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A Subramaniam

Publications and source records attributed to A Subramaniam.

At least 19 recordsLinked to original sources
PubMed2006

Aspects regarding measurement of thickness of intergranular glassy films.

Materials such as Si(3)N(4), SiC and SrTiO(3) can have grain boundaries characterized by the presence of a thin intergranular amorphous film of nearly constant thickness, in some cases (e.g. Si(3)N(4)) almost independent of the orientation of the bounding grains, but dependent on the composition of the ceramic. Microscopy techniques such as high-resolution lattice fringe imaging, Fresnel fringe imaging and diffuse dark field imaging have been applied to the study of intergranular glassy films. The theme of the current investigation is the use of Fresnel fringes and Fourier filtering for the measurement of the thickness of intergranular glassy films. Fresnel fringes hidden in high-resolution micrographs can be used to objectively demarcate the glass-crystal interface and to measure the thickness of intergranular glassy films. Image line profiles obtained from Fourier filtering the high-resolution micrographs can yield better estimates of the thickness. Using image simulation, various kinds of deviation from an ideal square-well potential profile and their effects on the Fresnel image contrast are considered. A method is also put forth to objectively retrieve Fresnel fringe spacing data by Fourier filtering Fresnel contrast images. Difficulties arising from the use of the standard Fresnel fringe extrapolation technique are outlined and an alternative method for the measurement of the thickness of intergranular glassy films, based on zero-defocus (in-focus) Fresnel contrast images is suggested. The experimental work is from two ceramic systems: Lu-Mg-doped Si(3)N(4) and SrTiO(3) (stoichiometric and nonstoichiometric). Further, a comparison is made between the standard high-resolution lattice fringe technique, the standard Fresnel fringe extrapolation technique and the methods of analyses introduced in the current work, to illustrate their utility and merits. Taking experimental difficulties into account, this work is intended to be a practical tool kit for the study of intergranular glassy films.

Journal Article↗
PubMed2003

Increased fatty acid oxidation in transgenic mice overexpressing UCP3 in skeletal muscle.

AIM: To determine the rates of substrate oxidation by skeletal muscle in vitro as well as tissue-specific glucose uptake in vivo in transgenic mice overexpressing uncoupling protein-3 (UCP3) in skeletal muscle. METHODS: Soleus muscle was isolated from transgenic mice overexpressing UCP3 in skeletal muscle and wild-type mice. Rates of [1-14C]-palmitate oxidation and [2-14C]-pyruvate oxidation were determined by in vitro incubation of the soleus muscle. Tissue glucose uptake rates were characterized during a glucose tolerance test using 2-deoxy-[1-3H]-glucose as a tracer. RESULTS: Oxidation of [1-14C]-palmitate to CO2 by isolated soleus muscle was increased in UCP3 transgenic mice (0.45 +/- 0.03 vs. 0.24 +/- 0.02 micro mol/h/g). [2-14C]-pyruvate oxidation, which is a measure of the activity of pyruvate carboxylase in introducing pyruvate carbon into the tricarboxylic acid cycle, was increased 1.4-fold in the presence of fatty acid in the UCP3 transgenic mice (3.84 +/- 0.28 vs. 5.36 +/- 0.29 micro mol/h/g). The plasma glucose concentration after an overnight fast was significantly lower in the UCP3 transgenic mice (3.56 +/- 0.37 vs. 5.11 +/- 0.33 m/mol). Only brown adipose tissue from the UCP3 transgenic mice showed increased tissue glucose uptake rates compared with the wild-type mice. Skeletal muscle uptake rates of 2-deoxyglucose were either unchanged (soleus and gastrocnemius) or reduced (diaphragm) in the UCP3 transgenic mice. CONCLUSIONS: The improved glucose tolerance in the UCP3 transgenic mice does not appear to be the result of increased uptake into peripheral tissues. The increased fatty acid oxidation in skeletal muscle of UCP3 transgenic mice supports the proposed role of UCP3 in the export of fatty acid anions from mitochondria during fatty acid oxidation.

Animals↗
PubMed1999

A new role for neurotrophins: involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control.

Neurotrophins exert many biological effects not directly targeted at neurons, including modulation of keratinocyte proliferation and apoptosis in vitro. Here we exploit the cyclic growth and regression activity of the murine hair follicle to explore potential nonneuronal functions of neurotrophins in the skin, and analyze the follicular expression and hair growth-modulatory function of BDNF, NT-4, and their high-affinity receptor, TrkB. The cutaneous expression of BDNF and NT-4 mRNA was strikingly hair cycle dependent and peaked during the spontaneous, apoptosis-driven hair follicle regression (catagen). During catagen, BDNF mRNA and immunoreactivity, as well as NT-4-immunoreactivity, were expressed in the regressing hair follicle compartments, whereas TrkB mRNA and immunoreactivity were seen in dermal papilla fibroblasts, epithelial strand, and hair germ. BDNF or NT-4 knockout mice showed significant catagen retardation, whereas BDNF-overexpressing mice displayed acceleration of catagen and significant shortening of hair length. Finally, BDNF and NT-4 accelerated catagen development in murine skin organ culture. Together, our data suggest that BDNF and NT-4 play a previously unrecognized role in skin physiology as agents of hair growth control. Thus, TrkB agonists and antagonists deserve exploration as novel hair growth-modulatory drugs for the management of common hair growth disorders.

Animals↗
PubMed1998

BDNF overexpression induces differential increases among subsets of sympathetic innervation in murine back skin.

Besides their recognized dependence on nerve growth factor (NGF) during development, the dependence of mature sympathetic ganglion neurons on other neurotrophins is still unclear. Here, we have investigated the sympathetic innervation of back skin in mice overexpressing brain-derived neurotrophic factor (BDNF) under the alpha-myosin heavy-chain promoter, as well as in BDNF knockout (-/-) mice. Compared with wild-type controls, the dorsal skin of BDNF overexpressing mice displayed a significantly enhanced number of adrenergic, tyrosine hydroxylase-immunoreactive (IR) nerve fibres, while cholinergic or peptidergic sensory nerve fibres appeared unaltered. The adrenergic hyperinnervation in dorsal skin of BDNF overexpressing mice was most pronounced in the arrector pili muscle of hair follicles, while no increase of tyrosine hydroxylase-or neuropeptide Y-IR fibres associated with subcutaneous blood vessels was found. Instead, back skin of BDNF knockout (-/-) mice contained significantly fewer tyrosine hydroxylase-IR dermal nerve fibres than wild-type animals. This suggests that BDNF plays an important role in the control of different subsets of adrenergic innervation in murine back skin, and indicates that paravertebral sympathetic ganglia display a previously unrecognized differential BDNF-dependence in vivo.

Adrenergic Fibers↗
PubMed1997

Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris.

Numerous heterologous proteins have been produced at greater than gram per liter levels in the methylotrophic yeast, Pichia pastoris, using the methanol oxidase promoter. The factors that drastically influence protein production in this system include: copy number of the expression cassette, site and mode of chromosomal integration of the expression cassette, mRNA 5'- and 3'-untranslated regions (UTR), translational start codon (AUG) context, A+T composition of cDNA, transcriptional and translational blocks, nature of secretion signal, endogenous protease activity, host strain physiology, media and growth conditions, and fermentation parameters. All these factors should be considered in designing an optimal production system. The inherent ability of P. pastoris to convert the zymogen (pro-enzyme) form of matrix metalloproteinases (MMP) into active mature forms (which tend to self-degrade, and in some instances also cause damage to cells), largely limits the use of this system for the production of MMP. However, this problem can be partly alleviated by co-expression of tissue inhibitor of MMP (TIMP-1).

Cloning, Molecular↗
PubMed1995

An in vivo analysis of transcriptional elements in the mouse alpha-myosin heavy chain gene promoter.

During development in the murine ventricle, there is a switch in myosin heavy chain gene (MyHC) transcription. The beta-MyHC is expressed in the ventricles during foetal development, but is shut down at or around birth, at which time alpha-MyHC transcription is activated. This antithetical switch is thought to be mediated by circulating levels of thyroid hormone (TH) and both low and high affinity thyroid response elements (TREs) have been identified in the proximal promoter region of the murine alpha-MyHC. Myosin gene expression in the atria is relatively unaffected by the TH status. Previously, we used site-directed mutagenesis of the promoter in a transgenic analysis to define those elements responsible for high levels of transcription in vivo. These analyses focused on the role(s) of two cis elements, TRE1 and TRE2 that are located at -129 to -149 and -102 to -120, respectively, on the alpha-MyHC promoter. Although the elements' ablation had differential effects on transgene expression, neither single mutation abolished transgene expression completely. Here, we show that mutating both elements results in a complete inactivation of the transgene in both ventricles and atria under euthyroid conditions. However, expression still can be detected in the hyperthyroid state, implying that, although the TRE1 and TRE2 elements are critical elements for high levels of alpha-MyHC transcription in vivo, other promoter sites can mediate at least some degree of transcriptional activation.

Animals↗
PubMed1993

Role of myocyte-specific enhancer-binding factor (MEF-2) in transcriptional regulation of the alpha-cardiac myosin heavy chain gene.

The intergenic region between the mouse alpha-cardiac myosin heavy chain and beta-myosin heavy chain genes has previously been shown to direct expression of the bacterial chloramphenicol acetyltransferase reporter gene in transgenic mice in a tissue-specific manner. Sequence analyses located a putative myocyte-specific enhancer-binding factor (MEF-2) site situated in the regulatory region of this gene proximal to the start site of transcription. The role of this element in directing the cardiac compartment-specific expression of the transgene was assessed. The polymerase chain reaction was used to perform substitution mutagenesis of the MEF-2 binding site, and lack of MEF-2 binding was confirmed by gel retardation assays. The resultant construct was used to generate transgenic mice. Surprisingly, transgene expression was not down-regulated, but was significantly increased in the hearts of the MEF-2 mutant mice. In addition, cardiac-specific expression of the transgene was perturbed with significant levels of ectopic expression occurring in the aorta.

Animals↗
PubMed1993

Transgenic analysis of the thyroid-responsive elements in the alpha-cardiac myosin heavy chain gene promoter.

The role of two putative, cis-acting thyroid hormone-responsive elements, TRE1 and TRE2, located at -129 to -149 and -102 to -120, respectively, on the murine alpha-myosin heavy chain (MHC) gene, has been investigated in transgenic mice. These motifs are present in a 4.5-kilobase fragment lying upstream of the transcriptional start site of the mouse alpha-MHC gene: this fragment directs appropriate expression of a reporter gene in transgenic mice (Subramaniam, A., Jones, W. K., Gulick, J., Wert, S., Neumann, J., and Robbins, J. (1991) J. Biol. Chem. 266, 24613-24620). Here, we independently mutate the TRE1 and TRE2 elements by base substitution. The mice were analyzed for transgene expression in different muscle and non-muscle tissues including the atria and ventricles. Normal levels of transgene expression were observed in euthyroid mice carrying a mutation in TRE1. In contrast to these results, mice in which TRE2 was mutated showed reduced levels of CAT activity in both the atria and ventricles, suggesting a previously undefined role for this element in the constitutive up-regulation of the alpha-MHC gene. In hypothyroid mice carrying either of these mutations, the complete cessation of ventricular expression of the chloramphenicol acetyltransferase transcripts that takes place in the alpha-5.5 (wild type) animals did not occur.

Animals↗
PubMed1993

Comparison of normal, hypodynamic, and hyperdynamic mouse hearts using isolated work-performing heart preparations.

It is now possible to manipulate the murine genome and produce transgenic mice in which genes encoding myocardial proteins have been ablated, resulting in an altered myocardial performance. In this study, we quantitate myocardial performance in work-performing mouse heart preparations from euthyroid, hypothyroid, and hyperthyroid mice. Our results show that time to peak pressure (TPP) and time to half-relaxation (RT1/2), together with first derivatives of intraventricular pressure (+/- dP/dt), are significant indicators of the quality and quantity of systolic contraction and relaxation. We compared the normal control indicators of contraction and relaxation of three different mouse strains at minimum afterloads (approximately 50 mmHg) and preloads (approximately 5 ml/min) and found them identical in range. All indicators of myocardial performance were significantly higher in the hyperthyroid and lower in the hypothyroid compared with normal mice. The cardiac myosin heavy chain isoform transcript shift (alpha-->beta) associated with hypothyroidism was observed. Because sympathetic activity is greatly enhanced with hyperthyroidism, we studied the effects of isoproterenol and the beta-blocker sotalol on cardiac contractility. Only approximately 50% of the myocardial hyperactivity displayed by hyperthyroid mice could be attributed to beta-adrenergic activity.

Animals↗
PubMed1992

Inhibition of interleukin 2 receptor expression in normal human T cells by cyclosporine. Demonstration at the mRNA, protein, and functional levels.

In view of the importance of the IL-2 receptors in the expression of antiallograft immunity and the currently existing controversy regarding the effect of CsA on the induction of IL-2 receptors, we explored the effect of cyclosporine on the induction of interleukin-2 receptor alpha and beta in normal human T cells. The effect of CsA on the induction of IL-2 receptors was examined at the levels of mRNA expression (with the aid of the polymerase chain reaction), protein (by SDS-PAGE analysis of chemically crosslinked 125I-IL-2 membrane protein complexes and by FACS), and function (by Scatchard analysis of 125I-IL-2 binding to T cells). The T cells were signaled with sn-1,2-dioctanoylglycerol and ionomycin or with crosslinked anti-CD3 and anti-CD2 mAbs. Our experimental design revealed that (A) CsA inhibits the induction of IL-2 receptor alpha and beta in normal human T cells, (B) the inhibitory activity is realized by a direct effect on T cells, and (C) the inhibitory activity is detectable at the pretranslational level--CsA significantly reduced the induction of mRNA encoding IL-2 receptor alpha and IL-2 receptor beta. These observations together persuasively demonstrate the ability of CsA to interrupt the emergence of IL-2 receptors on the surface of normal human T cells.

Antibodies, Monoclonal↗
PubMed1991

Tissue-specific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice.

The intergenic region between the mouse alpha-myosin heavy chain (MHC) and beta-MHC genes was analyzed in terms of its ability to drive gene expression in transgenic mice. Earlier, we reported that the entire intergenic region was sufficient to direct expression of the bacterial chloramphenicol acetyl transferase reporter gene in a tissue-specific and developmental stage-specific manner. Additional transgenic lines have been generated which include two deletions. The first deletion, alpha-3, which lacks the distal 2.5 kilobase pairs of the upstream region, is competent to direct tissue- and developmental-specific expression of the transgene. A larger deletion, in which only 138 base pairs upstream of the transcriptional start site remain, shows no chloramphenicol acetyltransferase activity in either muscle or non-muscle tissue. Tissue surveys of transgene expression indicated low levels of activity in the lung, and analyses via the polymerase chain reaction confirmed the presence of the endogenous alpha-MHC gene transcripts in this tissue. Subsequently, an alpha-MHC gene-specific riboprobe was used to detect the cognate transcripts in lung sections by in situ hybridization. The data show that, in the lung, the transcripts are localized to the thick intimal wall of the veins and venules.

Animals↗
PubMed1991

Isolation and characterization of the mouse cardiac myosin heavy chain genes.

Two mouse genomic libraries were probed in order to isolate the murine cardiac myosin heavy chain (MHC) genes. Two overlapping cosmid clones that encode the cardiac genes were isolated. One of them encompasses the entire alpha-cardiac MHC gene, its 5'-flanking region and approximately 10 kilobase pairs (kb) of the 3'-end of the beta-cardiac MHC gene which we determined is located approximately 4 kb upstream of the alpha-cardiac MHC gene. Four clones isolated from a bacteriophage library were found to overlap with the 5'-region of the cosmid clones, and sequence analysis confirmed that the 5'-end of the beta-cardiac MHC gene was contained within one of the clones. Primer extension and polymerase chain reaction (PCR) analyses were used to define the transcriptional start site and the 5'-organization of the alpha-cardiac MHC gene. This region exhibits greater than 90% homology to the corresponding nucleotides of the rat alpha-cardiac MHC gene. To assess the importance of the intergenic region in directing expression of the alpha-cardiac MHC gene, a fragment containing the 3'-end of the beta-cardiac gene and the 5'-end of the alpha-cardiac gene was linked to a chloramphenicol acetyltransferase gene and used to generate transgenic mice. Analyses of the chloramphenicol acetyltransferase (CAT) activity in two lines indicate that the intergenic region is sufficient to properly direct expression in a tissue-specific manner.

Animals↗
PubMed1991

Vagino-cornual cannulation and embryo recovery from crossbred (Bos indicusxBos taurus) heifers.

Successful vaginal and bicornual cannulations were performed with a tabular cannulator shaped like a giant hypodermic needle in crossbred (Bos indicusxBos taurus) heifers (n=7). A total of 58 embryos from a possible 73 were flushed out. The embryo recovery rate was 79.45% and average recovery time was 50 minutes. The wound healing at cannulation sites was rapid. All heifers except one conceived due to insemination during subsequent cycles. One heifer developed pyometra and did not settle.

Journal Article↗
PubMed1991

The molecular basis for the synergism between the CD3/alpha beta T cell receptor and the CD2 antigen-derived signals in promoting T-cell proliferation.

Northern blot analysis and a highly sensitive methodology for mRNA phenotyping, polymerase chain reaction (PCR), were used to explore the basis for the synergism between CD3/alpha beta T cell receptor (TCR) and the CD2 antigen-derived signals in promoting proliferation of T cells. Northern blotting of RNA isolated from highly purified normal human T cells revealed that crosslinking of anti-TCR-1 (a mAb directed at a framework determinant of the TCR) and OKT11 (a mAb directed at the SRBC-binding epitope of the CD2 antigen) induced the expression of the interleukin-2 gene and the gene for IL-2 receptor alpha, mRNA phenotyping by PCR revealed that crosslinkage of TCR with the CD2 antigen, and not independent crosslinking of TCR or the CD2 antigen, results in the induction of IL-2, IL-2 receptors alpha and beta, and IL-4-specific transcripts. Highly purified CD4+ T cells, as well as CD8+ T cells proliferated by crosslinking TCR with CD2 antigen. Moreover, crosslinkage of TCR with the CD2 antigen and not of either antigen with the CD4 antigen (on the surface of CD4+ T cells) or the CD8 antigen (on the surface of CD8+ T cells) resulted in marked proliferation. Our demonstration that the CD2 antigen-derived signal(s) contribute to the expression of growth promoting genes elicited via the TCR, and that the CD2 antigen is more efficient compared with the CD4 or CD8 antigen in evoking T cell proliferation, suggests that autoimmunity as well as alloimmunity might be regulated by targeting the CD2 antigen.

Antigens, CD↗
PubMed1991

Cardiac myosin heavy chain mRNA expression and myocardial function in the mouse heart.

The vertebrate heart contains two myosin heavy chain isoforms, alpha and beta, which are differentially expressed. To establish a murine model for gene-targeting experiments, we defined the precise temporal expression of the myosin isoforms during cardiogenesis and obtained quantitative measurements of cardiac performance. The relative levels of the alpha- and beta-cardiac transcripts were determined by isolating the RNA from the hearts of CD-1 mice during development and hybridizing the preparations to probes that detect specifically the alpha- or beta-cardiac myosin heavy chain mRNAs. The data indicate that, although both isoforms are present from the onset of cardiogenesis, the beta-isoform predominates during embryogenesis and fetal development. This relation is reversed after the first day of life with a significant drop in the absolute transcript levels during the switch; and alpha/beta ratio of 16:1 is maintained in the neonate, and the relatively high levels of the alpha-transcript remain throughout the adult stages. To be able to make functional comparisons between normal and transgenic mice, we obtained indexes of myocardial function in isolated retrogradely perfused and in work-performing heart preparations in normal and hypodynamic mouse hearts. We found that the physiology of the mouse heart is similar to the rat heart in that we observed a positive staircase in the force-frequency relation of the mouse Langendorff preparation. We also saw contractile responses of more than twice control induced by paired stimulation and persistent postextrasystolic potentiation. As is the case for the rat, in the work-performing mouse heart, afterload (Starling resistance, pressure) changes produced a steeper Starling function curve than did changes in preload (volume, venous return).

Animals↗
PubMed1990

Analysis of the upstream regulatory region of a chicken skeletal myosin heavy chain gene.

The organization of the cis-acting regulatory elements of a chick myosin heavy chain gene has been investigated. The data show that a gene which is transcribed in vivo in the fast white embryonic musculature is also the major transcript expressed during myotube differentiation of primary myoblasts derived from 12-day embryonic chick leg muscles. The upstream region of this gene consists of 7500 base pairs, and we have tested the ability of these sequences to drive expression of the chloramphenicol acetyltransferase gene in developing primary muscle cultures. Deletion analyses of the upstream region show that negative regulatory elements are present within 2000 base pairs of the basal promoter elements, the CCAAT and TAATA boxes. Removal of these elements reveals the presence of a strong positive element located near the start site of transcription. Sequence analysis showed that the region also contains a sequence characteristic of an enhancer found in the immunoglobulin heavy chain gene, ATGCAAAT, the "octa" element. Gel band-shift assays show that this octa sequence binds a transacting factor present in muscle nuclear extracts, although footprint analysis indicates a limited interaction. Transient assays carried out with a fragment in which the octa sequence has been mutated, with the subsequent abolition of protein binding, shows that the particular interaction probably plays a role in negatively modulating the action of the strong positive promoter element.

Animals↗