Myristoylated protein kinase C beta II peptide inhibitor exerts dose-dependent inhibition of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced leukocyte superoxide release

Protein kinase C (PKC) phosphorylation of leukocyte NADPH oxidase is essential to generatesuperoxide (SO) release. Inhibition of leukocyte SO release attenuates inflammation mediated vascular injury. However, the role of PKC isoforms mediating this response has not been fully elucidated. We hypothesize that PKC beta II (βII) isoform positively regulates leukocyte NADPH oxidase, and that a cell-permeable (myr)-PKC βII peptide inhibitor (N-myr-SLNPEWNET) would dose-dependently attenuate fMLP induced leukocyte SO release. fMLP is a leukocyte chemoattractant cell membrane receptor agonist. We isolated leukocytes by peritoneal lavage from male Sprague-Dawley rats using standard methods. fMLP (1 M)-induced leukocyte SO release was measured for 120 sec spectrophotometrically by reduction of ferricytochrome c in the presence/absence of myr-PKC βII peptide inhibitor (0.2 to 20 M) in 5 x 106 leukocytes. After each assay, cell viability was determined by 0.3% trypan blue exclusion. fMLP-induced leukocyte SO release increased peak absorbance to 0.18±0.03 in controls (n=20). This response was dose-dependently inhibited by myr-PKC βII peptide inhibitor at 0.17±0.05 (0.2 M; n=9), 0.14±0.05 (0.5 M; n=11), 0.1±0.05 (1 M; n=9), 0.05±0.03 (5 M; n=9), 0.04±0.03 (10 M; n=8) and 0.05±0.03 (20 µM; n=7) and was significantly attenuated in the 5 to 20 M range compared to controls (p\u3c0.05). Moreover, cell viability was \u3e 94±1% in all study groups. These results suggest that myr-PKC βII peptide inhibitor dose-dependently inhibits fMLP-induced leukocyte SO release in the 0.2 to 5 M dose-range and these effects are attributed to inhibition of PKC βII isoform

The Effects of Protein Kinase C Beta II Peptide Modulation on Superoxide Release in Rat Polymorphonuclear Leukocytes

Phorbol 12-myristate 13-acetate (PMA; a diacylglycerol mimetic) is known to augment polymorphonuclear leukocyte (PMN) superoxide (SO) release via protein kinase C (PKC) activation. However, the role of PKC beta II (βII) mediating this response is not known. It’s known that myristic acid (myr-) conjugation facilitates intracellular delivery of the cargo sequence, and that putative PKCβII activator and inhibitor peptides work by augmenting or attenuating PKCβII translocation to cell membrane substrates (e.g. NOX-2). Therefore, we hypothesize that myr- conjugated PKCβII peptide-activator (N-myr-SVEIWD; myr-PKCβ+) would increase PMA-induced rat PMN SO release, whereas, myr-PKCβII peptide-inhibitor (N-myr-SLNPEWNET; myr-PKCβ-) would attenuate this response compared to non-drug treated controls. Rat PMNs (5x106) were incubated for 15min at 370C in the presence/absence of myr-PKCβ+/- (20 μM) or SO dismutase (SOD;10μg/mL; n=8) as positive control. PMA (100nM) induced PMN SO release was measured spectrophotometrically at 550nm via reduction of ferricytochrome c for 390 sec. PMN SO release increased absorbance to 0.39±0.04 in non-drug treated controls (n=28), and 0.49±0.05 in myr-PKCβ+(n=16). This response was significantly increased from 180 seconds to 240 seconds (p\u3c0.05). By contrast, myr-PKCβ- (0.26±0.03; n=14) significantly attenuated PMA-induced SO release compared to non-drug controls and myr-PKCβ+ (p\u3c0.05). SOD-treated samples showed \u3e90% reduction of PMA-induced SO release and was significantly different from all groups (p\u3c0.01). Cell viability ranged between 94± to 98±2% in all groups as determined by 0.2% trypan blue exclusion. Preliminary results suggest that myr-PKCβ- significantly attenuates PMA-induced SO release, whereas myr-PKCβ+ significantly augments PMA-induced SO release, albeit transiently. Additional dose response and western blot experiments are planned with myr-PKCβ+/- in PMA-induced PMN SO release assays. This research was supported by the Department of Bio-Medical Sciences and the Division of Research at PCOM and by Young Therapeutics, LLC

Protein Kinase C Beta II Peptide Inhibitor Elicits Robust Effects on Attenuating Myocardial Ischemia/Reperfusion Injury

Reperfusion injury contributes to myocardial tissue damage following a heart attack partly due to the generation of reactive oxygen species (ROS) upon cardio-angioplasty. Protein kinase C beta II (PKCβII) inhibition during reperfusion with peptide inhibitor (N-myr-SLNPEWNET; PKCβII-) decreases ROS release and leukocyte infiltration in rat hind-limb and myocardial ischemia/reperfusion (I/R) studies, respectively. However, the role of activating PKCβII during reperfusion has not been previously determined. In this study, we hypothesize that myristoylated (myr)-PKCβII- will decrease infarct size and improve post-reperfused cardiac function compared to untreated controls, whereas PKCβII peptide activator (N-myr-SVEIWD; myr-PKCβII+) will show no improvement compared to control. Myristoylation of PKCβII peptides facilitate their entry into the cell in order to affect PKCβII activity by either augmenting or attenuating its translocation to cell membrane proteins, such as NOX-2. Isolated perfused rat hearts were subjected to global I(30min)/R(50min) and infused with myr-PKCβII+ (20μM; n=9), myr-PKCβII- (20µM; n=8), or plasma (control; n=9) at reperfusion. Hearts were frozen (-20oC), sectioned and stained using 1% triphenyltetrazolium chloride to differentiate necrotic tissue. The measurement of Left ventricular (LV) cardiac function was determined using a pressure transducer and infarct size was calculated as percent dead tissue vs. total heart tissue weight. Myr-PKCβII- significantly improved LV end-diastolic pressure 37±7 mmHg compared to control (58±5; p\u3c0.01) and myr-PKCβII+ (58±4; p\u3c0.01). Myr-PKCβII- significantly reduced infarct size to 14±3% compared to control (26±5%; p\u3c0.01), while myr-PKCβII+ (25±3%) showed no difference. The data indicate that myr-PKCβII- may be a putative treatment to reduce myocardial reperfusion injury when given to heart attack patients during cardio-angioplasty. Future studies are planned to determine infarct size by Image J analysis

Is There a Correlation Between Streptococcus Gallolyticus Bacteremia and Infectious Colitis?

Introduction: Streptococcus gallolyticus are catalase-negative gram-positive cocci; which typically presents as bacteremia in adults with infective endocarditis or colonic neoplasms. Among hospitalized patients, the approximately 5% streptococcal bloodstream isolates originate from Streptococcus gallolyticus. Currently, there are no documented cases of infectious colitis causing Streptococcus gallolyticus bacteremia. This case presentation outlines these possible Streptococcus gallolyticus colitis. Case description/methods: Patient is a 55-year-old with known history of cocaine abuse; who was admitted after being found unresponsive by his fiancée; and subsequently requiring intubation for ventilator-dependent respiratory failure. Patient was incidentally found to have Streptococcus gallolyticus bacteremia. Patient also had episodes of hematochezia likely secondary to colitis. Patient underwent colonoscopy; which showed mucus discontinuous ulceration and erythema without bleeding and ascending colon, transverse colon, and sigmoid colon. Pathology suggested that colitis was secondary to infectious etiology rather than ischemic. Transesophageal echocardiogram did not show valvular vegetations. Patient was subsequently started on Rocephin; and subsequently transitioned to Cefdinir complete 2 weeks of antibiotic therapy under Infectious Disease guidance. Discussion: This patient presented with colitis likely secondary to infectious source (Streptococcus gallolyticus). Unfortunately, stool cultures were not obtained. This would be required to determine if infectious colitis can indeed cause Streptococcus gallolyticus bacteremia. Pathology showed signs of infectious etiology. Other common etiologies for Streptococcus gallolyticus were negative (colonoscopy did not reveal colonic mass and echocardiogram is a not revealed vegetations). The antibiotic of choice is Rocephin for treatment Streptococcus gallolyticus

Protein kinase C beta II (PKC ßII) peptide inhibitor exerts cardioprotective effects in myocardial ischemia/reperfusion injury

During myocardial ischemia/reperfusion (I/R), the generation of reactive oxygen species (ROS) contributes to the post-reperfused cardiac injury and contractile dysfunction. Activation of Protein Kinase C beta II (PKC βII) has been associated with increased ROS release from myocardial I/R tissue, decreased endothelial-derived nitric oxide, and increased infarct size. We tested the hypothesis that using a cell permeable PKC βII peptide inhibitor (PKC βII-) (N-myr-SLNPEWNET, MW=1300 g/mol, 10µM) will attenuate infarct size and improve post-reperfused cardiac function compared to untreated controls in isolated perfused rat hearts subjected to I(30min)/R(45 or 90 min). The 90 min reperfusion group (n=9) showed significantly less recovery to the initial baselines in left ventricular developed pressure (LVDP) (38±6%) and maximal rate of LVDP (+dP/dtmax ) (28±4%), both p˂0.01. The 45 min reperfusion group (n=9) also showed significantly compromised LVDP (46±6%) and +dP/dtmax (35±4%) compared to initial baseline but to a lesser extent than the 90 min group. Conversely, PKC βII- treated hearts significantlyimproved cardiac function compared to controls (all p\u3c0.05). Similarly, 90 min reperfusion (n=7) showed a reduced recovery in LVDP (57±7%) and +dP/dtmax (48±5%) compared to 45 min reperfusion (LVDP: 70±6%; +dP/dtmax: 55±6%; n=7). Furthermore, PKC βII- treated hearts showed significant reduction in infarct size (24±3% and 29±3% for 45 and 90 min reperfusion, respectively) compared to controls (43±2% and 46±3% for 45 and 90 min reperfusion, respectively; [p˂0.01]). The results suggest that PKC βII- is effective in improving cardiac function and reducing infarct size and aids in clinical myocardial infarction/organ transplantation patient recovery

Protein Kinase C Beta II (PKC ßII) Peptide Inhibitor Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion Injury

Coronary heart disease is the leading cause of death worldwide, and is primarily attributable to the detrimental effects of tissue infarct after an ischemic insult, The most effective therapeutic intervention for reducing infarct size associated with myocardial ischemia injury is timely and effective reperfusion of blood flow back to the ischemic heart tissue. However, the reperfusion of blood itself can induce additional cardiomyocyte death that can account for up to 50% of the final infarction size. Currently, there are no effective clinical pharmacologic treatments to limit myocardial ischemia/reperfusion (MI/R) injury in heart attack patients. Reperfusion injury is initiated by decreased endothelial derived nitric oxide (NO) which occurs within 5 min of reperfusion, and may in part be explained by PKC ßII mediated activation of NADPH oxidase, which occurs upon cytokine release during MI/. PKC ßII activity is increased in animal models of MI/R and known to exacerbate tissue injury. PKC ßII is known to increase NADPH oxidase activity in leukocytes, endothelial cells and cardiac myocytes via phox47 phosphorylation, and decrease endothelial NO synthase (eNOS) activity via phosphorylation of Thr 495. NADPH oxidase produces superoxide (SO) and quenches endothelial derived NO in cardiac endothelial cells. Moreover, PKC ßII phosphorylation of p66Shc at Ser 36 leads to increased mitochondrial reactive active oxygen species (ROS) production, opening of the mitochondrial permeability transition pore (MPTP), and pro-apoptotic factors leading to cell death and increased infarct size. Therefore, using a pharmacologic agent that inhibits the rapid release of PKC ßII mediated ROS, would attenuate endothelial dysfunction and downstream pro apoptotic pathways when given during reperfusion and should be an ideal candidate to attenuate MI/R injury. PKC ßII peptide inhibitor mechanism of action is to inhibit PKC ßII translocation to cellular substrates such as eNOS, NADPH oxidase, and mitochondrial p66Shc protein that increase ROS leading to opening of the MPTP which in turn leads to consequent release of proapoptotic factors into the cytosol. We\u27ve previously shown that PKC ßII peptide inhibitor restored post-reperfused cardiac function and reduced polymorphornuclear leukocyte (PMN) infiltration in isolated rat hearts subjected to MI(20min)/R(45min) reperfused with PMNs. In addition, the use of PKC ßII peptide inhibitor (10-20 µM) correlated with the inhibition of SO release from isolated leukocytes suggesting that this dose range maybe effective in attenuating ROS production. We extended our research in the current study by using a MI (30min)/R (90min) isolated perfused rat heart model. A cell permeable PKC ßII peptide inhibitor (10-20 µM) was given at the beginning of reperfusion for five minutes. Post-reperfused cardiac function and infarct size were measured and compared to untreated control MI/R hearts