The influence of age and gender on remifentanil EC(50) for preventing rocuronium induced withdrawal movements.

BACKGROUND: Rocuronium-induced withdrawal movements can be harmful to patients during the induction period. Remifentanil has been reported to reduce these movements effectively. In this study, we determined the EC(50) of remifentanil for the prevention of rocuronium induced withdrawal movements in male, female, old and child group. METHODS: We included patients scheduled for general anesthesia and assigned them into 4 groups depending on their age and gender: male group (20-60 yr), female group (20-60 yr), old group (>65 yr) and child group (6-12 yr). Remifentanil was administered by target controlled infusion. Propofol 2 mg/kg was then administered after equilibration between the effect and plasma concentration of remifentanil was reached. After loss of consciousness, rocuronium 0.6 mg/kg was administered. Patient's response to the rocuronium was graded using a 4 point scale in a blinded manner. The EC(50) of remifentanil for preventing rocuronium induced withdrawal movements was determined using Dixon's up-and -down method. RESULTS: The EC(50) of remifentanil for preventing rocuronium induced withdrawal movements was 1.8 +/- 0.5 ng/ml [95% confidence interval 1.3-2.2] in the male group, 2.3 +/- 1.0 ng/ml [1.3-3.2] in the female group, 0.5 +/- 0.4 ng/ml [0.2-0.8] in the old group and 2.8 +/- 0.8 ng/ml [2.1-3.5] in the child group. CONCLUSIONS: The EC(50) of remifentanil for preventing rocuronium induced withdrawal movements was lowest in the elderly and higher in children than male adult patients. No difference in the EC(50) of remifentanil was seen between male and female adult patients.

Comparative hemodynamic effects of vasopressin and norepinephrine after milrinone-induced hypotension in off-pump coronary artery bypass surgical patients.

OBJECTIVE: Phosphodiesterase inhibitor is essential to the pharmacologic management of decompensated heart failure because it increases contractility and decreases afterload of right ventricle. It also improves hemodynamics and increases blood flow of the grafted internal mammary arteries and middle cerebral arteries during coronary artery bypass surgery. However, it induces vasodilation and necessitates the use of vasoconstrictors, such as norepinephrine. We hypothesized that vasopressin could recover hypotension induced by milrinone with less effect on pulmonary vascular resistance (PVR) compared to norepinephrine. METHODS: Fifty patients, undergoing coronary artery bypass graft (CABG) surgery, were assigned randomly in a double-blind manner to receive either vasopressin or norepinephrine. After baseline hemodynamic measurements, a loading dose of milrinone 50 microg/kg was infused slowly for 20 min followed by continuous infusion of 0.5 microg/(kg min). Immediately after the loading dose of milrinone, hemodynamic variables were measured, and vasopressin (VP group) or norepinephrine (NE groups) was infused. After being titrated until the mean arterial pressure was increased by 20%, hemodynamic variables were measured again. RESULTS: Milrinone infusion reduced both systemic vascular resistance (SVR, 1218+/-299 dynes/cm5 vs 838+/-209 dynes/cm5, 1345+/-299 dynes/cm5 vs 1011+/-195 dynes/cm5) and PVR (95+/-34 dynes/cm5 vs 72+/-30 dynes/cm5, 119+/-85 dynes/cm5 vs 87+/-33 dynes/cm5) in the VP and NE groups, respectively. Vasopressin and norepinephrine infusion increased both SVR (838+/-209 dynes/cm5 vs 1100+/-244 dynes/cm5, 1011+/-195 dynes/cm5 vs 1446+/-681 dynes/cm5, respectively) and PVR (72+/-30 dynes/cm5 vs 84+/-18 dynes/cm5, 87+/-33 dynes/cm5 vs 139+/-97 dynes/cm5, respectively). The PRV/SVR ratio was decreased after vasopressin infusion (0.10+/-0.03 vs 0.08+/-0.03), while no changes were found after norepinephrine infusion (0.09+/-0.02 vs 0.09+/-0.02). CONCLUSIONS: In the patients undergoing CABG surgery, both norepinephrine and low dose vasopressin were effective in restoring milrinone-induced decrease of SVR. However, only low-dose vasopressin decreased the PVR/SVR ratio that was increased by milrinone. Considering the importance of maintaining systemic perfusion pressure as well as reducing right heart afterload, milrinone-vasopressin may provide better hemodynamics than milrinone-norephinephrine during the management of right heart failure.

The homotrimeric structure of HtrA2 is indispensable for executing its serine protease activity.

Serine protease activity of high temperature requrement 2 (HtrA2) is essential for promoting cell death, as well as for protecting against cellular stresses. An X-ray crystallographic study described the formation of a pyramid shaped homotrimer that is a proteolytically competent form of HtrA2; however, little is known about effects of the trimeric structure of HtrA2 on the natural substrates. In this study, we generated the HtrA2 protein that has a single point mutation at the homotrimerization motif to assess relationship between structure and the proteolytic activity of HtrA2 on its substrates. Using gel filtration, a native gel electrophoresis system, and a co-precipitation assay, we confirm that phenylalanine 149 in HtrA2 is a crucial determinant for the formation of the HtrA2 homotrimeric structure. Moreover, we described that the HtrA2 monomeric form abolished not only autoproteolytic activity, but also the proteolytic activity against XIAP (X-linked inhibitor of apoptosis protein) known as the HtrA2 substrate. Taken together, the results indicate that the homotrimeric structure of HtrA2 is required for executing its serine protease activity.

Fine epitope mapping of monoclonal antibodies specific to human alpha-synuclein.

The neuronal phosphoprotein alpha-synuclein has been increasingly implicated in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative diseases; however, the exact function of alpha-synuclein still remains illusive. Suitable antibodies (Abs) specific for the gene of interest are indispensable for studying biological and immunological properties of the target gene. Here, we report not only the generation and characterization of monoclonal Abs, Syn-1 and Syn-17, against human alpha-synuclein, but also the epitope mapping by using recombinant synuclein family proteins and various GST fusion proteins of human alpha-synuclein domains. Syn-17 recognizes human and rodent alpha-synuclein, and its epitope is localized within residues 97-99 and 101 of alpha-synuclein. In contrast, the Syn-1 epitope is localized in residues 121 and 122 of human alpha-synuclein, and Syn-1 recognizes only human but not rodent alpha-synuclein, indicating that it can be utilized as a useful reagent for studying human alpha-synuclein transgenic mouse and zebrafish lines.

Prostaglandin A2 induces caspase-independent apoptosis in hepatocellular carcinoma cells.

BACKGROUND/AIMS: Prostaglandin (PG) A2 has been reported to inhibit the growth of hepatocellular carcinoma cells via activation of apoptosis, although the molecular mechanisms involved have not been clarified, yet. To investigate the mechanism of the PGA2-induced apoptosis, we analyzed the activation of caspases during the apoptosis of hepatoma cell lines. METHODS: Induction of apoptosis by PGA2 in hepatoma cell lines, Hep 3B and Hep G2, was assessed by DAPI staining of nuclei and agarose gel electrophoresis of genomic DNA. The involvement of caspases was analyzed by immunoblot analysis of poly ADP-ribose polymerase (PARP) and by checking the effect of caspase inhibitors on PGA2-induced apoptosis. RESULTS: PGA2 inhibited the growth of Hep 3B and Hep G2 cells, accompanying nuclear condensation and fragmentation, and genomic DNA laddering, which are the hallmarks of apoptosis. The PARP was not cleaved during the apoptosis of Hep 3B and Hep G2 cells and caspase inhibitors such as z-VAD-Fmk and z-DEVD-Fmk exerted no effect on the PGA2-induced apoptosis. CONCLUSIONS: These results suggest that PGA2 induces apoptosis in Hep 3B and Hep G2 cells via caspase-independent pathway.

Functional identification of the pro-apoptotic effector domain in human Sox4.

Recent studies provide evidence that Sox4 is involved in regulating apoptosis as well as tumorigenesis of various human cancers; however, its role in the apoptotic machinery is not fully understood. Here we describe that the central domain containing glycine-rich region in Sox4, named CD, is a pivotal pro-apoptotic domain to induce apoptotic cell death. Deletion of the DNA-binding domain or trans-activation domain in Sox4 did not significantly affect pro-apoptotic activity, whereas transient transfection of the high mobility group box or the serine-rich region abrogated the apoptotic activity. Moreover, overexpression of the CD construct (aa 166-342) revealed the apoptotic activity comparable to that of wild-type Sox4, approximately 60% of cell death. Our data suggest that the apoptotic activity of Sox4 can be dissociated from its transcriptional trans-activation and is mediated through its CD.

Autocatalytic processing of HtrA2/Omi is essential for induction of caspase-dependent cell death through antagonizing XIAP.

A mature form of nuclear-encoded mitochondrial serine protease HtrA2/Omi is pivotal in regulating apoptotic cell death; however, the underlying mechanism of the processing event of HtrA2/Omi and its relevant biological function remain to be clarified. Here, we describe that HtrA2/Omi is autocatalytically processed to the 36-kDa protein fragment, which is required for the cytochrome c-dependent caspase activation along with neutralizing XIAP-mediated inhibition of caspases through interaction with XIAP, eventually promoting apoptotic cell death. We have shown that the autocatalytic processing of HtrA2/Omi occurs via an intermolecular event, demonstrated by incubating an in vitro translated HtrA2/Omi (S306A) mutant with the enzymatically active glutathione S-transferase-HtrA2/Omi protein. Using N-terminal amino acid sequencing and mutational analysis, we identified that the autocatalytic cleavage site is the carboxyl side of alanine 133 of HtrA2/Omi, resulting in exposure of an inhibitor of apoptosis protein binding motif in its N terminus. Our study provides evidence that the autocatalytic processing of HtrA2/Omi is crucial for regulating HtrA2/Omi-mediated apoptotic cell death.

Alzheimer's disease-associated amyloid beta interacts with the human serine protease HtrA2/Omi.

Amyloid beta (Abeta), a principle component of the cerebral plaques found in the brains of patients with Alzheimer's disease (AD), is a pivotal factor implicated in the pathogenesis of AD. Recent reports show that not only extracellular Abeta but also intracellular Abeta induces neuronal apoptosis; however, the mechanism remains to be elucidated. Using yeast two-hybrid assays, we found that Abeta interacts with HtrA2/Omi, an essential human serine protease with proapoptotic activity. Additionally, we mapped the C-terminal region containing the PDZ domain of HtrA2/Omi as the binding determinant for Abeta? The interaction of Abeta with HtrA2/Omi was further confirmed through in vivo co-immunoprecipitation assay in HEK293 cells. This study suggests the possibility that the accumulation of intracellular Abeta and a function of proapoptotic protease, HtrA2/Omi are correlated.

N-terminal truncation circumvents proteolytic degradation of the human HtrA2/Omi serine protease in Escherichia coli: rapid purification of a proteolytically active HtrA2/Omi.

HtrA2/Omi, a mitochondrial trypsin-like serine protease, is pivotal in regulating apoptotic cell death; however, the underlying mechanism of HtrA2/Omi-mediated apoptosis remains to be elucidated. Using the pGEX bacterial expression system, we investigated the expression patterns of various forms of HtrA2/Omi. Full-length mouse HtrA2/Omi (mHtrA2/Omi) was successfully expressed in E. coli and purified as a proteolytically active protein. In contrast, the expression of full-length human HtrA2/Omi (hHtrA2/Omi) in E. coli was barely detected. On the basis of this result, we characterized further the expression patterns of N- or C-terminally truncated hHtrA2/Omi proteins. We found that three copies of the PRAXXTXXTP motif, which exist only in hHtrA2/Omi, might serve as a primary site that is highly susceptible to proteolytic degradation by host proteases. Removal of the N-terminal region containing the PRAXXTXXTP motifs produced a form resistant to proteolytic degradation during expression in E. coli and purification, consequently improving the production of a catalytically active, mature hHtrA2/Omi. Our study provides a method for generating useful reagents to investigate molecular mechanism by which HtrA2/Omi contributes to regulating apoptotic cell death and to identify natural substrates of HtrA2/Omi.

Antigenicity of the region encoded by exon8 of the human serine protease, HtrA2/Omi, is associated with its protein solubility.

HtrA2/Omi, a mitochondrial serine protease, is pivotal in regulating apoptotic cell death. To determine the location of antigenic determinants in HtrA2/Omi, we expressed a series of the N-terminally truncated HtrA2/Omi as GST fusion proteins in E. coli. We assessed protein solubility and antigenic reactivity of various N-terminally truncated HtrA2/Omi proteins by binding to glutathione beads and immunoblot analyses, respectively. We identified that the region encoded by exon8 of HtrA2/Omi was expressed as a highly soluble form and contains an antigenic determinant specifically recognized by a polyclonal serum against HtrA2/Omi. Our data provide evidence that protein solubility of the specific region in target proteins may contribute to the antigenicity.

Rapid purification and analysis of alpha-synuclein proteins: C-terminal truncation promotes the conversion of alpha-synuclein into a protease-sensitive form in Escherichia coli.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons and the formation of eosinophilic intracytoplasmic inclusion bodies known as Lewy bodies. Although alpha-synuclein is known to be a pivotal factor implicated in the pathogenesis of PD, its function remains to be elucidated. We used the pGEX expression system to develop a simple and rapid method for purifying alpha-synuclein proteins in suitable forms for biochemical studies of their functions. The wild-type alpha-synuclein protein was overexpressed in Escherichia coli and purified to approx. 80% purity with relatively high yields. We also used this expression system to investigate the expression pattern of the various domains of alpha-synuclein. With the exception of the alpha-synuclein protein that was truncated at amino acid residue 95, all domain constructs of alpha-synuclein were purified at similar levels with relatively high yields. Unexpectedly, removal of amino acid residues 96-140 in the C-terminal acidic region of alpha-synuclein promotes its conversion to a protease-sensitive form during expression and purification in E. coli. Our study suggests a method for generating useful reagents to investigate the molecular mechanism by which alpha-synuclein regulates the pathogenesis of PD.

alpha-Synuclein interacts with phospholipase D isozymes and inhibits pervanadate-induced phospholipase D activation in human embryonic kidney-293 cells.

alpha-Synuclein has been implicated in the pathogenesis of many neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Although the function of alpha-synuclein remains largely unknown, recent studies have demonstrated that this protein can interact with phospholipids. To address the role of alpha-synuclein in neurodegenerative disease, we have investigated whether it binds phospholipase D (PLD) and affects PLD activity in human embryonic kidney (HEK)-293 cells overexpressing wild type alpha-synuclein or the mutant forms of alpha-synuclein (A53T, A30P) associated with Parkinson's disease. Tyrosine phosphorylation of alpha-synuclein appears to play a modulatory role in the inhibition of PLD, because mutation of Tyr(125) to Phe slightly increases inhibitory effect of alpha-synuclein on PLD activity. Treatment with pervanadate or phorbol myristate acetate inhibits PLD more in HEK 293 cells overexpressing alpha-synuclein than in control cells. Binding of alpha-synuclein to PLD requires phox and pleckstrin homology domain of PLD and the amphipathic repeat region and non-Abeta component of alpha-synuclein. Although biologically important, co-transfection studies indicate that the interaction of alpha-synuclein with PLD does not influence the tendency of alpha-synuclein to form pathological inclusions. These results suggest that the association of alpha-synuclein with PLD, and modulation of PLD activity, is biologically important, but PLD does not appear to play an essential role in the pathophysiology of alpha-synuclein.