Therapeutic safety of high myocardial expression levels of the molecular inotrope S100A1 in a preclinical heart failure model.

Low levels of the molecular inotrope S100A1 are sufficient to rescue post-ischemic heart failure (HF). As a prerequisite to clinical application and to determine the safety of myocardial S100A1 DNA-based therapy, we investigated the effects of high myocardial S100A1 expression levels on the cardiac contractile function and occurrence of arrhythmia in a preclinical large animal HF model. At 2 weeks after myocardial infarction domestic pigs presented significant left ventricular (LV) contractile dysfunction. Retrograde application of AAV6-S100A1 (1.5 × 10(13) tvp) via the anterior cardiac vein (ACV) resulted in high-level myocardial S100A1 protein peak expression of up to 95-fold above control. At 14 weeks, pigs with high-level myocardial S100A1 protein overexpression did not show abnormalities in the electrocardiogram. Electrophysiological right ventricular stimulation ruled out an increased susceptibility to monomorphic ventricular arrhythmia. High-level S100A1 protein overexpression in the LV myocardium resulted in a significant increase in LV ejection fraction (LVEF), albeit to a lesser extent than previously reported with low S100A1 protein overexpression. Cardiac remodeling was, however, equally reversed. High myocardial S100A1 protein overexpression neither increases the occurrence of cardiac arrhythmia nor causes detrimental effects on myocardial contractile function in vivo. In contrast, this study demonstrates a broad therapeutic range of S100A1 gene therapy in post-ischemic HF using a preclinical large animal model.

Hemodynamics after endoscopic submucosal injection of epinephrine in a porcine model.

BACKGROUND AND STUDY AIMS: Endoscopic submucosal injection of epinephrine may cause systemic effects on the cardiovascular system. The aim of this experimental study was to assess systemic hemodynamic changes after submucosal injection of epinephrine during upper gastrointestinal endoscopy in a porcine model. METHODS: Measurements were taken from 12 pigs under general anesthesia. During gastroscopy 5 mL of normal saline, and 2.5 mL and 5 mL of epinephrine (1:10,000) were injected into the submucosal layers of the gastric antrum, corpus, and distal esophagus. After each injection, the cardiac index and global end diastolic volume index (GEDVI, reflecting preload) were measured every 3 minutes by transpulmonary thermodilution for a minimum of 12 minutes. The following parameters were also recorded: heart rate, mean arterial pressure (MAP), and systemic vascular resistance index (SVRI, reflecting afterload). RESULTS: Significant hemodynamic changes were observed after submucosal injection of epinephrine into the esophagus, including heart rate (maximum + 4 %) and MAP (maximum - 4%) after injection of 2.5 mL epinephrine, and stronger changes in heart rate (maximum +13%), cardiac index (maximum +21%), MAP (maximum -4%), and SVRI (maximum -12%) after the injection of 5 mL epinephrine. After submucosal injection of epinephrine into the gastric antrum and corpus, hemodynamic effects were less evident. Here significant changes were observed in heart rate (maximum +3%), MAP (maximum -2%), cardiac index (maximum +7%), and SVRI (maximum -8%) only after the injection of 5 mL epinephrine into the antrum. CONCLUSION: Endoscopic submucosal injection of epinephrine is associated with changes in systemic hemodynamic parameters, especially when performed in the esophagus, and the procedure might therefore induce harmful side effects.

Isolation, characterization and establishment of an equine retinal glial cell line: a prerequisite to investigate the physiological function of Müller cells in the retina.

Retinal Müller glial cells are of vital importance for maintaining a physiological environment within the retina. To this end, they provide highly specialized physiological properties to support neurons in structure, nutrition and metabolism. The purpose of this study was to isolate Müller cells from the equine retina, determine their characteristics and subsequently establish a stable equine Müller cell line (eqMC) that will provide a prerequisite for investigations on their physiological properties. Dissociated retinal cells were obtained from equine retinas by a papain digestion technique followed by trituration and a cell attachment method by which pure Müller cell cultures were achieved. Morphological examination was performed using phase-contrast microscopy, and further characterization of different subcultures was accomplished by immunocytochemistry. Cells of passage 1 showed distinct signals for glutamine synthetase and vimentin, whereas glial fibrillary acidic protein expression was almost absent. Characteristic expression patterns remained unaltered in all subcultures. Furthermore, cultured Müller cells stably expressed the microfilament alpha-smooth muscle actin, the proliferation marker Ki67 and the membrane channels Kir4.1 and aquaporin 4. The present study introduces the eqMC-7 that will facilitate studies investigating the physiological role of Müller cells within the equine retina.

Natural-orifice transluminal endoscopic surgery: low-pressure pneumoperitoneum is sufficient and is associated with an improved cardiopulmonary response (PressurePig Study).

BACKGROUND AND AIMS: The aim of this randomized trial in the acute porcine model was to compare the quality of transgastric peritoneoscopy with the use of low-pressure versus standard-pressure pneumoperitoneum and to evaluate the respective associated cardiopulmonary changes. METHODS: For transgastric peritoneoscopy, carbon dioxide was insufflated via the endoscope for a constant intraperitoneal pressure of 6 mmHg or 12 mmHg in 9 pigs each. The quality of transgastric peritoneoscopy was rated on a visual analog scale (0 mm, min.; 100 mm, max.) by the endoscopist, who was blinded to the intraperitoneal pressure. The cardiac index and global end-diastolic volume index (GEDVI, reflecting preload) were measured every 3 minutes by transpulmonary thermodilution. The following were also recorded: heart rate, mean arterial pressure (MAP), systemic vascular resistance index (SVRI, reflecting afterload), peak inspiratory pressure (PIP), pH, PCO (2), and PO (2). RESULTS: The quality of transgastric peritoneoscopy with the use of low-pressure pneumoperitoneum was not inferior to that obtained using standard-pressure pneumoperitoneum (87.0 mm vs. 87.3 mm; P<0.05). In both groups we observed a statistically significant rise in MAP and SVRI. The increase in SVRI was less pronounced during low-pressure peritoneum ( P=0.042), indicating a reduced stress response in comparison to standard-pressure peritoneum. There were no relevant differences between the groups in relation to cardiac index, GEDVI, and heart rate. An intra-abdominal pressure of 6 mmHg also led to better oxygenation ( P=0.031 for difference in PO (2) between the two groups) due to lower peak inspiratory pressure ( P<0.001 for difference). There were only slight differences between the groups with regard to pH and PCO (2). CONCLUSIONS: Pneumoperitoneum of 12-16 mmHg is used for standard laparoscopy. For NOTES, low-pressure pneumoperitoneum is sufficient and is associated with an improved cardiopulmonary response compared to standard-pressure pneumoperitoneum.

Transfer of the chemokine receptor CCR5 between cells by membrane-derived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection.

The release of microparticles from eukaryotic cells is a well-recognized phenomenon. We demonstrate here that the chemokine receptor CCR5, the principal co-receptor for macrophage-tropic human immunodeficiency virus (HIV)-1, can be released through microparticles from the surface of CCR5+ Chinese hamster ovary cells and peripheral blood mononuclear cells. Microparticles containing CCR5 can transfer the receptor to CCR5- cells and render them CCR5+. The CCR5 transfer to CCR5-deficient peripheral blood mononuclear cells homozygous for a 32-base-pair deletion in the CCR5 gene enabled infection of these cells with macrophage-tropic HIV-1. In monocytes, the transfer of CCR5 could be inhibited by cytochalasin D, and transferred CCR5 could be downmodulated by chemokines. A transfer of CCR5 from peripheral blood mononuclear cells to endothelial cells during transendothelial migration could be demonstrated. Thus, the transfer of CCR5 may lead to infection of tissues without endogenous CCR5 expression. Moreover, the intercellular transfer of membrane proteins by microparticles might have broader consequences for intercellular communication beyond the effects seen for HIV-1.

Aminooxypentane-RANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity.

CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH2-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.

Oncotic pressure in solid tumors is elevated.

Oncotic and hydrostatic pressure differences control the movement of fluid and large molecules across the microvascular wall of normal and tumor tissues. Recent studies have shown that the interstitial fluid pressure in tumors is elevated and is approximately equal to the microvascular pressure. Whereas oncotic pressure in blood plasma of various species is known, no data are available on the oncotic pressure in the interstitial space of tumors. We hypothesize that because of the leaky nature of tumor vessels, oncotic pressure in tumor interstitium should be close to that in plasma. To this end, we first developed a chronic wick method for the direct measurement of oncotic pressures in the interstitial fluid of tumors grown in mice. We found interstitial oncotic pressures in four human tumor xenografts to be higher than in s.c. tissue and comparable to that in plasma [rhabdomyosarcoma (RD), 24.2+/-4.7; squamous cell carcinoma (FaDu), 19.9+/-1.9; small cell lung carcinoma (54A), 21.1+/-2.8; colon adenocarcinoma (LIS174T), 16.7+/-3.0 mm Hg; s.c. tissue, 8.2+/-2.3; plasma, 20.0+/-1.6 mm Hg]. These results support our hypothesis that the oncotic pressure difference across the tumor microvascular wall is low. The high oncotic pressure in tumors is consistent with the elevated interstitial fluid pressure, and it contributes to the suboptimal delivery of large therapeutic agents to neoplastic cells.

Skeletal effects of low-dose cyclosporin A in aged male rats: lack of relationship to serum testosterone levels.

The aim of this study was to investigate the skeletal effects of cyclosporin A (CsA) in a dose range relevant to clinical medicine in lumbar vertebral cancellous bone of aged male rats and to correlate these effects with possible changes in serum testosterone levels. Thirty-one 18-month-old male Wistar rats were divided into four weight-matched groups and subcutaneously injected with either 0, 1, 3, or 5 mg of CsA/kg of body weight three times per week After 4 weeks of treatment, all rats were killed after in vivo fluorochrome labeling and the first lumbar vertebrae analyzed by quantitative histomorphometry. Serum was analyzed for total calcium, creatinine, alkaline phosphatase, osteocalcin, parathyroid hormone, total testosterone, and CsA levels. CsA administration resulted in a dose-dependent increase in serum osteocalcin levels and in histomorphometric indices of cancellous bone turnover in the axial skeleton. Furthermore, CsA-treated rats showed a deterioration of vertebral cancellous bone structure with increased discontinuity of the trabecular bone network due to trabecular plate perforations. Serum testosterone levels were not significantly changed by CsA treatment and were uncorrelated to all biochemical or histomorphometric indices of bone turnover. We conclude that the 4-week administration of CsA at doses that are close to those used in transplantation patients induced high turnover osteopenia in the axial skeleton of aged, 18-month-old male rats, and that these effects were likely not mediated by changes in serum testosterone levels.

1alpha-hydroxyvitamin D2 is less toxic but not bone selective relative to 1alpha-hydroxyvitamin D3 in ovariectomized rats.

Identification of bone selective vitamin D analogues would provide an interesting substance class for the treatment of osteoporosis. The synthetic prodrug 1alpha-hydroxyvitamin D2 [1alpha(OH)D2] has been shown to combine equal bone-preserving activity with distinctly reduced calcemic effects relative to 1alpha-hydroxyvitamin D3 [1alpha(OH)D3] in 3-month-old ovariectomized (OVX) rats. Therefore, 1alpha(OH)D2 may be a bone-selective compound. The aim of this study was to compare the bone protective and the calcemic activities of chronically administered 1alpha(OH)D2 and 1alpha(OH)D3 in 6-month-old OVX rats over a broad dose range from ineffective to toxic doses. Ninety-six female 6-month-old Fischer-344 rats were used for this experiment. Eighty rats were bilaterally OVX, 8 rats were sham-operated (SHAM), and 8 rats were killed at the time of surgery as a baseline control. Groups of OVX rats received vehicle alone (n = 16) or daily doses in the diet of 0.025, 0.05, 0.1, and 0.2 microg of 1alpha(OH)D2 or 1alpha(OH)D3 per kg body weight (BW) per day (n = 8 each). After calcein double-labeling, all animals were killed 3 months post-OVX. Orally administered 1alpha(OH)D2 was significantly less toxic compared with 1alpha(OH)D3 in terms of BW gain and kidney calcium content. The effects of 1alpha(OH)D2 and 1alpha(OH)D3 on serum calcium and urinary calcium excretion were generally similar at all doses in this study. Both 1alpha(OH)D2 and 1alpha(OH)D3 prevented the estrogen deficiency-induced bone loss in OVX rats, and induced profound bone anabolic effects at high dosages. 1alpha(OH)D3 and 1alpha(OH)D2 also dose-dependently increased total bone mineral density (BMD), cortical area, and cortical thickness in the tibial diaphysis of OVX rats. Bone resorption as assessed by osteoclast numbers (Oc.Ns) in vertebral cancellous bone and urinary excretion of deoxypyridinoline (DPD) was dose-dependently suppressed by 1alpha(OH)D2 and 1alpha(OH)D3. These data show that although 1alpha(OH)D2 was slightly but significantly less toxic compared with 1alpha(OH)D3, it did not have increased skeletal effects at any dose. Taken together, our findings argue against selective metabolic activation of 1alpha(OH)D2 in bone.

Therapeutic efficacy of 1alpha,25-dihydroxyvitamin D3 and calcium in osteopenic ovariectomized rats: evidence for a direct anabolic effect of 1alpha,25-dihydroxyvitamin D3 on bone.

It is an important question for clinical therapy of osteoporosis with vitamin D metabolites whether these compounds exert their beneficial effects on the skeleton indirectly through an increase in intestinal calcium absorption or whether there is also a major direct component of action on bone. In this study, female 6-month-old Fischer rats were either ovariectomized (OVX) or sham operated. One month before surgery, all rats were placed on a diet containing 0.25% calcium and were kept on this diet throughout the study. Beginning 3 months post-OVX, groups of OVX rats orally received vehicle, a calcium supplement, low dose (0.025 microg/kg x day) or high dose (0.1 microg/kg x day) 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or combinations of low and high dose 1,25-(OH)2D3 with the calcium supplement. By 3 months postsurgery, pretreatment OVX controls had lost 74% and 37% of tibial and vertebral cancellous bone, respectively. Two-way factorial ANOVA showed that a 3-month treatment of osteopenic OVX rats with 1,25-(OH)2D3 dose dependently increased vertebral and tibial cancellous bone mass (P < 0.001 and P = 0.021, respectively) and trabecular width (P < 0.001). Furthermore, 1,25-(OH)2D3 increased serum calcium (P = 0.028) and urinary calcium excretion (P < 0.001) and reduced serum PTH levels (P < 0.001), osteoclast numbers (P < 0.001), and urinary collagen cross-links excretion (P < 0.001). Calcium supplementation alone was without therapeutic effect, and there was no significant two-way interaction between the individual treatment effects of 1,25-(OH)2D3 and calcium on bone mass. These data indicate that the anabolic effects of 1,25-(OH)2D3 in osteopenic OVX rats are mediated through a direct activity on bone.

Helper activity of chicken V beta 1+ and V beta 2+ alpha beta T cells for in vitro IgA antibody synthesis.

Chickens have only two T cell receptor variable beta gene families: V beta 1 and V beta 2 (1). In our previous work we found that IgA production was almost completely suppressed in chickens depleted of V beta 1+ alpha beta T cells by treatment with a TCR V beta 1-specific monoclonal antibody (2), while IgM and IgG production was not affected. Our present results indicate that, in vitro, both V beta 1+ and V beta 2+ chicken cecal tonsil T cells provide help for the differentiation of cecal tonsil IgA B cells, suggesting that the failure of V beta 1+ T cell-depleted chickens to produce IgA is not caused by the inability of V beta 2+ T cells to provide help for IgA production by B cells, but rather by the scarcity of these T cells in mucosal tissues (3), where most IgA responses are induced (4).

Specific methods for the determination of radioactivity in D-(-)-3-hydroxybutyrate in blood plasma.

Two simple, high-yield rapid methods with good reproducibility are described, which permit the determination of radioactivity in plasma D-(-)-3-hydroxybutyrate. The compound is converted to acetoacetate, using a modified enzymatic method. In procedure 1, acetoacetate is reacted with 2,4-dinitrophenylhydrazine; the resulting hydrazone is oxidised by means of a sample oxidiser, and the product 14CO2 is collected in scintillation liquid and counted. In procedure 2, a Conway microdiffusion unit is applied. The acetoacetate is decarboxylated to acetone in the presence of o-phenylenediamine, and the acetone is then diffused into semicarbazide solution. This solution, containing the semicarbazone derivative of labelled acetone, is transferred to liquid scintillation and counted. In both procedures the radioactivity is measured simultaneously in a separate sample which was not subjected to the enzymatic conversion of D-(-)-3-hydroxybutyrate. The difference in radioactivity between the two samples is attributed to labelled (D-(-)-3-hydroxybutyrate.

Skeletal effects of zinc deficiency in growing rats.

There is ample evidence that zinc plays an important role in bone metabolism and zinc deficiency has been implicated as a risk factor in the development of osteoporosis. It was the aim of the present study to investigate the skeletal effects of alimentary zinc deficiency in growing rats using quantitative bone histomorphometry. Twenty-four male Sprague Dawley rats with a mean initial body weight of 101 +/- 2 g were allocated in two groups of 12 rats each and had free access to a semi-synthetic, casein-based, zinc-deficient diet (0.76 mg zinc/kg) or to the same diet supplemented with 60 mg zinc per kg. All rats were sacrificed 42 days after the start of the experiment and the right distal femur was removed for bone histomorphometry. Relative to controls (+Zn), the zinc-deficient rats (-Zn) had a significantly lower body weight and about an 80% reduction in plasma and femur zinc concentration. The histomorphometric evaluation of the distal femoral metaphysis showed that zinc deficiency led to a 45% reduction (p < 0.01) in cancellous bone mass and to a deterioration of trabecular bone architecture, with fewer and thinner trabeculae. The osteopenia in -Zn rats was accompanied by significant reductions in osteoid perimeter (-31%, p < 0.05), osteoblast perimeter (-30%, p < 0.05), and osteoclast number (-38%, p < 0.01) relative to +Zn controls. We conclude that zinc deficiency induced low turnover osteopenia in femoral cancellous bone of growing rats. These results support the hypothesis that zinc deficiency during growth may impair the accumulation of maximal bone mass in humans; additionally, they suggest that zinc deficiency may play a role as a risk factor in the pathogenesis of osteoporosis.

Adjuvant effects of various lipopeptides and interferon-gamma on the humoral immune response of chickens.

The adjuvant effects of various lipopeptides and recombinant chicken interferon gamma (IFN-gamma) on the humoral immune response of laying hens was investigated in four immunization studies. We used the lipopeptide Pam3Cys-Ser-(Lys)4 (PCSL), the conjugate P-Th1 consisting of the lipopeptide P3CS and the T-helper epitope Th1 (FISEAIIHVLHSRHPG), and the conjugate P-Th2 of the lipopeptide P3CSS and the T-helper epitope Th2, which corresponds to the peptide EWEFVNTPPLV, as adjuvants. Human serum albumin (HSA), recombinant bovine somatotropin (RBST), and human immunoglobulin G (IgG) served as antigens in the different experiments. All tested adjuvants enhanced the humoral immune response with various intensities. Chickens showed high antibody titers after the immunization with HSA even without adjuvant, but the adjuvant effects of PCSL and the combination of PCSL and recombinant chicken interferon-gamma (IFN-gamma) were much more pronounced using the antigens RBST and IgG. Especially after the third immunization, higher titers of antibodies were induced by the coadministration of P-Th1 and, to a greater extent, by the combination of PCSL and P-Th1 compared with the use of PCSL. Also, chickens that had received PCSL and P-Th2 showed the highest immune response, even after the second booster. The average concentrations of chicken immunoglobulin Y were significantly higher in 5-mo-old chickens (9.4 mg/mL serum and 10.1 mg/mL egg yolk) compared with 9-mo-old chickens (5.9 mg/mL serum and 5.1 mg/mL egg yolk). The specific serum antibody response was higher in the older chickens than in the younger chickens. Because chicken antibodies are likely to be used increasingly for diagnostic and therapy in the future, lipopeptides and recombinant chicken IFN-gamma may find many applications as adjuvants, thus contributing to the welfare of experimental animals.

Evaluation of various immunisation procedures in laying hens to induce high amounts of specific egg yolk antibodies.

The present study, involving 972 laying hens divided into 162 groups (n = 6), was aimed at the development of an immunisation protocol for laying hens to produce specific egg yolk antibodies. Recombinant bovine somatotropin (rbst), Escherichia coli pilus antigen K88 (K88), human serum immunoglobulin G (IgG), and low density lipoprotein (LDL) were used as antigens, each at four different doses (rbst, K88, LDL : 1µg, 10µg, 100µg, 1mg ; IgG : 0.5µg, 5µg, 50µg, 0.5mg). Three subcutaneous or intramuscular immunisations were performed at intervals of four weeks. The adjuvant used was either the lipopeptide Pam3Cys-Ser-(Lys)4 (PCSL) or Freund ` s incomplete adjuvant (FIA), in two different doses (PCSL: 0.1 and 0.25mg ; FIA: 0.1 and 0.25ml). In the four antigen control groups, hens were immunised without any adjuvant. In two negative control groups, only physiological saline was injected. The mean egg weight and egg yield were not influenced by the immunisation procedures. An antigen dose of 10-100g per injection was sufficient to induce high specific antibody titres in the egg yolk. The adjuvant efficacy of PCSL and FIA was proved to be the same (p < 0.05 versus antigen control). With PCSL as adjuvant, some groups showed a tendency to produce even higher specific antibody titres than did FIA groups. A second booster often caused a further significant increase in the amounts of specific antibodies, especially with PCSL. Subcutaneous administration of the antigen together with 250µg PCSL, resulted in a significantly higher immune response than when FIA was used.

The humoral immune response and the productivity of laying hens kept on the ground or in cages.

The effects of two different keeping systems on the humoral immune response and productivity were compared for 80 laying hens, divided into four groups. Two groups each of 20 hens were kept on the ground and two were kept in cages. All the birds were immunised subcutaneously with human serum immunoglobulin G (IgG) at a dose of 100(microg per injection. The immunisations were performed twice at 4-week intervals. The lipopeptide Pam(3)Cys-Ser-(Lys)(4) was used as an adjuvant at a dose of 0.25mg per injection in one group from each housing system. In the second group from each housing system, the hens were immunised without any adjuvant (antigen control groups). The mean egg yield was significantly higher in both the antigen control group and the adjuvant group, when laying hens were kept in cages. Total egg weight remained constant in both of the housing systems. Keeping hens in cages resulted in higher mean specific antibody titres and mean immunoglobulin Y concentrations in the egg yolk.

Protective effect of vitamin E in a rat model of focal cerebral ischemia.

Under certain pathological conditions such as cerebral ischemia and reperfusion the occurrence of free radicals is remarkably increased. However, only very little information is available on their quantitative relevance for the pathophysiology and final outcome of diseases. The aim of the present study was to evaluate the contribution of oxygen radicals in the pathogenesis of a stroke. For this purpose a rat model for stroke was used. Two of three vitamin E deficient groups were repleted with different dosages of DL-alpha-tocopherylacetate. No signs of vitamin E deficiency could be observed. However, the weight gain during repletion was increased in the vitamin E repleted groups. Brain infarction was created by occlusion of the right middle cerebral artery (MCAO) for two hours. After 24 hours the measurements of infarct volumes were taken. The infarct volume of the group with the highest repletion dosage was significantly reduced by 81%. This was also expressed in a higher rate of gait disturbances after MCAO of the deficient animals. The control of vitamin E status exhibited a similar repletion-dependent level in plasma and brain. These results strongly support the hypothesis that the generation of oxygen radicals occurring during reperfusion is an important aspect of the pathophysiological mechanism in brain infarction.

Effects of amount of intake and stage of forage maturity on urinary allantoin excretion and estimated microbial crude protein synthesis in the rumen of steers.

Using ruminally cannulated steers, we investigated how urinary allantoin excretion was related to variations in feed intake and stage of forage maturity. Further, different approaches were compared for predicting ruminal microbial crude protein (MCP) synthesis and its efficiency. Experimental diets were arranged in a replicated 3 x 3 Latin square design (experiment 1) and a 4 x 4 Latin square design (experiment 2). In experiment 1, a mixed diet [forage to concentrate, 68:32 on a dry matter (DM) basis] was fed at three intake levels corresponding to 1, 1.5 and 2 times maintenance energy requirements. In experiment 2, four silage-based diets were fed based on perennial ryegrass (Lolium perenneL.), which was harvested at four maturity stages. Both experiments demonstrated the influence of diet on microbial growth rate and by this on efficiency of MCP synthesis, although the magnitude of the effects differed between approaches used for estimating MCP. Linear functions satisfactorily characterised the relationship between urinary allantoin excretion (y) and digestible organic matter (OM) intake (x, kg/day; experiment 1: y = 7.94 + 17.34 x; R(2) = 0.785) or intake of OM effectively degraded in the rumen (x, kg/day; experiment 2; y = 22.32 + 5.93 x; R(2) = 0.695). Urinary excretion of allantoin permitted a semi-quantitative prediction of MCP synthesis: ranking of diets and magnitude of changes in MCP synthesis were reflected.

Quantitative measurement of gluconeogenesis from isobutyrate in sheep.

Experiments with continuous infusion of [14C] isobutyrate and single injection of [3H] glucose were performed in two sheep under fed and fasted conditions in order to investigate the contribution of isobutyrate to glucose synthesis. The pool size, total entry and irreversible loss of glucose in the fed sheep were 2.8 mmol/kg0.75, 1.70 and 1.43 mmol/h per kg0.75. After 72-h fasting these parameters decreased about 40% but recycling of glucose carbon increased from 16 to 38% of the total entry rate. Isobutyrate infused intravenously at a rate of 3.5 mmol/h contributed to a minimum of 3-5% of glucose entry indicating that at least 40-60% of the infused isobutyrate was used for net glucose synthesis. The efficiency of the glucogenic and energetic use of isobutyrate as compared to propionate is discussed.