Mutation of Aspartate 555 of the Sodium/Bicarbonate Transporter SLC4A4/NBCe1 Induces Chloride Transport

To understand the mechanism for ion transport through the sodium/bicarbonate transporter SLC4A4 (NBCe1), we examined amino acid residues, within transmembrane domains, that are conserved among electrogenic Na/HCO₃ transporters but are substituted with residues at the corresponding site of all electroneutral Na/HCO₃ transporters. Point mutants were constructed and expressed in Xenopus oocytes to assess function using two-electrode voltage clamp. Among the mutants, D555E (charge-conserved substitution of the aspartate at position 555 with a glutamate) produced decreasing HCO₃⁻ currents at more positive membrane voltages. Immunohistochemistry showed D555E protein expression in oocyte membranes. D555E induced Na/HCO₃-dependent pH recovery from a CO₂-induced acidification. Current-voltage relationships revealed that D555E produced an outwardly rectifying current in the nominally CO₂/HCO₃⁻-free solution that was abolished by Cl⁻ removal from the bath. In the presence of CO₂/HCO₃⁻, however, the outward current produced by D555E decreased only slightly after Cl⁻ removal. Starting from a Cl⁻-free condition, D555E produced dose-dependent outward currents in response to a series of chloride additions. The D555E-mediated chloride current decreased by 70% in the presence of CO₂/HCO₃⁻. The substitution of Asp⁵⁵⁵ with an asparagine also produced a Cl⁻ current. Anion selectivity experiments revealed that D555E was broadly permissive to other anions including NO₃⁻. Fluorescence measurements of chloride transport were done with human embryonic kidney HEK 293 cells expressing NBCe1 and D555E. A marked increase in chloride transport was detected in cells expressing D555E. We conclude that Asp⁵⁵⁵ plays a role in HCO₃⁻ selectivity.The electrogenic Na/HCO₃ cotransporter NBCe1 (SLC4A4) is one of the SLC4A gene family members transporting HCO₃⁻ across the plasma membrane (1–3). NBCe1 plays a role in transepithelial HCO₃⁻ movement and pH_i regulation in many tissues (4–6). NBCe1 is responsible for HCO₃⁻ reabsorption in the proximal tubules of the kidney (7). The proximal tubule cells reclaim HCO₃⁻ from the lumen through a series of reactions involving titration of HCO₃⁻ by H⁺ secretion via the apical Na/H exchanger, production of CO₂, and regeneration of HCO₃⁻ and H⁺ in the tubule cells. HCO₃⁻ then moves to the interstitium via the basolateral NBCe1. The essential feature driving this basolateral Na⁺/HCO₃⁻ exit is the stoichiometry of 1:3 Na⁺:HCO₃⁻, which makes the equilibrium potential for NBCe1 more positive than the resting membrane potential of the proximal tubule cells (8). The stoichiometry of 1Na⁺:1HCO₃⁻ or 1Na⁺:2HCO₃⁻ causes both ions to move into cells in other tissues such as pancreas, brain, and cardiovascular tissues (9, 10).Despite the importance of NBCe1 for basolateral HCO₃⁻ reabsorption in the proximal tubules, the mechanism of electrogenic Na/HCO₃ transport via the transporter is not well understood. Ion movement depends on loading ions at their translocation or binding sites that likely reside within the membrane field at some distance from the bath solution (11). This implies that the transmembrane domains (TMs)2 of NBCe1 and amino acid residues within TMs play critical roles in ion transport.Sequence analysis of different SLC4A proteins shows similar hydropathy plots, predicting that these proteins share structural elements of transport function (12). Such similarities have facilitated structure/function studies to define molecular domains or motifs responsible for conferring Na/HCO₃ transport of NBCe1. Abuladze et al. (13) performed a large scale mutagenesis on acidic and basic amino acids in non-TMs and found many residues affecting Na⁺-dependent base flux. McAlear et al. (14) identified amino acids in TM8 involving ion translocation. By a systematic approach of chimeric transporters between NBCe1 and the electroneutral Na/HCO₃ cotransporter NBCn1 (SLC4A7) (15), we and our colleagues (16) demonstrated that electrogenic Na/HCO₃ transport of NBCe1 requires interactions between the regions TM1–5 and TM6–13 of the protein. Zhu et al. (17) recently proposed TM1 as a domain lining the ion translocation pathway. On the other hand, Chang et al. (18) reported that the cytoplasmic N-terminal domain might contribute to HCO₃⁻ permeation.In the present study, we searched amino acid residues that are highly conserved among electrogenic Na/HCO₃ transporters but not among electroneutral Na/HCO₃ transporters and examined their role in electrogenic Na/HCO₃ transport. Nine candidate residues in human renal NBCe1-A (5, 19) were selected and mutated by replacement with the amino acids at the corresponding sites of NBCn1. Mutant transporters were expressed in Xenopus oocytes and assessed via two-electrode voltage clamp. Our data show that Asp⁵⁵⁵ of NBCe1 plays an important role in HCO₃⁻ selectivity.     

Characterization of microsatellite loci isolated from the wasp, Microstigmus nigrophthalmus (Hymenoptera)

Fifty‐two microsatellite loci were characterized in 22–31 unrelated females of the wasp (Microstigmus nigrophthalmus) collected from the Mata do Paraiso, Viçosa, M.G., Brazil. Fifty‐one of these loci were developed from a microsatellite‐enriched genomic library derived from M. nigrophthalmus and one was derived from the wasp, Ormyrus nitidulus. The genus Microstigmus represents an independent origin of social behaviour in the Hymenoptera and is thus of great potential in the study of social evolution.     

Nevirapine Resistance and Breast-Milk HIV Transmission: Effects of Single and Extended-Dose Nevirapine Prophylaxis in Subtype C HIV-Infected Infants

Background

Daily nevirapine (NVP) prophylaxis to HIV-exposed infants significantly reduces breast-milk HIV transmission. We assessed NVP-resistance in Indian infants enrolled in the “six-week extended-dose nevirapine” (SWEN) trial who received single-dose NVP (SD-NVP) or SWEN for prevention of breast-milk HIV transmission but who also acquired subtype C HIV infection during the first year of life.

Methods/Findings

Standard population sequencing and cloning for viral subpopulations present at ≥5% frequency were used to determine HIV genotypes from 94% of the 79 infected Indian infants studied. Timing of infection was defined based on when an infant''s blood sample first tested positive for HIV DNA. SWEN-exposed infants diagnosed with HIV by six weeks of age had a significantly higher prevalence of NVP-resistance than those who received SD-NVP, by both standard population sequencing (92% of 12 vs. 38% of 29; p = 0.002) and low frequency clonal analysis (92% of 12 vs. 59% of 29; p = 0.06). Likelihood of infection with NVP-resistant HIV through breast-milk among infants infected after age six weeks was substantial, but prevalence of NVP-resistance did not differ among SWEN or SD-NVP exposed infants by standard population sequencing (15% of 13 vs. 15% of 20; p = 1.00) and clonal analysis (31% of 13 vs. 40% of 20; p = 0.72). Types of NVP-resistance mutations and patterns of persistence at one year of age were similar between the two groups. NVP-resistance mutations did differ by timing of HIV infection; the Y181C variant was predominant among infants diagnosed in the first six weeks of life, compared to Y188C/H during late breast-milk transmission.

Conclusions/Significance

Use of SWEN to prevent breast-milk HIV transmission carries a high likelihood of resistance if infection occurs in the first six weeks of life. Moreover, there was a continued risk of transmission of NVP-resistant HIV through breastfeeding during the first year of life, but did not differ between SD-NVP and SWEN groups. As with SD-NVP, the value of preventing HIV infection in a large number of infants should be considered alongside the high risk of resistance associated with extended NVP prophylaxis.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00061321","term_id":"NCT00061321"}}NCT00061321     

Pre- and Postnatal Nutritional Histories Influence Reproductive Maturation and Ovarian Function in the Rat

Background

While prepubertal nutritional influences appear to play a role in sexual maturation, there is a need to clarify the potential contributions of maternal and childhood influences in setting the tempo of reproductive maturation. In the present study we employed an established model of nutritional programming to evaluate the relative influences of prenatal and postnatal nutrition on growth and ovarian function in female offspring.

Methods

Pregnant Wistar rats were fed either a calorie-restricted diet, a high fat diet, or a control diet during pregnancy and/or lactation. Offspring then were fed either a control or a high fat diet from the time of weaning to adulthood. Pubertal age was monitored and blood samples collected in adulthood for endocrine analyses.

Results

We report that in the female rat, pubertal timing and subsequent ovarian function is influenced by the animal''s nutritional status in utero, with both maternal caloric restriction and maternal high fat nutrition resulting in early pubertal onset. Depending on the offspring''s nutritional history during the prenatal and lactational periods, subsequent nutrition and body weight gain did not further influence offspring reproductive tempo, which was dominated by the effect of prenatal nutrition. Whereas maternal calorie restriction leads to early pubertal onset, it also leads to a reduction in adult progesterone levels later in life. In contrast, we found that maternal high fat feeding which also induces early maturation in offspring was associated with elevated progesterone concentrations.

Conclusions

These observations are suggestive of two distinct developmental pathways leading to the acceleration of pubertal timing but with different consequences for ovarian function. We suggest different adaptive explanations for these pathways and for their relationship to altered metabolic homeostasis.     

The Diagnostic and Prognostic Accuracy of Five Markers of Serious Bacterial Infection in Malawian Children with Signs of Severe Infection

Background

Early recognition and prompt and appropriate antibiotic treatment can significantly reduce mortality from serious bacterial infections (SBI). The aim of this study was to evaluate the utility of five markers of infection: C-reactive protein (CRP), procalcitonin (PCT), soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), CD163 and high mobility group box-1 (HMGB1), as markers of SBI in severely ill Malawian children.

Methodology and Principal Findings

Children presenting with a signs of meningitis (n = 282) or pneumonia (n = 95), were prospectively recruited. Plasma samples were taken on admission for CRP, PCT, sTREM-1 CD163 and HMGB1 and the performance characteristics of each test to diagnose SBI and to predict mortality were determined. Of 377 children, 279 (74%) had SBI and 83 (22%) died. Plasma CRP, PCT, CD163 and HMGB1 and were higher in HIV-infected children than in HIV-uninfected children (p<0.01). In HIV-infected children, CRP and PCT were higher in children with SBI compared to those with no detectable bacterial infection (p<0.0005), and PCT and CD163 were higher in non-survivors (p = 0.001, p = 0.05 respectively). In HIV-uninfected children, CRP and PCT were also higher in children with SBI compared to those with no detectable bacterial infection (p<0.0005), and CD163 was higher in non-survivors (p = 0.05). The best predictors of SBI were CRP and PCT, and areas under the curve (AUCs) were 0.81 (95% CI 0.73–0.89) and 0.86 (95% CI 0.79–0.92) respectively. The best marker for predicting death was PCT, AUC 0.61 (95% CI 0.50–0.71).

Conclusions

Admission PCT and CRP are useful markers of invasive bacterial infection in severely ill African children. The study of these markers using rapid tests in a less selected cohort would be important in this setting.     

Comparison of Overall Obesity and Body Fat Distribution in Predicting Risk of Mortality

Results of studies comparing overall obesity and abdominal adiposity or body fat distribution with risk of mortality have varied considerably. We compared the relative importance and joint association of overall obesity and body fat distribution in predicting risk of mortality. Participants included 5,799 men and 6,429 women aged 30–102 years enrolled in the third National Health and Nutrition Examination Survey who completed a baseline health examination during 1988–1994. During a 12‐year follow‐up (102,172 person‐years), 1,188 men and 925 women died. In multivariable‐adjusted analyses, waist‐to‐thigh ratio (WTR) in both sexes (P_trend <0.01 for both) and waist‐to‐hip ratio (WHR) in women (P_trend 0.001) were positively associated with mortality in middle‐aged adults (30–64 years), while BMI and waist circumference (WC) exhibited U‐ or J‐shaped associations. Risk of mortality increased with a higher WHR and WTR among normal weight (BMI 18.5–24.9 kg/m²) and obese (BMI ≥30.0 kg/m²) adults. In older adults (65–102 years), a higher BMI in both sexes (P_trend <0.05) and WC in men (P_trend 0.001) were associated with increased survival, while remaining measures of body fat distribution exhibited either no association or an inverse relation with mortality. In conclusion, ratio measures of body fat distribution are strongly and positively associated with mortality and offer additional prognostic information beyond BMI and WC in middle‐aged adults. A higher BMI in both sexes and WC in men were associated with increased survival in older adults, while a higher WHR or WTR either decreased or did not influence risk of death.     

The ubiquitin receptor Rad23: At the crossroads of nucleotide excision repair and proteasomal degradation

A protein that exemplifies the intimate link between the ubiquitin/proteasome system (UPS) and DNA repair is the yeast nucleotide excision repair (NER) protein Rad23 and its human orthologs hHR23A and hHR23B. Rad23, which was originally identified as an important factor involved in the recognition of DNA lesions, also plays a central role in targeting ubiquitylated proteins for proteasomal degradation, an activity that it shares with other ubiquitin receptors like Dsk2 and Ddi1. Although the finding that Rad23 serves as a ubiquitin receptor explains to a large extent its importance in proteasomal degradation, the precise mode of action of Rad23 in NER and the possible link with the UPS is less clear. In this review, we discuss our present knowledge on the functions of Rad23 in protein degradation and DNA repair and speculate on the importance of the dual roles of Rad23 for the cell's ability to cope with stress conditions.