Demonstration and Partial Characterization of a Bacterial Growth Enhancer in sera

During our research into the pathogenesis of Vibrio parahaemolyticus, we noticed that the concentration of serum added to the tissue culture medium (Dulbecco’s modified Eagle’s medium: DMEM) greatly affected its growth. Using gel filtration column chromatography, we clearly demonstrated that serum contains not only a bacterial growth inhibitor (BGI) but also a bacterial growth enhancer (BGE) for Vibrio parahaemolyticus. Our data indicate that the BGI is transferrin, whereas the BGE seems to be an undescribed small molecule (molecular weight of 1,000–3,000 Da) and is associated with magnesium and molybdenum ions. BGE activity was not decreased by heat treatment (at 60 or 100°C for 30 min) and affected the growth rate of a wide range of Gram-negative and Gram-positive bacteria. The addition of EDTA into DMEM lowered the growth rate, whereas the addition of BGE restored the growth activity. This study suggests that sera contain a previously undescribed small BGE molecule.     

Neurexin-1, a presynaptic adhesion molecule, localizes at the slit diaphragm of the glomerular podocytes in kidneys

The slit diaphragm connecting the adjacent foot processes of glomerular epithelial cells (podocytes) is the final barrier of the glomerular capillary wall and serves to prevent proteinuria. Podocytes are understood to be terminally differentiated cells and share some common features with neurons. Neurexin is a presynaptic adhesion molecule that plays a role in synaptic differentiation. Although neurexin has been understood to be specifically expressed in neuronal tissues, we found that neurexin was expressed in several organs. Several forms of splice variants of neurexin-1α were detected in the cerebrum, but only one form of neurexin-1α was detected in glomeruli. Immunohistochemical study showed that neurexin restrictedly expressed in the podocytes in kidneys. Dual-labeling analyses showed that neurexin was colocalized with CD2AP, an intracellular component of the slit diaphragm. Immunoprecipitation assay using glomerular lysate showed that neurexin interacted with CD2AP and CASK. These observations indicated that neurexin localized at the slit diaphragm area. The staining intensity of neurexin in podocytes was clearly lowered, and their staining pattern shifted to a more discontinuous patchy pattern in the disease models showing severe proteinuria. The expression and localization of neurexin in these models altered more clearly and rapidly than that of other slit diaphragm components. We propose that neurexin is available as an early diagnostic marker to detect podocyte injury. Neurexin coincided with nephrin, a key molecule of the slit diaphragm detected in a presumptive podocyte of the developing glomeruli and in the glomeruli for which the slit diaphragm is repairing injury. These observations suggest that neurexin is involved in the formation of the slit diaphragm and the maintenance of its function.     

Neutrophil-derived resistin release induced by Aggregatibacter actinomycetemcomitans

Resistin is an adipokine that induces insulin resistance in mice. In humans, resistin is not produced in adipocytes, but in various leukocytes instead, and it acts as a proinflammatory molecule. The present investigation demonstrated high levels of resistin in culture supernatants of neutrophils that are stimulated by a highly leukotoxic strain of Aggregatibacter actinomycetemcomitans. In contrast, the level of resistin was remarkably low when neutrophils were exposed to two other strains that produce minimal levels of leukotoxin and a further isogenic mutant strain incapable of producing leukotoxin. Pretreatment of neutrophils with a monoclonal antibody to CD18, β chain of lymphocyte function-associated molecule 1 (LFA-1), or an Src family tyrosine kinase inhibitor before incubation with the highly leukotoxic strain inhibited the release of resistin. These results show that A. actinomycetemcomitans-expressed leukotoxin induces extracellular release of human neutrophil-derived resistin by interacting with LFA-1 on the surface of neutrophils and, consequently, activating Src family tyrosine kinases.     

Intraspecies discrimination of Lactobacillus paraplantarum by PCR

Lactobacillus paraplantarum is a species phenotypically close to Lactobacillus plantarum. Several PCR methods were evaluated to discriminate L. paraplantarum strains and among them, a PCR using an enterobacterial repetitive intergenic consensus (ERIC) sequence differentiated L. paraplantarum from other Lactobacillus species. In addition, a combination of ERIC and random amplified polymorphic DNA (RAPD) analysis distinguished among seven strains of L. paraplantarum tested. ERIC-PCR profiles showed several strain-specific DNA fragments in L. paraplantarum, among them, a 2.2-kb ERIC marker, termed LpF1, found to be specific to strain FBA1, which improved the skin integrity in an animal model. The LpF1 encodes three proteins similar to Lactobacillus fermentum AroA, TyrA, and AroK, which are involved in the shikimate pathway. A primer pair specific to FBA1 based on the internal sequence of LpF1 amplified a 950-bp FBA1-specific fragment LpF2. Southern blot analysis of Dra I-digested genomic DNA of L. paraplantarum strains using LpF2 as a probe showed that LpF2 is distinctive of strain FBA1 among 16 L. paraplantarum strains. Because both ERIC- and RAPD-PCR are fast and technically simple methods, they are useful for the rapid discrimination of L. paraplantarum strains and for the development of new strain-specific DNA markers for identifying industrially important strains.     

RhoA activation participates in rearrangement of processing bodies and release of nucleated AU-rich mRNAs

Cytoplasmic ribonucleoprotein granules, known as processing bodies (P-bodies), contain a common set of conserved RNA-processing enzymes, and mRNAs with AU-rich elements (AREs) are delivered to P-bodies for translational silencing. Although the dynamics of P-bodies is physically linked to cytoskeletal network, it is unclear how small GTPases are involved in the P-body regulation and the ARE-mRNA metabolism. We found here that glucose depletion activates RhoA GTPase and alters the P-body dynamics in HeLa cells. These glucose-depleted effects are reproduced by the overexpression of the RhoA-subfamily GTPases and conversely abolished by the inhibition of RhoA activation. Interestingly, both RhoA activation and glucose depletion inhibit the mRNA accumulation and degradation. These findings indicate that RhoA participates in the stress-induced rearrangement of P-bodies and the release of nucleated ARE-mRNAs for their stabilization.     

mGluR7 Genetics and Alcohol: Intersection Yields Clues for Addiction

Development of addiction to alcohol or other substances can be attributed in part to exposure-dependent modifications at synaptic efficacy leading to an organism which functions at an altered homeostatic setpoint. Genetic factors may also influence setpoints and the stability of the homeostatic system of an organism. Quantitative genetic analysis of voluntary alcohol drinking, and mapping of the involved genes in the quasi-congenic Recombinant QTL Introgression strain system, identified Eac2 as a Quantitative Trait Locus (QTL) on mouse chromosome 6 which explained 18% of the variance with an effect size of 2.09 g/kg/day alcohol consumption, and Grm7 as a quantitative trait gene underlying Eac2 [Vadasz et al. in Neurochem Res 32:1099–1112, 100, Genomics 90:690–702, 102]. In earlier studies, the product of Grm7 mGluR7, a G protein-coupled receptor, has been implicated in stress systems [Mitsukawa et al. in Proc Natl Acad Sci USA 102:18712–18717, 63], anxiety-like behaviors [Cryan et al. in Eur J Neurosci 17:2409–2417, 14], memory [Holscher et al. in Learn Mem 12:450–455, 26], and psychiatric disorders (e.g., [Mick et al. in Am J Med Genet B Neuropsychiatr Genet 147B:1412–1418, 61; Ohtsuki et al. in Schizophr Res 101:9–16, 72; Pergadia et al. in Paper presented at the 38th Annual Meeting of the Behavior Genetics Association, Louisville, Kentucky, USA, 76]. Here, in experiments with mice, we show that (1) Grm7 knockout mice express increased alcohol consumption, (2) sub-congenic, and congenic mice carrying a Grm7 variant characterized by higher Grm7 mRNA drink less alcohol, and show a tendency for higher circadian dark phase motor activity in a wheel running paradigm, respectively, and (3) there are significant genetic differences in Grm7 mRNA abundance in the mouse brain between congenic and background mice identifying brain areas whose function is implicated in addiction related processes. We hypothesize that metabotropic glutamate receptors may function as regulators of homeostasis, and Grm7 (mGluR7) is involved in multiple processes (including stress, circadian activity, reward control, memory, etc.) which interact with substance use and the development of addiction. In conclusion, we suggest that mGluR7 is a significant new therapeutic target in addiction and related neurobehavioral disorders.     

Ceiling culture-derived proliferative adipocytes retain high adipogenic potential suitable for use as a vehicle for gene transduction therapy

Adipose tissue is expected to provide a source of proliferative cells for regenerative medicine and cell-transplantation therapies using gene transfer manipulation. We have recently identified ceiling culture-derived proliferative adipocytes (ccdPAs) from the mature adipocyte fraction as cells suitable as a therapeutic gene vehicle because of their stable proliferative capacity. In this study, we examined the capability of adipogenic differentiation of the ccdPAs compared with stromal vascular fraction (SVF)-derived progenitor cells (adipose-derived stem cells, ASCs) with regard to their multipotential ability to be converted to another lineage and therefore their potential to be used for regenerative medicine research. After in vitro passaging, the surface antigen profile and the basal levels of adipogenic marker genes of the ccdPAs were not obviously different from those of the ASCs. However, the ccdPAs showed increased lipid-droplet accumulation accompanied with higher adipogenic marker gene expression after stimulation of differentiation compared with the ASCs. The higher adipogenic potential of the ccdPAs than the ASCs from the SVF was maintained for 42 days in culture. Furthermore, the difference in the adipogenic response was enhanced after partial stimulation without indomethacin. These results indicate that the ccdPAs retain a high adipogenic potential even after in vitro passaging, thus suggesting the commitment of ccdPAs to stable mature adipocytes after autotransplantation, indicating that they may have potential for use in regenerative and gene-manipulated medicine.     

Using CRISPRs as a metagenomic tool to identify microbial hosts of a diffuse flow hydrothermal vent viral assemblage

Metagenomic analyses of viruses have revealed widespread diversity in the viriosphere, but it remains a challenge to identify specific hosts for a viral assemblage. To address this problem, we analyze the viral metagenome of a northeast Pacific hydrothermal vent with a comprehensive database of spacers derived from the clustered regularly interspaced short palindromic repeat (CRISPR) putative immune system. CRISPR spacer matches to the marine vent virome suggest that viruses infecting hosts from diverse taxonomic groups are present in this vent environment. Comparative virome analyses show that CRISPR spacers from vent isolates and from thermophiles in general have a higher percentage of matches to the vent virome than to other marine or terrestrial hot spring viromes. However, a high percentage of hits to spacers from mesophilic hosts, combined with a moderately high modeled alpha diversity, suggest that the marine vent virome is comprised of viruses that have the potential to infect diverse taxonomic groups of multiple thermal regimes in both the bacterial and the archaeal domains.