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</