Microorganisms

Angew Chem Int Ed Engl, 2001 Aug 3, 40(15), 2782 - 2807
Dioxygen Activation and Methane Hydroxylation by Soluble Methane Monooxygenase: A Tale of Two Irons and Three Proteins A list of abbreviations can be found in Section 7 .
Merkx M, Kopp DA, Sazinsky MH, Blazyk JL, Muller J, Lippard SJ.
Methanotrophic bacteria are capable of using methane as their sole source of carbon and energy . The first step in methane metabolism, the oxidation of methane to methanol, is catalyzed by a fascinating enzyme system called methane monooxygenase (MMO) . The selective oxidation of the very stable C-H bond in methane under ambient conditions is a remarkable feat that has not yet been repeated by synthetic catalysts and has attracted considerable scientific and commercial interest . The best studied MMO is a complex enzyme system that consists of three soluble protein components, all of which are required for efficient catalysis . Dioxygen activation and subsequent methane hydroxylation are catalyzed by a hydroxylase enzyme that contains a non-heme diiron site . A reductase protein accepts electrons from NADH and transfers them to the hydroxylase where they are used for the reductive activation of O(2) . The third protein component couples electron and dioxygen consumption with methane oxidation . In this review we examine different aspects of catalysis by the MMO proteins, including the mechanisms of dioxygen activation at the diiron site and substrate hydroxylation by the activated oxygen species . We also discuss the role of complex formation between the different protein components in regulating various aspects of catalysis.

Cytometry, 2001 Aug 1, 44(4), 361 - 8
Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein; Chan FK et al.; BACKGROUND: Fluorescence resonance energy transfer (FRET) is a powerful technique for measuring molecular interactions at Angstrom distances . We present a new method for FRET that utilizes the unique spectral properties of variants of the green fluorescent protein (GFP) for large-scale analysis by flow cytometry . METHODS: The proteins of interest are fused in frame separately to the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP) . FRET between these differentially tagged fusion proteins is analyzed using a dual-laser FACSVantage cytometer . RESULTS: We show that homotypic interactions between individual receptor chains of tumor necrosis factor receptor (TNFR) family members can be detected as FRET from CFP-tagged receptor chains to YFP-tagged receptor chains . Noncovalent molecular complexation can be detected as FRET between fusions of CFP and YFP to either the intracellular or extracellular regions of the receptor chains . The specificity of the assay is demonstrated by the absence of FRET between heterologous receptor pairs that do not biochemically associate with each other . Interaction between a TNFR-like receptor (Fas/CD95/Apo-1) and a downstream cytoplasmic signaling component (FADD) can also be demonstrated by flow cytometric FRET analysis . CONCLUSIONS: The utility of spectral variants of GFP in flow cytometric FRET analysis of membrane receptors is demonstrated . This method of analyzing FRET allows probing of noncovalent molecular interactions that involve both the intracellular and extracellular regions of membrane proteins as well as proteins within the cells . Unlike biochemical methods, FRET allows the quantitative determination of noncovalent molecular associations at Angstrom level in living cells . Moreover, flow cytometry allows quantitative analyses to be carried out on a cell-by-cell basis on large number of cells . Published 2001 Wiley-Liss, Inc.

Mem Inst Oswaldo Cruz, 2001 Jul, 96(5), 723 - 8
Biological activities of Curcuma longa L; Araujo CC et al.; There are several data in the literature indicating a great variety of pharmacological activities of Curcuma longa L . (Zingiberaceae), which exhibit anti-inflammatory, anti-human immunodeficiency virus, anti-bacteria, antioxidant effects and nematocidal activities . Curcumin is a major component in Curcuma longa L., being responsible for its biological actions . Other extracts of this plant has been showing potency too . In vitro, curcumin exhibits anti-parasitic, antispasmodic, anti-inflammatory and gastrointestinal effects; and also inhibits carcinogenesis and cancer growth . In vivo, there are experiments showing the anti-parasitic, anti-inflammatory potency of curcumin and extracts of C . longa L . by parenteral and oral application in animal models . In this present work we make an overview of the pharmacological activities of C . longa L., showing its importance.

Saudi Med J, 2000 Jun, 21(6), 569 - 73
Ultrastructural study of the gastric mucosa and Helicobacter pylori in duodenal ulcer patients; Al-Muhtaseb MH et al.; OBJECTIVE: To investigate the relationship between Helicobacter pylori and gastric mucosa in control and duodenal ulcer patients at the electron microscopic level . METHODS: Three antral biopsies were taken from each of 20 normal control volunteers and 30 duodenal ulcer patients presented to the gastroenterology unit at Jordan University Hospital for upper endoscopic examination . Each specimen was fixed and processed for electron microscopic study . RESULTS: Two types of Helicobacter pylori were observed and identified by their morphology at electron microscopy . The first one was characterized by double external smooth membranes and homogeneous cytoplasmic contents, and the second type with a characteristic ring-shaped intracytoplasmic vacuole . Electron microscopic examination of normal controls showed normal gastric mucosa and a small number of Helicobacter pylori in 12 out of 20 controls . However, in duodenal ulcer patients, 5 different patterns of interaction between the Helicobacter pylori and gastric mucosa were observed in relation to the severity of the disease . In duodenal ulcer patients, various types of epithelial damage was seen accompanied with a decrease or absence of mucous secretion and with more colonization of bacteria . CONCLUSION: The morphology and pathogenesis of Helicobacter pylori was described in duodenal ulcer patients, and 5 different patterns of contact between Helicobacter pylori and surface epithelium were recognized causing variable degrees of microvillous atrophy and reduced mucous secretion . The vacuolated type of Helicobacter pylori was more adherent to the damaged epithelium and there was a direct relationship between the epithelial damage and bacterial load . In the normal controls, no epithelial damage and scanty bacteria were observed . The various types of epithelial changes of gastric mucosa has initiated more research at electron microscopic level on the immune mechanism of the gastric mucosa to determine the underlying cause of the varying severity of the disease.

Plant Physiol, 2001 Aug, 126(4), 1358 - 69
The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants; Johanson U et al.; Major intrinsic proteins (MIPs) facilitate the passive transport of small polar molecules across membranes . MIPs constitute a very old family of proteins and different forms have been found in all kinds of living organisms, including bacteria, fungi, animals, and plants . In the genomic sequence of Arabidopsis, we have identified 35 different MIP-encoding genes . Based on sequence similarity, these 35 proteins are divided into four different subfamilies: plasma membrane intrinsic proteins, tonoplast intrinsic proteins, NOD26-like intrinsic proteins also called NOD26-like MIPs, and the recently discovered small basic intrinsic proteins . In Arabidopsis, there are 13 plasma membrane intrinsic proteins, 10 tonoplast intrinsic proteins, nine NOD26-like intrinsic proteins, and three small basic intrinsic proteins . The gene structure in general is conserved within each subfamily, although there is a tendency to lose introns . Based on phylogenetic comparisons of maize (Zea mays) and Arabidopsis MIPs (AtMIPs), it is argued that the general intron patterns in the subfamilies were formed before the split of monocotyledons and dicotyledons . Although the gene structure is unique for each subfamily, there is a common pattern in how transmembrane helices are encoded on the exons in three of the subfamilies . The nomenclature for plant MIPs varies widely between different species but also between subfamilies in the same species . Based on the phylogeny of all AtMIPs, a new and more consistent nomenclature is proposed . The complete set of AtMIPs, together with the new nomenclature, will facilitate the isolation, classification, and labeling of plant MIPs from other species.

Infect Immun, 2001 Sep, 69(9), 5857 - 63
In vivo and in vitro studies of cytosolic phospholipase A2 expression in Helicobacter pylori infection; Nardone G et al.; Modifications of mucosal phospholipids have been detected in samples from patients with Helicobacter pylori-positive gastritis . These alterations appear secondary to increased phospholipase A2 activity (PLA2) . The cytosolic form of this enzyme (cPLA2), normally involved in cellular signaling and growth, has been implicated in cancer pathogenesis . The aim of this study was to investigate cPLA2 expression and PLA2 activity in the gastric mucosae of patients with and without H . pylori infection . In gastric biopsies from 10 H . pylori-positive patients, cPLA2 levels, levels of mRNA as determined by reverse transcriptase PCR, levels of protein as determined by immunohistochemistry, and total PLA2 activity were higher than in 10 H . pylori-negative gastritis patients . To clarify whether H . pylori had a direct effect on the cellular expression of cPLA2, we studied cPLA2 expression in vitro with different human epithelial cell lines, one from a patient with larynx carcinoma (i.e., HEp-2 cells) and two from patients with gastric adenocarcinoma (i.e., AGS and MKN 28 cells), incubated with different H . pylori strains . The levels of cPLA2, mRNA, and protein expression were unchanged in Hep-2 cells independently of cellular adhesion or invasion of the bacteria . Moreover, no change in cPLA2 protein expression was observed in AGS or MKN 28 cells treated with wild-type H . pylori . In conclusion, our study shows increased cPLA2 expression and PLA2 activity in the gastric mucosae of patients with H . pylori infection and no change in epithelial cell lines exposed to H . pylori.

Infect Immun, 2001 Sep, 69(9), 5777 - 85
Identification and characterization of mycobacterial proteins differentially expressed under standing and shaking culture conditions, including Rv2623 from a novel class of putative ATP-binding proteins; Florczyk MA et al.; The environmental signals that affect gene regulation in Mycobacterium tuberculosis remain largely unknown despite their importance to tuberculosis pathogenesis . Other work has shown that several promoters, including acr (also known as hspX) (alpha-crystallin homolog), are upregulated in shallow standing cultures compared with constantly shaking cultures . Each of these promoters is also induced to a similar extent within macrophages . The present study used two-dimensional gel electrophoresis and mass spectrometry to further characterize differences in mycobacterial protein expression during growth under standing and shaking culture conditions . Metabolic labeling of M . bovis BCG showed that at least 45 proteins were differentially expressed under standing and shaking culture conditions . Rv2623, CysA2-CysA3, Gap, and Acr were identified from each of four spots or gel bands that were specifically increased in bacteria from standing cultures . An additional standing-induced spot contained two comigrating proteins, GlcB and KatG . The greatest induction was observed with Rv2623, a 32-kDa protein of unknown function that was strongly expressed under standing conditions and absent in shaking cultures . Analysis using PROBE, a multiple sequence alignment and database mining tool, classified M . tuberculosis Rv2623 as a member of a novel class of ATP-binding proteins that may be involved in M . tuberculosis's response to environmental signals . These studies demonstrate the power of combined proteomic and computational approaches and demonstrate that subtle differences in bacterial culture conditions may have important implications for the study of gene expression in mycobacteria.

Infect Immun, 2001 Sep, 69(9), 5752 - 9
Cytolethal distending toxin demonstrates genotoxic activity in a yeast model; Hassane DC et al.; Cytolethal distending toxins (CDTs) are multisubunit proteins produced by a variety of bacterial pathogens that cause enlargement, cell cycle arrest, and apoptosis in mammalian cells . While their function remains uncertain, recent studies suggest that they can act as intracellular DNases in mammalian cells . Here we establish a novel yeast model for understanding CDT-associated disease . Expression of the CdtB subunit in yeast causes a G2/M arrest, as seen in mammalian cells . CdtB toxicity is not circumvented in yeast genetically altered to lack DNA damage checkpoint control or that constitutively promote cell cycle progression via mutant Cdk1, because CdtB causes a permanent type of damage that results in loss of viability . Finally, we establish that CDTs are likely to be potent genotoxins, as indicated by in vivo degradation of chromosomal DNA associated with expression of CdtB-suggesting that the varied distribution of CDT in bacteria implicates many human pathogens as possessors of genotoxic activity.

Infect Immun, 2001 Sep, 69(9), 5698 - 708
Porphyromonas gingivalis traffics to autophagosomes in human coronary artery endothelial cells; Dorn BR et al.; Porphyromonas gingivalis is a periodontal pathogen that also localizes to atherosclerotic plaques . Our previous studies demonstrated that P . gingivalis is capable of invading endothelial cells and that intracellular bacteria are contained in vacuoles that resemble autophagosomes . In this study, we have examined the trafficking of P . gingivalis 381 to the autophagic pathway . P . gingivalis 381 internalized by human coronary artery endothelial (HCAE) cells is located within vacuoles morphologically identical to autophagosomes . The progression of P . gingivalis 381 through intracellular vacuoles was analyzed by immunofluorescence microscopy . Vacuoles containing P . gingivalis colocalize with Rab5 and HsGsa7p early after internalization . At later times, P . gingivalis colocalizes with BiP and then progresses to a vacuole that contains BiP and lysosomal glycoprotein 120 . Late endosomal markers and the lysosomal cathepsin L do not colocalize with P . gingivalis 381 . The intracellular survival of P . gingivalis 381 decreases over 8 h in HCAE cells pretreated with the autophagy inhibitors 3-methyladenine and wortmannin . In addition, the vacuole containing P . gingivalis 381 lacks BiP but contains cathepsin L in the presence of wortmannin . These results suggest that P . gingivalis 381 evades the endocytic pathway to lysosomes and instead traffics to the autophagosome.

Infect Immun, 2001 Sep, 69(9), 5661 - 70
Production of matrix metalloproteinases in response to mycobacterial infection; Quiding-Jarbrink M et al.; Matrix metalloproteinases (MMPs) constitute a large family of enzymes with specificity for the various proteins of the extracellular matrix which are implicated in tissue remodeling processes and chronic inflammatory conditions . To investigate the role of MMPs in immunity to mycobacterial infections, we incubated murine peritoneal macrophages with viable Mycobacterium bovis BCG or Mycobacterium tuberculosis H37Rv and assayed MMP activity in the supernatants by zymography . Resting macrophages secreted only small amounts of MMP-9 (gelatinase B), but secretion increased dramatically in a dose-dependent manner in response to either BCG or M . tuberculosis in vitro . Incubation with mycobacteria also induced increased MMP-2 (gelatinase A) activity . Neutralization of tumor necrosis alpha (TNF-alpha), and to a lesser extent interleukin 18 (IL-18), substantially reduced MMP production in response to mycobacteria . Exogenous addition of TNF-alpha or IL-18 induced macrophages to express MMPs, even in the absence of bacteria . The immunoregulatory cytokines gamma interferon (IFN-gamma), IL-4, and IL-10 all suppressed BCG-induced MMP production, but through different mechanisms . IFN-gamma treatment increased macrophage secretion of TNF-alpha but still reduced their MMP activity . Conversely, IL-4 and IL-10 seemed to act by reducing the amount of TNF-alpha available to the macrophages . Finally, infection of BALB/c or severe combined immunodeficiency (SCID) mice with either BCG or M . tuberculosis induced substantial increases in MMP-9 activity in infected tissues . In conclusion, we show that mycobacterial infection induces MMP-9 activity both in vitro and in vivo and that this is regulated by TNF-alpha, IL-18, and IFN-gamma . These findings indicate a possible contribution of MMPs to tissue remodeling processes that occur in mycobacterial infections.

Infect Immun, 2001 Sep, 69(9), 5417 - 22
Ontogeny of Th1 memory responses against a Brucella abortus conjugate; Scharf O et al.; Protective immune responses to intracellular pathogens such as Brucella abortus are characteristically Th1-like . Recently we demonstrated that heat-killed B . abortus (HKBa), a strong Th1 stimulus, conjugated to ovalbumin (HKBA-OVA), but not B . abortus alone, can alter the antigen-specific cytokine profile from Th2- to Th1-like . In this report we study the ability of a single injection of B . abortus to switch a Th2 to a Th1 response in immature mice . One-day- and 1-week-old mice were given a single injection of B . abortus in the absence or presence of OVA, and at maturity mice were challenged with an allergenic preparation, OVA with alum (OVA-A) . B . abortus given without OVA did not diminish the subsequent Th2 response in either age group . In contrast, mice receiving a single injection of B . abortus-OVA at the age of 1 week, but not those injected at the age of 1 day, had reversal of the ratio of OVA-specific Th1 to Th2 cells and decreased immunoglobulin E levels after allergen challenge as adults . Within 6 h both 1-day- and 1-week-old mice expressed interleukin-12 p40 mRNA following either B . abortus or B . abortus-OVA administration . However, only the 1-week-old mice exhibited increased expression of gamma interferon (IFN-gamma) mRNA . The absence of the early IFN-gamma response in 1-day-old mice may explain their inability to generate a Th1 memory response . These results suggest that at early stages of immune development, responses to intracellular bacteria may be Th2- rather than Th1-like . Furthermore, they suggest that the first encounter with antigen evokes either a Th1- or a Th2-like response which becomes imprinted, so that subsequent memory responses conform to the original Th bias . This has implications for protection against infectious agents and development of allergic responses.

Infect Immun, 2001 Sep, 69(9), 5286 - 93
Borrelia burgdorferi RevA antigen is a surface-exposed outer membrane protein whose expression is regulated in response to environmental temperature and pH; Carroll JA et al.; Borrelia burgdorferi, the causative agent of Lyme disease, produces RevA protein during the early stages of mammalian infection . B . burgdorferi apparently uses temperature as a cue to its location, producing proteins required for infection of warm-blooded animals at temperatures corresponding to host body temperature, but does not produce such virulence factors at cooler, ambient temperatures . We have observed that B . burgdorferi regulates expression of RevA in response to temperature, with the protein being synthesized by bacteria cultivated at 34 degrees C but not by those grown at 23 degrees C . Tissues encountered by B . burgdorferi during its infectious cycle vary in their pH values, and the level of RevA expression was also found to be dependent upon pH of the culture medium . The cellular localization of RevA was also analyzed . Borrelial inner and outer membranes were purified by isopycnic centrifugation, and membrane fractions were conclusively identified by immunoblot analysis using antibodies raised against the integral inner membrane protein MotB and outer membrane-associated Erp lipoproteins . Immunoblot analyses indicated that RevA is located in the B . burgdorferi outer membrane . These analyses also demonstrated that an earlier report (H . A . Bledsoe et al., Infect . Immun . 176:7447-7455, 1994) had misidentified such B . burgdorferi membrane fractions . RevA was further demonstrated to be exposed to the external environment, where it could facilitate interactions with host tissues.

EMBO J, 2001 Aug 15, 20(16), 4341 - 8
The Dictyostelium homologue of mammalian soluble adenylyl cyclase encodes a guanylyl cyclase; Roelofs J et al.; A new Dictyostelium discoideum cyclase gene was identified that encodes a protein (sGC) with 35% similarity to mammalian soluble adenylyl cyclase (sAC) . Gene disruption of sGC has no effect on adenylyl cyclase activity and results in a >10-fold reduction in guanylyl cyclase activity . The scg- null mutants show reduced chemotactic sensitivity and aggregate poorly under stringent conditions . With Mn(2+)/GTP as substrate, most of the sGC activity is soluble, but with the more physiological Mg(2+)/GTP the activity is detected in membranes and stimulated by GTPgammaS . Unexpectedly, orthologues of sGC and sAC are present in bacteria and vertebrates, but absent from Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae.

J Biotechnol, 2001 Aug 23, 89(2-3), 155 - 62
Batch tests for assessing decolourisation of azo dyes by methanogenic and mixed cultures; Bras R et al.; Most of the published studies on azo dye colour removal involve anaerobic mixed cultures and there is some interest in the knowledge of how dye reduction occurs, if by facultative, strictly anaerobic or both bacterial trophic groups present in classic anaerobic digestors . This paper describes the behaviour of methanogenic and mixed bacteria cultures on the colour removal in batch systems, of a commercial azo dye, C.I . Acid Orange 7, used in paper and textile industries . The aim of this study is to demonstrate, by analysing dye decolourisation, that it occurs with mixed cultures as well as with strictly anaerobic (methanogenic) cultures . Tests were performed with a range of dye concentrations between 60 and 300 mg x l(-1) . The influence of dye concentration on the carbon source removal and decolourisation processes was studied . The effect of carbon source concentration on colour removal was also analysed for both cultures . The degradation rates in mixed and methanogenic cultures were compared . The consumption of carbon source was monitored by COD analysis and dye degradation by ultraviolet-visible spectrophotometry and thin layer chromatography.

Transfus Clin Biol, 2001 Jun, 8(3), 163 - 99
Structural and functional diversity of blood group antigens; Cartron JP et al.; Biochemical and molecular genetic studies have revealed that blood group antigens are present on cell surface molecules of wide structural diversity, including carbohydrate epitopes on glycoproteins and/or glycolipids, and peptide antigens on proteins inserted within the membrane via single or multi-pass transmembrane domains, or via glycosylphosphatidylinositol linkages . These studies have also shown that some blood group antigens are carried by complexes consisting of several membrane components which may be lacking or severely deficient in rare blood group 'null' phenotypes . In addition, although all blood group antigens are serologically detectable on red blood cells (RBCs), most of them are also expressed in non-erythroid tissues, raising further questions on their physiological function under normal and pathological conditions . In addition to their structural diversity, blood group antigens also possess wide functional diversity, and can be schematically subdivided into five classes: i) transporters and channels; ii) receptors for ligands, viruses, bacteria and parasites; iii) adhesion molecules; iv) enzymes; and v) structural proteins . The purpose of this review is to summarize recent findings on these molecules, and in particular to illustrate the existing structure-function relationships.

Transfus Clin Biol, 2001 Jun, 8(3), 138 - 45
Inactivation of infectious pathogens in labile blood components: meeting the challenge; Corash L; Substantial improvement in the safety of blood transfusion has been achieved through the addition of new tests, such as nucleic acid tests, yet residual risk associated with transfusion of blood components persists . Transfusion of blood components has been implicated in the transmission of viruses, bacteria, and protozoa . While it is commonly recognized that hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), and the retroviruses, such as human immunodeficiency virus (HIV) and the human lymphotrophic viruses (HTLV) can be transmitted through cellular components, other pathogens are emerging as potentially significant transfusion-associated infectious agents . For example, transmission of protozoan infections due to trypanosomes and babesia have been reported . In addition to viral and protozoal infectious agents, bacterial contamination of platelet and red cell concentrates continues to be reported; and may be an under-reported transfusion complication . More importantly, new infectious agents may periodically enter the donor population before they can be definitively identified and tested for to maintain consistent safety of the blood supply . The paradigm for this possibility is the HIV pandemic, which erupted in 1979 . During the past decade a number of methods to inactivate infectious pathogens in labile blood components have been developed and have entered the advanced clinical trial phase.

Biochem Soc Trans, 2001 Aug, 29(Pt 4), 480 - 4
Internalization of the epidermal growth factor receptor: role in signalling; Sorkin A; The interaction of the activated epidermal growth factor (EGF) receptor (EGFR) with the Src homology 2 (SH2) domain of Grb2 (growth-factor-receptor-bound protein 2) initiates signalling through Ras and mitogen-activated protein kinase . Grb2 can bind EGFR directly or through another SH2-containing protein, Shc . Activation of EGFRs by ligand also triggers rapid endocytosis of EGF-receptor complexes . To analyse the spatial and temporal regulation of EGFR interactions with SH2 domains in living cells, we have combined imaging microscopy with a modified method of measuring fluorescence resonance energy transfer (FRET) on a pixel-by-pixel basis using EGFR fused to cyan fluorescent protein (CFP) in pair with Grb2 or Shc fused to yellow fluorescent protein (YFP) . Stimulation by EGF resulted in the recruitment of Grb2-YFP and YFP-Shc to cellular compartments that contained EGFR-CFP, and a large increase in the FRET signal . In particular, FRET measurements indicated that activated EGFR-CFP interacted with YFP-Shc and Grb2-YFP in membrane ruffles and endosomes . These results demonstrate that signalling via EGFRs can occur in the endosomal compartment . Moreover, in contrast with previous biochemical studies, FRET experiments show that a large pool of Grb2 and Shc is associated with EGFRs for a prolonged period after EGF stimulation.

Biochem Soc Trans, 2001 Aug, 29(Pt 4), 455 - 9
Auxiliary functions in photosynthesis: the role of the FtsH protease; Bailey S et al.; Oxygenic photosynthesis can be described effectively by using two long-standing models: the Z-scheme and the chemiosmotic hypothesis . However, these models do not reveal the dynamic nature of the thylakoid membrane and the four major complexes that it binds . The composition of the photosynthetic apparatus is continually changing in response to a range of environmental stimuli . In addition, many photosynthetic components have some of the highest turnover rates in Nature . Changes in composition and turnover of photosynthetic components require the degradation of existing and damaged polypeptides and the resynthesis and co-ordinated assembly of new polypeptides and their associated cofactors . This is achieved by several auxiliary functions, including proteolysis, protein targeting and the action of molecular chaperones . Some of the components involved in these functions, such as translocons, chaperones and proteases, have been identified but many of the auxiliary functions of photosynthesis remain uncharacterized . Among the proteases known to be associated with the thylakoids is the zinc metalloprotease FtsH, which might also act as a chaperone . Here we provide an overview of the thylakoid FtsH protease and discuss its role in the maintenance and assembly of the photosynthetic apparatus.

Biochem Soc Trans, 2001 Aug, 29(Pt 4), 427 - 30
Degradation of unassembled and damaged thylakoid proteins; Adam Z et al.; To study protein degradation in thylakoid membranes we identified, characterized and cloned thylakoid proteases, and then linked them to known proteolytic processes . Several families of chloroplast proteases were identified and characterized to different extents . FtsH, an ATP-dependent metalloprotease that belongs to the AAA-protein family, was found to be integral to the thylakoid membrane, facing the stroma . It is involved in both the degradation of unassembled subunits of membrane complexes, such as the Rieske Fe-S protein of the cytochrome complex, and the degradation of oxidatively damaged proteins such as the D1 protein of the photosystem II (PS II) reaction centre . Plant genomes contain multiple isomers of this protease but the functional significance of this multiplication is not clear yet . A second protease, the serine ATP-independent DegP, was found to be strongly associated with the luminal side of the thylakoid membrane . Although a specific role has not yet assigned for it, its location suggests that it can degrade luminal soluble proteins as well as luminally exposed regions of thylakoid membrane proteins.

Biochem Soc Trans, 2001 Aug, 29(Pt 4), 418 - 21
Incorporation of iron-sulphur clusters in membrane-bound proteins; Seidler A et al.; The completely sequenced genome of the cyanobacterium Synechocystis PCC 6803 contains several open reading frames, of which the deduced amino acid sequences show similarities to proteins known to be involved in FeS cluster synthesis of nitrogenase (Nif proteins) and other FeS proteins (Isc proteins) . In this article, the results of our studies on these proteins are summarized and discussed with respect to their relevance in FeS cluster incorporation in chloroplasts . In cyanobacteria, there appears to exist several pathways for FeS cluster synthesis.

Biochem Soc Trans, 2001 Aug, 29(Pt 4), 392 - 5
Basal and regulated transcription in Archaea; Bell SD et al.; The basal transcription machinery of Archaea is fundamentally related to the eucaryal RNA polymerase (RNAP) II apparatus . In addition to a 12-subunit RNAP, Archaea possess two general transcription factors, the activities of which are required for accurate and efficient in vitro transcription . These factors, TBP and TFB, are homologues of the eucaryal TATA-box binding protein and TFIIB respectively . Archaea also possess TFE, a homologue of the eucaryal RNAP II general transcription factor TFIIE . Although not absolutely required for transcription in vitro, TFE nonetheless plays a stimulatory role under conditions where promoter recognition by TBP is sub-optimal . The basal transcription apparatus of Archaea is closely related to that of Eucarya but archaeal transcriptional regulators resemble those of bacteria . The mode of action of two such regulators has been characterized to determine how these 'bacterial-like' regulators impinge on the 'eucaryal-like' basal machinery.

Nippon Yakurigaku Zasshi, 2001 Jul, 118(1), 15 - 22
{Histamine produced by macrophage and T lymphocyte: a new type of signal transducer}; Nakano K et al.; Macrophages (M phi) produce histamine (Hm) when activated by bacterial endotoxin (LPS) through induced histidine decarboxylase (HDC) . Among the cytokines tested, GM-CSF or IL-3 specifically augmented the LPS-dependent HDC induction by M phi . Hm formed by M phi regulates synthesis of cytokines such as IL-1, IL-6, G-CSF and M-CSF by the cells per se and may modulate immune reactions and division and differentiation of various hematopoietic cells . Kupffer cells, M phi-like cells in the liver, also synthesize Hm in mice injected with hepatotoxins such as tetradecanoylphorbol acetate or LPS . Hm thus produced by Kupffer cells may participate in the regeneration of the injured liver through induction of hepatocyte growth factor . Concanavalin A (Con A) enhanced Hm formation by T lymphocytes . GM-CSF or IL-3 also enhanced the Hm synthesis by CD4+ and CD8+ T cells . Hm formed by T cells regulates immune reactions such as lymphocyte blastogenesis . In animals infected with gram(-) bacteria Hm is produced by the M phi-T cell system and may regulate immune competence to the bacteria . In addition, Hm may act as a signal transducer between the peripheral immune system and hypothalamus-pituitary-adrenal system, leading to GC secretion, in order to prevent occurrence of tissue injury caused by excess immune reactions.

Water Sci Technol, 2001, 44(1), 161 - 6
Sludge digestion enhancement and nutrient removal from anaerobic supernatant by Mg(OH)2 application; Wu Q et al.; Anaerobic sludge digestion is a widely adopted process for sludge stabilization . Phosphate removal from anaerobic supernatant is necessary to limit the phosphate returned to the head of the treatment plant, thereby improving the overall treatment efficiency . In this study, magnesium hydroxide (Mg(OH)2) was used to improve the sludge digestion efficiency and to remove phosphorus from anaerobic supernatant . The anaerobic sludge digestion experiment was conducted at a pilot scale, and the results showed that applying Mg(OH)2 to anaerobic sludge digester resulted in a larger reduction in SS and COD, a higher biogas production rate, a lower level of phosphate and ammonia nitrogen concentrations in the sludge supernatant and an improved sludge dewaterability . Research results at both lab scale and pilot scale on phosphorus removal from anaerobic supernatant using Mg(OH)2 showed that a high removal of phosphorus can be achieved through the addition of Mg(OH)2 . The required reaction time depends on the initial phosphorus concentration and the Mg(OH)2 dosage.

J Allergy Clin Immunol, 2001 Aug, 108(2), 157 - 66
Endotoxin-stimulated innate immunity: A contributing factor for asthma; Reed CE et al.; Exposure to airborne endotoxin in infancy may protect against asthma by promoting enhanced T(H)1 response and tolerance to allergens . On the other hand, later in life, it adversely affects patients with asthma . Endotoxin binding to receptors on macrophages and other cells generates IL-12, which inhibits IgE responses . It also generates cytokines like IL-1, TNF-alpha, and IL-8, which cause inflammation . These signal transduction pathways resemble those leading to the generation of cytokines, such as IL-4, IL-13, and IL-5, which are responsible for the inflammation of IgE-mediated allergic disease . The main difference seems to be that endotoxin recruits neutrophils, but IgE recruits eosinophils, and the details of the tissue injury from these granulocytes differ . Sources of airborne endotoxin include many agricultural dusts, aerosols from contaminated water in many industrial plants, contaminated heating and air-conditioning systems, mist-generating humidifiers, and damp or water-damaged homes . Acute inhalation of high concentrations of endotoxin can cause fever, cough, and dyspnea . Chronic inhalation of lesser amounts causes chronic bronchitis and emphysema and is associated with airway hyperresponsiveness . Airborne endotoxin adversely affects patients with asthma in 3 ways: (1) by increasing the severity of the airway inflammation; (2) by increasing the susceptibility to rhinovirus-induced colds; and (3) by causing chronic bronchitis and emphysema with development of irreversible airway obstruction after chronic exposure of adults . The most effective management is mitigating exposure . The potential of drug treatments requires further clinical investigation.

Microbiology, 2001 Aug, 147(Pt 8), 2293 - 305
cDNA-RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG; Li MS et al.; Identifying genes that are differentially expressed by Mycobacterium bovis BCG after phagocytosis by macrophages will facilitate the understanding of the molecular mechanisms of host cell-intracellular pathogen interactions . To identify such genes a cDNA-total RNA subtractive hybridization strategy has been used that circumvents the problems both of limited availability of bacterial RNA from models of infection and the high rRNA backgrounds in total bacterial RNA . The subtraction products were used to screen a high-density gridded Mycobacterium tuberculosis genomic library . Sequence data were obtained from 19 differential clones, five of which contained overlapping sequences for the gene encoding mycocerosic acid synthase (mas) . Mas is an enzyme involved in the synthesis of multi-methylated long-chain fatty acids that are part of phthiocerol dimycocerosate, a major component of the complex mycobacterial cell wall . Northern blotting and primer extension data confirmed up-regulation of mas in intracellular mycobacteria and also revealed a putative extended -10 promoter structure and a long untranslated upstream region 5' of the mas transcripts, containing predicted double-stranded structures . Furthermore, clones containing overlapping sequences for furB, groEL-2, rplE and fadD28 were identified and the up-regulation of these genes was confirmed by Northern blot analysis . The cDNA-RNA subtractive hybridization enrichment and high density gridded library screening, combined with selective extraction of bacterial mRNA represents a valuable approach to the identification of genes expressed during intra-macrophage residence for bacteria such as M . bovis BCG and the pathogenic mycobacterium, M . tuberculosis.

Microbiology, 2001 Aug, 147(Pt 8), 2285 - 92
Characterization of the low-pH responses of Helicobacter pylori using genomic DNA arrays; Allan E et al.; Helicobacter pylori is unique among bacterial pathogens in its ability to persist in the acidic environment of the human stomach . To identify H . pylori genes responsive to low pH, the authors assembled a high-density array of PCR-amplified random genomic DNA . Hybridization of radiolabelled cDNA probes, prepared using total RNA from bacteria exposed to buffer at either pH 4.0 or pH 7.0, allowed both qualitative and quantitative information on differential gene expression to be obtained . A previously described low-pH-induced gene, cagA, was identified together with several novel genes that may have relevance to the survival and persistence of H . pylori in the gastric environment . These include genes encoding enzymes involved in LPS and phospholipid synthesis and secF, encoding a component of the protein export machinery . A hypothetical protein unique to H . pylori (HP0681) was also found to be acid induced . Genes down-regulated at pH 4.0 include those encoding a sugar nucleotide biosynthesis protein, a flagellar protein and an outer-membrane protein . Differential gene expression was confirmed by total RNA slot-blot hybridization.

Microbiology, 2001 Aug, 147(Pt 8), 2265 - 73
SigB, an alternative sigma factor of the myxobacterium Stigmatella aurantiaca, is synthesized during development and heat shock; Silakowski B et al.; Alternative sigma factors have been detected in the myxobacterium Stigmatella aurantiaca during indole-induced sporulation, fruiting body formation and heat shock using an antiserum raised against sigma factor SigB . The time course of sigB gene expression was analysed by RT-PCR and by determining beta-galactosidase activity during development in a merodiploid strain that harboured a sigB-lacZ fusion gene . Inactivation of the sigB gene by insertion of the neo gene resulted in the loss of one sigma factor as shown by Western analysis . Neither fruiting body formation nor sporulation, nor the production of possible SigB targets, such as DnaK, GroEL or HspA, were affected.

Microbiology, 2001 Aug, 147(Pt 8), 2141 - 8
Molecular characterization of a deletion/duplication rearrangement in tfd genes from Ralstonia eutropha JMP134(pJP4) that improves growth on 3-chlorobenzoic acid but abolishes growth on 2,4-dichlorophenoxyacetic acid; Clement P et al.; Ralstonia eutropha JMP134(pJP4) is able to grow on minimal media containing the pollutants 3-chlorobenzoate (3-CB) or 2,4-dichlorophenoxyacetate (2,4-D) . tfd genes from the 88 kb plasmid pJP4 encode enzymes involved in the degradation of these compounds . During growth of strain JMP134 in liquid medium containing 3-CB, a derivative strain harbouring a approximately 95 kb plasmid was isolated . This derivative, designated JMP134(pJP4-F3), had an improved ability to grow on 3-CB, but had lost the ability to grow on 2,4-D . Sequence analysis of pJP4-F3 indicated that the plasmid had undergone a deletion of approximately 16 kb, which included the tfdA-tfdS intergenic region, spanning the tfdA gene to a previously unreported IS1071 element . The loss of the tfdA gene explains the failure of the derivative to grow on 2,4-D . A approximately 23 kb duplication of the region spanning tfdR-tfdD(II)C(II)E(II)F(II)-tfdB(II)-tfdK-ISJP4-tfdT-tfdC(I)D(I)E(I)F(I)-tfdB(I), giving rise to a 51-kb-long inverted repeat, was also observed . The increase in gene copy number for the tfdCD(DC)EF gene cluster may provide an explanation for the derivative strain's improved growth on 3-CB . These observations are additional examples of the metabolic plasticity of R . eutropha JMP134, one of the more versatile pollutant-degrading bacteria.

Curr Opin Microbiol, 2001 Aug, 4(4), 435 - 41
Interactions between rotavirus and gastrointestinal cells; Ciarlet M et al.; Rotaviruses are the leading cause of life-threatening diarrheal disease in infants and in young animals worldwide . The outcome of rotavirus infection of intestinal epithelial cells is more complex and involves induction of more diverse cellular responses than initially appreciated . Similar to bacteria, the pathogenesis of rotavirus-induced disease involves an enterotoxin, activation of the enteric nervous system and malabsorption, suggesting that common mechanisms of pathogenesis may exist between viral and bacterial pathogens.

Curr Opin Struct Biol, 2001 Aug, 11(4), 420 - 6
Crystal structure of rhodopsin: implications for vision and beyond; Okada T et al.; A heptahelical transmembrane bundle is a common structural feature of G-protein-coupled receptors (GPCRs) and bacterial retinal-binding proteins, two functionally distinct groups of membrane proteins . Rhodopsin, a photoreceptor protein involved in photopic (rod) vision, is a prototypical GPCR that contains 11-cis-retinal as its intrinsic chromophore ligand . Therefore, uniquely, rhodopsin is a GPCR and also a retinal-binding protein, but is not found in bacteria . Rhodopsin functions as a typical GPCR in processes that are triggered by light and photoisomerization of its ligand . Bacteriorhodopsin is a light-driven proton pump with an all-trans-retinal chromophore that photoisomerizes to 13-cis-retinal . The recent crystal structure determination of bovine rhodopsin revealed a structure that is not similar to previously established bacteriorhodopsin structures . Both groups of proteins have a heptahelical transmembrane bundle structure, but the helices are arranged differently . The activation of rhodopsin involves rapid cis-trans photoisomerization of the chromophore, followed by slower and incompletely defined structural rearrangements . For rhodopsin and related receptors, a common mechanism is predicted for the formation of an active state intermediate that is capable of interacting with G proteins.

Curr Opin Struct Biol, 2001 Aug, 11(4), 408 - 14
Potassium channels: life in the post-structural world; Minor DL Jr; More than three years have passed since the first structure of a potassium channel protein revealed fundamental molecular details of a platform for ion-selective conduction . Recent efforts have turned to understanding what this structure tells us about potassium channel structure and function in general and, most importantly, which questions remain unanswered . Successes in solving membrane protein structures are still hard won and slow . High-resolution studies of cytoplasmic channel domains and channel-associated proteins, the most tractable entry points for dissecting large, complex eukaryotic channels, are revealing a modularity of function commonly seen in many other biological systems . Studies of these domains bring into sharp focus issues of channel regulation, how these domains and associated proteins are coupled to the transmembrane domains to influence channel function, and how ion channels are integrated into cellular signaling pathways.

Drug Metab Rev, 2001 May, 33(2), 125 - 47
The pharmacokinetics of glycyrrhizic acid evaluated by physiologically based pharmacokinetic modeling; Ploeger B et al.; Glycyrrhizic acid is widely applied as a sweetener in food products and chewing tobacco . In addition, it is of clinical interest for possible treatment of chronic hepatitis C . In some highly exposed subjects, side effects such as hypertension and symptoms associated with electrolyte disturbances have been reported . To analyze the relationship between the pharmacokinetics of glycyrrhizic acid in its toxicity, the kinetics of glycyrrhizic acid and its biologically active metabolite glycyrrhetic acid were evaluated . Glycyrrhizic acid is mainly absorbed after presystemic hydrolysis as glycyrrhetic acid . Because glycyrrhetic acid is a 200-1000 times more potent inhibitor of 11-beta-hydroxysteroid dehydrogenase compared to glycyrrhizic acid, the kinetics of glycyrrhetic acid are relevant in a toxicological perspective . Once absorbed, glycyrrhetic acid is transported, mainly taken up into the liver by capacity-limited carriers, where it is metabolized into glucuronide and sulfate conjugates . These conjugates are transported efficiently into the bile . After outflow of the bile into the duodenum, the conjugates are hydrolyzed to glycyrrhetic acid by commensal bacteria; glycyrrhetic acid is subsequently reabsorbed, causing a pronounced delay in the terminal plasma clearance . Physiologically based pharmacokinetic modeling indicated that, in humans, the transit rate of gastrointestinal contents through the small and large intestines predominantly determines to what extent glycyrrhetic acid conjugates will be reabsorbed . This parameter, which can be estimated noninvasively, may serve as a useful risk estimator for glycyrrhizic-acid-induced adverse effects, because in subjects with prolonged gastrointestinal transit times, glycyrrhetic acid might accumulate after repeated intake.

Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9494 - 8 Epub 2001 Aug 07.
Formation of a selenium-substituted rhodanese by reaction with selenite and glutathione: possible role of a protein perselenide in a selenium delivery system; Ogasawara Y et al.; Selenophosphate is the active selenium-donor compound required by bacteria and mammals for the specific synthesis of Secys-tRNA, the precursor of selenocysteine in selenoenzymes . Although free selenide can be used in vitro for the synthesis of selenophosphate, the actual physiological selenium substrate has not been identified . Rhodanese (EC ) normally occurs as a persulfide of a critical cysteine residue and is believed to function as a sulfur-delivery protein . Also, it has been demonstrated that a selenium-substituted rhodanese (E-Se form) can exist in vitro . In this study, we have prepared and characterized an E-Se rhodanese . Persulfide-free bovine-liver rhodanese (E form) did not react with SeO(3)(2-) directly, but in the presence of reduced glutathione (GSH) and SeO(3)(2-) E-Se rhodanese was generated . These results indicate that the intermediates produced from the reaction of GSH with SeO(3)(2-) are required for the formation of a selenium-substituted rhodanese . E-Se rhodanese was stable in the presence of excess GSH at neutral pH at 37 degrees C . E-Se rhodanese could effectively replace the high concentrations of selenide normally used in the selenophosphate synthetase in vitro assay in which the selenium-dependent hydrolysis of ATP is measured . These results show that a selenium-bound rhodanese could be used as the selenium donor in the in vitro selenophosphate synthetase assay.

J Biol Chem, 2001 Oct 26, 276(43), 40202 - 9 Epub 2001 Aug 07.
The iota-carrageenase of Alteromonas fortis . A beta-helix fold-containing enzyme for the degradation of a highly polyanionic polysaccharide; Michel G et al.; Carrageenans are gel-forming hydrocolloids extracted from the cell walls of marine red algae . They consist of d-galactose residues bound by alternate alpha(1-->3) and beta(1-->4) linkages and substituted by one (kappa-carrageenan), two (iota-carrageenan), or three (lambda-carrageenan) sulfate-ester groups per disaccharide repeating unit . Both the kappa- and iota-carrageenan chains adopt ordered conformations leading to the formation of highly ordered aggregates of double-stranded helices . Several kappa-carrageenases and iota-carrageenases have been cloned from marine bacteria . Kappa-carrageenases belong to family 16 of the glycoside hydrolases, which essentially encompasses polysaccharidases specialized in the hydrolysis of the neutral polysaccharides such as agarose, laminarin, lichenan, and xyloglucan . In contrast, iota-carrageenases constitute a novel glycoside hydrolase structural family . We report here the crystal structure of Alteromonas fortis iota-carrageenase at 1.6 A resolution . The enzyme folds into a right-handed parallel beta-helix of 10 complete turns with two additional C-terminal domains . Glu(245), Asp(247), or Glu(310), in the cleft of the enzyme, are proposed as candidate catalytic residues . The protein contains one sodium and one chloride binding site and three calcium binding sites shown to be involved in stabilizing the enzyme structure.

Br J Anaesth, 2001 Aug, 87(2), 291 - 4
Touch contamination levels during anaesthetic procedures and their relationship to hand hygiene procedures: a clinical audit; Merry AF et al.; After different methods of hand preparation, volunteers rolled segments of sterile central venous catheter between their fingertips, and bacterial transfer was evaluated by standardized quantitative culture . The number of bacteria transferred differed between methods (P<0.001) . Comparisons were made with the control group (no preparation at all; median, third quartile and maximum count=6.5, 24, 55) . Bacterial transfer was greatly increased with wet hands (1227, 1932, 3254; P<0.001) . It was reduced with a new rapid method, based on thorough drying with a combination of 10 s using a cloth towel followed by either 10 or 20 s with a hot-air towel (0, 3, 7 and 0, 4, 30, respectively; P=0.007 and 0.004, respectively) . When asked to follow their personal routines, 10 consultant anaesthetists used a range of methods . Collectively, these were not significantly better than control (7.5, 15, 55; P=0.73), and neither was an air towel alone (2.5, 15, 80; P=0.176) nor the hospital's standard procedure (0, 1, 500; P=0.035) . If hand preparation is needed, an adequate and validated method should be used, together with thorough hand drying.

Blood, 2001 Aug 15, 98(4), 1226 - 30
Role of the liver in regulating numbers of circulating neutrophils; Shi J et al.; Neutrophils (polymorphonuclear leukocytes {PMNs}) carry potent destructive enzymes that can destroy invasive bacteria or damage normal tissue . PMNs have a half-life of only 6 hours in the blood, but the details of this homeostasis are unknown . In a rat model of endotoxemia, P-selectin was selectively up-regulated in hepatic sinusoids and veins where it was necessary for phagocytosis of PMNs by Kupffer cells in the liver, as opposed to the spleen or the lungs . Apoptotic PMNs appeared in the lungs and spleen only after inactivation of Kupffer cells by gadolinium chloride (GdCl(3)) . Blocking of Fas protein reduced the number of apoptotic cells in the liver; binding of annexin V to phosphatidylserine (PS) reduced the number of PMNs phagocytosed by Kupffer cells . The results support a clearance pathway in which apoptosis and phagocytosis are effected by Kupffer cells after P-selectin-mediated sequestration . (Blood . 2001;98:1226-1230)

J Clin Periodontol, 2001 Sep, 28(9), 886 - 90
Prevalence of Actinobacillus actinomycetemcomitans in an ethnic adult Chinese population; Tan KS et al.; AIM: The aim of this study was to determine the prevalence and the structure of the leukotoxin promoter region of Actinobacillus actinomycetemcomitans in an ethnic Chinese population . METHOD: Subgingival plaque samples were collected from 42 patients with moderate to advanced periodontitis and 50 periodontally healthy patients . A . actinomycetemcomitans was detected directly from the crude subgingival plaque by PCR using leukotoxin gene specific primers . The presence of A . actinomycetemcomitans was determined by a single 285 bp PCR amplicon . RESULTS: A . actinomycetemcomitans was found to be present in the subgingival plaque of 68 out of a total of 92 patients examined (74%) . 29 out of the 42 periodontitis patients tested were carriers of A . actinomycetemcomitans (69%) . Among the periodontally healthy patients studied, 39 out of 50 subjects possessed the bacteria (78%) . PCR analysis of the promoter region of the ltx operon revealed that none of the 42 moderate to advanced periodontitis patients examined harboured A . actinomycetemcomitans strains with the JP2-like promoter of the ltx operon, known to enhance leukotoxin expression . 2 out of the 27 advanced periodontitis patients clinically diagnosed as suffering from rapidly progressive periodontitis were found to be carriers of the mildly toxic strain of A . actinomycetemcomitans with the characteristic 652-like promoter . CONCLUSIONS: The high prevalence of A . actinomycetemcomitans, regardless of whether the subgingival samples were analysed from patients with healthy or diseased periodontium suggests that this bacterial species is part of the normal oral flora of ethnic Chinese . Our preliminary results also suggested that subjects who harboured the mildly toxic strain of A . actinomycetemcomitans were potentially susceptible to aggressive forms of periodontitis.

J Clin Periodontol, 2001 Sep, 28(9), 840 - 7
Cytokine gene expression in chronic periodontitis; Bickel M et al.; BACKGROUND: Cytokines play an important role in controlling inflammatory processes and tissue homeostasis . Periodontitis, as any other chronic inflammatory disease, results from a disarrangement of host factors, mainly cytokines and the initiating agent . Modulation of the cytokines is not only controlled by the host but also by infecting bacteria and their products . AIM: In the present study, we examined the cytokine mRNA expression profiles in six patients, each presenting sites affected with (1) severe progressive periodontitis, (2) chronic, but stable periodontal lesions, and (3) with healthy sites . Analysis using a quantitative RT-PCR included IFN-gamma, IL-1beta, IL-2, IL-4, IL-5, IL-6, and TNF-alpha . MATERIAL AND METHODS: 6 patients with chronic periodontitis were following treatment observed for a period of six years for local sites staying healthy, local sites with periodontal pathology but without signs of progression of attachment loss and sites with verified progression were biopsied . The biopsies were lyzed and analyzed for levels of cytokine mRNAs . RESULTS: Results revealed considerable variation not only between patients, but also between individual sites . Each patient's site has thus to be looked at as an independent entity . CONCLUSIONS: The local action of cytokines, which is heavily dependent on recruitment, interaction and activation of immunocompetent cells can explain the site-specific nature of cytokine expression . Cytokine data from individual sites together with the local clinical status and data from the literature demonstrate the complexity of periodontal disease pathogenesis . To gain insight to specific mechanisms further studies are needed.

J Clin Periodontol, 2001 Sep, 28(9), 820 - 7
Subgingival debridement of root surfaces with a micro-brush: macroscopic and ultrastructural assessment; Carey HM et al.; AIM: The aim of this study was to assess the use of a micro-brush to remove plaque deposits from subgingival, periodontally involved root surfaces in vivo . METHODS: 30 periodontally involved teeth requiring extraction for periodontal or prosthetic reasons in 26 adult patients were utilised . For inclusion, teeth had to display at least 30% bone loss radiographically . Following the establishment of local anaesthesia, grooves were cut on the proximal root surface adjacent to the gingival margin at the line angles . For each tooth, 1 proximal root surface was rubbed with the micro-brush for 2 min to the depth of the pocket whilst the other root surface acted as an undebrided control . The teeth were then extracted, rinsed in 0.85% NaCl, stained with 2% erythrosine solution and photographed . The amount of erythrosine staining on each subgingival, periodontally involved root surface was assessed by tracing the areas of stain on a colour photograph and scanning the tracings into a computerised image tracing program . RESULTS: Results were expressed as the % of the periodontally involved root-surface area that exhibited staining . Stained areas were further examined with the scanning electron microscope (SEM) . The undebrided root surfaces each displayed 100% staining . The debrided surfaces (with probing pocket depths of 4-10 mm) displayed mean staining of 16.1% (SD +/-7.1%) of the proximal surface area . SEM assessment showed that undebrided root surfaces were covered with thick deposits of bacteria . On debrided surfaces, stain-free areas were free of plaque whilst areas of faint staining exhibited either no plaque, calculus deposits or scanty, isolated islands of bacteria . Bacteria had been partially removed from the surface of calculus in some areas . CONCLUSIONS: The findings indicate that subgingival debridement with a micro-brush is effective in removing plaque deposits from periodontally involved root surfaces.

Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1267 - 76
Description of Microbacterium foliorum sp . nov . and Microbacterium phyllosphaerae sp . nov., isolated from the phyllosphere of grasses and the surface litter after mulching the sward, and reclassification of Aureobacterium resistens (Funke et al . 1998) as Microbacterium resistens comb . nov.; Behrendt U et al.; The taxonomic position of a group of coryneform bacteria isolated from the phyllosphere of grasses and the surface litter after sward mulching was investigated . On the basis of restriction analyses of 16S rDNA, the isolates were divided into two genotypes . According to the 16S rDNA sequence analysis, representatives of both genotypes were related at a level of 99.2% similarity and clustered within the genus Microbacterium . Chemotaxonomic features (major menaquinones MK-12, MK-11 and MK-10; predominating iso- and anteiso-branched cellular fatty acids; G+C content 64-67 mol%; peptidoglycan-type B2beta with glycolyl residues) corresponded to this genus as well . DNA-DNA hybridization studies showed a reassociation value of less than 70% between representative strains of both subgroups, suggesting that two different species are represented . Although the extensive morphological and physiological analyses did not reveal any differentiating feature for the genotypes, differences in the presence of the cell-wall sugar mannose enabled the subgroups to be distinguished from one another . DNA-DNA hybridization with type strains of closely related Microbacterium spp . indicated that the isolates represent two individual species, which can also be differentiated from previously described species of Microbacterium on the basis of biochemical features . As a result of phenotypic and phylogenetic analyses, the species Microbacterium foliorum sp . nov., type strain P 333/02T (= DSM 12966T = LMG 19580T), and Microbacterium phyllosphaerae sp . nov., type strain P 369/06T (= DSM 13468T = LMG 19581T), are proposed . Furthermore, the reclassification of Aureobacterium resistens (Funke et al . 1998) as Microbacterium resistens (Funke et al . 1998) comb . nov . is proposed.

J Mol Biol, 2001 May 25, 309(1), 121 - 38
The RegB/RegA two-component regulatory system controls synthesis of photosynthesis and respiratory electron transfer components in Rhodobacter capsulatus; Swem LR et al.; Recently, we demonstrated that the RegB/RegA two-component regulatory system from Rhodobacter capsulatus functions as a global regulator of metabolic processes that either generate or consume reducing equivalents . For example, the RegB/RegA system controls expression of such energy generating processes as photosynthesis and hydrogen utilization . In addition, RegB/RegA also control nitrogen and carbon fixation pathways that utilize reducing equivalents . Here, we use a combination of DNase I protection and plasmid-based reporter expression studies to demonstrate that RegA directly controls synthesis of cytochrome cbb3 and ubiquinol oxidases that function as terminal electron acceptors in a branched respiratory chain . We also demonstrate that RegA controls expression of cytochromes c2, c(y) and the cytochrome bc1 complex that are involved in both photosynthetic and respiratory electron transfer events . These data provide evidence that the RegB/RegA two-component system has a major role in controlling the synthesis of numerous processes that affect reducing equivalents in Rhodobacter capsulatus.

Eur Respir J, 2001 Jun, 17(6), 1322 - 7
A small outbreak of Legionnaires' disease in a cargo ship under repair; Cayla JA et al.; It was reported that two mechanics working on a cargo ship under repair in the port of Barcelona had died after having fever . An investigation was made into the possibility of any additional cases and the presence of Legionella pneumophila in the ship they were repairing and in their hotel . The contaminated water system was treated with sodium hypochlorite . Both patients died after having been repeatedly diagnosed as having influenza . The two cases occurred among those who had been working with the pump of the ship's water system, while no cases were observed among the other workers (p = 0.02) . Various serogroups of L . pneumophila were isolated from the ship's water pump and distribution system . However, organism of serogroup 1, subgroup Pontiac (Knoxville) were identified with identical deoxyribonucleic acid (DNA) patterns in the lung tissue of one patient and in the cooling water circuit valve of the ship's water pump . The first postintervention control water samples showed no further growth of legionella, but serogroups 4 and 8 were identified 8 months later . This legionellosis outbreak, although small, was highly lethal, probably due to the high levels of bacteria to which the patients were exposed and also because of the failure of correct diagnosis . International recommendations on prevention and control of legionellosis, which include ships under repair, are required.

J Cell Biol, 2001 Aug 6, 154(3), 549 - 71
A protein interaction map for cell polarity development; Drees BL et al.; Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy . Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes . To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment . 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions . 191 protein-protein interactions were detected, of which 128 had not been described previously . 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development . Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization . Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis . Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery . In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

J Bacteriol, 2001 Sep, 183(17), 5209 - 12
Dehalogenation of dichloromethane by dichloromethane dehalogenase/glutathione S-transferase leads to formation of DNA adducts; Kayser MF et al.; Formation of DNA adducts following conversion of dichloromethane by bacterial dichloromethane dehalogenase/glutathione S-transferase was demonstrated . Adducts included dichloromethane carbon and glutathione sulfur atoms . A reaction with DNA occurred preferentially at guanine bases . Increased DNA degradation in a polA mutant of Methylobacterium dichloromethanicum DM4 grown with dichloromethane confirmed the genotoxicity associated with dichloromethane degradation, suggesting an important role of DNA repair in the metabolism of halogenated, DNA-alkylating compounds by bacteria.

J Bacteriol, 2001 Sep, 183(17), 5092 - 101
Differential regulation of ftsZ transcription during septation of Streptomyces griseus; Kwak J et al.; Streptomyces has been known to form two types of septa . The data in this research demonstrated that Streptomyces griseus forms another type of septum near the base of sporogenic hyphae (basal septum) . To understand the regulation of the septation machinery in S . griseus, we investigated the expression of the ftsZ gene . S1 nuclease protection assays revealed that four ftsZ transcripts were differentially expressed during morphological differentiation . The vegetative transcript (emanating from P(veg)) is present at a moderate level during vegetative growth, but is switched off within the first 2 h of sporulation . Two sporulation-specific transcripts predominantly accumulated, and the levels increased by approximately fivefold together shortly before sporulation septa begin to form . Consistently, the sporulation-specific transcripts were expressed much earlier and more abundantly in a group of nonsporulating mutants that form their sporulation septa prematurely . Promoter-probe studies with two different reporter systems confirmed the activities of the putative promoters identified from the 5' end point of the transcripts . The levels and expression timing of promoter activities were consistent with the results of nuclease protection assays . The aseptate phenotype of the P(spo) mutant indicated that the increased transcription from P(spo) is required for sporulation septation, but not for vegetative or basal septum formation.

J Bacteriol, 2001 Sep, 183(17), 5001 - 7
Proteolysis of the Caulobacter McpA chemoreceptor is cell cycle regulated by a ClpX-dependent pathway; Tsai JW et al.; Proteolysis is involved in cell differentiation and the progression through the cell cycle in Caulobacter crescentus . We have constitutively expressed the transmembrane chemoreceptor McpA from a multicopy plasmid to demonstrate that McpA degradation is modulated during the cell cycle . The level of McpA protein starts to decrease only when the swarmer cells differentiate into stalked cells . The reduction in McpA protein levels is maintained until the stalked cells develop into predivisional cells, at which point the level returns to that observed in swarmer cells . The cell-cycle-regulated degradation of McpA does not require the last 12 C-terminal amino acids, but it does require three amino acids (AAL) located 15 residues away from the C terminus . The ClpXP protease is essential in C . crescentus for viability, and thus, we tested McpA degradation in xylose conditional mutants . The effect on McpA degradation occurred within two generations from the start of ClpX depletion . The conditional mutants' growth rate was only slightly affected, suggesting that ClpX is directly involved in McpA proteolysis.

J Control Release, 2001 Jul 6, 74(1-3), 357 - 62
Phage derived peptides for targeting of doxorubicin conjugates to solid tumours; Schatzlein AG et al.; Barriers are frequently hampering targeting of drugs and toxins to solid tumours and their microenvironment . Nano-conjugates are low molecular weight conjugates of a small drug or toxin and a targeting ligand coupled through a cleavable linker group . They offer potential advantages for tumour specific delivery in diffusion-limited situations . We have exploited fd phage-derived peptides for the targeting of low molecular weight drug conjugates to solid tumours . As a model we have chosen doxorubicin conjugates targeted to the transferrin receptor (TfR) . A library of phage expressing a cyclic nona-peptide was panned against TfR . The apparent affinity of phages determined by surface plasmon resonance (SPR) increased with each cycle of the panning procedure . After five rounds approximately 80% of phages expressed the same peptide, which mediated a 30-50-fold increased receptor specific cellular uptake of the phages . The corresponding peptide was synthesised using solid phase peptide chemistry on a sulfonamide based safety catch resin . Crude mixtures of the peptide, as well as transferrin itself, were able to inhibit the phage uptake significantly . The doxorubicin conjugate of the peptide containing a cleavable linker was prepared and endosomal uptake confirmed by fluorescence microscopy.

Microbes Infect, 2001 Jul, 3(9), 729 - 38
Classification of Brucella spp . isolated from marine mammals by DNA polymorphism at the omp2 locus; Cloeckaert A et al.; A number of recent reports have described the isolation and characterization of Brucella strains from a wide variety of marine mammals such as seals, porpoises, dolphins and a minke whale . These strains were identified as brucellae by conventional typing tests . However, their overall characteristics were not assimilable to those of any of the six currently recognized Brucella species and it was suggested that they comprise a new nomen species to be called Brucella maris . In the present study we analysed DNA polymorphism at the omp2 locus of 33 marine mammal Brucella strains isolated from seals, dolphins, porpoises and an otter . The omp2 locus contains two gene copies (named omp2a and omp2b) coding for porin proteins and has been found particularly useful for molecular typing and identification of Brucella at the species, biovar, or strain level . PCR-restriction fragment length polymorphism (RFLP) and DNA sequencing showed that strains isolated from dolphins and porpoises carry two omp2b gene copies instead of one omp2a and one omp2b gene copy or two similar omp2a gene copies reported in the currently recognized species . This observation was also recently made for a minke whale Brucella isolate . The otter and all seal isolates except one were shown to carry one omp2a and one omp2b gene copy as encountered in isolates from terrestrial mammals . By PCR-RFLP of the omp2b gene, a specific marker was detected grouping the marine mammal Brucella isolates . Although marine mammal Brucella isolates may represent a separate group from terrestrial mammal isolates based on omp2b sequence constructed phylogenetic trees, the divergence found between their omp2b and also between their omp2a nucleotide sequences indicates that they form a more heterogeneous group than isolates from terrestrial mammals . Therefore, grouping the marine mammal Brucella isolates into one species Brucella maris seems inappropriate unless the currently recognized Brucella species are grouped . With respect to the current classification of brucellae according to the preferential host, brucellae isolated from such diverse marine mammal species as seals and dolphins could actually comprise more than one species, and at least two new species, B . pinnipediae and B . cetaceae, could be compatible with the classical criteria of host preferentialism and DNA polymorphism at their omp2 locus.

Mol Microbiol, 2001 Jul, 41(2), 423 - 37
The Mycobacterium tuberculosis ECF sigma factor sigmaE: role in global gene expression and survival in macrophages; Manganelli R et al.; In previously published work, we identified three Mycobacterium tuberculosis sigma (sigma) factor genes responding to heat shock (sigB, sigE and sigH) . Two of them (sigB and sigE) also responded to SDS exposure . As these responses to stress suggested that the sigma factors encoded by these genes could be involved in pathogenicity, we are studying their role in physiology and virulence . In this work, we characterize a sigE mutant of M . tuberculosis H37Rv . The sigE mutant strain was more sensitive than the wild-type strain to heat shock, SDS and various oxidative stresses . It was also defective in the ability to grow inside both human and murine unactivated macrophages and was more sensitive than the wild-type strain to the killing activity of activated murine macrophages . Using microarray technology and quantitative reverse transcription-polymerase chain reaction (RT-PCR), we started to define the sigmaE regulon of M . tuberculosis and its involvement in the global regulation of the stress induced by SDS . We showed the requirement for a functional sigE gene for full expression of sigB and for its induction after SDS exposure but not after heat shock . We also identified several genes that are no longer induced when sigmaE is absent . These genes encode proteins belonging to different classes including transcriptional regulators, enzymes involved in fatty acid degradation and classical heat shock proteins.

Heredity, 2001 Mar, 86(Pt 3), 325 - 32
Wolbachia endosymbiont responsible for cytoplasmic incompatibility in a terrestrial crustacean: effects in natural and foreign hosts; Moret Y et al.; Wolbachia bacteria are vertically transmitted endosymbionts that disturb the reproduction of many arthropods thereby enhancing their spread in host populations . Wolbachia are often responsible for changes of sex ratios in terrestrial isopods, a result of the feminization of genotypic males . Here we found that the Wolbachia hosted by Cylisticus convexus (wCc) caused unidirectional cytoplasmic incompatibility (CI), an effect commonly found in insects . To understand the diversity of Wolbachia-induced effects in isopods, wCc were experimentally transferred in a novel isopod host, Armadillidium vulgare . wCc conserved the ability to induce CI . However, Wolbachia were not transmitted to the eggs, so the capacity to restore the compatibility in crosses involving two transinfected individuals was lost . The feminizing Wolbachia hosted by A . vulgare was unable to rescue CI induced by wCc . These results showed that Wolbachia in isopods did not evolved broadly to induce feminization, and that CI and the feminizing effect are probably due to different mechanisms . In addition, wCc reduces the mating capacity of infected C . convexus males, suggesting that the bacteria might alter reproductive behaviour . The maintenance of wCc in host populations is discussed.

Eur Respir J, 2001 May, 17(5), 1049 - 51
Interleukin-12 as successful adjuvant in tuberculosis treatment; Greinert U et al.; Interleukin-12 (IL-12) proved to be an effective and successful adjuvant to a standard antituberculotic medication in a patient suffering from progressive clinical tuberculosis (TB) . IL-12 is a potent enhancer of interferon-gamma production which is necessary for killing intracellular bacteria like mycobacteria . This patient's TB was progressive, although sensitivity to first-line antituberculotics was proven and medication was given as directly observed therapy over more than 8 months . The 3-month adjuvant therapy with IL-12 significantly and convincingly improved results . It is believed that this case, the first in the literature to describe adjuvant interleukin-12 therapy in tuberculosis, strongly encourages the study of adjuvant interleukin-12 therapy on a more systematic basis.

Front Biosci, 2001 Aug 01, 6, D927 - 35
Features of the two gene pairs RD-SKI2W and DOM3Z-RP1 located between complement component genes factor B and C4 at the MHC class III region; Yang Z et al.; Located at the 30 kb genomic region between complement factor B and component C4 are four ubiquitously expressed genes RD, SKI2W, DOM3Z and RP1 . Besides RP1, the protein products of the other three genes each has highly conserved homologues or related proteins in lower eukaryotes, contains leucine zipper motifs for protein interaction, and plays important roles related to RNA metabolism . RD is a subunit of the negative transcription elongation factor, critical for the regulation of gene expression . It has an RNA recognition motif and 24 copies of Arg-Asp (RD) repeats . Ski2w is a nucleolar and cytoplasmic protein that has a putative RNA helicase domain . Fusion proteins of human Ski2w expressed in insect cells and bacteria have ATPase activity . The cytoplasmic protein of human Ski2w is associated with the polysomes and probably the 40S subunit of ribosomes . Ski2w is probably involved in the regulation of translation and RNA turnover . Dom3z is a nuclear protein whose yeast homologue forms a complex with an exoribonuclease . RP1 (or STK19) is a Ser/Thr nuclear protein kinase . No homologues of RP1 in lower eukaryotes have been discovered . Six polymorphic residues are present in human Ski2w and two in Dom3z . The potential roles of Ski2w and Dom3z on the clearance of degraded nuclear and cytoplasmic RNA raised their possibilities as susceptibility genes of systemic lupus erythematosus that is a disease with flawed processes in the removal of apoptotic materials.

J Immunoassay Immunochem, 2001, 22(2), 99 - 112
A specific and ultrasensitive chemiluminescent sandwich ELISA test for the detection and quantitation of pneumolysin; Cima-Cabal MD et al.; A chemiluminescent sandwich ELISA test has been developed for the detection and quantitation of pneumolysin . The test is based on a mouse monoclonal as the capture antibody and on rabbit polyclonal IgGs as detection antibodies, in combination with an anti-rabbit IgG alkaline phosphatase conjugate . The estimated detection limit of the purified recombinant toxin in phosphate-buffered saline with 0.05% Triton X-100 is around 5 pg ml(-1), with averaged intra- and inter-assay variation coefficients of 7% and 13.5%, respectively . The assay has been applied to the quantitation of pneumolysin in pneumococcal isolates, providing, for the first time, a direct measurement of the amount of the toxin produced by different strains, a variation has been found in their pneumolysin content . The test is highly specific as no other purified toxins or human pneumonia- or meningitis-associated bacteria yielded false-positive results . This specific and highly sensitive method could help in the diagnosis of human infections.

Clin Infect Dis, 2001 Sep 1, 33(5), 629 - 40 Epub 2001 Aug 06.
Infectious complications among 620 consecutive heart transplant patients at Stanford University Medical Center; Montoya JG et al.; A total of 1073 infectious episodes (IEs) that occurred in 620 consecutive heart transplantation patients at Stanford Medical Center between 16 December 1980 and 30 June 1996 were reviewed . Infectious complications were a major cause of morbidity and mortality, second only to rejection as the cause of early deaths and the most common cause of late deaths . Of the IEs, 468 (43.6%) were caused by bacteria, 447 (41.7%) by viruses, 109 (10.2%) by fungi, 43 (4.0%) by Pneumocystis carinii, and 6 (0.6%) by protozoa . The largest number of IEs occurred in the lungs (301 {28.1%}) . A significant reduction in the incidence of IEs and a delay in presentation after transplantation were observed; these were most likely related to the introduction of new chemoprophylactic regimens during the study period and prevention of significant disease caused by cytomegalovirus.

Hum Pathol, 2001 Jul, 32(7), 750 - 2
Leptospirosis mimicking acute cholecystitis among athletes participating in a triathlon; Guarner J et al.; Leptospirosis, a disease acquired by exposure to contaminated water, is characterized by fever accompanied by various symptoms, including abdominal pain . An acute febrile illness occurred in athletes who participated in an Illinois triathlon in which the swimming event took place in a freshwater lake . Of 876 athletes, 120 sought medical care and 22 were hospitalized . Two of the athletes had their gallbladders removed because of abdominal pain and clinical suspicion of acute cholecystitis . We applied an immunohistochemical test for leptospirosis to these gallbladders and demonstrated bacterial antigens staining (granular and filamentous patterns) around blood vessels of the serosa and muscle layer . Rare intact bacteria were seen in 1 case . These results show that leptospirosis can mimic the clinical symptoms of acute cholecystitis . If a cholecystectomy is performed in febrile patients with suspicious environmental or animal exposure, pathologic studies for leptospirosis on formalin-fixed, paraffin-embedded tissues may be of great value.

Science, 2001 Aug 3, 293(5531), 839 - 43
Biogenic methane, hydrogen escape, and the irreversible oxidation of early Earth; Catling DC et al.; The low O2 content of the Archean atmosphere implies that methane should have been present at levels approximately 10(2) to 10(3) parts per million volume (ppmv) (compared with 1.7 ppmv today) given a plausible biogenic source . CH4 is favored as the greenhouse gas that countered the lower luminosity of the early Sun . But abundant CH4 implies that hydrogen escapes to space (upward arrow space) orders of magnitude faster than today . Such reductant loss oxidizes the Earth . Photosynthesis splits water into O2 and H, and methanogenesis transfers the H into CH4 . Hydrogen escape after CH4 photolysis, therefore, causes a net gain of oxygen {CO2 + 2H2O --> CH4 + 2O2 --> CO2 + O2 + 4H(upward arrow space)} . Expected irreversible oxidation (approximately 10(12) to 10(13) moles oxygen per year) may help explain how Earth's surface environment became irreversibly oxidized.

Mol Cell Biol, 2001 Sep, 21(17), 6044 - 55
Intact lysosome transport and phagosome function despite kinectin deficiency; Plitz T et al.; The mechanism of cargo coupling to kinesin motor proteins is a fundamental issue in organelle transport along microtubules . Kinectin has been postulated to function as a membrane anchor protein that attaches various organelles to the prototype motor protein kinesin . To verify the biological relevance of kinectin in vivo, the murine kinectin gene was disrupted by homologous recombination . Unexpectedly, kinectin-deficient mice were viable and fertile, and no gross abnormalities were observed up to 1 year of age . The assembly of the endoplasmic reticulum was essentially unaffected in kinectin-deficient cells . Mitochondria appeared to be correctly distributed throughout the cytoplasm along the microtubules . Furthermore, the stationary distribution and the bidirectional movement of lysosomes did not depend on kinectin . Kinectin-deficient phagocytes internalized and cleared bacteria, indicating that phagosome trafficking and maturation are functional without kinectin . Thus, these data unequivocally indicate that kinectin is not essential for trafficking of lysosomes, phagosomes, and mitochondria in vivo.

J Photochem Photobiol B, 2001 Aug 15, 61(1-2), 35 - 45
Photodegradation of natural organic matter exposed to fluctuating levels of solar radiation; Zagarese HE et al.; Irradiation of natural water samples with natural or artificial UVR typically results in a progressive loss of color and decreased absorbance; a process often referred to as photobleaching . In a typical photobleaching experiment, samples are exposed to a relatively constant level of artificial or natural UVR . However, under most natural situations, the vertical mixing of the water within the upper mixed layer results in strong and periodic fluctuations in UV irradiance . In this paper, we present the results of an experiment in which natural lake water was exposed to solar radiation in quartz tubes that were incubated either at fixed depths or rotating within the water column . We found differences between rotating and fixed samples in (i) photobleaching, (ii) nutrient release, and (iii) subsequent use by algae and bacteria . The evidence presented in this study demonstrated that photochemical processes might be affected by vertical water motion . The reasons for such differences remain largely unknown . Although we offer a potential explanation for such differences, our proposed mechanism is based on a post-hoc analysis of the data and should be taken solely as a working hypothesis for future research.

Biotechnol Prog, 2001 Jul-Aug, 17(4), 752 - 9
Hyperaccumulation of nickel by hairy roots of alyssum species: comparison with whole regenerated plants; Nedelkoska TV et al.; Hairy roots were used to investigate nickel uptake by the hyperaccumulator species, Alyssum bertolonii, A . tenium, and A . troodii . The Ni biosorption capacity of A . tenium hairy roots was lower than for other types of biomass such as bacteria and algae; in short-term (9-h) equilibrium studies, the highest Ni content measured in the roots was 17 500 microg g(-1) dry weight at a liquid concentration of about 4000 ppm . Using long-term hairy root cultures, it was demonstrated that Ni tolerance and hyperaccumulation do not necessarily depend on the presence of shoots or root-shoot translocation . A . bertolonii hairy roots remained healthy in appearance and continued to grow in the presence of 20-100 ppm Ni, accumulating up to 7200 microg g(-1) dry weight Ni . In contrast, hairy roots of Nicotiana tabacum turned dark brown at 20 ppm Ni and growth was negligible . The ability to grow at high external Ni concentrations allowed hyperaccumulator hairy roots to remove much greater amounts of heavy metals from the culture liquid than nonhyperaccumulator hairy roots, even though biomass Ni concentrations were similar . Although hairy roots proved to be a useful tool for investigating Ni hyperaccumulation, there were significant differences in the Ni uptake capacity of hairy roots and whole plants . Regenerated plants of A . tenium were much more tolerant of Ni and capable of accumulating higher Ni concentrations than hairy roots of this species.

Southeast Asian J Trop Med Public Health, 2001 Mar, 32(1), 171 - 6
Cutaneous manifestations in HIV positive patients; Supanaranond W et al.; Cutaneous manifestations are common clinical findings among HIV positive patients . The causes may be bacteria, viruses, fungi and other non-infectious agents . This study was conducted at the Pramongkutklao Hospital skin clinic to determine the frequency distribution of cutaneous manifestations in HIV positive patients . A total of 147 patients with HIV seropositivity were recruited and divided into a retrospective group and a prospective study group . For the retrospective study, hospital records of 129 patients who attended from January 1995 to November 1998 were recruited . The prospective study was carried out from November 1998 to January 1999 and 18 patients were recruited . Cutaneous finding among patients in the two studies were evaluated . There were ten common cutaneous manifestations observed in the retrospective and prospective study including pruritic papular eruptions (PPE) (51.2%, 50%), oral candidiasis (16.7%, 21.7%), herpes zoster (10.9%, 5.6%), oral hairy leukoplakia (10%, 5.6%), unclassified eczema (9%, 11.1%), urticaria (5.6%, 3.1%), seborrheic dermatitis (4.7%, 16.7%), folliculitis (4.7%, 5.6%), prurigo simplex (4.7%, 5.6%), and Steven-Johnson syndrome (3.9%, 0%) . However, the distribution of cutaneous manifestations in the two studies were not significantly different . These findings may be useful as baseline data for common cutaneous manifestations in HIV positive patients.

Integr Physiol Behav Sci, 2001 Jan-Mar, 36(1), 75 - 83
Is there a role for psychology in ulcer disease?
Murison R.
The discovery of the importance of bacterial factors in the etiology of ulcer disease has led to a neglect of psychological factors . However, both earlier theoretical and empirical approaches implicating these factors are supported by more recent studies, both epidemiological and experimental . While not ignoring the unquestioned role of Helicobacter, it is important for future research to recognize the multi-factorial nature of ulcer disease by which several factors, including stress, bacteria and non-steroid antiinflammatory drugs, may interact to drive a pre-pathology (erosions or ulcerations) to a pathological state (ulcer) . Calls for general eradication programs should be cautioned in the light of possible unwanted side effects.

J Virol, 2001 Sep, 75(17), 8187 - 94
Expression of immunoregulatory cytokines by recombinant coxsackievirus B3 variants confers protection against virus-caused myocarditis; Henke A et al.; Clinical and laboratory investigations have demonstrated the involvement of viruses and bacteria as potential causative agents in cardiovascular disease and have specifically found coxsackievirus B3 (CVB3) to be a leading cause . Experimental data indicate that cytokines are involved in controlling CVB3 replication . Therefore, recombinant CVB3 (CVB3rec) variants expressing the T-helper-1 (T(H)1)-specific gamma interferon (IFN-gamma) or the T(H)2-specific interleukin-10 (IL-10) as well as the control virus CVB3(muIL-10), which produce only biologically inactive IL-10, were established . Coding regions of murine cytokines were cloned into the 5' end of the CVB3 wild type (CVB3wt) open reading frame and were supplied with an artificial viral 3Cpro-specific Q-G cleavage site . Correct processing releases active cytokines, and the concentration of IFN-gamma and IL-10 was analyzed by enzyme-linked immunosorbent assay and bioassays . In mice, CVB3wt was detectable in pancreas and heart tissue, causing massive destruction of the exocrine pancreas as well as myocardial inflammation and heart cell lysis . Most of the CVB3wt-infected mice revealed virus-associated symptoms, and some died within 28 days postinfection . In contrast, CVB3rec variants were present only in the pancreas of infected mice, causing local inflammation with subsequent healing . Four weeks after the first infection, surviving mice were challenged with the lethal CVB3H3 variant, causing casualties in the CVB3wt- and CVB3(muIL-10)-infected groups, whereas almost none of the CVB3(IFN-gamma)- and CVB3(IL-10)-infected mice died and no pathological disorders were detectable . This study demonstrates that expression of immunoregulatory cytokines during CVB3 replication simultaneously protects mice against a lethal disease and prevents virus-caused tissue destruction.

Vaccine, 2001 Aug 14, 19(31), 4465 - 72
Role of antibody to lipopolysaccharide in protection against low- and high-virulence strains of Francisella tularensis; Fulop M et al.; Mice immunised with lipopolysaccharide (LPS) from Francisella tularensis were protected against challenge with the live vaccine strain (LVS) . However, when similarly immunised mice were challenged using the fully virulent F . tularensis strain Schu4, only an increase in the time to death was observed . Passive transfer of serum from LPS-immunised mice to naive mice afforded protection against F . tularensis LVS . LPS-immunised mice depleted of either CD4+ or CD8+ T-cells survived a F . tularensis LVS challenge although the rate of clearance of bacteria from the spleen was significantly reduced in the CD8+ depleted group . LPS-immunised mice boosted with F . tularensis LVS were re-challenged with F . tularensis Schu4 . This cohort was significantly protected (LD(50) increased from <1 to >1000 CFU) . However, passive transfer of serum did not confer protection and mice depleted of CD4+ or CD8+ T-cells did not survive.

Environ Technol, 2001 Jun, 22(6), 661 - 72
Biological sulfide oxidation in a fluidized bed reactor; Annachhatre AP et al.; Feasibility of a laboratory scale fluidized bed process for biological sulfide oxidation to elemental sulfur and the formation of well-settleable sulfur sludge is demonstrated . Sulfide oxidation strongly depends upon oxygen concentration, sulfide loading rate and upflow velocity . At reactor dissolved oxygen concentrations (DOr) higher than 0.1 mg l(-1), sulfate was the main product of sulfide oxidation Upon increasing the sulfide loading rate, the sulfate production rate decreased as sulfide oxidation to sulfur showed marked increase . Low formation of sulfate could mean that sulfide was inhibitory to sulfate producing bacteria or that conversion of sulfide to sulfur was more favorable than sulfate production . Sulfide conversions higher than 90% were obtained at sulfide loading rates of 0.13-1.6 kgS mr(-3) d(-1) . At DOr less than 0.1 mg l(-1), sulfur was the major end product of the sulfide oxidation . Upflow velocity in the range of 16-26 m h(-1) and sulfide loading rate of 0.9-1.6 kgS mr(-3) d(-1) were necessary for generation of biogranules containing 65-76% of elemental sulfur . The elemental sulfur production of 76% was obtained at upflow velocity of 17 m h(-1) with sulfide loading rate up to 1.6 kgS mr(3)d(-1) . Morphological examination of the biogranules showed elemental sulfur deposition in the sludge granule and outside the bacterial cells.

Aust Endod J, 2001 Apr, 27(1), 12 - 21
Biocompatibility of root canal filling materials; Geurtsen W; Results of in vitro and in vivo studies clearly indicate that some endodontic sealers may cause local and systemic adverse effects . Though occasionally contradictory data has been reported from various authors, it may be concluded that zinc-oxide-eugenol sealers possess a marked cytotoxic and tissue-irritating potency . Most Ca(OH)2-based materials, however, were biocompatible . Genotoxic effects have been observed with sealers releasing paraformaldehyde or containing mutagenic substances, such as bisphenol-A-diglycidyl-ether or its derivatives . It cannot be excluded that these materials may pose a systemic risk because formaldehyde is rapidly distributed systemically following its application into the pulp cavity . Furthermore an increasing number of cases with an aspergillosis of the maxillary sinus have been observed which were mainly caused by zinc-releasing endodontic sealers . Overall, it is recommended that for endodontic practice, sealers that have been found to be biocompatible in a "mixed bag" of various in vitro and in vivo tests, be selected . From this point of view, ZnOE-sealers should no longer be used for root canal fillings . This recommendation applies also to sealers containing paraformaldehyde or generating this substance during their setting reaction . More experimental and clinical studies are necessary to elucidate whether new materials, such as mineral trioxide aggregate (MTA) or calcium phosphate cement, will be biocompatible alternatives in the future.

Naturwissenschaften, 2001 Apr, 88(4), 137 - 46
Climate change and temperature-dependent biogeography: oxygen limitation of thermal tolerance in animals; Portner HO; Recent years have shown a rise in mean global temperatures and a shift in the geographical distribution of ectothermic animals . For a cause and effect analysis the present paper discusses those physiological processes limiting thermal tolerance . The lower heat tolerance in metazoa compared with unicellular eukaryotes and bacteria suggests that a complex systemic rather than molecular process is limiting in metazoa . Whole-animal aerobic scope appears as the first process limited at low and high temperatures, linked to the progressively insufficient capacity of circulation and ventilation . Oxygen levels in body fluids may decrease, reflecting excessive oxygen demand at high temperatures or insufficient aerobic capacity of mitochondria at low temperatures . Aerobic scope falls at temperatures beyond the thermal optimum and vanishes at low or high critical temperatures when transition to an anaerobic mitochondrial metabolism occurs . The adjustment of mitochondrial densities on top of parallel molecular or membrane adjustments appears crucial for maintaining aerobic scope and for shifting thermal tolerance . In conclusion, the capacity of oxygen delivery matches full aerobic scope only within the thermal optimum . At temperatures outside this range, only time-limited survival is supported by residual aerobic scope, then anaerobic metabolism and finally molecular protection by heat shock proteins and antioxidative defence . In a cause and effect hierarchy, the progressive increase in oxygen limitation at extreme temperatures may even enhance oxidative and denaturation stress . As a corollary, capacity limitations at a complex level of organisation, the oxygen delivery system, define thermal tolerance limits before molecular functions become disturbed.

Leukemia, 2001 Aug, 15(8), 1248 - 55
The multi-organ origin of interleukin-5 in the mouse; Ryan PJ et al.; Murine Ba/F3 cells were transfected with cDNA for the alpha-chain of the murine interleukin-5 (IL-5) receptor and cloned lines of these cells were able to proliferate in response to as little as 2.5 pg/ml of IL-5 . The bioassay was demonstrated to be specific for IL-5 and was able to measure IL-5 produced in culture by organs from adult C57BL/6 and BALB/c mice . The highest levels of IL-5 were produced by lung tissue but thymus and bladder consistently produced IL-5 and more variable production was observed by the heart, spleen, muscle, bone shaft, uterus and testes . Bone marrow cells produced no detectable IL-5 . Observed levels of production of IL-5 were similar when using organs from mice lacking high-affinity receptors for IL-5 and from nu/nu, RAG-1-/- and NOD/SCID mice lacking T lymphocytes . In inflammatory peritoneal exudates induced by the injection of casein plus bacteria, levels of induced IL-5 were higher if the mice lacked high-affinity receptors for IL-5 . The data indicate that T lymphocytes are not the dominant cellular source of IL-5 in organ-conditioned media and that local IL-5 production can occur with a wide range of normal murine organs.

J Pediatr Surg, 2001 Aug, 36(8), 1122 - 9
Intestinal cytokine gene expression in infants with acute necrotizing enterocolitis: interleukin-11 mRNA expression inversely correlates with extent of disease; Nadler EP et al.; BACKGROUND/PURPOSE: The authors have shown previously that surgical specimens from infants with acute necrotizing enterocolitis (NEC) show upregulation of inducible nitric oxide (NO) synthase (iNOS) and interferon-gamma mRNA . However, the contribution of other inflammatory cytokines such as interleukin-8 (IL-8), IL-11, and IL-12 has not been defined . Likewise, the role of GTP-cyclohydrolase, the rate-limiting enzyme in tetrahydrobiopterin synthesis, and thus NO production by iNOS is unclear . In this study, the authors sought to further define the pattern of cytokine expression seen in infants with acute NEC . METHODS: The authors measured intestinal cytokine mRNA expression by semiquantitative reverse transcriptase polymerase chain reaction in 21 infants with histologically confirmed NEC, 18 with other inflammatory conditions, and in 9 patients without intestinal inflammation . Guanosine triphosphate-cyclohydrolase (GTP-CH) activity was measured by specific enzyme assay . Univariate exact logistic regression analysis was performed to identify predictors of outcome . RESULTS: IL-8 and IL-11 mRNA were upregulated in patients with acute NEC compared with those with other inflammatory conditions or those without disease; these levels returned to baseline at the time of stoma closure . Increased IL-11 mRNA decreased the likelihood of pan-necrosis (odds ratio, 0.93; P =.002) . Increased IL-12 levels (but not IL-8) seemed to protect against pan-necrosis (odds ratio, 0.70; P =.06) . CONCLUSIONS: Local upregulation of IL-11 may represent an adaptive response designed to limit the extent of intestinal damage in NEC . Decreased IL-12 levels may contribute to the pathogenesis of NEC by allowing bacteria to escape host defenses .

Arch Microbiol, 2001 Jul, 176(1-2), 62 - 8
Characterisation of the mob locus of Rhodobacter sphaeroides WS8: mobA is the only gene required for molybdopterin guanine dinucleotide synthesis; Buchanan G et al.; The mob genes of several bacteria have been implicated in the conversion of molybdopterin to molybdopterin guanine dinucleotide . The mob locus of Rhodobacter sphaeroides WS8 comprises three genes, mobABC . Chromosomal in-frame deletions in each of the mob genes have been constructed . The mobA mutant strain has inactive DMSO reductase and periplasmic nitrate reductase activities (both molybdopterin guanine dinucleotide-requiring enzymes), but the activity of xanthine dehydrogenase, a molybdopterin enzyme, is unaffected . The inability of a mobA mutant to synthesise molybdopterin guanine dinucleotide is confirmed by analysis of cell extracts of the mobA strain for molybdenum cofactor forms following iodine oxidation . Mutations in mobB and mobC are not impaired for molybdoenzyme activities and accumulate wild-type levels of molybdopterin and molybdopterin guanine dinucleotide, indicating they are not compromised in molybdenum cofactor synthesis . In the mobA mutant strain, the inactive DMSO reductase is found in the periplasm, suggesting that molybdenum cofactor insertion is not necessarily a pre-requisite for export.

Arch Microbiol, 2001 Jul, 176(1-2), 9 - 18
Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria; Li H et al.; The temporal and spatial accumulation of cyanophycin was studied in two unicellular strains of cyanobacteria, the diazotrophic Cyanothece sp . strain ATCC 51142 and the non-diazotrophic Synechocystis sp . strain PCC 6803 . Biochemistry and electron microscopy were used to monitor the dynamics of cyanophycin accumulation under nitrogen-sufficient and nitrogen-deficient conditions . In Cyanothece sp . ATCC 51142 grown under 12 h light/12 h dark nitrogen-fixing conditions, cyanophycin was temporally regulated relative to nitrogenase activity and accumulated in granules after nitrogenase activity commenced . Cyanophycin granules reached a maximum after the peak of nitrogenase activity and eventually were utilized completely . Knock-out mutants were constructed in Synechocystis sp . PCC 6803 cphA and cphB genes to analyze the function of these genes and cyanophycin accumulation under nitrogen-deficient growth conditions . The mutants grew under such conditions, but needed to degrade phycobilisomes as a nitrogen reserve . Granules could be seen in some wild-type cells after treatment with chloramphenicol, but were never found in Delta cphA and Delta cphB mutants . These results led to the conclusion that cyanophycin is temporally and spatially regulated in nitrogen-fixing strains such as Cyanothece sp . ATCC 51142 and represents a key nitrogen reserve in these organisms . However, cyanophycin appeared to play a less important role in the non-diazotrophic unicellular strains and phycobilisomes appeared to be the main nitrogen reserve.

Biochemistry, 2001 Aug 7, 40(31), 9238 - 46
Structural changes of pharaonis phoborhodopsin upon photoisomerization of the retinal chromophore: infrared spectral comparison with bacteriorhodopsin; Kandori H et al.; Archaeal rhodopsins possess a retinal molecule as their chromophores, and their light energy and light signal conversions are triggered by all-trans to 13-cis isomerization of the retinal chromophore . Relaxation through structural changes of the protein then leads to functional processes, proton pump in bacteriorhodopsin and transducer activation in sensory rhodopsins . In the present paper, low-temperature Fourier transform infrared spectroscopy is applied to phoborhodopsin from Natronobacterium pharaonis (ppR), a photoreceptor for the negative phototaxis of the bacteria, and infrared spectral changes before and after photoisomerization are compared with those of bacteriorhodopsin (BR) at 77 K . Spectral comparison of the C--C stretching vibrations of the retinal chromophore shows that chromophore conformation of the polyene chain is similar between ppR and BR . This fact implies that the unique chromophore-protein interaction in ppR, such as the blue-shifted absorption spectrum with vibrational fine structure, originates from both ends, the beta-ionone ring and the Schiff base regions . In fact, less planer ring structure and stronger hydrogen bond of the Schiff base were suggested for ppR . Similar frequency changes upon photoisomerization are observed for the C==N stretch of the retinal Schiff base and the stretch of the neighboring threonine side chain (Thr79 in ppR and Thr89 in BR), suggesting that photoisomerization in ppR is driven by the motion of the Schiff base like BR . Nevertheless, the structure of the K state after photoisomerization is different between ppR and BR . In BR, chromophore distortion is localized in the Schiff base region, as shown in its hydrogen out-of-plane vibrations . In contrast, more extended structural changes take place in ppR in view of chromophore distortion and protein structural changes . Such structure of the K intermediate of ppR is probably correlated with its high thermal stability . In fact, almost identical infrared spectra are obtained between 77 and 170 K in ppR . Unique chromophore-protein interaction and photoisomerization processes in ppR are discussed on the basis of the present infrared spectral comparison with BR.

J Mol Biol, 2001 Aug 10, 311(2), 297 - 310
Structure and mechanism of the RuvB Holliday junction branch migration motor; Putnam CD et al.; The RuvB hexamer is the chemomechanical motor of the RuvAB complex that migrates Holliday junction branch-points in DNA recombination and the rescue of stalled DNA replication forks . The 1.6 A crystal structure of Thermotoga maritima RuvB together with five mutant structures reveal that RuvB is an ATPase-associated with diverse cellular activities (AAA+-class ATPase) with a winged-helix DNA-binding domain . The RuvB-ADP complex structure and mutagenesis suggest how AAA+-class ATPases couple nucleotide binding and hydrolysis to interdomain conformational changes and asymmetry within the RuvB hexamer implied by the crystallographic packing and small-angle X-ray scattering in solution . ATP-driven domain motion is positioned to move double-stranded DNA through the hexamer and drive conformational changes between subunits by altering the complementary hydrophilic protein- protein interfaces . Structural and biochemical analysis of five motifs in the protein suggest that ATP binding is a strained conformation recognized both by sensors and the Walker motifs and that intersubunit activation occurs by an arginine finger motif reminiscent of the GTPase-activating proteins . Taken together, these results provide insights into how RuvB functions as a motor for branch migration of Holliday junctions .

J Investig Med, 2001 Jul, 49(4), 362 - 9
Monocyte chemoattractant protein-1 and interleukin-8 are increased in bronchopulmonary dysplasia: relation to isolation of Ureaplasma urealyticum; Baier RJ et al.; BACKGROUND: An exaggerated inflammatory response occurs in infants who subsequently develop bronchopulmonary dysplasia (BPD) . Ureaplasma urealyticum (Uu) is frequently isolated from cultures of tracheal secretions obtained from very low birth weight infants and is associated with an increased risk of BPD . METHODS: We examined the relationships between isolation of genital mycoplasmas, tracheal aspirate (TA) interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) concentrations and the development of BPD . Serial TAs were obtained prospectively from 35 very low birth weight infants, and IL-8 and MCP-1 concentrations were determined by enzyme-linked immunoadsorbent assay . Tracheal cultures for bacteria and genital mycoplasmas were performed on aspirates obtained during the first 2 days of life . RESULTS: Infants who developed BPD (n=18) were less mature (25.2+/-0.2 vs 27.8+/-0.5 weeks; P<0.001), of lower birth weight (746+/-28 vs 1052+/-41 g; P<0.001), and more likely to have a positive tracheal culture for Uu (39% vs 6%; P=0.026) than those who did not develop BPD (n=17) . Tracheal concentrations of IL-8 and MCP-1 were significantly increased in infants who developed BPD (IL-8: P=0.0001; MCP-1: P<0.001, analysis of variance) and correlated with duration of mechanical ventilation and oxygen treatment . Uu-positive infants had an increased incidence of BPD (88% in infants with Uu vs 42% in infants without Uu; P=0.020) and had TA concentrations of IL-8 and MCP-1 that were significantly increased compared with those of Uu-negative infants . CONCLUSIONS: Increased TA concentrations of IL-8 and MCP-1 during the first 2 weeks of life are associated with the development of BPD . Recovery of Uu from TAs is associated with a more robust inflammatory reaction and an increased risk of BPD.

Arch Immunol Ther Exp (Warsz), 2001, 49(3), 217 - 29
CD89: the human myeloid IgA Fc receptor; Morton HC et al.; CD89 (Fc alphaRI) is the human myeloid IgA Fc receptor expressed on cells, such as neutrophils, eosinophils and monocytes/macrophages . Cross-linking of CD89 on these cells, by IgA-opsonised particles (e.g . bacteria, viruses) or anti-CD89 monoclonal antibodies, can trigger various immunological effector functions which are generally protective but may also cause harm to the body . CD89 is a transmembrane glycoprotein that binds both subclasses of IgA in all its molecular forms (i.e . monomeric, dimeric and secretory IgA) via a region of its membrane-distal EC1 domain . DNA studies have shown that the CD89 gene is located within the newly described leukocyte receptor cluster (LRC) on chromosome 19 . CD89 is more closely related to the KIR and MIR proteins, whose genes are also found in the LRC, than to other human Fc receptors (FcRs) . On myeloid cells, CD89 is able to associate with the immunoreceptor tyrosine-based activation motif (ITAM)-containing the FcR gamma chain, which is responsible for intracellular signaling via CD89 . Recently, it has been suggested that some cells express CD89 in a form that does not associate with the FcR gamma chain . Although the biological relevance of this observation is not yet clear, it may explain certain anti-inflammatory/inhibitory effects attributed to IgA . Here we review current knowledge concerning the genetics, structure and biological function of CD89.

Environ Sci Technol, 2001 Jul 15, 35(14), 2942 - 8
Siderophore mediated plutonium accumulation by Microbacterium flavescens (JG-9); John SG et al.; Uptake of plutonium and uranium mediated by the siderophore desferrioxamine-B (DFOB) has been studied for the common soil aerobe Microbacterium flavescens(JG-9) . M . flavescens does not bind or take up nitrilotriacetic acid (NTA) complexes of U(VI), Fe(III), or Pu(IV) or U(VI)-DFOB but does take up Fe(III)-DFOB and Pu(IV)-DFOB . Pu(IV)-DFOB and Fe(III)-DFOB accumulations are similar: only living and metabolically active bacteria take up these metal-siderophore complexes . The Fe(III)-DFOB and Pu(IV)-DFOB complexes mutually inhibit uptake of the other, indicating that they compete for shared binding sites or uptake proteins . However, Pu uptake is much slower than Fe uptake, and cumulative Pu uptake is less than Fe, 1.0 nmol of Fe vs 0.25 nmol of Pu per mg of dry weight bacteria . The Pu(IV)-DFOB interactions with M . flavescens suggest that Pu-siderophore complexes could generally be recognized by Fe-siderophore uptake systems of many bacteria, fungi, or plants, thereby affecting Pu environmental mobility and distribution . The results also suggest that the siderophore complexes of tetravalent metals can be recognized by Fe-siderophore uptake proteins.

Rheumatology (Oxford), 2001 Jul, 40(7), 801 - 5
Indirect evidence of intra-articular immunoglobulin G synthesis in patients with Chlamydia trachomatis reactive arthritis; Bas S et al.; OBJECTIVES: To investigate whether B-cell stimulation occurs in joints of Chlamydia trachomatis reactive arthritis patients by comparing the immunoglobulin G (IgG) anti-C . trachomatis antibody responses in serum and synovial fluid (SF) . METHODS: The number and spectrum of C . trachomatis antigens recognized by paired serum and SF samples from 16 patients with C . trachomatis reactive arthritis and 20 patients with other inflammatory arthropathies independent of this bacteria, were studied by immunoblotting . The responses to five different Chlamydia antigens were also determined in enzyme-linked immunosorbent assays . RESULTS: In C . trachomatis reactive arthritis patients, a higher number of C . trachomatis antigens was recognized by SF (17.6+/-5.1) than by serum (11.1+/-6.3) IgG and a higher intensity of SF IgG binding to the outer membrane protein 2 (OMP2) was observed . CONCLUSIONS: These results suggest an intra-articular IgG production and a possible role of some Chlamydia antigens like OMP2 in the pathogenesis of C . trachomatis reactive arthritis.

J Biol Chem, 2001 Sep 28, 276(39), 36063 - 6 Epub 2001 Jul 26.
The gp91phox component of NADPH oxidase is not the voltage-gated proton channel in phagocytes, but it helps; DeCoursey TE et al.; During the "respiratory burst," the NADPH oxidase complex of phagocytes produces reactive oxygen species that kill bacteria and other invaders (Babior, B . M . (1999) Blood 93, 1464-1476) . Electron efflux through NADPH oxidase is electrogenic (Henderson, L . M., Chappell, J . B., and Jones, O . T . G . (1987) Biochem . J . 246, 325-329) and is compensated by H(+) efflux through proton channels that reportedly are contained within the gp91(phox) subunit of NADPH oxidase . To test whether gp91(phox) functions as a proton channel, we studied H(+) currents in granulocytes from X-linked chronic granulomatous disease patients lacking gp91(phox) (X-CGD), the human myelocytic PLB-985 cell line, PLB-985 cells in which gp91(phox) was knocked out by gene targeting (PLB(KO)), and PLB-985 knockout cells re-transfected with gp91(phox) (PLB(91)) . H(+) currents in unstimulated PLB(KO) cells had amplitude and gating kinetics similar to PLB(91) cells . Furthermore, stimulation with the phorbol ester phorbol 12-myristate 13-acetate increased H(+) currents to a similar extent in X-CGD, PLB(KO), and PLB(91) cells . Thus, gp91(phox) is not the proton channel in unstimulated phagocytes and does not directly mediate the increase of proton conductance during the respiratory burst . Changes in H(+) channel gating kinetics during NADPH oxidase activity are likely crucial to the activation of H(+) flux during the respiratory burst.

FEMS Immunol Med Microbiol, 2001 Jul, 31(1), 73 - 81
Genetic differences among the LPS biosynthetic loci of serovars of Leptospira interrogans and Leptospira borgpetersenii; de la Pena-Moctezuma A et al.; The gene organization in the lipopolysaccharide biosynthetic (rfb) locus was analyzed in seven Leptospira interrogans serovars within serogroup Icterohemorrhagiae, seven non-Icterohemorrhagiae serovars and one Leptospira borgpetersenii serovar . Two groups of loci were delineated based on DNA hybridization and sequence analysis . Group 1 contained the two Hardjo subtypes, Hardjoprajitno and Hardjobovis . Group 2 (containing Copenhageni, Pomona, Naam, Mwogolo, Smithi, Lai, Canicola, Autumnalis, Pyrogenes, Australis and Icterohemorrhagiae) differed from Group 1 in its organization upstream of orf11, where five ORFs (32, 33, 34, 35, 37) were identified that were not contained in the Group 1 loci . These ORFs encoded a putative epimerase (orf32), a glycosyltransferase (orf33), two integral membrane proteins (orfs 34 and 35), and a galactosyltransferase (orf37) . Serovars Australis, Pomona and Autumnalis did not contain orf37 . Serovar Bataviae was excluded from the grouping because of its unique genetic organization upstream of orf13 . In the Group 2 loci, comparison of the genetic layout at the 5' end revealed differences which included mutations disrupting reading frames in either or both orf34 and orf35 and apparent allelic differences between orf33 homologs that may be sufficient to account for the genetic basis of serovar identity.

Biochim Biophys Acta, 2001 Jul 27, 1537(1), 63 - 70
Investigation of the functional role of active site loop II in a hypoxanthine phosphoribosyltransferase; Lee CC et al.; Hypoxanthine phosphoribosyltransferases (HPRTs) are of biomedical interest because defects in the enzyme from humans can result in gouty arthritis or Lesch-Nyhan syndrome, and in parasites these enzymes are potential targets for antiparasite chemotherapy . In HPRTs, a long flexible loop (active site loop II) closes over the active site during the enzyme catalyzed reaction . Functional roles for this loop have been proposed but have yet to be substantiated . For the present study, seven amino acids were deleted from loop II of the HPRT from Trypanosoma cruzi to probe the functional role of this active site loop in catalysis . The mutant enzyme (Deltaloop II) was expressed in bacteria, purified by affinity chromatography, and kinetic constants were determined for substrates of both forward (purine salvage) and reverse (pyrophosphorolysis) reactions catalyzed by the enzyme . Loop II deletion resulted in moderate (0.6-2.7-fold) changes in the Michaelis constants (K(m)s) for substrates other than pyrophosphate (PP(i)), for which there was a 5.8-fold increase . In contrast, k(cat) values were severely affected by loop deletion, with rates that were 240-840-fold below those for the wild-type enzyme . Together with previously reported structural data, these results are consistent with active site loop II participating in transition-state stabilization by precise positioning of the substrates for in line nucleophilic attack and in the liberation of PP(i) as a product of the salvage reaction.

Trends Neurosci, 2001 Aug, 24(8), 450 - 5
Glial activation: a driving force for pathological pain; Watkins LR et al.; Pain is classically viewed as being mediated solely by neurons, as are other sensory phenomena . The discovery that spinal cord glia (microglia and astrocytes) amplify pain requires a change in this view . These glia express characteristics in common with immune cells in that they respond to viruses and bacteria, releasing proinflammatory cytokines, which create pathological pain . These spinal cord glia also become activated by certain sensory signals arriving from the periphery . Similar to spinal infection, these signals cause release of proinflammatory cytokines, thus creating pathological pain . Taken together, these findings suggest a new, dramatically different approach to pain control, as all clinical therapies are focused exclusively on altering neuronal, rather than glial, function.

Anal Biochem, 2001 Aug 1, 295(1), 1 - 8
Colorimetric silver detection of DNA microarrays; Alexandre I et al.; Development of microarrays has revolutionized gene expression analysis and molecular diagnosis through miniaturization and the multiparametric features . Critical factors affecting detection efficiency of targets hybridization on microarray are the design of capture probes, the way they are attached to the support, and the sensitivity of the detection method . Microarrays are currently detected in fluorescence using a sophisticated confocal laser-based scanner . In this work, we present a new colorimetric detection method which is intented to make the use of microarray a powerful procedure and a low-cost tool in research and clinical settings . The signal generated with this method results from the precipitation of silver onto nanogold particles bound to streptavidin, the latter being used for detecting biotinylated DNA . This colorimetric method has been compared to the Cy-3 fluorescence method . The detection limit of both methods was equivalent and corresponds to 1 amol of biotinylated DNA attached on an array . Scanning and data analysis of the array were obtained with a colorimetric-based workstation .

J Environ Qual, 2001 Jul-Aug, 30(4), 1271 - 7
Anaerobic degradation of atrazine and metolachlor and metabolite formation in wetland soil and water microcosms; Seybold CA et al.; The half-lives, degradation rates, and metabolite formation patterns of atrazine (6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) and metolachlor {2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide} were determined in an anaerobic wetland soil incubated at 24 degrees C for 112 d . At 0, 7, 14, 28, 42, 56, and 112 d, the soil and water were analyzed for atrazine and metolachlor, and their major metabolites . The soil oxidation-reduction potential reached -200 mV after 14 d . Degradation reaction rates were first-order for atrazine in anaerobic soil and for metolachlor in the aqueous phase . Zero-order reaction rates were best fit for atrazine in the aqueous phase and metolachlor in anaerobic soil . In anaerobic soil, the half-life was 38 d for atrazine and 62 d for metolachlor . In the aqueous phase above the soil, the half-life was 86 d for atrazine and 40 d for metolachlor . Metabolites detected in the anaerobic soil were hydroxyatrazine and deethylatrazine for atrazine, and relatively small amounts of ethanesulfonic acid and oxanilic acid for metolachlor . Metabolites detected in the aqueous phase above the soil were hydroxyatrazine, deethylatrazine, and deisopropylatrazine for atrazine, and ethanesulfonic acid and oxanilic acid for metolachlor . Concentrations of metabolites in the aqueous phase generally peaked within the first 25 d and then declined . Results indicate that atrazine and metolachlor can degrade under strongly reducing conditions found in wetland soils . Metolachlor metabolites, ethanesulfonic acid, and oxanilic acid are not significantly formed under anaerobic conditions.

Eur J Clin Microbiol Infect Dis, 2001 Jun, 20(6), 418 - 20
Implications of the simultaneous presence of metronidazole-susceptible and -resistant Helicobacter pylori colonies within a single biopsy specimen; Arents NL et al.; This study examined whether the simultaneous presence of metronidazole-susceptible and -resistant Helicobacter pylori colonies in a single biopsy specimen is caused by a multiple strain infection with a susceptible and a resistant strain or by two subpopulations within a single strain . Single colonies obtained from seven biopsy specimens known to harbour both susceptible and resistant Helicobacter pylori were fingerprinted by restricted fragment length polymorphism typing of the ureC gene and by the random amplified polymorphic DNA procedure . Metronidazole susceptibility was determined by the E test . The results indicated that the occurrence of metronidazole-resistant and metronidazole-susceptible bacteria within a single biopsy does not imply the presence of a multiple strain infection with one resistant and one sensitive strain.

J Bone Joint Surg Br, 2001 Jul, 83(5), 760 - 6
Spatial and temporal collagen gene expression in lumbar intertransverse fusion in the rabbit; Suzuki H et al.; We have examined the process of fusion of the intertransverse processes and bone graft in the rabbit by in situ hybridisation and evaluated the spatial and temporal expression of genes encoding pro-alpha1 (I) collagen (COL1A1), pro-alpha1 (II) collagen (COL2A1) and pro-alphal (X) collagen (COL10A1) . Beginning at two weeks after operation, osteogenesis and chondrogenesis occurred around the transverse process and the grafted bone at the central portion of the area of the fusion mass . Osteoblasts and osteocytes at the newly-formed woven bone expressed COL1A1 . At the cartilage, most chondrocytes expressed COL2A1 and some hypertrophic chondrocytes COL10A1 . In some regions, co-expression of COL1A1 and COL2A1 was observed . At four weeks, such expressions for COLlA1, COL2A1 and COL10A1 became prominent at the area of the fusion mass . From four to six weeks, bone remodelling progressed from the area of the transverse processes towards the central zone . Osteoblasts lining the trabeculae expressed a strong signal for COL1A1 . At the central portion of the area of the fusion mass, endochondral ossification progressed and chondrocytes expressed COL2A1 and COL10A1 . Our findings show that the fusion process begins with the synthesis of collagens around the transverse processes and around the grafted bone independently . Various spatial and temporal osteogenic and chondrogenic responses, including intramembranous, endochondral and transchondroid bone formation, progress after bone grafting at the intertransverse processes . Bone formation through cartilage may play an important role in posterolateral spinal fusion.

Ophthalmic Surg Lasers, 2001 Jul-Aug, 32(4), 300 - 4
Evaluation of glutaraldehyde and povidone iodine for sterilization of wide-field contact vitrectomy lenses; Das T et al.; BACKGROUND AND OBJECTIVE: Wide-field vitrectomy contact lenses are currently sterilized with ethylene oxide gas, and other lenses with autoclaving . To maintain a large inventory or possibly run the risk of loss of lens quality with repeated autoclaving, glutaraldehyde 2% and povidone iodine 5% solution were evaluated as possible sterilizing agents . MATERIALS AND METHODS: Ethylene oxide presterilized lenses were contaminated with known concentrations (10(5) organisms/mL) of bacteria (S . epidemidis, P . aeruginosa, B . subtilis), and fungi (A . flavus, C . albicans) for 5 minutes . The test lenses were treated with glutaraldehyde or povidone iodine for 5, 10, 30, 60, and 120 minutes, and controls with sterilized water for a similar duration . Following treatment, both test and control lenses were sampled with sterile cotton swabs . The swabs were cultured for bacteria (tryptone soya broth 48 hours), and fungi (Saubourd's dextrose broth 5 days) . RESULTS: The culture was negative for both glutaraldehyde- and povidone iodine-treated lenses against all organisms at all time points except B subtilis, which needed 120 minutes treatment . CONCLUSION: Two hours contact time with glutaraldehyde 2% or providone iodine 5% can sterilize vitrectomy contact lenses against common bacteria and fungi without affecting lens quality.

Evolution Int J Org Evolution, 2001 Jun, 55(6), 1136 - 45
Genetic variation in a host-parasite association: potential for coevolution and frequency-dependent selection; Carius HJ et al.; Models of host-parasite coevolution assume the presence of genetic variation for host resistance and parasite infectivity, as well as genotype-specific interactions . We used the freshwater crustacean Daphnia magna and its bacterial microparasite Pasteuria ramosa to study genetic variation for host susceptibility and parasite infectivity within each of two populations . We sought to answer the following questions: Do host clones differ in their susceptibility to parasite isolates? Do parasite isolates differ in their ability to infect different host clones? Are there host clone-parasite isolate interactions? The analysis revealed considerable variation in both host resistance and parasite infectivity . There were significant host clone-parasite isolate interactions, such that there was no single host clone that was superior to all other clones in the resistance to every parasite isolate . Likewise, there was no parasite isolate that was superior to all other isolates in infectivity to every host clone . This form of host clone-parasite isolate interaction indicates the potential for coevolution based on frequency-dependent selection . Infection success of original host clone-parasite isolate combinations (i.e., those combinations that were isolated together) was significantly higher than infection success of novel host clone-parasite isolate combinations (i.e., those combinations that were created in the laboratory) . This finding is consistent with the idea that parasites track specific host genotypes under natural conditions . In addition, correspondence analysis revealed that some host clones, although distinguishable with neutral genetic markers, were susceptible to the same set of parasite isolates and thus probably shared resistance genes.

Science, 2001 Jul 27, 293(5530), 629 - 37
Historical overfishing and the recent collapse of coastal ecosystems; Jackson JB et al.; Ecological extinction caused by overfishing precedes all other pervasive human disturbance to coastal ecosystems, including pollution, degradation of water quality, and anthropogenic climate change . Historical abundances of large consumer species were fantastically large in comparison with recent observations . Paleoecological, archaeological, and historical data show that time lags of decades to centuries occurred between the onset of overfishing and consequent changes in ecological communities, because unfished species of similar trophic level assumed the ecological roles of overfished species until they too were overfished or died of epidemic diseases related to overcrowding . Retrospective data not only help to clarify underlying causes and rates of ecological change, but they also demonstrate achievable goals for restoration and management of coastal ecosystems that could not even be contemplated based on the limited perspective of recent observations alone.

J Clin Microbiol, 2001 Aug, 39(8), 3009 - 12
Cariogenic actinomyces identified with a beta-glucosidase-dependent green color reaction to Gardenia jasminoides extract; Chen L et al.; The oral bacteria Actinomyces naeslundii and Actinomyces viscosus are known to contribute to the initiation and progression of human dental caries, especially root caries . We report that both A . naeslundii and A . viscosus react with a component in the Gardenia jasminoides extract to produce a distinct green product . This green color reaction was found to be dependent on the bacterial beta-glucosidase . The reaction is specific for cariogenic actinomyces, and it can detect as few as 10(4) cells of A . naeslundii and A . viscosus per ml.

Nature, 2001 Jul 26, 412(6845), 433 - 6
Mealybug beta-proteobacterial endosymbionts contain gamma-proteobacterial symbionts; von Dohlen CD et al.; Some insects have cultivated intimate relationships with mutualistic bacteria since their early evolutionary history . Most ancient 'primary' endosymbionts live within the cytoplasm of large, polyploid host cells of a specialized organ (bacteriome) . Within their large, ovoid bacteriomes, mealybugs (Pseudococcidae) package the intracellular endosymbionts into 'mucus-filled' spheres, which surround the host cell nucleus and occupy most of the cytoplasm . The genesis of symbiotic spheres has not been determined, and they are structurally unlike eukaryotic cell vesicles . Recent molecular phylogenetic and fluorescent in situ hybridization (FISH) studies suggested that two unrelated bacterial species may share individual host cells, and that bacteria within spheres comprise these two species . Here we show that mealybug host cells do indeed harbour both beta- and gamma-subdivision Proteobacteria, but they are not co-inhabitants of the spheres . Rather, we show that the symbiotic spheres themselves are beta-proteobacterial cells . Thus, gamma-Proteobacteria live symbiotically inside beta-Proteobacteria . This is the first report, to our knowledge, of an intracellular symbiosis involving two species of bacteria.

Diabetes, 2001 Aug, 50(8), 1749 - 54
Characterization of preparations of GAD65, proinsulin, and the islet tyrosine phosphatase IA-2 for use in detection of autoreactive T-cells in type 1 diabetes: report of phase II of the Second International Immunology of Diabetes Society Workshop for Standardization of T-cell assays in type 1 diabetes; Peakman M et al.; The identification, quantification, and characterization of T-cells reactive with the islet autoantigens GAD65, proinsulin (PI), and tyrosine phosphatase-like molecules IA-2 and phogrin are major research goals in type 1 diabetes . In the Immunology of Diabetes Society First Workshop on Autoreactive T-Cells, the quality of recombinant preparations of these autoantigens was identified as a significant weakness, a finding that may account for much of the inconsistency in published studies of peripheral blood T-cell reactivity to islet autoantigens . Poor antigen quality has also hampered the development of novel technologies for the detection of islet-reactive T-cells . For these reasons, in the present study, several preparations of GAD65, PI, and IA-2 were collected and evaluated for endotoxin content, ability to stimulate a panel of relevant T-cell clones, and inhibitory effects on proliferation to unrelated third-party antigens . Through this process, we have been able to identify preparations of GAD65 and IA-2, generated in insect cells using the baculovirus expression system, that stimulate relevant clones and display low inhibitory effects on third-party antigens . In addition, we characterized a PI preparation generated in bacteria as being free of effects on proliferation to third-party antigens and low in endotoxin content . These preparations are important to promote the development of robust and sensitive assays of islet-reactive T-cells in patients with type 1 diabetes or patients at high risk for developing the disease.

Appl Environ Microbiol, 2001 Aug, 67(8), 3728 - 31
Isolation and regulation of Sinorhizobium meliloti 1021 loci induced by oxygen limitation; Trzebiatowski JR et al.; Eleven Sinorhizobium meliloti 1021 loci whose expression was induced under low oxygen concentrations were identified in a collection of 5,000 strains carrying Tn5-1063 (luxAB) transcriptional reporter gene fusions . The 11 Tn5-1063-tagged loci were cloned and characterized . The dependence of the expression of the tagged loci on the FixL/FixJ oxygen-sensing two-component regulatory system was examined . Three of the loci were found to be dependent upon fixL and fixJ for their expression, while one locus showed a partial dependence . The remaining seven loci showed fixL- and fixJ-independent induction of expression in response to oxygen limitation . This suggests that in S . meliloti, additional regulatory system(s) exist that respond either directly or indirectly to oxygen limitation conditions.

Appl Environ Microbiol, 2001 Aug, 67(8), 3358 - 62
Growth of chitinolytic dune soil beta-subclass Proteobacteria in response to invading fungal hyphae; De Boer W et al.; It has frequently been reported that chitinolytic soil bacteria, in particular biocontrol strains, can lyse living fungal hyphae, thereby releasing potential growth substrate . However, the conditions used in such assays (high bacterial density, rich media, fragmented hyphae) make it difficult to determine whether mycolytic activity is actually of importance for the growth and survival of chitinolytic bacteria in soils . An unidentified group of beta-subclass Proteobacteria (CbetaPs) was most dominant among the culturable nonfilamentous chitinolytic bacteria isolated from Dutch sand dune soils . Here we demonstrate that the CbetaPs grew at the expense of extending fungal mycelium of three dune soil fungi (Chaetomium globosum, Fusarium culmorum, and Mucor hiemalis) under nutrient-limiting, soil-like conditions . Aggregates of CbetaPs were also often found attached to fungal hyphae . The growth of a control group of dominant nonchitinolytic dune soil bacteria (beta- and gamma-subclass Proteobacteria) was not stimulated in the mycelial zone, indicating that growth-supporting materials were not independently released in appreciable amounts by the extending hyphae . Therefore, mycolytic activities of CbetaPs have apparently been involved in allowing them to grow after exposure to living hyphae . The chitinase inhibitor allosamidin did not, in the case of Mucor, or only partially, in the cases of Chaetomium and Fusarium, repress mycolytic growth of the CbetaPs, indicating that chitinase activity alone could not explain the extent of bacterial proliferation . Chitinolytic Stenotrophomonas-like and Cytophaga-like bacteria, isolated from the same dune soils, were only slightly stimulated by exposure to fungal hyphae.

An Esp Pediatr, 2001 Aug, 55(2), 154 - 8
{Eosinophilic pustular folliculitis in childhood}; Luelmo Aguilar J et al.; BACKGROUND: Eosinophilic pustular folliculitis (EPF) is an inflammatory disorder of unknown etiology . In infants this disorder is characterized by recurrent episodes of sterile pustules primarily or exclusively involving the scalp with occasional involvement of the face, trunk and extremities . There are few reports of EPF in children . OBJECTIVE: To describe the clinical features and evolution of four pediatric patients and to discuss the main differential diagnoses . METHODS: Biopsy specimens were examined, pustules were cultured and laboratory tests were analyzed . RESULTS: Four patients (3 males and 1 female) aged 7-18 months presented with self limiting recurrent pruritic papules and pustules on the scalp . In one patient, the lesions were mainly localized on the extremities . Cultures for bacteria, fungi and viruses were negative . No systemic disease was found . Topical steroids were effective in three patients but pustules recurred after treatment was stopped . Cetirizine and Hydroxacen were administered in two corticoid-resistant patients with fair response . No other systemic therapy was administered . Peripheral eosinophilia was detected in three patients . CONCLUSIONS: EPF in infants seems to be a clearly defined entity . Although few cases have been described in children, this dermatosis is undoubtedly more frequent than suggested by the literature . Consequently, pediatricians should be aware of its existence.

Environ Microbiol, 2001 Jun, 3(6), 397 - 406
Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes; Allaway D et al.; The techniques of differential fluorescence induction (DFI) and optical trapping (OT) have been combined to allow the identification of environmentally induced genes in single bacterial cells . Designated DFI-OT, this technique allows the in situ isolation of genes driving the expression of green fluorescent protein (Gfp) using temporal and spatial criteria . A series of plasmid-based promoter probe vectors (pOT) was developed for the construction of random genomic libraries that are linked to gfpUV or egfp . Bacteria that do not express Gfp on laboratory medium (i.e . non-fluorescent) were inoculated into the environment, and induced genes were detected with a combined fluorescence/optical trapping microscope . Using this selection strategy, rhizosphere-induced genes with homology to thiamine pyrophosphorylase (thiE) and cyclic glucan synthase (ndvB) were isolated . Other genes were expressed late in the stationary phase or as a consequence of surface-dependent growth, including fixND and metX, and a putative ABC transporter of putrescine . This strategy provides a unique ability to combine spatial, temporal and physical information to identify environmental regulation of bacterial gene expression.

Clin Exp Immunol, 2001 Jun, 124(3), 398 - 405
Functional T lymphocytes infiltrate implanted polyvinyl alcohol foams during surgical wound closure therapy; Gouttefangeas C et al.; Vacuum-assisted closure involving the implantation of polyvinyl alcohol foam is a technique recently developed for the treatment of patients suffering from either wound infection or chronic wounds . This method has been shown to improve and accelerate wound healing . However, little is known about the cell populations that infiltrate the foam, and their potential role in resolving the infection and promoting granulation tissue formation . Our study demonstrates that wound-implanted foams are mainly infiltrated with granulocytes, but that mononuclear cells, including macrophages and minor populations of T, B and natural killer lymphocytes, are also present . We show that foam-infiltrating T cells, especially CD4(+) T cells, constitute a phenotypically and functionally heterogeneous population influenced by wound-infecting bacteria . Thus, T lymphocytes could play a role in wound cleansing . In addition, our data indicate that implanted polyvinyl alcohol foams might be suitable microenvironments for manipulating T cell-mediated immune responses in patients.

Curr Pharm Des, 2001 Sep, 7(13), 1297 - 301
Tubulin-interacting agents . Epilogue; Fahy J et al.; Besides the many recognised compounds described and detailed in the present issue including the Vinca alkaloids, the taxanes, certain cryptophycines, epothilones and eleutherobines, several new products interacting with tubulin are identified regularly in the literature . These products may have been isolated from natural sources (plants, marine organisms, bacteria), but also more recently combinatorial, or at least automatised chemistry, has provided new families of "small" molecules, which on occasions have been found by High Throughput Screening directed against tubulin as a specific target . A recent review has listed more than one hundred of such derivatives . Certain of these are in an advanced stage of pharmaceutical development, as reviewed by Li et al . and von Angerer . From a mechanistic point of view, these newer products may be classified into one of three main families, although exceptions to this rule are now also being reported on: microtubule stabilising compounds, Vinca alkaloid site interacting agents, colchicine site binders.

J Struct Biol, 2001 Feb-Mar, 133(2-3), 246 - 53
Structure analysis of the flagellar cap-filament complex by electron cryomicroscopy and single-particle image analysis; Yonekura K et al.; The cap of the bacterial flagellum plays an essential role in the growth of the long helical filament by promoting the efficient self-assembly of flagellin transported to the distal end through the narrow central channel of the flagellum . The structure of the cap-filament complex was analyzed by electron cryomicroscopy and single-particle image analysis to understand how the cap stays attached while allowing the flagellin insertion between the cap and the filament end and also allowing the HAP proteins to pass through . In the images of the complex, the projection pattern of the helical subunit array in the filament portion occupied the major fraction but was variable depending on the azimuthal orientation of the filament; therefore the images showed a strong tendency to be misaligned . Various methods had to be newly developed to correctly align the images by overcoming this misalignment problem . The structure thus obtained clearly demonstrated the pentameric structure of the cap and how the cap operates . The new methods of analysis presented here would be generally applicable to cap structures of various filaments that play biologically important roles in cellular activities .

J Infect Dis, 2001 Aug 15, 184(4), 439 - 45 Epub 2001 Jul 18.
Urease prevents adherence of Helicobacter pylori to Kato III gastric epithelial cells; Makristathis A et al.; The role of urease in Helicobacter pylori adherence to and internalization by Kato III cells was investigated . Kato III cells were incubated with wild-type strains (N6 or P1), with isogenic mutants lacking urease (N6ureB::TnKm or P1ureA::TnMax5) or producing the inactive apoprotein (N6ureG::TnKm), and with urease-positive clones recovered after complementation of N6ureB::TnKm with ureAB . Bacteria were stained with the green fluorescent dye PKH2, and the bacteria load of cells was analyzed by flow cytometry . With mutants lacking urease, the bacteria load was considerably increased, in comparison with the corresponding parental strains (P<.001) . With clone K2(3), producing larger amounts of urease than N6, a significant reduction of bacteria load was observed, in comparison with the wild type (P<.001) . N6ureG::TnKm showed adherence characteristics similar to those of N6 . The role of urease in internalization was not clear . Thus, urease significantly inhibits H . pylori adherence to Kato III cells by a mechanism largely independent of enzymatic activity.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2000 Dec, 14(4), 313 - 6
{Human Fab antibodies derived from phage display library neutralize hepatitis A virus in vitro}; Cao J et al.; OBJECTIVE: Development of recombinant human monoclonal antibody to hepatitis A virus as a emergent measure for prevention of hepatitis A virus infection . METHODS: Human neutralizing monoclonal antibody Fab fragments to HAV have been developed by using phage display technique . The heavy and light chains of human IgG Fab genes were amplified from a HAV patient in convalescent stage . The combinatorial phage antibody library was established by inserting both heavy and light chains of Fab genes into phage mid-vector pComb3 and followed by help phage infection after 4 rounds of panning with purified HAV as coated antigen . RESULTS: The human Fab fragments to HAV were selected and expressed in bacteria . CONCLUSIONS: The specific binding of Fab antibodies to HAV were demonstrated by their reaction with HAV antigen in ELISA . These results provide the basis for further development of a neutralizing recombinant human whole IgG molecule and hold promise for future use in the prophylaxis of HAV infection.

Am J Clin Nutr, 2001 Aug, 74(2), 160 - 3
Total parenteral nutrition: potion or poison?
Jeejeebhoy KN.
The role of nutritional support in clinical care has burgeoned over the past 40 y . Initially, total parenteral nutri-tion (TPN) was considered to be the standard of care . Later, the concept that enteral nutrition (EN) promoted gut function and prevented the translocation of intestinal bacteria resulted in EN becoming the standard of care . Furthermore, TPN was consid-ered to be a dangerous form of therapy . Critical review of the data suggests that, in humans, TPN does not cause mucosal atrophy or increase bacterial translocation . Increased sepsis with TPN can be ascribed to overfeeding; the dangers of TPN-induced complications have been exaggerated . TPN is an equally effective alternative to EN when a risk of malnutrition is present and EN is not tolerated or when gut failure is present.

Structure (Camb), 2001 Jul 3, 9(7), 571 - 86
Crystal structures of the MJ1267 ATP binding cassette reveal an induced-fit effect at the ATPase active site of an ABC transporter; Karpowich N et al.; BACKGROUND: ATP binding cassette (ABC) transporters are ubiquitously distributed transmembrane solute pumps that play a causative role in numerous diseases . Previous structures have defined the fold of the ABC and established the flexibility of its alpha-helical subdomain . But the nature of the mechanical changes that occur at each step of the chemical ATPase cycle have not been defined . RESULTS: Crystal structures were determined of the MJ1267 ABC from Methanococcus jannaschii in Mg-ADP-bound and nucleotide-free forms . Comparison of these structures reveals an induced-fit effect at the active site likely to be a consequence of nucleotide binding . In the Mg-ADP-bound structure, the loop following the Walker B moves toward the Walker A (P-loop) coupled to backbone conformational changes in the intervening "H-loop", which contains an invariant histidine . These changes affect the region believed to mediate intercassette interaction in the ABC transporter complex . Comparison of the Mg-ADP-bound structure of MJ1267 to the ATP-bound structure of HisP suggests that an outward rotation of the alpha-helical subdomain is coupled to the loss of a molecular contact between the gamma-phosphate of ATP and an invariant glutamine in a segment connecting this subdomain to the core of the cassette . CONCLUSIONS: The induced-fit effect and rotation of the alpha-helical subdomain may play a role in controlling the nucleotide-dependent change in cassette-cassette interaction affinity believed to represent the power-stroke of ABC transporters . Outward rotation of the alpha-helical subdomain also likely facilitates Mg-ADP release after hydrolysis . The MJ1267 structures therefore define features of the nucleotide-dependent conformational changes that drive transmembrane transport in ABC transporters.

FEMS Microbiol Lett, 2001 Jul 24, 201(2), 291 - 4
Effect of HEPES buffer systems upon the pH, growth and survival of Mycoplasma mycoides subsp . mycoides small colony (MmmSC) vaccine cultures; Waite ER et al.; The use of a buffer system based on N-{2-hydroxyethyl}piperazine-N'-{2-ethanesulfonic acid} (HEPES), in conjunction with standard Gourlay's culture medium was investigated for the growth and maintenance of Mycoplasma mycoides subsp . mycoides SC vaccine strain T(1)44 . When the initial pH of the culture medium was adjusted to 8.0, 0.075 M HEPES-NaOH was found to be sufficient to prevent the pH falling below 7.1 at any stage during the growth cycle, even in the presence of 0.5% glucose . Compared to growth in standard unbuffered Gourlay's medium, the final culture titre was found to be one log(10) higher, at 10(11) colour changing units (CCU) per ml, and considerably extended culture survival was observed at 37 degrees C . The titre remained above 10(10) CCU ml(-1) for 4 days, and above 10(8) CCU ml(-1) in excess of 1 month . After 4 month's storage at 37 degrees C the titre had fallen to 5x10(4) CCU ml(-1) . In contrast, no viable bacteria could be detected in standard unbuffered medium 3 days after the onset of stationary phase, at which point the pH had dropped to 5.4 . No significant difference in growth rate between the two media was observed . Adoption of a HEPES-NaOH buffer system by African vaccine manufacturers should require minimal changes to current formulations and procedures, and should enhance both the final titre and thermostability of freeze-dried and liquid broth vaccines against contagious bovine pleuropneumonia (CBPP).

FEMS Microbiol Lett, 2001 Jul 24, 201(2), 177 - 80
Role played by the response regulator Ris in Bordetella bronchiseptica resistance to macrophage killing; Zimna K et al.; Previous studies suggested that the persistence in eukaryotic cells of a Bordetella bronchiseptica mutant carrying an insertion in the locus encoding the response regulator RisAS is impaired . This suggested that ris-dependent products are required for the intracellular survival of bacteria . In this study we demonstrate that ris-regulated products play a role in B . bronchiseptica resistance against both phagosomal acidification and reactive oxygen intermediates.

Trop Med Int Health, 2001 Jul, 6(7), 529 - 34
The influence of prematurity and low birthweight on transplacental antibody transfer in a rural West African population; Okoko JB et al.; OBJECTIVE: To determine the influence of prematurity and low birthweight (LBW) on transplacental antibody transfer . METHOD: In a physician-blinded, cross-sectional study of 213 mother--baby pairs in the labour ward of Bansang Hospital, The Gambia, paired maternal and cord serum samples were tested for specific IgG antibody titres for measles virus (MeV), herpes simplex virus type 1 (HSV1), respiratory syncytial virus (RSV), varicella-zoster virus (VZV), tetanus toxoid (TT) and diphtheria toxoid (DT) antigens using enzyme linked immunosorbent assay (ELISA) . RESULTS: Prematurity was significantly associated with reduced placental antibody transfer for MeV, HSV1, TT, DT, RSV and VZV . Maternal antibody transfer for MeV, HSV1, TT, DT, RSV and VZV was significantly lower in neonates with LBW than in babies with adequate birthweight (ABW) . CONCLUSION: Materno--foetal transfer of antibodies is impaired in prematurity and LBW babies in this Gambian population . Reduction in antibody transfer may further predispose these already vulnerable neonates to bacteria and viral infections . Therefore, alternative vaccination strategies, including earlier vaccination schedules, are needed to provide better protection to these young infants.

J Mol Biol, 2001 Aug 3, 311(1), 183 - 93
Excluded volume in protein side-chain packing; Kussell E et al.; The excluded volume occupied by protein side-chains and the requirement of high packing density in the protein interior should severely limit the number of side-chain conformations compatible with a given native backbone . To examine the relationship between side-chain geometry and side-chain packing, we use an all-atom Monte Carlo simulation to sample the large space of side-chain conformations . We study three models of excluded volume and use umbrella sampling to effectively explore the entire space . We find that while excluded volume constraints reduce the size of conformational space by many orders of magnitude, the number of allowed conformations is still large . An average repacked conformation has 20 % of its chi angles in a non-native state, a marked reduction from the expected 67 % in the absence of excluded volume . Interestingly, well-packed conformations with up to 50 % non-native chi angles exist . The repacked conformations have native packing density as measured by a standard Voronoi procedure . Entropy is distributed non-uniformly over positions, and we partially explain the observed distribution using rotamer probabilities derived from the Protein Data Bank database . In several cases, native rotamers that occur infrequently in the database are seen with high probability in our simulation, indicating that sequence-specific excluded volume interactions can stabilize rotamers that are rare for a given backbone . In spite of our finding that 65 % of the native rotamers and 85 % of chi(1) angles can be predicted correctly on the basis of excluded volume only, 95 % of positions can accommodate more than one rotamer in simulation . We estimate that, in order to quench the side-chain entropy observed in the presence of excluded volume interactions, other interactions (hydrophobic, polar, electrostatic) must provide an additional stabilization of at least 0.6 kT per residue in order to single out the native state .

Rev Assoc Med Bras, 2001 Apr-Jun, 47(2), 129 - 36
{Pulmonary involvement in children with AIDS: clinical study and necropsy of 14 cases}; Yparraguirre IT et al.; BACKGROUND: To describe clinical, laboratorial, radiological, and histopathological lung findings from necroscopy of aidetic children with pulmonary disease . MATERIAL AND METHODS: Fourteen children admitted at the Hospital Universitario Antonio Pedro - Universidade Federal Fluminense, Niteroi, RJ, and Instituto de Puericultura e Pediatria Martagao Gesteira - Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, between 1989 and 1996, were revised in a retrospective survey . RESULTS: Eight were males (57%) . The median age was 1.2 years old (from three months to nine years) . Nine children (64.4%) were younger than 24 months old . The HIV transmission was vertical in 10 (71%) children . In these cases, five mothers were contaminated from heterosexual relations . Pneumonia (n=8), oral candidiasis (n=8), and diarrhea (n=5) were the most common previous conditions . The most frequent signs and symptoms on admission were fever (n=12), respiratory distress (n=10), cough (n=10), peripheral lymphadenopathy (n=11), hepatomegaly and/or splenomegaly (n=10), and malnutrition (n=9) . The chest x-rays findings were condensation (n=5) and diffuse infiltrates (n=6) patterns . The microscopic lung characteristics were compatibles with pneumonia by cytomegalovirus (CMV) (n=9), bacteria (n=8), Pneumocystis carinii (n=3), Toxoplasma gondii (n=1), Hystoplasma capsulatum (n=1) and Lymphocytic interstitial pneumonia (n=1) . The association between CMV and bacteria was observed in six cases . CONCLUSIONS: Age less than two years old, vertical transmission, inespecific clinical presentation of pneumonia, and infiltrates and condensation patterns at the x-rays were the predominant characteristics . Cytomegalovirus and bacteria were the most common etiologic agents, being their association frequent . Lymphocytic interstitial pneumonia and pneumonia by P . carinii were not common causes of pulmonary disease.

Biochemistry, 2001 Jul 31, 40(30), 8997 - 9004
Low-temperature Fourier transform infrared spectroscopy of photoactive yellow protein; Imamoto Y et al.; The photocycle intermediates of photoactive yellow protein (PYP) were characterized by low-temperature Fourier transform infrared spectroscopy . The difference FTIR spectra of PYP(B), PYP(H), PYP(L), and PYP(M) minus PYP were measured under the irradiation condition determined by UV-visible spectroscopy . Although the chromophore bands of PYP(B) were weak, intense sharp bands complementary to the 1163-cm(-1) band of PYP, which show the chromophore is deprotonated, were observed at 1168-1169 cm(-1) for PYP(H) and PYP(L), indicating that the proton at Glu46 is not transferred before formation of PYP(M) . Free trans-p-coumaric acid had a 1294-cm(-1) band, which was shifted to 1288 cm(-1) in the cis form . All the difference FTIR spectra obtained had the pair of bands corresponding to them, indicating that all the intermediates have the chromophore in the cis configuration . The characteristic vibrational modes at 1020-960 cm(-1) distinguished the intermediates . Because these modes were shifted by deuterium-labeling at the ethylene bond of the chromophore while labeling at the phenol part had no effect, they were attributed to the ethylene bond region . Hence, structural differences among the intermediates are present in this region . Bands at about 1730 cm(-1), which show that Glu46 is protonated, were observed for all intermediates except for PYP(M) . Because the frequency of this mode was constant in PYP(B), PYP(H), and PYP(L), the environment of Glu46 is conserved in these intermediates . The photocycle of PYP would therefore proceed by changing the structure of the twisted ethylene bond of the chromophore.

Can J Microbiol, 2001 Jun, 47(6), 567 - 73
Temporal effects on the composition of a population of Sinorhizobium meliloti associated with Medicago sativa and Melilotus alba; Bromfield ES et al.; An assessment was made of the impact of temporal separation on the composition of a population of Sinorhizobium meliloti associated with Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a single site that had no known history of alfalfa cultivation . Root nodules were sampled on six occasions over two seasons, and a total of 1620 isolates of S . meliloti were characterized on the basis of phage sensitivity using 16 typing phages . Plant infection tests indicated that symbiotic S . meliloti were deficient in the soil at the time of planting and that these bacteria were present at low density during the first season (<10(2)/g of soil); in the second season numbers increased markedly to about 10(5)/g of soil . Overall, 37 and 51 phage types, respectively, were encountered among the nodule isolates from M . sativa and M . alba . The data indicate significant temporal shifts in the frequency and diversity of types associated with the two legume species . Apparent temporal variation with respect to the frequency of types appeared largely unpredictable and was not attributable to any one sampling time . The results indicate an apparent reduction in phenotypic diversity over the course of the experiment . Differential host plant selection of specific types with respect to nodule occupancy was indicated by significant interactions between legume species and either the frequency or diversity of phage types . Isolates from M . sativa that were resistant to lysis by all typing phages (type 14) were unusual in that they were predominant on this host at all sampling times (between 53% and 82% nodule occupancy) and were relatively homogeneous on the basis of DNA hybridization with 98% of the isolates analysed sharing the same nod EFG hybridization profile . In contrast, those isolates from M . alba comprising type 14 were encountered at low total frequency (2%) and were genetically heterogeneous on the basis of Southern hybridization . The implications of the observed temporal and host plant variation for ecological studies are discussed.

Vet Pathol, 2001 Jul, 38(4), 460 - 3
Disseminated Mycobacterium celatum (type 3) infection in a domestic ferret (Mustela putorius furo); Valheim M et al.; A 4-year-old male ferret (Mustela putoriusfuro) had a 6-month history of weight loss and gradual development of depression and coughing . Necropsy findings included pale gray tissue around the distal trachea, multiple nodules in the lungs, a single nodule in the stomach wall, gray foci in the liver, and enlarged lymph nodes . Histologic examination revealed multifocal to coalescing granulomatous inflammation in the trachea, lungs, stomach, liver, and lymph nodes, with acid-fast bacteria in epithelioid cells and macrophages . The acid-fast bacteria were identified as Mycobacterium celatum (type 3) using DNA sequence analysis of the 16S ribosomal DNA gene . M . celatum is a recently described mycobacterium isolated mainly from immunocompromised humans . This is the first report of M . celatum infection in an animal.

Vet Pathol, 2001 Jul, 38(4), 390 - 5
Detection and localization of ApxI, -II and -III genes of Actinobacillus pleuropneumoniae in natural porcine pleuropneumonia in natural porcine pleuropneumonia by in situ hybridization; Choi C et al.; In situ hybridization techniques that employed a nonradioactive digoxigenin-labeled probe were used to detect and localize ApxI, II and III genes in tissue sections of pneumonic lung naturally infected with Actinobacillus pleuropneumoniae . In pigs infected with either serotype 2 or 6, a hybridization signal for apxIICA, apxIIICA, apxIBD, and apxIIIBD was detected, and in pigs infected with serotype 5, a hybridization signal for apxICA, apxIICA, and apxIBD was detected in the pneumonic lesions . A hybridization signal for apxIICA and apxIBD was detected in pigs infected with serotype 7 . A strong hybridization signal for apx genes was seen in streaming degenerate alveolar leukocytes bordering zones of coagulative necrosis . Simultaneous detection of hybridization signals for the apxCA and apxBD genes provided scientific evidence that the expression of the apx genes could be potential indicators of the production of corresponding Apx toxins . This study demonstrates the expression of ApxI, II, and III genes in pneumonic lesions caused by A . pleuropneumoniae.

Commun Dis Public Health, 2001 Mar, 4(1), 68 - 70
An outbreak of viral gastro-enteritis at a charity function; Steel N et al.; One hundred and thirty-eight people of 431 who attended a charity function fell ill with gastro-enteritis mainly between 12 and 72 hours later . Three people were ill at the event, and a further three became ill within two hours of the event . A cohort study undertaken using a postal questionnaire showed that illness was statistically associated with having eaten cream, mints, or profiteroles . The duration of illness ranged from two to 120 hours, with a median of 48 hours . Twenty-three of the cases sent a stool sample to the laboratory . Six of these were taken to the laboratory within three days of the onset of illness and examined for bacteria before being sent for virological examination by electron microscopy (EM) and polymerase chain reaction (PCR) . All six were positive for small round structured virus (SRSV) on PCR, and negative on EM . The illness was likely to have been transmitted both by foodborne transmission and person to person spread at the event.

Rapid Commun Mass Spectrom, 2001, 15(15), 1287 - 90
Characterisation of dissolved combined amino acids in marine waters; Sommerville K et al.; Dissolved combined amino acids (DCAA) are important constituents of the dissolved organic nitrogen (DON) pool in marine environments, although little is known about their sources, dynamics and sinks . The DCAA pool consists of various compounds including proteins and peptides, proteins linked to sugars and amino acids adsorbed to humic and fulvic acids, clays and other materials . The proportions of each of these components and the extent to which they are used by microplankton living within the photic zone are not known . An investigation was carried out, using (15)N isotope dilution techniques, to determine the concentration and composition of dissolved amino acid pools in the marine environment . A near-shore seawater sample was collected and split into fractions to determine the concentrations of dissolved free amino acids (DFAA), DCAA and a <3 kDa dissolved peptide fraction (DPEP; obtained by ultrafiltration) . DCAA and DPEP fractions were hydrolysed to yield free amino acids and all samples were analysed by gas chromatography/mass spectrometry (GC/MS) as isobutyloxycarbonyl/tert-butyldimethylsilyl derivatives . The DFAA was the smallest fraction representing approximately 1% of total dissolved amino acids . The majority of DCAA was contained in the low molecular weight DPEP fraction (90%) and was probably as a result of release from phytoplankton and degradation by heterotrophic bacteria .

J Mol Med, 2001 Jul, 79(7), 358 - 67
New approaches to dissect degeneracy and specificity in T cell antigen recognition; Jacobsen M et al.; The acquired immune system is a complex and very effective defense against invading pathogens such as bacteria and viruses . T cells are central to the acquired immune system by controlling B and T cell activation and induction of T cell effector functions . The key event for T cell activation is the recognition of a specific antigen by the T cell receptor . During the past decade antigen recognition of T cells has been investigated intensively leading to new insights into the molecular mechanisms of T cell activation . In addition to the resolution of the molecular structure of the trimolecular complex (T cell receptor, peptide, major histocompatibility complex) functional studies have demonstrated the flexibility of the T cell receptor interaction with its ligand . These observations have had strong implications for the understanding of T cell selection, maturation, and repertoire maintenance . In addition, the flexibility of the T cell receptor has provided the basis for novel methods to dissect antigen recognition and define the repertoire of ligands for a given receptor . Here, we summarize recent progress on T cell recognition and method innovations with respect to future studies in autoimmune diseases.

J Biol Chem, 2001 Sep 21, 276(38), 35558 - 63 Epub 2001 Jul 20.
Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation; Olteanu H et al.; Methionine synthase is a key enzyme in the methionine cycle that catalyzes the transmethylation of homocysteine to methionine in a cobalamin-dependent reaction that utilizes methyltetrahydrofolate as a methyl group donor . Cob(I)alamin, a supernucleophilic form of the cofactor, is an intermediate in this reaction, and its reactivity renders the enzyme susceptible to oxidative inactivation . In bacteria, an NADPH-dependent two-protein system comprising flavodoxin reductase and flavodoxin, transfers electrons during reactivation of methionine synthase . Until recently, the physiological reducing system in mammals was unknown . Identification of mutations in the gene encoding a putative methionine synthase reductase in the cblE class of patients with an isolated functional deficiency of methionine synthase suggested a role for this protein in activation (Leclerc, D., Wilson, A., Dumas, R., Gafuik, C., Song, D., Watkins, D., Heng, H . H . Q., Rommens, J . M., Scherer, S . W., Rosenblatt, D . S., and Gravel, R . A . (1998) Proc . Natl . Acad . Sci . U . S . A . 95, 3059-3064) . In this study, we have cloned and expressed the cDNA encoding human methionine synthase reductase and demonstrate that it is sufficient for supporting NADPH-dependent activity of methionine synthase at a level that is comparable with that seen in the in vitro assay that utilizes artificial reductants . Methionine synthase reductase is a soluble, monomeric protein with a molecular mass of 78 kDa . It is a member of the family of dual flavoproteins and is isolated with an equimolar concentration of FAD and FMN . Reduction by NADPH results in the formation of an air stable semiquinone similar to that observed with cytochrome P-450 reductase . Methionine synthase reductase reduces cytochrome c in an NADPH-dependent reaction at a rate (0.44 micromol min(-1) mg(-1) at 25 degrees C) that is comparable with that reported for NR1, a soluble dual flavoprotein of unknown function, but is approximately 100-fold slower than that of P-450 reductase . The K(m) for NADPH is 2.6 +/- 0.5 microm, and the K(act) for methionine synthase reductase is 80.7 +/- 13.7 nm for NADPH-dependent activity of methionine synthase.

J Bacteriol, 2001 Aug, 183(16), 4932 - 7
Iron-dependent transcription of the frpB gene of Helicobacter pylori is controlled by the Fur repressor protein; Delany I et al.; We have overexpressed and purified the Helicobacter pylori Fur protein and analyzed its interaction with the intergenic regions of divergent genes involved in iron uptake (frpB and ceuE) and oxygen radical detoxification (katA and tsaA) . DNase I footprint analysis showed that Fur binds specifically to a high-affinity site overlapping the P(frpB) promoter and to low-affinity sites located upstream from promoters within both the frpB-katA and ceuE-tsaA intergenic regions . Construction of an isogenic fur mutant indicated that Fur regulates transcription from the P(frpB) promoter in response to iron . In contrast, no effect by either Fur or iron was observed for the other promoters.

J Bacteriol, 2001 Aug, 183(16), 4839 - 47
Molecular, antigenic, and functional analyses of Omp2b porin size variants of Brucella spp; Paquet JY et al.; Omp2a and Omp2b are highly homologous porins present in the outer membrane of the bacteria from the genus Brucella, a facultative intracellular pathogen . The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions . In this work, we present the cloning, purification, and characterization of four Omp2b size variants found in various Brucella species, and we compare their antigenic and functional properties to the Omp2a and Omp2b porins of Brucella melitensis reference strain 16M . The variation of the Omp2a and Omp2b porin sequences among the various strains of the genus Brucella seems to result mostly from multiple gene conversions between the two highly homologous genes . As shown in this study, this phenomenon has led to the creation of natural Omp2a and Omp2b chimeric proteins in Omp2b porin size variants . The comparison by liposome swelling assay of the porins sugar permeability suggested a possible functional differences between Omp2a and Omp2b, with Omp2a showing a more efficient pore in sugar diffusion . The sequence variability in the Omp2b size variants was located in the predicted external loops of the porin . Several epitopes recognized by anti-Omp2b monoclonal antibodies were mapped by comparison of the Omp2b size variants antigenicity, and two of them were located in the most exposed surface loops . However, since variations are mostly driven by simple exchanges of conserved motifs between the two genes (except for an Omp2b version from an atypical strain of Brucella suis biovar 3), the porin variability does not result in major antigenic variability of the Brucella surface that could help the bacteria during the reinfection of a host . Porin variation in Brucella seems to result mainly in porin conductivity modifications.

Science, 2001 Aug 31, 293(5535), 1651 - 3 Epub 2001 Jul 19.
Regulation of transcriptional activation domain function by ubiquitin; Salghetti SE et al.; The ability of transcriptional activation domains (TADs) to signal ubiquitin-mediated proteolysis suggests an involvement of the ubiquitin-proteasome pathway in transcription . To probe this involvement, we asked how ubiquitylation regulates the activity of a transcription factor containing the VP16 TAD . We show that the VP16 TAD signals ubiquitylation through the Met30 ubiquitin-ligase and that Met30 is also required for the VP16 TAD to activate transcription . The requirement for Met30 in transcription is circumvented by fusion of ubiquitin to the VP16 activator, demonstrating that activator ubiquitylation is essential for transcriptional activation . We propose that ubiquitylation regulates TAD function by serving as a dual signal for activation and activator destruction.

Mol Cell Biol, 2001 Aug, 21(16), 5512 - 9
Nonsense-mediated decay of human HEXA mRNA; Rajavel KS et al.; Nonsense-mediated mRNA decay (NMD), the loss of mRNAs carrying premature stop codons, is a process by which cells recognize and degrade nonsense mRNAs to prevent possibly toxic effects of truncated peptides . Most mammalian nonsense mRNAs are degraded while associated with the nucleus, but a few are degraded in the cytoplasm; at either site, there is a requirement for translation and for an intron downstream of the early stop codon . We have examined the NMD of a mutant HEXA message in lymphoblasts derived from a Tay-Sachs disease patient homozygous for the common frameshift mutation 1278ins4 . The mutant mRNA was nearly undetectable in these cells and increased to approximately 40% of normal in the presence of the translation inhibitor cycloheximide . The stabilized transcript was found in the cytoplasm in association with polysomes . Within 5 h of cycloheximide removal, the polysome-associated nonsense message was completely degraded, while the normal message was stable . The increased lability of the polysome-associated mutant HEXA mRNA shows that NMD of this endogenous mRNA occurred in the cytoplasm . Transfection of Chinese hamster ovary cells showed that expression of an intronless HEXA minigene harboring the frameshift mutation or a closely located nonsense codon resulted in half the normal mRNA level . Inclusion of multiple downstream introns decreased the abundance further, to about 20% of normal . Thus, in contrast to other systems, introns are not absolutely required for NMD of HEXA mRNA, although they enhance the low-HEXA-mRNA phenotype.

Biophys J, 2001 Aug, 81(2), 725 - 36
Mesoscopic simulation of cell membrane damage, morphology change and rupture by nonionic surfactants; Groot RD et al.; A new simulation method, dissipative particle dynamics, is applied to model biological membranes . In this method, several atoms are united into a single simulation particle . The solubility and compressibility of the various liquid components are reproduced by the simulation model . When applied to a bilayer of phosphatidylethanolamine, the membrane structure obtained matches quantitatively with full atomistic simulations and with experiments reported in the literature . The method is applied to investigate the cause of cell death when bacteria are exposed to nonionic surfactants . Mixed bilayers of lipid and nonionic surfactant were studied, and the diffusion of water through the bilayer was monitored . Small transient holes are seen to appear at 40% mole-fraction C(9)E(8), which become permanent holes between 60 and 70% surfactant . When C(12)E(6) is applied, permanent holes only arise at 90% mole-fraction surfactant . Some simulations have been carried out to determine the rupture properties of mixed bilayers of phosphatidylethanolamine and C(12)E(6) . These simulations indicate that the area of a pure lipid bilayer can be increased by a factor 2 . The inclusion of surfactant considerably reduces both the extensibility and the maximum stress that the bilayer can withstand . This may explain why dividing cells are more at risk than static cells.

Tuberculosis (Edinb), 2001, 81(1-2), 133 - 9
Optimal models to evaluate the protective efficacy of tuberculosis vaccines; Griffin JF et al.; BCG has been used widely as a vaccine to prevent tuberculosis (TB) for 80 years, yet there is still considerable controversy about its efficacy . Many experimental variables have obscured the true efficacy of BCG . The absence of appropriate animal models for the study of protective efficacy and the lack of in vitro correlates of protective immunity have impeded progress . Laboratory animal studies, which have contributed to understanding the pathogenesis, heritability of resistance and immunology of TB, have failed to identify the immunological pathways necessary for protective immunity . In recent years, cattle and deer, which are naturally susceptible to TB, have been used to study protective immunity in vaccinated animals, challenged with virulent bacteria . A deer TB infection model has been developed that can measure protection against TB infection or the development of disease . Data from this model show that, providing live BCG is administered in a short interval prime-boost protocol, significant protection against infection and disease can be obtained . Single dose vaccine provides suboptimal protection that attenuates pathology but does not prevent infection . Low dose BCG vaccine (10(4)cfu), administered in a prime-boost protocol, sufficient to prevent infection, does not cause conversion to delayed type hypersensitivity or produce unacceptable side-effects . Immune memory for protection against infection persists at optimal levels for at least 12 months post vaccination . Used optimally, BCG produces good levels of protection against TB and improved protocols or its use should be explored, before attempts are made to replace it with new-generation vaccines . It is now possible to integrate the fundamental information obtained from laboratory animals with studies of functional immune protection in target host species . Justification for the use of TB vaccines for domestic livestock under field conditions, must be underpinned by scientific evidence that they provide acceptable levels of protection long term .

Bioessays, 2001 Jul, 23(7), 657 - 61
Oligotrophs versus copiotrophs; Koch AL; Bacteria can grow rapidly, yet there are some that grow slowly under apparent optimal conditions . These organisms are usually present in environments with low levels of nutrients, and are not found in conditions of more plentiful nutrients . They are known as "oligotrophs"in contrast to "copiotrophs", which are common in environments with greater nutritional opportunities . This essay asks why do the oligotrophs not occupy richer environments, and why are copiotrophs not more prevalent in chronic starvation environments?

Med Hypotheses, 2001 Aug, 57(2), 186 - 91
Application of genomeceuticals to the molecular and immunological aspects of autism; Brudnak MA; Autism is a developmental disease affecting as many as 1 in 300 children and is often characterized as a mental disorder originating in infancy that is associated with self-absorption, inability to interact socially, behavior, and language dysfunction (e.g . echolalia) . Current theories indicate an important role of diet in the development of disease . It is thought that, as a result of maldigestion of casein and gluten, opioid-type peptides, or exorphins, are produced . Additionally, because of the time-frame of development of the disease, there has been an association with childhood vaccination . Consequently, prevailing therapies attempt to address these causes in one, or a combination, of three ways: diet restriction (removing casein and gluten); supplementation with exogenous enzymes; and probiotic bacteria . Until recently, none of the therapies addressed the molecular mechanisms that may be at work in the development and progression of autism . This paper presents potential molecular and cellular mechanism related to autism as well as discusses their application to the treatment of the disease through the application of genomeceuticals . Additionally, a link between developmentally associated aberrant immune and inflammatory responses, and autism is suggested and explored .

Adv Parasitol, 2001, 49, 1 - 70
Antigenic variation in trypanosomes: enhanced phenotypic variation in a eukaryotic parasite; Barry JD et al.; African trypanosomes are unicellular, eukaryotic parasites that live extracellularly in a wide range of mammals, including humans . They have a surface coat, composed of variant surface glycoprotein (VSG), which probably is essential and acts as a defence against general innate immunity and against acquired immunity directed at invariant surface antigens . In effect, the VSG is the only antigen that the host can target, and each trypanosome expresses only one VSG . To counter specific antibodies against the VSG, trypanosomes periodically undergo antigenic variation, the change to expression of another VSG . Antigenic variation belongs to the general survival strategy of enhanced phenotypic variation, where a subset of 'contingency' genes of viruses, bacteria and parasites hypermutate, allowing rapid adaptation to hostile or changing environments . A fundamental feature of antigenic variation is its link with the population dynamics of trypanosomes within the single host . Antigenic variants appear hierarchically within the mammalian host, with a mixture of order and randomness . The underlying mechanisms of this are not understood, although differential VSG gene activation may play a prominent part . Trypanosome antigenic variation has evolved a second arm in which the infective metacyclic population in the tsetse fly expresses a defined mixture of VSGs, although again each trypanosome expresses a single VSG . Differential VSG expression enhances transmission to new hosts, in the case of bloodstream trypanosomes by prolonging infection, and in the metacyclic population by generating diversity that may counter existing partial immunity in reservoir hosts . Antigenic variation employs a huge repertoire of VSG genes . Only one is expressed at a time in bloodstream trypanosomes, as a result of transcription being restricted to a set of about 20 bloodstream expression sites (BESs), which are at chromosome telomeres . Only one BES is active at a time, probably through transcriptional elongation being inhibited in the silent BESs . Although transcriptional switching between BESs can effect a VSG switch, the most prolific switch route involves homologous recombination of deoxyribonucleic acid, usually by the copying of a silent gene into a BES . Hierarchical expression of VSGs may be dictated in part by the different types of locus occupied by VSG genes . The VSG genes expressed in the metacyclic population also occupy telomeric sites, which appear to be derived from BESs but have a simpler structure . Their differential expression is achieved by random transcriptional activation; the detailed story requires direct study of the metacyclic stage itself . Available evidence suggests that the VSG originated as a surface receptor, and it can be proposed that a number of selective events have contributed to the evolution of the complex, multisystem phenomenon that antigenic variation has become.

Photochem Photobiol, 2001 Jul, 74(1), 81 - 7
Thermal and structural changes of photosynthetic reaction centers characterized by photoacoustic detection with a broad frequency band hydrophone; Nagy L et al.; Photoacoustic measurements using a broad frequency band hydrophone were carried out in photosynthetic reaction centers (RC) isolated from Rhodobacter sphaeroides R-26 purple bacteria . Data were extracted on enthalpy and volume changes accompanying the primary steps after excitation in the range of 0-500 microseconds aimed at further characterizing the thermodynamic properties of the RC . Quinone titration showed that the volume contraction accompanying the electron transport is sensitive to the molecular species occupying the secondary quinone site . delta VM = 14.4, 7.7 and 4.3 cm3 molar volume contractions were calculated from the measured parameters for 1, 2 and 0.07 quinone/RC after light excitation . Comparing the enthalpy changes (delta H) to the Gibbs free energy data in the literature, a rather large (26%) entropic contribution to the free energy changes (delta G) is estimated for the P*QAQB-->P+QA-QB electron transport (where QA and QB represent primary and secondary quinones, respectively) . This is in contrast to previous estimations that delta G = delta H in these processes . On the other hand, only a small (4%) entropic contribution to the delta G of the P*QAQB-->P+QAQB- process is estimated, in agreement with the literature data . Our results are in good agreement with the data obtained earlier (Edens et al . {2000} J . Am . Chem . Soc . 122, 1479-1485).

Photochem Photobiol, 2001 Jul, 74(1), 72 - 80
Spectroscopic studies on self-aggregation of bacteriochlorophyll-e in nonpolar organic solvents: effects of stereoisomeric configuration at the 3(1)-position and alkyl substituents at the 8(1)-position; Saga Y et al.; Self-aggregation of naturally occurring bacteriochlorophyll (BChl)-e in nonpolar organic solvents was investigated by visible absorption, fluorescence emission and circular dichroism spectra . Cultured brown-colored photosynthetic bacteria have several BChl-e as light-harvesting antenna pigments . Three major BChl-e homologs were separated from the extracts of the culture by reverse-phase high-performance liquid chromatography (HPLC) and characterized by 1H-NMR and fast-atom bombardment mass spectroscopy: 8-ethyl-12-ethyl ({E,E})-, 8-propyl-12-ethyl- and 8-isobutyl-12-ethyl-BChl-e farnesyl esters . All the homologs consisted of a mixture of the 3(1)-epimers, and epimerically pure BChl-e were also given by HPLC separation . All the separated BChl-e epimers, the epimeric mixtures and the homologous mixtures formed self-aggregates in 2% dichloromethane/hexane, giving visible absorption spectra similar to that of the whole cells, which showed two peaks (or shoulders) around 430-450 and 520 nm at the Soret region as well as a red-shifted Qy band relative to the monomeric . The spectral properties of the Soret band were basically unchanged among the epimers or epimeric/homologous mixtures . In contrast, the Qy band of aggregates of epimeric mixtures (except {E,E}) and homologous mixtures red-shifted and broadened compared with the epimerically pure . The red-shift and broadening of the Qy band are advantageous for efficient energy transfer from BChl-e aggregates to BChl-a in a baseplate in chlorosomes because their spectral overlap increases.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2001 May, 36(5), 779 - 91
Bioaugmentation and anaerobic treatment of pharmaceutical effluent in fluidized bed reactor; Saravanane R et al.; The start-up of an anaerobic fluidized bed reactor was carried out using a single inoculum (supernatant of anaerobic digester) and later on with multiple inoculum (a mixture of supernatant of anaerobic digester and volatile fatty acid (VFA)) to achieve a faster start-up . Then regular experiments were carried out to study the effect of hydraulic retention time (HRT) on COD removal (%) and biogas production . The pharmaceutical effluent with COD of 2000 to 4000 mg/L was treated in a fluidized bed reactor using an enricher-reactor concept with a hydraulic retention times of 3 (Uf = 6 Umf) to 24 (Uf = 1.5 Umf) hr . The maximum COD removal (%) of 91.2 and a maximum biogas production of 5.62 L/d were obtained at 24 hr HRT for a maximum COD concentration of 4000 mg/L corresponding to a fluidization velocity (Uf) of 20 m/hr (1.5 Umf) using a granular activated carbon bed of average size 700 microns.

Int Immunopharmacol, 2001 Jul, 1(7), 1249 - 59
Development of immunoadjuvants for immunotherapy of cancer; Azuma I et al.; Previously, we have reported that cell-wall skeleton (CWS) fraction was the major adjuvant-active principle of mycobacterial cells which were used in Freund's complete adjuvant (FCA) . We have described the biochemical and immunological properties of CWS of mycobacteria and related bacteria, especially the CWS of Mycobacterium bovis BCG strain (BCG-CWS) in detail . The effectiveness of BCG-CWS for the cancer immunotherapy in patients was shown in several clinical trials . On the action mechanism of BCG-CWS on host immune cells, we have suggested that dendritic cells and macrophages express two sorts of receptors, Toll-like receptors, TLR-2 and TLR-4, and a putative binding receptor for BCG-CWS, whose signaling pathways lead to a sufficient antigen-presenting state in the activation of the innate immune system . We have also reported the usefulness of synthetic immunoadjuvants such as muramyldipeptide (MDP) derivatives, trehalose-dimycolates (TDM) and DNA fraction for the application for the cancer and infectious diseases in experimental systems and cancer patients.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8381 - 7
Defining the roles of individual residues in the single-stranded DNA binding site of PcrA helicase; Dillingham MS et al.; Crystal structures and biochemical analyses of PcrA helicase provide evidence for a model for processive DNA unwinding that involves coupling of single-stranded DNA (ssDNA) tracking to a duplex destabilization activity . The DNA tracking model invokes ATP-dependent flipping of bases between several pockets on the enzyme formed by conserved aromatic amino acid residues . We have used site-directed mutagenesis to confirm the requirement of all of these residues for helicase activity . We also demonstrate that the duplex unwinding defects correlate with an inability of certain mutant proteins to translocate effectively on ssDNA . Moreover, the results define an essential triad of residues within the ssDNA binding site that comprise the ATP-driven DNA motor itself.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8181 - 8
Rescue of arrested replication forks by homologous recombination; Michel B et al.; DNA synthesis is an accurate and very processive phenomenon; nevertheless, replication fork progression on chromosomes can be impeded by DNA lesions, DNA secondary structures, or DNA-bound proteins . Elements interfering with the progression of replication forks have been reported to induce rearrangements and/or render homologous recombination essential for viability, in all organisms from bacteria to human . Arrested replication forks may be the target of nucleases, thereby providing a substrate for double-strand break repair enzyme . For example in bacteria, direct fork breakage was proposed to occur at replication forks blocked by a bona fide replication terminator sequence, a specific site that arrests bacterial chromosome replication . Alternatively, an arrested replication fork may be transformed into a recombination substrate by reversal of the forked structures . In reversed forks, the last duplicated portions of the template strands reanneal, allowing the newly synthesized strands to pair . In bacteria, this reaction was proposed to occur in replication mutants, in which fork arrest is caused by a defect in a replication protein, and in UV irradiated cells . Recent studies suggest that it may also occur in eukaryote organisms . We will review here observations that link replication hindrance with DNA rearrangements and the possible underlying molecular processes.

Bioorg Med Chem Lett, 2001 Jul 23, 11(14), 1939 - 42
2-Amino-4,6-diarylpyridines as novel ligands for the estrogen receptor; Henke BR et al.; We have prepared a novel series of 2-amino-4,6-diarylpyridines that function as ligands of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) . These compounds bind to both ERalpha and ERbeta with a modest selectivity for the alpha subtype . The most potent of these analogues, compound 19, has a K(i)=20nM at ERalpha . These molecules represent a novel template for designing potentially useful ligands for the estrogen receptor.

J Am Chem Soc, 2001 Jul 25, 123(29), 6983 - 8
The total synthesis of lipid I; VanNieuwenhze MS et al.; A total synthesis of lipid I (4), a membrane-associated intermediate in the bacterial cell wall (peptidoglycan) biosynthesis pathway, is reported . This highly convergent synthesis will enable further studies on bacterial resistance mechanisms and may provide insight toward the development of new chemotherapeutic agents with novel modes of action.

Mol Cells, 2001 Jun 30, 11(3), 303 - 11
In vitro selection of RNA against kanamycin B; Kwon M et al.; Aminoglycosides are well-known antibiotics that function by interacting with ribosomal RNA in bacteria . In order to understand the molecular details between RNA and the drug, RNA aptamer was selected against kanamycin B . After 12 cycles of selection, RNA was cloned and sequenced . Among 9 clones, sequences of three clones were identical, suggesting the selected RNA was enriched . Among the cloned RNA molecules, the triplicated RNA was the maximum binding RNA . It showed a 180 nM affinity (KD) to the cognate aminoglycoside, as measured by a surface plasmon resonance, and a competition assay using a fluorescence anisotropy technique . The affinity of the maximum binding RNA to a similar aminoglycoside, tobramycin, was much stronger than 12 nM of KD . The binding site of the aminoglycoside in the maximum binding RNA was a stem loop located at the end of the 5' region . A stem loop structural motif, found in this study, was similar to those previously reported, even though the sequences of the RNA were totally different from the known sequences of the aminoglycoside binding site of other aptamers . The present study suggests that the aminoglycoside-binding region in RNA does not have a sequence specificity, but has a shape-specific bulged stem loop, even though it has a nanomolar affinity.

Nature, 2001 Jun 14, 411(6839), 826 - 33
Plant pathogens and integrated defence responses to infection; Dangl JL et al.; Plants cannot move to escape environmental challenges . Biotic stresses result from a battery of potential pathogens: fungi, bacteria, nematodes and insects intercept the photosynthate produced by plants, and viruses use replication machinery at the host's expense . Plants, in turn, have evolved sophisticated mechanisms to perceive such attacks, and to translate that perception into an adaptive response . Here, we review the current knowledge of recognition-dependent disease resistance in plants . We include a few crucial concepts to compare and contrast plant innate immunity with that more commonly associated with animals . There are appreciable differences, but also surprising parallels.

Planta Med, 2001 Jun, 67(4), 372 - 4
SC-1, an antimycotic spirostan saponin from Solanum chrysotrichum; Alvarez L et al.; A new antimycotic steroidal saponin named SC-1 has been isolated from the leaves of Solanum chrysotrichum by bioassay-guided fractionation . The structure of SC-1 was characterized as 3-O-{beta-quinovopyranosyl(1-->6)-beta-glucopyranosyl(1-->6)-beta- glucopyranosyl}chlorogenin on the basis of spectral analyses and chemical evidence.

J Biol Chem, 2001 Sep 28, 276(39), 36681 - 6 Epub 2001 Jul 16.
Helix proximity in OxlT, the oalate:formate antiporter of oxalobacter formigenes . Cross-linking between TM2 and TM11; Kim YM et al.; Experiments were designed to evaluate the proximity of transmembrane helices two (TM2) and eleven (TM11) in the tertiary structure of OxlT, the oxalate:formate exchange transporter of Oxalobacter formigenes . A tandem duplication of the Factor Xa protease cleavage site (IEGRIEGR) was inserted into the central cytoplasmic loop of an OxlT cysteine-less derivative in which an endogenous cleavage site had been eliminated by mutagenesis (R248Q) . Using this host, double cysteine derivatives were constructed so as to pair one of seventeen positions in TM2 with one of four positions in TM11 . Following treatment of membrane vesicles with Cu(II)(1,10-phenanthroline)(3), molecular iodine, or N,N'-o-phenylenedimaleimide, samples were exposed to Factor Xa, and disulfide bond formation was assessed after SDS-polyacrylamide gel electrophoresis by staining with antibody directed against the OxlT C terminus . In the absence of disulfide bond formation, exposure to Factor Xa revealed the expected C-terminal 22-kDa fragment, a result unaffected by the presence of reductant . By contrast, after disulfide formation, OxlT mobility remained at 35 kDa, and appearance of the 22-kDa fragment required addition of 200 mm dithiothreitol prior to electrophoresis . The four TM11 positions chosen for cysteine substitution lie on a helical face known to interact with substrate . Similarly, TM2 positions supporting disulfide trapping were also confined to a single helical face . We conclude that TM2 and TM11 are in close juxtaposition to one another in the tertiary structure of OxlT.

J Steroid Biochem Mol Biol, 2001 Jun, 77(4-5), 229 - 38
Common phytochemicals are ecdysteroid agonists and antagonists: a possible evolutionary link between vertebrate and invertebrate steroid hormones; Oberdorster E et al.; Many plant compounds are able to modulate growth and reproduction of herbivores by directly interacting with steroid hormone systems . In insects, several classes of phytochemicals, including the phytoestrogens, interfere with molting and reproduction . We investigated whether the anti-ecdysone activity may be due to interaction with the ecdysone receptor (EcR) using a reporter-gene assay and a cell differentiation assay of an ecdysone-responsive cell line, Cl.8+ . We tested rutin (delays molt in insects); four flavones: luteolin and quercetin (metabolites of rutin), and apigenin and chrysin; and three non-flavones, coumestrol and genistein (both estrogenic) and tomatine (alters molt in insects) . None of the phytochemicals tested were ecdysone agonists in the reporter-gene assay, but the flavones were able to significantly inhibit EcR-dependent gene transcription . In the Cl.8+ cells, quercetin and coumestrol were mixed agonists/antagonists, while genistein, tomatine and apigenin showed a synergistic effect with ecdysteroid in the reduction of cell growth . We suggest that the rutin effects on molting in insects are most likely due to the metabolites, luteolin or quercetin, while tomatine acts via a non-EcR pathway . Flavones not only interact with EcR and estrogen receptor (ER), but also signal nitrogen-fixing bacteria to form root nodules . The NodD protein which regulates this symbiosis has two ligand-binding domains similar to human ERalpha . The evolutionary significance of these findings are discussed.

Biochemistry, 2001 Jul 24, 40(29), 8452 - 62
Interaction between cytochrome c2 and the photosynthetic reaction center from Rhodobacter sphaeroides: effects of charge-modifying mutations on binding and electron transfer; Tetreault M et al.; The electrostatic interactions governing binding and electron transfer from cytochrome c(2) (cyt c(2)) to the reaction center (RC) from the photosynthetic bacteria Rhodobacter sphaeroides were studied by using site-directed mutagenesis to change the charges of residues on the RC surface . Charge-reversing mutations (acid --> Lys) decreased the binding affinity for cyt c(2) . Dissociation constants, K(D) (0.3--250 microM), were largest for mutations of Asp M184 and nearby acid residues, identifying the main region for electrostatic interaction with cyt c(2) . The second-order rate constants, k(2) (1--17 x 10(8) M(-1) s(-1)), increased with increasing binding affinity (log k(2) vs log 1/K(D) had a slope of approximately 0.4), indicating a transition state structurally related to the final complex . In contrast, first-order electron transfer rates, k(e), for the bound cyt did not change significantly (<3-fold), indicating that electron tunneling pathways were unchanged by mutation . Charge-neutralizing mutations (acid --> amide) showed changes in binding free energies of approximately 1/2 the free energy changes due to the corresponding charge-reversing mutations, suggesting that the charges in the docked complex remain well solvated . Charge-enhancing mutations (amide --> acid) produced free energy changes of the same magnitude (but opposite sign) as changes due to the charge-neutralizing mutations in the same region, indicating a diffuse electrostatic potential due to cyt c(2) . A two-domain model is proposed, consisting of an electrostatic docking domain with charged surfaces separated by a water layer and a hydrophobic tunneling domain with atomic contacts that provide an efficient pathway for electron transfer.

Cell Tissue Res, 2001 Jun, 304(3), 391 - 9
Cysteine proprotease colocalizes with vitellogenin in compound granules of the cockroach fat body; Yin L et al.; A cysteine proprotease has been identified in developing embryos of the cockroach Blattella germanica and found to be a maternally encoded gene product that is transferred endocytically to the oocyte . The present study aims at establishing how this maternally derived proprotease is synthesized, packaged, and secreted during vitellogenesis . To this end, proprotease was localized immunocytochemically in the fat body of postmating females and its localization compared with that of vitellogenin over the same developmental periods . Fat bodies in cockroaches are comprised of two different cell types: trophocytes and bacteriocytes . Data show that proprotease and vitellogenin come to colocalize in compound granules of the fat body trophocytes . While synthesis of vitellogenin can be traced back to granules resulting from the coalescence of Golgi-derived vesicles in the trophocyte cytoplasm, proprotease appears to be localized predominantly on the cytolysosomes of both trophocytes and bacteriocytes . When probed with an anti-proprotease antiserum, bacteria are also positively labeled, regardless of whether they are segregated inside the cytolysosomes or free in the bacteriocyte cytoplasm . Since vitellogenin and proprotease colocalize within the same cell organelle, it is assumed that Golgi-derived vesicles, which contain vitellogenin, may fuse with cytolysosomes bearing proprotease to yield compound secretory granules . To account for the present observations, the origin and role of proprotease are discussed in relation to the turnover of bacteria in the fat body and to the requirements of endosymbiosis.

J Eukaryot Microbiol, 2001 Jul-Aug, 48(4), 425 - 32
Confusing selective feeding with differential digestion in bacterivorous nanoflagellates; Boenigk J et al.; Food selectivity and the mechanisms of food selection were analyzed by video microscopy for three species (Spumella, Ochromonas, Cafeteria) of interception-feeding heterotrophic nanoflagellates . The fate of individual prey particles, either live bacteria and/or inert particles, was recorded during the different stages of the particle-flagellate-interaction, which included capture, ingestion, digestion, and egestion . The experiments revealed species-specific differences and new insights into the underlying mechanisms of particle selection by bacterivorous flagellates . When beads and bacteria were offered simultaneously, both particles were ingested unselectively at similar rates . However, the chrysomonads Spumella and Ochromonas egested the inert beads after a vacuole passage time of only 2-3 min, which resulted in an increasing proportion of bacteria in the food vacuoles . Vacuole passage time for starved flagellates was significantly longer compared to that of exponential-phase flagellates for Spumella and Ochromonas . The bicosoecid Cafeteria stored all ingested particles, beads as well as bacteria, in food vacuoles for more then 30 min . Therefore "selective digestion" is one main mechanism responsible for differential processing of prey particles . This selection mechanism may explain some discrepancies of former experiments using inert particles as bacterial surrogates for measuring bacterivory.

EMBO Rep, 2001 Jul, 2(7), 598 - 603 Epub 2001 Jul 03.
A genetic look at the active site of RNA polymerase III; Rozenfeld S et al.; rpc160-112, a mutant of the RNA polymerase III active site, is corrected in vivo by six second-site mutants obtained by random mutagenesis . These mutants introduce single-site amino acid replacements at the two large subunits of the enzyme . The mutated motifs are conserved in RNA polymerases I and II and, for some of them, in the bacterial enzyme, thus delineating key elements of the active site in eukaryotic RNA polymerases.

Mol Microbiol, 2001 Jul, 41(1), 117 - 30
Regulation of late flagellar gene transcription and cell division by flagellum assembly in Caulobacter crescentus; Muir RE et al.; Biogenesis of the single polar flagellum of Caulobacter crescentus is regulated by a complex interplay of cell cycle events and the progression of flagellum assembly . The expression of class III/IV flagellar genes requires the assembly of an early flagellar basal body structure, encoded by class II genes, and is activated by the transcription factor FlbD . Previous experiments indicated that the class II flagellar gene, flbE, encoded a trans-acting factor that was required for FlbD activity . Here, using mutant alleles of flbE we have determined that FlbE is either a structural component of the flagellum or is required for flagellar assembly and does not, as originally proposed, function as a trans-acting factor . We also demonstrate that two deleted derivatives of flbE have a dominant negative effect on the transcriptional activation of class III/IV flagellar genes that can be relieved by a gain-of-function mutation in flbD called bfa . This same mutation in flbD has been shown to restore class III/IV transcription in the absence of early class II flagellar assembly . These deleted mutants of flbE also exhibited a filamentous cell phenotype that was indistinguishable from that previously observed in class II flagellar mutants . Introduction of a flbD-bfa mutation into these cells expressing the deleted alleles of flbE, as well as several class II mutant strains, restored normal cell division and FtsZ localization . These results suggest that class III/IV transcription and a step in cell division are coupled to flagellar assembly by the same genetic pathway.

J Intern Med, 2001 Jul, 250(1), 67 - 71
Chlamydia pneumoniae and luminal narrowing after coronary angioplasty; Mattila KJ et al.; OBJECTIVES: Numerous studies have linked Chlamydia pneumoniae with atherosclerotic vessel disease and a trend for an association of the bacteria with restenosis after percutaneous transluminal coronary angioplasty (PTCA) has also been observed . The aim of this study was to assess the role of Chlamydia pneumoniae in the luminal narrowing taking place after PTCA . DESIGN: A noninterventional 6-month follow-up study . SETTING: A university hospital . SUBJECTS: A total of 122 patients with angiographically proven coronary heart disease (CHD) referred for PTCA . INTERVENTIONS: None . MAIN OUTCOME MEASURES: The degree of luminal narrowing in the coronary arteries following coronary angioplasty . RESULTS: The levels of C . pneumoniae antibodies (IgG, IgA and IgM classes) and immune complexes were not associated with luminal narrowing after PTCA in multivariate analyses whilst smoking, plasma endothelin levels and diabetes were . The serologic parameters did not change during the follow up either . CONCLUSIONS: These results do not support a role for C . pneumoniae in luminal narrowing following PTCA.

Eur J Biochem, 2001 Jul, 268(14), 3907 - 15
Concurrent maltodextrin and cellodextrin synthesis by Fibrobacter succinogenes S85 as identified by 2D NMR spectroscopy; Matulova M et al.; 1D and 2D NMR experiments were used to analyse the synthesis of various metabolites by resting cells of Fibrobacter succinogenes S85 when incubated with {1-(13)C}glucose, in both extracellular and cellular media . Besides the expected glycogen, succinate, acetate, glucose-1-P and glucose-6-P, maltodextrins and cellodextrins were detected . Maltodextrins were excreted into the external medium . They were found to have linear structures with a maximum degree of polymerization (DP) of about 6 or 7 units . Cellodextrins were located in the cells (cytoplasm and/or periplasm), and their DP was < or = 4 . Both labelled (1-(13)C and 6-(13)C) and unlabelled maltodextrins and cellodextrins were detected, showing the contribution of carbohydrate cycling in F . succinogenes, including the reversal of glycolysis and the futile cycle of glycogen . The mechanisms of these oligosaccharide syntheses are discussed.

Curr Genet, 2001 Jun, 39(4), 244 - 52
Identification and characterization of a gene encoding a subtilisin-like serine protease induced during the vegetative incompatibility reaction in Podospora anserina; Paoletti M et al.; In the filamentous fungi, cell fusion between unlike individuals generally triggers a cell-death reaction known as vegetative incompatibility . In Podospora anserina, it was shown that, during this cell-death reaction, there is a strong increase in proteolytic activity . Here, we report the purification of a 36-kDa protease that is induced during the incompatibility reaction . An internal peptide of this protein displayed a strong similarity with the PEPC subtilisin-like serine protease from Aspergillus niger . This led us to use the pepC gene as a probe to clone the homologous gene from P . anserina, which we called pspA . The expression of pspA is upregulated at the transcript level during the progress of the incompatibility reaction . This induction is diminished in strains bearing suppressors of the vegetative incompatibility reaction . The fact that pspA is homologous to PrB, a vacuolar protease involved in autophagy in yeast, suggests that the incompatibility cell-death reaction and autophagy might be related processes.

Biochim Biophys Acta, 1971 Dec 28, 251(3), 388 - 92
Anomalous behavior or ribonuclease A on Sephadex G-100; Mitch WE Jr et al.; Ribonuclease A was found to behave in an unusual fashion on a Sephadex gel column . Though ribonuclease A produces a single, well-defined protein peak on elution, enzyme activity can be detected several void volumes after the protein peak . A second unrelated protein added to the column will displace further activity as will 0.5 M phosphate buffer . This additional activity, apparently due to ribonuclease A or an active fragment of the enzyme, would appear to make this enzyme unsuitable for use as a standard in molecular weight determinations of other nucleases.

Rocz Panstw Zakl Hig, 2001, 52(1), 1 - 18
{Legionella sp.--laboratory diagnosis of infections in humans and detection in environmental water}; Stypulkowska-Misiurewicz H et al.; Legionella sp . is the etiological agent of Legionnaires Disease and the Pontiac Fever, one of the new emerging diseases . Legionella are common in natural environment but in low number of bacteria cells, especially in comparison to other bacterial flora . Because of special nutritious requirement and long time of Legionella generation, the isolation of bacteria and the diagnosis of legionellosis cause many problems . In this publication some methods for detection of Legionella sp . in environmental water and clinical samples are presented and evaluated.

Rinsho Ketsueki, 2001 May, 42(5), 414 - 9
{Hemophagocytic syndrome with hemophagocytes in the peripheral blood}; Kuwata K et al.; A 79-year-old man developed a high fever, facial erythema, anemia and thrombocytopenia during conservative therapy for ischemic colitis . Peripheral hemophagocytes (PHP) were identified in smear specimens of peripheral blood, and hemophagocytes also showed proliferation in the bone marrow . After treatment with steroid and antibiotics under a diagnosis of bacteria-associated hemophagocytic syndrome, the patient recovered rapidly . Although the prognosis of hemophagocytic syndrome (HPS) depends on the underlying disease, any delay in diagnosis can sometimes result in a poor outcome in cases of infection-associated hemophagocytic syndrome . In the present case, early diagnosis of bacteria-associated hemophagocytic syndrome was made by detection of PHP . The appearance of PHP in virus-associated hemophagocytic syndrome (VAHS) and after administration of macrophage colony stimulating factor has been described . However, the significance and cytological characteristics of PHP have been unknown . In this report, we propose that PHP can be a useful indicator for early diagnosis of HPS, and we report 7 additional cases in which the PHP was detected retrospectively . The cytological characteristics and biological significance of PHP are discussed.

Rev Inst Med Trop Sao Paulo, 2001 May-Jun, 43(3), 125 - 31
Clinical patterns and seasonal trends in respiratory syncytial virus hospitalizations in São Paulo, Brazil; Vieira SE et al.; The respiratory viruses are recognized as the most frequent lower respiratory tract pathogens for infants and young children in developed countries but less is known for developing populations . The authors conducted a prospective study to evaluate the occurrence, clinical patterns, and seasonal trends of viral infections among hospitalized children with lower respiratory tract disease (Group A) . The presence of respiratory viruses in children's nasopharyngeal was assessed at admission in a pediatric ward . Cell cultures and immunofluorescence assays were used for viral identification . Complementary tests included blood and pleural cultures conducted for bacterial investigation . Clinical data and radiological exams were recorded at admission and throughout the hospitalization period . To better evaluate the results, a non- respiratory group of patients (Group B) was also constituted for comparison . Starting in February 1995, during a period of 18 months, 414 children were included- 239 in Group A and 175 in Group B . In Group A, 111 children (46.4%) had 114 viruses detected while only 5 children (2.9%) presented viruses in Group B . Respiratory Syncytial Virus was detected in 100 children from Group A (41.8%), Adenovirus in 11 (4.6%), Influenza A virus in 2 (0.8%), and Parainfluenza virus in one child (0.4%) . In Group A, aerobic bacteria were found in 14 cases (5.8%) . Respiratory Syncytial Virus was associated to other viruses and/or bacteria in six cases . There were two seasonal trends for Respiratory Syncytial Virus cases, which peaked in May and June . All children affected by the virus were younger than 3 years of age, mostly less than one year old . Episodic diffuse bronchial commitment and/or focal alveolar condensation were the clinical patterns more often associated to Respiratory Syncytial Virus cases . All children from Group A survived . In conclusion, it was observed that Respiratory Syncytial Virus was the most frequent pathogen found in hospitalized children admitted for severe respiratory diseases . Affected children were predominantly infants and boys presenting bronchiolitis and focal pneumonias . Similarly to what occurs in other subtropical regions, the virus outbreaks peak in the fall and their occurrence extends to the winter, which parallels an increase in hospital admissions due to respiratory diseases.

FEMS Microbiol Ecol, 2001 Jul, 36(2-3), 185 - 191
Geographic distribution of Desulfitobacterium frappieri PCP-1 and Desulfitobacterium spp . in soils from the province of Quebec, Canada; Lanthier M et al.; The presence of indigenous Desulfitobacterium species in 44 soil samples taken from various sites in the southern part of the province of Quebec (Canada) and four from locations outside Quebec was investigated . Twenty-four of these soils were sampled from contaminated industrial sites . Indigenous Desulfitobacterium bacteria from soil samples were enriched by cultivation in anaerobic soil slurry culture . Total DNA was then extracted from these slurries and polymerase chain reaction (PCR) amplifications were performed with primers targeting 16S ribosomal RNA gene sequences of Desulfitobacterium spp . and of Desulfitobacterium frappieri PCP-1 . A positive PCR signal was obtained in 31 soil slurry cultures . Resolution of single-strand DNAs of some of the PCR products by a single-strand conformational polymorphism protocol suggests that more than one species of Desulfitobacterium were present in the corresponding slurry cultures . These results suggest that Desulfitobacterium are ubiquitous in soils in the province of Quebec, especially in soils from the St . Lawrence valley and the southern part of the province.

Clin Orthop, 2001 Jul, (388), 143 - 6
Fistula between a total hip arthroplasty and the rectum: a case report; Bach CM et al.; An 81-year-old man was referred to the authors for examination of the gastrointestinal tract . A proctoscopy revealed a draining sinus tract in the terminal rectum . Plain radiographs revealed a failed total hip arthroplasty that had migrated into the pelvis . Hip aspiration revealed an infection with bacteria commonly found in the gastrointestinal tract . A fistulogram confirmed a connection between the rectum and the hip replacement . The development of a fistula between the colon and the hip is extremely uncommon . A fistula between the hip and the rectum is a previously unreported complication of total hip arthroplasty.

Mikrobiologiia, 2001 May-Jun, 70(3), 405 - 11
{Effect or preliminary filtration on the functional characteristics of bacterioplankton from Lake Khanka}; Shchur LA et al.; The dependence of the functional characteristics of bacterioplankton from the loess of Lake Khanka on the pore size of filtering materials was investigated . Soluble organic matter (SOM), bacteria, and bacterial consumers adsorbed on particles suspended in the lake water were found to filter differently depending on the pore size of the filtering material . Filters with pore size 4.5 microns (filters II) retained up to 20% of SOM and 20-30% of bacterial cells . Filters III with pore size 2.87 microns retained almost 50% SOM and about 40% of bacteria . The double layer of gauze no . 72 (referred to as filter I) with pores size 40 microns was unable to completely retain bacterial consumers . In the case of filtrates I and II, the generation time of bacterioplankton decreased with its increasing average daily concentration . In the case of filtrate III, the generation time of bacterioplankton was minimum and did not depend on its concentration . Oxygen consumption rates per one bacterial cell and per unit biomass in filtrates increased with decreasing pore size of the filters through which they had passed . The bacterial biomass and oxygen consumption rate increased exponentially in filtrates III and logarithmically in filtrates I.

Mikrobiologiia, 2001 May-Jun, 70(3), 337 - 47
{Ultrastructure of the cell surface of heteromorphic Nostoc muscorum CALU 304 cocultured with Rauwolfia tissue}; Gorelova OA; The ultrastructure of the heteromorphic cells (HMCs) of the cyanobacterium Nostoc muscorum CALU 304 grown in pure culture, monoculture, and a mixed culture with the Rauwolfia callus tissue was studied . The comparative analysis of the cell surface of HMCs, the frequency of the generation of cell forms with defective cell walls (DCWFs), including protoplasts and spheroplasts, and the peculiarities of the cell surface ultrastructure under different growth conditions showed that, in the early terms of mixed incubation, the callus tissue acts to preserve the existing cyanobacterial DCWFs, but begins to promote their formation in the later incubation terms . DCWFs exhibited an integrity of their protoplasm and were metabolically active . It is suggested that structural alterations in the rigid layer of the cell wall may be due to the activation of the murolytic enzymes of cyanobacteria and the profound rearrangement of their peptidoglycan metabolism caused by the Rauwolfia metabolites diffused through the medium . These metabolites may also interfere with the functioning of the universal cell division protein of bacteria, FtsZ . In general, the Rauwolfia callus tissue promoted the unbalanced growth of the cyanobacterium N . muscorum CALU 304 and favored its viability in the mixed culture . The long-term incubation of the Rauwolfia tissue with the N . muscorum CALU 304 cells led to their transformation to L-forms.

Przegl Lek, 2001, 58(1), 31 - 3
{Is Helicobacter pylori infection a zoonosis?}; Mach T; On the basis of papers review there are presented the possibilities of Helicobacter pylori (H . pylori) infection in animals and the route of transmission . The discovery of bacteria has changed our approach to the etiopathogenesis and treatment of some digestive tract diseases . The prevalence of H . pylori in some populations and socioeconomic factors confirms the man-to-man mode of transmission with a human being as a main reservoir . There was confirmed the correlation between the stomach colonization of H . pylori in some animals, particularly those living in a human environment, and the route of transmission . Besides many studies performed on domestic animals and primate monkeys it is not clear, whether H . pylori infection can be accepted as a zoonosis . Many investigators confirmed the presence of bacteria in the animal stomach . There is a lack of data, whether the bacteria coming from other sources than food infected by the man could colonize the stomach, and whether it can cause the gastric mucosa changes by the viable forms presented in feces . If scientists are still interested in H . pylori the mentioned problems will probably be elucidated.

Vox Sang, 2001 Apr, 80(3), 135 - 41
Evaluation of donor arm disinfection techniques; McDonald CP et al.; BACKGROUND AND OBJECTIVE: To validate a standardized optimal national procedure for donor arm disinfection . MATERIALS AND METHODS: A direct swabbing and plating technique was used to enumerate bacteria present on the arm pre- and postdisinfection . Twelve donor arm disinfection techniques were evaluated . RESULTS: The Medi-Flex Adapted method, consisting of a two-stage process with an initial application of isopropyl alcohol followed by tincture of iodine, produced the best arm disinfection . A percentage reduction in bacterial counts of 99.79% (logarithmic reduction of 2.67) was obtained . Postdisinfection, 70% of donors had bacterial counts of zero, and 98% had counts of 10 or less . CONCLUSION: The Medi-Flex disinfection method offers the English National Blood Service a validated, optimal 'best practice' disinfection technique and should contribute significantly to the reduction in risk of transmission of bacteria by transfusion.

Dtsch Tierarztl Wochenschr, 2001 Jun, 108(6), 236 - 43
Intracellular trafficking of Mycobacterium avium ss . paratuberculosis in macrophages; Cheville NF et al.; The granulomatous enteric lesions of cattle with Johne's disease are composed of infected macrophages, and grow by accumulation, re-infection, and expansion of macrophage populations in the intestinal wall . We have examined the growth of bacteria in macrophages to define characteristics of intracellular trafficking for exocytosis, replication, and antigen presentation . Using immunocytochemical markers for light, confocal and electron microscopy, we have examined potential pathway tropisms using data for bacterial attachment, phagosomal acidification, phagolysosomal degradation and apoptosis . Our hypotheses are that pathogenic/wild-type strains block phagosomal acidification so that the phagosome fails to obtain markers of the late phagosome and phagolysosome, and this leads to the replication pathway within bacteriophorous vacuoles . Non-pathogenic strains appear to be processed to exocytosis, and avirulent mutant strains may be degraded and have preference of antigen processing pathways that involve transport vesicles bearing MHC II antigens . Pathogenicity in a nude mouse model of intestinal infection reveals lesion development and confirms pathway preferences of virulent strains for bacteriophorous vacuole formation.

Acad Med, 2001 Jul, 76(7), 684 - 92
Harvard v . Canada: the myc mouse that still squeaks in the maze of biopatent law; Deftos LJ; The Canadian Supreme Court will soon make a decision about Harvard University's long-standing application for a Canadian patent on a mouse transgenic for the myc oncogene . That decision could reignite in North America the controversy that continues in Europe and elsewhere to surround the patenting of life forms . The tortuous steps in this 15-year patent maze are marked by the arguments about life patents that attended the U.S . Supreme Court's decision in Diamond v . Chakrabarty . This patent dispute about oil-digesting bacteria cracked open the door for animal patents in the United States and other countries, even though the legal arguments involved were based on patent applications for corn seeds and oysters, not mammals . The patent challenge to the Harvard mouse by the Canadian government now threatens to close this door in Canada . The arguments against life patents are commonly based on moral and religious grounds that regard the sanctity of life and oppose its commodification . The most compelling arguments for such patents are based on the benefits they deliver through commercial exploitation of inventions . The debate about patenting animals has been more heated outside North America and cacaphonic in the Third World . However, the Canadian debate could be amplified by the U.S . Supreme Court's recent entry on the biopatent stage through the side door of a new corn seed patent dispute . A narrow legal analysis by the Canadian Supreme Court would award the mouse patent to Harvard, while a policy analysis might support the government's challenge of the patent . Although the impact of the Harvard mouse patent process in Canada could be just a squeak, opponents of patenting life can mount the myc mouse to once again roar their opposition to animal patents . And the sound could resonate through the arguments about both biopatents and human cloning, with potentially important effects for academia, industry, and the public.

Arch Virol, 2001, 146(5), 843 - 57
Frequency of morphological phage descriptions in the year 2000 . Brief review; Ackermann HW; Over 5100 bacterial viruses have been examined in the electron microscope since 1959 . About 4950 phages (96%) are tailed and only 186 phages (3.6%), are cubic, filamentous, or pleomorphic . Phages belong to 13 virus families and occur in over 140 bacterial genera . Phages are listed by morphotypes and host genera . Siphoviridae or phages with long, noncontractile tails comprise 61% of tailed phages . The distribution of phages in different bacterial phylogenetic divisions is shown.

J Bone Miner Metab, 2001, 19(4), 220 - 7
Establishment and characterization of macrophage-like cell lines expressing osteoclast-specific markers; Sakiyama H et al.; Osteoclasts differentiate from hematopoietic precursors of the monocyte/macrophage lineage to mononuclear preosteoclasts and multinuclear mature osteoclasts . In the present study, we report on the establishment of macrophage like cell lines from mouse bone marrow by coculturing bone marrow cells with mouse chondrocytes . Isolated clones (MLC-6 and MLC-7 cells) expressed fully differentiated osteoclast markers, such as calcitonin receptors, vitronectin receptors, tartrate-resistant acid phosphatase and vacuolar H+ -ATPase, in the absence of osteoclast differentiation factor/osteoprotegerin ligand/RANKL/TRANCE, which was essential for osteoclast differentiation . Most clones also maintained expression of a macrophage-associated protein, namely non-specific esterase . Both MLC-6 and MLC-7 cells released cathepsin K into the culture medium . Both clones resolved dentine slices when cocultured with the osteoblast cell line ST2 . The cloned cell lines are considered to be useful tools in the study of osteoclast differentiation.

Infect Dis Clin North Am, 2001 Jun, 15(2), 551 - 65, x
Infectious complications in asplenic hosts; Sumaraju V et al.; Hyposplenism, secondary to splenectomy or disease state, predisposes the host to overwhelming infection with certain bacteria, such as S . pneumoniae . Recognition of the hyposplenic state and preventive measures, including patient education and vaccination, appear to reduce the rate of this highly fatal infection . In addition to considering chemoprophylaxis, a clinician should promptly evaluate or empirically treat all febrile episodes in hyposplenic patients.

Infect Immun, 2001 Aug, 69(8), 5186 - 8
Effect of low-dose antigen exposure on development of immunity to Helicobacter pylori infection in mice; Radcliff FJ et al.; The effect of low-dose antigen exposure on the development of immunity to Helicobacter pylori infection was studied in outbred mice . Animals that were primed with a subinfectious number of H . pylori bacteria exhibited significantly lower bacterial loads after challenge with an infectious dose of pathogen (versus controls, P < 0.05).

Infect Immun, 2001 Aug, 69(8), 5121 - 30
Arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion; Lourbakos A et al.; Periodontitis is a chronic inflammatory disease affecting oral tissues . Oral epithelial cells represent the primary barrier against bacteria causing the disease . We examined the responses of such cells to an arginine-specific cysteine proteinase (RgpB) produced by a causative agent of periodontal disease, Porphyromonas gingivalis . This protease caused an intracellular calcium transient in an oral epithelial cell line (KB), which was dependent on its enzymatic activity . Since protease-activated receptors (PARs) might mediate such signaling, reverse transcription-PCR was used to characterize the range of these receptors expressed in the KB cells . The cells were found to express PAR-1, PAR-2, and PAR-3, but not PAR-4 . In immunohistochemical studies, human gingival epithelial cells were found to express PAR-1, PAR-2, and PAR-3 on their surface, but not PAR-4, indicating that the cell line was an effective model for the in vivo situation . PAR-1 and PAR-2 expression was confirmed in intracellular calcium mobilization assays by treatment of the cells with the relevant receptor agonist peptides . Desensitization experiments strongly indicated that signaling of the effects of RgpB was occurring through PAR-1 and PAR-2 . Studies with cells individually transfected with each of these two receptors confirmed that they were both activated by RgpB . Finally, it was shown that, in the oral epithelial cell line, PAR activation by the bacterial protease-stimulated secretion of interleukin-6 . This induction of a powerful proinflammatory cytokine suggests a mechanism whereby cysteine proteases from P . gingivalis might mediate inflammatory events associated with periodontal disease on first contact with a primary barrier of cells.

Infect Immun, 2001 Aug, 69(8), 5088 - 97
Bartonella henselae induces NF-kappaB-dependent upregulation of adhesion molecules in cultured human endothelial cells: possible role of outer membrane proteins as pathogenic factors; Fuhrmann O et al.; The endothelium is a specific target for Bartonella henselae, and endothelial cell infection represents an important step in the pathogenesis of cat scratch disease and bacillary angiomatosis . Mechanisms of Bartonella-endothelial cell interaction as well as signaling pathways involved in target cell activation were analyzed . B . henselae strain Berlin-1, isolated from bacillary angiomatosis lesions of a human immunodeficiency virus-infected patient, potently stimulated human umbilical cord vein endothelial cells (HUVEC), as determined by NF-kappaB activation and enhanced adhesion molecule expression . These effects were accompanied by increased PMN rolling on and adhesion to infected endothelial cell monolayers, as measured in a parallel-plate flow chamber assay . Monoclonal antibodies against E-selectin significantly reduced PMN rolling and adhesion . In our hands, B . henselae Berlin-1 was substantially more active than the typing strain B . henselae ATCC 49882 . E-selectin and ICAM-1 upregulation occurred for up to 9 days, as verified by Northern blotting and cell surface enzyme-linked immunosorbent assay . Induction of adhesion molecules was mediated via NF-kappaB activation and could be blocked by a specific NF-kappaB inhibitor . Additional studies indicated that B . henselae-induced effects did not require living bacteria or Bartonella lipopolysaccharides . Exposure of HUVEC to purified B . henselae outer membrane proteins (OMPs), however, reproduced all aspects of endothelial cell activation . In conclusion, B . henselae, the causative agent of cat scratch disease and bacillary angiomatosis, infects and activates endothelial cells . B . henselae OMPs are sufficient to induce NF-kappaB activation and adhesion molecule expression followed by enhanced rolling and adhesion of leukocytes . These observations identify important new properties of B . henselae, demonstrating its capacity to initiate a cascade of events culminating in a proinflammatory phenotype of infected endothelial cells.

Infect Immun, 2001 Aug, 69(8), 4808 - 15
Endogenous interleukin-12 is not required for resolution of Chlamydophila abortus (Chlamydia psittaci serotype 1) infection in mice; Del Rio L et al.; A Th1 immune response involving gamma interferon (IFN-gamma) production is required to eliminate Chlamydophila abortus infections . In this study, the role of interleukin-12 (IL-12) in protecting against C . abortus infection was investigated using IL-12(-/-) and wild-type (WT) C57BL/6 mice to determine the role of this Th1-promoting cytokine . IL-12(-/-) mice were able to eliminate the C . abortus infection in a primary infection . However, there was a delay in the clearance of bacteria when IL-12(-/-) mice were infected with a sublethal dose of C . abortus, the delay being associated with a lower production of IFN-gamma . The low level of IFN-gamma was essential for survival of IL-12(-/-) infected mice . Both WT and IL-12(-/-) mice developed a Th1 immune response against C . abortus infection, since they both produced IFN-gamma and immunoglobulin G2a antibody isotype . In addition, when mice were given a secondary infectious challenge with C . abortus, a protective host response which resolved the secondary infection was developed by both WT and IL-12(-/-) mice . The lack of IL-12 resulted in few infiltrating CD4(+) T cells in the liver relative to the number in WT mice, although the number of CD8(+) T cells was slightly higher . The more intense Th1 response presented by WT mice may have a pathogenic effect, as the animals showed higher morbidity after the infection . In conclusion, these results suggest that although IL-12 expedites the clearance of C . abortus infection, this cytokine is not essential for the establishment of a protective host response against the infection.

Nutr Res, 2001 Jul, 21(7), 1053 - 1066
The influence of dietary proteins on colon cancer risk; McIntosh GH et al.; Experimental evidence is accumulating from animal models and in vitro data which shows that dietary proteins can influence cancer expression, some having a promotional influence, others a preventative effect relative to an arbitrarily established standard diet . This result will to a degree be determined by the nature of the cancer model under study . Dairy proteins have been shown to be relatively protective when compared with defatted soybean meal and cooked red meat in the rat dimethylhydrazine-induced (DMH) colon cancer model . Some epidemiological evidence supports these experimental observations . Both protein and fat appear to be influencing outcome, with potential for interactive effects . A number of possible mechanisms have been postulated as to how these proteins and closely associated factors could be influencing colon cancer risk, an area that deserves more investigation . Combinations of foods such as dairy foods with cereals and/or probiotic bacteria provide potentially interesting alliances in reducing colon cancer risk . The combining of relatively protective agents deserves more investigation as to its potential, in devising functional foods and diets worthy of further evaluation, in animal models of cancer, and human intervention studies using relevant endpoint markers.

Chemistry, 2001 Jun 1, 7(11), 2449 - 58
Multipeptide-metalloporphyrin assembly on a dendrimer template and photoinduced electron transfer based on the dendrimer structure; Sakamoto M et al.; To construct an artificial photosynthetic system, peptide dendrimers {n-(X-HLY)PAMAMs: X = R, E; Y= L, F; n=4, 8, 16, 32 and 64 segments}, in which amphiphilic alpha-helix peptides (X-HLY: R-HLL, E-HLL and R-HLF) were introduced at the end groups of polyamidoamine dendrimers (PAMAMs), were designed and synthesized . The peptide dendrimers 64-(X-HLY)PAMAMs are novel synthetic biopolymers with an enormous molecular weight, about 160 kDa, and with a regulated amino acid sequence and three-dimensional conformation . The peptide dendrimers bound Fe(III)- or Zn(II)-mesoporphyrin IX per two alpha-helices; this afforded a multimetalloporphyrin assembly similar to the natural light-harvesting antennae in photosynthetic bacteria . Circular dichroism studies and peroxidase activity measurements revealed that metalloporphyrins were coordinated to the peptide dendrimers in a regulated manner and packed more densely with the growth of the dendrimer generation . Fluorescence quenching and photoreduction studies with methylviologen demonstrated that the photoinduced electron-transfer function with the peptide dendrimer-multi-Zn-MP was accomplished more effectively as the dendrimer generation increased . Thus, the three-dimensional assembly of metalloporphyrins and peptides in the dendrimer was an effective module for light-harvesting antennae in an artificial photosynthetic system.

Microbes Infect, 2001 Jul, 3(8), 655 - 77
Immunity to Bordetella pertussis; Mills KH; Bordetella pertussis exploits extracellular and intracellular niches in the respiratory tract and a variety of immune evasion strategies to prolong its survival in the host . This article reviews evidence of complementary roles for cellular and humoral immunity in protection . It discusses the effector mechanisms of bacterial elimination, the strategies employed by the bacteria to subvert protective immune responses and the immunological basis for systemic and neurological responses to infection and vaccination.

FEMS Microbiol Lett, 2001 Jul 10, 201(1), 59 - 64
Cloning and characterisation of a hyp gene cluster in the filamentous cyanobacterium Nostoc sp . strain PCC 73102; Hansel A et al.; Maturation of {NiFe}-hydrogenases requires the action of several groups of accessory genes . Homologues of one group of these genes, the so-called hyp genes, putatively encoding proteins participating in the formation of an active uptake hydrogenase in the filamentous, heterocyst-forming cyanobacterium Nostoc PCC 73102, were cloned . The cluster, consisting of hypF, hypC, hypD, hypE, hypA, and hypB, is located 3.8 kb upstream from the uptake hydrogenase-encoding hupSL . Gene expression analyses show that these hyp genes are, like hupL, transcribed under N(2)-fixing but not under non-N(2)-fixing growth conditions . Furthermore, the six hyp genes are transcribed together with an open reading frame upstream of hypF, as a single mRNA . Analysis of the DNA region upstream of the experimentally determined transcriptional start site revealed putative -10 and -35 sequence elements and putative binding sites for the global nitrogen regulator NtcA.

FEMS Microbiol Lett, 2001 Jul 10, 201(1), 21 - 7
Beta-galactofuranoside glycoconjugates on conidia and conidiophores of Aspergillus niger; Wallis GL et al.; Galactose in the furanoic conformation appears to be limited to bacteria and lower eukaryotes . Galactofuranoic (Galf)-containing glycoconjugates that occur in organisms pathogenic or allergenic to man are frequently antigenic and immunodominant . We have used an immunochemical approach, employing a monoclonal antibody that recognises Galf epitopes, to investigate the presence of Galf-containing glycoconjugates within conidia and conidiophores of Aspergillus niger . ELISA and immunofluorescence microscopy indicated that specific and saturable binding sites were found on both . Inhibition studies confirmed that this binding was to Galf-containing glycoconjugates . Interestingly, the conidiophore heads were particularly rich in these glycoconjugates . Western blotting identified a Galf glycoprotein of 150-200 kDa from disrupted conidia.

Water Sci Technol, 2001, 43(11), 315 - 22
Control and separation of algae particles from WSP effluent by using floating aquatic plant root mats; Kim Y et al.; In this paper, the potential uses of water hyacinth and its root mats for separating algae particles in the effluent from waste stabilization ponds (WSPs) were discussed . Pilot-scale integrated processes consisting of WSPs and multiple WHPs (water hyacinth ponds) were operated in order to extract effects of the root mats on the reduction of algae concentrations . Root mats in the bottom of WHPs separated significant amount of the algae cells through attachment as the effluent from WSPs passed through them . Attachment of the algae particles to the surface of live roots was found to be similar to adsorption phenomena but it lasted even at saturation, probably due to the continuous reproduction of active attachment sites by detachment and growth of the roots . Additionally, this paper discusses attachment mechanisms and other issues concerning design and polishing of the WSPs effluent by WHPs.

Water Sci Technol, 2001, 43(11), 267 - 74
Inhibition by chromium and cadmium of anaerobic acidogenesis; Yu HQ et al.; The effects of chromium (III) and cadmium on the anaerobic acidogenesis of a simulated dairy waste were examined using serum vials . At Cd dosages less than 20 mg/l, the acidogenesis process was enhanced by the dosage, resulting in a higher degree of acidification, protein conversion, and hydrogen production than the control . At dosages over 20-mg/l, Cd inhibited the acidogenesis . The Cr (III) dosage of 5 mg/l reduced overall volatile fatty acid and alcohol generation, degree of acidification, conversions of lactose, lipid and protein, and total biogas production, with the exception of accumulation of hydrogen and propionate . At dosages exceeding 5 mg/l, Cr (III) had a severe inhibition on the acidogenesis . The Cd concentrations which caused a 50% reduction in total volatile fatty acid and alcohol production, degree of acidification and cumulative gas production were higher than the corresponding values caused by Cr (III), suggesting that Cr (III) was more toxic to acidogenic bacteria than Cd.

Prikl Biokhim Mikrobiol, 2001 May-Jun, 37(3), 274 - 8
{Immunomodulatory properties of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate and search for its new derivatives}; Potapov VD et al.; A strong immunomodulatory effect of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC) responsible for the survival of bacteria was shown on isolated macrophages and in experimental infections in mice (typhoid and tularemia) . Derivatives of MEC were found by 1H-NMR spectroscopy under stress conditions in colorless mutants of the bacteria and isolated to be subsequently purified and used for modulation of the immune system of animals.

Cell Motil Cytoskeleton, 2001 Jun, 49(2), 104 - 11
Interaction of elongation factor 1alpha from Zea mays (ZmEF-1alpha) with F-actin and interplay with the maize actin severing protein, ZmADF3; Gungabissoon RA et al.; EF-1alpha is an abundant eukaryotic protein whose principle function appears to be to bind aminoacyl-tRNA to the ribosome . However, it is also known that EF-1alpha from other sources binds both microtubules and microfilaments . We report the expression of Zea mays EF-1alpha (ZmEF-1alpha) in bacteria and that this protein has similar actin-binding properties as other EF-1alpha members . ZmEF-1alpha bundles actin filaments at low pH (6.5) and inhibits the addition of monomer at both filament ends, possibly as a consequence . ZmEF-1alpha binds actin filaments at all pH values tested (pH 6.0-8.0), indicating that one actin binding site is not pH sensitive . One of the actin-binding sites was determined to reside within domain I (1-223) of ZmEF-1alpha, but this domain did not affect the kinetics of polymerisation . We show that the bundling activity of ZmEF-1alpha is modulated by ZmADF3 a (a Zea mays ADF/cofilin), an actin filament severing protein, in vitro . Bundling of actin filaments caused by ZmEF-1alpha was enhanced in the presence of ZmADF3 . The pH-dependent activities of both proteins in vitro suggests that they may work together to respond to temporal and spatial intracellular pH changes to regulate the pattern of the growth of plant cells.

J Infect Dis, 2001 Aug 1, 184(3), 385 - 9 Epub 2001 Jul 03.
Detection of serum IgG antibodies specific for Wolbachia surface protein in rhesus monkeys infected with Brugia malayi; Punkosdy GA et al.; The mechanism of lymphedema development in individuals with lymphatic filariasis is presently poorly understood . To investigate whether Wolbachia, symbiotic bacteria living within filarial nematodes, may be involved in disease progression, Wolbachia-specific immune responses were assayed in a group of Brugia malayi-infected rhesus monkeys . Serum IgG antibodies specific for a major Wolbachia surface protein (WSP) were detected in 2 of 12 infected monkeys . It is interesting that both of these monkeys developed lymphedema after becoming amicrofilaremic . WSP-specific antibody responses were temporally associated with increases in antifilarial IgG1 antibodies as well as lymphedema development . These findings suggest that Wolbachia may be important in understanding disease caused by filarial worms.

J Infect Dis, 2001 Aug 1, 184(3), 308 - 14 Epub 2001 Jul 10.
Protective anti-Helicobacter immunity is induced with aluminum hydroxide or complete Freund's adjuvant by systemic immunization; Gottwein JM et al.; To determine whether systemic immunization against Helicobacter pylori could be achieved with an adjuvant approved for human use, the efficacy of vaccination with Helicobacter antigen in combination with aluminum hydroxide (AlOH) was evaluated in a murine model of Helicobacter infection . Immunization with antigen and AlOH induced interleukin-5-secreting, antigen-specific T cells, and immunization with antigen and complete Freund's adjuvant induced interferon-gamma-secreting, antigen-specific T cells, as determined by ELISPOT assay . Both immune responses conferred protection after challenge with either H . pylori or H . felis, as confirmed by the complete absence of any bacteria, as assessed by both histology and culture of gastric biopsy samples . Protection was antibody independent, as demonstrated with antibody-deficient muMT mice (immunoglobulin-gene knockout mice), and CD4(+) spleen T cells from immunized mice were sufficient to transfer protective immunity to otherwise immunodeficient rag1(-/-) recipients . These results suggest an alternative and potentially more expeditious strategy for development of a human-use H . pylori vaccine.

J Infect Dis, 2001 Aug 1, 184(3), 278 - 84 Epub 2001 Jun 26.
Suburban transmission of Q fever in French Guiana: evidence of a wild reservoir; Gardon J et al.; The annual incidence of Q fever in French Guiana was found to have increased in 1996 and was 37/100,000 population over the last 4 years . Subsequent investigations in Cayenne and its suburbs indicated that a wild reservoir of the bacteria was responsible for the epidemiologic pattern . A case-control study showed that residence near a forest and occupations and activities that result in exposure to aerosols of dusts from the soil are risk factors for Q fever . By means of time-series analysis, a strong positive correlation between rainfall and the incidence of Q fever with a time lag of 1-3 months was found . The spatial distribution of the cases showed that transmission occurs widely throughout greater Cayenne, which is incompatible with a pinpoint source of contamination . Transmission from livestock and dissemination of the bacteria by the wind appeared to be unlikely, which strengthens the hypothesis that a wild reservoir is responsible for transmission.

J Mol Evol, 2001 Jun, 52(6), 471 - 89
Phylogenetic analysis of metabolic pathways; Forst CV et al.; The information provided by completely sequenced genomes can yield insights into the multi-level organization of organisms and their evolution . At the lowest level of molecular organization individual enzymes are formed, often through assembly of multiple polypeptides . At a higher level, sets of enzymes group into metabolic networks . Much has been learned about the relationship of species from phylogenetic trees comparing individual enzymes . In this article we extend conventional phylogenetic analysis of individual enzymes in different organisms to the organisms' metabolic networks . For this purpose we suggest a method that combines sequence information with information about the underlying reaction networks . A distance between pathways is defined as incorporating distances between substrates and distances between corresponding enzymes . The new analysis is applied to electron-transfer and amino acid biosynthesis networks yielding a more comprehensive understanding of similarities and differences between organisms.

Oral Microbiol Immunol, 2001 Aug, 16(4), 218 - 23
The recA gene in Porphyromonas gingivalis is expressed during infection of the murine host; Liu Y et al.; The recA gene in Porphyromonas gingivalis is involved in DNA repair . To further elucidate the importance of the recA locus in the pathogenesis of P . gingivalis, we assessed its ability for expression in an animal host . The promoterless xa-tetA(Q)2 cassette was used in heterodiploid mutants to study recA promoter activity during infection . P . gingivalis FLL118.1 had the xa-tetA(Q)2 cassette under the control of recA promoter whereas P . gingivalis FLL119 had the cassette in the opposite orientation . xa encodes a bifunctional xylosidase/arabinosidase enzyme (XA) and the tetA(Q)2 gene product confers tetracycline resistance . Intramuscular infection in a mouse model allowed the recovery of the bacteria from inguinal lymph nodes . Infusion of tetracycline in the animals permitted the enrichment P . gingivalis FLL118.1 over the wild-type strain, during a mixed infection . The xylosidase activity of FLL118.1 could be detected on agar plates in the presence of 5-methylumbellifiry-beta-D-xyloside . No such enrichment for xylosidase activity was detected when the mixture of P . gingivalis W83 and P . gingivalis FLL119 was used to infect the mouse or cultured in vitro . These results indicated that recA promoter was transcriptionally active during the infection of the murine host and further support the importance of this locus during the P . gingivalis infection process.

Oral Microbiol Immunol, 2001 Aug, 16(4), 193 - 201
Immunoglobulin G response of periodontitis patients to Porphyromonas gingivalis capsular carbohydrate and lipopolysaccharide antigens; Sims TJ et al.; Porphyromonas gingivalis clonal types that participate in periodontal infections express serologically distinct surface antigens . This investigation sought to determine whether serum antibodies titers against the serotype-specific capsular carbohydrate K antigen and lipopolysaccharide antigens of P . gingivalis might reveal which serotypes are most likely to be responsible for subgingival infections in subjects with adult periodontitis . Immunoglobulin G (IgG) titers to purified K antigen and lipopolysaccharide from different P . gingivalis strains were measured by ELISA for 28 healthy controls and 51 patients with periodontal pockets known to be infected with genetically and serologically distinct P . gingivalis clonal types . Titers to purified K antigen from strains W50, HG184, A7A1-28, 49417, HG1690 and HG1691, representing serotypes K1-K6, respectively, and lipopolysaccharide from strains 381, HG1691 and W50, representing serotypes O1-O3, respectively, were measured for all subjects . Chi-square likelihood ratios, Mann-Whitney tests and receiver-operating characteristic sensitivity-specificity plots were used to compare the accuracy with which titer results for different target antigens classified subjects with or without disease . Results from assays targeting K2, K3, K4, K5, O1 and O2 generally gave poor diagnostic accuracy, whether evaluated separately or as summed titer pairs corresponding to the K/O combinations actually expressed by the target antigen parent strains . Exceptions were O3 (from W50) and K5+O2 (both from HG1690), which gave moderate accuracy in classifying subjects . In contrast, highly significant diagnostic accuracy was achieved using individual K1 (W50) and K6 (HG1691) titer data and K1+O3 (W50) and K6+O2 (HG1691) titer sum values . These observations suggest that P . gingivalis clonal types expressing K/O serotypes matching those of W50 (K1/O3) and HG1691 (K6/O2) are more likely than others to participate in periodontal infections in adult periodontitis patients and thus are more likely than others to express relevant virulence factors.

Br J Haematol, 2001 Jun, 113(4), 915 - 24
Epitope mapping of factor VIII inhibitor antibodies of Chinese origin; Huang CC et al.; Epitopes recognized by factor VIII (FVIII) inhibitors of Chinese origin were analysed by immunoblotting with full-length recombinant FVIII (rFVIII), thrombin-activated FVIII (FVIIIa) and 16 FVIII fusion proteins synthesized by bacteria . Twenty-eight patients, 12 with haemophilia A and 16 with autoimmune diseases, were recruited . Antibodies from 22 patients showed reactivity with rFVIII, 20 with FVIIIa, and one reacted only with FVIII fusion proteins . Of these 22 cases, most were reactive with A2-a2 and A3-C1-C2 of FVIII(a) . Of the nine cases that depicted binding to the fusion proteins, three were reactive with the A domains, three with only the B domain, and the other three with both the A and B (or C) domains . An epitope for a neutralizing antibody of a haemophilia A patient, designated TWN-112, was localized to residues 323-390, specified by FVIII fusion proteins . The same epitope also appeared on an FVIII-expression phage library screening . Immunoabsorption of antibodies from TWN-112 with the epitope reduced the neutralizing activity of the inhibitor by 33% . The incidence of a1 of FVIII is higher, and that of a3 is lower, than previously reported . Two novel epitopes, reported for the first time in this paper, were localized on the 8B2 (amino acid residues 1022-1204) and 8A2(V) (residues 673-740) fusion proteins . These two epitopes were able to reduce inhibitory antibody activity by 24% and 25% respectively . Changes of FVIII fragment specificity were also observed in one of six patients for whom multiple samples, collected at different times, were available . Our initial finding showed that the FVIII inhibitors in these Chinese patients shared epitopes with those of patients from very different genetic backgrounds, suggesting a common mechanism for the development of FVIII inhibitors.

J Org Chem, 2001 Jul 13, 66(14), 4809 - 13
Synthesis of enantiomerically and diastereomerically pure 2(S)-amino-6(R)-hydroxy-1,7-heptanedioic acid dimethyl ester hydrochloride from cycloheptadiene; Shireman BT et al.; The complete carbon framework of enantiomerically and diastereomerically pure 2(S)-amino-6(R)-hydroxy-1,7-heptanedioic acid dimethyl ester hydrochloride was derived from cycloheptadiene in six steps utilizing an amino acid-derived acylnitroso Diels-Alder reaction as the key step . This versatile amino diester has been previously used to synthesize amino-differentiated diaminopimelic acid (DAP) and biologically active analogues . In addition, after formation of a novel aminoxy diketopiperazine, the newly formed carboxyl groups were differentiated by a novel transpeptidation of the amino acid that directed the stereochemistry of the initial cycloaddition.

Parasitology, 2001, 122 Suppl, S23 - 38
Concomitant infections, parasites and immune responses; Cox FE; Concomitant infections are common in nature and often involve parasites . A number of examples of the interactions between protozoa and viruses, protozoa and bacteria, protozoa and other protozoa, protozoa and helminths, helminths and viruses, helminths and bacteria, and helminths and other helminths are described . In mixed infections the burden of one or both the infectious agents may be increased, one or both may be suppressed or one may be increased and the other suppressed . It is now possible to explain many of these interactions in terms of the effects parasites have on the immune system, particularly parasite-induced immunodepression, and the effects of cytokines controlling polarization to the Th1 or Th2 arms of the immune response . In addition, parasites may be affected, directly or indirectly, by cytokines and other immune effector molecules and parasites may themselves produce factors that affect the cells of the immune system . Parasites are, therefore, affected when they themselves, or other organisms, interact with the immune response and, in particular, the cytokine network . The importance of such interactions is discussed in relation to clinical disease and the development and use of vaccines.

Science, 2001 Jul 6, 293(5527), 115 - 20
A transcriptionally {correction of transcriptively} active complex of APP with Fe65 and histone acetyltransferase Tip60; Cao X et al.; Amyloid-beta precursor protein (APP), a widely expressed cell-surface protein, is cleaved in the transmembrane region by gamma-secretase . gamma-Cleavage of APP produces the extracellular amyloid beta-peptide of Alzheimer's disease and releases an intracellular tail fragment of unknown physiological function . We now demonstrate that the cytoplasmic tail of APP forms a multimeric complex with the nuclear adaptor protein Fe65 and the histone acetyltransferase Tip60 . This complex potently stimulates transcription via heterologous Gal4- or LexA-DNA binding domains, suggesting that release of the cytoplasmic tail of APP by gamma-cleavage may function in gene expression.

J Immunol, 2001 Jul 15, 167(2), 682 - 90
The Peyer's patch microenvironment suppresses T cell responses to chemokines and other stimuli; Kellermann SA et al.; Immunosurveillance of mucosal sites presents immune cells with challenges not encountered in the periphery . T cells in the gut must distinguish enteric pathogens from innocuous non-self Ag derived from food or commensal bacteria . The mechanisms that regulate T cells in the gut remain incompletely understood . We assessed the effect of the Peyer's patch microenvironment on T cell responses to chemokines . Chemokines are believed to play an important role during T cell priming by facilitating T cell migration into and within lymphoid tissues as well as T cell encounter and interaction with APCs . We found a profound suppression of chemokine-stimulated T cell chemotaxis and actin polymerization in Peyer's patch relative to lymph node . Chemokine hyporesponsiveness is imposed upon T cells within hours of their entry into Peyer's patches and is reversed following their removal . Suppression was not restricted to chemokine stimulation, as T cell responses to Con A and PMA were also suppressed . The global nature of this defect is further underscored by an impairment in calcium mobilization . Evidence indicates that a soluble factor contributes to this hyporesponsiveness, and comparison of Peyer's patches and lymph nodes revealed striking differences in their chemokine and cytokine constitution, indicating a marked Th2 bias in the Peyer's patches . The role of the Th2 microenvironment in mediating suppression is suggested by the ability of Nippostrongylus brasiliensis to elicit hyporesponsiveness in lymph node T cells . The suppressive milieu encountered by T cells in Peyer's patches may be critical for discouraging undesired immune responses and promoting tolerance.

Infection, 2001 May-Jun, 29(3), 113 - 8
Chlamydia trachomatis in andrologic patients--direct and indirect detection; Bollmann R et al.; BACKGROUND: Chlamydia trachomatis is considered to be the most common sexually transmitted disease in Germany . It is currently unclear whether chlamydial infection causes pathological conditions of the male accessory glands with consequences for male infertility . PATIENTS AND METHODS: Within the framework of several prospective studies the association between sperm quality, male accessory gland function and infection with C . trachomatis was investigated in men of couples with unexplained infertility . Chlamydial infection was determined by serologic methods and by proof of Chlamydia-specific DNA . As a marker of infection the direct determination of granulocytes in the ejaculate or the measurement of the polymorphonuclear (PMN) elastase concentration was used . The male accessory gland function was evaluated using the markers fructose, citric acid and alpha-glucosidase in the seminal plasma . RESULTS: Chlamydia-specific DNA in the ejaculate was present in between 3-5% of the subjects, which corresponds to its prevalence in the normal population . Chlamydia IgA antibodies were demonstrated with a frequency of 38% in seminal plasma (n = 834) using a genus-specific test (rELISA) . Using other species-specific tests (MIF, SeroCT, IgA pELISA and ImmunoComb), Chlamydia IgA antibodies were found at frequencies of between 8 and 22% . CONCLUSION: Only in a few individual cases was it possible to show a connection between reduced sperm quality, disturbed male accessory gland function and indication of infection with Chlamydia, bacteria or Ureaplasma.

Biotech Histochem, 2001 Mar, 76(2), 89 - 95
Histological techniques and microscopic analysis of biological agents for preservation of human bone remains; Dore B et al.; Microscopy and histology techniques can be applied to morphological study of fungi and bacteria contaminating ancient human osteological remains . Undecalcified samples are cut with a diamond rotary blade microtome and an original technique was applied using adhesive tape to prevent damage to the bone surface during sectioning . We used light microscopy, polarized light microscopy, and epi-illumination fluorescence systems . Common dyes can be applied to 80 microm sections using classic staining techniques to reveal the architecture of bone and the presence of infecting biological agents.

Biotech Histochem, 2001 Mar, 76(2), 67 - 73
Modified silver staining of nucleolar organizer regions to improve the accuracy of image analysis; Orrea SC et al.; Silver staining of nucleolar organizer regions (NORs) and their subsequent quantification by image analysis are used increasingly in human pathological specimens and experimental models . Because certain conditions determined by the type of tissue and/or its fixation render AgNOR segmentation for image analysis difficult due to insufficient contrast or nonspecific silver precipitation, we propose three improvements to the original technique to overcome these difficulties . Pretreatment with 7% nitric acid produced very distinct dark brown images of AgNORs on a yellow background . The gradient of background colors allowed easy discrimination of nucleolar, nuclear and cytoplasmic structures . Seven morphometric parameters related to number, size and shape of AgNORs were evaluated quantitatively by image analysis on sections pretreated with nitric acid and on adjacent sections treated with citrate buffer in a wet autoclave according to the most widely accepted method for image analysis of AgNOR . Both methods yielded similar results . A second improvement was achieved by coating the slides with 7% celloidin solution in ethyl alcohol-ether prior to AgNOR staining and acid pretreatment . This coating prevented nonspecific silver deposition on argyrophilic bacteria and other tissue debris in human vaginal smears that could make visualizing AgNOR sites difficult . Finally, placing sections face down on the staining solution prevents the formation of nonspecific silver precipitates . These procedures can be applied together or separately according to the requirements of the material to be evaluated.

Br J Biomed Sci, 2001, 58(2), 66 - 75
Ammonia vapour in the mouth as a diagnostic marker for Helicobacter pylori infection: preliminary 'proof of principle' pharmacological investigations; Dun CDR et al.; Most current non-invasive tests for Helicobacter pylori depend on the conversion of labelled (13C or 14C) urea to labelled carbon dioxide (13CO2 or 14CO2) and ammonium (NH4+) by the enzyme urease, with the labelled CO2 detected in exhaled air . Despite suggestions going back over a number of years, the alternative possibility of using NH4+ (in the form of gaseous ammonia {NH3}) as the test parameter has received little or no attention . However, this approach is now being explored using a chemiresistive sensor detecting sub-parts per million concentrations of NH3 . An in vitro 'glass stomach' (containing various volumes of hydrochloric acid {HCl} and ammonium chloride {NH4Cl}) was used to evaluate the means of increasing 'gastric' pH to that of the NH4+-->NH3 transition that occurs significantly at pH 9.24 . This 'stomach' also was used to study mechanisms by which NH3 may be expelled in a pulse (as a surrogate belch), either by the in situ production of CO2 or through an exogenous source . On the basis of the protocols developed, H . pylori-negative subjects were tested before and after ingestion of 10 mg NH4Cl (as a surrogate for bacteria-produced NH4,), and H . pylori-positive subjects were tested without taking urea or NH4Cl . 'Intragastric' pH in the in vitro 'glass stomach' could be increased above pH 9.24 by adding a mixture of 15-30 mL magnesium hydroxide mixture (or the proprietary equivalent) and 50 mL water, and the resulting NH3 expelled by adding 100 mL CO2-saturated cold water (sparkling water) . In vivo, NH3 levels in the oral cavity of H . pylori-negative subjects were increased after ingestion of 10 mg NH4Cl; however, levels in the oral cavity of a small number of H . pylori-positive subjects were two- to threefold higher after magnesium hydroxide and sparkling water . On the basis of in vitro studies, an in vivo protocol was developed to increase gastric pH above that required for the NH4+-->NH3 transition, and a mechanism established to release the NH3 into the oral cavity . Preliminary in vivo data confirm the chemiresistive sensor is sufficiently sensitive to NH3 to distinguish H . pylori-negative subjects who have taken 10 mg NH4Cl from those who have not, and clearly distinguish H . pylori-negative subjects from H . pylori-positive subjects . Ingestion of urea or other labelled tracers is not required, nor is belching; and the sensor takes less than two minutes to reach a maximum response . The data provide good evidence that the chemiresistive detection of NH3 has considerable potential as a rapid, point-of-care diagnostic test for H . pylori infection.

Mol Biotechnol, 2001 May, 18(1), 71 - 7
Isolation and purification of chemokines from natural sources; Schroder JM; Chemokines (e.g., IL-8) were originally identified as chemotactic proteins obtained from various different natural sources . Today, using the genome walking strategy an ever-increasing number of novel genes encoding chemokines have been discovered that were expressed in bacteria or eukaryotic cells and subsequently tested for biological activity . Usually biological significance of the considered chemokine is extrapolated from these data . The increasing evidence, however, that post-translational modification of chemokines can dramatically affect its biological activity makes it necessary to identify the naturally occurring chemokines in order to identify its biological function . Furthermore, with the isolation of natural chemokines, evidence is provided that transcription of chemokine genes is really followed by translation into a bioactive molecule . Purification of chemokines from natural sources requires special strategies: The bioassay or immunoassay should allow screening of high-performance liquid chromatography fractions and detection of the required chemokine at low concentration . Parameters that affect the detection of bioactivity and immunoreactivity (giving either false positive or false negative results) should be carefully considered . In this article methods for molecular characterization of chemokines from both cell culture supernatants and human tissue (lesional inflammatory skin scales) will be described.

Proc Natl Acad Sci U S A, 2001 Jul 3, 98(14), 7958 - 63
Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis; Peters W et al.; Although the protective cellular immune response to Mycobacterium tuberculosis requires recruitment of macrophages and T lymphocytes to the site of infection, the signals that regulate this trafficking have not been defined . We investigated the role of C-C chemokine receptor 2 (CCR2)-dependent cell recruitment in the protective response to M . tuberculosis . CCR2(-/-) mice died early after infection and had 100-fold more bacteria in their lungs than did CCR2(+/+) mice . CCR2(-/-) mice exhibited an early defect in macrophage recruitment to the lung and a later defect in recruitment of dendritic cells and T cells to the lung . CCR2(-/-) mice also had fewer macrophages and dendritic cells recruited to the mediastinal lymph node (MLN) after infection . T cell migration through the MLN was similar in CCR2(-/-) and CCR2(+/+) mice . However, T cell priming was delayed in the MLNs of the CCR2(-/-) mice, and fewer CD4(+) and CD8(+) T cells primed to produce IFN-gamma accumulated in the lungs of the CCR2(-/-) mice . These data demonstrate that cellular responses mediated by activation of CCR2 are essential in the initial immune response and control of infection with M . tuberculosis.

Gastroenterology, 2001 Jul, 121(1), 118 - 23
Identification of oxysterols in human bile and pigment gallstones; Haigh WG et al.; BACKGROUND AND AIMS: Although cholesterol is the most abundant sterol in animal tissues, oxidized products of cholesterol (oxysterols) also occur in mammalian organs and blood and are cytotoxic, atherogenic, and carcinogenic . However, the presence of oxysterols in bile or gallstones has never been reported . METHODS: Fresh human bile and gallstones were collected . Sterol content and structure were analyzed using gas chromatography/mass spectrometry (GC/MS) . Bacterial DNA was extracted from human gallstones . RESULTS: GC/MS identified cholesta-4,6-diene-3-one and cholest-4-ene-3-one, with several as yet unidentified oxysterols in bile and stone samples . Several plant and fungal sterols were also present in gallstones . When 102 human gallstones were analyzed for oxysterols, they were markedly higher (as percent of total sterols) in pigment gallstones, where bacterial DNA is most abundant . CONCLUSIONS: These observations suggest biliary oxysterols are associated with the presence of bacteria and may play a role in the pathogenesis of gallstones and biliary tract cancers.

Cell Microbiol, 2001 Jul, 3(7), 487 - 97
Identification of Brucella spp . genes involved in intracellular trafficking; Delrue RM et al.; After uptake by host cells, the pathogen Brucella transits through early endosomes, evades phago-lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum (ER) . The molecular mechanisms underlying these processes are still poorly understood . To identify new bacterial factors involved in these processes, a library of 1800 Brucella melitensis 16M mini-Tn5catkm mutants was screened for intracellular survival and multiplication in HeLa cells and J774A.1 macrophages . Thirteen mutants were identified as defective for their intracellular survival in both cell types . In 12 of them, the transposon had inserted in the virB operon, which encodes a type IV-related secretion system . The preponderance of virB mutants demonstrates the importance of this secretion apparatus in the intracellular multiplication of B . melitensis . We also examined the intracellular fate of three virB mutants (virB2, virB4 and virB9) in HeLa cells by immunofluorescence . The three VirB proteins are not necessary for penetration and the inhibition of phago-lysosomal fusion within non-professional phagocytes . Rather, the virB mutants are unable to reach the replicative niche and reside in a membrane-bound vacuole expressing the late endosomal marker, LAMP1, and the sec61beta protein from the ER membrane, proteins that are present in autophagic vesicles originating from the ER.

J Comp Pathol, 2001 May, 124(4), 290 - 9
Natural paratuberculosis infection in rabbits in Scotland; Beard PM et al.; Natural paratuberculosis infection of rabbits (Oryctolagus cuniculus) was recently diagnosed in Scotland, and an investigation into the pathology of the disease in wild rabbits is reported in this paper . Evidence of Mycobacterium avium subsp . paratuberculosis (M.a . paratuberculosis) infection was detected in 22% of 110 rabbits; the organism was cultured from 17 of 110 rabbits, and histopathological lesions consistent with M.a . paratuberculosis infection were noted in 18 of 98 rabbits examined . No macroscopical lesions suggestive of M.a . paratuberculosis infection were observed . The histopathological lesions were either severe or mild . Severe lesions consisted of extensive macrophage granulomata and numerous giant cells, with many intracellular acid-fast bacteria in the small intestine . For the examination of formalin-fixed, paraffin wax-embedded tissues, neither immunohistochemistry nor the polymerase chain reaction was as sensitive a method of diagnosis as histopathology.

Plant Mol Biol, 2001 May, 46(1), 67 - 77
Activation of defence-related genes during senescence: a correlation between gene expression and cellular damage; Obregon P et al.; The correlation between activation of defence-related gene expression and plant senescence was investigated by evaluating the presence of specific transcripts in various leaves of tobacco senescing plants . Expression of most genes examined was found to be induced shortly after flowering; however, each gene had its own characteristic timing of expression and level of RNA accumulation . Studies of the symptoms developed in senescing leaves responding to bacterial inoculation suggest that the accumulation of defence-related transcripts in these tissues might be related with the mechanism of senescence rather than with protection of the plant against pathogen infection . We observed that the high level of GUS expression directed by the beta-1,3-glucanase gn1 promoter, in senescing leaves of transgenic tobacco plants . decreased after bacterial inoculation, in correlation with the formation of symptoms . Reduction of gene expression was likely to be the reflection of the additional damage caused by the bacteria in the senescent tissues inoculated.

Genetika, 2001 May, 37(5), 610 - 6
{Genetic diversity of a natural population of Sinorhizobium meliloti, detected during analysis of a cryptic plasmid and ISRm2011-2 fingerprints}; Andronov EE et al.; Fifty-six natural strains of alfalfa nodule bacteria were isolated from samples of the soil under wild legume and alfalfa in two different field sites of Irkutsk oblast . Based on the results of analysis of plasmid profile, 11 different types of strains were detected, and 43 types were identified based on the results of hybridization with the insertion sequence element ISRm2011-2 . Significant differences were found in the plasmid profile and IS fingerprints between strains isolated from the soil under alfalfa and the soil under legume . In contrast, strains growing at some distance from each other differed only in the IS fingerprints . From a comparison of results obtained in the assessment of plasmid profile and in analysis of IS fingerprints with results of RFLP analysis in strains, the conclusion about the transference of cryptic plasmids between strains and genetic rearrangements in strains of this population was drawn.

Mol Endocrinol, 2001 Jul, 15(7), 1077 - 92
Dynamic changes in subcellular localization of mineralocorticoid receptor in living cells: in comparison with glucocorticoid receptor using dual-color labeling with green fluorescent protein spectral variants; Nishi M et al.; Mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) are ligand-dependent transcription factors . Although it is generally accepted that GR is translocated into the nucleus from the cytoplasm only after ligand binding, the subcellular localization of MR is still quite controversial . We examined the intracellular trafficking of MR in living neurons and nonneural cells using a fusion protein of green fluorescent protein (GFP) and rat MR (GFP-MR) . Corticosterone (CORT) induced a rapid nuclear accumulation of GFP-MR, whereas in the absence of ligand, GFP-MR was distributed in both cytoplasm and nucleus in the majority of transfected cells . Given the differential action of MR and GR in the central nervous system, it is important to elucidate how the trafficking of these receptors between cytoplasm and nucleus is regulated by ligand . To examine the simultaneous trafficking of MR and GR within single living cells, we use different spectral variants of GFP, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP), linked to MR and GR, respectively . In COS-1 cells, expressing no endogenous corticosteroid receptors, the YFP-MR chimera was accumulated in the nucleus faster than the CFP-GR chimera in the presence of 10(-9) M CORT, while there was no significant difference in the nuclear accumulation rates in the presence of 10(-6) M CORT . On the other hand, in primary cultured hippocampal neurons expressing endogenous receptors, the nuclear accumulation rates of the YFP-MR chimera and CFP-GR chimera were nearly the same in the presence of both concentrations of CORT . These results suggest that CORT-induced nuclear translocation of MR and GR exhibits differential patterns depending on ligand concentrations or cell types.

Am J Respir Crit Care Med, 2001 Jul 1, 164(1), 97 - 102
Neutrophils, unopposed neutrophil elastase, and alpha1-antiprotease defenses following human lung transplantation; Meyer KC et al.; Neutrophils are sequestered in the newly transplanted lung after reperfusion or with infection, rejection, and chronic graft dysfunction . Because unopposed (free) neutrophil elastase (NE) released into bronchoalveolar secretions may injure the lung allograft and impair bacterial clearance, we assessed total neutrophil numbers, myeloperoxidase activity as an index of neutrophil influx and degranulation, alpha1-antiprotease (alpha1-AP) concentrations, and unopposed NE activity in bronchoalveolar secretions from lung transplant recipients . Unopposed NE activity was present in bronchoalveolar lavage fluid (BALF) from recipients transplanted for emphysema associated with alpha1-AP deficiency as well as recipients without such deficiency (171 of 2,137 BALF; 8%) . Ten of 17 (59%) recipients with alpha1-AP deficiency who were followed for at least 1 yr after transplant with multiple surveillance and diagnostic bronchoscopies had at least one BALF containing unopposed NE, usually associated with the presence of > or = 10(5) colony forming units/ml BALF of aerobic bacteria . In contrast, 19 of 58 (33%) with emphysema not associated with alpha1-AP deficiency, 8 of 32 (25%) recipients with cystic fibrosis (CF), 6 of 16 (38%) with idiopathic pulmonary fibrosis (IPF), and 11 of 36 (31%) with other indications for transplant had unopposed NE in BALF . alpha1-AP levels were significantly elevated in the early posttransplant time period and could be augmented considerably in alpha1-AP-deficient recipients with episodes of infection or rejection . Our findings indicate that unopposed NE activity can be found in both alpha1-AP-deficient and alpha1-AP-sufficient recipients after transplantation, usually in association with endobronchial bacterial infection.

Structure (Camb), 2001 Jun, 9(6), 473 - 81
Crystal structure of a novel-type archaeal rubisco with pentagonal symmetry; Kitano K et al.; BACKGROUND: Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme of the Calvin-Benson cycle and catalyzes the primary reaction of CO2 fixation in plants, algae, and bacteria . Rubiscos have been so far classified into two types . Type I is composed of eight large subunits (L subunits) and eight small subunits (S subunits) with tetragonal symmetry (L8S8), but type II is usually composed only of two L subunits (L2) . Recently, some genuinely active Rubiscos of unknown physiological function have been reported from archaea . RESULTS: The crystal structure of Rubisco from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-Rubisco) was determined at 2.8 A resolution . The enzyme is composed only of L subunits and showed a novel (L2)5 decameric structure . Compared to previously known type I enzymes, each L2 dimer is inclined approximately 16 degrees to form a toroid-shaped decamer with its unique L2-L2 interfaces . Differential scanning calorimetry (DSC), circular dichroism (CD), and gel permeation chromatography (GPC) showed that Tk-Rubisco maintains its secondary structure and decameric assembly even at high temperatures . CONCLUSIONS: The present study provides the first structure of an archaeal Rubisco, an unprecedented (L2)5 decamer . Biochemical studies indicate that Tk-Rubisco maintains its decameric structure at high temperatures . The structure is distinct from type I and type II Rubiscos and strongly supports that Tk-Rubisco should be classified as a novel type III Rubisco.

Environ Toxicol Chem, 2001 Jul, 20(7), 1474 - 82
Aircraft and runway deicers at General Mitchell International Airport, Milwaukee, Wisconsin, USA . 1 . Biochemical oxygen demand and dissolved oxygen in receiving streams; Corsi SR et al.; Aircraft and runway deicers are used during cold weather at many of the world's airports to facilitate safe air travel . Propylene glycol-, ethylene glycol-, and urea-based deicers are known to have very high biochemical oxygen demand . At General Mitchell International Airport (GMIA) in Milwaukee, Wisconsin, USA, deicer application, water chemistry, and dissolved oxygen (DO) data were collected for two deicing seasons in order to evaluate and define premanagement water quality parameters prior to the implementation of a glycol management program . Calculations using stream-monitoring data during a controlled release of deicer provided an estimate of 0.8/d for the first-order decay rate constant, substantially higher than published laboratory test results . For eight precipitation events with deicing activities, between 2.4 and 99% of propylene and ethylene glycol applied to aircraft was delivered directly to receiving streams . The percentage of glycol runoff during an event increased with increasing storm-flow volume . Elevated concentrations of glycol and biochemical oxygen demand were measured downstream from the airport . However, the frequency of low DO concentrations in the receiving streams is comparable with that at an upstream reference site . This is possibly due to slowed bacteria metabolism at low water temperatures, short travel times, and dilution from downstream tributaries.

Pol Merkuriusz Lek, 2001 Apr, 10(58), 216 - 8
{Peritonitis in children treated with automated peritoneal dialysis}; Zurowska A et al.; The rate of peritonitis, its etiology and efficacy of treatment was compared between 28 children treated with APD for a total of 439 dialysis months and 24 children on CAPD treated for 328 dialysis months . The peritonitis rate was significantly lower in children on APD (1 episode per 27 months) than in children on CAPD (1 episode per 17 months) (p = 0.005) . The etiology of peritonitis (68% v . 62% Gram + organisms), efficacy of treatment (100% v . 100%) and relapse rate did not differ between the machine operated and manual methods of peritoneal dialysis.

Orig Life Evol Biosph, 2001 Jun, 31(3), 271 - 85
A coupled ecosystem-climate model for predicting the methane concentration in the Archean atmosphere; Kasting JF et al.; A simple coupled ecosystem-climate model is described that can predict levels of atmospheric CH4, CO2, and H2 during the Late Archean, given observed constraints on Earth's surface temperature . We find that methanogenic bacteria should have converted most of the available atmospheric H2 into CH4, and that CH4 may have been equal in importance to CO2 as a greenhouse gas . Photolysis of this CH4 may have produced a hydrocarbon smog layer that would have shielded the surface from solar UV radiation . Methanotrophic bacteria would have consumed some of the atmospheric CH4, but they would have been incapable of reducing CH4 to modern levels . The rise of O2 around 2.3 Ga would have drastically reduced the atmospheric CH4 concentration and may thereby have triggered the Huronian glaciation.

Izv Akad Nauk Ser Biol, 2001 May-Jun, (3), 312 - 7
{clpP2 gene encoding peptidase in cyanobacteria Synechocystis sp . PCC 6803 controls the sensitivity of cells to photoinhibition}; Panichkin VB et al.; A homozygous insertion mutant with the inactivated clpP2 gene, which encodes the proteolytic subunit of ATP-dependent peptidase, was obtained in the unicellular cyanobacterium Synechocystis sp . PCC 6803 . The mutant cannot grow under photoautotrophic conditions, but cells grown under heterotrophic conditions in a glucose-containing medium have active photosystems I and II (PS I and PS II) . The loss of capacity for photoautotrophic growth is determined by a high sensitivity of mutant cells to the inactivating effect of light . Their incubation under light with an intensity above 10 microE m-2 s-1 inhibits cell growth in culture and causes degradation of photosynthetic pigments . It is proposed that the ClpP2 peptidase is involved in the protection of Synechocystis 6803 cells from photoinhibition.

Nat Rev Mol Cell Biol, 2001 Jul, 2(7), 514 - 20
The function and synthesis of ribosomes; Lafontaine DL et al.; Structural analyses of the large and small ribosomal subunits have allowed us to think about how they work in more detail than ever before . The mechanisms that underlie ribosomal synthesis, translocation and catalysis are now being unravelled, with practical implications for the design of antibiotics.

J Exp Bot, 2001 May, 52(358), 943 - 7
Trehalose becomes the most abundant non-structural carbohydrate during senescence of soybean nodules; Muller J et al.; Carbohydrate metabolism and symbiont survival were studied in nodules of soybean (G . max {L.} Merr . cv . Maple Arrow infected with Bradyrhizobium japonicum 61-A-101), induced to senesce simultaneously by application of the photosynthesis inhibitor dichloromethyl urea (DCMU) . The plant-borne carbohydrates sucrose and starch started to decline after 2 d and reached background levels after 8 d, in parallel with the decline of nitrogenase . However, the microsymbiont-borne disaccharide trehalose declined only by about 40% and subsequently remained at a constant level of c . 6 mg x g(-1) dry weight up to 14 d, when nodules softened and decayed . The number of re-isolated viable bacteria was not significantly decreased in senescent nodules as compared to control nodules . These results indicate that during terminal senescence of nodules an appreciable part of the bacteria conserve their trehalose pools and survive.

J Biochem (Tokyo), 2001 Jul, 130(1), 51 - 6
Amino acids in the N-terminal region regulate the photocycle of photoactive yellow protein; Harigai M et al.; The spectroscopic properties of photoactive yellow protein (PYP) partially digested by chymotrypsin were studied . Chymotrypsin yielded three major products that were yellow but distinguishable by SDS-PAGE . They were readily separated by DEAE-Sepharose column chromatography . Protein sequencing and mass spectrometry demonstrated that chymotrypsin cleaved the N-terminal 6, 15, or 23 amino acids (T6, T15, and T23) . The blue-shifts of the absorption maxima and the increases in the apparent pK(a) of the chromophores relative to those of intact PYP were less than 4 nm and 0.2, respectively . The absorption spectra of the near-UV intermediates produced from T6, T15, and T23 were identical to that of intact PYP, but with lifetimes that were 140, 2,300, and 4,500 times longer, respectively . These observations suggest that the recovery of the dark state of PYP from the near-UV intermediate is accelerated by the N-terminal region, and that this region acts as a regulatory factor for the photocycle of PYP.

FEMS Microbiol Lett, 2001 May 1, 198(2), 91 - 7
Group-specific 16S rRNA targeted probes for the detection of type I and type II methanotrophs by fluorescence in situ hybridisation; Eller G et al.; The study of methane-oxidising bacteria (methanotrophs) is of special interest, because of their role in the natural reduction of methane emissions from many different sources . Therefore new probes were developed to detect specifically either type I (Methylococcaceae) or type II methanotrophs (Methylocystaceae) . The probes have shown high specificity in fluorescence in situ hybridisations (FISH), as demonstrated by parallel hybridisation of target and reference strains as well as sequence data analysis . With these probes, methanotrophs were detected in soil and root samples from rice microcosms, demonstrating their applicability even in a complex environmental matrix.

FEMS Microbiol Lett, 2001 May 1, 198(2), 105 - 10
Complexes with truncated RNAs from the large domain of Archaeoglobus fulgidus signal recognition particle; Bhuiyan SH et al.; Protein SRP19 is an important component of the signal recognition particle (SRP) as it promotes assembly of protein SRP54 with SRP RNA and recognizes a tetranucleotide loop . Structural features and RNA binding activities of SRP19 of the hyperthermophilic archaeon Archaeoglobus fulgidus were investigated . An updated alignment of SRP19 sequences predicted three conserved regions and two alpha-helices . With Af-SRP RNA the Af-SRP54 protein assembled into an A . fulgidus SRP which remained intact for many hours . Stable complexes were formed between Af-SRP19 and truncated SRP RNAs, including a 36-residue fragment representing helix 6 of A . fulgidus SRP RNA.

Org Lett, 2001 Jan 11, 3(1), 57 - 9
Structure-activity relationships within a family of selectively cytotoxic macrolide natural products; Salomon AR et al.; {figure: see text} We describe a semi-synthetic deglycosylated derivative of apoptolidin that retains considerable activity against the mitochondrial ATPase but has greatly reduced cellular cytotoxicity . We also demonstrate that a related antifungal natural product, cytovaricin, inhibits the same molecular target . Structural comparison of these macrolides provides insights into their conserved features that are presumably important for biological activity and identifies promising avenues at the interface of organic synthesis and biosynthesis for the generation of new selective cytotoxic agents.

Proc R Soc Lond B Biol Sci, 2001 Jul 7, 268(1474), 1423 - 7
Recombination confounds interpretations of Wolbachia evolution; Jiggins FM et al.; Wolbachia are vertically transmitted bacteria known from arthropods and nematode worms, which are maintained in host populations because they either physiologically benefit infected individuals or parasitically manipulate their reproduction . The different manipulation phenotypes are scattered across the Wolbachia phylogeny, suggesting that there have been multiple evolutions of similar phenotypes . This conclusion relies on the assumption of an absence of recombination between bacterial strains, so that the gene used to reconstruct the phylogeny reflects the evolutionary history of the genes involved in the trait . We tested for recombination by reconstructing the phylogeny of two Wolbachia genes from seven B-subdivision strains . The two genes produced mutually incompatible topologies, indicating that these lineages are subject to genetic recombination . This means that many evolutionary patterns inferred from Wolbachia phylogenies must be re-evaluated . Furthermore, recombination may be an important feature both in the evolution of the manipulation phenotypes and avoidance of Muller's ratchet . Finally, we discuss the implications of recombination for attempts to genetically engineer Wolbachia for use in the control of crop pests and human pathogens.

Transpl Infect Dis, 2001 Mar, 3(1), 34 - 9
Human granulocytic ehrlichiosis in pancreas transplant recipients; Trofe J et al.; Human ehrlichioses are tick-borne infections caused by bacteria in the genus Ehrlichia of the family Rickettsiaceae . To date there have been three cases of ehrlichiosis reported in the transplant population, a human monocytic ehrlichiosis (HME) infection in a liver transplant recipient and two cases of human granulocytic ehrlichiosis (HGE) in kidney transplant recipients . We report three pancreas transplant patients who developed HGE in the last two years at a single southeastern center in the United States . All three patients had clinical, laboratory, and pathophysiologic findings on bone marrow biopsy and peripheral blood smears consistent with HGE, and responded to doxycycline therapy . In the setting of potent immunosuppression, ehrlichiosis should be considered in the differential diagnosis of transplant patients presenting with persistent fever, pancytopenia, and abnormal liver function . Patients with ehrlichiosis infection may be at risk for developing other opportunistic infections or lymphoproliferative disease.

J Mol Biol, 2001 Jul 6, 310(2), 299 - 309
Effect of mutations in the Mu-host junction region on transpososome assembly; Coros CJ et al.; Mu transposition occurs through a series of higher-order nucleoprotein complexes called transpososomes . The region where the Mu DNA joins the host DNA plays an integral role in the assembly of these transpososomes . We have created a series of point mutations at the Mu-host junction and characterized their effect on the Mu in vitro strand transfer reaction . Analysis of these mutant constructs revealed an inhibition in transpososome assembly at the point in the reaction pathway when the junction region is engaged by the transposase active site (i.e . the transition from LER to type 0) . We found that the degree of inhibition was dependent upon the particular base-pair change at each position and whether the substitution occurred at the left or right transposon end . The MuB transposition protein, an allosteric effector of MuA, was shown to suppress all of the inhibitory Mu-host junction mutants . Most of the mutant constructs were also suppressed, to varying degrees, by the substitution of Mg(2+) with Mn(2+) . Analysis of the mutant constructs has revealed hierarchical nucleotide preferences at positions -1 through +3 for transpososome assembly and suggests the possibility that specific metal ion-DNA base interactions are involved in DNA recognition and transpososome assembly .

J Zoo Wildl Med, 2000 Dec, 31(4), 552 - 7
Use of pyloroplasty (Y-U) to treat presumed delayed gastric emptying in a cheetah (Acinonyx jubatus); Neiffer DL et al.; A 4-yr-old cheetah (Acinonyx jubatus) with a 2-yr history of chronic intermittent vomiting and spiral bacteria-associated gastritis presented with dramatically increased vomiting frequency and marked intermittent abdominal distention . Physical examination revealed loss of muscle mass and poor fur coat quality . Contrast radiography was consistent with delayed gastric emptying due to presumed gastric outlet obstruction . Both Y-U pyloroplasty and incisional gastropexy were performed, and no subsequent vomiting has been observed for 3 yr with the exception of three episodes during the immediate postoperative period . The cause of delayed gastric emptying was not determined, although a gastric motility disorder associated with gastric bacterial infection and elevated gastrin levels was suspected.

Proc Natl Acad Sci U S A, 2001 Jul 3, 98(14), 7835 - 40 Epub 2001 Jun 26.
The complete genome of the crenarchaeon Sulfolobus solfataricus P2; She Q et al.; The genome of the crenarchaeon Sulfolobus solfataricus P2 contains 2,992,245 bp on a single chromosome and encodes 2,977 proteins and many RNAs . One-third of the encoded proteins have no detectable homologs in other sequenced genomes . Moreover, 40% appear to be archaeal-specific, and only 12% and 2.3% are shared exclusively with bacteria and eukarya, respectively . The genome shows a high level of plasticity with 200 diverse insertion sequence elements, many putative nonautonomous mobile elements, and evidence of integrase-mediated insertion events . There are also long clusters of regularly spaced tandem repeats . Different transfer systems are used for the uptake of inorganic and organic solutes, and a wealth of intracellular and extracellular proteases, sugar, and sulfur metabolizing enzymes are encoded, as well as enzymes of the central metabolic pathways and motility proteins . The major metabolic electron carrier is not NADH as in bacteria and eukarya but probably ferredoxin . The essential components required for DNA replication, DNA repair and recombination, the cell cycle, transcriptional initiation and translation, but not DNA folding, show a strong eukaryal character with many archaeal-specific features . The results illustrate major differences between crenarchaea and euryarchaea, especially for their DNA replication mechanism and cell cycle processes and their translational apparatus.

Plant Cell Physiol, 2001 Jun, 42(6), 555 - 9
Visualization of an FtsZ ring in chloroplasts of Lilium longiflorum leaves; Mori T et al.; FtsZ is a bacterial division protein which forms a ring at the leading edge of the cell division site . To date, a hypothesis that the plant FtsZ forms the same structure in chloroplast division is proposed, but has not been demonstrated yet . In this study, recombinant LlFtsZ (Lilium longiflorum FtsZ) protein was produced from a previously isolated ftsZ cDNA clone {Mori and Tanaka (2000) Protoplasma 214: 57} and used to raise polyclonal anti-LlFtsZ antibodies in rabbits . In immunoblot analysis with the total protein extracted from L . longiflorum leaves, purified antibodies specifically recognized LlFtsZ whose molecular mass was approximately 43 kDa . This size corresponded to that of the recombinant LlFtsZ protein lacking N-terminal sequence, which suggests that the full-length LlFtsZ translation product has a putative N-terminal signal peptide . Moreover, fluorescent and electron microscopy revealed that the anti-LlFtsZ antibodies recognized ring structures at stromal side of the constriction point of dividing chloroplasts . Here, we show direct evidence that FtsZ ring is involved in chloroplast division.

Biochim Biophys Acta, 2001 Jul 2, 1513(1), 1 - 24
Protein translocation across membranes; Agarraberes FA et al.; Cellular membranes act as semipermeable barriers to ions and macromolecules . Specialized mechanisms of transport of proteins across membranes have been developed during evolution . There are common mechanistic themes among protein translocation systems in bacteria and in eukaryotic cells . Here we review current understanding of mechanisms of protein transport across the bacterial plasma membrane as well as across several organelle membranes of yeast and mammalian cells . We consider a variety of organelles including the endoplasmic reticulum, outer and inner membranes of mitochondria, outer, inner, and thylakoid membranes of chloroplasts, peroxisomes, and lysosomes . Several common principles are evident: (a) multiple pathways of protein translocation across membranes exist, (b) molecular chaperones are required in the cytosol, inside the organelle, and often within the organelle membrane, (c) ATP and/or GTP hydrolysis is required, (d) a proton-motive force across the membrane is often required, and (e) protein translocation occurs through gated, aqueous channels . There are exceptions to each of these common principles indicating that our knowledge of how proteins translocate across membranes is not yet complete.

Microb Pathog, 2001 Jul, 31(1), 37 - 45
Intracellular signals triggered during association of Mycobacterium leprae and Mycobacterium bovis BCG with human monocytes; Lima CS et al.; To gain a better understanding of mycobacteria-host cell interaction, the present study compared the signal transduction events triggered during the interaction of Mycobacterium leprae (the causative agent of leprosy) and of Mycobacterium bovis BCG (an attenuated strain used as a vaccine against leprosy and tuberculosis) with human monocytes . The assays consisted of pre-treating or not THP-1 cells (a human monocytic cell line) with different kinase inhibitors, followed by incubation with fluorescein-labelled bacteria and analysis of bacterial association via fluorescence microscopy . The specific tyrosine kinase (TK) inhibitor tyrphostin AG126 provided the highest rates of association inhibition (>90% for BCG and >65% for M . leprae) . The early activation of TKs during mycobacteria-host cell interaction was confirmed by immunoblot analysis, demonstrating that in several host cell proteins mycobacteria stimulated tyrosine phosphorylation . The use of the drugs wortmannin and bisindolylmaleimide I which, respectively, inhibit phosphatidylinositide 3-kinase (PI 3-kinase) and protein kinase C (PKC), produced lower but consistent results within a 35--60% association inhibition range for both bacteria . Dose response curves with these inhibitors were obtained . Similar results were obtained when primary human monocytes were used as host cells, strongly suggesting that TK, PKC and PI 3-kinase signals are activated during the interaction of human monocytes with both pathogenic and attenuated species of mycobacteria .

Oncogene, 2000 Dec 27, 19(56), 6660 - 9
Replication-selective oncolytic adenoviruses: virotherapy aimed at genetic targets in cancer; Kirn D; Replication-selective oncolytic adenoviruses represent a novel cancer treatment platform . Clinical studies have demonstrated the safety and feasibility of the approach, including the delivery of adenovirus to tumors through the bloodstream (Heise et al., 1999b; Reid et al., 1999; Nemunaitis et al., 1999) . The inherent ability of replication-competent adenoviruses to sensitize tumor cells to chemotherapy was a novel discovery that has led to chemosensitization strategies . These data will support the further development of adenoviral agents, including second-generation constructs containing exogenous therapeuitc genes to enhance both local and systemic antitumoral activity (Heise and Kirn, 2000; Hermiston, 2000; Agha-Mohammadi and Lotze, 2000) . In addition to adenovirus, other viral species are being developed including herpesvirus, vaccinia, reovirus and measles virus (Kirn, 2000a; Martuza, 2000; Norman and Lee, 2000; Mastrangelo et al., 2000; Coffey et al., 1998; Martuza et al., 1991; Kirn, 2000b; Lattime et al., 1996) . Since intratumoral spread also appears to be a substantial hurdle for viral agents, inherently motile agents such as bacteria may hold great promise for this field (Low et al., 1999; Sznol et al., 2000) . Given the unknown predictive value of in vitro cell-based assays and murine tumor model systems for the efficacy and therapeutic index of replication-selective oncolytic adenoviruses in patients, we believe that encouraging adenoviral agents must be tested in well-designed clinical trials as soon as possible . Only then can the true therapeutic potential of these agents be realized.

J Biomed Mater Res, 2001 Sep 5, 56(3), 307 - 23
Binding and orientation of fibronectin on surfaces with collagen-related peptides . Student Research Award in the Masters Science Degree Candidate category, 27th annual meeting of the Society for Biomaterials, St . Paul, MN, April 24-29, 2001; Klueh U et al.; Although fibronectin (FN) has been used in a variety of in vitro studies to enhance cell and bacteria adhesion, relatively little is known about the molecular interactions of FN with surfaces, particularly the interactions that can control the binding, conformation, and functionality of FN on these surfaces . Even less is known about approaches needed to control binding, orientation, and functionality of FN bound on surfaces . To begin to fill this gap in our knowledge, we hypothesized that functional FN can be bound and specifically oriented on polystyrene surfaces with FN-specific collagen-related peptides (CRPs) . We further hypothesized that monoclonal antibodies that react with specific epitopes on FN can be used to quantify both FN binding and orientation on these surfaces . On the basis of these hypotheses, we initiated a systematic investigation of the binding and orientation of FN on polystyrene surfaces with CRPs . To bind FN to surfaces, we used two different CRPs: CRP-I (TLQPVYEYMVGV) and CRP-II (TGLPVGVGYVVTVLT) . The binding and orientation of the FN molecule to these immobilized CRPs was quantified with (125)I-FN and monoclonal antibodies . Monoclonal antibodies used for this study were reactive with specific regions of the FN molecule, that is, the amino (N) terminus (anti-N antibodies) and carboxyl (C) terminus (anti-C antibodies) . The results of our studies demonstrated that although CRP-I and CRP-II could be bound directly to polystyrene, these directly immobilized CRPs failed to bind (125)I-FN . Thus, to facilitate FN binding to the CRPs, we used bovine serum albumin (BSA) as a spacer to physically elevate the CRPs away from the polystyrene surface . Thus, CRP-I and CRP-II were covalently linked to BSA via the N and C termini of each CRP (CRP-I-BSA and CRP-II-BSA) . (125)I-CRP-BSAs were all found to bind to equivalent levels on polystyrene (1.60-2.60 microg/cm2) . When CRP-BSAs were immobilized on polystyrene, they all successfully bound (125)I-FN in a range of 34-72 ng/cm2 (mean) . Using monoclonal antibodies to FN to characterize the orientation of FN bound to the various CRP-BSAs, we demonstrated that (1) FN consistently bound to either CRP-I-BSA or CRP-II-BSA; (2) bound FN reacted significantly more with anti-C antibodies than with anti-N antibodies; and (3) the increased reactivity of bound FN to anti-C antibodies was consistent, whether FN was bound by CRP-I or CRP-II or the CRPs were bound to BSA by the C or N termini . These data demonstrated an enhanced binding of anti-C antibodies to immobilized CRP-BSA relative to anti-N antibodies . We interpreted the data to be the result of FN binding in an oriented fashion with N termini of FN bound tightly to the BSA-polystyrene surface . In this position, the C termini of FN are exposed and available for binding by the anti-C antibodies . Alternatively, in this orientation the N termini of the FN would not be available to bind the anti-N antibodies, thereby explaining the decreased reactivity of the CRP immobilized FN to the anti-N antibodies . These studies not only demonstrate the utility of peptides in binding and orienting large molecular weight proteins such as FN on surfaces but underscore the need for well-characterized reagents (e.g., monomeric/functional FN and antibodies) to specifically bind, orient, and characterize large molecular weight proteins immobilized on various surfaces.

Appl Environ Microbiol, 2001 Jul, 67(7), 3314 - 8
Congruent phylogenies of most common small-subunit rRNA and dissimilatory sulfite reductase gene sequences retrieved from estuarine sediments; Joulian C et al.; The diversity of sulfate-reducing bacteria (SRB) in brackish sediment was investigated using small-subunit rRNA and dissimilatory sulfite reductase (DSR) gene clone libraries and cultivation . The phylogenetic affiliation of the most commonly retrieved clones for both genes was strikingly similar and produced Desulfosarcina variabilis-like sequences from the inoculum but Desulfomicrobium baculatum-like sequences from a high dilution in natural media . Related organisms were subsequently cultivated from the site . PCR bias appear to be limited (or very similar) for the two primersets and target genes . However, the DSR primers showed a much higher phylogenetic specificity . DSR gene analysis is thus a promising and specific approach for investigating SRB diversity in complex habitats.

Environ Mol Mutagen, 2001, 37(4), 345 - 55
Direct separation of in vivo and in vitro am3 revertants in transgenic mice carrying the phiX174 am3, cs70 vector; Malling HV et al.; Target genes in most transgenic systems have higher spontaneous mutation frequencies than do endogenous mammalian genes . Spontaneous mutations in transgenes predominantly arise from three sources: (1) mutations fixed in the animals, (2) mutations arising from replication errors caused by damage to the DNA that may have occurred in vivo or in vitro and then was fixed during amplification of the vector in vitro, and (3) mutations arising during replication of non-revertant phages in non-permissive bacteria . An assay based on single bursts was developed to directly distinguish between the in vivo and in vitro origins of revertants . The size of the aliquot is determined by mutant frequency and is adjusted so that ideally no more than 10 to 20% of the aliquots contain a bacterial cell transformed with a mutant phage . Mutations are detected as revertants of an amber mutation (am3) in phiX174 am3, cs70 . The minimum burst size of non-revertant phiX am3, cs70 from splenic DNA on a permissive bacterial strain was larger than 30 plaque-forming units (pfu) . Based on this observation, a burst size of 31 plaque-forming revertants was chosen as the minimum burst size of a fixed mutation . The single burst assay was tested on DNA from spleens of animals that were treated with 150 mg/kg 1-ethyl-1 nitrosurea . Only the fraction of aliquots with single bursts of revertants (> 30) increased in the treated animals compared to the controls . In contrast, there was no difference between treated and control animals for revertant frequencies calculated for burst sizes < or =30 pfu . Among the spontaneous mutations, only 30% were caused by mutations fixed in animals (i.e . burst size >30 pfu) . Total average revertant frequency measured in DNA from treated animals was less than twofold more than the average spontaneous frequency (P = 0.048) . When frequencies were based on burst sizes >30, there was a 4.6-fold increase among treated animals compared with controls (P = 0.026) . The single burst-assay resulted in a more sensitive test for mutagenicity because it eliminated noise from in-vitro mutations.

J Ind Microbiol Biotechnol, 1999 Oct, 23(4-5), 273 - 283
Ecology, physiology, and phylogeny of deep subsurface Sphingomonas sp; Fredrickson JK et al.; Several new species of the genus Sphingomonas including S . aromaticivorans, S . stygia, and S . subterranea that have the capacity for degrading a broad range of aromatic compounds including toluene, naphthalene, xylenes, p-cresol, fluorene, biphenyl, and dibenzothiophene, were isolated from deeply-buried (>200 m) sediments of the US Atlantic coastal plain (ACP) . In S . aromaticivorans F199, many of the genes involved in the catabolism of these aromatic compounds are encoded on a 184-kb conjugative plasmid; some of the genes involved in aromatic catabolism are plasmid-encoded in the other strains as well . Members of the genus Sphingomonas were common among aerobic heterotrophic bacteria cultured from ACP sediments and have been detected in deep subsurface environments elsewhere . The major source of organic carbon for heterotrophic metabolism in ACP deep aquifers is lignite that originated from plant material buried with the sediments . We speculate that the ability of the subsurface Sphingomonas strains to degrade a wide array of aromatic compounds represents an adaptation for utilization of sedimentary lignite . These and related subsurface Sphingomonas spp may play an important role in the transformation of sedimentary organic carbon in the aerobic and microaerobic regions of the deep aquifers of the ACP.

Eur J Clin Nutr, 2001 Jun, 55(6), 509 - 12
Hydrogen excretion upon ingestion of dairy products in lactose-intolerant male subjects: importance of the live flora; Pelletier X et al.; OBJECTIVE: To assess the effects of the ingestion of milk, yoghurt (10(8) bacteria/ml), heat-treated yoghurt (<15 bacteria/ml) and two products obtained by dilution of yoghurt with heat-treated product (10(6) and 10(5) bacteria/ml) on hydrogen production and symptoms of lactose intolerance in lactose malabsorbers . DESIGN: Double-blind, randomised cross-over design . SETTING: The study was performed in the phase 1 clinical unit of OPTIMED, Nancy, France . SUBJECTS: Twenty-four male lactose malabsorbers were selected for the study . INTERVENTIONS: Hydrogen production and adverse events were followed during 8 h after ingestion of the products . RESULTS: The results clearly demonstrate that ingestion of yoghurt with 10(8) bacteria/ml leads to lower H(2) excretion and complaints than the other products . Results observed with the products containing a reduced population of live flora remain better than those observed with milk . CONCLUSIONS: The importance of a high population of the live flora is underlined.

Science, 2001 Jun 22, 292(5525), 2285 - 9
Common and contrasting themes of plant and animal diseases; Staskawicz BJ et al.; Recent studies in bacterial pathogenesis reveal common and contrasting mechanisms of pathogen virulence and host resistance in plant and animal diseases . This review presents recent developments in the study of plant and animal pathogenesis, with respect to bacterial colonization and the delivery of effector proteins to the host . Furthermore, host defense responses in both plants and animals are discussed in relation to mechanisms of pathogen recognition and defense signaling . Future studies will greatly add to our understanding of the molecular events defining host-pathogen interactions.

Science, 2001 Aug 10, 293(5532), 1139 - 42 Epub 2001 Jun 21.
Coupled transcription and translation within nuclei of mammalian cells; Iborra FJ et al.; It is widely assumed that the vital processes of transcription and translation are spatially separated in eukaryotes and that no translation occurs in nuclei . We localized translation sites by incubating permeabilized mammalian cells with {3H}lysine or lysyl-transfer RNA tagged with biotin or BODIPY; although most nascent polypeptides were cytoplasmic, some were found in discrete nuclear sites known as transcription "factories." Some of this nuclear translation also depends on concurrent transcription by RNA polymerase II . This coupling is simply explained if nuclear ribosomes translate nascent transcripts as those transcripts emerge from still-engaged RNA polymerases, much as they do in bacteria.

Environ Microbiol, 2001 May, 3(5), 304 - 11
Linking the composition of bacterioplankton to rapid turnover of dissolved dimethylsulphoniopropionate in an algal bloom in the North Sea; Zubkov MV et al.; The algal osmolyte, dimethylsulphoniopropionate (DMSP), is abundant in the surface oceans and is the major precursor of dimethyl sulphide (DMS), a gas involved in global climate regulation . Here, we report results from an in situ Lagrangian study that suggests a link between the microbially driven fluxes of dissolved DMSP (DMSPd) and specific members of the bacterioplankton community in a North Sea coccolithophore bloom . The bacterial population in the bloom was dominated by a single species related to the genus Roseobacter, which accounted for 24% of the bacterioplankton numbers and up to 50% of the biomass . The abundance of the Roseobacter cells showed significant paired correlation with DMSPd consumption and bacterioplankton production, whereas abundances of other bacteria did not . Consumed DMSPd (28 nM day(-1)) contributed 95% of the sulphur and up to 15% of the carbon demand of the total bacterial populations, suggesting the importance of DMSP as a substrate for the Roseobacter-dominated bacterioplankton . In dominating DMSPd flux, the Roseobacter species may exert a major control on DMS production . DMSPd turnover rate was 10 times that of DMS (2.7 nM day(-1)), indicating that DMSPd was probably the major source of DMS, but that most of the DMSPd was metabolized without DMS production . Our study suggests that single species of bacterioplankton may at times be important in metabolizing DMSP and regulating the generation of DMS in the sea.

Clin Exp Immunol, 2001 May, 124(2), 172 - 9
Slow recovery of follicular B cells and marginal zone B cells after chemotherapy: implications for humoral immunity; Zandvoort A et al.; Although most chemotherapeutic agents are known to cause primarily reduction or suppression of immune responses, surprisingly little is known about the influence of cytostatic agents on lymphoid tissue compartments such as the splenic marginal zone . The marginal zone plays an important role in the defence against encapsulated bacteria, which are potential candidates for postchemotherapeutic infections . We studied the effect of three different cytostatic agents (cisplatin, methotrexate, and cyclophosphamide) on B cell subpopulations in a rat model . Rats received a single dose of a single cytostatic agent and were sacrificed at different time points after treatment . Bone marrow, blood, mesenteric lymph nodes and spleens were analysed by flow-cytometry and immunohistochemistry . All three cytostatic agents showed severe bone marrow depression . CP and MTX showed only mild reduction of cell populations in the spleen . CyPh showed a severe reduction of recirculating follicular B (RF-B) cells and marginal zone B (MZ-B) cells . At day 24 most populations were already recovered, but RF-B cells and MZ-B cells were still reduced . The reduction of the marginal zone and late recovery may imply that, beside the overall increased infection risk due to neutropenia, patients treated with chemotherapy are at risk for developing infections from encapsulated bacteria for a considerable period of time after treatment, extending beyond the period of bone marrow depression.

Cell Microbiol, 2001 Jun, 3(6), 427 - 37
Low iron availability modulates the course of Chlamydia pneumoniae infection; Al-Younes HM et al.; Chlamydiae are obligate intracellular bacteria residing exclusively in host cell vesicles termed inclusions . We have investigated the effects of deferoxamine mesylate (DAM)-induced iron deficiency on the growth of Chlamydia pneumoniae and Chlamydia trachomatis serovar L2 . In epithelial cells subjected to iron starvation and infected with either C . pneumoniae or C . trachomatis L2, small inclusions were formed, and the infectivity of chlamydial progeny was impaired . Moreover, for C . trachomatis L2, we observed a delay in homotypic fusion of inclusions . The inhibitory effects of DAM were reversed by adding exogenous iron-saturated transferrin, which restored the production of infectious chlamydiae . Electron microscopy examination of iron-deprived specimens revealed that the small inclusions contained reduced numbers of C . pneumoniae that were mostly reticulate bodies . We have previously reported specific accumulation of transferrin receptors (TfRs) around C . pneumoniae inclusions within cells grown under normal conditions . Using confocal and electron microscopy, we show here a remarkable increase in the amount of TfRs surrounding the inclusions in iron-starved cultures . It has been shown that iron is an essential factor in the growth and survival of C . trachomatis . Here, we postulate that, for C . pneumoniae also, iron is an indispensable element and that Chlamydia may use iron transport pathways of the host by attracting TfR to the phagosome.

J Org Chem, 2001 Jun 29, 66(13), 4559 - 62
Facile enzymatic aldol reactions with dihydroxyacetone in the presence of arsenate; Schoevaart R et al.; Aldol reactions of in situ formed dihydroxyacetone arsenate with different aldehydes were catalyzed by bacterial D-fructose-1,6-bisphosphate aldolase (FruA) . Aldolases from bacteria were found to be much more stable and active than FruA from rabbit muscle . Arsenate acts as a phosphate mimic and can, in principle, be used in catalytic amounts . The use of inorganic arsenate and dihydroxyacetone afforded high yields with hydrophobic aldehydes . Cosolvents increased the solubility of hydrophobic aldehydes and afforded higher reaction rates and enzyme stability . Insight is given, for the first time, in the influence of arsenate on the stereoselectivity of the aldol reaction.

Res Microbiol, 2001 Apr-May, 152(3-4), 331 - 40
Mitochondrial ABC transporters; Lill R et al.; In contrast to bacteria, mitochondria contain only a few ATP binding cassette (ABC) transporters in their inner membrane . The known mitochondrial ABC proteins fall into two major classes that, in the yeast Saccharomyces cerevisiae, are represented by the half-transporter Atm1p and the two closely homologous proteins Mdl1p and Mdl2p . In humans two Atm1p orthologues (ABC7 and MTABC3) and two proteins homologous to Mdll/2p have been localized to mitochondria . The Atm1p-like proteins perform an important function in mitochondrial iron homeostasis and in the maturation of Fe/S proteins in the cytosol . Mutations in ABC7 are causative of hereditary X-linked sideroblastic anemia and cerebellar ataxia (XLSA/A) . MTABC3 may be a candidate gene for the lethal neonatal syndrome . The function of the mitochondrial Mdl1/2p-like proteins is not clear at present with the notable exception of murine ABC-me that may transport intermediates of heme biosynthesis from the matrix to the cytosol in erythroid tissues.

Res Microbiol, 2001 Apr-May, 152(3-4), 323 - 9
ABC transporters associated with cytochrome c biogenesis; Goldman BS et al.; It is generally agreed that cytochrome c biogenesis requires that the apocytochrome and heme be transported separately to their site of function and assembly . In bacteria, this is outside the cytoplasmic membrane, whereby the apocytochromes c use sec-dependent signals for their translocation . Two different hypotheses have recently emerged as to how heme is exported: one involves an helABCD-encoded ATP binding cassette (ABC) transporter complex and the second does not . The second hypothesis concludes that an (HelAB)2 heterodimeric ABC transporter does not transport heme but some other substrate for cytochrome c biogenesis . The evidence supporting each of these two hypotheses and the role of this ABC transporter is discussed.

Res Microbiol, 2001 Apr-May, 152(3-4), 245 - 58
Peptides and ATP binding cassette peptide transporters; Detmers FJ et al.; In this review our knowledge of ATP binding cassette (ABC) transporters specific for peptides is discussed . Besides serving a role in nutrition of the cell, the systems participate in various signaling processes that allow (micro)organisms to monitor the local environment . In bacteria, these include regulation of gene expression, competence development, sporulation, DNA transfer by conjugation, chemotaxis, and virulence development, and the role of ABC transporters in each of these processes is discussed . Particular attention is paid to the specificity determinants of peptide receptors and transporters in relation to their structure and to the mechanisms of peptide binding.

J Antimicrob Chemother, 2001 Jul, 48 Suppl 1, 87 - 102
Interpretative reading: recognizing the unusual and inferring resistance mechanisms from resistance phenotypes; Livermore DM et al.; If isolates are speciated and if a sufficient range of antibiotics is tested, underlying resistance mechanisms can often be inferred from the antibiogram data . This allows: (i) anomalous combinations of phenotype and organism to be reconsidered; (ii) prediction of further antibiotics that deserve testing; and (iii) the suppression of susceptibilities that are anomalous in the light of the inferred mechanism . This 'interpretative reading' is widely undertaken in France but is largely precluded in the UK by limited speciation and the testing of narrow ranges of antibiotics . Nevertheless, UK laboratories should be aware of: (i) grossly anomalous combinations of species and phenotype, demanding reference laboratory confirmation; (ii) useful indicator drugs, where resistance implies a mechanism conferring other resistances that may be less obvious in direct tests; and (iii) antibiotics that are prone to select resistant mutants of particular species during therapy . Details of these combinations of organism and resistance are presented . Relationships between antibiogram and mechanism are also presented to allow full interpretative reading for those testing wide panels of drugs versus speciated isolates.

J Mol Biol, 2001 Jun 29, 310(1), 83 - 91
Conformational changes of the ferric uptake regulation protein upon metal activation and DNA binding; first evidence of structural homologies with the diphtheria toxin repressor; Gonzalez de Peredo A et al.; Fur (ferric uptake regulation protein) is a bacterial global regulator that uses iron as a cofactor to bind to specific DNA sequences . It has been suggested that metal binding induces a conformational change in the protein, which is subsequently able to recognize DNA . This mechanism of activation has been investigated here using selective chemical modification monitored by mass spectrometry . The reactivity of each lysine residue of the Fur protein was studied, first in the apo form of the protein, then after metal activation and finally after DNA binding . Of particular interest is Lys76, which was shown to be highly protected from modification in the presence of target DNA . Hydrogen-deuterium exchange experiments were performed to map with higher resolution the conformational changes induced by metal binding . On the basis of these results, together with a secondary structure prediction, the presence in Fur of a non-classical helix-turn-helix motif is proposed . Experimental results show that activation upon metal binding induces conformational modification of this specific motif . The recognition helix, interacting directly with the major groove of the DNA, would include the domain {Y55-F61} . This helix would be followed by a small "wing" formed between two beta strands, containing Lys76, which might interact directly with DNA . These results suggest that Fur and DtxR (diphtheria toxin repressor), another bacterial repressor, share not only the function of being iron concentration regulators, and the structure of their DNA-binding domain .

Chemosphere, 2001 Jul, 44(1), 53 - 7
A comparative study of anaerobically digested and undigested sewage sludges in preparation of activated carbons; Tay JH et al.; Disposal of sewage sludge is an increasingly expensive and environmentally sensitive problem throughout the world . Preparation of activated carbon from sewage sludge offers an attractive re-use alternative to the traditional disposal routes . The objective of this research work was to compare anaerobically digested sewage sludge (DS) and undigested sewage sludge (US) as source materials in the preparation of activated carbons . Prior to the preparation the properties of the two types of sewage sludges were determined and compared . Subsequently the sludge samples were activated with 5 M ZnCl2 solution and thereafter pyrolysed at heating temperature of 650 degrees C for 2 h with the heating rate of 15 degrees C/min under a nitrogen atmosphere . The produced activated carbons were characterised by surface area and porosity analysis, CHN elemental composition and ash contents determination, and aqueous phase phenol adsorption tests . The results indicate that in comparison with the DS, the US had a higher carbon content and lower ash content, and accordingly yielded a better activated carbon with a higher BET surface area, pore volume, carbon content and phenol adsorption capacity.

Chemosphere, 2001 Jul, 44(1), 45 - 51
Optimising the preparation of activated carbon from digested sewage sludge and coconut husk; Tay JH et al.; Preparation of activated carbon from sewage sludge is a promising way to dispose of sewage sludge as well as to produce a low-cost adsorbent for pollutant removal . This research work aimed to optimise the condition for activated carbon preparation from anaerobically digested sewage sludge with the additive coconut husk . The sewage sludge sample was mixed with the additive coconut husk . The preparation condition variables investigated involved the concentration of the ZnCl2 solutions, heating temperature, dwell time and heating rate in pyrolysis and the mixing ratio of coconut husk to sewage sludge . Surface area, pore size distribution, aqueous phenol adsorption capacity and the production yield of the final products were determined and compared . Experimental results revealed that low concentrations of ZnCl2 solution tended to improve the microporosity of the final product . Heating temperature had a considerable impact on the surface area, pore size distribution and phenol adsorption capacity of the final products, whereas dwell time and heating rate performed comparatively insignificantly . The effect of increasing the mixing ratio of coconut husk to sewage sludge was principally to increase the microporosity of the final products . The activated carbon with the highest BET surface area was produced with the activation of 5 M ZnCl2 solution and, thereafter, pyrolysis at a heating temperature of 500 degrees C for 2 h with a heating rate of 10 degrees C/min . The mixing ratio of 1:4 in terms of coconut husk to sewage sludge based on their dried weights was found to be most cost effective.

Chemosphere, 2001 Jul, 44(1), 31 - 6
Enhanced sludge granulation in upflow anaerobic sludge blanket (UASB) reactors by aluminum chloride; Yu HQ et al.; Two upflow anaerobic sludge blanket (UASB) reactors were concurrently operated for 146 days to examine the effects of aluminum chloride (AlCl3) on the sludge granulation process during start-up . Sludge granulation (defined as that over 10% of granules were larger than 2.0 mm) was achieved in the control reactor (R1) in approximate three months . Introduction of Al3+ at a concentration of 300 mg/l reduced the sludge granulation time by approximate one month . Throughout the experiment the AlCl3-added reactor (R2) had a higher biomass concentration, e.g., 13.8 g-MLVSS/l versus 12.8 g-MLVSS/l on Day 146 . Granules became visible earlier in R2 compared with R1 (35 days versus 65 days) . The average size of granules from R2 was larger than that from R1 . The results demonstrated that AlCl3 enhanced the sludge granulation process in the UASB reactors.

J Chromatogr B Biomed Sci Appl, 2001 May 25, 756(1-2), 141 - 50
Investigations on the carbohydrate moieties of glycoprotein allergens; Petersen A et al.; Many allergens are glycoproteins and their carbohydrate structure can contribute to the IgE reactivity . Therefore it is of great interest to study the carbohydrate structures of these particular antigens . Here, we present an overview of methods combining basic procedures in glycochemistry with various applications of electrophoresis that allow investigating single allergens in crude extracts . Various allergen extracts, e.g . from tomato, grass pollen and bacteria were analysed and the suitability of the tests are discussed.

Sci Total Environ, 2001 Jun 12, 273(1-3), 27 - 40
Indoor air quality at nine shopping malls in Hong Kong; Li WM et al.; Hong Kong is one of the most attractive shopping paradises in the world . Many local people and international tourists favor to spend their time in shopping malls in Hong Kong . Good indoor air quality is, therefore, very essential to shoppers . In order to characterize the indoor air quality in shopping malls, nine shopping malls in Hong Kong were selected for this study . The indoor air pollutants included carbon dioxide (CO2), carbon monoxide (CO), total hydrocarbons (THC), formaldehyde (HCHO), respirable particulate matter (PM10) and total bacteria count (TBC) . More than 40% of the shopping malls had 1-h average CO2 levels above the 1000 ppm of the ASHRAE standard on both weekdays and weekends . Also, they had average weekday PM10 concentrations that exceeded the Hong Kong Indoor Air Quality Objective (HKIAQO) . The highest indoor PM10 level at a mall was 380 microg/m3 . Of the malls surveyed, 30% had indoor airborne bacteria levels above 1000 cfu/m3 set by the HKIAQO . The elevated indoor CO2 and bacteria levels could result from high occupancy combined with insufficient ventilation . The increased PM10 levels could be probably attributed to illegal smoking inside these establishments . In comparison, the shopping malls that contained internal public transport drop-off areas, where vehicles were parked with idling engines and had major entry doors close to heavy traffic roads had higher CO and PM10 indoor levels . In addition, the extensive use of cooking stoves without adequate ventilation inside food courts could increase indoor CO2, CO and PM10 levels.

J Immunol, 2001 Jul 1, 167(1), 228 - 34
Regulation of dendritic cell recruitment into resting and inflamed airway epithelium: use of alternative chemokine receptors as a function of inducing stimulus; Stumbles PA et al.; Dendritic cells (DC) were purified by flow cytometry from rat tracheal mucosa; they exhibited the phenotypic characteristics of immature DC including high endocytic activity, low CD80/86 expression, and in vitro responsiveness to a broad range of CC chemokines . Daily treatment of adult rats with the selective CCR1 and CCR5 antagonist Met-RANTES reduced baseline numbers of tracheal intraepithelial DC by 50-60%, and pretreatment of animals with Met-RANTES before inhalation of aerosol containing heat-killed bacteria abolished the rapid DC influx into the epithelium that occurred in untreated controls, implicating CCR1 and CCR5 and their ligands in recruitment of immature DC precursors into resting airway tissues and during acute bacterial-induced inflammation . Comparable levels of DC recruitment were observed during airway mucosal Sendai virus infection and after aerosol challenge of sensitized animals with the soluble recall Ag OVA . However, Met-RANTES did not affect these latter responses, indicating the use of alternative chemokine receptors/ligands for DC recruitment, or possibly attraction of different DC subsets, depending on the nature of the eliciting stimulus.

J Bacteriol, 2001 Jul, 183(14), 4217 - 26
Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells; York GM et al.; Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules . Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs . The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha . R . eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed . The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a phaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP . The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the phaP-gfp fusion . The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains . Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis . The results suggest that R . eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.

Gene, 2001 Jun 27, 271(2), 285 - 91
Characterization of two genes, Impa1 and Impa2 encoding mouse myo-inositol monophosphatases; Shamir A et al.; The enzyme myo-inositol monophosphatase (Impa) catalyzes the synthesis of free myo-inositol from various myo-inositol monophosphates in the phosphatidylinositol signaling system . Impa is a lithium-blockable enzyme that has been hypothesized to be the biological target for lithium-salts used as mood-stabilizing drugs in the treatment of manic-depressive (bipolar) illness . As an initial step to explore the functional consequences of reduced or absent Impa activity in an animal model we here report the isolation of two Impa-encoding mouse genes, Impa1 and Impa2 . Impa1 spans approximately 17.5 kb and contains nine exons of 46--1354 bp encoding a protein of 277 amino acids . Impa2 spans at least 19.5 kb and contains eight exons of 46--444 bp size encoding a protein of 290 amino acids . The genomic structure including the positions of the exon-intron splice sites seems to be conserved among myo-inositol monophosphatase genes in mammalian species . One or more Impa-like genes do also exist in evolutionary more distant species like invertebrates, plants and bacteria . The proteins encoded by the non-vertebrate genes seem to be equally related to Impa1 and Impa2 . We therefore suggest that the Impa1 and Impa2 genes duplicated from a common ancestral gene after the evolutionary divergence of vertebrates.

J Air Waste Manag Assoc, 2001 Jun, 51(6), 848 - 60
The importance of pathogenic organisms in sewage and sewage sludge; Dumontet S et al.; Deficient sanitation poses a serious threat to human and animal health, involving complex relationships between environments, animals, refuse, food, pathogens, parasites, and man . However, by sanitizing and stabilizing the organic matter of sewage sludge, agriculture can utilize it to maintain soil, water, and air quality . As ingredients in soil amendments, such bioresidues are a source of nutrients for plants . Stabilization and sanitation of sewage sludge safely couple its recycling and disposal . This coupling becomes increasingly important as economic and environmental constraints make strategies for waste disposal more difficult to apply . The occurrence of viruses, bacteria, yeasts, fungi, and zooparasites in sewage sludge is reviewed in this article, and consequential epidemiologic concerns that arise from sewage sludge recycling is also addressed.

Curr Top Microbiol Immunol, 2001, 257, 85 - 111
RTX toxin structure and function: a story of numerous anomalies and few analogies in toxin biology; Welch RA; It can be agreed that RTX toxins contribute to the pathogenesis of different diseases by causing dysfunction of the general cellular reactions of the immune response . The suggestion that RTX toxins induce cytokine production in nonimmune cells that would ultimately cause tissue damage is an expansion of their role in disease pathogenesis (Uhlen et al . 2000) . Investigators in the RTX toxin field may not agree with me, but precise and satisfactory answers to the following questions are not yet available . How do RTX toxins mechanistically damage a cell? Do RTX toxins have receptors in the classic sense, in which there is a reversible ligand and receptor complex? What is responsible for the common Ca2+ ion influx in affected cells? The recent observation that an RTX toxin stimulates host-cell-mediated Ca2+ ion oscillation in part challenges the long held concept that these toxins damage cells by the direct formation of pores . Are the Ca2+ ion fluxes truly the noxious cellular insult? What is the final molecular structure of RTX toxins at the time they cause cellular death? How does the common requirement for acyl modification among RTX toxins fit into the toxin structure and mechanism of cellular killing, particularly when mixtures of unusual fatty acids are used by some toxins? There are a number of outstanding laboratories throughout the world that are seeking answers to these questions . We can reasonably expect that during the next decade research on the structure and function of RTX toxins will lead to new chemotherapeutic targets and reagents for basic cell biology and biotechnology.

Proc R Soc Lond B Biol Sci, 2000 Oct 22, 267(1457), 2037 - 42
The cause of parasitic infection in natural populations of Daphnia (Crustacea: Cladocera): the role of host genetics; Little TJ et al.; Disease patterns in nature may be determined by genetic variation for resistance or by factors, genetic or environmental, which influence the host-parasite encounter rate . Elucidating the cause of natural infection patterns has been a major pursuit of parasitologists, but it also matters for evolutionary biologists because host resistance genes must influence the expression of disease if parasite-mediated selection is to occur . We used a model system in order to disentangle the strict genetic component from other causes of infection in the wild . Using the crustacean Daphnia magna and its sterilizing bacterial parasite Pasteuria ramosa, we tested whether genetic variation for resistance, as determined under controlled conditions, accounted for the distribution of infections within natural populations . Specifically, we compared whether the clonally produced great-granddaughters of those individuals that were infected in field samples (but were subsequently 'cured' with antibiotics) were more susceptible than were the great-granddaughters of those individuals that were healthy in field samples . High doses of parasite spores led to increased infection in all four study populations, indicating the importance of encounter rate . Host genetics appeared to be irrelevant to natural infection patterns in one population . However, in three other populations hosts that were healthy in the field had greater genetic-based resistance than hosts that were infected in the field, unambiguously showing the effect of host genetic factors on the expression of disease in the wild.

Biochem J, 2001 Jul 1, 357(Pt 1), 211 - 6
Characterization of the cyanobacterial ycf37: mutation decreases the photosystem I content; Wilde A et al.; We have constructed and analysed a cyanobacterial mutant that lacks the putative homologue of ycf37, the chloroplast open reading frame 37, which is conserved in different algae, but missing in the plastome of higher plants . In this report we show that Ycf37 of Synechocystis sp . PCC 6803 contains three tetratrico-peptide repeat (TPR) units resembling the structural organization of Ycf3, a protein that has been suggested to function as a chaperone during photosystem (PS) I complex formation . We demonstrate a light-activated transcript accumulation of this gene . Inactivation of ycf37 leads to a lower PSI/PSII ratio and a higher phycocyanin/chlorophyll ratio in Synechocystis cells . The observed alterations in the ycf37 mutants and the structural organization of the gene product suggest a functional role in PSI stability or assembly.

J Am Chem Soc, 2001 Jun 27, 123(25), 6164 - 71
Comparison of methyl rotation axis order parameters derived from model-free analyses of (2)H and (13)C longitudinal and transverse relaxation rates measured in the same protein sample; Ishima R et al.; Recombinant HIV-1 protease was obtained from bacteria grown on a 98% D(2)O medium containing 3-(13)C pyruvic acid as the sole source of (13)C and (1)H . The purified protein is highly deuterated at non-methyl carbons, but contains significant populations of (13)CHD(2) and (13)CH(2)D methyl isotopomers . This pattern of isotope labeling permitted measurements of (1)H and (13)C relaxation rates of (13)CHD(2) isotopomers and (2)H (D) relaxation rates of (13)CH(2)D isotopomers using a single sample . The order parameters S(axis)(2), which characterize the motions of the methyl rotation axes, were derived from model-free analyses of R(1) and R(2) data sets measured for (13)C and (2)H spins . Our primary goal was to compare the S(axis)(2) values derived from the two independent types of data sets to test our understanding of the relaxation mechanisms involved . However, S(axis)(2) values derived from the analyses depend strongly on the geometry of the methyl group, the sizes of the quadrupolar and dipolar couplings, and the effects of bond vibrations and librations on these couplings . Therefore uncertainties in these basic physical parameters complicate comparison of the order parameters . This problem was circumvented by using an experimental relationship, between the methyl quadrupolar, (13)C-(13)C and (13)C-(1)H dipolar couplings, derived from independent measurements of residual static couplings of weakly aligned proteins by Ottiger and Bax (J . Am . Chem . Soc . 1999, 121, 4690-4695) and Mittermaier and Kay (J . Am . Chem . Soc . 1999, 121, 10608-10613) . This approach placed a tight experimental restraint on the values of the (2)H quadrupolar and (13)C-(1)H dipolar interactions and greatly facilitated the accurate comparison of the relative values of the order parameters . When applied to our data this approach yielded satisfactory agreement between the S(axis)(2) values derived from the (13)C and (2)H data sets.

Appl Occup Environ Hyg, 2001 Jun, 16(6), 679 - 84
Survey of the Asp f 1 allergen in office environments; Ryan TJ et al.; Sick Building Syndrome remains a prevalent problem with patient complaints similar to typical allergy symptoms . Unlike household allergens typically found in domestic reservoirs, the allergen from a common fungus like Aspergillus fumigatus (i.e., Asp f 1) is conceivably widespread in the work environment . This project surveyed airborne levels of the Asp f 1 allergen in office and non-industrial occupational environments, as well as the dust reservoirs of A . fumigatus believed to be responsible for those levels . Airborne and bulk dust samples were collected, extracted, and assayed for Asp f 1 . Concurrently, bulk dusts collected from the same locations were selectively cultured for A . fumigatus, and mesophilic fungi and bacteria . Samples were collected during both wet and dry climatological conditions from paired wet and dry building locations to examine the possibility of Asp f 1 increases due to fungal growth blooms . Very low levels of Asp f 1 were detected but only in the airborne samples (2/120 positive samples, with 3.6 ng/m3 and 1.8 ng/m3; LOD < 1.2 ng/m3) . No dust samples showed even detectable traces of the allergen (LOD = 5 ng/g dust) . Although A . fumigatus counts from dusts fluctuated significantly with exterior moisture events, analysis of wet versus dry period samples showed no differences in Asp f 1 levels . These results indicate that even in the presence of measurable fungal concentrations, background levels of Asp f 1 are low . Nonindustrial office buildings devoid of indoor air quality issues were not observed to have significant levels of the Asp f 1 allergen in the geographical region studied.

Immunol Rev, 2001 Apr, 180, 100 - 11
CR1 and CR1-like: the primate immune adherence receptors; Birmingham DJ et al.; Immune adherence describes the phenomenon in which complement-opsonized substrates, such as immune complexes (IC), viruses, or bacteria, are bound by primate erythrocytes via erythrocyte complement receptors . In vivo studies have shown that this binding allows the erythrocyte to act as an inert shuttle, targeting IC to the monocyte phagocytic system and away from vulnerable tissue . Thus, immune adherence appears to play an integral role in the primate in promoting the safe clearance of circulating IC and preventing IC-mediated pathologies . The complement receptors that mediate immune adherence comprise two unique but closely related gene products, either the type one complement receptor (CRI) in humans or CRI-like in non-human primates . This review focuses on the structure, function, and physiological role of the primate immune adherence receptors.

Blood Rev, 2001 Jun, 15(2), 69 - 83
Non-infectious complications of transfusion therapy; Perrotta PL et al.; Blood transfusion is considered safe when the infused blood is tested using state of the art viral assays developed over the past several decades . Only rarely are known viruses like HIV and hepatitis C transmitted by transfusion when blood donors are screened using these sensitive laboratory tests . However, there are a variety of transfusion risks which still remain that cannot be entirely eliminated, many of which are non-infectious in nature . Predominantly immune-mediated complications include the rapid intravascular or slow extravascular destruction (hemolysis) of transfused red cells or extravascular removal of platelets by pre-formed antibodies carried by the transfusion recipient . Alternatively, red cells can be damaged when exposed to excessive heat or incompatible intravenous fluids before or during the transfusion . Common complications of blood transfusion that at least partly involve the immune system include febrile non-hemolytic and allergic reactions . While these are usually not life-threatening, they can hamper efforts to transfuse a patient . Other complications include circulatory overload, hypothermia and metabolic disturbances . Profound hypotensive episodes have been described in patients on angiotensin-converting enzyme (ACE) inhibitors who receive platelet transfusions through bedside leukoreduction filters . These curious reactions appear to involve dysmetabolism of the vasoactive substance bradykinin . Products contaminated by bacteria during blood collection and transfused can cause life-threatening septic reactions . A long-term complication of blood transfusion therapy unique to chronically transfused patients is iron overload . Less common - but serious - reactions more specific to blood transfusion include transfusion-associated graft-versus-host disease and transfusion-associated acute lung injury . Many of these complications of transfusion therapy can be prevented by adhering to well-established practice guidelines . In addition, individuals who administer blood transfusions should recognize these complications in order to be able to quickly provide appropriate treatment .

Farmaco, 2001 Mar, 56(3), 229 - 32
Synthesis, characterization and pharmacological activities of 5,6,11,12-tetrahydroindolo{2,3-alpha}carbazole derivatives; Balamurali R et al.; A series of new 5,6,11,12-tetrahydroindolo{2,3-alpha}carbazole derivatives (3a-h) was synthesized . Treatment of 8-methyl-1-oxo-1,2,3,4-tetrahydrocarbazole (1a) with phenylhydrazine hydrochloride in ethanol furnished the title compound (3a) in poor yield along with 8-methyl-1-phenylhydrazono-1,2,3,4-tetrahydrocarbazole (2a) . Better yields were obtained when 1a-h were treated with phenylhydrazine in glacial acetic acid . All the newly synthesized compounds were characterized on the basis of IR, NMR, mass-spectra and elemental analysis and screened for pharmacological activities.

J Gen Virol, 2001 Jul, 82(Pt 7), 1695 - 702
Identification of conformational neutralizing epitopes on the capsid protein of canine calicivirus; Matsuura Y et al.; Two neutralizing monoclonal antibodies (MAbs) against canine calicivirus (CaCV), which has a distinct antigenicity from feline calicivirus (FCV), were obtained . Both MAbs recognized conformational epitopes on the capsid protein of CaCV and were used to identify these epitopes . Neutralization-resistant variants of CaCV were selected in the presence of individual MAbs in a cell culture . Cross-neutralization tests using the variants indicated that the MAbs recognized functionally independent epitopes on the capsid protein . Recombinantly expressed ORF2 products (capsid precursors) of the variants showed no reactivity to the MAbs used for the selection, suggesting that the resistance was induced by a failing in binding of the MAbs to the variant capsid proteins . Several nucleotide changes resulting in amino acid substitutions in the capsid protein were found by sequence analysis . Reactivities of the MAbs to the revertant ORF2 products produced from each variant ORF2 by site-directed mutagenesis identified a single amino acid substitution in each variant capsid protein responsible for the failure of MAb binding . The amino acid residues related to forming the conformational neutralizing epitopes were located in regions equivalent to the 5' and 3' hypervariable regions of the FCV capsid protein, where antigenic sites were demonstrated in previous studies . The recombinant ORF2 products expressed in bacteria failed to induce neutralizing antibody, suggesting that neutralizing antibodies were only generated when properly folded capsid protein was used as an antigen . In CaCV, the conformational epitopes may play a more important role in neutralization than do linear epitopes.

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