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Biomedical subjects

C N Chang

Publications and source records attributed to C N Chang.

At least 109 records · Page 6Linked to original sources
PubMed1987

Oligodeoxynucleotides containing synthetic abasic sites. Model substrates for DNA polymerases and apurinic/apyrimidinic endonucleases.

A synthetic procedure has been developed by which stable abasic sites are introduced into oligodeoxynucleotides at any desired position in the sequence. A modified tetrahydrofuran moiety, isosteric with 2'-deoxyribofuranose, serves as a structural analog of the natural apurinic/apyrimidinic site. We have also prepared oligodeoxynucleotides that lack cyclic structure at the abasic site but retain the carbon atoms of the phosphodiester backbone. These synthetic oligodeoxynucleotides are cleaved on the 5' side of the abasic site by endonuclease IV and by exonuclease III; they serve also as templates for avian myeloblastosis virus reverse transcriptase, Escherichia coli DNA polymerase I (Klenow fragment), and calf thymus DNA polymerase-alpha. Extension of primed templates by these DNA polymerases is blocked initially at the position immediately 3' to the abasic site; nucleoside monophosphates are subsequently incorporated opposite the lesion. The nucleotide most frequently incorporated opposite all abasic sites, regardless of structure, is dAMP. Significant "readthrough" at the abasic site was observed in experiments using avian myeloblastosis virus reverse transcriptase and DNA polymerase-alpha and, to a much lesser degree, with DNA polymerase I. We conclude that a modified tetrahydrofuran group can serve as a stable structural analog of 2'-deoxyribose in the apurinic/apyrimidinic site. These modified oligodeoxynucleotides should prove useful for studies of chemical mutagenesis.

Animals↗
PubMed1987

High-level secretion of human growth hormone by Escherichia coli.

A gene encoding the mature form of human growth hormone (hGH) was fused to the secretion signal coding sequence of the Escherichia coli heat-stable enterotoxin II (STII). This hybrid gene was preceded by two Shine-Dalgarno sequences derived from the trp and STII-coding genes and was expressed in E. coli under the transcriptional control of the E. coli alkaline phosphatase (phoA) promoter. In low-phosphate growth media, cells synthesized about 15 to 25 micrograms of hGH/ml/1 A550 unit of cells. This represents 6 to 10% of total cellular protein. The majority of the hGH produced (more than 90%) was processed precisely and secreted into the periplasmic space. These results demonstrate that E. coli cells are able to synthesize and secrete high levels of this human protein using a prokaryotic signal sequence.

Chorionic Gonadotropin↗
PubMed1987

Scalp, skull, and dura reconstruction on an emergency basis.

Severe cranial trauma caused by an industrial accident resulted in loss of scalp, skull, dura, and brain tissue in the left frontal region of a 55-year-old man. Successful one-stage total reconstruction of the defect was accomplished using a patch of fascia lata, autogenous split rib graft, and a free forearm flap on an emergency basis. A ten-month follow-up showed uneventful recovery and a good cosmetic result.

Craniocerebral Trauma↗
PubMed1986

Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli.

The nucleotide sequence of the alkaline phosphatase (APase) gene (phoA) of Escherichia coli strain 294 has been determined. Pre-APase has a total of 471 amino acids (aa) including a signal sequence of 21 aa. The derived aa sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at positions 16 and 36, and glutamic acid instead of glutamine at position 197. Two open reading frames (ORF1 and ORF2) located downstream from phoA or upstream from proC have been found. ORF1 encodes a putative presecretory protein of 106 aa with a signal sequence of 21 or 22 aa. If this protein is actually produced, it may be one of the smallest periplasmic proteins in E. coli.

Alkaline Phosphatase↗
PubMed1986

Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides.

Synthetic oligonucleotides coding for the yeast invertase secretion signal peptide were fused to the gene for the mature form of human interferon (huIFN-alpha 2). Two plasmids (E3 and F2) were constructed. E3 contained the invertase signal codons in a reading frame with the mature huIFN-alpha 2 gene. F2 had a deletion of the codon for alanine at amino acid residue-5 in the invertase signal and an addition of a methionine codon located between the coding sequences for the invertase signal and mature huIFN-alpha 2. Both hybrid genes were located adjacent to the promoter from the 3-phosphoglycerate kinase gene on the multicopy yeast expression plasmid, YEp1PT. Yeast transformants containing these plasmids produced somewhat more IFN than did the same expression plasmid containing the IFN gene with its human secretion signal sequence. HuIFN-alpha 2, purified from the medium of yeast cells containing E3, was found to be processed at the correct site. The huIFN-alpha 2 made by plasmid F2 was found to be completely processed at the junction between the invertase signal (a variant) and the methionine of methionine-huIFN-alpha 2. These results strongly suggested that the invertase signal (or its variant) attached to huIFN was efficiently recognized by the presumed signal recognition particle and was cleaved by the signal peptidase in the yeast cells. These results also suggested that amino acid changes on the right side of the cleavage site did not necessarily prevent cleavage or secretion.

Amino Acid Sequence↗
PubMed1985

Surgical treatment of spontaneous cerebellar hemorrhage.

UNLABELLED: Spontaneous cerebellar hemorrhage accounts for 5%-10% of intracerebral hemorrhage in most series. From June 1979 to June 1983 we had 26 surgical cases of spontaneous cerebellar hemorrhage. There were 15 men and 11 women. The typical history was sudden onset of severe headache, vomiting, dizziness, and inability to walk. Disturbance of consciousness was usually a late feature. Common signs were truncal ataxia, nystagmus, conjugate eyeball deviation, small miotic pupils with or without light reflex and abducens palsy. Surgical indications are (a) disturbance of consciousness, (b) signs of brainstem compression and (c) hematoma with transverse diameter greater than 3 cm. The overall surgical mortality was 34.6%. Twenty-two patients underwent suboccipital craniectomy to evacuate hematomas with or without ventriculostomy; mortality rate was 27%. Four patients underwent ventriculostomy only; mortality was 75%. Causes of death were (a) brainstem failure, six patients; (b) airway obstruction, one patient; (c) chest infection, one patient; (d) chronic renal failure, one patient. CONCLUSION: (a) suboccipital craniectomy to evacuate the hematoma is the most effective procedure where treatment is indicated; (b) the clinical recovery of the survivors show that 31% return to work, 38% are moderately disabled but take care of themselves, and 31% remain dependent on others; (c) deeply comatose patients may still benefit from early operation.

Adult↗
PubMed1985

Stroke-in-evolution--an indication for an emergency extracranial-intracranial bypass.

The role of the emergency extracranial-intracranial bypass in the management of the stroke-in-evolution is controversial. A case is presented in which the patient's fluctuating clinical picture was influenced by hemodynamic factors. The site of occlusion was inaccessible and an emergency extracranial-intracranial bypass was made to restore cerebral blood flow. The patient made a dramatic recovery after the operation. An emergency extracranial-intracranial bypass may be indicated in carefully selected cases.

Adult↗
PubMed1985

Periplasmic production of correctly processed human growth hormone in Escherichia coli: natural and bacterial signal sequences are interchangeable.

We have studied the synthesis, secretion, and processing of human growth hormone (hGH) in Escherichia coli transformed with plasmids engineered for the expression of hGH as a secreted product. In one plasmid, pPreHGH207-2, the coding sequence of the natural hGH precursor (pre-hGH) is placed under the control of the E. coli trp promoter. In a second plasmid, pAPH-1, a DNA fragment containing the E. coli alkaline phosphatase promoter and signal sequence codons is fused to the mature hGH coding sequence (pho-hGH). Most of the hGH was present in the osmotic shock fluids of E. coli cells containing either plasmid, indicating transport to the periplasmic space. Amino acid sequencing of the N termini of the pre-hGH and pho-hGH gene products revealed that both were processed correctly. Electrophoretic analysis of these polypeptides on reducing and nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels indicates that periplasmic hGH is monomeric and contains the same two disulfide bonds as authentic hGH.

Cell Compartmentation↗
PubMed1983

Saccharomyces cerevisiae contains two discrete genes coding for the alpha-factor pheromone.

Two genes, MF alpha 1 and MF alpha 2, coding for the alpha-factor in yeast Saccharomyces cerevisiae were identified by in situ colony hybridization of synthetic probes to a yeast genomic library. The probes were designed on the basis of the known amino acid sequence of the tridecapeptide alpha-pheromone. The nucleotide sequence revealed that the two genes, though similar in their overall structure, differ from each other in several striking ways. MF alpha 1 gene contains 4 copies of the coding sequence for the alpha-factor, which are separated by 24 nucleotides encoding the octapeptide Lys-Arg-Glu-Ala-Glu(or Asp)-Ala-Glu-Ala. The first alpha-factor coding block is preceded by a sequence for the hexapeptide Lys-Arg-Glu-Ala and 83 additional amino acids. MF alpha 2 gene contains coding sequences for two copies of the alpha-factor that differ from each other and from alpha-factor encoded by MF alpha 1 gene by a Gln leads to Asn and a Lys leads to Arg substitution. The first copy of the alpha-factor is preceded by a sequence coding for 87 amino acids which ends with Lys-Arg-Glu-Ala-Val-Ala-Asp-Ala. The coding blocks of the two copies of the pheromone are separated by the sequence for Lys-Arg-Glu-Ala-Asn-Ala-Asp-Ala. Thus, the alpha-factor can be derived from 2 different precursor proteins of 165 and 120 amino acids containing, respectively, 4 and 2 copies of the pheromone.

Amino Acid Sequence↗
PubMed1983

The effect of alpha-adrenergic blockade on cardiac performance and tissue oxygen delivery during excision of pheochromocytoma.

To establish criteria for administration of the optimal dose of alpha-adrenergic receptor blocking drugs, we studied cardiac performance and tissue oxygen tension in three patients who underwent excision of pheochromocytoma. Subcutaneous oxygen tension was measured by the method of Niinikoski and Hunt. Cardiac function was assessed by thermodilution cardiac output, systemic and pulmonary arterial blood pressures, and continuous two-dimensional transesophageal echocardiography of a cross section of the left ventricle at the level of the papillary muscles. Despite large changes in cardiac output and systemic, pulmonary, and wedge pressures, intraoperative tissue oxygen tensions and ejection fractions remained normal (even at times of peak catecholamine excretion and very abnormal wedge pressures). Studies of healthy animals that received no alpha-adrenergic receptor blocking drugs showed major decrements of tissue oxygen in response to modest doses of epinephrine. We conclude that progressive administration of alpha-adrenergic receptor blocking drugs does not absolutely protect the patient from major changes in blood pressure during operation for pheochromocytoma, but that cardiac performance and oxygen supply to the tissues are unimpaired.

Adrenal Gland Neoplasms↗
PubMed1982

A bacterial secretory protein requires signal recognition particle for translocation across mammalian endoplasmic reticulum.

In vitro transcription of DNA from plasmid pBR322 was coupled to cell-free translation in a wheat germ system. The major translation product was pre-beta-lactamase. Upon addition of dog pancreas microsomes, the precursor was processed to authentic beta-lactamase as shown by partial NH2-terminal sequence analysis. Processing was linked to translocation into the microsomal vesicles. Salt-extracted microsomes did not process pre-beta lactamase but could be reactivated by purified signal recognition particle, which is the functional component of the salt wash (Walter, P., and Blobel, G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7112-7116). Signal recognition particle alone caused a drastic translation arrest that could be released by salt-depleted membranes. These data are consistent with those obtained for eukaryotic proteins and suggest that co-translational translocation of both bacterial and eukaryotic secretory proteins across the endoplasmic reticulum require identical components.

Animals↗
PubMed1982

Identification of the signal peptidase cleavage site in Bacillus licheniformis prepenicillinase.

The DNA sequence of the entire gene for penicillinase of Bacillus licheniforms 749 has recently been determined (Neugebauer, K., Sprengel, R., and Schaller, H. (1981) Nucleic Acid Res. 9, 2577-2588). Here we show that a primary translation product (Mr 35,000) can be synthesized in vitro by translation of B. licheniformis mRNA in a EScherichia coli cell-free system, or in vivo, after phenylethyl alcohol treatment of B. licheniformis. The partial NH2-terminal sequence of the in vivo synthesized primary translation product, termed prepenicillinase, was in agreement with the NH2-terminal sequence deduced from the DNA sequence. Furthermore, when a B. licheniformis membrane fraction plus the nonionic detergent Nikkol were present in the in vitro translation system, prepenicillinase was proteolytically processed to a polypeptide (Mr 31,250) that comigrated electrophoretically with the membrane-bound form of this enzyme. The partial NH2-terminal sequence of this processed form showed that it had lost 26 NH2-terminal residues present in prepenicillinase. We propose that the observed cleavage was due to membrane-associated and detergent-activated signal peptidase and consequently, that the 26-residue-long extension of nascent prepenicillinase functions in translocation across the prokaryotic plasma membrane. Based on our partial protein sequence data and the DNA sequence data, the signal peptidase-processed penicillinase starts with a Cys residue. Cotranslational and post-translational modifications of this NH2-terminal Cys, similar to those observed in the E. coli lipoprotein, might be the only means by which penicillinase can be anchored to the outer leaflet of the B. licheniformis plasma membrane.

Amino Acid Sequence↗