Wings of Change: aPKC/FoxP-dependent plasticity in steering motor neurons underlies operant self-learning in Drosophila.

Background: Motor learning is central to human existence, such as learning to speak or walk, sports moves, or rehabilitation after injury. Evidence suggests that all forms of motor learning share an evolutionarily conserved molecular plasticity pathway. Here, we present novel insights into the neural processes underlying operant self-learning, a form of motor learning in the fruit fly Drosophila. Methods: We operantly trained wild type and transgenic Drosophila fruit flies, tethered at the torque meter, in a motor learning task that required them to initiate and maintain turning maneuvers around their vertical body axis (yaw torque). We combined this behavioral experiment with transgenic peptide expression, CRISPR/Cas9-mediated, spatio-temporally controlled gene knock-out and confocal microscopy. Results: We find that expression of atypical protein kinase C (aPKC) in direct wing steering motoneurons co-expressing the transcription factor FoxP is necessary for this type of motor learning and that aPKC likely acts via non-canonical pathways. We also found that it takes more than a week for CRISPR/Cas9-mediated knockout of FoxP in adult animals to impair motor learning, suggesting that adult FoxP expression is required for operant self-learning. Conclusions: Our experiments suggest that, for operant self-learning, a type of motor learning in Drosophila, co-expression of atypical protein kinase C (aPKC) and the transcription factor FoxP is necessary in direct wing steering motoneurons. Some of these neurons control the wing beat amplitude when generating optomotor responses, and we have discovered modulation of optomotor behavior after operant self-learning. We also discovered that aPKC likely acts via non-canonical pathways and that FoxP expression is also required in adult flies.

Network and Experimental Pharmacology on Mechanism of Yixintai Regulates the TMAO/PKC/NF-κB Signaling Pathway in Treating Heart Failure.

Objective: This study aims to explore the mechanism of action of Yixintai in treating chronic ischemic heart failure by combining bioinformatics and experimental validation. Materials and Methods: Five potential drugs for treating heart failure were obtained from Yixintai (YXT) through early mass spectrometry detection. The targets of YXT for treating heart failure were obtained by a search of online databases. Gene ontology (GO) functional enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were conducted on the common targets using the DAVID database. A rat heart failure model was established by ligating the anterior descending branch of the left coronary artery. A small animal color Doppler ultrasound imaging system detected cardiac function indicators. Hematoxylin-eosin (HE), Masson's, and electron microscopy were used to observe the pathological morphology of the myocardium in rats with heart failure. The network pharmacology analysis results were validated by ELISA, qPCR, and Western blotting. Results: A total of 107 effective targets were obtained by combining compound targets and eliminating duplicate values. PPI analysis showed that inflammation-related proteins (TNF and IL1B) were key targets for treating heart failure, and KEGG enrichment suggested that NF-κB signaling pathway was a key pathway for YXT treatment of heart failure. Animal model validation results indicated the following: YXT can significantly reduce the content of intestinal microbiota metabolites such as trimethylamine oxide (TMAO) and improve heart failure by improving the EF and FS values of heart ultrasound in rats and reducing the levels of serum NT-proBNP, ANP, and BNP to improve heart failure. Together, YXT can inhibit cardiac muscle hypertrophy and fibrosis in rats and improve myocardial ultrastructure and serum IL-1ß, IL-6, and TNF-α levels. These effects are achieved by inhibiting the expressions of NF-κB and PKC. Conclusion: YXT regulates the TMAO/PKC/NF-κB signaling pathway in heart failure.

Amyloid Beta Leads to Decreased Acetylcholine Levels and Non-Small Cell Lung Cancer Cell Survival via a Mechanism That Involves p38 Mitogen-Activated Protein Kinase and Protein Kinase C in a p53-Dependent and -Independent Manner.

Several studies have shown an inverse correlation between the likelihood of developing a neurodegenerative disorder and cancer. We previously reported that the levels of amyloid beta (Aß), at the center of Alzheimer's disease pathophysiology, are regulated by acetylcholinesterase (AChE) in non-small cell lung cancer (NSCLC). Here, we examined the effect of Aß or its fragments on the levels of ACh in A549 (p53 wild-type) and H1299 (p53-null) NSCLC cell media. ACh levels were reduced by cell treatment with Aß 1-42, Aß 1-40, Aß 1-28, and Aß 25-35. AChE and p53 activities increased upon A549 cell treatment with Aß, while knockdown of p53 in A549 cells increased ACh levels, decreased AChE activity, and diminished the Aß effects. Aß increased the ratio of phospho/total p38 MAPK and decreased the activity of PKC. Inhibiting p38 MAPK reduced the activity of p53 in A549 cells and increased ACh levels in the media of both cell lines, while opposite effects were found upon inhibiting PKC. ACh decreased the activity of p53 in A549 cells, decreased p38 MAPK activity, increased PKC activity, and diminished the effect of Aß on those activities. Moreover, the negative effect of Aß on cell viability was diminished by cell co-treatment with ACh.

Omics-based pharmacological evaluation reveals Yuanhu Zhitong oral liquid ameliorates arthritis by regulating PKC/ERK/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Successful use of herbal medicine in the treatment of rheumatoid arthritis (RA) creates opportunities for alternative therapies. Yuanhu Zhitong oral liquid (YZOL) is an herbal preparation known for its potent analgesic and anti-inflammatory properties in traditional use. However, the pharmacological mechanism of YZOL for treating RA remains unclear. AIM OF THE STUDY: The aim of this study was to evaluate the efficacy of YZOL in the treatment of RA and to explore its potential mechanisms through omics analysis. MATERIALS AND METHODS: Type II collagen was used to induce an arthritis rat model. The effects of YZOL on paw swelling, inflammatory cytokines, oxidative stress, and histopathological changes were systematically investigated. A pathway-driven transcriptomic analysis was performed to identify key signaling pathways associated with YZOL therapy. The key alterations were validated by qRT-PCR, Western blot, and immunohistochemistry assays. RESULTS: YZOL significantly attenuated arthritis progression, reduced paw swelling rate, and lowered arthritis score in CIA rats. YZOL also inhibited systemic inflammation and associated oxidative stress during RA. Transcriptomic analysis identified 341 genes with significantly altered expression following YZOL treatment. These genes were enriched in inflammation-related pathways, particularly in the NF-κB and MAPK signaling pathways. In addition, we discovered that YZOL can alleviate inflammation in the local synovial tissue. The effect of YZOL was confirmed by the suppression of PKC/ERK/NF-κB p65 signaling at systemic and local levels. CONCLUSIONS: This study provides novel evidence that YZOL treatment ameliorates RA by suppressing the PKC/ERK/NF-κB pathway, suggesting its potential as an alternative therapy for RA.

Inactivity-induced phrenic motor facilitation requires PKCζ activity within phrenic motor neurons.

Prolonged inhibition of respiratory neural activity elicits a long-lasting increase in phrenic nerve amplitude once respiratory neural activity is restored. Such long-lasting facilitation represents a form of respiratory motor plasticity known as inactivity-induced phrenic motor facilitation (iPMF). Although facilitation also occurs in inspiratory intercostal nerve activity after diminished respiratory neural activity (iIMF), it is of shorter duration. Atypical PKC activity in the cervical spinal cord is necessary for iPMF and iIMF, but the site and specific isoform of the relevant atypical PKC are unknown. Here, we used RNA interference to test the hypothesis that the zeta atypical PKC isoform (PKCζ) within phrenic motor neurons is necessary for iPMF but PKCζ within intercostal motor neurons is unnecessary for transient iIMF. Intrapleural injections of siRNAs targeting PKCζ (siPKCζ) to knock down PKCζ mRNA within phrenic and intercostal motor neurons were made in rats. Control rats received a nontargeting siRNA (NTsi) or an active siRNA pool targeting a novel PKC isoform, PKCθ (siPKCθ), which is required for other forms of respiratory motor plasticity. Phrenic nerve burst amplitude and external intercostal (T2) electromyographic (EMG) activity were measured in anesthetized and mechanically ventilated rats exposed to 30 min of respiratory neural inactivity (i.e., neural apnea) created by modest hypocapnia (20 min) or a similar recording duration without neural apnea (time control). Phrenic burst amplitude was increased in rats treated with NTsi (68 ± 10% baseline) and siPKCθ (57 ± 8% baseline) 60 min after neural apnea vs. time control rats (-3 ± 3% baseline), demonstrating iPMF. In contrast, intrapleural siPKCζ virtually abolished iPMF (5 ± 4% baseline). iIMF was transient in all groups exposed to neural apnea; however, intrapleural siPKCζ attenuated iIMF 5 min after neural apnea (50 ± 21% baseline) vs. NTsi (97 ± 22% baseline) and siPKCθ (103 ± 20% baseline). Neural inactivity elevated the phrenic, but not intercostal, responses to hypercapnia, an effect that was blocked by siPKCζ. We conclude that PKCζ within phrenic motor neurons is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for, transient iIMF.NEW & NOTEWORTHY We report important new findings concerning the mechanisms regulating a form of spinal neuroplasticity elicited by prolonged inhibition of respiratory neural activity, inactivity-induced phrenic motor facilitation (iPMF). We demonstrate that the atypical PKC isoform PKCζ within phrenic motor neurons is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for, transient inspiratory intercostal facilitation. Our findings are novel and advance our understanding of mechanisms contributing to phrenic motor plasticity.

Seco-cyclic phorbol derivatives and their anti-HIV-1 activities.

Phorbol esters are recognized for their dual role as anti-HIV-1 agents and as activators of protein kinase C (PKC). The efficacy of phorbol esters in binding with PKC is attributed to the presence of oxygen groups at positions C20, C3/C4, and C9 of phorbol. Concurrently, the lipids located at positions C12/C13 are essential for both the anti-HIV-1 activity and the formation of the PKC-ligand complex. The influence of the cyclopropane ring at positions C13 and C14 in phorbol derivatives on their anti-HIV-1 activity requires further exploration. This research entailed the hydrolysis of phorbol, producing seco-cyclic phorbol derivatives. The anti-HIV-1 efficacy of these derivatives was assessed, and the affinity constant (Kd) for PKC-δ protein of selected seco-cyclic phorbol derivatives was determined through isothermal titration calorimetry. The findings suggest that the chemical modification of cyclopropanols could affect both the anti-HIV-1 activity and the PKC binding affinity. Remarkably, compound S11, with an EC50 of 0.27 µmol·L-1 and a CC50 of 153.92 µmol·L-1, demonstrated a potent inhibitory effect on the intermediate products of HIV-1 reverse transcription (ssDNA and 2LTR), likely acting at the viral entry stage, yet showed no affinity for the PKC-δ protein. These results position compound S11 as a potential candidate for further preclinical investigation and for studies aimed at elucidating the pharmacological mechanism underlying its anti-HIV-1 activity.

Nematicidal Activity of 20-Deoxyingenol-3-angelate from Euphorbia peplus Latex Through Protein Kinase C Isotype TPA-1.

The latex of Euphorbia peplus and its major component 20-deoxyingenol-3-angelate (DI3A) displayed significant nematicidal activity against Caenorhabditis elegans and Panagrellus redivivus. DI3A treatment inhibited the growth and development of nematodes and caused significantly negative effects on locomotion behavior, reproduction, and accumulation of reactive oxygen species. Transcriptome analysis indicated that differential expression genes in DI3A-treated C. elegans were mainly associated with the metabolism, growth, and development process, which were further confirmed by RT-qPCR experiments. The expression level of TPA-1 gene encoding a protein kinase C isotype was obviously upregulated by DI3A treatment, and knockdown of TPA-1 by RNAi technology in the nematode could relieve the growth-inhibitory effect of DI3A. Metabolic analysis indicated that DI3A was hardly metabolized by C. elegans, but a glycosylated indole derivative was specifically accumulated likely due to the activation of detoxification. Overall, our findings suggested that DI3A from E. peplus latex exerted a potent nematicidal effect through the gene TPA-1, which provides a potential target for the control of nematodes and also suggests the potential application value of E. peplus latex and DI3A as botanical nematicides.

A novel bivalent interaction mode underlies a non-catalytic mechanism for Pin1-mediated protein kinase C regulation.

Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms α and ßII via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of full-length Pin1 complexed to the C-terminal tail of PKCßII reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action. Specifically, Pin1 adopts a conformation in which it uses the WW and PPIase domains to engage two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, respectively. Hydrophobic motif is a non-canonical Pin1-interacting element. The structural information combined with the results of extensive binding studies and experiments in cultured cells suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates.

Monocyte to macrophage differentiation and changes in cellular redox homeostasis promote cell type-specific HIV latency reactivation.

HIV latency regulation in monocytes and macrophages can vary according to signals directing differentiation, polarization, and function. To investigate these processes, we generated an HIV latency model in THP-1 monocytes and showed differential levels of HIV reactivation among clonal populations. Monocyte-to-macrophage differentiation of HIV-infected primary human CD14+ and THP-1 cells induced HIV reactivation and showed that virus production increased concomitant with macrophage differentiation. We applied the HIV-infected THP-1 monocyte-to-macrophage (MLat) model to assess the biological mechanisms regulating HIV latency dynamics during monocyte-to-macrophage differentiation. We pinpointed protein kinase C signaling pathway activation and Cyclin T1 upregulation as inherent differentiation mechanisms that regulate HIV latency reactivation. Macrophage polarization regulated latency, revealing proinflammatory M1 macrophages suppressed HIV reactivation while anti-inflammatory M2 macrophages promoted HIV reactivation. Because macrophages rely on reactive-oxygen species (ROS) to exert numerous cellular functions, we disrupted redox pathways and found that inhibitors of the thioredoxin (Trx) system acted as latency-promoting agents in T-cells and monocytes, but opposingly acted as latency-reversing agents in macrophages. We explored this mechanism with Auranofin, a clinical candidate for reducing HIV reservoirs, and demonstrated Trx reductase inhibition led to ROS induced NF-κB activity, which promoted HIV reactivation in macrophages, but not in T-cells and monocytes. Collectively, cell type-specific differences in HIV latency regulation could pose a barrier to HIV eradication strategies.

Apelin-13 reverses bupivacaine-induced cardiotoxicity: an experimental study.

INTRODUCTION: Cardiac arrest or arrhythmia caused by bupivacaine may be refractory to treatment. Apelin has been reported to directly increase the frequency of spontaneous activation and the propagation of action potentials, ultimately promoting cardiac contractility. This study aimed to investigate the effects of apelin-13 in reversing cardiac suppression induced by bupivacaine in rats. METHODS: A rat model of cardiac suppression was established by a 3-min continuous intravenous infusion of bupivacaine at the rate of 5 mg.kg-1.min-1, and serial doses of apelin-13 (50, 150 and 450 µg.kg-1) were administered to rescue cardiac suppression to identify its dose-response relationship. We used F13A, an inhibitor of Angiotensin Receptor-Like 1 (APJ), and Protein Kinase C (PKC) inhibitor chelerythrine to reverse the effects of apelin-13. Moreover, the protein expressions of PKC, Nav1.5, and APJ in ventricular tissues were measured using Western blotting and immunofluorescence assay. RESULTS: Compared to the control rats, the rats subjected to continuous intravenous administration of bupivacaine had impaired hemodynamic stability. Administration of apelin-13, in a dose-dependent manner, significantly improved hemodynamic parameters in rats with bupivacaine-induced cardiac suppression (p < 0.05), and apelin-13 treatment also significantly upregulated the protein expressions of p-PKC and Nav1.5 (p < 0.05), these effects were abrogated by F13A or chelerythrine (p < 0.05). CONCLUSION: Exogenous apelin-13, at least in part, activates the PKC signaling pathway through the apelin/APJ system to improve cardiac function in a rat model of bupivacaine-induced cardiac suppression.

Protein kinase C-inhibition reduces critical weight loss and improves functional outcome after experimental subarachnoid haemorrhage.

OBJECTIVES: Subarachnoid haemorrhage (SAH) carries a high burden of morbidity and mortality. One in three patients develop vasospasm, which is associated with Delayed Cerebral Ischemia. The pathophysiology includes vasoconstrictor receptor upregulation in cerebral arteries. The protein kinase C - inhibitor RO-31-7549 reduces the expression of several vasoconstrictor receptors and normalizes cerebral blood flow in experimental SAH but functional and behavioural effects are unknown. This study was undertaken to analyse functional outcomes up to 14 days after experimental SAH. MATERIALS AND METHODS: 54 male rats were randomised to experimental SAH or sham, using the pre-chiasmatic, single injection model, and subsequent treatment or vehicle. 42 remained for final analysis. The animals were euthanized on day 14 or when reaching a humane endpoint. The primary endpoint was overall survival, defined as either spontaneous mortality or when reaching a predefined humane endpoint. The secondary outcomes were differences in the rotating pole test, weight, open field test, novel object recognition and qPCR of selected inflammatory markers. RESULTS: In the vehicle group 6/15 rats reached the humane endpoint of >20 % weight loss compared to 1/14 in the treatment group. This resulted in a significant reduced risk of early euthanasia due to >20 % weight loss of HR 0.15 (0.03-0.66, p = 0.04). Furthermore, the treatment group did significantly better on the rotating pole test, RR 0.64 (0.47-0.91, p = 0.02). CONCLUSION: RO-31-7549 improved outcomes in terms >20 % weight loss and rotating pole performance after experimental SAH and could be investigated.

A new invertebrate NPY-like polypeptide, ZoaNPY, from the Zoanthus sociatus, as a novel ligand of human NPY Y2 receptor rescues vascular insufficiency via PLC/PKC and Src- FAK-dependent signaling pathways.

Our recent multi-omics studies have revealed rich sources of novel bioactive proteins and polypeptides from marine organisms including cnidarians. In the present study, we initially conducted a transcriptomic analysis to review the composition profile of polypeptides from Zoanthus sociatus. Then, a newly discovered NPY-like polypeptide-ZoaNPY was selected for further in silico structural, binding and virtually pharmacological studies. To evaluate the pro-angiogenic effects of ZoaNPY, we employed an in vitro HUVECs model and an in vivo zebrafish model. Our results indicate that ZoaNPY, at 1-100 pmol, enhances cell survival, migration and tube formation in the endothelial cells. Besides, treatment with ZoaNPY could restore a chemically-induced vascular insufficiency in zebrafish embryos. Western blot results demonstrated the application of ZoaNPY could increase the phosphorylation of proteins related to angiogenesis signaling including PKC, PLC, FAK, Src, Akt, mTOR, MEK, and ERK1/2. Furthermore, through molecular docking and surface plasmon resonance (SPR) verification, ZoaNPY was shown to directly and physically interact with NPY Y2 receptor. In view of this, all evidence showed that the pro-angiogenic effects of ZoaNPY involve the activation of NPY Y2 receptor, thereby activating the Akt/mTOR, PLC/PKC, ERK/MEK and Src- FAK-dependent signaling pathways. Furthermore, in an excision wound model, the treatment with ZoaNPY was shown to accelerate the wound healing process in mice. Our findings provide new insights into the discovery and development of novel pro-angiogenic drugs derived from NPY-like polypeptides in the future.

Phosphorylated CPI-17 and MLC2 as Biomarkers of Coronary Artery Spasm-Induced Sudden Cardiac Death.

Coronary artery spasm (CAS) plays an important role in the pathogeneses of various ischemic heart diseases and has gradually become a common cause of life-threatening arrhythmia. The specific molecular mechanism of CAS has not been fully elucidated, nor are there any specific diagnostic markers for the condition. Therefore, this study aimed to examine the specific molecular mechanism underlying CAS, and screen for potential diagnostic markers. To this end, we successfully constructed a rat CAS model and achieved in vitro culture of a human coronary-artery smooth-muscle cell (hCASMC) contraction model. Possible molecular mechanisms by which protein kinase C (PKC) regulated CAS through the C kinase-potentiated protein phosphatase 1 inhibitor of 17 kDa (CPI-17)/myosin II regulatory light chain (MLC2) pathway were studied in vivo and in vitro to screen for potential molecular markers of CAS. We performed hematoxylin and eosin staining, myocardial zymogram, and transmission electron microscopy to determine myocardial and coronary artery injury in CAS rats. Then, using immunohistochemical staining, immunofluorescence staining, and Western blotting, we further demonstrated a potential molecular mechanism by which PKC regulated CAS via the CPI-17/MLC2 pathway. The results showed that membrane translocation of PKCα occurred in the coronary arteries of CAS rats. CPI-17/MLC2 signaling was observably activated in coronary arteries undergoing CAS. In addition, in vitro treatment of hCASMCs with angiotensin II (Ang II) increased PKCα membrane translocation while consistently activating CPI-17/MLC2 signaling. Conversely, GF-109203X and calphostin C, specific inhibitors of PKC, inactivated CPI-17/MLC2 signaling. We also collected the coronary artery tissues from deceased subjects suspected to have died of CAS and measured their levels of phosphorylated CPI-17 (p-CPI-17) and MLC2 (p-MLC2). Immunohistochemical staining was positive for p-CPI-17 and p-MLC2 in the tissues of these subjects. These findings suggest that PKCα induced CAS through the CPI-17/MLC2 pathway; therefore, p-CPI-17 and p-MLC2 could be used as potential markers for CAS. Our data provide novel evidence that therapeutic strategies against PKC or CPI-17/MLC2 signaling might be promising in the treatment of CAS.

PKC Inhibition Improves Human Penile Vascular Function and the NO/cGMP Pathway in Diabetic Erectile Dysfunction: The Role of NADPH Oxidase.

Erectile dysfunction (ED) is a frequent and difficult-to-treat condition in diabetic men. Protein kinase C (PKC) is involved in diabetes-related vascular and cavernosal alterations. We aimed to evaluate the role of PKC in endothelial dysfunction and NO/cGMP impairment associated with diabetic ED in the human corpus cavernosum (CC) and penile resistance arteries (PRAs) and the potential mechanisms involved. Functional responses were determined in the CC and PRAs in patients with non-diabetic ED and diabetic ED undergoing penile prosthesis insertion. PKC activator 12,13-phorbol-dibutyrate (PDBu) impaired endothelial relaxations and cGMP generation in response to acetylcholine in the CC from non-diabetic ED. PDBu also impaired responses to a PDE5 inhibitor, sildenafil, in non-diabetic ED patients. Conversely, a PKC inhibitor, GF109203X, improved endothelial, neurogenic, and PDE5-inhibitor-induced relaxations and cGMP generation only in the CC in diabetic ED patients. Endothelial and PDE5-inhibitor-induced vasodilations of PRAs were potentiated only in diabetes. Improvements in endothelial function in diabetes were also achieved with a specific inhibitor of the PKCß2 isoform or an NADPH-oxidase inhibitor, apocynin, which prevented PDBu-induced impairment in non-diabetic patients. PKC inhibition counteracted NO/cGMP impairment and endothelial dysfunction in diabetes-related ED, potentially improving response to PDE5 inhibition.

Negative Regulation of Autophagy during Macrophage Infection by Mycobacterium bovis BCG via Protein Kinase C Activation.

Mycobacterium tuberculosis (Mtb) employs various strategies to manipulate the host's cellular machinery, overriding critical molecular mechanisms such as phagosome-lysosome fusion, which are crucial for its destruction. The Protein Kinase C (PKC) signaling pathways play a key role in regulating phagocytosis. Recent research in Interferon-activated macrophages has unveiled that PKC phosphorylates Coronin-1, leading to a shift from phagocytosis to micropinocytosis, ultimately resulting in Mtb destruction. Therefore, this study aims to identify additional PKC targets that may facilitate Mycobacterium bovis (M. bovis) infection in macrophages. Protein extracts were obtained from THP-1 cells, both unstimulated and mycobacterial-stimulated, in the presence or absence of a general PKC inhibitor. We conducted an enrichment of phosphorylated peptides, followed by their identification through mass spectrometry (LC-MS/MS). Our analysis revealed 736 phosphorylated proteins, among which 153 exhibited alterations in their phosphorylation profiles in response to infection in a PKC-dependent manner. Among these 153 proteins, 55 are involved in various cellular processes, including endocytosis, vesicular traffic, autophagy, and programmed cell death. Importantly, our findings suggest that PKC may negatively regulate autophagy by phosphorylating proteins within the mTORC1 pathway (mTOR2/PKC/Raf-1/Tsc2/Raptor/Sequestosome-1) in response to M. bovis BCG infection, thereby promoting macrophage infection.

Histamine H1 Receptor-Mediated JNK Phosphorylation Is Regulated by Gq Protein-Dependent but Arrestin-Independent Pathways.

Arrestins are known to be involved not only in the desensitization and internalization of G protein-coupled receptors but also in the G protein-independent activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), to regulate cell proliferation and inflammation. Our previous study revealed that the histamine H1 receptor-mediated activation of ERK is dually regulated by Gq proteins and arrestins. In this study, we investigated the roles of Gq proteins and arrestins in the H1 receptor-mediated activation of JNK in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H1 receptors, the Gq protein-biased mutant S487TR, and the arrestin-biased mutant S487A. In these mutants, the Ser487 residue in the C-terminus region of the WT was truncated (S487TR) or mutated to alanine (S487A). Histamine significantly stimulated JNK phosphorylation in CHO cells expressing WT and S487TR but not S487A. Histamine-induced JNK phosphorylation in CHO cells expressing WT and S487TR was suppressed by inhibitors against H1 receptors (ketotifen and diphenhydramine), Gq proteins (YM-254890), and protein kinase C (PKC) (GF109203X) as well as an intracellular Ca2+ chelator (BAPTA-AM) but not by inhibitors against G protein-coupled receptor kinases (GRK2/3) (cmpd101), ß-arrestin2 (ß-arrestin2 siRNA), and clathrin (hypertonic sucrose). These results suggest that the H1 receptor-mediated phosphorylation of JNK is regulated by Gq-protein/Ca2+/PKC-dependent but GRK/arrestin/clathrin-independent pathways.

MIR96 Has Good Potential to Differentiate Human Bone Marrow-Derived Mesenchymal Stem Cells into Photoreceptor-Like Cells.

OBJECTIVES: MicroRNAs play an important role in the development and function of neuron cells. Among these, the miRNA known as MIR96 is abundantly expressed in mammalian retina and significantly affects differentiation, maturation, and survival of human photoreceptor cells. In this study, a mimic to miRNA-96 was transfected into human bone marrowderived mesenchymal stem cells to explore the biological functions of MIR96 at differentiation processing. MATERIALS AND METHODS: A mimic to miRNA-96 and a competitive control were transfected into human bone marrow-derived mesenchymal stem cells using Lipofectamine. After 24 and 48 hours, we evaluated changes in expression levels of genes associated with neural progenitor and photoreceptor differentiation (OTX2, NRL, protein kinase C, SLC1A1, and recoverin) by real-time polymerase chain reaction. In addition, we measured expression of mRNA and protein of the CRX gene (neuroretinal progenitor cell marker) and the RHO gene (terminal differentiation marker) using real-time polymerase chain reaction and immunocytochemistry, respectively. RESULTS: Real-time polymerase chain reaction results showed increased levels of RHO and recoverin mRNA after 24 hours in transfected cells. In addition, mRNA levels of OTX2, CRX, NRL, RHO, recoverin, and protein kinase C increased after 48 hours in transfected cells. Immunocytochemistry results confirmed these findings by demonstrating RHO and CRX at both 24 and 48 hours in transfected cells. CONCLUSIONS: Control of the expression of MIR96 can be a good strategy to promote cell differentiation and can be used in cell therapy for retinal degeneration. Our results showed that human bone marrow-derived mesenchymal stem cells can differentiate into photoreceptor cells after transfection with MIR96. These results support therapeutic use of MIR96 in retinal degeneration and suggest human bone marrowderived mesenchymal stem cells as a promising tool for interventions.

Lipid-Binding Regions within PKC-Related Serine/Threonine Protein Kinase N1 (PKN1) Required for Its Regulation.

PKC-related serine/threonine protein kinase N1 (PKN1) is a protease/lipid-activated protein kinase that acts downstream of the RhoA and Rac1 pathways. PKN1 comprises unique regulatory, hinge region, and PKC homologous catalytic domains. The regulatory domain harbors two homologous regions, i.e., HR1 and C2-like. HR1 consists of three heptad repeats (HR1a, HR1b, and HR1c), with PKN1-(HR1a) hosting an amphipathic high-affinity cardiolipin-binding site for phospholipid interactions. Cardiolipin and C18:1 oleic acid are the most potent lipid activators of PKN1. PKN1-(C2) contains a pseudosubstrate sequence overlapping that of C20:4 arachidonic acid. However, the cardiolipin-binding site(s) within PKN1-(C2) and the respective binding properties remain unclear. Herein, we reveal (i) that the primary PKN1-(C2) sequence contains conserved amphipathic cardiolipin-binding motif(s); (ii) that trimeric PKN1-(C2) predominantly adopts a ß-stranded conformation; (iii) that two distinct types of cardiolipin (or phosphatidic acid) binding occur, with the hydrophobic component playing a key role at higher salt levels; (iv) the multiplicity of C18 fatty acid binding to PKN1-(C2); and (v) the relevance of our lipid-binding parameters for PKN1-(C2) in terms of kinetic parameters previously determined for the full-length PKN1 enzyme. Thus, our discoveries create opportunities to design specific mammalian cell inhibitors that disrupt the localization of membrane-associated PKN1 signaling molecules.

Hypothermia Inhibits Dexmedetomidine-Induced Contractions in Isolated Rat Aortae.

Dexmedetomidine is widely used to induce sedation in the perioperative period. This study examined the effect of hypothermia (33 and 25 °C) on dexmedetomidine-induced contraction in an endothelium-intact aorta with or without the nitric oxide synthase inhibitor NW-nitro-L-arginine methyl ester (L-NAME). In addition, the effect of hypothermia on the contraction induced by dexmedetomidine in an endothelium-denuded aorta with or without a calcium-free Krebs solution was examined. The effects of hypothermia on the protein kinase C (PKC), myosin light chain (MLC20) phosphorylation, and Rho-kinase membrane translocation induced by dexmedetomidine were examined. Hypothermia inhibited dexmedetomidine-induced contraction in the endothelium-intact aorta with L-NAME or endothelium-denuded aorta. Hypothermia had almost no effect on the dexmedetomidine-induced contraction in the endothelium-denuded aorta with the calcium-free Krebs solution; however, the subsequent contraction induced by the addition of calcium was inhibited by hypothermia. Conversely, the transition from profound hypothermia back to normothermia reversed the hypothermia-induced inhibition of subsequent calcium-induced contractions. Hypothermia inhibited any contraction induced by KCl, PDBu, and NaF, as well as PKC and MLC20 phosphorylation and Rho-kinase membrane translocation induced by dexmedetomidine. These results suggest that hypothermia inhibits dexmedetomidine-induced contraction, which is mediated mainly by the impediment of calcium influx and partially by the attenuation of pathways involving PKC and Rho-kinase activation.

Binding of the plant-derived toxin simplexin to bovine protein kinase C: insights from molecular dynamics.

Pimelea poisoning of cattle is toxicologically linked to the activation of bovine protein kinase C (PKC) by the plant-derived toxin simplexin. To understand the affinity of PKC for simplexin, we performed molecular dynamics (MD) studies of simplexin, simplexin analogues, and several other activators of PKC. Binding enthalpy calculations indicated that simplexin had the strongest affinity for PKCα-C1B among the activators studied. Key to simplexin's affinity is its ability to form more hydrogen bonds to PKC, compared to the other activators. The C-3 carbonyl group and C-20 hydroxyl group of simplexin were identified as especially important for stabilizing the PKC binding interaction. The hydrophobic alkyl chain of simplexin induces deep membrane embedding of the PKC-simplexin complex, enhancing the protein-ligand hydrogen bonding. Our findings align with previous experiments on structure-activity relationships (SAR) for simplexin analogues, and provide insights that may guide the development of interventions or treatments for Pimelea poisoning.