Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) containing a predominant amount of 3-hydroxyvalerate by engineered Escherichia coli expressing propionate-CoA transferase.

AIMS: Of the biodegradable polyhydroxyalkanoates (PHAs), poly(hydroxybutyrate-co-hydroxyvalerate) (P(HB-co-HV)) is often considered for fabrication of biocompatible and absorbable medical devices and other applications. Depending on the application, however, specific mechanical or processing properties must be improved. To address these required properties, we sought to alter the monomer composition of the copolymer by a combination genetic engineering in an Escherichia coli host and carbon substrate feeding. METHODS AND RESULTS: We applied a new method of 3-hydroxyvalerate (3HV) monomer synthesis to produce a co-polymer by the introduction of a propionyl-CoA transferase gene (pct), along with PHA biosynthetic genes bktB, phaB and phaC from Ralstonia eutropha into engineered E. coli to produce P(HB-co-HV). The resulting strain successfully produced the copolymer containing an ultra-high 3HV monomer composition (over 80 wt%). CONCLUSIONS: To the best of our knowledge, the P(HB-co-HV) production strain constructed here synthesized polymer with the highest 3HV content of any engineered E. coli strain. This strain could also produce P(HB-co-HV) with the use of lower concentrations of propionate in the growth medium, compared to other reported strains, which could avoid the known growth inhibition from propionate in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyhydroxyalkanoates have been emphasized as a potential alternative for petroleum-based plastics by virtue of their physical properties and environmentally friendly characteristics. The copolymer produced in this work validates our genetic engineering approach and suggests that the Pct enzyme is a more efficient method for production of propionyl-CoA, the 3-hydroxyvaleryl-CoA precursor.

Cell engineering and molecular pharming for biopharmaceuticals.

Biopharmaceuticals are often produced by recombinant E. coli or mammalian cell lines. This is usually achieved by the introduction of a gene or cDNA coding for the protein of interest into a well-characterized strain of producer cells. Naturally, each recombinant production system has its own unique advantages and disadvantages. This paper examines the current practices, developments, and future trends in the production of biopharmaceuticals. Platform technologies for rapid screening and analyses of biosystems are reviewed. Strategies to improve productivity via metabolic and integrated engineering are also highlighted.

A bioactive polyacetylene compound isolated from Centella asiatica.

A polyacetylene compound was isolated from the aerial parts of Centella asiatica. The chemical structure of this new compound was identified as methyl 5-[(E)-9-hydroxy-1-(1-hydroxyhexyl)-2-methoxyundeca-3,10-diene-5,7-diynyloxy]pentanoate (cadiyenol). This compound induces apoptosis (63%) independent of cell cycle regimen in mouse lymphoma cells (P388D1) at 28 microM (IC (50) = 24 +/- 2 microM) in 24 hours. The compound also reduces nitric oxide production by 70 +/- 2% in lipopolysacharride-activated mouse macrophages at 24 microM with no measurable cytotoxicity.

Characterization of Streptomyces MITKK-103, a newly isolated actinomycin X2-producer.

A new actinomycete strain designated MITKK-103 was isolated from the soil of a flowerpot using a humic acid agar medium. The newly isolated strain was able to produce a large amount of actinomycin X2 even under nonoptimized growing conditions and serves as a promising source of this antibiotic. Actinomycin X2 has higher cytotoxicity toward cultured human leukemia (HL-60) cells than does actinomycin D, and it induces cell death via apoptosis. A nearly complete 16S ribosomal DNA (rDNA) sequence from the isolate was determined and found to have high identity (98.5-100%) with Streptomyces galbus, Streptomyces griseofuscus, and Streptomyces padanus, indicating that MITKK-103 belongs to the genus Streptomyces. The isolate clustered with species belonging to the S. padanus clade in a 16S-rDNA-based phylogenetic tree and showed 75% overall homology to S. padanus ATCC 25646 in DNA-DNA relatedness analysis. Although the growth of the isolate was somewhat different from the three species mentioned, the strain MITKK-103 most closely resembles S. padanus on the basis of the morphological and phenotypic characteristics, phylogenetic analysis, and genotypic data. As such, this is the first report of a strain of S. padanus capable of producing actinomycins.

Using queueing theory to determine operating room staffing needs.

BACKGROUND: To meet American College of Surgeons criteria, Level I and II trauma centers are required to have in-house operating room (OR) staff 24 hours per day. According to the number of emergency cases occurring, hospitals may have varying needs for OR staffing during the night shift. Queueing theory, the analysis of historic data to provide optimal service while minimizing waiting, is an objective method of determining staffing needs during any time period. This study was done to determine the need to activate a backup OR team during the night shift at a designated, verified Level II trauma center. METHODS: The basic queueing theory formula for a single-phase, single-channel system was applied to patients needing the services of the OR. The mean arrival rate was determined by dividing the number of actual cases by 2,920 hours in a year (8 hours per night x 365). The mean service rate is determined by averaging the length of the actual cases during the period studied. Using the mean arrival rate and the mean service rate, the probability of two or more patients needing the OR at the same time was determined. This probability was used to reflect the likelihood of needing to activate the backup OR team. Simulation was then used to calculate the same probability and validate the results obtained from the queueing model. RESULTS: All OR cases (n = 62) beginning after 11 PM and before 7 AM from July 1, 1996, through June 30, 1997, were analyzed. During the study period, the average arrival rate (A) was one patient every 5.9 days (0.0212 patient every hour), with an average service rate (mu) of 80.79 minutes per patient (0.7427 patients per hour). According to queueing theory, lambda = 0.0212 patients per hour, mu = 0.7427 patients per hour, lambda/mu = 0.0285, the probability of no patients being in the system (P0) = 0.9714, P1 = 0.0278, P> or =2 = 1 - (0.0278 + 0.9714) = 0.0008. The probability of two or more cases occurring simultaneously on the night shift is less than 0.1%. CONCLUSION: In our institution, activation of a second OR team is unnecessary when the first team is busy with a case on the night shift because the likelihood of two cases occurring concurrently is less than one in a thousand. Queueing theory can be a valuable tool to use in determining the staffing needs of many hospital departments. Trauma centers should apply this mathematical model in optimizing the use of their operational resource.

Relationships between mitotic delay and the dose rate of X radiation.

Upon exposure of cells to radiation delivered at a continuous low dose rate, cell proliferation may be sustained with the cells exhibiting a constant doubling time that is independent of the total dose. The doubling time or mitotic delay under these conditions has been shown to depend on the dose rate in HeLa, V79 and P388F cells (Mitchell et al., Radiat. Res. 79, 520-536, 1979; Fox and Gilbert, Int. J. Radiat. Biol. 11, 339-347, 1966). Reanalysis of the data for these particular cell lines shows that there is a threshold dose rate for mitotic delay, and that above the threshold there is a linear relationship between the length of mitotic delay and the logarithm of the dose rate which is referred to as the dose-rate response. We have observed the same relationships for L5178Y (LY)-R and LY-S cells exposed to low-dose-rate radiation. The threshold dose rates for LY-R, LY-S and P388F cells are similar (0.01-0.02 Gy/h) and are much lower than for V79 and HeLa cells. The slope of the dose-rate response curve is the greatest for HeLa cells, followed in order by LY-S, V79 and P388F cells, and finally by LY-R cells. The slopes for HeLa and LY-R cells differ by a factor of 35.

Hepatocyte transplantation: an alternative system for evaluating cell survival and immunoisolation.

To evaluate systems for barrier immunoisolation of transplanted hepatocytes, we used transgenic mouse hepatocytes that secrete HBsAg. Hepatocytes were rapidly encapsulated in chitosan, a cationic polymer derived by deacetylation of chitin. Chitosan was allowed to electrostatically bond with anionic sodium alginate for creating an outer bipolymer membrane of the capsules. After encapsulation, hepatocyte viability remained unchanged for seven days in vitro with secretion of HBsAg into the culture medium throughout this period. Following intraperitoneal transplantation of encapsulated hepatocytes, HBsAg promptly appeared in blood of recipients. In congeneic recipients, serum HBsAg peaked at two weeks. Hepatocytes were present in recovered chitosan capsules and expressed HBsAg mRNA. In allogeneic recipients, however, serum HBsAg disappeared within one week and recovered chitosan capsules showed lymphomononuclear cells but not hepatocytes. Transplantation of chitosan encapsulated HbsAg secreting hepatocytes failed to induce an anti-HBs response, suggesting modulation of the host immune response. These results indicate that transplantation systems using genetically modified hepatocytes which secrete gene products in the blood of recipients should facilitate evaluation of hepatocyte encapsulation.

Secondary structure of 11 S globulin in aqueous solution investigated by FT-IR derivative spectroscopy.

Secondary structure of 11 S globulin, a major storage protein of soybean seeds, has been investigated in aqueous solution by FT-IR spectroscopy. Conformational changes in the native protein upon thermal and chemical denaturation have been monitored by observing changes in the frequency position and peak intensity of the various bands. The frequency of the Amide I band of the native protein shifts by 4 cm-1 from 1,643 cm-1 to 1,647 cm-1 when denatured, while the corresponding intensity of the Amide I band compared to the native protein, decreases by 30 and 67%, respectively, for the urea and thermally denatured proteins, indicating gross conformational changes in the secondary structure. Trifluoroethanol, an alpha-helix promoter shifts the Amide I band from 1,643 cm-1 to 1,651 cm-1, typical of alpha-helix, with a corresponding increase in intensity by 14% relative to the native protein. Derivative spectroscopy, allowing resolution of overlapping bands, shows that the native protein mainly consists of beta-sheet, beta-turns and disordered structure with very little alpha-helix. On denaturation, beta-sheet disappeared almost completely with urea, while this is less so with thermal denaturation.

Pseudomembranous colitis caused by topical clindamycin phosphate.

Pseudomembranous colitis was observed on two occasions in the same patient and was associated with the topical administration of clindamycin phosphate. Assay for Clostridium difficile toxin was positive, and the patient was ultimately cured by oral vancomycin hydrochloride and the withdrawal of clindamycin therapy.

Secondary structural changes in globular protein induced by a surfactant: Fourier self-deconvolution of FT-IR spectra.

Conformational changes in ovalbumin, a globular protein, induced by an anionic surfactant, sodium dodecyl sulfate (SDS), have been monitored by an FT-IR spectrometer using ZnSe cylindrical internal reflection optics which allows high quality IR spectra to be obtained in water solution. The most notable change, on addition of SDS, occurs in the composite band of the Amide I absorption band and the vibrational frequency of the composite C = O bond shifts from 1639 cm-1 to 1652 cm-1. On the other hand, the position of the Amide II band remains fairly unchanged. Comparison of the various peak positions in the deconvoluted spectra for the native protein and the perturbed protein clearly shows the effect of SDS on the secondary structures of the protein. SDS unfolds the protein. It increases the helix content slightly. More importantly, it alerts the beta sheet structure, destroying it almost completely in the Amide I region, while retaining it in its neighbourhood. In the deconvoluted spectra of the perturbed protein, a band at 1531 cm-1 indicates generation of some beta turns. We used the second derivative of the deconvoluted spectra for fixing positions of minor peaks and shoulders. The results of this study indicate that the deconvolution of the normal IR spectra, consisting of composite bands, provides evidence for the specific secondary structures in a protein and for the way they are affected by changes in the environment, e.g., the addition of SDS. This makes it possible to relate conformational changes to specific secondary structures.

Synthesis of low-phenylalanine polypeptides.

Low-phenylalanine-peptides for dietotherapy of phenylketonuria (PKU) were prepared from soybean protein isolate. Soluble fraction of soybean protein isolate was hydrolysed by alpha-chymotrypsin then followed by carboxypeptidase-A. Molecular weight distribution and amino acid analysis were made on the resultant polypeptides. The chymotrypsin hydrolysate was divided into two fractions, Fraction I (molecular weight greater than 2500) and Fraction II (molecular weight between 1000 and 2500). The phenylalanine content of Fraction I (3.1%) was lower than that of Fraction II (5%), indicating the nonuniform distribution of phenylalanine in soy bean protein. Carboxypeptidase hydrolysis of Fraction I further reduced the phenylalanine concentration to 2.3%, approximately half of the original concentration in soybean protein isolate.