bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyadenylation site heterogeneity in mRNA encoding the precursor of the barley toxin β‐hordothionin
Authors: Hernández-Lucas Carlos , Royo Joaquin , Paz-Ares Javier , Ponz Fernando , García-Olmedo Francisco , Carbonero Pilar
Published: July 2002

Two cDNA clones, pTH2 and pTH3, encoding the precursor of the barley toxin β‐hordothionin have been identified and their nucleotide sequences determined. These sequences are identical, except that pTH2 is 22 bp longer than pTH3 at the 5'‐end and that the cleavage/poly(A) site of the mRNA represented by pTH3 is 4 positions further downstream from the single 5'‐AAUAAA‐3' polyadenylation signal than that of the pTH2 mRNA. In contrast, the cleavage/poly(A) site of the α‐hordothionin mRNA is 30 positions downstream from a second polyadenylation signal. The deduced amino acid sequence of the β‐hordothionin precursor differs from that of α‐hordothionin at 13 out of 127 positions.
Calcium‐dependent association of 33 kDa protein in polymorphonuclear leukocytes with phospholipid liposomes containing phosphatidylserine or cardiolipin
Authors: Utsumi Kozo , Sato Eisuke , Okimasu Eiji , Miyahara Masanobu , Takahashi Rutaro
Published: July 2002

Ca2+‐binding protein
Protein‐lipid interaction
Liposome
Polymorphonuclear leukocyte
Phosphatidylserine
Cardiolipin
Measles virus hemagglutinin
Authors: Varsanyi Tamas M. , Lundquist Gunilla , Norrby Erling , Jörnvall Hans
Published: July 2002

Measles virus hemagglutinin has been isolated by immunoadsorption. The total composition of the protein and its N‐terminal amino acid sequence give data matching the structure indirectly deduced from cDNA. However, direct analysis of the hemagglutinin also shows that the mature protein is proteolytically processed and has a partly heterogeneous N‐terminus. The initiator Met is removed, and non‐stoichiometrically also the second residue.
Effects of α‐amanitin on coordination of two mRNAs of ribulose‐bisphosphate carboxylase in greening pea leaves
Authors: Sasaki Yukiko
Published: July 2002

The effects of α‐amanitin on light induction of the two mRNAs of ribulose‐bisphosphate carboxylase/oxygenase (RuBisCO) in greening pea leaves were examined. Induction of the nuclear‐coded small‐subunit mRNA but not of the chloroplast‐coded large‐subunit mRNA was significantly reduced by α‐amanitin, indicating that the steady‐state level of the large‐subunit mRNA is not directly affected by that of the small subunit. This suggests that there is no direct link between nuclear transcription and light induction of the large‐subunit mRNA. The synthesis of RuBisCO was also slowed in proportion to the small‐subunit mRNA level, and there was not a large excess of the unassociated large subunit polypeptide. The synthesis of the two subunits is probably coordinated. Thus, the expression of the large subunit is partly controlled at the post‐transcriptional level.
Distinct physiological roles of animal succinate thiokinases Association of guanine nucleotide‐linked succinate thiokinase with ketone body utilization
Authors: Jenkins T.M. , Weitzman P.D.J.
Published: July 2002

Two distinct succinate thiokinases have recently been shown to exist in animal tissues, one specific for guanine nucleotide and the other for adenine nucleotide. Their physiological roles have here been investigated by comparing the levels of the two enzymes in liver and brain of normal and diabetic rats. A marked rise in the level of brain guanine nucleotide‐linked succinate thiokinase in the diabetic condition is consistent with an enhanced utilization of ketone bodies and hence with the associated elevated demand for succinyl‐CoA for the activation of acetoacetate. Taken together with the reported mitochondrial values of the ATP/ADP and GTP/GDP ratios, the results are interpreted to indicate that the adenine nucleotide‐linked enzyme functions as a component of the citric add cycle whereas the guanine nucleotide‐linked enzyme functions in the opposite metabolic direction to produce succinyl‐CoA from succinate.
Control of nucleation in microtubule self‐assembly
Authors: Bayley P.M. , Butler F.M.M. , Manser E.J.
Published: July 2002

The inhibition of the rate and amplitude of assembly of microtubule protein at low GTP concentration is shown by measurement of microtubule length distributions to be due to the suppression of microtubule nucleation. This inhibitory effect is enhanced by GDP added before assembly, but can be overcome by a number of molecules such as pyrophosphate or ADP. The selective inhibition of nucleation by GDP in vitro, which occurs in addition to inhibition of elongation, could provide a mechanism for the control of spontaneous microtubule nucleation in vivo.
Radiation inactivation analysis of kidney microvillar peptidases
Authors: Fulcher Ian S. , Ingram Jean , Kenny A.John
Published: July 2002

Five membrane peptidases were studied by radiation inactivation analysis of pig kidney microvillar membranes. One heterodimeric enzyme, γ‐glutamyl transferase, presented a target size corresponding to the dimeric M
r. The other enzymes are known to be homodimers. Three of these, aminopeptidase A, aminopep‐tidase N and dipeptidyl peptidase IV, gave results clearly indicating the monomer to be the target and, hence, in this group the association of the subunits was not essential for activity. The target size for endopep‐tidase‐24.11 was intermediate between those for monomer and dimer and its functional state was not resolved by the experiments.
Induction of cell surface insulin antigenicity in fibroblasts transfected with islet genomic DNA
Authors: Klein Benjamin Y. , Nesher Rafael , Cerasi Erol
Published: July 2002

Ltk−cells were transfected with total genomic DNA obtained from rat islets or from insulinoma cells mixed with DNA tagged with fluorescent dye. Flyorescence‐activated cell sorter (FACS) was used for initial selection of successful transfectants, monitoring fluorescent DNA incorporated into the cell. For subsequent selections the cells were treated with anti‐insulin antiserum labeled with fluorescent dye and selected by FACS. B cell DNA, tiut not DNA from murine leukemic cells, induced the appearance of cell surface insulin antigenicity in fibroblasts. This phenotypic expression was transient. Together with our previous demonstration of the induction of insulin secreation in Ltk− cells [(1986) Biochem. Biophys. Res. Commun. 136, 638‐644], these results indicate that B cell specific characteristics can be transfected to non‐endocrine cells by genomic DNA transfection.
Evidence for two distinct adenylate cyclase catalysts in rat brain
Authors: Coussen Francoise , Guermah Mohamed , d'Alayer Jacques , Monneron Ariane , Haiech Jacques , Cavadore Jean-Claude
Published: July 2002

The Lubrol‐soluble adenylate cyclase activity of brain synaptosomal membranes appeared, upon gel filtration or sucrose gradient centrifugation, as two overlapping peaks. Fractions corresponding to the peak of the largest Stokes radius (Biogel pool 1) or highest s value (gradient pool 1) contained an adenylate cyclase activity which could be detected whatever the enzyme assay conditions. In contrast, in fractions from the second peak (Biogel pool 2 or gradient pool 2), forskolin was needed to reveal adenylate cyclase activity. The enzyme activity of each Biogel pool was retained by forskolin‐agarose and eluted by forskolin with a 34–83% yield. A polypeptide of 155 kDa made up 80% of the forskolin‐agarose eluate 1, whereas it was almost absent from eluate 2. Since data from various groups point to the 155 kDa polypeptide as a brain adenylate cyclase catalyst, still another distinct catalyst of lower molecular mass is likely to be present in brain.
Neo‐kyotorphin, an analgesic peptide isolated from human lung carcinoma
Authors: Zhu Y.X. , Hsi K.L. , Chen Z.G. , Zhang H.L. , Wu S.X. , Zhang S.Y. , Fang P.F. , Guo S.Y. , Kao Y.S. , Tsou K.
Published: July 2002

A pentapeptide with analgesic activity has been isolated from human lung squamous cell carcinoma and from three other types of propagated tumors of human lung small‐cell carcinoma (SCC), adenoma (AD) and large‐cell carcinoma (LCC) in nude mice. The amino acid sequence of the peptide has been revealed to be H‐Thr‐Ser‐Lys‐Tyr‐Arg‐OH, which is exactly the same as that of neo‐kyotorphin, an analgesic peptide originally isolated from bovine brain [(1982) Life Sci. 31, 1733]. No neo‐kyotorphin could be isolated from normal lung tissue using the same procedures as those used for carcinomas. The results suggest that the presence of neo‐kyotorphin in the lung carcinoma may represent the ectopic expression of peptide hormone. Our findings constitute the first example of a human lung carcinoma producing analgesic peptide.
XANES spectroscopy of carp hemoglobin‐iron in correlation with the affinity changes of the protein for ligand
Authors: Pin Serge , Cortes Robert , Alpert Bernard
Published: July 2002

The strong variation of ligand‐binding properties with pH for carp hemoglobin is not reflected in the electronic distribution of the heme‐iron. Thus, we can suppose that hemoglobin affinity is directly controlled by the protein and not by some particular changes of the iron atom.
Identification of the site of iodide photooxidation in the photosystem II reaction center complex
Authors: Takahashi Yuichiro , Takahashi Masa-aki , Satoh Kimiyuki
Published: July 2002

The highly purified PS II reaction center complex with 47, 43, 32, 30 and 9 kDa (and 4.5 kDa) polypeptides was subjected to iodo‐labeling to identify the polypeptide(s) participating on the donor side of the photosystem. The 32 kDa component (D2) and the aggregate(s) of about 60 kDa were exclusively labeled in the dark, suggesting the presence of a dark‐stable oxidizing component on this polypeptide. Illumination of the complex in the presence of 2,6‐dichlorophenolindophenol and iodide resulted in the preferential iodination of the 30 kDa component (D1) and the aggregate(s), but not the 47 kDa polypeptide. This iodo‐labeling was completely inhibited by the addition of an electron donor, 1,5‐diphenylcarbazide. From these results, it is concluded that the site of photooxidation of iodide, and presumably the site of Z binding, is located on or near the herbicide‐binding polypeptide, D1, in the PS II reaction center complex.
A unique nucleoprotein structure associated with the Drosophila melanogaster 18–28 S rDNA nontranscribed spacer
Authors: Levinger Louis F. , Nass G.Scott
Published: July 2002

We have detected unique nucleoprotein particles specific for the 18–28 S rDNA nontranscribed spacer of Drosophila
melanogaster. The particles migrate between di‐ and trinucleosomes on nucleoprotein gels, and are between mono‐ and dinucleosomal in DNA length. These migration properties suggest that the nontranscribed spacer particles could have a protein component larger than a histone core. The variant nucleoprotein structures map primarily within the nontranscribed spacer 235 base pair internal subrepeat, which is AT‐rich and possesses a 50 base pair sequence homologous to the RNA polymerase I binding site.
Expression of a truncated v‐myb product in transformed chicken embryo fibroblasts
Authors: Kryceve-Martinerie Cécile , Soret Johann , Crochet Janine , Baluda Marcel , Perbal Bernard
Published: July 2002

Transformed cells have been isolated after transfection of chicken embryo fibroblasts (CEF) with the DNA of a recombinant clone (KXA 3457) in which the v‐myb sequences are flanked by the two AMV‐LTRs. Abnormal myb‐specific RNA species and myb‐related polypeptides were found to be expressed in these cells, suggesting that transformation of CEF by v‐myb might require alterations of the oncogene product.
Ca2+ uptake and IP3‐induced Ca2+ release in permeabilized human lymphocytes
Authors: Eberl G. , Schnell K.
Published: July 2002

The 45Ca2+ uptake and 45Ca2+ release in saponin‐permeabilized human lymphocytes were studied. An ATP‐dependent Ca2+ uptake into a nonmitochondrial, intracellular Ca2+ store is observed which is approx. 2 orders of magnitude greater than the ATP‐independent Ca2+ uptake. The Ca2+ uptake is inhibited by vanadate, but it is insensitive to oligomycin and ruthenium red. IP3 induces dose‐dependent 45Ca2+ release. For half‐maximum Ca2+ release 0.25–0.5 μM IP3 is required. The results of our studies suggest that 45Ca2+ is predominantly stored within the endoplasmic reticulum of the lymphocytes.
Investigations by picosecond polarized fluorescence spectrochronography of structural aspects of energy transfer in living cells of the green bacterium Chlorobium limicola
Authors: Fetisova Z.G. , Freiberg A.M. , Timpmann K.E.
Published: July 2002

The orientation of the long‐wavelength (Q
y) transition moments of the antenna bacterioviridin (BVr) was examined in living cells of Chlorobium limicola. Previous linear dichroism studies [(1986) FEBS Lett. 199, 234–236] indicated that in each individual chromatophore of C. limicola the Q
y, transition moment vectors of the whole chlorosome BVr are essentially parallel to each other and are practically ideally oriented along the long axis of the chlorosome. We measured the picosecond polarized fluorescence decay kinetics for antenna bacteriochlorophyll (BChl) emissions upon selective excitation with polarized light of the Q
y, transition of BVr. The polarization (p) of the BVr fluorescence is measured to be constant during the BVr excited‐state lifetime and to be equal to the limiting value of p achieved in monomeric BChl: P = + 0.42 ± 0.02. The results indicate convincingly that the excitation energy transfer within chlorosomes of C. limicola cells takes place between chromophores (or their coupled associates) with parallel transition moment vectors.
Characterization of human red cell Rh (Rhesus‐)specific polypeptides by limited proteolysis
Authors: Krahmer Martina , Prohaska Rainer
Published: July 2002

Human red cells of various Rh phenotypes were surface‐labelled with 125I and the Rh‐specific labelled polypeptides were isolated by preparative SDS‐PAGE. The polypeptides were subjected to limited proteolysis and the resulting fragments were analysed by SDS‐PAGE and autoradiography. Chymotryptic peptide maps of proteins obtained from Rh(D)‐positive and ‐negative types appeared completely identical, whereas tryptic peptide maps revealed a difference: a fragment of M
r 17 500 was associated with the Rh(D) antigen, and one of M
r 19 000 with the Rh(C/c,E/e) antigens. Treatment of Rh polypeptides with carboxypeptidase Y prior to tryptic digestion resulted in a shift of nearly all tryptic fragments, including a fragment of M
r 8000, indicating that the surface label was incorporated into the C‐terminal part of the molecule.
In situ distribution of EcoRI methylase and restriction endonuclease in cells of Escherichia coli Bs 5
Authors: Kohring Gert-Wieland , Mayer Frank
Published: July 2002

Specific IgG antibodies were raised in rabbits against purified EcoRI methylase and restriction endonuclease. Post embedding labeling experiments, using the protein A‐gold technique, were made with paraformaldehyde‐glutaraldehyde fixed cells, embedded in Lowicryl K4M resin at low temperatures. Labeling with methylase‐specific antibodies showed 60–70% of gold particles in the cytoplasm and 30–40% at the cell envelope, whereas the use of restriction enzyme‐specific antibodies led to a distribution of 10–30% in the cytoplasm and 70–90% in the cell envelope. The results coincide with the proposed function of the enzymes: in the cytoplasm methylase protects the cells' own DNA from self‐destruction, and the restriction endonuclease cuts foreign DNA when entering the cell.
Immunoblotting and ligand blotting of the low‐density lipoprotein receptor of human liver, HepG2 cells and HeLa cells
Authors: Havinga Johan R. , Lohse Pia , Beisiegel Ulrike
Published: July 2002

Low‐density lipoprotein receptors from adult human liver and the human hepatoblastoma cell line HepG2 were analyzed by polyacrylamide electrophoresis in SDS followed by immuno‐ and ligand botting. In both liver and HepG2 we detected a protein band with apparent relative molecular mass of 130 kDa, which is similar to that of the LDL receptor in fibroblasts. In addition we showed that HeLa cells also possess this LDL‐receptor protein.
Polyamines inhibit the yeast histone deacetylase
Authors: Vu Quang A. , Zhang Dong-er , Chroneos Zissis C. , Nelson Daniel A.
Published: July 2002

n‐Butyrate inhibits the histone deacetylase from higher cells, but has little effect on the enzyme activity in Saccharomyces cerevisiae. Spermine and spermidine were therefore tested as potential yeast deacetylase inhibitors and found to inhibit fully the enzyme at 2 and 5 mM, respectively. The utility of these inhibitors was demonstrated by showing that 2 mM spermine substantially increased the incorporation of [3H]acetate into histone in a yeast nuclear acetyltransferase assay.
Prediction of antigenic determinants and secondary structures of the major AIDS virus proteins
Authors: Sternberg Michael J.E. , Barton Geoffrey J. , Zvelebil Marketa J. , Cookson John , Coates Anthony R.M.
Published: July 2002

Criteria for the design of peptide vaccines to prevent AIDS are presented. The best vaccine candidates contain both B and T lymphocyte‐defined epitopes in regions conserved in sequence between viral isolates. We propose that attention should focus on proteins specified by the gag and, possibly, pol genes in addition to the env gene envelope glycoproteins being actively studied. The predictions of B‐ and T‐epitopes are refined by consideration of secondary structure prediction and inter‐isolate sequence variability to suggest peptides from env, gag and pol that would be the best vaccine candidates.
On the possible mechanism of recognition of DNA base sequence by steroid hormones
Authors: Chandrasekhar B. , Mrigank  , Kothekar V.
Published: July 2002

Geometry of the complex of a steroid hormone, dexamethasone, with a hexanucleotide sequence from the glucocorticoid responsive element d(TGTTCT)2, is optimised here using computer aided geometry simulation with an energy minimization technique. We have also optimised its geometries with genetically modified and arbitrarily chosen DNA sequences. The drug molecule is considered to have both intercalative as well as non‐intercalative binding. Comparison of energetics and stereochemical aspects, as well as the H‐bonding scheme, is used here to bring out salient features about the mechanism of DNA sequence recognition by steroid hormones.
Growth hormone gene expression in myoepithelial cells directed by various eucaryotic transcriptional regulatory sequences
Authors: Gorewit R.C. , Chen H.Y. , Kopchick J.J.
Published: July 2002

Mammary gland myoepithelial cells were isolated from cattle and cell lines were established. Cells were plated onto tissue culture dishes with or without collagen. Cells were transfected with bovine growth hormone rDNA containing one of the following eucaryotic transcriptional regulatory sequences: human cytomegalovirus immediate early promoter, simian virus 40 early promoter, mouse metallothionein I promoter and the Rous sarcoma virus long terminal repeat. These sequences were evaluated for their ability to direct recombinant bovine growth hormone DNA expression in myoepithelial cells. The most effective transcriptional regulatory sequences were the cytomegalovirus immediate early and simian virus 40 early promoters.
Impaired calcium sequestration activity in liver microsomes from fasted rats
Authors: Osuna Carmen , Galván Aurora , Lucas Miguel
Published: July 2002

Calcium uptake activity was assayed in liver microsomal vesicles from fed and fasted rats. This activity required ATP and was stimulated by the calcium trapping agent oxalate. The most striking feature was the low rate of calcium accumulation in liver microsomes from fasted rats. Maximal rate was inhibited up to 66 and 82% after 1 and 3 days starvation, respectively. This defective microsomal calcium handling suggests its possible involvement in the massive glycogen breakdown during starvation.
A new mutation in the gene coding for the herbicide‐binding protein in Chlamydomonas
Authors: Johanningmeier Udo , Bodner Ulrich , Wildner Günter F.
Published: July 2002

Sequence analysis of herbicide‐resistant Chlamydomonas mutants revealed a new mutation in the psb A gene coding for the herbicide‐binding (D1) protein. A point mutation at codon 251 leading to an amino acid substitution from alanine in wild‐type cells to valine in the mutant was identified by sequencing total cellular RNA. The folding pattern of D1 predicts Ala 251 to be part of the Qb‐binding niche.
7‐Mercaptoheptanoylthreonine phosphate functions as component B in ATP‐independent methane formation from methyl‐CoM with reduced cobalamin as electron donor
Authors: Ankel-Fuchs D. , Böcher R. , Thauer R.K. , Noll K.M. , Wolfe R.S.
Published: July 2002

Purified methyl‐CoM reductase of Methanobacterium thermoautotrophicum (strain Marburg) catalyzed the reduction of methyl‐CoM to methane with reduced cobalamin, when either synthetic 7‐mercaptoheptanoyl‐threonine phosphate (HS‐HTP) or naturally occurring component B was present. With both compounds the same maximal specific acitivity was obtained and ATP was neither required nor stimulatory. These findings indicate that HS‐HTP functions as component B and do not support the idea that HS‐HT is only active in an adenosine monophosphorylated form.
Purification of Rhodobacter capsulatus RNA polymerase and its use for in vitro transcription
Authors: Forrest Mary E. , Beatty J.Thomas
Published: July 2002

RNA polymerase from the photosynthetic bacterium Rhodobacter capsulatus has been purified to near homogeneity. The fractions that contained RNA polymerase activity were compared with Escherichia coli RNA polymerase in SDS‐PAGE. The predominant bands were putative β and β' subunits of about 150–160 kDa, a candidate σ subunit of about 70 kDa, and a potential α subunit of approx. 45 kDa. This preparation was active in a run‐off transcription assay with a linearized pUC13 plasmid as template, and specific transcripts were detected.
Organization of soluble enzymes in the cell Relay at the surface
Authors: Ryazanov Alexey G.
Published: July 2002

A model is proposed uniting two groups of facts: The adsorption of enzymes on subcellular structures and the direct (‘from hand to hand’) transfer of metabolites between enzymes. The basic idea is that the binding of metabolites (substrates and/or products) results in desorption of the enzymes from subcellular structures during each catalytic act. This makes the enzymes mobile and capable of directly (from hand to hand) transferring metabolites to other enzymes adsorbed on subcellular structures. The model leads to a mechanism by means of which soluble enzymes can be compartmentalized in defined regions of the cytoplasm.
The in vitro insertion of monoamine oxidase B into mitochondrial outer membranes
Authors: Zhuang Z. , Hogan M. , McCauley R.
Published: July 2002

Bovine monoamine oxidase (MAO) B has been synthesized in vitro using a reticulocyte lysate translation system directed by bovine liver poly(A)+ RNA. The newly synthesized enzyme apparently lacks a cleavable N‐terminal extension, but MAO B is readily incorporated into mitochondria or isolated mitochondrial outer membranes prepared from rat liver. ATP is not required for the binding of the newly synthesized enzyme to the outer membranes, but is necessary for the insertion of MAO B into these membrane vesicles. The ATP is not required to generate a mitochondrial membrane potential as assembly occurs under conditions that preclude either the formation or the maintenance of the potential. MAO B will bind to but not become incorporated into outer membrane vesicles which have been treated with trypsin, suggesting that the insertion of MAO B also depends on protein factors present on the outer membranes.
Insulin like growth factor‐I, protein kinase‐C, calcium and cyclic AMP: Partners in the regulation of chondrocyte mitogenesis and metabolism
Authors: Taylor A.M. , Dandona P. , Morrell D.J. , Preece M.A.
Published: July 2002

The possible role of protein kinase‐C (PKC), calcium and cyclic AMP (cAMP) in mediating the metabolic and mitogenic effects of insulin‐like growth factor‐I (IGF‐I) on chondrocytes was investigated using a PKC activator (phorbol ester 12,13‐dibutyrate, PDBU), a PKC inhibitor (compound H7), a calcium channel blocker, (verapamil) and a cAMP analogue (dibutyryl cAMP). IGF‐I and PDBU stimulated sulphate and thymidine incorporation by chondrocytes. Both of these effects were inhibited by compound H7. Verapamil inhibited IGF‐I and PDBU‐stimulated sulphate incorporation, but contrastingly stimulated basal and enhanced IGF‐I and PDBU stimulation of thymidine incorporation. Dibutyryl cAMP increased basal and IGF‐I‐stimulated sulphate incorporation but inhibited both basal and IGF‐I stimulation of thymidine incorporation. These results suggest a harmonic overlap between the activities of PKC and cAMP‐dependent PKA enzyme systems, and calcium balance in the mitogenic and metabolic process of the chondrocyte.
Organization of 5′proximal c‐myb exons in chicken DNA Implications for c‐myb tissue‐specific transcription
Authors: Soret Johann , Vellard Michel , Martinerie Cécile , Perbal Bernard
Published: July 2002

The organization of 5′‐proximal c‐myb exons in chicken DNA has been established by restriction enzyme mapping and nucleotide sequencing. Hybridization studies performed with cDNA probes revealed that yolk sac and thymic c‐myb RNAs differ in their 5′‐termini. A comparison of the genomic c‐myb sequence with that of cDNAs isolated from normal thymic and lymphoma avian cells suggests that different promoter regions are used to initiate c‐myb transcription in hematopoietic cells of different origins.
The reactivation of apodopamine β‐monooxygenase by vanadyl ions
Authors: Markossian K.A. , Paitian N.A. , Nalbandyan R.M.
Published: July 2002

Vanadyl ions may be used for reactivation of apodopamine β‐monooxygenase. Maximal activity of the enzyme was achieved at a 350–400‐fold molar excess of vanadyl ions, whereas for maximal reconstitution with copper, an 8–10‐fold molar excess of copper was necessary. At higher concentrations of vanadyl as well as of copper, inhibition was observed.
The presumption of proteolytic enzymes related to the formation of intermediates during the terminal differentiation
Authors: Katagata Yohtaro , Aso Kazuo
Published: July 2002

The [14C]Gly‐labelled keratin polypeptides extracted with 1% SDS and 10 mM DTT were made to undergo changes with an enzyme fraction (ammonium sulfate, 50–75% saturated fraction) prepared from a human epidermis in the presence of 1% Triton X‐100. In particular, 69‐67 kDa peptides were considerably decreased with the above enzyme fraction in the time course experiments, and the components strongly bound to the cell membrane had little effect on the above reaction. In addition, in the case of the [14C]Gly‐labelled keratin filament assembly, 69 and 62 kDa peptides were decreased and 55, 52 and 50 kDa peptides were increased with the same enzyme fraction in the time course experiments. From these results, we estimated that the proteolytic enzyme(s) may exist in the human epidermis, and may be processed to keratin intermediates from prekeratin during the initial stage of terminal differentiation in the human epidermis.
Fluorescence digital image analysis of the inositol trisphosphate‐mediated calcium transient in single permeabilized parietal cells
Authors: Tsunoda Yasuhiro , Yodozawa Susumu , Tashiro Yutaka
Published: July 2002

The myo‐inositol 1,4,5‐trisphosphate (IP3)‐induced Ca2+ mobilization in single saponin‐permeabilized and fura‐2‐loaded parietal cells was analysed by a fluorescence digital image processor. When the cells were incubated with ATP, free cytoplasmic Ca2+ concentration ([Ca2+]i) increased in some restricted cytoplasmic regions showing discontinuous plateau and in the peripheral cytoplasm showing continuous [Ca2+]i gradient towards the plasma membranes. When treated with IP3, the high plateau enlarged to the entire cytoplasm and (a) new higher plateau(s) appeared and enlarged again in a transient manner. The IP3‐induced Ca2+ transient was also observed by fluorescence microphotometry of the single cells or by fluorescence spectrophotometry and 45Ca2+ uptake experiment of the cell population.
Gene expression of fibroblast matrix proteins is altered by indomethacin
Authors: Mauviel A. , Kähäri V.-M. , Heino J. , Daireaux M. , Hartmann D.J. , Loyau G. , Pujol J.-P.
Published: July 2002

The role of indomethacin in the regulation of extracellular matrix synthesis was studied in dermal fibroblast cultures. Indomethacin (10 μM) blocked totally the prostaglandin secretion and markedly increased the synthesis of collagen. In parallel, measurement of fibronectin, type I and type III procollagen mRNA levels showed a substantial increase under the action of indomethacin. On the other hand, indomethacin did not modify the mRNA levels of dermatan sulfate proteoglycan core protein. Measurement of collagen production estimated as the amount of collagenase digestible protein and by specific radioimmunoassay indicated a good correlation with the corresponding mRNA levels. These results suggest that indomethacin can regulate the extracellular matrix deposition at a transcriptional level.
Adhesion defect of ascites cells corrected with membrane‐bound attachment molecules
Authors: Gordeeva L.V. , Bogdanov A.A. , Baibakov B.A. , Torchilin V.P. , Margolis L.B.
Published: July 2002

Adhesion is obligatory for cell proliferation in most types of cells. This function becomes defective after malignant transformation. An extreme example is ascites cells which proliferate in suspension. The nature of their defects remains obscure. Here we show that the linking of biotin molecules to the Ehrlich ascites carcinoma cells enables these cells to spread normally on an avidin‐coated substrate. The spreading was the result of specific avidin‐biotin interaction. The morphology of the spread cells and sensitivity to different inhibitors are similar to those of normal epithelia. Thus it is enough to supply appropriate substrate‐adhesive molecules to the ascites cell surface to normalize their adhesion.
Galanin and the endocrine pancreas
Authors: Ahrén Bo , Rorsman Patrik , Berggren Per-Olof
Published: July 2002

Galanin is a 29 amino acid peptide, initially isolated from the porcine small intestine. The peptide has been shown to occur in intrapancreatic nerves in close association to the islets. Its effects on islet hormone secretion and its possible mechanisms behind these effects are reviewed. Galanin has been shown to inhibit basal and stimulated insulin secretion both in vivo and in vitro under a variety of experimental conditions. The peptide has also been shown to inhibit somatostatin secretion and the secretion of pancreatic polypeptide (PP). With regard to glucagon secretion, however, results in the literature are not consistent since both stimulatory and inhibitory effects have been reported. A direct interaction with the pancreatic β‐cells has been proposed behind its inhibitory action on insulin secretion, since galanin inhibits insulin secretion from isolated β‐cells from obese, hyperglycaemic, mice. Galanin has thereby also been shown to induce repolarization and to reduce the free Ca2+ concentration, [Ca2+]i. The reduction in [Ca2+]i is probably not due to a direct interference with the voltage‐activated Ca2+ channels, since there is no effect of galanin when these channels are opened by depolarization induced by high concentrations of K+. Instead, preliminary studies indicate that galanin activates the K+ channels that are regulated by ATP, in turn inducing a repolarization‐induced reduction in [Ca2+]i resulting in reduced insulin secretion. However, the possibility that galanin inhibits the insulin secretory mechanism at a step distal to the regulation of cytoplasmic free Ca2+ concentration should not be overlooked.
Adsorption of cytochrome c to phospholipid monolayers studied by reflection spectroscopy
Authors: Kozarac Z. , Dhathathreyan A. , Möbius D.
Published: July 2002

The uptake of cytochrome c by charged and neutral lipid monolayers was studied by using reflection spectroscopy. The method was shown to be a very sensitive and useful technique in studies of lipid‐protein interactions. It was found that cytochrome c is preferentially bound to monolayers of negatively charged monolayers in the solid phase. Polarized light under oblique incidence was used to determine the average orientation of chromophores in cytochrome c bound to lipid monolayer. The transition moments of chromophore are oriented parallel to the monolayer plane.
Carrier‐mediated uptake of L‐(+)‐lactate in plasma membrane vesicles from rat liver
Authors: Quintana Irene , Felipe Antonio , Remesar Xavier , Pastor-Anglada Marçal
Published: July 2002

Plasma membrane vesicles from rat liver transported L‐lactate into the inner vesicular space. Kinetic analysis of L‐lactate uptake gave a K
m value of approx. 2.9 mM. Selective inhibition was found in a similar pattern to that described for the hepatic lactate carrier. L‐Lactate transport was enhanced when a pH gradient was created across the plasma membrane. Vesicles obtained from fasted rats showed a higher uptake of L‐lactate than those from fed rats, when incubated with physiological concentrations of L‐lactate.
Solubilization of immune precipitates by complement in the absence of properdin or factor D
Authors: Späth P.J. , Pascual M. , Meyer-Hänni Liselotte , Schaad U.B. , Schifferli J.A.
Published: July 2002

Various experiments have demonstrated that immune precipitates (IPs) are not solubilized by complement in the absence of alternative pathway function. To determine whether the characteristics of the IPs were responsible for these observations, we studied the solubilization (Sol) of IPs formed by bovine serum albumin (BSA)‐rabbit antiBSA and tetanus toxoid (TT)‐human antiTT. Sera deficient in properdin solubilized a fraction of BSA‐antiBSA precipitates, although only when the IPs were formed in antibody excess. The same sera solubilized TT‐antiTT precipitates with some delay but almost as efficiently as normal serum. Factor D‐depleted serum solubilized a fraction of TT‐antiTT precipitates too, indicating that Sol may proceed through activation of the classical pathway only. Thus, the requirements for complement‐mediated Sol depend on the characteristics of the IPs and do not necessarily include alternative pathway function.
Periportal and perivenous hepatocytes respond equally to glycogenolytic agonists
Authors: Keppens Stefaan , De Wulf Henri
Published: July 2002

We have used the technique of short‐term infusion with digitonin to obtain hepatocytes originating either from the periportal or the perivenous zone of the liver acinus [(1985) Biochem. J. 229, 221–226]. Total glycogen phosphorylase content and sensitivity to cyclic AMP‐dependent and calcium‐mediated glycogenolytic agonists were very similar for both cell sub‐populations and did not differ from the values obtained for control cells. We conclude therefore that there is an apparent absence of metabolic zonation as far as receptor‐mediated glycogenolysis and glycogenolytic potency is concerned.
Human proteins IEF 58 and 57a are associated with the Golgi apparatus
Authors: Celis Ariana , Madsen Peder , Nielsen Henrik V. , Rasmussen Hanne H. , Thiessen Hanne , Lauridsen Jette B. , van Deurs Bo , Celis Julio E.
Published: July 2002

A mouse monoclonal antibody (mAB 22‐II‐D8B) raised against lysed transformed human amnion cells (AMA) has been characterized. The mAB decorated the Golgi apparatus in growing and quiescent cultured monolayer cells (fibroblasts and epithelial cells) of various species as determined by double immunofluorescence labeling and colocalization with galactosyltransferase antibodies. It reacted with the acidic human proteins IEF 58 (M
r = 29 000) and 57a, respectively (M
r = 30 000) (HeLa protein catalogue number; [(1982) Clin. Chem. 28, 766]), Golgi staining was also observed in BS‐C‐1 cells microinjected with mAB 22‐II‐DSB suggesting that the epitopes recognized by the antibody are most likely located on the cytoplasmic face of the membranes. The precise localization of the antigens to the various cisternae of the Golgi apparatus could not be demonstrated by immunogold cytochemistry on ultrathin cryosections due to either weak reactivity of the antibody or low concentration of the antigens. Immunofluorescence staining with mAB 22‐II‐D8B of lymphoid human Molt‐4 cells and some human tissues failed to reveal any significant staining even though these expressed high levels of both IEF 58 and 57a. These results are taken to imply that the epitopes recognized by mAB 22‐II‐D8B may be masked in some cell types.
Mechanism of transport of L‐alanine by luminal‐membrane vesicles from pars recta of rabbit proximal tubule
Authors: Vorum Henrik , Jessen Henrik , Jørgensen Karl Evald , Sheikh M.Iqbal
Published: July 2002

The characteristics of renal transport of L‐alanine by luminal‐membrane vesicles from proximal straight tubules (pars recta) of rabbit kidney were investigated. The following picture emerges from transport studies. Two electrogenic and Na+ requiring systems confined to this region of the nephron exist for the transport of L‐alanine. In addition to Na+, the transport of L‐alanine was influenced by H+. However, H+ does not substitute for Na+, but instead potentiates the Na+ effect. Modification of histidyl residues of the intact luminal‐membrane vesicles by diethylpyrocarbonate (DEP), completely abolished the transient renal accumulation of L‐alanine. Substrate and Na+‐protection experiments suggest that histidyl residues may be at or close to the active site of the L‐alanine transporter in membrane vesicles from pars recta.
Effect of low density lipoproteins, high density lipoproteins, and cholesterol on apolipoprotein A‐I mRNA in Hep G2 cells
Authors: Monge Juan Carlos , Hoeg Jeffrey M. , Law Simon W. , Brewer H.Bryan
Published: July 2002

We have utilized the human hepatocellular carcinoma cell line, Hep G2, to study the effects of low density lipoproteins (LDL), high density lipoproteins (HDL), and free cholesterol on apolipoprotein (apo) A‐I mRNA levels. Incubation of the Hep G2 cells with LDL and free cholesterol led to a significant increase in the cellular content of cholesterol without any effect on the yield of total RNA or in the cellular protein content. Our studies established that incubation with LDL or free cholesterol increased the relative levels of apoA‐I mRNA in the Hep G2 cells. In contrast with cholesterol loading, HDL had the effect of lowering the levels of apoA‐I mRNA. These results indicate the LDL and HDL pathways as well as intracellular cholesterol may be important in apoA‐I gene expression and regulation.
Isolation and some properties of mammalian hepatic membrane lectins
Authors: Ali Najma , Salahuddin A.
Published: July 2002

Membrane lectins were isolated from sheep, goat, and buffalo liver by chromatography on an asialofetuin (ASF)‐Sepharose 4B column. The lectins moved as a single protein band in SDS‐PAGE with molecular masses of 42, 54 and 50 kDa, respectively, for sheep, goat and buffalo lectins. The molecular masses remained unchanged in 0.2 M 2‐mercaptoethanol. As judged from the inhibition of binding of the lectin to ASF gel, the three lectins were β‐galactoside‐specific. Sheep, goat and buffalo lectins were found to be sialoglycoproteins containing 18.6, 27 and 38.8 mol/mol lectin of neutral hexose, respectively; the corresponding values for the sialic acid content being 5.3, 8.7 and 11.8 mol/mol lectin. Thus goat and buffalo lectins are physico‐chemically different from many mammalian hepatic lectins described so far.
The isolation of high molecular mass DNA from the prochlorophyte Prochloron didemni
Authors: Lockhart Peter J. , Larkum Anthony W.D. , Beanland Timothy J. , Howe Christopher J.
Published: July 2002

Using a protocol known to inactivate endogenous nucleases in other organisms we have been able to isolate high molecular mass DNA from Prochloron didemni. This DNA is shown to be suitable for restriction endonuclease analysis and the generation of genomic libraries in the λ cloning vector EMBL3.
Effect of redox potential on rat liver F1‐ATPase
Authors: López-Zabalza M.J. , Flores F. , Santiago E. , López-Moratalla N.
Published: July 2002

Redox titration of F1‐ATPase from rat liver mitochondria referred to the modification of the hydrolytic activity on Mg‐ATP has resulted in a three‐step pattern, with three distinct jumps of activity separated by clear plateaus. The measured potentials ranged from −400 mV to +400 mV and were obtained by the addition of dithionite and ferricyanide. Electron exchange was facilitated with a mixture of different redox mediators. At pH 7.4 the midpoint potentials were +210 mV, +40 mV and −230 mV. These three midpoint potentials were displaced towards more negative values by 2,4‐dinitrophenol or by an increase of the pH of the medium. The titration curves were described by n=2 Nernst equations.
Interaction between bacteriophage T4 coded gene 32 protein and poly(rA)
Authors: Watanabe Fumiyuki
Published: July 2002

The cooperative binding of T4 gene 32 protein with polynucleotides, of which the quantitative aspects in the literature have not satisfied the requirements of thermodynamics, is studied by adopting a modified formula of the lattice theory. A moderate value is found for the cooperativity parameter (q ∼ 200 at 0.2 M NaCl), which is weakly dependent on salt concentration. The cation effect on the binding suggests that the shielding of negative charges of the protein or a loose cation bridge between the bound protein molecules plays a role in the cooperative binding process.
Squalene epoxide cyclase and microemulsion
Authors: Lee Kang Min , Duriatti Albert , Schuber Francis , Biellmann Jean-François
Published: July 2002

Squalene epoxide cyclase was extracted from microsomal preparations of rat liver using anionic, cationic and non‐ionic microemulsions. The anionic microemulsion was the best with respect to protein solubilisation, extracted cyclase activity and stability of this activity over time. The activity assay showed cyclase activity to be higher in anionic microemulsion than in buffer in the presence of surfactant. Calcium chloride in the anionic microemulsion had a stabilising effect and less total protein seemed to be extracted.
Structures and functions associated with the group of mammalian lectins containing collagen‐like sequences
Authors: Thiel Steffen , Reid Kenneth B.M.
Published: July 2002

The number of proteins found in body fluids and at cell surfaces, which are known to display carbohydrate‐binding properties, continues to increase rapidly. In these proteins, in addition to a domain associated with lectin properties, one or more, non‐lectin domains are present. It is possible that binding of sugar residues by the lectin domain may be important in triggering a variety of recognition and clearance mechanisms via the non‐lectin domains. The group of lectins containing collagen‐like sequences may provide some insight into structure/function relationships of the different domains in view of the well defined structures already available for several of these molecules.
Regulation of tetrahydrobiopterin synthesis during lectin stimulation of human peripheral blood lymphocytes
Authors: Kerler Franz , Ziegler Irmgard , Schwarzkopf Bruno , Bacher Adelbert
Published: July 2002

Lectin stimulation of human T lymphocytes causes a 7‐fold increase in the specific activities of GTP‐cyclohydrolase and a 4‐fold increase in the specific activities of sepiapterin reductase. GTP‐cyclohydrolase activities are maximal after 48 h and subsequently decline, whereas sepiapterin reductase activities continue to increase during the 72 h period measured. The specific activities of 6‐pyruvoyltetrahydropterin synthase remain unchanged upon stimulation. Tetrahydrobiopterin synthesis during blast transformation is thus directed by both GTP‐cyclohydrolase and sepiapterin reductase.
Energization of the membrane prevents the formation of tight inactive complexes of ATPase with MgADP in submitochondrial particles
Authors: Chernyak B.V. , Khodjaev E.Yu.
Published: July 2002

Mitochondrial ATPase binds ADP tightly, forming inactive complexes. Dissociation of the complexes is blocked by azide. Azide inhibits neither ATP hydrolysis at high δH+ nor ATP‐dependent NAD+ reduction, provided that the ADP concentration is higher than 10−4 M. At lower ADP levels, azide is inhibitory. These data suggest that in the presence of ATP δH+ prevents the fromation of one of the inactive complexes. In the absence of ATP at high δH+, azide‐sensitive complexes are not formed at any ADP concentrations tested (5 × 10−7‐5 × 10−4 M). The inactive E·ADP complexes can play a significant role in the regulation of ATPase in mitochondria, preventing futile ATP hydrolysis at low δH+.
Thyroid hormone effect on rat heart mitochondrial proteins and affinity labeling with N‐bromoacetyl‐3,3′,5‐triiodo‐L‐thyronine Lack of direct effect on the adenine nucleotide translocase
Authors: Rasmussen Ulla B. , Köhrle Josef , Rokos Hartmut , Hesch Rolf-Dieter
Published: July 2002

N‐bromoacetyl‐3,3′,5‐tri[3′‐125I]iodo‐L‐thyronine was used to label intact heart mitochondria from eu‐, hypo‐ and hyperthyroid rats in order to identify proteins involved in T3‐regulated mitochondrial processes. The results show strong labeling, competed for by T3 and other analogues, of two proteins with a molecular mass of 48 000 and 49 200 Da. No labeling is seen of the adenine nucleotide translocase, a likely target, neither at 0°C, at room temperature, nor after preincubation with the substrates or specific inhibitors. No difference in labeling intensity or distribution is seen in mitochondria from eu‐, hypo‐ or hyperthyroid rats, and the abundance of the adenine nucleotide translocase is unchanged, but five other proteins show differential abundance.
Evidence for a sodium/calcium exchanger and voltage‐dependent calcium channels in adipocytes
Authors: Pershadsingh Harrihar A. , Lee L.-Y. , Snowdowne Kenneth W.
Published: July 2002

The objective of these studies is to identify and characterize Ca2+‐transport systems that may be of potential importance in the action of Ca2+‐mobilizing hormones in the adipocyte. Using the Ca2+‐sensitive photoprotein, aequorin, [Ca2+]i was estimated to be 0.15 μM, assuming an intracellular [Mg2+] of 1 mM. Substitution of Na+ with choline+ caused a transient increase in [Ca2+]i which was inversely related to extracellular [Na+], consistent with operation of a mediated Na+‐Ca2+ exchange system. The stoichiometry was 3Na+:Ca2+. Elevation of extracellular K+ caused an increase in [Ca2+]i that was blocked by the Ca2+ channel antagonist, diltiazem, by omitting extracellular Ca2+, or by substituting Sr2+ for Ca2+. These findings indicate the presence of an Na+‐Ca2+ exchanger and voltage‐sensitive Ca2+ channels in adipocytes.
Inhibition by glucosamine of myristoylation in human H9 lymphocytes and rat liver cells
Authors: Jacobsen C. , Andreassen H. , Saermark T.
Published: July 2002

N‐Myristoyl transferase (NMT) activity was measured in rat liver and H9 cells using an in vitro assay based on acylation of synthetic peptides. Glucosamine was found to inhibit the NMT activity. Using a synthetic peptide mimicking the N‐terminus of HIV p27nef a K
m value of 2.4 μM and a V
max of 240 per h was found. In the presence of glucosamine the V
max was lowered indicating that glucosamine acted as a non‐competitive inhibitor. Glucosamine also inhibited incorporation of radiolabelled myristic acid into H9 cell proteins in vivo. In liver cells using a peptide from the N‐terminus of p60 SRC only the V
max was affected.