Sensitive and rapid detection of herpes simplex virus and varicella-zoster virus DNA by loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react rapidly and efficiently, with a high specificity, under isothermal conditions. We used a LAMP assay for the detection of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV). The virus specificities of primers were confirmed by using 50 HSV-1, 50 HSV-2, and 8 VZV strains. The assay was performed for 45 min at 65 degrees C. The LAMP assay had a 10-fold higher sensitivity than a PCR assay. An analysis of nucleotide sequence variations in the target and primer regions used for the LAMP assay indicated that 3 of 50 HSV-1 strains had single nucleotide polymorphisms. No HSV-2 or VZV strains had nucleotide polymorphisms. Regardless of the sequence variation, there were no differences in sensitivity with the HSV-1-specific LAMP assay. To evaluate the application of the LAMP assay for clinical diagnosis, we tested clinical samples from 40 genital herpes patients and 20 ocular herpes patients. With the LAMP assay, 41 samples with DNA extraction and 26 direct samples without DNA extraction were identified as positive for HSV-1 or HSV-2, although 37 samples with DNA extraction and just one without DNA extraction were positive by a PCR assay. Thus, the LAMP assay was less influenced than the PCR assay by the presence of inhibitory substances in clinical samples. These observations indicate that the LAMP assay is very useful for the diagnosis of HSV-1, HSV-2, and VZV infections.     

Inhibition of TRPC5 channels by Ca2+-binding protein 1 in Xenopus oocytes

The transient receptor potential canonical type 5 (TRPC5) channel is a member of the channels that has been implicated in neurite extension and growth cone morphology of hippocampal neurons. Although homomeric TRPC5 channels are activated following stimulation of G_q/11-coupled receptors, the exact mechanism for this activation remains unresolved. Using two-electrode voltage clamp recordings, we show that the activity of TRPC5 channels expressed in Xenopus oocytes is dependent on the presence of Ca²⁺ at the extracellular as well as the cytoplasmic side of the plasma membrane. TRPC5 was activated by the stimulation of coexpressed M5 muscarinic receptors or by ionomycin. The TRPC5 activity was detectable with the presence of submillimolar levels of extracellular Ca²⁺, but it was eliminated by the injection of 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid into the oocytes. Lanthanum could substitute for extracellular Ca²⁺ to support TRPC5 activity. Coexpression of Ca²⁺-binding protein 1 (CaBP1), but not calmodulin (CaM), inhibited the TRPC5 activity, without affecting the cell surface expression of TRPC5 proteins. Using in vitro binding assays, we demonstrated direction interactions between CaBP1 and TRPC5. The CaBP1-binding sites at the C terminus of TRPC5 are closely localized, but not identical, to CaM-binding sites. We conclude that TRPC5 is a Ca²⁺-regulated channel, and its activity is negatively controlled by CaBP1.     

Discharge patterns and recruitment order of identified motoneurons and internuclear neurons in the monkey abducens nucleus

1. Single neurons in the abducens nucleus were recorded extracellularly in alert rhesus macaques trained to make a variety of eye movements. An abducens neurons was identified as a motoneuron (MN) if its action potentials triggered an averaged EMG potential in the lateral rectus muscle. Abducens internuclear neurons (INNs) that project to the oculomotor nucleus were identified by collision block of spontaneous with antidromic action potentials evoked with a stimulating electrode placed in the medial rectus subdivision of the contralateral oculomotor nucleus. 2. All abducens MNs and INNs had qualitatively similar discharge patterns consisting of a burst of spikes for lateral saccades and a steady firing whose rate increased with lateral eye position in excess of a certain threshold. 3. For both MNs and INNs the firing rates associated with different, constant eye positions could be described accurately by a straight line with slope, K (the eye position sensitivity in spikes.s-1.deg-1), and intercept, T (the eye position threshold for steady firing). For different MNs, K increased as T varied from more medial to more lateral values. In contrast, the majority of INNs already were active for values of T more medial than 20 degrees and showed little evidence of recruitment according to K. 4. During horizontal sinusoidal smooth-pursuit eye movements, both MNs and INNs exhibited a sinusoidal modulation in firing rate whose peak preceded eye position. From these firing rate patterns, the component of firing rate related to eye velocity, R (the eye velocity sensitivity in spikes.s-1.deg-1.s-1), was determined. The R for INNs was, on average, 78% larger than that for MNs. Furthermore, R increased with T for MNs, whereas INNs showed no evidence of recruitment according to R. If, as in the cat, the INNs of monkeys provide the major input to medial rectus MNs and if simian medial rectus MNs behave like our abducens MNs, then recruitment order, which is absent in INNs, must be established at the MN pool itself. 5. Unexpectedly, the R of MNs decreased with the frequency of the smooth-pursuit movement. Furthermore, the eye position sensitivity, K, obtained during steady fixations was usually less than that determined during smooth pursuit. Therefore, conclusions about the roles of MNs and premotor neurons based on how their R and K values differ must be viewed with caution if the data have been obtained under different tracking conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunohistochemical analysis of centromere protein F expression in buccal and gingival squamous cell carcinoma

Centromere protein F (CENP-F) expression (localization and characteristics) in relation to tumor clinicopathological parameters was immunohistochemically examined and evaluated in 47 archival biopsy specimens of buccal and gingival squamous cell carcinomas (SCC). Centromere protein F expression was detected in 79% of the samples. An increase in the labeling index (LI) with WHO grading was obtained ( P  < 0.05). Correlations were obtained between the CENP-F LI and tumor size ( P  < 0.05). Immunoelectron microscopy showed CENP-F nuclear staining as punctate or fine dots. The present study shows that CENP-F expression and detection of a more specific cell subpopulation presents a theoretical advantage for the analysis of the precise cell cycle of G₂ to M cells, compared to Ki-67.     

Helicobacter pylori infection produces expression of a secretory component in gastric mucous cells

Helicobacter pylori infection induces the expression of a secretory component (SC) in gastric epithelial cells. We investigated the cell lineage of the SC- and immunoglobulin (Ig) A-expressing epithelial cells in H. pylori-infected gastric mucosa. Materials were obtained by means of gastric biopsy from H. pylori-infected patients (24 cases) before and after the eradication of H. pylori, from five normal uninfected volunteers, and from three gastrectomy cases. Acetic acid-ethanol-fixed and paraffin-embedded specimens were examined using histochemical staining for gastric mucins (periodic acid oxidation-thionine Schiff reaction-concanavalin A-horse radish peroxidase staining) by means of immunostaining for gastric mucins (45M1 and HIK1083), intestinal cells (MUC2 and CD10), Ki67, H. pylori, SC, and IgA. The SC and IgA were not found in normal gastric mucosa. The expressions of the SC and IgA in gastric surface mucous cells and mucous neck cells in the generating zone of the gastric mucosa of H. pylori-infected patients were significantly higher before eradication of H. pylori than after the eradication. These mucous cells have the potential for SC-mediated translocation of IgA into the gastric lumen, and this may act as part of the antibacterial defense system against H. pylori infection in the gastric generating zone.     

Propagation of action potentials from the soma to individual dendrite of cultured rat amacrine cells is regulated by local GABA input

Retinal amacrine cells are interneurons that make lateral and vertical connections in the inner plexiform layer of the retina. Amacrine cells do not possess a long axon, and this morphological feature is the origin of their naming. Their dendrites function as both presynaptic and postsynaptic sites. Half of all amacrine cells are GABAergic inhibitory neurons that mediate lateral inhibition, and their light-evoked response consists of graded voltage changes and regenerative action potentials. There is evidence that the amount of neurotransmitter release from presynaptic sites is increased by spike propagation into the dendrite. Thus understanding of how action potentials propagate in dendrites is important to elucidating the extent and strength of lateral inhibition. In the present study, we used the dual whole cell patch-clamp technique on the soma and the dendrite of cultured rat amacrine cells and directly demonstrated that the action potentials propagate into the dendrites. The action potential in the dendrite was TTX sensitive and was affected by the local membrane potential of the dendrite. Propagation of the action potential was suppressed by local application of GABA to the dendrite. Dual dendrite whole cell patch-clamp recordings showed that GABA suppresses the propagation of action potentials in one dendrite of an amacrine cell, while the action potentials propagate in the other dendrites. It is likely that the action potentials in the dendrites are susceptible to various external factors resulting in the nonuniform propagation of the action potential from the soma of an amacrine cell.     

Oxidative polymerization of 2,6-dimethylphenol with semiconducting organic polymeric metal complexes containing the bis(ethylene-1,2-dithiolato)copper (II) structure, 1

In the oxidative polymerization of 2,6-dimethylphenol ( 8 ) the catalytic activities of the following semiconducting organic polymeric metal complexes were studied: polymeric complexes containing bis(ethylene-1,2-dithiolato)Cu(II) or Fe(III) complexes and copper(II) phthalocyanine structures ( 3a – c ), catalysts consisting of bis(ethylene-1,2-dithiolato)-Cu(II) or Fe(III) complex structures and hemiporphyrazine type structures ( 5a – c ), and also polymers containing a copper complex of dimercaptomaleic acid monoamide ( 7 ). It was found that a suspension of the insoluble bis(ethylene-1,2-dithiolato)Cu(II) polymeric complexes of type 3 catalyzes the polymerization in the presence of oxygen in pyridine at room temperature producing poly[oxy-(2,6-dimethyl-1,4-phenylene)] ( 9 ) in very good yields. Polymeric complexes of type 5 were inactive by themselves. However, we showed that bis(ethylene-1,2-dithiolato)Cu(II) polymeric complexes of both the 3 and 5 type possess great activities if they are used in the presence of small amounts of copper(II)-pyridine complex in pyridine solution (the chosen concentration was so low that the Cu(II)-pyridine complex alone had only a small activity). Bis(ethylene-1,2-dithiolato)Fe(III) complexes of both the 3 and 5 type were inactive even in the presence of the Cu(II)-pyridine complex.     

Role of interleukin-18 (IL-18) in mycobacterial infection in IL-18-gene-disrupted mice

Immunity to mycobacterial infection is closely linked to the emergence of T cells that secrete cytokines, gamma interferon (IFN-gamma), interleukin-12 (IL-12), and tumor necrosis factor alpha (TNF-alpha), resulting in macrophage activation and recruitment of circulating monocytes to initiate chronic granuloma formation. The cytokine that mediates macrophage activation is IFN-gamma, and, like IL-12, IL-18 was shown to activate Th1 cells and induce IFN-gamma production by these cells. In order to investigate the role of IL-18 in mycobacterial infection, IL-18-deficient mice were infected with Mycobacterium tuberculosis and Mycobacterium bovis BCG Pasteur, and their capacities to control bacterial growth, granuloma formation, cytokine secretion, and NO production were examined. These mice developed marked granulomatous, but not necrotic, lesions in their lungs and spleens. Compared with the levels in wild-type mice, the splenic IFN-gamma levels were low but the IL-12 levels were normal in IL-18-deficient mice. The reduced IFN-gamma production was not secondary to reduced induction of IL-12 production. The levels of NO production by peritoneal macrophages of IL-18-deficient and wild-type mice did not differ significantly. Granulomatous lesion development by IL-18-deficient mice was inhibited significantly by treatment with exogenous recombinant IL-18. Therefore, IL-18 is important for the generation of protective immunity to mycobacteria, and its main function is the induction of IFN-gamma expression.     

Na+/H+ exchange regulates intracellular pH of rat gastric surface cells in vivo

Intracellular pH (pH_i) and viability of gastric surface cells of the rat stomach in response to luminal acidification, and the role of Na⁺/H⁺ exchange in maintaining pH_i homeostasis were studied in vivo using a fluorescent microscopic technique. pH_i was measured during superfusion with buffers of pH 1.2–7.4. When the pH of the superfusate was 7.4, baseline pH_i was unchanged. Superfusion with pH 3 buffer rapidly decreased pH_i to 6.7, with subsequent recovery to baseline pH_i within 15 min despite continuing acid exposure. Superfusion with buffers of pH 1.7 and 1.2 decreased pH_i continuously to below 6.2 with no recovery observed. Despite the relentless decline in pH_i during superfusion with pH-1.2 and –1.7 solutions, over 75% of the surface cells were still viable, as measured by exclusion of the vital dye propidium iodide. We then examined the role of Na⁺/H⁺ exchange in the regulation of pH_i. Superfusion with amiloride did not affect recovery of pH_i from intracellular acidification induced by a NH₄Cl prepulse. Exposure to the potent, lipophilic Na⁺/H⁺ exchange inhibitor 5-(N,N-hexaniethylene)-amiloride (HMA), either in the superfusate or by close arterial perfusion, decreased baseline pH_i from 7.1 to 6.8. Close arterial perfusion of HMA additionally attenuated the recovery of pH_i to baseline during superfusion with pH 3 buffer. We conclude that luminal protons permeate into the cytoplasm of gastric surface cells, where they are eliminated by an Na⁺/H⁺ exchanger, most probably localized to the basolateral membrane.     

Quasi-living surface graft polymerization with phosphorylcholine group(s) at the terminal end

A surface graft polymer with one or two phosphorylcholine (PC) polarheads at the terminus of the growing chain end was prepared by sequential reactions on a glass substrate. The dithiocarbamate group covalently bound to glass surfaces was derivatized with one or two PC groups and then irradiated with ultraviolet light in the presence of N,N-dimethylacrylamide (DMAAm). X-ray photoelectron spectroscopy, wettability measurements and dye staining experiment for the PC group showed that the resultant graft copolymers were produced via iniferter-based quasi-living radical polymerization, in which the polyDMAAm graft chain contains one or two PC groups at the terminal end of the graft chain. These polymer surface grafts may help provide biocompatibility.