Urethral stricture (US) is a urologic disorder with a high morbidity and recurrence rate, and may arise due to external trauma, surgical procedures or urethritis. The pathogenesis of US is closely associated with scar formation, including excessive collagen-rich connective tissues. Previous studies have shown that connective tissue growth factor (CTGF) plays a key role in the fibrosis process of many tissues and organs, such as kidney and lung. We investigated the variation of CTGF expression in urethral tissues from 12 patients with US and normal urethral tissues. The stricture length range was 1 to 2.5 cm, and 4 patients underwent repeated urethral dilatation. The normal urethral tissues were obtained from 6 patients with heart disease, who were suffering from brain death. The expression of CTGF mRNA and protein was analyzed by real-time PCR, immunohistochemistry, and western blotting analyses. Results showed that the expression level of CTGF mRNA was significantly increased in US patients compared with the control. In addition, US patients who have longer history and more repeated surgical procedures have extremely high CTGF mRNA levels and its protein expression, while the age of patients, position of injury and stricture length were not found obvious relation with CTGF expression. Moreover, the degree of collagen deposition and muscle fiber proliferation in the submucosa is consistent with the increase in CTGF expression. In conclusion, our data suggest that the up-regulation of CTGF expression may be responsible for the fibrosis process of urethral tissues in US patients.
Semicircular canals are sensory organs for balance, consisting of fluid-filled tubules that are arranged perpendicularly to each other in inner ear. The precise mechanism of the morphogenesis of this unique organ is still under investigation. Semicircular canals arise from the flattened pouch of epithelium. The centers of two apposing wall of the pouch approach each other and form a fusion plate. The clearing of the fusion plate makes a hole and leaves the remaining tissue as semicircular canals. Three mechanisms have been proposed for this clearing: programmed cell death, epithelial-mesenchymal transition, and retraction of the cells in the fusion plate to surrounding semicircular canals. Previous studies have revealed programmed cell death in the fusion plate, although other two hypotheses were not disproved. Here we examined the contribution of epithelial-mesenchymal transition and epithelial retraction to the morphogenesis of semicircular canals. We analyzed immunohistochemically the structural change in the epithelium of the developing fusion plate using molecular markers, basal lamina component laminin, cytoskeletal F-actin, and cellular junctional marker β-catenin. Our observation revealed that fusion plate epithelium lost its apico-basal polarity and intermingled with facing fusion plate cells, associated with the disruption of basal lamina. Moreover, there were several cells with mesenchymal appearance adjacent to the torn basal lamina. We also found the merging of apposing basal laminae at the border between forming canal and breaking fusion plate. These observations suggest that the epithelial-mesenchymal transition, rather than the epithelial retraction, may be responsible for clearing fusion plate cells.
Repeated heating of soy oil may promote lipid peroxidation. Oxidized unsaturated fatty acids may contribute to the pathogenesis of atherosclerosis, especially in estrogen-deficient states. This study was performed to explore the deleterious effects of repeatedly heated soy oil on the development of atherosclerosis using ovariectomized rats, which represent an estrogen-deficient state. Twenty-four female Sprague-Dawley rats were ovariectomized and were divided equally into four groups. The control group was fed with 2% cholesterol diet without any oil. The three treatment groups each received 2% cholesterol diet fortified with fresh, once-heated or five-times-heated (repeatedly heated) soy oil, respectively. Serum thiobarbituric acid reactive substances (TBARS), lipid profile and homocysteine levels were measured prior to ovariectomy and at the end of four months. Ovariectomized rats treated with repeatedly heated soy oil showed significant increases in lipid peroxidation and low-density lipoprotein (LDL) levels. Treatment with once-heated or repeatedly heated soy oil caused a significant increase in total cholesterol, while fresh soy oil caused significant reduction in homocysteine level as compared to other groups. Repeatedly heated soy oil caused significant increases in TBARS and LDL as compared to fresh oil. The higher level of homocysteine in the ovariectomized rats fed with repeatedly heated oil, as compared to those fed with fresh oil, also suggests the repeatedly heated oil contributes to the development of atherosclerosis. Importantly, the protective effect of the soy oil may be lost once it was being repeatedly heated. In conclusion, the consumption of repeatedly heated oil may predispose to atherosclerosis in estrogen-deficient states.
The human succinyl-CoA: 3-ketoacid CoA transferase (SCOT) gene encodes the ketolytic enzyme that functions in the mitochondrial matrix. The activation of acetoacetate to acetoacetyl-CoA by SCOT is essential for the use of ketone bodies as an energy source. The ketolytic capacity of tissues is proportional to their level of SCOT activity. Normal hepatocytes, the site of ketone body synthesis, have no detectable SCOT protein. The absence of SCOT in hepatocytes is an important element in energy metabolism, suppressing ketolysis in the liver. To study the tissue-specific silencing of SCOT expression, we analyzed the promoter function of SCOT gene in three different human cell lines. Immunoblot analysis showed that SCOT protein was detectable in HeLa cervical cancer cells and Chang liver cells. However, SCOT protein was not detected in HepG2 hepatoma cells and liver tissues, indicating that HepG2 hepatoma cells maintain the characteristics of liver cells in the ketone body metabolism. Luciferase reporter assays in HeLa and Chang liver cells showed that the 361-bp proximal region of the SCOT gene was responsible for the basal promoter activity and contained two GC boxes, each of which was bound in vitro by Sp1, a ubiquitously expressed transcription factor. These results suggest that these GC boxes may be important for SCOT gene expression. Moreover, the region between -2168 and -361 appeared to inhibit the SCOT promoter activity in HepG2 cells. Thus, liver-specific silencing of the SCOT gene expression may be mediated in part by its 5'-flanking sequence.
In Japan, one of the duties of the long-term care insurance system is the prevention of oral function degradation. Although various exercise programs for oral function have been developed and practiced in Japan, to date, no study has reported their effects. In the present study, we examined the effects of an exercise program on oral functions in healthy elderly people. Healthy elderly people participated in the study and were randomly divided into intervention and control groups (39 subjects/group). The exercise program consisted of four exercises: exercise for expression muscles, tongue, salivary glands, and swallowing. Before and after a six-month intervention period, we evaluated changes in oral functions, namely bite force, swallowing ability, and salivary flow rate. Fifty-four subjects completed the study protocol: 26 and 28 subjects from intervention and control groups, respectively. A significant improvement in all oral functions, including bite force, swallowing ability, as well as unstimulated and stimulated salivary flow rate, was observed in the intervention group after six months, whereas no improvement was observed in the control group. Further, among the intervention group, a significant improvement in oral functions was observed in 17 subjects with 20 or more remaining teeth, whereas no improvement was observed in the other nine with less than 20 teeth. Our results suggest that oral functions significantly improve after conducting a six-month exercise program for oral function. Further, this improvement appears to be influenced by the number of remaining teeth.
Infections with influenza virus type A and B present serious public health problems on a global scale. However, only influenza A virus has been reported to cause fatal pandemic in many species. To provide suitable clinical management and prevent further virus transmission, efficient and effective clinical diagnosis is essential. Therefore, we developed multiplex PCR assays for detecting influenza types A and B and the subtypes of influenza A virus (H1, H3 and H5). Upon performing multiplex PCR assays with type-specific primer sets, the clearly distinguishable products representing influenza A and B virus were separated by agarose gel electrophoresis. In addition, the subtypes of influenza A virus (H1, H3 and H5), which are most common in humans, can be readily distinguished by PCR with subtype-specific primer sets, yielding PCR products of different sizes depending on which subtype has been amplified. This method was tested on 46 influenza virus positive specimens of avian and mammalian (dog and human) origins collected between 2006 and 2008. The sensitivity of this method, tested against known concentrations of each type and subtype specific plasmid, was established to detect 103 copies/μl. The method's specificity was determined by testing against other subtypes of influenza A virus (H2, H4 and H6-H15) and respiratory pathogens commonly found in humans. None of them could be amplified, thus excluding cross reactivity. In conclusion, the multiplex PCR assays developed are advantageous as to rapidity, specificity, and cost effectiveness.
Activated neutrophils have been implicated in the development of ischemia/reperfusion (I/R)-induced renal failure. Cytokine-induced neutrophil chemoattractant-1 (CINC-1), a major factor in acute inflammation, is responsible for the activation of neutrophils and for neutrophil chemotaxis to sites of injury. Atrial natriuretic peptide (ANP), a hormone synthesized by the cardiac atria, was shown to possess anti-inflammatory potential due to its potency to inhibit the production of inflammatory mediators. We examined whether the human form of ANP attenuates I/R-induced renal injury by reducing neutrophil activation in a rat model. Male Wistar rats weighing 200-240 g were observed for 24 h after reperfusion following 45-min renal ischemia. Rats were intravenously administered alpha-human ANP (α-hANP, 0.2 μg/kg/min) beginning immediately after ischemia and continuing for 2 h after reperfusion. CINC-1 and myeloperoxidase (MPO) concentrations were measured to assess activation of the infiltrating neutrophil. Blood urea nitrogen and serum creatinine and urinary N-acetyl β-d-glucosaminidase (NAG) were measured as indicators of glomerular function and as a specific indicator of proximal tubular function, respectively. α-hANP significantly inhibited I/R-induced increases in renal CINC-1 and MPO concentrations. α-hANP also reduced I/R-induced increases in the concentrations of blood urea nitrogen and serum creatinine, and improved histopathologic changes, including acute tubular necrosis. These findings indicate that α-hANP attenuates I/R-induced acute renal injury, at least in part by reducing neutrophil activation, and may be useful in surgeries, associated with renal ischemia, as well as in renal transplantation.
Colorectal cancer (CRC) is a major cause of morbidity and mortality for cancer worldwide, but many patients with CRC are resistant to chemotherapy. We therefore investigated the therapeutic mechanism and clinical effect of combined chemotherapy of hydroxycampothecin (HCPT) with oxaliplatin (L-OHP) on CRC. HCPT represents a potential antitumor agent of Chinese herb. Mice carrying the xenografted human LS174T CRC cells were injected into peritoneal cavities with different drugs: HCPT + L-OHP (OH), HCPT, L-OHP, or saline. Treatment of mice with OH caused the decrease in the volume of tumor and the expression of p53, but increased the apoptotic rate and Fas-L expression, compared to those of animals treated with HCPT or L-OHP, or control animals. Thus, the combination of HCPT with L-OHP could more effectively induce the apoptosis of CRC cells. Furthermore, 56 patients with CRC were treated with HCPT and L-OHP (28 cases, OH group) or L-OHP plus leucovorin plus 5-fluorouracil (28 cases, OFL group), then reviewed the response rate, survival rate and toxicity. The one-year survival rate was 35.07% in OH group and 24.21% in OFL group. However, the occurrence of anemia (51.8%) or diarrhea (60.7%) was higher in OH group than that of 19.6% or 46.4% in OFL group. The clinical results suggest that HCPT plus L-OHP combined chemotherapy could increase the survival time of patients. Taken together, the present study indicates that the combined chemotherapy of HCPT with L-OHP could become a new adjuvant treatment for CRC.
Excitotoxicity is a major neurotoxic mechanism in cochlear disorders, including cochlear ischemic injury and acoustic injury. Kainic acid (KA), an excitatory amino acid, can damage glutamatergic neurons. The application of KA to the round window membrane, an opening of the cochlear bony labyrinth to the middle ear, induces excitotoxicity of cochlear afferent dendrites and significantly decreases the amplitude of compound action potential of the cochlea (CAP), a cochlear potential generated by activation of the auditory nerve fibers. On the other hand, muscimol, a gamma-aminobutyric acid (GABA)A receptor agonist, is neuroprotective in excitotoxicity in the central nervous system. Here we studied whether activation of GABA receptor decreased the excitotoxicity of the cochlea caused by KA. KA (10 mM) was applied to the round window membrane of guinea pigs for 30 minutes. Muscimol, bicuculline, a GABAA receptor antagonist, or baclofen, a GABAB receptor agonist, was given at the onset of KA application. The threshold shift of CAP was examined with chronically implanted electrodes. Application of KA significantly elevated the CAP threshold on day 1 and day 3 as compared with the pre-application level. Muscimol significantly decreased the KA-induced CAP threshold shifts. Furthermore, this protective effect of muscimol was inhibited by the application of bicuculline, a GABAA receptor antagonist. However, baclofen, a GABAB receptor agonist, did not affect the CAP threshold shifts caused by KA. These results suggest that activation of GABAA receptor could prevent cochlear excitotoxicity. GABAA rececptor activation may represent an effective treatment of cochlear ischemc injury and acoustic injury.
Muscle endurance is one of the important composite factors to health-related physical fitness. However, previous measurement techniques for evaluation of isometric muscle endurance have been problematic, including rapid increase of blood pressure and heart rate. The progressive workload method which gradually increases the load can reduce subject's physical and mental burden and is safe even for the elderly. This study aims to examine the relationship between exerted force and subjective muscle-fatigue sensation (SMS) of the antebrachial region as well as differences with respect to gender during sustained static gripping. Subjects consisted of 12 males (age 20.8 ± 1.6 years) and 15 females (age 20.5 ± 1.3 years) with no history of neuromuscular disorders. They performed sustained static gripping, in which the demand values are gradually increased: 10 sec for 20% maximal voluntary contraction (MVC), 20 sec each for 30, 40, 50, 60, and 70% MVC, and 10 sec for 80% MVC. Demand values (each %MVC) were determined based on each subject's MVC. The forces exerted during at the demand values of 70-80% MVC were significantly lower in males than in females. A gender difference in SMS between male and female subjects occurred at demand values of 40-60% MVC. In short, even when maintaining the same exertion force during demand values of 40-60% MVC, males may experience greater physical fatigue than females.