Effects of eupatilin on the contractility of corpus cavernosal smooth muscle through nitric oxide-independent pathways.

Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) is one of the main compounds present in Artemisia species. Eupatilin has both antioxidative and anti-inflammatory properties and a relaxation effect on vascular contraction regardless of endothelial function. We evaluated the relaxant effects of eupatilin on the corpus cavernosum (CC) of rabbits and the underlying mechanisms of its activity in human corpus cavernosum smooth muscle (CCSM) cells. Isolated rabbit CC strips were mounted in an organ bath system. A conventional whole-cell patch clamp technique was used to measure activation of calcium-sensitive K+ -channel currents in human CCSM cells. The relaxation effect of eupatilin was evaluated by cumulative addition (10-5  m ~ 3 × 10-4  m) to CC strips precontracted with 10-5  m phenylephrine. Western blotting analysis was performed to measure myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17-kDa (CPI-17) expression and to evaluate the effect of eupatilin on the RhoA/Rho-kinase pathway. Eupatilin effectively relaxed the phenylephrine-induced tone in the rabbit CC strips in a concentration-dependent manner with an estimated EC50 value of 1.2 ± 1.6 × 10-4  m (n = 8, p < 0.05). Iberiotoxin and tetraethylammonium significantly reduced the relaxation effect (n = 8, p < 0.001 and p = 0.003, respectively). Removal of the endothelium or the presence of L-NAME or indomethacin did not affect the relaxation effect of eupatilin. In CCSM cells, the extracellular application of eupatilin 10-4  m significantly increased the outward currents, and the eupatilin-stimulated currents were significantly attenuated by treatment with 10-7  m iberiotoxin (n = 13, p < 0.05). Eupatilin reduced the phosphorylation level of MYPT1 at Thr853 of MLCP and CPI-17 at Thr38. Eupatilin-induced relaxation of the CCSM cells via NO-independent pathways. The relaxation effects of eupatilin on CCSM cells were partially due to activation of BKCa channels and inhibition of RhoA/Rho-kinase.

Comparisons of apomorphine-induced erection and spontaneous erection in rats by telemetric assessment of intracavernosal pressure.

Although there are several methods for assessing erectile function in rats, the standard methods for telemetric monitoring have not been established. Theoretically assessment of spontaneous erection (SE) seems to be a physiologic method but it needs long measuring time and additional efforts. Apomorphine-induced erection (AIE) is one available and simple method; however, the correlation with SE has not been assessed. We compared erection profiles of AIE and SE in normal and two disease rat models using telemetric assessment of intracavernosal pressure (ICP). Seven-week-old male Sprague-Dawley rats were assigned to normal control, diabetes mellitus (DM) and hypercholesterolemia (HC) group. After 19 weeks a telemetric pressure sensor (C40; Data Sciences) was surgically implanted in the corpus cavernosum. One week later, ICP was recorded in freely moving rats after intraperitoneal apomorphine (100 µg/kg) injection (AIE) or during SE. Sexual events were visually identified and recorded. Only the pressure increases that occurred during sexual behavior were analyzed. We compared the erectile profiles such as duration, maximal ICP and the area under the curve (AUC, area under time × ICP curves). Two-way anova revealed no significant effect of the measuring methods on the mean AUC (F1,43 = 2.756, p-value = 0.104), but a significant effect of different disease models on mean AUC (two-way anova: F2,43 = 12.929, p-value < 0.001) was observed. The mean AUC of normal control rats was significantly higher than that of DM and HC rats (Bonferroni post hoc test: p < 0.001 and p = 0.001, respectively). ICP measurements using a telemetric device showed no significant difference in AUC between AIE and SE. AIE is easy and requires less time than SE measurements. Therefore, AIE could be a useful method to evaluate ICP in rats.

Increased expression of TRPC4 channels associated with erectile dysfunction in diabetes.

In recent reports, an association between altered TRPC channel function and the development of various diabetic complications has drawn the attention of many investigators. The aim of this study was to investigate the expression of TRPC4 channels of corpus smooth muscle (CSM) cells in diabetes, and to evaluate the association between erectile dysfunction (ED) and altered TRPC4 channel function. The expression of TRPC4 in the penile tissue of human, normal and diabetic rat was investigated using RT-PCR, western blotting and immunohistochemistry (IHC). In vivo gene transfer of dominant negative (DN) TRPC4 into the CSM of rat was conducted. In vivo pelvic nerve stimulation was performed to measure erectile function. Expression of TRPC1, TRPC3, TRPC4 and TRPC6 in human and rat CSM tissues was confirmed by RT-PCR, western blot and IHC. In the diabetic rat, the expression levels of mRNA and protein of the TRPC4, and TRPC6 were significantly increased compared to control rats (p < 0.05). The change in TRPC4 expression in the diabetic rats was higher than those of the other TRPC subunits (p < 0.05). The IHC showed that only TRPC4 expression had a higher intensity in the diabetes compared to normal rats (p < 0.05). Gene transfection with TRPC4(DN) into the diabetic rats restored erectile function to levels similar to that of normal controls. Gene expression of TRPC4(DN) in CSM tissue was confirmed by RT-PCR 2 weeks after transfection. This study demonstrated that TRPC4 channel expression increased in the penile CSM cells of diabetic rats. The down-regulation of TRPC4 with DN form restored erectile function in the diabetic rats. The alteration of TRPC4 channel is one of pathophysiology of ED and could be a target for drug development for ED.

The role of intracavernosal injection therapy and the reasons of withdrawal from therapy in patients with erectile dysfunction in the era of PDE5 inhibitors.

There has been little data regarding the role of intracavernosal injection (ICI) treatment, its discontinuation rate and the reasons of withdrawal in patients with erectile dysfunction (ED) in the era of phosphodiesterase type 5 (PDE5) inhibitors. The aim of this study was to investigate the rate of withdrawal and its associated reasons in patients undergoing ICI therapy. Patients who were prescribed with ICI treatment two times or more were included since the introduction of sildenafil in Korea in 1999. Telephone surveys were performed to evaluate intercourse rates, withdrawal rates and their associated reasons, adverse events and the patients' satisfaction with their sex lives after the ICI treatments. Two hundred and ninety-four men were contacted by telephone. The mean age was 61.8 ± 7.9 years with a follow-up duration of 25.6 ± 32.1 months. At the last follow-up, 79.9% had discontinued the treatment. Most patients had previously failed PDE5 inhibitor treatment prior to the ICI therapy, and more than half had two or more risk factors of ED. Adequate penile rigidity after ICI therapy was restored in 60.2% of patients. The reasons for discontinuation of ICI were poor response (43.1%), inconvenience of use (18.3%), switch to other treatments (10.7%), loss of libido (6.7%), adverse events (5.5%) and return of spontaneous erection (2.8%). Pain was the most common adverse event in the withdrawal group, whereas prolonged erection was most common in the continuing group. Following ICI treatment, PDE5 inhibitors were the most common therapeutic option (63.1%). The overall satisfaction rate regarding sex life was significantly high in the treatment-continuing group. In conclusion, patients on ICI treatment had severe ED and high withdrawal rates in the era of PDE5 inhibitors. The most common reason for treatment discontinuation was poor response. Before initiating ICI treatments, sufficient counselling is necessary.

Vitamin E status of infants at birth.

Preterm infants may be susceptible to chronic lung disease and retinopathy of prematurity because of deficient antioxidant mechanisms including deficiency of vitamin E. The aim of this study was to evaluate the status of the antioxidant vitamin E among preterm and term livebirths. Umbilical cord blood samples collected from 40 preterm and 180 term babies were analyzed for vitamin E levels using high performance liquid chromatography. Linear regression analysis was used to examine the relationship of vitamin E with gestational age, birth weight and appropriateness of weight for gestational age. The median vitamin E level of preterm babies (2.61 mg/L) was not significantly different from that of term babies (2.77 mg/L), p = 0.2. Linear regression analysis demonstrated a weak but statistically significant correlation between cord blood vitamin E levels and gestational age (r = 0.14, p = 0.046). Vitamin E levels did not correlate with birth weight or weight for gestational age. Preterm babies had a higher incidence of vitamin E deficiency compared to term babies (38% v 19%, p = 0.02). Our findings lead us to conclude that vitamin E accumulates in the fetus throughout the third trimester so that preterm infants are likely to have vitamin E deficiency.

Viral loads and E2/NS1 region sequences of hepatitis C virus in hepatocellular carcinoma and surrounding liver.

OBJECTIVES: Numerous epidemiologic data have documented that chronic infection by hepatitis C virus (HCV) is a major risk factor for the development of hepatocellular carcinoma (HCC). But the molecular mechanism underlying these strong epidemiologic associations between HCV and HCC has not be elucidated. We observed the changes of HCV in HCC to investigate the association of HCV with HCC. METHODS: We used competitive and quantitative polymerase chain reaction and dideoxy-nucleotide chain termination method to compare HCV titers and sequences of the hypervariable region of E2/NS1 region of four isolates from the HCC and surrounding cirrhotic liver tissues in tow anti-HCV positive patients. RESULTS: The copy numbers of HCV-RNA were 1 x 10(6) and 4 x 10(6)/gm wet weight of HCC, and 8 x 10(7) and 3.2 x 10(8)/gm wet weight of cirrhotic liver tissues from patient-1 and -2. The sequence differences between HCV RNA in HCC and in cirrhotic liver were two and five nucleotides in patient-1 and in patient-2 respectively. The amino acid sequences were changed in one and two site in each patient. CONCLUSION: These findings may suggest the possible etiological role of HCV in carcinogenesis of HCC, but complete sequence analysis of HCV including multiple isolates in the same patient, should be performed in many cases.

Association of hepatitis C virus particles with immunoglobulin: a mechanism for persistent infection.

The physical properties of hepatitis C virus (HCV) particles were determined by ultracentrifugation on 20-60% isopycnic sucrose density gradients. We report that (i) two populations of HCV particles were found in the sera of patients with chronic HCV infection [at high density (1.186-1.213 g/ml) and at low density (1.099-1.127 g/ml)], (ii) virus particles with high density values were associated with immunoglobulin, and (iii) virus particles with low density values accumulated base changes within a hypervariable region (HVR) of the E2 envelope domain of the RNA genome. The results indicate that base changes within the HVR of E2 lead to the accumulation of immunoglobulin-free virus particles. Therefore, these findings imply that persistent HCV infection is established as a consequence of sequence variation in the E2 envelope domain.

Inactivation of aminated horseradish peroxidase by interaction with S-Sepharose.

Horseradish peroxidase which had been aminated by periodate oxidation and reductive amination was purified by cation-exchange chromatography on S-Sepharose. Instead of the expected single peak of aminated enzyme, two distinct peaks of protein were eluted from the column. Evaluation of the protein in each of the two distributions showed that peak number 1 had spectral properties and specific activity similar to those of native enzyme. Distribution number 2 had a threefold reduction in the extinction in the Soret region at 404 nm and was completely devoid of enzymatic activity. This inactivation was caused by a specific interaction between the aminated peroxidase and the S-Sepharose matrix, resulting in a displacement of the heme prosthetic group out of its native orientation. The inactivation of the aminated peroxidase was found to be dependent on time, pH, and the support matrix itself. These results indicate that the S-Sepharose and Mono-S resins are not interchangeable, despite the chemical similarities of the two resins.

Head injuries in childhood: a 2-year survey.

A retrospective study was conducted of the 880 children with head injuries consecutively admitted to the Children's Hospital of Eastern Ontario in Ottawa from July 1976 to June 1978. It confirmed a boy:girl ratio of about 2:1, with a peak of 3.5:1 around 7 years of age. The largest number of head injuries was in children under 1 year of age. Injuries were most common in summer and spring, and most were caused by falls. The most common place for head injuries was in the home, but the single most common cause of injuries was bicycle accidents, which were responsible for 12% of all the head injuries. Skull fractures were found in 30% of all the patients. Of the 34 patients with severe head injuries 8 (24%) died, 9 (26%) had a moderate residual disability and 17 (50%) made a good recovery. There were no other deaths, so the mortality for the entire group of 880 patients was 0.9%.

A comparative study of epidural and cisternal pressure in dogs.

The epidural and cisternal pressure was recorded simultaneously in eight dogs. Epidural pressure was monitored with a Ladd fiberoptic sensor, and the cisternal pressure via a Statham transducer and a Grass polygraphy. Various pressure levels were compared when mock cerebrospinal fluid was injected into the cisterna magna. The results were analyzed statistically and, within a range of 0 to 70 mm Hg, a high degree of correlation was found between the pressures of the two compartments (r = 0.96 to 1.0).

Epidural hematoma: report of seven cases with delayed evolution of symptoms.

Epidural hematomas occasionally have a prolonged clinical course with gradual evolution of the neurologic symptoms. Seven such cases are reviewed in this report. Although the clinical course is insidious, there are certain features which should signal the presence of a slowly expanding hematoma. After an apparently minor head injury, the patient who is usually in the younger age group, develops headache. This persists and is accompanied by other non-specific neurologic symptoms which may lead to a mistaken diagnosis of "post-concussion syndrome". Papilledema and focal neurologic deficits eventually appear. Definitive diagnosis is made by the CT scan, although contrast enhancement may be necessary to confirm the nature of the lesion. The treatment is craniotomy and evacuation of the hematoma before serious neurologic deterioration occurs.

Complications of ventriculoatrial and ventriculoperitoneal shunts in a new children's hospital.

At a new children's hospital between 1974 and 1978, 125 patients underwent 243 shunt procedures performed by six neurosurgeons. In spite of diverse techniques used, the overall results were acceptable. Seventy-seven patients required 118 shunt revisions. The mortality related to the shunt procedures of 7.2% and the infection rate of 14.4% were comparable to those of other series reported during the same period. Ventriculoatrial shunts, although fewer in number, required fewer revisions (20 revisions of 18 shunts) than ventriculoperitoneal shunts (97 revisions of 58 shunts) for which the variation in technique was larger. A sharp decline in the complication rate was noted during the last year of the study.

Super giant globoid intracranial aneurysm in an infant. Case report.

The incidence of intracranial aneurysms in infancy is less than 1%. Intracranial arterial aneurysms are very rare in patients under 1 year of age. This 6-month-old baby girl presented with progressive macrocephaly and anemia. Computerized tomography and cerebral angiography demostrated a giant globoid aneurysm, 8 X 9 cm in size, arising from an anomalous posterior cerebral artery. The aneurysm was successfully clipped and excised in two stages. The child made an uneventful recovery. The congenital nature of the aneurysm is discussed, and the literature concerning intracranial aneurysms in patients under 1 year of age is reviewed.

Intracranial pressure monitoring with the fiberoptic transducer in children.

Observations on 52 patients with continuous intracranial pressure (ICP) monitoring are reported. The authors used a fiberoptic epidural transducer with digital readout and paper-recording facilities in the intensive care unit of a children's hospital. They confirm the reports of others that [1] ICP monitoring is an extremely useful aid in the management of patients in coma and that [2] epidural pressure measurement is a safe and accurate method. No infection occurred in this series nor were there any other complications arising from the epidural method of ICP monitoring. Apart from three episodes of breakage of the transducer cable, they did not encounter technical difficulties with the system.