In vitro activity of ramoplanin and comparator drugs against anaerobic intestinal bacteria from the perspective of potential utility in pathology involving bowel flora

Susceptibility of intestinal bacteria to various antimicrobial agents in vitro, together with levels of those agents achieved in the gut, provides information on the likely impact of the agents on the intestinal flora. Orally administered drugs that are poorly absorbed may be useful for treatment of intestinal infections and for certain other situations in which intestinal bacteria may play a role. The antimicrobial activity of ramoplanin (MDL 62,198) against 928 strains of intestinal anaerobic bacteria was determined using the NCCLS-approved Wadsworth brucella laked-blood agar dilution method. The activity of ramoplanin was compared with that of ampicillin, bacitracin, metronidazole, trimethoprim/sulfamethoxazole (TMP/SMX), and vancomycin. The organisms tested included Bacteroides fragilis group (n=89), other Bacteroides species (n=16), other anaerobic Gram-negative rods (n=56) anaerobic cocci (n=114), Clostridium species (n=426), and non-sporeforming anaerobic Gram-positive rods (n=227). The overall MIC(90)s of ramoplanin, ampicillin, bacitracin, metronidazole, and vancomycin were 256, 32, 128, 16, and 128 mcg/ml, respectively. Ramoplanin was almost always highly active vs. Gram-positive organisms and relatively poor in activity against Gram-negative organisms, particularly Bacteroides, Bilophila, Prevotella, and Veillonella. Vancomycin was quite similar to ramoplanin in its activity. Ampicillin was relatively poor in activity vs. organisms that often produce beta-lactamase, including most of the Gram-negative rods as well as Clostridium bolteae and C. clostridioforme. Bacitracin was relatively poor in activity against most anaerobic Gram-negative rods, but better vs. most Gram-positive organisms. Metronidazole was very active against all groups other than bifidobacteria and some strains of other types of non-sporeforming Gram-positive bacilli. TMP/SMX was very poorly active, with an MIC(90) of >2048 mcg/ml.     

A quality-controlled microarray method for gene expression profiling

Gene expression profiling on microarrays is widely used to measure the expression of large numbers of genes in a single experiment. Because of the high cost of this method, feasible numbers of replicates are limited, thus impairing the power of statistical analysis. As a step toward reducing technically induced variation, we developed a procedure of sample preparation and analysis that minimizes the number of sample manipulation steps, introduces quality control before array hybridization, and allows recovery of the prepared mRNA for independent validation of results. Sample preparation is based on mRNA separation using oligo(dT) magnetic beads, which are subsequently used for first-strand cDNA synthesis on the beads. cDNA covalently bound to the magnetic beads is used as template for second-strand cDNA synthesis, leaving the intact mRNA in solution for further analysis. The quality of the synthesized cDNA can be assessed by quantitative polymerase chain reaction using 3'- and 5'-specific primer pairs for housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase. Second-strand cDNA is chemically labeled with fluorescent dyes to avoid dye bias in enzymatic labeling reactions. After hybridization of two differently labeled samples to microarray slides, arrays are scanned and images analyzed automatically with high reproducibility. Quantile-normalized data from five biological replica display a coefficient of variation 45% for 90% of profiled genes, allowing detection of twofold changes with false positive and false negative rates of 10% each. We demonstrate successful application of the procedure for expression profiling in plant leaf tissue. However, the method could be easily adapted for samples from animal including human or from microbial origin.     

Prostaglandin e and f receptor expression and myometrial sensitivity at labor onset in the sheep

Prostaglandins (PGs) play a pivotal role in the initiation and progression of term and preterm labor. Uterine activity is stimulated primarily by PGE(2) and PGF(2alpha) acting on prostaglandin E (EP) and prostaglandin F (FP) receptors, respectively. Activation of FP receptors strongly stimulates the myometrium, whereas stimulation of EP receptors may lead to contraction or relaxation, depending on the EP subtype (EP1-4) expression. Thus, the relative expression of FP and EP1-4 may determine the responsiveness to PGE(2) and PGF(2alpha). The aims of this study were to characterize the expression of EP1-4 and FP in intrauterine tissues and placentome, together with myometrial responsiveness to PG, following the onset of dexamethasone-induced preterm and spontaneous term labor. Receptor mRNA expression was measured using quantitative real-time polymerase chain reaction using species-specific primers. There was no increase in myometrial contractile receptor expression at labor onset, nor was there a change in sensitivity to PGE(2) and PGF(2alpha). This suggests expression of these receptors reaches maximal levels by late gestation in sheep. Placental tissue showed a marked increase in EP2 and EP3 receptor expression, the functions of which are unknown at this time. Consistent with previous reports, these results suggest that PG synthesis is the main factor in the regulation of uterine contractility at labor. This is the first study to simultaneously report PG E and F receptor expression in the key gestational tissues of the sheep using species-specific primers at induced-preterm and spontaneous labor onset.     

High resolution 1H NMR-based metabolomics indicates a neurotransmitter cycling deficit in cerebral tissue from a mouse model of Batten disease

The neuronal ceroid lipofuscinoses (NCLs) constitute a range of progressive neurological disorders primarily affecting children. Although six of the causative genes have been characterized, the underlying disease pathogenesis for this family of disorders is unknown. Using a metabolomics approach based on high resolution 1H NMR spectroscopy of the cortex, cerebellum, and remaining regions of the brain in conjunction with statistical pattern recognition, we report metabolic deficits associated with juvenile NCL in a Cln3 knock-out mouse model. Tissue from Cln3 null mutant mice aged 1-6 months was characterized by an increased glutamate concentration and a decrease in -amino butyric acid (GABA) concentration in aqueous extracts from the three regions of the brain. These changes are consistent with the reported altered expression of genes involved in glutamate metabolism in older mice and imply a change in neurotransmitter cycling between glutamate/glutamine and the production of GABA. Further variations in myo-inositol, creatine, and N-acetyl-aspartate were also identified. These metabolic changes were distinct from the normal aging/developmental process. Together, these changes represent the first documented pre-symptomatic symptoms of the Cln3 mouse at 1 month of age and demonstrate the versatility of 1H NMR spectroscopy as a tool for phenotyping mouse models of disease.     

Induction of protein-like molecular architecture by monoalkyl hydrocarbon chains

Numerous approaches have been described for creating relatively small folded biomolecular structures. "Peptide-amphiphiles," whereby monoalkyl or dialkyl hydrocarbon chains are covalently linked to peptide sequences, have been shown previously to form specific molecular architecture of enhanced stability. The present study has examined the use of monoalkyl hydrocarbon chains as a more general method for inducing protein-like structures. Peptide and peptide-amphiphiles have been characterized by CD and one- and two-dimensional nmr spectroscopic techniques. We have examined two structural elements: alpha-helices and collagen-like triple helices. The alpha-helical propensity of a 16-residue peptide either unmodified or acylated with a C(6) or C(16) monoalkyl hydrocarbon chain has been examined initially. The 16-residue peptide alone does not form a distinct structure in solution, whereas the 16-residue peptide adopts predominantly an alpha-helical structure in solution when a C(6) or C(16) monoalkyl hydrocarbon chain is N-terminally acylated. The thermal stability of the alpha-helix is greater upon addition of the C(16) compared with the C(6) chain, which correlates to the extent of aggregation induced by the respective hydrocarbon chains. Very similar results are seen using a 39-residue triple-helical model peptide, in that structural thermal stability (a) is increasingly enhanced as alkyl chain length is increased and (b) correlates to the extent of peptide-amphiphile aggregation. Overall, structures as diverse as alpha-helices, triple helices, and turns/loops have been shown to be induced and/or stabilized by alkyl chains. Increasing alkyl chain length enhances stability of the structural element and induces aggregates of defined sizes. Hydrocarbon chains may be useful as general tools for protein-like structure initiation and stabilization as well as biomaterial modification.     

rgf1, a mutation reducing grain filling in maize through effects on basal endosperm and pedicel development

The maize cob presents an excellent opportunity to screen visually for mutations affecting assimilate partitioning in the developing kernel. We have identified a defective kernel mutant termed rgf1, reduced grain filling, with a final grain weight 30% of the wild type. In contrast with most defective endosperm mutants, rgf1 shows gene dosage-dependent expression in the endosperm. rgf1 kernels possess a small endosperm incompletely filling the papery pericarp, but embryo development is unaffected and the seeds are viable. The mutation conditions defective pedicel development and greatly reduces expression of endosperm transfer layer-specific markers. rgf1 exhibits striking morphological similarities to the mn1 mutant, but maps to a locus approximately 4 cM away from mn1 on chromosome 2 of maize. Despite reduced starch accumulation in the mutant, no obvious lesion in starch biosynthesis has been detected. Free sugar levels are unaltered in rgf1 endosperm. Rates of sugar uptake, measured over short (8 h) periods in cultured kernels, are increased in rgf1 compared to the wild type. rgf1 and wild-type kernels, excised at 5 DAP and cultured in vitro also develop differently in response to variations in sugar regime: glucose concentrations above 1% arrest placentochalazal development of rgf1 kernels, but have no effect on cultured wild-type kernels. These findings suggest that either uptake or perception of sugar(s) in endosperm cells at 5-10 DAP determines the rgf1 kernel phenotype.     

A cellular mechanism for the bidirectional pain-modulating actions of orphanin FQ/nociceptin

Orphanin FQ/nociceptin (OFQ/N) and its receptor share substantial structural features and cellular actions with classic opioid peptides and receptors, but have distinct pharmacological profiles and behavioral effects. Currently there is an active debate about whether OFQ/N produces hyperalgesia or analgesia. Using a well-defined brainstem pain-modulating circuit, we show that OFQ/N can cause either an apparent hyperalgesia by antagonizing mu opioid-induced analgesia or a net analgesic effect by reducing the hyperalgesia during opioid abstinence. It presumably produces these two opposite actions by inhibiting two distinct groups of neurons whose activation mediates the two effects of opioid administration. OFQ/N antagonism of the hyperalgesia may have significance for the treatment of opioid withdrawal and sensitized pain.     

Protein kinase C isozymes and the regulation of diverse cell responses

Individual protein kinase C (PKC) isozymes have been implicated in many cellular responses important in lung health and disease, including permeability, contraction, migration, hypertrophy, proliferation, apoptosis, and secretion. New ideas on mechanisms that regulate PKC activity, including the identification of a novel PKC kinase, 3-phosphoinositide-dependent kinase-1 (PDK-1), that regulates phosphorylation of PKC, have been advanced. The importance of targeted translocation of PKC and isozyme-specific binding proteins (like receptors for activated C-kinase and caveolins) is well established. Phosphorylation state and localization are now thought to be key determinants of isozyme activity and specificity. New concepts on the role of individual PKC isozymes in proliferation and apoptosis are emerging. Opposing roles for selected isozymes in the same cell system have been defined. Coupling to the Wnt signaling pathway has been described. Phenotypes for PKC knockout mice have recently been reported. More specific approaches for studying PKC isozymes and their role in cell responses have been developed. Strengths and weaknesses of different experimental strategies are reviewed. Future directions for investigation are identified.     

Evaluation of radiolabeled type IV collagen fragments as potential tumor imaging agents.

The objective of this study was to examine radiopharmaceuticals that target the alpha3beta1 integrin to determine if these agents target tumors for diagnostic imaging and/or targeted radiotherapy of cancer. Prior studies had shown that residues 531-542 from the alpha1 chain of type IV collagen bind a variety of tumor cell alpha3beta1 integrins. A peptide mimic of this sequence containing all D-amino acids (designated D-Hep-III) was synthesized by solid-phase methods. The tetraazamacrocyclic chelator, TETA, was conjugated to the peptide while it was resin-bound. TETA-D-Hep-III and D-Hep-III were radiolabeled with 64Cu and 125I, respectively, in high specific activity and radiochemical purity. Heterologous competitive binding assays between D-Hep-III and either 125I-D-Hep-III or 64Cu-TETA-D-Hep-III indicated low micromolar affinity of D-Hep-III. The biodistribution of each radiolabeled analogue of D-Hep-III was carried out in rats and tumor-bearing mice. Both analogues were rapidly cleared from the blood in normal rats, with the kidneys receiving the highest accumulation of each. SKOV3 human ovarian tumor cells, known to strongly express alpha3beta1, were xenografted in SCID mice. Localization of 125I-D-Hep III and 64Cu-TETA-D-Hep III in the xenografts were low (<2% ID/g), and in the case of 125I-D-Hep III, not inhibited by a competitive dose of D-Hep III. The low tumor accumulation is likely not due to receptor down-regulation, but rather due to the weak affinity of the radioligands for the alpha3beta1 integrin.