Chromosomal organization of adrenergic receptor genes.

The adrenergic receptors (ARs) (subtypes alpha 1, alpha 2, beta 1, and beta 2) are a prototypic family of guanine nucleotide binding regulatory protein-coupled receptors that mediate the physiological effects of the hormone epinephrine and the neurotransmitter norepinephrine. We have previously assigned the genes for beta 2- and alpha 2-AR to human chromosomes 5 and 10, respectively. By Southern analysis of somatic cell hybrids and in situ chromosomal hybridization, we have now mapped the alpha 1-AR gene to chromosome 5q32----q34, the same position as beta 2-AR, and the beta 1-AR gene to chromosome 10q24----q26, the region where alpha 2-AR is located. In mouse, both alpha 2- and beta 1-AR genes were assigned to chromosome 19, and the alpha 1-AR locus was localized to chromosome 11. Pulsed field gel electrophoresis has shown that the alpha 1- and beta 2-AR genes in humans are within 300 kilobases (kb) and the distance between the alpha 2- and beta 1-AR genes is less than 225 kb. The proximity of these two pairs of AR genes and the sequence similarity that exists among all the ARs strongly suggest that they are evolutionarily related. Moreover, they likely arose from a common ancestral receptor gene and subsequently diverged through gene duplication and chromosomal duplication to perform their distinctive roles in mediating the physiological effects of catecholamines. The AR genes thus provide a paradigm for understanding the evolution of such structurally conserved yet functionally divergent families of receptor molecules.

The origin and evolution of phototropins.

Plant phototropism, the ability to bend toward or away from light, is predominantly controlled by blue-light photoreceptors, the phototropins. Although phototropins have been well-characterized in Arabidopsis thaliana, their evolutionary history is largely unknown. In this study, we complete an in-depth survey of phototropin homologs across land plants and algae using newly available transcriptomic and genomic data. We show that phototropins originated in an ancestor of Viridiplantae (land plants + green algae). Phototropins repeatedly underwent independent duplications in most major land-plant lineages (mosses, lycophytes, ferns, and seed plants), but remained single-copy genes in liverworts and hornworts-an evolutionary pattern shared with another family of photoreceptors, the phytochromes. Following each major duplication event, the phototropins differentiated in parallel, resulting in two specialized, yet partially overlapping, functional forms that primarily mediate either low- or high-light responses. Our detailed phylogeny enables us to not only uncover new phototropin lineages, but also link our understanding of phototropin function in Arabidopsis with what is known in Adiantum and Physcomitrella (the major model organisms outside of flowering plants). We propose that the convergent functional divergences of phototropin paralogs likely contributed to the success of plants through time in adapting to habitats with diverse and heterogeneous light conditions.

Identification of dopamine receptors across the extant avian family tree and analysis with other clades uncovers a polyploid expansion among vertebrates.

Dopamine is an important central nervous system transmitter that functions through two classes of receptors (D1 and D2) to influence a diverse range of biological processes in vertebrates. With roles in regulating neural activity, behavior, and gene expression, there has been great interest in understanding the function and evolution dopamine and its receptors. In this study, we use a combination of sequence analyses, microsynteny analyses, and phylogenetic relationships to identify and characterize both the D1 (DRD1A, DRD1B, DRD1C, and DRD1E) and D2 (DRD2, DRD3, and DRD4) dopamine receptor gene families in 43 recently sequenced bird genomes representing the major ordinal lineages across the avian family tree. We show that the common ancestor of all birds possessed at least seven D1 and D2 receptors, followed by subsequent independent losses in some lineages of modern birds. Through comparisons with other vertebrate and invertebrate species we show that two of the D1 receptors, DRD1A and DRD1B, and two of the D2 receptors, DRD2 and DRD3, originated from a whole genome duplication event early in the vertebrate lineage, providing the first conclusive evidence of the origin of these highly conserved receptors. Our findings provide insight into the evolutionary development of an important modulatory component of the central nervous system in vertebrates, and will help further unravel the complex evolutionary and functional relationships among dopamine receptors.

An alpha 2-macroglobulin-like protein is the cue to gregarious settlement of the barnacle Balanus amphitrite

Many benthic marine invertebrates, like barnacles, have a planktonic larval stage whose primary purpose is dispersal. How these species colonize suitable substrata is fundamental to understanding their evolution, population biology, and wider community dynamics. Unlike larval dispersal, settlement occurs on a relatively small spatial scale and involves larval behavior in response to physical and chemical characteristics of the substratum. Biogenic chemical cues have been implicated in this process. Their identification, however, has proven challenging, no more so than for the chemical basis of barnacle gregariousness, which was first described >50 years ago. We now report that a biological cue to gregarious settlement, the settlement-inducing protein complex (SIPC), of the major fouling barnacle Balanus amphitrite is a previously undescribed glycoprotein. The SIPC shares a 30% sequence homology with the thioester-containing family of proteins that includes the alpha sub(2)-macroglobulins. The cDNA (5.2 kb) of the SIPC encodes a protein precursor comprising 1,547 aa with a 17-residue signal peptide region. A number of structural characteristics and the absence of a thioester bond in the SIPC suggest that this molecule is a previously undescribed protein that may have evolved by duplication from an ancestral alpha sub(2)-macroglobulin gene. Although the SIPC is regarded as an adult cue that is recognized by the cyprid at settlement, it is also expressed in the juvenile and in larvae, where it may function in larva-larva settlement interactions.

Intragenic SNP haplotypes associated with 84dup18 mutation in TNFRSF11A in four FEO pedigrees suggest three independent origins for this mutation.

Familial expansile osteolysis (FEO) is a rare disorder causing bone dysplasia. The clinical features of FEO include early-onset hearing loss, tooth destruction, and progressive lytic expansion within limb bones causing pain, fracture, and deformity. An 18-bp duplication in the first exon of the TNFRSF11A gene encoding RANK has been previously identified in four FEO pedigrees. Despite having the identical mutation, phenotypic variations among affected individuals of the same and different pedigrees were noted. Another 18-bp duplication, one base proximal to the duplication previously reported, was subsequently found in two unrelated FEO patients. Finally, mutations overlapping with the mutations found in the FEO pedigrees have been found in ESH and early-onset PDB pedigrees. An Iranian FEO pedigree that contains six affected individuals dispersed in three generations has previously been introduced; here, the clinical features of the proband are reported in greater detail, and the genetic defect of the pedigree is presented. Direct sequencing of the entire coding region and upstream and downstream noncoding regions of TNFRSF11A in her DNA revealed the same 18-bp duplication mutation as previously found in the four FEO pedigrees. Additionally, eight sequence variations as compared to the TNFRSF11A reference sequence were identified, and a haplotype linked to the mutation based on these variations was defined. Although the mutation in the Iranian and four of the previously described FEO pedigrees was the same, haplotypes based on the intragenic SNPs suggest that the mutations do not share a common descent.

Detection of the icaADBC gene cluster and biofilm formation in Staphylococcus epidermidis isolates from catheter-related urinary tract infections.

Cho SH, Naber K, Hacker J, Ziebuhr W. Institut für Molekulare Infektionsbiologie, Röntgenring 11, D-97070 Würzburg, Germany. Biofilm production in Staphylococcus epidermidis is an important virulence factor that is mediated by the expression of the icaADBC operon. In this study 41 S. epidermidis isolates obtained from catheter-related urinary tract infections were analyzed for the presence of the icaADBC operon and biofilm formation. Eighteen of 41 isolates (44%) were shown to carry ica-specific DNA, but only 11 isolates (27%) produced biofilms spontaneously under normal growth conditions. Upon induction by external stress or antibiotics, biofilm formation could be stimulated in five of seven ica-positive, biofilm-negative isolates, indicating that the icaADBC expression was down-regulated in these strains. Genetic analyses of the ica gene clusters of the remaining two ica-positive, biofilm-negative strains revealed a spontaneous ICAC::IS256 insertion in one strain. Insertion of the element caused a target site duplication of seven base pairs and a biofilm-negative phenotype. After repeated passages the insertion mutant was able to revert to a biofilm-forming phenotype which was due to the precise excision of IS256 from the icaC gene. The data show that icaC::IS256 integrations occur during S. epidermidis polymer-related infections and the results highlight the biological relevance of the IS256-mediated phase variation of biofilm production in S. epidermidis during an infection.

HOX genes: seductive science, mysterious mechanisms

HOX genes are evolutionarily highly conserved. The HOX proteins which they encode are master regulators of embryonic development and continue to be expressed throughout postnatal life. The 39 human HOX genes are located in four clusters (A-D) on different chromosomes at 7p15, 17q21 [corrected] 12q13, and 2q31 respectively and are assumed to have arisen by duplication and divergence from a primordial homeobox gene. Disorders of limb formation, such as hand-foot-genital syndrome, have been traced to mutations in HOXA13 and HOXD13. Evolutionary conservation provides unlimited scope for experimental investigation of the functional control of the Hox gene network which is providing important insights into human disease. Chromosomal translocations involving the MLL gene, the human homologue of the Drosophila gene trithorax, create fusion genes which exhibit gain of function and are associated with aggressive leukaemias in both adults and children. To date 39 partner genes for MLL have been cloned from patients with leukaemia. Models based on specific translocations of MLL and individual HOX genes are now the subject of intense research aimed at understanding the molecular programs involved, and ultimately the design of chemotherapeutic agents for leukaemia. Investigation of the role of HOX genes in cancer has led to the concept that oncology may recapitulate ontology, a challenging postulate for experimentalists in view of the functional redundancy implicit in the HOX gene network.

The DUB/USP17 deubiquitinating enzymes: A gene family within a tandemly repeated sequence, is also embedded within the copy number variable Beta-defensin cluste

Background: The DUB/USP17 subfamily of deubiquitinating enzymes were originally identified as immediate early genes induced in response to cytokine stimulation in mice (DUB-1, DUB-1A, DUB-2, DUB-2A). Subsequently we have identified a number of human family members and shown that one of these (DUB-3) is also cytokine inducible. We originally showed that constitutive expression of DUB-3 can block cell proliferation and more recently we have demonstrated that this is due to its regulation of the ubiquitination and activity of the 'CAAX' box protease RCE1.

Results: Here we demonstrate that the human DUB/USP17 family members are found on both chromosome 4p16.1, within a block of tandem repeats, and on chromosome 8p23.1, embedded within the copy number variable betadefensin cluster. In addition, we show that the multiple genes observed in humans and other distantly related mammals have arisen due to the independent expansion of an ancestral sequence within each species. However, it is also apparent when sequences from humans and the more closely related chimpanzee are compared, that duplication events have taken place prior to these species separating.

Conclusions: The observation that the DUB/USP17 genes, which can influence cell growth and survival, have evolved from an unstable ancestral sequence which has undergone multiple and varied duplications in the species examined marks this as a unique family. In addition, their presence within the beta-defensin repeat raises the question whether they may contribute to the influence of this repeat on immune related conditions.

Novel Venom Proteins Produced by Differential Domain-Expression Strategies in Beaded Lizards and Gila Monsters (genus Heloderma)

The origin and evolution of venom proteins in helodermatid lizards were investigated by multidisciplinary techniques. Our analyses elucidated novel toxin types resultant from three unique domain-expression processes: 1) The first full-length sequences of lethal toxin isoforms (helofensins) revealed this toxin type to be constructed by an ancestral monodomain, monoproduct gene (beta-defensin) that underwent three tandem domain duplications to encode a tetradomain, monoproduct with a possible novel protein fold; 2) an ancestral monodomain gene (encoding a natriuretic peptide) was medially extended to become a pentadomain, pentaproduct through the additional encoding of four tandemly repeated proline-rich peptides (helokinestatins), with the five discrete peptides liberated from each other by posttranslational proteolysis; and 3) an ancestral multidomain, multiproduct gene belonging to the vasoactive intestinal peptide (VIP)/glucagon family being mutated to encode for a monodomain, monoproduct (exendins) followed by duplication and diversification into two variant classes (exendins 1 and 2 and exendins 3 and 4). Bioactivity characterization of exendin and helokinestatin elucidated variable cardioactivity between isoforms within each class. These results highlight the importance of utilizing evolutionary-based search strategies for biodiscovery and the virtually unexplored potential of lizard venoms in drug design and discovery.

Trisomy 10p with clinical features of facio-auriculo-vertebral spectrum: a case report

We report a male child born with complete absence of his external ear, hemifacial microsomia of the right side, high arched palate, a down-turned upper lip and slightly upslanting palpebral fissures. The features were suggestive of facio-auriculo-vertebral spectrum. Investigations showed a tandem duplication of the short arm of one chromosome 10 with apparent breakpoints at p14 and p15. This case extends the list of chromosomal abnormalities associated with the facio-auriculo-vertebral phenotype and also adds useful clinical information to possible trisomy 10p phenotypes.

Establishing a minimum dataset for prospective registration of systematic reviews: an international consultation

Background: In response to growing recognition of the value of prospective registration of systematic review protocols, we planned to develop a web-based open access international register. In order for the register to fulfil its aims of reducing unplanned duplication, reducing publication bias, and providing greater transparency, it was important to ensure the appropriate data were collected. We therefore undertook a consultation process with experts in the field to identify a minimum dataset for registration. Methods and Findings: A two-round electronic modified Delphi survey design was used. The international panel surveyed included experts from areas relevant to systematic review including commissioners, clinical and academic researchers, methodologists, statisticians, information specialists, journal editors and users of systematic reviews. Direct invitations to participate were sent out to 315 people in the first round and 322 in the second round. Responses to an open invitation to participate were collected separately. There were 194 (143 invited and 51 open) respondents with a 100% completion rate in the first round and 209 (169 invited and 40 open) respondents with a 91% completion rate in the second round. In the second round, 113 (54%) of the participants reported having previously taken part in the first round. Participants were asked to indicate whether a series of potential items should be designated as optional or required registration items, or should not be included in the register. After the second round, a 70% or greater agreement was reached on the designation of 30 of 36 items. Conclusions: The results of the Delphi exercise have established a dataset of 22 required items for the prospective registration of systematic reviews, and 18 optional items. The dataset captures the key attributes of review design as well as the administrative details necessary for registration.

Home medication management practices and associated factors among patients with selected chronic diseases in a community pharmacy in Uganda

ABSTRACT: BACKGROUND: Chronic diseases are rapidly increasing and are currently the major cause of death and disability worldwide. Patients with chronic diseases experience many challenges including medicine-related problems. However, there is limited information about the home management of medicines among these patients. This study therefore was to determine home medication management practices and associated factors among patients with chronic diseases seeking care in a community pharmacy in Uganda. METHODS: A cross-sectional study was conducted in a community pharmacy in Kampala from June to July 2010. A total of 207 consenting chronic disease patients or caregivers of children with chronic disease were consecutively sampled. The patients were visited at home to evaluate their drug management practices and to check their medical forms for disease types and drugs prescribed. An interviewer-administered questionnaire and an observation checklist were used to collect the data. RESULTS: Overall home medication management was inappropriate for 70% (n = 145) of the participants (95% CI = 63.3-76.2) and was associated with perceived severity of disease (not severe OR =0.40, moderately severe OR = 0.35), duration of disease >5 years (OR = 2.15), and health worker not assessing for response to treatment (OR = 2.53). About 52% (n = 107) had inappropriate storage which was associated with inadequate information about the disease (OR = 2.39) and distance to the health facility >5 kilometres (OR = 2.82). Fifteen percent (n = 31) had no drug administration schedule and this was associated with increasing age (OR = 0.97), inadequate information about the disease (OR = 2.96), and missing last appointment for medical review (OR = 6.55). About 9% (n = 18) had actual medication duplication; 1.4% (n = 3) had expired medicines; while 18.4% (n = 38) had drug hoarding associated with increasing number of prescribers (OR = 1.34) and duration of disease (OR = 2.06). About 51% (n = 105) had multiple prescribers associated with perceiving the disease to be non severe (OR = 0.27), and having more than one chronic disease (OR = 2.37). CONCLUSIONS: Patients with chronic disease have poor home management of medicines. In order to limit the occurrence of poor outcomes of treatment or drug toxicity, health providers need to strengthen the education of patients with chronic disease on how to handle their medicines at home.

Helminth pathogen cathepsin proteases:it's a family affair

Helminth pathogens express papain-like cysteine peptidases, termed cathepsins, which have important roles in virulence, including host entry, tissue migration and the suppression of host immune responses. The liver fluke Fasciola hepatica, an emerging human pathogen, expresses the largest cathepsin L cysteine protease family yet described. Recent phylogenetic, biochemical and structural studies indicate that this family contains five separate clades, which exhibit overlapping but distinct substrate specificities created by a process of gene duplication followed by subtle residue divergence within the protease active site. The developmentally regulated expression of these proteases correlates with the passage of the parasite through host tissues and its encounters with different host macromolecules.

Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica:Expansion of a repertoire of virulence-associated factors

Cathepsin L proteases secreted by the helminth pathogen Fasciola hepatica have functions in parasite virulence including tissue invasion and suppression of host immune responses. Using proteomics methods alongside phylogenetic studies we characterized the profile of cathepsin L proteases secreted by adult F. hepatica and hence identified those involved in host-pathogen interaction. Phylogenetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) and two clades specific to infective juvenile stages (Clades 3 and 4). Consistent with these observations our proteomics studies identified representatives from Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F. hepatica secretory products. Clades 1 and 2 account for 67.39 and 27.63% of total secreted cathepsin Ls, respectively, suggesting that their expansion was positively driven and that these proteases are most critical for parasite survival and adaptation. Sequence comparison studies revealed that the expansion of cathepsin Ls by gene duplication was followed by residue changes in the S2 pocket of the active site. Our biochemical studies showed that these changes result in alterations in substrate binding and suggested that the divergence of the cathepsin L family produced a repertoire of enzymes with overlapping and complementary substrate specificities that could cleave host macromolecules more efficiently. Although the cathepsin Ls are produced as zymogens containing a prosegment and mature domain, all secreted enzymes identified by MS were processed to mature active enzymes. The prosegment region was highly conserved between the clades except at the boundary of prosegment and mature enzyme. Despite the lack of conservation at this section, sites for exogenous cleavage by asparaginyl endopeptidases and a Leu-Ser[downward arrow]His motif for autocatalytic cleavage by cathepsin Ls were preserved.

Secretion and processing of a novel multi-domain cystatin-like protein by intracellular stages of Trichinella spiralis

The excretory-secretory (ES) proteins of nematode parasites are of major interest as they function at the host-parasite interface and are likely to have roles crucial for successful parasitism. Furthermore, the ES proteins of intracellular nematodes such as Trichinella spiralis may also function to regulate gene expression in the host cell. In a recent proteomic analysis we identified a novel secreted cystatin-like protein from T. spiralis L1 muscle larva. Here we show that the protein, MCD-1 (multi-cystatin-like domain protein 1), contains three repeating cystatin-like domains and analysis of the mcd-1 gene structure suggests that the repeated domains arose from duplication of an ancestral cystatin gene. Cystatins are a diverse group of cysteine protease inhibitors and those secreted by parasitic nematodes are important immuno-modulatory factors. The cystatin superfamily also includes cystatin-like proteins that have no cysteine protease inhibitory activity. A recombinant MCD-1 protein expressed as a GST-fusion protein in Escherichia coli failed to inhibit papain in vitro suggesting that the T. spiralis protein is a new member of the non-inhibitory cystatin-related proteins. MCD-1 secreted from T. spiralis exists as high- and low-molecular weight isoforms and we show that a recombinant MCD-1 protein secreted by HeLa cells undergoes pH-dependent processing that may result in the release of individual cystatin-like domains. Furthermore, we found that mcd-1 gene expression is largely restricted to intracellular stages with the highest levels of expression in the adult worms. It is likely that the major role of the protein is during the intestinal stage of T. spiralis infections.