Morphology and immunotyping of AC-43 strain of Chlamydia pneumoniae isolated from a Japanese child]

A strain of Chlamydia pneumoniae (C. pneumoniae) which had been isolated from a Japanese child (AC-43) was examined morphologically and serologically using micro-immunofluorescent (micro-IF) test. Inclusions of AC-43 were stained by an indirect immunofluorescent method using C. pneumoniae specific monoclonal antibody. They were dense round inclusions which had been reported as a characteristic figure for C. pneumoniae. An elementary body (EB) of AC-43 was pear-shaped by electron micrograph, which was the same as previous reports for C. pneumoniae. We produced monoclonal antibodies using purified EB of AC-43 as antigen. Culture fluids of these clones reacted with C. pneumoniae antigens, but did not react with C. trachomatis or C. psittaci antigens by micro-IF tests. There was no difference in morphological and serological findings among standard strains and Japanese isolate of C. pneumoniae.     

Purification and characterization of human immunodeficiency virus type 1 nef gene product expressed by a recombinant baculovirus

We have constructed the recombinant baculovirus which expresses the human immunodeficiency virus type 1 negative factor (nef) gene. Spodoptera frugiperda cells infected with the recombinant virus produced a 27-kDa protein which reacted with rabbit antisera raised against a carboxy-terminal synthetic peptide of the Nef protein by immunoblot analysis. Labeling experiment showed that the recombinant Nef protein was myristoylated. The recombinant Nef protein was purified to near homogeneity by DEAE-Sephacel, phenyl-Sepharose 4B, blue-Sepharose, and Sephadex G-150 column chromatography. No detectable GTP binding activity was observed in the purified recombinant Nef product.     

Participation of rat liver cytochrome P450 2E1 in the activation of N-nitrosodimethylamine and N-nitrosodiethylamine to products genotoxic in an acetyltransferase-overexpressing Salmonella typhimurium strain (NM2009).

The possible roles of cytochrome P450 (P450) enzymes in the metabolic activation of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) by rat liver microsomes have been examined in a system containing the bacterial tester strain Salmonella typhimurium NM2009, a newly developed strain showing high O-acetyltransfer activities. The DNA-damaging activity could be determined by measuring expression of the umu gene in a plasmid containing the fused umuC-lacZ gene construct in the bacteria. The following lines of evidence support the view that both NDMA and NDEA are principally oxidized to reactive products by P450 2E1 in rat liver microsomes. First, NDMA and NDEA were activated by rat liver microsomes in a protein- and substrate-dependent manner and the former chemical was more active than the latter; both activities were induced in rats treated with P450 2E1 inducers such as ethanol, acetone and isoniazid and by starvation. Second, activation of NDMA and NDEA were both inhibited significantly by antibodies raised against rat P450 2E1 and by P450 2E1 inhibitors such as diethyldithiocarbamate and 4-methylpyrazole in rat liver microsomes. Finally, in reconstituted monooxygenase systems containing purified rat P450 enzymes, P450 2E1 gave the highest rates of the activation of both NDMA and NDEA; the addition of rabbit cytochrome b5 to the system caused about a 1.5-fold increase in both reactions. In separate experiments we also found that N-nitrosomethylacethoxymethylamine, a compound that reacts with DNA after ester cleavage, is more genotoxic in S.typhimurium NM2009 than in S.typhimurium NM2000, a strain that is defective in O-acetyltransferase activity. Part of the pathway involved in the activation of nitrosamines is suggested to be acetylation of alkyldiazohydroxides formed by P450 or acetylesterase, because the genotoxic activity of N-nitrosomethylacethoxymethylamine in S.typhimurium NM2009 could be inhibited by the O-acetyltransferase inhibitor pentachlorophenol. These results indicate that NDMA and NDEA are oxidized to gentoxoic products by rat liver microsomes and that a P450 2E1 enzyme plays a major role in the activation of these two potent carcinogens. The activation pathway of N-nitrosodialkylamines through acetylation by O-acetyltransferase has been proposed. This simple bacterial system for measuring genotoxicity should facilitate studies on the activation of N-nitroso alkylamines.     

Changes in water intake following hepatic vagotomy in young rats

Changes in water intake after hepatic vagotomy were examined in the body weights of 3 different rats (about 100, 200 and 280 g). The vagotomy reduced water intake only in the 100-g rats.     

Identification and viability assessment of dermatophytes infecting nail based on quantitative PCR of dermatophyte actin (ACT) mRNA]

The diagnosis and choice of treatment for dermatophytoses are usually based on the result of microscopic observation of hyphal elements and culture. However, false negative cultures have sometimes been encountered and appropriate timing of discontinuation of treatment has not been formulated. In this study, we attempted the identification and viability assessment of dermatophytes based on the quantitative measurement of dermatophyte actin (ACT) mRNA. An internal fragment of the ACT, 725 to 762 bp, was isolated by PCR from the genomic DNA of dermatophytes and sequenced. ACT intron-based primers were dermatophyte species-specific and primer pairs crossing the intron were dermatophyte genus-specific. The LightCycler (LC) instrument, employing the two-step RT-PCR/fluorescent hybridization system, was used to quantify the actin mRNA (ACT) of dermatophytes. A 669 bp ACT cDNA fragment was used as a quantification standard. Several mg of samples were collected from skin scales or nail plates before and after the treatment using oral terbinafine. The results indicated that quantification of ACT mRNA correlated with the results of culture and KOH examination and that copy numbers of dermatophyte ACT mRNA per mg sample decreased with progression of the therapy. This method comprises a sensitive (1 fg), specific, rapid (< 4 h) and quantitative assessment of the viability and identification of dermatophytes in skin tissue.     

Genetic alteration in carcinoid tumors of the lung.

Surgically resected specimens of 13 carcinoid tumors of the lung including nine typical carcinoids and four atypical carcinoids, and eight salivary gland type carcinomas (six mucoepidermoid carcinomas and two adenoid cystic carcinomas) were analyzed regarding p53 expression, loss of heterozygosity (LOH) in chromosome 3p, 9p, and K-ras mutation. The overexpression of p53 was identified in four atypical carcinoid tumors, one mucoepidermoid carcinoma, and one adenoid cystic carcinoma, however, none of typical carcinoids showed p53 immunoreactivity. LOH in 3p14 was demonstrated in three of seven informative cases in all tumors. LOH in 9p was demonstrated in two of five informative cases in all tumors. Two of three cases with LOH at 3p14 had a poor prognosis, one of which also had LOH at 9p. No mutation of the K-ras gene was observed in any of these tumors. These data thus indicate that p53 overexpression might distinguish atypical carcinoid tumors from typical tumors and might therefore be useful as an adjunct modality in the differential diagnosis of pulmonary carcinoid tumors. The presence of LOH at 3p14 or 9p may thus help to identify lung cancer patients with a poor prognosis.     

Ethylene vinylacetate copolymer particles dissolved in polyvinyl alcohol (2,000-mer) solution as an embolic material for vascular anomalies

We have prepared a new material for embolisation: ethylene vinylacetate copolymer dissolved in polyvinyl alcohol. When in contact with blood, polyvinyl alcohol rapidly becomes a soft gel, which is accompanied by wedging of the ethylene vinylacetate copolymer. We analysed the histopathology of intra-arterial microemboli in rats, after intracarotid injection of this material. We confirmed that it was applicable to embolisation for neurosurgical treatment.     

Intrathecal administration of N-methyl-D-aspartate receptor antagonist reduces the minimum alveolar anaesthetic concentration of isoflurane in rats

We have studied the effect of intrathecal administration of N-methyl-D- aspartate (NMDA) receptor antagonists on the minimum alveolar anaesthetic concentration (MAC) of isoflurane in rats. In Wistar rats fitted with indwelling intrathecal catheters, we determined the MAC of isoflurane after administration of a competitive NMDA receptor antagonist, APV (0.01, 0.1, 1.0, 10, 30 micrograms), a non-competitive NMDA receptor antagonist, MK801 (0.1, 1.0, 10, 30 micrograms). NMDA (0.01, 0.1, 1.0, 10, 30 micrograms) and saline. APV at all doses except 0.01 micrograms decreased MAC by 17.1-32% (P < 0.001 and P < 0.0001). Although MK801 at 10 and 30 micrograms reduced MAC by 24.3-31.7% (P < 0.001 and P < 0.0001), lower doses did not affect MAC. Intrathecal administration of NMDA reversed these decreases in MAC, but not to control values with APV 10 and 30 micrograms and MK801 30 micrograms. We suspect that NMDA and NMDA receptor antagonists play important roles in the spinal cord in determining the MAC of isoflurane.