Based upon existing methods of isolating fetal porcine islet tissue, a simple, reliable procedure was developed for the preparation of porcine neonatal islet cell aggregates with a reproducible and defined cellular composition. After 9 d of in vitro culture, tissue from one neonatal pig pancreas yielded ‫ف‬ 50,000 islet cell aggregates, consisting of primarily epithelial cells (57%) and pancreatic endocrine cells (35%). During the culture period, the total ␤ cell mass decreased initially, but subsequently increased 1.5-fold between days 3 and 9. Transplantation of grafts consisting of 3 ϫ

Acid resistance (AR) in Escherichia coli is defined as the ability to withstand an acid challenge of pH 2.5 or less and is a trait generally restricted to stationary-phase cells. Earlier reports described three AR systems in E. coli. In the present study, the genetics and control of these three systems have been more clearly defined. Expression of the first AR system (designated the oxidative or glucose-repressed AR system) was previously shown to require the alternative sigma factor RpoS. Consistent with glucose repression, this system also proved to be dependent in many situations on the cyclic AMP receptor protein. The second AR system required the addition of arginine during pH 2.5 acid challenge, the structural gene for arginine decarboxylase (adiA), and the regulatorcysB, confirming earlier reports. The third AR system required glutamate for protection at pH 2.5, one of two genes encoding glutamate decarboxylase (gadA or gadB), and the gene encoding the putative glutamate:γ-aminobutyric acid antiporter (gadC). Only one of the two glutamate decarboxylases was needed for protection at pH 2.5. However, survival at pH 2 required both glutamate decarboxylase isozymes. Stationary phase and acid pH regulation of the gad genes proved separable. Stationary-phase induction of gadA and gadBrequired the alternative sigma factor ςS encoded byrpoS. However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be ςS independent. Neither gad gene required the presence of volatile fatty acids for induction. The data also indicate that AR via the amino acid decarboxylase systems requires more than an inducible decarboxylase and antiporter. Another surprising finding was that the ςS-dependent oxidative system, originally thought to be acid induced, actually proved to be induced following entry into stationary phase regardless of the pH. However, an inhibitor produced at pH 8 somehow interferes with the activity of this system, giving the illusion of acid induction. The results also revealed that the AR system affording the most effective protection at pH 2 in complex medium (either Luria-Bertani broth or brain heart infusion broth plus 0.4% glucose) is the glutamate-dependent GAD system. Thus, E. coli possesses three overlapping acid survival systems whose various levels of control and differing requirements for activity ensure that at least one system will be available to protect the stationary-phase cell under naturally occurring acidic environments.

Degenerate oligonucleotides based on the published Escherichia coli glutamate decarboxylase (GAD) protein sequence were used in a polymerase chain reaction to generate a DNA probe for the E. coli GAD structural gene. Southern blots showed that there were two cross-hybridizing GAD genes, and both of these were cloned and sequenced. The two GAD structural genes, designated gadA and gadB, were found to be 98% similar at the nucleotide level. Each gene encoded a 466-residue polypeptide, named, respectively, GAD a and GAD 13, and these differed by only five amino acids. Both GAD a and GAD 13 contain amino acid residues which are highly conserved among pyridoxal-dependent decarboxylases, but otherwise the protein sequences were not homologous to any other known proteins. By restriction mapping and hybridization to the Kohara miniset library, the two GAD genes were located on the E. coli chromosome. gadA maps at 4046 kb and gadB at 1588 kb. Neither of these positions is in agreement with the current map position for gadS as determined by genetic means. Analysis of Southern blots indicated that two GAD genes were present in all E. coli strains examined, including representatives from the ECOR collection. However, no significant cross-hybridizing gene was found in Salmonella species. Information about the DNA sequences and map positions of gad4 and gadB should facilitate a genetic approach to elucidate the role of GAD in E. coli metabolism.The enzyme glutamate decarboxylase (GAD; also known as glutamic acid decarboxylase; EC 4.1.1.15) catalyzes the a-decaboxylation of glutamic acid to produce -y-aminobutyric acid. Within bacteria, GAD activity seems to be relatively unique to Escherichia coli (37). Gale (14) proposed a general role for the inducible bacterial amino acid decarboxylases, including GAD, in the maintenance of physiological pH under acidic conditions. E. coli GAD has been extensively characterized with respect to its biophysical and biochemical properties (1,14,30,31,(39)(40)(41)(42)(43)(44)(45)(46), and a partial protein sequence has been available for some time (42, 43). Based on genetic linkage studies in E. coli, the structural gene for GAD (gadS) and a potential regulatory gene (gadR) have been mapped between mtl at approximately 80.7 minutes and gltS at approximately 82.4 minutes (3,23,26,27), but these early results have not been followed up. For the purposes of our immunological studies, we were interested in obtaining the complete DNA and protein sequences of E. coli GAD. At the time this work was initiated, an extensive search of the literature and all available sequence data bases suggested that neither the complete protein sequence nor the gene sequence for E. coli GAD had been determined. To obtain this information, we generated a DNA probe for the gene based on a recent more extensive partial protein sequence (24). By using this probe on E. coli genomic DNA, we discovered that there were not one but two separate cross-hybridizing GAD genes. We report here the complete DNA sequences and map pos...

We evaluated CD8+ T cell responses against the dominant CTL epitope, OVA257–264, expressed by an acute (Listeria monocytogenes (LM) OVA) vs a chronic pathogen (Mycobacterium bovis bacillus Calmette-Guérin (BCG) OVA) to reveal the influence on CD8+ T cell memory and consequent protection against a challenge with OVA-expressing tumor cells. Infection with lower doses of both pathogens resulted in stronger bacterial growth but weaker T cell memory indicating that memory correlates with pathogen dose but not with bacterial expansion. The CD8+ T cell response induced by LM-OVA was helper T cell-independent and was characterized by a rapid effector response followed by a rapid, but massive, attrition. In contrast, BCG-OVA induced a delayed and weak response that was compensated for by a longer effector phase and reduced attrition. This response was partly dependent on CD4+ T cells. CD8+ T cell response induced by BCG-OVA, but not LM-OVA, was highly dependent on pathogen persistence to compensate for the weak initial CD8+ T cell priming. Despite a stronger initial T cell response with LM-OVA, BCG-OVA provided more effective tumor (B16OVA) control at both local and distal sites due to the induction of a persistently activated acquired, and a more potent innate, immunity.

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