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Biomedical subjects

S A Lehnert

Publications and source records attributed to S A Lehnert.

13 recordsLinked to original sources
PubMed2006

Gene expression profiling of bovine skeletal muscle in response to and during recovery from chronic and severe undernutrition.

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.

Animal Feed↗
PubMed2005

Gene expression profiling of muscle tissue in Brahman steers during nutritional restriction.

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.

Analysis of Variance↗
PubMed2004

Joint analysis of multiple cDNA microarray studies via multivariate mixed models applied to genetic improvement of beef cattle.

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.

Animals↗
PubMed2004

A mixed-model approach for the analysis of cDNA microarray gene expression data from extreme-performing pigs after infection with Actinobacillus pleuropneumoniae.

We proposed a novel statistical approach for the analysis of cDNA experiments based on mixed-model methodology combined with mixtures of distributions. Our objective was to detect genes that may be involved in conferring heritable differences in susceptibility to common infections in intensive pig production. We employed a microarray expression profiling strategy and a mixed-model approach to the analysis of the expression data. A cDNA microarray of pig with 6,420 probes from immune tissues and cells was used to compare gene expression in peripheral blood leukocytes of two pigs showing extreme performance in their response to infection with Actinobacillus pleuropneumoniae. Principal components analyses were used to identify the two most extreme-performing pigs after infection (i.e., pigs whose measured responses to infection fell at the extremes). Blood samples and expression profiles from 0 to 24 h after infection were compared using a bivariate, mixed-model approach, in which the effect gene x immunological status interaction was treated as a random effect. Bayesian model-based clustering via mixtures of normal distributions of the resulting BLUP of the random interaction was approached and resulted in a list of 307 differentially expressed genes, of which 179 were down-regulated in the susceptible pig. The majority of the differentially expressed genes were derived from a cDNA library of leukocytes of A. pleuropneumoniae-challenged pigs that were subtracted against leukocytes before the challenge. These results provide evidence that the proposed statistical approach was useful in enhancing the knowledge of the mechanisms involved in the genetics of the immune response.

Actinobacillus Infections↗
PubMed2003

A mixture model-based cluster analysis of DNA microarray gene expression data on Brahman and Brahman composite steers fed high-, medium-, and low-quality diets.

The objective of this study is to explore aspects of the statistical analysis of gene expression response at the muscle tissue level to varying levels of energy and protein in the diet. Eleven Brahman and Brahman composite steers (weighing 302 +/- 9.8 kg, on average) were allocated randomly into high- (HIGH), medium- (MED), and low- (LOW) quality forage diets for 27 d. After this period, a biopsy of the longissimus dorsi muscle was taken from each animal and total RNA was extracted to generate the labeled target for microarray experimentation. These targets were hybridized to a complementary DNA (cDNA) microarray of 9,274 probes from cattle muscle and subcutaneous fat cDNA libraries. After edits, 151,904 expression intensity levels of 4,747 genes were analyzed. Emphasis was given to the choice of power transformation of the intensity channel readings and to the consistency of readings within each diet quality group. The statistical approach to isolate differentially expressed genes was based on model-based clustering via a mixture of normal distributions estimated through maximal likelihood. The base-2 logarithm was found to be the optimal power transformation to normalize gene intensity levels. A two-sample t-statistic was defined as a measure of possible differential expression. For each of the three diet contrasts, HIGH vs. LOW, HIGH vs. MED, and MED vs. LOW, three clusters were found, two of which contained more than 94% genes with almost no altered gene expression levels, whereas the third cluster contained the remaining genes with a differential expression. Results from the HIGH vs. LOW contrast identified 27 genes with a greater than 95% posterior probability of belonging to the cluster of differentially expressed genes.

Animal Feed↗
PubMed2001

Mapping of 12 bovine ribosomal protein genes using a bovine radiation hybrid panel.

Twelve bovine ribosomal protein genes, for which sequence data had been acquired from complementary deoxyribonucleic acid (cDNA) clones isolated from a cattle skin cDNA library, were mapped. As ribosomal protein genes are a group of highly conserved house keeping genes, specific primers were designed to span the intron-exon splice sites and to amplify intronic sequences, in order to obtain bovine-specific polymerase chain reaction (PCR) products. Two of 12 ribosomal protein genes were genotyped in this way and the remaining 10 were mapped using additional primers designed from within the intron. Eleven previously unmapped ribosomal protein genes were localized and one previously reported ribosomal protein gene localization was confirmed. The 12 ribosomal protein genes mapped in this study are spread over 10 chromosomes, including the X chromosome. The locations show conservation of comparative map position in cattle and human.

Animals↗
PubMed1999

Isolation of a cDNA encoding a putative cellulase in the red claw crayfish Cherax quadricarinatus.

Amino acid sequences of cellulases have been determined in insects, nematodes, plants, slime moulds and bacteria but not in crustaceans. However, cellulase activity has been demonstrated in the hepatopancreas of the red claw crayfish, Cherax quadricarinatus. In order to obtain information on the nature of this cellulase, a C. quadricarinatus hepatopancreas cDNA library was screened with a PCR product generated using degenerate oligonucleotide primers derived from conserved regions of known cellulases. Two identical 1.56kb cDNAs with sequence similarities to known cellulases, particularly the termite endoglucanases, were identified and sequenced. The clones contain the complete cDNA open reading frame for an endo-1, 4-beta-glucanase of 469 amino acids termed Cherax quadricarinatus endoglucanase (CqEG). The endogenous origin of the gene was confirmed by PCR amplification and sequencing of a 1012bp PCR product from genomic DNA. This fragment contains four exon sequences identical to the cDNA and is interrupted by three introns of 371, 102, 194bp respectively, with one intron exhibiting typical eukaryotic splice sites. The isolation of an endo-1,4-beta-glucanase encoding cDNA from the crayfish C. quadricarinatus provides the first endogenous cellulase sequence in a crustacean species.

Amino Acid Sequence↗
PubMed1993

An engineered PGK promoter and lac operator-repressor system for the regulation of gene expression in mammalian cells.

Previous reports have demonstrated that the Escherichia coli lac repressor can operate effectively in mammalian cells to repress expression of genes driven by modified viral or metallothionein (MT) promoters. We have developed a more general expression system using the promoter from the PGK1 gene (encoding murine 3-phosphoglycerate kinase) which is widely expressed in almost all cell types, including early embryonic and ES (embryonic stem) cells. Firstly, we engineered the lac repressor to include a nuclear localisation signal and placed it under control of the PGK1 promoter. Efficient nuclear localisation of the repressor was demonstrated by mobility-shift assays and immunofluorescence detection. For the target vectors, we modified the wild-type (wt) PGK1 promoter to include lac operator (lacO) sites for binding of the lac repressor and compared a number of different lacO positions and arrangements based on proximity to the native start points for transcription (tsp) and translation. In the absence of repressor, we observed reduced expression of the neo reporter gene for some placements of the lacO, but wt expression for placements near the tsp. When both target and repressor were present in the cells, we observed that the expression of neo could be strongly suppressed and reversibly regulated by induction with IPTG. In particular, for a promoter which contained two spaced lacO replacing native sequence around the major tsp, we observed 90-95% repression by the lac repressor for the neo reporter gene and up to 98% repression for the cat reporter gene. Efficient derepression by IPTG was observed in both cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Transforming growth factor betas in mammalian embryogenesis.

Type beta transforming growth factors (TGF beta s) are members of a large superfamily of related proteins, each of which plays a pivotal role in embryonic processes. The TGF beta s per se are at least five in number, though only three isoforms have been identified in mammals. Here we will review the evidence, taken from in vitro studies on bioactivity and histochemical localization of RNAs and encoded proteins in vivo, that TGF beta 1, beta 2 and beta 3 are involved in several mammalian developmental processes, including control of growth, differentiation, tissue inductions and morphogenesis.

Animals↗
PubMed1990

TGF beta in murine morphogenetic processes: the early embryo and cardiogenesis.

The tissue distribution of TGF beta-1 RNA was examined within whole mouse embryos from implantation to 10.5 days gestational age and, in the developing heart, up to 8 days postpartum. The earliest high level expression of TGF beta-1 RNA is at 7.0 days postcoitum (p.c.) in the cardiac mesoderm. At 8.0 days gestational age, cardiac TGF beta-1 RNA expression is limited to endocardial cells. By 9.5 days p.c., this expression pattern becomes regionalized to those cells that overlie cardiac cushion tissue. High TGF beta-1 RNA levels continue to persist in endothelial cells of the heart valves until approximately one week postpartum. The TGF beta-1 RNA distribution was compared with the extracellular distributions of polypeptides for TGF beta and J1/tenascin. As previously reported, endothelial expression of TGF beta-1 RNA is correlated with mesenchymal expression of TGF beta polypeptide, suggesting a paracrine mode of action for this growth factor in cardiac development. Minor discrepancies in the distributions of TGF beta-1 RNA and the extracellular form of the TGF beta polypeptide suggest that translational or post-translational control of protein levels occurs and/or the possibility that the antibody used may also recognise other members of the TGF beta polypeptide family. A correlation between endothelial TGF beta-1 expression and distribution of J1/tenascin in the mesenchyme gives further support to the proposition that the biological effects of TGF beta-1 may, in part, be mediated by J1/tenascin.

Animals↗
PubMed1988

Embryonic expression pattern of TGF beta type-1 RNA suggests both paracrine and autocrine mechanisms of action.

The tissue distribution of TGF beta form 1 RNA within mouse embryos of 10.5 to 15.5 days gestational age was investigated using in situ hybridization. As predicted from the prevalence of TGF beta-1 protein in adult bone and platelets, the RNA is highly abundant in fetal bone and in fetal liver megakaryocytes. Our data also reveal previously undocumented sites of synthesis for TGF beta-1, namely epithelia overlying those mesenchymal tissues that are known to contain TGF beta protein as detected by immunohistochemical methods (Heine et al. 1987) and in the mesenchymal tissues of certain internal organs. From a combined knowledge of the distribution of the TGF beta polypeptide (Heine et al. 1987) and its mRNA, and a knowledge of the reported biological activities of TGF beta-1, we invoke both paracrine and autocrine mechanisms of action for this growth factor.

Animals↗