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Biomedical subjects

P T Chen

Publications and source records attributed to P T Chen.

10 recordsLinked to original sources
PubMed2002

High production of heterologous proteins in Escherichia coli using the thermo-regulated T7 expression system.

The exclusive use of isopropyl beta-D-thiogalactopyranoside to activate the T7 promoter for protein production has limited the general use of the expression system. We have sought an alternative by constructing a recombinant Escherichia coli strain, BL21 (G2), to carry a chromosomal copy of T7 gene 1 fused to the lambdaPL and lambdaP(R) tandem promoter. As a result, the recombinant strain harboring the carbamoylase gene from Agrobacterium radiobacter NRRL B11291 was shown to display various levels of.protein production in response to different degrees of heat shock. In particular, the system remained inactive at 30 degrees C and exhibited high sensitivity to heat such that a detectable carbamoylase activity could be measured after exposure to 33 degrees C. Moreover, heating in two steps - elevating the temperature from 30 degrees C to 39 degrees C and holding for a brief period, followed by reducing to 37 degrees C--was found to be the most potent method for protein production in this case. Using this approach, the recombinant protein accounted for 20% of total protein content of the cell. These results reveal the advantages of this expression system: responsiveness to thermal modulation and high-level production capability. In an attempt to enhance the total protein yield, a fed-batch fermentation process was carried out to control the cell growth rate by adjusting the substrate inflow. By applying the two-step temperature change. a carbamoylase yield with enzyme activity corresponding to 14,256 units was obtained. This production yield is a 10-fold increase in comparison with that at the batch-fermentation scale and 2,000-fold higher than that achieved at the shake-flask scale. Overall, it illustrates the promise of the newly constructed T7 system based on heat inducibility for industrial scale production of recombinant proteins.

Amidohydrolases↗
PubMed2001

Early epileptic encephalopathy with suppression burst electroencephalographic pattern--an analysis of eight Taiwanese patients.

Early epileptic encephalopathy with suppression burst (SB) comprises two distinct epileptic syndromes, early infantile epileptic encephalopathy (EIEE) and early myoclonic encephalopathy (EME). We reviewed etiologies, neurological outcome and clinico-electroencephalographic features of EIEE and EME. Chart records of early epileptic encephalopathy with SB from January 1997 to December 2000 were reviewed. These cases fulfilled the diagnostic criteria of EIEE and EME. Totally eight patients (four females, four males) were enrolled. They consisted of three cases of EIEE and five cases of EME. The follow-up periods ranged from 6 to 30 months. For EIEE, two cases had migrational disorders, and one was cryptogenic; for EME, three cases had non-ketotic hyperglycinemia (NKH), one was pyridoxine dependency and one was cryptogenic. The main initial seizure patterns were tonic spasms in EIEE, and were erratic myoclonus in EME. The age of seizure onset ranged from 26 h to 5 days after birth for EIEE, and 2 h to 7 days of life for EME. The SB pattern in the electroencephalography (EEG) was noted mainly during sleep state in EME, but in both awake and sleep states in EIEE. Asymmetric SB pattern and background activities in EEG were found in migrational disorders. The EEG in all cases of EIEE changed to hypsarrhythmia at 4-6 months of age. In EME, only the EEG in cases of NKH evolved to hypsarrhythmia. Response to anti-convulsants was generally poor. All had severe psychomotor retardation. Although EIEE and EME share several common features, differences in terms of seizure seminology and evolution, EEG patterns and etiologies still exist.

Anticonvulsants↗
PubMed2000

Otogenic brain abscess--a case report.

Brain abscess is one of the life-threatening complications of otitis media. Mortality and morbidity have decreased with the advent of antibiotic therapy. More frequently encountered in cases of acute otitis media in the preantibiotic era, in recent years otogenic brain abscess was noticed almost only in patients of chronic otitis media with cholesteatoma. A case of brain abscess in a 49-year-old female was initially diagnosed as a headache. A high resolution computed tomography (HRCT) scan of the temporal bones later revealed that there were two abscesses over the right side temporal lobe. A modified radical mastoidectomy was performed. Cultures of the middle ear cholesteatoma later grew Pseudomonas aeruginosa and Strenotrophomonas maltophilia. Antibiotic therapy was carried on for three months postoperatively. The patient improved but retained a conductive hearing loss.

Brain Abscess↗
PubMed1995

Characterization of a human immunodeficiency virus type 1 variant with reduced sensitivity to an aminodiol protease inhibitor.

Development of viral resistance to the aminodiol human immunodeficiency virus (HIV) protease inhibitor BMS 186,318 was studied by serial passage of HIV type 1 RF in MT-2 cells in the presence of increasing concentrations of compound. After 11 passages, an HIV variant that showed a 15-fold increase in 50% effective dose emerged. This HIV variant displays low-level cross-resistance to the C2 symmetric inhibitor A-77003 but remains sensitive to the protease inhibitors Ro 31-8959 and SC52151. Genetic analysis of the protease gene from a drug-resistant variant revealed an Ala-to-Thr change at amino acid residue 71 (A71T) and a Val-to-Ala change at residue 82 (V82A). To determine the effects of these mutations on protease and virus drug susceptibility, recombinant protease and proviral HIV type 1 clones containing the single mutations A71T and V82A or double mutation A71T/V82A were constructed. Subsequent drug sensitivity assays on the mutant proteases and viruses indicated that the V82A substitution was responsible for most of the resistance observed. Further genotypic analysis of the protease genes from earlier passages of virus indicated that the A71T mutation emerged prior to the V82A change. Finally, the level of resistance did not increase following continued passage in increasing concentrations of drug, and the resistant virus retained its drug susceptibility phenotype 34 days after drug withdrawal.

Amino Acid Sequence↗
PubMed1984

Inhibition of human cytomegalovirus replication by 9-(1,3-dihydroxy-2-propoxymethyl)guanine alone and in combination with human interferons.

The inhibitory action of 9-(1,3-dihydroxy-2-propoxymethyl)guanine on the replication of human cytomegalovirus was studied. Three laboratory strains (AD-169, Towne, and Davis) and three early passage (less than 10) clinical isolates were all inhibited in yield inhibition assays. In cultures infected with AD-169, virus yields could be inhibited if the drug was added as late as 3 days after the replication cycle had begun. The effects of the drug were fully reversible during the first 4 days of the viral replication cycle. Viral infectivity and viral DNA synthesis were reduced more than viral protein synthesis. Synergistic antiviral effects were observed with beta-cysteine, and to a lesser extent, with beta-serine recombinant interferons, but only over a narrow range of dose combinations.

Acyclovir↗
PubMed1984

Comparison of antiviral activities of cloned and native human interferons against herpes simplex virus types 1 and 2 and human cytomegalovirus.

Five recombinant alpha interferons and two recombinant beta interferons have been tested for their ability to inhibit yields of herpes simplex virus types 1 and 2 and human cytomegalovirus in human embryonic lung cells. All of the alpha species and both of the beta forms (cysteine and serine) were active against the herpesviruses tested in this study. Neither the recombinant alpha nor the recombinant beta interferons exceeded the activity of the native species against herpes simplex viruses types 1 and 2. However, the recombinant beta interferons inhibited cytomegalovirus more than either the native beta or the alpha interferon species with the exception of interferon alpha K (alpha 6).

Cloning, Molecular↗
PubMed1980

Surgical treatment of esophageal carcinoma.

Recent progress in the surgical treatment of carcinoma of the esophagus has changed the pessimistic outlook for this malignancy. With reasonably early diagnosis, it is feasible to achieve a resectability rate of 75 to 85 percent, an operative mortality of about 5 percent and a 5 year survival rate of about 30 percent. With careful selection of patients for operation and proper use of radiation and other adjunctive therapy, even more encouraging results are possible. Efforts for further improvement may be directed toward early diagnosis, refinement in operative technique and better pre- and postoperative care. The use of microsurgery and the development of mechanical suture apparatus in esophageal reconstruction after resection may further improve surgical treatment of cancer of the esophagus. Thoracic surgeons still have the responsibility to improve the management of the nonresectable cases of carcinoma of the esophagus.

China↗
PubMedDate not supplied

One-step production of D-p-hydroxyphenylglycine by recombinant Escherichia coli strains.

The gene encoding D-hydantoinase from Agrobacterium radiobacter NRRL B11291 was successfully cloned by use of polymerase chain reaction. A positive clone was scored, and its nucleotide sequence was further analyzed. The analysis by deleting various lengths of nucleotides from the amino terminus of the open reading frame revealed the putative regions for promoter and RBS site. By highly expressing both D-hydantoinase and carbamoylase, recombinant Escherichia coli strains were able to convert DL-hydroxyphenyl hydantoin (DL-HPH) to D-p-hydroxyphenylglycine (D-HPG) with a conversion yield of 97%, accounting for productivity 5 times higher than that obtained by A. radiobacter NRRL B11291. Immobilizing the recombinant cells with kappa-carrageenan could also achieve a conversion of 93%, while A. radiobacter NRRL B11291 attained 20% within the same period of reaction time. These results illustrate the feasibility in employing recombinant E. coli to accomplish one-step conversion of DL-HPH to D-HPG. In the process of improving D-HPG production, D-hydantoinase activity was increased 2.57-fold but carbamoylase activity remained constant, which resulted in only a 30% increase in the reaction rate. It suggests that carbamoylase is the step setting the pace of the reaction. Since the reaction substrate is highly insoluble, achieving sufficient agitation appears to be an important issue in this heterogeneous system. This view is further supported by the study on repeated use of cells, which shows that to reach a conversion of more than 90% free cells can be recycled six times, whereas immobilized cells can be used only twice. In conclusion, the poor reusability of immobilized cells is due to the fouling on the gel surface.

Amidohydrolases↗