The effect of rotator cuff physical exercise combined with electrically stimulated antagonist on shoulder rotator cuff strength.

BACKGROUND: An elastic band (EB) is generally used with a low load for rotator cuff physical exercise, but the resulting increase in muscle strength is insufficient. We assessed the efficacy on external rotator muscle strength of the shoulder joint; of a hybrid training system (HTS) that resists the motion of a volitionally contracting agonist muscle using the force generated by its electrically stimulated antagonist vs. general rotator cuff exercise with EB. METHODS: Twenty healthy men with no shoulder joint disorders were randomized to 6 weeks of triweekly 10-min rotator cuff exercise with HTS or EB in a clinical research laboratory. Isokinetic concentric external rotator muscle strength at angular velocities of 60°/s and 180°/s (CON60, CON180, respectively) and isokinetic eccentric external rotator muscle strength at an angular velocity of 60°/s (ECC60) were measured as rotator cuff function before and after 6 weeks of intervention. RESULTS: There were no significant intergroup differences in baseline characteristics. There were statistically significant differences (p = 0.0358, p = 0.0213, respectively) in the increase in CON180 (mean ± SD) and ECC60 between the HTS group (Δ6.0 ± 6.0Nm, p = 0.015; Δ7.5 ± 4.7Nm p = 0.0007, respectively) and the EB group (Δ0.3 ± 5.2Nm, p = 0.8589; Δ1.8 ± 5.3 Nm p = 0.3133, respectively). There was a trend toward CON60 increasing in the HTS group (Δ4.7 ± 6.5Nm, p = 0.0494) which was greater than in the control group (Δ-0.9 ± 6.3Nm, p = 0.6637) (inter-group, p = 0.0677). CONCLUSIONS: The results of this study support the conclusion that HTS is more effective for increasing external rotator muscle strength more effectively than EB. HTS would be useful for rotator cuff physical exercise.

The LMTK1-TBC1D9B-Rab11A Cascade Regulates Dendritic Spine Formation via Endosome Trafficking.

Dendritic spines are postsynaptic protrusions at excitatory synapses that are critical for proper neuronal synaptic transmission. While lipid and protein membrane components are necessary for spine formation, it is largely unknown how they are recruited to developing spines. Endosomal trafficking is one mechanism that may influence this development. We recently reported that Lemur kinase 1A (LMTK1A), a membrane-bound Ser/Thr kinase, regulates trafficking of endosomes in neurons. LMTK1 has been shown to be a p35 Cdk5 activator-binding protein and a substrate for Cdk5-p35; however, its neuronal function has not been sufficiently studied. Here, we investigate the role of LMTK1 in spine formation. Depletion of LMTK1 increases spine formation, maturation, and density in primary cultured neurons and in mouse brain of either sex. Additionally, expression of kinase-negative LMTK1 stimulates spine formation in primary neurons and in vivo LMTK1 controls spine formation through Rab11, a regulator of recycling endosome trafficking. We identify TBC1D9B, a Rab11A GTPase-activating protein (Rab11A GAP), as a LMTK1 binding protein, and find that TBC1D9B mediates LMTK1 activity on Rab11A. TBC1D9B inactivates Rab11A under the control of LMTK1A. Further, by analyzing the effect of decreased TBC1D9B expression in primary neurons, we demonstrate that TBC1D9B indeed regulates spine formation. This is the first demonstration of the biological function of TBC1D9B. Together, with the regulation of LMTK1 by Cdk5-p35, we propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel signaling mechanism regulating endosomal transport for synapse formation and function.SIGNIFICANCE STATEMENT Dendritic spines are postsynaptic specializations essential for synaptic transmission. However, it is not known how critical membrane components are recruited to spines for their formation. Endosomal trafficking is one such mechanism that may mediate this process. Here we investigate regulators of endosomal trafficking and their contribution to spine formation. We identify two novel factors, LMTK1 and TBC1D9B, which regulate spine formation upstream of Rab11A, a small GTPase. LMTK1 is a membrane bound Ser/Thr kinase regulated by Cdk5-p35, and TBC1D9B is a recently identified Rab11 GAP. LMTK1 controls the GAP activity of TBC1D9B on Rab11A, and TBC1D9B mediates the LMTK1 activity on Rab11A. We propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel mechanism controlling spine formation and function.

AGAP1 regulates subcellular localization of FilGAP and control cancer cell invasion.

The Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) regulate membrane trafficking and actin cytoskeleton. The molecular mechanism of how Arf GAPs regulate actin cytoskeleton remains to be elucidated. We identified AGAP1, a subtype of Arf GAP, as a binding protein of FilGAP, a Rac-specific GAP, in mammalian cells. AGAP1 binds to C-terminus of FilGAP whereas FilGAP binds to N-terminus of AGAP1 containing GLD domain. FilGAP co-localized with AGAP1 at intracellular vesicles and targeting of FilGAP at the vesicles requires its interaction with AGAP1. Consistently, depletion of endogenous AGAP1 induced the accumulation of endogenous FilGAP into paxillin-positive focal adhesions and actin cytoskeletal structures. Knockdown of endogenous AGAP1 suppressed cell spreading on collagen and the suppression was released by depletion of endogenous FilGAP. Moreover, depletion of AGAP1 in MDA-MB-231 cells promoted cell invasion in extracellular matrices and depletion of FilGAP blocked the invasion. Taken together, the present study suggests that AGAP1 may regulate subcellular localization of FilGAP and control cell migration and invasion through interaction with FilGAP.

FilGAP regulates distinct stages of epithelial tubulogenesis.

Epithelial cells form a globular organ-like multi-cellular structure called cyst when cultured in extracellular matrix. The cyst generates extension followed by cell chains and tubules in response to hepatocyte growth factor (HGF). The Rho family small GTPases play essential roles for tubulogenesis. FilGAP, a Rac specific Rho GTPase-activating protein, is highly expressed in kidney. In this study, we examined the role of FilGAP in the tubulogenesis of Madin-Darby Canine Kidney (MDCK) epithelial cells. HGF induces basolateral extensions from cysts. Depletion of FilGAP by siRNA increased the number of extensions in response to HGF, whereas forced expression of FilGAP decreased the number of the extensions. FilGAP is phosphorylated and activated downstream of Rho-ROCK-signaling. Overexpression of phospho-mimic FilGAP (ST/D) mutant blocked formation of the membrane extensions induced by HGF in the presence of ROCK inhibitor, Y-27632. On the other hand, treatment of the tubules with Y27632 induced scattering of the cells, but FilGAP (ST/D) blocked cell scattering and promoted lumen formation. Taken together, our study suggests that FilGAP may suppress formation of extensions whereas stabilize tubule formation downstream of Rho-ROCK-signaling.

FilGAP, a Rho-ROCK-regulated GAP for Rac, controls adherens junctions in MDCK cells.

Rho family small GTPases are essential for the formation of adherens junctions in epithelial cells. Here, we found that FilGAP (also known as ARHGAP24), a Rac-specific Rho GTPase-activating protein, promoted the formation of adherens junctions in Madin-Darby canine kidney (MDCK) cells. Knockdown of FilGAP by siRNA stimulated the disassembly and migration of MDCK cells induced by hepatocyte growth factor (HGF). By contrast, forced expression of FilGAP induced accumulation of E-cadherin at adherens junctions. Endogenous FilGAP colocalized with E-cadherin at adherens junctions, and depletion of FilGAP reduced the amount of E-cadherin expressed at the surface. The Rac GAP domain of FilGAP was necessary for the suppression of cell scattering induced by HGF. In agreement with this, siRNA-mediated knockdown of both Rac1 and FilGAP suppressed cell scattering induced by HGF. Forced expression of Rho kinase (ROCK, of which there are two isoforms ROCK1 and ROCK2) induced the accumulation of E-cadherin at the adherens junction, and depletion of FilGAP prevented the accumulation of E-cadherin. Moreover, wild-type FilGAP but not a non-phosphorylatable FilGAP mutant rescued the accumulation of E-cadherin at adherens junctions. These results suggest that FilGAP might regulate cell-cell adhesion through inactivation of Rac downstream of Rho-ROCK-signaling in MDCK cells.

Kinase activity of endosomal kinase LMTK1A regulates its cellular localization and interactions with cytoskeletons.

Neurite formation, a fundamental process in neuronal maturation, requires the coordinated regulation of cytoskeletal reorganization and membrane transport. Compared to the understanding of cytoskeletal functions, less is known about the supply of membranes to growing neurites. Lemur kinase 1A (LMTK1A) is an endosomal protein kinase that is highly expressed in neurons. We recently reported that LMTK1A regulates the trafficking of Rab11-positive recycling endosomes in growing axons and dendrites. Here, we used the kinase-negative (kn) mutant to investigate the role of the kinase activity of LMTK1A in its cellular localization and interactions with the cytoskeleton in Neuro2A and PC-12 cells. Kinase activity was required for the localization of LMTK1A in the perinuclear endocytic recycling compartment. Perinuclear accumulation was microtubule dependent, and LMTK1A wild type (wt) localized mainly on microtubules, whereas kn LMTK1A was found in the actin-rich cell periphery. In the neurites of PC-12 cells, LMTK1A showed contrasting distributions depending on the kinase activity, with wt being located in the microtubule-rich shaft and the kn form in the actin-rich tip. Taken together, these results suggest that the kinase activity of LMTK1A regulates the pathway for endosomal vesicles to transfer from microtubules to actin filaments at the tip of growing neurites.

Phosphorylation of Serine 402 Regulates RacGAP Protein Activity of FilGAP Protein.

FilGAP is a Rho GTPase-activating protein (GAP) that specifically regulates Rac. FilGAP is phosphorylated by ROCK, and this phosphorylation stimulates its RacGAP activity. However, it is unclear how phosphorylation regulates cellular functions and localization of FilGAP. We found that non-phosphorylatable FilGAP (ST/A) mutant is predominantly localized to the cytoskeleton along actin filaments and partially co-localized with vinculin around cell periphery, whereas phosphomimetic FilGAP (ST/D) mutant is diffusely cytoplasmic. Moreover, phosphorylated FilGAP detected by Phos-tag is also mainly localized in the cytoplasm. Of the six potential phosphorylation sites in FilGAP tested, only mutation of serine 402 to alanine (S402A) resulted in decreased cell spreading on fibronectin. FilGAP phosphorylated at Ser-402 is localized to the cytoplasm but not at the cytoskeleton. Although Ser-402 is highly phosphorylated in serum-starved quiescent cells, dephosphorylation of Ser-402 is accompanied with the cell spreading on fibronectin. Treatment of the cells expressing wild-type FilGAP with calyculin A, a Ser/Thr phosphatase inhibitor, suppressed cell spreading on fibronectin, whereas cells transfected with FilGAP S402A mutant were not affected by calyculin A. Expression of constitutively activate Arf6 Q67L mutant stimulated membrane blebbing activity of both non-phosphorylatable (ST/A) and phosphomimetic (ST/D) FilGAP mutants. Conversely, depletion of endogenous Arf6 suppressed membrane blebbing induced by FilGAP (ST/A) and (ST/D) mutants. Our study suggests that Arf6 and phosphorylation of FilGAP may regulate FilGAP, and phosphorylation of Ser-402 may play a role in the regulation of cell spreading on fibronectin.

Phosphorylation of cyclin-dependent kinase 5 (Cdk5) at Tyr-15 is inhibited by Cdk5 activators and does not contribute to the activation of Cdk5.

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities.

Isomerase Pin1 stimulates dephosphorylation of tau protein at cyclin-dependent kinase (Cdk5)-dependent Alzheimer phosphorylation sites.

Neurodegenerative diseases associated with the pathological aggregation of microtubule-associated protein Tau are classified as tauopathies. Alzheimer disease, the most common tauopathy, is characterized by neurofibrillary tangles that are mainly composed of abnormally phosphorylated Tau. Similar hyperphosphorylated Tau lesions are found in patients with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) that is induced by mutations within the tau gene. To further understand the etiology of tauopathies, it will be important to elucidate the mechanism underlying Tau hyperphosphorylation. Tau phosphorylation occurs mainly at proline-directed Ser/Thr sites, which are targeted by protein kinases such as GSK3ß and Cdk5. We reported previously that dephosphorylation of Tau at Cdk5-mediated sites was enhanced by Pin1, a peptidyl-prolyl isomerase that stimulates dephosphorylation at proline-directed sites by protein phosphatase 2A. Pin1 deficiency is suggested to cause Tau hyperphosphorylation in Alzheimer disease. Up to the present, Pin1 binding was only shown for two Tau phosphorylation sites (Thr-212 and Thr-231) despite the presence of many more hyperphosphorylated sites. Here, we analyzed the interaction of Pin1 with Tau phosphorylated by Cdk5-p25 using a GST pulldown assay and Biacore approach. We found that Pin1 binds and stimulates dephosphorylation of Tau at all Cdk5-mediated sites (Ser-202, Thr-205, Ser-235, and Ser-404). Furthermore, FTDP-17 mutant Tau (P301L or R406W) showed slightly weaker Pin1 binding than non-mutated Tau, suggesting that FTDP-17 mutations induce hyperphosphorylation by reducing the interaction between Pin1 and Tau. Together, these results indicate that Pin1 is generally involved in the regulation of Tau hyperphosphorylation and hence the etiology of tauopathies.

Salvage of severe ischemic lower limb having peak creatine phosphokinase level exceeding 200,000 IU/L treated by continuous hemodiafiltration.

We performed revascularization by an anti-anatomical bypass in a 40-year-old man with extended ischemia of both legs beyond 12 hr after onset because of traumatic aortic dissection. This patient developed myonephropathic metabolic syndrome, including renal and circulatory failure accompanied by a creatine phosphokinase level above 200,000 IU/L. Nevertheless, his bilateral affected limbs were salvaged by intensive care based on aggressive hemocatharsis with continuous hemodiafiltration with treatment for poor hemodynamics and respiratory distress.

RSK/GSK3-mediated phosphorylation of FilGAP regulates chemotactic cancer invasion.

Cell migration plays a crucial role in various biological processes, such as gastrulation, immune response, and cancer metastasis. In response to chemoattractant-like growth factors, cells form protrusions and migrate toward the source of the signal. Rho family small GTPase Rac is a key regulator of cell migration by stimulating actin polymerization to generate lamellipodia, flat membrane protrusions at the leading edge of migrating cells. FilGAP (ARHGAP24), a Rac-specific GTPase-activating protein (GAP), suppresses lamellipodia formation, and controls tumor cell migration. In this study, we found that FilGAP is phosphorylated downstream of epidermal growth factor (EGF) signaling. Upon EGF stimulation, FilGAP is phosphorylated at Ser625 by p90 ribosomal S6 kinase (RSK) and then at Ser621 by glycogen synthase kinase 3 (GSK3). Phosphorylation of FilGAP induces its dissociation from actin filaments. We identified a novel actin-localization domain of FilGAP that is essential for stabilizing cell adhesion. Additionally, we found that phosphorylation of FilGAP inhibits its lamellipodia suppression activity. Finally, we showed the expression of nonphosphorylatable FilGAP mutant, but not wild-type FilGAP, reduced cell migration speed and persistence toward the EGF gradient. Taken together, our results suggest that phosphorylation of FilGAP downstream of EGF-signaling plays a critical role in regulating chemotactic tumor cell migration by controlling cell-matrix adhesion and protrusion formation.

Transarterial embolization for an infected internal iliac artery aneurysm without recurrence for a long period: A case report.

We report an 85-year-old man who underwent transarterial embolization (TAE) for an infected internal iliac artery aneurysm. The patient presented with fever and left lower abdominal pain. Computed tomography (CT) revealed the expansion of a left internal iliac artery aneurysm. We planned surgical treatment for an infected internal iliac artery aneurysm; however, the patient's age and general condition made the surgery high-risk. Therefore, we performed emergency TAE. The patient was administered antibiotics for 4 weeks and discharged on day 33 after the procedure with good progression. A 3-year follow-up CT scan showed aneurysm reduction and no recurrent infections. This case report highlights that TAE can be a treatment option for patients with an infected artery aneurysm.

LMTK1/AATYK1 is a novel regulator of axonal outgrowth that acts via Rab11 in a Cdk5-dependent manner.

Axonal outgrowth is a coordinated process of cytoskeletal dynamics and membrane trafficking; however, little is known about proteins responsible for regulating the membrane supply. LMTK1 (lemur kinase 1)/AATYK1 (apoptosis-associated tyrosine kinase 1) is a serine/threonine kinase that is highly expressed in neurons. We recently reported that LMTK1 plays a role in recycling endosomal trafficking in CHO-K1 cells. Here we explore the role of LMTK1 in axonal outgrowth and its regulation by Cdk5 using mouse brain cortical neurons. LMTK1 was expressed and was phosphorylated at Ser34, the Cdk5 phosphorylation site, at the time of axonal outgrowth in culture and colocalized with Rab11A, the small GTPase that regulates recycling endosome traffic, at the perinuclear region and in the axon. Overexpression of the unphosphorylated mutant LMTK1-S34A dramatically promoted axonal outgrowth in cultured neurons. Enhanced axonal outgrowth was diminished by the inactivation of Rab11A, placing LMTK1 upstream of Rab11A. Unexpectedly, the downregulation of LMTK1 by knockdown or gene targeting also significantly enhanced axonal elongation. Rab11A-positive vesicles were transported anterogradely more quickly in the axons of LMTK1-deficient neurons than in those of wild-type neurons. The enhanced axonal outgrowth was reversed by LMTK1-WT or the LMTK1-S34D mutant, which mimics the phosphorylated state, but not by LMTK1-S34A. Thus, LMTK1 can negatively control axonal outgrowth by regulating Rab11A activity in a Cdk5-dependent manner, and Cdk5-LMTK1-Rab11 is a novel signaling pathway involved in axonal outgrowth.

FilGAP regulates tumor growth in Glioma through the regulation of mTORC1 and mTORC2.

The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that forms the two different protein complexes, known as mTORC1 and mTORC2. mTOR signaling is activated in a variety of tumors, including glioma that is one of the malignant brain tumors. FilGAP (ARHGAP24) is a negative regulator of Rac, a member of Rho family small GTPases. In this study, we found that FilGAP interacts with mTORC1/2 and is involved in tumor formation in glioma. FilGAP interacted with mTORC1 via Raptor and with mTORC2 via Rictor and Sin1. Depletion of FilGAP in KINGS-1 glioma cells decreased phosphorylation of S6K and AKT. Furthermore, overexpression of FilGAP increased phosphorylation of S6K and AKT, suggesting that FilGAP activates mTORC1/2. U-87MG, glioblastoma cells, showed higher mTOR activity than KINGS-1, and phosphorylation of S6K and AKT was not affected by suppression of FilGAP expression. However, in the presence of PI3K inhibitors, phosphorylation of S6K and AKT was also decreased in U-87MG by depletion of FilGAP, suggesting that FilGAP may also regulate mTORC2 in U-87MG. Finally, we showed that depletion of FilGAP in KINGS-1 and U-87MG cells significantly reduced spheroid growth. These results suggest that FilGAP may contribute to tumor growth in glioma by regulating mTORC1/2 activities.

In vitro study on the effect of fibrinogen γ-chain peptide-coated ADP-encapsulated liposomes on postcardiopulmonary bypass coagulopathy using patient blood.

BACKGROUND: Fibrinogen γ-chain peptide-coated, adenosine 5'-diphosphate (ADP)-encapsulated liposomes (H12-ADP-liposomes) are potent hemostatic adjuvants that promote platelet thrombi formation at bleeding sites. Although we have reported the efficacy of these liposomes in a rabbit model of cardiopulmonary bypass coagulopathy, we are yet to address the possibility of their hypercoagulative potential, especially in human beings. OBJECTIVES: Considering its future clinical applications, we herein investigated the safety of using H12-ADP-liposomes in vitro using blood samples from patients who had received platelet transfusion after cardiopulmonary bypass surgeries. METHODS: Ten patients receiving platelet transfusions after cardiopulmonary bypass surgery were enrolled. Blood samples were collected at the following 3 points: at the time of incision, at the end of the cardiopulmonary bypass, and immediately after platelet transfusion. After incubating the samples with H12-ADP-liposomes or phosphate-buffered saline (PBS, as a control), blood coagulation, platelet activation, and platelet-leukocyte aggregate formation were evaluated. RESULTS: Patients' blood incubated with H12-ADP-liposomes did not differ from that incubated with PBS in coagulation ability, degree of platelet activation, and platelet-leukocyte aggregation at any of the time points. CONCLUSION: H12-ADP-liposomes did not cause abnormal coagulation, platelet activation, or platelet-leukocyte aggregation in the blood of patients who received platelet transfusion after a cardiopulmonary bypass. These results suggest that H12-ADP-liposomes could likely be safely used in these patients, providing hemostasis at the bleeding sites without causing considerable adverse reactions. Future studies are needed to ensure robust safety in human beings.

Prophylactic use of intra-aortic balloon pumps in open heart surgery.

During a 1-year period, intra-aortic balloon pumps (IABPs) were used in open heart surgery on 57 patients. Indications were prophylactic usage for coronary artery bypass grafting (CABG) in 52 patients, prophylactic usage for valve replacement in three patients, and cardiopulmonary bypass (CPB) weaning during valve replacement in two patients. The 52 CABG patients comprised 94.5% of all CABG procedures during the period. Sheathless 8 Fr IABPs were used in all cases. The 57 patients using IABPs were analyzed. The mean duration of IABP use was 41.7 h. Morbidity was not associated with using IABPs. There was one case of balloon rupture. Hemostasis was performed easily after removing IABP catheters by compressing the groin for approximately 15 min. The lowest blood pressure during anastomosis or cardiac arrest was also assessed. The lowest peak pressure was 55.9 ± 17.3 mm Hg for patients with IABP still turned on, and the lowest mean pressure was 34.7 ± 6.5 mm Hg for patients with IABP temporarily turned off. Peak blood pressure after CPB was 73.8 ± 17.8 mm Hg. During open heart surgery under anesthesia with the low blood pressure presented by this series, use of IABPs enabled patients to tolerate the procedure. In conclusion, aggressive use of IABPs is easy, safe, and effective with no related morbidity.

Identification of tubulin deglutamylase among Caenorhabditis elegans and mammalian cytosolic carboxypeptidases (CCPs).

Tubulin polyglutamylation is a reversible post-translational modification, serving important roles in microtubule (MT)-related processes. Polyglutamylases of the tubulin tyrosine ligase-like (TTLL) family add glutamate moieties to specific tubulin glutamate residues, whereas as yet unknown deglutamylases shorten polyglutamate chains. First we investigated regulatory machinery of tubulin glutamylation in MT-based sensory cilia of the roundworm Caenorhabditis elegans. We found that ciliary MTs were polyglutamylated by a process requiring ttll-4. Conversely, loss of ccpp-6 gene function, which encodes one of two cytosolic carboxypeptidases (CCPs), resulted in elevated levels of ciliary MT polyglutamylation. Consistent with a deglutamylase function for ccpp-6, overexpression of this gene in ciliated cells decreased polyglutamylation signals. Similarly, we confirmed that overexpression of murine CCP5, one of two sequence orthologs of nematode ccpp-6, caused a dramatic loss of MT polyglutamylation in cultured mammalian cells. Finally, using an in vitro assay for tubulin glutamylation, we found that recombinantly expressed Myc-tagged CCP5 exhibited deglutamylase biochemical activities. Together, these data from two evolutionarily divergent systems identify C. elegans CCPP-6 and its mammalian ortholog CCP5 as a tubulin deglutamylase.

Actin interaction and regulation of cyclin-dependent kinase 5/p35 complex activity.

Cyclin-dependent kinase 5 (Cdk5) plays a critical role during neurodevelopment, synaptic plasticity, and neurodegeneration. Cdk5 activity depends on association with neuronal proteins p35 and p25, a proteolytic product of p35. Cdk5 regulates the actin cytoskeletal dynamics that are essential for neuronal migration, neuritic growth, and synaptogenesis. However, little is known about the interaction of actin and Cdk5 and its effect on neuronal Cdk5 activity. In a previous study, we observed that Cdk5/p35 activity is negatively correlated with co-immunoprecipitated F-actin (filamentous actin) amounts in the mouse brain, and suggested that F-actin inhibits the formation of the Cdk5/p35 complex [Journal of Neuroscience (2008) vol. 28, p. 14511]. The experiments reported here were undertaken to elucidate the relationship between actin and the formation of the Cdk5/p35 complex and its activity. Instead of an F-actin-mediated inhibition, we propose that G-actin (globular actin) in the F-actin preparations is responsible for inhibiting Cdk5/p35 and Cdk5/p25 kinase activity. We found that F-actin binds to p35 but not p25 or Cdk5. We have shown that G-actin binds directly to Cdk5 without disrupting the formation of the Cdk5/p35 or Cdk5/p25 complexes. G-actin potently suppressed Cdk5/p35 and Cdk5/p25 activity when either histone H1 or purified human tau protein were used as substrates, indicating a substrate-independent inhibitory effect of G-actin on Cdk5 activity. Finally, G-actin suppressed the activity of Cdk5 immunoprecipitated from wild type and p35-deficient mouse brain, suggesting that G-actin suppresses endogenous Cdk5 activity in a p35-independent manner. Together, these results suggest a novel mechanism of actin cytoskeletal regulation of Cdk5/p35 activity.

AATYK1A phosphorylation by Cdk5 regulates the recycling endosome pathway.

Trafficking of recycling endosomes (REs) is regulated by the small GTPase, Rab11A; however, the regulatory mechanism remains elusive. Apoptosis-associated tyrosine kinase 1A (AATYK1A) is a Ser/Thr kinase expressed highly in brain. We have recently shown that AATYK1A localizes to Rab11A-positive RE and is phosphorylated at Ser34 by cyclin-dependent kinase 5 (Cdk5). Here, we have investigated a role of AATYK1A and its phosphorylation in recycling endosomal trafficking using Chinese hamster ovary-K1 (CHO-K1) cells. AATYK1A localizes predominantly to Rab11A-positive pericentrosomal endocytic recycling compartment (ERC). Phosphorylation at Ser34 of AATYK1A disrupts its accumulation in the pericentrosomal ERC. Consistently, phosphorylation-mimic mutant (AATYK1A-S34D) did not accumulate in the ERC and additionally attenuated ERC formation. ERC formation suppression can be reversed by constitutively active Rab11A-Q70L, suggesting a functional link between AATYK1A phosphorylation and Rab11A activity. Although no direct interaction between AATYK1A and Rab11A could be detected, the exchange of guanine nucleotides bound to Rab11A was significantly reduced in the presence of the phosphorylation-mimic AATYK1A-S34D. Together, our results reveal a regulatory role for AATYK1A in the formation of pericentrosomal ERC. They furthermore indicate that Cdk5 can disrupt ERC formation via Ser34 phosphorylation of AATYK1A. Finally, our data suggest a mechanism by which AATYK1A signaling couples Cdk5 to Rab11A activity.

Total aortic arch replacement using the J-graft open stent graft for distal aortic arch aneurysm: report from two centres in Japan.

OBJECTIVES: The open-style stent graft technique has been changing the strategy for true distal arch aneurysms extending to the descending aorta. Our mid-term results of surgical repair using a J-graft open stent graft are presented. METHODS: Between May 2015 and June 2020, 69 patients with a distal arch aneurysm (53 males, median age 74 years) underwent total arch replacement combined with J-graft open stent deployment. All 59 surviving patients were followed for a median follow-up period of 1.8 (0.6-3.6) years. RESULTS: Antegrade deployment was successfully performed in all patients without any difficulties. The deployed device was securely fixed at the target area, and it initiated thrombus formation. The diameter of the excluded aneurysm was decreased in 54 patients (91.5%) during the follow-up period. There were no type I endoleaks, but there were 3 type II endoleaks; 2 of the 3 type II endoleaks disappeared during the follow-up period. Additional endovascular operations were performed in 3 patients. There were 10 in-hospital deaths (14.5%), and the incidences of stroke, spinal cord injury and distal embolism were 11.6%, 5.8% and 2.9%, respectively. The 1- and 3-year survival rates were 84.8% and 79.4%, respectively, and the 1- and 3-year freedom from reintervention rates were 97.2% and 81.3%, respectively. CONCLUSIONS: The J-graft open stent graft was easy to deploy, and it could shift the distal anastomosis to a more proximal side. The mid-term performance of this device was good. It has the potential to provide one-stage repair.