Detection of Salmonella typhi by polymerase chain reaction

A rapid and sensitive method for detection of Salmonella typhi would help in preventing the spread of outbreaks and in clinical diagnosis. In order to develop unique PCR primers to detect Salm. typhi , ribosomal RNA genes from Salm. typhi (Rawlings) were cloned in pUC18. The resulting clone was confirmed by sequencing. The cloned DNA fragment contained the 5S, part of the 23S rRNA genes and the 5S-23S spacer region (EMBL/GenBank accession No. U04734).
It was expected that the 5S-23S spacer region is divergent unlike the highly conserved 23S+5S genes. This was confirmed by comparison with the rRNA gene sequences in the EMBL/GenBank database. A pair of PCR primers specific for Salm. typhi was obtained, based on this spacer region sequence. The specificity of this pair of primers was tested with 54 Salm. typhi strains (of 27 different phage types). All these Salm. typhi strains showed the positive 300 bp PCR product with this pair of primers. Six other Salmonella species as well as six other non- Salmonella bacteria were tested and none showed the 300 bp PCR product. The sensitivity of the detection level was 0·1 pg of pure Salm. typhi genomic DNA, or approximately 40 Salm. typhi cells in a spiked food sample. This pair of primers therefore has the potential for development into a diagnostic tool for the rapid diagnosis of typhoid fever.     

Ligand recognition in μ opioid receptor: experimentally based modeling of μ opioid receptor binding sites and their testing by ligand docking

For three-dimensional understanding of the mechanisms that control potency and selectivity of the ligand binding at the atomic level, we have analysed opioid receptor-ligand interaction based on the receptor's 3D model. As a first step, we have constructed molecular models for the multiple opioid receptor subtypes using bacteriorhodopsin as a template. The S-activated dihydromorphine derivatives should serve as powerful tools in mapping the three-dimensional structure of the μ opioid receptor, including the nature of the agonist-mediated conformational change that permits G protein-coupling to ‘second messenger’ effector molecules, and in identifying specific ligand-binding contacts with the μ opioid receptor. The analyses of the interactions of some opioid ligands with the predicted ligand binding sites are consistent with the results of the affinity labeling experiments.     

The importance of carotenoids in signaling during aggressive interactions between male firemouth cichlids (Cichlasoma meeki)

Male firemouth cichlids, Cichlasoma meeki, have red pigmentationcovering large areas of their ventral surface, which is displayedduring aggressive interactions. We manipulated the levels ofred pigmentation by assigning the fish to one of two diets,which were as similar as possible except that one was high incarotenoids while the other was low in carotenoids. During diadictrials under white light, fish kept on the high carotenoid dietwon a higher proportion of contests than fish kept on the lowcarotenoid diet Under green light, where differences in rednesscannot be discriminated, there was no effect of diet on theoutcome of contests. These experiments demonstrate that it isthe effect of the diet on red pigmentation that is importantrather than some confounding variable such as differential growthrates. The weight of the two fish was also important; therewas a tendency for the heavier fish to win more contests. Themass effect was subordinate to color under white light but wasthe dominant factor under green light The nature of the contestsunder the different light conditions also varied; the displayin which the red pigmentation is most obvious was not used undergreen light, but was common under white light This suggeststhat the display strategies are flexible and can be alteredaccording to which displays are most effective in a given environmentPrevious studies of other species of fish and birds have shownthat the degree of redness influences mate choice and is affectedby parasite infestations. We propose that carotenoid pigmentationis likely to reflect a general quality, influenced by severalfactors, rather than a context-specific quality such as fightingability.     

cDNA cloning and characterization of a novel nucleolar protein.

In an initial study of anti-nuclear antibodies in the chronic inflammatory bladder disease interstitial cystitis, we reported that 7% of interstitial cystitis patients studied had autoantibodies to the nucleolus. We now report that, using an autoimmune serum from a patient with interstitial cystitis, we have identified and partially characterized a novel protein with an M(r) of approximately 55 kDa (hereafter referred to as No55) localized to the granular component of the nucleolus. No55 was initially characterized by diffuse nucleolar immunofluorescence staining in interphase cells and by Western blotting as a 55-kDa doublet on whole-cell extracts. During mitosis, No55 was associated with chromosomes and appeared in prenucleolar bodies during telophase, but it did not colocalize with p80-coilin in coiled bodies. Immunoelectron microscopy revealed that No55 was localized uniformly throughout the granular component of the nucleolus compared with a more peripheral localization of nucleolar granular component protein B23. On segregation of the nucleolus with actinomycin D, No55 remained with the granular component of the segregated nucleolus, whereas protein B23 was found predominantly in the nucleoplasm. Finally, a cDNA expression library was screened with the human autoantibody against No55, and a 2.4-kb insert was isolated, subcloned to homogeneity, and then sequenced. Analysis of this sequence showed an open reading frame of approximately 1.3 kb coding for 437 amino acids with a predicted molecular weight of 50 kDa. A search of the gene sequence database indicated homology with SC65, a rat synaptonemal complex protein. Therefore, on the basis of molecular weight, nucleolar sublocalization, response to actinomycin D, and cDNA sequence determination, No55 is a novel protein of the interphase nucleolus.     

Further characterization of nociception-related and arterial pressure-related neuronal responses in the nucleus reticularis gigantocellularis of the rat

The present study was undertaken to further characterize the nucleus reticularis gigantocellularis (NRGC) of the medulla oblongata in the central processing of nociceptive and cardiovascular signals, and its modulation by metenkephalin. In Sprague-Dawley rats anesthetized with pentobarbital sodium, we found that all 125 spontaneously active NRGC neurons that responded to noxious stimuli (tail clamp) also exhibited arterial pressure-relatedness. Forty neurons additionally manifested cardiac periodicity that persisted even during nociceptive responses. While maintaining their cardiovascular responsive characteristics, the nociception-related NRGC neuronal activity was blocked, naloxone-reversibly (0.5 mg/kg, i.v.), by morphine (5 mg/kg, i.v.). Microiontophoretically applied met-enkephalin suppressed the responsiveness of NRGC neurons to individually delivered tail clamp or transient hypertension induced by phenylephrine (5 µg/kg, i.v.). Interestingly, in NRGC neurons that manifested both nociception and arterial pressure relatedness, the preferential reduction in the response to noxious stimuli upon simultaneous elevation in systemic arterial pressure was reversed to one that favored nociception in the presence of met-enkephalin. All actions of met-enkephalin were discernibly blocked by the opioid receptor antagonist, naloxone. Our results suggest that individual NRGC neurons may participate in the processing of both nociceptive and cardiovascular information, or in the coordination of the necessary circulatory supports during nociception. In addition, neuropeptides such as met-enkephalin may exert differential modulation on neuronal responsiveness according to the prevailing physiologic status of the animal. They also showed that NRGC may be a central integrator for pain and cardiovascular-related functions.     

Human papillomavirus is associated with monoclonal gammopathies of unknown significance

When monoclonal gammopathies arise in persons without evidence of plasma cell malignancy or lymphoproliferative disease, the term monoclonal gammopathy of unknown significance (MGUS) can be used. MGUS is believed to be the preneoplastic phase of lymphoproliferative diseases because many of these patients eventually develop malignant disease, mainly multiple myeloma. We have previously identified human papillomavirus (HPV) in a chronic benign plasma cell tumor of the cervix and in the bone marrow of multiple-myeloma patients. In the following study, we expanded upon our initial observation by analyzing 14 patients with MGUS. Bone marrow biopsies of the patients were analyzed for HPV sequences using polymerase chain reaction (PCR) and in situ hybridization. Normal controls included 26 bone marrow specimens, 24 analyzed by PCR and two by in situ hybridization. A significant association was found to exist between HPV and MGUS (p=0.001). Among 14 patients iwth MGUS, HPV sequences have been identified in 10 of the bone marrow biopsies. These results suggest that HPV can reside in the bone marrow of a premalignant lymphoproliferative disease.     

Inhibitory effect of oxygen accumulation on the growth of Spirulina platensis

Summary The growth of Spirulina platensis was studied in a light-limited culture under various dissolved oxygen (DO) concentrations. At high DO concentration, e.g. at 1.25 mM DO, the growth rate was decreased up to 36 % compared with that of 0.063 mM DO. The retarded growth rate at high DO concentrations seemed to be coupled with the degeneration of photosynthetic activity in terms of O₂ evolution. Under higher DO concentrations, superoxide dismutase and ascorbate peroxidase activities tended to increase, while the contents of photosynthetic pigment, such phycocyanin, carotenoid and chlorophyll-a decreased distinctly.     

Water Soluble Pigments Containing Xylose and Glucose in Gametangia of the Green Alga, Bryopsis maxima

Several water-soluble pigments were purified from gametangiaof Bryopsis maxima by liquid chromatography and characterizedby pyridylamination and high-performance anion-exchange chromatography.The structure of the main red pigment is proposed based on thedata of infrared spectrum, Mass spectrum, ¹H and ¹³C NMR spectraand pyridylamino analysis. As a consequence, this pigment containeda tetrapyrrole with phytol and a sugar chain comprised of xyloseand glucose. The sequence of the sugars in the chain was determinedbased on its Mass spectrum. The pigment was similar to chlorophyll-originpigments observed in other plants. No aldehyde group, however,was present at C5 in the open tetrapyrrole chain. (Received August 3, 1994; Accepted November 10, 1994)     

Differential Effects of Hypoxia on Ligand Binding Properties of Nicotinic and Muscarinic Acetylcholine Receptors on Cultured Bovine Adrenal Chromaffin Cells

Abstract: Hypoxia is known to disturb neuronal signal transmission at the synapse. Presynaptically, hypoxia is reported to suppress the release of neurotransmitters, but its postsynaptic effects, especially on the function of neurotransmitter receptors, have not yet been elucidated. To clarify the postsynaptic effects, we used cultured bovine adrenal chromaffin cells as a model of postsynaptic neurons and examined specific binding of l -[³H]nicotine (an agonist for nicotinic acetylcholine receptors: nAChRs) and ²²Na⁺ flux under control and hypoxic conditions. Experiments were performed in media preequilibrated with a gas mixture of either 21% O₂/79% N₂ (control) or 100% N₂ (hypoxia). Scatchard analysis of the specific binding to the cells revealed that the K_D under hypoxic conditions was twice as large as that under control conditions, whereas the B _max was unchanged. When the specific [³H]nicotine binding was kinetically analyzed, the association constant ( k ₁) but not the dissociation constant ( k ₋₁) was decreased to 40% of the control value by hypoxia. When the binding assay was performed using the membrane fraction, these changes were not observed. Nicotine-evoked ²²Na⁺ flux into the cells was suppressed by hypoxia. In contrast, specific [³H]quinuclidinyl benzilate binding to the intact cells was unaffected by hypoxia. These results demonstrate that hypoxia specifically suppresses the function of nAChRs (and hence, neuronal signal transmission through nAChRs), primarily by acting intracellularly.