bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional sensitivity of polar surfaces on transmembrane helix 8 and cytoplasmic loop 8-9 of the Escherichia coli GABA (4-aminobutyrate) transporter encoded by gabP: mutagenic analysis of a consensus amphipathic region found in transporters from bacteria to mammals
Authors: HU A. Liaoyuan , KING C. Steven
Published: August 2015

The gab permease (GabP) catalyses transport of GABA (4-aminobutyrate) into Escherichia coli. Although GabP can recognize and transport many GABA analogues that exhibit activity at GABAergic synapses in the nervous system, the protein domains responsible for these transport and ligand recognition properties have not been studied. Here we report that an amphipathic domain extending through putative transmembrane helix 8 and into the adjoining cytoplasmic region (loop 8-9) contains a critical 20 residue zone within which mutagenesis of polar amino acids has a deleterious effect on [3H]GABA transport activity. This functionally important amphipathic domain is found to be highly conserved in the many APC family transporters that are homologous to GabP. And even though members of the GAT family of GABA transporters from the animal nervous system are not homologous to GabP, an analogous amphipathic structure is found in their loop 8-9 region. These results and observations suggest: (1) that the consensus amphipathic region (CAR) in the putative helix 8 and loop 8-9 region of GabP has functional significance, and (2) that nature has repeatedly used this CAR in transporters from bacteria to mammals.
Ethanol potentiates the stimulatory effects of insulin and phosphocholine on mitogenesis by a zinc-dependent and rapamycin-sensitive mechanism in fibroblasts and JB6 cells
Authors: KISS Zoltan , ANDERSON H. Wayne , MUKHERJEE J. Jagat
Published: August 2015

In most cellular systems ethanol inhibits growth factor-induced cell growth. Here we examined the effects of ethanol on DNA synthesis and cell proliferation induced by insulin and phosphocholine (PCho) in NIH 3T3 fibroblasts, Swiss 3T3 fibroblasts and mouse epidermal JB6 cells. In serum-starved low (12-18) passage NIH 3T3 fibroblasts, 60 mM ethanol enhanced the mitogenic effect of insulin in the absence or presence of 25 μM zinc about 2- or 12-fold, respectively. In contrast, in serum-starved high (30-47) passage NIH 3T3 cells 60 mM ethanol had large (20-40-fold) potentiating effects on insulin-induced DNA synthesis even in the absence of zinc. Furthermore, ethanol also enhanced the effects of PCho on DNA synthesis in both the absence and presence of insulin. The potentiating effects of ethanol on insulin- and PCho-induced DNA synthesis were associated with 1.2-1.3-fold stimulation of cell proliferation. Rapamycin, an inhibitor of p70 S6 kinase action, strongly inhibited the potentiating effects of ethanol on insulin- and PCho-induced mitogenesis. Unexpectedly, ethanol inhibited synergistic activation of p42/p44 mitogen-activated protein kinases by insulin and PCho. In both Swiss 3T3 and JB6 cells, ethanol potentiated insulin-induced DNA synthesis only in the presence of zinc. In these cells, ethanol also increased the effects of PCho on both DNA synthesis and cell proliferation in the co-presence of either insulin or ATP. The results indicate that in various cell lines physiologically relevant concentrations of ethanol can increase the ability of insulin and PCho to induce DNA synthesis and, to smaller extents, cell proliferation. In low passage NIH 3T3 cells as well as in Swiss 3T3 and JB6 cells potentiation of insulin-induced DNA synthesis by ethanol requires the presence of zinc.
Protein serine/threonine phosphatase inhibitors suppress phenobarbital-induced Cyp2b10 gene transcription in mouse primary hepatocytes
Authors: HONKAKOSKI Paavo , NEGISHI Masahiko
Published: August 2015

Using a primary hepatocyte culture in which the mouse Cyp2b10 gene transcription is activated by phenobarbital (PB)-type inducers, we examined the cellular signalling mechanisms associated with PB induction. Low nanomolar concentrations of protein serine/threonine phosphatase inhibitors okadaic acid (OA) and calyculin A blocked the induction of CYP2B10 mRNA. Nuclear run-on assays indicated that OA suppressed Cyp2b10 gene transcription. Pretreatment of the cells with an inhibitor of Ca2+/calmodulin-dependent protein kinases {1-[N,O-bis-(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62)} or with a flavonoid, naringin, were completely or partly protective respectively against the OA-mediated suppression of CYP2B10 mRNA. Several other established modulators of protein kinase activities did not greatly affect the induction of CYP2B10 mRNA, nor could they prevent the suppressive effect of OA. Our results indicate that specific protein phosphorylation-dephosphorylation is required for the induction of Cyp2b10 gene expression, which is modulated through multiple endogenous and exogenous signals.
Cleavage of native type I collagen by human neutrophil elastase
Authors: KAFIENAH Wa'el , BUTTLE J. David , BURNETT David , HOLLANDER P. Anthony
Published: August 2015

The ability of purified human neutrophil elastase (EC 3.4.21.37) to cleave native type I collagen has been investigated. Soluble human, bovine or rat type I collagen was incubated with neutrophil elastase for 16 h at 25 °C before catalysis was stopped with 3,4-dichloroisocoumarin. Analysis by SDS/PAGE of the collagen digests revealed 3/4-length fragments similar in size to those produced by interstitial collagenase. The collagenolytic activity was dose dependent and was not due to a contaminating metalloproteinase or cysteine proteinase, as it was not inhibited by 1,10-phenanthroline, EDTA or L-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane. The identity of the cleavage products was confirmed using a new antibody that recognizes the unwound α2(I)-chain. This detected the 3/4-length fragment of type I collagen following neutrophil elastase cleavage. In addition to cleaving soluble collagen, neutrophil elastase also cleaved reconstituted, radiolabelled type I collagen fibrils, at a rate of 16 μg/min per nmol. These results indicate that neutrophil elastase can cleave native type I collagen in the helix, an activity that might contribute to its roles in connective-tissue pathology.
Interaction in vitro of the product of the c-Crk-II proto-oncogene with the insulin-like growth factor I receptor
Authors: KOVAL P. Anatolii , BLAKESLEY A. Vicky , ROBERTS T. Charles , ZICK Yehiel , LeROITH Derek
Published: August 2015

The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein. We have previously demonstrated that Crk-II becomes rapidly tyrosine-phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) and might be involved in the IGF-I receptor signalling pathway. To determine whether this involvement includes the direct interaction of Crk-II with the cytoplasmic region of the receptor, studies were performed in vitro with glutathione S-transferase (GST) fusion proteins containing various domains of Crk-II. The kinase assay in vitro showed that activated IGF-I receptors efficiently phosphorylated the GST-Crk-II fusion protein. This phosphorylation was dependent on the presence of the SH2 domain and Tyr-221 located in the spacer region between the two SH3 domains. Mutation of Tyr-221 not only prevented phosphorylation of GST-Crk in vitro, but also significantly increased the ability of GST-Crk proteins to co-precipitate activated IGF-I receptors from total cell lysates. Additional binding experiments in vitro showed that Crk-II might interact with the phosphorylated IGF-I receptor through its SH2 domain. To elucidate which region of the IGF-I receptor interacts with Crk-II, a peptide association assay was used in vitro. Different domains of the IGF-I receptor were expressed as (His)6-tagged fusion peptides, phosphorylated with activated wheat germ agglutinin-purified IGF-I receptors and tested for association with GST-Crk-II fusion proteins. Using wild-type as well as mutated peptides, we showed that the SH2 domain of Crk-II preferentially binds the peptide encoding the juxtamembrane region of the IGF-I receptor. Phosphorylation of Tyr-950 and Tyr-943 of the receptor is important for this interaction. These findings allow us to propose a model of direct interaction of Crk-II and the IGF-I receptor in vivo. On activation of the IGF-I receptor, Crk-II binds to phosphorylated tyrosine residues, especially in the juxtamembrane region. As a result of this binding, the IGF-I receptor kinase phosphorylates Tyr-221 of Crk-II, resulting in a change in intramolecular folding and binding of the SH2 domain to the phosphorylated Tyr-221, which causes rapid disassociation of the Crk-II-IGF-I receptor complex.
Evidence for a model of integrated inositol phospholipid pools implies an essential role for lipid transport in the maintenance of receptor-mediated phospholipase C activity in 1321N1 cells
Authors: BATTY H. Ian , CURRIE A. Richard , DOWNES C. Peter
Published: August 2015

The compartmentation of inositol phospholipids was examined by using a combination of radiolabelling approaches in intact and permeabilized 1321N1 astrocytoma cells. A ‘chase’ protocol was developed with whole cells in which phosphoinositide (PI) pools were labelled to steady state with [3H]inositol and the cellular [3H]inositol pool was then diluted selectively with non-radioactive inositol. In these cells muscarinic-receptor-stimulated phospholipase C (PLC) hydrolysed [3H]PI at approx. 1-2%/min. However, after the chase procedure the relative specific radioactivity of [3H]Ins(1,3,4)P3, a rapidly metabolized and sensitive marker of PLC activity, decreased only after more than 5 min and over a time course similar to that during which the labelling of each [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 declined by at least 50%. These results demonstrate a large receptor-responsive [3H]PI pool that is accessed by stimulated PLC without apparent metabolic compartmentation, despite its probable distribution between different membrane fractions. Support for this was obtained in intact cells by using an acute [3H]inositol labelling method in which increases in the specific radioactivity of [3H]inositol phosphates stimulated by carbachol occurred only in parallel with similar increases in the labelling of the bulk of cellular [3H]PI. In [3H]inositol-prelabelled cells permeabilized to deplete cytosolic proteins, carbachol and guanosine 5ʹ-[γ-thio]triphosphate stimulated the endogenous PLC to degrade only approx. 5% of [3H]PI. This was increased to approx. 30% in the presence of exogenous PtdIns transfer protein, which, at a concentration approx. 5-10% of that in 1321N1 cell cytosol, was sufficient to support PLC activity comparable with that observed in response to carbachol in whole cells. These and earlier results in 1321N1 cells suggest a model of integrated PI pools involving an obligatory role for lipid transport. Given the multifunctional capacity of PI in cellular signalling mechanisms, this model has important implications, particularly for the hypothesis that the ability of Li+ ions to influence these selectively might account for its therapeutic actions.
Oestrogen and progesterone inhibit the stimulated production of endothelin-1
Authors: MOREY K. Anjali , RAZANDI Mahnaz , PEDRAM Ali , HU Ren-Ming , PRINS A. Bruce , LEVIN R. Ellis
Published: August 2015

Important vascular proteins such as endothelin-1 (ET-1) promote the development of cardiovascular diseases. Oestrogen, and perhaps progesterone, prevent the development of vascular disease in women through incompletely understood cellular mechanisms. We hypothesized that oestradiol or progesterone might regulate the production of ET-1 as a potential novel mechanism. We found that serum and angiotensin II (AII) significantly stimulated ET-1 secretion from cultured bovine aortic endothelial cells, inhibited 50-75% by oestradiol or by progesterone. Serum and AII stimulated ET-1 mRNA levels, inhibited at least 70% by oestradiol and by progesterone. Serum stimulated ET-1 transcription mainly through the first 43 nucleotides of the ET-1 promoter, but oestradiol and progesterone did not inhibit this. In contrast, AII stimulated ET-1 transcription through nucleotides -143 to -98, specifically involving an activator protein-1 (AP-1) site at -102. Oestradiol and progesterone caused a 60-70% inhibition of AII-stimulated wild-type construct -.143ET-1/CAT activity (CAT is chloramphenicol acyltransferase). AII-stimulation of ET-1 transcription was critically dependent on stimulation of mitogen-activated protein kinase (erk) activity, inhibited by oestradiol and progesterone. In summary, we found that sex steroids inhibit AII-induced erk signalling to the ET-1 transcriptional programme. This novel mechanism of negative transcriptional regulation by oestradiol and progesterone decreases the production of ET-1, potentially contributing to the vascular protective effects of these steroids.
Recombinant expression and biochemical characterization of an NADPH:flavin oxidoreductase from Entamoeba histolytica
Authors: BRUCHHAUS Iris , RICHTER Symi , TANNICH Egbert
Published: August 2015

The gene encoding a putative NADPH:flavin oxidoreductase of the protozoan parasite Entamoeba histolytica (Eh34) was recombinantly expressed in Escherichia coli. The purified recombinant protein (recEh34) has a molecular mass of about 35 kDa upon SDS/PAGE analysis, exhibits a flavoprotein-like absorption spectrum and contains 1 mol of non-covalently bound FMN per mol of protein. RecEh34 reveals two different enzymic activities. It catalyses the NADPH-dependent reduction of oxygen to hydrogen peroxide (H2O2), as well as of disulphides such as 5,5ʹ-dithiobis-(2-nitrobenzoic acid) (DTNB) and cystine. The disulphide reductase but not the H2O2-forming NADPH oxidase activity is inhibitable by sulphydryl-active compounds, indicating that a thiol component is part of the active site for the disulphide reductase activity, whereas for the H2O2-forming NADPH oxidase activity only the flavin is required. Compared with the recombinant protein, similar activities are present in amoebic extracts. Native Eh34 is active in a monomeric as well as in a dimeric state. In contrast to recEh34, no flavin was associated with the native protein. However, both NADPH oxidase as well as DTNB reductase activity were found to be dependent on the addition of FAD or FMN.
Human platelet heparanase: purification, characterization and catalytic activity
Authors: FREEMAN Craig , PARISH R. Christopher
Published: August 2015

Heparan sulphate (HS) is an important component of the extracellular matrix (ECM) and the vasculature basal lamina (BL) which functions as a barrier to the extravasation of metastatic and inflammatory cells. Platelet-tumour cell aggregation at the capillary endothelium results in activation and degranulation of platelets. Cleavage of HS by endoglycosidase or heparanase activity produced in relatively large amounts by the platelets and the invading cells may assist in the disassembly of the ECM and BL, and thereby facilitate cell migration. Using a recently published rapid, quantitative assay for heparanase activity towards HS [Freeman, C. and Parish, C. R. (1997), Biochem. J., 325, 229-237], human platelet heparanase has now been purified 1700-fold to homogeneity in 19% yield by a five column procedure, which consists of concanavalin A-Sepharose, Zn2+-chelating-Sepharose, Blue A-agarose, octyl-agarose and gel filtration chromatography. The enzyme, which was shown to be an endoglucuronidase that degrades both heparin and HS, has a native molecular mass of 50 kDa when analysed by gel filtration chromatography and by SDS/PAGE. Platelet heparanase degraded porcine mucosal HS in a stepwise fashion from a number average molecular mass of 18.5 to 13, to 8 and finally to 4.5 kDa fragments as determined by gel filtration analysis. Bovine lung heparin was degraded from 8.9 to 4.8 kDa while porcine mucosal heparin was degraded from 8.1 kDa to 3.8 and finally to 2.9 kDa fragments. Studies of the enzyme's substrate specificity using modified heparin analogues showed that substrate cleavage required the presence of carboxyl groups, but O- and N-sulphation were not essential. Inhibition studies demonstrated an absolute requirement for the presence of O-sulphate groups. Platelet heparanase was inhibited by heparin analogues which also inhibited tumour heparanase, suggesting that sulphated polysaccharides which inhibit tumour metastasis may act to prevent both tumour cell and platelet heparanase degradation of endothelial cell surface HS and the basal laminar.
α- and δ-tocopherol induce expression of hepatic α-tocopherol-transfer-protein mRNA
Authors: FECHNER Henry , SCHLAME Michael , GUTHMANN Florian , STEVENS Paul A. , RÜSTOW Bernd
Published: August 2015

α-Tocopherol transfer protein (α-TTP) supplements nascent very-low-density lipoprotein (VLDL) preferentially with α-tocopherol by selecting the α-isomers against other stereoisomers of tocopherol. It is exclusively expressed in liver. We investigated whether the expression of the hepatic α-TTP can be induced by dietary tocopherols. Vitamin E-depleted rats were fed with a diet containing α- and δ-tocopherol (ratio 1:3). The expression of α-TTP mRNA was measured in liver tissue. The ratio of tocopherol stereoisomers was determined in plasma, plasma lipoproteins and tissues to measure the metabolic action of α-TTP. Refeeding a diet containing either α- or δ-tocopherol, or both, caused a steady increase of the expression of α-TTP mRNA. In parallel the α/δ-tocopherol ratio increased in plasma, VLDL, high-density lipoprotein and low-density lipoprotein as well as in liver tissue, when the diet was fed containing both isomers. The α-tocopherol/δ-tocopherol ratio of heart, kidney, lung, lamellar bodies of lung and in lung lavage showed no or a comparatively low increase. The data show that both tocopherol isomers were able to induce α-TTP mRNA in rat liver and, thus, the ability of liver to select for the α-isomer. On the other hand, tocopherol depletion did not change the expression of hepatic α-TTP mRNA in the rat.
cDNA cloning reveals two mouse β5 integrin transcripts distinct in cytoplasmic domains as a result of alternative splicing
Authors: ZHANG Haifeng , TAN Suet M. , LU Jinhua
Published: August 2015

The integrin β5 subunit has only been found to form a heterodimer with subunit αv which acts as a vitronectin receptor. Integrin αvβ5 has been implicated in cell migration and growth factor-induced angiogenesis. In the present study, a mouse liver cDNA library was screened using a human β5 cDNA fragment obtained by reverse transcriptase PCR (RT-PCR). Three of the clones (MB5, MB15 and MB17) overlapped to give an open reading frame, called β5A, which is homologous to the human β5 subunit. The sequence of another clone (MB26), called β5B, was identical with β5A, except for a deletion of 29 bp near the 3´ end of the open reading frame. The 29 bp deletion resulted in an open-reading-frame shift and a completely different C-terminal sequence in β5B. β5A and β5B were shown, by RT-PCR, to be co-expressed in most mouse tissues tested, although β5B mRNA was detected at much lower levels than β5A. β5A and β5B mRNAs were also detected in the mouse monocytic cell line, J774, and in isolated mouse peritoneal macrophages. Adhesion of peritoneal macrophages has been shown to up-regulate the expression of both β5A and β5B mRNAs. The 29 bp sequence begins with a putative intron-splicing donor site (GTGAT …). A 3´ fragment of the mouse integrin β5 gene was cloned by PCR and sequenced showing that the 29 bp sequence was also immediately followed by an intron. Therefore, the 29 bp sequence was apparently expressed as part of the β5A mRNA but was spliced out as part of the downstream intron in β5B. Since the cytoplasmic domains of the integrin β subunits are important in cytoskeleton attachment and signalling, the two alternatively spliced β5 isoforms may have distinct roles in cell adhesion and other cellular functions.
Cystathionine γ-synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli
Authors: RAVANEL Stéphane , GAKIÈRE Bertrand , JOB Dominique , DOUCE Roland
Published: August 2015

Cystathionine γ-synthase catalyses the first reaction specific for methionine biosynthesis in plants, the γ-replacement of the phosphoryl substituent of O-phosphohomoserine by cysteine. A cDNA encoding cystathionine γ-synthase from Arabidopsis thalianahas been cloned and used to overexpress the enzyme in Escherichia coli.The native recombinant enzyme is a homotetramer composed of 53 kDa subunits, each being tightly associated with one molecule of pyridoxal 5´-phosphate that binds at lysine-379 of the protein precursor. The replacement reaction follows a Ping Pong mechanism with a Vmax of 33.6 units/mg and Km values of 2.5 mM and 460 µM for O-phosphohomoserine and cysteine respectively. The protective effect of O-phosphohomoserine against enzyme inactivation by propargylglycine indicated that the Kd for the substrate is approx. 1/2500 of its Km value. Thus most of these biochemical properties are similar to those previously reported for plant and bacterial cystathionine γ-synthases. However, the plant enzyme differs markedly from its enterobacterial counterparts because it catalyses a very faint γ-elimination of O-phosphohomoserine in the absence of cysteine, this process being about 1/2700 as fast as the γ-replacement reaction and approx. 1/1500 as fast as the γ-elimination catalysed by the E. colienzyme. This huge difference could be attributed to the inability of the A. thalianacystathionine γ-synthase to accumulate a long-wavelength-absorbing species that is characteristic for the efficient γ-elimination reaction catalysed by the enterobacterial enzyme.
Protein heterogeneity of spinach pullulanase results from the coexistence of interconvertible isomeric forms of the monomeric enzyme
Authors: HENKER Anette , SCHINDLER Ilka , RENZ Andreas , BECK Erwin
Published: August 2015

Purified pullulanase (starch-debranching enzyme, R-enzyme, EC 3.2.1.41) from spinach (Spinacia oleracea L.) chloroplasts separated into at least seven individual enzymically active proteins (isomers, numbered 1–7) on isoelectric focusing or column chromatofocusing. At their isoelectric points (between pH 4.7 and 5.2) these forms were rather stable. At slightly alkaline pH, each converted into the whole set of isomers. PAGE of the purified enzyme under denaturing or non-denaturing conditions resulted in one protein band. When substrate (amylopectin or pullulan) was included in the gel, the native enzyme as well as any of the individual isomers separated into two (sometimes three) bands (‘substrate-induced forms’, numbered I–III) with different specific activities, dissociation constants of the enzyme–substrate complexes and activation energies. Each substrate-induced form produced the whole set of seven isomers on isoelectric focusing. The specific activity of the total enzyme reflected the relative proportions of the substrate-induced forms. To some extent the relative proportions, as determined by crossed immunoelectrophoresis, could be shifted in favour of the more or the less active forms by reduction with dithiothreitol, and gentle oxidation respectively. Activation by dithiothreitol did not alter the mode of action of the enzyme but only increased the velocity of substrate degradation and extended its activity into the pH range of the chloroplast. As a consequence of isomer interconversion, microheterogeneity could serve to regulate pullulanase activity in a biochemical manner that shares some features with allosteric regulation.
Complementation studies with co-expressed fragments of human red cell band 3 (AE1): the assembly of the anion-transport domain in Xenopus oocytes and a cell-free translation system
Authors: GROVES Jonathan D. , WANG Lin , TANNER Michael J. A.
Published: August 2015

We examined the assembly of the membrane domain of the human red cell anion transporter (band 3; AE1) by co-expression of recombinant N- and C-terminal fragments in Xenopus oocytes and in cell-free translation with canine pancreatic microsomes. Co-immunoprecipitation was performed in non-denaturing detergent solutions using antibodies directed against the N- and C-termini of the membrane domain. Eleven of the twelve fragments were expressed stably in oocytes in the presence or absence of their respective partners. However, the fragment containing from putative span nine to the C-terminus could be detected in oocytes only when co-expressed with its complementary partner containing the first eight spans. Co-expression of pairs of fragments divided in the first, second, third and fourth exofacial loops and in the fourth cytoplasmic loop resulted in a concentration-dependent association, but a pair of fragments divided in the sixth cytoplasmic loop did not co-immunoprecipitate. When two complementary fragments were translated separately in the cell-free system and the purified microsomes were then mixed, co-immunoprecipitation was observed only if the membranes were first fused using polyethylene glycol. This shows that co-immunoprecipitation results from specific interactions within the membrane and is not an artefact of detergent solubilization or immunoprecipitation. We demonstrate that band 3 assembly can occur within the membrane after translation, insertion and initial folding of the individual fragments have been completed. We conclude that most band 3 fragments contain the necessary information to fold in the membrane and adopt a structure that is sufficiently similar to the native protein that it permits correct assembly with its complementary partner.
Characterization of the functional properties of smooth muscle caldesmon domain 4a: evidence for an independent inhibitory actin–tropomyosin binding domain
Authors: EL-MEZGUELDI Mohammed , COPELAND O'Neal , FRASER Iain D. C. , MARSTON Steven B. , HUBER Pia A. J.
Published: August 2015

Recent analysis has shown the presence of three sequences in the C-terminal 170 amino acids of human caldesmon (domain 4) which are involved in actin binding and tropomyosin-dependent inhibition of actomyosin ATPase. Two are in domain 4b (amino acids 715–793) and one is in domain 4a (amino acids 636–714). In the present work we have compared recombinant peptides containing either domain 4a or domain 4b to address the question as to whether domain 4a alone has any inhibitory activity. We have produced three new recombinant fragments containing domain 4a: H10 [622–708], H12 [506–708] and H13 [622–726] and we have characterized their functional properties. All three fragments bound to actin and tropomyosin. Caldesmon, but not domain 4b, was able to displace the fragments H10, H12 and H13 from actin. Thus the isolated caldesmon domain 4a peptides bind to the same region on actin as in the whole molecule while domains 4a and 4b occupy different sites on the actin molecule. Unlike domain 4b, none of the domain 4a fragments inhibited the actomyosin ATPase in the absence of tropomyosin. However both domain 4a and 4b fragments displayed an inhibitory activity in the presence of tropomyosin. H13 and H12 were more potent inhibitors than H10. Ca2+-calmodulin bound to H13 and reversed the inhibitory activity of this fragment but did not bind to H10 and H12. We conclude that domain 4a can act as an independent inhibitory actin–tropomyosin binding domain, but its properties are very different from the extreme C-terminal domain 4b.
Cadmium-induced differential accumulation of metallothionein isoforms in the Antarctic icefish, which exhibits no basal metallothionein protein but high endogenous mRNA levels
Authors: CARGINALE Vincenzo , SCUDIERO Rosaria , CAPASSO Clemente , CAPASSO Antonio , KILLE Peter , PRISCO Guido di , PARISI Elio
Published: August 2015

Reverse transcriptase-mediated PCR has been used to isolate two distinct metallothionein (MT) cDNA species from RNA extracted from icefish liver, namely MT-I and MT-II. Northern blot analysis with these cDNA species revealed that significant endogenous levels of MT mRNA were present in liver tissues of normal animals despite the fact that no MT protein could be found accumulating in the same tissue. However, multiple injections of CdCl2 induced high levels of both MT mRNA and MT protein. Sequence analysis of the cDNA species that were present after cadmium injection revealed the presence of both isoforms. Quantification of the MT-I and MT-II transcripts from normal and heavy-metal-treated fish showed an alteration in the ratio of the MT isoform transcripts. Endogenous transcripts consisted mostly of MT-II, whereas the MT-I transcript was preferentially accumulated only in response to the cadmium salt. The protein encoded by each cDNA isoform was isolated from the heavy-metal-treated fish and the availability of the specific MT mRNA for translation was demonstrated by translation in vitro. These results show that: (1) there is a discrepancy between the significant endogenous levels of MT mRNA and the absence of MT protein; (2) the accumulation of MT in icefish liver can be triggered by heavy metals; (3) genes encoding distinct MT isoforms are differentially regulated by heavy metals.
Quantification of cathepsins B and L in cells
Authors: XING Ruye , ADDINGTON Adele K. , MASON Robert W.
Published: August 2015

A method for quantifying active cysteine proteinases in mammalian cells has been developed using an active-site-directed inhibitor. Fluoren-9-ylmethoxycarbonyl(di-iodotyrosylalanyl)-diazomethane (Fmoc-[I2]Tyr-Ala-CHN2) was prepared and shown to react irreversibly with cathepsins B and L, but not with cathepsin S. The non- and mono-iodo forms of the inhibitor reacted with all three enzymes. These results demonstrate that, unlike cathepsins B and L, cathepsin S has a restricted S2-binding site that cannot accommodate the bulky di-iodotyrosine. Fmoc-[I2]Tyr-Ala-CHN2 was able to penetrate cells and react with active enzymes within the cells. A radiolabelled form of the inhibitor was synthesized and the concentration of functional inhibitor was established by titration with papain. This inhibitor was used to quantify active cysteine proteinases in cultured cells. Active cathepsin B was found to be expressed by all of the cells studied, consistently with a housekeeping role for this enzyme. Active forms of cathepsin L were also expressed by all of the cells, but in different quantities. Two additional proteins were labelled in some of the cells, and these may represent other non-characterized proteinases. Higher levels of active cathepsins B and L, and an unidentified protein of Mr 39000, were found in breast tumour cells that are invasive, compared with those that are not invasive. From the data obtained, it can be calculated that the concentrations of both active cathepsins B and L in lysosomes can be as high as 1 mM, each constituting up to 20% of total protein in the organelle. This new technique provides a more direct procedure for determining the proteolytic potential of cellular lysosomes.
Transcript heterogeneity of the human reduced folate carrier results from the use of multiple promoters and variable splicing of alternative upstream exons
Authors: ZHANG Long , WONG So C. , MATHERLY Larry H.
Published: August 2015

We previously identified three separate cDNAs (KS6, KS32 and KS43) for the human reduced folate carrier (RFC) with unique 5´ untranslated regions (5´ UTRs) [Wong, Proefke, Bhushan and Matherly (1995) J. Biol. Chem. 270, 17468–17475]. Multiple RFC transcripts were confirmed in CCRF-CEM cells and transport-up-regulated K562.4CF cells by 5´ rapid amplification of cDNA ends (5´ RACE) and/or primer extension analysis. Two groups of 5´ RACE clones were identified, one containing a variable length sequence identical with the KS43 cDNA 5´ UTR, and another consisting of variants of the KS32 5´ UTR, apparently generated by alternative splicing. The 5´ UTR for the KS6 cDNA was not detected. A single band was detected on Southern blots of CCRF-CEM genomic DNA probed with a 326 bp genomic fragment common to all three cDNA species. The unique 5´ UTRs for the KS43 and KS32 transcripts were localized to separate non-coding exons (exons 1 and 2 respectively), upstream from a large (approx. 3.42 kb) intron; the KS6 5´UTR also mapped to exon 1. Exons 1 and 2 were contiguous with 996 and 342 bp GC-rich 5´ flanking regions (designated Pro43 and Pro32 respectively) that contained multiple SP1 and AP2 but no TATA or CAAT boxes. Both Pro43 and Pro32 exhibited strong promoter activities when cloned in front of a luciferase reporter gene and transfected into HT1080 and K562 cells. By an analysis of promoter deletion mutants we identified two 89 bp tandem repeats that seemed to increase Pro32 activity, and a 240 bp distal sequence that repressed Pro43 activity. Taken together, our results show that multiple human RFC transcripts are encoded by a single gene locus and that the heterogeneous 5´ UTRs result from multiple transcriptional starts and variable splicing of alternative non-coding exons transcribed from separate promoters.
Nuclear factor 1 regulates the distal silencer of the human PIT1/GHF1 gene
Authors: RAJAS Fabienne , DELHASE Mireille , HOYA Miguel de La , VERDOOD Peggy , CASTRILLO José-Luis , HOOGHE-PETERS Elisabeth L.
Published: August 2015

Here we report the characterization of 12 kb genomic DNA upstream of the human PIT1/GHF1 promoter. Different regions involved in the modulation of human PIT1/GHF1 gene expression were defined by transient transfection studies. Two regions, one proximal (-7.1/-2.3) and one distal (-11.8/-10.9), presented an enhancer activity in pituitary cells when placed upstream of the SV40 promoter. The 0.9 kb distal region was analysed further and found to decrease the basal transcriptional activity of the human PIT1/GHF1 minimal promoter, indicating that this region behaves as a silencer for its own promoter. Three Pit-1/GHF-1-binding sites and two ubiquitous nuclear factor 1 (NF-1)-binding sites were identified by DNase I footprinting in the distal regulatory region. Deletion analysis indicated that NF-1 or NF-1-related protein(s) participate in the down-regulation of human PIT1/GHF1 gene expression by interacting with an NF-1-binding site within the distal regulatory region.

The nucleotide sequence data reported will appear in DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number X97489.
Membrane type 1 matrix metalloproteinase (MT1-MMP) cleaves the recombinant aggrecan substrate rAgg1mut at the ‘aggrecanase’ and the MMP sites
Authors: BÜTTNER Frank H. , HUGHES Clare E. , MARGERIE Daniel , LICHTE Andrea , TSCHESCHE Harald , CATERSON Bruce , BARTNIK Eckart
Published: August 2015

The recent detection of membrane type 1 matrix metalloproteinase (MT1-MMP) expression in human articular cartilage [Büttner, Chubinskaya, Margerie, Huch, Flechtenmacher, Cole, Kuettner, and Bartnik (1997) Arthritis Rheum. 40, 704–709] prompted our investigation of MT1-MMP's catabolic activity within the interglobular domain of aggrecan. For these studies we used rAgg1mut, a mutated form of the recombinant fusion protein (rAgg1) that has been used as a substrate to monitor ‘aggrecanase ’ catabolism in vitro [Hughes, Büttner, Eidenmüller, Caterson and Bartnik (1997) J. Biol. Chem. 272, 20269–20274]. The rAgg1 was mutated (G332 to A) to avoid the generation of a splice variant seen with the original genetic construct, which gave rise to heterogeneous glycoprotein products. This mutation yielded a homogeneous recombinant product. Studies in vitro with retinoic acid-stimulated rat chondrosarcoma cells indicated that the rAgg1mut substrate was cleaved at the ‘aggrecanase ’ site equivalent to Glu373-Ala374 (human aggrecan sequence enumeration) in its interglobular domain sequence segment. The differential catabolic activities of the recombinant catalytic domain (cd) of MT1-MMP and matrix metalloproteinases (MMPs) 3 and 8 were then compared by using this rAgg1mut as a substrate. Coomassie staining of rAgg1mut catabolites separated by SDS/PAGE showed similar patterns of degradation with all three recombinant enzymes. However, comparative immunodetection analysis, with neoepitope antibodies BC-3 (anti-ARGS …) and BC-14 (anti-FFGV …) to distinguish between ‘aggrecanase ’ and MMP-generated catabolites, indicated that the catalytic domain of MT1-MMP exhibited strong ‘aggrecanase ’ activity, cdMMP-8 weak activity and cdMMP-3 no activity. In contrast, cdMMP-3 and cdMMP-8 led to strongly BC-14-reactive catabolic fragments, whereas cdMT1-MMP resulted in weak BC-14 reactivity. N-terminal sequence analyses of the catabolites confirmed these results and also identified other potential minor cleavage sites within the interglobular domain of aggrecan. These results indicate that MT1-MMP is yet another candidate for ‘aggrecanase ’ activity in vivo.
Sp3 regulates Fas expression in lung epithelial cells
Authors: PANG Huiling , MIRANDA Kathleen , FINE Alan
Published: August 2015

By transducing an apoptotic signal in immune effector cells, Fas has been directly implicated in the control of immunological activity. Expression and functional results, however, have also suggested a role for Fas in regulating cell turnover in specific epithelial populations. To characterize factors responsible for Fas expression in epithelial cells, approximately 3 kb of the 5´ flanking region of the mouse Fas gene was isolated. By rapid amplification of cDNA ends and primer extension, transcriptional start sites were identified within 50 bp upstream of the translation start site. Transient transfection of promoter–luciferase constructs in a mouse lung epithelial cell line, MLE-15, localized promoter activity to the first 77 bp of upstream sequence. By using a 60 bp DNA probe (-18 to -77) in electrophoretic mobility-shift assays, three shifted complexes were found. Incubation with excess cold Sp1 oligonucleotide or an anti-Sp3 antibody inhibited complex formation. Site-directed mutagenesis of the Sp1 site resulted in 60–70% loss of promoter activity. In Drosophila SL-2 cells, promoter activity was markedly increased by co-transfection of an Sp3 expression construct. These results show that the Sp3 protein is involved in regulating Fas gene expression in lung epithelial cells.
Loss of the hepatic glycogen-binding subunit (GL) of protein phosphatase 1 underlies deficient glycogen synthesis in insulin-dependent diabetic rats and in adrenalectomized starved rats
Authors: DOHERTY Martin J. , CADEFAU Joan , STALMANS Willy , BOLLEN Mathieu , COHEN Patricia T. W.
Published: August 2015

Hepatic glycogen synthesis is impaired in insulin-dependent diabetic rats and in adrenalectomized starved rats, and although this is known to be due to defective activation of glycogen synthase by glycogen synthase phosphatase, the underlying molecular mechanism has not been delineated. Glycogen synthase phosphatase comprises the catalytic subunit of protein phosphatase 1 (PP1) complexed with the hepatic glycogen-binding subunit, termed GL. In liver extracts of insulin-dependent diabetic and adrenalectomized starved rats, the level of GL was shown by immunoblotting to be substantially reduced compared with that in control extracts, whereas the level of PP1 catalytic subunit was not affected by these treatments. Insulin administration to diabetic rats restored the level of GL and prolonged administration raised it above the control levels, whereas re-feeding partially restored the GL level in adrenalectomized starved rats. The regulation of GL protein levels by insulin and starvation/feeding was shown to correlate with changes in the level of the GL mRNA, indicating that the long-term regulation of the hepatic glycogen-associated form of PP1 by insulin, and hence the activity of hepatic glycogen synthase, is predominantly mediated through changes in the level of the GL mRNA.
Distribution of the mevalonate and glyceraldehyde phosphate/pyruvate pathways for isoprenoid biosynthesis in unicellular algae and the cyanobacterium Synechocystis PCC 6714
Authors: DISCH Andrea , SCHWENDER Jörg , MÜLLER Christian , LICHTENTHALER Hartmut K. , ROHMER Michel
Published: August 2015

Isopentenyl diphosphate, the universal isoprenoid precursor, can be produced by two different biosynthetic routes: either via the acetate/mevalonate (MVA) pathway, or via the more recently identified MVA-independent glyceraldehyde phosphate/pyruvate pathway. These two pathways are easily differentiated by incorporation of [1-13C]glucose and analysis of the resulting labelling patterns found in the isoprenoids. This method was successfully applied to several unicellular algae raised under heterotrophic growth conditions and allowed for the identification of the pathways that were utilized for isoprenoid biosynthesis. All isoprenoids examined (sterols, phytol, carotenoids) of the green algae Chlorella fusca and Chlamydomonas reinhardtii were synthesized via the GAP/pyruvate pathway, as in another previously investigated green alga, Scenedesmus obliquus, which was also shown in this study to synthesize ubiquinone by the same MVA-independent route. In the red alga Cyanidium caldarium and in the Chrysophyte Ochromonas danica a clear dichotomy was observed: as in higher plants, sterols were formed via the MVA route, whereas chloroplast isoprenoids (phytol in Cy. caldariumand O. danica and β-carotene in O. danica) were synthesized via the GAP/pyruvate route. In contrast, the Euglenophyte Euglena gracilis synthesized ergosterol, as well as phytol, via the acetate/MVA route. Similar feeding experiments were performed with the cyanobacterium SynechocystisPCC 6714 using [1-13C]- and [6-13C]-glucose. The two isoprenoids examined, phytol and β-carotene, were shown to have the typical labelling pattern derived from the GAP/pyruvate route.
Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase)
Authors: YU Jau-Song , CHEN Wei-Jen , NI Mei-Hui , CHAN Wen-Hsiung , YANG Shiaw-Der
Published: August 2015

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034–7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421–9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or γ-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both autophosphorylation/activation and protein phosphatase-mediated dephosphorylation/inactivation processes. Taken together, our results identify Thr402 as the regulatory autophosphorylation site of auto-kinase, which is a C-terminal catalytic fragment of PAK2.
The gene for the human tMDC I sperm surface protein is non-functional: implications for its proposed role in mammalian sperm–egg recognition
Authors: FRAYNE Jan , HALL Len
Published: August 2015

The sperm surface antigen tMDC I (also known as cyritestin) has been proposed to play a role in sperm–egg binding in the mouse via an interaction between its disintegrin-like domain and an integrin receptor on the oolemma plasma membrane. Here we report the cloning and sequence of human tMDC I transcripts and show that they are non-functional, owing to the presence of a variety of deletions, insertions and in-frame termination codons. The absence of a tMDC I protein is further supported by the lack of immunoreactivity on Western blots of human testis and sperm extracts probed with macaque (Macaca fascicularis) and human anti-tMDC I antisera.
The survival of ingested lactoferrin in the gastrointestinal tract of adult mice
Authors: KUWATA Hidefumi , YIP Tai-Tung , YAMAUCHI Koji , TERAGUCHI Susumu , HAYASAWA Hirotoshi , TOMITA Mamoru , HUTCHENS T. William
Published: August 2015

Lactoferrin is an 80 kDa major protein component of mammalian colostral whey. The antimicrobial active centre of lactoferrin, lactoferricin (Lfcin), may also be an important determinant of the interaction between lactoferrin and specific receptors on lymphocytes. We have documented the survival in vivo of ingested lactoferrin in the gastrointestinal tract of adult mice by surface-enhanced laser desorption/ionization affinity MS. Various kinds of degraded lactoferrin fragments were detected as molecular-ion peaks corresponding to Lfcin after being captured by an affinity capture device, hydrolysis in situ and laser desorption/ionization. No evident molecular-ion peaks of Lfcin were observed upon analysis of faeces from mice fed commercial milk, whereas lactoferrin fragments containing the Lfcin region were detected at concentrations in the order of at least pmol/g in the faeces of mice fed milk enriched with lactoferrin at 40 mg/ml. These results suggest that ingested lactoferrin would survive transit through the gastrointestinal tract as partially degraded forms containing the receptor-binding region(s) as well as the antimicrobial active centre.
Effects of polyamine levels on the degradation of short-lived and long-lived proteins in cultured L-132 human lung cells
Authors: CORELLA Dolores , GUILLÉN Marisa , HERNÁNDEZ José Miguel , HERNÁNDEZ-YAGO José
Published: August 2015

Biogenic polyamines have important regulatory functions in various biological processes and it has also been suggested that they could modulate intracellular protein degradation. For an overall assessment of the role of polyamines in this process, we have investigated the effect that the decrease in intracellular polyamine levels caused by inhibitors of polyamine biosynthesis brings about on the degradation of the pools of short- and long-lived proteins in cultured L-132 human lung cells. Treatment of cells with 100 µM (2R,5R)-δ-methyl acetylenic putrescine (MAP), a potent enzyme-activated irreversible inhibitor of ornithine decarboxylase, or with 100 µM MAP plus 50 µM N-butyl 1,3-diaminopropane, a specific inhibitor of spermine synthase, caused a similar decrease (65–70% of control) in the total intracellular levels of polyamines, although they affected the concentrations of spermidine and spermine differently. The effect of the two treatments on protein degradation was essentially the same. In polyamine-depleted cells we observed an inhibition of degradation in long-lived proteins of 16% (P< 0.05), with a significant increase in the half-life (t½) of this pool from 100.5 to 120.1 h. This was concomitant with an increase of 26% (P< 0.05) in degradation in short-lived proteins, with a significant decrease in the t½ of this pool from 0.85 to 0.67 h. Recovery of polyamine levels by the addition of 50 µM spermidine to polyamine-depleted cells resulted in a restoration of the degradation rates in both pools of proteins. The way(s) by which polyamines could modulate proteolysis are discussed.