T-cell development: a role for self-peptides in positive selection.

An important issue for immunologists is the difference between the two main processes that determine the mature repertoire of T-cell receptors, termed positive and negative selection. Recent papers have addressed the role of self-peptides in the process of positive selection.     

Isolation, identification and characterization of fluoride resistant bacteria: Possible role in bioremediation

Microorganisms found in industrial effluents and near the sites of the contamination can be used to indicate pollution and detoxify the contaminated water resources. Emergence of xenobiotic resistant bacteria among them might be potential application in bioremediation. The objective of this study was to isolate and characterize fluoride resistant bacteria from soil and water samples of different regions of India. Five isolates were recovered from different samples which were found to be fluoride resistant. Two of them effectively reduced the fluoride from their media. Through the current study it can be predicted that fluoride pollution results in selective pressure that leads to the development of fluoride resistant among bacterial populations, probably through the mechanism which involved high affinity anion binding compounds called ionophores. Resistant microbes may play a bioremediative role by transforming and concentrating these anions so that they are less available and less dangerous.     

Isolation, identification and characterization of fluoride resistant bacteria: possible role in bioremediation

Microorganisms found in industrial effluents and near the sites of the contamination can be used to indicate pollution and detoxify the contaminated water resources. Emergence of xenobiotic resistant bacteria among them might be potential application in bioremediation. The objective of this study was to isolate and characterize fluoride resistant bacteria from soil and water samples of different regions of India. Five isolates were recovered from different samples which were found to be fluoride resistant. Two of them effectively reduced the fluoride from their media. Through the current study it can be predicted that fluoride pollution results in selective pressure that leads to the development of fluoride resistant among bacterial populations, probably through the mechanism which involved high affinity anion binding compounds called ionophores. Resistant microbes may play a bioremediative role by transforming and concentrating these anions so that they are less available and less dangerous.     

MICE: multiple-peak identification, characterization, and estimation

MICE--multiple-peak identification, characterization, and estimation--is a procedure for estimating a lower bound of the number of frequency peaks and for estimating the frequency peak parameters. The leading application is protein structure determination using nuclear magnetic resonance (NMR) experiments. NMR frequency data are multiple-peak data, where each frequency peak corresponds to two connected atoms in the three-dimensional protein structure. We analyze the NMR frequency data through a series of steps: a preliminary step for separating the signal from the background followed by identification of local maxima up to a noise-level-dependent threshold, estimation of the frequency peak parameters using an iterative algorithm, and detection of mixtures of peaks using hypothesis testing.     

Heavy membrane-associated caspase 3: identification, isolation, and characterization

Heavy membrane preparations from 697 lymphoblastoid cells contain a tightly bound caspase zymogen. This heavy membrane-bound procaspase can be efficiently liberated from membrane preparations using detergents. Alternatively, the procaspase can be rapidly processed and activated from membrane preparations by caspase-1 without detergents. The activated caspase-3 was purified using affinity chromatography and characterized by amino acid sequencing and inhibitor specificity analysis. The sequence indicates that this heavy membrane bound caspase is caspase-3. The kinetic properties and inhibitor binding specificity also show that this purified caspase is enzymologically indistinguishable from cytoplasmic or recombinant caspase-3. However, the N-termini of activated heavy membrane-bound and cytoplasmic caspase-3 are slightly different; peptide sequencing data indicate that the heavy membrane caspase-3 begins at Lys 14, whereas the cytoplasmic enzyme begins at Ser 10. Implications of this structural difference are discussed.     

Exosomes in tears of healthy individuals: Isolation,identification, and characterization

Exosomes represent a sort of extracellular vesicles, which transfer molecular signals in the body and contain markers of the exosome-producing cell. This study was aimed at search of exosomes in the tears of healthy humans, validation of their nature and examination of their morphological and molecular-biological characteristics. Samples of the tears individually collected from 24 healthy donors (aged 45–60 years) were centrifuged at 20000 g for 15 min to pellet cell debris. The supernatants were examined in an electron microscope using negative staining and were also used for isolation and purification of exosomes by filtration (100 nm pore-size) and double ultracentrifugation (90 min at 100000 g, 4°C). Resultant pellets were investigated by electron microscopy and immunolabeling, RNA and DNA were isolated and their sizes were determined by capillary electrophoresis, concentration and localization of nucleic acids in the isolated exosomes were studied. DNA sequencing was performed using MiSeq (Illumina, USA), data were analyzed using CLC GW 7.5 (Qiagen, USA). Sequences were mapped on human genome (hg19). Supernatants of the tears contained cell debris, spherical microparticles (20–40 nm), and vesicles; some of the vesicles had morphology and sizes corresponding to exosomes. The pellets obtained after ultracentrifugation of tears contained microparticles (17%), spherical and cup-shaped vesicles (40–100 nm, 83%), which were positive for CD63, CD9, and CD24 receptors (specific markers of exosomes). The study revealed high concentrations of exosomes in human tears; these exosomes contained both RNA (of less than 200 nucleotides in size) and DNA (of 3–9 kb in size). DNA sequencing demonstrated that about 92% of the reads was mapped to human genome.     

Dynamical characterization of two differentially disease associated MHC class I proteins in complex with viral and self-peptides

Major histocompatibility complex (MHC) class I proteins are expressed on the cell surface where they present foreign and self-peptides to effector cells of the immune system. While an understanding of the structural prerequisites for antigen presentation has already been achieved, insight into subtype- or peptide-dependent dynamical characteristics of a peptide–MHC antigen is so far largely obscure. We approached this problem by employing 400-ns molecular dynamics simulations with two human MHC class I subtypes as model systems: the ankylosing spondylitis-associated HLA-B127:05 and the non-ankylosing spondylitis-associated HLA-B127:09. Both proteins differ only by a micropolymorphism at the floor of the peptide binding groove (Asp116His). A viral (pLMP2) and three self-peptides (pVIPR, pGR, and TIS) were evaluated. The stability of the binding grooves was found to be both subtype dependent and peptide dependent. A detachment from the C- and/or N-terminal pockets was observed for all peptides except TIS, resulting in a stabilization of the α1-helix in both TIS-displaying subtypes. Estimates of the entropy associated with the bound peptides showed an increased entropy for pLMP2 presented by B127:05 as compared to B127:09, in contrast to the self-peptides. Additionally, the flexibility of the α1-helix that is probably important for receptor binding to the B27:peptide epitope is significantly enhanced for B127:05. These in silico results show that the dynamic properties of peptide–MHC complexes are affected both by the bound peptide and by micropolymorphisms of the heavy chain. Our findings suggest a role for the conformational flexibility of MHC class I molecules in the context of recognition by receptors on effector cells.     

Interleukin 20: discovery, receptor identification, and role in epidermal function

A structural, profile-based algorithm was used to identify interleukin 20 (IL-20), a novel IL-10 homolog. Chromosomal localization of IL-20 led to the discovery of an IL-10 family cytokine cluster. Overexpression of IL-20 in transgenic (TG) mice causes neonatal lethality with skin abnormalities including aberrant epidermal differentiation. Recombinant IL-20 protein stimulates a signal transduction pathway through STAT3 in a keratinocyte cell line, demonstrating a direct action of this ligand. An IL-20 receptor was identified as a heterodimer of two orphan class II cytokine receptor subunits. Both receptor subunits are expressed in skin and are dramatically upregulated in psoriatic skin. Taken together, these results demonstrate a role in epidermal function and psoriasis for IL-20, a novel cytokine identified solely by bioinformatics analysis.     

Secretin in the rat hypothalamo-pituitary system: Localization,identification and characterization

Secretin-like immunoreactivity (SLI) has been identified and characterized in the pituitary of the rat. The concentration in the neurointermediate lobe is about 45 fold higher than the concentration of SLI observed in the anterior lobe. Transections of the pituitary stalk of the rat caused a significant depletion of SLI in the neurointermediate lobe without affecting the content in the anterior lobe. In view of the relatively high concentration of SLI reported to occur in the hypothalamus, it appears that there may be a secretinergic pathway between the brain and the neurointermediate lobe of the pituitary.     

Yeasts from kefir grains: isolation, identification, and probiotic characterization

Kefir—a traditional beverage whose consumption has been associated with health benefits—is a logical natural product to investigate for new probiotic strains. The aim of the present work was to isolate and identify kefir yeasts and select those with acid and bile tolerance to study their adhesion to epithelial cells and their transit through mouse gut. From 4 milky and 3 sugary kefir grains, 34 yeast strains were isolated and identified by means of classical microbiological and molecular-genetic methods (whole-cell protein pattern, internal-transcribed-spacer amplification, and analysis of restriction-fragment–length polymorphisms). We identified 4 species belonging to 3 genera—Saccharomyces cerevisiae (15 strains), Saccharomyces unisporus (6 strains), Issatchenkia occidentalis (4 strains), and Kluyveromyces marxianus (9 strains)—and selected 13 strains on the basis of resistance to low pH and bile salts. Among the strains selected, Kluyveromyces marxianus CIDCA 8154 and Saccharomyces cerevisiae CIDCA 8112 were further studied. Both strains evidenced the capacity to adhere to epithelial intestine-derived cells in vitro and to survive passage through the gastrointestinal tract of BALB/c mice. The investigation of the potential probiotic features of these kefir-yeast strains should be useful for the development of novel functional foods.     

Ecdysteroids in nemerteans: further characterization and identification

Ecdysteroids are a class of steroidal hormones thatare important in molting and reproduction inarthropods. These hormones have been recentlydetected in non-arthropodan groups, such assoft-bodied worms. To continue our efforts todetermine the presence of ecdysteroids in nemerteans,this study further documents the identification of20-hydroxyecdysone (20E), in Paranemertesperegrina, by gas chromatography/mass spectrometry(GC/MS). After C₁₈ Sep-Pak extraction,radioimmunoassay of the 40% and 80% methanolicfractions from an extract of 1000 worms indicated 98and 156 ng of ecdysteroids, respectively. Ecdysteroids of these two samples, as well as the 20Eand ecdysone standards, were N-trimethylsilyimidazole(TMSI)-derivatized before GC/MS analysis. Themethanolic samples contained a large number ofcompounds but only small, insignificant peaks in thearea where ecdysteroid standards eluted. However, thereconstruction ion current (RIC) chromatograms for m/z561 indicated the presence of 20E at the correctretention time of 12.48 min in the 40% methanolicfraction. Reanalysis of the samples under selectedion monitoring mode demonstrated the presence of 20Ein both the 40% and 80% fractions. These resultsdemonstrate conclusively the presence of an activeecdysteroid in the phylum Nemertea.     

Patients, pathogens, and protective immunity: the relevance of virus-induced alloreactivity in transplantation

Successful transplantation requires the establishment of an ongoing state in which there is simultaneous inhibition of the undesired T cell-dependent rejection response and yet retention of the ability to develop effective cell-mediated primary and memory responses to pathogens. The complexity of attaining such a precarious state is underscored by the growing body of evidence that alloreactivity can be profoundly influenced by infections that occur before, concurrent with, or subsequent to an organ transplant. In this review, we explore the growing list of mechanisms that have been identified by which pathogen-host interactions might influence rejection, including the degeneracy of TCR recognition leading to cross-reactive immune responses, the effects of pathogens on innate immune mechanisms, and the potential impact of virally induced lymphopenia.     

Critical role for T cells in Sephadex-induced airway inflammation: pharmacological and immunological characterization and molecular biomarker identification

Intratracheal instillation of Sephadex particles is a convenient model for assessing the impact of potential anti-inflammatory compounds on lung eosinophilia thought to be a key feature in asthma pathophysiology. However, the underlying cellular and molecular mechanisms involved are poorly understood. We have studied the time course of Sephadex-induced lung eosinophilia, changes in pulmonary T cell numbers, and gene and protein expression as well as the immunological and pharmacological modulation of these inflammatory indices in the Sprague Dawley rat. Sephadex increased T cell numbers (including CD4(+) T cells) and evoked a pulmonary eosinophilia that was associated with an increase in gene/protein expression of the Th2-type cytokines IL-4, IL-5, and IL-13 and eotaxin in lung tissue. Sephadex instillation also induced airway hyperreactivity to acetylcholine and bradykinin. A neutralizing Ab (R73) against the alphabeta-TCR caused 54% depletion of total (CD2(+)) pulmonary T cells accompanied by a significant inhibition of IL-4, IL-13 and eotaxin gene expression together with suppression (65% inhibition) of eosinophils in lung tissue 24 h after Sephadex treatment. Sephadex-induced eosinophilia and Th2 cytokine gene and/or protein expression were sensitive to cyclosporin A and budesonide, compounds that inhibit T cell function, suggesting a pivotal role for T cells in orchestrating Sephadex-induced inflammation in this model.     

Pea chloroplast topoisomerase I: purification,characterization, and role in replication

A DNA-relaxing enzyme was purified 5 000-fold to homogeneity from isolated chloroplasts of Pisum sativum. The enzyme consists of a single polypeptide of 112 kDa. The enzyme was able to relax negatively supercoiled DNA in the absence of ATP. It is resistant to nalidixic acid and novobiocin, and causes a unit change in the linkage number of supercoiled DNA. The enzyme shows optimum activity at 37°C with 50 mM KCl and 10 mM MgCl₂. From these properties, the enzyme can be classified as a prokaryotic type I topoisomerase.Using a partiall purified pea chloroplast DNA polymerase fraction devoid of topoisomerase I activity for in vitro replication on clones containing the pea chloroplast DNA origins of replication, a 2–6-fold stimulation of replication activity was obtained when the purified topoisomerase I was added to the reaction at 70–100 mM KCl. However, when the same reaction was carried out at 125 mM KCl, which does not affect DNA polymerase activity on calf thymus DNA but is completely inhibitory for topoisomerase I activity, a 4-fold drop in activity resulted. Novobiocin, an inhibitor of topoisomerase II, was not found to inhibit the in vitro replication of chloroplast DNA.     

The p55 TNF-alpha receptor plays a critical role in T cell alloreactivity

TNF-alpha is known to be an important mediator of tissue damage during allograft rejection and graft-vs-host disease (GVHD), but its role in supporting T cell responses to allogeneic Ags is unclear. We have studied this question by comparing normal mice with those lacking the p55 (p55 TNFR-/-) or p75 (p75 TNFR-/-) TNF-alpha receptors as donors in well-defined bone marrow transplant (BMT) models. Recipients of p55 TNFR-/- cells had significantly reduced mortality and morbidity from GVHD compared with the other two sources of T cells. In vitro, T cells lacking the p55 (but not the p75) TNF-alpha receptor exhibited decreased proliferation and production of Th1 cytokines in MLC. This defect was only partially restored by exogenous IL-2 and affected both CD4+ and CD8+ populations. CD8+ p55 TNFR-/- proliferation was impaired independently of IL-2 whereas CTL effector function was impaired in an IL-2-dependent fashion. Inhibition of TNF-alpha with TNFR:Fc in primary MLC also impaired the proliferation and Th1 differentiation of wild-type T cells. BMT mixing experiments demonstrated that the reduced ability of p55 TNFR-/- donor cells to induce GVHD was due to the absence of the p55 TNFR on T cells rather than bone marrow cells. These data highlight the importance of TNF-alpha in alloreactive T cell responses and suggest that inhibition of the T cell p55 TNF-alpha receptor may provide an additional useful therapeutic maneuver to inhibit alloreactive T cell responses following bone marrow and solid organ transplantation.     

c-kit protein, a transmembrane kinase: identification in tissues and characterization.

The proto-oncogene c-kit encodes a transmembrane kinase which is related to the receptors for colony-stimulating factor type 1 and platelet-derived growth factor, as well as to the immunoglobulin superfamily. Antibodies specific for the kinase domain of the P80 gag-kit protein of the Hardy-Zuckerman 4 feline sarcoma virus were prepared. These kit-specific antibodies were used to identify and characterize the c-kit protein in cat brain tissue. The c-kit protein product displays an autophosphorylating activity in immune complex kinase assays, and, in turn, this activity was used to identify the c-kit protein in different tissues. In cat brain, a single 145-kilodalton (kDa) glycoprotein was detected. Its N-linked carbohydrates were found to be sensitive to digestion with the endoglycosidases (neuraminidase, endoglycosidase F, and endoglycosidase H), indicating hybrid and/or complex and high-mannose structures. A partial purification of the c-kit protein was achieved by wheat germ agglutinin affinity chromatography, and the autophosphorylating activity of the partially purified c-kit protein was characterized and found to be specific for tyrosine. The kit antibodies cross-react with the murine c-kit protein product, and variant c-kit proteins in different mouse tissues were identified, with sizes of about 145 kDa (brain), 160 kDa (spleen), and 150 kDa (testis).