bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of glucose phosphorylation by fatty acids in the perfused rat heart
Authors: Chatham John , Gilbert Hiram.F. , Radda George K.
Published: July 2002

The flux of glucose entering the glycolytic pathway under various metabolic conditions has been indirectly monitored in the Langendorff perfused rat heart using 31P‐NMR spectroscopy. By totally inhibiting (>95%) glyceraldehyde‐3‐phos‐phate dehydrogenase with low concentrations of iodoacetic acid (0.2 mM) in the perfusion medium, active glycolysis results in the accumulation of sugar phosphate species (fructose 1,6‐bisphosphate, dihydroxyacetone phosphate, and glyceraldehyde 3‐phosphate) which can be observed in the 31P‐NMR spectrum. Using this technique, it has been shown that butyrate (10 mM) in the perfusion medium decreases the flux through the initial steps of the glycolytic pathway by at least 6‐fold and that both glucose phosphorylation and glycogenolysis are inhibited. Upon total global ischemia in the presence of both glucose and butyrate, the glycolysis rate is stimulated approx. 100‐fold.
The substrate determines the rate and pattern of neutral lipid synthesized by isolated human sebaceous glands
Authors: Middleton B. , Birdi I. , Heffron M. , Marsden J.R.
Published: July 2002

Lipogenesis from different substrates was determined in isolated human sebaceous glands after 17–20 h in culture. Rates of total lipogenesis were 1O03 ± 141, 842 ± 90, 481 ± 57 pmol·h−1·gland−1 ± SE from acetate, lactate and glucose, respectively, when present as sole substrates: the rate from glucose was significantly lower (P < 0.01). Squalene synthesis was greatest from acetate at 479 ± 44 pmol·h−1·gland−1; significantly higher than from lactate (281 ± 45 pmol·h−1·gland−1) or glucose at 119 ± 18 pmol·h−1·gland−1. Wax ester plus cholesterol ester synthesis showed similar dependence on substrate but triglyceride synthesis was unaffected. We conclude that the added substrate determines both the rate and pattern of non‐polar lipid synthesized by isolated human sebaceous glands.
Cloning and characterization of the human gene for the α‐subunit of Gi2, a GTP‐binding signal transduction protein
Authors: Weinstein L.S. , Spiegel A.M. , Carter A.D.
Published: July 2002

We isolated and characterized human genomic clones encompassing the gene for the α‐subunit of Gi2, a GTP‐binding signal transduction protein abundantly expressed in myeloid cells. The gene is divided into 9 exons and spans 23.5 kb. Exons 2, 6 and 7 encode putative guanine nucleotide‐binding domains that are highly conserved among GTP‐binding proteins. A polyadenylation signal located within exon 9 predicts an MRNA size (∼ 2.3 kb) several hundred bases longer than that of published cDNAs, and consistent with the size seen on RNA blot hybridization. Primer extension and S1 nuclease analysis determined a major and several minor transcriptional start sites. The first exon and 5′ flanking region are highly G+ C rich, contain several GC boxes (SP1 transcription factor binding sites), a CAAT box, and lack a TATA box. The presumptive promoter region is thus similar to that of ras and other widely expressed genes.
Tissue plasminogen activator mRNA in murine tissues
Authors: Rickles Richard J. , Strickland Sidney
Published: July 2002

The urokinase‐type and tissue‐type plasminogen activators are the two enzymes found in mammals, which specifically convert the zymogen plasminogen to plasmin. Using cDNA probes, we have assayed for the presence of the two types of plasminogen activator mRNAs in murine tissues. We demonstrate that tissue‐type plasminogen activator mRNA can be detected in a wide variety of tissues. In contrast, the accumulation of urokinase‐type plasminogen activator mRNA is observed in only a few of the tissues analyzed. Using an S1 nuclease assay, we demonstrate that the tPA mRNA detected contains the complete sequences encoding the non‐protease finger, growth‐factor and kringle domains.
Phosphorylation of D‐glyceraldehyde‐3‐phosphate dehydrogenase by Ca2+/calmodulin‐dependent protein kinase II
Authors: Ashmarina Lyudmila I. , Louzenko Serguei E. , Severin Serguei E. , Muronetz Vladimir I. , Nagradova Natalia K.
Published: July 2002

Rabbit muscle D‐glyceraldehyde‐3‐phosphate dehydrogenase was shown to serve as a substrate for Ca2+/calmodulin‐dependent protein kinase II with a K
m of 0.33 μM and a V
max of 2.63 μmol · min−1 · mg−1 at pH 7.5 and 30°C. In the absence of calmodulin, the V
max was halved and K
m unchanged. 0.99 mol of phosphate was incorporated per tetrameric molecule of D‐glyceraldehyde‐3‐phosphate dehydrogenase under the experimental conditions employed.
Isolation and characterization of a fraction from brain that inhibits 1,4‐[3H]dihydropyridine binding and L‐type calcium channel current
Authors: Janis R.A. , Shrikhande A.V. , Johnson D.E. , McCarthy R.T. , Howard A.D. , Greguski R. , Scriabine A.
Published: July 2002

Bovine brain was subjected to acid extraction and several purification steps. A fraction from brain that eluted from C18 reverse‐phase columns at 30–35% acetonitrile inhibited [3H]nitrendipine binding to cardiac membranes. Further purification of this fraction on a sizing column in the presence of 40% acetonitrile yielded a low molecular mass fraction (< 1 kDa) that produced a time‐ and voltage‐dependent inhibition of L‐type (but not T‐type) Ca2+‐channel current in GH3 cells. The results suggest that this fraction contains an endogenous substance that binds directly to slowly‐inactivating Ca2+ channels and thereby inhibits current flow.
Characterization and expression of the rat heart sarcoplasmic reticulum Ca2+‐ATPase mRNA
Authors: Lompre Anne-Marie , de la Bastie Diane , Boheler Kenneth R. , Schwartz Ketty
Published: July 2002

Sarcoplasmic reticulum Ca2+‐ATPase cDNA clones have been isolated from an adult rat heart cDNA library and the nucleotide sequence of the Ca2+‐ATPase mRNA determined. The sequence has an open reading frame of 997 codons. It is identical to a cDNA isolated from a rat stomach cDNA library and 90% isologous to the rabbit and human slow/ cardiac cDNAs. Nuclease S1 mapping analysis indicates that this sequence corresponds to the main Ca2+‐ATPase mRNA present in heart and in slow skeletal muscle and that it is expressed in various proportions in smooth and non‐muscle tissues, together with another isoform which differs from the cardiac form in the sequence of its 3′‐end.
Presence of immunoreactive endothelin in human plasma
Authors: Ando K. , Hirata Y. , Shichiri M. , Emori T. , Marumo F.
Published: July 2002

A highly specific and sensitive radioimmunoassay has been established for measurement of human endothelin (hET) in human plasma. After extraction of plasma with an octyl‐silica column, this assay allowed for detection of immunoreactive (IR) hET as low as 0.2 fmol/ml. In 16 healthy subjects, the mean concentration of plasma IR‐hET was 0.6 fmol/ml. Reverse‐phase HPLC coupled with radioimmunoassay revealed two major IR‐hET components, one corresponding to authentic hET(1–21) and another with more hydrophilicity than hET(1–21). These data indicate that ET is a circulating vasoconstrictor hormone in man.
Primary structure of rat liver elongation factor 2 deduced from the cDNA sequence
Authors: Oleinikov A.V. , Jokhadze G.G. , Alakhov Yu.B.
Published: July 2002

A number of cDNA clones in summary encoding 700 amino acid residues from the N‐end of rat liver elongation factor 2 (EF‐2) and including 49 nucleotides of the 5′‐untranslated mRNA region have been obtained. EF‐2 cDNA clones were isolated from gradually constructed small (1000–5000 clones) specific cDNA libraries using the primer extension method for synthesis of the first cDNA chain. The complete primary structure of cDNA and protein EF‐2 from rat liver was derived taking into account the primary structure of the 3′‐terminal region EF‐2 cDNA previously reported [(1986) Proc. Natl. Acad. Sci. USA 83, 4978–4982]. Comparison of this cDNA with hamster cDNA has shown that (i) the base sequences had a 89.7% homology while that of the 5′‐untranslated region was 73%; (ii) there are two amino acid replacements in rat liver EF‐2 as compared with hamster EF‐2.
The ADP/ATP carrier from yeast (AAC‐2) is uniquely suited for the assignment of the binding center by photoaffinity labeling
Authors: Mayinger Peter , Winkler Edith , Klingenberg Martin
Published: July 2002

The ADP/ATP carrier from yeast was photoaffinity‐labeled in mitochondria with 2‐azido‐[α‐32P]ATP in a binding‐center‐specific, i.e. carboxyatractylate‐sensitive, manner. After isolation, fragmentation possibilities unique for the yeast AAC‐2 could be exploited to assign the insertion to a narrow range of the sequence. The CNBr fragment 115–210 contained all the incorporated label which corresponds to the second domain within the triple‐domain primary structure of the AAC. With hydroxylamine cleavage directed to the Asn 171‐Gly 172 site, all the label was found in the C‐terminal 16 kDa fragment. Thus the 2‐azido‐ATP incorporation is clearly delimited to the 172–210 segment. 8‐Azido‐[α‐32P]ATP could be site‐specifically incorporated only in isolated AAC since it has a much lower affinity for AAC than 2‐azido‐ATP. The label was also exclusively found in the 172–210 region. With both forms no incorporation into the C‐terminal region was found, as claimed for bovine AAC. The labeled segment contains Lys 179 and 182 which are homologous to bovine Lys 162 and 165 and which have been proposed to be in the translocation path.
Neuropeptide Y inhibits vasoactive intestinal peptide‐ and dopamine‐induced cyclic AMP formation in human Ewing's sarcoma WE‐68 cells
Authors: van Valen F. , Keck E. , Jürgens H.
Published: July 2002

Neuropeptide Y (NPY) regulation of intracellular cyclic AMP accumulation was studied in human Ewing's sarcoma cell line, WE‐68. NPY inhibited vasoactive intestinal peptide (VIP)‐ and dopamine‐stimulated but not basal cyclic AMP formation. The peptide effect was rapid (<2 min), concentration‐dependent with a half‐maximal effective concentration (EC50) of 8 nM NPY, and maximal inhibition reaching 60–70% with 100 nM NPY. Prior exposure of WE‐68 cells to pertussis toxin completely abolished the inhibitory action of NPY. It is concluded that NPY attenuates agonist‐stimulated cyclic AMP formation in Ewing's sarcoma WE‐68 cells, and may do so via the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.
Brain natriuretic peptide‐like immunoreactivity is present in human plasma
Authors: Togashi Kazuyoshi , Hirata Yukio , Ando Kenji , Takei Yoshio , Kawakami Masaya , Marumo Fumiaki
Published: July 2002

A highly specific and sensitive radioimmunoassay (RIA) for a novel porcine brain natriuretic peptide (BNP) has been established to elucidate whether BNP‐like immunoreactivity (LI) is present in human plasma. The antibody used was specific for BNP without any crossreactivities with known human atrial natriuretic peptides (hANP). After extraction of human plasma with Sep‐Pak cartridge, this assay allowed for detection of BNP‐LI as low as 0.1 fmol/tube. In 12 healthy subjects, the mean concentrations of plasma BNP‐LI were 1.5 fmol/ml. Reverse‐phase HPLC coupled with BNP RIA revealed that the single major component with BNP‐LI, corresponding to porcine BNP(1–26), was apparently distinct from that of α‐hANP. These data indicate that a small molecular mass form with BNP‐LI circulates in human blood.
Conformational behavior of fragments of adrenocorticotropin and their antisense peptides determined by NMR spectroscopy and CD spectropolarimetry
Authors: Najem Elias S. , Corigliano-Murphy Angela , Ferretti James A.
Published: July 2002

An ‘antisense’ peptide (‘HTCA’), whose sequence was generated by reading the antisense RNA sequence corresponding to ACTH(1–24) was shown to bind ACTH(1–24) with a K
d of 0.3 nM in a solid‐matrix binding assay [(1986) Biochem. J. 234, 679–683]. Two‐dimensional NMR spectra were used to examine the conformational behavior in methanol and in water solution of two fragments of adrenocorticotropin, ACTH(1–24) and ACTH(1–13), as well as their antisense peptides, HTCA and HTCA(12–24). The conformations are extended chains in these solutions, both as isolated molecules and when mixed with their antisense complements. The K
d values are greater than 1 mM.
Synthesis and assembly of native myosin on muscle polyribosomes
Authors: Gagnon Jacques , Kurowski Thomas T. , Zak Radovan
Published: July 2002

We have used the overload‐induced growth of avian muscles to study the assembly of the newly synthesized myosins which were separated by non‐denaturing pyrophosphate‐polyacrylamide gel electrophoresis. Using this model, we have observed the appearance of fast‐like isomyosins in polyribosomes prepared from slow anterior latissimus dorsi muscle after 72 h of overload. These new isoforms comigrating with native myosin from fast posterior latissimus dorsi muscle were not yet present in cellular extracts from the same muscle. The in vitro translation system utilizing muscle specific polyribosomes directs the synthesis of the corresponding myosin isoforms. Under denaturing conditions, myosin heavy chains and light chains dissociate to the expected subunit composition of each specific isoform. The synthesis and assembly of native myosin on polyribosomes indicate that myosin exists as a single mature protein prior to the incorporation in the thick filament.
Secretion of endothelin and related peptides from renal epithelial cell lines
Authors: Shichiri M. , Hirata Y. , Emori T. , Ohta K. , Nakajima T. , Sato K. , Sato A. , Marumo F.
Published: July 2002

Using specific radioimmunoassays (RIAs) for endothelin (ET) and big ET, we have studied whether ET and related peptides are secreted from renal epithelial cell lines (LLCPK1 and MDCK) of non‐endothelial origin. Dilution curves of extracts of conditioned media from both LLCPK1 and MDCK cell lines were parallel to those of standard porcine (p) ET and big pET in each RIA. Both cell lines incubated in serum‐free medium secreted ET‐ and C‐terminal fragment (CTF)‐like immunoreactivity (LI) of big ET as a function of time. Reverse‐phase HPLC coupled with both RIAs of the extracted media from both cell lines revealed a single component with ET‐LI coeluting with pET(1–21) and several components with CTF‐LI, one corresponding to the elution position of big pET(1–39), one to its CTF(22–39), and the others eluting earlier than CTF. These data indicate that endothelin and related peptides are synthesized by and secreted from cells other than endothelial cells.
Lack of arginine‐ and polyphosphate‐storage pools in a vacuole‐deficient mutant (end1) of Saccharomyces cerevisiae
Authors: Westenberg Beate , Boller Thomas , Wiemken Andres
Published: July 2002

Yeast cells accumulate large amounts of arginine and polyphosphate in their vacuoles and utilize these compounds as endogenous nitrogen or phosphate sources under conditions of starvation. We examined a vacuoleless mutant, end1, and found that it stored virtually no arginine or polyphosphate when grown on a medium with arginine as the sole nitrogen source. When starved of nitrogen or phosphate it stopped growing much faster than the wildtype. Unlike the wildtype, end1 showed no accumulation of polyphosphate and, concomitantly, of arginine after a period of phosphate starvation. The results support the concept that vacuoles contain the main reserves of nitrogen and phosphate in fungi.
Formation of endothelin by cultured airway epithelial cells
Authors: Black P.N. , Ghatei M.A. , Takahashi K. , Bretherton-Watt D. , Krausz T. , Dollery C.T. , Bloom S.R.
Published: July 2002

Immunoreactivity to endothelin was detected in conditioned culture medium from both canine and porcine tracheal epithelial cells. Gel permeation chromatography and fast protein liquid chromatography were used to confirm the identity of the endothelin. The two peaks demonstrated on fast protein liquid chromatography co‐eluted with endothelin 1 and endothelin 3 respectively.
The intracellular localization of glycolate oxidoreductase in Escherichia coli
Authors: Sallal A.-K.J. , Nimer N.A.
Published: July 2002

Distribution of glycolate oxidoreductase in cell‐free extracts of E. coli grown in mineral medium containing sodium glycolate has been studied. The enzyme was found to be largely associated with the cytoplasmic membranes. Homologous antiserum to the membranes inhibited the enzyme activity completely after the solubilization of the intact membranes with Triton X‐100. Results on the effect of pronase on glycolate oxidoreductase activity confirmed that part of the enzyme is exposed to the surface of the membrane.
Prorenin is sorted into the regulated secretory pathway independent of its processing to renin in mouse pituitary AtT‐20 cells
Authors: Nakayama Kazuhisa , Nagahama Masami , Kim Won-Sin , Hatsuzawa Kiyotaka , Hashiba Kiyomi , Murakami Kazuo
Published: July 2002

Renin; Prohonnone processing; Gene expression; (AtT‐20 cell)
Brain natriuretic peptide is cosecreted with atrial natriuretic peptide from porcine cardiocytes
Authors: Iida Tatsuyoshi , Hirata Yukio , Takemura Naoyuki , Togashi Kazumi , Nakagawa Shigeki , Marumo Fumiaki
Published: July 2002

Using primary cultures of atrial cardiocytes from neonatal pig, the secretion of brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP)‐like immunoreactivities (LI) was studied in vitro. Porcine cardiocytes time‐dependently secreted both BNP‐LI and ANP‐LI into medium under a serum‐free condition, although the amount of BNP‐LI secreted was about one‐third that of ANP‐LI. Phorbol ester and Ca2+ ionophore had less stimulatory effects on secretion of BNP‐LI than that of ANP‐LI. Reverse‐phase HPLC of the conditioned medium revealed a single major BNP‐LI component corresponding to synthetic porcine BNP(1‐26). These data suggest that a small molecular weight form BNP, possibly BNP(1‐26), is cosecreted with ANP from porcine cardiocytes.
Effects of D600 on hypoxic suppresssion of K+ currents in isolated type I carotid body cells of the neonatal rat
Authors: Peers C.
Published: July 2002

K+ currents recorded from isolated type I carotid body cells were reversibly suppressed by hypoxia (pO2 25 Torr). Inhibition of Ca2+ influx using high Mg2+, low Ca2+ solutions abolished this effect, indicating hypoxia selectively inhibits the Ca2+‐activa ted component of K+ currents. The Ca2+ channel blockers D600 and verapamil reversed the suppressive effects of hypoxia. These drugs also partially inhibited Ca2+‐independent K+ currents, but this effect was relieved by hypoxia, causing apparent reversal of hypoxic suppression of the K+ currents seen under control conditions.
Generation of human endothelin by cathepsin E
Authors: Lees Wendy E. , Kalinka Siân , Meech John , Capper Stephen J. , Cook Neil D. , Kay John
Published: July 2002

Highly‐purified cathepsin D processed human big endothelin1–38 into endothelin‐like fragments but did not appear to generate endothelin1–21 under the conditions employed. By contrast, human cathepsin E specifically cleaved human big endothelin into endothelin1–21 and the C‐terminal fragment under identical conditions but did not degrade either product further.
mRNA levels and methylation patterns of the tyrosine aminotransferase gene in aging inbred rats
Authors: Slagboom P.Eline , de Leeuw Wiljo J.F. , Vijg Jan
Published: July 2002

We have examined the mRNA levels and methylation patterns of the liver‐specific tyrosine aminotransferase (TAT) gene in inbred female rats aged 6, 24 and 36 months. Northern hybridization analysis of total RNA showed a 65% decrease in the steady state transcript level of TAT in the liver of 24‐ and 36‐month old rats as compared to 6‐month old animals. The TAT gene as studied by Southern hybridization analysis using the isoschizomers Hpa II and Msp I was found to be hypomethylated in the liver as compared to spleen and brain at six CpG sites within the gene. Methylation at these sites remained unchanged during aging.
Identification and cDNA sequence of δ‐preprotachykinin, a fourth splicing variant of the rat substance P precursor
Authors: Harmar A.J. , Hyde V. , Chapman K.
Published: July 2002

The neuropeptides substance P and neurokinin A are synthesised from a family of precursor polypeptides encoded by the preprotachykinin A (PFT) gene. In addition to mRNA (β‐PPT) containing all 7 exons of the gene, alternatively spliced mRNAs lacking either exon 4 (γ‐PPT) or exon 6 (α‐PPT) have been identified. We have determined the sequences of cDNA clones encoding four variants of PPT mRNA from rat dorsal root ganglion (DRG), including a novel mRNA species (δ‐PPT) in which both exons 4 and 6 are absent. The sequence of δ‐PPT predicts the existence of a novel tachykinin precursor polypeptide.
The role of apoE in inhibitory effects of apoE‐rich HDL on platelet function
Authors: Higashihara Masaaki , Kinoshita Makoto , Teramoto Tamio , Kume Shoji , Kurokawa Kiyoshi
Published: July 2002

Apolipoprotein E‐ (ApoE‐) rich high‐density lipoprotein (HDL), which was prepared from the bound fraction of normolipemic volunteers on heparin‐Sepharose and from a hyperalphalipoproteinemic patient, potently inhibited aggregation of human platelets in a dose‐dependent fashion. Dimyristoyl phosphatidylcholine liposome with apoE (apoE · DMPC) also inhibited platelet aggregation, and incubation of washed platelets with apoE · DMPC resulted in the release of cholesterol into the supernatant in a time and dose‐dependent manner. These results suggest that apoE plays a major role in the inhibitory effect of apoE‐rich HDL in platelet function, presumably due to the release of cholesterol from the plasma membrane.
Molecular cloning of cDNA and a chromosomal gene encoding GPE1‐BP, a nuclear protein which binds to granulocyte colony‐stimulating factor promoter element 1
Authors: Nishizawa Mikio , Wakabayashi-Ito Noriko , Nagata Shigekazu
Published: July 2002

Granulocyte colony‐stimulating factor (G‐CSF) is produced from macrophages in response to lipopolysaccharide (LPS). GPE1, a cis‐acting element of the G‐CSF gene promoter, functioned as an LPS‐inducible element. We isolated cDNA and a chromosomal gene encoding the mouse GPEI‐binding protein (GPE1‐BP). A 150‐amino acid protein deduced from the cDNA has a basic domain and a leucine zipper motif, and seems to be identical to that of recently isolated Ig/EBP. The nuclear extract from COS cells transfected with the cDNA showed GPE1‐binding activity. Transcripts were ubiquitously detected, and may be spliced from two exons of a single gene.
Identification and characterization of a C‐terminally extended form of recombinant murine IL‐6
Authors: Danley Dennis E. , Strick Christine A. , James Larry C. , Lanzetti Anthony J. , Otterness Ivan G. , Grenett Hernan E. , Fuller Gerald M.
Published: July 2002

Murine interleukin‐6 (mIL‐6) was expressed inEscherichia coliin the insoluble fraction of cell lysates. Approximately equal amounts of two polypeptide species, reactive with anti‐IL‐6 antibodies, were produced. The two forms of mIL‐6 were isolated and found to have identical N‐terminal sequences initiated by Met‐Phe‐Pro‐Thr‐Ser‐Gin‐. Peptide mapping after endoproteinase glu‐C digestion led to isolation led to isolation and characterization of the C‐terminal peptides from each of the two forms and allowed the source of the heterogeneity to be identified as a C‐terminal addition of three amino acids, Gin‐Lys‐Leu, to authentic mIL‐6. Inspection of the nucleotide sequence of the plasmid containing the mIL‐6 gene and expression of the plasmid in other strains suggested that the addition of three amino acids was caused by a readthrough of the termination codon arising from an unexpected suppressor mutation in the original host strain. Although the C‐terminus of IL‐6 is critical for the activity of this cytokine, the IL‐6 variant with extended C‐terminus was fully active in two separate bioassays. This suggests that the additional amino acids do not disrupt the structure or function of this important region of the molecule.
A novel protein phosphatase inhibitor, tautomycin Effect on smooth muscle
Authors: Hori M. , Magae J. , Han Y.-G. , Hartshorne D.J. , Karaki H.
Published: July 2002

The antibiotic, tautomycin, was found to be a potent inhibitor of protein phosphatases and equally effective for the type‐1 and type‐2A enzymes. For the catalytic subunits of the type‐1 and type‐2A phosphatases the IC50 value was 22 to 32 nM. For the phosphatase activity present in chicken gizzard actomyosin the IC50 value was 6 nM. Tautomycin had no effect on myosin light chain kinase activity. Tautomycin induced a Ca2+‐independent contraction of intact and permeabilized smooth muscle fibers and this was accompanied by an increase in the level of myosin phosphorylation. Thus, tautomycin by virtue of its ability to inhibit phosphatase activity is a valuable addition for studying the role of protein phosphorylation.
Protein kinase C of a human megakaryoblastic leukemic cell line (MEG‐01) Analysis of subspecies and activation by diacylglycerol and free fatty acids
Authors: Kitagawa Yasuo , Matsuo Yoshinobu , Minowada Jun , Nishizuka Yasutomi
Published: July 2002

Protein kinase C (PKC) from a human megakaryoblastic leukemic cell line (MEG‐01) was resolved into two fractions by hydroxyapatite column chromatography, which are indistinguishable from the brain type II (βI/βII) and type III (α) subspecies, by biochemical and immunoblot analysis. In the presence of both phosphatidylserine and diacylglycerol, several free unsaturated fatty acids (FFA's), such as arachidonic, oleic, linoleic and linolenic acids, further enhanced the enzyme activation, and allowed the enzyme to exhibit almost full activity at nearly basal levels of Ca2+ concentration. The concentration of unsaturated FFA's giving rise to the maximum enzyme activation was around 2 × 10−5 M. Palmitic and stearic acids were inactive. The result implies that, in addition to diacylglycerol, the receptor‐mediated release of unsaturated FFA's from membrane phospholipids may also take part in the activation of PKC.
Immunological demonstrations of εPKC Murine tissue distribution, ontogeny, cellular localization and translocation
Authors: Gschwendt Michael , Leibersperger Hanno , Rincke Gabriele , Marks Friedrich
Published: July 2002

An antiserum raised against an εPKC‐specific peptide recognizes εPKC with an apparent molecular weight of 97 kDa in cytosol of mouse brain. No cross‐reaction with α,β,γPKC or the δPKC‐like p76‐kinase is observed. εPKC is mainly present in brain. Just traces of this PKC isoenzyme can be detected in some other murine tissues. Ontogenetic studies indicate that the amount of εPKC in murine brain increases constantly and reaches a maximal level at day 7 after birth. Upon TPA activation εPKC is translocated from the cytosol to the particulate fraction in a brain homogenate.
Differentiation of neuroblastoma cells correlates with an altered splicing pattern of tau RNA
Authors: de Garcini E.Montejo , Corrochano Luis , Wischik C.M. , Nido J.Diaz , Correas I. , Avila J.
Published: July 2002

Morphological differentiation of N2A nouroblastoma cells is associated with an altered splicing of the gene of the microtubule‐associated protein, tau. Two populations of RNA (coding for tau proteins containing three or four tubulin‐binding motifs) are present in a similar proportion in undifferentiated neuroblastoma cells while in differentiated cells the proportion is changed in favour of that population coding for tau protein containing four tubulin‐binding motifs. An increase in a high molecular weight tau isoforms correlates with the increase in the RNA population coding for four tubulin‐binding motifs. A possible consequence of expressing a higher proportion of the tau protein containing four tubulin‐binding motifs could be an increase in microtubule stability of differentiated neuroblastoma cells.
Solution conformation of a cyclic pentapeptide endothelin antagonist Comparison of structures obtained from constrained dynamics and conformational search
Authors: Krystek Stanley R. , Bassolino Donna A. , Bruccoleri Robert E. , Hunt John T. , Porubcan Michael A. , Wandler Charles F. , Andersen Niels H.
Published: July 2002

The structure of a cyclic pentapeptide, cyclo‐(d‐Trp‐d‐Asp‐l‐Pro‐d‐Val‐l‐Leu), that has high selectivity for the endothelin ETAA receptor has been determined by NMR spectroscopy using constrained molecular dynamics and conformational search procedures. Structures obtained using two methods of refinement, namely (i) constrained molecular dynamics; and (ii) systematic searches of conformational space for optimal satisfaction of distance constraints, were compared to those obtained from systematic searches of conformational space without NMR data. The two different procedures of refinement produce similar conformations that are consistent with the NMR distance constraints. Conformational searches for optimal energy without any NMR distance constraints produced several low‐energy structures, two of which have essentially the same backbone as those structures derived from distance‐constrained procedures and one of these even reproduces several side‐chain positions well. The pentapeptide backbone consists of a linked γ and β‐turn conformation, with the leucine and tryptophan as corner residues of the type II β‐turn. The side chains are highly ordered both in aqueous solvent and in dimethyl sulfoxide. In aqueous media the leucine side chain is directed towards the indole ring, presumably to reduce the non‐polar surface exposure, producing unusual upfield shifts for the methyls (and particularly Hγ). This structural feature was reproduced in one of the structures obtained from conformational searches performed without NMR data. Exhaustive conformational searches appear to provide an alternative method for structure generation for cyclic peptides.
Ca2+‐dependent ubiquitination of calmodulin in yeast
Authors: Jennissen H.P. , Botzet G. , Majetschak M. , Laub M. , Ziegenhagen R. , Demiroglou A.
Published: July 2002

Recently we were able to show that calmodulin from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca2+‐dependent manner by ubiquityl‐calmodulin synthetase (uCaM‐synthetase) from mammalian sources [R. Ziegenhagen and H.P. Jennissen (1990) FEBS Lett. 273, 253‐256]. It was therefore of high interest to investigate whether this covalent modification of calmodulin also occurs in one of the simplest eukaryotes, the unicellular Saccharomyces cerevisiae. Yeast calmodulin was therefore purified from bakers yeast. In contrast to calmodulin from spinach and N. crassa it does not activate phosphorylase kinase. Crude yeast uCaM‐synthetase conjugated ubiquitin Ca2+‐dependently to yeast and mammalian (bovine) calmodulin. Yeast calmodulin was also a substrate for mammalian (treticulocyte) uCaM‐synthetase. As estimated from autoradiograms the monoubiquitination product (first‐order conjugate) of yeast calmodulin has an apparent molecular mass of ca. 23–26 kDa and the second‐order conjugate an apparent molecular mass of ca. 28–32 kDa. Two to three ubiquitin molecules can be incorporated per yeast calmodulin. Experiments with methylated ubiquitin in the heterologous reticulocyte system indicate that, as with vertebrate calmodulins, only one lysine residue of yeast calmodulin reacts with ubiquitin so that the incorporation of multiple ubiquitin molecules will lead to a polyubiquitin chain. These results also indicate that the ability of coupling ubiquitin to calmodulin was acquired at a very early stage in evolution.
Mechanism of catalysis of Fe(II) oxidation by ferritin H chains
Authors: Treffry Amyra , Hirzmann Jorg , Yewdall Stephen J. , Harrison Pauline M.
Published: July 2002

Recombinant H chain ferritins bearing site‐directed amino acid substitutions at their ferroxidase centres have been used to study the mechanism of catalysis of Fe(II) oxidation by this protein. UV‐difference spectra have been obtained at various times after the aerobic addition of Fe(II) to the recombinants. These indicate that the first product of Fe(II) oxidation by wild type H chain apoferritin is an Fe(III) μ‐oxo‐bridged dimer. This suggests that fast oxidation is achieved by 2‐electron transfer from two Fe(II) to dioxygen. Modelling of Fe(III) dimer binding to human H chain apoferritin shows a solvent‐accessible site, which resembles that of ribonucleotide reductase in its ligands. Substitution of these ligands by other amino acids usually prevents dimer formation and leads to greatly reduced Fe(II) oxidation rates.
Apoptosis Biochemical events and relevance to cancer chemotherapy
Authors: Sen Soumitra , D'Incalci Maurizio
Published: July 2002

Two distinct pathways for cell death exist. Compared to necrotic death, physiological or apoptotic cell death is an active suicidal process that consists of a cascade of well‐regulated synthetic events. Participation of specific genes in apoptosis, and its possible molecular regulation, are considered in order to investigate the mechanism of cell death induced by some cancer chemotherapeutic agents.
Carbohydrate binding specificity of the Tn‐antigen binding lectin from Vicia villosa seeds (VVLB4)
Authors: Puri Kamal Deep , Gopalakrishnan B. , Surolia Avadhesha
Published: July 2002

2‐Dansylamino‐2‐deoxy‐d‐galactose (GalNDns) is a useful fluorescent probe to study the interaction of non‐fluorescent sugars with the B4 lectin from Vicia villosa seed; (VVLB4). Binding of the lectin to GalNDns leads to a 5.2‐fold increase in Dansyl fluorescence with a concomitant 10 nm blue shift in its emission maximum. The strong binding of GalNDns (K
u = 7.33 × 104 M− at 20°C) is due to a favourable entropic contribution to the association process. Among the other sugars studied, GalNAcαl‐O‐Ser followed by MeαGalNAc are the best ligands. 2‐Deoxygalactose, galactosamine and galactose are 2013, 469 and 130 times weaker ligands, respectively, as compared to GalNAc, whereas GalNDns is about 2.44 times more potent than GalNAc, indicating that substitutions at the C‐2 position of GalNAc have a considerable influence on the binding affinities. Equatorial orientation of the hydroxyl group at C‐3 and axial orientation at C‐4 as in galactose are important for the interaction with VVLB4. The C‐6 hydroxyl group is not indispensable. The binding site of the lectin is directed exclusively towards monosaccharides alone. Interestingly enough, despite its preference for MeαGalNAc over MeβGalNAc, in oligosaccharides, the lectin prefers terminal β‐linked GalNAc as compared to the α‐linked one.
One step purification and characterization of the pyrrolidone carboxyl peptidase of Streptococcus pyogenes over‐expressed in Escherichia coli
Authors: Awadé A. , Gonzalès Th. , Cleuziat Ph. , Robert-Baudouy J.
Published: July 2002

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp), an enzyme which selectively removes pyrrolidone carboxylic acid (PCA) from some PCA‐peptides and ‐proteins, was demonstrated in bacteria and in plant, animal and human tissues. In this paper we describe the purification to homogeneity of the enzyme of Streptococcus pyogenes, over‐expressed in Escherichia coli. This was achieved, for the first time in one step, by hydrophobic interaction chromatography. Analysis under non‐denaturing conditions revealed a molecular mass of 85 kDa and in the presence of sodium dodecyl sulfate gave a molecular mass of 23.5 kDa. Investigations on enzymatic properties showed that the Pcp over‐expressed in E. coli disclosed properties similar to those found for the enzyme extracted from S. pyogenes or for some other Pcps studied previously. Thus the over‐expressed enzyme should serve as a suitable source for N‐terminal unblocking prior to some PCA protein sequencing.
Reactive oxygen species are involved in the activation of cellular phospholipase A2
Authors: Goldman R. , Ferber E. , Zort U.
Published: July 2002

Vanadate (V) potentiated (4‐ to 10‐fold) the activation of cellular phospholipase A2 (PLA2) induced by H2O2 (H), a phorbol ester (T), a Ca2+‐ionophore (A) and opsonized zymosan in macrophages. V+H induced in intact cells the activation and translocation of PLA2 and protein kinase C(PKC) to the plasma membrane. V+H and V+T+A induced strong chemiluminescence (CL) which was abrogated by a specific NADPH oxidase inhibitor diphenylene iodonium (DPI). DPI markedly suppressed the stimulation of PLA2 by V+T+A and V+OZ. The results suggest that the formation of endogenous reactive oxygen species (ROS) is important for PLA2 activation.
Requirement of the calcium channel β subunit for functional conformation
Authors: Nishimura Seiichiro , Takeshima Hiroshi , Hofmann Franz , Flockerzi Veit , Imoto Keiji
Published: July 2002

The cardiac dihydropyridine‐sensitive L‐type calcium channel was stably expressed in Chinese hamster ovary cells by transfecting the rabbit cardiac calcium channel α1 subunit cDNA with or without coexpression of the β subunit of skeletal muscle calcium channel. Whereas coexpression of the β subunit significantly increased DHP binding activity and calcium channel activity, it did not affect the amount of the α1 subunit expressed, as judged by RNA blot hybridization analysis and immunoblotting analysis. The results suggest that association with the β subunit is necessary for the α1 subunit protein to take a proper conformation suitable for a functional calcium channel.
Lysophosphatidic acid induces tyrosine phosphorylation and activation of MAP‐kinase and focal adhesion kinase in cultured Swiss 3T3 cells
Authors: Kumagai Naokazu , Morii Narito , Fujisawa Kazuko , Yoshimasa Takaaki , Nakao Kazuwa , Narumiya Shuh
Published: July 2002

Lysophosphatidic acid (LPA) added to serum‐starved Swiss 3T3 cells induced, in a time‐ and concentration‐dependent manner, tyrosine phosphorylation of multiple proteins, including proteins of 43, 64, 88 kDa and a group of proteins between 110 and 130 kDa. Among them, two proteins, p43 and p120, were identified as mitogen‐activated protein kinase (MAP‐kinase) and focal adhesion kinase (FAK), respectively, by immunoprecipitation and immunoblot analysis. Tyrosine phosphorylation of p64 peaked at l min and declined rapidly, whereas that of MAP‐kinase and FAK peaked at 5 and 10 min after the addition of LPA, respectively. The activity of MAP‐kinase determined as phosphorylation of myelin basic protein increased transiently about 3‐fold at 5 min, and correlated with tyrosine phosphorylation. These results indicate that tyrosine phosphorylation of these proteins is a part of the signal transduction by LPA and may be involved in its mitogenic responses.
Expression, purification and crystallization of fully active, glycosylated human interleukin‐5
Authors: Guisez Yves , Oefner Christian , Winkler Fritz K. , Schlaeger Ernst-Jürgen , Zulauf Martin , Van der Heyden José , Plaetinck Geert , Cornells Sigrid , Tavernier Jan , Fiers Walter , Devos René , D'Arcy Allan
Published: July 2002

Recombinant human interleukin‐5 (hIL‐5) has been expressed at high levels and produced in large quantities in baculovirus infected Sf9 insect cells. The glycosylated protein was purified using immuno‐affinity chromatography and gel filtration. Purified hIL‐5 has been crystallized using standard vapour diffusion techniques with PEG as a coprecipitant. The crystals belong to the C2 space group and diffract to 2 Å.
Immediate‐early gene induction by repetitive mechanical but not electrical activity in adult rat cardiomyocytes
Authors: Kubisch Christian , Wollnik Bernd , Maass Alexander , Meyer Rainer , Vetter Hans , Neyses Ludwig
Published: July 2002

Mechanical factors are thought to play an important role in the induction of myocardial hypertrophy. Yet, it is not known whether active contraction induces genes that probably represent initial steps in the hypertrophic response in the adult myocardium ‐ and if so, whether the mechanical or the electrical component of the twitch governs this response. We therefore investigated whether electrical stimulation of contraction was able to induce the immediate‐early genes (IEGs) egr‐1 and c‐fos in adult rat cardiomyocytes. Cyclical contraction led to an increase in egr‐1 and c‐fos mRNA levels within 30 min. Full inhibition of contraction during electrostimulation by the Ca2+‐desensitizer 2,3‐butanedione monoxime (BDM) totally blocked this IEG‐response without altering membrane potential. These data suggest that in adult myocardium, the mechanical rather than the electrical activity is responsible for the IEG‐response during active twitch.
Triplet repeat sequences in human DNA can be detected by hybridization to a synthetic (5'‐CGG‐3')17 oligodeoxyribonucleotideag]
Authors: Behn-Krappa Annett , Mollenhauer Jan , Doerfler Walter
Published: July 2002

The seemingly autonomous amplification of naturally occurring triplet repeat sequences in the human genome has been implicated in the causation of human genetic disease, such as the fragile X (Martin‐Bell) syndrome, myotonic dystrophy (Curshmann‐Steinert), spinal and bulbar muscular atrophy (Kennedy's disease) and Huntington's disease. The molecular mechanisms underlying these triplet amplifications are still unknown. We demonstrate here that a synthetic (CGG)17 oligodeoxyribonucleotide can be utilized as hybridization probe to visualize some of the triplet repeats in the human genome. This technique may help in studies aimed at the elucidation of the amplification mechanism.
Long‐term regulation of pyruvate dehydrogenase kinase by high‐fat feeding
Authors: Orfali Karen A. , Fryer Lee G.D. , Holness Mark J. , Sugden Mary C.
Published: July 2002

The provision of the high‐fat diet (47% of calories as fat) for 28 days evoked a significant decline in cardiac PDHa, activity, together with marked increases in the activity of PDH kinase measured in isolated mitochondria and freshly‐prepared cardiomyocytes from adult rats. Plasma insulin concentrations in fat‐fed rats were not significantly different from control, but plasma NEFA concentrations were elevated. PDH kinase activity in cardiomyocytes from fat‐fed rats fell substantially in culture (25 h). This decline was prevented by the inclusion of n‐octanoate and DBcAMP in combination, but not individually, in the culture medium. The results are discussed in relation to the role for fatty acids and insulin in the long‐term modulation of cardiac PDH kinase activity by high‐fat feeding.
β‐Amyloid protein enhances the mitogen‐induced calcium response in circulating human lymphocytes
Authors: Eckert Anne , Hartmann Henrike , Müller Walter E.
Published: July 2002

The role of β‐amyloid in Alzheimer's disease and its cellular mechanism of action on neurons are still unclear. There is growing evidence that β‐amyloid or its fragment, 25–35, influence neuronal calcium regulation. To investigate the effects of β‐amyloid on calcium homeostasis in man we used peripheral human lymphocytes as a model system for central neurons. β‐Amyloid fragment 25–35 exposed to lymphocytes for 60 s elevates the phytohemagglutinin (PHA)‐induced Ca2+ rise in a dose‐dependent manner. Small effects were already seen at concentrations as low as 50 nmol/1. Similar effects were also observed with fragment 1–40, whereas fragments 1–28 or 12–28 did not affect the Ca2+ response after PHA stimulation. Our findings support the hypothesis of an enhanced calcium response as a general feature of β‐amyloid's neurotoxicity. The lymphocyte seems to be a valuable model to study this effect in man.
Dephosphorylation of distinct sites on microtubule‐associated protein MAP1B by protein phosphatases 1, 2A and 2B
Authors: Ulloa Luis , Dombrádi Viktor , Díaz-Nido Javier , Szücs Kornélia , Gergely Pál , Friedrich Peter , Avila Jesus
Published: July 2002

Rat brain microtubule‐associated protein MAP1 B has been tested as a substrate for Ser/Thr protein phosphatases (PP). The dephosphorylation reactions were followed by specific antibodies recognizing phosphorylated and phosphorylatable epitopes. One set of phosphorylation sites on MAPI B are referred to as mode I sites, and their phosphorylation is presumably catalyzed by proline‐directed protein kinases. These mode I sites are efficiently dephosphorylated by PP2B and 2A but not by PP1. Another set of phosphorylation sites on MAP1B are named mode II sites, and their phosphorylation is possibly due to casein kinase II. These mode II sites are dephosphorylated by PP2A and PP1, the PP2B being ineffective. The selectivity of phosphatases for different sites within the same protein indicates the complexity of the dephosphorylation reactions regulating the functionality of MAP1B in neurons.
Interferon‐γ and tumor necrosis factor‐α enhance p60src expression in human macrophages and myelomonocytic cell lines
Authors: Sorio Claudio , Melotti Paola , Dusi Stefano , Berton Giorgio
Published: July 2002

We investigated modulation of p60src expression in human mononuclear phagocytes. By analysis of [35S]methionine‐labelled cells we found that synthesis of p60src is higher in human monocytes compared to macrophages derived from in vitro cultivation of monocytes. Western blot analysis showed that expression of p60src in monocyte‐derived macrophages can be enhanced if monocytes are differentiated into macrophages in the presence of interferon‐γ (IFN‐γ), or tumor necrosis factor‐α (TNF‐α). Enhanced p60src expression caused by IFN‐γ or TNF‐a correlated with an enhanced autophosphorylating kinase activity assayed in anti‐p60src immune precipitates. In vivo phosphorylation of p60src and analysis of phosphopeptides by tryptic digestion showed that treatment with cytokines did not affect the pattern of phosphorylation of distinct phosphopeptides. The human monocytic cell lines, U937 and HL‐60, induced to differentiate along the monocytic pathway by IFN‐γ, or a combination of IFN‐γ and TNF‐α, expressed higher amounts of the p60src, but not of the p59fyn or p62yes, kinase activity. These findings show that p60src is modulated in the course of differentiation of human monocytes to macrophages, and that macrophage‐activating cytokines increase p60src expression in human monocyte‐derived macrophages.
Variable and constant regions in the C‐terminus of vinculin and metavinculin
Authors: Strasser Peter , Gimona Mario , Herzog Monika , Geiger Benjamin , Small J.Victor
Published: July 2002

Metavinculin differs from vinculin in having an additional insert of 68 to 79 amino acids in length in the C‐tenninal half of the molecule. Cross‐species comparison of metavinculin sequences from pig, man, chicken and frog reveals a division of the insert into two parts: the first variable and the second highly conserved. The longest insert, 79 amino acids, was found in Xenopus laevis. Three different C‐terminal constructs of vinculin and metavinculin over‐expressed in E. coli could be purified by column chromatography. Two‐dimensional gel electrophoresis and peptide analysis revealed pI values between 8.35 and 10.25 for the recombinant proteins. Biochemical and structural features of the metavinculin‐specific sequence and the conserved vinculin/metavinculin carboxy‐terminus are discussed.
Tyrosine phosphorylation is involved in the respiratory burst of electropermeabilized human neutrophils at a step before diacylglycerol formation by phospholipase C
Authors: Mitsuyama Takashi , Takeshige Koichiro , Minakami Shigeki
Published: July 2002

We studied a step where tyrosine phosphorylation is involved in a signaling pathway for the activation of the superoxide (O−
2)‐generating NADPH oxidase using electropermeabilized human neutrophils. The permeabilized cells produced O−
2 by the addition of a protein tyrosine phosphatase inhibitor, vanadate, as well as N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) and protein kinase C (PKC) activators such as phorbol myristate acetate (PMA) and l‐α‐ 1 ‐oleoyl‐2‐acetoyl‐sn‐3‐glycerol (OAG). The O−
2 production by the stimulants was completely inhibited by PKC inhibitors such as calphostin C and staurosporine and was not affected by 1 % ethanol, a metabolic modulator of phospholipase D (PLD). Furthermore, the O−
2 production by vanadate and fMLP, but not by OAG and PMA, was inhibited by both an inhibitor of phospholipase C (PLC), neomycin, and an inhibitor of tyrosine kinase, ST‐638. These findings suggest that tyrosine phosphorylation is involved in the activation of the oxidase at a step before diacylglycerol formation by PLC, and that PLD may not be involved in the signaling pathway in permeabilized cells.