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PubMed2002

Microbial beta-glucosidases: cloning, properties, and applications.

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.

Biotechnology↗
PubMed2005

The use of stable isotope probing techniques in bioreactor and field studies on bioremediation.

Stable isotope probing (SIP) is a molecular technique that allows investigators to follow the flow of atoms in isotopically enriched molecules through complex microbial communities into metabolically active microorganisms. Thus, SIP has immense promise for discovering microorganisms responsible for ecologically important biogeochemical reactions in nature. Applications of SIP to biodegradation and bioremediation processes are still in their infancy. In the past few years, approximately a dozen biodegradation studies using SIP based on the analysis of labeled DNA, RNA or phospholipid fatty acids have been completed. Results have begun to link biomarkers (especially sequences of 16S ribosomal RNA and functional genes) to biodegradation reactions in naturally occurring microbial communities. As extensive compilations of ecologically important genotypes and phenotypes accrue, predictive abilities for contaminant metabolism in particular habitats may be achieved.

Bacteria↗
PubMed1985

[Ultrastructural characteristics of the natural heteromorphic growth of Clostridium septicum].

Periodic cultures of C. septicum strain No. 59, growing in Pope's broth, have been studied by means of transmission electron microscopy on ultrathin sections. During the lagphase of growth the inoculated bacilliary cells are sequentially converted into giant filamentous multinucleate forms. These changes, reflecting the reaction of phenotypical adaptation to new environmental conditions, are followed by the restoration of the initial phenotype via the fragmentation of the giant cells. At the same time heteromorphic growth becomes, also spontaneously, atypical in some of the multinucleate cells. This atypical heteromorphic growth results in pronounced degenerative changes leading to bacteriolysis and the death of microbial cells due to disturbances in the coordination of the formation of structural and functional complexes in these cells at the period of their phenotypical adaptation. Such pathology of microbial cells leads to elimination of individual cells with developmental defects, which is finally conducive to the sanitation of the population.

Clostridium↗
PubMed1992

Differentiation of round cells in semen by means of monoclonal antibodies and relationship with male fertility.

OBJECTIVE: To differentiate round cells in semen samples of subfertile men and evaluate the clinical significance during infertility investigation. PATIENTS: One hundred and eight randomly chosen couples with a median duration of infertility of 4 (range, 1 to 20) years presenting at the outpatient infertility clinic of the University of Heidelberg, Germany. MAIN OUTCOME MEASURES: Differentiation of round cells in semen by means of monoclonal antibodies (mABs) and a streptavidin-biotin system for staining. Correlation of results with medical history, outcome of clinical examination, sperm analysis, microbial screening of both partners, evaluation of sperm functional capacity in vivo by means of the postcoital test (PCT) and in vitro with the standardized crossed sperm-cervical mucus penetration test (SCMPT) and the subsequent fertility in a prospective study. RESULTS: The method used for differentiation of round cells proved to be practical and suitable for routine use. The percentage of leukocytes ranged from 0% to 58% with a median of 3%. Number of round cells and percentage of leukocytes did not differ markedly with regard to andrologic history, clinical findings, for example, varicocele, results of standard sperm analysis, and microbial colonization of semen samples. However, high rates of leukocytes of the round cells correlated with reduced sperm count and morphology and results of PCT. Leukocyte-positive (> 15% leukocytes) specimens were also significantly more frequent in case of inadequate SCMPT and reduced sperm penetration ability in vitro. CONCLUSIONS: In asymptomatic patients (in terms of genital tract infection), the majority of round cells consist of immature germ cells and < 5% are white blood cells. The streptavidin-biotin system and the mABs used in this study proved to be useful to identify patients with elevated rates of leukocytes in semen possibly reflecting subclinical genital tract infection with influence on sperm functional capacity and subsequent fertility. Thus the procedure can be recommended to be included in a comprehensive evaluation of male fertility.

Adult↗
PubMed1993

Lipid metabolism in the rumen.

Recent advances in ruminal lipid metabolism have focused primarily on manipulation of physicochemical events in the rumen aimed at two practical outcomes: 1) control of antimicrobial effects of fatty acids so that additional fat can be fed to ruminants without disruption of ruminal fermentation and digestion and 2) regulation of microbial biohydrogenation to alter the absorption of selected fatty acids that might enhance performance or reduce saturation of meat and milk. Properties of lipids that determine their antimicrobial effects in the rumen include type of functional group, degree of unsaturation, formation of carboxylate salts, and physical association of lipids with surfaces of feed particles and microbes. The mechanism of how lipids interfere with ruminal fermentation is a complex model involving partitioning of lipid into the microbial cell membrane, potency of the lipid to disrupt membrane and cellular function, physical attachment of microbial cells to plant surfaces, and expression and activity of microbial hydrolytic enzymes. Lipolytic and hydrogenation rates vary with forage quality (stage of maturity and N content), surface area of feed particles in the rumen, and structural modifications of the lipid molecule that inhibit attack by bacterial isomerases.

Animals↗
PubMed1986

Phenotypic and cytotoxic characteristics of the immune cells of the human placenta.

Despite some functional impairment of the newborn's T-cell immune system, most infants survive the intrauterine and perinatal period without succumbing to infection or maternal lymphocyte engraftment. The placenta may play a crucial role in protecting the infant from microbial and histocompatibility antigens. Accordingly, we studied phenotypic and functional capacities of placental cells. Placentas were obtained from uncomplicated pregnancies. Matched cord blood and maternal peripheral blood were also obtained in many instances. Fresh minced placental tissue was washed and digested with collagenase and DNase and mononuclear cells were obtained by density gradient centrifugation. The average yield was 10(6) cells/g of tissue with greater than 80% viability. Chromosome analysis of five placental preparations indicated that these cells were of fetal rather than maternal origin. The isolated placental cells consisted of trophoblasts, lymphocytes (74 +/- 3%), monocytes (16 +/- 3%), and granulocytes (8 +/- 2%). E-rosette forming cells (T cells) made up 65 +/- 2% and surface membrane immunoglobulin positive cells made up 8 +/- 1% of the placental mononuclear cells. Fluorescent activated analysis of the mononuclear cells indicated less Leu 4-positive cells (Pan-T) 43 +/- 3%, and less Leu 3-positive (T-helper cells) (25 +/- 2%), than cord and maternal cell preparations. Leu-2, DR, and B1 positive cells were similar to those in cord and maternal blood. Leu 7 and especially Leu 11 positive cells, markers for natural killer cells, were abundant in placental cells, making up 4 +/- 0.7% and 20 +/- 3%, respectively. The Leu 7/Leu 11 ratio of the placental cells was different from either the maternal or cord blood cells. Natural killer activity of placental cells against a K562 natural killer target was low, despite the abundance of cells with NK markers. The K562 activity was low in the placental cells, similar to the low NK activity of maternal and cord cells. Molt 4f killer activity was near normal. Lectin-dependent cytotoxicity using an EL-4 cell target plus PHA was low in placentas, compared to normal, maternal, or cord cell cytotoxicity. Matched samples indicated that LDCC activity was mother greater than cord greater than placenta. Antibody-dependent cytotoxicity (Raji target) of placental cells showed low activity, and again the paired studies indicated that normal controls greater than maternal greater than cord greater than placenta cytotoxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed2005

Finding the missing link between diversity and activity using denitrifying bacteria as a model functional community.

The recent development and application of numerous methods mainly based on 16S rDNA analyses have brought insights into the questions of which and how many bacterial populations can be found in a given ecosystem. A new and challenging question for microbial ecologists has emerged from the exploration of this diversity: what is its significance for ecosystem functioning? We propose the denitrifying bacteria as a model microbial community for understanding the relationship between community structure and activity, and have summarized the recent progress in studies of this functional community.

Bacteria↗
PubMed2005

Functional genomics and gene expression profiling in sepsis: beyond class prediction.

Functional genomics involving genome-wide expression analyses is rapidly finding applications in clinical medicine. New technologies now permit the simultaneous analysis of mRNA levels for the entire human transcriptome from as few as 1000 cells. This approach is dramatically changing the way we define health and disease, allowing, for the first time, an unbiased view of the global changes in gene expression that are occurring. For the study of trauma biology and sepsis, this technology offers a powerful tool to develop molecular signatures for inflamed tissues and specific cell populations. At present, functional genomics is being used to classify the progress of disease and survival in response to traumatic and burn injury, sepsis and visceral ischemia, and reperfusion injury, as well as to describe patterns of gene expression in response to varying microbial pathogens. As the number of bioinformatics tools increases, functional genomics is beginning to reveal the underlying complexity of the biological response to a variety of inflammatory diseases and is providing new approaches for their exploration. Functional genomics is becoming a standard tool in inflammation research as a means to unravel the basic biological processes.

Animals↗
PubMed2005

Cationic fullerenes are effective and selective antimicrobial photosensitizers.

Fullerenes are soccer ball-shaped molecules composed of carbon atoms, and, when derivatized with functional groups, they become soluble and can act as photosensitizers. Antimicrobial photodynamic therapy combines a nontoxic photosensitizer with harmless visible light to generate reactive oxygen species that kill microbial cells. We have compared the antimicrobial activity of six functionalized C(60) compounds with one, two, or three hydrophilic or cationic groups in combination with white light against gram-positive bacteria, gram-negative bacteria, and fungi. After a 10 min incubation, the bis- and tris-cationic fullerenes were highly active in killing all tested microbes (4-6 logs) under conditions in which mammalian cells were comparatively unharmed. These compounds performed significantly better than a widely used antimicrobial photosensitizer, toluidine blue O. The high selectivity and efficacy exhibited by these photosensitizers encourage further testing for antimicrobial applications.

Animals↗
PubMed2000

From fold to function predictions: an apoptosis regulator protein BID.

With the rapidly increasing pace of genome sequencing projects and the resulting flood of predicted amino acid sequences of uncharacterized proteins, protein sequence analysis, and in particular, protein structure prediction is quickly gaining in importance. Prediction algorithms can be used for preliminary annotation of newly sequenced proteins and, at least in some cases, provide insights into their function and specific mode of action. Such annotations for several microbial genomes were performed by several groups and placed in public domain for evaluation. An example presented in this work comes from a related project of structural and functional predictions for proteins involved in the process of controlled cell death (apoptosis). The BID protein belongs to an important class of regulators of apoptosis identified by short sequence motifs. Here, several fold prediction methods are used to build a series of three-dimensional models. Structure analysis of the models with reference to the biological data available allows selection of the most appropriate model. It is found that the most likely structural model of BID is built on the structure of Bcl-X(L). The model is discussed in terms of experimental data on specific proteolytic cleavage of BID and its effect on BID interactions with other proteins and membranes.

Algorithms↗
PubMed1978

Acquired cell-mediated immunodepression in acute Chagas' disease.

In this study two groups of patients with acute Chagas' disease were identified. Group one consisted of five patients with apparent acute Chagas' disease. These patients showed symptoms and signals of an acute illness, such as high fever and enlarged spleen. One of these patients developed severe myocarditis and heart failure. Group two consisted of seven patients with inapparent acute Chagas' disease. This was a nonclinical entity, not perceived by the patient who did not seek medical care. The diagnosis was made by the shift of a serologic test which indicates the presence of immunoglobulin M antibodies to Trypanosoma cruzi. The patients with apparent acute Chagas' disease showed positive delayed-type skin response to T. cruzi antigen. Also, their leukocytes showed significant inhibition of migration in the presence of this antigen. By contrast, the patients with the inapparent acute Chagas' disease did not show positive delayed-type skin response to T. cruzi antigen and no significant inhibition was observed when their cells migrated in the presence of this antigen. Of interest, none of these patients was capable of developing contact sensitivity to 2,4-dinitrochlorobenzene. However, three out of five patients with the apparent acute disease and all the normal control subjects showed positive contact reaction after sensitization to this drug. The results of these experiments would suggest that the thymus-derived (T)-lymphocyte function is depressed in patients with the clinically inapparent acute Chagas' disease. This immunodepression seems to be acquired in the course of the T. cruzi infection because all patients showed positive delayed-type skin response to at least one ubiquitous microbial extract, thus indicating previously normal T-cell function. We hypothesize that T. cruzi antigens may directly stimulate T cells with the concomitant release of factors that might become supressive for T-cell responses. Furthermore, the suppressive effect might interfere with the T-cell response to other antigens, such as to 2,4-dinitrochlorobenzene.

Acute Disease↗
PubMed2005

Methane production and microbial community structure in single-stage batch and sequential batch systems anaerobically co-digesting food waste and biosolids.

Anaerobic co-digestion of food waste and biosolids was carried out in sequential batch and single-stage batch systems in four treatments. Methane yield, which was used as a functional process parameter, differed between treatments, with the single-stage batch system generating lower volumes than the sequential batch systems. Volatile fatty acid (VFA) concentrations and pH in the leachate also differed between treatments. VFA concentrations were highest and methane generation yields lowest in the single-stage batch system in comparison to the sequential batch systems. The anaerobic microbial community structure of the domains Archaea and Bacteria, determined by denaturing gradient gel electrophoresis, differed between treatments and was correlated to a number of environmental parameters such as pH, VFA concentration and methane generation rate. Methane generation rate was significantly correlated to the community structure of Bacteria but not Archaea. This indicated that the substrates that are produced by acetogens (Bacteria) are important for the growth and community structure of the methanogens (Archaea). Community structure of Archaea changed over time, but this had no observable effect on functional ability based on methane yields. Microbial diversity (H') was shown to be not important in developing a functionally successful anaerobic microbial community.

Anaerobiosis↗
PubMed2001

Regulation of midgut defensin production in the blood-sucking insect Stomoxys calcitrans.

The Stomoxys midgut defensin (Smd) family of genes are exclusively expressed in the anterior midgut of adult flies. Their putative function is protection of the stored bloodmeal from microbial attack. Smd genes are constitutively expressed, up-regulated in response to a bloodmeal and further up-regulated by immune stimulation per os but only in the presence of a bloodmeal not a sugar meal. Smd genes are down-regulated in response to a systemic immune challenge. Smd gene constructs transfected into l(2)mbn cells undertake constitutive expression but are not up-regulated by immune challenge. Electrophoretic mobility shift assays (EMSA) suggest the rel-like sites in the proximal promoter region of Smd genes do not bind midgut factors and so are non-functional.

Animals↗
PubMed2003

Micro-cantilever method for measuring the tensile strength of biofilms and microbial flocs.

Cohesive strength is an important factor in determining the structure and function of biofilm systems, and cohesive strength plays a key role in our ability to remove or control biofilms in engineered systems. A micro-mechanical device has been developed to directly measure the tensile strength of biofilms and other microbial aggregates. An important feature of this method is the combination of a direct measurement of force with particle separations that occur at a scale comparable to that observed for biofilm systems. The force required to separate an aggregate is determined directly from the deflection of cantilevered glass micropipettes with a 20-40-microm diameter. Combined with an estimate of the cross-sectional area of the aggregate at the point of separation this measurement indicates the cohesive strength of the aggregate. Samples of return activated sludge (RAS) and a Pseudomonas aeruginosa biofilm were tested using the device. The measured cohesive strengths of the RAS flocs ranged from 419 to 206,400 N/m(2), while many of the flocs exceeded the range of measurement of the device. Fragments of P. aeruginosa biofilm had cohesive strengths ranging from 395 to 15,640 N/m(2), with a median value of 3020 N/m(2). The median equivalent diameters of the particles detached from the aggregates were 32 microm for RAS and 30 microm for the P. aeruginosa biofilms.

Biofilms↗
PubMed1996

Granulocytes harvested following G-CSF-enhanced leukocyte recovery retain their functional capacity during in vitro culture for 72 hours.

The purpose of this study was to compare two different in vitro culture conditions for the preservation of human granulocytes. These cells could be used in patients with severe neutropenia following cytotoxic chemotherapy if the functional capacity was retained, and autologous transfusions of granulocytes would circumvent the risk of alloimmunization. Granulocytes were obtained from the peripheral blood of healthy donors and patients with hematologic malignancies who received cytotoxic chemotherapy supported by recombinant human granulocyte colony-stimulating factor (R-metHuG-CSF, 300 micrograms/day, s.c.). Granulocytes were either cultured for 72 h at 4 degrees C in the presence of 100 ng/ml G-CSF or cryopreserved at -196 degrees C. The viability, surface antigen expression, and function of the granulocytes were assessed. Since effective microbial killing involves the attachment of granulocytes to blood vessel walls, transmigration into tissues, chemotaxis, and phagocytosis, the surface expression of the adhesion molecules LFA-1 (CD11a/CD18) and gp 150,95 (CD11c/CD18) was measured. In addition, the IgG receptors Fc gamma RI (CD64), Fc gamma RII (CD32), and Fc gamma RIII (CD16), as well as the complement receptor CR3 (CD11b/CD18), were assessed. Dynamic superoxide anion release served as a measure of the metabolic pathway of the oxidative burst after f-Met-Leu-Phe (fMLP) and phorbol-12-myristate-13-acetate (PMA) stimulation. Substantial differences in the preservation of granulocyte integrity and function were observed between the two storage conditions. Cryopreservation abolished reactivity to extracellular stimuli and severely affected the cell phenotype. On the other hand, functional activity could be maintained for up to 72 h when in vivo primed granulocytes of patients were incubated at 4 degrees C in the presence of G-CSF. This storage modality may permit the use of granulocyte autotransfusion to reduce the risk of neutropenic fever.

Antigens, Surface↗
PubMed1993

Expression of functional papain precursor in Saccharomyces cerevisiae: rapid screening of mutants.

A microbial expression system for the study of the cysteine protease papain has been developed as a more useful alternative to the insect cell/baculovirus expression system we have previously used. A synthetic papain precursor (propapain) gene was expressed in the yeast Saccharomyces cerevisiae under the control of the alpha-factor promoter. Efficient expression required fusion of the propapain sequence with the yeast alpha-factor prepro region and a yeast host cell defective in the synthesis of vacuolar proteases. Surprisingly, the glycosylated form of the inactive papain precursor is not secreted, but accumulates within the yeast cell. Complete conversion of the intracellular zymogen into active mature papain could be achieved in vitro. Purified recombinant papain produced by the yeast system has kinetic characteristics similar to those of the natural enzyme. An advantage of the yeast expression system over the baculovirus/insect cell system is that we can perform mutagenesis and screening of papain mutants very efficiently. We have set up a 'one-tube' screening procedure for the simultaneous characterization of numerous mutants of the papain precursor. Yeast cells are grown and lysed in microtiter plate wells and the released papain precursor is then activated to mature papain. This assay allows easy discrimination between proteins with close to wild type properties and proteins that are not functional. We have applied this assay to investigate the spectrum of amino acids which are tolerated at Asn175 of papain using two independently derived libraries of mutants at this position. Many amino acid substitutions at this position are not accepted; only the reintroduction of Asn restored normal function.

Amino Acid Sequence↗
PubMed1999

Definition of dendritic cell subpopulations present in the spleen, Peyer's patches, lymph nodes, and skin of the mouse.

Dendritic cells (DC) are highly efficient antigen-presenting cells (APC) that have an essential function in the development of immune responses against microbial pathogens and tumors. Although during the past few years our understanding of DC biology has remarkably increased, a precise characterization of the different DC subpopulations remains to be achieved with regard to their phenotype and lineage relationships. In this report, we have extensively studied the DC subpopulations present in the thymus, spleen, Peyer's patches, lymph nodes (LN) and skin of the mouse. Thymus DC and 60% spleen DC have a lymphoid DC phenotype, ie, CD8(+) DEC-205(high) Mac-1(low), whereas 40% spleen DC have a myeloid DC phenotype, ie, CD8(-) DEC-205(low) Mac-1(high). Both CD8(+) and CD8(-) DC are leukocyte function-associated antigen-1 (LFA-1)high and highly adherent. Within Peyer's patches the majority of DC correspond to the CD8(+) DEC-205(high) Mac-1(low) lymphoid category. In the LN, together with CD8(+) and CD8(-) DC, an additional nonadherent CD8(int) LFA-1(int) subpopulation with lymphoid DC characteristics is described. Finally, in the skin both epidermal Langerhans cells (LC) and dermal DC are CD8(-)DEC-205(high) Mac-1 (high), and do not express LFA-1. Interestingly, LC migration experiments indicate that LC underwent the upregulation of CD8 and LFA-1 upon migration to the LN, supporting the hypothesis that LC belong to the CD8(+) lymphoid lineage.

Animals↗
PubMed2002

Genomic functional annotation using co-evolution profiles of gene clusters.

BACKGROUND: The current speed of sequencing already exceeds the capability of annotation, creating a potential bottleneck. A large proportion of the genes in microbial genomes remains uncharacterized. Here we propose a new method for functional annotation using the conservation patterns of gene clusters. If several gene clusters show the same coevolution pattern across different genomes it is reasonable to infer they are functionally related. The gene cluster phylogenetic profile integrates chromosomal proximity information and phylogenetic profile information and allows us to infer functional dependences between the gene clusters even at great distance on the chromosome. RESULTS: As a proof of concept, we applied our method to the genome of Escherichia coli K12 strain. Our method establishes functional relationships among 176 gene clusters, comprising 738 E. coli genes. The accuracy of pair phylogenetic profiles was compared with the single-gene phylogenetic profile and was shown to be higher. As a result, we are able to suggest functional roles for several previously unknown genes or unknown genomic regions in E. coli. We also examined the robustness of coevolution signals across a larger set of genomes and suggest a possible upper limit of accuracy for the phylogenetic profile methods. CONCLUSIONS: The higher-order phylogenetic profiles, such as the gene-pair phylogenetic profiles, can detect functional dependences that are missed by using conventional single-gene phylogenetic profile or the chromosomal proximity method only. We show that the gene-pair phylogenetic profile is more accurate than the single-gene phylogenetic profiles.

Alleles↗