Characterization of the stromal cell-derived factor-1alpha-heparin complex

The binding of chemokines to glycosaminoglycans is thought to play a crucial role in chemokine functions. It has recently been shown that stromal cell-derived factor-1alpha (SDF-1alpha), a CXC chemokine with potent anti-human immunodeficiency virus activity, binds to heparan sulfate through a typical consensus sequence for heparin recognition (BBXB, where B is a basic residue KHLK, amino acids 24-27). Calculation of the accessible surface, together with the electrostatic potential of the SDF-1alpha dimer, revealed that other amino acids (Arg-41 and Lys-43) are found in the same surface area and contribute to the creation of a positively charged crevice, located at the dimer interface. GRID calculations confirmed that this binding site will be the most energetically favored area for the interaction with sulfate groups. Site-directed mutagenesis and surface plasmon resonance-based binding assays were used to investigate the structural basis for SDF-1alpha binding to heparin. Among the residues clustered in this basic surface area, Lys-24 and Lys-27 have dominant roles and are essential for interaction with heparin. Amino acids Arg-41 and Lys-43 participate in the binding but are not strictly required for the interaction to take place. Direct binding assays and competition analysis with monoclonal antibodies also permitted us to show that the N-terminal residue (Lys-1), an amino acid critical for receptor activation, is involved in complex formation. Binding studies with selectively desulfated heparin, heparin oligosaccharides, and heparitinase-resistant heparan sulfate fragments showed that a minimum size of 12-14 monosaccharide units is required for efficient binding and that 2-O- and N-sulfate groups have a dominant role in the interaction. Finally, the heparin-binding site was identified on the crystal structure of SDF-1alpha, and a docking study was undertaken. During the energy minimization process, heparin lost its perfect ribbon shape and fitted the protein surface perfectly. In the model, Lys-1, Lys-24, Lys-27, and Arg-41 were found to have the major role in binding a polysaccharide fragment consisting of 13 monosaccharide units.     

In vivo subcellular target compartments of interferon-gamma and interferon-gamma receptor (alpha- and beta-chains) in mouse liver

IFN-gamma displays several effects on different tissues via its specific cell surface receptor (IFN-gammaR). In order to identify the target compartments of IFN-gamma and IFN-gammaR (alpha and beta-chains), we used a quantitative immunogold approach. In physiological conditions, IFN-gamma and IFN-gammaR immunoreactivities were detected in the plasma membrane, in the endoplasmic reticulum area, in the mitochondria and in the nucleus. After a single IFN-gamma injection, we observed, in a quantitative manner, an increase of signal density without modification of the subcellular distribution of IFN-gamma and IFN-gammaR subunits.     

Effects of poly(ADP-ribose) polymerase inhibition in bladder damage caused by cyclophosphamide in rats

It was previously shown that nitric oxide produced by inducible nitric oxide synthase (iNOS) and peroxynitrite are responsible for cyclophosphamide (CP)-induced cystitis. Since endogenous production of peroxynitrite is known to lead to poly(ADP-ribose) polymerase (PARP) activation, in this study, the aim was to evaluate whether the PARP activation pathway is also included in the pathogenesis of CP-induced bladder ulceration in rats. A total of 48 male albino Wistar rats were divided into 5 groups. Group 1 served as control and was given 2 ml saline; four groups received a single dose of CP (200 mg/kg) with the same time intervals. Group 2 received CP only; Group 3, selective iNOS inhibitor 1400W (20 mg/kg); Group 4, peroxynitrite scavenger ebselen (30 mg/kg); and Group 5, PARP inhibitor 3-aminobenzamide (20 mg/kg). CP injection resulted in severe cystitis with continuous macroscopic hemorrhage, strong edema, inflammation, and ulceration. Moreover, bladder iNOS activation and urine nitrite-nitrate levels were dramatically increased. Histologically, 1400W protected bladder against CP damage and decreased urine nitrite-nitrate levels and bladder iNOS induction. Ebselen has shown similar histologic results with 1400W without changing urinary nitrite-nitrate level and iNOS activity. Furthermore in the 3-aminobenzamide group, beneficial effects had also occurred including decreased ulceration. These results suggest that PARP activation involves pathogenesis of CP-induced bladder ulceration. Furthermore, PARP is not only important for ulceration but also for bladder edema, hemorrhage, and inflammation because of broken uroepithelial cellular integrity.     

The early protective thymus-independent antibody response to foot-and-mouth disease virus is mediated by splenic CD9+ B lymphocytes

Infection of mice with cytopathic foot-and-mouth disease virus (FMDV) induces a rapid and specific thymus-independent (TI) neutralizing antibody response that promptly clears the virus. Herein, it is shown that FMDV-infected dendritic cells (DCs) directly stimulate splenic innate-like CD9(+) B lymphocytes to rapidly (3 days) produce neutralizing anti-FMDV immunoglobulin M antibodies without T-lymphocyte collaboration. In contrast, neither follicular (CD9(-)) B lymphocytes from the spleen nor B lymphocytes from lymph nodes efficiently respond to stimulation with FMDV-infected DCs. The production of these protective neutralizing antibodies is dependent on DC-derived interleukin-6 (IL-6) and on CD9(+) cell-derived IL-10 secretion. In comparison, DCs loaded with UV-inactivated FMDV are significantly less efficient in directly stimulating B lymphocytes to secrete TI antibodies. A critical role of the spleen in the early production of anti-FMDV antibodies in infected mice was also demonstrated in vivo. Indeed, either splenectomy or functional disruption of the marginal zone of the spleen delays and reduces the magnitude of the TI anti-FMDV antibody response in infected mice. Together, these results indicate that in addition to virus localization, the FMDV-mediated modulation of DC functionality is a key parameter that collaborates in the induction of a rapid and protective TI antibody response against this virus.     

Exposure Time Related Oxidative Action of Hyperbaric Oxygen in Rat Brain

Hyperbaric oxygen (HBO) is known to cause oxidative stress in several organs and tissues. Due to its high rate of blood flow and oxygen consumption, the brain is one of the most sensitive organs to this effect. The present study was performed to elucidate the relation of HBO exposure time to its oxidative effects in rats’ brain cortex tissue. For this purpose, 49 rats were randomly divided into five groups. Except the control group, study groups were subjected to three atmospheres HBO for 30, 60, 90, and 120 min. Their cerebral cortex layer was taken immediately after exposure and used for analysis. Thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and nitrate–nitrite (NO_X) levels were determined. TBARS and SOD levels were found to increase in a time-dependent manner. GSH-Px activity reflected an inconsistent course. NO_X levels were found to be increased only in the 120 min exposed group. The results of this study suggests that HBO induced oxidative effects are strongly related with exposure time.     

A kinetics and modeling study of RANTES(9-68) binding to heparin reveals a mechanism of cooperative oligomerization

Heparan sulfate (HS) and heparin bind to virtually all chemokines and have been shown to play critical roles in the regulation of their activities. However, both binding mechanisms and structural features involved in chemokine-HS interactions remain poorly defined. In the study presented here, we analyzed the binding of heparin to RANTES(9-68), a N-terminally truncated form of the CC-chemokine RANTES. Using biochemical and surface plasmon resonance (BIAcore system) approaches, we showed that the RANTES(9-68)-heparin interaction was characterized by a complex binding model that involved dimerization of the chemokine through a mechanism of positive cooperativity. Since RANTES(9-68) remains monomeric in solution, we concluded that heparin induced chemokine dimerization. The structure of a complex involving a RANTES dimer and a heparin heptadecasaccharide was proposed by molecular modeling. This model was used to design a dimer of "head to head" coupled octasaccharides that would fit the internal symmetry of the chemokine dimer. This engineered oligosaccharide bound RANTES(9-68) much better than a natural heparin fragment of the same length, further supporting the interaction process and the proposed structural model. Altogether, the data reported here provide a basis for understanding the mechanisms by which HS modulates RANTES functions.     

Exogenously administered and endogenously produced melatonin reduce hyperbaric oxygen-induced oxidative stress in rat lung

Hyperbaric oxygen (HBO) is a widely used treatment modality in many diseases. A known side effect of HBO is the production of reactive oxygen species. Many antioxidants such as vitamins C and E, riboflavin and selenium have been used successfully to scavenge the reactive oxygen species caused by HBO administration. In this study, we aimed to see if melatonin, a newly discovered antioxidant, has a protective effect against the overproduction of reactive oxygen species produced by HBO in rat lung tissue. Sixty male Sprague-Dawley rats were divided into 5 groups as follows: control, daytime HBO (3 ATA, 120 min), daytime HBO plus melatonin (10 mg/kg), nighttime HBO and nighttime HBO (under light exposure). The MDA, SOD and CAT levels of daytime and nighttime HBO (under light exposure) increased significantly. This significance was not found in the daytime HBO plus melatonin and nighttime HBO groups when compared with the control. In this study, HBO caused oxidant stress, and melatonin decreased the levels of MDA, SOD and CAT. Moreover, endogenous melatonin was found to be a more effective antioxidant than exogenous 10 mg/kg melatonin.     

Internalization and nuclear translocation of IFN-gamma and IFN-gammaR: an ultrastructural approach

IFN-gamma signalling involves the Jak-STAT pathway. However, several hypothesis have been proposed where the receptor and its ligand itself took an active role within the cell. Using a quantitative immunogold approach, we found that both IFN-gamma and its receptor are rapidly internalized and translocated in the nucleus. We found that cell surface heparan sulfate, which binds IFN-gamma, delayed the nuclear accumulation of IFN-gamma suggesting that these molecules serve as storage depot around the cell for local delivery of the cytokine.     

Alpha-tocopherol, beta-carotene and melatonin administration protects cyclophosphamide-induced oxidative damage to bladder tissue in rats

Cyclophosphamide (CP) has potential urotoxicity such as hemorrhagic cystitis (HC). 2-Mercaptoethane sulfonate (mesna) has been widely used as an effective agent against CP-induced cystitis, but significant HC has still been encountered clinically. In recent studies, mesna was shown to be more effective if combined with antioxidants. The purpose of this study was to evaluate the effects of antioxidants, alpha-tocopherol, beta-carotene and melatonin on CP-induced bladder damage in rats, even if used without mesna administration. Male Sprague-Dawley rats weighing 180-210 g were divided into 5 groups. Four groups received a single dose of CP (100 mg/kg) intraperitoneally with the same time intervals. Group 2 received CP only, group 3 received beta-carotene (40 mg/kg/day), group 4 received alpha-tocopherol (40 mg/kg/day) and group 5 received melatonin (10 mg/kg/day) both before and the day after CP injection. Group 1 served as control. Bladder histopathology, as well as malondialdehyde (MDA) and iNOS levels, and excretion of nitrite-nitrates (NO(x)) in urine were evaluated. CP injection resulted in severe histological changes and macroscopic hematuria. alpha-Tocopherol and melatonin showed meaningful protection against bladder damage. Protection by beta-carotene was also significant but weaker. MDA levels increased significantly with CP injection and all antioxidants ameliorated this increase in bladder tissue. CP also elevated the NO(x) level in urine and iNOS activity in bladder. Only melatonin was able to decrease these parameters. In conclusion, there is no doubt that oxidants have a role in the pathogenesis of CP-cystitis. Antioxidants, especially melatonin and alpha-tocopherol, may help to ameliorate bladder damage induced by CP.