The short-term effects of soybean intake on oxidative and carbonyl stress in men and women.

Beyond other beneficial effects, a soy-rich diet has been shown to reduce the risk of cardiovascular diseases and diabetic complications. Reduction of oxidative and carbonyl stress has been proposed as the underlying mechanism, but the evidence for this is lacking. The aim of our study was to evaluate the effects of short-term increased soy intake on oxidative and carbonyl stress parameters in young volunteers. Young healthy probands (omnivores) of both genders (55 women, 33 men) were given soybeans (2 g/kg bodyweight daily) for one week. Markers of oxidative and carbonyl stress were measured in plasma at the beginning and at the end of one week soybean intake and after another week of a wash-out period. Total antioxidant capacity was increased by soybean intake in both genders. This led to decreased levels of advanced oxidation protein products in women, but not in men. On the contrary, in men, soybean intake increased lipoperoxidation. No effects on carbonyl stress markers (advanced glycation end products-specific fluorescence and fructosamine) were found. Soybean intake has gender-specific effects on oxidative stress in young healthy probands potentially due to divergent action and metabolism of phytoestrogens in men and women. Effects of soybean intake on carbonyl stress should be evaluated in longer studies.

Antioxidant vitamins prevent oxidative and carbonyl stress in an animal model of obstructive sleep apnea.

PURPOSE: The aim of our study was to analyze the effects of an antioxidant treatment on markers of oxidative and carbonyl stress in a rat model of obstructive sleep apnea. METHODS: Wistar rats were randomized into six groups-according to gender and intervention-sham, intermittent hypoxia, and intermittent hypoxia with treatment by vitamins C and E. Rats underwent tracheostomy. The tracheal cannula was closed for 12 s every minute for 1 h to simulate obstructive sleep apnea-related intermittent hypoxia. In the treatment group, rats received vitamin C and E 24 h prior to surgery. RESULTS: The intervention had a significant effect on advanced oxidation protein products (p = 0.008) and advanced glycation end products-specific fluorescence (p = 0.006) but no effect on malondialdehyde. Oxidation and glycation protein products were higher in intermittent hypoxia groups than in sham and in treated groups. CONCLUSIONS: Antioxidants alleviate oxidative and carbonyl stress in an experimental model of obstructive sleep apnea. Future studies will show whether such treatment has any clinical value regarding cardiovascular complications of sleep apnea syndrome, preferably in patients with low compliance to continuous positive airway pressure.

The effects of anti-inflammatory and anti-angiogenic DNA vaccination on diabetic nephropathy in rats.

Inflammation and angiogenesis play a crucial role in the pathomechanism of diabetic nephropathy. Monocyte chemoattractant protein 1 (MCP) is a key regulator of the immune system in kidneys, and its inhibition with a dominant-negative mutant lacking the N-terminal amino acids 2-8 (7ND) reduces renal fibrosis. Angiomotin (Amot) is a novel angiogenesis modulator. We studied the effects of inhibition of Amot and MCP using DNA vaccination on incipient diabetic nephropathy in rats. Plasmid DNA (with either 7ND or human Amot) was electroporated twice into hind-limb muscles of rats with streptozotocin-induced diabetes mellitus. Sham-electroporated diabetic rats and healthy animals served as controls. After 4 months, renal histology and biochemical analyses were performed. In sham-electroporated diabetic rats, glomerular histology revealed pathological changes. 7ND and Amot treatments reduced glomerular hypertrophy and periodic acid-Schiff positivity. In both treated groups, the expression of profibrotic (transforming growth factor-ß, collagen 1), proinflammatory (interleukin-6, tumor necrosis factor-α), and proangiogenic (vascular endothelial growth factor) genes in the renal cortex was lower than in the diabetic group without treatment. The mentioned renoprotective effects could be mediated via higher total antioxidant capacity and improved glycemic control. Anti-angiogenic and anti-inflammatory DNA vaccination ameliorates the progression of glomerular pathology in an animal model of diabetic nephropathy.

Oxidative and carbonyl stress in patients with obstructive sleep apnea treated with continuous positive airway pressure.

PURPOSE: The pathogenesis of cardiovascular complications of obstructive sleep apnea syndrome (OSAS) can be explained by oxidative and carbonyl stress due to oxygenation and reoxygenation injury during sleep. This hypothesis has yet to be proved experimentally, although several clinical observations have found increased oxidative damage in plasma. Continuous positive airway pressure (CPAP) improves symptoms and prognosis of patients with OSAS. METHODS: Patients with confirmed SAS (n = 89) underwent polysomnography and received CPAP treatment. Plasma and saliva samples were taken before CPAP therapy as well as after 1 and 6 months of CPAP treatment. Selected markers of oxidative and carbonyl stress were measured in plasma and saliva, and their dynamics was statistically analyzed. RESULTS: Plasma levels of thiobarbituric acid reacting substances-a marker of lipoperoxidation-and advanced glycation end products (AGEs)-a marker of carbonyl stress-were decreased by the CPAP therapy. The decrease of AGEs and fructosamine was also found in saliva. Interestingly, no gender differences and no changes of antioxidant status measured as total antioxidant capacity and ferrous reducing ability were found in either of the samples. CONCLUSION: Previous findings of lowered plasma markers of oxidative stress were confirmed. Plasma AGEs were lowered by CPAP therapy. This is the first study analyzing markers of oxidative and carbonyl stress in saliva. Non-invasive sampling of saliva makes it a very interesting source of information for repeated monitoring of therapy success. Salivary AGEs and fructosamine as markers of carbonyl stress were decreased by the CPAP therapy and might therefore have potential informative value for clinical observations, as well as for the understanding of the pathogenesis of OSAS complications.

Mutation analysis of PMP22 in Slovak patients with Charcot-Marie-Tooth disease and hereditary neuropathy with liability to pressure palsies.

Charcot-Marie-Tooth disease (CMT) and related peripheral neuropathies are the most commonly inherited neurological disorders in humans, characterized by clinical and genetic heterogeneity. The most prevalent clinical entities belonging to this group of disorders are CMT type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP). CMT1A and HNPP are predominantly caused by a 1.5 Mb duplication and deletion in the chromosomal region 17p11.2, respectively, and less frequently by other mutations in the peripheral myelin protein 22 (PMP22) gene. Despite being relatively common diseases, they haven't been previously studied in the Slovak population. Therefore, the aim of this study was to identify the spectrum and frequency of PMP22 mutations in the Slovak population by screening 119 families with CMT and 2 families with HNPP for causative mutations in this gene. The copy number determination of PMP22 resulted in the detection of CMT1A duplication in 40 families and the detection of HNPP deletion in 7 families, 6 of which were originally diagnosed as CMT. Consequent mutation screening of families without duplication or deletion using dHPLC and sequencing identified 6 single base changes (3 unpublished to date), from which only c.327C>A (Cys109X) present in one family was provably causative. These results confirm the leading role of PMP22 mutation analysis in the differential diagnosis of CMT and show that the spectrum and frequency of PMP22 mutations in the Slovak population is comparable to that seen in the global population.

Effect of unexpected sequence interruptions to conventional PCR and repeat primed PCR in myotonic dystrophy type 1 testing.

Myotonic dystrophy type 1 (DM1) is caused by expansion of the CTG trinucleotide repeat in the DMPK gene. Our study focuses on the effect of recently described unusual sequence interruptions inside the CTG tract on conventional polymerase chain reaction (PCR) and triplet repeat primed PCR (TP-PCR) amplifications, which are the methods now widely used in molecular testing for DM1. For molecular characterization of the CTG repeat tract, we used conventional fluorescent PCR with bidirectional labeling and both forward and reverse direction TP-PCR. Though the results of the methods are still unambiguous for most alleles, mistyping and false results may occur in the typing of some unordinary alleles carrying sequence interruptions. The presence of these interruptions may lead not only to altered TP-PCR profiles, as can be expected, but also to abnormal electrophoretic mobility of complementary strands produced by conventional amplification of such alleles. Our findings suggest that the simultaneous combination of bidirectionally labeled conventional PCR with TP-PCR performed in both directions may be necessary for increasing the reliability and accuracy of the TP-PCR-based assay for DM1 testing.

Salmonella-mediated gene therapy in experimental colitis in mice.

Bacterial gene therapy - bactofection is a simple and effective method to deliver plasmid DNA into target tissue. We hypothesize that oral in vivo bactofection can be an interesting approach to influence the course of inflammatory bowel diseases. The aim of this study was to prove the effects of antioxidative and anti-inflammatory bactofection in dextran sulfate sodium (DSS)-treated mice. Attenuated bacteria Salmonella Typhimurium SL7207 carrying plasmids with genes encoding Cu-Zn superoxide dismutase and an N-terminal deletion mutant of monocyte chemoattractant protein-1 were prepared. Male Balb/c mice had ad libitum access to 1% DSS solution in drinking water during 10 days (mild model of colitis). The animals were daily fed with 200 Mio bacteria via gastric gavage during the experiment. Fecal consistency, clinical status, food and water intake were monitored. After 10 days samples were taken and markers of oxidative stress and inflammatory cytokine levels were measured. Colonic tissue was scored histologically by a blinded investigator. DSS treatment significantly increased the levels of inflammatory cytokines and malondialdehyde as a marker of lipoperoxidation in the colon. Anti-inflammatory gene therapy improved the total antioxidative capacity. In comparison with the untreated group, bacterial gene therapy lowered the histological colitis score. Salmonella-mediated antioxidative and anti-inflammatory gene therapy alleviated colitis in mice. The effect seems to be mediated by increased antioxidative status. Further studies will show whether recombinant probiotics expressing therapeutic gene might be used for the therapy of inflammatory bowel diseases.

Oral in vivo bactofection in dextran sulfate sodium treated female Wistar rats.

Salmonella typhimurium SL7207 carrying Cu-Zn superoxide dismutase and an N-terminal deletion mutant of monocyte chemoattractant protein-1 genes was applied to dextran sodium sulfate treated female Wistar rats. Stool quality, food and water intake were monitored. Markers of oxidative stress, interleukin 1, interleukin 6 and tumor necrosis factor alpha were quantified. No differences were found in body weights, markers of oxidative stress in plasma and inflammatory markers in colon homogenates. Plasma concentrations of I11, I16 were lower in the treatment groups than in the dextran sodium sulfate group. However, dextran sodium sulfate induced inflammation could not be confirmed by plasma levels of I11, I16 and TNFalpha. Although some parameters showed a tendency to improve, the inflammation caused by administration of 4% dextran sodium sulfate during 7 days was low and contradictory to other studies. Results showed the potential synergic effect of combined bacteria-mediated antioxidative and anti-inflammatory gene therapy.

Renal and metabolic effects of three months of decarbonated cola beverages in rats.

Epidemiological studies have shown an association between the intake of cola beverages and chronic kidney diseases. Experimental evidence for the negative effects of cola intake on kidneys is lacking. Male Wistar rats had ad libitum access to water (control group) or three different sugar-sweetened cola beverages for three months. Despite very high cola intake (daily cca 140 mL), no differences were found in body weight, kidney weight, glomerular morphology, oxidative and carbonyl stress or expression of selected marker genes in the renal cortex. Interestingly, all groups consuming cola beverages had lower blood glucose levels during an oral glucose tolerance test, suggesting improved insulin sensitivity. Despite hyperfiltration (5-6-fold increase in diuresis), cola beverages had no effect on assessed parameters of renal function, histology, gene expression or oxidative stress. Moreover, cola intake seems to increase creatinine clearance and to decrease plasma levels of urea. In our study increased insulin sensitivity and altered renal functional parameters were observed in rats receiving cola beverages for three months. Whether the findings are due to the short duration of the study or interspecies metabolic differences should be uncovered in further studies. Even more interesting might be the analysis of effects of cola intake in animal models of diabetes.

Effects of orally administered bacteria carrying HIF-1α gene in an experimental model of intestinal ischemia.

BACKGROUND AND AIMS: Bactofection is delivery of therapeutic genes into target cells using bacteria penetrating the target cell membrane and releasing the gene into the cell. Hypoxia-inducible factor 1α (HIF-1α) represents a potential therapeutic gene to be used for gene delivery in ischemic diseases. The aim of this study was to prove the effects of bacteria-mediated transfer of hypoxia-inducible factor 1α (HIF-1α) in an experimental model of intestinal ischemia in rats. METHODS: Male Wistar rats with a surgically induced ischemia of colon (cecum) or sham-operated rats were treated by per os application of E. coli carrying therapeutic genes. After 1 week, samples were taken for measurement of oxidative stress markers and expression analyses. RESULTS: According to our observation, there were no signs or symptoms of ongoing ischemia in gastrointestinal tissue. Interestingly, all experimental groups treated by bacteria, regardless of their ability to invade cells or the presence of HIF-1α gene, showed decreased levels of vascular endothelial growth factor (VEGF) compared to control groups. Similarly, all treatment groups showed increased hematocrit. CONCLUSIONS: We conclude ineffectiveness of the bacterial gene delivery system. However, the effect of bacteria themselves was obvious. HIF-1 can be activated hypoxia-independently by the action of pathogenic bacteria in the rat intestine. We hypothesize that therapeutic bacterial strain used may compete with siderophore-expressing bacteria present in the gut of rats to force them out and prevent their ability to activate HIF-1 in a hypoxia-independent manner. This phenomenon should be analyzed in detail in further studies.

Continuous light and L-NAME-induced left ventricular remodelling: different protection with melatonin and captopril.

OBJECTIVE: Blood pressure enhancement induced by continuous light exposure represents an attractive but rarely investigated model of experimental hypertension. DESIGN AND METHODS: The aim of this study was to show whether the combination of continuous light (24 h/day) exposure and chronic N-nitro-L-arginine-methyl ester (L-NAME) treatment induces remodelling of the left ventricle and whether captopril or melatonin can modify these potential alterations. Six groups of 3-month-old Wistar rats (nine per group) were treated for 6 weeks: control (untreated), L-NAME (40 mg/kg per day), exposed to continuous light, L-NAME treated and exposed to continuous light (L24), L24 rats treated with either captopril 100 mg/kg per day, or melatonin (10 mg/kg/24 h). Systolic blood pressure (SBP), relative weights of the left ventricle, endothelial nitric oxide synthase (eNOS) and angiotensin-converting enzyme (ACE) expression in tissues, malondialdehyde and advanced oxidation protein product concentrations in the plasma and hydroxyproline levels in collagenous protein fractions were measured. RESULTS: The continuous light and L-NAME treatment led to hypertension, left ventricular hypertrophy (LVH) and fibrosis. An increase in SBP was completely prevented by captopril and partly by melatonin in the L24 group. Both drugs reduced oxidative damage and attenuated enhanced expression of ACE in the myocardium. Neither of the drugs prevented the attenuation of eNOS expression in the combined hypertensive model. Only captopril reduced LVH development in L24, whereas captopril and melatonin reduced left ventricular hydroxyproline concentrations in soluble and insoluble collagen, respectively. The total hydroxyproline concentration was reduced only by melatonin. CONCLUSION: In hypertension induced by a combination of continuous light and L-NAME treatment, melatonin and captopril protect the heart against pathological left ventricular remodelling differently.

Modulation of antioxidative response in the therapy of hypertension and other cardiovascular diseases.

OBJECTIVES: This paper reviews and compares major approaches and strategies to modulation of antioxidative response in the therapy of hypertension and cardiovascular diseases. DESIGN: There are two major strategies of modulation of antioxidative response in hypertension and cardiovascular diseases: (i) modulation of NO levels by NOS stimulation, increase of NO bioavailability, administration of NO, and NOS gene incorporation; (ii) scavenging of superoxide and suppression of oxidative stress by activation of antioxidant gene expression or by suppression of selected genes by RNA silencing. These strategies are accomplished by several concepts, including (1) delivery of external agents, (2) antioxidant gene therapy and RNA silencing, and (3) combined therapies and approaches. CONCLUSION: Combined therapies and approches often achieve multiplicative effects and are the most promising attitude in antioxidant-oriented therapy of hypertension and cardiovascular diseases.

Variability of thiobarbituric acid reacting substances in saliva.

INTRODUCTION: Salivary TBARS are a potential marker of oxidative stress in the oral cavity. Previous studies have found increased levels of salivary TBARS in various diseases. The aim of this study was to assess the variability of salivary TBARS in both genders. SUBJECTS & METHODS: Saliva samples from thirty-eight healthy volunteers (18F & 20M) were collected every day during 30 day period. TBARS levels were measured spectrophotometrically using a high-throughput 96-well plate method. Time series analysis was performed using standard statistical methods. RESULTS: Repeated measures ANOVA showed a significant variation of salivary TBARS within day and subjects (p < 0.001). The dynamics did not differ between genders. Intraindividual variability was very high in both genders with coefficients of variation of more than 60%. Interindividual variability was higher in men than in women (73% vs. 46%; p < 0.01). DISCUSSION: The relatively high intraindividual variability indicates that the use of salivary TBARS will be limited to research on a population level, although some informative value might be gained by repeated samplings. Factors influencing the biological variability of salivary TBARS should be identified in further studies.

On the physiology and pathophysiology of antimicrobial peptides.

Antimicrobial peptides (AMP) are a heterogeneous group of molecules involved in the nonspecific immune responses of a variety of organisms ranging from prokaryotes to mammals, including humans. AMP have various physical and biological properties, yet the most common feature is their antimicrobial effect. The majority of AMP disrupt the integrity of microbial cells by 1 of 3 known mechanisms--the barrel-stave pore model, the thoroidal pore model, or the carpet model. Results of growing numbers of descriptive and experimental studies show that altered expression of AMP in various tissues is important in the pathogenesis of several gastrointestinal, respiratory, and other diseases. We discuss novel approaches and strategies to further improve the promising future of therapeutic applications of AMP. The spread of antibiotic resistance increases the importance of developing a clinical role for AMP.

Biological and biomedical aspects of genetically modified food.

Genetically modified (GM) foods are the product of one of the most progressive fields of science-biotechnology. There are major concerns about GM foods in the public; some of them are reasonable, some of them are not. Biomedical risks of GM foods include problems regarding the potential allergenicity, horizontal gene transfer, but environmental side effects on biodiversity must also be recognized. Numerous methods have been developed to assess the potential risk of every GM food type. Benefits of the first generation of GM foods were oriented towards the production process and companies, the second generation of GM foods offers, on contrary, various advantages and added value for the consumer. This includes improved nutritional composition or even therapeutic effects. Recombinant probiotics and the principle of alternative gene therapy represent the latest approach of using GM organisms for biomedical applications. This article tries to summarize and to explain the problematic topic of GM food.

Vectors and delivery systems in gene therapy.

Gene therapy provides modern medicine with new perspectives that were unthinkable two decades ago. Progress in molecular biology and, especially, molecular medicine is now changing the basics of clinical medicine. Reports of long-term side effects of the first successful human gene therapy study have slowed the penetration of DNA usage into clinical routine. However, the main safety problem lies in the secure and efficient delivery of genes into target cells and tissues. A number of older and more recently discovered techniques have been developed for therapeutic gene transfer. A variety of viral and non-viral possibilities are available for basic and clinical research. This review summarizes the delivery routes and methods for gene transfer used in gene therapy. The best known are viral vectors derived from adenoviruses and adeno-associated viruses. Specific properties of lentiviruses and retroviruses ensure their status in current gene therapy procedures. Recent advances in the preparation of lipoplexes and greater safety support the use of naked DNA transfer. The latest approach, using bacteria as vectors for gene therapy or as in situ producers of therapeutic proteins (alternative gene therapy), must undergo critical evaluation in further experiments.

The use of transformed Escherichia coli for experimental angiogenesis induced by regulated in situ production of vascular endothelial growth factor--an alternative gene therapy.

BACKGROUND: Defects in angiogenesis (blood vessel formation) are responsible for two most important causes of death in developed countries (ischemic heart disease and cancer). Vascular endothelial growth factor (VEGF) plays a pivotal role in physiological and pathological regulation of angiogenesis. In the last years several studies have indicated the possibilities of VEGF in the therapy of ischemic heart disease. However, especially VEGF gene therapy (naked DNA, plasmids and adenovirus mediated) is associated with adverse side effects regarding the expression regulation. AIM: To prepare bacterial strains producing VEGF using plasmids containing the VEGF cDNA for the use in experimental angiogenesis. METHODS AND RESULTS: Escherichia coli strain BL21(DE3) was transformed with Bluescript vector containing the inserts with cDNA sequences coding VEGF-A isoforms (VEGF121, VEGF164, VEGF189). Selection of recombinants was achieved by cultivating E. coli cells on ampicillin-added medium. The expression of target genes in the T7 expression system was induced by isopropyl-beta-D-thiogalactoside (IPTG). Polyacrylamide gel electrophoresis of the cell lysates showed the presence of polypeptides of molecular weight corresponding with known values of VEGF isoforms. Blood vessel formation induced by bacterial VEGF production was proved in vivo in mice seven days after intraperitoneal injection of transformed bacteria by light microscopy. CONCLUSION AND HYPOTHESIS: In summary, E. coli strain expressing VEGF was prepared and its biological effect confirmed. Bacteria, which produce angiogenic factors, provide a new modality for experimental angiogenesis and may be also suitable for clinical use. The in situ production of therapeutic proteins using optimalized prokaryotic expression systems can represent a useful tool for treatment based on molecular biomedicine. The main advantage of the described approach lies in the enhanced regulation control--bacterial expression can be regulated positively (induction by exogenous low molecular weight agents) and negatively (application of antibiotics). The hypothesis of alternative gene therapy should be proved in further studies.