Detection and identification of groundwater bacteria capable of escaping entrapment on 0.45-micron-pore-size membrane filters.

Rural drinking water systems supplied by untreated groundwater were examined to determine whether coliform or heterotrophic plate count bacteria are capable of escaping entrapment on standard porosity (0.45-micron-pore-size) membrane filters. Filterable bacteria were present in 42% of the 24 groundwater sources examined by using nonselective media (R2A, full strength m-HPC, and 0.1x m-HPC agars). Pseudomonads were the most frequently identified group of filterable bacteria detected. Flavobacterium, Alcaligenes, Acinetobacter, and Achromobacter isolates were also identified. Total coliforms were not recovered from any of the 24 groundwater samples following filtration through 0.45-micron-pore-size membrane filters by using selective M-Endo LES agar or mT7 agar. In addition, none of the isolates identified from nonselective media were coliforms. Similarly, neither total coliforms nor specifically Escherichia coli were detected in these filtrates when Colilert P/A medium was used.     

The de novo and salvage pathways for the synthesis of pyrimidine residues of RNA predominate in different locations within the mouse doudenal epithelium

Summary RNA synthesis was examined by radioautography in mouse doudenal epithelium using ³H-uridine as a tracer of the salvage pathway and ³H-orotic acid as a tracer of the de novo pathway. The incorporation of the two precursors was estimated by counting silver grains in light-microscopic and electron-microscopic radioautographs at successive levels of crypt and villus. With both precursors, silver grains were found over all epithelial nuclei, but in numbers varying by location. Thus, after ³H-uridine injection, the number of grains was high over nucleolus and nucleoplasm in the base of the crypt, declined gradually in the middle and top of the crypt, and was low along the villus. After ³H-orotic acid, the number of grains was fairly low throughout, but peaked over the nucleoplasm in lower villus cells. The ³H-uridine reaction over nucleolus and nucleoplasm in crypt cells was interpreted as synthesis by the salvage pathway of ribosomal RNA and heterogeneous RNA, respectively, whereas the ³H-orotic acid reaction over the nucleoplasm of some villus cells indicated that these cells synthesized heterogeneous RNA by the de novo pathway.     

Influence of Environmental Stress on Enumeration of Indicator Bacteria from Natural Waters

The problems associated with recovery of pure cultures of Escherichia coli and Streptococcus faecalis from stream environments were examined utilizing membrane filter chambers. It was observed that upon exposure to the aquatic environment a significant proportion of cells lost their ability to produce colonies on a selective medium, yet retained this capability on a nutritionally rich, nonselective medium. Discrepancies in colony-forming units between nonselective and selective media indicated that a substantial portion of bacterial cells may become physiologically injured due to the environmental stress imposed by the aquatic environment. The extent of injury was observed to vary considerably among the eight different stream environments, since the amount of injury was not uniform for all types of water environments examined. It was observed that the injury acquired by a population of E. coli, during exposure to the aquatic environment, could be rapidly repaired in a nutritionally rich, nonselective medium. As the injured population of cells was exposed to the rich, nonselective broth, increasing proportions of cells were able to repair themselves such that they became insensitive to inhibitory agents in selective media.     

1,25-dihydroxyvitamin D3 stimulates the phosphorylation of two acidic membrane proteins of 42,000 and 48,000 daltons in rat colonocytes: An effect modulated by vitamin D status

Our laboratory has recently demonstrated that 1,25-dihydroxyvitamin D₃(1,25(OH)₂D₃) rapidly stimulated membrane polyphosphoinositide breakdown and increased intracellular calcium, as well as activated protein kinase C (PKC) in vitamin D-sufficient rat colonocytes. These effects of 1,25(OH)₂D₃ were, however, lost in vitamin D-insufficient rats and restored by the in vivo repletion of 1,25(OH)₂D₃. In the present studies we have examined the ability of 1,25(OH)₂D₃ to stimulate the phosphorylation of colonic membrane proteins in intact D-sufficient cells. In addition, we investigated the effects of vitamin D status on the phosphorylation of these membrane proteins in broken cell preparations. These studies demonstrated that 1,25(OH)₂D₃ increased the phosphorylation of at least two colonic membrane proteins with apparent molecular weights of 42,000 (pp42) and 48,000 (pp48) in intact cells of vitamin D-sufficient rats. Moreover, in vitamin D-sufficient rats, treatment of colonocytes with 1,25(OH)₂D₃ or 12-Otertradecanoyl phorbol 13-acetate (TPA), a known activator of PKC, significantly increased the phosphorylation of pp42 and pp48 in broken cell preparations. The kinetics of these phosphorylations in response to 1,25(OH)₂D₃ were both rapid and transient. In addition, PKC_19–36, a specific PKC inhibitor, decreased the phosphorylation of pp42 and pp48, whereas okadaic acid (OA), a type 1 and 2A protein phosphatase inhibitor, further augmented their phosphorylation in response to 1,25(OH)₂D₃. The isoelectric points of pp42 and pp48 were 5.79 and 5.97, respectively, and both were predominantly phosphorylated on threonine residues. In contrast to our findings in colonocytes from vitamin D-sufficient animals, basal phosphorylation of pp42 and pp48 were increased in membranes prepared from vitamin D-insufficient rats. Moreover, these phosphorylations failed to change in response to 1,25(OH)₂D₃-treatment of colonocytes from vitamin D-insufficient rats. The basal phosphorylation of each of these proteins was restored to control levels, as was their ability to respond to the direct addition of 1,25(OH)₂D₃ following the in vivo repletion of vitamin D-insufficient rats with this secosteroid. In summary, we have identified two acidic membrane proteins from rat colonocytes that are phosphorylated in both intact and broken cell preparations in response to 1,25(OH)₂D₃ treatment, an event modulated by vitamin D status and mediated, at least in part, by PKC. © 1995 Wiley-Liss, Inc.     

PlasmoSEP: Predicting surface‐exposed proteins on the malaria parasite using semisupervised self‐training and expert‐annotated data

Accurate and comprehensive identification of surface‐exposed proteins (SEPs) in parasites is a key step in developing novel subunit vaccines. However, the reliability of MS‐based high‐throughput methods for proteome‐wide mapping of SEPs continues to be limited due to high rates of false positives (i.e., proteins mistakenly identified as surface exposed) as well as false negatives (i.e., SEPs not detected due to low expression or other technical limitations). We propose a framework called PlasmoSEP for the reliable identification of SEPs using a novel semisupervised learning algorithm that combines SEPs identified by high‐throughput experiments and expert annotation of high‐throughput data to augment labeled data for training a predictive model. Our experiments using high‐throughput data from the Plasmodium falciparum surface‐exposed proteome provide several novel high‐confidence predictions of SEPs in P. falciparum and also confirm expert annotations for several others. Furthermore, PlasmoSEP predicts that 25 of 37 experimentally identified SEPs in Plasmodium yoelii salivary gland sporozoites are likely to be SEPs. Finally, PlasmoSEP predicts several novel SEPs in P. yoelii and Plasmodium vivax malaria parasites that can be validated for further vaccine studies. Our computational framework can be easily adapted to improve the interpretation of data from high‐throughput studies.     

SMIT2 mediates all myo-inositol uptake in apical membranes of rat small intestine

This study presents the characterization of myo-inositol (MI) uptake in rat intestine as evaluated by use of purified membrane preparations. Three secondary active MI cotransporters have been identified; two are Na(+) coupled (SMIT1 and SMIT2) and one is H(+) coupled (HMIT). Through inhibition studies using selective substrates such as d-chiro-inositol (DCI, specific for SMIT2) and l-fucose (specific for SMIT1), we show that SMIT2 is exclusively responsible for apical MI transport in rat intestine; rabbit intestine appears to lack apical transport of MI. Other sugar transport systems known to be present in apical membranes, such as SGLT1 or GLUT5, lacked any significant contribution to MI uptake. Functional analysis of rat SMIT2 activity, via electrophysiological studies in Xenopus oocytes, demonstrated similarities to the activities of SMIT2 from other species (rabbit and human) displaying high affinities for MI (0.150 +/- 0.040 mM), DCI (0.31 +/- 0.06 mM), and phlorizin (Pz; 0.016 +/- 0.007 mM); low affinity for glucose (36 +/- 7 mM); and no affinity for l-fucose. Although these functional characteristics essentially confirmed those found in rat intestinal apical membranes, a unique discrepancy was seen between the two systems studied in that the affinity constant for glucose was approximately 40-fold lower in vesicles (K(i) = 0.94 +/- 0.35 mM) than in oocytes. Finally, the transport system responsible for the basolateral efflux transporter of glucose in intestine, GLUT2, did not mediate any significant radiolabeled MI uptake in oocytes, indicating that this transport system does not participate in the basolateral exit of MI from small intestine.     

Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes

Over the past two decades, Xenopus laevis oocytes have been widely used as an expression system to investigate both physiological and pathological properties of membrane proteins such as channels and transporters. Past studies have clearly shown the key implications of mistargeting in relation to the pathogenesis of these proteins. To unambiguously determine the plasma membrane targeting of a protein, a thorough purification technique becomes essential. Unfortunately, available techniques are either too cumbersome, technically demanding, or require large amounts of material, all of which are not adequate when using oocytes individually injected with cRNA or DNA. In this article, we present a new technique that permits excellent purification of plasma membranes from X. laevis oocytes. This technique is fast, does not require particular skills such as peeling of vitelline membrane, and permits purification of multiple samples from as few as 10 and up to >100 oocytes. The procedure combines partial digestion of the vitelline membrane, polymerization of the plasma membrane, and low-speed centrifugations. We have validated this technique essentially with Western blot assays on three plasma membrane proteins [aquaporin (AQP)2, Na⁺-glucose cotransporter (SGLT)1, and transient receptor potential vanilloid (TRPV)5], using both wild-type and mistargeted forms of the proteins. Purified plasma membrane fractions were easily collected, and samples were found to be adequate for Western blot identification. expression studies; aquaporin 2 mutations