Association analysis of the ACTN3 R577X polymorphism with passive muscle stiffness and muscle strain injury.

Passive muscle stiffness is considered to be a major factor affecting joint flexibility and is thought to relate to the occurrence of muscle strain injury. In skinned muscle fiber experiments, the R577X polymorphism of the α-actinin-3 gene (ACTN3) has been associated with passive muscle stiffness. Our primary purpose was to clarify whether the ACTN3 R577X polymorphism influences passive stiffness of human muscle in vivo. We also examined whether the ACTN3 R577X polymorphism is associated with the occurrence of hamstring strain injury. Seventy-six healthy young male subjects were genotyped for the ACTN3 R577X (rs1815739) polymorphism. Shear modulus (an index of stiffness) of each hamstring muscle (biceps femoris, semitendinosus, and semimembranosus) was assessed using ultrasound shear wave elastography, and history of hamstring strain injury was collected via a questionnaire. The muscle shear moduli of the semitendinosus and semimembranosus were significantly higher in R-allele (RR + RX genotype) carriers than in XX genotype carriers, whereas the shear modulus of the biceps femoris did not differ among the ACTN3 R577X genotypes. Frequency of past hamstring strain injury also did not differ between the 3 genotypes nor between the R-allele and XX genotype carriers. This study indicates that RR and RX genotypes of the ACTN3 R577X polymorphism (corresponding to the presence of α-actinin-3 in type II muscle fibers) are associated with increased passive muscle stiffness of the human hamstring in vivo. However, this altered mechanical property might not affect the risk of hamstring muscle strain injury.

Effects of hamstring stretching on passive muscle stiffness vary between hip flexion and knee extension maneuvers.

The purpose of this study was to examine whether the effects of hamstring stretching on the passive stiffness of each of the long head of the biceps femoris (BFl), semitendinosus (ST), and semimembranosus (SM) vary between passive knee extension and hip flexion stretching maneuvers. In 12 male subjects, before and after five sets of 90 s static stretching, passive lengthening measurements where knee or hip joint was passively rotated to the maximal range of motion (ROM) were performed. During the passive lengthening, shear modulus of each muscle was measured by ultrasound shear wave elastography. Both stretching maneuvers significantly increased maximal ROM and decreased passive torque at a given joint angle. Passive knee extension stretching maneuver significantly reduced shear modulus at a given knee joint angle in all of BFl, ST, and SM. In contrast, the stretching effect by passive hip flexion maneuver was significant only in ST and SM. The present findings indicate that the effects of hamstring stretching on individual passive muscles' stiffness vary between passive knee extension and hip flexion stretching maneuvers. In terms of reducing the muscle stiffness of BFl, stretching of the hamstring should be performed by passive knee extension rather than hip flexion.

No graduated pressure profile in compression stockings still reduces muscle fatigue.

Most sporting compression stockings possess a graduated pressure profile. However, it remains unclear whether the graduated pressure profile is an essential feature for reducing the development of muscle fatigue. This study sought to examine the effect of the pressure profile of compression stockings on the degree of muscle fatigue of lower leg muscles induced by submaximal running exercise. 15 male subjects performed 30-min treadmill running in 1 control and 4 compression stocking conditions with the following profiles; 1) graduated low pressure, 2) graduated high pressure, 3) uniform pressure distribution, and 4) localized pressure just over the gastrocnemius muscle belly. Before and immediately after the exercise, T2-weighted magnetic resonance images of the right lower leg were obtained without testing garments. T2 values of the triceps surae and tibialis anterior were calculated from the images. T2 was significantly increased after the running in all conditions. The magnitude of T2 increase was significantly greater in the control than in other 3 conditions except for the one with graduated low pressure, whereas there were no significant differences among the latter 3 conditions. The findings suggest that a graduated pressure profile is not an essential feature of compression stockings for reducing the development of muscle fatigue during submaximal running exercise.

Visceral fat accumulation is associated with cerebral small vessel disease.

BACKGROUND AND PURPOSE: Obesity is associated with the risk of coronary artery disease and stroke. Visceral fat plays a significant role in the atherogenic effects of obesity. Whether visceral fat accumulation, as measured by computed tomography (CT), is an independent risk factor for the presence of cerebral small vessel disease (SVD) was investigated. METHODS: This study comprised 506 Japanese subjects 35-74 years of age (mean 55.3 years) without a history of symptomatic cerebrovascular disease who underwent health screening tests, including brain magnetic resonance imaging, carotid echography and measurements of the visceral fat area (VFA) and subcutaneous fat area (SFA) on abdominal CT. Visceral fat accumulation was defined as VFA ≥ 100 cm(2) . Logistic regression analysis was performed to examine the associations between visceral fat accumulation and cerebral SVD such as white matter lesions (WMLs) and silent lacunar infarction (SLI). RESULTS: The prevalence of WMLs and SLI but not carotid plaque were significantly higher in subjects with VFA ≥ 100 cm(2) than those with VFA < 100 cm(2) . A VFA ≥ 100 cm(2) was associated with WMLs and SLI independent of age, cardiovascular risk factors and other measurements of obesity, such as waist circumference and body mass index. A large waist circumference was independently associated with SLI. SFA, the combination of VFA and SFA, and body mass index were not associated with WMLs or SLI. CONCLUSIONS: Visceral fat accumulation was independently associated with the presence of cerebral SVD in subjects without a history of symptomatic cerebrovascular disease.

High-density lipoprotein cholesterol subfractions and lecithin: cholesterol acyltransferase activity in collegiate soccer players.

Many of the published data on the lipid profile of athletes is based on studies of endurance athletes. The data on soccer players are rare. The purpose of this study was to examine serum high-density lipoprotein cholesterol subfractions and lecithin:cholesterol acyltransferase activity in collegiate soccer players. 31 well-trained male collegiate soccer players were divided into 2 groups: 16 defenders and 15 offenders. They were compared with 16 sedentary controls. Dietary information was obtained with a food frequency questionnaire. The subjects were all non-smokers and were not taking any drug known to affect the lipid and lipoprotein metabolism. The offenders had significantly higher high-density lipoprotein cholesterol, high-density lipoprotein2 cholesterol, and apolipoprotein A-I than the defenders and controls, whereas the defenders had the significantly higher high-density lipoprotein2 cholesterol than the controls. Both groups of athletes had significantly higher lecithin:cholesterol acyltransferase activity than the controls. The results indicate that favorable lipid and lipoprotein profile could be obtained by vigorous soccer training.

A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2.

Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has been mapped to human chromosome 2q33. Here we report the identification of two independent deletion mutations linked to ALS2 in the coding exons of the new gene ALS2. These deletion mutations result in frameshifts that generate premature stop codons. ALS2 is expressed in various tissues and cells, including neurons throughout the brain and spinal cord, and encodes a protein containing multiple domains that have homology to RanGEF as well as RhoGEF. Deletion mutations are predicted to cause a loss of protein function, providing strong evidence that ALS2 is the causative gene underlying this form of ALS.

Activation of tyrosine hydroxylase prevents pneumonia in a rat chronic cerebral hypoperfusion model.

Pneumonia is a common complication with the highest attributable proportion of deaths in patients with stroke. Cilostazol is a potent type III phosphodiesterase inhibitor, approved as an anti-platelet aggregation agent. The present study was designed to determine the protective mechanism of cilostazol against post-stroke pneumonia using a rat chronic cerebral hypoperfusion model. Rats were subjected to bilateral common carotid artery ligation (LBCCA) and divided randomly into the vehicle group (n=72) and cilostazol group (n=72). Rats of each group were sacrificed at baseline and at days 14, 28 and 42 after LBCCA. Cilostazol significantly improved the swallowing reflex by shortening the latency to elicited swallowing and increasing the numbers of swallows (P<0.05) at 14 days of hypoperfusion. It also decreased the numbers of bacterial colonies grown in cultures from homogenized lungs. Cilostazol markedly upregulated cyclic AMP responsive element binding protein (CREB) phosphorylation, increased tyrosine hydroxylase (TH) expression in the substantial nigra, and maintained dopamine (84.7+/-2.3 vs. 79.2+/-4.1% control; P=0.0512) and substance P levels (86.6+/-7.9 vs. 73.9+/-6.5% control; P<0.05) in the striatum, compared with the vehicle group. Our results indicate that cilostazol improves the swallowing reflex by enhancing the expression of TH through the CREB phosphorylation signaling pathway, and suggest that cilostazol could be useful in preventing pneumonia in the chronic stage of stroke.

Strategic hand assistance for effective and safe retroperitoneoscopic live donor nephrectomy.

Hand-assisted laparoscopic live donor nephrectomy has been widely applied, because it enables safe dissection of the renal vessels, reducing warm ischemia time (WIT) during rapid extraction of the kidney. In the method described in the current series, the hand-port device was placed after the kidney was mostly mobilized using a pure retroperitoneoscopic procedure. After placement of the hand port, the ureter was completely dissected by an open procedure. Finally, the renal vessels were dissected and transected under the hand-assisted retroperitoneoscopic procedure, and the kidney removed through the hand port. We performed 66 retroperitoneoscopic live donor nephrectomies, including 14 right-sided and 52 left-sided procedures, with this original method of hand assistance. The mean operative time, WIT, blood loss, and renal vein length were 246 +/- 43 minutes, 209 +/- 124 seconds, 202 +/- 180 mL, and 17.4 +/- 6.4 mm, respectively. Comparison of the operative data between the initial 30 cases and the recent 36 cases using the established method showed significant differences in blood loss and WIT that approached statistical significance. No delayed graft function was observed in the current series. The technical and functional outcomes were acceptable. The site and timing of hand assistance minimize the disadvantage of a small working space during the retroperitoneoscopic procedure, making surgery easier and safer.

PlGF-1 and VEGFR-1 pathway regulation of the external epithelial hemato-ocular barrier. A model for retinal edema.

VEGF is considered as an important factor in the pathogenesis of macular edema. VEGF induces the rupture of the blood retinal barrier and may also influence the retinal pigment epithelial (RPE) outer retinal barrier. The aim of this work was to analyze the influence of the VEGF receptor pathways in the modulation of the RPE barrier breakdown in vitro and in vivo. The ARPE19 human junctions in culture are modulated by VEGF through VEGFR-1 but not through VEGFR-2. PlGF-1, that is a pure agonist of VEGFR-1, is produced in ARPE-19 cells under hypoxic conditions and mimics VEGF effects on the external retinal barrier as measured by TER and inulin flux. In vivo, the intravitreous injection of PlGF-1 induces a rupture of the external retinal barrier together with a retinal edema. This effect is reversible within 4 days. VEGF-E, that is a pure agonist of VEGFR-2, does not induce any acute effect on the RPE barrier. These results demonstrate that PlGF-1 can reproduce alterations of the RPE barrier occurring during diabetic retinopathy.

Transplantation of ABO-incompatible and living unrelated donor-recipient combinations.

INTRODUCTION: According to the Japanese renal transplant registry 2005, 834 transplantations were performed using living donors. Among them 199 (23.9%) kidneys were donated from spouses (husband/wife) and 174 (20.9%) from ABO-incompatible donors. This study summarized our experience of ABO-incompatible and living unrelated, especially spousal kidney transplantation. PATIENTS AND METHODS: We performed 44 cases of living donor kidney transplantation (LKT) between April 2003 and July 2007, including 14 (31.8%) from spouses (unrelated donor) who were divided into two groups: six patients (group 1; G1) from ABO-incompatible donors and eight patients (group 2; G2) from ABO-compatible donors. During the induction phase, tacrolimus or cyclosporine, mycophenolate mofetil, and methylprednisolone were used for immunosuppression. Basiliximab was administered on postoperative days 0 and 4. In all G1 patients plasmapheresis was performed to remove anti-AB antibodies prior to LKT, and splenectomy performed at the time of or before LKT. RESULTS: Among G1, no patient died. Among G2, one patient died with a functioning graft due to a traumatic subdural hematoma. Graft survival rate was 100% in both groups. The incidence of acute rejection was 33.3% and 25.0% in G1 and G2, respectively. No patient experienced a lethal infectious complication. CONCLUSIONS: Our results demonstrated that transplantation from an ABO-incompatible spousal donor was equivalent to transplantation from an ABO-compatible spousal donor. In response to the shortage of deceased donors, LKT between married couples and from ABO-incompatible donors will spread in Japan.

Preemptive kidney transplantation of living related or unrelated donor-recipient combinations.

INTRODUCTION: According to the Japanese renal transplant registry 2005, 834 transplantations were performed using living donors. Among them 112 (13.4%) patients were transplanted from living donors before the initiation of maintenance dialysis. Preemptive kidney transplantation (PreKTx) has been associated with improved allograft and patient survival rates compared to non-PreKTx. This study was designed to summarize our experience with PreKTx. PATIENTS AND METHODS: From April 2003 to July 2007, 44 living kidney transplantations were performed at our institution. We divided these 44 patients into two groups: 5 (11.4%) patients (group 1; G1) who underwent PreKTx and the other 39 patients (group 2; G2) who received kidneys after the institution of maintenance dialysis. Living unrelated donors were mostly spouses. During the induction phase, tacrolimus or cyclosporine, mycophenolate mofetil, and methylprednisolone were used for immunosuppression. In ABO-incompatible cases, plasmapheresis was performed to remove anti-AB antibodies prior to transplantation and splenectomy at the time of or before transplantation. RESULTS: Among G1, no patient died. Among G2, two patients died with functioning grafts, one due to a traumatic subdural hematoma and another due to malignant B cell lymphoma. Death-censored graft survival rates were 100% in both groups. The incidence of acute rejection was 20.0% and 20.5% in G1 and G2, respectively. CONCLUSIONS: Our results demonstrated that PreKTx from a living donor was equivalent to the non-PreKTx. However, there were also potential benefits to PreKTx in the long-term outcome, including avoidance of morbidity associated with dialysis and access procedures, as well as reduced cost. In response to the shortage of deceased donors, PreKTx from living donors will spread in Japan.

Structure of the two promoters of the human lck gene: differential accumulation of two classes of lck transcripts in T cells.

The human T-cell- or lymphocyte-specific gene, lck, encodes a tyrosine kinase and is a member of the src family. In this report we demonstrate that there are two classes of human lck transcripts (types I and II), containing different 5'-untranslated regions, which are expressed from two distinct promoters. No apparent sequence similarity was observed between the 5'-flanking regions of the two promoters. The expression of lck in human T-cell leukemia and carcinoma cell lines and in human peripheral blood T lymphocytes was examined by S1 nuclease and primer extension mapping and by Northern (RNA) blot analysis of total cellular RNA. The following results were obtained. (i) Two RNA start sites in the downstream promoter were used to generate type I transcripts. (ii) The major human type I start site has not been described for the mouse. (iii) At least five RNA start sites in the upstream promoter were used to generate type II transcripts. (iv) In T cells and in two colon carcinoma cell lines, type II transcripts were present in higher amounts than type I transcripts. (v) In T cells treated with phytohemagglutinin, tetradecanoylphorbol acetate, and cyclosporin A, the modulation of lck expression was associated primarily with changes in levels of type II transcripts. The above results suggest that the two human lck promoters are utilized differentially and may be regulated independently during certain physiological states.

Development of a long-slot microwave plasma source.

A 20 cm long 10 cm wide microwave plasma source was realized by inserting two 20 cm long 1.5 mm diameter rod antennas into the plasma. Plasma luminous distributions around the antennas were changed by magnetic field arrangement created by permanent magnets attached to the source. The distributions appeared homogeneous in one direction along the antenna when the spacing between the antenna and the source wall was 7.5 mm for the input microwave frequency of 2.45 GHz. Plasma density and temperature at a plane 20 cm downstream from the microwave shield were measured by a Langmuir probe array at 150 W microwave power input. The measured electron density and temperature varied over space from 3.0 × 10(9) cm(-3) to 5.8 × 10(9) cm(-3), and from 1.1 eV to 2.1 eV, respectively.

Sandwich organization of non-ionic surfactant liquid crystalline phases as induced by large inorganic K4Nb6O17 nanosheets.

While retaining its lamellar liquid crystal phase, K4Nb6O17 nanosheets were used as a template to sandwich and stabilize an alkylpoly(ethylene oxide) nonionic surfactant-water system showing monodomain (lamella) formation within the inorganic niobate sheets that appears to be not dependent on the surfactant liquid crystalline state in solution but more its concentration.

Dust particle diffusion in ion beam transport region.

Dust particles of µm size produced by a monoplasmatron ion source are observed by a laser light scattering. The scattered light signal from an incident laser at 532 nm wavelength indicates when and where a particle passes through the ion beam transport region. As the result, dusts with the size more than 10 µm are found to be distributed in the center of the ion beam, while dusts with the size less than 10 µm size are distributed along the edge of the ion beam. Floating potential and electron temperature at beam transport region are measured by an electrostatic probe. This observation can be explained by a charge up model of the dust in the plasma boundary region.

Myb and Ets related transcription factors are required for activity of the human lck type I promoter.

The lck gene, which encodes a lymphoid-specific Src family tyrosine kinase, is transcribed from two promoters that are differentially utilized during T cell development. We have shown previously that the human lck type I promoter, which is preferentially expressed in immature thymocytes, requires a binding site (-97 to -90) for the Ets family of transcription factors for its activity in Jurkat T leukemia cells. Three putative Myb binding sites (-86 to -82, -77 to -72 and -59 to -54) were analysed for their ability to activate the lck type I promoter. In vitro assays demonstrated specific binding of purified, bacterially expressed c-Myb DNA binding domain to the Myb (-59 to -54) site. Transient transfection assays using the site-directed mutants of the lck type I promoter in Jurkat cells revealed that mutation of the Myb (-59 to -54) site abolished transcriptional activity. In transiently transfected HeLa cells, the lck type I promoter was activated by co-transfection with a vector that expresses c-Myb. This c-Myb dependent activation required the presence of intact Myb and Ets binding sites, indicating that the expressed c-Myb functions with endogenous Ets related transcription factors to activate the lck type I promoter. This effect was further enhanced by co-transfection with vectors that express either Ets1 or Ets2. These results demonstrate that Myb and Ets related transcription factors synergistically activate the human lck type I promoter.

Requirement of an ETS-binding element for transcription of the human lck type I promoter.

The requirement for cis-acting DNA sequences for transcriptional activity of the human lck type I promoter was investigated in two human cell lines that express type I transcripts, the leukemic T-cell line, Jurkat, and the colon carcinoma line, SW620. Transient transfection assays in Jurkat and SW620 cells revealed negative and positive cis-acting regulatory elements in the lck type I promoter between -570 and -480 and between -128 and -63 respectively. For the latter, a triple point mutation of a sequence, GCAGGAAGT, from -99 and -91 resulted in complete loss of lck type I promoter activity in both Jurkat and SW620 cells. In vitro binding assays indicated that this sequence, denoted the ETS-binding element or EBE, can interact with the lymphoid-specific transcription factor ETS-1. Thus, a protein(s) in the ETS family appears to be required for transcription of the lck type I promoter in T cells and may be important for the activation of the lck gene in human colon carcinoma.

An alternative pathway for expression of p56lck from type I promoter transcripts in colon carcinoma.

The lymphoid-specific protein tyrosine kinase, p56lck which is essential for both T cell development and function, is aberrantly expressed in colon and small lung carcinoma lines. In this paper, we demonstrate p56lck is also expressed in colon tumour biopsies due predominantly or exclusively to the use of the lck type I promoter. In T leukaemia lines, the lck type I promoter requires binding sites for both Ets- and Myb-related transcription factors. In contrast, in colon tumour lines the activation of the lck type I promoter requires the Ets but not the Myb binding site. In these lines, a consensus binding site for HMG-related transcription factors, AACAAAG, is required for efficient lck type I promoter activity. Sox-4 is a candidate transcription factor for binding and activating the lck type l promoter in colon carcinoma cells. Co-expression of Ets-1 and Sox-4, but neither protein alone, was sufficient to activate the lck type l promoter in HeLa cells which do not normally express lck transcripts. These results suggest that aberrant expression of p56lck from the lck type l promoter in colon carcinoma arises from transcriptional activation mediated by Ets- and HMG-related transcription factors.

Transcriptional regulation of OCI-5/Glypican 3: elongation control of confluence-dependent induction.

OCI-5/Glypican 3, a member of the glypican family of proteoglycans, is the defective gene in the Simpson-Golabi-Behmel overgrowth syndrome. OCI-5 expression is developmentally regulated in the intestinal epithelium, and the mechanism of its regulation was studied in the rat intestinal epithelial cell line IEC-18. A large induction of OCI-5 transcript and protein was observed at high cell density. Among other glypican family members, kappa-glypican also exhibited a confluence-dependent induction in select cell types. Nuclear run-on analysis indicated that cell-density regulation of OCI-5 occurs at the level of transcription. The rat and mouse OCI-5 promoters were cloned and found to be highly conserved, located within CpG islands and contain regions of alternating purine and pyrimidine residues. No TATA-box or recognizable INR element was observed. Consensus binding sites for AP-2, SP-1, zeste and NF-1/CTF are conserved across human, mouse and rat promoters. 5' deletion mapping of the rat promoter identified regions which enhance and repress promoter activity, with no apparent confluence-dependence or tissue-specificity. Nuclear run-on analysis probing different regions of the gene suggests that elongation control plays a role in the induction of OCI-5 by confluence.

Mechanism of cytosine arabinoside toxicity to the blast cells of acute myeloblastic leukemia: involvement of free radicals.

Retinoic acid and hydrocortisone (HC) have been shown to regulate the drug sensitivity of the blast cells of acute myeloblastic leukemia (AML). We asked if the proto-oncogene bcl-2 played a role in this regulation. As target cells we used the continuous lines, OCI/AML-1, OCI/AML-2 or OCI/AML-5; expression of bcl-2 can be detected by Northern analysis of RNA from OCI/AML-2 or OCI/AML-5 cells; bcl-2 expression can be found in OCI/AML-1 cells only by using RT-PCR. Exposure of OCI/AML-2 or OCI/AML-5 cells to retinoic acid (all-trans retinoic acid, ATRA) led to a down-regulation of bcl-2 expression that was first seen after 2 h of exposure and was complete after a day. The down-regulation could be prevented by exposing the cells to ara-C either before or after ATRA; decrease in bcl-2 protein was moderate and only obvious after 36 h of ATRA treatment. Nuclear run-on experiments provided evidence that bcl-2 down-regulation was occurring at transcriptional and post-translational levels. Since bcl-2 is considered to have anti-oxidant activity, we tested the sensitivity of the three cell lines to H2O2; we found that OCI/AML-1, the line with very low bcl-2 expression, was a 100-fold more H2O2-sensitive than OCI/AML-2 or OCI/AML-5, where bcl-2 expression can be detected readily. We then asked if H2O2 sensitivity could be regulated. We found that exposure of cells to HC before H2O2 was protective while ATRA after peroxide treatment increased killing; this is the same pattern of regulation observed when AML blasts are exposed to HC before, or ATRA after ara-C. Finally, we asked whether N-acetylcysteine (NAC), a known radical scavenger would protect cells against ara-C killing. Significant protection was observed when NAC was given before drug, but not if given after drug. NAC protection against ara-C killing was seen for OCI/AML-1 and 2 cells, but not for OCI/AML-5 cells. We interpret the results as follows: ara-C kills cells in two ways: first, directly, by incorporation into DNA and chain termination; second, indirectly, by inducing the production of toxic radicals. Bcl-2 reduces the oxidant activity of such radicals, and is protective. ATRA regulates ara-C toxicity by its action on bcl-2. Left unexplained are the action of HC, which does not affect bcl-2 expression and the mechanism by which ara-C prevents down-regulation of bcl-2 by ATRA.