Cloning and primary structure of putative cytosolic and mitochondrial malate dehydrogenase from the mollusc Nucella lapillus (L.)

The evolutionary history of the malate dehydrogenase (MDH) gene family [NAD-dependent MDH; EC 1.1.1.37 and NAD(P)-dependent MDH; EC 1.1.1.82] has received much attention. MDHs have also featured extensively as electrophoretic markers in population genetics and evolutionary ecology, and in many cases, intraspecific variation in MDH has been correlated with environmental variables. However, while the amino acid residues essential for MDH function are known, no studies have examined intraspecific nucleotide variation despite evidence indicating that natural selection may be operating on this locus. This study presents two sets of degenerate oligonucleotide PCR primers to facilitate the cloning of cytosolic MDH (cMDH) and mitochondrial MDH (mMDH) from a broad range of animals (cMDH) and animals and plants (mMDH). These primers were used to obtain putative cMDH and mMDH cDNAs from the mollusc Nucella lapillus. The N. lapillus cMDH cDNA was found to encode a putative cMDH protein of 334aa and 36kDa, while the mMDH cDNA encoded a putative mature mMDH protein of 315aa and 33kDa. The putative amino acid sequences of the two compartmentalised N. lapillus MDHs are presented and compared to other known MDH sequences.     

Identification of contact residues and definition of the CAR-binding site of adenovirus type 5 fiber protein

The binding of adenovirus (Ad) fiber knob to its cellular receptor, the coxsackievirus and Ad receptor (CAR), promotes virus attachment to cells and is a major determinant of Ad tropism. Analysis of the kinetics of binding of Ad type 5 (Ad5) fiber knob to the soluble extracellular domains of CAR together (sCAR) and each immunoglobulin (Ig) domain (IgV and IgC2) independently by surface plasmon resonance demonstrated that the IgV domain is necessary and sufficient for binding, and no additional membrane components are required to confer high-affinity binding to Ad5 fiber knob. Four Ad5 fiber knob mutations, Ser408Glu and Pro409Lys in the AB loop, Tyr477Ala in the DG loop, and Leu485Lys in beta strand F, effectively abolished high-affinity binding to CAR, while Ala406Lys and Arg412Asp in the AB loop and Arg481Glu in beta strand E significantly reduced the level of binding. Circular dichroism spectroscopy showed that these mutations do not disorder the secondary structure of the protein, implicating Ser408, Pro409, Tyr477, and Leu485 as contact residues, with Ala406, Arg412, and Arg481 being peripherally or indirectly involved in CAR binding. The critical residues have exposed side chains that form a patch on the surface, which thus defines the high-affinity interface for CAR. Additional site-directed mutagenesis of Ad5 fiber knob suggests that the binding site does not extend to the adjacent subunit or toward the edge of the R sheet. These findings have implications for our understanding of the biology of Ad infection, the development of novel Ad vectors for targeted gene therapy, and the construction of peptide inhibitors of Ad infection.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Refined radiation hybrid map of mouse Chromosome 17

We have made a radiation hybrid map of mouse Chromosome (Chr) 17 with 75 microsatellite markers, including those from McCarthy et al. (Genome Res 7, 1153–1161, 1997). Seventy-four of the markers are linked at LOD > 9, and all link at LOD > 5. A LOD 3 framework of 18 markers was used to construct a placement map. The order obtained is in good agreement with genetic maps, and distance estimates give an idea of how recombination rates vary across the chromosome. Recombination is remarkably low with respect to RH break frequency in the region from the centromere to the end of H2. This is similar in interspecific and intersubspecific crosses despite the inversion of a substantial part of this region in Mus spretus with respect to Mus musculus. Received: 10 April 1998 / Accepted: 18 June 1998     

Evidence for pore-forming ability by Legionella pneumophila

Legionella pneumophila is the cause of Legionnaires' pneumonia. After internalization by macrophages, it bypasses the normal endocytic pathway and occupies a replicative phagosome bound by endoplasmic reticulum. Here, we show that lysis of macrophages and red blood cells by L . pneumophila was dependent on dotA and other loci known to be required for proper targeting of the phagosome and replication within the host cell. Cytotoxicity occurred rapidly during a high-multiplicity infection, required close association of the bacteria with the eukaryotic cell and was a form of necrotic cell death accompanied by osmotic lysis. The differential cytoprotective ability of high-molecular-weight polyethylene glycols suggested that osmotic lysis resulted from insertion of a pore less than 3 nm in diameter into the plasma membrane. Results concerning the uptake of membrane-impermeant fluorescent compounds of various sizes are consistent with the osmoprotection analysis. Therefore, kinetic and genetic evidence suggested that the apparent ability of L . pneumophila to insert a pore into eukaryotic membranes on initial contact may play a role in altering endocytic trafficking events within the host cell and in the establishment of a replicative vacuole.     

Bronchial airway deposition and retention of particles in inhaled boluses: effect of anatomic dead space

The fractionaldeposition of particles in boluses delivered to shallow lung depths andtheir subsequent retention in the airways may depend on the relativevolume and size of an individual's airways. To evaluate the effect ofvariable anatomic dead space (ADS) on aerosol bolus delivery we hadhealthy subjects inhale radiolabeled, monodisperse aerosol(^99mTc-iron oxide, 3.5 µm meanmondispersed aerosol diameter) boluses (40 ml) to a volumetric frontdepth of 70 ml into the lung at a lung volume of 70% total lungcapacity end inhalation. By using filter techniques, aerosolphotometry, and gamma camera analysis, we estimated the fraction of theinhaled boluses deposited in intrathoracic airways (IDF). ADS bysingle-breath N₂ washout was alsomeasured from 70% total lung capacity. Results showed that among allsubjects IDF was variable (range = 0.04-0.43, coefficient ofvariation = 0.54) and increased with decreasing ADS(r = 0.76, P = 0.001, n = 16). We found significantlygreater deposition in the left (L) vs. right (R) lungs; mean L/R (ratioof deposition in L lung to R lung, normalized to ratio of L-to-R lungvolume) was 1.58 ± 0.42 (SD; P < 0.001 for comparison with 1.0). Retention of deposited particles at 2 hwas independent of ADS or IDF. There was significant retention ofparticles at 24 h postdeposition (0.27 ± 0.05) andslow clearance of these particles continued through 48 hpostdeposition. Finally, analysis of central-to-peripheral ratios ofinitial deposition and 24-h-retention gamma-camera images suggestsignificant retention of insoluble particles in large bronchial airwaysat 24 h postdeposition (i.e., 24 h central-to-peripheral ratio = 1.40 ± 0.44 and 1.82 ± 0.54 in the R and L lung, respectively; P < 0.02 for comparison with 1.0).These data may prove useful for 1)designing aerosol delivery techniques to target bronchial airways and2) understanding airway retention ofinhaled particles.

     

Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19⁺CD1d^highCD5⁺ B cells (CD1d^highCD5⁺ Breg) obtained from Was gene knockout (WKO) mice and found that their numbers were significantly lower in these mice compared to wild type (WT) controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1d^highCD5⁺ Breg cells as compared to age-matched WT control mice. CD1d^highCD5⁺ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4⁺ T cells. Interestingly, CD1d^highCD5⁺ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS.     

Thymus-associated parathyroid hormone has two cellular origins with distinct endocrine and immunological functions

In mammals, parathyroid hormone (PTH) is a key regulator of extracellular calcium and inorganic phosphorus homeostasis. Although the parathyroid glands were thought to be the only source of PTH, extra-parathyroid PTH production in the thymus, which shares a common origin with parathyroids during organogenesis, has been proposed to provide an auxiliary source of PTH, resulting in a higher than expected survival rate for aparathyroid Gcm2 ^−/− mutants. However, the developmental ontogeny and cellular identity of these “thymic” PTH–expressing cells is unknown. We found that the lethality of aparathyroid Gcm2 ^−/− mutants was affected by genetic background without relation to serum PTH levels, suggesting a need to reconsider the physiological function of thymic PTH. We identified two sources of extra-parathyroid PTH in wild-type mice. Incomplete separation of the parathyroid and thymus organs during organogenesis resulted in misplaced, isolated parathyroid cells that were often attached to the thymus; this was the major source of thymic PTH in normal mice. Analysis of thymus and parathyroid organogenesis in human embryos showed a broadly similar result, indicating that these results may provide insight into human parathyroid development. In addition, medullary thymic epithelial cells (mTECs) express PTH in a Gcm2-independent manner that requires TEC differentiation and is consistent with expression as a self-antigen for negative selection. Genetic or surgical removal of the thymus indicated that thymus-derived PTH in Gcm2 ^−/− mutants did not provide auxiliary endocrine function. Our data show conclusively that the thymus does not serve as an auxiliary source of either serum PTH or parathyroid function. We further show that the normal process of parathyroid organogenesis in both mice and humans leads to the generation of multiple small parathyroid clusters in addition to the main parathyroid glands, that are the likely source of physiologically relevant “thymic PTH.”