Continuous ulnar nerve block at the forearm for early active mobilisation following flexor tendon reconstruction.

A 63-year-old woman had sustained a subcutaneous rupture of the flexor digitorum profundus tendon of the little finger due to osteoarthritis of the pisotriquetral joint. She underwent excision of the pisiform bone and reconstruction of the flexor digitorum profundus tendon of the little finger using an autogenous palmaris longus tendon graft. After surgery, a continuous ulnar nerve block was performed at the forearm under ultrasound and nerve stimulator guidance. During rehabilitation, she could not actively extend her little finger independently due to the block; however, she could actively extend it when the dorsum of the metacarpophalangeal joint was pressed by the occupational therapist, resulting in successful early active mobilisation. A continuous ulnar nerve block at the forearm may help to facilitate early active mobilisation after reconstructive surgery for little finger flexor tendon rupture. However, it may restrict the active extension of the little finger because the block does not spare the innervation of the intrinsic muscles responsible for little finger extension.

Intravoxel Incoherent Motion in Normal Pituitary Gland: Initial Study with Turbo Spin-Echo Diffusion-Weighted Imaging.

BACKGROUND AND PURPOSE: DWI with conventional single-shot EPI of the pituitary gland is hampered by strong susceptibility artifacts. Our purpose was to evaluate the feasibility of intravoxel incoherent motion assessment by using DWI based on TSE of the normal anterior pituitary lobe. MATERIALS AND METHODS: The intravoxel incoherent motion parameters, including the true diffusion coefficient (D), the perfusion fraction (f), and the pseudo-diffusion coefficient (D*), were obtained with TSE-DWI in 5 brain regions (the pons, the WM and GM of the vermis, and the genu and splenium of the corpus callosum) in 8 healthy volunteers, and their agreement with those obtained with EPI-DWI was evaluated by using the intraclass correlation coefficient. The 3 intravoxel incoherent motion parameters in the anterior pituitary lobe were compared with those in the brain regions by using the Dunnett test. RESULTS: The agreement between TSE-DWI and EPI-DWI was moderate (intraclass correlation coefficient = 0.571) for D, substantial (0.699) for f', but fair (0.405) for D*. D in the anterior pituitary lobe was significantly higher than in the 5 brain regions (P < .001). The f in the anterior pituitary lobe was significantly higher than in the 5 brain regions (P < .001), except for the vermian GM. The pituitary D* was not significantly different from that in the 5 brain regions. CONCLUSIONS: Our results demonstrated the feasibility of intravoxel incoherent motion assessment of the normal anterior pituitary lobe by using TSE-DWI. High D and f values in the anterior pituitary lobe were thought to reflect its microstructural and perfusion characteristics.

Distinguishing adrenal adenomas from non-adenomas on dynamic enhanced CT: a comparison of 5 and 10 min delays after intravenous contrast medium injection.

AIM: To evaluate the usefulness of several parameters of 5 min compared to 10 min delayed contrast-enhanced CT in distinguishing adenomas from non-adenomas. MATERIALS AND METHODS: The study population consisted of 94 patients (52 men and 42 women; mean age 62 years) with 103 adrenal lesions (75 adenomas and 28 non-adenomas). In each patient, unenhanced CT was followed by early, 5 and 10 min enhanced CT. Diagnostic parameters included delayed enhanced attenuation at 5 and 10 min, washout attenuation (WO) at 5 and 10 min, absolute percentage washout (APW) at 5 and 10 min, and relative percentage washout (RPW) at 5 and 10 min. The accuracy of each parameter for diagnosing adenomas from non-adenomas was calculated using receiver operating characteristic (ROC) analysis. RESULTS: Upon comparison between 5 and 10 min delayed contrast-enhanced CT for differentiating total adenomas or lipid-poor adenomas from non-adenomas, there was no significant difference in the area under the binomial ROC curve (Az) values of delayed enhanced attenuation (total adenomas versus non-adenomas, p = 0.164; lipid-poor adenomas versus non-adenomas, p = 0.178), WO (total adenomas versus non-adenomas, p = 0.216; lipid-poor adenomas versus non-adenomas, p = 0.230), APW (total adenomas versus non-adenomas, p = 0.401; lipid-poor adenomas versus non-adenomas, p = 0.870), or RPW (total adenomas versus non-adenomas, p = 0.160; lipid-poor adenomas versus non-adenomas, p = 0.780). CONCLUSION: Five minute contrast-enhanced CT was as useful as 10 min contrast-enhanced CT for differentiation of adrenal adenomas from non-adenomas.

Novel electric power-driven hydrodynamic injection system for gene delivery: safety and efficacy of human factor IX delivery in rats.

The development of a safe and reproducible gene delivery system is an essential step toward the clinical application of the hydrodynamic gene delivery (HGD) method. For this purpose, we have developed a novel electric power-driven injection system called the HydroJector-EM, which can replicate various time-pressure curves preloaded into the computer program before injection. The assessment of the reproducibility and safety of gene delivery system in vitro and in vivo demonstrated the precise replication of intravascular time-pressure curves and the reproducibility of gene delivery efficiency. The highest level of luciferase expression (272 pg luciferase per mg of proteins) was achieved safely using the time-pressure curve, which reaches 30 mm Hg in 10 s among various curves tested. Using this curve, the sustained expression of a therapeutic level of human factor IX protein (>500 ng ml(-1)) was maintained for 2 months after the HGD of the pBS-HCRHP-FIXIA plasmid. Other than a transient increase in liver enzymes that recovered in a few days, no adverse events were seen in rats. These results confirm the effectiveness of the HydroJector-EM for reproducible gene delivery and demonstrate that long-term therapeutic gene expression can be achieved by automatic computer-controlled hydrodynamic injection that can be performed by anyone.

Functions of chondroitin sulfate and heparan sulfate in the developing brain.

Chondroitin sulfate and heparan sulfate proteoglycans are major components of the cell surface and extracellular matrix in the brain. Both chondroitin sulfate and heparan sulfate are unbranched highly sulfated polysaccharides composed of repeating disaccharide units of glucuronic acid and N-acetylgalactosamine, and glucuronic acid and N-acetylglucosamine, respectively. During their biosynthesis in the Golgi apparatus, these glycosaminoglycans are highly modified by sulfation and C5 epimerization of glucuronic acid, leading to diverse heterogeneity in structure. Their structures are strictly regulated in a cell type-specific manner during development partly by the expression control of various glycosaminoglycan-modifying enzymes. It has been considered that specific combinations of glycosaminoglycan-modifying enzymes generate specific functional microdomains in the glycosaminoglycan chains, which bind selectively with various growth factors, morphogens, axon guidance molecules and extracellular matrix proteins. Recent studies have begun to reveal that the molecular interactions mediated by such glycosaminoglycan microdomains play critical roles in the various signaling pathways essential for the development of the brain.

Opposing functions of chondroitin sulfate and heparan sulfate during early neuronal polarization.

Axon-dendrite polarity of neurons is essential for information processing in the nervous system. Here we studied the functions of chondroitin sulfate (CS) and heparan sulfate (HS) in neuronal polarization using cultured dissociated hippocampal neurons. Immunohistochemical analyses of early cultured neurons indicated the distribution of these glycosaminoglycans to be quite different. While CS epitopes were accumulated in the focal contacts present in axons and cell bodies, those of HS were detected ubiquitously on the cell surface including on dendrites and axons. Treatment with chondroitinase (CHase) ABC, which degrades CS, and knockdown of a CS sulfotransferase, N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (4,6-ST), which is involved in the biosynthesis of oversulfated structures, induced the formation of multiple axons in hippocampal neurons. Time-lapse recordings revealed the multiple axons of CHase ABC-treated neurons to be highly unstable, extending and retracting, repeatedly. CHase ABC-treatments suggested that CS is involved in the formation of phosphorylated focal adhesion kinase-positive focal contacts. Thus, CS may enhance integrin signaling in the nascent axons, supporting axon specification. On the other hand, when neurons were treated with heparitinases that specifically degrade HS, neurons with a single axon increased. The axons of HSase-treated neurons extended steadily and showed almost no retraction. These results suggest that CS stabilizes and HS destabilizes the growth of axons in an opposing manner, contributing to early neuronal polarization.

Downregulation of HLA Class I molecules in the tumour is associated with a poor prognosis in patients with oesophageal squamous cell carcinoma.

As antigenic peptides in the context of human leukocyte antigen (HLA) class I molecules are recognised by cytotoxic T lymphocytes (CTL), the downregulation of HLA class I molecules is one of the reasons why tumour cells can evade CTL-mediated anti-tumour immunity. In this study, we investigated HLA class I expression in oesophageal squamous cell carcinoma (ESCC) (n=70) and in their metastatic lesions (lymph nodes (n=40) and liver (n=3)), by immunohistochemistry with anti-HLA class I monoclonal antibody (EMR8-5). As a result, the downregulation of HLA class I expression in primary lesions of ESCC was observed in 43%, and that in metastatic lymph nodes was noted in 90%. Furthermore, patients with preserved HLA class I expression in primary tumours showed a better survival in comparison to those with downregulated HLA class I molecules (P<0.01). Furthermore, multivariate analysis using Cox's proportional hazards model revealed that the downregulated expression of HLA class I in primary lesions was an independent, unfavourable prognostic factor (P<0.01). In conclusion, the downregulation of HLA class I expression frequently occurred in primary tumour and, to a greater extent, in metastatic lesions of patients with ESCC and was associated with patient survival.

Localisation pattern of Foxp3+ regulatory T cells is associated with clinical behaviour in gastric cancer.

It has been reported that the population of regulatory T cells (T regs) is increased in tumour-infiltrating lymphocytes in cancer-bearing hosts. Recently, forkhead/winged helix transcription factor p3, Foxp3, is thought to be the most reliable marker of T regs. In the present study, we investigated the prevalence and localisation pattern of Foxp3+ cells in gastric cancer (n=80) by immunohistochemistry, in relation to the clinical outcome of gastric cancer patients. Immunohistochemical staining was performed with anti-Foxp3 mAb, and Foxp3+ cells were semiquantified. We divided all cases into two groups: Foxp3+ -high (n=40) and Foxp3+ -low (n=40) groups, by the median size of the population of Foxp3+ cells. Furthermore, in terms of the localisation pattern of accumulating Foxp3+ cells in tumours, we classified all cases into three groups: a peri-tumour group (n=30), a diffuse group (n=40), and a follicular group (n=10). As a result, although the populations of Foxp3+ cells in stage IV were significantly larger than those in stage I (P<0.05), there was no significant difference in survival between the patients with high and low population levels of Foxp3+ cells. However, survival in patients with a diffuse pattern of Foxp3+ cells was significantly poorer than in those with a peri-tumoral pattern. In conclusion, the localisation pattern, but not the population size, of Foxp3+ cells was significantly related to patient survival.

Lack of Bcl11b tumor suppressor results in vulnerability to DNA replication stress and damages.

Bcl11b/Rit1 is involved in T-cell development and undergoes chromosomal rearrangements in human T-cell leukemias. Thymocytes of Bcl11b(-/-) newborn mice exhibit apoptosis at a certain developmental stage when thymocytes re-enter into the cell-cycle. Here, we show that Bcl11b-knockdown T-cell lines, when exposed to growth stimuli, exhibited apoptosis at the S phase with concomitant decreases in a cell-cycle inhibitor, p27 and an antiapoptotic protein, Bcl-xL, owing to transcriptional repression. This repression was a likely consequence of the impairment of Sirt1, a nicotinamide adenine dinucleotide-dependent deacetylase associating with Bcl11b. Activation of the apoptotic process cleaved the mediator protein, Claspin, and inhibited phosphorylation of cell-cycle checkpoint kinase 1 (Chk1) that plays a central role in sensing and responding to incomplete replication. Bcl11b(-/-) thymocytes also failed to phosphorylate Chk1 when UV irradiated. These results implicate Bcl11b in the remedy for DNA replication stress and maintenance of genomic integrity.

Multi-step lymphomagenesis deduced from DNA changes in thymic lymphomas and atrophic thymuses at various times after gamma-irradiation.

Whole-body gamma-irradiation to mice causes thymic atrophy where a population of precancerous cells with mutation can be found. Thus, clonal growth and DNA changes at Bcl11b, Ikaros, Pten, Notch1 and Myc were examined in not only thymic lymphomas but also in atrophic thymuses at various times after irradiation. Clonal expansion was detected from the distinct patterns of rearrangements at the TCRbeta receptor locus in a fraction of atrophic thymuses at as early as 30 days after irradiation. This expansion may be in part owing to the rearranged TCRbeta signaling because the transfer of bone marrow cells with the rearrangement and the wild-type locus into severe-combined immunodeficiency mice showed preferential growth of the rearranged thymocytes in atrophic thymus. Loss of heterozygosity (LOH) at Bcl11b and trisomy of Myc were found at high frequencies in both lymphomas and atrophic thymuses, and in contrast, LOH at Ikaros and Pten were rare in atrophic thymuses but prevalent in lymphomas. Notch1 activation was detected in lymphomas and in atrophic thymuses only at a late stage. Similar patterns of DNA changes were found in atrophic thymuses induced in Bcl11b(+/-) mice. These results suggest the order of genetic changes during lymphomagenesis, Bcl11b and Myc being at the early stage; whereas Ikaros, Pten and Notch1 at the late stage.

Volatilization of mercury under acidic conditions from mercury-polluted soil by a mercury-resistant Acidithiobacillus ferrooxidans SUG 2-2.

Volatilization of mercury under acidic conditions from soil polluted with mercuric chloride (1.5 mg Hg/kg soil) was studied with resting cells of a mercury-resistant strain, Acidithiobacillus ferrooxidans SUG 2-2. When resting cells of SUG 2-2 (0.01 mg of protein) were incubated for 10 d at 30 degrees C in 20 ml of 1.6 mM sulfuric acid (pH 2.5) with ferrous sulfate (3%) and mercury-polluted soil (1 g), which contained 7.5 nmol of Hg, approximately 4.1 nmol of mercury was volatilized, indicating that 54% of the total mercury in the soil was volatilized. The amount of mercury volatilized from the soil was dependent on the concentration of Fe2+ added to the medium. When elemental sulfur, sodium tetrathionate, and pyrite were used as an electron donor for the mercury reduction, 16, 2.4 and 0.84%, respectively, of the total mercury added to the solution were volatilized. The optimum pH and temperature for mercury volatilization were 2.5 and 30 degrees C. Approximately 92% of the total mercury in a salt solution (pH 2.5) with resting cells of SUG 2-2 (0.01 mg of protein), ferrous sulfate (3%) and mercury-polluted soil (1 g) was volatilized by further addition of both resting cells and Fe2+ and by incubating for 30 d at 30 degrees C.

Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis.

Roles of mast cells and histamine in mosquito bite-induced allergic itch-associated responses in mice.

We investigated itch-associated responses (scratching) to mosquito bites and the role of histamine and mast cells in mosquito-induced itching in mice. Although the first bites of mosquito Aedes albopictus did not increase scratching, repeated bites increased scratching. The response was not diminished even after an interval of 2 months. Similarly, repeated intradermal (i.d.) injections of salivary gland extract (SGE) from Aedes albopictus increased scratching after SGE injection itself and mosquito bites. The scratching peaked within 10 min and almost subsided by 60 min. The opioid antagonist naloxone (1 mg/kg, s.c.) inhibited scratching following SGE injection. Although the non-sedative H1-histamine-receptor antagonist terfenadine (30 mg/kg, p.o.) significantly suppressed scratching induced by histamine (100 nmol/site, i.d.) in either naive or mosquito-sensitized mice, it did not affect mosquito-induced scratching in mosquito-sensitized mice. Repeated injections of SGE increased scratching in mast cell-deficient (WBB6F1-W/Wv) mice as well as in normal (WBB6F1-+/+) littermates. Repeated exposure to mosquito bites roughly doubled serum concentrations of total IgE and IgG1, but not IgG2a. Repeated injections of SGE markedly increased plasma extravasation induced by mosquito bites and such an increase was almost completely suppressed by terfenadine (30 mg/kg, p.o.). The results show the presence of histamine-mediated and histamine-independent mechanisms in cutaneous itching and suggest that histamine probably released from mast cells does not play an important role in itching in immediate allergic reaction. Our murine model of mosquito itching may be useful for studying the mechanisms of immediate allergic itching.

Comparative sequences of two type 1 dengue virus strains possessing different growth characteristics in vitro.

The complete genome sequences of two dengue-1 virus strains having different growth characteristics (Mochizuki and A88) were compared with other published strains. The sequence analysis indicated several unique amino acid changes throughout the coding region of Mochizuki strain, mostly in envelope (E) protein. A unique amino acid, Ile-69 for Mochizuki strain at E protein resulted in the loss of an Asn-67-linked glycosylation site. A Thr substitution for Ala-114 at C protein and amino acid changes found in E, non-structural NS3, NS4a, and NS5 proteins were unique for A88 strain. These substitutions might be correlated to their different growth characteristics in vitro.

Mechanism of growth inhibition by tungsten in Acidithiobacillus ferrooxidans.

Cell growth of three hundred iron-oxidizing bacteria isolated from natural environments was inhibited strongly by 0.05 mM, and completely by 0.2 mM of sodium tungstate (Na2WO4), respectively. Since no great difference in the level of tungsten inhibition was observed among the 300 strains tested, the mechanism of inhibition by Na2WO4 was studied with Acidithiobacillus ferrooxidans strain AP19-3. When resting cells of AP19-3 were incubated in 0.1 M beta-alanine-SO4(2-) buffer (pH 3.0) with 0.1 mM Na2WO4 for 1 h, the amount of tungsten bound to the cells was 12 microg/mg protein. The optimum pH for tungsten binding to the resting cells was 2 to approximately 3. Approximately 2 times more tungsten bound to the cells at pH 3.0 than at pH 6.0. The tungsten binding was specifically inhibited by sodium molybdenum. However, copper, nickel, cadmium, zinc, manganese, cobalt, and vanadate did not disturb tungsten binding to the resting cells. The iron-oxidizing activity of AP19-3 was inhibited 24, 62, and 77% by 1, 5, and 10 mM of Na2WO4, respectively. Among the components of iron oxidation enzyme system, iron:cytochrome c oxidoreductase activity was not inhibited by 10 mM of Na2WO4. In contrast, the activity of cytochrome c oxidase purified highly from the strain was inhibited 50 and 72%, respectively, by 0.05 and 0.1 mM of Na2WO4. The amounts of tungsten bound to plasma membrane, cytosol fraction, and a purified cytochrome c oxidase were 8, 0.5, and 191 microg/mg protein, respectively. From the results, the growth inhibition by Na2WO4 observed in A. ferrooxidans is explained as follows: tungsten binds to cytochrome c oxidase in plasma membranes and inhibits cytochrome c oxidase activity, and as a results, the generation of energy needed for cell growth from the oxidation of Fe2+ is stopped.

L-lactic acid as a mosquito (Diptera: Culicidae) repellent on human and mouse skin.

The attraction of Aedes albopictus (Skuse) to hands and forearms of human subjects treated with several concentrations of L-LA solution were studied in a test chamber containing proboscis-amputated mosquitoes. Fewer mosquitoes alighted on L-LA treated human skin than on water-treated control skin. Similar results were found using normal mosquitoes following L-LA and water treatment of mouse skin. The relative repellent effects of L-LA varied with concentration. The minimum repellent concentration was lower than previously reported for human skin. The number of alightments decreased at increasing concentrations of L-LA, demonstrating the absolute repellency of L-LA. Unlike previous reports suggesting that L-LA attracted mosquitoes, our studies using human and mouse skin showed that L-LA exhibited both relative and absolute repellency.

A new iron oxidase from a moderately thermophilic iron oxidizing bacterium strain TI-1.

Iron oxidase was purified from plasma membranes of a moderately thermophilic iron oxidizing bacterium strain TI-1 in an electrophoretically homogeneous state. Spectrum analyses of purified enzyme showed the existence of cytochrome a, but not cytochrome b and c types. Iron oxidase was composed of five subunits with apparent molecular masses of 46 kDa (alpha), 28 kDa (beta), 24 kDa (gamma), 20 kDa (delta), and 17 kDa (epsilon). As the molecular mass of a native enzyme was estimated to be 263 kDa in the presence of 0.1% n-dodecyl-beta-D-maltopyranoside (DM), a native iron oxidase purified from strain TI-1 seems to be a homodimeric enzyme (alpha beta gamma delta epsilon)(2). Optimum pH and temperature for iron oxidation were pH 3.0 and 45 degrees C, respectively. The K(m) of iron oxidase for Fe(2+) was 1.06 mM and V(max) for O(2) uptake was 13.8 micromol x mg(-1) x min(-1). The activity was strongly inhibited by cyanide and azide. Purified enzyme from strain TI-1 is a new iron oxidase in which electrons of Fe(2+) were transferred to haem a and then to the molecular oxygen.

Purification and some properties of ubiquinol oxidase from obligately chemolithotrophic iron-oxidizing bacterium, Thiobacillus ferrooxidans NASF-1.

Ubiquinol-oxidizing activity was detected in an acidophilic chemolithotrophic iron-oxidizing bacterium, T. ferrooxidans. The ubiquinol oxidase was purified 79-fold from plasma membranes of T. ferrooxidans NASF-1 cells. The purified oxidase is composed of two polypeptides with apparent molecular masses of 32,600 and 50,100 Da, as measured by gel electrophoresis in the presence of sodium dodecyl sulfate. The absorption spectrum of the reduced enzyme at room temperature showed big peaks at 530 and 563, and a small broad peak at 635 nm, indicating the involvement of cytochromes b and d. Characteristic peaks of cytochromes a and c were not observed in the spectrum at around 600 and 550 nm, respectively. This enzyme combined with CO, and its CO-reduced minus reduced difference spectrum showed peaks at 409 nm and 563 nm and a trough at 431 nm. These results indicated that the oxidase contained cytochrome b, but the involvement of cytochrome d was not clear. The enzyme catalyzed the oxidations of ubiquinol-2 and reduced N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride. The ubiquinol oxidase activity was activated by the addition of albumin and lecithin to the reaction mixture and inhibited by the respiratory inhibitors KCN, HQNO, NaN3, and antimycin A1, although the enzyme was relatively resistant to KCN, and the divalent cation, Zn2+, compared with ubiquinol oxidases of E. coli.