Reversible unfolding of the major fraction of ovalbumin by guanidine hydrochloride

The guanidine hydrochloride induced unfolding of the major fraction of ovalbumin (i.e. A1 which contains two phosphate groups and constitutes about 77% of the total protein) was investigated systematically by difference spectran and viscosity measurements. As judged by the intrinsic viscosity (3.9 ml/g), the native protein conformation is compact and globular. Difference spectral results showed extensive disruption of the native structure by guanidine hydrochloride with and without 0.1 M beta-mercaptoethanol were 31.1 and 27.0 ml/g. These and optical rotation results indicated that the denatured protein existed in a cross-linked random coil conformation in 6 M guanidine hydrochloride alone. Strikingly, in contrast to whole ovalbumin, the denaturation of its A1 fraction by guanidine hydrochloride was fully reversible and obeyed first-order kinetic law under different experimental condit ions of pH, temperature, and the denaturant concentration. The monotonic variation of deltaH for the unfolding of ovalbumin A1 by guanidine hydrochloride with temperature, the coincidence of the two transition curves obtained by measuring two independent properties (namely reduced viscosity and difference in light absorption at 288 nm (or 293 nm) as a function of the denaturant concentration, and finally the adherence of the unfolding as well as refolding reactions to first-order kinetic law suggested that the transition of ovalbumin. A1 can reasonably be approximated by a two-state mode. Analysis of the equilibrium data obtained at pH 7.0 and 25 degrees C according to Aune and Tanford (Aune, K.C.,and Tanford, C. (1969), Biochemistry 8, 4586) showed that 12 additional binding sites for the denaturant with an association constant of 1.12 were freshly exposed by the unfolding process and that the native protein was marginally more stable (approximately 6 kcal/mol) than its unfolded form even under native condition. The temperature dependence of the equilibrium constant for the unfolding of ovalbumin A1 by guanidine hydrochloride which was studied in the range 10-60 degrees C at pH 7.0 can be described by assigning the following values of the thermodynamic parameters for the unfolding process: deltaH = 52 kcal/mol at 25 degrees C; deltaS = 153 cal deg-1 mol-1 at 25 degrees C; and delta Cp = 2700 +/- 400 cal deg-1 mol-1.     

Cardiodepression by tumor necrosis factor-alpha

A 75-year-old man with carcinoma of the prostate presented with a pruritic, erythematous plaque involving the scrotal skin. Histological examination revealed extramammary Paget's disease. The intraepidermal tumour cells expressed prostate-specific antigen in keeping with a prostatic origin.     

Effect of Tb(III) on the unfolding of ciliate Euplotes octocarinatus centrin induced by guanidine hydrochloride

To understand the unfolding of ciliate Euplotes octocarinatus centrin (EoCen), the glycine positioned at 115, the sixth residue of the loop of the protein's third EF-hand, was mutated into tryptophan (Trp). Intrinsic fluorescence and Tb(III) binding properties of wild type EoCen and G115W mutant were monitored by fluorescence spectra in 10 mmol/L Hepes. The emission maximum of EoCen was 306 nm and mutation had no impact on the Tb(III) binding properties. The properties of G115W were investigated by fluorescence, far-UV circular dichroism (CD) spectra and fluorescence decays in the absence or in the presence of 6 mol/L guanidine hydrochloride (GdnHCl). For the increase in polarity of micro-environment around Trp residue, the emission maximum of apoG115W at 343 nm is shifted to 359 nm in 6 mol/L GdnHCl. Also the secondary structure is lost nearly and fluorescence lifetime decreases in 6 mol/L GdnHCl. The unfolding of G115W induced by GdnHCl was assessed by using the model of structural element. The unfolding of proteins is a sequential reaction, namely two-transition, three-state process. The first transition belongs to the unfolding of the C-terminal domain, and the second transition is assigned to the unfolding of the N-terminal domain. The $\Delta 〈\Delta G_{\text{total}}^0\left({\text{H}}_2\text{O}\right)〉$ was used to determine the effect of Tb(III) on the stability of apoprotein. The $〈\Delta G_{\text{total}}^0\left({\text{H}}_2\text{O}\right)〉$ for Tb₂-G115W has a less increase of 0.68 kJ/mol compared with apoG115W, proving Tb(III) situated at C-terminal has negligible impact on the stability of protein. Whereas the $〈\Delta G_{\text{total}}^0\left({\text{H}}_2\text{O}\right)〉$ for Tb₄-G115W has a rise of 1.29 kJ/mol compared with Tb₂-G115W, manifesting Tb(III) located at low affinity sites has considerable influence on protein stability, mainly stabilizing the N-terminal domain.     

Cytotoxicity induced by recombinant human tumor necrosis factor-alpha dependent on the types of its receptors on canine cells

Based on the recent findings that show how recombinant human tumor necrosis factor (rh-TNF)-alpha has potent antitumor activity on human cancer patients when it locally administrated, we have tested the cytotoxicity of rh-TNF-alpha on 3 canine cultured cells: (1) canine kidney carcinoma (CKCa-1), (2) mastocytoma and (3) Mardin Darby canine kidney cells (MDCK). The cell surface expression of TNF-alpha receptors on these canine cells was also determined with anti-human TNF RI and RII polyclonal antibodies. Our data shows that on CKCa-1 which has TNF RI receptors rh-TNF-alpha induced cytotoxicity. By contrast, it exhibited no toxicity on canine mastocytoma which has mainly RII receptors. The data also suggest actinomycin D (ACT-D), an anticancer antibiotic, enhanced the cytotoxicity of rh-TNF-alpha. Combined with ACT-D, rh-TNF-alpha showed the cytotoxicity on MDCK which possessed both TNF RI and RII receptors. The results indicate that the cytotoxicity of rh-TNF-alpha depends on the presence of TNF RI receptors on canine tumor cells.     

Activation of protein kinase C by tumor necrosis factor-alpha in human non-pigmented ciliary epithelium

Previously, we have shown that tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, increases the synthesis and release of endothelin-1 (ET-1), a potent vasoactive peptide from human non-pigmented ciliary epithelial (HNPE) cells, in a protein kinase C (PKC)-dependent manner. Diacylglycerol (DAG) and intracellular calcium ([Ca2+]i) are well known activators of PKC. Some cytokines induce PKC activation by stimulating phospholipase C that hydrolyzes phosphatidylinositol bisphosphate (PIP2) into IP3 (intracellular calcium mobilizer) and DAG. In this study, the existence of a similar pathway was evaluated in HNPE cells treated with TNF-alpha, using intracellular calcium ([Ca2+]i) measurements, PKC translocation assays and thin-layer chromatography (TLC) for quantification of DAG. Incubation times for agonists and inhibitors ranged from 1-30 minutes. The increase in DAG levels with TNF-alpha treatment was consistent with the observed translocation of the calcium-dependent PKC alpha isoform from the cytosol to the plasma membrane. However, these observations were not accompanied by a concomitant increase in [Ca2+]i. Similar translocation responses were observed with phorbol ester (phorbol 12-myristate 13-acetate) treatment. Our results indicate that TNF-alpha-induced PKC activation in HNPE cells occurs as a result of elevated DAG levels and is not due to an increase in intracellular calcium. Activated PKC, could enhance the pro-inflammatory responses of TNF-alpha in part by increasing the production of endothelins in the eye.     

Apoptosis in cultured human colon cancer cells induced by combined treatments with 5-fluorouracil, tumor necrosis factor-alpha and interferon-alpha

An experimental study of Allergan's Oxysept Comfort system was performed by measuring the slight reddish hue that appears in the disinfecting solution, indicating to the users that their lenses are again ready to be worn. The temporal evolution of the color of the solution has been measured under standardized conditions and analyzed in the CIELAB system, from the perspective of the typical threshold discrimination of the human eye. Color differences between neutralized and non-neutralized solutions occurred in an appropriate direction of the color space to enhance discrimination and were clearly perceptible by normal observers (greater than 9.7 +/- 1.2 CIELAB units). Colorimetric analyses have been used to draw conclusions regarding observers with defective color vision. The color of the solution changes abruptly, approximately 25 min after the neutralization process begins, and remains nearly constant after about 60 min, this agreeing well with the temporal evolution of the hydrogen peroxide concentration.     

Influence of pentoxifylline on fevers induced by bacterial lipopolysaccharide and tumor necrosis factor-alpha in guinea pigs

In guinea pigs intraperitoneal (i.p.) injections of 50 mg/kg pentoxifylline had no influence on abdominal temperature while higher doses of pentoxifylline caused a hypothermic response lasting for 2-3 h. Administration of 50 mg/kg pentoxifylline 1 h before intramuscular (i.m.) injections of 20 micrograms/kg bacterial lipopolysaccharide reduced the lipopolysaccharide-induced production of endogenous tumor necrosis factor-alpha (TNF-alpha) by 68%. The second phase of lipopolysaccharide-induced fever was significantly attenuated by pretreatment with 50 mg/kg pentoxifylline, a dose which had, per se, no influence on core temperature of guinea pigs. The thermal response of guinea pigs to administration of exogenous TNF-alpha was not modulated by pretreatment with pentoxifylline. Intra-arterial infusions with 5 micrograms/kg TNF-alpha, a dose which yielded the same circulating TNF bioactivity as i.m. injections of 20 micrograms/kg lipopolysaccharide, induced a biphasic febrile response. The magnitude and duration of TNF-induced fever were the same whether guinea pigs were pretreated with pentoxifylline or with 0.9% saline. The results indicate that endogenous formation of TNF-alpha may contribute to the development of fever induced by lipopolysaccharide, but is not its only mediator, since the first phase of lipopolysaccharide-induced fever was not altered by the blockade of TNF production.     

Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin

Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of both acute and chronic inflammatory responses in many diseases. Tristetraprolin (TTP), the prototype of a class of Cys-Cys-Cys-His (CCCH) zinc finger proteins, inhibited TNF-alpha production from macrophages by destabilizing its messenger RNA. This effect appeared to result from direct TTP binding to the AU-rich element of the TNF-alpha messenger RNA. TTP is a cytosolic protein in these cells, and its biosynthesis was induced by the same agents that stimulate TNF-alpha production, including TNF-alpha itself. These findings identify TTP as a component of a negative feedback loop that interferes with TNF-alpha production by destabilizing its messenger RNA. This pathway represents a potential target for anti-TNF-alpha therapies.     

TNF-R55-specific form of human tumor necrosis factor-alpha induces collagenase gene expression by human skin fibroblasts

Tumor necrosis factor-alpha (TNF-alpha) is a potent inhibitor of connective tissue formation. The cellular effects of TNF-alpha are mediated by two distinct cell-surface receptors, TNF-R55 and TNF-R75, both present on various types of cells, including fibroblasts. In this study we wanted to elucidate the role of TNF-R55 as a mediator of the connective tissue effects of TNF-alpha by using a mutant, TNF-R55-specific form of human TNF-alpha. This mutant TNF-alpha markedly induced collagenase and stromelysin-1 gene expression in dermal fibroblasts, the maximal activation (up to 42-fold) being 65%-89% of that noted with wild-type human TNF-alpha. In addition, TNF-R55-specific TNF-alpha suppressed type I collagen mRNA levels as potently as wild-type TNF-alpha (by 60%). The enhancement of collagenase gene expression by TNF-R55-specific TNF-alpha was augmented by simultaneous treatment of normal and scleroderma skin fibroblasts with interferon-gamma, indicating specific enhancement of TNF-R55 signaling pathway by interferon-gamma. These results show that stimulation of the TNF-R55 signaling pathway is sufficient for the inhibitory effects of TNF-alpha on extracellular matrix formation by dermal fibroblasts. It is conceivable that due to reduced systemic toxicity, TNF-R55-specific forms of human TNF-alpha may prove to be feasible in the therapy of fibrotic disorders.     

Morphological change in tumor endothelial cells induced by natural-type human tumor necrosis factor

The effects of natural-type human tumor necrosis factor (nh-TNF) on tumor endothelial cells of experimental brain tumors were investigated electron microscopically. Tumor vessels with hypertrophic endothelial cells were observed 12 and 24 hr after an intralesional administration of 5,000 U of nh-TNF. Increased biosynthetic organelles such as the Golgi complex and rough endoplasmic reticulum were evident in the plump cytoplasms. These endothelial cells resembled those in high endothelial venules (HEV) functionally characterized by the high permeability of leukocytes. In addition, close interactions between these endothelial cells and leukocytes were observed. Our findings indicated that nh-TNF could promote the morphological change in tumor endothelial cells into HEV-like cells.     

The specific type IV phosphodiesterase inhibitor rolipram suppresses tumor necrosis factor-alpha production by human mononuclear cells

Compounds suppressing the production of tumor necrosis factor-alpha are protective in animal models of septic shock. Recent studies demonstrated a beneficial effect of xanthine derivatives, which suppress tumor necrosis factor-alpha production by acting as non-specific cAMP phosphodiesterase inhibitors. In this experiment we tested the effect of (+/-)-rolipram (racemate) and its enantiomers on human mononuclear cells stimulated with lipopolysaccharide (LPS). Rolipram has a phenyl-pyrrolidinone structure, unrelated to the methylxanthines, and acts as a specific inhibitor of the type IV phosphodiesterase. Our results identify rolipram as a remarkably potent suppressor of the LPS-induced synthesis of tumor necrosis factor-alpha. When compared to the non-specific inhibitor pentoxifylline, the IC50 of (+/-)-rolipram (130 nM) is more than 500 times lower. The influence of rolipram on tumor necrosis factor-alpha production depended on the steric configuration of the molecule, since the (-)-enantiomer exhibited a five times lower IC50 than the (+)-enantiomer. The inhibitory effect of all substances tested is selective for tumor necrosis factor-alpha rather than interleukin-1 beta, since interleukin-1 beta production is only slightly influenced.     

Nutritional parameters observed during 28-day infusion of recombinant human tumor necrosis factor-alpha

In conjunction with a Phase I investigation of the antineoplastic activity of recombinant human tumor necrosis factor-alpha (TNF-alpha), administered as a 28-day continuous infusion, selected nutritional parameters were evaluated to identify any effect that might be attributed to the TNF infusion. Seven clinically stable men with a variety of tumor types were studied. None had clinical or laboratory evidence of significant malnutrition before entry into the study. Five patients received 10 micrograms of recombinant human TNF-alpha per square meter per day and two patients received 25 micrograms/m2 per day. Indirect calorimetry assessment of resting energy expenditure, body weight, serum TNF concentration, and laboratory analysis of common nutritional markers (albumin, prealbumin, and triglycerides) were performed at baseline, day 14, day 28, and 2 weeks (day 42) after completion of the infusion. There were no statistically significant differences by analysis of variance observed in any parameter during the study period compared with baseline values and values on day 42. Also, there were no differences between any parameters when stratified by dose administered, although the number of patients studied was small. Measured serum TNF concentrations ranged from 0.02 to 1.56 ng/mL and did not correlate with study day or dose of TNF infused. No correlation was observed between serum TNF concentrations and resting energy expenditure. Although others have reported significant metabolic changes associated with acute administration of TNF in humans and animals, our experience does not support a hypermetabolic state in patients receiving low daily dose, long-term (28-day) continuous infusion of recombinant human TNF-alpha, a state that may be consistent with many neoplastic conditions.     

Expression and cleavage of tumor necrosis factor-alpha and tumor necrosis factor receptors by human monocytic cell lines upon direct contact with stimulated T cells

Tumor necrosis factor-alpha (TNF-alpha) is a potent cytokine in inflammatory processes. A variety of mechanisms that modulate its activity have been described, one being its binding to soluble receptors (sTNFR). In this study, we demonstrate that human monocytic cells such as THP-1 respond to direct contact with a membrane preparation of stimulated HUT-78 cells by producing TNF-alpha and by releasing sTNFR-p75, but not sTNFR-p55, with different kinetics. TNF-alpha concentration peaked after 12 h of contact and then decreased, whereas sTNFR-p75 production increased progressively upon cell/cell contact. The decrease in TNF-alpha concentration is not due to trapping of TNF-alpha by its soluble receptors or other soluble or cell-associated molecules, but rather to a proteolytic activity associated to THP-1 cells. On the other hand, the increase in sTNFR-p75 release does not result from an increase in the cleavage of pre-existing cell-associated sTNFR-p75 but from an increase in TNFR-p75 expression, immediately followed by the cleavage of its extracellular domain. Phenylmethylsulfonylfluoride, a serine protease inhibitor, has a negative effect on both TNF-alpha degradation and sTNFR-p75 release by THP-1 cells. Thus, there may be an enzymatic activity associated to THP-1 cells that plays an important role in the neutralization of TNF-alpha activity both by degrading the molecule and by cleaving its receptors at the cell surface.     

Epinephrine inhibits tumor necrosis factor-alpha and potentiates interleukin 10 production during human endotoxemia

Short-term preexposure of mononuclear cells to epinephrine inhibits LPS-induced production of TNF, whereas preexposure for 24 h results in increased TNF production. To assess the effects of epinephrine infusions of varying duration on in vivo responses to LPS, the following experiments were performed: (a) Blood obtained from eight subjects at 4-24 h after the start of a 24-h infusion of epinephrine (30 ng/kg per min) produced less TNF after ex vivo stimulation with LPS compared with blood drawn before the start of the infusion, and (b) 17 healthy men who were receiving a continuous infusion of epinephrine (30 ng/kg per min) started either 3 h (EPI-3; n = 5) or 24 h (EPI-24; n = 6) were studied after intravenous injection of LPS (2 ng/kg, lot EC-5). EPI-3 inhibited LPS-induced in vivo TNF appearance and also increased IL-10 release (both P < 0.005 versus LPS), whereas EPI-24 only attenuated TNF secretion (P = 0.05). In separate in vitro experiments in whole blood, epinephrine increased LPS-induced IL-10 release by a combined effect on alpha and beta adrenergic receptors. Further, in LPS-stimulated blood, the increase on IL-10 levels caused by epinephrine only marginally contributed to concurrent inhibition of TNF production. Epinephrine, either endogenously produced or administered as a component of sepsis treatment, may have a net antiinflammatory effect on the cytokine network early in the course of systemic infection.     

Expression and release of tumor necrosis factor-alpha by explants of mouse cornea

PURPOSE: To elucidate a possible target of immunosuppressive agents widely used in the treatment of corneal disorders, the authors determined whether corneal cells are capable of expressing and releasing tumor necrosis factor-alpha (TNF alpha) on lipopolysaccharide (LPS) stimulation, and they investigated whether TNF alpha production can be modulated by pharmacologic agents. METHODS: Trephined central corneas from C57BL/6 mice were kept in culture for 3 days. Release of TNF alpha after a 24-hour stimulation with LPS (1 microgram/ml) into the culture medium was determined both by bioassay and by enzyme-linked immunosorbent assay. Expression of TNF alpha mRNA after 6-hour stimulation was examined by polymerase chain reaction. Immunofluorescent staining on cryostat sections of cultured corneas was performed to localize TNF alpha in the tissue. Corneal explants were pretreated with immunosuppressive agents (prednisolone, budesonide, cyclosporin A) for 48 hours, followed by 6-or 24-hour stimulation with LPS in the continuous presence of the agents. RESULTS: Lipopolysaccharide stimulated TNF alpha release into the culture medium. The addition of budesonide (10(-7) M) or prednisolone (10(-6) M) significantly inhibited LPS-induced TNF alpha release, whereas cyclosporin A (10(-7) - 10(-5) M) had no marked effect. Levels of TNF alpha mRNA in corneal explants increased fivefold after stimulation with LPS. Immunohistochemical staining revealed that TNF alpha was expressed in the epithelial cells. Budesonide markedly decreased mRNA expression and abolished immunostaining of TNF alpha stimulated by LPS. CONCLUSIONS: TNF alpha is produced and released by the epithelial cells of mouse central cornea in response to LPS. Contrary to cyclosporin A, corticosteroids such as prednisolone and budesonide potently inhibit TNF alpha production.     

Role of tumor necrosis factor-alpha and platelet-activating factor in neoangiogenesis induced by synovial fluids of patients with rheumatoid arthritis

The aim of the present study was to investigate in vivo in a mouse model the stimulation of neoangiogenesis by synovial fluids of patients with rheumatoid arthritis (RA) and to determine the role of tumor necrosis factor (TNF)-alpha and platelet-activating factor (PAF) in the formation of new vessels. Angiogenesis was studied in a mouse model in which Matrigel, injected subcutaneously, was used as a vehicle for the delivery of potential angiogenic stimuli. Synovial fluids of patients with RA but not with osteoarthritis (OA) were shown to induce neoangiogenesis. Since synovial fluid of patients with RA contained significantly higher levels of TNF-alpha-like bioactivity and of PAF than that of patients with OA, the role of these mediators was evaluated by using an anti-TNF-alpha neutralizing monoclonal antibody (mAb) and a PAF receptor antagonist, WEB 2170. When added to Matrigel, anti-TNF-alpha mAb and particularly WEB 2170 significantly reduced neoangiogenesis induced by synovial fluids of RA patients. Moreover, PAF extracted and purified from synovial fluid induced angiogenesis. These results suggest that the neoangiogenesis observed in rheumatoid synovitis may be due, at least in part, to the angiogenic effect of locally produced TNF-alpha and PAF.     

Study of antiinfectious potential of recombinant tumor necrosis factor-alpha

46 patients with coronary heart disease with hypercholesterolemia were exposed to therapeutic plasmapheresis (TP) in combination with alpha-tocopherol treatment (AT). The results of 3-month follow-up with assessment of the clinical status, lipid spectrum, lipid peroxidation, concentration of ceruloplasmin indicated high hypolipidemic effectiveness of TP 2-3 weeks after the treatment as shown by inhibition of lipid peroxidation and antioxidant system. The addition of AT prolonged the hypolipidemic effect of TP, promoted optimization of plasma antioxidant potential (a rise in HDL, stabilization of ceruloplasmin levels).