In vitro biocompatibility of dextrin: the addition of a low concentration of dextrin in the medium promotes the cell activity of L929 mouse fibroblasts

To develop a bone substitute with shape-generating properties, we focused our attention on dextrin, which has a low viscosity. After considering methods of evaluation for research and development, we started by using cells that are widely used for safe biological evaluations in the field of dentistry and conducted in vitro evaluations. In this experiment, we variously added concentrations of 0.1, 1.0 and 10 mmol/l of dextrin to a culture medium in order to examine the effects on L929 mouse fibroblasts in vitro. As a result, the proliferative activity of the L929 cells was promoted during the culture period as the concentration of added dextrin became lower, and in particular, the 0.1 and 1 mmol/l addition group showed higher values than those of the control group. From the above results, it was revealed that the addition of a low concentration of dextrin in a medium promotes the cell proliferative activity.     

Human peroxisome assembly factor-2 (PAF-2): a gene responsible for group C peroxisome biogenesis disorder in humans.

Peroxisome-biogenesis disorders (PBD) are genetically heterogeneous and can be classified into at least ten complementation groups. We recently isolated the cDNA for rat peroxisome assembly factor-2 (PAF-2) by functional complementation using the peroxisome-deficient Chinese-hamster-ovary cell mutant, ZP92. To clarify the novel pathogenic gene of PBD, we cloned the full-length human PAF-2 cDNA that morphologically and biochemically restores peroxisomes of group C Zellweger fibroblasts (the same as group 4 in the Kennedy-Krieger Institute) and identified two pathogenic mutations in the PAF-2 gene in two patients with group C Zellweger syndrome. The 2,940-bp open reading frame of the human PAF-2 cDNA encodes a 980-amino-acid protein that shows 87.1% identity with rat PAF-2 and also restored the peroxisome assembly after gene transfer to fibroblasts of group C patients. Direct sequencing of the PAF-2 gene revealed a homozygous 1-bp insertion at nucleotide 511 (511 insT) in one patient with group C Zellweger syndrome (ZS), which introduces a premature termination codon in the PAF-2 gene, and, in the second patient, revealed a splice-site mutation in intron 3 (IVS3+1G-->A), which skipped exon 3, an event that leads to peroxisome deficiency. Chromosome mapping utilizing FISH indicates that PAF-2 is located on chromosome 6p21.1. These results confirm that human PAF-2 cDNA restores peroxisome of group C cells and that defects in the PAF-2 produce peroxisome deficiency of group C PBD.     

Release of H2O2 and phosphorylation of 30 kilodalton proteins as early responses of cell cycle-dependent inhibition of DNA synthesis by transforming growth factor beta 1.

Transforming growth factor beta 1 (TGF-beta 1) and H2O2 both inhibited DNA synthesis of mouse osteoblastic (MC3T3) cells in the late G1 phase of the cell cycle. TGF-beta 1 stimulated cells to release H2O2 in the late G1 phase, but not in the G0 phase, even though TGF-beta 1 receptors were present in both phases. The inhibition of DNA synthesis caused by TGF-beta 1 was partly decreased by the addition of catalase. TGF-beta 1 and H2O2 increased the phosphorylation of the same proteins with a molecular weight of 30,000 in cells in the late G1 phase, and the increase by TGF-beta 1 was abolished at least partly by catalase. The results suggest that H2O2 is one of the mediators of inhibition of DNA synthesis by TGF-beta 1.     

Intramolecular cross-linkage of lysozyme. Imidazole catalysis of the formation of the cross-link between lysine-13 (epsilon-amino) and leucine-129 (alpha-carboxyl) by carbodiimide reaction

The salt bridge between Lys-13 (epsilon-NH3+) and Leu-129 (alpha-COO-) in lysozyme was converted to an amide bond by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) reaction in the presence of imidazole (0.3-1 M) at pH 5 and room temperature, followed by dialysis at pH 10. Absence of imidazole under a similar condition did not give this intramolecularly cross-linked lysozyme derivative (CL-lysozyme) but resulted in the formation of intermolecularly cross-linked lysozyme oligomers. From the mechanistic studies on the formation of CL-lysozyme, imidazole was suggested to play the following three roles. (1) Some carboxyl groups activated by EDC in lysozyme were converted to acylimidazole groups which protected them from the reaction with amino groups in other lysozyme molecules at pH 5. These could be hydrolyzed at pH 10 to regenerate free carboxyls. (2) High concentrations of imidazole (pH 5) increased the ionic strength of the solution which weakened the salt bridge in lysozyme and facilitated the activation of the alpha-carboxyl group by EDC. (3) The alpha-carboxyl group activated by EDC was converted to an acylimidazole group which could react with the epsilon-amino group of Lys-13 in the same molecule to form an amide bond. The last step may involve some conformational change of the backbone of lysozyme and be slower than the hydrolysis reaction of the alpha-carboxyl group activated by EDC itself. However, acylimidazole groups are stable against hydrolysis at pH 5. This may afford enough time to allow the epsilon-amino group of Lys-13 to attack the acylimidazole group of Leu-129.     

Spatial Change of Cruciate Ligaments in Rat Embryo Knee Joint by Three-Dimensional Reconstruction

This study aimed to analyze the spatial developmental changes of rat cruciate ligaments by three-dimensional (3D) reconstruction using episcopic fluorescence image capture (EFIC). Cruciate ligaments of Wister rat embryos between embryonic day (E) 16 and E20 were analyzed. Samples were sectioned and visualized using EFIC. 3D reconstructions were generated using Amira software. The length of the cruciate ligaments, distances between attachment points to femur and tibia, angles of the cruciate ligaments and the cross angle of the cruciate ligaments were measured. The shape of cruciate ligaments was clearly visible at E17. The lengths of the anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) increased gradually from E17 to E19 and drastically at E20. Distances between attachment points to the femur and tibia gradually increased. The ACL angle and PCL angle gradually decreased. The cross angle of the cruciate ligaments changed in three planes. The primordium of the 3D structure of rat cruciate ligaments was constructed from the early stage, with the completion of the development of the structures occurring just before birth.     

'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.     

Structural basis for dynamic interdomain movement and RNA recognition of the selenocysteine-specific elongation factor SelB

Selenocysteine (Sec) is the "21st" amino acid and is genetically encoded by an unusual incorporation system. The stop codon UGA becomes a Sec codon when the selenocysteine insertion sequence (SECIS) exists downstream of UGA. Sec incorporation requires a specific elongation factor, SelB, which recognizes tRNA(Sec) via use of an EF-Tu-like domain and the SECIS mRNA hairpin via use of a C-terminal domain (SelB-C). SelB functions in multiple translational steps: binding to SECIS mRNA and tRNA(Sec), delivery of tRNA(Sec) onto an A site, GTP hydrolysis, and release from tRNA and mRNA. However, this dynamic mechanism remains to be revealed. Here, we report a large domain rearrangement in the structure of SelB-C complexed with RNA. Surprisingly, the interdomain region forms new interactions with the phosphate backbone of a neighboring RNA, distinct from SECIS RNA binding. This SelB-RNA interaction is sequence independent, possibly reflecting SelB-tRNA/-rRNA recognitions. Based on these data, the dynamic SelB-ribosome-mRNA-tRNA interactions will be discussed.     

Conjugational transfer system to shuttle giant DNA cloned by Bacillus subtilis genome (BGM) vector

The Bacillus subtilis GenoMe (BGM) vector was designed as a versatile vector for the cloning of giant DNA segments. Cloned DNA in the BGM can be retrieved to a plasmid using our Bacillus recombinational transfer (BReT) method that takes advantage of competent cell transformation. However, delivery of the plasmid to a different B. subtilis strain by the normal transformation method is hampered by DNA size-related inefficiency. Therefore, we designed a novel method, conjugational plasmid-mediated DNA retrieval and transfer (CReT) from the BGM vector, and investigated conjugational transmission to traverse DNA between cells to circumvent the transformation-induced size limitation. pLS20, a 65-kb plasmid capable of conjugational transfer between B. subtilis strains, was modified to retrieve DNA cloned in the BGM vector by homologous recombination during normal culture. As the plasmid copy number was estimated to be 3, the retrieval plasmid was selected using increased numbers of marker genes derived from the retrieved DNA. We applied this method to retrieve Synechocystis genome segments up to 90 kb in length. We observed retrieved plasmid transfers between B. subtilis strains by conjugation in the absence of structural alterations in the DNA fragment. Our observations extend DNA transfer protocols over previously exploited size ranges.     

Valproic Acid-Induced Anxiety and Depression Behaviors are Ameliorated in p39 Cdk5 Activator-Deficient Mice

Neurochemical Research - Valproic acid (VPA) is a drug used for the treatment of epilepsy, seizures, migraines, and bipolar disorders. Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase activated...     

Structural basis for acceptor‐substrate recognition of UDP‐glucose: anthocyanidin 3‐O‐glucosyltransferase from Clitoria ternatea

UDP‐glucose: anthocyanidin 3‐O‐glucosyltransferase (UGT78K6) from Clitoria ternatea catalyzes the transfer of glucose from UDP‐glucose to anthocyanidins such as delphinidin. After the acylation of the 3‐O‐glucosyl residue, the 3′‐ and 5′‐hydroxyl groups of the product are further glucosylated by a glucosyltransferase in the biosynthesis of ternatins, which are anthocyanin pigments. To understand the acceptor‐recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 Å, 2.55 Å, 2.70 Å, and 1.75 Å, respectively. The enzyme recognition of unstable anthocyanidin aglycones was initially observed in this structural determination. The anthocyanidin‐ and flavonol‐acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The 3‐hydroxyl groups of the acceptor substrates were located at hydrogen‐bonding distances to the Nε2 atom of the His17 catalytic residue, supporting a role for glucosyl transfer to the 3‐hydroxyl groups of anthocyanidins and flavonols. However, the molecular orientations of these three acceptors are different from those of the known flavonoid glycosyltransferases, VvGT1 and UGT78G1. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180° rotation about the O1–O3 axis of the flavonoid backbones observed in VvGT1 and UGT78G1; consequently, the 5‐ and 7‐hydroxyl groups are protected from glucosylation. These substrate recognition schemes are useful to understand the unique reaction mechanism of UGT78K6 for the ternatin biosynthesis, and suggest the potential for controlled synthesis of natural pigments.