Evidence against Na+-pump mediation of Ca2+-activated K+ transport and diuretic-sensitive (Na+/K+)-cotransport

Proteoliposomes reconstituted from purified Na⁺ pumps show neither Ca²⁺ activation nor bumetanide inhibition of Rb⁺ uptake, suggesting that the Na⁺ pump does not mediate these passive fluxes.     

Simultaneous determination of inosine, hypoxanthine, xanthine, and uric acid and the effect of metal chelators

We describe a sensitive, reproducible method for the simultaneous determination of the ATP catabolites inosine, hypoxanthine, xanthine, and uric acid in biological samples and organ perfusate using reverse-phase chromatography and multiwavelength detection at 254, 270, and 292 nm. Sample preparation includes precipitating proteins with perchloric acid, neutralizing the sample, passing the supernatant over a polyethyleneimine column, and analyzing the collected fractions by high-performance liquid chromatography. Addition of metal chelators to the perchloric acid resulted in increased values for xanthine, hypoxanthine, and uric acid. The method was sensitive (limit of detection, 0.08 nmol on column; S/N = 4) and linear over the range 0.5-30 microM. Precision and accuracy of the method were evaluated for lung tissue and lung perfusate. Coefficients of variation ranged from 2.8 to 6.1% for perfusate and from 1.7 to 12.6% for tissue. Recoveries for all compounds exceeded 90%. We applied this method to rat lung tissue, lung perfusate, and rat and human blood. Advantages of this method are simultaneous quantitation with excellent sensitivity of all compounds, simplified peak identification by using multiwavelength detection, and improved accuracy by preventing loss of compounds with metal chelators.     

β‐carotene suppresses Porphyromonas gingivalis lipopolysaccharide‐mediated cytokine production in THP‐1 monocytes cultured with high glucose condition

Periodontitis is associated with development of diabetes mellitus. Although lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg), a major pathogen of periodontitis, may lead the progression of diabetes complications, the precise mechanisms are unclear. We, therefore, investigated the effects of β‐carotene on production of Pg LPS‐induced inflammatory cytokines in human monocytes cultured high glucose (HG) condition. THP‐1 cells were cultured under 5.5 mM or 25 mM glucose conditions, and cells were stimulated with Pg LPS. To investigate the productivity of TNF‐α, IL‐6, and MCP‐1, cell supernatants were collected for ELISA. To examine the effects of NF‐kB signals on cytokine production, Bay11‐7082 was used. HG enhanced Pg LPS‐induced production of TNF‐α, IL‐6, and MCP‐1 via NF‐kB signals in THP‐1. β‐carotene suppressed the enhancement of the Pg LPS‐induced cytokine production in THP‐1 via NF‐κB inactivation. Our results suggest that β‐carotene might be a potential anti‐inflammatory nutrient for circulating Pg LPS‐mediated cytokine production in diabetic patients with periodontitis.     

Investigating factors associated with success of breastfeeding in first-time mothers undergoing epidural analgesia: a prospective cohort study

Background

We investigated the possible risk factors that could influence the likelihood of breastfeeding at 5 to 9 weeks postpartum with our primary aim being to analyse the associations between psychological vulnerabilities, such as peripartum depression and anxiety, and continued breastfeeding. Our secondary aim was to investigate other non-psychological factors’ influence on continued breastfeeding.

Methods

A prospective cohort study was conducted in KK Women’s and Children’s Hospital in Singapore. Healthy nulliparous parturients at ≥36 weeks gestation with a singleton fetus who received epidural analgesia were recruited. Demographic and anaesthetic data were obtained. Self-reported psychological and pain determinants such as anxiety (State-Trait Anxiety Inventory), depression (Edinburgh Postnatal Depression Scale), stress (Perceived Stress Scale), pain susceptibility (Pain Catastrophizing Scale) and pain perception (McGill Pain Questionnaire) were also recorded at baseline. A phone interview was then performed at 5 to 9 weeks postpartum to obtain information on breastfeeding status.

Results

329 participants were included into this study, of which 263 (79.9%) of them were still breastfeeding at 5 weeks postpartum. Multivariate logistic regression analysis showed that a higher State-Trait Anxiety Inventory score (Adjusted Odds Ratio [AOR] 0.97; 95% Confidence Interval [CI] 0.94, 1.00) at baseline, higher intrapartum blood loss (AOR 0.76; 95% CI 0.61, 0.93), and occurrence of fetal anomalies (AOR 0.15; 95% CI 0.03, 0.72) were associated with reduced likelihood of breastfeeding at 5 to 9 weeks postpartum. Indians (AOR 0.56; 95% CI 0.20, 1.53), Malays (AOR 0.30; 95% CI 0.14, 0.62) and other ethnicities (AOR 0.36; 95% CI 0.16, 0.83) were less likely to continue breastfeeding compared to Chinese participants. On the other hand, receiving any support services on breastfeeding during the participants’ hospital stay was 3.3 times more likely (AOR 3.30; 95% CI 1.21, 9.02) to increase the likelihood of breastfeeding at 5 to 9 weeks postpartum.

Conclusion

We identified 5 independent association factors that could have significant influences on breastfeeding at 5 to 9 weeks postpartum. Healthcare providers could utilize this risk stratification to identify parturients likely to have poorer breastfeeding outcomes and undertake interventions that may help safeguard optimization of breastfeeding outcomes and parturient care.

Trial registration

Clinicaltrials.gov NCT02278601. Registered 26 October 2014.

     

Interrelationships between Cytoplasmic Ca2+ Peaks, Pollen Hydration and Plasma Membrane Conductances during Compatible and Incompatible Pollinations of Brassica napus Papillae

We have investigated Ca²⁺-involving cell signaling, plasma membranepotentials and conductances and callose formation during earlystages of pollination of papillae of Brassica napus. Using fluorescenceimaging of calcium green-1, we found that application of a rangeof pollen types and controls all rapidly produced small localizedpeaks in papillar cytoplasmic [Ca²⁺]. This response was morefrequent in compatible than incompatible interactions and wascorrelated with subsequent hydration of the applied pollen grains,indicating that it may be a differential prerequisite of thecompatible signaling pathway leading to successful pollinations.In contrast, a slight trend to increased plasma membrane conductance(but with no indications of action potential-like responses)and also callose deposition in papillae adjacent to pollen grainsfollowed pollination in both SC and SI interactions, indicatingthat alterations in plasma membrane permeability and callosedeposition during early phases of pollination are not primarydeterminants of the fate of attempted pollinations. ²On leave of absence from A.N. Bach Institute of Biochemistry,Russian Academy of Sciences, Leninsky Pr., 33, Moscow, Russia,117071     

Second-messenger regulation of receptor association with clathrin-coated pits: a novel and selective mechanism in the control of CD4 endocytosis.

CD4, a member of the immunoglobulin superfamily, is not only expressed in T4 helper lymphocytes but also in myeloid cells. Receptor-mediated endocytosis plays a crucial role in the regulation of surface expression of adhesion molecules such as CD4. In T lymphocytes p56lck, a CD4-associated tyrosine kinase, prevents CD4 internalization, but in myeloid cells p56lck is not expressed and CD4 is constitutively internalized. In this study, we have investigated the role of cyclic AMP (cAMP) in the regulation of CD4 endocytosis in the myeloid cell line HL-60. Elevations of cellular cAMP were elicited by 1) cholera toxin, 2) pertussis toxin, 3) forskolin and IBMX, 4) NaF, or 5) the physiological receptor agonist prostaglandin E1. All five interventions led to an inhibition of CD4 internalization. Increased cAMP levels did not inhibit endocytosis per se, because internalization of insulin receptors and transferrin receptors and fluid phase endocytosis were either unchanged or slightly enhanced. The mechanism of cAMP inhibition was further analyzed at the ultrastructural level. CD4 internalization, followed either by quantitative electron microscopy autoradiography or by immunogold labeling, showed a rapid and temperature-dependent association of CD4 with clathrin-coated pits in control cells. This association was markedly inhibited in cells with elevated cAMP levels. Thus these findings suggest a second-messenger regulation of CD4 internalization through an inhibition of CD4 association with clathrin-coated pits in p56lck-negative cells.     

Bradykinin analogues with beta-amino acid substitutions reveal subtle differences in substrate specificity between the endopeptidases EC 3.4.24.15 and EC 3.4.24.16.

The closely related zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) cleave many common substrates, including bradykinin (BK). As such, there are few substrate-based inhibitors which are sufficiently selective to distinguish their activities. We have used BK analogues with either alanine or beta-amino acid (containing an additional carbon within the peptide backbone) substitutions to elucidate subtle differences in substrate specificity between the enzymes. The cleavage of the analogues by recombinant EP24.15 and EP24.16 was assessed, as well as their ability to inhibit the two enzymes. Alanine-substituted analogues were generally better substrates than BK itself, although differences between the peptidases were observed. Similarly, substitution of the four N-terminal residues with beta-glycine enhanced cleavage in some cases, but not others. beta-Glycine substitution at or near the scissile bond (Phe5-Ser6) completely prevented cleavage by either enzyme: interestingly, these analogues still acted as inhibitors, although with very different affinities for the two enzymes. Also of interest, beta-Gly8-BK was neither a substrate nor an inhibitor of EP24.15, yet could still interact with EP24.16. Finally, while both enzymes could be similarly inhibited by the D-stereoisomer of beta-C3-Phe5-BK (IC50 approximately 20 microM, compared to 8 microM for BK), EP24.16 was relatively insensitive to the L-isomer (IC50 12 approximately microM for EP24.15, >40 microM for EP24.16). These studies indicate subtle differences in substrate specificity between EP24.15 and EP24.16, and suggest that beta-amino acid analogues may be useful as templates for the design of selective inhibitors.