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Biomedical subjects

M Subramaniam

Publications and source records attributed to M Subramaniam.

At least 19 recordsLinked to original sources
PubMed1992

High-performance capillary electrophoretic analysis of chloramphenicol acetyl transferase activity.

This study highlights the potential utility of high-performance capillary electrophoresis (HPCE) for monitoring enzyme activity. Free-zone capillary electrophoresis is used to rapidly and reproducibly analyze the activity of the bacterial enzyme chloramphenicol acetyl transferase (CAT) which converts the substrates acetyl coenzyme A (CoA) and chloramphenicol to acetyl chloramphenicol and CoA. The results of this study indicate that HPCE may be an excellent tool for studying enzyme activities since it has several advantages over standard single parameters assays, most notably, the ability to monitor both loss of substrate and appearance of products simultaneously. Conditions have been identified for optimal separation of the substrate (chloramphenicol) from the products (acetylated derivatives). This presents a unique potential of HPCE for the analysis of enzymatic reactions that may be applied to areas of analytical research presently utilizing enzymatic reactions. One such analytical method is the CAT assay used for analysis of gene promoter activity. In this study, HPCE is shown to yield similar quantitative results with nonradiolabelled substrate in a fraction of the time. HPCE has several advantages over standard techniques including speed of analysis, no need for radiolabelled substrate, small sample volumes, high sensitivity/resolution and excellent quantitative capabilities.

Chloramphenicol O-Acetyltransferase↗
PubMed1992

Glucocorticoid regulation of alkaline phosphatase, osteocalcin, and proto-oncogenes in normal human osteoblast-like cells.

In humans, glucocorticoids are known to have marked effects on bone metabolism and function, including the significant regulation of osteoblast cells. To aid in the understanding of the mechanism of glucocorticoid action on normal human osteoblasts (hOB), confluent cells were analyzed for the presence of glucocorticoid receptors (GR) as well as for the effects of the glucocorticoid dexamethasone (Dex) on the expression of both the rapid responding nuclear proto-oncogenes and the late responding structural genes for bone matrix proteins. The interactions between Dex and 1,25 dihydroxy vitamin D3 (1,25 D3) on the gene expression in these cells were also examined. Using a functional receptor assay, a mean of 11,600 functional nuclear bound glucocorticoid receptors (range 6,000-22,000) was measured in fifteen separate cell strains. Northern blot analysis with a cDNA probe to the human GR was used to demonstrate the presence of a 7Kb transcript which is a candidate mRNA for GR in these cells. In agreement with previous studies, treatment of the hOB cells with Dex increased the steady state mRNA levels for alkaline phosphatase (AP) but displayed little or no effect on the mRNA levels for osteocalcin (OC) and glyceraldehyde phosphate dehydrogenase (GAPDH). Interestingly, the 1,25 D3 inductions of mRNA levels for OC were blocked by Dex but enhanced for AP. The above effects of Dex on AP and OC gene expression, including the interaction with 1,25 D3, were also shown to occur at the level of protein. The effect of Dex on the mRNA levels of the nuclear proto-oncogenes c-myc, c-fos, and c-jun was also investigated, since the oncoproteins (Fos/Jun) appear to play a role in the delayed glucocorticoid regulation of structural genes. Interestingly, Dex increased the steady state levels of c-myc, c-fos, and c-jun mRNAs in nonproliferating (confluent) hOB cells by 3.5-, 10-, and 2.0-fold, respectively, over control (untreated cells) values within one h of steroid treatment. The Dex-induced mRNA levels were transient and returned to basal values within 24 h of the steroid treatment. A reduced but qualitatively similar pattern of response was found in proliferating hOB cells. The pattern of response of these genes to glucocorticoids in hOB cells mimics the response in avian liver cells but not in reproductive cells. These results support the theory that hOB cells are target cells for glucocorticoids, and that as a primary event glucocorticoids rapidly regulate the expression of the nuclear oncoproteins Fos/Jun in these cells.

Alkaline Phosphatase↗
PubMed1992

The effect of 'conditioning' on hearing loss from a high frequency traumatic exposure.

The role of high frequency, low level 'conditioning' exposures as moderators of hearing loss from subsequent exposure to the same noise at a higher level was studied using monaural chinchillas. All the animals in the experimental groups were exposed to an octave band noise centered at 4 kHz at 85 dB SPL for 6 h a day for 10 days. One of the experimental groups was allowed to recover for 5 days and the other was allowed to recover for 18 h, prior to the higher level exposure at 100 dB for 48 h. A third group exposed only to the higher level constituted the control group. A comparison of threshold shifts and hair cell loss after 4 weeks of recovery across the three groups revealed: (a) the 5-day recovery group incurred greater threshold shifts than the other two groups; the hair cell loss in this group was greater than in the 18-h recovery group, but the same as in the control group and (b) the 18-h recovery group incurred considerably less threshold shift as well as hair cell loss than the other two groups. The results were also compared with the results from similar exposures using low frequency noise which indicated that the base vs. apex differences in the cochlea appear to extend to the effects of 'conditioning' exposures.

Animals↗
PubMed1991

Nuclear matrix localization and specific matrix DNA binding by receptor binding factor 1 of the avian oviduct progesterone receptor.

A chromatin acceptor protein for the avian oviduct progesterone receptor (PR), termed receptor binding factor 1 (RBF-1), has recently been shown to (1) be a component of the nuclear binding sites (acceptor sites) for PR and (2) generate high-affinity binding sites (termed the RBF-1 class of sites) on avian genomic DNA [Schuchard et al. (1991) Biochemistry 30, 4535-4542]. A second class of sites and its associated protein (termed RBF-2) were also identified. This paper demonstrates that RBF-1 and also the PR nuclear binding sites are localized in the oviduct nuclear matrix. RBF-1 is found in abundance in the nuclear matrix of liver but only in traces in the nuclear matrix of spleen. Extraction of the nuclear matrix with 4.0 M Gdn-HCl results in the complete removal of RBF-1 as occurs with whole chromatin. Interestingly, a second class of specific PR binding, termed RBF-2, remains on the nuclear matrix after the removal of all RBF-1. Southern blot analysis indicates that the nuclear matrix DNA contains sequences homologous with the 5'-flanking domains of the rapidly steroid regulated c-myc and c-jun protooncogenes and the beta-actin gene, but not genomic sequences of the late sex steroid regulated gene, ovalbumin, or the alpha-actin gene. A specific, small region in the 5'-flanking domain of the c-myc gene appears to be associated with the nuclear matrix. Southwestern blot analysis using partially purified RBF-1 shows a marked affinity and specificity of the RBF-1 for the nuclear matrix DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Enrichment of a second class of native acceptor sites for the avian oviduct progesterone receptor as intact chromatin fragments.

Several classes of specific progesterone receptor (PR) nuclear binding sites (acceptor sites) have previously been identified in avian oviduct chromatin on the basis of different binding affinities. Recently, two classes of acceptor proteins (AP) that are associated with these binding sites in the avian oviduct have been identified. These APs were termed receptor binding factors (RBF-1 and -2), and one (RBF-1) has been purified [Schuchard et al. (1991) Biochemistry 30, 4535-4542]. The RBF-1 is associated with the highest affinity class of sites in the intact chromatin, and the RBF-2 is associated with the second highest affinity class of sites. The PR binding sites and their associated RBF-2 protein remain with the residual chromatin fraction following extraction by 4 M Gdn-HCl. This Gdn-HCl-treated chromatin has been termed nucleoacidic protein (NAP). This paper describes the 200-fold enrichment of the native RBF-2 class of PR acceptor sites beginning with the DNase I digestion of NAP to obtain DNase-resistant fragment (NAPf) containing approximately 150 bp of DNA. The PR binding sites are further enriched by high-performance or fast protein liquid chromatography and chromatofocusing. Anti-RBF-1/RBF-2 protein antibodies identify antigens that coelute with the PR binding activity. Hybridization analysis of the DNAf from the enriched NAPf demonstrates sequence homologies with the nuclear matrix DNA as well as with genomic sequences of the rapid steroid responding nuclear protooncogenes c-myc and c-jun. However, comparative analyses of the whole genomic DNA with the nuclear matrix DNA indicate that the RBF-2 (NAPf) is largely nonnuclear matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Rapid induction of the c-jun protooncogene in the avian oviduct by the antiestrogen tamoxifen.

This report describes a rapid regulation of the expression of the c-jun protooncogene by the antiestrogen tamoxifen (Tam). The c-jun protooncogene codes for an important component of the AP-1 transcription factor complex, which regulates the expression of many unlinked genes. Repeated experiments have shown that Tam rapidly increases the steady-state c-jun mRNA levels in the avian oviduct but decreases the levels in the liver. The Tam effects are time- and dose-dependent. These results are supported by other studies that have demonstrated that 17 beta-estradiol decreases steady-state levels of c-jun protooncogene mRNA in oviducts of animals fully withdrawn from estradiol. The effect of Tam in the avian oviduct is in contrast to the reported effects of Tam on the expression of practically all other genes in the avian oviduct and other animal tissues. Transcription analyses using nuclear runoff experiments with oviduct nuclei demonstrate a decrease in the c-jun gene transcription within minutes after Tam treatment with a return to 75% of control values by 4 hr. The fact that Tam transiently decreases the transcription of the c-jun gene but increases the steady-state c-jun mRNA levels suggests that Tam must alter both transcriptional and post-transcriptional events. The results support a role of the c-jun protooncogene as a regulatory gene in the cascade model for steroid action whereby steroids rapidly regulate the regulatory genes, which in turn regulate many other structural genes.

Animals↗
PubMed1991

Cloning of murine tissue factor and regulation of gene expression by transforming growth factor type beta 1.

We have cloned a serum- and cycloheximide-inducible mRNA from AKR-2B murine fibroblasts which encodes a protein with significant sequence similarity to human tissue factor, a cellular initiator of the blood coagulation cascade. Information derived from this clone was used to establish the presence of a virtually identical sequence in mouse brain. Most importantly, cDNA-directed expression in a quail fibroblast cell line produced high levels of tissue factor procoagulant activity, confirming the identity of this protein as murine tissue factor. Additional studies demonstrate that transforming growth factor type beta 1 stimulates tissue factor gene transcription and is a potent inducer of tissue factor procoagulant activity in fibroblasts. Other tested mitogens such as platelet-derived growth factor, epidermal growth factor, and insulin were weak inducers. These results may reflect a role for transforming growth factor beta 1 in the maintenance of hemostasis or, alternatively, a role for tissue factor in cellular functions unrelated to blood coagulation.

Amino Acid Sequence↗
PubMed1991

The effect of 'conditioning' exposures on hearing loss from traumatic exposure.

The role of 'conditioning' exposures as moderators of hearing loss produced by exposure to a higher level noise was explored using chinchillas. Monaural chinchillas were exposed to an octave band of noise centered at 0.5 kHz at 95 dB for six hours/day for ten days. The subjects were allowed to recover to pre-exposure sensitivity and at five days after the last exposure they were re-exposed to the same noise at 106 dB. Thresholds recorded at various time intervals following the second exposure were compared with those recorded in a control group exposed only to the higher level noise. The experimental animals were found to have less threshold shift at all stages of recovery. Results are discussed in the light of results of other related studies and possible mechanisms involved are hypothesized.

Acoustic Stimulation↗
PubMed1991

Development of resistance to hearing loss from high frequency noise.

The effect of interrupted exposure on the development of progressive resistance to hearing loss from exposure to high frequency noise was studied using monaural chinchillas. The animals were exposed to an octave band noise centered at 4 kHz at 85 dB SPL for 6 h a day for ten consecutive days. Hearing thresholds were measured using evoked potential recording before and after each exposure. Results indicated a reduction in threshold shift with repeated exposures. This reduction in threshold shift or 'toughening' was more rapid for the high frequency exposures than the 'toughening' from similar low frequency exposures.

Acoustic Stimulation↗
PubMed1991

The effect of exposure level on the development of progressive resistance to noise.

The effect of exposure level on the development of progressive resistance to temporary threshold shift caused by exposures to an octave band of noise centered at 0.5 kHz was explored using chinchillas. The animals were exposed to either 85, 95 or 100 dB SPL for six hours a day for ten days. Hearing thresholds were recorded electrophysiologically, prior to and after each daily exposure. A trend toward decreasing threshold shift with increase in the number of exposures was seen at all the levels. The amount of threshold shift appeared to depend upon the level as well as the test frequency. The findings are discussed in the light of results of previous studies and possible mechanisms involved in the process of 'toughening' are hypothesized.

Adaptation, Physiological↗
PubMed1991

Impact noise: the importance of level, duration, and repetition rate.

The applicability of the equal energy hypothesis (EEH) to impact noise exposures was studied using chinchillas. Hearing thresholds were estimated by recording the evoked potentials from a chronic electrode implanted in the inferior colliculus. The animals were exposed to broadband impacts of 200-ms duration. The study was carried out in two parts. In experiment I, six exposure levels (107, 113, 119, 125, 131, and 137 dB SPL) and three repetition rates (4/s, 1/s and 1/4s) were employed. In the second experiment, the total duration of the exposure as well as the total energy were kept constant by trading level and rate. Results indicate that hearing loss resulting from exposure to impact noise does not conform to the predictions of the EEH. The permanent threshold shift as well as the hair cell loss are more or less equal across the lower peak exposure levels. However, both the hearing loss and the hair cell damage increase for exposures with higher peak levels. Furthermore, hearing loss and cochlear damage are dependent upon the rate of exposure. Thus the amount of hearing loss and hair cell damage appears to depend on the interaction of several factors including peak level, rate, and the susceptibility of the animal.

Animals↗
PubMed1991

Sporadic Koro and marital dysharmony.

A case of Koro is described in a Malaysian Chinese man in the setting of martial dysharmony and sexual rejection. A distinction is suggested between the epidemic form of Koro and the Koro symptom occurring sporadically. Existence of the sporadic Koro syndrome is discussed and a unified classificatory system is proposed.

Adult↗
PubMed1990

Rapid inhibition of the c-jun proto-oncogene expression in avian oviduct by estrogen.

The c-jun proto-oncogene codes for an important component of the AP-1 transcription factor complex which regulates the expression of many genes. This paper demonstrates that the steady state c-jun mRNA level in the avian oviduct is markedly decreased to 50% of control values within 30 minutes after estrogen injection into the animals. In the avian liver, the level is rapidly increased over 10 fold. Nuclear run-off transcription analysis demonstrates that the decrease in the c-jun mRNA levels in the avian oviduct occurs at least in part at the level of transcription. These changes are reproducible in repeat experiments. This response in the avian oviduct is unique since 1) it is the first demonstration of a steroid effect on c-jun expression in any animal system, 2) the changes in c-jun mRNA occur much more rapidly than most steroid responsive genes, and 3) it is the rare demonstration of an estrogen inhibition of the expression of a gene. A role for the c-jun proto-oncogene as an early regulatory gene in the cascade model for steroid action is proposed.

Animals↗
PubMed1990

Evoked-response tone-on-tone masking in the chinchilla: effect of masker frequency.

Tone-on-tone masking patterns were measured at 0.5, 1, and 4 kHz using the auditory-evoked response from the inferior colliculus of the chinchilla. Masking profiles obtained with a masking level of 30 dB SPL were relatively symmetrical; however, as masker level increased, masking spread toward the high frequencies, particularly with the 0.5-kHz masker. For masker frequencies of 1 and 4 kHz, a low-threshold notch was observed in the masking profile 2/3 of an octave above the frequency of the masker. The low-threshold notch may represent a response to the distortion tone 2F1-F2. The notch was absent in the 0.5-kHz masking profile. The masking profiles obtained with the evoked response are somewhat wider, but otherwise qualitatively similar to those measured psychophysically in humans. Thus, the evoked-response procedure may provide a convenient way of evaluating the spread of masking and the presence of distortion tones in difficult-to-test subjects.

Acoustic Stimulation↗
PubMed1989

Negative regulation of serum-responsive enhancer elements.

Transcription of the c-fos proto-oncogene and the cytoskeletal actin genes is induced within minutes of the addition of serum growth factors in a variety of cell types. Inhibitors of protein synthesis such as cycloheximide have been shown to dramatically potentiate the transcriptional response, an effect termed 'superinduction'. Although the stimulatory effect of serum has been shown to be transmitted through a cis-acting enhancer sequence termed a serum response element (SRE), the sequence element(s) responsible for mediating the effect of cycloheximide has not been identified. We now report that a synthetic copy of the c-fos SRE is sufficient to confer cycloheximide-dependent inducibility upon a heterologous promoter. This does not require the presence of serum, but several mutations in the SRE that impair serum-inducibility also impair cycloheximide-inducubility. These results imply that serum-responsive enhancer elements are negatively regulated by one or more labile proteins and that both positive and negative regulators of enhancer activity require a functional 'CArG box', a sequence domain previously implicated in muscle-specific transcription.

Actins↗
PubMed1988

Evidence that the functional beta-actin gene is single copy in most mice and is associated with 5' sequences capable of conferring serum- and cycloheximide-dependent regulation.

Hybridization to synthetic oligonucleotides representing conserved regions in the promoter and first intron of several vertebrate beta-actin genes was used to discriminate between what appears to be a single functional beta-actin gene and numerous pseudogenes in the mouse genome. Sequences derived from the 5' end of this gene were shown to confer serum-inducible expression upon a heterologous reporter gene when transfected into mouse fibroblasts. Moreover, these sequences rendered reporter gene expression superinducible by a combination of serum and cycloheximide. These experiments indicate that the 5' end of the mouse beta-actin gene contains sequence elements which mediate the stimulatory effects of serum growth factors and which are responsive to both positive and negative regulators of gene expression.

Acetyltransferases↗