bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear magnetic resonance spectroscopy to determine the micellar cholesterol in human bile
Authors: Ellul J.P.M. , Murphy G.M. , Parkes H.G. , Slapa R.Z. , Dowling R.H.
Published: July 2002

The cholesterol of gallstones comes from the vesicular rather than the micellar phase of bile. Progress in this field has been limited because conventional analytical methods disturb the distribution of cholesterol between the two phases. The resonance of the cholesterol C6 proton occurs at a chemical shift of 5.4 ppm, to be shown by 2D NMR to be specific for biliary cholesterol, and arises only from the micellar mode. Thus integration of C6 proton resonance peak area provides a direct non‐invasive determination of the cholesterol distribution in human bile.
SP‐40,40, a protein involved in the control of the complement pathway, possesses a unique array of disulphide bridges
Authors: Kirszbaum L. , Bozas S.E. , Walker I.D.
Published: July 2002

SP‐40,40 is a two‐chain serum protein which acts in vitro as a potent inhibitor of the assembly of the membrane attack complex of human complement. It contains 10 cysteine residues, the numbers and locations of which are conserved in several mammalian species. Evidence is presented that all the cysteine residues are involved in interchain (α—β) disulphide bonds. There are no free cysteine residues. The disulphide bond motif established in this study for SP‐40,40 is unique and bears no obvious homology to those complement components whose disulphide bonds have been assigned, nor is there any homology apparent between SP‐40,40 and other multi‐chain proteins containing disulphide bonds.
Di‐myo‐inositol‐1, 1′‐phosphate: A new inositol phosphate isolated from Pyrococcus woesei
Authors: Scholz Stefan , Sonnenbichler Johann , Schäfer Wolfram , Hensel Reinhard
Published: July 2002

A new inositol derivative could be isolated from the Archaeum Pyrococcus woesei and identified as di‐myo‐inositol‐1,1′‐phosphate by 1H, 31P NMR spectroscopy, mass spectrometry and thin layer chromatograpy. In P. woesel, this inositol phosphate represents the dominant counterion of K+ which ranges from 500 to 600 mM. The role of the potassium salt of di‐myo‐inositol‐1,1′‐phosphate as thermostabilizer is discussed.
A novel mutant (transthyretin Ile‐50) related to amyloid polyneuropathy Single‐strand conformation polymorphism as a new genetic marker
Authors: Saeki Yoshinaga , Ueno Satoshi , Takahashi Nobuyuki , Soga Fumihisa , Yanagihara Takehiko
Published: July 2002

DNA sequence polymorphisms in transthyretin (TrR) genes were investigated by single‐strand conformation polymorphism (SSCP) analysis of polymerase chain reaction products. The amplified DNA fragments that encode each exon of the normal TTR gene showed two bands, representing the two complementary single strands of DNA. In one patient with amyloid polyneuropathy, the exon 3 DNA showed a unique, aberrant migration pattern. Direct sequencing analysis of the amplified exon 3 revealed a single base change (G‐to‐T), resulting in a novel amino acid substitution (Ser‐50 → Ile). We also present the SSCP patterns for five known Japanese TTR variants.
Lipid interaction of Tetanus neurotoxin A calorimetric and fluorescence spectroscopy study
Authors: Calappi Emanuela , Masserini Massimo , Schiavo Giampietro , Montecucco Cesare , Tettamanti Guido
Published: July 2002

The interaction of Tetunus toxin with phospholipid vesicles containing gangliosides (GD1a, GD1b or GT1b) or phosphatidic acid has been investigated at neutral or acidic pH. Change in the thermotropic properties of the vesicles occurred only after addition of the toxin at acidic pH, and led to surface binding or membrane insertion of the protein, dependent on the physical state of the membrane. Most remarkably, toxin addition at acidic pH to dipalmitoyl‐phosphatidylcholine vesicles containing GT1b ganglioside, caused formation of ganglioside microdomains on the vesicle surface.
The molecular mechanism of the control of excitation energy dissipation in chloroplast membranes Inhibition of ΔpH‐dependent quenching of chlorophyll fluorescence by dicyclohexylcarbodiimide
Authors: Ruban A.V. , Walters R.G. , Horton P.
Published: July 2002

Non‐radiative dissipation of absorbed excitation energy in chloroplast membranes is induced in the presence of the trans‐thylakoid proton motive force; this dissipation is measured as high energy state quenching of chlorophyll fluorescence. qE. It has been suggested that this results from a low pH‐induced structural alteration in the light harvesting complex of photosystem II, LHCII [(1991) FEBS Letters 292, 1–4], The effect of the carboxyl‐modifying agent, dicyclohexylcarbodiimide (DCCD). on energy dissipation in chloroplast membranes has been investigated. At concentrations below that required to Inhibit electron transport. DCCD caused a decrease in the steady state ΔpH, completely inhibited qE and also inhibited the low pH‐dependent induction of qE. DCCD binding to polypeptides in the 22–28 kDa range correlated with inhibition of qE. It is suggested that DCCD reacts with amino acids residues in LHCII whose protonation is the primary event in the induction of energy dissipation. This LHCII domain may be identical to one forming a proton channel linking the site of PSII‐dependent water oxidation to the thylakoid lumen [(1990) Eur. J. Biochem. 193, 731–736].
Magnesium deficiency in vitro enhances free radical‐induced intracellular oxidation and cytotoxicity in endothelial cells
Authors: Dickens B.F. , Weglicki W.B. , Li Y.-S. , Mak I.T.
Published: July 2002

The effect of magnesium (Mg)‐deficient culture on endothelial cell susceptibility to oxidative stress was examined. Bovine endothelial cells were cultured in either control sufficient (0.8 mM) or deficient (0.4 mM) levels of MgCl2. Oxygen radicals were produced extracellularly by the addition of dihydroxyfumarate and Fe3+‐ADP. Isolated Mg‐deficient endothelial cells produced 2‐ to 3‐fold higher levels of thiobarbituric acid (TBA)‐reactive materials when incubated with this free radical system. Additional studies were performed using digitized video microscopy and 2′,7′‐dichlorofluorescin diacetate (DCFDA) as an intracellular indicator for oxidative events at the single cell level. In response to the exogenous oxidative Stress, endothelial cells exhibited a time‐dependent increase in fluorescence, suggestive of intracellular lipid peroxidation. The increase in cellular fluorescence began within 1 min or free radical addition; the Mg‐deficient cells exhibited a more rapid increase in fluoresence than that of Mg‐sufficient cells. In separate experiments, cellular viability was assessed using the Trypan blue exclusion assay. Mg deficiency increased cytotoxicity of the added oxyradicals, but the loss or cellular viability began to occur only after 15 min of free radical exposure, tagging behind the detection of intracellular oxidation products. These results suggest that increased oxidative endothelial cell injury may contribute to vascular injury during Mg deficiency.
Phosphate derivatives of AZT display enhanced selectivity of action against HIV1 by comparison to the parent nucleoside
Authors: McGuigan Christopher , Nickson Caleb , Petrik Juraj , Karpas Abraham
Published: July 2002

Novel phosphate derivatives of the anti‐HIV nucleoside analogue AZT have been prepared by phosphorochloridate chemistry. In particular, phosphates carrying ester‐containing side‐chains are described. These materials are designed to act as membrane‐soluble pro‐drugs of the bio‐active free nucleotides. In vitro evaluation revealed the compounds to have a pronounced, selective antiviral activity. In several cases the phosphate derivatives are more selective in their action than the parent nucleoside AZT. In particular, this arises from the low toxicity of the phosphate pro‐drugs by comparison to AZT. These data support the suggestion that the phosphate derivatives exert their biological effects via intracellular release of the nucleotide forms, and suggests that such pro‐drug, forms may be worthy of further study.
The fidelity of the reverse transcriptases of human immunodeficiency viruses and murine leukemia virus, exhibited by the mispair extension frequencies, is sequence dependent and enzyme related
Authors: Bakhanashvili Mary , Hizi Amnon
Published: July 2002

Sequence variations in HIV‐1 and HIV‐2 probably result in part from inaccurate DNA synthesis by viral reverse transcriptases (RTs). We have studied in vitro the fidelity of both the DNA‐ and RNA‐dependent DNA polymerization functions of the two HIV RTs, as compared to that of murine leukemia virus (MLV) RT. The two HIV RTs were less accurate than MLV RT. The mispair extension frequencies observed previously with ribosomal RNA (rRNA) template were higher than those detected with ØX174am3 DNA template with all three RTs. In the current study we have investigated whether the nature of the copied nucleic acid (RNA vs. DNA) or the template nucleotide sequences affect the accuracy of DNA synthesis. We have analyzed the fidelity of DNA synthesis with DNA sequences identical to those of the rRNA sequences previously employed for reverse transcription. The results indicate that the fidelity of DNA synthesis depends mainly on the nucleotide sequences copied by every given RT. Yet, fidelity of DNA synthesis depends not only on the sequences copied but also on the nature of the enzymes per se. It is possible that these factors are major contributors to the high mutation rates of the two human immunodeficiency viruses.
High sensitivity to site directed mutagenesis of the peptide segment connecting phosphorylation and Ca2+ binding domains in the Ca2+transport ATPase
Authors: Zhang Ziyu , Sumbilla Carlota , Lewis David , Inesi Giuseppe
Published: July 2002

Nine residues (Leu321, Lys329, Asn330, Val333, Arg334, Leu336, Pro337, Val339 and Glu340), within the peptide segment intervening between the catalytic domain and the Ca2+ binding domain of the sarcoplasmic reticulum (SERCA 1) ATPase, were individually mutated to Ala. The mutated proteins were recovered in the microsomal fraction of COS‐1 cells following transient expression, and exhibited inhibition of Ca2+ uptake and ATPase hydrolytic activity, while forming discernable levels of phosphorylated intermediate. Mutation of Glu340 to Gln (rather than to Ala) was much less effective, suggesting that the functional consequence of the mutation is related to structural perturbation, rather than loss of the acidic side chain. The high sensitivity of this peptide segment to single mutations suggests that its structural integrity is required for functional linkage of the phosphorylation and Ca2+ binding domains.
Identification of a second membrane‐active 13‐residue peptide segment in the antimicrobial protein, bovine seminalplasmin
Authors: Sitaram N. , Subbalakshmi C. , Nagaraj R.
Published: July 2002

Seminalplasmin (SPLN) is a 47‐residue protein from bovine seminalplasma having broad‐spectrum antibacterial activity. The protein has no hemolytic activity. SPLN interacts with lipid vesicles and its antibacterial activity appears to stem from its ability to permeabilize the bacterial plasma membrane. Analysis of SPLN's primary structure, with respect to its relative hydrophobicity and hydrophilicity, revealed a segment, PKLLETFLSKWIG, more hydrophobic than the rest of the protein. A synthetic peptide corresponding to this region had not only antibacterial activity but also hemolytic properties. Analysis of the SPLN sequence based on hydrophobic moment plots has revealed a second segment, SLSRYAKLANRLA, which could be membrane active. A synthetic peptide corresponding to this region shows only antibacterial activity with no hemolytic activity.
Role of the C‐terminal domain of the lysozyme of Clostridium acetobutylicum ATCC 824 in a chimeric pneumococcal‐clostridial cell wall lytic enzyme
Authors: Croux Christian , Ronda Concepción , López Rubens , L. García José
Published: July 2002

An active chimeric cell wall lytic enzyme has been constructed by domain substitution between the major autolysins of Clostridium acetobutylicum ATCC 824 and Streptococcus pneumoniae. The chimeric enzyme, built up by the fusion of the N‐terminal domain of the pneumococcal LYTA amidase and the C‐terminal domain of the clostridial LYC lysozyme, exhibited an amidase activity capable of hydrolyzing choline‐containing clostridial cell walls with an efficiency 250‐times higher than when tested on pneumococcal cell walls. This experimental approach demonstrates the basic role of the C‐terminal domain of the LYC lysozyme in substrate recognition and provides additional support to our hypothesis of modular evolution of these lytic enzymes. Moreover, the construction described here confirmed the role of the C‐terminal domains of the modular cell wall lytic enzymes on the optimal pH for catalytic activity. To our knowledge, this is the first example of the construction of an active chimeric lytic enzyme by fusing genes that lack nucleotide homology and are derived from different bacterial genera.
Somatostatin receptor subtype SSTR2 mediates the inhibition of high‐voltage‐activated calcium channels by somatostatin and its analogue SMS 201‐995
Authors: Fujii Yasukazu , Gonoi Tohru , Yamada Yuichiro , Chihara Kazuo , Inagaki Nobuya , Seino Susumu
Published: July 2002

Somatostatin and its anologue SMS 201‐995 inhibit high voltage‐activated (HVA) Ca2+ currents in the rat insulinoma cell line RINm5F which stably express cloned human somatostatin receptor subtype 2 (hSSTR2). In contrast, neither somatostatin nor SMS 201‐995 suppresses the HVA Ca2+ currents in RINm5F which stably express cloned hSSTR1. These results suggest that somatostatin‐induced inhibition of HVA Ca2+ currents is mediated by a specific receptor subtype and that inhibition of calcium influx through HVA Ca2+ channels is one of the mechanisms of SMS 201‐995 action on inhibitory processes of hormone secretion and cell proliferation.
Brazzein, a new high‐potency thermostable sweet protein from Pentadiplandra brazzeana B
Authors: Ming Ding , Hellekant Göran
Published: July 2002

We have discovered a new high‐potency thermostable sweet protein, which we name brazzein, in a wild African plant Pentadiplandra brazzeana Baillon. Brazzein is 2,000 times sweeter than sucrose in comparison to 2% sucrose aqueous solution and 500 times in comparison to 10% of the sugar. Its taste is more similar to sucrose than that of thaumatin. Its sweetness is not destroyed by 80°C for 4 h. Brazzein is comprised of 54 amino acid residues, corresponding to a molecular mass of 6,473 Da.
Histidine‐419 plays a role in energy coupling in the vesicular monoamine transporter from rat
Authors: Shirvan Anat , Laskar Orly , Steiner-Mordoch Sonia , Schuldiner Shimon
Published: July 2002

Vesicular monoamine transporters (VMAT) catalyze transport of serotonin, dopamine, epinephrine and norepinephrine into subcellular storage organelles in a variety of cells. Accumulation of the neurotransmitter depends on the proton electrochemical gradient across the organelle membrane and involves VMAT‐mediated exchange of two lumenal protons with one cytoplasmic amine. It has been suggested in the past that His residues play a role in H+ movement or in its coupling to active transport in H+‐symporters and antiporters. Indeed VMAT‐mediated transport is inhibited by reagents specific for His residues. We have identified one His residue in VMAT1 from rat which is conserved in other vesicular neurotransmitter transporters. Mutagenesis of this His (H419) to either Arg or Cys completely inhibits [3H]serotonin and [3H]dopamine accumulation. Mutagenesis also inhibits other H+‐dependent partial reactions of VMAT such as the acceleration of binding of the high affinity ligand reserpine, but does not inhibit the [3H]reserpine binding which is not dependent on H+ translocation. It is concluded that His‐419 plays a role in energy coupling in r‐VMAT1.
Protein uptake into E. coll during Bdellovibrio infection
Authors: Saier Milton H.
Published: July 2002

Bdellovibrio bacteriovorus is a small bacterial parasite that infects other Gram‐negative bacteria, resides in the periplasm of the host cell, and utilizes host macromolecules as a source of nutrients. Evidence is summarized suggesting that B. bacteriovorus secretes proteases and nucleases synthesized in its own cytoplasm that are targeted to the cytoplasm of the host cell. Possible mechanisms for this trans‐trimembrane protein transport process are discussed.
Glycosylation and stability of mature HIV envelope glycoprotein conformation under various conditions
Authors: Papandreou M.J. , Idziorek T. , Miquelis R. , Fenouilleta E.
Published: July 2002

The role of the glycans of the mature human immunodeficiency virus (HIV) envelope (gp160) in its stability in various conditions was studied. gp160 conformation was monitored through its subsequent ability to bind (125I]CD4. Treatment of glycosylated (CHO+) gp160 with (i) sodium dodecyl sulfate (SDS) concentrations above 0.01% impaired subsequent CD4 binding while 0.3% SDS abolished it; (ii) β‐mercaptoethanol (MSH) concentrations above 0.01% impaired CD4 binding while 0.03% MSH abolished it; (iii) 2 M guanidine‐HCI had no effect; (iv) temperatures between 50C and 80C altered CD4 binding while, above 80C, the binding was abolished; (v) CD4 binding was decreased by 50% by 2 freeze‐thaw cycles but was not further affected by subsequent (up to 15) cycles; (vi) gp160 incubation in serum or cell lysate had no effect on CD4 binding. Glycanase treated (CHO‐) gp160 binding activity was only 3‐fold lower than that of CHO+ gp160. Only 2 M guanidine‐HCI and heating at 70C differentially affected the binding of CHO+ and CHOgp160, the effects being larger for CHO‐gp160. CHO‐gp160 binding was impaired after incubation in either serum or cell lysate. Thus, glycans stabilize gp160 conformation in some environments. However, CHO‐gp160 appears to be resistant to denaturation as compared to other glycoproteins reported in the literature.
Dynamin and receptor‐mediated endocytosis
Authors: Damke Hanna
Published: July 2002

The internalization of receptor‐bound ligands involves concentration of cell surface receptors in specialized areas of the plasma membrane and subsequent formation of clathrin‐coated vesicles. The complex process of invagination, constriction and budding of clathrin‐coated vesicles employs the coordinated actions of several proteins. This review is focused on the GTPase dynamin, which plays a key role in the constriction of coated pits.
Concerns about depression
Authors: van Praag HM
Published: July 2002

Biological depression research can boast of a number of significant achievements over the past 35 years. Yet, in spite of those achievements, the field is in danger of desiccation. Five reasons are discussed herein: 1) short-comings of the DSM-based depression classification; 2) the ever increasing number of, generally poorly validated, diagnostic categories; 3) desubjectivation of psychiatric diagnosing; 4) the lack of a dimensional (better: functional) component in diagnosing depression; and 5) horizontalism, ie the absence of attempts to group symptoms “vertically” according to their diagnostic weight. The issues are in need of urgent scientific attention, lest biological depression research will stagnate and ultimately whither. We have indicated ways to approach the issues.
Anomalous RNA substrates for mammalian tRNA 3′ processing endoribonuclease
Authors: Nashimoto Masayuki
Published: July 2002

Mammalian tRNA 3′ processing endoribonuclease (3′ tRNase) is an enzyme responsible for the removal of a 3′ trailer from pre‐tRNA. The enzyme can also recognize and cleave any target RNA that forms a pre‐tRNA‐like complex with another RNA. To investigate the interaction between 3′ tRNase and substrates, we tested various anomalous pre‐tRNA‐like complexes for cleavage by pig 3′ tRNase. We examined how base mismatches in the acceptor stem affect 3′ tRNase cleavage of RNA complexes, and found that even one base mismatch in the acceptor stem drastically reduces the cleavage efficiency. Mammalian 3′ tRNase was able to recognize complexes between target RNAs and 5′‐half tDNAs, and cleave the target RNAs, although inefficiently, whereas the enzyme had no activity to cleave phosphodiester bonds of DNA. A relatively long RNA target, the Escherichia coli chloramphenicol acetyltransferase (CAT) mRNA, was cleaved by 3′ tRNase in the presence of appropriate 5′‐half tRNAs. We also demonstrated that an RNA complex of lin‐4 and lin‐14 from Caenorhabditis elegans can be recognized and cleaved by pig 3′ tRNase.
Ceramides induce a form of apoptosis in human acute lymphoblastic leukemia cells that is inhibited by Bcl‐2, but not by CrmA
Authors: Geley Stephan , Hartmann Bernd L , Kofler Reinhard
Published: July 2002

The generation of ceramides by the action of acidic and/or neutral sphingomyelinases has been implicated in many forms of apoptosis. We investigated whether exposure to ceramides is sufficient to induce apoptosis in human leukemia cells and, if so, what the characteristics of this form of apoptosis might be. Treatment of the acute lymphoblastic T‐cell line CEM‐C7H2 with short‐ and medium‐chain ceramide analogs (C2‐, C6‐, and C8‐ceramide) resulted in apoptosis, whereas the inactive C2‐dihydroceramide had no effect on cell survival. Induction of apoptosis was relatively slow (∼40% after 24 h) and required high concentrations of ceramide analogs (40–100 μM). To investigate a possible involvement of interleukin 1‐β‐converting enzyme (ICE) or ICE‐related proteases, we treated CEM‐C7H2 sublines constitutively expressing the vaccinia virus protease inhibitor crmA with ceramide analogs. Although such cells were completely resistant to apoptosis induced by antibodies to the Apo‐1/Fas surface receptor (a form of apoptosis known to be inhibitable by CrmA), they were not protected from ceramide‐induced cell death. In contrast, tetracycline‐regulated overexpression of Bcl‐2 protected CEM‐C7H2 sublines stably transfected with corresponding constructs from ceramide‐induced apoptosis. Thus, in these human leukemia cells, ceramides induce a relatively slow death response that can be prevented by Bcl‐2, but is independent of CrmA‐inhibitable proteases. These characteristics distinguish ceramide‐induced from other forms of apoptosis, such as Apo‐1/Fas‐induced cell death where ceramide production has been causally implicated.
Analysis of a regulatory element in the 5′‐untranslated region of the bcl‐2 gene
Authors: Körner Ines , Weber-Nordt Renate , Pfaff Peter , Finke Jürgen
Published: July 2002

The bcl‐2 gene is an important antagonist of apoptosis, the programmed cell death. Bcl‐2 is highly expressed in a variety of lymphomas. Lymphocytes of patients with chronic lymphocytic leukemia (CLL) express high amounts of bcl‐2 even in the absence of the t(14;18) translocation, resulting in a strong resistance towards corticosteroid induced apoptosis. Within the 5′‐untranslated region of the bcl‐2 gene a p53 dependent negative response element has been described. Genetic alterations within this element could lead to uncontrolled overexpression of bcl‐2 and subsequent resistance towards apoptosis. We therefore analyzed the mRNA from the 5′‐untranslated region −279 to −85 bp of the bcl‐2 gene by direct PCR sequencing from peripheral blood derived lymphocytes from patients with CLL and normal healthy donors. Compared to published sequences (Tsujimoto and Croce (1986) Proc. Natl. Acad. Sci. USA 83, 5214), we consistently found an exchange at position 1271 from A to G and at position 1284 from G to A in all CLL as well as normal donor derived samples analyzed. Thus, CLL specific alterations compared to normal cells could not be found and deregulated expression of bcl‐2 in CLL cells does not appear to be due to alterations in the p53 dependent negative response element of the bcl‐2 gene. However, our data add information to published sequence data of this region. © 1997 Federation of European Biochemical Societies.
Identification of a vitamin K‐dependent carboxylase in the venom duct of a Conus snail
Authors: Stanley Thomas B , Stafford Darrel W , Olivera Baldomero M , Bandyopadhyay Pradip K
Published: July 2002

Peptides from the venom ducts of cone snails (genus Conus) contain γ‐carboxyglutamate residues. The γ‐glutamyl carboxylase responsible for this post‐translational modification is localized in the microsomal fraction, strictly dependent on vitamin K, activated by ammonium sulfate, and is associated with endogenous substrate. The K
m of the enzyme for vitamin K is comparable to that for the bovine carboxylase. However, a propeptide containing substrate related to the blood coagulation protein factor IX, a highly efficient substrate for the bovine enzyme, was poorly carboxylated by the Conus enzyme, suggesting differences in γ‐carboxylase recognition signal sequences and/or structural requirements at the carboxylation site.
Genomic structure of a potassium channel toxin from Heteractis magnifica1
Authors: Gendeh Gurmil S , Chung Max C.M , Jeyaseelan Kandiah
Published: July 2002

We provide information on the gene encoding the K+ channel toxin, HmK, of the sea anemone Heteractis magnifica. A series of DNA amplifications by PCR, which included the amplification of the 5′‐untranslated region of the gene, showed that an intron of 402 nucleotides separated the sequence that encodes the matured toxin from the signal peptide sequence. A second 264 nucleotide intron interrupted the 5′‐untranslated region of the previously reported HmK cDNA. Two possible transcription‐initiation sites were identified by primer extension analysis. Corresponding TATA‐box consensus sequences, characteristic of a promoter region, were also located from PCR products of uncloned libraries of adaptor‐ligated genomic DNA fragments. The coding region for matured HmK is intronless. The same is also true for other sea anemone toxins reported thus far. More notably, a similar intron‐exon organization is present in other ion channel‐blocking toxins from scorpions implying that molecules having similar functions share a similar organization at the genomic level suggesting a common path of evolution.
NG108‐15 cells express neuregulin that induces AChR α‐subunit synthesis in cultured myotubes
Authors: Pun San , Yang Jie F , Ng Yu P , Tsim Karl W.K
Published: July 2002

A cholinergic neuroblastoma×glioma hybrid cell line NG108‐15 is able to form functional synapses, and contains both AChR‐aggregating and AChR‐inducing activities when co‐cultured with myotubes. Several lines of evidence indicate that the AChR‐inducing activity of NG108‐15 cells is derived from neuregulin. The conditioned medium of cultured NG108‐15 cells induced the expression of AChR α‐subunit as well as the tyrosine phosphorylation of erbB‐3 receptor. NG108‐15 cells expressed neuregulin with a protein of ∼100 kDa in size and transcripts of ∼6.8 kbp, ∼2.6 kbp and ∼1.8 kbp; mRNAs encoding β1 and α2 isoforms of neuregulin were revealed. NG108‐15 cells were induced to differentiate by chemicals, and the chemical‐induced differentiation of NG108‐15 cells increased the level of neuregulin mRNA expression ∼3‐fold while the expression of a housekeeping gene remained relatively unchanged. The activity of neuregulin in the conditioned medium of NG108‐15 cells was reduced by treating the medium with heparin and anti‐neuregulin antibody. In addition, NG108‐15 cells were transfected with antisense neuregulin cDNA and its expression of neuregulin was reduced, while its neuregulin‐induced tyrosine phosphorylation activity was markedly decreased. This is the first direct demonstration that the NG108‐15 cell‐induced AChR up‐regulation on cultured myotubes is mediated by neuron‐derived neuregulin.
Biological dynamics and distribution of 3‐hydroxy fatty acids in the yeast Dipodascopsis uninucleata as investigated by immunofluorescence microscopy. Evidence for a putative regulatory role in the sexual reproductive cycle1
Authors: Kock J.L.F , Venter P , Linke D , Schewe T , Nigam S
Published: July 2002

Dipodascopsis uninucleata has been recently shown to produce 3‐hydroxy polyenoic fatty acids from several exogenous polyenoic fatty acids. In order to examine whether endogenous 3‐hydroxy fatty acids (3‐OH‐FA) may be implicated in the developmental biology of this yeast, we mapped by immunofluorescence microscopy their occurrence in fixed cells with or without cell walls using an antibody raised against 3R‐hydroxy‐5Z,8Z,11Z,14Z‐eicosatetraenoic acid (3R‐HETE), the biotransformation product from arachidonic acid (AA). This antibody turned out to cross‐react with other 3‐OH‐FA. 3‐OH‐FA were detected in situ in gametangia, asci, as well as between released ascospores, and proved to be associated with the sexual reproductive stage of the life cycle of the yeast. Acetylsalicylic acid (1 mM), which is known to suppress the formation of 3‐OH‐FA from exogenous polyenoic fatty acids, inhibited the occurrence of immunoreactive material as well as the sexual phase of the life cycle suggesting a prominent regulatory role of 3‐OH‐FA for the latter.
`Tissue' transglutaminase in cell death: a downstream or a multifunctional upstream effector?
Authors: Melino Gerry , Piacentini Mauro
Published: July 2002

Apoptotic cells show morphological modifications which occur as the result of complex molecular mechanisms involving several proteins including `tissue' transglutaminase (tTG). Although tTG was originally thought to be responsible for the protein crosslinks which prevent the leakage of intracellular components, thereby reducing inflammation and autoimmunity, recent evidence indicates that tTG is a multifunctional enzyme involved in the complex upstream regulation of the apoptotic machinery: (i) it functions as a GTP‐binding protein to transduce signals; (ii) it binds/crosslinks only specific cytosolic and nuclear substrates, suggesting highly specific actions, e.g. on intermediate filaments and in cell cycle control; (iii) it is finely tuned by Ca2+, GTP, S‐nitrosylation, polyamines. In light of these recent discoveries, the role of tTG in the regulation of the crucial balance between survival and death is clearly complex.
Antioxidant enzymatic activities and lipid peroxidation in cultured human chondrocytes from vertebral plate cartilage
Authors: Grazioli Vittorio , Schiavo Roberta , Casari Erminia , Marzatico Fulvio , Rodriguez y Baena Riccardo , Gaetani Paolo
Published: July 2002

Total SOD activity increased, even if not significantly, between the 12th and the 18th day. A significant variation of GSHPx (P<0.01) and of catalase (P<0.001) activity was observed between the 3rd and the 6th day with no further variation until the 18th day. A significant increase (P<0.001) of lipid peroxidation from the 3rd to the 18th day was also observed. These results seem to indicate that only fresh human cultured chondrocytes are suitable to study, through in vitro models, the in vivo behavior of the antioxidative status of these cells.
Expression and functional analysis of an N‐truncated NifA protein of Herbaspirillum seropedicae
Authors: Monteiro R.A , Souza E.M , Funayama S , Yates M.G , Pedrosa F.O , Chubatsu L.S
Published: July 2002

In Herbaspirillum seropedicae, an endophytic diazotroph, nif gene expression is under the control of the transcriptional activator NifA. We have over‐expressed and purified a protein containing the central and C‐terminal domains of the H. seropedicae NifA protein, N‐truncated NifA, fused to a His‐Tag sequence. This fusion protein was found to be partially soluble and was purified by affinity chromatography. Band shift and footprinting assays showed that the N‐truncated NifA protein was able to bind specifically to the H. seropedicae nifB promoter region. In vivo analysis showed that this protein activated the nifH promoter of Klebsiella pneumoniae in Escherichia coli only in the absence of oxygen and this activation was not negatively controlled by ammonium ions.
Influenza hemagglutinin mediated fusion of membranes containing poly(ethylene‐glycol) grafted lipids: new insights into the fusion mechanism
Authors: Chams Vida , Bonnafous Pierre , Stegmann Toon
Published: July 2002

The influence of a hydrophilic layer covering the membrane on influenza hemagglutinin (HA) mediated fusion was investigated using membranes containing poly(ethylene‐glycol) grafted phosphatidylethanolamine (PEG‐2000‐PE). Steric inhibition of HA‐membrane interactions by these lipids affected virus fusion (half‐maximal inhibition at 0.8 mol% for lipids with 114 ethylene glycol residues, or at 3.2 mol% for 45 residues (PEG‐2000‐PE), concentrations at which the PEG moieties adopt a random coil structure). Reconstituted viral membranes containing 3 mol% PEG‐2000‐PE retained 40% of their fusion activity. Therefore, efficient fusion is possible with membranes completely covered by a hydrophilic layer of several nanometers, and fusogenic virosomes containing PEG‐PE are feasible.
Early growth determines longevity in male rats and may be related to telomere shortening in the kidney
Authors: Jennings B.J. , Ozanne S.E. , Dorling M.W. , Hales C.N.
Published: July 2002

Maternal protein undernutrition can influence the growth and longevity of male offspring in the rat. We tested the hypothesis that these differences in longevity were associated with changes in the rate of telomere shortening. We found age‐related shortening of telomeres in the liver and kidney but not in the brain of male rats. Growth retardation in postnatal life was associated with significantly longer kidney telomeres and an increased longevity. Conversely, growth retardation during the foetal life followed by postnatal catch‐up growth was associated with a shorter life span and shorter kidney telomeres. These findings may provide a mechanistic basis for epidemiological studies linking early growth retardation to adult degenerative diseases.
A RING‐H2 finger motif is essential for the function of Der3/Hrd1 in endoplasmic reticulum associated protein degradation in the yeast Saccharomyces cerevisiae
Authors: Bordallo Javier , Wolf Dieter H.
Published: July 2002

Der3/Hrd1p is a protein required for proper degradation of misfolded soluble and integral membrane proteins in the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae. It is located to the ER membrane and consists of a N‐terminal hydrophobic region with several transmembrane domains and a large hydrophilic tail oriented to the ER lumen containing a RING finger motif of the H2 class. We had previously reported that a truncated version of Der3p, Der3ΔRp, lacking 111 residues of the lumenal domain including the RING finger motif is not functional, suggesting the involvement of this domain in the function of the protein in ER degradation. We substantiated this hypothesis by constructing a mutated form of Der3/Hrd1p replacing the last cysteine of the motif with a serine. This mutated Der3C399S protein maintains the correct localization and topology of the wild‐type protein, however, is not able to support the degradation of soluble and integral membrane proteins. This point mutation altering the RING‐H2 motif behaves as a dominant allele especially when overexpressed from a 2μ plasmid by this increasing the half‐life of CPY* more than 6‐fold when compared with a wild‐type strain. Furthermore co‐expression of der3C399S
with the wild‐type allele is also able to partially suppress the temperature sensitive growth phenotype of a sec61‐2 strain. Finally we have shown that overexpression of Hrd3p suppresses the dominant effect of the der3C399S
mutation. These results could be explained by a competition between wild‐type and mutant Der3 protein for the interaction with some other component of the ER degradation pathway, probably Hrd3p.
Ubiquinone is reduced by lipoamide dehydrogenase and this reaction is potently stimulated by zinc
Authors: Olsson Jerker M , Xia Ling , Eriksson Lennart C , Björnstedt Mikael
Published: July 2002

Ubiquinol is an endogenously synthesized lipid‐soluble antioxidant. Regeneration of ubiquinol from the oxidized form is essential to the maintenance of its antioxidant function. We demonstrated that lipoamide dehydrogenase can reduce ubiquinone to ubiquinol. Zinc increased the rate of the NADPH‐dependent reduction more than 10‐fold. The concentration ubiquinone resulting in the half‐maximal rate of reduction was approximately 5 μM in the presence and 4 μM in the absence of zinc. These data may explain how ubiquinone is reduced to the active antioxidant ubiquinol, which plays such an important role in protecting against oxidative stress and lipid peroxidation.
Sequence and detailed organization of the human caveolin‐1 and ‐2 genes located near the D7S522 locus (7q31.1)
Authors: Engelman Jeffrey A , Zhang Xiao Lan , Lisanti Michael P
Published: July 2002

The CA microsatellite repeat marker, D7S522, is located at the center of a ∼1000 kb smallest common deleted region that is lost in many forms of human cancer. It has been proposed that a putative tumor suppressor gene lies in close proximity to D7S522, within this smallest common deleted region. However, the genes located in proximity to D7S522 have remained elusive. Recently, we identified five independent BAC clones (∼100–200 kb) containing D7S522 and the human genes encoding caveolins 1 and 2. Here, we present the detailed organization of the caveolin locus and its relationship to D7S522, as deduced using a shot‐gun sequencing approach. We derived two adjacent contigs for a total coverage of ∼250 kb. Analysis of these contigs reveals that D7S522 is located ∼67 kb upstream of the caveolin‐2 gene and that the caveolin‐2 gene is located ∼19 kb upstream of the caveolin‐1 gene, providing for the first time a detailed genetic map of this region. Further sequence analysis reveals many interesting features of the caveolin genes; these include the intron‐exon boundaries and several previously unrecognized CA repeats that lie within or in close proximity to the caveolin genes. The first and second exons of both caveolin genes are embedded within CpG islands. These results suggest that regulation of caveolin gene expression may be controlled, in part, by methylation of these CpG regions. In support of this notion, we show here that the CGs in the 5′ promoter region of the caveolin‐1 gene are functionally methylated in two human breast cancer cell lines (MCF7 and T‐47D) that fail to express the caveolin‐1 protein. In contrast, the same CGs in cultured normal human mammary epithelial cells (NHMECs) are non‐methylated and these cells express high levels of the caveolin‐1 protein. Comparison of the human locus with the same locus in the pufferfish Fugu rubripes reveals that the overall organization of the caveolin‐1/‐2 locus is conserved from pufferfish to man. In conclusion, our current studies provide a systematic basis for diagnostically evaluating the potential deletion, mutation, or methylation of the caveolin genes in a variety of human tumors.
Transient accumulation of elastic energy in proton translocating ATP synthase
Authors: Cherepanov Dmitry A. , Mulkidjanian Armen Y. , Junge Wolfgang
Published: July 2002

ATP synthase is conceived as a rotatory engine with two reversible drives, the proton‐transporting membrane portion, F0, and the catalytic peripheral portion, F1. They are mounted on a central shaft (subunit γ) and held together by an eccentric bearing. It is established that the hydrolysis of three molecules of ATP in F1 drives the shaft over a full circle in three steps of 120° each. Proton flow through F0 probably generates a 12‐stepped rotation of the shaft so that four proton‐translocating steps of 30° each drive the synthesis of one molecule of ATP. We addressed the elasticity of the transmission between F0 and F1 in a model where the four smaller steps in F0 load a torsional spring which is only released under liberation of ATP from F1. The kinetic model of an elastic ATP synthase described a wealth of published data on the synthesis/hydrolysis of ATP by F0F1 and on proton conduction by F0 as function of the pH and the protonmotive force. The pK values of the proton‐carrying group interacting with the acidic and basic sides of the membrane were estimated as 5.3–6.4 and 8.0–8.3, respectively.
Engineering a central metabolic pathway: glycolysis with no net phosphorylation in an Escherichia coli gap mutant complemented with a plant GapN gene
Authors: Valverde Federico , Losada Manuel , Serrano Aurelio
Published: July 2002

A cDNA fragment containing the Pisum sativum GapN gene, which encodes the non‐phosphorylating glyceraldehyde‐3‐phosphate dehydrogenase, was cloned in a prokaryote expression vector. This construct enabled Escherichia coli strain W3CG, a mutant which lacks the glycolytic phosphorylating G3P dehydrogenase, to grow aerobically on sugars. The functionally complemented mutant exhibited high levels of the catalytically active plant enzyme, which renders 3‐phosphoglycerate and NADPH, thus bypassing the first substrate level phosphorylation step of the glycolysis. As expected if such a glycolytic bypass would be operative in vivo, this clone failed to grow anaerobically on sugars in contrast to W3CG clones complemented with phosphorylating glyceraldehyde‐3‐phosphate dehydrogenases. According to the irreversible catabolic character of the non‐phosphorylating reaction, the GapN‐complemented clone was unable to grow on gluconeogenic substrates. This metabolic engineering approach demonstrates that a pure catabolic Embden‐Meyerhof pathway with no net energy yield is feasible.
Assessment of uncoupling activity of uncoupling protein 3 using a yeast heterologous expression system
Authors: Zhang Chen-Yu , Hagen Thilo , Mootha Vamsi K , Slieker Lawrence J , Lowell Bradford B
Published: July 2002

Uncoupling protein 3L, uncoupling protein 1 and the mitochondrial oxoglutarate carrier were expressed in Saccharomyces cerevisae. Effects on different parameters related to the energy expenditure were studied. Both uncoupling protein 3L and uncoupling protein 1 reduced the growth rate by 49% and 32% and increased the whole yeast O2 consumption by 31% and 19%, respectively. In isolated mitochondria, uncoupling protein 1 increased the state 4 respiration by 1.8‐fold, while uncoupling protein 3L increased the state 4 respiration by 1.2‐fold. Interestingly, mutant uncoupling protein 1 carrying the H145Q and H147N mutations, previously shown to markedly decrease the H+ transport activity of uncoupling protein 1 when assessed using a proteoliposome system (Bienengraeber et al. (1998) Biochem. 37, 3–8), uncoupled the mitochondrial respiration to almost the same degree as wild‐type uncoupling protein 1. Thus, absence of this histidine pair in uncoupling protein 2 and uncoupling protein 3 does not by itself rule out the possibility that these carriers have an uncoupling function. The oxoglutarate carrier had no effect on any of the studied parameters. In summary, a discordance exists between the magnitude of effects of uncoupling protein 3L and uncoupling protein 1 in whole yeast versus isolated mitochondria, with uncoupling protein 3L having greater effects in whole yeast and a smaller effect on the state 4 respiration in isolated mitochondria. These findings suggest that uncoupling protein 3L, like uncoupling protein 1, has an uncoupling activity. However, the mechanism of action and/or regulation of the activity of uncoupling protein 3L is likely to be different.
Neurotoxicity of acetylcholinesterase amyloid β‐peptide aggregates is dependent on the type of Aβ peptide and the AChE concentration present in the complexes
Authors: Muñoz Francisco J. , Inestrosa Nibaldo C.
Published: July 2002

Alzheimer's disease (AD) is a neurodegenerative disorder whose hallmark is the presence of senile plaques and neurofibrillary tangles. Senile plaques are mainly composed of amyloid β‐peptide (Aβ) fibrils and several proteins including acetylcholinesterase (AChE). AChE has been previously shown to stimulate the aggregation of Aβ1–40 into amyloid fibrils. In the present work, the neurotoxicity of different amyloid aggregates formed in the absence or presence of AChE was evaluated in rat pheochromocytoma PC12 cells. Stable AChE‐Aβ complexes were found to be more toxic than those formed without the enzyme, for Aβ1–40 and Aβ1–42, but not for amyloid fibrils formed with AβVal18→Ala, a synthetic variant of the Aβ1–40 peptide. Of all the AChE‐Aβ complexes tested the one containing the Aβ1–40 peptide was the most toxic. When increasing concentrations of AChE were used to aggregate the Aβ1–40 peptide, the neurotoxicity of the complexes increased as a function of the amount of enzyme bound to each complex. Our results show that AChE‐Aβ1–40 aggregates are more toxic than those of AChE‐Aβ1–42 and that the neurotoxicity depends on the amount of AChE bound to the complexes, suggesting that AChE may play a key role in the neurodegeneration observed in Alzheimer brain.
Direct involvement of p53 in the base excision repair pathway of the DNA repair machinery
Authors: Offer Hagai , Wolkowicz Roland , Matas Devorah , Blumenstein Sara , Livneh Zvi , Rotter Varda
Published: July 2002

The p53 tumor suppressor that plays a central role in the cellular response to genotoxic stress was suggested to be associated with the DNA repair machinery which mostly involves nucleotide excision repair (NER). In the present study we show for the first time that p53 is also directly involved in base excision repair (BER). These experiments were performed with p53 temperature‐sensitive (ts) mutants that were previously studied in in vivo experimental models. We report here that p53 ts mutants can also acquire wild‐type activity under in vitro conditions. Using ts mutants of murine and human origin, it was observed that cell extracts overexpressing p53 exhibited an augmented BER activity measured in an in vitro assay. Depletion of p53 from the nuclear extracts abolished this enhanced activity. Together, this suggests that p53 is involved in more than one DNA repair pathway.
Assembly of paired helical filaments from mouse tau: implications for the neurofibrillary pathology in transgenic mouse models for Alzheimer's disease
Authors: Kampers T. , Pangalos M. , Geerts H. , Wiech H. , Mandelkow E.
Published: July 2002

In Alzheimer's disease and related dementias, human tau protein aggregates into paired helical filaments and neurofibrillary tangles. However, such tau aggregates have not yet been demonstrated in transgenic mouse models of the disease. One of the possible explanations would be that mouse tau has different properties which prevents it from aggregating. We have cloned several murine tau isoforms, containing three or four repeats and different combinations of inserts, expressed them in Escherichia coli and show here that they can all be assembled into paired helical filaments similar to those in Alzheimer's disease, using the same protocols as with human tau. Therefore, the absence of pathologically aggregated tau in transgenic mice cannot be explained by intrinsic differences in mouse tau protein and instead must be explained by other as yet unknown factors.
Characterization of the actin binding properties of the vasodilator‐stimulated phosphoprotein VASP
Authors: Hüttelmaier Stefan , Harbeck Birgit , Steffens Nils Ole , Meßerschmidt Tania , Illenberger Susanne , Jockusch Brigitte M
Published: July 2002

The vasodilator‐stimulated phosphoprotein (VASP) colocalizes with the ends of stress fibers in cell‐matrix and cell‐cell contacts. We report here that bacterially expressed murine VASP directly interacts with skeletal muscle actin in several test systems including cosedimentation, viscometry and polymerization assays. It nucleates actin polymerization and tightly bundles actin filaments. The interaction with actin is salt‐sensitive, indicating that the complex formation is primarily based on electrostatic interactions. Actin binding is confined to the C‐terminal domain of VASP (EVH2). This domain, when expressed as a fusion protein with EGFP, associates with stress fibers in transiently transfected cells.