Hypoxia-induced left ventricular dysfunction in myoglobin-deficient mice

Myoglobin-deficient mice are viable and have preserved cardiac function due to their ability to mount a complex compensatory response involving increased vascularization and the induction of the hypoxia gene program (hypoxia-inducible factor-1alpha, endothelial PAS, heat shock protein27, etc.). To further define and explore functional roles for myoglobin, we challenged age- and gender-matched wild-type and myoglobin-null mice to chronic hypoxia (10% oxygen for 1 day to 3 wk). We observed a 30% reduction in cardiac systolic function in the myoglobin mutant mice exposed to chronic hypoxia with no changes observed in the wild-type control hearts. The cardiac dysfunction observed in the hypoxic myoglobin-null mice was reversible with reexposure to normoxic conditions and could be prevented with treatment of an inhibitor of nitric oxide (NO) synthases. These results support the conclusion that hypoxia-induced cardiac dysfunction in myoglobin-null mice occurs via a NO-mediated mechanism. Utilizing enzymatic assays for NO synthases and immunohistochemical analyses, we observed a marked induction of inducible NO synthase in the hypoxic myoglobin mutant ventricle compared with the wild-type hypoxic control ventricle. These new data establish that myoglobin is an important cytoplasmic cardiac hemoprotein that functions in regulating NO homeostasis within cardiomyocytes.     

Bryostatin-1 enhances barrier function in T84 epithelia through PKC-dependent regulation of tight junction proteins

Protein kinase C (PKC) is known to regulate epithelial barrier function. However, the effect of specific PKC isozymes, and their mechanism of action, are largely unknown. We determined that the nonphorbol ester PKC agonist bryostatin-1 increased transepithelial electrical resistance (TER), a marker of barrier function, in confluent T84 epithelia. Bryostatin-1, which has been shown to selectively activate PKC-, -, and - (34), was associated with a shift in the subcellular distribution of the tight junction proteins claudin-1 and ZO-2 from a detergent-soluble fraction into a detergent-insoluble fraction. Bryostatin-1 also led to the appearance of a higher-molecular-weight form of occludin previously shown to correspond to protein phosphorylation. These changes were attenuated by the conventional and novel PKC inhibitor Gö-6850 but not the conventional PKC inhibitor Gö-6976 or the PKC- inhibitor röttlerin, implicating a novel isozyme, likely PKC-. The results suggest that enhanced epithelial barrier function induced by bryostatin-1 involves a PKC--dependent signaling pathway leading to recruitment of claudin-1 and ZO-2, and phosphorylation of occludin, into the tight junctional complex. protein kinase C; epithelial barrier function     

Epigenetic regulation of insulin-like growth factor binding protein-3 (IGFBP-3) in cancer

Epigenetics refers to heritable changes in gene expression that are independent of alterations in DNA sequence. It is now accepted that disruption of epigenetic mechanisms plays a key role in the pathogenesis of cancer: culminating in altered gene function and malignant cellular transformation. DNA methylation and histone modifications are the most widely studied changes but non-coding RNAs such as miRNAs are also considered part of the epigenetic machinery. The insulin-like growth factor (IGF) axis is composed of two ligands, IGF-I and –II, their receptors and six high affinity IGF binding proteins (IGFBPs). The IGF axis plays a key role in cancer development and progression. As IGFBP genes have consistently been identified among the most common to be aberrantly altered in tumours, this review will focus on epigenetic regulation of IGFBP-3 in cancer for which the majority of evidence has been obtained.     

Impaired Innate COPD Alveolar Macrophage Responses and Toll-Like Receptor-9 Polymorphisms

Background

Dysfunctional innate responses of alveolar macrophages to nontypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae contribute to morbidity in chronic obstructive pulmonary disease (COPD). Our earlier studies discovered impaired COPD alveolar macrophage responses to Toll-like receptor (TLR) ligands of nontypeable H. influenzae and provide rationale for further evaluation of TLR signaling. While the role of TLR single nucleotide polymorphisms is increasingly recognized in inflammatory diseases, TLR single nucleotide polymorphisms in COPD have only recently been explored. We hypothesized that specific TLR polymorphisms are associated with dysfunctional innate immune COPD alveolar macrophage responses and investigated polymorphisms of TLR2(Arg753Gln), TLR4(Thr399Ile; Asp299Gly), and TLR9(T1486C; T1237C).

Methods

DNA was purified from cells of 1) healthy nonsmokers (n = 20); 2) COPD ex-smokers (n = 83); 3) COPD active smokers (n = 93). DNA amplifications (polymerase chain reaction) were performed for each SNP. Alveolar macrophages from each group were incubated with nontypeable H. influenzae, M. catarrhalis and S. pneumoniae. Cytokine induction of macrophage supernatants was measured and the association with TLR single nucleotide polymorphism expression was determined.

Results

No significant inter-group differences in frequency of any TLR SNP existed. However both TLR9 single nucleotide polymorphisms were expressed in high frequency. Among COPD ex-smokers, diminished IL-8 responsiveness to nontypeable H. influenzae, M. catarrhalis and S. pneumoniae was strongly associated with carriage of TLR9(T1237C) (p = 0.02; p = 0.008; p = 0.02), but not TLR9(T1486C). Carriage of TLR9(T1237C), but not TLR9(T1486C), correlated with diminished FEV₁%predicted (p = 0.037).

Conclusion

Our results demonstrate a notable association of TLR9(T1237C) expression with dysfunctional innate alveolar macrophage responses to respiratory pathogens and with severity of COPD.     

Synthesis of site-specific methotrexate-IgG conjugates

Summary With a view to increasing drug incorporation without loss of antibody activity, tritium-labeled methotrexate (MTX) was covalently linked to a polyclonal rabbit IgG antibody against bovine serum albumin and a monoclonal mouse IgG antibody against human renal cancer (Dal K20) by a site-specific method based on hydrazone bond formation between MTX hydrazide and the aldehyde groups generated by periodate oxidation of carbohydrate moieties in IgG (which are uncommon in the antigen-binding region). These conjugates were compared with the corresponding non-site-specific MTX-IgG conjugates produced by the N-hydroxysuccinimide active-ester method with regard to synthesis, stability, retention of antibody activity, inhibition of the target enzyme dihydrofolate reductase and antitumor effect. Incorporation levels achieved with the hydrazide method were no greater than with the active-ester method, typically 6–7 mol MTX/mol IgG. Approximately the same dihydrofolate-reductase-inhibitory capacity was observed for MTX bound by either method. Hydrazide conjugates lost bound drug more rapidly than active-ester conjugates on freezing and thawing, on incubation at 37° C and 51° C, and in the presence of serum or rat liver homogenates. Exposure to rat liver homogenates at 37° C, pH 4.6, for 24 h led to the loss of 50%–60% of the bound drug from hydrazide conjugates compared to 20%–30% from the active ester conjugates. Bio-Gel P-2 chromatography of low-molecular-mass fractions, obtained after exposure of each of the conjugates to liver homogenates, revealed the presence of a compound that had the same elution volume and R _F on thin-layer chromatography as free MTX. Enzyme-linked immunosorbent assay showed loss of antibody activity of both types of conjugates at 51° C and on freezing and thawing. In a clonogenic assay, the active-ester conjugate of Dal K20 appeared to be equally effective or slightly better as a tumor inhibitor than the corresponding hydrazide conjugate. The hydrazide method may be useful in linking MTX to those monoclonal antibodies that tend to denature when subjected to the active-ester method of linkage. Abbreviations used: aBSA, rabbit anti-(bovine serum albumin) IgG; EDCI, 3-ethyl-1-(3-dimethylaminopropyl)carbodiimide; ELISA, enzyme-linked immunosorbant assay; IC₅₀, concentration giving 50% inhibition; MTX, methotrexate; MTXAE, N-hydroxy-succinimide-based active ester of MTX; MTXAE-IgG, MTX-IgG conjugate prepared by the active-ester method; MTXH, methotrexate hydrazide; MTXH-IgG, MTX-IgG conjugate prepared by the hydrazide method; PBS, 0.01 M sodium phosphate, pH 7.1, containing 0.145 M sodium chloride; TLC, thin-layer chromatography     

The usage of marker traits in plants for evaluation of resistance of the winter wheat to pest insects

A new method of selection of the winter wheat varieties has been tested for resistance to the pest insects' complex by the traits of plants that are the markers of plant resistance. It makes it possible to use this method from year to year independently of the pests' density.     

The Spatial Dynamics of Dengue Virus in Kamphaeng Phet,Thailand

Background

Dengue is endemic to the rural province of Kamphaeng Phet, Northern Thailand. A decade of prospective cohort studies has provided important insights into the dengue viruses and their generated disease. However, as elsewhere, spatial dynamics of the pathogen remain poorly understood. In particular, the spatial scale of transmission and the scale of clustering are poorly characterized. This information is critical for effective deployment of spatially targeted interventions and for understanding the mechanisms that drive the dispersal of the virus.

Methodology/Principal Findings

We geocoded the home locations of 4,768 confirmed dengue cases admitted to the main hospital in Kamphaeng Phet province between 1994 and 2008. We used the phi clustering statistic to characterize short-term spatial dependence between cases. Further, to see if clustering of cases led to similar temporal patterns of disease across villages, we calculated the correlation in the long-term epidemic curves between communities. We found that cases were 2.9 times (95% confidence interval 2.7–3.2) more likely to live in the same village and be infected within the same month than expected given the underlying spatial and temporal distribution of cases. This fell to 1.4 times (1.2–1.7) for individuals living in villages 1 km apart. Significant clustering was observed up to 5 km. We found a steadily decreasing trend in the correlation in epidemics curves by distance: communities separated by up to 5 km had a mean correlation of 0.28 falling to 0.16 for communities separated between 20 km and 25 km. A potential explanation for these patterns is a role for human movement in spreading the pathogen between communities. Gravity style models, which attempt to capture population movement, outperformed competing models in describing the observed correlations.

Conclusions

There exists significant short-term clustering of cases within individual villages. Effective spatially and temporally targeted interventions deployed within villages may target ongoing transmission and reduce infection risk.     

Effect of Hypoxia on Expression of Selected Proteins Involved in Regulation of Apoptotic Activity in Striatum of Newborn Piglets

The levels of selected neuroregulatory proteins that inhibit or promote apoptotic cell death were measured in the striatum of piglets subjected to precisely controlled 1 h hypoxic insult followed by 0, 2 and 4 h recovery and compared to sham operated animals. The anti-apoptotic proteins: there were increases in Survivin at 0 (157%, P = 0.031) and 4 h (171%, P = 0.033), in Bcl-XL at 0 (138%, P = 0.028) and 4 h (143%, P = 0.007), in VEGF at 4 h (185%, P = 0.019) and Hsp27 at 2 h (144%, P = 0.05) and 4 h (143%, P = 0.05). The pro-apoptotic proteins: caspases-1 and 7 increased at 4 h (135%, P = 0.05) and (129%, P = 0.038), respectively. Bim increased after 4 h (115%, P = 0.028), Apoptosis Inducing Factor after 2 h (127%, P = 0.048) and Calpain after 4 h (143% of control, P = 0.04). Hypoxia causes increase in levels of both anti- and pro-apoptotic proteins. Their relative activity determines the outcome in terms of cell damage and neuronal deficit.