Development and evaluation of a loop-mediated isothermal amplification method for the rapid detection of Chlamydophila pneumoniae.

We developed a loop-mediated isothermal amplification (LAMP) method to detect Chlamydophila pneumoniae infection. This assay exclusively amplified C. pneumoniae sequences and no cross-reactivity was observed for other Chlamydia species. The detection limit for this assay was found to be ten elementary bodies in 25 min, as observed in a real-time turbidimeter and electrophoretic analysis. The specificity of the LAMP reaction was confirmed by restriction endonuclease analysis, as well as direct sequencing of the amplified product. Among nasopharyngeal swab specimens from 120 patients with acute respiratory tract infections and 40 healthy individuals, the LAMP results showed 100% agreement with the results of real-time polymerase chain reaction (PCR) assays.

Evaluation of myocardial sympathetic nerve function in patients with mitral valve prolapse using iodine-123-metaiodobenzylguanidine myocardial scintigraphy.

Mitral valve prolapse (MVP) is closely related to myocardial sympathetic nerve function. This study evaluated the presence of impaired myocardial sympathetic nerve function by Iodine-123-metaiodobenzylguanidine (MIBG) scintigraphy in ten patients with MVP. For comparison, 15 healthy subjects without heart disease were investigated (control group). Single photon emission computed tomography (SPECT) and anterior planar myocardial scintigraphy were performed 15 min (initial images) and 3 hours (delayed images) after injection of MIBG (111 MBq). The location and degrees of reduced tracer uptake were evaluated. Myocardial MIBG uptake was quantified by uptake ratio of the heart (H) to upper mediastinum (M) on the anterior planar images (H/M). Percentage washout of MIBG in nine sectors of all oblique slices along the short-axis was calculated. The washout rates were higher at the inferoposterior and septal segments in patients with anterior leaflet prolapse, and at inferoposterior and lateral segments in patients with posterior leaflet prolapse. The bull's eye map showed increased washout rate in the apical and posteroseptal basal segments. There was no significant difference in the H/M ratio between MVP patients and the control group. These results indicate that MIBG can be used to evaluate localized myocardial sympathetic nerve function in MVP.

Iron-dependent regulation of the divalent metal ion transporter.

The first step in intestinal iron absorption is mediated by the H(+)-coupled Fe(2+) transporter called divalent cation transporter 1/divalent metal ion transporter 1 (DCT1/DMT1) (also known as natural resistance-associated macrophage protein 2). DCT1/DMT1 mRNA levels in the duodenum strongly increase in response to iron depletion. To study the mechanism of iron-dependent DCT1/DMT1 mRNA regulation, we investigated the endogenous expression of DCT1/DMT1 mRNA in various cell types. We found that only the iron responsive element (IRE)-containing form, which corresponds to one of two splice forms of DCT1/DMT1, is responsive to iron treatment and this responsiveness was cell type specific. We also examined the interaction of the putative 3'-UTR IRE with iron responsive binding proteins (IRP1 and IRP2), and found that IRP1 binds to the DCT1/DMT1-IRE with higher affinity compared to IRP2. This differential binding of IRP1 and IRP2 was also reported for the IREs of transferrin receptors, erythroid 5-aminolevulinate synthase and mitochondrial aconitase. We propose that regulation of DCT1/DMT1 mRNA by iron involves post-transcriptional regulation through the binding of IRP1 to the transporter's IRE, as well as other as yet unknown factors.

Novel forms of squamous cell carcinoma antigen transcripts produced by alternative splicing.

Squamous cell carcinoma antigen (SCCA) is a member of the ovalbumin serine protease inhibitor family, and the serum level of SCCA is a tumor marker of squamous cell carcinoma. Reverse transcription (RT)-PCR of the squamous cell carcinoma cell line showed the existence of a 156 base shorter transcript compared with that of SCCA1 cDNA. By inverse PCR, we cloned the full length cDNA of this SCCA (SCCA1b). Sequence analysis of the complete 1541 bp SCCA1b cDNA showed that it coded for 338 amino acids and had no typical signal sequence in the NH(2) terminus. The cDNA was expressed in Escherichia coli and the product was detected using Western blotting with antibodies against SCCA. Furthermore, RT-PCR of the full coding region of SCCA2 cDNA from cancer tissue showed the existence of a 63 base short transcript (SCCA2b). A comparison of SCCA1b and SCCA2b cDNA with the SCCA1 and SCCA2 genes showed that these messages were derived from each gene by an alternative splicing mechanism.

Cloning and characterization of adenylate kinase from Chlamydia pneumoniae.

Chlamydiae proliferate only within the infected host cells and are thought to be "energy parasites," because they take up ATP from the host cell as an energy source. In the present study, we isolated from Chlamydia pneumoniae the gene encoding adenylate kinase (AK). Using the enzyme produced in Escherichia coli, its properties were characterized. K(m) values for AMP and for ADP of the purified C. pneumoniae AK (AKcpn) were each 330 microm, which is significantly higher than the reported values of other AKs, whereas K(m) for ATP was 24 microm, which was rather lower than others. AKcpn contains 1 g atom of zinc/mol of 24,000-dalton protein. Mass spectrometric analysis of AKcpn and analysis of properties of mutated AKcpn strongly suggested that zinc is associated with four cysteine residues in the LID domain of the enzyme. The apo-AKcpn that lost zinc retained AK activity, although K(m) for AMP of apo-AKcpn increased about 2-fold and V(max) decreased about one-half from that of holo-AKcpn. The apo-AKcpn was more thermolabile and sensitive to trypsin digestion than the holo-AKcpn. Moreover, the recovery in vitro of the AK activity during the renaturation process of the denatured apo-AKcpn was dependent on zinc. A mutated protein in which cysteine residues in the LID domain were substituted by other amino acids lost both zinc and enzyme activity. The mutated protein was more sensitive to protease than the apo-AKcpn. These results indicate that zinc in AKcpn, although not essential for the catalysis, stabilizes the enzyme and probably plays a crucial role in proper folding of the protein. Furthermore, the catalytic properties of AKcpn suggest a distinctive regulatory mechanism in the metabolism compared with AKs in other organisms.

Two putative tumor suppressor genes on chromosome arm 8p may play different roles in prostate cancer.

Although loss of heterozygosity (LOH) on the short arm of chromosome 8 has been frequently observed in human prostate cancer, the relationship between LOH and clinical background is poorly understood. Fluorescence in situ hybridization (FISH) was employed to evaluate the chromosomal deletion on 8p in 42 prostate cancers using a centromeric probe for chromosome 8, in combination with 4 cosmid probes spanning 8p12 to 8p22. Deletions for at least one locus on the 8p were observed in 29 (69.0%) tumors. The most frequently deleted regions were 8p22 (54.8%) and 8p21.3 (52.4%), in almost the same frequency. The second most frequently deleted region was 8p21.1-p21.2 (38.1%). Deletions of 8p22 and 8p21.3 significantly correlated with tumor grade (P=0.0034, Fisher's exact probability test). A significantly higher frequency of the deletion on 8p21.1-p21.2 was observed in advanced prostate cancer (beyond capsular penetration or positive nodal metastases) than in localized prostate cancer (P=0.0033). In particular, deletion of 8p21.1-p21.2 was more frequently observed in the cases with lymph node metastases than without them (P=0.0029). No clinicopathological parameters had significant relation to deletions on 8p12. These results suggest that deletions on 8p22-p21.3 play an important role in tumor differentiation, while an 8p21.1-p21.2 deletion plays a role in the progression of prostate cancer.

Specific detection and quantitation of SCC antigen 1 and SCC antigen 2 mRNAs by fluorescence-based asymmetric semi-nested reverse transcription PCR.

Squamous cell carcinoma antigen (SCCA) is expressed in normal squamous epithelia and malignant squamous cell tissues. The serum level of SCCA has been used to evaluate treatment efficacy, clinical course of disease, and recurrence. SCCA is produced by at least two genes (SCCA1 and SCCA2); both of them have been located on chromosome 18q21.3. It has been difficult to examine the expression levels of SCCA1 and SCCA2 mRNAs separately because of their high homology at nucleotide level. In the present study, asymmetric semi-nested reverse transcription PCR, based on the principle of fluorescence energy transfer, enabled to quantitate the copy numbers of both SCCA1 and SCCA2 mRNAs. Using this method, the expression levels of these mRNAs were evaluated in normal and malignant squamous tissues. The copy number of SCCA2 mRNA was higher in malignant tissues than in normal tissues, while those of SCCA1 mRNA did not significantly differ between normal and malignant tissues. These data indicate that specific quantitation of the expression level of SCCA2 mRNA may be useful for the diagnosis and management of patients with squamous cell carcinoma.

Comparison of outer membrane protein genes omp and pmp in the whole genome sequences of Chlamydia pneumoniae isolates from Japan and the United States.

Chlamydia pneumoniae is a widespread pathogen of the respiratory tract that is also associated with atherosclerosis. The whole genome sequence was determined for a Japanese isolate, C. pneumoniae strain J138. The sequence predicted a variety of genes encoding outer membrane proteins (OMPs) including ompA and porB, another 10 predicted omp genes, and 27 pmp genes. All were detected in the whole genome sequence of strain CWL029, a strain isolated and sequenced in the United States. A comparative study of the OMPs of the two strains revealed a nucleotide sequence identity of 89.6%-100% (deduced amino acid sequence identity, 71.1%-100%). The overall genomic organization and location of genes are identical in both strains. Thus, a few unique sequences of the OMPs may be essential for specific attributes that define the differential biology of two C. pneumoniae strains.

Comparison of whole genome sequences of Chlamydia pneumoniae J138 from Japan and CWL029 from USA.

Chlamydia pneumoniae is a widespread pathogen of humans causing pneumonia and bronchitis. There are many reports of an association between C.PNEUMONIAE: infection and atherosclerosis. We determined the whole genome sequence of C.PNEUMONIAE: strain J138 isolated in Japan in 1994 and compared it with the sequence of strain CWL029 isolated in the USA before 1987. The J138 circular chromosome consists of 1 226 565 nt (40.7% G+C) with 1072 likely protein-coding genes that is 3665 nt shorter than the CWL029 genome. Plasmids, phage- or transposon-like sequences were not identified. The overall genomic organization, gene order and predicted proteomes of the two strains are very similar, suggesting a high level of structural and functional conservation between the two unrelated isolates. The most conspicuous differences in the J138 genome relative to the CWL029 genome are the absence of five DNA segments, ranging in size from 89 to 1649 nt, and the presence of three DNA segments, ranging from 27 to 84 nt. The complex organization of these 'different zones' may be attributable to a unique system of recombination.

Human NRAMP2/DMT1, which mediates iron transport across endosomal membranes, is localized to late endosomes and lysosomes in HEp-2 cells.

NRAMP2 (natural resistance-associated macrophage protein 2)/DMT1 (divalent metal transporter 1) is a divalent metal transporter conserved from prokaryotes to higher eukaryotes that exhibits an unusually broad substrate range, including Fe(2+), Zn(2+), Mn(2+), Cu(2+), Cd(2+), Co(2+), Ni(2+), and Pb(2+), and mediates active proton-coupled transport. Recently, it has been shown that the microcytic anemia (mk) mouse and the Belgrade (b) rat, which have inherited defects in iron transport that result in iron deficiency anemia, have the same missense mutation (G185R) in Nramp2. These findings strongly suggested that NRAMP2 is the apical membrane iron transporter in intestinal epithelial cells and the endosomal iron transporter in transferrin cycle endosomes of other cells. To investigate the cellular functions of NRAMP2, we generated a polyclonal antibody against the N-terminal cytoplasmic domain of human NRAMP2. The affinity-purified anti-NRAMP2 N-terminal antibody recognized a 90-116-kDa membrane-associated protein, and this band was shifted to 50 kDa by deglycosylation with peptide N-glycosidase F. Subcellular fractionation revealed that NRAMP2 co-sedimented with the late endosomal and lysosomal membrane proteins and LAMP-1 (lysosome-associated membrane protein 1), but not with the transferrin receptor in early endosomes. The intracellular localization of endogenous NRAMP2 and recombinant green fluorescent protein (GFP)-NRAMP2 was examined by immunofluorescence staining and by native fluorescence of GFP, respectively. Both endogenous and GFP-NRAMP2 were detected in vesicular structures and were colocalized with LAMP-2, but not with EEA1 (early endosome antigen 1) or the transferrin receptor. These results indicated that NRAMP2 is localized to the late endosomes and lysosomes, where NRAMP2 may function to transfer the endosomal free Fe(2+) into the cytoplasm in the transferrin cycle.

Dynamics of actin and alpha-actinin in nascent myofibrils and stress fibers.

Actin labeled with fluorescein isothiocyanate (FITC) and alpha-actinin labeled with rhodamine (rh) were co-injected into chick embryonic cardiac myocytes and fibroblasts. In cardiomyocytes, FITC-actin was distributed in nonstriated lines, linearly arranged punctate structures with short intervals, and cross-striated bands with regular sarcomeric intervals. rh-alpha-Actinin was seen to be distributed in the same pattern in the former two portions, and in the center of each striation in the latter portion. Photobleaching of structures incorporated with these fluorescent analogs revealed that the fluorescent recovery rate of actin decreased in the order of nonstriated > punctated > striated portions, while that of alpha-actinin was low and stable at all portions. During the transition phase from punctate to regular sarcomere structures of these proteins, short spaced alpha-actinin dots adjoined each other and aligned with Z bands of neighboring myofibrils. It appears that both the difference in exchangeability between actin and alpha-actinin molecules and the movement of alpha-actinin dots during this phase of myofibrillogenesis are related to sarcomere lengthening and I-Z-I brush formation; adjoining dots of low-exchangeable alpha-actinin may provide favorable situations for exchangeable actin molecules in filaments to elongate and/or rearrange. In fibroblasts, both FITC-actin and rh-alpha-actinin formed nonstriated lines. In these cells, exchangeabilities of both proteins were high and similar in rate. This seems to indicate that stress fibers are constantly exchanging their components for motile and other vital functions of these cells. The high exchangeabilities of both proteins in stress fibers showthat these fibers are clearly different from nonstriated, stress-fiber like structures of nascent myofibrils.

[An analysis of nutritional status and pulmonary hypertension in patients with sequela of pulmonary tuberculosis and chronic obstructive pulmonary disease].

The prognostic value of hypercapnia and/or pulmonary hypertension differs in patients with sequela of pulmonary tuberculosis (TBseq) and those with chronic obstructive pulmonary disease (COPD) who are receiving home oxygen therapy (HOT). In an attempt to identify the factors, if any, that might explain this difference, we first compared nutritional status, respiratory function test results, dyspnea indexes, and other data for hypercapnic patients (PaCO2 > or = 45 Torr) and normocapnic patients (PaCO2 < 45 Torr) receiving HOT. Second, we examined the relationship between the degree of pulmonary hypertension and several respiratory function parameters for patients in each disease category. In 44 patients with TBseq, nutritional status estimated by body mass index and serum albumin was significantly better in the hypercapnic patients than in the normocapnic patients. However, this difference was not observed in 37 patients with COPD. In 30 patients with TBseq, the degree of pulmonary hypertension correlated significantly only with PaO2; in 32 patients with COPD, however, significant correlations were observed not only with PaO2 but also with PaCO2, %VC, and FEV1. These differences distinguishing groups of patients with the 2 diseases may provide an explanatory basis for the difference in prognostic value of hypercapnia and/or pulmonary hypertension in patients receiving HOT.

Functional analysis of the human NRAMP family expressed in fission yeast.

The Bcg/Ity/Lsh locus in the mouse genome regulates macrophage activation for antimicrobial activity against intracellular pathogens, and the positional cloning of this locus identified the Nramp1 (natural resistance-associated macrophage protein) gene. Nramp2 was initially isolated as a homologue of Nramp1. Recently, the rat divalent metal transporter DMT1 was identified electrophysiologically, and was found to be an isoform of Nramp2, a mutation which was subsequently identified in rats suffering from hereditary iron-deficiency anaemia. Despite the 64% amino acid sequence identity of Nramp1 and Nramp2, no divalent metal transport activity has yet been detected from Nramp1, and the function of Nramp1 on the molecular level is still unclear. To investigate the divalent metal transport activity of NRAMP molecules, we constructed four chimeric NRAMP genes by swapping the domains of human NRAMP1 and NRAMP2 with each other. The functional characteristics of wild-type NRAMP1, NRAMP2 and their chimeras were determined by expression in the divalent metal transporter-disrupted strain of fission yeast, pdt1Delta, and we analysed the divalent metal transport activity by complementation of the EGTA- and pH-sensitive phenotype of pdt1Delta. Replacement of the N-terminal cytoplasmic domain of NRAMP2 with the NRAMP1 counterpart resulted in inactive chimeras, indicating that the functional difference between NRAMP1 and NRAMP2 is located in this region. However, results obtained with the reverse construct and other chimeras indicated that these regions are not solely responsible for the differences in EGTA- and pH-sensitivity of NRAMP1 and NRAMP2. These findings indicate that NRAMP1 itself cannot represent the divalent metal transport activity in S. pombe and the additional protein segments of the molecules located elsewhere in NRAMP1 are also functionally distinct from their NRAMP2 counterparts.

Dinucleotide repeat polymorphism in the third intron of the NRAMP2/DMT1 gene.

Nramp1 (natural resistance-associated macrophage protein 1), encoding a polytropic integral membrane protein, was isolated as a candidate of the mouse Lsh/Ity/Bcg locus, which regulates macrophage activation for anti-microbial activity against intracellular pathogens. The NRAMP2 gene was cloned from human genome as a homologue of NRAMP1. We found a polymorphic dinucleotide repeat in the third intron of the NRAMP2 gene. This polymorphism will be a useful genetic marker to study disease associated with susceptibility to infection with intracellular pathogens.

Structural analysis of human SCC antigen 2 promoter.

The squamous cell carcinoma antigen (SCCA) has been used as a circulating tumor marker for the management of squamous cell carcinoma. SCCA consists of a small gene family of at least two in human genome (SCCA1 and SCCA2), which are tandemly arrayed on chromosome 18q21.3 and share 92% identical residues. SCCA expressions are tightly controlled in a tissue-specific manner. To investigate the role of SCCA2 in the cancer cells, we first isolated the human genomic clones, containing the promoter region of SCCA2 gene, and determined the nucleotide sequence surrounding the exon 1. The transcription start site was mapped by primer extension analysis, and a putative TATA box element was found in the 5'-flanking region. Other putative regulatory sequences, which include Ets binding sequence, NF-IL6 binding sequence and IRE consensus sequence, were also found in the region. Analysis of luciferase reporter gene expression in transient transfection showed that the promoter region of SCCA2 gene was located within the region from -424 to +47.

Human natural resistance-associated macrophage protein 2: gene cloning and protein identification.

The Lsh/Ity/Bcg locus in the mouse genome regulates macrophage activation for antimicrobial activity against intracellular pathogens, and mouse Nramp1 (natural resistance-associated macrophage protein) gene was isolated as its candidate. The human NRAMP1 gene was subsequently isolated and its gene product was identified in macrophage/monocyte cells. Recently, a second Nramp gene, Nramp2, was found in mouse and human genomes. In the present study, we report the cloning and characterization of the human NRAMP2 gene, which is approximately 42 kb in length, containing 16 exons. The transcription start site was determined by 5'-RACE method, and the promoter was located between -246 bp to 145 bp in a region relative to the transcription start site, able to drive the luciferase reporter gene in HeLa cells. We also raised a polyclonal antibody against the glutathione S-transferase fusion protein containing the NH2-terminal 86 amino acids of human NRAMP2. The protein product of the human NRAMP2 gene is apparently present in human cultured cell lines as a 64 kDa protein recognized by this antibody, which is consistent with the molecular mass deduced from the human NRAMP2 cDNA.

Histamine H2-receptor-mediated nitric oxide release from porcine endothelial cells.

The involvement of histamine-receptor subtypes in histamine-induced release of nitric oxide (NO) from porcine aortic endothelial cells was studied. NO release was measured directly by using an NO electrode and by electron paramagnetic resonance (EPR) spin trapping. NO release induced by histamine (200 microM) was reduced in the presence of 2 microM cimetidine, an H2-receptor antagonist, but not altered by 2 microM pyrilamine, an H1-receptor antagonist. Histamine-induced NO release was significantly reduced by the addition of 20 microM of the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), a membrane-permeable antagonist of cyclic adenosine monophosphate (cAMP). Application of 100 microM forskolin, an activator of adenylate cyclase, induced NO release from porcine aortic endothelial cells. Fura-2 acetoxymethylester (fura-2/AM) studies showed that addition of 100 microM histamine did not produce any significant increase in the use of free concentration of intracellular Ca2+. These results suggest that in porcine aortic endothelial cells, NO-mediated vasodilation might be caused by production of cAMP initiated through the histamine H2-receptor.