Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

Human haplotype block sizes are negatively correlated with recombination rates

The International Haplotype Map ("HapMap") Project is motivated, in part, by the belief that the organization of the human genome, the mechanics of recombination, and the population-level behavior of alleles at adjacent loci should allow researchers to parse the genome into small segments, or "blocks," that show strong linkage disequilibrium (LD) between alleles at loci within those segments. The discovery and evidence for these blocks is to be based solely on the observed LD strength and patterns between alleles at adjacent loci throughout the genome. Although there are many factors that contribute to LD strength, we assessed the correlation between block structure, in terms of length and percentage of the genome assembled into blocks within a region, and recombination rate obtained from two independent sources. We found evidence of a striking negative correlation between the average recombination rate and average block length, suggesting that recombination rate is a strong contributor to haplotype block structure within the genome. We discuss the potential implications of this negative correlation in the context of the organization, properties, and potential ubiquity of a block-like structure in the human genome.     

Bruceine B, A Potent Inhibitor of Leukocyte-Endothelial Cell Adhesion

Leukocyte adhesion to vascular endothelial cells is an essential step in the development of inflammatory diseases. We have searched for inhibitors of leukocyte-endothelial cell adhesion that could be used as anti-inflammatory drugs and found that bruceine B (0.2 g/ml; 0.44 M) inhibited human neutrophil or T cell adhesion to tumor necrosis factor- (TNF) stimulated human umbilical vein endothelial cells (HUVEC). The inhibition of neutrophil adhesion to TNF-stimulated HUVEC by bruceine B was not derived from cytotoxic effects, as determined by measurement of the level of lactate dehydrogenase (LDH) activity in conditioned medium. The effect of bruceine B on neutrophil adhesion to HUVEC was not seen when the neutrophils were preincubated with bruceine B. However, inhibitory effects were evident when the HUVEC were preincubated with bruceine B. Bruceine B also inhibited neutrophil adhesion to lipopolysaccharide-stimulated HUVEC and T cell adhesion to TNF-stimulated HUVEC. These findings suggest that bruceine B may have anti-inflammatory activity.     

Variable Regions 1 and 2 (VR1 and VR2) in JSRV gag Are Not Responsible for the Endogenous JSRV Particle Release Defect

Jaagsiekte sheep retrovirus (JSRV) is a betaretrovirus causing ovine pulmonary adenocarcinoma, a transmissible lung tumor of sheep. A very closely related endogenous retrovirus (enJSRV) occurs as 15 to 20 copies in the genome of all sheep, and is not known to be linked to pathogenesis. We previously localized a particle release defect of the full-length endogenous-derived expression construct pCMV2enJS56A1 to the amino-terminal region of gag that incorporates the two variable regions VR1 and VR2, which harbor the main sequence differences between endogenous and exogenous JSRV in this part of gag. Here, we tested the hypothesis that either or both of these variable regions are responsible for the observed particle release defect in enJS56A1. We found that the PPPPPPPS motif of the exogenous VR1 is neither necessary nor sufficient for particle release. Furthermore, the precise substitution of VR1 and VR2 in the exogenous JSRV expression plasmid pCMV2JS ₂₁, using their enJS56A1-derived counterparts, did not abrogate the ability of the resulting constructs to release particles. The particle release defect of enJS56A1 is therefore not determined exclusively by either VR1 or VR2. These results point to a small number of amino acids lying outside of VR1 and VR2 that may be responsible for the particle defect of enJS56A1 Gag.     

Concentrations of endometrial protein PP 14 and CA-125 in uterine flushings performed in natural and stimulated cycles

BACKGROUND: Impaired implantation in assisted reproduction cycles with high serum estradiol (E(2)) concentrations may be attributed to abnormal endometrial development. This study compared concentrations of endometrial proteins in uterine flushings of infertile patients between natural and stimulated cycles. METHODS: Patients received a standard regimen of ovarian stimulation. Seven days after the LH surge in natural cycles or the hCG injection in stimulated cycles, uterine flushings were performed by slowly injecting and aspirating normal saline through a paediatric Foley catheter. Natural cycles were considered as group A whereas stimulated cycles with serum E(2) <20 000 pmol/l and serum E(2) >20 000 pmol/l were classified as groups B and C respectively. PP 14 and CA-125 in uterine flushings were measured and expressed per total protein content. RESULTS: Concentrations of the total protein, PP 14 and CA-125 in the uterine flushings were similar among the three groups. PP 14 per total protein in the uterine flushings was significantly correlated with serum E(2) on the day of hCG (r = 0.459; P = 0.009) in natural cycles only but not in stimulated cycles. CONCLUSION: There was no significant difference between natural and stimulated cycles in concentrations of PP 14 and CA-125 in uterine flushings performed in the mid-luteal phase.     

Evaluation of RGS4 as a candidate gene for schizophrenia.

Several studies have suggested that the regulator of G-protein signaling 4 (RGS4) may be a positional and functional candidate gene for schizophrenia. Three single nucleotide polymorphisms (SNP) located at the promoter region (SNP4 and SNP7) and the intron 1 (SNP18) of RGS4 have been verified in different ethnic groups. Positive results have been reported in these SNPs with different numbers of SNP combinatory haplotypes. In this study, these three SNP markers were genotyped in 218 schizophrenia pedigrees of Taiwan (864 individuals) for association analysis. Among these three SNPs, neither SNP4, SNP7, SNP18 has shown significant association with schizophrenia in single locus association analysis, nor any compositions of the three SNP haplotypes has shown significantly associations with the DSM-IV diagnosed schizophrenia. Our results fail to support the RGS4 as a candidate gene for schizophrenia when evaluated from these three SNP markers.     

Isolation and characterization of proteins from Rous sarcoma virus

When the Bryan high-titer strain of Rous sarcoma virus RSV (RAV-1) labeled with a mixture of ¹⁴C amino acids was dissociated with a neutral detergent (Brij 35), mercaptoethanol, and urea and analyzed by isoelectric focusing in urea, seven radioactive peaks with pIs between 3.5 and 9.9 were found. The peaks were further analyzed by polyacrylamide gel electrophoresis in SDS to determine the number and molecular weight of the individual protein components in each peak. A total of eight amino acid-¹⁴C-labeled proteins were identified in RSV (RAV-1) with molecular weights between 14,000 and 96,000 daltons. When ³H-glucosamine labeled RSV (RAV-1) was dissociated with SDS and the viral components separated by SDS-gel electrophoresis, four radioactive components were observed. Analysis of glucosamine-³H-labeled virus together with amino acid-¹⁴C-labeled virus by dissociation with Brij 35, urea, and mercaptoethanol and separation of components by isoelectric focusing followed by electrophoresis in SDS-containing gels revealed that three of the glucosamine labeled components were glycoproteins corresponding to the three largest amino acid-labeled proteins. The fourth glucosamine-labeled component did did not contain radioactive amino acids suggesting that it was protein-free carbohydrate. Separation of virion components on Bio-Gel columns revealed that the glycoproteins were released from virus with Brij 35, urea, and mercaptoethanol in a large complex which was further dissociated into smaller units by SDS. Two of the eight protein components had high complement fixation titers and formed crossing precipitin lines in agar gel diffusion with hamster antiserum to the avian tumor virus group-specific antigen.