Regulatory Architecture of the Neuronal Cacng2/Tarpγ2 Gene Promoter: Multiple Repressive Domains, a Polymorphic Regulatory Short Tandem Repeat, and Bidirectional Organization with Co-regulated lncRNAs.

CACNG2 (TARPγ2, Stargazin) is a multi-functional regulator of excitatory neurotransmission and has been implicated in the pathological processes of several brain diseases. Cacng2 function is dependent upon expression level, but currently, little is known about the molecular mechanisms that control expression of this gene. To address this deficit and investigate disease-related gene variants, we have cloned and characterized the rat Cacng2 promoter and have defined three major features: (i) multiple repressive domains that include an array of RE-1 silencing transcription factor (REST) elements, and a calcium regulatory element-binding factor (CaRF) element, (ii) a (poly-GA) short tandem repeat (STR), and (iii) bidirectional organization with expressed lncRNAs. Functional activity of the promoter was demonstrated in transfected neuronal cell lines (HT22 and PC12), but although selective removal of REST and CaRF domains was shown to enhance promoter-driven transcription, the enhanced Cacng2 promoter constructs were still about fivefold weaker than a comparable rat Synapsin-1 promoter sequence. Direct evidence of REST activity at the Cacng2 promoter was obtained through co-transfection with an established dominant-negative REST (DNR) construct. Investigation of the GA-repeat STR revealed polymorphism across both animal strains and species, and size variation was also observed in absence epilepsy disease model cohorts (Genetic Absence Epilepsy Rats, Strasbourg [GAERS] and non-epileptic control [NEC] rats). These data provide evidence of a genotype (STR)-phenotype correlation that may be unique with respect to proximal gene regulatory sequence in the demonstrated absence of other promoter, or 3' UTR variants in GAERS rats. However, although transcriptional regulatory activity of the STR was demonstrated in further transfection studies, we did not find a GAERS vs. NEC difference, indicating that this specific STR length variation may only be relevant in the context of other (Cacna1h and Kcnk9) gene variants in this disease model. Additional studies revealed further (bidirectional) complexity at the Cacng2 promoter, and we identified novel, co-regulated, antisense rat lncRNAs that are paired with Cacng2 mRNA. These studies have provided novel insights into the organization of a synaptic protein gene promoter, describing multiple repressive and modulatory domains that can mediate diverse regulatory inputs.

Selecting an appropriate method for expressing S locus F-box-S2 recombinant protein.

A single locus (S locus) including at least two linked genes (female and male determinants) genetically controls the gametophytic self-incompatibility (GSI) in apple, which has evolved to avoid self-fertilization. There has been extensive work done on the female determinant of self-incompatibility, which has led to the determination of the tertiary structure of S-RNase. However, the tertiary structure of male determinant (S locus F-box, SLF/SFB) remains unresolved, which could mainly be due to difficulties associated with its expression in the recombinant expression systems. In addressing this, we have evaluated several in vivo (prokaryotic and eukaryotic) and in vitro expression systems for their efficiency in the expression of apple SLF2. The most successful expression of SLF2 (1 mg/ml) was achieved in E. coli using the synthesized gene in a high salt culture and applying heat shock before induction of culture. We therefore present an approach for the efficient expression of S locus F-box recombinant proteins for future functional and structural studies.

A simple, high-throughput modeling approach reveals insights into the mechanism of gametophytic self-incompatibility.

Specificity in the GSI response results from the S-haplotype-specific molecular interaction of S-locus F-box (SLF/SFB) and SRNase proteins in the self-incompatibility locus (S-locus). The answer to the question of how these two components of the S-locus (SRNase and SLF/SFB) interact has been gathered from several models. Since there is not enough evidence as to which one is the definitive model, none of them can be ruled out. Despite the identification of interacting protein elements, the mechanism by which SLF/SFB and SRNase interact to differently trigger the self-incompatibility among families and subfamilies remain uncertain. The high-throughput modeling approach demonstrates structural visions into the possible existence of a Collaborative Non-Self Recognition model in apple. These findings postulate several prospects for future investigation providing useful information to guide the implementation of breeding strategies.

The Critical Role Of VP1 In Forming The Necessary Cavities For Receptor-mediated Entry Of FMDV To The Host Cell.

The antigenic inconsistency of the foot-and-mouth disease virus (FMDV) is very broad, such that a vaccine made from one isolate will not offer protection against infection with other isolates from the same serotype. Viral particles (VPs) or surface exposed capsid proteins, VP1-VP3, of FMDV determine both the antigenicity of the virus and its receptor-mediated entry into the host cell. Therefore, modifications of these structural proteins may alter the properties of the virus. Here we show putative cavities on the FMDV-SAT1 (FMDV Southern African Territories1) capsid as possible binding sites for the receptor-mediated viral entry into the host cell. We identified three possible cavities on the FMDV capsid surface, from which the largest one (C2) is shaped in the contact regions of VP1-VP3. Our results demonstrate the significance of VP1, in the formation of FMDV-SAT1 surface cavities, which is the main component in all the identified cavities. Our findings can have profound implications in the protein engineering of FMDV in the contact region of VP1-VP3 found to be embedded in several cavities. Such information is of great significance in the context of vaccine design, as it provides the ground for future improvement of synthetic vaccines to control FMD caused by FMDV-SAT1 serotypes.

Tropically adapted cattle of Africa: perspectives on potential role of copy number variations.

Africa is host to diverse and locally adapted cattle breeds that are expected to survive the harsh and extreme tropical environments associated with diseases and parasite infections, heat stress and episodes of feed and water scarcity. Genomic copy number variations (CNVs) are considered to be primary role players in cattle breed formation and adaptation where isolation and genetic drift together with subsequent mutations have created an enormous diversity of local populations. CNVs are modifications in DNA structure comprising deletions, duplications and insertions that are >1 kb in size. Despite attracting much attention, the frequency and pattern of bovine CNV events, especially in African cattle breeds, are for the most part largely unknown. Characterization of genetic variation in the indigenous cattle of Africa will be a vital step toward dissecting the molecular mechanisms underlying phenotypic variation and local adaptation. This review therefore aims to describe the current knowledge regarding bovine CNVs and the implications and potentials they encompass for dissecting genetic adaptation and the genotypic skeleton of tropical African cattle populations.

A Comprehensive Study of Molecular Evolution at the Self-Incompatibility Locus of Rosaceae.

The family Rosaceae includes a range of important fruit trees, most of which have the S-RNase-based self-incompatibility (SI). Several models have been developed to explain how pollen (SLF) and pistil (S-RNase) components of the S-locus interact. It was discovered in 2010 that additional SLF proteins are involved in pollen specificity, and a Collaborative Non-Self Recognition model has been proposed for SI in Solanaceae; however, the validity of such model remains to be elucidated for other species. The results of this study support the divergent evolution of the S-locus genes from two Rosaceae subfamilies, Prunoideae/Amygdaloideae and Maloideae, The difference identified in the selective pressures between the two lineages provides evidence for positive selection at specific sites in both the S-RNase and the SLF proteins. The evolutionary findings of this study support the role of multiple SLF proteins leading to a Collaborative Non-Self Recognition model for SI in the Maloideae. Furthermore, the identification of the sites responsible for SI specificity determination and the mapping of these sites onto the modelled tertiary structure of ancestor proteins provide useful information for rational functional redesign and protein engineering for the future engineering of new functional alleles providing increased diversity in the SI system in the Maloideae.

In vitro cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea leaves and bulbs.

ETHNOPHARMACOLOGICAL RELEVANCE: Infusions of Tulbaghia violacea (wild garlic) in water are used in traditional medicine in Southern Africa to treat numerous diseases, including cancer. Several studies have previously demonstrated the cytotoxic activities of extracts of T. violacea in cultured cancer cells. Their findings support the potential anti-cancer properties of this plant. However, these studies made use of organic solvent extraction methods, while the traditional use of the plant involves the preparation of infusions in water. MATERIALS AND METHODS: In the current study, we investigated the potential anti-cancer properties of infusions of T. violacea. We also performed a comparative study investigating the cytotoxic activities of T. violacea bulbs and leaves. A panel of four cancer cell lines (HepG2, MCF7, H157, and HT29) and one non-cancerous cell line (KMST6) was treated with the two extracts and the effects of the extracts on the growth of the cells were evaluated. We also investigated whether the growth inhibitory effects were associated with the induction of apoptosis and whether the mechanism of cell death is the result of oxidative stress and the activation of caspase-3. RESULT: We found that extracts of the leaves and not the bulbs have growth inhibitory effects and that this is the result of the induction of apoptosis, which is associated with the production of Reactive Oxygen Species (ROS) and the activation of caspase-3. The leaf extract demonstrated variable selective toxicity towards the cancer lines. Although the extract also induced cell death in the non-cancerous cell line (KMST6), we found that the levels of toxicity were lower in this cell line. CONCLUSION: this study confirms that infusions of T. violacea have potential anti-cancer activity and that this bioactivity is contained in the leaf extract. This study lends support to claims that this plant can be used to treat cancer.

Combination of HIF-1α gene transfection and HIF-1-activated bone marrow-derived angiogenic cell infusion improves burn wound healing in aged mice.

Impaired burn wound healing in the elderly represents a major clinical problem. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator that orchestrates the cellular response to hypoxia. Its actions in dermal wounds promote angiogenesis and improve healing. In a murine burn wound model, aged mice had impaired wound healing associated with reduced levels of HIF-1. When gene therapy with HIF-1 alone did not correct these deficits, we explored the potential benefit of HIF-1 gene therapy combined with the intravenous infusion of bone marrow-derived angiogenic cells (BMDACs) cultured with dimethyloxalylglycine (DMOG). DMOG is known to reduce oxidative degradation of HIF-1. The mice treated with a plasmid DNA construct expressing a stabilized mutant form of HIF-1α (CA5-HIF-1α)+BMDACs had more rapid wound closure. By day 17, there were more mice with completely closed wounds in the treated group (χ(2), P=0.05). The dermal blood flow measured by laser Doppler showed significantly increased wound perfusion on day 11. Homing of BMDACs to the burn wound was dramatically enhanced by CA5-HIF-1α gene therapy. HIF-1α mRNA expression in the burn wound was increased after transfection with CA5-HIF-1α plasmid. Our findings offer insight into the pathophysiology of burns in the elderly and point to potential targets for developing new therapeutic strategies.

Mitochondrial DNA, ecology and morphology: interpreting the phylogeography of the Nesotes (Coleoptera: Tenebrionidae) of Gran Canaria (Canary Islands).

The genus Nesotes (Coleoptera: Tenebrionidae) is represented in the Canary Islands by 19 endemic species, the majority of which are single island endemics. Nesotes conformis and N. fusculus are described on four and three islands, respectively, but each forms a paraphyletic assemblage between Gran Canaria and the other islands. The other described species for Gran Canaria are N. quadratus, N. lindbergi and N. piliger. Thirty-six individuals representing the five species on Gran Canaria have been sequenced for 675 bp of the mitochondrial DNA (mtDNA) cytochrome oxidase II gene. Neighbour-joining analysis of maximum likelihood distances resulted in five distinct mtDNA lineages for N. quadratus, two of which also include mitotypes of N. conformis. Each of the other three species is found on only one mtDNA lineage. We propose from the molecular data that differentiation in a widespread N. quadratus-type ancestor was followed by morphological adaptation to coastal, pine and laurel forest habitats.

Reconciling gene trees with organism history: the mtDNA phylogeography of three Nesotes species (Coleoptera: Tenebrionidae) on the western Canary Islands.

The processes of island colonization and speciation are investigated through mtDNA studies on Canary Island beetles. The genus Nesotes (Coleoptera: Tenebrionidae) is represented by 19 endemic species on the Canary Islands, the majority of which are single island endemics. Nesotes conformis is the most widespread, occurring on Gran Canaria, Tenerife, La Palma and El Hierro. Nesotes conformis forms a paraphyletic assemblage, with a split between Gran Canaria and the other three islands. Nesotes conformis of the western Canary Islands cluster with Nesotes altivagans and Nesotes elliptipennis from Tenerife. Fifty-two individuals from this western islands species complex have been sequenced for 675 base pairs of the mtDNA cytochrome oxidase II gene, representing Tenerife, La Palma and El Hierro. A neighbour joining analysis of maximum likelihood distances resulted in three distinct mtDNA lineages for N. conformis, two of which also include mitotypes of N. altivagans and N. elliptipennis. Through application of parametric bootstrap tests, we are able to reject hypotheses of monophyly for both N. conformis and N. altivagans. Nesotes altivagans and N. elliptipennis are poorly separated morphologically and mtDNA sequence data adds support to this being one species with a highly variable morphology. We propose that N. altivagans/N. elliptipennis is recently derived from two ancestral mtDNA lineages within N. conformis from the Teno region of Tenerife. We further propose colonization of the younger islands of La Palma and El Hierro by N. conformis from a mitochondrial lineage within the Teno massif (colonization; diversification; mitochondrial DNA; Canary Islands; Coleoptera).

Sequence analysis or rat integrin alphaE1 and alphaE2 subunits: tissue expression reveals phenotypic similarities between intraepithelial lymphocytes and dendritic cells in lymph.

The integrin alphaOX-62 subunit is defined by the OX-62 monoclonal antibody that was raised against rat dendritic cells in lymph (veiled cells) and shows properties similar to those of human alphaE that is predominantly expressed on intraepithelial lymphocytes. To clone alphaOX-62, rat probes generated using primers specific for the human alphaE sequence were used to screen rat T cell cDNA libraries. cDNA clones encoding two similar but not identical alpha subunits that are closely related but distinct from human alphaE were isolated. alphaE1 is predicted to be the rat homolog of mouse alphaM290 and alphaE2 corresponds to rat alphaOX-62. Immunofluorescence analysis revealed that mouse alphaE1 and rat alphaE2 are expressed in dendritic epidermal T cells in the skin, intraepithelial lymphocytes in the small intestine and in cells with a dendritic morphology present at sites where gammadelta T cells occur in lymphoid organs. Unexpectedly, in veiled cells alphaE2 is co-expressed with intracellular CD3-delta and a 33-kDa CD3 chain but not the T cell receptor. These findings suggest that veiled cells may be derived from a lymphoid precursor. Furthermore, veiled cells show phenotypic similarities to intraepithelial lymphocytes.

The Talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation.

The beta subunit cytoplasmic domains of integrin adhesion receptors are necessary for the connection of these receptors to the actin cytoskeleton. The cytoplasmic protein, talin, binds to beta integrin cytoplasmic tails and actin filaments, hence forming an integrin-cytoskeletal linkage. We used recombinant structural mimics of beta(1)A, beta(1)D and beta(3) integrin cytoplasmic tails to characterize integrin-binding sites within talin. Here we report that an integrin-binding site is localized within the N-terminal talin head domain. The binding of the talin head domain to integrin beta tails is specific in that it is abrogated by a single point mutation that disrupts integrin localization to talin-rich focal adhesions. Integrin-cytoskeletal interactions regulate integrin affinity for ligands (activation). Overexpression of a fragment of talin containing the head domain led to activation of integrin alpha(IIb)beta(3); activation was dependent on the presence of both the talin head domain and the integrin beta(3) cytoplasmic tail. The head domain of talin thus binds to integrins to form a link to the actin cytoskeleton and can thus regulate integrin function.

Sequence analysis of rat integrin alpha E1 and alpha E2 subunits: tissue expression reveals phenotypic similarities between intraepithelial lymphocytes and dendritic cells in lymph.

The integrin alpha OX-62 subunit is defined by the OX-62 monoclonal antibody that was raised against rat dendritic cells in lymph (veiled cells) and shows properties similar to those of human alpha E2 that is predominantly expressed on intraepithelial lymphocytes. To clone alpha OX-62, rat probes generated using primers specific for the human alpha E sequence were used to screen rat T cell cDNA libraries. cDNA clones encoding two similar but not identical alpha subunits that are closely related to but distinct from human alpha E were isolated. alpha E1 is predicted to be the rat homolog of mouse alpha M290 and alpha E2 corresponds to rat alpha OX-62. Immunofluorescence analysis revealed that mouse alpha E1 and rat alpha E2 are expressed in dendritic epidermal T cells in the skin, intraepithelial lymphocytes in the small intestine and in cells with a dendritic morphology present at sites where gamma delta T cells occur in lymphoid organs. Unexpectedly, alpha E2 is co-expressed with intracellular CD3-delta and a 33-kDa CD3 chain but not the T cell receptor in veiled cells. These findings suggest that veiled cells may be derived from a lymphoid precursor. Furthermore, veiled cells show phenotypic similarities to intraepithelial lymphocytes.

Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site.

We have determined the sequence of chicken talin (2,541 amino acids, M(r) 271,881) which is very similar (89% identity) to that of the mouse protein. Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent. By expressing overlapping talin polypeptides as fusion proteins, we have identified at least three regions of the protein which can bind F-actin: residues 102-497, 951-1,327 and 2,269-2,541. The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p. Each of the actin-binding sites is close to, but distinct from a binding site for vinculin, a protein which also binds actin. The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin. Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localised weakly to stress fibres, whereas one containing the C-terminal site initially localised predominantly to focal adhesions. The former was readily solubilised, and the latter was resistant to Triton extraction. The N-terminal talin polypeptide eventually disrupted actin stress fibres whereas the C-terminal polypeptide was without effect. However, a larger C-terminal fusion protein also containing a vinculin-binding site did disrupt stress fibres and focal adhesions. The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct.

Ca2+ binding to the first epidermal growth factor-like domain of human blood coagulation factor IX promotes enzyme activity and factor VIII light chain binding.

Ca2+ binding to the first epidermal growth factor (EGF)-like domain of factor IX is known to be required for biological activity, but the mechanism by which Ca2+ contributes to factor IX function has remained unclear. We have studied recombinant factor IX mutants which lack Ca2+ binding to the first EGF-like domain, due to a replacement of Asp64 by Glu, Lys, or Val. The purified mutants (factors IX D64E, D64K, and D64V), were compared to plasma-derived and recombinant wild-type factor IX with regard to a number of metal-ion dependent functional parameters. In the presence of Mg2+, the activated mutants were indistinguishable from normal factor IXa in hydrolyzing the synthetic substrate CH3-SO2-Leu-Gly-Arg-p-nitroanilide. Replacing Mg2+ by Ca2+ further stimulated the activity of normal factor IXa but not of mutant factor IXa. In factor VIII-independent factor X activation, factor IXa D64K and D64E displayed reduced catalytic activity compared to normal factor IXa (apparent kcat/Km approximately 1, 2, and 4 x 10(3) M-1 s-1, respectively). In the presence of factor VIIIa, factor X activation rates by normal and mutant factor IXa were stimulated by factor VIIIa to a different extent ( approximately700- and 200-fold, respectively), indicating that Asp64 replacements affect the interaction with factor VIIIa. This possibility was addressed in inhibition studies employing synthetic peptides comprising the factor IXa-binding motifs of factor VIII heavy or light chains. Whereas the heavy chain peptide (Ser558-Gln565) inhibited factor VIII-dependent factor X activation by normal and mutant factor IXa with similar efficiency, the light chain peptide (Lys1804-Lys1818) inhibited normal factor IXa 2-3-fold more efficiently than did mutant factor IXa. This indicates that the reduced response to factor VIIIa may be due to impaired binding of mutant factor IXa to the factor VIII light chain. This was further explored in direct binding studies. In the presence of Mg2+, normal and mutant factor IXa were similar in binding to the factor VIII light chain. However, in the presence of Ca2+, factor IXa mutants were less efficient than normal factor IXa, which was illustrated by a 4-5-fold lower affinity than normal factor IXa for factor VIII light chain. Collectively, our data demonstrate that a number of factor IXa functions, including enzymatic activity and assembly into the factor IXa-factor VIIIa complex, are dependent on Ca2+ binding to the first EGF-like domain of factor IX.

Isolation and sequence analysis of a cDNA encoding the c subunit of a vacuolar-type H(+)-ATPase from the CAM plant Kalanchoë daigremontiana.

We report the sequence of a cDNA clone encoding the c ("16 kDa') subunit of a vacuolar-type H(+)-ATPase (V-ATPase) from Kalanchoë daigremontiana, a plant in which the cell vacuole plays a pivotal role in crassulacean acid metabolism. The clone, pKVA211, was isolated from a K. daigremontiana leaf cDNA library constructed in lambda ZAP II using a homologous PCR-generated cDNA probe for the V-ATPase c subunit. The KVA211 cDNA was 839 nucleotides long and included a 20 bp poly(A)+ tail together with a complete 495 bp coding region for a polypeptide with a predicted molecular mass of 16659 Da. The deduced amino acid sequence was highly conserved across the wide range of eukaryotes (vertebrates, invertebrates, fungi, plants and protozoa) in which this gene has now been identified. Sequence comparison of several PCR products and genomic Southern analysis indicated that the V-ATPase c subunit in K. daigremontiana is encoded by a small multi-gene family. Steady-state levels of the KVA211 mRNA were much higher in leaves than in roots or flowers, and expression of this transcript in leaves was shown to be strongly light-dependent.

Analysis of repeated motifs in the talin rod.

The amino acid sequence of the rod portion of talin contains strong periodic patterns with a long period of 32 to 34 residues superimposed on short periods of 7 and 7/2 residues. The rod includes 50 to 60 copies of an irregular repeated motif approximately 34 residues long. The motif itself consists of three sections: a short "leader" segment of about six residues, which has a high proportion of the prolines and acidic residues; a relatively well-conserved hydrophobic "core" pattern of approximately 21 residues; and a highly variable "linker" region of seven residues which joins onto the next leader. The core section sequence has many of the characteristics of an amphipathic helix. The extensive hydrophobic side of this postulated helix has a characteristic surface pattern of large and small hydrophobic residues (mainly Leu and Ala), with a strong periodicity of seven residues. It also has a narrow hydrophilic edge with a highly variable sequence. The core sequence is unlike either a normal helical coiled coil or a leucine zipper, because it contains several helical ridges and grooves. The helical cores probably form a tightly packed hydrophobic central strand for the fibrous tail. The leader and linker sections are highly variable in length, so that the spacing between the starting points of adjacent cores varies between 20 and 40 residues. The most common spacing is 34, and many spacings are close to this length.

The cytoskeletal protein talin contains at least two distinct vinculin binding domains.

We have mapped the vinculin-binding sites in the cytoskeletal protein talin as well as those sequences which target the talin molecule to focal contacts. Using a series of overlapping talin-fusion proteins expressed in E. coli and 125I-vinculin in both gel-overlay and microtitre well binding assays, we present evidence for three separable binding sites for vinculin. All three are in the tail segment of talin (residues 434-2541) and are recognized by the same fragment of vinculin (residues 1-258). Two sites are adjacent to each other and span residues 498-950, and the third site is more than 700 residues distant in the primary sequence. Scatchard analysis of 125I-vinculin binding to talin also indicates three sites, each with a similar affinity (Kd = 2-6 x 10(-7) M). We also detect a substoichiometric interaction of higher affinity (Kd = 3 x 10(-8) M) which remains unexplained. By expressing regions of the chicken talin molecule in heterologous cells, we have shown that the sequences required to target talin to focal contacts overlap those which bind vinculin.