Phosphotyrosine proteomics in cells synchronized at monopolar cytokinesis reveals EphA2 as functioning in cytokinesis.

Cytokinesis is the final step of the cell division in which cellular components are separated into two daughter cells. This process is regulated through the phosphorylation of different classes of proteins by serine/threonine (Ser/Thr) kinases such as Aurora B and Polo-like kinase 1 (PLK1). Conversely, the role of phosphorylation at tyrosine residues during cytokinesis has not been studied in detail yet. In this study, we performed a phosphotyrosine proteomic analysis of cells undergoing monopolar cytokinesis synchronized by using the Eg5 inhibitor (+)-S-trityl-l-cysteine (STLC) and the CDK1 inhibitor RO-3306. Phosphotyrosine proteomics gave 362 tyrosine-phosphorylated peptides. Western blot analysis of proteins revealed tyrosine phosphorylation in mitogen-activated protein kinase 14 (MAPK14), vimentin, ephrin type-A receptor 2 (EphA2), and myelin protein zero-like protein 1 (MPZL1) during monopolar cytokinesis. Additionally, we demonstrated that EphA2, a protein with unknown function during cytokinesis, is involved in cytokinesis. EphA2 knockdown accelerated epithelial cell transforming 2 (Ect2) knockdown-induced multinucleation, suggesting that EphA2 plays a role in cytokinesis in a particular situation. The list also included many proteins previously reported to play roles during cytokinesis. These results evidence that the identified phosphopeptides facilitate the identification of novel tyrosine phosphorylation signaling involved in regulating cytokinesis.

Characterization of glutamate-cysteine ligase and glutathione synthetase from the δ-proteobacterium Myxococcus xanthus.

Glutathione (GSH) is synthesized in two ATP-dependent reactions by glutamate-cysteine ligase (Gcl) and glutathione synthetase (Gs). Myxococcus xanthus, a gram-negative bacterium belonging to δ-proteobacteria, possesses mxGcl and mxGs, which have high sequence identity with the enzymes from plants and bacteria, respectively. MxGcl2 was activated by Mn2+ , but not by Mg2+ , and stabilized in the presence of 5 mM Mn2+ or Mg2+ . Sequence comparison of mxGcl2 and Brassica juncea Gcl indicated that they have the same active site residues, except for Tyr330, which interacts with Cys and which in mxGcl2 is represented by Leu267. The substitution of Leu267 with Tyr resulted in the loss of mxGcl2 activity, but that with Met (found in cyanobacterial Gcls) increased the mxGcl2 affinity for Cys. GSH and its oxidized form GSSG equally inhibited the activity of mxGcl2; the inhibition was augmented by ATP at concentrations >3 mM. Buthionine sulfoximine inactivated mxGcl2 with Ki  = 2.1 µM, which was lower than those for Gcls from other organisms. The mxGcl2 activity was also suppressed by pyrophosphate and polyphosphates. MxGs was a dimer, and its activity was induced by Mg2+ but strongly inhibited by Mn2+ even in the presence of 10 mM Mg2+ . MxGs was inhibited by GSSG at Ki  = 3.6 mM. Approximately 1 mM GSH was generated with 3 units of mxGcl2 and 6 units of mxGs from 5 mM Glu, Cys, and Gly, and 10 mM ATP. Our results suggest that GSH production in M. xanthus mostly depends on mxGcl2 activity.

Primary cell culture of canine corneal endothelial cells.

OBJECTIVE: To establish a primary cell culture and clarify the characteristics of canine corneal endothelial cells in vitro. PROCEDURES: The eyes were enucleated from dogs that were euthanized for reasons unrelated to this study. Enucleated canine eyes were dissected, and the intact corneas were isolated from the globes. Using enzymes, the corneal endothelial cells were dispersed from the cornea. The obtained canine corneal endothelial cells were cultured in a cell culture dish. Cultured corneal endothelial cells were morphologically evaluated using phase-contrast microscopy. Immunohistochemical analysis of the cultured cells, particularly of the corneal endothelial cell marker, zonula occludens-1 (ZO-1), Na+ /K+ -ATPase, and vimentin, was performed to clarify whether the cultured cells were actually corneal endothelial cells. Furthermore, the post-passage morphology of cultured cells was evaluated. RESULTS: Canine primary cultured corneal endothelial cells showed morphologically small, cobblestone-like structures. The isolated cells had proliferative ability in vitro and demonstrated positive expression of the corneal endothelial cell markers, ZO-1, Na+ /K+ -ATPase, and vimentin. However, repeated passages resulted in larger cell sizes as assessed by phase-contrast microscopy. Repeated passages also resulted in lower cell density. CONCLUSIONS: This study demonstrated the successful culture of canine corneal endothelial cells. This might enhance the understanding of corneal endothelial cell characteristics in dogs.

Using a radiopaque marker with radiography for evaluating colonic transit by geometric center in conscious rats: A novel method.

This study developed a new method using radiopaque markers under X-ray to measure rat colonic transit by geometric center repeatedly and/or over a time series in the same individually. Additionally, the utility of this method was shown by elucidating the innervation of the autonomic nerve on colonic transit in detail with a pharmacological technique in conscious rats. An in-dwelling silastic cannula was inserted into the cecum and the proximal part was moved through the abdominal wall, where it was fixed to the posterior neck skin. Twenty markers were administered from the cannula to the proximal colon with saline on the fifth day after surgery. The markers were observed with soft X-ray before required repeated short anesthesia. Experimentation 1: Rats were measured colonic transit twice over 2 days with no administration. Experimentation 2: Rats were administered saline on the first day and pharmacology on the second day intraperitoneally before measurement. Experimentation 1: The markers administrated from the cannula and transited from proximal colon to distal colon over a time series. It showed no significant difference in complication rates between 2 days. Experimentation 2: The colonic transit was increasingly accelerated by neostigmine and phentolamine but not propranolol. Significant changes in 1.0 mg/kg atropine were noted although no differences were found between control and 0.05 mg/kg atropine and between each other's. We have presented the method using radiopaque markers under X-ray with short anesthesia for evaluating the colonic transit. The methods could show rat colonic transit changes in detail with a pharmacological technique.

Effect of acupuncture on the haemodynamic system in men.

BACKGROUND: Acupuncture stimulation decreases heart rate (HR) through somato-autonomic reflexes. However, the mechanisms responsible for other cardiovascular changes induced by acupuncture, such as its effects on stroke volume (SV) and blood pressure (BP), remain obscure. OBJECTIVE: To evaluate continuously the comprehensive cardiovascular changes occurring during acupuncture. METHOD: 20 healthy men participated in the study. HR, SV and BP were measured in the supine position using electrocardiogram, transthoracic impedance cardiography and continuous non-invasive finger blood pressure, respectively. Manual acupuncture stimulation using a stainless steel needle was performed at LI10 for 60 s after resting periods of approximately 15 min. RESULTS: HR was reduced and SV increased, in parallel, during the period of acupuncture stimulation (P<0.01, respectively). Diastolic blood pressure (DBP) decreased in the 10 s period of acupuncture stimulation compared with the 120 s pre-stimulation period (P<0.01) and recovered close to the pre-stimulation reading instantly after the transient reduction. No change was observed in cardiac output (CO) derived from HR and SV. CONCLUSIONS: This study indicates that HR reduction during acupuncture does occur, as previous reports have indicated. SV increased during acupuncture stimulation in parallel with HR reduction and CO was maintained during these changes. Any reduction in DBP caused by acupuncture recovered to baseline, likely due to baroreflexes.

Mechanism of Electroacupuncture on Postoperative Ileus Induced by Surgical Stress in Rats.

Objectives: Acupuncture has been used for treating gastrointestinal (GI) disorders such as postoperative nausea and vomiting. Electroacupuncture (EA) accelerates GI transit following surgery and ameliorates postoperative ileus (POI) to restore colonic transit (CT); however, the mechanisms of this EA-induced restoration remain unclear. The aims of this study were to show CT following surgery and the effects of EA at ST 36 on POI induced by surgical stress (SS) in 45 conscious, male Sprague-Dawley rats. Materials and Methods: An operation was performed in each rat, setting a cannula into the cecum to connect the proximal colon to inject markers. On the day after surgery, 20 metal radiopaque markers were administered to the proximal colon of each rat. These markers were visible throughout the GI tract on soft X-ray immediately after administration and up to 240 minutes afterward. The rats were divided into 5 groups with 9 rats in each group: (1) SS; (2) 5 days post surgery (POST-5D); (3) SS + phentolamine; (4) EA alone; and (5) EA + atropine. The EA was performed at ST 36 for 20 minutes at a frequency of 10 Hz and agents were administered in the appropriate groups before markers were administered and measurements were taken. Measurements were performed the day after surgery except in the POST 5-D group. CT was calculated by the geometric center on the images showing the CT for each rat. Results: CT after surgery was delayed significantly and phentolamine accelerated CT. EA restored CT following surgery and atropine abolished the effect of EA on CT. Conclusions: The current study demonstrated that surgery induced a delay in CT through the sympathetic pathway via α-adrenoreceptors; CT was restored by EA. These results suggest that EA can be used to treat POI through mediation of the autonomic nervous system.