Risk factors for Nipah virus encephalitis in Bangladesh.

Nipah virus (NiV) is a paramyxovirus that causes severe encephalitis in humans. During January 2004, twelve patients with NiV encephalitis (NiVE) were identified in west-central Bangladesh. A case-control study was conducted to identify factors associated with NiV infection. NiVE patients from the outbreak were enrolled in a matched case-control study. Exact odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by using a matched analysis. Climbing trees (83% of cases vs. 51% of controls, OR 8.2, 95% CI 1.25-infinity) and contact with another NiVE patient (67% of cases vs. 9% of controls, OR 21.4, 95% CI 2.78-966.1) were associated with infection. We did not identify an increased risk for NiV infection among persons who had contact with a potential intermediate host. Although we cannot rule out person-to-person transmission, case-patients were likely infected from contact with fruit bats or their secretions.

Mitochondrial DNA and Y chromosome-specific polymorphisms in the Seminole Tribe of Florida.

Mitochondrial DNA (mtDNA) sequence variation was examined in 37 Seminoles from Florida by polymerase chain reaction amplification and high resolution restriction endonuclease analysis. The Y chromosome TaqI restriction fragment length polymorphisms detected by the probes 49a, 49f, and 12f2 were examined in the 26 males of this group. Analysis of the mtDNA revealed that all four Native American haplogroups (A, B, C and D) were present in the Seminoles encompassing about 95% of the Seminole mtDNAs. No European mtDNAs were found among the Seminoles, but two mtDNAs (about 5%) were members of the African-specific haplogroup L1, thus indicating that a limited number of African women were incorporated in the Seminole tribe. Analysis of Y chromosome haplotypes supports the hypothesis that haplotypes 18 and 63 are the most likely founding Native American Y chromosome haplotypes from Asia. However, 11% of the Seminole Y chromosomes represented haplotypes generally attributed to Europeans, though none harbored standard African haplotypes. These findings support historical evidence that the Seminole tribe has integrated individuals of European and African ancestry, but suggests that the sex ratio of nonnatives from different continents may have varied.

Mitochondrial DNA sequence analysis of four Alzheimer's and Parkinson's disease patients.

The mitochondrial DNA (mtDNA) sequence was determined on 3 patients with Alzheimer's disease (AD) exhibiting AD plus Parkinson's disease (PD) neuropathologic changes and one patient with PD. Patient mtDNA sequences were compared to the standard Cambridge sequence to identify base changes. In the first AD+PD patient, 2 of the 15 nucleotide substitutions may contribute to the neuropathology, a nucleotide pair (np) 4336 transition in the tRNA(Gln) gene found 7.4 times more frequently in patients than in controls, and a unique np 721 transition in the 12S rRNA gene which was not found in 70 other patients or 905 controls. In the second AD+PD patient, 27 nucleotide substitutions were detected, including an np 3397 transition in the ND1 gene which converts a conserved methionine to a valine. In the third AD+PD patient, 2 polymorphic base substitutions frequently found at increased frequency in Leber's hereditary optic neuropathy patients were observed, an np 4216 transition in ND1 and an np 13708 transition in the ND5 gene. For the PD patient, 2 novel variants were observed among 25 base substitutions, an np 1709 substitution in the 16S rRNA gene and an np 15851 missense mutation in the cytb gene. Further studies will be required to demonstrate a causal role for these base substitutions in neurodegenerative disease.

Isolation, characterization, and transcription of the gene encoding mouse mast cell protease 7.

A gene that encodes mouse mast cell protease (mMCP) 7 (also known as mouse mast cell tryptase 2) was isolated by genomic cloning with a cDNA that encodes mMCP-6, a tryptase in serosal mast cells. cDNAs encoding mMCP-7 were isolated from a bone-marrow-derived mast cell cDNA library. The mMCP-7 gene spans 2.3 kilobases and contains five exons rather than six, as found in the mMCP-6 and human mast cell tryptase I genes. Comparison of the 5' end of the transcript with the genomic sequence indicated that the region corresponding to the first intron in the mMCP-6 and human tryptase I genes is not spliced during transcription of mMCP-7 mRNA because of a point mutation at the intron 1 acceptor splice site; this results in a 5' untranslated region of 195 nucleotides, which is longer than that of any other known mast cell-specific transcript. mMCP-7 is 71-76% homologous with mMCP-6 and with dog and human mast cell tryptases, and it is the most acidic mast cell protease, with an overall net charge of -10. RNA blot analyses revealed that the mMCP-7 gene is transcribed in bone-marrow-derived mast cells but is not transcribed in mature serosal mast cells or in mucosal mast cell-enriched intestinal tissue of Trichinella spiralis-infected mice. Transcription of the mMCP-7 gene by differentiating bone-marrow-derived mast cells occurred within 1 week of bone-marrow culture but decreased dramatically after 3 weeks. Thus, the mMCP-7 gene displays a number of unusual structural characteristics and is distinctive in its transient and selective expression in immature mast cells maintained in interleukin 3-enriched medium.

Cloning and characterization of the novel gene for mast cell carboxypeptidase A.

No gene for a hematopoietic cell carboxypeptidase has previously been characterized. Mast cell carboxypeptidase A (MC-CPA) is a prominent secretory granule marker of mast cell differentiation and phenotype. The 32-kb human MC-CPA gene was isolated, localized to chromosome 3, and found to contain 11 exons. No significant homology was found between the 5' flanking region of the MC-CPA gene and those of three rat pancreatic carboxypeptidase genes (carboxypeptidase A1 and A2, and carboxypeptidase B [CPB]). In contrast, the intron/exon organization of the MC-CPA gene was conserved, most closely resembling the CPB gene. MC-CPA is unique among carboxypeptidases in having a CPA-like substrate-binding pocket and enzymatic activity despite overall protein and gene structures more similar to CPB. Evolutionary tree analysis of the carboxypeptidase gene family showed that, before the mammalian species radiation, a common MC-CPA/CPB ancestor diverged by gene duplication from the lineage leading to CPA, and then underwent another gene duplication to form separate but similar gene structures for MC-CPA and CPB. MC-CPA mRNA was prominent in dispersed lung cells enriched for mast cells but was undetectable in other nontransformed populations of several lineages, demonstrating that transcription of MC-CPA, a novel carboxypeptidase gene, provides a specific molecular marker for mast cells among normal hematopoietic cell populations.

Cloning of the cDNA and gene of mouse mast cell protease-6. Transcription by progenitor mast cells and mast cells of the connective tissue subclass.

The cDNA and gene for mouse mast cell protease-6 (MMCP-6) have been sequenced and show MMCP-6 to be translated as a prepro-enzyme with a 21-amino acid hydrophobic leader peptide, a 10-amino acid activation peptide, and a 245-amino acid mature enzyme. The mature form of the enzyme has 73% amino acid sequence identity with human and dog mast cell tryptases. The MMCP-6 gene includes 6 exons, with a total span of 1.8 kilobases. A 208-base pair intron was defined which separates the 5'-untranslated sequence of MMCP-6 from the translation initiation codon, thereby presenting a gene organization which distinguishes tryptic serine proteases from chymotryptic serine proteases of the mast cell secretory granule. By RNA blot analysis with a gene-specific probe, MMCP-6 has a unique subclass distribution in being transcribed in mouse connective tissue mast cells but undetectable in mucosal mast cells. MMCP-6 is the first serine protease of any class to be shown to be significantly transcribed in progenitor, bone marrow-derived mast cells, which can reconstitute both mucosal mast cell and connective tissue mast cell populations in mast cell-deficient mice.

Cloning of cDNAs that encode human mast cell carboxypeptidase A, and comparison of the protein with mouse mast cell carboxypeptidase A and rat pancreatic carboxypeptidases.

Human skin and lung mast cells and rodent peritoneal mast cells contain a carboxypeptidase in their secretory granules. We have screened human lung cDNA libraries with a mouse mast cell carboxypeptidase A (MC-CPA) cDNA probe to isolate a near-full-length cDNA that encodes human MC-CPA. The 5' end of the human MC-CPA transcript was defined by direct mRNA sequencing and by isolation and partial sequencing of the human MC-CPA gene. Human MC-CPA is predicted to be translated as a 417 amino acid preproenzyme which includes a 15 amino acid signal peptide and a 94 amino acid activation peptide. The mature human MC-CPA enzyme has a predicted size of 36.1 kDa, a net positive charge of 16 at neutral pH, and 86% amino acid sequence identity with mouse MC-CPA. DNA blot analyses showed that human MC-CPA mRNA is transcribed from a single locus in the human genome. Comparison of the human MC-CPA with mouse MC-CPA and with three rat pancreatic carboxypeptidases shows that these enzymes are encoded by distinct but homologous genes.

Isolation and molecular cloning of mast cell carboxypeptidase A. A novel member of the carboxypeptidase gene family.

Mast cell carboxypeptidase A has been isolated from the secretory granules of mouse peritoneal connective tissue mast cells (CTMC) and from a mouse Kirsten sarcoma virus-immortalized mast cell line (KiSV-MC), and a cDNA that encodes this exopeptidase has been cloned from a KiSV-MC-derived cDNA library. KiSV-MC-derived mast cell carboxypeptidase A was purified with a potato-derived carboxypeptidase-inhibitor affinity column and was found by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a Mr 36,000 protein. Secretory granule proteins from KiSV-MC and from mouse peritoneal CTMC were then resolved by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted to polyvinylidine difluoride membranes. Identical aminoterminal amino acid sequences were obtained for the prominent Mr 36,000 protein present in the granules of both cell types. Based on the amino-terminal sequence, an oligonucleotide probe was synthesized and used to isolate a 1,470-base pair cDNA that encodes this mouse exopeptidase. The deduced amino acid sequence revealed that, after cleavage of a 15-amino acid hydrophobic signal peptide and a 94-amino acid activation peptide from a 417-amino acid preproenzyme, the mature mast cell carboxypeptidase A protein core has a predicted Mr of 35,780 and a high positive charge [Lys + Arg) - (Asp + Glu) = 17) at neutral pH. Although critical zinc-binding amino acids (His67, Glu70, His195), substrate-binding amino acids (Arg69, Asn142, Arg143, Tyr197, Asp255, Phe278), and cysteine residues that participate in intrachain disulfide bonds (Cys64-Cys77, Cys136-Cys159) of pancreatic carboxypeptidases were also present in mast cell carboxypeptidase A, the overall amino acid sequence identities for mouse mast cell carboxypeptidase A relative to rat pancreatic carboxypeptidases A1, A2, and B were only 43, 41, and 53%, respectively. RNA and DNA blot analyses revealed that mouse peritoneal CTMC, KiSV-MC, and bone marrow-derived mast cells all express a prominent 1.5-kilobase mast cell carboxypeptidase A mRNA which is transcribed from a single gene. We conclude that mouse mast cell carboxypeptidase A is a prominent secretory granule enzyme of mast cells of the CTMC subclass and represents a novel addition to the carboxypeptidase gene family.