Synergistic effects of endothelin-1 (ET-1) and transforming growth factor alpha (TGF-α) or epidermal growth factor (EGF) on DNA replication and G1 to S phase transition

The cooperative cell kinetic actions of ET-1 with TGF- or EGF in normal rat kidney fibroblasts (NRK-49F) and KNRK cells (Kirsten MSV transformed) were analyzed by [³H]-thymidine incorporation assay and flow cytometry. A marked synergistic effect of TGF- and ET-1 (or EGF and ET-1) on DNA synthesis and G1 to S transition was observed in NRK cells; 15–20% S for TGF- and 12% S for ET-1 alone but 45–50% S in combination. There was no detectable effect on cell cycle kinetics by TGF- (1 ng/ml) or EGF (1 ng/ml) plus ET-1 (1 ng/ml) in KNRK cells treated for 22 hours. Insulin, insulin-like growth factor I (IGF-I), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), and transforming growth factor (TGF-) were also tested and found to have no significant synergistic effects on ET-1 actions. Our findings suggest that the combination of TGF- (EGF) and ET-1 is an important part of an intricate network which coordinates progression of G1 to S phase in normal cells.     

Potential role of follicle-stimulating hormone (FSH) and transforming growth factor (TGFβ1) in the regulation of ovarian angiogenesis

Angiogenesis occurs during ovarian follicle development and luteinization. Pituitary secreted FSH was reported to stimulate the expression of endothelial mitogen VEGF in granulosa cells. And, intraovarian cytokine transforming growth factor (TGF)β1 is known to facilitate FSH‐induced differentiation of ovarian granulosa cells. This intrigues us to investigate the potential role of FSH and TGFβ1 regulation of granulosa cell function in relation to ovarian angiogenesis. Granulosa cells were isolated from gonadotropin‐primed immature rats and treated once with FSH and/or TGFβ1 for 48 h, and the angiogenic potential of conditioned media (granulosa cell culture conditioned media; GCCM) was determined using an in vitro assay with aortic ring embedded in collagen gel and immunoblotting. FSH and TGFβ1 increased the secreted angiogenic activity in granulosa cells (FSH + TGFβ1 > FSH ≈ TGFβ1 > control) that was partly attributed to the increased secretion of pro‐angiogenic factors VEGF and PDGF‐B. This is further supported by the evidence that pre‐treatment with inhibitor of VEGF receptor‐2 (Ki8751) or PDGF receptor (AG1296) throughout or only during the first 2‐day aortic ring culture period suppressed microvessel growth in GCCM‐treated groups, and also inhibited the FSH + TGFβ1‐GCCM‐stimulated release of matrix remodeling‐associated gelatinase activities. Interestingly, pre‐treatment of AG1296 at late stage suppressed GCCM‐induced microvessel growth and stability with demise of endothelial and mural cells. Together, we provide original findings that both FSH and TGFβ1 increased the secretion of VEGF and PDGF‐B, and that in turn up‐regulated the angiogenic activity in rat ovarian granulosa cells. This implicates that FSH and TGFβ1 play important roles in regulation of ovarian angiogenesis during follicle development. J. Cell. Physiol. 226: 1608–1619, 2011. © 2010 Wiley‐Liss, Inc.     

Involvement of polyamines in epidermal growth factor (EGF), transforming growth factor (TGF)-α and -β1 action on culture of L6 and fetal bovine myoblasts

• 1.1. α-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase significantly abolished stimulation of protein synthesis evoked by EGF, TGF-α or -β 1 in L6 and fetal bovine myoblasts.
• 2.2. The participation of polyamines in early events evoked by growth factors was shown by a significant stimulation of ornithine decarboxylase and Sdenosylmethionine decarboxylase activity as well as increased concentration of spermidine and spermine in L6 cells exposed to TGF-α and EGF.
• 3.3. TGF-β 1 at a high concentration (1 ng/ml) increased protein synthesis in L6 myoblasts but inhibited it in fetal bovine myoblasts. Metabolic effects of TGF-β 1 in L6 cells was associated with an enhancement of decarboxylase activities, however there were no significant changes in cellular polyamine concentrations. Presented data suggest that polyamines are involved in the signal transduction pathway of EGF, TGF-α, and -β 1 in L6 and fetal bovine myoblasts.

     

Comparison of transforming growth factor β and a human tumour-derived suppressor factor

Summary Serum-free supernatants from the human melanoma cell line G361 contain a factor that can potently suppress the generation of tumouricidal lymphokine-activated killer (LAK) cells in response to interleukin-2. To characterise the suppressive factor of tumour origin we performed a number of physicochemical and functional comparisons with another immunosuppressive protein, transforming growth factor (TGF). The bioactivity of tumour-derived suppressor factor (TDSF), assayed by suppression of LAK cell generation, was unaffected by a reducing agent but lost when denatured with a chaotropic agent. In contrast, TGF was inactivated by reduction but not denaturation. TDSF lost bioactivity in conditions of pH less than 4, whereas TGF showed no loss of activity. The TDSF moiety has an estimated pI of 4.3 and a molecular mass of 69–87 kDa. This differs from published values of pI 9.5, and 25 kDa molecular mass for TGF. Anti-TGF antiserum reversed the effects of TGF but did not affect the suppression of LAK cell generation caused by TDSF. These findings provide compelling evidence that the TDSF moiety is not TGF, and may be a novel immunoregulatory cytokine.     

Oral glucosamine increases expression of transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) mRNA in rat cartilage and kidney: implications for human efficacy and toxicity

Glucosamine is used for alleviating pain in osteoarthritis. Clinical trials have reported that glucosamine has equivocal efficacy. Glucosamine is also used in cell cultures to stimulate hexosamine flux and protein O-glycosylation, but at many-fold greater concentrations than those in human plasma following oral dosing. Lean Zucker rats were dosed orally for 6 weeks with glucosamine hydrochloride at doses (0–600 mg/kg/day) that produced peak serum concentrations of <1–35 μM, spanning the human exposure range. Relative expression of both TGFβ1 and CTGF mRNA were significantly increased up to 2.3-fold in liver, kidney and articular cartilage when evaluated 4 h after final dose. Apparent threshold serum glucosamine (C_max) concentration required to increase TGFβ1 expression in cartilage was 10–20 μM. These increases were associated with significant increases in UDP-N-acetylglucosamine concentrations suggesting increased hexosamine flux. Both TGFβ1 and CTGF are mediators of chondrocyte proliferation and cartilage repair. Study demonstrates that oral glucosamine doses that produce clinically relevant serum glucosamine concentrations can induce tissue TGFβ1 and CTGF expression in vivo and provides a mechanistic rationale for reported beneficial effects of glucosamine therapy. Induction of renal TGFβ1 and CTGF mRNA suggests that potential sclerotic side-effects may occur following consumption of potent glucosamine preparations.     

Molecular cloning of rock bream's (Oplegnathus fasciatus) tumor necrosis factor receptor-associated factor 2 and its role in NF-κB activiation

Tumor necrosis factor receptor-associated factor 2 (TRAF2) acts as a transducer of tumor necrosis factor-α (TNF-α)-triggered cell signals which results in inflammation, cell proliferation and antiapoptotic response. In this study, we have cloned cDNA of rock bream (Oplegnathus fasciatus) TRAF2, and analyzed its function in activation of NF-κB. The full length cDNA of rock bream TRAF2 consisted of 95 bp 5' UTR, 335 bp 3' UTR, and 1563 bp ORF encoding 520 amino acids that contained N-terminal RING-type and TRAF-type zinc finger domains and a C-terminal TRAF domain. The deduced amino acid sequence of rock bream TRAF2 showed more than 75% identity with other fish TRAF2s, and even as high as 56% identity with mouse and human TRAF2 proteins. To know whether the rock bream TRAF2 involves in NF-κB activation, Epithelioma papulosum cyprini (EPC) cells harboring an NF-κB reporting vector were transfected with a vector expressing rock bream TRAF2 or a control empty vector. NF-κB activity of EPC cells was significantly increased by exposure to the rock bream recombinant TNF-α. EPC cells transfected with the vector expressing rock bream TRAF2 showed significantly higher NF-κB activity by stimulation with the recombinant TNF-α than cells transfected with a control empty vector, suggesting the present rock bream TRAF2 acts as a transducer of TNF-α-mediated cell signals that enhance NF-κB activation.     

Effects of transforming growth factor β1 and basic fibroblast growth factor on lipoprotein lipase activity in primary cultures of chicken (Gallus domesticus) adipocyte precursors

• 1.1. The effect of TGF-β and bFGF on lipoprotein lipase activity in chicken adipocyte precursors was investigated.
• 2.2. Lipoprotein lipase activity was reduced by up to 80% by incubation with TGF-β whereas bFGF had no effect.
• 3.3. Contrary to that found with the 3T3-L1 preadipocyte cell line it was not necessary for TGF-β to be present prior to the start of differentiation in order to be effective.
• 4.4. Incubation of adipocyte precursors with actinomycin D abolished the effect of TGF-β suggesting that synthesis of a protein effector is required.
• 5.5. These results indicate differences in responsiveness to TGF-β and bFGF between primary chicken adipocyte precursors and some preadipocyte cell lines.

     

Synthesis of (±)-Homosarkomycin and (±)-Rosaprostol

(±)-Homosarkomycin (2) and (±)-rosaprostol (3) were synthesized from (±)-methyl 2-oxo-bicyclo[3.1.0]hexane-1-carboxylate (1) by using the nucleophilic ring opening reaction on the double-activated cyclopropane ring as the key step.     

Synthesis of (±)-Lamprolobine and (±)-Epilamprolobine

(±)-Lamprolobine, the (+)-enatiomer of which was isolated from the leaves of Lamprolobium fruticosum, and (±)-epilamprolobine were synthesized from δ-valerolactam.     

Immunogenicity of mannophosphoinositides of mycobacteria: Effect of cord factor (trehalose-6,6′-dimycolate)

Attempts were made to enhance the immunogenicity of mannophosphoinositides of mycobacteria against experimental tuberculosis. It was found that immunization of mice with a combination of mannosides and cord factor, coupled to methylated bovine serum albumin (MBSA) could not further alter the protective efficacy of mannoside-MBSA complexes against challenge with a LD₅₀ dose ofM. tuberculosis H₃₇Rv as seen by survival-to-mortality rate, root-specific lung mass, lung density and bacterial counts recovered from the infected organs.     

Relationship of tumor necrosis factor α expression with activation of sphingomyelinase and lipid peroxidation after removal of cholestatic factor

The goal of this work was to study the expression of tumor necrosis factor α (TNFα), sphingomyelin cycle activation, and lipid peroxidation (LPO) processes after the removal of a cholestatic factor in the liver subjected to different durations of cholestasis. Restored bile flow after a 9-day hepatic cholestasis normalized sphingomyelinase (SMase) activity and levels of TNFα and LPO products. The removal of a cholestatic factor after a 12-day cholestasis did not normalize the studied parameters: SMase activity and the levels of TNFα and LPO products remained much higher compared to control. A significant positive correlation between TNFα expression, SMase activity, and LPO rate has been revealed. The obtained data indicate that hepatocyte apoptosis after bile outflow restoration in late cholestasis can be due to the activation of the sphingomyelin cycle, LPO, and TNFα expression. The synergistic interaction can sharply increase the proapoptotic capacity of each of these factors since TNFα activates SMase and LPO, SMase activity depends on the LPO rate, while ceramide, an SMase-produced secondary messenger of apoptosis, can induce oxidative stress.