Search

Purpose Water use in the livestock sector has featured in the debate about sustainable food systems. Most evidence has come from virtual water calculations which lack impact assessment and adequate consideration of the heterogeneity in livestock production. This study sought new evidence, using a recently developed life cycle impact assessment method for water use to assess six geographically defined beef cattle production systems in New South Wales, Australia, a major production region.

This study determined the extent to which bovine longissimus lumborum muscle (LLM) myofibers are influenced by nutrition for 120 d from weaning and the time-course of recovery after severe postweaning nutritional restriction. After weaning, 3 groups of Belmont Red cattle, a tropically adapted breed, were fed to achieve rapid growth (RG,or =0.6 kg of BW gain/d; n = 16), slow growth (SG, 0.2 kg of BW gain/d; n = 17), or BW loss (WL, 10% loss of weaning weight; n = 17) over 120 d. They were then grazed as 1 group at pasture with forage supplementation for 600 d until slaughter at approximately 500 kg of BW. Samples of LLM were taken from 8 to 12 animals per treatment 6 d before (baseline) and 115, 204, 324, and 476 d after commencement of the study and from all cattle at slaughter (d 721). Myofiber characteristics were determined by immunocytochemical staining of myosin heavy chains. Cross-sectional areas (CSA) of the major myofiber types 1, 2A, and 2X in WL were reduced at d 115 compared with baseline and with the growth groups (all P0.001); however, there was little difference in the percentage of the different myofiber types (all P0.10). Differences in CSA of the major myofiber types between WL and the growth groups at 115 d were smallest for type 1 (slow oxidative) and greatest for type 2X (fast glycolytic). Consequently, the relative area (percentage of total myofiber area) of type 1 myofibers in WL was significantly greater at 115 d than in the growth groups (P0.001). During recovery from postweaning nutritional restriction, significant differences in major myofiber type percentages were not evident (all P0.10), and by 721 d CSA of myofiber types differed little between the treatment groups, although SG had greater CSA of type 1 (P0.05) and type 2A (P0.01) myofibers than WL and RG. At 721 d, the relative area of type 2A myofibers was less in WL compared with SG (P0.01) and RG (P0.05) and of type 2X myofibers greater (P0.05) in WL compared with SG. It is concluded that in the LLM of cattle undergoing severe nutritional restriction immediately postweaning, the size of the more glycolytic fiber types is more adversely affected than the more oxidative types, resulting in an increased relative area of type 1, slow oxidative myofibers. However, given adequate time and nutriment at pasture, LLM myofiber characteristics of cattle recovered to near normal after severe, chronic nutritional restriction immediately postweaning, consistent with earlier findings for beef quality.

Vitamin A plays a critical role in many essential life processes. In herbivores, it is either derived from plant β-carotene or directly as a dietary supplement. In cattle, vitamin A has the potential to influence various carcass traits that are sought by specific beef markets. A group of 20 Angus steers was removed from pasture and fed a low β-carotene and vitamin A cereal-based ration on a feedlot for 308 days. Ten of the steers were supplemented with vitamin A (retinyl palmitate, 60 IU of vitamin A/100 kg body weight/day) and the other ten received no supplement. The results demonstrated that restriction of vitamin A intake changed intramuscular fat deposition without changing subcutaneous fat depots. Angus steers that had been depleted of vitamin A showed increased intramuscular fat in the longissimus thoracis et lumborum (LTL) by 35% (P < 0.026) and seam fat area at the quartering site by 33% (P < 0.0273), when compared with cattle supplemented with vitamin A. There were no changes in intramuscular fat in the semitendinosus. Visually assessed marbling scores were also higher (19%; P < 0.094) in the non-supplemented, depleted group. There was no effect of vitamin A depletion on cattle growth and other meat traits (eye muscle area, meat colour, pH, meat cut weight), meat eating attributes (tenderness, cooking loss) or muscle fibre diameter. The only difference (P < 0.0177) among the meat traits was fat colour where depleted animals had whiter fat than the controls. Moreover, the fat from the vitamin A depleted group was softer with a lower melting point. We conclude that the reduced vitamin A consumption, leading to vitamin A depletion, increases intramuscular fat. On the other hand, the vitamin A depletion did not increase subcutaneous fat depth or change other meat quality traits, suggesting that marbling and these other traits are not invariably related.

Green-striped burrowing frogs, Cyclorana alboguttata, survive droughts by entering a metabolic depression called aestivation, characterised by a reduction in resting oxygen consumption by 80%. Aestivation in C. alboguttata is manifest by transcriptional silencing of skeletal muscle bioenergetic genes, such as NADH ubiquinone oxidoreductase 1, ATP synthase and superoxide dismutase 2. In this study, we hypothesised that aestivation is associated with epigenetic change in frog muscle. We assessed mRNA transcript abundance of seven genes that code for proteins with established roles in epigenetically-mediated gene silencing [transcriptional co-repressor SIN3A, DNA (cytosine-5-) methyltransferase 1, methyl CpG binding protein 2, chromodomain helicase DNA binding protein 4, histone binding protein rbbp4, histone deacetylase 1 and nuclear receptor co-repressor 2] using qRT-PCR. These seven genes showed a modest (1.1-3.5-fold) but coordinated upregulation in 6-month aestivating muscle. This reached significance for SIN3A and DNA cytosine-5-methyltransferase 1 in standard pair-wise comparisons (p < 0.05), and the candidates as a whole when analysed by Fisher's combined probability test (p < 0.01). These data are consistent with the hypothesis that the transcriptional silencing and metabolic depression that occurs during seasonal dormancy are associated with chromatin remodelling, and present a novel example of an environmentally induced epigenetic modification in an adult vertebrate.

Gene expression profiles of LM from beef cattle that underwent significant postweaning undernutrition were studied using complementary DNA (cDNA) microarrays. After 114 d of undernutrition, the RNA from LM showed 2- to 6-fold less expression of many genes from the classes of muscle structural proteins, muscle metabolic enzymes, and extracellular matrix compared with animals on a rapid growth diet. The expression levels of these genes had mostly returned to pretreatment levels after 84 d of realimentation. The gene expression changes associated with undernutrition and BW loss showed an emphasis on downregulation of gene expression specific to fast-twitch fibers, typical of starving mammals, with a preferential atrophy of glycolytic fast-twitch fibers. We also identified a small group of genes that showed 2- to 5-fold elevated expression in LM after 114 d of undernutrition. Putative roles for these genes in atrophying skeletal muscle are regulation of myogenic differentiation (CSRP3), maintenance of mesenchymal stem cells (CYR61), modulation of membrane function (TM4SF2), prevention of oxidative damage (SESN1), and regulation of muscle protein degradation (SQSTM1). A significant increase in stearoyl-CoA desaturase (SCD) gene expression was observed in atrophying muscle, suggesting either that increased fatty acid synthesis is part of the tissue response to caloric restriction, or that SCD plays another role in energy metabolism in the mixed cellular environment of bovine skeletal muscle.

The high molecular weight glycosaminoglycan hyaluronan (HA) is an essential component of the extracellular matrix (ECM), however, the link between HA regulation and development of the adipocyte ECM, which is essential for differentiation, remains undefined. Hyaluronan synthase gene expression, HA synthetic rate and molecular weight during differentiation of 3T3-L1 pre-adipocytes were compared to undifferentiated 3T3-L1 pre-adipocytes and non-adipogenic NIH/3T3 fibroblasts. In the 3T3-L1 pre-adipocytes, the predominant genes associated with HA metabolism were found to be HA synthase-2 (Has-2) and hyaluronidase-2 (Hyal-2) demonstrating a co-regulation of expression which was stimulated by adipogenic induction consequently resulting in increased synthesis of high molecular weight HA (>10 MDa) and its simultaneous degradation. Accumulation of HA correlated positively with cell number, although synthetic rate was inversely related suggesting a regulatory feedback mechanism. Within 24 h post-induction, pre-adipocytes responded with a higher HA synthetic rate and later, accumulated cytoplasmic lipid. In contrast, undifferentiated pre-adipocytes had a reduced HA synthetic rate during clonal expansion and did not accumulate lipid. HA was continuously and rapidly metabolised throughout 3T3-L1 adipogenesis, where terminal differentiation coincided with the increased generation of low molecular weight, angiogenic HA fragments, a likely prerequisite for concurrent neovascularisation of adipose tissue. This study has highlighted a relationship between HA metabolism and adipocyte differentiation, suggesting that the balance between the formation and regulation of the adipocyte extracellular matrix is finely coordinated in a growth phase-specific dependent manner.

A group of Angus beef cattle was removed from temperate pastures and fed a very low beta-carotene cereal-based ration in a feedlot for over 300 d. Half the group was supplemented weekly with retinyl palmitate (at the rate of 60,000 IU vitamin A/100 live weight (LW)/day), sufficient to offset clinical vitamin A deficiency; the other half received no supplement. Blood was sampled from all animals at biweekly intervals to assess beta-carotene and vitamin A status. Adipose tissue was sampled by biopsy on three occasions throughout the experimental period and at slaughter to assess FA composition. Muscle was sampled at slaughter to determine the intramuscular fat content. The mean plasma concentration of beta-carotene of all animals fell from an initial value of 20.1 to 5.2 microg/mL at 14 d, to 1.4 microg/mL at 35 d, and to zero at 105 d. Mean vitamin A in plasma was not significantly different between the treatment groups initially. The values then rose to almost twice their initial values by 35 d, but subsequently fell to below initial values by day 119. Thereafter, plasma vitamin A of the supplemented group was significantly greater than that of the unsupplemented group (P0.05). Muscle samples at slaughter from supplemented animals contained significantly (P0.01) more intramuscular lipid (13.0 vs. 9.6%). Major changes occurred over time in FA composition in both groups. Saturated FA decreased as monounsaturated FA increased over the first 60 d. An index of desaturation of FA was significantly lower (P0.001) in the vitamin A-supplemented group than in the nonsupplemented group. M.P. of the adipose tissue of nonsupplemented animals was 32.3 degrees C, significantly less (P0.05) than that of supplemented animals (34.1 degrees C). Feeding vitamin A was associated with less intramuscular fat but with a less desirable (less unsaturated, more solid) FA profile.

We used a 9.6 K cattle muscle/fat cDNA microarray to study gene expression differences between the longuissimus dorsi (LD) muscle of Japanese Black (JB) and Holstein (HOL) cattle. JB cattle exhibit an unusual ability to accumulate intramuscular adipose tissue with fat melting points lower than that in other breeds. The LD biopsies from three JB (Tajima strain) and three HOL animals were used in this breed comparison. Seventeen genes were identified as preferentially expressed in LD samples from JB and seven genes were found to be expressed more highly in HOL. The expression of six selected differentially expressed genes was confirmed by quantitative real-time PCR. The genes more highly expressed in JB are associated with unsaturated fatty acid synthesis, fat deposition, and the thyroid hormone pathway. These results are consistent with the increased amounts and proportions of monounsaturated fatty acids observed in the muscle of JB animals. By discovering as yet uncharacterized genes that are differentially regulated in this comparison, the work may lead us to a better understanding of the regulatory pathways involved in the development of intramuscular adipose tissue.

Expression profiling using microarrays allows for the detailed characterization of the gene networks that regulate an animal's response to environmental stresses. During nutritional restriction, processes such as protein turnover, connective tissue remodeling, and muscle atrophy take place in the skeletal muscle of the animal. These processes and their regulation are of interest in the context of managing livestock for optimal production efficiency and product quality. Here we expand on recent research applying complementary DNA (cDNA) microarray technology to the study of the effect of nutritional restriction on bovine skeletal muscle. Using a custom cDNA microarray of 9,274 probes from cattle muscle and s.c. fat libraries, we examined the differential gene expression profile of the LM from 10 Brahman steers under three different dietary treatments. The statistical approach was based on mixed-model ANOVA and model-based clustering of the BLUP solutions for the gene x diet interaction effect. From the results, we defined a transcript profile of 156 differentially expressed array elements between the weight loss and weight gain diet substrates. After sequence and annotation analyses, the 57 upregulated elements represented 29 unique genes, and the 99 downregulated elements represented 28 unique genes. Most of these co-regulated genes cluster into groups with distinct biological function related to protein turnover and cytoskeletal metabolism and contribute to our mechanistic understanding of the processes associated with remodeling of muscle tissue in response to nutritional stress.

In functional genomic laboratories, it is common to use the same microarray slide across studies, each investigating a unique biological question, and each analyzed separately due to computational limitations and/or because there is no hybridization of samples from different studies on one slide. However, the question of analyzing data from multiple studies is a major current issue in microarray data analysis because there are gains to be made in the accuracy of estimated effects by exploiting a covariance structure between gene expression data across studies. We propose an approach for combining multiple studies using multivariate mixed models, with the assumption of a nonzero correlation among genes across experiments, while imposing a null residual covariance. We applied this method to jointly analyze three experiments in genetics of cattle with a total of 54 arrays, each with 19,200 spots and 7,638 elements. The resulting seven-variate model contains 752,476 equations and 56 covariances. To identify differentially expressed genes, we applied model-based clustering to a linear combination of the random gene x variety interaction effect. We enhanced the biological interpretation of the results by applying an iterative algorithm to identify the gene ontology classes that significantly changed in each experiment. We found 118 elements with coordinate expression that clustered into distinct biological functions such as adipogenesis and protein turnover. These results contribute to our understanding of the mechanistic processes involved in adipogenesis and nutrient partitioning.

The effect of cattle breed on stearoyl-CoA desaturase (SCD) gene expression was investigated in this study. Detailed comparisons of SCD mRNA level were made among three steers each of Japanese Black, Holstein and their crossbreed which were age-matched had been fed the same diet and were sampled by biopsy of the longissimus dorsi (LD) muscle and subcutaneous fat. The levels of SCD mRNA were measured in samples of muscle and subcutaneous fat. The levels of SCD mRNA demonstrated a breed effect in each tissue, though the relative expression was higher (P

The ability of cells to decorate glycosaminoglycans (GAGs) with sulphate in highly specific patterns is important to extracellular matrix biogenesis and placing appropriate glycosulphated ligands on the cell surface. We have examined sulphate metabolism in two pancreatic duct epithelial cell lines ^ PANC-1 and CFPAC-1 (derived from a cystic fibrosis patient) with a view to understanding how pancreatic cells utilise intracellular sulphate. [ 35 S]Sulphate uptake was rapid and reached near steady state levels within 10 min. However, the intracellular specific activity of [ 35 S]sulphate for PANC-1 and CFPAC-1 reached only 35 and 10%, respectively, of the medium specific activity at 10 min. Therefore, sulphate appears to reside within two compartments; a rapidly exchangeable sulphate pool (RESP) and a slowly exchangeable sulphate pool (SESP). Reducing chloride in the medium, increased the specific activity of [ 35 S]sulphate within cells and increased the size of the inorganic sulphate pool, suggesting that the RESP was enlarged. Sulphate pools were not different in size between the two cell lines in physiological NaCl. Increasing the size of the sulphate pool had no effect on [ 35 S]sulphate :[ 3 H]glucosamine ratios incorporated into glycosaminoglycans (GAGs); however, stimulating the synthesis of GAGs with 4-methylumbelliferyl-L-D-xyloside, stably elevated [ 35 S]:[ 3 H] ratios. This was due to higher [ 35 S]sulphate incorporation. [ 35 S]Cysteine contributed less than 0.1% of the cells’ sulphate requirements. We conclude that in the face of elevated demand for sulphate, pancreatic cells appear to channel a greater proportion through the RESP. fl 1999 Elsevier Science B.V. All rights reserved.

The growth paths of 36, nine-month-old Brahman-cross steers were modified to determine the effect of their growth history on tenderness of the semitendinosus (ST) muscle. Steers were assigned to one of three treatment groups. One group of steers (uninterrupted group) was grazed on improved tropical pasture for 257 days and had an average weight gain of 0.6 kg day−1. The other two groups were fed a restricted diet of low-quality grass hay and lost on average ~ 13% of their initial live weight over 100 days. These groups were then regrown for 157 days on either pasture (pasture finished) with the uninterrupted group, or on a grain-based feedlot diet (grain finished). Growth rates of the previously restricted groups during the regrowth phase were indicative of compensatory growth and were significantly different (p < 0.05) at 0.76 (sem 0.03) kg day−1 and 1.22 (0.05) kg day−1, pasture and grain finished groups, respectively. Growth rates for both restricted groups were significantly different (p < 0.05) from the uninterrupted group [0.55 (0.02) kg day−1]. At slaughter, the grain finished group had heavier carcases, higher dressing percentages and more fat coverage, than either the uninterrupted or pasture finished groups, the latter being significantly lighter than the uninterrupted group. Tenderness was assessed by shear, compression (C) and adhesion (ADH) measurements. Shear peak force (PF) values of cooked ST samples did not differ significantly between groups. However, PF values of pressure-heat treated ST samples from the grain finished group were significantly less (p < 0.05) than comparable values from the uninterrupted group suggesting a reduced contribution of connective tissue to toughness. The pasture finished group mean PF value was not significantly different from either the uninterrupted group or grain finished group means. C and ADH values were significantly less (p < 0.05) in the grain finished group compared to the pasture finished groups values, again indicating a reduced connective tissue contribution to toughness. We conclude that the physical properties of the connective tissue component of the ST muscle may be altered by rapid compensatory growth after a weight loss phase and reduce the connective tissue contribution to toughness which may enhance meat tenderness.

Cystic fibrosis (CF) is a fatal inherited disease caused by the loss of function of a plasma membrane chloride channel-the cystic fibrosis transmembrane conductance regulator (CFTR). It is characterized by viscous mucous secretions which have abnormal glycosylation and sulfation. The development of a CFTR knockout mouse has allowed in vivo experiments aimed at investigating the over-sulfation phenomenon reported for CF glycoconjugates. Four CF and five control mice injected with [35S]sulfate were examined for differences in the sulfation of glycosaminoglycans (GAGs) synthesized by 12 tissues after 48 h. The liver and pancreas of CF mice incorporated significantly higher amounts of [35S]sulfate into GAGs (dpm/microg) than the controls, while the ileum, jejunum, colon, cecum, spleen, trachea, and gall bladder of CF mice exhibited higher incorporation levels that were not significant. The lung and nasal septum were not different, and the nasal mucosa of CF mice was significantly lower (P0.05). Structural analysis of the chondroitin/dermatan sulfate component by strong anion-exchange HPLC revealed that the liver and ileum of CF mice incorporated significantly more total sulfate than controls. However, for other organs, the explanation for higher isotope incorporation was a 40-50% higher specific activity of [35S]sulfate within GAGs. This finding implied different uptake kinetics of sulfate from the circulation or that CF mice have altered sulfate pools. CF mice also had altered proportions of chondroitin/dermatan sulfate to heparan sulfate in the ileum and gall bladder (P0.05). We conclude that extracellular matrix architecture in some CF organs may be abnormal and that sulfation of glycoconjugates by some organs and sulfate utilization in others have been affected by the loss of CFTR. This study provides the first in vivo evidence for an influence of CFTR on glycoconjugate sulfation and suggests other secondary manifestations of CFTR dysfunction associated with abnormalities of the extracellular matrix.

The 12D3 antigen of Babesia bovis was found to be synthesised rapidly in cultured parasites, and localised to both the apical complex of the merozoite and the cytoplasm of the parasitised erythrocyte. Amino-terminal sequencing suggested that the nascent protein had been processed and differences between the predicted and measured molecular weights suggested post-translational modification. The major proportion of 12D3 appeared in the soluble compartment of the parasitised erythrocytes with a molecular weight consistent with no further processing. A significant proportion of the protein required extraction by sodium carbonate, suggesting association with membranous components. The timing of release of soluble 12D3 was coincident with haemoglobin release and this probably reflects a non-specific lysis of the erythrocyte. Synthesis of recombinant BV12D3 was achieved in baculovirus-infected SF9 insect epithelial cells. The product was of the same molecular weight as the native 12D3 and polyclonal antibodies raised against the recombinant protein reacted with both the recombinant and native forms of the antigen.

Stand-alone environmental indicators based on life cycle assessment (LCA), such as the carbon footprint and water footprint, are becoming increasingly popular as a means of directing sustainable production and consumption. However, individually, these metrics violate the principle of LCA known as comprehensiveness and do not necessarily provide an indication of overall environmental impact. In this study, the carbon footprints for six diverse beef cattle production systems in southern Australia were calculated and found to range from 10.1 to 12.7 kg CO 2 e kg −1 live weight (cradle to farm gate). This compared to water footprints, which ranged from 3.3 to 221 L H 2 Oe kg −1 live weight. For these systems, the life cycle impacts of greenhouse gas (GHG) emissions and water use were subsequently modelled using endpoint indicators and aggregated to enable comparison. In all cases, impacts from GHG emissions were most important, representing 93 to 99% of the combined scores. As such, the industry’s existing priority of GHG emissions reduction is affirmed. In an attempt to balance the demands of comprehensiveness and simplicity, to achieve reliable public reporting of the environmental impacts of a large number of products across the economy, a multi-indicator approach based on combined midpoint and endpoint life cycle impact assessment modelling is proposed. For agri-food products, impacts from land use should also be included as tradeoffs between GHG emissions, water use and land use are common.

A number of technologies that increase feed efficiency and lean tissue deposition while decreasing fat deposition have been developed in an effort to improve profitability of animal production. In general, the mode of action of these metabolic modifiers is to increase muscle deposition while often simultaneously reducing fat deposition. However, there have been some concerns that the focus on increasing production efficiency and lean meat yield has been to the detriment of meat quality. The aim of this review is to collate data on the effects of these metabolic modifiers on meat quality, and then discuss these overall effects. When data from the literature are collated and subject to meta-analyses it appears that conservative use of each of these technologies will result in a 5-10% (0.3-0.5kg) increase in shear force with a similar reduction in perception of tenderness. However, it should be borne in mind that the magnitude of these increases are similar to those observed with similar increases in carcass leanness obtained through other means (e.g. nutritional, genetic selection) and may be an inherent consequence of the production of leaner meat. To counter this, there are some other metabolic factors and dietary additives that offer some potential to improve meat quality (for example immuncastration) and it is possible that these can be used on their own or in conjunction with somatotropin, approved β-agonists, anabolic implants and CLA to maintain or improve meat quality.

High-titre stocks of an amphotropic retrovirus, constructed so as to express a full-length cDNA encoding the human lysosomal enzyme N-acetylgalactosamine-4-sulphatase (4-sulphatase) from the cytomegalovirus immediate early promoter, were used to infect skin fibroblasts from a clinically severe mucopolysaccharidosis type VI (MPS VI) patient. The infected MPS VI cells showed correction of the enzymic defect with the enzyme being expressed at high levels and in the correct subcellular compartment. Surprisingly this did not result in correction of glycosaminoglycan turnover as measured by accumulation of 35S in metabolically labelled cells. We demonstrate that this is apparently caused by an induced reduction of the activities of other lysosomal sulphatases, presumably due to competition for a sulphatase-specific processing mechanism by the over-expressed 4-sulphatase. The level of steroid sulphatase, which is a microsomal sulphatase, was also reduced. Infection of skin fibroblasts from a second, clinically mildly affected, MPS VI patient with the same virus also resulted in no significant change in the level of glycosaminoglycan storage. However, in this case the cause of the observed phenomenon was less clear. These results are of obvious practical importance when considering gene therapy for a sulphatase deficiency such as MPS VI and also provide possible new avenues for exploration of the processes involved in sulphatase synthesis and genetically determined multiple sulphatase deficiency.

SUMMARYWe investigated the effect of prolonged immobilisation of six and nine months duration on the morphology and antioxidant biochemistry of skeletal muscles in the amphibian aestivator Cyclorana alboguttata. We hypothesised that, in the event of atrophy occurring during aestivation,larger jumping muscles were more likely to be preserved over smaller non-jumping muscles. Whole muscle mass (g), muscle cross-sectional area (CSA)(μm2), water content (%) and myofibre number (per mm2) remained unchanged in the cruralis muscle after six to nine months of aestivation; however, myofibre area (μm2) was significantly reduced. Whole muscle mass, water content, myofibre number and myofibre CSA remained unchanged in the gastrocnemius muscle after six to nine months of aestivation. However, iliofibularis dry muscle mass, whole muscle CSA and myofibre CSA was significantly reduced during aestivation. Similarly,sartorius dry muscle mass, water content and whole muscle CSA was significantly reduced during aestivation. Endogenous antioxidants were maintained at control levels throughout aestivation in all four muscles. The results suggest changes to muscle morphology during aestivation may occur when lipid reserves have been depleted and protein becomes the primary fuel substrate for preserving basal metabolic processes. Muscle atrophy as a result of this protein catabolism may be correlated with locomotor function, with smaller non-jumping muscles preferentially used as a protein source during fasting over larger jumping muscles. Higher levels of endogenous antioxidants in the jumping muscles may confer a protective advantage against oxidative damage during aestivation; however, it is not clear whether they play a role during aestivation or upon resumption of normal metabolic activity.

Human obesity is considered a consequence of a thrifty or economic metabolism. In this hypothesis, we apply an established economic design theory, called symmorphosis, to help explain the known association between obesity and low oxidative capacity skeletal muscle. Symmorphosis reflects an engineering principle, and predicts that physiological systems are most economically designed when unnecessary spare capacity is eliminated. This is because the structural/functional adaptations accounting for spare capacity themselves bear energetic costs of construction, maintenance and load. As oxidation of feed energy occurs in mitochondria, and because skeletal muscle accounts for 30% of resting metabolism, we focus on skeletal muscle mitochondria. In the same way that the most economically designed elevator is supported by a cable that is strong enough, but not too strong, symmorphosis predicts that the most economically designed feed converters should have enough, but not too much mitochondrial oxidative (fuel burning) capacity. While ATP demand is clearly more efficiently met by oxidative (38 molecules of ATP) rather than glycolytic (2 molecules of ATP) metabolism, symmorphosis predicts that having excess oxidative capacity actually reduces feed efficiency. This inefficiency is manifest by having to maintain, ultimately using feed energy, the expensive inner mitochondrial proton gradient in the superfluous mitochondria. On this basis, we predict that established molecular controllers of mitochondrial biogenesis and oxidative capacity such as eNOS, SIN3 co-repressor, TFAM and PPARgamma may yield useful DNA markers and therapeutic targets for issues relating to frugal energetics, namely predisposition to obesity and starvation resilience.

Connective tissue has recently been found to play a role in mediating mammalian skeletal muscle atrophy. We investigated connective tissue remodelling in the skeletal muscle of a species of the Australian burrowing frog, Cyclorana alboguttata. Despite being inactive whilst aestivating, the frog shows an inhibition of muscle atrophy. Connective tissue size and distribution was measured in histological sections of the cruralis muscle of control and aestivating C. alboguttata. Using a custom written software application we could detect no significant difference in any connective tissue morphological parameter between the two treatment groups. Biochemical measurements of gelatinase activity showed 2-fold higher activity in aestivating gastrocnemius muscle than in controls (p < 0.001). We measured the messenger RNA transcript levels for C. alboguttata metalloproteinase 2 (MMP2) and tissue inhibitor of metalloproteinase 2 (TIMP2) in cruralis skeletal muscle using quantitative real-time PCR. The trend of reduced expression of the two genes in the aestivators did not meet statistical significance. This work indicates that aestivation in C. alboguttata leads to subtle and specific changes in some extracellular matrix remodelling factors. Their main impact is to maintain proportional representation of extracellular matrix components of skeletal muscle and therefore preserve the active frog phenotype.

We present the application of large-scale multivariate mixed-model equations to the joint analysis of nine gene expression experiments in beef cattle muscle and fat tissues with a total of 147 hybridizations, and we explore 47 experimental conditions or treatments. Using a correlation-based method, we constructed a gene network for 822 genes. Modules of muscle structural proteins and enzymes, extracellular matrix, fat metabolism, and protein synthesis were clearly evident. Detailed analysis of the network identified groupings of proteins on the basis of physical association. For example, expression of three components of the z-disk, MYOZ1, TCAP, and PDLIM3, was significantly correlated. In contrast, expression of these z-disk proteins was not highly correlated with the expression of a cluster of thick (myosins) and thin (actin and tropomyosins) filament proteins or of titin, the third major filament system. However, expression of titin was itself not significantly correlated with the cluster of thick and thin filament proteins and enzymes. Correlation in expression of many fast-twitch muscle structural proteins and enzymes was observed, but slow-twitch-specific proteins were not correlated with the fast-twitch proteins or with each other. In addition, a number of significant associations between genes and transcription factors were also identified. Our results not only recapitulate the known biology of muscle but have also started to reveal some of the underlying associations between and within the structural components of skeletal muscle.

This paper discusses the effects of severe nutritional restriction, both pre- and post-weaning, on development of skeletal muscle in food animals. Given recent predictions about growth in demand for muscle-foods in developing countries, the global community will need to face the food-feed dilemma, and balance efficiency of production against the quality-of-life aspects of local livestock husbandry. It is likely that production animals will be grown in successively more marginal environments and at higher stocking rates on unimproved pastures. Understanding the nutritional limits to animal growth at the level of muscle gene networks will help us find optima for nutrition, growth rate and meat yield. Genomic approaches give us unprecedented capacity to map the networks of control under nutritionally restricted conditions, though the challenges remain of identifying steps that regulate substrate flux. The paper describes some approaches currently being taken to understanding muscle development, and concludes that the genes contributing to two ruminant phenotypes should be mapped and characterized. These are: the capacity to depress metabolic rate in response to nutritional restriction; and the capacity to exhibit compensatory growth after restriction is relieved.

Long (6- to 9-mo) bouts of estivation in green-striped burrowing frogs lead to 28% atrophy of cruralis oxidative fibers ( P < 0.05) and some impairment of in vitro gastrocnemius endurance ( P < 0.05) but no significant deficit in maximal twitch force production. These data suggest the preferential atrophy of oxidative fibers at a rate slower than, but comparable to, laboratory disuse models. We tested the hypothesis that the frog limits atrophy by modulating oxidative stress. We assayed various proteins at the transcript level and verified these results for antioxidant enzymes at the biochemical level. Transcript data for NADH ubiquinone oxidoreductase subunit 1 (71% downregulated, P < 0.05) and ATP synthase (67% downregulated, P < 0.05) are consistent with mitochondrial quiescence and reduced oxidant production. Meanwhile, uncoupling protein type 2 transcription ( P = 0.31), which is thought to reduce mitochondrial leakage of reactive oxygen species, was maintained. Total antioxidant defense of water-soluble (22.3 ± 1.7 and 23.8 ± 1.5 μM/μg total protein in control and estivator, respectively, P = 0.53) and membrane-bound proteins (31.5 ± 1.9 and 42.1 ± 7.3 μM/μg total protein in control and estivator, respectively, P = 0.18) was maintained, equivalent to a bolstering of defense relative to oxygen insult. This probably decelerates muscle atrophy by preventing accumulation of oxidative damage in static protein reserves. Transcripts of the mitochondrially encoded antioxidant superoxide dismutase type 2 (67% downregulated, P < 0.05) paralleled mitochondrial activity, whereas nuclear-encoded catalase and glutathione peroxidase were maintained at control values ( P = 0.42 and P = 0.231), suggesting a dissonance between mitochondrial and nuclear antioxidant expression. Pyruvate dehydrogenase kinase 4 transcription was fourfold lower in estivators ( P = 0.11), implying that, in contrast to mammalian hibernators, this enzyme does not drive the combustion of lipids that helps spare hypometabolic muscle.

We propose a data-driven reverse engineering approach to isolate the components of a gene interaction and regulatory network. We apply this method to the construction of a network for bovine skeletal muscle. Key nodes in the network include muscle-specific genes and transcription factors. muscle-specific genes are identified from data mining the USA National Cancer Institute, Cancer Genome Anatomy Project database, while transcription factors are predicted by accurate function annotation. A total of 5 microarray studies spanning 78 hybridisations and 23 different experimental conditions provided raw expression data. A recently-reported analytical method based on multivariate mixed-model equations is used to compute gene co-expression measures across 624 genes. The resulting network included 102 genes (of which 40 were muscle-specific genes and 7 were transcription factors) that clustered in 7 distinct modules with clear biological interpretation.

The effect of growth path, as opposed to advancing age, on the biophysical and biochemical properties of muscle connective tissue was investigated. Nine-month old Brahman-cross steers were grown across either an uninterrupted path, or paths that incorporated weight-loss and then weight gain on two different diets: one group was realimented on pasture, whilst the other was realimented on a grain-based diet. Biophysical attributes of connective tissue toughness (Compression and Adhesion) in the semitendinosus muscle, were significantly reduced by treatment (P

Cystic fibrosis is associated with mutations of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated plasma membrane chloride channel. Cystic fibrosis patients have been reported to possess elevated sulfation of glycoconjugates, which may contribute to the pathogenesis of the disease. Sulfation of glycosaminoglycans by a cystic fibrosis pancreatic adenocarcinoma cell line homozygous for ΔF 508 (CFPAC-1), a control pancreatic cell line (PANC-1), two CFPAC-1 cell lines transfected with the gene for CFTR (PLJ-CFTR-4.7, TR20), and a mock-transfected CFPAC-1 control (PLJ-6) was investigated. Cells were radiolabeled with [ 35 S]sulfate and [ 3 H]glucosamine, and glycosaminoglycans secreted into the medium after 24 and 72 h were isolated. Chondroitinase ABC digestion of chondroitin/dermatan sulfate allowed the recovery of disaccharides which were analyzed for their degree of sulfation by strong anion-exchange HPLC. No differences in the extent of sulfation by any of the cell lines were noted. However, glycoaminoglycans synthesized by cystic fibrosis cells consistently exhibited twofold higher [ 35 S]-sulfate:[ 3 H]glucosamine ratios than the controls. We conclude that CFTR plays no role in the sulfation of chondroitin/dermatan sulfate by pancreatic cells and that isotope incorporation ratios alone are insufficient evidence of changes in sulfation levels.

Studies using lysosomal membrane vesicles have suggested that efflux of the sulfate that results from lysosomal glycosaminoglycan degradation is carrier-mediated. In this study, glycosaminoglycan degradation and sulfate efflux were examined using cultured skin fibroblasts and lysosomes deficient in the lysosomal enzyme N-acetylgalactosamine-4-sulfatase. Such fibroblasts store dermatan sulfate lysosomally, which could be labelled biosynthetically with Na2(35)SO4. The addition of recombinant N-acetylgalactosamine-4-sulfatase to the media of 35S labelled fibroblasts degraded up to 82% of the stored dermatan [35S] sulfate over a subsequent 96 h chase and released inorganic [35S] sulfate into the medium. In the presence of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), sulfate was reused to a minor extent in newly synthesized proteoglycan. Isolated granules from recombinant enzyme supplemented fibroblasts degraded stored dermatan [35S]sulfate to sulfate which was rapidly released into the medium at a rate that was reduced by the extra-lysosomal presence of the lysosomal sulfate transport inhibitors SITS, Na2SO4 and Na2MoO4. SITS also inhibited dermatan sulfate turnover, although it had no effect on the action of purified recombinant enzyme in vitro. These data imply that sulfate clearance occurred concomitantly with dermatan sulfate turnover in the lysosome even at high substrate loading, and that lysosome-derived sulfate, while available, is reutilized minimally in synthetic pathways.

Based on an association with extracellular matrix remodelling, mitosis, proliferation and adipogenic differentiation, the glycosaminoglycan hyaluronan (HA) was assessed as a marker for intramuscular fat (IMF) development (marbling) in bovine loin muscle (longissimus dorsi, LD). Loin samples collected from the quartering site of feedlot-finished Wagyu–Angus and Jersey–Limousin steers were assayed for percentage IMF (IMF%) and HA after assignment of AUS-MEAT marbling scores. There was a moderate phenotypic correlation (r2 = 0.69) between IMF% and marbling score but little variance was explained by HA concentration. Breed was not a significant factor in marbling score or IMF% but did influence the HA concentration of the LD, with Wagyu–Angus steers having 2-fold more HA than Jersey–Limousin steers at the same marbling score. The non-linear decline in fat-adjusted HA levels as marbling score increased suggests that HA concentration was associated with lean growth potential of the muscle rather than adipogenesis. Using a different experimental approach, differences in distribution and amount of HA could not be discerned in histological sections of LD from age-matched Wagyu–Hereford heifers allocated to a low (score 1) or medium (score 3) marbling score group. These findings were consistent with the absence of differences between the two groups for other indicators of fatness (IMF% and P8 fat depth), maturity and myofibre characteristics despite an increase in oxidative capacity of the muscle with age. The data support the conclusion that the concentration of HA in the LD alone was not predictive of development of intramuscular fat.

A full-length human N-acetylgalactosamine-4-sulphatase (4-sulphatase) cDNA clone was constructed and expressed in CHO-DK1 cells under the transcriptional control of the Rous sarcoma virus long terminal repeat. A clonal cell line expressing high activities of human 4-sulphatase was isolated. The maturation and processing of the human enzyme in this transfected CHO cell line showed it to be identical with that seen in normal human skin fibroblasts. The high-uptake precursor form of the recombinant enzyme was purified from the medium of the transfected cells treated with NH4Cl and was shown to be efficiently endocytosed by control fibroblasts and by fibroblasts from a mucopolysaccharidosis type-VI (MPS VI) patient. Enzyme uptake was inhibitable by mannose 6-phosphate. After uptake, the enzyme was processed normally in both normal and MPS VI fibroblasts and was shown both to correct the enzymic defect and to initiate degradation of [35S]sulphated dermatan sulphate in MPS VI fibroblasts. The stabilities of the recombinant enzyme and enzyme from human fibroblasts appeared to be similar after uptake. However, endocytosed enzyme has a significantly shorter half-life than endogenous human enzyme. The purified precursor 4-sulphatase had a similar pH optimum and catalytic parameters to the mature form of 4-sulphatase isolated from human liver.

The glycoconjugate component of cystic fibrosis (CF) epithelial secretions is abnormally sulfated. Previous studies have suggested that some but not all CF fibroblasts express this secondary defect. We tested the hypothesis that the major CF mutation ( Δ F 508 Δ F 508 ) is correlated with elevated sulfate transport, by measuring the rates of saturable and nonsaturable [35S]SO42− uptake in skin fibroblasts isolated from CF patients of known genotype. No significant differences were apparent between normal and CF fibroblasts.

Mucopolysaccharidosis type I (MPS I: McKusick 25280) is a clinically heterogenous lysosomal storage disorder which is caused by a variable deficiency in alpha-L-iduronidase activity (alpha-L-iduronide iduronohydrolase, EC 3.2.1.76). Cultured fibroblasts from an MPS I patient (cell line 2827) with a severe clinical phenotype (Hurler syndrome) have been characterized using immunochemical and biochemical techniques. Using a specific immunoquantification assay, we have demonstrated that cell line 2827 had an alpha-L-iduronidase protein content (189 ng/mg of extracted cell protein) at least six times greater than the mean level found in normal control fibroblasts (30 ng/mg of extracted cell protein). This was the only MPS I cell line, from a group of 23 MPS I patients, that contained greater than 7% of the mean level of alpha-L-iduronidase protein detected in normal controls. Cell line 2827 had very low alpha-L-iduronidase activity toward the fluorogenic substrate 4-methylumbelliferyl-alpha-L-iduronide, and a radiolabeled disaccharide substrate derived from heparin. Maturation studies of alpha-L-iduronidase in cell line 2827 showed apparently normal levels of alpha-L-iduronidase synthesis with delayed processing to the mature form. Subcellular fractionation experiments demonstrated alpha-L-iduronidase protein in lysosomal-enriched fractions isolated from cell line 2827, suggesting a normal cell distribution and supporting the proposed delayed processing. It is proposed that the MPS I patient described has an alpha-L-iduronidase gene mutation which affects both the active site and post-translational processing of the enzyme. This mutation must be structurally conservative because it does not result in instability either during maturation or in the lysosome.

The purpose of this study was to determine if estimated breeding value (EBV) of an animal’s sire and or the animal’s nutrition affected the structure of its M. longissimus thoracis et lumborum (LL) and, hence, the eating quality of meat derived from its carcass. Lambs were chosen based on the EBV of their sires in terms of post-weaning live weight (PWWT), post weaning fat at the C-site (PFAT), and post-weaning eye muscle depth (PEMD). Morphometric techniques were used to characterise muscle structure in terms of the distribution of intramuscular connective tissue; the variables together are called fascicular structure. Perimysial seam thickness and fascicular width were both influenced by sire estimated breeding values for PWWT, PFAT, and PEMD. Variation in fascicular structure was associated with an interaction between PEMD-EBV and PFAT-EBV of the sire. Fascicular width decreased with increased PEMD-EBV and increased with PFAT-EBV, but was not affected by PWWT-EBV. When the total seam thickness was adjusted to a common fascicular width, the lambs on a low plane of nutrition had relatively more intramuscular connective tissue than those on a high plane. The total seam thickness was negatively associated with PFAT-EBV and positively associated with PEMD-EBV. Warner Bratzler shear (WBS) peak force (PF) and initial yield were not associated with differences in sire EBV. The residual WBS shear force, peak force minus initial yield (PFIY), and compression values were negatively associated with nutrition but were positively associated with PWWT-EBV and PEMD-EBV of the sires. These latter 2 effects were moderated by nutrition. The data support the hypothesis that morphological characteristics of perimysium are genetically determined and nutritionally responsive. Variance in morphology accounted for some variance in the biophysical attributes of meat and may help explain why sheep with high muscling potential have tougher meat.

The effects of post-weaning nutrient restriction on growth, carcass characteristics and beef quality were determined. Belmont Red weaner steers (n = 100) were allocated to an initial slaughter group and 3 treatment groups of 120 days duration: rapid growth, slow growth and weight loss. The average daily gain of the groups were (mean ± s.e.): 0.81 ± 0.02, 0.29 ± 0.02 and –0.22 ± 0.01 kg/day, for the rapid growth, slow growth and weight loss groups, respectively. At the end of the treatment period, rapid growth steers had significantly (P

Japanese Black cattle are characterised by a unique ability to deposit intramuscular fat with lower melting temperature. In this study, 3 consecutive biopsies from Longissimus muscle tissue were taken and RNA isolated from 3 Japanese Black (Tajima strain) and 3 Holstein animals at age 11–20 months. The gene expression changes in these samples were analysed using a bovine fat/muscle cDNA microarray. A mixed-ANOVA model was fitted to the intensity signals. A total of 335 (4.8%) array elements were identified as differentially expressed genes in this breed × time comparison study. Genes preferentially expressed in Japanese Black are associated with mono-unsaturated fatty acid synthesis, fat deposition, adipogenesis development and muscle regulation, while examples of genes preferentially expressed in Holstein come from functional classes involved in connective tissue and skeletal muscle development. The gene expression differences detected between the Longissimus muscle of the 2 breeds give important clues to the molecular basis for the unique features of the Japanese Black breed, such as the onset and rate of adipose tissue development, metabolic differences, and signalling pathways involved in converting carbohydrate to lipid during lipogenesis. These findings will impact on industry management strategies designed to manipulate intramuscular adipose development at different development stages to gain maximum return for beef products.

Marbling is an important meat quality trait, in that it contributes directly to the value of beef on international markets. The development of marbling is not well understood, though there have been some significant recent discoveries regarding adipogenesis in general. This article describes a working hypothesis around the early events of marbling. It attempts to rationalise findings from several mammalian experimental systems on hyperplastic growth of adipocyte precursor cells.

This review describes the pattern of intramuscular fat accretion in cattle and the potential for its manipulation during both the pasture (or backgrounding) and intensive grain-finishing phases of development. A growth curve for the development of marbling in British and Japanese Black type breeds is discussed with the conclusion that 3 phases of development exist: (i) a period of growth up to ~200 kg hot carcass weight where intramuscular fat does not increase; (ii) a period of linear development as carcass weight increases from 200 to 450�kg; and (iii) the attainment of mature body size (~500 kg carcass weight depending on genotype) at which intramuscular fat content appears to reachea maximum. Data are also presented to show that the intramuscular and other fat depots develop at similar rates indicating that intramuscular fat is not a late maturing depot. Pre-finishing growth checks reduce the initial intramuscular fat at the start of finishing and this is translated into lower levels at the end of finishing. It is argued that the greatest potential for the manipulation of intramuscular fat accretion during fattening is via an increase in the net energy of the ration. Increasing net energy can be achieved by increasing the cereal grain content of the diet (grain v. grass); by feeding processed cereal grain, which allows both maximal rumen fermentation and small intestinal digestion of starch, and by increasing the lipid content of the diet. In addition it is proposed that the substrate supply or hormonal milieu can also be optimised, along with the availability of net energy to maximise fat accretion. The role of lipolysis (fat turnover) as a regulator of fat accretion is also discussed.

This study investigates pre-weaning growth of cattle and its effect on biochemical and histochemical markers of muscle development and subsequent biophysical attributes of eating quality. Combinations of cow (late pregnancy to mid-lactation) and pre-weaning (varying duration of access to a high-energy ration) supplementation were used to vary calf growth to weaning in 6 treatment groups. After weaning, calves were grazed together on pasture (backgrounding) and then grown rapidly on a feedlot ration (finishing) until slaughter. Biochemical and myofibre characteristics were determined in semitendinosus muscle samples collected just prior to weaning (7 months), at the end of backgrounding (13 months), and at slaughter (17 months). The concentration of sarcoplasmic protein and the activity of lactate dehydrogenase in the muscle at weaning were associated with differences in pre-weaning growth and both variables correlated positively with liveweight at weaning. Isocitrate dehydrogenase activity varied with sex, not treatment, at weaning and at the end of backgrounding. The size of myofibres at weaning related to differences in growth path and correlated positively with liveweight. Pre-weaning growth effects on these characteristics were not evident at slaughter. Biophysical properties of the meat were not affected by earlier growth path treatment, and were not correlated with biochemical characteristics or myofibre type profile. Variation in both shear peak force and adhesion was related to sex. We conclude that the effects of divergent early life growth do not persist 10 months after weaning, at least in meat quality characteristics.

The intrinsic properties (those extant at the time of slaughter) of bovine skeletal muscle as they relate to the subsequent quality attributes of beef are reviewed here. Attributes of bovine skeletal muscle that ultimately affect toughness, colour, fat content, flavour, juiciness, and nutritive value of beef are discussed. The dynamic nature of muscle development, particularly with regard to structure and composition, is highlighted. Variation in development of muscle structure and composition due to inherited (genetic) factors and environment (particularly nutrient supply) are described. Examples are given of the implications of sources of variation due to animal genotype, age, nutrient supply, and hormonal environment on muscle cellularity and growth, fibre type, connective tissue composition and structure as they affect meat quality attributes. Key intrinsic properties of muscle include muscle type, cellularity, size, myofibre type, connective tissue composition and structure, glycogen and fat content and proteolytic activity. Activity of the calpain system at slaughter is seen as an important attribute. Regulation of myofibrillar and connective tissue proteolysis in vivo are discussed together with implications for subsequent meat quality. Amongst the on-farm environmental factors, nutritional history and developmental pathway are identified as factors that can be responsible for significant variation in the intrinsic properties of muscle that contribute to variation in toughness, colour and fat content, and thus consumer liking of beef.

This review focuses on recent developments in the field of muscle biology that reflect on the problem of toughness in beef. Meat science has shown that post-mortem processing can make a large contribution to beef tenderness. However live-animal factors such as growth path and genotype also influence the toughness of beef either directly or indirectly through interactions with processing technologies. This review sets out to integrate recent developments in the field of meat science into a mechanistic overview of toughness, while still highlighting the biology of some important contributors. These contributors are discussed at several levels of order between the molecular and the whole animal. The myofibrillar component of muscle is identified as the major contributor to initial toughness particularly through the effects of variation in sarcomere length. Muscle fibre-types whilst important to the growth and development of the animal are yet to be linked convincingly with toughness. Connective tissue is seen to play a dominant role in the sensation of toughness in muscles where its content is high. In muscles that are generally used for table beef, the contribution of connective tissue is less significant. In either case its contribution to measurable toughness cannot be easily separated from that of the myofibrillar component and the review discusses various levels of interaction between these 2 major components of beef. The review covers aspects of muscle ultrastructure as far as they are pertinent to the problem of beef toughness. In particular it deals with current knowledge of post-mortem metabolism of muscle and the degradation of costameric structures. Molecules are considered that are likely to propagate tensional forces from sarcomeres across the sarcolemma to the extracellular molecules of the endomysium. While much of the research around these molecules has not been performed by meat scientists, the insights developed are likely to be important to our understanding of beef toughness.Technological approaches to the objective measurement of toughness are discussed, as well as recent developments in the field. The review takes an integrative approach to features of the life of the bovine that might impact on the toughness of beef derived from its carcass. Features of the animal's pre-slaughter experience, including stress and physical activity, have been shown to influence markedly the toughness of beef through mechanisms that are described at the tissue level. Features of the growth path that the animal followed during its development have also recently been shown to significantly reduce the toughness of beef and properties of the connective tissue component have been implicated. Areas of strategic research are identified that, in the author's opinion, will facilitate commercial- scale improvements in the tenderness of beef.

Lowe (oculocerebrorenal) syndrome (LS) is an X-linked disorder characterized by congenital cataracts, generalized hypotonia, mental retardation, and renal Fanconi syndrome. The basic defect remains unknown, but the possibility that fibroblasts express reduced sulfation of glycosaminoglycans has been studied in several laboratories. A mechanism involving overproduction of an enzyme (nucleotide pyrophosphatase) active against adenosine 3'-phosphate, 5'-phosphosulfate (PAPS) has been postulated. Decreased synthesis of normally sulfated glycosaminoglycans was also reported. We measured the synthesis of proteoglycans and glycosaminoglycans by incorporation of [3H]glucosamine and Na2(35)SO4 into cultured fibroblasts from four LS patients and related it directly to the synthesis in six normal fibroblast cultures. We found that the rate of synthesis varied greatly among the normal cultures (cv, 30%), but not significantly between LS and the normal. The LS fibroblasts' ability to sulfate glycosaminoglycans was assayed as the amount of 3H-glycosaminoglycan eluting at low ionic strength on anion exchange chromatography, the amount of non-sulfated disaccharide present in chondroitinase digests of labeled proteoglycans, and the ratio of 35S to 3H incorporation into proteoglycans. Each parameter suggested that the LS cells were synthesizing normally sulfated glycosaminoglycans (e.g. % delta Di-0S, 21 +/- 6 in normal; 27 +/- 6 in LS). The cells' ability to sulfate glycosaminoglycans was tested under conditions of markedly stimulated glycosaminoglycan synthesis, by treating the cultures with a beta-D-xyloside. LS and normal cells responded to the treatment by elevating the rate of synthesis of normally sulfated glycosaminoglycans (3.5-6-fold in normal, 3-7-fold in LS). Nucleotide pyrophosphatase activities were found to be elevated in each of our four LS cell strains as in the previous studies, excluding genetic heterogeneity as an explanation for our findings. We conclude that LS fibroblasts do not express defects in sulfation of glycosaminoglycans or in synthesis of proteoglycans.

Sorted muscle cells, cultured from a patient with nephropathic cystinosis, stored 100 times normal amounts of cystine. Subcellular fractionation and density-gradient centrifugation confirmed that the cystine was located in a lysosomal compartment. 2. Myoblasts from cystinotic patients in culture underwent fusion to myotubes in a normal fashion. 3. The free thiol cysteamine effectively depleted cystinotic-muscle cells of cystine. 4. Cultured myoblast and myotubes offered a unique system for investigating the effects of lysosomal storage on differentiated cell functions.

Monoiodotyrosine (MIT) crosses the lysosomal membrane of rat FRTL-5 thyroid cells by a carrier-mediated process. In egress studies, MIT lost from inside lysosomes was quantitatively recovered outside lysosomes as MIT, indicating that the compound was transported intact across the lysosomal membrane. In uptake studies, [125I]MIT entry required intact lysosomes and exhibited saturation kinetics. The apparent Km for MIT was approximately 1.5 microM and the Vmax was approximately 0.24 pmol/unit hexosaminidase/min. Countertransport of MIT was demonstrated, with an initial velocity of [125I]MIT uptake which reached a maximum at high intralysosomal MIT loading. Nonradioactive MIT and diiodotyrosine competed to approximately equivalent extents with [125I]MIT for uptake in countertransport experiments. The existence of a lysosomal MIT carrier in thyroid cells may explain how this product of thyroglobulin catabolism is transported to the cytosol for iodine salvage and reutilization.

Tyrosine countertransport was used to demonstrate the existence of a carrier system for neutral amino acids in the lysosomal membrane of FRTL-5 thyroid cells. In addition to tyrosine, the carrier system recognized the neutral amino acids leucine, histidine, phenylalanine, and tryptophan. Cystine and lysine, amino acids for which a lysosomal carrier system has been demonstrated, showed no competition with tyrosine for countertransport. The tyrosine system showed stereospecificity and cation independence. It did not require an acidic lysosome or the availability of free thiols. The apparent Km for tyrosine was approximately 100 microM; the energy of activation of the system was approximately 9.7 kcal/mol. This new lysosomal membrane carrier system for neutral amino acids resembles the plasma membrane L system in 3T3 Chinese hamster ovary cells and melanoma B-16 cells.

Viscometry and gel chromatography of mixtures of proteoglycans with other linear flexible polymers suggest that proteoglycans shrink as the concentration of the linear polymer is increased. Similar behavior was observed for binary proteoglycan solutions using a differential viscometry procedure. The shrinkage does not involve specific chemical properties of the linear polymer, but rather is a consequence of purely entropic excluded volume interactions with the proteoglycans. Comparison with a hydrodynamic model supports this conclusion. The polydisperse proteoglycan preparation was subfractionated, and the individual fractions were tested for shrinkage. Fractions of lower molecular weight were found to shrink to a greater extent, suggesting that the molecules are more flexible when they contain fewer glycosaminoglycan chains.

Tyrosine countertransport was used to demonstrate the hormonal stimulation of neutral amino acid transport across the lysosomal membrane of FRTL-5 cells. Cells grown with thyrotropin (1 X 10(-10) M) had 7-fold (+/- S.E.) higher tyrosine countertransport activity in their lysosomes than cells grown without thyrotropin. Thyrotropin also stimulated the uptake into tyrosine-loaded lysosomes of other neutral amino acids recognized by the tyrosine carrier, namely, phenylalanine (3-fold) and leucine (6-fold). In contrast lysosomal cystine countertransport was not affected by thyrotropin. Addition of thyrotropin to cells grown without thyrotropin showed that the stimulation of tyrosine counter-transport (a) required at least 48 h to reach the level of the thyrotropin-supplemented cells, (b) depended upon protein synthesis, since cycloheximide (20 microM) was inhibitory, and (c) depended upon RNA synthesis, since actinomycin D (1 nM) was inhibitory. Cells grown without thyrotropin but with dibutyryl cyclic AMP (1 mM) or cholera toxin (1 nM) exhibited enhanced lysosomal countertransport of tyrosine, suggesting that cyclic AMP may act as a messenger. This represents the first demonstration of hormonal responsiveness in a lysosomal transport system and may reflect the importance of salvage and reutilization of lysosomal degradation products for the thyroid epithelial cell.

The rapid transport of cells has been shown to occur by ordered countercurrent convection. This convection can be created by mixtures of macromolecules which make up the extracellular matrix and by the degradation and aggregation products of these macromolecules. The ordered countercurrent convection is manifested in the form of structured flows and arises in isothermal systems with small concentration gradients of solutes. The flows are gravity driven but may rapidly move at angles close to the horizontal axis if they are mechanically constrained to do so. These flows have been shown to rapidly transport cells at rates ranging from 1 to 100 mm h-1, depending on the conditions of the experiment. The transport of cells is nonspecific in that various cell types (chondrocytes, fibroblasts, endothelial cells, and red blood cells) as well as inert particles of similar size (latex beads 6-microns diam) are transported at similar rates. Latex bead transport by structured flow has also been demonstrated to occur in confined spaces in the form of Teflon tubing down to 200 microns in diameter and at angles in the range of 45-90 degrees to the horizontal axis. The flows may also occur over relatively long distances for a prolonged period of time. The conditions for flow formation are simple and widespread. It is suggested that it may contribute to the forces involved in the movement of cells in the extracellular matrix in vivo especially during remodeling and embryogenesis.

Egress of free NeuAc from normal lysosome-rich granular fractions was assessed at NeuAc concentrations of up to 221 pmol/hexosaminidase unit, achieved by exposure of growing fibroblasts to 40-125 nM N-acetylmannosamine for up to 7 days. The normal velocity of NeuAc egress increased with NeuAc loading and with temperature, exhibiting a Q10 of 2.4, characteristic of carrier-mediated transport. Fibroblasts cultured from five patients with infantile free sialic acid storage disease (ISSD) contained approximately 139 nmol of free NeuAc/mg of whole cell protein, or 100 times the normal level. Differential centrifugation, as well as density gradient analysis using 25% Percoll, showed that the stored NeuAc cosedimented with the lysosomal enzyme beta-hexosaminidase. The velocity of appearance of free NeuAc outside ISSD granular fractions was negligible, even at initial loading levels of up to 3500 pmol/hexosaminidase unit. The lack of egress from ISSD granular fractions was found for both endogenous and N-acetylmannosamine-derived NeuAc. Fibroblasts from ISSD parents did not accumulate excess free NeuAc and did not display a velocity of NeuAc egress significantly different from normal. The defect in ISSD, like that in Salla disease, appears to be an impairment of carrier-mediated transport of free NeuAc across the lysosomal membrane. Clinical and biochemical differences between Salla disease and ISSD may reflect differences in the amount of residual NeuAc transport capacity.

Proteoglycans synthesized by cultured human muscle cells were separated by ion-exchange high-performance liquid chromatography using a Bio-gel TSK DEAE 5-PW analytical column. The procedure requires only 40 min to complete. The same analytical size column can be used for either analytical or semipreparative scale separations without significant loss of resolution. Proteoglycans elute from the TSK column with a similar recovery and at similar elution ionic strengths when compared to the established cellulose-based chromatographic gel, DEAE-Sephacel. The technique has been applied to the analysis of chondroitinase-digested samples and is particularly useful for rapid screening of large numbers of cultures for both biosynthetic rate studies and analysis of patterns of proteoglycan synthesis.

The mutual diffusion coefficient of the bovine nasal cartilage proteoglycan subunit is found to increase rapidly with increasing concentration and decreasing ionic strength. These results have been obtained by analysis of the boundary relaxation of concentration gradients in the analytical ultracentrifuge by schlieren optics. The diffusion behavior can be understood in terms of the nonideality of the proteoglycan. The magnitude of the nonideality is dominated by charge interactions, whereas the influence of molecular size and associated excluded-volume interactions is small. The concentration dependence of the apparent diffusion coefficient of the proteoglycan subunit from dynamic light scattering was found, in contrast, to decrease with increasing concentration. Computer simulation of the dynamic light scattering suggests that the presence of a small population of aggregates may account for the difference in the two types of diffusion measurement due to their marked influence on the scattering.