aerobic treatment of pig slurry, e.g . separated solids, liquid effluent and sludge, was monitored in three different farm-scale units . Samples of raw slurry, solid products, aerated slurry, liquid effluent and sludge were characterised and analysed for their concentration in total phosphorus, nitrogen content and heavy metals (Cu and Zn) . At each treatment stage, nitrogen, phosphorus and heavy metals mass balance between input and output was established . Moreover, liquid products were characterised and analysed both for their total and dissolved ortho-phosphate content . Separated solids, sludge and liquid effluent represented 5%, 15-40% and 75-83% of the mass of the raw slurry, respectively . A mechanical separation step prior to aeration allowed the export of 25-30% of total phosphorus for further use as organic fertiliser . A large amount of total phosphorus (e.g . 60-70%) was located in sludge while phosphorus remaining in liquid effluent was about 15-25% . Raw slurry separation and sufficient aeration allowed phosphorus to concentrate in the sludge . Insufficient aeration resulted in the release of phosphorus as dissolved ortho-phosphate within the liquid effluent . Finally, relevance of the agronomic use of the products was discussed and improvements of biological aerobic treatment to enhance phosphorus removal and/or recovery were considered.

J Wildl Dis, 2003 Oct, 39(4), 762 - 71
Pathogens of house mice on arid Boullanger Island and subantarctic Macquarie Island, Australia; Moro D et al.; Studies on island populations of house mice (Mus domesticus) and their viruses reveal insights into viral persistence in isolated communities . We surveyed the ectoparasites, endoparasites, and antiviral antibodies for 11 murine viruses and two bacteria of house mice inhabiting two islands off Australia . House mice on Boullanger Island were seropositive to two viruses, murine cytomegalovirus and epizootic diarrhea of infant mice . On subantarctic Macquarie Island, house mice were seropositive for five viruses: murine cytomegalovirus, lymphocytic choriomeningitis virus, mouse parvovirus, epizootic diarrhea of infant mice, and Theiler's murine encephalomyelitis virus . The diversity of antiviral antibodies was lower among populations of house mice on islands than those inhabiting mainland Australia . The decreased diversity of viruses in island populations of house mice may be a function of which agent the founder mice transfer to the island and related to the low densities which the host population may periodically reach over time.

Acta Biochim Biophys Sin (Shanghai), 2004 Jan, 36(1), 16 - 20
DNAskew: statistical analysis of base compositional asymmetry and prediction of replication boundaries in the genome sequences; Ma XR et al.; Sueoka and Lobry declared respectively that, in the absence of bias between the two DNA strands for mutation and selection, the base composition within each strand should be A=T and C=G (this state is called Parity Rule type 2, PR2) . However, the genome sequences of many bacteria, vertebrates and viruses showed asymmetries in base composition and gene direction . To determine the relationship of base composition skews with replication orientation, gene function, codon usage biases and phylogenetic evolution, in this paper a program called DNAskew was developed for the statistical analysis of strand asymmetry and codon composition bias in the DNA sequence . In addition, the program can also be used to predict the replication boundaries of genome sequences . The method builds on the fact that there are compositional asymmetries between the leading and the lagging strand for replication . DNAskew was written in Perl script language and implemented on the LINUX operating system . It works quickly with annotated or unannotated sequences in GBFF (GenBank flatfile) or fasta format . The source code is freely available for academic use at http://www.epizooty.com/pub/stat/DNAskew.

Proc Natl Acad Sci U S A, 2004 Jan 27, 101(4), 1033 - 8 Epub 2004 Jan 19.
Direct interactions between Epstein-Barr virus leader protein LP and the EBNA2 acidic domain underlie coordinate transcriptional regulation; Peng CW et al.; The Epstein-Barr virus nuclear leader protein LP (EBNALP) and EBNA2 are expressed first in lymphocyte infection, coordinately regulate cell and viral gene transcription, and are critical for lymphocyte outgrowth into lymphoblastoid cell lines (LCLs) . We have now found that EBNALP readily associated with EBNA2 or with the EBNA2 C-terminal acidic activation domain (E2AD) when both components were expressed by bacteria . In lymphoblasts, EBNALP and EBNA2 did not stably associate . However, EBNALP deleted for only 10 C-terminal amino acids stably associated with EBNA2 in lymphoblasts or with EBNA2 acidic activating domain from bacteria . The E2AD was essential for EBNALP coactivation of the latent membrane protein 1 promoter in lymphoblasts; EBNALP could coactivate with a deficient mutant EBNA2, EBNA2W(454)T, but not with EBNA2 deleted for E2AD . Moreover, EBNALP 31 amino acids (dW2Y1) with 24 C- or N-terminal amino acids was a specific and efficient affinity matrix for EBNA2 or EBNALP . Even an EBNALP 22-aa peptide, dW2, specifically bound EBNALP or EBNA2 . These biochemical interactions between EBNALP and EBNA2 enable coordinated transcriptional regulation of cell and viral gene expression in lymphoblasts only when the interaction is unstable; deletion of the EBNALP C-terminal 10 aa stabilized association with EBNA2 and prevented coactivation . Because EBNALPd10 dominantly inhibited EBNALP coactivation with EBNA2, EBNALPd10 expression in LCLs may be useful in assessing the role of EBNALP coactivation in LCL growth or survival.

Trends Cell Biol, 1995 Mar, 5(3), 109 - 13
Regulation of the phagocyte respiratory burst by small GTP-binding proteins; Bokoch GM; Bacteria phagocytosed by leukocytes are killed and degraded by toxic oxygen metabolites produced in the phagosome via an NADPH oxidase . NADPH oxidase activity is regulated by small GTP-binding proteins in response to phagocytic stimuli . In this review, Gary Bokoch focuses on the role of Rac in regulating this important phagocytic process.

Trends Cell Biol, 1992 Jul, 2(7), 183 - 5
Toxin entry: how reversible is the secretory pathway?
Pelham HR, Roberts LM, Lord JM.
A number of proteins produced by plants and bacteria are extremely toxic to eukaryotic cells . Their potency arises from their ability to catalyse the modification of crucial cellular components . Only a few toxin molecules are required to kill a cell, but to do so they must first reach the cytosol . How such proteins are translocated across the target cell membrane is poorly understood, but we argue here that some toxins may travel the secretory pathway in reverse, passing all the way from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol.

J Microsc, 2004 Feb, 213(Pt 2), 180 - 97
Magnetosomal matrix: ultrafine structure may template biomineralization of magnetosomes; Taylor AP et al.; The organic matrix surrounding bullet-shaped, cubo-octahedral, D-shaped, irregular arrowhead-shaped, and truncated hexa-octahedral magnetosomes was analysed in a variety of uncultured magnetotactic bacteria . The matrix was examined using low- (80 kV) and intermediate- (400 kV) voltage TEM . It encapsulated magnetosomes in dehydrated cells, ultraviolet-B-irradiated dehydrated cells and stained resin-embedded fixed cells, so the apparent structure of the matrix does not appear to be an artefact of specimen preparation . High-resolution images revealed lattice fringes in the matrix surrounding magnetite and greigite magnetosomes that were aligned with lattice fringes in the encapsulated magnetosomes . In all except one case, the lattice fringes had widths equal to or twice the width of the corresponding lattice fringes in the magnetosomes . The lattice fringes in the matrix were aligned with the {311}, {220}, {331}, {111} and {391} related lattice planes of magnetite and the {222} lattice plane of greigite . An unidentified material, possibly an iron hydroxide, was detected in two immature magnetosomes containing magnetite . The unidentified phase had a structure similar to that of the matrix as it contained {311}, {220} and {111} lattice fringes, which indicates that the matrix acts as a template for the spatially controlled biomineralization of the unidentified phase, which itself transforms into magnetite . The unidentified phase was thus called pre-magnetite . The presence of the magnetosomal matrix explains all of the five properties of the biosignature of the magnetosomal chain proposed previously by Friedmann et al . and supports their claim that some of the magnetite particles in the carbonate globules in the Martian meteorite ALH84001 are biogenic . Two new morphologies of magnetite magnetosomes are also reported here (i.e . tooth-shaped and hexa-octahedral magnetosomes) . Tooth-shaped magnetite magnetosomes elongated in the {110} direction are reported, and are distinct from arrowhead-shaped and bullet-shaped magnetosomes . Elongation of magnetite magnetosomes in the {110} direction has not been reported previously . A Martian hexa-octahedral magnetite particle was previously characterized by Thomas-Keptra et al . and compared with truncated hexa-octahedral magnetite magnetosomes . Hexa-octahedral magnetite magnetosomes with the same morphology and similar sizes and axial ratios as those reported by Thomas-Keptra et al . are characterized here . These observations support their claim that ALH84001 contains evidence for a past Martian biota.

Biochemistry, 2004 Jan 27, 43(3), 808 - 18
Superoxide reductase from Desulfoarculus baarsii: identification of protonation steps in the enzymatic mechanism; Niviere V et al.; Superoxide reductase (SOR) is a metalloenzyme that catalyzes the reduction of O2*- to H2O2 and provides an antioxidant mechanism in some anaerobic and microaerophilic bacteria . Its active site contains an unusual mononuclear ferrous center (center II) . Protonation processes are essential for the reaction catalyzed by SOR, since two protons are required for the formation of H2O2 . We have investigated the acido-basic and pH dependence of the redox properties of the active site of SOR from Desulfoarculus baarsii, both in the absence and in the presence of O2*- . In the absence of O2*-, the reduction potential and the absorption spectrum of the iron center II exhibit a pH transition . This is consistent with the presence of a base (BH) in close proximity to the iron center which modulates its reduction properties . Studies of mutants of the closest charged residues to the iron center II (E47A and K48I) show that neither of these residues are the base responsible for the pH transitions . However, they both interact with this base and modulate its pKa value . By pulse radiolysis, we confirm that the reaction of SOR with O2*- involves two reaction intermediates that were characterized by their absorption spectra . The precise step of the catalytic cycle in which one protonation takes place was identified . The formation of the first reaction intermediate, from a bimolecular reaction of SOR with O2*-, does not involve proton transfer as a rate-limiting step, since the rate constant k1 does not vary between pH 5 and pH 9.5 . On the other hand, the rate constant k2 for the formation of the second reaction intermediate is proportional to the H+ concentration in solution, suggesting that the proton arises directly from the solvent . In fact, BH, E47, and K48 have no role in this step . This is consistent with the first intermediate being an iron(III)-peroxo species and the second one being an iron(III)-hydroperoxo species . We propose that BH may be involved in the second protonation process corresponding to the release of H2O2 from the iron(III)-hydroperoxo species.

Biochemistry, 2004 Jan 27, 43(3), 700 - 9
Divergent roles of hepatitis B virus X-associated protein 2 (XAP2) in human versus mouse Ah receptor complexes; Ramadoss P et al.; The aryl hydrocarbon receptor (AhR) mediates the toxicologic and carcinogenic properties of 2,3,7,8-tetrachlorodibenzo-p-dioxin . In the cytoplasm, the AhR is complexed with a dimer of hsp90, and the hepatitis B virus X-associated protein 2 (XAP2) . Most studies that have examined the ability of XAP2 to modulate the AhR have characterized the mouse receptor (mAhR) . However, the amino acid sequence of mAhR is significantly different from human AhR (hAhR) in the carboxy terminal half of the protein, and this could lead to differences in the behavior of the two receptors . mAhR-yellow fluorescent protein (YFP) and hAhR-YFP were used to compare nucleocytoplasmic shuttling properties and the ability of XAP2 to modulate their activity . As reported previously, mAhR localized predominantly in the nucleus and was redistributed to the cytoplasm by coexpression of XAP2 in COS-1 cells . Leptomycin B treatment revealed that XAP2 blocked mAhR-YFP translocation to the nucleus in the absence of ligand . In contrast, hAhR-YFP localized predominantly in the cytoplasm, and coexpression of XAP2 did not affect this localization, and did not block nuclear accumulation in the presence of leptomycin B . An XAP2 fusion protein with a nuclear localization signal fused to the carboxy terminus (XAP2-NLS) was utilized to test whether this protein could drag the AhR into the nucleus . Coexpression of mAhR-YFP and XAP2-NLS caused cytoplasmic localization of the mAhR, while hAhR-YFP was partially localized in the nucleus, suggesting that XAP2 remains bound to the hAhR during nucleocytoplasmic shuttling . The presence of XAP2 in the ligand-bound hAhR complex enhanced the rate of nuclear translocation but repressed transcriptional activity . Together, these results suggest that the hAhR differs biochemically from the mAhR.

Nucleic Acids Res, 2004 Jan 16, 32(1), 386 - 96 Print 2004.
CIRCE is not involved in heat-dependent transcription of groESL but in stabilization of the mRNA 5'-end in Rhodobacter capsulatus; Jager S et al.; The CIRCE element, an inverted DNA repeat, is known to be involved in the temperature-dependent regulation of genes for heat shock proteins in a variety of organisms . The CIRCE element was identified as the target for the HrcA protein, which represses transcription of heat shock genes under normal growth temperature . Our data reveal that the CIRCE element is not involved in the temperature-dependent transcription of the groESL genes in Rhodobacter capsulatus . Apparently, R.capsulatus does not harbour an HrcA protein . The mechanisms of heat shock regulation of the groESL genes in R.capsulatus therefore diverge significantly from the regulatory pathway identified in other organisms . A structural analysis of the CIRCE RNA element revealed a stem of 11 nt pairs and a loop of only 5 nt . This folding differs from a structure with a 9 nt loop suggested previously on the basis of computer analysis . The RNA structure leads to a slight stabilization of the groESL mRNA that is more pronounced at normal growth temperature than under heat shock conditions.

J Bacteriol, 2004 Feb, 186(3), 777 - 84
The RecA protein of Helicobacter pylori requires a posttranslational modification for full activity; Fischer W et al.; The RecA protein is a central component of the homologous recombination machinery and of the SOS system in most bacteria . In performing these functions, it is involved in DNA repair processes and plays an important role in natural transformation competence . This may be especially important in Helicobacter pylori, where an unusually high degree of microdiversity among strains is generated by homologous recombination . We have suggested previously that the H . pylori RecA protein is subject to posttranslational modifications that result in a slight shift in its electrophoretic mobility . Here we show that at least two genes downstream of recA are involved in this modification and that this process is dependent on genes involved in glycosylation and lipopolysaccharide biosynthesis . Site-directed mutagenesis of a putative glycosylation site results in production of an unmodified RecA protein . This posttranslational modification is not involved in membrane targeting or cell division functions but is necessary for the full function of RecA in DNA repair . Thus, it might be an adaptation to the specific requirements of H . pylori in its natural environment.

Trends Biochem Sci, 2004 Jan, 29(1), 7 - 10
Resuscitation-promoting factors possess a lysozyme-like domain; Cohen-Gonsaud M et al.; The novel bacterial cytokine family--resuscitation-promoting factors (Rpfs)--share a conserved domain of uncharacterized function . Predicting the structure of this domain suggests that Rpfs possess a lysozyme-like domain . The model highlights the good conservation of residues involved in catalysis and substrate binding . A lysozyme-like function makes sense for this domain in the light of experimental characterization of the biological function of Rpfs.

Trends Biochem Sci, 2004 Jan, 29(1), 4 - 7
HTTM, a horizontally transferred transmembrane domain; Schultz J; Sequence analysis of vitamin K-dependent gamma-carboxylases (VKGC) has revealed the presence of a novel domain, HTTM (for horizontally transferred transmembrane) in its N terminus . In contrast to most known domains, HTTM contains four transmembrane regions . Its occurrence in eukaryotes, bacteria and archaea is probably caused by horizontal gene transfer rather than by early evolution . The conservation of VKGC catalytic sites also indicates an enzymatic function for the other family members.

Trends Plant Sci, 2004 Jan, 9(1), 18 - 25
Evolution of oxygenic photosynthesis: genome-wide analysis of the OEC extrinsic proteins; De Las Rivas J et al.; The appearance of oxygenic photosynthesis was a key event in the evolution of our green biosphere . Oxygen in the atmosphere is generally believed to come from the biomolecular water-splitting reaction that occurs in oxyphotosynthetic organisms catalysed by the oxygen evolving centre (OEC) of Photosystem II . Using knowledge from complete genomes and current databases, we have investigated the nature and composition of the extrinsic proteins forming the OECs of different organisms, with particular focus on the manganese stabilizing protein that is present in all known oxyphototrophs . This analysis traces the evolution of the extrinsic proteins from ancient cyanobacteria to higher plants and gives hints about the ancestral form of the OEC.

Dermatol Ther, 2004, 17 Suppl 1, 26 - 34
Role of mild cleansing in the management of patient skin; Subramanyan K; Routine everyday care of skin is an essential part of optimal patient management . Common problems such as xerosis, dermatitis, eczema, psoriasis, acne, rosacea, and photodamage leave the skin vulnerable to external insults, partly as a result of varying levels of barrier dysfunction . Cosmetic surgery procedures also typically damage the stratum corneum (SC) and leave skin with a very weak barrier during recovery phase . Cleansing is an important aspect of any skin care, since it not only removes unwanted dirt, soil, and bacteria from skin, but also removes dead surface cells, preparing skin to better absorb topically applied drugs/medication . Care must be taken to minimize any further weakening of the SC barrier during cleansing . Cleansers based on mild synthetic surfactants and/or emollients that cause minimal barrier perturbation are ideal for these patients . The present paper is a brief review of four clinical trials that evaluated the efficacy and compatibility of either mild syndet bars or cleansers in patients with atopic dermatitis, acne, rosacea, or patients who had received chemical peels or Retin-A(R) (tretinoin) treatment for sustained photodamage.

AMIA Annu Symp Proc . 2003;:907.
Creating public health standard vocabularies: mapping a set of CDC's pathogen codes to SNOMED concepts; Li W et al.; CDC and its public health partners are conducting pilot projects designed to capture data directly from healthcare providers or to exchange existing data electronically.1 The translation of proprietary standards into the accepted national standards is a key part of this effort . This study mapped a set of pathogens within CDC's hospital infection control activities to The Systematized Nomenclature of Medicine (SNOMED) concepts and investigated the differences between the two.

Science, 2004 Jan 16, 303(5656), 373 - 7
Heterodimeric GTPase core of the SRP targeting complex; Focia PJ et al.; Two structurally homologous guanosine triphosphatase (GTPase) domains interact directly during signal recognition particle (SRP)-mediated cotranslational targeting of proteins to the membrane . The 2.05 angstrom structure of a complex of the NG GTPase domains of Ffh and FtsY reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides . The structure explains the coordinate activation of the two GTPases . Conformational changes coupled to formation of their extensive interface may function allosterically to signal formation of the targeting complex to the signal-sequence binding site and the translocon . We propose that the complex represents a molecular "latch" and that its disengagement is regulated by completion of assembly of the GTPase active site.

J Leukoc Biol, 2004 Apr, 75(4), 612 - 23 Epub 2004 Jan 14.
Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene; Burnett SH et al.; Transgenic mice expressing an inducible suicide gene, which allows systemic and reversible elimination of macrophages, were developed . A macrophage-specific c-fms promoter was used to express enhanced green fluorescent protein and a drug-inducible suicide gene that leads to Fas-mediated apoptosis in resting and cycling cells of the macrophage lineage . Transgenic mice were fertile, of normal weight, and showed no abnormal phenotype before drug exposure . The transgene was expressed constitutively in macrophages and dendritic cells (DC) but not significantly in T cells or B cells . Induction of the suicide gene led to depletion of 70-95% of macrophages and DC in nearly all tissues examined . Depletion reduced the ability to clear bacteria from the blood and led to increased bacterial growth in the liver . Depleted mice displayed several abnormalities, including splenomegaly, lymphadenopathy, thymic atrophy, extramedullary hematopoiesis, and development of peritoneal adhesions . This new, transgenic line will be useful in investigating the role of macrophages and DC.

Biochim Biophys Acta, 2004 Jan 14, 1696(1), 113 - 9
Structural features of the initiator of replication protein RepB encoded by the promiscuous plasmid pMV158; Ruiz-Maso JA et al.; The promiscuous rolling circle (RC)-replicating plasmid pMV158 encodes the 210-amino-acid initiator of replication protein, RepB . The protein relaxes supercoiled cognate DNA in a topoisomeraseI-like manner . A new vector and procedure for overproduction, scaling-up, and purification of the protein has been developed . RepB purified as a hexamer in solution, as shown by analytical ultracentrifugation assays . Circular dichroism (CD) of RepB indicated that the protein has an estimated content of around 33% alpha-helices and 20% beta-strands . Characterisation of temperature-induced transitions of the protein showed an irreversible change in the spectra when the temperature was raised above 35 degrees C, indicating that the protein undergoes a conformational change that could account for the relatively high optimal temperature of the RepB-mediated cleavage.

Biochimie, 2003 Nov, 85(11), 1185 - 93
Human diseases deficient in RecQ helicases; Harrigan JA et al.; RecQ helicases are conserved from bacteria to man . Mutations in three of the human RecQ family members give rise to genetic disorders characterized by genomic instability and a predisposition to cancer . RecQ helicases are therefore caretakers of the genome, and although they do not directly regulate tumorigenesis, they influence stability and the rate of accumulation of genetic alterations, which in turn, result in tumorigenesis . Maintenance of genome stability by RecQ helicases likely involves their participation in DNA replication, recombination, and repair pathways.

Int J Pediatr Otorhinolaryngol, 2004 Feb, 68(2), 205 - 10
Presence of human herpesviruses in young children with acute otitis media; Shinogami M et al.; Some herpesviruses have been detected in middle ear fluid (MEF) of patients with acute otitis media (AOM), but their role in middle ear disease is unknown . We examined 73 middle ear fluid samples from 73 children with acute otitis media for the presence of four major herpesviral DNA, respiratory viral genomes, and bacterial DNA by multiplex polymerase chain reaction (PCR) . Herpesviruses were detected in 16 specimens (22%), with 18 viral infections were identified overall . Respiratory viruses were detected in 35 specimens (48%), 39 viral infections overall . Bacterial DNA was detected in 51 specimens (70%), 60 bacterial infections overall . Clinical outcome was compared in patients with and without herpesvirus DNA, respiratory viral genomes, or bacterial DNA . Progression to otitis media with effusion (OME) was more common when herpesviral DNA was present . Presence of herpesvirus DNA may reflect an immunocompromised state that may make it difficult to eliminate bacteria from the middle ear after infection.

Brain Res, 2004 Feb 13, 998(1), 1 - 12
Comparison between the influence of shocks and endotoxemia on the activation of brain cells that contain nitric oxide; Seo DO et al.; We sought to identify the brain circuitry that underlies the stimulatory role of nitric oxide (NO) role on the hypothalamic-pituitary-adrenal (HPA) axis . Specifically, we determined whether electrofootshocks (60 min) or the intravenous administration of lipopolysaccharide (LPS, 100 microg/kg)-activated neurocircuitries that express either neuronal NO synthase (nNOS), a constitutive enzyme responsible for NO formation, or L-citrulline, an amino acid that is produced in equimolar amounts with NO . Shocks significantly increased the number of cells showing Fos immunoreactivity (ir) in the paraventricular nucleus (PVN) of the hypothalamus, the lateral hypothalamus (LH), amygdaloid complex (AD) and thalamus (TH), and to a lesser extent, in the hippocampus (HP), caudate putamen (CP) and frontal cortex (FC) . However, shocks did not alter the number of nNOS-positive cells nor increased citrulline signals in these brain regions . LPS significantly upregulated the number of cells with fos-like ir in the PVN, LH, AD, TH, HP, CP and FC, but only increased the number of cells positive for citrulline in the PVN, 87% of which co-expressed Fos . Thus, while shocks did not alter nNOS gene expression or citrulline levels in the brain regions studied, LPS significantly increased the number of PVN cells expressing citrulline without concomitant changes in other brain areas . Endotoxemia also upregulated significantly more PVN cells that co-expressed Fos and nNOS, compared to shocks . As NO stimulates the PVN circuitries that participate in shocks- and LPS-induced ACTH release, the lack of changes in nNOS or citrulline levels due to shocks suggests that, in this model, constitutively formed NO may modulate HPA axis activity in the absence of changes in its synthesis.

Mar Pollut Bull, 2004 Jan, 48(1-2), 132 - 6
Impacts of beach closures on perceptions of swimming-related health risk in Orange County, California; Turbow D et al.; Following a major beach closure due to bacterial contamination, a survey of beachgoers was conducted in Huntington Beach, California in 1999 to assess perceived health risk from swimming . Responses were compared to those of beachgoers at the unaffected Laguna Main Beach . No significant differences were found in risk perception . Respondents were aware of the closures (83%), yet the majority (83%) felt the water was safe for swimming . Proximity of residence to the beach was strongly associated with closure awareness (Spearman's rho=0.427, p<0.0001) . Although 83% of respondents felt that water quality was important in deciding to go to the beach, only 25% of respondents who did not plan to swim cited pollution or contamination as a reason not to swim . Most respondents (70%) trusted local health agency officials' decisions about when to open/close beaches . Surveyed beach visitors were likely to bathe (84%) and were not highly concerned with swimming-related health risks.

Arch Oral Biol, 2004 Mar, 49(3), 177 - 81
Human minor and major gland saliva proteins and ability to mediate Actinomyces naeslundii adherence; Carlen A et al.; Bacteria-binding components and the ability to mediate bacterial adhesion to the tooth surface have been thoroughly studied in major salivary gland secretions . Our knowledge on the bacteria binding activity in minor gland saliva is, however, limited . In this study, proteins were examined in parallel in minor (palatal, buccal and labial) and major (parotid and submandibular/sublingual) salivary gland secretions in one subject using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting . The adherence of early colonizing Actinomyces naeslundii to pellicles formed from the secretions on hydroxyapatite beads was also examined . Amylase, IgA, proline-rich proteins and the high-molecular-weight glycoproteins, agglutinins, were detected in all saliva tested . Carbohydrate-reactive antibodies recognized the low-molecular weight mucin, MUC 7 in submandibular/sublingual saliva only . A . naeslundii strain 12104 adhered to all pellicles and especially to the buccal gland saliva pellicles . Strain LY7 adhered in highest numbers to the submandibular/sublingual saliva pellicles . It also bound in considerable numbers to parotid and palatal saliva pellicles but not to the ones formed from buccal and labial gland saliva . Our findings indicate that several bacteria-binding components are secreted in both minor and major gland saliva . The adherence-promoting ability of the various gland secretions differs, however.

Bioorg Med Chem, 2004 Jan 15, 12(2), 405 - 16
Synthesis, biological, and chiroptical activity of 3-phenyl-clavams; Cierpucha M et al.; The {2+2}cycloaddition of chlorosulfonyl isocyanate to simple vinyl ethers derived from the 2-O-sulfonylated (R) and (S) 1-phenyl-1,2-ethanediol leads to 4-alkoxy-azetidin-2-ones with a moderate stereoselectivity . The cycloaddition to analogous (Z)-propenyl ethers proceeds stereospecifically with the retention of the olefin configuration . The intramolecular alkylation of beta-lactam nitrogen atom furnished all possible stereoisomers of 3-phenyl- and 6-methyl-3-phenyl-clavams . The biological and chiroptical activity of synthesized clavams was investigated . The (3R,5R)-diastereomer 30 showed higher inhibition of bacterial enzymes than other related compounds.

Water Res, 2004 Feb, 38(3), 688 - 92
Seawater induced release and transformation of organic and inorganic phosphorus from river sediments; Gardolinski PC et al.; This paper reports an investigation of the release of organic and inorganic phosphorus from a riverine sediment subjected to salinity conditions typical of estuarine mixing . Freshwater sediment was mixed with filtered river water in a thermostatted reactor, and allowed to equilibrate under aerobic conditions for 3 days . Salinity was then increased in a stepwise manner by addition of filtered low-nutrient seawater over a period of 4 days . A control experiment was performed in a second reactor by substituting ultrapure water for seawater . Using a flow injection analysis method for measurement of filterable reactive phosphorous (FRP, the so-called inorganic fraction) and filterable organic phosphorous (FOP), it was found that rapid releases of both FOP and FRP occurred at salinities of >/=10 per thousand . Over the 4-day experimental period, sediment release increased the filterable P concentration by approx . 50 microgL(-1), and of this, nearly half was initially present as FOP, which subsequently underwent rapid mineralisation to FRP . The observed behaviour may be explained by a combination of salinity induced plasmolysis of sediment bacteria and ion exchange by suspended sediment particles.

BMC Genomics . 2004 Jan 15;5(1):7.
Molecular cloning and characterization of the mouse Acdp gene family; Wang CY et al.; BACKGROUND: We have recently cloned and characterized a novel gene family named ancient conserved domain protein (ACDP) in humans . To facilitate the functional study of this novel gene family, we have cloned and characterized Acdp, the mouse homologue of the human ACDP gene family . RESULTS: The four Acdp genes (Acdp1, Acdp2, Acdp3 and Acdp4) contain 3,631 bp, 3,244 bp, 2,684 bp and 2,743 bp of cDNA sequences, and encode deduced proteins of 951, 874, 713 and 771 amino acids, respectively . The mouse Acdp genes showed very strong homologies (>90%) in both nucleotide and amino acid sequences to their human counterparts . In addition, both nucleotide and amino acid sequences within the Ancient Conserved Domain (ACD) are highly conserved in many different taxonomic species . Particularly, Acdp proteins showed very strong AA homologies to the bacteria CorC protein (35% AA identity with 55% homology), which is involved in magnesium and cobalt efflux . The Acdp genes are widely expressed in all tissues tested except for Acdp1, which is only highly expressed in the brain with low levels of expression in kidney and testis . Immunostaining of Acdp1 in hippocampus neurons revealed a predominant localization on the plasma membrane . CONCLUSION: The Acdp genes are evolutionarily conserved in diverse species and ubiquitously expressed throughout development and adult tissues suggesting that Acdp may be an essential gene . Acdp showed strong homology to bacteria CorC protein and predominantly localized on the plasma membrane . These results suggest that Acdp is probably a family of proteins involved in ion transport in mammalian cells

J Infect Dis, 2004 Jan 15, 189(2), 339 - 45 Epub 2004 Jan 09.
Effect of sex on Coxiella burnetii infection: protective role of 17beta-estradiol; Leone M et al.; Q fever is a zoonosis caused by Coxiella burnetii and recently has been recognized as a potential agent of bioterrorism . In Q fever, men are symptomatic more often than women, despite equal seroprevalence . We hypothesized that sex hormones play a role in the pathogenesis of C . burnetii infection . When C57/BL6 mice were injected with C . burnetii, bacteria load and granuloma numbers were lower in females than in males . Ovarectomized mice showed increased bacteria load in the spleen and the liver, similar to that found in males . The granuloma number was also increased in ovarectomized mice and reached the levels found in males . Tissue infection and granulomatous response are largely under the control of estrogens: treatment of ovarectomized mice with 17beta-estradiol reduced both bacteria loads and granuloma numbers . These results show that sex hormones control host response to C . burnetii infection and may account for host-dependent clinical presentation of Q fever.

Cell Tissue Res, 2004 Mar, 315(3), 311 - 9 Epub 2004 Jan 14.
An immunocytochemical study of pulpal responses to cavity preparation by laser ablation in rat molars by using antibodies to heat shock protein (Hsp) 25 and class II MHC antigen; Suzuki T et al.; Initial responses of odontoblasts and immunocompetent cells to cavity preparation by laser ablation were investigated in rat molars . In untreated control teeth, intense heat shock protein (Hsp) 25 immunoreactivity was found in the cell bodies of odontoblasts, whereas cells immunopositive for the class II major histocompatibility complex (MHC) antigen were predominantly located beneath the odontoblast layer in the dental pulp . Cavity preparation caused the destruction of the odontoblast layer and the shift of most class-II-MHC-positive cells from the pulp-dentin border toward the pulp core at the affected site . Twelve hours after cavity preparation, numerous class-II-MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, but subsequently disappeared from the pulp-dentin border together with Hsp-25-immunopositive cells by 24 h after the operation . By 3-5 days postoperation, distinct abscess formation consisting of polymorphonuclear leukocytes was found in the dental pulp . The penetration of masses of oral bacteria was recognizable in the dentinal tubules beneath the prepared cavity . These findings indicate that cavity preparation by laser ablation induces remarkable inflammation by continuous bacterial infections via dentinal tubules in this experimental model, thereby delaying pulpal regeneration.

Microb Ecol, 2003 Nov, 46(4), 454 - 64 Epub 2003 Aug 14.
Population dynamics of Gluconacetobacter diazotrophicus in sugarcane cultivars and its effect on plant growth; Munoz-Rojas J et al.; Different experiments have estimated that the contribution of biological nitrogen fixation (BNF) is largely variable among sugarcane cultivars . Which bacteria are the most important in sugarcane-associated BNF is unknown . However, Gluconacetobacter diazotrophicus has been suggested as a strong candidate responsible for the BNF observed . In the present study, bacteria-free micropropagated plantlets of five sugarcane cultivars were inoculated with three G . diazotrophicus strains belonging to different genotypes . Bacterial colonization was monitored under different nitrogen fertilization levels and at different stages of plant growth . Analysis of the population dynamics of G . diazotrophicus strains in the different sugarcane varieties showed that the bacterial populations decreased drastically in relation to plant age, regardless of the nitrogen fertilization level, bacterial genotype or sugarcane cultivars . However, the persistence of the three strains was significantly longer in some cultivars (e.g., MEX 57-473) than in others (e.g., MY 55-14) . In addition, some strains (e.g., PAl 5(T)) persisted for longer periods in higher numbers than other strains (e.g., PAl 3) inside plants of all the cultivars tested . Indeed, the study showed that the inoculation of G . diazotrophicus may be beneficial for sugarcane plant growth, but this response is dependent both on the G . diazotrophicus genotype and the sugarcane variety . The most positive response to inoculation was observed with the combination of strain PAl 5(T) and the variety MEX 57-473 . Although the positive effect on sugarcane growth apparently occurred by mechanisms other than nitrogen fixation, the results show the importance of the sugarcane variety for the persistence of the plant-bacteria interaction, and it could explain the different rates of BNF estimated among sugarcane cultivars.

Chemosphere, 2004 Apr, 55(1), 101 - 7
Bioleaching of heavy metals from anaerobically digested sewage sludge using FeS2 as an energy source; Wong JW et al.; The effect of using FeS2 as an energy source, on the bioleaching of heavy metals (Zn, Cr, Cu, Pb and Ni) and nutrients (nitrogen and phosphorus) from anaerobically digested sludge using isolated indigenous iron-oxidizing bacteria was investigated in this paper . Addition of FeS2 in the range of 0.5-4.0 g l(-1) accelerated the acidification of sludge and raised the oxidation-reduction potential of sludge medium with an inoculation of 15% (v/v) of active bacteria, thus resulting in an overall increase in metal dissolution efficiency . After 16 days of bioleaching at 28 degrees C and an initial pH of 3.0, up to 99% of Zn, 65% of Cr, 74% of Cu, 58% of Pb and 84% of Ni can be removed from the sludge . In contrast, only 94% of Zn, 12% of Cr, 21% of Cu, 32% of Pb and 38% of Ni were leached out in the control without inoculation of iron-oxidizing bacteria and the addition of FeS2 . Less than 15% of nitrogen and 6% of phosphorous were lost after 16 days of bioleaching when using FeS2 as the energy source . Comparing to 39% and 45% loss respectively for these two nutrients when using FeSO4.7H2O as the energy source, FeS2 appears to be a more suitable energy source for preserving nutrients in sludge while removing heavy metals from sludge.

Am J Respir Med, 2003, 2(3), 261 - 73
Management of malignancy-associated pleural effusion: current and future treatment strategies; Marchi E et al.; Management of recurrent malignant pleural effusion, a common complication of malignancy, poses a challenge to clinicians . Although almost one century has elapsed since the introduction of the pleurodesis procedure, the ideal approach and best agent are still to be defined . Optimally, pleurodesis should be done at the bedside with a minimally invasive procedure, and suitable agents to achieve pleural symphysis should be inexpensive, available worldwide and free of adverse effects . To date, no substance completely fulfills these requirements . Silver nitrate should be considered for pleurodesis because of its low cost and ease of handling . Although talc has been used most frequently to induce pleurodesis, reports of death due to acute respiratory failure have raised concerns about the safety of this agent . Tetracycline, an effective alternative used in the past, is no longer commercially available . This agent has been substituted with derivatives of tetracycline, such as minocycline and doxycycline with success rates similar to those with tetracycline . Several antineoplastic agents have been injected into the pleural space with the aim of producing pleural symphysis, the most representative of this group being bleomycin . Recent knowledge of the molecular mechanisms involved in pleural inflammation has brought into focus new substances, such as transforming growth factor beta and vascular endothelial growth factor, which may be used as pleurodesis agents in the future . Nevertheless, more studies are necessary to better define the potential of these substances in the induction of pleural symphysis.Ideally, a sclerosing agent should be cost-effective, available worldwide and easily administered . Talc will probably stand as the preferred agent to be used for pleurodesis in malignant pleural effusion because of its efficacy, easy manipulation and handling . However, further investigation is necessary to minimize adverse effects related to talc.

Huan Jing Ke Xue, 2003 Sep, 24(5), 81 - 5
{Improvement of biological pretreatment performance by addition of phosphorus}; Sang J et al.; Addition of phosphorus as a novel way to improve the performance of bio-ceramic filter for biological pretreatment of source water was conducted . More bacteria grown in raw water and the BDOC of raw water increased when 50 micrograms/L PO4(3-)-P (NaH2PO4) was added alone were clear evidence of phosphorus limitation on bacterial growth . It shown that about 4.7%, 3.6% and 5.7% more of COD(Mn), UV254 and TOC from the raw water investigated in the experiment on the average was removed respectively when 25 micrograms/L PO4(3-)-P (H3PO4) was added to the influent of bioceramic filter . The role of phosphorus in raw water and drinking water should be paid more attention.

Huan Jing Ke Xue, 2003 Sep, 24(5), 54 - 9
{Multimedia fate modeling with spatial resolution for phenanthrene in Tianjin}; Cao H et al.; Behavior and fate of phenanthrene in various phases in Tianjin were calculated using a multimedia model with spatial resolution under steady-state assumption . Spatial variation of two parameters, namely soil organic carbon content and emission from fossil fuel combustion, were taken into consideration . Both soil and air phases were further divided into 3113 sub-compartments and 6226 equations in total were solved simultaneously under mass-balance assumption . A number of output parameters, therefore, were generated and were used for mapping of phenanthrene concentrations in soil and air, as well as transfer fluxes between compartments . The model was evaluated in two ways . 1 . Comparison between the calculated and the observed average concentrations in bulk compartments, and 2 . Comparison of spatial distribution of the calculated and the observed phenanthrene concentrations in surface soil . In both cases, the predicted results are in fair agreement with the independently measured values . As the results of the modeling, it was demonstrated that soil and sediment, especially the later, is the primary sink of phenanthrene in the area over 70% of the chemical accumulated in sediment . The spatial distribution pattern of phenanthrene in surface soil depends on content of soil organic matter which prevent the degradation of the material by bacteria . For distribution in ambient air, the dominant controlling factor is the emission.

Semin Dial, 2004 Jan-Feb, 17(1), 57 - 60
Intravenous iron and the risk of infection in end-stage renal disease patients; Brewster UC et al.; Oral iron is typically insufficient for the iron deficiency of hemodialysis patients . Intravenous (IV) iron is well tolerated by most patients and non-dextran-containing iron preparations are associated with few allergic reactions . However, there is the potential for an increased risk of infection with IV iron that appears to increase bacterial growth as well as inhibit the host's innate immune response to bacterial infection . Clinical studies suggest a link between iron therapy and infection . Practicing nephrologists should be aware of this issue, but should not hesitate to use IV iron in iron-deficient patients while avoiding the development of iron overload and administration of iron to patients who have active infection.

Dermatol Clin, 2003 Oct, 21(4), 601 - 7
Epidemiology of skin diseases in ethnic populations; Taylor SC; The spectrum of cutaneous disease occurring in ethnic populations is as broad and diverse as the ethnic populations themselves . Many skin diseases are seemingly common to most of the ethnic populations, however, including blacks, Hispanics, Asians, and Native Americans . These diseases include acne vulgaris; pigmentary disorders; eczematous dermatitis; and infection caused by bacteria, fungi, and viruses . Diseases of a more cosmetic nature have emerged over recent years and include the pigmentary disorders melasma and postinflammatory pigmentation, acne keloidalis nuchae, scalp and facial folliculitis, keloidal scarring, alopecia, and photoaging . The identification of cutaneous diseases affecting the rapidly increasing ethnic populations serves to focus resources both research and clinical in these areas.

Environ Sci Technol, 2003 Dec 15, 37(24), 5671 - 7
Surface chemical heterogeneity of bacteriogenic iron oxides from a subterranean environment; Martinez RE et al.; This study quantifies the surface chemical heterogeneity of bacteriogenic iron oxides (BIOS) and its end-members (2-line ferrihydrite and intermixed intact and fragmented bacteria) . On a dry weight basis, BIOS consisted of 64.5 +/- 1.8% ferrihydrite and 34.5 +/- 1.8% organic matter . Enrichment of Al, Cu, Cr, Mn, Sr, and Zn was shown in the solid versus the aqueous phase (1.9 < log Kd < 4.2) . Within the solid-phase Al (69.5%), Cu (78.7%), and Zn (77.9%) were associated with the bacteria, whereas Cr (59.8%), Mn (99.8%), and Sr (79.4%) preferred ferrihydrite . Acid-base titration data from the BIOS and bacteria were fitted using FOCUS pKa spectroscopy . The bacteria spectrum with pKa's of 4.18 +/- 0.37, 4.80 +/- 0.54, 6.98 +/- 0.45, and 9.75 +/- 0.68 was similar to discrete and continuous spectra for intact and fragmented bacteria . The BIOS spectrum recorded pKa's of 4.27 +/- 0.51, 6.61 +/- 0.51, 7.89 +/- 1.10, and 9.65 +/- 0.66 and was deconvoluted to remove overlapping binding site contributions from the bacteria . The resulting residual iron oxide spectrum coincided with discrete MUSIC spectra for goethite and lepidocrocite with pKa values of 4.10 +/- 0.43, 6.53 +/- 0.45, 7.81 +/- 0.76, and 9.51 +/- 0.68 . Surface site density analysis showed that acidic sites (pKa < 6) were contributed by the bacteria (37%), whereas neutral sites (6 < pKa < 8) were characteristic of the iron oxide fraction (35%) . Basic sites (8 < pKa) were higher in the bacteria (57%), than in the BIOS (44%) or iron oxide fractions (47%) . This analysis suggested a high degree of bacterial group masking and a similarity between the BIOS and goethite surface reactivity . An understanding of the BIOS surface chemical heterogeneity and inherent proton and metal binding capacity was obtained through the use of FOCUS apparent pKa spectroscopy.

Zh Mikrobiol Epidemiol Immunobiol, 2003 Nov-Dec, (6), 37 - 42
{Improvement of methods for indication of the causative agents of natural focal infections under conditions of emergency situations with epidemiologic consequences}; Efremenko VI et al.; The improvement of the laboratory diagnostics of quarantine infections (QI) is aimed at obtaining the result at a shorter time, the simplification of making serological reactions, enhancing the specificity and sensitivity of the results due to the introduction of new = generation diagnostic preparations into practical use . The wide practical use of erythrocytic and immunofluorescent diagnostic preparations has shown that they cannot completely meet all requirements simultaneously . The optimized scheme of the indication of the causative agents of QI must ensure the combined use, at different stages of the analysis, of magneto-immunosorbents, enzyme immunoassay, as well as immuno-suspension preparations for the simplification and acceleration of obtaining results with sufficient sensitivity and specificity provided.

FASEB J, 2004 Mar, 18(3), 537 - 9 Epub 2004 Jan 08.
Evidence of melatonin synthesis by human lymphocytes and its physiological significance: possible role as intracrine, autocrine, and/or paracrine substance; Carrillo-Vico A et al.; It has been historically assumed that the pineal gland is the major source of melatonin (N-acetyl-5-methoxytryptamine) in vertebrates . Melatonin plays a central role in fine-tuning circadian rhythms in vertebrate physiology . In addition, melatonin shows a remarkable functional versatility exhibiting antioxidant, oncostatic, antiaging, and immunomodulatory properties . Melatonin has been identified in a wide range of organisms from bacteria to human beings . Its biosynthesis from tryptophan involves four well-defined intracellular steps catalyzed by tryptophan hydroxylase, aromatic amino acid decarboxylase, serotonin-N-acetyltransferase, and hydroxyindole-O-methyltransferase . Here, for the first time, we document that both resting and phytohemagglutinin-stimulated human lymphocytes synthesize and release large amounts of melatonin, with the melatonin concentration in the medium increasing up to five times the nocturnal physiological levels in human serum . Moreover, we show that the necessary machinery to synthesize melatonin is present in human lymphocytes . Furthermore, melatonin released to the culture medium is synthesized in the cells, because blocking the enzymes required for its biosynthesis or inhibiting protein synthesis in general produced a significant reduction in melatonin release . Moreover, this inhibition caused a decrease in IL-2 production, which was restored by adding exogenous melatonin . These findings indicate that in addition to pineal gland, human lymphoid cells are an important physiological source of melatonin and that this melatonin could be involved in the regulation of the human immune system, possibly by acting as an intracrine, autocrine, and/or paracrine substance.

J Virol Methods, 2004 Mar 1, 116(1), 55 - 61
Recombinant VP9-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Banna virus (genus Seadornavirus); Mohd Jaafar F et al.; Banna virus (BAV, genus Seadornavirus, family Reoviridae) is an arbovirus suspected to be responsible for encephalitis in humans . Two genotypes of this virus are distinguishable: A (Chinese isolate, BAV-Ch) and B (Indonesian isolate, BAV-In6969) which exhibit only 41% amino-acid identity in the sequence of their VP9.The VP7 to VP12 of BAV-Ch and VP9 of BAV-In6969 were expressed in bacteria using pGEX-4T-2 vector . VP9 was chosen to establish an ELISA for BAV, based mainly on two observations: (i) . VP9 is a major protein in virus-infected cells and is a capsid protein (ii) . among all the proteins expressed, VP9 was obtained in high amount and showed the highest immuno-reactivity to anti-BAV ascitic fluid.The VP9s ELISA was evaluated in three populations: French blood donors and two populations (blood donors and patients with a neurological syndrome) from Malaysia, representing the region where the virus was isolated in the past.The specificity of this ELISA was >98% . In mice injected with live BAV, the assay detected IgG-antibody to BAV infection 21 days post-injection, which was confirmed by Western blot using BAV-infected cells.The VP9 ELISA permits to determine the sero-status of a population without special safety precautions and without any requirements to propagate the BAV . This test should be a useful tool for epidemiological survey of BAV.

J Vector Ecol, 2003 Dec, 28(2), 184 - 9
Survey of ticks collected in Mississippi for Rickettsia, Ehrlichia, and Borrelia species; Goddard J et al.; From November 1999 through October 2000, we tested ticks collected from vegetation as well as from deer, dogs, and humans for spotted fever group (SFG) rickettsiae, Ehrlichia chaffeensis, and Borrelia spp . spirochetes . A total of 149 adult ticks representing four species was collected from 11 collection sites from southwestern to northern Mississippi . Amblyomma americanum was most commonly collected (n=68), followed by Ixodes scapularis (n=53) . The bird tick, Ixodes brunneus (usually rare), was the third most commonly collected tick (n=17) . Eleven Dermacentor variabilis were also collected . Ticks were cut longitudinally to make smears on three microscope slides . The remaining body parts were frozen at -65 degrees C for additional testing . Tick smears were stained by direct immunofluorescence assays (DFA) for Rickettsia spp . and Borrelia spp., while indirect immunofluorescence assays (IFA) were used for Ehrlichia spp . The corresponding tick for each positive smear was evaluated using PCR analysis . None of the 149 ticks tested was DFA positive for Borrelia spp . However, smears of 30 (20%) and 32 (22%) ticks reacted with anti-E . chaffeensis sera and anti-R . rickettsii conjugate (known to react with several members of the spotted fever group), respectively . None of the ticks staining with the IFA for Ehrlichia was positive for E . chaffeensis using PCR . However, 23 (72%) of 32 FA-positive ticks for SFG rickettsiae yielded amplicons of the appropriate size when tested using a PCR assay for SFG rickettsiae, corresponding to an overall infection rate with SFG rickettsiae among the collected ticks of 15% . Smears of 12 (71%) of 17 I . brunneus revealed abundant bacilliform bacteria . PCR amplification of DNA from a single I . brunneus containing these bacteria was performed using universal primers for the 16S rRNA gene as well as Borrelia-specific primers . The predominant sequence obtained using the universal primers did not match any sequence in GenBank, but it showed 91% identity with an endosymbiont of Acanthoamoeba . Other sequences represented in the top 50 Basic Local Alignment Search (BLAST) scores were primarily from soil bacteria, although some similarity to several Anaplasma species and Ehrlichia risticii was indicated . The significance of this finding remains undetermined.

Biofizika, 2003 Nov-Dec, 48(6), 1052 - 70
{Electrostatic interactions in catalytic centers of F1-ATPase}; Tikhonov AN et al.; The first part of this paper is a brief review of works concerned with the mechanisms of functioning of F0F1-ATP synthases . F0F1-ATP syntheses operate as rotating molecular machines that provide the synthesis of ATP from ADP and inorganic phosphate (Pi) in mitochondria, chloroplasts, and bacteria at the expense of the energy of electrochemical gradient of hydrogen ions generated across energy-transducing mitochondrial, chloroplast or, bacterial membranes . A distinguishing feature of these enzymes is that they operate as rotary molecular motors . In the second part of the work, we calculated the contribution of electrostatic interactions between charged groups of a substrate (MgATP), reaction products (MgADP and Pi), and charged amino acid residues of the F1-ATPase molecule to energy changes associated with the binding of ATP and its chemical transformations in the catalytic centers located at the interface of the alpha- and beta-subunits of the enzyme (oligomer complex alpha 3 beta 3 gamma of bovine mitochondrial ATPase) . The catalytic cycle of ATP hydrolysis considered in the work includes conformational changes of alpha- and beta-subunits caused by unidirectional rotations of the central gamma-subunit . The results of our calculations are consistent with the idea that the energetically favorable process of ATP binding to the "open" catalytic center of F1-ATPase initiates the rotation of the gamma-subunit followed by ATP hydrolysis in another ("closed") catalytic center of the enzyme.

Parasitol Res, 2004 Mar, 92(4), 270 - 84 Epub 2004 Jan 09.
Factors affecting the component community structure of haemoparasites in common voles ( Microtus arvalis) from the Mazury Lake District region of Poland; Pawelczyk A et al.; The prevalence and abundance of infections with haemoparasites were studied over a 4-year period in Microtus arvalis (common vole, n =321) sampled from fallow grassland sites in north-eastern Poland . Total species richness was five (prevalence= Haemobartonella sp . 63.9%, Bartonella spp . 27.7%, Babesia microti 9.0%, Trypanosoma sp . 8.4% and and Hepatozoon lavieri 3.1%) with 76.9% of the voles carrying at least one species and a mean infracommunity species richness of 1.1 . Variation in species richness was determined primarily by season and year, the interaction of these factors, and that of year with host age . The observed frequency distribution of infracommunity species richness did not differ from that predicted by a null model, suggesting that there were no marked associations between the species . Analyses of prevalence and abundance of infection with each species in turn, revealed that overall the principal causes of variation were temporal and seasonal, their interaction, and interactions with intrinsic factors (age and sex), the latter playing only a minor role in their own right . However, the relative importance of these combinations varied and was distinct for each of the species in the study . Prevalence data revealed eight sets of two- and three-way associations between species, mostly dependent to some extent on one of the intrinsic and extrinsic factors in the model . Analysis of quantitative associations suggested two sets of positive two-way interactions, none of which remained after controlling for the effect of extrinsic and intrinsic factors on the abundance of each species . These data are discussed in the context of the changing ecological profiles in this region of Eastern Europe and, in a wider context, in relation to current understanding of the factors that shape component community structures of haemoparasites in wild rodents.

Environ Toxicol Chem, 2003 Dec, 22(12), 3049 - 55
The effects of metals and food availability on the behavior of Caenorhabditis elegans; Boyd WA et al.; Caenorhabditis elegans, a nonparasitic soil nematode, was used to assess the combined effects of metal exposures and food availability on behavior . Movement was monitored using a computer tracking system after exposures to Cu, Pb, or Cd while feeding was measured as a change in optical density (deltaOD) of bacteria suspensions over the exposure period . After 24-h exposures at high and low bacteria concentrations, movement was decreased in a concentration-dependent fashion by Pb and Cd but feeding reductions were not directly proportional to exposure concentrations . Copper exposure induced concentration-dependent declines in feeding and movement regardless of bacteria concentration . The impact of 24-h metal exposures was apparently reduced by increasing food availability . Therefore, exposures were shortened to 4 h in an attempt to minimize starvation effects on movement . Although nematodes were immobilized following 24 h of food depravation, worms deprived of food during the 4-h exposure continued to feed and move after exposure . A bead-ingestion assay after 4-h exposures was also used as an additional means of assessing the effects of metals on feeding behavior . Ingestion was significantly reduced by all concentrations of metals tested, indicating its sensitivity as a sublethal assay . Feeding (deltaOD) during exposures exhibited similar trends as ingestion but was slightly less sensitive, while movement was the least sensitive assay of 4-h metal exposures to C . elegans . Assessment of multiple sublethal endpoints allowed for the determination of the separate and interactive effects of metals and food availability on C . elegans behavior.

Environ Toxicol Chem, 2003 Dec, 22(12), 2986 - 94
Immunocompetence of juvenile chinook salmon against Listonella anguillarum following dietary exposure to polycyclic aromatic hydrocarbons; Palm RC Jr et al.; Juvenile chinook salmon (Oncorhynchus tschawytscha) were fed a mixture of 14 polycyclic aromatic hydrocarbon (PAH) compounds that reflected the PAH composition of salmon stomach contents in an urban estuary of Puget Sound, Washington (USA) . Following a 28-d dietary exposure, a standardized Listonella anguillarum challenge model was used to determine whether PAH exposure (16, 64, and 252 mg/kg wet wt feed) causes reduced disease resistance under the conditions examined in this study . To assess innate immunity, five replicate groups of fish per dose were acclimated for one week, exposed to a lethal concentration 60 of bacteria, and monitored for 14 d . In a parallel experiment, the effects of PAH exposure on the acquired immune response were examined by immersion vaccinating fish against L . anguillarum and allowing specific immunity to develop for three weeks prior to challenge . All mortalities were aseptically sampled to confirm L . anguillarum infections . No significant differences in fish length, weight, or coefficient of condition were observed . These controlled laboratory experiments suggest that dietary exposures to an environmentally relevant mixture of PAH compounds do not alter the immunocompetence or growth of juvenile chinook salmon.

Int Rev Cytol, 2003, 231, 197 - 258
Regulation by RNA; Szymanski M et al.; In recent years, noncoding RNAs (ncRNAs) have been shown to constitute key elements implicated in a number of regulatory mechanisms in the cell . They are present in bacteria and eukaryotes . The ncRNAs are involved in regulation of expression at both transcriptional and posttranscriptional levels, by mediating chromatin modifications, modulating transcription factor activity, and influencing mRNA stability, processing, and translation . Noncoding RNAs play a key role in genetic imprinting, dosage compensation of X-chromosome-linked genes, and many processes of differentiation and development.

Eur J Clin Microbiol Infect Dis, 2004 Feb, 23(2), 119 - 22 Epub 2004 Jan 08.
Semiquantitative culture of open surgical wounds for diagnosis of surgical site infection; Bouza E et al.; The aim of this study was to assess the performance of a semiquantitative culture method and traditional qualitative culture for the diagnosis of surgical site infections resulting in an open wound . Results were correlated with the definitions for the diagnosis of SSI proposed by the Centers for Disease Control and Prevention, Atlanta, Ga., USA . The sensitivity, specificity, positive and negative predictive values of the qualitative culture method were 96%, 61%, 84% and 89%, respectively . For the semiquantitative technique with a breakpoint of 15 colony forming units/plate these values were 91%, 96%, 98% and 83%, respectively . Analysis of the receiver operating characteristic curve revealed an area under the curve of 94.44% (95% confidence interval, 88.5%-99%) for the semiquantitative method . This result indicates that our easy-to-perform semiquantitative culture technique for open surgical wounds correlates better with the diagnosis of SSI than the traditional qualitative method.

Appl Environ Microbiol, 2004 Jan, 70(1), 413 - 20
Periplasmic cytochrome c3 of Desulfovibrio vulgaris is directly involved in H2-mediated metal but not sulfate reduction; Elias DA et al.; Kinetic parameters and the role of cytochrome c(3) in sulfate, Fe(III), and U(VI) reduction were investigated in Desulfovibrio vulgaris Hildenborough . While sulfate reduction followed Michaelis-Menten kinetics (K(m) = 220 micro M), loss of Fe(III) and U(VI) was first-order at all concentrations tested . Initial reduction rates of all electron acceptors were similar for cells grown with H(2) and sulfate, while cultures grown using lactate and sulfate had similar rates of metal loss but lower sulfate reduction activities . The similarities in metal, but not sulfate, reduction with H(2) and lactate suggest divergent pathways . Respiration assays and reduced minus oxidized spectra were carried out to determine c-type cytochrome involvement in electron acceptor reduction . c-type cytochrome oxidation was immediate with Fe(III) and U(VI) in the presence of H(2), lactate, or pyruvate . Sulfidogenesis occurred with all three electron donors and effectively oxidized the c-type cytochrome in lactate- or pyruvate-reduced, but not H(2)-reduced cells . Correspondingly, electron acceptor competition assays with lactate or pyruvate as electron donors showed that Fe(III) inhibited U(VI) reduction, and U(VI) inhibited sulfate loss . However, sulfate reduction was slowed but not halted when H(2) was the electron donor in the presence of Fe(III) or U(VI) . U(VI) loss was still impeded by Fe(III) when H(2) was used . Hence, we propose a modified pathway for the reduction of sulfate, Fe(III), and U(VI) which helps explain why these bacteria cannot grow using these metals . We further propose that cytochrome c(3) is an electron carrier involved in lactate and pyruvate oxidation and is the reductase for alternate electron acceptors with higher redox potentials than sulfate.

Appl Environ Microbiol, 2004 Jan, 70(1), 214 - 23
Within- and between-lake variability in the composition of bacterioplankton communities: investigations using multiple spatial scales; Yannarell AC et al.; This study examined the similarity of epilimnetic bacterial community composition (BCC) across several within- and among-lake spatial scales, and the environmental factors giving rise to similar bacterial communities in different lakes were also explored . Samples were collected from 13 northern and southern Wisconsin lakes representing gradients in lake size, productivity, dissolved organic carbon and humic acid contents, and pH . Hypotheses regarding patchy distribution of bacterial communities in lakes were tested by comparing samples collected from nearby (tens of meters) and distant (hundreds of meters) sampling sites in the same lake . BCC was characterized by using a molecular fingerprinting technique, automated ribosomal intergenic spacer analysis (ARISA) . Overall, samples collected at the 10-m, 100-m, and between-lake scales differed by 13, 17, and 75%, respectively . Variation at these last two scales was significant . The development of within-lake variation in BCC appeared to depend on the isolation of water by lake shoreline features such as bays or narrow constrictions . ARISA profiles from northern lakes had fewer peaks and were less similar to each other than were those of the southern lakes, suggesting that regional features do not necessarily lead to the development of similar bacterial communities . Lakes at similar positions on productivity and dissolved organic carbon concentration gradients had similar bacterial communities, and bacterial diversity was positively correlated with lake productivity and water temperature . Factorial studies taking into account these gradients, as well as regional spatial scales, should provide much insight into the nature of aquatic bacterial biogeography.

Adv Cancer Res, 2003, 90, 63 - 89
Persistent infection with Helicobacter pylori and the development of gastric cancer; Normark S et al.; Gastric malignancies have been closely linked to infection of the gastric mucosa with Helicobacter pylori, but the individual factors involved in the multistage process of tumor development are still poorly understood . H . pylori evades the host defense system and causes persistent infection and chronic inflammation . Immune activation leads to DNA damage by the release of oxygen and nitrogen radicals . Ongoing tissue repair mechanisms and the secretion of cytokines and growth factors, as well as bacterial effector molecules, cause disturbances in the balance between epithelial cell proliferation and apoptosis, promote the accumulation of potential oncogenic mutations, and support neovascularization and tumor growth . In addition, H . pylori might hamper the development of an efficient antitumor immunity and provoke immune-mediated pathology . This review summarizes the recent progress in the understanding of the intimate bacteria-host relationship and the mechanisms by which H . pylori may promote the process of tumor development.

J Vet Med Sci, 2003 Dec, 65(12), 1377 - 80
The detection of bovine lactoferrin binding protein on Toxoplasma gondii; Tanaka T et al.; Lactoferrin (LF), a member of the transferrin (TF) protein family, is an iron-binding protein that is known to interact with bacteria through a specific receptor . We examined the binding of bovine LF (bLF), bovine TF (bTF), and ovotransferrin (OTF) by Toxoplasma gondii using a fluorescence test and the streptavidin-biotin (SAB) method using biotin-streptavidin, and found that bLF, bTF, and OTF bound to the protein components of T . gondii . Furthermore, we confirmed that bLF, bTF, and OTF bound a 42 kDa soluble protein of T . gondii by far Western blot method . These results demonstrated that bLF binding proteins are present on T . gondii.

Biosens Bioelectron, 2004 Feb 15, 19(7), 721 - 6
A biosensor for the analysis of acetonitrile; Hakansson K et al.; A biosensor for monitoring acetonitrile was constructed . A mixed culture was taken from a degradation reactor and mounted on top of a Clark electrode . The amperometric biosensor was placed in a flow-through cell and integrated into a flow injection system . The metabolic response in terms of oxygen consumption was well correlated to the concentration of acetonitrile in standard solutions . However, when the reaction products, acetic acid and ammonia, were also present, the response was erratic, due to the additional metabolic reaction on acetate . By introducing a hydrophobic barrier it was possible to eliminate the negative influence of these charged products and thus to improve the operational selectivity of the sensor.The biosensor showed good stability for analysis during at least 6 days and future work will focus on using it for monitoring and control of degradation processes.

Microb Ecol, 2003 Aug, 46(2), 270 - 8
Analysis of methanogen diversity in a hypereutrophic lake using PCR-RFLP analysis of mcr sequences; Earl J et al.; The incidence and diversity of methanogens in Priest Pot, a dynamic and active lake, were monitored by analysing mcrA gene sequences generated from total DNA samples obtained at different times of the year and amplified using the polymerase chain reaction . A number of mcrA clones were analysed by developing an RFLP-based protocol to generate a number of restriction patterns that were assigned to a number of classes . The RFLP patterns for each class were compared with published sequence information for mcrA from cultured methanogens as well as with those from other experimental studies . They could be used to assign tentative identification for some of the Priest Pot clones and also revealed the presence of a number of clones that could not be affiliated to any known methanogens . The limitations of using RFLP profiles of mcrA gene sequences for studying methanogen ecology are discussed.

Microb Ecol, 2003 Aug, 46(2), 257 - 69
Diversity of Geobacteraceae species inhabiting metal-polluted freshwater lake sediments ascertained by 16S rDNA analyses; Cummings DE et al.; The abundance, distribution, and phylogenetic diversity of members of the Fe(III)-reducing family Geobacteraceae were studied along a gradient of metal contaminants in Lake Coeur d'Alene, Idaho . Partial 16S rRNA gene fragments were amplified by PCR using primers directed toward conserved regions of the gene within the family Geobacteraceae . Analysis of amplicons separated by denaturing gradient gel electrophoresis (DGGE) suggested within-site variation was as great as between-site variation . Amplicons were cloned and grouped by RFLP type and DGGE migration distance and representatives were sequenced . Grouping clones with 3% or less sequence dissimilarity, 15 distinct phylotypes were identified compared to 16 distinct DGGE bands . Only 1 phylotype was recovered from all sites . This clone, B14, is most closely related to Geobacter metallireducens and constituted a greater portion of the pristine community than of the contaminated communities . A second phylotype, Q2, predominated in the contaminated communities and was notably absent from the pristine libraries . Clone Q2 presents a high degree of sequence similarity to two Geobacter spp . previously isolated from this region of Lake Coeur d'Alene . Six phylotypes were unique to the contaminated sediments, whereas two were found only in the pristine sediments . Indices of diversity (Shannon and Simpson) were consistently higher when calculated with DGGE data than when clone library data were used . Most-probable-number PCR and real-time PCR suggested that the Geobacteraceae phylotypes were spread relatively evenly across all three sites along the gradient . Our data indicate that the Geobacteraceae are diverse and abundant in Lake Coeur d'Alene sediments, regardless of metals content . These results provide insight into the ability of dissimilatory Fe(III)-reducing bacteria to colonize habitats with elevated metal concentrations, and they have important implications for the management and remediation of metal-contaminated sites.

Microb Ecol, 2003 Aug, 46(2), 238 - 48
Archaeal nucleic acids in picoplankton from great lakes on three continents; Keough BP et al.; Phylogenetic analysis of PCR-amplified 16S rRNA genes revealed the presence of archaea in picoplankton collected from the Laurentian Great Lakes in North America, Africa's Lake Victoria, and Lakes Ladoga and Onega in northeastern Eurasia . From 1 to 10% of the rRNA extracted from size-fractionated picoplankton (>0.2 microm but <1.2 microm) collected in the epilimnion and hypolimnion of these lakes was specific to the Archaea, whereas the majority of rRNA was derived from Bacteria . Analysis of the 16S rRNA genes cloned from these samples indicated they were closely related to crenarchaeal sequences that have been widely characterized from marine environments . The presence of nearly identical 16S rDNA clones in several of these geographically disparate lakes suggests a cosmopolitan distribution of specific subgroups of these Archaea in freshwater environments . Despite their abundance in the water column of freshwater lakes, we have no representatives of these crenarchaea in pure culture, and so their physiological characteristics and ecological role remain unknown.

J Mol Evol, 2003 Oct, 57(4), 383 - 96
Secondary structure and molecular evolution of the mitochondrial small subunit ribosomal RNA in Agaricales (Euagarics clade, Homobasidiomycota); Barroso G et al.; The complete sequences and secondary structures of the mitochondrial small subunit (SSU) ribosomal RNAs of both mostly cultivated mushrooms Agaricus bisporus (1930 nt) and Lentinula edodes (2164 nt) were achieved . These secondary structures and that of Schizophyllum commune (1872 nt) were compared to that previously established for Agrocybe aegerita . The four structures are near the model established for Archae, Bacteria, plastids, and mitochondria; particularly the helices 23 and 37, described as specific to bacteria, are present . Within the four Agaricales (Homobasidiomycota), the SSU-rRNA "core" is conserved in size (966 to 1009 nt) with the exception of an unusual extension of 40 nt in the H17 helix of S . commune . The four core sequences possess 76% of conserved positions and a cluster of C in their 3' end, which could constitute a signal involved in the RNA maturation process . Among the nine putative variable domains, three (V3, V5, V7) do not show significant length variations and possess similar percentages of conserved positions (69%) than the core . The other six variable domains show important length variations, due to independent large size inserted/deleted sequences, and higher rates of nucleotide substitutions than the core (only 31% of conserved positions between the four species) . Interestingly, the inserted/deleted sequences are located in few preferential sites (hot spots for insertion/deletion) where they seem to arise or disappear haphazardly during evolution . These sites are located on the surface of the tertiary structure of the 30S ribosomal subunit, at the beginning of hairpin loops; the insertions lead to a lengthening of existing hairpins or to branching loops bearing up to five additional helices.

Acta Gastroenterol Latinoam, 2003, 33(2), 73 - 6
Evaluation of a liquid urease test (LUT) for detection of Helicobacter pylori; Montes H et al.; The aim of our study was to develop a rapid diagnostic urease test to demonstrate the presence of Helicobacter pylori in the Endoscopy room . MATERIALS AND METHODS: 200 consecutive patients referred to gastroscopy for different indications, were included in this study . One antral biopsy sample was obtained to be immersed in our test . The same sample was used for histological evaluation, considered to be the gold standard method for diagnose of Helicobacter pylori infection . RESULTS: 135 patients (67.5%) were found positives and 65 patients (32.5%) were negatives in our test . 128 patients (64%) showed Helicobacter pylori on histological examination . Our test showed a sensitivity of 91%, specificity of 88.1%, and positive and negative predictive values of 95% and 80% respectively . A remarkable correlation between density of Helicobacter pylori and reading time was also observed, where a high density of the bacteria reduced the reaction time in this liquid test . Furthermore, an overall accuracy of 90% was shown, which is comparable with other available commercial tests . CONCLUSION: LUT is easy to handle, cost effective and fast, with a high positive predictive value.

J Soc Biol, 2003, 197(3), 205 - 10
{Cellular lipid dynamics}; Baumann N et al.; During the past years, the notion of microdomains at the surface of cellular membranes has been developed . These are constituted by lipid rafts which involve sphingoglycolipids and cholesterol . To these rafts are associated proteins which have a lipid anchor or are transmembrane proteins . These lipid rafts target specific proteins at the plasma membrane surface and can remain associated with them . They are present in surface receptors and endocytosis occurs upon binding of the specific ligands . Thus these rafts participate to major aspects of cellular dynamics . These rafts are complex structures, insoluble in non-ionic detergents . According to the detergent used, many types of rafts can be isolated . Any alteration of cholesterol, sphingoglycolipids, or abnormalities of the proteins themselves, can lead to abnormal targeting at the membrane surface . It is possible that specific sphingoglycolipids are necessary to target specific proteins at the membrane surface . This may explain the complexity of the sphingoglycolipid molecules, both in relation to their oligosaccharide and to their ceramide structures . There is both a cellular and a tissue specificity of these constituents . Complex sphingoglycolipids are involved in cellular differentiation, cellular polarization, and modified in relation to cancer . Virus and bacteria can be linked to the sphingoglycolipids of these microdomains and alter cellular signaling and function . Sphingoglycolipids are involved in autoimmune diseases as antibody targets and in neurolipidoses which are genetic diseases involving their catabolism . The dynamics of the lipid rafts, in relation to cholesterol, can be altered in Niemann-Pick's disease type C and in Alzheimer's disease . Thus these microdomains are involved in many aspects related to normal and pathological cellular dynamics.

Biotechnol Bioeng, 2003 Dec 30, 84(7), 822 - 33
Insights into the relation between mRNA and protein expression patterns: I . Theoretical considerations; Mehra A et al.; Translation is a central cellular process in every organism and understanding translation from the systems (genome-wide) perspective is very important for medical and biochemical engineering applications . Moreover, recent advances in cell-wide monitoring tools for both mRNA and protein levels have necessitated the development of such a model to identify parameters and conditions that influence the mapping between mRNA and protein expression . Experimental studies show a lack of correspondence between mRNA and protein expression profiles . In this study, we describe a mechanistic genome-wide model for translation that provides mapping between changes in mRNA levels and changes in protein levels . We use our model to study the system in detail and identify the key parameters that affect this mapping . Our results show that the correlation between mRNA and protein levels is a function of both the kinetic parameters and concentration of ribosomes at the reference state . In particular, changes in concentration of free and total ribosomes in response to a perturbation; changes in initiation and elongation kinetics due to competition for aminoacyl tRNAs; changes in termination kinetics; average changes in mRNA levels in response to the perturbation; and changes in protein stability are all important determinants of the mapping between mRNA and protein expression .

Mol Cancer Ther, 2003 Dec, 2(12), 1341 - 50
Targeted delivery of human pro-apoptotic enzymes to tumor cells: In vitro studies describing a novel class of recombinant highly cytotoxic agents; Liu Y et al.; The serine protease granzyme B (GrB, 25 kDa) can initiate apoptosis by multiple mechanisms including directly activating caspases, inducing DNA fragmentation, activating the mitochondrial death pathway, and directly cleaving the nuclear matrix . The purpose of this study was to determine whether a recombinant antibody could deliver sufficient amounts of GrB to target cells to generate an apoptotic signal . The gene sequence encoding GrB was attached to the single-chain anti-melanoma antibody scFvMEL (anti-gp240) via a flexible (G(4)S) tether . The 53-kDa GrB/scFvMEL fusion protein was expressed in bacteria and purified by metal affinity chromatography . Western blotting confirmed presence of both scFvMEL and GrB proteins . The fusion construct displayed intact GrB enzymatic activity (specific activity = 2.6 x 10(5) units/ micro mol) similar to native GrB (specific activity = 4.8 x 10(5) units/ micro mol) . The construct bound specifically to human A375-M melanoma cells and delivered GrB to the cytosol as assessed by confocal microscopy . Against log-phase melanoma cells, GrB/scFvMEL demonstrated an IC(50) of 20 nM and minimal cytotoxicity to non-target cells at doses of up to 1 micro M . Coadministration of exogenous perforin (PFN) to cells resulted in a slight increase in the cytotoxic effects of the GrB/scFvMEL construct on A375 target cells and a significant increase in cytotoxicity to SKBR3 (non-target) cells . The cytotoxic effects of this fusion construct on target cells were similar to those of the previously described MEL sFv/rGel fusion toxin (IC(50) approximately 20 nM) . The construct produced impressive apoptotic effects by 8 h after treatment of target cells . Mediation of the apoptotic effects of GrB/scFvMEL included caspase-3 cleavage and release of cytochrome c into the cytosolic compartment from the mitochondrial compartment . These studies demonstrate that delivery of the human pro-apoptotic pathway enzyme GrB to tumor cells may have significant therapeutic potential for cancer treatment and represents a new class of targeted therapeutic agents with a defined mechanism of action.

J Microbiol Methods, 2004 Jan, 56(1), 107 - 18
Development of degenerate and specific PCR primers for the detection and isolation of known and putative chloroethene reductive dehalogenase genes; Regeard C et al.; Degenerate and specific PCR primers were designed for the detection of chloroethene reductive dehalogenases (CE-RDase), the key enzymes of chloroethene dehalorespiration, based on sequence information of three CE-RDases and three chlorophenol (CP) RDases . For the design of the degenerate primers, seven conserved amino-acid blocks identified with different bioinformatic tools were used . For one block degenerate, primers containing a 5'-consensus clamp region specific for CE-RDases and a 3'-end degenerate core region specific for RDases in general were designed using the Consensus-Degenerate Hybrid Oligonucleotide Primer (CDHOP) design method . Applying the degenerate primers to genomic DNA of Sulfurospirillum multivorans strain K, Dehalobacter restrictus strain PER-K23, and Desulfitobacterium sp . strain PCE1 led to the isolation of the known CE-RDase genes and three new genes encoding putative reductive dehalogenases that cluster with CE-RDases and not with CP-RDases . In addition, primers designed to be specific for the three known CE-RDase genes, namely pceA of S . multivorans, pceA of D . restrictus, and tceA of Dehalococcoides ethenogenes were successfully tested on genomic DNA of different chloroethene-dehalorespiring bacteria . Nested PCR using degenerate primers followed by a PCR with specific primers allowed a sensitive detection of only 10(2) copies per reaction.

J Microbiol Methods, 2004 Jan, 56(1), 73 - 82
Fourier transform infrared spectroscopy as a new tool for characterization of mollicutes; Melin AM et al.; Fourier transform infrared (FT-IR) spectroscopy is a convenient physico-chemical technique to investigate various cell materials . Bacteria of class Mollicutes, identified by conventional methods, as Mycoplasma, Acholeplasma and Ureaplasma genera were characterized using this method . A data set of 74 independent experiments corresponding to fourteen reference strains of Mollicutes was examined by FT-IR spectroscopy to attempt a spectral characterization based on the biomolecular structures . In addition to the separation of Mollicutes within the lipidic region into five main clusters corresponding to the three phylogenetic groups tested, FT-IR spectroscopy allowed a fine discrimination between strains belonging to the same species by using selective spectral windows, particularly in the 1200-900 cm(-1) saccharide range . The results obtained by FT-IR were in good agreement with both taxonomic and phylogenetic classifications of tested strains . Thus, this technique appears to be a useful tool and an accurate mean for a rapid characterization of Mollicutes observed in humans.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2003 Dec, 11(6), 650 - 3
{Effects of leukocyte elimination before storage on quality of red blood cell concentration}; Wang HB et al.; The objective of this study was to explore the possible effects of leukocyte elimination by filteration before storage on the quality of red blood cell concentrations (RCC) that prepared through two procedures . Eight units of red blood cell concentrations derived from whole blood after plasma separated (RCC1) and eight units of red blood cell concentrations derived from whole blood after platelet-rich plasma separated (RCC2) were divided randomly into filtered group and control group respectively . The RCC of filtered group were filtered by leukocyte deplete filter before storage . The control group didn't have any other treatments . These two groups were stored for five weeks at 4 degrees C according to AABB standard . Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and plasma concentration of K(+) and lactate dehydrogenase (LDH), free hemoglobin (FHb), adenosine triphosphate (ATP) of red blood cell of all RCC were evaluated weekly, and bacteria contamination of all RCC was also detected after five weeks of storage . The results showed that there was no difference of MCV, MCH and MCHC and ATP level of red blood cell in all RCC of two groups, the ATP of red blood cell was lower than the control group on week 4 and 5 . The average concentration of K(+) of the filtered group was less than the control group . The differences are significant except that of RCC1 stored till the third week . The plasma LDH concentration of filtered group was less than the control group, and the differences were exacerbate during the storing time prolonged . FHb release in the filtered group of RCC2 was significant less than that of control, but no significant difference was found between the two groups of RCC1 . It was concluded that leukocyte elimination by filter before storage could be benefit to RCC preservation.

Cell Microbiol, 2004 Feb, 6(2), 167 - 74
Helicobacter pylori toxin VacA is transferred to host cells via a novel contact-dependent mechanism; Ilver D et al.; Helicobacter pylori is the causative agent of peptic ulcer disease . A major virulence factor of H . pylori is VacA, a toxin that causes massive vacuolization of epithelial cell lines in vitro and gastric epithelial erosion in vivo . Although VacA is exported over the outer membrane and is released from the bacteria, a portion of the toxin remains associated with the bacterial surface . We have found surface-associated toxin to be biologically active and spatially organized into distinct toxin-rich domains on the bacterial surface . Upon bacterial contact with host cells, toxin clusters are transferred directly from the bacterial surface to the host cell surface at the bacteria-cell interface, followed by uptake and intoxication . This contact-dependent transfer of VacA represents a cost-efficient route for delivery of VacA and potentially other bacterial effector molecules to target cells.

J Agric Food Chem, 2003 Jul 30, 51(16), 4487 - 503
Systemic acquired resistance in crop protection: from nature to a chemical approach; Gozzo F; Plant natural resistance to potential parasites is regulated by two fundamental mechanisms: the "nonhost" and the "gene-for-gene" resistance, respectively . The latter is relevant when a cultivar resistant (R) gene product recognizes an avirulence gene product in the attacking pathogen and triggers an array of biochemical reactions that halt the pathogen around the site of attempted invasion . To cope with virulent pathogens, plants may benefit by some temporary immunity after a challenge triggering such an array of defense reactions, following a localized necrotizing infection as a possible consequence of a hypersensitive response (HR) . This process, mediated by accumulation of endogenous salicylic acid (SA), is called systemic acquired resistance (SAR) and provides resistance, to a certain extent even against unrelated pathogens, such as viruses, bacteria, and fungi, for a relatively long-lasting period . SAR may be more potently activated in plants pretreated with chemical inducers, most of which appear to act as functional analogues of SA . This review summarizes the complex aspects of SAR as a way to prevent crop diseases by activating the plants' own natural defenses . The following outline is taken: (1) introduction through the historical insight of the phenomenon; (2) oxidative burst, which produces high levels of oxygen reactive species in a way similar to the inflammation state in animals and precedes the HR to the pathogen attack; (3) SAR as a coordinate action of several gene products leading to the expression of defenses well beyond the time and space limits of the HR; (4) jasmonic acid (JA) and ethylene as other endogenous factors mediating a different pathway of induced resistance; (5) pathogenesis related proteins (PR proteins) de novo synthesized as specific markers of SAR; (6) exogenous inducers of SAR, which include both synthetic chemicals and natural products; (7) the pathway of signal transduction between sensitization by inducers and PR expression, as inferred by mutageneses, a process that is still, to a large extent, not completely elucidated; (8) prospects and costs; (9) final remarks on the state-of-the-art of the topic reflecting the chemical view of the author, based on the more authoritative ones expressed by the authors of the reviewed papers.

Proteins, 2004 Jan 1, 54(1), 135 - 52
Molecular basis for redox-Bohr and cooperative effects in cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774: crystallographic and modeling studies of oxidized and reduced high-resolution structures at pH 7.6; Bento I et al.; The tetraheme cytochrome c3 is a small metalloprotein with ca . 13,000 Da found in sulfate-reducing bacteria, which is believed to act as a partner of hydrogenase . The three-dimensional structure of the oxidized and reduced forms of cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774 at pH 7.6 were determined using high-resolution X-ray crystallography and were compared with the previously determined oxidized form at pH 4.0 . Theoretical calculations were performed with both structures, using continuum electrostatic calculations and Monte Carlo sampling of protonation and redox states, in order to understand the molecular basis of the redox-Bohr and cooperativity effects related to the coupled transfer of electrons and protons . We were able to identify groups that showed redox-linked conformational changes . In particular, Glu61, His76, and propionate D of heme II showed important contributions to the redox-cooperativity, whereas His76, propionate A of heme I, and propionate D of heme IV were the key residues for the redox-Bohr effect . Upon reduction, an important movement of the backbone region surrounding hemes I and II was also identified, that, together with a few redox-linked conformational changes in side-chain residues, results in a significant decrease in the solvent accessibility of hemes I and II .

Water Environ Res, 2003 Nov-Dec, 75(6), 549 - 52
Evaluation of different methods for the determination of maximum heterotrophic growth rates; Stasinakis AS et al.; This work evaluated the most commonly used methods for determining maximum heterotrophic specific growth rates (mum) in batch reactors . Parallel batch experiments were conducted under various initial substrate-to-biomass (S0/X0) ratios and values of the solids retention time (thetac) . The maximum specific growth rate, mum, was determined simultaneously according to measurements of oxygen consumption (i.e., oxygen uptake rate, OUR) and volatile suspended solids (VSS) increase . The S0X0 ratio was found to significantly influence mum values . Under high S0/X0 ratios (= 20), fast-growing bacteria seemed to gain a competitive advantage resulting in higher mum values than those obtained under low S0/X0 ratios (= 1.5) . The OUR-based estimate of mum (mum(OUR)), under certain circumstances, is differentiated from mum that is based on exponential bacterial growth (mum(VSS)), and seems to be more a measure of substrate oxidation than a measure of bacterial growth . At high S0/X0 ratio and low thetac, mum(OUR) was significantly higher than the mum(VSS), indicating that considerable uncoupling between anabolism and catabolism was occurring under these conditions . Batch experiments conducted at high S0/X0 ratios seemed to be a more sensitive method for determining mum values in the presence of an inhibitor than tests conducted at low S0/X0 ratios.

Zhonghua Kou Qiang Yi Xue Za Zhi, 2003 Nov, 38(6), 423 - 5
{Effect of chewing sugar-free gum after sucrose challenge on dental plaque pH in situ}; Dong YM et al.; OBJECTIVE: To evaluate the effect of chewing sugar-free gum after sucrose challenge on dental plaque pH in situ . METHODS: 16 healthy volunteers aged 23 - 32 years were screened as subjects . The pH of 48-hour dental plaque was measured using a Beetrode pH microelectrode when subjects chewed Extra sugar-free gum after sucrose challenge . RESULTS: Dental plaque pH maintained at resting plaque pH when immediately chewed sugar-free gum after sucrose challenge . Chewing sugar-free gum at 5 min after sucrose challenge, dental plaque pH was raised from 5.59 (measured at 5 min after sucrose challenge) to 6.98 (measured at 10 min after sucrose challenge) . CONCLUSIONS: Chewing sugar-free gum after sucrose challenge can neutralize organic acid produced by bacteria in dental plaque and rapidly rise plaque pH.

Can Vet J, 2003 Dec, 44(12), 982 - 3
Evaluation of deficiencies in labeling of commercial probiotics; Weese JS; Labels of 44 human or veterinary probiotics were scrutinized . Organisms were improperly identified in 9/21 (43%) human and 8/23 (35%) veterinary products . Contents of 5/20 (25%) human and 3/17 (18%) veterinary products were misspelled . In only 9 human and 2 veterinary products were the contents adequately identified.

Trans R Soc Trop Med Hyg, 2004 Jan, 98(1), 3 - 11
Mycetoma: a thorn in the flesh; Fahal AH; Mycetoma is a chronic, granulomatous, subcutaneous, inflammatory disease caused by true fungi (eumycetoma) or filamentous bacteria (actinomycetoma) . It occurs in the mycetoma belt stretching between the latitudes of 15 degrees South and 30 degrees North and is endemic in relatively arid areas . The organisms are present in the soil and may enter the subcutaneous tissue by traumatic inoculation . Mycetoma commonly affects adults aged 20 to 40 years, predominantly males . The foot is most commonly affected . Both forms of mycetoma present as a progressive, subcutaneous swelling, although actinomycetoma has a more rapid course . Multiple nodules develop which may suppurate and drain through sinuses, discharging grains during the active phase of the disease . Diagnosis may involve radiology, ultrasonic imaging, cytology, culture, histology or immunodiagnosis . Actinomycetoma is amenable to treatment by antibiotics, preferably by combined drug therapy for long periods . Eumycetoma is usually treated by aggressive surgical excision combined with medical treatment.

Histol Histopathol, 2004 Jan, 19(1), 259 - 70
The mast cell: an active participant or an innocent bystander?
Crivellato E, Beltrami CA, Mallardi F, Ribatti D.
Mast cells (MC) are phylogenetically old cells which are distributed throughout the human organism and, on the whole, occupy roughly the volume of the spleen . MC have long been recognized as key cells of type I hypersensitivity reactions . Several lines of evidence, however, indicate that they not only express critical effector functions in classic IgE-associated allergic disorders, but also play important roles in host defence against parasites, bacteria and perhaps even viruses . Indeed, it is now clear that MC can contribute to host defence in the context of either acquired or innate immune responses through the release of a myriad of pro-inflammatory and immunoregulatory molecules and the expression of a wide spectrum of surface receptors for cytokines and chemokines . Moreover, there is growing evidence that MC exert distinct non-immunological functions, playing a relevant role in tissue homeostasis, remodeling and fibrosis as well as in the processes of tissue angiogenesis . In this review, we provide a small insight into the biology of human MC and their potential implications in clinical pathology.

J Infect Dis, 2004 Jan 1, 189(1), 120 - 7 Epub 2003 Dec 22.
Most of the response elicited against Wolbachia surface protein in filarial nematode infection is due to the infective larval stage; Lamb TJ et al.; Immune responses to the intracellular Wolbachia bacteria of filarial nematodes are thought to contribute to the pathologic process of filarial infection . Here, we compare antibody responses of subjects living in an area where lymphatic filariasis is endemic with antibody responses elicited in a murine model of filarial infection, to provide evidence that the infective larval stage (L3), not adult nematodes, are the primary inducer of responses against Wolbachia . In human subjects, antibody responses to Brugia malayi Wolbachia surface protein (WSP) are most often correlated with antibody responses to the L3 stage of B . malayi . Analysis of anti-WSP responses induced in mice by different stages of the rodent filariae Litomosoides sigmodontis shows that the strongest anti-WSP response is elicited by the L3 stage . Although adult filarial nematode death may play a role in the generation of an anti-WSP response, it is the L3 stage that is the major source of immunogenic material, and incoming L3 provide a continual boosting of the anti-WSP response . Significant exposure to the endosymbiotic bacteria may occur earlier in nematode infection than previously thought, and the level of exposure to infective insect bites may be a key determinant of disease progression.

J Clin Invest, 2004 Jan, 113(1), 106 - 14
Nitrite in saliva increases gastric mucosal blood flow and mucus thickness; Bjorne H H et al.; Salivary nitrate from dietary or endogenous sources is reduced to nitrite by oral bacteria . In the acidic stomach, nitrite is further reduced to NO and related compounds, which have potential biological activity . We used an in vivo rat model as a bioassay to test effects of human saliva on gastric mucosal blood flow and mucus thickness . Gastric mucosal blood flow and mucus thickness were measured after topical administration of human saliva in HCl . The saliva was collected either after fasting (low in nitrite) or after ingestion of sodium nitrate (high in nitrite) . In additional experiments, saliva was exchanged for sodium nitrite at different doses . Mucosal blood flow was increased after luminal application of nitrite-rich saliva, whereas fasting saliva had no effects . Also, mucus thickness increased in response to nitrite-rich saliva . The effects of nitrite-rich saliva were similar to those of topically applied sodium nitrite . Nitrite-mediated effects were associated with generation of NO and S-nitrosothiols . In addition, pretreatment with an inhibitor of guanylyl cyclase markedly inhibited nitrite-mediated effects on blood flow . We conclude that nitrite-containing human saliva given luminally increases gastric mucosal blood flow and mucus thickness in the rat . These effects are likely mediated through nonenzymatic generation of NO via activation of guanylyl cyclase . This supports a gastroprotective role of salivary nitrate/nitrite.

Chem Biol, 2003 Dec, 10(12), 1205 - 13
A new H/D exchange- and mass spectrometry-based method for thermodynamic analysis of protein-DNA interactions; Ma L et al.; The application of SUPREX (stability of unpurified proteins from rates of H/D exchange) to the thermodynamic analysis of protein-DNA complexes is described . A series of five model protein-DNA complexes involving two known DNA binding proteins, Arc repressor and CopG, were analyzed in order to determine the accuracy, precision, and generality of the SUPREX technique for quantifying the strength of protein-DNA interactions . For protein-DNA complexes that reversibly unfold in a two-state manner, we demonstrate that reasonably precise Kd values in agreement with those determined by conventional techniques can be determined by SUPREX . In the case of protein-DNA complexes that are not well modeled by a two-state unfolding mechanism, we find that relative binding affinities can be determined in the SUPREX experiment.

Trends Genet, 2004 Jan, 20(1), 44 - 50
Riboswitches: the oldest mechanism for the regulation of gene expression?
Vitreschak AG, Rodionov DA, Mironov AA, Gelfand MS.
Riboswitches are structures that form in mRNA and regulate gene expression in bacteria . Unlike other known RNA regulatory structures, they are directly bound by small ligands . The mechanism by which gene expression is regulated involves the formation of alternative structures that, in the repressing conformation, cause premature termination of transcription or inhibition of translation initiation . Riboswitches regulate several metabolic pathways including the biosynthesis of vitamins (e.g . riboflavin, thiamin and cobalamin) and the metabolism of methionine, lysine and purines . Candidate riboswitches have also been observed in archaea and eukaryotes . The taxonomic diversity of genomes containing riboswitches and the diversity of molecular mechanisms of regulation, in addition to the fact that direct interaction of riboswitches with their effectors does not require additional factors, suggest that riboswitches represent one of the oldest regulatory systems.

J Mol Biol, 2004 Jan 23, 335(4), 1131 - 41
Crystal structure of MshB from Mycobacterium tuberculosis, a deacetylase involved in mycothiol biosynthesis; McCarthy AA et al.; All living species require protection against the damaging effects of the reactive oxygen species that are a natural by-product of aerobic life . In most organisms, glutathione is a critical component of these defences, maintaining a reducing environment inside cells . Some bacteria, however, including pathogenic mycobacteria, use an alternative low molecular mass thiol compound called mycothiol (MSH) for this purpose . Enzymes that synthesize MSH are attractive candidates for the design of novel anti-TB drugs because of the importance of MSH for mycobacterial life and the absence of such enzymes in humans . We have determined the three-dimensional structure of MshB (Rv1170), a metal-dependent deacetylase from Mycobacterium tuberculosis that catalyses the second step in MSH biosynthesis . The structure, determined at 1.9A resolution by X-ray crystallography (R=19.0%, R(free)=21.4%), reveals an alpha/beta fold in which helices pack against a seven-stranded mostly parallel beta-sheet . Large loops emanating from the C termini of the beta-strands enclose a deep cavity, which is the location of the putative active site . At the bottom of this cavity is a metal-binding site associated with a sequence motif AHPDDE that is invariant in all homologues . An adventitiously bound beta-octylglucoside molecule, used in crystallization, enables us to model the binding of the true substrate and propose a metal-dependent mechanistic model for deacetylation . Sequence comparisons indicate that MshB is representative of a wider family of enzymes that act on substituted N-acetylglucosamine residues, including a deacetylase involved in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors in eukaryotes.

J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Feb 5, 800(1-2), 303 - 7
Multivariate analysis of fatty acids in spores of higher basidiomycetes: a new method for chemotaxonomical classification of fungi; Brondz I et al.; The aim of the present work was to study the possibility of using the fatty acid content in the basidiospores as a taxonomic tool . Basidiospores of Armillaria borealis, Amanita muscaria, Agaricus sylvicola, Hypholoma capnoides, Cortinarius nemorensis and Russula delica were used . The content of fatty acids as well as other substances may vary to a certain degree depending on the part (pileus, stipe, lamella) or stage of development of the actual basidiocarp analysed . Moreover, substances from fungivorous invertebrates, parasitic fungi or bacteria may be found in the chemical analyses of the basidiocarps . Chemotaxonomic conclusions may, therefore, be burdened with serious uncertainties . On the other hand, the ripe basidiospores are terminated structures and belong to the most homogenous structures encountered from a basidiocarp . Their shape, size, colour and ornamentation are considerably homogenous within an actual species . Therefore, the basidiospores are often used as a reliable differentiating characteristic separating species as well as taxa of higher categories . From a practical point of view, ripe spores are easy to obtain in relatively large quantities with simple techniques, and they are not so prone to decay as the carpophore tissue . In the present study, gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), after methanolysis of the fungus spores, were used to map essential fatty acids in basidiomycetes . Gas chromatography and gas chromatography-mass spectrometry revealed the presence of fatty acids of C12:0-C24:0 size in the basidiospores of these higher basidiomycetes . The major fatty acid in H . capnoides is C18:2, and the major fatty acid in the other species is C18:1 . The basidiospores proved to be a good source of fatty acids for chemotaxonomic investigations of agarics.

Int J Food Microbiol, 2004 Jan 15, 90(2), 139 - 59
Flavours of cheese products: metabolic pathways, analytical tools and identification of producing strains; Marilley L et al.; Aroma development in cheese products results from the metabolic activities of cheese bacteria, by glycolysis, lipolysis and proteolysis . To respond to the increasing demand for products with improved aroma characteristics, the use of bacterial strains for cheese ripening with enhanced flavour production is seen as promising . In this review, the catabolism of amino acids, presumably the origin of some major aroma compounds, is discussed . The techniques of detection of flavour-producing strains are then presented . Their detection may be achieved either by genotyping, by enzymatic analysis, or by physico-chemical analysis such as HPLC, TLC, GC, and electronic nose.

Am J Gastroenterol, 2003 Dec, 98(12 Suppl), S6 - S17
Maintenance therapy for inflammatory bowel disease; Feagan BG; Crohn's disease and ulcerative colitis frequently recur after effective induction therapy . Accordingly, improved maintenance therapy remains the greatest unmet medical need in treating these disorders . Although 5-aminosalicylate compounds are widely used to maintain remission in Crohn's disease, the scientific evidence supporting this practice is poor . Antimetabolites (azathioprine, 6-mercaptopurine, and methotrexate) and infliximab are moderately effective in high-risk patients; however, these drugs might cause significant adverse effects . For patients with ulcerative colitis, maintenance therapy with 5-aminosalicylate compounds is a mainstay of treatment, being both effective and safe . However, this approach is not effective in a minority of patients . Although the purine metabolites can be used to treat these patients, strong scientific evidence that supports this practice is lacking . An initial experience with probiotic bacteria for maintenance of remission in ulcerative colitis seems promising . This article reviews the data from randomized, controlled trials of maintenance therapy in inflammatory bowel disease.

Anal Chem, 2004 Jan 1, 76(1), 9 - 14
Real-time nucleic acid sequence-based amplification in nanoliter volumes; Gulliksen A et al.; Real-time nucleic acid sequence-based amplification (NASBA) is an isothermal method specifically designed for amplification of RNA . Fluorescent molecular beacon probes enable real-time monitoring of the amplification process . Successful identification, utilizing the real-time NASBA technology, was performed on a microchip with oligonucleotides at a concentration of 1.0 and 0.1 microM, in 10- and 50-nL reaction chambers, respectively . The microchip was developed in a silicon-glass structure . An instrument providing thermal control and an optical detection system was built for amplification readout . Experimental results demonstrate distinct amplification processes . Miniaturized real-time NASBA in microchips makes high-throughput diagnostics of bacteria, viruses, and cancer markers possible, at reduced cost and without contamination.

Angew Chem Int Ed Engl, 2004 Jan, 43(2), 150 - 8
Bioinspired molecular design of light-harvesting multiporphyrin arrays; Choi MS et al.; Recent progress in fundamental studies on multiporphyrin arrays has provided structural parameters for the molecular design of artificial light-harvesting antennae which mimic the wheel-like antenna complexes of photosynthetic purple bacteria . Covalent and noncovalent approaches have been employed for the construction of artificial light-harvesting multiporphyrin arrays . Such arrays are categorized into ring-shaped, windmill-shaped, star-shaped, and dendritic architectures . In particular, dendritic multiporphyrin arrays have been proven to be promising candidates for both providing a large absorption cross-section and enabling the vectorial transfer of energy over a long distance to a designated point . Such molecular and supramolecular systems are also expected to be potent components for molecular electronics and photonic devices.

Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi, 2002 Feb, 20(1), 19 - 22
{Hygienic investigation of indoor environment in air-conditioned passenger trains}; Zhang X et al.; OBJECTIVE: Explore the hygienic status of indoor environment in railway air-conditioned passenger trains . METHODS: We carried out a hygienic investigation of various air-conditioned passenger trains on total 10 railway lines in Shanghai, Chengtu, Kunming, Harbin, Urumchi and Guangzhou railway bureaus for two years between July, 1997 and Oct., 1999 . The monitoring was conducted on difference train cars at different time, different railway status . The control groups were selected in the same condition . RESULTS: The results of temperature and wind speed in air-conditioned trains both meet with the national standards GB 9673-1996 in summer and winter . The humidity in summer exceeded the national standards . Of different railways the status, the noise in four different cars except dining cars exceeded the national standards . The results of shock demonstrated that all measure up to the standards of ISO 2631/1-85 . As to indoor air quality, the concentration of CO in air-conditioned trains in difference seasons(summer, autumn, winter) all met with the national standards, the ratio was 100% . In summer, the concentrations of CO2 in hard seat cars and soft berth car exceeded the national standard with a statistic significance(P < 0.01) . In winter, the concentrations of CO2 in soft berth and hard berth cars both exceeded the same standard . The results of inhalable particulate matter(IP) and total count of bacteria all satisfied the standards, meanwhile the natural lighting and artificial illumination in air-conditioned train cars all were superior to that in ordinary trains . CONCLUSION: Humidity in summer and noise in dining car exceeded the standards, the concentration of CO2 in different cars in summer and winter exceeded the standards in some degrees.

Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi, 2002 Dec, 20(6), 449 - 51
{Study on the method for demonstrating dust granules in phagocytes with Warthin-Starry special staining}; Liu H; OBJECTIVE: To search a method for showing dust granules in phagocytes . METHODS: The autopsy samples of 10 cases who died of non-pulmonary diseases(group A), 6 cases with silicosis(group B) and 5 cases of dead fetus(group C) were collected . The samples of lung, spleen and lymphnodes were stained by methods of (1)HE staining; (2)Warthin-Starry staining(W-S staining) and modified W-S staining; (3) CD68 (as a first antibody) immunohistochemical staining . The ultrastructure and chemical component of the dust granules in the pulmonary phagocytes(dust cells) of group A(3 cases) and group B(3 cases) were observed and analyzed by transmission electron microscope(TEM) and analytic electron microscope(AEM) . RESULTS: In group A and B smaller or minute dust granules which could not be shown by HE staining method were clearly shown by W-S staining and modified W-S staining methods . There were no positive granules found in group C . The CD68 marker of the dust cells was positive . The size, shape, density and chemical elements of the dust granules of group A were different from those of group B under TEM and AEM, excluding bacteria and other intracellular contents . CONCLUSION: The dust granules in the phagocytes could be shown by W-S staining method, its staining effect for minute dust granules is superior to general HE staining . However, the determination of chemical elements of dusts must depend on AEM.

Mol Biol Evol, 2004 Mar, 21(3), 541 - 54 Epub 2003 Dec 23.
The natural history of nitrogen fixation; Raymond J et al.; In recent years, our understanding of biological nitrogen fixation has been bolstered by a diverse array of scientific techniques . Still, the origin and extant distribution of nitrogen fixation has been perplexing from a phylogenetic perspective, largely because of factors that confound molecular phylogeny such as sequence divergence, paralogy, and horizontal gene transfer . Here, we make use of 110 publicly available complete genome sequences to understand how the core components of nitrogenase, including NifH, NifD, NifK, NifE, and NifN proteins, have evolved . These genes are universal in nitrogen fixing organisms-typically found within highly conserved operons-and, overall, have remarkably congruent phylogenetic histories . Additional clues to the early origins of this system are available from two distinct clades of nitrogenase paralogs: a group composed of genes essential to photosynthetic pigment biosynthesis and a group of uncharacterized genes present in methanogens and in some photosynthetic bacteria . We explore the complex genetic history of the nitrogenase family, which is replete with gene duplication, recruitment, fusion, and horizontal gene transfer and discuss these events in light of the hypothesized presence of nitrogenase in the last common ancestor of modern organisms, as well as the additional possibility that nitrogen fixation might have evolved later, perhaps in methanogenic archaea, and was subsequently transferred into the bacterial domain.

Br J Ophthalmol, 2004 Jan, 88(1), 142 - 4
Diagnosis of herpes simplex virus-1 keratitis using Giemsa stain, immunofluorescence assay, and polymerase chain reaction assay on corneal scrapings; Farhatullah S et al.; AIMS: To evaluate three tests used routinely for the diagnosis of herpes simplex virus (HSV) keratitis . METHODS: Corneal scrapings from 28 patients with clinically typical dendritic corneal ulcer suggestive of HSV keratitis, and 30 patients with clinically non-viral corneal ulcers, were tested by (i) Giemsa stain for multinucleated giant cells, (ii) immunofluorescence assay (IFA) for HSV-1 antigen, and (iii) polymerase chain reaction (PCR) for HSV-1 DNA, by investigators masked to clinical diagnosis . The control subjects were also investigated by smears and cultures for bacteria, fungus, and Acanthamoeba . RESULTS: The specificity and positive predictive values of all three tests for the diagnosis of HSV keratitis were between 95-100% . The sensitivity of IFA and PCR was 78.6% and 81.2%, respectively, and the difference was not significant; however, their sensitivity and negative predictive value were significantly higher than Giemsa stain . CONCLUSIONS: While a combination of IFA and PCR constitute the choice of tests in clinically suspected cases of HSV keratitis, multinucleated giant cells in Giemsa stain can pre-empt testing by IFA and PCR in otherwise atypical cases of HSV keratitis.

Bioresour Technol, 2004 Apr, 92(2), 151 - 5
Removal of chromium by mucilaginous seeds of Ocimum basilicum; Melo JS et al.; Polysaccharides bound to bacteria or in isolated form have been shown to bind heavy metals . A limitation of this technology can be overcome by immobilization . In view of this Ocimum basilicum seeds which swell upon wetting could serve as natural immobilized source of agriculturally-based polysaccharides . The seeds consist of an inner hard core and a pectinous fibrillar outer layer . Pretreating the seeds with acid, alkali, periodate or boiling in water was found to alter the metal binding capacity . Of the various treatments given, seeds boiled in water were found to be superior in terms of mechanical stability and exhibited fairly optimal Cr(VI) uptake kinetics . The maximum adsorption capacity as calculated from the Langmuir isotherm was 205 mg Cr/g dry seeds . Biosorption of Cr(VI) was found to be pH dependent with maximum uptake at pH 1.5 wherein sorption was not affected by the presence of other metal ions such as Cd(2+), Cu(2+), Ca(2+) and Na(+) . Seeds were used in a packed bed reactor for the continuous removal of Cr(VI) . Thus O . basilicum seeds may have application as a potential bioresource in tropical countries such as India where they are widely available.

Bioresour Technol, 2004 Apr, 92(2), 121 - 31
Advanced bioconversion of biowaste for production of a peat substitute and renewable energy; Veeken A et al.; Traditional composting systems for biowaste generally produce low quality composts that may endanger recycling . A pilot-scale bioconversion process yielding quality compost and renewable energy was designed and tested . The process consisted of a set of wet physical separation units, composting and anaerobic digestion . Biowaste was divided in four streams by physical separation: (1) organic fraction >2 mm, (2) inorganic fraction 0.05-2 mm, (3) residual fraction composed of organics 0.05-2 mm and the fraction <0.05 mm and (4) a fraction solubilised in the washing water . The organic fraction >2 mm was composted and the compost, high in organic matter and low in EC and heavy metals, aimed at replacing peat in horticulture . The inorganic fraction 0.05-2 mm was completely made up of sand and can be used as a construction material . Solubilised organic matter in the washing water was converted to CH(4) by anaerobic digestion . The residual fraction can be used as landfill cover material.

J Comp Pathol, 2004 Jan, 130(1), 48 - 57
Natural killer (NK) cells play a critical role in the early innate immune response to Chlamydophila abortus infection in mice; Buendia AJ et al.; Chlamydophila abortus, the aetiological agent of ovine enzootic abortion, induces a strong inflammatory reaction that leads to the T helper cell (Th1) specific immune response necessary for the clearance of infection . Because the role of natural killer (NK) cells during the first stages of this response has received little attention, this study focused on determining the function of these cells in a mouse model of infection . The location of NK cells in the liver and spleen of infected mice was examined immunohistochemically with an anti-Ly49G monoclonal antibody . The number of NK cells increased during the infection both in spleen and liver . In subsequent experiments, an anti-asialo GM1 polyclonal antibody was injected to deplete the NK cells . NK-depleted mice showed a substantial increase in their susceptibility to C . abortus infection, with high mortality rates and an increased burden of bacteria in the liver . Histopathological studies showed that inflammatory foci, composed mainly of neutrophils, were greater in size and number in depleted mice, while numerous chlamydial inclusions were associated with the foci . Serum concentrations of IFN-gamma, a key cytokine in the control of C . abortus infection, were substantially reduced in the NK-depleted mice . To establish the relationship between NK cells and other components of the innate immune response, neutrophils were depleted with the RB6-8C5 antibody . These cells were shown to be crucial in the recruitment of NK cells to the inflammatory foci.

Leuk Lymphoma, 2003 Oct, 44(10), 1697 - 703
Current concepts of the immunobiology and immunotherapy of cutaneous T cell lymphoma: insights gained through cross-talk between the clinic and the bench; Berger CL et al.; An understanding of the immunologic features of cutaneous T cell lymphoma (CTCL) has led to insights into the life cycle of the malignancy . The identification of the T cell lineage of the neoplastic CTCL cells has allowed unification of diverse clinical presentations under a single entity . The CD4 inducer T cell phenotype of the malignant cells has provided an understanding of the patient's ability to resist infection with certain bacteria . The clonality of the tumor cells, beyond its diagnostic implications, has made them a valuable resource for studying both normal and neoplastic T cell biology . The recently identified immunosuppressive features of the malignant T cells and their dependency for survival on an interaction with immature dendritic cells have explained previously cryptic clinical observations and identified new targets for immunotherapy . Future insights gained both from the bedside and the bench will provide not only an understanding of the immunobiology of the malignancy but also open new avenues for therapeutic intervention.

J Proteome Res, 2003 Nov-Dec, 2(6), 665 - 6
Peripheral neuropathy and light-preliminary report indicating prevalence of nanobacteria in HIV; Sommer AP; Peripheral neuropathy is a common condition in HIV-positive patients and is often experienced in diabetes mellitus . The primary mechanism of the disease, which can considerably aggravate the patient's state, is unknown . The perineurium of patients with peripheral neuropathy is frequently enveloped by apatite . Nanobacteria (NB) are protectd by a mineral shell consisting of apatite . Light has been shown to elevate the vitality level of cells, and was predicted to inhibit deposition of stressed NB in the cardiovascular system . Results indicate that light can durably restore the condition of patients with severe peripheral neuropathy.

Biopolymers, 2004 Jan, 73(1), 79 - 89
Roles of protein subunits in RNA-protein complexes: lessons from ribonuclease P; Hsieh J et al.; Ribonucleoproteins (RNP) are involved in many essential processes in life . However, the roles of RNA and protein subunits in an RNP complex are often hard to dissect . In many RNP complexes, including the ribosome and the Group II introns, one main function of the protein subunits is to facilitate RNA folding . However, in other systems, the protein subunits may perform additional functions, and can affect the biological activities of the RNP complexes . In this review, we use ribonuclease P (RNase P) as an example to illustrate how the protein subunit of this RNP affects different aspects of catalysis . RNase P plays an essential role in the processing of the precursor to transfer RNA (pre-tRNA) and is found in all three domains of life . While every cell has an RNase P (ribonuclease P) enzyme, only the bacterial and some of the archaeal RNase P RNAs (RNA component of RNase P) are active in vitro in the absence of the RNase P protein . RNase P is a remarkable enzyme in the fact that it has a conserved catalytic core composed of RNA around which a diverse array of protein(s) interact to create the RNase P holoenzyme . This combination of highly conserved RNA and altered protein components is a puzzle that allows the dissection of the functional roles of protein subunits in these RNP complexes .

Biochemistry, 2003 Dec 30, 42(51), 15114 - 23
Role of the C-terminal extrinsic region of the alpha polypeptide of the light-harvesting 2 complex of Rhodobacter sphaeroides: a domain swap study; Olsen JD et al.; The LH1 and LH2 complexes of Rhodobacter sphaeroides form ring structures of 16 and 9 protomers, respectively, comprising alpha and beta polypeptides, bacteriochlorophylls (Bchl), and carotenoids . Using the LH2 complex as a starting point, two chimeric LH complexes were constructed incorporating the alphaC-terminal domain of either the Rb . sphaeroides LH1 complex or the Rhodospirillum molischianum LH2 complex . The LH1 domain swap produced a new red-shifted component that comprised approximately 30% of the total absorbance . In the LH1alpha C-terminal mutant this new red-shifted species acts as the terminal emitter, with the new emission maximum located 10 nm further to the red than for the WT . Raman spectroscopy indicates that a fraction of the B850 Bchls is involved in relatively weak H-bonds, possibly involving the alphaTrp(+11) residue within the new alphaC-terminus, consistent with a more LH1-like character for one of the Bchls . The CD data indicate that the domain swaps have perturbed the native arrangement of the B850 Bchls, including the site energy difference between the alpha- and beta-bound Bchls . Thus, the normal energetic structure of the ring system has been disrupted, with one component blue shifted due to the presumed loss of an H-bond donor and the other red shifted by the influence of the new alphaC-terminal domain . The dichotomous response of the mutants to the carotenoids incorporated, spheroidenone or neurosporene, strongly suggests that the C-terminal region of the alpha polypeptide is involved in binding a carotenoid . The projection map of the LH1alpha C-terminal mutant complex was determined in negative stain at 25 A resolution, and it shows a diameter of 53 A, compared to 50 A for the WT . Hence these new spectral properties have not been accompanied by an alteration in ring size.

Acta Derm Venereol, 2003, 83(6), 445 - 50
Atopic dermatitis is increased following vaccination for measles, mumps and rubella or measles infection; Olesen AB et al.; The prevalence of atopic dermatitis increased markedly in the period 1960s to the 1990s . Earlier findings indicate that infections acquired in early life enhance or suppress the expression of atopic disease as a result of a change in immune reactivity . Our objectives were to examine the association between measles, mumps and rubella vaccination, measles infection and the risk of atopic dermatitis . A random sample of 9,744 children were followed up from birth to 3-15 years . Their parents responded to a questionnaire including highly structured questions on atopic dermatitis, measles, mumps and rubella vaccination and measles infection . Information on parental educational level was obtained from Statistics Denmark . The cumulative incidence of atopic dermatitis at age 14 was 19.7% . The confounder adjusted incidence ratio of atopic dermatitis among measles, mumps and rubella vaccinated children versus children not subjected to measles, mumps and rubella vaccination and measles infection was 1.86 (95% CI 1.25-2.79); the incidence ratio for measles-infected children was similar . The incidence of atopic dermatitis increased after measles, mumps and rubella vaccination and measles infection, which is surprising in view of the hygiene hypothesis . We suggest further study of the possible short-term and long-term effects of virus and bacteria on the immune responses and expression of atopic disease.

Environ Sci Pollut Res Int, 2003, 10(6), 357 - 60
Persistence of methyl tertiary butyl ether (MTBE) against metabolism by Danish vegetation; Trapp S et al.; BACKGROUND: Methyl tertiary butyl ether (MTBE) is the second most highly produced industrial chemical in the US and a frequent groundwater pollutant . At the same time, MTBE is quite persistent to biotic and abiotic decomposition . The goal of this study was to find plant species that could degrade MTBE and might be used in phytoremediation . METHODS: Excised roots and leaves (0.3 g) from more than 24 Danish plant species out of 15 families were kept in glass vessels with 25 ml spiked aqueous solution for 2 to 4 days . MTBE concentrations were 1 to 5 mg/L . Samples were taken directly from the solution with a needle and injected to a purge and trap unit . MTBE and the main metabolite, TBA, were measured by GC/FID . RESULTS AND DISCUSSION: Solutions with roots of poplar (Populus robusta) and a willow hybrid (Salix viminalis x schwerinii) produced TBA in trace amounts, probably stemming from bacteria . Significant MTBE reduction (> 10%) was not observed in any of the tests . Leaves from none of the species (trees, grasses and herbs) reduced the concentration of MTBE in the solution and no TBA, nor any other known metabolite of MTBE, was detected . CONCLUSION: It was not possible to find plants capable of efficiently degrading MTBE . This gives rise to the conclusion that plants probably cannot degrade MTBE at all, or only very slowly . RECOMMENDATIONS AND OUTLOOK: For phytoremediation projects, this has, as consequence, that the volatilization by plants (except with genetically engineered plants) is the only relevant removal process for MTBE . For risk assessment of MTBE, degradation by the plant empire is not a relevant sink process.

Nippon Ronen Igakkai Zasshi, 2003 Nov, 40(6), 593 - 5
{DNA damage, repair and aging}; Yasui A et al.; Oxidative DNA damage has been shown to accumulate with age in the nuclear and mitochondrial genome and cause cancer . Among DNA lesions produced by reactive oxygen species, base lesions and single-strand breaks are most frequently produced and cause mutation and cell death . However, these lesions are effectively repaired by base excision repair, which is very well conserved from bacteria to human . Since many proteins are involved in the repair process, understanding of their functions and the effects of repair deficiency will provide the relation between DNA damage and aging-related diseases . For this purpose we analyzed the proteins involved in the repair of oxidative DNA damage and found novel mechanisms protecting mammals against oxidative stresses.

Oecologia, 2004 Mar, 138(4), 584 - 91 Epub 2003 Dec 19.
Experimental evidence that terrestrial carbon subsidies increase CO2 flux from lake ecosystems; Lennon JT; Subsidies are donor-controlled inputs of nutrients and energy that can affect ecosystem-level processes in a recipient environment . Lake ecosystems receive large inputs of terrestrial carbon (C) in the form of dissolved organic matter (DOM) . DOM inputs may energetically subsidize heterotrophic bacteria and determine whether lakes function as sources or sinks of atmospheric CO(2) . I experimentally tested this hypothesis using a series of mesocosm experiments in New England lakes . In the first experiment, I observed that CO(2) flux increased by 160% 4 days following a 1,000 microM C addition in the form of DOM . However, this response was relatively short lived, as there was no effect of DOM enrichment on CO(2) flux beyond 8 days . In a second experiment, I demonstrated that peak CO(2) flux from mesocosms in two lakes increased linearly over a broad DOM gradient (slope for both lakes=0.02+/-0.001 mM CO(2).m(-2) day(-1) per microM DOC, mean+/-SE) . Concomitant changes in bacterial productivity and dissolved oxygen strengthen the inference that increasing CO(2) flux resulted from the metabolism of DOM . I conducted two additional studies to test whether DOM-correlated attributes were responsible for the observed change in plankton metabolism along the subsidy gradient . First, terrestrial DOM reduced light transmittance, but experimental shading revealed that this was not responsible for the observed patterns of CO(2) flux . Second, organically bound nitrogen (N) and phosphorus (P) accompanied DOM inputs, but experimental nutrient additions (without organic C) caused mesocosms to be saturated with CO(2) . Together, these results suggest that C content of terrestrial DOM may be an important subsidy for freshwater bacteria that can influence whether recipient aquatic ecosystems are sources or sinks of atmospheric CO(2).

Arch Microbiol, 2004 Feb, 181(2), 144 - 54 Epub 2003 Dec 20.
The effect of FITA mutations on the symbiotic properties of Sinorhizobium fredii varies in a chromosomal-background-dependent manner; Vinardell JM et al.; nodD1 of Sinorhizobium fredii HH103, which is identical to that of S . fredii USDA257 and USDA191, repressed its own expression . Spontaneous flavonoid-independent transcription activation (FITA) mutants of S . fredii HH103 M (=HH103 RifR pSym::Tn 5-Mob) showing constitutive expression of nod genes were isolated . No differences were found among soybean cultivar Williams plants inoculated with FITA mutants SVQ250 or SVQ253 or with the parental strain HH103M . Soybean plants inoculated with mutant SVQ255 formed more nodules, and those inoculated with mutant SVQ251 had symptoms of nitrogen starvation . Sequence analyses showed that all of the FITA mutants carried a point mutation in their nodD1 coding region . Mutants SVQ251 and SVQ253 carried the same mutation, but only the former was symbiotically impaired, which indicated the presence of an additional mutation elsewhere in the genome of mutant SVQ251 . Mutants SVQ251 and SVQ255 were outcompeted by the parental strain for nodulation of soybean cultivar Williams . The symbiotic plasmids of mutants SVQ251 and SVQ255 (pSym251 and pSym255, respectively) and that (pSymHH103M) of the parental strain were transferred to pSym-cured derivatives of S . fredii USDA192 and USDA193 (USDA192C and USDA193C, respectively) . Soybean responses to inoculation with S . fredii USDA192C and USDA193C transconjugants carrying pSym251 and pSymHH103M were not significantly different, whereas more nodules were formed after inoculation with transconjugants carrying pSym255 . Only transconjugant USDA192C(pSym255) produced a significant increase in soybean dry weight.

J Biomed Biotechnol, 2003, 2003(5), 267 - 290
Proteomics in Vaccinology and Immunobiology: An Informatics Perspective of the Immunone; Doytchinova IA et al.; The postgenomic era, as manifest, inter alia, by proteomics, offers unparalleled opportunities for the efficient discovery of safe, efficacious, and novel subunit vaccines targeting a tranche of modern major diseases . A negative corollary of this opportunity is the risk of becoming overwhelmed by this embarrassment of riches . Informatics techniques, working to address issues of both data management and through prediction to shortcut the experimental process, can be of enormous benefit in leveraging the proteomic revolution . In this disquisition, we evaluate proteomic approaches to the discovery of subunit vaccines, focussing on viral, bacterial, fungal, and parasite systems . We also adumbrate the impact that proteomic analysis of host-pathogen interactions can have . Finally, we review relevant methods to the prediction of immunome, with special emphasis on quantitative methods, and the subcellular localization of proteins within bacteria.

J Immunol, 2004 Jan 1, 172(1), 138 - 43
APC-independent activation of NK cells by the Toll-like receptor 3 agonist double-stranded RNA; Schmidt KN et al.; Toll-like receptors (TLRs) play a fundamental role in the recognition of bacteria and viruses . TLR3 is activated by viral dsRNA and polyinosinic-polycytidylic acid (poly(I:C)), a synthetic mimetic of viral RNA . We show that NK cells, known for their capacity to eliminate virally infected cells, express TLR3 and up-regulate TLR3 mRNA upon poly(I:C) stimulation . Treatment of highly purified NK cells with poly(I:C) significantly augments NK cell-mediated cytotoxicity . Poly(I:C) stimulation also leads to up-regulation of activation marker CD69 on NK cells . Furthermore, NK cells respond to poly(I:C) by producing proinflammatory cytokines like IL-6 and IL-8, as well as the antiviral cytokine IFN-gamma . The induction of cytokine production by NK cells was preceded by activation of NF-kappaB . We conclude that the ability of NK cells to directly recognize and respond to viral products is important in mounting effective antiviral responses.

Plant Cell, 2004 Jan, 16(1), 201 - 14 Epub 2003 Dec 19.
Maize mutants lacking chloroplast FtsY exhibit pleiotropic defects in the biogenesis of thylakoid membranes; Asakura Y et al.; A chloroplast signal recognition particle (SRP) that is related to the SRP involved in secretion in bacteria and eukaryotic cells is used for the insertion of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membranes . A conserved component of the SRP mechanism is a membrane-bound SRP receptor, denoted FtsY in bacteria . Plant genomes encode FtsY homologs that are targeted to the chloroplast (cpFtsY) . To investigate the in vivo roles of cpFtsY, we characterized maize cpFtsY and maize mutants having a Mu transposon insertion in the corresponding gene (chloroplast SRP receptor1, or csr1) . Maize cpFtsY accumulates to much higher levels in leaf tissue than in roots and stems . Interestingly, it is present at similar levels in etiolated and green leaf tissue and was found to bind the prolamellar bodies of etioplasts . A null cpFtsY mutant, csr1-1, showed a substantial loss of leaf chlorophyll, whereas a "leaky" allele, csr1-3, conditioned a more moderate chlorophyll deficiency . Both alleles caused the loss of various LHCPs and the thylakoid-bound photosynthetic enzyme complexes and were seedling lethal . By contrast, levels of the membrane-bound components of the thylakoid protein transport machineries were not altered . The thylakoid membranes in csr1-1 chloroplasts were unstacked and reduced in abundance, but the prolamellar bodies in mutant etioplasts appeared normal . These results demonstrate the essentiality of cpFtsY for the biogenesis not only of the LHCPs but also for the assembly of the other membrane-bound components of the photosynthetic apparatus.

J Biol Chem, 2004 Mar 26, 279(13), 12769 - 76 Epub 2003 Dec 18.
Potent anti-tumor effects of an active site mutant of human manganese-superoxide dismutase . Evolutionary conservation of product inhibition; Davis CA et al.; Mn-SOD serves as the primary cellular defense against oxidative damage by converting superoxide radicals (O(2)(-)) to O(2) and H(2)O(2) . A unique characteristic of this mitochondrial anti-oxidant enzyme is the conservation from bacteria to man of a rapidly formed product inhibited state . Using site-directed mutagenesis, we have generated an active site mutant (H30N) of human Mn-SOD, which exhibits significantly reduced product inhibition and increased enzymatic efficiency . Overexpression of the H30N enzyme causes anti-proliferative effects in vitro and anti-tumor effects in vivo . Our results provide a teleological basis for the phylogenetically invariant nature of position His-30 and the evolutionary conservation of product inhibition . These data also provide more direct intracellular evidence for the signaling role associated with H(2)O(2).

FASEB J, 2004 Feb, 18(2), 412 - 4 Epub 2003 Dec 19.
Neuronal injury mediated via stimulation of microglial toll-like receptor-9 (TLR9); Iliev AI et al.; Innate immune cells express toll-like receptor-9 (TLR9) and respond to unmethylated, CG dinucleotide motif-rich DNA released from bacteria during infection or endogenous cells during autoimmune tissue injury . Oligonucleotides containing CG dinucleotide (CpG-DNA) mimic the effect of unmethylated DNA and stimulate TLR9 . CpG-DNA was cytotoxic to neurons in organotypic brain cultures . Neurotoxicity of CpG-DNA was mediated via microglial cells and started primarily from neurites as determined by time-lapse imaging of enhanced green fluorescent protein (EGFP)-transfected neurons . Cultured brain microglial cells expressed TLR9 and responded to CpG-DNA by production of the inflammatory mediators nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) . Blockade of NO synthase and TNF-alpha prevented damage of neurites and neurotoxicity of CpG-DNA . The data suggest that stimulation of microglia via TLR9 and subsequent release of NO and TNF-alpha is a major source of neurotoxicity in bacterial and autoimmune brain tissue injury.

Aquat Toxicol, 2004 Jan 7, 66(1), 39 - 53
Trophic transfer and in vivo immunotoxicological effects of tributyltin (TBT) in polar seastar Leptasterias polaris; Bekri K et al.; This study investigated the potential in vivo immunotoxic effects of tributyltin (TBT) on amoebocytes of 6-armed seastar Leptasterias polaris . Tested animals were contaminated by trophic transfer via alive contaminated prey consisting of blue mussels (3microg TBT g(-1) wet weight (WW) tissue) exposed to seawater containing dissolved TBT . Four biomarkers of immunotoxicological effects were monitored over 45 days at different sampling times (9, 24, 48 and 72h, 11, 18, 25, 32 and 45 days): amoebocytes count (AC), cell viability using Trypan blue exclusion test, phagocytic activity (PA) using a suspension of dead bacteria labelled with fluorescein isothiocyanate (FITC) and injected directly in the coelomic fluid of the animals, and lysosomal integrity (LI) using the neutral red (NR) retention test . Data showed that TBT and its metabolites (DBT and MBT) bioaccumulated preferentially in pyloric caeca, whereas gonads contained only small quantities . Despite the differences in exposure periods to the contaminated diet and in burdens of butyltins (BTs) ingested by the various contaminated groups, there were no significant differences in body burdens of BTs . Only 6.2+/-2.0% of total ingested BTs were retained in soft tissues of seastars . Even if butyltins were not detected in the coelomic fluid (CF), their detrimental effects have been detected in the phagocytic activity of amoebocytes and their lysosomal retention of neutral red, but no effects were observed on amoebocytes count and their viability . These results show that seastar L . polaris possesses adequate mechanisms to depurate ingested TBT without supporting major disturbances of its immune defence system . By their ability to digest whole contaminated prey and eliminate only dissolved metabolites, L . polaris and other seastars with the same preying mode could play a role of "recycling organisms" in coastal environments where toxicants, such as butyltins and other metallic species are accumulated by bivalves and particularly blue mussels.

Neurochem Int, 2004 May, 44(6), 381 - 92
Inhibition of cytokines expression in human microglia infected by virulent and non-virulent mycobacteria; Curto M et al.; The pathogenesis of tuberculosis (TBC) meningitis is still unknown . As shown by previous studies, human microglia can be the target of mycobacteria, but no data are available about their cellular response to infection . Consequently, we studied the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and IL-10 in human microglia pure cultures infected with the two variants of Mycobacterium avium (domed-opaque (SmD) and transparent (SmT)) and with Mycobacterium tuberculosis . Results showed that microglia was productively infected by mycobacteria which could grow inside the cells . Mycobacteria internalization was more rapid for M . avium, but M . tuberculosis infection turned out to be more efficient due to the incorporation of densely packed bacteria . TNF-alpha expression was not affected by M . avium, whereas an increase followed by a decrease was observed in M . tuberculosis . Both IL-1 and IL-10 cytokine expression was rapidly inhibited by infection with the more virulent bacteria, whereas the non-pathogenic one had almost no effect . Also, the expression of the co-stimulatory molecule CD137, a member of tumor necrosis factor receptor family, was affected by infection with virulent mycobacteria.Our results show that microglia response to mycobacterial infection is modulated in correlation with virulence, mainly toward inhibition of inflammatory response . This observation might be one of the mechanisms by which non-pathogenic mycobacteria are quickly eliminated, explaining one of the bases of virulence.

J Mol Biol, 2004 Jan 16, 335(3), 667 - 78
Phage integrases: biology and applications; Groth AC et al.; Phage integrases are enzymes that mediate unidirectional site-specific recombination between two DNA recognition sequences, the phage attachment site, attP, and the bacterial attachment site, attB . Integrases may be grouped into two major families, the tyrosine recombinases and the serine recombinases, based on their mode of catalysis . Tyrosine family integrases, such as lambda integrase, utilize a catalytic tyrosine to mediate strand cleavage, tend to recognize longer attP sequences, and require other proteins encoded by the phage or the host bacteria . Phage integrases from the serine family are larger, use a catalytic serine for strand cleavage, recognize shorter attP sequences, and do not require host cofactors . Phage integrases mediate efficient site-specific recombination between two different sequences that are relatively short, yet long enough to be specific on a genomic scale . These properties give phage integrases growing importance for the genetic manipulation of living eukaryotic cells, especially those with large genomes such as mammals and most plants, for which there are few tools for precise manipulation of the genome . Integrases of the serine family have been shown to work efficiently in mammalian cells, mediating efficient integration at introduced att sites or native sequences that have partial identity to att sites . This reaction has applications in areas such as gene therapy, construction of transgenic organisms, and manipulation of cell lines . Directed evolution can be used to increase further the affinity of an integrase for a particular native sequence, opening up additional applications for genomic modification.

Environ Microbiol, 2004 Jan, 6(1), 35 - 44
Detection and phylogenetic analysis of Wolbachia in Collembola; Czarnetzki AB et al.; Wolbachia are obligatory, cytoplasmatically inherited alpha-Proteobacteria which are known for infecting the reproductive tissues of many arthropods . Their prevalence in the large group of Collembola, however, is not known, except for PCR detection in the parthenogenetically reproducing species Folsomia candida (Order: Entomobryomorpha; Family: Isotomidae) . In this study, fluorescence in situ hybridization on microscopic sections of F . candida specimens indicated that Wolbachia-related bacteria were restricted to tissues of the ovary and brain . PCR with primers designed to detect 16S rRNA genes of Wolbachia were positive with specimens from all of five geographically independent F . candida breeding stocks and with three parthenogenetic species from another order (Poduromorpha; Family Tullbergiidae), i.e . Mesaphorura italica, M . macrochaeta and Paratullbergia callipygos . In contrast, negative results were obtained with the two sexually reproducing species, Isotoma viridis (Isotomidae) and Protaphorura fimata (Poduromorpha; Onychiuridae) . The ftsZ gene of Wolbachia could be PCR-amplified from all Wolbachia-positive hosts with the exception of M . macrochaeta . The phylogenetic distances of the ftsZ and 16S rRNA gene sequences reflected the phylogenetic distances of the host organisms but the sequences of Wolbachia were relatively closely related, indicating that Wolbachia infections took place after the Collembola had diversified . Our study confirms a monophyletic branch (supergroup E) of Collembola colonizing Wolbachia and indicates that this group is a sister group of supergroup A, the latter harbouring a high diversity of host organisms within the group of insects.

J Neurosci, 2003 Dec 17, 23(37), 11692 - 7
Tyrosinase expression during neuroblast divisions affects later pathfinding by retinal ganglion cells; Cronin CA et al.; Occulocutaneous albinism is caused by mutations in the gene encoding the enzyme tyrosinase . Individuals with this disorder are predisposed to visual system deficits . We determined the critical period during development when tyrosinase expression is essential for the appropriate pathfinding of ganglion cell axons from the retina to the dorsal lateral geniculate nucleus . We used a line of mice with a Tyrosinase transgene, the expression of which is regulatable with the lac operator-repressor system, to restrict tyrosinase activity to discrete periods of embryogenesis . When tyrosinase was expressed throughout the period of neuroblast divisions that produce the ipsilaterally projecting ganglion cells, axonal projections innervated the same volume of the ipsilateral dorsal lateral geniculate nucleus of the thalamus as in normal mice . If tyrosinase expression ceased before the end of neuroblast divisions, or was not initiated until after they had begun, the degree of ipsilateral innervation was smaller, as in albino mice . Tyrosinase expression was not required during the entire period of pathfinding itself or during final maturation of the retinogeniculate pathway . Thus, tyrosinase appears to set up a signal early in visual system development that determines the pathway taken later by ganglion cell axons.

Gut, 2004 Jan, 53(1), 1 - 4
Dysbiosis in inflammatory bowel disease; Tamboli CP et al.; Abundant data have incriminated intestinal bacteria in the initiation and amplification stages of inflammatory bowel diseases . However, the precise role of intestinal bacteria remains elusive . One theory has suggested a breakdown in the balance between putative species of "protective" versus "harmful" intestinal bacteria--this concept has been termed "dysbiosis" . Arguments in support of this concept are discussed.

Org Lett, 2003 Dec 25, 5(26), 4935 - 8
Proofs of macrocyclization of gable porphyrins as mimics of photosynthetic light-harvesting complexes; Ikeda C et al.; Porphyrin macrocycles composed of five and six units of m-gable imidazolylporphyrinatozinc (1-Zn) were synthesized by self-assembled cyclization followed by ring-closing metathesis linkings . Each porphyrin macrocycle was isolated by GPC chromatography, and their molecular weights were determined by MALDI-TOF mass spectroscopy . These structures represent mimics of light-harvesting complexes in photosynthetic bacteria . {structure: see text}

Plant Mol Biol, 2003 Aug, 52(6), 1169 - 80
Auxin distribution in Lotus japonicus during root nodule development; Pacios-Bras C et al.; For this work, Lotus japonicus transgenic plants were constructed expressing a fusion reporter gene consisting of the genes beta-glucuronidase (gus) and green fluorescent protein (gfp) under control of the soybean auxin-responsive promoter GH3 . These plants expressed GUS and GFP in the vascular bundle of shoots, roots and leafs . Root sections showed that in mature parts of the roots GUS is mainly expressed in phloem and vascular parenchyma of the vascular cylinder . By detecting GUS activity, we describe the auxin distribution pattern in the root of the determinate nodulating legume L . japonicus during the development of nodulation and also after inoculation with purified Nod factors, N-naphthylphthalamic acid (NPA) and indoleacetic acid (IAA) . Differently than white clover, which forms indeterminate nodules, L . japonicus presented a strong GUS activity at the dividing outer cortical cells during the first nodule cell divisions . This suggests different auxin distribution pattern between the determinate and indeterminate nodulating legumes that may be responsible of the differences in nodule development between these groups . By measuring of the GFP fluorescence expressed 21 days after treatment with Nod factors or bacteria we were able to quantify the differences in GH3 expression levels in single living roots . In order to correlate these data with auxin transport capacity we measured the auxin transport levels by a previously described radioactive method . At 48 h after inoculation with Nod factors, auxin transport showed to be increased in the middle root segment . The results obtained indicate that L . japonicus transformed lines expressing the GFP and GUS reporters under the control of the GH3 promoter are suitable for the study of auxin distribution in this legume.

Water Sci Technol, 2003, 48(8), 255 - 9
Elucidation mechanism of organic acids production from organic matter (grass) using digested and partially digested cattle feed; Sonakya V et al.; The process of anaerobic digestion is highly influenced by the environmental and operational factors like organic acids concentration and the reactor volume occupied by the feed material . The optimum level of organic acids is commonly assumed to be in the range between 2,500 and 3,500 mg/l for the anaerobic digestion process . It was observed that the production of total organic acids during hydrolysis of grass using cattle dung slurry (CDS) as the inoculum reached up to 4,850 mg/l in 6 days, while on the other hand it reached 5,700 mg/l within 4 days when rumen content was used as inoculum . The organic acids production continued to the 30th day in the case of rumen content, while in the case of CDS it stopped within 10 days because of pH drop . As compared to CDS the anaerobic digestion of grass with rumen content showed better degradation and biogas production with nearly 80% of methane and up to 80 and 95% reduction in chemical oxygen demand and organic acids respectively.

Water Sci Technol, 2003, 48(8), 151 - 8
Effects of ferric ion on bioleaching of heavy metals from contaminated sediment; Chen SY et al.; Bioleaching is one of the promising procedures for removal of heavy metals from contaminated sediments . The advantages of this biotechnology are that it is easy, efficient, less costly and friendly to the environment . However, the principal disadvantage of this process is its slow kinetics in metal solubilization, which may limit practical application of the bioleaching process . In order to enhance the rate and efficiency of metal solubilization, the ferric ion was used as a catalytic agent in the bioleaching process . It was found that the sediment pH apparently decreased in the bioleaching after addition of ferric ion . The metal solubilization increased quickly after the addition of ferric ion . The rate of metal solubilization was enhanced by the addition of ferric ion, especially for Cr and Pb . An increase in the amount of ferric ion added increased the final efficiency of metal solubilization . The highest final efficiency of metal solubilization was obtained in the bioleaching with 1 g/L of ferric ion . Besides, the growth of sulfur-oxidizing bacteria was not affected by addition of ferric ion in the bioleaching . It was concluded that the kinetics of metal solubilization were enhanced by addition of ferric ion in the bioleaching process.

Biotechniques, 2003 Dec, 35(6), 1216 - 21
Oligo Design: a computer program for development of probes for oligonucleotide microarrays; Herold KE et al.; Oligonucleotide microarrays have demonstrated potential for the analysis of gene expression, genotyping, and mutational analysis . Our work focuses primarily on the detection and identification of bacteria based on known short sequences of DNA . Oligo Design, the software described here, automates several design aspects that enable the improved selection of oligonucleotides for use with microarrays for these applications . Two major features of the program are: (i) a tiling algorithm for the design of short overlapping temperature-matched oligonucleotides of variable length, which are useful for the analysis of single nucleotide polymorphisms and (ii) a set of tools for the analysis of multiple alignments of gene families and related short DNA sequences, which allow for the identification of conserved DNA sequences for PCR primer selection and variable DNA sequences for the selection of unique probes for identification . Note that the program does not address the full genome perspective but, instead, is focused on the genetic analysis of short segments of DNA . The program is Internet-enabled and includes a built-in browser and the automated ability to download sequences from GenBank by specifying the GI number . The program also includes several utilities, including audio recital of a DNA sequence (useful for verifying sequences against a written document), a random sequence generator that provides insight into the relationship between melting temperature and GC content, and a PCR calculator.

RNA, 2004 Jan, 10(1), 59 - 65
The 5'-3' exoribonuclease xrn-1 is essential for ventral epithelial enclosure during C . elegans embryogenesis; Newbury S et al.; Ribonucleases have been studied in yeast and bacteria, but their biological significance to multicellular organisms is virtually unknown . However, there is increasing evidence that specific, timed transcript degradation is critical for regulation of many cellular processes, including early development and RNA interference . In this report we have investigated the effects of the 5'-3' exoribonuclease xrn-1 on the development of the nematode worm Caenorhabditis elegans . Silencing of xrn-1 expression using RNA interference results in embryos that fail to complete ventral enclosure, where the outer layer of cells normally closes over the mesoderm in a purse-string movement . Our data suggest that xrn-1 is involved in a critical aspect of epithelial movement and reveal an unexpected link between RNA stability and morphogenesis . Because xrn-1 is highly conserved in all eukaryotes, it is possible that it plays a role in similar morphological processes such as dorsal or thorax closure in Drosophila and wound healing in humans . In contrast to work in human tissue culture cells, where the 3'-5' pathway has been shown to be the most important for degradation of mRNAs, our work shows that the 5'-3' degradation pathway is crucially important at a critical stage of development in C . elegans . We have also investigated whether xrn-1 can influence the response of C . elegans to RNA interference . Our data indicate that xrn-1 plays a facilitating, but not crucial role in this process.

Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D307 - 10
MolliGen, a database dedicated to the comparative genomics of Mollicutes; Barre A et al.; Bacteria belonging to the class Mollicutes were among the first ones to be selected for complete genome sequencing because of the minimal size of their genomes and their pathogenicity for humans and a broad range of animals and plants . At this time six genome sequences have been publicly released (Mycoplasma genitalium, Mycoplasma pneumoniae, Ureaplasma urealyticum-parvum, Mycoplasma pulmonis, Mycoplasma penetrans and Mycoplasma gallisepticum) and as the number of available mollicute genomes increases, comparative genomics analysis within this model group of organisms becomes more and more instructive . However, such an analysis is difficult to carry out without a suitable platform gathering not only the original annotations but also relevant information available in public databases or obtained by applying common bioinformatics methods . With the aim of solving these difficulties, we have developed a web-accessible database named MolliGen . After selecting a set of genomes the user can launch various types of search based on annotation, position on the chromosomes or sequence similarity . In addition, relationships of putative orthology have been precomputed to allow differential genome queries . The results are presented in table format with multiple links to public databases and to bioinformatic analyses such as multiple alignments or BLAST search . Specific tools were also developed for the graphical visualization of the results, including a multi- genome browser for displaying dynamic pictures with clickable objects and for viewing relationships of precomputed similarity . MolliGen is designed to integrate all the complete genomes of mollicutes as they become available.

Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D153 - 5
KinG: a database of protein kinases in genomes; Krupa A et al.; The KinG database is a comprehensive collection of serine/threonine/tyrosine-specific kinases and their homologues identified in various completed genomes using sequence and profile search methods . The database hosted at mbu.iisc.ernet.in/ approximately king provides the amino acid sequences, functional domain assignments and classification of gene products containing protein kinase domains . A search tool enabling the retrieval of protein kinases with specified subfamily and domain combinations is one of the key features of the resource . Identification of a kinase catalytic domain in the user's query sequence is possible using another search tool . The occurrence and location of critical catalytic residues if the query has a catalytic kinase domain, recognition of non-kinase domains in the sequence and subfamily classification of the kinase in the query will help in deciphering the biological role of the kinase . This online compilation can also be used to compare the protein kinases of a given subfamily and domain combinations across various genomes . Another exclusive feature of the database is the collection of the Ser/Thr/Tyr protein kinases and similar sequences encoded in the genomes of archaea and bacteria.

Nucleic Acids Res, 2004 Jan 1, 32 Database issue, D104 - 8
The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts; Gueneau de Novoa P et al.; tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes . Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website , a collection of sequences, alignments, secondary structures and other information . Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only . Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages . The new sequences include the shortest known tmRNA sequence . While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions . The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps . Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.

J Biol Chem, 2004 Mar 5, 279(10), 8808 - 19 Epub 2003 Dec 16.
Differential mode of regulation of the checkpoint kinases CHK1 and CHK2 by their regulatory domains; Ng CP et al.; CHK1 and CHK2 are key mediators that link the machineries that monitor DNA integrity to components of the cell cycle engine . Despite the similarity and potential redundancy in their functions, CHK1 and CHK2 are unrelated protein kinases, each having a distinctive regulatory domain . Here we compare how the regulatory domains of human CHK1 and CHK2 modulate the respective kinase activities . Recombinant CHK1 has only low basal activity when expressed in cultured cells . Surprisingly, disruption of the C-terminal regulatory domain activates CHK1 even in the absence of stress . Unlike the full-length protein, C-terminally truncated CHK1 displays autophosphorylation, phosphorylates CDC25C on Ser(216), and delays cell cycle progression . Intriguingly, enzymatic activity decreases when the entire regulatory domain is removed, suggesting that the regulatory domain contains both inhibitory and stimulatory elements . Conversely, the kinase domain suppresses Ser(345) phosphorylation, a major ATM/ATR phosphorylation site in the regulatory domain . In marked contrast, CHK2 expressed in either mammalian cells or in bacteria is already active as a kinase against itself and CDC25C and can delay cell cycle progression . Unlike CHK1, disruption of the regulatory domain of CHK2 abolishes its kinase activity . Moreover, the regulatory domain of CHK2, but not that of CHK1, can oligomerize . Finally, CHK1 but not CHK2 is phosphorylated during the spindle assembly checkpoint, which correlates with the inhibition of the kinase . The mitotic phosphorylation of CHK1 requires the regulatory domain, does not involve Ser(345), and is independent on ATM . Collectively, these data reveal the very different mode of regulation between CHK1 and CHK2.

J Biol Chem, 2004 Mar 19, 279(12), 11489 - 94 Epub 2003 Dec 17.
Developmental cell death in dictyostelium does not require paracaspase; Roisin-Bouffay C et al.; Apoptotic cell death often requires caspases . Caspases are part of a family of related molecules including also paracaspases and metacaspases . Are molecules of this family generally involved in cell death? More specifically, do non-apoptotic caspase-independent types of cell death require paracaspases or metacaspases? Dictyostelium discoideum lends itself well to answering these questions because 1) it undergoes non-apoptotic developmental cell death of a vacuolar autophagic type and 2) it bears neither caspase nor metacaspase genes and apparently only one paracaspase gene . This only paracaspase gene can be inactivated by homologous recombination . Paracaspase-null clones were thus obtained in each of four distinct Dictyostelium strains . These clones were tested in two systems, developmental stalk cell death in vivo and vacuolar autophagic cell death in a monolayer system mimicking developmental cell death . Compared with parent cells, all of the paracaspase-null cells showed unaltered cell death in both test systems . In addition, paracaspase inactivation led to no alteration in development or interaction with a range of bacteria . Thus, in Dictyostelium, vacuolar programmed cell death in development and in a monolayer model in vitro would seem not to require paracaspase . To our knowledge, this is the first instance of developmental programmed cell death shown to be independent of any caspase, paracaspase or metacaspase . These results have implications as to the relationship in evolution between cell death and the caspase family.

Biochem Biophys Res Commun, 2003 Dec 19, 312(3), 747 - 54
Identification and characterization of mouse GTPBP3 gene encoding a mitochondrial GTP-binding protein involved in tRNA modification; Li X et al.; We report here the identification and characterization of mouse GTPBP3 encoding a mitochondrial GTPase . A full-length GTPBP3 cDNA has been isolated and the genomic organization of GTPBP3 has been elucidated . The mouse GTPBP3 gene containing 9 exons encodes a 486 residue protein with a strong homology to the GTPBP3-like proteins of bacteria, yeast, and other homologs, related to tRNA modification . The mouse GTPBP3 is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, and brain . Surprisingly, this gene, unlike its human homolog, exhibited a low expression in skeletal muscle . Furthermore, immunofluorescence analysis of NIH3T3 cells expressing GTPBP3-GFP fusion protein demonstrated that the mouse Gtpbp3 localizes in mitochondrion . These observations suggest that the mouse Gtpbp3 is an evolutionarily conserved mitochondrial GTP-binding protein involved in the tRNA modification . Thus, it may modulate the translational efficiency and accuracy of codon-anticodon base pairings on the decoding region of mitochondrial ribosomes.

FEMS Microbiol Lett, 2003 Dec 12, 229(2), 189 - 94
Production of free and organic iodine by Roseovarius spp; Fuse H et al.; Two strains of iodine-producing bacteria were isolated from marine samples . 16S rRNA gene sequences indicated the strains were most closely related to Roseovarius tolerans, and phylogenetic analysis indicated both belong to the same genus . 5 mM iodide inhibited the growth of strain 2S5-2 almost completely, and of strain S6V slightly . Both strains produced free iodine and organic iodine from iodide . CH2I2, CHI3 and CH2ClI were the main organic iodines produced by strain 2S5-2, and CHI3 and CH2I2 by strain S6V . Experiments using cells and spent media suggested that the organic iodines were produced from the compounds released or contained in the media and cells were necessary for the considerable production of CH2I2 and CH2ClI, though CHI3 was produced by spent media with H2O2 or free iodine.

Chronobiol Int, 2003 Nov, 20(6), 901 - 19
Adaptive significance of circadian clocks; Sharma VK; Circadian clocks are ubiquitous and are found in organisms ranging from bacteria to mammals . This ubiquity of occurrence implies adaptive significance, but to date there has been no rigorous empirical evidence to support this . It is believed that an organism possessing circadian clocks gains fitness advantage in two ways: (i) by synchronizing its behavioral and physiological processes to cyclic environmental factors (extrinsic adaptive value); (ii) by coordinating its internal metabolic processes (intrinsic adaptive value) . There is preliminary circumstantial evidence to support both . Several studies using organisms living in constant environments have shown that these organisms possess functional circadian clocks, suggesting that circadian clocks may have some intrinsic adaptive value . Studies to assess the adaptive value of circadian clocks in periodic environments suggest that organisms may have a fitness advantage in those periodic environments, which closely match their own intrinsic periodicity . Furthermore, evidence from organisms living in the wild, selection studies, and studies on latitudinal clines suggest that circadian clocks may have an extrinsic adaptive value as well . In this paper, I have presented several hypotheses for the emergence of circadian clocks and have reviewed some major empirical studies suggesting adaptive significance of circadian clocks.

J Bacteriol, 2004 Jan, 186(1), 51 - 60
Analysis of differences in the functional properties of the substrate binding proteins of the Borrelia burgdorferi oligopeptide permease (Opp) operon; Wang XG et al.; The Borrelia burgdorferi genome encodes five orthologues of the substrate binding protein oligopeptide permease A (OppA) . It was previously shown that these genes are under the control of separate promoters and are differentially expressed under various environmental conditions . We were interested in determining whether there are also differences in substrate specificities among the proteins . The substrate specificities of recombinant proteins were determined by screening for high-affinity peptides by use of a combinatorial phage display heptapeptide library . Different heptapeptides with high affinities for OppA-1, OppA-2, and OppA-3 were identified . No heptapeptide binding OppA-4 or OppA-5 could be identified . Competitive binding assays were performed under various conditions to determine the substrate preferences of the OppA proteins . OppA-1 retained maximal activity over a broad range of pHs (5.5 to 7.5), whereas OppA-2 and OppA-3 showed peak activities at pHs below 5.5 . OppA-1 and OppA-2 showed preferences for tripeptides over dipeptides and longer-chain peptides . Although a wide variety of amino acyl side chains were tolerated by all three OppA proteins, OppA-1 showed the broadest substrate specificity and was able to accommodate peptides composed of bulky hydrophobic residues; OppA-2 and OppA-3 showed preferences for peptides composed of small nonpolar amino acids . All three OppA proteins showed preferences for peptides composed of L- rather than D-amino acids . OppA-3 showed the greatest tolerance for changes in stereochemistry . Substantial differences in the substrate specificities of the OppA proteins of B . burgdorferi suggest that they may have distinct functions in the organism.

J Bacteriol, 2004 Jan, 186(1), 22 - 8
Multiple formate dehydrogenase enzymes in the facultative methylotroph Methylobacterium extorquens AM1 are dispensable for growth on methanol; Chistoserdova L et al.; Formate dehydrogenase has traditionally been assumed to play an essential role in energy generation during growth on C(1) compounds . However, this assumption has not yet been experimentally tested in methylotrophic bacteria . In this study, a whole-genome analysis approach was used to identify three different formate dehydrogenase systems in the facultative methylotroph Methylobacterium extorquens AM1 whose expression is affected by either molybdenum or tungsten . A complete set of single, double, and triple mutants was generated, and their phenotypes were analyzed . The growth phenotypes of the mutants suggest that any one of the three formate dehydrogenases is sufficient to sustain growth of M . extorquens AM1 on formate, while surprisingly, none is required for growth on methanol or methylamine . Nuclear magnetic resonance analysis of the fate of {(13)C}methanol revealed that while cells of wild-type M . extorquens AM1 as well as cells of all the single and the double mutants continuously produced {(13)C}bicarbonate and (13)CO(2), cells of the triple mutant accumulated {(13)C}formate instead . Further studies of the triple mutant showed that formate was not produced quantitatively and was consumed later in growth . These results demonstrated that all three formate dehydrogenase systems must be inactivated in order to disrupt the formate-oxidizing capacity of the organism but that an alternative formate-consuming capacity exists in the triple mutant.

J Biol Chem, 2004 Mar 12, 279(11), 10323 - 30 Epub 2003 Dec 16.
Ca2+-dependent protein kinase C isoforms induce cholestasis in rat liver; Kubitz R et al.; Bile secretion is regulated by different signaling transduction pathways including protein kinase C (PKC) . However, the role of different PKC isoforms for bile formation is still controversial . This study investigates the effects of PKC isoform selective activators and inhibitors on PKC translocation, bile secretion, bile acid uptake, and subcellular transporter localization in rat liver, isolated rat hepatocytes and in HepG2 cells . In rat liver activation of Ca(2+)-dependent cPKCalpha and Ca(2+)-independent PKCepsilon by phorbol 12-myristate 13-acetate (PMA, 10nmol/liter) is associated with their translocation to the plasma membrane . PMA also induced translocation of the cloned rat PKCepsilon fused to a yellow fluorescent protein (YFP), which was transfected into HepG2 cells . In the perfused liver, PMA induced marked cholestasis . The PKC inhibitors Go6850 (1 micromol/liter) and Go6976 (0.2 micromol/liter), a selective inhibitor of Ca(2+)-dependent PKC isoforms, diminished the PMA effect by 50 and 60%, respectively . Thymeleatoxin (Ttx,) a selective activator of Ca(2+)-dependent cPKCs, did not translocate rat PKCepsilon-YFP transfected in HepG2 cells . However, Ttx (0.5-10 nmol/liter) induced cholestasis similar to PMA and led to a retrieval of Bsep from the canalicular membrane in rat liver while taurocholate-uptake in isolated hepatocytes was not affected . Go6976 completely blocked the cholestatic effect of Ttx but had no effect on tauroursodeoxycholate-induced choleresis . The data identify Ca(2+)-dependent PKC isoforms as inducers of cholestasis . This is mainly due to inhibition of taurocholate excretion involving transporter retrieval from the canalicular membrane.

J Biol Chem, 2004 Mar 12, 279(11), 10364 - 73 Epub 2003 Dec 16.
CD95-tyrosine nitration inhibits hyperosmotic and CD95 ligand-induced CD95 activation in rat hepatocytes; Reinehr R et al.; Epidermal growth factor receptor-dependent CD95-tyrosine phosphorylation was recently identified as an early step in apoptosis induction via the CD95 system (Reinehr, R., Schliess, F., and Haussinger, D . (2003) FASEB J . 17, 731-733) . The effect of peroxynitrite (ONOO(-)) on modulation of the hyperosmotic and CD95 ligand (CD95L)-induced CD95 activation process was studied . Pretreatment of hepatocytes with ONOO(-) inhibited CD95L- and hyperosmolarity-induced CD95 membrane trafficking and formation of the death-inducing signaling complex, but not epidermal growth factor receptor activation and its association with CD95 . Under these conditions, however, no tyrosine phosphorylation of CD95 occurred; instead, CD95 was tyrosine-nitrated . When ONOO(-) was added after induction of CD95-tyrosine phosphorylation by CD95L or hyperosmolarity, tyrosine nitration of CD95 was largely prevented and death-inducing signaling complex formation occurred . CD95-tyrosine nitration abolished the hyperosmotic sensitization of hepatocytes toward CD95L-induced apoptosis . Additionally, in CD95-yellow fluorescent protein-transfected Huh7-hepatoma cells, ONOO(-) induced CD95 Tyr nitration and prevented CD95L-induced Tyr phosphorylation and apoptosis . Tyrosine-nitrated CD95 was also found in rat livers derived from an in vivo model of endotoxinemia . The data suggest that CD95-tyrosine nitration prevents CD95 activation by inhibiting CD95-tyrosine phosphorylation . Apparently, CD95-tyrosine phosphorylation and nitration are mutually exclusive . The data identify critical tyrosine residues of CD95 as another target of the anti-apoptotic action of NO.

J Clin Invest, 2003 Dec, 112(12), 1862 - 70
Chemokine-mediated recruitment of NK cells is a critical host defense mechanism in invasive aspergillosis; Morrison BE et al.; Invasive aspergillosis is a severe pneumonia that is usually fatal despite currently available therapy . The disease disproportionately afflicts immunocompromised patients, indicating the critical importance of the immune status of the host in this infection, but the defense mechanisms against this pathogen remain incompletely understood . In the current study, we hypothesized that the chemokine ligand monocyte chemotactic protein-1, also designated CC chemokine ligand-2 (MCP-1/CCL2) is necessary for effective host defense against invasive aspergillosis in immunocompromised hosts . We found a rapid and marked induction of MCP-1/CCL2 in the lungs of neutropenic mice with invasive aspergillosis . Neutralizing MCP-1/CCL2 resulted in twofold greater mortality and greater than threefold increase in pathogen burden in the lungs . Neutralization of MCP-1/CCL2 also resulted in reduced recruitment of NK cells to the lungs at early time points, but did not affect the number of other leukocyte effector cells in the lungs . Ab-mediated depletion of NK cells similarly resulted in impaired defenses against the infection, resulting in a greater than twofold increase in mortality and impaired clearance of the pathogen from the lungs . These data establish MCP-1/CCL2-mediated recruitment of NK cells to the lungs as a critical early host defense mechanism in invasive aspergillosis and demonstrate NK cells to be an important and previously unrecognized effector cell in this infection.

Astrobiology, 2003 Fall, 3(3), 565 - 79
Why Raman spectroscopy on Mars?--a case of the right tool for the right job; Ellery A et al.; We provide a scientific rationale for the astrobiological investigation of Mars . We suggest that, given practical constraints, the most promising locations for the search for former life on Mars are palaeolake craters and the evaporite deposits that may reside within them . We suggest that Raman spectroscopy offers a promising tool for the detection of evidence of former (or extant) biota on Mars . In particular, we highlight the detection of hopanoids as long-lived bacterial cell wall products and photosynthetic pigments as the most promising targets . We further suggest that Raman spectroscopy as a fibre optic-based instrument lends itself to flexible planetary deployment.

Hybrid Hybridomics, 2003 Oct, 22(5), 293 - 9
New monoclonal antibodies to non-glycosylated domains of the secreted mucins MUC5B and MUC7; Rousseau K et al.; The separation and characterization of salivary mucins is not straightforward because of their large size, heterogeneity, and molecular interactions . The MUC5B and MUC7 mucins are major glycoprotein components of saliva that are thought to play a vital role in maintaining oral health . MUC5B is also a major component of respiratory mucus and is produced by the tracheal and bronchial glands, while MUC7 has a more limited pattern of expression in the bronchial tree . MUC5B is a gel-forming mucin and thus confers viscosity, whereas MUC7 is much smaller . MUC7 has anti-fungal activity, and both mucins interact with bacteria . The aim of this work was to produce new monoclonal antibodies that can be used to quantify and characterize these mucins by standard laboratory procedures . Peptide sequences in non-conserved and non-glycosylated regions were selected and monoclonal antibodies produced by an efficient immunization and cloning strategy, and screening against purified mucins . Three new antibodies-EU-MUC5Ba and EU-MUC5Bb (against MUC5B) and EU-MUC7a (against MUC7)-were isolated that do not show cross-reactivity with other gel-forming mucins . All work on immunohistochemistry can be used for semi-quantitative immunoblotting after agarose gel electrophoresis . These reagents are valuable tools to study changes in these mucins in oral and respiratory disease, and unlike other monoclonal antibodies to these mucins they recognize epitopes that are not affected by glycosylation.

J Microsc, 2004 Jan, 213(Pt 1), 46 - 62
Optimizing imaging parameters for the separation of multiple labels in a fluorescence image; Neher R et al.; A theoretical analysis is presented on how to separate the contributions from individual, simultaneously present fluorophores in a spectrally resolved image . Equations are derived that allow the calculation of the signal-to-noise ratio of the estimates for such contributions, given the spectral information on the individual fluorophores, the excitation wavelengths and intensities, and the number and widths of the spectral detection channels . We then ask how such imaging parameters have to be chosen for optimal fluorophore separation . We optimize the signal-to-noise ratio or optimize a newly defined 'figure of merit', which is a measure of efficiency in the use of emitted photons . The influence of photobleaching on the resolution and on the choice of imaging parameters is discussed, as well as the additional resolution gained by including fluorescence lifetime information . A surprisingly small number of spectral channels are required for an almost optimal resolution, if the borders of these channels are optimally selected . The detailed consideration of photobleaching is found to be essential, whenever there is significant bleaching . Consideration of fluorescence lifetime information (in addition to spectral information) improves results, particularly when lifetimes differ by more than a factor of two.

J Microsc, 2004 Jan, 213(Pt 1), 29 - 38
phiFLIM: a new method to avoid aliasing in frequency-domain fluorescence lifetime imaging microscopy; Van Munster EB et al.; In conventional wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM), excitation light is intensity-modulated at megahertz frequencies . Emitted fluorescence is recorded by a CCD camera through an image intensifier, which is modulated at the same frequency . From images recorded at various phase differences between excitation and intensifier gain modulation, the phase and modulation depth of the emitted light is obtained . The fluorescence lifetime is determined from the delay and the decrease in modulation depth of the emission relative to the excitation . A minimum of three images is required, but in this case measurements become susceptible to aliasing caused by the presence of higher harmonics . Taking more images to avoid this is not always possible owing to phototoxicity or movement . A method is introduced, phiFLIM, requiring only three recordings that is not susceptible to aliasing . The phase difference between the excitation and the intensifier is scanned over the entire 360 degrees range following a predefined phase profile, during which the image produced by the intensifier is integrated onto the CCD camera, yielding a single image . Three different images are produced following this procedure, each with a different phase profile . Measurements were performed with a conventional wide-field frequency-domain FLIM system based on an acousto-optic modulator for modulation of the excitation and a microchannel-plate image intensifier coupled to a CCD camera for the detection . By analysis of the harmonic content of measured signals it was found that the third harmonic was effectively the highest present . Using the conventional method with three recordings, phase errors due to aliasing of up to +/- 29 degrees and modulation depth errors of up to 30% were found . Errors in lifetimes of YFP-transfected HeLa cells were as high as 100% . With phiFLIM, using the same specimen and settings, systematic errors due to aliasing did not occur.

Cell Microbiol, 2004 Jan, 6(1), 33 - 48
Activation of caspase-3 by the Dot/Icm virulence system is essential for arrested biogenesis of the Legionella-containing phagosome; Molmeret M et al.; The Dot/Icm type IV secretion system of Legionella pneumophila is essential for evasion of endocytic fusion and for activation of caspase-3 during early stages of infection of macrophages, but the mechanisms of manipulating these host cell processes are not known . Here, we show that caspase-3 activation by L . pneumophila is independent of all the known apoptotic pathways that converge on the activation of caspase-3 . The cytoplasmic proteins IcmS, IcmR and IcmQ, which are involved in secretion of Dot/Icm effectors, are required for caspase-3 activation . Pretreatment of U937 macrophages and human peripheral blood monocytes (hPBM) with the capase-3 inhibitor (DEVD-fmk) or the paninhibitor of caspases (Z-VAD-fmk) before infection blocks intracellular replication of L . pneumophila in a dose-dependent manner . Inhibition of caspase-3 results in co-localization of the L . pneumophila-containing phagosome (LCP) with the late endosomal/lysosomal marker Lamp-2, and the LCP contains lysosomal enzymes, similar to the dotA mutant, which is defective in caspase-3 activation . However, activation of caspase-3 before infection does not rescue the replication defect of the dotA mutant . Interestingly, inhibition of caspase-3 after a 15 or 30 min infection period by the parental strain has no detectable effect on the formation of a replicative niche . The Dot/Icm-mediated activation of caspase-3 by L . pneumophila specifically cleaves, in a dose- and time-dependent manner, the Rab5 effector Rabaptin-5, which maintains Rab5-GTP on the endosomal membrane . In addition, PI3 kinase, which is a crucial effector of Rab5 downstream of Rababptin-5, is not required for intracellular replication . Using single-cell analysis, we show that apoptosis is not evident in the infected cell until bacterial replication results in > 20 bacteria per cell . We conclude that activation of caspase-3 by the Dot/Icm virulence system of L . pneumophila is essential for halting biogenesis of the LCP through the endosomal/lysosomal pathway, and that this is associated with the cleavage of Rabpatin-5.

Immunology, 2004 Jan, 111(1), 91 - 9
Surfactant protein A, an innate immune factor, is expressed in the vaginal mucosa and is present in vaginal lavage fluid; MacNeill C et al.; Surfactant protein A (SP-A), first identified as a component of the lung surfactant system, is now recognized to be an important contributor to host defence mechanisms . SP-A can facilitate phagocytosis by opsonizing bacteria, fungi and viruses, stimulate the oxidative burst by phagocytes and modulate pro-inflammatory cytokine production by phagocytic cells . SP-A can also provide a link between innate and adaptive immune responses by promoting differentiation and chemotaxis of dendritic cells . Because of the obvious relevance of these mechanisms to the host defence and 'gate keeping' functions of the lower genital tract, we examined human vaginal mucosa for SP-A protein and transcripts and analysed vaginal lavage fluid for SP-A . By immunocytochemistry, SP-A was identified in two layers of the vaginal epithelium: the deep intermediate layer (the site of newly differentiated epithelial cells); and the superficial layer (comprising dead epithelial cells), where SP-A is probably extracellular and associated with a glycocalyx . Transcripts of SP-A were identified by Northern blot analysis in RNA isolated from vaginal wall and shown, by sequencing of reverse transcription-polymerase chain reaction products, to be derived from each of the two closely related SP-A genes, SP-A1 and SP-A2 . SP-A was identified in vaginal lavage fluid by two-dimensional gel electrophoresis, and confirmed by mass spectrometry . This study provides evidence, for the first time, that SP-A is produced in a squamous epithelium, namely the vaginal mucosa, and has a localization that would allow it to contribute to both the innate and adaptive immune response . The findings support the hypothesis that in the vagina, as in lung, SP-A is an essential component of the host-defence system . A corollary hypothesis is that qualitative and quantitative alterations of normal SP-A may play a role in the pathogenesis of lower genital tract inflammatory conditions.

Immunology, 2004 Jan, 111(1), 27 - 34
Efficient induction of T helper type 1-mediated immune responses in antigen-primed mice by anti-CD3 single-chain Fv/interleukin-18 fusion DNA; Kim EJ et al.; Two types of T helper (Th) cells - Th1 and Th2 - play different roles in protection and immunopathology . The Th1 cell-mediated immune response plays an important role in inducing the host defence against intracellular bacteria and also in cancer immunotherapy . To effectively induce Th1 immune responses, we constructed a mammalian expression plasmid (pAnti-CD3sFv/IL-18) carrying a fusion gene in which anti-CD3 single-chain Fv (sFv) cDNA, the smallest unit of antibody recognizing the CD3 epsilon moiety of the T-cell receptor, was covalently linked to mature interleukin (IL)-18 cDNA . Intramuscular injection of ovalbumin (OVA)-sensitized BALB/c mice with pAnti-CD3sFv/IL-18 DNA efficiently increased the production of both OVA-specific interferon-gamma and anti-OVA immunoglobulin G2a, compared to injection with pAnti-CD3sFv DNA . In addition, pAnti-CD3sFv/IL-18 was more efficient than a mixture of pAnti-CD3sFv + pIL-18 in inducing OVA-specific, Th1 immune responses and also in inhibiting OVA-specific, IL-4 production . These studies indicate that vaccination with pAnti-CD3sFv/IL-18 fusion DNA efficiently induces the Th1 immune response in antigen-sensitized mice.

APMIS, 2003 Dec, 111(12), 1114 - 6
Increased sensitivity of Mycobacterium tuberculosis Cobas Amplicor PCR following brief incubation of tissue samples on Löwenstein-Jensen substrate; Fernstrom MC et al.; The sensitivity of Mycobacterium tuberculosis Cobas Amplicor PCR (MTB-PCR) is considerably lower for tissue than for airway samples, depending on both lower bacterial content and presence of inhibitory substances in tissues . The aim of this study was to improve the sensitivity of MTB-PCR in inhibiting biopsy samples . A brief-culture method was applied to 33 inhibitory tissue samples out of 356 obtained in Laboratory I, and compared with 44/197 such samples treated by dilution in Laboratory II . We found that 2-3 days' incubation on Lowenstein-Jensen substrate (LJ) significantly increased the sensitivity of MTB-PCR in samples exerting PCR inhibition . Sensitivity was 63% before and 92% following brief-culture of inhibitory tissue samples in Lab I, compared to 46% and 50%, respectively, with dilution in Lab II . Thus, dilution did not significantly increase sensitivity in inhibiting samples . Specificities were 99.4/99.4 and 99.2/98.2, respectively . The higher sensitivity attained by the LJ-method was probably due to diffusion of inhibiting substances into the substrate, as well as to increase in numbers of bacteria after the brief-culture . This method adds substantially to the value of MTB-PCR of biopsy material.

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