EzrA prevents aberrant cell division by modulating assembly of the cytoskeletal protein FtsZ

In response to a cell cycle signal, the cytoskeletal protein FtsZ assembles into a ring structure that establishes the location of the division site and serves as a framework for assembly of the division machinery. A battery of factors control FtsZ assembly to ensure that the ring forms in the correct position and at the precise time. EzrA, a negative regulator of FtsZ ring formation, is important for ensuring that the ring forms only once per cell cycle and that cytokinesis is restricted to mid-cell. EzrA is distributed throughout the plasma membrane and localizes to the ring in an FtsZ-dependent manner, suggesting that it interacts directly with FtsZ to modulate assembly. We have performed a series of experiments examining the interaction between EzrA and FtsZ. As little as twofold overexpression of EzrA blocks FtsZ ring formation in a sensitized genetic background, consistent with its predicted function. A purified EzrA fusion protein interacts directly with FtsZ to block assembly in vitro. Although EzrA is able to inhibit FtsZ assembly, it is unable to disassemble preformed polymers. These data support a model in which EzrA interacts directly with FtsZ at the plasma membrane to prevent polymerization and aberrant FtsZ ring formation.     

Cellulolysis is severely affected in Clostridium cellulolyticum strain cipCMut1

Progress towards understanding the molecular basis of cellulolysis by Clostridium cellulolyticm was obtained through the study of the first cellulolysis defective mutant strain, namely cipCMut1. In this mutant, a 2 659 bp insertion element, disrupts the cipC gene at the sequence encoding the seventh cohesin of the scaffoldin CipC. cipC is the first gene in a large 'cel' gene cluster, encoding several enzymatic subunits of the cellulosomes, including the processive cellulase Cel48F, which is the major component. Physiological and biochemical studies showed that the mutant strain was affected in cellulosome synthesis and severely impaired in its ability to degrade crystalline cellulose. It produced small amounts of a truncated CipC protein (P120), which had functional cohesin domains and assembled complexes which did not contain any of the enzymes encoded by genes of the 'cel' cluster. The mutant cellulolytic system was mainly composed of three proteins designated P98, P105 and P125. Their N-termini did not match any of the known cellulase sequences from C. cellulolyticum. A large amount of entire CipC produced in the cipCMut1 strain by trans-complementation with plasmid pSOScipC did not restore the cellulolytic phenotype, in spite of the assembly of a larger amount of complexes. The complexes produced in the mutant and complemented strains contained at least 12 different dockerin-containing proteins encoded by genes located outside of the 'cel' cluster. The disturbances observed in the mutant and trans-complemented strains were the result of a strong polar effect resulting from the cipC gene disruption. In conclusion, this study provided genetic evidence that the cellulases encoded by the genes located in the 'cel' cluster are essential for the building of cellulosomes efficient in crystalline cellulose degradation.     

Epithelial trafficking: new routes to familiar places

Research carried out in mammalian epithelial cell systems over the past 25 years has delineated pathways and sorting signals involved in polarized delivery of plasma membrane proteins. Recently some progress has been made in the identification of mechanisms underlying this polarized trafficking and in the visualization of trafficking routes in live cells. A promising area of research is the study of trafficking functions of novel polarity genes identified in Drosophila and Caenorhabditis elegans.     

Current management of severe homozygous hypercholesterolaemias

PURPOSE OF REVIEW: This review focuses on recent advances in the management of patients with homozygous familial hypercholesterolaemia, autosomal recessive hypercholesterolaemia and familial defective apolipoprotein B. RECENT FINDINGS: Autosomal recessive hypercholesterolaemia has been described as a 'phenocopy' of homozygous familial hypercholesterolaemia. Although the clinical phenotypes are similar, autosomal recessive hypercholesterolaemia seems to be less severe, more variable within a single family, and more responsive to lipid-lowering drug therapy. The cardiovascular complications of premature atherosclerosis are delayed in some individuals and involvement of the aortic root and valve is less common than in homozygous familial hypercholesterolaemia. Apheresis is still the treatment of choice in homozygous familial hypercholesterolaemia and in autosomal recessive hypercholesterolaemia patients in whom maximal drug therapy does not achieve adequate control. In addition to the profound cholesterol-lowering effects of apheresis, other potentially beneficial phenomena have been documented: improved vascular endothelial function and haemorheology, reduction in lipoprotein (a) and procoagulatory status, and a decrease in adhesion molecules and C-reactive protein. SUMMARY: Patients with severe homozygous hypercholesterolaemia illustrate the natural history of atherosclerosis within a condensed timeframe. Effective cholesterol-lowering treatment started in early childhood is essential to prevent onset of life-threatening atherosclerotic involvement of the aortic root and valve, and the coronary arteries. Noninvasive methods for regular monitoring of the major sites involved in the atherosclerotic process are necessary in patients with no symptoms or signs of ischaemia. Management of patients with severe homozygous hypercholesterolaemia continues to be a major challenge.     

Comparison of a prototype magnetoresistive biosensor to standard fluorescent DNA detection

We present a comparative analysis of a magnetoresistive biosensor to standard fluorescent DNA detection. The biosensor consists of giant magnetoresistive (GMR) type Cu/Ni(80)Fe(20) multilayers in the second antiferromagnetic coupling maximum. Each of the 206 elements of the magnetoresistive biosensor is patterned into a spiral-shaped line that can cover the area of a typical DNA spot (70 microm diameter). The probe DNA is assembled on top of the sensor elements in different concentrations ranging from 16 pg/microl to 10 ng/microl. Complementary biotin-labeled analyte DNA is hybridized to the probe DNA at a concentration of 10 ng/microl. A number of different commercially available magnetic microspheres are investigated to determine the most appropriate markers. The experimental comparison shows that the relative sensitivity of the magnetoresistive biosensor is superior to the fluorescent detection at low probe DNA concentrations.     

Neuroprotection by tempol in a model of iron-induced oxidative stress in acute ischemic stroke

Studies suggest iron exacerbates the damage caused by ischemic stroke. Our aim was to elucidate the effect of iron overload on infarct size after middle cerebral artery occlusion (MCAO) and to evaluate the efficacy of tempol, a superoxide dismutase mimetic, as a neuroprotective agent. Rats were administered iron +/- tempol before MCAO; control rats received saline. The middle cerebral artery was occluded for 24 h, and the size of the resultant infarct was assessed and expressed as the percentage of the hemisphere infracted (%HI). Iron treatment increased infarct size compared with control (51.83 +/- 3.55 vs. 27.56 +/- 3.28%HI iron treated vs. control, P = 0.01); pretreatment with tempol reversed this (51.83 +/- 3.55 vs. 26.09 +/- 9.57%HI iron treated vs. iron + tempol treated, P = 0.02). We hypothesized that reactive oxygen species (ROS) were responsible for the iron-induced damage. We measured ROS generated by exogenous iron in brain and peripheral vasculature from rats that had not undergone MCAO. There was no increase in ROS production in the brain of iron-treated rats or in brain slices incubated with iron citrate. However, ROS generation in carotid arteries incubated with iron citrate was significantly increased. ROS generation from the brain was assessed after MCAO by dihydroethidine staining; there was a dramatic increase in the ROS generation by the brain in the iron-treated rats compared with control 30 min after MCAO. We propose that iron-induced ROS generation in the cerebral vasculature adds to oxidative stress during an ischemic episode after the disruption of the blood-brain barrier.     

Augmented systolic response to the calcium sensitizer EMD-57033 in a transgenic model with troponin I truncation

Myocardial stunning is a form of acute reversible cardiac dysfunction that occurs after brief periods of ischemia and reperfusion. In several animal models, stunning is associated with proteolytic truncation of troponin I (TnI). Mice expressing the same proteolytic TnI fragment [TnI-(1-193)] demonstrate cardiac depression with a decreased maximal calcium-activated tension. We therefore hypothesized preferential improvement in mice expressing TnI-(1-193) treated with the calcium-sensitizing drug EMD-57033. TnI-(1-193) and nontransgenic myofibrils exhibited significant sensitization to calcium in Mg-ATPase assays after EMD-57033 exposure. However, only transgenic myofibrils exhibited an increase in maximal activity (P = 0.023). EMD-57033 also increased maximal calcium-activated force in TnI-(1-193) muscle, such that it was comparable to nontransgenic cardiac muscle. EMD-57033 enhanced in vivo systolic function modestly in controls but had a marked effect in transgenic mice, with an almost threefold greater leftward shift of the end-systolic pressure-volume relation (P = 0.0005). These data indicate a targeted efficacy of EMD-57033 in offsetting the contractile defect in TnI-(1-193) mice, and this may have therapeutic implications in models displaying this myofilament defect.     

Hepatocyte growth factor induces MDCK cell morphogenesis without causing loss of tight junction functional integrity

Hepatocyte growth factor (HGF) induces mitogenesis, motogenesis, and tubulogenesis of cultured Madin-Darby canine kidney (MDCK) epithelial cells. We report that in addition to these effects HGF stimulates morphogenesis of tight, polarized MDCK cell monolayers into pseudostratified layers without loss of tight junction (TJ) functional integrity. We tested TJ functional integrity during formation of pseudostratified layers. In response to HGF, the TJ marker ZO-1 remained in morphologically complete rings and functional barriers to paracellular diffusion of ruthenium red were maintained in pseudostratified layers. Transepithelial resistance (TER) increased transiently two- to threefold during the morphogenetic transition from monolayers to pseudostratified layers and then declined to baseline levels once pseudostratified layers were formed. In MDCK cells expressing the trk/met chimera, both HGF and NGF at concentrations of 2.5 ng/ml induced scattering. However, 2.5 ng/ml HGF did not affect TER. The peak effect of HGF on TER was at a concentration of 100 ng/ml. In contrast, NGF at concentrations as high as 25 µg/ml had no effect on TER or pseudostratified layer morphogenesis of trk/met-expressing cultures. These results suggest that altered presentation of the stimulus, such as through HGF interaction with low-affinity sites, may change the downstream signaling response. In addition, our results demonstrate that HGF stimulates pseudostratified layer morphogenesis while inducing an increase in TER and maintaining the overall tightness of the epithelial layer. Stimulation of epithelial cell movements by HGF without loss of functional TJs may be important for maintaining epithelial integrity during morphogenetic events such as formation of pseudostratified epithelia, organ regeneration, and tissue repair. c-met protooncogene; transepithelial resistance; Madin-Darby canine kidney cell     

IL-10 inhibits vascular smooth muscle cell activation in vitro and in vivo

The anti-inflammatory cytokine IL-10 inhibits intimal hyperplasia after stent implantation via a powerful inactivation of monocytes. We tested the hypothesis that IL-10 may also inhibit vascular smooth muscle cell (SMC) activation via the inhibition of the NF-kappaB/I-kappaB system. The IL-10 receptor was detected in rat SMCs in vitro and in vivo. In LPS-stimulated rat SMCs, 1 ng/ml recombinant murine IL-10 (mIL-10) reduced I-kappaBalpha and I-kappaBbeta degradation, NF-kappaB activation, as well as the expression of the NF-kappaB-dependent gene IL-6 by 32%, 31%, 75%, and 19%, respectively (P < 0.05 for all). Similar results were obtained in vivo 6 h and 4 days after balloon abrasion of the rat aorta, a model in which intimal hyperplasia results essentially from SMC activation. Moreover, mIL-10 reduced SMC proliferation and migration in vitro (by 60% for both, P < 0.0001), resulting in reduced SMC proliferation and intimal growth 14 days after balloon abrasion of the rat aorta (by 76% and 75%, respectively; P < 0.005). In conclusion, mIL-10 has a direct inhibitory effect on SMCs in vitro and in vivo. This effect is mediated in part by NF-kappaB inactivation and may participate in the overall protective effect of IL-10 on postangioplasty restenosis.     

Alexander Disease: A Leukodystrophy Caused by a Mutation in GFAP

Alexander disease, a rare fatal disorder of the central nervous system, causes progressive loss of motor and mental function. Until recently it was of unknown etiology, almost all cases were sporadic, and there was no effective treatment. It was most common in an infantile form, somewhat less so in a juvenile form, and was rarely seen in an adult-onset form. A number of investigators have now shown that almost all cases of Alexander disease have a dominant mutation in one allele of the gene for glial fibrillary acidic protein (GFAP) that causes replacement of one amino acid for another. Only in very rare cases of the adult-onset form is the mutation present in either parent. Thus, in almost all cases, the mutation arises as a spontaneous event, possibly in the germ cell of one parent.