Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+ exchange

Intestinal neutral NaCl absorption, which is made up ofbrush-border (BB)Na⁺/H⁺exchange linked to BBCl/HCO₃exchange, is up- and downregulated as part of digestion and diarrhealdiseases. Glucocorticoids stimulate ileal NaCl absorption and BBNa⁺/H⁺exchange. Intestinal BB contains twoNa⁺/H⁺exchanger isoforms, NHE2 and NHE3, but their relative roles in rabbitileal BBNa⁺/H⁺exchange has not been determined. A technique to separate the contribution of NHE2 and NHE3 to ileal BBNa⁺/H⁺exchange activity was standardized by using an amiloride-related compound, HOE-694. Under basal conditions, both NHE2 and NHE3 contribute ~50% to ilealNa⁺/H⁺exchange. Glucocorticoids (methylprednisolone) increase BBNa⁺/H⁺exchange (2.5 times) but increase only ileal NHE3 activity (4.1 times),without an effect on NHE2 activity. Thus ileal BBNa⁺/H⁺exchange in animals treated with glucocorticoids is 69% via NHE3. Aquantitative Western analysis for NHE3 was developed, using as aninternal standard a fusion protein of the COOH-terminal 85 amino acidsof NHE3 and maltose binding protein. Glucocorticoid treatment increasedthe amount of BB NHE3. The quantitative Western analysis showed thatNHE3 makes up 0.018% of ileal BB protein in control rabbits and0.042% (2.3 times as much) in methylprednisolone-treated rabbits.Methylprednisolone treatment did not alter the amount of ileal BB NHE2protein. NHE3 turnover number was estimated to be 458 cycles/s underbasal conditions and 708 cycles/s in glucocorticoid-treated ileum. Thusmethylprednisolone stimulates ileal BBNa⁺/H⁺exchange activity only by an effect on NHE3 and not on NHE2; it does soprimarily by increasing the amount of BB NHE3, although it alsoincreases the NHE3 turnover number.

     

<Emphasis Type="Italic">CBS</Emphasis> mutations and <Emphasis Type="Italic">MTFHR</Emphasis> SNPs causative of hyperhomocysteinemia in Pakistani children

Three index patients with hyperhomocysteinemia and ocular anomalies were screened for cystathionine beta synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR) polymorphisms. Genotyping of hyperhomocysteinemia associated MTHFR polymorphisms C677T (rs1801133) and A1298C (rs1801131) was done by PCR-restriction fragment length polymorphism. Sanger sequencing was performed for CBS exonic sequences along with consensus splice sites. In the case of MTHFR polymorphisms, all the patients were heterozygous CT for the single nucleotide polymorphism (SNP) C677T and were therefore carriers of the risk allele (T), while the patients were homozygous CC for the risk genotype of the SNP A1298C. CBS sequencing resulted in the identification of two novel mutations, a missense change (c.467T>C; p.Leu156Pro) in exon 7 and an in-frame deletion (c.808_810del; p.Glu270del) in exon 10. In addition, a recurrent missense mutation (c.770C>T; p.Thr257Met) in exon 10 of the gene was also identified. The mutations were present homozygously in the patients and were inherited from the carrier parents. This is the first report from Pakistan where novel as well as recurrent CBS mutations causing hyperhomocysteinemia and lens dislocation in three patients from different families are being reported with the predicted effect of the risk allele of the MTHFR SNP in causing hyperhomocysteinemia.     

Regulation of cell motility by tyrosine phosphorylated villin

Temporal and spatial regulation of the actin cytoskeleton is vital for cell migration. Here, we show that an epithelial cell actin-binding protein, villin, plays a crucial role in this process. Overexpression of villin in doxycyline-regulated HeLa cells enhanced cell migration. Villin-induced cell migration was modestly augmented by growth factors. In contrast, tyrosine phosphorylation of villin and villin-induced cell migration was significantly inhibited by the src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) as well as by overexpression of a dominant negative mutant of c-src. These data suggest that phosphorylation of villin by c-src is involved in the actin cytoskeleton remodeling necessary for cell migration. We have previously shown that villin is tyrosine phosphorylated at four major sites. To further investigate the role of tyrosine phosphorylated villin in cell migration, we used phosphorylation site mutants (tyrosine to phenylalanine or tyrosine to glutamic acid) in HeLa cells. We determined that tyrosine phosphorylation at residues 60, 81, and 256 of human villin played an essential role in cell migration as well as in the reorganization of the actin cytoskeleton. Collectively, these studies define how biophysical events such as cell migration are actuated by biochemical signaling pathways involving tyrosine phosphorylation of actin binding proteins, in this case villin.     

Obligatory role for phospholipase C-gamma(1) in villin-induced epithelial cell migration

While there is circumstantial evidence to suggest a requirement for phospholipase C-₁ (PLC-₁) in actin reorganization and cell migration, few studies have examined the direct mechanisms that link regulators of the actin cytoskeleton with this crucial signaling molecule. This study was aimed to examine the role that villin, an epithelial cell-specific actin-binding protein, and its ligand PLC-₁ play in migration in intestinal and renal epithelial cell lines that endogenously or ectopically express human villin. Basal as well as epidermal growth factor (EGF)-stimulated cell migration was accompanied by tyrosine phosphorylation of villin and its association with PLC-₁. Inhibition of villin phosphorylation prevented villin-PLC-₁ complex formation as well as villin-induced cell migration. The absolute requirement for PLC-₁ in villin-induced cell migration was demonstrated by measuring cell motility in PLC-₁^–/– cells and by downregulation of endogenous PLC-₁. EGF-stimulated direct interaction of villin with the Src homology domain 2 domain of PLC-₁ at the plasma membrane was demonstrated in living cells by using fluorescence resonance energy transfer. These results demonstrate that villin provides an important link between the activation of phosphoinositide signal transduction pathway and epithelial cell migration. fluorescence resonance energy transfer; actin     

Synthesis,characterization and properties of a new polymerisable surfactant: 12-Methacryloyl dodecylphosphocholine

A new polymerizable surfactant, 12-methacryloyl dodecylphosphocholine (MDPC), has been synthesized using a three-step procedure in moderate yield. Phase transitions were characterized by DSC and phase behavior in water was determined by surface tension and polarizing microscopy. MDPC showed typical surfactant behavior and self-aggregated to micelles above a distinct concentration. The critical micelle concentration (CMC) of MDPC was determined to be 5 × 10^?4 mol/L. MDPC showed mesomorphic properties between 75 and 86 °C as studied by differential scanning calorimetry (DSC). The formation of black lipid membranes was further investigated. The methacrylate functionalized MDPC could form a bilayer membrane (BLM) although it was very unstable (collapsed after 10–30 s). However, it was possible to form stable BLMs in mixture with non-polymerizable two chain phospholipids, i.e. asolectin and diphytanoyl phosphatidylcholine (DPhPC). Stable bilayers could be obtained up to a MDPC content of 50 mol%. Gramicidin A was incorporated into MDPC/DPhPC membranes and exhibited ion-channel activity shown by single channel conductivity measurements.     

Identification of the dominant translation start site in the attB1 sequence of the pET-DEST42 Gateway vector

Gateway technology is a powerful system for converting a single entry vector into a wide variety of expression vectors. We expressed recombinant influenza matrix protein M1 (FMP), a potent antigen for cytotoxic T cells, using the Gateway vector pET-DEST42 containing the FMP cDNA, and purified the expressed FMP as a single 32 kDa recombinant protein. N-terminal and internal protein sequencing, however, showed that the recombinant FMP contained an extra 10 amino acids fused to the N-terminal of native FMP. Further investigation of the DNA sequence adjacent to the 5'-FMP cDNA indicated that the "TTG" in the attB1 site (30 bp upstream of the "ATG" in the 5'-FMP cDNA) behaved as a dominant translation start site, resulting in a 10 amino acid extension of the recombinant FMP. Thus, it is possible that recombinant proteins produced by this Gateway vector contain unexpected vector-derived peptides, which may affect experimental outcomes.     

RNA tertiary interactions mediate native collapse of a bacterial group I ribozyme

Large RNAs collapse into compact intermediates in the presence of counterions before folding to the native state. We previously found that collapse of a bacterial group I ribozyme correlates with the formation of helices within the ribozyme core, but occurs at Mg2+ concentrations too low to support stable tertiary structure and catalytic activity. Here, using small-angle X-ray scattering, we show that Mg2+-induced collapse is a cooperative folding transition that can be fit by a two-state model. The Mg2+ dependence of collapse is similar to the Mg2+ dependence of helix assembly measured by partial ribonuclease T1 digestion and of an unfolding transition measured by UV hypochromicity. The correspondence between multiple probes of RNA structure further supports a two-state model. A mutation that disrupts tertiary contacts between the L9 tetraloop and its helical receptor destabilized the compact state by 0.8 kcal/mol, while mutations in the central triplex were less destabilizing. These results show that native tertiary interactions stabilize the compact folding intermediates under conditions in which the RNA backbone remains accessible to solvent.     

Chamber Bioaerosol Study: Outdoor Air and Human Occupants as Sources of Indoor Airborne Microbes

Human occupants are an important source of microbes in indoor environments. In this study, we used DNA sequencing of filter samples to assess the fungal and bacterial composition of air in an environmental chamber under different levels of occupancy, activity, and exposed or covered carpeting. In this office-like, mechanically ventilated environment, results showed a strong influence of outdoor-derived particles, with the indoor microbial composition tracking that of outdoor air for the 2-hour sampling periods. The number of occupants and their activity played a significant but smaller role influencing the composition of indoor bioaerosols. Human-associated taxa were observed but were not particularly abundant, except in the case of one fungus that appeared to be transported into the chamber on the clothing of a study participant. Overall, this study revealed a smaller signature of human body-associated taxa than had been expected based on recent studies of indoor microbiomes, suggesting that occupants may not exert a strong influence on bioaerosol microbial composition in a space that, like many offices, is well ventilated with air that is moderately filtered and moderately occupied.