Further studies on short-term adaptations in the expression of lipogenic genes in broilers.

This experiment was conducted to determine possible relationships between certain indices of lipid metabolism and specific gene expression in chickens fed graded levels of dietary crude protein. Male, broiler chickens (Gallus gallus) growing from 7 to 28days of age were fed diets containing 12 or 30% protein ad libitum. Both groups were then switched to the diets containing the opposite level of protein. Birds were sampled at 0, 6, 9, 12, 18 and 24h following the switch in protein levels. Measurements taken included in vitro lipogenesis (IVL), malic enzyme (ME), aspartate aminotransferase (AAT) and isocitrate dehydrogenase (NADP) (ICD) activities. In addition, ME, AAT, ICD, fatty acid synthase (FAS), and acetyl coenzyme carboxylase (ACC) gene expression rates were determined. IVL and ME activities were inversely related to dietary protein levels (12 to 30%) and to acute changes from 12 to 30%. In contrast, expression of ME, FAS and ACC genes was decreased by feeding a 30% protein diet (acute or chronic feeding). Results of the present study demonstrate a continued role for protein in the regulation of broiler metabolism. It should be pointed out; however, that metabolic regulation at the gene level only occurs when feeding very high or very low levels of dietary protein.

The role of feeding regimens in regulating metabolism of sexually mature broiler breeders.

A trial was conducted to determine the effects of different rearing feed regimens on plasma hormone and metabolite levels and hepatic lipid metabolism and gene expression on sexually mature broiler breeders. Cobb 500 birds were divided into 2 groups at 4 wk and fed either an everyday (ED) or skip-a-day (SKP) regimen. At 24 wk of age, all birds were switched over to an ED regimen. At 26.4 wk, breeder hens were randomly selected and killed at intervals after feeding. Livers were sampled from 4 hens at 4-h intervals for 24 h for a total of 28 samples per treatment. Blood was sampled from 4 hens per sampling time; sampling times were 0, 30, and 60 min and 2 and 4 h after feeding and then every 4 h up to 24 h for a total of 36 samples per treatment. Main feeding regimen, time, and interaction effects were analyzed. Significant interaction effects were found between time and feeding regimen for acetyl-coenzyme A carboxylase and malic enzyme mRNA expression. The peak for acetyl-coenzyme A carboxylase expression was higher in ED-reared birds, whereas the peak for malic enzyme expression was higher in SKP-reared birds. Overall, plasma levels of insulin-like growth factor-II were higher in SKP-reared birds. Overall, plasma corticosterone levels were also higher in SKP-reared birds and significant interaction effects between time and feeding regimen were seen. The expression of apolipoprotein A1 was significantly higher in ED-reared birds: significant interaction effects were also noted. Other researchers also found some of the differences observed in the present study in 16-wk-old pullets. In summary, different feeding regimens alter metabolic responses, some of which carry over into sexual maturity.

Expression and activity of the 5'-adenosine monophosphate-activated protein kinase pathway in selected tissues during chicken embryonic development.

The 5'-adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved serine-threonine protein kinase and a key part of a kinase-signaling cascade that senses cellular energy status (adenosine monophosphate:adenosine triphosphate ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating metabolic pathways. The objective of this study was to investigate aspects of the AMPK pathway in the liver, brain, breast muscle, and heart from d 12 of incubation through hatch in chickens. We first determined mRNA and protein expression profiles for a major upstream AMPK kinase, LKB1, which is known to activate (phosphorylate) AMPK in response to increases in the adenosine monophosphate:adenosine triphosphate ratio. Expression of LKB1 protein was greatest in the brain, which demonstrated tissue-specific patterns for phosphorylation. Next, AMPK subunit mRNA and protein expression profiles were determined. Significant changes in AMPK subunit mRNA expression occurred in all tissues from d 12 of incubation to hatch. Differences in the levels of active (phosphorylated) AMPK as well as alpha and beta subunit proteins were observed in all 4 tissues during embryonic development. Finally, we determined the protein level and phosphorylation status of an important downstream target for AMPK, acetyl-coenzyme A carboxylase. The expression of acetyl-co-enzyme A carboxylase and phosphorylated acetyl-coenzyme A was greater in the brain than the liver, but was undetectable by Western blotting in the breast muscle and heart throughout the period of study. Together, our results are the first to demonstrate the expression and activity of the AMPK pathway in key tissues during the transition from embryonic to posthatch development in chickens.

Uncoupling protein expression in skeletal muscle and adipose tissue in response to in vivo porcine somatotropin treatment.

These experiments examined the potential roles of somatropin (pST) and IGF-I in the regulation of uncoupling protein (UCP)2 and UCP3 and their regulatory proteins peroxisome proliferator activated receptor (PPAR) alpha, gamma and delta using in vivo pST treatment of swine and in vitro supplementation of pST or IGF-I to adipose slices. Six, 90kg barrows were treated with recombinant pST (10mg) for 2 week while another six pigs were injected with buffer. Total RNA from outer subcutaneous adipose (OSQ) and middle subcutaneous adipose (MSQ) tissues, leaf fat, liver and longissimus (LM) was amplified by reverse transcription-PCR with quantification of transcripts by capillary electrophoresis with laser-induced fluorescence detection. UCP2 mRNA abundance increased in liver (P<0.001) and all three adipose tissues by pST treatment (P<0.05). Administration of pST increased UCP3 mRNA abundance by 42% in LM (P<0.01). PPARalpha mRNA abundance increased with pST treatment by 29% in liver (P<0.05), while decreasing 25% in LM (P<0.05). PPARgamma mRNA abundance decreased 32% (P<0.01) while PPARdelta increased 48% in LM (P<0.01) with pST administration. In vitro, pST reduced UCP2 mRNA abundance in OSQ and MSQ tissue slices (P<0.05). UCP3 mRNA abundance decreased in OSQ (P<0.05) but increased in MSQ (P<0.05) with pST. In contrast, IGF-I increased UCP2 and UCP3 mRNA abundance in both MSQ and OSQ slices (P<0.05). These experiments suggest pST, IGF-I and metabolic adaptations to pST contribute to regulating UCP2 and UCP3.

Finishing steers with diets based on corn, high-tannin sorghum or a mix of both: Color and lipid oxidation in beef.

We tested the hypothesis that feeding high-tannin sorghum (HTS) to steers would produce beef more resistant to oxidative deterioration. We observed lower thiobarbituric acid-reactive substances (TBARS) in Gluteus medius of steers fed HTS before it was displayed (P=0.028), which could be explained by a reduced response to stress in these animals. While steers finished with corn and corn+HTS had elevated plasma cortisol at the end of the feeding period (P=0.047 and 0.093, respectively), animals fed HTS and corn+vitamin E did not. However, feeding HTS increased the rate of discoloration and TBARS accumulation after aerobic display of Longissimus lumborum and Gluteus medius. Diet did not affect the activity of oxidation-related enzymes and fatty acid composition of muscle. The accelerated rate of lipid oxidation during display of beef could be partially explained by a numerically lower concentration of tocopherols in the tissue.

Administration of adrenocorticotropic hormone during chicken embryonic development prematurely induces pituitary growth hormone cells.

Treatment of fetal rats and embryonic chickens with exogenous glucocorticoids induces premature GH cell differentiation. However, it is unknown whether the developing adrenal gland is capable of mounting this response autonomously. The present study determined whether stimulation of the adrenal gland in developing chicken embryos through administration of ACTH could induce a premature increase in GH cells. We found that plasma corticosterone and ACTH levels increased between embryonic day (e) 11 and e17, consistent with GH cell (somatotroph) ontogeny. Injection of ACTH into eggs on e9, e10, or e11 increased somatotrophs on e14. In contrast, thyroid-stimulating hormone, CRH, alpha-MSH, GHRH, and TRH were ineffective. Culture of e11 pituitary cells with ACTH failed to induce somatotrophs, suggesting an indirect action of ACTH on GH cells in vivo. Intravenous administration of ACTH dramatically increased plasma levels of corticosterone within 1 h and increased the percentage of pituitary somatotrophs within 24 h. Although ACTH administration increased the relative abundance of pituitary GH cells, there was no effect on plasma levels of GH, IGF-I, or IGF-II, or in hepatic expression of IGF-I or IGF-II mRNA. We conclude that ACTH administration can increase the population of GH cells in the embryonic pituitary. However, this treatment alone does not lead to downstream activation of hepatic IGF production. These findings indicate that the embryonic adrenal gland, and ultimately anterior pituitary corticotrophs, may function to regulate pituitary GH cell differentiation during embryonic development.

Investigation of the insulin-like growth factor system in the avian epiphyseal growth plate.

Components of the insulin-like growth factor (IGF) system were investigated in chondrocytes isolated from the avian growth plate. The genes for IGF-I, IGF-II, type 1 IGF receptor (IGF-R), IGF binding protein-2 (IGFBP-2), IGFBP-3, IGFBP-5 and IGFBP-7 were found to be expressed in both proliferative and hypertrophic chondrocytes. The expression of IGF-II in proliferative chondrocytes was extremely high relative to IGF-I. Although IGF-I expression was significantly increased in hypertrophic chondrocytes, the level was still low relative to IGF-II. In cell culture, IGF-I stimulated proteoglycan synthesis and increased the expression of Indian hedgehog (Ihh) and type X collagen, markers of chondrocyte differentiation. IGF-II was found to be equally efficacious in stimulating proteoglycan biosynthesis. These observations suggest that IGF-II may play a significant role in avian growth plate physiology, which is consistent with several reports on mammalian endochondral bone growth.

Mechanisms regulating feed intake, energy expenditure, and body weight in poultry.

To achieve energy balance and maintain a constant BW, changes in feed intake and energy expenditure must be coordinated and tightly regulated. This may not hold true for some poultry species intensively selected for such economically important traits as growth and meat production. For example, the modern commercial broiler breeder does not adequately control voluntary feed intake to meet its energy requirements and maintain energy balance. As a consequence, feeding must be limited in these birds to avoid overconsumption and excessive fattening during production. It is important to determine a genetic basis to help explain this situation and to offer potential strategies for producing more efficient poultry. This review summarizes what is currently known about the control of feed intake and energy expenditure at the gene level in birds. Highly integrated regulatory systems have been identified that link the control of feeding with the sensing of energy status. How such systems function in poultry is currently being explored. One example recently identified in chickens is the adenosine monophosphate-activated protein kinase pathway that links energy sensing with modulation of metabolic activity to maintain energy homeostasis at the cellular level. In the hypothalamus, this same pathway may also play an important role in regulating feed intake and energy expenditure commensurate with perceived whole body energy needs. Genes encoding key regulatory factors such as hormones, neuropeptides, receptors, enzymes, and transcription factors produce the molecular components that make up intricate and interconnected neural, endocrine, and metabolic pathway networks linking peripheral tissues with the central nervous system. Moreover, coordinate expression of specific gene groups can establish functional pathways that respond to and are regulated by such factors as hormones, nutrients, and metabolites. Thus, with a better understanding of the genetic and molecular basis for regulating feed intake and energy expenditure in birds important progress can be made in developing, evaluating, and managing more efficient commercial poultry lines.

The effect of blood removal on oxidation and shelf life of broiler breast meat.

Blood components, especially hemoglobin, are powerful promoters of lipid oxidation and may decrease the shelf life of meat products. Therefore, this study examined different slaughter techniques to determine their effects on pH (24 h), color (L*a*b* values at 24 h), lipid oxidation, residual hemoglobin concentration (24 h), and sensory evaluation (d 1 and 4 postmortem; PM) in broiler breast fillets. The treatments included 1) CO(2) slaughter and not bled, 2) no stunning and bled, 3) electrical stunning (ES) and bled, 4) CO(2) stunning and bled, and 5) ES and decapitation. The birds were conventionally processed, and analyses were performed at 24 h PM except residual hemoglobin for which the samples were frozen (-80 degrees C) until analyses ( < 2 mo). There were no significant differences in pH or b* values at 24 h PM among any of the treatments. L* values were significantly higher, indicating lighter fillets in the ES and decapitated birds compared with the darker fillets from the CO(2) stunned and bled birds. The CO(2) slaughter and not bled birds had significantly higher a* values, indicating more red color, when compared with the ES and bled and decapitated birds. There were no significant differences in the residual hemoglobin contents in the broiler breast muscle when comparing all of the treatments except CO(2) slaughter and not bled, which was significantly (around 15%) greater. Overall TBA-reactive substances (TBARS; raw, cooked at 24 h, and cooked at 72 h PM) indicated that ES and bled birds had the lowest TBARS when compared with the remaining treatments. Consumer panels detected increased aroma (chicken meaty and warmed-over aromas) and flavor (chicken meaty and warmed-over flavors) in not bled samples at 24 h PM. By 72 h PM, however, there were no significant differences in aroma or flavor. Therefore, different slaughter and bleeding method may affect color and sensory properties of the broiler breast fillets, and the ES and decapitation method had the most favorable results for sensory quality.

An examination of the role of feeding regimens in regulating metabolism during the broiler breeder grower period. 1. Hepatic lipid metabolism.

A trial was conducted to determine the effects of feeding regimens on hepatic lipid metabolism in 16-wk-old broiler breeder pullets. A flock of 350 Cobb 500 breeder pullets was divided into 2 at 4 wk of age and fed either every day (ED) or skip-a-day (SKIP) from 4 to 16 wk of age. Total feed intake did not differ between the 2 groups. At 112 d, 52 randomly selected ED-fed pullets, and 76 SKIP-fed pullets were individually caged and fed a 74-g (ED) or 148-g (SKIP) meal. Four pullets from each group were killed at intervals after feeding and livers were collected, weighed, and snap-frozen for determination of lipogenic gene expression. Total RNA was isolated from livers using Trizol reagent and then quantitatively measured by noting the optical density 260:280 ratio and qualitatively measured by gel electrophoresis. The expression of certain regulatory genes in metabolism [acetyl coenzyme A carboxylase; fatty acid synthase; malic enzyme (MAE); isocitrate dehydrogenase (ICDH); and aspartate aminotransferase (AAT)] were determined by real-time reverse-transcription PCR. Remaining liver portions were analyzed for enzyme activity of MAE, ICDH, and AAT as well as glycogen and lipid contents. Liver weight was higher in SKIP than in ED birds. Feeding caused dramatic increases in liver weight, glycogen, and lipids of SKIP birds. Expression of acetyl coenzyme A carboxylase, FAS, and MAE genes were increased in SKIP birds 12 and 24 h after feeding, with the increases in MAE expression from 0 to 24 h after feeding being of the greatest magnitude. In contrast, SKIP decreased ICDH and AAT gene expression, which parallels findings noted in fasting-refeeding experiments conducted with much younger birds. Skip-a-day feeding resulted in far greater changes in gene expression compared with ED, which was indicative of the inconsistent supply of nutrients in such regimens. Enzyme activity of MAE, ICDH, and AAT was reflective of noted changes in gene expression. In summary, the feeding regimen greatly affected hepatic gene expression in breeder pullets.

Investigation of mechanisms by which sodium citrate reduces the pink color defect in cooked ground turkey.

The principal mechanism by which sodium citrate reduces the pink color defect in cooked ground turkey was investigated. Sodium citrate (SC; 0, 0.125, 0.25, 0.5, 1.0, 2.0M), sodium nitrite (0.01, 0.1M), and nicotinamide (0.5, 0.75M) were combined in solutions of bovine hemin to determine SCs ability to bind heme iron and competitively inhibit pink-color-generating ligands from binding. Additionally, the effects of sodium erythorbate (0, 275, 550ppm), ferrous iron chloride (0, 0.3, 3.0, 30ppm), and ferric iron chloride (0, 0.3, 3.0, 30ppm) on SCs ability to reduce pink cooked color was examined. Absorbance curves of hemin+nitrite and hemin+nicotinamide were relatively unaffected by SC, therefore whether or not SC bound heme iron, that did not appear to be a mechanism for inhibiting the pink color defect. Both ferrous and ferric iron chloride had minimal effects on color values, possibly due to sodium tripolyphosphate chelation ability in the meat system and thus their presence did not enhance SCs ability to reduce the pink color defect. However, sodium erythorbate, a reducing agent, inhibited SCs ability to decrease the pink color defect in samples induced pink with sodium nitrite and nicotinamide. Therefore, it appears SC requires the presence of oxygen and may participate in oxidative processes to reduce the pink color defect.

Molecular cloning, genomic organization, and expression of three chicken 5'-AMP-activated protein kinase gamma subunit genes.

The 5'-AMP-activated protein kinase (AMPK) plays a key role in regulating cellular energy homeostasis. The AMPK is a heterotrimeric enzyme complex that consists of 1 catalytic (alpha) and 2 regulatory (beta and gamma) subunits. Mutations of the gamma subunit genes are known to affect AMPK functioning. In this study, we characterized the genomic organization and expression of 3 chicken AMPK gamma subunit genes (cPRKAG). Alternative splicing of the second exon of the cPRKAG1 gene resulted in 2 transcript variants that code for predicted proteins of 298 and 276 amino acids. Use of an alternate promoter and alternative splicing of the cPRKAG2 gene resulted in 4 transcript variants that code for predicted proteins of 567, 452, 328, and 158 amino acids. Alternative splicing of exon 3 of the cPRKAG3 gene resulted in the production of "long" and "short" transcript variants that code for predicted proteins of 382 and 378 amino acids, respectively. We found evidence for differential expression of individual gamma subunit gene transcript variants and, in some cases, tissue-specific expression was observed. The cPRKAG subunit genes displayed similar structural features and high sequence homology compared with corresponding mammalian gamma subunit gene homologues.

The relationship of body composition, feed intake, and metabolic hormones for broiler breeder females.

Three hundred twenty Cobb 500 broiler breeder pullets at 21 wk of age were selected from a flock fed according to Cobb Breeder Management Guide specifications. One hundred sixty pullets at 21 wk of age were switched to ad libitum feeding, and the remaining 160 pullets continued to be control-fed. The pullets were photostimulated at 22 wk and maintained until 36.5 wk. Plasma samples were obtained, BW was determined, and hens were killed for determination of body composition at the following periods: 24 h prior to photostimulation, 2.5 wk after photostimulation, 24 h after first egg, and 36.5 wk following peak egg production. Compared with ad libitum-fed breeders, the restricted breeders had a higher percentage carcass protein and lower percentage carcass fat at all sampling periods. Total egg numbers were greater, and abnormal eggs were less for the restricted pullets compared with the ad libitum-fed pullets at 36.5 wk. Carcass percentage fat of ad libitum-fed pullets was positively related to plasma glucagon, insulin-like growth factor-II (IGF-II), and 17beta-estradiol but negatively related to plasma insulin, insulin/glucagon M ratio, insulin-like growth factor-I (IGF-I), thyroxine (T4), and triiodothyronine (T3). Carcass percentage fat of feed-restricted pullets was negatively related to IGF-I, IGF-II, and T4. The T4 was the most important hormone for predicting the percentage carcass fat in ad libitum-fed pullets, and IGF-I was the most important hormone for predicting the percentage carcass fat in feed-restricted pullets. The percentage carcass protein for ad libitum-fed breeders was positively correlated to IGF-I, T4, T3, insulin/glucagon M ratio, and insulin. Carcass percentage protein for feed-restricted breeders was positively correlated to IGF-I, IGF-II, T4, and glucagon. Stepwise regressions for predicting percentage carcass protein for breeders fed by both systems shows that T3 and IGF-I concentrations were the most important for ad libitum-fed breeders, whereas IGF-II and T4 were best for feed-restricted breeders. The hormone status of breeders may be a key indicator to help predict the body composition and thus support management decisions for maintaining optimum production.

Hormonal regulation of leptin receptor expression in primary cultures of porcine hepatocytes.

A study was conducted to elucidate hormonal control of leptin receptor gene expression in primary cultures of porcine hepatocytes. Hepatocytes were isolated from pigs (52 kg) and seeded into collagen-coated T-25 flasks. Monolayer cultures were established in medium containing fetal bovine serum for 1 day and switched to a serum-free medium for the remainder of the 3-day culture period. To establish basal conditions hepatocytes were maintained in serum-free William's E medium containing 10 nM dexamethasone and 1 ng/ml insulin. For the final 24 h, insulin (1 or 100 ng/ml) or glucagon (100 ng/ml), were added in the presence or absence of 100 nM triiodothyronine (T3). RNA was extracted and quantitative RT-PCR was performed with primers specific for the long form and total porcine leptin receptors. Leptin receptor expression was calculated relative to co-amplified 18S rRNA. Expression of the long form of the leptin receptor was confirmed under basal conditions. Insulin, glucagon and synthetic human proteins (ghrelin and GLP-1) at 100 ng/ml had no influence on leptin receptor expression; the addition of T3 was associated with a marked increase (P < 0.001) in expression of total and long forms of the leptin receptor by 1.6 and 2.4-fold, respectively. Addition of leptin to cells which were pre-treated with T3 for 24 h (to up-regulate leptin receptor expression), confirmed the lack of a direct effect of leptin on glucagon-induced glycogen turnover and cAMP production. These data suggest that porcine hepatocytes may be insensitive to leptin stimulation even when leptin receptor expression is enhanced by T3.

Changes in the tibial growth plates of chickens with thiram-induced dyschondroplasia.

Tibial dyschondroplasia (TD) is a metabolic cartilage disease of young poultry in which endochondral bone formation is disrupted leading to the retention of a non-calcified, avascular plug of cartilage in the tibial growth plate. Chicks aged 7 days were fed either a control diet or one containing thiram 100 ppm for 48 h to induce TD. Cell multiplication in the growth plate was determined thereafter with bromodeoxyuridine (BrdU) labelling, and metabolic changes by measuring alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), and glutathione (GSH) activities. The effect on chondrocyte maturation was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of gene expression. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and DNA fragmentation were used to determine the effects of thiram on cell survival. The results showed that thiram-induced TD was not due to the multiplication of cells in the post-proliferative zones. Thiram did not affect ALP activity, which would have indicated a loss of calcification potential, but it reduced both TRAP and the glutathione concentrations, suggesting that the growth plate metabolism and remodelling functions were adversely affected. Thiram appeared to have no effect on the expression of type X collagen, transglutaminase, RUNX2, or matrix metalloproteinase-2 (MMP) genes suggesting that it did not alter the maturation potential of chondrocytes. On the contrary, the expressions of MMP-13 and vascular endothelial growth factor (VEGF) genes were "up-regulated," suggesting that thiram has pro-angiogenic activity. However, TUNEL assay showed that thiram induced endothelial cell apoptosis in the capillary vessels of the growth plates, as early as 10 days of age, when TD was not visually evident. The vascular death increased on subsequent days accompanied by massive death of chondrocytes in the transition zone of the growth plate. The induction of apoptosis in the growth plate was also demonstrated by DNA fragmentation. It was concluded that thiram induced TD not through an increase in the multiplication of chondrocytes in the transition zone and not by altering the expression of genes causing the arrest of chondrocytes in a prehypertrophic state, but by creating a metabolic dysfunction which led to the destruction of blood capillaries in the transition zone chondrocytes.

Analysis of metallothionein isoforms by capillary electrophoresis: optimisation of protein separation conditions using micellar electrokinetic capillary chromatography.

Capillary electrophoresis (CE) techniques have been successfully applied to the separation of metallothionein (MT) isoforms and have proved to be rapid, practical and economical. Study of a variety of different electrolytes and capillaries has shown that electrolyte buffer composition and capillary wall surface modifications can have considerable influence on isoform separation and resolution. Ionic surfactants such as sodium dodecyl sulphate (SDS) form micelles at elevated concentrations and the partitioning of molecules between the hydrophobic micelle phase and the aqueous phase and their resulting migration in an electric field is the basis of the technique known as micellar electrokinetic capillary chromatography (MECC). In the present work, we have used sheep and rabbit MT to optimise MECC conditions for analysis of MT isoforms. Capillaries of 57 cm gave much better separations than shorter columns although analysis times were increased to about 12 min. Changing the buffer and SDS concentration or the pH affected the selectivity of isoform separation and up to 5 isoforms in sheep MT and 6 in rabbit MT were completely or partially resolved. Comparing different diameter capillaries we conclude that 25 microns I.D. columns give better separations than 50 or 75 microns I.D. columns although sensitivity is reduced by a factor of about 3 and 5, respectively. Using our MECC conditions, columns coated with C1 or C18 hydrophobic material were not found to be useful in improving MT separation or resolution although further evaluation of these columns is in progress.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of metallothionein isoforms by micellar electrokinetic capillary chromatography.

Current techniques for the separation and quantification of metallothionein isoforms have limited value for routine analysis. Isoforms having a similar charge have been separated successfully using reversed-phase HPLC but this technique suffers from a slow sample turnover time. The use of the surfactant sodium dodecyl sulphate for the separation of metallothionein isoforms by micellar electrokinetic capillary chromatography (MECC) is described. The charge-different isoforms MT-1 and MT-2 from rats, rabbits and sheep were separated within 9-12 min. In addition, a varying degree of heterogeneity was observed in purified samples of human MT-1, rat MT-2, rabbit MT-1, rabbit MT-2 and sheep MT-1. The behaviour of chicken MT was different from that of any other species. The separation of sheep liver extracts indicated the potential of MECC as the basis for a quantitative assay for both charge-different and charge-similar metallothionein isoforms.

Analysis of leptin gene expression in chickens using reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection.

Leptin is a peptide hormone product of the obese (ob) gene that functions in the regulation of appetite, energy expenditure and reproduction in animals and humans. We have developed a technique using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for the analysis of chicken leptin (261 base pairs, bp) and beta-actin (612 bp) double-stranded DNA products from reverse transcription polymerase chain reaction (RT-PCR) assays. Amplicons were separated using a DB-1 coated capillary (27 cm x 100 microns I.D.) at a field strength of 300 V/cm in a replaceable sieving matrix consisting of 0.5% hydroxypropylmethylcellulose (HPMC) in 1X TBE (89 mM Tris-base, 89 mM boric acid, 2 mM EDTA, pH 8.3) buffer with 0.5 microgram/ml EnhanCE fluorescent intercalating dye. RT-PCR samples (1-2 microliters) were diluted 1:100 with deionized water and introduced into the capillary by electrokinetic injection. Separations were completed in less than 6 min and the total time required per sample, including capillary conditioning, was 8 min. We have applied RT-PCR-CE-LIF to determine the effects of insulin and estrogen treatment on leptin gene expression relative to that of beta-actin in chicken liver and adipose tissue. In addition, we have constructed a chicken leptin mRNA competitor (234 bp amplicon) and evaluated it for use as an internal standard in the development of a quantitative-competitive RT-PCR assay. Our findings represent the first reported application of capillary electrophoresis to the analysis of leptin gene expression by RT-PCR.

A brief review of the archaeological evidence for Palaeolithic and Neolithic subsistence.

Knowledge of our ancestor's diets is becoming increasingly important in evolutionary medicine, as researchers have argued that we have evolved to specific type of 'Palaeolithic' diet, and many modern nutritional disorders relate to the mismatch between the diet to which we have evolved, and the relatively newer agricultural-based 'Neolithic' diets. However, what is the archaeological evidence for pre-agricultural diets and how have they changed over the four million years of hominid evolution? This paper briefly introduces the three lines of evidence we have for Palaeolithic and Neolithic diets; morphological changes, archaeological material evidence, and direct measurement of diet from bone chemistry. The morphological changes, increasing gracilization of the mandible and increasing brain size have been interpreted (based on analogies with living primates) as the move from plants to higher-quality, more digestible, animal meat, although this is debated. The archaeological evidence is especially weak, as many organic materials, especially plants, do not survive well, and are therefore invisible in the archaeological record. Artefacts, such as stone tools which are likely to be used for hunting and animal bones with evidence of human processing and butchering do indicate that hunting did occur at many times in the past, but it is impossible to judge the frequency. Direct evidence from bone chemistry, such as the measurement of the stable isotopes of carbon and nitrogen, do provide direct evidence of past diet, and limited studies on five Neanderthals from three sites, as well as a number of modern Palaeolithic and Mesolithic humans indicates the importance of animal protein in diets. There is a significant change in the archaeological record associated with the introduction of agriculture worldwide, and an associated general decline in health in some areas. However, there is an rapid increase in population associated with domestication of plants, so although in some regions individual health suffers after the Neolithic revolution, as a species humans have greatly expanded their population worldwide.

Design and application of a polyclonal peptide antiserum for the universal detection of leptin protein.

An epitope-specific polyclonal antiserum was produced in rabbits immunized against a synthetic 15 amino acid peptide (QRVTGLDFIPGLHPV) derived from the coding sequence reported for the porcine leptin gene (GenBank Accession No. U59894). This peptide contains a core sequence comprised of eight amino acids (GLDFIPGL) that is totally conserved in all leptin proteins studied to date. Purified recombinant human, mouse, rat, pig, and chicken leptin proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and electro-blotted onto PVDF membranes. Western blots were developed employing the leptin-specific peptide antiserum with an alkaline-phosphatase-conjugated anti-rabbit IgG second antibody chromogenic system. The peptide antiserum was found to be highly specific for leptin which exhibited an estimated molecular weight of about 16 kDa for all species analyzed. The sensitivity of the Western blot assay was not sufficient to permit the direct detection of leptin in chicken serum or plasma. However, with this assay we were able to detect native leptin protein in an enriched fraction prepared from chicken plasma using a combination of gel filtration and ion exchange column chromatography. Slot blots indicated a potential application of the immunostaining technique for quantitative analysis of leptin protein. Finally, the peptide antiserum was successfully employed to localize leptin protein by immunohistochemical staining of thin sections prepared from adipose (chicken and pig) and liver (chicken) tissue samples. This study is the first to report a polyclonal peptide antiserum that apparently recognizes intact leptin protein, both native and recombinant, regardless of the species of origin.