Heliorhodopsin Helps Photolyase to Enhance the DNA Repair Capacity.

Light quality is a significant factor for living organisms that have photosensory systems, such as rhodopsin, a seven alpha-helical transmembrane protein with the retinal chromophore. Here, we report, for the first time, the function of new rhodopsin, which is an inverted 7-transmembrane protein, isolated from Trichococcus flocculiformis. T. flocculiformis heliorhodopsin (TfHeR) works as a regulatory helper rhodopsin that binds with class 2 cyclobutane pyrimidine dimer (CPDII) photolyase to broaden the spectrum and upregulate DNA repair activity. We have confirmed their interaction through isothermal titration calorimetry (dissociation constant of 21.7 µM) and identified the charged residues for the interaction. Based on in vivo and in vitro experiments, we showed that the binding of heliorhodopsin with photolyase improved photolyase activity by about 3-fold to repair UV-caused DNA damage. Also, the DNA repair activity of TfHeR/T. flocculiformis photolyase (TfPHR) was observed in the presence of green light. Our results suggested that heliorhodopsin directly controls the activity of photolyase and coevolves to broaden the activity spectrum by protein-protein interaction. IMPORTANCE This study reports a function for Heliorhodopsin working as a regulatory helper rhodopsin that with CPDII photolyase to broaden the spectrum and upregulating the DNA repair activity. Our results suggested that heliorhodopsin directly controls photolyase activity and coevolves to broaden the DNA repair capacity by protein-protein interaction.

The role of carotenoids in proton-pumping rhodopsin as a primitive solar energy conversion system.

Rhodopsin and carotenoids are two molecules that certain bacteria use to absorb and utilize light. Type I rhodopsin, the simplest active proton transporter, converts light energy into an electrochemical potential. Light produces a proton gradient, which is known as the proton motive force across the cell membrane. Some carotenoids are involved in light absorbance and transfer of absorbed energy to chlorophyll during photosynthesis. A previous study in Salinibacter ruber has shown that carotenoids act as antennae to harvest light and transfer energy to retinal in xanthorhodopsin (XR). Here, we describe the role of canthaxanthin (CAN), a carotenoid, as an antenna for Gloeobacter rhodopsin (GR). The non-covalent complex formed by the interaction between CAN and GR doubled the proton pumping speed and improved the pumping capacity by 1.5-fold. The complex also tripled the proton pumping speed and improved the pumping capacity by 5-fold in the presence of strong and weak light, respectively. Interestingly, when canthaxanthin was bound to Gloeobacter rhodopsin, it showed a 126-fold increase in heat resistance, and it survived better under drought conditions than Gloeobacter rhodopsin. The results suggest direct complementation of Gloeobacter rhodopsin with a carotenoid for primitive solar energy harvesting in cyanobacteria.

Color-tuning of natural variants of heliorhodopsin.

Microbial rhodopsins are distributed through many microorganisms. Heliorhodopsins are newly discovered but have an unclear function. They have seven transmembrane helices similar to type-I and type-II rhodopsins, but they are different in that the N-terminal region of heliorhodopsin is cytoplasmic. We chose 13 representative heliorhodopsins from various microorganisms, expressed and purified with an N-terminal His tag, and measured the absorption spectra. The 13 natural variants had an absorption maximum (λmax) in the range 530-556 nm similar to proteorhodopsin (λmax = 490-525 nm). We selected several candidate residues that influence rhodopsin color-tuning based on sequence alignment and constructed mutants via site-directed mutagenesis to confirm the spectral changes. We found two important residues located near retinal chromophore that influence λmax. We also predict the 3D structure via homology-modeling of Thermoplasmatales heliorhodopsin. The results indicate that the color-tuning mechanism of type-I rhodopsin can be applied to understand the color-tuning of heliorhodopsin.

Use of steel slag as a catalyst in CO2-cofeeding pyrolysis of pine sawdust.

To seek an innovative way for simultaneous waste management and energy recovery, two waste materials (pine sawdust: PSD and steel slag: SS) were used in the pyrolysis process. PSD was used as a carbonaceous material for pyrolysis, and SS was used as a catalyst. Also, to achieve a more sustainable conversion system, a viable use of carbon dioxide (CO2) as a raw material in the non-catalytic/catalytic pyrolysis process was evaluated. Hence, the present study laid great stress on the CO2 effects. The present study pointed the optimistic technical features in line with the use of CO2 in the pyrolysis process. Exploiting CO2 in pyrolysis of PSD offered a strategic way to control carbon reallocation from liquid to gaseous pyrolysates by the gas phase reactions (GPRs). The reactions of CO2 and volatile pyrolysates led to CO enhancement, which was only observed at ≥ 600 °C due to the slow reaction kinetics of the GPRs of volatile pyrolysates and CO2. Such the slow reaction kinetics was expedited remarkably when SS was acted as a catalyst. Moreover, CO2 expedited thermal cracking of volatile pyrolysates including dehydrogenation, which led to the enhanced formation of CH4 and H2.

Temperature Dependency of Proton Pumping Activity for Marine Microbial Rhodopsin from Antartic Ocean.

Proteorhodopsin (PR) is discovered from marine bacteria and it has proton pumping activity from inside to outside of the cell using light energy. In general, PR classified into two groups by the maximum absorption spectra. In this study, we isolated the two of a full sequence of opsin homologues by PCR from the seawater sample near King George Island, Antarctica. One was the same sequence as the first reported GPR (Green-light absorbing PR) from Monterey Bay. Another named HSG119 was a newly discovered sequence which shows high sequence similarity with BPR (Blue-light absorbing PR). HSG119 has an absorption maximum at 493 nm with broader spectrum at pH7.0 and it can pump protons out of the cell membrane. Interestingly, it showed a similar temperature dependence to GPR(Y200N) that isolated near the North pole.

Photochemical characterization of flavobacterial rhodopsin: The importance of the helix E region for heat stability.

Although many microbial rhodopsins have been discovered many of organisms in a variety of habitats, little is known about the property and diversity of rhodopsin in flavobacteria. Recent studies discovered that many proteorhodopsin (PR)-like proteins exist in genomes of flavobacteria. Following the isolation of a flavobacterial rhodopsins (FR) from the flavobacteria IMCC1997 from the East Sea of Korea, we characterized its photochemical features. We confirmed that the FR expression is induced by light in the IMCC1997 cell. Upon receiving light energy in vitro, the proton acceptor (D83) and donor (E94) of the FR translocate protons from intracellular to extracellular regions. Compared with proteorhodopsin (PR), the FR from IMCC 1997 cells is very unstable, which may be explained by their primary sequence differences. The ratio of all trans/13-cis retinal conformation does not influence this stability. To measure the stability of FR, we tested heat endurance at 70 °C and found that the heat endurance time of some FR mutants increased. Based upon these results, we found the helix E of this protein to be critical for the unstability of FR.

Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system.

Inside cells, complex metabolic reactions are distributed across the modular compartments of organelles. Reactions in organelles have been recapitulated in vitro by reconstituting functional protein machineries into membrane systems. However, maintaining and controlling these reactions is challenging. Here we designed, built, and tested a switchable, light-harvesting organelle that provides both a sustainable energy source and a means of directing intravesicular reactions. An ATP (ATP) synthase and two photoconverters (plant-derived photosystem II and bacteria-derived proteorhodopsin) enable ATP synthesis. Independent optical activation of the two photoconverters allows dynamic control of ATP synthesis: red light facilitates and green light impedes ATP synthesis. We encapsulated the photosynthetic organelles in a giant vesicle to form a protocellular system and demonstrated optical control of two ATP-dependent reactions, carbon fixation and actin polymerization, with the latter altering outer vesicle morphology. Switchable photosynthetic organelles may enable the development of biomimetic vesicle systems with regulatory networks that exhibit homeostasis and complex cellular behaviors.

Outcome of radiosurgery for recurrent malignant gliomas: assessment of treatment response using relative cerebral blood volume.

Gamma knife radiosurgery (GKS) is efficacious for treating recurrent malignant gliomas as a salvage treatment. However, contrast enhancement alone on MR imaging remains difficult to determine the treatment response following GKS. The purpose of this study was to evaluate the radiosurgical effect for recurrent malignant gliomas and to clarify if relative cerebral blood volume (rCBV) derived from dynamic susceptibility-weighted contrast-enhanced (DSC) perfusion MR imaging could represent the treatment response. Between March 2006 and December 2008, 38 patients underwent GKS for recurrent malignant gliomas. Before and after GKS, DSC perfusion MR imaging datasets were retrospectively reprocessed and regions of interest were drawn around the contrast-enhancing region targeted with GKS. DSC-perfusion MR scans were assessed at a regular interval of two months. Following GKS for the recurrent lesions, MR images showed response (stable disease or partial response) in 26 of 38 patients (68.4 %) at post-GKS 2 months and 18 of 38 patients (47.3 %) at post-GKS 4 months. Initial mean rCBV value was 2.552 (0.586-6.178) at the pre-GKS MRI. In the response group, mean rCBV value was significantly decreased (P < 0.05) at the follow up of 2 and 4 months. However, in the treatment-failure group, mean rCBV value had no significant change. We suggest that GKS is an alternative treatment choice for the recurrent glioma. DSC-perfusion MR images are helpful to predict the treatment response after GKS.

Spontaneous Thrombolysis of Multiple Thrombi at Distal Region of Hypoplastic Vertebral Artery After Stent-assisted Angioplasty on Vertebral Artery Origin Stenosis: Angiographic Follow-up.

Vertebral artery hypoplasia (VAH) can be easily overlooked if the contralateral side vertebral artery is intact, because of compensation by the contralateral artery or cerebral collateral network. The clinical relevance and hemodynamic impact of VAH is still controversial. However, VAH has recently been considered a risk factor for posterior circulation ischemia. Ischemic stroke is seldom caused by free floating thrombi (FFT) in the artery. Pathophysiology of FFT has not yet been clarified. The state of reduced blood flow such as a vertebral artery origin stenosis may cause FFT. Their instability may make them sources of recurrent artery to artery embolism. Patients with FFT will require appropriate medical and endovascular treatment. The current case illustrates a short-term angiographic change of spontaneous thrombolysis of VAH and multiple thrombi at the distal region of the stenosed lesion after stent-assisted angioplasty for a vertebral artery origin stenosis.

The cardiovascular effects of midazolam co-induction to propofol for induction in aged patients.

BACKGROUND: The aim of this study was to investigate whether a small dose of midazolam and lessening the propofol dosage could prevent cardiovascular change at tracheal intubation for induction in aged patients. METHODS: Eighty patients over 65 years (ASA physical status 1, 2) scheduled for elective surgery received general anesthesia with remifentanil and propofol or midazolam. Patients in group P (n = 40) were induced with 0.9% NaCl 0.03 ml/kg, propofol 1. 2 mg/kg and remifentanil. Patients in group MP (n = 40) were induced with midazolam 0.03 mg/kg, propofol 0.8 mg/kg and remifentanil. The time taken to reach loss of consciousness (LOC) and the value of bispectral index score (BIS) at LOC were recorded. After LOC, 0.8 mg/kg of rocuronium was given and tracheal intubation was performed. The mean blood pressure (MBP) and heart rate (HR) were recorded before induction as the base value, before intubation, immediately post-intubation and 3 minutes after intubation. RESULTS: Compared with the base values, MBP at before intubation and 3 minutes after intubation was significantly decreased in group P and group MP (P < 0.05). Compared with group P, the decrease of MBP was significantly less at before intubation, immediately after intubation and 3 minutes after intubation in group MP (P < 0.05). The time taken to reach LOC was significantly decreased in group MP compared with that in group P (P < 0.05). There were no significant differences of HR at any time between the two groups. CONCLUSIONS: Co-induction with midazolam and propofol could prevent a marked BP decrease at tracheal intubation for induction in aged patients.

Impact of maximal safe resection on the clinical outcome of adults with craniopharyngiomas.

Recent studies suggest that subtotal resection (STR) followed by adjuvant radiation therapy is an appealing alternative to gross total resection (GTR) for craniopharyngioma, as STR provides similar tumor control without the associated endocrinological and behavioral morbidity. We have examined the impact of maximal safe resection on the clinical outcome of patients with craniopharyngioma. A total of 90 patients underwent surgical resection of craniopharyngioma at a single institution between January 1995 and April 2009. Sixty-one patients underwent GTR alone, four underwent GTR followed by adjuvant radiotherapy, 15 underwent STR alone, and 10 underwent partial removal followed by adjuvant radiotherapy. We analyzed and compared the clinical and endocrinological outcomes and radiological follow-up data of these patients. During the follow-up period, tumor recurrence following the initial resection occurred in 36 of 90 patients (40%). The repeat resection rate was higher in the STR group than the GTR group. Recurrence occurred in 20 of 61 patients (32.8%) from the GTR alone group, in 11 of 15 patients (73.3%) from the STR alone group, and in five of 10 (50%) patients from the STR with adjuvant radiation, such as radiotherapy or stereotactic radiosurgery, group (p=0.030). Maximal safe resection of craniopharyngioma leads to excellent local control. STR with adjuvant radiation therapy does not assure preservation of endocrine function, although it provides better local control than STR alone.

Doxorubicin-induced platelet procoagulant activities: an important clue for chemotherapy-associated thrombosis.

Thrombotic risk associated with chemotherapy including doxorubicin (DOX) has been frequently reported; yet, the exact mechanism is not fully understood. Here, we report that DOX can induce procoagulant activity in platelets, an important contributor to thrombus formation. In human platelets, DOX increased phosphatidylserine (PS) exposure and PS-bearing microparticle (MP) generation. Consistently, DOX-treated platelets and generated MPs induced thrombin generation, a representative marker for procoagulant activity. DOX-induced PS exposure appeared to be from intracellular Ca²âº increase and ATP depletion, which resulted in the activation of scramblase and inhibition of flippase. Along with this, apoptosis was induced by DOX as determined by the dissipation of mitochondrial membrane potential (Δψ), cytochrome c release, Bax translocation, and caspase-3 activation. A Ca²âº chelator ethylene glycol tetraacetic acid, caspase inhibitor Q-VD-OPh, and antioxidants (vitamin C and trolox) can attenuate DOX-induced PS exposure and procoagulant activity significantly, suggesting that Ca²âº, apoptosis, and reactive oxygen species (ROS) were involved in DOX-enhanced procoagulant activity. Importantly, rat in vivo thrombosis model demonstrated that DOX could manifest prothrombotic effects through the mediation of platelet procoagulant activity, which was accompanied by increased PS exposure and Δψ dissipation in platelets.

Challenging reconstructive techniques for skull base defect following endoscopic endonasal approaches.

OBJECTIVE: We assessed the outcomes of various reconstructive methods for skull base defect after endoscopic endonasal approaches (EEA) depending on the degree of intraoperative cerebrospinal fluid (CSF) leaks. METHODS: Between Jan. 2008 and Sep. 2009, 122 consecutive patients underwent 124 EEA for sellar and extra-sellar lesions. Intraoperative CSF leaks were classified as grade 0, no intraoperative CSF leak; grade 1, low output; and grade 2, high-output based on the degree of CSF leakage and size of opening in the arachnoid membrane (<5 or ≥5 mm). RESULTS: Postoperative CSF leaks or meningitis occurred in 13 of 124 cases (10.5%). In 77 patients with grade 0, there was no postoperative CSF leak. Among 20 patients with grade 1 CSF leaks, four patients developed meningitis or postoperative CSF leak. Postoperative CSF leaks occurred in nine of 26 patients (34.6%) with grade 2 leaks. Comparison of reconstructive methods revealed that gasket-seal method provided better control of CSF leaks than free-fat graft in patients with grade 2 leaks (11.8% vs. 66.7%, p = 0.028). However, in grades 0 and 1, we found no difference among the various reconstructive methods. CONCLUSION: The selection of reconstructive methods for skull base defects should be determined by the degree of CSF leaks. Although grade 0 or 1 leak requires relatively conservative management such as simple closure or free-tissue grafting, a more aggressive reconstructive technique is required to prevent postoperative complication in grade 2 CSF leak.

[A case of peritoneal mesothelioma with direct invasion to gastric mucosa].

Mesothelioma is a rare aggressive tumor arising from the mesothelial cell and regarded as universally fatal disease with average survival around 1 year. The incidence rate is varied from one to forty per million in different countries and increasing by the year. The most common site of tumor origin is the pleura and only 20% to 33% of mesothelioma arise from the peritoneum. There are increasing reports of malignant mesothelioma with forty to fifty fatal cases per year in Korea. Histological studies with immunohistochemical stain is helpful for the diagnosis of peritoneal mesothelioma and imaging modality alone is not sufficient for diagnosis, so it is difficult to confirm diagnosis. A 64-year-old male patient was admitted to the hospital with a palpable mass on abdomen. The 6x6 cm sized huge mass was seen on the body of stomach adjacent to the peritoneum. We report a case of malignant peritoneal mesothelioma without evident exposure to asbestos, of which direct invasion to the gastric mucosa was confirmed by endoscopic biopsy and immunohistochemical stain.

Difference of the hemodynamic changes induced by tracheal intubation using remifentanil between smokers and nonsmokers.

BACKGROUND: It was well-known that smoking affects the cardiovascular system, and remifentanil can suppress the sympathetic stimulations induced by tracheal intubation. The purpose of this study was to investigate whether there was any difference in the hemodynamic changes induced by tracheal intubation with using remifentanil between smokers and nonsmokers. METHODS: EIGHTY PATIENTS WERE ENROLLED: male smokers (MS), male nonsmokers (MN), female smokers (FS) and female nonsmokers (FN). Anesthesia was induced with diluted remifentanil (20 microg/ml) at a rate of 10 microg/kg/hr using an infusion pump, and 2 min later, midazolam 0.05 mg/kg and propofol 0.8 mg/kg were injected for achieving unconsciousness. Rocuronium 1 mg/kg was used for muscle relaxation, and tracheal intubation was performed 2 min after rocuronium injection. After tracheal intubation, the remifentanil was decreased to 2 microg/kg/hr. The mean arterial pressure (MAP) and heart rate (HR) were checked before induction, on unconsciousness, just before intubation, just after intubation and 1, 2 and 3 minutes after intubation, and these values were compared between the groups. RESULTS: In men, the MAP and HR just after intubation and at 1, 2 and 3 minutes after intubation in Group MS were significantly higher than those of Group MN (P < 0.05). For the women, the HR in both groups (the FS and FN groups) were increased just after intubation and 1, 2 and 3 minutes after intubation compared with that at the baseline, respectively, but there was no difference between the two groups. CONCLUSIONS: There was a difference of the hemodynamic changes induced by tracheal intubation with using remifentanil between the male smokers and nonsmokers, but not in women.

The expression of survivin and its related genes in adipocyte-derived stem cell by demethylation.

BACKGROUND: Survivin is thought to contribute to stem cell maintenance partly by a hypomethylation mechanism. This study attempted to elucidate the signal transduction pathway of adipocyte-derived stem cells (ASCs) by using a demethylating agent, 5-aza-2'-deoxycytidine (ADC), to analyze the survivin, MEK/ERK, c-Myc and p53 gene expression. METHODS: Demethylation in the ASCs was induced by 1 microM ADC treatment. RT-PCR for survivin mRNA was preformed, before and 24, 48 and 72 hours (hr) after ADC treatment. Western blotting analysis was performed for p53, survivin, unphosphorylated and phosphorylated (p)-MEK, and p-ERK. Immunohistochemistry for ERK and survivin was done to evaluate the localization of the proteins. RESULTS: ADC inhibited the population growth of the ASCs and it increased the number of apoptotic cells 24, 48, and 72 hr after treatment. ADC treatment slightly decreased the expression of survivin mRNA after 48 hr and its level was restored after 72 hr of treatment. Otherwise, the level of survivin protein gradually increased up to 48 hr and it was decreased at 72 hr. The levels of p-MEK and p53 were increased time-dependently. c-Myc and p-ERK were elevated after ADC treatment and their highest levels were seen 48 hr after treatment. The ADC treatment increased the nuclear expression of ERK and survivin in the ASCs. CONCLUSIONS: The overexpression of p-MEK/ERK, p53, and c-Myc increased the survivin protein expression of the demethylated ASCs. These results suggest that demethylation could alter the expression of survivin, and p53, c-Myc and the MAPK (MEK/ERK) pathway might play a role in survivin's regulation in ASCs.

[A case of sclerosed hemangioma mimicking intrahepatic cholangiocarcinoma].

Hemangioma is one of the most frequently encountered benign hepatic neoplasm which can develop secondary degeneration. Sclerosed hemangioma is a rare disease histologically characterized by large amount of collagen and elastic fibril between sclerosed small vessels. Its differential diagnosis is very difficult. It should be included in the differential diagnosis of other hepatic lesions such as hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and metastatic hepatic tumor. A 77-year old male was admitted with upper abdominal discomfort. Abdominal ultrasonography revealed GB stone, dilated common bile duct with bile duct stone, and a 4.6 cm sized hyperechoic mass at segment 5 and 6 of the liver. Abdominal dynamic computed tomography demonstrated dilated intrahepatic bile ducts and a 5 x 5 cm sized mass which showed minimally delayed enhancement. Abdominal magnetic resonance imaging revealed the mass with low signal intensity in T1 weighted image, high signal intensity and focal low signal in T2 weighted image which showed minimal enhancement. We removed common bile duct stone with endoscopic retrograde cholangiopancreatography then decided to undergo right lower segmentectomy of liver due to possibility of cholangiocarcinoma. Histopathological examination of hepatic mass showed large amount of fibrous tissue with occasional residual vascular channels. We describe one case of sclerosed hemangioma mimicking cholangiocarcinoma.

Trivalent methylated arsenical-induced phosphatidylserine exposure and apoptosis in platelets may lead to increased thrombus formation.

Trivalent methylated metabolites of arsenic, monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), have been found highly reactive and toxic in various cells and in vivo animal models, suggesting their roles in the arsenic-associated toxicity. However, their effects on cardiovascular system including blood cells, one of the most important targets for arsenic toxicity, remain poorly understood. Here we found that MMA(III) and DMA(III) could induce procoagulant activity and apoptosis in platelets, which play key roles in the development of various cardiovascular diseases (CVDs) through excessive thrombus formation. In freshly isolated human platelets, treatment of MMA(III) resulted in phosphatidylserine (PS) exposure, a hallmark of procoagulant activation, accompanied by distinctive apoptotic features including mitochondrial membrane potential disruption, cytochrome c release, and caspase-3 activation. These procoagulant activation and apoptotic features were found to be mediated by the depletion of protein thiol and intracellular ATP, and flippase inhibition by MMA(III), while the intracellular calcium increase or reactive oxygen species generation was not involved. Importantly, increased platelet procoagulant activity by MMA(III) resulted in enhanced blood coagulation and excessive thrombus formation in a rat in vivo venous thrombosis model. DMA(III) also induced PS-exposure with apoptotic features mediated by protein thiol depletion, which resulted in enhanced thrombin generation. In summary, we believe that this study provides an important evidence for the role of trivalent methylated arsenic metabolites in arsenic-associated CVDs, giving a novel insight into the role of platelet apoptosis in toxicant-induced cardiovascular toxicity.

The effects of remifentanil and lidocaine on the cardiovascular responses to tracheal intubation in hypertensive patients.

BACKGROUND: Hypertensive patients are more prone to exhibit an exaggerated hemodynamic responses to laryngoscopy and tracheal intubation. We compared the effects of remifentanil and lidocaine on the cardiovascular responses during intubation in hypertensive patients. METHODS: Fifty adult hypertensive patients (>140/90 mmHg) were divided into 2 groups (remifentanil group; 1.0 microgram/kg, n = 25, lidocaine group; 1.5 mg/kg, n = 25). After 3 minutes'oxygenation, anesthesia was induced using 0.2 mg glycopyrrolate, 1.5 mg/kg propofol, and 1 of experimental drugs (30 seconds) bolus administration. Tracheal intubation was facilitated 90 seconds after administration of rocuronium; anesthesia was maintained with 2% sevoflurane and air in oxygen. Arterial blood pressure and heart rate were measured at the following times: resting state (baseline), after oxygenation; before intubation; just after intubation; and at 1, 3, and 5 minutes after intubation. RESULTS: There was significant attenuation of mean systolic and diastolic arterial blood pressures, after intubation of the remifentanil group compared to the lidocaine group at just after intubation and at 1 minute after intubation (P < .05). The difference in heart rate was not significant between both groups. CONCLUSIONS: Bolus administration of remifentanil was found to be superior to lidocaine in the attenuation of the blood pressure during intubation in hypertensive patients.