Characterization of troponin T dilated cardiomyopathy mutations in the fetal troponin isoform

The major goal of this study was to elucidate how troponin T (TnT) dilated cardiomyopathy (DCM) mutations in fetal TnT and fetal troponin affect the functional properties of the fetal heart that lead to infantile cardiomyopathy. The DCM mutations R141W and DeltaK210 were created in the TnT1 isoform, the primary isoform of cardiac TnT in the embryonic heart. In addition to a different TnT isoform, a different troponin I (TnI) isoform, slow skeletal TnI (ssTnI), is the dominant isoform in the embryonic heart. In skinned fiber studies, TnT1-wild-type (WT)-treated fibers reconstituted with cardiac TnI.troponin C (TnC) or ssTnI.TnC significantly increased Ca(2+) sensitivity of force development when compared with TnT3-WT-treated fibers at both pH 7.0 and pH 6.5. Porcine cardiac fibers treated with TnT1 that contained the DCM mutations (R141W and DeltaK210), when reconstituted with either cardiac TnI.TnC or ssTnI.TnC, significantly decreased Ca(2+) sensitivity of force development compared with TnT1-WT at both pH values. The R141W mutation, which showed no significant change in the Ca(2+) sensitivity of force development in the TnT3 isoform, caused a significant decrease in the TnT1 isoform. The DeltaK210 mutation caused a greater decrease in Ca(2+) sensitivity and maximal isometric force development compared with the R141W mutation in both the fetal and adult TnT isoforms. When complexed with cardiac TnI.TnC or ssTnI.TnC, both TnT1 DCM mutations strongly decreased maximal actomyosin ATPase activity as compared with TnT1-WT. Our results suggest that a decrease in maximal actomyosin ATPase activity in conjunction with decreased Ca(2+) sensitivity of force development may cause a severe DCM phenotype in infants with the mutations.     

subito encodes a kinesin-like protein required for meiotic spindle pole formation in Drosophila melanogaster

The female meiotic spindle lacks a centrosome or microtubule-organizing center in many organisms. During cell division, these spindles are organized by the chromosomes and microtubule-associated proteins. Previous studies in Drosophila melanogaster implicated at least one kinesin motor protein, NCD, in tapering the microtubules into a bipolar spindle. We have identified a second Drosophila kinesin-like protein, SUB, that is required for meiotic spindle function. At meiosis I in males and females, sub mutations affect only the segregation of homologous chromosomes. In female meiosis, sub mutations have a similar phenotype to ncd; even though chromosomes are joined by chiasmata they fail to segregate at meiosis I. Cytological analyses have revealed that sub is required for bipolar spindle formation. In sub mutations, we observed spindles that were unipolar, multipolar, or frayed with no defined poles. On the basis of these phenotypes and the observation that sub mutations genetically interact with ncd, we propose that SUB is one member of a group of microtubule-associated proteins required for bipolar spindle assembly in the absence of the centrosomes. sub is also required for the early embryonic divisions but is otherwise dispensable for most mitotic divisions.     

Selection of the sex‐linked inhibitor of apoptosis in mountain pine beetle (Dendroctonus ponderosae) driven by enhanced expression during early overwintering

The mountain pine beetle (Dendroctonus ponderosae) is an insect native to western North America; however, its geographical range has recently expanded north in BC and east into Alberta. To understand the population structure in the areas of expansion, 16 gene‐linked microsatellites were screened and compared to neutral microsatellites using outlier analyses of F_st and F_ct values. One sex‐linked gene, inhibitor of apoptosis (IAP), showed a strong signature of positive selection for neo‐X alleles and was analyzed for evidence of adaptive variation. Alleles of IAP were sequenced, and differences between the neo‐X and neo‐Y alleles were consistent with neutral evolution suggesting that the neo‐Y allele may not be under functional constraints. Neo‐Y alleles were amplified from gDNA, but not effectively from cDNA, suggesting that there was little IAP expression from neo‐Y alleles. There were no differences in overall IAP expression between males and females with the common northern neo‐X allele suggesting that the neo‐X allele in males compensates for the reduced expression of neo‐Y alleles. However, males lacking the most common northern neo‐X allele thought to be selected for in northern populations had reduced overall IAP expression in early October—at a time when beetles are preparing for overwintering. This suggests that the most common allele may have more rapid upregulation. The reduced function of neo‐Y alleles of IAP suggested by both sequence differences and lower levels of expression may foster a highly selective environment for neo‐X alleles such as the common northern allele with more efficient upregulation.     

Unequal evolutionary rates in the human immunodeficiency virus type 1 (HIV-1) pandemic: the evolutionary rate of HIV-1 slows down when the epidemic rate increases

HIV-1 sequences in intravenous drug user (IDU) networks are highly homogenous even after several years, while this is not observed in most sexual epidemics. To address this disparity, we examined the human immunodeficiency virus type 1 (HIV-1) evolutionary rate on the population level for IDU and heterosexual transmissions. All available HIV-1 env V3 sequences from IDU outbreaks and heterosexual epidemics with known sampling dates were collected from the Los Alamos HIV sequence database. Evolutionary rates were calculated using phylogenetic trees with a t test root optimization of dated samples. The evolutionary rate of HIV-1 subtype A1 was found to be 8.4 times lower in fast spread among IDUs in the former Soviet Union (FSU) than in slow spread among heterosexual individuals in Africa. Mixed epidemics (IDU and heterosexual) showed intermediate evolutionary rates, indicating a combination of fast- and slow-spread patterns. Hence, if transmissions occur repeatedly during the initial stage of host infection, before selective pressures of the immune system have much impact, the rate of HIV-1 evolution on the population level will decrease. Conversely, in slow spread, where HIV-1 evolves under the pressure of the immune system before a donor infects a recipient, the virus evolution at the population level will increase. Epidemiological modeling confirmed that the evolutionary rate of HIV-1 depends on the rate of spread and predicted that the HIV-1 evolutionary rate in a fast-spreading epidemic, e.g., for IDUs in the FSU, will increase as the population becomes saturated with infections and the virus starts to spread to other risk groups.     

Temperature Dependent Soret Spectral Band Shifts Accompany Human CN-Mesohemoglobin Assembly

The interaction between human apohemoglobin A and CN-Mesohemin, a monomeric non-native heme derivative, was probed by Soret spectrophotometric titrations in 0.05 M potassium phosphate buffer, pH 7 at varied temperatures. Hypsochromic shifts in the absorbance maxima were observed at all temperatures below 10°C. First derivative spectroscopy of CN-Mesohemin titrations was used to provide further evidence of a spectral shift upon CN-Mesohemoglobin assembly. Findings of Soret Spectral shifts demonstrate a preference for the α chain heme site by CN-Mesohemin indicative of semi-α-hemoglobin intermediate formation. CN-Mesohemin, a derivative with peripheral 2,4 ethyl groups, does not possess the extended conjugation seen for native CN-Protohemin with its 2,4 vinyl groups. Indeed, reduced polarity of CN-Mesohemin over that of CN-Protohemin resulted in distinct temperature dependencies. Molecular visualization and protein-ligand interaction analysis targeted a functionally diverse residue unique to the α-chain. Tyrosine-42 (a polar/non-polar amino acid) appeared to play a prominent role in the assembly process.     

A conserved carboxylesterase is a SUPPRESSOR OF AVRBST-ELICITED RESISTANCE in Arabidopsis

AvrBsT is a type III effector from Xanthomonas campestris pv vesicatoria that is translocated into plant cells during infection. AvrBsT is predicted to encode a Cys protease that targets intracellular host proteins. To dissect AvrBsT function and recognition in Arabidopsis thaliana, 71 ecotypes were screened to identify lines that elicit an AvrBsT-dependent hypersensitive response (HR) after Xanthomonas campestris pv campestris (Xcc) infection. The HR was observed only in the Pi-0 ecotype infected with Xcc strain 8004 expressing AvrBsT. To create a robust pathosystem to study AvrBsT immunity in Arabidopsis, the foliar pathogen Pseudomonas syringae pv tomato (Pst) strain DC3000 was engineered to translocate AvrBsT into Arabidopsis by the Pseudomonas type III secretion (T3S) system. Pi-0 leaves infected with Pst DC3000 expressing a Pst T3S signal fused to AvrBsT-HA (AvrBsTHYB-HA) elicited HR and limited pathogen growth, confirming that the HR leads to defense. Resistance in Pi-0 is caused by a recessive mutation predicted to inactivate a carboxylesterase known to hydrolyze lysophospholipids and acylated proteins in eukaryotes. Transgenic Pi-0 plants expressing the wild-type Columbia allele are susceptible to Pst DC3000 AvrBsTHYB-HA infection. Furthermore, wild-type recombinant protein cleaves synthetic p-nitrophenyl ester substrates in vitro. These data indicate that the carboxylesterase inhibits AvrBsT-triggered phenotypes in Arabidopsis. Here, we present the cloning and characterization of the SUPPRESSOR OF AVRBST-ELICITED RESISTANCE1.     

Characterization of Novel Lactobacillus fermentum from Curd Samples of Indigenous Cows from Malnad Region,Karnataka, for their Aflatoxin B1 Binding and Probiotic Properties

Thirty-four isolates of Lactobacillus spp. (LAB) from 34 curd samples were evaluated for their aflatoxin B₁ (AFB₁) binding and probiotic properties. Upon characterization, four LAB isolates (LC3/a, LC4/c, LC/5a, and LM13/b) were found to be effective in removing AFB₁ from culture media with a capacity of above 75%. Staining reaction, biochemical tests, pattern of sugar utilization, and 16s rRNA gene sequence analysis revealed the identity of all the four isolates as L. fermentum. All of them could tolerate acidic pH, salt, and bile, which promise the use of these probiotic bacterial isolates for human applications. These isolates showed poor hydrophobicity and higher auto-aggregation properties. All L. fermentum isolates were found susceptible to gentamycin, chloramphenicol, cefoperazone, ampicillin, and resistant to ciprofloxacin and vancomycin. Results of hemolytic and DNase activity indicated their nonpathogenic nature. Though all L. fermentum isolates found inhibiting the growth of Salmonella ebony, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, maximum inhibition was obtained with isolate LC5/a. Kinetic studies revealed that all four bacteria required a minimum of 2 h to reach stationary phase of AFB₁ binding. AFB₁ binding ability varied from 66 to 85.2% among these four isolates. Bile (0.4%) was significant (P ≤ 0.05) in reducing the AFB₁ binding property of isolates LC3/a, LC4/c, and LM13/b, while increased AFB₁ binding ability was recorded at acidic pH (2.0). AFB₁ binding properties of isolate LC5/a were found least affected by acidic pH and bile. The findings of our study revealed the higher efficiency of L. fermentum isolate LC5/a in reducing the bioavailability of AFB1 in gut, and additionally, it improves the consumers’ health by its various probiotic characters. These beneficial characters, L. fermentum isolates, promise them to use as probiotic formulations alone or in combinations with other beneficial probiotic-bacterial isolates.

     

Soret Spectroscopic and Molecular Graphic Analysis of Human Semi-β-Hemoglobin Formation

The interaction of heme-free α (α^o) and heme-containing β (β^h) chains of human hemoglobin has been monitored in 0.1 M potassium phosphate buffer, pH 7 or 8, at [5°C. Soret zero and first-derivative spectra were consistent with a uniform association reaction. Stopped-flow investigations demonstrated association rates on the order of 10⁷ M^?1 s^?1. This was 100-fold more rapid than the reported rate of combination of α^h and β^h proteins. This encounter-like rate of semi-β-hemoglobin (α^oβ^h) formation was increased by raising the pH from 7 to 8. pH change is known to affect the spatial arrangement of AB—GH helical entities. Molecular graphic analysis of modeled α^o protein superimposed over native α^h protein revealed an apo Mb-like structure with well-defined AB—GH segments. Repositioning of these core helical segments, resulting in increased conformational freedom of the α₁β₁ interface, was apparently responsible for the enhanced association properties of the α^o protein.