Temperature acclimation alters oxidative capacities and composition of membrane lipids without influencing activities of enzymatic antioxidants or susceptibility to lipid peroxidation in fish muscle.

Cold acclimation of ectotherms results typically in enhanced oxidative capacities and lipid remodeling, changes that should increase the risk of lipid peroxidation (LPO). It is unclear whether activities of antioxidant enzymes may respond in a manner to mitigate the increased potential for LPO. The current study addresses these questions using killifish (Fundulus heteroclitus macrolepidotus) and bluegill (Lepomis macrochirus) acclimated to 5 and 25 degrees C for 9 days and 2 months, respectively. Because the effects of temperature acclimation on pro- and antioxidant metabolism may be confounded by variable activity levels among temperature groups, one species (killifish) was also subjected to a 9-day exercise acclimation. Oxidative capacity of glycolytic (skeletal) muscle (indicated by the activity of cytochrome c oxidase) was elevated by 1.5-fold in killifish, following cold acclimation, but was unchanged in cardiac muscle and also unaffected by exercise acclimation in either tissue. No changes in citrate synthase activity were detected in either tissue following temperature acclimation. Enzymatic antioxidants (catalase and superoxide dismutase) of either muscle type were unaltered by temperature or exercise acclimation. Mitochondria from glycolytic muscle of cold-acclimated killifish were enriched in highly oxidizable polyunsaturated fatty acids (PUFA), including diacyl phospholipids (total carbons:total double bonds) 40:8 and 44:12. Increased oxidative capacity, coupled with elevated PUFA content in mitochondria from cold-acclimated animals did not, however, impact LPO susceptibility when measured with C11-BODIPY. The apparent mismatch between oxidative capacity and enzymatic antioxidants following temperature acclimation will be addressed in future studies.

Characterization of the hypoxia-inducible factor-1 pathway in hearts of Antarctic notothenioid fishes.

The ability of Antarctic notothenioid fishes to mount a robust molecular response to hypoxia is largely unknown. The transcription factor, hypoxia-inducible factor-1 (HIF-1), a heterodimer of HIF-1α and HIF-1ß subunits, is the master regulator of oxygen homeostasis in most metazoans. We sought to determine if, in the hearts of Antarctic notothenioids, HIF-1 is activated and functional in response to either an acute heat stress or hypoxia. The red-blooded Notothenia coriiceps and the hemoglobinless icefish, Chaenocephalus aceratus, were exposed to their critical thermal maximum (CTMAX) or hypoxia (5.0 ± 0.3 mg of O2 L-1) for 2 h. Additionally, N. coriiceps was exposed to 2.3 ± 0.3 mg of O2 L-1 for 12 h, and red-blooded Gobionotothen gibberifrons was exposed to both levels of hypoxia. Levels of HIF-1α were quantified in nuclei isolated from heart ventricles using western blotting. Transcript levels of genes involved in anaerobic metabolism, and known to be regulated by HIF-1, were quantified by real-time PCR, and lactate levels were measured in heart ventricles. Protein levels of HIF-1α increase in nuclei of hearts of N. coriiceps and C. aceratus in response to exposure to CTMAX and in hearts of N. coriiceps exposed to severe hypoxia, yet mRNA levels of anaerobic metabolic genes do not increase in any species, nor do lactate levels increase, suggesting that HIF-1 does not stimulate metabolic remodeling in hearts of notothenioids under these conditions. Together, these data suggest that Antarctic notothenioids may be vulnerable to hypoxic events, which are likely to increase with climate warming.

Biochemical control and toxicity for favorable- and intermediate-risk patients using real-time intraoperative inverse optimization prostate seed implant: Less is more!

PURPOSE: To report the biochemical control rate and clinical outcomes with real-time inverse planning (inverse optimization prostate seed implant [IO-PSI]) for favorable-risk (FR) and intermediate-risk (IR) prostate adenocarcinoma in a community practice setting. This analysis is an extended followup of our initial report, with favorable early biochemical control rate (biochemical nonevidence of disease) of 97% at 4 years. METHODS AND MATERIALS: Three hundred fifty-seven evaluable patients with FR and IR prostate cancer underwent real-time IO-PSI (iodine-125/145 Gy or palladium-103/120 Gy) between 2001 and 2013. RESULTS: With a median followup of 54 months (range, 24-110 months), the absolute biochemical failure free survival of disease was 96%. The 8-year actuarial probability of prostate-specific antigen failure-free survival for FR and IR cohorts was 92.4% and 87%, respectively. Late genitourinary and gastrointestinal toxicity remained low. Late Grade 2 and Grade 3 genitourinary toxicity was 19% and 1%, respectively. Late Grade 2 and 3 rectal bleeding rates were 1% and 0%, respectively. No difference in biochemical control was observed with preimplant short course androgen deprivation or between Gleason score 3 + 4 vs. 4 + 3 patients. No dosimetric parameter was predictive of biochemical failure. Patients with FR had a significantly decreased risk of failure (hazard ratio = 0.26; 95% confidence interval = 0.09-0.78; p = 0.02) compared with those with IR. Patients with a prostate-specific antigen nadir >0.4 ng/mL had an increased risk of failure (hazard ratio = 1.37; 95% confidence interval = 1.27-1.47; p < 0.0001). CONCLUSIONS: Our initial biochemical and clinical outcomes using real-time IO-PSI persisted with extended followup and support our original hypothesis for use of a reduced number of sources, needles, and total activity, suggesting that with IO, less is more.

Cholesterol levels explain inverse compensation of membrane order in brush border but not homeoviscous adaptation in basolateral membranes from the intestinal epithelia of rainbow trout

The role of cholesterol in the thermal adaptation of biological membranes is explored. Physical and chemical responses of membranes to acclimation temperature were evaluated using plasma membrane domains (basolateral and brush border) prepared from intestinal epithelia of 5- and 20 °C-acclimated rainbow trout (Oncorhynchus mykiss). Basolateral membranes (BLMs) exhibit perfect homeoviscous efficacy (indicated by fluorescence depolarization using 1,6-diphenyl-1,3,5-hexatriene), although cholesterol content does not change with acclimation temperature (molar ratios of cholesterol to phospholipid are 0.23± 0.01 from 5 °C-acclimated fish and 0.25±0.02 from 20°C-acclimated fish; mean ± s.e.m.). Reductions (greater than 30 %) in each of the two major saturated fatty acids (16:0 and 18:0), and a 42 % increase in the polyunsaturate 22:6 (n-3) are found in BLMs from fish acclimated to 5 °C compared with membranes from warm-acclimated animals, suggesting that the phospholipid acyl chain composition determines the physical properties of BLMs. In marked contrast, brush-border membranes (BBMs) display opposite trends. BBMs from 5 °C-acclimated fish are more ordered than BBMs from 20 °C-acclimated fish (inverse compensation). Cholesterol content expressed relative to protein or relative to total polar lipid (phospholipid plus glycolipid) is significantly higher in cold- than in warm-acclimated fish, and nearly so (P=0.15) relative to phospholipid (0.31±0.03 in 5 °C-acclimated animals and 0.25±0.02 in 20 °C-acclimated animals). Only minor changes in the acyl composition of BBMs are induced by temperature acclimation. These results suggest that bile, a constituent of the apical microenvironment, may impose unusual requirements for membrane order and/or stability in the brush border.

Hepatic ischemia/reperfusion injury in P-selectin and intercellular adhesion molecule-1 double-mutant mice.

Neutrophil adhesion and recruitment represents one of the early cellular events that occur during hepatic ischemia/reperfusion (IR) injury and plays a critical role in determining the extent of tissue damage. The adhesion molecules, such as selectins and intercellular adhesion molecules (ICAM), are important in mediating neutrophil-endothelial cell interactions and neutrophil emigration. The goal of this study was to evaluate the role of P-selectin and ICAM-1 in hepatic IR injury. Male wild-type and P-selectin/ICAM-1-deficient (P/I null) mice underwent 90 minutes of partial hepatic ischemia followed by reperfusion at various time points (0, 1.5, 3, and 6 hours). Reperfusion caused a time-dependent hepatocellular injury in both wild-type and P/I null mice as judged by plasma alanine aminotransferase (ALT) levels and liver histopathology examination. Although ALT levels were slightly lower in the P/I null mice compared with the wild-type mice the differences were not statistically significant. Neutrophil infiltration to the ischemic liver was observed in both mouse groups after 6 hours of reperfusion; however, the infiltration to the midzonal region of the ischemic liver was more pronounced in the wild-type group. This study suggests that hepatocellular injury induced after hepatic IR was independent of P-selectin and ICAM-1 in this model of acute inflammatory tissue injury.

Effects of acclimation temperature on enzymatic capacities and mitochondrial membranes from the body wall of the earthworm Lumbricus terrestris.

Many ectotherms respond to low temperature by adjusting capacities of enzymes from energy metabolism, restructuring membrane phospholipids and modulating membrane fluidity. Although much is known about the temperature biology of earthworms, it is not known to what extent earthworms employ compensatory changes in enzymatic capacities and membrane physical properties after exposure to low temperature. We examined activities of enzymes from glycolysis and central oxidative pathways as well as fluidity and phospholipid fatty acid composition of mitochondrial membranes prepared from the body wall of the temperate oligochaete Lumbricus terrestris after a one month acclimation to 5 degrees and 15 degrees C. No compensation occurs in central pathways of oxidative metabolism since activities of cytochrome-c oxidase and citrate synthase, when measured at a common temperature, are similar for 5 degrees C and 15 degrees C-acclimated animals. In contrast, activity of pyruvate kinase is elevated 1.3-fold after acclimation to 5 degrees C. Mitochondrial membranes display inverse compensation with respect to temperature (membranes from 5 degrees C animals are more ordered than membranes from 15 degrees C animals). Our results, in combination with earlier reports, indicate that routine metabolism in L. terrestris may be maintained at reduced temperatures with little or no change in enzymatic capacities and inverse compensation of mitochondrial membranes.

Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage.

The objective of this study was to determine if centrifugation and partial removal of seminal plasma would improve spermatozoal motility in semen from stallions whose whole ejaculates have poor tolerance to cooling and storage. Stallions were divided into two groups (n = 5/group) based on the ability of their extended semen to maintain spermatozoal motility after cooling and storage. Group 1 stallions ("good coolers") produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by < or = 30% of progressive motility prior to storage. Group 2 stallions ("poor coolers") produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by > or = 40% of progressive motility prior to storage. The sperm-rich portion of each ejaculate was divided into 4 aliquots. Two aliquots underwent standard processing for cooled transported semen and were examined after 24 and 48 h of cooling and storage in an Equitainer. The remaining two aliquots were diluted 1:1 with semen extender, then centrifuged at 400 x g for 12 min at room temperature. After centrifugation, approximately 90% of the seminal plasma was removed, and the sperm pellet was resuspended in extender to a final concentration of 25 to 50 x 10(6) sperm/mL. These aliquots were then packaged as for the non-centrifuged aliquots and examined after 24 and 48 h of storage. The spermatozoal motion characteristics in fresh semen and after 24 and 48 h of cooling and storage was determined via computer-assisted semen analysis. Centrifugation and partial removal of seminal plasma increased the percentage of progressively motile spermatozoa and limited the reduction in progressive spermatozoal motility of "poor cooling" stallions after 48 h of cooling and storage. Results of this study indicate that centrifugation and partial removal of seminal plasma is beneficial for stallions whose ejaculates have poor tolerance to cooling and storage with routine semen dilution and packaging techniques, especially if the semen is stored for > 24 h.

Effect of cooling of equine spermatozoa before freezing on post-thaw motility: preliminary results.

The ability to ship cooled stallion semen to a facility that specializes in cryopreservation of spermatozoa would permit stallions to remain at home while their semen is cryopreserved at facilities having the equipment and expertise to freeze the semen properly. To accomplish this goal, methods must be developed to freeze cooled shipped semen. Three experiments were conducted to determine the most appropriate spermatozoal extender, package, time of centrifugation, spermatozoal concentration and length of time after collection that spermatozoa can be cooled before cryopreservation. In the first experiment, spermatozoa were centrifuged to remove seminal plasma, resuspended in either a skim milk extender, a skim milk-egg yolk-sugar extender or a skim milk-egg yolk-salt extender, cooled to 5 degreesC and frozen in 0.5- or 2.5-mL straws either 2.5 or 24 h after cooling. Samples frozen 2.5 h after cooling had higher percentages of progressively motile (PM) spermatozoa (27%) than samples frozen 24 h after cooling (10%; P < 0.05). Samples frozen 2.5 h after cooling in skim milk extenders containing egg yolk had higher percentages of PM spermatozoa (average 32%) than did spermatozoa frozen in extender containing skim milk alone (average 16%; P < 0.05). The percentages of PM spermatozoa frozen in 0.5- or 2.5-mL straws were similar (21 and 28%, respectively; P > 0.05). In the second experiment, spermatozoa were centrifuged to remove seminal plasma either before (25 degreesC) or after cooling (5 degreesC), and spermatozoa were frozen after being cooled to 5 degreesC for 2, 6, or 12 h. The percentages of PM spermatozoa were higher (P < 0.05) for spermatozoa centrifuged before cooling (30%) than for spermatozoa centrifuged after cooling (19%). Spermatozoa centrifuged at 25 degreesC then cooled for 12 h to 5 degreesC had higher (P < 0.05) post-thaw progressive motility (23%) compared to spermatozoa cooled for 12 h and centrifuged at 5 degreesC (13%). In the third experiment, spermatozoa were centrifuged for seminal plasma removal, resuspended at spermatozoal concentrations of 50,250 or 500 x 10(6)/mL, cooled to 5 degreesC for 12 h and then frozen. Samples with spermatozoa packaged at 50 or 250 x 10(6)/mL had higher (P < 0.05 percentages of PM spermatozoa (25 and 23%) after freezing than did samples packaged at 500 x 10(6) spermatozoa/mL (17%). We recommend that semen be centrifuged at 25 degreesC to remove seminal plasma, suspended to 250 x 10(6) spermatozoa/ml and held at 5 degreesC for 12 h prior to freezing.

[Evolution of serum gammaglutamyl transpeptidase activity in treated hyperthyroid and hypothyroid patients]. / Evolution de l'activité sérique gammaglutamyl transpeptidase (GGT) au cours des hyperthyroïdies et des hypothyroïdies traitées.

The pattern of gammaglutamyl transpeptidase levels was studied in the sera of 25 subjects with hyperthyroidism and 11 subjects with hypothyroidism, before and after treatment, and in 14 age- and sex-matched control subjects. Gammaglutamyl transpeptidase levels were significantly increased in hyperthyroidism (65 +/- 59 U/l) (p less than 0.01) and significantly decreased under treatment (40 +/- 27 U/l) (p less than 0.001). Before treatment, gammaglutamyl transpeptidase levels correlated with alkaline phosphatase levels and 5'-nucleotidase levels, the correlation persisting after treatment with 5'-nucleotidase. Alkaline phosphatase levels significantly increased under treatment (p less than 0.01). The percentages of gammaglutamyl transpeptidase variation correlated with thyroxine (r = 0.44, p less than 0.03), triiodothyronine (r = 0.47, p less than 0.02) and latent fixation capacity (r = 0.44, p less than 0.03) variations. Subjects with hypothyroidism had significantly decreased gammaglutamyl transpeptidase levels before treatment (18 +/- 9 U/l, p less than 0.01). Alkaline phosphatase levels were significantly decreased before treatment, and significantly increased after treatment. For all subjects with hyperthyroidism of hypothyroidism, the percentages of gammaglutamyl transpeptidase variations correlated with thyroxine (r = 0.48, p less than 0.003) and triiodothyronine (r = 0.39, p less than 0.016) variations. These results suggest that variations in gammaglutamyl transpeptidase levels in hyperthyroidism and hypothyroidism are, at least in part, in relation with variations in thyroid hormone levels.

Sensitive assay for cholesterol in biological membranes reveals membrane-specific differences in kinetics of cholesterol oxidase.

Quantification of cholesterol in biological membranes from a variety of sources is an important step toward understanding cholesterol's roles in membrane function. We extend to biological membranes the fluorometric/enzymatic approach (cholesterol oxidase) to measure cholesterol, originally described for whole cells (Heider and Boyett [1978] J. Lipid Res., 19:514-518; Gamble et al. [1978] J. Lipid Res., 19:1068-1070) and serum (Huang et al. [1975] Clin Chem., 21:1605-1608). This method has a detection limit of 0.3 microgram cholesterol. As revealed by comparison with high-performance liquid chromatography, the fluorometric/enzymatic method with biological membranes is accurate (within 4% and 8% for intestinal and hepatic plasma membranes, respectively). The assay may be completed within 3 to 4 hours and requires neither lipid extraction nor chromatographic techniques. Kinetics of the cholesterol oxidase reaction are membrane-specific, and first-order rate constants (k) are positively correlated with membrane order.

Peroxisomal beta-oxidation is a significant pathway for catabolism of fatty acids in a marine teleost.

Hepatic beta-oxidation is characterized in a marine teleost, Myoxocephalus octodecimspinosus, to determine mitochondrial and peroxisomal substrate selectivity as well as metabolic partitioning. Substrate selectivity is broad for peroxisomal beta-oxidation. Acyl CoA oxidase activities, with all unsaturated substrates measured, are at least 35% of activity with palmitoyl CoA (16:0), a saturated substrate. Mitochondrial selectivities are more pronounced. Carnitine palmitoyltransferase activity with a monounsaturate, palmitoleoyl CoA (16:1), is nearly 40% greater than activity with palmitoyl CoA, whereas activities with two polyunsaturates are < 10% of activity with the saturate. The presence of polyunsaturated acyl CoA esters inhibits up to 70% the oxidation of palmitoyl CoA by intact peroxisomes. Acyl CoA hydrolase activity is localized to peroxisomal fractions prepared by density-gradient centrifugation. Hydrolytic activity in these fractions is nearly twofold the activity of beta-oxidation. Estimates for metabolic partitioning suggest that at least 50% of hepatic beta-oxidation may be initiated by the peroxisomal compartment.

Substrate selectivities differ for hepatic mitochondrial and peroxisomal beta-oxidation in an Antarctic fish, Notothenia gibberifrons.

Hepatic mitochondrial and peroxisomal beta-oxidation were examined in an Antarctic marine teleost, Notothenia gibberifrons. Enzymic profiles and rates of beta-oxidation by intact organelles were determined by using a range of fatty acyl-CoA substrates to evaluate substrate preferences. Partitioning of beta-oxidation between organelles was estimated. Substrate selectivities are broader for peroxisomal beta-oxidation than for mitochondrial beta-oxidation. Mitochondria show marked preference for the oxidation of a monounsaturated substrate, palmitoleoyl-CoA (C16:1), and two polyunsaturates, eicosapentaenoyl-CoA (C20:5) and docosahexaenoyl-CoA (C22:6). Carnitine palmitoyltransferase activities with palmitoleoyl-CoA (C16:1) are 2.4-fold higher than activities with palmitoyl-CoA (C16:0). Most polyunsaturated acyl-CoA esters measured appear to inhibit by over 40% the oxidation of palmitoyl-CoA by peroxisomes. Our findings suggest that the polyunsaturates, eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), found in high concentrations in Antarctic fishes [Lund and Sidell (1992) Mar. Biol. 112, 377-382], are utilized as fuels to support aerobic energy metabolism. Metabolic capacities of rate-limiting enzymes and beta-oxidation rates by intact organelles indicate that up to 30% of hepatic beta-oxidation in N. gibberifrons can be initiated by the peroxisomal pathway.

A survey of helminth parasites of game animals in Kainji Lake National Park of Nigeria.

Between January and April 1983, the Borgu sector of the Kainji Lake National Park was visited and faecal droppings of several game animals belonging to the groups of Carnivora, Artiodactyla, Proboscidae and Primates were collected and processed for the presence of helminth ova and larvae. Faeces were also collected from the rectum of domestic ruminants which had grazed on the periphery of the park. Post mortem examinations were conducted on a baboon and an adult Monitor Lizard. Most of the game animals possessed multiple helminth infection but the egg-counts were generally low. Larvae of Haemonchus, Oesophagostomum and Trichostrongylus were common to wild and domestic ruminants while larvae of hook- and lungworms were mostly restricted to carnivores and primates. Some of the helminths found in the stomach and intestine of baboon during post-mortem were of public health interest.