Voltage-activated K(+) (K(V)) channels play an important role in regulating the membrane potential in excitable cells. In gastrointestinal (GI) smooth muscles, these channels are particularly ...important in modulating spontaneous electrical activities. The purpose of this study was to identify the molecular components that may be responsible for the K(V) currents found in the canine GI tract. In this report, we have examined the qualitative expression of eighteen different K(V) channel genes in canine GI smooth muscle cells at the transcriptional level using RT-PCR analysis. Our results demonstrate the expression of K(V)1.4, K(V)1.5, K(V)1.6, K(V)2.2, and K(V)4.3 transcripts in all regions of the GI tract examined. Transcripts encoding K(V)1.2, K(V)beta1.1, and K(V)beta1.2 subunits were differentially expressed. K(V)1.1, K(V)1.3, K(V)2.1, K(V)3.1, K(V)3.2, K(V)3.4, K(V)4.1, K(V)4.2, and K(V)beta2.1 transcripts were not detected in any GI smooth muscle cells. We have also determined the protein expression for a subset of these K(V) channel subunits using specific antibodies by immunoblotting and immunohistochemistry. Immunoblotting and immunohistochemistry demonstrated that K(V)1.2, K(V)1.4, K(V)1.5, and K(V)2.2 are expressed at the protein level in GI tissues and smooth muscle cells. K(V)2.1 was not detected in any regions of the GI tract examined. These results suggest that the wide array of electrical activity found in different regions of the canine GI tract may be due in part to the differential expression of K(V) channel subunits.
Scanning alanine mutagenesis has been used to study the structural determinants of several activities of bothropstoxin-I (BthTx-I), a lysine 49 Phospholipases A(2) from the venom of Bothrops ...jararacussu. A total of 31 mutants were generated in the interfacial recognition site and C-terminal loop regions of the protein. The effects of mutagenesis on the in vivo myotoxic activity, the cytolytic activity against cultured C2C12 myoblasts, the bactericidal activity, and the Ca(2+)-independent membrane damaging activity against liposome membranes were compared. Residues 116-119 and 122-125 in the C-terminal loop region are structural determinants for these activities, indicating that membrane permeabilization by the BthTx-I is an important general property in all the measured effects. The structural determinants of myotoxicity and myoblast membrane permeabilization are highly correlated, demonstrating that cultured C2C12 myoblasts are a good model for the myotoxic effect. However, comparison of the structural determinants for all activities revealed several differences in the structural determinants between the effects against myoblast and bacterial membranes, and further differences when compared to the liposome membrane damaging effect. These membrane dependent effects are interpreted to be the consequence of differences in the activation of the membrane bound form of the protein on biological and artificial membranes.
Calreticulin (CRT) is located predominantly in the endoplasmic reticulum (ER) of cells, where it functions as a quality control controller of protein folding. However, CRT is also a prevalent ...autoantigen in patients with systemic lupus erythematosus (SLE), where its release from the cell may arise as a results of dysfunctional apoptosis and inefficient removal of ER vesicles, which are an abundant source of CRT and other autoantigens. Indicative of this is the presence of autoantibodies against CRT in the sera of 40−60% of all SLE patients. Once released into the circulation, CRT might bind directly to C1q and we have suggested that this association may result in a defect in C1q-mediated clearance of antigen−antibody complexes. It has been previously shown that CRT under physiological salt conditions binds to the globular head of C1q. It is known that the globular head region of C1q binds to the CH2 domain in the Fc portion of immunoglobulin γ (IgG). The N-terminal half of CRT contains a number of short regions of 7−10 amino acids that show sequence similarity to the putative C1q binding region in the CH2 domain of IgG. By use of a series of 92 overlapping CRT synthetic peptides, a number of C1q binding sites on the CRT molecule have been identified, including several containing a CH2 -like motif similar to the ExKxKx C1q binding motif found in the CH2 domain of IgG. A number of these peptides were shown to inhibit binding of C1q to IgG and reduce binding of native CRT to C1q. Moreover, several of the peptides were capable of inhibiting the classical pathway of complement activation. These studies have identified specific binding sites on the CRT molecule for C1q and lend support to the hypothesis that interaction of CRT with C1q may interfere with the ability of C1q to associate with immune complexes in autoimmune-related disorders.
Voltage-activated K+ (KV) channels play an important role in regulating the membrane potential in excitable cells. In gastrointestinal (GI) smooth muscles, these channels are particularly important ...in modulating spontaneous electrical activities.
•Ongoing endothelial activation is common in SCD children despite disease-modifying therapy with hydroxycarbamide or blood transfusion.•Persistent VWF-ADAMTS13 axis dysfunction identifies a sub-group ...of treated SCD children at increased risk for vaso-occlusive complications.
Previous studies have reported elevated von Willebrand factor (VWF) levels in patients with sickle cell disease (SCD) and demonstrated a key role for the VWF-ADAMTS13 axis in the pathobiology of SCD vaso-occlusion. Although blood transfusion is the gold standard for stroke prevention in SCD, the biological mechanisms underpinning its improved efficacy compared to hydroxycarbamide are not fully understood. We hypothesized that the improved clinical efficacy of blood transfusion might relate to differences in endothelial cell activation and VWF-ADAMTS13 axis dysfunction. One-hundred-eighty children with a confirmed diagnosis of SCD (HbSS) on hydroxycarbamide (n=96) or blood transfusion (n=84) were included. Despite disease-modifying treatment, plasma VWF and VWF propeptide were elevated in a significant proportion of SCD children (33% and 47% respectively). Crucially, all VWF parameters were significantly higher in the hydroxycarbamide compared to the blood transfusion cohort (p<0.05). Additionally, increased levels of other Weibel-Palade-body-stored proteins, including factor VIII (FVIII), angiopoietin-2, and osteoprotegerin were observed, indicated ongoing endothelial cell activation. Children treated with hydroxycarbamide also had higher FVIII activity and enhanced thrombin generation compared to the blood transfusion cohort (p<0.001), contributing to their thrombotic risk. Finally, hemolysis markers strongly correlated with VWF levels (p<0.001) and significantly reduced in the blood transfusion cohort (p<0.001). Cumulatively, our findings demonstrate for the first time that despite treatment, ongoing dysfunction of the VWF-ADAMTS13 axis is present in a significant sub-group of pediatric SCD patients, especially those treated with hydroxycarbamide. Since VWF plays an important role in vaso-occlusion pathogenesis, these data are of direct translational relevance.
The metabolic activity of H. influenzae can be followed quantitatively by measurement of the nitrite produced in a medium containing 0.2 per cent potassium or sodium nitrate. When X-factor, or hemin, ...and other specific substances required for the optimum growth of H. influenzae, are present in excess, the nitrite produced by this organism is quantitatively related to the concentration of V-factor, or total coenzyme. This quantitative relationship has been demonstrated for five strains of H. influenzae. It has been shown that various media, which in the past have been used for the determination of coenzyme by growth of H. influenzae, have in many instances been deficient in X-factor and that this substance rather than coenzyme has been the specific factor limiting growth. When 0.5 per cent blood is added to a basal proteose-peptone medium the specific requirements for optimum growth and metabolic activity of H. influenzae, other than coenzyme, are met, and a large number of specific biocatalysts and nutritive substances added to this medium are without effect in stimulating further growth. The foregoing studies have formed the basis for a quantitative method for the determination of total coenzyme in blood and tissue. This method is being described elsewhere.
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