TEMPO EC for the enumeration of Escherichia coli in foods: collaborative study.

The automated method for enumeration of Escherichia coli, TEMPO EC, in foods uses a dehydrated culture medium and enumeration card containing 48 wells across three different dilutions for the automatic determination of the most probable number (MPN). The alternative method was compared in a multilaboratory collaborative study to AOAC Official Method 966.24. Six food types were artificially contaminated with E. coli: raw ground beef, bagged lettuce, cooked chicken, pasteurized crabmeat, frozen green beans, and pasteurized whole milk. All foods were analyzed for E. coli counts by 11 collaborating laboratories throughout the United States. Test portions from the six food types each contaminated at four different contamination levels were evaluated. The study demonstrated that the TEMPO EC method is a reliable, automated assay for the enumeration of E. coli in foods.

Mature green waste compost enhances growth and nitrogen uptake in wheat (Triticum aestivum L.) and oilseed rape (Brassica napus L.) through the action of water-extractable factors.

A series of field and pot trials were carried out to determine the effects of growing wheat and oilseed rape in soils supplemented with green waste composts and provided with additional fertilisers. It was shown consistently that the response of wheat and rape to compost and fertiliser applied together was greater than the responses to the individual additives, but only when very stable compost was used (>10 months processing). Experiments with 15N-labelled fertiliser showed that wheat was able to utilise the applied N more efficiently when cultivated in the stable compost. The enhanced growth was also demonstrated in hydroponic culture of oilseed rape with water extracts of green waste compost in the presence of compound fertiliser. However the effect was rapidly lost at higher dilutions of compost extract (>3). It was concluded that water-extractable growth promoters are present in stable green waste compost, but these only have measurable activity at high concentrations. The identity of the growth promoting factors remains to be found, but the literature suggests that water-extractable humic substances or cytokinins may be involved.

5,5-Diaryl-2-amino-4-pentenoates as novel, potent, and selective glycine transporter type-2 reuptake inhibitors.

A novel series of 5,5-diaryl-2-amino-4-pentenoates was synthesized and found to be potent and selective glycine transporter type-2 reuptake inhibitors.

6-Bicyclopiperazinyl-1-arylsulfonylindoles and 6-bicyclopiperidinyl-1-arylsulfonylindoles derivatives as novel, potent, and selective 5-HT6 receptor antagonists.

A novel series of 6-bicyclopiperazinyl-1-arylsulfonylindoles and 6-bicyclopiperidinyl-1-arylsulfonylindoles derivatives was synthesized and found to be potent and selective 5-HT6 receptor antagonists.

5-Thienyltryptamine derivatives as serotonin 5-HT1B/1D receptor agonists: potential treatments for migraine.

A series of 5-(2- or 3-thienyl)tryptamine derivatives (9) has been synthesized and shown to be potent and selective 5-HT1D versus 5-HT1B receptor agonists and, therefore, potential treatments for migraine.

Pyrrolo[3,2,1-ij]quinoline derivatives, a 5-HT2c receptor agonist with selectivity over the 5-HT2a receptor: potential therapeutic applications for epilepsy and obesity.

A series of pyrrolo[3,2,1-ij]quinoline derivatives was synthesized, evaluated for their activity against the 5-HT2c and 5-HT2a, receptors and found to be agonists at 5-HT2c with selectivity over 5-HT2a.

The Junior Temperament and Character Inventory: preliminary validation of a child self-report measure.

A preliminary effort to validate the Junior Temperament and Character Inventory with a convenience sample of 322 children ages 9 to 12 years is described.

Prototypic G protein-coupled receptor for the intestinotrophic factor glucagon-like peptide 2.

Glucagon-like peptide 2 (GLP-2) is a 33-aa proglucagon-derived peptide produced by intestinal enteroendocrine cells. GLP-2 stimulates intestinal growth and up-regulates villus height in the small intestine, concomitant with increased crypt cell proliferation and decreased enterocyte apoptosis. Moreover, GLP-2 prevents intestinal hypoplasia resulting from total parenteral nutrition. However, the mechanism underlying these actions has remained unclear. Here we report the cloning and characterization of cDNAs encoding rat and human GLP-2 receptors (GLP-2R), a G protein-coupled receptor superfamily member expressed in the gut and closely related to the glucagon and GLP-1 receptors. The human GLP-2R gene maps to chromosome 17p13.3. Cells expressing the GLP-2R responded to GLP-2, but not GLP-1 or related peptides, with increased cAMP production (EC50 = 0.58 nM) and displayed saturable high-affinity radioligand binding (Kd = 0.57 nM), which could be displaced by synthetic rat GLP-2 (Ki = 0.06 nM). GLP-2 analogs that activated GLP-2R signal transduction in vitro displayed intestinotrophic activity in vivo. These results strongly suggest that GLP-2, like glucagon and GLP-1, exerts its actions through a distinct and specific novel receptor expressed in its principal target tissue, the gastrointestinal tract.

Computerized interviews as a method of assessing psychopathology in children.

A computerized version of the Diagnostic Interview for Children and Adolescents-Revised (DICA-R) has been developed. There are three versions of the interview, one for children aged 6 to 12, one for adolescents aged 13 to 18, and one for parents of children aged 6 to 18. Acceptability and reliability studies of the child and adolescent versions were performed on 93 subjects. The results show that children of all ages enjoyed the computer, and most were able to complete the interview with very little help. Reasonable reliability was found for both age groups despite difficulty with certain diagnostic criteria.

Design and synthesis of RNA miniduplexes via a synthetic linker approach. 2. Generation of covalently closed, double-stranded cyclic HIV-1 TAR RNA analogs with high Tat-binding affinity.

We recently developed an approach which allows rapid generation of short, double-stranded oligonucleotides whereby one end of the duplex was joined and stabilized by a synthetic linker of specific design (miniduplexes)(6). Model miniduplexes based on the HIV-1 TAR RNA hairpin were shown to be thermodynamically stable and good substrates for binding by the HIV-1 Tat protein which normally bind to natural TAR (6). In this study, we have extended our studies to the design, synthesis and analysis of the binding properties of covalently closed, double-stranded, cyclic RNA miniduplexes. A strategy using automated chemical synthesis and T4 RNA ligase-catalyzed cyclization was employed to generate cyclic oligoribonucleotides. When both ends of a shortened, wild-type TAR RNA stem (9 bp) were covalently linked through either nucleotidic loops (4-6 nt) or synthetic linkers (derivatized from hexaethylene glycol), the resulting cyclic TAR RNA analogs were good substrates for binding by both Tat-derived peptide or full-length Tat protein. Interestingly, the cyclic TAR analogs failed to show any binding if the synthetic linker was reduced in length (e.g. derivatized from triethylene glycol), although such linkers are acceptable in the hairpin-shaped miniduplexes series (6). This implies that RNA conformational changes are required for Tat binding and that these changes are restricted in certain cyclic variants. Our findings suggest that covalently-closed nucleic acid miniduplexes may be useful both to study nucleic acid-protein interactions as well as to provide a basis for therapeutic intervention as transcription decoys.

Phylogenetic diversity of subsurface marine microbial communities from the Atlantic and Pacific Oceans.

The extent of the diversity of marine prokaryotes is not well known, primarily because of poor cultivability. However, new techniques permit the characterization of such organisms without culturing, via 16S rRNA sequences obtained directly from biomass. We performed such an analysis by polymerase chain reaction amplification with universal primers on five oligotrophic open-ocean samples: from 100-m (three samples) and 500-m depths in the western California Current (Pacific Ocean) and from a 10-m depth in the Atlantic Ocean near Bermuda. Of 61 clones, 90% were in clusters of two or more related marine clones obtained by ourselves or others. We report 15 clones related to clone SAR 11 found earlier near Bermuda (S. J. Giovannoni, T. B. Britschgi, C. L. Moyer, and K. G. Field, Nature [London] 345:60-63, 1990), 11 related to marine cyanobacteria, 9 clustered in a group affiliated with gram-positive bacteria, 9 in an archaeal cluster we recently described (mostly from the 500-m sample), 4 in a novel gamma-proteobacterial cluster, and 6 in three two-membered clusters (including other archaea). One clone was related to flavobacteria. Only the cyanobacteria plus one other clone, related to Roseobacter denitrificans (formerly Erythrobacter longus Och114), were within 10% sequence identity to any previously sequenced cultured organism in a major data base. We never found more than two occurrences of the same sequence in a sample, although four times we found identical sequences between samples, two of which were between oceans; one of these sequences was also identical to SAR 11.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel major archaebacterial group from marine plankton.

Marine bacteria often dominate the plankton biomass and are responsible for much of the cycling of organic matter, but bacterial diversity is poorly understood because conventional identification methods (requiring culturing) miss about 99% of the organisms. Recent advances permit characterization of microbial communities by analysis of 16S ribosomal RNA gene sequences directly from biomass without the need to culture the organisms; such studies from surface ocean samples have found only eubacteria, not archaebacteria (or Archaea), which are profoundly different. Here we report 16S rRNA sequences obtained from Pacific Ocean bacterioplankton samples collected from depths of 100 m and 500 m. Among these we found sequences only distantly related to those of any organisms previously characterized by 16S rRNA sequences, with similarities to the nearest such relatives (extreme thermophiles) approximately the same as those between animals and plants. We suggest that these sequences are from a previously undescribed archaebacterial group that may have diverged from the ancestors of characterized organisms very early in evolution.

Developmental, structural, and clinical approach to narcissistic and antisocial personalities.

The conception of personality disorders (PDs) as distinct units of mental disorders is neither precise nor useful. At least some PDs, classified as separate units, reflect different behavioral expression of the same personality deviation. In this article we describe structural, developmental, and clinical continuum between relatively distinct entities of antisocial PD and narcissistic PD. The two disorders represent different endpoints sharing a borderline level of personality organization and pathological narcissism. We propose a spectrum relation for antisocial and narcissistic PD because the disorders tend to co-occur in the same individual and to run in the same family more often than expected by chance.

Antisocial behavior and personality disorders.

Present classifications fall short of helping clinicians to systematically approach syndromes of antisocial (A-S) behavior. Various clinical forms of A-S behavior derive from different levels of personality organization (normal, neurotic, and borderline level) whereas certain personality disorders (PD) display specific antisocial "profiles" and form the horizontal continuum of antisocial behavior. The borderline level of personality and pathological narcissism stand behind A-S PD, Narcissistic PD, and Histrionic PD. The authors propose that the disorders should be regarded as spectrum disorders. Paranoid PD and "pure" Borderline PD complete the list of PDs manifesting A-S behaviors. Finally, diagnostic instruments for clinical approach to and research of A-S PD are presented.

The rcsA gene of Klebsiella pneumoniae O1:K20 is involved in expression of the serotype-specific K (capsular) antigen.

In Klebsiella pneumoniae, the ability to synthesize large amounts of capsular polysaccharide is an important correlate of virulence. We report the cloning of rcsA from K. pneumoniae serotype O1:K20 and demonstrate that rcsA is involved in the expression of the K antigen capsule. We have determined the nucleotide sequence for the rcsA gene from K. pneumoniae K20 and shown it to be identical to the sequence reported previously for rcsA from strain K21 (Allen et al., J. Gen. Microbiol. 133:331-340, 1987). Southern hybridization results indicate that this gene is widely distributed among different Klebsiella K serotypes. When cloned into Escherichia coli K-12, the K. pneumoniae rcsA gene caused a mucoid phenotype, resulting from the activation of colanic acid synthesis. Activation of colanic acid synthesis was not dependent on growth at low temperatures (less than or equal to 30 degrees C). The K. pneumoniae rcsA gene complemented E. coli K-12 rcsA mutations but could not complement defects in rcsB, suggesting that RcsA may be functionally homologous in these bacteria. The cloned rcsA gene also complemented a defect in nonmucoid strain K20 derivatives that normally produced only trace amounts of K20 antigen and were unable to assemble a wild-type capsular structure. Mutants that were K20-deficient were not complemented. The K antigen capsule of K. pneumoniae therefore joins a growing list of polysaccharide-synthetic systems in which "RcsA-like" proteins are involved.

A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing.

The lipopolysaccharide (LPS) O-antigen side chains of Klebsiella serotype O1 have been studied by using mutants selected by resistance to a Klebsiella bacteriophage designated O1-A. Two classes of LPS mutants were identified. The major group (90%) synthesized rough LPS. The remaining 10% of the mutants produced a novel LPS profile that lacked the highest-molecular-weight O-substituted molecules (HMW-LPS) but still produced lower-molecular-weight O-substituted species (LMW-LPS). By using antisera raised against mutant Klebsiella strains and antiserum specific for Pasteurella haemolytica serotype 4, it was demonstrated that HMW-LPS and LMW-LPS contain shared epitopes. HMW-LPS also contained an epitope absent in LMW-LPS. This unique epitope was recognized by a monoclonal antibody (O1-52.6) and appears to be responsible for the serological cross-reaction between the O antigens of Klebsiella O1 and Escherichia coli O19. This HMW-LPS epitope was present in eight other Klebsiella O1 isolates which were examined. Electron microscopy demonstrated that HMW-LPS excluded overlying capsular polysaccharide for a distance of 25 to 40 nm. The distance was reduced to 10 to 18 nm in strains which synthesized only LMW-LPS and to zero in rough LPS strains. The HMW-LPS of Klebsiella O1 was shown to be an important virulence determinant, since this molecule was responsible for the resistance of the bacterium to nonspecific, complement-mediated serum killing.

Hot flushes are induced by thermogenic stimuli.

Hot flushes were caused by hot drinks, alcohol, radiant heaters and thermal blankets in men undergoing treatment for carcinoma of the prostate and in menopausal women. Avoiding or changing these commonplace stimuli appears to reduce the frequency of flushing.

Use of a bacteriophage-encoded glycanase enzyme in the generation of lipopolysaccharide O side chain deficient mutants of Escherichia coli O9:K30 and Klebsiella O1:K20: role of O and K antigens in resistance to complement-mediated serum killing.

Coliphage K30 lysates contain free and phage-associated forms of a bacteriophage-encoded capsule depolymerase (glycanase) enzyme, active against the serotype K30 capsular polysaccharide of Escherichia coli. The free glycanase has been purified to apparent homogeneity. The molecular weight of the enzyme was estimated at 450,000, and when heated in SDS at 100 degrees C, the enzyme dissociated into two subunits of 90,000 and 52,000. The glycanase enzyme was used as a reagent to reversibly degrade the capsular layers on cells of Escherichia coli O9:K30 and Klebsiella O1:K20. This treatment rendered these bacteria sensitive to their respective lipopolysaccharide-specific bacteriophages, coliphage O9-1 and Klebsiella phage O1-3. This novel approach facilitated isolation of lipopolysaccharide O antigen side chain deficient mutants which retained the ability to synthesize the capsule. The response of defined mutants, O+:K-, O-:K+, and O-:K-, to exposure to nonimmune rabbit serum was measured. Results showed that the primary barrier against complement-mediated serum killing in both Escherichia coli O9:K30 and Klebsiella O1:K20 was the O antigen side chains of the lipopolysaccharide molecules. In both strains, the capsule played no role in the determination of serum resistance.