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PubMed2005

A better global resolution function and a novel iterative stochastic search method for optimization of high-performance liquid chromatographic separation.

HPLC optimization strategy consists of four elements; experimental design, retention modeling, quality criteria function, and optimum search method. In this paper we present a simple, superior alternative to general classes of classical resolution functions (S function) and a novel optimum search algorithm (iterative stochastic search, ISS) for HPLC optimization. Comparison of S with general classes of resolution-based quality criteria functions (Rs, Rp, and Rmin) shows superior features such as correct assessment of favorable separation conditions, preservation of peak pair contributions, elimination of arbitrary cut-off values, and a unique capability to interpret absolute significance of function values through a simple inequality. The proposed ISS algorithm is more robust than standard methods and it is easily applicable to hyperdimensional optimization. ISS also shows clear advantages in its ability to correctly identify the global optimum (instead of local optimum), with higher precision, with more efficient use of computation cycles, and with easier implementation. Successful application of S and ISS to HPLC optimization was demonstrated in the separation of representative functionalities (sugars, alcohols, and organic acids) present in microbial fermentations. Both the optimal and pathological (worst) conditions were successfully predicted and experimentally verified.

Algorithms↗
PubMed2005

Toll-like receptor 2 ligands activate human basophils for both IgE-dependent and IgE-independent secretion.

BACKGROUND: Toll-like receptor (TLR) molecules play a critical role in directing the course of acquired immunity, including that associated with allergic disease, by recognizing specific microbial products that activate immune cells for effector functions. OBJECTIVE: We investigated whether human basophils express 2 such molecules (TLR2 and TLR4), and assessed whether putative ligands for these receptors activate nuclear factor kappaB (NFkappaB) and modulate mediator release and cytokine secretion either alone or in response to stimulation. METHODS: Toll-like receptor expression was assessed by using RT-PCR and flow cytometry. Immunoblotting detected nuclear NFkappaB. Automated fluorometry, RIA, and ELISA detected concurrent changes in histamine, leukotriene C 4 , and cytokine, respectively, after culture with specific ligands. RESULTS: mRNA and protein for TLR2 and TLR4 were detected in basophils. However, in assessing nuclear localization of NFkappaB as a measure of functional receptor responses, basophils selectively reacted only to peptidoglycan, a TLR2 ligand, and not to LPS, a TLR4 ligand. Likewise, basophils secreted both IL-4 and IL-13 in direct response to peptidoglycan but not to LPS. Although neither ligand induced histamine or leukotriene C 4 release, several TLR2-specific ligands augmented the secretion of these mediators (and cytokine) in response to IgE-dependent activation and of IL-13 in response to IgE-independent stimulation. Finally, a selective inhibitor of NFkappaB did not prevent these enhancing effects mediated by TLR2 ligands. CONCLUSION: These data suggest that innate immune responses mediated through TLR2 play a role in augmenting allergic reactions, in part by modulating basophil cytokine secretion and mediator release independently of NFkappaB activation.

Basophils↗
PubMed2004

A powerful combination: the use of positional scanning libraries and biometrical analysis to identify cross-reactive T cell epitopes.

Studies on the elucidation of the specificity of the T cell receptor (TCR) at the antigen and peptide level have contributed to the current understanding of T cell cross-reactivity. Historically, most studies of T cell specificity and degeneracy have relied on the determination of the effects on T cell recognition of amino acid changes at individual positions or MHC binding residues, and thus they have been limited to a small set of possible ligands. Synthetic combinatorial libraries (SCLs), and in particular positional scanning synthetic combinatorial libraries (PS-SCLs) represent collections of millions to trillions of peptides which allow the unbiased elucidation of T cell ligands that stimulate clones of both known and unknown specificity. PS-SCLs have been used successfully to study T cell recognition and to identify and optimize T cell clone (TCC) epitopes in infectious diseases, autoimmune disorders and tumor antigens. PS-SCL-based biometrical analysis represents a further refinement in the analysis of the data derived from the screening of a library with a TCC. It combines this data with information derived from protein sequence databases to identify natural peptide ligands. PS-SCL-based biometrical analysis provides a method for the determination of new microbial antigen and autoantigen sequences based solely on functional data rather than sequence homology or motifs, making the method ideally suited for the prediction and identification of both native and cross-reactive epitopes by virtue of its ability to integrate the examination of trillions of peptides in a systematic manner with all of the protein sequences in a given database. We review here the application of PS-SCLs and biometrical analysis to identify cross-reactive T cell epitopes, as well as the current efforts to refine this strategy.

Biometry↗
PubMed2006

Type III effector proteins: doppelgangers of bacterial virulence.

Bacterial pathogens have co-evolved with their hosts in their ongoing quest for advantage in the resulting interaction. These intimate associations have resulted in remarkable adaptations of prokaryotic virulence proteins and their eukaryotic molecular targets. An important strategy used by microbial pathogens of animals to manipulate host cellular functions is structural mimicry of eukaryotic proteins. Recent evidence demonstrates that plant pathogens also use structural mimicry of host factors as a virulence strategy. Nearly all virulence proteins from phytopathogenic bacteria have eluded functional annotation on the basis of primary amino-acid sequence. Recent efforts to determine their three-dimensional structures are, however, revealing important clues about the mechanisms of bacterial virulence in plants.

Bacteria↗
PubMed1998

The ABC family of multidrug transporters in microorganisms.

Multidrug transporters are membrane proteins that are able to expel a broad range of toxic molecules from the cell. In humans, the overexpression of the multidrug resistance P-glycoprotein (Pgp) and the multidrug resistance-associated protein MRP1 (MRP) is a principal cause of resistance of cancers to chemotherapy. These multidrug transporters belong to the ATP-binding cassette (ABC) family of transport proteins that utilize the energy of ATP hydrolysis for activity. In microorganisms, multidrug transporters play an important role in conferring antibiotic resistance on pathogens. In the last decade, homologs of human Pgp and MRP have been found in microorganisms such as Plasmodium falciparum, Candida albicans, Saccharomyces cerevisiae and, more recently, in Lactococcus lactis. In this review, we will summarize the current state of knowledge on three major aspects of microbial ABC-type multidrug transporters: (i) the functional and structural similarities among these proteins in prokaryotic and eukaryotic cells, (ii) the molecular mechanism of these transporters, and (iii) their potential physiological role.

ATP-Binding Cassette Transporters↗
PubMed2002

The inhibition of marine nitrification by ocean disposal of carbon dioxide.

In an attempt to reduce the threat of global warming, it has been proposed that the rise of atmospheric carbon dioxide concentrations be reduced by the ocean disposal of CO2 from the flue gases of fossil fuel-fired power plants. The release of large amounts of CO2 into mid or deep ocean waters will result in large plumes of acidified seawater with pH values ranging from 6 to 8. In an effort to determine whether these CO2-induced pH changes have any effect on marine nitrification processes, surficial (euphotic zone) and deep (aphotic zone) seawater samples were sparged with CO2 for varying time durations to achieve a specified pH reduction, and the rate of microbial ammonia oxidation was measured spectrophotometrically as a function of pH using an inhibitor technique. For both seawater samples taken from either the euphotic or aphotic zone, the nitrification rates dropped drastically with decreasing pH. Relative to nitrification rates in the original seawater at pH 8, nitrification rates were reduced by ca. 50% at pH 7 and more than 90% at pH 6.5. Nitrification was essentially completely inhibited at pH 6. These findings suggest that the disposal of CO2 into mid or deep oceans will most likely result in a drastic reduction of ammonia oxidation rates within the pH plume and the concomitant accumulation of ammonia instead of nitrate. It is unlikely that ammonia will reach the high concentration levels at which marine aquatic organisms are known to be negatively affected. However, if the ammonia-rich seawater from inside the pH plume is upwelled into the euphotic zone, it is likely that changes in phytoplankton abundance and community structure will occur. Finally, the large-scale inhibition of nitrification and the subsequent reduction of nitrite and nitrate concentrations could also result in a decrease of denitrification rates which, in turn, could lead to the buildup of nitrogen and unpredictable eutrophication phenomena. Clearly, more research on the environmental effects of ocean disposal of CO2 is needed to determine whether the potential costs related to marine ecosystem disturbance and disruption can be justified in terms of the perceived benefits that may be achieved by temporarily delaying global warming.

Ammonia↗
PubMed1998

Functional expression of a mammalian acetylcholinesterase in Pichia pastoris: comparison to acetylcholinesterase, expressed and reconstituted from Escherichia coli.

The mature rat brain acetylcholinesterase gene (T subunit, AChE) was subcloned downstream of the temperature-inducible lambda promoter PL and fused to the signal peptide of the OmpA protein. Three different expression vectors were constructed: (i) pCompmA containing the mature AChE, (ii) pComp delta TA containing a truncated AChE and (iii) pComp delta TAH containing the truncated AChE C-terminal fused to a 6xHis-tag. With all expression vectors the overexpression of AChE in Escherichia coli resulted mainly in cytoplasmic inclusion bodies (IB). However, some activity was found in the periplasmic space. The inclusion bodies were refolded in vitro, yielding up to 1.42 U/mg IB of active AChE. The refolded AChE was partially purified (approx. 300-fold) by affinity chromatography with a specific activity of approx. 250 U/mg. Removing the cysteine residue near the C-terminus (truncated AChE, delta TAChE) assuming to affect the refolding, did not increase the amount of active enzyme obtained after refolding. Purification of denatured delta TAChE-6xHis prior to refolding by Ni-NTA-chromatography increased the refolding efficiency by a factor of 1.5. Functional expression and secretion of rat brain acetylcholinesterase into the medium was achieved in Pichia pastoris. By optimizing the culture conditions, 100 mU/ml AChE in the medium was produced. In this work we are describing the functional expression of a mammalian AChE in a microbial host in good yields for the first time. The physico-chemical properties of both, the bacterial and yeast expressed AChE were compared with those of the native AChE. The properties of the yeast expressed AChE and the native AChE were similar, whereas the E. coli expressed enzyme was found to be less stable and had different inhibition properties.

Acetylcholinesterase↗
PubMed1997

Diseases and surgery of the cecum.

The cecum is an important site of water and electrolyte absorption, as well as microbial digestion of soluble and insoluble carbohydrates. These functions of the cecum and ascending colon require that ingesta be mixed constantly and retained long enough to complete the digestion of cellulose. Parasites, changes in regional blood flow, diet, and various pharmacologic agents are associated with alterations in normal cecal motility patterns.

Anastomosis, Surgical↗
PubMed2000

Detection of genes essential in specific niches by signature-tagged mutagenesis.

Variations of the signature-tagged mutagenesis (STM) technique are now possible and the method can be applied to most pathogens that have an STM-selectable phenotype in a host system. STM screening of 15,040 mutants from 11 bacterial species identified 323 in vivo attenuated mutants. As a genome-scanning tool, STM will yield information about genes with unknown functions as well as information crucial for understanding microbial pathogenesis.

Animals↗
PubMed2003

Analysis of the logistic function model: derivation and applications specific to batch cultured microorganisms.

Mathematical models are useful for describing microbial growth, both in natural ecosystems and under research conditions. To this end, a rate expression that accounted for depletion of nutrients was used to derive the logistic function model for batch cultures. Statistical analysis was used to demonstrate the suitability of this model for growth curve data. Two linear forms of the model and two procedures for calculating growth rate constants were derived to facilitate statistical evaluation of growth curves. The procedures for calculating growth rate constants were found to be useful for calculation of growth rate constants at each time point, or for estimating growth rate constants from early growth curve data. The utility of the logistic function model and its alternative forms is discussed with respect to planning experiments, analyzing growth curves for the effects of factors other than nutrient limitation, and developing more complete descriptions of cell proliferation.

Bacteria↗
PubMed2006

Neopikromycin and novapikromycin from the pikromycin biosynthetic pathway of Streptomyces venezuelae.

Two new macrolides from the pikromycin biosynthetic pathway of Streptomyces venezuelae, neopikromycin (9) and novapikromycin (10), were identified and structurally characterized through mass spectrometry and NMR spectroscopy. The established structures showed that 9 and 10 have hydroxyl groups at C-14 (9) and at both C-12 and C-14 (10), on the basis of a comparison with narbomycin (7). The purified PikC cytochrome P450 monooxygenase catalyzes the in vitro hydroxylation of 7 and pikromycin (8) to yield 9 and 10, respectively, thus expanding the substrate- and regio-flexibility of this enzyme.

Anti-Bacterial Agents↗
PubMedDate not supplied

Dendritic cells as inducers of antimicrobial immunity in vivo.

Models of infection have provided important insight into the function of dendritic cells (DC) in vivo. Several microbial products induce DC maturation via Toll-like receptors, a process that is crucial for the ability of DC to initiate adaptive immune responses. Splenic DC have also been shown to produce IL-12 during infection in vivo. This DC-derived IL-12 might be important to skew T cell responses towards Th1. Microbial infections also induce changes in the DC populations of lymphoid organs, often in a subset-specific manner, manifested as an accumulation and redistribution of DC. Furthermore, data are emerging pointing at an absolute requirement of DC in priming of naïve T cells in vivo.

Animals↗
PubMed2000

The TEA/ATTS transcription factor CaTec1p regulates hyphal development and virulence in Candida albicans.

The temporal and spatial expression of stage-specific genes during morphological development of fungi and higher eukaryotes is controlled by transcription factors. In this study, we report the cloning and functional analysis of the Candida albicans TEC1 (CaTEC1) gene, a new member of the TEA/ATTS family of transcription factors that regulates C. albicans virulence. The promoters of the type 4, 5 and 6 proteinase isogenes (SAP4-6) contain repetitive TEA/ATTS consensus sequence motifs. This finding suggests a possible role for a homologue of Saccharomyces cerevisiae TEC1 during the activation of proteinase gene expression in C. albicans. CaTEC1 is predominantly expressed in the hyphal form of C. albicans. In vitro, serum-induced hyphal formation as well as evasion from MPhi after phagocytosis is suppressed in catec1/catec1 mutant cells. Furthermore, expression of the proteinase isogenes SAP4-6 is no longer inducible in these mutant cells. The deletion of the CaTEC1 gene attenuates virulence of C. albicans in a systemic model of murine candidiasis, although both mutant and revertant cells that were prepared from infected tissues or the vaginal mucosa grew in a hyphal morphology in vivo. CaTEC1 complements the pseudohyphal and invasive growth defect of haploid and diploid S. cerevisiae tec1/tec1 mutant cells and strongly activates the promoter of FLO11, a gene required for pseudohyphal growth. This study provides the first evidence pointing to an essential role for a member of the TEA/ATTS transcription factor family that had so far only been ascribed to function during development as a virulence regulator in microbial pathogenesis.

Animals↗
PubMedDate not supplied

Intestinal function and body growth of broiler chickens on diets based on maize dried at different temperatures and supplemented with a microbial enzyme.

A study was conducted to evaluate the effects of varying drying temperature (Fresh, 85, 95 or 105 degrees C) on the nutritive value of maize and response of broiler chickens to diets based on such grain, and supplemented with a microbial enzyme (Avizyme 1500). The chemical composition of the grain was affected by drying temperature. Starch and amylopectin contents were increased while there was a reduction in amylose content. These changes were expected to underlie the response of chicks to the diets. Total feed intake over 28 days was increased (P < 0.05) as a result of heat-treating the maize up to 95 degrees C. The final body weight of chicks on the diet based on fresh maize was improved (P < 0.05) by the microbial enzyme supplement (MES). There was no effect of the enzyme supplement on body weight when assessed at earlier ages. Over the entire feeding period, feed conversion efficiency (FCE) declined (P < 0.001) with increasing oven temperature, regardless of the supplementation with the microbial enzyme. Body weight was influenced (P < 0.05) by the microbial enzyme only when assessed over the entire trial period. The weight of visceral organs, protein content and activities of pancreatic and jejunal digestive enzymes were unaffected by grain heat treatment or MES. The ileal digestibility of calcium was reduced (P < 0.001) on diets based on fresh maize and maize that was oven-dried at 105 degrees C. Heat-treatment also improved (P < 0.05) the ileal digestibility of phosphorus in chicks on the diets without MES. There were no effects of grain heat treatment or MES on the ileal digestibility of energy, protein, Ca and amino acids. The results indicate some variations in grain quality as a result of heat treatment but the differences were not significant enough to stimulate major responses to the MES. Further studies should examine samples from commercial drying processes or samples obtained from a closer simulation of commercial conditions, to arrive at more practical conclusions.

Animal Feed↗
PubMed2002

Diagnosis of irritable bowel syndrome.

Irritable bowel syndrome (IBS) is the most common disorder seen in gastroenterology practice. It is also a large component of primary care practices. Although the classic IBS symptoms of lower abdominal pain, bloating, and alteration of bowel habits is easily recognizable to most physicians, diagnosing IBS remains a challenge. This is in part caused by the absence of anatomic or physiologic markers. For this reason, the diagnosis of IBS currently needs to be made on clinical grounds. A number of symptom-based diagnostic criteria have been proposed over the last 15 years. The most recent of these, the Rome II criteria, seem to show reasonable sensitivity and specificity in diagnosing IBS. However, the role of the Rome II criteria in clinical practice remains ill defined. A review of the literature shows that, in patients with no alarm symptoms, the Rome criteria have a positive predictive value of approximately 98%, and that additional diagnostic tests have a yield of 2% or less. Diagnostic evaluation should also include a psychosocial assessment specifically addressing any history of sexual or physical abuse because these issues significantly influence management strategies and treatment success.

Breath Tests↗
PubMed1999

Antimicrobial peptides from platelets.

The fact that platelets play a key role in host defense against infection has been demonstrated by the following observations(1): (a) platelets rapidly respond to sites of endovascular trauma and chemotactic stimuli associated with microbial colonization, and they are the earliest and predominant cells at sites of microbial colonization of vascular endothelium; (b) platelets have surface receptors and cytoplasmic granules comparable in structure and function to those of neutrophils, monocytes, or macrophages; (c) platelets adhere directly to, and may internalize, microbial pathogens, thereby enhancing their clearance from the bloodstream and limiting their potential for hematogenous dissemination; (d) bacterial, fungal, and protozoal pathogens are damaged or killed by activated platelets in vitro; (e) platelets are capable of initiating or amplifying complement fixation in the presence of microorganisms; (f) platelets generate oxygen metabolites which likely contribute to their antimicrobial activity; (g) platelets and leukocytes interact synergistically to exert enhanced antimicrobial functions in vitro; (h) thrombocytopenia increases susceptibility to and severity of certain infections. Importantly, rabbit and human platelets are now known to contain and release microbicidal proteins (termed platelet microbicidal proteins [PMPs] or thrombin-induced PMPs [tPMPs]) when stimulated with microorganisms or platelet agonists associated with infection in vitro. It is hypothesized that these microbicidal peptides accumulate locally at sites of endovascular damage or infection. Recent investigations have confirmed that tPMP-susceptible pathogens are less capable of proliferation or hematogenous dissemination in vivo as compared with their isogenic counterpart strains that are resistant to PMPs. Collectively, the above observations strongly suggest that platelets play key and multi-faceted roles in antimicrobial host defense which appear to be significantly mediated by PMPs and tPMPs. Copyright 1999 Harcourt Publishers Ltd.

Journal Article↗