Expression of NO-synthase in cells of foreign-body and BCG-induced granulomata in mice: influence of L-NAME on the evolution of the lesion

Plasmodium inui (Halberstaedter and von Prowazek, 1907), a malarial parasite of Old World monkeys that occurs in isolated pockets throughout the Celebes, Indonesia, Malaysia, and the Philippines, has traditionally been considered to be related more closely to Plasmodium malariae of humans (and its primate counterpart Plasmodium brasilianum), than to other primate Plasmodium species. This inference was made in part because of the similarities in the periodicities or duration of the asexual cycle in the blood, the extended sporogonic cycle, and the longer period of time for development of the pre-erythrocytic stages in the liver. Both P. inui and P. malariae have quartan (72 hr) periodicities associated with their asexual cycle, whereas other primate malarias, such as Plasmodium fragile and Plasmodium cynomolgi, are associated with tertian periodicities (48 hr), and Plasmodiumn knowlesi, with a quotidian (24 hr) periodicity. Phylogenetic analyses of portions of orthologous small subunit ribosomal genes reveal that P. inui is actually more closely related to the Plasmodium species of the "vivax-type" lineage than to P. malariae. Ribosomal sequence analysis of many different, geographically isolated, antigenically distinct P. inui isolates reveals that the isolates are nearly identical in sequence and thus members of the same species.     

Comparison of circumsporozoite proteins from avian and mammalian malarias: biological and phylogenetic implications

The circumsporozoite (CS) protein of malaria parasites (Plasmodium) covers the surface of sporozoites that invade hepatocytes in mammalian hosts and macrophages in avian hosts. CS genes have been characterized from many Plasmodium that infect mammals; two domains of the corresponding proteins, identified initially by their conservation (region I and region II), have been implicated in binding to hepatocytes. The CS gene from the avian parasite Plasmodium gallinaceum was characterized to compare these functional domains to those of mammalian Plasmodium and for the study of Plasmodium evolution. The P. gallinaceum protein has the characteristics of CS proteins, including a secretory signal sequence, central repeat region, regions of charged amino acids, and an anchor sequence. Comparison with CS signal sequences reveals four distinct groupings, with P. gallinaceum most closely related to the human malaria Plasmodium falciparum. The 5-amino acid sequence designated region I, which is identical in all mammalian CS and implicated in hepatocyte invasion, is different in the avian protein. The P. gallinaceum repeat region consists of 9-amino acid repeats with the consensus sequence QP(A/V)GGNGG(A/V). The conserved motif designated region II-plus, which is associated with targeting the invasion of liver cells, is also conserved in the avian protein. Phylogenetic analysis of the aligned Plasmodium CS sequences yields a tree with a topology similar to the one obtained using sequence data from the small subunit rRNA gene. The phylogeny using the CS gene supports the proposal that the human malaria P. falciparum is significantly more related to avian parasites than to other parasites infecting mammals, although the biology of sporozoite invasion is different between the avian and mammalian species.     

Hyperlactatemia during acute severe asthma

The prevalence of the four human malaria parasites was investigated among malaria patients at northern, central and southern towns in Thailand along the border with Myanmar between September 1995 and May 1996. Thin smears obtained from 548 Thai and Burmese patients were reviewed by an acridine orange staining method, and many mixed infections with two to four species, including P. malariae and P. ovale, were detected. These diagnostic results were compared with those by two PCR-based diagnoses, microtitre plate hybridization (MPH) and a nested PCR method, both of which targets the same, species-specific regions in the 18S rRNA genes. In both PCR diagnoses, many P. malariae and P. ovale infections were also detected. Detection sensitivity of P. malariae infection was higher in nested PCR than MPH, and a total prevalence of P. malariae infection estimated by nested PCR reached 24.3% (133/548). In 16 of them, the size of PCR products amplified by the P. malariae-specific primer was about 20-bp shorter than the expected size of 115-bp. Four of 16 possessed two different bands with normal and shorter sizes, suggesting that P. malariae isolates may be separated into two types, and that those with shorter products may be new variant form (s) with a nucleotide deletion in the target region. On the other hand, 21 P. ovale infections (3.8%) were detected by nested PCR, but four of them were MPH-negative because of the sequence variation at the probe region. These results indicated that the prevalence of P. malariae and P. ovale along the Thai-Myanmar border may be substantially higher than previously reported.     

An unusual pattern of mutation in the duplicated portion of PKD1 is revealed by use of a novel strategy for mutation detection

The gene for the most common and severe form of autosomal dominant polycystic kidney disease, PKD1, encodes a 14 kb mRNA that is predicted to result in an integral membrane protein of 4302 amino acids. The major challenge faced by researchers attempting to complete mutation analysis of the PKD1 gene has been the presence of several homologous loci also located on chromosome 16. Because the sequence of PKD1 and its homologs is nearly identical in the 5' region of the gene, most traditional approaches to mutation analysis cannot distinguish sequence variants occurring uniquely in PKD1. Therefore, only a small number of mutations have been identified to date and these have all been found in the 3', unique portion of the gene. In order to begin analysis of the duplicated region of PKD1, we have devised a novel strategy that depends on long-range PCR and a single gene-specific primer from the unique region of the gene to amplify a PKD1-specific template that spans exons 23-34. This 10 kb template, amplified from genomic DNA, can be employed for mutation analysis using a wide variety of sequence-based approaches. We have used our long-range PCR strategy to begin screening for sequence variants with heteroduplex analysis, and several affected individuals were discovered to have clusters of base pair substitutions in exons 23 and 25. In two patients, these changes, identified in exon 23, would be predicted to result in multiple amino acid substitutions in a short stretch of the protein. This clustering of base pair substitutions is unusual and suggests that mutation may result from unique structural features of the PKD1 gene.     

The prevalence of Chlamydia pneumoniae in atherosclerotic and nonatherosclerotic blood vessels of patients attending for redo and first time coronary artery bypass graft surgery

The current genetic strategies used to identify Tropheryma whippelii, the putative agent of Whipple's disease, are based on PCR-mediated amplification of a part of its 16S rRNA gene (16S rDNA). Because there is very little intraspecies variation in these molecules, they are not suitable as targets for epidemiologic investigations. However, the intergenic spacer region between the 16S and 23S rDNAs is usually much more variable and has repeatedly been used for epidemiologic purposes. We have therefore amplified the spacer region of T. whippelii directly from clinical specimens from nine independent Swiss patients with Whipple's disease by PCR with primers complementary to the 3' and 5' ends of the 16S and 23S rDNAs, respectively. The amplicons were directly sequenced and the sequences were compared to the T. whippelii reference sequence in GenBank/EMBL (accession no. X99636). Complete sequence homogeneity was found between the samples from our nine patients; the spacer sequence was also identical to the reference sequence. However, the sequences corresponding to the 3' and 5' ends of the 16S and the 23S rDNAs of T. whippelii, respectively, differed from the respective sequences in GenBank/EMBL. The same sequence found in our patients was then found in a sample from the German patient from which the published sequence had been derived. We conclude that the 16S-23S rDNA spacer region seems to be very conserved in T. whippelii and that the respective reference entry in public databases should be revised.     

Cloning and characterization of the human and Caenorhabditis elegans homologs of the Saccharomyces cerevisiae MSH5 gene

Genetic variation among malaria parasites has important consequences with regard to drug resistance, pathogenicity, immunity, transmission, and speciation. In this regard, malaria parasites have been shown to display a high degree of inter- and intra-species genetic divergence. The nuclear genomes of Plasmodium falciparum, Plasmodium yoelii, and Plasmodium gallinaceum are vastly divergent yet share a similar codon usage and total A/T content of approximately 82%. This is in contrast to other primate-specific species including P. vivax which have an A/T content of approximately 67%. To assess the effects of this evolutionary divergence on the conservation of gene content, organization, and codon usage in the mitochondrial DNA (mtDNA) of malaria parasites, we have cloned and sequenced the mitochondrial genome of Plasmodium vivax, and compared it with the mtDNAs of P. falciparum, P. yoelii, and P. gallinaceum. The P. vivax mitochondrial genome was found to be 5990 base pairs in length, and displayed a gene organization identical to that of P. falciparum, P. yoelii, and P. gallinaceum. Furthermore, there was a remarkable 90% conservation of sequence identity between the mitochondrial genomes of all four species. As an example of intra-species conservation, comparison of mtDNAs from two independently cloned P. falciparum isolates, Malay Camp and C10, revealed only a single nucleotide substitution. A/T content of the P. vivax mitochondrial genome was found to be identical to other species of Plasmodium, hence, we have postulated that the mitochondrial genomes of malaria parasites were refractory to the evolutionary shifts in nucleotide content seen among the nuclear genomes of malaria parasites. Among different Plasmodium species, the second position of mitochondrial codons were found to be the least prone to substitutions and displayed a significant bias in pyrimidines. These aspects of mitochondrial codon usage were distinct from the nuclear genome and may reflect functional aspects of decoding by the mitochondrial translational system.     

The short 3''-end region of complementary DNAs as PCR-based polymorphic markers for an expression map of the mouse genome

All of the protein-encoding exons and the 3' flanking region of the human decorin gene have been cloned and partially sequenced. The locations of the intron-exon junctions within the coding portion of the gene were identical to those found for the homologous human gene, biglycan. The sizes of the introns in the decorin gene, however, were substantially larger than those of the same introns of the biglycan gene. Portions of introns 1, 2, and 3 as well as exon 1 were not found during our extensive screening process. The 5' end of intron 2 was found to have an AG-rich region followed immediately by a CT-rich region. Furthermore, the 5' end of intron 3 was very rich in thymidine, whereas the 3' end of intron 7 was rich in adenosine. Several cDNA clones constructed from cultured human bone cell mRNA were found to contain a different sequence at the 5' end compared to that previously published for mRNA from a human embryonic fibroblast cell line. We were also unable to find the alternate 3' flanking region of the previously published cDNA sequence. We have mapped the human decorin gene by in situ methods to chromosome 12q21.3.     

Citrus viroid Ia is a derivative of citrus bent leaf viroid (CVd-Ib) by partial sequence duplications in the right terminal region

Nucleotide sequences of group I citrus viroids Ia (CVd-Ia) and citrus bent leaf viroid (CBLVd, formerly designated CVd-Ib) isolated from citrus plants in Japan, the Philippines and China have been determined. Citrus samples in Japan and the Philippines contained CVd-Ia, which consists of 328 nucleotides(nt). Although 10 nt longer than the type CBLVd-225A in Israel they share 94% identity in overall nucleotide sequence. The Philippines sample also contained a 329-nt long CVd-Ia sequence variant, in which one base insertion and three substitutions were observed. A citrus in China contained CBLVd, which consists of 318 nt and shares 98% identity to CBLVd-225A. CVd-Ia was clearly separated from CBLVd by two 5-nt insertions located in upper (5'-AGCUG-3') and the lower (5'-CUUCU-3') strand of the right terminal region (which is also designated T2 domain) in rod-like secondary structure. Since both of the additional 5-nt sequences are similar to the adjacent sequences (5'-AGUUG-3' and 5'-CUUCU-3'), we hypothesize that CVd-Ia is a derivative of CBLVd caused by partial sequence duplications and substitutions taking place in the right terminal region.     

Functional neuroimaging of hallucinations in schizophrenia: toward an integration of bottom-up and top-down approaches

Phenol sulfotransferases catalyze the transfer of a sulfonate moiety from 3'-phosphoadenosyl 5'-phosphosulfate to a phenolic group of lipophylic substrates to generate soluble sulfate esters. Using a phenol sulfotransferase cDNA as probe to screen a human leukocyte genomic DNA library constructed in lambda EMBL3, we obtained a clone containing a complete gene sequence. Comparison of the gene sequence with that of the corresponding cDNAs, namely phenol-sulfating phenol sulfotransferase (P-PST) or thermostable sulfotransferase (TS-PST), and human aryl sulfotransferase 1 and 2 (HAST1 and HAST2) indicates that the gene possesses eight short exons separated by seven introns included in approximately 5 kb. HAST2 has a different 5' untranslated sequence, and thus is encoded by a different mRNA species. While the nucleotide sequence corresponding to the 5' noncoding region of P-PST (TS-PST and HAST1) is included in the exon I, the 5' untranslated sequence of HAST2 is located in the beginning of exon IIa. The remaining sequence in exon II that is identical to both P-PST and HAST2 was termed exon IIb. Exons III to VIII, which cover the coding region and the 3' untranslated region, are almost identical in all types of PST or AST cDNAs. These results suggest that the phenol sulfotransferase gene possesses two alternate promoters that drive the expression of the two different mRNA species in a tissue-specific manner. Transfection of chloramphenicol acetyl transferase (CAT) reporter gene vectors containing the 5'-flanking sequence upstream from exon I and exon II, respectively, in transformed human embryonal kidney (293) cells indicate that both sequences possess promoter activity with higher activity for promoter 1. RNA blot analysis indicates that human phenol sulfotransferase gene is expressed in kidney, liver, lung, leukocyte, colon, small intestine, and spleen.     

A novel gene expressed in rat ameloblasts codes for proteins with cell binding domains

Two variants of an mRNA sequence are identified that are expressed at high levels in rat ameloblasts during the formation of the enamel matrix. The sequences contain open reading frames for 407 and 324 amino acid residues, respectively. The encoded proteins, which we call amelins, are rich in proline, glycine, leucine, and alanine residues and contain the peptide domain DGEA, an integrin recognition sequence. The sequences coding for the C-terminal 305 amino acid residues, the 3' nontranslated part, and a microsatellite repeat at the nontranslated 5' region are identical in both mRNA variants. The remaining 5' regions contain 338 nucleotides unique to the long variant, 54 common nucleotides, and 46 nucleotides present only in the short variant. Eleven nucleotides have the potential to code for 5 amino acids of both proteins in different reading frames. The reading frame of the longer variant includes codons for a typical N-terminal signal peptide. The amelins are likely to be constituents of the enamel matrix and the only proteins that have so far been implicated in binding interactions between the ameloblast surface and its extracellular matrix.     

Alternative splicing of a Caenorhabditis elegans gene produces two novel inhibitory amino acid receptor subunits with identical ligand binding domains but different ion channels

Two full-length cDNAs, gbr-2A and gbr-2B, encoding inhibitory amino acid receptor subunits have been amplified and cloned from Caenorhabditis elegans mRNA. The 5' 732 bp of the two cDNAs, encoding 237 amino acids, are identical. The 3' 758 bp of the gbr-2B cDNA are present within the 3' untranslated region of the gbr-2A clone. As a result, the two cDNAs are predicted to encode subunits which share a common extracellular N-terminal sequence of 237 amino acids, but different, though closely related, C-terminal sequences which include four predicted membrane-spanning regions. A search of the EMBL database revealed that the sequences of the two subunits are most closely related to the alpha-subunit of the C. elegans avermectin receptor. Northern blot analysis showed the presence of two related mRNAs of approximately 2.2 and 1.5 kb in a developmentally mixed population of C. elegans. The genomic DNA sequence confirms that both mRNAs were transcribed from the same gene, gbr-2, suggesting that the closely related 3' sequences have arisen as a result of a partial gene duplication event. We propose that C. elegans is utilising alternative splicing to generate receptor subunits with identical extracellular, ligand-binding domains but different transmembrane, channel forming domains.     

Complete cDNA sequence, genomic structure, and chromosomal localization of the LPA receptor gene, lpA1/vzg-1/Gpcr26

The lpA1/Gpcr26 locus encodes the first cloned and identified G-protein-coupled receptor that specifically interacts with lysophosphatidic acid. A murine full-length cDNA of size consistent with that seen on Northern blots (3.7 kb) was determined using 3' rapid amplification of cDNA ends. Analysis of genomic clones revealed that the gene is divided into five exons, with one intron inserted in the coding region for transmembrane domain VI and one exon encoding the divergent 5' sequence in another published cDNA clone variant (orphan receptor mrec1.3). This structure differs from the intronless coding region for a homologous receptor, Edg1, but is identical to another more similar orphan receptor (lpA2) that has been deposited with GenBank. Using backcross analysis, both exons 1 and 4 mapped to a proximal region of murine Chromosome 4 indistinguishable from the vacillans gene. Exon 4 also mapped to a second locus on proximal Chromosome 6 in Mus spretus, and this partial duplication was confirmed by Southern blot. The genomic structure indicates a distinct, divergent evolutionary lineage for the vzg-1/lpA1 subfamily of receptors compared to those of homologous orphan receptor genes.     

Improved preparation of Xenopus oocytes for patch-clamp recording

The testis-determining gene SRY (sex determining region, Y) is located on the short arm of the Y chromosome and consists of a single exon, the central third of which is predicted to encode a conserved motif with DNA binding/bending properties. We describe the screening of 26 patients who presented with 46,XY partial or complete gonadal dysgenesis for mutations in both the SRY open reading frame (ORF) and in 3.8 kb of Y-specific flanking sequences. DNA samples were screened by using the fluorescence-assisted mismatch analysis (FAMA) method. In two patients, de novo mutations causing complete gonadal dysgenesis were detected in the SRY ORF. One was a nonsense mutation 5' to the HMG box, whereas the other was a missense substitution located at the C terminus of the conserved motif and identical to one previously detected in an unrelated patient. In addition, two Y-specific polymorphisms were found 5' to the SRY gene, and a sequence variant was identified 3' to the SRY polyadenylation site. No duplications of the DSS region in 20 of these patients were detected.