Bacteriological

Environ Int, 2003 Mar, 28(8), 779 - 82
Effect of heavy metals and PAH on soil assessed via dehydrogenase assay; Irha N et al.; Dehydrogenase enzyme activity (DHA) assay method using resazurin was accommodated for measuring of toxicity of compound contaminants on uncharacterized microbial communities present in any given soil . The method was used to compare the toxic effect of heavy metal and polycyclic aromatic hydrocarbon (PAH) contaminant mixture (Cr, Pb, Cu, Cd, Pyrene) on four typical Estonian soils covering a range of compositions . The method proved to be useable on all soils; the sensitivity of soil microbiology to toxic effect of contaminants was found to have a negative correlation with Ca and organic matter (OM) content and a positive correlation with amorphous phase content of soils.

Pharmacogenomics, 2003 Mar, 4(2), 163 - 70
Applications of pharmacogenomics in general dental practice; Slavkin HC; The human oral cavity represents a complex ecology of approximately 500 microbial species existing as commensals, and interacting with human/host factors . Perturbation of this ecosystem can lead to diseases of the oral cavity . The oral cavity also acts as a mirror of complex systemic diseases . Unique challenges and opportunities in oral healthcare exist in the diagnosis and treatment of primary disorders of the oral cavity or manifestations of systemic diseases . The completion of human and microbial genome projects has provided a wealth of information that will permit the application of pharmacogenomics - how genetic variations within the commensal and the host will impact the efficacy of drugs, and the diagnosis and treatment of oral disease . This review highlights opportunities focusing on oral health where pharmacogenomics can be applied.

Biosens Bioelectron, 2003 Apr, 18(4), 327 - 34
Operational parameters affecting the performannce of a mediator-less microbial fuel cell; Gil GC et al.; A mediator-less microbial fuel cell was optimized in terms of various operating conditions . Current generation was dependent on several factors such as pH, resistance, electrolyte used, and dissolved oxygen concentration in the cathode compartment . The highest current was generated at pH 7 . Under the operating conditions, the resistance was the rate-determining factor at over 500 omega . With resistance lower than 500 omega, proton transfer and dissolved oxygen (DO) supply limited the cathode reaction . A high strength buffer reduced the proton limitation to some extent . The DO concentration was around 6 mg l(-1) at the DO limited condition . The fact that oxygen limitation was observed at high DO concentration is believed to be due to the poor oxygen reducing activity of the electrode used, graphite . The current showed linear relationship with the fuel added at low concentration, and the electronic charge was well correlated with substrate concentration from up to 400 mg l(-1) of COD(cr) . The microbial fuel cell might be used as a biochemical oxygen demand (BOD) sensor .

J Invest Dermatol, 2003 Mar, 120(3), 379 - 89
The cathelicidin anti-microbial peptide LL-37 is involved in re-epithelialization of human skin wounds and is lacking in chronic ulcer epithelium; Heilborn JD et al.; The human cathelicidin anti-microbial protein, hCAP18 is a component of the innate immune system and has broad anti-microbial activity conferred by its C-terminal fragment LL-37 . hCAP18 is constitutively produced in leukocytes and is induced in barrier organs upon inflammation and infection . We demonstrate here a novel role for this peptide in re-epithelialization of skin wounds . We show that high levels of hCAP18 are produced in skin in vivo upon wounding . The highest hCAP18 levels are attained at 48 h post-injury, declining to pre-injury levels upon wound closure . hCAP18 is detected in the inflammatory infiltrate and in the epithelium migrating over the wound bed . In chronic ulcers, however, hCAP18 levels are low and immunoreactivity for hCAP18/LL-37 is absent in ulcer edge epithelium . Using a noninflammatory ex vivo wound healing model, composed of organ-cultured human skin, we show that hCAP18 is strongly expressed in healing skin epithelium, and that treatment with antibodies raised and affinity purified against LL-37, inhibits re-epithelialization in a concentration-dependent manner . Immunoreactivity for the proliferation marker Ki67 is absent in the epithelium of such inhibited wounds, suggesting that LL-37 may play a part in epithelial cell proliferation . Thus, we suggest that, in addition to being an anti-microbial peptide, LL-37 also plays a part in wound closure and that its reduction in chronic wounds impairs re-epithelialization and may contribute to their failure to heal.

Immunology, 2003 Mar, 108(3), 321 - 8
Immunomodulatory effects of cyclosporin A on human peripheral blood dendritic cell subsets; Tajima K et al.; Cyclosporin A (CsA) is a potent immuno-suppressant and is approved for the treatment of various disease conditions . The molecular biological mechanism of CsA has been investigated intensively in T cells and has been shown to involve the intracellular calcineurin pathway . Recently, it was reported that CsA has capacities to affect not only T cells but also antigen-presenting cells such as B cells and dendritic cells (DCs) . DCs are a master regulator of immune responses that have an integral capacity to prime naive T cells . In the present study, we investigated the biological effects of CsA on human peripheral blood DC subsets: CD11c+ myeloid and CD11c- lymphoid subsets . CsA inhibited the up-regulation of co-stimulatory molecules induced with or without microbial stimuli and CD40L on both CD11c+ and CD11c- subsets . In addition, CsA negatively regulated the endocytic activity of CD11c+ DC during the immature state . CsA inhibited the interleukin-12 (IL-12) production, but augmented the IL-10 production from the LPS-stimulated CD11c+ subset, whereas CsA reduced the interferon-alpha (IFN-alpha) production from the CD11c- subset infected with Sendai virus (SV) . Both the LPS-stimulated CD11c+ subset and SV-infected CD11c- subset preferentially induced the development of IFN-gamma-producing T helper-type 1 (Th1) cells . Pretreatment of these DC subsets with CsA inhibited the Th1 skewing . These findings suggested a DC-mediated mechanism of immunosuppression by CsA.

Infect Control Hosp Epidemiol, 2003 Feb, 24(2), 142 - 4
Monsel's solution: a potential vector for nosocomial infection?
Rupp ME, Medcalf SJ, Fey PD, Handke LD, Marion ND.
Monsel's solution is a common topically applied hemostatic agent used in minor dermatologic and gynecologic surgery . Clinically, because it is often stored for long periods and dispensed from a common source for multiple patients, Monsel's solution is a potential vector for transmission of infection . However, microbiologic inoculation studies and contamination surveys indicate that Monsel's solution has properties that prohibit microbial growth, making it an unlikely vector for nosocomial infection.

Ann Allergy Asthma Immunol, 2003 Feb, 90(2), 265 - 70
Hypersensitivity pneumonia-nonspecific interstitial pneumonia/fibrosis histopathologic presentation: a study in diagnosis and long-term management; Jacobs RL et al.; BACKGROUND: Nonspecific interstitial pneumonia/fibrosis (NSIP) has been classified a form of idiopathic interstitial pneumonia/fibrosis . We have shown that cases of NSIP without demonstrable serum precipitins may be caused by inhalation of high levels of mold and/or bacteria in closed environments . OBJECTIVE: We report a patient with a clinical and histopathologic diagnosis of NSIP without serum precipitins caused by a microbial contamination in her home . Her case was converted from an acute to an insidious clinical presentation by inadequate remediation . A prolonged avoidance-challenge technique demonstrated that this case of NSIP was a form of hypersensitivity pneumonia that was reversible by effective remediation . METHODS: The patient was identified by compatible signs and symptoms, roentgenographic studies, pulmonary function tests, and a transbronchial lung biopsy . She was further evaluated with a detailed environmental history, serologic tests, and investigation of the home environment . An environmental avoidance and challenge technique was performed to confirm cause and effect and to determine that remediation had been effective . RESULTS: Review of the biopsy showed NSIP and failed to reveal any non-caseating granuloma formation . Investigation of the home revealed a Cladosporium species contamination of the air conditioning system and Penicillium species beneath an entryway carpet . Serum precipitins to commercial antigens of common mold to the south Texas area were negative . Avoidance and challenge techniques confirmed the home as the causative environment in this case of NSIP . The patient has been free of signs and symptoms and has taken no medication for interstitial lung disease over the past 30 months . CONCLUSIONS: Some cases of NSIP may be caused by inhalation of microbial antigen(s) in a closed environment . An environmental challenge technique was an effective method to determine the causative environment and confirm that remediation had been effective . Inadequate remediation may lead to symptomatic improvement, but may convert a patient from an acute to an insidious presenter . The environmental challenge obviates a need for specific challenges to determine specific causation . Remediation of or moving from an environmental contamination to achieve reversibility or prevent progression was the treatment of choice to avoid use of long-term immunosuppressive agents.

Phytother Res, 2003 Feb, 17(2), 97 - 106
Garlic as an antioxidant: the good, the bad and the ugly; Banerjee SK et al.; Garlic has played an important dietary and medicinal role throughout the history of mankind . In some Western countries, the sale of garlic preparations ranks with those of leading prescription drugs . The therapeutic efficacy of garlic encompasses a wide variety of ailments, including cardiovascular, cancer, hepatic and microbial infections to name but a few . However, the elucidation of its mechanism for therapeutic action has proved to be more elusive and a unifying theory, which could account for its reported multifarious activities, is yet to emerge . Reactive oxygen species (ROS) seem to be at the core of many disease processes and it is an attractive and convenient hypothesis that garlic might exert its activities through modulatory effects on ROS . A literature search on garlic and its antioxidant potential churned up a surprisingly large amount of data, some of it good, some bad and some of its definitely ugly.Various preparations of garlic, mainly aged garlic extract (AGE), have been shown to have promising antioxidant potential . However, the presence of more than one compounds in garlic, with apparently opposite biological effects, has added to the complexity of the subject . Raw garlic homogenate has been reported to exert antioxidant potential but higher doses have been shown to be toxic to the heart, liver and kidney.So where do we stand today on this issue of garlic? Is garlic always good for health? How safe is it? Is it necessary to isolate the antioxidant compounds for its medicinal use in a more effective way? These issues are addressed in this review .

Soc Sci Med, 2003 Mar, 56(6), 1279 - 93
Different frames, different fears: communicating about chlorinated drinking water and cancer in the Canadian media; Driedger SM et al.; Risk issues become complicated when scientific evidence concerning a potential environmental exposure is equivocal; particularly when many argue that the public health benefits of a policy action outweigh any potential negative health effects . Chlorinated drinking water, and chlorinated disinfection byproducts (CDBPs) that are formed during the disinfection process, represent a useful case-study for examining these complications . We conduct a media analysis of chlorinated drinking water stories in the Canadian print media from 1977 to 2000 . We examine media presentations of science compared to framings by scientists, regulators, the chlorine industry, water utility representatives, and non-governmental organizations of the CDBP issue based on key informant interviews . We argue that there are two main framings of the debate, each of which are powerful in constructing risk perceptions . On the one hand, many frame the debate as a 'voluntary' risk: we choose chlorine disinfection to protect against microbial risks with a possible adverse consequence of that protection . On the other hand, others frame the issue as an 'involuntary' risk: chlorine disinfection was a 'choice' imposed by public health and water utility officials; a choice that carries a potential cancer risk, and alternative disinfection technologies are advocated . We demonstrate these different frames by examining metaphorical constructs of water, chlorine and cancer contained within them.

AIDS, 2003 Mar 7, 17(4), 495 - 504
Interferon-alpha restores HIV-induced alteration of natural killer cell perforin expression in vivo; Portales P et al.; OBJECTIVE: The percentage and the activity of natural killer (NK) cells are known to be decreased in HIV-infected patients . However, the mechanisms responsible for this NK deficiency are poorly understood . Because of the role of NK cells in the host defence against microbial infections, this defect contributes to the virus-induced immune deficiency . The aim of the present study was to better understand this defect in order to be able to restore NK function in HIV infection . DESIGN AND METHODS: The expression of the cytolytic mediators perforin and granzyme A was analysed by flow cytometry, the lytic activity of peripheral blood NK cells of HIV-infected patients was analysed by cytotoxic assay, and the expression of perforin was followed during administration of interferon (IFN)alpha attached to polyethylene glycol (PEG)-IFNalpha . RESULTS: The lytic activity and the expression of perforin and granzyme A was low in NK cells of infected individuals in comparison with normal control volunteers . In both groups NK cytotoxic capacity was linked to perforin expression . The low perforin expression in HIV-infected subjects negatively correlated with HIV RNA plasma level . administration of PEG-IFNalpha restored perforin expression even in patients whose viral load was not reduced by this treatment . CONCLUSIONS: These results suggest that HIV-induced NK deficiency could be partly mediated by a defect in perforin and granzyme A expression, and that PEG-IFNalpha could be used in infected subjects to directly improve their natural immunity in addition to eventually reducing their viraemia.

Microsc Microanal, 2001 Nov, 7(6), 470 - 485
In Situ Environmental Cell-Transmission Electron Microscopy Study of Microbial Reduction of Chromium(VI) Using Electron Energy Loss Spectroscopy; Daulton TL et al.; Reduction of Cr(VI) by the bacterium, Shewanella oneidensis (previously classified Shewanella putrefaciens strain MR-1), was studied by absorption spectrophotometry and in situ, environmental cell-transmission electron microscopy (EC-TEM) coupled with electron energy loss spectroscopy (EELS) . Bacteria from rinsed cultures were placed directly in the environmental cell of the transmission electron microscope and examined under 100 Torr pressure . Bright field EC-TEM images show two distinct populations of S . oneidensis in incubated cultures containing Cr(VI)O2- 4: those that exhibit low image contrast and heavily precipitate-encrusted cells exhibiting high image contrast . Several EELS techniques were applied to determine the oxidation state of Cr associated with encrusted cells . The encrusted cells are shown to contain a reduced form of Cr in oxidation state +3 or lower . These results demonstrate the capability to determine the chemistry and valence state of reduction products associated with unfixed, hydrated bacteria in an environmental cell transmission electron microscope.

Crit Care Nurs Clin North Am, 2003 Mar, 15(1), 89 - 96, ix
Economics of antibiotic administration; Sommers BD; This article examines several elements of antibiotic administration that make it worthy of policy analysis, including microbial resistance, contagion, competing brand and generic drugs, and formulary restrictions by insurers and hospitals . These topics are explored using two concepts from health economics, cost-effectiveness and externalities, revealing theoretical and empirical evidence that society may not be using antibiotics as efficiently as it could.

Life Sci Space Res, 1977, 15, 59 - 63
Determinations of microbial loads associated with microscopic-size particles of Kennedy Space Center soil; Ruschmeyer OR et al.; Plate counts for six fractions, of micrometer-size, of Kennedy Space Center soil provided estimates of aerobic, mesophilic, heterotrophic, microbial loads on single soil particles . Analyses included unheated particles, particles subjected to wet heat at 80 degrees C for 20 min, and particles subjected to dry heat at 110 degrees C for 1 hr . Unheated particles yielded mean counts ranging from 6 colonies per particle for the smallest (44-53 micrometers) soil fraction to approximately 55 colonies per particle for the largest size (105-125 micrometers) soil fraction tested . Mean counts for heat-resistant forms ranged from 2 colonies per particle for the smaller particles to 12-15 colonies for the largest particles analyzed.

Neurochirurgie, 2002 Dec, 48(6), 516 - 21
{Nocardia farcinica brain abscess: clinical and specific radiological findings and management . Report of two cases in immunononcompromised patients}; Vialle R et al.; Nocardia spp . cerebral abscesses are rare and usually occur in immunocompromised patients . We report two recent cases of cerebral abscesses due to Nocardia farcinica in immunocompetent patients and review the literature about diagnosis and therapeutic issues . Outcome was good for the two patients following an early identification of the bacteria . Stereotactic biopsy was performed in one case and craniotomy with excision of the abscess in the other case . In both cases, complete identification of the bacteria could be achieved, followed by prolonged antibiotic therapy . Exposure to the germ (mainly telluric) is often difficult to suspect from the past medical history of the patient . Diagnosis is also unexpected . Despite the typically and characteristic aspect on CT and MRI, specific identification and anti-microbial sensitivity profiles are necessary to optimize treatment . In some rare cases, unusual species like Nocardia farcinica, can be resistant to numerous antibiotics requiring adjustments of medical management . Early identification of the bacteria is necessary to achieve good outcome in immunocompetents patients.

Philos Trans R Soc Lond B Biol Sci, 2003 Jan 29, 358(1429), 191 - 201; discussion 201-2
Mitochondria and hydrogenosomes are two forms of the same fundamental organelle; Embley TM et al.; Published data suggest that hydrogenosomes, organelles found in diverse anaerobic eukaryotes that make energy and hydrogen, were once mitochondria . As hydrogenosomes generally lack a genome, the conversion is probably one way . The sources of the key hydrogenosomal enzymes, pyruvate : ferredoxin oxidoreductase (PFO) and hydrogenase, are not resolved by current phylogenetic analyses, but it is likely that both were present at an early stage of eukaryotic evolution . Once thought to be restricted to a few unusual anaerobic eukaryotes, the proteins are intimately integrated into the fabric of diverse eukaryotic cells, where they are targeted to different cell compartments, and not just hydrogenosomes . There is no evidence supporting the view that PFO and hydrogenase originated from the mitochondrial endosymbiont, as posited by the hydrogen hypothesis for eukaryogenesis . Other organelles derived from mitochondria have now been described in anaerobic and parasitic microbial eukaryotes, including species that were once thought to have diverged before the mitochondrial symbiosis . It thus seems possible that all eukaryotes may eventually be shown to contain an organelle of mitochondrial ancestry, to which different types of biochemistry can be targeted . It remains to be seen if, despite their obvious differences, this family of organelles shares a common function of importance for the eukaryotic cell, other than energy production, that might provide the underlying selection pressure for organelle retention.

Eur J Immunol, 2003 Jan, 33(1), 101 - 7
The ability of murine dendritic cell subsets to direct T helper cell differentiation is dependent on microbial signals; Manickasingham SP et al.; Dendritic cells (DC) initiate T cell responses and direct the class of T cell immunity through the production of Th-polarizing cytokines . In the mouse, immunization with CD8alpha(+) DC has led to Th1 priming whereas immunization with CD8alpha(-) DC has been associated with Th2 induction . Here, we use a direct T cell priming assay in vitro to re-examine the Th-directing potential of total DC or purified CD4(+) DC, CD8alpha(+) DC or CD4(-) CD8alpha(-) (double-negative; DN) DC subsets from mouse spleen . We show that the default Th effector phenotype induced by priming with DC depends on the protocol used for T cell purification, the T cell:antigen-presenting cell ratio and the antigen dose but is only marginally affected by DC subtype . All DC subsets can direct increased Th1 development in response to microbial stimuli known to elicit IL-12 production . Similarly, all subsets can suppress Th1 development and allow Th2 cellsto expand upon exposure to IL-10-inducing microbial agents . The flexibility of DC in directing Th development in function of microbial signals argues against the notion of pre-determined "DC1" and "DC2" subsets and suggests that multiple DC subtypes can direct an appropriate Th response to different classes of infectious agents.

J Immunol, 2003 Mar 1, 170(5), 2409 - 16
The 19-kDa Mycobacterium tuberculosis protein induces macrophage apoptosis through Toll-like receptor-2; Lopez M et al.; Macrophages infected with Mycobacterium tuberculosis undergo increased rates of apoptosis . Important objectives are to define the microbial factors that cause apoptosis, the mechanisms involved and the impact on infection . The 19-kDa M . tuberculosis glycolipoprotein (p19) is both cell wall-associated and secreted and is a candidate virulence factor . We investigated the potential of recombinant, His-tagged p19 lacking the secretion/acylation signal to induce macrophage apoptosis . The TUNEL assay and annexin V binding to membrane phosphatidylserine were used to measure apoptosis . The results show that p19 does act to induce apoptosis in differentiated THP-1 cells and monocyte-derived macrophages and that this effect is both dose- and time-dependent . Furthermore, this effect of p19 is Toll-like receptor (TLR)-2-mediated because preincubation of either THP-1 cells or TLR-2-expressing CHO cells with anti-TLR-2 mAb inhibited apoptosis induced by p19 . Apoptosis of macrophages in response to p19 was found to be caspase-8 dependent and caspase-9 independent consistent with a transmembrane pathway signaling cell death through TLR-2 . The viability of M . tuberculosis in cells undergoing apoptosis induced by p19 was significantly reduced suggesting the possibility that this may favor containment of infection . Although native p19 is a mycobacterial glycolipoprotein, based upon the use of recombinant p19 where the acylation signal had been removed, we conclude that it is the polypeptide component of p19 that is responsible for signaling through TLR-2 and that the lipid moiety is not required.

J Periodontol, 2003 Jan, 74(1), 90 - 6
Molecular interaction of Porphyromonas gingivalis with host cells: implication for the microbial pathogenesis of periodontal disease; Amano A; Porphyromonas gingivalis is a predominant periodontal pathogen, which expresses a number of potential virulence factors involved in the pathogenesis of periodontitis . Among them, fimbriae are a critical factor to mediate the bacterial interaction with host tissues, which promotes the bacterial adhesion to and invasion of the targeted sites . Fimbriae are capable of binding to human salivary components, commensal bacteria, and a variety of host cells including macrophages, epithelial cells, and fibroblasts . Human extracellular matrix (ECM) proteins such as vitronectin and fibronectin play important roles in cellular signal transduction via binding to receptor integrins . Fimbriae showed significant binding affinity to ECM proteins and clearly inhibited the molecular interactions between vitronectin/fibronectin and their receptor alphavbeta3 and alpha5beta1 integrins overexpressed on Chinese hamster ovary (CHO) cell strain . P . gingivalis fimbriae are likely to interrupt the cellular signaling via ECM proteins/integrins in periodontal regions . Fimbriae are also thought to be critically important in invasive events of the organism to host cells . The fimA genes, encoding FimA (a subunit of fimbriae), of P . gingivalis strains are classified into 5 types, I to V . Recent clinical investigations demonstrated the close relationship between the organisms with type II fimA and periodontitis development . Recombinant FimA (rFimA) proteins of types I to V were generated to compare their adhesion/invasion abilities to human gingival fibroblasts (HGF) and a human epithelial cell line (HEp-2 cells), respectively . There were no significant differences in the adhesion ability of microspheres (MS) coated with these rFimAs to HGF; however, the adhesion of type II rFimA-MS to HEp-2 cells was significantly greater than that of other rFimA types . It was also observed that the type II rFimA-MS markedly invaded the epithelial cells and accumulated around the nuclei . Collectively, these findings suggest that fimbriae of P . gingivalis, especially type II, are involved in the initiation and progression of human periodontitis.

DNA Seq, 2002 Oct, 13(5), 301 - 6
Cloning and characterization of a novel human alcohol dehydrogenase gene (ADHFe1); Deng Y et al.; There are three types of NAD(P)(+)-dependent alcohol dehydrogenase: "medium-chain" zinc-containing enzymes, "short-chain" zinc-lacking enzymes and iron-activated enzymes . Although the medium-chain family and the short-chain family have been characterized in human, the iron-activated alcohol dehydrogenase family seems to exist only in microbial organisms . We have now cloned and characterized an iron-activated alcohol dehydrogenase gene, Fe-containing alcohol dehydrogenase 1 (ADHFe1) in human . The cDNA was isolated from the human fetal brain cDNA library . It contains a long open reading frame, which is not homologous with the other alcohol dehydrogenases isoenzymes characterized in human . The hypothetical alcohol dehydrogenase does, however, show strong homology to the iron-activated alcohol dehydrogenases from microbial organisms . Northern blotting analysis only detected expression in adult liver tissue . At least two different splicing variants were screened by PCR using the multiple tissue cDNA panels as templates.

Dermatology, 2003, 206(2), 96 - 105
The role of innate immunity in the pathogenesis of acne; Koreck A et al.; Acne is a multifactorial disease of the pilosebaceous follicle . The most significant pathogenetic factors of acne are: abnormal ductal keratinization, increased sebum secretion, abnormalities of the microbial flora and inflammation . The pilosebaceous unit is an immunocompetent organ . Keratinocytes and sebocytes may act as immune cells capable of pathogen recognition and abnormal lipid presentation, and they might have an important role in initiating and perpetuating the activation of both innate and adaptive immune responses . The elements of the skin immune system are involved in the development of both noninflammatory and inflammatory acne lesions .

Phytochemistry, 2003 Apr, 62(7), 1101 - 4
Microbial transformation of zaluzanin-D; Kumari GN et al.; Microbial transformation of zaluzanin-D using different fungi gave 11,13-dihydrozaluzanin-C, zaluzanin-C, 4,16,11,13 - tetrahydro zaluzanin-C, estafiatone, dihydroestafiatol and dihydroestafiatone.

J Agric Food Chem, 2003 Feb 26, 51(5), 1460 - 3
Factors Influencing texture retention of salt-free, acidified, red bell peppers during storage; Papageorge LM et al.; Red bell peppers were stored in a salt-free, acidified cover solution with sulfite as a microbial preservative . The texture retention of stored peppers was evaluated as a function of pH, acid, calcium, blanch treatment, and growing conditions of the peppers . Field-grown peppers softened at a faster rate than greenhouse-grown peppers and exhibited more variability in their ability to maintain texture during storage . Improved firmness retention of red peppers stored at 30 degrees C was observed when the pH was adjusted to be in the range of 3.4-3.8, when at least 5 mM calcium was added to the peppers, and when red peppers were blanched at 75 degrees C for at least 1 min.

J Agric Food Chem, 2003 Feb 26, 51(5), 1347 - 52
Fipronil metabolism and dissipation in a simplified aquatic ecosystem; Aajoud A et al.; Several phenylpyrazole derivatives are selective inhibitors of chloride channel activities in insects . In this chemical family, fipronil is a powerful insecticide now widely used for several purposes . The dissipation of this molecule in a simplified aquatic ecosystem has been studied for 3 months, using (14)C-labeled fipronil . The main features of the complex process leading to fipronil transformation in this system were the following . The fipronil aqueous solution was submitted to two chemical transformations: the photodependent desulfuration of the side chain bound to the 4-position of the heterocyclic ring and the chemical hydrolysis of the nitrile function bound to the 3-position . Fipronil, rapidly transferred from the water solution to the organic matter, was protected from the previously mentioned chemical transformations but evolved to give two main metabolites, which were either reduced or oxidized in the side chain on the 4-position . These derivatives were powerful insecticides as shown by LC(50) measurements on Aedes aegypti larvae (LC(50) for CF(3)-S-R and CF(3)-SO(2)-R = 8.8 nM) . During the course of this experiment, nitrile hydrolysis took place slowly, originating either from the chemical hydrolysis in the aqueous solution or from enzymatic hydrolysis inside the microbial biomass . The fipronil-amide (3-NH(2)-CO-R') derivative, although much more polar than fipronil itself, was mostly bound to the organic matter . Other more polar derivatives were also detected but in very small amounts . No (14)CO emission was observed during the experiment.

Clin Chim Acta, 2003 Mar, 329(1-2), 95 - 102
A new method to evaluate spontaneous platelet aggregation in type 2 diabetes by Cellfacts; Gabbianelli R et al.; BACKGROUND: The alterations in the functional activities of platelets in diabetes produce an increase of spontaneous platelet aggregation (SPA) and release of platelet-derived microparticles . Platelet-derived microparticles are shed from platelets during activation by high shear stress, collagen and certain agonists . Although the physiologic role of microparticles has been difficult to assess, the characterization of their biological activity is of interest in view of a possible role in hemostasis and coagulation and their reported involvement in thrombotic disease . METHODS: We propose a new, simple method to evaluate spontaneous platelet aggregation and release of platelet-derived microparticles by the Cellfacts analyser (Microbial System Limited (MSL), Coventry, England) that uses electrical sensing flow impedance determination to detect the size particles and the cells in a conductive fluid . Platelet-rich plasma (PRP) from type 2 diabetes was employed for this study . The importance of platelet-activating factor (PAF) on spontaneous platelet aggregation was evaluated and the effect of vitamin E and WEB 2086-BS, an antagonist of platelet-activating factor, was measured . RESULTS AND CONCLUSIONS: Data presented show that Cellfacts could be an easy and fast instrument to check the state of platelets in patients with alterations in the functionality of platelets, and to follow the effect of pharmacological therapy on spontaneous platelet aggregation and the release of platelet-derived microparticles.

Biochem Biophys Res Commun, 2003 Feb 21, 301(4), 1062 - 8
Inhibition of Epstein-Barr virus lytic cycle by (-)-epigallocatechin gallate; Chang LK et al.; (-)-Epigallocatechin gallate (EGCG), abundant in green tea, is a potent anti-microbial and anti-tumor compound . This investigation used immunoblot, flow cytometry, microarray, and indirect immunofluorescence analyses to show that at concentrations exceeding 50 microM, EGCG inhibits the expression of Epstein-Barr virus (EBV) lytic proteins, including Rta, Zta, and EA-D, but does not affect the expression of EBNA-1 . Moreover, DNA microarray and transient transfection analyses demonstrated that EGCG blocks EBV lytic cycle by inhibiting the transcription of immediate-early genes, thus inhibiting the initiation of EBV lytic cascade.

Oral Microbiol Immunol, 2003 Feb, 18(1), 59 - 65
Denaturing gradient gel electrophoresis analysis to study bacterial community structure in pockets of periodontitis patients; Zijnge V et al.; Bacteria are involved in the onset and progression of periodontitis . A promising molecular technique, denaturing gradient gel electrophoresis (DGGE), to study microbial population dynamics in the subgingival pocket is presented . Twenty-three samples were taken from the subgingival pockets of nine patients and six healthy family members . From four periodontitis patients, 12 samples were evaluated before, 1 day after and 3 months after treatment . Part of the 16S rRNA gene of all bacteria was amplified by PCR and separated by DGGE, creating banding patterns representative of the community structure . Shifts in composition and diversity of the microbial population could be determined semiquantitatively, and this showed that treatment resulted in a decrease in the diversity of the population . After 3 months a microbial population 33-47% different from the population before treatment had re-established . Intense bands representing Exiguobacterium aurantiacum were present in 13 out of 25 samples, indicating that this species may play a role in periodontal disease.

FEBS Lett, 2003 Feb 11, 536(1-3), 77 - 84
Unexpected catalytic site variation in phosphoprotein phosphatase homologues of cofactor-dependent phosphoglycerate mutase; Rigden DJ; The cofactor-dependent phosphoglycerate mutase (dPGM) superfamily contains, besides mutases, a variety of phosphatases, both broadly and narrowly substrate-specific . Distant dPGM homologues, conspicuously abundant in microbial genomes, represent a challenge for functional annotation based on sequence comparison alone . Here we carry out sequence analysis and molecular modelling of two families of bacterial dPGM homologues, one the SixA phosphoprotein phosphatases, the other containing various proteins of no known molecular function . The models show how SixA proteins have adapted to phosphoprotein substrate and suggest that the second family may also encode phosphoprotein phosphatases . Unexpected variation in catalytic and substrate-binding residues is observed in the models.

Syst Appl Microbiol, 2002 Dec, 25(4), 555 - 71
Improvement of ciliate identification and quantification: a new protocol for fluorescence in situ hybridization (FISH) in combination with silver stain techniques; Fried J et al.; A new protocol for taxon specific probe based fluorescent in situ hybridization was developed for the identification and quantification of ciliates in microbial communities . Various fixatives and experimental parameters were evaluated and optimized with respect to cell permeability and morphological preservation . Optimum results were adaption by obatined of a modified fixation method using Bouin's solution . Furthermore, conventional staining procedures such as different Protargol stain techniques and a silver nitrate impregnation method were modified and can now be applied in combination with fluorescence in situ hybridization . The new protocol allows a rapid and reliable identification as well as quantification of ciliates based upon classical morphological aspects and rRNA based phylogenetic relationships performed in one experiment . Furthermore, a set of specific probes targeting different regions of the 18S rRNA was designed for Glaucoma scintillans Ehrenberg, 1830 and tested by applying this new approach of combining in situ cell hybridization with conventional staining techniques.

Chirality, 2003 Mar, 15(3), 220 - 6
Lipase regio- and stereoselectivities toward three enantiomeric pairs of didecanoyl-deoxyamino-O methyl glycerol: a kinetic study by the monomolecular film technique; Douchet I et al.; A kinetic study was carried out on the regio- and stereoselectivities of 12 lipases of animal and microbial origin . For this purpose, monomolecular films consisting of three pairs of enantiomers (didecanoyl-deoxyamino-O methyl glycerol, DDG) containing a single hydrolyzable decanoyl ester bond and two lipase-resistant groups were spread at the air-water interface . Each of the lipases tested displayed a particular type of behavior, on the basis of which they were classified in two groups, depending on their ability to hydrolyze the sn-2 position . From the qualitative point of view, the sn-2 preference measured on triacylglycerides and DDG were in good agreement . The inductive chemical effect might explain why a greater level of hydrolytic activity was observed with the diglycerides than with DDG . With most of the lipases tested, it was observed that the enantiomeric pair having two distal acyl chains was more clearly differentiated stereochemically than the two homologous pairs with two adjacent acyl chains . This finding is consistent with the hypothesis that during the chiral recognition process two of the three attachment points may be the external (distal) hydrophobic chains, which is in line with the hypothesis of a tuning fork conformation of a triglyceride in the lipase active site .

AIDS Res Hum Retroviruses, 2003 Jan 1, 19(1), 21 - 9
HIV-mediated gammadelta T cell depletion is specific for Vgamma2+ cells expressing the Jgamma1.2 segment; Enders PJ et al.; Circulating Vgamma2/Vdelta2(+) T cells, normally constituting 3-6% of all CD3(+) T cells in blood, are severely depleted after HIV infection . The mechanism(s) for Vgamma2/Vdelta2(+) T cell depletion are unknown, partly because these cells are CD4(-) and resistant to HIV infection . To determine whether this cell depletion was general for all Vgamma2(+) cells or specific for an individual subset, we analyzed the Vgamma2 repertoire and found consistent differences between HIV(+) and uninfected control samples . The change in Vgamma2 repertoire was the result of preferentially depleting only those Vgamma2 cells that express the Jgamma1.2 segment . The specific loss of Vgamma2-Jgamma1.2(+) cells was polyclonal, as the Vgamma subset retained normal diversity even after HIV infection, and loss occurred without significant changes in the paired chain (Vdelta2) repertoire, or in the alternate Vdelta1 chain repertoire . Specific depletion of Vgamma2-Jgamma1.2/Vdelta2 T cells is the first evidence of a common, T cell receptor-dependent cell loss in HIV disease and it provides a clear example of bystander cell depletion . Vgamma2-Jgamma1.2/Vdelta2 T cells mediate potent responses to microbial pathogens including HIV, and loss of this subset is an important aspect of AIDS pathogenesis.

Lett Appl Microbiol, 2003, 36(3), 140 - 4
Pulsed electric fields cause sublethal injury in Escherichia coli; Garcia D et al.; AIMS: The objective was to investigate the occurrence of sublethal injury in Escherichia coli by pulsed electric fields (PEF) at different pH values . METHODS AND RESULTS: The occurrence of sublethal injury in PEF-treated E . coli cells depended on the pH of the treatment medium . Whereas a slight sublethal injury was detected at pH 7, 99.95% of survivors were injured when cells were treated at pH 4 for 400 micros at 19 kV . The PEF-injured cells were progressively inactivated by a subsequent holding at pH 4 . CONCLUSIONS: PEF cause sublethal injury in E . coli . The measurement of sublethal injury using a selective medium plating technique allowed prediction of the number of cells that would be inactivated by subsequent storage in acidic conditions . SIGNIFICANCE AND IMPACT OF THE STUDY: This work could be useful for improving food preservation by PEF technology and contributes to the knowledge of the mechanism of microbial inactivation by PEF.

Poult Sci, 2003 Jan, 82(1), 92 - 9
Effects of phytase supplementation on the performance, egg quality, and phosphorous excretion of laying hens fed different levels of dietary calcium and nonphytate phosphorous; Lim HS et al.; An experiment employing a factorial arrangement of two levels (3.0 and 4.0%) of Ca, two levels (0.15 and 0.25%) of nonphytate phosphorus (NPP), and two levels (0 and 300 U/kg diet) of microbial phytase was carried out with 960 ISA-brown layers from 21 to 41 wk of age . There was a significant interaction between NPP level and phytase for egg production . High NPP level and phytase supplementation increased egg production only in the second 10-wk period (31 to 41 wk) . High NPP and low Ca increased feed intake, and a significant interaction between levels of NPP and Ca was observed in the first 10 wk . High NPP improved feed efficiency only in the second 10-wk period . Low NPP improved egg specific gravity and eggshell thickness but decreased Haugh units in the first 10-wk period; high NPP decreased the percentage of broken and soft-shell eggs in the second period . Low Ca decreased egg specific gravity, eggshell strength, and eggshell thickness in both periods and increased Haugh units in the second 10-wk period . Phytase supplementation decreased the percentage of broken and soft-shell eggs . High NPP increased fiber availability but decreased Ca availability . High Ca decreased Ca availability, whereas phytase increased availability of dry matter, fiber, and P . High NPP increased retention of P and Fe but also increased excretion of P . High Ca decreased retention of Zn and Fe . Phytase supplementation increased P retention, resulting in decrease of P excretion . In conclusion, supplementation of microbial phytase at a level of 300 U per kg diet of laying hens can improve egg production, decrease broken and soft egg production rate, and P excretion . The effects of phytase supplementation are significantly modified by the level of Ca and NPP.

Int J Mol Med, 2003 Mar, 11(3), 395 - 400
Stem cell transplantation for treatment of liver diseases: from biological foundations to clinical experience (review); Sukhikh GT et al.; The insufficiency of liver functions remains one of the major causes of death in different regions of the world . This syndrome is an ultimate outcome of irreversible damage of liver parenchyma by a variety of stimuli such as environmental toxins and microbial metabolites . The acute and/or chronic inflammation caused by these agents leads to massive apoptosis of hepatocytes, highly specialized polyfunctional epithelial cells . A decrease in the number of hepatocytes below the critical level dictates the necessity of supplanting the exogenous viable cells . Such cells must fulfill two major requirements: i) they should be capable of proliferating in the recipient's liver, and ii) they should differentiate into mature (i.e, functionally competent) hepatocytes . Evidence is growing in support of the role of early pluripotent precursors (stem cells of various origin) as a unique source for transplantation . This review analyses the state-of-the-art in the fields of developmental biology, cytology, molecular physiology and biochemistry of fetal stem cells with the emphasis on their use in experimental models of liver insufficiency . These biological considerations validate the therapeutic manipulations with fetal cells in clinical trials.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2001 Jun, 9(2), 153 - 159
{Operating Procedure of Collection, Processing and Preservation of 3000 Units Umbilical Cord Blood in Shangdong Cord Blood Bank}; Zhou SL et al.; The experience with the umbilical cord blood (UCB) stem cells for unrelated transplantation from our 3 000 UCB storage was described . UCB, collected from closed blood bags, were mixed with hydroxyethyl starch for nucleated cell (NC) enrichment . After finishing CD34 analysis, culture of hematopoietic progenitors (CFU-GM and CFU-GEMM) assays, microbial culture, HLA Class I (A, B) serology and class II (DR) low resolution SSP typing, cord blood units are stored in the liquid nitrogen for clinical applicatoin . Cord blood contained an average of nuclear cell (NC) (1.2 +/- 0.6) x 10(9), CD34(+) cells (3.0 +/- 3.7) x 10(6), CFU-GM (1.1 +/- 0.7) x 10(6) and CFU-GEMM (1.1 +/- 1.2) x 10(6) for storage and the recovery rates were 91%, 88%, 85% and 82%, respectively . The recovery rates for red blood cell and Hb were (39 +/- 9)% and (40 +/- 8)%, respectively . The storage volume was (35.1 +/- 7.1) ml in a 50 ml storage bags . The mean time from collection to processing of 15 hours (range 4 - 24 hours) had no influence on cell viability . The cell viability before processing is more than 95% and 92% after UCB thawing . The recovery rates of NC, CD34(+) cells and CFU-GM post-thawing were 96%, 90% and 91%, respectively . There were no HIV antibody (HIVAb) positive in all of UCB units . For an incidence of processed samples, infection with syphilis, HBsAg, HBcAb, HCVAb, CMV, bacterial contamination and abnormal hemoglobin were 0.1%, 0.8%, 3.2%, 0.2%, 87.1%, 1.2% and 0.1%, respectively . More than 3 HLA loci matched can be found for random patients in our cord blood bank and 6 HLA loci matched have 5% . For transplantation with nucleated cell counts of > 2.7 x 10(7) cells/kg, our cord blood bank will be able to provide all of the umbilical cord blood stem cell samples for children and 50% of units can be used for some of adult recipients transplantation in the country . It is concluded that: (1) The large cord blood banking for 20 000 UCB storage is feasible in China . (2) Our system of whole procedure and methods is functionable for supplying qualified cord blood units in transplantation . (3) The volume for collection is critical to the yield of CD34(+) cells or hematopoietic progenitor cells, however cord blood NC is also important and proportional with CD34(+) cells . Only the units containing more than 8 x 10(8) cells and more than 60 ml of cord blood can be in the procession for storage.

J Heart Valve Dis, 2003 Jan, 12(1), 68 - 75
Persistence of Chlamydia pneumoniae in degenerative aortic valve stenosis indicated by heat shock protein 60 homologues; Skowasch D et al.; BACKGROUND AND AIMS OF THE STUDY: Based on the concept of chronic persistent infections with Chlamydia pneumoniae among variable stressors for aortic valve degeneration, the study aim was to assess the presence of chlamydial heat shock protein (cHSP) 60 and its human homologue (hHSP60) in diseased valvular tissue . METHODS: Surgical specimens of high-grade stenosed, native (n = 33) and bioprosthetic (n = 10) aortic valves were examined immunohistochemically for the localization of cHSP60, hHSP60 and macrophages (CD68), supplemented by polymerase chain reaction (PCR) and electron microscopy to prove microbial presence . RESULTS: Degenerated valves showed specific immunostaining of cHSP60 in 27 cases (65%), of hHSP60 in 26 (63%), and of CD68 in 36 (84%) . Both HSP60 homologues were predominantly detected in valvular fibrosa, consistently co-localized with macrophages and, quantitatively, showed a strong correlation (r = 0.81, p < 0.001) . Presence of C . pneumoniae was demonstrated by PCR in a subset of 11 of 18 valves (61%) . Microbial persistence was confirmed by ultrastructural analysis . Degenerated prosthetic valves revealed markedly higher macrophage infiltration and cHSP60 signaling compared with degenerated native valves (each p < 0.05) . CONCLUSION: Beyond detection of C . pneumoniae, the present data on co-localization and valvular predilection sites (fibrosa) of both HSP60 homologues indicate the presence of chronic persistent C . pneumoniae infection as well as regional stressor effects, and suggest their involvement in native and prosthetic valve degeneration.

J Lipid Res, 2003 Feb, 44(2), 342 - 8 Epub 2002 Nov 04.
Effect of GM2 activator protein on the enzymatic hydrolysis of phospholipids and sphingomyelin; Shimada Y et al.; GM2 activator protein (GM2AP) is a specific protein cofactor that stimulates the enzymatic hydrolysis of the GalNAc from GM2, a sialic acid containing glycosphingolipid, both in vitro and in lysosomes . While phospholipids together with glycosphingolipids are important membrane constituents, little is known about the possible effect of GM2AP on the hydrolysis of phospholipids . Several recent reports suggest that GM2AP might have functions other than stimulating the conversion of GM2 into GM3 by beta-hexosaminidase A, such as inhibiting the activity of platelet activating factor and enhancing the degradation of phosphatidylcholine by phospholipase D (PLD) . We therefore examined the effect of GM2AP on the in vitro hydrolyses of a number of phospholipids and sphingomyelin by microbial (Streptomyces chromofuscus) and plant (cabbage) PLD . GM2AP, at the concentration as low as 1.08 microM (1 microg/50 microl) was found to inhibit about 70% of the hydrolyses of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol by PLD, whereas the same concentration of GM2AP only inhibited about 20-25% of the hydrolysis of sphingomyelin by sphingomyelinase and had no effect on the hydrolysis of sphingosylphosphorylcholine by PLD . Thus, GM2AP exerts strong and broad inhibitory effects on the hydrolysis of phospholipids carried out by plant and microbial PLDs . High ammonium sulfate concentration (1.6 M or 21.1%) masks this inhibitory effect, possibly due to the alteration of the ionic property of GM2AP.

Int J Immunopathol Pharmacol, 2002 Sep, 15(3), 245 - 248
Prevalence of IgG antibodies against Borrelia Burgdorferi s.l . and Ehrlichia Phagocytophila in sera of patients presenting symptoms of Lyme disease in a central region of Italy; Santino I et al.; The aim of this study was to evaluate the prevalence (seroprevalence) of antibodies against Borrelia burgdorferi and Ehrlichia phagocytophila among patients resident in Lazio, a region of central Italy . Of a sample of 1,050 patients, which presented clinical manifestations related to Lyme disease, 34 (3.2%) were Borrelia-seropositive (Lyme index value >/= 1.2) . The sera of 25 out of the 34 patients that were Borrelia-positive were also analysed for the presence of antibodies against E . phagocytophila and 3 (12%) were found Ehrlichia-positive (titres >1:64) . No Ehrlichia positive samples were found among sera of 250 Borrelia-negative patients . Since both B . burgdorferi s.l . and Ehrlichia species share the same tick vector (Ixodes ricinus), our results indicate that concurrent transmission of these microbial pathogens might have been occurred among the patients included in this study.

J Immunol, 2003 Feb 15, 170(4), 1763 - 9
Expression of IFN-gamma upon triggering of activating Ly49D NK receptors in vitro and in vivo: costimulation with IL-12 or IL-18 overrides inhibitory receptors; Ortaldo JR et al.; NK cells can express both activating and inhibitory Ly49 receptors on their cell surface . When cells expressing both receptors are presented with a ligand, inhibition dominates the functional outcome . In this report we demonstrate that costimulation of the activating Ly49D murine NK cell receptor with IL-12 or IL-18 is capable of over-riding the inhibitory Ly49G2 receptor blockade for cytokine production both in vitro and in vivo . This synergy is mediated by and dependent upon Ly49D-expressing NK cells and results in significant systemic expression of IFN-gamma . This would place NK cells and their activating Ly-49 receptors as important initiators of microbial, antiviral, and antitumor immunity and provide a mechanism for the release of activating Ly49 receptors from inhibitory receptor blockade.

Appl Occup Environ Hyg, 2003 Mar, 18(3), 193 - 9
Extent of fungal growth on fiberglass duct liners with and without biocides under challenging environmental conditions; Samimi BS et al.; Eight brands of fiberglass duct liners, including three that contained biocides, were exposed to challenging environmental conditions that would promote fungal growth . Twenty-four rectangular sheet metal ducts in three groups of eight ducts per group were lined with the eight selected liners . Each group of ducts was exposed to one of the three test conditions within an environmental chamber for a period of 15 days . These conditions were a) 75 percent RH, b) 75 percent RH plus water spray, c) 75 percent RH plus dry nutrient, and d) 75 percent RH plus water plus nutrient . Viable spores of Aspergillus niger were aerosolized into each duct as seed . On the 16th day, air and surface samples for fungal spores were collected from inside ducts . The results of air sampling using N6 sampler and visual inspection indicated that two out of three biocide-containing liners, Permacote and Toughgard, inhibited fungal growth but only under condition A . The third biocide-containing liner, Aeroflex Plus, was effective even when it was wet (conditions A and B) . All three biocide-containing liners failed to inhibit fungal growth under conditions C and D . Among the five other types of liners that did not contain biocides, ATCO Flex with a smooth Mylar coating was more preferable, exhibiting lower fungal activity during conditions A, B, and C . All liners failed under condition D when nutrient and water were added together . Surface sampling using adhesive tape failed to produce representative results, apparently due to rough/porous surface of duct liners . It was concluded that duct liners with biocide treatment could be less promoting to microbial growth under high humidity as long as their surfaces remain clean and water-free . A liner with an impermeable and smooth surface seems to be less subject to microbial growth under most conditions than biocide-containing liners having porous and/or rough surfaces.

Biometals, 2003 Jun, 16(2), 359 - 68
Early onset of virus infection and up-regulation of cytokines in mice treated with cadmium and manganese; Seth P et al.; A substantial database indicates that a large number of environmental pollutants, chemicals and therapeutic agents to which organisms are exposed cause immunotoxicity . The suppression of immune functions may cause increased susceptibility of the host to a variety of microbial pathogens potentially resulting in a life-threatening state . Evaluation of the immunotoxic potential of chemical xenobiotics is of great concern and, therefore, we have investigated the impact of exposure of inorganic metals, specifically cadmium (Cd) and manganese (Mn) on Encephalomyocarditis virus (EMCV), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEEV) infection . Pretreatment with a single, oral dose of Cd or Mn increased the susceptibility of mice to a sub-lethal infection of these viruses as observed by increased severity of symptoms and mortality compared to untreated controls . An early onset of virus infection was found in brains of Cd and Mn treated animals . Histopathological observations of the brain indicate evidence of inflammation and greater tissue pathology in Cd-or Mn-exposed mice compared to control animals . Meningitis and vascular congestion was seen in virus infected mice in all the metal treated groups, and further, the perivascular inflammation appeared earlier in treated mice compared to control . Encephalitis was maximum in Cd pretreated mice . Widespread environmental contamination of metals and the potential for their exposure and subsequent infection of humans or animals is indicative that further studies of these and all other metals are important to understand the effect of environmental pollution on human health.

J Matern Fetal Neonatal Med, 2002 Oct, 12(4), 237 - 46
Neutrophil elastase and secretory leukocyte protease inhibitor in prelabor rupture of membranes, parturition and intra-amniotic infection; Helmig BR et al.; OBJECTIVE: Neutrophil elastase (NE), a multifunctional serine protease stored in azurophilic granules of mature neutrophils, is capable of intracellular degradation of proteins during phagocytosis and extracellular degradation of connective tissue during an inflammatory process . Secretory leukocyte protease inhibitor (SLPI) is a natural NE inhibitor present in amniotic fluid, fetal membranes and cervical mucus . An imbalance between NE and SLPI has been implicated as a mechanism of abnormal tissue destruction in chronic inflammatory diseases . The purpose of this study was to determine if parturition, premature rupture of the membranes (PROM) and microbial invasion of the amniotic cavity are associated with changes in amniotic fluid concentrations of NE and SLPI . STUDY DESIGN: Amniotic fluid was retrieved by amniocentesis from 380 patients in the following groups: (1) preterm labor and intact membranes without microbial invasion of the amniotic cavity who delivered at term (n = 13) or prematurely (n = 26), and preterm labor with microbial invasion of the amniotic cavity (n = 9); (2) preterm PROM with (n = 34) and without (n = 51) microbial invasion of the amniotic cavity; and (3) term gestation without microbial invasion of the amniotic cavity with intact membranes not in labor (n = 63), in labor (n = 158), and with rupture of membranes not in labor (n = 26) . Microbial invasion of the amniotic cavity was determined by a positive amniotic fluid culture for micro-organisms including aerobic, anaerobic and Mycoplasma species . NE and SLPI amniotic fluid levels were determined by highly specific and sensitive immunoassays . RESULTS: Preterm PROM was associated with a significant increase in the amniotic fluid concentration of NE . Microbial invasion of the amniotic cavity was associated with a significant increase in the amniotic fluid concentration of NE in women with preterm labor and intact membranes, as well as in women with preterm PROM . Term and preterm parturition was associated with a significant increase in the amniotic fluid concentration of NE . In the absence of microbial invasion of the amniotic cavity, preterm and term PROM were associated with a significant reduction in the amniotic fluid concentration of SLPI . CONCLUSION: Preterm PROM, microbial invasion of the amniotic cavity, and parturition at term and preterm are associated with a significant increase in the amniotic fluid concentration of NE . PROM is associated with a reduced amniotic fluid concentration of SLPI.

J Matern Fetal Neonatal Med, 2002 Oct, 12(4), 231 - 6
Protein kinase C stimulates release of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by human decidual cells; Edwin SS et al.; OBJECTIVE: Increased concentrations of amniotic fluid matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 have been observed in the context of premature rupture of membranes (PROM) and microbial invasion of the amniotic cavity . However, the source of the stimuli that contribute to the accumulation of these proteins in amniotic fluid remains to be identified . The present study was conducted to investigate MMP-2, MMP-9 and TIMP-1 secretion by decidual cells in response to activated protein kinase C (PKC) . METHODS: Decidual cells were isolated from term placentae, grown to confluence and incubated with control media or 10(-11) to 10(-8) mol/l concentrations of phorbol 12-myristate 13-acetate (PMA) . Concentrations of MMP-2, MMP-9 and TIMP-1 in the culture supernatant were determined using sensitive and specific immunoassays . Substrate zymography was conducted to confirm MMP-9 assays . RESULTS: PMA induced a concentration-dependent stimulation of release of MMP-9 (control vs . PMA l0(-9) and 10(-8) mol/l; p < 0.01) and TIMP-1 (control vs . PMA 10(-9) and 10(-8) mol/l; p < 0.001), but not MMP-2 . A direct positive correlation was observed between MMP-9 and TIMP-1 release (r = 0.645; p < 0.001) . Substrate zymography confirmed increased release of MMP-9 in response to PMA (control vs . PMA 10(-8) and PMA 10(-7) mol/l; p < 0.01) . CONCLUSIONS: Activation of PKC within the decidua will result in enhanced MMP-9 release, which upon activation could contribute to degradation of matrices within fetal membranes leading to PROM.

Appl Environ Microbiol, 2003 Feb, 69(2), 1320 - 4
Dibenzyl sulfide metabolism by white rot fungi; Van Hamme JD et al.; Microbial metabolism of organosulfur compounds is of interest in the petroleum industry for in-field viscosity reduction and desulfurization . Here, dibenzyl sulfide (DBS) metabolism in white rot fungi was studied . Trametes trogii UAMH 8156, Trametes hirsuta UAMH 8165, Phanerochaete chrysosporium ATCC 24725, Trametes versicolor IFO 30340 (formerly Coriolus sp.), and Tyromyces palustris IFO 30339 all oxidized DBS to dibenzyl sulfoxide prior to oxidation to dibenzyl sulfone . The cytochrome P-450 inhibitor 1-aminobenzotriazole eliminated dibenzyl sulfoxide oxidation . Laccase activity (0.15 U/ml) was detected in the Trametes cultures, and concentrated culture supernatant and pure laccase catalyzed DBS oxidation to dibenzyl sulfoxide more efficiently in the presence of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) than in its absence . These data suggest that the first oxidation step is catalyzed by extracellular enzymes but that subsequent metabolism is cytochrome P-450 mediated.

Appl Environ Microbiol, 2003 Feb, 69(2), 1299 - 304
High rate of uptake of organic nitrogen compounds by Prochlorococcus cyanobacteria as a key to their dominance in oligotrophic oceanic waters; Zubkov MV et al.; Direct evidence that marine cyanobacteria take up organic nitrogen compounds in situ at high rates is reported . About 33% of the total bacterioplankton turnover of amino acids, determined with a representative {(35)S}methionine precursor and flow sorting, can be assigned to Prochlorococcus spp . and 3% can be assigned to Synechococcus spp . in the oligotrophic and mesotrophic parts of the Arabian Sea, respectively . This finding may provide a mechanism for Prochlorococcus' competitive dominance over both strictly autotrophic algae and other bacteria in oligotrophic regions sustained by nutrient remineralization via a microbial loop.

Appl Environ Microbiol, 2003 Feb, 69(2), 827 - 34
In-field spatial variability in the degradation of the phenyl-urea herbicide isoproturon is the result of interactions between degradative Sphingomonas spp . and soil pH; Bending GD et al.; Substantial spatial variability in the degradation rate of the phenyl-urea herbicide isoproturon (IPU) {3-(4-isopropylphenyl)-1,1-dimethylurea} has been shown to occur within agricultural fields, with implications for the longevity of the compound in the soil, and its movement to ground- and surface water . The microbial mechanisms underlying such spatial variability in degradation rate were investigated at Deep Slade field in Warwickshire, United Kingdom . Most-probable-number analysis showed that rapid degradation of IPU was associated with proliferation of IPU-degrading organisms . Slow degradation of IPU was linked to either a delay in the proliferation of IPU-degrading organisms or apparent cometabolic degradation . Using enrichment techniques, an IPU-degrading bacterial culture (designated strain F35) was isolated from fast-degrading soil, and partial 16S rRNA sequencing placed it within the Sphingomonas group . Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial community 16S rRNA revealed two bands that increased in intensity in soil during growth-linked metabolism of IPU, and sequencing of the excised bands showed high sequence homology to the Sphingomonas group . However, while F35 was not closely related to either DGGE band, one of the DGGE bands showed 100% partial 16S rRNA sequence homology to an IPU-degrading Sphingomonas sp . (strain SRS2) isolated from Deep Slade field in an earlier study . Experiments with strains SRS2 and F35 in soil and liquid culture showed that the isolates had a narrow pH optimum (7 to 7.5) for metabolism of IPU . The pH requirements of IPU-degrading strains of Sphingomonas spp . could largely account for the spatial variation of IPU degradation rates across the field.

Curr Drug Targets Infect Disord, 2003 Mar, 3(1), 66 - 75
New approaches for anti-infective drug discovery: antibiotics, vaccines and beyond; Cheng Q et al.; Infectious disease is the leading cause of death worldwide, and billions of dollars are invested every year in developing anti-infective drugs . In the meantime, resistant bacteria are on the steady rise and render many once effective drugs useless . The tremendous funding and the urgent need to treat the resistant bacterial infections lead to the rapid progress on development of new drugs and potential new drug targets . New discoveries are being made that increase our understanding of microbial pathogenesis . Technological advancement is also being made to accelerate the drug discovery process . This review will mainly focus on discussing novel strategies on the development of antibiotics and vaccines for treating bacterial infections . Details of how some of the emerging technologies such as genomics and bioinformatics are accelerating the drug discovery process will be highlighted . Newly emerging concepts in controlling bacterial infections such as the use of probiotics and enzybiotics will also be briefly described.

Curr Drug Targets Immune Endocr Metabol Disord, 2003 Mar, 3(1), 1 - 22
Controlled modulation of inflammatory, stress and apoptotic responses in macrophages; Malyshev IY et al.; An outstanding question of current immunology is to define the mechanisms by which microbial products influence the immunopathologic host response elements in the early stages of infection . Macrophages are now well recognized to have a critical role in both innate and acquired immunity . In order to adjust promptly to continuous changes in microenvironment and maintain the immunologic balance, macrophages adequately respond by activating one of the numerous immunologic programs . However, sustained macrophage activation and excessive production of inflammatory mediators can perpetuate the numerous pathological processes and contribute to induction of stress response and even apoptosis . Therefore, selective modulation of macrophage activity represents an important strategy for prevention and treatment of inappropriate inflammatory responses in order to minimize the unwanted side-effects of the immunity . Macrophages can be selectively reprogrammed for a specific phenotype of immune response, e.g . cytokine or nitric oxide (NO), by relatively short-term exposure of the cells to substimulatory concentrations of different microbial components, including LPS . These LPS-dependent reprogramming effects are mediated by IFN-gamma-independent autocrine cytokine regulatory mechanisms that also controlled at the transcriptional level . Furthermore, LPS reprogrammed macrophages exhibit differential capacity to resist experimentally induced apoptosis and to produce heat shock proteins . Complete analysis of, and appreciation for, the immunoregulatory mechanisms implicated in LPS-dependent reprogramming of immune responses in macrophages can be expected to increase our understanding of the host innate response, as well as allow investigators to utilize emerging immunologic technologies in effective treatment of infections and chronic inflammatory diseases.

Curr Med Chem, 2003 Mar, 10(6), 505 - 24
Beta-propellers: associated functions and their role in human diseases; Pons T et al.; The beta-propeller fold appears as a very fascinating architecture based on four-stranded antiparallel and twisted beta-sheets, radially arranged around a central tunnel . Similar to the alpha/beta-barrel (TIM-barrel) fold, the beta-propeller has a wide range of different functions, and is gaining substantial attention . Some proteins containing beta-propeller domains have been implicated in the pathogenesis of a variety of diseases such as cancer, Alzheimer, Huntington, arthritis, familial hypercholesterolemia, retinitis pigmentosa, osteogenesis, hypertension, and microbial and viral infections . This article reviews some aspects of 3D structure, amino acids sequence regularities, and biological functions of the proteins containing beta-propeller domains . Major emphasis has been laid on beta-propellers whose functions are associated to human diseases . Recent research efforts reported in the fields of protein engineering, drug design, and protein structure-function relationship studies, concerning the beta-propeller architecture, have also been discussed.

Phys Rev Lett . 2003 Jan 10;90(1):018101 . Epub 2003 Jan 03.
Minimal model for genome evolution and growth; Hsieh LC et al.; Textual analysis of typical microbial genomes reveals that they have the statistical characteristics of a DNA sequence of a much shorter length . This peculiar property supports an evolutionary model in which a genome evolves by random mutation but primarily grows by random segmental duplication . That genomes grew mostly by duplication is consistent with the observation that repeat sequences in all genomes are widespread and intragenomic and intergenomic homologous genes are preponderant across all life forms.

Tidsskr Nor Laegeforen, 2002 Dec 10, 122(30), 2891 - 4
{Mobilization of mesenchymal infection defense}; Seljelid R et al.; BACKGROUND: Most infections are arrested in epithelial and superficial connective tissues long before antibodies and antigen specific killer cells have been induced; i.e . before the specific defence system has had time enough to come to the rescue . Microbial substances that activate and modulate this non-specific first-line defence in and near the body surfaces may enhance disease resistance, mainly by stimulating the production of anti-microbial substances by epithelia and by local activation of tissue macrophages . MATERIAL AND METHODS: There are many different microbial substances that can activate macrophages . Beta-1,3-glucans from yeast and mushrooms are the most obvious candidates for pharmaceutical development because their chemical composition and mode of action has been clarified in great detail . RESULTS: Beta-1,3-glucans in purified form provide efficient protection of animals against infections by virus, bacteria, fungi and parasites . Such enhanced protection is obtained after injection as well as after oral or mucosal administration . Beta-1,3-glucans also counteract the toxic effects of bacterial endotoxins and enhance the body's capacity to destroy cancer cells . INTERPRETATION: Activation of non-specific immunity in epithelia and in connective tissues by purified microbial substances corresponds to early events in a natural infection process and renders animals more resistant to infections . This way to enhance resistance to microbial infections has been applied with success in animal husbandry with beta-1,3-glucans administrated orally or onto mucosal surfaces . Corresponding use in human medicine is a realistic possibility, in addition to the use of microbial immune modulators as adjuvants in mucosal vaccines.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1999 Apr, 21(2), 140 - 5
{Establishment of a microbial assay for screening anti-human immunodeficiency virus type-1 protease inhibitors}; Leng Q et al.; OBJECTIVE: This study was to establish a microbial assay of human immunodeficiency virus type-1 protease (HIV-1 PR) activity for screening anti-HIV PR inhibitors . METHODS: A 24 bp synthetic oligonucleotide fragment that encodes the HIV-1 PR recognition sequence was inserted into the tetr gene of pBR322 (mtetr) . Escherichia coli containing HIV-1 PR expression vector-pPOLO was transformed with pACYC184M containing modified mtetr gene . The transformant could express both HIV-1 PR and the modified Tet protein . RESULTS: The growth of engineered E . coli was prevented in the presence of tetracycline because the resistance Tet protein was degraded by HIV-1 PR . However inhibition of the HIV-1 PR restored tetracycline resistance . 31 chemical synthetic compounds were tested by the microbial assay . CONCLUSIONS: A microbial assay method of HIV-1 PR activity was established through a engineered E . coli . 5 mumol/L saqunavir-a special HIV-1 PR inhibitor showed inhibitory effect on the engineered E . coli . That means this model could be used as a initial screening model for anti-HIV PR agents.

J Microencapsul, 2002 Nov-Dec, 19(6), 797 - 809
Reacetylated chitosan microspheres for controlled delivery of anti-microbial agents to the gastric mucosa; Portero A et al.; The high aqueous solubility of chitosan restricts the utility of chitosan microspheres for gastric drug delivery . This paper describes the preparation of reacetylated chitosan microspheres with suitable properties for the controlled release of active anti-microbial agents, such as amoxycillin and metronidazole, in the gastric cavity . Two different microencapsulation approaches were developed and optimized in order to encapsulate hydrophilic (amoxycillin) and hydrophobic (metronidazole) compounds efficiently . The reacetylated chitosan microspheres exhibited a controlled water swelling capacity and gelified at acidic pH, resulting in prolonged release of the encapsulated antibiotics . The reacetylation time was found to be a key factor that affects not only drug release, but also encapsulation efficiency and anti-microbial activity of the encapsulated compound . The last two parameters were also dependent on drug solubility in the reacetylating agent . Using short reacetylation time periods, it was possible to efficiently control the release of both hydrophilic and lipophilic antibiotics while maintaining their activity against different bacteria . Consequently, reacetylated chitosan microspheres are promising vehicles for the controlled delivery of anti-microbial agents to the gastric cavity and, hence, for the eradication of Helicobacter pylori, a pathogen strongly associated with gastric ulcers and possibly gastric carcinoma.

Cytotherapy, 2002, 4(6), 539 - 49
Catastrophic failures of freezing bags for cellular therapy products: description, cause, and consequences; Khuu HM et al.; BACKGROUND: Container integrity is critical for maintaining sterility of cryopreserved cellular therapy products . We investigated a series of catastrophic bag failures, first noticed in early 2001 . METHODS: Process records were reviewed for all PBPC and lymphocyte products cryopreserved in bags from January 2000 through April 2002 . Patient charts were also reviewed . RESULTS: One thousand two hundred and four bags were removed from storage for infusion to 261 patients . All products had been cryopreserved in Cryocyte poly(ethylene co-vinyl acetate) (EVA) bags in either 10% DMSO or 5% DMSO and 6% pentastarch . Product volumes were 25-75 mL, and bags were stored with overwrap bags in a liquid nitrogen tank . From January 2000 to April 2001, failure occurred in 10 of 599 (1.7%) bags . From May 2001 to April 2002, 58 of 605 (9.6%) bags failed, typically with extensive fractures that were visible before thaw . Of the 58 that failed, 24 were salvaged by aseptic methods and infused to patients under antibiotic coverage; 10 of those 24 (42%) had positive bacterial cultures . Bag failures were not related to product type, cryoprotectant solution, liquid versus vapor storage, or freezer location . Failures were linked to use of four Cryocyte bag lots manufactured in 2000 and 2001 . After replacing these lots with a 1999 Cryocyte lot and with KryoSafe polyfluoroethylene polyfluoropropylene (FEP) bags, no more failures occurred in 75 and 102 bags, respectively, thawed through April 2002 . DISCUSSION: High rates of bag failure were associated with four Cryocyte bag lots . No serious adverse patient effects occurred, but bag failures led to microbial contamination, increased product preparation time, increased antibiotic use, and increased resource expenditure to replace products.

Br J Nutr, 2003 Jan, 89(1), 97 - 103
Effects of feed intake on composition of sheep rumen contents and their microbial population size; Rodriguez CA et al.; The present study was conducted to determine the effect of feed intake on the composition of the rumen contents of sheep and on their bacterial densities . Whole rumen contents were sampled after a period of continuous inter-rumen infusion of 15NH3 from four rumen-cannulated wethers successively fed on a hay-concentrate diet (2:1, w/w on a DM basis) at two rates of feed intake: 40 and 80 g DM/kg body weight0.75 . Total weight and chemical composition of rumen contents, as well as the distribution by size and chemical composition of particles, were determined . The populations of bacteria associated with the liquid (liquid-associated bacteria, LAB) and solid (solid-associated bacteria, SAB) fractions of rumen digesta and the distribution of SAB according to feed particle size were also examined . The greater feed intake caused an increase in the mass of the rumen contents, while its chemical composition did not change, except for a higher content of organic matter (P=0.023) . The distribution of feed particles by size was similar at both levels of intake . The concentrations of neutral- and acid-detergent fibre in feed particles decreased and those of total, dietary, and microbial N increased, both with a quadratic response (P=0.001), as particle size decreased . The proportion of LAB in the microbial biomass of rumen digesta reached only 8.0 % . This proportion and the density of LAB were unaffected by the level of feed intake, whereas an apparent reduction (10.4 %) occurred with the SAB biomass in whole rumen contents . A systematic, but not significant, reduction (mean value 11.9 %) in the level of microbial colonisation in the different particle fractions with the increase of feed intake was also observed.

Hautarzt, 2003 Jan, 54(1), 45 - 52 Epub 2002 Dec 20.
{Significance of immunologic tolerance in dermatology}; Steinbrink K et al.; The immunological tolerance processes enable the organism to distinguish between self and non-self and are, therefore, critical for an efficient immune system . Exogenous or endogenous factors that disturb tolerance mechanisms induce uncontrolled activation of the immune system and the development of autoimmune diseases . In the field of dermatology, the most relevant autoimmune diseases are connective tissue diseases and autoimmune bullous skin disorders . In contrast, increased activity of tolerance shuts down parts of the normal immune response and thus facilitates the development of neoplasia and microbial infections in the skin and other organs . Immunological mechanisms for the induction of tolerance have been studied with the help of experimental models of tolerance to contact allergens . T- and B-cells, as well as antigen presenting cells, in particular dendritic cells, are involved in the immunological mechanisms of tolerance . The modification of autologous immune cells of patients with malignant tumors, allergic and autoimmune diseases might have potential for the development of new therapies.

Curr Microbiol, 2003 Mar, 46(3), 233 - 4
A thermodynamic interpretation of the Monod equation; Liu Y et al.; The Monod equation for microbial growth is purely empirical, and the theoretical basis of this model stays unclear . Similar to any chemical reactions, overall microbial growth process is dependent upon the changes in free energy . This study showed that the Monod equation could be interpreted in a thermodynamic sense very well . It was probably for the first time demonstrated that the Monod constant ( K(s)) was inversely related to the equilibrium constant of the overall microbial growth process.

Genome Res, 2003 Feb, 13(2), 206 - 15
A microarray-based antibiotic screen identifies a regulatory role for supercoiling in the osmotic stress response of Escherichia coli; Cheung KJ et al.; Changes in DNA supercoiling are induced by a wide range of environmental stresses in Escherichia coli, but the physiological significance of these responses remains unclear . We now demonstrate that an increase in negative supercoiling is necessary for transcriptional activation of a large subset of osmotic stress-response genes . Using a microarray-based approach, we have characterized supercoiling-dependent gene transcription by expression profiling under conditions of high salt, in conjunction with the microbial antibiotics novobiocin, pefloxacin, and chloramphenicol . Algorithmic clustering and statistical measures for gauging cellular function show that this subset is enriched for genes critical in osmoprotectant transport/synthesis and rpoS-driven stationary phase adaptation . Transcription factor binding site analysis also supports regulation by the global stress sigma factor rpoS . In addition, these studies implicate 60 uncharacterized genes in the osmotic stress regulon, and offer evidence for a broader role for supercoiling in the control of stress-induced transcription.

Nat Rev Mol Cell Biol, 2003 Feb, 4(2), 95 - 104
NALPs: a novel protein family involved in inflammation; Tschopp J et al.; A newly discovered family of cytoplasmic proteins--the NALPs--has been implicated in the activation of caspase-1 by the Toll-like receptors (TLRs) during the cell's response to microbial infection . Like the structurally related apoptotic protease-activating factor-1 (APAF-1), which is responsible for the activation of caspase-9, the NALP1 protein forms a large, signal-induced multiprotein complex, the inflammasome, resulting in the activation of pro-inflammatory caspases.

Wei Sheng Yan Jiu, 2001 Nov, 30(6), 357 - 8, 361
{Survey on the microbial pollution of pure water in water dispenser and its disinfection}; Chen X et al.; The counts of bacteria, total coliforms and fungi of pure water in the water dispensers were detected and the regular pattern of bacterial reproduction in water were studied after the dispensers and water were disinfected . The results showed the bacterial pollution in the water dispensers was serious and the bacteria counts increased with the time of using dispensers lasted . The disinfectant could killed effectively the bacteria in the water sources but it could not control the new bacteria polluted and reproduced while the water dispensers were in use . It was suggested that the bacteria polluted from air should be prevented by taking effective methods and selecting appropriate organic materials for making dispensers.

Arch Microbiol, 2003 Jan-Feb, 179(2), 75 - 82 Epub 2002 Dec 06.
Relevance of airborne fungi and their secondary metabolites for environmental, occupational and indoor hygiene; Fischer G et al.; Airborne fungal contaminants are increasingly gaining importance in view of health hazards caused by the spores themselves or by microbial metabolites . In addition to the risk for infection, the allergenic and toxigenic properties, as well as the inflammatory effects are discussed in this review as possible health impacts of bioaerosols . A major problem is the lack of threshold values for pathogenic and non-pathogenic fungi, both in the workplace and in outdoor air . While the relevance of mycotoxins has been intensely studied in connection with contamination of food and feed, the possible respiratory uptake of mycotoxins from the air has so far not been sufficiently taken into account . Toxic secondary metabolites are expected to be present in airborne spores, and may thus occur in airborne dust and bioaerosols . Potential health risks cannot be estimated reliably unless exposure to mycotoxins is determined qualitatively and quantitatively . Microbial volatile organic compounds (MVOC) have been suggested to affect human health, causing lethargy, headache, and irritation of the eyes and mucous membranes of the nose and throat . The production of MVOC by fungi has been discussed in connection with domestic indoor microbial pollution, but the relevance of fungal metabolites in working environments remains insufficiently studied.

J Mol Biol, 2003 Feb 14, 326(2), 517 - 27
Crystal structures of Tritrichomonasfoetus inosine monophosphate dehydrogenase in complex with substrate, cofactor and analogs: a structural basis for the random-in ordered-out kinetic mechanism; Prosise GL et al.; The enzyme inosine monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide biosynthesis . Because it is up-regulated in rapidly proliferating cells, human type II IMPDH is actively targeted for immunosuppressive, anticancer, and antiviral chemotherapy . The enzyme employs a random-in ordered-out kinetic mechanism where substrate or cofactor can bind first but product is only released after the cofactor leaves . Due to structural and kinetic differences between mammalian and microbial enzymes, most drugs that are successful in the inhibition of mammalian IMPDH are far less effective against the microbial forms of the enzyme . It is possible that with greater knowledge of the structural mechanism of the microbial enzymes, an effective and selective inhibitor of microbial IMPDH will be developed for use as a drug against multi-drug resistant bacteria and protists . The high-resolution crystal structures of four different complexes of IMPDH from the protozoan parasite Tritrichomonas foetus have been solved: with its substrate IMP, IMP and the inhibitor mycophenolic acid (MPA), the product XMP with MPA, and XMP with the cofactor NAD(+) . In addition, a potassium ion has been located at the dimer interface . A structural model for the kinetic mechanism is proposed.

Carbohydr Res, 2003 Feb 14, 338(5), 423 - 31
Isolation and characterisation of the homogalacturonan from type II cell walls of the commelinoid monocot wheat using HF-solvolysis; Wietholter N et al.; In contrast to the typical type I cell wall of the dicot plants, the type II cell wall of the commelinoid monocot plants is known to be relatively poor in pectins . Assuming a critical role for the remaining pectins in terms of cell wall architecture and/or as a reservoir of signalling molecules, we have compared different protocols for the isolation of the main pectin polymer, homogalacturonan, from wheat leaf cell walls . Pectin was detected in these cell walls immunochemically using the monoclonal antibodies JIM5 and JIM7, and biochemically by monosaccharide analysis . The Ca(++)-chelators CDTA and imidazole extracted a pectin rich fraction from isolated cell walls which was however contaminated with significant amounts of hemicelluloses . Pretreatment of the cell walls with anhydrous hydrogen fluoride at controlled low temperatures followed by HF/ether- and water-extraction prior to imidazole-extraction of pectins yielded a purer homogalacturonan fraction . The near absence of rhamnosyl residues proved that the isolated homogalacturonan fraction was free of rhamnogalacturonans . If HF-solvolysis was performed at -23 degrees C, the resulting homogalacturonan had a degree of methyl esterification identical to that of the pectins in the initial wheat cell wall . The antibodies JIM5 and JIM7 as well as PAM1 and LM5 proved that the isolated homogalacturonan had a low methyl ester content, was polymeric and free of galactan side chains . We can thus isolate native homogalacturonan from the type II wheat cell walls with the original in muro pattern of methyl esterification still intact, to further investigate e.g., its degradability by plant or microbial pectic enzymes.

J Periodontal Res, 2003 Feb, 38(1), 104 - 8
Relationship between fibrinolytic activity and gingival inflammatory reaction in young individuals; Olofsson A et al.; The fibrinolytic system (the plasminogen activating system) is involved in several physiological and pathological processes . Through the transformation of plasminogen to the aggressive broad spectrum protease plasmin, potent enzymatic activity is released . Plasmin acts directly on connective tissue components, and indirectly by activating proforms of the metalloproteinases . The destructive potential of the fibrinolytic system may thus be of importance for the initiation and progression of periodontal diseases . Earlier studies have shown high concentrations of the plasminogen activator t-PA and its inhibitor PAI-2 in gingival crevicular fluid (GCF) as well as enhanced concentrations in areas of gingival inflammation . The aim of this study was to investigate a possible relationship between the gingival inflammatory reactivity and the fibrinolytic activity in gingival crevicular fluid . Thirty-one young individuals took part in the study . Gingival Index scores and Plaque Index scores were assessed and used to formulate a score expressing an individuals' inflammatory response to microbial plaque levels (Relative G/P score) . The fibrinolytic activity of GCF was assessed with a fibrin gel lysis assay, and the levels of t-PA and PAI-2 were assayed with ELISAs . All samples showed fibrinolytic activity . A positive correlation between the fibrinolytic activity and Relative G/P score was found . Thus, in individuals with an enhanced reactivity to dental plaque, a higher plasminogen activating activity in GCF was seen . This indicates a higher potential for tissue proteolysis in these individuals, possibly facilitating spread and deeper involvement of the lesions.

Environ Microbiol, 2003 Feb, 5(2), 92 - 102
Physiological and molecular characterization of anaerobic benzene-degrading mixed cultures; Ulrich AC et al.; Nine distinct anaerobic benzene-degrading cultures were enriched from sediment samples from four different sites . These cultures used nitrate, sulphate or CO2 as electron acceptors . The shortest doubling times were observed in nitrate-reducing cultures, although cell yield was lowest in these cultures . The highest substrate concentration utilized and maximum absolute rates of benzene degraded (in micro M day-1) were observed in methanogenic cultures . The microbial compositions of a methanogenic and nitrate-reducing culture were determined from a clone library of 16S rRNA genes . Five Bacterial 16S rRNA sequences, one of which resembled a clone previously found in a sulphate-reducing, benzene-degrading culture and four Archaeal 16S rRNA sequences were identified in a methanogenic culture . Four Bacterial and no Archaeal 16S rRNA sequences were identified in a nitrate-reducing culture . The relative abundance of the four nitrate-reducing putative species was determined by slot blot hybridization . Two green sulphur bacteria together formed 52% of the clone library, but were found to be less than 4% of the culture by slot blot analysis . One of the cloned 16S rRNA gene sequences comprised 70% of the culture and was phylogenetically 93% similar to both Azoarcus and Dechloromonas species, which have been shown to degrade aromatic compounds, including benzene, under nitrate-reducing conditions.

Pest Manag Sci, 2003 Jan, 59(1), 114 - 8
Dissipation of the herbicide dithiopyr in soil and residues in wheat (Triticum aestivum L) grain under Indian tropical conditions; Saikia N et al.; Dissipation of dithiopyr in soil was monitored after application to wheat crop as pre- or post-emergence applications at two rates, viz 100 and 200 g AI ha(-1) . The level of dithiopyr in the soil was assessed by gas chromatography, and its disappearence was found to follow a first-order decay curve irrespective of rate or method of application . The half-life in soil ranged between 17.3 and 25.0 days and residues at harvest (150 days after application) ranged between 4.0 and 8.8% of amounts applied . Investigation of microbial degradation of dithiopyr was conducted in minimal salt and Czapek Dox media in which 80% of the compound degraded within 15 days . Residues were not detected in wheat grain at harvest.

J Egypt Soc Parasitol, 2001 Apr, 31(1), 21 - 35
Laboratory evaluation of a Helicosporidium sp . (Protozoa: Helicosporida) as an agent for the microbial control of mosquitoes; Seif AI et al.; A Helicosporidium sp . isolated from diseased larvae of Culex pipiens L . in Egypt was assessed for its microbial control potential against mosquito larvae . Dose infectivity and dose mortality of the pathogen was determined for each larval instar of Cx . pipiens . Helicosporidium sp was infectious to Aedes caspius (Pallas), C . antennatus (Becker), C . perexiguus Theobald, and Culiseta longiareolata (Macquart) . Using 24-hr exposures of first instar larvae to a range of concentrations between 50 and 5 x 10(3) spores/ml, Cx . antennatus and Cx . perexiguus were more susceptible than Cx . pipiens, Ae . caspius and Cs . longiareolata with LC50's of 5 x 10(2), 5 x 10(2), 1.6 x 10(3), 1.4 x 10(3), and 1.7 x 10(3), respectively . Current experiments shown that Helicosporidium spores were tolerant of freezing and storage at 5 degrees C, but not to desiccation . Production trails indicated that up to 6.2 x 10(8) spores could be routinely produced in individual cotton leaf worms, Spodoptera littolaris (Boisd) (Lepidoptera: Noctuidae).

Wei Sheng Wu Xue Bao, 2002 Aug, 42(4), 484 - 9
{Asymmetric microbial reduction of organosilyl ketone with immobilized Saccharomyces cerevisiae cells}; Lou W et al.; Asymmetric microbial reduction of acetyltrimethylsilane with immobilized Saccharomyces cerevisiae cells was studied . The effects of shake speeds, substrate concentration, immobilized cell concentration, pH and reaction temperature on the initial reaction rate, maximum yield and optical purity of the product were systematically studied . All above mentioned parameters have important effects on the raction . The optimum shake speed, substrate concentration, immobilized cell concentration, pH and reaction temperature were found to be 150 r/min, 14 mmol/L, 0.15 g/mL, 7.3 and 25 degrees C-30 degrees C respectively for the reaction, under which the maximum yield and optical purity of the product were as high as 84.9% and 90.2% ee.

Biotechnol Bioeng, 2003 Mar 30, 81(7), 775 - 82
Bioprocess considerations for expanded-bed chromatography of crude canola extract: sample preparation and adsorbent reuse; Bai Y et al.; Compared to the conventional microbial and mammalian systems, transgenic plants produce proteins in a different matrix . This provides opportunities and challenges for downstream processing . In the context of the plant host Brassica napus (canola), this work addresses the bioprocessing challenges of solid fractionation, resin fouling by native plant components (e.g., oil, phenolics, etc.), hydrodynamic stability, and resin reuse for expanded bed adsorption for product capture . Plant tissue processing and subsequent protein extraction typically result in an extract with a high content of solids containing a wide particle-size distribution . Without removal of larger particles, the column inlet distributor plugged . The larger particles (> 50 microm) were easily removed through centrifugal settling comparable to that attainable with a scroll decanter . The remaining solids did not affect the column performance . Less than 4% of the lipids and phenolics in the fed extract bound to STREAMLINE trade mark DEAE resin, and this small proportion could be satisfactorily removed using recommended clean-in-place (CIP) procedures . Hydrodynamic expansion and adsorption kinetics of the STREAMLINE trade mark DEAE resin were maintained throughout 10 cycles of reuse, as was the structural integrity of the resin beads . No significant accumulation of N-rich (e.g., proteins) and C/O-rich components (e.g., oil and phenolics) occurred over the same period .

Gastroenterology, 2003 Feb, 124(2), 521 - 36
The genetics of inflammatory bowel disease; Bonen DK et al.; The inflammatory bowel diseases (IBD) comprise complex genetic disorders, with multiple contributing genes . Linkage studies have implicated several genomic regions as likely containing IBD susceptibility genes, with some observed uniquely in Crohn's disease (CD) or ulcerative colitis (UC), and others common to both disorders . The best replicated linkage region, IBD1, on chromosome 16q contains the CD susceptibility gene, NOD2/CARD15 . NOD2/CARD15 is expressed in peripheral blood monocytes and is structurally related to the plant R proteins, which mediate host resistance to microbial pathogens . Three major coding region polymorphisms within NOD2/CARD15 have been highly associated with CD among patients of European descent . Having one copy of the risk alleles confers a 2-4-fold risk for developing CD, whereas double-dose carriage increases the risk 20-40-fold . All 3 major CD variants exhibit a deficit in NF-kappaB activation in response to bacterial components . Carriage of NOD2/CARD15 risk alleles is associated with ileal location, earlier disease onset, and stricturing phenotype . Other IBD genomic regions include IBD2 on chromosome 12q (observed more in UC), and IBD3, containing the major histocompatibility complex region . A short genomic region has been associated with CD on chromosome 5q, but the precise contributing gene is as yet unidentified . The characterization of additional IBD susceptibility genes could potentially lead to the identification of novel therapeutic agents for IBD, make possible a molecular reclassification of disease, and increase understanding of the contribution of environmental factors (notably, tobacco and the intestinal microbial milieu) to intestinal inflammation.

Arch Tierernahr, 2002 Oct, 56(5), 327 - 37
Effect of dietary supplementation with alpha-galactosidase preparation and stachyose on growth performance, nutrient digestibility and intestinal bacterial populations of piglets; Pan B et al.; Two experiments were conducted to investigate the effect of dietary supplementation with alpha-galactosidase preparation and stachyose on growth performance, nutrient digestibility and intestinal bacterial populations of piglets . In Experiment I 72 crossbred piglets were allotted to three treatments, i.e . 1) control (basal) diet, 2) alpha-Gal1 and 3) alpha-Gal5, the basal diet supplemented with 0.01% and 0.05% alpha-galactosidase preparation (alpha-Gal), respectively . Average daily gain, average daily feed intake and feed conversion ratio were not influenced by alpha-Gal supplementation . In Experiment II 72 crossbred weanling piglets were randomly assigned to four treatments with a complete factorial design, i.e . 1) basal diet, 2) basal diet with 0.01% alpha-Gal supplementation, 3) basal diet with 1% stachyose supplementation and 4) basal diet with 1% stachyose and 0.01% alpha-Gal supplementation . Average daily gain and feed conversion ratio were influenced by alpha-Gal and stachyose supplementation . The ileal digestibility of stachyose, raffinose, gross energy and crude protein was improved significantly by alpha-Gal supplementation . The microbial populations in the intestine were modified by both alpha-Gal and stachyose supplementation . The data suggested that stachyose supplementation had an adverse effect on the growth performance of piglets and alpha-Gal supplementation could ameliorate it, especially in younger pigs.

Ann Chim, 2002 Nov-Dec, 92(11-12), 1057 - 65
Presence and racemisation of amino acids in calcium oxalate patinas . A case study from the Baptistery in Parma, Italy; Casoli A et al.; The organic material present in oxalate patinas, collected from the stone external surfaces of the Baptistery in Parma (Italy), was analysed by GC-MS . The high racemisation levels determined for several specific amino acids indicate that microbial activity could contribute to the formation and modification of the organic material leading to the calcium oxalate patinas.

Eur J Immunol, 2002 Nov, 32(11), 3235 - 45
Combined dendritic cell- and CpG oligonucleotide-based immune therapy cures large murine tumors that resist chemotherapy; Heckelsmiller K et al.; The use of dendritic cells (DC) loaded with tumor antigen is one of the most advanced approaches in cancer immunotherapy . CpG motifs within microbial DNA detected by toll-like receptor 9 are responsible for the favorable properties of CpG oligodeoxynucleotides (ODN) as immune modulators . In this study, mature antigen-pulsed DC or peritumoral injections of CpG ODN, both effective for the treatment of small established tumors, were almost ineffective against large established tumors (1-cm diameter) in a syngeneic murine colon carcinoma model . For large tumors, the antitumor activity of mature antigen-pulsed DC was strongly increased by coinjection of CpG ODN, resulting in a transient control of tumor growth . Rejection of large tumors and long-term cure of mice was achieved by combining injection of antigen-pulsed DC plus CpG ODN at a site distant to the tumor with peritumoral injections of CpG ODN . Depletion of CD8 T cells abrogated the therapeutic activity . Large numbers of DEC-205-positive DC infiltrated the tumor in treated mice . Therapy with 5-fluorouracil and leucovorin was unable to control tumors of the same size . In conclusion, we demonstrate that the immune system, provided that appropriate stimulation with DC and CpG ODN is given, has the potential to cure animals of large solid tumors in situations where even chemotherapy is not efficient.

J Toxicol Environ Health A, 2003 Feb 28, 66(4), 345 - 63
Ecotoxicological risks associated with land treatment of petrochemical wastes . III . Immune function and hematology of cotton rats; Wilson J et al.; Landfarming is a widely used method of treating petrochemical waste through microbial bio-degradation . The effects of residual petrochemical contamination on wildlife, especially terrestrial mammals, are poorly understood . The effects of contaminants on the immune system and hematology of cotton rats (Sigmodon hispidus) living on five abandoned petrochemical landfarms (units 1-5) in Oklahoma were studied . Cotton rats were sampled seasonally (summer and winter) from each landfarm and from five ecologically matched reference sites for 2 yr (1998-2000) and returned to the laboratory for immunological and hematological assays . Overall analysis indicated that rats inhabiting landfarms exhibited decreased relative spleen size compared to rats collected from reference sites, with the landfarm at unit 1 showing the greatest reduction . Cotton rats collected from landfarms also had increased hemoglobin, hematocrit, and platelet levels and decreased blood leukocytes during summer . During winter, an increase in the number of popliteal node white blood cells was observed from rats collected on landfarms . No marked difference was detected for lymphocyte proliferation in response to concanavalin A, pokeweed, or interleukin-2 . Lymphokine-activated killer cell lytic ability showed a seasonal pattern, but no treatment differences . No differences between landfarm and reference sites were detected in the hypersensitivity reaction of rats given an intradermal injection of phytohemagluttinin (PHA-P) . Comparisons within individual sites indicated that two sites (units 1 and 3) had the greatest effects on immune function and hematology of cotton rats . The results of this study suggest that residual petrochemical waste affects the immune system and hematology of cotton rats living on abandoned landfarms during summer and is complicated by variation in the contaminants found on individual petroleum sites.

Water Res, 2003 Mar, 37(5), 1064 - 70
Impact of UV disinfection on microbially available phosphorus, organic carbon, and microbial growth in drinking water; Lehtola MJ et al.; UV irradiation at a wavelength of 253.7 nm (UV(254)) is commonly used for drinking water disinfection . UV radiation is known to convert organically combined phosphorus to orthophosphate and to degrade natural organic matter . We studied if UV disinfection increases the amount of microbially available forms of organic carbon and phosphorus in drinking waters with different characteristics, and if these changes in water chemical quality could enhance the microbial growth in drinking water . The UV(254) dose (15-50 mWs/cm(2)) used in waterworks reduced the concentration of assimilable organic carbon and the sum of the molecular size fractions . The release of microbially available phosphorus needed higher doses (204 mWs/cm(2)) of UV(254) radiation . Of bacteria in drinking water, 90% were inactivated with UV(254)-irradiation doses below 50 mWs/cm(2) . A high dose (501 mWs/cm(2)) of UV(254) radiation inhibited the microbial growth in water.

Dis Aquat Organ, 2002 Dec 10, 52(3), 217 - 31
Continuous in vitro culture of the carpet shell clam Tapes decussatus protozoan parasite Perkinsus atlanticus; Casas SM et al.; Continuous in vitro cultures of the clam Tapes decussatus parasite Perkinsus atlanticus were established from infected gill fragments, infected haemolymph and parasite hypnospores isolated from infected gill fragments following incubation in Ray's fluid thioglycollate medium (RFTM) . No continuous cultures could be initiated from P . atlanticus zoospores . Cultures initiated from hypnospores yielded the highest percentage of continuous cultures (100%, 6/6), followed by cultures initiated from gill fragments (93%, 43/46) and from haemolymph (30%, 3/10) . Failures to establish continuous cultures were due to microbial contamination . The source of parasite influenced the success rate, the time taken to establish cultures and the size of cultured cells . In vitro proliferation of parasite cells was mainly by vegetative multiplication . Zoosporulation, yielding motile biflagellated zoospores, was observed at a low frequency (< 1% of dividing cells) in every culture . Morphology of cultured cells examined with light and transmission electron microscopy corresponded to that of P . atlanticus found in clam tissues . Cultured cells enlarged in RFTM and stained blue-black with Lugol's solution, which are characteristics of the Perkinsus species cells . DNA sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex matched those of P . atlanticus . All cultures were established in a medium designated JL-ODRP-2A that was similar in composition to the culture medium JL-ODRP-1 originally used to propagate Perkinsus marinus in vitro . Proliferation of P . atlanticus in vitro could be supported by the commercial culture medium (1:2 v/v) DME:Ham's F-12 with fetuin.

Wei Sheng Wu Xue Bao, 2001 Aug, 41(4), 505 - 9
{Effect of drying and temperature on the sporulation of Pandora delphacis mycelia gelatinized}; Liu Z et al.; The entomophthoraceous fungus, Pandora delphacis, is a microbial agent highly potential for control of sucking-type insects . In this study, effort was made of gelatinizing the mycelia of the isolate F95129 from submerged culture using polyacrylamide-starch gel pwder and sodium alginate . The resulting film-like gel of the mycelia sporulated very well, indicating that the materials used for gelatinization of the mycelia was biologically compatible with P . delphacis . Then, the gelatinized mycelia were slowly dried in silica chamber at 4, 14, and 24 degrees C, respectively . After 35-day drying and storage in the chamber, however, a 22.7%-92.8% decrease of sporulation capacity occurred in the film-like gels, which then had different water content . The nutrient-supplemented polyacrylamide-starch gel stored at 14 degrees C with water content of 6.1%-7.4% was best for largely decreasing the loss of sporulation capacity among the combinations of gel, nutrition, water content and temperature . The results indicate that it could be possible to choose a desirable gel for longer biological activity of gelatinized P . delphacis mycelia.

Se Pu, 1999 Sep, 17(5), 483 - 5
{Analysis of organic acids in human dental plaque by means of gas chromatography/mass spectrometry}; Li J et al.; The purpose of this study was to investigate the composition of plaque fatty acids in the healthy population . The study was made on 10 volunteers over the age of 18 who were divided into three sub-groups (3-4 individuals) . Neither subject exhibited clinical evidence of salivary gland disorder and any medication affecting salivary functions were not used . A sensitive GC/MS method with VG7070E mass spectrometer was developed in our study . The sample separation was carried out on a fused silica capillary column with OV-1 . The column size was 23 m x 0.23 mm . The temperature program was as follows: from 40 degrees C to 120 degrees C fast, then from 120 degrees C to 240 degrees C at 6 degrees C/min . The results showed that there were 14 organic acids and isomers present in plaque . They were C12:0, C14:0, C15:0, C16:0, C16:1, C18:0, C18:1, C18:2, C20:0 and phenylacetic acid, phenylpropionic acid . The higher content of fatty acids in the sample were C16:0, C18:0, and C18:1 . The aromatic acids were detected only in some samples . The odd-numbers fatty acids and aromatic acids were for the first time detected . The origin of organic acids in plaque is an ongoing area of research . Our data clearly identify the bacterial contribution to the organic acids of plaque matrix, which may have a special relationship with bacteria metabolism . The research will help us to understand fatty acids metabolism of dental plaque and to determine their role in the microbial homeostasis of dental plaque.

Wei Sheng Wu Xue Bao, 2001 Dec, 41(6), 745 - 9
{Microbial reduction ability of various iron oxides in pure culture experiment}; Qu D et al.; The microbial reduction of ferrihydrite, lepidocrocite, hematite, goethite and aluminum-substituted iron oxides were examined by iron-reducer GS-15 under anaerobic pure culture condition . The results indicated that the ferrihydrite and lepidocrocite can be rapidly reduced by iron-reducer, and the percentage of microbial reduction are respectively 95.4% and 95.8% after 4 days incubation at 25 degrees C . The other iron oxides like hematite, Al-hematite, goethite and Al-goethite are very difficult to reduce during short-term incubation . The amounts of Fe(III)-reducing, Fe(II)-producing and Fe-reducing calculated by acetate consumption are identical under the conditions of lower concentration iron oxides addition.

J Environ Qual, 2003 Jan-Feb, 32(1), 317 - 24
Microbial reduction of hexavalent chromium under vadose zone conditions; Oliver DS et al.; Hexavalent chromium {Cr(VI)} is a common contaminant associated with nuclear reactors and fuel processing . Improper disposal at facilities in and and semiarid regions has contaminated underlying vadose zones and aquifers . The objectives of this study were to assess the potential for immobilizing Cr(VI) using a native microbial community to reduce soluble Cr(VI) to insoluble Cr(III) under conditions similar to those in the vadose zone, and to evaluate the potential for enhancing biological Cr(VI) reduction through nutrient addition . Batch microcosm and unsaturated flow column experiments were performed . Native microbial communities in subsurface sediments with no prior Cr(VI) exposure were shown to be capable of Cr(VI) reduction . In both the batch and column experiments, Cr(VI) reduction and loss from the aqueous phase were enhanced by adding high levels of both nitrate (NO3-) and organic C (molasses) . Nutrient amendments resulted in up to 87% reduction of the initial 67 mg L(-1) Cr(VI) in an unsaturated batch experiment . Molasses and nitrate additions to 15 cm long unsaturated flow columns receiving 65 mg L(-1) Cr(VI) resulted in microbially mediated reduction and immobilization of 10% of the Cr during a 45-d experiment . All of the immobilized Cr was in the form of Cr(III), as shown by XANES analysis . This suggests that biostimulation of microbial Cr(VI) reduction in vadose zones by nutrient amendment is a promising strategy, and that immobilization of close to 100% of Cr contamination could be achieved in a thick vadose zone with longer flow paths and longer contact times than in this experiment.

J Environ Qual, 2003 Jan-Feb, 32(1), 84 - 91
Herbicide loading to shallow ground water beneath Nebraska's Management Systems Evaluation Area; Spalding RF et al.; Better management practices can counter deterioration of ground water quality . From 1991 through 1996 the influence of improved irrigation practices on ground water pesticide contamination was assessed at the Nebraska Management Systems Evaluation Area . Three 13.4-ha corn (Zea mays L.) fields were studied: a conventional furrow-irrigated field, a surge-irrigated field and a center pivot-irrigated field, and a center pivot-irrigated alfalfa (Medicago sativa L.) field . The corn fields received one identical banded application of Bicep (atrazine {6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4,-diamine} + metolachlor {2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamidel) annually; the alfalfa field was untreated . Ground water samples were collected three times annually from 16 depths of 31 multilevel samplers . Six years of sample data indicated that a greater than 50% reduction in irrigation water on the corn management fields lowered average atrazine concentrations in the upper 1.5 m of the aquifer downgradient of the corn fields from approximately 5.5 to <0.5 microg L(-1) . Increases in deethylatrazine (DEA; 2-chloro-4-amino-6-isopropylamino-s-triazine) to atrazine molar ratios indicated that reducing water applications enhanced microbial degradation of atrazine in soil zones . The occurrence of peak herbicide loading in ground water was unpredictable but usually was associated with heavy precipitation within days of herbicide application . Focused recharge of storm runoff that ponded in the surge-irrigated field drainage ditch, in the upgradient road ditch, and at the downgradient end of the conventionally irrigated field was a major mechanism for vertical transport . Sprinkler irrigation technology limited areas for focused recharge and promoted significantly more soil microbial degradation of atrazine than furrow irrigation techniques and, thereby, improved ground water quality.

J Environ Qual, 2003 Jan-Feb, 32(1), 23 - 32
Greenhouse gas emissions and soil indicators four years after manure and compost applications; Ginting D et al.; Understanding how carbon, nitrogen, and key soil attributes affect gas emissions from soil is crucial for alleviating their undesirable residual effects that can linger for years after termination of manure and compost applications . This study was conducted to evaluate the emission of soil CO2, N2O, and CH4 and soil C and N indicators four years after manure and compost application had stopped . Experimental plots were treated with annual synthetic N fertilizer (FRT), annual and biennial manure (MN1 and MN2, respectively), and compost (CP1 and CP2, respectively) from 1992 to 1995 based on removal of 151 kg N ha(-1) yr(-1) by continuous corn (Zea mays L.) . The control (CTL) plots received no input . After 1995, only the FRT plots received N fertilizer in the spring of 1999 . In 1999, the emissions of CO2 were similar between control and other treatments . The average annual carbon input in the CTL and FRT plots were similar to soil CO2-C emission (4.4 and 5.1 Mg C ha(-1) yr(-1), respectively) . Manure and compost resulted in positive C and N balances in the soil four years after application . Fluxes of CH4-C and N2O-N were nearly zero, which indicated that the residual effects of manure and compost four years after application had no negative influence on soil C and N storage and global warming . Residual effects of compost and manure resulted in 20 to 40% higher soil microbial biomass C, 42 to 74% higher potentially mineralizable N, and 0.5 unit higher pH compared with the FRT treatment . Residual effects of manure and compost on CO2, N20, and CH4 emissions were minimal and their benefits on soil C and N indicators were more favorable than that of N fertilizer.

Wei Sheng Wu Xue Bao, 1998 Oct, 38(5), 365 - 70
{Studies on microbial factor on color change of Dunhuang mural . III . Effect of Cladosporium sp . on color-change of red lead in the surface of mural}; Feng Q et al.; The conditions isolated Cladosporium sp . from Mural of Dunhuang grottoe germinated on the surface of imitated mural were as follows: 20 degrees C RH 60% or 30 degrees C RH 50%, which could be reached occasionally in some grottoes . Bone glue had protective function on red lead, while Cladosporium sp . could decompose bone glue and form some metabolite (such as oxalic acid, etc) as it grows . Then it could form a special chemical circumstance, which promoted the change from red lead to while lead.

Fish Shellfish Immunol, 2003 Jan, 14(1), 39 - 54
In vivo effects of beta-glucan and LPS on regulation of lysozyme activity and mRNA expression in Atlantic salmon (Salmo salar L.); Paulsen SM et al.; The present study was undertaken to compare the effects of intraperitoneally injected bacterial lipopolysaccharide (LPS) and yeast beta-glucan on lysozyme activity in Atlantic salmon, and to explore what organ(s) are responsible for the increase in plasma lysozyme activity induced by the compounds . The results indicated that LPS stimulates plasma lysozyme activity at least as efficiently as beta-glucan . The lysozyme gene was shown to be transcribed in head kidney, spleen, liver and intestine, and accumulation of transcript was demonstrated in response to both beta-glucan and LPS in all of these organs . Intracellular lysozyme activity was detected in the same organs and in isolated blood polymorphonuclear cells (PMN) and lymphocytes . Increased lysozyme activity in response to both beta-glucan and LPS was demonstrated in blood PMN and cells isolated from head kidney and intestine . In spleen and liver on the other hand, there was no increase in lysozyme activity in response to the stimulants . Based on previous work and the present results it is suggested that plasma lysozyme induced by LPS and beta-glucan originate from macrophages in the different organs . The head kidney is likely to be the main supplier of plasma lysozyme considering its high contents of macrophages . This work supports the notion that microbial compounds containing phylogenetically conserved structures (beta-glucan and LPS) are able to stimulate the non-specific defence of animals against infection by enhancing the lysozyme expression.

Chemosphere, 2003 Mar, 50(9), 1243 - 7
Fate of isoxaben in a containerized plant rhizosphere system; Drakeford CE et al.; Commercial production of ornamental plants is an important industry in the United States and involves a complex technology that includes the use of herbicides . Isoxaben{N-{3-(1-ethyl-1-methylpropyl)-5-isoxazolyl}-2,6-dimethoxybenzamide} is a pre-emergence herbicide used for controlling weeds in many areas including containerized ornamental plants . Degradation was studied in potting mix (80% bark, 20% sand) with three different regimes (sterile, bulk and rhizosphere) . The rhizosphere regime contained Switch Grass (Panicum virgatum), and plants were allowed to grow for 14 days before adding isoxaben (10 microg/g potting mix) . Isoxaben was degraded to 0.5 microg/g in 60 days giving a half-life of 7 days . Two degradation products were detected: 3-nitrophthalic acid in the rhizosphere and bulk regimes and 4-methoxyphenol in the sterile regime . Microbial population shifts were determined by fatty acid methyl ester profile analysis and were influenced by the introduction of a plant (rhizosphere regime) and by isoxaben addition.

Biotechniques, 2003 Jan, 34(1), 92 - 4, 96-7
Extraction of PCR-quality plant and microbial DNA from total rumen contents; Sharma R et al.; DNA from rumen digesta has several diagnostic applications such as studying microbial community dynamics, transgene/DNA stability, and population typing of various rumen bacteria . Several DNA extraction procedures are described in the literature for rumen digesta, which describe the removal of tannins, polysaccharides, and other PCR inhibitors . Some of these protocols are time-consuming and impractical when handling a large number of samples routinely . Here we describe a rapid method for the extraction of PCR-quality plant and microbial DNA from total rumen contents that is based on modifications in the cetyltrimethylammonium bromide procedure followed by cleanup using a Qiagen column . This procedure is highly reproducible and relatively short, once the initial grinding of the samples is performed, and it consistently yields PCR-quality DNA.

Microb Ecol, 2003 Feb, 45(2), 173 - 82 Epub 2003 Jan 28.
Antagonism between bacteria and fungi on decomposing aquatic plant litter; Mille-Lindblom C et al.; Bacterial and fungal decomposers of aquatic plant litter may exhibit either synergistic or antagonistic interactions, which are likely to influence microbial growth as well as the decomposition of litter and, eventually, the carbon metabolism of aquatic systems . To elucidate such interactions, we inoculated decomposing Phragmites culms in microcosms with fungal isolates and with natural communities of bacteria and fungi in different combinations . The development of fungal and bacterial biomass and the carbon dynamics were studied during several months of degradation . The results show a bilateral antagonistic relationship between bacteria and fungi . After 3 months, fungal biomass accumulation was approximately 12 times higher in the absence than in the presence of bacteria . Bacterial biomass accumulation was about double in the absence of fungi compared to when fungi were present . Similar interactions developed between a natural assemblage of bacteria and five different fungal strains isolated from Phragmites litter (three identified hyphomycetes and two unidentified strains) . Despite the great difference in biomass development between the treatments, the carbon metabolism was similar regardless of whether fungi and/or bacteria were present alone or in coexistence . We suggest that the antagonism between bacteria and fungi is an important controlling factor for microbial colonization and growth on aquatic plant litter.

Microb Ecol, 2003 Feb, 45(2), 191 - 202 Epub 2003 Jan 28.
Microbial diversity and resistance to copper in metal-contaminated lake sediment; Konstantinidis KT et al.; Contamination of habitats with heavy metals has become a worldwide problem . We describe herein the analysis of lake sediment contaminated with high concentrations of copper as a consequence of mine milling disposal over a 100-year period . Copper concentrations in the sediment were found to vary with depth and ranged from 200 to 5500 ppm . Analysis of the microbial community with T-RFLP identified a minimum of 20 operational taxonomic units (OTU) . T-RFLP analysis along a depth profile detected as many as nine shared OTUs across 15 centimeters, suggesting a conservation of community structure over this range . Only two genera, Arthrobacter and Ralstonia, were detected among 50 aerobic copper-resistant isolates cultivated on R2A, one of which (Ralstonia sp.) was characterized by the sequestration of copper, identified by electron diffraction scanning, in growing colonies . Scanning electron microscopy showed changes to the outer envelope of the cells when grown in the presence of copper . The copper-resistant Ralstonia isolates were also resistant to Ni, Cd, and Zn, showing two patterns of phenotypic resistant to these three metals in which either resistance to Zn or Ni was expressed in an isolate but never both.

Microb Ecol, 2003 Feb, 45(2), 119 - 27 Epub 2003 Jan 28.
Viral lysis, flagellate grazing potential, and bacterial production in Lake Pavin; Bettarel Y et al.; Abundances of different compartments of the microbial loop (i.e., viruses, heterotrophic bacteria, nonpigmented nanoflagellates, and pigmented nanoflagellates), bacterial heterotrophic production (BHP), viral lysis, and potential flagellate grazing impacts on the bacterial assemblages were estimated during a short-term study (24 h) conducted in June 1998 in the epilimnion (5 m) and metalimnion (10 m) of a moderate-altitude oligomesotrophic lake (Lake Pavin, France) . Viral and bacterial abundances were higher in the metalimnion than in the epilimnion, whereas pigmented and nonpigmented nanoflagellates were more numerous in the epilimnion . The control of the BHP due to viral lysis (determined by examination of viral-containing bacteria using a transmission electron microscope) was significantly higher in the meta- (range = 6.0-33.7%, mean = 15.6%) than in the epilimnion (3.5-10.3%, 6.4%) . The same was for the losses of BHP from the potential predation by nanoflagellates which ranged from 0.5 to 115.4% (mean = 38.7%) in the epilimnion, and from 0.7 to 97.5% (mean = 66.7%) in the metalimnion . Finally, estimated viral mediated mortality rates from the percentage of visibly infected cells and potential nanoflagellate grazing rates based on assumed clearance rates suggest that flagellates consumed a larger proportion of bacterial production than was lost to viral lysis.

Genet Med, 2002 Nov-Dec, 4(6 Suppl), 21S - 26S
The crucial role of the public health sciences in the postgenomic era; Omenn GS; Genomic sequence information and gene and protein expression patterns must be linked with information about diet and metabolism, lifestyle behaviors, diseases and medications, and microbial, chemical, and physical exposures . These linkages depend upon the public health sciences: epidemiology, biostatistics, environmental health sciences, pathobiology, health sciences research, and clinical prevention trials.

J Clin Gastroenterol, 2003 Feb, 36(2), 111 - 9
Use of probiotics in the treatment of inflammatory bowel disease; Hart AL et al.; The gut flora plays a fundamental role in maintaining normal intestinal function . A disturbance of this flora, or the host response to this flora, has been clearly demonstrated to play a role in the pathogenesis of inflammatory bowel disease (IBD) . This has led to attempts to modify the bacterial flora with "live non-pathogenic organisms that confer health benefits by improving the microbial balance," otherwise known as probiotics . Recent attention has focused on this potential strategy to treat or prevent IBD . The potential therapeutic benefit is enhanced by the natural and apparently safe approach that probiotics offer . Animal models of colitis have provided the proof of principle that probiotics can prevent and treat established intestinal inflammation . Controlled clinical studies have demonstrated the efficacy of probiotics in the maintenance of remission of pouchitis, prophylaxis of pouchitis after the formation of an ileoanal reservoir, maintenance of remission of ulcerative colitis, and treatment of Crohn's disease . However, large controlled trials are needed to definitively establish the place for probiotics in the treatment of IBD and resolve issues such as the dose, duration, frequency of treatment, and use of single or multiple strains . Research is focusing on establishing the mechanism of action, so that treatments with individually tailored properties are developed and innovative approaches are explored.

Ann N Y Acad Sci, 2002 Dec, 979, 166 - 77; discussion 188-96
De novo lymph node formation in chronic inflammation of the human leg; Olszewski WL; Organized lymphoid tissue is the first line of antigenic defense . Recruited by antigen located in the non-lymphoid tissues, the infiltrating lymphocytes often organize themselves as follicle-like structures that contain germinal centers, similar to those in secondary lymphoid follicles of lymph nodes . These extranodal tertiary lymphoid follicles are found in various autoimmune diseases . We investigated 153 patients with protracted lymph stasis of the lower limb, caused by lymphatic damage incurred through soft tissue bacterial inflammation or mechanical trauma of soft tissues and bones . In 10% of patients with post-inflammatory, and in 25% with post-traumatic lymph stasis, "newly-formed" lymph nodes were detected by means of lymphoscintigraphy . They were located along the large calf and thigh veins . Although scattered nodes are normally detected in these areas, the number and total mass of visualized nodes substantially exceeded those seen in healthy subjects . The calculated surface area of "newly formed" nodes attained 50-70% of the area of ipsilateral inguinal nodes . Histological evaluation of nodal biopsy specimens in three such patients revealed, in one, a lymph node structure without differentiation into cortical and medullary areas, and in a second, a follicle-like structure within a dilated lymph vessel . Lymph clot removed from another dilated vessel contained a lymphocyte/dendritic cell aggregate . The "newly formed" nodes likely originate from primordial lymphoid follicles and/or lymphoid cell aggregates formed in response to chronic stimulation by microbial products and self-antigens from the damaged tissues . Detection of "newly-formed" lymph nodes in the limb is evidence of an ongoing inflammatory process and requires appropriate therapy.

Curr Allergy Asthma Rep, 2003 Jan, 3(1), 79 - 85
Eosinophilic gastroenteritis: a problem of the mucosal immune system?
Kweon MN, Kiyono H.
The gastrointestinal immune system is a major component of the mucosal barrier, which provides an appropriate immunologic homeostasis between host and numerous foreign antigens, including microbial and dietary antigens . However, under certain pathological circumstances created by disturbance of the immunologic balance, allergic responses associated with the gastrointestinal tract can be triggered by abnormal immune responses against selected food protein antigens . Among the several types of immune competent cells, eosinophils are generally considered to play a central role for the development of allergic diseases in gastrointestinal tissue . Although most research has been focused on the molecular and cellular understanding of eosinophils in the peripheral tissues and lung, recent studies elucidate the unique trafficking and regulation mechanisms of eosinophils in the gastrointestinal tissues . In this review, we summarize current findings in the regulatory mechanism of gastrointestinal eosinophils . Furthermore, several unique murine models for eosinophilic gastroenteritis, which can be applied for the elucidation of underlying mechanisms of eosinophil-mediated gastrointestinal allergy, and the development of new mucosal immune therapy for the control of food allergy are reviewed.

Environ Microbiol, 2003 Jan, 5(1), 55 - 63
Diversity of microbial communities correlated to physiochemical parameters in a digestion basin of a zero-discharge mariculture system; Cytryn E et al.; Bacterial community structure and physiochemical parameters were examined in a sedimentation basin of a zero-discharge mariculture system . The system consisted of an intensively stocked fish basin from which water was recirculated through two separate treatment loops . Surface water from the basin was pumped over a trickling filter in one loop while bottom-water was recirculated through a sedimentation basin followed by a fluidized bed reactor in the other . Ammonia oxidation to nitrate in the trickling filter and organic matter digestion together with nitrate reduction in the sedimentation basin and fluidized bed reactor, allowed zero-discharge operation of the system . Relatively high concentrations of oxygen, nitrate, sulphate and organic matter detected simultaneously in the digestion basin suggested the potential for a wide range of microbially-mediated transformation processes . In this study, physiochemical parameters were correlated to bacterial diversity and distribution in horizontal and vertical profiles within this basin in an effort to obtain a basic understanding of the chemical and microbial processes in this system . Chemical activity and microbial diversity, the latter measured by denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR) amplified 16S rDNA fragments, were higher in the sludge layer than in the overlying aqueous layer of the basin . Chemical parameters in sludge samples close to the basin inlet suggested enhanced microbial activity relative to other sampling areas with evidence of both nitrate and sulphate reduction . Four of the nine DGGE bands identified in this zone were affiliated with the Bacteroidetes phylum . Detected sequences closely related to sequences of organisms involved in the sulphur cycle included Desulfovibrio, Dethiosulfovibrio and apparent sulphur oxidizers from the gamma-proteobacteria . In addition, a number of sequences from the beta and alpha-proteobacteria were identified.

FEMS Microbiol Ecol, 2002 Oct, 42(1), 71 - 80
A geostatistical analysis of small-scale spatial variability in bacterial abundance and community structure in salt marsh creek bank sediments; Franklin RB et al.; Small-scale variations in bacterial abundance and community structure were examined in salt marsh sediments from Virginia's eastern shore . Samples were collected at 5 cm intervals (horizontally) along a 50 cm elevation gradient, over a 215 cm horizontal transect . For each sample, bacterial abundance was determined using acridine orange direct counts and community structure was analyzed using randomly amplified polymorphic DNA fingerprinting of whole-community DNA extracts . A geostatistical analysis was used to determine the degree of spatial autocorrelation among the samples, for each variable and each direction (horizontal and vertical) . The proportion of variance in bacterial abundance that could be accounted for by the spatial model was quite high (vertical: 60%, horizontal: 73%); significant autocorrelation was found among samples separated by 25 cm in the vertical direction and up to 115 cm horizontally . In contrast, most of the variability in community structure was not accounted for by simply considering the spatial separation of samples (vertical: 11%, horizontal: 22%), and must reflect variability from other parameters (e.g., variation at other spatial scales, experimental error, or environmental heterogeneity) . Microbial community patch size based upon overall similarity in community structure varied between 17 cm (vertical) and 35 cm (horizontal) . Overall, variability due to horizontal position (distance from the creek bank) was much smaller than that due to vertical position (elevation) for both community properties assayed . This suggests that processes more correlated with elevation (e.g., drainage and redox potential) vary at a smaller scale (therefore producing smaller patch sizes) than processes controlled by distance from the creek bank . c2002 Federation of European Microbiological Societies . Published by Elsevier Science B.V . All rights reserved.

Annu Rev Pharmacol Toxicol, 2003, 43, 609 - 28 Epub 2002 Jan 10.
Innate immune responses to microbial poisons: discovery and function of the Toll-like receptors; Beutler B; There are many circumstances under which a toxin exploits an endogenous receptor or another protein of host origin to work its untoward effects . In most instances, the receptor normally fulfills a function that has nothing to do with the toxin per se; that is, the toxin is not the "natural" ligand . The situation with endotoxin, however, is a remarkable one . The endotoxin receptor evolved to detect endotoxin . Why have mammals maintained a gene that can undermine their survival? The search for the endotoxin receptor answered this question and also revealed the essential function and biological strategy of the Toll-like receptors: principal sensors of the innate immune system.

J Immunol, 2003 Feb 1, 170(3), 1462 - 5
Differential expression of the murine mannose-binding lectins A and C in lymphoid and nonlymphoid organs and tissues; Wagner S et al.; Mannose-binding lectin (MBL), a member of the collectin family, binds to carbohydrate structures on the surfaces of micro-organisms and may serve as a recognition molecule of the lectin pathway of complement activation . In rodents two forms, MBL-A and MBL-C, were described and shown to be products of two related, but uncoupled, genes . The liver is the main source of MBL biosynthesis . For rat MBL-A, expression has also been described in the kidney . Here we report that the two forms of murine MBL are differentially expressed in a number of nonhepatic tissues . Real-time RT-PCR revealed that the liver is the major site of expression for both MBL genes . Lower copy numbers were found in kidney, brain, spleen, and muscle . In testis, only the MBL-A gene is expressed, whereas MBL-C is exclusively expressed in small intestine . Using in situ hybridization and immunohistochemistry, we demonstrate that both MBLs are synthesized by hepatocytes and show MBL expression in cells of the monocyte/macrophage lineage . In the kidney MBL-A, but not MBL-C, was found to be synthesized . Vice versa, only MBL-C biosynthesis was detected in endothelial cells of the small intestine . The latter finding may support the view that MBL-C, as part of the innate immune system, may be a counterpart of secretory IgA of the acquired immune system in preventing, for example, microbial invasion and colonization . Our findings demonstrate that MBL-A and MBL-C are differentially expressed, implying distinct biological roles for both recognition molecules of the murine lectin pathway of complement.

Int Immunopharmacol, 2003 Jan, 3(1), 119 - 28
Innate recognition of lipopolysaccharide by CD14 and toll-like receptor 4-MD-2: unique roles for MD-2; Miyake K; Adaptive immunity generally refers to the ability of lymphocytes to recognize microbial, viral and fungal proteins via T cell receptors and antibodies . More ancestral and widespread innate immune mechanisms include those responsible for recognition of microbial glycolipids . Lipopolysaccharide (LPS) is the best studied, and arguably one of the most important of bacterial products because of its role in innate immune responses and endotoxin-mediated sepsis . Converging studies in two independent fields have recently led to the identification of LPS recognition molecules utilized by mammalian cells . Toll-like receptor 4 (TLR4) was identified as a mammalian homologue of the Toll receptor, which recognized fungi in the Drosophila's immune system . Spontaneous and targeted mutations identified TLR4 as an LPS recognition molecule . Separate studies of a Radioprotective 105 (RP105) and MD-1 heterodimer expressed by cells led to the identification of MD-2 as a molecule associated with TLR4 . Very recent in vivo studies have now revealed an essential contribution of MD-2 to LPS recognition . These findings further our understanding of protective, as well as detrimental innate immune mechanisms and may lead to new therapies for endotoxin shock .

J Endotoxin Res, 2002, 8(5), 329 - 335
LPS in microbial pathogenesis: promise and fulfilment; Beutler B; Since LPS was discovered, all inquiry in the field was motivated by two articles of faith . First, it was held that LPS was an important factor in microbial pathogenesis . Second, it was held that the responses elicited by numerous molecules of microbial origin might be formally similar to the responses elicited by LPS . The identification of TLR4 as the core transducer of LPS responses, and the added discovery that other TLR paralogs sense other microbial products, has strongly validated these cherished beliefs . Moreover, it has amalgamated many separate lines of inquiry, and focused attention on the how the innate immune system perceives infection.

Environ Pollut, 2003, 122(1), 1 - 5
Preliminary evidence that copper inhibits the degradation of DDT to DDE in pip and stonefruit orchard soils in the Auckland region, New Zealand; Gaw SK et al.; Orchards (n=13) were sampled as part of a larger survey investigating agrichemical residues (pesticides and trace elements) in cropping soils in the Auckland region, New Zealand . SigmaDDT concentrations in orchard soils ranged from <0.03 to 24.41 mg kg(-1) . DDT (o,p'- and p,p'-) comprised at least 40% of the SigmaDDT residues in 67% of orchards in which DDT residues were detected . There was a highly significant negative correlation (-0.924, P<0.001) between copper concentration (21-490 mg kg-1) and the ratio of DDE:DDT (0.4-5.2) in pip and stonefruit orchard soils . In further investigations involving five pip and stone fruit orchard sites and one grazing paddock it was found that soil respiration and the ratio of soil microbial carbon to soil carbon (%Cmic/Org-C) in orchard soils decreased with increasing copper concentration . These findings are consistent with the conclusion that elevated soil copper concentrations in pip and stone fruit orchard soils in the Auckland region may have reduced the ability of the indigenous soil microbial community to degrade DDT to DDE

Mol Cell, 2003 Jan, 11(1), 35 - 47
A novel assay system implicates PtdIns(3,4)P(2), PtdIns(3)P, and PKC delta in intracellular production of reactive oxygen species by the NADPH oxidase; Brown GE et al.; Activated neutrophils assemble an NADPH oxidase enzyme complex to produce superoxide for microbial killing . Much of the initial oxidase assembly occurs on intracellular granules, followed by movement of the oxidase to phagolysosomes and the plasma membrane . We have developed a novel assay system using Streptolysin-O permeabilized neutrophils that recapitulates the initial intracellular activation process while maintaining the ultrastructural features of this granulocytic cell type . Using this system, we biochemically dissect molecular events and signaling pathways involved in NADPH oxidase assembly and demonstrate specific roles for PKC delta, PI(3,4)P(2)/PI(3,4,5)P(3), and PI(3)P in the PMA-dependent intracellular activation process . This system should be of great utility for the study of cell signaling events that regulate the intracellular production of reactive oxygen species by neutrophils.

J Anim Physiol Anim Nutr (Berl), 2002 Dec, 86(11-12), 422 - 32
Characterization of 18:1 and 18:2 isomers produced during microbial biohydrogenation of unsaturated fatty acids from canola and soya bean oil in the rumen of lactating cows; Loor JJ et al.; Ruminal production of biohydrogenation intermediates in response to unsaturated oils was assessed using 24 Jersey cows fed a control diet or the control diet supplemented at 35 g/kg dry matter (DM) with canola, soya bean, or a mixture of equal amounts of canola plus soya bean oil for 4-weeks . Total fatty acid content averaged 63 or 35 g/kg DM for oil-supplemented diets or control . Oleic acid accounted for 6, 29, 21 or 12 g/kg DM in the control, canola, mixture, or soya bean oil diet, respectively . Linoleic acid averaged 17, 19, 26, or 33 g/kg DM and linolenic acid 5, 5, 6 or 8 g/kg DM for control, canola, mixture, or soya bean oil . Concentrations of cis12-, trans11-, trans13+14, and trans15-18:1 were 0.81, 2.99, 2.24, and 0.73 mg/g rumen fluid, respectively, in response to soya bean oil and were 126, 90, 45, and 38% greater compared with other diets . Trans11cis15-, cis9trans11- and cis9 cis11-18:2 also were greater when soya bean oil (0.30, 0.34 and 0.01 mg/g, respectively) was fed compared with other treatments (0.12, 0.21 and 0.004 mg/g, respectively) . Feeding canola oil resulted in greater concentrations of trans4-, trans5-, trans6+7+8-, trans9- and trans10-18:1 (0.20, 0.25, 0.87, 0.39 and 0.70 mg/g, respectively) compared with other diets (0.09, 0.15, 0.36, 0.20 and 0.46 mg/g, respectively) . Trans10cis12-18:2 concentration did not differ as a result of diet and averaged 0.002 mg/g rumen contents . The pattern of 18:1 and 18:2 isomers formed during ruminal biohydrogenation depends greatly on dietary profile of unsaturated fatty acids.

Med Trop (Mars), 2002, 62(4), 396 - 400
{Access for all to quality drugs}; Videau JY; The implementation of essential drugs policies and widespread use of generic products in humanitarian programs has ensured access to medication for poverty-stricken populations . However rigorous drug selection according to origin is necessary to guarantee quality . A drug is more than a mixture of chemical components, it depends on a complex balance that can be affected by numerous factors . To be beneficial to the patient, a drug must be manufactured, purchased, distributed and administered in a strictly professional and responsible manner . An obvious danger for drugs such as antibiotics is that use of poor quality products producing insufficient bioavailability will promote development of microbial resistance . This could become a worldwide public health problem with particularly dramatic consequences for the treatment of tuberculosis and retroviruses.

Medicina (Kaunas), 2002, 38(11), 1106 - 10
{Development of "Saules" tincture technology}; Velziene S et al.; The article deals with the production of the "Saules" tincture in a way of selecting an extractive agent concentration, the size of particles of the raw material and the method of extraction . The experiment indicates that the optimum concentration of ethanol in the tincture is 70% V/V, the particles' size is in the range from 1 to 2 mm; the production method recommended for use is percolation . The quality of the tincture was evaluated by determining the quantity of flavonoids and dry residue, the concentration of the ethanol, the density and the microbial contamination . The presence of quercetin, rutin and hyperoside was confirmed using high-pressure liquid chromatography . The stability of the tincture was evaluated.

Medicina (Kaunas), 2002, 38(10), 1009 - 13
{Development and analysis of revitalizing tincture}; Kalveniene Z et al.; The article deals with the production of the complex tincture of Hyperici herba 80.0 g, Cinnamomi cortex 20.0 g, Rosmarini folium 20.0 g, Lavandulae flos 20.0 g, Zingiberis rhizoma 60.0 g and the selection of an extracting liquid phase, and the choice of extraction method . The experimentally determined optimal conditions for tincture preparation were as follows: concentration of ethanol--70% (V/V), the particle size of the leaves, herb, flowers--2.0-3.0 mm, for the bark and the roots--0.3-0.5 mm, the extraction method--percolation . The quality of the tincture was evaluated by analyzing the sum of flavonoids (recalculated into equivalent amount of rutin), dry residue, the concentration of ethanol, density of the extractive preparation, and microbial contamination . Also the overall appearance was checked, the identification of flavonoids was performed by color reactions and high-pressure liquid chromatography.

Indian J Ophthalmol, 2002 Dec, 50(4), 265 - 82
Complications of laser-in-situ-keratomileusis; Sridhar MS et al.; Laser-in-situ-keratomileusis (LASIK) has become a popular technique of refractive surgery because of lower postoperative discomfort, early visual rehabilitation and decreased postoperative haze . Compared to photorefractive keratectomy (PRK), LASIK involves an additional procedure of creating a corneal flap . This may result in complications related to the flap, interface and underlying stromal bed . The common flap-related complications include thin flap, button holing, free caps, flap dislocation and flap striae . The interface complications of diffuse lamellar keratitis, epithelial ingrowth and microbial keratitis are potentially sight threatening . Compared to PRK, there is less inflammation and faster healing after LASIK, but there is a longer period of sensory denervation leading to the complication of dry eyes . The refractive complications include undercorrection, regression, irregular astigmatism, decentration and visual aberrations . Honest and unbiased reporting is important to understand the aetiology and redefine the management.

Science, 2003 Feb 14, 299(5609), 1033 - 6 Epub 2003 Jan 16.
Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells; Pasare C et al.; Toll-like receptors (TLRs) control activation of adaptive immune responses by antigen-presenting cells (APCs) . However, initiation of adaptive immune responses is also controlled by regulatory T cells (TR cells), which act to prevent activation of autoreactive T cells . Here we describe a second mechanism of immune induction by TLRs, which is independent of effects on costimulation . Microbial induction of the Toll pathway blocked the suppressive effect of CD4+CD25+ TR cells, allowing activation of pathogen-specific adaptive immune responses . This block of suppressor activity was dependent in part on interleukin-6, which was induced by TLRs upon recognition of microbial products.

Blood, 2003 May 1, 101(9), 3495 - 500 Epub 2003 Jan 16.
Role of the MyD88 transduction signaling pathway in endothelial activation by antiphospholipid antibodies; Raschi E et al.; Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the persistent presence of antiphospholipid antibodies (aPLs) and recurrent thrombosis or fetal loss . The thrombophilic state has been partially related to the induction of a proinflammatory and procoagulant endothelial cell (EC) phenotype induced by anti-beta(2)-glycoprotein I (beta(2)-GPI) antibodies that bind beta(2)-GPI expressed on the EC surface . Anti-beta(2)-GPI antibody binding has been shown to induce nuclear factor-kappa B (NF-kappa B) translocation leading to a proinflammatory EC phenotype similar to that elicited by interaction with microbial products (lipopolysaccharide {LPS}) and proinflammatory cytokines (interleukin 1 beta {IL-1 beta}, tumor necrosis factor alpha {TNF-alpha}) . However, the upstream signaling events are not characterized yet . To investigate the endothelial signaling cascade activated by anti-beta(2)-GPI antibodies, we transiently cotransfected immortalized human microvascular endothelial cells (HMEC-1) with dominant-negative constructs of different components of the pathway (Delta TRAF2, Delta TRAF6, Delta MyD88) together with reporter genes (NF-kappa B luciferase and pCMV-beta-galactosidase) . Results showed that both human anti-beta(2)-GPI IgM monoclonal antibodies as well as polyclonal affinity-purified anti-beta(2)-GPI IgG display a signaling cascade comparable to that activated by LPS or IL-1 . Delta TRAF6 and Delta MyD88 significantly abrogate antibody-induced as well as IL-1- or LPS-induced NF-kappa B activation, whereas Delta TRAF2 (involved in NF-kappa B activation by TNF) does not affect it . Moreover, anti- beta(2)-GPI antibodies and LPS followed the same time kinetic of IL-1 receptor-activated kinase (IRAK) phosphorylation, suggesting an involvement of the toll-like receptor (TLR) family . Our findings demonstrate that anti-beta(2)-GPI antibodies react with their antigen likely associated to a member of the TLR/IL-1 receptor family on the EC surface and directly induce activation.

Vaccine, 2003 Jan 17, 21(5-6), 419 - 30
Memory T cells and vaccines; Esser MT et al.; T lymphocytes play a central role in the generation of a protective immune response in many microbial infections . After immunization, dendritic cells take up microbial antigens and traffic to draining lymph nodes where they present processed antigens to naive T cells . These naive T cells are stimulated to proliferate and differentiate into effector and memory T cells . Activated, effector and memory T cells provide B cell help in the lymph nodes and traffic to sites of infection where they secrete anti-microbial cytokines and kill infected cells . At least two types of memory cells have been defined in humans based on their functional and migratory properties . T central-memory (T(CM)) cells are found predominantly in lymphoid organs and can not be immediately activated, whereas T effector-memory (T(EM)) cells are found predominantly in peripheral tissue and sites of inflammation and exhibit rapid effector function . Most currently licensed vaccines induce antibody responses capable of mediating long-term protection against lytic viruses such as influenza and small pox . In contrast, vaccines against chronic pathogens that require cell-mediated immune responses to control, such as malaria, Mycobacterium tuberculosis (TB), human immunodeficiency virus (HIV) and hepatitis C virus (HCV), are currently not available or are ineffective . Understanding the mechanisms by which long-lived cellular immune responses are generated following vaccination should facilitate the development of safe and effective vaccines against these emerging diseases . Here, we review the current literature with respect to memory T cells and their implications to vaccine development.

Vaccine, 2003 Jan 17, 21(5-6), 354 - 60
Anticipating rotavirus vaccines: epidemiology and surveillance of rotavirus in South Africa; Steele AD et al.; Rotavirus infection is associated with acute infantile gastroenteritis in infants and young children globally . In South Africa, rotavirus infection has been shown to be associated with approximately one-quarter of all diarrhoeal admissions to hospital . Rotavirus infection predominantly occurs in infants less than 12 months of age (75%) and has a peak of shedding during the cooler, drier months of the year . A secondary peak during the spring has been observed . Multiple infections with rotavirus and at least one other microbial agent are common . The circulating VP7 serotypes and VP4 genotypes have been determined in various regions of South Africa and show a geographic specific distribution . A decade previously, P{8}G1 or G4 strains predominated, and P{4}G2 strains occurred in an epidemic pattern in one region . More recently, rotavirus strains with P{6} genotype have become common and novel VP7/VP4 genotype combinations are occurring across the country . G9 strains have been reported from Cape Town to Vendaland . The circulating rotavirus types observed in this study add to the knowledge of the natural history of rotavirus infection and provide the groundwork to consider future vaccine strategies.

J Microbiol Methods, 2003 Mar, 52(3), 325 - 32
Use of gas chromatography-mass spectrometry/solid phase microextraction for the identification of MVOCs from moldy building materials; Wady L et al.; Gas chromatography-mass spectrometry/solid phase microextraction (GC-MS/SPME) was applied to identify microbial volatile organic compounds (MVOCs) in water-damaged, mold-infested building materials (gypsum board papers (n=2), mineral wool, and masonite) and in cultivated molds (Aspergillus penicillioides, Stachybotrys chartarum, and Chaetomium globosum) . Three SPME fibers (65-microm PDMS-DVB, 75-microm Carboxen-PDMS, and 70-microm Carbowax-stableflex) designed for automated injection were used of which the latter showed best performance . A number of previously reported MVOCs were detected both in the building materials and the cultivated molds . In addition, methyl benzoate was identified both in the S . chartarum and A . penicillioides cultures and in the building materials . SPME combined with GC-MS may be a useful method for the determination of MVOCs emitted from mold-infested building materials.

Drug Resist Updat, 2002 Dec, 5(6), 219 - 23
HIV mutagenesis and the evolution of antiretroviral drug resistance; Mansky LM; The development of antiretroviral drug resistance is a major threat to the effective treatment of human immunodeficiency virus type 1 (HIV-1) infection . Drug treatment failure is associated with accumulation of drug resistance mutations and the evolution of drug resistance . Studies from microbial systems provide evidence for a correlation between drug resistance development and increased pathogen mutation rates . Recent studies with HIV-1 have shown that drugs targeted against reverse transcriptase (RT) as well as drug-resistant RT can increase HIV-1 mutation frequencies . Furthermore, combinations of drug and drug-resistant RT have been found to increase virus mutation frequencies in a multiplicative manner . The correlation of increased HIV-1 mutation rates with the evolution of antiretroviral drug resistance indicates that drug failure could increase the likelihood of further resistance evolving from subsequent drug regimens.

Paediatr Respir Rev, 2000 Sep, 1(3), 235 - 40
Cellular and humoral defence mechanisms against bacteria; Fleer A; Host defence to bacterial infection is a complex process of highly efficient effector systems that co-operate to eliminate microbial invaders . The systems not only co-operate, they are also interdependent, as deficiency of one system may cause dysfunction in another . As is so often the case in medicine, the discovery of the various effector systems and their components originated from studies of patients with unusually severe, frequently recurring infections . Modern molecular biology has greatly aided in elucidation of the basic disorders and identification of the corresponding gene defect . This knowledge will hopefully provide us with better tools for causal therapy in the future; at present the only available "cure" is bone marrow transplantation, a treatment modality with many problems of its own.

Lipids, 2002 Oct, 37(10), 997 - 1006
Lipase-catalyzed hydrolysis of TG containing acetylenic FA; Jie MS et al.; Hydrolysis of symmetrical acetylenic TG of type AAA {viz., glycerol tri-(4-decynoate), glycerol tri-(6-octadecynoate), glycerol tri-(9-octadecynoate), glycerol tri-(10-undecynoate), and glycerol tri-(13-docosynoate)} in the presence of eight microbial lipases was studied . Novozyme 435 (Candida antarctica), an efficient enzyme for esterification, showed a significant resistance in the hydrolysis of glycerol tri-(9-octadecynoate) and glycerol tri-(13-docosynoate) . Hydrolysis of acetylenic TG with Lipolase 100T (Humicola lanuginosa) was rapidly accomplished . Lipase PS-D (Pseudomonas cepacia) showed a fair resistance toward the hydrolysis of glycerol tri-(6-octadecynoate) only, which reflected its ability to recognize the delta6 positional isomer of 18:1 . Lipase CCL (Candida cylindracea, syn . C . rugosa) and AY-30 (C . rugosa) were able to catalyze the release of 10-undecynoic acid and 9-octadecynoic acid from the corresponding TG, but less readily the 13-docosynoic acid in the case of glycerol tri-(13-docosynoate) . The two lipases CCL and AY-30 were able to distinguish the small difference in structure of fatty acyl moieties in the TG substrate . To confirm this trend, three regioisomers of mixed acetylenic TG of type ABC (containing one each of delta6, delta9, and delta13 acetylenic FA in various positions) were prepared and hydrolyzed with CCL and AY-40 . The results reconfirmed the observation that AY-30 and CCL were able to distinguish the slight differences in the molecular structure (position of the acetylenic bond and chain length) of the acyl groups in the TG during the hydrolysis of such TG substrates.

Astrobiology, 2002 Fall, 2(3), 271 - 80
Molecular identification of cyanobacteria associated with stromatolites from distinct geographical locations; Neilan BA et al.; Modern stromatolites represent a significant resource for studying microbial ecology and evolution . A preliminary investigation was undertaken employing specific genetic probes to characterize the cyanobacteria responsible for stromatolite construction in a range of environments, including microbial mats found in Australia not previously examined with molecular methods . Isolates of cyanobacteria were collected from stromatolites in thermal springs, hypersaline lakes, and oceanic fringes on two continents . A polymerase chain reaction specific for DNA of cyanobacterial 16S rRNA was developed, the resulting products of the DNA amplification reaction were sequenced, and the data were used to infer relatedness between the isolates studied and other members of the cyanobacterial radiation . Complete sequence was generated for the region from position 27 to 408 for 13 strains of cyanobacteria associated with stromatolites . All stromatolite-derived sequences were most closely related to cyanobacteria, as indicated by local sequence alignment . It was possible to correlate genetic identity with morphological nomenclatures and to expand the phylogeny of benthic cyanobacteria . These inferences were also expanded to temporal variation in the dominant resident cyanobacterial species based on sampling of surface and core sinter laminations . Under the methods employed, only one cyanobacterial strain was detected in each sample, suggesting the possible dominance of a specific clonal population of cyanobacteria at any one time in the biota of the samples tested . The data indicate that internal core samples of a stromatolite at least 10 years old can be successfully analyzed by DNA-based methods to identify preserved cyanobacteria.

Insect Biochem Mol Biol, 2002 Nov, 32(11), 1457 - 67
Molecular characterization of the insect immune protein hemolin and its high induction during embryonic diapause in the gypsy moth, Lymantria dispar; Lee KY et al.; During the embryonic (pharate first instar) diapause of the gypsy moth, Lymantria dispar, a 55 kDa protein is highly up-regulated in the gut . We now identify that protein as hemolin, an immune protein in the immunoglobulin superfamily . We isolated a gypsy moth hemolin cDNA and demonstrated a high degree of similarity with hemolins from three other moth species . Hemolin mRNA levels increased at the time of diapause initiation and remained high throughout the mandatory period of chilling required to terminate diapause in this species, and then dropped in late diapause . This mRNA pattern reflects the pattern of protein synthesis . These results suggest that hemolin is developmentally up-regulated in the gut during diapause . Diapause in this species can be prevented using KK-42, an imidazole derivative known to inhibit ecdysteroid biosynthesis, and gypsy moths treated in this manner failed to elevate hemolin mRNA . Conversely, this diapause appears to be initiated and maintained by the steroid hormone, 20-hydroxyecdysone, and the addition of 20-hydroxyecdysone to the culture medium elevated hemolin mRNA in the gut . Our results thus indicate a role for 20-hydroxyecdysone in the elevation of hemolin mRNA during diapause . Presumably, hemolin functions to protect the gypsy moth from microbial infection during its long, overwintering diapause.

Mol Hum Reprod, 2003 Jan, 9(1), 53 - 8
Interrelationships of interleukin-8 with interleukin-1beta and neutrophils in vaginal fluid of healthy and bacterial vaginosis positive women; Cauci S et al.; Vaginal innate immunity in response to microbial perturbation is still poorly understood and could be crucial for protection from adverse outcomes . We investigated the relationship between interleukin (IL)-8, IL-1beta and neutrophils in vaginal fluid obtained from 60 healthy women and 51 women who were bacterial vaginosis (BV) positive . Concentrations of IL-8 and IL-1beta were highly correlated with counts of neutrophils in vaginal fluid of the entire population examined (111 subjects) . Vaginal IL-1beta concentrations were significantly higher (P < 0.001) in BV positive women . There was no significant difference in IL-8 levels or number of neutrophils between healthy controls and BV positive women . None of the healthy controls with high neutrophil counts (> or =75th percentile, 14 average count per field) had high concentrations of IL-1beta (> or =75th percentile, 220 pg/ml), whereas 84% of BV positive women with high neutrophil counts had high IL-1beta concentrations (P < 0.001) . On the contrary, no difference in the percentage of subjects with elevated concentrations of IL-8 (> or =75th percentile, 2842 pg/ml) was found between healthy and BV positive women with high numbers of neutrophils (55.5% of healthy versus 53% of BV positive women) . Our findings show that BV causes a large increase in IL-1beta concentrations which is not paralleled by an increase in IL-8 concentrations in vaginal fluid, suggesting that BV-associated factors more specifically dampen IL-8 rather than IL-1beta . The lack of an increase in IL-8 may explain the absence of an increase in neutrophil numbers in most women exposed to abnormal vaginal colonization (BV).

J Biomol Struct Dyn, 2003 Feb, 20(4), 589 - 94
Exploring the interaction energies for the binding of hydroxydiphenyl ethers to enoyl-acyl carrier protein reductases; Muralidharan J et al.; It is now well established that the potent anti-microbial compound, triclosan, interrupts the type II fatty acid synthesis by inhibiting the enzyme enoyl-ACP reductase in a number of organisms . Existence of a high degree of similarity between the recently discovered enoyl-ACP reductase from P . falciparum and B . napus enzyme permitted building of a satisfactory model for the former enzyme that explained some of the key aspects of the enzyme such as its specificity for binding to the cofactor and the inhibitor . We now report the interaction energies between triclosan and other hydroxydiphenyl ethers with the enzymes from B . napus, E . coli and P . falciparum . Examination of the triclosan-enzyme interactions revealed that subtle differences exist in the ligand binding sites of the enzymes from different sources i.e., B . napus, E . coli and P . falciparum . A comparison of their binding propensities thus determined should aid in the design of effective inhibitors for the respective enzymes.

FEBS Lett, 2003 Jan 16, 534(1-3), 197 - 201
Tautomeric state of alpha-sarcin histidines . Ndelta tautomers are a common feature in the active site of extracellular microbial ribonucleases; Perez-Canadilllas JM et al.; Extracellular fungal RNases, including ribotoxins such as alpha-sarcin, constitute a family of structurally related proteins represented by RNase T1 . The tautomeric preferences of the alpha-sarcin imidazole side chains have been determined by nuclear magnetic resonance and electrostatic calculations . Histidine residues at the active site, H50 and H137, adopt the Ndelta tautomer, which is less common in short peptides, as has been found for RNase T1 . Comparison with tautomers predicted from crystal structures of other ribonucleases suggests that two active site histidine residues with the Ndelta tautomer are a conserved feature of microbial ribonucleases and that this is related to their ribonucleolytic function.

Immunol Lett, 2003 Jan 22, 85(2), 85 - 95
Recognition of pathogen-associated molecular patterns by TLR family; Akira S et al.; Toll-like receptors (TLRs) are type I transmembrane proteins involved in innate immunity by recognizing microbial conserved structures . Recent studies have shown that TLR3 recognizes dsRNA, a viral product, whereas TLR9 recognizes unmethylated CpG motifs frequently found in the genome of bacteria and viruses, but not vertebrates . TLR7 recognizes small synthetic immune modifiers including imiquimod, R-848, loxoribine, and bropirimine, all of which are already applied or promising for clinical use against viral infections and cancers . Plasmacytoid dendritic cells express TLR7 and TLR9, and respond to TLR7 and TLR9 ligands by producing a large amount of interferon (IFN-alpha) . These results indicate that TLR3, TLR7 and TLR9 may play an important role in detecting and combating viral infections.

Theriogenology, 2003 Mar, 59(5-6), 1157 - 70
Fertility of ram semen frozen in Bioexcell and used for cervical artificial insemination; Gil J et al.; The current use of ingredients of animal origin, such as egg yolk, in semen extenders presents a risk of microbial contamination, and has led to the search for alternatives . Such an extender is commercially available for bull semen (Bioexcell), IMV, L'Aigle, France), and it has previously been tested in vitro for freezing ram semen, with satisfactory results . The aim of the present study was to compare the fertility results of ewes in Uruguay, after cervical insemination with ram semen that was frozen in Bioexcell versus semen frozen in a conventional milk-egg yolk extender (control) . Semen from five Corriedale rams was frozen, using a split sample design, in either milk-egg yolk or Bioexcell extender, using a two-step extension method . The sperm parameters assessed after thawing were subjective motility, membrane integrity (SYBR-14/PI), and capacitation status (CTC) . Thawed semen was inseminated intracervically once during spontaneous estrus in 970 Corriedale ewes that grazed in natural pastures, under extensive management conditions . Fertility was recorded as nonreturn rates at 21 days (NRR-21) and 36 days (NRR-36) after artificial insemination (AI), as well as pregnancy rate (PR-US, diagnosed ultrasonographically 50 days after AI of the last ewe) . Subjective motility was slightly higher in Bioexcell than in the milk extender (47 vs . 46.5%; NS), as was membrane integrity (38 vs . 37.7%; NS) and the percentage of uncapacitated spermatozoa (28.5 vs . 26.3%; NS) . There were no statistically significant differences in fertility rates found between Bioexcell and the control extender: NRR-21 (35.9 vs . 33.2%), NRR-36 (34.8 vs . 32.6%), and PR-US (28.4 vs . 27.2%) . In conclusion, Bioexcell appears to be an alternative to the conventional milk-egg yolk extender for freezing ram semen, and provides similar fertility results after cervical AI under extensive management conditions . Thus, Bioexcell, containing no additives of animal origin, can offer a safer alternative when frozen semen is used for introducing new genetic material into a flock or a country.

Bioelectromagnetics, 2003 Feb, 24(2), 91 - 102
Growth and yield of winter wheat (Triticum aestivum L.) and corn (Zea mays L.) near a high voltage transmission line; Soja G et al.; The objective of this study was to determine the effects of an electromagnetic field from a high voltage transmission line on the yield of agricultural crops cultivated underneath and near the transmission line . For 5 years, experiments with winter wheat and corn were carried out near the 380 kV transmission line Durnrohr (Austria)-Slavetice (Czech Republic) . Different field strengths were tested by planting the crops at different distances from the transmission line . The plants were grown in experimental plots (1.77 m2), aligned to equal electric field strengths, and were cultivated according to standard agricultural practice . The soil for all plots was homogenized layer-specifically to a depth of 0.5 m to guarantee uniform soil conditions in the plant root environment . The soil was sampled annually for determinations of carbon content and the behavior of microbial biomass . During development of the vegetation, samples were collected at regular intervals for growth rate analyses . At physiological maturity, the plots (n = 8) were harvested for grain and straw yield determinations . The average electric and magnetic field strengths at four distances from the transmission line (nominal distances: 40, 14, 8, and 2 m) were between 0.2 and 4.0 kV/m and between 0.4 and 4.5 micro T, respectively . No effect of the field exposures on soil microbial biomass could be detected . The wheat grain yields were 7% higher (average of 5 years) in the plots with the lowest field exposure than in the plots nearer to the transmission line (P <.10) . The responses of the plants were more pronounced in years with drought episodes during grain filling than in humid years . No significant yield differences were found for corn yields . The extent of the yield variations attributed to the distance from the transmission line was small compared to the observed annual variations in climatic or soil specific site characteristics .

Eur J Pharmacol, 2003 Jan 17, 459(2-3), 131 - 8
Effects of rhizoxin, a microbial angiogenesis inhibitor, on angiogenic endothelial cell functions; Aoki K et al.; Our previous study revealed that rhizoxin ({1S-{1R*,3R*,5S*,8R*(1R*,2S*,3E,5E,7E),10R*,11S*,13S*,14E,16S*,17S*}}-10-hydroxy-8-{2-methoxy-1,3,7-trimethyl-8-(2-methyl-4-oxazolyl)-3,5,7-octatrienyl}-11,16-dimethyl-4,7,12,18-tetraoxatetracyclo{15.3.1.03,5.011,13}heneicos-14-ene-6,19-dione) has a potent inhibitory effect on in vivo angiogenesis . However, little is known regarding the mechanism by which rhizoxin exhibits antiangiogenic activity . In this study, we examined its effects on the functions of endothelial cells associated with neovascular formation in vivo, using cultured vascular endothelial cells . Rhizoxin concentration-dependently inhibited the proliferation of bovine carotid artery endothelial cells, human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the IC(50) values being 7, 5 and 0.4 nM, respectively . In addition, it reduced the extracellular plasminogen activator level in bovine vascular endothelial cells in the low nM range, and suppressed the migration of human dermal microvascular endothelial cells in the pM range . Furthermore, it blocked the tubular morphogenesis of human umbilical vein endothelial cells and human dermal microvascular endothelial cells on Matrigel in a concentration-dependent manner; the IC(50) values being 40 and 130 pM, respectively . These results suggest that rhizoxin exhibits antiangiogenic activity through the combined inhibition of some functions of endothelial cells responsible for induction of in vivo angiogenesis.

Biomacromolecules, 2003 Jan-Feb, 4(1), 1 - 6
General trend of lipase to self-assemble giving bimolecular aggregates greatly modifies the enzyme functionality; Palomo JM et al.; Three microbial lipases (those from Candida rugosa, Humicola lanuginosa, and Mucor miehei) have been found to exhibit a tendency to form bimolecular aggregates in solution even at very low enzyme concentrations (44 microg/mL) in the absence of a detergent, as detected by gel filtration . The monomolecular form of the enzymes was found as unique only at low enzyme concentration and in the presence of detergents . However, in the case of the lipase B from Candida antarctica, no bimolecular form could be identified even at enzyme concentrations as high as 1.2 mg/mL in the absence of detergent . It has been stated that bimolecular and monomolecular structures display very different functional properties: (i) the enzyme specific activity decreased when the lipase concentration increased; (ii) the bimolecular form was much more stable than the monomeric one yielding a higher optimal T (increasing between 5 and 10 degrees C) and higher stability in inactivation experiments (the dimer half-life became several orders of magnitude higher than that of the monomer); (iii) the enantioselectivity depended on the enzyme concentration even after immobilization . For example, with use of the lipase from H . lanuginosa, the enantiomeric excess of the remaining ester in the hydrolysis of fully soluble ethyl ester of (R,S)-2-hydroxy-4-phenylbutanoic acid varied from 4 to 57 when the concentrated or diluted enzyme immobilized on PEI support, respectively, was used . It seems that the bimolecular structure of lipases might be formed by two open lipase molecules (interfacially activating each other) in very close contact and hence with a very altered active center.

Water Sci Technol, 2002, 46(11-12), 277 - 84
Progress and data gaps in quantitative microbial risk assessment; Haas CN; Quantitative microbial risk assessment (QMRA) has emerged as a useful tool to develop criteria for human exposures to pathogens . There is opportunity to extend the usefulness of this tool in water and other applications if new fundamental information can be obtained to complement existing data . Such information includes effects of strain and host differences, population level disease dynamics, and ability of animal data to serve as a predictor of human potency . This paper reviews the development of QMRA and outlines the nature of additional data that would be useful for its development.

Scand J Gastroenterol, 2002 Dec, 37(12), 1449 - 53
Outcome of necrosectomy in acute pancreatitis: the case for continued vigilance; Beattie GC et al.; BACKGROUND: Surgery for pancreatic necrosis complicating acute severe pancreatitis carries a high risk of mortality and may be influenced by a range of variables including patterns of referral, case selection and quality of care . METHODS: An observational study of a consecutive series of 54 patients undergoing pancreatic necrosectomy in a specialist Hepatobiliary unit over an 8-year study period . Principal outcomes were organ dysfunction and physiological derangement in relation to surgery, microbial colonization of necrosis and relation to outcome, re-operation rates, requirement for peri-operative nutritional support, trends in mortality and survival analysis . RESULTS: Necrosectomy was associated with statistically significant deterioration in immediate postoperative organ dysfunction scores (ANOVA P < 0.01) . Infected necrosis was present in 36 (68%) . Fungal colonization of necrosis was present in 5 (9%) . Mortality in this subgroup was 80% (4 deaths) . There was no association between bacterial colonization of necrosis and death in this study (P = 0.77; Fisher exact test; relative risk 0.9,95% confidence interval 0.54-1.54) . Twenty patients (37%) required further surgical intervention with an average of 1.5 surgical procedures per patient . Twenty-three patients (43%) died . Patient survival to discharge was best predicted by admission APACHE-II score with relative risk of death increasing 14% for each unit increase in APACHE-II score at admission . CONCLUSIONS: The results of the present study illustrate that there is no place for complacency in the surgical management of patients with severe acute pancreatitis . A clinical governance approach would promote pre-defined protocols between admitting hospitals and tertiary referral centres . Future research should target new interventions in patients with high admission APACHE-II scores in whom prognosis is particularly poor and explore the role of infection of necrotic tissue.

Adv Biochem Eng Biotechnol, 2003, 79, 113 - 36
The threonine story; Debabov VG; L-Threonine is an essential amino acid which has recently been brought into agricultural industry for balancing the livestock feed . L-Threonine is produced by microbial synthesis using glucose or sucrose as substrates . For the process to be cost-effective, the microbial strain must be capable of threonine overproduction . This paper reviews the biochemical pathways of L-threonine synthesis in bacteria and the regulation of these pathways, the principles and the techniques of constructing high-producing strains, and the most efficient strains thus developed.

Vopr Pitan, 2002, 71(6), 19 - 23
{The modern approaches to the complex treatment of functional constipation syndrome}; Blokhina LV et al.; The high abundance of functional disorders of a gastrointestinal tract stipulates an urgency of studies dedicated to their complex treatment and preventenance . The complex treatment of functional snaps is necessary to base on the system of measures which are powering up medical power supply, a fissile way of life, recovering of regularity of the act of defecation, normalization of the emotional status of the patients . The article considers characteristics of different kinds of probiotical substances and factors which provide normalization of intestinal microbial balance and related to probiotical action.

Proc Natl Acad Sci U S A, 2003 Jan 21, 100(2), 697 - 702 Epub 2003 Jan 09.
Autochthonous eukaryotic diversity in hydrothermal sediment and experimental microcolonizers at the Mid-Atlantic Ridge; Lopez-Garcia P et al.; The diversity and mode of life of microbial eukaryotes in hydrothermal systems is very poorly known . We carried out a molecular survey based on 18S ribosomal RNA genes of eukaryotes present in different hydrothermal niches at the Mid-Atlantic Ridge . These included metal-rich and rare-earth-element-rich hydrothermal sediments of the Rainbow site, fluid-seawater mixing regions, and colonization devices (microcolonizers) containing organic, iron-rich, and porous mineral substrates that were exposed for 15 days to a fluid source . We identified considerable phylogenetic diversity, both at kingdom level and within kinetoplastids and alveolates . None of our sequences affiliates to photosynthesizing lineages, suggesting that we are targeting only autochthonous deep-sea communities . Although sediment harbored most phylogenetic diversity, microcolonizers predominantly contained bodonids and ciliates, indicating that these protists pioneer the colonization process . Given the large variety of divergent lineages detected within the alveolates in deep-sea plankton, hydrothermal sediments, and vents, alveolates seem to dominate the deep ocean in terms of diversity . Compared with data from the Pacific Guaymas basin, some protist lineages seem ubiquitous in hydrothermal areas, whereas others, notably kinetoplastid lineages, very abundant and diverse in our samples, so far have been detected only in Atlantic systems . Unexpectedly, although alvinellid polychaetes are considered endemic of Pacific vents, we detected alvinellid-related sequences at the fluid-seawater interface and in microcolonizers . This finding can boost further studies on deep-sea vent animal biology and biogeography.

Clin Diagn Lab Immunol, 2003 Jan, 10(1), 30 - 7
Systemic immunoresponses in mice after repeated exposure of lungs to spores of Streptomyces californicus; Jussila J et al.; Microbial growth in moisture-damaged buildings is associated with respiratory and other symptoms in the occupants . Streptomyces spp . are frequently isolated from such buildings . In the present study, we evaluated the responses of mice after repeated exposure to spores of Streptomyces californicus . Mice were exposed via intratracheal instillation to six doses (at 7-day intervals) of the spores of S . californicus, originally isolated from the indoor air of a moisture-damaged building, at three dose levels (2 x 10(3), 2 x 10(5), and 2 x 10(7) spores) . Inflammation and toxicity, including changes in cell populations in the lungs, lymph nodes, and spleen, were evaluated 24 h after the last dosage . The exposure provoked a dose-dependent inflammatory cell response, as detected by the intense recruitment of neutrophils, but the numbers of macrophages and lymphocytes in the airways also increased . The cellular responses corresponded to the dose-dependent increases in inflammation- and cytotoxicity-associated biochemical markers (i.e., levels of albumin, total protein, and lactate dehydrogenase) in bronchoalveolar lavage fluid . The spore exposure increased the number of both activated and nonactivated T lymphocytes . Also, the amounts of CD3(-) CD4(-) and unconventional CD3(-) CD4(+) lymphocytes in the lung tissue were augmented . Interestingly, the spore exposure decreased cells in the spleen . This effect was strongest at the dose of 2 x 10(5) spores . These results indicate that the spores of S . californicus are capable of provoking both immunostimulation in lungs (inflammation) and systemic immunotoxicity, especially in the spleen . The immunotoxic effect resembled that caused by chemotherapeutic agents, originally isolated from Streptomyces spp . Thus, S . californicus must be considered a microbial species with potential to cause systemic adverse health effects in occupants of moisture-damaged buildings.

Biodegradation, 2002, 13(4), 273 - 83
Degradation of petroleum model compounds immobilized on clay by a hypersaline microbial mat; Grotzschel S et al.; In this study the degradation of hydrophobic petroleum model compounds (phenanthrene, pristane, octadecane and dibenzothiophene) added to a submersed hypersaline microbial mat was investigated . Montmorillonite with an artificially altered, hydrophobic surface was used as carrier material, forming an organo-clay complex (OCC) with the attached mixture of petroleum model compounds . 6 mg/cm2 OCC were applied to cyanobacterial mat pieces, containing approximately 33.3 microg/mg OCC of each compound . The degradation experiment was performed under controlled laboratory conditions and accompanied by chemical analyses by GC/GC-MS, molecular analyses by PCR and DGGE as well as functional analyses by microsensor measurements of oxygen, photosynthesis, sulfide, pH and light . All applied model compounds were degraded, but residues were still present after 18 weeks . The aromatic compounds phenanthrene (5.1 microg/mg OCC) and dibenzothiophene (4.3 microg/mg OCC) were preferentially degraded compared to the alkanes pristane (12.4 microg/mg OCC) and n-octadecane (13.4 microg/mg OCC) . Metabolic changes during the degradation process could not be detected by microsensor measurements . The molecular population analyses did not reveal any significant community changes concomitant with the decrease of the petroleum model compounds . We conclude, that the pristine mats represent an intact, robust ecosystem in which the enzymatic requirements for the degradation of the applied pollutants exist . The slow degradation process did not affect the usual high internal turnover rates and did not favor a certain population in the community of the mats.

Biodegradation, 2002, 13(4), 221 - 7
Combining photolysis and bioprocesses for mineralization of high molecular weight polyacrylamides; El-Mamouni R et al.; The influence of ultraviolet photolysis as a pretreatment to the aerobic and anaerobic biological mineralization of a 14C-polyacrylamide was assessed using a series of radiorespirometry bioassays . The polyacrylamide studied was non-ionic with molecular weights ranging between 100,000 and 1 million . Aerobic and anaerobic biomineralization of the unphotolysed (raw) polyacrylamide was found to be only 0.60% and 0.70%, respectively, after 6 weeks of incubation, and hence indicative of the natural recalcitrance of polyacrylamide to microbial degradation . The effectiveness of UV irradiation in the physical breakdown of the polyacrylamide chain into oligomers was demonstrated by the shift in the molecular weight distribution and the positive correlation between the time of irradiation and the degree of its biological mineralization . The molecular weight fraction below 3 kD, which represents only 2% of the raw polyacrylamide, was increased to 41, 60 and 80% after 12, 24 and 48 hours of photolysis, respectively . This in turn, yielded, after 6 weeks of incubation, an aerobic mineralization of 5, 17 and 29% of 150 mg/L polyacrylamide, respectively, and an anaerobic mineralization of 3, 5 and 17%, respectively . Biomass acclimation substantially improved the specific initial rate of biomineralization of the photolysed polyacrylamides, but not the overall percentage of polyacrylamides mineralized.

Nat Prod Rep, 2002 Dec, 19(6), 742 - 60
Quinoline, quinazoline and acridone alkaloids; Michael JP; This review covers the isolation, structure determination, synthesis and biological activity of quinoline, quinazoline and acridone alkaloids from plant, microbial and animal sources . The literature from July 2000 to June 2001 is reviewed, and 119 references are cited.

Nat Prod Rep, 2002 Dec, 19(6), 719 - 41
Indolizidine and quinolizidine alkaloids; Michael JP; This review covers the isolation, structure determination, synthesis and biological activity of indolizidine and quinolizidine alkaloids from microbial, plant and animal sources . Included in the review are slaframine; hydroxylated indolizidines and their analogues; alkaloids from ants and amphibians; metabolites of the genera Prosopis, Streptomyces and Nuphar and the Lythraceae; phenanthroindolizidines and related alkaloids; lupin alkaloids; and alkaloids from sponges . tunicates and coccinellid beetles . The literature from July 2000 to June 2001 is reviewed, and 172 references are cited.

Chem Pharm Bull (Tokyo), 2003 Jan, 51(1), 71 - 4
Transformation of Cinchona alkaloids into 1-N-oxide derivatives by endophytic Xylaria sp isolated from Cinchona pubescens; Shibuya H et al.; The microbial transformation of four Cinchona alkaloids (quinine, quinidine, cinchonidine, and cinchonine) by endophytic fungi isolated from Cinchona pubescens was investigated . The endophytic filamentous fungus Xylaria sp . was found to transform the Cinchona alkaloids into their 1-N-oxide derivatives.

Nucleic Acids Res, 2003 Jan 1, 31(1), 58 - 62
MBGD: microbial genome database for comparative analysis; Uchiyama I; MBGD is a workbench system for comparative analysis of completely sequenced microbial genomes . The central function of MBGD is to create an orthologous gene classification table using precomputed all-against-all similarity relationships among genes in multiple genomes . In MBGD, an automated classification algorithm has been implemented so that users can create their own classification table by specifying a set of organisms and parameters . This feature is especially useful when the user's interest is focused on some taxonomically related organisms . The created classification table is stored into the database and can be explored combining with the data of individual genomes as well as similarity relationships among genomes . Using these data, users can carry out comparative analyses from various points of view, such as phylogenetic pattern analysis, gene order comparison and detailed gene structure comparison . MBGD is accessible at http://mbgd.genome.ad.jp/.

Immunology, 2003 Jan, 108(1), 10 - 5
Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue; Ochoa MT et al.; To determine how distinct receptors of the immune system can contribute to innate immunity, we investigated the pattern of Toll-like receptor 1 (TLR1) and TLR2 expression in human lymphoid tissue . We found that TLR1 and TLR2 were co-expressed on cells of the innate immune system, including macrophages and dendritic cells . In addition, TLR1 and TLR2 were expressed in mucosa-associated lymphoid tissue on tonsillar crypt epithelium . Of the lymphoid tissue examined, spleen expressed the highest levels of TLR2 . Although TLR1- and TLR2-positive cells were in close proximity to T lymphocytes in vivo, lymphocytes themselves were devoid of TLR1 and TLR2 expression . The co-expression of TLR1 and TLR2 on myeloid cells in lymphoid tissue provides the host with the ability to respond to a variety of microbial ligands at sites conducive to the generation of an immune response.

Proc Natl Acad Sci U S A, 2003 Jan 7, 100(1), 26 - 31 Epub 2002 Dec 23.
Formation and character of an ancient 19-m ice cover and underlying trapped brine in an "ice-sealed" east Antarctic lake; Doran PT et al.; Lake Vida, one of the largest lakes in the McMurdo Dry Valleys of Antarctica, was previously believed to be shallow (<10 m) and frozen to its bed year-round . New ice-core analysis and temperature data show that beneath 19 m of ice is a water column composed of a NaCl brine with a salinity seven times that of seawater that remains liquid below -10 degrees C . The ice cover thickens at both its base and surface, sealing concentrated brine beneath . The ice cover is stabilized by a negative feedback between ice growth and the freezing-point depression of the brine . The ice cover contains frozen microbial mats throughout that are viable after thawing and has a history that extends to at least 2,800 (14)C years B.P., suggesting that the brine has been isolated from the atmosphere for as long . To our knowledge, Lake Vida has the thickest subaerial lake ice cover recorded and may represent a previously undiscovered end-member lacustrine ecosystem on Earth.

J Immunol, 2003 Jan 15, 170(2), 947 - 60
Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to identification of mimicry epitopes for glutamic acid decarboxylase 65-specific TCRs; Uemura Y et al.; Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed . Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process . A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult . We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4(+) T cell clones, established from type I diabetes patients . We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains . The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously . Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs . Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes . Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones . Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.

J Clin Microbiol, 2003 Jan, 41(1), 100 - 5
Molecular assays for detection of human metapneumovirus; Mackay IM et al.; The recent description of the respiratory pathogen human metapneumovirus (hMPV) has highlighted a deficiency in current diagnostic techniques for viral agents associated with acute lower respiratory tract infections . We describe two novel approaches to the detection of viral RNA by use of reverse transcriptase PCR (RT-PCR) . The PCR products were identified after capture onto a solid-phase medium by hybridization with a sequence-specific, biotinylated oligonucleotide probe . The assay was applied to the screening of 329 nasopharyngeal aspirates sampled from patients suffering from respiratory tract disease . These samples were negative for other common microbial causes of respiratory tract disease . We were able to detect hMPV sequences in 32 (9.7%) samples collected from Australian patients during 2001 . To further reduce result turnaround times we designed a fluorogenic TaqMan oligoprobe and combined it with the existing primers for use on the LightCycler platform . The real-time RT-PCR proved to be highly reproducible and detected hMPV in an additional 6 out of 62 samples (9.6%) tested during the comparison of the two diagnostic approaches . We found the real-time RT-PCR to be the test of choice for future investigation of samples for hMPV due to its speed, reproducibility, specificity, and sensitivity.

Eur Cytokine Netw, 2002 Oct-Dec, 13(4), 447 - 55
Prolactin-cytokine network in the defence against Acanthamoeba castellanii in murine microglia {corrected}; Benedetto N et al.; In the central nervous system, cytokine-primed microglia play a pivotal role in host defence against parasite infections . In this study, the effect of recombinant (r) prolactin (rPRL) and rIFN-gamma on A . castellanii infection in murine microglia was examined . Priming of microglia with rPRL and rIFN-gamma synergistically triggered, in a dose-dependent manner, amebastatic activity and the release of endogenous IL-1alpha, IL-1beta, IL-6 and TNF-alpha . More than 53, 57, 49, 89 or 96 % of amebastatic activity was abrogated by anti-PRLR, IFN-gammaR, IL-1beta (but not IL-1alpha), IL-6 or TNF-alpha antibodies, suggesting that these two receptors and proinflammatory cytokines participate in the anti-microbial function . Inasmuch as DPI and AET, both inhibitors of NO synthase, blocked amebastatic activity only during the priming process, the NO-dependent pathway itself appears not to be directly involved in the anti-parasitic capacity . These data suggest that the PRL/PRLR and IFN-gamma/IFN-gamma/R complexes, by the induction of the IFN-gammaR on microglia, up-regulate the release of endogenous TNF-alpha, IL-6 and IL-1beta by these cells, which could trigger anti-microbial activity against A . castellanii infection in the brain {corrected}.

Structure (Camb), 2003 Jan, 11(1), 55 - 65
The crystal structure of a cyanobacterial water-soluble carotenoid binding protein; Kerfeld CA et al.; Carotenoids undergo a wide range of photochemical reactions in animal, plant, and microbial systems . In photosynthetic organisms, in addition to light harvesting, they perform an essential role in protecting against light-induced damage by quenching singlet oxygen, superoxide anion radicals, or triplet-state chlorophyll . We have determined the crystal structure of a water-soluble orange carotenoid protein (OCP) isolated from the cyanobacterium Arthrospira maxima at a resolution of 2.1 A . OCP forms a homodimer with one carotenoid molecule per monomer . The carotenoid binding site is lined by a striking number of methionine residues . The structure reveals several possible ways in which the protein environment influences the spectral properties of the pigment and provides insight into how the OCP carries out its putative functions in photoprotection.

Structure (Camb), 2003 Jan, 11(1), 7 - 9
Twenty thousand ORFan microbial protein families for the biologist?
Siew N, Fischer D.
The genomes of most newly sequenced organisms contain a significant fraction of ORFs (open reading frames) that match no other sequence in the databases . We refer to these singleton ORFs as sequence ORFans . Because little can be learned about ORFans by homology, the origin and functions of ORFans remain a mystery . However, in this era of full genome sequencing, it seems that ORFans have been underemphasized . In this minireview, we draw attention to the increasing number of ORFans and to the consequences of this growth to biological research in the postgenomic era.

Eur J Immunol, 2002 Dec, 32(12), 3628 - 37
CpG-ODN-induced inflammation is sufficient to cause T-cell-mediated autoaggression against hepatocytes; Sacher T et al.; Autoimmune diseases are often associated with microbial infections . Molecular mimicry between microbial antigens and self-epitopes has been suggested as a mechanism for breaking self-tolerance and induction of autoimmunity . Since infections also cause inflammatory responses we explored the role of local inflammation in organ-specific autoimmunity . For this purpose, transgenic mice were used expressing the MHC class I molecule Kb exclusively on hepatocytes . These mice exhibit Kb-specific tolerance as exemplified by the acceptance of Kb+ grafts . Inflammatory reactions were induced by injection of immunostimulatory cytosine-phosphorothioate-guanine (CpG)-rich oligodeoxynucleotides (ODN) . Application of CpG-ODN is sufficient to break tolerance in vivo, and to cause activation of Kb-specific CD8+ T cells and subsequent autoaggression against hepatocytes . The CpG-ODN-induced inflammation appears to have two major effects . First, it causes infiltration of T cells into the liver parenchyma . Second, adhesion and costimulatory molecules are up-regulated on hepatocytes so that the infiltrating CD8+ T cells encounter Kb on hepatocytes, which display an APC-like phenotype, resulting in activation and tissue damage . Autoimmune hepatitis can be maintained for at least eight weeks by repeated application of CpG-ODN but subsides after termination of the inflammatory stimulus, suggesting the requirement of additional factors for a self-perpetuation of autoimmunity . These observations describe an additional pathway for the induction of autoimmunity, i.e . in the absence of microbial antigens inflammatory reactions alone can lead to infiltration of T cells into organs, resulting in breaking of tolerance and autoaggression . Moreover, the results provide evidence that T cell activation can take place not only in draining lymph nodes but also directly on parenchymal cells.

Plant Cell Physiol, 2002 Dec, 43(12), 1436 - 44
The growing world of expansins; Cosgrove DJ et al.; Expansins are cell wall proteins that induce pH-dependent wall extension and stress relaxation in a characteristic and unique manner . Two families of expansins are known, named alpha- and beta-expansins, and they comprise large multigene families whose members show diverse organ-, tissue- and cell-specific expression patterns . Other genes that bear distant sequence similarity to expansins are also represented in the sequence databases, but their biological and biochemical functions have not yet been uncovered . Expansin appears to weaken glucan-glucan binding, but its detailed mechanism of action is not well established . The biological roles of expansins are diverse, but can be related to the action of expansins to loosen cell walls, for example during cell enlargement, fruit softening, pollen tube and root hair growth, and abscission . Expansin-like proteins have also been identified in bacteria and fungi, where they may aid microbial invasion of the plant body.

Appl Environ Microbiol, 2003 Jan, 69(1), 625 - 33
Phylogenetic position and in situ identification of ectosymbiotic spirochetes on protists in the termite gut; Noda S et al.; Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists . Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters . In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N . koshunensis . The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population . The absence of cluster II spirochetes in N . koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I . Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp . in N . koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes . The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes . The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment . Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species . The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.

Appl Environ Microbiol, 2003 Jan, 69(1), 607 - 15
Bacteria associated with cysts of the soybean cyst nematode (Heterodera glycines); Nour SM et al.; The soybean cyst nematode (SCN), Heterodera glycines, causes economically significant damage to soybeans (Glycine max) in many parts of the world . The cysts of this nematode can remain quiescent in soils for many years as a reservoir of infection for future crops . To investigate bacterial communities associated with SCN cysts, cysts were obtained from eight SCN-infested farms in southern Ontario, Canada, and analyzed by culture-dependent and -independent means . Confocal laser scanning microscopy observations of cyst contents revealed a microbial flora located on the cyst exterior, within a polymer plug region and within the cyst . Microscopic counts using 5-(4,6-dichlorotriazine-2-yl)aminofluorescein staining and in situ hybridization (EUB 338) indicated that the cysts contained (2.6 +/- 0.5) x 10(5) bacteria (mean +/- standard deviation) with various cellular morphologies . Filamentous fungi were also observed . Live-dead staining indicated that the majority of cyst bacteria were viable . The probe Nile red also bound to the interior polymer, indicating that it is lipid rich in nature . Bacterial community profiles determined by denaturing gradient gel electrophoresis analysis were simple in composition . Bands shared by all eight samples included the actinobacterium genera Actinomadura and STREPTOMYCES: A collection of 290 bacteria were obtained by plating macerated surface-sterilized cysts onto nutrient broth yeast extract agar or on actinomycete medium . These were clustered into groups of siblings by repetitive extragenic palindromic PCR fingerprinting, and representative isolates were tentatively identified on the basis of 16S rRNA gene sequence . Thirty phylotypes were detected, with the collection dominated by Lysobacter and Variovorax spp . This study has revealed the cysts of this important plant pathogen to be rich in a variety of bacteria, some of which could presumably play a role in the ecology of SCN or have potential as biocontrol agents.

Appl Environ Microbiol, 2003 Jan, 69(1), 583 - 92
Influences of pond geochemistry, temperature, and freeze-thaw on terminal anaerobic processes occurring in sediments of six ponds of the McMurdo Ice Shelf, near Bratina Island, Antarctica; Mountfort DO et al.; The effects of freeze-thaw, freezing and sediment geochemistry on terminal anaerobic processes occurring in sediments taken from below cyanobacterial mats in meltwater ponds of the McMurdo Ice Shelf in Antarctica were investigated . Depending on the geochemical and physical status of the sediments (i.e., frozen or thawed), as well as passage of sediment through a freeze-thaw cycle, terminal carbon and electron flow shifted in which the proportions of hydrogen and acetate utilized for methanogenesis and sulfate reduction changed . Thus, in low-sulfate (or chloride) sediment which was thawed and incubated at 4 degrees C, total carbon and electron flow were mediated by acetate-driven sulfate reduction and H(2)-driven methanogenesis . When the same sediments were incubated frozen, both methanogenesis and sulfate reduction decreased . However, under these conditions methanogenesis was favored over sulfate reduction, and carbon flow from acetate to methane increased relative to sulfate reduction; >70% of methane was contributed by acetate, and more than 80% of acetate was oxidized by pathways not coupled to sulfate reduction . In high-sulfate pond sediments, sulfate reduction was a major process mediating terminal carbon and electron flow in both unfrozen and frozen incubations . However, as with low-sulfate sediments, acetate oxidation became uncoupled from sulfate reduction with freezing . Geochemical and temperature effects could be expressed by linear models in which the log (methanogenesis to sulfate reduction) was negative log linear with respect to either temperature or the log of the sulfate (or chloride) concentration . From these relationships it was possible to predict the ratio for a given temperature (low-sulfate sediments) or sulfate (chloride) concentration . Small transitory changes, such as elevated sulfate reduction coupled to increased acetate turnover, resulted from application of a freeze-thaw cycle to low-salinity pond sediments . The results demonstrate how ecophysiological processes may change in anaerobic systems under extreme conditions (e.g., freezing) and provide new insights into microbial events occurring under these conditions.

Appl Environ Microbiol, 2003 Jan, 69(1), 568 - 76
2-n-Pentyl-4-quinolinol produced by a marine Alteromonas sp . and its potential ecological and biogeochemical roles; Long RA et al.; Bacterium-bacterium interactions occur at intimate spatial scales on the order of micrometers, but our knowledge of interactions at this level is rudimentary . Antagonism is a potential interaction in such microenvironments . To study the ecological role of antibiosis, we developed a model system involving an antibiotic-producing isolate (SWAT5) derived from a marine particle and its dominant antibiotic product, 2-n-pentyl-4-quinolinol (PQ) . This system was used to address questions about the significance of this antibiotic for microbial ecology and carbon cycling on particles . We characterized the chemical and inhibitory properties of PQ in relation to the mechanisms used by particle-associated bacteria in interacting with particles and with other attached bacteria . PQ was produced by SWAT5 only on surfaces . When SWAT5 was grown in polysaccharide matrices, PQ diffused within the matrices but not into the surrounding seawater . SWAT5 might thus be able to generate a localized zone of high antibiotic concentration on particles suspended or sinking through seawater . Target bacterial respiration was most sensitive to PQ (75 nM), while inhibition of DNA synthesis, protein synthesis, and bacterial motility required higher (micromolar) PQ levels . The presence of PQ altered the composition of the bacterial community that colonized and developed in a model particle system . PQ also inhibited Synechococcus and phytoplankton growth . Our results suggest that antibiosis may significantly influence community composition and activities of attached bacterial and thus regulate the biogeochemical fate of particulate organic matter in the ocean.

Appl Environ Microbiol, 2003 Jan, 69(1), 483 - 9
Changes in microbial community composition and function during a polyaromatic hydrocarbon phytoremediation field trial; Siciliano SD et al.; The purpose of this study was to investigate the mechanism by which phytoremediation systems promote hydrocarbon degradation in soil . The composition and degradation capacity of the bulk soil microbial community during the phytoremediation of soil contaminated with aged hydrocarbons was assessed . In the bulk soil, the level of catabolic genes involved in hydrocarbon degradation (ndoB, alkB, and xylE) as well as the mineralization of hexadecane and phenanthrene was higher in planted treatment cells than in treatment cells with no plants . There was no detectable shift in the 16S ribosomal DNA (rDNA) composition of the bulk soil community between treatments, but there were plant-specific and -selective effects on specific catabolic gene prevalence . Tall Fescue (Festuca arundinacea) increased the prevalence of ndoB, alkB, and xylE as well as naphthalene mineralization in rhizosphere soil compared to that in bulk soil . In contrast, Rose Clover (Trifolium hirtum) decreased catabolic gene prevalence and naphthalene mineralization in rhizosphere soil . The results demonstrated that phytoremediation systems increase the catabolic potential of rhizosphere soil by altering the functional composition of the microbial community . This change in composition was not detectable by 16S rDNA but was linked to specific functional genotypes with relevance to petroleum hydrocarbon degradation.

Appl Environ Microbiol, 2003 Jan, 69(1), 461 - 7
In situ exposure to low herbicide concentrations affects microbial population composition and catabolic gene frequency in an aerobic shallow aquifer; de Lipthay JR et al.; The aim of this study was to evaluate how the in situ exposure of a Danish subsurface aquifer to phenoxy acid herbicides at low concentrations (<40 micro g l(-1)) changes the microbial community composition . Sediment and groundwater samples were collected inside and outside the herbicide-exposed area and were analyzed for the presence of general microbial populations, Pseudomonas bacteria, and specific phenoxy acid degraders . Both culture-dependent and culture-independent methods were applied . The abundance of microbial phenoxy acid degraders (10(0) to 10(4) g(-1) sediment) was determined by most probable number assays, and their presence was only detected in herbicide-exposed sediments . Similarly, PCR analysis showed that the 2,4-dichlorophenoxyacetic acid degradation pathway genes tfdA and tfdB (10(2) to 10(3) gene copies g(-1) sediment) were only detected in sediments from contaminated areas of the aquifer . PCR-restriction fragment length polymorphism measurements demonstrated the presence of different populations of tfd genes, suggesting that the in situ herbicide degradation was caused by the activity of a heterogeneous population of phenoxy acid degraders . The number of Pseudomonas bacteria measured by either PCR or plating on selective agar media was higher in sediments subjected to high levels of phenoxy acid . Furthermore, high numbers of CFU compared to direct counting of 4',6-diamidino-2-phenylindole-stained cells in the microscope suggested an increased culturability of the indigenous microbial communities from acclimated sediments . The findings of this study demonstrate that continuous exposure to low herbicide concentrations can markedly change the bacterial community composition of a subsurface aquifer.

Appl Environ Microbiol, 2003 Jan, 69(1), 367 - 72
A labeling study to elucidate the biosynthesis of 4-(4-hydroxyphenyl)-butan-2-one (raspberry ketone) by Nidula niveo-tomentosa; Zorn H et al.; Submerged cells of the basidiomycete Nidula niveo-tomentosa, a microbial producer of 4-(4-hydroxyphenyl)-butan-2-one, were supplemented with (13)C-labeled L-phenylalanines and with {1-(13)C}glucose . Labeled transformation products were detected by a novel method of analyzing stable isotope-labeled metabolites, gas chromatography (GC) coupled to an atomic emission detector, and by GC-mass spectrometry . A benzoate moiety was side chain elongated according to the poly-beta-keto scheme . The presence of an acetyl coenzyme A-carboxylase inhibitor shifted the spectrum of products to benzyl compounds . Hence, the fungal pathway differs from the one established for plant tissues.

Appl Environ Microbiol, 2003 Jan, 69(1), 220 - 6
Culture-independent analysis of probiotic products by denaturing gradient gel electrophoresis; Temmerman R et al.; In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial . Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming . In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis . The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel . Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level . This whole culture-independent approach can be performed in less than 30 h . Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner . Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential.

J Dairy Sci, 2002 Dec, 85(12), 3420 - 9
The proportion of the diet to which fibrolytic enzymes are added affects nutrient digestion by lactating dairy cows; Bowman GR et al.; Eight lactating Holstein cows, four with ruminal cannulas, were used in a duplicated 4 x 4 Latin square design to investigate a fibrolytic enzyme product characterized by xylanase and cellulase activities (Promote N.E.T . Agribrands International, St . Louis, MO) . The diet consisted of concentrate containing rolled barley and supplement, barley silage and alfalfa haylage (55% to 45% DM basis, forage to concentrate ratio) and differed in enzyme application: 1) control, 2) enzyme applied to concentrate (45% of TMR), 3) enzyme applied to supplement (4% of TMR), and 4) enzyme applied to premix (0.2% of TMR) . All diets that were supplemented with the enzyme product delivered about 1.0 grams per cow per day . Digestibility of OM, NDF and ADF in the total tract was increased in comparison to the control when enzymes were added to the entire concentrate . Enzyme treatments that were applied to a smaller portion of the diet showed only numerical increases in digestibility over the control . However, there was an increase in microbial N synthesis for cows fed enzymes added to the premix . The effects of enzyme supplementation on milk production and composition were not statistically significant, but cows receiving the enzyme product added to the concentrate had a numerically higher FCM compared to the control cows . These results indicate that enzyme supplementation increases total tract digestibility of organic matter and fiber . The proportion of the diet to which the enzyme is applied must be maximized to ensure a beneficial response.

Nat Cell Biol, 2003 Jan, 5(1), 16 - 9
Eukaryotic cells and microbial pathogens: a familiar couple take centre stage; Roy CR et al.; The interaction of a microbial pathogen with its host is a highly dynamic process shaped by evolution . Understanding cellular responses to microbial products is essential for a complete understanding of virulence . Similarly, studies aimed at determining how a particular virulence factor functions have helped unravel molecular mechanisms that govern eukaryotic cellular events . These two seemingly diverse areas were brought together on the beautiful Spanish Costa Brava in a recent meeting (October 13-17, 2002) sponsored by European Research Conferences (EURESCO) and the European Molecular Biology Organization (EMBO).

Arch Pharm Res, 2002 Dec, 25(6), 770 - 80
Airway mucus: its components and function; Lillehoj ER et al.; The airway surface liquid (ASL), often referred to as mucus, is a thin layer of fluid covering the luminal surface of the airway . The major function of mucus is to protect the lung through mucociliary clearance against foreign particles and chemicals entering the lung . The mucus is comprised of water, ions, and various kinds of macromolecules some of which possess the protective functions such as anti-microbial, anti-protease, and anti-oxidant activity . Mucus glycoproteins or mucins are mainly responsible for the viscoelastic property of mucus, which is crucial for the effective mucociliary clearance . There are at least eight mucin genes identified in the human airways, which will potentially generate various kinds of mucin molecules . At present, neither the exact structures of mucin proteins nor their regulation are understood although it seems likely that different types of mucins are involved in different functions and might also be associated with certain airway diseases . The fact that mucins are tightly associated with various macromolecules present in ASL seems to suggest that the defensive role of ASL is determined not only by these individual components but rather by a combination of these components . Collectively, mucins in ASL may be compared to aircraft carriers carrying various types of weapons in defense of airbome enemies.

Yakugaku Zasshi, 2002 Dec, 122(12), 1153 - 8
{Effect of admixture of betamethasone butyrate propionate ointment on preservative efficacy}; Ohtani M et al.; Twenty percent of dermatologists have experienced a separation of water or deterioration of topical corticosteroids mixed with commercially available ointments and/or creams . However, few investigations of this deterioration of admixtures have been reported . To assess the effects of preservatives in preventing microbial contamination of these admixtures, we attempted to investigate the concentration of preservative agents in admixtures and the microbial contamination of these admixtures with a topical corticosteroid ointment (Antebate) . The concentration of parabens was reduced by half using an admixture of corticosteroid ointment with four types of moisturizing creams, Urepearl, Pastaronsoft, Hirudoid, and Hirudoidsoft . After a further 3 months, no decrease in parabens was seen . No microbial contamination was found in any admixture stored at room temperature for 1 week and touched two times daily with a finger . The concentration and ratio of the parabens in the aqueous phase and oil phase were entirely different in the admixtures before being centrifuged . The aqueous phase of the admixtures of the oil/water (O/W)-type emulsions of Urepearl and Hirudoid was not found to have microbial contamination immediately after being centrifuged . All aqueous phases stored at room temperature or in a refrigerator for 1 week and touched with a finger twice daily exhibited microbial contamination . These experiments demonstrated that O/W-type emulsions, in which the water easily separates from the bases, should be thoroughly mixed to prevent microbial contamination.

Nat Rev Genet, 2003 Jan, 4(1), 50 - 60
The evolutionary genomics of pathogen recombination; Awadalla P; A pressing problem in studying the evolution of microbial pathogens is to determine the extent to which these genomes recombine . This information is essential for locating pathogenicity loci by using association studies or population genetic approaches . Recombination also complicates the use of phylogenetic approaches to estimate evolutionary parameters such as selection pressures . Reliable methods that detect and estimate the rate of recombination are, therefore, vital . This article reviews the approaches that are available for detecting and estimating recombination in microbial pathogens and how they can be used to understand pathogen evolution and to identify medically relevant loci.

Clin Exp Rheumatol, 2002 Nov-Dec, 20(6), 833 - 6
A cluster of inflammatory rheumatic diseases in a moisture-damaged office; Myllykangas-Luosujarvi R et al.; OBJECTIVE: To describe a cluster of inflammatory rheumatic diseases in an office workplace that suggests the presence of an environmental trigger . METHODS: There had been an indoor air problem in the workplace since the early 1990s . Large areas of the outer walls of the building were found to be moisture-damaged and contaminated by microbial growth . Case histories of the personnel were studied, and their working areas were related to the areas with highest microbial contamination . The incidence of inflammatory rheumatic diseases was compared with the statistics of the same geographic area . RESULTS: Ten patients with inflammatory rheumatic diseases (3 rheumatoid arthritis, 4 ankylosing spondylitis, 2 Sjogren's syndrome, and one of psoriatic arthritis) entitled to specially reimbursed medication were diagnosed in 1987-2000 (seven cases in 1995-1998) . The incidence density ratio computed for the period 1987-2000 was 6.8 (95% confidence interval 3.6-13.0) for all office personnel and 13.2 (6.0-29.0) for those working close to the wall sustaining the worst damage . CONCLUSION: The accumulation of chronic inflammatory rheumatic diseases in a single workplace suggests that some environmental exposure in this damp office had triggered the diseases.

J Neuroimmunol, 2003 Jan, 134(1-2), 25 - 34
Autoreactive T cells promote post-traumatic healing in the central nervous system; Hofstetter HH et al.; In general, autoimmune responses are considered harmful to the host . In the best-defined model of autoimmune disease, murine experimental allergic encephalomyelitis (EAE), for example, brain-protein-specific autoimmune responses of both major classes, type-1 and type-2, have been implicated in causing brain pathology . We induced type-1 and type-2 autoimmunity to myelin oligodendrocyte protein (MOG) in C57.BL/6 mice . Instead of using pertussis toxin (PTX) to open the blood-brain barrier (BBB), which is the classic procedure, we set an aseptic cerebral injury (ACI) to see what the consequences of pre-primed, autoreactive type-1 and type-2 memory T cells gaining access to the brain in the course of sterile tissue injury would be . Neither of these autoimmune response types induced pathology; on the contrary, both accelerated re-vascularization and post-traumatic healing . The data suggest that induction of either type-1 or type-2 autoimmune responses is not inherently noxious to the host, but can have beneficial effects on tissue repair . Autoimmune pathology may develop only if molecules of microbial origin such as pertussis toxin additionally induce the "infectious nonself/danger" reaction in the antigen-presenting cells (APC) of the target organ itself.

J Invertebr Pathol, 2002 Nov, 81(3), 175 - 84
Hirsutella thompsonii and Metarhizium anisopliae as potential microbial control agents of Varroa destructor, a honey bee parasite; Kanga LH et al.; The potential of Hirsutella thompsonii Fisher and Metarhizium anisopliae (Metschinkoff) as biological control agents of the parasitic mite, Varroa destructor Anderson and Trueman was evaluated in the laboratory and in observation hives . In the laboratory, time required for 90% cumulative mortality of mites (LT(90)) was 4.16 (3.98-4.42) days for H . thompsonii and 5.85 (5.48-7.43) days for M . anisopliae at 1.1 x 10(3) conidia mm(-2) . At a temperature (34+/-1 degrees C) similar to that of the broodnest in a honey bee colony, Apis mellifera L., H . thompsonii {LC(90)=9.90 x 10(1) (5.86-19.35) conidia mm(-2) at Day 7} and M . anisopliae {LC(90)=7.13 x 10(3) (2.80-23.45) conidia mm(-2) at Day 7} both showed significant virulence against V . destructor . The applications of H . thompsonii to observation hives resulted in significant mortality of mites, and reduction of the number of mites per bee 21 and 42 days post-treatments . The treatments did not significantly affect the mite population in sealed brood . However, the fungus must have persisted because infected mites were still observed {82.97+/-(0.6)%} 42 days post-treatment . In addition, the fungus was found to sporulate on the host . A small percentage {2.86+/-(0.2)%} of dead mites found in the control hives also showed fungal infection, suggesting that adult bees drifted between hives and disseminated the fungus . H . thompsonii was harmless to the honey bees at the concentrations applied and did not have any deleterious effects on the fecundity of the queens . Microbial control with fungal pathogens provides promising new avenues for control of V . destructor and could be a useful component of an integrated pest management program for the honey bee industry.

J Invertebr Pathol, 2002 Nov, 81(3), 131 - 47
DNA polymerase gene sequences indicate western and forest tent caterpillar viruses form a new taxonomic group within baculoviruses; Nielsen CB et al.; Baculoviruses infect larval lepidopterans, and thus have potential value as microbial controls of agricultural and forest pests . Understanding their genetic relatedness and host specificity is relevant to the risk assessment of viral insecticides if non-target impacts are to be avoided . DNA polymerase gene sequences have been demonstrated to be useful for inferring genetic relatedness among dsDNA viruses . We have adopted this approach to examine the relatedness among natural isolates of two uncharacterized caterpillar-infecting baculoviruses, Malacosoma californicum pluviale nucleopolyhedrovirus (McplMNPV) and Malacosoma disstria nucleopolyhedrovirus (MadiMNPV), which infect two closely related host species with little to no cross-infectivity . We designed two degenerate primers (BVP1 and BVP2) based on protein motifs conserved among baculoviruses . McplMNPV and MadiMNPV viral DNA was obtained from naturally infected caterpillars collected from geographically distinct sites in the Southern Gulf Islands and Prince George regions of British Columbia, Canada . Sequencing of 0.9 kb PCR amplicons from six McplMNPV and six MadiMNPV isolates obtained from a total of eight sites, revealed very low nucleotide variation among McplMNPV isolates (99.2-100% nucleotide identity) and among MadiMNPV isolates (98.9-100% nucleotide identity) . Greater nucleotide variation was observed between viral isolates from the two different caterpillar species (only 84.7-86.1% nucleotide identity) . Both maximum parsimony and maximum likelihood phylogenetic analyses support placement of McplMNPV and MadiMNPV in a clade that is distinct from other groups of baculoviruses.

Wien Med Wochenschr, 2002, 152(21-22), 552 - 4
Advances in research of the inflammatory response: the importance of toll-like receptors; Knapp S et al.; The innate immune system provides the first line of defence against invading pathogens . Cells that mediate the innate immune response express a variety of receptors that permit recognition of pathogen-associated motifs, subsequently resulting in a cascade of responses that initiates inflammation . Recently, the members of the Toll-like receptor family (TLR) have emerged as key receptors responsible not only for the detection of a variety of microbial cell-wall components and bacterial DNA but also for the initiation of signal transduction events eventually leading to the production of various proinflammatory mediators.

Wien Med Wochenschr, 2002, 152(21-22), 547 - 51
Innate immune sensing of microbial infection: the mechanism and the therapeutic challenge; Beutler B; Studies of sepsis conducted over the century have led to an understanding of many of the molecular events that take place during a severe infection . But what are the first events? Very recent genetic analyses have provided an answer to this question . Genetic studies have disclosed that bacterial endotoxin is sensed through a solitary biochemical pathway . At the heart of this pathway is the Toll-like receptor 4 (TLR4): one member of an ancient receptor family dedicated to the detection of infectious organisms . Most and perhaps all of the untoward effects of infection are initiated by the TLRs, ten of which are represented in humans . At the same time, it is known that TLRs are required to sense infection at its earliest stages, and thereby defeat it . The means to block TLR signal transduction is now at hand . Will this do good or harm?

Toxicology, 2002 Dec 27, 181-182, 305 - 10
Respiratory allergy: what are the uncertainties?
Karol MH.
The prevalence of asthma is increasing worldwide . The increase has been found for both sexes, for all races and age groups . The factors responsible are unclear . The short period of increase makes it unlikely that a genetic change is responsible but rather suggests that either air pollutants or a behavioral change may be involved . Behavioral changes may include decreased exercise and outdoor activity due to, for example increased usage of television and computers . What is the role of allergy in the development of asthma? In allergic sensitization, allergens initiate the inflammation and IgE antibodies are typically present . Once asthma has developed, factors such as irritants, infections and exercise may result in acute symptomatology . Infancy is a high risk period for allergic sensitization since natural defense mechanisms are not fully developed . Epidemiologic evidence suggests that microbial stimuli during early childhood can influence induction of atopic diseases . In animal studies, pre-infection with respiratory virus has resulted in enhanced response to allergens . Another factor implicated in the surge of allergic disease is airborne particulates . Evidence has been obtained for an association of environmental tobacco smoke (ETS) with development of allergic sensitization . ETS enhances IgE production as does diesel and aluminum silicate, the latter a component of fly ash . What are the mechanisms responsible for the environmental influences on development of allergy? Th2 cytokine responses, with suppression of Th1 cytokines, are prominent in children . Th1 maturation appears to be promoted by microbial exposure . Increased understanding of the complex interactions of environmental factors with the developing immune system is essential to reverse the current upward trend in allergic respiratory disease.

FEBS Lett, 2003 Jan 2, 533(1-3), 105 - 9
Different heat shock protein 60 species share pro-inflammatory activity but not binding sites on macrophages; Habich C et al.; In a study of seven different hsp60 species, we found that all mammalian and microbial proteins shared the property of eliciting an inflammatory response in mouse macrophages . In all cases, TNFalpha production was induced by 0.1 microM concentrations of hsp60 . However, the different hsp60 preparations did not compete for the same binding site . The binding of fluorescence-labeled human hsp60 was inhibited by excess unlabeled human, rat or mouse hsp60, but not hamster, Escherichia coli, Chlamydia pneumoniae or Mycobacterium bovis hsp60 . We conclude that phylogenetically separate hsp60 species interact with innate immune cells via different recognition pathways.

Curr Opin Struct Biol, 2002 Dec, 12(6), 704 - 8
Novel proteases: common themes and surprising features; Vandeputte-Rutten L et al.; Proteases perform a wide variety of functions, inside and outside cells, regulating many biological processes . Recent years have witnessed a number of significant advances in the structural biology of proteases, including aspects of intracellular protein and peptide degradation by self-compartmentalizing proteases, activation of proteases in proteolytic cascades of regulatory pathways, and mechanisms of microbial proteases in pathogenicity.

Biomaterials, 2003 Mar, 24(6), 1041 - 5
Polyhydroxyalkanoate (PHA) scaffolds with good mechanical properties and biocompatibility; Zhao K et al.; Blending microbial polyesters polyhydroxyalkanoates containing polyhydroxybutyrate (PHB) and poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) were turned into films and scaffolds . The films made from blending polyesters showed that the elongation to break of the blending PHBHHx/PHB film increased from 15% to 106% when PHBHHx content in the blend increased from 40% to 60% . Scaffolds made of PHBHHx/PHB consisting of 60 wt% PHBHHx showed strong growth and proliferation of chondrocytes on the blending materials under scanning electron microscope . Energy dispersive X-ray analysis of the extra cellular matrix on the scaffolds demonstrated a high level of calcium and phosphorus elements in a molar ratio of Ca/P at 1.66, this is approximately equal to that of natural material hydroxyapatite which has a Ca/P ratio of 1.67 . This suggested that the chondrocyte cells grown on PHBHHx/PHB scaffolds presented effective physiological function for generation of cartilage.

Environ Toxicol Chem, 2003 Jan, 22(1), 35 - 9
Effects of linear alkylbenzene sulfonates on functional diversity of microbial communities in soil; Vinther FP et al.; Linear alkylbenzene sulfonates (LAS) often occur in sewage sludge that is applied to agricultural soil . Here LAS may affect the microbial activity, which is an important basis for nutrient cycling . Previous studies have shown that single bacterial species and specific soil processes can be very sensitive to LAS . Here we report that two levels of LAS, 22 and 174 mg/kg dry weight soil, had little or no significant influence of the functional diversity of bacteria in a sandy soil, as tested by community-level physiological profiles . Briefly, these profiles are a characterization of the microbial communities based on the pattern of substrate utilization in 96-well microtiter plates (Biolog EcoPlates) . Sandy agricultural soil was incubated in duplicate 1-L mesocosms with or without LAS, and bacteria were extracted after one, two, and four weeks . During incubation, more than 98 and 93% of LAS added to 22 and 174 mg/kg dry weight soil was degraded, respectively . The presence of LAS at 174 mg/kg dry weight caused a higher number of bacteria in the soil extracts, maximally corresponding to 2.5 times the numbers in LAS-free soil (1.8 x 10(7) cells/g dry wt soil) after four weeks of incubation . No inhibitory effect of LAS was observed when the substrate utilization data were analyzed for substrate richness and diversity (Shannon-Weaver indices) . Principal component analysis, however, showed that the pattern of substrate utilization in soil with the highest LAS content (174 mg/kg dry wt) could be distinguished from control soil and soil with 22 mg LAS/kg dry weight . Yet the overall conclusion was that the functional diversity of the aerobic, heterotrophic bacterial community was rather insensitive to LAS.

J Eukaryot Microbiol, 2002 Nov-Dec, 49(6), 478 - 84
Identification and phylogenetic analysis of morphologically similar naked amoebae using small subunit ribosomal RNA; Sims GP et al.; Fan-shaped, naked amoebae are commonly encountered in samples from freshwater and marine habitats suggesting that they are an important component of the microbial food web . However, there are considerable problems in both detecting these amoebae and identifying them, given their morphological similarity . In this study we used restriction analysis and partial sequence analysis of the small-subunit 18S ribosomal RNA gene to examine the phylogenetic relationships between nine "fan-shaped" Vannella and Platyamoeba species . The molecular phylogeny showed that the marine Vannella and Platyamoeba isolates are closely related, whereas the freshwater isolates are disparate . Thus, the current reliance on the fine structure of the cell coat (glycocalyx) used to separate these genera is not justified . The study also highlights sequence elements that might be targeted by fluorescent probes for the direct detection of these amoebae in field samples . The molecular data were also used to aid the identification of three unknown fan-shaped isolates . All three unknowns resembled Vannella or Platyamoeba . However, one of the strains (a small < 10 microm, benthic, fan-shaped amoeba) probably represents a new genus.

J Agric Food Chem, 2003 Jan 1, 51(1), 236 - 43
Effects of Ca2+ and sulfhydryl reductant on the polymerization of soybean glycinin catalyzed by mammalian and microbial transglutaminases; Zhang G et al.; Two types of transglutaminases (TGases), Ca(2+)-dependent TGase derived from guinea pig liver (GTGase) and Ca(2+)-independent TGase derived from a variant of Streptoverticillium mobaraense (MTGase), were used to study the cross-linking of soybean 11S globulin (glycinin) . The effects of sulfhydryl reductant (dithiothreitol, DTT) and Ca(2+) on the conformation and TGase-catalyzed polymerization of glycinin were investigated . The conformational change of glycinin was probed by spectral methods . The degree of cross-linking and the polymer (aggregate) formation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dynamic light scattering, respectively . Addition of DTT stimulated the TGase-catalyzed cross-linking reactions without destroying the secondary and tertiary structure of glycinin but did not influence the polymer or aggregate formation . It was found that Ca(2+) caused the formation of larger size polymers at lower concentrations, while it suppressed the polymerization at higher concentrations . In addition, the cross-linking behaviors of glycinin were shown to be different between MTGase- and GTGase-catalyzed systems.

Water Res, 2003 Jan, 37(2), 409 - 15
Temperature effect on microbial community of enhanced biological phosphorus removal system; Panswad T et al.; Microbial population dynamics to gradual temperature change in an enhanced biological phosphorus removal system was kinetically investigated . As the temperature rose from 20.0 degrees C to 30.0 degrees C, and to 35.5 degrees C, the predominant microbial group changed from the phosphorus-accumulating organisms, PAOs (47-70% of total VSS) to the glycogen-accumulating organisms (64-75% of total VSS), and to the ordinary heterotrophs (90% of total VSS), respectively . Despite the species alteration, the phosphorus contents of the PAOs appeared to be steady within 0.182-0.308 mg/mg VSS(PAO) regardless of the temperature level . The initial specific phosphorus release rates, which are solely due to the PAOs activities, increased with the temperature from 37.5-55.9 to 51.8-61.3, 52.0-76.9, 147.2-210.3, and 374.2-756.3 mgP/gmVSS(PAO) h, at 20.0 degrees C, 25.0 degrees C, 30.0 degrees C, 32.5 degrees C, and 35.0 degrees C, respectively . On the other hand, mean initial specific phosphorus uptake rates of the biomass decreased as the temperature increased; however, the data implied that the rate of the PAOs was higher than the other two microbial groups . These results indicate that the PAOs are lower-range mesophiles or possibly psychrophiles . As the temperature rises, the portion of energy required for maintenance increases substantially which reduces the energy availability for cell reproduction; hence, the PAOs are washed out from the system.

Hua Xi Yi Ke Da Xue Xue Bao, 2000 Mar, 31(1), 46 - 8
{Observation of the modulatiny effect of histamine on intestinal barrier function}; Chen X et al.; The model of CaCo2 cell monolayer system has been generally accepted as a standard method for in vitro study of the relationship between the epithelium and microbial invasion . In this study, we set up the model with a simplified method and used it to observe if histamine, which is an important inflammatory factor in gastroenteric tract, can offer some barrier protection from E . coli invasion . After two weeks' routine incubation, the established CaCo2 cells monolayer systems were co-cultured with 1 x 10(-8) mmol/L, 1 x 10(-7) mmol/L and 1 x 10(-6) mmol/L histamine DMEM for 2 hours, and with frank DMEM as control for the same length of the time . For observation on time course effect of histamine, the co-cultures with 1 x 10(-6) mmol/L histamine DMEM were kept up for 0.5, 1.0, 2.0, 4.0 and 18.0 hours . Bacterial invasion ws assessed by quantitating the number of E . coli within the coultured epithelial cells . The results showed that histamine significantly inhibited the invasion of E . coli to epithelial cells(P < 0.05) . The colony counts in co-cultures with 1 x 10(-7) mmol/L and 1 x 10(-8) mmol/L histamine DMEM were 52.5, 30.3 and 91.3 respectively, compared with 563 in control group . In the study of the time course effect of histamine, the colony conuts of co-culture with histamine DMEM for 0.5, 1.0, 2.0, 4.0 and 18.0 hours were 135.5, 64.0, 29.5, 36.0 and 22.5 respectively . It was concluded that histamine can enhance the protective barrier function of intestinal epithelium against E . coli invasion.

Schweiz Rundsch Med Prax, 2002 Nov 20, 91(47), 2023 - 8
{Inflammatory bowel diseases: diagnosis (including new procedures for small intestine examination)}; Hahne M et al.; Medical history, clinical examination, general laboratory data and microbial stool examinations are basic diagnostics in inflammatory bowel diseases . Specific immunologic parameters can make classification easier in indefinite disease, their use is still in an experimental state . Ultrasound and endoscopic examinations have largely replaced conventional radiological procedures . For the experienced examiner, ultrasound allows assessment of small bowel and colon as well as other organs . Ileo-colonoscopy with biopsies remains the gold standard in the diagnosis of inflammatory bowel diseases . For small bowel examination, modern methods like push-enteroscopy, capsule endoscopy or the Hydro-MRI can be employed . At the moment, their usage is limited to specialized centers . Because of its broad availability small bowel follow through is still the standard in small bowel examination . After 8 years of colitis ulcerosa, ileo-colonoscopy with biopsy should be performed every year according to the guidelines . If these recommendations also apply to Crohn's disease is not sure yet.

J Gen Appl Microbiol, 1998 Apr, 44(2), 119 - 127
Use of 16S rDNA community fingerprints to study cricket hindgut microbial communities; Santo Domingo JW; A nucleic acid-based method was evaluated in the course of a study of microbial community structure in the cricket hindgut . Genomic DNA was extracted from the hindgut microbial community of Acheta domesticus and used as a template in the polymerase chain reaction (PCR) method, using primers that align to well conserved regions of the 16S rRNA gene . The rDNA-PCR product was used as a community probe to generate restriction fragment length polymorphisms (RFLPs) of hindgut bacterial isolates and gut microbial communities of insects fed different diets . Fingerprints of the bacterial isolates consisted of several bands suggesting multiple rRNA operons . In contrast with soil communities, hindgut community RFLP contained distinguishable band patterns . However, community rDNA fingerprints were complex and varied among insects fed similar diets, suggesting considerable intrinsic variability in the hindgut microbial community structure between crickets regardless of dietary regime . These results suggest that community RFLP methods using broad-specific phylogenetic probes do not have the resolution or specificity required to ascertain the effect of diet on the cricket hindgut microbial community structure.

Crit Rev Immunol, 2002, 22(3), 217 - 50
Signal integration following Toll-like receptor triggering; Dalpke A et al.; Innate immune cells sense infectious danger through recognition of pathogen-associated molecular patterns (PAMP) . Toll-like receptors (TLRs) play a pivotal role in the recognition of PAMP . Although various TLRs with distinct ligand specificities are expressed almost completely and contemporaneously by innate immune cells, and although central key signal transduction pathways of TLRs are shared, the innate response pattern is not uniform . Innate immune cells organize their response dependent on spatial and temporal requirements and mount a pathogen-specific and microbial-adopted reaction . Thus, after triggering of TLRs, complex signaling interactions take place that control and shape the reactivity patterns of innate immune cells . Here we discuss the central signaling pathways of TLRs and their interconnection with other signaling pathways, with emphasis on Janus kinases/signal transducers and activators of transcription JAK/STAT)-dependent intracellular signals . Cis and trans acting signal convergence mechanisms and mutual influence of TLR and cytokine signaling pathways determine the response profile . Hence, in innate immune cells, integration and processing of microbial-dependent and immunologic signals is achieved within individual cells to ascertain an appropriate immune response . Knowledge of these complex mechanisms will help to clarify the critical events during initiation of infection, which in turn may lead to new strategies to control and overcome infectious disease.

Int Rev Neurobiol, 2002, 52, 93 - 131
Brain-immune interactions in sleep; Marshall L et al.; This chapter discusses various levels of interactions between the brain and the immune system in sleep . Sleep-wake behavior and the architecture of sleep are influenced by microbial products and cytokines . On the other hand, sleep processes, and perhaps also specific sleep states, appear to promote the production and/or release of certain cytokines . The effects of immune factors such as endotoxin and cytokines on sleep reveal species specificity and usually strong dependence on parameters such as substance concentration, time relative to administration or infection with microbial products, and phase relation to sleep and/or the light-dark cycle . For instance, endotoxin increased SWS and EEG SWA in humans only at very low concentrations, whereas higher concentrations increased sleep stage 2 only, but not SWS . In animals, increases in NREM sleep and SWA were more consistent over a wide range of endotoxin doses . Also, administration of pro-inflammatory cytokines such as IL-6 and IFN-alpha in humans acutely disturbed sleep while in rats such cytokines enhanced SWS and sleep . Overall, the findings in humans indicate that strong nonspecific immune responses are acutely linked to an arousing effect . Although subjects feel subjectively tired, their sleep flattens . However, some observations indicate a delayed enhancing effect on sleep which could be related to the induction of secondary, perhaps T-cell-related factors . This would also fit with results in animals in which the T-cell-derived cytokine IL-2 enhanced sleep while cytokines with immunosuppressive functions like IL-4 and L-10 suppressed sleep . The most straightforward similarity in the cascade of events inducing sleep in both animals and humans is the enhancing effect of GHRH on SWS, and possibly the involvement of the pro-inflammatory cytokine systems of IL-1 beta and TNF-alpha . The precise mechanisms through which administered cytokines influence the central nervous system sleep processes are still unclear, although extensive research has identified the involvement of various molecular intermediates, neuropeptides, and neurotransmitters (cp . Fig . 5, Section III.B) . Cytokines are not only released and found in peripheral blood mononuclear cells, but also in peripheral nerves and the brain (e.g., Hansen and Krueger, 1997; Marz et al., 1998) . Cytokines are thereby able to influence the central nervous system sleep processes through different routes . In addition, neuronal and glial sources have been reported for various cytokines as well as for their soluble receptors (e.g., Kubota et al., 2001a) . Links between the immune and endocrine systems represent a further important route through which cytokines influence sleep and, vice versa, sleep-associated processes, including variations in neurotransmitter and neuronal activity may influence cytokine levels . The ability of sleep to enhance the release and/or production of certain cytokines was also discussed . Most consistent results were found for IL-2, which may indicate a sleep-associated increase in activity of the specific immune system . Furthermore, in humans the primary response to antigens following viral challenge is enhanced by sleep . In animals results are less consistent and have focused on the secondary response . The sleep-associated modulation in cytokine levels may be mediated by endocrine parameters . Patterns of endocrine activity during sleep are probably essential for the enhancement of IL-2 and T-cell diurnal functions seen in humans: Whereas prolactin and GH release stimulate Th1-derived cytokines such as IL-2, cortisol which is decreased during the beginning of nocturnal sleep inhibits Th1-derived cytokines . The immunological function of neurotrophins, in particular NGF and BDNF, has received great interest . Effects of sleep and sleep deprivation on this cytokine family are particularly relevant in view of the effects these endogenous neurotrophins can have not only on specific immune functions and the development of immunological memories, but also on synaptic reorganization and neuronal memory formation.

Int Microbiol, 2002 Dec, 5(4), 209 - 14 Epub 2002 Aug 22.
The biomineralization of magnetosomes in Magnetospirillum gryphiswaldense; Schuler D; Magnetotactic bacteria (MTB) are major constituents of natural microbial communities in sediments and chemically stratified water columns . The ability of MTB to migrate along magnetic field lines is based on specific intracellular structures, the magnetosomes, which, in most MTB, are nanometer-sized, membrane-bound magnetic particles consisting of the iron mineral magnetite (Fe(3)O(4)) . A broad diversity of morphological forms has been found in various MTB . The unique characteristics of bacterial magnetosomes have attracted a broad interdisciplinary research interest . The magnetosome membrane (MM) in Magnetospirillum gryphiswaldense contains a number of specific Mam proteins . Several mam genes were analyzed and assigned to different genomic regions . Many of the Mam proteins are highly conserved in other MTB but display low sequence similarity to any proteins from nonmagnetic organisms.

Biochemistry (Mosc), 2002 Nov, 67(11), 1276 - 82
Variability of hydrocarbon and fatty acid components in cultures of the filamentous cyanobacterium Scytonema sp . isolated from microbial community "black cover" of limestone walls in Jerusalem; Dembitsky VM et al.; The hydrocarbon and lipid components of four strains of the filamentous cyanobacterium Scytonema sp . isolated from microbial community "Black Cover" of limestone walls in Jerusalem were identified by gas chromatography-mass spectrometry using serially coupled capillary columns . The dominant compounds were: 1-heptadecyne (1.5-8%), hexadecanoic acid (14-36%), (Z,Z)-9,12-octadecadienoic acid (12-30%), (Z,Z,Z)-9,12,15-octadecatrienoic acid (6-12%), n-heptadecane (4-16%), and 1-heptadecene (1.5-8%) . In addition to unsaturated alkanes and fatty acids, the very long-chain (C30-C32) hydrocarbons, squalene (2.4-3.0%), and branched 4,8,12-trimethyl-C13:0 acid were also isolated . Two major hydrocarbons were detected in the cyanobacteria species using GC-MS and 13C-NMR.

Avian Dis, 2002 Oct-Dec, 46(4), 964 - 70
Comparison of two formaldehyde administration methods of in ovo-injected eggs; Steinlage SJ et al.; Formaldehyde administration in the hatchery can be very useful in decreasing microbial numbers . However, its use is controversial because of the adverse effects that can occur to chicks and people . This study was designed to look at alternative methods of application of formaldehyde in the hatchery . In addition, the study compared the effects of these methods of application on in ovo-and non-in ovo-injected eggs . All in ovo-injected eggs were given diluent only with no vaccine or antibiotic added . In hatchers containing both in ovo-injected eggs and non-in ovo-injected eggs, formaldehyde was administered two ways, dose (DOSE) and constant rate infusion (CRI) . In the DOSE hatcher, 12 ml of formaldehyde was administered at one time every 12 hr, whereas in the CRI hatcher, the same volume was administered at a rate of 1 ml/hr over a 12-hr period . A control (CONT) hatcher received 12 ml of distilled water at the same time that the DOSE hatcher was given formaldehyde . In the DOSE hatcher, a peak concentration of formaldehyde of 102 ppm was reached . The CRI was maintained at approximately 20 ppm of formaldehyde . At pipping, the aerosol bacterial load in the hatchers receiving formaldehyde (DOSE, 130 colony-forming units {CFU}/m3; CRI, 82.5 CFU/m3) was significantly less than in the CONT hatcher (235 CFU/m3) . At hatch, the CRI (337.5 CFU/m3) was not able to control bacterial levels and only the DOSE hatcher (150 CFU/m3) had a significantly lower aerosol bacterial count . The CRI non-in ovo-injected eggs (93.39%) had a significantly higher percentage of hatch of fertile compared with non-in ovo-injected eggs exposed to water (84.27%) . In ovo-injected eggs in CONT and DOSE treatment groups contained significantly higher percentages of visual contamination than non-in on-injected eggs in the same hatchers . This difference had numerical significance only in the treatment groups within the CRI hatcher . The chicks were then placed into replicate treatment groups and grown for 14 days . Chicks from the CRI in ovo-injected eggs had a statistically significant improvement in feed conversion ratio (1.24) at 14 days when compared with chicks from CONT non-in ovo-injected eggs (1.29) . All formaldehyde-exposed chicks had numerically lower feed conversion ratios compared with the CONT exposed chicks.

J Food Prot, 2002 Dec, 65(12), 1903 - 8
Composition and physiological profiling of sprout-associated microbial communities; Matos A et al.; The native microfloras of various types of sprouts (alfalfa, clover, sunflower, mung bean, and broccoli sprouts) were examined to assess the relative effects of sprout type and inoculum factors (i.e., sprout-growing facility, seed lot, and inoculation with sprout-derived inocula) on the microbial community structure of sprouts . Sprouts were sonicated for 7 min or hand shaken with glass beads for 2 min to recover native microfloras from the surface, and the resulting suspensions were diluted and plated . The culturable fraction was characterized by the density (log CFU/g), richness (e.g., number of types of bacteria), and diversity (e.g., microbial richness and evenness) of colonies on tryptic soy agar plates incubated for 48 h at 30 degrees C . The relative similarity between sprout-associated microbial communities was assessed with the use of community-level physiological profiles (CLPPs) based on patterns of utilization of 95 separate carbon sources . Aerobic plate counts of 7.96 +/- 0.91 log CFU/g of sprout tissue (fresh weight) were observed, with no statistically significant differences in microbial cell density, richness, or diversity due to sprout type, sprout-growing facility, or seed lot . CLPP analyses revealed that the microbial communities associated with alfalfa and clover sprouts are more similar than those associated with the other sprout types tested . Variability among sprout types was more extensive than any differences between microbial communities associated with alfalfa and clover sprouts from different sprout-growing facilities and seed lots . These results indicate that the subsequent testing of biocontrol agents should focus on similar organisms for alfalfa and clover, but alternative types may be most suitable for the other sprout types tested . The inoculation of alfalfa sprouts with communities derived from various sprout types had a significant, source-independent effect on microbial community structure, indicating that the process of inoculation alters the dynamics of community development regardless of the types of organisms involved.

J Food Prot, 2002 Dec, 65(12), 1894 - 902
Distribution patterns of Escherichia coli O157:H7 in ground beef produced by a laboratory-scale grinder; Flores RA et al.; This study determined the distribution patterns of Escherichia coli O157:1H7 in ground beef when a contaminated beef trim was introduced into a batch of uncontaminated beef trims prior to grinding in a small-scale laboratory grinder . A beef trim (15.3 +/- 2 g) was inoculated with a rifampicin-resistant strain of E . coli O157:H7 (E . coli O157:H7rif) and introduced into a stream of noncontaminated beef (322 +/- 33 g) prior to grinding . Seven inoculum levels (6, 5, and 4 total log CFU {high}; and 3, 2, 1, and 0 total log CFU {low}) were studied in triplicate . E . coli O157:H7rif was not detected in 3.1 to 43% of the ground beef inoculated with the high levels or in 3.4 to 96.9% of the ground beef inoculated with the low levels . For all inoculum levels studied, the five ground beef fractions (each 7.8 +/- 0.6 g) with the highest pathogen levels accounted for 59 to 100% of the total pathogens detected . For all inoculum levels, there was a linear relationship between the quantity of ground beef containing E . coli O157:H7rif and the inoculum level . The quantity of E . coli O157:H7rif in the beef remaining in the grinder was proportional to the inoculum level and was related to the location in the grinder . Different components of the grinder accumulated E . coli O157:H7rif in different quantities, with the most significant accumulation being in the nut (collar) that attaches the die to the blade . This study determined specific distribution patterns of E . coli O157:H7rif after the grinding of a contaminated beef trim along with uncontaminated trims, and the results indicate that the grinding operation should be regarded as a means of distribution of microbial contamination in risk analyses of ground beef operations.

Biomed Tech (Berl), 2002 Nov, 47(11), 290 - 3
{A new method of testing the effectiveness of sterile packaging in general practice}; Dunkelberg H et al.; The aim of our study was to develop a practical test for assessing the effectiveness of the microbial barrier of packaging materials for sterile products . The suitability of the test was verified in the exemplary case of double-wrapped sterilized trays . During testing, the bacterial count of the ambient air was 35 and 440 colony-forming units/cubic metre . The test is based on the co-sterilization in the sterile packing of petri dishes containing CASO agar, which at the end of the test were investigated for re-contamination . The petri dishes covered the sterilizing sieves as completely as possible . After sterilization, the packaging was loaded 300 to 900 times at a pressure of 1 kg (5x/min) . This was followed by incubation for 48 hours at 37 degrees C, and evaluation (No . of colonies) . The ability of the agar to culture colonies of bacteria was preserved unchanged for a period of at least 3 weeks after sterilization . For double-wrapped trays it was shown that re-contamination increases with intensity of mechanical loading and the atmospheric bacterial count . Since the package was breached only for the analysis, confounding factors due to removal of contents for examination, were effectively excluded . This test procedure is characterised by simplicity in handling and high specificity . As a final pack test it effectively closes a gap in the quality assurance chain for sterile materials.

Kekkaku, 2002 Nov, 77(11), 735 - 40
Current problems of drug-resistant tuberculosis and its control; Kim SJ; Drug resistance emerges wherever and whenever the microbial environment favors the selective growth of drug-resistant mutants . The programmatic errors that lead to the development of drug resistance are inappropriate regimens, non-adherence to therapy by the patients, the sale and availability of over-the-counter drugs, an interruption in the drug supply, and the unavailability of free diagnosis and treatment . As a result, drug-resistant organisms are spread in the community, generating secondary cases with primary drug resistance, which in turn can spread and generate further cases . The uninterrupted cycle of creation and spreading is responsible for increases in DR . According to global data on anti-tuberculosis drug resistance appearing in the WHO monographs, drug resistance is so ubiquitous as to be encountered in every country . Anti-TB DR among previously treated cases was found to be very high in some countries, while it remained relatively low in others . The median of at least one drug resistance among four major drugs tested, was 25.2% (ranging from 8.3 to 68.5%), and the median MDR-TB was 8.7% (ranging from 0 to 48.2%) among those previously treated patients . Drug resistance among new cases infected with drug-resistant organisms from patients with acquired or primary drug resistance was found to be higher in some areas than in others . The median of at least one drug resistance was 10.9% (ranging from 1.7 to 40.6%) and MDR-TB of 1.1% (ranging from 0 to 14.1%) . Twelve geographic areas had levels of 3% or more of MDR-TB among new cases . The majority of MDR-TB cases end up as incurable and spread these deadly organisms in the community, as indicated by several outbreaks in certain areas . Increased MDR-TB will seriously threaten TB control programs in the future . We must therefore exert our best efforts to prevent the further generation of such cases by improving the cure rate of newly diagnosed cases, and by aggressively intervening to identify and remove MDR-TB cases as soon as possible.

Biol Reprod, 2003 Jan, 68(1), 290 - 301
Cloning and characterization of a novel sperm-associated isoantigen (E-3) with defensin- and lectin-like motifs expressed in rat epididymis; Rao J et al.; In the present study we report the identification of a novel epididymis-specific secretory glycoprotein, E-3, which is a sperm-associated isoantigen containing defensin- and lectin-like motifs . E-3 was detected in rat epididymal fluid and in sperm extracts by two-dimensional (2-D) Western blotting using rat hyperimmune sera raised against rat sperm . The immunoreactive spot of approximately 28 kDa with an isoelectric point (pI) of 3.5 was cored from silver-stained gels . Microsequencing by tandem mass spectrometry and database searches revealed several peptides to be novel sequences . Degenerate deoxyinosine-containing primers corresponding to the novel peptides were used in rapid amplification of cDNA ends and polymerase chain reaction to clone E-3 from a rat epididymal cDNA library . A 449-base pair nucleotide sequence was subsequently obtained consisting of a complete open reading frame (ORF) of 111 amino acids, which showed similarity to the defensin and lectin families . The first 21 amino acids constituted a putative signal peptide, suggesting that E-3 is a secretory protein . Mature E-3 protein corresponding to amino acids 22-111 was expressed in E . coli, and chickens were immunized with recombinant E-3 (rE-3) . The resulting anti-rE-3 antisera recognized the recombinant immunogen as well as a "native" protein of 28 kDa, pI 2.5-3.5 in both epididymal fluid and in sperm extracts on 2-D Western blots . Northern hybridization indicated that E-3 mRNA was present in the epididymis but not in testis or other tissues, and that E-3 mRNA was predominantly expressed in the corpus and cauda of the epididymis, but not in the initial segment or caput . Similarly, Western blots detected the E-3 protein only in the epididymal fluid and sperm from the corpus and caudal regions . Finally, indirect immunofluorescence localized E-3 on the entire tail, and with less intensity on the head of the sperm . These observations indicate that E-3 is a secreted epididymal protein that becomes associated with the sperm as it transits through the corpus and cauda . The presence of a defensin-like motif suggests that E-3 may play a role in protecting the sperm from microbial infections in the epididymis and in the female reproductive tract.

J Hazard Mater, 2003 Jan 31, 96(2-3), 331 - 40
Microbial stability evaluation of cement-based waste forms at different waste to cement ratio; Idachaba MA et al.; An evaluation of the effect of differences in chromium nitrate to cement ratio on the microbial stability of a chromium nitrate/cement waste form, as reflected in the leaching of chromium, calcium, magnesium and aluminum; was carried out in this study . An increase in the proportion of chromium in the waste form from 4.8 to 8.7% had no noticeable effect on microbial stability, with the total chromium leached essentially unchanged . Further increases in the proportion of chromium in the waste form from 8.7 to 10.7%, and from 10.7 to 15.9% resulted in a substantial decrease in microbial stability, with 3-fold and 1.3-fold increase in the total chromium leached, respectively, observed . For calcium, increases in the chromium proportion were accompanied with increases in the total calcium leached even though the increases were not in direct proportion to the increases in chromium proportion . For magnesium and aluminum, increases in the proportion of chromium within the range 4.8-10.7% were accompanied with increases in the total respective metals leached, with minor variation for each metal . On the whole, the maximum percentage chromium leached from the different waste forms was substantially lower than those of the other metals.

Mol Ecol, 2003 Jan, 12(1), 251 - 62
Bacterial diversity in maize rhizospheres: conclusions on the use of genetic profiles based on PCR-amplified partial small subunit rRNA genes in ecological studies; Schmalenberger A et al.; A cultivation-independent approach based on polymerase chain reaction (PCR)-amplified partial small subunit rRNA genes and genetic profiling by single-strand conformation polymorphism (SSCP) was used to characterize the bacterial diversity inhabiting the rhizosphere of maize plants grown on an agricultural field . The community structures of two cultivars, a genetically engineered and a nonengineered variety, different herbicide regimes and soil tillage were compared with each other at two sampling dates . SSCP-profiles were generated with DNA from bacterial cell consortia with primers hybridizing to evolutionarily highly conserved rRNA gene regions . On silver-stained gels, each profile consisted of approx . 50 distinguishable bands . Similarity analyses of patterns recorded by digital image analyses could not detect any difference between cultivars or treatments that was greater than the variability between replicates . A total of 54 sequences recovered from different bands were identified and grouped into operational taxonomical units (OTUs) . Surprisingly, only five of 40 OTUs contained sequences of both samplings . Three different bands from a profile were selected to test whether this small overlap was due to an incomplete recovery of sequences . From a faint band, two different OTUs were found when 12 clones were analysed, and from two strong bands 24 and 22 OTUs were detected from a total of 26 and 36 clones, respectively . The OTUs belonged to phylogenetically different groups of bacteria . Gene probes that were developed to target different bands of the profiles, however, indicated in Southern blot analyses that patterns between treatments, replicates and samplings, and even from two different growing seasons were highly conserved . Our study demonstrates that community profiles can consist of more sequences than detectable by staining and that gene probes in Southern blot can be a useful control to investigate the composition of microbial communities by genetic profiles.

Aliment Pharmacol Ther, 2003 Jan, 17(1), 125 - 9
Human recombinant lactoferrin is ineffective in the treatment of human Helicobacter pylori infection; Guttner Y et al.; BACKGROUND: Lactoferrin, a multifunctional glycoprotein, is known to have anti-microbial actions . Bovine lactoferrin and recombinant human lactoferrin have been shown to inhibit Helicobacter pylori, and more recently recombinant human lactoferrin was found to significantly increase the eradication rate of H . pylori when added to standard triple therapy . AIM: To determine the efficacy, safety and tolerability of recombinant human lactoferrin as a therapy in suppressing or eliminating H . pylori infection in subjects with minimal upper gastrointestinal symptoms who have not previously been treated . SUBJECTS AND METHODS: Nine healthy subjects with minimal upper gastrointestinal symptoms and a positive urea breath test were recruited . None of the volunteers had previously been treated for H . pylori . Subjects received 5 x 1.0 g human recombinant lactoferrin daily for 5 or 14 days . Breath tests were repeated during therapy and shortly after to check for eradication . The safety and tolerability of the drug were assessed by physical examination, by monitoring adverse events, and clinical laboratory evaluation . RESULTS: No conversion of the urea breath test from positive to negative was observed and there was no consistent change in urea breath test count to indicate a possible suppression of H . pylori . CONCLUSION: Lactoferrin, given as a single agent, does not eradicate H . pylori infection.

Microb Ecol, 2003 Feb, 45(2), 156 - 62 Epub 2002 Dec 20.
In situ identification by fluorescently labeled oligonucleotide probes of morphologically similar, closely related ciliate species; Petroni G et al.; Ciliate protozoa are important members of microbial communities in which they play specific ecological roles . The determination of single species distribution is fundamental for food web analysis, but species recognition, which is mainly based on morphological characters, is often difficult between closely related species . The use of species-specific, purposely designed, fluorescently labeled probes for in situ hybridization is here presented as an easy and fast identification method for three closely related species belonging to the widespread genus Euplotes, namely E . crassus, E . vannus, and E . minuta, that in spite of their remarkable morphological similarity have significant metabolic and ecological differences . These three species can be detected simultaneously, provided the probes employed are bound to different fluorescent dyes: in this way their relative abundance and their population dynamics in the natural environment can be evaluated . As more ciliate sequences become available in databases, species-specific probes can be designed for other ciliates, thus rendering the application of the method of more general importance . The probes used in this study may also provide a tool to prevent erroneous species identification in future studies.

Nature, 2002 Dec 19-26, 420(6917), 853 - 9
The inflammatory reflex; Tracey KJ; Inflammation is a local, protective response to microbial invasion or injury . It must be fine-tuned and regulated precisely, because deficiencies or excesses of the inflammatory response cause morbidity and shorten lifespan . The discovery that cholinergic neurons inhibit acute inflammation has qualitatively expanded our understanding of how the nervous system modulates immune responses . The nervous system reflexively regulates the inflammatory response in real time, just as it controls heart rate and other vital functions . The opportunity now exists to apply this insight to the treatment of inflammation through selective and reversible 'hard-wired' neural systems.

Nature, 2002 Dec 19-26, 420(6917), 806 - 10
SAR11 clade dominates ocean surface bacterioplankton communities; Morris RM et al.; The most abundant class of bacterial ribosomal RNA genes detected in seawater DNA by gene cloning belongs to SAR11-an alpha-proteobacterial clade . Other than indications of their prevalence in seawater, little is known about these organisms . Here we report quantitative measurements of the cellular abundance of the SAR11 clade in northwestern Sargasso Sea waters to 3,000 m and in Oregon coastal surface waters . On average, the SAR11 clade accounts for a third of the cells present in surface waters and nearly a fifth of the cells present in the mesopelagic zone . In some regions, members of the SAR11 clade represent as much as 50% of the total surface microbial community and 25% of the subeuphotic microbial community . By extrapolation, we estimate that globally there are 2.4 x 10(28) SAR11 cells in the oceans, half of which are located in the euphotic zone . Although the biogeochemical role of the SAR11 clade remains uncertain, these data support the conclusion that this microbial group is among the most successful organisms on Earth.

Chemosphere, 2002 Dec, 49(10), 1295 - 9
Occurrence and microbial degradation of phthalate esters in Taiwan river sediments; Yuan SY et al.; Concentrations and microbial degradation rates were measured for eight phthalate esters (PAEs) found in 14 surface water and six sediment samples taken from rivers in Taiwan . The tested PAEs were diethyl phthalate (DEP), dipropyl phthalate (DPP), di-n-butyl phthalate (DBP), diphenyl phthalate (DPhP), benzylbutyl phthalate (BBP), dihexyl phthalate (DHP), dicyclohexyl phthalate (DCP), and di-(2-ethylhexyl) phthalate (DEHP) . In all samples, concentrations of DEHP and DBP were found to be higher than the other six PAEs . DEHP concentrations in the water and sediment samples ranged from ND to 18.5 microg/l and 0.5 to 23.9 microg/g, respectively; for DBP the concentration ranges were 1.0-13.5 microg/l and 0.3-30.3 microg/g, respectively . Concentrations of DHP, BBP, DCP and DPhP were below detection limits . Under aerobic conditions, average degradation half-lives for DEP, DPP, DBP, DPhP, BBP, DHP, DCP and DEHP were measured as 2.5, 2.8, 2.9, 2.6, 3.1, 9.7, 11.1 and 14.8 days, respectively; under anaerobic conditions, respective average half-lives were measured as 33.6, 25.7, 14.4, 14.6, 19.3, 24.1, 26.4 and 34.7 days . In other words, under aerobic conditions we found that DEP, DPP, DBP, DPhP and BBP were easily degraded, but DEHP was difficult to degrade; under anaerobic conditions, DBP, DPhP and BBP were easily degraded, but DEP and DEHP were difficult to degrade . Aerobic degradation rates were up to 10 times faster than anaerobic degradation rates.

Chemosphere, 2002 Dec, 49(10), 1239 - 46
Behavior of PAHs during cold storage of historically contaminated soil samples; Rost H et al.; The stability of historically polycyclic aromatic hydrocarbon (PAH)-contaminated soils during cold storage was investigated . Samples from two former manufactured gas plants exhibited quantitative recoveries of PAHs over the whole period of sample holding at 4 degrees C in the dark (8-10 months), whereas significant losses of PAHs were observed for soils received from a former railroad sleeper preservation plant with low molecular weight compounds being notably more affected compared to heavier PAHs . Already after 2 weeks of holding time, 3-ring PAHs in one of theses samples were down to 29-73% of the initial concentration and significant losses were observed for up to 5-ring compounds . Dissipation of PAHs was found to be predominantly due to aerobic microbial metabolism since sodium azide poisoned samples showed quantitative recoveries for all PAHs over the entire storage time of 3 months . A similar stabilizing effect was observed for freezing at -20 degrees C as means of preservation . Except for acenaphthene, no significant loss for any of the PAHs was observed over 6 weeks of holding time . Eventually, selected chemical, physical, and biological parameters of two soils were investigated and identified as potential indicators for the stability of PAH-contaminated soil samples.

J Dairy Sci, 2002 Nov, 85(11), 3059 - 61
Technical note: effect of removal of microbial cells by centrifugation on peptide and alpha-amino nitrogen concentrations in ruminal fluid; Ives SE et al.; We evaluated the effect of centrifuging rumen fluid prior to analysis on concentrations of alpha-amino N (AAN) and peptides . Rumen fluid was collected from steers fed grain-based diets at either various times after feeding or after dosing the rumens with solubilized casein . Fluid was either directly processed for peptide analysis by acidifying 10 ml of rumen fluid with 0.5 ml of 70% (wt/wt) perchloric acid, or first centrifuged at 500 x g for 20 min to remove protozoa and then at 30,000 x g for 15 min to remove bacterial cells prior to further processing . By removing microbial cells, intracellular AAN and peptides were not included in subsequent analyses . Concentrations of AAN were determinedusing an automated trinitrobenzene sulfonic acid assay, and peptides were determined as the increase in AAN following acid hydrolysis of the samples . When casein was not dosed, removal of microbial cells prior to analysis decreased concentrations of both AAN andpeptides, and the decrease was greater for AAN (2.2 mM)than for peptides (1.2 mM) . Dosing with casein led to much higher concentrations of ruminal peptides and AAN . After casein dosing, decreases in AAN and peptidecon-centrations due to prior centrifugation (2.1 mM and 1.0 mM for AAN a nd pept ides, respectively) were similar to the decreases observed before the casein dosing . Results suggest that the contribution of intracellular AAN and peptides to the concentrations in ruminal fluid are relatively constant across broad ranges of dietary protein supply for cattle fed corn-based diets.

Crit Rev Biotechnol, 2002, 22(4), 375 - 407
Microbial beta-glucosidases: cloning, properties, and applications; Bhatia Y et al.; Beta-glucosidases constitute a major group among glycosylhydrolase enzymes . Out of the 82 families classified under glycosylhydrolase category, these belong to family 1 and family 3 and catalyze the selective cleavage of glucosidic bonds . This function is pivotal in many crucial biological pathways, such as degradation of structural and storage polysaccharides, cellular signaling, oncogenesis, host-pathogen interactions, as well as in a number of biotechnological applications . In recent years, interest in these enzymes has gained momentum owing to their biosynthetic abilities . The enzymes exhibit utility in syntheses of diverse oligosaccharides, glycoconjugates, alkyl- and aminoglucosides . Attempts are being made to understand the structure-function relationship of these versatile biocatalysts . Earlier reviews described the sources and properties of microbial beta-glucosidases, yeast beta-glucosidases, thermostable fungal beta-glucosidase, and the physiological functions, characteristics, and catalytic action of native beta-glucosidases from various plant, animal, and microbial sources . Recent efforts have been directed towards molecular cloning, sequencing, mutagenesis, and crystallography of the enzymes . The aim of the present article is to describe the sources and properties of recombinant beta-glucosidases, their classification schemes based on similarity at the structural and molecular levels, elucidation of structure-function relationships, directed evolution of existing enzymes toward enhanced thermostability, substrate range, biosynthetic properties, and applications.

Ann Allergy Asthma Immunol, 2002 Dec, 89(6 Suppl 1), 75 - 82
Inventing probiotic functional foods for patients with allergic disease; Laiho K et al.; OBJECTIVE: The primary objective of this review is to discuss the rationale for and applicability of probiotics as part of the nutritional approach to the prevention and management of allergic disease . DATA SOURCES: This review represents a synthesis of recent literature published in the fields of probiotics, nutrition, and allergic disease . STUDY SELECTION: The expert opinions of the authors were used to select the relevant data for the review . RESULTS: The hygiene hypothesis suggests that allergic disease may arise from a lack of counterbalancing microbial exposure at an early age . Thus, the initial compositional development of gut microbiota is considered a key determinant in the development of both the immune responder phenotype and the normal gut barrier functions . A wide range of data documents the specific actions of selected probiotics . The regulatory role of probiotics in allergic disease has been demonstrated as improving the clinical course or preventing the development of atopic eczema in infants given a probiotic-supplemented diet . In addition, certain types of fatty acids and antioxidants have been proposed for protective effects against the development of allergy and to diminish the inflammatory response in allergic disease . CONCLUSIONS: The increase in the prevalence of allergic disease during the past decades is likely to be explained by changes in the environment, including reduced microbial exposure and altered food consumption . Scientifically composed functional foods containing probiotics and other functional components offer a nutritional strategy for both the prevention and the management of allergic disease . Further research is needed to characterize the gut microbiota and to clarify the mechanisms of action that control specific physiologic processes not only in the evolution of allergic disease in at-risk populations but also in the management of allergic diseases.

Methods Cell Sci, 2001, 23(4), 197 - 204
Modification of cell perifusion for extended study of hormone release in the rat pituitary; Vella S et al.; We use a flow-through cell perifusion system allowing continuous sampling of Luteinizing Hormone (LH) release from excised pituitary tissue . With this system, we are able to sample from viable tissue in primary culture for three days as opposed to a more typical of primary culture of 6-12 hours . Rat pituitary tissue was perifused and challenged with a physiological dose of LHRH . Samples were collected for 3.5 hours on each of 3 consecutive days in a refrigerated fraction collector and assayed for LH concentration by radioimmunoassay . In these experiments we tested two types of media, each with or without fetal calf serum supplementation, for their ability to support pituitary tissue for extended lengths of perifusion . In addition, we broadened the use of aseptic technique, sterile media and equipment to limit microbial contamination . Our results indicate that perifusion of pituitary tissue can be done successfully for 3 or more days at a modest cost, greatly increasing the amount of information that can be collected from each piece of tissue harvested.

J Exp Med, 2002 Dec 16, 196(12), 1645 - 51
Lipopolysaccharide-enhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen; Eisenbarth SC et al.; Allergic asthma is an inflammatory lung disease initiated and directed by T helper cells type 2 (Th2) . The mechanism involved in generation of Th2 responses to inert inhaled antigens, however, is unknown . Epidemiological evidence suggests that exposure to lipopolysaccharide (LPS) or other microbial products can influence the development and severity of asthma . However, the mechanism by which LPS influences asthma pathogenesis remains undefined . Although it is known that signaling through Toll-like receptors (TLR) is required for adaptive T helper cell type 1 (Th1) responses, it is unclear if TLRs are needed for Th2 priming . Here, we report that low level inhaled LPS signaling through TLR4 is necessary to induce Th2 responses to inhaled antigens in a mouse model of allergic sensitization . The mechanism by which LPS signaling results in Th2 sensitization involves the activation of antigen-containing dendritic cells . In contrast to low levels, inhalation of high levels of LPS with antigen results in Th1 responses . These studies suggest that the level of LPS exposure can determine the type of inflammatory response generated and provide a potential mechanistic explanation of epidemiological data on endotoxin exposure and asthma prevalence.

J Exp Med, 2002 Dec 16, 196(12), 1575 - 84
CD1-mediated gamma/delta T cell maturation of dendritic cells; Leslie DS et al.; Immature myeloid dendritic cells (DCs) express only low levels of major histocompatibility complex (MHC) class II but express high levels of CD1 a, b, and c antigen-presenting molecules at the cell surface . As Vdelta1+ gamma/delta T cells are the main tissue subset of gamma/delta T cells and they are known to recognize CD1c in the absence of specific foreign antigen recognition, we examined the possible interaction of these T cells with immature DCs . We show that CD1-restricted gamma/delta T cells can mediate the maturation of DCs . DC maturation required cell-cell contact and could be blocked by antibodies against CD1c . The maturation process was partially mediated by tumor necrosis factor alpha . Importantly, immature DCs matured in the presence of lipopolysaccharide and CD1-restricted gamma/delta T cells produced bioactive interleukin-12p70 . In addition, these DCs were able to efficiently present peptide antigens to naive CD4+ T cells . CD1-restricted gamma/delta T cell recognition of immature DCs provides the human immune system with the capacity to rapidly generate a pool of mature DCs early during microbial invasion . This may be an important source of critical host signals for T helper type 1 polarization of antigen-specific naive T cells and the subsequent adaptive immune response.

EMBO J, 2002 Dec 16, 21(24), 6681 - 8
Pep-13, a plant defense-inducing pathogen-associated pattern from Phytophthora transglutaminases; Brunner F et al.; Innate immunity, an ancient form of defense against microbial infection, is well described for animals and is also suggested to be important for plants . Discrimination from self is achieved through receptors that recognize pathogen-associated molecular patterns (PAMPs) not found in the host . PAMPs are evolutionarily conserved structures which are functionally important and, thus, not subject to frequent mutation . Here we report that the previously described peptide elicitor of defense responses in parsley, Pep-13, constitutes a surface-exposed fragment within a novel calcium-dependent cell wall transglutaminase (TGase) from Phytophthora sojae . TGase transcripts and TGase activity are detectable in all Phytophthora species analyzed, among which are some of the most destructive plant pathogens . Mutational analysis within Pep-13 identified the same amino acids indispensable for both TGase and defense-eliciting activity . Pep-13, conserved among Phytophthora TGases, activates defense in parsley and potato, suggesting its function as a genus-specific recognition determinant for the activation of plant defense in host and non-host plants . In summary, plants may recognize PAMPs with characteristics resembling those known to trigger innate immune responses in animals.

J Gen Appl Microbiol, 2001 Aug, 47(4), 149 - 160
Microbial toxins in plant-pathogen interactions: Biosynthesis, resistance mechanisms, and significance; Kimura M et al.; In the history of phytopathology, microbial toxins have been the objects of extensive studies as possible pathogenicity or virulence factors for the producer pathogens . The recent development of molecular genetic techniques provided an experimental basis to thoroughly test the role of these secondary metabolites in pathogenesis . Some of them did prove to be highly associated with disease initiation or enhanced virulence in certain plant-pathogen interactions . In this review, we describe recent progresses in the field of plant-pathogen interactions focusing on two toxins; i.e., tabtoxin from Pseudomonas syringae and trichothecenes from Fusarium and other fungi . These microbial toxins have convincingly been shown to play causal roles in plant disease development . Studies on the biosynthesis and resistance mechanisms of these producers are outlined, and the significance of this knowledge is discussed in relation to practical applications in agriculture.

J Ind Microbiol Biotechnol, 2002 Dec, 29(6), 326 - 30
Possible implications of biocide accumulation in the environment on the prevalence of bacterial antibiotic resistance; McBain AJ et al.; The lethality of biocides depends upon their interaction with a number of distinct biochemical targets . This often reflects reactive chemistry for any given agent, such as thiol oxidation . Susceptibility may vary markedly between different target organisms, and changes within the more sensitive targets can alter the inhibitory effect . The multiplicity of potential targets, however, usually dictates against the development of overt resistance to concentrations used for hygienic applications . Similarly, although changes in cellular permeability toward such agents, mediated either by envelope modification or the induction of efflux-pumps may reduce susceptibility, they rarely influence the outcome of treatments at use-concentration . It has recently been proposed that chronic exposure of the environment to biocides used in a variety of commercial products might expose some microbial communities to subeffective concentrations causing emergence of resistant clones . Such resistance might relate to mutational changes in the most susceptible target or to regulatory mutants that cause the constitutive expression of certain efflux pumps . Although selection of organisms with such modifications is unlikely to influence the effectiveness of the biocides, changes in their susceptibility to third-party antibiotics can be postulated . This is particularly the case where a cellular target is shared between a biocide and an antibiotic, or where induction of efflux is sufficient to confer antibiotic resistance in the clinic . Although such linkage has been demonstrated in the laboratory in pure culture, it has not been documented in environments commonly exposed to biocides . In nature, the effects of chronic stressing with biocides are complicated by competition between microbial community members that may result in clonal expansion of naturally insusceptible clones.

Curr Opin Biotechnol, 2002 Dec, 13(6), 572 - 7
Screening for novel enzymes for biocatalytic processes: accessing the metagenome as a resource of novel functional sequence space; Lorenz P et al.; Historically, biotechnology has missed up to 99% of existing microbial resources by using traditional screening techniques . Strategies of directly cloning 'environmental DNA' comprising the genetic blueprints of entire microbial consortia (the so-called 'metagenome') provide molecular sequence space that along with ingenious in vitro evolution technologies will act synergistically to bring a maximum of available sequence-space into biocatalytic application.

Ecotoxicol Environ Saf, 2002 Sep, 53(1), 93 - 103
Responses of microbial activity and decomposer organisms to contamination in microcosms containing coniferous forest soil; Salminen J et al.; Soil respiration from microcosms contaminated with pentachlorophenol, 2-ethanolhexanoate, creosote, CuSO4, and benomyl was measured in order to evaluate usefulness of soil microcosms and microbial respiration rate monitoring as a toxicity test in soils with high organic matter content . Coniferous forest soil and its organisms were used as test objects . In addition, how a short-term low temperature period including frost affects respiration dynamics in stressed soils was studied, i.e., whether contaminants reduce resistance of the community to other (also natural) stresses . In addition, at the end of the experiment, effects of contaminants on faunal and microbial community structures were analyzed . Soil respiration measurements from the microcosms appeared to be a sensitive parameter for testing community-level effects of chemicals in the soil with high organic matter content . An 84-day exposure had acute effects, long-term effects, delaying effects, and total recovery of community respiration . Direct negative and indirect positive effects of chemical contamination on the community of soil organisms were found . Responses to contamination of soil respiration rate and structure of the soil community were parallel . Addition of pentachlorophenol, 2-ethanolhexane, and Cu into the soil reduced frost resistance of the decomposer community . It was concluded that soil respiration monitoring of artificially contaminated soil microcosms seems to be a useful tool for testing community-level toxic effects of chemicals.

Trends Biotechnol, 2003 Jan, 21(1), 45 - 52
Microbial cell-surface display; Lee SY et al.; Cell-surface display allows peptides and proteins to be displayed on the surface of microbial cells by fusing them with the anchoring motifs . The protein to be displayed - the passenger protein - can be fused to an anchoring motif - the carrier protein - by N-terminal fusion, C-terminal fusion or sandwich fusion . The characteristics of carrier protein, passenger protein and host cell, and fusion method all affect the efficiency of surface display of proteins . Microbial cell-surface display has many potential applications, including live vaccine development, peptide library screening, bioconversion using whole cell biocatalyst and bioadsorption.

Hum Immunol, 2002 Dec, 63(12), 1201 - 5
Involvement of plasmacytoid dendritic cells in human diseases; Jahnsen FL et al.; In vitro studies have reported that plasmacytoid dendritic cells (PDCs) exert multiple functions, including production of interferon (IFN)-alpha as effector cells and regulation of T-cell responses as mature DCs . Here we review recent data obtained in situ showing that PDCs accumulate in lesions of type I IFN-related disorders (virus infections and lupus erythematosus), Th2 cell-dominated allergic reactions, and ovarian carcinoma . These results demonstrate that PDCs do migrate to peripheral tissues during inflammation, which lends further support to the view that PDCs most likely are important players in innate and adaptive immunity in vivo . Future research should aim at defining the exact pathogenic or defense roles of PDCs in such disorders and determine whether these cells are potential targets for therapeutic intervention in microbial infections, allergy, autoimmunity, or cancer.

Hum Immunol, 2002 Dec, 63(12), 1126 - 32
Natural type I interferon-producing cells as a link between innate and adaptive immunity; Kadowaki N et al.; Type I interferons (IFNs) are promptly produced upon invasion of pathogens, and activate a broad range of effector cells in the innate and adaptive immune system . Lin(-)CD4(+)CD11c(-) plasmacytoid dendritic cell precursors (plasmacytoid pre-DCs) produce enormous amounts of type I IFNs in response to viruses and CpG DNA, thus corresponding to the previously described but not fully defined natural type I IFN-producing cells (IPCs) . Plasmacytoid pre-DCs strongly express toll-like receptor (TLR) 7 and TLR9, in contrast to monocytes, which mainly express TLR1, 2, 4, 5, and 8, suggesting that these two DC precursors recognize different microbial molecules and that they may have developed through different evolutionary trails . Three different stimuli, CpG DNA plus CD40 ligand, interleukin-3 (IL-3), and herpes simplex virus, stimulate plasmacytoid pre-DCs to differentiate into DCs that induce distinct types of T helper cells, i.e., Th1, Th2, and IFN-gamma- and IL-10-producing T cells, respectively . The remarkable versatility of plasmacytoid pre-DCs distinguishes them from other cell types in the immune system that have only limited functions, and suggests that these cells may play a key role in integrating the innate and adaptive aspects of various immune responses.

Hum Immunol, 2002 Dec, 63(12), 1111 - 9
Plasmacytoid dendritic cells: the key to CpG; Rothenfusser S et al.; The vertebrate immune system has established TLR9 to detect microbial DNA based on unmethylated CG dinucleotides within certain sequence contexts (CpG motifs) . In humans, the expression of toll-like receptor 9 (TLR9) is restricted to B cells and plasmacytoid dendritic cells (PDC) . The PDC is characterized by the ability to rapidly synthesize large amounts of type I IFN (IFN-alpha and IFN-beta) in response to viral infection . In contrast to other dendritic cell subsets which express a broad profile of TLRs, the TLR profile in PDC is restricted to TLR7 and TLR9 . So far, CpG DNA is the only defined microbial molecule recognized by PDC . An intriguing feature of PDC is its ability to simultaneously produce the two major Th1-inducing cytokines in humans, IFN-alpha and IL-12, both at high levels . The ratio of IFN-alpha versus IL-12 and the quantity of these cytokines are regulated by T helper cell-mediated costimulation via CD40 ligation . The ratio also depends on the differentiation stage of the PDC at the time of stimulation and the type of CpG ODN used . We propose a model in which the establishment of Th1 responses in vivo is improved by appropriately stimulated PDC that otherwise - in the absence of CpG DNA--support Th2 or Th0 responses and thus have been called DC2.

J Periodontol, 2002 Nov, 73(11), 1260 - 6
In vitro effect of laser irradiation on cementum-bound endotoxin isolated from periodontally diseased roots; Liu CM et al.; BACKGROUND: In a previous study, we evaluated the in vivo effects of an Nd:YAG laser on periodontal disease by measuring crevicular interleukin (IL)-1beta levels before and after laser application . It was found that laser therapy was less effective than traditional scaling and root planing . These results might be due to incomplete removal of microbial residues and cementum-bound endotoxin on root surfaces by the laser . In this study, we explored the in vitro effectiveness of an Nd:YAG laser for the elimination of cementum-bound endotoxin by measuring IL-1beta changes in stimulated monocytes . METHODS: Fresh human monocytes were harvested from adults without periodontitis and grown in RPMI 1640 medium . Diseased cementum particles were collected and prepared from teeth with untreated periodontitis and were irradiated with 5 levels of laser energy . Cementum particles were subjected to endotoxin testing by a limulus amebocyte lysate (LAL) assay and then were incubated with cultured monocytes . Production of IL-1beta in stimulated monocytes was measured by enzyme-linked immunosorbent assay and quantified by spectrophotometry . RESULTS: The endotoxin unit (EU) of diseased cementum was 18.4 EU/mg, which seemed to be remarkably lower than that of common periodontal pathogens including Porphyromonas gingivalis (381) at 15,300 EU/mg/ml, Prevotella intermedia (ATCC 25611) at 227 EU/mg/ml, and Fusobacterium nucleatum (ATCC 25586) at 1,987 EU/mg/ml . Monocytes subjected to stimulation by diseased cementum particles without laser irradiation produced 124 to 145 pg/ml IL-1beta, 9- to 18-fold higher than that of unstimulated monocytes (7.07 to 15.95 pg/ml) . Diseased cementum particles after irradiation with various energy levels of the Nd:YAG laser could still stimulate monocytes to secrete 89 to 129 pg/ml IL-1beta . No statistically significant difference was found in the production of IL-1beta induced by diseased-bound cementum with or without laser irradiation . CONCLUSIONS: The Nd:YAG laser varying from 50 mJ, 10 pps to 150 mJ, 20 pps, for 2 minutes, did not seem to be effective in destroying diseased cementum endotoxin.

Brain, 2003 Jan, 126(Pt 1), 20 - 31
Molecular tracking of antigen-specific T cell clones in neurological immune-mediated disorders; Muraro PA et al.; T cells recognizing self or microbial antigens may trigger or reactivate immune-mediated diseases . Monitoring the frequency of specific T cell clonotypes to assess a possible link with the course of disease has been a difficult task with currently available technology . Our goal was to track individual candidate pathogenic T cell clones, selected on the basis of previous extensive studies from patients with immune-mediated disorders of the CNS, including multiple sclerosis, HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) and chronic Lyme neuroborreliosis . We developed and applied a highly specific and sensitive technique to track single CD4(+) and CD8(+) T cell clones through the detection and quantification of T cell receptor (TCR) alpha or beta chain complementarity-determining region 3 transcripts by real-time reverse transcriptase (RT)-PCR . We examined the frequency of the candidate pathogenic T cell clones in the peripheral blood and CSF during the course of neurological disease . Using this approach, we detected variations of clonal frequencies that appeared to be related to clinical course, significant enrichment in the CSF, or both . By integrating clonotype tracking with direct visualization of antigen-specific staining, we showed that a single T cell clone contributed substantially to the overall recognition of the viral peptide/MHC complex in a patient with HAM/TSP . T cell clonotype tracking is a powerful new technology enabling further elucidation of the dynamics of expansion of autoreactive or pathogen-specific T cells that mediate pathological or protective immune responses in neurological disorders.

Curr Drug Targets Immune Endocr Metabol Disord, 2002 Apr, 2(1), 83 - 95
Mucosal immunity--basic principles, ontogeny, cystic fibrosis and mucosal vaccination; Vancikova Z; The mucosal immune system is an integral part of the whole-body immune system, however its regulation, maturation and function are to a great degree independent . Mucosal lymphoid tissue is the largest immune organ of the body, that stands in the first line of defence against foreign invaders . The goal of the immune system is immunity, however immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, because the organism must tolerate thousands of ingested and inhaled harmless food and bacterial antigens . The phenomenon of oral tolerance is the unique feature of the mucosal immune system . If abrogated, severe autoimmune diseases like Crohn's disease, ulcerative colitis or coeliac sprue can develop . The quality of mucosal immune responses during newborn and infant age strongly influences the immune reactivity later in life . The most important factors influencing the development of mucosal immune reactivity are the feeding practices and microbial colonization . Manipulation of the mucosal immune system offers interesting possibilities to prevent infection as well as autoimmune diseases directly in the affected tissue, without participation of the whole-body immune system . In this review we present the most recent basic information about the mechanisms of mucosal immunity, ontogeny of mucosal immunity, mucosal tolerance and immunisation and the role of mucosal immunity in an inherited disease in which the main battlefield is the lung mucosa-cystic fibrosis.

Curr Drug Targets Immune Endocr Metabol Disord, 2002 Apr, 2(1), 13 - 25
Hormone mediation of immune responses in the progression of diabetes, rheumatoid arthritis and periodontal diseases; Soory M; The crucial role of the immune response is common to diabetes mellitus (DM), rheumatoid arthritis (RA) and periodontal disease . This review identifies advances in this field and exciting paradigms in their management . Uncontrolled hyperglycaemia in diabetic patients results in the formation of advanced glycation end products (AGEs), which are detrimental to cell structure and function . Altered host resistance such as defective migration of PMN, impaired phagocytosis and an exaggerated inflammatory response to microbial products also compromises healing in uncontrolled diabetic patients, further compromised in smokers . Nicotine has well documented effects on the immune response, cell adhesion proteins and apoptosis which affect the severity of disease presentation and response to treatment . Rheumatoid arthritis is a multifactorial disease that results in severe destruction of synovial cartilage and bone . Local secretion of large amounts of TNF-alpha and IL-1 due to activation of immunocompetent cells characterises the pathophysiology of RA . This has lead to the emergence of TNF-alpha inhibitors such as etanercept and infliximab in its management . Periodontal disease has a microbial aetiology . But it is similar to RA, in its cyclical pattern of destruction associated with high levels of pro-inflammatory cytokines, which can persist after removal of the antigenic stimulus . Non steroidal anti-inflammatory agents (NSAIDs) have been used as an adjunct to mechanical removal of bacterial antigen, in the management of periodontal disease . The non-reproductive functions of steroid hormones include effects on immunocompetent cells, fibroblasts and osteoblasts, which affect the initiation and progression of inflammatory diseases . Hormone replacement therapy could be another facet in a multifaceted treatment approach in these patients, where indicated.

Mikrobiyol Bul, 2002 Jan, 36(1), 105 - 14
{Ecological factors for host tissue colonization}; Yurdakul P; The first stage of microbial infection is colonization; the establishment of the pathogen at the appropriate portal of the entry . Organisms that have the ability to colonize host tissues, have usually developed tissue adherence mechanisms and some ability to overcome or withstand the constant pressure of host defense mechanisms . There are namely 3 adaptations that are important for bacteria to colonize human hosts: Coaggregation, osmo-adaptation, acid tolerance and resistance . In this review article, these adaptation mechanisms have been discussed under the light of literature.

Inhal Toxicol, 2003 Jan, 15(1), 23 - 38
Inflammatory and cytotoxic potential of the airborne particle material assessed by nasal lavage and cell exposure methods; Roponen M et al.; Exposure to bioaerosols in moisture-damaged indoor environments has been shown to be a potential health risk . The aim of the present study was to evaluate the inflammatory and cytotoxic potential of airborne particle material using both the nasal lavage (NAL) method and a cell exposure study . A 24-h sample collection for airborne particles was performed using personal sampling and microenvironmental measurements in homes and an 8-h sample collection in the working places of the studied subjects . At the end of the sampling period, the production of nitric oxide, tumor necrosis factor alpha, interleukin (IL)-1 beta, IL-4, and IL-6 was analyzed in the NAL samples of the subjects . The same mediators, excluding IL-4, were measured in the cell culture medium of mouse RAW264.7 macrophages, which were exposed to the pooled filter extracts representing personal, home, and workplace exposure of each individual during the 24 h before the NAL . Samplings were repeated after 2 wk . The subjects were divided into groups of "low exposure" and "high exposure" according to the concentrations of viable fungi, viable bacteria, or total microbial amount in the pooled extract . Cytokine levels in the NAL samples of subjects with high microbial exposure were slightly increased compared to the corresponding values of the subjects with low exposure . Filter samples collected from the subjects with high microbial exposure induced a significant increase in the production of cytokines in the RAW264.7 macrophages, as compared to those from the subjects with low exposure . The within-subject variation was low in all of the cytokine measurements, but the correlation between the studied methods was poor . In conclusion, both of the methods discriminate at the group level between subjects with high and low microbial exposure . Sampling of airborne particle material and exposure of the mammalian cells to the obtained samples seems to be highly applicable in the environmental monitoring, whereas examination of the exposed subjects directly, for example by using the NAL method, is essential when association between exposure and health effects is evaluated.

J Chem Ecol, 2002 Oct, 28(10), 1987 - 2000
Chemical defense of early life stages of benthic marine invertebrates; Lindquist N; Accurate knowledge of factors affecting the survival of early life stages of marine invertebrates is critically important for understanding their population dynamics and the evolution of their diverse reproductive and life-history characteristics . Chemical defense is an important determinant of survival for adult stages of many sessile benthic invertebrates, yet relatively little consideration has been given to chemical defenses at the early life stages . This review examines the taxonomic breadth of early life-stage chemical defense in relation to various life-history and reproductive characteristics, as well as possible constraints on the expression of chemical defense at certain life stages . Data on the localization of defensive secondary metabolites in larvae and the fitness-related consequences of consuming even a small amount of toxic secondary metabolites underpin proposals regarding the potential for Mullerian and Batesian mimicry to occur among marine larvae . The involvement of microbial symbionts in the chemical defense of early life stages illustrates its complexity for some species . As our knowledge of chemical defenses in early life stages grows, we will be able to more rigorously examine connections among phylogeny, chemical defenses, and the evolution of reproductive and life-history characteristics among marine invertebrates.

Methods Enzymol, 2002, 358, 43 - 66
Green fluorescent protein as a marker for conditional gene expression in bacterial cells; Bongaerts RJ et al.; To date, the majority of studies of bacterial gene expression have been carried out on large communities, as techniques for analysis of expression in individual cells have not been available . Recent developments now allow us to use reporter genes to monitor gene expression in individual bacterial cells . Conventional reporters are not suitable for studies of living single cells . However, variants of GFP have proved to be ideal for the study of development, cell biology, and pathogenesis and are now the reporters of choice for microbial studies . In combination with techniques such as DFI and IVET and the use of flow cytometry and advanced fluorescence microscopy, the latest generation of GFP reporters allows the investigation of gene expression in individual bacterial cells within particular environments . These studies promise to bring a new level of understanding to the fields of bacterial pathogenesis and environmental microbiology.

Biotechnol Bioeng, 2003 Feb 5, 81(3), 356 - 69
Kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP+-dependent dehydrogenases using N6-linked immobilized NADP+ derivatives: Studies with mammalian and microbial glutamate dehydrogenases; McMahon M et al.; This study is concerned with the development and application of kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP(+)-dependent dehydrogenases, specifically (1) the synthesis and characterization of highly substituted N(6)-linked immobilized NADP(+) derivatives using a rapid solid-phase modular approach; (2) the evaluation of the N(6)-linked immobilized NADP(+) derivatives for use with the kinetic locking-on strategy for bioaffinity purification of NADP(+)-dependent dehydrogenases: Model bioaffinity chromatographic studies with glutamate dehydrogenase from bovine liver (GDH with dual cofactor specificity, EC 1.4.1.3) and glutamate dehydrogenase from Candida utilis (GDH which is NADP(+)-specific, EC 1.4.1.4); (3) the selection of an effective "stripping ligand" for NADP(+)-dehydrogenase bioaffinity purifications using N(6)-linked immobilized NADP(+) derivatives in the locking-on mode; and (4) the application of the developed bioaffinity chromatographic system to the purification of C . utilis GDH from a crude cellular extract.Results confirm that the newly developed N(6)-linked immobilized NADP(+) derivatives are suitable for the one-step bioaffinity purification of NADP(+)-dependent GDH provided that they are used in the locking-on mode, steps are taken to inhibit alkaline phosphatase activity in crude cellular extracts, and 2',5'-ADP is used as the stripping ligand during chromatography . The general principles described here are supported by a specific sample enzyme purification; the purification of C . utilis GDH to electrophoretic homogeneity in a single bioaffinity chromatographic step (specific activity, 9.12 micromol/min/mg; purification factor, 83.7; yield 88%) . The potential for development of analogous bioaffinity systems for other NADP(+)-dependent dehydrogenases is also discussed .

Tissue Antigens, 2002 Oct, 60(4), 299 - 308
Conserved TCR beta chain usage in reactive arthritis; evidence for selection by a putative HLA-B27-associated autoantigen; May E et al.; Previous work suggested that expanded CD8+ T-cell clones in the synovial fluid (SF) of HLA-B27+ patients with reactive arthritis (ReA) preferentially use the T-cell receptor variable region (TCRBV) 1, similar CDR3 sequences, and joining region (BJ) 2S3 . To determine the range of conservation and disease-specificity of CDR3-sequences, we analyzed the TCRBV1-J2S3 repertoire from 33 healthy HLA-B27+ individuals, patients with various types of spondyloarthropathies (SpA), and with rheumatoid arthritis (RA) by CDR3-spectratyping . After collection and database submission of all available TCRB-CDR3 from HLA-B27-restricted or SpA-derived T cells, we systematically screened the entire human sequence database for sequences similar to the B27/SpA-related CDR3 . Spectratyping revealed expanded T cell clones using conserved TCRBV1J2S3 in the SF from 5/6 of the patients with acute ReA but not among the controls . In database searches, 50 HLA-B27 or SpA-related CDR3-sequences generated similar clusters of matched sequences, and matched reciprocally . Identical or closely related sequences were identified in 15 different individuals and a canonical ReA-associated TCRB was defined {BV1-CASSVG(V/I/L)(Y/F)STDTQYF-J2S3} . All but one patient-derived conserved sequences originated from acute stage ReA-patients, and were not present among approximately 3800 other human TCRB sequences in the database . Five of the conserved sequences originated from T cell clones that recognized uninfected cells in an HLA-B27-restricted fashion, implying a role of HLA-B27-restricted CD8+ T cells specific for a ubiquitous self- or cross-reactive microbial determinant in the early phase of ReA . Related sequences were independently identified in four different laboratories . The consensus TCRB motif could be a helpful diagnostic marker in HLA-B27-associated 'undifferentiated arthritis'.

J Immunol, 2002 Dec 15, 169(12), 6951 - 8
Conservation of CD1 intracellular trafficking patterns between mammalian species; Dascher CC et al.; Dendritic cells (DC) are potent APCs that sample Ags from the surrounding environment and present them to naive T cells using cell surface Ag-presenting molecules . The DC in both lymphoid and nonlymphoid tissues express high levels of CD1, a cell surface glycoprotein capable of presenting lipids and glycolipids to T cells . Distinct group 1 CD1 isoforms (CD1a, -b, -c) in man are known to traffic to different parts of the endocytic system where microbial Ags may be sampled . Guinea pigs are the only known rodent species that express the group 1 CD1 proteins . Therefore, we examined the expression and trafficking of guinea pig CD1 (gpCD1) isoforms on isolated DC . Confocal microscopy using mAbs specific for individual gpCD1 isoforms revealed differential trafficking of two distinct CD1b isoforms within DC . Colocalization of MHC class II was observed with the gpCD1b1 isoform, consistent with localization in the late endosomes of DC . In contrast, the gpCD1b3 isoform lacks an endosomal sorting motif and remains on the cell surface . Following incubation with Mycobacterium tuberculosis lipoarabinomannan, colocalization of endocytosed lipoarabinomannan with the gpCD1b1 isoform was observed but not with the gpCD1b3 isoform, which remained primarily on the cell surface . These data demonstrate that guinea pig DC express CD1 isoforms with unique trafficking patterns that recapitulate the patterns seen for human CD1 isoforms . This suggests evolutionary pressure for a conserved mechanism in mammals that allows CD1 to sample lipid Ags from various subcompartments of the endocytic system.

J Biol Chem, 2003 Feb 21, 278(8), 6201 - 8 Epub 2002 Dec 05.
Molecular sensing of bacteria in plants . The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco; Felix G et al.; To detect microbial infection multicellular organisms have evolved sensing systems for pathogen-associated molecular patterns (PAMPs) . Here, we identify bacterial cold shock protein (CSP) as a new such PAMP that acts as a highly active elicitor of defense responses in tobacco . Tobacco cells perceive a conserved domain of CSP and synthetic peptides representing 15 amino acids of this domain-induced responses at subnanomolar concentrations . Central to the elicitor-active domain is the RNP-1 motif KGFGFITP, a motif conserved also in many RNA- and DNA-binding proteins of eukaryotes . Csp15-Nsyl, a peptide representing the domain with highest homology to csp15 in a protein of Nicotiana sylvestris exhibited only weak activity in tobacco cells . Crystallographic and genetic data from the literature show that the RNP-1 domain of bacterial CSPs resides on a protruding loop and exposes a series of aromatic and basic side chains to the surface that are essential for the nucleotide-binding activity of CSPs . Similarly, these side chains were also essential for elicitor activity and replacement of single residues in csp15 with Ala strongly reduced or abolished activity . Most strikingly, csp15-Ala10, a peptide with the RNP-1 motif modified to KGAGFITP, lacked elicitor activity but acted as a competitive antagonist for CSP-related elicitors . Bacteria commonly have a small family of CSP-like proteins including both cold-inducible and noninducible members, and Csp-related elicitor activity was detected in extracts from all bacteria tested . Thus, the CSP domain containing the RNP-1 motif provides a structure characteristic for bacteria in general, and tobacco plants have evolved a highly sensitive chemoperception system to detect this bacterial PAMP.

Curr Cancer Drug Targets, 2002 Dec, 2(4), 279 - 308
Natural products and derivatives as leads to cell cycle pathway targets in cancer chemotherapy; Newman DJ et al.; The influence of natural products upon drug discovery in general has been quite impressive; one only has to look at the number of clinically active drugs that are in use in cancer therapy to see how many either are natural products or have a natural pro-duct pharmacophore . What is now becoming quite apparent is that materials from natural sources are excellent probes (indicators) for cellular targets that when modulated, may well have a deleterious effect upon the cycling of a tumor cell through the conventional cell cycle . If the particular target is not expressed in normal cell cycling, then a directed "perturbation" of the tumor cell's cycle may well lead to a novel method of treatment for specific tumor types . In this review we have not attempted to be exhaustive but have given a current overview of how natural products from marine, microbial and plant sources have permitted in-depth analyses of various parts of the cell cycle under varying conditions with the ultimate aims of attempting to "control or perturb" the cycling of tumor cells in a fashion that permits their ultimate removal via cellular death, with a minimum of trauma to the host.

J Environ Qual, 2002 Nov-Dec, 31(6), 2074 - 85
Seasonal variation in microbial communities and organic malodor indicator compound concentrations in various types of swine manure storage systems; Merrill L et al.; Anaerobic manure storage systems are one of the major contributors to the odor and environmental pollution associated with swine (Sus scrofa) production systems . The microbial ecology of manure storage systems and the relationships between microbial communities and odor production are largely unknown . In this study, we used community fatty acid methyl ester (FAME) analysis to generate lipid profiles to assess seasonal differences among microbial communities inhabiting various types of outdoor swine manure storage systems . Concurrently, we measured manure concentrations of several malodor indicator compounds as well as pH, temperature, and solids content . Principal components analysis (PCA) showed that there are differences in FAME profiles among the swine manure storage systems examined and most of the variation was in the relative abundance of 18:0, 18:1omega7t, 18:1omega7c/omega9t/omega12t, and 16:1omega7t/i15:0 2OH FAMEs . The PCA of the FAME profiles revealed that the phototrophic systems were more similar to each other and that the nonphototrophic systems were more similar to each other than they were to phototrophic lagoons . There were seasonal changes in the FAME profiles in the phototrophic systems and the concrete nonphototrophic basin (CNPB), and in one phototrophic system, the FAME profiles more closely resembled a CNPB FAME profile during the winter than the other phototrophic lagoons . In the phototrophic lagoon systems, there was a direct correlation between the abundance of the FAMEs identified in the PCA and manure concentrations of phenol, p-cresol, and 4-ethyl phenol . In the CNPB, there was a negative correlation between the total phenolics concentration and the 18:1omega7t FAME . Our results indicate that community FAME profiles could be used as a diagnostic tool for obtaining preliminary evidence that management practices are altering the system's microbial community to one that favors less air pollution potential.

J Environ Qual, 2002 Nov-Dec, 31(6), 1831 - 41
Low-temperature chromium(VI) biotransformation in soil with varying electron acceptors; Tseng JK et al.; Effective and low-cost strategies for remediating chromium (Cr)-contaminated soil are needed . Chromium(VI) leaching from contaminated soil into ground water and surface water threatens water supplies and the environment . This study tested indigenous Cr(VI) microbial transformation in batch systems at 10 degrees C in the presence of various electron acceptors . The effects of carbon addition, spiked Cr(VI), and mixing highly contaminated soil with less contaminated soil were investigated . The results indicated that Cr(VI) can be biotransformed in the presence of different electron acceptors including oxygen, nitrate, sulfate, and iron . Sugar addition had the greatest effect on enhancing Cr(VI) removal . Less dissolved organic carbon (DOC) was consumed per amount of Cr(VI) transformed under anaerobic conditions {0.8-93 mg DOC/mg Cr(VI)} compared with aerobic conditions {1.4-265 mg DOC/mg Cr(VI)} . Toxicity of high concentrations (< 160 mg/L) of spiked Cr(VI) were not evident . At Cr(VI) concentrations > 40 mg/L, aerobic conditions promoted faster Cr(VI) reduction than anaerobic conditions with nitrate or sulfate present . Biotransformation of Cr(VI) in highly contaminated soil (22,000 mg Cr/kg) was facilitated by mixing with less-contaminated soil . The study results provide a framework for evaluating indigenous Cr(VI) microbial transformation and enhance the ability to develop strategies for soil treatment.

J Gen Appl Microbiol, 2002 Aug, 48(4), 211 - 22
Phylogenetic characterization of microbial mats and streamers from a Japanese alkaline hot spring with a thermal gradient; Nakagawa T et al.; Dense microbial mats and streamers of various colors developed in an alkaline-hot spring water at 48-76 degrees C and ~0.077 mm sulfide in Nakabusa, Japan . The microbial community structures with a thermal gradient were compared by denaturing gradient gel electrophoresis (DGGE) analysis of the PCR-amplified 16S ribosomal RNA gene fragments . The sequence analysis revealed that a predominant cyanobacterial DGGE band phylogenetically related to Synechococcus elongatus was detected only from green mats at 48 degrees C . Four DGGE bands were detected commonly from green mats at 48 degrees C, orange mats at 58 degrees C and brown mats at 60 degrees C . The sequence analysis revealed that these were phylogenetically related to Chloroflexaceae group, Rhodothermus group, a candidate division OP10, and an unclassified bacterium . On the other hand, Aquificae-, Thermodesulfobacteria-, Thermus group-, and Crenarchaeota-like sequences were detected as a predominant component of DGGE profiling from the streamers only at temperatures over 66 degrees C, but no phototrophic bacterial bands were detected . Thus, the microbial community structure above 60 degrees C was drastically different from that at the lower temperatures . After the addition of hydrogen into in vitro gray streamers with in situ spring water, sulfide production markedly occurred in the presence of ambient sulfate at 66 degrees C . This result suggests that in situ sulfide is partly produced by Thermodesulfobacteria-like sulfate-reducing bacteria in the streamers.

Microb Ecol, 2003 Jan, 45(1), 63 - 71 Epub 2002 Dec 10.
Influence of drying-rewetting frequency on soil bacterial community structure; Fierer N et al.; Soil drying and rewetting represents a common physiological stress for the microbial communities residing in surface soils . A drying-rewetting cycle may induce lysis in a significant proportion of the microbial biomass and, for a number of reasons, may directly or indirectly influence microbial community composition . Few studies have explicitly examined the role of drying-rewetting frequency in shaping soil microbial community structure . In this experiment, we manipulated soil water stress in the laboratory by exposing two different soil types to 0, 1, 2, 4, 6, 9, or 15 drying-rewetting cycles over a 2-month period . The two soils used for the experiment were both collected from the Sedgwick Ranch Natural Reserve in Santa Ynez, CA, one from an annual grassland, the other from underneath an oak canopy . The average soil moisture content over the course of the incubation was the same for all samples, compensating for the number of drying-rewetting cycles . At the end of the 2-month incubation we extracted DNA from soil samples and characterized the soil bacterial communities using the terminal restriction fragment length polymorphism (T-RFLP) method . We found that drying-rewetting regimes can influence bacterial community composition in oak but not in grass soils . The two soils have inherently different bacterial communities; only the bacteria residing in the oak soil, which are less frequently exposed to moisture stress in their natural environment, were significantly affected by drying-rewetting cycles . The community indices of taxonomic diversity and richness were relatively insensitive to drying-rewetting frequency . We hypothesize that drying-rewetting induced shifts in bacterial community composition may partly explain the changes in C mineralization rates that are commonly observed following exposure to numerous drying-rewetting cycles . Microbial community composition may influence soil processes, particularly in soils exposed to a significant level of environmental stress.

Curr Top Microbiol Immunol, 2002, 270, 169 - 84
Heat shock proteins as ligands of toll-like receptors; Vabulas RM et al.; Toll-like receptors (TLRs) have been described as sensors for pathogen-associated molecular patterns crucial for the initiation of an innate immune response . These mechanisms were developed long before the adaptive immune system evolved . The latest additions to the growing list of TLR ligands are heat shock proteins (HSPs) . Interestingly, not only bacterial but also mammalian HSPs interact with TLRs demonstrating that the exclusive association of TLRs with microbial ligands is obsolete . Human HSP60 and Gp96 are the first examples of non-pathogen derived ligands of TLRs . More importantly, Gp96 provides the first example of how the innate and adaptive immune system can be stimulated simultaneously by the same molecule which is released under physiological conditions from necrotic cells . Understanding the mechanisms of innate immune system interaction with HSPs will make it possible to rationally modulate immune responses, either towards immunity or towards tolerance.

Curr Top Microbiol Immunol, 2002, 270, 109 - 20
TLR4 as the mammalian endotoxin sensor; Beutler B; For more than a century, the ability to sense endotoxin (later known also as lipopolysaccharide; LPS) stood as the archetypal innate immune response: even before the phrase 'innate immunity' became popular . Yet the mechanism by which LPS initiated a signal remained unknown . The problem was solved in 1998 by positional cloning, which revealed that Toll-like receptor (TLR) 4, one of ten mammalian paralogues with homology to the Drosophila protein Toll, is the central component of the LPS receptor . During the 3 years that followed, gene knockout work supported the view that the TLRs perceive a number of indispensable molecular structures shared by diverse representatives of the microbial world . The highly specific LPS-sensing function of TLR4 is remarkable for its prevalence in Mammalia, which to the present time is the only class of the phylum Chordata known to have a gene encoding TLR4, and known to display exquisite sensitivity to LPS . The fact that LPS signals are elicited through a single biochemical pathway has raised important pharmacotherapeutic opportunities as well.

Curr Top Microbiol Immunol, 2002, 270, 1 - 21
Evolution of the TIR, tolls and TLRs: functional inferences from computational biology; Beutler B et al.; The mammalian toll-like receptors (TLRs) are products of an evolutionary process that began prior to the separation of plants and animals . The most conserved protein motif within the TLRs is the TIR, which denotes Toll, the Interleukin-1 receptor, and plant disease Resistance genes . To trace the ancestry of the TLRs, it is desirable to draw upon the sequences of TIR domains from TLRs of diverse vertebrate species, including species with known dates of divergence (i.e., representatives of Mammalia and Aves) in order to establish a relationship between time and genetic divergence . It appears that a gene ancestral to modern TLRs 1 and 6 duplicated approximately 130 million years ago, only shortly before the speciation event that led to humans and mice . Though it is not represented in mice, TLR10 split from the TLR{1/6} precursor about 300 million years ago . The origins of other TLRs are more ancient, dating to the origins of vertebrate life, and some present-day vertebrate species appear to have many more TLRs than others . Moreover, the patterns of TLR expression are quite variable at the level of tissues, even among closely related species . A given TLR in species that are related by descent from a common ancestor may acquire different duties within each descendant line, so that some microbial inducers are avidly recognized in one species but not in others; likewise the intensity and the antomic location of an innate immune response may vary considerably . In this review, we discuss the computational methods used to analyze divergence of the TIR, and the conclusions that may be safely drawn.

Astrobiology, 2001 Summer, 1(2), 165 - 84
Brines in seepage channels as eluants for subsurface relict biomolecules on Mars?
Wynn-Williams DD, Cabrol NA, Grin EA, Haberle RM, Stoker CR.
Water, vital for life, not only maintains the integrity of structural and metabolic biomolecules, it also transports them in solution or colloidal suspension . Any flow of water through a dormant or fossilized microbial community elutes molecules that are potentially recognizable as biomarkers . We hypothesize that the surface seepage channels emanating from crater walls and cliffs in Mars Orbiter Camera images results from fluvial erosion of the regolith as low-temperature hypersaline brines . We propose that, if such flows passed through extensive subsurface catchments containing buried and fossilized remains of microbial communities from the wet Hesperian period of early Mars (approximately 3.5 Ga ago), they would have eluted and concentrated relict biomolecules and delivered them to the surface . Life-supporting low-temperature hypersaline brines in Antarctic desert habitats provide a terrestrial analog for such a scenario . As in the Antarctic, salts would likely have accumulated in water-filled depressions on Mars by seasonal influx and evaporation . Liquid water in the Antarctic cold desert analogs occurs at -80 degrees C in the interstices of shallow hypersaline soils and at -50 degrees C in salt-saturated ponds . Similarly, hypersaline brines on Mars could have freezing points depressed below -50 degrees C . The presence of hypersaline brines on Mars would have extended the amount of time during which life might have evolved . Phototrophic communities are especially important for the search for life because the distinctive structures and longevity of their pigments make excellent biomarkers . The surface seepage channels are therefore not only of geomorphological significance, but also provide potential repositories for biomolecules that could be accessed by landers.

Aust Vet J, 2002 Oct, 80(10), 617 - 9
Pea mania: deranged behaviour in cattle grazing a pea crop (Pisum sativum var arvense); Reardon CJ et al.; Hypersensitivity to external stimuli, progressing in some animals to manic behaviour, occurred in a cattle herd that grazed a crop of field peas (Pisum sativum var arvense) in the pre-flowering stage . Haematological and biochemical analyses eliminated hypomagnesaemia and ketosis as diagnoses . Other than two steers euthanased due to injuries sustained during manic episodes, all affected animals survived, recovering over 3 days when moved to alternative pasture . No necropsies were conducted . No microbial pathogens or endophytes were found on or in the plants . A previously reported incident in Victoria in 1987 in cattle grazing peas appeared to be of a similar nature . Environmental factors leading to these incidents were not clearly identified.

Med J Aust, 2002 Dec 2-16, 177(11-12), 609 - 13
Water and the environment: a natural resource or a limited luxury?
Leder K, Sinclair MI, McNeil JJ.
The risk of contamination of drinking water supplies with microbial pathogens is minimised by modern approaches to water management, but continues to be the major public health concern . Chemical contaminants usually pose little health risk except at very high levels, but debate continues over the potential adverse health effects of low-level, chronic exposure to compounds such as disinfection byproducts . Recreational water contact can be associated with adverse health outcomes either from microbial infections or exposure to cyanobacterial toxins . Environmental issues such as increasing salinity and global warming are likely to affect the sustainability of our current drinking water supplies and increase the threat of waterborne disease outbreaks . New technologies, use of alternative water sources, such as rainwater tanks, water reuse and restrictions will undoubtedly be part of the solution to our diminishing water resources, but have the potential to introduce new health threats.

BioDrugs, 2002, 16(6), 419 - 31
CpG DNA in the prevention and treatment of infections; Dalpke A et al.; Microbial infection is sensed by Toll-like receptors (TLRs) on innate immune cells . Among the ten so far defined TLRs, TLR9 and its ligand are peculiar . TLR9 recognises bacterial DNA characterised by the abundance of unmethylated CpG dinucleotides, which distinguish bacterial DNA (CpG DNA) from mammalian DNA . Moreover, TLR9 shows a restricted cellular and subcellular pattern of expression . In contrast to other TLR agonists, CpG DNA is superior in activation of dendritic dells and induction of costimulatory cytokines such as interleukin (IL)-12 and IL-18 . This qualifies CpG DNA as a Th1-promoting adjuvant . During infection, recognition of CpG DNA of intracellular pathogens skews and fine-tunes the ongoing immune response and induces long-lasting Th1 milieus . Thus, CpG DNA might play an important role in driving the immune system to a Th1 profile, preventing undesired Th2 milieus that might favour induction of allergic responses . Since CpG DNA can be synthesised with high purity and sequence fidelity, synthetic CpG DNA will become an important agent for Th1 instruction and be an effective adjuvant during vaccination.

Environ Toxicol Chem, 2002 Dec, 21(12), 2600 - 5
Degradation of chlorpyrifos, fenamiphos, and chlorothalonil alone and in combination and their effects on soil microbial activity; Singh BK et al.; The effects of repeated application and of combinations of pesticides on their degradation rates in soil and on some soil microbial properties were studied . Repeated application of chlorpyrifos did not modify its degradation rate, whereas repeated applications of fenamiphos and chlorothalonil suppressed their own rates of degradation . When applied in combination, the presence of chlorothalonil reduced the degradation rate of both chlorpyrifos and fenamiphos, and the half-life of chlorothalonil was extended in the presence of chlorpyrifos . The dynamics of residues of the major metabolites of the different compounds were also affected by the pesticide combinations and, particularly, by the presence of chlorothalonil . The measured soil microbial parameters (enzyme activities and total microbial biomass) were stable in the pesticide-free control soils throughout the 90-d incubation period, but they were all adversely affected by the presence of chlorothalonil in the soil . The effects from fenamiphos or chlorpyrifos on the soil microbial characteristics were either very small or insignificant.

Clin Exp Rheumatol, 2002 Nov-Dec, 20(6 Suppl 28), S50 - 1
Treatment of the SpA-like disease in HLA B27 transgenic animals; Breban M et al.; A major involvement of the immune system and of microbial flora in the HLA-B27 transgenic rat model of spondylarthropathy was demonstrated . The role of inflammatory pathways, such as cytokines and inducible nitric oxide synthase (iNOS) was investigated . Treatment with IL-10 failed to improve established disease, whereas such improvement was achieved with IL-11 . In contrast, aggravation was observed after treatment with selective inhibitor of iNOS.

Curr Mol Med, 2002 Dec, 2(8), 769 - 81
Innate T-cell immunity in HIV infection: the role of Vgamma9Vdelta2 T lymphocytes; Poccia F et al.; There is growing interest in the use of innate immune reactions in the therapy and prophylaxis of various diseases . Natural T (NT) lymphocytes that recognize infected cells or microbial compounds without the classical genetic restriction by polymorphic MHC molecules are crucial components of innate immunity . NT cells bearing the Vgamma9Vdelta2 T-cell receptor (TCR) are broadly reactive against intracellular pathogens, can lyse human immunodeficiency virus (HIV) infected cells, and release cytokines capable of regulating HIV replication . The potent antiviral activities of Vgamma9Vdelta2 T cells may help to contain viral spread during acute HIV infection and/or to prevent the establishment of viral persistence . Substantial changes in the composition and function of circulating gammadelta T-cell pools occur in HIV-infected patients . These changes a) may contribute to the etiopathogenesis of opportunistic infections and neoplasms, and b) are partly reversed by highly active anti-retroviral therapy (HAART) . In addition to direct antiviral activities, activated gammadelta T cells influence dendritic cell maturation and the adaptive alphabeta T-cell response . Vgamma9Vdelta2 T cells can be stimulated in vivo and in vitro by various nonpeptidic antigens (NpAgs) and recent animal experimental data suggest that activated Vgamma9Vdelta2 T cells may help to control SIV replication . Currently, NpAgs are being assessed as potential therapeutic agents in AIDS, tuberculosis and certain cancers susceptible to Vgamma9Vdelta2 T-cell effector mechanisms.

J Anim Sci, 2002 Nov, 80(11), 2967 - 77
Influence of rumen protein degradability and supplementation frequency on steers consuming low-quality forage: I . Site of digestion and microbial efficiency; Bohnert DW et al.; Seven cannulated (rumen and duodenal) Angus x Hereford steers (264 +/- 8 kg BW) consuming low-quality forage (5% CP; 61% NDF; 31% ADF) were used to determine the influence of CP degradability and supplementation frequency (SF) on DMI and nutrient digestion . Treatments included an unsupplemented control and degradable intake protein (DIP) or undegradable intake protein (UIP) provided daily, every 3 d, or every 6 d . The DIP treatments (18% UIP) were calculated to provide 100% of the DIP requirement, while the UIP treatments (60% UIP) were provided on an isonitrogenous basis compared with DIP . Forage DMI was not affected by treatment . Total DM and N intake, duodenal N flow, and intestinal N disappearance increased (P < 0.01) with supplementation . Dry matter intake and duodenal N flow responded quadratically (P < 0.04; greatest values on the every-third-day treatments) as SF decreased . However, no differences in N intake or intestinal N disappearance were observed because of CP degradability or SF . Duodenal bacterial N flow and true bacterial N synthesis (g bacterial N/kg of OM truly digested in the rumen) were increased (P < 0.05) with supplementation . Also, duodenal bacterial N flow was greater (P < 0.05) for DIP compared with UIP . Duodenal nonbacterial N flow was increased (P = 0.02) with CP supplementation and for UIP compared with DIP (P < 0.01) . Supplemental CP increased (P < 0.01) total tract DM and N digestibility with no difference due to CP degradability or SF . Results suggest CP supplements consisting of 20 to 60% UIP can be effectively used by steers consuming low-quality forage without adversely affecting DMI, nutrient digestibility, or bacterial CP synthesis, even when provided as infrequently as once every 6 d.

Med Pediatr Oncol, 2003 Feb, 40(2), 93 - 8
Ciprofloxacin and amoxicillin as continuation treatment of febrile neutropenia in pediatric cancer patients; Park JR et al.; BACKGROUND: The empiric administration of anti-microbial therapy significantly reduces the morbidity and mortality associated with febrile neutropenic episodes in oncology patients . Outpatient empiric antibiotic therapy can be safely administered to a subset of febrile neutropenic patients at low risk for clinical complications . PROCEDURE: Pediatric cancer patients presenting with febrile neutropenia after non-myeloablative chemotherapy and who met institutional criteria for early hospital discharge following a minimum of 48-hr inpatient empiric intravenous ceftazidime were eligible for the study . The feasibility and efficacy of an outpatient continuation therapy of oral ciprofloxacin (CPR) 25-30 mg/kg/day divided BID and amoxicillin (AMX) 30-50 mg/kg/day divided TID was assessed . RESULTS: Thirty febrile neutropenic episodes in 26 patients were treated with outpatient oral CPR/AMX therapy . Oral CPR/AMX therapy was feasible in 28 (93%) and efficacious in 26 (87%) of treatment episodes . CPR/AMX was discontinued due to abdominal pain and diarrhea (n = 2), recurrent fever (n = 3), or gastrointestinal bleeding (n = 1) . No patient developed new bacteremia or cardiopulmonary decompensation . Bone/joint pain or gastrointestinal symptoms occurred in 27% of treatment episodes . Duration of neutropenia, lower absolute neutrophil count (ANC) (< 100/mm(3)) at start of oral antibiotic therapy and active malignant disease were associated with failure of oral antibiotic therapy . CONCLUSIONS: It is feasible to administer oral CPR/AMX as continuation antibiotic therapy for a selected subgroup of febrile neutropenic episodes defined after initial hospitalization and empiric antibiotic therapy . Prospectively randomized trials will be required to analyze adequately the efficacy of an oral CPR/AMX outpatient antibiotic regimen for treatment of febrile neutropenia in pediatric oncology patients .

Environ Microbiol, 2002 Nov, 4(11), 683 - 95
Metabolic shifts in hypersaline microbial mats upon addition of organic substrates; Grotzschel S et al.; The responses of hypersaline microbial mats to the addition of acetate, glycolate or glucose were investigated using oxygen, pH and sulphide microsensors . Changes in community structure were investigated with molecular techniques . Acetate addition inhibited respiration in the photic zone, stimulated respiration in the aphotic zone and had no effect on gross photosynthesis . Glycolate addition strongly increased both respiration and gross photosynthesis in the photic zone . Thus, glycolate and acetate were probably consumed in those regions of the mat where these substrates are usually formed . Moreover, photosynthesis was only stimulated by increased respiration and concomitant CO2 production in the photic zone which indicates that the photosynthetic and respiratory populations must be present in close proximity to each other . Glucose addition had an unexpected negative effect on the microbial population, strongly inhibiting both respiration and gross photosynthesis within hours . After four days, oxygen profiles in the light were equal to those measured in the dark . After replacing the water phase with unamended water, photosynthesis and respiration recovered within a week . None of the physiological changes were accompanied by detectable shifts in the cyanobacterial or the overall microbial community . The mechanism of inhibition of photosynthesis by glucose requires further investigation.

Environ Microbiol, 2002 Nov, 4(11), 634 - 43
Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns; Fromin N et al.; Technical developments in molecular biology have found extensive applications in the field of microbial ecology . Among these techniques, fingerprinting methods such as denaturing gel electrophoresis (DGE, including the three options: DGGE, TGGE and TTGE) has been applied to environmental samples over this last decade . Microbial ecologists took advantage of this technique, originally developed for the detection of single mutations, for the analysis of whole bacterial communities . However, until recently, the results of these high quality fingerprinting patterns were restricted to a visual interpretation, neglecting the analytical potential of the method in terms of statistical significance and ecological interpretation . A brief recall is presented here about the principles and limitations of DGE fingerprinting analysis, with an emphasis on the need of standardization of the whole analytical process . The main content focuses on statistical strategies for analysing the gel patterns, from single band examination to the analysis of whole fingerprinting profiles . Applying statistical method make the DGE fingerprinting technique a promising tool . Numerous samples can be analysed simultaneously, permitting the monitoring of microbial communities or simply bacterial groups for which occurrence and relative frequency are affected by any environmental parameter . As previously applied in the fields of plant and animal ecology, the use of statistics provides a significant advantage for the non-ambiguous interpretation of the spatial and temporal functioning of microbial communities.

Immunology, 2002 Dec, 107(4), 507 - 16
Opposing roles of activator protein-1 and CCAAT/enhancer binding protein beta in the regulation of inducible granulysin gene expression in a human monocytic cell line, THP-1; Kida Y et al.; We previously reported that inducible granulysin gene expression in a human monocytic cell line, THP-1 is dominantly dependent on transcription factor activator protein-1 (AP-1) . Here, we further examined the precise regulatory mechanisms underlying granulysin gene expression using THP-1 cells treated with Acholeplasma laidlawii . Transfection of reporter gene constructs into THP-1 cells indicated that the presence of a positive regulatory element(s) is located from -329 to -85 base pairs, containing two distinct AP-1 binding sites and one nuclear factor-kappaB (NF-kappaB) binding site . Deletion or mutation of the NF-kappaB binding site failed to affect inducible promoter activity, whereas deletion or mutation of both the AP-1 binding sites abrogated the promoter activity . Interestingly, deletion of the putative CCAAT/enhancer binding protein beta (C/EBPbeta) binding site upstream of the positive regulatory element induced the augmentation of granulysin promoter activity . Electrophoretic mobility shift assays demonstrated that nuclear extract prepared from A . laidlawii-treated THP-1 cells generated a specific binding to oligonucleotides, including AP-1, C/EBPbeta, and NF-kappaB element . Furthermore, over-expression of liver-enriched transcriptional activator protein, a subunit of C/EBPbeta, augmented A . laidlawii-induced granulysin promoter activity, whereas over-expression of liver-enriched transcriptional inhibitory protein inhibited the promoter activity . NF-kappaB p50 homodimer had no transactivation property, although it bound to the NF-kappaB site . These results indicate that AP-1 and C/EBPbeta, but not NF-kappaB participate in the regulation of inducible granulysin gene expression in THP-1 cells . Moreover, the Toll-like receptor 2-dependent signalling pathway may be involved in A . laidlawii-induced transactivation of the granulysin promoter . Thus, these results suggest that the gene expression of granulysin in macrophages would be exquisitely regulated by positive and negative transcription factors when microbial invasion occurs.

FEBS Lett, 2002 Dec 4, 532(1-2), 171 - 6
Integrin-nucleated Toll-like receptor (TLR) dimerization reveals subcellular targeting of TLRs and distinct mechanisms of TLR4 activation and signaling; Zhang H et al.; Toll-like receptors (TLRs) are activated by microbial structures . To investigate the mechanisms of TLR activation, the 10 human TLRs were expressed as chimeras with the integrin alphav and beta5 subunits . Co-expression of the alphav-TLR and beta5-TLR chimeras in 293T cells generated 10 TLR homodimers, but only TLR4/4 could effectively activate NF-kappaB . TLR4 monomers also activated NF-kappaB but it was enhanced upon homodimerization . The TLR homodimers showed differential surface/intracellular expression . In TLR heterodimers, only TLR2/1 and TLR2/6 were potent in NF-kappaB activation . NF-kappaB activation by TLR2/1, TLR2/6 and the TLR4 monomer, but not TLR4/4, was completely inhibited by dominant negative MyD88, suggesting that TLR4 homodimers and monomers could activate NF-kappaB through different mechanisms.

J Microbiol Methods, 2003 Feb, 52(2), 149 - 82
Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes; Denich TJ et al.; Membranes lipids are one of the most adaptable molecules in response to perturbations . Even subtle changes of the composition of acyl chains or head groups can alter the packing arrangements of lipids within the bilayer . This changes the balance between bilayer and nonbilayer lipids, serving to affect bilayer stability and fluidity, as well as altering lipid-protein interactions . External factors can also change membrane fluidity and lipid composition; including temperature, chemicals, ions, pressure, nutrients and the growth phase of the microbial culture . Various biophysical techniques have been used to monitor fluidity changes within the bacterial membrane . In this review, bacterial cytoplasmic membrane changes and related functional effects will be examined as well as the use of fluorescence polarization methods and examples of data obtained from research with bacteria .

Med Immunol . 2002 Nov 21;1(1):4.
Optimal clinical trial designs for immune-based therapies in persistent viral infections; Smith KA; There is now effective therapy for infection by the Human Immunodeficiency Virus (HIV), but there is no cure . Consequently, antiviral drugs must be administered continuously to suppress viral replication . Recently, a large phase III international immune-based therapy trial was discontinued because it is difficult to measure clinical endpoints while antivirals are administered . Since the immune system has evolved under the selective force of microbial infections, the immune reaction is antiviral . This commentary explores the rationale of using "Diagnostic Treatment Interruptions" of antiviral therapies to determine efficacies of immune-based therapies.

Pharm Res, 2002 Nov, 19(11), 1736 - 44
A novel hepatic-targeting system for therapeutic cytokines that delivers to the hepatic asialoglycoprotein receptor, but avoids receptor-mediated endocytosis; Sato H et al.; PURPOSE: To demonstrate the utilities of a synthetic low-affinity ligand ((Gal)3) for the asialoglycoprotein receptor (ASGP-R) as a hepatic targeting device for therapeutic cytokines . METHODS: The site-specific incorporation of (Gal)3 or a typical high-affinity ligand (GaINAc)3 into IL-2 was catalyzed by microbial transglutaminase . The anti-tumor activities, pharmacokinetic profiles and receptor-mediated endocytosis in hepatocytes of the ligand-IL-2 conjugates were examined in mouse . RESULTS: The (Gal)3 has approximately 50 times lower affinity to ASGP-R than (GalNAc)3 . Nevertheless, the antitumor effects were in the order of (Gal)3-IL-2 > unmodified IL-2 > (GalNAc)3-IL-2 . The systemic elimination and the hepatic uptake of (GalNAc)374L-2 were more rapid than (Gal)3-IL-2 . The ratio of the rate constant representing dissociation from the cell-surface receptor (k(off) to that representing endocytosis of the ligand (k(int) was greater for (Gal)3-IL-2 than (GalNAc)s-IL-2, suggesting that (Gal)3-IL-2 preferably avoids internalization due to its lower affinity to the receptor . The simulation studies demonstrated that (Gal)3-L-2 was present in the hepatic extracellular space for a longer period than (GaINAc)3 IL-2 . CONCLUSIONS: The (Gal)3 ligand increases the therapeutic efficacy of IL-2 by enhancing its exposure to the cell-surface . The k(off)/k(int) affects the targeting efficacy of the conjugates to ASGP-R.

J Chromatogr B Analyt Technol Biomed Life Sci, 2002 Dec 25, 782(1-2), 67 - 72
Pyrosequencing for microbial typing; Ronaghi M et al.; Pyrosequencing is a real-time DNA sequencing technique generating short reads rapidly and inexpensively . This technology has the potential advantage of accuracy, ease-of-use, high flexibility and is now emerging as a popular platform for microbial typing . Here, we review the methodology and the use of this technique for viral typing, bacterial typing, and fungal typing . In addition, we describe how to use multiplexing for accurate and rapid typing.

Microbiol Mol Biol Rev, 2002 Dec, 66(4), 592 - 616, table of contents
Microbial biodiversity: approaches to experimental design and hypothesis testing in primary scientific literature from 1975 to 1999; Morris CE et al.; Research interest in microbial biodiversity over the past 25 years has increased markedly as microbiologists have become interested in the significance of biodiversity for ecological processes and as the industrial, medical, and agricultural applications of this diversity have evolved . One major challenge for studies of microbial habitats is how to account for the diversity of extremely large and heterogeneous populations with samples that represent only a very small fraction of these populations . This review presents an analysis of the way in which the field of microbial biodiversity has exploited sampling, experimental design, and the process of hypothesis testing to meet this challenge . This review is based on a systematic analysis of 753 publications randomly sampled from the primary scientific literature from 1975 to 1999 concerning the microbial biodiversity of eight habitats related to water, soil, plants, and food . These publications illustrate a dominant and growing interest in questions concerning the effect of specific environmental factors on microbial biodiversity, the spatial and temporal heterogeneity of this biodiversity, and quantitative measures of population structure for most of the habitats covered here . Nevertheless, our analysis reveals that descriptions of sampling strategies or other information concerning the representativeness of the sample are often missing from publications, that there is very limited use of statistical tests of hypotheses, and that only a very few publications report the results of multiple independent tests of hypotheses . Examples are cited of different approaches and constraints to experimental design and hypothesis testing in studies of microbial biodiversity . To prompt a more rigorous approach to unambiguous evaluation of the impact of microbial biodiversity on ecological processes, we present guidelines for reporting information about experimental design, sampling strategies, and analyses of results in publications concerning microbial biodiversity.

J Chromatogr A, 2002 Nov 15, 977(1), 27 - 38
Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns; Arvidsson P et al.; Continuous supermacroporous chromatographic columns with anion-exchange ligands {2-(dimethylamino)ethyl group} and immobilized metal affinity (IMA) ligands (Cu2+-loaded iminodiacetic acid) have been developed allowing binding of Escherichia coli cells and the elution of bound cells with high recoveries . These poly(acrylamide)-based continuous supermacroporous columns have been produced by radical co-polymerization of monomers in aqueous solution frozen inside a column (cryo-polymerization) . After thawing, the column contains a continuous matrix (so-called cryogel) with interconnected pores of 10-100 microm in size . The large pore size of the matrix makes it possible for E . coli cells to pass unhindered through a plain column containing no ligands . E . coli cells bound to an ion-exchange column at low ionic strength were eluted with 70-80% recovery at NaCl concentrations of 0.35-0.40 M, while cells bound to an IMA-column were eluted with around 80% recovery using either 10 mM imidazole or 20 mM EDTA solutions, respectively . The cells maintain their viability after the binding/elution procedure . These preliminary results indicate that microbial cells can be handled in a chromatographic mode using supermacroporous continuous columns . These columns are easy to manufacture from cheap and readily available starting materials, which make the columns suitable for single-time use.

J Appl Microbiol, 1997 Mar, 82(3), 317 - 24
Reduced oxidation of fresh pork in the presence of exogenous hydrolases and bacteria at 2 degrees C; Chung-Wang YJ et al.; Exogenous lipase and phospholipase A2 added to ground pork released free fatty acids (FFA) and reduced lipid oxidation as indicated by TBA values during storage at 2 degrees C . Bovine serum albumin (BSA) added to ground pork to bind FFA accelerated lipid oxidation . Both lipase and phospholipase-producing bacteria increased in numbers, and pseudomonads that produced lipase and phospholipase became the predominant bacteria growing in pork stored at 2 degrees C . The TBA values of ground pork, which were exposed to the growth of natural microbial flora, were reduced up to 84% during storage at 2 degrees C for 16 d . Pseudomonasfragi K1 inoculated into sterilized ground pork reduced TBA values and oxidation volatiles including saturated aldehydes, unsaturated aldehydes, alcohols and ketones.

J Appl Microbiol, 1997 Mar, 82(3), 287 - 91
Contamination of microbial foreign bodies in bottled mineral water in Tokyo, Japan; Fujikawa H et al.; A total of 292 imported and domestic bottled mineral waters (90 brands) obtained from consumers and retailers were examined, by eye, for observable microbial foreign bodies . Fungal and bacterial foreign contaminants were found in 45 samples of water (20 brands) and in 14 samples of water (10 brands), respectively . Of the samples of water found to be contaminated, 41 (22 brands) were imported and 18 (8 brands) were produced domestically . Of 22 brands that were contaminated, 20 (91%) had been sterilized by at least one method . Forty-eight (98%) of 49 samples confirmed with foreign bodies were less than 1 year old . Among the moulds isolated the most predominant genus was Penicillium, followed by Acremonium and Cladosporium . The samples that contained fungi were less contaminated by bacteria than those that contained observable bacterial foreign bodies.

Inhal Toxicol, 2002 Dec, 14(12), 1261 - 77
Spores of Aspergillus versicolor isolated from indoor air of a moisture-damaged building provoke acute inflammation in mouse lungs; Jussila J et al.; Microbial growth in moisture-damaged buildings has been associated with respiratory health effects, and the spores of the mycotoxin producing fungus Aspergillus versicolor are frequently present in the indoor air . To characterize the potential of these spores to cause harmful respiratory effects, mice were exposed via intratracheal instillation to a single dose of the spores of A . versicolor (1 x 10(5), 1 x 10(6), 5 x 10(6), 1 x 10(7), or 1 x 10(8) spores), isolated from the indoor air of a moisture-damaged building . Inflammation and toxicity in lungs were evaluated 24 h later by assessment of biochemical markers and histopathology . The time course of the effects was investigated with the dose of 5 x 10(6) spores for up to 28 days . The exposure to the spores increased transiently proinflammatory cytokine levels (tumor necrosis factor {TNF} alpha and interleukin {IL}-6) in bronchoalveolar lavage fluid (BALF) . The cytokine responses were dose and time dependent . The highest cytokine concentrations were measured at 6 h after the dose, and they returned to the control level by 3 days . Moreover, the spores of A . versicolor recruited inflammatory cells into airways: Neutrophils peaked transiently at 24 h, macrophages at 3 days, and lymphocytes at 7 days after the dosing . The inflammatory cell response did not completely disappear during the subsequent 28 days, though no histopathological changes were seen at that time point . The spores did not induce expression of inducible nitric oxide synthase in lavaged cells . Only the highest spore dose (1 x 10(8)) markedly increased serum IL-6, increased vascular leakage, and caused cytotoxicity (i.e., increased levels of albumin, total protein, lactate dehydrogenase {LDH}, and hemoglobin in BALF) in the airways . In summary, the spores of A . versicolor caused acute inflammation in mouse lungs . This indicates that they have potential to provoke adverse health effects in the occupants of moisture-damaged buildings.

QJM, 2002 Dec, 95(12), 797 - 802
Liver abscess in adults: ten years experience in a UK centre; Mohsen AH et al.; BACKGROUND: The epidemiology and management of liver abscess (LA) have evolved over time . Aim: To examine our experience over 10 years in a UK teaching centre . DESIGN: Retrospective review of patient records . METHODS: We reviewed the records of all patients aged >16 years discharged from Royal Hallamshire Hospital with a diagnosis of LA between April 1988 and December 1999 . RESULTS: There were 69 patients with LA (65 pyogenic, 4 amoebic), giving a crude annual incidence rate of 2.3/100,000/year (18.15/100,000 hospital admissions) . Median age was 64 years . Single lesions were found in 41 patients, multiple lesions in 28 . Pre-admission, patients were symptomatic for a median 14 days, with the most common symptoms and signs being fever and abdominal pain/tenderness . Pathogens were identified in 74% and predisposing aetiology in 92% of those undergoing investigation . Spread of infection to the liver via the portal venous system was the commonest route of infection (46%), most frequently in patients aged >/=60 years (p=0.019) . Abdominal ultrasound (US) was diagnostic for LA in >90% of cases . Treatment with anti-microbial therapy plus interventional radiology was optimal . The case fatality rate was 12.3%, mainly from associated underlying pathology . DISCUSSION: LA is commonly associated with underlying gastrointestinal pathology . Seeking out this underlying aetiology is an integral part of management . We recommend US as the first-line diagnostic tool with guided intervention plus antibiotic(s) as first-line treatment . Prognosis depends chiefly on the underlying pathology.

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