Demetylation of the sex-determining region Y gene promoter and incidence of disorder of sex development in cloned dog males.

Canine cloning is occasionally accompanied by abnormal sexual development. Some male donor cells produce cloned pups with female external genitalia and complete male gonadal dysgenesis, which is classified as an XY disorder of sex development (XY DSD). In this study, we examine the potential of 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, to reduce the phenotypic abnormality XY DSD in somatic cell nuclear transfer (SCNT)- derived pups. We used a 9-year-old normal male German Shepherd dog as a cell donor. Donor cells were treated with 10 nM 5-aza-dC for 4 days before being used for SCNT. At the same stage of cell development, significantly lower levels of DNA methylation of the sex-determining region Y (SRY) promoter was observed in the treated donor cells compared to that in the untreated cells (95.2% versus 53.3% on day 4 for the control and treated groups, respectively). No significant differences were observed in the control or treatment groups concerning fusion rate, pregnancy rate (30 days or entire period), the number of pups, or the incidence of XY DSD. However, more XY DSD dogs were observed in the control group (31.25%) than in the treatment group (14.29%). Hypermethylation of the SRY promoter was observed in the XY DSD cloned pups in both the treatment (84.8%) and control groups (91.1 ± 1.4%) compared to the methylation level in the phenotypically normal male pups of the treatment (23.2 ± 20.9%) and control groups (39.1 ± 20.1%). These results suggest that 5-aza-dC treatment of donor cells can reduce the methylation level of the SRY promoter in donor cells, and thus, 5-aza-dC is advantageous for reducing the incidence of XY DSD in canine cloning.

Short communication: retinoic acid plus prolactin to synergistically increase specific casein gene expression in MAC-T cells.

Mammary alveolar (MAC-T) cells, an established bovine mammary epithelial cell line, are frequently used to investigate differentiation. A lactogenic phenotype in these cells is induced by treatment with a combination of hydrocortisone, insulin, and prolactin (PRL). The effect of the vitamin A derivative retinoic acid (RA), which induces differentiation in many cells, has not been studied in MAC-T cells. The objective of this study was to evaluate the differentiation potential of RA (1 µM) in MAC-T cells and to examine the effect of combined treatment with RA (1 µM) and PRL (5 µg/mL). Although RA treatment alone inhibited MAC-T cell proliferation, co-treatment of RA with PRL increased cell growth compared with the control group (treated with 1 µg/mL hydrocortisone and 5 µg/mL insulin). The ratio of Bcl to Bax mRNA was decreased in the RA treatment compared with RA+PRL or control. Retinoic acid-induced differentiation of MAC-T cells was associated with an increase in the mRNA expression of αS1-casein (3.9-fold), αS2-casein (4.5-fold), and ß-casein (4.4-fold) compared with the control group. Expression of αS1-casein, αS2-casein, and ß-casein was increased 12.9-fold, 11.9-fold, and 19.3-fold, respectively, following treatment with RA and PRL combined compared with the control group. These results demonstrate that RA induces differentiation of MAC-T cells and acts synergistically with PRL to increase specific casein gene expression.

Implanted bone marrow-derived mesenchymal stem cells fail to metabolically stabilize or recover electromechanical function in infarcted hearts.

Mesenchymal stem cells (MSCs) have been used with success in several clinical applications for clinical treatment of ischemic hearts. However, the reported effects of MSC-based therapy on myocardial infarction (MI) are inconsistent. In particular, the preventive effects of MSC-based therapy on arrhythmic sudden death and metabolic disorders after infarction remain controversial. Here, we investigated the effects of MSCs on reverse remodeling in an infarcted myocardium, and found that MSC-therapy failed to achieve the complete regeneration of infarcted myocardium. Histological analyses showed that although infarct size and interstitial fibrosis induced by MI recovered significantly after MSC treatment, these improvements were marginal, indicating that a significant amount of damaged tissue was still present. Furthermore, transplanted MSCs had slight anti-apoptotic and anti-inflammatory effects in MSC-implanted regions and no significant improvements in cardiac function were observed, suggesting that naïve MSCs might not be the right cell type to treat myocardial infarction. Furthermore, small ion profiling using ToF-SIMS revealed that the metabolic stabilization provided by the MSCs implantation was not significant compared to the sham group. Together, these results indicate that pretreatment of MSCs is needed to enhance the benefits of MSCs, particularly when MSCs are used to treat arrhythmogenicity and metabolically stabilize infarcted myocardium.

Regulation of xanthine oxidase activity by substrates at active sites via cooperative interactions between catalytic subunits: implication to drug pharmacokinetics.

Three xanthine oxidase substrates (i.e., xanthine, adenine, and 2-amino-4-hydroxypterin) show a "substrate inhibition" pattern (i.e., slower turnover rates at higher substrate concentrations), whereas another two substrates (i.e., xanthopterin and lumazine) show a "substrate activation" pattern (i.e., higher turnover rates at higher substrate concentrations). Binding of a 6-formylpterin at one of the two xanthine oxidase active sites slows down the turnover rate of xanthine at the adjacent active site from 17.0 s(-1) to 10.5 s(-1), and converts the V-[S] plot from "substrate inhibition" pattern to a classical Michaelis-Menten hyperbolic saturation pattern. In contrast, binding of xanthine at an active site accelerates the turnover rate of 6-formylpterin at the neighboring active site. The experimental results demonstrate that a substrate can regulate the activity of xanthine oxidase via binding at the active sites; or a xanthine oxidase catalytic subunit can simultaneously serve as a regulatory unit. Theoretical simulation based on the velocity equation derived from the extended Michaelis-Menten model shows that the substrate inhibition and the substrate activation behavior in the V-[S] plots could be obtained by introducing cooperative interactions between two catalytic subunits in homodimeric enzymes. The current work confirms that there exist very strong cooperative interactions between the two catalytic subunits of xanthine oxidase.

Mechanics of nanowire/nanotube in-surface buckling on elastomeric substrates.

A continuum mechanics theory is established for the in-surface buckling of one-dimensional nanomaterials on compliant substrates, such as silicon nanowires on elastomeric substrates observed in experiments. Simple analytical expressions are obtained for the buckling wavelength, amplitude and critical buckling strain in terms of the bending and tension stiffness of the nanomaterial and the substrate elastic properties. The analysis is applied to silicon nanowires, single-walled carbon nanotubes, multi-walled carbon nanotubes, and carbon nanotube bundles. For silicon nanowires, the measured buckling wavelength gives Young's modulus to be 140 GPa, which agrees well with the prior experimental studies. It is shown that the energy for in-surface buckling is lower than that for normal (out-of-surface) buckling, and is therefore energetically favorable.

Identification and characterization of a novel mouse and human MOPT gene containing MORN-motif protein in testis.

A novel testis-derived membrane occupation and recognition nexus (MORN)-motif protein was identified in mouse testis (MOPT) by subtraction screening methods and found to be localized on chromosome 17E3, spanning approximately 7kb. Sequence analysis showed that MOPT contains 669 base pair nucleotides of open reading frame and the corresponding 79 amino acids. The protein is predicted to have theoretical molecular mass of 9000 Da and an expected isoelectric point of 5.8 and seems to have unique sequences except for MORN-motif domain. The transcript of MOPT is highly and specifically expressed in adult testis as well as skeletal muscle. Moreover, MOPT transcript and protein are confined mainly to round and elongated spermatids, except for a few individual dispersed spermatocytes, and increase in abundance at subsequent stages. MOPT first appeared in the proacrosomic vesicles of the early Golgi phase spermatids and was translocated from the head cap of elongated spermatid to the nucleus of mature spermatozoa at the final stage of spermiogenesis. Our study suggests that MOPT may play an important role in dynamic regulation of acrosome biogenesis during late spermiogenesis.

Stiffness and thickness of boron-nitride nanotubes.

We establish an analytic approach to determine the tensile and bending stiffness of a hexagonal boron-nitride (h-BN) monolayer and single- and multi-wall boron-nitride nanotubes (BNNTs) directly from the interatomic potential. Such an approach enables one to bypass atomistic simulations and to give the tensile and bending stiffness in terms of the parameters in the potential. For single- and multi-wall BNNTs, the stiffness also depends on the (inner most or outer most) wall radius and the number of the walls. The thickness of h-BN monolayer is also discussed.

Mechanical properties of functionalized carbon nanotubes.

Carbon nanotubes (CNTs) used to reinforce polymer matrix composites are functionalized to form covalent bonds with the polymer in order to enhance the CNT/polymer interfaces. These bonds destroy the perfect atomic structures of a CNT and degrade its mechanical properties. We use atomistic simulations to study the effect of hydrogenization on the mechanical properties of single-wall carbon nanotubes. The elastic modulus of CNTs gradually decreases with the increasing functionalization (percentage of C-H bonds). However, both the strength and ductility drop sharply at a small percentage of functionalization, reflecting their sensitivity to C-H bonds. The cluster C-H bonds forming two rings leads to a significant reduction in the strength and ductility. The effect of carbonization has essentially the same effect as hydrogenization.

Continuum modeling of boron nitride nanotubes.

Boron nitride nanotubes display unique properties and have many potential applications. A finite-deformation shell theory is developed for boron nitride nanotubes directly from the interatomic potential to account for the effect of bending and curvature. Its constitutive relation accounts for the nonlinear, multi-body atomistic interactions, and therefore can model the important effect of tube chirality and radius. The theory is then used to determine whether a single-wall boron nitride nanotube can be modeled as a linear elastic isotropic shell. Instabilities of boron nitride nanotubes under different loadings (e.g., tension, compression, and torsion) are also studied. It is shown that the tension instability of boron nitride nanotubes is material instability, while the compression and torsion instabilities are structural instabilities.

Seroprevalence and risk factors for human papillomavirus in Taiwan.

The aim of the study was to tailor a future Human papillomavirus (HPV) vaccine campaign and to help perform early primary prevention of HPV infection in Taiwan, where the incidence of cervical cancer is high. A cross-sectional survey was conducted of 826 female students, ages 10, 13, 16 and 19-22 years. A self-administered questionnaire was used to collect information on risk factors for HPV infection. Serum samples were tested for antibodies to HPV 16 capsids using a virus-like particle-based enzyme-linked immunosorbence assay. The age-adjusted odds ratio of HPV seropositivity was calculated for each risk factor by multiple logistic regression analysis. HPV 16 antibodies were detected in 13 (1.6%) of 826 participants. The HPV 16 seroprevalence was 0.35% (1/287), 0.85% (2/235), 3.2% (6/185) and 3.4% (4/119), respectively, for age groups of 10, 13, 16 and 19-22 years. In the multiple regression analysis, the history of having sexual activity was the most significant risk predictor for HPV 16 seropositivity. The seroprevalence of HPV 16 increased dramatically among high school seniors and university students, and was significantly associated with sexual activity. Vaccination against HPV is suggested to be undertaken in early adolescence, before 16 years of age and prior to sexual debut.

Long-term follow-up of porcine male germ cells transplanted into mouse testes.

This study investigated the effect of increased phylogenetic distance on the outcome of spermatogonial transplantation, with porcine donors and mice recipients. It was designed to develop a technique for detecting foreign donor cells in recipient animals. Porcine male germ cells were harvested from postnatal male testes and incubated with the lipophilic membrane dye PKH-26. For transplantation, approximately 10(6) PKH-26-labelled porcine male germ cells were injected into the efferent ducts of mouse testes. Animals were sacrificed at post-graft days 1, 10, 30, 45, 60 and 150 (n = 5 each). Serial frozen sections of explanted testes were prepared for detecting labelled cells. Transplanted porcine donor cells were easily detected in the recipient tubules for 8 weeks. After transplantation, we could detect both incorporation into the basement membrane and differentiation of grafted porcine donor cells by our double detection system, using PKH staining and slide PCR. However, our RT-PCR and apoptosis results revealed that most of the grafted porcine male donor cells could not differentiate past early-meiotic spermatocytes. We could induce partial differentiation of xenografted porcine donor cells in mouse testes, but not full induction of spermatogenesis. We have developed a very reliable technique for detecting foreign donor cells in recipient animals using a combination of PKH staining and slide PCR methods. Our results provide a valuable experimental model for applying and evaluating this technology in other species.

Impact of obesity on coronary artery calcification examined by electron beam computed tomographic scan.

AIM: Obesity is highly associated with cardiovascular disease (CVD). The early and non-invasive diagnosis method for asymptomatic obese is desirable. The aim of this study was to examine the impact of obesity on coronary artery calcification (CAC) by electron beam computed tomographic (EBCT) scan. METHODS: A total of 465 subjects (i) aged between 40 and 65 years, (ii) being Chinese, (iii) without clinical or historical angiographic obstruction or arrhythmia and (iv) without family history of CVD were enrolled in this study. All the subjects were assigned to one of the EBCT CAC score categories according to the quartiles: quartile 1 (<25%), quartile 2 (25-49%), quartile 3 (50-75%) and quartile 4 (>75%), for further assessment and comparison. The main outcome evaluated is the difference in CAC scores between obese [body mass index (BMI): > or =30 kg/m(2)] and healthy (BMI: 18.5-25.0 g/m(2)) BMI groups. The trends of the characteristics in CAC quartile groups and the odds ratios (ORs) were also evaluated. RESULTS: The mean of CAC scores between the obese and the healthy BMI groups showed significant difference (p = 0.05). The obese subjects had higher ORs (1.0-5.8 times) than those with BMI < 23 kg/m(2), and male had higher ORs (1.1-3.6 times) than female, to develop the high CAC score quartile group. CONCLUSION: This study demonstrated that the obese BMI group has a higher mean of CAC scores than the healthy BMI group of middle-aged, asymptomatic, Chinese adults. The obese males have higher risk of developing high CAC scores, which might induce CVD.

Collapse and stability of single- and multi-wall carbon nanotubes.

The collapse and stability of carbon nanotubes (CNTs) have important implications for their synthesis and applications. While nanotube collapse has been observed experimentally, the conditions for the collapse, especially its dependence on tube structures, are not clear. We have studied the energetics of the collapse of single- and multi-wall CNTs via atomistic simulations. The collapse is governed by the number of walls and the radius of the inner-most wall. The collapsed structure is energetically favored about a certain diameter, which is 4.12, 4.96 and 5.76 nm for single-, double- and triple-wall CNTs, respectively. The CNT chirality also has a strong influence on the collapsed structure, leading to flat, warped and twisted CNTs, depending on the chiral angle.

Shock resuscitation with acupuncture: case report.

A 77 year old aboriginal woman in an isolated village became drowsy and shocked. Poor weather conditions delayed the arrival of the medical and support team--the roads had been seriously destroyed by torrential rains and helicopter was the only means for delivering critical medical care and support. While waiting for the arrival of the helicopter, and in the absence of the necessary emergency medical equipment, the patient's condition deteriorated. Administration of persistent emergency acupuncture stimulation for 80 minutes helped maintain the patient's vital signs until successful transfer of the patient to hospital. She recovered without any complications of shock and was discharged six days later.

Arsenic burden survey among refuse incinerator workers.

BACKGROUND: Incinerator workers are not considered to have arsenic overexposure although they have the risk of overexposure to other heavy metals. AIM: To examine the relationship between arsenic burden and risk of occupational exposure in employees working at a municipal refuse incinerator by determining the concentrations of arsenic in the blood and urine. SETTINGS AND DESIGN: The workers were divided into three groups based on their probability of contact with combustion-generated residues, namely Group 1: indirect contact, Group 2: direct contact and Group 3: no contact. Healthy age- and sex-matched residents living in the vicinity were enrolled as the control group. MATERIALS AND METHODS: Heavy metal concentrations were measured by atomic absorption spectrophotometer. Downstream rivers and drinking water of the residents were examined for environmental arsenic pollution. A questionnaire survey concerning the contact history of arsenic was simultaneously conducted. STATISTICAL ANALYSIS: Non-parametric tests, cross-tabulation and multinomial logistic regression. RESULTS: This study recruited 122 incinerator workers. The urine and blood arsenic concentrations as well as incidences of overexposure were significantly higher in the workers than in control subjects. The workers who had indirect or no contact with combustion-generated residues had significantly higher blood arsenic level. Arsenic contact history could not explain the difference. Airborne and waterborne arsenic pollution were not detected. CONCLUSION: Incinerator workers run the risk of being exposed to arsenic pollution, especially those who have incomplete protection in the workplace even though they only have indirect or no contact with combustion-generated pollutants.

Effects of electroacupuncture in reducing weight and waist circumference in obese women: a randomized crossover trial.

OBJECTIVE: To compare the effects of weight control on simple obese women between electroacupuncture and sit-up exercise. DESIGN: Randomized and crossover trial conducted from 1 January 2002 to 31 December 2002. The subjects were randomly divided into groups A and B. Group A received electroacupuncture treatment first while group B received sit-up exercise treatment first. After 6 weeks of treatment and 7 days of washout, group A switched to sit-up exercise treatment and group B received electroacupuncture treatment for another 6 weeks. PATIENTS: In total, 54 simple obese women, with waist circumference (WC)>90 cm and body mass index (BMI)>30 kg/m(2), and who had not received any other weight control maneuver within the last 3 months. MEASUREMENT: The measurements of body weight (BW), BMI and WC were performed at the beginning, 6, 8 and 13 weeks. The data at different time periods were compared and expressed as % reductions. RESULTS: Electroacupuncture (n=46) showed significant differences in the % reductions in BW (P=0.001), BMI (P=0.003) and WC (P=0.005) compared with sit-up exercise. At the end of 13 weeks, there were no significant difference between groups A (n=24) and B (n=22) in all the measurements. At the end of the study, groups A and B showed significant differences in the % reductions in BW (P=0.004; 0.001), BMI (P=0.003; 0.021) and WC (P< or =0.001; 0.001) compared with the initial values. CONCLUSIONS: Electroacupuncture treatment is more effective than sit-up exercise in reducing weight and WC, making it an alternative treatment option for weight and WC control on obese women.

Fullerene derivative attenuates ischemia-reperfusion-induced lung injury.

Reactive oxygen species are the major contributing factors to lung ischemia-reperfusion (IR) injury. In this study, we tested whether a water soluble antioxidant fullerene derivative [C(60)(ONO(2))(7 +/- 2)] attenuates IR lung injury. Young Wistar rats were divided into two groups: control and C(60)(ONO(2))(7 +/- 2). Under ventilation with 95% air-5% CO(2) gas mixture and a 2.5 cm H(2)O end-expiratory pressure, the isolated lungs were perfused with a physiological solution. The experimental protocol included three periods: baseline (10 min), ischemia (45 min) and reperfusion (60 min, ventilated with 95% O(2)-5% CO(2) gas mixture). Before and after ischemia, we measured pulmonary arterial pressure (Ppa), pulmonary venous pressure and lung weight (W). Then, pulmonary capillary pressure and filtration coefficient (K(fc)) were calculated. Ischemia caused increases in Ppa, W and K(fc) in the control group. For most cases, the above ischemia-induced increases were attenuated by the C(60)(ONO(2))(7 +/- 2) pretreatment. Our results suggest that the antioxidant C(60)(ONO(2))(7 +/- 2) attenuates IR-induced lung injury.

Implication of the small GTPase Rac1 in the apoptosis induced by UV in Rat-2 fibroblasts.

Exposure of mammalian cells to ultraviolet (UV) light elicits a cellular response and also lead to apoptotic cell death. However, the role of Rac, a member of Rho family GTPases, in the UV-induced apoptosis has never been examined. In UV-irradiated Rat-2 fibroblasts, nuclear fragmentation began to be observed within 2 h and the total viability of Rat-2 cells were only about 15% at 6 h following by UV irradiation, whereas the total viability in Rat2-Rac(N17) cells stably expressing RacN17, a dominant negative Rac1 mutant, was almost close to 67%. Pretreatment with SB203580, a specific inhibitor of p38 kinase, likewise attenuated UV-induced cell death, but PD98059, a MEK inhibitor, did not. Thus, Rac1 and p38 kinase appear to be components in the apoptotic signaling pathway induced by UV irradiation in Rat-2 fibroblasts. In addition, our results show that p38 kinase stimulation by UV is dramatically inhibited by RacN17, suggesting that p38 kinase is situated downstream of Rac1 in the UV signaling to apoptosis.

A novel recombinant basic fibroblast growth factor and its secretion.

Basic fibroblast growth factor (FGF-2) is a pleiotropic mitogen which plays an important role in cell growth, differentiation, migration, and survival in different cells and organ systems. Recently, several clinical applications for FGF-2 gene transfer are being evaluated in wound healing and collateral artery development to relieve myocardial and peripheral ischemia due to the ability of FGF-2 to regulate the growth and function of vascular cells. However, FGF-2 lacks a classical hydrophobic secretion signal peptide, the FGF-2 chimeras containing various signal sequences have been explored. In this study, a novel recombinant 4sFGF-2 was constructed by replacing nine residues from the amino-terminus of native FGF-2 (Met1 to Leu9) with eight amino acid residues of signal peptide of FGF-4 (Met1 to Ala8) to better increase the secretion level of FGF-2. When the recombinant FGF-2 gene, cloned into the expression vector with CMV promoter, was expressed in COS-7 cells, the recombinant 4sFGF-2 was highly secreted into the media. The secreted 4sFGF-2 showed the same biological activity as the native FGF-2 in the dose-response effects on DNA synthesis and cell growth of rat aortic smooth muscle cells (RASMCs) and NIH3T3 cells. The 4sFGF-2 also was able to activate MAPK as wild FGF-2 in RASMCs. These results indicate that a novel recombinant 4sFGF-2 may be useful as clinical applicability of angiogenic growth factor gene transfer.

Photoreactivation of alloxanthine-inhibited xanthine oxidase.

Alloxanthine-inhibited xanthine oxidase (XOD) was found to be photoreactivated by irradiation of light of wavelengths in the range of 340-430 nm. The enzyme activity can be fully controlled to be on or off by many dark-light cycles. Electron spin resonance measurement shows the appearance of the molybdenum (V) ion and the reduced form of flavin adenine dinucleotide (FADH.) radical signals after irradiation of the alloxanthine-XOD complex. Electronic-absorption spectrum also shows the bleaching of Fe/S and flavin adenine dinucleotide chromophores at 375 and 450 nm as well as broad-band absorption of FADH. in the range of 500-700 nm. The quantum yield of photoreactivation of the enzyme activity is approximately 0.06. A photoinduced intraenzyme electron-transfer model is proposed to rationalize the photoreactivation process.