Involvement of the SCF Complex in the Control of Cdh1 Degradation in S-phase

The anaphase promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that acts as a key regulator in the progression through mitosis (when mostly in complex with Cdc20) and as a stabilizer of the G1 phase (when in complex with Cdh1). Cdh1 is an activator of APC/C, and it has previously been reported that it is capable of mediating its own degradation during Go and G1. Herein, we show that the SCF complex (Skp1/Cul1/F-box protein/Roc1) intervenes in the surveillance of Cdh1 cellular abundance in S-phase.     

Adiabatic Heteronuclear Decoupling in Rotating Solids

In magic angle spinning solid state NMR experiments the potential of heteronuclear (1)H decoupling employing a continuous train of adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements. It is shown that, with a (1)H RF field strength of approximately 100 kHz that is typically available in MAS NMR probes, it is possible to achieve efficient adiabatic (1)H decoupling at low magic angle spinning frequencies. It is pointed out that in the presence of H (1) inhomogeneities it will be advantageous to employ adiabatic decoupling in MAS solid state NMR experiments.     

Phases in potassium transport and their regulation under near-equilibrium conditions in wheat seedlings

Potassium uptake and release in roots and translocation to the shoots were studied in 14-day-old winter wheat ( Tritictum aestivum L. cv. Martonvásári 8) of different K status. Transport processes were measured in the growth solutions for 5 h ensuring near-equilibrium conditions. The uptake showed three phases: (1) at low external K⁺ concentrations it increased with increasing concentrations and culminated at 0.1 m M : (2) between 0.1 and 1 m M it decreased, and (3) it increased again above 1 m M : The release of K⁺ showed a constant low level below 1 m M while paralleling the uptake above that. The uncoupler 2,4-dinitrophenol inhibited uptake phases (1) and (2), whereas it did not affect either phase (3) or K⁺ release. Translocation showed similar patterns. It is concluded that phases (1) and (2) depend on metabolic energy while phase (3) is mostly passive. It is emphasized that different types of regulation seem to operate in the transport mechanism: i.e. limitation by transport sites, control by negative feedback and by K⁺/K⁺ exchange, respectively.     

Mutations in the rod outer segment membrane guanylate cyclase in a cone-rod dystrophy cause defects in calcium signaling.

Rod outer segment guanylate cyclase 1 (ROS-GC1) is a member of the subfamily of Ca(2+)-regulated membrane guanylate cyclases; and it is pivotal for vertebrate phototransduction. Two opposing regulatory modes control the activity of ROS-GC1. At nanomolar concentrations of Ca(2+), ROS-GC1 is activated by Ca(2+)-binding proteins named guanylate cyclase activating proteins (GCAPs). However, at micromolar concentrations of Ca(2+), ROS-GC1 is stimulated by S100beta [also named calcium-dependent (CD) GCAP]. This mode is not linked with phototransduction; instead, it is predicted to be involved in retinal synaptic activity. Two point mutations, E786D and R787C, in ROS-GC1 have been connected with cone-rod dystrophy (CORD6), with only one type of point mutation occurring in each family. The present study shows that the E786D mutation has no effect on the basal catalytic activity of ROS-GC1 and on its activation by GCAP1 and S100beta; however, the mutated cyclase becomes more activated by GCAP2. The R787C mutation has three consequences: (1) it causes major damage to the basal cyclase activity, (2) it makes the cyclase 5-fold more sensitive to activation by GCAP1; and 3) converts the cyclase into a form that is less sensitive to activation by GCAP2 and S100beta. Thus, the two CORD6-linked mutations in ROS-GC1, which occur at adjacent positions, result in vastly different biochemical phenotypes, and they are connected with very specific molecular defects in the Ca(2+) switching components of the cyclase. These defects, in turn, are proposed to have a profound effect on both the machinery of phototransduction and the retinal synapse. The study for the first time defines the biochemistry of CORD6 pathology in precise molecular terms.     

The role of protein-tyrosine phosphatase 1B in integrin signaling

Protein-tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and leptin signaling and a novel therapeutic target for the treatment of type 2 diabetes, obesity, and other associated metabolic syndromes. Because PTP1B regulates multiple signal pathways and it can both enhance and antagonize a cellular event, it is important to establish the physiological relevance of PTP1B in these processes. In this study, we utilize potent and selective PTP1B inhibitors to delineate the role of PTP1B in integrin signaling. We show that down-regulation of PTP1B activity with small molecule inhibitors suppresses cell spreading and migration to fibronectin, increases Tyr(527) phosphorylation in Src, and decreases phosphorylation of FAK, p130(Cas), and ERK1/2. In addition, PTP1B "substrate-trapping" mutants bind Tyr(527)-phosphorylated Src and protect it from dephosphorylation by endogenous PTP1B. These results establish that PTP1B promotes integrin-mediated responses in fibroblasts by dephosphorylating the inhibitory pTyr(527) and thereby activating the Src kinase. We also show that PTP1B forms a complex with Src and p130(Cas), and that the proline-rich motif PPRPPK (residues 309-314) in PTP1B is essential for the complex formation. We suggest that the specificity of PTP1B for Src pTyr(527) is mediated by protein-protein interactions involving the docking protein p130(Cas) with both Src and PTP1B in addition to the interactions between the PTP1B active site and the pTyr(527) motif.     

Anti-cytokine therapy in chronic destructive arthritis

Tumor necrosis factor (TNF) and interleukin-1 (IL-1) are considered to be master cytokines in chronic, destructive arthritis. Therapeutic approaches in rheumatoid arthritis (RA) patients have so far focused mainly on TNF, which is a major inflammatory mediator in RA and a potent inducer of IL-1; anti-TNF therapy shows great efficacy in RA patients. However, it is not effective in all patients, nor does it fully control the arthritic process in affected joints of good responders. Directed therapy for IL-1, with IL-1 receptor antagonist, mainly reduces erosions and is marginally anti-inflammatory. It is as yet unclear whether the limited effect is akin to the RA process or linked to suboptimal blocking of IL-1. Analysis of cytokine patterns in early synovial biopsies of RA patients reveals a marked heterogeneity, with variable staining of TNF and IL-1 beta, indicative of TNF-independent IL-1 production in at least some patients. Evidence for this pathway emerged from experimental arthritises in rodents, and is summarized in this review. If elements of the models apply to the arthritic process in RA patients, it is necessary to block IL-1 beta in addition to TNF.     

Anti-cytokine therapy in chronic destructive arthritis

Tumor necrosis factor (TNF) and interleukin-1 (IL-1) are considered to be master cytokines in chronic, destructive arthritis. Therapeutic approaches in rheumatoid arthritis (RA) patients have so far focused mainly on TNF, which is a major inflammatory mediator in RA and a potent inducer of IL-1; anti-TNF therapy shows great efficacy in RA patients. However, it is not effective in all patients, nor does it fully control the arthritic process in affected joints of good responders. Directed therapy for IL-1, with IL-1 receptor antagonist, mainly reduces erosions and is marginally anti-inflammatory. It is as yet unclear whether the limited effect is akin to the RA process or linked to suboptimal blocking of IL-1. Analysis of cytokine patterns in early synovial biopsies of RA patients reveals a marked heterogeneity, with variable staining of TNF and IL-1β, indicative of TNF-independent IL-1 production in at least some patients. Evidence for this pathway emerged from experimental arthritises in rodents, and is summarized in this review. If elements of the models apply to the arthritic process in RA patients, it is necessary to block IL-1β in addition to TNF.     

A New Hybrid Rescue Allele in Drosophila melanogaster

Crosses of Drosophila melanogaster females to males of its sibling species Drosophila simulans, Drosophila mauritiana and Drosophila sechellia produce no sons and daughters that are viable only at low temperatures. We describe here a novel rescue allele Df(1)EP307-1-2 isolated on the basis of its suppression of high temperature hybrid female lethality. Df(1)EP307-1-2 also rescues hybrid males to the pharate adult stage, the same stage at which it is lethal to D. melanogaster pure species males. Molecular analysis indicates that Df(1)EP307-1-2 is associated with a deletion of about 61 kb in the 9D region of the X chromosome. The structure of Df(1)EP307-1-2 suggests that it was formed by a process similar to P-element induced male recombination.     

Improvement in the bioreactor specific productivity by coupling continuous reactor with repeated fed-batch reactor for acetone-butanol-ethanol production

In comparison to the different fermentation modes for the production of acetone, butanol and ethanol (ABE) researched to date, the continuous fermentation is the most economically favored. Continuous fermentation with two or more reactor cascade is reported to be the most efficient as it results in a more stable solvent production process. In this work, it is shown that a continuous (first-stage) reactor coupled to a repeated fed-batch (second stage) is superior to batch and fed-batch fermentations, including two-stage continuous fermentation. This is due to the efficient catalyst use, reported through the specific product rate and rapid glucose consumption rate. High solvents are produced at 19.4 g(ABE) l?1, with volumetric productivities of 0.92 g(butanol) l?1 h?1 and 1.47 g(ABE) l ?1 h?1. The bioreactor specific productivities of 0.62 and 0.39 g g?1(cdw) h?1 obtained show a high catalyst activity. This new process mode has not been reported before in the development of ABE fermentation and it shows great potential and superiority to the existing fermentation methods.     

Lipase maturation factor 1: a lipase chaperone involved in lipid metabolism

Mutations in lipase maturation factor 1 (LMF1) are associated with severe hypertriglyceridemia in mice and human subjects. The underlying cause is impaired lipid clearance due to lipase deficiency. LMF1 is a chaperone of the endoplasmic reticulum (ER) and it is critically required for the post-translational activation of three vascular lipases: lipoprotein lipase (LPL), hepatic lipase (HL) and endothelial lipase (EL). As LMF1 is only required for the maturation of homodimeric, but not monomeric, lipases, it is likely involved in the assembly of inactive lipase subunits into active enzymes and/or the stabilization of active dimers. Herein, we provide an overview of current understanding of LMF1 function and propose that it may play a regulatory role in lipase activation and lipid metabolism. Further studies will be required to test this hypothesis and elucidate the full spectrum of phenotypes in combined lipase deficiency. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

Regulation of protein kinase CK1alphaLS by dephosphorylation in response to hydrogen peroxide

Low levels of hydrogen peroxide (H(2)O(2)) are mitogenic to mammalian cells and stimulate the hyperphosphorylation of heterogeneous nuclear ribonucleoprotein C (hnRNP-C) by protein kinase CK1alpha. However, the mechanisms by which CK1alpha is regulated have been unclear. Here it is demonstrated that low levels of H(2)O(2) stimulate the rapid dephosphorylation of CK1alphaLS, a nuclear splice form of CK1alpha. Furthermore, it is demonstrated that either treatment of endothelial cells with H(2)O(2), or dephosphorylation of CK1alphaLS in vitro enhances the association of CK1alphaLS with hnRNP-C. In addition, dephosphorylation of CK1alphaLS in vitro enhances the kinase's ability to phosphorylate hnRNP-C. While CK1alpha appears to be present in all metazoans, analysis of CK1alpha genomic sequences from several species reveals that the alternatively spliced nuclear localizing L-insert is unique to vertebrates, as is the case for hnRNP-C. These observations indicate that CK1alphaLS and hnRNP-C represent conserved components of a vertebrate-specific H(2)O(2)-responsive nuclear signaling pathway.     

Role of gangliosides in Alzheimer's disease

One of the fundamental questions regarding the pathogenesis of Alzheimer's disease (AD) is how the monomeric, nontoxic amyloid beta-protein (Abeta) is converted to its toxic assemblies in the brain. A unique Abeta species was identified previously in an AD brain, which is characterized by its binding to the GM1 ganglioside (GM1). On the basis of the molecular characteristics of this GM1-bound Abeta (GAbeta), it was hypothesized that Abeta adopts an altered conformation through its binding to GM1, and GAbeta acts as a seed for Abeta fibrillogenesis in an AD brain. To date, various in vitro and in vivo studies of GAbeta have been performed, and their results support the hypothesis. Using a novel monoclonal antibody specific to GAbeta, it was confirmed that GAbeta is endogenously generated in the brain. Regarding the role of gangliosides in the facilitation of Abeta assembly, it has recently been reported that region-specific deposition of hereditary variant-type Abetas is determined by local gangliosides in the brain. Furthermore, it is likely that risk factors for AD, including aging and the expression of apolipoprotein E4, alter GM1 distribution on the neuronal surface, leading to GAbeta generation.     

Overexpression of type 1 angiotensin II receptors impairs excitation-contraction coupling in the mouse heart

Transgenic mice that overexpress human type 1 angiotensin II receptor (AT(1)R) in the heart develop cardiac hypertrophy. Previously, we have shown that in 6-mo AT(1)R mice, which exhibit significant cardiac remodeling, fractional shortening is decreased. However, it is not clear whether altered contractility is attributable to AT(1)R overexpression or is secondary to cardiac hypertrophy/remodeling. Thus the present study characterized the effects of AT(1)R overexpression on ventricular L-type Ca(2+) currents (I(CaL)), cell shortening, and Ca(2+) handling in 50-day and 6-mo-old male AT(1)R mice. Echocardiography showed there was no evidence of cardiac hypertrophy in 50-day AT(1)R mice but that fractional shortening was decreased. Cellular experiments showed that cell shortening, I(CaL), and Ca(v)1.2 mRNA expression were significantly reduced in 50-day and 6-mo-old AT(1)R mice compared with controls. In addition, Ca(2+) transients and caffeine-induced Ca(2+) transients were reduced whereas the time to 90% Ca(2+) transient decay was prolonged in both age groups of AT(1)R mice. Western blot analysis revealed that sarcoplasmic reticulum Ca(2+)-ATPase and Na(+)/Ca(2+) exchanger protein expression was significantly decreased in 50-day and 6-mo AT(1)R mice. Overall, the data show that cardiac contractility and the mechanisms that underlie excitation-contraction coupling are altered in AT(1)R mice. Furthermore, since the alterations in contractility occur before the development of cardiac hypertrophy, it is likely that these changes are attributable to the increased activity of the renin-angiotensin system brought about by AT(1)R overexpression. Thus it is possible that AT(1)R blockade may help maintain cardiac contractility in individuals with heart disease.     

Direct evidence in vivo of impaired macrophage-specific reverse cholesterol transport in ATP-binding cassette transporter A1-deficient mice

The ATP-binding cassette transporter A1 (ABCA1) is a key regulator of high-density lipoprotein (HDL) metabolism. There is strong evidence that ABCA1 is a key regulator of reverse cholesterol transport (RCT). However, this could not be proved in vivo since hepatobiliary cholesterol transport was unchanged in ABCA1-deficient mice (ABCA1-/-). We used ABCA1-/- mice to test the hypothesis that ABCA1 is a critical determinant of macrophage-specific RCT. Although this cell-specific RCT only accounts for a tiny part of total RCT, it is widely accepted that it may have a major impact on atherosclerosis susceptibility. [(3)H]cholesterol-labeled endogenous macrophages were injected intraperitoneally into wild-type ABCA1+/+, ABCA1+/- and ABCA1-/- mice maintained on a chow diet. A direct relationship was observed between ABCA1 gene dose and plasma [(3)H]cholesterol at 24 and 48 h after the injection of tracer into the mice. Forty-eight hours after this injection, ABCA1-/- mice had significantly reduced [(3)H]cholesterol in liver (2.8-fold), small intestine enterocytes (1.7-fold) and feces (2-fold). To our knowledge, this is the first direct in vivo quantitative evidence that ABCA1 is a critical determinant of macrophage-specific RCT.     

Immune responses to tuberculosis in developing countries: implications for new vaccines

Tuberculosis is out of control in developing countries, where it is killing millions of people every year. In these areas, the present vaccine - Mycobacterium bovis bacillus Calmette-Guérin (BCG) - is failing. Progressive tuberculosis occurs because the potentially protective T helper 1 (T(H)1)-cell response is converted to an immunopathological response that fails to eliminate the bacteria. Here, we discuss the data indicating that the problem in developing countries is not a lack of adequate T(H)1-cell responses but, instead, an exaggerated tendency to switch to immunopathological responses. We propose that a successful vaccine needs to block this immunopathology, because it is not the quantity of T(H)1-cell activity that matters but, rather, its context.     

A role for hTRPC1 and lipid raft domains in store-mediated calcium entry in human platelets

We have previously suggested that store-mediated Ca2+ entry (SMCE) in human platelets may be activated by a secretion-like coupling model, involving de novo coupling of the type II inositol 1,4,5-trisphosphate receptor (IP(3)RII) to the putative Ca2+ entry channel, hTRPC1. In other cells, hTRPC1 has been reported to be associated with cholesterol-rich lipid raft domains (LRDs) in the plasma membrane. Here we have shown that hTRPC1 is largely associated with detergent-resistant platelet membranes, from which it is partially released when the cells are depleted of cholesterol by treatment with methyl-beta-cyclodextrin (MBCD). MBCD treatment inhibited thapsigargin (TG)-evoked SMCE in a concentration-dependent manner, reducing it to 38.1+/-4.1% at a concentration of 10mM. Similarly, the Ca2+ entry evoked by thrombin (1unit/ml) was reduced to 48.2+/-4.5% of control following MBCD (10mM) treatment. Thrombin- and TG-evoked coupling between IP(3)RII and hTRPC1 was also reduced following cholesterol depletion. These results suggest that hTRPC1 is associated with LRDs in human platelets and that these domains are important for its participation in SMCE.     

Aspartyl aminopeptidase is imported from the cytoplasm to the vacuole by selective autophagy in Saccharomyces cerevisiae

Macroautophagy is a catabolic process by which cytosolic components are sequestered by double membrane vesicles called autophagosomes and sorted to the lysosomes/vacuoles to be degraded. Saccharomyces cerevisiae has adapted this mechanism for constitutive transport of the specific vacuolar hydrolases aminopeptidase I (Ape1) and α-mannosidase (Ams1); this process is called the cytoplasm to vacuole targeting (Cvt) pathway. The precursor form of Ape1 self-assembles into an aggregate-like structure in the cytosol that is then recognized by Atg19 in a propeptide-dependent manner. The interaction between Atg19 and autophagosome-forming machineries allows selective packaging of the Ape1-Atg19 complex by the autophagosome-like Cvt vesicle. Ams1 also forms oligomers and utilizes the Ape1 transport system by interacting with Atg19. Although the mechanism of selective transport of the Cvt cargoes has been well studied, it is unclear whether proteins other than Ape1 and Ams1 are transported via the Cvt pathway. We describe here that aspartyl aminopeptidase (Yhr113w/Ape4) is the third Cvt cargo, which is similar in primary structure and subunit organization to Ape1. Ape4 has no propeptide, and it does not self-assemble into aggregates. However, it binds to Atg19 in a site distinct from the Ape1- and Ams1-binding sites, allowing it to "piggyback" on the Ape1 transport system. In growing conditions, a small portion of Ape4 localizes in the vacuole, but its vacuolar transport is accelerated by nutrient starvation, and it stably resides in the vacuole lumen. We propose that the cytosolic Ape4 is redistributed to the vacuole when yeast cells need more active vacuolar degradation.     

PSO4: a novel gene involved in error-prone repair in Saccharomyces cerevisiae

The haploid xs9 mutant, originally selected for on the basis of a slight sensitivity to the lethal effect of X-rays, was found to be extremely sensitive to inactivation by 8-methoxypsoralen (8MOP) photoaddition, especially when cells are treated in the G2 phase of the cell cycle. As the xs9 mutation showed no allelism with any of the 3 known pso mutations, it was now given the name of pso4-1. Regarding inactivation, the pso4-1 mutant is also sensitive to mono- (HN1) or bi-functional (HN2) nitrogen mustards, it is slightly sensitive to 254 nm UV radiation (UV), and shows nearly normal sensitivity to 3-carbethoxypsoralen (3-CPs) photoaddition or methyl methanesulfonate (MMS). Regarding mutagenesis, the pso4-1 mutation completely blocks reverse and forward mutations induced by either 8MOP or 3CPs photoaddition, or by gamma-rays. In the cases of UV, HN1, HN2 or MMS treatments, while reversion induction is still completely abolished, forward mutagenesis is only partially inhibited for UV, HN1, or MMS, and it is unaffected for HN2. Besides severely inhibiting induced mutagenesis, the pso4-1 mutation was found to be semi-dominant, to block sporulation, to abolish the diploid resistance effect, and to block induced mitotic recombination, which indicates that the PSO4 gene is involved in a recombinational pathway of error-prone repair, comparable to the E. coli SOS repair pathway.     

Anion exchanger 1 in red blood cells and kidney: Band 3's in a pod

The bicarbonate/chloride exchanger 1 (AE1, Band 3) is abundantly expressed in the red blood cell membrane, where it is involved in gas exchange and functions as a major site of cytoskeletal attachment to the erythrocyte membrane. A truncated kidney isoform (kAE1) is highly expressed in type A intercalated cells of the distal tubules, where it is vital for urinary acidification. Recently, kAE1 has emerged as a novel physiologically significant protein in the kidney glomerulus. This minireview will discuss the known interactions of kAE1 in the podocytes and the possible mechanisms whereby this important multispanning membrane protein may contribute to the function of the glomerular filtration barrier and prevent proteinuria.