Selection of cellular genetic markers for the detection of infectious poliovirus.

AIMS: Cellular responses of an established cell line from human intestinal epithelial cells (INT-407 cells) against poliovirus (PV) infections were investigated in order to find cellular genetic markers for infectious PV detection. METHODS AND RESULTS: Gene expression profile of INT-407 cells was analysed by DNA microarray technique when cells were infected with poliovirus 1 (PV1) (sabin) at multiplicity of infection of 10-3 and incubated for 12 h. Poliovirus infection significantly altered the gene expressions of two ion channels, KCNJ4 and SCN7A. The expression profile of KCNJ4 gene was further investigated by real-time RT-qPCR, and it was found that KCNJ4 gene was significantly regulated at 24 h postinfection of PV1. CONCLUSIONS: KCNJ4 gene, coding a potassium channel protein, is proposed as a cellular genetic marker for infectious PV detection. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to show the availability of cellular responses to detect infectious PV. The selection of cellular genetic markers for infectious viruses using DNA microarray and RT-qPCR can be applicable for the other enteric viruses.

Feeding increases 5-hydroxytryptamine and norepinephrine within the hypothalamus of chicks.

It is thought that hypothalamic 5-hydroxytryptamine (5HT) and norepinephrine (NE) are involved in the regulation of feeding in chicks. The present study was conducted to elucidate changes in the levels of extracellular 5HT and NE in the hypothalamus during feeding of chicks. In order to measure 5HT, NE and 4-hydroxy-3-methoxyphenylglycol (MHPG), which is a major metabolite of NE, we used brain microdialysis and high-pressure liquid chromatography with an electrochemical detector. After collecting samples to determine the basal levels of 5HT, NE and MHPG, food-deprived birds were given access to food. 5HT levels in the medial hypothalamus (MH) and lateral hypothalamus (LH) increased during the first 30 min of feeding, and then returned to basal levels. NE and MHPG in the LH increased during feeding, and remained elevated throughout the experiment. This study supports an idea that hypothalamic monoamines in the chick brain are involved in the regulation of feeding.

Catecholamines and serotonin are differently regulated by tetrahydrobiopterin. A study from 6-pyruvoyltetrahydropterin synthase knockout mice.

(6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase (TH), tryptophan hydroxylase, phenylalanine hydroxylase, and nitric-oxide synthase. These enzymes synthesize neurotransmitters, e.g. catecholamines, serotonin, and nitric oxide (NO). We established mice unable to synthesize BH4 by disruption of the 6-pyruvoyltetrahydropterin synthase gene, the encoded protein of which catalyzes the second step of BH4 biosynthesis. Homozygous mice were born at the almost expected Mendelian ratio, but died within 48 h after birth. In the brain of homozygous mutant neonates, levels of biopterin, catecholamines, and serotonin were extremely low. The number of TH molecules was highly dependent on the intracellular concentration of BH4 at nerve terminals. Alteration of the TH protein level by modulation of the BH4 content is a novel regulatory mechanism. Our data showing that catecholaminergic, serotonergic, and NO systems were differently affected by BH4 starvation suggest the possible involvement of BH4 synthesis in the etiology of monoamine-based neurological and neuropsychiatric disorders.

Turgor pressure regulation and the orientation of cortical microtubules in Spirogyra cells.

Microtubules (MTs) of cells of Spirogyra sp. were depolymerized by treatment with amiprophos-methyl (APM) for 1 h and then reorganized in 0.30 M mannitol solution. The reorganized MTs after 1.5 h incubation showed an oblique/longitudinal orientation and then became transversely oriented as the incubation was prolonged. During this incubation, the osmotic pressure of cells was measured by the plasmolysis method. The cell osmotic pressure increased with time. The calculated turgor pressure at 1.5 h was 0.11 M (mannitol equivalent) and, at 13.5 h, 0.25 M. Similar changes in MT orientation and recovery of the turgor pressure were also observed in 0.30 M sorbitol solution. These results suggest that the MT orientation may be correlated with the turgor pressure. Among fresh water algae sensitive to a saline environment, this Spirogyra was the first species shown to have a turgor regulating mechanism, although the recovery of turgor pressure was incomplete. The recovery of turgor pressure in mannitol solutions was also observed without APM treatment.

Different cytoplasmic calcium contents among three species of Characeae.

Internodal cells of three species of Characeae, Nitella flexilis, Nitella axilliformis and Chara corallina, were analyzed for the contents of Ca(2+ )and Mg(2+) in the cytoplasm. To avoid contamination of Ca(2+) from the cell wall and vacuole, the vacuolar sap was replaced with a sorbitol solution containing Sr(2+) by the vacuolar perfusion method after the cell had been treated with Sr(2+). No significant difference in the cytoplasmic content of Mg(2+) was found among three species of Characeae, but significant differences in the cytoplasmic content of Ca(2+) were observed among them. The cytoplasmic Ca(2+) content of N. flexilis was 2.0 times that of N. axilliformis and 3.3 times that of C. corallina. The cytoplasmic drop was furthermore separated into two fractions: a chloroplast-free fraction and a chloroplast fraction. In the chloroplast-free fraction the Ca(2+) content of N. flexilis was 2.3 times that of C. corallina and 2.0 times that of N. axilliformis, while the Mg(2+) content was the same among the three species. In the chloroplast fraction N. flexilis contained about seven times more Ca(2+) and about two times more Mg(2+) than C. corallina. The difference in the cytoplasmic Ca(2+ )content was discussed in relation to the difference in the capacity for the hydration-induced Ca(2+) release existing among the three species.

Mechanosensitive Ca2+ release from intracellular stores in Nitella flexilis.

We found previously that the cytoplasmic drop isolated from internodal cells of Nitella flexilis releases Ca2+ in response to hypotonic treatment and named the phenomenon hydration-induced Ca2+ release (HICR). The HICR is assumed to be a result of activation of Ca2+ permeable channels in the membrane of Ca2+ stores in a stretch-activated manner. To prove this idea, mechanical stimulus was applied to the drop by means of shooting isotonic/hypnotic medium or silicon oil into the drop, or compressing the drop. All these mechanical stimuli induced a rapid increase in the Ca2+ concentration of the drop. The chloroplast fraction isolated from the cytoplasmic drop released Ca2+ on compression, while the chloroplast-free cytoplasm did not. In Chara corallina, the cytoplasmic drop, which shows a very weak HICR, also responded weakly to the mechanical stimulus, but the chloroplast fraction was inert. When chloroplasts from Chara were added to the chloroplast-free cytoplasm of N. flexilis, the cytoplasm recovered the mechanoresponse. Starch grains were as effective as chloroplasts. The data indicate that Ca2+ permeable channels in the membrane of Ca2+ stores in N. flexilis are really mechano-sensitive.

Onion root water transport sensitive to water channel and K+ channel inhibitors.

Transroot osmotic water flux (Jos) and radial hydraulic conductivity (Lpr) in onion roots were greatly increased by three means; infiltration of roots by pressurization, repetition of osmosis and chilling at 5 degrees C. Jos was strongly reduced by the water channel inhibitor HgCl2 (91%) and the K+ channel inhibitor nonyltriethylammonium (C9, 75%), which actually made the membrane potential of root cells less sensitive to K+. C9 decreased the rate of turgor reduction induced by sorbitol solution to the same extent as HgCl2. Thus, C9 is assumed to decrease the hydraulic conductivity (Lp) of the plasma membrane by blocking water channels, although possible inhibition of the plasmodesmata of the root symplast by C9 cannot be excluded. Onion roots transported water from the tip to the base in the absence of the osmotic gradient. This non-osmotic water flux (Jnos) was equivalent to Jos induced by 0.029 M sorbitol. Jnos increased when Jos was increased by repetition of osmosis and decreased when Jos was decreased by either HgCl2 or by C9. The correlation between Jnos and Jos suggests that non-osmotic water transport occurs via the same pathways as those for osmotic water transport.

GTP cyclohydrolase I from Tetrahymena pyriformis: cloning of cDNA and expression.

A full-length cDNA clone for GTP cyclohydrolase I (EC 3.5.4.16) was isolated from a Tetrahymena pyriformis cDNA library by plaque hybridization. The nucleotide sequence determination revealed that the length of the cDNA insert was 1516 bp. The coding region encoded a protein of 223 amino acid residues with a calculated molecular mass of 25 416 Da. The deduced amino acid sequence of Tetrahrymena GTP cyclohydrolase I showed sequence identity with that of Escherichia coli (55%). The identity of T. pyriformis GTP cyclohydrolase I with sequences of Dictyostelium discoideum, Saccharomyces cerevisiae, Drosophila melanogaster, mouse, rat, and human enzymes was less marked and was 30, 30, 25, 28, 28, and 27%, respectively. RNA blot analysis showed a single mRNA species of 2.1 kb in this protozoan. The mRNA level of GTP cyclohydrolase I increased during synchronous cell division induced by intermittent heat treatment. The results suggest that the mRNA expression is associated with the cell cycle of T. pyriformis.

Enzymes related to catecholamine biosynthesis in Tetrahymena pyriformis. Presence of GTP cyclohydrolase I.

We first identified GTP cyclohydrolase I activity (EC 3.5.4.16) in the ciliated protozoa, Tetrahymena pyriformis. The Vmax value of the enzyme in the cellular extract of T. pyriformis was 255 pmol mg-1 protein h-1. Michaelis-Menten kinetics indicated a positive cooperative binding of GTP to the enzyme. The GTP concentration producing half-maximal velocity was 0.8 mM. By high-performance liquid chromatography (HPLC) with fluorescence detection, a major peak corresponding to D-monapterin (2-amino-4-hydroxy-6-[(1'R,2'R)-1',2',3'-trihydroxypropyl]pteridin e, D-threo-neopterin) and minor peaks of D-erythro-neopterin and L-erythro-biopterin were found to be present in the cellular extract of Tetrahymena. Thus, it is strongly suggested that Tetrahymena converts GTP into unconjugated pteridine derivatives. In this study, dopamine was detected as the major catecholamine, while neither epinephrine nor norepinephrine was identified. Indeed, this protozoa was shown to possess the activity of a dopamine synthesizing enzyme, aromatic L-amino acid decarboxylase. On the other hand, activities of tyrosine hydroxylase or tyrosinase which converts tyrosine into dopa, the substrate of aromatic L-amino acid decarboxylase, could not be detected in this protozoa. Furthermore, neither dopamine beta-hydroxylase activity nor phenylethanolamine N-methyltransferase activity could be identified by the HPLC methods.

Optical constants of V(1-x)W(x)O(2) Films.

The spectral complex optical constants in the visible and the near-infrared region of VO(2) and V(1-x)W(x)O(2) films deposited on glass substrates were determined from observed reflectance and transmittance spectra for which the least-squares method was used. In the metallic phase, the optical properties were characterized by the Drude model in wavelength regions longer than 750 nm.

[Long-term follow-up study of children with minimal change nephrotic syndrome].

We conducted a long-term follow-up study of 37 children with biopsy-proved minimal change nephrotic syndrome during a period of over 6 years from onset to adulthood. These patients were classified into 4 groups of 13 infrequent relapsers, 17 frequent relapsers, 3 non-responders and 4 no-relapsers according to the International Study of Kidney Disease in Children (ISKDC). All patients were treated with conventional prednisolone therapy. Two cases of infrequent relapsers, 7 cases of frequent relapsers and 1 case of non responders relapsed in adult life. Two cases of infrequent relapsers and 1 case of frequent relapsers relapsed in adult life after remission for 5 or more years. We concluded that minimal change nephrotic syndromes in childhood should be followed up over a long duration in adult life, evenly in cases with good steroid responsiveness.

Antibodies to hepatitis C virus and elevated transaminase levels in a town of hyperendemicity in Iwate, Japan.

In an annual survey for liver function tests in persons aged > 35 years in Iwate Prefecture, Japan, a town was identified where high levels of alanine aminotransferase abounded. Of 5152 inhabitants aged > 35 years in this town, antibodies to hepatitis C virus (HCV) determined by enzyme-linked immunosorbent assay with HCV core peptides were detected in 798 (15.5%) people, hepatitis B surface antigen was detected in 47 (0.9%) people and antibodies to hepatitis B surface antigen and/or hepatitis B core antigen unaccompanied by surface antigenaemia were detected in 1748 (33.9%) people. Antibodies to HCV core peptides correlated closely with the antibodies detected by passive haemagglutination with recombinant HCV proteins of the second generation. Antibodies to HCV core peptides were particularly common in one of the nine districts of the town (district 8), where 276 (45.3%) of 609 inhabitants tested positive; there were no differences in the frequency of serological markers of hepatitis B virus (HBV) infection among residents in the nine districts. Of the 798 individuals with antibodies to HCV core peptides, 257 (32.2%) were found to have elevated transaminase levels. A history of transfusion was associated with a high frequency of antibodies to HCV core peptides, except in district 8 where such antibodies were equally frequent in residents irrespective of transfusions. These results indicate an epidemiology of HCV distinct from that of HBV in this town and a very local spread of HCV, as well as a high frequency of hepatic injuries in individuals with antibodies to HCV.

[Exercise-induced acute renal failure observed in a boy with idiopathic renal hypouricemia caused by postsecretary reabsorption defect of uric acid].

Acute renal failure without oliguria developed in an 11-year boy after running exercise. With improvement of his renal function, marked hypouricemia became apparent (0.8-0.9 mg/dl). Increased excretion of uric acid into the urine, increased clearance ratio of uric acid against creatinine (CUA/CCr), normal concentration of plasma xanthine and hypoxanthine, and suppression of CUA/CCr ratio by pyrazinamide loading but not by probenecid, were observed in the patient and his two siblings, suggesting that hereditary abnormalities of reabsorption of uric acid after secretion from the renal tubules resulted in the hypouricemia. The mechanism of acute renal failure in this disease remains unknown.

Characean cells as a tool for studying electrophysiological characteristics of plant cells.

Characean cells have contributed significantly to various areas of plant cell biology such as cell motility and membrane transport. Since characean cells are very large, various kinds of operations can easily be applied to them. Development of techniques of intracellular perfusion and permeabilization of plasma membrane has facilitated studies on functions of the plasma membrane and the vacuolar membrane (or tonoplast) which is specific to plant cells. The present article is aimed at reviewing the contribution of characean cells to the study of electrophysiological characteristics of plant membranes. Our attention was mainly focused on experiments using plasma membrane-permeabilized cells and intracellularly perfused cells.

Far-infrared spectra of magnesium oxide.

High-resolution spectra from 20 to 200 cm(-1) for temperatures from 10 to 300 K give interference fringes that cannot be fitted by previous analysis in terms of four Lorentz oscillators. Parameters in the theory are revised to give a better fit both to the far IR and to the reststrahlen spectra.

Immunocytochemical localization of substance P and neurofilament proteins in the guinea pig vestibular ganglion.

To examine the cytochemical features of primary vestibular afferent neurons, we examined the immunocytochemical distribution of substance P(SP) and neurofilament proteins (NFP) in the guinea pig vestibular endorgans and ganglion. 84.9% of vestibular ganglion cells showed SP-like immunoreactivity. SP immunoreactive cells were small in diameter (25.4 microns), and randomly distributed in the ganglia. NFP immunoreactive cells (34.2%) were relatively large in diameter (31.1 microns). By double-staining immunocytochemistry it was possible to distinguish three different cell types: SP-positive/NFP-negative, SP-negative/NFP-positive, and SP/NFP-positive vestibular ganglion cells. Vestibular ganglion cells can thus be classified on the basis of size and immunocytochemical characteristics. These chemically distinct subpopulations of vestibular ganglion cells may be an indication of functional differences within vestibular afferent neurons.

Role of cytoplasmic inorganic phosphate in light-induced activation of H(+)-pumps in the plasma membrane and tonoplast of Chara corallina.

A unique variant strain of Chara corallina, which contains little inorganic phosphate in the vacuole ([Pi]v) was isolated. The level of cytoplasmic inorganic phosphate ([Pi]c) in these cells was the same as that in normal cells. Using these unique cells, we studied the change in [Pi]c and the effect of Pi on the activities of electrogenic H(+)-pumps associated with the plasma membrane and tonoplast. Upon illumination, the plasma membrane of C. corallina became hyperpolarized by 15 mV, the pH of the vacuolar sap decreased by 0.5 unit, and [Pi]c decreased by 30% with a similar time course. The activities of the electrogenic H (+)-pump in the plasma membrane and the ATP and PPi-dependent H(+)-transport in the tonoplast were noncompetitively inhibited by Pi with Ki values of, in the order given, 21.3 mM, 22.1 mM and 37.7 mM. From the kinetics study we calculated that the electrogenic H(+)-pump in the plasma membrane and the ATP and PPi-dependent H(+) transport in the tonoplast were activated by, again in this order, 13%, 13% and 9%, in accordance with the decrease in [Pi]c. We propose that the change in [Pi]c is one of the regulators of photosynthesis-mediated activation of the H(+)-pumps in the plasma membrane and the tonoplast in C. corallina upon illumination.

Localization of substance P-like immunoreactivity in guinea pig vestibular endorgans and the vestibular ganglion.

The immunocytochemical distribution of substance P (SP) in guinea pig vestibular endorgans and the vestibular ganglion was investigated. Two kinds of SP-immunoreactive fibers were distinguished. Most were thick, and found around or beneath sensory hair cells. These SP-immunoreactive fibers were distributed predominantly on the slope of the crista and the peripheral region of the macula. By electron microscopy, we confirmed this type of SP-like immunoreactivity to be restricted within primary afferent neurons. Some vestibular ganglion cells also showed SP-like immunoreactivity, suggesting that SP is present in some primary afferent neurons, and is involved in afferent neurotransmission. The characteristic distribution of SP may indicate functional differences within each endorgan. The other group of immunoreactive nerve fibers, varicous thin fibers, could be found in the stroma of vestibular endorgans, nerve trunk, vestibular ganglion, and along blood vessels of the vestibular ganglion. These fibers may have a different origin, and have an influence on blood flow and certain other functions.