Anti‐adhesive property of P‐selectin glycoprotein ligand‐1 (PSGL‐1) due to steric hindrance effect

P‐selectin glycoprotein ligand‐1 (PSGL‐1) is an adhesive molecule that is known to be a ligand for P‐selectin. An anti‐adhesive property of PSGL‐1 has not been previously reported. In this study, we show that PSGL‐1 expression is anti‐adhesive for adherent cells and we have elucidated the underlying mechanism. Overexpression of PSGL‐1 induced cell rounding and floating in HEK293T cells. Similar phenomena were demonstrated in other adherent cell lines with overexpression of PSGL‐1. PSGL‐1 overexpression inhibits access of antibodies to cell surface molecules such as integrins, HLA and CD25. Cells transfected with PSGL‐1 deletion mutants that lack a large part of the extracellular domain and chimeric construct expressing extracellular CD86 and intracellular PSGL‐1 only showed rounded morphology, but there are no floating cells. These results indicated that PSGL‐1 causes steric hindrance due to the extended structure of its extracellular domain that is highly O‐glycosylated, but intracellular domain also has some effect on cell rounding. This study implies that PSGL‐1 has Janus‐faced functions, being both adhesive and anti‐adhesive. J. Cell. Biochem. 114: 1271–1285, 2013. © 2013 Wiley Periodicals, Inc.     

Cloning of the Genes Concerned in Phenylalanine Biosynthesis in Corynebacterium glutamicum and its Application to Breeding of a Phenylalanine Producing Strain

The chorismate mutase and prephenate dehydratase genes of phenylalanine producing Corynebacterium glutamicum K38, which is resistant to p-fluorophenylalanine and m-fluorophenylalanine, were cloned into plasmid pCE53 in C. glutamicum KY9456, which lacks chorismate mutase and prephenate dehydratase. One of the resultant plasmids, pCmB4, contained a 9.4kb BamHI DNA fragment inserted into the unique BamHl site of pCE53. Plasmid pCmB4 complemented a phenylalanine and tyrosine double auxotroph of C. glutamicum KY9456. Introduction of pCmB4 into C. glutamicum RRL5 resulted in an about ten times increase in chorismate mutase activity. C. glutamicum K38 carrying the plasmid accumulated 19.0mg/ml of phenylalanine (50% increase over the yield of K38).     

L-Tyrosine production by Polyauxotrophic Mutants of Corynebacterium glutamicum

Polyauxotrophic mutants of Corynebacterium glutamicum which have additional requirements to L-phenylalanine were derived from L-tyrosine producing strains of phenylalanine auxotrophs, C. glutamicum KY 9189 and C. glutamicum KY 10233, and screened for L-tyrosine production. The increase of L-tyrosine production was noted in many auxotrophic mutants derived from both strains. Especially some double auxotrophs which require phenylalanine and purine, phenylalanine and histidine, or phenylalanine and cysteine produced significantly higher amounts of L-tyrosine compared to the parents, A phenylalanine and purine double auxotrophic strain LM–96 produced L-tyrosine at a concentration of 15.1 mg per ml in the medium containing 20% sucrose. L-Tyrosine production by the strain decreased at high concentrations of L-phenylalanine.     

Structures of Gibberellins A39, A48, A49 and a New Kaurenolide in Cucurbita pepo L.

The structures of three new gibberellins A₃₀, A₄₈ and A₄₉ and a new kaurenolide, isolated from seeds of Cucurbita pepo L., were elucidated. The structures of GA₃₉, GA₄₈ and GA₄₉ were shown to be ent-3α,12β-dihydroxygibberell-16-ene-7,19,20-trioic acid (1), ent-2α,3α,10,12α-tetrahydroxy-20-norgibberell-16-ene-7,19-dioic acid 19,10-lactone (5) and the epimer at C–12 of GA₄₈ (8), respectively. The kaurenolide was shown to have the structure: ent-6β,7α,12β-trihydroxykaur-16-en-19-oic acid 19,6-lactone (14).     

A Questionnaire Survey of the Type of Support Required by <Emphasis Type="Italic">Yogo</Emphasis> Teachers to Effectively Manage Students Suspected of Having an Eating Disorder

Background

Many studies have focused on the decreasing age of onset of eating disorders (EDs). Because school-age children with EDs are likely to suffer worse physical effects than adults, early detection and appropriate support are important. The cooperation of Yogo teachers is essential in helping these students to find appropriate care. To assist Yogo teachers, it is helpful to clarify the encounter rates (the proportion of Yogo teachers who have encountered ED students) and kinds of requested support (which Yogo teachers felt necessary to support ED students). There are no studies that have surveyed the prevalence rates of ED children by ED type as defined by the Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5), nor were we able to find any quantitative study surveying the kinds of support Yogo teachers feel helpful to support ED students.

Methods

A questionnaire survey was administered to 655 Yogo teachers working at elementary/junior high/senior high/special needs schools in Chiba Prefecture. The questionnaire asked if the respondents had encountered students with each of the ED types described in DSM-5 (anorexia nervosa (AN), bulimia nervosa (BN), binge eating disorder (BED), avoidant/restrictive food intake disorder (ARFID), and other types of EDs (Others)), and the kinds of support they felt necessary to support these students. The encounter rates and the kinds of requested were obtained and compared, taking their confidence intervals into consideration.

Results

The encounter rates for AN, BN, BED, ARFID, and Others were 48.4, 14.0, 8.4, 10.7, and 4.6 %, respectively. When classified by school type, AN, BN, BED, and ARFID had their highest encounter rates in senior high schools. Special needs schools had the highest rate for Others. The support most required for all ED types was “a list of medical/consultation institutions.”

Conclusions

Our results have clarified how to support Yogo teachers in the early detection and support of ED students. We found that the encounter rate of AN was the highest, and that it is effective to offer “a list of medical/consultation institutions” to junior and senior high schools where the encounter rates for AN are high.

     

Depression of Complement Regulatory Factors in Rat and Human Renal Grafts Is Associated with the Progress of Acute T-Cell Mediated Rejection

Background

The association of complement with the progression of acute T cell mediated rejection (ATCMR) is not well understood. We investigated the production of complement components and the expression of complement regulatory proteins (Cregs) in acute T-cell mediated rejection using rat and human renal allografts.

Methods

We prepared rat allograft and syngeneic graft models of renal transplantation. The expression of Complement components and Cregs was assessed in the rat grafts using quantitative real-time PCR (qRT-PCR) and immunofluorescent staining. We also administered anti-Crry and anti-CD59 antibodies to the rat allograft model. Further, we assessed the relationship between the expression of membrane cofactor protein (MCP) by immunohistochemical staining in human renal grafts and their clinical course.

Results

qRT-PCR results showed that the expression of Cregs, CD59 and rodent-specific complement regulator complement receptor 1-related gene/protein-y (Crry), was diminished in the rat allograft model especially on day 5 after transplantation in comparison with the syngeneic model. In contrast, the expression of complement components and receptors: C3, C3a receptor, C5a receptor, Factor B, C9, C1q, was increased, but not the expression of C4 and C5, indicating a possible activation of the alternative pathway. When anti-Crry and anti-CD59 mAbs were administered to the allograft, the survival period for each group was shortened. In the human ATCMR cases, the group with higher MCP expression in the grafts showed improved serum creatinine levels after the ATCMR treatment as well as a better 5-year graft survival rate.

Conclusions

We conclude that the expression of Cregs in allografts is connected with ATCMR. Our results suggest that controlling complement activation in renal grafts can be a new strategy for the treatment of ATCMR.     

TGF-beta mediated Msx2 expression controls occipital somites-derived caudal region of skull development

Craniofacial development involves cranial neural crest (CNC) and mesoderm-derived cells. TGF-beta signaling plays a critical role in instructing CNC cells to form the craniofacial skeleton. However, it is not known how TGF-beta signaling regulates the fate of mesoderm-derived cells during craniofacial development. In this study, we show that occipital somites contribute to the caudal region of mammalian skull development. Conditional inactivation of Tgfbr2 in mesoderm-derived cells results in defects of the supraoccipital bone with meningoencephalocele and discontinuity of the neural arch of the C1 vertebra. At the cellular level, loss of TGF-beta signaling causes decreased chondrocyte proliferation and premature differentiation of cartilage to bone. Expression of Msx2, a critical factor in the formation of the dorsoventral axis, is diminished in the Tgfbr2 mutant. Significantly, overexpression of Msx2 in Myf5-Cre;Tgfbr2flox/flox mice partially rescues supraoccipital bone development. These results suggest that the TGF-beta/Msx2 signaling cascade is critical for development of the caudal region of the skull.     

Blood-flow route from the hepatic artery and portal vein to the sinusoid in normal human liver observed by confocal laser scanning microscopy

OBJECTIVE: To observe the microvasculature in normal human liver. STUDY DESIGN: Four autopsy livers cut into 50-micron-thick sections were observed by confocal laser scanning microscopy. Immunofluorescence was performed using anti-alpha smooth muscle actin (alpha-SMA) antibody. In addition, double immunofluorescence was performed on the other sections using antilysozyme antibody. The routes from the portal vein branches and hepatic artery branches to the sinusoids were defined as follows: portal venule, septal branch, inlet venule, hepatic arteriole and terminal hepatic arteriole. RESULTS: The reactivity of the walls of septal branches and inlet venule was positive for alpha-SMA. Lysozyme-positive cells (Kupffer cells) were dense in the sinusoids but were sparse in the septal branches and absent from the inlet venules. Terminal hepatic arterioles were observed along the septal branch, and the anastomoses between them were observed at the peripheral portion. No routes opening directly from the terminal hepatic arteriole into the sinusoids or arterioportal anastomoses in the portal tract were observed on alpha-SMA-stained sections. CONCLUSION: Regulation of the microcirculation in human liver may be performed by the smooth muscle layer of both peripheral portal and hepatic arterial routes.