Rec8 Guides Canonical Spo11 Distribution along Yeast Meiotic Chromosomes

Spo11-mediated DNA double-strand breaks (DSBs) that initiate meiotic recombination are temporally and spatially controlled. The meiotic cohesin Rec8 has been implicated in regulating DSB formation, but little is known about the features of their interplay. To elucidate this point, we investigated the genome-wide localization of Spo11 in budding yeast during early meiosis by chromatin immunoprecipitation using high-density tiling arrays. We found that Spo11 is dynamically localized to meiotic chromosomes. Spo11 initially accumulated around centromeres and thereafter localized to arm regions as premeiotic S phase proceeded. During this stage, a substantial proportion of Spo11 bound to Rec8 binding sites. Eventually, some of Spo11 further bound to both DSB and Rec8 sites. We also showed that such a change in a distribution of Spo11 is affected by hydroxyurea treatment. Interestingly, deletion of REC8 influences the localization of Spo11 to centromeres and in some of the intervals of the chromosomal arms. Thus, we observed a lack of DSB formation in a region-specific manner. These observations suggest that Rec8 would prearrange the distribution of Spo11 along chromosomes and will provide clues to understanding temporal and spatial regulation of DSB formation.     

Microtubule affinity–regulating kinase 4 with an Alzheimer's disease-related mutation promotes tau accumulation and exacerbates neurodegeneration

Accumulation of the microtubule-associated protein tau is associated with Alzheimer''s disease (AD). In AD brain, tau is abnormally phosphorylated at many sites, and phosphorylation at Ser-262 and Ser-356 plays critical roles in tau accumulation and toxicity. Microtubule affinity–regulating kinase 4 (MARK4) phosphorylates tau at those sites, and a double de novo mutation in the linker region of MARK4, ΔG316E317D, is associated with an elevated risk of AD. However, it remains unclear how this mutation affects phosphorylation, aggregation, and accumulation of tau and tau-induced neurodegeneration. Here, we report that MARK4^ΔG316E317D increases the abundance of highly phosphorylated, insoluble tau species and exacerbates neurodegeneration via Ser-262/356–dependent and –independent mechanisms. Using transgenic Drosophila expressing human MARK4 (MARK4^wt) or a mutant version of MARK4 (MARK4^ΔG316E317D), we found that coexpression of MARK4^wt and MARK4^ΔG316E317D increased total tau levels and enhanced tau-induced neurodegeneration and that MARK4^ΔG316E317D had more potent effects than MARK4^wt. Interestingly, the in vitro kinase activities of MARK4^wt and MARK4^ΔG316E317D were similar. When tau phosphorylation at Ser-262 and Ser-356 was blocked by alanine substitutions, MARK4^wt did not promote tau accumulation or exacerbate neurodegeneration, whereas coexpression of MARK4^ΔG316E317D did. Both MARK4^wt and MARK4^ΔG316E317D increased the levels of oligomeric forms of tau; however, only MARK4^ΔG316E317D further increased the detergent insolubility of tau in vivo. Together, these findings suggest that MARK4^ΔG316E317D increases tau levels and exacerbates tau toxicity via a novel gain-of-function mechanism and that modification in this region of MARK4 may affect disease pathogenesis.     

An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.     

Is Poverty Driving Borana Herders in Southern Ethiopia to Crop Cultivation?

This study addresses whether or not crop cultivation by Borana herders in southern Ethiopia is motivated by poverty since 80% of the households belong to poor wealth classes (i.e., poor, very poor and destitute). Yet our findings showed little evidence that Borana communities have become self-sufficient in grain production. Compared to wealthy households, poor households generally cultivated the least land and sampled households, producing yields only 31% of the Ethiopian national average. Grain per capita met only 26% of the annual requirement per person, equivalent to three to four months of self-sufficiency per household. The livelihood response model (LRM) developed for testing the relationship between extent of croplands and household wealth showed that poverty alone cannot be motivating herders to cultivate crops. Factors such as shortage of labor, lack of sufficient traction animals, and unreliable rainfall also need to be considered. Crop cultivation has not enabled self-sufficiency, but it has resulted in fragmented grazing lands. Future policies address changes in land use, including improving soil fertility through manure-nutrient transfers, by promoting better integration of crop cultivation and pastoralism. Research is needed to (a) understand household time allocation between crop cultivation and livestock management, (b) improve the LRM by considering temporal variability in the wealth of households and extent of cultivated lands, and (c) understand the role of poverty in motivating the adoption of alternative livelihood coping strategies.     

Involvement of the 3’ Untranslated Region in Encapsidation of the Hepatitis C Virus

Although information regarding morphogenesis of the hepatitis C virus (HCV) is accumulating, the mechanism(s) by which the HCV genome encapsidated remains unknown. In the present study, in cell cultures producing HCV, the molecular ratios of 3’ end- to 5’ end-regions of the viral RNA population in the culture medium were markedly higher than those in the cells, and the ratio was highest in the virion-rich fraction. The interaction of the 3’ untranslated region (UTR) with Core in vitro was stronger than that of the interaction of other stable RNA structure elements across the HCV genome. A foreign gene flanked by the 3’ UTR was encapsidated by supplying both viral NS3-NS5B proteins and Core-NS2 in trans. Mutations within the conserved stem-loops of the 3’ UTR were observed to dramatically diminish packaging efficiency, suggesting that the conserved apical motifs of the 3´ X region are important for HCV genome packaging. This study provides evidence of selective packaging of the HCV genome into viral particles and identified that the 3’ UTR acts as a cis-acting element for encapsidation.     

X-ray crystal structure and molecular dynamics simulation of bovine pancreas phospholipase A2–n-dodecylphosphorylcholine complex

The crystal structure of n-dodecylphosphorylcholine (n-C₁₂PC)–bovine pancreas phospholipase A₂ (PLA₂) complex provided the following structural.characteristics: (1) the dodecyl chain of n-C₁₂PC was located at the PLA₂ N -terminal helical region by hydrophobic interactions, which corresponds to the binding pocket of 2-acyl fatty acid chain (β-chain) of the substrate phospholipid, (2) the region from Lys-53 to Lys-56 creates a cholinereceiving pocket of n-C₁₂PC and (3) the N-termillal group of Ala-1 shifts significantly toward the Tyr-52 OH group by the binding of the n-C1₂PC inhibitor. Since the accuracy of the X-ray analysis (R = 0.275 at 2.3 Å resolution) was insufficient to establish these important X-ray insights, the complex structure was further investigated through the molecular dynamics (M D) simulation, assuming a system in aqueous solution at 310K. The M D simulation covering 176 ps showed that the structural characteristics observed by X-ray analysis are intrinsic and also stable in the dynamic state. Furthermore, the M D simulation made clear that the PLA₂ binding pocket is large enough to permit the conformational fluctuation of the n-C₁₂PC hydrocarbon chain. © 1994 Wiley-Liss, Inc. © 1994 Wiley-Liss, Inc.     

Effect of auxin on Mg++-activated and -inhibited ATPases from mung bean hypocotyls

Mg⁺⁺-activated (A) and -inhibited (B) ATPases were isolatedfrom dark-grown mung bean hypocotyls and the effect of in vivoand in vitro IAA-treatment on these ATPases was observed. The activity of the A-ATPase, which was localized in the plasmamembrane-rich fractions, was enhanced by exogenously added l0^–10M IAA (ca. 35%). The stimulatory effect of in vitro IAA-treatmentwas particularly found in the A ATPase isolated from sectionstreated with 10^–5 M IAA for 60 min (ca. 280%). The activity of the B-ATPase, which was localized in the solublefraction of the cell homogenate, was markedly increased by invivo treatment with 10^–7 M IAA for 60 min (ca. 360%).The stimulatory effect of IAA-treatment was observed even ifprotein synthesis was inhibited by cycloheximide. These results suggest that the enhancement of the activitiesof the A- and B-ATPases caused by in vivo and in vitro IAA-treatmentsoccur as a result of the activation of already present enzymes,rather than of the synthesis of these enzymes. ¹Present address: Institute for Plant Virus Research, 959 Aobacho,Chiba 280, Japan. (Received March 27, 1974; )     

Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: Comparison of wild-type protein and active-site mutant D27E

To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl₂ concentration-dependence of the ¹H–¹⁵N HSQC spectra of the wild-type DHFR–folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased K_m and K_d values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250 MPa together with negative activation volumes of ? 4.0 or ? 4.8 mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7 mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme.