bluuebunny/crossref_metadata_2025 huggingface dataset:
Phencyclidine (PCP) blocks glutamate‐activated postsynaptic currents
Authors: Idriss M. , Albuquerque E.X.
Published: July 2002

Phencyclidine
Glutamate receptor
Locust
Neuromuscular transmission
Excitatory postsynaptic current
Channel blacker
Effect of netropsin on plasmid DNA cleavage by BAL 31 nuclease
Authors: Sakaguchi Reiko , Joho Ken-ichi , Shishido Kazuo
Published: July 2002

BAL 31 nuclease is known to possess two sorts of catalytic activities: one is a single‐strand‐specific endonuclease activity that converts negatively supercoiled UNA to the unit‐length linear form, the other being a quasi‐processive exonuclease activity that simultaneously degrades both 3'‐ and 5'‐termini of linear dúplex DNA. Netropsin, a bactericidal and antiviral compound, was found to enhance the former activity but inhibit the latter. Netropsin‐bound supercoiled plasmid DNA had a tendency to be fragmented by BAL 31 into several species of linear DNAs smaller than full‐length size. Size reduction of linear plasmid DNA by BAL 31 was significantly inhibited by netropsin binding.
Effect of pH on the induction of competence and progression to the S‐phase in mouse fibroblasts
Authors: Bravo Rodrigo , Macdonald-Bravo Heather
Published: July 2002

The pH dependence of competence induction and progression to the S‐phase in quiescent stimulated cells has been studied. The results show that: (i) induction of competence by fibroblast growth factor in these cells is relatively independent of the external pH between pH 5.6–7.6; (ii) progression of cells to the S‐phase is highly sensitive to pH and shows a dramatic increase between pH 6.8–7.2. These observations suggest that the intracellular alkalinization triggered by growth factors is fundamental for progression but not for competence induction.
In vivo synthesis and processing of rat hypothalamic prosomatostatin
Authors: Camier Maryse , Barre Nicole , Morel Alain , Cohen Paul
Published: July 2002

The in vivo incorporation of [3H]phenylalanine into an apparent 15 kDa prosomatostatin was observed in the hypothalamus of rats injected with the labeled amino acid in the third ventricle. Precursor‐product relationships were established between this newly synthesized material and both somatostatin‐28 and ‐14.
On the presence of HMG proteins in yeast
Authors: Estruch F. , Pérez-Ortín J.E. , José M. , Puigdomènech P. , Franco L.
Published: July 2002

Two polypeptides antigenically related to mammalian HMG1/2 have been detected in the yeast Saccharomyces cerevisiae. One exhibits an electrophoretic mobility in the range of mammalian HMG1/2 whereas the second polypeptide comigrates with yeast HMG S4. Evidence for the presence of HMG14/17 in yeast has not been obtained by immunological or nuclease digestion‐based methods, although their presence cannot be excluded.
Phorbol ester and 1,2‐diolein are not fully equivalent activators of protein kinase C in respect to phosphorylation of membrane proteins in vitro
Authors: Kiss Zoltan , Luo Yuan
Published: July 2002

Phosphorylation of liver plasma membrane proteins by protein kinase C was studied by using the two best known activators of the enzyme, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and 1,2‐diolein. While the effects of TPA and diolein were almost identical on two proteins and similar in magnitude on four proteins, the phosphorylation of an additional four proteins was increased only by TPA. We conclude that in respect to phosphorylation of membrane proteins, TPA and diglycerides are not fully equivalent activators of kinase C.
Insulin antagonises the growth hormone‐mediated increase in the activity of phosphatidate phosphohydrolase in isolated rat hepatocytes
Authors: Pittner Richard A. , Bracken Paul , Fears Robin , Brindley David N.
Published: July 2002

Rat hepatocytes were incubated in monolayer culture, under serum‐free conditions for 8 h. Rat growth hormone (up to 100 nM) increased the activity of phosphatidate phosphohydrolase by up to 41%. Insulin (500 pM or 35 nM), cycloheximide or actinomycin D reversed this effect. The ability of growth hormone to modify the effects of insulin is discussed in relation to the control of the phosphohydrolase activity and glycerolipid synthesis.
Isolation of the haemopexin‐haem receptor from pig liver cells
Authors: Majuri R. , Gräsbeck R.
Published: July 2002

Isolated pig liver plasma membranes interact specifically with the haemopexin‐haem complex (K
d 4.4 × 10−7 M). Affinity chromatography was used to isolate a membrane component which binds this complex with high affinity. Pig serum haemopexin was first isolated by affinity chromatography on haemin‐Sepharose followed by HPLC gel filtration. Liver membranes solubilized with Triton X‐100 were incubated with haemin‐Sepharose saturated with haemopexin, and as a control, with affinity gel lacking haemopexin. SDS‐polyacrylamide gel electrophoresis of the eluted protein indicated that from the haemin‐Sepharose emerglow‐molecular‐mass haemin‐binding proteins whereas the eluate from haemopexin‐haemin‐Sepharose contained an additional 71 kDa protein, which did not bind free haemin. This protein appears to represent the haemo‐pexin‐haem receptor or a part of it. Haem from the haemopexin complex, as also free haemin, was accepted by a binder in the plasma membrane, which in gel filtration behaved like an 80 kDa molecule. This component probably represents a second functional subunit of the haemopexin‐haem receptor.
Studies on the mechanism of MPTP oxidation by human liver monoamine oxidase B
Authors: Gessner Wieslaw , Brossi Arnold , Bembenek Michael E. , Fritz Richard R. , Abell Creed W.
Published: July 2002

The neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and its deuterated analogues were oxidized to their corresponding dihydropyridinium species (MPDP+) by preparations of pure human liver MAO B : monoclonal antibody complex to investigate the mechanism of MPTP activation. Lineweaver‐Burk plots of initial reaction rates revealed that the K
m,app values for the various deuterated MPTP analogues were similar to those of MPTP. In contrast, V
max,app values were substantially decreased by substitution of deuterium for hydrogen on the tetrahydropyridinium ring, especially at C‐6. Deuterium substitution on the N‐methyl group alone did not significantly reduce V
max,app. These studies support the interpretation that oxidation of MPTP at the C‐6 position on the tetrahydropyridine ring is a major rate‐determining step in its biotransformation by MAO B.
A water‐soluble form of penicillin‐binding protein 2 of Escherichia coli constructed by site‐directed mutagenesis
Authors: Adachi Hiroyuki , Ohta Takahisa , Matsuzawa Hiroshi
Published: July 2002

Penicillin‐binding protein (PBP) 2 of Escherichia coli is located in the cytoplasmic membrane. The N‐terminal hydrophobic segment (31 amino acids, residues 15–45) of PBP2 was removed by a deletion in the PBP2 gene by site‐directed mutagenesis, resulting in the production of a water‐soluble form of PBP2 (called PBP2*). PBP2* retained the penicillin‐binding activity, was localized in the cytoplasm and was overproduced under the control of the lpp‐lac promoter. This indicates that the removed hydrophobic segment is an uncleaved signal sequence required for translocation of PBP2 across the cytoplasmic membrane, and also suggests that the segment anchors the protein to the membrane.
Endogenously generated 5‐hydroperoxyeicosatetraenoic acid is the preferred substrate for human leukocyte leukotriene A4 synthase activity
Authors: Puustinen Tapio , Scheffer Mark M. , Samuelsson Bengt
Published: July 2002

A single protein from human leukocytes possesses both 5‐lipoxygenase and leukotriene A4 (LTA4) synthase activities. It has been reported that LTA4 production is more efficient when the enzyme utilizes arachidonic acid, than when 5‐HPETE is exogenously supplied as substrate. In the present study, human leukocyte homogenate 100 000 × g supernatant was incubated with 100 μM octadeuterated arachidonic acid and exogenous 5‐HPETE (0–80 μM), and the isotopic composition of LTA4 hydrolysis products was determined by gas chromatography‐mass spectrometry. Even though 100 μM deuterated arachidonic acid results in 20–30 μM deuterated 5‐HPETE, 80 μM exogenous 5‐HPETE in the incubation could reduce the amount of deuterated LTA4 by only approx. 20%. The present study would thus indicate that the arachidonic acid moiety is preferentially converted to LTA4 in a concerted reaction without dissociation of a 5‐HPETE intermediate.
A quantitative analysis of the metabolic pathways of hepatic glucose synthesis in vivo with 13C‐labeled substrates
Authors: Kalderon B. , Gopher A. , Lapidot A.
Published: July 2002

A quantitative analysis of the major metabolic pathways of hepatic glucose synthesis in fasted rats was conducted. [2‐13C]Acetate was administered intraintestinally into awake fasted rats. 13C NMR and GC‐MS analysis were used to quantitate the isotopic enrichments of glutamate, glutamine, lactate, alanine and the newly synthesized liver glucose. By measuring the ratio of carbon atoms in glutamate molecules derived from acetyl‐CoA to carbon atoms in the glucose molecule derived from oxaloacetate and gluconeogenic substrates, such as lactate and alanine, the relative activities of the Krebs cycle and gluconeogenesis were quantified. Our results indicate that the percentage of glucose carbons originating by ‘metabolic exchange’ with the oxaloacetate pool, via the Krebs cycle, is less than 7%.
Luminescent Ag12‐metallothionein: Dependence of emission intensity on silver‐thiolate cluster formation
Authors: Stillman Martin J. , Zelazowski Andrew J. , Gasyna Zbigniew
Published: July 2002

We report the observation of emission intensity at 77 K that is a function of Ag(I)‐thiolate bonds formation within the protein metallothionein. The emission characteristics (a large, 250 nm, Stokes shift and long emission lifetime) suggests that the transition occurs from the excited triplet state. The emission intensity and circular dichroism both indicate that silver(I) clusters form with stoichiometric ratios of 12 Ag(I) to the 20 thiolate sulfur groups that are present in the protein. These data are the first to show that Ag(I)‐metallothionein complexes are luminescent and that a specific Ag12‐MT species forms.
Phorbol ester‐responsive H‐ras1 gene promoter contains multiple TPA‐inducible/AP‐1‐binding consensus sequence elements
Authors: Spandidos Demetrios A. , Nichols Rosely A.B. , Wilkie Neil M. , Pintzas Alex
Published: July 2002

We have constructed recombinant DNA plasmids which carry both the aminoglycoside phosphotransferase (aph) gene and the chloramphenicol acetyl‐transferase (CAT) gene linked to the human normal or mutant T24 H‐ras1 promoter. We have transfected these plasmids into rat 208F fibroblasts using the calcium phosphate technique and selected for stable transformants by geneticin resistance. These transformants expressed CAT activity at low levels. However, when treated with the phorbol ester TPA, CAT levels increased substantially. Cells transfected with recombinant plasmids carrying a promoterless CAT gene did not respond to TPA. We have noted four motifs in the H‐ras1 promoter region which resemble TPA‐inducible and AP‐1‐binding consensus sequences. We suggest that AP‐1‐like proteins may play a role in control of H‐ras1 transcription.
An improved purification of lactose permease
Authors: Dornmair Klaus
Published: July 2002

The integral membrane protein lactose permease of Escherichia coli was purified to homogeneity in detergent micelles. No other proteins could be detected in the final product. The molar ratio of lactose permease to lipid was less than 0.2 in detergent solution, thus the preparation was essentially lipid‐free. All molecules were functionally active as shown by substrate binding.
Binding of benzyl penicilloyl to human serum albumin Evidence for a highly reactive region at the junction of domains 1 and 2 of the albumin molecule
Authors: Yvon Mireille , Anglade Patricia , Wal Jean-Michel
Published: July 2002

Tryptic digests of fragment C124–298 of penicilloylated serum albumin, obtained from a penicillin‐treated patient or prepared by in vitro conjugation, were analyzed by HPLC. Determinations of benzyl penicilloyl groups (BPO) were performed on the different fractions. Three BPO‐containing peptides were identified by their amino acid sequence and the bound BPO was located on lysines 190, 195 and 199 and serine 193. These four main BPO‐binding sites are all located on a very short region (10 amino acid residues) of the albumin molecule at the junction of domains 1 and 2.
Loss of calcium sensitivity of plasma gelsolin is associated with the presence of calcium ions during preparation
Authors: Pope B. , Gooch J. , Hinssen H. , Weeds A.G.
Published: July 2002

Gelsolin is a calcium‐dependent actin severing and capping protein. Calcium ‘opens’ the molecule to make actin binding sites accessible, but removal of calcium from the medium does not necessarily fully reverse this process. The calcium sensitivity of actin monomer binding and actin filament severing is here shown to vary considerably with the source of gelsolin and conditions of preparation. Plasma gelsolin undergoes irreversible loss of calcium sensitivity when prepared in the presence of calcium ions. This is not due solely to effects of bound calcium, because purified human plasma gelsolin expressed in E. coli and stored in calcium shows no comparable loss of calcium sensitivity when prepared or stored in calcium. These results suggest the presence of factors in plasma which, in the presence of calcium, promote an irreversible structural change in gelsolin resulting in permanent loss of calcium sensitivity.
Inhibition of microtubule assembly in vitro by anti‐tubulin monoclonal antibodies
Authors: Dráber P. , Vater W. , Böhm K.J. , Kuklova E. , Unger E.
Published: July 2002

Five monoclonal antibodies against N‐terminal domains of α‐ or β‐tubulin were tested for their ability to interfere with the in vitro formation of microtubules. Although all the antibodies exhibited similar association constants for immobilized tubulin, they differed in their inhibitory effect on microtubule assembly. For the most potent antibody, TU‐13, the antibody/tubulin molar ratio of about 1:320 was sufficient for a 50% inhibition. The data indicate that the surface regions of N‐terminal domains of tubulin are involved in the formation of microtubules.
Regulation of phosphoinositide hydrolysis induced by histamine and guanine nucleotides in human HeLa carcinoma cells
Authors: Tilly Ben C. , Lambrechts A.Caro , Tertoolen Leon G.J. , de Laat Siegfried W. , Moolenaar Wouter H.
Published: July 2002

The regulation of phospholipase C has been investigated in both intact and streptolysin‐O permeabilized human HeLa carcinoma cells. Stimulation of phospholipase C by histamine and guanosine‐5'‐O‐thiotriphosphate (GTP[S]) requires the presence of at least 10 nM free Ca2+, but is not significantly further increased by raising [Ca2+]i to > 10−6 M. The pH optimum of the inositol phosphate response is at pH 6.8, while small changes in intracellular pH, as occur during hormonal stimulation (0.2‐0.4 unit) attenuate the histamine/GTP[S]‐induced stimulation of phospholipase C. Increasing cellular cAMP levels, either through addition of cell permeable cAMP analogues to intact cells or by stimulation with isoproterenol, does not affect histamine responsiveness, arguing against cross‐talk between both signalling pathways. In contrast, we found that the response to histamine and/or GTP[S] is largely inhibited after brief pretreatment of the cells with phorbol esters or synthetic diacylglycerol prior to permeabilization, suggesting that protein kinase C exerts feedback inhibition at the level of, or downstream from, the putative GTP‐binding protein.
Protein kinase C activity in rat skeletal muscle Apparent relation to body weight and muscle growth
Authors: Richter Erik A. , Nielsen Niss B.S.
Published: July 2002

Protein kinase C (PKC) may be involved in growth regulation. In the present study the relationship between body weight, and thereby age, and the activity of PKC in muscle as well as in rapidly growing overloaded muscle were investigated. PKC activity in music was linearly inversely correlated to rat weight in both soleus (r = −0.59, P < 0.05) and in plantaris (r = −0.74, P < 0.01) muscles. During compensatory hypertrophy. PKC activity per muscle was maximally increased compared with the contralateral control muscles after 4days in both soleus(126%)and in plantaris (105%) but had returned to basal levels by the 9th day. The data are in agreement with a role for PKC in muscle growth.
A Saporin‐6 cDNA containing a precursor sequence coding for a carboxyl‐terminal extension
Authors: Benatti Luca , Nitti Gianpaolo , Solinas Michela , Valsasina Barbara , Vitale Alessandro , Ceriotti Aldo , Soria Marco R.
Published: July 2002

Saporin‐6 is a single‐chain ribosome inactivating protein (RIP) from the seeds and the leaves of Saponaria officinalis (Caryophyllaceae). Here we have identified the COOH‐terminal end of mature Saporin‐6 and, by cDNA sequencing, the predicted carboxyl‐terminal sequence of a leaf saporin‐6 primary translation product. Our data indicate that the characterized cDNA codes for a precursor contiaing a 22 amino acid carboxyl‐terminal extension, not present in mature Saporin‐6, that shows similarity to carboxyl‐terminal propeptides of vacuolar proteins, suggesting that it may be involved in protein trafficking.
Distribution of 4a‐hydroxytetrahydropterin dehydratase in rat tissues Comparison with the aromatic amino acid hydroxylases
Authors: Davis Michael D. , Kaufman Seymour , Milstien Sheldon
Published: July 2002

A 4a‐carbinolamine intermediate is generated stoichiometrically during the tetrahydrobiopterin‐dependent phenylalanine hydroxylation reaction catalyzed by phenylalanine hydroxylase. The dehydration of the carbinolamine is catalyzed by the enzyme, 4a‐hydroxytetrahydropterin dehydratase. We have now examined the distribution of the dehydratase activity in various rat tissues by activity measurements and by immunoblot analysis to explore the possibility that the dehydratase may also play a role in tyrosine and tryptophan hydroxylation. The only two tissues that express relatively high dehydratase activity are liver and kidney, which are also the only two tissues that express phenylalanine hydroxylase activity. The dehydratase activity was generally very low in those tissues which contain high levels of tyrosine and tryptophan hydroxylase activity, except for the pineal gland. These results suggest that the dehydratase may not play an important role in the regulation of the synthesis of those neurotransmitters which are derived from the hydroxylated aromatic amino acids.
Correlation between carbohydrate‐binding specificity and amino acid sequence of carbohydrate‐binding regions of Cytisus‐type anti‐H(O) lectins
Authors: Konami Yukiko , Yamamoto Kazuo , Osawa Toshiaki , Irimura Taturo
Published: July 2002

A carbohydrate‐binding peptide of the di‐N‐acetylchitoblose‐binding Cytisus sessilifolius anti‐H(O) lectin I (CSA‐I) was isolated from the endoproteinase Asp‐N digest of CSA‐I by affinity chromatography on a column of N‐acetyl‐d‐glucosamine oligomer‐Sepharose (GIcNAc oligomer‐Sepharose). The amino acid sequence of the carbohydrate‐binding peptide of CSA‐I was determined to be DTYFGKTYNPW using a gas‐phase protein sequencer. This sequence corresponds to the sequence from Asp‐129 to Trp‐139 based on the primary structure of CSA‐I, and shows a high degree of homology to those of the putative carbohydrate‐binding peptide of the Laburnum alpinum lectin I (LAA‐I) (DTYFGKAYNPW) and of the Ulex europaeus lectin II (UEA‐II) (DSYFGKTYNPW). The binding of these three anti‐H(O) lectins is known to be inhibited by di‐N‐acetylchitobiose but not by l‐fucose. These results strongly suggest that there is a good correlation between the carbohydrate‐binding specificity and the amino acid sequence of the carbohydrate‐binding regions of di‐N‐acetylchitobiose‐binding lectins.
Expression of myogenic factors in skeletal muscle and electric organ of Torpedo californica
Authors: Neville Craig M. , Schmidt Jakob
Published: July 2002

Fish electric organ is a skeletal muscle homolog in which many muscle‐specific genes are inhibited while acetylcholine receptor is expressed at high levels. The molecular mechanisms underlying this discoordinate regulation have not yet been explored. We have obtained partial sequences for MyoD, myogenin, and myf5 from Torpedo californica and have measured their mRNAs in several organs, using ribonuclease protection. We have found that MyoD and myf5 are expressed at comparable levels in muscle and electric organ, whereas myogenin transcripts could not be detected in either tissue. Acetylcholine receptor α subunit mRNA, on the other hand, is two orders or magnitude more abundant in electric tissue. We conclude that neither the loss of contractile proteins from, nor the enhanced expression of acetylcholine receptor genes in, the differentiating electrocyte is a simple consequence of the abundance of myogenic factor messages.
The non‐equivalence of binding sites of coenzyme quinone and rotenone in mitochondrial NADH‐CoQ reductase
Authors: Ahmed Ishak , Krishnamoorthy G.
Published: July 2002

The fluorescent probe erythrosine 5′‐iodoacetamide (ER) binds to mitochondrial NADH‐CoQ reductase (Complex‐I) accompanied by an enhancement of the fluorescence intensity. The binding of the CoQ analogue, 2,3‐dimethoxy‐5‐methyl‐6‐decyl‐1,4‐benzoquinone (DB), decreased the fluorescence intensity of the ER:Complex‐I system. The ‘site 1’ inhibitor rotenone did not decrease the fluorescence intensity showing the non‐identical nature of the binding sites of DB and rotenone. Also, the reduced form of DB did not decrease the fluorescence intensity. The decrease of the fluorescence intensity by DB was shown to be due to the removal of bound ER by DB. The rapid kinetics of ER binding was studied by temperature‐jump relaxation. While DB caused complete elimination of the relaxation process in the ER:Complex‐I system, rotenone caused only a decrease in the relaxation rate, suggesting conformational change. The relaxation rate showed a pH dependence with a maximum around pH 7.5.
Slow, tight binding to human renin of some nonpeptidic renin inhibitors containing a 4‐methoxymethoxypiperidinylamide at the P4 position
Authors: Stein Herman H. , Fung Anthony K.L. , Cohen Jerome , Baker William R. , Rosenberg Saul H. , Boyd Steven A. , Dayton Brian D. , Armiger Yoek Lin S.-T. , Condon Stephen L. , Mantei Robert A. , Kleinert Hollis D.
Published: July 2002

A series of nonpeptidic human renin inhibitors with a 4‐methoxymethoxypiperidinylamide at the P4 position of the molecule exhibited slow tight binding to the enzyme. Replacement of the methoxymethoxy moiety on the piperidine ring with H, OH, methoxyethyl, propyloxy or n‐butyl eliminated the effect. The inhibition was partially reversed by prolonged dialysis at 4°C, arguing against formation of a covalent bond in the tightened complex.
The oxygen reactive species of cytochrome‐c‐oxidase: An alternate view
Authors: Brunori Maurizio , Antonini Giovanni , Malatesta Francesco , Sarti Paolo , Wilson Michael T.
Published: July 2002

In a recent review article Babcok and Wikström (Nature, 1992, 356, 301–309) proposed that the species of cytochrome‐c‐oxidase which binds molecular oxygen during turnover is the so‐called mixed valence enzyme, in which the binuclear center cytochrome a
3‐Cu
B is reduced, while the cytochrome a/Cu
A sites are oxidized. This proposal is based on earlier work (Morgan and Wikström, Biochemistry 1991, 30, 948–958) in which it was found that the steady‐state reduction levels of cytochrome c and cytochrome a in respiring rat liver mitochondria (sustained by ascorbate and TMPD) are quite different, the latter being much more oxidized than the former; evaluation of the steady‐state reduction levels demanded a large correction due to the optical contribution of oxidized TMPD+ which overlaps with the cytochromes. We report below that application of transient spectroscopy and SVD analysis to respiring rat heart myocytes, under conditions in which the contribution of TMPD+ is very small or absent, allows to show that the steady‐state reduction levels of cytochrome c and cytochrome a are comparable at all times accessible to measurement in the rapid‐scanning stopped‐flow spectrophotometer. Our conclusion, in agreement with previous results, is that mixed valence cytochrome‐c‐oxidase as defined above is not the prevailing oxygen binding species of cytochrome‐c‐oxidase, unless electron donation to cytochrome c becomes rate limiting.
Evidence for the tetraplex structure of the d(GT)n repetitive sequences in solution
Authors: Borisova Olga F. , Shchyolkina Anna K. , Timofeev Edward N. , Florentiev Vladimir L.
Published: July 2002

The ability of oligonucleotides 3′‐d(GT)5pO(CH2)6Opd(GT)5‐5′(anti[d(GT)]) and 3′‐d(GT)5pO(CH2)6Opd(GT)5‐3′(par[d(GT)]) to form tertiary structures has been studied. Circular dichroism (CD) as well as the fluorescence of the ethidium bromide (E1Br) complexes with oligonucleotides and hydrodynamic volume measurements in solutions containing 0.01 M phosphate buffer, pH 7 and NACl in concentrations from 0.1 M to 1 M, have been used. The data obtained in the temperature interval from 30°C to 10°C are in good agreement with the structure suggested earlier [1] where the par[d(GT)] and anti[d(GT)] form structures with four parallel strands in which layers of four G‐residues alternate with unpaired bulged‐out T‐residues. Ethidium bromide interacts with the structure in a cooperative manner. Two ethidium bromide molecules intercalate between two layers of four G‐residues.
Bleomycin‐reactive iron in patients with acute non‐lymphocytic leukemia
Authors: Gordeuk Victor R. , Brittenham Gary M.
Published: July 2002

Bleomycin‐reactive iron was detected in the sera of six out of nine adults undergoing intensive chemotherapy for acute non‐lymphocytic leukemia. In these individuals the corresponding transferrin saturation ranged from 96% to 113% and the serum ferritin from 775 to 9975 μg/l. Nontransferrin‐bound iron has been postulated to be a factor in organ toxicity in iron overload conditions such as beta thalassemia and hereditary hemochromatosis by facilitating the production of tissue‐damaging free radicals. We propose that bleomycin‐reactive iron should be considered as a possible factor in organ dysfunction seen with intensive cancer chemotherapy.
Facile synthesis of chiral 2‐hydroxy acids catalyzed by a stable duck ε‐crystallin with endogenous l‐lactate dehydrogenase activity
Authors: Wu Chi-Yue , Chen Shui-Tein , Chiou Shyh-Horng , Wang Kung-Tsung
Published: July 2002

Duck ε‐crystallin, an abundant structural protein in lenses of some avian species, was shown to possess a genuine and stable l‐lactate dehydrogenase (l‐LDH, EC 1.1.1.27) activity suitable for the application to enzyme technology as a catalyst for the synthesis of chiral α‐hydroxy acids. Two pharmaceutically important intermediates, 2‐hydroxy acids (S)‐2‐hydroxybutanoic acid (S)‐2‐hydroxypentanoic acid) have been synthesized in high yields and Optical purity utilizing an in situ NADH regeneration system of duck ε‐crystallin coupled with formate/formate dehydrogenase. This enzyme system is also shown to offer some advantages over the conventional l‐LDH sources from several mammalian species.
Rat liver BiP/GRP78 is down‐regulated by a peroxisome‐proliferator, clofibrate
Authors: Motojima Kiyoto , Goto Sataro
Published: July 2002

Administration of clofibrate in rat results in down‐regulation of several liver proteins and a vast induction of peroxisomal proteins. One protein was identified as BiP/GRP78 using antibodies and cDNA cloning. The level of mRNA was reduced by the drug.
Processing of the chloroplast transit peptide of pea carbonic anhydrase in chloroplasts and in Escherichia coli Identification of two cleavage sites
Authors: Johansson Inga-Maj , Forsman Cecilia
Published: July 2002

The chloroplast transit peptide (cTP) of pea carbonic anhydrase was shown to be processed at two different sites, giving protein subunits of two sizes. The cleavage sites were identified and found to be localized immediately before and after a highly charged part, containing 8 acidic and 6 basic residues, of the cTP. Properties of pea carbonic anhydrase produced in Escherichia coli show that folding, oligomerization and catalytic activity do not depend on the presence of the acidic part or the rest of the cTP. The pattern of processing of the cTP in E. coli indicates that cleavage at site I is specific for a chloroplastic stromal peptidase and that cleavage at site I prevents processing at site II.
Phorbol ester markedly increases the sensitivity of intestinal epithelial cells to 1,25‐dihydroxyvitamin D3
Authors: Armbrecht H.James , Hodam Tara L. , Boltz Monica A. , Chen Ming L.
Published: July 2002

We have used a clonal intestinal epithelial cell line (IEC‐18) to study the mechanism of action of 1,25‐dihydroxyvitamin D3 (1,25(OH)2D) in vitro. 1,25(OH)2D (10−7 M) elevated by over 10‐fold the mRNA levels for the cytochrome P450 component (P450cc24) of the 1,25(OH)2D‐24‐hydroxylase. Increased P450cc24 mRNA levels were detectable at 6 h and peaked at 36 h. Below a concentration of 10−7 M, 1,25(OH)2D had almost no effect. However, addition of phorbol ester for 2 h made the intestine responsive to 1,25(OH)2D concentrations as low as 10−9 M.
Substitution of potential metal‐coordinating amino acid residues in the zinc‐binding site of endopeptidase‐24.11
Authors: Le Moual Hervé , Roques Bernard P. , Crine Philippe , Boileau Guy
Published: July 2002

Neutral endopeptidase (EC 3.4.24.11; NEP) is a membrane‐bound zinc‐metallopeptidase. The catalytic zinc ion is coordinated to three amino acid residues (His538, Hi587 and Glu646) and a water molecule. Here, we have systematically substituted potential metal‐coordinating amino acid residues (His, Glu, Asp, Cys, Tyr, Ser) for each of the three zinc ligands of NEP using a recombinant polymerase chain reaction procedure. NEP mutants at positions 583 and 587 were devoid of catalytic activity. However, Glu587 NEP and Cys583 NEP were able to bind partially a tritiated inhibitor, the binding of which is dependent on the presence of the zinc atom. At position 646, the aspartate and cysteine mutants exhibited activity. For both mutants K
m values were unaltered but K
cat values were decreased by about 20‐fold. Both mutants bound the tritiated inhibitor with K
d values similar to that of the wild‐type enzyme. Our data suggest that neither histidine‐583 nor ‐587 can be replaced by any other ligands. On the other hand, the glutamic acid at position 646 can be converted to an aspartic acid or a cysteine indicating the importance of a negative charge at this position.
Cloning, expression and purification of a recombinant poly‐histidine‐linked HIV‐1 protease
Authors: Leuthardt Andreas , Roesel Johannes L.
Published: July 2002

The gene coding for the HIV‐1 protease was cloned in an Escherichia coli expression vector adding three‐histidine codons to the amino and carboxy terminus of the protease sequence. Expression of the protease from this construct led to the accumulation of high amounts of insoluble histidine‐linked protease entrapped in inclusion bodies. The histidine‐linked protease could be efficiently released from purified inclusion bodies with 6 M guanidine hydrochloride and further purified by metal chelate affinity chromatography. The refolded protease cleaved synthetic peptide substrates and the viral polyprotein p55 with the same specificity as the wild type protease. It displays a specific activity of 4.4 μmol/min/mg.
Light signals are transduced to the phosphorylation of 15 kDa proteins in Neurospora crassa
Authors: Oda Kazushi , Hasunuma Kohji
Published: July 2002

A microsomal fraction prepared from the mycelia of the band (bd) strain of Neurospora crassa showed enhanced phosphorylation of two small proteins with molecular masses of around 15 kDa (ps15) by the irradiation of the reaction mixture containing [γ‐32P]ATP at 0°C for 1 s with blue light (450 nm, 6 μmol/m2/s or 420 nm, 80 μmol/m2/s). The reaction was stopped at 5 s of incubation at 0°C after blue light irradiation. The light effect could not be detected in ps15, when a microsomal fraction from a blind mutant, wc‐1 or wc‐2 was used. The mixing followed by homogenization of the microsomal fractions from wc‐1 and wc‐2 restored the activity to stimulate the phosphorylation of ps15 by blue light. The phosphorylated amino acid residue of ps15 was unstable when the proteins on a nylon membrane were exposed to an acid or alkaline solution, suggesting that the phosphorylated residue was aspartic acid. The other phosphorylated protein with a molecular mass of 70 kDa (p70) showed no light effect in the phosphorylation and the phosphorylated residue was estimated to be histidine, since it was stable in alkaline solution.
Luffin‐S—a small novel ribosome‐inactivating protein from Luffa cylindrica
Authors: Gao Wenda , Ling Jun , Zhong Xuemei , Liu Wangyi , Zhang auHailing Yang Ruping , Cao Huiting , Zhang Zuchuan
Published: July 2002

We purified and characterized a novel RIP (ribosome inactivating protein), Luffin‐S from the seeds of Luffa cylindrica. Different from Luffin‐A and B, which are RNA N‐glycosidases with molecular weights of 27 and 28 kDa, respectively, Luffin‐S has an M.W. of only approx. 10 kDa, much smaller than any other RIPs so far investigated. Its abundant resources, toxicity similar to TCS in a cell‐free protein synthesis system and unique mechanism as phosphodiesterase, like α‐sarcin, promisingly make it a potential toxic moiety of immunotoxin.
Swimming speed and chemokinetic response of Rhodobacter sphaeroides investigated by natural manipulation of the membrane potential
Authors: Harrison David M. , Packer Helen L. , Armitage Judith P.
Published: July 2002

The ΔΨ of R. sphaeroides, grown under high light to reduce the levels of light‐harvesting bacteriochlorophyll, was naturally manipulated using light intensity. The relationship between ΔΨ and the swimming speed of free swimming populations of cells was investigated. After de‐energisation by incubation in the dark there was an apparent threshold of about −13 mV which had to be overcome before functional motor rotation could resume and at −45 mV the motor saturated. Further increases in ΔΨ over −45 mV did not increase the free swimming velocity. However, when a chemokinetic effector was added there was an increase in swimming speed, even though the ΔΨ was well above saturation, indicating that the chemokinetic response is independent of normal relationship between motor rotation and ΔΨ.
Kinetic studies on the formation of acridine‐linked DNA triple helices
Authors: Fox Keith R.
Published: July 2002

We have used DNase I footprinting to measure the rate of intermolecular triple helix formation at the target sites A6G6 · C6T6 and G6A6 · T6C6 with the acridine‐linked oligonucleotides Acr‐T5C5, and Acr‐C5T5 respectively. Under pseudo first‐order reaction conditions we find that the reactions are slow, with half‐lives of several minutes. The rates are dependent on the concentration of the third strand oligonucleotide and yield bimolecular association rate constants of 300 M−1 · s−1 for Acr‐T5C5 binding to A6G6 · C6T6 and 2000 M−1 · s−1 for the interaction of Acr‐C5T5 with G6A6 · T6C6.
Translocation of an SH2‐containing protein tyrosine phosphatase (SH‐PTP1) to the cytoskeleton of thrombin‐activated platelets
Authors: Li Ruo Ya , Gaits Frédérique , Ragab Ashraf , Ragab-Thomas Jeannie M.F. , Chap Hugues
Published: July 2002

A significant protein tyrosine phosphatase (PTP) activity was found to be associated with the cytoskeleton of thrombin‐stimulated platelets. Translocation of the enzyme became maximal within 1‐2 min of thrombin stimulation and was suppressed by cytochalasin D or upon inhibition of aggregation. Immunoblotting as well as immunoprecipitation revealed that a PTP with two SH2 domains (SH‐PTP1) displayed the same behaviour, translocation to the cytoskeleton showing the same time course as that observed for pp60
c‐src
. We conclude that SH‐PTP1 might represent a critical enzyme in the complex interplay between the various proteins regulating protein tyrosine phosphorylation in the cytoskeletal matrix.
Insulin and ATP stimulate actin polymerization in U937 cells by a wortmannin‐sensitive mechanism
Authors: Walters Rhodri J. , Hawkins Phill , Cooke Frank T. , Eguinoa Alicia , Stephens Len R.
Published: July 2002

ATP and insulin stimulate increases in phosphatidylinositol (3,4,5)‐trisphosphate levels in myeloid‐derived U937 cells. Quantification of FITC‐phalloidin binding by fluorescence‐activated cell sorting reveals that both ATP and insulin stimulate actin polymerization with distinctive kinetics in U937 cells. The response to ATP is rapid and dose‐dependent with an EC50 of 200 nM, and is abolished by pre‐incubation with the Ca2+ chelator BAPTA‐AM. At 800 nM concentration, wortmannin, a potent inhibitor of phosphoinositide 3‐kinase (PI3K), blocks the late, but not the early phase of actin polymerization stimulated by 100 nM ATP. Responses elicited by 10 μg/ml insulin are slower, smaller and more transient than responses to ATP, and are inhibited by preincubation with 100 nM wortmannin. Actin polymerization can also be stimulated by thapsigargin, but not by phorbol ester, providing further evidence for a role for Ca2+ in actin polymerization. These data implicate distinct Ca2+ and PI3K‐mediated pathways in the regulation of actin polymerization.
Dependence of cranial motor neuron formation on ventromedial brain stem
Authors: Sohal G.S. , Ali M.M. , Tsai N.T.
Published: July 2002

The formation of motor neurons in the spinal cord is dependent on inductive signals from the floor plate and notochord. Motor neurons in the brain stem, on the other hand, develop in the absence of both structures. This suggests that either the germinal epithelium is specified intrinsically to form specific cranial motor nuclei or that the inductive signals for the formation of cranial motor neurons arise from some other structure. These possibilities were investigated experimentally by using the formation of trochlear motor neurons in the midbrain of duck embryos as a model system. The trochlear motor neurons, which form the nucleus of the fourth cranial nerve, developed normally after early damage to the prospective germinal epithelium, suggesting that it is unlikely to be specified intrinsically to form these cranial motor neurons. Instead, their development was found to be dependent on the cells within, or associated with, the ventromedial region of the brain stem, as the extirpation of this region results in the absence of motor neuron formation. These results show that structures other than the floor plate and notochord provide inductive signals for the cellular differentiation and patterning of the developing central nervous system. They raise the possibility that the inductive signals for motor neuron differentiation in the spinal cord and the brain stem may not be necessarily identical.
Effects of nerve growth factor on splenic norepinephrine and pineal N‐acetyl‐transferase in neonate rats exposed to alcohol in utero: Neuroimmune correlates
Authors: Gottesfeld Zehava , Simpson Shane , Yuwiler Arthur , Perez‐Polo J. Regino
Published: July 2002

Prenatal alcohol exposure (FAE) has been associated with multiple anomalies, including a selective developmental delay of sympathetic innervation in lymphoid organs. Sympathetic neurons require nerve growth factor (NGF) for their development and maintenance, and recent evidence has suggested that alcohol impairs the synthesis and/or biological activity of NGF in selected central and peripheral neurons. Thus, the present study examined the hypothesis that NGF administration to FAE rats during early postnatal development would reverse some of the peripheral sympathetic deficits. Neonate rats, FAE and the corresponding control cohorts, received daily treatments of NGF or cytochrome C (0.3 mg/kg; s.c.) for various time intervals, and were killed 24hr or 10 days after the last treatment. The measured parameters included norepinephrine (NE) concentrations in the spleen and heart, which receive noradrenergic innervation from the coeliac ganglion and the superior cervical ganglion (SCG), respectively. In addition, we measured the activity of pineal N‐acetyltransferase (NAT), the rate‐limiting enzyme of melatonin biosynthesis, which depends on sympathetic innervation from the SCG. The data show that chronic, but not acute, NGF treatments reversed the FAE‐related deficits in splenic NE concentrations as well as in pineal NAT activity in a time‐ and age‐dependent manner. Sympathetic neurons play an important role in immune modulation. Thus, the altered splenic NE levels and pineal NAT activity may play a role in immune deficits associated with exposure to alcohol in utero.
Functional properties of the R154X HNF‐4α protein generated by a mutation associated with maturity‐onset diabetes of the young, type 1
Authors: Laine B. , Eeckhoute J. , Suaud L. , Briche I. , Furuta H. , Bell G.I. , Formstecher P.
Published: July 2002

Mutations in the hepatocyte nuclear factor 4α (HNF‐4α) gene are associated with one form of maturity‐onset diabetes of the young (MODY1). The R154X mutation generates a protein lacking the E‐domain which is required for normal HNF‐4α functions. Since pancreatic β‐cell dysfunction is a feature of MODY1 patients, we compared the functional properties of the R154X mutant in insulin‐secreting pancreatic β‐cells and non‐β‐cells. The R154X mutation did not affect nuclear localisation in β‐cells and non‐β‐cells. However, it did lead to a greater impairment of HNF‐4α function in β‐cells compared to non‐β‐cells, including a complete loss of transactivation activity and a dominant‐negative behaviour.
Colecystokinin octapeptide CCK‐8 and carbachol reduce [32P]orthophosphate labeling of phosphatidylcholine without modifying phospholipase D activity in rat pancreatic acini
Authors: Sarri Elisabet , Ramos Belén , Salido Ginés , Claro Enrique
Published: July 2002

We have studied phospholipase D activation in [32P]orthophosphoric acid‐prelabeled rat pancreatic acini by measuring the formation of 32P‐phosphatidylalcohols as stimulated in the presence of ethanol or butanol. A small but significant and time‐dependent basal accumulation of [32P]phosphatidylethanol and [32P]phosphatidylbutanol was detected, which was further stimulated by phorbol myristate acetate, orthovanadate and pervanadate. However, the secretagogues cholecystokinin octapeptide and carbachol did not enhance basal accumulation of 32P‐phosphatidylalcohol, yet they decreased [32P]phosphatidylcholine content and stimulated the generation of [32P]phosphatidic acid. Our results stress the need to examine the transphosphatidylation reaction as well as agonist effects on the synthesis of phosphatidylcholine in order to assess unambiguously phospholipase D activity.
Enzymatic synthesis of a novel trehalose derivative, 3,3′‐diketotrehalose, and its potential application as the trehalase enzyme inhibitor
Authors: Sode Koji , Akaike Eri , Sugiura Hiroto , Tsugawa Wakako
Published: July 2002

We reported the preparation of a novel trehalose derivative based on enzymatic oxidation of trehalose by water‐soluble glucose‐3‐dehydrogenase (G3DH) from marine bacterium Halomonas sp. α‐15 cells. The product of G3DH enzymatic conversion was 3,3′‐diketotrehalose (3,3′dkT), a novel trehalose derivative of which both third hydroxy groups of glucopyranosides were oxidized. 3,3′dkT was revealed to show an inhibitory effect toward pig‐kidney and Bombyx mori trehalases. The IC50 values of 3,3′dkT were 0.8 and 2.5 mM and K
i values were 0.2 and 0.6 mM for pig‐kidney and for B. mori trehalases, respectively. In addition, 3,3′dkT did not show any inhibitory effect on both maltase and mannosidase activities. Therefore, 3,3′dkT was a specific inhibitor of trehalases.
Brain‐to‐blood active transport of β‐alanine across the blood–brain barrier
Authors: Komura Junko , Tamai Ikumi , Senmaru Mizuho , Terasaki Tetsuya , Sai Yoshimichi , Tsuji Akira
Published: July 2002

A high‐affinity antiluminal uptake system for β‐alanine was demonstrated in primary cultured bovine brain capillary endothelial cells (BCEC) for which K
t is 66.9 μM. β‐alanine uptake was energy‐, sodium‐ and chloride ion‐dependent. β‐amino acids strongly inhibited the uptake, while α‐ and γ‐amino acids had a little or no inhibitory effect. In ATP‐depleted cells, the uptake was stimulated by preloading β‐alanine or taurine but not by l‐leucine. These results suggest that β‐alanine is actively transported across the antiluminal membrane of BCECs that is common to β‐amino acids. The system may function for the efflux from the brain to blood.
The interleukin 1β‐converting enzyme inhibitor CrmA prevents Apo1/Fas‐ but not glucocorticoid‐induced poly(ADP‐ribose) polymerase cleavage and apoptosis in lymphoblastic leukemia cells
Authors: Geley Stephan , Hartmann Bernd L , Kapelari Klaus , Egle Alexander , Villunger Andreas , Heidacher Daniela , Greil Richard , Auer Bernhard , Kofler Reinhard
Published: July 2002

Glucocorticoids (GC) induce programmed cell death (apoptosis) in immature lymphocytes and are an essential component in the therapy of acute lymphatic leukemia. The mechanism underlying GC‐induced apoptosis particularly in leukemia cells is, however, not well understood. Most forms of apoptosis seem to employ a common final effector pathway characterized by specific proteolytic events mediated by interleukin 1β‐converting enzyme (ICE) and/or other ICE‐like cysteine proteases. These events may result in the morphologic changes characteristic of apoptosis. To determine whether a similar proteolytic pathway is activated during GC‐induced leukemia cell apoptosis, we investigated poly(ADP‐ribose) polymerase (PARP), a typical target of ICE‐like proteases, during GC‐induced apoptosis of the human acute T‐cell leukemic cell line CEM‐C7H2. Our studies showed proteolytic PARP cleavage suggestive of activation of ICE‐like proteases that preceeded morphologic signs of apoptosis. We further established stably transfected CEM‐C7H2 sublines expressing the cowpox virus protein CrmA that inhibits some, but not all, ICE‐like proteases. GC‐induced PARP cleavage and apoptosis were neither inhibited nor delayed in crmA‐expressing cell lines. In contrast, crmA expression rendered the same lines resistant to Apo1/Fas‐induced PARP cleavage and apoptosis. Thus, different proteases might be activated during the effector phases of GC‐ and Apo1/Fas‐induced apoptosis in human leukemia cells.
cGMP as second messenger during Dictyostelium chemotaxis
Authors: van Haastert Peter J.M , Kuwayama Hidekazu
Published: July 2002

The chemoattractant cAMP induces directed cell locomotion in Dictyostelium cells. Several second messenger pathways are activated upon binding of cAMP to G‐protein‐coupled receptors, including adenylyl cyclase, guanylyl cyclase, phospholipase C, and the opening of plasma membrane Ca2+ channels. These second messenger responses are unaltered in many chemotactic mutants, except for the cGMP response. Activation of guanylyl cyclase depends on G‐proteins and is regulated by a cGMP‐binding protein in a complex manner. This cGMP‐binding protein also mediates intracellular functions of cGMP to activate a PKC‐related kinase that phosphorylates myosin II heavy chain, thereby allowing myosin filaments to rearrange during cell movement.