Molecular cloning and characterization of dullard: a novel gene required for neural development

In a screen for genes expressed in neural tissues and pronephroi, we isolated a novel gene, named dullard. Dullard protein contains the C-terminal conserved domain of NLI-IF (Nuclear LIM Interactor-Interacting Factor), a protein whose function is not yet characterized. Dullard mRNA was maternally derived and localized to the animal hemisphere. At neurula stages, the expression was in neural regions and subsequently localized to neural tissues, branchial arches, and pronephroi. Using antisense morpholino oligonucleotide-mediated inhibition, we showed that dullard was required for neural development. The translational knock-down of dullard resulted in failure of neural tube development and the embryos consequently showed a reduction of head development. Expression of neural marker genes in dullard-inhibited embryos was also suppressed. These results suggest that dullard is necessary for neural development.     

Construction of a high-performance human fetal liver-derived lentiviral cDNA library

The gene transduction method is a very powerful tool, not only in basic science but also in clinical medicine. Regenerative medicine is one field that has close connection with both basic and clinical. Recently, it has been reported that induced pluripotent stem (iPS) cells can be produced from somatic cells by a three or four gene transduction. We have also recently reported that lentiviral gene transfer of the tal1/scl gene can efficiently differentiate non-human primate common marmoset ES cells into hematopoietic cells without the support of stromal cells. In this study, we constructed a high-performance human fetal liver-derived lentiviral expression library, which contains a high number of individual clones, in order to develop a very helpful tool for understanding early hematopoiesis and/or hepatocytosis for future regenerative medicine. Our lentiviral cDNA library consisted of more than 8 x 10(7) individual clones, and their average insert size was >2 kb. DNA sequence analysis for each individual inserted cDNAs revealed that >60% contained the full-length protein-coding regions for many genes including cytokine receptors, cytoplasmic proteins, protein inhibitors, and nuclear factors. The transduction efficiency on 293T cells was 100% and the average size of an integrated cDNA was ~1.1 kb. These results suggest that our lentiviral human fetal liver cDNA expression library could be a very helpful tool for accelerating the discovery of novel genes that are involved in early hematopoiesis and hepatopoiesis and to make the use of iPS cells more efficient in the field of regenerative medicine.     

Crystal structure of the 64M-2 antibody Fab fragment in complex with a DNA dT(6-4)T photoproduct formed by ultraviolet radiation

DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts formed by ultraviolet radiation are implicated in mutagenesis and cancer, particularly skin cancer. The crystal structure of the Fab fragment of the murine 64M-2 antibody specific to DNA T(6-4)T photoproducts is determined as a complex with dT(6-4)T, a (6-4) pyrimidine-pyrimidone photodimer of dTpT, at 2.4 A resolution to a crystallographic R-factor of 0.199 and an R(free) value of 0.279. The 64M-2 Fab molecule is in an extended arrangement with an elbow angle of 174 degrees, and its five complementarity-determining regions, except L2, are involved in the ligand binding. The bound dT(6-4)T ligand adopting a ring structure with (6-4) linked 5' thymine-3' pyrimidone bases is fully accommodated in an antigen-binding pocket of about 15 Ax10 A. The 5'-thymine and 3'-pyrimidone bases are in half-chair and planar conformations, respectively, and are nearly perpendicular to each other. The 5'-thymine base is hydrogen-bonded to Arg95H and Ser96H, and is in van der Waals contact with Tyr100iH. The 3'-pyrimidone base is hydrogen-bonded to His35H, and is in contact with Trp33H. Three water molecules are located at the interface between the bases and the Fab residues. Hydrogen bonds involving these water molecules also contribute to Fab recognition of the dT(6-4)T bases. The sugar-phosphate backbone connecting the bases is surrounded by residues His27dL, Tyr32L, Ser92L, Trp33H, and Ser58H, but is not hydrogen-bonded to these residues.     

Crystal structure of a bacterial protein proteinase inhibitor (Streptomyces subtilisin inhibitor) at 2.6 A resolution

The crystal structure of a bacterial protein proteinase inhibitor (Streptomyces subtilisin inhibitor) was solved at 2·6 Å resolution. Each subunit of the dimeric inhibitor has a five-stranded antiparallel β-sheet and two short α-helices. The subunit-subunit interface formed by a stack of two β-sheets provided by the two subunits resembles the dimer-dimer interface of concanavalin A. Conformation of the reactive site around the scissible bond Met73-Val74 seems very rigid. Between bovine pancreatic trypsin inhibitor (Kunitz) and the Streptomyces inhibitor, the reactive site conformations are almost identical with each other from the P₂ to P₂′ residues, while between the soybean trypsin inhibitor (Kunitz) and the Streptomyces inhibitor they are similar from the P₂ to P₁′ residues. There are overall similarities in conformation extending from the P₃ to P₂′ residues between the Streptomyces inhibitor and a hypothetical substrate presumed (Robertus et al., 1972b) to be bound to subtilisin BPN′ in a productive binding mode. Apart from the reactive site, there seems to be no structural relationship among the Streptomyces, bovine pancreatic and soybean inhibitors, suggesting their convergent evolution from separate ancestral proteins.     

Naftopidil reduced the proliferation of lung fibroblasts and bleomycin‐induced lung fibrosis in mice

Naftopidil, an α‐1 adrenoceptor antagonist with few adverse effects, is prescribed for prostate hyperplasia. Naftopidil inhibits prostate fibroblast proliferation; however, its effects on lung fibroblasts and fibrosis remain largely unknown. Two normal and one idiopathic pulmonary fibrosis human lung fibroblast lines were cultured with various naftopidil concentrations with or without phenoxybenzamine, an irreversible α‐1 adrenoceptor inhibitor. We examined the incorporation of 5‐bromo‐2?‐deoxyuridine into DNA and lactic acid dehydrogenase release by enzyme‐linked immunosorbent assay, cell cycle analysis by flow cytometry, scratch wound‐healing assay, and mRNA expressions of type IV collagen and α‐smooth muscle actin by polymerase chain reaction. Effects of naftopidil on bleomycin‐induced lung fibrosis in mice were evaluated using histology, micro‐computed tomography, and surfactant protein‐D levels in serum. Naftopidil, dose‐dependently but independently of phenoxybenzamine, inhibited 5‐bromo‐2?‐deoxyuridine incorporation in lung fibroblasts. Naftopidil induced G1 cell cycle arrest, but lactic acid dehydrogenase release and migration ability of lung fibroblasts were unaffected. Naftopidil decreased mRNA expressions of type IV collagen and α‐smooth muscle actin in one normal lung fibroblast line. Histological and micro‐computed tomography examination revealed that naftopidil attenuated lung fibrosis and decreased serum surfactant protein‐D levels in bleomycin‐induced lung fibrosis in mice. In conclusion, naftopidil may have therapeutic effects on lung fibrosis.     

Allelic variation of the dopamine receptor D4 gene polymorphic region in gibbons

We examined the tandem repeat sequence of the dopamine receptor D4 (DRD4) gene in 73 individuals derived from 8 species of gibbons (genusHylobates) in an attempt to assess the variability of this gene in gibbon species.H. syndactylus (subgenusSymphalangus) andH. concolor (subgenusNomascus), which were inferred to have diverged at an early time within the family Hylobatidae, shared only long repeat (7–8) alleles. On the other hand, DRD4 was highly polymorphic in gibbons of the subgenusHylobates, with 4-, 5-, 6-, 7-, and 8-repeat alleles being recognized. In this subgenus, 4- and 5-repeat alleles were found in the species distributed mainly in the southern islands such as Sumatra, Java, and Borneo but not in the species inhabiting the Asian continent. Sequence analysis indicated that the repeat structure of the gibbon DRD4 gene was quite complex but most of the 48-bp units could be classified into several groups across the species based on sequence similarities. However, the sequence of the 7-repeat allele ofH. muelleri was unique, since the repeat units had low similarities to other units of gibbons.     

Detection of Staphylococci in Mouse Phagocytic Cells by in Situ Hybridization Using Biotinylated Dna Probes

In situ hybridization was used to detect intracellular Staphylococcus aureus and S. epidermidis in mouse phagocytic cells after experimental infection of C3H mice with Staphylococci via abdominal or intravenous injection. Isolated ascites or whole blood were tested by the phagocyte smear technique, using bacteriolytic enzymes to preserve phagocytic cell morphology. The exposed bacterial DNA was visualized as intracellular hybridized signals by use of biotinylated DNA probes and by immunocytochemistry using streptavidin-alkaline phosphatase conjugates as detector molecules. These DNA probes, prepared from randomly cloned genomic DNA fragments of S. aureus and S. epidermidis, were strain-specific and did not cross-hybridize either in situ or on dot-blot hybridization. This technique of in situ hybridization with phagocyte smears is useful for detection and diagnosis of intracellular bacteria regardless of viability.