Phosphoinositide 3-kinase is required for intracellular Listeria monocytogenes actin-based motility and filopod formation

Motile nonmuscle cells concentrate phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in areas of new actin filament assembly. There is great interest in assessing the in vivo functional significance of these phosphoinositides, and we have used Listeria monocytogenes to explore the contribution of PtdIns(3,4,5)P3 and PtdIns(4,5)P2 to its actin-based motility. In Listeria-infected PtK2 cells Akt-pleckstrin homology (PH)-green fluorescent protein (GFP) and phospholipase C delta (PLC delta)-PH-GFP both first concentrate at the front of motile Listeria, subsequently surrounding the bacterium and then concentrating in the actin filament tail. Surprisingly, Listeria ActA mutant strains lacking the putative phosphoinositide binding site are also able to concentrate these probes. Reduction of available PtdIns(3,4,5)P3 by expression of Akt-PH-GFP and available PtdIns(4,5)P2 by expression of PLC delta-PH-GFP both significantly slow Listeria actin-based movement. Treatment of cells with the PI 3-kinase inhibitor, LY294002, dissociates Akt-PH but not PLC delta-PH, from the bacterial surface and cell membranes, and results in near complete inhibition of Listeria actin-based motility and filopod formation. Removal of LY294002 results in rapid and full recovery of Akt-PH localization, Listeria actin-based motility, and filopod formation. These findings suggest that PtdIns(4,5)P2 is concentrated at the surface of Listeria and serves as the substrate for PtdIns(3,4,5)P3 production, indicating a central role for PI 3-kinases in Listeria intracellular actin-based motility and filopod formation.     

A Single-Dose PLGA Encapsulated Protective Antigen Domain 4 Nanoformulation Protects Mice against Bacillus anthracis Spore Challenge

Bacillus anthracis, the etiological agent of anthrax, is a major bioterror agent. Vaccination is the most effective prophylactic measure available against anthrax. Currently available anthrax vaccines have issues of the multiple booster dose requirement, adjuvant-associated side effects and stability. Use of biocompatible and biodegradable nanoparticles to deliver the antigens to immune cells could solve the issues associated with anthrax vaccines. We hypothesized that the delivery of a stable immunogenic domain 4 of protective antigen (PAD4) of Bacillus anthracis encapsulated in a poly (lactide-co-glycolide) (PLGA) - an FDA approved biocompatible and biodegradable material, may alleviate the problems of booster dose, adjuvant toxicity and stability associated with anthrax vaccines. We made a PLGA based protective antigen domain 4 nanoparticle (PAD4-NP) formulation using water/oil/water solvent evaporation method. Nanoparticles were characterized for antigen content, morphology, size, polydispersity and zeta potential. The immune correlates and protective efficacy of the nanoparticle formulation was evaluated in Swiss Webster outbred mice. Mice were immunized with single dose of PAD4-NP or recombinant PAD4. The PAD4-NP elicited a robust IgG response with mixed IgG1 and IgG2a subtypes, whereas the control PAD4 immunized mice elicited low IgG response with predominant IgG1 subtype. The PAD4-NP generated mixed Th1/Th2 response, whereas PAD4 elicited predominantly Th2 response. When we compared the efficacy of this single-dose vaccine nanoformulation PAD4-NP with that of the recombinant PAD4 in providing protective immunity against a lethal challenge with Bacillus anthracis spores, the median survival of PAD4-NP immunized mice was 6 days as compared to 1 day for PAD4 immunized mice (p<0.001). Thus, we demonstrate, for the first time, the possibility of the development of a single-dose and adjuvant-free protective antigen based anthrax vaccine in the form of PAD4-NP. Further work in this direction may produce a better and safer candidate anthrax vaccine.     

DNA sequencing and expression of the formyl coenzyme A transferase gene, frc, from Oxalobacter formigenes.

Oxalic acid, a highly toxic by-product of metabolism, is catabolized by a limited number of bacterial species utilizing an activation-decarboxylation reaction which yields formate and CO2. frc, the gene encoding formyl coenzyme A transferase, an enzyme which transfers a coenzyme A moiety to activate oxalic acid, was cloned from the bacterium Oxalobacter formigenes. DNA sequencing revealed a single open reading frame of 1,284 bp capable of encoding a 428-amino-acid protein. A presumed promoter region and a rho-independent termination sequence suggest that this gene is part of a monocistronic operon. A PCR fragment containing the open reading frame, when overexpressed in Escherichia coli, produced a product exhibiting enzymatic activity similar to the purified native enzyme. With this, the two genes necessary for bacterial catabolism of oxalate, frc and oxc, have now been cloned, sequenced, and expressed.     

Effects on intestinal nutrient uptake and brush border membrane enzymes in response to atherogenic diet in rhesus monkeys

Transport of nutrients and kinetic parameters (Vmax and Km) of brush border membrane (BBM) enzymes were studied in duodenum, jejunum, and ileum from atherogenic diet-fed monkeys. The Km remained unaltered while feeding of atherogenic diet resulted in higher Vmax of sucrase, maltase, and alkaline phosphatase and lower Vmax of gamma-glutamyltranspeptidase and leucine-aminopeptidase compared to controls. Na+-dependent D-glucose transport was higher in duodenum and jejunum and unaltered in ileum. In contrast to D-glucose transport, the transport of amino acids was decreased in all three intestinal segments from atherogenic diet-fed monkeys.     

Absorption of glyoxylate and oxalate in thiamine and pyridoxine deficient rat intestine

Dietary deficiency of thiamine or pyridoxine has been shown to produce hyperoxaluria and renal stone formation in man and experimental animals. To determine the possible contribution of exogenous glyoxylate and oxalate, the intestinal transport of [14C] - oxalate and [14C] - glyoxylate was measured in vitamin B1 and B6 deficient rats and their respective pair-fed controls. Results indicate that glyoxylate and oxalate are passively diffused from lumen to lamina propria in thiamine deficient and their pair-fed controls with no significant change in the rate of uptake of both the substrates. However B6 deficient rats showed a significant enhancement in the rate of oxalate uptake due to development of a new biphasic transport system. The rate of glyoxylate uptake by simple passive diffusion remained unaltered in pyridoxine deficiency.     

Pattern of dry matter accumulation in developing fruit parts of early- and late-maturing pigeon peas (Cajanus cajan (L.)Millsp.)

Pigeon pea cultivars AL 15 (early-) and T₂₁ (late-maturing) were compared for dry matter accumulation in fruit parts pod wall (PW), seed coats (SC) and seed at various seed developmental stages. Significant water loss and dry matter accumulation in the fruit parts commenced much earlier in cv. AL 15 as compared with cv. T₂₁. The pod wall accumulated starch, reducing sugars and N-substances up to 21 and 28 days after anthesis (DAA) in cultivars AL 15 and T₂₁, respectively, which was later distributed to the seed. Growth of pod wall and seed was sequential, not concurrent, as the pod wall lost significant dry matter when the seeds within them reached their maximum dry matter. The fruit parts of cv. AL 15 accumulated more photosynthates than cv. T₂₁ at all comparable stages.     

Isolation and characterization of new facultatively alkalophilic strains of Bacillus species.

Four facultatively alkalophilic isolates were purified from enrichment cultures initiated with lime-treated garden soil. Four isolates, OF1, OF3, OF4, and OF6, were obligately aerobic, spore-forming, gram-positive, motile rods which were capable of growth at both pH 7.5 and pH 10.5. Strains OF1 and OF6 grew best at the lower pH value; and whereas growth of these strains at pH 10.5 was completely dependent on added Na+, growth at pH 7.5 was only partially dependent on added Na+. Strains OF3 and OF4 grew better at pH 10.5 than at pH 7.5, with strain OF3 growing modestly over its entire pH range, while OF4 grew well. Growth of OF3 and OF4 was completely dependent on added Na+ at both pH 7.5 and pH 10.5. DNA-DNA hybridization studies indicated that OF1 and OF6 are closely related strains but are not related to the other isolates, Bacillus subtilis, or two previously studied obligately alkalophilic bacilli. OF3 was unrelated to any of the other organisms examined in the study, whereas OF4 showed complete homology with obligately alkalophilic Bacillus firmus RAB. All four isolates maintained a cytoplasmic pH that was considerably lower than the external pH when the latter was 10.5. Although substantial transmembrane electrical potentials were observed, the total electrochemical proton gradient (delta mu H+) was low at pH 10.5 in all the strains. By contrast, delta mu H+ was substantial at pH 7.5 and at that pH was composed entirely of an electrical potential. These results are in contrast to previous findings that obligately alkalophilic bacilli generate only small electrical potentials at near neutral pH. All the isolates exhibited substantial rates of respiration as measured by oxygen consumption. Neither respiration nor NADH oxidation by everted membrane vesicles was significantly stimulated by Na+. Analyses of reduced versus oxidized difference spectra of membranes from OF4 showed that the total membrane cytochrome content was considerably higher in cells grown at pH 10.5 than at pH 7.5, with the levels of c- and a-type cytochromes exhibiting the largest pH-dependent differences. Initial examination of membrane protein profiles on gel electrophoresis also indicated a number of changes in pattern in each isolate, depending on the growth pH.     

Peroxidase isozyme patterns in primary trisomics of pearl millet

Summary The peroxidases zymogram phenotypes of seven primary trisomics of pearl millet (Pennisetum typhoides) and their disomic sibs were determined. It was found that each of the trisomics can be differentiated from its disomic sib and from other trisomics on the basis of the isozyme band intensities.This research was supported in part by the USDA under grant PL-480     

In vivo autoradiographic demonstration of beta-adrenergic binding sites in adult rat type II alveolar epithelial cells

Adult male rats were injected intravenously with the muscarinic binding probe 3H-Quinuclidinyl benzilate (QNB) or the beta-adrenergic probe 3H-dihydroalprenolol (DHA). Other rats were pre-treated with an intraperitoneal injection of a 500-fold excess of L-isoproterenol prior to the DHA. Light microscopic autoradiography of 0.5 micron sections of lung from the QNB group demonstrated very little labelling even after 6 months of exposure. In contrast, trachealis smooth muscle from these animals contained substantial labelling. Autoradiographs of lung from rats injected with DHA demonstrated labelling which was well localized over alveolar septa and concentrated over the cytoplasm of type II cells. Quantitative analysis of labelling in the DHA groups indicated a significant reduction of labelling in animals treated with L-isoproterenol prior to DHA, in both the alveolar parenchyma in general and over type II cells. The results of this study provide morphologic evidence for the uptake and specific binding of beta-adrenergic antagonists by the adult lung in vivo, while failing to demonstrate similar binding of a muscarinic probe. In addition, the results demonstrate specific beta-adrenergic receptors on type II cells in vivo and substantiate the view of a direct effect of beta-adrenergic agonists on alveolar type II cells.