Differential expression of transforming growth factor-beta 1, -beta 2, and -beta 3 by glioblastoma cells, astrocytes, and microglia.

The type beta transforming growth factors (TGF) are potent regulators of the growth and functions of lymphocytes and macrophages. Recently the human glioblastoma cell line 308 was shown to produce TGF-beta 2. The relevance of this finding was evaluated further by comparing human glioblastoma cells with their nontransformed animal counterpart, astrocytes, with regard to the production of the three TGF-beta isoforms observed so far in mammals. In this report astrocytes are demonstrated to secrete also TGF-beta 2 and to express TGF-beta 1, -beta 2, and -beta 3 mRNA in vitro. In contrast, cultured murine brain macrophages release TGF-beta 1 and are positive for TGF-beta 1 mRNA only. Glia cell-derived TGF-beta 1 and -beta 2 are detected in latent form whereas both latent and active TGF-beta are identified in the supernatant of three human glioblastoma cell lines tested. These cell lines, however, show heterogeneity in regard to the isoform of TGF-beta expressed but share with astrocytes the inability to release TGF-beta 3. Provided production and activation of latent TGF-beta occur in vivo, astrocytes and microglia may then be expected to exert regulatory influences on immune mediated diseases of the central nervous system.     

Transforming growth factor-beta 1, -beta 2 and -beta 3 in cartilage and bone cells during endochondral ossification in the chick.

The localization of TGF-beta 1, -beta 2 and -beta 3 was studied in the growth plate, epiphysis and metaphysis of the tibiotarsus of three-week-old chicks. The different TGF-beta isoforms were localized to hypertrophic chondrocytes, chondroclasts, osteoblasts and osteoclasts using immunohistochemical staining analysis with specific TGF-beta antibodies. TGF-betas in osteoclasts and chondroclasts were restricted to those cells located on the respective matrices. TGF-beta 3 localization was mainly cytoplasmic in the transitional (early hypertrophic) chondrocytes, but nuclear staining was also detected in some proliferating chondrocytes. The cell-specific localization of these TGF-beta isoforms supports the hypothesis that TGF-beta has a role in the coupling of new bone formation to bone and cartilage matrix resorption during osteochondral development and suggests that TGF-beta may be a marker of chondrocyte differentiation. TGF-beta localization preceded a marked increase in type II collagen mRNA expression in transitional chondrocytes, suggesting a role for TGF-beta in the induction of synthesis of extracellular matrix.     

Characterization of the binding of transforming growth factor-beta 1, -beta 2, and -beta 3 to recombinant beta 1-latency-associated peptide.

Preprotransforming growth factor-beta 1 (TGF beta 1) is a 390-amino acid precursor polypeptide that undergoes a number of processing steps to yield mature TGF beta 1 (amino acid residues 279-390) and a pro portion (residues 30-278) termed beta 1-latency-associated peptide (beta 1LAP). The dimeric form of beta 1LAP has been shown to associate noncovalently with the mature growth factor, resulting in inactivation of biological activity. To further characterize this interaction, the mature TGF beta 1 was radioiodinated and used to determine dissociation constants. A cross-linking method using the bifunctional covalent cross-linker bis-(sulfosuccinimidyl)suberate was found to be the best approach for measuring the amount of bound growth factor. The efficiency of cross-linking was constant within each experiment and varied between 45-55%. Saturation plots and their associated Scatchard analyses indicate apparent Kd values between 1.1-1.8 nM. Competition of TGF beta 1 binding to beta 1LAP by TGF beta 2 and TGF beta 3 (two closely related growth factors) revealed that the latter also bind beta 1LAP tightly, with apparent Kd values of 1.9 and 0.4 nM, respectively.     

Epitope-dependent localization of estrogen receptor-alpha,but not -beta,in en face arterial endothelium

Rapid, nongenomic effects of 17 beta-estradiol (E(2)) in endothelial cells are postulated to arise from membrane-associated estrogen receptors (ERs), which have not been visualized in vascular tissue. To identify membrane ERs, we used multiple site-directed ER alpha or ER beta antibodies to label en face rat cerebral and coronary arterial endothelia. Western blots revealed a novel 55-kDa ER alpha isoform. Three-dimensional images of cells labeled with these antibodies and markers for the nucleus and caveolin-1 were acquired with a wide-field microscope, deconvolved, and numerically analyzed. We found ER alpha in the nucleus and cell periphery, where one-third colocalized with caveolin-1. The receptor location was dependent on the epitope of the antibody. Human ovarian surface epithelium produced similar results; but in rat myometrium, the distribution was epitope independent and nuclear. ER beta distribution was predominantly intranuclear and epitope independent. A small amount of ER alpha colocalized with ER beta within the nucleus. The results were identical in both arterial preparations and insensitive to E(2). We postulate that the different ER alpha conformations at the membrane, in the nucleus, and between different cell types allow E(2) to trigger cell- and location-specific signaling cascades.     

Polymorphism of chicken CD8-alpha, but not CD8-beta

 We report here the structural basis of CD8 polymorphism in the chicken. Three chicken strains (RPRL Line 7, H.B15.H7, and H.B15.H12) have 14 nucleotide differences in the CD8A cDNA sequence causing eight amino acid replacements in the extracellular part of the molecule. Only two amino acid replacements and four silent mutations were observed in the CD8B cDNA sequence in one (H7) of the strains. Substitutions in CD8α were solely responsible for the binding of CD8-specific monoclonal antibodies, as detected by cDNA expression in COS cells. The majority of the amino acid substitutions are located in the immunoglobulin V-like domain and three of the changes (residues 30, 34, and 58) are situated in the putative major histocompatibility complex class I binding CDR1 and CDR2 regions of the chicken CD8α. CD8A polymorphism has not been reported in other species and this suggests that CD8A and CD8B have evolved under different selective pressures in the chicken. Received: 28 February 1997 / Revised: 19 April 1997     

Recombinant interferon-alpha, -beta, and -gamma enhance the proliferative response of human B cells

Recombinant interferons (IFN-alpha, -beta, and -gamma) were examined for their effects on B cell activation. Relatively small IgM+ B cells from human blood samples were isolated by fluorescence-activated cell sorting and were used as target cells. Although the interferons themselves were nonmitogenic, each enhanced the proliferative response induced by a mitogenic anti-mu monoclonal antibody, with IFN-beta usually showing the greatest enhancement and IFN-gamma the least. Pretreatment with the interferons primed resting B cells to undergo enhanced DNA synthesis in response to the anti-mu antibody DA4. Conversely, anti-mu pretreatment, followed by IFN treatment, did not induce B cells to enter the S phase. Time-course analysis revealed that IFN could augment the anti-mu response even when added as late as the final 24 hr of a 3-day culture interval. Combinations of IFN-gamma plus IFN-alpha or -beta were synergistic in the anti-mu response, whereas the IFN-alpha plus IFN-beta combination was not. The data suggest that interferons produced by both lymphocytes (IFN-gamma) and nonlymphoid inflammatory cells (IFN-alpha and -beta) can enhance B cell growth via different mechanisms.     

转化生长因子β

TGFβ广泛存在于动物体多种组织和细胞内,由二条相同的、含112个氨基酸的肽链组成,是细胞的多功能双重调节因子。它对不同组织类型的细胞,可促进生长、分化,也可抑制生长、分化,并直接参与组织修复、胚胎发育等过程,调节细胞外基质形成。     

Transforming growth factor-beta 1, -beta 2, and -beta 3 secreted by a human glioblastoma cell line. Identification of small and different forms of large latent complexes.

Transforming growth factor-beta 1 (TGF-beta 1) has been found to occur as latent high molecular weight complexes, with or without an associated component denoted latent TGF-beta 1-binding protein (LTBP). We show here that a human glioblastoma cell line (U-1240 MG) secretes all isoforms of TGF-beta s found in mammalian cells (TGF-beta 1, -beta 2, and -beta 3). Approximately 26% of the secreted TGF-beta is in an active form. Latent TGF-beta s were partially purified from medium conditioned by the U-1240 MG cell line using anion exchange chromatography. Analysis of the different fractions by immunoblotting using antisera against precursor parts of the different TGF-beta isoforms, and against LTBP, revealed that not only TGF-beta 1 but also other isoforms of TGF-beta may occur in high molecular weight forms containing LTBP. In addition, each one of the TGF-beta isoforms occurred in smaller forms not containing LTBP. Interestingly, each of the TGF-beta isoforms was also seen in complexes of about 210 kDa containing associated component(s) distinct from LTBP. These results indicate that each of the different isoforms of TGF-beta is synthesized and secreted by this glioblastoma cell line in several different high molecular weight latent forms; the biological importance of the various latent TGF-beta complexes is discussed.     

Preparation of 9-alpha,11-xi-tritiated 17-alpha-ethynylestradiol, mestranol, estradiol-alpha-17-beta, and norethindrone

The preparation of 9alpha, 11xi-tritiated 17alpha-ethinyl estradiol, mestranol, estradiol-17beta, and norethindrone are described. Estrone-3-methyl ether was employed as starting material, and ethinylation with lithium acetylide-ethylene diamine resulted in 95% mestranol. Demethylation of mestranol with boron tribromide at 0 degrees resulted in 92% 17alpha-ethinyl estradiol. Dimethylsulfoxide was the choice of reagent for the condensation reaction which was complete at room temperature in about 4 hours. The usually less than 3% of unreacted 17-oxo product was removed by Girard separation. Demethylation of methyl ether with boran tribromide in methylene chloride resulted in an excellent yield of 17alpha-ethinyl estradiol-9alpha, 11xi-tritium. 3-methoxyestra-1,3,5-trien-17-one-9alpha, 11xi-tritium was reduced with sodium bis(2-methoxyethoxy) aluminum hydride to the 17beta-hydroxy compound and subsequent demethylation resulted in estradiol-9alpha, 11xi-tritium. The general method of Ringold et al was employed for the preparation of 17beta-hydroxy-17alpha-ethinylestr-4-en-3-one. Improvements for small scale radiosynthesis are also presented.     

Adrenal 11-beta hydroxysteroid dehydrogenase activity in response to stress

This work studied the effect of stresses produced by simulated gavage or gavage with 200 mmol/L HCl two hours before adrenal extraction, on the activities of the 11beta-hydroxysteroid dehydrogenase 1 and 11beta-hydroxysteroid dehydrogenase 2 isoforms present in the rat adrenal gland. These activities were determined on immediately prepared adrenal microsomes following incubations with 3H-corticosterone and NAD+ or NADP+. 11-dehydrocorticosterone was measured as an end-product by TLC, and controls were adrenal microsomes from rats kept under basal (unstressed) conditions. 11beta-hydroxysteroid dehydrogenase 1 activity, but not 11beta-hydroxysteroid dehydrogenase 2 activity, was increased under both stress-conditions. Homeostatically, the stimulation of 11beta-hydroxysteroid dehydrogenase 1 activity would increase the supply of glucocorticoids. These, in turn, would activate the enzyme phenylethanolamine N-methyl transferase, thereby improving the synthesis of epinephrine as part of the stress-response.