Expression of the Klebsiella pneumoniae CG21 acetoin reductase gene in Clostridium acetobutylicum ATCC 824.

Acetoin reductase catalyzes the production of 2,3-butanediol from acetoin. The gene encoding the acetoin reductase of Klebsiella pneumoniae CG21 was cloned and expressed in Escherichia coli and Clostridium acetobutylicum ATCC 824. The nucleotide sequence of the gene encoding the enzyme was determined to be 768 bp long. Expression of the K. pneumoniae acetoin reductase gene in E. coli revealed that the enzyme has a molecular mass of about 31,000 Da based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The K. pneumoniae acetoin reductase gene was cloned into a clostridial/E. coli shuttle vector, and expression of the gene resulted in detectable levels of acetoin reductase activity in both E. coli and C. acetobutylicum. While acetoin, the natural substrate of acetoin reductase, is a typical product of fermentation by C. acetobutylicum, 2,3-butanediol is not. Analysis of culture supernatants by gas chromatography revealed that introduction of the K. pneumoniae acetoin reductase gene into C. acetobutylicum was not sufficient for 2,3-butanediol production even though the cultures were producing acetoin. 2,3-Butanediol was produced by cultures of C. acetobutylicum containing the gene only when commercial acetoin was added.

Molecular screening of donor corneas for fungi before excision.

PURPOSE: To develop panfungal and Candida albicans species-specific polymerase chain reaction (PCR) assays to screen donor eyes for fungal contamination before corneal excision. METHODS: PCR primers were designed for either the broad-spectrum detection of fungal DNA or the specific detection of C. albicans DNA. Their sequences were based on rDNA regions highly conserved among and specific to fungi and C. albicans, respectively. PCR conditions with the two primer sets were optimized and tested for sensitivity using purified C. albicans genomic DNA and a plasmid containing the relevant region of C. albicans DNA. The specificity of the primer sets was established using higher eukaryotic, fungal, prokaryotic, and viral DNAs as PCR templates. Donor eye swab specimens were collected before corneal excision. DNA was extracted from the specimens and tested by both PCR assays. RESULTS: The lower limit of detection for both primer sets was consistently 10(3) genome equivalents, when using genomic DNA as a template and 10(2) copies of plasmid. The fungal PCR assay amplified DNA from all fungal species tested but did not amplify any of the selected mammalian, bacterial, or viral DNA. The C. albicans PCR detected the C. albicans DNA but was negative for all other DNA substrates, including the other fungal templates. Thirty-five percent of the donor eye samples tested were positive for fungus, and 19% were positive for C. albicans DNA. CONCLUSIONS: The PCR assays allowed the rapid screening of DNA extracted from specimens collected from corneal donors for potential fungal contamination. The assay was highly sensitive and specific for screening corneal surfaces. The results suggest that approximately one-third of donor eyes tested harbor fungi on the ocular surface.

Electrotransformation of Clostridium acetobutylicum ATCC 824 using high-voltage radio frequency modulated square pulses.

Molecular biological improvement of industrial solventogenic clostridia could be enhanced by a higher efficiency of electrotransformation. In this research, we used a new approach to determine the frequency spontaneously generated by Clostridium acetobutylicum ATCC 824 cells during the application of a square high-voltage pulse. Once the frequency of 100 kHz was determined we transformed clostridial cells with pSOS84 plasmid DNA using radio-frequency modulated high-voltage square pulses (electric field strength 12 kVcm-1; pulse duration 22.5 ms; frequency of pulse modulation 100 kHz) to reach an efficiency exceeding 106 transformants microg-1 of plasmid DNA. We propose a possible role for cellular membrane structures in affecting the transformation yield.

Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum.

A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery.

Effect of butyrate and corticosteroids on retinoblastoma in vitro and in vivo.

The effect of sodium butyrate (NaB) alone and in combination with a glucocorticoid was studied on Y-79 retinoblastoma cells both in vitro and in vivo. Both NaB (0.5 and 4 mM) and hydrocortisone (1-100 microM) added separately to cells grown in suspension resulted in a small growth inhibition (less than 15%). A marked synergistic effect (75-77% growth inhibition) was observed in vitro when NaB and hydrocortisone were added in combination at all the concentrations tested. With in vivo experiments using the nude mouse model of retinoblastoma, the combination of NaB and methylprednisolone did not inhibit tumor growth.

The value of nucleolar organizer regions in uveal melanoma. The Collaborative Ocular Melanoma Study Group.

Silver staining of nucleolar organizer regions is an objective method for evaluating the malignancy of a variety of tumors. We studied 126 ciliochoroidal melanomas, three coincidental nevi that occurred in eyes with melanomas, and one magnocellular nevus collected from the Collaborative Ocular Melanoma Study to determine the effectiveness of the silver-stained nucleolar organizer region technique in assessing the malignant potential of these tumors. Malignant lesions demonstrated higher mean silver-stained nucleolar organizer region counts (4.347) than benign nevi (1.855) (P less than or equal to .0001). Among malignant melanomas, mixed-cell melanomas had slightly higher counts than spindle-cell melanomas (P less than or equal to .0001), but this difference was not important clinically. Results were also compared to other histopathologic variables, which disclosed correlation of silver-stained nucleolar organizer regions with mitoses and tumor size. Comparison with computerized cytomorphometric analyses of prognosis also disclosed significant correlation. This technique may prove to be a useful adjunct in the assessment of malignancy and treatment response of uveal melanomas.