Microorganisms

Mol Cell Biol, 2001 Jan, 21(1), 354 - 66
Translational and structural requirements of the early nodulin gene enod40, a short-open reading frame-containing RNA, for elicitation of a cell-specific growth response in the alfalfa root cortex; Sousa C et al.; A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes . The early nodulin gene enod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria . Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells . Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5' and 3' regions of enod40 was required for this biological activity . These sORFs may be translated in roots via a reinitiation mechanism . In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids . Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity . Our data reveal a complex regulation of enod40 action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.

J Biol Chem, 2001 Mar 16, 276(11), 8427 - 35 Epub 2000 Dec 11.
The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi; Hellwage J et al.; Spirochete bacteria of the Borrelia burgdorferi sensu lato complex cause Lyme borreliosis . The three pathogenic subspecies Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto differ in their disease profiles and susceptibility to complement lysis . We investigated whether complement resistance of Borreliae could be due to acquisition of the main soluble inhibitors of the alternative complement pathway, factor H and the factor H-like protein 1 . When exposed to nonimmune EDTA-plasma, the serum-resistant B . afzelii and B . burgdorferi sensu stricto strains bound factor H/factor H-like protein 1 to their surfaces . Assays with radiolabeled proteins showed that factor H bound strongly to the B . burgdorferi sensu stricto strain . To identify factor H ligands on the borrelial surface, we analyzed a panel of outer surface proteins of B . burgdorferi sensu stricto with the surface plasmon resonance technique . The outer surface lipoprotein OspE was identified as a specific ligand for factor H . Using recombinant constructs of factor H, the binding site for OspE was localized to the C-terminal short consensus repeat domains 15-20 . Specific binding of factor H to B . burgdorferi sensu stricto OspE may help the pathogen to evade complement attack and phagocytosis.

J Cell Sci, 2001 Jan, 114(Pt 1), 173 - 185
A novel nucleolar G-protein conserved in eukaryotes; Park JH et al.; We describe here a novel, evolutionarily conserved set of predicted G-proteins . The founding member of this family, TbNOG1, was identified in a two-hybrid screen as a protein that interacts with NOPP44/46, a nucleolar phosphoprotein of Trypanosoma brucei . The biological relevance of the interaction was verified by co-localization and co-immunoprecipitation . TbNOG1 localized to the trypanosome nucleolus and interacted with domains of NOPP44/46 that are found in several other nucleolar proteins . Genes encoding proteins highly related to TbNOG1 are present in yeast and metazoa, and related G domains are found in bacteria . We show that NOG1 proteins in humans and Saccharomyces cerevisae are also nucleolar . The S . cerevisae NOG1 gene is essential for cell viability, and mutations in the predicted G motifs abrogate function . Together these data suggest that NOG1 may play an important role in nucleolar functions . The GTP-binding region of TbNOG1 is similar to those of Obg and DRG proteins, which, together with NOG, form a newly recognized family of G-proteins, herein named ODN . The ODN family differs significantly from other G-protein families, and shows several diagnostic sequence characteristics . All organisms appear to possess an ODN gene, pointing to the biological significance of this family of G-proteins.

J Invertebr Pathol, 2000 Nov, 76(4), 249 - 56
Light and electron microscopic study of a Rickettsiella species from the cockroach Blatta orientalis; Radek R; An infection with Rickettsiella sp . was responsible for an illness causing heavy body swelling in the Oriental cockroach Blatta orientalis . Reproduction of the colony stagnated . Vacuoles with parasitic bacteria occurred mainly in the fat body, but also in nearly all other organs, such as gut epithelium, Malpighian tubules, blood cells, and ovarioles . The parasites clearly differed from the symbiotic bacteria of the genus Blattabacterium, which regularly occur in the mycetocytes of B . orientalis . The vacuoles contained four stages of Rickettsiella: (1) infectious, electron-dense, rod-like elementary bodies (mean size 300 x 145 nm); (2) an electron-dense, flat intermedium stage, called flat body (mean size 515 x 255 x 125 nm); (3) an electron-light, spherical intermedium stage, called condensing sphere (mean size 340 nm); portions of cytoplasm condensed crescent-like at the border or in the center of the cell; and (4) large, spherical, electron-light initial bodies that multiplied by binary fission (mean size 600 nm) . The initial bodies had a three-layered cell boundary, but all other stages had a five-layered cell boundary . Elementary and flat bodies contained an electron-light, oblique lamella and an oval structure with an array of ribosome-like granules, respectively . In contrast to other species of Rickettsiella, crystal formation or multiple division did not occur . The described species of Rickettsiella is different from "R . blattae," which belongs to the R . popilliae group . Instead, it shares more similarities with the R . chironomi group . To avoid confusion, it was provisionally named "R . crassificans."

J Oral Sci, 2000 Sep, 42(3), 139 - 46
Topography of periodontally involved human root surfaces after different chemical treatment modalities: an in vitro scanning electron microscopic study; Okte E et al.; The significance of chemical and conservative treatments of cemental tissue proximal to periodontal pockets has been pointed out in recent years . This in vitro scanning electron microscopy (SEM) study aimed to investigate the surface effects of topical applications of 0.1% cetylpyridinium chloride (CPC) and 2% sodium lauryl sulfate (SLS) and polishing on the periodontally involved root surfaces of human teeth . Ten single-rooted teeth from 8 patients with advanced adult periodontitis were included . Following extraction, any calculus was removed with extreme care to preserve as much cementum as possible . Eighty root specimens were prepared . Fresh solutions of CPC and SLS were applied for 1, 3 and 5 minutes each to 10 segments of root cementum . A total of 20 segments formed the polished (P) and control (C) groups, respectively . The results showed that the surfaces treated with CPC or SLS differed considerably from polished and control specimens . Depending on time, the surface coating was partly or wholly removed, leaving a nodular cementum structure, uncovering a fibrillar collagen substrate and the openings of dentinal tubules . Scarce debris was present on both control and polished surfaces, whereas bacteria were observed only on the control specimens . In view of these results, further definitive in vitro and in vivo research must be done to determine the advantages of chemical treatment and its effect on periodontal regeneration.

Vox Sang, 2000, 79(3), 168 - 74
An experiment with glycerol-frozen red blood cells stored at -80 degrees C for up to 37 years; Valeri CR et al.; BACKGROUND AND OBJECTIVES: Red cells frozen using 40% W/V glycerol are currently FDA approved for frozen storage at -80 degrees C for up to 10 years . MATERIALS AND METHODS: Red cells frozen with 40% W/V glycerol and stored at -80 degrees C for up to 37 years were thawed, deglycerolized, and stored at 4 degrees C for 24 h . RESULTS: Red cells frozen for up to 37 years had mean freeze-thaw-wash recovery values of 75%, less than 1% hemolysis, and normal ATP, 2,3-DPG and P50 levels, and 60% of normal RBC K(+) levels . CONCLUSIONS: Red cells frozen with 40% W/V glycerol can be stored at -80 degrees C for up to 37 years with acceptable in vitro results .

Biochim Biophys Acta, 2000 Dec 15, 1529(1-3), 63 - 88
Sterol methyl transferase: enzymology and inhibition; Nes WD; Sterol C-methylations catalyzed by the (S)-adenosyl-L-methionine: Delta(24)-sterol methyl transferase (SMT) have provided the focus for study of electrophilic alkylations, a reaction type of functional importance in C-C bond formation of natural products . SMTs occur generally in nature, but do not occur in animal systems, suggesting that the difference in sterol synthetic pathways can be exploited therapeutically and in insect-plant interactions . The SMT genes from several plants and fungi have been cloned, sequenced and expressed in bacteria or yeast and bioengineered into tobacco or tomato plants . These enzymes share significant amino acid sequence similarity in the putative sterol and AdoMet binding sites . Investigations of the molecular recognition of sterol fitness and studies with stereospecifically labeled substrates as well as various sterol analogs assayed with native or mutant SMTs from fungi and plants have been carried out recently in our own and other laboratories . These analyses have led to an active-site model, referred to as the 'steric-electric plug' model, which is consistent with a non-covalent mechanism involving the intermediacy of a 24beta-methyl (or ethyl) sterol bound to the ternary complex . Despite the seeming differences between fungal and plant SMT activities the recent data indicate that a distinct SMT or family of SMTs exist in these organisms which bind and transform sterols according to a similar mechanistic plan . Vascular plants have been found to express different complements of C(1)/C(2)-activities in the form of at least three SMT isoforms . This enzyme multiplicity can be a target of regulatory control to affect phytosterol homeostasis in transgenic plants . The state of our current understanding of SMT enzymology and inhibition is presented.

Biochim Biophys Acta, 2000 Dec 15, 1529(1-3), 33 - 48
Isoprenyl diphosphate synthases; Wang KC et al.; Isoprenyl diphosphate synthases catalyze consecutive condensations of isopentenyl diphosphates with allylic primer substrates to form linear backbones for all isoprenoid compounds including cholesterol . These synthases are classified according to the final chain length of their end products and the stereochemistry of the newly formed double bonds . Mutagenesis and X-ray crystallography data have uncovered the basic catalytic and chain length determination mechanisms of E-isoprenyl diphosphate synthases and shed light on their possible evolutionary course . Although much less is known about the Z-isoprenyl diphosphate synthase family, successful cloning and subsequent crystallizations in the near future will no doubt bring more insight as researchers begin to unravel the essential components and precise reaction mechanisms of this cellular machinery.

Mol Biol Evol, 2000 Dec, 17(12), 1956 - 70
Evolution of two-component signal transduction; Koretke KK et al.; Two-component signal transduction (TCST) systems are the principal means for coordinating responses to environmental changes in bacteria as well as some plants, fungi, protozoa, and archaea . These systems typically consist of a receptor histidine kinase, which reacts to an extracellular signal by phosphorylating a cytoplasmic response regulator, causing a change in cellular behavior . Although several model systems, including sporulation and chemotaxis, have been extensively studied, the evolutionary relationships between specific TCST systems are not well understood, and the ancestry of the signal transduction components is unclear . Phylogenetic trees of TCST components from 14 complete and 6 partial genomes, containing 183 histidine kinases and 220 response regulators, were constructed using distance methods . The trees showed extensive congruence in the positions of 11 recognizable phylogenetic clusters . Eukaryotic sequences were found almost exclusively in one cluster, which also showed the greatest extent of domain variability in its component proteins, and archaeal sequences mainly formed species-specific clusters . Three clusters in different parts of the kinase tree contained proteins with serine-phosphorylating activity . All kinases were found to be monophyletic with respect to other members of their superfamily, such as type II topoisomerases and Hsp90 . Structural analysis further revealed significant similarity to the ATP-binding domain of eukaryotic protein kinases . TCST systems are of bacterial origin and radiated into archaea and eukaryotes by lateral gene transfer . Their components show extensive coevolution, suggesting that recombination has not been a major factor in their differentiation . Although histidine kinase activity is prevalent, serine kinases have evolved multiple times independently within this family, accompanied by a loss of the cognate response regulator(s) . The structural and functional similarity between TCST kinases and eukaryotic protein kinases raises the possibility of a distant evolutionary relationship.

Mol Biol Evol, 2000 Dec, 17(12), 1807 - 15
Reconstructing genealogies of serial samples under the assumption of a molecular clock using serial-sample UPGMA; Drummond A et al.; Reconstruction of evolutionary relationships from noncontemporaneous molecular samples provides a new challenge for phylogenetic reconstruction methods . With recent biotechnological advances there has been an increase in molecular sequencing throughput, and the potential to obtain serial samples of sequences from populations, including rapidly evolving pathogens, is fast being realized . A new method called the serial-sample unweighted pair grouping method with arithmetic means (sUPGMA) is presented that reconstructs a genealogy or phylogeny of sequences sampled serially in time using a matrix of pairwise distances . The resulting tree depicts the terminal lineages of each sample ending at a different level consistent with the sample's temporal order . Since sUPGMA is a variant of UPGMA, it will perform best when sequences have evolved at a constant rate (i.e., according to a molecular clock) . On simulated data, this new method performs better than standard cluster analysis under a variety of longitudinal sampling strategies . Serial-sample UPGMA is particularly useful for analysis of longitudinal samples of viruses and bacteria, as well as ancient DNA samples, with the minimal requirement that samples of sequences be ordered in time.

J Biol Chem, 2001 Mar 9, 276(10), 7437 - 41 Epub 2000 Dec 07.
Direct demonstration that homotetrameric chaperone SecB undergoes a dynamic dimer-tetramer equilibrium; Topping TB et al.; We have shown here that the cytosolic bacterial chaperone SecB is a structural dimer of dimers that undergoes a dynamic equilibrium between dimer and tetramer in the native state . We demonstrated this equilibrium by mixing two tetrameric species of SecB that can be distinguished by size . We showed that the homotetrameric species exchanged dimers, because when the mixture was analyzed both by size exclusion chromatography and native polyacrylamide gel electrophoresis a third hybrid tetrameric species was detected . Furthermore, treatment of SecB with 5,5'-dithiobis-(2-nitrobenzoic acid), which modifies the sulfhydryl group on cysteines, caused irreversible dissociation to a dimer indicating that cysteine must be involved in the stabilizing interactions at the dimer interface . It is clear that the two dimer-dimer interfaces of the SecB tetramer are differentially stable . Dissociation at one interface allows for a dynamic dimer-tetramer equilibrium . Because only dimers were exchanged it is clear that the other interface between dimers is significantly more stable, otherwise oligomers should have formed with a random distribution of monomers.

ASAIO J, 2000 Nov-Dec, 46(6), S2 - 5
Physiology of wound healing and surgical wound care; Phillips SJ; Wound healing is a systemic process, which occurs stepwise and involves the stages of hemostasis, inflammation, and repair . Hemostasis with fibrin formation creates a protective wound scab . The scab provides a surface beneath which cell migration and movement of the wound edges can occur . Inflammation brings nutrients to the area of the wound, removes debris and bacteria, and provides chemical stimuli for wound repair . Repair begins immediately after wounding and proceeds rapidly through the processes of epithelialization, fibroplasia, and capillary proliferation into the healing area . Different tissues have their own normal rates of growth during the process of healing . The optimal rate of healing is approached when factors advantageous to healing are present and factors having the ability to disturb or retard the healing processes are controlled or absent . These factors are discussed.

Przegl Lek, 2000, 57(7-8), 424 - 6
{Tick spirochetosis--Lyme borreliosis}; Nowakowski G et al.; Lyme boreliosis is currently the most common tick-borne infection . It may cause various clinical symptoms depending on organ localization and duration of the infection . The disease may be symptomless, subclinical or with full clinical manifestation . Usually three clinical stages may be distinguished . In stage I erythema migrans and flu-like symptoms usually develop . In stage II, connected with the infection spreading with blood and lymph, beside joint pains, neuroboreliosis appears, sometimes the disease involves other organs such as heart, eyes, testicles, joints . Stage III, chronic in its character, usually develops in patients who had previously reported joint and neurological complaints . Encephalopathy and fibromyalgia accompany joint involvement . Diagnostics of Lyme borreliosis is based on clinical evaluation and laboratory test including culture of the bacteria obtained from biopsies and serological tests . There are no established standards of the treatment--some examples of the therapy are presented in the paper . The disease if not treated has a progressive course in most causes, however in some patients it can resolve spontaneously even with no treatment.

J Public Health Dent, 2000 Summer, 60(3), 197 - 206; discussion 207-9
Dietary determinants of dental caries and dietary recommendations for preschool children; Tinanoff N et al.; OBJECTIVES: The purpose of this review, commissioned by the Administration for Children and Families, the Health Resources and Services Administration, the Health Care Financing Administration, and the Department of Agriculture's Food and Nutrition Service, was to update the evidence of the dietary factors that affect dental caries, and subsequently formulate dietary recommendations for preschool children based on principles of cariology . METHODS: Literature on the dental caries process, dietary factors affecting dental caries initiation and progression, and nutrition education and counseling were reviewed and synthesized . Dietary guidelines for children at various ages were then constructed based on the review . RESULTS: Dental caries in preschool children is due to a combination of factors, including colonization of teeth with cariogenic bacteria, type of foods and frequency of exposure of these foods to the cariogenic bacteria, and susceptible teeth . Caries risk is greatest if sugars are consumed at high frequency and are in a form that is retained in the mouth for long periods . Sucrose is the most cariogenic sugar because it can form glucan that enables firm bacterial adhesion to teeth and limits diffusion of acid and buffers in the plaque . There is emerging interest in the effects of tooth development and its role in the future dental caries risk of the child . CONCLUSIONS: Nutrition education and counseling for the purposes of reducing caries in children is aimed at teaching parents the importance of reducing high frequency exposures to obvious and hidden sugars . Guidelines include: avoiding frequent consumption of juice or other sugar-containing drinks in the bottle or sippy cup, discouraging the behavior of a child sleeping with a bottle, promoting noncariogenic foods for snacks, fostering eating patterns consistent with the Food Guide Pyramid, limiting cariogenic foods to mealtimes, rapidly clearing cariogenic foods from the child's oral cavity either by toothbrushing or by consumption of protective foods, and restricting sugar-containing snacks that are slowly eaten (e.g., candy, cough drops, lollipops, suckers) . Along with nutritional factors, a comprehensive approach to preventing dental caries in preschool children must include improved general dietary habits, good oral hygiene, appropriate use of fluorides, and access to preventive and restorative dental care.

Diagn Microbiol Infect Dis, 2000 Nov, 38(3), 169 - 70
Feculent meningitis: polymicrobial meningitis in colorectal surgery; Garcia-Lechuz JM et al.; Polymicrobial anaerobic meningitis is a rare event secondary to a contiguous infection in the head or neck . Anaerobic meningitis due to a meningo-intestinal fistula is a rare event with only two cases reported in the literature . We describe a new case of adult polymicrobial anaerobic meningitis after colorectal surgery and radiotherapy and review the previous two cases.

FEBS Lett, 2000 Dec 1, 486(1), 52 - 6
Isolation, reconstitution and functional characterisation of the Rhodobacter sphaeroides photoactive yellow protein; Haker A et al.; We report the isolation, functional reconstitution and photophysical characterisation of Rhodobacter sphaeroides photoactive yellow protein (PYP), of which the gene was recently cloned . Reconstitution of the his-tagged purified apo-protein with 4-hydroxy-cinnamic acid yields the characteristic blue absorbance at 446 nm, but surprisingly also an absorbance peak at 360 nm . This additional peak is not caused by binding of a second chromophore, as confirmed with mass spectroscopy . Moreover, reconstitution with the 'locked' analogue 7-hydroxy-coumarin-3-carboxylic acid yields only a single absorbance peak at 441 nm . The 446 nm and 360 nm species are part of a temperature- and pH-dependent equilibrium . Photoactivation of the protein leads to formation of a blue-shifted intermediate as in other PYPs, with a 100-fold increased groundstate recovery rate (k(pB-->pG)=500 s(-1)) compared to E-PYP.

Int Microbiol, 2000 Mar, 3(1), 45 - 9
DNA fluorescent stain accumulates in the Golgi but not in the kinetosomes of amitochondriate protists; Dolan MF; Hindgut symbiotic trichomonads (uninucleate Caduceia versatilis, and multinucleate Stephanonympha sp . and Snyderella tabogae) from the dry-wood-eating termite Cryptotermes cavifrons (Kalotermitidae) accumulate DAPI (4,6diamidino-2-phenylindole) in the membranous sacs of the Golgi complex . This form of Golgi complex, typical of protists in the class Parabasalia, is called a parabasal body . Trichomonads contain organellar systems, mastigonts, that consist of four undulipodia (e.g . eukaryotic flagella and cilia), axostylar microtubules, a parabasal body and other structures . These cells bear from one (in the case of Caduceia) to hundreds (in the case of Snyderella) of mastigonts . These features are characteristic of their protist class (Parabasalia) . The nuclei of all three species stained with DNA-specific stains: DAPI, SYTOX, acridine orange, propidium iodide, ethidium bromide and Feulgen, at optimal concentrations, but kinetosomes failed to stain at all . The nuclei, parabasal bodies and symbiotic bacteria (but no microtubular structures) fluoresced in glutaraldehyde-fixed cells stained with 1.45 microM DAPI . Parabasal bodies of Snyderella and Caduceia treated to remove lipids with Triton X-100, or treated with 5% trichloroacetic acid, lacked DAPI-fluorescence . I conclude that DNA, present as expected in nuclei and bacterial symbionts, is absent from and not associated with calonymphid kinetosomes . The reason for DNA-RNA stain accumulation in the Golgi cistemae is not clear.

Int Microbiol, 2000 Mar, 3(1), 17 - 24
Biosorption: a solution to pollution?
Vieira RH, Volesky B.
To solve the water pollution problem by toxic heavy metal contamination resulting from humans technological activities has for long presented a challenge . Biosorption can be a part of the solution . Some types of biosorbents such as seaweeds, molds, yeasts, bacteria or crab shells are examples of biomass tested for metal biosorption with very encouraging results . The uptake of heavy metals by biomass can in some cases reach up to 50% of the biomass dry weight . New biosorbents can be manipulated for better efficiency and multiple re-use to increase their economic attractiveness.

Int Microbiol, 1999 Sep, 2(3), 155 - 60
Molecular mechanisms of malaria sporozoite motility and invasion of host cells; Sultan AA; Malaria sporozoites have the unique capacity to invade two entirely different types of target cell in the mosquito vector and the vertebrate host during the course of the parasite's life cycle . Although little is known about the specific interaction of the sporozoite with its target cells, two sporozoite proteins, circumsporozoite (CS) and thrombospondin-related adhesive protein (TRAP), have been shown to play important roles in the invasion of both cell types . CS protein is a multifunctional protein involved in sporogony, invasion of the salivary glands, the specific arrest of sporozoites in the liver sinusoid, gliding motility of the sporozoite, and hepatocyte recognition and entry . TRAP has been shown to be critical for sporozoite infection of the mosquito salivary glands and liver cells, and is essential for sporozoite gliding motility . This review will focus on the involvement of these molecules in sporozoite motility and the invasion of host cells.

Int Microbiol, 1999 Sep, 2(3), 137 - 44
Molecular aspects of Bordetella pertussis pathogenesis; Locht C; The molecular mechanisms of Bordetella virulence are now well understood, and many virulence factors have been identified and characterized at the molecular level . These virulence factors can be grouped into two major categories: adhesins, such as filamentous hemagglutinin, pertactin and fimbriae, and toxins, such as pertussis toxin, adenylate cyclase, dermonecrotic toxin and tracheal cytotoxin . The production of most virulence factors is coordinately regulated by a two-component signal transduction system composed of the regulator BvgA and the sensor protein BvgS . The adhesins and toxins act in concert to establish infection . Some adhesins exert their effects synergically or are redundant functioning only in the absence of another adhesin, illustrating the importance of adhesion in infection . Most virulence factors are secreted into the culture supernatant or exposed at the surface of the bacterial cell . A notable exception is dermonecrotic toxin, which remains in the cytoplasmic compartment of bacterial cells . Most virulence factors are produced by all of the three major Bordetella species, B . pertussis, B . parapertussis and B . bronchiseptica . However, some, such as pertussis toxin and the tracheal colonization factor, are only produced by B . pertussis . Our understanding of Bordetella virulence at the molecular level has led to the development of new acellular vaccines against whooping cough, and of genetically attenuated B . pertussis strains to be used as recombinant live bacterial vaccine vectors for homologous and heterologous protection.

Int Microbiol, 1998 Dec, 1(4), 319 - 26
Morphogenesis by symbiogenesis; Chapman MJ et al.; Here we review cases where initiation of morphogenesis, including the differentiation of specialized cells and tissues, has clearly evolved due to cyclical symbiont integration . For reasons of space, our examples are drawn chiefly from the plant, fungal and bacterial kingdoms . Partners live in symbioses and show unique morphological specializations that result when they directly and cyclically interact . We include here brief citations to relevant literature where plant, bacterial or fungal partners alternate independent with entirely integrated living . The independent, or at least physically unassociated stages, are correlated with the appearance of distinctive morphologies that can be traced to the simultaneous presence and strong interaction of the plant with individuals that represent different taxa.

Evolution Int J Org Evolution, 2000 Oct, 54(5), 1661 - 72
Expression of cytoplasmic incompatibility in Drosophila simulans and its impact on infection frequencies and distribution of Wolbachia pipientis; James AC et al.; The aim of this study is to examine the expression of cytoplasmic incompatibility and investigate the distribution and population frequencies of Wolbachia pipientis strains in Drosophila simulans . Nucleotide sequence data from 16S rDNA and a Wolbachia surface protein coding sequence and cytoplasmic incompatibility assays identify four distinct Wolbachia strains: wHa, wRi, wMa, and wAu . The levels of cytoplasmic incompatibility between six lines carrying these strains of bacteria and three control lines without bacteria are characterized . Flies infected with wHa and wRi are bidirectionally incompatible, and males that carry either strain can only successfully produce normal numbers of offspring with females carrying the same bacterial strain . Males infected with wAu do not express incompatibility . Males infected with the wMa strain express intermediate incompatibility when mated to females with no bacteria and no incompatibility with females with any other Wolbachia strain . We conduct polymerase chain reaction/restriction fragment length polymorphism assays to distinguish the strain of Wolbachia and the mitochondrial haplotype to survey populations for each type and associations between them . Drosophila simulans is known to have three major mitochondrial haplotypes (siI, sill, and siIII) and two subtypes (siIIA and siIIB) . All infected lines of the sil haplotype carry wHa, wNo, or both; wMa and wNo are closely related and it is not clear whether they are distinct strains or variants of the same strain . Infected lines with the silIA haplotype harbor wRi and the siIIB haplotype carries wAu . The wMa infection is found in siIII haplotype lines . The phenotypic expression of cytoplasmic incompatibility and its relation to between-population differences in frequencies of Wolbachia infection are discussed.

Cytokines Cell Mol Ther, 2000 Jun, 6(2), 89 - 95
Use of G-CSF for granulocyte transfusion therapy; Hubel K et al.; Patients with neutropenia, especially neutropenia following aggressive myeloablative therapy, are at high risk for developing infectious complications caused by bacteria and opportunistic fungi . Infections remain one of the leading causes of treatment failure in patients with cancer . Thus, new and innovative therapeutic strategies are needed for management of neutropenic patients with infection . Because neutrophils represent the first line of host defense, granulocyte transfusion therapy should be a logical therapeutic approach . Although such therapy has been employed sporadically for several decades, clinical benefit has been compromised by technical problems and low granulocyte yields resulting from inadequate donor stimulation . The discovery of granulocyte colony-stimulating factor (G-CSF) as a means to elevate blood neutrophil counts when administered to normal donors has rekindled interest in granulocyte transfusion therapy . Extensive experience has been gained worldwide with G-CSF in clinical practice, and adverse events have been minimal when G-CSF has been administered to patients or healthy persons in human trials . This review focuses on the use of G-CSF in granulocyte transfusion therapy, including technical considerations of granulocyte leukapheresis and storage, donor selection and stimulation, as well as treatment results and associated risks.

Angiology, 2000 Oct, 51(10), 867 - 71
Abdominal aortic aneurysm infected with Helicobacter pylori--a case report; Hirose H et al.; The authors present a case of an abdominal aortic aneurysm infected with Helicobacter pylori bacteria . In their literature search, the authors found no other report of the Helicobacter pylori involved in an infected abdominal aortic aneurysm.

Aust N Z J Med, 2000 Oct, 30(5), 578 - 84
Non-urease producing Helicobacter pylori in chronic gastritis; Ren Z et al.; BACKGROUND: Helicobacter pylori infection is the commonest cause of gastritis . Different patterns of immune response to H . pylori infection and characteristics of bacteria are considered to contribute to clinical outcomes . AIM: To determine characteristics of the host H . pylori relationship in subjects with non-ulcer dyspepsia and a histological diagnosis of gastritis . METHODS: Thirty-five subjects with chronic gastritis undergoing endoscopy (mean age 53 years, range 24-82, 14 male and 21 female) were studied, none of whom was on nonsteroidal anti-inflammatory drugs or antibiotics . H . pylori infection was determined by rapid urease test (CLOtest), culture, antibody and RT-PCR for Ure C, Cag A and 26 kDa gene and histology . Cytokine production of mucosal IL-6 and IL-8 were measured by ELISA . RESULTS: Fifteen subjects were positive by CLOtest and/or bacterial culture . In these subjects histology showed numerous helical forms of H . pylori (Group I) . Nine subjects were negative by CLOtest, bacterial culture, and mRNA for urease C fragment, but positive by PCR for the 26 kDa protein encoding gene . Histology in these subjects showed the presence of either coccoid forms (four), or scant helical forms (two), or mixed coccoid/helical forms (three) (Group II) . Eleven subjects were negative by all methods of detection (Group III) . IgG and IgA antibody levels in serum (p<0.05) and gastric tissue culture supernatant (p<0.001) were significantly higher in Group I than those in Group II or III . There were significant differences in the IgG serum and IgA supernatant antibody levels (p<0.01 and p<0.05) when Group II was compared to Group III . Supernatant IL-6 levels were significantly higher in Group I (p<0.01) than those from Groups II and III . IL-8 levels were higher in Group I (p<0.01) and Group II (p<0.05) when compared to Group III . CONCLUSIONS: 'H . pylori-negative' gastritis can be associated with a non-urease producing form of H . pylori, with a reduction in both local and systemic antibody levels and mucosal pro-inflammatory cytokines.

J Electron Microsc (Tokyo), 2000, 49(2), 371 - 8
Reproducibility and applicability of gallium replication as evaluated by biological specimen use; Adachi E et al.; Structures of biological surfaces pressed on to a pure liquid gallium surface were successfully traced on to the gallium surface by quick-freezing below the melting point (28.78 degrees C) in air or water for replication in scanning electron microscopy . Gallium's high surface tension (approximately 700 mN m(-1) at 30 degrees C) deteriorates the spatial resolution of replicas and destroys some types of specimens . Five different biological surfaces were replicated on to gallium surfaces to evaluate spatial resolution and specimen resistance, i.e . reproducibility and applicability . Gallium replication of jewel beetle wing and human hair demonstrated submicron spatial resolution in the horizontal direction at least . Trials of protozoa, bacteria, and culture cell replication showed that protozoa are suited to replication because the cell membrane has characteristic structures with sufficient resistance to the gallium surface.

Photochem Photobiol, 2000 Nov, 72(5), 639 - 44
Probing the primary event in the photocycle of photoactive yellow protein using photochemical hole-burning technique; Masciangioli T et al.; Photochemical hole-burning spectroscopy was used to study the excited-state electronic structure of the 4-hydroxycinnamyl chromophore in photoactive yellow protein (PYP) . This system is known to undergo a trans-to-cis isomerization process on a femtosecond-to-picosecond time scale, similar to membrane-bound rhodopsins, and is characterized by a broad featureless absorbance at 446 nm . Resolved vibronic structure was observed for the hole-burned spectra obtained when PYP in phosphate buffer at pH 7 was frozen at low temperature and irradiated with narrow bandwidth laser light at 431 nm . The approximate homogeneous width of 752 cm-1 could be calculated from the deconvolution of the hole-burned spectra leading to an estimated dephasing time of approximately 14 fs for the PYP excited-state structure . The resolved vibronic structure also enabled us to obtain an estimated change in the C=C stretching frequency, from 1663 cm-1 in the ground state to approximately 1429 cm-1 upon photoexcitation . The results obtained allowed us to speculate about the excited-state structure of PYP . We discuss the data for PYP in relation to the excited-state model proposed for the photosynthetic membrane protein bacteriorhodopsin, and use it to explain the primary event in the function of photoactive biological protein systems . Photoexcitation was also carried out at 475 nm . The vibronic structure obtained was quite different both in terms of the frequencies and Franck-Condon envelope . The origin of this spectrum was tentatively assigned.

Med Dosw Mikrobiol, 2000, 52(1), 67 - 74
{Use of PCR methods for detection of selected genes of Mycoplasma pneumoniae}; Rastawicki W et al.; The aim of this study was standardization of PCR for the detection of gene encoding the P1 protein, 16S rRNA and elongation factor Tu of M . pneumoniae . A total of 13 strains of M . pneumoniae, 28 strains of other mycoplasmas and 14 strains of different bacteria causing respiratory tract infections were tested . In all of tested M . pneumoniae strains the presence of the sought genes was confirmed . The specificity of DNA was confirmed by the restriction endonuclease analysis with enzymes Hind III, Alu I and Hha I . With none of primers specific for the M . pneumoniae genes amplification of DNA from other bacteria was noted . The PCR method with the selected primers allowed to detect from 10(2) to 10(4) cfu M . pneumoniae/ml suspended in broth . The obtained results indicate that the PCR method can be used for detection of M . pneumoniae genes . A very good sensitivity and specificity predestine++ PCR as a potential quick diagnostic method for identification of M . pneumoniae in clinical specimens.

Biochim Biophys Acta, 2000 Nov 20, 1460(2-3), 353 - 74
Interactions between redox partners in various cytochrome P450 systems: functional and structural aspects; Lewis DF et al.; The various types of redox partner interactions employed in cytochrome P450 systems are described . The similarities and differences between the redox components in the major categories of P450 systems present in bacteria, mitochondria and microsomes are discussed in the light of the accumulated evidence from X-ray crystallographic and NMR spectroscopic determinations . Molecular modeling of the interactions between the redox components in various P450 mono-oxygenase systems is proposed on the basis of structural and mutagenesis information, together with experimental findings based on chemical modification of key residues likely to be associated with complementary binding sites on certain typical P450 isoforms and their respective redox partners.

Microbiol Mol Biol Rev, 2000 Dec, 64(4), 786 - 820
Origin and evolution of the mitochondrial proteome; Kurland CG et al.; The endosymbiotic theory for the origin of mitochondria requires substantial modification . The three identifiable ancestral sources to the proteome of mitochondria are proteins descended from the ancestral alpha-proteobacteria symbiont, proteins with no homology to bacterial orthologs, and diverse proteins with bacterial affinities not derived from alpha-proteobacteria . Random mutations in the form of deletions large and small seem to have eliminated nonessential genes from the endosymbiont-mitochondrial genome lineages . This process, together with the transfer of genes from the endosymbiont-mitochondrial genome to nuclei, has led to a marked reduction in the size of mitochondrial genomes . All proteins of bacterial descent that are encoded by nuclear genes were probably transferred by the same mechanism, involving the disintegration of mitochondria or bacteria by the intracellular membranous vacuoles of cells to release nucleic acid fragments that transform the nuclear genome . This ongoing process has intermittently introduced bacterial genes to nuclear genomes . The genomes of the last common ancestor of all organisms, in particular of mitochondria, encoded cytochrome oxidase homologues . There are no phylogenetic indications either in the mitochondrial proteome or in the nuclear genomes that the initial or subsequent function of the ancestor to the mitochondria was anaerobic . In contrast, there are indications that relatively advanced eukaryotes adapted to anaerobiosis by dismantling their mitochondria and refitting them as hydrogenosomes . Accordingly, a continuous history of aerobic respiration seems to have been the fate of most mitochondrial lineages . The initial phases of this history may have involved aerobic respiration by the symbiont functioning as a scavenger of toxic oxygen . The transition to mitochondria capable of active ATP export to the host cell seems to have required recruitment of eukaryotic ATP transport proteins from the nucleus . The identity of the ancestral host of the alpha-proteobacterial endosymbiont is unclear, but there is no indication that it was an autotroph . There are no indications of a specific alpha-proteobacterial origin to genes for glycolysis . In the absence of data to the contrary, it is assumed that the ancestral host cell was a heterotroph.

Biochem J, 2000 Dec 15, 352 Pt 3, 659 - 66
Characterization of the unique function of a reduced amide bond in a cytolytic peptide that acts on phospholipid membranes; Oh JE et al.; The incorporation of a reduced amide bond, psi(CH(2)NH), into peptide results in an increase in the net positive charge and the perturbation of alpha-helical structure . By using this characteristic of the reduced amide bond, we designed and synthesized novel pseudopeptides containing reduced amide bonds, which had a great selectivity between bacterial and mammalian cells . A structure-activity relationship study on pseudopeptides indicated that the decrease in alpha-helicity and the increase in net positive charge in the backbone, caused by the incorporation of a reduced amide bond into the peptide, both contributed to an improvement in the selectivity between lipid membranes with various surface charges . However, activity results in vitro indicated that a perturbation of alpha-helical structure rather than an increase in net positive charge in the backbone is more important in the selectivity between bacterial and mammalian cells . The present result revealed that the backbone of membrane-active peptides were important not only in maintaining the secondary structure for the interactions with lipid membranes but also in direct interactions with lipid membranes . The present study showed the unique function of a reduced amide bond in cytolytic peptides and a direction for developing novel anti-bacterial agents from cytolytic peptides that act on the lipid membrane of micro-organisms.

J Surg Res, 2000 Dec, 94(2), 178 - 84
Surgical stress induces phospholipid degradation in the intestinal brush border membrane; Prabhu R et al.; BACKGROUND: Surgical stress can lead to translocation of bacteria from the intestine into the systemic circulation . The intestinal brush border membrane (BBM) plays an important role in defense against such invasion by luminal bacteria and endotoxin . Our earlier work has shown the development of oxidative stress in the intestine after surgical stress and since the BBM is sensitive to free radical attack, this study examined the effect of surgical stress on the structure and function of intestinal BBM . METHODS: Intestinal BBM were isolated from control and after surgical stress and compared for structural and functional alterations . Surgical stress was also carried out following pretreatment with the xanthine oxidase inhibitor allopurinol or the nitric oxide donor l-arginine, to study the protection offered by these compounds . RESULTS: Surgical stress affected intestinal BBM structure as well as function . A decrease in alkaline phosphatase activity and alpha-tocopherol content, accompanied by an increase in lipid peroxidation, was seen . Surgical stress induced phospholipid degradation with generation of arachidonic acid . Functional impairment with a decrease in glucose transport ability was also seen . These changes are prevented by inhibition of xanthine oxidase by allopurinol pretreatment but not by NO . CONCLUSION: Surgical stress in the small intestine causes structural and functional alterations in the BBM through oxidative stress . This damage could affect gut barrier integrity and generation of arachidonic acid might mediate distal organ dysfunction .

Braz J Infect Dis, 1997 Dec, 1(6), 306 - 313
Molecular Epidemiology of Lyme Disease Spirochetes Based on a Probe Complementary to Ribosomal RNA; dos Santos MA et al.; Lyme disease is caused by the spirochete, Borrelia burgdorferi, a bacteria which infects many vertebrates including humans . Borrelia have been isolated from many parts of the world, and there is interest to identify common genetic markers to improve molecular methods of diagnosis, and to aid in understanding varied manifestations of the disease . A total of 48 Borrelia burgdorferi strains, including: 38 isolated from ticks (Ixodes dammini, I . persulcatus, I . ricinus and I . pacificus), 3 from animals (dog, bird and hamster), and 7 from human clinical cases (skin, CSF, plasma and blood) from different geographic areas, were studied by DNA/DNA hybridization and rRNA gene restriction patterns by using a biotinylated pKK3535 probe (Altewegg M., Mayer L.W., 1989) . The migration patterns of rRNA gene-restriction fragments after clevage by Hind III separate these strains into 5 ribotypes of Borrelia burgdorferi: Type I (38 American,2 European strains); Type II (13 American strains); Type III (3 Asian and 1 European strains); Type IV (1 European and 2 Asian strains) and Type V (1 Asian strain) . The use of ribotyping has provided an additional tool to investigate the differences or common patterns which cause various Lyme disease syndromes.

J Dairy Sci, 2000 Nov, 83(11), 2585 - 95
Nitrogen metabolism of early lactation cows fed diets with two different levels of protein and different amino acid profiles; Bach A et al.; Four multiparous Holstein cows (569+/-122 kg) surgically prepared with indwelling catheters in the mesenteric, portal, and hepatic veins and carotid artery were allocated in a 4 x 4 Latin square to determine the effects of dietary crude protein (CP) level and amino acid (AA) profile on N metabolism during early lactation (from 25 to 65 d in milk) . Cows received their diets in two equal meals and were milked twice daily . The dietary treatments were: 18% CP with a high (18H) or a low (18L) quality AA profile, and 15% CP with a high (15H) or a low (15L) quality AA profile . The four diets were similar in net energy for lactation (1.75 NEL Mcal/kg) and contained the same amount of RUP (34% of CP) . The quality of the AA profile pertained only to the essential AA (EAA), and was assessed by comparison with the EAA profile of casein and considered the potential contribution of EAA from ruminal bacteria . The 18H and 15H diets were supplemented with 50 and 25 g/d of ruminally protected Met, respectively . After 10 d on treatment, a blood flow marker (p-amino-hippurate) was infused into a mesenteric vein, and arterial, portal, hepatic, and mammary blood samples were obtained at 3, 6, and 12 h after feeding . Dry matter intake was similar across treatments (23.4+/-0.5 kg/d) . Amino acid oxidation, and consequent urea production, in the liver were numerically greater with the 18% CP rations, and, as a result, arterial urea concentrations were greatest (P < 0.01) with these rations . The amount of total AA extracted by the mammary gland tended to be greater with the H than with the L diets (21.4 vs . 18.2 mmol/ h, respectively) . Milk yield tended to be greater (P = 0.16) with the 18H and 15H diets (47.7 and 46.3 kg/d, respectively) compared with the 18L and 15L diets (45.9 and 44.6 kg/d, respectively) . Also, milk CP and casein contents were greatest (P = 0.09) with the H diets compared with the L diets . Milk and plasma urea N were greatest (P < 0.01) with the 18% CP diets . The efficiency of N utilization for milk protein synthesis was greatest (P < 0.09) with the 15% CP diets . It is concluded that milk protein production during early lactation is less susceptible to variations in dietary CP contents than variations in the AA profile of the dietary protein.

Acta Cardiol, 2000 Oct, 55(5), 295 - 300
Chlamydia and atherosclerotic coronary arterial disease in Turkey; Ozsan M et al.; OBJECTIVE: Chlamydia pneumoniae, which is a Gram(-) intracellular bacteria, besides being a respiratory pathogen, is thought to play an active role in the progress of acute myocardial infarction and chronic coronary artery disease . In this study we aim to determine the frequency of C . pneumoniae in coronary artery lesions of Turkish people . METHODS AND RESULTS: The atherosclerotic material taken from 8 cases by directional atherectomy and from 23 cases by surgical endarterectomy and examined by indirect immunofluorescence (IIFA) test and polymerase chain reaction (PCR) . C . pneumoniae positivity was 32.3% (10/31) by IIFA and 29.0% (9/31) by PCR while the evaluation of the methods together yield a positivity of 35.5% (11/31) . CONCLUSIONS: A statistically significant difference could not be established between C . pneumoniae positive and negative groups according to age and the classical atherosclerotic risk factors such as diabetes mellitus, smoking, hypercholesterolaemia, hypertension, family history; besides, a statistically significant difference could not be found between the presence of C . pneumoniae and the severity and clinical picture of coronary artery disease.

Curr Opin Biotechnol, 2000 Dec, 11(6), 540 - 6
Biodesulfurization and the upgrading of petroleum distillates; Monticello DJ; Biotechnology offers an alternative way to process fossil fuels . There have been several important advances in the elucidation of the mechanisms of biodesulfurization and the development of a biocatalytic desulfurization process . These include a detailed analysis of the rate and extent of desulfurization of real target molecules in a diesel matrix, the directed evolution of rate- and extent-limiting enzymes for better performance and the expression of the genes in alternative hosts . Process innovations include new reactor designs, separations and recovery strategies and the production of value-added byproducts during desulfurization.

Curr Opin Immunol, 2000 Dec, 12(6), 632 - 40
The influence of infections on the development and severity of allergic disorders; Herz U et al.; The frequency and severity of atopic disorders are steadily increasing, particularly in developing countries . The reason for this observation is not clear . Recent studies indicate that infections with viruses and especially with bacteria early in life may help to inhibit allergic Th2 responses by skewing the immune system towards Th1 responses . However, infections can also lead to the exacerbation of atopic disorders.

Nat Biotechnol, 2000 Dec, 18(12), 1311 - 4
Stable correction of a genetic deficiency in human cells by an episome carrying a 115 kb genomic transgene; Wade-Martins R et al.; Persistent expression of a transgene at therapeutic levels is required for successful gene therapy, but many small vectors with heterologous promoters are prone to vector loss and transcriptional silencing . The delivery of genomic DNA would enable genes to be transferred as complete loci, including regulatory sequences, introns, and native promoter elements . These elements may be critical to ensure prolonged, regulated, and tissue-specific transgene expression . Many studies point to considerable advantages to be gained by using complete genomic loci in gene expression . Large-insert vectors incorporating elements of the bacterial artificial chromosome (BAC) cloning system, and the episomal maintenance mechanisms of Epstein-Barr virus (EBV), can shuttle between bacteria and mammalian cells, allowing large genomic loci to be manipulated conveniently . We now demonstrate the potential utility of such vectors by stably correcting a human genetic deficiency in vitro . When the complete hypoxanthine phosphoribosyltransferase (HPRT) locus of 115 kilobases (kb) was introduced into deficient human cells, the transgene was both maintained as an episome and expressed stably for six months in rapidly dividing cell cultures . The results demonstrate for the first time that gene expression from an episomal genomic transgene can correct a cell culture disease phenotype for a prolonged period.

J Clin Microbiol, 2000 Dec, 38(12), 4665 - 7
Comparison of iodophor and alcohol pledgets with the Medi-Flex blood culture prep kit II for preventing contamination of blood cultures; Wilson ML et al.; Iodophor and alcohol pledgets were compared with the Medi-Flex Prep Kit II for skin disinfection before venipuncture . Of 12,367 blood cultures collected, 6,362 were done with conventional pledgets and 6, 005 were done with Medi-Flex kits . Contamination occurred in 351 of 6,362 blood cultures (5.5%; range, 3.7 to 8.1%) with conventional pledgets versus 328 of 6,005 (5.5%; range, 3.5 to 7.5%) with Medi-Flex kits.

J Biomol NMR, 2000 Oct, 18(2), 83 - 100
Lipari-Szabo mapping: A graphical approach to Lipari-Szabo analysis of NMR relaxation data using reduced spectral density mapping; Andrec M et al.; In this paper, we explore connections between the Lipari-Szabo formalism and reduced spectral density mapping, and show how spectral density estimates can be associated with Lipari-Szabo parameters via a simple geometric construction which we call Lipari-Szabo mapping . This relationship can be used to estimate Lipari-Szabo parameters from spectral density estimates without the need for nonlinear optimization, and to perform 'model selection' in a graphical manner . The Lipari-Szabo map also provides insight into the Lipari-Szabo model, and allows us to determine when a given set of experimental spectral densities are inconsistent with the Lipari-Szabo formalism . Practical applications of Lipari-Szabo mapping in conjunction with more traditional analysis methods are discussed.

J Gastroenterol Hepatol, 2000 Oct, 15 Suppl, G73 - 7
Reprocessing of flexible endoscopes; Leung JW; Proper reprocessing of endoscopes prevents the risk of transmission of infection between patients . Meticulous mechanical cleaning is the most important step as it removes the majority of the contaminating bacteria . It should be performed before manual or automatic disinfection . High-level disinfection involves total immersion of the endoscope in a liquid chemical germicide (LCG) at a preset temperature and concentration for a pre-determined period of time . Subsequent rinsing and drying are essential steps to remove the chemical solution and prevent bacterial colonization during storage . Endoscopy units that are used for more than 50 procedures per week may benefit from cleaning in an automatic endoscope reprocessor (AER) . This allows automated exposure of the endoscope to the LCG with subsequent flushing and drying of the channels, and minimizes staff exposure to the LCG . Reprocessing should be performed by trained and accredited personnel according to written guidelines or standards of practice as defined by professional societies . Regular monitoring of the reprocessing process is important for quality control and in ensuring patients' safety.

J Antibiot (Tokyo), 2000 Sep, 53(9), 879 - 85
Tubulysins, new cytostatic peptides from myxobacteria acting on microtubuli . Production, isolation, physico-chemical and biological properties; Sasse F et al.; New cytostatic compounds, tubulysins, were isolated from the culture broth of strains of the myxobacteria Archangium gephyra and Angiococcus disciformis . The compounds are peptides partly consisting of unusual amino acids and are distantly related to the dolastatins . The tubulysins were not active against bacteria and only little against fungi, but showed high cytostatic activity against mammalian cell lines with IC50 values in the picomolar range . An incubation with 50 ng/ml tubulysin A led to a complete disappearance of the microtubuli network of the cells within 24 hours . The more active tubulysin D induced multipolar spindles: At 0.5 ng/ml all mitotic cells showed more than four spindle poles.

Scand J Gastroenterol, 2000 Oct, 35(10), 1048 - 52
Fructose- and sorbitol-reduced diet improves mood and gastrointestinal disturbances in fructose malabsorbers; Ledochowski M et al.; BACKGROUND: Fructose malabsorption is characterized by the inability to absorb fructose efficiently . As a consequence fructose reaches the colon where it is broken down by bacteria to short fatty acids, CO2 and H2 . Bloating, cramps, osmotic diarrhea and other symptoms of irritable bowel syndrome are the consequences and can be seen in about 50% of fructose malabsorbers . We have previously shown that fructose malabsorption is associated with early signs of mental depression and low serum tryptophan concentrations . It was therefore of interest whether a fructose-reduced diet could not only improve gastrointestinal complaints but also depressive signs seen in fructose malabsorbers . METHODS: Fifty-three adults (12 males, 41 females), who were identified as fructose malabsorbers according to their breath-H2 concentrations, filled out a Beck's depression inventory-questionnaire, and a questionnaire with arbitrary scales for measurement of meteorism, stool frequency and quality of life for a 4-week period before dietary intervention and 4 weeks after dietary change as for fructose- and sorbitol-reduced diet . RESULTS: Depression scores were reduced by 65.2% after 4 weeks of diet (P < 0.0001), and there was a significant reduction of meteorism (P < 0.0001) and stool frequency (P < 0.01) . Improvement of signs of depression and of meteorism was more pronounced in females than in males . CONCLUSION: Fructose- and sorbitol-reduced diet in subjects with fructose malabsorption does not only reduce gastrointestinal symptoms but also improves mood and early signs of depression.

Nature, 2000 Nov 16, 408(6810), 325 - 30
Functional genomic analysis of C . elegans chromosome I by systematic RNA interference; Fraser AG et al.; Complete genomic sequence is known for two multicellular eukaryotes, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, and it will soon be known for humans . However, biological function has been assigned to only a small proportion of the predicted genes in any animal . Here we have used RNA-mediated interference (RNAi) to target nearly 90% of predicted genes on C . elegans chromosome I by feeding worms with bacteria that express double-stranded RNA . We have assigned function to 13.9% of the genes analysed, increasing the number of sequenced genes with known phenotypes on chromosome I from 70 to 378 . Although most genes with sterile or embryonic lethal RNAi phenotypes are involved in basal cell metabolism, many genes giving post-embryonic phenotypes have conserved sequences but unknown function . In addition, conserved genes are significantly more likely to have an RNAi phenotype than are genes with no conservation . We have constructed a reusable library of bacterial clones that will permit unlimited RNAi screens in the future; this should help develop a more complete view of the relationships between the genome, gene function and the environment.

J Microw Power Electromagn Energy, 2000, 35(3), 179 - 84
Complex high-frequency technology for protection of grain against pests; Mishenko AA et al.; The results of experimental investigation of physical methods are presented for suppressing of biological activity of grain and grain product pests: harmful insects at each developmental stage except eggs (Insecta), mites (Arachnida, Acariformes) and microscopic fungi and bacteria . The technologies under development for disinfestation and disinfection of grain are based on irradiation of grain by modulated pulses of high-frequency (HF) electromagnetic fields and on simultaneous action of a complex of factors: vacuum and HF-field induced plasma . The threshold value of the electric field intensity for total insect mortality was found to be E = 4.0-5.0 kV/cm in the pulse mode at the base frequency of 47.5 MHz . When the combined technology is used, conditions are created in the irradiation chamber for HF-discharge and plasma formation, which are very strong factors influencing the biological organisms . These raise the energy (and cost) efficiency (approximately $2-3 per tonne of grain) of the combined technology for destruction of grain pests with complete environmental safety.

DNA Cell Biol, 2000 Nov, 19(11), 689 - 96
Structure of the gene for uteroferrin; Vallet JL et al.; The published structure of the gene for uteroferrin differs from that of the human and mouse tartrate-resistant acid phosphatase (TRAP) genes . Polymerase chain reaction using genomic DNA as template and primers designed from exon 2 of the porcine uteroferrin gene amplified a product containing two previously undescribed introns . Because of these discrepancies, we cloned an EcoRI fragment from a porcine genomic BAC library containing the uteroferrin gene, and the region containing the uteroferrin gene was completely sequenced . The uteroferrin gene spanned 2.5 kb and contained five exons, which is similar to the structure previously reported for human and mouse TRAP genes but different from the published structure of the uteroferrin gene . Southern blotting of porcine genomic DNA digested with a variety of enzymes was consistent with the sequence that we obtained . The most likely explanation for the differing results is that the previously reported structure for the uteroferrin gene was the result of artifactual elimination of introns 2 and 3 by bacteria and artifactual recombination of the region upstream of the transcription start site of this gene.

Appl Environ Microbiol, 2000 Dec, 66(12), 5353 - 9
A homologue of the tryptophan-rich sensory protein TspO and FixL regulate a novel nutrient deprivation-induced Sinorhizobium meliloti locus; Davey ME et al.; A nutrient deprivation-induced locus in Sinorhizobium meliloti strain 1021 was identified by use of a Tn5-luxAB reporter gene transposon . The tagged locus is comprised of two open reading frames (ORFs) designated ndiA and ndiB for nutrient deprivation-induced genes A and B . Comparison of the deduced amino acid sequences of both ndiA and ndiB to the protein databases failed to reveal similarity to any known genes . The expression of the ndi locus was found to be induced by carbon and nitrogen deprivation, osmotic stress, and oxygen limitation and during entry into stationary phase . To identify regulatory components involved in the control of ndi gene expression, a second round of mutagenesis was performed on the primary ndiB::Tn5-luxAB-tagged strain (C22) with transposon Tn1721 . A double-mutant strain was obtained that lacked ndi locus transcriptional activity under all of the inducing conditions tested . The Tn1721-tagged gene showed a high degree of similarity to tryptophan-rich sensory protein TspO from Rhodobacter sphaeroides, as well as to mitochondrial benzodiazepine receptor pK18 from mammals . Induction of the ndi::Tn5-luxAB reporter gene fusion was restored under all inducing conditions by introducing the tspO coding region, from either S . meliloti or R . sphaeroides, in trans . Furthermore, it was found that, in addition to tspO, fixL, which encodes the sensor protein of an oxygen-sensing two-component system, is required for full expression of the ndi locus, but only under low oxygen tension.

Appl Environ Microbiol, 2000 Dec, 66(12), 5116 - 22
Community composition of marine bacterioplankton determined by 16S rRNA gene clone libraries and fluorescence in situ hybridization; Cottrell MT et al.; We determined the compositions of bacterioplankton communities in surface waters of coastal California using clone libraries of 16S rRNA genes and fluorescence in situ hybridization (FISH) in order to compare the community structures inferred from these two culture-independent approaches . The compositions of two clone libraries were quite similar to those of clone libraries of marine bacterioplankton examined by previous studies . Clones from gamma-proteobacteria comprised ca . 28% of the libraries, while approximately 55% of the clones came from alpha-proteobacteria, which dominated the clone libraries . The Cytophaga-Flavobacter group and three others each comprised 10% or fewer of the clone libraries . The community composition determined by FISH differed substantially from the composition implied by the clone libraries . The Cytophaga-Flavobacter group dominated 8 of the 11 communities assayed by FISH, including the two communities assayed using clone libraries . On average only 10% of DAPI (4', 6'-diamidino-2-phenylindole)-stained bacteria were detected by FISH with a probe for alpha-proteobacteria, but 30% of DAPI-stained bacteria appeared to be in the Cytophaga-Flavobacter group as determined by FISH . alpha-Proteobacteria were greatly overrepresented in clone libraries compared to their relative abundance determined by FISH, while the Cytophaga-Flavobacter group was underrepresented in clone libraries . Our data show that the Cytophaga-Flavobacter group can be a numerically dominant component of coastal marine bacterioplankton communities.

Nucleosides Nucleotides Nucleic Acids, 2000 Aug, 19(8), 1249 - 64
The interaction of drugs with DNA gyrase: a model for the molecular basis of quinolone action; Heddle JG et al.; DNA gyrase supercoils DNA in bacteria . The fact that it is essential in all bacteria and absent from eukaryotes makes it an ideal drug target . We discuss the action of coumarin and quinolone drugs on gyrase . In the case of coumarins, the drugs are known to be competitive inhibitors of the gyrase ATPase reaction . From a combination of structural and biochemical studies, the molecular details of the gyrase-coumarin complex are well established . In the case of quinolones, the drugs are thought to act by stabilising a cleavage complex between gyrase and DNA that arrests polymerases in vivo . The exact nature of the gyrase-quinolone-DNA complex is not known; we propose a model for this complex based on structural and biochemical data.

Clin Infect Dis, 2000 Dec, 31(6), 1343 - 8 Epub 2000 Nov 29.
Endotoxin, cytokines, and procalcitonin in febrile patients admitted to the hospital: identification of subjects at high risk of mortality; van Langevelde P et al.; We prospectively examined 464 febrile patients (median age, 61 years) for predictors of in-hospital death, by use of univariate and multivariate logistic regression using clinical data (age, underlying disease, duration of fever, chills, and shock on admission) and plasma endotoxin, TNF-alpha, IL-6, IL-10, and procalcitonin levels . The mortality rate was 4.6-fold higher (95% confidence interval {CI}, 1.8-12) in 31 patients with shock on admission, 7 of whom died; the strongest association with mortality was the endotoxin concentration (relative risk, 13.7; 95% CI, 1 . 4-136), which predicted 5 of the deaths with a 5% false-positive rate . For 433 patients without shock on admission, mortality (26 deaths) was associated with age and underlying disease: clinical data predicted 30% of the deaths, whereas IL-6 and procalcitonin levels identified an extra 10% with a 5% false-positive rate . When febrile patients are screened on hospital admission to identify those with a high risk for mortality, clinical judgment on the basis of age, underlying disease, and recent history outweighs the predictive value of endotoxin, cytokine, and procalcitonin levels . Only in patients who present with shock will measurement of endotoxin levels help predict those who will likely die at the cost of few false-positive results.

Am J Hum Genet, 2001 Jan, 68(1), 208 - 13 Epub 2000 Nov 28.
A mutation in the gene for the neurotransmitter receptor-clustering protein gephyrin causes a novel form of molybdenum cofactor deficiency; Reiss J et al.; Gephyrin was originally identified as a membrane-associated protein that is essential for the postsynaptic localization of receptors for the neurotransmitters glycine and GABA(A) . A sequence comparison revealed homologies between gephyrin and proteins necessary for the biosynthesis of the universal molybdenum cofactor (MoCo) . Because gephyrin expression can rescue a MoCo-deficient mutation in bacteria, plants, and a murine cell line, it became clear that gephyrin also plays a role in MoCo biosynthesis . Human MoCo deficiency is a fatal disease resulting in severe neurological damage and death in early childhood . Most patients harbor MOCS1 mutations, which prohibit formation of a precursor, or carry MOCS2 mutations, which abrogate precursor conversion to molybdopterin . The present report describes the identification of a gephyrin gene (GEPH) deletion in a patient with symptoms typical of MoCo deficiency . Biochemical studies of the patient's fibroblasts demonstrate that gephyrin catalyzes the insertion of molybdenum into molybdopterin and suggest that this novel form of MoCo deficiency might be curable by molybdate supplementation.

Proc Natl Acad Sci U S A, 2000 Dec 5, 97(25), 13561 - 6
Crystal structures of photosynthetic reaction center and high-potential iron-sulfur protein from Thermochromatium tepidum: thermostability and electron transfer; Nogi T et al.; The reaction center (RC) of photosynthetic bacteria is a membrane protein complex that promotes a light-induced charge separation during the primary process of photosynthesis . In the photosynthetic electron transfer chain, the soluble electron carrier proteins transport electrons to the RC and reduce the photo-oxidized special-pair of bacteriochlorophyll . The high-potential iron-sulfur protein (HiPIP) is known to serve as an electron donor to the RC in some species, where the c-type cytochrome subunit, the peripheral subunit of the RC, directly accepts electrons from the HiPIP . Here we report the crystal structures of the RC and the HiPIP from Thermochromatium (Tch.) tepidum, at 2.2-A and 1.5-A resolution, respectively . Tch . tepidum can grow at the highest temperature of all known purple bacteria, and the Tch . tepidum RC shows some degree of stability to high temperature . Comparison with the RCs of mesophiles, such as Blastochloris viridis, has shown that the Tch . tepidum RC possesses more Arg residues at the membrane surface, which might contribute to the stability of this membrane protein . The RC and the HiPIP both possess hydrophobic patches on their respective surfaces, and the HiPIP is expected to interact with the cytochrome subunit by hydrophobic interactions near the heme-1, the most distal heme to the special-pair.

Nucleic Acids Res, 2000 Dec 1, 28(23), 4623 - 33
Divergent mechanisms of 5' 23S rRNA IVS processing in the alpha-proteobacteria; Zahn K et al.; Widespread occurrence of a separate small RNA derived from the 5'-end of 23S rRNA and of an intervening sequence (IVS) which separates this domain from the main segment of 23S rRNA in the alpha-proteobacteria implies that processing reactions which act to excise the IVS are also maintained in this group . We previously characterized the first example of processing of this IVS in Rhodopseudomonas palustris, which is classified with the Bradyrhizobia In this case, IVS excision occurs by a multistep process and RNase III appears to act at an early step . Here, we characterize in vivo and in vitro IVS processing in two other related, but phenotypically distinct, Bradyrhizobia We also examine in vivo and in vitro processing of rRNA precursors from a more distantly related alpha-proteobacterium, Rhodobacter sphaeroides which produces a separate 5' 23S rRNA domain but has different sequences in the 5' 23S rRNA IVS . The details of the in vivo processing of all of the Bradyrhizobial rRNAs closely resemble the R . palustris example and in vitro studies suggest that all of the Bradyrhizobia utilize RNase III in the first step of IVS cleavage . Remarkably, in vivo and in vitro studies with R.sphaeroides indicate that initial IVS cleavage uses a different mechanism . While the mechanism of IVS cleavage differs among these alpha-proteobacteria, in all of these cases the limits of the internal segments processed in vivo are almost identical and occur far beyond the initial cleavage sites within the IVSs . We propose that these bacteria possess common secondary maturation pathways which enable them to generate similarly processed 23S rRNA 5'- and 3'-ends.

Cornea, 2000 Nov, 19(6), 864 - 9
Infiltrative keratitis associated with extended wear of hydrogel lenses and Abiotrophia defectiva; Keay L et al.; PURPOSE: Infiltrative keratitis is a common complication associated with extended wear of hydrogel lenses . Causative bacteria are often isolated from the lens at the time of an event . We report a case where three repeated occurrences of infiltrative keratitis were associated with contamination of the contact lenses by Abiotrophia defectiva . METHODS: A 34-year-old man participating in a clinical trial of extended wear hydrogel contact lenses experienced three episodes of infiltrative keratitis . The clinical presentation was observed using a biomicroscope . At the time of each event, the contact lenses were removed aseptically and ocular swabs were taken for bacterial identification and enumeration . The condition was monitored until full resolution . RESULTS: The condition was characterized by irritation, marked bulbar and limbal injection, and multiple focal subepithelial infiltrates . Many of the infiltrates also showed overlying staining with fluorescein . In each of the three events of infiltrative keratitis, A . defectiva was cultured from the contact lens and ocular swabs . CONCLUSION: This is the first reported occurrence of infiltrative keratitis associated with A . defectiva contamination of contact lenses.

FEBS Lett, 2000 Nov 24, 485(2-3), 113 - 6
Iron starvation leads to increased expression of Cu/Zn-superoxide dismutase in Aspergillus; Oberegger H et al.; In a search for iron-regulated proteins of Aspergillus nidulans and Aspergillus fumigatus a 16-kDa protein was identified which is about 5-fold upregulated during iron starvation in both species and which can be approximately 500-fold enriched by simple one-step chromatography on Amberlite XAD-16 resin . N-terminal protein sequence analysis and cloning of the respective A . nidulans cDNA identified this protein as a Cu/Zn-superoxide dismutase (SODA) . Northern analysis revealed that upregulation of sodA expression occurs at the level of transcript accumulation . This seems to be a specific low iron response and not a general starvation answer since sodA transcript levels do not respond to carbon or nitrogen starvation . In contrast, copper depletion leads to transcriptional downregulation of sodA . Furthermore, sodA expression was found still to be subject to iron regulation in an A . nidulans mutant lacking SREA, a regulator of iron homeostasis, indicating that sodA expression is regulated by an SREA-independent mechanism . The data presented suggest that SODA plays a protective role under iron deplete conditions.

J Biol Chem, 2001 Mar 16, 276(11), 7721 - 6 Epub 2000 Nov 28.
Interaction between the N-terminal domain of gastric H,K-ATPase and the spectrin binding domain of ankyrin III; Festy F et al.; We screened a cDNA bank of rabbit gastric fundic mucosa by two-hybrid assays looking for binding partners of the N-terminal domain of the rabbit gastric H,K-ATPase . We extracted five clones sharing more than 90% sequence identity . The longest clone codes for a protein sharing a high identity (96 and 96.8%, respectively) with a fragment of the membrane domain, from Arg-835 to Ser-873, plus the major part of the "spectrin binding domain" going from Glu-874 to Leu-1455 of human and mouse ankyrin III . We conclude that the membrane and spectrin binding domains of the rabbit ankyrin III are candidates for the binding partner of the N-terminal domain of the rabbit gastric H,K-ATPase . To validate the ankyrin-ATPase interaction and to test its specificity, we produced both domains in yeast and bacteria, coimmunoprecipitated them with an anti-ATPase antibody, and copurified them by affinity chromatography . The sequence of rabbit ankyrin III was not known, and this is the first report demonstrating that the ankyrin III and the H,K-ATPase interact with no intermediate . The interaction involves the N-terminal domain of the ATPase on one hand and the spectrin binding domain of the ankyrin on the other.

Carbohydr Res, 2000 Oct 6, 328(4), 605 - 10
Synthesis of furanose derivatives of 3-deoxy-D-erythro-2-hexulosonic acid and their 3-bromo and 3-deuterio analogs; Di Nardo C et al.; Methanolysis of 2,4,6-tri-O-benzoyl-2,3-dibromo-3-deoxy-D-altrono-1,5-lactone gave methyl 3-bromo-3-deoxy-2,4,6-tri-O-benzoyl-alpha-D-ribo-hex-2-ulofuranosonat e (3) and the anomeric mixture of the analogous 4,6-di-O-benzoyl derivative, having HO-2 free . Compound 3 was subjected to debromination with tributyltin hydride and tributyltin deuteride in the presence of 2,2'-azo-bisisobutyronitrile affording, respectively, the corresponding derivatives of 3-deoxy-D-erythro-2-hexulosonic acid and its 3-deuterio analog . The structure of the products and intermediates was established by spectroscopic methods and chemical transformations.

Expert Opin Investig Drugs, 2000 Dec, 9(12), 2783 - 97
Antineoplastic agents from natural sources: achievements and future directions; Cragg GM et al.; The influence of natural products upon anticancer drug discovery and design cannot be overestimated . Approximately 60% of all drugs now in clinical trials for the multiplicity of cancers are either natural products, compounds derived from natural products, contain pharmacophores derived from active natural products or are 'old drugs in new clothes', where (modified) natural products are attached to targeting systems . This review covers those materials that the authors are aware of as being in clinical trials through early 2000 and demonstrates how, even today, in the presence of massive numbers of agents from combinatorial libraries, the compounds produced by 'Mother Nature' are still in the forefront of cancer chemotherapeutics as sources of active chemotypes.

Jpn J Cancer Res, 2000 Nov, 91(11), 1161 - 8
Induction of apoptosis and cell cycle arrest in mouse colon 26 cells by benastatin A; Kakizaki I et al.; Benastatin A, isolated from Streptomyces bacteria, is reported to inhibit mammalian glutathione transferases (GSTs) . Since GST inhibitors such as ethacrynic acid are suggested to induce apoptosis in some cell lines, the effect of benastatin A on the survival of mouse colon 26 adenocarcinoma cells was compared with that of ethacrynic acid . When cells in stationary phase were treated with benastatin A, viable cells were found to be dose-dependently decreased after 3 days . In the case of ethacrynic acid, this became apparent within 24 h . Electrophoretic analysis revealed DNA fragmentation, indicating that cell loss was due to apoptosis in both cases . The dominant GST in colon 26 cells was identified as the class Pi-form (GST-II), and the activities in crude extracts as well as purified GST-II were almost completely inhibited by 50 microM ethacrynic acid . Immunoblot and northern blot analyses revealed increased GST-II protein and mRNA levels in cells treated with ethacrynic acid . Benastatin A did not significantly affect the activity in the crude extract even at 20 microM, a 10-fold higher concentration than that which almost completely inhibited the activity of purified GST-II . However, GST activity and GST-II protein were decreased in colon 26 cells treated with benastatin A for 5 days, no significant activity being detected in the range of 16 - 20 microM . In addition, beta-actin and bax mRNAs were also decreased in a dose-dependent manner . Furthermore, flow cytometric analysis of colon 26 cells revealed that benastatin A blocked the cell cycle at the G1/G0 phase . Thus, benastatin A also induces apoptosis of colon 26 cells, but this is unlikely to be due to inhibition of GST activity.

Acta Crystallogr D Biol Crystallogr, 2000 Dec, 56 Pt 12, 1634 - 7
Crystallization of the Mycobacterium tuberculosis cell-division protein FtsZ; Leung AK et al.; Mycobacterium tuberculosis FtsZ (MtbFtsZ), an essential protein in bacterial cell division, has been crystallized in the presence of a new inhibitor of MtbFtsZ polymerization and GTPase activity, ethyl (6-amino-2,3-dihydro-4-phenyl-1H-pyrido{4,3-b}{1, 4}diazepin-8-yl)carbamate (SRI-7614) . Crystals of the MtbFtsZ-SRI-7614 complex (form I, 30% polyethylene glycol 4000, 0.1 M sodium citrate pH 5.6, 0.2 M NH(4)OAc, 293 K) belong to space group P6(1) or P6(5), with unit-cell parameters a = 88.78, c = 178 . 02 A, and diffract to 2.3 A resolution . A second crystal form, of the GDP complex, grows in the presence or absence of Mg(2+) from PEG 4000 at 277 K or from (NH(4))(2)SO(4) at 293 K, respectively (form II, space group P6(2)22 or P6(4)22, with unit-cell parameters a = 135.02, c = 328.97 A or a = 129.30, c = 327.97 A, respectively) . Complete data sets to approximately 7 A resolution have been collected from both . Exceptional form II crystals diffract to at least 4.5 A resolution . Determination of the MtbFtsZ structure may advance the design of improved inhibitors of FtsZ polymerization.

Acta Crystallogr D Biol Crystallogr, 2000 Dec, 56 Pt 12, 1554 - 9
Ab initio phasing of a 4189-atom protein structure at 1.2 A resolution; Tame JR; The phase problem remains a key rate-limiting step in the determination of macromolecular X-ray structures . Direct methods, applying probability theory to the native data set, can routinely solve structures of up to about 200 non-H atoms, although much larger structures have been solved given sufficiently high resolution data and the presence of heavy atoms . Here it is shown that maximum-likelihood refinement of free-atom models with ARP/wARP can solve ab initio a much larger metalloprotein structure than the largest so far solved by conventional direct methods . The protein, OppA, is not naturally associated with metal ions but was co-crystallized with uranium.

Annu Rev Genet, 2000, 34, 359 - 399
DNA mismatch repair and genetic instability; Harfe BD et al.; Mismatch repair (MMR) systems play a central role in promoting genetic stability by repairing DNA replication errors, inhibiting recombination between non-identical DNA sequences and participating in responses to DNA damage . The discovery of a link between human cancer and MMR defects has led to an explosion of research on eukaryotic MMR . The key proteins in MMR are highly conserved from bacteria to mammals, and this conservation has been critical for defining the components of eukaryotic MMR systems . In eukaryotes, there are multiple homologs of the key bacterial MutS and MutL MMR proteins, and these homologs form heterodimers that have discrete roles in MMR-related processes . This review describes the genetic and biochemical approaches used to study MMR, and summarizes the diverse roles that MMR proteins play in maintaining genetic stability.

Angew Chem Int Ed Engl, 2000 Nov 17, 39(22), 4004 - 4032
Piezoelectric Mass-Sensing Devices as Biosensors-An Alternative to Optical Biosensors?
Janshoff A, Galla HJ, Steinem C.
In the early days of electronic communication-as a result of the limited number of quartz resonators available-frequency adjustment was accomplished by a pencil mark depositing a foreign mass layer on the crystal . In 1959, Sauerbrey showed that the shift in resonance frequency of thickness-shear-mode resonators is proportional to the deposited mass . This was the starting point for the development of a new generation of piezoelectric mass-sensitive devices . However, it was the development of new powerful oscillator circuits that were capable of operating thickness shear mode resonators in fluids that enabled this technique to be introduced into bioanalytic applications . In the last decade adsorption of biomolecules on functionalized surfaces turned in to one of the paramount applications of piezoelectric transducers . These applications include the study of the interaction of DNA and RNA with complementary strands, specific recognition of protein ligands by immobilized receptors, the detection of virus capsids, bacteria, mammalian cells, and last but not least the development of complete immunosensors . Piezoelectric transducers allow a label-free detection of molecules; they are more than mere mass sensors since the sensor response is also influenced by interfacial phenomena, viscoelastic properties of the adhered biomaterial, surface charges of adsorbed molecules, and surface roughness . These new insights have recently been used to investigate the adhesion of cells, liposomes, and proteins onto surfaces, thus allowing the determination of the morphological changes of cells as a response to pharmacological substances and changes in the water content of biopolymers without employing labor-intense techniques . However, the future will show whether the quartz-crystal microbalance will assert itself against established label-free sensor devices such as surface plasmon resonance spectroscopy and interferometry.

Autoimmunity, 2000 Oct, 32(3), 153 - 60
Do immune complexes formed with autoantibodies have a role in the maintenance of immune homeostasis through interaction with FC receptors; Guarnotta G et al.; Natural autoantibodies play an important regulatory role in the maintenance of immune homeostasis . They act as a first line of defense against environmental pathogens like toxins, bacteria and erythrocytes . In humans they are mainly produced by CD5+ B cells that are under the control of a regulatory T cell population . Fc-gamma receptors are involved in antigen recognition and signal transduction and tuning, and some of the members of the FcR family have structural similarity to MHC molecules; they may interact with multiple Ig ligands and with non-Ig ligands . We discuss the interactions between immune-complexes formed with natural autoantibodies and Fc-gamma receptors and suggest that such interactions may affect self-recognition in the thymus and regulate immune homeostasis.

Toxicology, 2000 Nov 2, 152(1-3), 47 - 52
(1-->3)-beta-D-glucan - relationship to indoor air-related symptoms, allergy and asthma; Rylander R et al.; (1-->3)-beta-D-glucan is a polyglucose structure in the cell wall of moulds, some bacteria and plants . Due to its unique (1-->3)-beta linkage it binds to specific receptors on phagocytosing cells and induces changes in their metabolism . Under realistic environmental concentrations, available data suggest that these changes express themselves as alterations of the defense mechanisms to other agents . Inhalation of (1-->3)-beta-D-glucan in humans causes symptoms from the upper respiratory tract and induction of cytokines in blood monocytes . (1-->3)-beta-D-glucan can be used as a marker of mould biomass in field studies . Relationships between the amount of (1-->3)-beta-D-glucan and the extent of symptoms as well as lung function changes and inflammatory markers have been described . In view of the mechanisms involved in the normal development of the immune system, children seem to be a particular group at risk due to (1-->3)-beta-D-glucan exposure.

Vaccine, 2000 Nov 8, 19(6), 637 - 43
Investigation of cellular and humoral immune responses to whole cell and acellular pertussis vaccines; Canthaboo C et al.; New generation acellular pertussis vaccines were compared with the established whole cell pertussis vaccine for the induction of humoral and cellular immune-responses in mice . At the same time, the in vivo protective effect of these two types of vaccine was also compared in both intracerebral (ic) and aerosol challenge models . In general, whole cell vaccine induced lower antibody titres to pertussis toxin, filamentous haemagglutinin and pertactin than the acellular vaccine . Nitric oxide concentration in macrophage cultures was used as a marker for macrophage activation . The nitric oxide concentrations in the macrophage cultures from mice following immunisation with the whole cell vaccine were higher than those from mice immunised with the acellular vaccine, which indicated that the whole cell vaccine was more effective than the acellular vaccine in activating macrophages . This was associated with better protection in vivo after challenge . After ic challenge of mice following immunisation with whole cell or acellular vaccine, 90% of the whole cell vaccine group survived compared with 40% of the acellular vaccine group at the vaccine dose selected . Following aerosol challenge, mice in the whole cell vaccine group showed faster clearance of bacteria from the lungs than those in the acellular vaccine group . Our findings suggest that the different types of pertussis vaccines may achieve protection in different ways and that CMI may play an important role in eliminating bacteria which escape humoral defence mechanisms.

Aquat Toxicol, 2001 Feb, 51(4), 405 - 17
Effects of Al(3+) ions and Cu(2+) ions on microcosms with three different biological complexities; Sugiura K; Cu(2+) ions or Al(3+) ions were added to microcosms containing three, four and eight species (S-3, S-4, S-8) at the beginning of the culture, or at the 15th day from the beginning of the culture to determine the production rates (P), the respiration rates (R), and P/R ratios . There was a balance between the oxygen production rates and the oxygen consumption rates in the microcosms (S-4) and (S-8), whereas there was no balance between two quantities in the microcosm (S-3) . There were no significant differences in the Cu(2+) and Al(3+) ion concentrations influencing the material cycles and the metabolic balance between the case when the number of species was four and the case when the number of species was eight . It was assumed that the effects concentrations of the chemical substances are not significantly related to the degree of diversity of the constituent species in the microcosms having a balance between production and consumption.

J Virol, 2000 Dec, 74(24), 11548 - 56
Integrase-lexA fusion proteins incorporated into human immunodeficiency virus type 1 that contains a catalytically inactive integrase gene are functional to mediate integration; Holmes-Son ML et al.; Purified fusion proteins made up of a retroviral integrase and a sequence-specific DNA-binding protein have been tested in in vitro assays for their ability to direct integration into specific target sites . To determine whether these fusion proteins can be incorporated into human immunodeficiency virus type 1 (HIV-1) and are functional to mediate integration, we used an in trans approach to deliver various integrase-LexA proteins to an integrase-defective virus containing an integrase mutation at aspartate residue 64 . Integrase-LexA, integrase-LexA DNA-binding domain, or N- or C-terminally truncated integrase-LexA proteins were fused to the HIV-1 accessory protein, Vpr . Coexpression of the Vpr fusion proteins and an integrase-defective HIV-1 molecular clone by a producer cell line resulted in efficient incorporation of the fusion protein into the integrase-mutated virus . In addition, each of these viruses was infectious and capable of performing integration, as determined by two independent cellular assays that measure reporter gene expression . With the exception of the N-terminally truncated integrase fused to LexA, which was at about 1%, all of the fusion proteins restored integration to a similar level, at 17 to 24% of that of the wild-type virus . The low level observed with the N-terminally truncated integrase fused to LexA is consistent with previous results implying that the N terminus of integrase is involved in multiple steps of the retroviral life cycle . These data indicate that the integrase-fusion proteins retain catalytic function in the integrase-mutated viruses and demonstrate the feasibility of incorporating integrase fusion proteins into HIV-1 for the development of site-directed retroviral vectors.

Eur J Cell Biol, 2000 Oct, 79(10), 735 - 49
Disruption of the actin filament network affects delivery of endocytic contents marker to phagosomes with early endosome characteristics: the case of phagosomes with pathogenic mycobacteria; Guerin I et al.; Phagosomes containing live virulent mycobacteria undergo fusion with early endosomes, but they are unable to mature normally . Accordingly, they do not fuse with lysosomes . Although M . avium-containing phagosomes retain fusion and intermingling characteristics of early endosomes indefinitely, fusions with early endosomes are increasingly restricted as bacteria multiply . In addition, when endocytic tracers, such as horseradish peroxidase (HRP), are added to M . avium-infected macrophages at 1 or up to 15 days after infection, an atypical time course of acquisition of the tracer by the phagosomes is observed, i.e., a 10 to 20 min lag, instead of immediate acquisition as is typical for early endosomes (and phagosomes with early endosome characteristics) . These events coincide with a marked disorganization of the actin filament network in M . avium-infected macrophages . In the present study, we have therefore addressed the following question: Do actin filaments play a role in fusion and intermingling of contents between early endosomes and immature phagosomes that undergo homotypic fusion with early endosomes? We examined the time course of acquisition of subsequently internalized endocytic marker (HRP) by early endosome-like preexisting phagosomes, i.e . 2 hour-old phagosomes with either hydrophobic latex particles, virulent or avirulent M . avium, after depolymerization of the actin filament network with cytochalasin D or after repolymerization of the actin filament network with jasplakinolide, in cases where the network had been depolymerized (macrophages infected with M . avium, at 1 or up to 7 days after infection) . By direct morphological observation at the electron microscope level and by a kinetic approach, we show here that depolymerization of the actin filament network with cytochalasin D delays acquisition of HRP whereas repolymerization restores immediate acquisition of the marker . We conclude that the actin filament network is involved in fusion and intermingling of endocytic contents between early endosomes and early endosome-like phagosomes, and that disruption of this network by M . avium is the cause for the atypical acquisition of content marker by phagosomes containing these pathogenic mycobacteria.

Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics, 2000 Oct, 62(4 Pt B), 5353 - 9
Model for water transport into powdered xanthan combining gel swelling and vapor diffusion; Goerke U et al.; Water ingress into xanthan powder compressed to various packing densities has been studied using nuclear magnetic resonance stray field imaging (STRAFI) . A foot is observed ahead of the main water ingress front which is attributed to vapor transport around the particles . The main development of the reported work is an analytical model which describes the coupling of vapor transport through the pore space and liquid transport through the progressively swelling gel which gradually occludes the vapor path . Good agreement between theory and experiment is found over a wide range of packing densities with the model requiring only one adjustable parameter, the water diffusivity in the gel measured in a constant polymer mass reference frame . It is suggested that the results are of considerable relevance to situations where the polymer is produced at low concentration by bacteria such as in the rhizosphere and aerial bio films.

Protein Expr Purif, 2000 Dec, 20(3), 421 - 34
Synthesis of a highly substituted N(6)-linked immobilized NAD(+) derivative using a rapid solid-phase modular approach: suitability for use with the kinetic locking-on tactic for bioaffinity purification of NAD(+)-dependent dehydrogenases; Tynan J et al.; This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD(+)-dependent dehydrogenases . Specifically, the synthesis of highly substituted N(6)-linked immobilized NAD(+) derivatives is described using a rapid solid-phase modular approach . Other modifications of the N(6)-linked immobilized NAD(+) derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 A) in an attempt to minimize nonbiospecific interactions . Analysis of the N(6)-linked NAD(+) derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S(6)-linked immobilized NAD(+) derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis . Model locking-on bioaffinity chromatographic studies were carried out with bovine heart l-lactate dehydrogenase (l-LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp . l-phenylalanine dehydrogenase (l-PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and d-phenylalanine, respectively, as locking-on ligands . Surprisingly, two of these test NAD(+)-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S(6)-linked immobilized cofactor derivatives than for the new N(6)-linked derivatives . In contrast, the NAD(+)-dependent phenylalanine dehydrogenase showed no affinity for the S(6)-linked immobilized NAD(+) derivative, but was locked-on strongly to the N(6)-linked immobilized derivative . That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N(6)-linked immobilized NADP(+) derivative in the presence of d-phenylalanine . This differential locking-on of NAD(+)-dependent dehydrogenases to N(6)-linked and S(6)-linked immobilized NAD(+) derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD(+) immobilized via N(6)-linkage as compared to thiol-linkage .

J Nat Prod, 2000 Nov, 63(11), 1570 - 2
New butenolides from two marine streptomycetes; Mukku VJ et al.; Chemical examination of two marine Streptomycetes has resulted in the isolation of four new butenolides, namely 4, 10-dihydroxy-10-methyl-dodec-2-en-1,4-olide (1), two diastereomeric 4,11-dihydroxy-10-methyl-dodec-2-en-1,4-olides (2/3), and 4-hydroxy-10-methyl-11-oxo-dodec-2-en-1,4-olide (4) . The structures were identified by interpretation of the 2D NMR and mass spectral data.

Ann Med, 2000 Oct, 32(7), 475 - 84
Efforts in minimizing risk of viral transmission through viral inactivation; Horowitz B et al.; The viral safety of blood and blood products has improved substantially over the last decade on account of the development of new viral screening and virucidal procedures . For nearly 15 years, virally inactivated blood derivatives, prepared by using advanced virucidal procedures, have amassed an extraordinary safety record with respect to hepatitis B and C and HIV . This record of safety has spawned the development of newer virucidal procedures designed to eliminate nonenveloped viruses from blood derivatives and viruses and other pathogens from blood components, including cellular components . Ongoing tests that include clinical studies will demonstrate how close we are to achieving a blood supply that is free of viruses, bacteria, and parasites.

Vet Q, 2000 Oct, 22(4), 204 - 8
Defence mechanisms against viral infection in poultry: a review; Jeurissen SH et al.; Defence against viral infections in poultry consists of innate and adaptive mechanisms . The innate defence is mainly formed by natural killer cells, granulocytes, and macrophages and their secreted products, such as nitric oxide and various cytokines . The innate defence is of crucial importance early in viral infections . Natural killer cell activity can be routinely determined in chickens of 4 weeks and older using the RP9 tumour cell line . In vitro assays to determine the phagocytosis and killing activity of granulocytes and macrophages towards bacteria have been developed for chickens, but they have not been used with respect to virally infected animals . Cytokines, such as interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)-alpha, are indicators of macrophage activity during viral infections, and assays to measure IL-1 and IL-6 have been applied to chicken-derived materials . The adaptive defence can be divided into humoral and cellular immunity and both take time to develop and thus are more important later on during viral infections . Various enzyme-linked immunosorbent assays (ELISAs) to measure humoral immunity specific for the viruses that most commonly infect poultry in the field are now commercially available . These ELISAs are based on a coating of a certain virus on the plate . After incubation with chicken sera, the bound virus-specific antibodies are recognized by conjugates specific for chicken IgM and IgG . Cytotoxic T lymphocyte activity can be measured using a recently developed in vitro assay based on reticuloendotheliosis virus-transformed target cells that are loaded with viral antigens, e.g . Newcastle disease virus . This assay is still in an experimental stage, but will offer great opportunities in the near future for research into the cellular defence mechanisms during viral infections.

J Immunol, 2000 Dec 1, 165(11), 6148 - 55
Vaccination with empty plasmid DNA or CpG oligonucleotide inhibits diabetes in nonobese diabetic mice: modulation of spontaneous 60-kDa heat shock protein autoimmunity; Quintana FJ et al.; Nonobese diabetic (NOD) mice develop insulitis and diabetes through a process involving autoimmunity to the 60-kDa heat shock protein (HSP60) . Treatment of NOD mice with HSP60 or with peptides derived from HSP60 inhibits this diabetogenic process . We now report that NOD diabetes can be inhibited by vaccination with a DNA construct encoding human HSP60, with the pcDNA3 empty vector, or with an oligonucleotide containing the CpG motif . Prevention of diabetes was associated with a decrease in the degree of insulitis and with down-regulation of spontaneous proliferative T cell responses to HSP60 and its peptide p277 . Moreover, both the pcDNA3 vector and the CpG oligonucleotide induced specific Abs, primarily of the IgG2b isotype, to HSP60 and p277, and not to other islet Ags (glutamic acid decarboxylase or insulin) or to an unrelated recombinant Ag expressed in bacteria (GST) . The IgG2b isotype of the specific Abs together with the decrease in T cell proliferative responses indicate a shift of the autoimmune process to a Th2 type in treated mice . These results suggest that immunostimulation by bacterial DNA motifs can modulate spontaneous HSP60 autoimmunity and inhibit NOD diabetes.

Biochem J, 2000 Dec 1, 352 Pt 2, 343 - 51
Cloning and expression of a cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase C; Dewaste V et al.; Inositol 1,4,5-trisphosphate {Ins(1,4,5)P(3)} 3-kinase catalyses the phosphorylation of Ins(1,4,5)P(3) to Ins(1,3,4,5)P(4) . cDNAs encoding two isoenzymes of Ins(1,4,5)P(3) 3-kinase (3-kinases A and B) have been described previously . In the present study, we report the cloning of a full-length 2052 bp cDNA encoding a third human isoenzyme of the Ins(1,4,5)P(3) 3-kinase family, referred to as isoform C . This novel enzyme has a calculated molecular mass of 75 . 207 kDa and a K(m) for Ins(1,4,5)P(3) of 6 microM . Northern-blot analysis showed the presence of a transcript of approx . 3.9 kb in various human tissues . Inositol trisphosphate 3-kinase C demonstrates enzymic activity when expressed in DH5alphaF' bacteria or COS-7 cells . Calcium alone decreases the Ins(1,4,5)P(3) 3-kinase activity of the 3-kinase C isoenzyme in transfected COS-7 cells . This inhibitory effect is reversed in the presence of calmodulin . The recombinant bacterial 3-kinase C can be adsorbed on calmodulin-Sepharose in the presence of calcium . The present data show that Ins(1,4,5)P(3) 3-kinase C: (i) shares a conserved catalytic domain of about 275 amino acids with the two other mammalian isoforms, (ii) could be purified on a calmodulin-Sepharose column and (iii) could be distinguished from the A and B isoenzymes by the effects of calcium and of calmodulin.

J Submicrosc Cytol Pathol, 2000 Apr, 32(2), 159 - 67
Structure of lamina propria lymphoid follicles and associated epithelium in the gastric mucosa during Helicobacter pylori infection in ulcer-bearing Mongolian gerbils; Wada R et al.; To develop a gerbil model of Helicobacter pylori-induced chronic active gastritis comparable in severity to human lesions, we made acetic acid-induced ulcer in the anterior antral wall and concurrently challenged 1 x 10(8) colony-forming units bacteria per os . At 30 and 60 days after inoculation, the number of viable bacteria colonizing on the surface epithelium of the gastric mucosa was larger in ulcer-bearing animals compared to non-bearing ones . Furthermore, in the former animals, neutrophil and mononuclear cell infiltration as well as lymphoid follicle formation in the lamina propria was more prominent . Electron microscopically, lymphoid follicle-associated epithelium displayed specialized structures . Namely, brush cells interposed between mucous epithelial cells and characterized by prominent microfilament bundles and many apical vesicles or caveola specifically embraced the cluster of intraepithelially invading lymphocytes and macrophage-like cells by the attenuated cytoplasm in an analogous manner to M cells in Peyer's patches . The present study has demonstrated that ulcer formation enhances both H . pylori colonization and lamina propria lymphoid follicle formation and suggested that follicle-associated epithelium might play roles in the delivery of intraluminal antigen.

Vet Clin North Am Food Anim Pract, 2000 Nov, 16(3), 497 - 509, vii
Ionophore use and toxicosis in cattle; Hall JO; Ionophores comprise a rapidly expanding class of antibiotics produced by filamentous branching bacteria of the order Actinomycetales . The use of ionophores as coccidiostats and growth promotants has resulted in the occurrence of toxicoses in target and nontarget species . Clinical and pathologic effects of ionophore poisoning are caused by bioactivity and damage to excitable tissues such as cardiac muscle, skeletal muscle, smooth muscle, and the nervous system . Ionophore toxicoses are often related to errors in feed mixing, so the practitioner should give primary importance to the removal of suspect feeds and testing to confirm excessive exposure.

Vet Clin North Am Food Anim Pract, 2000 Nov, 16(3), 455 - 64
Current knowledge of water quality and safety for livestock; Carson TL; Basic laboratory evaluation of water quality for livestock should include measurement of TDS, sulfate, nitrate-nitrite, and coliform bacteria . Supplementary water tests may include pH, sodium, iron, magnesium, chloride, calcium, potassium, manganese, and contaminants specific to the situation . Using the best-quality drinking water available contributes to the optimal production of livestock . Restricted quantity of drinking water or drinking water containing excessive levels of nitrate, TDS, sulfate, and other constituents can affect growth and production of all classes of animals . Drinking-water quality and availability should be evaluated as a cause of poor performance or nonspecific disease conditions in livestock . It is important that attempts to evaluate water quality include obtaining a thorough history, making astute observations, and asking intelligent questions . A thorough laboratory examination of animal specimens and water samples should be evaluated in view of existing standards for livestock drinking-water quality.

Curr Biol, 2000 Nov 2, 10(21), R804 - 8
ATP synthase: what dictates the size of a ring?
Ferguson SJ.
Recent data suggest the source of F(0)F(1) ATP synthase determines a significant and surprising difference in the size of a putative rotating ring of integral membrane subunits of F(0); this can be correlated with biochemical data suggesting there is variation in the number of protons translocated per ATP synthesised.

Infect Immun, 2000 Dec, 68(12), 7152 - 5
Neutralizing antibodies to adenylate cyclase toxin promote phagocytosis of Bordetella pertussis by human neutrophils; Weingart CL et al.; A previous study showed that opsonization with human immune serum could either promote or antagonize phagocytosis of Bordetella pertussis by human neutrophils depending on whether the bacteria expressed adenylate cyclase toxin . Opsonization of the wild-type strain inhibited phagocytosis relative to unopsonized controls . In contrast, mutants lacking adenylate cyclase toxin were efficiently phagocytosed when opsonized with human immune serum . In this study, we examined opsonization in the presence or absence of monoclonal antibodies to adenylate cyclase toxin . Addition of neutralizing monoclonal antibodies to adenylate cyclase toxin converted a serum that previously inhibited both attachment and phagocytosis of the wild-type strain to one that increased both attachment and phagocytosis compared to the no-serum control . Monoclonal antibodies that recognize the adenylate cyclase toxin but fail to neutralize activity were without effect . These results suggest that adenylate cyclase toxin inhibits both Fc receptor-mediated attachment and phagocytosis of B . pertussis by neutrophils.

Infect Immun, 2000 Dec, 68(12), 6997 - 7002
Identification of mycobacterial surface proteins released into subcellular compartments of infected macrophages; Beatty WL et al.; Considerable effort has focused on the identification of proteins secreted from Mycobacterium spp . that contribute to the development of protective immunity . Little is known, however, about the release of mycobacterial proteins from the bacterial phagosome and the potential role of these molecules in chronically infected macrophages . In the present study, the release of mycobacterial surface proteins from the bacterial phagosome into subcellular compartments of infected macrophages was analyzed . Mycobacterium bovis BCG was surface labeled with fluorescein-tagged succinimidyl ester, an amine-reactive probe . The fluorescein tag was then used as a marker for the release of bacterial proteins in infected macrophages . Fractionation studies revealed bacterial proteins within subcellular compartments distinct from mycobacteria and mycobacterial phagosomes . To identify these proteins, subcellular fractions free of bacteria were probed with mycobacterium-specific antibodies . The fibronectin attachment protein and proteins of the antigen 85-kDa complex were identified among the mycobacterial proteins released from the bacterial phagosome.

Infect Immun, 2000 Dec, 68(12), 6826 - 32
Naive human T cells develop into Th1 effectors after stimulation with Mycobacterium tuberculosis-infected macrophages or recombinant Ag85 proteins; Russo DM et al.; Most studies of human T-cell responses in tuberculosis have focused on persons with either active disease or latent infection . Although this work has been critical in defining T-cell correlates of successful versus failed host containment, little is known about the development of Mycobacterium-specific T-cell responses in uninfected persons . To explore this issue, naive T cells from uninfected donors were sensitized in vitro with avirulent Mycobacterium tuberculosis-infected autologous macrophages . T-cell lines primed in this manner proliferated and produced cytokines after challenge with mycobacterial antigens . Of 11 such lines, 8 were high Th1 responders, 2 were low Th1 responders, and 1 was a Th2 responder . Furthermore, similar patterns and magnitudes of proliferative and cytokine responses were seen when Mycobacterium infection-primed lines were challenged with recombinant antigen 85 (Ag85) proteins . The addition of interleukin 12 (IL-12) during the initial sensitization increased the magnitude of Th1 responses; however, antibody to IL-12 did not eliminate Th1 responses, suggesting that additional factors contributed to the differentiation of these cells . Finally, in the presence of IL-12, recombinant Ag85B was able to prime naive T cells for Th1 responses upon challenge with Mycobacterium-infected macrophages or Ag85B . Therefore, under the appropriate conditions, priming with whole bacteria or a subunit antigen can stimulate Mycobacterium-specific Th1 effector cell development . Further definition of the antigens and conditions required to drive naive human T cells to differentiate into Th1 effectors should facilitate the development of an improved tuberculosis vaccine.

Infect Immun, 2000 Dec, 68(12), 6737 - 43
Gamma interferon influences intestinal epithelial hyperplasia caused by Lawsonia intracellularis infection in mice; Smith DG et al.; Lawsonia intracellularis is a recently identified bacterial pathogen which causes disease in a broad range of animals . Invasion of intestinal epithelial cells and the resultant hyperplasia of infected cells are central processes in disease pathogenesis . In this study, we aimed to establish whether immunocompetent mice were susceptible to infection and whether gamma interferon (IFN-gamma) contributed to the pathogenesis of infection . Wild-type 129-Sv-Ev mice (129 mice) and IFN-gamma receptor knockout mice based on the 129 background (IFN-gammaR(-)) were challenged orally with approximately 5.5 x 10(7) L . intracellularis cells . Both 129 and IFN-gammaR(-) mice became infected, although the extent of infection (as determined by the proportion of infected crypts) was substantially lower in 129 mice than in IFN-gammaR(-) mice . Despite these differences, infected crypts showed characteristics typical of proliferative enteropathies of other animals, i.e., intracellular colonization of epithelial cells by L . intracellularis with resultant epithelial hyperplasia . Infection in 129 mice was cleared between days 21 and 28 postchallenge, whereas infection in IFN-gammaR(-) mice was evident in 100% of animals from day 21 onward . Additionally, in IFN-gammaR(-) mice the infection was so extensive that fatalities resulted . IFN-gamma therefore plays a significant role in limiting intracellular infection and increased cellular proliferation associated with L . intracellularis . L . intracellularis infection is generally associated with modest cellular infiltration; therefore, further comparative examinations will be necessary to determine pathogenicity factors and define the role of IFN-gamma in controlling this infection.

Infect Immun, 2000 Dec, 68(12), 6670 - 6
Analysis of urease expression in Actinomyces naeslundii WVU45; Morou-Bermudez E et al.; The hydrolysis of urea by ureases of oral bacteria in dental plaque can cause a considerable increase in plaque pH, which can inhibit the development of dental caries . There is also indirect evidence that urea metabolism may promote the formation of calculus and that ammonia release from urea could exacerbate periodontal diseases . Actinomyces naeslundii, an early colonizer of the oral cavity and a numerically significant plaque constituent, demonstrates comparatively low levels of urease activity on isolation, so this organism has not been considered a major contributor to total oral urease activity . In this study it was observed that urease activity and urease-specific mRNA levels in A . naeslundii WVU45 can increase up to 50-fold during growth under nitrogen-limiting conditions . Using primer extension analysis, a putative, proximal, nitrogen-regulated promoter of the A . naeslundii urease gene cluster was identified . The functionality and nitrogen responsiveness of this promoter were confirmed using reporter gene fusions and 5' deletion analysis . The data indicated that regulation of urease expression by nitrogen availability in A . naeslundii may require a positive transcriptional activator . Plaque bacteria may experience nitrogen limitation when carbohydrates are present in excess . Therefore, based on the results of this study and in contrast to previous beliefs, strains of A . naeslundii may have the potential to be significant contributors to total plaque ureolysis, particularly during periods when there is an increased risk for caries development.

Infect Immun, 2000 Dec, 68(12), 6567 - 73
Immunomodulatory role of endogenous interleukin-18 in gamma interferon-mediated resolution of replicative Legionella pneumophila lung infection; Brieland JK et al.; The in vivo role of endogenous interleukin-18 (IL-18) in modulating gamma interferon (IFN-gamma)-mediated resolution of replicative Legionella pneumophila lung infection was assessed using a murine model of Legionnaires' disease . Intratracheal inoculation of A/J mice with virulent bacteria (10(6) L . pneumophila organisms per mouse) resulted in induction of IL-18 protein in bronchoalveolar lavage fluid (BALF) and intrapulmonary expression of IL-18 mRNA . Real-time quantitative RT-PCR analysis of infected lung tissue demonstrated that induction of IL-18 in BALF preceded induction of IL-12 and IFN-gamma mRNAs in the lung . Blocking intrapulmonary IL-18 activity by administration of a monoclonal antibody (MAb) to the IL-18 receptor (anti-IL-18R MAb) prior to L . pneumophila infection inhibited induction of intrapulmonary IFN-gamma production but did not significantly alter resolution of replicative L . pneumophila lung infection . In contrast, blocking endogenous IL-12 activity by administration of anti-IL-12 MAb) alone or in combination with anti-IL-18R MAb inhibited induction of intrapulmonary IFN-gamma and resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection . Taken together, these results demonstrate that IL-18 plays a key role in modulating induction of IFN-gamma in the lung in response to L . pneumophila and that together with IL-12, IL-18 regulates intrapulmonary growth of the bacteria.

Infect Control Hosp Epidemiol, 2000 Oct, 21(10), 649 - 51
Comparison of blood-culture contamination rates in a pediatric emergency room: newly inserted intravenous catheters versus venipuncture; Ramsook C et al.; We compared contamination rates of blood cultures obtained either from newly inserted intravenous catheters or via venipuncture . Of 2,431 blood cultures, the overall contamination rate was 2.7% (intravenous catheter, 3.4%; venipuncture, 2.0%; P=.043) . The site of lowest contamination was the antecubital fossa, making this the optimal choice for blood-culture sampling.

Reprod Nutr Dev, 2000 Jul-Aug, 40(4), 383 - 91
Relationship between embryo recovery rate and uterine lavage fluid composition in postpartum mares; Reilas T et al.; The aim of the study was to determine whether neutrophil numbers (PMN), trypsin-inhibitor capacity (TIC), lysozyme, N-acetyl-beta-D-glucosaminidase (NAGase), beta-glucuronidase (B-Gase), total protein, and plasmin in uterine lavage fluid of postpartum (p.p.) mares, either at the time of foal heat insemination or around the time of arrival of the embryo in the uterus, could be used in predicting conception . Fifteen mares were inseminated within 13 h after the first p.p . ovulation . Uterine lavage fluids were successfully collected from 9 out of 12 mares before insemination and from all 15 mares before embryo recovery 7 to 8 days after insemination . The embryo recovery rate was 53% (8/15) . Prior to insemination, PMN, TIC and lysozyme levels were elevated in 3/4 mares not producing embryos . However, only 1/5, 1/5 and 0/5 mares producing embryos had elevated levels of PMN, TIC, and lysozyme, respectively . None of the parameters was significantly different in mares with or without embryos, but lysozyme was the closest to significance (p = 0.07) . In both groups of mares, activities of NAGase (p < 0.01) and B-Gase (p < 0.05) were significantly higher in dioestrus than immediately after ovulation . At embryo recovery, NAGase was higher in mares not producing embryos (p < 0.05) . The results suggest that a long-lasting inflammation is the best explanation for low pregnancy rates during the first p.p . oestrus . Further research is needed to establish whether lysozyme, or possibly TIC, could be used in predicting conception at foal heat.

Pol Merkuriusz Lek, 2000 Aug, 9(50), 579 - 83
{Selected aspects of immunopathogenesis in Lyme disease}; Zajkowska JM et al.; Abilities of B . burgdorferi to infect and induce illness is connected with immunopathogenic factors as: immunomodulatory elements of tick saliva abilities of B . burgdorferi to move in extracellular matrix, connecting and activation of proteolitic zymogen, inducing endothelium to production of adhesive molecules, chemokines and acute phase proteins . Important elements in pathogenesis and course of Lyme disease are organ tropism, producing by lymphocytes T proinflammatory cytokines (Th1 and Th2 profile), phagocytic abilities of infected organisms . In pathogenesis of chronic and recurrent cases difficult to treat is essential is survive of metabolic inactive bacteria, antigens B . burgdorferi as form "blebs", cystic L-form or insoluble complexes antigen-antibody or possibility of intracellular survive of B . burgdorferi . Tissue and organs damage is caused not only directly by B . burgdorferi antigens . Undoubtedly in the late stages there are immunologic disregulation and molecular mimicry . In some cases important are concommitant other infections . All this factors and results of therapy have influence on prognosis of Lyme disease . Knowledge of pathogenesis of Lyme disease has an important meaning in interpretation of laboratory tests and estimation of clinical signs and choosing of treatment.

Structure Fold Des, 2000 Oct 15, 8(10), 1037 - 47
Crystal structure of saccharopine reductase from Magnaporthe grisea, an enzyme of the alpha-aminoadipate pathway of lysine biosynthesis; Johansson E et al.; BACKGROUND: The biosynthesis of the essential amino acid lysine in higher fungi and cyanobacteria occurs via the alpha-aminoadipate pathway, which is completely different from the lysine biosynthetic pathway found in plants and bacteria . The penultimate reaction in the alpha-aminoadipate pathway is catalysed by NADPH-dependent saccharopine reductase . We set out to determine the structure of this enzyme as a first step in exploring the structural biology of fungal lysine biosynthesis . RESULTS: We have determined the three-dimensional structure of saccharopine reductase from the plant pathogen Magnaporthe grisea in its apo form to 2.0 A resolution and as a ternary complex with NADPH and saccharopine to 2.1 A resolution . Saccharopine reductase is a homodimer, and each subunit consists of three domains, which are not consecutive in amino acid sequence . Domain I contains a variant of the Rossmann fold that binds NADPH . Domain II folds into a mixed seven-stranded beta sheet flanked by alpha helices and is involved in substrate binding and dimer formation . Domain III is all-helical . The structure analysis of the ternary complex reveals a large movement of domain III upon ligand binding . The active site is positioned in a cleft between the NADPH-binding domain and the second alpha/beta domain . Saccharopine is tightly bound to the enzyme via a number of hydrogen bonds to invariant amino acid residues . CONCLUSIONS: On the basis of the structure of the ternary complex of saccharopine reductase, an enzymatic mechanism is proposed that includes the formation of a Schiff base as a key intermediate . Despite the lack of overall sequence homology, the fold of saccharopine reductase is similar to that observed in some enzymes of the diaminopimelate pathway of lysine biosynthesis in bacteria . These structural similarities suggest an evolutionary relationship between two different major families of amino acid biosynthetic pathway, the glutamate and aspartate families.

EMBO J, 2000 Nov 15, 19(22), 6041 - 50
Signal transduction to the Azotobacter vinelandii NIFL-NIFA regulatory system is influenced directly by interaction with 2-oxoglutarate and the PII regulatory protein; Little R et al.; PII-like signal transduction proteins, which respond to the nitrogen status via covalent modification and signal the carbon status through the binding of 2-oxoglutarate, have been implicated in the regulation of nitrogen fixation in several diazotrophs . The NIFL-NIFA two-component regulatory system, which integrates metabolic signals to fine-tune regulation of nitrogenase synthesis in Azotobacter vinelandii, is a potential target for PII-mediated signal transduction . Here we demonstrate that the inhibitory activity of the A.vinelandii NIFL protein is stimulated by interaction with the non-uridylylated form of PII-like regulatory proteins . We also observe that the NIFL-NIFA system is directly responsive to 2-oxoglutarate . We propose that the PII protein signals the nitrogen status by interaction with the NIFL-NIFA system under conditions of nitrogen excess, and that the inhibitory activity of NIFL is relieved by elevated levels of 2-oxoglutarate when PII is uridylylated under conditions of nitrogen limitation . Our observations suggest a model for signal transduction to the NIFL-NIFA system in response to carbon and nitrogen status which is clearly distinct from that suggested from studies on other diazotrophs.

EMBO J, 2000 Nov 15, 19(22), 5962 - 70
Crystal structure of the Xrcc4 DNA repair protein and implications for end joining; Junop MS et al.; XRCC4 is essential for carrying out non-homologous DNA end joining (NHEJ) in all eukaryotes and, in particular, V(D)J recombination in vertebrates . Xrcc4 protein forms a complex with DNA ligase IV that rejoins two DNA ends in the last step of V(D)J recombination and NHEJ to repair double strand breaks . XRCC4-defective cells are extremely sensitive to ionizing radiation, and disruption of the XRCC4 gene results in embryonic lethality in mice . Here we report the crystal structure of a functional fragment of Xrcc4 at 2.7 A resolution . Xrcc4 protein forms a strikingly elongated dumb-bell-like tetramer . Each of the N-terminal globular head domains consists of a beta-sandwich and a potentially DNA-binding helix- turn-helix motif . The C-terminal stalk comprising a single alpha-helix >120 A in length is partly incorporated into a four-helix bundle in the Xrcc4 tetramer and partly involved in interacting with ligase IV . The Xrcc4 structure suggests a possible mode of coupling ligase IV association with DNA binding for effective ligation of DNA ends.

Antisense Nucleic Acid Drug Dev, 2000 Oct, 10(5), 381 - 9
Requirements for effective inhibition of immunostimulatory CpG motifs by neutralizing motifs; Zhao H et al.; The DNA of bacteria and many viruses contain unmethylated CpG dinucleotides in particular sequence contexts that activate vertebrate immune cells . A subset of these CpG motifs was previously found to oppose the effects of immunostimulatory (CpG-S) motifs and has been termed neutralizing (CpG-N) motifs . Here we show that oligodeoxynucleotides (ODNs) composed of clusters of CpG-N motifs could partially inhibit the induction of interleukin-12 (IK-12) from mouse spleen cells by ODN containing CpG-S motifs . However, non-CpG-containing ODN were also inhibitory, suggesting that neutralization of CpG-S ODNs by CpG-N ODNs in trans was nonspecific . Neutralization of CpG-S motifs by CpG-N motifs in cis was specific, but the degree of inhibition was strongly dependent on the particular CpG-S motif being neutralized, with motifs having an A residue 5' to the CG being much more resistant to inhibition than motifs having a T residue 5' to the CG . The degree of inhibition was dependent on the spacing between the CpG-S and CpG-N motifs, with the ability to neutralize inversely correlating with distance . In addition, whereas ODNs containing extended clusters of CpG-N motifs were nonstimulatory, isolated CpG-N motifs remained stimulatory in most sequence contexts . Finally, CpG-N ODNs were shown to be nonstimulatory when instilled into the lungs of BALB/c mice, but the ability of CpG-N motifs to neutralize CpG-S motifs in cis was not observed . These results show that there are precise and fairly complex interactions between immunostimulatory and inhibitory sequence motifs that govern whether a given DNA is able to activate the vertebrate immune system.

Cell Mol Life Sci, 2000 Sep, 57(10), 1423 - 39
Enzymatic in vitro synthesis of I-branches of mammalian polylactosamines: generation of scaffolds for multiple selectin-binding saccharide determinants; Renkonen O; Polylactosamines are covalent monosaccharide assemblies of the animal kingdom and some bacteria, and are characterized by backbones of interlinked N-acetyllactosamine units (Galbeta1-4GlcNAc, LacNAc) . The mammalian LacNAc arrays are linear (blood group i-type) and branched (blood group I-type), and are linked to the core elements of glycolipids as well as O- and N-glycans of glycoproteins and keratan sulfate proteoglycans . Generation of I-branches to linear i-type polylactosamines is initiated by two kinds of beta6GlcNAc transferases . One type of the enzymes transfers to Glc-NAcbeta 1-3Galbeta 1-OR of growing i-chains at the peridistal (underlined) Gal; these enzymes are called dIGnT (d for 'distally acting') . The other enzymes transfer to internal Gal units of preformed i-chains; they are called cIGnT (c for 'centrally acting') . Purified natural and recombinant enzymes of both types have been described . The structures of I-type polylactosamines result from a collaboration of dIGnTs, cIGnTs, beta4GalTs and the i-chain-elongating iGnTs . At present, the interplay of these enzymes in vivo is poorly understood . By contrast, enzyme-assisted in vitro synthesis of branched polylactosamines representing distinct LacNAc arrays that are multiply capped by a variety of decorations is possible . Some of the synthetic polylactosamines reduce the lymphocyte-endothelium adhesion in a tissue-specific mode, raising the possibility of achieving local immunosuppression in the future . Useful applications of multiply decorated I-type polylactosamines may also be found in prevention of mammalian gamete adhesion and in inhibition of bacterial and viral adhesion to host tissues.

FEMS Microbiol Rev, 2000 Dec, 24(5), 601 - 14
Recent advances in exploring physiology and biodiversity of ectomycorrhizas highlight the functioning of these symbioses in ecosystems; Buscot F et al.; Ectomycorrhizas, the dominating mycorrhizal symbiosis in boreal, temperate and some tropical forests, are formed by 5000-6000 species of the asco- and basidiomycetes . This high diversity of fungal partners allows optimal foraging and mobilisation of various nitrogen and phosphorus forms from organic soil layers . In this review, two approaches to study the functioning of this multitude of symbiotic associations are presented . On selected culture models, physiological and molecular investigations have shown that the supply of hexoses has a key function in controlling the plant-fungus interaction via partner-specific regulation of gene expression . Environmental factors which affect fungal carbon supply, such as increased nitrogen availability, also affect mycorrhiza formation . Based on such laboratory results, the adaptative capability of ectomycorrhizas to changing field conditions is discussed . The second approach consists of analysing the distribution of mycorrhizas in ecosystem compartments and to relate distribution patterns to variations of ecological factors . Recent advances in identification of fungal partners in ectomycorrhizas by analysing the internal transcribed spacer of ribosomal DNA are presented, which can help to resolve sampling problems in field studies . The limits of the laboratory and the field approaches are discussed . Despite some problems, this combined approach is the most promising . Direct investigation of gene expression, which has been introduced for soil bacteria, will be difficult in the case of mycorrhizal fungi which constitute organisms with functionally varying structures.

J Cell Biol, 2000 Nov 13, 151(4), 945 - 50
Visualization of a cytoskeleton-like FtsZ network in chloroplasts; Kiessling J et al.; It has been a long-standing dogma in life sciences that only eukaryotic organisms possess a cytoskeleton . Recently, this belief was questioned by the finding that the bacterial cell division protein FtsZ resembles tubulin in sequence and structure and, thus, may be the progenitor of this major eukaryotic cytoskeletal element . Here, we report two nuclear-encoded plant ftsZ genes which are highly conserved in coding sequence and intron structure . Both their encoded proteins are imported into plastids and there, like in bacteria, they act on the division process in a dose-dependent manner . Whereas in bacteria FtsZ only transiently polymerizes to a ring-like structure, in chloroplasts we identified persistent, highly organized filamentous scaffolds that are most likely involved in the maintenance of plastid integrity and in plastid division . As these networks resemble the eukaryotic cytoskeleton in form and function, we suggest the term "plastoskeleton" for this newly described subcellular structure.

J Org Chem, 2000 Nov 3, 65(22), 7422 - 31
Tethered bisubstrate derivatives as probes for mechanism and as inhibitors of aminoglycoside 3'-phosphotransferases; Liu M et al.; Aminoglycoside 3'-phosphotransferases {APH(3')s} phosphorylate aminoglycoside antibiotics, a reaction that inactivates the antibiotics . These enzymes are the primary cause of resistance to aminoglycosides in bacteria . APH(3')-Ia operates by a random-equilibrium BiBi mechanism, whereas APH(3')-IIIa catalyzes its reaction by the Theorell-Chance mechanism, a form of ordered BiBi mechanism . Hence, both substrates have to be present in the active site prior to the transfer of phosphate by both mechanisms . Four bisubstrate analogues, compounds 1-4, were designed and synthesized as inhibitors for APH(3')s . These compounds are made of adenosine linked covalently to the 3'-hydroxyl of neamine (an aminoglycoside) via all-methylene tethers of 5-8 carbons . The K(i) values measured for these compounds indicated that affinities of APH(3')-Ia and APH(3')-IIa for compounds 2 and 3 (six- and seven-carbon tethers, respectively) were the best, and the inhibition constants for the two were comparable.

Biochemistry, 2000 Nov 14, 39(45), 13625 - 32
Mechanistic diversity in a metalloenzyme superfamily; Armstrong RN; It is now appreciated that the relationships of proteins, particularly enzymes, within a protein superfamily can be understood not only in terms of their sequence similarities and three-dimensional structures but also by chemical threads that relate their functional attributes . The mechanistic ties among superfamily members can often be traced to a common transition state for the rate-limiting step of the reactions being catalyzed . This paper presents an analysis of a metalloenzyme superfamily, the members of which catalyze a very diverse set of reactions with unrelated transition states but a more general common mechanistic imperative . The vicinal oxygen chelate (VOC) superfamily is composed of structurally related proteins with paired beta alpha beta beta beta motifs that provide a metal coordination environment with two or three open or readily accessible coordination sites to promote direct electrophilic participation of the metal ion in catalysis . The known types of reactions that are catalyzed include isomerizations (glyoxalase I), epimerizations (methylmalonyl-CoA epimerase), oxidative cleavage of C-C bonds (extradiol dioxygenase), and nucleophilic substitutions (fosfomycin resistance proteins) . The remarkable access to mechanism space that is provided by the VOC superfamily appears to derive from a simple, pseudosymmetric structural fold that maximizes the catalytic versatility of the metal center.

Przegl Epidemiol, 2000, 54(1-2), 75 - 84
{Meningitis and encephalitis in 1998}; Zabicka J et al.; In 1998--3,024 cases of meningitis and 581 cases of encephalitis were reported . It was 1,436 cases less then in 1997 . A significant decrease of enteroviral meningitidis was observed and strains of ECHO30 was not dominant, it was Cox B5 . Among bacterial factors the most common was N . meningitidis . 131 sporadic cases of meningitis caused by this bacteria were reported with serotype B dominating (96%) . There were 103 cases of bacterial meningitis caused by S . pneumoniae and 101 cases caused by H . influenzae b among those with confirmed diagnosis . There were 208 cases of tick-borne encephalitis, diagnosed mainly in endemic areas of Bialystok and Suwalki voivodeships.

Biol Chem, 2000 Sep-Oct, 381(9-10), 865 - 76
The role of Se, Mo and Fe in the structure and function of carbon monoxide dehydrogenase; Meyer O et al.; CO dehydrogenase (EC 1.2.99.2) catalyzes the oxidation of CO according to the following equation: CO + H2O-->CO2 + 2 e- + 2 H+ . It is a selenium-containing molybdo-iron-sulfur-flavoenzyme, which has been crystallized and structurally characterized in its oxidized state from the aerobic CO utilizing bacteria Oligotropha carboxidovorans and Hydrogenophaga pseudoflava . Both CO dehydrogenase structures show only minor differences, and the enzymes are dimers of two heterotrimers . Each heterotrimer is composed of a molybdoprotein, a flavoprotein, and an iron-sulfur protein . CO oxidation takes place at the molybdoprotein which contains a 1:1 mononuclear complex of molybdopterin-cytosine dinucleotide and a Mo-ion, along with a catalytically essential S-selanylcysteine . The latter is appropriately positioned in the SeMo-active site by a unique VAYRCSFR active site loop . In H . pseudoflava the arginine preceeding the cysteine in the active site loop is modified to a Cgamma-hydroxy arginine residue which has no obvious function . The substituents in the first coordination sphere of the Mo-ion are the enedithiolate sulfur atoms of the molybdopterin-cytosine dinucleotide, two oxo- and a sulfido-group . Extended X-ray absorption fine structure spectroscopy (EXAFS), along with the crystal structure of CO dehydrogenase (23.2 U mg(-1)) at 1.85 A resolution, have identified a sulfur atom at 2.3 A from the Mo-ion . The sulfur reacts with cyanide yielding thiocyanate . The corresponding inactive desulfo-CO dehydrogenase shows a typical desulfo inhibited-type of Mo-electron paramagnetic resonance (EPR) spectrum . Structural changes at the SeMo-site during catalysis are suggested by the Mo to Se distance of 3.7 A and the Mo-S-Se angle of 113 degrees in the oxidized enzyme which increase to 4.1 A, and 121 degrees, respectively, in the reduced enzyme . The intramolecular electron transport chain in CO dehydrogenase involves the following prosthetic groups and minimal distances: CO-->{Mo of the molybdenum cofactor} - 14.6 A - {2Fe-2S}I - 12.4 A - {2Fe-2S}II - 8.7 A - {FAD}.

J Mol Microbiol Biotechnol, 2000 Oct, 2(4), 473 - 81
Transcriptional regulation in spirochetes; Indest KJ et al.; Spirochetes belong to a widely diverse family of bacteria . Several species in this family can cause a variety of illnesses including syphilis and Lyme disease . Despite the fact that the complete genome sequence of two species, Borrelia burgdorferi and Treponema pallidum, have been deciphered, much remains to be understood about spirochetal gene regulation . In this review we focus on the environmental transitions that spirochetes undergo during their life cycles and the mechanisms of transcriptional regulation that might possibly mediate spirochetal adaptations to such changes.

J Mol Microbiol Biotechnol, 2000 Oct, 2(4), 433 - 42
DNA exchange and insertional inactivation in spirochetes; Tilly K et al.; Spirochetes have complex life cycles and are associated with a number of diseases in humans and animals . Despite their significance as pathogens, spirochete genetics are in their early stages . However, gene inactivation has been achieved in Borrelia burgdorferi, Brachyspira hyodysenteriae, and Treponema denticola . Here, we review methods that have been used in spirochetes for gene inactivation and DNA exchange, with a primary focus on B . burgdorferi . We also describe factors influencing electrotransformation in B . burgdorferi . In summary, optimal transformation frequencies are obtained with log phase bacteria, large amounts of DNA (up to 50 microg per transformation), and high field strength (12.5-37.5 kV/cm) . Infectious B . burgdorferi isolates transform with frequencies 100-fold lower than those found for high passage, non-infectious strains . Surface characteristics of the bacteria, which often correlate with infectivity, are among the obstacles to effective transformation by electroporation.

J Mol Microbiol Biotechnol, 2000 Oct, 2(4), 411 - 22
Repetition, conservation, and variation: the multiple cp32 plasmids of Borrelia species; Stevenson B et al.; Members of the spirochete genus Borrelia contain large numbers of extrachromosomal DNAs . Sequence analysis of the B . burgdorferi strain B31 genome indicated that its many plasmids contain large quantities of repeated sequences, the most obvious of which are the cp32 plasmid family . Individual spirochetes may carry nine or more different, but homologous, cp32 plasmids . Every other species of Borrelia examined thus far also contains multiple plasmids related to the B . burgdorferi cp32s . These plasmids are arguably the best characterized of all the borrelial plasmids, and epitomize the apparent redundancy evident in the many plasmids carried by these bacteria . Despite their extensive similarities, cp32 plasmids contain some open reading frames whose sequences often vary between plasmids, and which encode proteins synthesized by the bacteria during vertebrate infection . In this review, we analyze the hypervariable and conserved regions of the cp32 plasmid family, and discuss possible reasons why borreliae harbor multiple gene paralogs.

J Mol Microbiol Biotechnol, 2000 Oct, 2(4), 387 - 92
Genome analyses of spirochetes: a study of the protein structures, functions and metabolic pathways in Treponema pallidum and Borrelia burgdorferi; Das R et al.; We perform a comprehensive genome analysis on two spirochetes, T . pallidum and B . burgdorferi . First, we focus on the occurrence of protein structures in these organisms . We find that there are only a few spirochete-specific folds, relative to those in other types of bacteria . The most common fold, by far, in the spirochetes is the P-loop NTP hydrolase, followed by the TIM barrel . These folds also happen to be amongst the most multifunctional of the known folds . We also survey the membrane-protein structures in T . pallidum and find a notable large family with twelve transmembrane (TM) helices, reflecting the prevalence of 12-TM transporters in bacteria . Then we move to analysis of the metabolic pathways and overall metabolism in the spirochetes, using the metabolic-flux-balancing method . We find that the lipid biosynthesis pathway is absent from the spirochetes . This strongly limits the degree to which these organisms can metabolize NADPH . In turn, we find that the spirochetes distribute flux disproportionately through the glycolytic pathway instead of the NADPH-providing pentose phosphate pathway . Further information is available at http://bioinfo.mbb.yale.edu

J Mol Microbiol Biotechnol, 2000 Oct, 2(4), 345 - 54
Spirochete periplasmic flagella and motility; Li C et al.; Spirochetes have a unique structure, and as a result their motility is different from that of other bacteria . They also have a special attribute: spirochetes can swim in a highly viscous, gel-like medium, such as that found in connective tissue, that inhibits the motility of most other bacteria . In spirochetes, the organelles for motility, the periplasmic flagella, reside inside the cell within the periplasmic space . A given periplasmic flagellum is attached only at one end of the cell, and depending on the species, may or may not overlap in the center of the cell with those attached at the other end . The number of periplasmic flagella varies from species to species . These structures have been shown to be directly involved in spirochete motility, and they function by rotating within the periplasmic space . The mechanics of motility also vary among the spirochetes . In Leptospira, a motility model developed several years ago has been extensively tested, and the evidence supporting this model is convincing . Borrelia burgdorferi swims differently, and a model of its motility has been recently put forward . This model is based on analyzing the motion of swimming cells, high voltage electron microscopy of fixed cells, and mutant analysis . To better understand spirochete motility on a more molecular level, the proteins and genes involved in motility are being analyzed . Spirochete periplasmic flagellar filaments are among the most complex of bacterial flagella . They are composed of the FlaA sheath proteins, and in many species, multiple FlaB core proteins . Allelic exchange mutagenesis of the genes which encode these proteins is beginning to yield important information with respect to periplasmic flagellar structure and function . Although we are at an early stage with respect to analyzing the function, organization, and regulation of many of the genes involved in spirochete motility, unique aspects have already become evident . Future studies on spirochete motility should be exciting, as only recently have complete genome sequences and tools for allelic exchange mutagenesis become available.

J Mol Microbiol Biotechnol, 2000 Oct, 2(4), 341 - 4
Phylogenetic foundation of spirochetes; Paster BJ et al.; The spirochetes are free-living or host-associated, helical bacteria, some of which are pathogenic to man and animal . Comparisons of 16S rRNA sequences demonstrate that the spirochetes represent a monophyletic phylum within the bacteria . The spirochetes are presently classified in the Class Spirochaetes in the order Spirochetales and are divided into three major phylogenetic groupings, or families . The first family Spirochaetaceae contains species of the genera Borrelia, Brevinema, Cristispira, Spirochaeta, Spironema, and Treponema . The second family Brachyspiraceae contains the genus Brachyspira (Serpulina) . The third family Leptospiraceae contains species of the genera Leptonema and Leptospira . Novel spirochetal species, or phylotypes, that can not be presently cultivated in vitro, have been identified from the human oral cavity, the termite gut, and other host-associated or free-living sources . There are now over 200 spirochetal species or phylotypes, of which more than half is presently not cultivable . It is likely that there is still a significant unrecognized spirochetal diversity that should be evaluated.

Proc R Soc Lond B Biol Sci, 2000 Oct 7, 267(1456), 1931 - 7
Wolbachia-induced 'hybrid breakdown' in the two-spotted spider mite Tetranychus urticae Koch; Vala F et al.; The most common post-zygotic isolation mechanism between populations of the phytophagous mite Tetranychus urticae is 'hybrid breakdown', i.e . when individuals from two different populations are crossed, F1 hybrid females are produced, but F2 recombinant male offspring suffer increased mortality . Two-spotted spider mites collected from two populations, one on rose and the other on cucumber plants, were infected with Wolbachia bacteria . These bacteria may induce cytoplasmic incompatibility in their hosts: uninfected (U) females become reproductively incompatible with infected (W) males . We report on the effect of Wolbachia infections in intra- and interstrain crosses on (i) F1 mortality and sex ratios (a test for cytoplasmic incompatibility), and (ii) the number of haploid offspring and mortality in clutches of F1 virgins (a test for hybrid breakdown) . U x W crosses within the rose strain exhibited partial cvtoplasmic incompatibility . More interestingly, F2 males suffered increased mortality, a result identical to the hybrid breakdown phenomenon . The experiments were repeated using females from the cucumber strain . In interstrain U x W and U x U crosses, hybrid breakdown was much stronger in the former (80 versus 26%) . This is the first report of a Wolbachia infection causing a hybrid breakdown phenotype . Our results show that Wolbhachia infections can contribute to reproductive incompatibility between populations of T . urticae.

Haematologica, 2000 Nov, 85(11), 1172 - 206
Antitumor vaccination: where we stand; Bocchia M et al.; BACKGROUND AND OBJECTIVES: Vaccination is an effective medical procedure of preventive medicine based on the induction of a long-lasting immunologic memory characterized by mechanisms endowed with high destructive potential and specificity . In the last few years, identification of tumor-associated antigens (TAA) has prompted the development of different strategies for antitumor vaccination, aimed at inducing specific recognition of TAA in order to elicit a persistent immune memory that may eliminate residual tumor cells and protect recipients from relapses . In this review characterization of TAA, different potential means of vaccination in experimental models and preliminary data from clinical trials in humans have been examined by the Working Group on Hematopoietic Cells . EVIDENCE AND INFORMATION SOURCES: The method employed for preparing this review was that of informal consensus development . Members of the Working Group met four times and discussed the single points, previously assigned by the chairman, in order to achieve an agreement on different opinions and approve the final manuscript . Some of the authors of the present review have been working in the field of antitumor immunotherapy and have contributed original papers to peer-reviewed journals . In addition, the material examined in the present review includes articles and abstracts published in journals covered by the Science Citation Index and Medline . STATE OF THE ART: The cellular basis of antitumor immune memory consists in the generation and extended persistence of expanded populations of T- and B-lymphocytes that specifically recognize and react against TAA . The efficacy of the memory can be modulated by compounds, called "adjuvants", such as certain bacterial products and mineral oils, cytokines, chemokines, by monoclonal antibodies triggering co-stimulatory receptors . Strategies that have been shown in preclinical models to be efficient in protecting from tumor engraftment, or in preventing a tumor rechallenge, include vaccination by means of soluble proteins or peptides, recombinant viruses or bacteria as TAA genes vectors, DNA injection, tumor cells genetically modified to express co-stimulatory molecules and/or cytokines . The use of professional antigen-presenting cells, namely dendritic cells, either pulsed with TAA or transduced with tumor-specific genes, provides a useful alternative for inducing antitumor cytotoxic activity . Some of these approaches have been tested in phase I/II clinical trials in hematologic malignancies, such as lymphoproliferative diseases or chronic myeloid leukemia, and in solid tumors, such as melanoma, colon cancer, prostate cancer and renal cell carcinoma . Different types of vaccines, use of adjuvants, timing of vaccination as well as selection of patients eligible for this procedure are discussed in this review . PERSPECTIVES: Experimental models demonstrate the possibility of curing cancer through the active induction of a specific immune response to TAA . However, while pre-clinical research has identified several possible targets and strategies for tumor vaccination the clinical scenario is far more complex for a number of possible reasons . Since experimental data suggest that vaccination is more likely to be effective on small tumor burden, such as a minimal residual disease after conventional treatments, or tumors at an early stage of disease, better selection of patients will allow more reliable clinical results to be obtained . Moreover, a poor correlation is frequently observed between the ability of TAA to induce a T-cell response in vitro and clinical responses . Controversial findings may also be due to the techniques used for monitoring the immune status . Therefore, the development of reliable assays for efficient monitoring of the state of immunization of cancer patients against TAA is an important goal that will markedly improve the progress of antitumor vaccines . (ABSTRACT TRUNCATED)

Prostate, 2000 Nov 1, 45(3), 201 - 6
Rat model of experimentally induced abacterial prostatitis; Lang MD et al.; BACKGROUND: An experimental model in rats was developed to investigate the significance of mucosal integrity in abacterial prostatitis . METHODS: Ethanol was instilled into the ventral prostates of male rats to reduce mucosal integrity; dinitrobenzenesulfonic acid (DNBS) was added as an irritant to induce inflammation . Controls received no treatment, ethanol only, DNBS only, or a suspension of bacteria . After various time points, rats were sacrificed, and their prostates were assayed for gross morphology, histological appearance, and cytokine levels . RESULTS: Prostates subjected to ethanol plus DNBS showed significant inflammation, most notably after 12, 24, and 48 hr . Inflammation judged by gross and histological observations and interleukin-1beta levels correlated well at these times . Rats given only ethanol, DNBS, or no treatment, acting as negative controls, displayed little or no inflammation; rats given a bacterial suspension, acting as positive controls, showed inflammation consistent with past studies . Cytokine assays revealed raised interleukin-1beta levels in this model, while tumor necrosis factor-alpha remained at a basal level . CONCLUSIONS: The loss of an intact mucosal surface in the prostate resulted in inflammation caused by an irritant . Interleukin-1beta appears to play a role in this inflammation, while tumor necrosis factor-alpha does not .

J Bacteriol, 2000 Dec, 182(23), 6850 - 3
Secretion of an acid phosphatase (SapM) by Mycobacterium tuberculosis that is similar to eukaryotic acid phosphatases; Saleh MT et al.; Mycobacterium tuberculosis secretes a large number of polypeptides with broad biological and immunological functions . We describe here the characterization of a 28-kDa acid phosphatase of M . tuberculosis (SapM) localized to the culture filtrate . The mature protein demonstrated biochemical characteristics similar to those of the bacterial nonspecific acid phosphatases . However, SapM yielded significant sequence homology to fungal acid phosphatases and not those of bacteria . Thus, SapM may represent a new class of bacterial nonspecific acid phosphatases.

J Bacteriol, 2000 Dec, 182(23), 6834 - 41
Minimal and contributing sequence determinants of the cis-acting locus of transfer (clt) of streptomycete plasmid pIJ101 occur within an intrinsically curved plasmid region; Ducote MJ et al.; Efficient interbacterial transfer of streptomycete plasmid pIJ101 requires the pIJ101 tra gene, as well as a cis-acting plasmid function known as clt . Here we show that the minimal pIJ101 clt locus consists of a sequence no greater than 54 bp in size that includes essential inverted-repeat and direct-repeat sequences and is located in close proximity to the 3' end of the korB regulatory gene . Evidence that sequences extending beyond the minimal locus and into the korB open reading frame influence clt transfer function and demonstration that clt-korB sequences are intrinsically curved raise the possibility that higher-order structuring of DNA and protein within this plasmid region may be an inherent feature of efficient pIJ101 transfer.

J Bacteriol, 2000 Dec, 182(23), 6645 - 50
Novel formaldehyde-activating enzyme in Methylobacterium extorquens AM1 required for growth on methanol; Vorholt JA et al.; Formaldehyde is toxic for all organisms from bacteria to humans due to its reactivity with biological macromolecules . Organisms that grow aerobically on single-carbon compounds such as methanol and methane face a special challenge in this regard because formaldehyde is a central metabolic intermediate during methylotrophic growth . In the alpha-proteobacterium Methylobacterium extorquens AM1, we found a previously unknown enzyme that efficiently catalyzes the removal of formaldehyde: it catalyzes the condensation of formaldehyde and tetrahydromethanopterin to methylene tetrahydromethanopterin, a reaction which also proceeds spontaneously, but at a lower rate than that of the enzyme-catalyzed reaction . Formaldehyde-activating enzyme (Fae) was purified from M . extorquens AM1 and found to be one of the major proteins in the cytoplasm . The encoding gene is located within a cluster of genes for enzymes involved in the further oxidation of methylene tetrahydromethanopterin to CO(2) . Mutants of M . extorquens AM1 defective in Fae were able to grow on succinate but not on methanol and were much more sensitive toward methanol and formaldehyde . Uncharacterized orthologs to this enzyme are predicted to be encoded by uncharacterized genes from archaea, indicating that this type of enzyme occurs outside the methylotrophic bacteria.

J Am Soc Mass Spectrom, 2000 Nov, 11(11), 1023 - 6
Competitive binding to the oligopeptide binding protein, OppA: in-trap cleanup in an Fourier transform ion cyclotron resonance mass spectrometer; Freitas MA et al.; This communication demonstrates that gentle infrared laser heating can remove unwanted buffer adducts from a gas-phase protein complex without dissociating the complex itself . Specifically, noncovalent complexes of the oligopeptide-binding protein, OppA, bound to either (Ala)3 or LysTrpLys were electrosprayed from aqueous buffer solution into a 9.4 tesla Fourier transform ion cyclotron resonance mass spectrometer . In addition to the intact complexes, several additional buffer adduct species were produced under the conditions of the experiment . Irradiation of the trapped ion population with a continuous-wave infrared CO2 laser at relatively low power (2.5 W) for 1 s dissociated the buffer adducts but retained the intact protein:peptide complexes . Adduct-free complex(es) were then readily identified, and signal-to-noise ratio also increased by an order of magnitude because the same number of protein ions are distributed over fewer species . Higher IR power (5 W for 1 s) dissociated the adduct-free complex(es) without internal fragmentation . The present in-trap clean-up technique may prove especially useful for identifying and screening the combinatorial library ligands most strongly bound to a receptor in the gas phase.

Adv Drug Deliv Rev, 2000 Nov 15, 44(2-3), 119 - 34
The role of CpG motifs in immunostimulation and gene therapy; Scheule RK; The mammalian immune system has evolved mechanisms to recognize and respond to 'danger' signals arising from pathogens . Among those danger signals are the unmethylated CpG dinucleotide motifs found in bacteria . At least some of the recognition of these sequences is through cellular components of the innate immune system, such as macrophages . Cytokines released by these cells in response to CpG motifs in turn activate other immune cells, such as NK cells and T cells, and can drive the development of adaptive immune responses . These proinflammatory, Th1 responses can also be generated intentionally with small oligodeoxynucleotides containing stimulatory CpG motifs, and have beneficial properties as vaccine adjuvants and in cancer immunotherapy . These proinflammatory responses have also been seen in gene therapy applications, especially in systemic delivery systems in which plasmid DNA vectors have been introduced with a vehicle such as a cationic lipid . For many gene therapy applications, finding ways to counter the immunostimulatory properties of plasmid DNA vectors is an important approach designed to enhance the vector safety profile, thereby increasing its effective therapeutic index.

AIHAJ, 2000 Sep-Oct, 61(5), 658 - 68
Autocorrelation and variability of indoor air quality measurements; Luoma M et al.; Measurements of gaseous and particulate concentrations are used to characterize the indoor environment, but such measurements may reflect temporary conditions that are not representative of longer time periods . Moreover, indoor air quality (IAQ) measurements are autocorrelated, a result of limited mixing and air exchange, cyclic emissions, HVAC operation, and other factors . This article analyzes the autocorrelation and variability of IAQ measurements using time series analysis techniques in conjunction with a simple IAQ model . Autocorrelations may be estimated using the air exchange rate (alpha) and ventilation effectiveness (epsilon) of the building or room under study, or estimated from pollutant measurements . From this, the variability, required sample size, and other sampling parameters are estimated . The method is tested in a case study in which particle number, fungi, bacteria, and carbon dioxide concentrations were continuously measured in an office building over a 1-week period . The estimated air exchange rate (1.4/hr) for area studied was predicted to yield autocorrelation coefficients of approximately 0.5 for measurements collected on 30-min intervals . Autocorrelation coefficients based on airborne measurements (lag 0.5 hr) ranged from 0.5 to 0.7 for 1-25 microm diameter particles, fungi, and CO2, but near zero for particles < or =1 microm diameter and bacteria . As expected, the variability of measurements with the lowest autocorrelation decreased the most at long sampling times . The implications for spaces with low alpha * epsilon products are that measurements may not benefit significantly from longer averaging periods, measurements on any single day may not be representative, and day-to-day variability may be significant . Steps to determine sample sizes, averaging times, and sampling strategies that can improve the representativeness of IAQ measurements are discussed.

J Can Dent Assoc, 2000 Oct, 66(9), 488 - 91
Periodontal medicine: a new paradigm; Matthews DC; Recent evidence indicates that we need to change how we think about the etiology and pathogenesis of periodontal disease . Although bacteria are a necessary factor in the equation, the reaction of the host's immuno-inflammatory system is responsible for most of the destruction found in periodontal disease . Thus, it makes sense that a number of environmental and acquired factors may modify a patient's risk of developing periodontal disease . This paper reviews the scientific evidence for a number of these risk factors including age, genetics, smoking, diabetes mellitus, stress and osteoporosis.

Plant J, 2000 Oct, 24(2), 265 - 73
Technical advance: An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants; Zuo J et al.; We have developed an estrogen receptor-based chemical-inducible system for use in transgenic plants . A chimeric transcription activator, XVE, was assembled by fusion of the DNA-binding domain of the bacterial repressor LexA (X), the acidic transactivating domain of VP16 (V) and the regulatory region of the human estrogen receptor (E; ER) . The transactivating activity of the chimeric XVE factor, whose expression was controlled by the strong constitutive promoter G10-90, was strictly regulated by estrogens . In transgenic Arabidopsis and tobacco plants, estradiol-activated XVE can stimulate expression of a GFP reporter gene controlled by the target promoter, which consists of eight copies of the LexA operator fused upstream of the -46 35S minimal promoter . Upon induction by estradiol, GFP expression levels can be eightfold higher than that transcribed from a 35S promoter, whereas the uninduced controls have no detectable GFP transcripts, as monitored by Northern blot analysis . Neither toxic nor adverse physiological effects of the XVE system have been observed in transgenic Arabidopsis plants under all the conditions tested . The XVE system thus appears to be a reliable and efficient chemical-inducible system for regulating transgene expression in plants.

Plant J, 2000 Oct, 24(2), 219 - 29
A DNA helicase from Pisum sativum is homologous to translation initiation factor and stimulates topoisomerase I activity; Pham XH et al.; DNA helicases play an essential role in all aspects of nucleic acid metabolism, by providing a duplex-unwinding function . This is the first report of the isolation of a cDNA (1.6 kb) clone encoding functional DNA helicase from a plant (pea, Pisum sativum) . The deduced amino-acid sequence has eight conserved helicase motifs of the DEAD-box protein family . It is a unique member of this family, containing DESD and SRT motifs instead of DEAD/H and SAT . The encoded 45.5 kDa protein has been overexpressed in bacteria and purified to homogeneity . The purified protein contains ATP-dependent DNA and RNA helicase, DNA-dependent ATPase, and ATP-binding activities . The protein sequence contains striking homology with eIF-4A, which has not so far been reported as DNA helicase . The antibodies against pea helicase inhibit in vitro translation . The gene is expressed as 1.6 kb mRNA in different organs of pea . The enzyme is localized in the nucleus and cytosol, and unwinds DNA in the 3' to 5' direction . The pea helicase interacts with pea topoisomerase I protein and stimulates its activity . These results suggest that pea DNA helicase could be an important multifunctional protein involved in protein synthesis, maintaining the basic activities of the cell, and in upregulation of topoisomerase I activity . The discovery of such a protein with intrinsic multiple activity should make an important contribution to our better understanding of DNA and RNA transactions in plants.

Mol Microbiol, 2000 Oct, 38(2), 381 - 91
The role of Mycobacterium avium complex fibronectin attachment protein in adherence to the human respiratory mucosa; Middleton AM et al.; Mycobacterium avium complex (MAC) are opportunistic respiratory pathogens that infect non-immunocompromised patients with established lung disease, although they can also cause primary infections . The ability to bind fibronectin is conserved among many mycobacterial species . We have investigated the adherence of a sputum isolate of MAC to the mucosa of organ cultures constructed with human tissue and the contribution of M . avium fibronectin attachment protein (FAP) to the process . MAC adhered to fibrous, but not globular mucus, and to extracellular matrix (ECM) in areas of epithelial damage, but not to intact extruded cells and collagen fibres . Bacteria occasionally adhered to healthy unciliated epithelium and to cells that had degenerated exposing their contents, but never to ciliated cells . The results obtained with different respiratory tissues were similar . Two ATCC strains of MAC gave similar results . There was a significant reduction (P < 0.05) in the number of bacteria adhering to ECM after preincubation of bacteria with fibronectin and after preincubation of the tissue with M . avium FAP in a concentration-dependant manner . The number of bacteria adhering to fibrous mucus was unchanged . Immunogold labelling demonstrated fibronectin in ECM as well as in other areas of epithelial damage, but only ECM bound FAP . A Mycobacterium smegmatis strain had the same pattern of adherence to the mucosa as MAC . When the FAP gene was deleted, the strain demonstrated reduced adherence to ECM, and adherence was restored when the strain was transfected with an M . avium FAP expression construct . We conclude that MAC adheres to ECM in areas of epithelial damage via FAP and to mucus with a fibrous appearance via another adhesin . Epithelial damage exposing ECM and poor mucus clearance will predispose to MAC airway infection.

Mol Microbiol, 2000 Oct, 38(2), 276 - 88
Occurrence of three PII-like signal transmitter proteins in the diazotrophic proteobacterium Azoarcus sp . BH72; Martin DE et al.; PII-like signal transmitter proteins are involved in the regulation of ammonium assimilation and nitrogen fixation . We report the identification of three PII-like proteins in the diazotrophic, endophytic proteobacterium Azoarcus sp . BH72, encoded by glnB (monocistronically transcribed) or in the glnKamtB and glnYamtY operons . Phylogenetic analysis revealed that glnB, glnK and glnY represent distinct lineages within the Proteobacteria . A combined approach of two-dimensional gel electrophoresis, Western blotting with paralogue-specific antibodies, N-terminal sequencing and marker exchange mutagenesis allowed us to analyse PII protein expression of Azoarcus sp . BH72 in vivo . GlnK and GlnB were present on all nitrogen sources . Knock-out mutant analysis revealed that GlnB was the only detectable PII protein in a glnK- background, whereas GlnY was only present in a glnK/glnB- double mutant . Nitrogen limitation enhanced transcript abundance of glnK strongly, glnY moderately and glnB not at all in wild-type, glnB-/glnK- or glnK- backgrounds respectively . Phenotypic characterization of knock-out mutants revealed that, unlike in other Proteobacteria, neither glnK nor glnB were essential for nitrogen fixation . As the growth of a double mutant was drastically impaired only on minimal media, both proteins are probably involved in the control of ammonium and nitrate assimilation . The PII-like proteins differed from each other in details of N-sensing . They were covalently modified by uridylylation upon nitrogen limitation, as shown by mass spectrometry; however, the modification patterns in relation to the supplied nitrogen source differed . The novel paralogue GlnY was unusual, as it only occurred in the uridylylated state in vivo and thus lacked a deuridylylation response to nitrogen excess.

Mol Microbiol, 2000 Oct, 38(2), 232 - 41
Control of directionality in the site-specific recombination system of the Streptomyces phage phiC31; Thorpe HM et al.; The genome of the Streptomyces temperate phage phiC31 integrates into the host chromosome via a recombinase belonging to a novel group of phage integrases related to the resolvase/invertase enzymes . Previously, it was demonstrated that, in an in vitro recombination assay, phiC31 integrase catalyses integration (attP/attB recombination) but not excision (attL/attR) . The mechanism responsible for this recombination site selectivity was therefore investigated . Purified integrase was shown to bind with similar apparent binding affinities to between 46 bp and 54 bp of DNA at each of the attachment sites, attP, attB, attL and attR . Assays using recombination sites of 50 bp and 51 bp for attP and attB, respectively, showed that these fragments were functional in attP/attB recombination and maintained strict site selectivity, i.e . no recombination between non-permissive sites, such as attP/attP, attB/attL, etc., was observed . Using bandshifts and supershift assays in which permissive and non-permissive combinations of att sites were used in the presence of integrase, only the attP/attB combination could generate supershifts . Recombination products were isolated from the supershifted complexes . It was concluded that these supershifted complexes contained the recombination synapse and that site specificity, and therefore directionality, is determined at the level of stable synapse formation.

J Invest Dermatol, 2000 Nov, 115(5), 813 - 23
Human peptidylarginine deiminase type III: molecular cloning and nucleotide sequence of the cDNA, properties of the recombinant enzyme, and immunohistochemical localization in human skin; Kanno T et al.; Peptidylarginine deiminase catalyzes the post-translational modification of proteins through the conversion of arginine to citrulline in the presence of calcium ions . In rodents, peptidylarginine deiminase has been classified into four isoforms, types I, II, III, and IV, which are distinct in their molecular weights, substrate specificities, and tissue localization . Of these isoforms, only type III was detected in epidermis and hair follicles . Although the role of this enzyme in these tissues is not yet clear, indirect data have shown that several structural proteins such as filaggrin, trichohyalin, and keratin are substrates for peptidylarginine deiminase . In this study, we cloned the full-length cDNA of human peptidylarginine deiminase type III (3142 bp) from cultured human keratinocytes by reverse transcription-polymerase chain reaction and by rapid amplification of cDNA ends methods . This cDNA contained a 1995 bp open reading frame encoding 664 amino acids (Mr = 74 770) . To explore the physicochemical and enzymatic properties of human peptidylarginine deiminase type III, we constructed a plasmid for producing a recombinant human peptidylarginine deiminase type III in bacteria . The enzymatic characteristics of the recombinant enzyme were very similar to those of the rodent peptidylarginine deiminase type III . The recombinant enzyme showed the catalytic activities toward structural proteins of epidermis and hair follicle, filaggrin and trichohyalin, in which the deiminations maxima of about 60% and 13% arginine residues were observed in filaggrin and trichohyalin, respectively . An immunohistochemical study of human scalp skin with a monospecific anti-peptidyl-arginine deiminase type III antibody revealed that the type III enzyme was localized to the inner root sheath and outer root sheath of hair follicles . Peptidylarginine deiminase type III in the inner root sheath was notable between supramatrix and keratogenous zone and was scarcely detected in cornified hair zone . The enzyme was also expressed in the cuticle layer of hair . On the other hand, expression of the enzyme in the epidermis was very low . These data imply that human peptidylarginine deiminase type III is the predominant isoform in hair follicles and may function as a modulator of hair structural proteins, including trichohyalin during hair and hair follicle formation.

Can J Microbiol, 2000 Oct, 46(10), 952 - 5
A common molecular signature unifies the chitosanases belonging to families 46 and 80 of glycoside hydrolases; Tremblay H et al.; The 3D structure-oriented alignment of the primary sequences of fourteen chitosanases, mainly of bacterial origin and belonging to families 46 and 80 of glycoside hydrolases, resulted in the identification of the following pattern common to all these enzymes: E-{DNQ}-x(8,17)-Y-x(7)-D-x-{RD}-{GP}-x-{TS}-x(3)-{AIVFLY}-G- x(5,11)-D . This pattern is proposed as the molecular signature of the chitosanases from families 46 and 80 . It includes several amino acids essential for enzyme activity and (or) stability as shown by site-directed mutagenesis studies on the chitosanase from Streptomyces sp . N174 . In particular, it includes two carboxylic residues directly involved in catalysis . We suggest that there is a continuum of sequence similarity between all the analyzed chitosanases, and that all these enzymes should probably be classified in one family.

Can J Microbiol, 2000 Oct, 46(10), 885 - 91
Differential expression of IL-1 and TNF receptors in murine macrophages infected with virulent vs . avirulent Legionella pneumophila; McHugh S et al.; Infection of macrophages from genetically susceptible A/J mice with Legionella pneumophila induces high levels of various cytokines in serum as well as in cultures of spleen or peritoneal cells from the mice . However, modulation of receptor expression for these cytokines during infection has not been studied in detail, even though these receptors on macrophages have a critical role in inflammatory responses during the infection . In the present study, the differential expression of mRNA for TNF and IL-1 receptors as well as receptor antigens during infection of macrophages with virulent vs . avirulent L . pneumophila was investigated . Mouse thioglycollate-elicited peritoneal macrophages showed by RT-PCR constitutive steady-state levels of mRNA for TNF-type I and -type II receptors as well as IL-1 type I receptor . However, IL-1 type II receptor mRNA was not expressed in thioglycollate-elicited macrophages . Infection of macrophages with virulent bacteria caused an upregulation of IL-1 type I and TNF type I receptor mRNA, but had no effect on TNF type II receptor message . Avirulent L . pneumophila infection caused much less induction of these receptor mRNAs . The amount of receptor antigen of IL-1 type I on the surface of macrophages was also increased by infection with virulent L . pneumophila determined by flow cytometric analysis . These results indicate that L . pneumophila infection not only causes induction of various cytokines, but also modulation of certain cytokine receptors, which may regulate the susceptibility to infection.

FEBS Lett, 2000 Nov 3, 484(2), 144 - 8
Replacement of tyr62 by trp in the designer protein milk bundle-1 results in significant improvement of conformational stability; Gagnon MC et al.; Protein design is currently used for the creation of new proteins with desirable traits . In our lab, we focus on the synthesis of proteins with high essential amino acid content, having potential application in animal nutrition . One of the limitations we face in this endeavor is the achievement of stable proteins in spite of a highly biased amino acid content . We report here the synthesis and characterization of MB-1Trp, a protein with a tailored content in selected essential amino acids . The protein is a Tyr62-Trp mutant of the parent molecule MB-1 described earlier . The new protein is largely helical as per design, is well folded, and has a melting temperature of 55 degrees C . Its resistance to proteolytic degradation compares to that of cytochrome c, a protein of similar size . Design strategy used for MB-1Trp is discussed with regards to its applicability toward the creation of efficient nutritional proteins.

J Exp Med, 2000 Nov 6, 192(9), 1261 - 72
Legionella pneumophila replication vacuoles mature into acidic, endocytic organelles; Sturgill-Koszycki S et al.; After ingestion by macrophages, Legionella pneumophila inhibits acidification and maturation of its phagosome . After a 6-10-h lag period, the bacteria replicate for 10-14 h until macrophage lysis releases dozens of progeny . To examine whether the growth phase of intracellular L . pneumophila determines the fate of its phagosome, interactions between the endosomal network and pathogen vacuoles were analyzed throughout the primary infection period . Surprisingly, as L . pneumophila replicated exponentially, a significant proportion of the vacuoles acquired lysosomal characteristics . By 18 h, 70% contained lysosomal-associated membrane protein 1 (LAMP-1) and 40% contained cathepsin D; 50% of the vacuoles could be labeled by endocytosis, and the pH of this population of vacuoles averaged 5.6 . Moreover, L . pneumophila appeared to survive and replicate within lysosomal compartments: vacuoles harboring more than five bacteria also contained LAMP-1, inhibition of vacuole acidification and maturation by bafilomycin A1 inhibited bacterial replication, bacteria within endosomal vacuoles responded to a metabolic inducer by expressing a gfp reporter gene, and replicating bacteria obtained from macrophages, but not broth, were acid resistant . Understanding how L . pneumophila first evades and then exploits the endosomal pathway to replicate within macrophages may reveal the mechanisms governing phagosome maturation, a process also manipulated by Mycobacteria, Leishmania, and Coxiella.

J Biol Chem, 2001 Jan 26, 276(4), 2427 - 31 Epub 2000 Nov 07.
The role of disulfide bridge in the folding and stability of the recombinant human prion protein; Maiti NR et al.; It is believed that the critical step in the pathogenesis of transmissible spongiform encephalopathies is a transition of prion protein (PrP) from an alpha-helical conformation, PrP(C), to a beta-sheet-rich form, PrP(Sc) . Native prion protein contains a single disulfide bond linking Cys residues at positions 179 and 214 . To elucidate the role of this bridge in the stability and folding of the protein, we studied the reduced form of the recombinant human PrP as well as the variant of PrP in which cysteines were replaced with alanine residues . At neutral pH, the reduced prion protein and the Cys-free mutant were insoluble and formed amorphous aggregates . However, the proteins could be refolded in a monomeric form under the conditions of mildly acidic pH . Spectroscopic experiments indicate that the monomeric Cys-free and reduced PrP have molten globule-like properties, i.e . they are characterized by compromised tertiary interactions, an increased exposure of hydrophobic surfaces, lack of cooperative unfolding transition in urea, and partial loss of native (alpha-helical) secondary structure . In the presence of sodium chloride, these partially unfolded proteins undergo a transition to a beta-sheet-rich structure . However, this transition is invariably associated with protein oligomerization . The present data argue against the notion that reduced prion protein can exist in a stable monomeric form that is rich in beta-sheet structure.

Microbiology, 2000 Nov, 146 ( Pt 11), 2937 - 46
Transcriptional co-regulation of five chitinase genes scattered on the Streptomyces coelicolor A3(2) chromosome; Saito A et al.; Streptomyces coelicolor A3(2) strain M145 has eight chitinase genes scattered on the chromosome: six genes for family 18 (chiA, B, C, D, E and H) and two for family 19 (chiF and G) . In this study, the expression and regulation of these genes were investigated . The transcription of five of the genes (chiA, B, C, D and F) was induced in the presence of colloidal chitin while that of the other three genes (chiE, G and H) was not . The transcripts of the five induced chi genes increased and reached their maximum at 4 h after the addition of colloidal chitin, all showing the same temporal patterns . The induced levels of the transcripts of chiB were significantly lower than those of the other four genes . Dynamic analysis of the transcripts of the chi genes indicated that chiA and chiC were induced more strongly than chiD and chiF . Addition of chitobiose also induced transcription of the chi genes, but significantly earlier than did colloidal chitin . When cells were cultured in the presence of colloidal chitin, an exponential increase of chitobiose concentration in the culture supernatant was observed prior to the induced transcription of the chi genes . This result, together with the immediate effect of chitobiose on the induction, suggests that chitobiose produced from colloidal chitin is involved in the induction of transcription of the chi genes . The transcription of the five chi genes was repressed by glucose . This repression was apparently mediated by the glucose kinase gene glkA.

Rev Neurosci, 2000, 11 Spec No, 213 - 328
A unifying hypothesis of Alzheimer's disease . IV . Causation and sequence of events; Heininger K; Contrary to common concepts, the brain in Alzheimer's disease (AD) does not follow a suicide but a rescue program . Widely shared features of metabolism in starvation, hibernation and various conditions of energy deprivation, e.g . ischemia, allow the definition of a deprivation syndrome which is a phylogenetically conserved adaptive response to energetic stress . It is characterized by hypometabolism, oxidative stress and adjustments of the glucose-fatty acid cycle . Cumulative evidence suggests that the brain in aging and AD actively adapts to the progressive fuel deprivation . The counterregulatory mechanisms aim to preserve glucose for anabolic needs and promote the oxidative utilization of ketone bodies . The agent mediating the metabolic switch is soluble Abeta which inhibits glucose utilization and stimulates ketone body utilization at various levels . These processes, which are initiated during normal aging, include inhibition of pro-glycolytic neurohormones, cholinergic transmission, and pyruvate dehydrogenase, the key transmitter and effector systems regulating glucose metabolism . Hormonal and effector systems which promote ketone body utilization, such as glucocorticosteroid and galanin activity, GABAergic transmission, nitric oxide, lipid transport, Ca2+ elevation, and ketone body metabolizing enzymes, are enhanced . A multitude of risk factors feed into this pathophysiological cascade at a variety of levels . Taking into account its pleiotropic regulatory actions in the deprivation response, a new name for Abeta is suggested: deprivin . On the other hand, cumulative evidence, taken together compelling, suggests that senile plaques are the dump rather than the driving force of AD . Moreover, the neurotoxic action of fibrillar Abeta is a likely in vitro artifact but does not contribute significantly to the in vivo pathophysiological events . This archaic program, conserved from bacteria to man, aims to ensure the survival of a deprived organism and controls such divergent processes as sporulation, hibernation, aging and aging-related diseases . In contrast to the immature brain, ketone body utilization of the aged brain is no longer sufficient to meet the energetic demands and is later supplemented by lactate, thus recapitulating in reverse order the sequential fuel utilization of the immature brain . The transduction pathways which operate to switch metabolism also convey the programming and balancing of the de-/redifferentiation/apoptosis cell cycle decisions . This encompasses the reiteration of developmental processes such as transcription factor activation, tau hyperphosphorylation, and establishment of growth factor independence by means of Ca2+ set point shift . Thus, the increasing energetic insufficiency results in the progressive centralization of metabolic activity to the neuronal soma, leading to pruning of the axonal/dendritic trees, loss of neuronal polarity, downregulation of neuronal plasticity and, eventually, depending on the Ca2+ -energy-redox homeostasis, degeneration of vulnerable neurons . Finally, it is outlined that genetic (e.g . Down's syndrome, APP and presenilin mutations and apoE4) and environmental risk factors represent progeroid factors which accelerate the aging process and precipitate the manifestation of AD as a progeroid systemic disease . Aging and AD are related to each other by threshold phenomena, corresponding to stage 2, the stage of resistance, and stage 3, exhaustion, of a metabolic stress response.

FEMS Microbiol Lett, 2000 Nov 15, 192(2), 217 - 21
The transcriptional activator NtrC controls the expression and activity of glutamine synthetase in Herbaspirillum seropedicae; Persuhn DC et al.; The role of the Ntr system in Herbaspirillum seropedicae was determined via ntrB and ntrC mutants . Three phenotypes were identified in these mutants: Nif(-), deficiency in growth using nitrate, and low glutamine synthetase (GS) activity . All phenotypes were restored by the plasmid pKRT1 containing the intact glnA, ntrB and ntrC genes of H . seropedicae . The promoter region of glnA was subcloned into a beta-galactosidase fusion vector and the results suggested that NtrC positively regulates the glnA promoter in response to low nitrogen . The H . seropedicae ntrC and ntrB mutant strains showed a deficiency of adenylylation/deadenylylation of GS, indicating that NtrC and NtrB are involved in both transcription and activity control of GS in this organism.

Biochemistry, 2000 Nov 7, 39(44), 13487 - 95
Defining the bilin lyase domain: lessons from the extended phytochrome superfamily; Wu SH et al.; Through pattern searches of genomic databases, new members of the growing family of phytochrome-related genes were identified and used to construct a 130-180 amino acid motif that delimits the bilin lyase domain, a subdomain of the extended phytochrome family that is sufficient for covalent attachment of linear tetrapyrroles (bilins) . To test this hypothesis, portions of locus sll0821, a novel phytochrome-related gene from Synechocystis sp . PCC6803 that encodes a large protein with two potential bilin binding sites, were amplified, and the recombinant apoproteins were tested for bilin binding and phytochrome photoactivity . Our experiments indicated that both sites of this protein, termed Cph2 for cyanobacterial phytochrome 2, possessed bilin lyase activity, revealing two distinct classes of bilin lyase domains--those whose bilin adducts are red, far-red reversible and a second class whose bilin adducts are nonphotochromic . Spectroscopic analysis of photochromic phycocyanobilin and fluorescent phycoerythrobilin adducts of a 24-kDa fragment of Cph2 definitively established that the motif identified by pattern searches represents a bona fide bilin lyase domain . Site-directed mutagenesis of highly conserved charged residues within bilin lyase domains of nearly all members of the extended phytochrome superfamily has identified a glutamate residue critical for bilin binding.

Biochemistry, 2000 Nov 7, 39(44), 13478 - 86
Coupling of hydrogen bonding to chromophore conformation and function in photoactive yellow protein; Brudler R et al.; To understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal, we are characterizing photoactive yellow protein (PYP), a water-soluble, 14 kDa blue-light receptor which undergoes a photocycle upon illumination . The active site residues glutamic acid 46, arginine 52, tyrosine 42, and threonine 50 form a hydrogen bond network with the anionic p-hydroxycinnamoyl cysteine 69 chromophore in the PYP ground state, suggesting an essential role for these residues for the maintenance of the chromophore's negative charge, the photocycle kinetics, the signaling mechanism, and the protein stability . Here, we describe the role of T50 and Y42 by use of site-specific mutants . T50 and Y42 are involved in fine-tuning the chromophore's absorption maximum . The high-resolution X-ray structures show that the hydrogen-bonding interactions between the protein and the chromophore are weakened in the mutants, leading to increased electron density on the chromophore's aromatic ring and consequently to a red shift of its absorption maximum from 446 nm to 457 and 458 nm in the mutants T50V and Y42F, respectively . Both mutants have slightly perturbed photocycle kinetics and, similar to the R52A mutant, are bleached more rapidly and recover more slowly than the wild type . The effect of pH on the kinetics is similar to wild-type PYP, suggesting that T50 and Y42 are not directly involved in any protonation or deprotonation events that control the speed of the light cycle . The unfolding energies, 26.8 and 25.1 kJ/mol for T50V and Y42F, respectively, are decreased when compared to that of the wild type (29.7 kJ/mol) . In the mutant Y42F, the reduced protein stability gives rise to a second PYP population with an altered chromophore conformation as shown by UV/visible and FT Raman spectroscopy . The second chromophore conformation gives rise to a shoulder at 391 nm in the UV/visible absorption spectrum and indicates that the hydrogen bond between Y42 and the chromophore is crucial for the stabilization of the native chromophore and protein conformation . The two conformations in the Y42F mutant can be interconverted by chaotropic and kosmotropic agents, respectively, according to the Hofmeister series . The FT Raman spectra and the acid titration curves suggest that the 391 nm form of the chromophore is not fully protonated . The fluorescence quantum yield of the mutant Y42F is 1.8% and is increased by an order of magnitude when compared to the wild type.

J Periodontol, 2000 Oct, 71(10), 1535 - 45
Levels of interleukin-1 beta, -8, and -10 and RANTES in gingival crevicular fluid and cell populations in adult periodontitis patients and the effect of periodontal treatment; Gamonal J et al.; BACKGROUND: Various cytokines have been identified at sites of chronic inflammation such as periodontitis . Cytokines are synthesized in response to bacteria and their products, inducing and maintaining an inflammatory response in the periodontium . The purpose of the present study was to investigate the involvement of interleukin-1 beta (IL-1 beta), IL-8, and IL-10 and RANTES (regulated on activation, normally T cell expressed and secreted) and the cell populations associated with the immune response in destructive periodontitis, as well as the effect of periodontal therapy on cytokine levels in gingival crevicular fluid (GCF) . METHODS: Data were obtained from 12 patients with moderate to advanced periodontitis and 6 healthy controls . Patients presenting at least 2 sites with > or =2 mm clinical attachment loss were included in the destructive periodontitis group . After monitoring for 4 months, only 6 patients showed destructive periodontitis and GCF samples and soft tissues biopsies were collected from these patients . GCF samples and biopsies were collected both from active (12 CGF samples and 6 biopsies) and inactive (12 CGF samples and 6 biopsies) sites . The comparison with healthy controls was carried out by collecting GCF samples from 6 healthy volunteers (12 samples) and biopsies during the surgical removal of wisdom teeth . In periodontal patients, clinical data and GCF samples were obtained prior to periodontal treatment (72 samples) and 2 months after periodontal therapy (72 samples) . GCF was collected using a paper strip; eluted and enzyme-linked immunoabsorbent assays (ELISA) were performed to determine cytokine levels . The inflammatory infiltrate was analyzed by immunohistochemistry of gingival biopsy samples with monoclonal antibodies against CD3, CD8, CD4, CD11c, and CD19 antigens . RESULTS: Cellular components of the inflammatory infiltrate include B and T lymphocytes and monocyte/macrophages . Active sites contained a higher number of B lymphocytes and macrophages . IL-8 and IL-1 beta and RANTES in GCF were detected in the majority of sites from periodontal patients (100%, 94% and 87%, respectively); IL-10 was found in only 43% . IL-8 was the only cytokine detected in the GCF (75%) of the control group . Moreover, IL-1 beta levels were significantly higher in active sites versus inactive sites (P <0.05) . IL-8 and IL-10 and RANTES were increased in active sites; however, differences were not significant (P>0.05) . A positive correlation between the IL-8 and RANTES (r = 0.677, P<0.05) was observed in periodontitis patients . Periodontal therapy reduced the total amount of IL-1 beta, IL-8, and IL-10 and RANTES . Data showed a weak correlation between the clinical parameters and the total amount of cytokines in periodontitis . CONCLUSIONS: These data suggest that the amount of crevicular IL-1 beta, IL-8, and IL-10 and RANTES is associated with periodontal status . Removal of the bacterial plaque reduces the antigenic stimuli and consequently could modulate the chemokines present in GCF . We propose that the dynamic interactions between cytokines, their production rates, and their quantity could represent factors controlling the induction, perpetuation, and collapse of the cytokine network present in the periodontal disease.

Connect Tissue Res, 1998, 38(1-4), 207 - 14; discussion 241-6
Biochemical characterization of recombinant mouse amelogenins: protein quantitation, proton absorption, and relative affinity for enamel crystals; Ryu OH et al.; Four recombinant mouse amelogenins, which varied by the presence or absence of the exon 4 encoded segment as well as the carboxyl-terminus were heterologously expressed and purified from bacteria . The rM193 and rM179 contain the carboxyl-terminus, whereas the rM180 and rM166 do not . The rM193 and rM180 contain the polypeptide segment encoded by exon 4 of the amelogenin gene . A precisely weighed sample of purified rM179 was quantified by Lowry, Bicinchoninic Acid and Bradford assays . It was determined that these protein quantification methods characteristically under or overestimate the amount of amelogenin . The calculated correction factors were: Lowry (x 1.35), BCA (x 1.96), and Bradford (x 0.78) . Recombinant mouse amelogenin (rM179) was characterized with respect to its hydrogen ion binding properties . The protein absorbs 11.9 +/- 1.7 protons during a pH change from 8.0 to 5.0, suggesting that amelogenins buffer the enamel fluid in vivo . Crystal binding experiments were performed using rM193, rM180, rM179 and rM166 . The carboxyl-terminus enhanced the binding of amelogenin to enamel crystals while the exon 4 encoded segment did not appreciably affect crystal binding.

Virology, 2000 Nov 10, 277(1), 193 - 203
Identification and genomic mapping of the ORF3 and VPg proteins in feline calicivirus virions; Sosnovtsev SV et al.; Two minor proteins with molecular masses of 8.5 and 15.5 kDa were identified in feline calicivirus (FCV) virions . Direct sequence analysis showed that the N-terminal sequence of the 8.5-kDa protein was identical to that of the predicted protein encoded by open reading frame 3 (ORF3) of the FCV genome . The N-terminal sequence of the 15.5-kDa protein corresponded to amino acids 961-980 of the FCV ORF1 polyprotein and mapped to the genomic location of the calicivirus VPg . Antisera raised against recombinant ORF3 protein or the N-terminal 20 amino acids of the putative VPg reacted with the corresponding proteins present in both a Western blot analysis of purified FCV virions and an immunofluorescence assay of FCV-infected cells . A comparative analysis of radioactivity incorporated into virion proteins during in vivo labeling experiments indicated that the ORF3 protein is likely present in one or two copies per virion . The mobility of the ORF3 protein present in virions was similar to that of the ORF3 protein found in FCV-infected cells or expressed in bacteria . Direct N- and C-terminal sequence analysis of the purified ORF3 protein obtained by expression in bacteria demonstrated the presence of intact, uncleaved termini, suggesting that the observed difference between the calculated and the apparent masses in SDS-PAGE was not due to proteolytic processing of the protein.

Expert Opin Investig Drugs, 2000 Aug, 9(8), 1767 - 75
Inhibitors of aminoacyl-tRNA synthetases as novel anti-infectives; Tao J et al.; Resistance to existing antibiotics has emerged as a major problem in healthcare . Novel antibiotics for which bacteria have not yet acquired resistance need to be developed to combat drug-resistant pathogens . Aminoacyl-tRNA synthetases are leading targets for novel anti-infectives . The validation of aminoacyl-tRNA synthetases as drug targets for anti-infectives has been established in an animal system . Using several conceptually distinct approaches, new inhibitors of synthetases have been developed as drug prototypes.

Bioelectrochemistry, 2000 Sep, 52(1), 17 - 21
Electromagnetic field-induced protection of chick embryos against hypoxia exhibits characteristics of temporal sensing; Di Carlo AL et al.; We previously studied the response of mammalian cultured cells to weak, 60 Hz-electromagnetic (EM) fields . Two time constants, similar to those observed in chemotaxis, were found to govern the cellular response to the field . We concluded that a system of temporal sensing, similar to that employed in chemotaxis by motile bacteria, was operative . We termed the shorter time (approximately 0.1 s) the "sensing" time, and the longer time (approximately 10 s) the "memory" time . To investigate the possibility that temporal sensing was a general property of EM field-cell interaction, the temporal properties of another EM field-induced effect was studied . The EM field-induced protection against the effects of extreme hypoxia was examined in chick embryos . Embryos were exposed to 60 Hz-magnetic fields, the amplitudes of which were regularly altered throughout the 20-min exposure . Alteration was accomplished either by turning the field off and on at regular intervals (1-50 s), or by introducing brief (10 or 100 ms), zero amplitude gaps, once each second, throughout exposure . When the field was turned on and off at 0.1 s intervals, the protective effect conferred by a constant field was lost . At progressively longer on/off intervals, protection was progressively restored, maximizing at intervals of 10-30 s . Gapping the magnetic field for 10 ms, each second of exposure conferred the same protection as that observed for an uninterrupted field, but gapping the field at 100 ms each second produced a significant reduction in protection . These data exhibit remarkable consistency with those obtained in similar temporal studies of the magnetic field-induced enhancement of ornithine decarboxylase activity in L929 fibroblasts . It appears that temporal sensing is a general feature of the EM field-cell interaction.

Schweiz Med Wochenschr, 2000 Sep 30, 130(39), 1352 - 60
{Indications for lung transplantation in advanced cystic fibrosis}; Speich R et al.; Lung transplantation has become a valid therapeutic option for cystic fibrosis patients with end-stage lung disease . The indication for transplantation does not rely on strict criteria only but must be evaluated case by case . In particular, the dynamics of the clinical course need to be considered with regard to impaired physical performance, recurrent infections, decline in pulmonary function and weight loss . Important risk factors are a poor nutritional status, osteoporosis, liver involvement, previous pleurodesis and the occurrence of multiresistant bacteria . Management and assessment of cystic fibrosis patients for lung transplantation is complex . Therefore patients should be referred to specialised centres at an early stage.

Int Immunol, 2000 Nov, 12(11), 1539 - 46
Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway; Basu S et al.; Dendritic cells (DC) are key components of innate and adaptive immune responses . The identity of endogenous signals that activate DC is a crucial and unresolved question . We report here that heat shock proteins (HSP), the most abundant and conserved mammalian molecules, constitute such an internal signal . Necrotic but not apoptotic cell death leads to release of HSP gp96, calreticulin, hsp90 and hsp70 . HSP stimulate macrophages to secrete cytokines, and induce expression of antigen-presenting and co-stimulatory molecules on the DC . The HSP gp96 and hsp70 act differentially, and each induces some but not all molecules . HSP interact with these antigen-presenting cells through the highly conserved NF-kappa B pathway . As HSP are intracellular, abundant and soluble, their presence in the extra-cellular milieu and the consequent activation of antigen-presenting cells (APC) constitutes an excellent mechanism for response to cell death . As HSP are conserved from bacteria to mammals, the ability of HSP to activate APC provides a unified mechanism for response to internal and external stimuli.

J Biol Chem, 2001 Feb 2, 276(5), 3683 - 90 Epub 2000 Nov 01.
Induction of rapid histone degradation by the cytotoxic T lymphocyte protease Granzyme A; Zhang D et al.; The cytotoxic T lymphocyte protease granzyme A induces caspase-independent cell death in which DNA single-strand nicking is observed instead of oligonucleosomal fragmentation . Granzyme A is a specific tryptase that concentrates in the nucleus of targeted cells and synergistically enhances DNA fragmentation induced by the caspase activator granzyme B . Here we show that granzyme A treatment of isolated nuclei enhances DNA accessibility to exogenous endonucleases . In vitro and after cell loading with perforin, GrnA completely degrades histone H1 and cleaves core histones into approximately 16-kDa fragments . Histone digestion provides a mechanism for unfolding compacted chromatin and facilitating endogenous DNase access to DNA during T cell and natural killer cell granule-mediated apoptosis.

Nucleic Acids Res, 2000 Nov 1, 28(21), 4332 - 9
The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts; Gamper HB et al.; Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folded by complementarity into a double hairpin conformation, have been shown to alter or repair single bases in plant and animal genomes . An uninterrupted stretch of DNA bases within the chimera is known to be active in the sequence alteration while RNA residues aid in complex stability . In this study, the two strands were separated in the hope of defining the role each plays in conversion . Using a series of single-stranded oligonucleotides, comprised of all RNA or DNA residues and various mixtures, several new structures have emerged as viable molecules in nucleotide conversion . When extracts from mammalian and plant cells and a genetic readout assay in bacteria are used, single-stranded oligonucleotides, containing a defined number of thioate backbone modifications, were found to be more active than the original chimera structure in the process of gene repair . Single-stranded oligonucleotides containing fully modified backbones were found to have low repair activity and in fact induce mutation . Molecules containing various lengths of modified RNA bases (2'-O-methyl) were also found to possess low activity . Taken together, these results confirm the directionality of nucleotide conversion by the DNA strand of the chimera and further present a novel, modified single-stranded DNA molecule that directs conversion in plant and animal cell-free extracts.

Nucleic Acids Res, 2000 Nov 1, 28(21), 4166 - 71
An imperfect inverted repeat is critical for DNA binding of the response regulator RegR of Bradyrhizobium japonicum; Emmerich R et al.; RegR is the response regulator of the RegSR two-component regulatory system in Bradyrhizobium japonicum . The only target known so far is the fixR-nifA operon, encoding the redox-responsive transcription factor NifA, which activates many genes required for symbiotic nitrogen fixation in soybean nodules . In previous in vivo studies, we identified a 32 bp upstream activating sequence located around position -68, which is essential for RegR-dependent expression of the fixR-nifA operon . Here, we used an in vitro binding-site selection assay (SELEX) to more precisely define the DNA-binding specificity of RegR . The selected sequences comprised an imperfect inverted repeat (GCGGC-N(5)-GTCGC) which is highly similar to an imperfect inverted repeat in the fixR UAS (GCGAC-N(5)-GACGC) . In a parallel approach, band-shift experiments were performed with oligonucleotides comprising defined point or deletion mutations in the fixR UAS . This led to the identification of 11 critical nucleotides within a 17 bp minimal RegR binding site centered at position -64 upstream of the fixR-nifA transcription start site . Notably, all 11 critical nucleotides were located either within the half sites of the inverted repeat (four nucleotides in each half site) or in the 5 bp spacer that separates the half sites (three nucleotides) . Based on these results, we defined a DNA motif comprising those nucleotides that are critical for RegR binding (RegR box; 5'-GNG(A)(G)C(A)(G)TTNNGNCGC-3') . A comparison of the RegR box with functional binding sites of the RegR-like regulator RegA of Rhodobacter capsulatus revealed considerable similarities . Thus, the RegR box may assist in the identification of new RegR target genes not only in B.japonicum but also in other alpha-proteobacteria possessing RegR-like response regulators.

Extremophiles, 2000 Oct, 4(5), 267 - 74
Genetic diversity analysis of Rhodothermus reflects geographical origin of the isolates; Petursdottir SK et al.; The genetic diversity and relationships of 81 Rhodothermus isolates from different geothermal environments in Iceland were examined by analysis of electrophoretically demonstrable allelic variation of 13 genes encoding enzymes . All the enzymes were polymorphic . A total of 71 distinctive multilocus genotypes (electrophoretic types, ETs) were identified . The mean genetic diversity per locus (H1) was 0.586 . The relatively high genetic variance observed within Rhodothermus isolates from different locations is most likely the result of genetic changes occurring independently in the locations studied . A high Gst value (0.284) indicates that a considerable part of the variance observed is due to differences between locations . Cluster analysis revealed two major groups of ET clusters diverging at a genetic distance of 0.75, reflecting strongly the geographic origin of isolates . Estimation of the association index (I(A)) indicates that Rhodothermus marinus is a clonal species in which recombination events occur rarely . Partial or whole sequencing of the 16S rRNA genes of Rhodothermus isolates grouping at genetic distance of 0.40 confirmed that all the isolates belonged to the species Rhodothermus marinus . The results of this study confirm that, despite phylogenetic and phenotypic similarity, genetic diversity within Rhodothermus marinus is quite high.

Chemosphere, 2000 Nov, 41(10), 1643 - 9
Toxicology of benzyl alcohols: a QSAR analysis; Kapur S et al.; There is an evidence that benzyl alcohols may exhibit toxicity via a radical mechanism . To test this possibility, we studied the toxicity of para substituted benzyl alcohols on rapidly dividing cancer cells (L1210 leukemia) . This system has previously found utility in studying the apparent radical toxicity of a variety of phenols . However, no evidence could be found for an electronic effect and the cellular toxicity was associated primarily with hydrophobicity . Comparison of this quantitative structure-activity relationships (QSAR) with others for the reactions of benzyl alcohols in diverse systems provides insight into mechanisms of action . A QSAR for the interaction of benzyl alcohols with protozoa yields an equation that is dependent on both hydrophobicity and acidity of the OH group versus a mixture of bacteria and fungi, the critical dependence on hydrophobicity prevails with a small dependence on a resonance-stabilized, radical mediated electronic effect . The chloramphenicols provide an instructive example, where the radical mediated electronic effect overshadows the hydrophobic contribution to bacterial toxicity . These various QSAR for benzyl alcohols indicate that mechanisms of growth inhibition in vitro vary depending on cell/organism type, the strength of the bond and lability of the hydrogen, and the strength of the initiating radical reagent.

Yi Chuan Xue Bao, 2000, 27(6), 556 - 62
{FruA, a transcription factor in Myxococus xamthus, regulates transcription of target genes in collaboration with the associated protein FruB}; Mao XH et al.; Many developmental genes are regulated by FruA, a transcription factor essential for the development of Myxococus xamthus . Another protein, designated FruB, was purified from myxobacteria by its affinity to FruA . FruB could be phosphorylated by protein kinase(s) located in cell membrane . Gel shift assay showed that FruA regulates transcription of target genes in collaboration with phosphorylated FruB . This study may shed light on the molecular mechanisms of regulatory network involved in the development of Myxococus xamthus.

Biotechniques . 2000 Oct;29(4):800, 802, 804, 806 passim.
Fluorescent protein vector for directional selection of PCR clones; Dabrowski S et al.; Green fluorescent protein (GFP) has become a convenient and versatile tool as a reporter protein in many aspects of science . Here, we show that the enhanced yellow fluorescent protein (EYFP) variant may be used advantageously as a reporter system for directional cloning of blunt-ended PCR products . We have constructed a pUC18-derived plasmid containing a reporter gene coding EYFP cloned into the BamHI/HindIII sites . The blunt-ended PCR product is cloned into the SmaI site of that plasmid . A reverse PCR primer must be designed with extra bases on the 5' end that are required to introduce a ribosome binding site (rbs) for EYFP expression . The reporter gene coding EYFP is not expressed unless an rbs is introduced in the proper orientation at the 3' end of the cloned PCR insert . The results of this cloning procedure may be analyzed by simple visual inspection using a transilluminator . In most cases, successful directional cloning results in white fluorescent colonies . The proposed procedure is a convenient method that can reduce the time- and labor-intensive analysis of the clones obtained during blunt-ended PCR product cloning.

FEBS Lett, 2000 Oct 27, 484(1), 29 - 32
The N-terminal domain of the Caulobacter crescentus CgtA protein does not function as a guanine nucleotide exchange factor; Lin B et al.; The Caulobacter crescentus GTP binding protein CgtA is a member of the Obg/GTP1 subfamily of monomeric GTP binding proteins . In vitro, CgtA displays moderate affinity for both GDP and GTP, and rapid exchange rate constants for either nucleotide . One possible explanation for the observed rapid guanine nucleotide exchange {corrected} rates is that CgtA is a bimodal protein with a C-terminal GTP binding domain and an N-terminal GEF domain . In this study we demonstrate that although the N-terminus of CgtA is required for function in vivo, this domain plays no significant role in the guanine nucleotide binding, exchange or GTPase activity.

Neuron, 2000 Sep, 27(3), 447 - 59
A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons; Kuner T et al.; We constructed a novel optical indicator for chloride ions by fusing the chloride-sensitive yellow fluorescent protein with the chloride-insensitive cyan fluorescent protein . The ratio of FRET-dependent emission of these fluorophores varied in proportion to the concentration of Cl and was used to measure intracellular chloride concentration ({Cl-}i) in cultured hippocampal neurons . {Cl-}i decreased during neuronal development, consistent with the shift from excitation to inhibition during maturation of GABAergic synapses . Focal activation of GABAA receptors caused large changes in {Cl-}i that could underlie use-dependent depression of GABA-dependent synaptic transmission . GABA-induced changes in somatic {Cl-}i spread into dendrites, suggesting that {Cl-}i can signal the location of synaptic activity . This genetically encoded indicator will permit new approaches ranging from high-throughput drug screening to direct recordings of synaptic Cl- signals in vivo.

Yi Chuan Xue Bao, 2000, 27(8), 742 - 50
{Improvement of nitrogen fixation efficiency and plasmid stability in Bradyrhizobium japonicum by the introduction of dctABD and parCBA/DE genes}; Li YG et al.; A recombinant plasmid pHN207 containing C4-dicarboxylic acid transport genes (dctABD) from Sinorhizobium meliloti, parCBA/DE genes from pTR102 and reporter genes luxAB from pDB30 was constructed by using pLAFR3 as the vector . The pHN207 was then introduced into the Bradyrhizobium japonicum TA11 and CB1809 by bi-parental mating . It was confirmed that parCBA/DE genes could increase the stability of pLAFR3 in the transconjugants under both free-living and symbiotic condition . The results of plant pot experiment indicated that the introduction of dctABD genes could significantly improve the symbiotic nitrogen fixation efficiency of TA11 and CB1809 with soybean varieties of Heilong 33, Ningzhen No . 1 and Yudou No . 1 . Compared with the control, the shoot dry weight (biomass) and total nitrogen content of the plants tested were significantly increased.

J Endocrinol, 2000 Nov, 167(2), 247 - 52
Regulation of rat magnocellular neurosecretory system by D-aspartate: evidence for biological role(s) of a naturally occurring free D-amino acid in mammals; Wang H et al.; Little evidence is available for the physiological function of D-amino acids in species other than bacteria . Here we demonstrate that naturally occurring freed -aspartate (D-Asp) is present in all magnocellular neurons of rat hypothalamus . The levels of this naturally occurring D-amino acid were elevated during lactation and returned to normal thereafter in the magnocellular neurosecretory system, which produces oxytocin, a hormone responsible for milk ejection during lactation . Intraperitoneal injections of D-Asp reproducibly increased oxytocin gene expression and decreased the concentration of circulating oxytocin in vivo . Similar changes were observed in the vasopressin system . These results provide evidence for the role(s) of naturally occurring free D-Asp in mammalian physiology . The findings argue against the conventional concept that only L-stereoisomers of amino acids are functional in higher species.

ORL J Otorhinolaryngol Relat Spec, 2000 Nov-Dec, 62(6), 335 - 7
Antrolithiasis in the frontal sinus; Mori S et al.; A very rare case of sinusitis with antrolithiasis in the left frontal sinus of a 63-year-old male patient is reported . Various conservative treatments had no effect on the decrease of his left frontal pain and of postnasal drip . Neither bacteria nor fungus were detected in the discharge . Computed tomographic scanning revealed several high-dense spots in an isodense shadow in the left frontal sinus . At first, endoscopic sinus surgery (ESS) was employed and a stony mass was detected in the nasofrontal duct and sinus . Patency of the nasofrontal duct was insured and a sticky paste and small masses were removed as well as possible . However, the flow of discharge from the frontal duct continued after surgery . We performed a second operation with extranasal approach and additional stones in the sinus were successfully removed . Most cases of antrolithiasis are caused by a foreign body or caseous sinusitis with fungus . The maxillary sinus is the most common site of disease in antrolithiasis . It is unknown why the present case of antrolithiasis was in the frontal sinus . In such cases of antrolithiasis or cases having pastelike contents in the frontal sinuses, we conclude that ESS may be an unsuccessful treatment and a classical surgical approach may be required .

Dig Surg, 2000, 17(4), 379 - 87; discussion 387-9
Effects of N-acetylcysteine on pulmonary macrophage activity after intestinal ischemia and reperfusion in rats / with invited commentaries; Borjesson A et al.; BACKGROUND/AIMS: Intestinal ischemia and reperfusion (I/R) is considered to be a critical and triggering event in the development of distal organ dysfunction after a variety of insults . It appears that activated leukocytes, especially polymorphonuclear granulocytes (PMNs), and reactive oxygen species are important mediators in the process . In the present study, the aim was to evaluate the behavior of pulmonary macrophages, acute lung injury and pulmonary endothelial permeability after intestinal I/R, together with potential alterations in pulmonary endothelial and epithelial ultrastructure and cellular membrane system integrity . METHODS: Intestinal ischemia for 40 min was followed by reperfusion for 12 h in the rat . Macrophage uptake of radiolabeled bacteria, levels of pulmonary blood content assessed by radiolabeled red blood cells and pulmonary endothelial permeability of radiolabeled albumin, as well as pulmonary endothelial and epithelial ultrastructure and cellular membrane system integrity by the use of scanning electron microscopy and a tracer was evaluated after 12 h reperfusion . Treatment with the free radical scavenger N-acetylcysteine (NAC) administered prior to reperfusion was evaluated . RESULTS: Overactivation of pulmonary macrophages was noted after intestinal I/R, as was a significant decrease in pulmonary blood content . No increase in pulmonary albumin leakage or increase in pulmonary water content was found after intestinal I/R as compared to controls . Treatment with NAC prevented against intestinal I/R-induced overactivation of pulmonary macrophages and a decrease in pulmonary blood content . CONCLUSION: Reactive oxygen species may be involved in the regulation of pulmonary macrophage function and pulmonary circulation after intestinal I/R .

Acta Crystallogr D Biol Crystallogr, 2000 Nov, 56 ( Pt 11), 1432 - 3
Crystallization and preliminary crystallographic study of an extremophile cytochrome c4 from Thiobacillus ferrooxidans; Abergel C et al.; Soluble periplasmic dihaemic cytochrome c(4), of 21 293 Da molecular mass, has been characterized from Thiobacillus ferrooxidans, an acidophilic bacteria . The native cytochrome has been purified from the bacteria using ion-exchange chromatography and crystallized using solution 27 of the Hampton Research Crystal Screen II . The crystals belong to the hexagonal space group P6(2)22 or P6(4)22, with unit-cell parameters a = 101.59, b = 101.59, c = 151.59 A . Frozen crystals diffract to 2.17 A resolution . The MAD method is currently being used (four Fe atoms per asymmetric unit) to solve the protein structure.

J Bacteriol, 2000 Nov, 182(22), 6358 - 65
Detection of mRNA transcripts and active transcription in persistent Mycobacterium tuberculosis induced by exposure to rifampin or pyrazinamide; Hu Y et al.; Mycobacterium tuberculosis can persist in an altered physiological state for many years after initial infection, and it may reactivate to cause active disease . An analogous persistent state, possibly consisting of several different subpopulations of bacteria, may arise during chemotherapy; this state is thought to be responsible for the prolonged period required for effective chemotherapy . Using two models of drug-induced persistence, we show that both microaerophilic stationary-phase M . tuberculosis treated with a high dose of rifampin in vitro and pyrazinamide-induced persistent bacteria in mice are nonculturable yet still contain 16S rRNA and mRNA transcripts . Also, the in vitro persistent, plate culture-negative bacteria incorporate radioactive uridine into their RNA in the presence of rifampin and can rapidly up-regulate gene transcription after the replacement of the drug with fresh medium and in response to heat shock . Our results show that persistent M . tuberculosis has transcriptional activity . This finding provides a molecular basis for the rational design of drugs targeted at persistent bacteria.

Am J Physiol Lung Cell Mol Physiol, 2000 Nov, 279(5), L790 - 8
Complement-mediated host defense in the lung; Watford WT et al.; Complement is a system of plasma proteins that aids in the elimination of pathogens from the body . We hypothesized that there is a functional complement system present in the lung that aids in the removal of pathogens . Western blot analysis revealed complement proteins of the alternative and classical pathways of complement in bronchoalveolar lavage fluids (BALF) from healthy volunteers . Functional classical pathway activity was detected in human BALF, but there was no significant alternative pathway activity in lavage fluid, a finding that correlates with the low level of the alternative pathway protein, factor B, in these samples . Although the classical pathway of complement was functional in lavage fluid, the level of the classical pathway activator C1q was very low . We tested the ability of the lung- specific surfactant proteins, surfactant protein A (SP-A) and surfactant protein D (SP-D), to substitute for C1q in classical pathway activation, since they have structural homology to C1q . However, neither SP-A nor SP-D restored classical pathway activity to C1q-depleted serum . These data suggest that the classical pathway of complement is functionally active in the lung where it may play a role in the recognition and clearance of bacteria.

Am J Physiol Gastrointest Liver Physiol, 2000 Nov, 279(5), G918 - 24
Butyrate upregulates stromelysin-1 production by intestinal mesenchymal cells; Pender SL et al.; Nutritional factors and resident bacteria participate in the pathogenesis of intestinal inflammation . However, the ways in which bacteria and complex diets might modulate matrix metalloproteinase (MMP) production are unknown . We hypothesized that butyrate might enhance production of MMPs, thus amplifying their response to signals in inflammatory conditions . Human mesenchymal cells were incubated with butyrate and then stimulated with cytokines . MMPs and inhibitors were studied by Western blotting and quantitative RT-PCR . Acetylation of histones was examined in Triton X acetic acid-urea gels by PAGE . We showed that butyrate selectively enhanced the protein production and mRNA expression of stromelysin-1 in tumor necrosis factor-alpha- or interleukin-1beta-stimulated mesenchymal cells . Butyrate alone did not induce any change in MMP production or mRNA expression . It increased the acetylation of histones in mesenchymal cells . Furthermore, acetylation of histones (induced by trichostatin A) reproduced the effects of butyrate . Although butyrate is a major source of nutrient for the colonic epithelial cells, it modulates intestinal inflammation through the secretion of stromelysin-1 in stimulated stromal cells via the inhibition of histone deacetylase.

Science, 2000 Oct 27, 290(5492), 791 - 5
A low temperature transfer of ALH84001 from Mars to Earth; Weiss BP et al.; The ejection of material from Mars is thought to be caused by large impacts that would heat much of the ejecta to high temperatures . Images of the magnetic field of martian meteorite ALH84001 reveal a spatially heterogeneous pattern of magnetization associated with fractures and rock fragments . Heating the meteorite to 40 degrees C reduces the intensity of some magnetic features, indicating that the interior of the rock has not been above this temperature since before its ejection from the surface of Mars . Because this temperature cannot sterilize most bacteria or eukarya, these data support the hypothesis that meteorites could transfer life between planets in the solar system.

J Agric Food Chem, 2000 Oct, 48(10), 5074 - 8
Antimutagenic activity of cacao: inhibitory effect of cacao liquor polyphenols on the mutagenic action of heterocyclic amines; Yamagishi M et al.; We investigated the effect of polyphenols derived from cacao liquor on the mutagenic action of heterocyclic amines (HCAs) in vitro and ex vivo . In the Ames test, the cacao liquor polyphenols showed antimutagenic effects in bacteria treated with HCA in the presence of an S-9 mixture; however, they showed less efficacy than quercetin . On the other hand, the cacao liquor polyphenols showed potent antimutagenic activity in bacteria treated with activated forms of HCA, compared with quercetin . We also evaluated the effect of these compounds on enzymatic activation of HCA . They weakly suppressed the production of activated HCA . In the host-mediated assay in mice, a method used to estimate the potential carcinogenicity of chemicals ex vivo, oral administration of the cacao liquor polyphenols, reduced the number of colonies of revertant bacteria recovered from the liver . These data suggest that the cacao liquor polyphenols have an antimutagenic effect not only in vitro, but also ex vivo.

Dig Dis Sci, 2000 Sep, 45(9), 1769 - 73
Helicobacter pylori reduces intracellular glutathione in gastric epithelial cells; Beil W et al.; Helicobacter pylori infection has been associated with stimulation of gastric mucosal reactive oxygen species (ROS) production, and it was postulated that ROS production is due to neutrophil infiltration and activation . The aim of this study was to investigate the direct effect of H . pylori on ROS formation in gastric epithelial cells in vitro . The human gastric cancer cell line HM02 was incubated with H . pylori for 24 hr, and the effects on cell number and the intracellular radical scavenger reduced glutathione (GSH) were assessed . H . pylori caused a concentration-dependent reduction of cellular GSH concentrations over a broad bacteria-to-cell ratio (1.4-42) in the absence of cell necrosis . The radical scavengers MnTBAP (a cell permeable superoxide dismutase) and ebselen provided protection against H . pylori-induced decrease in cellular GSH concentrations . We conclude that H . pylori directly decreases cellular GSH concentrations in gastric epithelial cells . We suggest that this effect is caused by the release of ROS by H . pylori.

Acta Biochim Pol, 2000, 47(2), 469 - 80
Ubiquinone . Biosynthesis of quinone ring and its isoprenoid side chain . Intracellular localization; Szkopinska A; Ubiquinone, known as coenzyme Q, was shown to be the part of the metabolic pathways by Crane et al . in 1957 . Its function as a component of the mitochondrial respiratory chain is well established . However, ubiquinone has recently attracted increasing attention with regard to its function, in the reduced form, as an antioxidant . In ubiquinone synthesis the para-hydroxybenzoate ring (which is the derivative of tyrosine or phenylalanine) is condensed with a hydrophobic polyisoprenoid side chain, whose length varies from 6 to 10 isoprene units depending on the organism . para-Hydroxybenzoate (PHB) polyprenyltransferase that catalyzes the condensation of PHB with polyprenyl diphosphate has a broad substrate specificity . Most of the genes encoding (all-E)-prenyltransferases which synthesize polyisoprenoid chains, have been cloned . Their structure is either homo- or heterodimeric . Genes that encode prenyltransferases catalysing the transfer of the isoprenoid chain to para-hydroxybenzoate were also cloned in bacteria and yeast . To form ubiquinone, prenylated PHB undergoes several modifications such as hydroxylations, O-methylations, methylations and decarboxylation . In eukaryotes ubiquinones were found in the inner mitochondrial membrane and in other membranes such as the endoplasmic reticulum, Golgi vesicles, lysosomes and peroxisomes . Still, the subcellular site of their biosynthesis remains unclear . Considering the diversity of functions of ubiquinones, and their multistep biosynthesis, identification of factors regulating their cellular level remains an elusive task.

Immunol Cell Biol, 2000 Oct, 78(5), 496 - 501
Special feature for the Olympics: effects of exercise on the immune system: exercise effects on systemic immunity; Nieman DC; Heavy exertion has acute and chronic influences on systemic immunity . In the resting state, the immune systems of athletes and non-athletes are more similar than disparate with the exception of NK cell activity, which tends to be elevated in athletes . Many components of the immune system exhibit adverse change after prolonged, heavy exertion . These immune changes occur in several compartments of the immune system and body (e.g . the skin, upper respiratory tract mucosal tissue, lung, blood and muscle) . Although still open to interpretation, most exercise immunologists believe that during this 'open window' of impaired immunity (which may last between 3 and 72 h, depending on the immune measure) viruses and bacteria may gain a foothold, increasing the risk of subclinical and clinical infection . The infection risk may be amplified when other factors related to immune function are present, including exposure to novel pathogens during travel, lack of sleep, severe mental stress, malnutrition or weight loss.

Curr Opin Microbiol, 2000 Oct, 3(5), 481 - 6
Origins of hydrogenosomes and mitochondria; Rotte C et al.; Complete genome sequences for many oxygen-respiring mitochondria, as well as for some bacteria, leave no doubt that mitochondria are descendants of alpha-proteobacteria, a finding for which the endosymbiont hypothesis can easily account . Yet a wealth of data indicate that mitochondria and hydrogenosomes - the ATP-producing organelles of many anaerobic protists - share a common ancestry, a finding that traditional formulations of the endosymbiont hypothesis less readily accommodates . Available evidence suggests that a more in-depth understanding of the origins of eukaryotes and their organelles will hinge upon data from the genomes of protists that synthesize ATP without the need for oxygen.

Trends Genet, 2000 Oct, 16(10), 461 - 8
It takes two transposons to tango: transposable-element-mediated chromosomal rearrangements; Gray YH; Transposable elements (TEs) promote various chromosomal rearrangements more efficiently, and often more specifically, than other cellular processes(1-3) . One explanation of such events is homologous recombination between multiple copies of a TE present in a genome . Although this does occur, strong evidence from a number of TE systems in bacteria, plants and animals suggests that another mechanism - alternative transposition - induces a large proportion of TE-associated chromosomal rearrangements . This paper reviews evidence for alternative transposition from a number of unrelated but structurally similar TEs . The similarities between alternative transposition and V(D)J recombination are also discussed, as is the use of alternative transposition as a genetic tool.

Proc Natl Acad Sci U S A, 2000 Oct 24, 97(22), 11996 - 2001
Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine); Heikal AA et al.; Gene expression of intrinsically fluorescent proteins in biological systems offers new noninvasive windows into cellular function, but optimization of these probes relies on understanding their molecular spectroscopy, dynamics, and structure . Here, the photophysics of red fluorescent protein (dsRed) from discosoma (coral), providing desired longer emission/absorption wavelengths, and an improved yellow fluorescent protein mutant (Citrine) (S65G/V68L/Q69 M/S72A/T203Y) for significant comparison, are characterized by using fluorescence correlation spectroscopy and time-correlated single-photon counting . dsRed fluorescence decays as a single exponential with a 3.65 +/- 0.07-ns time constant, indicating a single emitting state/species independent of pH 4.4-9.0, in contrast with Citrine . However, laser excitation drives reversible fluorescence flicker at 10(3)-10(4) Hz between dark and bright states with a constant partition fraction f(1) = 0.42 +/- 0.06 and quantum yield of approximately 3 x 10(-3) . Unlike Citrine (pKa approximately 5.7), pH-dependent proton binding is negligible (pH 3 . 9-11) in dsRed . Time-resolved anisotropy of dsRed reveals rapid depolarization (211 +/- 6 ps) plus slow rotational motion (53 +/- 8 ns), in contrast with a single rotational time (16 +/- 2 ns) for Citrine . The molecular dimensions, calculated from rotational and translational diffusion, indicate that dsRed is hydrodynamically 3.8 +/- 0.4 times larger than predicted for a monomer, which suggests an oligomer (possibly a tetramer) configuration even at approximately 10(-9) M . The fast depolarization is attributed to intraoligomer energy transfer between mobile nonparallel chromophores with the initial anisotropy implying a 24 +/- 3 degrees depolarization angle . Large two-photon excitation cross sections ( approximately 100 GM at 990 nm for dsRed and approximately 50 GM at 970 nm for Citrine), advantageous for two-photon-fluorescence imaging in cells, are measured.

J Immunol, 2000 Nov 1, 165(9), 4957 - 63
TGF-beta 1 and IFN-gamma direct macrophage activation by TNF-alpha to osteoclastic or cytocidal phenotype; Fox SW et al.; TNF-related activation-induced cytokine (TRANCE; also called receptor activator of NF-kappaB ligand (RANKL), osteoclast differentiation factor (ODF), osteoprotegerin ligand (OPGL), and TNFSF11) induces the differentiation of progenitors of the mononuclear phagocyte lineage into osteoclasts in the presence of M-CSF . Surprisingly, in view of its potent ability to induce inflammation and activate macrophage cytocidal function, TNF-alpha has also been found to induce osteoclast-like cells in vitro under similar conditions . This raises questions concerning both the nature of osteoclasts and the mechanism of lineage choice in mononuclear phagocytes . We found that, as with TRANCE, the macrophage deactivator TGF-beta(1) strongly promoted TNF-alpha-induced osteoclast-like cell formation from immature bone marrow macrophages . This was abolished by IFN-gamma . However, TRANCE did not share the ability of TNF-alpha to activate NO production or heighten respiratory burst potential by macrophages, or induce inflammation on s.c . injection into mice . This suggests that TGF-beta(1) promotes osteoclast formation not only by inhibiting cytocidal behavior, but also by actively directing TNF-alpha activation of precursors toward osteoclasts . The osteoclast appears to be an equivalent, alternative destiny for precursors to that of cytocidal macrophage, and may represent an activated variant of scavenger macrophage.

J Pediatr Gastroenterol Nutr, 2000 Oct, 31(4), 433 - 8
Lactose-{13C}ureide breath test: a new, noninvasive technique to determine orocecal transit time in children; Van Den Driessche M et al.; BACKGROUND: The lactose-{13C}ureide breath test (LUBT) is a novel, noninvasive test to determine orocecal transit time . Lactose ureide resists the action of brush border enzymes and is metabolized by colonic bacteria . The purpose of the present study was to adapt this breath test for children of various age groups and to determine whether it can be applied in infants, newborns, and preterms to study the development of small intestinal motility . METHODS: In a group of 20 children (3-17 years) in vitro stool analysis and in vivo LUBT results were compared . From each subject a blank stool sample and a sample produced after induction with unlabeled lactose ureide were incubated with 10 mg lactose-{13C}ureide in small, closed bottles . Ten-milliliter CO2 samples were aspirated from the bottles using a needle and a syringe every 30 minutes for 24 hours . All children performed the breath test after induction of 500 mg unlabeled lactose ureide three times the prior day . A liquid test meal (chocolate milk) with 250 mg lactose-{13C}ureide was ingested . Breath samples were collected every 15 minutes for 10 hours . In a second group of 32 children (age range, 0-3 years) consisting of 6 children between 1 and 3 years of age, 6 infants between 6 and 12 months, 13 infants between 0 and 6 months, and 7 preterm infants, only the in vitro stool analysis was performed . Stools were collected for stool incubation, as described . RESULTS: The mean orocecal transit time in the group of 20 children aged 3 to 17 years was 255 minutes (range, 165-390 minutes) . Stool incubations demonstrated a clear 13CO2 peak in all infants aged more than 8 months, indicating that their colonic bacterial enzymic activity hydrolyses lactose ureide . However, in all infants aged less than 6 months and in preterm infants, the 13CO2 signal was absent, indicating that those subjects were unable to hydrolyze lactose ureide . CONCLUSION: Infants aged less than 6 months do not host the appropriate bacterial enzymic activity for splitting lactose ureide . The authors conclude that the LUBT can be applied in infants aged more than 8 months, after weaning to solid foods, to determine orocecal transit time.

J Microbiol Methods, 2000 Nov, 42(3), 225 - 32
A comparative study on the disintegration of filamentous fungi; Taubert J et al.; Different methods for cell disintegration were tested for their efficacy on filamentous fungi, including percussion grinding, homogenization using an Ultra-Turrax, chemical treatment and lyophylization . The release of protein from Ganoderma applanatum and Pycnoporus cinnabarinus and the activity of cytoplasmatic glucose-6-phosphate dehydrogenase in the crude extracts were monitored to determine the efficiency of each disintegration technique used . Fungal cells proved to be particularly resistant towards some disintegration methods commonly used for yeasts and bacteria . Best results were obtained using a percussion grinder, if necessary, in combination with an Ultra-Turrax pretreatment.

Biochem Soc Trans, 2000 Oct, 28(5), 545 - 50
Serine proteases of the complement system; Sim RB et al.; The complement system in blood plasma is a major mediator of innate immune defence . The function of complement is to recognize, then opsonize or lyse, particulate materials, including bacteria, yeasts and other microrganisms, host cell debris and altered host cells . Recognition occurs by binding of complement proteins to charge or saccharide arrays . After recognition, a series of serine proteases is activated, culminating in the assembly of complex unstable proteases called C3/C5 convertases . These activate the complement protein C3, which acts as an opsonin . The complement serine proteases include the closely related C1r, C1s, MASPs 1-3 (80-90 kDa), C2 and Factor B (100 kDa), Factor D (25 kDa) and Factor I (85 kDa) . Each of these has unusually restricted specificity and low enzymic activity . The C1r, C1s and MASP group occur as proenzymes . When activated, they are regulated, like many plasma serine proteases, by a serpin, C1-inhibitor . C2 and Factor B, however, have complex multiple regulation by a group of complement proteins called the Regulation of Complement Activation (or RCA) proteins, whereas Factors I and D appear to have no natural inhibitors . Advances in structure determination and protein-protein interaction properties are leading to a more detailed understanding of the complement-system proteases, and are indicating possible new routes for potential therapeutic control of complement.

Br J Surg, 2000 Oct, 87(10), 1346 - 51
Interleukin 10-deficient colitis: new similarities to human inflammatory bowel disease; Kennedy RJ et al.; BACKGROUND: Interleukin (IL) 10 is a potent anti-inflammatory cytokine . Disruption of the IL-10 gene in C57/Black6 mice results in enterocolitis in the presence of intestinal bacteria . This study investigated gut mucosal barrier function sequentially during the development of colitis in this model . METHODS: Animals were bred in specific pathogen-free conditions and transferred to conventional housing at 4 weeks . Mice were evaluated at 6, 8, 10, 12, 14 and 15 weeks of age . Barrier function was assessed by measuring intestinal permeability and antibody response to systemic endotoxaemia (antibody to the core glycolipid region of lipopolysaccharide; EndoCAb) . Colons were harvested and a histological injury score (HIS) was calculated . RESULTS: The HIS increased progressively until 12 weeks, with an associated increase in intestinal permeability, and immunoglobulin (Ig) M and IgG EndoCAb . The HIS correlated positively with both intestinal permeability and IgM and IgG EndoCAb . Intestinal permeability showed a positive correlation with EndoCAb . CONCLUSION: IL-10 knockout mice develop colitis with an associated disturbance in gut mucosal barrier function, as measured by increased permeability and endotoxaemia . The colitis found in the IL-10 knockout mouse shares these histological, physiological and biochemical features with human inflammatory bowel disease and is therefore suitable for therapeutic trials . A measure of endotoxaemia correlated directly with intestinal permeability in this model.

Eur J Cell Biol, 2000 Sep, 79(9), 621 - 30
The receptor tyrosine phosphatase-like protein ICA512 binds the PDZ domains of beta2-syntrophin and nNOS in pancreatic beta-cells; Ort T et al.; Islet cell autoantigen (ICA) 512 of type I diabetes is a receptor tyrosine phosphatase-like protein associated with the secretory granules of neurons and endocrine cells including insulin-secreting beta-cells of the pancreas . Here we show that in a yeast two-hybrid assay its cytoplasmic domain binds beta2-syntrophin, a modular adapter which in muscle cells interacts with members of the dystrophin family including utrophin, as well as the signaling molecule neuronal nitric oxide synthase (nNOS) . The cDNA isolated by two-hybrid screening corresponded to a novel beta2-syntrophin isoform with a predicted molecular mass of 28 kDa . This isoform included the PDZ domain, but not the C-terminal region, which in full-length beta2-syntrophin is responsible for binding dystrophin-related proteins . In vitro binding of the beta2-syntrophin PDZ domain to ICA512 required both ICA512's C-terminal region and an internal polypeptide preceding its tyrosine phosphatase-like domain . Immunomicroscopy and co-immunoprecipitations from insulinoma INS-1 cells confirmed the occurrence of ICA512-beta2-syntrophin complexes in vivo . ICA512 also interacted in vitro with the PDZ domain of nNOS and ICA512-nNOS complexes were co-immunoprecipitated from INS-1 cells . Finally, we show that INS-1 cells, like muscle cells, contain beta2-syntrophin-utrophin oligomers . Thus, we propose that ICA512, through beta2-syntrophin and nNOS, links secretory granules with the actin cytoskeleton and signaling pathways involving nitric oxide.

Am J Phys Anthropol, 2000 Nov, 113(3), 293 - 304
Molecular evidence for different stages of tuberculosis in ancient bone samples from Hungary; Haas CJ et al.; This paleomicrobiologic study was conducted on osseous tissue specimens from ancient Hungarian skeletal samples from the 7-8th and the 17th centuries AD with typical macromorphologic evidence of osseous tuberculosis (n = 3), morphologic alterations probably due to tuberculosis (n = 6), or with nontypical osseous changes of vertebral bodies suggestive of inflammatory reaction (n = 5) . From these bone samples, DNA was extracted and amplified by polymerase chain reaction (PCR) by using various primer pairs recognizing DNA segments of different mycobacterial species . To confirm specificity of the analysis, the amplification products of several samples were subjected to restriction enzyme digestion and/or direct sequencing . Of the analyzed 14 cases, 8 were unambiguously positive for mycobacterial DNA of the Mycobacterium tuberculosis complex, as shown by the amplification of the IS6110 sequence . In 13 cases we found a PCR product with primers specific for the 65-kDa antigen gene, including 2 cases without genomic DNA . We conclude that the application of other mycobacterial DNA primers may reveal contamination of bones with atypical saprophytic mycobacteria . A positive result for typical mycobacteria was seen in 2 of 3 cases with typical morphologic signs of tuberculosis and amplifiable DNA, in 3 of 6 probable cases, but also in 3 of 6 cases with nontypical bone changes . This indicates that minor osseous reactions of the surface of vertebral bodies may be due-at least in several cases-to infections with bacteria of the M . tuberculosis complex . In these cases the disease may have proceeded rapidly, and the morphologic osseous changes may represent "early" stages of tuberculous infection of the vertebrae .

Trends Endocrinol Metab, 2000 Nov, 11(9), 368 - 75
RAGE: a new target for the prevention and treatment of the vascular and inflammatory complications of diabetes; Schmidt AM et al.; Although the underlying causes of hyperglycemia are multiple, a common thread associated with high levels of blood sugar is the development of a range of vascular and inflammatory complications that might seriously limit the quality and duration of life in affected individuals . Despite multiple aggressive efforts to prevent complications, diabetes remains the leading disease consuming healthcare dollars in the USA . This review focuses on the role of advanced glycation endproducts (AGEs) and their interaction with their signal-transduction AGE receptor (RAGE), in vascular and inflammatory cell perturbation and the chronic activation that underlies diabetes . Our studies provide mechanistic insights into complications within the macrovasculature and those ensuing from an exaggerated host response to invading bacteria, and suggest that blockade of RAGE might provide a potent and safe strategy for the prevention of complications that typify long-term diabetes.

Genome Res, 2000 Oct, 10(10), 1468 - 84
Biosynthesis of isoprenoids via mevalonate in Archaea: the lost pathway; Smit A et al.; Isoprenoid compounds are ubiquitous in living species and diverse in biological function . Isoprenoid side chains of the membrane lipids are biochemical markers distinguishing archaea from the rest of living forms . The mevalonate pathway of isoprenoid biosynthesis has been defined completely in yeast, while the alternative, deoxy-D-xylulose phosphate synthase pathway is found in many bacteria . In archaea, some enzymes of the mevalonate pathway are found, but the orthologs of three yeast proteins, accounting for the route from phosphomevalonate to geranyl pyrophosphate, are missing, as are the enzymes from the alternative pathway . To understand the evolution of isoprenoid biosynthesis, as well as the mechanism of lipid biosynthesis in archaea, sequence motifs in the known enzymes of the two pathways of isoprenoid biosynthesis were analyzed . New sequence relationships were detected, including similarities between diphosphomevalonate decarboxylase and kinases of the galactokinase superfamily, between the metazoan phosphomevalonate kinase and the nucleoside monophosphate kinase superfamily, and between isopentenyl pyrophosphate isomerases and MutT pyrophosphohydrolases . Based on these findings, orphan members of the galactokinase, nucleoside monophosphate kinase, and pyrophosphohydrolase families in archaeal genomes were evaluated as candidate enzymes for the three missing steps . Alternative methods of finding these missing links were explored, including physical linkage of open reading frames and patterns of ortholog distribution in different species . Combining these approaches resulted in the generation of a short list of 13 candidate genes for the three missing functions in archaea, whose participation in isoprenoid biosynthesis is amenable to biochemical and genetic investigation.

Plant Cell, 2000 Oct, 12(10), 1917 - 26
HRT gene function requires interaction between a NAC protein and viral capsid protein to confer resistance to turnip crinkle virus; Ren T et al.; An Arabidopsis protein was found to interact specifically with the capsid protein (CP) of turnip crinkle virus (TCV) through yeast two-hybrid screening . This protein, designated TIP (for TCV-interacting protein), was found to be a member of the recently recognized NAC family of proteins . NAC proteins have been implicated in the regulation of development of plant embryos and flowers . TIP alone was able to activate expression of reporter genes in yeast if fused to a DNA binding domain, suggesting that it may be a transcriptional activator . The TIP binding region in the TCV CP has been mapped to the N-terminal 25 amino acids . Site-directed mutagenesis within this region revealed that loss of the TIP-CP interaction in the yeast two-hybrid assay correlated with loss of the ability of TCV to induce the hypersensitive response and resistance in the TCV-resistant Arabidopsis ecotype Dijon (Di-0 and its inbred line Di-17) . These data suggest that TIP is an essential component in the TCV resistance response pathway.

Biochemistry, 2000 Oct 24, 39(42), 12778 - 88
Mapping the binding of synthetic disaccharides representing epitopes of chlamydial lipopolysaccharide to antibodies with NMR; Maaheimo H et al.; A NMR study of the binding of the synthetic disaccharides alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl 1 (Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid) and alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl 2, representing partial structures of the lipopolysaccharide epitope of the intracellular bacteria Chlamydia, to corresponding monoclonal antibodies (mAbs) S23-24, S25-39, and S25-2 is presented . The conformations of 1 bound to mAbs S25-39 and of 2 bound to mAbs S23-24 and S25-39 were analyzed by employing transfer-NOESY (trNOESY) and QUIET-trNOESY experiments . A quantitative analysis of QUIET-trNOESY buildup curves clearly showed that S25-39 recognized a conformation of 1 that was similar to the global energy minimum of 1, and significantly deviated from the conformation of 1 bound to mAb S25-2 . For disaccharide 2, only a qualitative analysis was possible because of severe spectral overlap . Nevertheless, the analysis showed that all mAbs most likely bound to only one conformational family of 2 . Saturation transfer difference (STD) NMR experiments were then employed to analyze the binding epitopes of the disaccharide ligands 1 and 2 when binding to mAbs S23-24, S25-39, and S25-2 . It was found that the nonreducing pyranose unit was the major binding epitope, irrespective of the mAb and the disaccharide that were employed . Individual differences were related to the engagement of other portions of the disaccharide ligands.

Hum Reprod, 2000 Jul, 15 Suppl 2, 18 - 27
Genetic control of oxidative phosphorylation and experimental models of defects; Trounce I; Energy in the form of ATP is continually produced by all cells for normal growth and function . Anaerobic glycolysis can provide enough ATP for some cells, but energetic cells such as cardiomyocytes and neurons require a more efficient ATP supply, which can only be provided by mitochondrial oxidative phosphorylation . Invented by bacteria that became symbiotically associated with other bacteria to form eukaryotic cells billions of years ago, oxidative phosphorylation carries with it a genetic legacy that is unique . The mitochondrial oxidative phosphorylation complexes are assembled from protein subunits encoded by both the mitochondrial genome (mtDNA) and the nuclear genome (nDNA, located in the chromosomes) . The mtDNA is a remnant genome of the bacterial progenitor of mitochondria, and (unlike the biparental diploidy that characterizes the nuclear genome) is present in thousands of copies per cell, is replicated through life, and is inherited (cytoplasmically) only from the female parent . Oxidative phosphorylation comprises five multimeric enzyme complexes that act as a redox pathway, passing electrons from oxidizable intermediates produced by the metabolism of food to molecular oxygen in the mitochondrial matrix, while producing an electrochemical gradient by pumping protons into the intermembranal space . The proton (hydrogen ion) gradient across the inner mitochondrial membrane is used by the H+-transporting ATP synthase to produce ATP from ADP and inorganic phosphate, with the protons released into the mitochondrial matrix then combining with electronated oxygen to form water . Many of the details regarding the control of the synthesis of oxidative phosphorylation enzyme complexes remain to be elucidated . Transmitochondrial cell culture systems have been developed so that defective oxidative phosphorylation can be studied in a controlled nuclear background . Such systems may soon enable the development of mtDNA 'knockout' mice in order to better model mtDNA transmission and mitochondrial disease.

Pigment Cell Res, 2000, 13 Suppl 8, 94 - 7
Tanning as part of the eukaryotic SOS response; Eller MS et al.; We have determined that DNA damage is at least one of the signals generated by ultraviolet radiation that stimulates pigmentation (tanning) in human skin . This photoprotective response is functionally similar to the SOS response described in bacteria . Here we present evidence that DNA damage stimulates pigmentation, at least in part, through up-regulation of tyrosinase mRNA and protein levels . Furthermore, this response can be induced in the absence of DNA damage by treatment of melanocytic cells and intact skin with small DNA fragments, particularly thymidine dinucleotides, pTpT . Topical application of these DNA fragments should provide a photoprotective tan to human skin without the harmful effects of ultraviolet radiation.

Arch Microbiol, 2000 Sep, 174(3), 152 - 61
Characterization of novel bacteriochlorophyll-a-containing red filaments from alkaline hot springs in Yellowstone National Park; Boomer SM et al.; Novel red, filamentous, gliding bacteria formed deep red layers in several alkaline hot springs in Yellowstone National Park . Filaments contained densely layered intracellular membranes and bacteriochlorophyll a . The in vivo absorption spectrum of the red layer filaments was distinct from other phototrophs, with unusual bacteriochlorophyll a signature peaks in the near-infrared (IR) region (807 nm and 911 nm) . These absorption peaks were similar to the wavelengths penetrating to the red layer of the mats as measured with in situ spectroradiometry . The filaments also demonstrated maximal photosynthetic uptake of radiolabeled carbon sources at these wavelengths . The red layer filaments displayed anoxygenic photoheterotrophy, as evidenced by the specific incorporation of acetate, not bicarbonate, and by the absence of oxygen production . Photoheterotrophy was unaffected by sulfide and oxygen, but was diminished by high-intensity visible light . Near-IR radiation supported photoheterotrophy . Morphologically and spectrally similar filaments were observed in several springs in Yellowstone National Park, including Octopus Spring . Taken together, these data suggest that the red layer filaments are most similar to the photoheterotroph, Heliothrix oregonensis . Notable differences include mat position and coloration, absorption spectra, and prominent intracellular membranes.

J Neurosci Methods, 2000 Jul 31, 100(1-2), 63 - 9
Combined use of the green and yellow fluorescent proteins and fluorescence-activated cell sorting to select populations of transiently transfected PC12 cells; Espinet C et al.; One of the more time-consuming procedures in the study of exogenously expressed proteins in cell lines is the selection of individual transfected clones . In recent years, green fluorescent protein variants with excitation/emission spectra matching the typical flow cytometer configurations have been generated and are in common use . We employed PC12 cells transfected with vectors encoding fluorescent proteins and a fluorescence selection procedure using a fluorescence-activated cell-sorter . In order to select the optimal co-electroporation and sorting conditions, we used the simultaneous detection of two variants of the green fluorescent protein, that possess separable emission peaks when excited at 488 nm . Using these variants and the adequate combination of band-pass filters, we were able to analyze and establish the conditions for identifying and sorting cells transfected with enhanced green fluorescent protein, that simultaneously express another plasmid of interest . Using this procedure, the cells sorted that express both plasmids exceeded 90% . The whole procedure did not alter the physiological responsiveness of the transfected cells to growth factors, and has been successfully applied to the constitutive activation of the mitogen-activated protein kinase pathway, resulting in the spontaneous differentiation of PC12 cells . Also, this procedure has been used with other set of expression vectors encoding proteins that protect PC12 cells from apoptosis caused by different stimuli . The method that we present here provides an easy and fast procedure to obtain a high proportion of positively transfected populations of PC12 cells.

J Mol Evol, 2000 Oct, 51(4), 374 - 7
Addition of wsp sequences to the Wolbachia phylogenetic tree and stability of the classification; Pintureau B et al.; Wolbachia are symbiotic bacteria altering reproductive characters of numerous arthropods . Their most recent phylogeny and classification are based on sequences of the wsp gene . We sequenced wsp gene from six Wolbachia strains infecting six Trichogramma species that live as egg parasitoids on many insects . This allows us to test the effect of the addition of sequences on the Wolbachia phylogeny and to check the classification of Wolbachia infecting Trichogramma . The six Wolbachia studied are classified in the B supergroup . They confirm the monophyletic structure of the B Wolbachia in Trichogramma but introduce small differences in the Wolbachia classification . Modifications include the definition of a new group, Sem, for Wolbachia of T . semblidis and the merging of the two closely related groups, Sib and Kay . Specific primers were determined and tested for the Sem group.

J Mol Evol, 2000 Oct, 51(4), 353 - 62
Are noncoding sequences of Rickettsia prowazekii remnants of "neutralized" genes?
Holste D, Weiss O, Grosse I, Herzel H.
It has been hypothesized that a large fraction of 24% noncoding DNA in R . prowazekii consists of degraded genes . This hypothesis has been based on the relatively high G+C content of noncoding DNA . However, a comparison with other genomes also having a low overall G+C content shows that this argument would also apply to other bacteria . To test this hypothesis, we study the coding potential in sets of genes, pseudogenes, and intergenic regions . We find that the correlation function and the chi(2)-measure are clearly indicative of the coding function of genes and pseudogenes . However, both coding potentials make almost no indication of a preexisting reading frame in the remaining 23% of noncoding DNA . We simulate the degradation of genes due to single-nucleotide substitutions and insertions/deletions and quantify the number of mutations required to remove indications of the reading frame . We discuss a reduced selection pressure as another possible origin of this comparatively large fraction of noncoding sequences.

Arch Dis Child, 2000 Nov, 83(5), 429 - 34
An association between sudden infant death syndrome (SIDS) and Helicobacter pylori infection; Kerr JR et al.; BACKGROUND: Helicobacter pylori has recently been detected in the stomach and trachea of cases of sudden infant death syndrome (SIDS) and proposed as a cause of SIDS . AIMS: To establish the incidence of H pylori in the stomach, trachea, and lung of cases of SIDS and controls . METHODS: Stomach, trachea, and lung tissues from 32 cases of SIDS and eight control cases were examined retrospectively . Diagnosis of SIDS was based on established criteria . Controls were defined by death within 1 year of age and an identifiable cause of death . Tissues were examined histologically for the presence of bacteria . Extracted DNA from these tissues was tested for H pylori ureC and cagA sequences by nested polymerase chain reaction and amplicons detected by enzyme linked immunosorbent assay (ELISA) . The cut off for each ELISA for each of the tissue types was taken as the mean optical density plus two times the standard deviation of a range of negative controls . RESULTS: Ages of SIDS cases ranged from 2 to 28 weeks . Ages of controls ranged from 3 to 44 weeks . For the ureC gene, 25 SIDS cases were positive in one or more tissues compared with one of the controls . For the cagA gene, 25 SIDS cases were positive in one or more tissues compared with one of the controls . CONCLUSIONS: There is a highly significant association between H pylori ureC and cagA genes in the stomach, trachea, and lung of cases of SIDS when compared with controls.

Virology, 2000 Oct 25, 276(2), 404 - 16
The GroEL protein of the whitefly Bemisia tabaci interacts with the coat protein of transmissible and nontransmissible begomoviruses in the yeast two-hybrid system; Morin S et al.; We have previously suggested that a GroEL homolog produced by the whitefly Bemisia tabaci endosymbiotic bacteria interacts in the insect hemolymph with particles of Tomato yellow leaf curl virus from Israel (TYLCV-Is), ensuring the safe circulative transmission of the virus . We have now addressed the question of whether the nontransmissibility of Abutilon mosaic virus from Israel (AbMV-Is) is related to a lack of association between GroEL and the virus coat protein (CP) . Translocation analysis has shown that, whereas TYLCV-Is DNA is conspicuous in the digestive tract, hemolymph, and salivary glands of B . tabaci 8 h after acquisition feeding started, AbMV-Is DNA was detected only in the insect digestive tract, even after 96 h . To determine whether AbMV-Is particles were rapidly degraded in the hemolymph as a result of their inability to interact with GroEL, we have isolated a GroEL gene from B . tabaci and used a yeast two-hybrid assay to compare binding of the CP of TYLCV-Is and AbMV-Is to the insect GroEL . The yeast assay showed that the CPs of the two viruses are able to bind efficiently to GroEL . We therefore suggest that, although GroEL-CP interaction in the hemolymph is a necessary condition for circulative transmission, the nontransmissibility of AbMV-Is is not the result of lack of binding to GroEL in the B . tabaci hemolymph, but most likely results from an inability to cross the gut/hemolymph barrier .

Rev Belge Med Dent, 2000, 55(2), 93 - 103
{Should the dentin smear layer be preserved or eliminated? (Review of the literature)}; Wauters T et al.; The smear layer is a direct consequence of instrumentation of the root canal wall . Hand instrumentation as well as ultrasonic instrumentation produce a smear layer on the canal wall . This smear layer is composed of dentine, remnants of pulp tissue and odontoblastic processes and bacteria in an infected canal . Removal of the smear layer is accomplished by the irrigation of root canals with EDTA (17%) followed by NaOCl (5.25%) . Permeability of the dentine is increased by the removal of smear layer . In this way the bacteria within the infected tubuli can be more easily destroyed by an intracanal dressing . Whether the removal of smear layer decreases the apical leakage is uncertain . To establish the clinical consequences from removal or preservation of the smear layer, further research is needed.

J Dent Res, 1988 Feb, 67(2), 438 - 46
Analysis of the buffering systems in dental plaque; Shellis RP et al.; A semi-micro method was used for investigation of the buffering properties of whole plaque, plaque fluid, and washed plaque bacteria . Artifacts associated with titration of samples containing live bacteria were noted and their effects estimated . All three sample types showed minimal buffering in the region of neutrality, with much stronger buffering in the regions pH 4-5.5 and pH 8-9 . For the range pH 4-7, almost 90% of the total buffer capacity of plaque appeared to be accounted for by macromolecules of bacterial cell walls and plaque matrix . Extracellular buffers in plaque fluid removable by centrifugation contributed up to 11% . These buffers (probably soluble proteins, peptides, organic acids, and phosphate) are, potentially at least, capable of exchange with saliva . In vitro, bicarbonate (dissolved in the extracellular fluid) contributed only 2-5% of total buffering; there was no evidence of formation of carbamino compounds . However, in vivo, salivary bicarbonate may be important as a continually replenished source of additional buffering.

Food Chem Toxicol, 2000 Nov, 38(11), 991 - 5
Inhibition by curcumin of diethylnitrosamine-induced hepatic hyperplasia, inflammation, cellular gene products and cell-cycle-related proteins in rats; Chuang SE et al.; Curcumin (CCM), a major yellow pigment of turmeric obtained from powdered rhizomes of the plant Curcuma longa Linn, is commonly used as coloring agent in foods, drugs and cosmetics . In this study we report that gavage administration of 200 mg/kg or 600 mg/kg CCM effectively suppressed diethylnitrosamine (DEN)-induced liver inflammation and hyperplasia in rats, as evidenced by histopathological examination . Immunoblotting analysis showed that CCM strongly inhibited DEN-mediated the increased expression of oncogenic p21(ras) and p53 proteins in liver tissues of rats . In cell-cycle-related proteins, CCM selectively reduced the expression of proliferating cell nuclear antigen (PCNA), cyclin E and p34(cdc2), but not Cdk2 or cyclin D1 . Moreover, CCM also inhibited the DEN-induced increase of transcriptional factor NF-kappa B . However, CCM failed to affect DEN-induced c-Jun and c-Fos expression . It has become widely recognized that the development of human hepatocellular carcinoma (HCC) is predominantly due to the chronic inflammation by virus, bacteria or chemical . Our results suggest a potential role for CCM in the prevention of HCC.

Laryngoscope, 2000 Oct, 110(10 Pt 1), 1745 - 9
Complement C3 cleavage and cytokines interleukin-1beta and tumor necrosis factor-alpha in otitis media with effusion; Narkio-Makela M et al.; OBJECTIVES: To analyze whether complement C3a anaphylatoxin, other C3 fragments, interleukin-1beta (IL-1beta), or tumor necrosis factor-alpha (TNF-alpha) contributes to inflammation in chronic otitis media with effusion (OME) . METHODS: The amount of C3a was measured by enzyme-linked immunoassay . Further breakdown of C3 was analyzed by Western blotting . IL-1beta and TNF-alpha concentrations were measured by radioimmunoassay . Bacteria were analyzed by culture and polymerase chain reaction . RESULTS: Highly elevated levels of C3a and other C3 cleavage fragments were found in all middle ear effusion (MEE) samples . The mean values (+/- SEM, n = 26) for C3a, IL-1beta, and TNF-alpha were 5,973 +/- 1,124 ng/mL, 1,043 +/- 490 pg/mL, and 79 +/- 14.3 pg/mL, respectively . Comparison to an average C3 level of 555 (+/-108) microg/mL indicated that at least 40.5% +/- 6% of total C3 had become activated within the MEE . C3a concentrations were higher in the group in which the effusion had been present in the middle ear for a prolonged period (> or =4 mo) (P = .04) . Children with multiple tube insertions had higher C3 (P = .006) and TNF-alpha (P = .04) concentrations in their MEE samples than those receiving their first tubes . C3 and C3a concentrations in MEE correlated to each other (correlation coefficient {r} = 0.513, P = .0056), as did concentrations of IL-1beta and TNF-alpha (r = 0.7016, P < .0001) . No significant correlation was found between complement C3 or C3a levels and IL-1beta, TNF-alpha, or bacterial growth . CONCLUSIONS: Highly elevated levels of C3a in MEE indicate ongoing complement activation, which is stronger than in almost any other disease demonstrated previously . Elevated C3a levels contribute to chemotactic and inflammatory potential in the MEE and correlate with the chronicity of the disease.

Sci Total Environ, 2000 Oct 16, 261(1-3), 33 - 41
The use of stable carbon isotopes to evaluate the importance of fine suspended particulate matter in the transfer of methylmercury to biota in boreal flooded environments; Montgomery S et al.; Applying the classic geochemical technique of stable carbon isotope ratios (delta13C), we confirmed the existence of a trophic link between fine particulate matter (FPM) and zooplankton in freshwater ecosystems, and examined possible reasons for the elevated MeHg concentrations ({MeHg}) in hydroelectric reservoirs as compared to natural lakes . Comparing natural and flooded environments, the delta13C and {MeHg} values for FPM and zooplankton differ significantly . Using a mixing model to calculate the contribution of terrestrial carbon to FPM, the differences in delta13C between natural and flooded sites are explained by an increasingly important autochthonous component in reservoirs . The stable isotopic evidence presented here strongly suggests that, despite the much greater abundance of detrital vascular-plant carbon, microalgae are important in supporting aquatic food webs in the oligotrophic flooded systems studied . Due to a significant inverse relationship between {MeHg} in FPM and the percentage of terrestrial carbon (r2 = 0.87), we propose that the higher {MeHg} in the zooplankton of flooded sites as compared to lakes are the result of proportionally higher levels of autochthonous material (algae/bacteria; i.e . potential sources/methylators of Hg) in the FPM of reservoirs.

J Histochem Cytochem, 2000 Nov, 48(11), 1469 - 78
Distribution of group II phospholipase A2 protein and mRNA in rat tissues; Nyman KM et al.; Group II phospholipase A2 (PLA2) is an acute-phase protein and an important component of the host defense against bacteria . In this study we investigated the distribution of PLA2 protein by immunohistochemistry and the distribution of mRNA of PLA2 by Northern blotting and in situ hybridization in rat tissues . PLA2 protein was localized in the Paneth cells of the intestinal mucosa, chondrocytes and the matrix of cartilage, and megakaryocytes in the spleen . By Northern blotting, mRNA of PLA2 was found in the gastrointestinal tract, lung, heart, and spleen . By in situ hybridization, PLA2 mRNA was localized in the Paneth cells of the small intestinal mucosa but in no other cell types . Our results show specific distribution of PLA2 in a limited number of cell types in rat tissues . The reagents developed in this study (the anti-rat PLA2 antibody and probes for Northern blotting and in situ hybridization of mRNA of rat PLA2) will provide useful tools for future studies concerning the role of PLA2 in various experimental disease models.

J Plant Growth Regul, 2000 Jun, 19(2), 167 - 182
The Actinorhizal Symbiosis; Wall LG; The term "actinorhiza" refers both to the filamentous bacteria Frankia, an actinomycete, and to the root location of nitrogen-fixing nodules . Actinorhizal plants are classified into four subclasses, eight families, and 25 genera comprising more than 220 species . Although ontogenically related to lateral roots, actinorhizal nodules are characterized by differentially expressed genes, supporting the idea of the uniqueness of this new organ . Two pathways for root infection have been described for compatible Frankia interactions: root hair infection or intercellular penetration . Molecular phylogeny groupings of host plants correlate with morphologic and anatomic features of actinorhizal nodules . Four clades of actinorhizal plants have been defined, whereas Frankia bacteria are classified into three major phylogenetic groups . Although the phylogenies of the symbionts are not fully congruent, a close relationship exists between plant and bacterial groups . A model for actinorhizal specificity is proposed that includes different levels or degrees of specificity of host-symbiont interactions, from fully compatible to incompatible . Intermediate, compatible, but delayed or limited interactions are also discussed . Actinorhizal plants undergo feedback regulation of symbiosis involving at least two different and consecutive signals that lead to a mechanism controlling root nodulation . These signals mediate the opening or closing of the window of susceptibility for infection and inhibit infection and nodule development in the growing root, independently of infection mechanism . The requirement for at least two molecular recognition steps in the development of actinorhizal symbioses is discussed.

Infect Immun, 2000 Nov, 68(11), 6265 - 72
Modulation of innate cytokine responses by products of Helicobacter pylori; Meyer F et al.; The gastric inflammatory and immune response in Helicobacter pylori infection may be due to the effect of different H . pylori products on innate immune mechanisms . The aim of this study was to determine whether bacterial components could modulate cytokine production in vitro and thus contribute to Th1 polarization of the gastric immune response observed in vivo . The effect of H . pylori recombinant urease, bacterial lysate, intact bacteria, and bacterial DNA on proliferation and cytokine production by peripheral blood mononuclear cells (PBMCs) from H . pylori-negative donors was examined as a model for innate cytokine responses . Each of the different H . pylori preparations induced gamma interferon (IFN-gamma) and interleukin-12p40 (IL-12p40), but not IL-2 or IL-5, production, and all but H . pylori DNA stimulated release of IL-10 . Addition of anti-IL-12 antibody to cultures partially inhibited IFN-gamma production . In addition, each bacterial product inhibited mitogen-stimulated IL-2 production by PBMCs and Jurkat T cells . The inhibitory effect of bacterial products on IL-2 production correlated with inhibition of mitogen-stimulated lymphocyte proliferation, although urease inhibited IL-2 production without inhibiting proliferation, suggesting that inhibition of IL-2 production alone is not sufficient to inhibit lymphocyte proliferation . The results of these studies demonstrate that Th1 polarization of the gastric immune response may be due in part to the direct effects of multiple different H . pylori components that enhance IFN-gamma and IL-12 production while inhibiting both IL-2 production and cell proliferation that may be necessary for Th2 responses.

Infect Immun, 2000 Nov, 68(11), 6101 - 7
NO contributes to proliferative suppression in a murine model of filariasis; O'Connor RA et al.; Infection of BALB/c mice with microfilariae (mf) of Brugia pahangi leads to the suppression of antigen (Ag)-specific proliferative responses in the spleen . The proliferative defect is dependent on inducible nitric oxide synthase (iNOS) activity, since inhibition of iNOS with either L-N-monomethyl arginine (L-NMMA) or aminoguanidine reversed defective proliferation . Splenocytes from mf-infected animals produce high levels of gamma interferon (IFN-gamma) upon in vitro restimulation with Ag, and experiments in IFN-gamma receptor-deficient (IFN-gamma R(-/-)) mice demonstrated that signaling via the IFN-gamma R is essential in the induction of NO production and subsequent proliferative suppression . Restimulation of splenocytes from mf-infected animals with an extract of Acanthocheilonema viteae, a related filarial worm which lacks endosymbiotic bacteria, also resulted in NO production and proliferative suppression, demonstrating that lipopolysaccharide of bacterial origin is not essential to the induction of iNOS activity . These results extend previous observations that infection with different life cycle stages of Brugia leads to the development of differentially polarized immune responses and demonstrate one method by which these differences may exert their effects on the proliferative potential of cells from infected animals.

FEMS Microbiol Lett, 2000 Sep 15, 190(2), 267 - 72
Changes in the virulence of Mycobacterium avium after passage through embryonated hens' eggs; Long EG et al.; Eight-day-old embryonated hen's eggs were used as a model to study Mycobacterium avium virulence . Strains isolated from human patients caused 20-90% mortality when eggs were infected by injection of bacterial suspensions into the amniotic sac . Virulence of examined strains subsequently decreased with passage through eggs to between 0 and 40% mortality in four passages . Virulence of the egg-attenuated strains could be restored by passage through human peripheral blood mononuclear cells . The site of infection in the egg was usually the mesodermal layer of the chorioallantoic membrane . A few small granulomas containing acid-fast bacteria were seen in the liver, but not in other organs . Death of chicken embryos may have resulted from destruction of the mesodermal layer of the chorioallantoic membrane with consequent respiratory failure . PBMCs infected with less virulent egg-passaged strains of M . avium produced higher levels of tumor necrosis factor-alpha than did peripheral blood mononuclear cells infected with more virulent nonpassaged strains.

Nature, 2000 Oct 5, 407(6804), 626 - 9
Modern freshwater microbialite analogues for ancient dendritic reef structures; Laval B et al.; Microbialites are organosedimentary structures that can be constructed by a variety of metabolically distinct taxa . Consequently, microbialite structures abound in the fossil record, although the exact nature of the biogeochemical processes that produced them is often unknown . One such class of ancient calcareous structures, Epiphyton and Girvanella, appear in great abundance during the Early Cambrian . Together with Archeocyathids, stromatolites and thrombolites, they formed major Cambrian reef belts . To a large extent, Middle to Late Cambrian reefs are similar to Precambrian reefs, with the exception that the latter, including terminal Proterozoic reefs, do not contain Epiphyton or Girvanella . Here we report the discovery in Pavilion Lake, British Columbia, Canada, of a distinctive assemblage of freshwater calcite microbialites, some of which display microstructures similar to the fabrics displayed by Epiphyton and Girvanella . The morphologies of the modern microbialites vary with depth, and dendritic microstructures of the deep water (> 30 m) mounds indicate that they may be modern analogues for the ancient calcareous structures . These microbialites thus provide an opportunity to study the biogeochemical interactions that produce fabrics similar to those of some enigmatic Early Cambrian reef structures.

J Clin Periodontol, 2000 Oct, 27(10), 758 - 62
Cytokine, elastase and oxygen radical release by Fusobacterium nucleatum-activated leukocytes: a possible pathogenic factor in periodontitis; Sheikhi M et al.; Periodontitis is characterised by tissue destruction caused by reactive oxygen species (ROS) and proteolytic enzymes, which are released by the interaction between bacteria and phagocytes . We estimated the ability of Fusobacterium species to induce release of tissue destructive and proinflammatory mediators from in vitro activated peripheral leukocytes . ROS was measured with the nitroblue tetrazolium (NBT) method, elastase with a specific chromogenic substrate and cytokines, including interleukin 1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha), and interleukin 8 (IL-8) with a sandwich ELISA method . Various clinical isolates of unopsonized Fusobacterium species stimulated the neutrophils to an increased NBT- reduction . IL-1beta, TNFalpha, IL-8 and elastase were released in significantly higher levels from neutrophils stimulated by Fusobacterium species . In conclusion, unopsonized Fusobacterium species can induce increased production of oxygen radicals, cytokines and elastase from leukocytes activated in vitro.

J Immunol Methods, 2000 Oct 20, 244(1-2), 133 - 7
Real time monitoring of lymphocyte proliferation by an impedance method; Upadhyay P et al.; Impedance methods are routinely used to estimate the concentration of viable bacteria in a culture . We have adapted an impedance method to monitor the growth of lymphocytes and used it to monitor lymphocyte proliferation in real time . In this method lymphocytes were cultured in modified micro-well strips with transparent indium-titanium oxide coated electrodes at the bottom . The assay was totally automated and did not involve handling of radioactive chemicals . As the method uses a non destructive method of recording the proliferation, the cells could be used for other studies after the proliferation assay . Due to the real time monitoring of proliferation, results could be obtained within 30 h and additional information such as the rate of proliferation and the limiting rate at time-->0 could also be calculated instantly.

J Biol Chem, 2001 Jan 12, 276(2), 1417 - 23
A novel superoxide-producing NAD(P)H oxidase in kidney; Shiose A et al.; During phagocytosis, gp91(phox), the catalytic subunit of the phagocyte NADPH oxidase, becomes activated to produce superoxide, a precursor of microbicidal oxidants . Currently increasing evidence suggests that nonphagocytic cells contain similar superoxide-producing oxidases, which are proposed to play crucial roles in various events such as cell proliferation and oxygen sensing for erythropoiesis . Here we describe the cloning of human cDNA that encodes a novel NAD(P)H oxidase, designated NOX4 . The NOX4 protein of 578 amino acids exhibits 39% identity to gp91(phox) with special conservation in membrane-spanning regions and binding sites for heme, FAD, and NAD(P)H, indicative of its function as a superoxide-producing NAD(P)H oxidase . The membrane fraction of kidney-derived human embryonic kidney (HEK) 293 cells, expressing NOX4, exhibits NADH- and NADPH-dependent superoxide-producing activities, both of which are inhibited by diphenylene iodonium, an agent known to block oxygen sensing, and decreased in cells expressing antisense NOX4 mRNA . The human NOX4 gene, comprising 18 exons, is located on chromosome 11q14.2-q21, and its expression is almost exclusively restricted to adult and fetal kidneys . In human renal cortex, high amounts of the NOX4 protein are present in distal tubular cells, which reside near erythropoietin-producing cells . In addition, overexpression of NOX4 in cultured cells leads to increased superoxide production and decreased rate of growth . The present findings thus suggest that the novel NAD(P)H oxidase NOX4 may serve as an oxygen sensor and/or a regulator of cell growth in kidney.

EMBO J, 2000 Oct 16, 19(20), 5300 - 7
Crystal structure of the cell division protein FtsA from Thermotoga maritima; van den Ent F et al.; Bacterial cell division requires formation of a septal ring . A key step in septum formation is polymerization of FtsZ . FtsA directly interacts with FtsZ and probably targets other proteins to the septum . We have solved the crystal structure of FtsA from Thermotoga maritima in the apo and ATP-bound form . FtsA consists of two domains with the nucleotide-binding site in the interdomain cleft . Both domains have a common core that is also found in the actin family of proteins . Structurally, FtsA is most homologous to actin and heat-shock cognate protein (Hsc70) . An important difference between FtsA and the actin family of proteins is the insertion of a subdomain in FtsA . Movement of this subdomain partially encloses a groove, which could bind the C-terminus of FtsZ . FtsZ is the bacterial homologue of tubulin, and the FtsZ ring is functionally similar to the contractile ring in dividing eukaryotic cells . Elucidation of the crystal structure of FtsA shows that another bacterial protein involved in cytokinesis is structurally related to a eukaryotic cytoskeletal protein involved in cytokinesis.

EMBO J, 2000 Oct 16, 19(20), 5288 - 99
Structure of the GAF domain, a ubiquitous signaling motif and a new class of cyclic GMP receptor; Ho YS et al.; GAF domains are ubiquitous motifs present in cyclic GMP (cGMP)-regulated cyclic nucleotide phosphodiesterases, certain adenylyl cyclases, the bacterial transcription factor FhlA, and hundreds of other signaling and sensory proteins from all three kingdoms of life . The crystal structure of the Saccharomyces cerevisiae YKG9 protein was determined at 1.9 A resolution . The structure revealed a fold that resembles the PAS domain, another ubiquitous signaling and sensory transducer . YKG9 does not bind cGMP, but the isolated first GAF domain of phosphodiesterase 5 binds with K:(d) = 650 nM . The cGMP binding site of the phosphodiesterase GAF domain was identified by homology modeling and site-directed mutagenesis, and consists of conserved Arg, Asn, Lys and Asp residues . The structural and binding studies taken together show that the cGMP binding GAF domains form a new class of cyclic nucleotide receptors distinct from the regulatory domains of cyclic nucleotide-regulated protein kinases and ion channels.

Biochem Biophys Res Commun, 2000 Oct 22, 277(2), 436 - 42
Protein translocation within chloroplast is similar in Euglena and higher plants; Inagaki J et al.; It is currently thought that chloroplasts of higher plants were derived from endosymbiont oxygenic photosynthetic bacteria (primary endosymbiosis), while Euglena, a photosynthetic protista, gained chloroplasts by secondary endosymbiosis (i.e., incorporation of a photosynthetic eukaryote into heterotrophic eukaryotic host) . To examine if the protein transport inside chloroplasts is similar between these organisms, we carried out heterologous protein import experiments with Euglena precursor proteins and spinach chloroplasts . The precursor of a 30-kDa subunit of the oxygen-evolving complex (OEC30) from the thylakoid lumen of Euglena chloroplasts contained the N-terminal signal, stroma targeting, and thylakoid transfer domains . Truncated preOEC30s lacking the N-terminal domain were post-translationally imported into spinach chloroplasts, transported into the thylakoid lumen, and processed to a mature protein . These results showed that protein translocations within chloroplasts in Euglena and higher plants are similar and supported the hypothesis that Euglena chloroplasts are derived from the ancestral Chlorophyta .

Sci Total Environ, 2000 Oct 9, 260(1-3), 57 - 71
Mercury mine drainage and processes that control its environmental impact; Rytuba JJ; Mine drainage from mercury mines in the California Coast Range mercury mineral belt is an environmental concern because of its acidity and high sulfate, mercury, and methylmercury concentrations . Two types of mercury deposits are present in the mineral belt, silica-carbonate and hot-spring type . Mine drainage is associated with both deposit types but more commonly with the silica-carbonate type because of the extensive underground workings present at these mines . Mercury ores consisting primarily of cinnabar were processed in rotary furnaces and retorts and elemental mercury recovered from condensing systems . During the roasting process mercury phases more soluble than cinnabar are formed and concentrated in the mine tailings, commonly termed calcines . Differences in mineralogy and trace metal geochemistry between the two deposit types are reflected in mine drainage composition . Silica-carbonate type deposits have higher iron sulfide content than hot-spring type deposits and mine drainage from these deposits may have extreme acidity and very high concentrations of iron and sulfate . Mercury and methylmercury concentrations in mine drainage are relatively low at the point of discharge from mine workings . The concentration of both mercury species increases significantly in mine drainage that flows through and reacts with calcines . The soluble mercury phases in the calcines are dissolved and sulfate is added such that methylation of mercury by sulfate reducing bacteria is enhanced in calcines that are saturated with mine drainage . Where mercury mine drainage enters and first mixes with stream water, the addition of high concentrations of mercury and sulfate generates a favorable environment for methylation of mercury . Mixing of oxygenated stream water with mine drainage causes oxidation of dissolved iron(II) and precipitation of iron oxyhydroxide that accumulates in the streambed . Both mercury and methylmercury are strongly adsorbed onto iron oxyhydroxide over the pH range of 3.2-7.1 in streams impacted by mine drainage . The dissolved fraction of both mercury species is depleted and concentrated in iron oxyhydroxide such that the amount of iron oxyhydroxide in the water column reflects the concentration of mercury species . In streams impacted by mine drainage, mercury and methylmercury are transported and adsorbed onto particulate phases . During periods of low stream flow, fine-grained iron hydroxide sediment accumulates in the bed load of the stream and adsorbs mercury and methylmercury such that both forms of mercury become highly enriched in the iron oxyhydroxide sediment . During high-flow events, mercury- and methylmercury-enriched iron hydroxide sediment is transported into larger aquatic systems producing a high flux of bioavailable mercury.

Arch Environ Contam Toxicol, 2000 Nov, 39(4), 452 - 61
An evaluation of the toxicity of contaminated sediments from Waukegan Harbor, Illinois, following remediation; Kemble NE et al.; Waukegan Harbor in Illinois was designated as a Great Lakes Area of Concern due to high concentrations of sediment-associated polychlorinated biphenyls (PCBs) . The objective of this study was to evaluate the toxicity of 20 sediment samples collected after remediation (primarily dredging) of Waukegan Harbor for PCBs . A 42-day whole sediment toxicity test with the amphipod Hyalella azteca (28-day sediment exposure followed by a 14-day reproductive phase) and sediment toxicity tests with Microtox(R) were conducted to evaluate sediments from Waukegan Harbor . Endpoints measured were survival, growth, and reproduction (amphipods) and luminescent light emission (bacteria) . Survival of amphipods was significantly reduced in 6 of the 20 sediment samples relative to the control . Growth of amphipods (either length or weight) was significantly reduced relative to the control in all samples . However, reproduction of amphipods identified only two samples as toxic relative to the control . The Microtox basic test, conducted with organic extracts of sediments identified only one site as toxic . In contrast, the Microtox solid-phase test identified about 50% of the samples as toxic . A significant negative correlation was observed between reproduction and the concentration of three polynuclear aromatic hydrocarbons (PAHs) normalized to total organic carbon . Sediment chemistry and toxicity data were evaluated using sediment quality guidelines (consensus-based probable effect concentrations, PECs) . Results of these analyses indicate that sediment samples from Waukegan Harbor were toxic to H . azteca contaminated at similar contaminant concentrations as sediment samples that were toxic to H . azteca from other areas of the United States . The relationship between PECs and the observed toxicity was not as strong for the Microtox test . The results of this study indicate that the first phase of sediment remediation in Waukegan Harbor successfully lowered concentrations of PCBs at the site . Though the sediments were generally not lethal, there were still sublethal effects of contaminants in sediments at this site observed on amphipods in long-term exposures (associated with elevated concentrations of metals, PCBs, and PAHs).

Microb Pathog, 2000 Nov, 29(5), 279 - 87
Fumarate reductase is essential for Helicobacter pylori colonization of the mouse stomach; Ge Z et al.; Fumarate reductase (FRD) is the key enzyme in fumarate respiration induced by anaerobic growth of bacteria . In Helicobacter pylori, this enzyme appears to be constitutively expressed under microaerobic conditions and is not essential for its survival in vitro . In this study, the role of FRD in the colonization of H . pylori was investigated using a mouse model . The frdA gene coding for subunit A of FRD, and two control genes, copA and copP associated with the export of copper out of H . pylori, were inactivated by insertion of the chloramphenicol acetyltransferase cassette into these individual genes . The isogenic mutants of H . pylori strain AH244 were obtained by natural transformation . Seventy-five ICR mice (15 mice/group) were orogastrically dosed with either the wild type H . pylori strain AH244, its isogenic mutants, or Brucella broth (negative control) . Five mice from each group were killed at 2, 4 and 8 weeks post-inoculation (WPI), respectively . H . pylori colonization was not detected in mouse gastric mucosa infected with the frdA mutant at any time point in the study by both quantitative culture and PCR . In contrast, the mice inoculated with either wild type AH244, copA or copPH . pylori mutants became readily infected . These data indicate that FRD plays a crucial role in H . pylori survival in the gastric mucosa of mice . Given that FRD, present in all H . pylori strains, is immunogenic in H . pylori -infected patients and H . pylori growth in vitro can be inhibited by three anthelmintics (morantel, oxantel and thiabendazole), this enzyme could potentially be used both as a novel drug target as well as in the development of vaccines for H . pylori prevention and eradication .

Appl Microbiol Biotechnol, 2000 Sep, 54(3), 335 - 40
Immobilisation of Thiobacillus ferrooxidans cells on nickel alloy fibre for ferrous sulfate oxidation; Gomez JM et al.; The immobilisation of the iron-oxidising bacteria Thiobacillus ferrooxidans on nickel alloy fibre as support is described . This matrix showed promise for application in iron oxidation under strongly acidic conditions . The influence on the colonisation process of T . ferrooxidans exerted by the initial pH of the medium and by temperature has also been studied . Results showed that immobilisation of T . ferrooxidans cells was affected by changes of temperature between 30 degrees C and 40 degrees C and in pH from 1.4 to 2.0.

Plant J, 2000 Oct, 24(1), 1 - 9
Knockout of the regulatory site of 3-ketoacyl-ACP synthase III enhances short- and medium-chain acyl-ACP synthesis; Abbadi A et al.; 3-ketoacyl-acyl carrier protein synthase (KAS) III catalyses the first condensing step of the fatty acid synthase (FAS) type II reaction in plants and bacteria, using acetyl CoA and malonyl-acyl carrier protein (ACP) as substrates . Enzymatic characterization of recombinant KAS III from Cuphea wrightii embryo shows that this enzyme is strongly inhibited by medium-chain acyl-ACP end products of the FAS reaction, i.e . inhibition by lauroyl-ACP was uncompetitive towards acetyl CoA and non-competitive with regard to malonyl-ACP . This indicated a distinct attachment site for regulatory acyl-ACPs . Based on alignment of primary structures of various KAS IIIs and 3-ketoacyl CoA synthases, we suspected the motif G290NTSAAS296 to be responsible for binding of regulatory acyl-ACPs . Deletion of the tetrapeptide G290NTS293 led to a change of secondary structure and complete loss of KAS III condensing activity . Exchange of asparagine291 to aspartate, alanine294 to serine and alanine295 to proline, however, produced mutant enzymes with slightly reduced condensing activity, yet with insensitivity towards acyl-ACPs . To assess the potential of unregulated KAS III as tool in oil production, we designed in vitro experiments employing FAS preparations from medium-chain fatty acid-producing Cuphea lanceolata seeds and long-chain fatty acid-producing rape seeds, each supplemented with a fivefold excess of the N291D KAS III mutant . High amounts of short-chain acyl-ACPs in the case of C . lanceolata, and of medium-chain acyl-ACPs in the case of rape seed preparations, were obtained . This approach targets regulation and offers new possibilities to derive transgenic or non-transgenic plants for production of seed oils with new qualities.

Mol Microbiol, 2000 Oct, 38(1), 114 - 25
Comparison of the sequences of the Aspergillus nidulans hxB and Drosophila melanogaster ma-l genes with nifS from Azotobacter vinelandii suggests a mechanism for the insertion of the terminal sulphur atom in the molybdopterin cofactor; Amrani L et al.; The molybdopterin cofactor (MoCF) is required for the activity of a variety of oxidoreductases . The xanthine oxidase class of molybdoenzymes requires the MoCF to have a terminal, cyanolysable sulphur ligand . In the sulphite oxidase/nitrate reductase class, an oxygen is present in the same position . Mutations in both the ma-l gene of Drosophila melanogaster and the hxB gene of Aspergillus nidulans result in loss of activities of all molybdoenzymes that necessitate a cyanolysable sulphur in the active centre . The ma-l and hxB genes encode highly similar proteins containing domains common to pyridoxal phosphate-dependent cysteine transulphurases, including the cofactor binding site and a conserved cysteine, which is the putative sulphur donor . Key similarities were found with NifS, the enzyme involved in the generation of the iron-sulphur centres in nitrogenase . These similarities suggest an analogous mechanism for the generation of the terminal molybdenum-bound sulphur ligand . We have identified putative homologues of these genes in a variety of organisms, including humans . The human homologue is located in chromosome 18.q12.

Mol Microbiol, 2000 Oct, 38(1), 20 - 30
Regulation of carbon metabolism in Chlamydia trachomatis; Iliffe-Lee ER et al.; The biological significance of glycogen accumulation and how the process is regulated in Chlamydia trachomatis remains poorly defined . C . trachomatis-infected HeLa cells were cultured in medium containing various glucose concentrations (0, 0.1, 1 or 10 mg ml-1) or in the presence of gluconeogenic carbon sources (20 mM glutamate, 20 mM malate, 20 mM alpha-ketoglutarate or 20 mM oxaloacetate), and the effects of these different culture conditions on the production of infectious chlamydial elementary bodies and glycogen accumulation were monitored . When chlamydiae were cultured in glucose concentrations greater than 1 mg ml-1, optimal growth and maximal glycogen accumulation occurred . In contrast to uninfected HeLa cells, which increased their glycogen stores when grown in the presence of high glucose concentrations, chlamydial glycogen accumulation remained essentially constant . When cultured in medium supplemented with either reduced glucose concentrations or any of the gluconeogenic carbon sources, chlamydiae still grew; however, the yield of elementary bodies was substantially decreased, and there was no significant amount of glycogen accumulated by host HeLa cells or C . trachomatis . This suggests that glycogen accumulation may not be essential for chlamydial survival . Reverse transcriptase-polymerase chain reaction (RT-PCR) results indicated that, despite the fact that the source and amount of carbon available in the medium affected chlamydial glycogen accumulation, the expression of genes required for glycogen metabolism was not significantly changed . Similarly, the expression of several genes encoding key enzymes of central metabolism was not affected by alterations in carbon source or availability . Taken together, the data suggest that, unlike most free-living bacteria, chlamydia are unable to alter the expression of genes involved in carbon metabolism in response to changes in environmental conditions.

Mol Microbiol, 2000 Oct, 38(1), 1 - 7
A pot-pourri of plasmid paradoxes: effects of a second copy; Yarmolinsky MB; Bacterial plasmids are exemplary subjects for study, being conveniently isolated, dissected, reassembled, and introduced into various hosts . Their versatility and power make them eminently worthy of our attention . In what follows I consider some consequences of simply doubling the dosage of particular plasmid genes or of forming a plasmid dimer . These consequences can be perverse, paradoxical, or informative . They bear on questions of cell viability, copy number limitation, clonal homogeneity, check-point control, and the recovery of mutants . They have relevance to biotechnology, evolution and medicine . In reviewing these effects, my motivation is largely to share my enthusiasm for certain kinds of biological narratives, the nature of which is best left for the reader to discern.

Insect Mol Biol, 2000 Oct, 9(5), 539 - 43
Ultrastructural and molecular identification of a Wolbachia endosymbiont in a spider, Nephila clavata; Oh HW et al.; Wolbachia-like bacteria were observed in the egg cells of golden orb-weaving spider, Nephila clavata, by means of transmission electron microscopy . The bacteria exhibited the typical morphology of Wolbachia, including three enveloping membranes . Based on the amplification and sequencing of partial 16S rDNA and ftsZ gene, the bacteria were identified as Wolbachia, intracellular, transovarially inherited alpha-proteobacteria in invertebrates . Phylogenetic analysis based on 16S rDNA and ftsZ gene sequences invariably indicated that the intracellular bacteria from N . clavata belonged to group A Wolbachia, which were found only from insects . Clustering of Wolbachia from N . clavata with group A Wolbachia indicates that the bacteria were probably transferred horizontally between insects and the spider.

J Bacteriol, 2000 Nov, 182(21), 6177 - 82
A genetic mechanism for deletion of the ser2 gene cluster and formation of rough morphological variants of Mycobacterium avium; Eckstein TM et al.; A major phenotypic trait of the Mycobacterium avium complex is the ability to produce rough and smooth colony variants . The chemical basis of this morphological variation is the loss of an antigenic surface structure, termed glycopeptidolipid (GPL), by rough variants . Using M . avium serovar 2 strain 2151 as a model system, this laboratory previously reported that rough variants arise via the deletion of large genomic regions encoding GPL biosynthesis . One such deletion encompasses the gene cluster (ser2) responsible for production of the serovar 2 GPL haptenic oligosaccharide . In this study, nucleotide sequencing revealed that both ends of the ser2 gene cluster are flanked by a novel insertion sequence (IS1601) oriented as direct repeats . Detailed analyses of the site of deletion in the genome of M . avium 2151 Rg-1 demonstrated that a single copy of IS1601 remained and that the ser2 gene cluster was deleted by homologous recombination . This same deletion pattern was observed for 10 out of 15 rough colony variants tested . Additionally, these studies revealed that IS1601 contains portions of three independent insertion sequences . This report is the first to define the precise genetic basis of colony variation in Mycobacterium spp . and provides further evidence that homologous recombination between insertion sequence elements can be a primary determinant of genome plasticity in these bacteria.

J Bacteriol, 2000 Nov, 182(21), 5997 - 6004
Characterization of the hydrogen-deuterium exchange activities of the energy-transducing HupSL hydrogenase and H(2)-signaling HupUV hydrogenase in Rhodobacter capsulatus; Vignais PM et al.; Rhodobacter capsulatus synthesizes two homologous protein complexes capable of activating molecular H(2), a membrane-bound {NiFe} hydrogenase (HupSL) linked to the respiratory chain, and an H(2) sensor encoded by the hupUV genes . The activities of hydrogen-deuterium (H-D) exchange catalyzed by the hupSL-encoded and the hupUV-encoded enzymes in the presence of D(2) and H(2)O were studied comparatively . Whereas HupSL is in the membranes, HupUV activity was localized in the soluble cytoplasmic fraction . Since the hydrogenase gene cluster of R . capsulatus contains a gene homologous to hoxH, which encodes the large subunit of NAD-linked tetrameric soluble hydrogenases, the chromosomal hoxH gene was inactivated and hoxH mutants were used to demonstrate the H-D exchange activity of the cytoplasmic HupUV protein complex . The H-D exchange reaction catalyzed by HupSL hydrogenase was maximal at pH 4 . 5 and inhibited by acetylene and oxygen, whereas the H-D exchange catalyzed by the HupUV protein complex was insensitive to acetylene and oxygen and did not vary significantly between pH 4 and pH 11 . Based on these properties, the product of the accessory hypD gene was shown to be necessary for the synthesis of active HupUV enzyme . The kinetics of HD and H(2) formed in exchange with D(2) by HupUV point to a restricted access of protons and gasses to the active site . Measurement of concentration changes in D(2), HD, and H(2) by mass spectrometry showed that, besides the H-D exchange reaction, HupUV oxidized H(2) with benzyl viologen, produced H(2) with reduced methyl viologen, and demonstrated true hydrogenase activity . Therefore, not only with respect to its H(2) signaling function in the cell, but also to its catalytic properties, the HupUV enzyme represents a distinct class of hydrogenases.

Am J Respir Crit Care Med, 2000 Oct, 162(4 Pt 2), S185 - 9
Control of experimental inflammatory bowel disease by regulatory T cells; Asseman C et al.; A helper T cell type 1-mediated colitis driven by enteric bacteria develops in severe combined immunodeficient mice after transfer of CD45RB(high)CD4(+) T cells . Development of disease can be prevented by cotransfer of the reciprocal CD45RB(low) subset . Analysis of the mechanism of immune suppression transferred by CD45RB(low)CD4(+) cells revealed essential roles for both IL-10 and TGF-beta . These data indicate that a functionally specialized population of regulatory T (Treg) cells exists in normal mice and that these can prevent the development of pathogenic responses toward commensal bacteria . The role of Treg cells in the control of the immune response is discussed.

Am J Infect Control, 2000 Oct, 28(5), 340 - 6
Effect of hand sanitizer use on elementary school absenteeism; Hammond B et al.; BACKGROUND: Several studies have indicated a connection between handwashing and illness-related absenteeism in school settings . The difficulty of ensuring consistent and effective handwashing among student populations has also been noted . The purpose of this study was to assess the effectiveness of the use of an alcohol gel hand sanitizer in the classroom to help decrease the illness-related absentee rate for elementary school students . METHODS: This study involved 5 individual school districts, 16 individual schools, and more than 6000 students in Delaware, Ohio, Tennessee, and California . Individual schools in each district were paired into product and control groups . In the product group schools, an alcohol gel hand sanitizer was used by the students and staff when entering and leaving the classroom . Absenteeism due to infection was recorded, and the data were statistically analyzed . RESULTS: The overall reduction in absenteeism due to infection in the schools included in this study was 19.8% for schools that used an alcohol gel hand sanitizer compared with the control schools (P <.05) . Data from the school system with the largest teacher population (n = 246) showed that teacher absenteeism decreased 10.1% (trend) in the schools where sanitizer was used . CONCLUSION: Elementary school absenteeism due to infection is significantly reduced when an alcohol gel hand sanitizer is used in the classroom as part of a hand hygiene program.

Nature, 2000 Sep 28, 407(6803), 508 - 13
The genome sequence of the thermoacidophilic scavenger Thermoplasma acidophilum; Ruepp A et al.; Thermoplasma acidophilum is a thermoacidophilic archaeon that thrives at 59 degrees C and pH 2, which was isolated from self-heating coal refuse piles and solfatara fields . Species of the genus Thermoplasma do not possess a rigid cell wall, but are only delimited by a plasma membrane . Many macromolecular assemblies from Thermoplasma, primarily proteases and chaperones, have been pivotal in elucidating the structure and function of their more complex eukaryotic homologues . Our interest in protein folding and degradation led us to seek a more complete representation of the proteins involved in these pathways by determining the genome sequence of the organism . Here we have sequenced the 1,564,905-base-pair genome in just 7,855 sequencing reactions by using a new strategy . The 1,509 open reading frames identify Thermoplasma as a typical euryarchaeon with a substantial complement of bacteria-related genes; however, evidence indicates that there has been much lateral gene transfer between Thermoplasma and Sulfolobus solfataricus, a phylogenetically distant crenarchaeon inhabiting the same environment . At least 252 open reading frames, including a complete protein degradation pathway and various transport proteins, resemble Sulfolobus proteins most closely.

Biochem Biophys Res Commun, 2000 Oct 14, 277(1), 159 - 63
Suppressive effects of DNA vaccines encoding heat shock protein on Helicobacter pylori-induced gastritis in mice; Todoroki I et al.; We investigated the effect of DNA vaccines encoding H . pylori-heat shock protein A and B (pcDNA3.1-hspA and -hspB) on inducing immune responses against H . pylori in mice . C57BL/six mice aged 5 weeks were immunized by single injection of 10 microg of pcDNA3.1-hspA and pcDNA3.1-hspB into intracutaneous tissue . Plasmid DNA lacking the inserted hsp were injected as a control . Three months after vaccination, significant specific antibodies against H . pylori were detected by ELISA in the sera of vaccinated mice . Antibody isotypes were predominantly IgG2a (Th1-like) with pcDNA3.1-hspA and mixed IgG1/IgG2a (Th0-like) with pcDNA3.1-hspB . DNA vaccination dramatically suppressed colonies of bacteria in stomach of vaccinated mice (28,400 +/- 21,600/mm(2) for pcDNA3.1-hspA and 6800 +/- 3470/mm(2) for pcDNA3.1-hspB) compared to control mice (128,000 +/- 42,200/mm(2)) . Histological analysis of the gastric mucosa demonstrated that the degree of gastritis was significantly lower in the vaccinated mice than in control mice . These results demonstrated that DNA vaccines encoding H . pylori-Hsp induce significant immune response against H . pylori to decrease gastric mucosal inflammation, indicating that a DNA vaccine can be a new approach against H . pylori in humans .

Mol Cell Biol, 2000 Nov, 20(21), 7980 - 90
Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells; De Silva IU et al.; The mechanisms by which DNA interstrand cross-links (ICLs) are repaired in mammalian cells are unclear . Studies in bacteria and yeasts indicate that both nucleotide excision repair (NER) and recombination are required for their removal and that double-strand breaks are produced as repair intermediates in yeast cells . The role of NER and recombination in the repair of ICLs induced by nitrogen mustard (HN2) was investigated using Chinese hamster ovary mutant cell lines . XPF and ERCC1 mutants (defective in genes required for NER and some types of recombination) and XRCC2 and XRCC3 mutants (defective in RAD51-related homologous recombination genes) were highly sensitive to HN2 . Cell lines defective in other genes involved in NER (XPB, XPD, and XPG), together with a mutant defective in nonhomologous end joining (XRCC5), showed only mild sensitivity . In agreement with their extreme sensitivity, the XPF and ERCC1 mutants were defective in the incision or "unhooking" step of ICL repair . In contrast, the other mutants defective in NER activities, the XRCC2 and XRCC3 mutants, and the XRCC5 mutant all showed normal unhooking kinetics . Using pulsed-field gel electrophoresis, DNA double-strand breaks (DSBs) were found to be induced following nitrogen mustard treatment . DSB induction and repair were normal in all the NER mutants, including XPF and ERCC1 . The XRCC2, XRCC3, and XRCC5 mutants also showed normal induction kinetics . The XRCC2 and XRCC3 homologous recombination mutants were, however, severely impaired in the repair of DSBs . These results define a role for XPF and ERCC1 in the excision of ICLs, but not in the recombinational components of cross-link repair . In addition, homologous recombination but not nonhomologous end joining appears to play an important role in the repair of DSBs resulting from nitrogen mustard treatment.

Skeletal Radiol, 2000 Aug, 29(8), 425 - 38
MRI in inflammatory myopathies; Garcia J; Inflammatory myopathies encompass a group of acquired muscle disorders caused by infectious agents (bacteria, viruses, fungi and parasitic agents) or autoimmune processes (polymyositis, dermatomyositis and other types) . In suspected infection sonography, CT and MRI are all able to show edema and fluid collections in soft tissues and muscles; sonography and CT may help guidance of a needle aspiration to establish a correct diagnosis . By offering better tissue differentiation, MRI appears to be more efficient than sonography and CT in diagnosing and managing autoimmune myopathies . MRI is indeed very sensitive to the presence of water and edema, and appears to be a very good indicator for an early diagnosis of diseases . MRI may also help to evaluate the extent and number of lesions, to guide a biopsy in an area of active disease and finally to follow the evolution under therapy.

Curr Opin Biotechnol, 2000 Oct, 11(5), 505 - 8
Gene transfer from genetically modified food; Gasson MJ; The current debate about the safety of genetically modified food includes some important scientific issues where more scientific data would aid the robustness of safety evaluation . One example is the possibility of gene transfer, especially from genetically modified plant material.

Curr Opin Biotechnol, 2000 Oct, 11(5), 440 - 4
In vivo expression technology strategies: valuable tools for biotechnology; Rainey PB et al.; Whole genome sequences have shown that bacteria possess a significant number of genes that have no known function . It is probable that many of these are required for survival in environments other than the agar plate . In vivo selection strategies provide a means of obtaining genes active in complex natural environments . Direct access to these genes is essential for understanding ecological performance and provides novel opportunities for biotechnology.

FASEB J, 2000 Oct, 14(13), 1889 - 900
Nitrosation and oxidation in the regulation of gene expression; Marshall HE et al.; A growing body of evidence suggests that the cellular response to oxidative and nitrosative stress is primarily regulated at the level of transcription . Posttranslational modification of transcription factors may provide a mechanism by which cells sense these redox changes . In bacteria, for example, OxyR senses redox-related changes via oxidation or nitrosylation of a free thiol in the DNA binding region . This mode of regulation may serve as a paradigm for redox-sensing by eukaryotic transcription factors as most-including NF-kappaB, AP-1, and p53-contain reactive thiols in their DNA binding regions, the modification of which alters binding in vitro . Several of these transcription factors have been found to be sensitive to both reactive oxygen species and nitric oxide-related species in vivo . It remains entirely unclear, however, if oxidation or nitrosylation of eukaryotic transcription factors is an important mode of regulation, or whether transcriptional activating pathways are principally controlled at other redox-sensitive levels.-Marshall, H . E., Merchant, K., Stamler, J . S . Nitrosation and oxidation in the regulation of gene expression.

Microb Ecol, 2000 Aug, 40(2), 94 - 103
Bio-optical Characteristics and the Vertical Distribution of Photosynthetic Pigments and Photosynthesis in an Artificial Cyanobacterial Mat; Kuhl M et al.; Zonations of photosynthesis and photopigments in artificial cyanobacterial mats were studied with (i) oxygen and pH microsensors, (ii) fiber-optic microprobes for field radiance, scalar irradiance, and PSII fluorescence, and (iii) a light microscope equipped with a spectrometer for spectral absorbance and fluorescence measurements . Our analysis revealed the presence of several distinct 1-2 mm thick cyanobacterial layers mixed with patches of anoxygenic photosynthetic bacteria . Strong attenuation of visible light confined the euphotic zone to the uppermost 3 mm of the mat, where oxygen levels of 3-4 times air saturation and a pH peak of up to pH 8.8 were observed under saturating irradiance (413 micromol photon m(-2) s(-1)) . Oxygen penetration was 5 mm in light and decreased to 1 mm in darkness . Volumetric oxygen consumption in the photic and aphotic zones of illuminated mat was 5.5 and 2.9 times higher, respectively, than oxygen consumption in dark incubated mats . Scalar irradiance reached 100-150% of incident irradiance in the upper 0.5 mm of the mat due to intense scattering in the matrix of cells, exopolymers, and carbonate precipitates . In deeper mat layers scalar irradiance decreased nearly exponentially, and highest attenuation coefficients of 6-7 mm(-1) were found in cyanobacterial layers, where photosynthesis and photopigment fluorescence also peaked . Visible light was attenuated >100 times more strongly than near infrared light . Microscope spectrometry on thin sections of mats allowed detailed spectral absorbance and fluorescence measurements at defined positions relative to the mat surface . Besides strong spectral signals of cyanobacterial photopigments (Chl a and phycobiliproteins), the presence of both green and purple photosynthetic bacteria was evident from spectral signals of Bchl a and Bchl c . Microprofiles of photopigment absorbance correlated well with microdistributions of phototrophs determined in an accompanying study.

Microb Ecol, 2000 Aug, 40(2), 85 - 93
Artificial Cyanobacterial Mats: Growth, Structure, and Vertical Zonation Patterns; Fenchel T et al.; The formation of cyanobacterial mats (originally induced by incubation of sediment cores in which metazoans and most other eukaryotes had been removed) was followed over approximately 2.6 years . The thickness of the mats increased at a rate of 2-3 mm per year because of accumulation of empty cyanobacterial sheaths and as a result of carbonate deposition; the fraction of living biomass remained relatively constant over at least 2 years, but there was a slow accumulation of nonliving organic C ( approximately 1 mmol yr(-1)) . Biota composition (dominated by five types of filamentous cyanobacteria, unicellular cyanobacteria, diatoms, anoxygenic phototrophs, and heterotrophic bacteria) and vertical zonation patterns in the upper 2-3 mm of the mats were also almost constant over time . Using transmission electron microscopy and stereological analysis it was possible to quantify the vertical distribution of major groups of organisms.

Dis Aquat Organ, 2000 Aug 31, 42(2), 101 - 10
Glycogen granules in resting and inflammatory rainbow trout phagocytes--an ultrastructural study; Afonso A et al.; The ultrastructural image of glycogen granules in the cytoplasm of rainbow trout phagocytes in sections stained by the conventional lead or uranyl-lead stains is highly dependent on fixation conditions, the granules being visible only when adequate fixation protocols are used . Morphometry of samples processed for the detection of peroxidase or esterase activities (to specifically label neutrophils and macrophages, respectively), and simultaneously stained for the specific detection of glycogen, showed that inflammatory peritoneal neutrophils were richer in glycogen granules than resting neutrophils . This increase in glycogen content occurs after the migration from the haematopoietic tissues and peripheral blood to the inflamed foci . Glycogen granules could not be found in resting peritoneal macrophages but were found in inflammatory macrophages . The macrophage granules occurred in smaller amounts than in neutrophils, and consisted of granules identical to those of neutrophils together with significantly smaller granules . No evidence for the utilization of glycogen by neutrophils phagocytosing bacteria within the peritoneal cavity was found.

J Med Microbiol, 2000 Oct, 49(10), 861 - 74
Enumeration of Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in subgingival plaque samples by a quantitative-competitive PCR method; Doungudomdacha S et al.; Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans are believed to play an important role in adult periodontitis, but the significance of their relative numbers and progress of the disease is still unclear . Traditional quantitative methods are generally time-consuming and inaccurate . The aim of this study was to develop a sensitive, quantitative PCR technique that would be useful for enumerating P . gingivalis, Pr . intermedia and A . actinomycetemcomitans in subgingival plaque samples from subjects with adult periodontitis . Primers to the following genes were employed: the fimbrial gene (fimA) of P . gingivalis, the 16S rRNA gene of Pr . intermedia and the leukotoxin-A (lktA) gene of A . actinomycetemcomitans . Competitive templates were constructed either by sequence deletion between primer binding sites or by annealing of the primer binding sites to an appropriate DNA core so as to yield products of a different size from that obtained with the target template . Coamplification of target and competitive templates yielded products of expected size and non-specific recognition by the primers was not found . The sensitivity of the designed primers was 100 cells of P . gingivalis, 100 cells of Pr . intermedia and 10 cells of A . actinomycetemcomitans . The three species were found in subgingival plaque samples collected from both healthy and diseased sites by the quantitative-competitive (QC)-PCR method and the technique was more sensitive than cultural methods . For determining the proportions of each of the three periodontopathogens, the total number of bacteria in the samples was enumerated by quantitative-PCR with 16S rRNA universal primers (27f and 342r) . The findings indicate that QC-PCR is a useful method for enumerating bacteria in clinical oral specimens and the technique could play a role in the investigation of disease progression.

J Gastroenterol Hepatol, 2000 Aug, 15(8), 910 - 4
Efficacy of a 1-week pantoprazole triple therapy in eradicating Helicobacter pylori in Asian patients; Goh K et al.; BACKGROUND: The aim of the present paper was to determine the efficacy and tolerability of a 1-week treatment regimen consisting of pantoprazole and two antibiotics: clarithromycin and amoxycillin, in the eradication of Helicobacter pylori . METHODS: The patients selected had unequivocal evidence of H . pylori infection based on urease test, culture and histology on antral and corpus biopsies obtained at endoscopy . Patients received pantoprazole 40 mg twice a day, clarithromycin 500 mg twice a day and amoxycillin 1 g twice a day for 1 week and were assessed for successful eradication at least 4 weeks after completion of therapy by repeat gastroscopy and gastric biopsies . Eradication was defined as absence of bacteria in both antral and corpus biopsies tested by culture, histology and urease test . RESULTS: One hundred and six patients were recruited for the study.The mean age was 48.0 years (range: 23-74 years) . Four patients defaulted follow up and five patients were not compliant (taking less than 85%) with medications . Eradication rates on per-protocol analysis were: 88/97 (90.7%; 95% CI: 83.1-95.7); and on intention-to-treat analysis they were: 88/106 (83.0%; 95% CI: 75.9-90.2) . Side-effects were in general mild and tolerable: 57 of 106 (53.7%) patients complained of a bitter taste; 15 (14.1%) complained of giddiness; 10 (9.4%) complained of increased abdominal pain; 11 (11.5%) complained of lethargy and 16 (15.1%) complained of loose motions . Pre-treatment metronidazole resistance was encountered in 57/74 strains (77.0%) . Clarithromycin resistance was not encountered in any of the strains . CONCLUSIONS: The pantoprazole 1-week triple therapy with amoxycillin and clarithromycin is effective in H . pylori eradication . The treatment was well tolerated by patients . Metronidazole resistance was reported in a high percentage of strains isolated from patients . Clarithromycin resistance was, however, not detected in any of the strains.

J Mol Biol, 2000 Oct 13, 303(1), 25 - 34
Archaeal histone selection of nucleosome positioning sequences and the procaryotic origin of histone-dependent genome evolution; Bailey KA et al.; Archaeal histones and the eucaryal (eucaryotic) nucleosome core histones have almost identical histone folds . Here, we show that DNA molecules selectively incorporated by rHMfB (recombinant archaeal histone B from Methanothermus fervidus) into archaeal nucleosomes from a mixture of approximately 10(14) random sequence molecules contain sequence motifs shown previously to direct eucaryal nucleosome positioning . The dinucleotides GC, AA (=TT) and TA are repeated at approximately 10 bp intervals, with the GC harmonic displaced approximately 5 bp from the AA and TA harmonics {(GCN(3)AA or TA)(n)} . AT and CG were not strongly selected, indicating that TA not equalAT and GC not equalCG in terms of facilitating archaeal nucleosome assembly . The selected molecules have affinities for rHMfB ranging from approximately 9 to 18-fold higher than the level of affinity of the starting population, and direct the positioned assembly of archaeal nucleosomes . Fourier-transform analyses have revealed that AA dinucleotides are much enriched at approximately 10 . 1 bp intervals, the helical repeat of DNA wrapped around a nucleosome, in the genomes of Eucarya and the histone-containing Euryarchaeota, but not in the genomes of Bacteria and Crenarchaeota, procaryotes that do not have histones . Facilitating histone packaging of genomic DNA has apparently therefore imposed constraints on genome sequence evolution, and since archaeal histones have no structure in addition to the histone fold, these constraints must result predominantly from histone fold-DNA contacts . Based on the three-domain universal phylogeny, histones and histone-dependent genome sequence evolution most likely evolved after the bacterial-archaeal divergence but before the archaeal-eucaryal divergence, and were subsequently lost in the Crenarchaeota . However, with lateral gene transfer, the first histone fold could alternatively have evolved after the archaeal-eucaryal divergence, early in either the euryarchaeal or eucaryal lineages .

Gene Ther, 2000 Sep, 7(18), 1562 - 9
Cotransduction of nondividing cells using lentiviral vectors; Frimpong K et al.; Diseases such as AIDS and cancers may require the introduction of multiple genes into either stem cells or nondividing cells, among others, for therapeutic purposes . Such genes may act at different points of the disease pathway, or may constitute a regulatory loop to bypass or rectify the defective gene or pathway underpinning the disease . Ideally, the therapeutic genes must be transduced together in diverse combinations, and the introduction should occur without constraints . Since lentiviral vectors can transduce both dividing and nondividing cells, they are ideal vehicles to investigate combinatorial gene transfer into diverse cells . In this study, we demonstrate that by using two independent lentiviral vectors, pseudotyped with the protein g of vesicular stomatitis virus, up to four genes can be introduced simultaneously into single dividing and nondividing cells . Up to 45% and 73% of dividing and nondividing cells, respectively, could be transduced with two lentiviral vectors . The efficiency of cotransducing a single cell was the product of the individual transduction efficiencies and suggested the absence of viral interference . Multiple and combinatorial gene transduction using lentiviral vectors may prove useful in gene therapy.

J Appl Microbiol, 2000 Sep, 89(3), 486 - 93
Production of B-group vitamins by two Azotobacter strains with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions; Revillas JJ et al.; Azotobacter vinelandii strain ATCC 12837 and A . chroococcum strain H23 (CECT 4435) were able to grow on N-free or NH4Cl-amended chemically-defined (Burk's) media, with protocatechuic acid (1-2 mmol 1(-1)) or sodium p-hydroxybenzoate (1-10 mmol 1(-1)) as sole carbon (C) sources . At a concentration of 2 mmol 1(-1), both substrates supported nitrogen fixation (acetylene reduction assay) at similar or higher rates than bacteria grown in control media amended with 2 mmol 1(-1) sodium succinate as C source . The two strains produced the B-group vitamins niacin, pantothenic acid, thiamine, riboflavin and biotin after 72 h of growth in chemically-defined media with 2 mmol 1(-1) protocatechuic acid, sodium phydroxybenzoate or sodium succinate as sole C source, either in N-free media or in media amended with 0.1% NH4Cl . Quantitative production of all vitamins was affected by the use of the different C and N substrates.

Biochem Pharmacol, 2000 Nov 15, 60(10), 1425 - 34
Inhibition of cellular action of thrombin by N3-cyclopropyl-7-{{4-(1-methylethyl)phenyl}methyl}-7H-pyrrolo{3, 2-f}quinazoline-1,3-diamine (SCH 79797), a nonpeptide thrombin receptor antagonist; Ahn HS et al.; A growing body of evidence suggests an important contribution of the cellular actions of thrombin to thrombosis and restenosis following angioplasty . Recently we reported on SCH 79797 (N3-cyclopropyl-7- inverted question mark{4-(1-methylethyl)phenyl}methyl inverted question mark-7H-pyrrolo{3, 2-f}quinazoline-1,3-diamine) and its analogs as new potent, nonpeptide thrombin receptor antagonists . This study further characterizes the biochemical and pharmacological actions of pyrroloquinazoline inhibitors of protease activated receptor-1 (PAR-1) in human platelets and coronary artery smooth muscle cells (hCASMC) . SCH 79797 and its N-methyl analog (SCH 203099) inhibited binding of a high-affinity thrombin receptor-activating peptide ({(3)H}haTRAP, Ala-Phe(p-F)-Arg-ChA-HArg-{(3)H}Tyr-NH(2)) to PAR-1 with IC(50) values of 70 and 45 nM, respectively . SCH 79797 inhibited {(3)H}haTRAP binding in a competitive manner . SCH 79797 and SCH 203099 inhibited alpha-thrombin- and haTRAP-induced aggregation of human platelets, but did not inhibit human platelet aggregation induced by the tethered ligand agonist for protease-activated receptor-4 (PAR-4), gamma-thrombin, ADP, or collagen . SCH 203099 inhibited surface expression of P-selectin induced by haTRAP and thrombin, and it did not increase P-selectin expression or prevent thrombin cleavage of the receptor . Thrombin and TFLLRNPNDK-NH(2) (TK), a PAR-1-selective agonist, produced transient increases in cytosolic free Ca(2+) concentration ({Ca(2+)}(i)) in hCASMC . This increase in {Ca(2+)}(i) was inhibited effectively by SCH 79797 . However, the Ca(2+) transients induced by SLIGKV-NH(2,) a PAR-2-selective agonist, were not inhibited by SCH 79797 . Thrombin- and TK-stimulated {(3)H}thymidine incorporation also was inhibited completely by SCH 79797 . The results of this study demonstrate that SCH 79797 and SCH 203099 are potent, selective antagonists of PAR-1 in human platelets and hCASMC . These data also suggest that the thrombin stimulation of Ca(2+) transients and mitogenesis in hCASMC is mediated primarily through activation of PAR-1.

Rev Prat, 2000 Sep 1, 50(13), 1431 - 6
{Gastritis, dyspepsia and Helicobacter pylori}; de Korwin JD; Helicobacter pylori is a major culprit in chronic gastritis . Interactions between the infected host, the bacteria and the environment, influence the type of gastritis and the occurrence of specific diseases (peptic ulcer, gastric lymphoma and adenocarcinoma) associated with the infection in a minority of cases . The benefit of H . pylori eradication is not proved in isolated gastritis, except for rare forms such as hypertrophic or lymphocytic gastritis . In spite of a high prevalence in functional dyspepsia with normal gastroduodenal endoscopy, H . pylori is not the main cause of symptoms in dyspeptic patients, but the gastritis could be involved in the appearance of ulcer-like symptoms . Strategies for non-invasive H . pylori detection for primary eradication are developed to reduce the endoscopy workload in the management of uninvestigated dyspepsia.

Mutat Res, 2000 Oct, 463(3), 247 - 83
Mammalian class theta GST and differential susceptibility to carcinogens: a review; Landi S; Glutathione S-transferases (GSTs) are an important part of the cellular detoxification system and, perhaps, evolved to protect cells against reactive oxygen metabolites . Theta is considered the most ancient among the GSTs and theta-like GSTs are found in mammals, fish, insects, plants, unicellular algae, and bacteria . It is thought that an ancestral theta-gene underwent an early duplication before the divergence of fungi and animals and further duplications generated the variety of the other classes of GSTs (alpha, mu, phi, etc.) . The comparison of the aminoacidic homologies among mammals suggests that a duplication of an ancient GST theta occurred before the speciation of mammals and resulted in the subunits GSTT1 and GSTT2 . The ancestral GST theta has a dehalogenase activity towards several halogenated compounds, such as the dichloromethane . In fact, some aerobic and anaerobic methylotrophic bacteria can use these molecules as the sole carbon and energy source . The mammalian GST theta cannot sustain the growth of bacteria but still retains the dehalogenating activity . Therefore, although mammalian GST theta behaves as a scavenger towards electrophiles, such as epoxides, it acts also as metabolic activator for halogenated compounds, producing a variety of intermediates potentially dangerous for DNA and cells . For example, mice exposed to dichloromethane show a dose-dependent incidence of cancer via the GSTT1-1 pathway . Because GSTT1-1 is polymorphic in humans, with about 20% of Caucasians and 80% of Asians lacking the enzyme, the relationship between the phenotype and the incidence of cancer has been investigated extensively in order to detect GSTT1-1-associated differential susceptibility towards endogenous or exogenous carcinogens . The lack of the enzyme is related to a slightly increased risk of cancer of the bladder, gastro-intestinal tract, and for tobacco-related tumors (lung or oral cavity) . More pronounced risks were found in males with the GSTT1-null genotype for brain diseases and skin basal cell carcinomas not related to sunlight exposures . Moreover, there was an increased risk of kidney and liver tumors in humans with the GSTT1-1 positive genotype following exposures to halogenated solvents . Interestingly, the liver and kidney are two organs that express the highest level of GST theta in the human body . Thus, the GSTT1-1 genotype is suspected to confer decreased or increased risk of cancer in relation to the source of exposure; in vitro studies, mostly conducted on metabolites of butadiene, confirm the protective action of GSTT1-1, whereas, thus far, experimental studies prove that the increasing risk is limited.

Mol Biol Evol, 2000 Oct, 17(10), 1456 - 66
Origin and evolution of eukaryotic chaperonins: phylogenetic evidence for ancient duplications in CCT genes; Archibald JM et al.; Chaperonins are oligomeric protein-folding complexes which are divided into two distantly related structural classes . Group I chaperonins (called GroEL/cpn60/hsp60) are found in bacteria and eukaryotic organelles, while group II chaperonins are present in archaea and the cytoplasm of eukaryotes (called CCT/TriC) . While archaea possess one to three chaperonin subunit-encoding genes, eight distinct CCT gene families (paralogs) have been characterized in eukaryotes . We are interested in determining when during eukaryotic evolution the multiple gene duplications producing the CCT subunits occurred . We describe the sequence and phylogenetic analysis of five CCT genes from TRICHOMONAS: vaginalis and seven from GIARDIA: lamblia, representatives of amitochondriate protist lineages thought to have diverged early from other eukaryotes . Our data show that the gene duplications producing the eight CCT paralogs took place prior to the organismal divergence of TRICHOMONAS: and GIARDIA: from other eukaryotes . Thus, these divergent protists likely possess completely hetero-oligomeric CCT complexes like those in yeast and mammalian cells . No close phylogenetic relationship between the archaeal chaperonins and specific CCT subunits was observed, suggesting that none of the CCT gene duplications predate the divergence of archaea and eukaryotes . The duplications producing the CCTdelta and CCTepsilon subunits, as well as CCTalpha, CCTbeta, and CCTeta, are the most recent in the CCT gene family . Our analyses show significant differences in the rates of evolution of archaeal chaperonins compared with the eukaryotic CCTs, as well as among the different CCT subunits themselves . We discuss these results in light of current views on the origin, evolution, and function of CCT complexes.

Environ Health Perspect, 2000 Sep, 108(9), A412 - 4
Go with the flow: an updated tool for detecting molecules; Frazer L; In the early 1970, researchers at Los Alamos National Laboratories developed the flow cytometer, a device that allows for the identification of unknown cells . In a flow cytometer, a single-cell suspension is passed in a continuous flow through a laser beam, with each cell scattering the light in a characteristic manner . A few years ago, researchers at Los Alamos began another project, refining the capabilities of the flow cytometer so that it could analyze not a single cell but a single molecule, allowing scientists to study bacteria at the molecular level, differentiating between individual strains more quickly and with greater accuracy than before . The new flow cytometer shows great promise in a variety of fields where single-molecule study would be valuable, such as genomics and disease transmission.

Ann Epidemiol . 2000 Oct 1;10(7):471.
An epidemic without illness . Using dna markers to model infection; Lawson M et al.; PURPOSE: Combining molecular biology with infection control interventions can increase compliance and allow objective measurement of effectiveness . We developed a group of PCR detectable non-infectious DNA markers that can be used to model infection and provide immediate feedback on hygiene practices in institutional settings . In previous studies, we illustrated that the markers were spread in the environment in the same manner as infectious particles.METHODS: We are conducting a prospective study in 10 child care centers in order to 1) confirm that the DNA markers are valid surrogates for bacteria and viruses; 2) identify specific foci of contamination and modes of transmission; 3) illustrate the effectiveness of infection control programs utilizing the DNA markers . Centers are randomized to receive an interactive educational infection control intervention or a standard immunization intervention . The DNA markers are introduced into the center and the rate of dispersion of the DNA markers is compared with directly observed changes in hygiene behavior among the staff.RESULTS: Initial results indicate that the markers can be removed mechanically by hand washing and that common over-the-counter cleaners are effective in inactivating the markers . Toys, countertops and doorknobs appear to be more important as infectious reservoirs than brief casual contact . Data from the prospective study will be available prior to September, 2000.CONCLUSIONS: This novel approach utilizing an objective measurement will be used to identify the interaction between behavior and environmental reservoirs of infection and drive future strategies for infection control.

J Cell Sci, 2000 Oct, 113 (Pt 20), 3543 - 4
"I'll have a genome with chips, please"
Caveman A.
An occasional column, in which Caveman and other troglodytes involved in cell science emerge to share their views on various aspects of life-science research . Messages for Caveman and other contributors can be left at caveman@biologists.com . Any correspondence may be published in forthcoming issues . Previous Sticky Wickets can be viewed at: com/JCS/caveman/index.html A new era of science has dawned . The completion of DNA sequences of whole genomes of plants, large and small eukaryotes, bacteria and viruses is providing an unprecedented amount of information on the genes that define complex organisms . Together with this veritable tsunami of data come new words (genomics, proteomics, data mining), new types of biologists (bio-informaticians), and the appearance of (biotech) companys that specialize in the development of software to analyze and display genomic information . Perhaps the most measurable change that access to this vast quantity of genomic information is having on the average scientist is the potential to identify genes, and I mean all genes, in a given metabolic, developmental or oncogenic pathway - we are able to examine gene expression on a genome-wide level . The As Ts, Gs and Cs will be presented to us on a computer screen in an easy-to-read format; complex characterization and cross-matching of references are just a few keyboard strokes away . The equipment budget for most labs will provide faster and more powerful computers to store, access and manipulate this information . What are the potential consequences of having all the genes laid out for us like a book both for the way that we do science and for how others think that we should do science? With proteomics and consensus-sequence alignments, we may be able to identify protein functions (coiled-coil domains for protein-protein interactions, kinase domains, sites for phosphorylation, etc.) and, possibly, a representation of the encoded protein's three-dimensional structure (based on similarities to proteins with the same domain organization) . No more fussing around with genetic tests; out with the suppressor screens; gone are probing complex pathways of tissue development; thank goodness we won't have to consider how to purifying protein complexes; two-hybrid analysis begone . But, as everything is laid out like a book, what need is there for an old-fashioned, bench-trained scientist who develops hypotheses (remember the old catch-phrase, "No Hypothesis, No Science!") and performs 'wet' experiments (purify proteins, run gels, localize mRNA and proteins in tissues and cells) . Where is the hypothesis if one scans a genome for a family of proteins and then systematically ablates the function of each of them (knockouts, RNAi) in an organism and hopes for an effect? Genome-wide analyses are also available in a 'carry-out' format - the seemingly ubiquitous CHIP . Here, the genome is laid out in a matrix and can be 'read' with an automatic analyzer (for a princely sum of money) . All you need to do is supply the manipulation (heat shock, a drug, serum) and mRNA, and, 'hey presto', presented to you in glorious greens and reds are the ranges of changes in gene expression (up, down, no change) for all the genes in that organism . (This is a minor point, but who chose the colors? With our heightened sense of equal rights for all, what about those who are color-blind? Who is looking after their interests in this emerging area?) But, I digress . These experiments are very easy to perform, assuming that you have access to the CHIPs, a reader and have the money to buy everything ($60,000, or EU25 billion, for a full set of human CHIPs, and that's before all of the genes have been identified! However, I assume that market forces and competition will increase availability and drive down the prices.) Easy experiments are very attractive to many scientists . (ABSTRACT TRUNCATED)

Antonie Van Leeuwenhoek, 2000 Jul, 78(1), 51 - 61
Renibacterium salmoninarum isolates from different sources possess two highly conserved copies of the rRNA operon ; Grayson TH et al.; The nucleotide sequences of the rRNA genes and the 5' flanking region were determined for R . salmoninarum ATCC 33209T from overlapping products generated by PCR amplification from the genomic DNA . Comparison of the sequences with rRNA genes from a variety of bacteria demonstrated the close relatedness between R . salmoninarum and the high G+C group of the actinobacteria, in particular, Arthrobacter species . A regulatory element within the 5' leader of the rRNA operon was identical to an element, CL2, described for mycobacteria . PCR, DNA sequence analysis, and DNA hybridisation were performed to examine variation between isolates from diverse sources which represented the four 16S-23S rRNA intergenic spacer sequevars previously described for R . salmoninarum . Two 23S-5S rRNA intergenic spacer sequevars of identical length were found . DNA hybridisation using probes complementary to 23S rDNA and 16S rDNA identified two rRNA operons which were identical or nearly identical amongst 40 isolates sourced from a variety of countries.

J Clin Microbiol, 2000 Oct, 38(10), 3646 - 51
Helicobacter pylori: clonal population structure and restricted transmission within families revealed by molecular typing; Han SR et al.; Helicobacter pylori infects up to 50% of the human population worldwide . The infection occurs predominantly in childhood and persists for decades or a lifetime . H . pylori is believed to be transmitted from person to person . However, tremendous genetic diversity has been reported for these bacteria . In order to gain insight into the epidemiological basis of this phenomenon, we performed molecular typing of H . pylori isolates from different families . Fifty-nine H . pylori isolates from 27 members of nine families were characterized by using restriction fragment length polymorphism analysis of five PCR-amplified genes, by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA, and by vacA and cagA genotyping . The 16S rRNA gene exhibited little allelic variation, as expected for a unique bacterial species . In contrast, the vacA, flaA, ureAB, and lspA-glmM genes were highly polymorphic, with a mean genetic diversity of 0.83, which exceeds the levels recorded for all other bacterial species . In conjunction with PFGE, 59 H . pylori isolates could be differentiated into 21 clonal types . Each individual harbored only one clone, occasionally with a clonal variant . Identical strains were always found either between siblings or between a mother and her children . Statistical analysis revealed clonality of population structure in all isolates . The results of this study suggest the possible coexistence of a large array of clonal lineages that are evolving in each individual in isolation from one another . Transmission appears to occur primarily from mother to child and perhaps between siblings.

Curr Microbiol, 2000 Nov, 41(5), 341 - 5
Evaluation of phospholipid and fatty acid compositions as chemotaxonomic markers of Alteromonas-like proteobacteria; Ivanova EP et al.; The cellular phospholipids (PLs) and fatty acids (FAs) were investigated in type and environmental strains of Pseudoalteromonas, Alteromonas macleodii, A . infernus, and in three type strains of Marinomonas, M . communis, M . vaga, M . mediterranea . A total of 40 strains (19 strains in this study and 21 reported in previous papers), including Idiomarina abyssalis, I . zobellii, and Glaciecola punicea, G . pallidula, aerobic Alteromonas-like proteobacteria showed genus-characteristic patterns of phospholipids and fatty acids useful for genera discrimination . The PL patterns of surface cultures of alteromonads, pseudoalteromonads, and marinomonads consisted almost entirely of phosphatidyl ethanolamine and phosphatidyl glycerol presented in different proportions . Neither diphosphatidyl glycerol nor glycophospholipids were found in bacteria studied . In addition, the minor amount of a glycolipid was found in all strains studied . Bacteria of the genera Marinomonas, Idiomarina, and Glaciecola were clearly distinguished by presence of one of the major FAs: 18:1 (n-7), i15:0, and 16:1 (n-7), respectively . The amounts of these FAs reached up to 40-60% of total FAs . Members of Alteromonas and Pseudoalteromonas were characterized by different ratio of the following major FAs:16:1(n-7), 16:0, 17:1 (n-8), and 18:1 (n-7).

Neurosurgery, 2000 Oct, 47(4), 969 - 72
Tuberculosis of the axis in a patient with systemic sarcoidosis: technique of posterior open biopsy of the dens: case report; Belanger E et al.; OBJECTIVE AND IMPORTANCE: This case report illustrates the importance of obtaining tissue from a destructive lesion of the dens in a patient with systemic sarcoidosis . Although sarcoidosis can involve the axial skeleton, tissue obtained at the time of C1-C2 fusion demonstrated unsuspected pathological features, which dramatically altered the subsequent medical treatment . The technique of open posterior biopsy of the dens is illustrated, and the advantages of the approach are discussed . CLINICAL PRESENTATION: A 40-year-old woman with systemic sarcoidosis developed neck pain and atlantoaxial instability . Imaging revealed multiple thoracic and cervical vertebral abnormalities, including a destructive enhancing lesion involving the base of the dens . INTERVENTION: At the time of posterior C1-C2 fusion, we elected to perform an open biopsy of the base of the dens . A 16-gauge biopsy needle was introduced along the medial portion of the left C2 pars, aiming medially toward the base of the odontoid process . This procedure was performed under direct observation, with fluoroscopic guidance . The biopsy specimen contained caseating granulomas, and cultures were positive for Mycobacterium tuberculosis . CONCLUSION: The unusual presentation, the technique, and the importance of obtaining tissue to confirm the diagnosis of tuberculous involvement of the dens are emphasized . The relationship between sarcoidosis and tuberculosis reported in the literature is reviewed . In the current case, cell wall-positive tuberculous bacteria were cultured, confirming the presence of two separate diseases in the same patient.

Microb Comp Genomics, 1998, 3(1), 21 - 9
On the presence and organization of open reading frames of the nonmotile pathogen Brucella abortus similar to class II, III, and IV flagellar genes and to LcrD virulence superfamily; Halling SM; Brucellae are pathogenic, nonmotile bacteria that are facultative intracellular parasites . Little is known about the genetics of these bacteria . Open reading frames from Brucella abortus with similarity to the flagellin, M-ring, and hook of related bacteria were discovered . The open reading frames encode proteins of three of the four flagellum gene classes, namely II, III, and IV . A homolog of the LcrD virulence superfamily was also found . This superfamily is involved in type III protein secretion . B . abortus has the potential for motility and type III secretion.

Cancer, 2000 Oct 1, 89(7), 1431 - 9
Detection of Helicobacter species in the liver of patients with and without primary liver carcinoma; Avenaud P et al.; BACKGROUND: Several studies have shown the presence of Helicobacter species in the human biliary tract and in the intestinal tract of animals . In this study, the presence of Helicobacter species in liver samples from patients with primary hepatic carcinomas was evaluated . METHODS: Sixteen liver specimens were studied (8 from patients with primary liver carcinoma and 8 from patients without primary liver carcinoma) . Histology with standard stains, culture, and polymerase chain reaction (PCR) amplification using two sets of primers located in the 16S ribosomal DNA (rDNA) were used to detect the presence of bacteria . Amplified products were sequenced to determine the genus and species of the bacteria . A search for other genes that were specific for Helicobacter pylori also was carried out by PCR . RESULTS: PCR performed with the 16S rDNA primers revealed the presence of bacteria from the genus Helicobacter in all of the liver specimens from patients with primary liver carcinoma (eight of eight patients) and in one specimen from a patient without primary liver carcinoma (one of eight patients) . When the nucleotide sequence of > 80% of the 16S rDNA was determined, the closest similarity was with the 16S rDNA from H . pylori in eight patients . In 1 patient sample from which only 398 nucleotides were sequenced, the closest match was Helicobacter felis . CONCLUSIONS: The results presented in this study indicate that Helicobacter species can be present in the liver of patients with primary hepatic carcinoma, but their eventual role in the carcinogenesis process, although it is plausible, remains to be proven . Based on sequence similarity, it seems that Helicobacter species that are related closely to H . pylori but are distinct from it have been found.

EMBO J, 2000 Oct 2, 19(19), 5041 - 50
Differential electron flow around photosystem I by two C(4)-photosynthetic-cell-specific ferredoxins; Kimata-Ariga Y et al.; In the C(4) plant maize (Zea mays L.), two ferredoxin isoproteins, Fd I and Fd II, are expressed specifically in mesophyll and bundle-sheath cells, respectively . cDNAs for these ferredoxins were introduced separately into the cyanobacterium Plectonema boryanum with a disrupted endogenous ferredoxin gene, yielding TM202 and KM2-9 strains expressing Fd I and Fd II . The growth of TM202 was retarded under high light (130 micromol/m(2)/s), whereas KM2-9 grew at a normal rate but exhibited a nitrogen-deficient phenotype . Measurement of photosynthetic O(2) evolution revealed that the reducing power was not efficiently partitioned into nitrogen assimilation in KM2-9 . After starvation of the cells in darkness, the P700 oxidation level under far-red illumination increased significantly in TM202 . However, it remained low in KM2-9, indicating an active cyclic electron flow . In accordance with this, the cellular ratio of ATP/ADP increased and that of NADPH/NADP(+) decreased in KM2-9 as compared with TM202 . These results demonstrated that the two cell type-specific ferredoxins differentially modulate electron flow around photosystem I.

J Theor Biol, 2000 Oct 21, 206(4), 539 - 60
Cytokine-modulated regulation of helper T cell populations; Yates A et al.; Helper T (Th) cells are a crucial component of the adaptive immune system and are of fundamental importance in orchestrating the appropriate response to pathogenic challenge . They fall into two broad categories defined by the cytokines each produces . Th1 cells produce interferon- gamma and are required for effective immunity to intracellular bacteria, viruses and protozoa whereas Th2 produce IL-4 and are required for optimal antibody production to T-dependent antigens . A great deal of experimental data on the regulation of Th1 and Th2 differentiation have been obtained but many essential features of this complex system are still not understood . Here we present a mathematical model of Th1/Th2 differentiation and cross regulation . We model Fas-mediated activation-induced cell death (AICD) as this process has been identified as an important mechanism for limiting clonal expansion and resolving T cell responses . We conclude that Th2 susceptibility to AICD is important for stabilizing the two polarized arms of the T helper response, and that cell-cell killing, not suicide, is the dominant mechanism for Fas-mediated death of Th1 effectors . We find that the combination of the anti-proliferative effect of the cytokine TGF- beta and the inhibiting influence of IL-10 on T cell activation are crucial controls for Th2 populations . We see that the strengths of the activation signals for each T helper cell subset, which are dependent on the antigen dose, co-stimulatory signals and the cytokine environment, critically determine the dominant helper subset . Switches from Th1- to Th2-dominance may be important in chronic infection and we show that this phenomenon can arise from differential AICD susceptibility of T helper subsets, and asymmetries in the nature of the cross-suppressive cytokine interactions . Our model suggests that in some senses a predominantly type 2 reaction may well be the "default" pathway for an antigen-specific immune response, due to these asymmetries .

Heredity, 2000 Aug, 85 ( Pt 2), 191 - 8
Wolbachia segregation rate in Drosophila simulans naturally bi-infected cytoplasmic lineages; Poinsot D et al.; Wolbachia are maternally transmitted endocellular bacteria infecting several arthropod species . In order to study Wolbachia segregation rate, Drosophila simulans females from an Indo-Pacific population (Seychelles) bi-infected by the two Wolbachia variants wHa and wNo were backcrossed to uninfected males in two conditions . In the first case, Seychelles males from a stock cured from its Wolbachia by tetracycline treatment were used . In the second case, the males came from a naturally uninfected Tunisian population . It was found that (i) the two Wolbachia variants can segregate, so that bi-infected females can produce a few offspring infected only by wHa or wNo . This occurs in both backcross conditions . (ii) Segregation leads more frequently to wHa than to wNo mono-infection . (iii) Wolbachia transmission is lower when the Seychelles genome is introgressed by the Tunisian genome, suggesting that host genomic factors might influence infection fate.

Heredity, 2000 Aug, 85 ( Pt 2), 130 - 5
Transinfection of Wolbachia in the mediterranean flour moth, Ephestia kuehniella, by embryonic microinjection; Sasaki T et al.; Wolbachia are maternally transmitted intracellular bacteria found in many arthropod species . They cause a reproductive incompatibility called cytoplasmic incompatibility (CI) in several hosts, including the Mediterranean flour moth, Ephestia kuehniella . Two strains of E . kuehniella, one from Yokohama city and the other from Tsuchiura city, express different levels of CI: the Yokohama strain expresses CI at a higher level than the Tsuchiura strain . In order to determine whether the difference of CI levels depends on Wolbachia or the host, we performed transinfection experiments in E . kuehniella by means of embryonic microinjection, and successfully transferred Wolbachia carried by the Yokohama strain into the Tsuchiura strain, from which the original Wolbachia had been removed by tetracycline treatment . The resulting transinfected strain expressed CI at a level near that of the Yokohama strain, suggesting that, in these strains of E . kuehniella, the level of CI is determined by Wolbachia rather than by the host.

Eur J Biochem, 2000 Oct, 267(20), 6102 - 9
Physiological functions of thioredoxin and thioredoxin reductase; Arner ES et al.; Thioredoxin, thioredoxin reductase and NADPH, the thioredoxin system, is ubiquitous from Archea to man . Thioredoxins, with a dithiol/disulfide active site (CGPC) are the major cellular protein disulfide reductases; they therefore also serve as electron donors for enzymes such as ribonucleotide reductases, thioredoxin peroxidases (peroxiredoxins) and methionine sulfoxide reductases . Glutaredoxins catalyze glutathione-disulfide oxidoreductions overlapping the functions of thioredoxins and using electrons from NADPH via glutathione reductase . Thioredoxin isoforms are present in most organisms and mitochondria have a separate thioredoxin system . Plants have chloroplast thioredoxins, which via ferredoxin-thioredoxin reductase regulates photosynthetic enzymes by light . Thioredoxins are critical for redox regulation of protein function and signaling via thiol redox control . A growing number of transcription factors including NF-kappaB or the Ref-1-dependent AP1 require thioredoxin reduction for DNA binding . The cytosolic mammalian thioredoxin, lack of which is embryonically lethal, has numerous functions in defense against oxidative stress, control of growth and apoptosis, but is also secreted and has co-cytokine and chemokine activities . Thioredoxin reductase is a specific dimeric 70-kDa flavoprotein in bacteria, fungi and plants with a redox active site disulfide/dithiol . In contrast, thioredoxin reductases of higher eukaryotes are larger (112-130 kDa), selenium-dependent dimeric flavoproteins with a broad substrate specificity that also reduce nondisulfide substrates such as hydroperoxides, vitamin C or selenite . All mammalian thioredoxin reductase isozymes are homologous to glutathione reductase and contain a conserved C-terminal elongation with a cysteine-selenocysteine sequence forming a redox-active selenenylsulfide/selenolthiol active site and are inhibited by goldthioglucose (aurothioglucose) and other clinically used drugs.

Microb Comp Genomics, 2000, 5(1), 25 - 39
Sequencing of the Francisella tularensis strain Schu 4 genome reveals the shikimate and purine metabolic pathways, targets for the construction of a rationally attenuated auxotrophic vaccine; Karlsson J et al.; Francisella tularensis is the etiological agent of tularemia, a serious disease in several Northern hemisphere countries . The organism has fastidious growth requirements and is very poorly understood at the genetic and molecular levels . Given the lack of data on this organism, we undertook the sample sequencing of its genome . A random library of DNA fragments from a highly virulent strain (Schu 4) of F . tularensis was constructed and the nucleotide sequences of 13,904 cloned fragments were determined and assembled into 353 contigs . A total of 1.83 Mb of nucleotide sequence was obtained that had a G+C content of 33.2% . Genes located on plasmids pOM1 and pNFL10, which had been previously isolated from low virulence strains of F . tularensis, were absent but all of the other known F . tularensis genes were represented in the assembled data . F . tularensis Schu4 was able to grow in the absence of aromatic amino acids and orthologues of genes which could encode enzymes in the shikimate pathway in other bacteria were identified in the assembled data . Genes that could encode all of the enzymes in the purine biosynthetic and most of the en- zymes in the purine salvage pathways were also identified . This data will be used to develop defined rationally attenuated mutants of F . tularensis, which could be used as replacements for the existing genetically undefined live vaccine strain.

Ann Dermatol Venereol, 2000 Aug-Sep, 127(8-9), 729 - 31
{Microsporum canis mycetoma of the scalp}; Vezon G et al.; BACKGROUND: Mycetoma is a chronic subcutaneous tumefaction with presence of grains or granules . Etiological agents include bacteria or filamentous fungi . Mycetoma due to dermatophytes is uncommon, mainly occurring in Africa . To our knowledge, no case has been reported in the West Indies . Only two observations of Micosporum canis mycetoma in humans have been reported in the literature . We report a third case of mycetoma of the scalp caused by this fungus . CASE REPORT: A 22-year-old woman from Martinique, French West Indies, presented with an indolent tumefaction of the scalp evolving over five years . She had mental retardation due to congenital adrenal hyperplasia with 21-hydroxylase deficiency . The lesion was extracted surgically . Pathology and mycology examinations showed features of Microsporum canis mycetoma . Two months later, the scalp lesion recurred and the patient was treated with griseofulvin after surgical extraction . DISCUSSION: Mycetoma due to dermatophytes is very uncommon, mainly observed on the scalp and nape of the neck . A history of a skin lesion is frequent, leading to transcutaneous penetration of the fungus and mycetoma formation . Several dermatophyte species have been identified as causal agents (Microsporum ferrugineum, Trichophyton rubrum, Trichophyton verrucosum, Trichophyton mentagrophytes, Microsporum audouinii, Microsporum langeronii) . Microsporum canis is rarely demonstrated in humans: two cases in children in Africa and Australia . Our observation was similar to the two cases in the literature: indolent and mobile tumefaction of the scalp, in a child or young adult, suggestive of lipoma or epidermal cyst, with excision leading to diagnosis . Association with tinea capitis and skin or nail involvement can also be observed.

Q Rev Biol, 2000 Sep, 75(3), 261 - 75
Kin selection and parasite evolution: higher and lower virulence with hard and soft selection; Chao L et al.; Conventional models predict that low genetic relatedness among parasites that coinfect the same host leads to the evolution of high parasite virulence . Such models assume adaptive responses to hard selection only . We show that if soft selection is allowed to operate, low relatedness leads instead to the evolution of low virulence . With both hard and soft selection, low relatedness increases the conflict among coinfecting parasites . Although parasites can only respond to hard selection by evolving higher virulence and overexploiting their host, they can respond to soft selection by evolving other adaptations, such as interference, that prevent overexploitation . Because interference can entail a cost, the host may actually be underexploited, and virulence will decrease as a result of soft selection . Our analysis also shows that responses to soft selection can have a much stronger effect than responses to hard selection . After hard selection has raised virulence to a level that is an evolutionarily stable strategy, the population, as expected, cannot be invaded by more virulent phenotypes that respond only to hard selection . The population remains susceptible to invasion by a less virulent phenotype that responds to soft selection, however . Thus, hard and soft selection are not just alternatives . Rather, soft selection is expected to prevail and often thwart the evolution of virulence in parasites . We review evidence from several parasite systems and find support for soft selection . Most of the examples involve interference mechanisms that indirectly prevent the evolution of higher virulence . We recognize that hard selection for virulence is more difficult to document, but we take our results to suggest that a kin selection model with soft selection may have general applicability.

Mikrobiologiia, 2000 Jul-Aug, 69(4), 559 - 64
{Characteristics of bacterioplankton in the Loess-containing Lake Khanka}; Shchur LA et al.; Some characteristics of bacterioplankton--generation time, daily (P) and specific (P/B) bacterioplankton production, and bacterial metabolic coefficient K2--in the loess-containing Lake Khanka were determined using five modifications of the bacterial-count procedure with the fluorescent dyes fluorescamin and erythrosin . Experiments showed that the organomineral complex (OMC) in this lake is broken down by chemoorganoheterotrophic bacteria . The increase in the loess content of the lake water intensified bacterial growth and the cycles of potassium, silicon, and other biogenic elements . The addition of starch to a loess suspension activated the breakdown of OMC due to the adsorption of starch on the OMC/water interface and stimulation of the metabolism of attached bacteria.

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