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R Bickerstaffe

Publications and source records attributed to R Bickerstaffe.

At least 19 recordsLinked to original sources
PubMed2001

A polymorphic marker for the human cathepsin B gene.

Human cathepsin B (CTSB) is a proteolytic enzyme implicated in tumor invasion and metastasis. We describe a PCR-based polymorphic marker for this gene comprising two amplimers differing in length by 19 consecutive nucleotides in intron 7, near the exon 8 splice acceptor site, identifying two gene alleles (A and B). Allele frequencies were 0.614 for A and 0.386 for the B allele, with an observed heterozygosity of 0.457 in a cohort of 70 non-related Australian blood donors. One additional nucleotide difference was also revealed through sequencing. The human CTSB gene is located on chromosome 8 and the alleles described here can potentially be used as markers in linkage and association studies of cancers and other diseases.

Alleles↗
PubMed2001

Differential expression of a gene homologous to a G-alpha protein gene in neonatal mouse skin during development of hair follicles.

The development of mouse hair follicles depends on the proliferation, differentiation and migration of epithelial matrix cells in the follicle bulb. In particular, induction of the proliferation of epithelial cells is thought to be signalled by the dermal papilla at the base of the bulb. Neonatal mouse skin is useful for studying changes in gene expression during development of the follicles, as the mitotic activity of skin cells changes shortly after birth. Using RNA differential display, a 248-bp message has been identified, which is expressed in the skin, specifically on day 2 and day 3 but not on day 4 after birth. Confirmation of expression of this gene by ribonuclease protection assay showed that strong expression is seen on day 2 and day 3, but weak expression is also shown on day 1, day 4 and day 5. In situ hybridization data revealed that it is mainly localized in the dermal papilla. Analysis of its nucleotide sequence showed 99% identity between nucleotide 2 and 232 of the mouse uncoupled S49 cell mRNA for stimulatory GTP-binding protein (G(S)) alpha subunit, suggesting it is a segment of G(S)alpha. As the G(S)alpha subunit is involved in transducing extracellular signals across the cell, the finding of its expression in the papilla suggests it may be a molecular signal to the induction of epithelial proliferation in the follicle bulb. Evidence of strong expression on day 2, at the time when the mitotic activity of epithelial matrix cells starts to increase, also suggests that the G(S)alpha is a potential candidate for involvement in the initiation of follicle growth.

Aging↗
PubMed2001

Effect of preslaughter feed withdrawal period on longissimus tenderness and the expression of calpains in the ovine.

The objective was to study the role of calpains in meat tenderness. Lambs were fasted for various periods of time to generate differences in meat tenderness and to determine in tandem the expression of calpain 1, calpain 2, calpain 3, and calpastatin. The assumption has been that increased calpain expression associated with an increase in tenderness indicates a role for calpain in the tenderization process and vice versa. Fasting lambs for 1 day caused a significant improvement in longissimus (LD) tenderness compared to the control. Correlations between the tenderness of the LD and the expression of the calpains and calpastatin were significant for calpains 1 and 3 but not for calpain 2 or calpastatin. Consequently, this study supports a role for calpains 1 and 3, but not for calpain 2, in the tenderization of the LD from fasted lambs during post-mortem aging.

Animals↗
PubMed2001

Intermuscular variation in tenderness: association with the ubiquitous and muscle-specific calpains.

The biochemistry of intermuscular variation in tenderness is not fully understood. To investigate the role of the calpains in this process we performed two experiments using bovine and ovine species. In the bovine experiment, two distinct muscles, longissimus thoracis et lumborum (LT) and psoas major (PM), were used. In the ovine experiment, four muscles, LT, PM, semimembranosus (SM), and semitendinosus (ST), were used. Muscles were sampled at death for the determination of the steady-state mRNA level of calpains and calpastatin and the activities of calpain 1, 2, and calpastatin. Muscles were also sampled to determine the temporal changes in pH, tenderness, and the activity of the ubiquitous calpain system during postmortem aging. The results of the relative rate of tenderization in both species was found to be related to muscle type; LT had the highest value in both species. Within species, the mRNA steady-state levels of calpain 1 and calpastatin were similar in various bovine and ovine muscles. Bovine calpain 2 mRNA level was significantly lower in the LT than in the PM. Ovine calpain 2 mRNA level was lower, but not significantly different, in the LT compared to the other muscles. The mRNA level of bovine calpain 3 was significantly higher in the LT muscle than in the PM. In the ovine, the mRNA level of calpain 3 was highest in the LT, followed by SM, PM, and ST. Results on the activity of the ubiquitous calpain system in various muscles at death were dependent on muscle type and species. Temporal changes in the activity of calpains and calpastatin during the first 24 h of postmortem aging were similar in the muscles studied: calpain 1 and calpastatin declined significantly and calpain 2 remained relatively unchanged. The temporal changes in muscle pH in both experiments indicated that the extent and rate of pH decline during aging was related to muscle type. Correlation analysis between the relative rate of tenderization and mRNA expression of calpains revealed a strong relationship with calpain 3 in both species.

Animals↗
PubMed2000

Single nucleotide polymorphisms in an intron of the ovine calpastatin gene.

Calpastatin is the specific inhibitor of the ubiquitous calcium-dependent proteases mu-calpain and m-calpain. Enzyme assay data from sheep and cattle inversely correlates post-mortem muscle calpastatin levels with ultimate meat tenderness. Genetic markers of meat quality may therefore be found linked to the calpastatin gene (CAST). A three-allele system detected by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) has been observed in the ovine CAST. The three allele amplimers have been fully nucleotide sequenced and their differences in terms of single nucleotide polymorphism (SNPs) in the intron region of the amplimer are reported and compared to a consensus sequence of the orthologous region of the cattle CAST. A PCR-RFLP for more rapid CAST genotyping of all three ovine alleles was also developed.

Alleles↗
PubMed2000

Rigor temperature and meat quality characteristics of lamb longissimus muscle.

The present experiment was conducted to determine the effect of muscle temperature during the prerigor and early postrigor period on meat tenderness, postmortem proteolysis, calpain system activity, water-holding capacity, and color. Lamb longissimus muscle (n = 14) from the right and left carcass sides was excised immediately after dressing, divided into an anterior and posterior sample, vacuum-packaged, and stored overnight at 5 to 35 degrees C. Further storage, up to 14 d postmortem, was at 2 degrees C. Tenderness at 1 d postmortem, tenderization during further storage, and postmortem proteolysis were negatively affected by overnight incubation above 25 degrees C. This effect could be explained by an effect of temperature on muscle contraction and activity of the calpain system. Muscle contraction was at a minimum after incubation at 15 degrees C. Water-holding capacity was negatively affected by incubation above 25 degrees C. Color scores improved with increasing incubation temperature at 1 d postmortem. However, after 14 d of postmortem storage, no differences in color scores were observed. Based on the present results and results of other groups, a temperature around 15 degrees C at the onset of rigor seems optimal to maximize tenderness without having detrimental effects on water-holding capacity or color.

Animals↗
PubMed1999

Increased vibrissa growth in transgenic mice expressing insulin-like growth factor 1.

Insulin-like growth factor 1 (IGF-1) mediates many of the actions of growth hormone. Overexpression of IGF-1 has been reported to have endocrine and paracrine/autocrine effects on somatic growth in transgenic mice. To study the paracrine/autocrine effects of IGF-1 in hair follicles, transgenic mice were produced by pronuclear microinjection of a construct containing a mouse ultra-high sulfur keratin (UHS-KER) promoter linked to an ovine IGF-1 cDNA. This UHS-KER promoter has previously been shown to direct expression of a reporter gene to the hair follicles of transgenic mice. Four transgenic mouse lines were established as a result of microinjection of 435 embryos. Transgene expression was found in skin at day 8 and day 15 of age in three of the lines. Progeny tests were carried out by mating two of the transgenic expressing males to nontransgenic females. Mice from one line were all nonexpressors while four of the 12 mice from the other showed integration of the transgene and three expressed transgene IGF-1 mRNA in the skin. Vibrissa growth at 11-21 d of age was significantly greater in transgenic expressors than in their nontransgenic littermates. Specifically, the increase in vibrissa length for transgenics at days 11-16 (20.5%) is approximately 2-fold compared with days 16-21 (11.9%). These results demonstrate that local overexpression of IGF-1 in transgenic mice is capable of stimulating vibrissa growth during the first neonatal hair cycle.

Animals↗
PubMed1999

Quantification of ovine and bovine calpain I, calpain II, and calpastatin mRNA by ribonuclease protection assay.

We developed a ribonuclease protection assay (RPA) to quantify the mRNA mass of calpastatin and the catalytic subunits of calpains I and II in ovine and bovine tissues. The method is based on constructing standard curves using predetermined amounts of in vitro synthesized sense cRNA of the calpain system, hybridized with excess radiolabeled antisense counterprobes. This is possible because the vectors used for riboprobe preparation can be used to transcribe the sense cRNA required to generate the standard curves to quantify absolute calpain I, calpain II, and calpastatin mRNA levels. We used the RPA to study calpain I, calpain II, and calpastatin gene expression in ovine liver, heart, and skeletal muscle. The results revealed that calpain II gene expression was similar in the three tissues. However, the expression of calpain I and calpastatin genes indicates that each tissue has its unique pattern. We also analyzed the activity of calpain I, calpain II, and calpastatin by the conventional DEAE chromatographic method for comparison. The results indicated that the RPA is more repeatable than the DEAE method. Special features of the RPA as compared with DEAE chromatography are as follows; 1) the RPA is a reliable method for quantifying the expression of calpains in all tissues because it is not affected by the presence of inhibitors or activators, 2) the RPA method can be expanded to analyze the expression of the tissue-specific calpains simply by designing specific probes for them, and 3) the RPA requires a small amount of tissue. The described method will facilitate future studies on the gene expression of calpains and will contribute to determining their physiological functions.

Animals↗
PubMed1999

Insulin-like growth factor 1 and hair growth.

Insulin-like growth factor 1 (IGF-1) has been identified as an important growth factor in many biological systems.[1] It shares considerable structural homology with insulin and exerts insulin-like effects on food intake and glucose metabolism. Recently it has been suggested to play a role in regulating cellular proliferation and migration during the development of hair follicles. [2,3] To exert its biological effects, the IGF-1 is required to activate cells by binding to specific cell-surface receptors. The type I IGF receptor (IGF-1R) is the only IGF receptor to have IGF-mediated signaling functions.[1] In circulation, this growth factor mediates endocrine action of growth hormone (GH) on somatic growth and is bound to specific binding proteins (BPs). The latter control IGF transport, efflux from vascular compartments and association with cell surface receptors.[4] In tissues, IGF-1 is produced by mesenchymal type cells and acts in a paracrine and autocrine fashion by binding to the IGF-1R. This binding activates the receptor tyrosine kinase (RTK) that triggers the downstream responses and finally stimulates cell division.[5] IGF-1 may therefore be able to stimulate the proliferation of hair follicle cells through cellular signaling pathways of its receptors. Local infusion of IGF-1 into sheep has been reported to be capable of stimulating protein synthesis in the skin.[6] It may also increase the production of wool keratin. Recently, transgenic mice overexpressing IGF-1 in the skin have been shown to have earlier hair follicle development than controls.[7] In addition, this growth factor plays an important role in many cell types as a survival factor to prevent cell death.[8] This anti-apoptotic function of IGF-1 may be important to the development of follicle cells as follicles undergo a growth cycle where the regressive, catagen phase is apoptosis driven. In this review, the effects of IGF-1 on follicle cell proliferation and differentiation are discussed. In particular, the paracrine versus endocrine action of IGF-1 on hair growth and the targeting of expression of the growth factor to the follicles of transgenic animals will be emphasized. The anti-apoptotic role of IGF-1 in hair follicles is also reviewed. Prospects for future studies on hair and fiber growth by IGF-1 are discussed.

Androgens↗
PubMed1996

The insulin status of sheep with genetic differences in glucose clearance.

Lines of sheep have been selected for Slow or Fast glucose clearance after a glucose tolerance test. The aim of this work was to establish what characteristics of the insulin status were altered by the breeding program. Six animals from each line with consistently Slow (T-half > 70 min) or Fast (T-half < 60 min) decreases in plasma glucose concentration were studied in three different experiments. After the injection of [125I]insulin, blood was sampled for 300 min. The change in radioactivity with time was used in a three-compartment series model to estimate theoretical insulin pool sizes and flow rates between pools. All three pools were significantly (P < 0.05) larger in the Slow (61, 115, and 191 mU) than in the Fast glucose clearance animals (45, 82, and 112 mU). Flow rates between the pools were not significantly different. A euglycemic clamp experiment was performed at two insulin infusion rates, each for 4 hr. A significantly higher glucose infusion rate was required to maintain blood glucose at basal levels in the Slow (3 and 9 mg of glucose/kg liveweight [lwt]0.75 per min) than in the Fast glucose clearance animals (1 and 5 mg/kg lwt0.75 per min). The increase in glucose infusion rate when the insulin infusion rate was increased from 0.63 to 3.46 mU/kg lwt0.75 per min (insulin sensitivity index) was significantly greater in Slow than in Fast glucose clearance animals (0.68 vs. 0.35 mU of insulin/kg lwt0.75 per min). There was no difference between the lines in insulin binding to membranes isolated from muscle or adipose tissue. It is concluded that selection for Slow or Fast glucose clearance has altered several aspects of insulin status, but further work is required to identify the primary difference between the lines.

Adipose Tissue↗
PubMed1995

Wool growth rate in vitro is independent of host animal nutrition, season, and the potential mediators of photoperiod, melatonin and prolactin.

In most sheep breeds, wool growth varies seasonally and is correlated with changes in photoperiod, temperature, and nutrition. Wool growth regulation by these factors may result from systemic changes, or to a response localized at the wool follicle. Possible roles of systemic and local factors have been examined here by comparison of wool growth responses in vitro and in vitro. Wool follicles were isolated and maintained under conditions facilitating fibre growth for at least 4 days. The wool growth rate by follicles isolated regularly over a 10 month period was invariate (P > 0.10), in contrast to a seasonally varying wool growth rate by follicles from the same animals in vivo (P < 0.001). Although a high energy, high protein diet increased wool growth in vivo (P < 0.05), cultured follicles isolated from these animals showed no change in growth rate (P > 0.10). These observations suggest that the regulation of wool growth by environmental factors is extrafollicular and that there is no carryover of the in vivo growth rate when follicles are isolated and systemic signals are therefore removed. Additional work discussed here examined the regulation of pelage by prolactin and melatonin, potential endocrine mediators of photoperiod. The fibre production rate by isolated follicles maintained in control media was not different to that of prolactin or melatonin supplemented follicles (P > 0.10). It is concluded that selective breeding of domesticated sheep has suppressed the response of follicles to regulation by prolactin and melatonin. Studies currently underway using in situ RT-PCR will provide further characterization of this conclusion.

Animal Nutritional Physiological Phenomena↗
PubMed1994

Polymorphism in two genes for B2 high sulfur proteins of wool.

Variation in the nucleotide sequence of the B2 high-sulfur protein genes has not been reported previously. This paper reports 15 nucleotide substitutions in each of the genes for the B2A and B2C proteins and a length of polymorphism in the B2A gene which translates to the insertion/deletion of one 30-nucleotide repeat sequence. Evidence is presented for gene conversion occurring within the B2 high-sulfur multigene family. These DNA polymorphisms may account for some of the microheterogeneity observed in the B2 high-sulfur proteins and may also be useful genetic markers of the B2 high-sulfur protein gene loci for future use in analysing wool fibre characteristics.

Alleles↗
PubMed1989

The influence of rumen volatile fatty acids on protein metabolism in growing lambs.

The effect of acetic or propionic acid rumen fermentation patterns on whole-body protein turnover, tissue protein synthetic rates and body composition was investigated in growing lambs. Protein turnover was assessed using a continuous intravenous infusion of [2,3-3H]tyrosine and tissue protein fractional synthetic rates (FSR) from the specific activities of plasma free, intracellular free and tissue bound tyrosine. Only the FSR of muscle tissue approached significance. The high FSR in the propionic group was attributed to the high plasma insulin concentration. Values for whole-body protein synthesis, corrected for tyrosine oxidation, were similar to those obtained by summating protein synthesis in individual tissues, confirming that tyrosine oxidation should be measured accurately if reliable whole-body protein synthesis values are required. Tyrosine oxidation and flux were high in the acetic acid group, suggesting that amino acids are used for gluconeogenesis. The high protein turnover rate probably ensures an adequate supply of gluconeogenic amino acids and that the penalty of mobilizing body proteins for gluconeogenic amino acids is minimal. In the propionic acid group, high plasma glucose and insulin concentrations were associated with a low protein turnover rate, high ratio of deposited: synthesized protein and a high body fat content. It is concluded that changing the proportion of ruminal volatile fatty acids influences protein turnover, protein synthesis and the efficiency of protein retention. Such factors probably contribute, indirectly, to the observed differences in body composition.

Acetates↗