bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stimulation of Ca2+ uptake into epididymal bull spermatozoa by analogues of amiloride
Authors: BREITBART Haim , CRAGOE Edward J. , LARDY Henry A. 
Published: March 2005

Certain amiloride analogues 3′,4′‐dichlorobenzamil 2′,4′‐dimethylbenzamil and α′,2′‐benzobenzamil hydrochloride (ATBB) stimulate calcium accumulation and motility by epididymal bovine spermatozoa. This stimulation can be seen at a range of 0.1–0.4 mM, while at higher concentration there is inhibition of calcium uptake by these amiloride analogues. The amiloride derivative 5‐(4‐chlorobenzyl)‐2′,4′‐dimethylbenzamil (CBDMB), which bears a 4‐chlorobenzyl substituent on the 5‐amino nitrogen atom, did not stimulate calcium uptake. The amiloride analogue 3′,4′‐dichlorobenzamil inhibits the Na+/Ca2+ ‐exchange activity in isolated plasma membrane vesicles, and the stimulatory effect of 3′,4′‐dichlorobenzamil on calcium uptake into epididymal sperm could be seen in Na+ ‐free medium. Thus, the stimulation of Ca2+ accumulation in the cells caused by 3′,4′‐dichlorobenzamil is not a result of inhibiting the Na−‐dependent Ca2‐ clearance. There is no stimulation of Ca2+ uptake into ejaculated cells by adding 3′,4′‐dichlorobenzamil, which is not due to the presence of the calcium‐transport inhibitor (caltrin) in these cells [Rufo, G. A., Schoff, P. K. & Lardy, H. A. (1984) J. Biol. Chem. 259, 2547–2552].The stimulatory effect of 3′,4′‐dichlorobenzamil on Ca2+ uptake is inhibited by the voltage‐dependent Ca2+‐channel blockers nifedipin and diltiazem. This indicates that the stimulation of Ca2+ uptake by the amiloride analogues is due to the activation of a voltage‐dependent Ca2+ channel of the plasma membrane.
The complete primary structure of two distinct forms of human α(IX) collagen chains
Authors: MURAGAKI Yasuteru , KIMURA Tomoatsu , NINOMIYA Yoshifumi , OLSEN Bjorn Reino 
Published: March 2005

Type IX collagen molecules contain three genetically distinct subunits. One of the subunits, α2(IX), contains a covalently attached glycosaminoglycan side chain. A second subunit, α1(IX), has been found to be synthesized in two forms. The two forms are generated by the alternative use of two transcription start sites and splice patterns. The two forms have been found in chicken, mouse and human but cDNAs encoding both forms have only been reported for chicken.In the present report we describe the isolation of cDNA clones encoding the complete translated portion of both forms of human α1(IX) collagen chains. Nucleotide sequence analysis has permitted the determination of the primary structure of both forms. These probes and sequences should prove useful in future studies of chondrodysplasias involving type IX collagen.
Construction and characterization of a recombinant murine monoclonal antibody directed against human fibrin fragment‐D dimer
Authors: VANDAMME Anne‐Mieke , BULENS Frank , BERNAR Hilde , NELLES Luc , LIJNEN Roger H. , COLLEN Désiré 
Published: March 2005

cDNA libraries in λ phage were generated from the murine hybridoma secreting mAb‐15C5, a monoclonal antibody directed against fragment‐D dimer of crosslinked human fibrin [Holvoet et al. (1989) Thromb. Haemo‐stasis 61, 307–313], and clones encoding fragments of the heavy (γ1) and the light (k) chain were isolated. The κ‐chain cDNA was reconstructed from two overlapping clones encoding 20 amino acids of signal sequence and the 214 amino acids of the mature protein chain. The γ1‐chain cDNA was reconstructed from the mAb‐15C5 κ‐chain signal sequence, the mAb‐15C5 γ1 variable‐domain coding sequence and murine γ1‐gene and γ1‐chain cDNA fragments encoding the constant domains. These cDNAs were expressed in Chinese hamster ovary cells, selected cell lines were scaled up in roller bottle culture, and recombinant mAb‐15C5 was purified from the conditoned medium by chromatography on Zn‐chelate–Sepharose, protein‐A–Sepharose and insolubilized fragment‐D dimer, with a yield of 50 μg/1 and a recovery of 20%. SDS‐gel electrophoresis without reduction revealed a homogeneous band, and after reduction a light‐chain band with identical and a heavy‐chain band with a somewhat slower mobility than that of the natural mAb‐15C5. Competitive binding revealed a comparable affinity of natural and recombinant mAb‐15C5, for fibrin fragment‐D dimer.Thus recombinant mAb‐15C5, obtained by co‐expression of the reconstructed cDNAs of the k and γ1 chain in Chinese hamster ovary cells, has very similar properties to natural mAb‐15C5. These recombinant mAb‐15C5 cDNAs may be useful for the construction of a humanized monoclonal antibody for thrombus imaging, and for targeting of thrombolytic agents to fibrin.
In vivo13C‐NMR studies on the metabolism of the lugworm Arenicola marina
Authors: JURETSCHKE Hans‐Paul , KAMP Günter 
Published: March 2005

13C‐NMR natural‐abundance spectra of specimens of Arenicola marina obtained, showed seasonal changes in the concentration of some metabolites, with the osmolite alanine as well as triacylglyceride storage compounds present at high concentrations. Glycogen was sometimes only barely detectable due to the low natural abundance level of 13C.Glycogenic metabolism of the lugworm A. marina was studied in vivo by 13C‐NMR spectroscopy using 13C‐labelled glucose. During recovery from a hypoxic period [1‐13C]glucose was incorporated into glycogen. [1‐13C]Glucose was injected 5 h after the end of hypoxia to guarantee sufficient and reliable 13C labelling of glycogen. An earlier injection of [1‐13C]glucose led to considerably diminished incorporation of 13C‐labelled glucosyl units into glycogen, probably due to the consumption of the available glucose as fuel for ATP production. No scrambling of 13C into the C6 position of glycogen was observed, indicating a lack of gluconeogenic activity. 13C was also incorporated into the C3 positions of alanine and alanopine. To assign correctly this last 13C‐NMR resonance, the compound was synthesized biochemically.No labelling of glycogen was observed when [3‐13C]alanine was injected into the coelomic cavity with similar incubation conditions being used.The 13C of [1‐13C]glucose, incorporated into glycogen, showed a very low turnover rate in normoxic lugworms as shown by two 13C{1H}‐NMR spectra, one obtained 48 h after the other. On the other hand, in hypoxia lugworms the signal due to 13C‐labelled glycogen decreased very rapidly proving a high turnover rate. The disappearance of 13C from glycogen during the first 24 h of hypoxia indicates that the last glycosyl units to be synthesized are the first to be utilized.Lugworms were quite sensitive to the 1H‐decoupling field used for obtaining the 13C{1H}‐NMR spectra, especially at 11.7 T. Using bi‐level composite‐pulse decoupling and long relaxation delays, no tissue damage or stress‐dependent phosphagen mobilization, as judged by 31P‐NMR spectroscopy, was observed.
Comparison of three classes of human liver alcohol dehydrogenase
Authors: EKLUND Hans , MÜLLER‐WILLE Per , HORJALES Eduardo , FUTER Olga , HOLMQUIST Barton , VALLEE Bert L. , HÖÖG Jan‐Olov , KAISER Rudolf , JÖRNVALL Hans 
Published: March 2005

Conformational models of the three characterized classes of mammalian liver alcohol dehydrogenase were constructed using computer graphics based on the known three‐dimensional structure of the E subunit of the horse enzyme (class I) and the primary structures of the three human enzyme classes. This correlates the substrate‐binding pockets of the class I subunits (α, β and γ in the human enzyme) with those of the class II and III subunits (π and χ, respectively) for three enzymes that differ in substrate specificity, inhibition pattern and many other properties. The substrate‐binding sites exhibit pronounced differences in both shape and properties. Comparing human class I subunits with those of class II and III subunits there are no less than 8 and 10 replacements, respectively, out of 11 residues in the substrate pocket, while in the human class I isozyme variants, only 1–3 of these 11 positions differ. A single residue, Va1294, is conserved throughout. The liver alcohol dehydrogenases, with different substrate‐specificity pockets, resemble the patterns of other enzyme families such as the pancreatic serine proteases.The inner part of the substrate cleft in the class II and III enzymes is smaller than in the horse class I enzyme, because both Ser48 and Phe93 are replaced by larger residues, Thr and Tyr, respectively. In class II, the residues in the substrate pocket are larger in about half of the positions. It is rich in aromatic residues, four Phe and one Tyr, making the substrate site distinctly smaller than in the class I subunits. In class III, the inner part of the substrate cleft is narrow but the outer part considerably wider and more polar than in the class I and II enzymes. In addition, Ser (or Thr) and Tyr in class II and III instead of His51 may influence proton abstraction/donation at the active site.
Distribution of the early light‐inducible protein in the thylakoids of developing pea chloroplasts
Authors: CRONSHAGEN Udo , HERZFELD Frank 
Published: March 2005

The distribution of the early light‐inducible protein (ELIP) of pea (Pisum sativum) between grana and stroma thylakoids was studied. An antibody raised against a bacterial‐expressed fusion protein containing ELIP sequences was used.Illumination of dark‐grown pea seedlings causes an accumulation of the ELIP in the thylakoid membranes with a maximum level at 16 h. During continuous illumination exceeding 16 h the level decreases again. The fractionation of thylakoid membranes of 48‐h‐illuminated pea seedlings in grana and stroma thylakoids reveals that there is no uniform distribution of ELIP in the thylakoids. Rather 60–70% of ELIP was found in the stroma thylakoids and 30–40% in the grana thylakoids. This distribution is in accordance with that of photosystem I but not with that of photosystem II. After Triton‐X‐100 solubilization almost all ELIP is found in the photosystem‐I‐containing fraction. This also supports an association of ELIP with photosystem I.
Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles
Authors: JAKUBOWSKI Josef , JAKOB Andrea 
Published: March 2005

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/ H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase‐mediated transport was determined from initial velocities of vanadate‐sensitive uptake of 22Na+, the Na+‐dependence of Na+/H+ exchange from initial velocities of amiloride‐sensitive uptake.By studying vanadate‐sensitive Na+ transport, high‐affinity binding sites for ATP with an apparent Km(ATP) of 15 ± 1 μM were observed at low concentrations of Na+ (1 mM) and K+ (1 mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 ± 25 μM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate‐sensitive transport by 3.8‐fold and 1.9‐fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 ± 7 μM and 76 ± 10 μM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6 – 1.7‐fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone.The velocity of amiloride‐sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6‐fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13‐fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride‐sensitive Na+ transport.The results show that hormone receptor‐mediated effects on (Na+,K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.
Myosin heavy chain isoform composition in striated muscle after denervation and self‐reinnervation
Authors: JAKUBIEC‐PUKA Anna , KORDOWSKA Jolanta , CATANI Claudia , CARRARO Ugo 
Published: March 2005

The total content of myosin heavy chains (MHC) and their isoform pattern were studied by biochemical methods in the slow‐twitch (soleus) and fast‐twitch (extensor digitorum longus) muscles of adult rat during atrophy after denervation and recovery after self‐reinnervation. The pattern of fibre types, in terms of ultrastructure, was studied in parallel.After denervation, total MHC content decreased sooner in the slow‐twitch muscle than in the fast‐twitch. The ratio of MHC‐1 and the MHC‐2B isoforms to the MHC‐2A isoform decreased in the slow and the fast denervated muscles, respectively. After reinnervation of the slow muscle, the normal pattern of MHC recovered within 10 days and the type 1 isoform increased above the normal. In the reinnervated fast muscle, the 2B/2A isoform ratio continued to decrease. Traces of the embryonic MHC isoform, identified by immunochemistry, were found in both denervated and reinnervated slow and fast muscles. A shift in fibre types was similar to that found in the MHC isoforms. Within 2 months of recovery a tendency to normalization was observed.The results show that (a) MHC‐2B isoform and the morphological characteristics of the 2B‐type muscle fibres are susceptible to lack of innervation, similar to those of type 1, (b) during muscle recovery induced by reinnervation the MHC isoforms and muscle fibres shift transiently to type 1 in the soleus and to type 2A in the extensor digitorum longus muscles, and (c) the embryonic isoform of MHC may appear in the adult skeletal muscles if innervation is disturbed.
Characterization of a novel calcium‐binding 90‐kDa glycoprotein (BM‐90) shared by basement membranes and serum
Authors: KLUGE Matthias , MANN Karlheinz , DZIADEK Marie , TIMPL Rupert 
Published: March 2005

The protein BM‐90 was solubilized from the mouse Engelbreth‐Holm‐Swarm tumor with neutral buffers in molar yields lower (15–30%) than found for other basement membrane proteins (e.g. laminin, BM‐40). The purified protein was shown to be rich in cysteine (5 mol%) and to change in SDS electrophoresis from an 84‐kDa position to a 95‐kDa one upon reduction. BM‐90 was also shown to be a calcium‐binding protein. The N‐terminal sequence of BM‐90, as well as those of several internal peptides, showed no identity with any known protein sequences, indicating that it is a new protein. Specific radioimmunoassays showed no or only minor cross‐reactions with other known basement membrane proteins.Immunological assays demonstrated BM‐90 to be present in neutral salt extracts from mouse heart and kidney, in serum (20–40 μ/ml) and in the medium of various cultured cells (0.1–1 μ/ml). The protein in these samples was identical in size to BM‐90 purified from the tumor, indicating that negligible degradation occurs during purification. An extracellular matrix localization of BM‐90 was shown by immunofluorescence for Reichert's membrane, lens capsules and other basement membranes. Thus, BM‐90 appears to be a novel basement membrane protein whose functions remain to be studied.
Drosophila contains three genes that encode distinct isoforms of protein phosphatase 1
Authors: DOMBRÁDI Viktor , AXTON J. Myles , BREWIS Neil D. , DA CRUZ E SILVA Edgar F. , ALPHEY Luke , COHEN Patricia T. W. 
Published: March 2005

The sequences of two Drosophila and one rabbit protein phosphatase (PP) 1 catalytic subunits were determined from their cDNA. The sequence of Drosophila PP1α1 was deduced from a 2.2‐kb cDNA purified from an embryonic cDNA library, while that for Drosophila PP1β was obtained from overlapping clones isolated from both a head cDNA library and an eye imaginal disc cDNA library. The gene for Drosophila PP1α1 is at 96A2–5 on chrõmosome 3 and encodes a protein of 327 amino acids with a calculated molecular mass of 37.3 kDa. The gene for Drosophila PP1β is localized at 9C1–2 on the X chromosome and encodes a protein of 330 amino acids with a predicted molecular mass of 37.8 kDa. PP1α1 shows 96% amino acid sequence identity to PP1α2 (302 amino acids), an isoform whose gene is located in the 87B6–12 region of chromsome 3 [Dombrádi, V., Axton, J. M., Glover, D. M. Cohen, P. T. W. (1989) Eur. J. Biochem. 183, 603–610]. PP1β shows 85% identity to PP1α1 and PP1α2 over the 302 homologous amino acids. These results demonstrate that at least three genes are present in Drosophila that encode different isoforms of PP1. Drosophila PP1α1 and PP1β show 89% amino acid sequence identity to rabbit PP1α (330 amino acids) [Cohen, P. T. W. (1988) FEBS Lett. 232, 17–23] and PP1β (327 amino acids), respectively, demonstrating that the structures of both isoforms are among the most conserved proteins known throughout the evolution of the animal kingdom. The presence of characteristic structural differences between PP1α and PP1β, which have been preserved from insects to mammals, implies that the α and β isoforms may have distinct biological functions.
Effector‐dependent conformational changes in protein kinase Cγ through epitope mapping with inhibitory monoclonal antibodies
Authors: CAZAUBON Sylvie , WEBSTER Catherine , CAMOIN Luc , STROSBERG A. Donny , PARKER Peter 
Published: March 2005

Three monoclonal antibodies (mAb) directed against the regulatory domain of the protein kinase Cγ (PKCγ); 15G4, 5A2 and 36G9, were shown to display distinct properties with respect to PKCγ kinase activity [Cazaubon, S., Marais, R., Parker, P. & Strosberg, A.D. (1989) Eur. J. Biochem. 182, 401–406]. The mAb 5A2 and 36G9, which act as potent inhibitors of the cofactor‐dependent kinase activity, can no longer bind PKCγ in the presence of phosphatidylserine and phosphatidylserine/phorbol ester, respectively; 15G4 binding is not influenced by effectors. Due to this functional relationship between the inhibitory mAb‐ and cofactor‐binding sites, we sought to localize the mAb epitopes with respect to the functional sites of PKCγ. For this purpose, several deletions were introduced at the 5′ end of the PKCγ cDNA and the mutant proteins were expressed in Escherichia coli. The determination of the immunoreactivity of the deleted PKCγ proteins shows that the amino acid residues essential to the binding of 5A2 and 36G9 are directly adjacent to the second cysteine‐rich motif: these are contained in the sequences at positions 151–163 and 164–197, respectively. In addition, various deletions around the C1 region of the regulatory domain allowed the identification of the second cysteine‐rich motif as a functional binding site for phorbol dibutyrate. These deletion studies thus demonstrate that the epitopes recognized by the inhibitory mAbs 5A2 and 36G9 are distinct from the cofactor‐binding sites. This suggests that the binding of phosphatidylserine and phorbol ester induce conformational changes in the regulatory domain of PKC, which are thus responsible for the loss of the 5A2 and 36G9 immunoreactivity of the native protein. In this conformational state, PKCγ conserves its ability to interact with the non‐inhibitory mAb 15G4. By using synthetic peptides, the 15G4 epitope was localized to the sequence 297–310 in the V3 variable region. This indicates that the flexibility of the V3 region, which delimits the C‐terminus of the regulatory domain, may not be necessary for the allosteric activation of PKC.In view of these results, we propose that PKC activation by its cofactors results in intramolecular changes which allow the enzyme to bind exogenous substrates.
Metabolism of [2‐13C]succinate in renal cells determined by 13C NMR
Authors: JANS Arnold W. H. , WILLEM Rudolph 
Published: March 2005

[2‐13C]Succinate has been used to examine the metabolic carbon flux from the Krebs cycle in rat renal proximal convoluted tubular (PCT) cells under physiological and pathophysiological conditions. Therefore, we developed a mathematical model that enabled us to determine the metabolic fluxes of the Krebs cycle. A mathematical model for the calculation of flux from [2‐13C]succinate was used to determine fluxes in rat PCT cells during chronic acidosis in the presence and absence of 0.1 mM angiotensin II. The relative carbon efflux via glutamate dehydrogenase in rat renal PCT cells increases during chronic acidosis from 0.27 to 0.39, whereas this carbon flux is not affected by the presence of peptide hormone angiotensin II in the incubation medium.The fraction of intermediate 13C‐labelled oxaloacetate transformed into the phosphoenolpyruvate and aspartate pools increases significantly from 0.41 to 0.57 in the case of chronic acidosis. The carbon efflux is not affected by angiotensin II. The 13C‐NMR data also show that the carbon efflux through phosphoenolpyruvate carboxykinase increases from 0.35 to 0.56 in rat renal PCT cells derived from chronic acidotic animals, as well as in the presence of angiotensin II. The present results indicate that angiotensin II affects only the flux through phosphoenolcarboxykinase, whereas chronic acidosis increases the flux through phosphoenolpyruvate carboxykinase as well as the gluconeogenic flux.