Diacylglycerol kinase synthesized by commensal Lactobacillus reuteri diminishes protein kinase C phosphorylation and histamine-mediated signaling in the mammalian intestinal epithelium.

Lactobacillus reuteri 6475 (Lr) of the human microbiome synthesizes histamine and can suppress inflammation via type 2 histamine receptor (H2R) activation in the mammalian intestine. Gut microbes such as Lr promote H2R signaling and may suppress H1R proinflammatory signaling pathways in parallel by unknown mechanisms. In this study, we identified a soluble bacterial enzyme known as diacylglycerol kinase (Dgk) from Lr that is secreted into the extracellular milieu and presumably into the intestinal lumen. DgK diminishes diacylglycerol (DAG) quantities in mammalian cells by promoting its metabolic conversion and causing reduced protein kinase C phosphorylation (pPKC) as a net effect in mammalian cells. We demonstrated that histamine synthesized by gut microbes (Lr) activates both mammalian H1R and H2R, but Lr-derived Dgk suppresses the H1R signaling pathway. Phospho-PKC and IκBα were diminished within the intestinal epithelium of mice and humans treated by wild-type (WT) Lr, but pPKC and IκBα were not decreased in treatment with ΔdgkA Lr. Mucosal IL-6 and systemic interleukin (IL)-1α, eotaxin, and granulocyte colony-stimulating factor (G-CSF) were suppressed in WT Lr, but not in ΔdgkA Lr colonized mice. Collectively, the commensal microbe Lr may act as a "microbial antihistamine" by suppressing intestinal H1R-mediated proinflammatory responses via diminished pPKC-mediated mammalian cell signaling.

Stem Cell-Soluble Signals Enhance Multilumen Formation in SMG Cell Clusters.

Saliva plays a major role in maintaining oral health. Patients with salivary hypofunction exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease, and microbial infections. At this time, treatments for hyposalivation are limited to medications (e.g., muscarinic receptor agonists: pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells as well as artificial salivary substitutes. Therefore, advancement of restorative treatments is necessary to improve the quality of life in these patients. Our previous studies indicated that salivary cells are able to form polarized 3-dimensional structures when grown on growth factor-reduced Matrigel. This basement membrane is rich in laminin-III (L1), which plays a critical role in salivary gland formation. Mitotically inactive feeder layers have been used previously to support the growth of many different cell types, as they provide factors necessary for cell growth and organization. The goal of this study was to improve salivary gland cell differentiation in primary cultures by using a combination of L1 and a feeder layer of human hair follicle-derived mesenchymal stem cells (hHF-MSCs). Our results indicated that the direct contact of mouse submandibular (mSMG) cell clusters and hHF-MSCs was not required for mSMG cells to form acinar and ductal structures. However, the hHF-MSC conditioned medium enhanced cell organization and multilumen formation, indicating that soluble signals secreted by hHF-MSCs play a role in promoting these features.

Expression of resolvin D1 biosynthetic pathways in salivary epithelium.

Resolvins are potent anti-inflammatory mediators derived from ω-3 fatty acids. Results from our previous studies indicated that resolvin D1 (RvD1) blocks pro-inflammatory responses in salivary glands. Furthermore, RvD1 enhances salivary epithelial integrity, demonstrating its potential use for the restoration of salivary gland function in Sjögren's syndrome (SS). We investigated whether the RvD1 biosynthetic machinery (e.g., cytosolic phospholipase A2, calcium-independent phospholipase A2, 12/15 and 5-lipoxygenase) is expressed in mouse submandibular glands (mSMG), using qPCR and Western blot analyses. Additionally, we determined the localization of RvD1 biosynthetic machinery in mSMG and human minor salivary glands (hMSG), with and without SS, using confocal microscopy. Finally, we measured RvD1 levels in cell supernatants from mSMG cell cultures and freshly isolated mSMG cells, with and without SS, using ELISA. Our results indicate that: (1) RvD1 machinery is expressed in mouse and human salivary glands; (2) polar distribution of RvD1 biosynthetic machinery is lost in hMSG with SS; (3) RvD1 levels in mSMG cell culture supernatants increased with time; and (4) RvD1 levels in mSMG cell supernatants, with and without SS, were similar. These studies demonstrate that the RvD1 biosynthesis machinery is expressed and functional in salivary glands with and without SS.

Mechanism of the sex difference in neuronal ischemic cell death.

BACKGROUND: Stroke risk and outcome are different in men and women. We hypothesized that this is partly due to an inherent difference in susceptibility to ischemia between neurons from male vs. female brains. We tested whether neurons from male rodents are more susceptible to in-vitro ischemia than cells from females, and if this is related to increased expression of soluble epoxide hydrolase (sEH). sEH contributes to neuronal cell death by inactivating neuroprotective epoxyeicosatrienoic acids (EETs). METHODS: Rodent cortical neurons were cultured, and exposed to oxygen-glucose deprivation (OGD); then cell death was measured. EETs levels were determined by LC-MS/MS. Expression of sEH-encoding ephx2 was determined by qRT-PCR. Western blotting, immunocytochemistry, and hydrolase activity assay assessed protein expression and activity. RESULTS: Cell death after OGD was higher in neurons from males vs. females, which correlated with higher ephx2 mRNA and stronger sEH immunoreactivity. However, EETs levels were similar in both sexes and pharmacological inhibition of the hydrolase domain of sEH did not abolish the sex difference in cell death. Genetic knockout of sEH in mice abolished the sex difference observed in neurons isolated from these mice after OGD. CONCLUSIONS: Cultured cortical neurons from females are more resistant to ischemia than neurons from males. Neurons from females have less sEH activity compared to neurons from males at baseline, although sEH levels were not measured after OGD. While pharmacological inhibition of the hydrolase domain of sEH does not affect cell death, knockout of the gene encoding sEH eradicates the sex difference seen in wild-type neurons, suggesting a role for further study of the lesser-known phosphatase domain of sEH and its role in sexual dimorphism in neuronal sensitivity to ischemia.

Association of endocrine disruptors and obesity: perspectives from epidemiological studies.

Although changes in diet and physical activity are undoubtedly key causal factors related to the increase in obesity, there is growing interest in the possibility that endocrine disrupting chemicals (EDCs) may affect obesity-related pathways by altering cell signalling involved in weight and lipid homeostasis. Proposed mechanisms that could underlie associations between EDCs and obesity include effects on thyroid and steroid hormones, and activation of peroxisome proliferator-activated receptors, which play a major role in adipocyte differentiation and energy storage. Most evidence supporting the hypothesis that EDCs affect obesity comes from laboratory studies. We summarize the limited epidemiological literature on the topic, including prospective studies of human prenatal exposure to EDCs. We also present findings from a cross-sectional study of levels of six phthalate metabolites and body mass index (BMI) and waist circumference (WC), using data from the U.S. National Health and Nutrition Examination Survey. We found positive associations between BMI and WC among adult males for most phthalate metabolites. For example, in males aged 20-59, the adjusted mean BMI across quartiles of mono-benzyl phthalate was 26.7, 27.2, 28.4, 29.0 (p-trend = 0.0002). In females, BMI and WC increased with quartiles of mono-ethyl phthalate in 12-19 year olds (adjusted mean BMI = 22.9, 23.8, 24.1, 24.7, p-trend = 0.03), and a similar but less strong pattern was seen in 20-59 year olds. By contrast, higher levels of mono-2-ethylhexyl phthalate were associated with lower BMI in adolescent girls and females aged 20-59. This exploratory analysis found several associations between phthalate metabolites and obesity, including notable differences by gender. However, the cross-sectional data are a limitation. Additional prospective studies of the association between exposures to EDCs, especially during development, and obesity are warranted. As this field of research advances, there are challenging methodological questions that must be considered by both epidemiologists and toxicologists.

A call to arms: the cytokine selection service.

The process by which naïve T helper (T(H)) cells differentiate into the T(H)1 and T(H)2 subtypes has been well studied. However, there remain some unresolved issues pertaining to the requirements for the initial step of T(H) cell differentiation. Much debate exists about whether the roles of cytokines include the forcing of the initial steps of differentiation on naïve T(H) cells, termed "instruction," or whether cytokines act in a supportive role, termed "selection," whereby newly differentiating T(H) cells are given the proper signals for survival and proliferation. A recent paper by Mullen et al., which helps delineate the role of cytokines in T(H)1 cell development, is addressed by Nelson; it appears that cytokines act in the selection stage of T(H) cell maturation.

A study to determine the accuracy of a computerized algorithm for interpretation of EEGs.

The main use of computerized EEG has been in sleep studies. A comprehensive system of interpreting routine EEGs by computers has not yet been developed and is technically difficult. We have tried to incorporate computers in the analysis and interpretation of EEGs by using information obtained from visual analysis of EEG in the present work. The purpose of this study was to determine the accuracy of such an algorithm. An electroencephalographer visually analyzed routine EEGs and the data was entered into an EEG Worksheet. The electroencephalographer then interpreted the data and a report was dictated and transcribed. Data from the EEG Worksheet was entered into a computer for interpretation, clinical correlation, and report preparation. Results indicate that the algorithm used with the EEG Worksheet can correctly interpret and clinically correlate visually-analyzed EEG data entered into a computer and reduce time for EEG report generation.

Reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome.

An RP-HPLC assay was developed for a recombinant adenovirus type 5. During chromatography, intact adenovirus dissociated into its structural components (DNA and proteins) and the viral proteome was separated yielding a characteristic fingerprint. The individual components were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, N-terminal sequencing and amino acid composition. The assay was utilized to measure adenovirus particle concentration through quantification of structural proteins. Each structural protein provided independent measurement of virus concentration allowing verification of accuracy. The assay sensitivity is at or below 2 x 10(8) particles. Contrary to the benchmark spectrophotometric assay, the RP-HPLC assay was shown to be insensitive to contaminants common for partially purified adenovirus preparations.

Janus kinases and their role in growth and disease.

Janus kinases (JAK) play a crucial role in the initial steps of cytokine signaling. Each of the four members (JAK1, JAK2, JAK3, TYK2) of this non-receptor tyrosine kinase family is indispensable for the effects of distinct cytokines. Moreover, recent reports have added to our knowledge on their highly specific functions: JAK3 knockout mice and JAK3 deficient patients cannot signal through the interleukin-2,4,7,9, or 15 receptors and suffer from severe combined immunodeficiency (SCID). JAK1 and JAK2 knockout mice do not survive, their cells again showing distinct patterns of cytokine signaling deficits. At the other end of the spectrum, JAK fusion proteins have been shown to play a role in leukemias. In addition, a new class of JAK-specific inhibitors was described by several groups, the CIS/SOCS/Jab family. This review on the rapidly growing field focuses on JAK function and regulation, and on their emerging role in development and human disease.

A novel bioassay for the determination of neutralizing antibodies to IFN-beta1b.

We have adapted the new MxA gene-induction bioassay to measure neutralizing antibodies to interferon-beta1b (IFN-beta1b, the active ingredient in Betaseron) in sera from patients treated with Betaseron. This antibody assay has been validated to quantify neutralizing titers of 1:20 and above, with a precision of +/- 0.20 in log10. We have used this MxA gene-induction antibody assay to reinvestigate serum samples from multiple sclerosis (MS) patients treated with Betaseron. The titers measured were closely comparable to those obtained in antiviral assays. Data obtained by both methods show that neutralizing antibodies may appear and subsequently disappear over time in the sera of some patients treated with Betaseron. Sera from some patients contain binding antibodies to IFN-beta1b. It was shown that binding antibody titers do not correlate quantitatively or qualitatively with neutralizing antibody titers, and indeed, a number of patients develop high levels of binding antibodies but never form measurable levels of neutralizing antibodies.

ATP and SH3 binding sites in the protein kinase of the large subunit of herpes simplex virus type 2 of ribonucleotide reductase (ICP10).

The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a multifunctional protein. It consists of a ribonucleotide reductase and a serine/threonine protein kinase (PK) domain, which has three proline-rich motifs consistent with SH3-binding sites at positions 140, 149, and 396. We used site-directed mutagenesis to identify amino acids required for kinase activity and interaction with signaling proteins. Mutation of Lys176 or Lys259 reduced PK activity (5-8-fold) and binding of the 14C-labeled ATP analog rho-fluorosulfonylbenzoyl 5'-adenosine (FSBA) but did not abrogate them. Enzymatic activity and FSBA binding were abrogated by mutation of both Lys residues, suggesting that either one can bind ATP. Mutation of Glu209 (PK catalytic motif III) virtually abrogated kinase activity in the presence of Mg2+ or Mn2+ ions, suggesting that Glu209 functions in ion-dependent PK activity. ICP10 bound the adaptor protein Grb2 in vitro. Mutation of the ICP10 proline-rich motifs at positions 396 and 149 reduced Grb2 binding 20- and 2-fold, respectively. Binding was abrogated by mutation of both motifs. Grb2 binding to wild type ICP10 was competed by a peptide for the Grb2 C-terminal SH3 motif, indicating that it involves the Grb2 C-terminal SH3.

The novel protein kinase of the RR1 subunit of herpes simplex virus has autophosphorylation and transphosphorylation activity that differs in its ATP requirements for HSV-1 and HSV-2.

The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR1) designated ICP6 and ICP10 for HSV-1 and HSV-2, respectively, has a novel protein kinase (PK) enzymatic activity. ICP10 is localized on the cell surface, a localization that depends on an intact transmembrane (TM) segment. We used immunocomplex PK assays to examine the PK activity of ICP10 in stably transfected eukaryotic cells. Activity was distinct from that of casein kinase II (CKII) in that it did not require monovalent ions and was not inhibited by zinc sulfate. PK activity was eliminated by deletion of the conserved PK catalytic motifs or of the TM segment and it was significantly impaired by mutation of the invariant Lys (Lys176). Loss of PK activity by Lys176 mutation resulted in the failure to bind ATP. A truncated ICP10 PK expressed in bacteria (pp29 1a1) retained auto- and transphosphorylating activity (for calmodulin) after purification to apparent homogeneity. PK activity was also absent in cells infected with a recombinant virus (ICP10 delta PK) deleted in the ICP10 PK catalytic motifs. In cells infected with HSV-1 or HSV-2, RR1 had auto- and transphosphorylating activity for the small subunit of HSV ribonucleotide reductase (RR2) and immunoglobulin G (IgG). Comparing the PK activity of ICP6 and ICP10 we found that ICP6 requires five-fold higher concentrations of [gamma-32P]ATP than ICP10 and both enzymes are Mn2+ dependent, which is also different from CKII that is primarily Mg2+-dependent. Similar results were obtained for various HSV strains and in different cell lines. The data are consistent with the conclusion that the RR1 PK activity is intrinsic.

Extensive modifications for methionine enhancement in the beta-barrels do not alter the structural stability of the bean seed storage protein phaseolin.

Common beans are widely utilized as a food source, yet are low in the essential amino acid methionine. As an initial step to overcome this defect the methionine content of the primary bean seed storage protein phaseolin was increased by replacing 20 evolutionarily variant hydrophobic residues with methionine and inserting short, methionine-rich sequences into turn and loop regions of the protein structure. Methionine enhancement ranged from 5 to 30 residues. An Escherichia coli expression system was developed to characterize the structural stability of the mutant proteins. Proteins of expected sizes were obtained for all constructs except for negative controls, which were rapidly degraded in E. coli. Thermal denaturation of the purified proteins demonstrated that both wild-type and mutant phaseolin proteins denatured reversibly at approximately 61 degrees C. In addition, urea denaturation experiments of the wild-type and a mutant protein (with 30 additional methionines) confirmed that the structural stability of the proteins was very similar. Remarkably, these results indicate that the phaseolin protein tolerates extensive modifications, including 20 substitutions and two loop inserts for methionine enhancement in the beta-barrel and loop structures, with extremely small effects on protein stability.

Burkholderia cepacia and cystic fibrosis: do natural environments present a potential hazard?

An environmental survey of 55 sites yielded only 12 Burkholderia cepacia isolates, none of which displayed the phenotypic properties of a multiresistant epidemic strain associated with pulmonary colonization in patients with cystic fibrosis. Although the environment probably poses a low risk for patients with cystic fibrosis as a source of B. cepacia, the pathogenic potential of individual environmental strains remains unclear. We advise caution in the development of B. cepacia as a biocontrol agent.

Role of glycine 212 in the allosteric behavior of phosphofructokinase from Bacillus stearothermophilus.

Crystallographic studies indicate that the loop between alpha-helix 8 and beta-strand H (the 8H loop) which borders the effector site of Bacillus stearothermophilus phosphofructokinase (BsPFK) is involved in the allosteric mechanism of the enzyme [Schirmer, T., and Evans, P.R. (1990) Nature 343, 140-145]. The residue at one end of this loop, glycine 212, has been proposed to be a pivot about which the loop hinges. Using site-directed mutagenesis, glycine 212 was replaced with valine (G212V). Steady-state kinetic analysis and ligand binding studies on the altered and native PFKs showed that the G212V substitution resulted in discernible changes at the effector site. The mutated PFK required a 3-fold higher concentration of the allosteric inhibitor phosphoenolpyruvate than did the native enzyme to cause the same level of inhibition. The altered PFK had a 2-fold higher dissociation constant for the allosteric activator GDP than the wild-type enzyme. More importantly, whereas the native PFK was fully activated by 1 mM GDP from its PEP-inhibited T-state, the altered enzyme was only marginally activated. On the other hand, the G212V mutation resulted in no changes at the catalytic site of BsPFK. The catalytic rate constant kcat remained unchanged. The altered PFK had the same Km values for ATP and fructose-6-phosphate (Fru-6-P) as did the wild-type enzyme. Furthermore, starting from the same PEP-inhibited T-state, both enzymes gave identical sigmoidal responses to increasing Fru-6-P concentration, indicating that Fru-6-P can activate both to the R-state.

Helix formation in model peptides based on nucleolin TPAKK motifs.

The structures formed by peptide models of the N-terminal domain of the nucleolar protein nucleolin were studied by CD and nmr. The sequences of the peptides are based on the putative nucleic acid binding sequence motif TPAKK. The peptides TP1 and TP2 have the sequence acetyl-G(ATPAKKAA)nG-amide, with n = 1 and 2, respectively. CD measurements indicate structural changes in both peptides when the lysine side chains are uncharged by increasing the pH or acetylation of the side-chain amines. When trifluoroethanol (TFE) is added, more extensive structural changes are observed, resembling helical structure based on nmr nuclear Overhauser effect (NOE) and C alpha proton chemical shift changes, and CD spectra. The structure formed in 0.5M NaClO4 as observed by nmr is similar to that when the lysine side chains are acetylated, due presumably to interactions of perchlorate ion with side-chain charges on lysines. The helical structure observed in TPAKK motifs may be stabilized via N-capping interactions involving threonine. The structures observed in TFE suggest that the Thr-Pro sequence initiates short helical segments in TPAKK motifs, and these helical structures might interact with nucleic acids, presumably via interactions between lysines and threonines of nucleolin.

Multi-resistance isolates possessing characteristics of both Burkholderia (Pseudomonas) cepacia and Burkholderia gladioli from patients with cystic fibrosis.

Multi-resistant strains from three UK centres, previously identified as Burkholderia (formerly Pseudomonas) cepacia, and associated with morbidity, mortality and transmission among patients with cystic fibrosis have been further characterised. Biochemical tests and fatty acid analyses indicate these strains to possess some characteristics atypical of B. cepacia but bearing close resemblance to Burkholderia gladioli, an organism previously regarded solely as a plant pathogen and a hindrance to the identification of B. cepacia. In contrast to the majority of reference strains, all multi-resistant clinical isolates possessed rough lipopolysaccharide which may be a major factor responsible for their increased antibiotic resistance and virulence. In view of the potential clinical and social problems in CF patients posed by these multi-resistant strains, it would seem prudent to consider the isolation of either B. cepacia or B. gladioli as of equal significance.

Serum sensitivity of Burkholderia (Pseudomonas) cepacia isolates from patients with cystic fibrosis.

Bacterial strains which are sensitive to the bactericidal activity of serum are generally considered to be less virulent than serum-resistant strains and are seldom associated with bacteraemia. Burkholderia (Pseudomonas) cepacia is an important pathogen in cystic fibrosis and is associated with rapid fatal pulmonary decline and bacteraemia in 20% of colonised patients. In this study 19 isolates of B. cepacia expressing either rough or smooth LPS were investigated to determine the degree of serum sensitivity. Strains expressing rough-LPS were serum-sensitive: these included a highly transmissible strain of B. cepacia isolated from approximately 50 cystic fibrosis patients attending various U.K. regional centres and associated with cases of bacteraemia.

One-disulfide intermediates of apamin exhibit native-like structure.

The conformations of three peptide models of the one-disulfide and fully reduced forms of apamin were characterized by using nuclear magnetic resonance (NMR) spectroscopy. Apa-2 contains the native disulfide bond between Cys3 and Cys15 in apamin with the other two cysteines replaced by alanines. Apa-1 contains the native disulfide bond between Cys1 and Cys11. Apa-S has all cysteines replaced with serines, mimicking fully reduced apamin. Comparing NOESY cross peaks and coupling constants for amide protons in the peptide analogs with those in native apamin indicates that a significant portion of Apa-2 possesses native-like structural elements of apamin in addition to some random coil conformations. Apa-1 contains a short helical structure from Ala9 to Arg13, corresponding to the N-terminal portion of the alpha-helix observed in the native structure, along with some local and probably flexible secondary structures corresponding to the reverse turn region in native apamin. A larger portion of Apa-1 exists in the form of random coil conformations compared to Apa-2. Apa-S displays mainly random coil conformations with some localized helical structures from Glu7 to Arg14 which are similar to the "nascent helices" proposed by Wright et al. [Wright, P. E., Dyson, H. J., & Lerner, R. A. (1988) Biochemistry 27, 7167-7175]. Formation of the first disulfide bond in apamin seems to be important in the folding process by stabilizing native-like structure, presumably by reducing the conformational freedom and initiating formation of structure.(ABSTRACT TRUNCATED AT 250 WORDS)