Simultaneous gene transfer of bone morphogenetic protein (BMP)-2 and BMP-7 by in vivo electroporation induces rapid bone formation and BMP-4 expression

Background: Transcutaneous in vivo electroporation is expected to be an effective gene-transfer method for promoting bone regeneration using the BMP-2 plasmid vector. To promote enhanced osteoinduction using this method, we simultaneously transferred cDNAs for BMP-2 and BMP-7, as inserts in the non-viral vector pCAGGS. Methods: First, an in vitro study was carried out to confirm the expression of BMP-2 and BMP-7 following the double-gene transfer. Next, the individual BMP-2 and BMP-7 plasmids or both together were injected into rat calf muscles, and transcutaneous electroporation was applied 8 times at 100 V, 50 msec. Results: In the culture system, the simultaneous transfer of the BMP-2 and BMP-7 genes led to a much higher ALP activity in C2C12 cells than did the transfer of either gene alone. In vivo, ten days after the treatment, soft X-ray analysis showed that muscles that received both pCAGGS-BMP-2 and pCAGGS-BMP-7 had better-defined opacities than those receiving a single gene. Histological examination showed advanced ossification in calf muscles that received the double-gene transfer. BMP-4 mRNA was also expressed, and RT-PCR showed that its level increased for 3 days in a timedependent manner in the double-gene transfer group. Immunohistochemistry confirmed that BMP- 4-expressing cells resided in the matrix between muscle fibers. Conclusion: The simultaneous transfer of BMP-2 and BMP-7 genes using in vivo electroporation induces more rapid bone formation than the transfer of either gene alone, and the increased expression of endogenous BMP-4 suggests that the rapid ossification is related to the induction of BMP-4.</p

Genetic diversity of group A rotaviruses associated with repeated outbreaks of diarrhea in a farrow-to-finish farm: identification of a porcine rotavirus strain bearing a novel VP7 genotype, G26

Group A rotaviruses (GARs) are one of the most common causes of diarrhea in suckling pigs. Although a number of G and P genotypes have been identified in porcine GARs, few attempts have been made to study the molecular epidemiology of these viruses associated with diarrhea outbreaks within a farm over an extended period of time. Here, we investigated the molecular characteristics of GARs that caused four outbreaks of diarrhea among suckling pigs in a farrow-to-finish farm over the course of a year. G and P genotyping of GARs detected at each outbreak demonstrated genetic diversity in this farm as follows: G9P[23] was detected at the first outbreak, G9P[13]/[22] and G9P[23] at the second, G3P[7] at the third, and G9P[23], G5P[13]/[22], and P[7] combined with an untypeable G genotype at the fourth. Sequence analysis of the detected GARs revealed that such genetic diversity could have resulted not only from the introduction of new GAR strains, but also from gene reassortment between GAR strains within the farm. Further, the GAR strain carrying the untypeable G genotype was shown to be a novel porcine GAR bearing a new G26 genotype, as confirmed by the Rotavirus Classification Working Group

LOW BMI IS THE RISK OF CARDIO-VASCULAR MORTALITY WITHOUT PROGRESSION OF CKD

The paradoxical risk of BMI on mortality is known in CKD as well in dialysis populations, but studies of CVD risk in CKD including underweight is limited. We hypothesized lean CKD increase the CVD risk, contributing different factors from obese. 2,676 CKD patients recruited from 11 outpatients’ hospitals. BMI and estimated GFR (eGFR) were calculated, and change of eGFR and CVD mortality during 2 years were collected. Patients were divided by BMI under cut off value of normal, thus 7% grouped in lean subjects (BMI <18.5). Systolic blood pressure (sBP), albumin, hemoglobin, age and prevalence of diabetes were lower in lean BMI group compared to other subjects. However CVD history, urinary protein, baseline eGFR and smoking didn't differ between the groups. The lean BMI increased significantly the risk of CVD mortality, in spite of low prevalence of comorbidities and young age in unadjusted model (HR 2.38, 95%CI 1.49-5.21, p<0.01). This significance remained after adjusted for CVD risk factors, such as primary disease of CKD, age, sex, smoking, albumin, cholesterol, sBP and eGFR. On the other hand, BMI was not associated with the decline rate of eGFR. We concluded that BMI less than 18.5 was an independent predictor of CVD, and that BMI did not effect on CKD progression rate in Japanese CKD

PKCα mediates TGFβ-induced growth inhibition of human keratinocytes via phosphorylation of S100C/A11

Growth regulation of epithelial cells is of major concern because most human cancers arise from them. We demonstrated previously a novel signal pathway involving S100C/A11 for high Ca2+-induced growth inhibition of normal human keratinocytes (Sakaguchi, M., M. Miyazaki, M. Takaishi, Y. Sakaguchi, E. Makino, N. Kataoka, H. Yamada, M. Namba, and N.H. Huh. 2003. J. Cell Biol. 163:825–835). This paper addresses a question whether transforming growth factor β (TGFβ) shares the pathway with high Ca2+. On exposure of the cells to TGFβ1, S100C/A11 was phosphorylated, bound to nucleolin, and transferred to the nucleus, resulting in induction of p21WAF1/CIP1 and p15INK4B through activation of Sp1. Protein kinase C α (PKCα) was shown to phosphorylate 10Thr of S100C/A11, which is a critical event for the signal transduction. The TGFβ1-induced growth inhibition was almost completely mitigated when PKCα activity was blocked or when S100C/A11 was functionally sequestered. These results indicate that, in addition to the well-characterized Smad-mediated pathway, the PKCα–S100C/A11-mediated pathway is involved in and essential for the growth inhibition of normal human keratinocytes cells by TGFβ1

Determination of natural estrogens in the sediment of coastal area in Japan

The concentration of natural estrogens such as 17β-estradiol (E2) and estrone (E1) in the sediment of coastal areas in Japan was determined using an enzyme-1inked immunosorbent assay (ELISA). The samples of sediment were collected in Osaka Bay and Otsuchi Bay, Yamada Bay and Ofunato Bay, in Iwate Prefecture, Japan. In addition, core samples were taken in Otsuchi Bay and Yamada Bay, Estrogens in sediments were extracted by sonication in acetone followed by liquid-1iquid extraction with dichloromethane. The levels of E1 were generally higher than those of E2 in sediments, whereas the concentrations of E2 were higher than E1 at some sampling sites. The levels of estrogens in the sites located near estuaries tended to be higher, and levels decreased at the sampling stations nearer to the mouth of the bay However, the estrogen concentration in the sediment collected from near the mouth of bay was highest of all samples in Osaka Bay, both in summer and winter. Core samples were sliced every 2cm from the surface to 20cm deep for the measurement of estrogen. Although the concentrations of estrogens decreased with the depth of core samples, fairly high levels of estrogens were again noticed at the layer deeper than 16cm

Regulation of Epithelial Sodium Transport via Epithelial Na+ Channel

Renal epithelial Na+ transport plays an important role in homeostasis of our body fluid content and blood pressure. Further, the Na+ transport in alveolar epithelial cells essentially controls the amount of alveolar fluid that should be kept at an appropriate level for normal gas exchange. The epithelial Na+ transport is generally mediated through two steps: (1) the entry step of Na+ via epithelial Na+ channel (ENaC) at the apical membrane and (2) the extrusion step of Na+ via the Na+, K+-ATPase at the basolateral membrane. In general, the Na+ entry via ENaC is the rate-limiting step. Therefore, the regulation of ENaC plays an essential role in control of blood pressure and normal gas exchange. In this paper, we discuss two major factors in ENaC regulation: (1) activity of individual ENaC and (2) number of ENaC located at the apical membrane

Microbiological Investigation of the Iron-Containing Floculent Mats in Various Deep Sea Environments

Conference abstract (August 14-19, 2011, Goldschmidt 2011 in Prague, Czech Republic)http://www.godac.jamstec.go.jp/darwin/cruise/natsushima/nt10-13_leg2/ehttp://www.godac.jamstec.go.jp/darwin/cruise/yokosuka/yk10-10/