Amino acid utilization by the hindlimb of warmblood horses at rest and following low intensity exercise.

BACKGROUND: In particular branched-chain amino acids might limit muscle protein loss in pathological conditions. Little is known on basic amino acid utilization of muscle in horses. OBJECTIVE: To assess amino acid utilization by the hindlimb of horses at rest and following low intensity exercise. ANIMALS & METHODS: Amino acid uptake by the hindlimb was investigated using the arteriovenous difference technique. Blood from six warmblood mares (mean age 12 ± 3 (SD) years and weighing 538 ± 39 kg) was collected simultaneously from the (transverse) facial artery and from the caudal vena cava. Food was withheld for 12 hours prior to exercise. Exercise comprised a standardized treadmill protocol consisting of 5 minutes of walk, 20 minutes of trot, and thereafter another 5 minutes of walk. Amino acids were determined quantitatively by means of anion exchange chromatography. Statistical analysis was performed using a general linear mixed model. RESULTS: Amino acids with the largest average extraction at rest were citrulline (11.1 ± 9%), cystine (8.3 ± 36%), serine (7.9 ± 11%), and leucine (5.9 ± 9%). Of the 25 amino acids studied, none showed a significant difference following exercise. Glycine (485 ± 65 µmol/L), glutamine (281 ± 40 µmol/L), valine (183 ± 26 µmol/L), and serine (165 ± 22 µmol/L) showed highest plasma concentrations. The average extraction for α-aminobutyric acid at rest was 18.2 ± 26%. Arterial plasma citrulline concentration was higher than venously. CONCLUSION: Citrulline, cystine, serine, and leucine might be regarded as most important amino acids at rest in warmblood mares. CLINICAL IMPORTANCE: Further investigation is necessary into the specific role of leucine supplementation to preserve or restore body protein in horses.

Cryopreservation with α-tocopherol and Sephadex filtration improved the quality of boar sperm.

The objectives were to evaluate postthaw sperm quality and the response to an inducer of in vitro sperm capacitation in boar sperm, cryopreserved with (T) or without (C) α-tocopherol. Boar sperm frozen in 0.2-mL pellets were thawed and washed (W) or selected by three methods: Percoll discontinuous gradient (PS) or Sephadex (Sigma-Aldrich, St. Louis, MO, USA) (neutral [S] or with ion exchange [S+IO] columns). All separation methods enhanced sperm motility, plasma membrane integrity, and functionality and acrosome integrity for both C and T samples (P < 0.05). The best results were obtained with S and ionic Sephadex column. There was a decrease (P < 0.05) in capacitation-like changes in C samples separated with Sephadex (W: 19 ± 0.9%, PS: 22 ± 2.5%, S: 17 ± 1.2%, and S+IO: 17 ± 2.0%). Cryopreservation with α-tocopherol decreased (P < 0.05) the percentage of cryocapacitated sperm (W: 14 ± 0.7%, PS: 14 ± 1.0%, S: 13 ± 1.0%, and S+IO: 14 ± 0.9%) compared with C samples, without differences among selection techniques. Freezing with α-tocopherol and subsequent selection decreased lipid peroxidation (W: 20.79 ± 2.64 nmol thiobarbituric acid reactive substances (TBARS)/10(8) sperm; PS: 13.15 ± 2.39 nmol TBARS/10(8) sperm; S: 13.20 ± 2.18 nmol TBARS/10(8) sperm, and S+IO: 13.62 ± 2.76 nmol TBARS/10(8) sperm), with respect to washed and selected C samples (W: 37.69 ± 5.34 nmol TBARS/10(8) sperm, PS: 25.61 ± 5.85 nmol TBARS/10(8) sperm, S: 19.16 ± 3.28 nmol TBARS/10(8) sperm, and S+IO: 22.16 ± 6.09 nmol TBARS/10(8) sperm). In vitro capacitation levels were significantly higher for neutral Sephadex-selected T samples in comparison with C and unselected samples. These results were confirmed with a follicular fluid-induced acrosome reaction. In conclusion, cryopreserved sperm with α-tocopherol and subsequent Sephadex selection, improved postthaw quality and functionality of boar sperm, which could be useful for assisted reproductive techniques.

Erratum: Purification of a toxic cysteine protease produced by pathogenic Aeromonas hydrophila isolated from rainbow trout.

An extracellular lethal toxin produced by Aeromonas hydrophila strain RT860715K originally isolated from diseased rainbow trout (Oncorhynchus mykiss) was purified by using Fast Protein Liquid Chromatography system with hydrophobic interaction chromatography and anion exchange columns. The toxin was a cysteine protease, inhibited by L-cysteine, iodoacetic acid, N-ethylamleimide, P-chloromercuibenzene-sulfonic acid and N-α-p-tosyl-1-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0. The molecular weight of the purified enzyme proved to be 94 kDa as estimated by SDS-PAGE. In addition, the toxin was also completely inhibited by HgCl2 but partially inhibited by ethylenediamine tetraacetic acid (EDTA) and CuCl2. Both the extracellular products of Aeromonas hydrophila RT860715K and the purified protease were lethal to rainbow trout (weighing 18 g) with LD50 values of 2.87 and 0.93 µg protein g-1 fish body weight, respectively. The addition of L-cysteine completely inhibited the lethal toxicity of the purified protease, indicating that this cysteine protease was a lethal toxin produced by the bacterium.

Progesterone and the distribution of pituitary gonadotropin isoforms in cattle.

The objective of the present study was to determine the relative proportion of gonadotropin isoforms in bovine pituitary glands affected by progesterone. Twelve postpubertal heifers (Swiss-Zebu) were assigned to three groups (n=4): intact animals in the luteal phase of the estrous cycle (diestrus group); ovariectomized heifers with (OVXP) or without progesterone treatment (OVX). Prior to pituitary gland collection, a blood sample was taken from each animal to determine the circulating progesterone concentration. Pituitary protein extractions processed by chromatofocusing were eluted with a pH gradient ranging from 10.5 to 3.5. The LH and FSH eluent was grouped on the basis of the following three criteria: (1) as either a basic (pH>or=7.5), neutral (pH 7.4-6.5) and acid (pHor=10.5-3.5); (3) on the basis of distinct isoforms 12 peaks of which (A-L) were identified for LH and 11 (I-XI) for FSH. The analysis by range of pH and by pH of elution in the OVX and OVXP groups showed no difference in the LH and FSH isoform ratio, but diestrus cattle differs having a greater ratio (p<0.05) of basic LH isoforms (87.5+/-0.4%) and lesser ratio (p<0.05) of acid isoforms (5.4+/-0.7%). In the diestrus group, the ratio of acid FSH isoform increased (62.1+/-1.7%), while neutral isoforms decreased (5.7+/-0.4%, P<0.05). The analysis by isoform type of LH revealed a greater proportion of isoforms C (pH 9.4) and E (pH 9.0) in the groups with circulating progesterone when compared to the OVX group. The heterogeneity of FSH was quantitatively similar in most isoforms in the three groups, with the exception of the predominant isoform (VIII, pH 4.9) that was more abundant in the diestrus group (p<0.05). These results indicate that progesterone with other gonad factors influence the pituitary glicosylation altering the relative proportions of gonadotropin isoforms.

Partial characterization and isolation of 130 kDa antigenic protein of Dicrocoelium dendriticum adults.

The study focused on characterizing and isolating Dicrocoelium dendriticum antigens or their fractions that could be used for the immunological diagnosis of dicrocoeliosis. Somatic (SoAg) and excretory-secretory antigens (ESAg) were analyzed by SDS-PAGE and their specificity was evaluated by Western blot with homologous and heterologous sera. The antigens were partially purified by chromatographic techniques of gel-filtration (Sephacryl S-300) and ion exchange (Hitrap-DEAE-Sepharose). Western blot analysis using sera of ovine infected with D. dendriticum revealed eight main antigenic polypeptides ranging from 24 to 205 kDa for SoAg and seven for ESAg with apparent molecular mass in the range of 26-205 kDa. We detected a specific parasite protein with an approximate molecular weight of 130 kDa in SDS-PAGE gels, arranged as a 450 kDa tetramer in native conditions. It also showed strong immunoreactivity by Western blot against ovine sera experimentally infected with D. dendriticum. Gel filtration chromatography (Sephacryl S-300) also showed other specific proteins, one of about 24 kDa in SoAg and another of about 42 kDa in ESAg. The elution conditions of 450 kDa protein (130 kDa monomer) by DEAE chromatography were similar to those from the somatic antigen (pH 7.2, 0.1M NaCl, in 29-34 ml fractions) and from the excretion-secretion antigen (pH 8.0, 0.1M NaCl, in 29-35 ml fractions). The suitability of 130 kDa polypeptide for D. dendriticum infection diagnosis was confirmed by Western blot using a pool of sera as well as individual serum samples from experimentally infected sheep. The sequence of amino termini of 130 kDa polypeptide from both fractions was the same and identical to that reported for a peptide from D. dendriticum described as a globin. This sequence also revealed an appreciable similarity with the amino end of globins from some phylogenetically related worms.

Pattern of circulating luteinizing hormone isoforms during the estrous and luteal phases in Holstein heifers.

The pattern of distribution of circulating luteinizing hormone (LH) isoforms in cattle during estrus and the luteal phase was investigated. In each stage, the stage of the estrous cycle was synchronized in seven Holstein heifers with a prostaglandin analogue. After estrus was detected, blood samples were taken at 2-h intervals for 24h. In the luteal phase, animals received 250 microg i.v. of GnRH and blood samples were collected every 15 min for 5h. LH concentration in the samples was determined. Samples with the greatest LH concentration in estrus (pre-ovulatory peak) and those collected 60 min after GnRH administration (luteal phase) were analyzed by chromatofocusing, eluted with a pH gradient from 10.5 to 3.5. Eluted LH was grouped into basic (pH > or = 7.5), neutral (pH 7.4-6.5) and acidic isoforms (pH < or = 6.4) as well as by pH unit. In both phases, basic forms were the most abundant, and these were greater (P < 0.05) during the luteal phase (78.4 +/- 4.2%) as compared with during estrus (57.1 +/- 6.2%); the proportion of neutral and acidic isoforms in estrus (13.7 +/- 2.6%; 28.5 +/- 2.8%) was greater (P < 0.05) as compared with the luteal phase (3.0 +/- 0.7; 18.7 +/- 3.4). These results indicate that the relative proportion of LH isoforms secreted by the adenohypophysis differ by stage of estrous cycle. The addition of excess of NaCl to the column modifies the antigen-antibody binding in the RIA, and the proteins eluted are erroneously quantified as LH; this is an artifact of the technique.

Feline dirofilariosis: antibody response to antigenic fractions containing specific 20 to 30 kDa polypeptides from the adult Dirofilaria immitis somatic antigen.

Fractions from the adult somatic antigen (SA) Dirofilaria immitis complex, containing polypeptides from 20 to 30kDa, previously identified as molecular markers of feline dirofilariosis are isolated by sequential application of gel filtration and anion exchange chromatography. Indirect enzyme-linked immunosorbent assays, employing these fractions (20-26kDa/ELISAF1 and 30kDa/ELISAF7) show multivalent diagnostic capacities: they were able to detect pre-patent infections 2 months after infection, infections in clinical phase, and the fall of antibodies after the worms were removed from the heart, or the application of a ivermectin treatment. The results obtained by the two tests correlated well, in spite of the fact that ELISAF1 was most useful to detect antibodies in sera from cats in the clinical phase, while ELISAF7 has more sensitivity for the early detection of the infections. Both ELISAs were useful in the detection of the decrease of antibodies after the worms were removed by surgery or pharmacological treatment.

Functional expression of the catalytic domains of two cysteine proteinases from Trypanosoma congolense.

The catalytic domains of two closely related cysteine proteinases (CP1 and CP2) from Trypanosoma congolense, referred to as C1 and C2, were expressed as proforms in Escherichia coli (C1) and in the baculovirus system (C1 and C2). While the bacterial expression system did not allow recovery of active C1, the baculovirus system led to secretion of inactive zymogens which could be processed at acidic pH into mature enzymes. Active C1 and C2 were purified from serum-free culture supernatants by anion-exchange chromatography and characterised. Their kinetic parameters and pH activity profiles confirmed the relatedness between C2 and native CP2 (congopain). These properties also underline major functional differences between C1 and C2, that appear to relate to discrete but essential sequence differences. It is likely that these two enzymes perform distinct roles in vivo, in the parasite and/or in the host-parasite relationships.

Heterogeneity of caprine kappa-casein macropeptide.

The heterogeneity of caprine caseinmacropeptide (CMP) was determined by means of treatments with neuraminidase and acid phosphatase and analyses by anion exchange FPLC and reversed-phase (RP)-HPLC, with on-line and off-line electrospray ionization mass spectrometry. The main CMP components were two non-glycosylated and di-phosphorylated forms, as well as two other mono-phosphorylated species, each corresponding to a genetic variant of caprine kappa-casein due to the silent substitution Ile/Val at position 119. Asialo-aglyco mono- and di-phosphorylated forms were found in the ratios 8-14% and 86-92%, respectively. Approximately 36% of caprine CMP was glycosylated. Based on the obtained molecular masses, the occurrence of tri-, di- and monosaccharide-containing di-phosphorylated CMP are reported, assuming that N-acetylgalactosamine, galactose, N-acetyl and N-glycolylneuraminic acids would constitute the main monosaccharides of caprine CMP. CMP microheterogeneity due to the genetic polymorphism was also observed in the glycosylated forms.

Can betaine partially replace or enhance the effect of methionine by improving broiler growth and carcase characteristics?

1. Growth rates and carcase characteristics were measured in male broiler chickens fed on a control diet deficient in methionine (c. 2.8 g/kg methionine) or a series of diets containing graded levels of betaine or DL-methionine or both additives. 2. We aimed to answer 2 main questions. First, can betaine replace part of the methionine in a broiler ration? Secondly is there a synergism between methionine and betaine? 3. Birds given the control diet or that supplemented only with betaine ate less, grew more slowly, had higher food convension ratio (FCR) and varied more in mass at 42 d than birds fed diets with DL-methionine. Adding 1.2 g/kg DL-methionine to the control ration produced the heaviest birds at 42 d (2500 g) with the 2nd heaviest breast muscle (366 g). 4. After correcting for treatment differences in body mass (analysis of convariance), birds fed on the control diet and the diet supplemented with betaine only, had relatively lighter breast muscles but relatively heavier abdominal fat pads than those of birds given diets supplemented with DL-methionine. However, adding betaine to diets containing added methionine further improved the relative breast muscle yield. 5. After correcting for differences in body mass between treatments, birds fed on diets containing most methionine had lighter viscera than birds fed diets deficient in methionine. This demonstrated gut plasticity, suggesting that the viscera enlarged to sequester methionine from low-methionine diets. 6. Our data refute the hypothesis that betaine can substitute for methionine in broilers fed diets that are marginally deficient in methionine plus cystine. However, betaine may improve carcase composition, especially breast meat yield.

Purification of bovine urinary beta2-microglobulin and its biochemical characteristics.

In this study, bovine beta2-m was purified from urine by ion-exchange chromatography and gel chromatography, and the characteristics were compared with those of colostral beta2-m by the immunological reactivity, isoelectric points, peptide map, and amino acid sequence. The characteristics of purified urinary beta2-m were consistent with those of the colostral beta2-m. The urinary and colostral beta2-m possessed the same polypeptide chain consisting of 98 amino acids, and its molecular weight is 11.8 kDa. Furthermore, four isoforms of beta2-m were found. The isoelectric points were different from each other.

Ionic strength-induced inactivation of mu-calpain in postmortem muscle.

The present study was conducted to study the stability of autolyzed mu-calpain activity and determine whether measurement of mu-calpain activity after anion exchange chromatography accurately reflects its activity in postmortem muscle. Ionic strength and pH affected the stability of partially autolyzed mu-calpain. Complete loss of activity was observed as a result of binding of autolyzed mu-calpain to DEAE-Sephacel when the large subunit of mu-calpain was autolyzed from 80 to 76 kDa. Therefore, determination of mu-calpain by standard anion exchange chromatography may underestimate mu-calpain activity in postmortem muscle. The activity of autolyzed mu-calpain was stabilized by inclusion of glycerol in the buffers, and this permitted us to investigate whether the apparent loss of mu-calpain activity in postmortem muscle is an artifact of the methodology. Despite the inclusion of glycerol in the buffers, a decrease in mu-calpain activity was observed during postmortem storage of muscle, even though the autolyzed enzyme was readily detectable by Western blotting in muscle extracts and column eluates. This result indicates that instability of autolyzed mu-calpain is a major cause for the decline in mu-calpain activity in postmortem muscle.

Biological and biochemical characterization of isolates of Trypanosoma evansi from Pantanal of Matogrosso--Brazil.

Ten isolates of Trypanosoma evansi from the Pantanal region of Brazil, recently derived from coati (Nasua nasua, carnivora, Procyonidae), horses and dogs, were characterized on the basis of biological (experimental infections in Wistar rats) and biochemical (multilocus enzyme eletrophoresis) data. Biological data were analyzed by Nested analysis of variance and Kruskal-Wallis. Marked heterogeneity in virulence was observed in the isolates. Some of the isolates showed an undulating parasitaemia, typical for African trypanosomes. This biological heterogeneity did not correspond with the biochemical homogeneity observed in the T. evansi isolates. T. evansi has one of the widest distributions and greatest range of mammalian hosts and is widely recognized to have evolved from Trypanosoma brucei. Adaptability of T. evansi was not reflected in the variability of biochemical and molecular parameters studied to date. The variability in virulence was very significant, but not correlated with the host from which it was derived. These data suggested that, in the region studied, T. evansi is transmitted among both domestic and sylvatic animals in one single transmission cycle.

Purification and embryotropic roles of tissue inhibitor of metalloproteinase-1 in development of "HanWoo" (Bos taurus coreanae) oocytes co-cultured with bovine oviduct epithelial cells.

This study was conducted to purify a tissue inhibitor of metalloproteinase (TIMP)-1 in a serum-free medium conditioned with bovine oviduct epithelial cells (BOEC) and to evaluate its effect on development of "HanWoo" (Bos taurus coreanae) embryos to the blastocyst stage. In the first study using SDS-PAGE electrophoresis, the presence of 32 kDa proteins, which contains TIMP-1, was detected in the medium conditioned with BOEC, and TIMP-1 was then purified from the medium by gel filtration and HPLC techniques. When examined TIMP-1 secretion, fluorescent foci indicating the secretion of TIMP-1 were found after stained BOEC with fluorescein isothiocyanate. In the next experiment, two-cell embryos derived from in vitro-fertilization were cultured in a serum-free medium, to which 0, 1.25, 2.5 or 5 microg/ml of purified TIMP-1 was supplemented. More (P<0.05) embryos developed to the morula and blastocyst stages after the addition of 2.5 microg/ml to culture medium than after no addition. In conclusion, our data indicate that BOEC secrete TIMP-1 and this glycoprotein promotes the prehatched development of "HanWoo" embryos derived from in vitro-fertilization.

Canine rheumatoid arthritis and inflammatory cytokines.

Bioassays were developed to detect canine pro-inflammatory cytokines. These enabled characterisation of these cytokines and their isoforms and provided means for their assay in the joints of dogs with different naturally occurring arthropathies. Canine IL-1 was detected by its induction of proliferation of D10(N4)M cell line, whilst IL-6 had a proliferative effect on B9 cell line. TNFalpha had a cytotoxic effect on WEHI 164 (13) cells. Partial purification of the cytokines was achieved by FPLC ion-exchange chromatography and two isoforms of IL-1 were shown, possibly corresponding to IL-1alpha and IL-1beta. TNFalpha only appeared as one isoform whereas IL-6 showed at least five isoforms, possibly corresponding to the other molecules in the IL-6 family, such as IL-11 and oncostatin M. Analysis of synovial fluids from dogs with osteoarthritis (OA) and rheumatoid arthritis (RA) showed that IL-1 and TNFalpha bioactivity was not readily detectable at increased levels in diseased joints but that IL-6 was significantly increased in both diseases. It is now important to determine the role of IL-6 in OA and RA in the dog, particularly in the induction of proteolytic enzymes which lead to cartilage loss.

Purification and characterization of protease produced by Staphylococcus aureus isolated from a diseased chicken.

A protease produced by Staphylococcus aureus, isolated from a chicken suffering from dermatitis, was purified by successive precipitation with ammonium sulfate, ion-exchange chromatography on Q-Sepharose FF, Sp-Sepharose FF and Mono-Q columns. By Mono-Q column chromatography, two proteases (protease 1 and 2) were obtained. The molecular weights of protease 1 and 2 were estimated at 23.1 and 22.7 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. Their isoelectric points were 5.85 and 5.55, respectively, and they possessed antigenic similarity when examined by the immunoblotting. The N-terminal amino acid sequences of both the proteases were identical (RAQYVNQLKNFKIRETQ). The activities of both the proteases were strongly increased by reducing agents such as L-cysteine and sodium thioglycolate. Their activity was inhibited by thiol protease inhibitors, but was not inhibited by metalloprotease or serine protease inhibitors. From the results, it seems likely that these proteases, produced by S. aureus from diseased chickens, might belong to the thiol protease group.

Dietary protein restriction stress in the domestic fowl (Gallus gallus domesticus) alters adrenocorticotropin-transmembranous signaling and corticosterone negative feedback in adrenal steroidogenic cells.

Previous work with growing chickens (Gallus gallus domesticus) indicates that transient dietary protein restriction induces long-term enhancement of adrenal steroidogenic function in response to adrenocorticotropin (ACTH). The present study investigated two possible cellular functions mediating this enhanced response: (a) ACTH signal transduction and dissemination and (b) short-loop feedback inhibition of ACTH-induced corticosterone production by exogenous corticosterone. Cockerels (2 weeks old) were fed isocaloric synthetic diets containing either 20% (control) or 8% (restriction) soy protein for 4 weeks. Adrenal glands were processed for the isolation of adrenal steroidogenic cells nearly devoid of chromaffin cells ( approximately 90% adrenal steroidogenic cells). Results of experiments to assess signal transduction and dissemination indicated that protein restriction selectively enhanced ACTH-induced corticosterone production mediated by the cyclic AMP (cAMP)-dependent pathway. In addition, protein restriction substantially counteracted exogenous corticosterone-dependent inhibition of acute ACTH-induced corticosterone production (by 40.7% vs control). The proximal portion of the cAMP pathway seemed most affected by this stressor. Protein-restricted cells exhibited enhanced homologous sensitization to ACTH (136% greater than that of control cells) which appeared to be localized at a step(s) prior to or at the formation to cAMP. Also, maximal ACTH-induced cAMP production and sensitivity to ACTH in terms of cAMP production by protein-restricted cells were, respectively, 2.2 and 15.8 times those of control cells. However, variable results were obtained from other experiments designed to pinpoint the altered early steps in ACTH-transmembranous signaling. For example, with intact cells, cAMP responses to cholera toxin (CT) and forskolin (FSK) did not corroborate the results suggesting an augmentation of ACTH-signal transduction induced by protein restriction. Furthermore, basal and stimulatable (by ACTH, CT, FSK, and NaF) adenylyl cyclase activities from membranes from protein-restricted cells were, respectively, 47.2 and 40.2% less than those from control cells (normalized to 10(7) cell equivalents of crude membranes). Collectively, these findings suggest that protein restriction stress potentiates ACTH-induced corticosterone secretion by chicken adrenal steroidogenic cells in at least two ways: (1) on the proximal end, by modulating unknown factors which enhance cellular sensitivity to ACTH, ACTH receptor-adenylyl cyclase coupling, and adenylyl cyclase activity, and (2) on the distal end, by suppressing end-product corticosterone negative feedback, thus facilitating an increase in net corticosterone secretion.

Development and application of a compartmental model of 3-methylhistidine metabolism in humans and domestic animals.

Measurement of urinary 3-methylhistidine (3MH) excretion is the primary in vivo method to measure skeletal muscle (myofibrillar) protein breakdown. This method requires quantitative collection of urine and is based on the assumption that no metabolism of 3MH occurs once it is released from actin and myosin. This is true in most species, but in sheep and swine a proportion is retained in muscle as a dipeptide, balenine. In neither of these species does urine 3MH yield any data on the metabolism of 3MH. We have conducted studies that propose that 3MH metabolism in humans, cattle, dogs, swine, and sheep can be defined from a single bolus infusion of a stable isotope 3-[methyl-2H3]-methylhistidine. Following the bolus dose of the stable isotope tracer, serial blood samples and/or urine was collected over three to five days. A minimum of three exponentials were required to describe the plasma decay curve adequately. The kinetic linear-time-invariant models of 3MH metabolism in the whole animal were constructed by using the SAAM/CONSAM modeling program. Three different configurations of a three-compartment model are described: (A) A simple three-compartment model for humans, cattle, and dogs, in which plasma kinetics (3-[methyl-2H3]-MH/3MH) are described by compartment 1 and with one urinary exit from compartment 1. (B) A plasma-urinary kinetic three-compartment model with two exits was used for sheep with a urinary exit out of compartment 1 and a balenine exit out of a tissue compartment 3. (C) A plasma three-compartment model was used in swine with an exit out of a tissue compartment 3. The kinetic parameters reflect the differences in known physiology of humans, cattle, and dogs as compared to sheep and swine that do not quantitatively excrete 3MH into the urine. Steady-state model calculations define masses and fluxes of 3MH between three compartments and, importantly, the de novo production of 3MH. The de novo production of 3MH for humans, cattle, dogs, sheep, and swine are 3.1, 6.0, 12.1, 10.3, and 7.2 mumol x kg-1 x d-1, respectively. The de novo production of 3MH as calculated by the compartmental model was not different when compared to 3MH production as calculated via traditional urinary collection. Additionally, data suggest that steady-state compartment masses and mass transfer rates may be related to fat free mass and muscle mass in humans and swine, respectively. In conclusion, models of 3MH metabolism have been developed in numerous species, and these models can be used for the assessment of muscle proteolysis and 3MH kinetics without the collection of urine. This methodology is less evasive and will be useful in testing further experimental designs that alter myofibrillar protein breakdown.

Order of amino acid limitation in meat and bone meal.

Three experiments were conducted to determine the order of amino acid (AA) limitation in meat and bone meal (MBM) using AA addition and deletion assays. In two addition assays, various individual and combined additions of eight AA were made to semipurified basal diets containing 16% protein solely from a MBM. The two MBM had previously been determined to vary greatly in protein quality. In the deletion assay, a semipurified basal diet containing 13.5% CP provided solely by MBM was supplemented with L-Thr, L-Val, DL-Met, L-Leu, L-Ile, L-Phe, L-Tyr, L-Lys-HCl, L-His, and L-Trp to fulfill the Illinois Ideal Protein or AA pattern on a digestible basis. Each AA was then deleted individually from the basal diet, and the effect of its deletion on growth performance was assessed. In both addition assays, supplementation of the MBM basal diet with Trp and Cys together yielded a large increase in growth performance, whereas supplementation with these AA individually had no effect. These results indicated that Trp and sulfur AA (with a primary need for Cys) were equally first limiting in the MBM. The order of limitation for the other AA was unclear. The results of the deletion assay showed that deletion of Trp, Thr, Phe + Tyr, Ile, Met, Lys, Val, or His significantly depressed growth performance. The results of the combined addition and deletion experiments indicated that the order of AA limitation in MBM was 1) Trp and sulfur AA, 2) Thr, 3) Ile and Phe + Tyr, 4) Met, 5) Lys, and 6) Val and His. The deletion assay using diets formulated on an ideal protein basis was more effective than the addition assay for determining the order of AA limitation in MBM.

Purification and activity of recombinant chicken stem cell factor produced by using a baculovirus vector.

Stem cell factor is a pleiotropic cytokine that plays an essential role in the development of hematopoietic cells, germ cells, and melanocytes. To obtain recombinant soluble chicken stem cell factor (chSCF), a baculovirus containing the cDNA encoding chSCF polypeptide from amino acids -25 to 170 was constructed. The chSCF produced in insect cells infected with the virus was purified by ion exchange column chromatography. The ability of the purified protein to induce the outgrowth of neurites from chicken dorsal root ganglia cultured in vitro was demonstrated.