Homozygosity haplotype allows a genomewide search for the autosomal segments shared among patients

A promising strategy for identifying disease susceptibility genes for both single- and multiple-gene diseases is to search patients' autosomes for shared chromosomal segments derived from a common ancestor. Such segments are characterized by the distinct identity of their haplotype. The methods and algorithms currently available have only a limited capability for determining a high-resolution haplotype genomewide. We herein introduce the homozygosity haplotype (HH), a haplotype described by the homozygous SNPs that are easily obtained from high-density SNP genotyping data. The HH represents haplotypes of both copies of homologous autosomes, allowing for direct comparisons of the autosomes among multiple patients and enabling the identification of the shared segments. The HH successfully detected the shared segments from members of a large family with Marfan syndrome, which is an autosomal dominant, single-gene disease. It also detected the shared segments from patients with model multigene diseases originating with common ancestors who lived 10-25 generations ago. The HH is therefore considered to be useful for the identification of disease susceptibility genes in both single- and multiple-gene diseases.     

X-ray crystallographic and biochemical characterization of the inhibitory action of an imidazole-dioxolane compound on heme oxygenase

Heme oxygenase (HO) catalyzes the regiospecific cleavage of the porphyrin ring of heme using reducing equivalents and O2 to produce biliverdin, iron, and CO. Because CO has a cytoprotective effect through the p38-MAPK pathway, HO is a potential therapeutic target in cancer. In fact, inhibition of the HO isoform HO-1 reduces Kaposi sarcoma tumor growth. Imidazole-dioxolane compounds have recently attracted attention because they have been reported to specifically inhibit HO-1, but not HO-2, unlike Cr-containing protoporphyrin IX, a classical inhibitor of HO, that inhibits not only both HO isoforms but also other hemoproteins. The inhibitory mechanism of imidazole-dioxolane compounds, however, has not yet been characterized. Here, we determine the crystal structure of the ternary complex of rat HO-1, heme, and an imidazole-dioxolane compound, 2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane. This compound bound on the distal side of the heme iron, where the imidazole and 4-chlorophenyl groups were bound to the heme iron and the hydrophobic cavity in HO, respectively. Binding of the bulky inhibitor in the narrow distal pocket shifted the distal helix to open the distal site and moved both the heme and the proximal helix. Furthermore, the biochemical characterization revealed that the catalytic reactions of both HO-1 and HO-2 were completely stopped after the formation of verdoheme in the presence of the imidazole-dioxolane compound. This result should be mainly due to the lower reactivity of the inhibitor-bound verdoheme with O2 compared to the reactivity of the inhibitor-bound heme with O2.     

Functional and intracellular signaling differences associated with the Helicobacter pylori AlpAB adhesin from Western and East Asian strains

Following adhesion of Helicobacter pylori to gastric epithelial cells, intracellular signaling leads to cytokine production, which causes H. pylori-related gastric injury. Two adjacent homologous genes (alpA and alpB), which encode H. pylori outer membrane proteins, are thought to be associated with adhesion and cytokine induction. We co-cultured gastric epithelial cells with wild type H. pylori strains and their corresponding alpA/alpB-deleted mutants (DeltaalpAB). Results were confirmed by complementation. Flow cytometry confirmed that AlpAB was involved in cellular adhesion. Deletion of alpAB reduced interleukin (IL)-6 induction in gastric epithelial cells. Deletion of alpAB reduced IL-8 induction with East Asian but not with Western strains. All AlpAB-positive strains tested activated the extracellular signal-regulated kinase, c-Fos, and cAMP-responsive element-binding protein. Activation of the Jun-N-terminal kinase, c-Jun, and NF-kappaB was exclusive to AlpAB from East Asian strains. DeltaalpAB mutants poorly colonized the stomachs of C57BL/6 mice and were associated with lower mucosal levels of KC and IL-6. Our results suggest that AlpAB may induce gastric injury by mediating adherence to gastric epithelial cells and by modulating proinflammatory intracellular signaling cascades. Known geographical differences in H. pylori-related clinical outcomes may relate to differential effects of East Asian and Western types of AlpAB on NF-kappaB-related proinflammatory signaling pathways.     

Physiological changes of premotor nonspiking interneurons in the central compensation of eyestalk posture following unilateral sensory ablation in crayfish

We investigated how the physiological characteristics and synaptic activities of nonspiking giant interneurons (NGIs), which integrate sensory inputs in the brain and send synaptic outputs to oculomotor neurons innervating eyestalk muscles, changed after unilateral ablation of the statocyst in order to clarify neuronal mechanisms underlying the central compensation process in crayfish. The input resistance and membrane time constant in recovered animals that restored the original symmetrical eyestalk posture 2 weeks after operation were significantly greater than those immediately after operation on the operated side whereas in non-recovered animals only the membrane time constant showed a significant increase. On the intact side, both recovered and non-recovered animals showed no difference. The frequency of synaptic activity showed a complex pattern of change on both sides depending on the polarity of the synaptic potential. The synaptic activity returned to the bilaterally symmetrical level in recovered animals while bilateral asymmetry remained in non-recovered ones. These results suggest that the central compensation of eyestalk posture following unilateral impairment of the statocyst is subserved by not only changes in the physiological characteristics of the NGI membrane but also the activity of neuronal circuits presynaptic to NGIs.     

In vivo selection for the directed evolution of L-rhamnulose aldolase from L-rhamnulose-1-phosphate aldolase (RhaD)

Dihydroxyacetone phosphate (DHAP)-dependent aldolases have been widely used for organic synthesis. The major drawback of DHAP-dependent aldolases is their strict donor substrate specificity toward DHAP, which is expensive and unstable. Here we report the development of an in vivo selection system for the directed evolution of the DHAP-dependent aldolase, L-rhamnulose-1-phosphate aldolase (RhaD), to alter its donor substrate specificity from DHAP to dihydroxyacetone (DHA). We also report preliminary results on mutants that were discovered with this screen. A strain deficient in the L-rhamnose metabolic pathway in Escherichia coli (DeltarhaDAB, DE3) was constructed and used as a selection host strain. Co-expression of L-rhamnose isomerase (rhaA) and rhaD in the selection host did not restore its growth on minimal plate supplemented with L-rhamnose as a sole carbon source, because of the lack of L-rhamnulose kinase (RhaB) activity and the inability of WT RhaD aldolase to use unphosphorylated L-rhamnulose as a substrate. Use of this selection host and co-expression vector system gives us an in vivo selection for the desired mutant RhaD which can cleave unphosphorylated L-rhamnulose and allow the mutant to grow in the minimal media. An error-prone PCR (ep-PCR) library of rhaD gene on the co-expression vector was constructed and introduced into the rha-mutant, and survivors were selected in minimal media with l-rhamnose (MMRha media). An initial round of screening gave mutants allowing the selection strain to grow on MMRha plates. This in vivo selection system allows rapid screening of mutated aldolases that can utilize dihydroxyacetone as a donor substrate.     

Production of 2-phenylethanol in roses as the dominant floral scent compound from L-phenylalanine by two key enzymes, a PLP-dependent decarboxylase and a phenylacetaldehyde reductase

We investigated the biosynthetic pathway for 2-phenylethanol, the dominant floral scent compound in roses, using enzyme assays. L-[(2)H8] Phenylalanine was converted to [(2)H8] phenylacetaldehyde and [(2)H8]-2-phenylethanol by two enzymes derived from the flower petals of R. 'Hoh-Jun,' these being identified as pyridoxal-5'-phosphate-dependent L-aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). The activity of rose petal AADC to yield phenylacetaldehyde was nine times higher toward L-phenylalanine than toward its D-isomer, and this conversion was not inhibited by iproniazid, a specific inhibitor of monoamine oxidase. Under aerobic conditions, rose petal AADC stoichiometrically produced NH3 together with phenylacetaldehyde during the course of decarboxylation and oxidation, followed by the hydrolysis of L-phenylalanine. Phenylacetaldehyde was subsequently converted to 2-phenylethanol by the action of PAR. PAR showed specificity toward several volatile aldehydes.     

Control of cell polarity and motility by the PtdIns(3,4,5)P3 phosphatase SHIP1

Proper neutrophil migration into inflammatory sites ensures host defense without tissue damage. Phosphoinositide 3-kinase (PI(3)K) and its lipid product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) regulate cell migration, but the role of PtdIns(3,4,5)P(3)-degrading enzymes in this process is poorly understood. Here, we show that Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1), a PtdIns(3,4,5)P(3) phosphatase, is a key regulator of neutrophil migration. Genetic inactivation of SHIP1 led to severe defects in neutrophil polarization and motility. In contrast, loss of the PtdIns(3,4,5)P(3) phosphatase PTEN had no impact on neutrophil chemotaxis. To study PtdIns(3,4,5)P(3) metabolism in living primary cells, we generated a novel transgenic mouse (AktPH-GFP Tg) expressing a bioprobe for PtdIns(3,4,5)P(3.) Time-lapse footage showed rapid, localized binding of AktPH-GFP to the leading edge membrane of chemotaxing ship1(+/+)AktPH-GFP Tg neutrophils, but only diffuse localization in ship1(-/-)AktPH-GFP Tg neutrophils. By directing where PtdIns(3,4,5)P(3) accumulates, SHIP1 governs the formation of the leading edge and polarization required for chemotaxis.