Membrane Association via an Amino-terminal Amphipathic Helix Is Required for the Cellular Organization and Function of RNase II

The subcellular localization of the exoribonuclease RNase II is not known despite the advanced biochemical characterization of the enzyme. Here we report that RNase II is organized into cellular structures that appear to coil around the Escherichia coli cell periphery and that RNase II is associated with the cytoplasmic membrane by its amino-terminal amphipathic helix. The helix also acts as an autonomous transplantable membrane binding domain capable of directing normally cytoplasmic proteins to the membrane. Assembly of the organized cellular structures of RNase II required the RNase II amphipathic membrane binding domain. Co-immunoprecipitation of the protein from cell extracts indicated that RNase II interacts with itself. The RNase II self-interaction and the ability of the protein to assemble into organized cellular structures required the membrane binding domain. The ability of RNase II to maintain cell viability in the absence of the exoribonuclease polynucleotide phosphorylase was markedly diminished when the RNase II cellular structures were lost due to changes in the amphipathicity of the amino-terminal helix, suggesting that membrane association and assembly of RNase II into organized cellular structures play an important role in the normal function of the protein within the bacterial cell.     

Comparative phylogeography of six herpetofauna species in Cyprus: late Miocene to Pleistocene colonization routes

The colonization patterns of oceanic islands are often interpreted through transmarine dispersal. However, in islands with intense human activities and unclear geological history, this inference may be inappropriate. Cyprus is such an island, whose geotectonic evolution has not been clarified yet to the desired level for biogeographical reconstructions, leaving the questions of ‘how the Cypriote biota arrived’ and ‘does the dispersal have the formative role in patterns of its diversification’ unanswered. Here, we address these issues through a reconstruction of the evolutionary history of six herptiles (Ablepharus budaki, Ophisops elegans, Acanthodactylus schreiberi, Telescopus fallax, Pelophylax cf. bedriagae, and Hyla savignyi) by means of mitochondrial DNA (cytochrome b and 16S rRNA), applying a Bayesian phylogenetic, biogeographical, and chronophylogenetic analyses. The phylogeographical analyses show that the colonization history of those species in Cyprus started in the late Miocene and extended into the Pliocene and Pleistocene, with geodispersal, transmarine dispersal, and human‐mediated dispersal having their share in shaping the diversification of Cypriote herptiles. The revealed patterns could be divided into three biogeographical categories: old colonizers that arrived in Cyprus during the late Miocene or early Pliocene either by a land bridge (geodispersal) which connected Cyprus with the mainland or by transmarine dispersal, younger colonizers that reached the island through transmarine dispersal from the Middle East, and new settlers that arrived through human‐induced (voluntary or not) introductions. This work advances our knowledge of the biogeography of Cyprus and highlights the need to consider both geo‐ and transmarine dispersal when dealing with islands whose associations do not have a straightforward interpretation. © 2013 The Linnean Society of London     

Selection of optimal stage for protocorm-like bodies and production of artificial seeds for direct regeneration on different media and short term storage of Dendrobium Shavin White

Micropropagation is currently the most popular method for orchid propagation through the production of protocorm-like bodies (PLBs). It is suggested that converting the PLBs into artificial seeds by encapsulation with sodium alginate can be useful for short-term preservation and distribution to the laboratories and commercial nurseries. Prior to the production of artificial seeds, the best developmental stage of PLBs based on sizes for increased conversion to plantlet was determined. PLBs were categorized based on size and presence of shoot namely ≤2 mm (S1), >2–4 mm (S2), >4–6 mm (S3), >2–4 mm with shoot (S4) and >4–6 mm with shoot (S5). S4 and S5 gave significantly higher conversion percentage (85 and 90 %, respectively) as compared to the PLBs without shoot (S1, S2 and S3). Thus, for uniformity PLBs of 3–5 mm with shoot were used for encapsulation with sodium alginate to form artificial seeds. The feasibility of germinating artificial seeds of Dendrobium Shavin White in different substrates namely; M1 (semi-solid ½ Murashige and Skoog (1962) basal medium), M2 (cotton irrigated with sterilized liquid ½ MS basal medium), M3 (cotton irrigated with sterilized distilled water) and M4 (cotton irrigated with non-sterilized distilled water) was tested. The encapsulated PLBs regenerated well in M1 where 96 % of encapsulated PLBs germinated after 12 days of inoculation and 76 % of them converted into plantlet after 37 days of inoculation while PLBs subjected to sterile distilled water gave 56 % germination and 44 % conversion after 42 and 167 days of inoculation respectively. The ability to store encapsulated PLBs would be advantageous for transport of planting materials. Encapsulated PLBs survived longer when stored at 25 ± 2 °C compared to 4 °C, 10 °C and 30 ± 2 °C whereby storage up to 75 days retained 80–92 % survival. Further storage up to 135 days retained 52 % survival. All plantlets survived after acclimatization when transferred to charcoal media under shade.     

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP)

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.     

Novel 3-hydroxy vinylboronates influence sphingolipid metabolism,cause apoptosis in Jurkat cells and prevent tumor development in nude mice

A series of novel 3-hydroxy vinylboronates which share structural similarities with sphingolipids were synthesized and tested in vitro and in vivo as anticancer agents. The molecules reduced cancer cell survival in vitro by influencing their sphingolipid metabolism. In a cancer model in nude mice the lead compound E7 prevented the development of tumor as long as the treatment period continued. Moreover, it delayed tumor growth after the treatment was finished.     

Effect of sulfonamides as carbonic anhydrase VA and VB inhibitors on mitochondrial metabolic energy conversion

Obesity is quickly becoming an increasing problem in the developed world. One of the major fundamental causes of obesity and diabetes is mitochondria dysfunction due to faulty metabolic pathways which alter the metabolic substrate flux resulting in the development of these diseases. This paper examines the role of mitochondrial carbonic anhydrase (CA) isozymes in the metabolism of pyruvate, acetate, and succinate when specific isozyme inhibitors are present. Using a sensitive electrochemical approach of wired mitochondria to analytically measure metabolic energy conversion, we determine the resulting metabolic difference after addition of an inhibitory compound. We found that certain sulfonamide analogues displayed broad spectrum inhibition of metabolism, where others only had significant effect on some metabolic pathways. Pyruvate metabolism always displayed the most dramatically affected metabolism by the sulfonamides followed by fatty acid metabolism, and then finally succinate metabolism. This allows for the possibility of using designed sulfonamide analogues to target specific mitochondrial CA isozymes in order to subtly shift metabolism and glucogenesis flux to treat obesity and diabetes.     

Phylogenetic relationships of the populations of Iranolacerta brandtii (de Filippi, 1863) (Squamata: Lacertidae) recently found in Eastern Anatolia,Turkey

The Persian Lizard, Iranolacerta brandtii, was until recently considered to be restricted to north-western Iran (Azerbaijan and Esfahan provinces). However, two recent studies have revealed the existence of populations in Eastern Anatolia, extending the range of this species for about 230?km westwards. The fragmented distribution of this species has been considered to be a consequence of the climatic oscillations during the Pleistocene and Holocene, which created events of alternating contact and isolation of populations in distinct glacial refugia. According to our obtained genealogy derived from three mitochondrial fragments (12S rRNA, 16S rRNA and cytb), the Turkish specimens cluster together but form an independent clade, sister to the individuals from Tabriz in Iran. The separation of these two clades is concurrent with the cladogenesis between the Esfahan and Ardabil clades, estimated to have taken place during the late Holocene.     

Microsatellite markers from expressed sequence tags (ESTs) of seaweeds in differentiating various Gracilaria species

Gracilaria is a red seaweed that has been cultivated worldwide and is commercially used for food, fertilizers, animal fodder, and phycocolloids. However, the high morphological plasticity of seaweeds often leads to the misidentification in the traditional identification of Gracilaria species. Molecular markers are important especially in the correct identification of Gracilaria species with high economic value. Microsatellite markers were developed from the expressed sequence tags of seaweeds deposited at the National Center for Biotechnology Information database and used for differentiating Gracilaria changii collected at various localities and two other Gracilaria species. Out of 33 primer pairs, only one primer pair gave significant results that can distinguish between three different Gracilaria species as well as G. changii from various localities based on the variation in repeated nucleotides. The unweighted pair group method using arithmetic mean dendrogram analysis grouped Gracilaria species into five main clades: (a) G. changii from Batu Besar (Malacca), Sandakan (Sabah), Bintulu (Sarawak), Batu Tengah (Malacca), Gua Tanah (Malacca), Middle Banks (Penang), Sungai (Sg.) Merbok (Kedah), Teluk Pelandok (Negeri Sembilan), Pantai Dickson (Negeri Sembilan), Sg. Kong-Kong (Johore), and Sg. Pulai (Johore); (b) Gracilaria manilaensis from Cebu, Philippines; (c) G. changii from Morib (Selangor); (d) Gracilaria fisheri from Pattani, Thailand; and (e) G. changii from Pantai Dickson (Negeri Sembilan), Gua Tanah (Malacca), Sg. Merbok (Kedah), Sg. Kong-Kong (Johore), and Sg. Pulai (Johore). This result shows that this primer pair was able to distinguish between three different species, which are G. changii from Morib (Malaysia), G. fisheri from Pattani (Thailand), and G. manilaensis from Cebu (Philippines), and also between different genotypes of G. changii. This suggested that the simple sequence repeat primer we developed was suitable for differentiating between different Gracilaria species due to the polymorphisms caused by the variability in the number of tandem repeats.     

Oral Ingestion of Transgenic RIDL Ae. aegypti Larvae Has No Negative Effect on Two Predator Toxorhynchites Species

Dengue is the most important mosquito-borne viral disease. No specific treatment or vaccine is currently available; traditional vector control methods can rarely achieve adequate control. Recently, the RIDL (Release of Insect carrying Dominant Lethality) approach has been developed, based on the sterile insect technique, in which genetically engineered ‘sterile’ homozygous RIDL male insects are released to mate wild females; the offspring inherit a copy of the RIDL construct and die. A RIDL strain of the dengue mosquito, Aedes aegypti, OX513A, expresses a fluorescent marker gene for identification (DsRed2) and a protein (tTAV) that causes the offspring to die. We examined whether these proteins could adversely affect predators that may feed on the insect. Aedes aegypti is a peri-domestic mosquito that typically breeds in small, rain-water-filled containers and has no specific predators. Toxorhynchites larvae feed on small aquatic organisms and are easily reared in the laboratory where they can be fed exclusively on mosquito larvae. To evaluate the effect of a predator feeding on a diet of RIDL insects, OX513A Ae. aegypti larvae were fed to two different species of Toxorhynchites (Tx. splendens and Tx. amboinensis) and effects on life table parameters of all life stages were compared to being fed on wild type larvae. No significant negative effect was observed on any life table parameter studied; this outcome and the benign nature of the expressed proteins (tTAV and DsRed2) indicate that Ae. aegypti OX513A RIDL strain is unlikely to have any adverse effects on predators in the environment.