PCR-based rapid genotyping of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis>isolates

Background  

All bacterial genomes contain repetitive sequences which are members of specific DNA families. Such repeats may occur as single units, or found clustered in multiple copies in a head-to-tail configuration at specific loci. The number of clustered units per locus is a strain-defining parameter. Assessing the length variability of clusters of repeats is a versatile typing methodology known as multilocus variable number of tandem repeat analysis (MLVA).     

A comparison of sugar indicators enables a universal high-throughput sugar-1-phosphate nucleotidyltransferase assay

A systematic comparison of six sugar indicators for their sensitivity, specificity, cross-reactivity, and suitability in the context of crude lysates revealed para-hydroxybenzoic acid hydrazide (pHBH) to be best suited for application in a plate-based phosphatase-assisted universal sugar-1-phosphate nucleotidyltransferase assay. The addition of a general phosphatase to nucleotidyltransferase reaction aliquots enabled the conversion of remaining sugar-1-phosphate to free sugar, the concentration of which could be rapidly assessed via the pHBH assay. The assay was validated using the model glucose-1-phosphate thymidylyltransferase from Salmonella enterica (RmlA) and compared favorably with a previously reported HPLC assay. This coupled discontinuous assay is quantitative, high throughput, and robust; relies only on commercially available enzymes and reagents; does not require chromatography, specialized detectors (e.g., mass or evaporative light scattering detectors), or radioisotopes; and is capable of detecting less than 5 nmol of sugar-1-phosphate. It is anticipated that this high-throughput assay system will greatly facilitate nucleotidyltransferase mechanistic and directed evolution/engineering studies.     

Proteolysis in miniature cheddar-type cheeses manufactured using extracts from the crustacean Munida as coagulant

Miniature (20 g) Cheddar-type cheeses were manufactured using enzymes extracted from the crustacean Munida or chymosin as coagulant. Cheeses were ripened at 8 degrees C and samples were collected for analysis after 2, 6 and 12 weeks. Proteolysis was assessed by urea-polyacrylamide gel electrophoresis, which showed that cheeses manufactured with the Munida extracts had a higher extent of degradation of beta-casein than cheeses made using chymosin as coagulant. Patterns of proteolysis were also obtained by reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption ionisation-time of flight (MALDI-ToF) mass spectrometry. In general, the products of proteolysis were more complex in cheese made using the Munida extracts than in cheese made by chymosin as coagulant. Statistical analysis of results clearly discriminated the cheeses on the basis of coagulant used. Molecular mass of peptides found in cheese made using Munida extracts were similar to those of peptides commonly detected in cheeses made using chymosin as coagulant.     

Redox options in two-dimensional electrophoresis

Summary. Two-dimensional electrophoresis is usually run on fully reduced samples. Under these conditions even covalently bound oligomers are dissociated and individual polypeptide chains may be fully unfolded by both, urea and SDS, which maximizes the number of resolved components and allows their pI and M_r to be most accurately evaluated. However, various electrophoretic protocols for protein structure investigation require a combination of steps under varying redox conditions. We review here some of the applications of these procedures. We also present some original data about a few related samples – serum from four species: Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus – which we run under fully unreduced and fully reduced conditions as well as with reduction between first and second dimension. We demonstrate that in many cases the unreduced proteins migrate with a better resolution than reduced proteins, mostly in the crowded -globulin area of pI 4.5–6 and M_r 50–70kDa.     

Immunohistochemical localisation of PDE5 in Leydig and myoid cells of prepuberal and adult rat testis

Expression of phosphodiesterase 5 (PDE5) in the rat testis at several pre and postnatal developmental stages was investigated by immunohistochemistry. The enzyme was localised in vascular smooth muscle cells, as well as in Leydig and peritubular cells. The latter were identified as myoid, based on their immunoreactivity to desmin and α-smooth muscle actin. The presence of PDE5 in myoid cells was confirmed by Western blot analysis and immunohistochemistry performed on highly purified cell fractions, obtained from 16-day-old rats. The expression of PDE5 in these somatic cells of rat testis is discussed in view of the roles played by cGMP signal transduction pathways in the mammalian male reproductive function.     

CD11c+ dendritic cells are required for survival in murine polymicrobial sepsis

CD11c+ dendritic cells (DCs) are APCs that link innate and adaptive immunity. Although DCs are lost from spleen and lymph nodes in sepsis, their role in outcome remains unclear. Transgenic mice (B6.FVB-Tg(.Itgax-DTR/EGFP.57)Lan/J) expressing the diphtheria toxin (DT) receptor on the CD11c promoter (DCKO mice) received 4 ng/kg DT, which resulted in depletion of 88-95% of mature myeloid and lymphoid DCs, with less depletion (75%) of plasmacytoid DCs. Pretreatment of DCKO mice with DT resulted in reduced survival in sepsis compared with saline-pretreated DCKO mice (0 vs 54%; p < 0.05) or DT-treated wild-type littermates (0 vs 54%; p < 0.05). This increased mortality was not associated with either increased bacteremia or plasma cytokine concentrations. Intravenous injection of 10(7) wild-type DCs improved survival in DCKO mice (42 vs 0%; p = 0.05). These data confirm that DCs are essential in the septic response and suggest that strategies to maintain DC numbers or function may improve outcome.     

Impaired assembly results in the accumulation of multiple HLA-C heavy chain folding intermediates

Class I MHC H chains assemble with beta2-microglobulin (beta2m) and are loaded with peptide Ags through multiple folding steps. When free of beta2m, human H chains react with Abs to linear epitopes, such as L31. Immunodepletion and coimmunoprecipitation experiments, performed in this study, detected a preferential association of L31-reactive, beta2m-free H chains with calnexin in beta2m-defective cells, and with calreticulin and TAP in beta2m-expressing cells. In beta2m-defective cells, the accumulation of calnexin-bound H chains stoichiometrically exceeded their overall accumulation, a finding that supports both chaperoning preferences and distinct sorting abilities for different class I folds. No peptide species, in a mass range compatible with that of the classical class I ligands, could be detected by mass spectrometry of acidic eluates from L31-reactive HLA-Cw1 H chains. In vitro assembly experiments in TAP-defective T2 cells, and in cells expressing an intact Ag-processing machinery, demonstrated that L31 H chains are not only free of, but also unreceptive to, peptides. L31 and HC10, which bind nearly adjacent linear epitopes of the alpha1 domain alpha helix, reciprocally immunodepleted free HLA-C H chains, indicating the existence of a local un-/mis-folding involving the N-terminal end of the alpha1 domain alpha helix and peptide-anchoring residues of the class I H chain. Thus, unlike certain murine free H chains, L31-reactive H chains are not the immediate precursors of conformed class I molecules. A model inferring their precursor-product relationships with other known class I intermediates is presented.     

Kinetics of fibrinopeptide release by thrombin as a function of CaCl2 concentration: different susceptibility of FPA and FPB and evidence for a fibrinogen isoform-specific effect at physiological Ca2+ concentration

The kinetics of release of fibrinopeptide A (FPA) and B (FPB) by thrombin were investigated on unfractionated fibrinogen samples as a function of CaCl(2) concentration. A 50 mM Tris, 104 mM NaCl, pH 7.4 (TBS) buffer, to which 1 mM EDTA-Na(2) (TBE) or 2.5 (TBC2.5), 14 (TBC14), and 30 mM CaCl(2) (TBC30) was alternatively added, was employed. The % FPA versus time curves were fitted with single stretched-exponential growth functions, where the stretch parameter beta likely reflects substrate polydispersity (beta = 1, monodisperse). For TBE, TBS, TBC14, and TBC30, we found beta approximately 1, with corresponding normalized rate constants (K(a)) of 3.8, 4.2, 2.7, and 1.9 x 10(-5) [(NIHu/L)s](-1). Surprisingly, in TBC2.5 we found beta = 0.69, with an "average" K(a) of 3.5 x 10(-5) [(NIHu/L)s](-1). This effect disappeared [beta = 0.97, K(a) = 2.7 x 10(-5) [(NIHu/L)s](-1)] with an increase in the ionic strength I to that of TBC30 with 186 mM NaCl (TBCaNa buffer). FPB releases were instead consistent with a nonstretched consecutive exponential growth function, except in TBC30 where some FPB appeared to be cleaved independently. Log-log plots of K(a) versus Ca(2+) concentration, Cl(-) concentration, or I showed a strong linear correlation with only the latter two except in TBCaNa, again suggesting specific effects of the physiological Ca(2+) concentration and I on FPA release. The corresponding K(b) plots showed instead that both total depletion and high Ca(2+) hampered FPB release. To further investigate the TBC2.5 beta = 0.69 effect, FG polydispersity was assessed by Western blot analyses. The thrombin-binding gamma'-chain isoform was approximately 4%, resulting in a bound:free thrombin ratio of approximately 25:75. With regard to the C-terminal ends of the Aalpha-chains, approximately 45% were either intact or lightly degraded, while the remaining approximately 55% were more degraded. Fitting the % FPA release data in TBC2.5 with a sum of two exponentials resulted in a faster component and a slower component (K(a1)/K(a2) approximately 6), with a ratio of approximately 48:52. While a role for the gamma'-chain isoform cannot be excluded, this good correlation with the C-terminal degradation of the Aalpha-chains suggests their calcium-dependent involvement in FPA release.