K+ channel KVLQT1 located in the basolateral membrane of distal colonic epithelium is not essential for activating Cl- secretion

The cellular mechanism for Cl^– and K⁺ secretion in the colonic epithelium requires K⁺ channels in the basolateral and apical membranes. Colonic mucosa from guinea pig and rat were fixed, sectioned, and then probed with antibodies to the K⁺ channel proteins K_VLQT1 (Kcnq1) and minK-related peptide 2 (MiRP2, Kcne3). Immunofluorescence labeling for Kcnq1 was most prominent in the lateral membrane of crypt cells in rat colon. The guinea pig distal colon had distinct lateral membrane immunoreactivity for Kcnq1 in crypt and surface cells. In addition, Kcne3, an auxiliary subunit for Kcnq1, was detected in the lateral membrane of crypt and surface cells in guinea pig distal colon. Transepithelial short-circuit current (I_sc) and transepithelial conductance (G_t) were measured for colonic mucosa during secretory activation by epinephrine (EPI), prostaglandin E₂ (PGE₂), and carbachol (CCh). HMR1556 (10 µM), an inhibitor of Kcnq1 channels (Gerlach U, Brendel J, Lang HJ, Paulus EF, Weidmann K, Brüggemann A, Busch A, Suessbrich H, Bleich M, and Greger R. J Med Chem 44: 3831–3837, 2001), partially (50%) inhibited Cl^– secretory I_sc and G_t activated by PGE₂ and CCh in rat colon with an IC₅₀ of 55 nM, but in guinea pig distal colon Cl^– secretory I_sc and G_t were unaltered. EPI-activated K⁺-secretory I_sc and G_t also were essentially unaltered by HMR1556 in both rat and guinea pig colon. Although immunofluorescence labeling with a Kcnq1 antibody supported the basolateral membrane presence in colonic epithelium of the guinea pig as well as the rat, the Kcnq1 K⁺ channel is not an essential component for producing Cl^– secretion. Other K⁺ channels present in the basolateral membrane presumably must also contribute directly to the K⁺ conductance necessary for K⁺ exit during activation of Cl^– secretion in the colonic mucosa. HMR1556; K⁺ secretion; epinephrine; prostaglandin E₂; cholinergic     

The variability of the hepatitis B virus genome: statistical analysis and biological implications

A statistical analysis of the nucleotide sequence variability in 14 published hepatitis B virus (HBV) genomes was carried out using parametric and nonparametric methods. A parametric statistical model revealed that the different regions of the genome differed significantly in their variability. The conclusion was supported by a nonparametric kernel-density model of the HBV genome. Genes S, C, and P, region X, the precore region, and the pre-S2/pre-S1 regions were ranked in order of increasing variability. In many instances, conserved regions of the genome identified with sequences of known function in HBV biology. However, other characterized regions (such as pre-S) showed much variability despite the involvement of their encoded peptides in specific functions. Point mutations that may result in the formation of stop codons and amino acid changes may affect the clinical picture of HBV infection and may be reflected in atypical serological patterns.      

Chromosome localization of genes for soluble proteins in hexaploid wheat (triticum aestivum L.)

An electrophoretic spectra of proteins, extracted with tris-HCI buffer, pH 8.3 are studied. The ditelosomic lines of the Chinese Spring common wheat cultivar are analysed by the chromosomes of the B genome and of the ditelosomic lines of the same cultivar by first and third chromosomes of the D genome. It is found that structural genes for the synthesis of components Nos. 7, 8, 9 and 10 are localized in 1BL, 2BS, 4BS and 5B chromosomes respectively. The genetic control of the component No. 3 is realized by genes, localized in 1BL and 3D chromosomes, while for component No. 2, in the 3D chromosome.     

Traction force on a kinetochore at metaphase acts as a linear function of kinetochore fiber length

We are investigating the relation between the force pulling a kinetochore poleward and the length of the corresponding kinetochore fiber. It was recognized by Ostergren in 1950 (Hereditas 36:1-19) that the metaphase position of a chromosome could be achieved by a balance of traction forces were proportional to the distance from kinetochore to pole. For the typical chromosome (i.e., a meiotic bivalent or mitotic chromosome) with a single kinetochore fiber extending to each pole, the resultant force (RF) would equal zero when the chromosome lay at the midpoint between the two poles. For special chromosomes that have unequal numbers of kinetochore fibers extending towards opposite poles. For special chromosomes that have unequal numbers of kinetochore fibers extending towards opposite poles. For special chromosomes that have unequal numbers of kinetochore fibers extending towards opposite poles, Ostergren’s proposal suggests that RF = 0 when the chromosome is shifted closer to the pole toward which the greater number of kinetochore fibers are pulling. We have measured the force-length relationship in living spindles by analyzing the metaphase positions of experimentally generated multivalent chromosomes having three or four kinetochore fibers. Multivalent chromosomes of varied configurations were generated by γ-irradiation of nymphs of the grasshopper melanoplus differentialis, and their behavior was analyzed in living first meiotic spermocytes. The lengths of kinetochore fibers were determined from time-lapse photographs by measuring the kinetochore-to-pole distances for fully congressed chromosomes just before the onset of anaphase. In our analysis, force (F) along a single kinetochore fiber is expressed by: F = kL(exp), where k is a length-independent proportionality constant, L represents the kinetochore fiber length, and exp is an unknown exponent. The RF on a chromosome is then given by: RF = σk(i)L(i)(exp), where kinetochore fiber lengths in opposite half- spindles are given opposite sign. If forces on a metaphase chromosome are at equilibrium (RF = 0), then for asymmetrical orientations of multivalents we can measure the individual kinetochore fiber lengths (L(i)) and solve for the exponent that yields a resultant force of zero. The value of the exponent relates how the magnitude of force along a kinetochore fiber varies with its length. For six trivalents and one naturally occurring quadrivalent we calculated an average value of exp = 1.06 +/- 0.18. This result is consistent with Ostergren’s hypothesis and indicates that the magnitude of poleward traction force along a kinetochore fiber is directly proportional to the length of the fiber. Our finding suggests that the balance of forces along a kinetochore fiber may be a major factor regulating the extent of kinetochore microtubule assembly.     

Selective modification of CaaX peptides with ortho-substituted anilinogeranyl lipids by protein farnesyl transferase: competitive substrates and potent inhibitors from a library of farnesyl diphosphate analogues

Protein farnesyl transferase (FTase) catalyzes transfer of a 15-carbon farnesyl group from farnesyl diphosphate (FPP) to a conserved cysteine in the C-terminal Ca1a2X motif of a range of proteins ("C" refers to the cysteine, "a" to any aliphatic amino acid, and "X" to any amino acid), and the lipid chain interacts with, and forms part of, the Ca1a2X peptide binding site. Here, we employed a library of anilinogeranyl diphosphate (AGPP) derivatives to examine whether altering the interacting surface between the two substrates could be exploited to generate Ca1a2X peptide selective FPP analogues. Analysis of transfer kinetics to dansyl-GCVLS peptide revealed that AGPP analogues with substituents smaller than or equal in size to a thiomethyl group supported FTase function, while analogues with larger substituents did not. Analogues with small meta-substitutions on the aniline ring such as iodo and cyano increased reactivity with dansyl-GCVLS and provided analogues that were effective FPP competitors. Other analogues with ortho-substitutions on the aniline were potent dansyl-GCVLS modification FTase inhibitors (Ki in the 2.4-18 nM range). Both meta- and para-trifluoromethoxy-AGPP are transferred to dansyl-GCVLS while the ortho-substituted isomer was a potent farnesyl transferase inhibitor (FTI) with an inhibition constant Ki = 3.0 nM. In contrast, ortho-trifluoromethoxy-AGPP was efficiently transferred to dansyl-GCVIM. Competition for dansyl-GCVLS and dansyl-GCVIM peptides by FPP and ortho-trifluoromethoxy-AGPP gave both analogue and farnesyl modified dansyl-GCVIM but only farnesylated dansyl-GCVLS. We provide evidence that competitive modification of dansyl-GCVIM by ortho-trifluoromethoxy-AGPP stems from a prechemical step discrimination between the competing peptides by the FTase-analogue complex. These results show that subtle changes engineered into the isoprenoid structure can alter the reactivity and FPP competitiveness of the analogues, which may be important for the development of prenylated protein function inhibitors.     

Soluble Human Cytomegalovirus gH/gL/pUL128–131 Pentameric Complex,but Not gH/gL,Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes

Congenital infection of human cytomegalovirus (HCMV) is one of the leading causes of nongenetic birth defects, and development of a prophylactic vaccine against HCMV is of high priority for public health. The gH/gL/pUL128–131 pentameric complex mediates HCMV entry into endothelial and epithelial cells, and it is a major target for neutralizing antibody responses. To better understand the mechanism by which antibodies interact with the epitopes of the gH/gL/pUL128–131 pentameric complex resulting in viral neutralization, we expressed and purified soluble gH/gL/pUL128–131 pentameric complex and gH/gL from Chinese hamster ovary cells to >95% purity. The soluble gH/gL, which exists predominantly as (gH/gL)₂ homodimer with a molecular mass of 220 kDa in solution, has a stoichiometry of 1:1 and a pI of 6.0–6.5. The pentameric complex has a molecular mass of 160 kDa, a stoichiometry of 1:1:1:1:1, and a pI of 7.4–8.1. The soluble pentameric complex, but not gH/gL, adsorbs 76% of neutralizing activities in HCMV human hyperimmune globulin, consistent with earlier reports that the most potent neutralizing epitopes for blocking epithelial infection are unique to the pentameric complex. Functionally, the soluble pentameric complex, but not gH/gL, blocks viral entry to epithelial cells in culture. Our results highlight the importance of the gH/gL/pUL128–131 pentameric complex in HCMV vaccine design and emphasize the necessity to monitor the integrity of the pentameric complex during the vaccine manufacturing process.     

Molecular docking analysis of glycogen phosphorylase with inhibitors from Cissampelos pareira Linn.

Cissampelos pareira Linn. is a climbing herb known in Indian traditional medicine as laghupatha. It belongs to the Menispermaceae family. The enzyme glycogen phosphorylase (GP) is a promising target for the treatment of type-2 diabetes (T2DM). A variety of natural product inhibitors with both pharmaceutical and nutraceutical potential have been reported in the search for powerful, selective and drug-like GP inhibitors that could lead to hypoglycemic medicines. Therefore, it is of interest to document the molecular docking analysis data of glycogen phosphorylase with compounds from Cissampelos pareira Linn. We report the optimal binding features of 4 compounds namely Trans-N-feruloyltyramine, Coclaurine, Magnoflorine, and Curine with the target protein for further consideration in the context of T2DM.     

Delayed-type hypersensitivity to Babesia microti-infected erythrocytes in mice

Strong delayed-type hypersensitivity (DTH) to Babesia microti was elicited when intraerythrocytic parasites (IEP) were inoculated subcutaneously into the flank of normal mice 6 to 14 days before challenge in the ipsilateral footpad with 10(8) IEP. Intraperitoneal or intravenous administration of antigen did not sensitize mice for DTH. When challenge was given 21 days after immunization, the response was approximately half of the maximum and then rose again slowly over the next 3 weeks to levels that were not significantly different from those maximal values. The response was similar in seven strains of mice, regardless of sex. The response was classified as a true DTH reaction on the basis of kinetics, histology, and the transfer of responsiveness with immune T lymphocytes of the Ly 1+ phenotype, but not with serum. The reaction was specific for IEP since control groups given two injections of red blood cells from uninfected syngeneic mice (NRBC) or one injection of NRBC or sheep red blood cells (SRBC) and one of IEP never developed significant footpad swelling. Freed parasites obtained by osmotic rupture, density gradient sedimentation, and lethally irradiated IEP were also effective for elicitation of DTH. Anti-IEP DTH was expressed in a dose-dependent fashion with 10(6), 10(7), or 10(8) parasites sufficing for immunizing inoculum as long as 10(8) parasites were used as the challenge dose. Mice immunized and challenged with 10(8) lethally irradiated IEP (60 krad, 60Co), were protected against subsequent intraperitoneal challenge with 10(8) viable IEP. If mice were infected intraperitoneally with 10(8) IEP at any time between 21 days before immunization to 2 hr after challenge, their ability to respond to immunization and challenge was profoundly depressed. These data suggest that development of a strong anti-parasite DTH response can occur in parallel with resistance to infection, but is not a rapid sequela of bloodborne infection.