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Biomedical subjects

S Merchant

Publications and source records attributed to S Merchant.

At least 19 recordsLinked to original sources
PubMed1992

Two metal-dependent steps in the biosynthesis of Scenedesmus obliques plastocyanin. Differential mRNA accumulation and holoprotein formation.

The accumulation of the interchangeable electron transfer catalysts plastocyanin and cytochrome c6 (cyt c6) in Scenedesmus obliquus is reciprocally regulated by the amount of copper ions in the medium. In copper-deficient cells, plastocyanin levels are severely reduced, whereas cyt c6 levels are high. Western blot analysis indicates that neither pre-apoplastocyanin nor apoplastocyanin accumulate to significant extents in copper-deficient Scenedesmus cells, and time course studies indicate that upon provision of copper salts to copper-deficient cells, the accumulation of plastocyanin to the levels maintained in copper-sufficient cells takes about 12-24 h. By 1) Northern hybridization analysis of Scenedesmus obliquus mRNA and 2) in vitro translation of polyadenylylated mRNA followed by immunoprecipitation of a 19.2-kilodalton precursor to plastocyanin, we demonstrate that the regulation of plastocyanin synthesis by copper must occur primarily at the level of mRNA accumulation. These results suggest that copper-dependent stimulation of holoplastocyanin accumulation requires de novo synthesis of the pre-apoprotein and contradict the conclusion of Bohner, H., Bohme, H., and Boger, P. (1981) FEBS Lett. 131, 386-388 that high levels of apoplastocyanin and a precursor to plastocyanin accumulate in copper-deficient Scenedesmus cells. We note also that although metal ions other than copper (e.g. silver) insert into Scenedesmus obliquus plastocyanin in vitro, synthesis of holoplastocyanin in vivo is specific for copper versus silver or mercury as it is in Chlamydomonas reinhardtii. Finally, the very different electrophoretic mobility and immunoreactivity of apoplastocyanin compared with holoplastocyanin suggests rather significant differences in structure between the copper-protein and the metal-free protein.

Blotting, Northern↗
PubMed1992

The biosynthesis of membrane and soluble plastidic c-type cytochromes of Chlamydomonas reinhardtii is dependent on multiple common gene products.

Cytochrome c6 functions in the thylakoid lumen to catalyze electron transfer from reduced cytochrome f of the cytochrome b6f complex to P700+ of photosystem I. The biogenesis of mature cyt c6 from cytosolically translated pre-apocytochrome c6 involves numerous post-translational modifications including the proteolytic removal of a transit sequence and the covalent attachment of heme to two cysteinyl thiols on the apoprotein. Here, we report on the characterization of a previously unrecognized class of non-allelic mutants of Chlamydomonas reinhardtii that are blocked at the conversion of apocyt c6 to holocyt c6. The mutants are acetate requiring since they are also deficient in cyt f, cyt b and the Rieske FeS protein. Pulse-chase studies indicate that heme attachment is not required for the two-step processing of pre-apocytochrome c6 to apocyt c6, but is required for the stability of the mature protein. This is in contrast to the biosynthesis of mitochondrial cyt c1 where heme attachment is required for the second processing step. We propose that the assembly of both holocytochrome c6 and the cytochrome b6f complex are dependent on common gene products, possibly those involved in heme delivery or metabolism. This is the first suggestion that multiple loci are involved in the biosynthesis of both plastidic c-type cytochromes.

Animals↗
PubMed1992

Improved myocardial ischemic response and enhanced collateral circulation with long repetitive coronary occlusion during angioplasty: a prospective study.

OBJECTIVES: The goal of the study was to evaluate the progressive increase in ischemic threshold with multiple sequential transient coronary occlusions and to assess the role of the collateral circulation in adaptation to ischemia. BACKGROUND: It has been observed that the duration of balloon inflations during coronary angioplasty can be gradually prolonged during subsequent dilations with a reduction in patient symptoms and diminished ischemic electrocardiographic (ECG) changes. Although the mechanism has not been fully explained, recruitment of coronary collateral circulation induced by repeated coronary occlusion has been reported. The stimuli for recruitment and the natural history of coronary collateral circulation are not understood. METHODS: Seventeen patients with isolated stenosis of the left anterior descending coronary artery and a normal left ventricle were enrolled. Angioplasty consisted of five successive prolonged inflations. Sequential changes in clinical, intracoronary ECG and left ventricular indexes of myocardial ischemia were examined. Coronary collateral channels were evaluated during balloon inflations by ipsilateral and contralateral injections of contrast medium and hemodynamically by occlusion pressure. RESULTS: An improved tolerance to myocardial ischemia with repetitive coronary occlusions was demonstrated by a significant reduction of angina, ST segment deviation, left ventricular filling pressure and less impairment of ejection fraction. Left ventricular wall motion abnormalities remained unchanged. Collateral angiographic grade did not change in 7 patients and increased in 10. CONCLUSIONS: This study confirms a progressive adaptation of myocardial ischemia to repetitive coronary occlusions and supports the concept that sequential episodes of myocardial ischemia are a stimulating factor for the recruitment of collateral channels in humans. These results also suggest that enhancement of recruitable collateral circulation might be an underlying mechanism of myocardial ischemic preconditioning.

Adult↗
PubMed1992

Myocardial dissection following successful chemical ablation of ventricular tachycardia.

We describe a case of fatal myocardial rupture and tamponade following a successful transcoronary chemical ablation of incessant ventricular tachycardia. Pathological examination showed a subepicardial dissection of the heart at the ablation site with fibrous and fatty degeneration of the myocardium. The present report calls for caution, underlying a possible lethal complication of ethanol ablation which has not been described before.

Aged↗
PubMed1991

Isolation and structural characterization of the Chlamydomonas reinhardtii gene for cytochrome c6. Analysis of the kinetics and metal specificity of its copper-responsive expression.

We have isolated a 5-kilobase pair fragment of genomic DNA containing the entire coding region for the Chlamydomonas reinhardtii gene encoding the copper-repressible Cyt c6. A region comprising 2.6 kilobase pairs contains the entire transcribed region plus 852 nucleotides upstream of the Cyt c6 transcription start site and 495 nucleotides downstream of the conserved C. reinhardtii polyadenylation signal. Comparison of the genomic sequence with the cDNA sequence (Merchant, S., and Bogorad, L. (1987) J. Biol. Chem. 262, 9062-9067) revealed that the coding region is interrupted by two introns, each of which is flanked by C. reinhardtii consensus intron/exon boundaries. Primer extension and S1 nuclease protection analyses identified the 5' border of the Cyt c6 mRNA at approximately 79 base pairs upstream from the initiator methionine. Analysis of the 5' upstream region reveals no significant similarity to sequences found in upstream regions of other copper-regulated genes. Time-course studies indicate that 1) the mature Cyt c6 mRNA has a half-life of approximately 45-60 min and is completely lost within 4 h, and 2) the primary, unspliced transcript has a half-life of approximately 10 min and is completely lost within 30 min after the addition of copper ions to copper-depleted cells. These results indicate that the response to copper occurs very rapidly upon elevation of extracellular copper levels. Although this gene is unresponsive to silver ions in vivo, in contrast to the yeast copper-responsive CUP1 gene (Furst, P., Hu, S., Hackett, R., and Hamer, D. (1988) Cell 55, 705-717), it does respond to mercury ions, albeit with less sensitivity. Mercury ions cannot, however, substitute for copper in allowing the accumulation of plastocyanin in vivo.

Amino Acid Sequence↗
PubMed1991

Dynamic interplay between two copper-titrating components in the transcriptional regulation of cyt c6.

The algal plastidic cytochrome c (cyt c6) is a biochemical equivalent of the copper-containing protein plastocyanin in photosynthetic electron transfer. But generally, cyt c6 accumulates and functions only under conditions (e.g. Cu-deficiency) where holoplastocyanin cannot be synthesized. In studying the regulation of Chlamydomonas reinhardtii cyt c6 expression by Cu we have determined that repression of cyt c6 accumulation occurs at the transcriptional level, and specifically in response to Cu as the metal ion regulator. Complete and sustained repression of cyt c6 transcription requires approximately 9 x 10(6) Cu ions in the medium/cell. Based on the estimated plastocyanin content of algal cells (8 x 10(6) molecules/cell) and the observation that lower ratios of Cu per cell result in only transient repression of cyt c6 transcription, we propose that Cu-dependent transcriptional repression of the gene encoding cyt c6 requires a Cu-binding factor which is titrated by Cu only after the alternate electron transfer catalyst, plastocyanin, has accumulated to the stoichiometry required for photosynthesis. The precise and highly metal-specific, autoregulatory control of cyt c6 levels--directly by Cu, and indirectly by holoplastocyanin--is in keeping with the functional role of cyt c6 as an alternate, although perhaps less preferred, electron transfer catalyst.

Blotting, Northern↗
PubMed1991

Congenital cholesteatoma of the middle ear in children: a clinical and histopathological report.

Forty-one children with congenital cholesteatoma of the middle ear seen from 1978 through 1989 are reviewed. The most common presentation was that of an asymptomatic white mass behind a normal intact tympanic membrane. Computed tomography (CT) scan was useful in documenting extension beyond the mesotympanum. Surgical removal was performed using an extended tympanotomy for lesions in the middle ear and tympanomastoidectomy for those that had extended into attic and mastoid air cells. Observation over an average 3.1-year period indicated that 80% of children were free of disease after initial surgery. Residual disease that required further surgery was present in 20%. The importance of early diagnosis of congenital cholesteatoma is strongly advocated. The prognosis is better when the cholesteatoma is confined to the anterosuperior quadrant of the middle ear. Seventeen patients in this study had such a lesion, and extended tympanotomy allowed removal of an encapsulated closed cholesteatoma with normal postoperative hearing and no residual cholesteatoma. The average age was 2.3 years. Temporal bone histopathological studies of three cases of congenital cholesteatoma demonstrate two distinct pathological types of congenital cholesteatoma. A "closed" keratotic cyst in the anterior mesotympanum, which is easily removed, and an "open" infiltrative type in which there is no containment of the keratotic debris and the cholesteatoma matrix is in direct continuity with middle ear mucosa. Surgical extirpation of the "open" type is difficult and more likely to be associated with residual disease.

Adolescent↗
PubMed1991

Developmental anomalies associated with hypospadias.

Hundred and thirty patients with hypospadias were studied between 1983 and 1990, with an aim of analysing the associated anomalies in these patients. Urogenital anomalies were observed in 30% and extra-urogenital anomalies were noted in 16%. Undescended testis was the most frequently seen anomaly, having been noted in 6.9% of 130 cases. Vesico-ureteral reflux was present in 5.4% and inguinal hernia in 3.8%. The incidence of these anomalies increased in proportion to the severity of penile deformity. The present article emphasizes the need for systemic urological and endocrinal examination, especially in severe cases of hypospadias.

Abnormalities, Multiple↗
PubMed1990

Isolation and characterization of a complementary DNA clone for an algal pre-apoplastocyanin.

We have isolated a cDNA clone for the Chlamydomonas reinhardtii pre-apoplastocyanin. The sequence contains codons for the complete pre-protein including a two-domain, lumen-targeting transit sequence and the mature apoprotein. The transit sequence (47 amino acids) is the shortest one described for chloroplast lumenal proteins, and like other C. reinhardtii lumen-targeting transit sequences appears to lack an uncharged amino-terminal domain usually present in plant lumen-directing sequences. The mature protein is deduced to be 98 amino acids in length and shows highest primary sequence similarity (74-76% identity) to other unicellular algal plastocyanins. Southern hybridization analysis of C. reinhardtii genomic DNA indicates the presence of a single nuclear gene, as is the case for all other plastocyanin genes characterized to date, although the algal gene might be interrupted. Codon usage in this gene reflects the high GC content of C. reinhardtii nuclear DNA, but is more highly biased than that found in the C. reinhardtii copper-repressible gene for the functionally equivalent pre-apocytochrome c552 (perhaps contributing to the more efficient synthesis in vivo of plastocyanin over cytochrome c552). The deduced physical properties of this plastocyanin are compared to those of the C. reinhardtii plastidic cytochrome c552.

Amino Acid Sequence↗
PubMed1990

Darkroom transplant.

Manual film processing systems produce their own types of artefacts. The authors report accidental transfer of an image-bearing emulsion from one film to another. The films were lying in close apposition in the darkroom washing tank. Forcible separation of the two films resulted in the "transplant".

Adult↗
PubMed1988

Isolation of a cDNA clone for the gamma subunit of the chloroplast ATP synthase of Chlamydomonas reinhardtii: import and cleavage of the precursor protein.

A cDNA library from Chlamydomonas reinhardtii, constructed in the phage expression vector lambda gt11, was probed with antiserum directed against the nuclear-encoded gamma subunit of the chloroplast H+-transporting ATP synthase [ATP phosphohydrolase (H+-transporting) or chloroplast coupling factors 0 and 1, EC 3.6.1.34] of C. reinhardtii. A cDNA was isolated and transcribed in vitro. The transcript was translated in vitro and immunoprecipitated with anti-gamma-subunit serum to yield a product that coelectrophoresed with the immunoprecipitated product from in vitro-translated polyadenylylated RNA. These proteins were larger than the mature gamma subunit, either immunoprecipitated as chloroplast coupling factor 1 or as the individual subunit. Thus, the gamma subunit is synthesized as a precursor of greater molecular weight in C. reinhardtii. Furthermore, the precursor protein encoded by the cDNA is imported into pea chloroplasts and processed to a lower molecular weight polypeptide that coelectrophoreses with mature C. reinhardtii gamma subunit. The largest cDNA isolated is about the same length as the corresponding mRNA (approximately equal to 1900 bases long) and probably contains the entire coding region. Southern blot analyses revealed restriction fragment length polymorphisms and that the gamma subunit is probably encoded by an intron-containing single-copy gene.

Biological Transport↗
PubMed1987

The Cu(II)-repressible plastidic cytochrome c. Cloning and sequence of a complementary DNA for the pre-apoprotein.

We have cloned a complementary DNA for pre-apocytochrome c-552 from Chlamydomonas reinhardtii. The deduced sequence of the mature protein shows high homology to those of cytochromes c-553 from cyanobacteria. Its homology to mitochondrial cytochrome c or bacterial photosynthetic cytochrome c2 is lower and appears to be concentrated in sequences around amino acids involved in the interaction with heme. With respect to primary sequence, the "transit sequence" for cytochrome c-552 appears to show no homology to other transit sequences for nuclear encoded chloroplast proteins. However, based on analogy to transit sequences for other proteins (Daldal, F., Cheng, S., Applebaum, J., Davidson, E., and Prince, R. C. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2012-2016; Goldschmidt-Clermont, M., and Rahire, M. (1986) J. Mol. Biol. 191, 421-432; Smeekens, S., de Groot, M., van Binsbergen, J., and Weisbeek, P. (1986) Cell 46, 365-375) the transit sequence of cytochrome c-552 can be divided into envelope-traversing and thylakoid-traversing domains. Cytochrome c-552 appears to encoded by a single nuclear gene in C. reinhardtii. The gene is expressed exclusively in Cu(II)-deficient cells.

Amino Acid Sequence↗
PubMed1987

Metal ion regulated gene expression: use of a plastocyanin-less mutant of Chlamydomonas reinhardtii to study the Cu(II)-dependent expression of cytochrome c-552.

Ac-208, a plastocyanin-deficient strain of Chlamydomonas reinhardtii, lacks the mRNA for pre-apoplastocyanin although Southern analyses indicate no gross deletion in the single gene for pre-apoplastocyanin. The alternative electron carrier, cytochrome c-552, is expressed only in Cu(II)-deficient cells of the mutant. Thus, the mutant strain can grow photoautotrophically in Cu(II)-deficient but not in Cu(II)-sufficient medium. This indicates that the absence of plastocyanin is the only defect (with respect to photosynthesis) in ac-208. Phenotypic revertants of ac-208 are capable of photoautotrophic growth by virtue of constitutive [with respect to Cu(II)] synthesis of cytochrome c-552. The revertant phenotype is attributed to a decreased internal concentration of Cu(II). This indicates that the Cu(II)-dependent expression of cytochrome c-552 synthesis is affected directly by the concentration of Cu(II) rather than indirectly by the level of plastocyanin in the cell.

Chlamydomonas↗
PubMed1986

Rapid degradation of apoplastocyanin in Cu(II)-deficient cells of Chlamydomonas reinhardtii.

Although plastocyanin is not detected in Cu(II)-deficient cells of Chlamydomonas reinhardtii, accumulation of messenger RNA for pre-apoplastocyanin is independent of the concentration of Cu(II) in the medium (Merchant, S., and Bogorad, L. (1986) Mol. Cell. Biol. 6, 462-469). This work shows that the synthesis, transport, and processing of pre-apoplastocyanin also appear to be unaffected in cells grown in Cu(II)-deficient medium. However, the mature protein, presumably formed after import of the precursor into the chloroplast, is rapidly degraded in Cu(II)-deficient cells. The half-life of the mature protein is estimated to be between 16 and 18 min in cells grown in Cu(II)-deficient medium. In cells grown in medium containing Cu(II), the mature protein is stable. The proteolytic activity thus appears to be specific for apoplastocyanin versus plastocyanin and thereby accounts for the absence of accumulated plastocyanin in Cu(II)-deficient cells. This process may be part of a general mechanism designed to remove chloroplast proteins which cannot be utilized.

Apoproteins↗