In vitro assembly of gap junctions

Gap junction structures were assembled in vitro from octyl-β- -glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = B = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg²⁺ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.     

Phosphorylation of the rat Ins(1,4,5)P3 receptor at T930 within the coupling domain decreases its affinity to Ins(1,4,5)P3

The Ins(1,4,5)P₃ receptor acts as a central hub for Ca²⁺ signaling by integrating multiple signaling modalities into Ca²⁺ release from intracellular stores downstream of G-protein and tyrosine kinase-coupled receptor stimulation. As such, the Ins(1,4,5)P₃ receptor plays fundamental roles in cellular physiology. The regulation of the Ins(1,4,5)P₃ receptor is complex and involves protein-protein interactions, post-translational modifications, allosteric modulation, and regulation of its sub-cellular distribution. Phosphorylation has been implicated in the sensitization of Ins(1,4,5)P₃-dependent Ca²⁺ release observed during oocyte maturation. Here we investigate the role of phosphorylation at T-930, a residue phosphorylated specifically during meiosis. We show that a phosphomimetic mutation at T-930 of the rat Ins(1,4,5)P₃ receptor results in decreased Ins(1,4,5)P₃-dependent Ca²⁺ release and lowers the Ins(1,4,5)P₃ binding affinity of the receptor. These data, coupled to the sensitization of Ins(1,4,5)P₃-dependent Ca²⁺ release during meiosis, argue that phosphorylation within the coupling domain of the Ins(1,4,5)P₃ receptor acts in a combinatorial fashion to regulate Ins(1,4,5)P₃ receptor function.     

The Expansion of the Pulmonary Rib Cage during Breath Stacking Is Influenced by Age in Obese Women

Objective

To analyze in obese women the acute effects of the breath stacking technique on thoraco-abdominal expansion.

Design and Methods

Nineteen obese women (BMI≥30 kg/m²) were evaluated by anthropometry, spirometry and maximal respiratory muscle pressures and successively analyzed by Opto-Electronic Plethysmography and a Wright respirometer during quiet breathing and breath stacking maneuvers and compared with a group of 15 normal-weighted healthy women. The acute effects of the maneuvers were assessed in terms of total and compartmental chest wall volumes at baseline, end of the breath stacking maneuver and after the maneuver. Obese subjects were successively classified into two groups, accordingly to the response during the maneuver, group 1 = prevalent rib cage or group 2 = abdominal expansion.

Results

Age was significantly lower in group 1 than group 2. When considering the two obese groups, FEV₁ was lower and minute ventilation was higher only in group 2 compared to controls group. During breath stacking, inspiratory capacity was significant differences in obese subjects with a smaller expansion of the pulmonary rib cage and a greater expansion of the abdomen compared to controls and also between groups 1 and 2. A significant inverse linear relationship was found between age and inspiratory capacity of the pulmonary rib cage but not of the abdomen.

Conclusions

In obese women the maximal expansion of the rib cage and abdomen is influenced by age and breath stacking maneuver could be a possible therapy for preventing respiratory complications.     

Lactose repressor hinge domain independently binds DNA

The short 8–10 amino acid “hinge” sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer‐binding domains. Structural studies of full‐length or truncated LacI‐operator DNA complexes demonstrate insertion of the dimeric helical “hinge” structure at the center of the operator sequence. This association bends the DNA ～40° and aligns flanking semi‐symmetric DNA sites for optimal contact by the N‐terminal helix‐turn‐helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1–50 to remove the HtH DNA binding domain or residues 1–58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix‐turn‐helix domain with its highly positive charge. LacI missing residues 1–50 binds to DNA with ～4‐fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1–58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences.     

Concanavalin A reveals olfactory receptors which discriminate between alkane odorants on the basis of size.

For certain odorants, the amplitude of the rat electro-olfactogram is reduced if the olfactory epithelium is treated with the lectin concanavalin A. When normal and cycloalkanes of one to ten carbon atoms are used as odorants at equimolar concentration, the maximum reduction in amplitude is found to correlate with the size of the stimulus molecule. This observation is consistent with the notion that concanavalin A disables an olfactory receptor molecule which normally responds to the alkyl moiety of odorants in a particular size range. That moiety may thus represent a 'primary' quality-determining component in odour discrimination.     

Smith degradation of gum exudates from some prosopis species

The polysaccharide of P. hymantophora has been shown to be composed of (1→4)-linked galactopyranosyl, (1→3)-linked galactopyranosyl, (1→3)-linked galactopyranosyl 2- and 4-sulphate and 2,6-disulphate residues. The (1→3)- and (1→4)-linked units are present in approximately equal amounts. The polysaccharide of P. hieroglyphica has been shown to possess (1→4)-linked galactopyranosyl, (1→3)-linked galactopyranosyl, and (1→3)-linked galactopyranosyl 2- and 4-sulphate residues. The (1→3)- and (1→4)-linked units are present in a 4:1 ratio. Both polysaccharides contain small proportions of non-reducing xylosyl end-groups.     

Dietary Omega-3 Fatty Acid Supplementation Reduces Inflammation in Obese Pregnant Women: A Randomized Double-Blind Controlled Clinical Trial

Objective

Long-chain omega 3 fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) exert potent anti-inflammatory properties in humans. This study characterized the effects of omega-3 ω-3 fatty acids supplements (ω-3 FA) on the inflammatory status in the placenta and adipose tissue of overweight/obese pregnant women.

Study Design

A randomized, double-masked controlled trial was conducted in overweight/obese pregnant women that were randomly assigned to receive DHA plus EPA (2g/day) or the equivalent of a placebo twice a day from week 10–16 to term. Inflammatory pathways were characterized in: 1) adipose tissue and placenta of treated vs. untreated women; and 2) adipose and trophoblast cells cultured with long chain FAs.

Results

The sum of plasma DHA and EPA increased by 5.8 fold and ω-3 FA/ ω-6 FA ratio was 1.5 in treated vs. untreated women (p< 0.005). Plasma CRP concentrations were reduced (p<0.001). The adipose tissue and placenta of treated women exhibited a significant decrease in TLR4 adipose and placental expression as well as IL6, IL8, and TNFα In vitro, EPA and DHA suppressed the activation of TLR4, IL6, IL8 induced by palmitate in culture of adipose and trophoblast cells.

Conclusion

Supplementation of overweight/obese pregnant women with dietary ω-3 FAs for >25 weeks reduced inflammation in maternal adipose and the placental tissue. TLR4 appears as a central target of the anti-inflammatory effects at the cellular level.

Trial Registration

ClinicalTrials.gov NCT00957476     

Mutations of Francisella novicida that Alter the Mechanism of Its Phagocytosis by Murine Macrophages

Infection with the bacterial pathogen Francisella tularensis tularensis (F. tularensis) causes tularemia, a serious and debilitating disease. Francisella tularensis novicida strain U112 (abbreviated F. novicida), which is closely related to F. tularensis, is pathogenic for mice but not for man, making it an ideal model system for tularemia. Intracellular pathogens like Francisella inhibit the innate immune response, thereby avoiding immune recognition and death of the infected cell. Because activation of inflammatory pathways may lead to cell death, we reasoned that we could identify bacterial genes involved in inhibiting inflammation by isolating mutants that killed infected cells faster than the wild-type parent. We screened a comprehensive transposon library of F. novicida for mutant strains that increased the rate of cell death following infection in J774 macrophage-like cells, as compared to wild-type F. novicida. Mutations in 28 genes were identified as being hypercytotoxic to both J774 and primary macrophages of which 12 were less virulent in a mouse infection model. Surprisingly, we found that F. novicida with mutations in four genes (lpcC, manB, manC and kdtA) were taken up by and killed macrophages at a much higher rate than the parent strain, even upon treatment with cytochalasin D (cytD), a classic inhibitor of macrophage phagocytosis. At least 10-fold more mutant bacteria were internalized by macrophages as compared to the parent strain if the bacteria were first fixed with formaldehyde, suggesting a surface structure is required for the high phagocytosis rate. However, bacteria were required to be viable for macrophage toxicity. The four mutant strains do not make a complete LPS but instead have an exposed lipid A. Interestingly, other mutations that result in an exposed LPS core were not taken up at increased frequency nor did they kill host cells more than the parent. These results suggest an alternative, more efficient macrophage uptake mechanism for Francisella that requires exposure of a specific bacterial surface structure(s) but results in increased cell death following internalization of live bacteria.     

Transcriptomic Identification of ADH1B as a Novel Candidate Gene for Obesity and Insulin Resistance in Human Adipose Tissue in Mexican Americans from the Veterans Administration Genetic Epidemiology Study (VAGES)

Type 2 diabetes (T2D) is a complex metabolic disease that is more prevalent in ethnic groups such as Mexican Americans, and is strongly associated with the risk factors obesity and insulin resistance. The goal of this study was to perform whole genome gene expression profiling in adipose tissue to detect common patterns of gene regulation associated with obesity and insulin resistance. We used phenotypic and genotypic data from 308 Mexican American participants from the Veterans Administration Genetic Epidemiology Study (VAGES). Basal fasting RNA was extracted from adipose tissue biopsies from a subset of 75 unrelated individuals, and gene expression data generated on the Illumina BeadArray platform. The number of gene probes with significant expression above baseline was approximately 31,000. We performed multiple regression analysis of all probes with 15 metabolic traits. Adipose tissue had 3,012 genes significantly associated with the traits of interest (false discovery rate, FDR ≤ 0.05). The significance of gene expression changes was used to select 52 genes with significant (FDR ≤ 10^-4) gene expression changes across multiple traits. Gene sets/Pathways analysis identified one gene, alcohol dehydrogenase 1B (ADH1B) that was significantly enriched (P < 10^-60) as a prime candidate for involvement in multiple relevant metabolic pathways. Illumina BeadChip derived ADH1B expression data was consistent with quantitative real time PCR data. We observed significant inverse correlations with waist circumference (2.8 x 10^-9), BMI (5.4 x 10^-6), and fasting plasma insulin (P < 0.001). These findings are consistent with a central role for ADH1B in obesity and insulin resistance and provide evidence for a novel genetic regulatory mechanism for human metabolic diseases related to these traits.