Identification of a membrane-associated 1-0-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine hydrolyzing phospholipase A2 in guinea pig 1 epidermis. Implications in the cutaneous biosynthesis of platelet-activating factor.

A membrane-associated 1-0-alkyl-2-arachidonoyl-GPC hydrolyzing phospholipase A2 was identified in guinea pig epidermis. It is regio-specific (associated with the particulate microsomal fraction) and specific for the hydrolysis of 1-0-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine. It is sensitive to low calcium concentrations suggesting that it may be activated by increasing intracellular calcium. Since ether-linked phospholipids are known to exist in the epidermis, further understanding of the properties of this 1-0-alkyl-arachidonoyl-hydrolyzing PLA2 may allow us to control the generation of 1-0-alkyl-2-lyso-sn-glycero-3-phosphocholine, a key substrate for the generation of the platelet-activating factor in the tissue.     

CD4-like molecules in human sperm

The expression of a molecule recognized by CD4 monoclonal antibodies was investigated on human sperm using immunolabelling, biochemical and immunochemical methods. Flow cytometry detected a significant fluorescence signal. SDS-PAGE analysis and Western blotting identified a molecule of 60 kDa, consistent with a CD4-like structure as confirmed after selective immunoseparation. Additional bands reacting with anti-CD4 were found in sperm extracts (73 kDa) and seminal fluid (90 kDa). These data indicate that sperm express a molecule similar to the receptor for HIV described on mononuclear cells.     

Bulge loops used to measure the helical twist of RNA in solution.

Bulge loops are commonly found in helical segments of cellular RNAs. When incorporated into long double-stranded RNAs, they may introduce points of flexibility or permanent bend that can be detected by the altered electrophoretic gel mobility of the RNA. We find that a single An or Un bulge loop near the middle of a long RNA helix significantly retards the RNA during polyacrylamide gel electrophoresis if n greater than or equal to 2. The mobility of an RNA containing two A2 bulges various periodically with the number of base pairs between the bulges. We interpret this to mean that A2 bulges varies periodically with the number of base pairs between the bulges. We interpret this to mean that Z2 bulges form torsionally stiff bends in the helix; the gel mobility reaches a minimum when the total helical twist between the bulges rotates the arms of the molecule into a cis conformation. The gel mobilities are proportional to the predicted end-to-end distance of the RNA if the average RNA helical repeat is 11.8 +/- 0.2 bp/turn and there is no helical twist (3 +/- 9 degrees) associated with the bulge (data obtained in 0.15 M Na+). Other sizes and sequences of bulges have very different effects on RNA helix conformation and flexibility. U2 bulges bend the helix to a much smaller degree than A2 bulges, while longer A or U bulge sequences probably allow bends of 90 degrees or more; all of these may be fairly flexible joints.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for cytosolic benzo(a)pyrene carrier proteins which function in cytochrome P450 oxidation in rat liver

Solutions of cytosolic proteins from rat liver contain benzo(a)pyrene solubilizing activity capable of serving as a carrier between solid state benzo(a)pyrene and microsomal cytochrome P₄₅₀. Fractionation of benzo(a)pyrene-saturated cytosolic proteins on a Sephadex G-100 column or by sucrose density gradients produced benzo(a)pyrene peaks of about 46,000 daltons and a very high molecular weight material. The protein-bound benzo(a)pyrene obtained in both peaks was oxidized rapidly by microsomes in the presence of NADPH, indicating that the benzo(a)pyrene carrier activity is capable of presenting the substrate to the cytochrome P₄₅₀. Liver cytosolic proteins from rats receiving intraperitoneal injection of [¹⁴C] benzo(a)pyrene was chromatographed on a column of Sephadex G-75. Radioactivity eluted at the same positions of the chromatogram as did the carrier activities described above. These results indicate that these benzo(a)pyrene carrier proteins may have an $\text{in}\text{vivo}$ role in the metabolism of benzo(a)pyrene.     

The C termini of Arabidopsis cryptochromes mediate a constitutive light response

Cryptochrome blue light photoreceptors share sequence similarity to photolyases, flavoproteins that mediate light-dependent DNA repair. However, cryptochromes lack photolyase activity and are characterized by distinguishing C-terminal domains. Here we show that the signaling mechanism of Arabidopsis cryptochrome is mediated through the C terminus. On fusion with beta-glucuronidase (GUS), both the Arabidopsis CRY1 C-terminal domain (CCT1) and the CRY2 C-terminal domain (CCT2) mediate a constitutive light response. This constitutive photomorphogenic (COP) phenotype was not observed for mutants of cct1 corresponding to previously described cry1 alleles. We propose that the C-terminal domain of Arabidopsis cryptochrome is maintained in an inactive state in the dark. Irradiation with blue light relieves this repression, presumably through an intra- or intermolecular redox reaction mediated through the flavin bound to the N-terminal photolyase-like domain.     

Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats

OBJECTIVE: The aim of this study was to develop a feasible approach to promote bone healing in osteoporotic rats using autogenous bone tissue-engineering and gene transfection of human bone morphogenetic protein 2 (hBMP-2). METHODS: Bone marrow stromal cells (BMSCs) from the left tibia of osteoporotic rats were transfected with the hBMP-2 gene in vitro which was confirmed by immunohistochemistry, in situ hybridization and Western blotting. Autogenous transfected or untransfected BMSCs were seeded on macroporous coral hydroxyapatite (CHA) scaffolds. Each cell-scaffold construct was implanted into a defect site which was created in the ramus of the mandible of osteoporotic rats. Four or eight weeks after implantation in situ hybridization was performed in BMSCs transfected with hBMP-2, X-ray examinations, histological and histomorphological analyses were used to evaluate the effect of tissue-engineered bone on osseous defect repair. RESULTS: Newly formed bone was observed at the margin of the defect 4 weeks after implantation with BMSCs transfected with BMP-2. Mature bone was observed 8 weeks after treatment. In the control group there was considerably less new bone and some adipose tissue was observed at the defect margins 8 weeks after implantation. CONCLUSIONS: Autogenous cells transfected with hBMP-2 promote bone formation in osteoporotic rats. BMSC-mediated BMP-2 gene therapy used in conjunction with bone tissue engineering may be used to successfully treat bone defects in osteoporotic rats. This method provides a powerful tool for bone regeneration and other tissue engineering.