Role of antioxidants in the protection against oxidative stress in cattle--trace elements and enzymatic mechanisms (Part 3).

The most important enzymatic mechanisms which protect an organism against oxidative stress are superoxide dismutase (SOD), peroxidase (Px), e.g. glutathione peroxidase (GSH-Px) and ascorbate peroxidase, catalase and glutathione reductase. Their activity depends on many trace elements. Enzymatic mechanisms, functioning under physiological conditions, prevent the spread of free radical reactions. New and reoccurring metabolic and infectious diseases of cattle emerge when there is a disproportion in the balance between reactive oxygen species and antioxidative enzymatic barrier.

Bacterial populations on teat ends of dairy cows housed in free stalls and bedded with either sand or sawdust.

The main objectives of the experiment were: 1) to compare bacterial populations of mastitis-causing organisms on the teats of lactating dairy cattle housed on sand and sawdust bedding and, 2) to examine the relationship between bacterial counts present in the 2 bedding types with those on teat ends. Sixteen lactating Holstein cows were housed on either sand or sawdust-bedded free stalls using a crossover design with 3 wk per bedding type. Bedding samples were collected on d 0 (prior to animals lying on the bedding), 1, 2, and 6. Teat ends were sampled prior to the morning milking on d 1, 2, and 6. All samples were analyzed to determine coliform, Klebsiella spp., and Streptococcus spp. populations. There were 2 times more coliforms and 6 times more Klebsiella bacteria on teat ends of cows housed on sawdust compared with those housed on sand. In contrast, there were 10 times more Streptococcus spp. bacteria on teat ends of cows when housed on sand compared with sawdust. In both sawdust and sand bedding, coliforms, Klebsiella and Streptococcus counts increased over each experimental week, although patterns varied with bedding and bacteria type. Bacterial counts on teat ends were correlated with bacterial counts in sawdust (r = 0.47, 0.69, and 0.60 for coliforms, Klebsiella spp., and streptococci, respectively) and in sand (r = 0.35 for coliforms and r = 0.40 for Klebsiella spp.). In conclusion, coliforms and Klebsiella spp. on teat ends were more numerous when cows were housed on sawdust bedding, but Streptococcus spp. were more numerous on teat ends of cows housed on sand.

Role of the antioxidants in the protection against oxidative stress in cattle--nonenzymatic mechanisms (Part 2).

Antioxidative status consists of two mechanisms: nonenzymatic and enzymatic mechanisms. Nonenzymatic mechanisms are composed of antioxidants, scavengers of free radicals, transition metal ions, sequester transition metal ions, albumins, ceruloplasmin, and metallothioneins. On the other hand, enzymatic mechanisms are composed of superoxide dismutase (SOD), peroxidase, catalase and reductase. In cattle, characteristics of these mechanisms depend on the nutritional status of anti-oxidant minerals, especially copper, zinc, iron, selenium, silicon, and manganese. The nutritional status of the cattle in different regions of the world and in Poland is often characterised by the lack of these minerals; therefore, there is a great potential for changes in the activity of defence mechanisms against free radicals.

Role of transition metals ion and reactive oxygen species in biological oxidation in cattle (part 1).

Technological advances in last decades of XX centuries were well utilised in the studies of biological oxidation processes. Biological oxidation can lead to the oxidative stress and subsequently to the cell damage of animal organisms leading to many diseases. Free radical processes taking place in cattle under pathological conditions. Metal ions are often responsible for the damage of biological systems. Fenton and the Fenton like reactions can play the central role in the oxidative stress. Transition metal ions and their complexes catalyse Fenton and the Fenton like reactions.

Outpatient commitment: what, why, and for whom.

The authors describe studies showing the effectiveness of involuntary outpatient commitment in improving treatment compliance, reducing hospital readmission, and reducing episodes of violence among persons with severe psychiatric illnesses. They point out that because of its role in enhancing compliance with treatment, outpatient commitment can be regarded as a form of assisted treatment, such as assertive case management, representative payeeship, and mental health courts. The authors argue that such assisted treatment is necessary for persons with severe psychiatric illnesses who are noncompliant with their medication regimens because many lack awareness of their illnesses because of biologically based cognitive deficits. They recommend outpatient commitment for any individual with a severe psychiatric disorder who has impaired awareness of his or her illness and is at risk of becoming homeless, incarcerated, or violent or of committing suicide, and they provide case examples. The authors conclude by addressing eight of the most common objections to outpatient commitment by mental health professionals and civil liberties groups that oppose outpatient commitment.

Analysis of Cx43alpha1 promoter function in the developing zebrafish embryo.

The Cx43alpha1 gap junctions play an important role in cardiovascular development. Studies using transgenic mouse models have indicated that this involves an essential role for Cx43alpha1 in modulating neural crest cell motility. We previously showed that a 6.8 kb mouse genomic sequence containing the promoter and upstream regulatory sequences of the Cx43alpha1 gene can drive lacZ reporter gene expression in all neural crest cell lineages in the mouse embryo. To obtain further insights into the sequence motifs and regulatory pathways involved in targeting Cx43alpha1 gene expression in neural crest cells, we assayed the activity of the mouse Cx43alpha1 promoter in evolutionarily distantly related zebrafish embryos. For these studies, the 6.8kb Cx43alpha1 genomic sequence and various deletion derivatives were used to generate GFP or lacZ expression vectors. The transcriptional activities of these constructs were analyzed in vivo after microinjection into one- or two- cell stage zebrafish embryos. These studies indicated that the mouse Cx43alpha1 promoter can drive lacZ expression in neural crest cells in the zebrafish embryos. Analysis by whole mount in situ hybridization showed that the endogenous zebrafish Cx43alpha1 gene is expressed maternally and zygotically, and expression is observed in regions where neural crest cells are found. To further elucidate the developmental regulation of Cx43alpha1 gene expression, we screened a zebrafish BAC library and identified a clone containing the entire zebrafish Cx43alpha1 gene and flanking upstream and downstream sequences. The upstrean Cx43alpha1 promoter sequences from zebrafish, mouse, and human were analyzed for evolutionarily conserved DNA motifs. Overall these studies suggest that the sequence motifs and transcriptional regulation involved in the targeting Cx43alpha1 expression to neural crest cells are evolutionarily conserved in zebrafish and mouse embryos.

A novel role for cardiac neural crest in heart development.

Ablation of premigratory cardiac neural crest results in defective development of the cardiac outflow tract. The purpose of the present study was to correlate the earliest functional and morphological changes in heart development after cardiac neural crest ablation. Within 24 hours after neural crest ablation, the external morphology of the hearts showed straight outflow limbs, tighter heart loops, and variable dilations. Incorporation of bromodeoxyuridine in myocytes, an indication of proliferation, was doubled after cardiac neural crest ablation. The myocardial calcium transients, which are a measure of excitation-contraction coupling, were depressed by 50% in both the inflow and outflow portions of the looped heart tube. The myocardial transients could be rescued by replacing the cardiac neural crest. The cardiac jelly produced by the myocardium was distributed in an uneven, rather than uniform, pattern. An extreme variability in external morphology could be attributed to the uneven distribution of cardiac jelly. In the absence of cardiac neural crest, the myocardium was characterized by somewhat disorganized myofibrils that may be a result of abnormally elevated proliferation. In contrast, endocardial development appeared normal, as evidenced by normal expression of fibrillin-2 protein (JB3 antigen) and normal formation of cushion mesenchyme and trabeculae. The signs of abnormal myocardial development coincident with normal endocardium suggest that the presence of cardiac neural crest cells is necessary for normal differentiation and function of the myocardium during early heart development. These results indicate a novel role for neural crest cells in myocardial maturation.

Use of a repetitive mouse B2 element to identify transplanted mouse cells in mouse-chick chimeras.

Monitoring the migrations of cells during embryonic development requires a system in which cells can be identified in situ during locomotion. One promising system involves the generation of chimeras by transplanting mouse cells into chick embryos in ovo to exploit the wealth of mouse genetic variants. The success of this technique relies on the ability to detect individual mouse cells in a chick environment with high specificity. The murine B2 family of short interspersed elements is present in the mouse genome at copy numbers in excess of 10(5), whereas this sequence is absent in the chick genome based on hybridization techniques. This differential of five orders of magnitude produces signals in mouse cells that are easily identified, even in an environment that is predominantly chick. Thus, the B2 repeat probe is highly effective for the purpose of identifying mouse cells in mouse-chick chimeras.

Insulin-like growth factor-I and high protein diet decrease calpain-mediated proteolysis in murine muscular dystrophy.

In muscular dystrophy (MD) the imbalance between muscle protein synthesis and degradation may be an important factor leading to muscle wasting. The three major pathways of muscle proteolysis identified in skeletal muscle are: the lysosomal cathepsin pathway, the calcium-dependent calpain pathway, and the ATP-dependent ubiquitin pathway. Insulin-like growth factor I (IGF-I) and a high-protein diet (HPD) have been shown to reduce proteolysis in skeletal muscle. We examined the effect of 6 weeks of recombinant human IGF-I (rhIGF-I) alone or in combination with HPD treatment on the proteolytic pathways in skeletal muscle of 129 ReJ dystrophic (dy) mice. (A group of normal (Norm) nondystrophic (129 J) mice were included as controls). Untreated dy mice exhibited increased net proteolysis (P < 0.05), elevated net calpain activity (P < 0.01), and increased ubiquitin levels when compared to control mice (P < 0.05). Our evidence suggests that HPD and rhIGF-I decrease proteolysis in the 129 ReJ dy mouse. This effect appears attributable, at least in part, to reduced calpain-mediated myofibrillar breakdown (P < 0.05) due to decreased calpain autolysis or increased calpastatin levels. In contrast to calpain, cathepsin B activity was increased in HPD and rhIGF-I + HPD-treated dy muscle (P < 0.05) and unaltered in the rhIGF-I treated animals. Levels of free and protein-conjugated ubiquitin were also increased in rhIGF-I, and rhIGF-I + HPD treated dyanimals (P < 0.05). The amelioration of muscle wasting in the 129 ReJ dy model by HPD and/or rhIGF-I may have potential implications in the treatment of human MD.

The role of dietary zinc in modifying the onset and severity of spontaneous diabetes in the BB Wistar rat.

The goal of this study was to determine whether zinc supplementation in the diet of diabetes-prone BB Wistar rats will delay or prevent the onset of overt diabetes. Male Wistar BB rats were fed diets containing either 1000 ppm (HZ), 50 ppm (NZ), or 1 ppm zinc (LZ) starting at 30 days of age. Non-diabetes-prone rats were fed NZ and designated as controls (NORM). Beginning at 60 days, the rats were checked for glycosuria and, if positive, were given an i.p. glucose tolerance test (IPGTT). All remaining animals underwent an IPGTT at 100 days and were sacrificed. At 90 days of age HZ rats had a lower incidence of diabetes (19%) than NZ (53%) or LZ (44%) animals (P < 0.015). By age 100 days, for the HZ group, there was a 60% reduction in the number of expected overt diabetic rats. HZ animals also had higher concentrations of both pancreatic and serum insulin and exhibited lower serum glucose and triglycerides. Immunohistochemistry of HZ rats was clearly different from NZ rats and showed evidence of nearly normal pancreatic endocrine activity. Data indicate that dietary treatment of diabetes-prone BB Wistar rats with zinc appears to be an effective approach for delaying or preventing the onset of diabetes in genetically predisposed rodents. This finding may suggest further experimental studies regarding dietary means for preservation of pancreatic function.

Metabolic and structural effects of insulin-like growth factor-I and high-protein diet on dystrophic hamster skeletal muscle.

In muscular dystrophy (MD) there is an imbalance between muscle protein synthesis and protein degradation, which results in a net muscle catabolism, along with muscle wasting and weakness. Using a dystrophic hamster model (BIO 53.58), we examined the chronic (8 weeks) effects of two factors that may enhance muscle protein synthesis and inhibit protein degradation, namely, insulin-like growth factor-I (rhIGF-I) and high-protein diet (HPD). Protein synthesis was determined by measuring the incorporation of 14C phenylalanine into perfused leg muscle, while protein degradation was calculated from the release of tyrosine from the same perfused muscle. Urinary 3-methylhistidine excretion was used as an indicator of myofibrillar degradation. Treatment of dystrophic hamsters with rhIGF-I, HPD, or a combination of the two for 8 weeks resulted in significant decreases in total and myofibrillar degradation when compared with untreated dystrophic animals (P < 0.05) but had minimal effects on protein synthesis. Significant morphologic improvements (P < 0.05), including a normalization and greater uniformity of muscle fibers, were also seen in rhIGF-I- and rhIGF-I + HPD-treated animals. rhIGF-I and HPD were effective in reducing the excessive proteolysis seen in dystrophic muscle, and this reduced proteolysis resulted in improvement of muscle morphology.

Expression of myogenic regulatory factors in normal and dystrophic mice: effects of IGF-1 treatment.

Myogenic regulatory factors (MRFs) promote differentiation of muscle cells from fibroblasts and are induced by insulin-like growth factor I (IGF-1). Prior studies have shown synthesis of new muscle protein and improved muscle morphology when mature dy mice with muscular dystrophy are treated with IGF-1. We investigated whether these salutary effects of IGF-1 might be attributable to stimulation of MRFs. Male dy (129ReJ) mice and controls (129J) were assigned to IGF-1 treatment (10 micrograms twice daily) or non-treatment at about 5 weeks of life and sacrificed 6 weeks later. RNA was extracted from skeletal muscles, reverse transcribed, and amplified by polymerase chain reaction (PCR) using primers specific for each MRF. Competitive PCR was performed to quantify MyoD expression in response to IGF-1 treatment. Transcripts for myf-5, MRF4, and myogenin were detected in both control and dy mouse muscles; no apparent differences were observed between treatment groups. Quantitative analysis of transcripts for MyoD indicated no significant basal differences between control and dy mice. There was, however, significantly higher MyoD expression in the dy group, and a trend toward significance in the control group, following IGF-1 treatment. These data suggest that IGF-1 exerts its in vivo effects in postembryonal muscle by stimulating MRFs.

Effect of insulin-like growth factor I in murine muscular dystrophy.

In muscular dystrophy there is an imbalance between muscle protein synthesis and protein degradation, resulting in net muscle catabolism and progressive muscle weakness and wasting. Both insulin and insulin-like growth factor I (IGF-I) are known to have an anabolic effect on skeletal muscle, which is believed to be enhanced in the presence of elevated concentrations of amino acids. We examined the effects of 4-week administration of recombinant human IGF-I (rhIGF-I), both alone and supplemented with a high protein diet (HPD), on muscle metabolism, morphology, and function in the 129 ReJ dystrophic mouse. rhIGF-I significantly reduced muscle protein degradation (P < 0.001), increased muscle protein content (P < 0.05), decreased fiber area variability (P < 0.01), and increased hind limb utilization (P < 0.01). Supplementation of rhIGF-I therapy with a HPD resulted in a significant increase in muscle protein synthesis (P < 0.05) in addition to a further increase in the above parameters. We conclude that rhIGF-I causes an improvement in muscle metabolism, morphology, and function in dystrophic mice, and this effect is further enhanced by the presence of a HPD.

High protein diet has beneficial effects in murine muscular dystrophy.

In normal muscle there is a delicate balance between muscle protein synthesis and protein degradation. It is believed that this balance is disturbed in muscular dystrophy (MD) by decreased muscle protein synthesis and/or increased muscle protein degradation, resulting in net catabolism. In an attempt to reduce or reverse this catabolism, a high protein diet (HPD, 50% protein) was fed to dystrophic mice (129/ReJ dy) for 4 wk. The effects on muscle biochemistry, muscle function and muscle morphology were compared with those in dystrophic mice fed a normal diet (NPD, 20% protein) and in nondystrophic mice (NORM) also fed the 20% protein diet. Compared with NORM mice, NPD mice demonstrated greater rates of muscle protein synthesis (P < 0.05) as measured by the incorporation of labeled phenylalanine into muscle, greater protein degradation (P < 0.01) as measured by urinary 3-methylhistidine excretion, and lower muscle protein concentration (P < 0.01). When dystrophic mice were fed HPD for 4 wk, protein degradation was lower (P < 0.01) and muscle protein concentration greater (P < 0.01) than in NPD mice. These biochemical improvements were accompanied by greater morphological uniformity of muscle fibers, higher volume density of muscle fibers per unit area of muscle (P < 0.01), and lower shape factor (P < 0.01). Functionally, HPD led to improved muscle endurance (P < 0.01) and increased hind-limb utilization (P 0.01). We conclude that in murine dystrophy, HPD decreases net muscle catabolism, principally by decreasing muscle protein degradation, resulting in improvement in muscle morphology, strength and function.

Short- and long-term effect of oral salbutamol on growth hormone secretion in prepubertal asthmatic children.

Salbutamol, a beta 2-adrenergic agonist, is being extensively used in Venezuela as a brochodilator in the treatment of asthma in children. Previous reports have shown oral salbutamol either to inhibit or not to affect growth hormone (GH) secretion. We evaluated the effect of oral salbutamol (0.1 mg/kg every 6 hours for 3 months) on GH secretion in eight prepubertal short children with mild asthma. Levels of GH during sleep (samples taken every 30 minutes from 9 PM to 6 AM) and after GH-releasing hormone ([GHRH] 1 microgram/kg intravenously [IV]) were measured before, at 24 hours, and at 3 months of salbutamol treatment. Overnight integrated concentrations of GH and peak GH levels following GHRH diminished significantly after 24 hours of salbutamol therapy (from 4.5 +/- 1.3 to 3.4 +/- 0.8 micrograms/L and from 46.6 +/- 47.3 to 16.2 +/- 7.9 micrograms/L, respectively, P < .05). However, GH levels after 3 months of salbutamol were not different from basal levels (4.5 +/- 1.3 v 5.1 +/- 5.1 +/- 2.9 micrograms/L during the overnight studies and 46.6 +/- 47.3 v 37.8 +/- 30.4 micrograms/L after GHRH). Our data suggest an inhibition of both spontaneous and stimulated GH secretion following short-term oral salbutamol ingestion, but this suppressive effect is not maintained with its long-term use.

Modification of insulin resistance by diazoxide in obese Zucker rats.

Hyperinsulinism, insulin resistance, and decreased number of insulin receptors are characteristic of obesity in both humans and experimental animals. To assess the role of insulin in developing obesity, diazoxide (DZ), an inhibitor of glucose-stimulated insulin secretion, was administered for 8 weeks to 7-week-old female Zucker rats in two concentrations, 50 mg/kg.day (LD-DZ), and 100 mg/kg.day (HD-DZ). The obese and lean rats were divided into three subgroups: diazoxide (DZ), pair-fed (PF), and control (C) groups (n = 6 rats/subgroup-genotype). Diazoxide-treated obese and lean animals showed significantly lower postabsorptive plasma insulin concentrations (P < 0.005) than their respective obese and lean PF and C subgroups. HD-DZ obese rats consumed more calories (P < 0.001), yet gained less weight (P < 0.05) than PF and C rats. The plasma glucose concentrations in the postabsorptive state and during glucose tolerance tests in HD-DZ obese rats were significantly lower than those in PF and C rats (P < 0.01) despite a decrease in their plasma insulin concentrations (P < 0.01), whereas HD-DZ lean rats displayed a diabetic response (P < 0.01). The adipocyte-specific insulin receptor binding was dose-dependently increased in both lean and obese DZ animals (P < 0.01). DZ had a dual effect on insulin metabolism; it decreased insulin secretion and increased insulin receptor binding. This dual effect was associated with improved glucose tolerance and a decrease in weight gain in obese rats.

Protective role of magnesium in catecholamine-induced arrhythmia and toxicity in vitro.

The present study examined the arrhythmogenic and toxic liability of isoprenaline, noradrenaline and adrenaline using a model of spontaneously beating, cultured rat cardiomyocytes. The cardioprotective role of magnesium (Mg) was also evaluated. Following two hours of exposure to 0.1-2.7 mM doses of isoprenaline, noradrenaline or adrenaline, there were dose-dependent increases in the incidence of arrhythmia and decreases in beating activity. Myocyte LDH release increased 64-189% while total myocyte K+ and Mg2+ decreased 13-60% at high catecholamine doses. Levels of the secondary messengers IP3 and cAMP were also increased. Equimolar Mg (0.9 mM) significantly reduced the incidence of catecholamine arrhythmia while preserving beating activity, membrane integrity and electrolyte levels. Mg proved to be in vitro a potent antiarrhythmic and cardioprotective agent which is likely to exert its beneficial effects via antagonism of calcium.

Magnesium protection against anthracycline toxicity in vitro.

The clinical usefulness of the antitumour agents daunomycin (DAU) and adriamycin (ADR) is limited by their secondary cardiotoxicity. The anthracycline compounds have a number of detrimental effects on the biochemical and morphological integrity of the cardiac cell which may be related to the accumulation of cellular calcium. Using a model of spontaneously beating, cultured neonatal rat cardiomyocytes, we examined the cardioprotective role of magnesium during 2 hours of exposure to 10, 25 or 50 micrograms/ml DAU. A significant preservation of myocyte membrane integrity and cellular morphology was observed with the addition of equimolar magnesium. Magnesium opposes the actions of calcium in a number of tissues and it may be this calcium antagonist action that makes magnesium effective in DAU toxicity.

Improved water and sodium absorption from oral rehydration solutions based on rice syrup in a rat model of osmotic diarrhea.

Rice syrup solids, rice protein, and casein hydrolysate were added to experimental oral rehydration solutions in various combinations and tested in a rat intestinal perfusion system. Chronic osmotic diarrhea was induced in juvenile rats by supplying the cathartic agents, magnesium citrate and phenolphthalein, in their drinking water for 1 week. The experimental oral rehydration solutions were compared with standard oral rehydration solutions containing 20 gm/L or 30 gm/L of glucose and with each other to determine if there were significant differences in net water, sodium, or potassium absorption. An oral rehydration solution containing 30 gm/L of rice syrup solids had a net water absorption rate significantly higher than that of the standard 20 gm/L glucose-based oral rehydration solution (2.1 +/- 0.62 versus 1.5 +/- 0.48 microliters/[min x cm], p less than 0.05). Casein hydrolysate did not significantly affect net water absorption. However, combinations of 30 gm/L rice syrup solids and 5 gm/L casein hydrolysate significantly increased (p less than 0.05) net sodium and potassium absorption compared with the 20 gm/L glucose-based oral rehydration solution but not versus rice syrup solids alone. Oral rehydration solutions containing 30 gm/L rice syrup solids plus 5 gm/L rice protein, and 30 gm/L rice syrup solids plus 5 gm/L casein hydrolysate, had net water absorption rates significantly higher than the rate of a 30 gm/L glucose-based oral rehydration solution (2.5 +/- 0.36 and 2.4 +/- 0.38, respectively, versus 0.87 +/- 0.40 microliters/[min x cm], p less than 0.05). Rice protein and casein hydrolysate, however, did not significantly affect net water, sodium, or potassium absorption when added to rice protein glucose-based oral rehydration solutions. An inverse correlation between osmolality and net water absorption was observed (r = -0.653, p less than 0.02). The data suggest that substitution of rice syrup solids for glucose in oral rehydration solutions will improve water absorption and that rice syrup solids in combination with protein hydrolysates may, in addition, promote better sodium and potassium uptake.

Alanine stimulation of water and sodium absorption in a model of secretory diarrhea.

We investigated the effectiveness of L-alanine (Ala) addition to oral hydration solutions (OHSs) during secretory conditions induced by ileal instillation of 10 mM theophylline in anesthetized rats using a perfusion procedure, and monitoring water and sodium transport. Ala was added to two hypotonic OHSs in which the sodium:glucose ratio was 2:1, and compared with the OHS recommended by the World Health Organization (WHO), which has a sodium:glucose ratio of 0.81:1. Theophylline had the expected secretory effect on water and sodium absorption in the WHO-recommended OHS, and on sodium transport in a formula containing 60 mM sodium and 30 mM glucose. However, an OHS with 90 mM sodium and 45 mM glucose canceled the secretory effect of theophylline and yielded a greater rate of net water absorption than the WHO formula. Addition to this solution of either 15 or 30 mM Ala enhanced water and sodium absorption of both control and theophylline-treated rats. In the hypotonic OHS with 60 mM sodium and 30 mM glucose, Ala had little effect on both sodium and water transport. Therefore, the data support the view that Ala added to solutions with 90 mM sodium, containing sufficient glucose to maintain a sodium:glucose ratio of not less than 2:1, is most effective at compensating fluid and sodium losses under secretory conditions. Ala presumably exerts its sodium-sparing effect because of its cotransport with sodium and the consequent water influx into the intestinal cells.