Characterization of the aegA locus of Escherichia coli: control of gene expression in response to anaerobiosis and nitrate.

Analysis of the DNA sequence upstream of the narQ gene, which encodes the second nitrate-responsive sensor-transmitter protein in Escherichia coli, revealed an open reading frame (ORF) whose product shows a high degree of similarity to a number of iron-sulfur proteins as well as to the beta subunit of glutamate synthase (gltD) of E. coli. This ORF, located at 53.0 min on the E. coli chromosome, is divergently transcribed and is separated by 206 bp from the narQ gene. Because of the small size of the intergenic region, we reasoned that the genes may be of related function and/or regulated in a similar fashion. An aegA-lacZ gene fusion was constructed and examined in vivo; aegA expression was induced 11-fold by anaerobiosis and repressed 5-fold by nitrate. This control was mediated by the fnr, narX, narQ, and narL gene products. Analysis of an aegA mutant indicated that the aegA gene product is not essential for cell respiration or fermentation or for the utilization of ammonium or the amino acids L-alanine, L-arginine, L-glutamic acid, glycine, and DL-serine as sole nitrogen sources. The ORF was designated aegA to reflect that it is an anaerobically expressed gene. The structural properties of the predicted AegA amino acid sequence and the regulation of aegA are discussed with regard to the possible function of aegA in E. coli.     

Translational control of programmed cell death: eukaryotic translation initiation factor 4E blocks apoptosis in growth-factor-restricted fibroblasts with physiologically expressed or deregulated Myc.

There is increasing evidence that cell cycle transit is potentially lethal, with survival depending on the activation of metabolic pathways which block apoptosis. However, the identities of those pathways coupling cell cycle transit to survival remain undefined. Here we show that the eukaryotic translation initiation factor 4E (eIF4E) can mediate both proliferative and survival signaling. Overexpression of eIF4E completely substituted for serum or individual growth factors in preserving the viability of established NIH 3T3 fibroblasts. An eIF4E mutant (Ser-53 changed to Ala) defective in mediating its growth-factor-regulated functions was also defective in its survival signaling. Survival signaling by enforced expression of eIF4E did not result from autocrine release of survival factors, nor did it lead to increased expression of the apoptosis antagonists Bcl-2 and Bcl-XL. In addition, the execution apparatus of the apoptotic response in eIF4E-overexpressing cells was found to be intact. Increased expression of eIF4E was sufficient to inhibit apoptosis in serum-restricted primary fibroblasts with enforced expression of Myc. In contrast, activation of Ha-Ras, which is required for eIF4E proliferative signaling, did not suppress Myc-induced apoptosis. These data suggest that the eIF4E-activated pathways leading to survival and cell cycle progression are distinct. This dual signaling of proliferation and survival might be the basis for the potency of eIF4E as an inducer of neoplastic transformation.     

Phytoplankton and periphyton responses to in situ experimental nutrient enrichment in a shallow subtropical lake

Experiments were performed in situ in shallow, subtropical LakeOkeechobee (Florida. USA) to quantify and compare the responsesof phytoplanklon (in 20 I clear polycarbonate carboys) and periphyton(on nutrient-diffusing clay substrates) to additions of nitrogenand/or phosphorus. During early and late summer. 1994, bothassemblages were nitrogen limited or co-limited by nitrogenand phosphorus, indicating the potential for competition betweenbenthic and planktonic communities. During late summer, therewas evidence that high phytoplankton biomass reduced light penetrationthrough the water column and may have suppressed periphytongrowth. The similar phytoplankton and periphyton taxonomic structures,both dominated by Lyngbya sp. and pennate diatoms, suggestedthat in shallow regions of this lake, resuspended meroplanktonmight account for a large portion of phytoplankton biomass.This phenomenon has been observed in other shallow, wind-drivenFlorida lakes.     

Multidrug resistance evaluation by confocal microscopy in primary urothelial cancer explant colonies

Assessing functional multidrug resistance (MDR) status in clinical biopsy material using drug autofluorescence has potential applications to clinical management. The small size of many cystoscopy specimens has led us to develop, as an alternative to flow cytometry, a protocol for studying epirubicin accumulation in adherent colonies of primary bladder cancer cells viewed live andin situ by confocal microscopy. The limitations to quantitation inherent in this technique are compensated for by preservation of cellular organisation and the elimination of non-malignant cells. Biopsy material is disaggregated and explanted into culture-grade petri dishes. After incubation for three to seven days plaques of epithelial cells have developed. Classical patterns of sensitive and resistant drug distribution are observed. Cells of the rolled edges of the colony accumulate more drug than those of the inner epithelial monolayer. Some central areas of larger colonies give the appearance of drug arrested at the intercellular junctions to give a fenestrated pattern. These observations contribute to the understanding of mechanisms in MDR as well as forming the basis for a clinical urological MDR evaluation protocol.     

Application of DNA amplification fingerprinting (DAF) to mixed culture bioreactors

Summary The use of DNA amplification fingerprinting (DAF) as a tool for monitoring mixed microbial populations in bioreactors was evaluated. Short (8-mer or 10-mer) oligonucleotides were used to prime DNA extracts from various biological reactors during polymerase chain reaction (PCR) amplification. The reactors examined in this study included two sets of anaerobic stirred tank continuous flow bioreactors. One set of anaerobic reactors was operated under methanogenic conditions and one set was operated under sulfate-reducing conditions. The anaerobic reactor communities in the methanol-fed reactors showed extensive DAF homology. DAF was also applied to a fixed-film azo dye degrading reactor to examine the degree of uniformity of colonization of the substratum in representative regions of the reactor. This method is a quick and relatively inexpensive means of monitoring microbial community structure during biological processes. Since no cultivation of the sample is involved, the genetic profile of the community is not biased by outgrowth conditions. DAF profiles may be useful for comparisons of population changes over time or of bench-scale vs pilot-scale reactors but not adequate for assessing community diversity.     

Surfactant Effects on Lipase-Catalyzed Hydrolysis of Olive Oil in AOT/ISOOCTANE Reverse Micelles

The effects of surfactant concentration on the hydrolytic activity of Candida rugosa lipase in AOT/isooctane reverse micelles with olive oil as the substrate has been investigated. A noncompetitive inhibition by the surfactant on the enzyme was observed. Strong dependences of the kinetic constants k_cat and k_M, but not k_I on the water-to-surfactant ratio (R value) have been identified. The benefits of carrying out the hydrolysis at higher surfactant and water concentrations were demonstrated from the improvement of the initial rate and time course of conversion.     

Expression of a Synthetic Gene Encoding the Anticoagulant-Antimetastatic Protein Ghilanten by the Methylotropic YeastPichia pastoris

Ghilantens are a family of cysteine-rich inhibitors of the clotting enzyme, factor Xa, that are produced in the salivary glands of the South American leechHaementeria ghilianii.In this study, a gene, designed from the amino acid sequence of a specific ghilanten isoform, was assembled from eight double-stranded oligonucleotide fragments. A yeast expression plasmid, pPIC9HG-2, was constructed by making an in-frame fusion of the ghilanten-coding sequences with the region encoding the pre-pro α-mating factor signal sequence for secretion. The expression of ghilanten in pPIC9HG-2 was under the control of the methanol-inducible, alcohol oxidase (AOX1) promoter.Pichia pastorisyeast strains KM 71 and SMD 1168 were transformed with linearized pPIC9HG-2 to target integration of the plasmid to the chromosomal 5′-AOX1locus via homologous recombination. Both strains yielded His⁺transformants that secreted a potent anticoagulant activity into the medium. Product yield was improved by using buffered media (pH 6.0) supplemented with either casamino acids or a mixture of yeast extract and peptone. The protease-deficient strain, SMD 1168, secreted about a twofold higher level ofr-ghilanten than KM 71. Significant clonal variation in the expression ofr-ghilanten was found among the His⁺transformants. A high producing clone was selected for production at the 2-liter shake flask and 10-liter bioreactor scales.r-Ghilanten was recovered from the fermentation broths in a single step by heparin Sepharose affinity chromatography. Protein sequence analysis of the amino terminus showed that the correct processing to yield mature ghilanten varied with the fermentation conditions.