Epithelium specific ETS transcription factor, ESE-3, of Protobothrops flavoviridis snake venom gland transactivates the promoters of venom phospholipase A2 isozyme genes.

Protobothrops flavoviridis (habu) (Crotalinae, Viperidae) is a Japanese venomous snake, and its venom contains the enzymes with a variety of physiological activities. The phospholipases A2 (PLA2s) are the major components and exert various toxic effects. They are expressed abundantly in the venom gland. It is thought that the venom gland-specific transcription factors play a key role for activation of PLA2 genes specifically expressed in the venom gland. Thus, the full-length cDNA library for P. flavoviridis venom gland after milking of the venom was made to explore the transcription factors therein. As a result, three cDNAs encoding epithelium-specific ETS transcription factors (ESE)-1, -2, and -3 were obtained. Among them, ESE-3 was specifically expressed in the venom gland and activated the proximal promoters of venom PLA2 genes, which are possibly regarded as the representatives of the venom gland-specific protein genes in P. flavoviridis. Interestingly, the binding specificity of ESE-3 to the ETS binding motif located near TATA box is well correlated with transcriptional activities for the venom PLA2 genes. This is the first report that venom gland-specific transcription factor could actually activate the promoters of the venom protein genes.

Discovery of a novel vascular endothelial growth factor (VEGF) with no affinity to heparin in Gloydius tsushimaensis venom.

Strong vascular permeability enhancing activity was found only in the venom of Gloydius tsushimaensis, in Tsushima island, Japan, when examined together with the venoms of G. blomhoffii snakes in several areas of Japan and of G. ussuriensis in South Korea. The active protein purified by using Superdex 75 and Mono Q columns showed no affinity to heparin, and migrated on SDS-PAGE with molecular weights of 26 and 13 kDa under nonreducing and reducing conditions, respectively, showing that it exists as homodimer. Its N-terminal amino acid sequence was highly homologous to those of snake venom vascular endothelial growth factors (VEGFs). The sequence of this protein, named GtVF, was inferred from the one base-substituted two cDNAs (438 bp) obtained via the 3' RACE. The phylogenetic analysis suggested the presence of a new type of snake venom VEGFs including GtVF with no affinity to heparin in addition to the known three types of snake venom VEGFs with high affinity to heparin. Since the vascular permeability enhancement by GtVF was inhibited by the antibody against kinase insert domain-containing receptor (KDR), the vascular permeability enhancing activity of GtVF arises through KDR but without heparin binding.

Identification of missing proteins in the neXtProt database and unregistered phosphopeptides in the PhosphoSitePlus database as part of the Chromosome-centric Human Proteome Project.

The Chromosome-Centric Human Proteome Project (C-HPP) is an international effort for creating an annotated proteomic catalog for each chromosome. The first step of the C-HPP project is to find evidence of expression of all proteins encoded on each chromosome. C-HPP also prioritizes particular protein subsets, such as those with post-translational modifications (PTMs) and those found in low abundance. As participants in C-HPP, we integrated proteomic and phosphoproteomic analysis results from chromosome-independent biomarker discovery research to create a chromosome-based list of proteins and phosphorylation sites. Data were integrated from five independent colorectal cancer (CRC) samples (three types of clinical tissue and two types of cell lines) and lead to the identification of 11,278 proteins, including 8,305 phosphoproteins and 28,205 phosphorylation sites; all of these were categorized on a chromosome-by-chromosome basis. In total, 3,033 "missing proteins", i.e., proteins that currently lack evidence by mass spectrometry, in the neXtProt database and 12,852 unknown phosphorylation sites not registered in the PhosphoSitePlus database were identified. Our in-depth phosphoproteomic study represents a significant contribution to C-HPP. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000089.

A strategy for large-scale phosphoproteomics and SRM-based validation of human breast cancer tissue samples.

Protein phosphorylation is a key mechanism of cellular signaling pathways and aberrant phosphorylation has been implicated in a number of human diseases. Thus, approaches in phosphoproteomics can contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets. Moreover, careful validation of large-scale phosphoproteome analysis, which is lacking in the current protein-based biomarker discovery, significantly increases the value of identified biomarkers. Here, we performed large-scale differential phosphoproteome analysis using IMAC coupled with the isobaric tag for relative quantification (iTRAQ) technique and subsequent validation by selected/multiple reaction monitoring (SRM/MRM) of human breast cancer tissues in high- and low-risk recurrence groups. We identified 8309 phosphorylation sites on 3401 proteins, of which 3766 phosphopeptides (1927 phosphoproteins) were able to be quantified and 133 phosphopeptides (117 phosphoproteins) were differentially expressed between the two groups. Among them, 19 phosphopeptides were selected for further verification and 15 were successfully quantified by SRM using stable isotope peptides as a reference. The ratio of phosphopeptides between high- and low-risk groups quantified by SRM was well correlated with iTRAQ-based quantification with a few exceptions. These results suggest that large-scale phosphoproteome quantification coupled with SRM-based validation is a powerful tool for biomarker discovery using clinical samples.

A [Lys49]phospholipase A2 from Protobothrops flavoviridis venom induces caspase-independent apoptotic cell death accompanied by rapid plasma-membrane rupture in human leukemia cells.

Protobothrops flavoviridis venom contains plural phospholipase A(2) (PLA(2)) isozymes. A [Lys(49)]PLA(2) called BPII induced cell death in human leukemia cells. PLA2, an [Asp(49)]PLA(2) that has much stronger lipolytic activity than BPII, failed to induce cell death. BPII-treated cells showed morphological changes, DNA fragmentation, and nuclear condensation. This BPII-induced apoptotic cell death was neither inhibited by inhibitors of caspases 3 and 6 nor accompanied by activation of procaspase 3, indicating that BPII-induced cell death is caspase independent. Since inactive p-bromophenacylated BPII induced cell death, BPII-induced apoptotic cell death is independent of PLA(2) lipolytic activity. Rapid externalization of phosphatidylserine in BPII-treated cells was observed for fluorescein isothiocyanate (FITC)-labeled annexin V. In the cells treated with BPII, this spread over the cell membranes, implying that the cell toxicity of BPII is mediated via its cell-surface receptor.

Identification and evolution of venom phospholipase A2 inhibitors from Protobothrops elegans serum.

The cDNAs encoding venom phospholipase A(2) (PLA(2)) inhibitors (PLIs), named Protobothrops elegans (Pe)γPLI-A, PeγPLI-B, PeαPLI-A, and PeαPLI-B, were cloned from the P. elegans liver cDNA library. They were further divided into several constituents due to nucleotide substitutions in their open reading frames. For PeαPLI-A, two constituents, PeαPLI-A(a) and PeαPLI-A(b), were identified due to three nonsynonymous substitutions in exon 3. Far-western blot and mass-spectrometry analysis of the P. elegans serum proteins showed the presence of γPLIs, and αPLIs, which can bind venom PLA(2)s. In αPLIs from Protobothrops sera, A or B subtype-specific amino acid substitutions are concentrated only in exon 3. A comparison of γPLIs showed that γPLI-As are conserved and γPLI-Bs diversified. Mathematical analysis of the nucleotide sequences of Protobothrops γPLI-B cDNAs revealed that the particular loops in the three-finger motifs diversified by accelerated evolution. Such evolutionary features should have made serum PLIs acquire their respective inhibitory activities to adapt to venom PLA(2) isozymes.

Inhibitory effects of landiolol and nicardipine on thiopental-induced yawning in humans.

PURPOSE: Either the calcium (Ca(2+))-channel blocker nicardipine or the beta(1)-adrenoceptor antagonist landiolol may be intravenously (IV) administered to reduce the hemodynamic responses to tracheal intubation. In this study, we examined the effects of these drugs on the yawning response elicited by intravenous thiopental in humans. METHODS: After Institutional Review Board approval, 180 consenting American Society of Anesthesiologists (ASA) I or II patients undergoing elective surgery were recruited. In a double-blind, randomized design, three groups of 60 patients each received one of the following intravenous injections: (1) landiolol 0.1 mg/kg (L-group), (2) nicardipine 0.02 mg/kg (N-group), or (3) saline (S-group). In all patients, anesthesia was subsequently induced IV with 4 mg/kg thiopental. Thereafter, the occurrence of the yawning response (characterized by mouth opening) was continuously assessed as the only clinical endpoint for 1 min. Throughout the study, mean arterial blood pressure and heart rate were also recorded at 1-min intervals. RESULTS: The incidence of the yawning response was lower in both the L-group (6.7%) and the N-group (16.7%) than in the S-group (46.7%) (each, P < 0.01). CONCLUSIONS: Prior intravenous administration of either a Ca(2+)-channel blocker or a beta(1)-adrenoceptor antagonist can greatly reduce the thiopental-induced yawning response in humans.

Island specific expression of a novel [Lys(49)]phospholipase A(2) (BPIII) in Protobothrops flavoviridis venom in Amami-Oshima, Japan.

In search of the transcripts expressed in Protobothrops flavoviridis venom gland, 466 expressed sequence tags (ESTs) were generated from the venom gland cDNA library of P. flavoviridis in Amami-Oshima, Japan. The sequencing of randomly selected cDNA clones followed by identification in similarity search against existing databases led to the finding of a novel lysine-49-phospholipase A(2) ([Lys(49)]PLA(2)) clone. It coded for one amino acid-substituted BPII homologue or two amino acids-substituted BPI homologue in which BPII and BPI are [Lys(49)]PLA(2)s contained in Amami-Oshima and Tokunoshima P. flavoviridis venoms. This isozyme, named BPIII, was isolated from Amami-Oshima P. flavoviridis venom. BPIII gave a specific [M+2H](2+) peak of m/z 736.3 on mass spectrometry (MS) analysis after S-carboxamidomethylation and trypsin digestion when compared with BPII. It became evident from MS analysis after S-carboxamidomethylation and trypsin digestion of the mixed protein peaks ranging from BPI to BPII obtained by fractionation on a carboxymethyl cellulose column of Amami-Oshima and Tokunoshima P. flavoviridis venoms that BPIII protein is contained in Amami-Oshima P. flavoviridis venom but not in Tokunoshima P. flavoviridis venom. It is for the first time that a protein present in Amami-Oshima P. flavoviridis venom is not found in Tokunoshima P. flavoviridis venom.

Identification of independent risk factors for fentanyl-induced cough.

PURPOSE: To determine how the probability of fentanyl-induced cough is affected by patient characteristics and/or anesthetic technique. METHODS: We analyzed data from a cohort of 1,311 adult patients undergoing elective surgery under general anesthesia, accompanied by i.v. fentanyl. The following data were collected: patient demographics, history of cigarette smoking, presence of bronchial asthma or chronic obstructive pulmonary disease, administration of angiotensin converting enzyme inhibitors; and anesthetic technique, including: preanesthetic anxiolytic medication, prior use of atropine, epidural lidocaine, a priming dose of vecuronium, and the dose of i.v. fentanyl. Associations between individual variables in the clinical evaluation model and the likelihood of fentanyl-induced cough were characterized by calculating odds ratios. Multiple logistic regression analysis was used to examine the independent contribution of each variable while controlling for all variables. RESULTS: Fentanyl-induced cough was independently associated with the following: aging, cigarette smoking, a prior epidural injection of lidocaine, and a priming dose of vecuronium. Fentanyl-induced cough was unaffected by gender, the presence of either bronchial asthma or chronic obstructive pulmonary disease, or prior use of atropine. CONCLUSIONS: Fentanyl-induced cough may be suppressed by aging, cigarette smoking, prior epidural injection of lidocaine, or a priming dose of vecuronium. These findings may allow insights into the mechanism of this phenomenon, thereby leading to its prevention.

Does yawning represent a transient arousal-shift during intravenous induction of general anesthesia?

UNLABELLED: Although yawning occurs frequently during the IV induction of general anesthesia, the significance of this response remains unknown. In this study, we induced 30 surgical patients with 4 mg/kg thiopental IV, and 30 patients with 2 mg/kg propofol IV. Thereafter, the occurrence of yawning was continuously assessed, as the only clinical end-point, for 1 min. The electroencephalographic bispectral index was monitored throughout the observation period. The criterion for an arousal response was a transient increase during a continuing decrease in the bispectral index value. On the basis of this criterion, the sensitivity and specificity of the yawning response as an arousal sign were 77% and 80%, respectively. If a patient exhibited a yawning response, the chance of arousal was 84% (positive predictive value). With no yawning response, the chance of nonarousal was 71% (negative predictive value). According to simple logistic regression, the yawning response was predictive of a transient arousal-shift with an odds ratio of 13.5 (95% confidence interval: 3.8-48; P < 0.001). The occurrence of a yawning response during IV induction may be a clinical indicator of a transient arousal-shift during progressive loss of consciousness. IMPLICATIONS: Yawning elicited by IV anesthetic induction was related to a transient increase during the continuing decrease in the electroencephalographic bispectral index value (sensitivity and specificity, 77% and 80%, respectively). This type of yawning may be a clinical indicator of a transient arousal-shift during progressive loss of consciousness.