bluuebunny/crossref_metadata_2025 huggingface dataset:
Endocrine cells share expression of N‐CAM with neurones
Authors: Langley O.K. , Aletsee M.C. , Gratzl M.
Published: July 2002

The expression of the neural cell adhesion molecule, N‐CAM, was examined in the anterior lobe of rat hypophysis by immunocytochemistry at light and electron microscope levels. In addition, N‐CAM antigenic determinants present in adrenal medulla, anterior hypophysis and PC12 cells were compared by immunoblotting with those found in cerebellum. All secretory cells in the anterior hypophysis were found to be N‐CAM positive on their surfaces, but not all of the three polypeptide determinants typical of cerebellum were present in the endocrine tissues or cell line tested. In addition, a new N‐CAM determinant of 49 kDa not present in cerebellum was found in adrenal medulla and hypophysis, although it was absent from PC12 cells. The possible implications of these data are discussed.
RNA polymerase molecules initiating transcription at tandem promoters can collide and cause premature transcription termination
Authors: Ponnambalam Sreenivasan , Busby Stephen
Published: July 2002

Using purified E. coli RNA polymerase we have studied the transcription in vitro of a series of DNA fragments carrying two tandemly arranged promoters, where the corresponding transcription start points were separated by 263, 138, 83 and 78 base pairs. In the case where the transcription start points are 83 base pairs apart, there is an interaction between RNA polymerase molecules transcribing from the two promoters. This interaction results in premature termination of the upstream transcript at a precise site. We propose that this is the result of RNA polymerase transcribing from the upstream promoter bumping into polymerase at the downstream promoter. The interaction between the two polymerase molecules is crucially dependent on the distance between the two promoters.
Phorbol esters inhibit inositol phosphate and diacylglycerol formation in proliferating HL60 cells Relationship to differentiation
Authors: Geny Blandine , LePeuch Christian , Cost Helene , Basset Michele , Cockcroft Shamshad
Published: July 2002

Phorbol 12‐myristate 13‐acetate (PMA) induces the differentiation of the human promyelocytic cell line, HL60, towards adherent macrophage‐like cells within 2 days. We have examined the early effects of PMA on inositol phosphates and on diacylglycerol production, two second messengers derived from inositol lipids. In proliferating HL60 cells, PMA induced a time‐ and concentration‐dependent decrease in inositol phosphate levels. Maximal effects were seen after 1 h at 10 nM PMA. PMA also induced the translocation of protein kinase C from the cytosol to the membrane. Comparison between the differentiating effects of several phorbol esters and of 1‐oleoyl‐2‐acetylglycerol with their ability to inhibit inositol phosphate formation suggests that the two effects are correlated.
Sphingomyelinases in human, bovine and porcine seminal plasma
Authors: Vanha-Perttula Tapani
Published: July 2002

The seminal plasma of man, boar and bull was found to have a sphingomyelinase (SMase) activity hydrolysing [N‐methyl‐14C]sphingomyelin. The human and porcine enzymes had an acid pH optimum and were not influenced by divalent metal ions or chelating agents. They were closely similar with the lysosomal enzyme in many tissues. The bovine seminal plasma SMase was partially purified. The enzyme was a glycoprotein with pH optimum at 6.5, a broad pI 4.2–4.8 and molecular mass of 160 and 60 kDa, respectively, in native and SDS‐PAGE. The enzyme was activated by Co > Mn > Cd > Ni and inhibited by chelating agents, Cu, Fe, Pb and Zn. The enzyme was clearly distinct from the acid lysosomal SMase and the previously described neutral Mg2+‐dependent and independent activities. It had a wide distribution in the bull reproductive tissues.
Phase II of carotenoid bandshift is mainly due to the electrogenic protonation of the secondary quinone acceptor
Authors: Drachev Lel A. , Mamedov Mahir D. , Mulkidjanian Armen Ya. , Semenov Alexey Yu. , Shinkarev Vladimir P. , Verkhovsky Mikhail I.
Published: July 2002

The flash‐induced kinetics of transmembrane electric potential (Δψ) generation with a rise‐time of 0.1–0.2 ms were investigated in Rhodobacter sphaeroides chromatophores by electrometry and by monitoring the electrochromic absorbance changes of carotenoids. An analysis of the results obtained electrometrically shows that this electrogenic phase is observed only after even‐numbered flashes. The sensitivity of this phase to o‐phenanthroline, the flash‐number dependence of its amplitude and the decrease of its rise‐time with decreasing pH indicate that it is due to the dismutation of Q−
A and Q−
B and to subsequent protonation of a doubly reduced ubiquinone QB molecule. The phase of the carotenoid bandshift with τ∼0.1 ms (the so‐called phase II) was shown to be sensitive to o‐phenanthroline, with its rise‐time decreasing with decreasing pH. It is concluded that a considerable part of phase II of the electrochromic carotenoid changes with τ∼0.1 ms is caused by the electrogenic reaction Q−
AQ−
B+2H+ → QAQBH2, but not solely by the oxidation of cytochrome c
2, as proposed earlier [(1973) Biochim. Biophys. Acta 325, 102–113].
Polyamines inhibit the ATP‐dependent proteolytic pathway in rabbit reticulocyte lysates
Authors: Wajnberg Ewa F. , Fagan Julie M.
Published: July 2002

Reticulocytes contain a soluble nonlysosomal proteolytic pathway that requires ATP and ubiquitin. Polyamines at physiological concentrations were found to inhibit rapidly the ATP‐dependent proteolytic system in reticulocyte lysates; spermidine and putrescine inhibited this process by 26–72% and spermine by 71–96%. Spermine had little effect on the ATP‐independent breakdown of oxidant‐treated hemoglobin. By fractionating the ATP‐dependent system, we show that polyamines inhibit the ATP‐dependent degradation of ubiquitin‐protein conjugates.
ATP stimulates secretion in human neutrophils and HL60 cells via a pertussis toxin‐sensitive guanine nucleotide‐binding protein coupled to phospholipase C
Authors: Cockcroft Shamshad , Stutchfield Jane
Published: July 2002

Human neutrophils and HL60 cells respond to extracellular ATP by causing exocytotic secretion. Secretion is accompanied by increases in inositol phosphates and a rise in cytosol Ca2+. The responses to ATP are blocked by pertussis toxin pretreatment, indicating the involvement of a guanine nucleotide regulatory protein. Other nucleotides that are active in promoting secretion are ATPγS, UTP, ITP and AppNHp, whilst 8‐bromo‐ATP, AppCH2p, ADP, AMP and adenosine are inactive.
Carbachol‐induced reverse transformation of Chinese hamster ovary cells transfected with and expressing the m5 muscarinic acetylcholine receptor
Authors: Felder Christian C. , Ma Alice L. , Conklin Bruce R.
Published: July 2002

Reverse transformation was induced in Chinese hamster ovary (CHO) cells transfected with and stably expressing the m5 subtype of the muscarinic acetylcholine receptor when stimulated with the muscarinic agonist, carbachol. Atropine, a muscarinic antagonist, blocked the carbachol‐stimulated reverse transformation. CHO cells not transfected with the muscarinic receptor did not change with added carbachol. PMA induced reverse transformation without increasing cAMP accumulation in CHO cells. Carbachol, prostaglandin E2, and cholecystokinin increased cAMP accumulation but only carbachol caused reverse transformation. Carbachol‐stimulated cAMP accumulation occurred at a higher concentration (EC50 10 μM) than did carbachol‐stimulated reverse transformation (EC50 63 nM). Muscarinic m5 acetylcholine receptor transfected into CHO cells can induce reverse transformation which may be independent of cAMP.
Nucleotide sequence of the aliphatic amidase regulator gene (amiR) of Pseudomonas aeruginosa
Authors: Lowe N. , Rice P.M. , Drew R.E.
Published: July 2002

The nucleotide sequence of a 1001 bp ClaI/XhoI DNA fragment encoding the amidase regulator gene (amiR) from Pseudomonas aeruginosa has been determined. The sequence derives from strain PAC433, a constitutive high expressing amidase mutant, and contains two overlapping open reading frames. Analysis of the sequence has identified one of the reading frames as amiR. The gene encodes a 196 amino acid polypeptide which shows a strong bias towards codons with G or C in the third position. The amiR gene shows no sequence homology with other bacterial regulator proteins.
Binding of murine monoclonal antibodies to the active and inactive configurations of aequorin
Authors: Watanabe Michiko , Kurihara Satoshi , Inouye Satoshi , Ohno Tsuneya
Published: July 2002

Murine monoclonal IgG1 antibodies (MAb), designated Aq‐11 and Aq‐12, were prepared against the photoprotein aequorin from jelly fish. Aequorin is a calcium‐sensitive photoprotein which consists of a single polypeptide chain, apoaequorin, and a functional chromophore, coelenterazine. Native aequorin consists of two species with molecular masses of 25 and 23.5 kDa. MAb Aq‐12 was found by immunoblot analysis to bind specifically to the 25 kDa species, while MAb Aq‐11 reacted with the 23.5 kDa protein. Activation of apoaequorin with coelenterazine was associated with a shift of the 23.5 kDa molecule to the 25 kDa species. In contrast, treatment with calcium ions induced a shift back to the 23.5 kDa form. These changes between the active and inactive forms were identified by reactivity with MAbs Aq‐11 and Aq‐12. The results thus indicate that these MAbs should be useful in monitoring activation of this photoprotein.
Degradation of quinaldine by Alcaligenes sp. and by Arthrobacter sp.
Authors: Dembek Gunnar , Rommel Theobald , Lingens Franz , Höke Hartmut
Published: July 2002

Two bacterial strains, which grew aerobically in mineral salt medium with quinaldine as sole carbon source, were isolated. These strains could be identified as Alcaligenes sp. and Arthrobacter sp. Alcaligenes sp. was adapted to higher concentrations of quinaldine in a continuous culture. Both strains accumulated one compound, identified as quinisatin, which decomposed chemically to anthranilic acid and isatin. Quinisatin spontaneously resulted from 3,4‐dihydroxy‐2‐oxo‐1,2‐di‐hydroquinoline.
Lectin‐like proteins accumulate as fragmentation products in bean seed protein bodies
Authors: Ceriotti Aldo , Vitale Alessandro , Bollini Roberto
Published: July 2002

The gene sequence of the Phaseolus vulgaris L. seed lectin‐like protein (LLP) and its precursor (∼ 40 kDa) which is associated with the endoplasmic reticulum has been described, while the molecular characteristics of the mature protein and its cellular localization are still unknown. Our data, obtained using antibodies raised against a fusion protein, which was produced in Escherichia coli and contained most of the LLP sequence, indicate that mature LLP accumulates in the protein bodies of cotyledon cells. LLP consists of several polypeptides in the M
r range 15 000 to 20 000, which are a result of proteolytic fragmentation of the protein precursor.
ENDOR study on the position of hydrogens close to the manganese cluster in S2 state of photosystem II
Authors: Kawamori Asako , Inui Takashi , Ono Takaaki , Inoue Yorinao
Published: July 2002

Proton matrix ENDOR of the manganese multiline in the S2, state of photosystem II membranes from spinach has been investigated. The spectral structure over a range of frequencies of ± 2 MHz centered on the position for a free proton was analyzed through employing a simulation method based on the dipolar interaction between electron and proton magnetic moments. The 6 pairs of lines resolved were attributed to the surrounding proton populations at distances varying within the range 2.7–6.0 Å from the putative manganese center. Two of the 6 pairs, namely those corresponding to protons at distances of 2.7 and 3.2 Å from the manganese center were eliminated on washing of the membranes with deuterated buffer. This suggests that these protons belong to the water molecules coordinating to the manganese cluster. The presence of ethylene glycol instead of glycerol and sucrose in the buffer broadened both EPR and ENDOR spectra. This suggests that ethylene glycol molecules are also accessible to the two‐manganese cluster. A possible model of water association to the manganese ions in the photosynthetic water‐oxidation system is proposed.
Crystallization and preliminary X‐ray crystallographic study of the quinoprotein methanol dehydrogenase from bacterium W3A1
Authors: Xia Zong-Xiang , Hao Zhi-Ping , Mathews F.Scott , Davidson Victor L.
Published: July 2002

Methanol dehydrogenase from bacterium W3A1 has been crystallized by the macroseeding method to give single crystals suitable for three‐dimensional structural study at resolution greater than 3 Å. The crystals belong to the space group P21 and have unit cell dimensions a = 124.13 Å, b = 62.87 Å, c = 84.71 Å, and β = 92.89°. There is one dimeric molecule of 114600 Da per asymmetric unit.
Cyclic nucleotides and GTP analogues stimulate light‐induced phosphorylation of octopus rhodopsin
Authors: Tsuda Motoyuki , Tsuda Tomoko , Hirata Hajime
Published: July 2002

Rhodopsin; Rhodopsin kinase; Kinase, A‐; Protein, G‐; Photoreceptor; (Octopus, Invertebrate)
Cromakalim, a vasodilator, differentially inhibits Ca2+ currents in NG108‐15 neuroblastoma × glioma hybrid cells
Authors: Ito Yuji , Miyamori Isamu , Matsubara Takao , Takeda Ryoyu , Higashida Haruhiro
Published: July 2002

Extracellular perfusion with the antihypertensive agent cromakalim produced an inhibition of 22–66% in the low‐threshold transient Ca2+ (T‐like) current in NG108‐15 hybrid cells. Cromakalim suppressed the high‐threshold and long‐lasting Ba2+ current (L‐like Ca2+ current) by 29–73%, but had almost no effect on the high‐threshold and inactivating Ba2+ current (N‐like Ca2+ current). IC50 for T‐like and L‐like currents was the same at about 100 μM. The inhibitory effect developed relatively fast and was reversible. These results indicate that cromakalim can selectively inhibit the activity of inward Ca2+ currents.
Vasopressin gene expression is stimulated by cyclic AMP in homologous and heterologous expression systems
Authors: Verbeeck Marleen A.E. , Adan Roger A.H. , Burbach J.Peter H.
Published: July 2002

The possible role of cyclic AMP (cAMP) in the regulation of the vasopressin (VP) gene was tested in two cellular expression systems: one cell line with endogenous VP expression and the other which was transiently with a VP promoter‐luciferase fusion gene. 8, Bromo‐cAMP stimulated the VP mRNA content about 4‐fold in the human VP‐expressing small cell lung carcinoma cell line GLC‐8. The luciferase activity in P19 embryonal carcinoma cells which were transiently transfected with − 174 to +44 of the 5'‐flanking region of the human VP gene linked to the firefly luciferase gene, was stimulated about 2‐fold by the cAMP analogue. The results indicate that cAMP plays a role in the upregulation of the VP gene and hence point to several putative nucleotide motives in the promoter functionally conferring this response.
The importance of the C‐terminal amide structure of rat pancreastatin to inhibit pancreatic exocrine secretion
Authors: Miyasaka Kyoko , Funakoshi Akihiro , Kitani Kenichi , Tamamura Hirokazu , Fujii Nobutaka , Funakoshi Susumu
Published: July 2002

A C‐terminal fragment of rat pancreastatin, a 26 residue peptide amide and a fragment without a C‐terminal amide were synthesized by Fmoc‐based solid phase methods and their biological activities were compared. The rat C‐terminal fragment inhibited pancreatic exocrine secretions produced by the intravenous injection of 2‐deoxy‐D‐glucose (a central vagal nerve stimulation), whereas the fragment without a C‐terminal amide showed no effect on pancreas. These results indicate that the C‐terminal amide of this peptide is necessary to reveal its biological activity.
An inhibitor of protein synthesis initiation from Alhagi kirgisorum S
Authors: Smailov Salim K. , Mukhamedzhanov Bektas G. , Lee Anatoly V. , Iskakov Bulat K. , Denisenko Oleg N.
Published: July 2002

Polyproanthocyanidin ‐ a plant phenolic compound from Alhagi kirgisorum S. effectively inhibited protein synthesis in rabbit reticulocyte and wheat germ cell‐free systems. Poly‐proanthocyanidin inhibited translation only at the level of initiation and not at the clongation level and aminoacylation of tRNA. The inhibitory effect of the phenolic compound is due to the blockage of the ternary complex formation of cIF‐2 with GTP and initiator Met‐tRNA.
Reactions of Fe(II)‐ATP and Fe(II)‐citrate complexes with t‐butyl hydroperoxide and cumyl hydroperoxide
Authors: Rush J.D. , Koppenol W.H.
Published: July 2002

Rate constants for the reactions of cumyl hydroperoxide and t‐butyl hydroperoxide with ferrous complexes of ATP and citrate were measured at pH 7.4. These ligands are potential chelators of iron(II) in the low‐molecular weight iron pool that may catalyze oxidative degradation of biomolecules. The second‐order rate constants for the reduction of cumyl hydroperoxide and t‐butyl hydroperoxide by ferrous ATP are 3.1 × 103 and 1.3 × 103 M−1·s−1, respectively, at 25°C and 0.11 M ionic strength. Rates of reduction by ferrous citrate are similar. Activation enthalpies for these reactions average 10 kcal/mol.
Analysis of the protein products encoded by variant glucokinase transcripts via expression in bacteria
Authors: Quaade Christian , Hughes Steven D. , Coats Ward S. , Sestak Andrea L. , Iynedjian Patrick B. , Newgard Christopher B.
Published: July 2002

Five variant transcripts of the single rat glucokinase gene have been described that are naturally expressed in islets of Langerhans, liver and anterior pituitary. Four of these were prepared as cDNA and expressed in bacteria in order to begin to address their physiological roles. Expression of constructs pGKB1 (normal islet/pituitary glucokinase) and pGKL1 (normal liver glucokinase) resulted in glucose‐dependent, glucokinase‐like activity, 7‐fold and 45‐fold, respectively, above background. Expression of pGKB3 (variant islet/pituitary glucokinase) and pGKL2 (variant liver glucokinase) in contrast, did not result in any glucokinase‐like activity.
The inhibition of restriction endonucleases due to Z‐DNA in negatively supercoiled plasmid
Authors: Lesnik E.A. , Bes'chetnikova Zh.A. , Maslova R.N. , Varschavsky Ja.M.
Published: July 2002

Plasmid pGC20 containing the (dGC)??? insert in Sma1 recognition site has been used to study the inhibition of cleavage by different restriction endonucleases due to Z‐DNA formation in (dCG)b10 sequence of the negatively supercoiled plasmid. Data obtained indicate the different sensitivity of restriction endonucleases to DNA conformational perturbations resulted from the Z‐DNA formation. Therefore, the inhibition of DNA cleavage by a particular restriction endonucleases cannot serve as a criterion for the estimation of the length of B—Z junctions in circular supercoiled DNAs
Conformation of endothelin in aqueous ethylene glycol determined by 1H‐NMR and molecular dynamics simulations
Authors: Krystek Stanley R.
Published: July 2002

The solution conformation of a 21‐residue vasoconstrictor peptide endothelin‐1 (ET‐1) in water‐ethylene glycol has been determined by two‐dimensional 1H‐NMR spectroscopy and constrained molecular dynamics simulations. The N‐terminus (residues 1–4) appears to undergo conformational averaging and no single structure consistent with the NMR constraints could be found for this region. Residues 5–8 form a turn, and residues 9–16 exist in a helical conformation. A flexible ‘hinge’ between residues 8–9 allows various orientations of the turn relative to the helix. Another ‘hinge’ at residue 17 connects the extended C‐terminus to the bicyclic core region (residues 1–15). Residues important for binding and biological activity form a contiguous surface on one side of the helix, with the two disulfides extending from the other side of the helix.
The preparation of biotinyl‐ε‐aminocaproylated forms of the vasoactive intestinal polypeptide (VIP) as probes for the VIP receptor
Authors: Andersson Mats , Marie Jean-Claude , Carlquist Mats , Mutt Viktor
Published: July 2002

Vasoactive intestinal polypeptide (VIP) was biotinyl‐ε‐aminocaproylated using sulfosuccinimidyl‐β‐(biotinamido) hexanoate thereby producing a series of products that were separated by high performance liquid chromatography (HPLC). Seven VIP‐derivatives were isolated and the number and location of biotinyl‐ε‐aminocaproylation was determined by a combination of enzymatic degradation and plasma desorption mass spectrometry (PDMS). Receptor binding experiments with the VIP biotinyl‐ε‐aminocaproylated derivatives revealed IC10 values for the monobiotinyl‐ε‐aminocaproylated peptides that were 1.3–3.2 times higher than for natural VIP. All isolated biotinyl‐ε‐aminocaproylated derivatives possess VIP‐like bioactivity as shown by an assay measuring pancreatic juice secretion in cat, VIP biotinyl‐ε‐aminocaproylated in position lysine15 being almost equipotent with natural VIP.
After microinjection hemimethylated DNA is converted into symmetrically methylated DNA before DNA replication
Authors: Sandberg G. , Guhl E. , Graessmann M. , Graessmann A.
Published: July 2002

In this investigation we analysed the maintenance methylation activity of the mammalian cell DNA methyltransferase by microinjection of hemimethylated HSV‐tk DNA into thymidine kinase‐negative rat 2 cells. We found that the hemimethylated DNA was efficiently converted into symmetrical methylated molecules before DNA replication. Furthermore, integration of the trans‐DNA into the host genome is an early event after gene transfer.
Protein export in prokaryotes and eukaryotes Theme with variations
Authors: Wiech Hans , Klappa Peter , Zimmerman Richard
Published: July 2002

Protein export in prokaryotes as well as in eukaryotes can be defined as protein transport across the plasma membrane. In both types of organisms there are various apparently ATP‐dependent transport mechanisms which can be distinguished from one another and which show similarities when the prokaryotic mechanism is compared with the respective eukaryotic mechanism. First, one can distinguish between transport mechanisms which involve so‐called signal or leader peptides and those which do not. The latter mechanisms seem to employ ATP‐dependent transport systems which belong to the family of oligopeptide permeases and multiple drug resistance proteins. Second, in signal or leader peptide‐dependent transport one can distinguish between transport mechanisms which involve ribonucleoparticles and those which employ molecular chaperones. Both mechanisms appear to converge at the level of ATP‐dependent translocases.
Signal transduction mechanism of interleukin 6 in cultured rat mesangial cells
Authors: Fukunaga Megumu , Fujiwara Yoshihiro , Fujibayashi Mari , Ochi Satoshi , Yokoyama Kenji , Ando Akio , Hirano Toshio , Ueda Naohiko , Kamada Takenobu
Published: July 2002

Interleukin 6 (IL‐6) is one of the potent autocrine growth factors for mesangial cells. We investigated the signal transduction mechanism or IL‐6 in cultured rat mesangial cells. IL‐6 induced a transient increase of inositol 1,4,5‐trisphosphate (Ins 1,4,5‐P3) followed by a transient and sustained increase of intracellular calcium concentration, suggesting that IL‐6 stimulates phosphoinositide turnover. IL‐6 also stimulated prostaglandin E2 (PGE2) production. The IL‐6‐concentration dependency in PGE2 production was similar to that in Ins 1,4,5‐P3 production. We concluded that the action of IL‐6 on mesangial cells is exerted at least partially through the enhancement of phosphoinositide turnover and PGE2 production.
Detection of small GTP‐binding proteins in the outer envelope membrane of pea chloroplasts
Authors: Sasaki Yukiko , Sekiguchi Kazumasa , Nagano Yukio , Matsuno Ryuichi
Published: July 2002

We found small GTP‐binding proteins in the outer envelope membrane of pea chloroplasts. The proteins in this membrane were separated by SDS‐PAGE, transferred to a nitrocellulose filter, and incubated with [α‐23P]GTP. Three GTP‐binding proteins with the molecular weight of 24 000 were found. Binding was prevented by 10−8—10−7 M GTP or by 10−7 M guanosine 5′‐[γ‐thio]triphosphate or GDP; binding was unaffected by 10−8—10−6 M ATP. Thermolysin treatment of intact chloroplasts resulted in the loss of GTP‐binding activity, suggesting that these proteins were in the cytosolic side of the outer envelope membrane.
Characterization of the RDC1 gene which encodes the canine omolog of a proposed human VIP receptor Expression does not correlate with an increase in VIP binding sites
Authors: Cook Jonathan S. , Wolsing Dana Hance , Lameh Jelveh , Olson Christy A. , Correa Paul E. , Sadee Wolfgang , Blumenthal Edward M. , Rosenbaum Jan S.
Published: July 2002

We have isolated a portion of the canine gene encoding the orphan receptor RDC1 [1]. The complete coding sequence is contained in a single exon, and an intron divides the 5′ untranslated region of RDC1 mRNA. The RDC1 protein is 94% homologous to the gene product of GPRN1, which has been proposed to serve as a VIP receptor when expressed in CHO‐K1 and COS‐7 cells (Sreedharan, S.P. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 4986–4990). Northern analysis indicates that CHO‐K1 cells endogenously express a 2.1 kb RDC1 mRNA. However, while CHO‐K1 cells possess detectable low affinity [125I]VIP binding sites, VIP binding is not altered in membranes of CHO‐K1 cells expressing varying amounts of the RDC1 gene construct. Further, endogenous VIP binding is not increased by transient expression of RDC1 in COS‐7 cells. Taken together, the data suggest that RDC1 is not a canine homolog of the proposed VIP receptor.
Molecular analysis of a new cytoplasmic male sterile genotype in sunflower
Authors: Spassova Mariana , Christov Michail , Bohorova Natasha , Petrov Peter , Dudov Kalin , Atanassov Atanas , Nijkamp H.John J. , Hille Jaques
Published: July 2002

Mitochondrial DNA from 1 fertile and 6 cytoplasmic male sterile (CMS) sunflower genotypes was studied. The CMS genotypes had been obtained either by specific crosses between different Helianthus species or by mutagenesis. CMS‐associated restriction fragment length polymorphisms (RFLPs) were found in the vicinity of the atpA locus, generated by various restriction enzymes. The organization of the mitochondrial genes 26S rRNA, 18S+5S rRNA and coxII was investigated by Southern blot analysis. These genes have similar structures in fertile and all studied sterile sources. Using the atpA probe, 5 from the 6 investigated CMS genotypes showed identical hybridization patterns to the Petiolaris CMS line, which is used in all commercial sunflower hybrids. Only 1 cytoplasm derived from an open pollination of Helianthus annuus ssp. texanus, known as ANT1, contained a unique mithochondrial DNA fragment, which is distinguishable from the fertile and sterile Petiolaris genotypes and from all investigated CMS genotypes. Male fertility restoration and male sterility maintenance of the ANT1 line are different from the Petiolaris CMS system, which is a confirmation that a novel CMS genotype in sunflower has been identified.
Effects of solubilization and vanadate/glutathione complex on inhibitor potencies against eosinophil cyclic AMP‐specific phosphodiesterase
Authors: Souness John E. , Maslen Christopher , Scott Lisa C.
Published: July 2002

Treatment of membranes from guinea‐pig peritoneal eosinophils with deoxycholate and NaCl solubilized >95% of the particulate cyclic AMP‐specific phosphodiesterase (PDE IV). Solubilized PDE IV was at least 10 times more potently inhibited by selective PDE IV inhibitors (e.g. rolipram, denbufylline) than bound enzyme. Vanadate/glutathione complex (V/GSH) activated membrane‐bound PDE IV and also increased potencies of these same inhibitors by at least 10‐fold. Neither solubilization nor V/GSH markedly influenced the inhibitory activities of non‐selective inhibitors (e.g. trequinsin, dipyridamole). Inhibitor effects on solubilized PDE IV and cyclic AMP accumulation in intact cells were strongly correlated. These results suggest a biologically important site on eosinophil PDE IV which is concealed or partially concealed in freshly prepared membranes and is exposed by solubilization or V/GSH.
Sequence analysis of pigeon δ‐crystallin gene and its deduced primary structure Comparison of avian δ‐crystallins with and without endogenous argininosuccinate lyase activity
Authors: Lin Chou-Wen , Chiou Shyh-Horng
Published: July 2002

δ‐Crystallin is a major lens protein present in the avian and reptilian lenses. To facilitate the cloning of the δ‐crystallin gene, cDNA was constructed from the poly(A)+ RNA of pigeon lenses, amplified by the polymerase chain reaction (PCR). The PCR product was then subcloned into pUC19 vector and transformed into E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by the dideoxynucleotide chain‐termination method. Sequencing two clones, containing 1.4 kb DNA inserts coding for δ‐crystallin allowed the construction of a complete, full‐length reading frame of 1,417 bp covering a deduced protein sequence of 466 amino acids, including the universal translation‐initiating methionine. The pigeon δ‐crystallin shows 88, 83 and 69% sequence identity to duck δ2, chicken δ1 crystallins and human argininosuccinate lyase respectively. It is also shown that, in contrast to duck δ2 crystallin which has a high argininosuccinate lyase activity, pigeon δ‐crystallin appears to contain very low activity of this enzyme, despite the fact that they share a highly homologous structure. A structural comparison of δ‐crystallins with or without enzymatic activity suggested several amino acid replacements which may account for the loss of argininosuccinate lyase activity in the lenses of certain avian species.
Purification of the complex of cathepsin L and the MHC class II‐associated invariant chain fragment from human kidney
Authors: Ogrinc Tadeja , Dolenc Iztok , Ritonja Anka , Turk Vito
Published: July 2002

The complex of cathepsin L and the fragment of the MHC class II‐associated invariant chain was purified from human kidney. M
r, of the complex, as determined by gel filtration, is about 40,000. Both components were identified by amino acid and sequence analyses. The bound invariant chain fragment is almost identical to the additional segment found in p41, but not in the p31 form of the invariant chain. The complex has significantly enhanced stability at neutral and slightly alkaline pH, and reduced proteolytic activity against the synthetic substrate Z‐Phe‐Arg‐MCA compared to free cathepsin L. The complex exhibits no enzymatic activity against the protein substrate azocasein. For the first time, the invariant chain was found in a complex with a protein, which was not an MHC molecule.
Confirmation and an unusual quality of the differentiated keratin peptide (K1) in cultured human squamous cell carcinomas
Authors: Katagata Yohtaro , Yoshida Tadashi
Published: July 2002

This finding may have important implications regarding evaluation of keratin extracted from normal epidermal and cultured keratinocytes.
The electronic structure of P840+• The primary donor of the Chlorobium limicola f. sp. thiosulphatophilum photosynthetic reaction centre
Authors: Rigby Stephen E.J. , Thapar Rajive , Evans Michael C.W. , Heathcote Peter
Published: July 2002

The radical cation P840+• was studied in frozen suspensions of Chlorobium limicola f. sp. thiosulphatophilum membranes using ENDOR and Special TRIPLE spectroscopies. The spectra show that P840+• arises from a bacteriochlorophyll a ‘special’ pair with a highly symmetrical distribution of electron spin density between the constituent bacteriochlorophylls. Special TRIPLE spectroscopy has resolved the separate contributions of the two halves of the pair and revealed small deviations from a 1:1 electron spin density distribution. Nevertheless P840+• appears to come the closest yet to the symmetrical ‘dimer’ originally proposed for the structure of the primary donor radical cation (P870+•) in purple non‐sulphur photosynthetic bacteria.
Nystatin changes the properties of transporters for arginine and sugars An in vitro study
Authors: Opekarová Miroslava , Tanner Widmar
Published: July 2002

In ergosterol‐containing energized yeast plasma membrane vesicles nystatin (5–10 μg/mg total lipid) caused a massive efflux of pre‐accumulated arginine while the membrane potential (the principal driving force; −110 mV) decreased by only 10–30 mV. Neither the substrate fluxes nor the membrane potential was influenced by nystatin when the permease was reconstituted in ergosterol‐free phospholipid vesicles. The same effect of nystatin was found with the reconstituted sugar transporter front Chlorella kessleri. It is suggested that nystatin binding to ergosterol in the vicinity of the permease releases the transport protein from its coupling to energy and converts it to a facilitator.
The mammary factor MPBF is a prolactin‐induced transcriptional regulator which binds to STAT factor recognition sites
Authors: Burdon Thomas G. , Demmer Jerome , Clark A.John , Watson Christine J.
Published: July 2002

Site‐directed mutagenesis of the three binding sites for the mammary factor MPBF in the β‐lactoglobulin (BLG) promoter demonstrates that MPBF is a transcriptional activator of the BLG gene in mammary cells. MPBF requires phosphorylation on tyrosine for maximum binding activity and binds to GAS (interferon γ‐activation site) elements which are similar to the MPBF binding sites. Prolactin induces MPBF binding activity in CHO cells and is not antigenically related to Stat1 (p91) and Stat2 (p113), suggesting that this transcription factor is likely to be another member of the STAT family of cytokine/growth factor‐induced transcription factors.
Iron—sulfur cluster‐containing l‐serine dehydratase from Peptostreptococcus asaccharolyticus: Correlation of the cluster type with enzymatic activity
Authors: Hofmeister A.E.M. , Albracht S.P.J. , Buckel W.
Published: July 2002

Investigations were performed with regard to the function of the iron—sulfur cluster of l‐serine dehydratase from Peptostreptococcus
asaccharolyticus, an enzyme which is novel in the class of deaminating hydro‐lyases in that it lacks pyridoxal‐5′‐phosphate. Anaerobically purified l‐serine dehydratase from P. asaccharolyticus revealed EPR spectra characteristic of a [3Fe–4S]+ cluster constituting 1% of the total enzyme concentration. Upon incubation of the enzyme under air the intensity of the [3Fe–4S]+ signal increased correlating with the loss of enzymatic activity. Addition of l‐serine prevented this. Hence, active l‐serine dehydratase probably contains a diamagnetic [4Fe–4S]2+ cluster which is converted by oxidation and loss of one iron ion to a paramagnetic [3Fe–4S]+ cluster, resulting in inactivation of the enzyme. In analogy to the mechanism elucidated for aconitase, it is proposed that l‐serine is coordinated via its hydroxyl and carboxyl groups to the labile iron atom of the [4Fe–4S]2+ cluster.
Does the solid‐state structure of endothelin‐1 provide insights concerning the solution‐state conformational equilibrium?
Authors: Lee Gregory M. , Chen Chinpan , Marschner Thomas M. , Andersen Niels H.
Published: July 2002

Additional NMR data (local NOE ratios and chemical shifts) for endothelin‐1 supporting the existence of a relatively regular helix initiated abruptly at Lys9 (with Asp8 as an N‐cap) and extending in all cases to Cys15 (and in a frayed form to Asp18 in some analogs) is presented. The recent solid‐state structure [Janes et al. (1994), Nature Struct. Biol. 1, 311–319], in contrast, places the helix in the extreme C‐terminal section of the structure and the Lys9—Tyr13 segment is not helical. The X‐ray structure does not predict the NOEs or chemical shifts observed for endothelins in aqueous media containing polar organic co‐solvents. An analysis of the chemical shift data for reporter groups indicates that the helical conformational preference of endothelins is not significantly altered by the addition of acetonitrile, acetic acid, or ethylene glycol. The validity of the analytic strategy is supported by results for both more rigid and less helical analogs. We conclude that the structure observed in crystals obtained from purely aqueous media is influenced by intermolecular interactions in the solid state and is not a significant contributor to the conformational equilibrium observed for monomeric ET‐1.
The kinetics of conformational changes of α2‐macroglobulin determined by time resolved X‐ray solution scattering
Authors: Arakawa Hideo , Urisaka Takuji , Tsuruta Hirotsugu , Amemiya Yoshiyuki , Kihara Hiroshi , Ikaia Atsushi
Published: July 2002

The rate of gross conformational change of α2‐macroglobulin (α2M) during its proteinase trapping was directly determined for the first time using time‐resolved X‐ray solution scattering. Decrease of radius of gyration was observed under pseudo‐first‐order conditions with excess proteinases, which exhibited a monophasic timecourse. The rate constants were 0.5 ± 0.1 s−1 and 0.8 ± 0.2 s−1 for the reaction with chymotrypsin and trypsin, respectively. There was no concentration dependence of the observed rate constants. Therefore, the rate‐limiting step of the gross conformational change was not the bimolecular encounter reaction between α2M and proteinases, which requires a new proposal of pre‐trapping of proteinases before the gross conformational change.
Sam68 from an immortalised B‐cell line associates with a subset of SH3 domains
Authors: Finan Peter M. , Hall Alison , Kellie Stuart
Published: July 2002

The binding of proteins from an immortalised B‐cell line to a panel of SH3 domains was investigated in vitro. One of the most prominent SH3 domain binding proteins was a 68 kD polypeptide which strongly associated with the SH3 domains of c‐src, p85α and p47phox and weakly with the SH3 domain of PLCγ and n‐src with undetectable binding to the other SH3 domains tested. Immunoblotting identified this protein as human Sam68. The ability of proline‐rich peptides homologous to the Sam68 primary sequence to inhibit the binding of Sam68 to SH3 domains was investigated. Only one peptide inhibited binding of Sam68 to the p85a SH3 domain, whereas several peptides inhibited binding of Sam68 to c‐src SH3 domain, suggesting that Sam68 uses different proline‐rich motifs to bind to different SH3 domains. A peptide derived from residues 32–44 of Sam68 which fits the class H SH3 domain binding consensus sequence inhibited binding of Sam68 to both p85a SH3 domain and c‐src SH3 domain, but with differential potency, suggesting a differential affinity of these SH3 domains for this proline‐rich motif.
Successful management of congenital protein C deficiency with recurrent pregnancy loss
Authors: Ogasawara M. , Sasa H. , Kajiura S. , Aoki K. , Yagami Y.
Published: July 2002

A 29‐year‐old woman experienced unexplained intrauterine fetal death (IUFD) three times, at 16, 27 and 27 gestational weeks. She was found to be protein C‐deficient and the existence of infarction in the placenta was suspected at 20 weeks' gestation during her fourth pregnancy. Both heparin and antithrombin III were administered until delivery of a small‐for‐dates but live baby. Massive infarction in the placenta was evident at term. Anticoagulation with heparin is a useful treatment approach for cases of recurrent IUFD with protein C deficiency.
Allo‐non‐specific elevation of maternal killer cell activity in intrauterine growth restriction
Authors: Fujii T. , Tsushima R. , Okai T. , Shibata Y. , Taketani Y.
Published: July 2002

Objective: In order to gain insight into the pathogenesis of intrauterine growth restriction (IUGR) of the fetus, we evaluated the cytotoxic activity of lymphocytes towards fetal cells in pregnancies complicated by IUGR. Methods: We performed a cytotoxic assay using the maternal lymphocyte‐derived lymphokine activated killer (LAK) cells as the effector cells. Cord vessel endothelial cells were used as the target cells. Results: LAK cells in pregnancies complicated by IUGR demonstrated higher killer cell activities against IUGR fetus‐derived endothelial cells compared with LAK cells from uncomplicated pregnancies against the same endothelial cells. However, when non‐IUGR fetus‐derived endothelial cells were used as the target cells, LAK cells of pregnant women with IUGR exhibited essentially the same activities as those of normal pregnant women. Conclusion: The results suggest that lymphocytes in pregnancies complicated by IUGR may have enhanced killer cell activities towards the IUGR fetus in an allo‐non‐specific way.
Reproductive outcome after metroplasty
Authors: Zorlu C.G. , Yalçin H. , Uḡur M. , Özden S. , Kara‐Soysal S. , Gökmen O.
Published: July 2002

Objective: To evaluate the impact of abdominal metroplasty for symmetric uterine anomalies on reproductive performance. Method: A retrospective analysis of 24 patients who conceived following metroplasty for symmetric congenital anomalies during a 5‐year period. Results: In patients with poor obstetric histories before surgery, the overall fetal survival rate increased from 2.5%. before treatment to 73.3% after treatment and 90.5% of women gave birth to a surviving infant. The remaining three patients suffering from infertility delivered a full‐term infant after metroplasty. Antenatal complications occurred in 56.7% of the pregnancies. Conclusion: Abdominal metroplasty clearly improves fetal survival particularly in women suffering from a history of poor obstetric outcome associated with symmetric malformations.
Interferon αβ‐induced abnormalities in adipocytes of suckling mice
Authors: Sbarbati Andrea , Leclercq Françoise , Osculati Francesco , Gresser Ion
Published: July 2002

Summary— The modification induced by interferon (IFN) in brown adipose tissue (BAT) was studied by high spatial resolution magnetic resonance imaging (MRI), histology, and transmission electron microscopy (TEM). In IFN‐treated mice, at MRI, the interscapular BAT was slightly enlarged and showed non‐homogeneous areas of lipid accumulation. The thickness of the subcutaneous white adipose tissue was reduced with respect to control mice. In the liver, MRI showed a lipid accumulation. In IFN‐treated mice, by light microscopy, brown adipocytes showed a larger lipid deposit with respect to control mice. At TEM, in BAT, the mitochondria were reduced in number, smaller and the number of cristae was also significantly reduced with respect to the controls (9.1 ± 1.5 vs 20.1 ± 1.9, P < 0.01). The inclusions in the mitochondrial matrix were significantly less numerous in IFN‐treated than in control animals (1.9 ± 0.7 vs 0.9 ± 0.7 for mitochondrial section, P < 0.01). Abnormalities of endoplasmic reticulum described in hepatocytes were not found in brown adipocytes of IFN‐treated mice. The present work demonstrates that, in the BAT of sucking mice, IFN‐treatment induces morphologic alterations and that brown adipocytes have MRI and TEM features resembling those found in the lipid laden BAT of aged animals.
CD44v6 cell surface expression is a common feature of macrophages and macrophage‐like cells – implication for a natural macrophage extravasation mechanism mimicked by tumor cells
Authors: Braumüller Heidi , Gansauge Susanne , Ramadani Marco , Gansauge Frank
Published: July 2002

Soluble CD44standard (sCD44s) and CD44v6 (sCD44v6) cannot only be detected in sera of patients with pancreatic carcinoma but also of healthy blood donors. To investigate whether sCD44s and sCD44v6 are derived from white blood cells, we stimulated whole blood with phytohemagglutinin and interleukin‐2, which induced expression of CD44v6 only on monocytes. For further investigations, we used the promyelocytic leukemia cell line Hl‐60. Only Hl‐60 cells differentiating along the macrophage pathway showed increased expression of CD44s and CD44v6. Furthermore, only macrophages showed increased secretion of sCD44s and sCD44v6. Our data suggest that CD44s and CD44v6 are common adhesion molecules on macrophages and macrophage‐like cells.
The influence of aspartate 26 on the tautomeric forms of folate bound to Lactobacillus casei dihydrofolate reductase
Authors: Birdsall Berry , Casarotto Marco G , Cheung H.T.Andrew , Basran Jaswir , Roberts Gordon C.K , Feeney James
Published: July 2002

The ternary complex of Lactobacillus casei dihydrofolate reductase (DHFR) with folate and NADP+ exists as a mixture of three interconverting forms (I, IIa and IIb) whose relative populations are pH dependent, with an effective pK of approx. 6. To investigate the role of Asp26 in this pH dependence we have measured the 13C chemical shifts of [2,4a,7,9‐13C4]folate in its complex with the mutant DHFR Asp26→Asn and NADP+. Only a single form of the complex is detected and this has the characteristics of form I, an enol form with its N1 unprotonated. A study of the pH dependence of the 13C chemical shifts of DHFR selectively labelled with [4‐13C]aspartic acid in its complex with folate and NADP+ indicates that no Asp residue has a pK value greater than 5.4. Two of the Asp CO−
2 signals appear as non‐integral signals with chemical shifts typical of non‐ionised COOH groups and with a pH dependence characteristic of the slow exchange equilibria previously characterised for signals in forms I and IIb (or IIa). It is proposed that the protonation/deprotonation controlling the equilibria involves the O4 position of the folate and that Asp26 influences this indirectly by binding in its CO−
2 form to the protonated N1 group of folate in forms I and IIa thus reducing the pK involving protonation at the O4 position to approx. 6. These findings indicate that, in forms I and IIa of the ternary complex, folate binds to DHFR in a very similar way to methotrexate.
Detection of secretory phospholipase A2s related but not identical to type IIA isozyme in cultured mast cells
Authors: Murakami Makoto , Tada Kinji , Shimbara Satoko , Kambe Terumi , Sawada Hisashi , Kudo Ichiro
Published: July 2002

We previously reported that BALB/cJ mouse‐derived bone marrow‐derived mast cells (BMMC) exhibited two sequential phases of prostaglandin D2 (PGD2) generation in response to Fcε receptor I (FcεRI) crosslinking and cytokine stimulation, the late phase of which was suppressed by an antibody raised against type IIA secretory phospholipase A2 (sPLA2). Here we report that BMMC derived from C57BL/6J mice, which are genetically deficient in type IIA sPLA2, display both immediate and delayed PGD2 generation normally. Lysates of C57BL/6J‐derived BMMC contained a Ca2+‐dependent PLA2 that was absorbed to a column conjugated with anti‐type IIA sPLA2 antibody and had a similar molecular mass of 14 kDa, as assessed by immunoblotting. Therefore we speculate that a sPLA2 similar to, but distinct from, type IIA sPLA2 would compensate for type IIA sPLA2 deficiency in C57BL/6J‐derived BMMC. We found that the two type IIA‐related sPLA2 family members, type V and type IIC sPLA2s, were expressed in BMMC as well as in rat mastocytoma RBL‐2H3 cells.
Promoter structure of the MxA gene that confers resistance to influenza virus
Authors: Nakade Kohji , Handa Hiroshi , Nagata Kyosuke
Published: July 2002

The human MxA protein is one of the interferon‐inducible proteins that inhibits multiplication of influenza virus and other viruses. To clarify the control mechanism of its expression, we prepared a series of mutant MxA promoters and identified a 30 nucleotides long cis‐acting interferon‐responsive element by transient transfection assay. Its nucleotide sequence is somewhat similar to that of ISRE (interferon‐stimulated response element), suggesting that the regulation of MxA mRNA synthesis is under the control of some ISRE binding factor such as ISGF‐3 (interferon‐stimulated gene factor‐3).