Osteoblasts synthesize and respond to transforming growth factor-type beta (TGF-beta) in vitro

Transforming growth factor-type beta (TGF-beta) has been identified as a constituent of bone matrix (Seyedin, S. M., A. Y. Thompson, H. Bentz, D. M. Rosen, J. M. McPherson, A. Conti, N. R. Siegel, G. R. Gallupi, and K. A. Piez, 1986, J. Biol. Chem. 261:5693-5695). We used both developing bone and bone-forming cells in vitro to demonstrate the cellular origin of this peptide. TGF-beta mRNA was detected by Northern analysis in both developing bone tissue and fetal bovine bone-forming cells using human cDNA probes. TGF-beta was shown to be synthesized and secreted by metabolically labeled bone cell cultures by immunoprecipitation from the medium. Further, TGF-beta activity was demonstrated in conditioned media from these cultures by competitive radioreceptor and growth promotion assays. Fetal bovine bone cells (FBBC) were found to have relatively few TGF-beta receptors (5,800/cell) with an extremely low Kd of 2.2 pM (high binding affinity). In contrast to its inhibitory effects on the growth of many cell types including osteosarcoma cell lines, TGF-beta stimulated the growth of subconfluent cultures of FBBC; it had little effect on the production of collagen by these cells. We conclude that bone-forming cells are a source for the TGF-beta that is found in bone, and that these cells may be modulated by this factor in an autocrine fashion.     

Fetal bovine bone cells synthesize bone-specific matrix proteins

We isolated cells from both calvaria and the outer cortices of long bones from 3- to 5-mo bovine fetuses. The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type 1 collagen. In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by immunoprecipitation and fluorography of SDS polyacrylamide gels. Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (approximately 94%) type I collagen, with small amounts of types III and V collagens. In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and beta-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen. These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro.     

Interspecies conservation of structure of interphotoreceptor retinoid-binding protein. Similarities and differences as adjudged by peptide mapping and N-terminal sequencing.

Structural properties of the retinal extracellular-matrix glycolipoprotein interphotoreceptor retinoid-binding protein (IRBP) from human, monkey and bovine retinas have been compared. SDS/polyacrylamide-gel-electrophoretic analysis of limited tryptic and Staphylococcus aureus-V8-proteinase digests show virtually identical patterns for the monkey and human proteins, whereas both sets differ considerably from the bovine protein pattern. Time-course digestion shows monkey IRBP to be more readily cleaved than bovine IRBP and also cleaved to smaller fragments. Also, reversed-phase h.p.l.c. of complete tryptic digests of the IRBPs indicate that, although they have in common a similar preponderance of hydrophobic peptides, all three proteins differ extensively in their fine structure. The N-terminal sequences of monkey and bovine IRBPs have been extended beyond those presented in our previous report [Redmond, Wiggert, Robey, Nguyen, Lewis, Lee & Chader (1985) Biochemistry 24, 787-793] to over 30 residues each. The sequences yet show extensive homology, differing at only two positions, although the major monkey sequence has an additional five amino acid residues at its N-terminus ('n + 5' sequence) not observed with bovine IRBP ('n' sequence). The newly determined N-terminal sequence of human IRBP demonstrates the presence of equal amounts of the 'n' and 'n+5' sequences that are qualitatively identical with those of the monkey. The presence of the five-amino-acid-residue extension in primate, but not bovine, IRBP may indicate variation in post-translational processing.     

Induction of glycinebetaine uptake into Xenopus oocytes by injection of poly(A)+ RNA from renal cells exposed to high extracellular NaCl

Madin-Darby canine kidney (MDCK) cells accumulate glycinebetaine via Na(+)-dependent transport in response to hypertonic stress. When extracellular tonicity is increased by the addition of NaCl, Vmax for glycinebetaine transport increases without an associated change in Km, consistent with an increase in the number of functioning transporters. To test whether increased transport activity results from increased gene expression, we injected poly(A)+ RNA (mRNA) from MDCK cells into Xenopus oocytes and assayed for glycinebetaine uptake in ovo. RNA-induced Na(+)-dependent uptake is observed in oocytes injected with mRNA from cells exposed to high extracellular NaCl, but not in oocytes injected with either water or mRNA from cells maintained in isotonic medium. Unfractionated mRNA induces glycinebetaine uptake in ovo at a rate which is approximately 3-fold higher than in water-injected controls. Size-fractionated mRNA (median size 2.8 kilobases) induces uptake at a rate which is approximately 7-fold higher than controls. Such RNA-induced transport activity in ovo is consistent with heterologous expression of Na(+)/glucinebetaine cotransporters encoded by renal mRNA. Increased transporter mRNA in cells exposed to hypertonicity probably underlies the pattern of expression observed in ovo. This can account for the observed rise in MDCK cell glycinebetaine transport during hypertonic stress.     

Commitment to the First Cortical Reorganization During Conjugation in Stylonychia mytilus: An Argument for Homology with Cortical Development During Binary Fission

The asexual nature of the first cortical reorganization of conjugation in Stylonychia was analyzed by comparing the effect of amputation performed at different stages of early conjugation to that performed on vegetative cells at different stages of the cell cycle. Amputation of vegetative cells delineated a point of commitment to binary fission at 0.51–0.57 of the cell cycle. Cells amputated before this point were induced to undergo the regenerative mode of asexual development, but those amputated after this point continued with binary fission. In parallel, during conjugation a similar commitment was made around the time of formation of tight mating-pairs: early conjugants amputated around this time might undergo regeneration, and those operated on after this stage continued with the first cortical reorganization as in typical conjugants. The two mates of a pair might differ in their response to amputation, suggesting that the timing of commitment to the first cortical reorganization is not related to the events of conjugation, but rather is individually determined in the vegetative cycle of the cells before they pair up in mating. These observations provide support for the notion that the first cortical reorganization of conjugants is homologous to the asexual mode of cortical development in dividers, according to the theory of developmental heterochrony in the sexual reproduction of hypotrichs. The timing of commitment to the first cortical reorganization was found to temporally correlate with the entrance of the micronuclei into meiosis. Since the first cortical reorganization can proceed without the micronucleus, this raises the possibility that initiation of micronuclear meiosis is closely coupled with, and may be determined by, the commitment to the first cortical reorganization.     

Lysine tRNA''s associated with avian myeloblastosis virus 70S RNA.

The lysine tRNA released from the 70S RNA of avian myeloblastosis virus was separated by reversed-phase chromatography. All of the AAG-coding lysine tRNA's were present in the 70S-associated fraction; however, the AAA-coding lysine tRNA could not be detected. Chromatography of the lysine tRNA released at various temperatures did not show any preferential release of one AAG-coding species over another.     

A correlation between BCL-2 modifying factor,p53 and livin gene expressions in cancer colon patients

Accumulating evidence has revealed that livin gene and BCL-2 modifying factor (BMF) gene are closely associated with the initiation and progression of colon carcinoma by activating or suppressing multiple malignant processes. Those genes that can detect colon - cancer are a promising approach for cancer screening and diagnosis. This study aimed to evaluate correlation between livin, BMF and p53 genes expression in colon cancer tissues of patients included in the study, and their relationship with clinicopathological features and survival outcome in those patients. In this study, 50 pathologically diagnosed early cancer colon patients included and their tissue biopsy with 50 matched adjacent normal tissue, and 50 adenoma tissue specimens were analyzed for livin gene and BMF gene expressions using real time PCR. The relationship of those genes expressions with clinicopathological features, tumor markers, Time to Progression and overall survival for those patients were correlated in cancer colon group. In this study, there was a significant a reciprocal relationship between over expression of livin gene and down regulation of BMF and p53 genes in colon cancer cells. Livin mRNA was significantly higher, while BMF and p53 mRNA were significantly lower in colorectal cancer tissue compared to benign and normal colon tissue specimens (P < 0.001), however, this finding was absent between colon adenomas and normal mucosa. There was a significant association between up regulation of livin and down regulation of BMF and p53 expressions with more aggressive tumor (advanced TNM stage), rapid progression with metastasis and decreased overall survival in cancer colon patients, hence these genes can serve as significant prognostic markers of poor outcome in colon cancer patients. This work highlights the role of livin, BMF and p53 genes in colorectal tumorigenesis and the applicability of using those genes as a diagnostic and prognostic markers in patients with colon carcinoma and as a good target for cancer colon treatment in the future.     

Location of the CD8 T Cell Epitope within the Antigenic Precursor Determines Immunogenicity and Protection against the Toxoplasma gondii Parasite

CD8 T cells protect the host from disease caused by intracellular pathogens, such as the Toxoplasma gondii (T. gondii) protozoan parasite. Despite the complexity of the T. gondii proteome, CD8 T cell responses are restricted to only a small number of peptide epitopes derived from a limited set of antigenic precursors. This phenomenon is known as immunodominance and is key to effective vaccine design. However, the mechanisms that determine the immunogenicity and immunodominance hierarchy of parasite antigens are not well understood.Here, using genetically modified parasites, we show that parasite burden is controlled by the immunodominant GRA6-specific CD8 T cell response but not by responses to the subdominant GRA4- and ROP7-derived epitopes. Remarkably, optimal processing and immunodominance were determined by the location of the peptide epitope at the C-terminus of the GRA6 antigenic precursor. In contrast, immunodominance could not be explained by the peptide affinity for the MHC I molecule or the frequency of T cell precursors in the naive animals. Our results reveal the molecular requirements for optimal presentation of an intracellular parasite antigen and for eliciting protective CD8 T cells.