No abstract

A microbial screen using a yeast strain with conditional deficiency in the GPA1 gene was carried out to search for inhibitors of protein farnesyltransferase (PFT). A strain of Streptomyces was found to produce active compounds named UCF1-A, UCF1-B, and UCF1-C. Structural determination of these compounds revealed that UCF1-C is identical to the known antibiotic, manumycin, whereas UCF1-A and UCF1-B are structurally related to manumycin. All three UCF1 compounds suppress the lethality of gpa1 disruption, with UCF1-C exhibiting the strongest activity. UCF1 inhibits yeast as well as rat brain PFT. Fifty percent inhibition of yeast PFT activity is observed with 5 microM UCF1-C. Kinetic analyses of the inhibition suggest that UCF1-C acts as a competitive inhibitor of PFT with respect to farnesyl pyrophosphate, exhibiting a Ki of 1.2 microM, whereas the same compound appears to act as a noncompetitive inhibitor of PFT with respect to the farnesyl acceptor, the Ras protein. UCF1-C shows significant activity to inhibit the growth of Ki-ras-transformed fibrosarcoma, raising the possibility of its use as an antitumor drug.

Necrotizing enterocolitis (NEC) is a serious intestinal disease that occurs in newborn infants. It is associated with major morbidity and affects 5% of all infants admitted to neonatal intensive care units. Probiotics have variable efficacy in preventing necrotizing enterocolitis. Tight junctions (TJ) are protein complexes that maintain epithelial barrier integrity. We hypothesized that the probiotics Lactobacillus rhamnosus and Lactobacillus plantarum strengthen intestinal barrier function, promote TJ integrity, and protect against experimental NEC. Both an in vitro and an in vivo experimental model of NEC were studied. Cultured human intestinal Caco-2 cells were pretreated with L. rhamnosus and L. plantarum probiotics. TJ were then disrupted by EGTA calcium switch or LPS to mimic NEC in vitro. Trans-epithelial resistance (TER) and flux of fluorescein isothiocynate dextran was measured. TJ structure was evaluated by ZO-1 immunofluorescence. In vivo effects of ingested probiotics on intestinal injury and ZO-1 expression were assessed in a rat model of NEC infected with Cronobacter sakazakii (CS). Caco-2 cells treated with individual probiotics demonstrated higher TER and lower permeability compared to untreated cells (p<0.0001). ZO-1 immunofluorescence confirmed TJ stability in treated cells. Rat pups fed probiotics alone had more intestinal injury compared with controls (p=0.0106). Probiotics were protective against injury when given in combination with CS, with no difference in intestinal injury compared to controls (p=0.21). Increased permeability was observed in the probiotic and CS groups (p=0.03, p=0.05), but not in the probiotic plus CS group (p=0.79). Lactobacillus sp. strengthened intestinal barrier function and preserved TJ integrity in an in vitro experimental model of NEC. In vivo, probiotic bacteria were not beneficial when given alone, but were protective in the presence of CS in a rat model of NEC.

Using a sensitive single isotope enzymatic assay we measured bronchoalveolar lavage (BAL) fluid histamine in asymptomatic normal (nonallergic), allergic rhinitic, and allergic asthmatic subjects. Normal subjects were found to have little or no detectable amounts of histamine in BAL fluid (11±11 pg/ml), and few BAL fluid mast cells. In comparison, the allergic rhinitics and allergic asthmatics had much higher amounts of BAL fluid histamine (113±53 and 188±42 pg/ml, respectively), and a significantly greater number of BAL fluid mast cells. Furthermore, despite having equivalent baseline pulmonary function values, allergic asthmatics with BAL fluid histamine levels > 100 pg/ ml required only 7±2 breath units of methacholine to induce a 20% drop in forced expiratory volume in 1 s (FEV1) (PD" FEV1) while asthmatics with BAL fluid histamine levels < 100 pg/ml required 49±19 breath units (P < 0.05). These data suggest that allergic asthmatics have ongoing lung mast cell degranulation that might contribute to the etiology of airway hyperresponsiveness.

The ras GTPase-activating protein (GAP), identified and characterized in mammalian cells, stimulates the intrinsic GTPase activity of ras proteins. We have previously proposed that the IRA genes, negative regulators of RAS genes in Saccharomyces cerevisiae, encode yeast homologs of the mammalian GAP. In this paper, we present the following evidence that a product of the IRA2 gene exhibits GAP activity similar to that of the mammalian GAP protein. Previously, we have reported on the identification of two Saccharomyces cerevisiae genes, IRAI and IRA2, which encode proteins with domains homologous to the mammalian GAP protein (8, 9). Genetic results suggest that these genes fpnction as upstream negative regulators of yeast RAS genes (8,9). A mutation in either IRA] or IRA2 produces a set of phenotypes that are also observed in strains carrying an activated mutation of RAS2, such as RAS2vall9. These phenotypes include heat shock sensitivity, nitrogen starvation sensitivity, and sporulation deficiency.The following observations further support the idea that IRA proteins have functions similar to those of the mammalian GAP. First, expression of mammalian GAP suppresses the heat shock-sensitive phenotype of irq mutants (10, 11).Second, the fraction of RAS proteins in the GTP-bound form is increased in ira mutants (10).We report here that IRA2 indeed possesses a GAP activity. This provides evidence that IRA proteins are yeast homologs of the mammalian GAP proteins. Since IRA genes are upstream regulators of RAS genes, the mammalian GAP may also have an upstream regulatory function. MATERIALS AND METHODSStrains and Medium. The yeast strain TK161-R2V (MATa ura3 leu2 trpl his3 ade8 RAS2vl19) (12) was used as the host for transformation. This RAS2vall9 strain was chosen because the putative GAP activity of IRA2 may be positively feedback-regulated by cAMP-dependent protein phosphorylation (9). Transformants were grown in SDA medium, which is 2% dextrose/0.5% ammonium sulfate/0.17% yeast nitrogen base without amino acids and ammonium sulfate (Difco)/0.5% Casamino acid (Difco)/0.63 mg ofadenine sulfate per ml/0.25 mg of L-tryptophan per ml.Plasmids. The 5183-base-pair (bp) Pvu II/Pvu II fragment of IRA2 was placed into the plasmid pKT10 (9) *To whom reprint requests should be addressed. 468The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.