Voluntary activation failure is detectable in some myositis patients with persisting quadriceps femoris weakness: an observational study

This cross-sectional, observational study was undertaken to examine whether voluntary activation failure could contribute to the persisting weakness observed in some patients with treated idiopathic inflammatory myositis. In 20 patients with myositis of more than six months' duration (5 males, 15 females; mean [± 1 SD] age 53 [11] years) and 102 normal subjects (44 males, 58 females; mean age 32 [8] years), isometric maximum voluntary contractions (MVCs) of the dominant quadriceps femoris (QF) were quantified. Absolute MVC results of normal subjects and patients were then normalised with respect to lean body mass (force per units of lean body mass), giving a result in Newtons per kilogram. Based on mass-normalised force data of normal subjects, patients were arbitrarily stratified into "weak" and "not weak" subgroups. During further MVC attempts, the "twitch interpolation" technique was used to assess whether the QF voluntary activation of patients was complete. This technique relies on the fact that, because muscle activation is incomplete during submaximal voluntary contractions, electrical stimulation of the muscle can induce force increments superimposed on the submaximal voluntary force being generated. No between-gender differences were seen in the mass-normalised MVC results of healthy subjects, so the gender-combined results of 6.6 (1.5) N/kg were used for patient stratification. No between-gender difference was found for mass-normalised MVCs in patients: males 5.4 (3.2) and females 3.0 (1.7) N/kg (p > 0.05). Mass-normalised MVCs of male patients were as great as those of normal subjects (p > 0.05), but mass-normalised MVCs of female patients were significantly smaller than those of the normal subjects (p < 0.001). Only one of the six "not weak" patients exhibited interpolated twitches during electrical stimulation, but six of the 14 "weak" patients did, the biggest twitches being seen in the weakest patient. That interpolated twitches can be induced in some myositis patients with ongoing QF weakness during supposed MVCs clearly suggests that voluntary activation failure does contribute to QF weakness in those patients.     

Crystalline structure of poly(hexamethylene adipate). Study on the morphology and the enzymatic degradation of single crystals

The crystalline structure of polyester 6 6 was studied by means of X-ray and electron diffraction and real-space electron microscopy. An orthorhombic unit cell containing eight chain segments with a quasi planar zigzag conformation was derived. The chain axis projection could be defined by a small rectangular cell containing only two molecular segments. Simulation of electron diffraction patterns indicates that molecular segments were arranged with azimuthal angles close to +/-46 degrees . X-ray diffraction patterns suggested that the large dimensions of the unit cell were a consequence of a slight shift between neighboring chains that improved the electrostatic interactions. Chain-folded lamellar crystals were obtained by isothermal crystallization of dilute n-hexanol or n-octanol solutions. The crystalline habit was studied, and the influence of temperature was evaluated. A regular folding surface was observed by using polyethylene decoration techniques. Lamellar crystals were easily degraded with different lipases. A preferential enzymatic attack was, in some cases, observed to occur in the crystal edges, giving rise to highly irregular borders with a fringed texture.     

Presence of interleukin 10 in the serum and blister fluid of patients with pemphigus vulgaris and pemphigoid

Interleukin 10 (IL-10) is an immunoregulatory cytokine produced by T lymphocytes and macrophages. Recently, it has been suggested that IL-10 may be involved in the pathogenesis of various inflammatory and autoimmune diseases. Using an ELISA we investigated the presence of IL-10 in the serum and blister fluid of pemphigus vulgaris (PV) patients with active disease and those in prolonged clinical remission compared with normal controls. Sera from patients with bullous pemphigoid (BP), ocular cicatricial pemphigoid (OCP), oral pemphigoid (OP) and blister fluid from five patients with BP were also studied. Increased levels of IL-10 were detected in the sera of 87.5% of patients with active PV and were statistically significant (P=0.0003) when compared with levels in normal human serum. Lower levels of IL-10 were detected in 12.5% PV patients in remission and were statistically significant (P=0.0001) when compared with levels in patients with active disease. Levels of IL-10 were detected in sera of 4.6% (1 of 24) of the normal controls. The levels of IL-10 were approximately four times higher in blister fluids than levels in the serum in the same PV patients. This difference was highly statistically significant (P=0.0008). A correlation was observed between serum levels of IL-10 and titres of pemphigus autoantibodies and with disease severity. Elevated level of IL-10 was detected in the blister fluid from five BP patients. Levels of IL-10 in the sera of patients with BP, OCP and OP were not significantly increased. These preliminary data suggest that IL-10 in concert with other cytokines may play an important role in the pathogenesis of PV and BP.     

Novel oxazolidinone based compounds as inhibitors of aromatase and the use of the substrate-heme complex approach in the rationalisation of these compounds

The synthesis, biochemical evaluation and molecular modelling of a series of N-alkylated 4-(4(')-aminobenzyl)-2-oxazolidinones is described involving the derivatisation of the starting R- or S-enantiomer of 4-benzyl-2-oxazolidinones. The compounds were tested for human placental aromatase (AR) inhibition in vitro and were found, in general, to be more potent than the standard compound, aminoglutethimide (AG). The inhibitory activity of the compounds was rationalised through the use of the novel substrate-heme complex (SHC) approach and suggests that the S-enantiomer based compounds protrude beyond the C(13), C(17), and C(16) area of the steroid backbone, resulting in steric hindrance with the active site of AR and thus reduced inhibitory activity. The R-enantiomer based compounds do not protrude in the same area and as such are not thought to undergo any steric hindrance and in comparison to the S-enantiomer, possess greater inhibitory activity.     

Synthesis of C-8 alkylamino substituted pyrrolo[2,1-c][1,4]benzodiazepines as potential anti-cancer agents

The design and facile synthesis of C-8 alkylamino substituted pyrrolo[2,1-c][1,4]benzodiazepines is described. These have been prepared by linking the amines at C-8 position with propane spacer to improve solubility in water, and their in vitro cytotoxicity studies have been carried out.     

Cell rounding in cultured human astrocytes and vascular endothelial cells upon inhibition of CK2 is mediated by actomyosin cytoskeleton alterations

Protein kinase CK2 participates in a wide range of cellular events, including the regulation of cellular morphology and migration, and may be an important mediator of angiogenesis. We previously showed that in the retina, CK2 immunolocalizes mostly to vascular endothelium and astrocytes in association with the cytoskeleton. Additionally, CK2 inhibitors significantly reduced retinal neovascularization and stem cell recruitment in the mouse model of oxygen-induced proliferative retinopathy. We have also shown that CK2 and F-actin co-localized in actin stress fibers in microvascular endothelial cells, and that highly specific CK2 inhibitors caused cell rounding in astrocytes and microvascular endothelial cells, which was alleviated by serum that promotes spreading by Rho/Rho-kinase (RhoK) activation of myosin II. Therefore, we examined a possible role of CK2 in the regulation of actin-myosin II-based contractility. Treatment with CK2 inhibitors correlated with disassembly of actomyosin stress fibers and cell shape changes, including cytoplasmic retraction and process formation that were similar to those occurring during astrocyte stellation. Low doses of specific inhibitors of kinases (RhoK and MLCK) that phosphorylate myosin light chain (MLC) enhanced the effect of suboptimal CK2 inhibition on cell shape. Such striking stellation-like alteration was accompanied by decreased level of phospho-MLC, thus implying a CK2 role in regulation of actomyosin cytoskeleton. Our results suggest an important role of CK2 in the control of cell contractility and motility, which may account for suppressing effect of CK2 inhibition on retinal neovascularization. Together, our data implicate protein kinase CK2 for the first time in stellation-like morphological transformation.     

The C-terminal region of E1A: a molecular tool for cellular cartography

The adenovirus E1A proteins function via protein-protein interactions. By making many connections with the cellular protein network, individual modules of this virally encoded hub reprogram numerous aspects of cell function and behavior. Although many of these interactions have been thoroughly studied, those mediated by the C-terminal region of E1A are less well understood. This review focuses on how this region of E1A affects cell cycle progression, apoptosis, senescence, transformation, and conversion of cells to an epithelial state through interactions with CTBP1/2, DYRK1A/B, FOXK1/2, and importin-α. Furthermore, novel potential pathways that the C-terminus of E1A influences through these connections with the cellular interaction network are discussed.     

Defective nitric oxide production impairs angiotensin II-induced Na-K-ATPase regulation in spontaneously hypertensive rats

Angiotensin (ANG) II via ANG II type 1 receptors (AT1R) activates renal sodium transporters including Na-K-ATPase and regulates sodium homeostasis and blood pressure. It is reported that at a high concentration, ANG II either inhibits or fails to stimulate Na-K-ATPase. However, the mechanisms for these phenomena are not clear. Here, we identified the signaling molecules involved in regulation of renal proximal tubular Na-K-ATPase at high ANG II concentrations. Proximal tubules from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were incubated with low concentrations of ANG II (pM), which activated Na-K-ATPase in both the groups; however, the stimulation was more robust in SHR. A high concentration of ANG II (μM) failed to stimulate Na-K-ATPase in WKY rats. However, in SHR ANG II (μM) continued to stimulate Na-K-ATPase, which was sensitive to the AT1R antagonist candesartan. In the presence of N(G)-nitro-l-arginine methyl ester (l-NAME), a nitric oxide (NO) synthase (NOS) inhibitor, ANG II (μM) caused stimulation of Na-K-ATPase in proximal tubules of WKY rats while having no further stimulatory effect in SHR. ANG II (μM), via AT1R, increased proximal tubular NO levels in WKY rats but not in SHR. In SHR, NOS was uncoupled as incubation of proximal tubules with ANG II and l-arginine, a NOS substrate, caused superoxide generation only in SHR and not in WKY rats. The superoxide production in SHR was sensitive to l-NAME. There was exaggerated proximal tubular AT1R-G protein coupling and NAD(P)H oxidase activation in response to ANG II (μM) in proximal tubules of SHR compared with WKY rats. In SHR, inhibition of NADPH oxidase restored NOS coupling and ANG II-induced NO accumulation. In conclusion, at a high concentration ANG II (μM) activates renal NO signaling, which prevents stimulation of Na-K-ATPase in WKY rats. However, in SHR ANG II (μM) overstimulates NADPH oxidase, which impairs the NO system and leads to continued Na-K-ATPase activation.     

YajL, prokaryotic homolog of parkinsonism-associated protein DJ-1, functions as a covalent chaperone for thiol proteome

YajL is the closest Escherichia coli homolog of the Parkinsonism-associated protein DJ-1, a multifunctional oxidative stress response protein whose biochemical function remains unclear. We recently reported the aggregation of proteins in a yajL mutant in an oxidative stress-dependent manner and that YajL exhibits chaperone activity. Here, we show that YajL displays covalent chaperone and weak protein oxidoreductase activities that are dependent on its exposed cysteine 106. It catalyzes reduced RNase oxidation and scrambled RNase isomerization and insulin reduction and forms mixed disulfides with many cellular proteins upon oxidative stress. The formation of mixed disulfides was detected by immunoblotting bacterial extracts with anti-YajL antibodies under nonreducing conditions. Disulfides were purified from bacterial extracts on a YajL affinity column, separated by nonreducing-reducing SDS-PAGE, and identified by mass spectrometry. Covalent YajL substrates included ribosomal proteins, aminoacyl-tRNA synthetases, chaperones, catalases, peroxidases, and other proteins containing cysteines essential for catalysis or FeS cluster binding, such as glyceraldehyde-3-phosphate dehydrogenase, aldehyde dehydrogenase, aconitase, and FeS cluster-containing subunits of respiratory chains. In addition, we show that DJ-1 also forms mixed disulfides with cytoplasmic proteins upon oxidative stress. These results shed light on the oxidative stress-dependent chaperone function of YajL and identify YajL substrates involved in translation, stress protection, protein solubilization, and metabolism. They reveal a crucial role for cysteine 106 and suggest that DJ-1 also functions as a covalent chaperone. These findings are consistent with several defects observed in yajL or DJ-1 mutants, including translational defects, protein aggregation, oxidative stress sensitivity, and metabolic deficiencies.