BK Treatment Research Articles

PLGA/BK microspheres targeting the bradykinin signaling pathway as a therapeutic strategy to delay intervertebral disc degeneration

Intervertebral disc degeneration(IVDD) is a common spinal condition with limited effective treatments available. This study aims to investigate the impact of poly(lactic-co-glycolic acid)/Bradykinin (PLGA/BK) microspheres on IVDD and its underlying mechanisms. We collected nucleus pulposus samples from both healthy and degenerated human intervertebral disks and conducted immunohistochemical analyses, revealing reduced BK expression in degenerated tissues. Subsequently, we used BK to treat nucleus pulposus cells and conducted Bulk RNA sequencing (RNA-seq), identifying BK’s involvement in cellular senescence, extracellular matrix metabolism, and the PI3K signaling pathway. Further experiments using tert-butyl hydroperoxide (TBHP)-induced cell senescence showed that BK treatment reduced senescence, enhanced extracellular matrix synthesis, and inhibited degradation, along with activation of the PI3K pathway. These effects were mediated through B2R (BK receptor 2) and the downstream PI3K pathway. Following this, we developed sustained-release BK microspheres with an optimized manufacturing process. In vitro co-culture experiments showed no observable toxicity. We established an IVDD model in rat tail vertebrae through fine needle puncture, administering local injections of BK sustained-release microspheres. Using various experimental methods, including X-ray, MRI, histopathology, and immunohistochemistry, we found that these microspheres could slow the progression of IVDD. This study highlights the potential of injectable PLGA/BK microspheres to regulate cellular senescence and extracellular matrix metabolism via the B2R and PI3K pathways, ultimately delaying IVDD.

Interleukin-6 Modulates Action Potential Generation in Freshly Isolated Rat Dorsal Root Ganglia Neurons And its Stimulating Effect Is Exacerbated in Polycystic Kidney Disease

Our laboratory has demonstrated a central role for increased afferent nerve renal activity (ARNA) in the progression of cystogenesis in the PCK rat model of polycystic kidney disease (PKD). The underlying cause for the pathological increase in ARNA is unknown. Inflammatory cell infiltration and increased proinflammatory cytokine content, such as interleukin 6 (IL-6), have been reported in PKD. We hypothesized inflammatory cytokines directly increase sensory nerve activity, and this response is exacerbated in PKD. We measured action potential generation (AP) in freshly isolated dorsal root ganglion (DRG) neurons in PCK rats and non-cystic controls, and their response to IL-6. DRGs (T10-L1) were isolated from Sprague-Dawley (SD) or PCK rat (Age: 10-26 weeks) spinal cords, enzymatically digested and mechanically dissociated. Neurons were plated on coverslips, and AP were recorded within 24-hours (whole-cell patch-clamp). We tested the effects of IL-6 (0.04 μg/ml) and bradykinin (BK; 40μg/ml) on AP under 100, 200 pA, and 300 pA current clamp steps. IL-6 elicited either an excitatory response or inhibitory response in AP. In excitatory cells (EC), AP was normalized to BK peak response at +300 pA. In inhibited cells (IC), AP was normalized to baseline at +300 pA. PCK and IL-6 effects were assessed by two-way ANOVA (Bonferroni post hoc test;*p<.05); data as mean ± SEM. Proportion of ECs and ICs did not differ between SD and PCK rats. IL-6 increased AP at 300 pA from 0.48±0.28 (baseline) to 0.95±0.05 (p<.05) in SD ECs cells. BK further increased AP, showing no cell fatigue by IL-6 exposure. In PCK ECs, IL-6 increased AP at 100 pA (0.07±0.05 vs. 0.52±0.25), at 200 pA (0.47±0.27 vs. 1.39±0.81), and 300 pA (0.54±0.25 vs. 1.11±0.47). BK further increased AP at 100 pA to 0.70± 0.28 but decreased AP at 200 pA to 1.21±0.40 and at +300 pA to 1.00±0.00, indicating that neurons activity was likely plateaued by IL-6 at +200 and +300 pA. In both SD and PCK ICs, there was a main effect of IL-6 and BK treatment across all current steps (p<0.05). In SD ICs, IL-6 decreased AP at 200 pA (1.03±0.67 vs. 0.28±0.24) and 300 pA (1.00±0.00 vs. 0.28±0.24). BK had no effect at +200 pA and partially reversed the effect of IL-6 at +300 pA (AP increase to 0.53±0.27). In PCK ICs, IL-6 decreased AP at +200 pA (0.21±0.08 vs. 0.11±0.09) and +300 pA (1.00±0.00 vs. 0.42±0.24). BK increased AP at +200 pA (0.67±0.37) and +300 pA (0.71±0.33). These data partially support our hypothesis that IL-6 increases sensory nerve discharge. This effect was not observed uniformly across cells, as there were inhibitory and excitatory responses in both PCK and SD rats. We conclude that pro-inflammatory cytokine IL-6 can increase a subset of DRG neuron activity and responsiveness, an effect potentiated in PKD. This neuro-immune interaction might contribute to the underlying increase in ARNA and cystogenesis in PKD. Further experimentation is underway to test neuronal sensitivity to IL-6 and other inflammatory cytokines, as well as the posited potentiated effects in PKD. Supported in part by the PKD Foundation Research Award 1022534 (CB); NIDDK/PKD RRC Pilot and Feasibility Award U24DK126110 (CB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BK Channelopathies and KCNMA1-Linked Disease Models.

Novel KCNMA1 variants, encoding the BK K+ channel, are associated with a debilitating dyskinesia and epilepsy syndrome. Neurodevelopmental delay, cognitive disability, and brain and structural malformations are also diagnosed at lower incidence. More than half of affected individuals present with a rare negative episodic motor disorder, paroxysmal nonkinesigenic dyskinesia (PNKD3). The mechanistic relationship of PNKD3 to epilepsy and the broader spectrum of KCNMA1-associated symptomology is unknown. This review summarizes patient-associated KCNMA1 variants within the BK channel structure, functional classifications, genotype-phenotype associations, disease models, and treatment. Patient and transgenic animal data suggest delineation of gain-of-function (GOF) and loss-of-function KCNMA1 neurogenetic disease, validating two heterozygous alleles encoding GOF BK channels (D434G and N999S) as causing seizure and PNKD3. This discovery led to a variant-defined therapeutic approach for PNKD3, providing initial insight into the neurological basis. A comprehensive clinical definition of monogenic KCNMA1-linked disease and the neuronal mechanisms currently remain priorities for continued investigation.

BK Virus Infection and Its Management in Renal Transplantation: An Update

BK virus (BKV) is a common opportunistic pathogen in kidney transplant recipients and one of the most challenging causes of allograft dysfunction and loss. Although over-immunosuppression remains the primary risk factor for BKV infection after transplantation, male gender, older recipient age, prior rejection episodes, degree of human leukocyte antigen mismatching, prolonged cold ischemia time, BK virus serostatus and ureteral stent placement have all been implicated as risk factors. Routine screening post-renal transplant is important to prevent allograft loss in patients with BK viruria or viremia. Reduction of immunosuppression remains the mainstay of BKV nephropathy treatment and is the most studied intervention. In this review, we are going to discuss the epidemiology of BK virus infection, screening strategies, treatment options and new studies or evidence in the future.

The role of kinins in the proliferation of fibroblast primed with TNF in scratch wound assay: Kinins and cell proliferation

The aim of this study was to evaluate the involvement of both B1 and B2 kinins receptors (B1R and B2R) in the fibroblast proliferation induced by the cytokine tumour necrosis factor (TNF) attempting to establish an in vitro model of wound healing. Murine fibroblasts L-929 were cultivated in 24 wells plaque until total confluence (DMEM (Vitrocell®); 5% fetal bovine serum, 5% CO2, 37 °C) and then submitted to the scratch assay. The cells were treated with PBS, TNF (2 ng/mL) and/or mr-TNF antibody (200 μg/mL), or PDTC. The cells received the second set of treatment (3 h later): PBS; 1 μM HOE-140; 1 μM des-Arg9-Leu8-BK (DALBK) or 100 μM PDTC. TNF was able to increase the cell proliferation when compared with the group treated with PBS. The co-treatment with the TNF antibody completely reversed the TNF effect. The TNF-proliferative effect was blocked by B1 (DALBK) and B2 (HOE-140) kinin receptor antagonists administered separately or along, suggesting the involvement of both receptors in the TNF mechanism of action. Furthermore, the treatment with a NF-ĸB inhibitor PDTC completely blocked the cell proliferation. The TNF cell proliferation was incremented with BK (1 μM) treatment, and its effect was totally reversed by HOE-140 treatment. No effect was observed for TNF plus DABK. Eventually, TNF treatment was able to increase TNF level in the growing medium; however, this increase was suppressed by BK treatment. These results suggest that TNF induces cell proliferation and the induced signalling cascade has the B2R participation. All these events seem to be totally dependent on the NF-ĸB activation. These inflammatory mediators can improve the wound healing in the resolution of inflammation.

High-Dose Intravenous Immunoglobulin Treatment of Polyomavirus Nephropathy Developing After T Cell–Mediated Rejection Treatment: A Case Report

BackgroundReactivation of BK polyomavirus causes destructive virus allograft nephropathy; however, treatment options are limited. Herein, we report a case in which a patient with T cell–mediated rejection was treated with steroid therapy. The patient subsequently developed BK viremia and was successfully treated by using intravenous immunoglobulin (IVIG) after failing to respond to conventional treatment. Case PresentationA 54-year-old man had been undergoing peritoneal dialysis for 3 years before kidney transplantation. He had an elevated serum creatinine level (2.26 mg/dL; normal range, 1.2–1.4 mg/dL) and reduced urine output 2 months after transplantation. Suspecting T cell–mediated rejection, steroid pulse therapy (methylprednisolone 250 mg twice daily) was performed for 3 days. Despite treatment, there was a recurrence of increased serum creatinine, and real-time quantitative polymerase chain reaction (serum samples) indicated BK viremia (>5.5 × 105 copies/mL). Results of a kidney biopsy revealed polyomavirus nephropathy (BK virus positive and C4d negative). Thus, the patient's tacrolimus dosage was reduced (from 2.75 mg twice daily to 2 mg once daily), he discontinued mycophenolate mofetil, and he was administered ciprofloxacin and leflunomide. However, the BK viremia showed no improvement, even after 3 months of treatment. Thus, he was administered high-dose IVIG (1 g/kg, 5 times over 5 weeks). The viremia load (blood specimen) decreased to 5197 copies/mL, and the patient's graft function stabilized. His serum creatinine decreased to 2.68 mg/dL. The patient is currently being followed up. ConclusionsOptimal BK treatment methods have not been established, and IVIG treatment remains controversial. However, the present case provides an example of successful treatment using high-dose IVIG.

Effect of electron radiation on vasomotor function of the left anterior descending coronary artery

The left anterior descending (LAD, interventricular) coronary artery provides the blood supply to the mid-region of the heart and is a major site of vessel stenosis. Changes in LAD function can have major effects on heart function. In this report, we examined the effect of electron simulated solar particle event (eSPE) radiation on LAD function in a porcine animal model. Vasodilatory responses to adenosine diphosphate (ADP; 10−9–10−4 M), bradykinin (BK; 10−11–10−6 M), and sodium nitroprusside (SNP; 10−10–10−4 M) were assessed. The LAD arteries from Control (non-irradiated) and the eSPE (irradiated) animals were isolated and exhibited a similar relaxation response following treatment with either ADP or SNP. In contrast, a significantly reduced relaxation response to BK treatment was observed in the eSPE irradiated group, compared to the control group. These data demonstrate that simulated SPE radiation exposure alters LAD function.

BK virus infection: an update on diagnosis and treatment

BK virus, first isolated in 1971, is a significant risk factor for renal transplant dysfunction and allograft loss. Unfortunately, treatment options for BK virus infection are limited, and there is no effective prophylaxis. Although overimmunosuppression remains the primary risk factor for BK infection after transplantation, male gender, older recipient age, prior rejection episodes, degree of human leukocyte antigen mismatching, prolonged cold ischemia time, BK serostatus and ureteral stent placement have all been implicated as risk factors. Routine screening for BK has been shown to be effective in preventing allograft loss in patients with BK viruria or viremia. Reduction of immunosuppression remains the mainstay of BK nephropathy treatment and is the best studied intervention. Laboratory-based methods such as ELISPOT assays have provided new insights into the immune response to BK and may help guide therapy in the future. In this review, we will discuss the epidemiology of BK virus infection, screening strategies, treatment options and future research directions.

Effects of single or combined dietary supplementation of β-glucan and kefir on growth performance, blood characteristics and meat quality in broilers

1. This study was conducted to evaluate the effects of dietary β-glucan and kefir (a fermented milk product) on growth performance, blood profiles, relative organ weight and meat quality in broilers.2. A total of 375 day-of-hatch mixed sex ROSS 308 broilers (BW of 46 ± 0.1 g) were used in a 5-week experiment and randomly allotted to one of the following dietary treatments: (1) NC, basal diet; (2) PC, basal diet + 40 mg/kg of avilamycin; (3) B, NC + 0.1% β-glucan; (4) K, NC + 0.1% kefir; (5) BK, NC + 0.1% β-glucan + 0.1% kefir.3. During weeks 0–3, broilers in B, K and BK treatments had higher body weight gain (BWG) than those in NC treatment. During weeks 4–5, BK treatment had a higher BWG than NC treatment. Overall, broilers given PC, K and BK diets had higher BWG than those given NC diet. The feed efficiency ratio (FCR) was improved by PC treatment.4. Relative liver weight was increased by B treatment, whereas the relative weight of breast meat and gizzard was higher in BK group than that in NC group. Broilers given PC, B and BK diets had greater breast meat redness value and reduced drip loss at d 5 and d 7. The cooking loss was also reduced by B and BK treatments compared with NC treatment.5. In conclusion, the results suggested that inclusion of 0.1% β-glucan and 0.1% kefir, either individually or combined, would improve growth performance and benefit meat quality in broiler chickens.

Low Rates of Short- and Long-term Graft Loss After Kidney-Pancreas Transplant From a Single Center

Since the 1980s, pancreas transplant has become the most effective treatment strategy to restore euglycemia in patients with type 1 diabetes mellitus. However, technical complications and BK virus nephropathy continue to be important causes of early and late graft loss. These and other complications lead to cited 1- and 3-year graft survival rates of 74% and 67% (pancreas) and 81% and 73% (kidney). To examine our center's outcomes with pancreas-kidney transplant and early BK virus screening and treatment. Prospective study from August 2004 to January 2012. University medical center. Sixty-five patients with type 1 diabetes who underwent simultaneous kidney and pancreas, pancreas after kidney, or pancreas transplant alone at a single center. Pancreas transplant. Pancreas and kidney survival; patient survival; and kidney loss due to BK virus nephropathy. Patient survival at 1, 3, and 5 years was 100%, 98.4%, and 98.4%, respectively. Of 2 early pancreatic allograft losses, 1 was due to thrombosis (1.6%). One- and 5-year pancreas graft survival rates were 95.4% and 92.3%; losses after more than 1 year were due to rejection. Kidney survival rates were 100% and 95.2% at 1 and 5 years; losses were due to nephropathy and noncompliance, with 1 death with function. BK virus incidence was 29.2%, with no graft losses due to BK infection. While pancreas transplant can be complicated by early graft loss, our results suggest that excellent outcomes at 5 years can be achieved. Posttransplant BK virus screening and treatment are essential tools to long-term success.

The use of filter media to determine filter cleanliness

It is general believed that a sand filter starts its life with new, perfectly clean media, which becomes gradually clogged with each filtration cycle, eventually getting to a point where either head loss or filtrate quality starts to deteriorate. At this point the backwash cycle is initiated and, through the combined action of air and water, returns the media to its original perfectly clean state. Reality, however, dictates otherwise. Many treatment plants visited a decade or more after commissioning are found to have unacceptably dirty filter sand and backwash systems incapable of returning the filter media to a desired state of cleanliness. In some cases, these problems are common ones encountered in filtration plants but many reasons for media deterioration remain elusive, falling outside of these common problems.The South African conditions of highly eutrophic surface waters at high temperatures, however, exacerbate the problems with dirty filter media. Such conditions often lead to the formation of biofilm in the filter media, which is shown to inhibit the effective backwashing of sand and carbon filters. A systematic investigation into filter media cleanliness was therefore started in 2002, ending in 2005, at the University of Johannesburg (the then Rand Afrikaans University). This involved media from eight South African Water Treatment Plants, varying between sand and sand-anthracite combinations and raw water types from eutrophic through turbid to low-turbidity waters.Five states of cleanliness and four fractions of specific deposit were identified relating to in situ washing, column washing, cylinder inversion and acid-immersion techniques. These were measured and the results compared to acceptable limits for specific deposit, as determined in previous studies, though expressed in kg/m3. These values were used to determine the state of the filters. In order to gain greater insight into the composition of the specific deposits stripped from the media, a four-point characterisation step was introduced for the resultant suspensions based on acid-solubility and volatility.Results showed that a reasonably effective backwash removed a median specific deposit of 0.89kg/m3. Further washing in a laboratory column removed a median specific deposit of 1.34kg/m3. Media subjected to a standardised cylinder inversion procedure removed a median specific deposit of 2.41kg/m3. Immersion in a strong acid removed a median specific deposit of 35.2kg/m3.The four-point characterisation step showed that the soluble-volatile fraction was consistently small in relation to the other fractions. The organic fraction was quite high at the RG treatment plant and the soluble-non-volatile fraction was particularly high at the BK treatment plant.

Late-Onset Hemorrhagic Cystitis in Children After Hematopoietic Stem Cell Transplantation (HSCT) for Thalassemia and Sickle Cell Anemia: A Prospective Evaluation of Polyoma (BK) Virus Infection and Treatment with Cidofovir (CDV).

Abstract 1133Poster Board I-155 BackgroundHemorrhagic cystitis (HC) is a significant cause of morbidity after allogeneic HSCT. BK virus infection has been associated with development of HC after HSCT, however most studies detected the virus at the time of cystitis, therefore not allowing estimation of the relationship between BK reactivation and HC. Furthermore little is known about development of late-onset HC in children following HSCT, its association with BK virus and treatment with Cidofovir. Therefore we prospectively investigated BK virus reactivation in pts receiving HSCT from HLA-identical related donors, risk factors for development of HC and treatment efficacy with CDV. Patients and Methods117 pts with thalassemia (n=107) and sickle cell anemia (n=10) with median age of 9 years (range, 1.7-17) were enrolled in this study. All pts received BUCY ± Thiotepa containing conditioning regimens. GVHD prophylaxis was cyclosporine and short MTX ± Thymoglobulin-ATG (in 26 pts). Before August 2006 qualitative BK-PCR was performed on urine samples in pts with HC. Since then we prospectively performed qualitative and quantitative PCR on blood and urine samples collected before conditioning regimen and weekly thereafter until at least 100 days post transplant in 64 pts. The quantitative BK virus assay was performed with Real Time Alert Q-PCR-Nanogen kit. Risk factors for the development of HC were evaluated on univariate and multivariate analysis using cumulative incidence curves and Competing risk regression analysis respectively. The cumulative incidence of HC was estimated considering death without HC as competing event. Nineteen pts with HC were given CDV at 1.5 mg/kg/day 3 times/week (n= 10) or 5 mg/kg/week (n=9). Results60 out of 64 pts (94%) had at least 1 positive and 52 of them (81%) 2 or more positive samples for BK viruria. 34 pts (53%) showed al least 1 and 18 of them (28%) 2 or more positive samples for BK viremia. The number of viral copies varied from <556 to >55 million copies. Median time to platelet engraftment was 23 days (range,8-163) and median number of platelets at onset of HC was 81×109/l (range, 2-274 ×109/l). Thirty pts (26%) developed clinically significant (grade 2 to 4) HC within 1 year after HSCT at a median of 38 days (range, 13- 114). All pts with HC had BK viruria. Coexisting viral infections were found in 3 pts: CMV in 2 and adenovirus in 1. The severity of HC was grade 2 in 24 pts, grade 3 in 2 and grade 4 in 4. The 4 pts with grade 4 HC had moderate or severe hydronephrosis along with partial ureteral obstruction which necessitated ureteral stent placement. The cumulative incidence of grade 2 or 3-4 HC was 21%(95% CI 13%-28%) and 5%(95%CI 2%-10%) respectively. In univariate analysis the use of ATG, peak BK viruria, GVHD and age >8 years were associated with HC. Multivariate analysis confirmed the prognostic importance of ATG (HR=10.5; p=0.001), peak BK viruria >100,000 copies (HR=6.2; p=0.004), and acute GVHD (HR=5.3; p=0.007), but not age for HC development. The cumulative incidence of HC in pts who had 2 adverse factors was 64%, compared with 31% (or 53%) and 10 % who had either one (GVHD or ATG) or none of these factors (p<0.0001). However, there were 10 pts who had at 2 or more time points BK viruria >6 millions copies without developing HC. With a median follow-up among survivors of 35 months (range, 5-61) HC did not have a significant impact on survival.Both dosing schedules of CDV were well tolerated and no cases of dose-limiting nephrotoxicity were observed. The median duration of HC was 17 days (range 9-53). The median duration of therapy was 27 days (range,21-180) with a median of 9 doses given (range,6-22). All pts had clinical response but only 6 pts (32%) had microbiological response at 2 weeks after therapy. None of these patients cleared BK viruria when a complete clinical response was achieved. The median time from clinical response to BK viruria clearance was 74 days (range, 14-176). Ten pts with grade 2 and one with grade 3 HC occurred before prospective trial of CDV had spontaneous resolution of HC. The median duration of HC in these pts was similar to those treated with CDV. ConclusionBK viruria is common after HSCT and high viral load is a risk factor for HC. However, even higher-level BK replication alone is not sufficient to cause HC. Coexisting factors such as the use of ATG and GVHD significantly contribute to the development of HC. Cidofovir may have some activity against BK virus-related HC, although our data showed that spontaneous resolution of HC could also occur. DisclosureNo relevant conflicts of interest to declare DisclosuresOff Label Use: Cidofovir as antiviral drug for treatment of BK virus-related hemorrhagic cystitis.

Bradykinin selectively modulates the blood–tumor barrier via calcium‐induced calcium release

To investigate the underlying mechanism for the selective modulation of permeability of the blood-tumor barrier by small dose of BK, we established cell lines of rat brain microvascular endothelial cells (BMECs) and astrocytes by primary culture from neonatal rats. BMECs, astrocytes, or C6 glioma cells were treated with BK, and changes of intracellular NO and intracellular calcium level were measured with a fluorescent spectrophotometer. Similarly to the observations in astrocytes, although the initial application of BK easily triggered a ryanodine-mediated calcium-induced calcium release (CICR), we also detected a long-lasting intracellular nitric oxide (NO) elevation in C6 glioma cells upon BK treatment. However, BMECs are not the direct target of BK. Further study showed that ryanodine-mediated CICR contributes greatly to the secondary NO elevation induced by BK treatment. With an in vitro blood-tumor barrier (BTB) model, we demonstrated that NO generated in C6 glioma cells might act as an intercellular messenger and play an important role in the selective modulation of permeability of BMECs by BK. In conclusion, BK triggered CICR in C6 glioma cells, and the associated NO generation might be the underlying mechanism for the selective modulation of BTB permeability by BK.

A129. Bradykinin cardioprotection is mediated by receptor internalization and mitoKATP opening

Bradykinin (Bk) mimics the cardioprotective effects of ischemic preconditioning (IPC) by a complex series of cellular signaling events that include activation of protein kinase G (PKG), opening of mitochondrial ATP-sensitive K+-channels (mitoKATP), and increased production of reactive oxygen species (ROS). In this study, we investigate the hypothesis that the bradykinin B2 receptor is internalized within a signaling platform that mediates the opening of mitoKATP and cardioprotection. We describe a procedure in which hearts are perfused with Bk in a Langendorff apparatus. Following perfusion, mitochondria are isolated and subjected to purification in a Percoll density gradient. This yields a low-density fraction (henceforth termed the light-layer) and a purified mitochondrial fraction. As determined by western blot analysis, the light-layer contains caveolin-1 and -3, the bradykinin B2 receptor, and PKG as well as other proteins. We find that addition of the light-layer to untreated mitochondria opens mitoKATP similar to KATP openers such as diazoxide, as measured by changes in matrix volume. MitoKATP opening by the light-layer does not require the addition of cyclic-GMP but is sensitive to the mitoKATP blocker 5-hydroxydecanoate, the PKCε peptide antagonist εV1–2, the serine/threonine phosphatase PP2A and the PKG-specific blocker KT5823, indicating that PKG was activated by Bk treatment. Inhibition of the open state was not observed upon addition of the Src kinase inhibitor PP2, the tyrosine phosphatase PTP2B, MEK inhibitor I or the ERK inhibitor 3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione, HCl. From this data, we conclude that the light-layer from Bk perfused hearts contains a cardioprotective signaling platform that migrates to mitochondria and opens mitoKATP, resulting in a protected state.

Polyomavirus BK nephropathy in a kidney transplant recipient: Critical issues of diagnosis and management

Diagnosis of polyomavirus BK nephropathy and treatment by low-dose immunosuppression may be optimized by using surrogate markers, such as the detection of viral inclusion bearing cells in the urine and polyomavirus BK DNA in plasma by polymerase chain reaction. These markers were used prospectively in the management of a 44-year-old woman and led to the diagnosis of polyomavirus BK nephropathy at an early stage. The management was complicated by the concurrence of acute allograft rejection. Two treatment steps were initiated: antirejection therapy consisting of methylprednisolone for 3 days followed by lowering of the maintenance immunosuppression. This treatment resulted in a return of the serum creatinine concentration to the baseline of 1.6 mg/L, clearance of polyomavirus BK from plasma, and disappearance of viral inclusion bearing cells from the urine. After 2 months of stable allograft function, a control biopsy confirmed the resolution of polyomavirus BK nephropathy. Histologic signs of acute interstitial rejection were found and preemptively treated by methylprednisolone without altering the baseline regimen. Allograft function remained stable without evidence of recurrent polyomavirus BK nephropathy. This case shows the value of surrogate markers used in a prospective fashion for diagnosis and management of polyomavirus BK nephropathy with concurrent rejection. © 2001 by the National Kidney Foundation, Inc.

Stimulation of receptor-mediated nitric oxide production by vanadate.

Nitric oxide (NO) production by endothelial cells in response to bradykinin (Bk) treatment was markedly and synergistically enhanced by cotreatment with sodium orthovanadate (vanadate), a phosphotyrosine phosphatase inhibitor. This enhancement was blocked by tyrosine kinase inhibitors. Calcium ionophore- (A23187) activated production of NO was also enhanced by cotreatment with vanadate. No significant changes were found in total endothelial NO synthase (eNOS) protein or in eNOS distribution between membrane (caveolae) and cytosolic fractions in response to the various treatments. Vanadate had no direct effect on eNOS activity, and lysates prepared from cells treated with vanadate showed little change in specific activity of eNOS. Western blots of immunoprecipitated eNOS showed the presence of a major tyrosine-phosphorylated protein band at a mass corresponding to approximately 125 kDa and 2 minor bands corresponding to approximately 105 and 75 kDa after treatment with vanadate/Bk. No tyrosine phosphorylation of eNOS after treatment with vanadate/Bk was observed. Geldanamycin, an inhibitor of heat shock protein 90, also inhibited the enhancement of NO production by vanadate/Bk or vanadate/A23187, and there was an increase in the amount of heat shock protein 90 that coimmunoprecipitated with eNOS after treatment with vanadate/Bk. These results show that there is a clear link between tyrosine phosphorylation and stimulation of eNO production, which does not appear to involve direct modification of eNOS, changes in eNOS levels, or compartmentation, but rather appears to be due to changes in proteins associating with eNOS, thereby enhancing the state of activation of eNOS.

Multiple intracellular Ca 2+ pools exist in human foreskin fibroblast cells: the effect of BK on release and filling of the non-cytosolic Ca 2+ pools

Previously, we have used the classical approach to examine intracellular calcium stores in human foreskin fibroblasts (HSWP) cells. In this classical protocol cells are first permeabilized and then allowed to fill their Ca 2+ reservoirs with 45Ca 2+ in the presence of ATP. In this paper we present an alternative method to examine intracellular calcium pools. In this alternate protocol, whole cells are loaded to isotopic steady-state with 45Ca 2+ and then permeabilized using digitonin. Comparison of the Ca 2+ content of cells treated with these two methodologies reveals that cells treated with the alternate protocol have a Ca 2+ content 3 orders of magnitude higher than those treated with the classical protocol. Using this alternative technique we demonstrate that there are 3 intracellular calcium pools in HSWP cells. These pools are: (1) an IP 3-sensitive, thapsigargin-sensitive Ca 2+ pool; (ii) an IP 3-insensitive, thapsigargin-sensitive Ca 2+ pool; and (iii) an ionomycin sensitive Ca 2+ pool. The relationship between the Ca 2+ pool mobilized by BK treatment and by IP 3 treatment is also explored. Microinjection data shown here suggest that IP 3 can mobilize all of the intracellular Ca 2+ mobilized by BK. However, in the permeabilized system BK pretreatment followed by IP 3 treatment can release more Ca 2+ than can be release by IP 3 treatment alone. We suggest one plausible explanation for this observation: when cells are treated using the alternative permeabilization protocol, communication occurs between an IP 3-sensitive and an IP 3-insensitive calcium pool. Thus BK pretreatment would empty the IP 3-sensitive Ca 2+ pool. This pool would subsequently be refilled with Ca 2+ from a previously untapped, IP 3-insensitive, Ca 2+ reservoir and more Ca 2+ would be available for subsequent release by IP3 treatment.

Theoretical study of electron spin resonance parameters: H2CN and H2CO+

The hyperfine structure of the two isoelectronic molecules H2CN and H2CO+ in their electronic ground state (X 2B2) is studied. The influence of the atomic orbital (AO) basis sets, of the correlation treatment, and of the equilibrium geometry on the obtained hyperfine properties is investigated. It is found that the multireference double excitation-configuration interaction (MRD-CI)/BK treatment in which an MRD-CI wave function is corrected by a modified BK method yields equivalent results to quadratic CI [QCISD(T)], coupled cluster single doubles [CCSD(T)], or Brueckner doubles [BD(T)]. Uncertainties in the equilibrium geometries are found to be the major source for discrepancies between theoretically and experimentally determined isotropic hyperfine coupling constants (hfccs). For the heavier centers, the calculated values of the isotropic hfccs agrees nearly perfectly with experimental values (≊1%–2%). The calculated values for the hydrogens are too low, but using the equilibrium structure suggested by Yamamoto and Sato [J. Chem. Phys. 96, 4157 (1992)], the best estimate deviates by less than 3%.