One-Step Affinity Purification of Leucine-Rich α2-Glycoproteins from Snake Sera and Characterization of Their Phospholipase A2-Inhibitory Activities as ß-Type Phospholipase A2 Inhibitors.

Snakes contain three types of phospholipase A2 (PLA2)-inhibitory proteins in their blood, PLIα, ß, and γ, which protect them from their own venom, PLA2. PLIß is the snake ortholog of leucine-rich α2 glycoprotein (LRG). Since autologous cytochrome c (Cyt c) serves as an endogenous ligand for LRG, in this study, we purified snake LRGs from various snake serum samples using Cyt c affinity chromatography. All purified snake LRGs were found to be dimers linked by disulfide bonds. Laticauda semifasciata and Naja kaouthia LRGs showed no inhibitory activity against L. semifasciata PLA2 and weak inhibitory activity against Gloydius brevicauda basic PLA2. Elaphe climacophora PLIß had weaker inhibitory activity against G. brevicauda basic PLA2 than G. brevicauda and Elaphe quadrivirgata PLIs, which are abundant in blood and known to neutralize G. brevicauda basic PLA2. Protobothrops flavoviridis LRG showed no inhibitory activity against basic venom PLA2, PL-X, or G. brevicauda basic PLA2. Binding analysis of P. flavoviridis LRG using surface plasmon resonance showed very strong binding to snake Cyt c, followed by that to horse Cyt c, weak binding to yeast Cyt c, and no binding to P. flavoviridis PL-X or BPI/II. We also deduced the amino acid sequences of L. semifasciata and P. flavoviridis LRG by means of cDNA sequencing and compared them with those of other known sequences of PLIs and LRGs. This study concluded that snake LRG can potentially inhibit basic PLA2, but, whether it actually functions as a PLA2-inhibitory protein, PLIß, depends on the snake.

First clinical experiences of robotic gastrectomy for gastric cancer using the hinotori™ surgical robot system.

BACKGROUND: Although the da Vinci™ Surgical System is the most predominantly used surgical robot worldwide, other surgical robots are being developed. The Japanese surgical robot hinotori™ Surgical Robot System was launched and approved for clinical use in Japan in November 2022. We performed the first robotic gastrectomy for gastric cancer using hinotori in the world. Here, we report our initial experience and evaluation of the feasibility and safety of robotic gastrectomy for gastric cancer using hinotori. METHODS: A single-institution retrospective study was conducted. Between November 2022 and October 2023, 24 patients with gastric cancer underwent robotic gastrectomy with hinotori. Five ports, including one for an assistant, were placed in the upper abdomen, and gastric resection with standard lymphadenectomy and intracorporeal reconstruction were performed. The primary endpoint was the postoperative complication rate within 30 days after surgery. The secondary outcomes were surgical outcomes, including intraoperative adverse events, operative time, blood loss, and the number of dissected nodes. RESULTS: Of the 24 patients, 16 (66.7%) were male. The median age and body mass index were 73.5 years and 22.9 kg/m2, respectively. Twenty-three patients (95.8%) had tumors in the middle to lower stomach. Sixteen (66.7%) and seven (29.2%) patients had clinical stage I and II diseases, respectively. Twenty-three (95.8%) patients underwent distal gastrectomy. No patient had postoperative complications of Clavien-Dindo classification IIIa or higher, whereas two (8.3%) had the grade II complications (enteritis and pneumonia). No intraoperative adverse events, including conversion to other approaches, were observed. All patients received R0 resection. The median operative and console times were 400 and 305 min, respectively. The median blood loss was 14.5 mL, and the number of lymph nodes dissected was 51.5. CONCLUSIONS: This study found that robotic gastrectomy with standard lymphadenectomy for gastric cancer using hinotori can be safely performed.

Capturing temperature changes on the ocular surface along with estrus and ovulation using infrared thermography in Japanese Black cows.

Pre-ovulatory follicles are cooler than the neighboring reproductive organs in cows. Thus, measuring the temperature of reproductive organs could be a useful method for predicting estrus and ovulation in cows, and the establishment of a non-invasive technique is required. In this study, we used infrared thermography (IRT) to measure ocular surface temperature as a potential surrogate for reproductive organ temperature. Five Japanese Black cows with synchronized estrus were subjected to temperature measurements in five regions of the ocular surface, including the nasal conjunctiva, nasal limbus, center cornea, temporal limbus, and temporal conjunctiva, twice a day (0800 h and 1600 h) during the experimental period. The temperatures in the five regions significantly declined in cows from estrus to ovulation. To the best of our knowledge, this study is the first to use IRT to show a temperature decrease in the ocular surface along with estrus to ovulation in Japanese Black cows.

Melphalan Febrile Neutropenia Risk Factors.

This study aimed to identify the risk factors of febrile neutropenia(FN)onset associated with melphalan(L-PAM)therapy. Thirty-nine patients(21 men, 18 women)were administered L-PAM intravenously for multiple myeloma(MM)from April 2011 to February 2022 at the Department of Hematology of Gifu Municipal Hospital. Patients were classified into those with and without FN(Grade 3 or higher), complete blood count and liver function tests were performed immediately before starting therapy. Univariate analysis with Fisher's exact probability test was performed. Factors with p<0.2 were considered as independent variables for multivariate analysis in the multiple logistic regression analysis. A multivariate analysis with 2 independent variables, lactate dehydrogenase(LD)level>222 U/L(upper limit of the facility reference value)and white <3.3×103/µL(lower limit of the facility reference value)from the univariate analysis, and FN onset(Grade 3 or higher)as the dependent variable showed that LD level>222 U/L(odds ratio: 6.33, 95% confidence interval: 1.12-35.8, p=0.037)was a significant factor. In conclusion, patients with LD levels >222 U/L immediately before starting therapy require adequate monitoring for FN onset following L-PAM administration.

A 77-Year-Old Man with a Pulse Granuloma of the Descending Colon Identified by Fluorodeoxyglucose-Positron Emission Tomography (FDG-PET) Imaging 19 Months Following Surgical Resection for Rectal Carcinoma.

BACKGROUND Food particles may sometime lodge in the intestinal wall, resulting in a granuloma. Pulse granuloma is associated with the seed of a legume and has a characteristic appearance on histology. This report describes a case of pulse granuloma of the descending colon identified by fluorodeoxyglucose-positron emission tomography (FDG-PET) imaging. Imaging was done 19 months after surgical resection for rectal carcinoma, and the results of imaging alone suggested a tumor metastasis. CASE REPORT A 77-year-old man underwent sigmoid colostomy for sigmoid colon perforation due to obstruction by rectal cancer affecting the upper rectum approximately 2 years ago. Two months later, after his general condition improved, he underwent laparoscopic low anterior resection. On postoperative pathological examination, the lesion was diagnosed as stage II. Nineteen months later, computed tomography showed an irregular nodule on the dorsolateral side of the descending colon. FDG-PET revealed positive results, and peritoneal dissemination was suspected. Because the lesion was localized and there was no other evidence of metastasis, resection was performed. A pathological examination revealed a pulse granuloma with a central legume seed, and no obvious malignant findings were observed. CONCLUSIONS This report has highlighted the importance of imaging and histopathology in cases in which a solitary nodule is present in the bowel in a patient with previous successful treatment for malignancy. Pulse granuloma, or other types of granuloma associated with impacted food material, may be a cause of a solitary nodule, or pseudotumor, in the bowel wall.

[Risk Factors for Anti-PD1 Antibody-Induced Skin Eruptions].

Skin complication caused by anti-programmed cell death-1(PD1)antibody is a typical immune-related adverse event. We designed this study to clarify the correlation between risk factors(patient's background and laboratory data)and skin toxicity( rash and eruption, excluding itch)after administration of either nivolumab or pembrolizumab. From February 2016 to January 2018, we evaluated the clinical outcomes of 54 patients who were administered anti-PD1 antibody. The patients were divided into 2 groups: 9 patients with skin eruption caused by anti-PD1 antibody(skin eruption group)and 45 patients without skin eruption caused by anti-PD1 antibody(non-skin eruption group). Univariate analysis revealed a significant difference in eosinophil counts in both the groups before anti-PD1 antibody administration(>300/µL)(p=0.020). Factors with p<0.2 in the univariate analysis and 4 factors, age(<65 years of age), sex(male), allergy(+), and pembrolizumab, likely to be related to the appearance of skin eruption, were examined by multivariate analysis. Consequently, eosinophil count before anti-PD1 antibody administration(>300/µL)was identified as a risk factor (odds ratio: 9.530, 95% confidence interval: 1.260-71.80). In conclusion, we suggest that cases with an increased eosinophil count before anti-PD1 antibody administration(>300/µL)may be associated with the appearance of skin eruption.

[Predictors of Nivolumab-Induced Skin Reactions].

Skin reactions to nivolumab are typical immune-related adverse events. We investigated the relation between patient background and test values before nivolumab administration and skin reactions. From February 2016 to February 2017, we evaluated the clinical outcomes of 21 patients who were administered nivolumab. Patients were divided into 2 groups: 3 cases of skin reactions to nivolumab(skin reaction group)and 18 cases without skin reactions to nivolumab(non-skin reaction group). In the skin reaction group, the numbers of eosinophils and basophils before nivolumab administration were significantly higher than those in the non-skin reaction group(p=0.0015 and p=0.0075, respectively). It was suggested that the numbers of eosinophils or basophils before nivolumab administration might be associated with the appearance of skin reactions.

Impact of pharmacist counseling on reducing instances of adverse events that can affect the quality of life of chemotherapy outpatients with breast Cancer.

BACKGROUND: In recent years, cancer chemotherapy is being conducted at outpatient clinics, wherein pharmacists are involved with patient guidance and management of adverse events as experts in medication therapy. Therefore, we clarified the influence of interventions by pharmacists during counseling of patients with cancer on patients' quality of life. METHODS: To determine this influence, we conducted a survey to assess the quality of life of 39 patients with breast cancer who underwent their initial course of outpatient cancer chemotherapy at Gifu Municipal Hospital. A quality of life survey was conducted before the 1st, 2nd, and 3rd courses of treatment and was based on a method obtained from a survey paper entitled, "Quality of Life Questionnaire for Cancer Patients Treated with Anticancer Drugs." RESULTS: Twenty patients were assigned to the intervention group, which received pharmacist counseling, and nineteen patients were assigned to the non-intervention group, which received no pharmacist counseling. Both groups were compared immediately before the 1st course and 2nd course. Regarding the subscale of social relationships, a significant difference was observed for malaise (p = 0.043), with the non-intervention group experiencing them to a greater degree than the intervention group. Regarding the change between immediately before the 1st course and the 3rd course, a significant difference was observed in the subscale of social relationships for nausea (p = 0.017), with the non-intervention group experiencing it to a greater degree than the intervention group. CONCLUSIONS: The results suggest that receiving pharmacists' guidance on adverse events and individually adjusted prescriptions tailored to address the occurrence of adverse events improved the treatment environment and enhanced the quality of life in the intervention group. These findings are beneficial in maintaining patients' quality of life during cancer treatment. TRIAL REGISTRATION: No. UMIN000027171, Registration date: Apr 27, 2017. Retrospectively registered.

[A Case of Duodenal Gastrointestinal Stromal Tumor Resection with Preservation of the Pancreatic Head Following Chemotherapy with Imatinib].

A 60-year-old male was admitted to our hospital on account of a duodenal submucosal tumor on the opposite side of Vater's papilla identified on a screening gastroduodenoscopy. The tumor was diagnosed as a duodenal gastrointestinal stromal tumor(GIST)due to positive c-kit and DOG1tests. Gastroduodenography and enhanced computed tomography showed a 50mm tumor widely invadingthe descendingportion of the duodenum close to the papilla of Vater and indicated the need for extended resection includingpancreaticoduodenectomy to achieve curation. Preoperative chemotherapy was initiated with imatinib mesylate(imatinib)to preserve the pancreas. A 16 month course of imatinib shrunk the tumor up to 21mm and laparoscopic partial duodenectomy was performed as a curative resection. So far, no local tumor recurrence has been seen for 26 months after the surgery. Herein, we report this case with a review of the literature.

Inhibition of Inwardly Rectifying Potassium (Kir) 4.1 Channels Facilitates Brain-Derived Neurotrophic Factor (BDNF) Expression in Astrocytes.

Inwardly rectifying potassium (Kir) 4.1 channels in astrocytes regulate neuronal excitability by mediating spatial potassium buffering. Although dysfunction of astrocytic Kir4.1 channels is implicated in the development of epileptic seizures, the functional mechanisms of Kir4.1 channels in modulating epileptogenesis remain unknown. We herein evaluated the effects of Kir4.1 inhibition (blockade and knockdown) on expression of brain-derived neurotrophic factor (BDNF), a key modulator of epileptogenesis, in the primary cultures of mouse astrocytes. For blockade of Kir4.1 channels, we tested several antidepressant agents which reportedly bound to and blocked Kir4.1 channels in a subunit-specific manner. Treatment of astrocytes with fluoxetine enhanced BDNF mRNA expression in a concentration-dependent manner and increased the BDNF protein level. Other antidepressants (e.g., sertraline and imipramine) also increased the expression of BDNF mRNA with relative potencies similar to those for inhibition of Kir4.1 channels. In addition, suppression of Kir4.1 expression by the transfection of small interfering RNA (siRNA) targeting Kir4.1 significantly increased the mRNA and protein levels of BDNF. The BDNF induction by Kir4.1 siRNA transfection was suppressed by the MEK1/2 inhibitor U0126, but not by the p38 MAPK inhibitor SB202190 or the JNK inhibitor SP600125. The present results demonstrated that inhibition of Kir4.1 channels facilitates BDNF expression in astrocytes primarily by activating the Ras/Raf/MEK/ERK pathway, which may be linked to the development of epilepsy and other neuropsychiatric disorders.

Efficacy of transarterial embolisation in the management of post-partum haemorrhage and its impact on subsequent pregnancies.

BACKGROUND: Transarterial embolisation (TAE) has been attracting attention as a treatment option for post-partum haemorrhage (PPH). AIMS: We investigated cases in which TAE was performed for PPH to determine the efficacy of TAE for the treatment of PPH and its impact on subsequent pregnancies and childbirth in a multi-institutional case-series study. MATERIALS AND METHODS: A total of 211 women who underwent emergency (n=161) or prophylactic (n=50) TAE for the management of PPH from 2002 to 2011 in the Chugoku and Shikoku regions of Japan were evaluated. Data on maternal background, treatment efficacy, adverse events in 113 cases followed up after TAE and subsequent pregnancies in 76 women were obtained. RESULTS: The overall success rate of emergency and prophylactic TAE was 91.9 and 96.0%, respectively. The rate of complications such as infection, uterine necrosis and amenorrhoea was 13.3% among women followed up after TAE. Forty women became pregnant after TAE, and the pregnancy rate among those who desired fertility was 52.6%. Twenty-eight gave birth, including four preterm deliveries. Five (16.7%) were associated with a hysterectomy due to placenta accreta. CONCLUSIONS: To reduce haemorrhage and avoid hysterectomy, TAE may be considered as a treatment option for PPH, however short and long terms complications are not uncommon.

Three cases of struma ovarii underwent laparoscopic surgery with definite preoperative diagnosis.

Struma ovarii is a rare neoplasm that accounts for approximately 0.3% of ovarian tumors. Due to its ultrasound morphology, which is quite similar to that of malignant ovarian carcinoma, most struma ovarii cases are open operated with laparotomy rather than laparoscopy. We present 3 cases of struma ovarii, which were diagnosed preoperatively by imaging studies and removed by laparoscopic surgery. All patients were premenopausal women between ages 31‒50. The magnetic resonance imaging (MRI) findings were complex masses composed of multiple cysts and solid components with T2-hypointense regions as well as multiple T1-hyperintense cystic areas, findings that are typical for struma ovarii. A combination of plain computed tomography (CT), positron emission tomography (PET)-CT, and scintigraphy was useful for diagnosis. Laboratory examination revealed elevated serum thyroglobulin, which led to the diagnosis of struma ovarii. Laparoscopic surgeries were performed without rupturing the tumors. Although it has been difficult to differentiate between struma ovarii and malignant tumors by conventional methods, recently MRI techniques appear make it possible to diagnose struma ovarii preoperatively from the abovementioned imaging characteristic, together with laboratory data. As for treatment, we think laparoscopy could be successful for struma ovarii, but the surgeon must be careful not to rupture the tumor intra-abdominally in order to prevent dissemination, which could lead to malignancy.

Pregnancy and delivery in patients with Fontan circulation: a report of two cases.

Fontan operation is performed to provide palliation for patients with many forms of highly complex congenital heart disease that cannot support a biventricular circulation. Increasing numbers of women who have undergone these connections in childhood are now reaching their childbearing years, and some are becoming pregnant. The low flow and fixed cardiac output of a Fontan circulation poses a number of problems during pregnancy. Here, we report two cases of pregnancy and delivery with Fontan circulation. Case 1, who underwent Fontan procedure for congenital pulmonary atresia with intact vertical septum at age 7, delivered a male infant weighing 1073 g by cesarean section at 28(6/7) weeks due to massive genital bleeding. Case 2 underwent Fontan procedure for double inlet left ventricle and delivered by vacuum extraction a male infant weighing 2142 g, while monitoring central venous pressure at 37(5/7) weeks. The former had ascites and dose of diuretic had to be added at early pregnancy, and the latter had no adverse cardiac and obstetric events. These cases suggest that patients after adequate Fontan palliation could complete pregnancy without long-term cardiac sequelae, but might be complicated with cardiac or obstetrical events. Intensive care should be required with specialists, including a neonatologist, anesthesiologist and cardiologist. We have added a literature review of pregnancy with Fontan circulation, referring to previous reports.

Superimposed preeclampsia in women with chronic kidney disease.

AIM: To evaluate whether pregnant women with chronic kidney disease (CKD) adapt poorly to increases in renal blood flow. This can exacerbate renal function and impair perinatal outcome, as there is a major interplay between CKD and preeclampsia (PE). METHODS: We analyzed the outcomes of 90 pregnant women with preexisting CKD. The estimated glomerular filtration rate (eGFR) was measured along with the levels of angiogenic factors, soluble fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor, which might act in the pathophysiology of PE. RESULTS: In pregnancies with CKD, PE and preterm delivery were increased and the increased blood pressure worsened the perinatal outcomes much more than the increased proteinuria. All pregnancies with severe renal insufficiency were delivered preterm because of impaired renal function. The eGFR was correlated significantly with 24-hour creatinine clearance (r = 0.830). Significant differences in sFlt-1 and placental growth factor levels were found between severe PE without any complications and severe superimposed PE (p < 0.05), and between women with and without declining renal function in superimposed PE (p < 0.01). CONCLUSION: Pregnancies with CKD have a high risk of obstetrical complications. The eGFR might serve for evaluating renal function during pregnancy. Angiogenic factors might be potential markers for a differential diagnosis between PE and worsening renal function.

Potential interaction of brain natriuretic peptide with hyperadiponectinemia in preeclampsia.

Adiponectin was reported recently to have roles in the pathophysiology of preeclampsia. Moreover, elevation of adiponectin and brain natriuretic peptide (BNP) has been observed in preeclampsia. We examined the possible links between adiponectin and BNP in the pathophysiology of preeclampsia. We performed a cross-sectional study in 56 preeclampsia patients and 56 controls matched for gestational age and body mass index. The BNP, leptin, and adiponectin levels were measured by ELISA, and their mRNA expressions were evaluated in omental adipose tissue by real-time PCR. The effects of BNP on adiponectin and leptin mRNA expression and secretion were investigated in primary cultures of adipocytes from obese and normal-weight women. The BNP, adiponectin, and leptin levels were significantly higher in preeclampsia patients compared with controls. The adiponectin level was increased significantly in normal-weight preeclampsia patients compared with overweight preeclampsia patients. Adiponectin mRNA expression was increased significantly in adipose tissues of preeclampsia patients compared with controls and was also increased significantly in normal-weight preeclampsia patients compared with overweight preeclampsia patients, whereas leptin was not. BNP and adiponectin showed significant positive correlations in both normal-weight and overweight preeclampsia patients. BNP had a significantly weaker effect on adiponectin in overweight compared with normal-weight preeclampsia patients. Moreover, BNP had a weaker effect on adiponectin production in adipocytes from overweight women compared with adipocytes from normal-weight women using primary culture of human adipocytes. These data suggested that BNP may play a role in hyperadiponectinemia of preeclampsia patients. The weaker effect of BNP on adiponectin production may participate in the pathophysiology of overweight preeclampsia patients.

Accessing the transport properties of graphene and its multilayers at high carrier density.

We present a comparative study of high carrier density transport in mono-, bi-, and trilayer graphene using electric double-layer transistors to continuously tune the carrier density up to values exceeding 10(14) cm(-2). Whereas in monolayer the conductivity saturates, in bi- and trilayer filling of the higher-energy bands is observed to cause a nonmonotonic behavior of the conductivity and a large increase in the quantum capacitance. These systematic trends not only show how the intrinsic high-density transport properties of graphene can be accessed by field effect, but also demonstrate the robustness of ion-gated graphene, which is crucial for possible future applications.

Effects of intermittent high glucose on BeWo choriocarcinoma cells in culture.

AIM: The aim of this study was to investigate the cellular effects of intermittent high glucose on the human BeWo placental choriocarcinoma cell line, used as a model of the effects of glucose fluctuation in diabetic pregnancies. MATERIALS AND METHODS: BeWo cells were subjected to three different glucose conditions for 48 h: 7 mmol/L (control), 42 mmol/L (high glucose), or 7 and 42 mmol/L glucose (intermittent, alternated every 6 h). Cell viability was assessed using cell counts, a cell proliferation assay, and a cell viability assay. Apoptosis was also studied using a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and by immunocytochemistry of fractin, the N-terminal fragment of actin, which can distinguish apoptotic from necrotic cells. Furthermore, the expression of the well-known survival factors of trophoblast cells, heparin-binding epidermal growth factor-like growth factor and leptin, was evaluated by real-time polymerase chain reaction and Western blot analyses. RESULTS: Intermittent high-glucose conditions significantly decreased cell viability and enhanced apoptosis compared with control or continuous high-glucose conditions. Furthermore, the expression of heparin-binding epidermal growth factor-like growth factor, but not that of leptin, was significantly increased under intermittent high-glucose conditions compared to its expression under either control or continuous high-glucose conditions. CONCLUSIONS: These data indicate that intermittent high glucose is more deleterious to BeWo cells than continuous high-glucose conditions. Although further in vitro and in vivo study is necessary, excess fluctuation of glucose levels in the placental circulation might be involved in the impairment of placental development leading to the placental dysfunction.

Retinol-binding protein 4 and insulin resistance in preeclampsia.

Preeclampsia is characterized by the onset of high blood pressure and proteinuria during pregnancy, which results in substantial maternal and neonatal morbidity and mortality. Insulin resistance has been observed before the onset of preeclampsia, and is implicated in its pathophysiology. Recently, retinol-binding protein 4 (RBP4), which carries retinol in circulation, has been shown to be a potential regulator of insulin resistance originating from adipose tissue. Here we measured insulin resistance and RBP-4 levels in patients with preeclampsia and in women with normal pregnancies matched for gestational age and body mass index at Okayama University Hospital. Our aim was to examine the potential role of RBP4 in the pathophysiology of this disorder. There were no significant differences in RBP4 levels between all patients with preeclampsia and controls. However, the RBP4 level and homeostasis model assessment as an index of insulin resistance (HOMA-IR) in overweight patients with late-onset preeclampsia were significantly higher than in overweight controls carrying normal pregnancies and in normal weight women with late-onset preeclampsia. In contrast, there were no significant differences between the overweight and normal weight groups among patients with early-onset preeclampsia and in healthy pregnant women. These data suggest that RBP4 might act in the pathophysiology of late-onset preeclampsia via increased insulin resistance in obese women.

Inhibitory activities of the heterotrimers formed from two α-type phospholipase A2 inhibitory proteins with different enzyme affinities and importance of the intersubunit electrostatic interaction in trimer formation.

α-type phospholipase A2 inhibitory protein (PLIα) isolated from the serum of the venomous snake Glyoidius brevicaudus, GbPLIα, is a homotrimer of subunits having a C-type lectin-like domain. The serum protein from nonvenomous snake Elaphe quadrivirgata, EqPLIα-LP, is homologous to GbPLIα, but it does not show any inhibitory activity against PLA2s. When a mixture of denaturant-treated monomeric forms of GbPLIα and EqPLIα-LP was used to reconstitute their trimers, no significant amounts of heterotrimers composed of GbPLIα and EqPLIα-LP subunits could be formed. On the other hand, when a mixture of denaturant-treated monomeric forms of GbPLIα and the recombinant chimeric EqPLIα-LP, Eq13Gb37Eq, in which the residues 13-36 were replaced by those of GbPLIα, was used to reconstitute their trimers, significant amounts of their heterotrimers were observed. Furthermore, when a mixture of denaturant-treated monomeric forms of EqPLIα-LP and the recombinant chimeric GbPLIα, Gb13Eq37Gb, in which the residues 13-36 were replaced by those of EqPLIα-LP, was used, significant amounts of their heterotrimers were observed. By comparison of the respective inhibitory activities of the heterotrimeric subspecies, it was suggested that the inhibitory activity of the trimer was governed by one subunit with the highest activity, and not affected by the number of these subunits. The intermolecular electrostatic interactions between Glu23 and Lys28 of GbPLIα were also suggested to be important in stabilizing the trimeric structure. The importance of the electrostatic interaction was supported by the less stability of the homotrimeric structure of a mutant GbPLIα with a single amino acid substitution, GbPLIα(K28E).

Transient signaling of Erk1/2, Akt and PLCgamma induced by nerve growth factor in brain capillary endothelial cells.

Cumulative evidences suggest that nerve growth factor (NGF) promotes angiogenic effects such as proliferation and migration of endothelial cells (ECs) from different vascular beds, induces capillary sprouting in chorioallantoic membrane and improves in vivo vascularization in a hind-limb ischemic model. In the present study, we sought to investigate the signaling properties of NGF in a microcapillary ECs model compared to those of a neuronal model. NGF-induced phosphorylation of signaling molecules Erk1/2, Akt and PLCgamma were measured using Western blotting and compared between mouse NGF (mNGF) and snake venom NGF analogues. NGFs-induced signaling was TrkA mediated as evident by inhibition with the TrkA antagonist K252a. NGF and its analogues-induced signaling in ECs were characterized by a transient effect in contrast to a prolonged stimulation in neuronal cells. The potency of mouse, cobra and viper NGFs to induce Erk1/2 phosphorylation in ECs was higher than in neurons. In ECs, mNGF exhibited the highest efficacy of stimulation of Erk1/2 phosphorylation, followed by viper and cobra NGFs. The efficacy of stimulation of Erk1/2 phosphorylation measured with neurons was opposite from that in ECs. NGF-induced temporal signaling differences between ECs and neurons may explain the dual vascular and neurotrophic effects of this growth factor.