bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism and localization of cardiolipin biosynthesis revisited: evidence for the identical mechanism and different localization in mitochondrial and submitochrondrial membranes isolated from guinea pig and rat liver
Authors: Stuhne-Sekalec Lidija , Stanacev Nikola Z.
Published: December 2009

The mechanism of cardiolipin (diphosphatidylglycerol) biosynthesis was examined in mitochondria and outer and inner mitochondrial membranes prepared from guinea pig and rat livers to determine whether this formation from phosphatidylglycerol was absolutely dependent on cytidinediphosphodiglyceride, as previously reported for intact mitochondria. Experimental results confirmed that the biosynthesis of cardiolipin, from the membrane-bound radioactive phosphatidylglycerol in intact mitochondria isolated from guinea pig and rat liver, was absolutely dependent on CDP-diglycerides and required the addition of divalent cations. Furthermore, the same mechanism for the biosynthesis of cardiolipin was operational in the outer and inner mitochondrial membranes. This biosynthesis was associated with both the outer and inner mitochondrial membranes prepared from guinea pig liver, but only with the inner mitochondrial membranes prepared from rat liver. The release of radioactive glycerol was also measured, but the amount obtained did not satisfy the stoichiometric requirement for CDP-diglyceride-independent biosynthesis of cardiolipin from 2 mol of phosphatidylglycerol with the liberation of 1 mol of glycerol. Therefore, it was concluded that this mechanism is not involved in the biosynthesis of cardiolipin in mitochondrial and submitochondrial membranes prepared from guinea pig and rat liver.Key words: mitochondria, outer mitochondrial membranes, inner mitochondrial membranes, cardiolipin, biosynthesis.
DNA methylation pattern during the encystment of Physarum flavicomum
Authors: Zhu Chengming , Henney Jr. Henry R.
Published: December 2009

In Physarum flavicomum Berk., haploid myxamoebae convert to dormant microcysts under conditions of nutrient imbalance. Exogenous adenine increases the intracellular content of S-adenosylmethionine (SAM) and inhibits this process. However, treatments that reduce the intracellular SAM levels relieve the inhibition of encystment induced by adenine. SAM plays a major metabolic role in cellular transmethylation reactions. In this study, we compared the DNA methylation patterns of growing cells, encysting cells, adenine-inhibited cells, and cysts using three different approaches: incubation of the cells with [14C]methylmethionine and detection of the labeled methyl group in purified DNA samples; analyses of DNA base composition by high performance liquid chromatography; and restriction endonuclease analyses of DNA. We found that DNA from the adenine-treated cells was labelled 1.3 times more with [14C]methylmethionine than was the DNA of untreated encysting cells. The DNA G + C content of this species was about 41%. The DNA of growing cells had the highest 5-methylcytosine (5MC) content, while DNA from the cysts had the lowest (about 27% that of growing cells). Adenine-inhibited cells had about 1.2 times more DNA-5MC than did encysting cells. Using the restriction enzymes SmaI, PvuI, and XhoI (which are inhibited by C residue methylation), we found that cyst DNA had more cutting sites than did amoebal DNA. By using the restriction enzyme DpnI which cuts DNA at GmATC sites, we found that cyst DNA, but not growing cell DNA, contained N6-methyladenine.Key words: amoebae, cysts, methylation, 5-methylcytosine, N6-methyladenine, DNA, encystment, Physarum flavicomum, development, inhibition.
Distribution of rDNA and 28S, 18S, and 5S rRNA in micronuclei containing a single chromosome
Authors: Labidi B. , Broders F. , Meyer J. L. , Hernandez-Verdun D.
Published: December 2009

Investigating genes and their transcription products in nuclear compartments corresponding to one mammalian chromosome, the ribosomal genes 18S–28S and 5S were localized in PtK1 micronucleated cells and rRNA was characterized in sorted micronuclei containing single identified chromosomes. In situ hybridization revealed the presence of 18S–28S rRNA genes in two micronuclei per cell and 5S rRNA genes in four micronuclei per cell. Flow cytometry histograms of isolated micronuclei stained with Hoechst 33342 exhibited five peaks (a–e) in which peaks b and c, respectively, corresponded to chromosomes 4 and X. Restricted genomic DNA from sorted peak c micronuclei showed the presence of 28S gene sequences. Direct sorting of the micronuclei from each peak on nitrocellulose and their hybridization with the 18S–28S rDNA probe revealed that the rRNA genes were exclusively located in micronuclei containing X chromosomes. Northern blotting showed the presence of 18S–28S and 5S rRNAs in peak c micronuclei and their absence from peak b micronuclei. Consequently, these procedures allowed us to show the presence of ribosomal genes and the corresponding rRNA in micronuclei containing single X chromosomes, and the absence of rRNA from micronuclei that do not contain the ribosomal genes. In regards to the transcription of these genes, the micronuclei from peak c can be considered as functional interphase X chromosomes.Key words: micronuclei, rDNA, rRNA, single chromosome, in situ hybridization.
Heat-stress stimulation of electron flow in a photosystem I submembrane fraction
Authors: Boucher Natalie , Harnois Johanne , Carpentier Robert
Published: December 2009

Oxygen uptake using methyl viologen as the terminal electron acceptor was recorded in digitonin-derived photosystem I submembrane fractions incubated at either 25 or 50 °C. A two- to four-fold heat-stress stimulation of electron flow was detected at 50 °C when reduced 2,6-dichlorophenol-indophenol was used as the primary electron donor. However, no stimulation was seen with N,N,N′,N′ -tetramethylphenylenediamine as the donor. The stimulation was enhanced by specific cations (Mg2+, Na+, K+), but not by Mn2 or Ca2+. The enhancement obtained with Mg2+ could be eliminated by incubating for a prolonged period. It is proposed that the observed heat-stress stimulation is due to a conformational change at the level of the cytochrome b6–f complex. This change increased the affinity of the protein complex for 2,6-dichlorophenol-indophenol at its oxidation sites. The involvement of a conformational modification is demonstrated by the absence of heat-stress stimulation in submembrane fractions immobilized in an albumin–glutaraldehyde cross-linked matrix.Key words: electron transport, photosystem I, heat stress, cytochrome b6–f complex, conformational changes, electron donor.
Escherichia coli β-galactosidase unexpectedly cleaves the hexasaccharide Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAc without branch specificity
Authors: Renkonen Ossi , Helin Jari , Vainio Anja , Niemelä Ritva , Penttilä Leena , Hilden Pekka
Published: December 2009

The branch specificity of Escherichia coli β-galactosidase (EC 3.2.1.23) was studied by analyzing the cleavage of the branched hexasaccharide Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)[14C(U)]Galβ1-4GlcNAc (1). This hexasaccharide was cleaved to pentasaccharides Galβ1-4GlcNAcβ1-3(GlcNAcβ1-6)[14C(U)]Galβ1-4GlcNAc (3) and GlcNAcβ1-3(Gal-β1-4GlcNAcβ1-6)[14C(U)]Galβ1-4GlcNAc (4) without any appreciable branch specificity. Even the further conversions of the pentasaccharides 3 and 4 into the tetrasaccharide GlcNAcβ1-3(GlcNAcβ1-6)[14C(U)]Galβ1-4GlcNAc seemed to proceed at similar rates, without any appreciable branch specificity. In marked contrast to the hexasaccharide 1, the pentasaccharide Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)[14C(U)]Gal (2), missing the reducing end GlcNAc, is known to be cleaved selectively at the 6-branch; this finding was confirmed in the present study. The different behaviour of hexasaccharide 1 and pentasaccharide 2 reflects differences in the reactivity of their 6-branches; the preferred conformations of these closely related molecules may be quite different.Key words: Escherichia coli β-galactosidase, branch-specific cleavage, random cleavage, oligo-N-acetyllactosaminoglycans, enzymatic in vitro synthesis.
A soluble fatty acyl – acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi
Authors: Byers David M. , Holmes Christine G.
Published: December 2009

An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi. Acyl–ACP synthetase activity (optimal pH 7.5–8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100. ACP from either Escherichia coli (apparent Km = 20 μM) or V. harveyi was used as a substrate. Of the [14C]fatty acids tested as substrates (8–18 carbons), a preference for fatty acids ≤ 14 carbons in length was observed. Vibrio harveyi acyl–ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay. The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture. Acyl–ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum. The cytosolic nature and enzymatic properties of V. harveyi acyl–ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E. coli, which has been implicated in phosphatidylethanolamine turnover. Acyl–ACP synthetase activity in V. harveyi could be involved in the intracellular activation and elongation of exogeneous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase.Key words: acyl carrier protein, acyl – acyl carrier protein synthetase, fatty acid activation, bioluminescent bacteria.
Hydrolysis of 2-acyl-sn-glycero-3-phosphocholines in guinea pig heart mitochondria
Authors: Badiani Ketan , Page Leona , Arthur Gilbert
Published: December 2009

Although both 2-acyl-sn-glycero-3-phosphocholine and 1-acyl-sn-glycero-3-phosphocholine may be produced from phosphatidylcholine hydrolysis, studies on the former have lagged behind that of the latter. In this study a lysophospholipase A2 that hydrolyses 2-acyl-sn-glycero-3-phosphocholine has been characterized in guinea pig heart mitochondria. The lysophospholipase A2 activity was not dependent on Ca2+ and was inhibited differentially by saturated and unsaturated fatty acids. This lysophospholipase A2 activity was able to discriminate among different molecular species of 2-acyl-sn-glycero-3-phosphocholines when they were presented individually or in pairs. The order of decreasing rates of hydrolysis of different molecular species of 2-lysophosphatidylcholines, when the substrates were presented singly, was 18:2 > 20:4 > 18:1 > 16:0. A differential inhibition of the rate of hydrolysis of the individual substrates was observed when the substrates were presented in pairs. The degree of inhibition was dependent on the molar ratio of the mixed substrates. The characteristics of the enzyme suggest that involvement in the selective release of fatty acids from mitochondrial phosphatidylcholine would depend on a high selectivity of phospholipase A1 for different molecular species of phosphatidylcholine. A lysophospholipase A1 activity was also characterized in the mitochondria with a distinct acyl specificity from the lysophospholipase A2. Other characteristics of the two lysophospholipases suggest that the two reactions are not catalysed by the same enzyme.Key words: lysophospholipases, mitochondria, fatty acid relase, heart.
High molecular weight soluble neutral maltase-glucoamylases in the intestine of the suckling rat
Authors: Lee L. , Forstner G.
Published: December 2009

Previous work from our laboratory has shown that the intestine of the suckling rat, unlike adult rat intestine, contains abundant quantities of at least two soluble neutral maltase-glucoamylases. These enzymes are related antigenically to membrane-bound maltase-glucoamylase, which predominates in adult intestine, but are either more easily solubilized or occupy a different cellular locus. To study the soluble enzymes further, we attempted their isolation from the intestine of 11-day-old suckling rats. Initial attempts were complicated by proteolytic degradation, despite the addition of phenylmethylsulfonyl fluoride, N-ethylmaleimide, leupeptin, pepstatin, and EDTA to buffers used for homogenization and column chromatography. Addition of aprotinin, amastatin, bestatin, and phosphoramidone resulted, however, in the isolation of two stable, high molecular weight maltases (HM1 and HM2). Both enzymes eluted before a papain-solubilized membrane-derived maltase-glucoamylase on Sepharose 4B and were separable by DE-52 and Sepharose 6B – Tris affinity columns. They were further purified on a lentil lectin – Sepharose 4B column. Substrate specificities were almost the same and characteristic of maltase-glucoamylases. Hydrophobic binding properties and pH optima of HM1 and HM2 were also similar. HM1 was resolved by sodium dodecyl sulfate – polyacrylamide gel electrophoresis into approximately equal portions of an endo-β-N-acetylglucosaminidase H sensitive enzyme of molecular weight (MW) 200 000 and an endo-β-N-acetylglucosaminidase H resistant but endo-β-acetylglucosaminidase F sensitive enzyme of MW 400 000. In contrast, most of HM2 consisted of a doublet of MW 200 000 – 210 000 that was endo-β-N-acetylglucosaminidase H sensitive. The intestine of the suckling rat, therefore, contains two soluble maltase–glucoamylase fractions, with a major portion of high mannose rather than complex oligosaccharides. In HM1, it is possible that the high mannose derivative is transformed during trafficking into the complex endo-β-N-acetylglucosaminidase H resistant form. HM2 appears to undergo little oligosaccharide processing.Key words: soluble maltase-glucoamylases, suckling rats.
Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol
Authors: Kihn Lee , Rutkowski Dorothy , Stinson Robert A.
Published: December 2009

As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 × slower in liposomes and 100 × slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.Key words: alkaline phosphatase, liposome, phosphatidylinositol, membrane anchor.
Isolation of a phospholipase-A2-like protein from human fetal intestine. Lysis of erythroid cells from bovine liver by this protein and porcine phospholipase A2
Authors: Congote L. Fernando , Blacker Russell , Esch Fred
Published: December 2009

Extracts of human fetal intestine contain factors that can stimulate or inhibit thymidine incorporation into fetal bovine erythroid cells. An inhibitory factor was purified to homogeneity by gel-permeation and reversed-phase high performance liquid chromatography. The inhibitory action was due to cell lysis. The first 25 amino acids of the N-terminal segment were identical to the human lung and pancreatic phospholipase A2. The isolated protein released arachidonic acid from 2-arachidonyl phosphatidylcholine. Porcine phospholipase A2 had the same effects as the intestinal protein, including its tissue-specific lysis of fetal bovine liver erythroid cells. No decrease of thymidine incorporation was seen in fetal bovine intestinal cells, 3T3 cells, or K562 cells incubated with the porcine enzyme. No release of hemoglobin or cell lysis was observed with human erythrocytes or fetal bovine erythrocytes. Porcine and bee phospholipases, which have low sequence homology, are nearly equipotent in inhibiting thymidine incorporation, whereas melittin and β-bungarotoxin were less active than the pancreatic enzyme. These results support the tissue-specific effects observed with other phospholipases A2. The high sensitivity of liver erythroid cells towards some phospholipases A2 suggest that these enzymes may be involved in the elimination of hepatic erythroid cells at the end of gestation.Key words: erythropoiesis, tissue remodeling, fetal liver, extracellular phospholipase A2, erythroblast maturation.
fatty acids modulate calcium-induced calcium release from skeletal muscle heavy sarcoplasmic reticulum fractions: implications for mallignant hyperthermia
Authors: Fletcher Jeffrey E. , Tripolitis Linda , Erwin Kirsten , Hanson Susan , Rosenberg Henry , Conti Pierre A. , Beech Jill
Published: December 2009

Based on studies in swine, the malignant hyperthermia syndrome has been postulated to result from an enhanced sensitivity (low threshold) of the Ca2+-induced Ca2+-release process. However, fatty acid production is elevated in homogenates of skeletal muscle from pigs and humans susceptible to malignant hyperthermia. In the present study, we demonstrate that the threshold of Ca2+-induced Ca2+ release is normal in susceptible humans and in susceptible swine depleted of triglycerides. Exogenously added unsaturated fatty acids decreased the threshold of Ca2+-induced Ca2+ release to a much greater extent in porcine and equine muscle than in human muscle. When triglyceride and free fatty acid values were reduced to about 40 and 60%, respectively, of control values, malignant hyperthermia-susceptible swine did not exhibit muscle rigidity when challenged in vivo with halothane and succinylcholine and the threshold of the Ca2+-induced Ca2+-release process in heavy sarcoplasmic reticulum fractions was normal. Despite the reduced triglyceride and fatty acid levels, these swine had a positive in vitro contracture test for malignant hyperthermia. A low Ca2+-induced Ca2+-release threshold is not essential for malignant hyperthermia susceptibility, but appears to be the result of excessive free fatty acids produced during organelle isolation.Key words: calcium release, fatty acids, malignant hyperthermia, triglycerides.
Ontogeny of pyruvate dehydrogenase complex and key enzymes involved in glycolysis and tricarboxylic acid cycle in rabbit fetal lung, heart, and liver
Authors: Bhavnani Bhagu R. , Wallace Duncan G.
Published: December 2009

The metabolic pathways by which the glycogen is utilized by fetal tissues is not well established. In the present study the ontogeny of seven key enzymes involved in glycolysis and the tricarboxylic acid cycle has been established for rabbit fetal lung, heart, and liver. In the fetal lung the activities of phosphofructokinase, pyruvate kinase, lactic dehydrogenase, citrate synthase, and malate dehydrogenase increase from day 21 to 25. Thereafter the levels either drop to day 19 levels or do not change. The isocitrate dehydrogenase activity continues to increase from day 19 of gestation to maximum level on day 31 of gestation. In fetal heart the pattern of activity is similar, but in fetal liver most of the enzymes reach maximum levels earlier and, with the exception of pyruvate kinase, do not show a significant fall in activity near term. The pattern of development of pyruvate dehydrogenase complex is different; maximum activity is observed on day 27 in fetal lung and heart and on day 21 in fetal liver. These results indicate that all three fetal tissues can oxidize glucose. Also, the accumulation of glycogen, particularly in fetal lung, appears to ensure that at specific times during gestation adequate quantities of energy (ATP) and substrates, required for surfactant phospholipid synthesis, are available independent of maternal supply of glucose or during brief episodes of hypoxia.Key words: glycogen, glycolysis, tricarboxylic acid cycle, pyruvate dehydrogenase, surfactant.
Heat shock response in Neurospora crassa: purification and some properties of HSP 80
Authors: Roychowdhury H. S. , Kapoor M.
Published: December 2009

The heat shock response of Neurospora crassa was investigated. A 80-kilodalton heat shock protein (HSP 80) was purified to near homogeneity from heat-shocked mycelial extracts employing ammonium sulphate fractionation, gel filtration, and ion-exchange and affinity chromatography. It was observed to migrate as a single band on one-dimensional sodium dodecyl sulphate – polyacrylamide gels, with a molecular mass of ~ 83 kilodaltons (kDa). On two-dimensional gels it resolved into four polypeptide species with isoelectric points in the acidic range, which on staining with periodic acid – Schiff method were demonstrated to be glycosylated. In the native state, HSP 80 had a molecular size of ~610 kDa.Key words: Neurospora, heat shock, fast protein liquid chromatography, 80-kilodalton heat shock protein, glycoprotein.
A plasma-membrane associated ATPase from the acidophilic bacteriu Acidiphilium cryptum
Authors: Bhattacharyya S. , Banerjee P. C. , Das P. K.
Published: December 2009

A membrane-bound ATPase of Acidiphilium cryptum, an acidophilic bacterium of mine origin, has been studied. The enzyme has a pH optimum of 8.4. Mg2+ is required for its activity and could be replaced by Mn2+, but not by Ca2+.The enzyme shows a strong preference for ATP as substrate, with the apparent Km of about 0.2 mM. Sulphite ion significantly stimulated the enzyme activity. N,N′ -Dicyclohexylcarbodiimide, oligomycin, and azide strongly inhibited the enzyme, whereas vanadate was without effect, suggesting that the A. cryptum ATPase might be of FOF1 type.Key words: Acidiphilium cryptum, ATPase, bacterial membrane.
Comparison of D-β-hydroxybutyrate dehydrogenase from rat liver and brain mitochondria
Authors: Zhang Wei-Wei , Redman Kent , Churchill Sharon , Churchill Perry
Published: December 2009

The properties of D-β-hydroxybutyrate dehydrogenase (BDH) from rat liver and brain mitochondria were compared to determine if isozymes of this enzyme exist in these tissues. The BDHs from these tissues behaved similarly during the purification process. The enzymes were indistinguishable by sodium dodecyl sulfate – polyacrylamide or acid-urea – polyacrylamide gel electrophoresis and they had identical isoelectric points. The BDHs from rat liver and brain were also quite similar in functional parameters determined by kinetic analysis and phospholipid activation of apo-BDH (i.e., the lipid-free enzyme). Antiserum against rat liver BDH inhibited both enzymes to an equivalent extent in a titration assay. The enzymes had similar patterns of peptide mapping by partial digestion with Staphylococcus aureus V8 protease, followed by immunoblotting using antiserum against the liver enzyme. These results suggest that the BDHs in rat liver and brain are very similar and possibly identical.Key words: D-β-hydroxybutyrate dehydrogenase, membrane-bound enzyme, lipid activation.
Decreased expressions of c-myc and H-ras oncogenes in vitamin E succinate induced morphologically differentiated murine B-16 melanoma cells in culture
Authors: Prasad Kedar N. , Cohrs Randall J. , Sharma Opendra K.
Published: December 2009

Treatment of B-16 melanoma cells in culture with d-α-tocopheryl succinate (vitamin E succinate) at concentrations of 11.3 and 15.1 μM inhibited growth and induced cell differentiation in culture. Vitamin E succinate treatment decreased the levels of c-myc and H-ras specific mRNAs in melanoma cells. Similar results were obtained by the vitamin retinoic acid and the nonvitamin agents R020-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), an inhibitor of cyclic nucleotide phosphodiesterase (0.72 mM), and sodium butyrate (1 mM), which induced differentiation and (or) inhibited growth of melanoma cells in culture. The extent of inhibition of c-myc mRNA was greater than that of H-ras mRNA. These results indicate that vitamin E succinate induced reduction of the levels of c-myc and H-ras mRNAs is related to growth inhibition of melanoma cells in culture.Key words: melanoma, vitamin E succinate, differentiation, c-myc, H-ras.
Synapsin-1 is found in a microtubule-associated complex of proteins isolated from bovine brain
Authors: Farrell Karen P. , Keates Robert A. B.
Published: December 2009

We have prepared microtubules from brain tissue by stabilizing the cellular microtubules in 6.7 M glycerol buffer, instead of the usual procedure which extracts the solubilized protein and then reassembles microtubules in vitro at some later time. There are substantial differences in the microtubule associated proteins obtained by the two methods, and brain spectrin is a major component of the stabilized microtubules. We have now modified the buffer used for the isolation of stabilized microtubules to minimize their tendency to aggregate. When the stabilized microtubules were further purified by sucrose density gradient centrifugation, we were able to distinguish previously unidentified polypeptides at 49, 74 (doublet), and 100 kilodaltons (doublet). These bands maintained staining intensity in the same proportion to tubulin as in the original homogenate, whereas background proteins were diminished in staining intensity. We now report the identification of the 74-kilodalton doublet polypeptides as synapsin-1 by peptide mapping. Synapsin-1 is a protein known to bind to brain spectrin and also to microtubules, and may thus serve as a linker between these cytoskeletal components.Key words: microtubule-associated protein, synapsin, spectrin, tubulin, cytoskeleton.
Antigenic determinant of a monoclonal antibody: extracellular domain at the M3–M4 junction of the α-subunit of Na,K-ATPase
Authors: Kano Itsu , Satoh Kanako , Nagai Fumiko , Ushiyama Keiko , Nakao Toshiko , Hara Yukichi , Kano Kazutaka
Published: December 2009

The binding site of a monoclonal antibody, M45-80, against the α-subunit of horse Na,K-ATPase was determined. Various sizes of DNA fragments derived from rat Na,K-ATPase α1-subunit cDNA were cloned into pUC19 expression vector and some fragments of horse genomic DNA were cloned into pUC18. Escherichia coli JM83 cells harboring the plasmids were grown and the cell lysates were used as antigens. An enzyme-linked immunosorbent assay revealed that M45-80 recognizes the hexapeptide Glu-Tyr-Thr-Trp-Leu-Glu (which is identical to the rat and horse α1-subunits) at the M3–M4 junction located on the extracellular side. The ouabain-binding site is discussed in relation to the recognition site of M45-80.Key words: Na,K-ATPase α-subunit, monoclonal antibody, pUC19 expression vector, antibody-binding site, ouabain-binding site.
Complete analysis of the trace elements of the kidney
Authors: Béliveau R. , Gélinas Y. , Ferraris J. , Schmit J. P.
Published: December 2009

The distribution of trace elements in kidney cortex and medulla and in their subcellular fractions was studied by inductively coupled plasma mass spectrometry (ICP-MS). This technology allows the analysis of almost all the elements of the periodic table, along with their isotopes, in the same experiment. Acid digestion of the tissue (samples) was required before ICP-MS elemental analysis. Mineralization in 8 N nitric acid for 2 min in a Parr microwave acid digestion bomb, inside a regular microwave oven working at medium power, gave good sensitivity and reproducible results. Trace element determinations could be precisely performed, despite the presence of a considerable amount of organic matter. Cortex/plasma and medulla/plasma inorganic ratios were taken as indicators of the inorganic bioaccumulation. The highest enrichment factors of elements found in the cortex were Mn > Co > Tl > Zn > Rb > Mo > Cu and the highest element enrichments of the medulla were Tl > Mn > Co > Rb > Zn > Bi > Mo > Ni > Cu. Subcellular fragmentation of the kidney cortex and medulla indicated that the majority of the trace elements was located in the cytosolic fraction. The membrane fraction of the medulla contained significant amounts of Hg and Al: 120.7 and 32.0 mg/g protein, respectively. Cu and Zn were the most abundant trace elements found in the brush border membrane from the cortex: 120.7 and 68.2 mg/g protein, respectively. These quantities are largely in excess of what is bound, for example, to alkaline phosphatase. This suggests additional roles for trace elements in this membrane. The endogenous location of trace elements in the kidney could reflect a biochemical regulation related to metallic cofactors.Key words: brush border membrane, kidney, inorganic tissue content, element determination, inductively coupled plasma mass spectrometry.
Protein kinase cascades in meiotic and mitotic cell cycle control
Authors: Pelech Steven L. , Sanghera Jasbinder S. , Daya-Makin Maleki
Published: December 2009

Eukaryotic cell cycle progression during meiosis and mitosis is extensively regulated by reversible protein phosphorylation. Many cell surface receptors for mitogens are ligand-stimulated protein-tyrosine kinases that control the activation of a network of cytoplasmic and nuclear protein-serine(threonine) kinases. Over 30 plasma membrane associated protein-tyrosine kinases are encoded by proto-oncogenes, i.e., genes that have the potential to facilitate cancer when disregulated. Proteins such as ribosomal protein S6, microtubule-associated protein-2, myelin basic protein, and casein have been used to detect intracellular protein-serine(threonine) kinases that are activated further downstream in growth factor signalling transduction cascades. Genetic analysis of yeast cell division control (cdc) mutants has revealed another 20 or so protein-serine(threonine) kinases. One of these, specified by the cdc-2 gene in Schizosaccharomyces pombe, has homologs that are stimulated during M phase in maturing sea star and frog oocytes and mammalian somatic cells. Furthermore, during meiotic maturation in these echinoderm and amphibian oocytes, this is followed by activation of many of the same protein-serine(threonine) kinases that are stimulated when quiescent mammalian somatic cells are prompted with mitogens to traverse from G0 to G1 phase. These findings imply that a similar protein kinase cascade may oversee progression at multiple points in the cell cycle.Key words: protein kinases, mitosis, meiosis, oncogenes, cell division control.
Microtubules, mitochondria, and molecular chaperones: a new hypothesis for in vivo assembly of microtubules
Authors: Gupta Radhey S.
Published: December 2009

In Chinese hamster ovary cells, a number of independent mutants selected for resistance to antimitotic drugs have been found to be specifically altered in two major cellular proteins, designated P1 (relative mass (Mr) ≈ 60–63 kilodaltons (kDa)) and P2 (Mr ≈ 69–70 kDa), which appeared microtubule related by a number of genetic and biochemical criteria. Antibodies to P1 have been found to bind specifically to mitochondria that showed specific association with microtubules in interphase cells. Biochemical and cDNA sequence studies on P1 showed that this protein, which is localized in the matrix compartment, is the mammalian homolog of the highly conserved chaperonin family of proteins (other members include the GroEL protein of Escherichia coli, the 60-kDa heat-shock protein of yeast, and the rubisco subunit binding protein of plant chloroplasts). The chaperonin proteins in various systems play a transient but essential molecular chaperone role in the proper folding of polypeptide chains and their assembly into oligomeric protein complexes. Our studies on P2 protein established that it corresponds to the constitutive form of the major 70-kDa heat-shock protein of mammalian cells (i.e., hsc70), which also acts as a molecular chaperone in the intracellular transport of nascent proteins to organelles such as mitochondria and endoplasmic reticulum. To account for the above, as well as a number of other observations (e.g., binding of fluorescent-labeled antimitotic drugs to mitochondria, association of tubulin with mitochondria as well as other membranes, and high affinity binding of antimitotic drugs to free tubulin but not to assembled microtubules), a new model for the in vivo assembly of interphase microtubules is proposed. The model ascribes a central role to the mitochondrially localized chaperonin (i.e., P1) protein in the intracellular formation of tubulin dimers and in their addition to the growth sites in microtubules. The proposed model also explains a number of other observations related to microtubule assembly in the literature.Key words: microtubule assembly, mitochondria, molecular chaperones, heat-shock proteins, antimitotic drugs.
Ornithine and S-adenosylmethionine decarboxylase activities and polyamine contents in developing muscle tissues and primary cultures of normal and polymyopathic hamsters
Authors: Sauro Victor S. , Klamut Henry J. , Dick William F. , Oey Daniella N. , Strickland Kenneth P.
Published: December 2009

Polyamine (putrescine, spermidine, and spermine) contents and ornithine (ODC) and S-adenosylmethionine (SAMDC) decarboxylase activities have been assessed in an age-dependent manner, in normal and polymyopathic (dystrophic) hamster skeletal muscle, heart, and tongue extracts and in primary tongue myoblast and skin fibroblast cultures. At 2 weeks of age, polyamine contents were significantly elevated in all of the dystrophic hamster tissues studied when compared with their age-matched controls. The degree of this elevation decreased with the age of the animals, generally, to a level where no significant difference in polyamine contents could be noted between normal and dystrophic hamster tissues. ODC and SAMDC activities in whole tissue extracts were consistently highest in 2-week-old muscle extracts and also declined with age. However, no significant changes in ODC or SAMDC activities were evident in any of the dystrophic muscle tissues studied when compared with their age-matched controls. Polyamine contents in dystrophic hamster myoblast and fibroblast primary cultures were also significantly elevated during proliferation (1 and 2 days after the initial seeding) compared with cultures prepared from normal hamsters. ODC and SAMDC activities in primary myoblast and fibroblast cultures clearly reflected the rate of cell proliferation, with highest activities found in subconfluent cell cultures. However, in general, no significant dystrophic-related abnormality in ODC or SAMDC activity was evident in proliferating myoblast or fibroblast cultures. These results suggest that the elevated polyamine contents of dystrophic hamster tissues and primary cultures may be due to a deficiency in polyamine catabolism or transport.Key words: polyamine contents, polyamine synthesis, polymyopathic hamsters.
The glycoprotein-processing inhibitors bromoconduritol and N-methyl-1-deoxynojirimycin alter the adhesion phenotype of skeletal myoblasts
Authors: Trudel Geralyn C. , Holland Paul C.
Published: December 2009

Treatment of chick myoblasts with the glucosidase inhibitors bromoconduritol (BCD) or N-methyl-1-deoxynojirimycin (MDJN), but not the mannosidase I inhibitor 1-deoxymannojirimycin (ManDJN), decreased their rate of adhesion to fibronectin and laminin and increased their rate of adhesion to collagen types I and IV. The adhesion of chick myoblasts to fibronectin, collagen type IV, and laminin was predominantly mediated by β1-type integrin(s) as judged by inhibition of adhesion with the β1-specific monoclonal antibody JG22. Collagen binding in inhibitor-treated cells remained JG22-sensitive suggesting the inhibitors promote increased activity of a β1-type collagen-selective integrin. The effects of BCD, MDJN, and ManDJN on myoblast β1-integrin detectable at the myoblast cell surface with JG22 antibody correlated well with their effects on adhesion to fibronectin and laminin, and paralleled the previously reported effects of these agents on myogenesis. Interaction of integrin with the extracellular matrix appears to be required for myoblast terminal differentiation. We found that Mn2+ ions increased the adhesion of myoblasts to extracellular matrix proteins and antagonized the effect of BCD and MDJN on myoblast differentiation, supporting a role for cell–matrix interactions in myogenesis. Inhibition of myogenesis by BCD or MDJN was not reversed by growth under low serum conditions, suggesting these agents do not act by maintaining myoblasts in a proliferative state.Key words: myoblast, myogenesis, integrin, cell adhesion, glycoprotein processing.
Alterations of band 3 transport protein by cellular aging and disease: erythrocyte band 3 and glucose transporter share a functional relationship
Authors: Bosman Gieljan J. C. G. M. , Kay Marguerite M. B.
Published: December 2009

Structural changes in human erythrocyte band 3 that affect anion transport are correlated with changes in glucose transport in situ. Breakdown of band 3, observed during normal erythrocyte aging in situ and in some diseases involving erythrocytes, is associated with an increase in Km and a decrease in Vmax of sulfate self-exchange, and with an increase in Km and Vmax of glucose efflux. Erythrocytes containing a high molecular weight form of band 3 exhibit an increase in Vmax of sulfate exchange and a decrease in Vmax of glucose efflux. Identical transport characteristics are observed in abnormal band-3-containing erythrocytes from individuals with familial amyotrophic chorea with acanthocytosis. A third band 3 alteration, fast-aging band 3, exhibits decreased Vmax of sulfate exchange and an increase in Km and decrease in Vmax of glucose efflux. Changes in band 3 structure that are the result of unstable hemoglobin or a deficiency in glucose-6-phosphate dehydrogenase and that do not affect anion transport have no effect on glucose transport characteristics. These data indicate the existence of a functional relationship between the membrane-spanning, anion-transport domain of band 3 and glucose transport in human erythrocytes. Antibodies to synthetic peptides reveal structural changes in membranes from the three inborn band 3 alterations and in band 3 itself in membranes from fast-aging band 3. Thus, immunological data suggests a structural relationship between anion and glucose transporters.Key words: red cell, anion transport, membrane proteins, aging, choreoacanthocytosis, anemia.
Control points in eucaryotic ribosome biogenesis
Authors: Larson D. E. , Zahradka P. , Sells B. H.
Published: December 2009

Ribosome biogenesis in eucaryotic cells involves the coordinated synthesis of four rRNA species, transcribed by RNA polymerase I (18S, 28S, 5.8S) and RNA polymerase III (5S), and approximately 80 ribosomal proteins translated from mRNAs synthesized by RNA polymerase II. Assembly of the ribosomal subunits in the nucleolus, the site of 45S rRNA precursor gene transcription, requires the movement of 5S rRNA and ribosomal proteins from the nucleoplasm and cytoplasm, respectively, to this structure. To integrate these events and ensure the balanced production of individual ribosomal components, different strategies have been developed by eucaryotic organisms in response to a variety of physiological changes. This review presents an overview of the mechanisms modulating the production of ribosomal precursor molecules and the rate of ribosome biogenesis in various biological systems.Key words: rRNA, ribosomal proteins, nucleolus, ribosome.
Timing of the appearance of ubiquitinated histones in developing new macronuclei of Tetrahymena thermophila
Authors: Davie James R. , Lin Rueyling , Allis C. David
Published: December 2009

Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inert micronucleus. During vegetative growth, macronuclear histones H2A and H2B and micronuclear H2A are ubiquitinated. Despite differences in function, macro- and micro-nuclei are related. During conjugation (the sexual phase of the life cycle in Tetrahymena), postzygotic division products of micronuclei give rise to new micro- and macro-nuclei. Using an anti-ubiquitin antibody in Western blotting experiments, we determined the levels of ubiquitinated histones in new macro- and micro-nuclei at various times during conjugation. Very soon after the second postzygotic division (approximately 8 h) when new macronuclei begin to synthesize RNA, ubiquitinated H2B and polyubiquitinated H2A are present. At this time micronuclei have only low levels of ubiquitinated H2A. During later stages of conjugation (15 h), the level of polyubiquitinated H2A decreases, while ubiquitinated H2B increases in developing new macronuclei, attaining levels of ubiquitinated H2B approaching that of parental macronuclei. Ubiquitinated histones are not detectable in the 15-h micronuclei. These results show that ubiquitination of H2B coincides with the transformation of an inert germinal nucleus into that of a transcriptionally active somatic nucleus, suggesting that ubiquitinated H2B has a role in maintaining the transcriptionally active chromatin state.Key words: histone ubiquitination, Tetrahymena thermophila, chromatin, transcription.
Structure of the glycan chain from the surface layer glycoprotein of Bacillus alvei CCM 2051
Authors: Altman Eleonora , Brisson Jean-Robert , Messner Paul , Sleytr Uwe B.
Published: December 2009

The cell surface of the mesophilic eubacterium Bacillus alvei CCM 2051 is covered by an oblique arranged surface layer glycoprotein. The subunits revealed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis were distinct bands of molecular masses 140 000, 128 000, and 127 000. Proteolytic degradation of the purified S-layer glycoprotein yielded a single glycopeptide fraction with an apparent molecular mass of ca. 25 000. Methylation analysis in conjunction with two-dimensional nuclear magnetic resonance experiments at 500 MHz established the branched trisaccharide[Formula: see text]as the repeating unit for this glycan chain.Key words: surface layer, eubacteria, glycoprotein, nuclear magnetic resonance, structure.
Cultured human nasal epithelial multicellular spheroids: polar cyst-like model tissues
Authors: Bridges Michael A. , Walker David C. , Harris Robert A. , Wilson Bruce R. , Davidson A. George F.
Published: December 2009

We report here a new readily cultured nonadherent hollow spheroidal epithelial tissue model: human nasal epithelial multicellular spheroids, prepared from brushings of human nasal epithelium in vivo. Although cultured cyst-like epithelial models developed from embryonic, transformed, or polypoid tissues have been reported previously, human nasal epithelial multicellular spheroids are derived from normal mature nontransformed human airway epithelial cells. In our studies, spheroids ranged in size from 50 to 700 μm diameter (averaging approximately 250 μm). Cells of the spheroid displayed morphological polarity and formed junctional complexes. Transcellular electrolyte transport may underlie the increase in spheroid size which occurred in culture. The ease and simplicity of the brushing and culture procedures reported here render normal and diseased human cell populations more readily accessible to investigation. We believe human nasal epithelial multicellular spheroids may have important applications in the study of electrolyte and fluid transport processes, ciliary motility, epithelial polarity, cellular metabolism, and drug cytotoxicity in normal and pathophysiological states of the human respiratory tract (e.g., cystic fibrosis).Key words: cultured airway epithelial cells, electrolyte and fluid transport, spheroid, cyst, cystic fibrosis.
The synthesis of phosphoenolpyruvate carboxylase in imbibing sorghum seeds
Authors: Khayat Eli , Dumbroff Erwin B. , Glick Bernard R.
Published: December 2009

When sorghum seeds were imbibed either in the light or in the dark, the presence of newly synthesized phosphoenolpyruvate carboxylase (PEPC) could be detected immunologically after approximately 6 h. In addition, both PEPC mRNA and enzyme activity were detected in extracts of dry seeds prior to imbibition. By contrast, ribulose-1,5-bisphosphate carboxylase mRNA, protein, and activity, as well as chlorophyll, were not detected even after 24 h of imbibition. These observations suggest that the nonphotosynthetic form of PEPC is synthesized during seed development and may play an important role in the germinative process.Key words: phosphoenolpyruvate carboxylase, sorghum seeds, germination, ribulose-1,5-bisphosphate carboxylase.
Trolox and ascorbate: are they synergistic in protecting liver cells in vitro and in vivo?
Authors: Zeng L.-H. , Wu Jun , Carey Doug , Wu T.-W.
Published: December 2009

From in vitro studies involving multilamellar liposomes or other artificial systems, several groups of workers have deduced that Trolox (a water-soluble analogue of vitamin E) and ascorbate are synergistic antioxidants. Here, we demonstrate that while Trolox and ascorbate individually protect cultured hepatocytes against oxyradicals generated either with xanthine oxidase plus hypoxanthine or with hydrogen peroxide, the two antioxidants do not appear to be synergistic when used in equimolar combinations. Also, in a rat model of hepatic ischemia–reperfusion, we observed that infusion of Trolox or ascorbate (7.5–10 μmol/kg body weight) into the postischemic liver reduced the reperfusion injury by 76 or 67%, respectively. However, when both compounds were used together (each at the same dose as used separately), the organ salvage amounted to only 79%. Therefore, there is no evidence of synergism between Trolox and ascorbate in our in vitro and especially in vivo systems.Key words: synergism, vitamin C, Trolox, antioxidants.
Snake venom cardiotoxins and bee venom melittin activate phospholipase C activity in primary cultures of skeletal muscle
Authors: Fletcher Jeffrey E. , Jiang Ming-Shi , Gong Qi-Hua , Smith Leonard A.
Published: December 2009

The effects of cardiotoxin fractions from Naja naja kaouthia and Naja naja atra snake venoms and synthetic melittin peptide were examined on lipolytic activity in red blood cells and primary skeletal muscle cultures. Both native cardiotoxin fractions caused considerable producion of free fatty acids in red blood cells. This production was abolished when the fractions were first treated with p-bromophenacyl bromide to reduce the venom phospholipase A2 activity contamination. In equine and human primary cultures of skeletal muscle, the N. n. kaouthia cardiotoxin (10 μM) and melittin (2 μM) caused a breakdown of phospholipids and production of free fatty acids and diacylglycerol in the absence of lysophospholipid formation. Additionally, melittin at higher concentrations (10 μM) caused triglyceride breakdown. These studies do not support the suggestion that snake venom cardiotoxins and melittin selectively activate endogenous phospholipase A2 activity. Instead, the toxins primarily activate endogenous phospholipase C activity and, in the case of melittin at high concentrations, triglyceride lipase activity.Key words: fatty acids, diacylglycerol, cytotoxins, phospholipase A2, phospholipase C.