Different regulatory sequences control creatine kinase-M gene expression in directly injected skeletal and cardiac muscle.

Regulatory sequences of the M isozyme of the creatine kinase (MCK) gene have been extensively mapped in skeletal muscle, but little is known about the sequences that control cardiac-specific expression. The promoter and enhancer sequences required for MCK gene expression were assayed by the direct injection of plasmid DNA constructs into adult rat cardiac and skeletal muscle. A 700-nucleotide fragment containing the enhancer and promoter of the rabbit MCK gene activated the expression of a downstream reporter gene in both muscle tissues. Deletion of the enhancer significantly decreased expression in skeletal muscle but had no detectable effect on expression in cardiac muscle. Further deletions revealed a CArG sequence motif at position -179 within the promoter that was essential for cardiac-specific expression. The CArG element of the MCK promoter bound to the recombinant serum response factor and YY1, transcription factors which control expression from structurally similar elements in the skeletal actin and c-fos promoters. MCK-CArG-binding activities that were similar or identical to serum response factor and YY1 were also detected in extracts from adult cardiac muscle. These data suggest that the MCK gene is controlled by different regulatory programs in adult cardiac and skeletal muscle.

Molecular cloning of a diverged homeobox gene that is rapidly down-regulated during the G0/G1 transition in vascular smooth muscle cells.

Adult vascular smooth muscle cells dedifferentiate and reenter the cell cycle in response to growth factor stimulation. Here we describe the molecular cloning from vascular smooth muscle, the structure, and the chromosomal location of a diverged homeobox gene, Gax, whose expression is largely confined to the cardiovascular tissues of the adult. In quiescent adult rat vascular smooth muscle cells, Gax mRNA levels are down-regulated as much as 15-fold within 2 h when these cells are induced to proliferate with platelet-derived growth factor (PDGF) or serum growth factors. This reduction in Gax mRNA is transient, with levels beginning to rise between 8 and 24 h after mitogen stimulation and returning to near normal by 24 to 48 h. The Gax down-regulation is dose dependent and can be correlated with the mitogen's ability to stimulate DNA synthesis. PDGF-AA, a weak mitogen for rat vascular smooth muscle cells, did not affect Gax transcript levels, while PDGF-AB and -BB, potent mitogens for these cells, were nearly as effective as fetal bovine serum. The removal of serum from growing cells induced Gax expression fivefold within 24 h. These data suggest that Gax is likely to have a regulatory function in the G0-to-G1 transition of the cell cycle in vascular smooth muscle cells.

Homeobox transcription factor regulation in the cardiovascular system.

Tissue remodeling and alterations in cellular differentiation occur in the cardiovascular system during normal development and pathologic conditions such as cardiac hypertrophy and atherosclerosis. Homeobox genes encode transcription factors that have been shown to be regulators of body-plan formation, cell growth, differentiation, and region-specific cell migration in developing embryos. Recently, several homeobox genes have been either isolated from or detected in cardiovascular tissues, and evidence suggests that some of them may have a regulatory function in the cardiovascular system. We review here what is known about the role of homeobox transcription factors in the control of development and gene expression in the cardiovascular system.

A double-blind comparison of parenteral dipyrone and pethidine in the treatment of post-operative pain.

A double-blind trial was carried out in 100 patients with moderate to severe post-operative pain to compare the analgesic effectiveness over a 6-hour period of single intramuscular injections of 2.5 g dipyrone and 100 mg pethidine. Maximum pain relief was seen 2 hours after drug administration in both groups and there was no statistically significant difference in responses. No side-effects were reported.

Development stage-specific expression of fibroin in the silk worm Bombyx mori is regulated translationally.

The contents of fibroin H RNA as a function of development have been quantitated in the posterior silk glands of Bombyx mori larvae on different days of 4th and 5th instars. The fibroin RNA levels increased during the feeding stages of larvae and the RNA got completely degraded during the interim moult. The patterns of accumulation of fibroin RNA were similar in both the instars. Although there was considerable increase in the fibroin RNA content during the 5th larval instar, the relative abundance of fibroin RNA in the total RNA was fairly constant during the 4th and 5th instars. The increased content of fibroin RNA in 5th instar was the consequence of an overall increase in transcription accompanying the development progress, rather than specific increase only in fibroin transcription. The contents of fibroin protein in the 4th and 5th instars of development have also been quantitated making use of a sensitive radioimmune assay with a purified, antifibroin antibody. There were substantial differences between 4th and 5th instars in the absolute fibroin contents as well as the relative proportion of fibroin in the total proteins. These results implied that although the fibroin gene was transcribed at the same efficiency during the 4th and 5th instars, the translational efficiency was much lower during the 4th instar. The extent of polyadenylation of fibroin RNA was similar in both instars. However, there was a two-fold increase in the polysome association of fibroin RNA in the 5th instar. Over and above this, there was substantial increase during the 5th instar in the contents of those tRNAs. (e.g. Gly, Ala and Ser) which are abundantly represented in fibroin and therefore directly related to the expression of fibroin. The increased polysome association of fibroin mRNA and the adequate supply of cognate tRNAs in the 5th instar, together contributes to the translational regulation of fibroin in a developmental stage-specific manner. Based on these observations, we propose that translational regulation plays a major role in the development stage-specific synthesis of fibroin in Bombyx mori.

Determination of trace amounts of 5-methylcytosine in DNA by reverse-phase high-performance liquid chromatography.

A high-performance liquid chromatographic method to separate five major bases (cytosine, thymine, guanine, adenine, and uracil) and three minor methylated bases (5-methylcytosine, N6-methyladenine, and 7-methylguanine) has been developed using a volatile mobile phase under isocratic conditions. It is extended to quantitate 5-methylcytosine in trace amounts (1 in 20,000 cytosine residues). The suitability of the method has been verified by estimating 5-methylcytosine in DNAs of phi X174 and pBR322. The method has been applied to quantitate the extent of cytosine methylation in DNA of larval silk glands of Bombyx mori. Our results confirm that the pupal DNA of Drosophila melanogaster does not contain detectable amounts of 5-methylcytosine.

A rapid and sensitive method for the estimation of individual tRNA pools.

A rapid and sensitive method is described to quantitatively compare tRNA pools for individual aminoacids in a single experiment. The procedure comprises of: charging of total tRNA with a mixture of radiolabeled aminoacids, deacylation of the esterified tRNA with a volatile base and the recovery of the labeled aminoacid, derivatisation of the aminoacid with phenylisothiocyanate after mixing with excess of nonradioactive aminoacids, baseline separation of the phenylthiocarbamyl aminoacids by reverse phase high performance liquid chromatography monitored by A254nm and quantitation of the radioactivity in individual aminoacid peaks. The radioactivity in the aminoacid peak corresponds to the quantity of the aminoacylated tRNA. The method has been successfully applied to quantitate the individual tRNA pools in the developing silk glands of Bombyx mori, a functionally adapted tissue which undergoes considerable variations in tRNA content.

Experience with the pectoralis major myocutaneous flap for head and neck reconstruction in a general surgical unit.

The pectoralis major myocutaneous (PMMC) flap or its modification was used in 19 cases after resectional surgery for malignancy of the oral cavity with minimal morbidity and no mortality. The resection as well as reconstruction was done by the same team consisting only of general surgeons. The final functional and cosmetic results were satisfactory. The pectoralis major myocutaneous flap is a hardy flap and can be performed with relative ease even by those not specialised in plastic surgery. This makes it an important tool for a general surgeon practicing in a country like India with its high incidence of head and neck malignancy.

Obstructed Morgagni's hernia (a case report).

A forty-year-old male patient was admitted with acute intestinal obstruction, plain X-ray abdomen and chest revealing air fluid levels on the right side of chest. A successful operation was carried out and the patient made an uneventful recovery. Obstructed Morgagni's Hernia is an uncommon case and hence the presentation.

Endothelial cells express a novel, tumor necrosis factor-alpha-regulated variant of HOXA9.

The expression of the class 1 homeobox (HOX) family of "master control" transcription factors has been studied principally in embryogenesis and neoplasia in which HOX genes play a critical role in cell proliferation, migration, and differentiation. We wished to test whether HOX family members were also involved in a differentiation-like process occurring in normal, diploid adult cells, that is, cytokine-induced activation of endothelial cells (EC). Screening of a human EC cDNA library yielded several members of the A and B groups of HOX transcription factors. One clone represented a novel, alternatively spliced variant of the human HOXA9 gene containing a new exon and the expression of which was driven by a novel promoter. This variant termed HOXA9EC appeared restricted to cells of endothelial lineage, i.e. expressed by human EC from multiple sources, but not by fibroblasts, smooth muscle cells, or several transformed cell lines. HOXA9EC mRNA was rapidly down-regulated in EC in response to tumor necrosis factor-alpha due to an apparent reduction in transcriptional rate. Reporter construct studies showed that the 400 base pairs of genomic DNA directly 5' to the transcription initiation site of HOXA9EC contained the information required for both up-regulation in response to cotransfection with a HOXA9EC expression vector and tumor necrosis factor-alpha-dependent down-regulation of this gene. These results provide evidence of a novel HOX family member that may participate in either the suppression or the genesis of EC activation.

Molecular cloning of a homeobox transcription factor from adult aortic smooth muscle.

We report here the cloning of a cDNA encoding a homeobox transcription factor from vascular smooth muscle and describe its unique pattern of mRNA expression at different stages in development. The cDNA isolated is 1576 base pairs in length not including the poly(A) tail and contains an open reading frame coding for a predicted 372-amino acid homeobox protein. During early embryogenesis, expression was detected in the neural tube with a sharp expression boundary occurring at an anterior position, in the myelencephalon, in the third and fourth branchial arches, and in vessels leading from the heart. In adults, however, transcripts were only detected in aortic smooth muscle and lung but were undetectable in cardiac or skeletal muscle, visceral smooth muscle, and many other tissues including brain. In neonates, expression was detected in the outflow tracts of the heart as well as in the cardiomyocytes. The expression pattern of this gene suggests that, although it likely has multiple roles during development, in the adult, it may participate in the control of vascular smooth muscle differentiation and proliferation.

PACT, a stress-modulated cellular activator of interferon-induced double-stranded RNA-activated protein kinase, PKR.

The interferon (IFN)-induced, double-stranded (ds)RNA-activated serine-threonine protein kinase, PKR, is a key mediator of the antiviral activities of IFNs. In addition, PKR activity is also involved in regulation of cell proliferation, apoptosis, and signal transduction. In virally infected cells, dsRNA has been shown to bind and activate PKR kinase function. Implication of PKR activity in normal cellular processes has invoked activators other than dsRNA because RNAs with perfectly duplexed regions of sufficient length that are able to activate PKR are absent in cellular RNAs. We have recently reported cloning of PACT, a novel protein activator of PKR. PACT heterodimerizes with PKR and activates it by direct protein-protein interaction. Overexpression of PACT in mammalian cells leads to phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha), the cellular substrate for PKR, and leads to inhibition of protein synthesis. Here, we present evidence that endogenous PACT acts as a protein activator of PKR in response to diverse stress signals such as serum starvation, and peroxide or arsenite treatment. Following exposure of cells to these stress agents, PACT is phosphorylated and associates with PKR with increased affinity. PACT-mediated activation of PKR leads to enhanced eIF2alpha phosphorylation followed by apoptosis. Based on the results presented here, we propose that PACT is a novel stress-modulated physiological activator of PKR.

Actinomycotic pseudo-tumour of the mid-cervical region (a case report).

Cervicofacial actinomycosis is today a rare disease in our country. Isolated actinomycotic neck masses are extremely rare. A case of young man with an isolated midcervical tumour like actinomycotic granuloma without sinuses or discharging granules is reported here.