Central role of protein kinase Cepsilon in constitutive activation of ERK1/2 and Rac1 in the malignant cells of hairy cell leukemia

We have previously identified the presence of Ras/Raf-independent constitutive activation of extracellular signal-regulated kinase (ERK) in the hairy cells (HCs) of hairy cell leukemia. The aim of the present study was to characterize the signaling components involved in this activation and their relationship to the reported activation of Rac1. We found that both Rac1 and ERK activation in HCs are downstream of active Src and protein kinase C (PKC). Inhibition with toxin B showed that Rac1 plays no role in ERK activation in HCs. However, toxin B inhibited p60src and the Rac1-GEF Vav, demonstrating a positive feedback/activation of p60src by Rac1. Treatment with specific small interfering RNA for various PKC isoforms, or with PKC isoform-specific inhibitors, demonstrated a central role for PKCepsilon in the constitutive activation of Rac1 and ERK in HCs. PKCepsilon and active ERK were mutually associated and co-localized with mitochondria in HCs. Furthermore, active PKCepsilon was nitrated on tyrosine, pointing to a reactive oxygen species-dependent mechanism of activation. By being involved in activation of ERK and Rac1, PKCepsilon plays roles in both the survival of HCs and in the cytoskeletal dynamics responsible for the distinctive morphology and tissue homing of these cells. Our study therefore describes novel aspects of signaling important for the pathogenesis of hairy cell leukemia.     

Effect of prolonged unweighting of human skeletal muscle on neuromotor force control

The purpose of this study was to determine the effect of 4 weeks of unilateral lower limb suspension (ULLS) on the fluctuations in motor output and the associated physiological changes. Subjects (n = 17) performed steady isometric plantarflexion (PF) and knee extension (KE) tasks, and KE shortening and lengthening contractions (intensity = 25% maximum). Spinal excitability of the soleus muscle was assessed via the H-reflex, muscle cross-sectional area (CSA) via MRI, along with EMG activity during the PF tasks. Following ULLS, isometric force fluctuations increased ∼12% for the PF, and 22% for the KE (P < 0.05), with no difference in the pattern of PF muscle activation (P = 0.46). The unsteadiness of lengthening KE contractions increased 25% following ULLS (P = 0.03), while KE steadiness during shortening contractions was not altered (P = 0.98). Significant correlations were observed between the percent changes in PF isometric force fluctuations and H-reflex (r = 0.49, P = 0.04), and between the PF isometric force fluctuations and PF CSA (r = −0.61, P < 0.01). These findings suggest the effects of unweighting on neuromotor performance are muscle group and contraction type dependent, and that the disuse-paradigm altering muscle CSA and spinal excitability may serve to mediate the associated loss of steadiness. Data for this project were collected in the Musculoskeletal Research Laboratory at Syracuse University.     

Clostridium difficile toxins A and B directly stimulate human mast cells

Clostridium difficile toxins A and B (TcdA and TcdB) are the causative agents of antibiotic-associated pseudomembranous colitis. Mucosal mast cells play a crucial role in the inflammatory processes underlying this disease. We studied the direct effects of TcdA and TcdB on the human mast cell line HMC-1 with respect to degranulation, cytokine release, and the activation of proinflammatory signal pathways. TcdA and TcdB inactivate Rho GTPases, the master regulators of the actin cytoskeleton. The inactivation of Rho GTPases induced a reorganization of the actin cytoskeleton accompanied by morphological changes of cells. The TcdB-induced reorganization of the actin cytoskeleton in HMC-1 cells reduced the number of electron-dense mast cell-specific granules. Accordingly, TcdB induced the release of hexosaminidase, a marker for degranulation, in HMC-1 cells. The actin rearrangement was found to be responsible for degranulation since latrunculin B induced a comparable hexosaminidase release. In addition, TcdB as well as latrunculin B induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 and also resulted in a p38 MAPK-dependent increased formation of prostaglandins D(2) and E(2). The autocrine stimulation of HMC-1 cells by prostaglandins partially contributed to the degranulation. Interestingly, TcdB-treated HMC-1 cells, but not latrunculin B-treated HMC-1 cells, showed a strong p38 MAPK-dependent increase in interleukin-8 release. Differences in the mast cell responses to TcdB and latrunculin B are probably due to the presence of functionally inactive Rho GTPases in toxin-treated cells. Thus, the HMC-1 cell line is a promising model for studying the direct effects of C. difficile toxins on mast cells independently of the tissue context.     

Serum endocan as a survival predictor for patients with liver cirrhosis

BACKGROUND:

The relationship between endocan expression and outcome in patients with chronic liver disease is not fully understood.

OBJECTIVE:

To examine whether serum endocan level is predictive of outcome in patients with liver cirrhosis.

METHODS:

A total of 68 patients with liver cirrhosis were enrolled. Outcome predictors were analyzed using the Cox proportional hazards model. The overall survival rates were calculated using the Kaplan-Meier method, and differences were evaluated using the log-rank test.

RESULTS:

During the median follow-up period (7.1 years), nine patients had hepatocellular carcinoma (HCC) and 10 patients died. Of the deceased patients, nine died due to hepatic decompensation or associated conditions. No significant factors were found to be predictive of the occurrence of HCC. In contrast, an elevated serum endocan level (≥2.0 ng/mL; HR 2.34 [95% CI 1.05 to 7.03]; P=0.037) and high Child-Pugh grade B/C (HR 2.65 [95% CI 1.30 to 6.89; P=0.006) were predictive of poor survival. Kaplan-Meier analysis revealed that the respective cumulative survival rates at five and 10 years were 97.1% and 87.4% in patients with serum endocan levels <2.0 ng/mL and 85.8% and 64.4% in patients with levels ≥2.0 ng/mL (P=0.009), respectively. Moreover, the cumulative survival rates were significantly different among the patient groups divided according to serum endocan level and Child-Pugh grade (P=0.002).

CONCLUSION:

These findings suggest that serum endocan level may be a survival predictor for patients with liver cirrhosis.     

Analysis of a surface protein of Streptococcus pneumoniae recognised by protective monoclonal antibodies

Using two monoclonal antibodies which protect mice from a fatal challenge with S. pneumoniae, we have identified a surface protein antigen on the pneumococcus. These antibodies recognised components of 84 and 76 kD in a cell wall extract of the nonencapsulated strain, R36A, against which they were made. Absorption experiments indicated that both of the antibodies recognised the same two proteins. The proteins detected by the antibodies in the encapsulated type 2 strain D39 and type 3 strain WU2, exhibited different molecular weights than those proteins detected from R36A. Using a colony blot procedure and a quantitative ELISA, we have shown that these antibodies react with 6 of the 21 pneumococcal strains tested. There was no association between reactivity with these anti-protein antibodies and the capsular serotype of the pneumococcal isolates tested.     

Visna DNA synthesis and the tempo of infection in vitro

We have examined the effect of reovirus infection on the CV-1 cytoskeleton. Reovirus infection produces a major disruption of vimentin filaments without producing a discernable disorganization of microtubules or microfilament bundles in CV-1 cells. In addition to disrupting the organization of vimentin filaments, reovirus infection appears to cause a reorganization of vimentin filaments. Viral inclusions contain vimentin filamentous structures. Viral infection also alters the cytoplasmic distribution of mitochondria, consistent with the proposed role of vimentin filaments in determining the distribution of mitochondria.     

Home Versus Clinic Blood Pressure Measurements

To evaluate the cost effectiveness of home versus clinic blood pressure 11 patients with mild untreated hypertension were instructed in self-measurement of blood pressure with mercury manometers. The lower total cost of using home readings as well as problems of bias and blinding are discussed. Home blood pressure is a promising technique for clinical and epidemiologic research.