Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases

Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the β-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because Bacillus subtilis has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from B. subtilis, yesZ, into Escherichia coli and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed B. subtilis mutants and showed that the increased β-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ. As further demonstration, we overexpressed the LacA and GalA proteins in E. coli and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 β-galactosidases. Similar functions for other β-galactosidases in both GHFs 2 and 42 are suggested by genomic data.     

Pulmonary responses to acute ozone exposure in fasted mice: effect of leptin administration.

Leptin is a satiety hormone that also has proinflammatory effects, including augmentation of ozone-induced pulmonary inflammation. The purpose of this study was to determine whether reductions in endogenous levels of leptin can attenuate pulmonary responses to ozone. To reduce serum leptin, we fasted mice overnight before ozone exposure. Fasting caused a marked reduction in serum leptin to approximately one-sixth the levels observed in fed mice, and continuous infusion of leptin via Alzet micro-osmotic pumps restored serum leptin to, but not above, fed levels. Ozone exposure (2 ppm for 3 h) caused a significant, approximately 40% increase in pulmonary resistance (P < 0.01) and increased airway responsiveness in fasted but not in fed mice. The increased effect of ozone on pulmonary mechanics and airway responsiveness in fasted mice was not observed when leptin was restored via continuous infusion. Ozone exposure caused pulmonary inflammation, as evident by increases in bronchoalveolar lavage cells, protein, and soluble tumor necrosis factor receptors. There was no effect of fasting status on ozone-induced changes in the bronchoalveolar lavage inflammatory profile, and leptin treatment did not alter these responses. Our results indicate that fasting augments ozone-induced changes in pulmonary mechanics and airway responsiveness in mice. These effects of fasting are the result of declines in serum leptin. The mechanistic basis for this protective effect of leptin in fasted mice remains to be determined but is not related to effects on ozone-induced inflammation.     

Stimulatory pathways of the Calcium-sensing receptor on acid secretion in freshly isolated human gastric glands.

Gastric acid secretion is not only stimulated via the classical known neuronal and hormonal pathways but also by the Ca(2+)-Sensing Receptor (CaSR) located at the basolateral membrane of the acid-secretory gastric parietal cell. Stimulation of CaSR with divalent cations or the potent agonist Gd(3+) leads to activation of the H(+)/K(+)-ATPase and subsequently to gastric acid secretion. Here we investigated the intracellular mechanism(s) mediating the effects of the CaSR on H(+)/K(+)-ATPase activity in freshly isolated human gastric glands. Inhibition of heterotrimeric G-proteins (G(i) and G(o)) with pertussis toxin during stimulation of the CaSR with Gd(3+) only partly reduced the observed stimulatory effect. A similar effect was observed with the PLC inhibitor U73122. The reduction of the H(+)/K(+)-ATPase activity measured after incubation of gastric glands with BAPTA-AM, a chelator of intracellular Ca(2+), showed that intracellular Ca(2+) plays an important role in the signalling cascade. TMB-8, a ER Ca(2+)store release inhibitor, prevented the stimulation of H(+)/K(+)-ATPase activity. Also verapamil, an inhibitor of L-type Ca(2+)-channels reduced stimulation suggesting that both the release of intracellular Ca(2+) from the ER as well as Ca(2+) influx into the cell are involved in CaSR-mediated H(+)/K(+)-ATPase activation. Chelerythrine, a general inhibitor of protein kinase C, and Go 6976 which selectively inhibits Ca(2+)-dependent PKC(alpha) and PKC(betaI)-isozymes completely abolished the stimulatory effect of Gd(3+). In contrast, Ro 31-8220, a selective inhibitor of the Ca(2+)-independent PKCepsilon and PKC-delta isoforms reduced the stimulatory effect of Gd(3+) only about 60 %. On the other hand, activation of PKC with DOG led to an activation of H(+)/K(+)-ATPase activity which was only about 60 % of the effect observed with Gd(3+). Incubation of the parietal cells with PD 098059 to inhibit ERK1/2 MAP-kinases showed a significant reduction of the Gd(3+) effect. Thus, in the human gastric parietal cell the CaSR is coupled to pertussis toxin sensitive heterotrimeric G-Proteins and requires calcium to enhance the activity of the proton-pump. PLC, ERK 1/2 MAP-kinases as well as Ca(2+) dependent and Ca(2+)-independent PKC isoforms are part of the down-stream signalling cascade.     

Colon cancer cell vaccine prepared with replication-deficient vaccinia viruses encoding B7.1 and interleukin-2 induce antitumor response in syngeneic mice

 A replication-deficient recombinant vaccinia virus, NYVAC, was developed by deleting 18 open reading frames in the vaccinia virus genome. Recombinant NYVAC, encoding the murine T cell co-stimulatory gene B7.1 (CD 80) (NYVAC-B7.1) and the murine interleukin-2 gene (NYVAC-IL-2), were prepared and the expression of B7.1 and the secretion of IL-2 were respectively confirmed in vitro. The use of these viruses to prepare a potent tumor cell vaccine was studied in a syngeneic murine CC-36 colon adenocarcinoma model. Mice were immunized on days 1 and 8 with 10⁶ irradiated CC-36 cells that were infected with 10⁷ plaque-forming units of either NYVAC-B7.1, NYVAC-IL-2 or a control virus, NYVAC-HR, which encodes a vaccinia virus host-range gene. These mice were then challenged with 10⁸ viable CC-36 tumor cells on day 15. All mice (10/10) in a group that had received no vaccination and all mice (20/20) in a group that had received a control vaccine of CC-36/NYVAC-HR developed tumor 4-weeks after tumor cell challenge. Interestingly, only 16/20 mice in a group that had received CC-36/NYVAC-B7.1 showed the development of tumor after the same interval. The protection against tumor development and the reduction in tumor burden (as mean tumor diameter, 4 weeks after tumor challenge) were significant in this group when compared to groups that were either unvaccinated or vaccinated with CC-36/NYVAC-HR (mean tumor diameter = 6.51±3.2 mm compared to 26.5±0.9 mm or 26.2±1.8 mm respectively) (P = < 0.05). The protection against tumor in a group of mice that received CC-36/NYVAC-IL-2 vaccination was similar to that in the unvaccinated group or the group receiving a CC-36/NYVAC-HR control vaccination. However, in a survival experiment, mice that received either CC36/NYVAC-B7.1 or CC-36/NYVAC-IL-2 vaccination on the day of tumor transplantation survived significantly longer than mice that had not been vaccinated (median survival 60+ days, 60+ days or 23.5 days respectively) (P = <0.05). Interestingly, when a therapeutic tumor vaccination was performed on day 4 after tumor transplantation, mice that had been vaccinated with either CC36/NYVAC-B7.1 or CC-36/NYVAC-IL-2 did not show an improved survival when compared to mice in the control that had not been vaccinated (median survival 28 days compared to 26 days or 25 days respectively). However, mice that had received a therapeutic vaccination with CC-36 cells infected with both NYVAC-B7.1 and NYVAC-IL-2, 4 days after tumor transplantation, survived significantly longer than control mice that had not received any vaccination (median survival 29.5 days compared to 25 days respectively) (P<0.05). These results suggest that a replication-deficient recombinant NYVAC encoding the B7.1 gene and NYVAC encoding the IL-2 gene can be used to produce an effective vaccinia-virus-augmented tumor cell vaccine. Received: 2 March 1998 / Accepted 23 March 1998     

FISH studies of the sperm of fathers of paternally derived cases of trisomy 21: no evidence for an increase in aneuploidy

Paternal nondisjunction accounts for approximately 5% of cases of trisomy 21. To test the hypothesis that, in some such cases, the fathers might be predisposed to meiotic nondisjunction, we utilized fluorescence in situ hybridization (FISH) to screen for aneuploidy in sperm. We analyzed sperm samples from ten males with a trisomy 21 offspring of paternal origin. Among these individuals, the overall frequency of disomy 21 was 0.15%, comparable to estimates of disomy 21 in the general male population. Furthermore, none of the ten fathers of trisomy 21 individuals had significantly elevated levels of disomic sperm. Thus, our results provide no evidence that the occurrence of a trisomy 21 conceptus of paternal origin imparts an increased risk of trisomy in subsequent pregnancies. Received: 9 September 1998 / Accepted: 30 September 1998