p34 is a novel regulator of the oncogenic behavior of NEDD4-1 and PTEN.

PTEN is one of the most frequently mutated or deleted tumor suppressors in human cancers. NEDD4-1 was recently identified as the E3 ubiquitin ligase for PTEN; however, a number of important questions remain regarding the role of ubiquitination in regulating PTEN function and the mechanisms by which PTEN ubiquitination is regulated. In the present study, we demonstrated that p34, which was identified as a binding partner of NEDD4-1, controls PTEN ubiquitination by regulating NEDD4-1 protein stability. p34 interacts with the WW1 domain of NEDD4-1, an interaction that enhances NEDD4-1 stability. Expression of p34 promotes PTEN poly-ubiquitination, leading to PTEN protein degradation, whereas p34 knockdown results in PTEN mono-ubiquitination. Notably, an inverse correlation between PTEN and p34/NEDD4-1 levels was confirmed in tumor samples from colon cancer patients. Thus, p34 acts as a key regulator of the oncogenic behavior of NEDD4-1 and PTEN.

Emergence of Enterococcus species in the infectious microorganisms cultured from patients with endophthalmitis in South Korea.

PURPOSE: To investigate the microorganisms in culture-proven endophthalmitis and their susceptibilities to antimicrobial agents commonly used in South Korea. METHODS: Medical records of consecutive patients with culture-proven endophthalmitis at eight institutions between 1 January 2004 and 31 July 31 2010 were reviewed. Four categories of endophthalmitis were studied: postoperative, posttraumatic, endogenous, and unspecified. Outcome measures were culture-proven infectious organisms, antimicrobial susceptibilities, and final visual acuity in the patients. RESULTS: A total of 93 microorganisms were identified from 103 patients during the study period. The positive culture rate was 59.2 % (103/174). The most common organisms identified were Enterococcus faecalis (in 20.8 % of patients, 20/96), Staphylococcus epidermidis (18.8 %, 18/96), other coagulase-negative staphylococci (10.4 %, 10/96), Pseudomonas aeruginosa (6.3 %, 6/96), and Klebsiella pneumoniae (6.3 %, 6/96). Two cases of Enterococcus faecium (2.1 %) were recognized. Overall, 70 of 96 (73.0 %) isolates were Gram-positive bacteria, 22 (23.0 %) were Gram-negative bacteria, and 4 (4.2 %) were fungi. The most common organisms resulting in reduced light perception were E. faecalis and K. pneumoniae. CONCLUSIONS: The emergence of E. faecalis in endophthalmitis is mainly caused by the high incidence of E. faecalis in postoperative endophthalmitis. This increase also impacts the final visual acuity of the patients.

Nitric oxide is involved in the immunomodulating activities of acidic polysaccharide from Panax ginseng.

The effects of an acidic polysaccharide isolated from the ethanol-insoluble and water-soluble fraction of Panax ginseng C. A. Meyer on immunomodulating activities were investigated. A high output nitric oxide synthase (iNOS) was shown in female BALB/c mice administered intraperitoneally with the acidic polysaccharide from ginseng. Newly synthesized iNOS protein was also observed in peritoneal macrophages cultured with interferon-gamma and the acidic polysaccharide. Spleen cells from acidic polysaccharide-treated mice did not proliferate in response to concanavalin A, but restored the responsiveness by the cotreatment of NG-monomethyl-L-arginine (NMMA) with concanavalin A. The treatment of mice with aminoguanidine, a specific iNOS inhibitor, alleviated the acidic polysaccharide-induced suppression of antibody response to sheep red blood cells. Present results suggest that the immunomodulating activities of the acidic polysaccharide were mediated by the production of nitric oxide.

Solution structure of the catalytic domain of human collagenase-3 (MMP-13) complexed to a potent non-peptidic sulfonamide inhibitor: binding comparison with stromelysin-1 and collagenase-1.

The full three-dimensional structure of the catalytic domain of human collagenase-3 (MMP-13) complexed to a potent, sulfonamide hydroxamic acid inhibitor (CGS 27023) has been determined by NMR spectroscopy. The results reveal a core domain for the protein consisting of three alpha-helices and five beta-sheet strands with an overall tertiary fold similar to the catalytic domains of other matrix metalloproteinase family members. The S1' pocket, which is the major site of hydrophobic binding interaction, was found to be a wide cleft spanning the length of the protein and presenting facile opportunity for inhibitor extension deep into the pocket. Comparison with the reported X-ray structure of collagenase-3 showed evidence of flexibility for the loop region flanking the S1' pocket in both NMR and X-ray data. This flexibility was corroborated by NMR dynamics studies. Inhibitor binding placed the methoxy phenyl ring in the S1' pocket with the remainder of the molecule primarily solvent-exposed. The binding mode for this inhibitor was found to be similar with respect to stromelysin-1 and collagenase-1; however, subtle comparative differences in the interactions between inhibitor and enzyme were observed for the three MMPs that were consistent with their respective binding potencies.

Effect of Korea red ginseng on the blood pressure in conscious hypertensive rats.

The change of blood pressure and heart rate after intravenous injection of Korea red ginseng (KRG) were studied in the conscious normotensive and one-kidney, one-clip Goldblatt hypertensive (1K, 1C-GBH) rats. Crude saponin (CS) of KRG (50, 100 mg/kg i.v.) induced a hypotensive effect and bradycardia in a dose-dependent manner in the anesthetized rats. On the other hand, CS of KRG (100 mg/kg) induced a hypotensive effect and reflex tachycardia in the conscious rats. Saponin-free fraction (SFF) of KRG did not affect them in the anesthetized normotensive rats (P>.05). The maximal hypotensive effect by CS of KRG in the conscious 1K, 1C-GBH hypertensive rats and L-nitroarginine methyl ester (L-NAME, 40 mg/kg)-treated conscious hypertensive rats was not different from that of conscious normotensive rats (Delta 31.6+/-6.3, Delta 27.5+/-5.8 vs. Delta 26.7+/-4.3 mmHg, P>.05). However, pretreatment of L-NAME significantly inhibited the reflex tachycardia by CS of KRG (70.8+/-7.0 vs. 30.6+/-15.0 bpm, P<.05). Hemolysate-sensitive nitric oxide (NO) current by the CS of KRG was greater than that of the SFF of KRG (651.9+/-128.2 pA for CS and 164.9+/-92.5 pA for SFF, P<.001). These findings suggest that KRG has a hypotensive effect and its effect may be due to saponin fraction of KRG in the conscious rats. The releasing effect of NO of KRG, like NO donor, may be partly contributed to the hypotensive effect of KRG.

Effect of Korean red ginseng on blood pressure and nitric oxide production.

AIM: To investigate the effect of crude saponin and nonsaponin fraction of Korean red ginseng (KRG) on the blood pressure and nitric oxide (NO) production in the conscious rats and cultured endothelial cell line, ECV 304 cells. METHODS: Systolic blood pressure and heart rate were monitored in the conscious rats. Nitric oxide levels and the expression of nitric oxide synthase were measured by a spectrophotometric assay using Griess reagents and Western blotting, respectively. Nitric-oxide synthase activity was measured based on the conversion rate of [3H]arginine to [3H]citrulline. RESULTS: Systolic blood pressure was decreased by crude saponin (100 mg/kg, i.v.) of KRG in the conscious control and one-kidney, one-clip Goldblatt hypertensive (1K, 1C-GBH) rats. The hypotensive effect induced by crude saponin of KRG reached maximum at 2-4 min and slowly recovered after 20 min to the initial level in both groups. Crude saponin of KRG induced tachycardia in the conscious rats but induced bradycardia in the anesthetized rats. In contrast to crude saponin of KRG, hypotensive effect induced by saponin-free fraction was minimal. Nitric oxide concentrations were increased by the treatment of crude saponin in conscious rats as well as in the cultured ECV 304 cells. The protein expression level of endothelial constitutive nitric-oxide synthase (eNOS) in the aorta of rats was not increased by crude saponin (100 mg/kg, i.p. for 3 d). However, nitric-oxide synthase activity was increased by crude saponin of KRG in the aortic homogenate of rats. CONCLUSION: The hypotensive effect of red ginseng is mainly due to saponin fraction of KRG in the conscious rats, and this effect may be due to an increase in the nitric-oxide production by KRG.

Korean red ginseng saponins with low ratios of protopanaxadiol and protopanaxatriol saponin improve scopolamine-induced learning disability and spatial working memory in mice.

The effects of two ginseng saponins having a different ratio of protopanaxadiol (PD) and protopanaxatriol saponins (PT) on the learning impairment induced by scopolamine, and learning and memory in mice were investigated in a passive avoidance task and a Morris water maze task. The ratio of PD and PT was 1.24 and 1.46, respectively. Before training, the ginseng saponins were administered intraperitoneally at doses of 50 and 100 mg/kg. The two saponins improved the scopolamine-induced learning impairment at different dosages in mice, 50 and 100 mg/kg, respectively. However, the two saponins did not show a favorable effect on learning and memory in normal mice. Korean red ginseng saponin with a low PD/PT ratio had an improving effect on spatial working memory, but the saponin with a high PD/PT ratio did not. This finding suggests that the PD/PT ratio of the ginseng saponins may be an important factor in the pharmacological role of red ginseng as a medicinal herb.

Ginsenosides that produce differential antinociception in mice.

Ginsenoside Rc, Rd, and Re induced antinociception in writhing and formalin tests among five representative ginsenosides: Rb1, Rc, Rd, Re, and Rg1. However, these ginsenosides had no effect in the tail-flick test. The antinociceptive effects induced by three ginsenosides were dose dependent. ED50 was 20.5 (7.3-57.4 mg/kg) for Rc, 17 (11.0-27.6 mg/kg) for Rd, and 3.5 (1-12 mg/ kg) for Re in the writhing test and 62 (42-90 mg/kg) for Rc, 45 (20.5-99.0 mg/kg) for Rd, and 82 (48-139 mg/kg) for Re in the second phase of the formalin test. The antinociceptive effects were not blocked by the opioid receptor antagonist naloxone in the writhing and formalin tests. These three ginsenosides did not affect motor function. Ginsenoside Rc and Rd induced hypothermia for 30 to 60 min, and ginsenoside Rc induced hyperthemia after 150 min of treatment at doses of 100 mg/kg. These results suggest that ginsenosides such as Rc, Rd, or Re inhibit mainly chemogenic pain rather than thermal pain by the nonopioid system in mice.

Effect of red ginseng on blood pressure in patients with essential hypertension and white coat hypertension.

The objective of this study is to evaluate the changes of diurnal blood pressure pattern after 8 weeks of red ginseng medication (4.5 g/day) by 24 hour ambulatory blood pressure monitoring. In 26 subjects with essential hypertension, 24 hour mean systolic blood pressure decreased significantly (p = 0.03) while diastolic blood pressure only showed a tendency of decline (p = 0.17). The decrease in pressures were observed at daytime (8 A.M.-6 P.M.) and dawn (5 A.M.-7 A.M.). In 8 subjects with white coat hypertension, no significant blood pressure change was observed. We suggest that red ginseng might be useful as a relatively safe medication adjuvant to current antihypertensive medications.

Determination of ginsenoside Rf and Rg2 from Panax ginseng using enzyme immunoassay.

We have developed an enzyme immunoassay (EIA) to quantify trace amounts of ginsenoside Rf (Rf), one of the glycosides of protopanaxatriol from Panax ginseng. A carrier protein of bovine serum albumin (BSA) was coupled to the carbohydrate component of Rf using the periodate oxidation method. Antibodies were raised in rabbits using Rf-BSA conjugate as the immunogen and competitive indirect EIA was used for the determination of Rf. The working range was 0.01-10 ng per assay. The anti-Rf antiserum cross-reacted with ginsenoside Rg2 (105%), which is also a component of Panax ginseng and has a very similar chemical structure to Rf. These results suggest that the anti-Rf antiserum could also be used for the quantitation of ginsenoside Rg2 as well as ginsenoside Rf. In a comparison of EIA and HPLC the linear regression equation and correlation coefficient for the two methods were y(EIA) = 1.31x (HPLC)-11.48 and 0.98, respectively.

Ginsenosides induce differential antinociception and inhibit substance P induced-nociceptive response in mice.

Ginsenosides are main pharmacoactive molecules of ginseng. The antinociceptive activity of ginsenosides after intrathecal (i.t.) injection was examined in formalin test. We also investigated the effects of ginsenosides on substance P (SP) induced-pain behaviors by i.t. treatment using mice. Pretreatment of ginsenosides by i.t. induced the inhibition of biting and licking of hind paw injected with 1% formalin with dose-dependent manner. The ED50 was 23 (19-28, 95% C.I.) microg/mouse for acute phase and 15 (9-23, 95% C.I.) microg/mouse for tonic phase. Interestingly, cotreatment of ginsenosides with SP also inhibited SP-induced pain behaviors (scratching, licking or biting of hind portion of body) with dose-dependent manner. The ED50 for the inhibition of SP-induced pain behavior by ginsenosides was 30 (11-85, 95% C.I.) microg/mouse. These results suggest that ginsenosides have antinociceptive activity in formalin test and this effect is due to blocking of SP-induced nociceptive information to postsynaptic site(s) at the spinal level.

Sealing effects of (-)-epigallocatechin gallate on protein kinase C and protein phosphatase 2A.

(-)-Epigallocatechin gallate (EGCG) was reported to inhibit protein kinase C (PKC) activation by 12-O-tetradecanoylphorbol-13-acetate (TPA), and inhibit interaction of tumor promoter with its receptors, named 'a sealing effect'. In order to clarify the sealing effect of EGCG, we prepared liposomes and examined inhibition of PKC activation by various concentrations of EGCG dispersed in the liposome. EGCG added to a liposome dispersion existed either in a buffer solution as aggregates or in phospholipid bilayer membranes, and EGCG disturbed membrane structure. The potency of inhibitory effect of EGCG on PKC activation was dependent on the nature of liposomes, indicating that interaction of EGCG with phospholipid bilayer membrane affects PKC activation. Moreover, EGCG prevented the binding of adenosine 5'-triphosphate and TPA to PKC, resulting in inhibition of PKC activation. On the other hand, the activity of protein phosphatase 2A (PP2A) was suppressed in the presence of liposomes, but was not influenced by EGCG. Moreover, EGCG recovered phosphatase activity of PP2A in a buffer solution, the activity of which was inhibited by okadaic acid. All the results indicated that EGCG possesses sealing effects in terms of PKC and PP2A, by inhibiting interaction of various ligands with proteins.

ATP regeneration by thermostable ATP synthase.

We investigated the possibility of using thermostable ATP synthase (TF(0)F(1)) for a new ATP regeneration method. TF(0)F(1) was purified from a thermophilic bacterium, PS3, and reconstituted into liposomes. ATP synthesis experiments showed that TF(0)F(1) liposomes could synthesize ATP in micromole concentrations by acid-base change. The acid-base change was repeated six times over an 11-day period with no detectable loss of activity at the reaction temperature (45 degrees C). Given these encouraging results, we conceptualized and modeled a system to synthesize ATP using ATP synthase with energy supplied by acid-base change. In this system, liposomes containing ATP synthase are immobilized on small glass spheres that facilitate separation of buffers from the liposomes after the acid-base change. Compared to an alternate system that uses membranes to separate the buffers from the liposomes, the glass spheres reduce inefficient mixing of acidic and basic buffers during the acid-base change. To increase the ATP synthesis yield, this system uses electrodialysis to regenerate a potassium gradient after the acid-base change. It also employs water-splitting electrodialysis to regenerate KOH and HCl required to adjust the pH of acidic and basic buffers. All reagents are recycled, so electrical energy is the only required input.

Effect of non-saponin fraction from Panax ginseng on cGMP and thromboxane A2 in human platelet aggregation.

The non-saponin fraction (NSF; lipophilic fraction) from the roots of Panax ginseng inhibited the aggregation of human platelets induced by thrombin (0.1 units/ml) in a dose-dependent manner. NSF induced the elevation of cGMP concentration in human platelets in a similar manner to molsidomine, a known vasodilator. NSF also inhibited Ca(2+)-influx into platelets. While verapamil, a Ca(2+)-antagonist, increased the cAMP level in platelets stimulated by thrombin, NSF had little effect on cAMP formation. Instead, NSF potently inhibited the thromboxane A2 (TXA2) production. The results suggest that NSF may regulate the levels of cGMP and TXA2 to inhibit platelet aggregation induced by thrombin.

Blockade by ginseng total saponin of the development of cocaine induced reverse tolerance and dopamine receptor supersensitivity in mice.

Daily repeated administration of cocaine (15 mg/kg, over a 7-day period) developed reverse tolerance to the ambulation-accelerating effect of cocaine. Intraperitoneal administration of ginseng total saponin (GTS, 100 and 200 mg/kg of body weight) prior to and during chronic administration of cocaine inhibited the development of reverse tolerance. Dopamine receptor supersensitivity was also developed in reverse tolerant mice that had received the same cocaine. The development of dopamine receptor supersensitivity was evidenced by the enhanced hypothermic response to apomorphine (1 mg/kg) and the enhanced ambulatory activity of apomorphine (4 mg/kg). GTS also prevented the development of dopamine receptor supersensitivity induced by the chronic administration of cocaine. These results provide that GTS may be useful for the prevention and therapy of the adverse action of cocaine. It is concluded that the development of reverse tolerance to the ambulation-accelerating effect of cocaine may be associated with the enhanced dopamine receptor sensitivity because both phenomena were blocked by GTS.

Permeability of a non-TPA-type tumor promoter, okadaic acid, through lipid bilayer membrane.

The interaction of okadaic acid (OA) with lipid bilayer membranes was studied to obtain information on its incorporation into the target cell. OA, which possesses a polyether structure with a carboxylic acid, was extracted with a chloroform or n-octanol solution from a buffer solution, indicating the hydrophobicity of OA. However, the distribution of OA to dipalmitoylphosphatidylcholine membrane was so low that OA did not strongly induce perturbation in the membrane structure. On the other hand, OA permeated freely through the lipid membrane in a liquid-crystalline state. It was therefore suggested that OA permeates through cell membrane and binds to the receptor, for example, protein phosphatase, which exists either in the cytosol or in the cell membrane.

Modulation of phospholipase A2 activity by the tumour promoters phorbol esters and teleocidin.

The effects of tumour promoters, namely phorbol esters and teleocidin, on the activity of porcine pancreatic phospholipase A2 (PLA2) was investigated by using a system of small unilamellar vesicles composed of dipalmitoyl-phosphatidylcholine (DPPC). DPPC vesicles encapsulating Quin 2 (Quin 2/DPPC vesicles) were suspended in a medium containing Ca2+. The addition of PLA2 to Quin 2/DPPC vesicles increased the fluorescence intensity of Quin 2. This increase was due to chelation of Quin 2 with Ca2+, which resulted from an increase in the permeability of the phospholipid bilayer caused by the hydrolytic activity of PLA2. The tumour promoters phorbol 12-myristate 13-acetate (PMA) and teleocidin, at low concentrations, enhanced PLA2 activity at temperatures below the phase-transition temperature of the membrane, but, in contrast, high concentrations of the tumour promoters suppressed PLA2 activity. Phorbol 12-myristate (PM) also had a similar effect on PLA2 activity. PMA and PM disturbed the membrane structure markedly, which was indicated by the enhanced leakage of carboxyfluorescein (CF) from DPPC vesicles encapsulating CF. On the other hand, phorbol 12,13-didecanoate and 4 alpha-phorbol 12,13-didecanoate, which did not disturb the membrane structure to the same extent, had an insignificant effect on PLA2 activity. It is therefore concluded that PLA2 catalyses the hydrolysis of phospholipids in bilayer vesicles which contain a moderate degree of structural defects. However, the effects of tumour promoters on PLA2 activity was not related to their potencies as inflammatory and tumour-promoting agents.

Distribution of tumor promoters in lipid membranes and changes in membrane structure.

The interaction of tumor promoters differing in molecular structure, namely, 12-O-tetradecanoylphorbol 13-acetate (TPA) and teleocidin, with dipalmitoylphosphatidylcholine (DPPC) vesicles was studied. Investigation by Fourier transform infrared spectroscopy clarified the differences between the tumor promoters in the mode of interaction with lipid bilayer membranes. The temperature dependence of the bandwidth of the C-H or C = O stretching absorption of lipid molecules in the presence of tumor promoters relative to that in pure DPPC vesicles indicated that TPA is incorporated into the hydrophobic core of the lipid bilayer membrane whilst teleocidin binds predominantly to the membrane surface. However, both tumor promoters tend to restrict the motion of lipid molecules in membranes. The same conclusion was derived from measurements of steady-state fluorescence polarization, which showed that tumor promoters decreased the membrane fluidity. On the other hand, carboxyfluorescein (CF) leakage from vesicles was enhanced by the addition of TPA below the phase-transition temperature, whereas the effect of teleocidin on steady-state CF leakage was not as significant. It is considered that the difference in the profile of the TPA-induced increase in CF leakage compared to that of teleocidin might be ascribable to a different binding site for each tumor promoter in the membranes.