Structural bases of the redox-dependent conformational switch in the serpin PAI-2

Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open beta-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the beta-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54A to the bottom of PAI-2 so that the C79-C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.     

Alterations in myocardial energy metabolism induced by the anti-cancer drug doxorubicin

Doxorubicin and other anthracyclines are among the most potent chemotherapeutic drugs for the treatment of acute leukaemia, lymphomas and different types of solid tumours such as breast, liver and lung cancers. Their clinical use is, however, limited by the risk of severe cardiotoxicity, which can lead to irreversible congestive heart failure. There is increasing evidence that essential components of myocardial energy metabolism are among the highly sensitive and early targets of doxorubicin-induced damage. Here we review doxorubicin-induced detrimental changes in cardiac energetics, with an emphasis on the emerging importance of defects in energy-transferring and -signalling systems, like creatine kinase and AMP-activated protein kinase.     

Protein kinase C mediates erythrocyte "programmed cell death" following glucose depletion

Glucose depletion of erythrocytes leads to activation of Ca²⁺-permeable cation channels, Ca²⁺ entry, activation of a Ca²⁺-sensitive erythrocyte scramblase, and subsequent exposure of phosphatidylserine at the erythrocyte surface. Ca²⁺ entry into erythrocytes was previously shown to be stimulated by phorbol esters and to be inhibited by staurosporine and chelerythrine and is thus thought to be regulated by protein phosphorylation/dephosphorylation, presumably via protein kinase C (PKC) and the corresponding phosphoserine/threonine phosphatases. The present experiments explored whether PKC could contribute to effects of energy depletion on erythrocyte phosphatidylserine exposure and cell volume. Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorter analysis. Removal of extracellular glucose led to depletion of cellular ATP, stimulated PKC activity, led to translocation of PKC, enhanced serine phosphorylation of membrane proteins, decreased cell volume, and increased annexin binding, the latter effect being blunted but not abolished in the presence of 1 µM staurosporine or 50 nM calphostin C. The PKC stimulator phorbol-12-myristate-13-acetate (3 µM) and the phosphatase inhibitor okadaic acid (1–10 µM) mimicked the effect of glucose depletion and similarly led to translocation of PKC and enhanced serine phosphorylation, increased annexin binding, and decreased forward scatter, the latter effects being abrogated by PKC inhibitor staurosporine (1 µM). Fluo-3 fluorescence measurements revealed that okadaic acid also enhanced erythrocyte Ca²⁺ activity. The present observations suggest that protein phosphorylation and dephosphorylation via PKC and the corresponding protein phosphatases contribute to phosphatidylserine exposure and cell shrinkage after energy depletion. cell volume; eryptosis; calcium; okadaic acid; staurosporine     

Genetic disregulation of TNF alpha and TNF alpha type II receptors in colon cancer at the II and III stage of disease

The expression of the genes coding TNFalpha and TNF RII receptors (TNF RII: TNFR2 membrane and soluble domain, TNFR2/R7 soluble domain) was analysed in colon cancer at the II and III stage of disease, by estimation of mRNA expression. The study included 80 patients with histopathologically confirmed adenocarcinoma. The number of TNFalpha mRNA, TNFR2 mRNA and TNFR2/R7 mRNA copies were estimated in tumour and healthy tissue. The highest number of mRNA TNF-alpha copies were investigated in all samples of tissue and independently of the stage of disease. Simultaneously, we noticed the largest number of mRNA copies for TNFalpha and TNF R2/R7 in healthy cells at stage III of the disease. It is possible to draw a hypothetical line separating the anti-cancer activity of TNFalpha and its influence on cancer progression.     

The Effect of a Spinal Cord Hemisection on Changes in Nitric Oxide Synthase Pools in the Site of Injury and in Regions Located Far Away from the Injured Site

1. The present study was designed to examine the nitric oxide synthase activities (constitutive and inducible) in the site of injury in response to Th10-Th11 spinal cord hemisection and, to determine whether unilateral disconnection of the spinal cord influences the NOS pools on the contra- and ipsilateral sides in segments located far away from the epicentre of injury.2. A radioassay detection was used to determine Ca²⁺-dependent and inducible nitric oxide synthase activities. Somal, axonal and neuropil neuronal nitric oxide synthase was assessed by immunocytochemical study. A quantitative assessment of neuronal nitric oxide synthase immunoreactivity was made by an image analyser. The level of neuronal nitric oxide synthase protein was measured by the Western blot analysis.3. Our data show the increase of inducible nitric oxide synthase activity and a decrease of Ca²⁺-dependent nitric oxide synthase activity in the injured site analysed 1 and 7 days after surgery. In segments remote from the epicentre of injury the inducible nitric oxide synthase activity was increased at both time points. Ca²⁺-dependent nitric oxide synthase activity had decreased in L5-S1 segments in a group of animals surviving for 7 days. A hemisection performed at thoracic level did not cause significant difference in the nitric oxide synthase activities and in the level of neuronal nitric oxide synthase protein between the contra- and ipsilateral sides in C6-Th1 and L5-S1 segments taken as a whole. Significant differences were observed, but only when the spinal cord was analysed segment by segment, and/or was divided into dorsal and ventral parts. The cell counts in the cervicothoracic (C7-Th1) and lumbosacral (L5-S1) enlargements revealed changes in neuronal nitric oxide synthase immunoreactivity on the ipsilateral side of the injury. The densitometric area measurements confirmed the reduction of somal, neuropil and axonal neuronal nitric oxide synthase immunoreactive staining in the ventral part of rostrally oriented segments.4. Our findings provide evidence that the changes in nitric oxide synthase pools are limited not only to impact zone, but spread outside the original lesion. The regional distribution of nitric oxide synthase activity and neuronal nitric oxide synthase immunoreactivity, measured segment by segment shows that nitric oxide may play a significant role in the stepping cycle in the quadrupeds.     

Purification and preconcentration of genomic DNA from whole cell lysates using photoactivated polycarbonate (PPC) microfluidic chips

We discuss the use of a photoactivated polycarbonate (PPC) microfluidic chip for the solid-phase, reversible immobilization (SPRI) and purification of genomic DNA (gDNA) from whole cell lysates. The surface of polycarbonate was activated by UV radiation resulting in a photo-oxidation reaction, which produced a channel surface containing carboxylate groups. The gDNA was selectively captured on this photoactivated surface in an immobilization buffer, which consisted of 3% polyethylene glycol, 0.4 M NaCl and 70% ethanol. The methodology reported herein is similar to conventional SPRI in that surface-confined carboxylate groups are used for the selective immobilization of DNA; however, no magnetic beads or a magnetic field are required. As observed by UV spectroscopy, a load of ~7.6 ± 1.6 µg/ml of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be 85 ± 5%. The immobilization and purification assay using this PPC microchip could be performed within ~25 min as follows: (i) DNA immobilization ~6 min, (ii) chip washout with ethanol 10 min, and (iii) drying and gDNA desorption ~6 min. The PPC microchip could also be used for subsequent assays with no substantial loss in recovery, no observable carryover and no need for ‘reactivation’ of the PC surface with UV light.     

Urodynamic parameters and plasma LH/FSH in spayed Beagle bitches before and 8 weeks after GnRH depot analogue treatment

The pathophysiology of urinary incontinence due to spaying remains unknown. Incontinent bitches can be treated successfully with depot preparations of GnRH-analogues and there are differences in plasma gonadotropin levels between continent and incontinent spayed bitches. It is therefore assumed that the supraordinated hormones, GnRH, FSH, and/or LH, have an effect on the urodynamic parameters. In this study, the potential influence of these hormones on the lower urinary tract was investigated by measuring urethral pressure profiles and cystometry. Simultaneously, plasma concentrations in 10 spayed Beagle bitches were determined 5 weeks prior to and 8 weeks after treatment with the GnRH analogue leuprolide. Within 1 week of GnRH analogue administration, plasma FSH and LH levels decreased from 72.5 and 7.7 to 7.75 and 0.72ng/mL, respectively. These plasma gonadotropin levels correspond with those of intact bitches during anoestrus. Urethral pressure profiles indicated that the treatment had no significant effect on maximum urethral closure pressure, functional and total length of the urethra, or area of the closure pressure curve. The data obtained by cystometry regarding mean bladder threshold volume showed a significant increase from 109 to 172mL. The improvement in bladder function after the application of GnRH-application is presumably a direct effect of the GnRH as a relationship between the plasma gonadotropin levels and the urodynamic parameters could not demonstrated.     

Enzymes in Bast Fibrous Plant Processing

The program COST Action 847 Textile Quality and Biotechnology (2000–2005) has given an excellent chance to review the possibilities of the research, aiming at development of the industrial application of enzymes for bast fibrous plant degumming and primary processing. The recent advancements in enzymatic processing of bast fibrous plants (flax, hemp, jute, ramie and alike plants) and related textiles are given. The performance of enzymes in degumming, modification of bast fibres, roving, yarn, related fabrics as well as enzymatic bonding of lignocellulosic composites is provided.