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.

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.

Immunocytochemical study of the GABA system in chicken vestibular endorgans and the vestibular ganglion.

The immunocytochemical distribution of gamma-aminobutyric acid (GABA), GABA synthesizing enzyme; L-glutamate decarboxylase (GAD) and degradative enzyme; GABA transaminase (GABA-T) in the chicken vestibular endorgans and the vestibular ganglion was investigated. GABA and GAD-like immunoreactivity were confined to the sensory hair cell cytoplasm, suggesting that GAD synthesizes GABA in the hair cell. GABA-T-like immunoreactivity, indicative of GABA degradation, was found around hair cells, along nerve fibers running through the stroma and within the ganglion cell. These immunocytochemical findings indicate that the GABAergic system exists in the chicken vestibular endorgans and that GABA may function as an afferent neurotransmitter at the level of hair cells.

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.

Partial purification and some characteristics of proteinase(s) induced by staphylococcal exfoliative toxin.

After 8 hrs incubation with epidermis of newborn mice and exfoliative toxin, a marked increase in caseinolytic activity was detected, which reached a maximum at 12 hrs. Casein-hydrolyzing enzyme(s) induced by ET were partially purified by chromatography. A substantial increase in caseinolytic activity was detected in the fractions obtained from Sephadex G-50, while practically no caseinolytic activity was observed when the extract obtained from the epidermis incubated without ET was applied. The caseinolytic activity appeared as a single peak eluted from DEAE-Sepharose CL-6B and Sephadex G-75. However, on SDS-PAGE, the partially purified fractions exhibited several protein bands. The molecular weights of these band were estimated as 78 KD, 68 KD, 45 KD, 14.5 KD and 8.8 KD. When the partially purified enzyme(s) was preincubated with EGTA or EDTA, substantial inhibition of the activity was observed; however, no recovery of the activity was detected after the addition of CaCl2. Treatment of the enzyme(s) with PMSF and NEM caused little inactivation of the activity. Enzyme activity retained about 57% of the initial activity following 3 min incubation at 60 degrees C, but was completely inactivated after 4 min.

[A case of systemic lupus erythematosus associated with an aneurysm of the lenticulostriate artery].

We describe a patient with established systemic lupus erythematosus (SLE) in whom an intracerebral hemorrhage developed secondary to a ruptured aneurysm of the lenticulostriate artery (LSA). A 24-year-old woman with a four-year history of SLE was admitted to the department of internal medicine of Iwate Medical University for the treatment of lupus nephritis in 1985. She suddenly complained of severe headache and nausea, and soon lost consciousness. The computed tomographic scan revealed intracerebral hemorrhage in the left front-temporal region and subarachnoid hemorrhage. Left common carotid angiography demonstrated a 3 X 3 mm aneurysm of the LSA and displacement of other LSAs and anterior cerebral artery. The incidence of intracerebral hemorrhage in SLE was about ten percent in the reported central nervous system SLE, and it seemed that the prognosis of SLE with intracerebral hemorrhage was poor. The mechanisms of the intracerebral hemorrhage and the aneurysmal formation in SLE seemed to be due to lupus angiitis, but without clinical, radiologic and pathologic correlation. In operation, a ruptured aneurysm without neck was found in LSA and extirpated. In the pathological study, there was transmural angiitis, which fibrinoid necrosis, elastic tissue disruption and infiltration of inflammatory cells were found. Inflammatory cells were chiefly lymphohistiocytic with some polymorphonuclear leukocytes. It seemed that pathologic studies confirmed transmural angiitis with secondary aneurysm formation.

[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.

[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.

Tonoplast action potential in Nitella in relation to vacuolar chloride concentration.

The action potential of Nitella internode was studied in relation to K+ and C1- concentrations in the vacuole. When the vacuole of Nitella pulchella was filled with an artificial solution with extremely low C1- concentration, a diphasic action potential (DAP) was observed. T he first phase consists of a rapid depolarization followed by a relatively rapid repolarization, and the second one consists of a strong hyperpolarization followed by a gradual return to the resting potential. When the cell was stimulated immediately after the generation of DAP, a monophasic action potential which resembles an action potential of the natural cell was observed, indicating that the DAP consists of two components with different refractory periods. The refractory period of the component responsible for the depolarizing is shorter than that of a component responsible for the hyperpolarizing phase. Measuring the plasmalemma potential and vascuolar potential separately, it was demonstrated that the hyperpolarizing component of DAP originates from the tonoplast. The action potential of the tonoplast, in contrast with that of the plasmalemma, could be generated independently of concentration of K+ in the vasuole. Since the maximum amplitude of hyperpolarization decreased significantly by increasing C1- concentration of the vacuole, it is concluded that the tonoplast is very sensitive to C1- during excitation.

Nature of the water channels in the internodal cells of Nitellopsis.

The hydraulic resistance was measured on internodal cells of Nitellopsis obtusa using the method of transcellular osmosis. The hydraulic resistance was approximately 2.65 pm-1 sec Pa, which corresponds to an osmotic permeability of 101.75 microns sec-1 (at 20 degrees C). p-Chloromercuriphenyl sulfonic acid (pCMPS) (0.1-1 mM, 60 min) reversibly increases the hydraulic resistance in a concentration-dependent manner. pCMPS does not have any effect on the cellular osmotic pressure. pCMPS increases the activation energy of water movement from 16.84 to 32.64 kJ mol-1, indicating that it inhibits water movement by modifying a low resistance pathway. pCMPS specifically increases the hydraulic resistance to exosmosis, but does not influence endosmosis. By contrast, nonyltriethylammonium (C9), a blocking agent of K+ channels, increases the hydraulic resistance to endosmosis, but does not affect that to exosmosis. These data support the hypothesis that water moves through membrane proteins in characean internodal cells and further that the polarity of water movement may be a consequence of the differential gating of membrane proteins on the endo- and exoosmotic ends.

Degradation of Proteins Artificially Introduced into Vacuoles of Chara australis.

When an exogenous protein, bovine serum albumin, was introduced into the vacuole of a Chara australis internodal cell, it was degraded with time. This degradation proceeded only in the vacuole as far as could be observed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Degradation was inhibited by protease inhibitors such as antipain and leupeptin. Endogenous proteins introduced into the vacuole were also degraded there. Furthermore, intravacuolar cytoplasmic drops, which were often formed by cell ligation, seemed to be degraded in the vacuole. However, bovine serum albumin degradation did not proceed when mixed with isolated vacuolar sap. These results show that the vacuole in the Chara internodal cell has the capacity to degrade cellular proteins, but that cytoplasmic support is needed for this degrading activity to be maintained.

Metabolic Conversion of Amino Acids Loaded in the Vacuole of Chara australis Internodal Cells.

Vacuoles of internodal cells of Chara australis (or Chara corallina) were loaded with a 10 millimolar amount of various amino acids by a perfusion method and incubated under continuous light. After 20 to 24 hours, the cell sap was collected, and free amino acids in it and the rest of the cell (cytoplasm) were analyzed. The only amino acid metabolized completely was alanine. About 40 to 80% of the aspartic acid, glutamine, serine, and glycine were metabolized, whereas less than 30% of the threonine, asparagine, isoasparagine, isoleucine, phenylalanine, gamma-aminobutyric acid, lysine, and arginine were metabolized. The figure for glutamic acid fluctuated between 10 and 100%. The main metabolites of alanine were glutamine, glycine and ammonia, which accumulated in the vacuole. Alanine utilization was not affected by l-methionine-d,l-sulfoximine or azaserine, but was strongly inhibited by aminooxyacetate. The cell extract contained enough alanine aminotransferase activity to account for the rate of alanine metabolism.