<Emphasis Type="Italic">HLA-A</Emphasis> allele associations with viral <Emphasis Type="Italic">MER9-LTR</Emphasis> nucleotide sequences at two distinct loci within the <Emphasis Type="Italic">MHC alpha</Emphasis> block

The study of the association of the Human Leukocyte Antigen (HLA) alleles and polymorphic retrotransposons such as Alu, HERV, and LTR at various loci within the Major Histocompatibility Complex allows for a better identification and stratification of disease associations and the origins of HLA haplotypes in different populations. This paper provides sequence and association data on two structurally polymorphic MER9-LTR retrotransposons that are located 54 kb apart and in close proximity to the multiallelic HLA-A gene involved in the regulation of the human immune system. Direct DNA sequencing and analysis of the PCR products identified DNA nucleotide variations between the MER9-LTR sequences at the two loci and their associations with HLA-A alleles as potential haplotype and evolutionary markers. All MER9-LTR sequences were haplotypic when associated with common HLA-A alleles. The number of SNP loci was 2.5 times greater for the solo LTR at the AK locus, which is located closer to the HLA-A gene than the solo or 3′ LTR at the HG locus. Our study shows that the nucleotide variations of the MER9-LTR DNA sequences are additional informative markers in fine mapping HLA-A genomic haplotypes for future population, evolutionary, and disease studies.     

Semaphorin 3E Initiates Antiangiogenic Signaling through Plexin D1 by Regulating Arf6 and R-Ras

Recent studies revealed that a class III semaphorin, semaphorin 3E (Sema3E), acts through a single-pass transmembrane receptor, plexin D1, to provide a repulsive cue for plexin D1-expressing endothelial cells, thus providing a highly conserved and developmentally regulated signaling system guiding the growth of blood vessels. We show here that Sema3E acts as a potent inhibitor of adult and tumor-induced angiogenesis. Activation of plexin D1 by Sema3E causes the rapid disassembly of integrin-mediated adhesive structures, thereby inhibiting endothelial cell adhesion to the extracellular matrix (ECM) and causing the retraction of filopodia in endothelial tip cells. Sema3E acts on plexin D1 to initiate a two-pronged mechanism involving R-Ras inactivation and Arf6 stimulation, which affect the status of activation of integrins and their intracellular trafficking, respectively. Ultimately, our present study provides a molecular framework for antiangiogenesis signaling, thus impinging on a myriad of pathological conditions that are characterized by aberrant increase in neovessel formation, including cancer.Pathological angiogenesis characterizes numerous human diseases, ranging from chronic inflammation, atherosclerosis, diabetic retinopathy, and age-related macular degeneration to cancer (5, 11, 30). Thus, elucidating the mechanisms underlying normal and aberrant blood vessel growth may provide new therapeutic options for many highly prevalent disease conditions. Ultimately, normal angiogenesis results from a precise balance between pro- and antiangiogenic mediators. Among the former, the family of vascular endothelial growth factors (VEGFs), basic fibroblastic growth factor (bFGF), sphingosine-1-phosphate (S1P), and the chemokines interleukin-8/CXCL8 and SDF-1/CXCL12 and their receptors are some of the most widely investigated (reviewed in references 3, 5, 8, and 17). The best-known angiogenesis inhibitors are proteolytic cleavage products of extracellular matrix (ECM) or serum components, such as endostatin, angiostatin, arresten, and tumstatin (reviewed in references 11 and 20). Antiangiogenic cytokines have also been described, including interferons and certain interleukins, which appear to act indirectly by limiting the expression of proangiogenic mediators or inducing antiangiogenic molecules (reviewed in references 11 and 20). In contrast, there are few known developmentally regulated, naturally occurring antiangiogenic molecules, which include platelet factor 4 (18), thrombospondin 1 (14), and pigment epithelium-derived factor (PEDF) (9). Their precise mechanism of action is not fully understood, thus limiting the ability to design new molecularly based antiangiogenic strategies.Emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including members of the netrin, slit, eph, and semaphorin families, also play a critical role in blood vessel guidance during physiological and pathological blood vessel development (6). For example, multiple secreted class III semaphorins, which regulate developmental axonal growth (23, 27), are now known to act through their receptors, the A family plexins (plexins A1, A2, and A3), and their coreceptors, neuropilin 1 and neuropilin 2, to initiate pro- and antiangiogenic responses (reviewed in references 6 and 19). However, neuropilins also act as coreceptors for multiple angiogenic factors, such as VEGF, thus limiting our ability to distinguish the downstream signaling events initiated by semaphorins from those resulting from their interplay with endothelial growth and motility factors (19). In this regard, recent studies revealed that a class III semaphorin, semaphorin 3E (Sema3E), acts through a single-pass transmembrane receptor, plexin D1, independently of neuropilins to control endothelial cell (EC) positioning and patterning of the developing vasculature (13, 15). These findings prompted us to explore whether Sema3E acts as a natural antiangiogenic molecule and, if so, to investigate the underlying molecular mechanism. Indeed, we show here that Sema3E is a potent inhibitor of adult and tumor-induced angiogenesis. Sema3E causes filopodial retraction in endothelial tip cells and inhibits endothelial cell adhesion by disrupting integrin-mediated adhesive structures. At the molecular level, this process involves the stimulation of plexin D1 by Sema3E, which in turn interferes with R-Ras function and leads to the rapid activation of Arf6, thus revealing a novel physiological antiangiogenic signaling route.     

G146V mutation at the hinge region of actin reveals a myosin class-specific requirement of actin conformations for motility

The G146V mutation in actin is dominant lethal in yeast. G146V actin filaments bind cofilin only minimally, presumably because cofilin binding requires the large and small actin domains to twist with respect to one another around the hinge region containing Gly-146, and the mutation inhibits that twisting motion. A number of studies have suggested that force generation by myosin also requires actin filaments to undergo conformational changes. This prompted us to examine the effects of the G146V mutation on myosin motility. When compared with wild-type actin filaments, G146V filaments showed a 78% slower gliding velocity and a 70% smaller stall force on surfaces coated with skeletal heavy meromyosin. In contrast, the G146V mutation had no effect on either gliding velocity or stall force on myosin V surfaces. Kinetic analyses of actin-myosin binding and ATPase activity indicated that the weaker affinity of actin filaments for myosin heads carrying ADP, as well as reduced actin-activated ATPase activity, are the cause of the diminished motility seen with skeletal myosin. Interestingly, the G146V mutation disrupted cooperative binding of myosin II heads to actin filaments. These data suggest that myosin-induced conformational changes in the actin filaments, presumably around the hinge region, are involved in mediating the motility of skeletal myosin but not myosin V and that the specific structural requirements for the actin subunits, and thus the mechanism of motility, differ among myosin classes.     

Use of a competitive probe in assay design for genotyping of the UGT1A1 *28 microsatellite polymorphism by the smart amplification process

A key feature of the smart amplification process version 2 (SMAP-2) is the ability to suppress mismatch amplification by using a unique asymmetric primer design and Thermus aquaticus MutS (Taq MutS). However we report here that use of SMAP-2 for polymorphism determination of the UGT1A1 *28 allele required a further ancillary approach for complete background suppression. The UGT1A1 *28 allele is a microsatellite copy number polymorphism. This is the first reported SMAP-2 assay designed for genotyping genetic variations of microsatellites. We found that by the addition of a primer to the amplification reaction, called a competitive probe (CP), assay specificity could be significantly enhanced. Including sample preparation time and use of a CP-enhanced SMAP-2 assay, we could rapidly detect the UGT1A1 *28 polymorphism within 60 min. To test our method, we compared results from PCR sequencing and the CP-enhanced SMAP-2 assay on 116 human blood samples for UGT1A1 *28 polymorphism and demonstrated perfect concordance. These results illustrate the versatility of SMAP-2 for molecular diagnostics and provide a new approach for enhancing SMAP-2 assay specificity.     

Molecular and functional analyses of rice NHX-type Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter genes

We previously cloned a vacuolar Na⁺/H⁺ antiporter gene (OsNHX1) from rice (Oryza sativa). Here we identified four additional NHX-type antiporter genes in rice (OsNHX2 through OsNHX5) and performed molecular and functional analyses of those genes. The exon–intron structure of the OsNHX genes and the phylogenetic tree of the OsNHX proteins suggest that the OsNHX proteins are categorized into two subgroups (OsNHX1 through OsNHX4 and OsNHX5). OsNHX1, OsNHX2, OsNHX3, and OsNHX5 can suppress the Na⁺, Li⁺, and hygromycin sensitivity of yeast nhx1 mutants and their sensitivity to a high K⁺ concentration. The expression of OsNHX1, OsNHX2, OsNHX3, and OsNHX5 is regulated differently in rice tissues and is increased by salt stress, hyperosmotic stress, and ABA. When we studied the expression of β-glucuronidase (GUS) driven by either the OsNHX1 or the OsNHX5 promoter, we observed activity in the stele, the emerging part of lateral roots, the vascular bundle, the water pore, and the basal part of seedling shoots with both promoters. In addition, each promoter had a unique expression pattern. OsNHX1 promoter–GUS activity only was localized to the guard cells and trichome, whereas OsNHX5 promoter–GUS activity only was localized to the root tip and pollen grains. Our results suggest that the members of this gene family play important roles in the compartmentalization into vacuoles of the Na⁺ and K⁺ that accumulate in the cytoplasm and that the differential regulation of antiporter gene expression in different rice tissues may be an important factor determining salt tolerance in rice.     

cDNA Cloning of the Human Homologues of the MouseKe4andKe6Genes at the Centromeric End of the Human MHC Region

cDNA clones corresponding to theHKE4andHKE6genes at the centromeric end of the HLA region on human chromosome 6p21.3 were isolated and characterized. The predicted amino acid sequences of HKE4 and HKE6 exhibited 81.5 and 85.6% identity to the mouse homologues, Ke4 and Ke6, respectively.HKE4may encode a membrane protein with histidine-rich charge clusters. HKE6 possesses remarkable amino acid sequence conservation with several bacterial proteins with oxidoreductase function and also shows significant homology with the two unique functional domains containing the nucleotide cofactor binding site and the consensus motif characteristic of the members of the superfamily of short-chain alcohol dehydrogenases such as human and rat steroid and prostaglandin dehydrogenases.     

A glycine-rich sequence in the catalytic site of F-type ATPase

Affinity labeling and genetic studies on the glycine-rich sequence of the subunit ofE. coli F-type ATPase are discussed. A model of the structure of the enzyme near the phosphate moiety is proposed.     

The Ingenious Structure of Central Rotor Apparatus in VoV1; Key for Both Complex Disassembly and Energy Coupling between V1 and Vo

Vacuolar type rotary H⁺-ATPases (V_oV₁) couple ATP synthesis/hydrolysis by V₁ with proton translocation by V_o via rotation of a central rotor apparatus composed of the V₁-DF rotor shaft, a socket-like V_o-C (eukaryotic V_o-d) and the hydrophobic rotor ring. Reconstitution experiments using subcomplexes revealed a weak binding affinity of V₁-DF to V_o-C despite the fact that torque needs to be transmitted between V₁-DF and V_o-C for the tight energy coupling between V₁ and V_o. Mutation of a short helix at the tip of V₁-DF caused intramolecular uncoupling of V_oV₁, suggesting that proper fitting of the short helix of V₁-D into the socket of V_o-C is required for tight energy coupling between V₁ and V_o. To account for the apparently contradictory properties of the interaction between V₁-DF and V_o-C (weak binding affinity but strict requirement for torque transmission), we propose a model in which the relationship between V₁-DF and V_o-C corresponds to that between a slotted screwdriver and a head of slotted screw. This model is consistent with our previous result in which the central rotor apparatus is not the major factor for the association of V₁ with V_o (Kishikawa and Yokoyama, J Biol Chem. 2012 24597-24603).     

Development of infant common marmosets’ (<Emphasis Type="Italic">Callithrix jacchus</Emphasis>) preference for their parents over adults from another group

Parent–offspring attachment is important for animals which have offspring that require parental care for their development. Infant attachment to the mother has been examined in macaques, but it remains poorly understood in common marmosets. Here, we examined the abilities of 14 common marmoset infants to show preference for their parents over adults from another group at the ages of 4, 10, and 15 weeks. Each infant was exposed to its parent and an adult from another group in an I-shaped maze. Although 4-week-old infants did not show a significant difference between approach behaviors toward their parents and other adults, 10- and 15-week-old infants approached and stayed longer near their parents than adults from another group. These results suggest selective approach behavior develops in marmosets by the age of 10 weeks.