The Differences in the Cellular and Plasma Antioxidative Capacity Between Transient and Defined Focal Brain Ischemia: Does it Suggest Supporting Time-Dependent Neuroprotection Therapy?

There are many opened questions about the precocious role of oxidative stress in the physiopathology of the early stage of transitory ischemic attack (TIA) and defined focal brain ischemia, as well as about its correlation with clinical severity, short-lasting and clinical outcome prediction in these conditions. The study evaluates the values of glutathione (GSH), glutathione peroxidase, and superoxide dismutase (SOD) in hemolysates and total thiol content (–SH), advanced oxidation protein products (AOPP), SOD, and malondialdehyde (MDA) in plasma, in TIA and stroke patients in the early stage of their neurological onset. The results are interpreted in view of the potential relationship between tested parameters and clinical severity and clinical outcome prediction. Better hemolysates’ and total antioxidant profile with higher values of AOPP were observed in TIA compared to stroke patients (p < 0.05). The stroke patients with initially better clinical presentation showed better antioxidant profile with lower values of AOPP (p < 0.05). In TIA patients, this was observed for GSH, –SH content, and AOPP (p < 0.05), which correlated with a short risk for stroke occurrence in this group (p < 0.01). Beyond MDA values, all tested parameters showed correlation with clinical outcome in stroke patients (p < 0.05). The measurement of oxidative stress in TIA and stroke patients would be important for identifying patients’ subgroups which might receive supporting therapy providing better neurological recovery and clinical outcome. That approach might give us an additional view of a short-lasting risk of stroke occurrence after TIA, and its clinical outcome and prognosis.     

Effects of Metformin on Tissue Oxidative and Dicarbonyl Stress in Transgenic Spontaneously Hypertensive Rats Expressing Human C-Reactive Protein

Inflammation and oxidative and dicarbonyl stress play important roles in the pathogenesis of type 2 diabetes. Metformin is the first-line drug of choice for the treatment of type 2 diabetes because it effectively suppresses gluconeogenesis in the liver. However, its “pleiotropic” effects remain controversial. In the current study, we tested the effects of metformin on inflammation, oxidative and dicarbonyl stress in an animal model of inflammation and metabolic syndrome, using spontaneously hypertensive rats that transgenically express human C-reactive protein (SHR-CRP). We treated 8-month-old male transgenic SHR-CRP rats with metformin (5 mg/kg/day) mixed as part of a standard diet for 4 weeks. A corresponding untreated control group of male transgenic SHR-CRP rats were fed a standard diet without metformin. In a similar fashion, we studied a group of nontransgenic SHR treated with metformin and an untreated group of nontransgenic SHR controls. In each group, we studied 6 animals. Parameters of glucose and lipid metabolism and oxidative and dicarbonyl stress were measured using standard methods. Gene expression profiles were determined using Affymetrix GeneChip Arrays. Statistical significance was evaluated by two-way ANOVA. In the SHR-CRP transgenic strain, we found that metformin treatment decreased circulating levels of inflammatory response marker IL-6, TNFα and MCP-1 while levels of human CRP remained unchanged. Metformin significantly reduced oxidative stress (levels of conjugated dienes and TBARS) and dicarbonyl stress (levels of methylglyoxal) in left ventricles, but not in kidneys. No significant effects of metformin on oxidative and dicarbonyl stress were observed in SHR controls. In addition, metformin treatment reduced adipose tissue lipolysis associated with human CRP. Possible molecular mechanisms of metformin action–studied by gene expression profiling in the liver–revealed deregulated genes from inflammatory and insulin signaling, AMP-activated protein kinase (AMPK) signaling and gluconeogenesis pathways. It can be concluded that in the presence of high levels of human CRP, metformin protects against inflammation and oxidative and dicarbonyl stress in the heart, but not in the kidney. Accordingly, these cardioprotective effects of metformin might be especially effective in diabetic patients with high levels of CRP.     

STIM1/ORAI1-mediated Ca2+ Influx Regulates Enolase-1 Exteriorization

Tumor cells use broad spectrum proteolytic activity of plasmin to invade tissue and form metastatic foci. Cell surface-associated enolase-1 (ENO-1) enhances plasmin formation and thus participates in the regulation of pericellular proteolysis. Although increased levels of cell surface bound ENO-1 have been described in different types of cancer, the molecular mechanism responsible for ENO-1 exteriorization remains elusive. In the present study, increased ENO-1 protein levels were found in ductal breast carcinoma and on the cell surface of highly metastatic breast cancer cell line MDA-MB-231. Elevated cell surface-associated ENO-1 expression correlated with augmented MDA-MB-231 cell migratory and invasive properties. Exposure of MDA-MB-231 cells to LPS potentiated translocation of ENO-1 to the cell surface and its release into the extracellular space in the form of exosomes. These effects were independent of de novo protein synthesis and did not require the classical endoplasmic reticulum/Golgi pathway. LPS-triggered ENO-1 exteriorization was suppressed by pretreatment of MDA-MB-231 cells with the Ca²⁺ chelator BAPTA or an inhibitor of endoplasmic reticulum Ca²⁺-ATPase pump, cyclopiazonic acid. In line with these observations, the stromal interaction molecule (STIM) 1 and the calcium release-activated calcium modulator (ORAI) 1-mediated store-operated Ca²⁺ entry were found to regulate LPS-induced ENO-1 exteriorization. Pharmacological blockage or knockdown of STIM1 or ORAI1 reduced ENO-1-dependent migration of MDA-MB-231 cells. Collectively, our results demonstrate the pivotal role of store-operated Ca²⁺ channel-mediated Ca²⁺ influx in the regulation of ENO-1 exteriorization and thus in the modulation of cancer cell migratory and invasive properties.     

Heparan Sulfate Proteoglycans Mediate Factor XIIa Binding to the Cell Surface

Hageman factor (FXIIa) initiates the intrinsic coagulation pathway and triggers the kallikrein-kinin and the complement systems. In addition, it functions as a growth factor by expressing promitogenic activities toward several cell types. FXIIa binds to the cell surface via a number of structurally unrelated surface receptors; however, the underlying mechanisms are not yet fully understood. Here, we demonstrate that FXIIa utilizes cell membrane-bound glycosaminoglycans to interact with the cell surface of human lung fibroblasts (HLF). The combination of enzymatic, inhibitory, and overexpression approaches identified a heparan sulfate (HS) component of proteoglycans as an important determinant of the FXIIa binding capacity of HLF. Moreover, cell-free assays and competition experiments revealed preferential binding of FXIIa to HS and heparin over dextran sulfate, dermatan sulfate, and chondroitin sulfate A and C. Finally, we demonstrate that fibroblasts isolated from the lungs of the patients suffering from idiopathic pulmonary fibrosis (IPF) exhibit enhanced FXIIa binding capacity. Increased sulfation of HS resulting from elevated HS 6-O-sulfotransferase-1 expression in IPF HLF accounted, in part, for this phenomenon. Application of RNA interference technology and inhibitors of intracellular sulfation revealed the cooperative action of cell surface-associated HS and urokinase-type plasminogen activator receptor in the accumulation of FXIIa on the cell surface of IPF HLF. Moreover, FXIIa stimulated IPF HLF migration, which was abrogated by pretreatment of cells with heparinase I. Collectively, our study uncovers a novel role of HS-type glycosaminoglycans in a local accumulation of FXIIa on the cell membrane. The enhanced association of FXIIa with IPF HLF suggests its contribution to fibrogenesis.     

Vitamin E analogues as a novel group of mitocans: anti-cancer agents that act by targeting mitochondria

Mitochondria have recently emerged as new and promising targets for cancer prevention and therapy. One of the reasons for this is that mitochondria are instrumental to many types of cell death and often lie downstream from the initial actions of anti-cancer drugs. Unlike the tumour suppressor gene encoding p53 that is notoriously prone to inactivating mutations but whose function is essential for induction of apoptosis by DNA-targeting agents (such as doxorubicin or 5-fluorouracil), mitochondria present targets that are not so compromised by genetic mutation and whose targeting overcomes problems with mutations of upstream targets such as p53. We have recently proposed a novel class of anti-cancer agents, mitocans that exert their anti-cancer activity by destabilising mitochondria, promoting the selective induction of apoptotic death in tumour cells. In this communication, we review recent findings on mitocans and propose a common basis for their mode of action in inducing apoptosis of cancer cells. We use as an example the analogues of vitamin E that are proving to be cancer cell-specific and may soon be developed into efficient anti-cancer drugs.     

Proteomics of the aqueous humor in healthy New Zealand rabbits

There are several physiological roles postulated for aqueous humor, a liquid located in the anterior and posterior chamber of the eye, such as maintenance of the intraocular pressure, provision of nutrients, and removal of metabolic waste from neighboring tissues and provision of an immune response and protection during inflammation and infection. To link these function to specific or classes of proteins, identification of the aqueous humor proteome is essential. Aqueous humor obtained from healthy New Zealand white rabbits was analyzed using three synergistic protein separation methods: 1-D gel electrophoresis, 2-DE, and 1-DLC (RPLC) prior to protein identification by MS. As each of these separation methods separates intact proteins based on different physical properties (pIs, molecular weights, hydrophobicity, solubility, etc.) the proteome coverage is expanded. This was confirmed, since overlap between all three separation technologies was only about 8.2% with many proteins found uniquely by a single method. Although the most dominant protein presented in normal aqueous humor is albumin, by using this extensive separation/MS strategy, additional proteins were identified in total amount of 98 nonredundant proteins (plus an additional ten proteins for consideration). This expands the current protein identifications by approximately 65%. The aqueous humor proteome comprises a specific selection of cellular and plasma based proteins and can almost exclusively be divided into four functional groups: cell-cell interactions/wound healing, proteases and protease inhibitors, antioxidant protection, and antibacterial/anti-inflammatory proteins.     

A modified procedure for the detection of microbial producers of extracellular proteolytic enzymes

Summary A simple procedure for the detection of microbial producers of proteolytic enzymes using dyed gelatin microcarrier particles incorporated into appropriate nutrient agar is described. Extracellular proteinases produced by the tested microbial strains hydrolyzed the substrate and clear dyed zones around and under the colonies were formed.