Remarkable genetic diversity detected at river buffalo prolactin receptor (PRLR) gene and association studies with milk fatty acid composition

Prolactin is an anterior pituitary peptide hormone involved in many different endocrine activities and is essential for reproductive performance. This action is mediated by its receptor, the prolactin receptor, encoded by the PRLR gene. In this study, we sequenced and characterized the Mediterranean river buffalo PRLR gene (from exon 3 to 10), and we found remarkable genetic diversity. In particular, we found 24 intronic polymorphisms and 13 exonic SNPs, seven of which were non‐synonymous. Furthermore, the polymorphisms identified in the 3′‐UTR were investigated to establish their possible influence on microRNA binding sites. Considering all the amino acid changes and the observed allelic combinations, it is possible to deduce at least six different translations of the buffalo prolactin receptor and, consequently, the presence at the PRLR gene of at least six alleles. Furthermore, we identified a deletion of a CACTACC heptamer between nucleotides 1102 and 1103 of exon 10 (3′‐UTR), and we developed an allele‐specific PCR to identify the carriers of this genetic marker. Finally, the SNP g.11188A>G, detected in exon 10 and responsible for the amino acid replacement p.His328Arg, was genotyped in 308 Italian Mediterranean river buffaloes, and an association study with milk fat traits was carried out. The statistical analysis showed a tendency that approached significance for the AA genotype with higher contents of odd branched‐chain fatty acids. Thus, our results suggest that the PRLR gene is a good candidate for gene association studies with qualitative traits related to buffalo milk production.     

Synthetic analogues of durantoside I from Citharexylum spinosum L. and their cytotoxic activity

New iridoid glycoside derivatives from durantoside I, the latter from the dried flowers and leaves of Citharexylum spinosum, were synthesized by variously modifying a sugar moiety by silylation or acetylation and/or removal of cinnamate group at C-7 position and subsequent screening for comparative cytotoxicity against several cancer cell lines. Addition of alkylsilane to durantoside I and removal of cinnamate group were most effective in improving cytotoxicity.     

Qualitative and quantitative insights into the 3D microanatomy of the nervous ganglia of Scrobicularia plana (Bivalvia: Tellinoidea: Semelidae)

The nervous system of bivalves is bilaterally symmetrical and consists of interconnected cerebropleural, pedal and visceral ganglia, which may be partially to totally fused. We studied the microanatomy of the ganglia of Scrobicularia plana using three-dimensional (3D) reconstruction. We also examined whether intersex differences in the neural structure exist. Each type of ganglion had a characteristic 3D shape, and the cerebropleural ganglia shape was slightly asymmetrical. The visceral, pedal and cerebropleural ganglia are progressively smaller in volume, but only the pedal ganglion volume was positively correlated with the animal’s length, height or width; suggesting functional implications. As to total surface area, correlations were found for the cerebropleural and visceral ganglia, but it was the visceral that consistently showed strong positive correlations with each biometric parameter. The medulla may often penetrate the cortex and touch the capsule in areas that (contrary to what might be expected) are not connected with emerging nerves. Despite the differences in volume and surface area among ganglia, the volume ratio of cortex/medulla is fairly stable (c. 1.5), suggesting a functional optimum. Finally, we conclude that the ganglia of males and females do not show significant quantitative differences.     

Synthesis of 14-deoxy-11,12-didehydroandrographolide analogues as potential cytotoxic agents for cholangiocarcinoma

A series of 14-deoxy-11,12-didehydroandrographolide analogues were synthesized from naturally occurring andrographolide and their cytotoxicity evaluated against nine cancer cell lines including cholangiocarcinoma. Analogues 5a and 5b exhibited the most potent cytotoxicity with ED₅₀s of 3.37 and 3.08?μM on KKU-M213 cell lines and 2.93 and 3.27?μM on KKU-100 cell lines, respectively. Selective cytotoxicity on cholangiocarcinoma cell lines identified in this study highlight the importance of structural modification in the development of drugs for this cancer.     

Structural variation in bacterial glyoxalase I enzymes: investigation of the metalloenzyme glyoxalase I from Clostridium acetobutylicum

The glyoxalase system catalyzes the conversion of toxic, metabolically produced α-ketoaldehydes, such as methylglyoxal, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the glyoxalase system, glyoxalase I (GlxI), from yeast, protozoa, animals, humans, plants, and Gram-negative bacteria, have suggested two metal activation classes, Zn(2+) and non-Zn(2+) activation. Here, we report a biochemical and structural investigation of the GlxI from Clostridium acetobutylicum, which is the first GlxI enzyme from Gram-positive bacteria that has been fully characterized as to its three-dimensional structure and its detailed metal specificity. It is a Ni(2+)/Co(2+)-activated enzyme, in which the active site geometry forms an octahedral coordination with one metal atom, two water molecules, and four metal-binding ligands, although its inactive Zn(2+)-bound form possesses a trigonal bipyramidal geometry with only one water molecule liganded to the metal center. This enzyme also possesses a unique dimeric molecular structure. Unlike other small homodimeric GlxI where two active sites are located at the dimeric interface, the C. acetobutylicum dimeric GlxI enzyme also forms two active sites but each within single subunits. Interestingly, even though this enzyme possesses a different dimeric structure from previously studied GlxI, its metal activation characteristics are consistent with properties of other GlxI. These findings indicate that metal activation profiles in this class of enzyme hold true across diverse quaternary structure arrangements.     

New substituted C-19-andrographolide analogues with potent cytotoxic activities

Andrographolide, the major diterpenoid lactone from Andrographis paniculata, is toxic against cancer cells. In the present study, we investigated the structure-activity relationships (SARs) of 19 andrographolide analogues which were synthesized by modification at the three hydroxyl groups. A number of the andrographolide analogues showed much higher cytotoxic activities than that of the parent compound on cancer cells including P-388, KB, COL-2, MCF-7, LU-1 and ASK cells. SAR studies of the synthetic analogues indicated that the introduction of silyl ether or triphenylmethyl ether group into C-19 of the parent compound led to increase in toxicity against the cancer cells. The 19-O-triphenylmethyl ether analogue 18 showed higher cytotoxic activity than the potent anticancer drug ellipticine, and this analogue may serve as a potential structure lead for the development of new anticancer drugs.     

An efficient method for the selective synthesis of 2-deoxy-2-iodo-glycosides by O-glycosidation of D-glucal using I₂-Cu(OAc)₂

An efficient and convenient method for the synthesis of 2-deoxy-2-iodo-O-glycosides from tri-O-acetyl-d-glucal with various alcohols by using I₂-Cu(OAc)₂ is described. The 21 examples of corresponding glycosides were obtained in high yields, with good anomeric selectivity.     

In vitro culture of glochidia from the freshwater mussel Anodonta cygnea

Abstract. Larvae of the freshwater swan mussel, Anodonta cygnea , were cultured in artificial media at the controlled temperature of 23°±2°C, with successful metamorphosis for the first time. The artificial medium contained a mixture of M199, common carp plasma, and antibiotics/antimycotics. Glochidia were reared to the juvenile stage in the medium after 10–11 d of culture. After 15 d of controlled feeding with phytoplankton, the juveniles showed an elongated shell with several growth lines. Larval survival was 34.3±9.3%, whereas the proportion undergoing metamorphosis was ≤60.8±4.2%. The ultrastructure of early developmental stages was observed by scanning electron microscopy, from the glochidial to the juvenile stage. Glochidia had a hooked shell, with two equal triangular valves formed by a calcareous layer with numerous pores and covered by a thin cuticle of chitin–keratin. The appearance of the complete foot within 11 d of in vitro culture was considered the final feature of metamorphosis to the juvenile stage. The main alteration during juvenile development was the formation, under the glochidial shell, of a new periostracum with growth lines. The prominent foot, gradually covered by long, dense cilia, showed rhythmical movements involved in the capture of particulate matter. Similarly, cilia and microvilli present in the mantle also performed the same role. Longer cilia, sparsely distributed in the mantle, may function as chemotactile sensors.     

Molecular identification of the economically important freshwater mussels (Mollusca–Bivalvia–Unionoida) of Thailand: developing species‐specific markers from AFLPs

Shells of certain freshwater mussel (Unionoida) species are highly demanded and serve as raw material for a range of decorative and pharmaceutical products. In Thailand, most animals for this purpose are currently harvested from wild populations, with unionoid culture still being in its infancy. Whilst reliable species identification is a prerequisite for developing a large‐scale industry, identification by morphological means is hampered by extensive phenotypic plasticity and poor knowledge of species delimitations. To facilitate alternative molecular identification, we developed species‐specific markers for the three Thai unionoids with considerable economic potential (CEP): that is, Chamberlainia hainesiana, Hyriopsis desowitzi and Hyriopsis myersiana. For this purpose, amplified fragment length polymorphism (AFLP) fingerprints using 24 specific primer pairs were generated for eight samples of each CEP species and four samples of the closely related, non‐CEP species Contradens contradens. Cloning and sequencing of 13 CEP species‐specific AFLP bands revealed fragment collision at three occasions. In total, 16 species‐specific primer pairs were designed and tested on 92 Thai specimens spanning seven species and four genera. Thereby, specificity of (1) three primers to C. hainesiana, (2) one primer to H. desowitzi + Hyriopsis bialata, (3) one primer to H. myersiana + H. bialata and (4) four primers to all three Hyriopsis species tested was confirmed. Respective multiplex PCR protocols are provided. The developed primers enable cheap, quick and reliable identification of the Thai CEP species by one to three PCRs and offer a tool for a range of additional applications within mussel culture and ecological and evolutionary research on these important organisms.     

Immunohistochemical and gene expression analysis of stem-cell-associated markers in rat dental pulp

Stem cells in the dental pulp comprise rare populations lacking definitive cytological markers and thus are poorly characterized in vivo, especially in rat species. To gain more insight into the phenotypical characteristics and tissue distribution of these cells, we examined the distribution of stem-cell-associated marker-expressing cells and mRNA expression levels of stem-cell-associated markers in the rat molar. CD146-positive cells co-expressing microtubule-associated protein 1B were counted following double-labeling immunoperoxidase staining and their density in the coronal pulp, root pulp and periodontal ligament was compared. Moreover, mRNA expression levels of CD146, CD105, CD166 and secreted phosphoprotein 1 (SPP1; also known as osteopontin, a negative regulatory element of the stem cell niche) were analyzed in these regions by using real time polymerase chain reaction. The double-positive cells could be clearly distinguished from non-stem cells single-stained by either of the markers and showed a significantly higher density in the coronal pulp compared with the other regions (P<0.05). Moreover, mRNA expression levels of CD146, CD105 and CD166 were significantly higher in the coronal pulp than in the other regions (P<0.05). On the other hand, SPP1 mRNA expression was significantly higher in the periodontal ligament than in the pulp. Thus, the density of stem-cell-associated marker-expressing cells and stem-cell-associated gene expression levels are higher in the coronal pulp than in the root pulp and periodontal ligament, suggesting that the coronal pulp harbors more stem cells than the other regions.