In vitro denaturation-renaturation of fibronectin. Formation of multimers disulfide-linked and shuffling of intramolecular disulfide bonds

It is well established that fibronectin into extracellular matrix undergoes repeated tensions applied by cells, resulting into dramatic structural changes which reflect its elastic properties. However, there is currently no study reporting with precision the consequences of this elasticity on fibronectin structure and conformation. In the present work, we investigated fibronectin structural and conformational reorganization in vitro through a denaturation-renaturation approach. The similarities and differences between "refolded fibronectin" and "native fibronectin" were investigated using various spectroscopic methods, hydrodynamic characterization, molecular imaging and biochemical characterization. In the refolded form, secondary structure elements as well as local tyrosine and tryptophan environment are identical compared to the native form. Interestingly, some differences in global tertiary structure organization and molecular conformation were observed. These differences are due to the reactivity of the two free cysteines, which are buried in the native state but become accessible during the unfolding process. First, oxidation of these residues leading to the formation of intermolecular disulfide bonds results in formation of stabilized multimer. Second, some illegitimate intramolecular disulfide bonds are formed. The presence of iodoacetamide, the sulfhydryl alkylating agent, during the unfolding-refolding process prevents all these events. This study clearly demonstrates that, under near physiological conditions, competitive renaturation pathways occur, involving free cysteines in either multimer formation or intermolecular shuffling of disulfide bonds. These findings might have important implications for future studies and be helpful to develop a deeper understanding of fibronectin morphology.     

Antioxidant potential of C-phycocyanin isolated from cyanobacterial species Lyngbya, Phormidium and Spirulina spp

The antioxidant activity of C-Phycocyanin (C-PC) isolated from three cyanobacterial species Lyngbya (marine), Phormidium (marine) and Spirulina (fresh water) was studied in vitro. The results demonstrate that C-PCs from Lyngbya, Phormidium and Spirulina spp. are able to scavenge peroxyl radicals (determined by crocin bleaching assay) with relative rate constant ratio of 3.13, 1.89 and 1.8, respectively. C-PCs also scavenge hydroxyl radicals (determined by deoxyribose degradation assay) with second order rate constant values of 7.87 x 10(10), 9.58 x 10(10) and 6.42 x 10(10), respectively. Interestingly, Lyngbya C-PC is found to be an effective inhibitor of peroxyl radicals (IC50 6.63 microM), as compared to Spirulina (IC50 12.15 microM) and Phormidium C-PC (IC50 12.74 microM) and is close to uric acid (IC50 2.15 microM). Further, the studies suggest that the covalently-linked tetrapyrrole chromophore phycocyanobilin is involved in the radical scavenging activity of C-PC. The electron spin resonance (ESR) spectra of C-PCs indicate the presence of free radical active sites, which may play an important role in its radical scavenging property. This is the first report on the ESR activity of native C-PCs without perturbations that can cause radical formation.     

Treatment with dehydroepiandrosterone (DHEA) stimulates oxidative energy metabolism in the liver mitochondria from developing rats

Effects of treatment with DHEA (0.2 mg or 1.0 mg / kg body weight for 7 days) on oxidative energy metabolism on liver mitochondria from developing and young adult rats were examined. Treatment with DHEA resulted in a progressive dose-dependent increase in the liver weights of the developing animals without change in the body weight. In the young adult rats treatment with 1.0 mg DHEA showed increase only in the body weight. Treatment with DHEA stimulated state 3 and state 4~respiration rates in developing as well as young adult rats in dose-dependent manner with all the substrates used; magnitude of stimulation was age-dependent. In young adults the extent of simulation of state 3 respiration rates declined at higher dose (1.0~mg) of DHEA with glutamate and succinate as substrates. Stimulation of state 3 respiration rates was accompanied by increase in contents of cytochrome aa_3, b and c + c₁ and stimulation of ATPase and dehydrogenases activities in dose- and age-dependent manner.     

Interaction between glutamate and GABA systems in the integration of sympathetic outflow by the paraventricular nucleus of the hypothalamus

The paraventricular nucleus (PVN) of the hypothalamus is a central site known to modulate sympathetic outflow. Excitatory and inhibitory neurotransmitters within the PVN dictate final outflow. The goal of the present study was to examine the role of the interaction between the excitatory neurotransmitter glutamate and the inhibitory neurotransmitter GABA in the regulation of sympathetic activity. In alpha-chloralose- and urethane-anesthetized rats, microinjection of glutamate and N-methyl-D-aspartate (NMDA; 50, 100, and 200 pmol) into the PVN produced dose-dependent increases in renal sympathetic nerve activity, blood pressure, and heart rate. These responses were blocked by the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP-5). Microinjection of bicuculline, a GABA(A) receptor antagonist, into the PVN (50, 100, and 200 pmol) also produced significant, dose-dependent increases in renal sympathetic nerve activity, blood pressure, and heart rate; AP-5 also blocked these responses. Using microdialysis and HPLC/electrochemical detection techniques, we observed that bicuculline infusion into the PVN increased glutamate release. Using an in vitro hypothalamic slice preparation, we found that bicuculline increased the frequency of glutamate-mediated excitatory postsynaptic currents in PVN-rostral ventrolateral medullary projecting neurons, supporting a GABA(A)-mediated tonic inhibition of this excitatory input into these neurons. Together, these data indicate that 1) glutamate, via NMDA receptors, excites the presympathetic neurons within the PVN and increases sympathetic outflow and 2) this glutamate excitatory input is tonically inhibited by a GABA(A)-mediated mechanism.     

Time sensing by NAADP receptors

NAADP (nicotinic acid-adenine dinucleotide phosphate) is a newly described intracellular messenger molecule that mediates Ca2+ increases in a variety of cells. However, little is known of the mechanism whereby ligand binding regulates the target protein. We report in the present paper that NAADP receptors from sea urchin eggs undergo an unusual stabilization process that appears to be dependent upon the time during which receptors are exposed to their ligand. We demonstrate that receptors 'tagged' with NAADP for short periods were more readily dissociated following subsequent delipidation than those labelled for longer. Stabilization of NAADP receptors by their ligand was delayed relative to ligand association taking on the order of minutes to develop at picomolar concentrations. The stabilizing effects of NAADP did not require cytosolic factors or the continued presence of NAADP and persisted upon solubilization. NAADP receptors, however, failed to stabilize at reduced temperature. We conclude that NAADP receptors possess a simple molecular memory endowing them with the remarkable ability to detect the duration of their activation.     

Weight loss and race modulate nitric oxide metabolism in overweight women

Weight reduction is associated with a decrease in the risk of developing cardiovascular disease. We hypothesized that, given the central role of reactive oxygen and nitrogen species in vascular biology, changes in nitric oxide (NO) metabolism contribute to benefits of weight loss. In a controlled weight loss trial involving overweight (body mass index (BMI) = 27-30 kg/m(2)), otherwise healthy premenopausal Caucasian and African-American women, serum levels of nitrite and nitrate, as an index of NO production, and protein 3-nitrotyrosine and myeloperoxidase (MPO), as markers of inflammation, were determined. Testing was performed before and after reduction to normal body weight (BMI < 25) under standardized conditions, with controlled diet, and following 1 month of weight maintenance. After weight loss there was an increase in nitrite and nitrate, and levels were higher among African-American women relative to Caucasian counterparts. Whereas weight loss was associated with a decrease in 3-nitrotyrosine in Caucasian women, no change was observed among African-Americans. Furthermore, MPO levels increased in response to weight loss for African-Americans, but did not change in Caucasian women. These data indicate that vascular production of reactive nitrogen species can be modulated by race and weight loss and highlight important racial differences in these responses and are discussed in the context of risk for developing vascular disease.     

Cytokine dysregulation induced by apoptotic cells is a shared characteristic of macrophages from nonobese diabetic and systemic lupus erythematosus-prone mice

Macrophages from nonobese diabetic (NOD) mice, which spontaneously develop type I diabetes, share a defect in elicited cytokine production with macrophages from multiple diverse strains of systemic lupus erythematosus (SLE)-prone mice. We have previously shown that, in SLE-prone mice, this defect is triggered by exposure to apoptotic cells. We report in this work that macrophages from prediseased NOD mice also respond abnormally to apoptotic cells, mimicking closely the apoptotic cell-dependent abnormality that we have observed in multiple SLE-prone strains. This defect is characterized by the underexpression of IL-1 beta and multiple other cytokines. In the presence of apoptotic cells or FBS, elicited expression of IL-1 beta by NOD macrophages is markedly reduced compared with that by macrophages from control mice, including three strains of mice that develop type II (nonautoimmune) diabetes. Given the increasing role of apoptotic cells in tolerance and autoimmunity, a macrophage defect triggered by apoptotic cells has broad potential to upset the balance between tolerance and immunity. The concordance of this defect among so many diverse autoimmune-prone strains suggests that the genetic basis for this abnormality may constitute a permissive background for autoimmunity.     

The plant nuclear envelope

This review summarizes our present knowledge about the composition and function of the plant nuclear envelope. Compared with animals or yeast, our molecular understanding of the nuclear envelope in higher plants is in its infancy. However, fundamental differences in the structure and function of the plant and animal nuclear envelope have already been found. Here, we compare and contrast these differences with respect to nuclear pore complexes, targeting of Ran signaling to the nuclear envelope, inner nuclear envelope proteins, and the role and fate of the nuclear envelope during mitosis. Further investigation of the emerging fundamental differences as well as the similarities between kingdoms might illuminate why there appears to be more than one blueprint for building a nucleus.Abbreviations GFP Green fluorescent protein - INE Inner nuclear envelope - LAP Lamina-associated polypeptide - LBR Lamin B receptor - MTOC Microtubule-organizing center - NE Nuclear envelope - NPC Nuclear pore complex - ONE Outer nuclear envelope - RanBP Ran-binding protein - RanGAP Ran GTPase-activating protein - WPP domain Tryptophan–proline–proline domain