The role of tendon stiffness in development of equine locomotion with age.

REASONS FOR PERFORMING STUDY: The flexor tendons support the metacarpophalangeal (MCP) and distal interphalangeal (DIP) joints during stance phase and since tendon stiffness and strain changes with age, it is likely that kinematics are also age-dependent. HYPOTHESIS: Maximum MCP and DIP angles decrease in the young horse, plateau in the mature horse and increase towards senescence. METHODS: The distal limbs of 57 walking horses age 3-212 months were filmed and digitised with an automated tracking system. Maximum MCP and DIP angles during stance phase were used to calculate strain in the superficial and deep digital flexor tendons. Horses were divided into 3 age groups; young (3-35 months), mature (36-99 months) and older horses (100-212 months). Pearson's correlation coefficients were calculated to determine the relationship between age and kinematics. RESULTS: Tendon strain decreased in young horses, stayed constant in mature horses and increased in older horses. Joint angles showed significant negative correlation in young horses, with coefficients of -0.88 (MCP) and -0.81 (DIP). In mature horses, correlations were not significant (P = 0.2 for MCP; P = 0.5 for DIP). In older horses, angles showed significant positive correlation, with coefficients of 0.62 (MCP) and 0.48 (DIP). CONCLUSIONS: Joint angles decreased in the young horse as tendon stiffness increases, remained constant in the mature horse where tendon stiffness is constant and increased in older horses as tendons weakens and stiffness decreases. Strain patterns were similar to those found in vitro. POTENTIAL RELEVANCE: Changing tendon stiffness appeared to influence the development and degeneration of gait. This has implications for studying musculoskeletal development, especially for identification of normal and pathological development.

Brain morphology and immunohistochemical localization of the gonadotropin-releasing hormone in the bluefin tuna, Thunnus thynnus.

The present study was focused on the morphology of the diencephalic nuclei (likely involved in reproductive functions) as well as on the distribution of GnRH (gonadotropin-releasing hormone) in the rhinencephalon, telencephalon and the diencephalon of the brain of bluefin tuna (Thunnus thynnus) by means of immunohistochemistry. Bluefin tuna has an encephalization quotient (QE) similar to that of other large pelagic fish. Its brain exhibits well-developed optic tecta and corpus cerebelli. The diencephalic neuron cell bodies involved in reproductive functions are grouped in two main nuclei: the nucleus preopticus-periventricularis and the nucleus lateralis tuberis. The nucleus preopticus-periventricularis consists of the nucleus periventricularis and the nucleus preopticus consisting of a few sparse multipolar neurons in the rostral part and numerous cells closely packed and arranged in several layers in its aboral part. The nucleus lateralis tuberis is located in the ventral-lateral area of the diencephalon and is made up of a number of large multipolar neurones. Four different polyclonal primary antibodies against salmon (s)GnRH, chicken (c)GnRH-II (cGnRH-II 675, cGnRH-II 6) and sea bream (sb)GnRH were employed in the immunohistochemical experiments. No immunoreactive structures were found with anti sbGnRH serum. sGnRH and cGnRH-II antisera revealed immunoreactivity in the perikarya of the olfactory bulbs, preopticus-periventricular nucleus, oculomotor nucleus and midbrain tegmentum. The nucleus lateralis tuberis showed immunostaining only with anti-sGnRH serum. Nerve fibres immunoreactive to cGnRH and sGnRH sera were found in the olfactory bulbs, olfactory nerve and neurohypophysis. The significance of the distribution of the GnRH-immunoreactive neuronal structures is discussed.

Effects of postexsanguination vascular infusion of carcasses with calcium chloride or a solution of saccharides, sodium chloride, and phosphates on beef display-color stability.

Hereford x Angus crossbred steers (n = 36) were stunned, exsanguinated, and infused via the carotid artery either with an aqueous solution containing 98.52% water, 0.97% saccharides, 0.23% sodium chloride, and 0.28% phosphates (MPSC; n = 12) or with 0.3 M CaCl2 (n = 12). The remaining 12 steers served as noninfused controls. At 48 h postmortem, the quadriceps muscles and subcutaneous fat were removed from the carcasses, frozen, and later made into ground beef (18 to 20% fat). The longissimus lumborum (LL), semimembranosus, and psoas major (PM) also were removed, vacuum packaged, aged until 14 d postmortem, and then one steak was sliced from each muscle for visual and instrumental color evaluations. The inside (ISM) and outside (OSM) portions of the SM were evaluated separately. The LL and OSM steaks from MPSC-infused carcasses had a lighter red (P < 0.05) initial appearance than steaks from the other treatments. The LL steaks from noninfused carcasses had the most (P < 0.05) uniform color; the MPSC treatment was intermediate, and the CaCl2 treatment was the most two-toned. Steaks from both infusion treatments had higher (P < 0.05) L* values for the LL, ISM, and OSM muscles compared with noninfused carcasses. In general, the LL from CaCl2-infused carcasses had lower (P < 0.05) a* values, saturation indices, and 630 nm to 580 nm reflectance values, and had larger (P < 0.05) hue angles. Infusion with MPSC increased (P < 0.05) hue angles in the LL and OSM. Display color stability was lowest (P < 0.05) for LL steaks from CaCl2-infused carcasses, whereas steaks from MPSC-infused carcasses were lighter red in initial color, but otherwise had display color stability similar to those from noninfused carcasses. No differences (P > 0.05) due to infusion were found for any color traits for the PM muscle and ground beef. Carotid artery vascular infusion of carcasses with CaCl2 resulted in undesirable meat colors, whereas the MPSC solution lightened loin and inside round color in a desirable way, but the color stability was slightly less compared to muscle from noninfused carcasses. Infusion effects were not consistent among muscles, and further research will be needed to determine what caused these differences.

Analyses of nuclear ldhA gene and mtDNA control region sequences of Atlantic northern bluefin tuna populations.

There has been considerable debate about whether the Atlantic northern bluefin tuna exist as a single panmictic unit. We have addressed this issue by examining both mitochondrial DNA control region nucleotide sequences and nuclear gene ldhA allele frequencies in replicate size or year class samples of northern bluefin tuna from the Mediterranean Sea and the northwestern Atlantic Ocean. Pairwise comparisons of multiple year class samples from the 2 regions provided no evidence for population subdivision. Similarly, analyses of molecular variance of both mitochondrial and ldhA data revealed no significant differences among or between samples from the 2 regions. These results demonstrate the importance of analyzing multiple year classes and large sample sizes to obtain accurate estimates when using allele frequencies to characterize a population. It is important to note that the absence of genetic evidence for population substructure does not unilaterally constitute evidence of a single panmictic population, as genetic differentiation can be prevented by large population sizes and by migration.

Effects of vascular infusion with a solution of saccharides, sodium chloride, and phosphates with or without vitamin C on carcass traits, Warner-Bratzler shear force, flavor-profile, and descriptive-attribute characteristics of steaks and ground beef from Charolais cattle.

Two groups of 18 grain-finished steers were utilized. Nine from one group were infused via the carotid artery immediately after jugular vein exsanguination with an aqueous solution containing saccharides, NaCl, and phosphates (MPSC; MPSC, Inc., Eden Prairie, MN, USA). Nine steers served as non-infused controls (CON). An additional 18 steers were infused with either MPSC (n=9) or MPSC plus 1000 ppm vitamin C (MPSC+C, n=9) solutions. Steers infused with MPSC had higher dressing percentages and organ weights than CON steers. Vascular infusion with MPSC had no effects on USDA yield or quality grade traits, descriptive-attribute sensory panel evaluations, or Warner-Bratzler shear force of longissimus lumborum and semitendinosus muscles. Vascular infusion with MPSC resulted in some significant, but inconsistent effects on flavor-profile characteristics of cooked beef. The addition of vitamin C to the MPSC solution did not provide any benefit.

Effects of postexsanguination vascular infusion of cattle with a solution of saccharides, sodium chloride, phosphates, and vitamins C, E, or C+E on meat display-color stability.

Grain-finished, high-percentage Charolais steers (n = 36) were selected for uniformity. Immediately after jugular vein exsanguination, 27 steers were infused at 10% of live weight via the carotid artery with a solution developed by MPSC, Inc. (St. Paul, MN) consisting of 98.52% water, 0.97% saccharides, 0.23% sodium chloride, and 0.28% phosphate blend plus either 500 ppm vitamin C (MPSC+C; n = 9), 500 ppm vitamin E (MPSC+E; n = 9), or 500 ppm vitamin C + 500 ppm vitamin E (MPSC+C+E; n = 9). Uninfused controls (CON) were exsanguinated conventionally. Carcasses were fabricated at 48 h postmortem. Longissimus thoracis (LT), psoas major (PM), and semimembranosus (SM) muscles were removed, vacuum-packaged, and stored at 2 degrees C until 14 d postmortem. Then, steaks 2.54 cm thick were sliced from the three muscles, placed on foam trays, and overwrapped with polyvinyl chloride film. Ground beef (GB) was formulated from the quadriceps femoris to contain 20% fat, mounded into 0.45-kg portions, placed on styrofoam trays, and wrapped with polyvinyl chloride film. Steaks were visually evaluated for uniformity and initial color on display d 0. Instrumental color measurements of L*, a*, b* and trained sensory panel color evaluations were obtained daily for 4 d (PM and GB) or 5 d (LT and SM) of display. No display time x treatment interaction existed for L*, a*, or b* values. The LT from CON cattle had more uniform color (P < 0.05) and was more cherry red than that from all infused cattle on d 0. Visual scores indicated that GB from MPSC+E cattle was more red (P < 0.05) than that from MPSC+C infused cattle throughout display, and GB from MPSC+E cattle was more red (P < 0.05) than that from CON cattle for the last 3 d of display. The vascular infusion solutions generally did not improve color or display-color stability of steaks, but the infusion solution with vitamin E did improve display-color stability of GB.

Novel HPLC analysis of tocopherols, tocotrienols, and cholesterol in tissue.

Tocopherols and tocotrienols are being increasingly recognized to have an important role in the prevention of atherosclerosis. It has been reported that they protect low-density lipoprotein (LDL) and tissues from oxidative stress and that tocotrienols can reduce plasma cholesterol levels. Two isocratic high-performance liquid chromatography (HPLC) methods for simultaneous analysis of tocopherols, tocotrienols, and cholesterol in muscle tissue were developed. Method A involves basic saponification of the sample, but causes losses of the gamma- and delta-homologs of vitamin E. Method B does not involve saponification, thereby protecting the more sensitive homologs. Both permit rapid analysis of multiple samples and neither requires specialized equipment. These methods may provide techniques useful in simultaneous assessment of oxidative stress status (OSS) and cholesterol levels.

Cell-mediated biotransformation of S-nitrosoglutathione.

Spontaneous release of nitric oxide (NO) from S-nitrosothiols cannot explain their bioactivity, suggesting a role for cellular metabolism or receptors. Using immortalised cells and human platelets, we have identified a cell-mediated mechanism for the biotransformation of the physiological S-nitrosothiol compound S-nitrosoglutathione (GSNO) into nitrite. We suggest the name "GSNO lyase" for this activity. GSNO lyase activity varied between cell types, being highest in a fibroblast cell line and lowest in platelets. In NRK 49F fibroblasts, GSNO lyase mediated a saturable, GSNO concentration-dependent accumulation of nitrite in conditioned medium, which was inhibited both by transition metal chelators, and by subjecting cells to oxidative stress using a combination of the thiol oxidant diamide and Zn2+, a glutathione reductase inhibitor. Activity was resistant, however, to both acivicin, an inhibitor of gamma-glutamyl transpeptidase (EC 2.3.2.2), and to ethacrynic acid, an inhibitor of Pi class glutathione-S-transferases (EC 2.5.1.18), thus neither of these enzymes could account for NO release. Although GSNO lyase does not explain the platelet-selective pharmacological properties of GSNO, cellular biotransformation suggests therapeutic avenues for targeted delivery of NO to other tissues.

Constitutional trisomy 8 as first mutation in multistep carcinogenesis: clinical, cytogenetic, and molecular data on three cases.

Three patients, with constitutional trisomy 8 mosaicism (CT8M), who developed a malignancy are reported. The diagnoses were refractory anaemia, acute lymphoblastic leukaemia, and idiopathic myelofibrosis. In the child with acute leukaemia, the CT8M was diagnosed at birth due to severe dysmorphisms and malformations; the other two patients showed a milder phenotype, and the CT8M was diagnosed only after the finding of trisomy 8 in neoplastic cells. The review of eight similar, previously reported cases and the clinical, cytogenetic, and molecular studies performed in our patients led us to make the following observations: (I) CT8M predisposes to neoplasms, preferentially to myelo- or lymphoproliferative diseases; (2) a gene dosage effect for glutathione reductase in red blood cells was seen in two of our patients; (3) the wide phenotypic variation of CT8M was confirmed: trisomy 8 in neoplastic cells of phenotypically near-normal cases may be misinterpreted as acquired; and (4) molecular studies suggested a postzygotic origin of the trisomy in our three cases, with the supernumerary chromosome being of paternal origin in one case and of maternal origin in the other two. We postulate that the trisomy 8 in neoplasms may often occur by mitotic nondisjunction in an early embryonic multipotent cell and that what is usually interpreted as an acquired trisomy 8 may in fact be CT8M. The constitutional trisomy 8 would act as a pathogenetically important first mutation in multistep carcinogenesis. Whenever trisomy 8 is found in malignancies, the patient should be reevaluated clinically to exclude CT8M, and CT8M patients should be monitored for the possible development of malignancies.

Biological effects of oxysterols: current status.

A review of relevant literature on biological activities of oxysterols (OS) and cholesterol is presented. The data clearly demonstrate manifold biological activities, often detrimental, for OS compared with little or no such activity of a deleterious nature for cholesterol itself. Cholesterol is perhaps the single most important compound in animal tissue and, as such, it is difficult to imagine it as a toxin or hazard. In contrast, OS exhibit cytotoxicity to a wide variety of cells leading to angiotoxic and atherogenic effects; alter vascular permeability to albumin; alter prostaglandin synthesis and stimulate platelet aggregation, an important process facilitating atherosclerosis and thrombosis; alter the functionality of low density lipoprotein (LDL) receptors, possibly stimulating hypercholesterolaemia; modify cholesteryl ester accumulation in various cells, inducing foam cell formation; and enrich the LDL particle in cholesteryl esters, possibly increasing its atherogenicity. Furthermore, OS are mutagenic and carcinogenic, although some have been studied as antitumour agents based on their cytotoxic properties. Moreover, numerous studies have implicated OS in membrane and enzyme alterations that are interrelated with many of the foregoing effects. The authors find that OS deserve much more attention than cholesterol itself in terms of research activity but that unfortunately the reverse is true with regard to funding.

Atherogenic and anti-atherogenic factors in the human diet.

New atherosclerosis causative factors and preventive modalities have been identified. Atherogenic factors include lipid oxidation products, such as cholesterol oxidation products, malonaldehyde and other aldehydes; trans-fatty acids; some saturated fatty acids (lauric, myristic and possibly palmitic acids); and myristic acid plus cholesterol. Lipid oxidation products are well suited to induce arterial damage, based on their known cytotoxic effects; evidence also indicates the possibility of plaque promotion and stimulation of thrombogenesis. Anti-atherogenic factors include antioxidants, fish oils and other polyunsaturates (if protected from oxidation), fibre and trace minerals such as copper, manganese, selenium and zinc. Iron is unique, being considered as both a potential promoter of atherosclerosis (component of ferritin, conceivably inducing lipid oxidation) and a possible anti-atherogenic component (of antioxidant enzyme catalase). It is apparent that an entire new series of research challenges has been uncovered.

HPLC method for quantitation of cholesterol and four of its major oxidation products in muscle and liver tissues.

A fast, sensitive, high performance liquid chromatographic method was developed for the quantitation of cholesterol and four of its major oxidation products: 3 beta-hydroxycholest-5-en-7-one (7-ketocholesterol), cholest-5-ene-3 beta, 7 alpha-diol (7 alpha-hydroxycholesterol), cholest-5-ene-3 beta, 7 beta-diol (7 beta-hydroxycholesterol), and cholest-5-ene-3 beta,25-diol (25-hydroxycholesterol). In this procedure 2:1 chloroform:methanol (v/v) extracts of tissue homogenate were combined, dried over anhydrous Na2SO4, filtered, evaporated to dryness under N2 and dissolved with a mobile phase of either 97:3 or 93:7 hexane:isopropanol (v/v). After membrane filtration and without further purification, aliquots were directly injected onto a 10-microns pore size, 30 X 0.39 cm mu-Porasil normal phase column. The separation of cholesterol and its oxidation products was monitored by a UV detector at 206 and 233 nm. This method was successfully applied to pork muscle as well as mouse liver tissues and was able to detect cholesterol oxidation products (COP) in the ppm range. The identity of the COP was confirmed by mass spectroscopy.

Capillary GC quantification of cholesterol oxidation products in plasma lipoproteins of fasted humans.

Cholesterol oxidation products have been hypothesized to be important factors in atherosclerosis, a process which can culminate in myocardial infarction. The relative importance of exogenous or in vivo sources of cholesterol oxidation products has not been determined. However, methodology used for cholesterol oxidation products analysis of foods is applicable to the determination of cholesterol oxidation products in human plasma lipoproteins. Such methodology, outlined in this report, permits numerous critical experiments to be conducted on the possible role of cholesterol oxidation products in coronary heart disease.

Quantification of Cholesterol Oxidation Products in a Variety of Foods.

Cholesterol oxidation products (COPS) were estimated in a variety of foods by gas chromatography. Substantial quantities of α- and ß-epoxide (2 to 46 and 0 to 111 ppm, respectively) and lesser quantities of 7ß-hydroxy- and 7-ketocholesterol were found in powdered egg products. Other foods found to contain COPS included dehydrated chicken, turkey and beef (0 to 43 ppm); and Cheddar, Blue, Romano and Parmesan cheese powders (0 to 17 ppm). Powdered infant formulas and dehydrated baby foods displayed several peaks coinciding to COPS but none were confirmed by mass spectroscopy. COPS were either not detected or noted at < 15 ppm in fresh dairy products.

Temperature-dependent abnormalities of the erythrocyte membrane in porcine malignant hyperthermia.

The temperature dependence of ATPase activities and stearic acid spin label motion in red blood cells of normal and MH-susceptible pigs have been examined. Arrhenius plots of red blood cell ghost Ca-ATPase and calmodulin-stimulable Ca-ATPase activities were identical for both normal and MH erythrocyte ghosts. Arrhenius plots of Mg-ATPase activity exhibited a break (defined as a change in slope) at 24 degrees C in both MH and normal erythrocyte ghosts. However, below 24 degrees C the apparent activation energy for this activity was less in MH than normal ghosts. To determine whether breaks in ATPase Arrhenius plots could be correlated with changes in the physical state of the red blood cell membrane, the spin label 16-doxyl-stearate was introduced into the bilayer of both erythrocyte ghosts and red blood cells. With both ghosts and intact cells, at each temperature examined, the mobility of the probe in the lipid bilayer, as measured by electron paramagnetic resonance, was greater in normal than in MH membranes. While there were no breaks in Arrhenius plots for probe motion in the erythrocyte ghosts, the apparent activation energy for probe motion was significantly greater in normal than in MH ghost membranes. While there was no break in the Arrhenius plot of probe motion in normal intact red blood cell membranes, there were breaks in the Arrhenius plot of probe motion at both 24 and 33 degrees C in intact MH red blood cell membranes. Based on the altered temperature dependence of Mg-ATPase activity and spin probe motion in membranes derived from MH red blood cells, we conclude that there may be a generalized membrane defect in MH pigs which is reflected in the red blood cell as an altered membrane composition or organization.

Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia.

To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg2+-ATPase activities, the (Na+, K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca2+-ATPase activity exhibited low- and high-affinity Ca2+-binding sites in both types of erythrocyte ghost. While the Km for Ca2+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca2+-binding site, the reverse was true at the low-affinity Ca2+-binding site. Irrespective of the type of calcium binding site occupied, the Vmax for normal erythrocyte ghost Ca2+-ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca2+ or the Vmax of the Ca2+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are unlikely to result in an impaired ability of MHS erythrocytes to regulate their cytosolic Ca2+ concentration.

Occurrence of lipid oxidation products in foods.

Lipid oxidation products are ubiquitous in foods, although much variation exists in the levels present. Although these levels are generally low, the problem of lipid oxidation severely compromises the quality of some foods and limits the shelf-life of others. Lipid oxidation represents a key barrier in the development of new food products and processes, especially convenience items and processes required to manufacture them. Deleterious changes in foods caused by lipid oxidation include loss of flavour, development of off-flavours, loss of colour, nutrient value and functionally, and the accumulation of compounds which may be detrimental to the health of consumers. All foods that contain lipids are susceptible to oxidation but especially affected are foods which are dehydrated, subjected to high temperatures or cooked and subsequently stored, e.g. dehydrated eggs, cheeses and meats, foods fried in frying oils, and cooked (uncured) meats. Specific examples of compounds which are of health concern include lipid peroxides and the free radicals involved in their formation and propagation, malonaldehyde, and several cholesterol oxidation products. Coronary artery disease (CAD) may be in part caused by the consumption of lipid oxidation products.

Capillary column gas-liquid chromatographic resolution of oxidized cholesterol derivatives.

Fused-silica capillary columns were evaluated for the resolution of oxidized cholesterol derivatives. Thermal instability of diol derivatives, epimeric 7 alpha- and 7 beta-hydroxy, 4 beta-hydroxy, and 25-hydroxycholesterol, was observed during gas chromatography. After derivatization as trimethylsilyl ethers the foregoing diols, alpha-epoxide, cholestane-triol, 7-ketocholesterol, and cholesta-3,5-dien-7-one were completely resolved on a DB-1 column. Each oxidized sterol revealed excellent response linearity as the trimethylsilylated sterol, enabling reliable quantification. The identity of each derivatized sterol was confirmed by mass spectrometry.