Site specific regulation in the kidney of endothelin and its receptor subtypes by cyclosporine

Site selective regulation in the kidney of endothelin and its receptor subtypes by cyclosporine. Endothelin (Et) has been suggested by us and others to play a role in glomerular dysfunction that characterizes cyclosporine (Cs)-associated nephrotoxicity. Since Et exerts its actions through at least two receptor subtypes, and because these receptor subtypes have particular distributions in the renal parenchyma, we investigated changes in mRNA expression for Et and its receptor subtypes in glomeruli and medulla of rats treated with Cs. Polymerase chain reaction coupled with reverse transcription (RT-PCR) method was used to assess prepro-Et-1, type A (EtA) and type B (EtB) receptor mRNA at 1, 3, 6, and 24 hours after Cs (20 mg/kg body wt i.v.). Results were normalized to the expression of β-actin as an internal standard. Compared with control rats, glomerular mRNA expression for prepro-Et-1 was not affected by Cs. Similarly, Cs did not significantly change the glomerular mRNA expression of either EtA or EtB receptor subtypes. By contrast, in the medulla there was a marked and persistent increase in the expression for prepro-Et-1 and the EtB receptor subtype: prepro-Et-1 at 1, 3, 6, and 24 hours was 336 ± 61, 295 ± 65, 339 ± 73,440 ± 123% of controls, respectively (P < 0.05 compared with controls at each time point). The EtB receptor mRNA at 1, 3, 6, 24 hours was 164 ± 22, 157 ± 15, 148 ± 14, 116 ± 18% (compared with controls, P < 0.01 at 3hr and P < 0.05 at 1 and 6 hr), while the mRNA expression for EtA was not affected by Cs treatment. These results demonstrate that, in vivo, Cs selectively modulates renal mRNA expression for Et peptide and one of its receptor subtypes. Furthermore, the modulation is site specific. These changes are most conspicuous in the renal medulla, such that mRNA expression for prepro-Et-1 and EtB increases and remains elevated for at least six hours after Cs administration. These alterations may contribute to the vasospastic as well as proliferative abnormalities which characterize Cs nephrotoxicity

Flocculation of Artemia induced by East Asian common Octopus octopus sinensis paralarvae under culture conditions

Artemia are potential food organisms for the mass culture of common octopus paralarvae but cause poor paralarval growth and mortality. To understand problems arising from Artemia use, we focused on Artemia flocculation in paralarval culture tanks; Artemia get caught up with each other, exhibit disrupted swimming, are deposited on the tank bottom and eventually die. To clarify whether paralarvae induce the flocculation of food organisms or not, we cultured newly hatched Artemia nauplii, 3-day-old metanauplii and decapod crustacean zoeae with or without paralarvae at different growth stages (weight). Flocculation occurred only when Artemia were cultured with paralarvae; metanauplii had a higher susceptibility for flocculation than nauplii. Flocculated Artemia proportion increased with increasing paralarval weight. Scanning electron microscopy revealed that flocculated metanauplii had deformed setules on their setae, with hook-shaped tips and adhesion of neighbouring tips, suggesting that flocculation may occur via a mechanism similar to the ‘hook-and-loop fastener’. As octopus paralarvae exhibit external digestion, digestive enzymes secreted by paralarvae may deform Artemia setules and result in flocculation. As flocculation did not occur when metanauplii were cultured in water in which paralarvae were cultured and then removed, causative enzymes were probably rapidly inactivated after secretion

Cyanobacterial Cell Lineage Analysis of the Spatiotemporal hetR Expression Profile during Heterocyst Pattern Formation in Anabaena sp. PCC 7120

Diazotrophic heterocyst formation in the filamentous cyanobacterium, Anabaena sp. PCC 7120, is one of the simplest pattern formations known to occur in cell differentiation. Most previous studies on heterocyst patterning were based on statistical analysis using cells collected or observed at different times from a liquid culture, which would mask stochastic fluctuations affecting the process of pattern formation dynamics in a single bacterial filament. In order to analyze the spatiotemporal dynamics of heterocyst formation at the single filament level, here we developed a culture system to monitor simultaneously bacterial development, gene expression, and phycobilisome fluorescence. We also developed micro-liquid chamber arrays to analyze multiple Anabaena filaments at the same time. Cell lineage analyses demonstrated that the initial distributions of hetR::gfp and phycobilisome fluorescence signals at nitrogen step-down were not correlated with the resulting distribution of developed heterocysts. Time-lapse observations also revealed a dynamic hetR expression profile at the single-filament level, including transient upregulation accompanying cell division, which did not always lead to heterocyst development. In addition, some cells differentiated into heterocysts without cell division after nitrogen step-down, suggesting that cell division in the mother cells is not an essential requirement for heterocyst differentiation

A Method for Rapid Demineralization of Teeth and Bones

Tooth and bone specimen require extensive demineralization for careful analysis of cell morphology, as well as gene and protein expression levels. The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate. This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest. To analyze LacZ activity, and the expression of other genes and their protein products in teeth and bones, it is necessary to carry out a complete demineralization of the specimen before cutting sections. However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase. Therefore, most protocols currently use mild acids such as 0.1 M ethylene diamine tetra-acetic acid (EDTA) for demineralization of tooth and bone specimen, which require a longer period of treatment for complete demineralization. A method by which hard tissue specimens such as teeth and bones can be rapidly, but gently, decalcified is necessary to save time and effort. Here, we report a suitable method for rapid demineralization of mouse teeth in 0.1M EDTA at 42˚C without any loss of ß-galactosidase activity

Somatic Primary piRNA Biogenesis Driven by cis-Acting RNA Elements and trans-Acting Yb

Primary piRNAs in Drosophila ovarian somatic cells arise from piRNA cluster transcripts and the 3′ UTRs of a subset of mRNAs, including Traffic jam (Tj) mRNA. However, it is unclear how these RNAs are determined as primary piRNA sources. Here, we identify a cis-acting 100-nt fragment in the Tj 3′ UTR that is sufficient for producing artificial piRNAs from unintegrated DNA. These artificial piRNAs were effective in endogenous gene transcriptional silencing. Yb, a core component of primary piRNA biogenesis center Yb bodies, directly bound the Tj-cis element. Disruption of this interaction markedly reduced piRNA production. Thus, Yb is the trans-acting partner of the Tj-cis element. Yb-CLIP revealed that Yb binding correlated with somatic piRNA production but Tj-cis element downstream sequences produced few artificial piRNAs. We thus propose that Yb determines primary piRNA sources through two modes of action: primary binding to cis elements to specify substrates and secondary binding to downstream regions to increase diversity in piRNA populations

Normal and high-normal blood pressures, but not body mass index, are risk factors for the subsequent occurrence of both preeclampsia and gestational hypertension : A retrospective cohort study

Blood pressure (BP) levels and body mass index (BMI) are known as risk factors for preeclampsia and gestational hypertension. However, there have been few investigations regarding the effects of BID and BMI levels on preeclampsia and gestational hypertension in the same cohort. In the present study, we conducted a retrospective cohort study using multiple logistic regression analysis. The cohort included 1,518 patients without nephritis. The unadjusted odds ratios (ORs) of preeclampsia and gestational hypertension were increased in pregnant women with normal BP (120-129 mmHg systolic or 80-84 mmHg diastolic), high-normal BID and hypertension in the second trimester compared to those with optimal BP. The unadjusted ORs of preeclampsia and gestational hypertension were also increased in obese women in the pre-pregnancy period compared to women with normal range BML When adjustment was made for both the BP levels and pre-pregnancy BMI levels, the ORs (95% confidence intervals) of normal BID, high-normal BID, hypertension and obesity for the subsequent occurrence of preeclampsia were 5.1 (2.2-12), 8.3 (3.1-22), 16 (5.0-50) and 2.0 (0.67-5.9), and those for the subsequent occurrence of gestational hypertension were 7.0 (2.6-19), 7.4 (2.1-25), 22 (6.1-83) and 1.3 (0.33-4.8), respectively. For the subsequent occurrence of preeclampsia or gestational hypertension, normal BID, high-normal BP and hypertension in the second trimester may be independent risk factors. Obesity in the pre-pregnancy period, however, may not be an independent risk factor