A novel transmembrane MSP-containing protein that plays a role in right ventricle development

We have identified and characterized a gene, Mospd3 on mouse chromosome 5 using gene trapping in ES cells. MOSPD3 is part of a family of proteins, including MOSPD1, which is defined by the presence of a major sperm protein (MSP) domain and two transmembrane domains. Interestingly Mospd3 is mammalian specific and highly conserved between mouse and man. Insertion of the gene trap vector at the Mospd3 locus is mutagenic and breeding to homozygosity results in a characteristic right ventricle defect and neonatal lethality in 50% of mice. The phenotypic defect is dependent on the genetic background, indicating the presence of genetic modifier loci. We speculate that the further characterization of Mospd3 will shed light on the complex genetic interactions involved in cardiac development and disease.     

Impact of Malaria Control on Mortality and Anemia among Tanzanian Children Less than Five Years of Age, 1999–2010

Background

Mainland Tanzania scaled up multiple malaria control interventions between 1999 and 2010. We evaluated whether, and to what extent, reductions in all-cause under-five child mortality (U5CM) tracked with malaria control intensification during this period.

Methods

Four nationally representative household surveys permitted trend analysis for malaria intervention coverage, severe anemia (hemoglobin <8 g/dL) prevalence (SAP) among children 6–59 months, and U5CM rates stratified by background characteristics, age, and malaria endemicity. Prevalence of contextual factors (e.g., vaccination, nutrition) likely to influence U5CM were also assessed. Population attributable risk percentage (PAR%) estimates for malaria interventions and contextual factors that changed over time were used to estimate magnitude of impact on U5CM.

Results

Household ownership of insecticide-treated nets (ITNs) rose from near zero in 1999 to 64% (95% CI, 61.7–65.2) in 2010. Intermittent preventive treatment of malaria in pregnancy reached 26% (95% CI, 23.6–28.0) by 2010. Sulfadoxine-pyrimethamine replaced chloroquine in 2002 and artemisinin-based combination therapy was introduced in 2007. SAP among children 6–59 months declined 50% between 2005 (11.1%; 95% CI, 10.0–12.3%) and 2010 (5.5%; 95% CI, 4.7–6.4%) and U5CM declined by 45% between baseline (1995–9) and endpoint (2005–9), from 148 to 81 deaths/1000 live births, respectively. Mortality declined 55% among children 1–23 months of age in higher malaria endemicity areas. A large reduction in U5CM was attributable to ITNs (PAR% = 11) with other malaria interventions adding further gains. Multiple contextual factors also contributed to survival gains.

Conclusion

Marked declines in U5CM occurred in Tanzania between 1999 and 2010 with high impact from ITNs and ACTs. High-risk children (1–24 months of age in high malaria endemicity) experienced the greatest declines in mortality and SAP. Malaria control should remain a policy priority to sustain and further accelerate progress in child survival.     

Latent Transforming Growth Factor-β Binding Protein Domains Involved in Activation and Transglutaminase-dependent Cross-Linking of Latent Transforming Growth Factor-β

Transforming growth factor-β (TGF-β) is secreted by many cell types as part of a large latent complex composed of three subunits: TGF-β, the TGF-β propeptide, and the latent TGF-β binding protein (LTBP). To interact with its cell surface receptors, TGF-β must be released from the latent complex by disrupting noncovalent interactions between mature TGF-β and its propeptide. Previously, we identified LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGF-β. In this study, we demonstrate that LTBP-1 and large latent complex are substrates for transglutaminase. Furthermore, we show that the covalent association between LTBP-1 and the extracellular matrix is transglutaminase dependent, as little LTBP-1 is recovered from matrix digests prepared from cultures treated with transglutaminase inhibitors. Three polyclonal antisera to glutathione S–transferase fusion proteins containing amino, middle, or carboxyl regions of LTBP-1S were used to identify domains of LTBP-1 involved in crosslinking and formation of TGF-β by transglutaminase. Antibodies to the amino and carboxyl regions of LTBP-1S abrogate TGF-β generation by vascular cell cocultures or macrophages. However, only antibodies to the amino-terminal region of LTBP-1 block transglutaminase-dependent cross-linking of large latent complex or LTBP-1. To further identify transglutaminase-reactive domains within the amino-terminal region of LTBP-1S, mutants of LTBP-1S with deletions of either the amino-terminal 293 (ΔN293) or 441 (ΔN441) amino acids were expressed transiently in CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that ΔN293 LTBP-1S was matrix associated via a transglutaminasedependent reaction, whereas ΔN441 LTBP-1S was not. This suggests that residues 294–441 are critical to the transglutaminase reactivity of LTBP-1S.     

Effect of neutrophil elastase and its inhibitor EPI-hNE4 on transepithelial sodium transport across normal and cystic fibrosis human nasal epithelial cells

Background

Hyperactivity of the epithelial sodium (Na⁺) channel (ENaC) and increased Na⁺ absorption by airway epithelial cells leading to airway surface liquid dehydration and impaired mucociliary clearance are thought to play an important role in the pathogenesis of cystic fibrosis (CF) pulmonary disease. In airway epithelial cells, ENaC is constitutively activated by endogenous trypsin-like serine proteases such as Channel-Activating Proteases (CAPs). It was recently reported that ENaC activity could also be stimulated by apical treatment with human neutrophil elastase (hNE) in a human airway epithelial cell line, suggesting that hNE inhibition could represent a novel therapeutic approach for CF lung disease. However, whether hNE can also activate Na⁺ reabsorption in primary human nasal epithelial cells (HNEC) from control or CF patients is currently unknown.

Methods

We evaluated by short-circuit current (I_sc) measurements the effects of hNE and EPI-hNE4, a specific hNE inhibitor, on ENaC activity in primary cultures of HNEC obtained from control (9) and CF (4) patients.

Results

Neither hNE nor EPI-hNE4 treatments did modify I_sc in control and CF HNEC. Incubation with aprotinin, a Kunitz-type serine protease inhibitor that blocks the activity of endogenous CAPs, decreased I_sc by 27.6% and 54% in control and CF HNEC, respectively. In control and CF HNEC pretreated with aprotinin, hNE did significantly stimulate I_sc, an effect which was blocked by EPI-hNE4.

Conclusions

These results indicate that hNE does activate ENaC and transepithelial Na⁺ transport in both normal and CF HNEC, on condition that the activity of endogenous CAPs is first inhibited. The potent inhibitory effect of EPI-hNE4 on hNE-mediated ENaC activation observed in our experiments highlights that the use of EPI-hNE4 could be of interest to reduce ENaC hyperactivity in CF airways.     

Aquatic Ecosystem Responses to Rapid Recovery from Extreme Acidification and Metal Contamination in Lakes Near Wawa, Ontario

In the region northeast of Wawa, Ontario (Canada), many circumneutral lakes downwind of a nearby iron-sintering plant were strongly acidified (pH 3–4) in response to the emissions of large amounts of sulfur dioxide from 1939–1998. Following closure of the plant in 1998, lakewater pH has returned to circumneutral conditions due to the high buffering capacity of the local geological substrate. Prior paleolimnological analyses of dated sediment cores have detected some biological recovery among algal communities (diatoms and chrysophytes), although they have not returned to their pre-impact assemblages. Here we take a broader ecosystem approach, and build upon the algal analyses by examining cladoceran sedimentary assemblages, and spectrally-inferred chlorophyll a and dissolved organic carbon (DOC) from the same dated sediment cores. Similar to the algal communities, recent cladoceran sedimentary assemblages from three impacted lakes remain in an altered state relative to the pre-impact period (for example, increased relative abundances of Chydorus brevilabris and reduced cladoceran density in sediments). However, trends in the spectrally-inferred chlorophyll a and DOC were mixed, with long-term decreases in the study lake closest to the plant and long-term increases within the other lakes. Collectively, the multi-proxy paleolimnological analyses of these markedly acidified lakes demonstrate the delayed biological recovery from acidification (and differences in timing) across multiple trophic levels, despite the near-elimination of acid deposition almost a decade previously, which led to a striking recovery in lakewater pH and increased food availability.     

Long-term survival of Legionella pneumophila serogroup 1 under low-nutrient conditions and associated morphological changes

Abstract Extended survival of Legionella pneumophila , using both a clinical and an environmental isolate, was studied in drinking water, creek water, and estuarine water microcosms. Legionella populations were monitored by acridine orange direct counts (AODC) and viable count on buffered charcoal yeast extract agar amended with alpha-ketoglutarate (BCYEα). Initial colony counts of the clinical isolate in drinking and creek water microcosms were 2 × 10⁸ cfu/ml and, after incubation for 1.5 years, the plate counts decreased to 3 × 10⁶ cfu/ml. The AODC counts, however, did not change significantly. The clinical isolate in estuarine water decreased in plate counts to 10² (cfu/ml) over the same period. After incubation for 1.5 years at 15°C in the microcosms, Legionella plate counts of creek and drinking water decreased by two logs. Direct microscopic examination of aliquots removed from all microcosms revealed the presence of small bacilli, large bacilli and rare filamentous cells. The environmental isolate demonstrated only one colony morphology upon culture on BCYEα. Interestingly, after four months incubation in the microcosm, upon plating the clinical isolate on BCYEα, two distinct colony types were evident. Examination by immunofluorescent staining employing a monoclonal antibody against L. pneumophila revealed both bacillus and filamentous forms. The total cellular proteins of both morphotypes were examined by sodium dodecyl sulfate polyacrylyamide gel electrophoresis (SDS-PAGE), demonstrating identical protein patterns. Those Legionella cells remaining culturable during 1.5 years of incubation grew rapidly when transferred to BCYEα. Incubation was continued and it was found that some strains of L. pneumophila serogroup 1 can remain viable for longer than 2.4 years under low-nutrient conditions.     

The Microenvironment of Cervical Carcinoma Xenografts: Associations with Lymph Node Metastasis and Its Assessment by DCE-MRI

Poor disease-free and overall survival rates in locally advanced cervical cancer are associated with a tumor micro-environment characterized by extensive hypoxia, interstitial hypertension, and high lactate concentrations. The potential of gadolinium diethylenetriamine pentaacetic acid-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing the microenvironment and microenvironment-associated aggressiveness of cervical carcinomas was investigated in this preclinical study. CK-160 and TS-415 cervical carcinoma xenografts were used as tumor models. DCE-MRI was carried out at 1.5 T, and parametric images of K^trans and v_e were produced by pharmacokinetic analysis of the DCE-MRI series. Pimonidazole was used as a marker of hypoxia. A Millar catheter was used to measure tumor interstitial fluid pressure (IFP). The concentrations of glucose, adenosine triphosphate (ATP), and lactate were measured by induced metabolic bioluminescence imaging. High incidence of lymph node metastases was associated with high hypoxic fraction and high lactate concentration in CK-160 tumors and with high IFP and high lactate concentration in TS-415 tumors. Low K^trans was associated with high hypoxic fraction, low glucose concentration, and high lactate concentration in tumors of both lines and with high incidence of metastases in CK-160 tumors. Associations between v_e and microenvironmental parameters or metastatic propensity were not detected in any of the tumor lines. Taken together, this preclinical study suggests that K^trans is a potentially useful biomarker for poor outcome of treatment in advanced cervical carcinoma. The possibility that K^trans may be used to identify patients with cervical cancer who are likely to benefit from particularly aggressive treatment merits thorough clinical investigations.