ABSTRACT
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 P < 0.001); however, there was little difference in the percentage of the different myofiber types (all P > 0.10). 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 (P < 0.001). During recovery from postweaning nutritional restriction, significant differences in major myofiber type percentages were not evident (all P > 0.10), and by 721 d CSA of myofiber types differed little between the treatment groups, although SG had greater CSA of type 1 (P < 0.05) and type 2A (P < 0.01) myofibers than WL and RG. At 721 d, the relative area of type 2A myofibers was less in WL compared with SG (P < 0.01) and RG (P < 0.05) and of type 2X myofibers greater (P < 0.05) 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.
Subject(s)
Animal Nutritional Physiological Phenomena/physiology , Cattle/physiology , Muscle Fibers, Skeletal/physiology , Animals , Australia , Biopsy/veterinary , Body Weight/physiology , Immunohistochemistry/veterinary , Meat , Random Allocation , Tropical ClimateABSTRACT
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.
Subject(s)
Animal Nutritional Physiological Phenomena , Cattle/metabolism , Gene Expression Profiling , Malnutrition/genetics , Muscle, Skeletal/metabolism , Animal Feed , Animals , Diet , Gene Expression Regulation , Male , Muscular Atrophy/metabolism , Random Allocation , Time Factors , Weight Gain/genetics , Weight Loss/geneticsABSTRACT
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 (P < 0.05). Muscle samples at slaughter from supplemented animals contained significantly (P < 0.01) 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 (P < 0.001) 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 (P< 0.05) 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.
Subject(s)
Adipose Tissue/metabolism , Fatty Acids/metabolism , Vitamin A/metabolism , Vitamin A/pharmacology , Animal Feed , Animal Husbandry/methods , Animals , Cattle , Male , Vitamin A Deficiency/metabolism , Vitamin A Deficiency/veterinary , beta Carotene/bloodABSTRACT
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 ß-agonists, anabolic implants and CLA to maintain or improve meat quality.
ABSTRACT
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.
Subject(s)
Animal Nutritional Physiological Phenomena , Cattle/genetics , Food Deprivation/physiology , Gene Expression Profiling/veterinary , Muscle, Skeletal/physiology , Analysis of Variance , Animal Husbandry , Animals , Cattle/physiology , DNA Primers/chemistry , Diet/veterinary , Down-Regulation/physiology , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation/physiology , Glyceraldehyde-3-Phosphate Dehydrogenases/physiology , Likelihood Functions , Male , Models, Genetic , Muscle Proteins/genetics , Muscle Proteins/metabolism , Oligonucleotide Array Sequence Analysis/veterinary , Peptide Fragments/physiology , RNA, Antisense/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Up-Regulation/physiologyABSTRACT
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.
Subject(s)
Cattle/genetics , Oligonucleotide Array Sequence Analysis/veterinary , Animals , Breeding , DNA, Complementary , Gene Expression Profiling/methods , Gene Expression Profiling/veterinary , Gene Expression Regulation , Multivariate Analysis , Oligonucleotide Array Sequence Analysis/methodsABSTRACT
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. [35S]Sulphate uptake was rapid and reached near steady state levels within 10 min. However, the intracellular specific activity of [35S]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 [35S]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 [35S]sulphate:[3H]glucosamine ratios incorporated into glycosaminoglycans (GAGs); however, stimulating the synthesis of GAGs with 4-methylumbelliferyl-beta-d-xyloside, stably elevated [35S]:[3H] ratios. This was due to higher [35S]sulphate incorporation. [35S]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.
Subject(s)
Glycosaminoglycans/biosynthesis , Pancreas/metabolism , Sulfates/metabolism , Cell Line , Cystic Fibrosis/metabolism , Epithelial Cells/metabolism , Extracellular Matrix/metabolism , Humans , Hymecromone/analogs & derivatives , Sodium Chloride/pharmacologyABSTRACT
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<0.05): weight loss with grain realimentation being the least tough in regard to the connective tissue component. Variance within the biophysical attributes was modelled statistically. Statistically significant models included terms for the post-slaughter connective tissue content as well as tissue contents of the enzymes lactate dehydrogenase and isocitrate dehydrogenase. The data suggest that biochemical measurements made up to 100 days prior to slaughter, may have value as indicators of final connective tissue toughness.
ABSTRACT
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.
ABSTRACT
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 DeltaF508 (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 [35S]sulfate and [3H]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 [35S]-sulfate:[3H]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.
Subject(s)
Chondroitin Sulfates/metabolism , Cystic Fibrosis/metabolism , Dermatan Sulfate/metabolism , Pancreatic Ducts/metabolism , Cell Line , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , HumansABSTRACT
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 (P < 0.05). 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 (P < 0.05). 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.
Subject(s)
Cystic Fibrosis/metabolism , Extracellular Matrix/metabolism , Glycosaminoglycans/metabolism , Animals , Chondroitin Sulfates/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Dermatan Sulfate/metabolism , Female , Heparitin Sulfate/metabolism , Male , Mice , Organ Specificity , Sulfates/metabolismABSTRACT
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.
Subject(s)
Antigens, Protozoan/biosynthesis , Babesia bovis/immunology , Erythrocytes/parasitology , Animals , Antibodies, Protozoan , Antigens, Protozoan/blood , Antigens, Protozoan/chemistry , Babesia bovis/physiology , Cattle , Cell Line , Fluorescent Antibody Technique, Indirect , Kinetics , Mice , Microscopy, Confocal , Oligosaccharides/chemistry , Oligosaccharides/isolation & purification , Recombinant Proteins/biosynthesis , Spodoptera , TransfectionABSTRACT
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.
Subject(s)
Chondro-4-Sulfatase/pharmacology , Glycosaminoglycans/metabolism , Lysosomes/metabolism , Mucopolysaccharidosis VI/metabolism , Sulfates/metabolism , Cells, Cultured , Chondro-4-Sulfatase/deficiency , Fibroblasts/metabolism , Humans , In Vitro Techniques , Sulfur RadioisotopesABSTRACT
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.
Subject(s)
Chondro-4-Sulfatase/metabolism , Lysosomes/enzymology , Mucopolysaccharidosis VI/enzymology , Chondro-4-Sulfatase/genetics , Genetic Therapy , Glycosaminoglycans/metabolism , Humans , Recombinant Proteins/metabolism , Skin/enzymology , TransfectionABSTRACT
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.
Subject(s)
Iduronidase/analysis , Mucopolysaccharidosis I/metabolism , Proteins/analysis , Cells, Cultured , Child, Preschool , Fibroblasts/metabolism , Humans , Lysosomes/enzymology , Male , Subcellular Fractions/metabolismABSTRACT
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 (delta F508/delta F508) is correlated with elevated sulfate transport, by measuring the rates of saturable and nonsaturable [35S]SO4(2-) uptake in skin fibroblasts isolated from CF patients of known genotype. No significant differences were apparent between normal and CF fibroblasts.
Subject(s)
Cystic Fibrosis/metabolism , Sulfates/metabolism , Biological Transport , Cells, Cultured , Fibroblasts/cytology , Humans , Sulfates/analysis , Sulfur RadioisotopesABSTRACT
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.
Subject(s)
Chondro-4-Sulfatase/genetics , Mucopolysaccharidosis VI/metabolism , Skin/enzymology , Alkaline Phosphatase/genetics , Animals , Avian Sarcoma Viruses/genetics , Base Sequence , CHO Cells , Cells, Cultured , Chondro-4-Sulfatase/deficiency , Chondro-4-Sulfatase/metabolism , Cloning, Molecular , Cricetinae , DNA/genetics , DNA/isolation & purification , Endocytosis , Fibroblasts/enzymology , Glycoconjugates/metabolism , Humans , Molecular Sequence Data , Molecular Weight , Oligodeoxyribonucleotides , Recombinant Proteins/metabolism , Repetitive Sequences, Nucleic Acid , Restriction Mapping , Transcription, Genetic , TransfectionABSTRACT
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.
Subject(s)
Carbohydrate Metabolism, Inborn Errors/metabolism , Lysosomes/metabolism , Sialic Acids/metabolism , Carbohydrate Metabolism, Inborn Errors/genetics , Cell Fractionation/methods , Cells, Cultured , Centrifugation, Density Gradient/methods , Fibroblasts/metabolism , Genetic Carrier Screening , Humans , Infant , Kinetics , Lysosomes/ultrastructure , N-Acetylneuraminic Acid , Reference ValuesABSTRACT
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.
Subject(s)
Carrier Proteins/physiology , Lysosomes/metabolism , Monoiodotyrosine/metabolism , Thyroid Gland/metabolism , Animals , Biological Transport , Cell Line , Cell-Free System , Diiodotyrosine/metabolism , Kinetics , RatsABSTRACT
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.