bluuebunny/crossref_metadata_2025 huggingface dataset:
Defensin-like peptide-2 from platypus venom: member of a class of peptides with a distinct structural fold
Authors: TORRES Allan M. , DE PLATER Greg M. , DOVERSKOG Magnus , BIRINYI-STRACHAN Liesl C. , NICHOLSON Graham M. , GALLAGHER Clifford H. , KUCHEL Philip W.
Published: August 2015

The venom of the male Australian duck-billed platypus contains a family of four polypeptides of appox. 5 kDa, which are referred to as defensin-like peptides (DLPs). They are unique in that their amino acid sequences have no significant similarities to those of any known peptides; however, the tertiary structure of one of them, DLP-1, has recently been shown to be similar to β-defensin-12 and to the sodium neurotoxin peptide ShI (Stichodactyla helianthus neurotoxin I). Although DLPs are the major peptides in the platypus venom, little is known about their biological roles. In this study, we determined the three-dimensional structure of DLP-2 by NMR spectroscopy, with the aim of gaining insights into the natural function of the DLPs in platypus venom. The DLP-2 structure was found to incorporate a short helix that spans residues 9-12, and an antiparallel β-sheet defined by residues 15-18 and 37-40. The overall fold and cysteine-pairing pattern of DLP-2 were found to be similar to those of DLP-1, and hence β-defensin-12; however, the sequence similarities between the three molecules are relatively small. The distinct structural fold of the DLP-1, DLP-2, and β-defensin-12 is based upon several key residues that include six cysteines. DLP-3 and DLP-4 are also likely to be folded similarly since they have high sequence similarity with DLP-2. The DLPs, and β-defensin-12 may thus be grouped together into a class of polypeptide molecules which have a common or very similar global fold. The fact that the DLPs did not display antimicrobial, myotoxic, or cell-growth-promoting activities implies that the nature of the side chains in this group of peptides is likely to play an important role in defining the biological function(s).
Activation of extracellular signal-regulated protein kinase1,2 results in down-regulation of decorin expression in fibroblasts
Authors: LAINE Pauliina , REUNANEN Niina , RAVANTI Laura , FOSCHI Marco , SANTRA Manoranjan , IOZZO V. Renato , KÄHÄRI Veli-Matti
Published: August 2015

Decorin is a small leucine-rich extracellular matrix proteoglycan, the expression of which is down-regulated in proliferating and malignantly transformed cells. In the present study we show that the expression of decorin in fibroblasts is suppressed by epidermal growth factor (EGF) and PMA, and that the effect of both is potently inhibited by blocking the extracellular signal-regulated protein kinase (ERK)1,2 signalling pathway (Raf/MEK1,2/ERK1,2) with the specific MAPK/ERK kinase (MEK)1,2 inhibitor, PD98059. In addition, specific activation of ERK1,2 by adenovirus-mediated expression of constitutively active MEK1 in dermal fibroblasts results in marked reduction in decorin mRNA abundance and production. Co-transfection of NIH-3T3 fibroblasts with human decorin promoter/chloramphenicol acetyltransferase (CAT) construct (pDEC-879/CAT) in combination with the expression vectors for constitutively active Raf-1 and MEK1 markedly suppressed decorin promoter activity. Co-transfections of human decorin promoter 5ʹ-deletion constructs with constitutively active MEK1 expression vector identified the region -278 to -188 as essential for ERK1,2 mediated down-regulation of decorin promoter activity. These results show that activation of the ERK1,2 signalling pathway by a mitogenic growth factor, a tumour promoter or transformation suppresses decorin gene expression in fibroblasts, which in turn may promote proliferation and migration of normal and malignant cells.
Identification of a nucleoside/nucleobase transporter from Plasmodium falciparum, a novel target for anti-malarial chemotherapy
Authors: PARKER Marie D. , HYDE Ralph J. , YAO Sylvia Y. M. , MCROBERT Louisa , CASS Carol E. , YOUNG James D. , MCCONKEY Glenn A. , BALDWIN Stephen A.
Published: August 2015

Plasmodium, the aetiologic agent of malaria, cannot synthesize purines de novo, and hence depends upon salvage from the host. Here we describe the molecular cloning and functional expression in Xenopus oocytes of the first purine transporter to be identified in this parasite. This 422-residue protein, which we designate PfENT1, is predicted to contain 11 membrane-spanning segments and is a distantly related member of the widely distributed eukaryotic protein family the equilibrative nucleoside transporters (ENTs). However, it differs profoundly at the sequence and functional levels from its homologous counterparts in the human host. The parasite protein exhibits a broad substrate specificity for natural nucleosides, but transports the purine nucleoside adenosine with a considerably higher apparent affinity (Km 0.32±0.05 mM) than the pyrimidine nucleoside uridine (Km 3.5±1.1 mM). It also efficiently transports nucleobases such as adenine (Km 0.32±0.10 mM) and hypoxanthine (Km 0.41±0.1 mM), and anti-viral 3ʹ-deoxynucleoside analogues. Moreover, it is not sensitive to classical inhibitors of mammalian ENTs, including NBMPR {6-[(4-nitrobenzyl)thio]-9-β-D-ribofuranosylpurine, or nitrobenzylthioinosine} and the coronary vasoactive drugs, dipyridamole, dilazep and draflazine. These unique properties suggest that PfENT1 might be a viable target for the development of novel anti-malarial drugs.
Apolipoprotein E includes a binding site which is recognized by several amyloidogenic polypeptides
Authors: BAUMANN Marc H. , KALLIJÄRVI Jukka , LANKINEN Hilkka , SOTO Claudio , HALTIA Matti
Published: August 2015

Inheritance of the apolipoprotein E (apoE) ϵ4 allele is a risk factor for late-onset Alzheimer's disease (AD). Biochemically apoE is present in AD plaques and neurofibrillary tangles of the AD brain. There is a high avidity and specific binding of apoE and the amyloid β-peptide (Aβ). In addition to AD apoE is also present in many other cerebral and systemic amyloidoses, Down's syndrome and prion diseases but the pathophysiological basis for its presence is still unknown. In the present study we have compared the interaction of apoE with Aβ, the gelsolin-derived amyloid fragment AGel183-210 and the amyloidogenic prion fragments PrP109-122 and PrP109-141. We show that, similar to Aβ, also AGel and PrP fragments can form a complex with apoE, and that the interaction between apoE and the amyloidogenic protein fragments is mediated through the same binding site on apoE. We also show that apoE increases the thioflavin-T fluorescence of PrP and AGel and that apoE influences the content of β-sheet conformation of these amyloidogenic fragments. Our results indicate that amyloids and amyloidogenic prion fragments share a similar structural motif, which is recognized by apoE, possibly through a single binding site, and that this motif is also responsible for the amyloidogenicity of these fragments.
Indirect induction of suppressor of cytokine signalling-1 in macrophages stimulated with bacterial lipopolysaccharide: partial role of autocrine/paracrine interferon-α/β
Authors: CRESPO Ana , FILLA Michael B. , RUSSELL Stephen W. , MURPHY William J.
Published: August 2015

It has previously been reported by us that a brief prior exposure of mouse bone marrow culture-derived macrophages to bacterial lipopolysaccharide (LPS) resulted in a dramatic reduction in their ability to produce NO in response to a subsequent stimulus with either interferon-γ (IFN-γ) or IFN-γ plus LPS. We show here that this brief exposure to LPS results in an impaired response to subsequently added IFN-γ. A 2-4 h pretreatment with LPS leads to a dramatic reduction in the IFN-γ-induced DNA-binding of the transcription factor, signal transducer and activator of transcription 1α (STAT1α). This loss in ability to activate STAT1α temporally correlates with the LPS-induced accumulation of mRNA encoding the suppressor of cytokine signalling-1 (SOCS-1). However, LPS does not directly induce the synthesis of SOCS-1. Rather, LPS induces the synthesis of autocrine/paracrine factors that are the true mediators of SOCS-1 induction. IFN-α/β is one of these mediators, but plays only a partial role in the induction of SOCS-1 because neutralization of LPS-induced IFN-α/β production incompletely inhibits the induction of SOCS-1. We show that mouse IFN-β directly induces the synthesis of SOCS-1, without the need for prior protein synthesis, and does so with faster kinetics than does LPS. Our results are consistent with the non-specific nature of LPS-induced tolerance and provide a mechanistic insight into nonspecificity; LPS indirectly induces the synthesis of a protein mediator, SOCS-1, which inhibits the signalling that is induced by IFN-γ.
The short prodomain influences caspase-3 activation in HeLa cells
Authors: MEERGANS Thomas , HILDEBRANDT Ann-Kristin , HORAK Daniel , HAENISCH Christina , WENDEL Albrecht
Published: August 2015

Proteolytic activation of caspases is a key step in the process of apoptosis. According to their primary structure, caspases can be divided into a group with a long prodomain and a group with a short prodomain. Whereas long prodomains play a role in autocatalytic processing, little is known about the function of the short prodomain, for example the prodomain of caspase-3. We constructed caspase-3 variants lacking the prodomain and overexpressed these in HeLa and yeast cells. We found that removal of the caspase-3 prodomain resulted in spontaneous proteolytic activation of the protein when expressed in HeLa cells. This processing was only partially autocatalytic, as demonstrated by a catalytically inactive caspase-3 mutant. Co-expression of the anti-apoptotic protein XIAP (X-chromosome-linked inhibitor of apoptosis protein) completely blocked the observed spontaneous activation, which excluded a direct involvement of caspase-8. Our findings indicate that the short prodomain of caspase-3 serves as a silencing component in mammalian cells by retaining this executioner caspase in an inactive state.
Identification and characterization of a novel cytochrome c3 from Shewanella frigidimarina that is involved in Fe(III) respiration
Authors: GORDON Euan H. J. , PIKE Andrew D. , HILL Anne E. , CUTHBERTSON Pauline M. , CHAPMAN Stephen K. , REID Graeme A.
Published: August 2015

Shewanella frigidimarina NCIMB400 is a non-fermenting, facultative anaerobe from the gamma group of proteobacteria. When grown anaerobically this organism produces a wide variety of periplasmic c-type cytochromes, mostly of unknown function. We have purified a small, acidic, low-potential tetrahaem cytochrome with similarities to the cytochromes c3 from sulphate-reducing bacteria. The N-terminal sequence was used to design PCR primers and the cctA gene encoding cytochrome c3 was isolated and sequenced. The EPR spectrum of purified cytochrome c3 indicates that all four haem irons are ligated by two histidine residues, a conclusion supported by the presence of eight histidine residues in the polypeptide sequence, each of which is conserved in a related cytochrome c3 and in the cytochrome domains of flavocytochromes c3. All four haems exhibit low midpoint redox potentials that range from -207 to -58 mV at pH 7; these values are not significantly influenced by pH changes. Shewanella cytochrome c3 consists of a mere 86 amino acid residues with a predicted molecular mass of 11780 Da, including the four attached haem groups. This corresponds closely to the value of 11778 Da estimated by electrospray MS. To examine the function of this novel cytochrome c3 we constructed a null mutant by gene disruption. S. frigidimarina lacking cytochrome c3 grows well aerobically and its growth rate under anaerobiosis with a variety of electron acceptors is indistinguishable from that of the wild-type parent strain, except that respiration with Fe(III) as sole acceptor is severely, although not completely, impaired.
The pepsin residue glycine-76 contributes to active-site loop flexibility and participates in catalysis
Authors: OKONIEWSKA Monika , TANAKA Takuji , YADA Rickey Y.
Published: August 2015

Glycine residues are known to contribute to conformational flexibility of polypeptide chains, and have been found to contribute to flexibility of some loops associated with enzymic catalysis. A comparison of porcine pepsin in zymogen, mature and inhibited forms revealed that a loop (a flap), consisting of residues 71-80, located near the active site changed its position upon substrate binding. The loop residue, glycine-76, has been implicated in the catalytic process and thought to participate in a hydrogen-bond network aligning the substrate. This study investigated the role of glycine-76 using site-directed mutagenesis. Three mutants, G76A, G76V and G76S, were constructed to increase conformational restriction of a polypeptide chain. In addition, the serine mutant introduced a hydrogen-bonding potential at position 76 similar to that observed in human renin. All the mutants, regardless of amino acid size and polarity, had lower catalytic efficiency and activated more slowly than the wild-type enzyme. The slower activation process was associated directly with altered proteolytic activity. Consequently, it was proposed that a proteolytic cleavage represents a limiting step of the activation process. Lower catalytic efficiency of the mutants was explained as a decrease in the flap flexibility and, therefore, a different pattern of hydrogen bonds responsible for substrate alignment and flap conformation. The results demonstrated that flap flexibility is essential for efficient catalytic and activation processes.
Investigation of the slow inhibition of almond β-glucosidase and yeast isomaltase by 1-azasugar inhibitors: evidence for the ‘direct binding’ model
Authors: LOHSE Anders , HARDLEI Tore , JENSEN Astrid , PLESNER Igor W. , BOLS Mikael
Published: August 2015

(-)-1-Azafagomine [(3R,4R,5R)-4,5-dihydroxy-3-hydroxymethylhexahydropyridazine; inhibitor 1] is a potent glycosidase inhibitor designed to mimic the transition state of a substrate undergoing glycoside cleavage. The inhibition of glycosidases by inhbitor 1 and analogues has been found to be a relatively slow process. This ‘slow inhibition’ process was investigated in the inhibition of almond β-glucosidase and yeast isomaltase by inhibitor 1 and analogues. Progress-curve experiments established that the time-dependent inhibition of both enzymes by inhibitor 1 was a consequence of relatively slow dissociation and association of the inhibitor from and to the enzyme, and not a result of slow interchanges between protein conformations. A number of hydrazine-containing analogues of inhibitor 1 also inhibited β-glucosidase and isomaltase slowly, while the amine isofagomine [(3R,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine; inhibitor 5] only inhibited β-glucosidase slowly. Inhibitor 1 and related inhibitors were found to leave almond β-glucosidase with almost identical rate constants, so that the difference in Ki values depended almost entirely on changes in the binding rate constant, kon. The same trend was observed for the inhibition of yeast isomaltase by inhibitor 1 and a related inhibitor. The values of the rate constants were obtained at 25 °C and at pH 6.8.
Kinetic properties of missense mutations in patients with glutathione synthetase deficiency
Authors: NJÅLSSON Runa , CARLSSON Katarina , OLIN Birgit , CARLSSON Birgit , WHITBREAD Lel , POLEKHINA Galina , PARKER Michael W. , NORGREN Svante , MANNERVIK Bengt , BOARD Philip G. , LARSSON Agne
Published: August 2015

Patients with hereditary glutathione synthetase (GS) (EC 6.3.2.3) deficiency present with variable clinical pictures, presumably related to the nature of the mutations involved. In order to elucidate the relationship between genotype, enzyme function and clinical phenotype, we have characterized enzyme kinetic parameters of missense mutations R125C, R267W, R330C and G464V from patients with GS deficiency. One of the mutations predominantly affected the Km value, with decreased affinity for glycine, two mutations influenced both Km and Vmax values, and one mutation reduced the stability of the enzyme. This characterization agrees well with predictions based on the recently reported crystal structure of human GS. Thus our data indicate that different mutations can affect the catalytic capacity of GS by decreasing substrate affinity, maximal velocity or enzyme stability.
Targeting motifs and functional parameters governing the assembly of connexins into gap junctions
Authors: MARTIN Patricia E. M. , STEGGLES James , WILSON Claire , AHMAD Shoeb , EVANS W. Howard
Published: August 2015

To study the assembly of gap junctions, connexin-green-fluorescent-protein (Cx-GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26- and Cx32-GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32-GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx-GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.
Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency
Authors: MEININGER Cynthia J. , MARINOS Rebecca S. , HATAKEYAMA Kazuyuki , MARTINEZ-ZAGUILAN Raul , ROJAS Jose D. , KELLY Katherine A. , WU Guoyao
Published: August 2015

Endothelial cells (EC) from diabetic BioBreeding (BB) rats have an impaired ability to produce NO. This deficiency is not due to a defect in the constitutive isoform of NO synthase in EC (ecNOS) or alterations in intracellular calcium, calmodulin, NADPH or arginine levels. Instead, ecNOS cannot produce sufficient NO because of a deficiency in tetrahydrobiopterin (BH4), a cofactor necessary for enzyme activity. EC from diabetic rats exhibited only 12% of the BH4 levels found in EC from normal animals or diabetes-prone animals which did not develop disease. As a result, NO synthesis by EC of diabetic rats was only 18% of that for normal animals. Increasing BH4 levels with sepiapterin increased NO production, suggesting that BH4 deficiency is a metabolic basis for impaired endothelial NO synthesis in diabetic BB rats. This deficiency is due to decreased activity of GTP-cyclohydrolase I, the first and rate-limiting enzyme in the de novo biosynthesis of BH4. GTP-cyclohydrolase activity was low because of a decreased expression of the protein in the diabetic cells.
Interferons activate the p42/44 mitogen-activated protein kinase and JAK-STAT (Janus kinase-signal transducer and activator transcription factor) signalling pathways in hepatocytes: differential regulation by acute ethanol via a protein kinase C-dependent mechanism
Authors: NGUYEN Van-Anh T. , CHEN Jianping , HONG Feng , ISHAC Edward J. N. , GAO Bin
Published: August 2015

Interferons (IFNs) have been used in the treatment of viral hepatitis. However, their effectiveness is much reduced (< 10%) in alcoholics. The mechanism underlying this resistance remains unknown. Here, we report that IFN-α/β and IFN-γ rapidly activate the JAK-STAT1 (Janus kinase-signal transducer and activator transcription factor 1) and p42/44 mitogen-activatedprotein kinase (p42/44 MAPK) in freshly isolated rat hepatocytes. Treatment of hepatocytes with 25-100 mM ethanol for 30 min inhibited IFN-β- or IFN-γ-induced STAT1 activation and tyrosine phosphorylation. The inhibitory effect of ethanol was not reversed by pretreatment with either sodium vanadate, a non-selective tyrosine phosphatase inhibitor, or with MG132, a specific proteasome inhibitor. This suggests that protein tyrosine phosphatases or the ubiquitin-proteasome pathway are not involved in the inhibitory action of ethanol. In contrast with the JAK-STAT signalling pathway, acute ethanol exposure significantly potentiated IFN-β or IFN-γ-induced activation of p42/44 MAPK, and caused marked activation of protein kinase C (PKC). Inhibition of PKC partially antagonized ethanol attenuation of IFN-induced STAT1 activation, suggesting that PKC may be involved. Taken together, these findings suggest that the ability of biologically relevant concentrations of ethanol (less than 100 mM) to markedly inhibit IFN-activated STAT1 is one of the cellular mechanisms responsible for the observed resistance of IFN therapy in alcoholics.
Link peptide cartilage growth factor is degraded by membrane proteinases
Authors: DEAN Michael F. , SANSOM Paul
Published: August 2015

The peptide DHLSDNYTLDHDRAIH (Link N), cleaved from the N-terminus of the link protein component of cartilage proteoglycan aggregates by the action of stromelysin, can act as a growth factor and stimulate synthesis of proteoglycans and collagen in articular cartilage [McKenna, Liu, Sansom and Dean (1998) Arthritis Rheum. 41, 157-161]. The mechanism by which this biologically active peptide is degraded and inactivated was investigated using U937 monocytes as a model cell. Time-course experiments showed that two major proteases, an initial serine proteinase followed by a metalloproteinase, acted in sequence. Analysis of the resulting fragments showed that the serine endopeptidase cleavage was at the Leu3-Ser4 bond to produce the peptide SDNYTLDHDRAIH. The terminal serine could then be removed from the resulting peptide by an aminopeptidase. A second metallopeptidase liberated the peptides SDNYTL or DNYTL from DHDRAIH by cleavage at the Leu9-Asp10 bond. The DNYTL peptide intermediate was degraded too rapidly to allow sequencing and sequential aminopeptidase cleavages removed further amino acids from the N-terminus of the remaining DHDRAIH peptide. The identical patterns of breakdown that occurred when either whole cells or purified plasma membranes were used indicated that proteolysis and inactivation of Link N was carried out entirely by membrane-associated enzymes.
The human breast carcinoma cell line HBL-100 acquires exogenous cholesterol from high-density lipoprotein via CLA-1 (CD-36 and LIMPII analogous 1)-mediated selective cholesteryl ester uptake
Authors: PUSSINEN Pirkko J. , KARTEN Barbara , WINTERSPERGER Andrea , REICHER Helga , MCLEAN Mark , MALLE Ernst , SATTLER Wolfgang
Published: August 2015

Aberrant cell proliferation is one of the hallmarks of carcinogenesis, and cholesterol is thought to play an important role during cell proliferation and cancer progression. In the present study we examined the pathways that could contribute to enhanced proliferation rates of HBL-100 cells in the presence of apolipoprotein E-depleted high-density lipoprotein subclass 3 (HDL3). When HBL-100 cells were cultivated in the presence of HDL3 (up to 200 μg/ml HDL3 protein), the growth rates and cellular cholesterol content were directly related to the concentrations of HDL3 in the culture medium. In principle, two pathways can contribute to cholesterol/cholesteryl ester (CE) uptake from HDL3, (i) holoparticle- and (ii) scavenger-receptor BI (SR-BI)-mediated selective uptake of HDL3-associated CEs. Northern- and Western-blot analyses revealed the expression of CLA-1 (CD-36 and LIMPII analogous 1), the human homologue of the rodent HDL receptor SR-BI. In line with CLA-1 expression, selective uptake of HDL3-CEs exceeded HDL3-holoparticle uptake between 12- and 58-fold. Competition experiments demonstrated that CLA-1 ligands (oxidized HDL, oxidized and acetylated low-density lipoprotein and phosphatidylserine) inhibited selective HDL3-CE uptake. In line with the ligand-binding specificity of CLA-1, phosphatidylcholine did not compete for selective HDL3-CE uptake. Selective uptake was regulated by the availability of exogenous cholesterol and PMA, but not by adrenocorticotropic hormone. HPLC analysis revealed that a substantial part of HDL3-CE, which was taken up selectively, was subjected to intracellular hydrolysis. A potential candidate facilitating extralysosomal hydrolysis of HDL3-CE is hormone-sensitive lipase, an enzyme which was identified in HBL-100 cells by Western blots. Our findings demonstrate that HBL-100 cells are able to acquire HDL-CEs via selective uptake. Subsequent partial hydrolysis by hormone-sensitive lipase could provide ‘free’ cholesterol that is available for the synthesis of cellular membranes during proliferation of cancer cells.
Vacuolar proton pyrophosphatase activity and pyrophosphate (PPi) in Toxoplasma gondii as possible chemotherapeutic targets
Authors: RODRIGUES Claudia O. , SCOTT David A. , BAILEY Brian N. , SOUZA Wanderley DE , BENCHIMOL Marlene , MORENO Ben , URBINA Julio A. , OLDFIELD Eric , MORENO Silvia N. J.
Published: August 2015

The addition of PPi promoted the acidification of a subcellular compartment in cell homogenates of Toxoplasma gondii tachyzoites, implying the presence of a proton-translocating pyrophosphatase. The proton gradient was collapsed by addition of the K+/H+ antiporter nigericin, and was also inhibited by addition of the PPi analogue aminomethylenediphosphonate (AMDP). Both proton transport and PPi hydrolysis were dependent upon K+, but Na+ caused partial inhibition of these activities. PPi hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, NaF and to the thiol reagent N-ethylmaleimide. This activity was unaffected by common inhibitors of phosphohydrolases, except that NaO3V (sodium orthovanadate) stimulated the activity by 87%. Immunofluorescence microscopy, using antisera raised against conserved peptide sequences of a plant vacuolar pyrophosphatase, suggested that the pyrophosphatase in T. gondii tachyzoites was located in the plasma membrane and intracellular vacuoles of the parasite. High-field 31P-NMR spectroscopy showed that PPi was more abundant than ATP in tachyzoites. Bisphosphonates (PPi analogues), drugs that are used in the treatment of bone diseases, inhibited proton transport and PPi hydrolysis in tachyzoite homogenates, and also inhibited intracellular proliferation of tachyzoites in tissue culture cells.
Conformational changes in activated protein C caused by binding of the first epidermal growth factor-like module of protein S
Authors: HACKENG Tilman M. , YEGNESWARAN Subramanian , JOHNSON Arthur E. , GRIFFIN John H.
Published: August 2015

The first epidermal growth factor-like module of human plasma protein S (EGF1, residues 76–116) was chemically synthesized and tested for its ability to inhibit the anticoagulant cofactor activity of protein S for the anticoagulant protease, activated protein C (APC). EGF1 completely inhibited the stimulation of APC activity by protein S in plasma coagulation assays, with 50% inhibition at approx. 1µM EGF1, suggesting direct binding of EGF1 to APC. To investigate a direct interaction between EGF1 and APC, fluorescence resonance energy transfer (FRET) experiments were employed. APC labelled in the active site with fluorescein as the donor, and phospholipid vesicles containing octadecylrhodamine as the acceptor, showed that EGF1 association with APC caused an increase in energy transfer consistent with a relocation of the active site of APC from 94Å (9.4nm) to 85Å above the phospholipid surface (assuming κ2 = 2/3). An identical increase in energy transfer between the APC active site-bound fluorescein and phospholipid-bound rhodamine was obtained upon association of protein S or protein S–C4b-binding protein complex with APC. The latter suggests the presence of a ternary complex of protein S–C4b-binding protein with APC on the phospholipid surface. To confirm a direct interaction of EGF1 with APC, rhodamine was covalently attached to the α-N-terminus of EGF1, and binding of the labelled EGF1 to APC was directly demonstrated using FRET. The data suggested a separation between the active site of APC and the N-terminus of EGF1 of 76Å (κ2 = 2/3), placing the APC-bound protein S-EGF1 close to, but above, the phospholipid surface and near the two EGF domains of APC. Thus we provide direct evidence for binding of protein S-EGF1 to APC and show that it induces a conformational change in APC.
Inhibition of smooth-muscle myosin-light-chain phosphatase by Ruthenium Red
Authors: YAMADA Aki , SATO Osamu , WATANABE Minoru , WALSH Michael P. , OGAWA Yasuo , IMAIZUMI Yuji
Published: August 2015

Ruthenium Red (RuR) is widely used as an inhibitor of ryanodine receptor Ca2+ release channels, but has additional effects such as the induction of Ca2+ sensitization of contraction of permeabilized smooth muscles. To address the mechanism underlying this process, we examined the effects of RuR on contractility in permeabilized guinea-pig ileum and on the activity of myosin-light-chain phosphatase (MP). RuR increased the force at submaximal [Ca2+] (pCa 6.3) approx. 4-fold. This effect was not observed after thiophosphorylation of MP. RuR also seemed capable of preventing the thiophosphorylation of MP, suggesting a direct interaction of RuR with MP. Consistent with this possibility, smooth-muscle MP was inhibited by RuR in a concentration-dependent manner (IC50 23µM). Exogenous calmodulin significantly increased RuR-induced contraction at pCa 6.3 but had little effect on contraction induced by microcystin at this [Ca2+]. Ca2+-independent contraction was induced by RuR (EC50 843µM) and by microcystin (EC50 59nM) but the maximal force induced by RuR was smaller than that induced by microcystin. The addition of 300µM RuR enhanced the contraction induced by 30nM microcystin but markedly decreased that induced by 1µM microcystin. Such a dual action of RuR on microcystin-induced effects was not observed in experiments using purified MP. We conclude that the RuR-induced Ca2+ sensitization of smooth-muscle contraction is due to the direct inhibition of MP by RuR.
Inhibition of mitogen-activated protein kinase by a Drosophila dual-specific phosphatase
Authors: LEE Won-Jae , KIM Sun-Hong , KIM Yong-Sik , HAN Sung-Jun , PARK Ki-Sook , RYU Ji-Hwan , HUR Man-Wook , CHOI Kang-Yell
Published: August 2015

The Drosophila extracellular signal-regulated kinase (DERK) mitogen-activated protein kinase (MAPK) is involved in the regulation of multiple differentiation and developmental processes. Tight control of MAPK activity is critical for normal cell behaviour. We identified a novel Drosophila MAPK phosphatase (DMKP) cDNA from the expressed-sequence-tag database and characterized it. Analysis of the nucleotide sequence revealed an open reading frame encoding the 203-amino acid protein, with a calculated molecular mass of 23kDa, which has a high amino acid sequence similarity with ‘VH1-like’dual-specific phosphatases at the broad region near the catalytic sites. The expression of DMKP mRNA occurs from the late larval stages to adulthood in Drosophila development. The recombinant DMKP protein produced in yeast retained its phosphatase activity. When expressed in Schneider cells, DMKP dose-dependently inhibited DERK and Drosophila c-Jun N-terminal kinase activities with high selectivity towards DERK. However, DMKP did not have any affect on Drosophila p38 activity. When DMKP was expressed in yeast, it down-regulated the fus1-lacZ trans-reporter gene of the pheromone MAPK pathway without any significant effect on the high-osmolarity-glycerol-response pathway.
Methylation and expression of amplified esterase genes in the aphid Myzus persicae (Sulzer)
Authors: FIELD Linda M.
Published: August 2015

Most populations of the aphid Myzus persicae have amplified genes (up to 80 copies) encoding the insecticide-detoxifying esterase E4. This paper reports the analysis of methylation of the E4 gene and its flanking DNA with the use of methylation-sensitive restriction enzymes, CpG profiling and bisulphite sequencing. In combination these show that E4 has 5-methylcytosine confined to CpG doublets, as previously shown for vertebrate genomes; this is the first such report for an insect gene. The methylation is present within the gene but absent from upstream regions, including the 5´ CpG-rich region around the start of transcription, and from 3´ flanking DNA. Methylated E4 genes are expressed; loss of the 5-methylcytosine is correlated with a loss of transcription, although this is not accompanied by a global loss of the 5-methylcytosine present in the aphid genome. These results suggest that the methylation of E4 has a positive role in expression, and call into question the widely held view that methylation in invertebrate genomes is confined to regions that do not contain genes and that methylation is always associated with gene silencing.
Band 3 mutations, renal tubular acidosis and South-East Asian ovalocytosis in Malaysia and Papua New Guinea: loss of up to 95% band 3 transport in red cells
Authors: BRUCE Lesley J. , WRONG Oliver , TOYE Ashley M. , YOUNG Mark T. , OGLE Graham , ISMAIL Zulkifli , SINHA Ashim K. , MCMASTER Paddy , HWAIHWANJE Ilomo , NASH Gerard B. , HART Sarah , LAVU Evelyn , PALMER Ronald , OTHMAN Ainoon , UNWIN Robert J. , TANNER Michael J. A.
Published: August 2015

We describe three mutations of the red-cell anion exchanger band 3 (AE1, SLC4A1) gene associated with distal renal tubular acidosis (dRTA) in families from Malaysia and Papua New Guinea: Gly701 → Asp (G701D), Ala858 → Asp (A858D) and deletion of Val850 (δV850). The mutations A858D and ∆V850 are novel; all three mutations seem to be restricted to South-East Asian populations. South-East Asian ovalocytosis (SAO), resulting from the band 3 deletion of residues 400–408, occurred in many of the families but did not itself result in dRTA. Compound heterozygotes of each of the dRTA mutations with SAO all had dRTA, evidence of haemolytic anaemia and abnormal red-cell properties. The A858D mutation showed dominant inheritance and the recessive ∆V850 and G701D mutations showed a pseudo-dominant phenotype when the transport-inactive SAO allele was also present. Red-cell and Xenopus oocyte expression studies showed that the ∆V850 and A858D mutant proteins have greatly decreased anion transport when present as compound heterozygotes (∆V850/A858D, ∆V850/SAO or A858D/SAO). Red cells with A858D/SAO had only 3% of the SO42- efflux of normal cells, the lowest anion transport activity so far reported for human red cells. The results suggest dRTA might arise by a different mechanism for each mutation. We confirm that the G701D mutant protein has an absolute requirement for glycophorin A for movement to the cell surface. We suggest that the dominant A858D mutant protein is possibly mis-targeted to an inappropriate plasma membrane domain in the renal tubular cell, and that the recessive ∆V850 mutation might give dRTA because of its decreased anion transport activity.
Mutagenesis studies on the sensitivity of Escherichia coli acetohydroxyacid synthase II to herbicides and valine
Authors: LEE Yu-Ting , DUGGLEBY Ronald G.
Published: August 2015

Acetohydroxyacid synthase (EC 4.1.3.18, also known as acetolactate synthase) isoenzyme II from Escherichia coli is inhibited by sulphonylurea and imidazolinone herbicides, although it is much less sensitive than the plant enzyme. This isoenzyme is also unusual in that it is not inhibited by valine. Mutating S100 (Ser100 in one-letter amino acid notation) of the catalytic subunit to proline increases its sensitivity to sulphonylureas, but not to imidazolinones. Mutating P536 to serine, as found in the plant enzyme, had little effect on the properties of the enzyme. Mutating E14 of the regulatory subunit to glycine, either alone or in combination with the H29N (His29 → Asn) change, did not affect valine-sensitivity.
Effects of modification of the hydrophobic C-1–C-16 segment of tautomycin on its affinity to type-1 and type-2A protein phosphatases
Authors: TAKAI Akira , TSUBOI Katsunori , KOYASU Masayoshi , ISOBE Minoru
Published: August 2015

Among the naturally occurring toxins that are known to have specific inhibitory effects on type-1 and type-2A protein phosphatases (PP1 and PP2A), tautomycin (TM) is unique in that it exhibits significantly higher affinity to PP1 than to PP2A. The ratio of the dissociation constant for the PP1–TM interaction to that for the PP2A–TM interaction (the PP1/PP2A ratio) is 0.01–0.03. The aim of the present study was to evaluate the possible contributions of the C-1–C-16 segment of TM to the affinity characteristics of the toxin. The relatively hydrophobic segment contains a spiroketal motif whose enantiomeric form is present in okadaic acid (OA), which exhibits exceedingly higher affinity to PP2A than to PP1. We therefore synthesized two TM analogues: TM1 in which the side chains of the spiroketal motif of TM were removed but its absolute configuration was retained, and TM2 in which the spiroketal motif of TM1 was replaced with its enantiomeric form. The effects of TM, TM1 and TM2 on the activities of the native catalytic subunits of PP1 (PP1C) and PP2A and a recombinant γ isoform of PP1 (PP1γ) were examined. The PP1/PP2A ratio determined thereby was 0.2–0.5 for TM1 and 5–10 for TM2. Both the presence of the side chains and the stereochemistry of the spiroketal moieties may be major determining factors for the affinity characteristics of TM. We also show that a monoclonal antibody raised against OA binds to TM2 albeit with much lower affinity than to OA, whereas it exhibits no measurable affinities to TM and TM1.
The diffusive component of intestinal glucose absorption is mediated by the glucose-induced recruitment of GLUT2 to the brush-border membrane
Authors: KELLETT George L. , HELLIWELL Philip A.
Published: August 2015

We have investigated the mechanism responsible for the diffusive component of intestinal glucose absorption, the major route by which glucose is absorbed. In perfused rat jejunum in vivo, absorption was strongly inhibited by phloretin, an inhibitor of GLUT2. The GLUT2 level at the brush-border membrane increased some 2-fold when the luminal glucose concentration was changed from 0 to 100mM. The phloretin-sensitive or diffusive component of absorption appeared superficially linear and consistent with simple diffusion, but was in fact carrier-mediated and co-operative (n = 1.6, [G1/2] = 56mM; where [G1/2] is the glucose concentration at half Vmax) because of the glucose-induced activation and recruitment of GLUT2 to the brush-border membrane. Diffusive transport by paracellular flow was negligible. The phloretin-insensitive, SGLT1-mediated, component of glucose absorption showed simple saturation kinetics with [G1/2] = 27mM: the activation of protein kinase C (PKC) βII, the isoenzyme of PKC that most probably controls GLUT2 trafficking [Helliwell, Richardson, Affleck and Kellett (2000) Biochem. J. 350, 149–154], also showed simple saturation kinetics, with [G1/2] = 21mM. We conclude that the principal route for glucose absorption is by GLUT2-mediated facilitated diffusion across the brush-border membrane, which is up to 3-fold greater than that by SGLT1; the magnitude of the diffusive component at any given glucose concentration correlates with the SGLT1-dependent activation of PKC βII. The implications of these findings for the assimilation of sugars immediately after a meal are discussed.
The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells
Authors: DIMMER Kai-Stefan , FRIEDRICH Björn , LANG Florian , DEITMER Joachim W. , BRÖER Stefan
Published: August 2015

Transport of lactate and other monocarboxylates in mammalian cells is mediated by a family of transporters, designated monocarboxylate transporters (MCTs). The MCT4 member of this family has recently been identified as the major isoform of white muscle cells, mediating lactate efflux out of glycolytically active myocytes [Wilson, Jackson, Heddle, Price, Pilegaard, Juel, Bonen, Montgomery, Hutter and Halestrap (1998) J. Biol. Chem. 273, 15920–15926]. To analyse the functional properties of this transporter, rat MCT4 was expressed in Xenopus laevis oocytes and transport activity was monitored by flux measurements with radioactive tracers and by changes of the cytosolic pH using pH-sensitive microelectrodes. Similar to other members of this family, monocarboxylate transport via MCT4 is accompanied by the transport of H+ across the plasma membrane. Uptake of lactate strongly increased with decreasing extracellular pH, which resulted from a concomitant drop in the Km value. MCT4 could be distinguished from the other isoforms mainly in two respects. First, MCT4 is a low-affinity MCT: for l-lactate Km values of 17±3mM (pH-electrode) and 34±5mM (flux measurements with l-[U-14C]lactate) were determined. Secondly, lactate is the preferred substrate of MCT4. Km values of other monocarboxylates were either similar to the Km value for lactate (pyruvate, 2-oxoisohexanoate, 2-oxoisopentanoate, acetoacetate) or displayed much lower affinity for the transporter (β-hydroxybutyrate and short-chain fatty acids). Under physiological conditions, rat MCT will therefore preferentially transport lactate. Monocarboxylate transport via MCT4 could be competitively inhibited by α-cyano-4-hydroxycinnamate, phloretin and partly by 4,4´-di-isothiocyanostilbene-2,2´-disulphonic acid. Similar to MCT1, monocarboxylate transport via MCT4 was sensitive to inhibition by the thiol reagent p-chloromercuribenzoesulphonic acid.
Structural basis for the functional difference between Smad2 and Smad3 in FAST-2 (forkhead activin signal transducer-2)-mediated transcription
Authors: NAGARAJAN Raman P. , CHEN Yan
Published: August 2015

Smad2 and Smad3 are signalling proteins that are involved in mediating the transcriptional regulation of target genes downstream of transforming growth factor-β and activin receptors. Although they are structurally very similar, Smad2 and Smad3 have some functional differences in transducing signals for these receptors. In FAST-2 (forkhead activin signal transducer-2)-mediated transcriptional regulation using the activin-responsive element derived from Xenopus Mix.2 promoter as a reporter, Smad3 but not Smad2 alone was able to stimulate the transcription. In addition, Smad3 was able to inhibit the transactivation of the promoter induced by co-expression of Smad2, Smad4 and an active activin type-I receptor. We used a series of chimaeras between Smad1 and Smad3 and found that the Mad homology 1 (MH1) domain of Smad3 was indispensable for the dual regulatory function of Smad3. However, this Smad3-specific function could not be manifested in Smad2 mutants that were devoid of the two amino acid insertions (at the MH1 domain) that comprise the major structural difference between Smad2 and Smad3, indicating that other structural motifs are involved in determining the regulatory activity of Smad3. By using chimaeras between Smad2 and Smad3, we found that the most N-terminal portion of Smad3 was crucial for its function. Taken together, these results suggest that, as compared with Smad2, the unique function of Smad3 in modulating the FAST-2-mediated transcription is contributed to by a subtle difference in the structural features at the MH1 domain.
Thrombospondin 1 does not activate transforming growth factor β1 in a chemically defined system or in smooth-muscle-cell cultures
Authors: GRAINGER David J. , FROW Emma K.
Published: August 2015

The cytokine transforming growth factor β1 (TGF-β1) is secreted in a latent form that has no known biological activity. The conversion of latent TGF-β1 into its biologically active 25kDa form is thought to be an important step in the regulation of TGF-β activity both in cell culture and in vivo. Thrombospondin (TSP)-1, a 360kDa platelet α-granule and extracellular matrix protein, has been shown to participate in TGF-β1 activation. We have used a chemically defined system to examine the mechanism of TSP-1-mediated TGF-β1 activation. However, the addition of two different preparations of TSP-1 to recombinant small latent TGF-β1 in the test tube resulted in only a very small increase in the proportion of the TGF-β1 able to bind to the TGF-β type II receptor: from 0.1% to a maximum of 0.4%. This small effect was not specific for TSP-1: matrix metalloproteinase 2, tissue inhibitor of matrix metalloproteinase 2 and active plasminogen activator inhibitor 1, but not transglutaminase, human serum albumin or immunoglobulin, had quantitatively similar effects on latent TGF-β1. Furthermore, no change in the activity associated with small latent TGF-β1 was noted in either mink lung epithelial cell or rat aortic smooth-muscle cell culture systems in the presence of TSP-1 (or TSP-1-derived peptides). We conclude that TSP-1, either alone or in the presence of cultured smooth-muscle cells (a cell type known to activate latent TGF-β in vitro and in vivo) is unable to activate latent TGF-β1. Any TSP-mediated activation of TGF-β1 must depend on additional factor(s) not present in our systems.
Sac phosphatase domain proteins
Authors: HUGHES William E. , COOKE Frank T. , PARKER Peter J.
Published: August 2015

Advances in our understanding of the roles of phosphatidylinositol phosphates in controlling cellular functions such as endocytosis, exocytosis and the actin cytoskeleton have included new insights into the phosphatases that are responsible for the interconversion of these lipids. One of these is an entirely novel class of phosphatase domain found in a number of well characterized proteins. Proteins containing this Sac phosphatase domain include the yeast Saccharomyces cerevisiae proteins Sac1p and Fig4p. The Sac phosphatase domain is also found within the mammalian phosphoinositide 5-phosphatase synaptojanin and the yeast synaptojanin homologues Inp51p, Inp52p and Inp53p. These proteins therefore contain both Sac phosphatase and 5-phosphatase domains. This review describes the Sac phosphatase domain-containing proteins and their actions, with particular reference to the genetic and biochemical insights provided by study of the yeast Saccharomyces cerevisiae.