An ana2/ctp/mud complex regulates spindle orientation in Drosophila neuroblasts

Drosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. Here, we identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. We isolated two ana2 alleles that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/maintain the association of Mud with Pins at the apical cortex. Our findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function to?orient the mitotic spindle during NB asymmetric division.     

Characterization of acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme of human small intestine

Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme plays a significant role in dietary triacylglycerol (TAG) absorption in the small intestine. However, the characteristics of human intestinal DGAT enzyme have not been examined in detail. The aim of our study was to characterize the human intestinal DGAT enzyme by examining acyl-CoA specificity, temperature dependency, and selectivity for 1,2-diacylglycerol (DAG) or 1,3-DAG. We detected DGAT activity of human intestinal microsome and found that the acyl-CoA specificity and temperature dependency of intestinal DGAT coincided with those of recombinant human DGAT1. To elucidate the selectivity of human intestinal DGAT to 1,2-DAG or 1,3-DAG, we conducted acyl-coenzyme A:monoacylglycerol acyltransferase assays using 1- or 2-monoacylglycerol (MAG) as substrates. When 2-MAG was used as acyl acceptor, both 1,2-DAG and TAG were generated; however, when 1-MAG was used, 1,3-DAG was predominantly observed and little TAG was detected. These findings suggest that human small intestinal DGAT, which is mainly encoded by DGAT1, utilizes 1,2-DAG as the substrate to form TAG. This study will contribute to understand the lipid absorption profile in the small intestine.     

Endogenous synthesis of corticosteroids in the hippocampus

Background

Brain synthesis of steroids including sex-steroids is attracting much attention. The endogenous synthesis of corticosteroids in the hippocampus, however, has been doubted because of the inability to detect deoxycorticosterone (DOC) synthase, cytochrome P450(c21).

Methodology/Principal Findings

The expression of P450(c21) was demonstrated using mRNA analysis and immmunogold electron microscopic analysis in the adult male rat hippocampus. DOC production from progesterone (PROG) was demonstrated by metabolism analysis of ³H-steroids. All the enzymes required for corticosteroid synthesis including P450(c21), P450(2D4), P450(11β1) and 3β-hydroxysteroid dehydrogenase (3β-HSD) were localized in the hippocampal principal neurons as shown via in situ hybridization and immunoelectron microscopic analysis. Accurate corticosteroid concentrations in rat hippocampus were determined by liquid chromatography-tandem mass spectrometry. In adrenalectomized rats, net hippocampus-synthesized corticosterone (CORT) and DOC were determined to 6.9 and 5.8 nM, respectively. Enhanced spinogenesis was observed in the hippocampus following application of low nanomolar (10 nM) doses of CORT for 1 h.

Conclusions/Significance

These results imply the complete pathway of corticosteroid synthesis of ‘pregnenolone →PROG→DOC→CORT’ in the hippocampal neurons. Both P450(c21) and P450(2D4) can catalyze conversion of PROG to DOC. The low nanomolar level of CORT synthesized in hippocampal neurons may play a role in modulation of synaptic plasticity, in contrast to the stress effects by micromolar CORT from adrenal glands.     

Clonal spread of antimicrobial-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates among pups in two kennels

Although the dog breeding industry is common in many countries, the presence of antimicrobial resistant bacteria among pups in kennels has been infrequently investigated. This study was conducted to better understand the epidemiology of antimicrobial-resistant Escherichia coli isolates from kennel pups not treated with antimicrobials. We investigated susceptibilities to 11 antimicrobials, and prevalence of extended-spectrum β-lactamase (ESBL) in 86 faecal E. coli isolates from 43 pups in two kennels. Genetic relatedness among all isolates was assessed using pulsed-field gel electrophoresis (PFGE). Susceptibility tests revealed that 76% of the isolates were resistant to one or more of tested antimicrobials, with resistance to dihydrostreptomycin most frequently encountered (66.3%) followed by ampicillin (60.5%), trimethoprim-sulfamethoxazole (41.9%), oxytetracycline (26.7%), and chloramphenicol (26.7%). Multidrug resistance, defined as resistance against two or more classes of antimicrobials, was observed in 52 (60.5%) isolates. Three pups in one kennel harboured SHV-12 ESBL-producing isolates. A comparison between the two kennels showed that frequencies of resistance against seven antimicrobials and the variation in resistant phenotypes differed significantly. Analysis by PFGE revealed that clone sharing rates among pups of the same litters were not significantly different in both kennels (64.0% vs. 88.9%), whereas the rates among pups from different litters were significantly different between the two kennels (72.0% vs. 33.3%, P < 0.05). The pups in the two kennels had antimicrobial-resistant E. coli clones, including multidrug-resistant and ESBL-producing clones. It is likely that resistant and susceptible bacteria can clonally spread among the same and/or different litters thus affecting the resistance prevalence.     

A review of spatial-explicit factors determining spatial distribution of land use/land-use change

Land development is necessary for human progress, but its impact has resulted in the degradation of ecosystem services not only locally and regionally, but globally as well. Human behavior toward land use/land-use change (LULUC) must be examined and fully understood in order to achieve better land management. Several studies were recently conducted on LULUC patterns, suggesting a relationship between spatial distribution of LULUC and land attributes. We reviewed these studies and listed the factors determining spatial distribution of LULUC, and then we categorized them into: (1) socioeconomic factors, subcategorized into accessibility, local community development, spatial configuration, and political restrictions; and (2) natural environmental factors, subcategorized into topography and productivity. Here, we discuss the effects of these factors, especially road construction as a socioeconomic, accessibility factor, and slope as a natural environmental, topography factor. We also discuss the future work required to provide the tools for better land management.     

Intestinal and renal adaptation to a low-Pi diet of type II NaPi cotransporters in vitamin D receptor- and 1alphaOHase-deficient mice

Intake of a low-phosphate diet stimulates transepithelial transport of P_i in small intestine as well as in renal proximal tubules. In both organs, this is paralleled by a change in the abundance of the apically localized NaP_i cotransporters NaP_i type IIa (NaP_i-IIa) and NaP_i type IIb (NaP_i-IIb), respectively. Low-P_i diet, via stimulation of the activity of the renal 25-hydroxyvitamin-D₃-1-hydroxylase (1OHase), leads to an increase in the level of 1,25-dihydroxy-vitamin D₃ [1,25(OH)₂D]. Regulation of the intestinal absorption of P_i and the abundance of NaP_i-IIb by 1,25(OH)₂D has been supposed to involve the vitamin D receptor (VDR). In this study, we investigated the adaptation to a low-P_i diet of NaP_i-IIb in small intestine as well as NaP_i-IIa in kidneys of either VDR- or 1OHase-deficient mice. In both mouse models, upregulation by a low-P_i diet of the NaP_i cotransporters NaP_i-IIa and NaP_i-IIb was normal, i.e., similar to that observed in the wild types. Also, in small intestines of VDR- and 1OHase-deficient mice, the same changes in NaP_i-IIb mRNA found in wild-type mice were observed. On the basis of the results, we conclude that the regulation of NaP_i cotransport in small intestine (via NaP_i-IIb) and kidney (via NaP_i-IIa) by low dietary intake of P_i cannot be explained by the 1,25(OH)₂D-VDR axis. NaP_i type IIb; vitamin D₃     

Structural mechanism for coordination of proofreading and polymerase activities in archaeal DNA polymerases

A novel mechanism for controlling the proofreading and polymerase activities of archaeal DNA polymerases was studied. The 3'-5'exonuclease (proofreading) activity and PCR performance of the family B DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus kodakaraensis KOD1) were altered efficiently by mutation of a "unique loop" in the exonuclease domain. Interestingly, eight different H147 mutants showed considerable variations in respect to their 3'-5'exonuclease activity, from 9% to 276%, as against that of the wild-type (WT) enzyme. We determined the 2.75A crystal structure of the H147E mutant of KOD DNA polymerase that shows 30% of the 3'-5'exonuclease activity, excellent PCR performance and WT-like fidelity. The structural data indicate that the properties of the H147E mutant were altered by a conformational change of the Editing-cleft caused by an interaction between the unique loop and the Thumb domain. Our data suggest that electrostatic and hydrophobic interactions between the unique loop of the exonuclease domain and the tip of the Thumb domain are essential for determining the properties of these DNA polymerases.     

Production of offspring using current income and reserves: size-number trade-off and optimal offspring size

To analyse the effects of current income on the nature of size-number trade-off and optimal offspring size, we developed a model in which offspring grow by absorbing current income and reserves. The offspring continue to grow while the current income is available or the reserves exist, and they cease to grow when the reserves are depleted and the current income ceases. We showed that the size-number trade-off is nonlinear in the region where the number of offspring is smaller than the critical number and linear in the region where the number of offspring is greater than the critical number. In the former region, the reserves are not depleted by the time the current income ceases and the offspring cease to grow when the reserves are depleted, whereas in the latter region, the reserves are depleted before the current income ceases and the offspring production is completed when the current income ceases. The optimal offspring size is the same as that shown in Sakai and Harada (Evolution 55 (2001) 467) if this optimal size is realized in the region of nonlinear trade-off, whereas the optimal offspring size is the same as that shown in Smith and Fretwell (Am. Natur. 108 (1974) 499) if this optimal size is realized in the region of linear trade-off.     

Dexamethasone influences human clock gene expression in bronchial epithelium and peripheral blood mononuclear cells in vitro

We determined whether human peripheral blood mononuclear cells (PBMCs) could be used to analyze clock genes by studying their mRNA expressions in human bronchial epithelium (BEAS-2B) and PBMCs following stimulation by the glucocorticoid homologue dexamethasone (DEX) in vitro. PBMCs were obtained at 10:00 h from two diurnally active (∼07:00 to 23:00 h) healthy volunteers and were evaluated for hPer1 mRNA expression following DEX stimulation in vitro using real time-PCR analysis. DEX stimulation of human BEAS-2B cells and PBMCs in vitro led to a remarkable increase of hPer1 mRNA. The glucocorticoid rapidly affected the expression of hPer1 mRNA in PBMCs, suggesting that human PBMCs may be a useful surrogate marker for the investigation of drug effects on clock genes.