Free dorsoulnar perforator flap transfers for the reconstruction of severely injured digits

The aim of this study was to investigate the feasibility of transferring the free dorsoulnar perforator flap nourished by the cutaneous perforator branched dorsoulnar artery to reconstruct severely injured fingers under upper arm anesthesia. Between April of 2001 and April of 2002, 13 free dorsoulnar perforator flaps were used in 13 patients. There were 11 men and two women ranging in age from 18 to 64 years, with an average age of 38 years. The affected fingers were one thumb, four index fingers, five middle fingers, two ring fingers, and one little finger. All cases were performed under upper arm anesthesia combined with intravenous local anesthesia. The operative time ranged from 103 to 140 minutes, with an average time of 120 minutes. The flap size ranged from 1 x 3 to 3 x 4 cm, and was transferred from the same forearm of the injured finger. All donor sites were closed primarily without a skin graft. The aim of reconstruction for fingers was to repair a traumatic defect (five cases), partial necrosis following replantation (two cases), and soft-tissue defects resulting from resection of a scar (three cases) and to revascularize ischemic fingers (three cases). All flaps survived completely. After repair of the flow-through circulation of the common digital artery and ischemic finger, a postoperative angiogram showed the vascular patency and hypervascularity of the reconstructed fingers, and the patients' complaints were reduced. The free dorsoulnar perforator flap under regional anesthesia is first reported; it may become one valuable option as a very small flap for the treatment of repairing intercalated or segmental defects as a flow-through flap for soft-tissue defects and ischemic fingers.     

The role of conformational flexibility of enzymes in the discrimination between amino acid and ester substrates for the subtilisin-catalyzed reaction in organic solvents

To investigate how the conformational flexibility of subtilisin affects its ability to discriminate between enantiomeric amino acid and ester substrates for the subtilisin-catalyzed reaction in an organic solvent, the flexibility around the active site and the surface of subtilisin was estimated from the mobility of a spin label bound to subtilisin by ESR spectroscopy. Many studies on enzyme flexibility focus on the active site. Both the surface and active site flexibility play an important role in the enantioselectivity enhancement of the enzyme-catalyzed reaction. It was found, however, that the different behavior observed for the enantioselectivity between the amino acid and ester substrates could be correlated with the flexibility around the surface rather than the flexibility at the active site of subtilisin. In other words, for the ester substrates, the greater flexibility around the surface of subtilisin induced by a conformational change resulting from the presence of an additive such as DMSO is essential for the enantioselectivity enhancement. This model is also supported by the Michaelis-Menten kinetic parameters for each enantiomeric substrate. Our findings provide insight into the enantioselectivity enhancement for the resolution of enantiomers for enzyme-catalyzed reactions in organic solvents.     

Mitochondrial genomes and phylogeny of the ocean sunfishes (Tetraodontiformes: Molidae)

We determined the complete nucleotide sequences of the mitochondrial genomes for the three currently recognized species of ocean sunfish: Mola mola, Masturus lanceolatus, and Ranzania laevis (Tetraodontiformes: Molidae). Each genome contained the 37 genes as found in teleosts, with the typical gene order in teleosts. Bayesian, maximum-likelihood, and maximum-parsimony analyses were conducted with the data set comprising concatenated nucleotide sequences from 36 genes (excluding the ND6 gene) of three molids and four outgroups (three tetraodontiforms plus a caproid). The resultant trees supported monophyly of the Molidae and its intrarelationships ((Mola, Masturus), Ranzania), which were congruent with previous morphology-based hypotheses.     

Free radical theory of apoptosis and metamorphosis

Reactive oxygen species (ROS) are the major factors that induce oxidative modification of DNA and gene mutation. ROS can elicit oxidative stress and affect a wide variety of physiological and pathological processes including embryonal development, maturation and aging.     

Enhanced Th2 cell-mediated allergic inflammation in Tyk2-deficient mice

Allergic inflammation is mediated by Th2 cell-derived cytokines, including IL-4, IL-5, and IL-13, and down-regulated by IFN-gamma and IL-12. Tyk2 is a member of the Janus family of protein tyrosine kinases and is activated by a variety of cytokines: IFN-alphabeta, IL-6, IL-10, IL-12, and IL-13. In this study, we investigated the role of Tyk2 in the regulation of Ag-induced Th cell differentiation and Ag-induced allergic inflammation in the airways using Tyk2-deficient (Tyk2(-/-)) mice. When splenocytes were stimulated with antigenic peptide, IL-12-mediated Th1 cell differentiation was decreased, but IL-4-mediated Th2 cell differentiation was increased in Tyk2(-/-) mice. In vivo, Ag-specific IgE and IgG1 production was increased, but Ag-specific IgG2a production was decreased in Tyk2(-/-) mice as compared with those in control mice. In addition, Ag-induced eosinophil and CD4(+) T cell recruitment, as well as the production of Th2 cytokines in the airways, was increased in Tyk2(-/-) mice. Adoptive transfer experiments revealed that CD4(+) T cells were responsible for the enhanced Ag-induced eosinophil recruitment in Tyk2(-/-) mice. In contrast, although the level of IL-13 was increased in the airways of Tyk2(-/-) mice after Ag inhalation, the number of goblet cells, as well as Muc5ac mRNA expression, was decreased in Tyk2(-/-) mice. Together, these results indicate that Tyk2 plays a bilateral role in the regulation of allergic inflammation in the airways: Tyk2 plays a role in the down-regulation of Th2 cell-mediated Ab production and eosinophil recruitment in the airways by regulating Th1/Th2 balance toward Th1-type, while Tyk2 is necessary for the induction of IL-13-mediated goblet cell hyperplasia in the airways.     

p57KIP2 modulates stress-activated signaling by inhibiting c-Jun NH2-terminal kinase/stress-activated protein Kinase

p57KIP2, a member of the Cip/Kip family of enzymes that inhibit several cyclin-dependent kinases, plays a role in many biological events including cell proliferation, differentiation, apoptosis, tumorigenesis and developmental changes. The human p57KIP2 gene is located in chromosome 11p15.5, a region implicated in sporadic cancers and Beckwith-Wiedemann syndrome. We here report that p57KIP2 physically interacts with and inhibits c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK). The carboxyl-terminal QT domain of p57KIP2 is crucial for the inhibition of JNK/SAPK. Overexpressed p57KIP2 also suppressed UV- and MEKK1-induced apoptotic cell death. p57KIP2 expression during C2C12 myoblast differentiation resulted in repression of the JNK activity stimulated by UV light. Furthermore, UV-stimulated JNK1 activity was higher in mouse embryonic fibroblasts derived from p57-/- mice than in the cells from wild-type mice. Taken together, these findings suggest that p57KIP2 modulates stress-activated signaling by functioning as an endogenous inhibitor of JNK/SAPK.     

Absence of superoxide dismutase activity in a soluble cellular isoform of prion protein produced by baculovirus expression system

A method for expression and purification of a soluble form of histidine (HIS)-tagged murine prion protein (bacMuPrP), which lacks the entire C-terminal cleavage and glycosyl phosphatidyl inositol (GPI) addition site, has been developed using a recombinant baculovirus expression system and purification with Ni-NTA agarose affinity chromatography. In mammalian sources, PrP(C) is attached to the cell membrane by a GPI anchor. However, in our system, bacMuPrP was secreted into the media, enabling its easy purification in abundance. Indirect immunofluorescence studies and immunoblot analysis localized not in cell membrane but in the perinuclear endoplasmic reticulum region in cells and is secreted into the media. Tunicamycin treatment revealed non-glycosylated proteins were secreted into the media, suggesting that glycosylation is not necessary for bacMuPrP secretion. Density-gradient sedimentation analysis demonstrated a sedimentation coefficient of secretory bacMuPrP as 2.3 S, indicating a monomeric form. Although affinity-purified PrP from mouse brain or recombinant prion protein (PrP) produced by Escherichia coli and refolded in the presence of copper has been reported to display superoxide dismutase (SOD) activity, bacMuPrP did not show SOD activity. These results suggest that bacMuPrP has a different biochemical and biophysical characterization from mammalian and bacterial-derived PrP. Furthermore, this simple expression system may provide an adequate source for structural, functional, and biochemical analyses of PrP.     

Characterization of the mouse gene for the U-box-type ubiquitin ligase UFD2a

UFD2a is a mammalian homolog of Saccharomyces cerevisiae Ufd2, originally described as an E4 ubiquitination factor. UFD2a belongs to the U-box family of ubiquitin ligases (E3s) and likely functions as both an E3 and E4. We have isolated and characterized the mouse gene (Ube4b) for UFD2a. A full-length (approximately 5700 bp) Ube4b cDNA was isolated and the corresponding gene spans >100 kb, comprising 27 exons. Luciferase reporter gene analysis of the 5(') flanking region of Ube4b revealed that nucleotides -1018 to -943 (relative to the translation initiation site) possess promoter activity. This functional sequence contains two putative Sp1 binding sites but not a TATA box. Immunoblot and immunohistochemical analyses revealed that UFD2a is expressed predominantly in the neuronal tissues. We also show that UFD2a interacts with VCP (a AAA-family ATPase) that is thought to mediate protein folding. These data implicate UFD2a in the degradation of neuronal proteins by the ubiquitin-proteasome pathway.     

Akt is an endogenous inhibitor toward tumor necrosis factor-related apoptosis inducing ligand-mediated apoptosis in rheumatoid synovial cells

Akt is known to be activated in the rheumatoid synovial tissues. We examined here functional role of Akt during tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in rheumatoid synovial cells. Rheumatoid synovial cells in vitro were rapidly committed to apoptosis in response to TRAIL in mitochondria-dependent manner whereas Akt and extracellular signal-regulated kinase (ERK) were also phosphorylated. TRAIL-mediated apoptosis in synovial cells was significantly increased through inactivation of Akt by LY294002, however, that process was not so changed by adding ERK inhibitor, PD98059. Platelet-derived growth factor (PDGF) clearly phosphorylated both Akt and ERK in synovial cells, and PDGF pretreatment markedly suppressed TRAIL-mediated synovial cell apoptosis. The use of not PD98059 but LY294002 abrogated PDGF-mediated inhibitory effect toward TRAIL-induced apoptosis in synovial cells. The above protective effect of Akt was confirmed by the use of short interfering RNA (siRNA)-directed inhibition of Akt. Our data suggest that Akt is an endogenous inhibitor during TRAIL-mediated synovial cell apoptotic pathway, which may explain that synovial cells in situ of the rheumatoid synovial tissues are resistant toward apoptotic cell death in spite of death receptor expression.