Differential regulation of a family of apyrase genes from Medicago truncatula

Four putative apyrase genes were identified from the model legume Medicago truncatula. Two of the genes identified from M. truncatula (Mtapy1 and Mtapy4) are expressed in roots and are inducible within 3 h after inoculation with Sinorhizobium meliloti. The level of mRNA expression of the other two putative apyrases, Mtapy2 and Mtapy3, was unaffected by rhizobial inoculation. Screening of a bacterial artificial chromosome library of M. truncatula genomic DNA showed that Mtapy1, Mtapy3, and Mtapy4 are present on a single bacterial artificial chromosome clone. This apyrase cluster was mapped to linkage group seven. A syntenic region on soybean linkage group J was found to contain at least two apyrase genes. Screening of nodulation deficient mutants of M. truncatula revealed that two such mutants do not express apyrases to any detectable level. The data suggest a role for apyrases early in the nodulation response before the involvement of root cortical cell division leading to the nodule structure.     

Lymphonodular cryptococcosis diagnosed by fine needle aspiration cytology in hyper-IgM syndrome. A case report

BACKGROUND: Most cases of cryptococcosis are diagnosed when signs of meningitis have appeared. We report a case of lymphonodular cryptococcosis that was diagnosed by fine needle aspiration cytology (FNAC), excisional biopsy of a cervical lymph node and culture of aspirated material. CASE: An 11-year-old boy presented with a history of fever and enlarged bilateral cervical lymph nodes of two weeks' duration. Past medical history included immunoglobulin replacement for hyper-IgM syndrome for the previous eight years. FNAC smears from a cervical lymph node showed numerous yeasts of various sizes, ranging from 5 to 15 microns in diameter, located in the cytoplasm of multinucleated giant cells and in the background. In air-dried, Diff-Quik-stained slides, the yeasts stained blue and were surrounded by clear halos. Aspirated material collected in the syringe was cultured, and Cryptococcus neoformans was isolated. CONCLUSION: This case report suggests that a combination of FNAC and culture is a simple and useful method of diagnosing fungal infections. Early diagnosis by FNAC makes possible the early initiation of treatment.     

Dramatically accelerated growth and extraordinary gigantism of transgenic mud loach Misgurnus mizolepis

Transgenic mud loaches (Misgurnus mizolepis), in which the entire transgene originated from the same species, have been generated by microinjecting the mud loach growth hormone (mlGH) gene fused to the mud loach -actin promoter. Out of 4,100 eggs injected, 7.5% fish derived from the injected eggs showed dramatically accelerated growth, with a maximum of 35-fold faster growth than their non-transgenic siblings. Many fast-growing transgenic individuals showed extraordinary gigantism: their body weight and total length (largest fish attained to 413g and 41.5cm) were larger and longer than even those of 12-year-old normal broodstock (maximum size reached to 89g and 28cm). Of 46 transgenic founders tested, 30 individuals transmitted the transgene to next generation with a wide range of germ-line transmission frequencies ranging from 2% to 33%. The growth performance of the subsequent generation (F₁) was also dramatically accelerated up to 35-fold, although the levels of enhanced growth were variable among transgenic lines. Three transgenic germ-lines up to F₄ were established, showing the expected Mendelian inheritance of the transgene. Expression of GH mRNA in many tissues was detected by RT-PCR analyses. The time required to attain marketable size (10g) in these transgenic lines was only 30–50 days after fertilization, while at least 6 months in non-transgenic fish. Besides growth enhancement, significantly improved feed-conversion efficiency up to 1.9-fold was also observed.     

The erythropoietin receptor cytosolic juxtamembrane domain contains an essential, precisely oriented, hydrophobic motif

We report that the erythropoietin receptor cytosolic juxtamembrane region is conformationally rigid and contains a hydrophobic motif, composed of residues L253, I257, and W258, that is crucial for Janus kinase 2 (JAK2) activation and receptor signaling. Alanine insertion mutagenesis shows that the orientation of this motif and not its distance from the membrane bilayer is critical. Intragenic complementation studies suggest that L253 is contained within an alpha helix functionally continuous to the transmembrane alpha helix. The alpha-helical orientation of L53 is required not for JAK2 activation but for activated JAK2 to induce phosphorylation of the erythropoietin receptor. This motif is highly conserved among cytokine receptors and couples ligand-induced conformational changes in the receptor to intracellular activation of JAK2.     

Cloning of Japanese flounder Paralichthys olivaceus CD3 cDNA and gene, and analysis of its expression

Two distinct CD3 homologue cDNAs, CD3-1 and CD3-2, were isolated from a Japanese flounder leukocyte cDNA library. CD3-1 consisted of 961 bp encoding 178 amino acid residues, and CD3-2 consisted of 927 bp encoding 182 amino acid residues. The two deduced amino acid sequences had an identity of 95.1%, and neither had N-linked glycosylation sites. The identities between the Japanese flounder CD3s and previously reported CD3s (CD3 epsilon, CD3 gamma, or CD3 delta) of Xenopus laevis, chicken, and various mammals were approximately 25%. The Japanese flounder CD3s had an extracellular domain, a CXXCXE motif, and an immunoreceptor tyrosine-based activation motif (ITAM), each of which are important characteristics of CD3 chains. Furthermore, the positions of four cysteine residues in the extracellular domain were preserved in both of the Japanese flounder CD3s. A phylogenetic tree based on the amino acid sequences confirmed that the Japanese flounder CD3s are closer to CD3 epsilon than to CD3 gamma and CD3 delta. However, the gene structure of Japanese flounder CD3 is identical to the chicken and Xenopus CD3 gamma/delta genes and the mammalian CD3 delta gene. Southern blot hybridization and the DNA sequence of the CD3 gene of homocloned Japanese flounder indicated that the CD3 gene exists as a single copy. Southern blot hybridization also showed the presence of a polymorphic variant of Japanese flounder CD3. An RT-PCR analysis detected Japanese flounder CD3 mRNA in several organs that contained lymphocytes. The proportion of CD3-positive cells in the peripheral blood leukocytes was 34.9%.     

Contribution of the hydrogen-bond network involving a tyrosine triad in the active site to the structure and function of a highly proficient ketosteroid isomerase from Pseudomonas putida biotype B

Delta(5)-3-Ketosteroid isomerase from Pseudomonas putida biotype B is one of the most proficient enzymes catalyzing an allylic isomerization reaction at rates comparable to the diffusion limit. The hydrogen-bond network (Asp99... Wat504...Tyr14...Tyr55...Tyr30) which links the two catalytic residues, Tyr14 and Asp99, to Tyr30, Tyr55, and a water molecule in the highly apolar active site has been characterized in an effort to identify its roles in function and stability. The DeltaG(U)(H2O) determined from equilibrium unfolding experiments reveals that the elimination of the hydroxyl group of Tyr14 or Tyr55 or the replacement of Asp99 with leucine results in a loss of conformational stability of 3.5-4.4 kcal/mol, suggesting that the hydrogen bonds of Tyr14, Tyr55, and Asp99 contribute significantly to stability. While decreasing the stability by about 6.5-7.9 kcal/mol, the Y55F/D99L or Y30F/D99L double mutation also reduced activity significantly, exhibiting a synergistic effect on k(cat) relative to the respective single mutations. These results indicate that the hydrogen-bond network is important for both stability and function. Additionally, they suggest that Tyr14 cannot function efficiently alone without additional support from the hydrogen bonds of Tyr55 and Asp99. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30. This observation supports the role of Tyr55 OH in positioning Tyr14 properly to optimize the hydrogen bond between Tyr14 and C3-O of the steroid substrate. No significant structural changes were observed in the crystal structures of Y30F and Y30F/Y55F, which allowed us to estimate approximately the interaction energies mediated by the hydrogen bonds Tyr30...Tyr55 and Tyr14...Tyr55. Taken together, our results demonstrate that the hydrogen-bond network provides the structural support that is needed for the enzyme to maintain the active-site geometry optimized for both function and stability.     

SPECIES PHYLOGENY OF COSMARIUM AND STAURASTRUM (DESMIDIACEAE) BASED ON RBC L SEQUENCES

Cosmarium and Staurastrum are the two most diverse genera of placoderm desmids (Family Desmidiaceae), with approximately 1100 and 800 species, respectively. Phylogenetic analysis of relationships of species has been extremely difficult. In a monograph of North American placoderm desmids, Prescott et al. described early phylogenetic work that concluded Staurastrum to be polyphyletic and certainly polymorphic. Likewise, Cosmarium has also been viewed as polyphyletic, and a number of workers have proposed splitting these genera. The classical view of West and West grouped species within each genus into two divisions and 6–8 sections based on wall features and semicell shape. We sequenced rbc L from 18 species of Cosmarium (2 divisions, 7 sections) and 12 species of Staurastrum (2 divisions and 7 sections) and performed a phylogenetic analysis (parsimony, maximum likelihood, bootstrap) using other placoderm desmids and Zygnematales as outgroups. The results exhibit little support for the monophyly of sections or divisions of the two genera. Furthermore, although there is support for the monophyly of clades within each genus, there is also support for a separate clade containing species from both genera.     

Utilization of DNA marker-assisted selection in Korean native animals

The recent progress of DNA technologies including DNA fingerprinting (DFP) and random amplified DNA polymorphism (RAPD) analysis make it possible to identify the specific genetic traits of animals and to analyze the genetic diversity and relatedness between or within species or populations. Using those techniques, some efforts to identify and develop the specific DNA markers based on DNA polymorphism, which are related with economic traits for Korean native animals, Hanwoo (Korean native cattle), Korean native pig and Korean native chicken, have been made in Korea for recent a few years. The developed specific DNA markers successfully characterize the Korean native animals as the unique Korean genetic sources, distinctively from other imported breeds. Some of these DNA markers have been related to some important economic traits for domestic animals, for example, growth rate and marbling for Hanwoo, growth rate and back fat thickness for native pig, and growth rate, egg weight and egg productivity for native chicken. This means that those markers can be used in important marker-assisted selection (MAS) of Korean native domestic animals and further contribute to genetically improve and breed them.