Antimicrobial resistance of Enterococcus species isolated from produce

The purpose of this study was to characterize the antibiotic resistance profiles of Enterococcus species isolated from fresh produce harvested in the southwestern United States. Among the 185 Enterococcus isolates obtained, 97 (52%) were Enterococcus faecium, 38 (21%) were Enterococcus faecalis, and 50 (27%) were other Enterococcus species. Of human clinical importance, E. faecium strains had a much higher prevalence of resistance to ciprofloxacin, tetracycline, and nitrofurantoin than E. faecalis. E. faecalis strains had a low prevalence of resistance to antibiotics used to treat E. faecalis infections of both clinical and of agricultural relevance, excluding its intrinsic resistance patterns. Thirty-four percent of the isolates had multiple-drug-resistance patterns, excluding intrinsic resistance. Data on the prevalence and types of antibiotic resistance in Enterococcus species isolated from fresh produce may be used to describe baseline antibiotic susceptibility profiles associated with Enterococcus spp. isolated from the environment. The data collected may also help elucidate the role of foods in the transmission of antibiotic-resistant strains to human populations.     

Effect of a therapeutic lifestyle change diet on immune functions of moderately hypercholesterolemic humans

Hypercholesterolemia is a risk factor for coronary heart disease (CHD) and also could contribute to impaired immune response. The National Cholesterol Education Program Expert Panel recommends a therapeutic lifestyle change (TLC) diet to reduce the risk for CHD. We investigated the effects of changing from a high-fat Western diet to a low-fat diet in accordance with a TLC diet on immune functions of older adults with hypercholesterolemia to determine whether improving the lipid profile via dietary intervention would have beneficial effects on immune functions. In a double-blind study, 18 subjects consumed both a Western diet (38% fat) and a TLC diet (28% fat) for 32 days in a randomized order. Measures of cellular immune responses, including delayed-type hypersensitivity (DTH) response, in vitro lymphocyte proliferation, and interleukin (IL)-2 production, and production of proinflammatory mediators, including tumor necrosis factor-alpha, IL-6, IL-1beta, and prostaglandin E2, were determined. DTH response and lymphocyte proliferative response increased significantly (29% and 27%, respectively) after consumption of a TLC diet. Our results indicate that consumption of a TLC diet enhances T cell-mediated immune functions in older adults with elevated cholesterol level. This might be a clinically important benefit, considering the decline of T cell-mediated immune functions with aging and evidence of impaired immune function associated with hypercholesterolemia.     

Copper Modulation of Ion Channels of PrP[106–126] Mutant Prion Peptide Fragments

We have shown previously that the protease-resistant and neurotoxic prion peptide fragment PrP[106-126] of human PrP incorporates into lipid bilayer membranes to form heterogeneous ion channels, one of which is a Cu(2+)-sensitive fast cation channel. To investigate the role of PrP[106-126]'s hydrophobic core, AGAAAAGA, on its ability to form ion channels and their regulation with Cu(2+), we used the lipid-bilayer technique to examine membrane currents induced as a result of PrP[106-126] (AA/SS) and PrP[106-126] (VVAA/SSSS) interaction with lipid membranes and channel formation. Channel analysis of the mutant (VVAAA/SSS), which has a reduced hydrophobicity due to substitution of hydrophobic residues with the hydrophilic serine residue, showed a significant change in channel activity, which reflects a decrease in the beta-sheet structure, as shown by CD spectroscopy. One of the channels formed by the PrP[106-126] mutant has fast kinetics with three modes: burst, open and spike. The biophysical properties of this channel are similar to those of channels formed with other aggregation-prone amyloids, indicating their ability to form the common beta sheet-based channel structure. The current-voltage (I-V) relationship of the fast cation channel, which had a reversal potential, E(rev), between -40 and -10 mV, close to the equilibrium potential for K(+) ( E(K) = -35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (g(max)) was 58 pS at positive potentials between 0 and 140 mV. Cu(2+) shifted the kinetics of the channel from being in the open and "burst" states to the spike mode. Cu(2+) reduced the probability of the channel being open (P(o)) and the mean open time (T(o)) and increased the channel's opening frequency (F(o)) and the mean closed time (T(c)) at a membrane potential ( V(m)) between +20 and + 140 mV. The fact that Cu(2+) induced changes in the kinetics of this channel with no changes in its conductance, indicates that Cu(2+) binds at the mouth of the channel via a fast channel block mechanism. The Cu(2+)-induced changes in the kinetic parameters of this channel suggest that the hydrophobic core is not a ligand Cu(2+) site, and they are in agreement with the suggestion that the Cu(2+)-binding site is located at M(109) and H(111) of this prion fragment. Although the data indicate that the hydrophobic core sequence plays a role in PrP[106-126] channel formation, it is not a binding site for Cu(2+). We suggest that the role of the hydrophobic region in modulating PrP toxicity is to influence PrP assembly into neurotoxic channel conformations. Such conformations may underlie toxicity observed in prion diseases. We further suggest that the conversions of the normal cellular isoform of prion protein (PrP(c)) to abnormal scrapie isoform (PrP(Sc)) and intermediates represent conversions to protease-resistant neurotoxic channel conformations.     

Exaggerated effect of fluvoxamine in heterozygote serotonin transporter knockout mice

Clearance rates for serotonin (5-HT) in heterozygote (+/-) and homozygote (-/-) serotonin transporter (5-HTT) knockout (KO) mice have not been determined in vivo. Moreover, the effect of selective serotonin reuptake inhibitors (SSRIs) on 5-HT clearance in these mice has not been examined. In this study, the rate of clearance of exogenously applied 5-HT was measured in the CA3 region of the hippocampus of anesthetized mice using high-speed chronoamperometry. Compared with wild-type mice, the maximal rate of 5-HT clearance from extracellular fluid (ECF) was decreased in heterozygotes and more markedly so in KO mice. Heterozygote mice were more sensitive to the 5-HT uptake inhibitor, fluvoxamine, resulting in longer clearance times for 5-HT than in wild-type mice; as expected, the KO mice were completely unresponsive to fluvoxamine. There were no associated changes in norepinephrine transporter density, nor was there an effect of the norepinephrine uptake inhibitor, desipramine, on 5-HT clearance in any genotype. Thus, adaptive changes in the norepinephrine transport system do not occur in the CA3 region of hippocampus as a consequence of 5-HTT KO. These data highlight the potential of the heterozygote 5-HTT mutant mice to model the dynamic in vivo consequences of the human 5-HTT polymorphism.     

Native human TATA-binding protein simultaneously binds and bends promoter DNA without a slow isomerization step or TFIIB requirement

The association of TATA-binding protein (TBP) with promoter DNA is central to the initiation and regulation of eukaryotic protein synthesis. Our laboratory has previously conducted detailed investigations of this interaction using yeast TBP and seven consensus and variant TATA sequences. We have now investigated this key interaction using human TBP and the TATA sequence from the adenovirus major late promoter (AdMLP). Recombinant native human protein was used together with fluorescently labeled DNA, allowing real time data acquisition in solution. We find that the wild-type hTBP-DNAAdMLP reaction is characterized by high affinity (Kd < or = 5 nm), simultaneous binding and DNA bending, and rapid formation of a stable human TBP-DNA complex having DNA bent approximately 100 degrees. These data allow, for the first time, a direct comparison of the reactions of the full-length, native human and yeast TBPs with a consensus promoter, studied under identical conditions. The general reaction characteristics are similar for the human and yeast proteins, although the details differ and the hTBPwt-induced bend is more severe. This directly measured hTBPwt-DNAAdMLP interaction differs fundamentally from a recently published hTBPwt-DNAAdMLP model characterized by low affinity (microM) binding and an unstable complex requiring either a 30-min isomerization or TFIIB to achieve DNA bending. Possible sources of these significant differences are discussed.     

Metal ions,pH, and cholesterol regulate the interactions of Alzheimer's disease amyloid-beta peptide with membrane lipid

The interaction of A beta peptides with the lipid matrix of neuronal cell membranes plays an important role in the pathogenesis of Alzheimer's disease. By using EPR and CD spectroscopy, we found that in the presence of Cu(2+) or Zn(2+), pH, cholesterol, and the length of the peptide chain influenced the interaction of these peptides with lipid bilayers. In the presence of Zn(2+), A beta 40 and A beta 42 both inserted into the bilayer over the pH range 5.5-7.5, as did A beta 42 in the presence of Cu(2+). However, A beta 40 only penetrated the lipid bilayer in the presence of Cu(2+) at pH 5.5-6.5; at higher pH there was a change in the Cu(2+) coordination sphere that inhibited membrane insertion. In the absence of the metals, insertion of both peptides only occurred at pH < 5.5. Raising cholesterol to 0.2 mol fraction of the total lipid inhibited insertion of both peptides under all conditions investigated. Membrane insertion was accompanied by the formation of alpha-helical structures. The nature of these structures was the same irrespective of the conditions used, indicating a single low energy structure for A beta in membranes. Peptides that did not insert into the membrane formed beta-sheet structures on the surface of the lipid.     

Sphingosine-dependent protein kinase-1, directed to 14-3-3, is identified as the kinase domain of protein kinase C delta

Some protein kinases are known to be activated by d-erythro-sphingosine (Sph) or N,N-dimethyl-d-erythro-sphingosine (DMS), but not by ceramide, Sph-1-P, other sphingolipids, or phospholipids. Among these, a specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, 14-3-3eta, or 14-3-3zeta, respectively, was termed "sphingosine-dependent protein kinase-1" (SDK1) (Megidish, T., Cooper, J., Zhang, L., Fu, H., and Hakomori, S. (1998) J. Biol. Chem. 273, 21834-21845). We have now identified SDK1 as a protein having the C-terminal half kinase domain of protein kinase Cdelta (PKCdelta) based on the following observations. (i). Large-scale preparation and purification of proteins showing SDK1 activity from rat liver (by six steps of chromatography) gave a final fraction with an enhanced level of an approximately 40-kDa protein band. This fraction had SDK1 activity approximately 50000-fold higher than that in the initial extract. (ii). This protein had approximately 53% sequence identity to the Ser/Thr kinase domain of PKCdelta based on peptide mapping using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry data. (iii). A search for amino acid homology based on the BLAST algorithm indicated that the only protein with high homology to the approximately 40-kDa band is the kinase domain of PKCdelta. The kinase activity of PKCdelta did not depend on Sph or DMS; rather, it was inhibited by these sphingoid bases, i.e. PKCdelta did not display any SDK1 activity. However, strong SDK1 activity became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40-kDa kinase domain. PKCdelta and SDK1 showed different lipid requirements and substrate specificity, although both kinase activities were inhibited by common PKC inhibitors. The high susceptibility of SDK1 to Sph and DMS accounts for their important modulatory role in signal transduction.     

A sphingosine-dependent protein kinase that specifically phosphorylates 14-3-3 (SDK1) is identified as the kinase domain of PKCdelta: a preliminary note

A specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, eta, or zeta, respectively, only in the presence of sphingosine (Sph) or N,N-dimethyl-Sph (DMS), was termed "sphingosine-dependent protein kinase-1" (SDK1) [J. Biol. Chem. 273(34) (1998) 21834]. We have now identified SDK1 as a protein having the same amino acid sequence as in the C-terminal-half kinase domain of PKCdelta, with approximately 40 kDa molecular mass, based on large-scale purification of a protein from rat liver, and partial sequence using three different combinations of LC-MS or LC-MS/MS with respective search engine. PKCdelta did not display any SDK1 activity and PKCdelta activity was inhibited by Sph and DMS. However, strong SDK1 activity, only in the presence of Sph or DMS, became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40 kDa kinase domain.     

Molecular mechanisms underlying a unique intermediate phase of memory in aplysia

Short- and long-term synaptic facilitation induced by serotonin at Aplysia sensory-motor (SN-MN) synapses has been widely used as a cellular model of short- and long-term memory for sensitization. In recent years, a distinct intermediate phase of synaptic facilitation (ITF) has been described at SN-MN synapses. Here, we identify a novel intermediate phase of behavioral memory (ITM) for sensitization in Aplysia and demonstrate that it shares the temporal and mechanistic features of ITF in the intact CNS: (1) it declines completely prior to the onset of LTM, (2) its induction requires protein but not RNA synthesis, and (3) its expression requires the persistent activation of protein kinase A. Thus, in Aplysia, the same temporal and molecular characteristics that distinguish ITF from other phases of synaptic plasticity distinguish ITM from other phases of behavioral memory.     

Molecular ordering of cellulose after extraction of polysaccharides from primary cell walls of Arabidopsis thaliana: a solid-state CP/MAS (13)C NMR study

Solid-state CP/MAS 13C NMR spectroscopy was used to determine the effects of three different sequential extraction procedures, used to remove non-cellulosic polysaccharides, on the molecular ordering of cellulose in a cell-wall preparation containing mostly primary cell walls obtained from the leaves of the model dicotyledon, Arabidopsis thaliana. The extractions were 50 mM trans-1,2-diaminocyclohexane N,N,N',N'-tetraacetic acid (CDTA) and 50 mM sodium carbonate (giving Residue 1); 50 mM CDTA, 50 mM sodium carbonate and 1 M KOH (giving Residue 2); and 50 mM CDTA, 50 mM sodium carbonate and 4 M KOH (giving Residue 3). The molecular ordering of cellulose in Residue 1 was similar to that in unextracted walls: the cellulose was almost all crystalline, with 43% of molecules contained in crystallite interiors and similar proportions of the triclinic (I(alpha)) and monoclinic (I(beta)) crystal forms. Residue 2 was partly decrystallized and the remaining crystallites were mostly in the I(beta) form. Residue 3 was a mixture of cellulose II, cellulose I and amorphous cellulose. The presence of signals at 100.0 and 102.3 ppm in the spectra of Residues 1 and 2, but not of unextracted cell walls, suggested that the extractions giving these residues caused some of the non-cellulosic polysaccharides, possibly xyloglucans and galactoglucomannans, to become relatively well ordered, for example through interactions with cellulose crystallite surfaces.