Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for activation of p38alpha and p38delta MAPK isoforms by TGF-beta 1 in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix (ECM) synthesis that leads to renal fibrosis. Intracellular signaling mechanisms involved in this process remain incompletely understood. Mitogen-activated protein kinase (MAPK) is a major stress signal-transducing pathway, and we have previously reported activation of p38 MAPK by TGF-beta1 in rat mesangial cells and its role in the stimulation of pro-alpha1(I) collagen. In this study, we further investigated the mechanism of p38 MAPK activation by TGF-beta1 and the role of MKK3, an upstream MAPK kinase of p38 MAPK, by examining the effect of targeted disruption of the Mkk3 gene. We first isolated glomerular mesangial cells from MKK3-null (Mkk3-/-) and wild-type (Mkk3+/+) control mice. Treatment with TGF-beta1 induced rapid phosphorylation of MKK3 as well as p38 MAPK within 15 min in cultured wild-type (Mkk3+/+) mouse mesangial cells. In contrast, TGF-beta1 failed to induce phosphorylation of either MKK3 or p38 MAPK in MKK3-deficient (Mkk3-/-) mouse mesangial cells, indicating that MKK3 is required for TGF-beta1-induced p38 MAPK activation. TGF-beta1 selectively activated the p38 MAPK isoforms p38alpha and p38delta in wild-type (Mkk3+/+) mesangial cells, but not in MKK3-deficient (Mkk3-/-) mesangial cells. Thus, activation of p38alpha and p38delta is dependent on the activation of upstream MKK3 by TGF-beta1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-beta1-stimulated up-regulation of pro-alpha1(I) collagen expression but not TGF-beta1 induction of fibronectin and PAI-1. These data demonstrate that the MKK3 is a critical component of the TGF-beta1 signaling pathway, and its activation is required for subsequent p38alpha and p38delta MAPK activation and collagen stimulation by TGF-beta1.     

Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway

Trypsin and mast cell tryptase can signal to epithelial cells, myocytes, and nerve fibers of the respiratory tract by cleaving proteinase-activated receptor 2 (PAR2). Since tryptase inhibitors are under development to treat asthma, a precise understanding of the contribution of PAR2 to airway inflammation is required. We examined the role of PAR2 in allergic inflammation of the airway by comparing OVA-sensitized and -challenged mice lacking or overexpressing PAR2. In wild-type mice, immunoreactive PAR2 was detected in airway epithelial cells and myocytes, and intranasal administration of a PAR2 agonist stimulated macrophage infiltration into bronchoalveolar lavage fluid. OVA challenge of immunized wild-type mice stimulated infiltration of leukocytes into bronchoalveolar lavage and induced airway hyperreactivity to inhaled methacholine. Compared with wild-type animals, eosinophil infiltration was inhibited by 73% in mice lacking PAR2 and increased by 88% in mice overexpressing PAR2. Similarly, compared with wild-type animals, airway hyperreactivity to inhaled methacholine (40 micro g/ml) was diminished 38% in mice lacking PAR2 and increased by 52% in mice overexpressing PAR2. PAR2 deletion also reduced IgE levels to OVA sensitization by 4-fold compared with those of wild-type animals. Thus, PAR2 contributes to the development of immunity and to allergic inflammation of the airway. Our results support the proposal that tryptase inhibitors and PAR2 antagonists may be useful therapies for inflammatory airway disease.     

Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference

Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle.     

The apomyoglobin folding pathway revisited: structural heterogeneity in the kinetic burst phase intermediate

Extensive analysis of accurate quench-flow hydrogen exchange results indicates that the burst phase kinetic intermediate in the folding of apomyoglobin (apoMb) from urea is structurally heterogeneous. The structural variability is associated with the partial folding of the E helix during the burst phase (<6.4ms) of the folding process. Analysis of the effects of exchange-out of amide proton labels during the labeling pulse ( approximately pH 10) of the quench-flow process indicates that three of the amide protons in the E helix are in fact largely protected in the burst phase of folding, while the remainder of the E helix has a substantial complement of amide protons that show biphasic kinetics, i.e. are protected partly during the burst phase and partly during the slow phase of folding. The locations of these amide protons can be used to map the sites of structural heterogeneity in the kinetic molten globule. These sites include, besides the E helix, the ends of the A and B helices and part of the C helix. Our results give significant support to the hypothesis that the kinetic molten globule intermediate of apoMb is native-like.     

Adipose Depletion and Apoptosis Induced by Trans-10, Cis-12 Conjugated Linoleic Acid in Mice*

Objective: To compare the effectiveness of a conjugated linoleic acid (CLA) isomer mixture (mCLA) with each main isomer [trans-10,cis-12 CLA (CLA10,12) and cis-9,trans-11 CLA (CLA9,11)] in causing body lipid loss and adipose tissue apoptosis. Research Methods and Procedures: Mice selected over 16 generations for high (MH) or low (ML) energy expenditure and a control group (MC) were fed diets containing either soy oil or soy oil plus mCLA, CLA10,12, or CLA9,11 for 5 days in one study and 14 days in a second study. Results: Mice fed mCLA or CLA10,12 had less body lipid (p < 0.05), smaller retroperitoneal fat pads (p < 0.05), and ate less (p < 0.01) than mice fed no CLA or CLA9,11 for 5 days. Mice consuming 1% mCLA or 0.5% CLA10,12 gained less weight (p < 0.01) and had less body lipid (p < 0.05) and smaller epididymal (p < 0.05) and retroperitoneal fat pads (p < 0.01) than mice consuming either control or 0.5% CLA9,11-containing diets for 14 days. Only mCLA and CLA10,12 increased apoptosis in retroperitoneal fat pads (p < 0.01). The effects of mCLA and CLA10,12 were independent of genetic line except for the effect on adipocyte apoptosis. Mice of the MH line were slightly less sensitive than MC or ML mice to CLA-induced adipose tissue apoptosis. Discussion: CLA10,12, but not CLA9,11, can induce both body fat loss and adipose apoptosis. Although mice of a genotype with less body fat and greater metabolic rate and feed intake appear less sensitive, these CLA effects are robust for mice of varying metabolic background.     

The effects of genetic drift in experimental evolution

Experimental evolution is characterized by exponential or logistic growth and periodic population bottlenecks. The fate of a rare beneficial mutation in these systems is influenced both by the bottleneck effect and by genetic drift. This paper explores the effects of incorporating genetic drift into models of fixation probability in populations with periodic bottlenecks. To model the inherent stochasticity during the growth phase in these populations, we assume a Poisson distribution of offspring. An analytical solution is developed to calculate the fixation probability and a computer simulation is used to verify the results. We find that the fixation rate of a favourable mutant is significantly lower when genetic drift is considered; fixation probability is reduced by over 25% for realistic experimental protocols. Our method is valid for both weak and strong selection; since very large selection coefficients have been reported in the experimental literature, this is an important improvement over previous results.     

Integrated effects of multiple modulators on human liver glycogen phosphorylase a

Hepatic glucose production is increased in people with type 2 diabetes. Glucose released from storage in liver glycogen by phosphorylase accounts for approximately 50% of the glucose produced after an overnight fast. Therefore, understanding how glycogenolysis in the liver is regulated is of great importance. Toward this goal, we have determined the kinetic characteristics of recombinant human liver glycogen phosphorylase a (HLGPa) (active form) and compared them with those of the purified rat enzyme (RLGPa). The Michaelis-Menten constant (K(m)) of HLGPa for P(i), 5 mM, was about fivefold greater than the K(m) of RLGPa. Two P(i) (substrate) concentrations were used (1 and 5 mM) to cover the physiological range for P(i). Other effectors were added at estimated intracellular concentrations. When added individually, AMP stimulated, whereas ADP, ATP and glucose inhibited, activity. These results were similar to those of the RLGPa. However, glucose inhibition was about twofold more potent with the human enzyme. UDP-glucose, glucose 6-phosphate, and fructose 1-phosphate were only minor inhibitors of both enzymes. We reported previously that when all known effectors were present in combination at physiological concentrations, the net effect was no change in RLGPa activity. However, the same combination reduced HLGPa activity, and the inhibition was glucose dependent. We conclude that a combination of the known effectors of phosphorylase a activity, when present at estimated intracellular concentrations, is inhibitory. Of these effectors, only glucose changes greatly in vivo. Thus it may be the major regulator of HLGPa activity.     

Quality improvement report: Safety and efficacy of nurse initiated thrombolysis in patients with acute myocardial infarction

ProblemDelay in starting thrombolytic treatment in patients arriving at hospital with chest pain who are diagnosed as having acute myocardial infarction.DesignAudit of “door to needle times” for patients presenting with chest pain and an electrocardiogram on admission that confirmed acute myocardial infarction. A one year period in each of three phases of development was studied.

Background and setting

The goal of the national service framework for coronary heart disease is that by April 2002, 75% of eligible patients should receive thrombolysis within 30 minutes of arriving at hospital. A district general hospital introduced a strategy to improve door to needle times. In phase 1 (1989-95), patients with suspected acute myocardial infarction, referred by general practitioners, were assessed in the coronary care unit; all other patients were seen first in the accident and emergency department. In phase 2 (1995-7), all patients with suspected acute myocardial infarction were transferred directly to a fast track area within the coronary care unit, where nurses assess patients and doctors started treatment.

Key measures for improvement

Median door to needle time in phase 1 of 45 minutes (range 5-300 minutes), with 38% of patients treated within 30 minutes. Median door to needle time in phase 2 of 40 minutes (range 5-180 minutes), with 47% treated within 30 minutes

Strategies for change

In phase 3 (1997-2001), all patients with suspected acute myocardial infarction were transferred directly to the fast track area and assessed by a “coronary care thrombolysis nurse.” If electrocardiography confirmed the diagnosis of acute myocardial infarction, the nurse could initiate thrombolytic therapy (subject to guidelines and exclusions determined by the consultant cardiologists).

Effects of change

Median door to needle time in phase 3 of 15 minutes (range 5-70 minutes), with 80% of patients treated within 30 minutes. Systematic clinical review showed no cases in which a nurse initiated inappropriate thrombolysis.

Lessons learnt

Thrombolysis started by nurses is safe and effective in patients with acute myocardial infarction. It may provide a way by which the national service framework''s targets for door to needle times can be achieved.     

Precursors and inhibitors of hydrogen sulfide synthesis affect acute hypoxic pulmonary vasoconstriction in the intact lung

The effects of hydrogen sulfide (H(2)S) and acute hypoxia are similar in isolated pulmonary arteries from various species. However, the involvement of H(2)S in hypoxic pulmonary vasoconstriction (HPV) has not been studied in the intact lung. The present study used an intact, isolated, perfused rat lung preparation to examine whether adding compounds essential to H(2)S synthesis or to its inhibition would result in a corresponding increase or decrease in the magnitude of HPV. Western blots performed in lung tissue identified the presence of the H(2)S-synthesizing enzymes, cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST), but not cystathionine β-synthase (CBS). Adding three H(2)S synthesis precursors, cysteine and oxidized or reduced glutathione, to the perfusate significantly increased peak arterial pressure during hypoxia compared with control (P < 0.05). Adding α-ketoglutarate to enhance the 3-MST enzyme pathway also resulted in an increase (P < 0.05). Both aspartate, which inhibits the 3-MST synthesis pathway, and propargylglycine (PPG), which inhibits the CSE pathway, significantly reduced the increases in arterial pressure during hypoxia. Diethylmaleate (DEM), which conjugates sulfhydryls, also reduced the peak hypoxic arterial pressure at concentrations >2 mM. Finally, H(2)S concentrations as measured with a specially designed polarographic electrode decreased markedly in lung tissue homogenate and in small pulmonary arteries when air was added to the hypoxic environment of the measurement chamber. The results of this study provide evidence that the rate of H(2)S synthesis plays a role in the magnitude of acute HPV in the isolated perfused rat lung.