Efficient isolation of human metapneumovirus using MNT‐1, a human malignant melanoma cell line with early and distinct cytopathic effects

Isolation of human metapneumovirus (HMPV) from clinical specimens is currently inefficient because of the lack of a cell culture system in which a distinct cytopathic effect (CPE) occurs. The cell lines LLC‐MK2, Vero and Vero E6 are used for isolation of HMPV; however, the CPE in these cell lines is subtle and usually requires a long observation period and sometimes blind passages. Thus, a cell line in which an early and distinct CPE occurs following HMPV inoculation is highly desired by clinical virology laboratories. In this study, it was demonstrated that, in the human malignant melanoma cell line MNT‐1, obvious syncytium formation occurs shortly after inoculation with HMPV‐positive clinical specimens. In addition, the growth and efficiency of isolation of HMPV were greater using MNT‐1 than using any other conventional cell line. Addition of this cell line to our routine viral isolation system for clinical specimens markedly enhanced isolation frequency, allowing isolation‐based surveillance. MNT‐1 has the potential to facilitate clinical and epidemiological studies of HMPV.

     

Control of IkappaBalpha proteolysis by the ubiquitin-proteasome pathway

It has recently been determined that the proteolytic destruction of IkappaB (inhibitor of NF-kappaB) by the ubiquitin-proteasome system plays a key role in the immediate elimination of IkappaB from the IkappaB-(NF-kappaB) complex which allows nuclear translocation of free NF-kappaB, thus leading to activation of a multitude of target genes. The SCF(Fbw1) (composed of Skp1, Cul-1, Roc1, and Fbw1) complex, identified as an IkappaBalpha-E3 ligase, binds and ubiquitylates IkappaBalpha phosphorylated by IkappaB kinase that has been activated in response to extracellular signals. The generating poly-ubiquitin chain is finally recognized by the 26S proteasome for ultimate degradation. In this NF-kappaB signalling pathway, it becomes clear that the SCF(Fbw1) activity is enhanced by a ubiquitin-like protein NEDD8 (equivalent to Rub1) that modifies Cul-1 in a manner analogous to ubiquitylation, and consequently, IkappaBalpha proteolysis is induced. NEDD8 is a new regulator of the SCF ubiquitin-ligase, functioning as a covalent modifier for proteolytic targeting at a physiological level.     

Noncompetitive, reversible inhibition of aminoacylase-1 by a series of L-alpha-hydroxyl and L-alpha-fluoro fatty acids: ligand specificity of aspergillus oryzae and porcine kidney enzymes

L-lactate and L-beta-phenyllactate have been identified in the culture broth of Streptomyces sp. KY-11 as reversible noncompetitive inhibitors of Aspergillus oryzae aminoacylase-1 and porcine kidney aminoacylase I. A series of alpha-hydroxyl acids (DL-R-CH(OH)-COOH, R = Et, n-pro, n-butyl, n-pentyl, n-hexyl) also inhibited the two enzymes in reversible noncompetitive kinetics, and the inhibition potency (-log K(i)) increased with the increased hydrophobicity of the R group. The two eukaryotic enzymes showed distinct preferences to the ligand alpha-alkyl group, and the fungus enzyme was inhibited by L-beta-phenyllactate (R = benzyl) 10(3)-fold more potently than the mammalian enzyme. L-alpha-Fluoro-beta-phenyl-propionate and its D-isomer were used to show that the L-configuration of the alpha-substituent was important for potent inhibition of both the enzymes. The fungus aminoacylase-1 steeply decreased the affinity to alpha-fluoro- and alpha-hydroxy-n-caproate as pH was raised from 7 to 11, whereas the mammalian enzyme retained the affinity to these ligands under alkaline conditions. These results suggest that A. oryzae aminoacylase-1 has an acidic residue that interacts with -OH or -F, while the mammalian enzyme would have a basic residue that recognizes the alpha-substituents.     

Changes in the Amounts of the Molt-Inhibiting Hormone in Sinus Glands during the Molt Cycle of the American Crayfish, Procambarus clarkii

The purposes of this study are to determine the molt cycle of the American crayfish, Procambarus clarkii, and to quantify the amounts of the molt-inhibiting hormone (Prc-MIH) in the hemolymph and neurohemal sinus glands during the molt cycle of the American crayfish. The molt cycle was classified into six stages based on the changes in volumes of gastroliths in the stomach and ecdysteroid titers in the hemolymph. A sandwich-type enzyme immunoassay using specific antibodies raised against N-terminal and C-terminal segments of Prc-MIH was developed for the Prc-MIH assay. It is sensitive to as little as 0.5 fmol of Prc-MIH (3.3 x10(-12) M). In the hemolymph, no Prc-MIH could be detected at any of the molt stages tested. However, in the sinus gland, it was demonstrated that the amount of Prc-MIH changes in a molt-stage-specific manner during the molt cycle. It was particularly noteworthy that the initiation of a molting sequence (i.e., entering the early premolt stage) corresponded to the increase in Prc-MIH content in the sinus gland, because the finding is consistent with the hypothesis that crustaceans enter the premolt stage when the MIH secretion from the sinus gland is reduced or ceases.     

Survey of Spirometra erinaceieuropaei in frogs in Taiwan and its experimental infection in cats

Eighteen of 56 (32.1%) wild Rana limnocharis from central and south Taiwan were found to contain plerocercoids of Spirometra erinaceieuropaei. This is the first report of S. erinaceieuropaei infections in frogs in Taiwan, with the plerocercoids being recovered from the thigh and back muscles or under the skin. Other species of frogs examined, including nine wild R. latouchii, one wild Buergeria robustus and 110 cultured R. rugulosa were free of infection. The plerocercoids were orally inoculated into four cats; three of which were each given a single plerocercoid and one a dose of three plerocercoids. Daily faecal examination showed that two cats started shedding eggs of S. erinaceieuropaei on day 8 postinfection (PI) and the other two on day 10 PI. The highest eggs per gram and eggs per day for a single worm was found to be 428,000 and 14,416,000 respectively. Only the cat inoculated with three plerocercoids shed proglottids in its faeces during the 2 month observation period.     

Novel archaeal alanine:glyoxylate aminotransferase from Thermococcus litoralis

A novel alanine:glyoxylate aminotransferase was found in a hyperthermophilic archaeon, Thermococcus litoralis. The amino acid sequence of the enzyme did not show a similarity to any alanine:glyoxylate aminotransferases reported so far. Homologues of the enzyme appear to be present in almost all hyperthermophilic archaea whose whole genomes have been sequenced.     

Sclerosing polycystic adenosis of the left parotid gland: report of a case with fine needle aspiration cytology

BACKGROUND: Sclerosing polycystic adenosis (SPCA) of major salivary glands is a rare recently described entity. We report a case of SPCA of the left parotid gland, including the cytologic and histopathologic findings. CASE: A 20-year-old man presented with a left parotid mass that had been growing slowly for 3 years. Fine needle aspiration cytology showed many syncytial cell clusters of variable size and some ductal structures with an inflammatory background. The cells forming syncytial clusters were large and polygonal, with abundant, eosinophilic, granular or lacelike cytoplasm. Apocrine differentiation with decapitation secretion was commonly seen. The ductal cells had a relatively high nuclear/cytoplasmic ratio, with granular cytoplasm. Grossly, the 5-cm lesion was a discrete, pale, cystic nodule embedded within the parotid gland parenchyma. Microscopically, the lesion was a nonencapsulated, circumscribed mass of sclerotic and hyalinized, collagenous tissue with lymphoplasmacytic infiltration. Sclerosing adenosis and cystic ducts with frequent apocrinelike cells were commonly seen. Some acinar cells contained eosinophilic, intracytoplasmic granules of various sizes. CONCLUSION: The presence of syncytial clusters with apocrine metaplasia and ductal structures in a lymphoplasmacytic background should suggest a diagnosis of SPCA of a major salivary gland.