Potential role of Diploscapter sp. strain LKC25, a bacterivorous nematode from soil, as a vector of food-borne pathogenic bacteria to preharvest fruits and vegetables

Diploscapter, a thermotolerant, free-living soil bacterial-feeding nematode commonly found in compost, sewage, and agricultural soil in the United States, was studied to determine its potential role as a vehicle of Salmonella enterica serotype Poona, enterohemorrhagic Escherichia coli O157:H7, and Listeria monocytogenes in contaminating preharvest fruits and vegetables. The ability of Diploscapter sp. strain LKC25 to survive on agar media, in cow manure, and in composted turkey manure and to be attracted to, ingest, and disperse food-borne pathogens inoculated into soil or a mixture of soil and composted turkey manure was investigated. Diploscapter sp. strain LKC25 survived and reproduced in lawns of S. enterica serotype Poona, E. coli O157:H7, and L. monocytogenes on agar media and in cow manure and composted turkey manure. Attraction of Diploscapter sp. strain LKC25 to colonies of pathogenic bacteria on tryptic soy agar within 10, 20, 30, and 60 min and 24 h was determined. At least 85% of the worms initially placed 0.5 to 1 cm away from bacterial colonies migrated to the colonies within 1 h. Within 24 h, > or =90% of the worms were embedded in colonies. The potential of Diploscapter sp. strain LKC25 to shed pathogenic bacteria after exposure to bacteria inoculated into soil or a mixture of soil and composted turkey manure was investigated. Results indicate that Diploscapter sp. strain LKC25 can shed pathogenic bacteria after exposure to pathogens in these milieus. They also demonstrate its potential to serve as a vector of food-borne pathogenic bacteria in soil, with or without amendment with compost, to the surface of preharvest fruits and vegetables in contact with soil.     

Intracellular screen to identify metagenomic clones that induce or inhibit a quorum-sensing biosensor

The goal of this study was to design and evaluate a rapid screen to identify metagenomic clones that produce biologically active small molecules. We built metagenomic libraries with DNA from soil on the floodplain of the Tanana River in Alaska. We extracted DNA directly from the soil and cloned it into fosmid and bacterial artificial chromosome vectors, constructing eight metagenomic libraries that contain 53,000 clones with inserts ranging from 1 to 190 kb. To identify clones of interest, we designed a high throughput "intracellular" screen, designated METREX, in which metagenomic DNA is in a host cell containing a biosensor for compounds that induce bacterial quorum sensing. If the metagenomic clone produces a quorum-sensing inducer, the cell produces green fluorescent protein (GFP) and can be identified by fluorescence microscopy or captured by fluorescence-activated cell sorting. Our initial screen identified 11 clones that induce and two that inhibit expression of GFP. The intracellular screen detected quorum-sensing inducers among metagenomic clones that a traditional overlay screen would not. One inducing clone carries a LuxI homologue that directs the synthesis of an N-acyl homoserine lactone quorum-sensing signal molecule. The LuxI homologue has 62% amino acid sequence identity to its closest match in GenBank, AmfI from Pseudomonas fluorescens, and is on a 78-kb insert that contains 67 open reading frames. Another inducing clone carries a gene with homology to homocitrate synthase. Our results demonstrate the power of an intracellular screen to identify functionally active clones and biologically active small molecules in metagenomic libraries.     

Cyclins D2 and D1 are essential for postnatal pancreatic beta-cell growth

Regulation of adult beta-cell mass in pancreatic islets is essential to preserve sufficient insulin secretion in order to appropriately regulate glucose homeostasis. In many tissues mitogens influence development by stimulating D-type cyclins (D1, D2, or D3) and activating cyclin-dependent kinases (CDK4 or CDK6), which results in progression through the G(1) phase of the cell cycle. Here we show that cyclins D2 and D1 are essential for normal postnatal islet growth. In adult murine islets basal cyclin D2 mRNA expression was easily detected, while cyclin D1 was expressed at lower levels and cyclin D3 was nearly undetectable. Prenatal islet development occurred normally in cyclin D2(-/-) or cyclin D1(+/-) D2(-/-) mice. However, beta-cell proliferation, adult mass, and glucose tolerance were decreased in adult cyclin D2(-/-) mice, causing glucose intolerance that progressed to diabetes by 12 months of age. Although cyclin D1(+/-) mice never developed diabetes, life-threatening diabetes developed in 3-month-old cyclin D1(-/+) D2(-/-) mice as beta-cell mass decreased after birth. Thus, cyclins D2 and D1 were essential for beta-cell expansion in adult mice. Strategies to tightly regulate D-type cyclin activity in beta cells could prevent or cure diabetes.     

Normal cell cycle and checkpoint responses in mice and cells lacking Cdc25B and Cdc25C protein phosphatases

The Cdc25 family of protein phosphatases positively regulates cell division by activating cyclin-dependent protein kinases (CDKs). In humans and rodents, there are three Cdc25 family members--denoted Cdc25A, Cdc25B, and Cdc25C--that can be distinguished based on their subcellular compartmentalizations, their abundances and/or activities throughout the cell cycle, the CDKs that they target for activation, and whether they are overexpressed in human cancers. In addition, murine forms of Cdc25 exhibit distinct patterns of expression throughout development and in adult tissues. These properties suggest that individual Cdc25 family members contribute distinct biological functions in embryonic and adult cell cycles of mammals. Interestingly, mice with Cdc25C disrupted are healthy, and cells derived from these mice exhibit normal cell cycles and checkpoint responses. Cdc25B-/- mice are also generally normal (although females are sterile), and cells derived from Cdc25B-/- mice have normal cell cycles. Here we report that mice lacking both Cdc25B and Cdc25C are obtained at the expected Mendelian ratios, indicating that Cdc25B and Cdc25C are not required for mouse development or mitotic entry. Furthermore, cell cycles, DNA damage responses, and Cdc25A regulation are normal in cells lacking Cdc25B and Cdc25C. These findings indicate that Cdc25A, or possibly other phosphatases, is able to functionally compensate for the loss of Cdc25B and Cdc25C in mice.     

Electroencephalographic Biofeedback in the Treatment of Attention-Deficit/Hyperactivity Disorder

Historically, pharmacological treatments for attention-deficit/hyperactivity disorder (ADHD) have been considered to be the only type of interventions effective for reducing the core symptoms of this condition. However, during the past three decades, a series of case and controlled group studies examining the effects of EEG biofeedback have reported improved attention and behavioral control, increased cortical activation on quantitative electroencephalographic examination, and gains on tests of intelligence and academic achievement in response to this type of treatment. This review paper critically examines the empirical evidence, applying the efficacy guidelines jointly established by the Association for Applied Psychophysiology and Biofeedback (AAPB) and the International Society for Neuronal Regulation (ISNR). On the basis of these scientific principles, EEG biofeedback was determined to be “probably efficacious” for the treatment of ADHD. Although significant clinical improvement was reported in approximately 75% of the patients in each of the published research studies, additional randomized, controlled group studies are needed in order to provide a better estimate of the percentage of patients with ADHD who will demonstrate such gains in clinical practice.     

SCHIP: statistics for chromosome interphase positioning based on interchange data

MOTIVATION: The position of chromosomes in the interphase nucleus is believed to be associated with a number of biological processes. Here, we present a web-based application that helps analyze the relative position of chromosomes during interphase in human cells, based on observed radiogenic chromosome aberrations. The inputs of the program are a table of yields of pairwise chromosome interchanges and a proposed chromosome geometric cluster. Each can either be uploaded or selected from provided datasets. The main outputs are P-values for the proposed chromosome clusters. SCHIP is designed to be used by a number of scientific communities interested in nuclear architecture, including cancer and cell biologists, radiation biologists and mathematical/computational biologists.     

Drosophila myosin V is required for larval development and spermatid individualization

Class V myosins are multifunctional molecular motors implicated in vesicular traffic, RNA transport, and mechanochemical coupling of the actin and microtubule-based cytoskeletons. To assess the function of the single myosin V gene in Drosophila (MyoV), we have characterized both deletion and truncation alleles. Mutant animals exhibit no detectable defects during embryogenesis but are delayed in larval development; most die prior to 3rd instar. MyoV protein is widely distributed; however, there are no obvious cytological defects in mutant larval tissues where MyoV was normally highly expressed. Of the few adult MyoV mutant escapers, females were fertile but males were not. We examined the expression of MyoV during spermatogenesis. MyoV was associated with membranes, microtubule, and actin structures required for spermatid maturation; MyoV was strongly associated with the sperm nuclei during the maturation of the actin-rich investment cones that package spermatids in individual membranes. In MyoV mutant escaper males, the early stages of spermatogenesis were normal; however, in the later stages, the investment cones stained weakly for actin and their registration was disrupted; no mature sperm were produced. Our results suggest that MyoV contributes to the formation of the actin-based investment cones and acts to coordinate and/or anchor these structures and other components of the individualization complex.     

Proliferative effects of apical, but not basal, matrix metalloproteinase-7 activity in polarized MDCK cells

Matrix metalloproteinase-7 (MMP-7) is primarily expressed in glandular epithelium. Therefore, its mechanism of action may be influenced by its regulated vectorial release to either the apical and/or basolateral compartments, where it would act on its various substrates. To gain a better understanding of where MMP-7 is released in polarized epithelium, we have analyzed its pattern of secretion in polarized MDCK cells expressing stably transfected human MMP-7 (MDCK-MMP-7), and HCA-7 and Caco2 human colon cancer cell lines. In all cell lines, latent MMP-7 was secreted to both cellular compartments, but was 1.5- to 3-fold more abundant in the basolateral compartment as compared to the apical. However, studies in the MDCK system demonstrated that MMP-7 activity was 2-fold greater in the apical compartment of MDCK-MMP-7(HIGH)-polarized monolayers, which suggests the apical co-release of an MMP-7 activator. In functional assays, MMP-7 over-expression increased cell saturation density as a result of increased cell proliferation with no effect on apoptosis. Apical MMP-7 activity was shown to be responsible for the proliferative effect, which occurred, as demonstrated by media transfer experiments, through cleavage of an apical substrate and not through the generation of a soluble factor. Taken together, our findings demonstrate the importance of MMP-7 secretion in relation to its mechanism of action when expressed in a polarized epithelium.     

LosA, a key glycosyltransferase involved in the biosynthesis of a novel family of glycosylated acyltrehalose lipooligosaccharides from Mycobacterium marinum

Members of the genus Mycobacterium are characterized by cell envelopes rich in unusual free lipids, interacting with a covalently anchored mycolyl-arabinogalactan matrix. Previous studies have shown that Mycobacterium marinum produces large amounts of a diacylglycosylphenolphthiocerol, "phenolic" glycolipid. When cultivated on liquid Sauton medium, traces of a polar lipooligosaccharide (LOS) glycolipid antigen were also previously indicated. In this study, it was found that growth of the type strain of M. marinum on solid Sauton or Middlebrook 7H10 agar gave substantial, but different, amounts of a family of four major trehalose-based LOSs. The core pentasaccharide LOS-I was a rhamnosyl diglucosyl-acylated trehalose. The heptasaccharide, LOS-II, was derived from LOS-I by adding xylose accompanied by a novel sugar (X); repeated addition of this sugar unit X gave the octasaccharide LOS-III. LOS-IV has a decasaccharide component with two additional unusual sugar units, YZ. In a recent study (Alexander, D. C., Jones, J. R., Tan, T., Chen, J. M., and Liu, J. (2004) J. Biol. Chem. 279, 18824-18833), chromatographically similar glycolipids were assigned to the family of phosphatidylinositol mannosides (PIMs) and a "PimF" (Rv1500) glycosyltransferase implicated in the conversion of a supposed "PIM5" to a "PIM7." The present study indicates that these putative PIMs are in fact members of the phosphorus-free LOS family of glycolipids and that the protein product of Rv1500, which we have now termed LosA, is a glycosyltransferase involved in transferring sugars to LOS-III to form LOS-IV of M. marinum.