bluuebunny/crossref_metadata_2025 huggingface dataset:
Coagulation factor Va is an actin filament binding and cross-linking protein
Authors: Furmaniak-Kazmierczak Emilia , Nesheim Michael E. , Côté Graham P.
Published: December 2009

Bovine coagulation cofactor factor Va is shown to bind to filaments of skeletal muscle actin with a dissociation constant of 40–50 nM in the presence of 50 mM NaCl. At saturation, approximately one molecule of factor Va was bound for every two actin molecules. The binding of factor Va to F-actin was inhibited by increasing ionic strength, being approximately 20-fold weaker at 150 mM NaCl. Addition of factor Va dramatically increased both the low-speed sedimentation and the low-shear viscosity of actin filament solutions, indicating that factor Va cross-links actin filaments. Factor Va also bound to actin filaments saturated with myosin. The isolated 74-kilodalton light chain of factor Va displayed actin binding and cross-linking properties indistinguishable from those of intact factor Va. The procofactor factor V bound weakly to F-actin, indicating that proteolytic activation is required to uncover the actin binding sites within the light chain domain. Actin filaments had only a slight inhibitory effect on the prothombinase activity of the factor Va – factor Xa – phospholipid complex. Since high concentrations of actin filaments can be exposed to the circulation when cells are damaged, the interaction of factor Va with actin may be of physiological relevance.Key words: blood coagulation, factor V, actin.
Two basic motifs of reovirus σ3 protein are involved in double-stranded RNA binding
Authors: Mabrouk T. , Danis C. , Lemay G.
Published: December 2009

It has been reported that the σ3 protein of reovirus can exert an inhibitory effect on the cellular double-stranded RNA (dsRNA) activated protein kinase. Activation of this kinase is thought to be a general mechanism mediating a cellular antiviral response. This enzyme can also be activated upon transfection, resulting in translational inhibition of plasmid-encoded mRNAs. σ3 has an affinity for dsRNA postulated to be responsible for antikinase activity. In the present study, site-directed mutagenesis was performed on two basic regions previously suggested as dsRNA-binding motifs and the mutant σ3 proteins were then expressed in COS cells. These experiments revealed that both motifs are involved in σ3 attachment to RNA. Expression of the mutants lacking RNA-binding capability is stimulated by coexpression of another dsRNA-binding protein, the E3L vaccinia virus protein. These results support a model in which the attachment to dsRNA is directly responsible for the trans-stimulating effect of σ3 on expression of cotransfected genes.Key words: reovirus, PKR, protein synthesis, RNA binding.
Characteristics of exogenous lipid uptake by renal and intestinal brush-border membrane vesicles
Authors: Bellemare François , Noël Josette , Malo Christiane
Published: December 2009

The transfer of radioactive phosphatidylcholine (PC*) from liposomes to rabbit jejunal and renal brush-border membrane vesicles (BBMVs) was measured with a fast-sampling, rapid-filtration apparatus. PC* uptake by jejunal and renal BBMVs was favoured when liposomes were made from soybean phosphatidylcholine (azolectin, AZO), whereas PC* uptake could not be quantitatively assessed from egg yolk phosphatidylcholine (PC) liposomes even after a 22-h period of incubation. The increased turbidity of BBMV dispersion following the addition of CaCl2or HCl to AZO-treated BBMVs suggested that negatively charged lipids and phosphatidylethanolamine are transferred during the process. These data and the analysis of PC*-uptake time measurements, using an algorithm simulating aggregation phenomena, indicated that the reaction mechanism involved liposome aggregation to BBMVs rather than specific lipid transfer. The constants of the dimerization reaction between AZO liposomes and BBMVs were evaluated to be 0.016 ± 0.006 min−1for jejunal and 0.095 ± 0.02 min−1for renal preparations. Intravesicular D-Aspartic acid accumulation in the presence of a Na+gradient indicated that vesicles were still closed after coincubation with liposomes. In contrast, 70–85% of rabbit jejunal and renal Na+-D-glucose cotransporter activities were lost after overnight incubation with either AZO liposomes or buffered solution. Further, H+–ATPase activity in rabbit renal BBMVs largely decreased after coincubation with AZO liposomes, while brush-border membrane associated enzymes remained stable. These results demonstrate that coincubation of BBMV with liposomes of different composition may represent a useful approach to study the influence of lipidic environment on various membrane protein functions.Key words: liposomes, brush-border membrane vesicles, transport functions, H+–ATPase, membrane fusion, phosphatidylcholine.
Vasopressin activates phospholipase D through pertussis toxin-insensitive GTP-binding protein in aortic smooth muscle cells: function of Ca2+/calmodulin
Authors: Miwa Masaichi , Suzuki Atsushi , Watanabe Yasuko , Shinoda Junji , Oiso Yutaka , Kozawa Osamu
Published: December 2009

In the present study, we examined the effect of vasopressin (AVP) on phosphatidylcholine-hydrolyzing phospholipase D activity in primary cultured rat aortic smooth muscle cells. AVP stimulation of choline formation was dose dependent. The time-course was quite different from those of inositol phosphates. The effect of AVP on the formation of inositol phosphates (EC50was 3 nM) was more potent than that on the formation of choline (EC50was 30 nM). 12-O-Tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC), stimulated the formation of choline. However, 4α-phorbol 12,13-didecanoate, which is inactive for PKC, had little effect. Staurosporine, an inhibitor of protein kinases, which inhibited the TPA-induced formation of choline, had little effect on the AVP-induced formation of choline. Neither calphostin C, a highly specific PKC inhibitor, nor PKC down-regulation with TPA affected AVP-induced formation of choline. A combination of AVP and TPA additively stimulated the formation of choline. The depletion of extracellular Ca2+by (ethylenebis(oxyethylenenitrilo))tetraacetic acid significantly reduced the AVP-induced formation of choline. W-7, an antagonist of calmodulin, inhibited the AVP-induced formation of choline in a dose-dependent manner. NaF, an activator for GTP-binding protein (G-protein), stimulated the formation of choline. However, the formation of choline by a combination of AVP and NaF was not additive. Pertussis toxin had little effect on the AVP-induced formation of choline. These results strongly suggest that AVP stimulates phospholipase D in a Ca2+/calmodulin-dependent manner in aortic smooth muscle cells, that a pertussis-toxin-insensitive G-protein is involved in the AVP-induced phospholipase D activation, and furthermore, that PKC is not essential for the activation.Key words: vasopressin, phospholipase D, protein kinase C, calmodulin, GTP-binding protein, aortic smooth muscle cells.
Calmodulin-dependent protein kinase II is required for G1/S progression in HeLa cells
Authors: Rasmussen Grace , Rasmussen Colin
Published: December 2009

Calmodulin (CaM) has been previously shown to be essential for cell cycle progression in eukaryotic cells, being required at the G1/S,G2/M, and metaphase–anaphase transitions. Little is known about the specific CaM-dependent enzymes that mediate Ca2+/CaM signaling to affect cell proliferation. In this study we show that inhibition of calmodulin kinase II (CaMKII) in HeLa cells using the CaMKII inhibitor KN-93 causes cell cycle arrest, demonstrating that CaMKII is required for cell cycle progression. Detailed analysis of arrest cells suggests that CaMKII is required for the initiation of DNA synthesis. Cells treated with KN-93 arrest with a G1DNA content, but with elevated cyclin-dependent histone H1 kinase activity, suggesting that CaMKII may act at a point very close to the onset of DNA synthesis in mammalian cells.Key words: calmodulin, protein kinase, cell cycle, HeLa.
X-ray diffraction studies of oriented lipid bilayers
Authors: Katsaras J.
Published: December 2009

This brief review of the X-ray diffraction technique used to study oriented lipid bilayer systems is primarily intended to demonstrate to the nonspecialist in the lipid field the amount of detailed information that can be obtained simply by visually inspecting the diffraction pattern and making some measurements with a ruler and protractor. The information that can be extracted from X-ray diffraction data is illustrated by selected examples of our most recent work.Key words: X-ray diffraction, oriented multibilayers, electron density profiles, subgel phase, phase problem.
Relationship between quenching of variable fluorescence and thermal dissipation in isolated thylakoid membranes: similar terminology and mathematical treatments may be used
Authors: Markovic Dejan Z. , Carpentier Robert
Published: December 2009

Simultaneous measurements of chlorophyll fluorescence and thermal emission using photoacoustic spectroscopy have been done in isolated thylakoid membranes to study the relationship between the photochemical quenching of fluorescence (qPF) and energy storage measured in photoacoustic experiments. It is shown that energy storage can be interpreted as the photochemical quenching of a variable component of thermal dissipation termed qPH. The parameters qPF and qPH were similarly sensitive to light intensity as demonstrated by their half-saturation light intensity. However, the nonvariable part of thermal dissipation (Ho) represented a greater proportion of the maximal thermal dissipation yield in comparison with the corresponding non-variable component of fluorescence (Fo) as a result of the thermal energy losses occurring during electron transport. A residual qPH found when qPF was removed indicated the participation of cyclic photosystem I or photosystem II in the measured qPH. The participation of cyclic photosystem I was also suggested by a low constant K, representing the quasi equilibria between (re)oxidized and reduced photosystem II quinone acceptors as determined from the logarithmic plots of the hyperbolic relationship obtained between qPH and light intensity. It is finally concluded that the terminology and mathematical treatments used for fluorescence measurements can also be applied to thermal dissipation.Key words: photosynthesis, thylakoid membranes, thermal dissipation, fluorescence quenching, plastoquinone.
Transfection of Na,K-ATPase α-subunit: regulation of enzyme abundance
Authors: Shanbaky Nabila M. , Pressley Thomas A.
Published: December 2009

DNA-mediated gene transfer into mammalian cells was used as a model for investigating the regulation of Na,K-ATPase abundance. Complementary DNA encoding the catalytic α1-subunit from rat was introduced into ouabain-sensitive monkey kidney cells, and transfectants were selected by their ability to survive in normally cytotoxic concentrations of ouabain. The overall specific activity of Na,K-ATPase in the membranes of transfectants was not significantly different from that in control cells, suggesting that there was a partial replacement, rather than an addition, of introduced α1for the endogenous subunit in the functional enzyme. Immunoblotting with specific antibodies confirmed the similarities in overall α abundance between control and transfected cells. Hybridization analysis of total RNA, however, revealed a higher abundance of the mRNA encoding total α1in transfected cells. The results suggest that endogenous and introduced α-subunit compete for a limited amount of β, with rapid degradation of unassembled subunits.Key words: DNA-mediated gene transfer, immunoblots, protein abundance, subunit assembly.
Oleate stimulation of incorporation of exogenous glycerol into cardiolipin in isolated perfused rat heart does not involve direct activation of the CDP–DG pathway
Authors: Cao Shu Guang , Hatch Grant M.
Published: December 2009

Oleate has been shown previously to stimulate the in vitro activity of phosphatidylglycerol-phosphatase, an important enzyme in the cytidine-5′-diphosphate – 1,2-diacyl-sn-glycerol pathway of phosphatidylglycerol and cardiolipin biosynthesis. In this study the in vivo effect of oleate on the biosynthesis of new phosphatidylglycerol and cardiolipin was investigated in the heart. Hearts were perfused for 60 min with Krebs–Henseleit buffer containing [1,3-3H]glycerol and 0.6 mM albumin in the absence or presence of 0.6 or 1.2 mM oleate. Total incorporation of radioactivity was higher in the oleate-treated hearts compared with controls and this was due to an exclusive incorporation of radioactive glycerol into the organic phase. Also, the radioactivity incorporated into phosphatidylglycerol and cardiolipin was higher in the oleate-treated hearts compared with controls; however, the increase was greater in hearts perfused with 0.6 mM oleate compared with 1.2 mM oleate, indicating that pathophysiological concentrations of oleate may attenuate the oleate-induced stimulation of glycerol incorporation into polyglycerophospholipids. The pool size of phosphatidyl-glycerol and cardiolipin were unchanged in oleate-perfused hearts compared with controls. To investigate if the biosynthesis of phosphatidylglycerol and cardiolipin via the cytidine-5′-diphosphate – 1,2-diacyl-sn-glycerol pathway was authentically stimulated by oleate, hearts were pulse labeled for 15 min with 0.1 mM [1,3-3H]glycerol and subsequently chased for 60 min with 0.1 mM glycerol in the absence or presence of 0.6 mM oleate in the perfusate. Radioactivity incorporated into phosphatidylglycerol and cardiolipin was unchanged compared with controls. Our data indicate that oleate increases the incorporation of exogenous glycerol into polyglycerophospholipids but does not accelerate synthesis from prelabeled precursor pools. Accordingly, oleate does not appear to stimulate directly enzymes of the cytidine-5′-diphosphate – 1,2-diacyl-sn-glycerol pathway in vivo.Key words: Cardiolipin, biosynthesis, heart, perfusion, oleate.
Patterns of spontaneous motility in videomicrographs of human epidermal keratinocytes (HEK)
Authors: Alt Wolfgang , Brosteanu Oana , Hinz Boris , Kaiser Hans Wilhelm
Published: December 2009

The subject of our observations was the spontaneous behaviour of normal and transfected human epidermal keratinocytes. Cell movements were recorded on video micrographs and analyzed by a mathematical approach, using new methods of image processing and statistical correlation analysis. Protrusive activity of single lamellae was examined using one-dimensional analysis of phase-contrast image sequences along section lines transversal to the cell edge. This method revealed high periodicity and correlation in the motility patterns of lamellae and ruffles. Two-dimensional correlation analysis of automatically digitized cell outlines was applied to detect spatiotemporal patterns and coordination of lamellar extension and retraction. Most cells showed regularly alternating pulsations of lamellar protrusions. In some extreme cases, extension waves rotating around the cell periphery were observed. The results were compared with computer simulations of two simple models for lamellar dynamics and shape deformation, based on few assumptions about chemical kinetics of F-actin and cytomechanical properties of the actin network, neglecting regulatory effects of actin-associated proteins or extracellular-stimulations. The simulation results reproduced the main dynamical features of the observed real cells, indicating the possibility that the basic universal mechanism for lateral coordination of lamellipodial protrusion is the interplay between hydrostatic pressure and viscocontractile tension in the cortical F-actin – plasma membrane complex.Key words: keratinocytes, cytomechanics, actin network, statistical correlation analysis, mathematical models.
Characterization of endothelial cell locomotion using a Markov chain model
Authors: Lee Yih , Mclntire Larry V. , Zygourakis Kyriacos , Markenscoff Pauline A.
Published: December 2009

A Markov chain model was developed to characterize the two-dimensional locomotion of bovine pulmonary artery endothelial (BPAE) cells cultured with or without basic fibroblast growth factor (bFGF). This model provides a detailed description of the migration process by computing the following locomotory parameters: (i) the speed of cell locomotion; (ii) the expected duration of cell movement in any given direction; (iii) the probability distribution of turn angles that will decide the next direction of cell movement; (iv) the frequency of cell stops; and (v) the duration of cell stops. Eight directional states and a stationary state were used in our Markov analysis. From cell trajectory data, the transition probabilities among the various states and the waiting times for the directional and the stationary states were computed. The steady-state probabilities were also calculated to obtain the ultimate direction of cell motion and, thus, determine whether cell motion was random. Our results showed how the addition of bFGF enhanced the locomotory capability of BPAE cells. Cells cultured with 30 ng/mL bFGF had lower probability of moving to the stationary state than those cultured without bFGF In addition, cells cultured with 30 ng/mL bFGF remained in the stationary state for shorter periods of time than cells cultured without bFGF. In both these cases, however, the transition probabilities from the stationary state to any directional state were uniformly distributed and were not affected by the presence of bFGF.Key words: Markov chain model, stochastic process, cell locomotion, endothelial cells.
Novel intron-encoded small nucleolar RNAs with long sequence complementarities to mature rRNAs involved in ribosome biogenesis
Authors: Bachellerie Jean-Pierre , Nicoloso Monique , Qu Liang-Hu , Michot Bernard , Caizergues-Ferrer Michèle , Cavaille Jérome , Renalier Marie-Hélène
Published: December 2009

Recently, several new snoRNAs encoded in introns of genes coding for ribosomal, ribosome-associated, or nucleolar proteins have been discovered. We are presently studying four of these intronic snoRNAs. Three of them, U20, U21, and U24, are closely related to each other on a structural basis. They are included in genes encoding nucleolin and ribosomal proteins L5 and L7a, respectively, in warm-blooded vertebrates. These three metabolically stable snoRNAs interact with nucleolar protein fibrillarin. In addition, they display common features that make them strikingly related to snoRNA U14. U14 contains two tracts of complementarity to 18S rRNA, which are required for the production of 18S rRNA. U20 displays a 21 nucleotide (nt) long complementarity to 18S rRNA. U21 contains a 13 nt complementarity to an invariant sequence in eukaryotic 28S rRNA. U24 has two separate 12 nt long complementarities to a highly conserved tract of 28S rRNA. Phylogenetic evidences support the fundamental importance of the pairings of these three snoRNAs to pre-rRNA, which could be involved in a control of pre-rRNA folding during preribosome assembly. By transfection of mouse cells, we have also analyzed the processing of U20 and found that the -cis acting signals for its processing from intronic RNA are restricted to the mature snoRNA sequence. Finally, we have documented changes of host genes for these three intronic snoRNAs during the evolution of eukaryotes.Key words: snoRNA, pre-rRNA, folding, genes, introns.
Genetic probes of ribosomal RNA function
Authors: O'Connor Michael , Brunelli Carleen A. , Firpo Matthew A. , Gregory Steven T. , Lieberman Kathy R. , Lodmell J. Stephen , Moine Hervé , Ryk Donald I. Van , Dahlberg Albert E.
Published: December 2009

We have used a genetic approach to uncover the functional roles of rRNA in protein synthesis. Mutations were constructed in a cloned rrn operon by site-directed mutagenesis or isolated by genetic selections following random mutagenesis. We have identified mutations that affect each step in the process of translation. The data are consistent with the results of biochemical and phylogenetic analyses but, in addition, have provided novel information on regions of rRNA not previously investigated.Key words: decoding, peptidyltransferase, streptomycin, paromomycin, suppression, 4.5S RNA.
Location and domain structure ofEscherichia coliribosomal protein L7/L12: site specific cysteine cross-linking and attachment of fluorescent probes
Authors: Traut Robert R. , Dey Debendranath , Bochkariov Dmitry E. , Oleinikov Andrew V. , Jokhadze George G. , Hamman Brian , Jameson David
Published: December 2009

Five different variants of L7/L12 containing single cysteine substitutions, two in the N-terminal (NTD) and three in the C-terminal domain (CTD), were produced, modified with [125I]N-[4-(p-azidosalicylamido)butyl]-3-(2′-pyridyldithio) propionamide ([125I]APDP), a sulfhydryl-specific, heterobifunctional, cleavable photo-cross-linking reagent, and reconstituted into ribosomes. These were irradiated, the total proteins were extracted and reductively cleaved, and the cross-linked proteins were identified. The effect of zero-length disulfide cross-linking on binding and activity was also determined. The same sites in L7/L12 were used to attach a rhodamine dye. The formation of ground-state rhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the free protein and in complexes with L10. The three sites in the CTD, but not the N-terminal sites, cross-linked to L2 and L5 and to 30S proteins S2, S3, S7, S14, and S18 in a manner influenced by elongation factors. Binding to the ribosome and, therefore, function were blocked by zero-length cross-linking within the NTD, but not the CTD. Binding also disrupted rhodamine dimers in the NTD. No rhodamine dimers formed in the CTD.Key words: ribosomes; L7/L12; cross-links; rhodamine dimers.
Structures of prokaryotic ribosomal proteins: implications for RNA binding and evolution
Authors: Ramakrishnan V. , Gerchman Sue Ellen , Kycia Jadwiga H. , Davies Christopher , Golden Barbara L. , Hoffmann David W. , Jaishree T. N. , Porter Stephanie , White Stephen W.
Published: December 2009

After along hiatus, the pace of determination of the structures of ribosomal proteins has accelerated dramatically. We discuss here the structures of five ribosomal proteins from Bacillus stearothermophilus: S5, S17, L6, L9, and L14. These structures represent several new motifs. Each of these structures has revealed new insights, and we have developed criteria for recognizing RNA-binding regions of each protein and correlating the structures with such properties as antibiotic resistance. The information here should also prove invaluable in an eventual high-resolution picture of the intact ribosome.Key words: ribosome, ribosomal proteins, ribosomal RNA, X-ray crystallography, NMR.
Structure and function of ribosomal RNA
Authors: Noller Harry F. , Green Rachel , Heilek Gabriele , Hoffarth Vernita , Hüttenhofer Alexander , Joseph Simpson , Lee Inho , Lieberman Kate , Mankin Alexander , Merryman Chuck , Powers Ted , Puglisi Elisabetta Viani , Samaha Raymond R. , Weiser Bryn
Published: December 2009

A refined model has been developed for the folding of 16S rRNA in the 30S subunit, based on additional constraints obtained from new experimental approaches. One set of constraints comes from hydroxyl radical footprinting of each of the individual 30S ribosomal proteins, using free Fe2+–EDTA complex. A second approach uses localized hydroxyl radical cleavage from a single Fe2+tethered to unique positions on the surface of single proteins in the 30S subunit. This has been carried out for one position on the surface of protein S4, two on S17, and three on S5. Nucleotides in 16S rRNA that are essential for P-site tRNA binding were identified by a modification interference strategy. Ribosomal subunits were partially inactivated by chemical modification at a low level. Active, partially modified subunits were separated from inactive ones by binding 3′-biotin-derivatized tRNA to the 30S subunits and captured with streptavidin beads. Essential bases are those that are unmodified in the active population but modified in the total population. The four essential bases, G926, 2mG966, G1338, and G1401 are a subset of those that are protected from modification by P-site tRNA. They are all located in the cleft of our 30S subunit model. The rRNA neighborhood of the acceptor end of tRNA was probed by hydroxyl radical probing from Fe2+tethered to the 5′ end of tRNA via an EDTA linker. Cleavage was detected in domains IV, V, and VI of 23S rRNA, but not in 5S or 16S rRNA. The sites were all found to be near bases that were protected from modification by the CCA end of tRNA in earlier experiments, except for a set of E-site cleavages in domain IV and a set of A-site cleavages in the α-sarcin loop of domain VI. In vitro genetics was used to demonstrate a base-pairing interaction between tRNA and 23S rRNA. Mutations were introduced at positions C74 and C75 of tRNA and positions 2252 and 2253 of 23S rRNA. Interaction of the CCA end of tRNA with mutant ribosomes was tested using chemical probing in conjunction with allele-specific primer extension. The interaction occurred only when there was a Watson–Crick pairing relationship between positions 74 of tRNA and 2252 of 23S rRNA. Using a novel chimeric in vitro reconstitution method, it was shown that the peptidyl transferase reaction depends on this same Watson–Crick base pair.Key words: rRNA, ribosome, tRNA, hydroxyl radical, ribosomal protein.
Identification of ribosome–ligand interactions using cleavage reagents
Authors: Hill Walter E. , Bucklin Douglas J. , Bullard James M. , Galbraith Alana L. , Jammi Narasimham V. , Rettberg Charles C. , Sawyer Brennan S. , Waes Michael A. Van
Published: December 2009

To characterize ribosome–ligand interactions, we have used a cleavage reagent, 1,10-orthopenanthroline–Cu(II), tethered to various ligands, to cleave nearby regions of rRNA. The phenanthroline is tethered to the ligand using either an internal 4-thiouridine or a terminal thiophosphate. When Cu2+and a reducing agent, such as mercaptopropionic acid, are present, cleavage of nearby nucleic acids occurs. The cleavage sites can be identified using primer-extension analysis. We have identified rRNA cleavage sites resulting from transcribed tRNAPhehaving randomly placed phenanthroline–Cu(II), tRNAPhewith phenanthroline–Cu(II) at position 8, and a DNA oligomer complementary to positions 2655–2667 (α-sarcin region) with phenanthroline–Cu(II) placed at the 5′ end. These results provide important new information on the structure of the rRNA within ribosomal subunits and on the proximity of rRNA neighborhoods to these bound ligands.Key words: ribosomal RNA structure, cleavage, phenanthroline, tRNA interactions, ribosome structure.
Upstream stimulators for recoding
Authors: Larsen B. , Brady K. , Atkins J. F. , Peden J. , Matsufuji S. , Matsufuji T. , Maldonado R. , Wills N. M. , Gesteland R. F. , Fayet O.
Published: December 2009

Recent progress in elucidation of 5′ stimulatory elements for translational recoding is reviewed. A 5′ Shine–Dalgarno sequence increases both +1 and −1 frameshift efficiency in several genes; examples cited include the E. coli prfB gene encoding release factor 2 and the dnaX gene encoding the γ and τ subunits of DNA polymerase III holoenzyme. The spacing between the Shine–Dalgarno sequence and the shift site is critical in both the +1 and −1 frameshift cassettes; however, the optimal spacing is quite different in the two cases. A frameshift in a mammalian chromosomal gene, ornithine decarboxylase antizyme, has recently been reported; 5′ sequences have been shown to be vital for this frameshift event. Escherichia coli bacteriophage T4 gene 60 encodes a subunit of its type II DNA topoisomerase. The mature gene 60 mRNA contains an internal 50 nucleotide region that appears to be bypassed during translation. A 16 amino acid domain of the nascent peptide is necessary for this bypass to occur.Key words: recoding, frameshifting, peptide factor, stimulatory elements.
Crystallographic studies of elongation factor G
Authors: Liljas Anders , Ævarsson Arnthor , Al-Karadaghi Salam , Garber Maria , Zheltonosova Julia , Brazhnikov Evgeni
Published: December 2009

The elongation factors G (EF-G) and Tu (EF-Tu) go through a number of conformation states in their functional cycles. Since they both are GTPases, have similar G domains and domains II, and have similar interactions with the nucleotides, then GTP hydrolysis must occur in similar ways. The crystal structures of two conformational states are known for EF-G and three are known for EF-Tu. The conformations of EF-G∙GDP and EF-Tu∙GTP are closely related. EF-Tu goes through a large conformational change upon GTP cleavage. This conformational change is to a large extent due to an altered interaction between the G domain and domains II and III. A number of kirromycin-resistant mutations are situated at the interface between domains I and III. The interface between the G domain and domain V in EF-G corresponds with this dynamic interface in EF-Tu. The contact area in EF-G is small and dominated by interactions between charged amino acids, which are part of a system that is observed to undergo conformational changes. Furthermore, a number of fusidic acid resistant mutants have been identified in this area. All of this evidence makes it likely that EF-G undergoes a large conformational change in its functional cycle. If the structures and conformational states of the elongation factors are related to a scheme in which the ribosome oscillates between two conformations, the pretranslocational and posttranslocational states, a model is arrived at in which EF-Tu drives the reaction in one direction and EF-G in the opposite. This may lead to the consequence that the GTP state of one factor is similar to the GDP state of the other. At the GTP hydrolysis state, the structures of the factors will be close to superimposable.Key words: elongation factor G, elongation factor Tu, crystal structures, conformational changes, ribosomal conformation.
Characterization, chromosomal mapping, and expression of different ubiquitin fusion protein genes in tissues from control and heat-shocked maize seedlings
Authors: Liu Ling , Frappier J. Roger H. , d'Ailly Karen , Atkinson Burr G. , Maillet Daniel S. , Walden David B.
Published: December 2009

Organisms possess at least two multigene families of ubiquitins: the polyubiquitins, with few to several repeat units, which encode a ubiquitin monomer, and the ubiquitin fusion (or extension) protein genes, which encode a single ubiquitin monomer and a specific protein. This report provides details about two ubiquitin fusion protein genes in maize referred to as MubG7 (uwo 1) and MubG10 (uwo 2). Each has one nearly identical ubiquitin coding unit fused without an intervening nucleotide to an unrelated, 237-nucleotide sequence that encodes for a 79 amino acid protein. The derived amino acid sequences of the two fusion proteins show that they differ by five amino acids (substitution by either a serine or threonine). MubG7 maps to chromosome 8L162 and MubG10 maps to chromosome 1L131. Analyses of the role(s) of these genes in response to heat shock (1 h at 42.5 °C) reveal that the level of these fusion protein mRNAs in the radicles or plumules from 2-day-old seedlings does not change; however, heat shock does cause a marked reduction in the accumulation of these same gene-specific mRNAs in the radicles and plumules of 5-day-old seedlings. These data confirm the suggestion from our earlier work that there is precise modulation, in a gene-specific manner, of the response to developmental as well as environmental signals.Key words: ubiquitin, ubiquitin extension (or fusion) protein, maize, heat shock, heat shock proteins, gene expression, chromosome map.
Purification and characterization of the double-stranded DNA-activated protein kinase, DNA-PK, from human placenta
Authors: Chan Doug W. , Ting Nicholas S. Y. , Lees-Miller Susan P. , Mody Christopher H.
Published: December 2009

The double-stranded DNA-activated protein kinase (DNA-PK) is a serine–threonine protein kinase that is composed of a large catalytic subunit (p350) and a DNA-binding protein of 70 and 80 kDa subunits known as the Ku autoantigen. When targeted to DNA by free DNA ends, DNA-PK phosphorylates many DNA- binding proteins and transcription factors. Previously, DNA-PK had only been purified and characterized from transformed human tissue culture cells. Here we report that DNA-PK is an abundant protein in human placenta and lymphocytes. We have purified the placental DNA-PK to homogeneity and show that its biochemical properties are similar to those of the HeLa cell enzyme.Key words: DNA-activated protein kinase.
Purification, characterization, and partial amino acid sequencing of an amphibian liver fatty acid binding protein
Authors: Schleicher Christian H. , Santomé José A.
Published: December 2009

The fatty acid binding protein (FABP) from toad liver cytosol was purified to homogeneity by a procedure involving gel filtration and anion exchange chromatography. The protein presented a molecular mass of 13 987 ± 2 daltons determined by electrospray mass spectrometry. Native isoelectric focusing of the purified liver FABP revealed a single pI 6.8 band. On the other hand, the toad heart FABP showed a different mobility than that of toad liver FABP by both sodium dodecyl sulfate – polyacrylamide gel electrophoresis and isoelectric focusing. Moreover, toad liver FABP cross-reacted with antisera to mammalian liver FABP but not with antisera to heart FABP. The difference between toad liver and heart FABPs was further confirmed by partial amino acid sequencing. As the N-terminus of toad liver FABP was blocked, the protein was chemically and enzymatically cleaved and the resulting peptides were subjected to automated Edman degradation. Partial amino acid sequencing showed that the toad liver FABP is related to that of mammalian liver and is clearly different from the amphibian heart FABP as well as from the amphibian intestine FABP.Key words: Fatty acid binding protein, liver, toad, Bufo arenarum, partial amino acid sequencing.
In vitro expression of a mouse tissue specific glutathione-peroxidase-like protein lacking the selenocysteine can protect stably transfected mammalian cells against oxidative damage
Authors: Vernet Patrick , Rigaudiére Nicole , Ghyselinck Norbert , Dufaure Jean Pierre , Drevet Joël R.
Published: December 2009

The complete sequence of the mouse epididymal protein (MEP24) was cloned. It contains a 663 bp open-reading frame that, after conceptual translation, shows extensive identity with proteins belonging to the glutathione peroxidase (GPX) family. However, a major difference between GPX5 (MEP24) and other known GPXs concerns a protein domain known to be critical for GPX function. To find out what could be the physiological function of such a protein in the mouse epididymis, we have used a mammalian expression system to overexpress the GPX5 protein. Cells constitutively expressing the GPX5 protein were generated and assayed for their ability to metabolize regular substrates of GPX enzymes. Data presented here show that the GPX5-expressing cells can metabolize hydrogen peroxide in a manner that is consistent with a peroxidase activity. However, the substrate preference of the GPX5-expressing cells and their apparent insensitivity to a regular inhibitor of GPX enzymes suggest that the GPX5 protein belongs to a particular class of GPX proteins. Involvement of this protein in the physiology of the mouse epididymis is discussed.Key words: glutathione peroxidase (GPX), mouse epididymis, MEP24, GPX5 cDNA, selenocysteine, GPX5 polyclonal antibody, spermatozoa, CHO-KI cells.
Basement membrane induced differentiation of HEC-1B(L) endometrial adenocarcinoma cells affects both morphology and gene expression
Authors: Hopfer Helmut , Vollmer Günter , Rinehart Jr. Clifford A. , Kaufman David G.
Published: December 2009

In vitro studies of endometrial carcinogenesis have been hampered by dedifferentiation of the cells in culture. Using the endometrial carcinoma cell line HEC-1B(L), we aimed to establish and characterize culture conditions mat preserve a more differentiated state of the tumor cells. HEC-1B(L) cells grown in a serum-free defined medium on plastic (PL/SFDM) on top of a reconstituted basement membrane (Matrigel™, MG/SFDM) or in a thick layer of Matrigel showed pronounced morphological differentiation as compared with HEC-1B(L) cells cultured on plastic in a medium containing serum (PL/10% FCS). Features of differentiation included cuboidal to columnar cell shape and an increase of rough endoplastic reticulum in Matrigel cultures. Gene expression of HEC-1B(L) cells was studied by metabolic [35S]methionine labeling and SDS–gel electrophoresis. HEC-1B(L) cells cultured in the presence of Matrigel showed two additional secretory proteins approximately 31 kD and 77 kD in size. rt-PCR was used to screen cell cultures for the presence of estrogen receptor, progesterone receptor, and lactoferrin–mRNA, genes typically expressed by normal endometrial epithelium. We found no expression of the estrogen receptor or progesterone receptor. Lactoferrin–mRNA was present under all culture conditions tested. Our results suggest a regulatory role of the extracellular matrix for the differentiation of the HEC-1B(L) cell line.Key words: endometrial carcinogenesis, basal membrane, differentiation, genetic expression, morphology.
Expression and epitopic conservation of calponin in different smooth muscles and during development
Authors: Jin Jian-Ping , Walsh Michael P. , Resek Mary E. , McMartin Gail A.
Published: December 2009

Calponin is a thin filament associated protein found in smooth muscle as a potential modulator of contraction. Five mouse monoclonal antibodies (mAbs CP1, CP3, CP4, CP7, and CP8) were prepared against chicken gizzard α-calponin. The CP1 epitopic structure is conserved in smooth muscles across vertebrate phyla and is highly sensitive to CNBr cleavage in contrast with the chicken-specific CP4 and the avian–mammalian-specific CP8 epitopes that are resistant to CNBr fragmentation. Using this panel of mAbs against multiple epitopes, only α-calponin was detected in adult chicken smooth muscles and throughout development of the gizzard. Western blotting showed that the calponin content varied among different smooth muscle tissues and correlated with that of h-caldesmon. In contrast with the constitutive expression of calponin in phasic smooth muscle of the digestive tract, very low levels of calponin were detected in adult avian tracheas and no calponin expression was detected in embryonic and young chick tracheas. These results provide information on the structural conservation of calponins and suggest a relationship between calponin expression and smooth muscle functional states.Key words: smooth muscle calponin, caldesmon, expression, development, chicken trachea.
Modelling of purine nucleoside metabolism during mouse embryonic development. Relative routes of adenosine, deoxyadenosine, and deoxyguanosine metabolism
Authors: Jenuth Jack P. , Mably Ellen R. , Snyder Floyd F.
Published: December 2009

The individual activities for adenosine kinase, deoxyadenosine kinase, adenosine deaminase, deoxyguanosine kinase, and purine nucleoside phosphorylase were determined during days 7 to 13 of mouse embryonic development. Adenosine deaminase increased 74-fold between days 7 and 9; deoxyadenosine kinase increased 5.4-fold during the same interval. Adenosine kinase, deoxyguanosine kinase, and purine nucleoside phosphorylase exhibited less than 2-fold changes in activity between days 7 and 13. Using Michaelis constants for each enzyme and the maximal velocities determined from enzyme assay, the relative routes of adenosine and deoxyadenosine metabolism via phosphorylation or deamination were modeled as a function of nucleoside concentration for days 7 through 13. For days 7 and 8, phosphorylation of adenosine is the principle route of metabolism at physiological concentrations. A switch occurred at day 9 and following where deamination is at least 5-fold greater than phosphorylation at all substrate concentrations. Deoxyadenosine phosphorylation was at most 10% of deamination at day 7 and then declined to less than 1% for days 9 to 13. Phosphorolysis was the principle route of deoxyguanosine metabolism through the 7 to 13 day period. Thus catabolism rather than phosphorylation was the principle pathway for purine deoxynucleoside metabolism during this period.Key words: mouse embryo, purine nucleoside metabolism.
Effects of aging and diet on the accumulation of dolichol in rat tissues
Authors: Ishinaga Masataka
Published: December 2009

A diet containing 15% (w/w) fat and 20% (w/w) of either casein or soy protein was fed to rats between 1 and 18 months of age. The effects of these dietary proteins on the accumulation of cholesterol and dolichols in kidney, spleen, brain, and heart were studied. The amount of cholesterol in these tissues was not influenced by the diet. In kidney and spleen, the amount of dolichols in rats fed the experimental diets was 50–70% higher than those in rats fed the lab chow diet. The contents of spleen dolichols in rats fed the soy protein diet tended to be higher than those in rats fed the casein diet. The amount of dolichols in heart and brain was not influenced by the diet. The proportion of spleen dolichyl fatty ester in rats fed the experimental diets was higher than that in rats fed the lab chow. The distribution of the dolichol isoprenologues was not influenced by the diet. There was a shift in the dolichol isoprenologues in kidney and spleen toward ones of lower chain length until 2 months of age, and after that there was no change. However, in heart and brain they shifted toward ones of lower chain length with aging. Our results suggested that dolichol metabolism may be influenced by fat content in the diets and differed among rat tissues.Key words: dolichol, cholesterol, dietary protein, aging, rat.
Hyperthyroidism impairs early repair in normal but not dystrophicmdxmouse tibialis anterior muscle. An in vivo study
Authors: Pernitsky A. N. , McIntosh L. M. , Anderson J. E.
Published: December 2009

The effect of hyperthyroidism on muscle repair was examined in mdx and control mice injected with triiodothyronine (T3) for 4 weeks. On day 24 of treatment, the right tibialis anterior (TA) muscle was crush-injured; 3 days later, mice received intraperitoneal [3H]thymidine to label newly synthesized DNA. One day later, muscles from both limbs were removed to study the severity of dystrophy (uncrushed muscle) and the regeneration response (crushed muscle). In uncrushed TA muscle, the area of active dystrophy (fiber damage and infiltration as a proportion of muscle cross-sectional area) was reduced by half after T3 treatment. Uncrushed muscle fiber diameter was lower in T3-treated control muscles. In crushed muscles, the diameter of new myotubes was larger in mdx mice than in controls and was reduced after T3 treatment in control regenerating muscle. In the same muscles, developmental myosin heavy chain was present in new myotubes and in small numbers of mononuclear cells (possibly differentiating myoblasts) near new myotubes and surviving fibers. Myotube density in the regenerating muscles was not changed by T3 treatment, although the number of myotube nuclei per field was decreased in control and increased in mdx T3-treated mice. Results extend previous reports of T3 effects on dystrophy and the strain difference in muscle precursor cell (mpc) proliferation. The results also suggest the hypothesis that excess T3 affects muscle regeneration either by reducing mpc proliferation or by increasing mpc fusion early in regeneration in control and mdx muscle.Key words: hypothyroid, muscle regeneration, crush injury, proliferation, mdx mouse.
Activation of human pp60c-src
Authors: Bjorge Jeffrey D. , O'Connor Tracy J. , Fujita Donald J.
Published: December 2009

pp60c-srcand the structurally related members of the Src family are non-receptor tyrosine kinases that reside within the cell associated with cell membranes and appear to transduce signals from transmembrane receptors to the cell interior. Many intracellular pathways can be stimulated upon Src activation, and a variety of cellular consequences can result, including morphological changes and cell proliferation. pp60c-srcactivity is normally suppressed by phosphorylation on its carboxy-terminal tail by an enzyme known as CSK. Various cellular stimuli or mutations within pp60c-srccan activate its endogenous kinase activity. In this paper, we review aspects of pp60c-srcactivation and regulation and discuss results obtained in our laboratory in two experimental systems: (i) in melanoma cell lines and primary pigmented normal human melanocytes and (ii) using activated mutant forms of purified human pp60c-srcprotein.Key words: Src, tyrosine, kinase, oncogene, phosphorylation.
Protein kinase C mediation of Ca2+-independent contractions of vascular smooth muscle
Authors: Walsh Michael P. , Clément-Chomienne Odile , Andrea Jacquelyn E. , Allen Bruce G. , Horowitz Arie , Morgan Kathleen G.
Published: December 2009

Tumour-promoting phorbol esters induce slow, sustained contractions of vascular smooth muscle, suggesting that protein kinase C (PKC) may play a role in the regulation of smooth muscle contractility. In some cases, e.g., ferret aortic smooth muscle, phorbol ester induced contractions occur without a change in [Ca2+]ior myosin phosphorylation. Direct evidence for the involvement of PKC came from the use of single saponin-permeabilized ferret aortic cells. A constitutively active catalytic fragment of PKC induced a slow, sustained contraction similar to that triggered by phenylephrine. Both responses were abolished by a peptide inhibitor of PKC. Contractions of similar magnitude occurred even when the [Ca2+] was reduced to close to zero, implicating a Ca2+-independent isoenzyme of PKC. Of the two Ca2+-independent PKC isoenzymes, ε and ζ, identified in ferret aorta, PKCε is more likely to mediate the contractile response because (i) PKCε, but not PKCζ, is responsive to phorbol esters; (ii) upon stimulation with phenylephrine, PKCε translocates from the sarcoplasm to the sarcolemma, whereas PKCζ translocates from a perinuclear localization to the interior of the nucleus; and (iii) when added to permeabilized single cells of the ferret aorta at pCa 9, PKCε, but not PKCζ, induced a contractile response similar to that induced by phenylephrine. A possible substrate of PKCε is the smooth muscle specific, thin filament associated protein, calponin. Calponin is phosphorylated in intact smooth muscle strips in response to carbachol, endothelin-1, phorbol esters, or okadaic acid. Phosphorylation of calponin in vitro by PKC (a mixture of α, β, and γ isoenzymes) dramatically reduces its affinity for F-actin and alleviates its inhibition of the cross-bridge cycling rate. Calponin is phosphorylated in vitro by PKCε but is a very poor substrate of PKCζ. A signal transduction pathway is proposed to explain Ca2+-independent contraction of ferret aorta whereby extracellular signals trigger diacylglycerol production without a Ca2+transient. The consequent activation of PKCε would result in calponin phosphorylation, its release from the thin filaments, and alleviation of inhibition of cross-bridge cycling. Slow, sustained contraction then results from a slow rate of cross-bridge cycling because of the basal level of myosin light chain phosphorylation (≈0.1 mol Pi/mol light chain). We also suggest that signal transduction through PKCε is a component of contractile responses triggered by agonists that activate phosphoinositide turnover; this may explain why smooth muscles often develop more force in response, e.g., to α1-adrenergic agonists than to K+.Key words: smooth muscle, protein kinase C, calponin.