bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of tissue inhibitor of metalloproteinase‐1 (TIMP‐1) in human hepatoma cells (HepG2) Up‐regulation by interleukin‐6 and transforming growth factor β1
Authors: Kordula Tomasz , Güttgemann Ines , Rose-John Stefan , Roeb Elke , Osthues Anja , Tschesche Harald , Koj Aleksander , Heinrich Peter C. , Graeve Lutz
Published: July 2002

Metalloproteinases and their specific inhibitors, believed to play a role in extracellular matrix metabolism, are regulated by inflammatory cytokines. Here we have addressed the question of whether liver, the major site of Synthesis of plasma proteinase inhibitors, is also capable of synthesizing the tissue inhibitor of metalloproteinase‐1 (TIMP‐1). We show at mRNA and protein levels that TIMP‐1 is expressed in differentiated human hepatoma cells (HepG2) and that its synthesis is up‐regulated by interleukin‐6 (IL‐6), transforming growth factor β1 and phorbol 12‐myristate 13‐acetate. The physiological role of this phenomenon is underlined by the fact that lipopolysaccharide administration into rats in vivo, as well as IL‐6‐stimulation of rat hepatocytes in primary culture, also leads to an increase of TIMP‐1 mRNA in liver cells.
Identification of a MAP kinase kinase kinase in phaeochromocytoma (PC12) cells
Authors: Gomez Nestor , Traverse Sarah , Cohen Philip
Published: July 2002

A MAP kinase kinase kinase (MAPKKK) was identified in phaeochromocytoma (PC12) cells which reactivated homogeneous MAP kinase kinase (MAPKK) from rabbit skeletal muscle that had been inactivated by incubation with protein phosphatase 2A. Reactivation was accompanied by stoichiometric phosphorylation of MAPKK on a serine residue(s). Following stimulation of PC12 cells with nerve growth factor and chromatography of the extracts on Mono Q, MAP kinase and MAPKK were detected as active phosphorylated enzymes, whereas MAPKKK was inactive and only activated after prolonged storage at 4°C. The results suggest that the activation of MAPKKK by growth factors is likely to occur by a non‐covalent mechanism.
Changes in the level of chloroplast transcripts in pumpkin cotyledons during heat shock
Authors: Kusnetsov V.V. , Mikulovich T.P. , Kukina I.M. , Cherepneva G.N. , Herrmann R.G. , Kulaeva O.N.
Published: July 2002

The levels of plastid gene transcripts are shown to be controlled by temperature in isolated pumpkin cotyledons. The temperature at which maximum transcript accumulation occurs varies between 38 and 42°C for the genes studied (rbcL, psaA, psbA, psbB, psbC, psbD, psbE, atpA). Heat shock‐induced transcript accumulation is transitory with a maximum after an approximately 3 h exposure at high temperatures. On the other hand, the accumulation of rbcL transcript was only moderately thermosensitive. A temperature increase to 46–48°C induces a sharp decrease of transcript levels which correlates with damage to the plant. Relatively little correlation has been noted between RNA and protein patterns. However, there is a remarkable coincidence between temperature dependence of the accumulation of transcripts and the temperature dependence (28–46°C) of the synthesis of chloroplast‐located heat shock proteins indicating that both processes may be related.
Purification of recombinant hepatitis delta antigen expressed in E. coli cells
Authors: Calogero R. , Barbieri U. , Borla M. , Osborne S. , Poisson F. , Bonelli F.
Published: July 2002

Recombinant DNA technology enables the massive production of recombinant hepatitis delta antigen (recHDAg) retaining immunological properties and transport functions. However, purification procedures of the recombinant delta antigen have, to date, not been described in the literature. We present a purification procedure allowing one to obtain highly purified recHDAg from bacterial cells expressing the hepatitis delta antigen.
The induction of early response genes in rat smooth muscle cells by PDGF‐AA is not sufficient to stimulate DNA‐synthesis
Authors: Sachinidis Agapios , Schulte Kerstin , Ko Yon , Meyer zu Brickwedde Maria Katharina , Hoppe Viviane , Hoppe Jürgen , Vetter Hans
Published: July 2002

The effect of the three platelet‐derived growth factor (PDGF) isoforms AA, AB and BB on the induction of the early growth response genes c‐fos, egr‐1 and c‐myc mRNA in vascular smooth muscle cells from rat was compared with their respective mitogenic potency. The three PDGF isoforms strongly stimulated the induction to a similar extent. In contrast, PDGF‐AB and ‐BB provoked a marked DNA synthesis whereas PDGF‐AA exerted only a poor mitogenic effect in smooth muscle cells. PDGF‐AA‐stimulated receptor autophosphorylation was not detectable in comparison with the strong effect elicited by PDGF‐AB or ‐BB and correlated with its low mitogenicity but not with the almost equal induction of the early response genes. It is discussed that no or only very low receptor phosphorylation is required to link receptor activation to the induction of c‐fos, egr‐1 or c‐myc. Furthermore the induction of the investigated gene does not seem to be sufficient for an optimal mitogenic response.
Purification of macrophage migration inhibitory factor (MIF) from bovine brain cytosol
Authors: Galat Andrzej , Rivière Sylvie , Bouet Françoise
Published: July 2002

Two isoforms of the macrophage migration inhibitory factor (MIF) have been isolated to homogeneity from bovine brain cytosol. In agreement with the cDNA sequence of their human counterpart, they both have an apparent molecular weight of 12 kDa and are characterized by the following N‐terminal amino acid sequence NH2‐PMFVVNTNVPRASVPDGLLSELTQQLAQATGKPPQYIAV‐. CD spectra revealed that bovine MIF contains 42% (±3%) α‐helix and 21% (±3%) β‐structure. CD‐constrained prediction of the secondary structure assigned MIF to the α/β‐class of proteins.
Heterocomplexes of tick‐borne encephalitis structural and non‐structural proteins
Authors: Pressman E.K.
Published: July 2002

The existence of the complex of structural and non‐structural proteins in the tick‐borne encephalitis (TBE) virus is shown. The complex was isolated from virus‐containing cultural medium by immunoaffinity chromatography on monoclonal antibodies (MAbs). By enzyme immunoassay and immunoblotting with the use of appropriate MAbs it was demonstrated that this complex consists of structural (protein E), and non‐structural (NS1) glycoproteins. Also, the trimer E‐NS1‐NS3 can be isolated. It is proposed that this trimer is the viral replicative complex.
Lysophosphatidylcholine mediates the mode of insertion of the NH2‐terminal SIV fusion peptide into the lipid bilayer
Authors: Martin I. , Dubois M.-C. , Saermark T. , Epand R.M. , Ruysschaert J.-M.
Published: July 2002

We report here on the interaction of a synthetic 12 residue peptide corresponding to the N‐terminal sequence of gp32 from SIV with phospholipid bilayers. This peptide has been shown to induce lipid mixing of PC/PE/SM/Chol LUV (large unilamellar vesicles) at pH 7.4 and 37°C [(1992) in: Advances in Membrane Fluidity, vol. 6, pp. 365‐376, Wiley‐Liss]. In the present study, this fusion process was inhibited by the addition of lysophosphatidylcholine (lysoPC) to the lipid bilayer of PC/PE/SM/Chol LUV. Fourier transform infrared spectroscopy (FTIR) reveals that the orientation of the SIV fusion peptide with respect to the lipid acyl chains depends on the presence of lysoPC in the lipid bilayer but that the peptide secondary structure and the amount of lipid‐associated peptides do not depend on the lipid composition. The peptide is obliquely inserted into the lipid bilayer of vesicles without lysoPC, whereas it is oriented parallel to the lipid‐water interface in the vesicles containing lysoPC. The data provide evidence that the orientation of the SIV fusion peptide depends on the lipid composition, and that this mediates its fusogenic activity.
Electrophoretic analysis of myosin heavy chain isoform patterns in extraocular muscles of the rat
Authors: Asmussen Gerhard , Traub Irmtrud , Pette Dirk
Published: July 2002

Six oculorotatory muscles and the levator palpebrae muscle of the rat were analysed by SDS‐PAGE for their myosin heavy chain (MHC) isoform patterns. Oculorotatory muscles display a marked predominance of fast MHC isoforms. They contain, in addition to the slow (MHCI) and fast (MHCIIb, MHCIId, MHCIIa) skeletal MHCs, the neonatal MHCneo and the extraocular MHCeom. The levator palpebrae, generally assumed to be a member of the extraocular muscle group because of its innervation by the ocolumotor nerve, does not contain MHCneo and MHCeom. It resembles a fast‐twitch skeletal muscle with a predominance of MHCIId.
Molecular cloning of a putative homolog of proline/arginine‐rich antibacterial peptides from porcine bone marrow
Authors: Pungerc̆ar Joz̆e , Štrukelj Borut , Kopitar Gregor , Renko Metka , Lenarc̆ic̆ Brigita , Gubens̆ek Franc , Turk Vito
Published: July 2002

Screening of a porcine bone marrow cDNA library with a PCR‐derived probe from rabbit LPS‐binding protein CAP18 led to the discovery of two closely related clones. The longer, full‐length cDNA clone encodes a 228 amino acid residue protein similar to the family of antibacterial/LPS‐binding cationic peptides. In contrast to other hitherto discovered precursors of Pro/Arg‐rich peptides from this family, they have a novel, unique structure of the C‐terminal region of 100 amino acid residues with a repeating sequence often residues (FPPPNXPGPR, where X = V or F). These precursors could represent a part of the antibacterial peptide repertoire of porcine bone marrow.
Crystal structure of apo‐glycolate oxidase
Authors: Sandalova Tatyana , Lindqvist Ylva
Published: July 2002

The crystal structure of the apoform of the α/β‐barrel enzyme glycolate oxidase has been determined to 2.6 Å resolution. Removal of the tightly bound cofactor FMN has a very strong influence on the protein structure; it is converted into a very flexible state, verging on a molten globule type of structure. The asymmetric unit contains two subunits with different conformations to each other and to the holo‐enzyme. The secondary structures are preserved, but their mutual arrangement has changed to some extent introducing cavities into the protein. The largest structural shifts are, however, found in the loops.
Tissue‐specific isoforms of the bovine mitochondrial ATP synthase γ‐subunit
Authors: Matsuda Chie , Endo Hitoshi , Hirata Hajime , Morosawa Hideaki , Nakanishi Makoto , Kagawa Yasuo
Published: July 2002

Tissue‐specific isoforms of the γ‐subunit of the bovine F0F1‐ATP synthase were identified for the first time. The two isoforms, heart and liver type, were generated by alternative splicing, the liver‐type RNA transcript containing a 37‐nucleotide sequence as a cassette exon. Protein sequencing of the C‐terminal fragments of the two isoforms of the F1γ‐subunit indicated that the liver‐type isoform had an additional aspartate residue at the C terminus, not present in the heart type one.
Effects of β6 amino acid hydrophobicity on stability and solubility of hemoglobin tetramers
Authors: Adachi K. , Kim J.Y. , Konitzer P. , Asakura T. , Saviola B. , Surrey S.
Published: July 2002

The relationship between different amino acids at theβ6 position of hemoglobin and tetramer stability was addressed by a site‐directed mutagenesis approach. Precipitation rates during mechanical agitation of oxyhemoglobins with Gln, Ala, Val, Leu and Trp at the β6 position increased 2, 5, 13, 21 and 53 times, respectively, compared with that for Hb A. There was a linear relationship between the log of the precipitation rate constant and amino acid hydrophobicity at the β6 position, suggesting that enhanced precipitation of oxy Hb S during mechanical agitation results in part from increased hydrophobicity of β6 Val. Deoxyhemoglobin solubility increased in the order of β6 Ile, Leu, Val, Trp, Gln, Ala and Glu suggesting that hydrophobic interactions between β6 Val and the acceptor site of another hemoglobin molecule during deoxy‐Hb S polymerization not only depend on hydrophobicity but also on stereospecificity of the amino acid side chain at the β6 position. Furthermore, our results indicate that hydrophobic amino acids at the β6 position which promote tetramer instability in the oxy form do not necessarily promote polymerization in the deoxy form.
An EPR investigation of non‐haem iron sites in Escherichia coli bacterioferritin and their interaction with phosphate
Authors: Le Brun Nick E. , Cheesman Myles R. , Thomson Andrew J. , Moore Geoffrey R. , Andrews Simon C. , Guest John R. , Harrison Pauline M.
Published: July 2002

EPR studies of bacterioferritin (BFR), an iron‐storage protein of Escherichia coli [1993, Biochem. J. 292, 47‐56.], have revealed the presence of non‐haem iron (III) (NHI) sites within the protein coat which may be involved in iron uptake and release. When nitric oxide was used as an EPR spin probe of the Fe(II) state of the NHI sites, two distinct mononuclear NHI species were found. Under certain conditions, an iron dimer was also observed. The reaction of phosphate with NHI species has been investigated. Results point to a function for this anion in core nucleation.
Purification and functional characterization of β‐adrenergic receptor kinase expressed in insect cells
Authors: Sohlemann Peter , Hekman Mirko , Buchen Claudia , Elce John S. , Lohse Martin J.
Published: July 2002

The β‐adrenergic receptor kinase mediates agonist‐dependent phosphorylation of β‐adrenergic receptors, which is thought to represent the first step of homologous desensitization. We have expressed bovine and human βARK1 in Sf9 cells and purified them to apparent homogeneity in milligram quantities. The K
m‐values of the enzyme were 3.8 μM for rhodopsin and 22 μM for ATP; the V
max‐value was 9.9 mol phosphate/mol βARK/min. These data indicate that the two recombinant kinases were at least as active as preparations previously obtained from bovine brain. There were no differences in the functional activity of human and bovine βARK.
Two different promoters direct expression of two distinct forms of mRNAs of human platelet‐activating factor receptor
Authors: Mutoh Hiroyuki , Bito Haruhiko , Minami Michiko , Nakamura Motonao , Honda Zenichiro , Izumi Takashi , Nakata Ryo , Kurachi Yoshihisa , Terano Akira , Shimizu Takao
Published: July 2002

The human platelet‐activating factor (PAF) receptor gene exists as a single copy on chromosome 1. We identified two 5′‐noncoding exons, each of which has distinct transcriptional initiation sites. These exons are alternatively spliced to a common splice acceptor site on a third exon that contains the total open reading frame to yield two different species of functional mRNA (Transcript 1 and 2). Transcript 1 has consensus sequences for transcription factor NF‐κB and Sp‐1, and the Initiator (Inr) sequence homologous to the murine terminal deoxynucleotidyltransferase gene. Transcript 2 also contains consensus sequences for transcription factor AP‐1, AP‐2, and Sp‐1. Transcripts 1 and 2 were both detected in heart, lung, spleen, and kidney, whereas only Transcript 1 was found in peripheral leukocytes, a differentiated human eosinophilic cell line (EoL‐1 cells), and brain. Existence of distinct promoters was thus suggested to play a role in the regulatory control of PAF receptor gene expression in different human tissues and cells.
The MAP kinase‐activated protein kinase 2 contains a proline‐rich SH3‐binding domain
Authors: Engel Katrin , Plath Kathrin , Gaestel Matthias
Published: July 2002

The protein sequence of MAP kinase‐activated protein kinase 2 (MAPKAP kinase 2) deduced from mouse cDNA sequence reveals structural features of the enzyme, which could be of importance for its function: a proline‐rich SH3‐binding domain N‐terminal to the catalytic region, a MAP kinase phosphorylation site and a bipartite nuclear targeting sequence located C‐terminal to the catalytic region. The catalytic domain itself has the strongest homology to calcium/calmodulin‐dependent protein kinase II. Northern blot analysis demonstrates a 3.5 kb MAPKAP kinase 2 transcript which is ubiquitously expressed and, hence, co‐expressed with the mRNA of the recently identified substrate Hsp25 in all tissues analysed. However, the functional consequences of the nuclear targeting sequence present in MAPKAP kinase 2 suggest the existence of further substrates for the enzyme in the nucleus.
Progesterone action on the human sperm surface is potentiated by an egg‐associated acrosin activator
Authors: Mendoza C. , Moos J. , Tesarik J.
Published: July 2002

The progesterone‐induced Ca2+ influx and acrosomal exocytosis in human sperm are recently described examples of non‐genomic steroid action on the cell surface. These progesterone effects are known to be inhibited by synthetic protease inhibitors. In this study we tested a hypothesis that a physiological activator of the sperm protease acrosin modulates the sperm response to progesterone. It was found that the activator augments the amplitude of the progesterone‐induced Ca2+ transient and accelerates the progesterone‐induced acrosomal exocytosis. These observations suggest a physiological significance of the modulation of nongenomic steroid effects by protease regulators.
Immunodetection and enzymatic characterization of the α3‐isoform of Na,K‐ATPase in dog heart
Authors: Berrebi-Bertrand I. , Maixent J.M.
Published: July 2002

The expression of the canine α 2 and 3 subunit isoenzymes of NA,K‐ATPase has been investigated in plasma membranes isolated from dog heart, brain and kidney by immunoblotting, employing polyclonal anti rat fusion protein, and enzymological techniques. Western blot analysis revealed with purified dog membrane Na,K‐ATPase preparations, one immunoreactive signal with rat specific α3 antisera in cardiac tissues, and two immunoreactive signals with rat α3 and α3 antisera in cerebral tissues. These findings suggested the specific expression of α3 polypeptide in dog heart (99 kDa), whereas dog brain expressed the α 2 and 3 polypeptides. The stained bands were superimposed. The antibody to rat brain α1 fusion protein did not cross‐react with dog antigens whatever the three tissues tested. Expression of the α3‐subunit isoform in dog heart membranes was consistent with a high affinity digitoxigenin‐sensitive class of Na,K‐ATPase (IC50 = 7 ± 2 nM). A single component with low affinity to digitoxigenin (IC50 = 110 ± 10 nM) characterized the α1 kidney form. The mixture of α2 and α3 isoforms in dog brain exhibited an apparent affinity for digitoxigenin (IC50 = 17 ± 5 nM) lower than the heart. The sodium dependences of the high affinity digitoxigenin sites were for the cardiac α3 form (K
0.5 = 10 ± 1.9 mM) and for the cerebral α2 and α3 mixture (K
5.0 19.6 ± 4.9 mM). The sensitivities for Na+ of the low affinity sites (α1) were: 6.7 ± 1.4 mM, 6.3 ± 1.2 mM and 11.6 ± 2.9 mM in heart, brain and kidney respectively. This is the first report of the catalytic characteristics of the α3 subunit isoenzyme in canine cardiac plasma membranes.
Functional expression of 2‐amino‐4‐phosphonobutyrate (APB) receptors in Xenopus laevis oocytes by injection of poly(A)+ RNA from quail brain
Authors: Moon C. , Fraser S.P. , Barrett P. , Morgan P.J. , Djamgoz M.B.A.
Published: July 2002

The glutamate analogue 2‐amino‐4‐phosphonobutyrate (APB) is known to activate a subtype of metabotropic glutamate receptor in the central nervous system, including the retina. In the present study, APB receptors were studied using the Xenopus oocyte expression system. No endogenous APB sensitivity was detected in control oocytes. In contrast, microinjection of mRNA, extracted from quail brain, into Xenopus oocytes resulted in the functional expression of APB receptors after 3–5 days incubation. Application of 50 μM‐1 mM APB to injected oocytes voltage clamped at a holding potential of −60 mV produced a sustained outward current which was associated with a significant decrease in membrane conductance; the reversal potential was around −11 mV. The response to APB was dose‐dependent and non‐desensitizing. This is the first demonstration of the expression of a conductance‐decreasing receptor mechanism in Xenopus oocytes.
Interaction of the calcium and calmodulin regulated eEF‐2 kinase with heat shock protein 90
Authors: Palmquist Kim , Riis Bent , Nilsson Anders , Nygård Odd
Published: July 2002

Immunoadsorbents containing the 8D3 anti‐heat shock protein 90 monoclonal antibodies were prepared. Partly purified preparations of the Ca2+ and calmodulin dependent eukaryotic elongation factor eEF‐2 specific kinase and crude rabbit reticulocyte lysates were mixed with the immunoadsorbent. After removal of unbound proteins the adsorbed material was released by increasing the salt concentration in the buffer. Analysis of the bound material showed that the eEF‐2 kinase was bound to the immunoadsorbent together with hsp 90. The adsorption of the kinase was found to depend on the presence of hsp 90.
Endogenous DHP‐sensitive Ca2+ channels in Pleurodeles oocytes
Authors: Ouadid H. , Browaeys-Poly E. , Vilain J.P. , Guilbault P.
Published: July 2002

The double electrode voltage‐clamp technique was used to study voltage‐dependent Ca2+ channels in Pleurodeles oocytes. From a holding potential of −80 mV, Ba‐current (I
Ba) (recorded in Cl‐free solution, Ba2+ = 40 mM) activated at −36.7 ± 4 mV, peaked at −11.6 ± 4 mV and reversed at 55 ± 7 mV (n = 24). This current activated slowly (rise time was 0.98 ± 0.2 s; n = 14 at −10 mV) and was not inactivated. Cadmium (Cd2+, 500 μM) completely inhibited I
Ba. The effect of Cd2+ was dose‐dependent (EC50 = 37 ± 5 μM; n = 5). Moreover, I
Ba was insensitive to ω‐conotoxin (10 μM) but interestingly this I
Ba. displayed dihydropyridine (DHP) sensitivity. Bay K 8644 (5 μM), a DHP activator, increased the peak current amplitude in a dose‐dependent manner (EC50 = 5.9 ± 0.6 μM; n = 10) and shifted the threshold and the maximum of current/voltage relationship towards negative potentials by − 10 mV. Nifedipine (5 μM), a DHP antagonist, decreased I
Ba by 80% at HP of −80 mV (EC50 = 1.2 ± 0.2 μM; n = 6). We concluded that Pleurodeles oocytes possess High‐Voltage Activated Ca2+ channels with properties similar to L‐type Ca2+ channels.
Blockage of the urokinase receptor on the cell surface: Construction and characterization of a hybrid protein consisting of the N‐terminal fragment of human urokinase and human albumin
Authors: Lu He , Yeh Patrice , Guitton Jean-Dominique , Mabilat Christelle , Desanlis Francine , Maury Isabelle , Legrand Yves , Soria Jeannette , Soria Claudine
Published: July 2002

Receptor‐bound urokinase is likely to be a crucial determinant in both tumor invasion and angiogenesis. We report here that a yeast‐derived genetic conjugate between human serum albumin and the 1–135 N‐terminal residues of urokinase (u‐PA) competitively inhibits the binding of exogenous and endogenous u‐PA to its cell‐anchored receptor (u‐PAR). This hybrid molecule (ATF‐HSA) also inhibits in vitro pro‐urokinase‐dependent plasminogen activation in the presence of u‐PAR bearing cells. These effects are probably responsible for the observed in vitro inhibition of tumor cell invasion in a reconstituted basement membrane extract (Matrigel).
Site‐specific labelling of the oligosaccharide chains of antibodies
Authors: Schwarz Uwe , Wunderlich Gerd , Brossmer Reinhard
Published: July 2002

This paper presents a new method for site‐specific labelling of antibodies employing enzymatic reactions without oxidizing or reducing agents. IgG was first treated with immobilized sialidase from Clostridium perfringens to cleave bound NeuAc. CMP‐9‐deoxy‐9‐salizoyl‐NeuAc, an activated sialic acid analogue, was labelled with 131I via the iodogen‐method in high yields (> 95%). Then the oligosaccharide chains of antibodies were labelled yield with the radioactive NeuAc analogue by transfer using α‐2,6‐sialyltransferase from rat liver in 50%.
How does NO activate hemeproteins?
Authors: Tsai Ah-lim
Published: July 2002

NO was reported to activate guanylate cyclase and, recently, prostaglandin H synthase. NO interaction with the heme component in different hemeproteins is determined by ligand property, electronic configuration of the heme iron and the specific effects contributed by the protein structure. It is found that although NO interaction with the free heme provides some common rules of interaction, the consequences of NO binding to different hemeproteins should be dealt with individually.
Low‐dose follicle‐stimulating hormone treatment for polycystic ovarian disease
Authors: Grigoriou O. , Antoniou G. , Antonaki V. , Patsouras C. , Zioris C. , Karakitsos P.
Published: July 2002

Objective: An attempt was made to induce ovulation with low‐dose follicle‐stimulating hormone (FSH) in clomifene‐resistant women with anovulatory polycystic ovarian disease (PCOD). Methods: Twenty‐two PCOD patients were treated with a low‐dose protocol of purified urinary FSH, starting with 75 IU/day and increasing every 7 days by 37.5 IU/day, if necessary. Monitoring was based on ultrasound scanning and estradiol measurements. Results: Twenty‐eight of the thirty‐one cycles induced were ovulatory, the majority being uniovulatory (58%). No multiple pregnancies occurred. There was a small number of cancelled cycles (12.9%). The prevalence of complications was low, with one case of ovarian hyperstimulation syndrome. Ovulation was induced by a small amount of FSH (15.1 ± 4.9 ampules). Conclusion: The use of the low‐dose protocol permitted induction of ovulation safely and successfully in a selected group of PCOD patients. This therapy was associated with a high incidence of single dominant follicles and a very low multiple pregnancy rate.
Rare occurrence of ovarian hyperstimulation syndrome in oocyte donors
Authors: Sauer M.V. , Paulson R.J. , Lobo R.A.
Published: July 2002

Objectives: To define the incidence and severity of ovarian hyperstimulation syndrome (OHSS) occurring in oocyte donors. Methods: Women (n = 149) aged 31.3 ± 4.8 years (mean ± S.D., range 21–41 years) participated as designated oocyte donors and underwent 400 consecutive cycles of controlled ovarian stimulation using human menopausal gonadotropin following pituitary downregulation with gonadotropin‐releasing agonist. Patients were monitored by serial transvaginal ultrasound examinations and serum estradiol (E2) determinations. Oocytes (15.6 ± 7.5 per aspiration; range 2–57) were harvested by ultrasound‐directed transvaginal follicle aspiration 36 h following the intramuscular injection of human chorionic gonadotropin (hCG). Follow‐up examination occurred 1 and 2 weeks post‐aspiration. Results: On the day of hCG injection E2 levels ranged from 512 to 13 502 pg/ml (mean 2902.7 ± 1486.9 pg/ml). Over the next few weeks the degree of hyperstimulation in donors was staged: mild 65% (grade I, n = 98; grade II, n = 162); moderate 33.5% (grade III, n = 120; grade IV, n = 14); severe 1.5% (grade V, n = 6; grade VI, n = 0). Associated preaspiration E2 levels were: grade I, 1120 ± 424 pg/ml; grade II, 2084 ± 613 pg/ml; grade III, 3785 ± 1713 pg/ml; grade IV, 5370 ± 1264 pg/ml; grade V, 4286 ± 1100 pg/ml. Worsening OHSS was associated with increasing levels of E2. There were no serious complications and hospitalization was not required. All symptoms resolved within 30 days of aspiration, disappearing by the time of the first menstrual flow in women of grade‐III or lower stage. Conclusion: Although oocyte donors commonly experienced exaggerated levels of serum E2 they rarely (<2%) developed severe OHSS. This may be attributable to their lack of embryo transfer which avoids exacerbating the illness.
Management of stage‐I borderline ovarian tumors
Authors: Ji H. , Yliskoski M. , Anttila M. , Syrjänen K. , Saarikoski S.
Published: July 2002

Objective: To evaluate the surgical management and outcome of stage‐I borderline ovarian tumors. Methods: A series of 95 cases of FIGO stage‐I borderline ovarian tumors was retrospectively reviewed. Results: The age of the patients ranged from 17.6 to 88.9 years (mean 52.7 years). Serous and mucinous tumors were equally represented (47.5% and 47.4%, respectively), endometrioid and clear cell tumors comprising the remaining three (3.2%) and two (2.1%) cases. Extirpative primary surgery was performed on all patients. Three patients received postoperative chemotherapy. Nineteen of the 28 patients (67.9%) who were younger than 40 years underwent conservative surgery in which ovarian tissue and the uterus were preserved in order to maintain reproductive potential, and nine of them had successful pregnancies later. Ninety patients were followed regularly for 0.75–21.3 years (average 7.3 years). Seven patients suffered a tumor relapse (7.8%) within 0.6–4.3 years, and four (57.1%) of them (three with mucinous tumors, one with clear cell tumor) died of the disease within 1–10.8 years. The 5‐ and 10‐year overall survival rates were 97.0% and 86.3%, respectively, and the disease‐free survival rates were 90.8% in both 5‐ and 10‐year follow‐ups. Conclusions: Even though the prognosis of surgically treated stage‐I borderline ovarian tumors is excellent, mucinous and clear cell sub‐types are associated with potential mortality.
Stillbirths and intrauterine infection, histologic chorioamnionitis and microbiological findings
Authors: Moyo S.R. , Hägerstrand I. , Nyström L. , Tswana S.A. , Blomberg J. , Bergström S. , Ljungh Å.
Published: July 2002

Objective: To analyze whether placental inflammation is associated with stillbirth in Zimbabwe. Method: Placentas from 66 stillbirths (> 22 weeks' gestation; patients with congenital malformations, diabetes or preeclampsia were excluded) and 66 term live births were studied for the presence and severity of chorioamnionitis. The morphological results were compared with earlier presented microbiological findings in the same material. Results: Chorioamnionitis was present in 79% of stillbirths and 30% of live births (O.R. 8.5, 95% C.I. 4.0–18). Nine percent of stillbirths but no live births presented vasculitis of the chorionic plate, which verified an inflammatory response from the infant (O.R. 14, 95% C.I. 2.8–72). The same types of microorganisms were isolated from stillbirths and liveborns, but Escherichia coli and group B streptococci were more frequent among stillbirths. Conclusions: Morphological chorioamnionitis occurred 2.6 times more often in women with stillbirths than in women with live births. In 9% of stillbirths the infant showed an inflammatory response. Thus the infant was alive when the infection occurred and it is therefore reasonable to assume that infection was the cause of death.
Both native conformers of rabbit muscle adenylate kinase are active
Authors: Li X , Pan X.M
Published: July 2002

There are two forms of rabbit muscle adenylate kinase (AK) with different 8‐anilino‐1‐naphthalenesulfonic acid (ANS) binding properties in equilibrium solution. One form (about 70%, denoted N1) binds rapidly with ANS, whereas the other (about 30%, denoted N2) does not. Furthermore, native forms of AK should adopt different conformations for binding with substrates and products, which should be pre‐existing for performing its catalytic function. The present experiments demonstrate both forms of AK distinguished by ANS probe are active. The activity of N2 is about 0.8 fold higher than N1 and shows higher susceptibility to proteolysis by trypsin. This means that the native state of AK might be an ensemble of kinetically attainable conformers and the energy landscapes of AK folding should be rugged with more than one local minimum.
A mouse bone marrow stromal cell line, TBR‐B, shows inducible expression of smooth muscle‐specific genes
Authors: Arakawa Emi , Hasegawa Kazuhide , Yanai Nobuaki , Obinata Masuo , Matsuda Yuzuru
Published: July 2002

We established an in vitro culture system which mimicked the differentiation pathway of smooth muscle cell, using TBR‐B, a bone marrow stromal cell line derived from transgenic mice harboring temperature‐sensitive SV40 large T‐antigen gene. TBR‐B cells have the potential to express smooth muscle‐specific genes including h1‐calponin, h‐caldesmon, SM22α and α‐actin, only after cultured in the differentiation medium for 2 weeks. The differentiation state of TBR‐B was well controlled by using different culture medium. Using this cell line, we also found that ascorbic acid is a potent factor inducing the expression of h1‐calponin and α‐actin. TBR‐B cells will serve as a useful tool for elucidating the regulatory mechanisms of smooth muscle‐specific gene expression, and for identifying compounds that regulate the differentiation state of vascular smooth muscle cells.
Electroporation‐mediated gene transfer in free‐swimming embryonic Xenopus laevis
Authors: Eide Fernette F , Eisenberg Staci R , Sanders Timothy A
Published: July 2002

Xenopus laevis are a rich resource for vertebrate embryology and cell biology. Transplantation and transgenesis have provided much information about the developmental mechanisms of embryogenesis and molecule function, however existing methods have faced limitations regarding either the precise localization of gene expression or flexibility in the timing of gene transfer. Here we have found that electroporation of tailbud (stage 29/30) embryos is a rapid and efficient method of combining cell‐specific expression with variation in temporal delivery. At the low voltages required for electroporation, embryos resumed normal swimming behavior and development. We conclude that electroporation has wide experimental application to Xenopus developmental and cell biology.
Genomic Exploration of the Hemiascomycetous Yeasts: 15. Pichia sorbitophila
Authors: de Montigny Jacky , Spehner Catherine , Souciet Jean-Luc , Tekaia Fredj , Dujon Bernard , Wincker Patrick , Artiguenave François , Potier Serge
Published: July 2002

This paper reports the genomic analysis of the strain CBS7064 of Pichia sorbitophila, a homothallic diploid yeast. We sequenced 4829 random sequence tags of about 1 kb and compared them to the Saccharomyces cerevisiae gene products. Approximately 1300 nuclear genes, 22 tRNAs, the rDNA locus, and six mitochondrial genes have been identified. The analysis of the rDNA genes has permitted to classify this organism close to the Candida species. Accession numbers from AL414896 to AL419724 at EMBL databank.
Consensus predictions of membrane protein topology
Authors: Nilsson Johan , Persson Bengt , von Heijne Gunnar
Published: July 2002

We have explored the possibility that consensus predictions of membrane protein topology might provide a means to estimate the reliability of a predicted topology. Using five current topology prediction methods and a test set of 60 Escherichia coli inner membrane proteins with experimentally determined topologies, we find that prediction performance varies strongly with the number of methods that agree, and that the topology of nearly half of all E. coli inner membrane proteins can be predicted with high reliability (>90% correct predictions) by a simple majority‐vote approach.
Endothelin‐1[1–31], acting as an ETA‐receptor selective agonist, stimulates proliferation of cultured rat zona glomerulosa cells
Authors: Mazzocchi Giuseppina , Rossi Gian Paolo , Malendowicz Ludwik K. , Champion Hunter C. , Nussdorfer Gastone G.
Published: July 2002

Endothelin‐1 (ET‐1)[1–31] is a novel hypertensive peptide that mimics many of the vascular effects of the classic 21 amino acid peptide ET‐1[1–21]. However, at variance with ET‐1[1–21] that enhances aldosterone secretion from cultured rat zona glomerulosa (ZG) cells by acting via ETB receptors, ET‐1[1–31] did not elicit such effect. Both ET‐1[1–21] and ET‐1[1–31] raised the proliferation rate of cultured ZG cells, the maximal effective concentration being 10−8 M. This effect was blocked by the ETA‐receptor antagonist BQ‐123 and unaffected by the ETB‐receptor antagonist BQ‐788. Quantitative autoradiography showed that ET‐1[1–21] displaced both [125I]PD‐151242 binding to ETA receptors and [125I]BQ‐3020 binding to ETB receptors in both rat ZG and adrenal medulla, while ET‐1[1–31] displaced only [125I]BQ‐3020 binding. The tyrosine kinase (TK) inhibitor tyrphostin‐23 and the p42/p44 mitogen‐activated protein kinase (MAPK) inhibitor PD‐98059 abolished the proliferogenic effect of ET‐1[1–31], while the protein kinase‐C (PKC) inhibitor calphostin‐C significantly reduced it. ET‐1[1–31] (10−8 M) stimulated TK and MAPK activity of dispersed ZG cells, an effect that was blocked by BQ‐123. The stimulatory action of ET‐1[1–31] on TK activity was annulled by tyrphostin‐23, while that on MAPK activity was reduced by calphostin‐C and abolished by either tyrphostin‐23 and PD‐98059. These data suggest that ET‐1[1–31] is a selective agonist of the ETA‐receptor subtype, and enhances proliferation of cultured rat ZG cells through the PKC‐ and TK‐dependent activation of p42/p44 MAPK cascade.
A potential role of chlorophylls b and c in assembly of light‐harvesting complexes
Authors: Hoober J.Kenneth , Eggink Laura L
Published: July 2002

Chlorophyll (Chl)‐containing light‐harvesting complexes (LHCs) in chloroplasts of plant and algal cells usually include an oxidized Chl (Chl b or c) in addition to Chl a. Oxidation of peripheral groups on the tetrapyrrole structure increases the Lewis acid strength of the central Mg atom. We propose that the resulting stronger coordination bonds between oxidized Chls and ligands in LHC apoproteins (LHCPs) stabilize the initial intermediates and thus promote assembly of LHCs within the chloroplast envelope.
Sub‐micellar phospholipid accelerates amyloid formation by apolipoprotein C‐II
Authors: Hatters Danny M. , Lawrence Lynne J. , Howlett Geoffrey J.
Published: July 2002

Lipid‐free human apolipoprotein C‐II (apoC‐II) forms amyloid fibrils with characteristic β‐structure. This conformation is distinct from the α‐helical fold of lipid‐bound apoC‐II. We have investigated the effect of the short‐chain phospholipid, dihexanoylphosphatidylcholine (DHPC) on amyloid formation by apoC‐II. The α‐helical content of apoC‐II increases in the presence of micellar DHPC (16 mM) and amyloid formation is inhibited. However, at sub‐micellar DHPC concentrations (below 8 mM) amyloid formation is accelerated 6 fold. These results suggest that individual phospholipid molecules in vivo may exert significant effects on amyloid folding pathways.
Hypochlorite induces the formation of LDL−, a potentially atherogenic low density lipoprotein subspecies
Authors: Fabjan Judith S. , Abuja Peter M. , Schaur R.Joerg , Sevanian Alex
Published: July 2002

Oxidation of low density lipoprotein (LDL) induced by hypochlorous acid (HOCl) leading to LDL−, a minimally oxidized subspecies of LDL, was investigated. LDL− is characterized by its greater electronegativity and oxidative status, and is found in plasma in vivo. Its concentration was found to be elevated under conditions that predispose humans to atherosclerosis. We found that HOCl also converts LDL rapidly to an even more oxidized state, identified as LDL2−, which is more electronegative than LDL−. After milder oxidation for short durations, formation of LDL− takes place while less LDL2− is formed. Under these conditions, addition of methionine not only suppressed further oxidation of LDL but also favored the formation of LDL− over LDL2−, possibly by removing chloramines at lysyl residues of LDL. The presence of lipoprotein‐deficient plasma did not prevent HOCl‐mediated conversion of LDL to more electronegative species. It is concluded that the HOCl‐mediated conversion of LDL into more electronegative species might be physiologically relevant.
In vitro selection of other proteins than antibodies by means of ribosome display
Authors: Lamla Thorsten , Erdmann Volker A
Published: July 2002

The in vitro selection and simultaneous evolution of proteins is feasible by means of ribosome display. Here, we describe the use of a protein bearing a binding property without being an antibody for affinity enrichment of the ternary complex, consisting of a protein, a ribosome and a encoding mRNA. The binding property was a simple affinity tag, namely Strep‐tag II and His‐tag. We could demonstrate that the selection of a specific mRNA encoding a shortened bovine heart fatty acid binding protein with a N‐terminal His‐tag was possible. After nine cycles of transcription, translation, affinity selection and reverse transcription PCR the protein with the His‐tag could be enriched 108‐fold.
Transcriptional regulation of apolipoprotein E expression by cyclic AMP
Authors: Andreani-Mangeney Marise , Vandenbrouck Yves , Janvier Brigitte , Girlich Delphine , Bereziat Gilbert
Published: July 2002

Incubation of HepG2 cells in the presence of dibutyryl cAMP (db‐cAMP), a cell permeable analogue of cyclic AMP, or forskolin, an agent which elevates intracellular cAMP, resulted in a 50% decrease in apoE mRNA levels within 24 h. Results of nuclear run‐on transcription assays showed that db‐cAMP down‐regulates apoE gene expression at the transcriptional level. By transfection analysis with a plasmid containing the human apoE gene fused to the secreted placental alkaline phosphatase (SPAP) reporter gene, we showed that the SPAP activity was decreased by 50% when HepG2 cells were incubated in the presence of db‐cAMP or forskolin, indicating that this promoter region mediated this negative effect. In contrast, when the smaller fragment of apoE promoter was linked to the CAT reporter gene, db‐cAMP treatment of HepG2 cells resulted in a 2‐fold increase in CAT activity, suggesting that positive cAMP‐responsive elements were present in the proximal apoE promoter. These data indicate that transcriptional modulation of apoE gene expression by agents known to elevate the intracellular cAMP level is complex and involves several negative and positive elements located in the −614 to +804 region of the apoE gene whose global effect is negative on apoE gene transcription.
A complex of the soluble interleukin‐6 receptor and interleukin‐6 is internalized via the signal transducer gp130
Authors: Graeve Lutz , Korolenko Tatjana A. , Hemmann Ulrike , Weiergräber Oliver , Dittrich Elke , Heinrich Peter C.
Published: July 2002

In human body fluids a soluble form of the interleukin‐6 receptor (sIL‐6R) has been found which together with interleukin‐6 (IL‐6) acts agonistically on cells expressing the signal transducer gp130. The means by which the sIL‐6R is removed from the circulation is unknown. Here, we show that a complex of 125I‐labelled recombinant sIL‐6R and IL‐6 is internalized by MDCK cells stably transfected with gp130 and by human hepatoma cells HepG2 that endogenously express the IL‐6R and gp130. We further show that most of the internalized sIL‐6R is degraded within lysosomes. Our studies suggest that cells expressing gp130 are capable of endocytosing an IL‐6/sIL6R complex, thereby removing both from the circulation.
Identification of Ser‐1275 and Ser‐1309 as autophosphorylation sites of the insulin receptor1
Authors: Al-Hasani Hadi , Eisermann Bernd , Tennagels Norbert , Magg Claudia , Paßlack Waltraud , Koenen Marlis , Müller-Wieland Dirk , Meyer Helmut E , Klein Helmut W
Published: July 2002

We have identified Ser‐1275 and Ser‐1309 as novel serine autophosphorylation sites by direct sequencing of HPLC‐purified tryptic phosphopeptides of the histidine‐tagged insulin receptor kinase IRKD‐HIS. The corresponding peptides (Ser‐1275, amino acids 1272–1292; Ser‐1309, amino acids 1305–1313) have been detected in the HPLC profiles of both the soluble kinase IRKD, which contains the entire cytoplasmic domain of the insulin receptor β‐subunit, and the insulin receptor purified from human placenta. In contrast, a kinase negative mutant, IRKD‐K1018A, did not undergo phosphorylation at either the tyrosine or serine residues, strongly suggesting that insulin receptor kinase has an intrinsic activity to autophosphorylate serine residues.
Effect of mevalonate availability on the association of G‐protein α‐subunits with the plasma membrane in GH4C1 cells
Authors: Chiloeches Antonio , Usera Fernado , Lasa Marina , Ropero Santiago , Montes Agustı́n , Toro Marı́a J
Published: July 2002

We show that the levels and activity of the α‐subunits of Gs and Gi proteins in plasma membrane of GH4C1 cells are regulated by the availability of mevalonate (MVA), and not by changes in cholesterol cell content. Changes in the levels of MVA, induced by modulation of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase, determine the amount of both membrane‐bound Gα‐subunits, which correlated with the activity of their effector adenylyl cyclase. Lipoprotein deficient serum (LPDS) decreases cholesterol content and increases both HMG‐CoA reductase activity and Gα‐subunits in the membrane. Cholesterol and 25‐hydroxycholesterol (25‐HC) each repress HMG‐CoA reductase and diminish Gα‐subunit levels. However, while cholesterol cell content is also decreased by 25‐HC, exogenous cholesterol increases it. In addition, the decrease of both Gα‐subunits is reversed by the presence of MVA. This regulation appears to be mediated by nonsterol products generated from MVA. We assume that the first is the prenylation of the γ‐subunits, since the attachment of Gα‐subunits to the membrane is dependent on this modification. However, as neither of our treatments completely abolished protein prenylation, we conclude that another MVA derivative is required in addition to prenyl residues to the presence and activity of α‐subunits in the membrane.
Differential splicing of the growth hormone‐releasing hormone gene in rat placenta generates a novel pre‐proGHRH mRNA that encodes a different C‐terminal flanking peptide1
Authors: Pérez-Riba Mercè , González-Crespo Sergio , Boronat Albert
Published: July 2002

We have isolated and characterized a novel rat placental pre‐proGHRH mRNA (pre‐proGHRH‐2 mRNA). This mRNA is generated by an alternative splicing process which results in the presence of an additional exon of 156 bp (designated exon 4.5) located between exons 4 and 5 of the previously reported hypothalamic and placental pre‐proGHRH mRNA (pre‐proGHRH‐1 mRNA). Since the sequences encoding mature GHRH are included within exons 3 and 4, the processing of pre‐proGHRH‐2 would not affect the synthesis of mature GHRH but would generate a C‐terminal peptide (designated GCTP‐2) different from that previously reported in the hypothalamus and placenta (GCTP‐1). The putative GCTP‐2 has 64 amino acids, and the first 18 N‐terminal residues are identical to those present in GCTP‐1 (30 amino acids long). Pre‐proGHRH‐2 mRNA has not been detected in the hypothalamus.
Myosin as cofactor and substrate in fibrinolysis
Authors: Machovich Raymund , Ajtai Katalin , Kolev Krasimir , Owen Whyte G.
Published: July 2002

Myosin accelerates plasminogen activation by tissue‐type plasminogen activator (tPA), and is degraded extensively by plasmin. Myosin binds both tPA and plasminogen, and enhances activation of des1‐77‐plasminogen by tPA but not by urokinase‐type plasminogen activator (uPA). Myosin decreases K
M and increases k
cat for des1‐77‐plasminogen activation by tPA, to yield catalytic efficiencies in excess of 8000 M−1 s−1. The effect of myosin is attributed to its C‐terminal portion, the myosin rod. With a K
M of 3 μM, myosin is a high‐affinity substrate for plasmin. The findings indicate that myosin is a cofactor for plasminogen activation and a substrate for plasmin.