bluuebunny/crossref_metadata_2025 huggingface dataset:
The cytochrome oxidase inhibitor azide also inhibits the alternative pathway of Neurospora crassa
Authors: Bridge Peter J. , Bertrand Helmut
Published: December 2009

Mutant strains of Neurospora crassa were used to investigate the effects of the inhibitor azide upon respiration mediated by either the cyanide-insensitive alternative mitochondrial respiratory pathway or the "standard" cyanide-sensitive cytochrome pathway. The use of appropriate cytochrome-oxidase-deficient and alternative-oxidase-deficient mutants permitted respiration to be restricted to the pathway of choice. In addition to its known effect upon cytochrome oxidase, azide was found to inhibit the alternative pathway of Neurospora with half-maximal inhibition occurring at 1.3 mM azide. This finding indicates that the activities of at least some of the "novel" azide-sensitive oxidases that have been discovered recently in organisms with inducible or constitutive cyanide-insensitive respiratory systems might be attributable to the "standard" salicylhydroxamate-sensitive mitochondrial alternative pathway.
Modification of biological activities of Ricinus communis agglutinin by cross-linking with formaldehyde
Authors: Turpin E. , Wantyghem J. , Beaudry P. , Néel D. , Goussault Y.
Published: December 2009

Formaldehyde treatment of Ricinus communis agglutinin, a nonmitogenic toxic 120 000 molecular weight (MW) lectin, yielded two distinct protein fractions: one was heterogeneous and contained high molecular weight lectin polymers (> 120 000), and the other was a homogeneous 120 000 MW protein. Both fractions lost their cytotoxicity after formaldehyde treatment and stimulated thymidine incorporation into lymphocytes. Binding of both treated lectin fractions to lymphocytes exhibited positive cooperativity, whereas binding of untreated lectin did not.
A genetical approach to the physical mapping of chloroplast genes in Chlamydomonas
Authors: Lemieux Claude , Turmel Monique , Seligy Verner L. , Lee Robert W.
Published: December 2009

Among the 100 potential chloroplast genes encoding polypeptides in higher plants, only 8 have been identified and mapped so far. Physical mapping of additional polypeptide genes for which there is no molecular probe can be facilitated by taking advantage of chloroplast genetics. We have demonstrated that it is possible to use a combination of genetical and molecular approaches to map directly a gene locus on the chloroplast genome of the interfertile green algae Chlamydomonas eugametos and Chlamydomonas moewusii. Because of the extensive recombination of chloroplast DNA and the biparental inheritance of chloroplast genetic markers detected in C. eugametos – C. moewusii hybrids, the inheritance of such genetic markers can be correlated with that of certain chloroplast DNA restriction fragments characteristic of one or the other parent. In 41 hybrids, a strict correlation was noted between the inheritance of a streptomycin resistance marker and that of chloroplast DNA restriction fragments encoding the two 16S rRNA genes. These results, therefore, support the view that the streptomycin resistance locus resides in each of the two 16S rRNA genes.
Mechanism of antibody stimulation of hexose transport in rat myoblasts
Authors: Lo Theodore C. Y. , Duronio Vincent
Published: December 2009

We have recently demonstrated that exposure of rat myoblasts to anti-rat myoblast antiserum results in two- to three-fold activation of hexose transport. The present communication reports the possible mechanism(s) by which specific antibody can bring about such activation. Studies with Fab and Fc fragments indicate that the binding of Fab to specific cell surface component(s) is not sufficient to trigger activation of hexose transport; the immunoglobulin G (IgG) mediated dimerization of membrane components is required for this process. Although cytochalasin D has no effect on hexose transport in control and antibody-treated cells, pretreatment of cells with this inhibitor prevents antibody-mediated activation of hexose transport. It may be inferred from this observation that proper disposition of membrane components is required for the dimerization of membrane receptors. Since this activation of hexose transport is an irreversible process, it is possible that covalent modification of membrane components may have occurred as a result of antibody treatment. Pretreatment of cells with ammonium chloride or methylamine is found to abolish the antibody-mediated activation of hexose transport, even though these inhibitors have no effect on hexose transport in control and antibody-treated cells. These inhibitors may be acting on transglutaminase and (or) on some other proteins involved in the activation process. Several lines of evidence suggest that limited proteolytic cleavage of membrane components may be involved in the antibody-mediated activation of hexose transport. First, pretreatment with several protease inhibitors prevents activation of hexose transport. Second, several cell surface proteins are missing in antibody-treated cells. Third, limited proteolysis of cell surface proteins with trypsin can also bring about activation of hexose transport. In view of the fact that proteolytic activity cannot be detected in various IgG and serum preparations, it seems likely that endogenous membrane associated proteases may be involved in this activation process.
Direction of synthesis of the message for a 35 000 polypeptide of herpes simplex virus type 2
Authors: Frost Eric , Filion Mario , Suh Martha
Published: December 2009

A restriction endonuclease map of the coding sequences for the transformation-related 35 000 polypeptide of herpes simplex virus type 2 is reported, using 11 enzymes that cut DNA infrequently. Hybrid-arrested translation with small DNA fragments was used to locate the 5′ terminus of these coding sequences. A novel procedure involving translation of the 35 000 mRNA selected with cloned viral DNA that had been digested with exonuclease was employed to confirm that this message is transcribed from left to right. The 35 000 polypeptide can thus be assigned to a 1.2-kilobase mRNA recently reported with the same starting point and direction.
Transcription of a trout protamine gene in vitro: the effects of alteration of promoters
Authors: Jankowski Jacek M. , Dixon Gordon H.
Published: December 2009

An in vitro approach has been used to study trout protamine gene expression using various recombinant plasmids containing trout protamine genes as templates in the HeLa cell lysate transcription system. The specific RNA transcript which is protected against S1 nuclease digestion by hybridization to the protamine gene sequence is α-amanitin sensitive (1 μg/mL), showing that RNA polymerase II is involved. The sizes of transcripts from templates linearized with Bam HI, Rsa I, and Hpa II (all downstream from the putative TATA box) are consistent with those predicted from the known sequence of the protamine gene. Digestion at an Alu I site only 14 base pairs (bp) upstream from TATA box has no effect on the accuracy of transcription in vitro; however, cutting at an Ava II site 9 bp downstream from the TATA box (reading from the first T) abolishes transcription. Chimeric plasmids, in which a herpes simplex virus (HSV-1) thymidine kinase (tk) promoter is tandemly inserted upstream from the trout protamine DNA sequences or as a replacement of the natural protamine promoter, were constructed. Use of these plasmids allowed an examination in a single assay of eight different putative promoter sequences (TATAAAA, TATAAA, TACAAA, TATATA, TATTTAA, CATATTA, TATATTAT, and TATTTAT) that are localized in either the protamine or the tk genes. The canonical TATAAAA promoter (the natural protamine promoter) was the strongest one and, in its presence, none of the others were used significantly for transcription. However, when this promoter was removed the weaker promoters were able to promote transcription.
Resolution and quantitation of diacylglycerol moieties of natural glycerophospholipids by reversed-phase liquid chromatography with direct liquid inlet mass spectrometry
Authors: Pind S. , Kuksis A. , Myher J. J. , Marai L.
Published: December 2009

The sn-1,2-diacylglycerol moieties of natural phosphatidylcholines as the tertiary-butyldimethylsilyl ethers were resolved on the basis of their carbon number and degree of unsaturation by high pressure liquid chromatography on reversed-phase C18 columns. Using acetonitrile and propionitrile as eluting solvents and reagent gases, the yields of both quasi molecular and fragment ions were found to vary with the degree of unsaturation and positional distribution of the fatty acids in the diacylglycerol molecules, and appropriate calibration factors were necessary for accurate quantitation. In the absence of pure structural isomer and mixed acid standards, we have determined preliminary calibration factors for total and specific ion current responses, by comparing the peak area ratios obtained by liquid chromatography – mass spectrometry with the weight and mole proportions of molecular species known to be present in the samples from detailed analyses by capillary gas–liquid chromatography on polar liquid phases. It was found that the total chemical ionization current response agreed closely with the weight composition of the molecular species. The relative yields of the (MH – 132)+ ions varied over one- to three-fold, while those of the (MH – RCOOH)+ ions varied over a three- to four-fold range of intensities. After suitable calibration of the relative ion response, it was possible to determine the identity and quantity of all common molecular species in the test diacylglycerophospholipids. Although the derived factors include both chromatographic and mass spectrometrie effects and are obtained with a gradient of reagent gases, they appear to be generally applicable.
Wheat-embryo ribonucleates. XV. Characterization of a fraction of RNA subject to conspicuous terminal labeling during early germination of wheat embryos
Authors: Kennedy Theresa D. , Lane Byron G.
Published: December 2009

Various techniques have been used to characterize the rapidly migrating electrophoretic components (RMEC) in an RNA fraction which is subject to conspicuous terminal labeling (mostly adenosine) when wheat embryos are pulse labeled with 3H-labeled nucleosides during early germination. Detection of terminally labeled RMEC RNA is most favoured during early germination when labeling of 3′-hydroxyl termini in preexisting RNA, catalyzed by RNA nucleotidyltransferases, is greatest in relation to nonterminal labeling of nascent RNA, catalyzed by RNA polymerases. The RMEC RNA is tenaciously associated with a fraction of high-molecular-weight RNA and it can be subdivided and classified into two fractions, RMEC-1 and RMEC-2, by electrophoresis in polyacrylamide, gel filtration through Sephadex, or assay of amino acid acceptance. The RMEC-1 RNA and bulk wheat-embryo tRNA have similar capacities to accept a wide variety of amino acids, but RMEC-2 RNA does not accept any of the amino acids tested. RMEC-2 RNA is broadly heterodisperse and one of its component polynucleotides has been identified by sequence analysis as a tridecanucleotide fragment from the 3′-hydroxyl end of 5.8S rRNA. This study was undertaken as part of a broader program in which isotopic labeling and analysis of nucleates and proteins in germinating wheat embryos have been used to detect signal events and to evaluate their physiological significance. It is concluded that conspicuous terminal labeling of RMEC RNA during early germination of wheat embryos is unlikely to be of physiological importance. However, isotopic labeling and analysis of the nucleates and proteins in germinating embryos has uncovered other signal events which appear to be of physiological importance. Discovery of the existence of a number of developmentally regulated proteins and the way in which discovery of these proteins is expected to direct the course of future investigations in the laboratory are subjects of a brief discussion.
3-Aminobenzamide does not increase repair patch size in mammalian cells
Authors: Walker Ian G. , Th'ng John P. H. , Schrader Timothy J. , Norry Thomas W.
Published: December 2009

Several groups of investigators have reported that the extent of repair replication following treatment of mammalian cells with dimethyl sulfate is increased by 3-aminobenzamide (3AB) over that occurring in its absence. Two plausable explanations for this phenomenon were tested. The first is that the number of nucleotides inserted per repair site, that is the repair patch size, is increased and the second is that the rate of repair is increased. Human T98G cells were treated with dimethyl sulfate and allowed to repair their DNA in the presence or absence of 3AB. It was found that the presence of 3AB did not increase the repair patch size nor the rate of removal of methylation products from the DNA, which is assumed to equal the rate of repair. The results of further experiments suggested that the 3AB effect can be explained by changes in nucleotide precursor pools.
Regulation of phosphatidylcholine synthesis and the activity of CTP:cholinephosphate cytidylyltransferase in myoblasts by 12-O-tetradecanoylphorbol-13-acetate
Authors: Hill Stephen A. , McMurray William C. , Sanwal Bishnu D.
Published: December 2009

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increases rate of incorporation of 32P into phosphatidylcholine and phosphatidylethanolamine about twofold in a line of rat skeletal myoblasts (L6). This effect is specific for TPA and is not caused by the nontumor-promoting analogue 4α-phorbol didecanoate (α-PDD). Cycloheximide and actinomycin D have no effect on the TPA-dependent increase in labelling. There is no significant difference in the rate of disappearance of [14C]glycerol from phospholipids in cells exposed to TPA. Several enzymes of phospholipid synthesis in myoblasts exposed to TPA are unaltered in activity, except CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) whose activity is enhanced 1.5- to 2-fold compared with cells exposed to α-PDD. Exposure of cells for only 15 min to a concentration of 1 × 10−6 M TPA is sufficient to cause maximum activation of the enzyme. The cytidylyltransferase activity is associated largely with the particulate fraction in the cell-free extracts of myoblasts. Following gel filtration on Bio-Gel A-1.5m, the enzyme activity appears in the void volume and smaller molecular weight forms are not discernible. The kinetic parameters of cytidylyltransferase in preparations from treated cells are unaltered except for an increased Vmax. The apparent Km value for CTP for the enzyme from treated and untreated cells is about 0.9 mM. Both enzyme preparations are weakly inhibited by lysophosphatidylcholine, but are unaffected by several other phospholipids.
Biosynthetic utilization of photoreactive fatty acids by rat liver microsomes
Authors: Leblanc Pierre , Gerber Gerhard E.
Published: December 2009

The photoreactive ω-diazirinophenoxy derivatives of nonanoate, undecanoate, tridecanoate, and pentadecanoate were shown to be activated by rat liver microsomes to the corresponding acyl-CoA derivatives. The Km and Vmax for these fatty acid analogues were determined; the values obtained indicate that the addition of a photoreactive group to an alkyl chain has an effect similar to that of elongation of the chain by about seven carbons. Incubation of microsomes in the presence of lysophospholipids resulted in the incorporation of the photoreactive fatty acids into the corresponding phospholipids. The ability of mammalian systems to utilize these photoreactive fatty acids for phospholipid synthesis establishes their suitability as photoaffinity analogues of fatty acids.
An increased incorporation of fatty acid into triacylglycerols of neuronal nuclei in vitro in the presence of CMP and EGTA
Authors: Baker R. Roy , Chang Huu-Yi
Published: December 2009

Neuronal nuclei (N1) were isolated from cerebral cortices of 15-day-old rabbits. With the addition of both EGTA and CMP the incorporation of radioactive oleate into N1 triacylglycerols in vitro (in the presence of ATP, CoA, and MgCl2) was increased threefold. The same large increase could not be achieved using citrate or EDTA in the presence of CMP or using AMP, UMP, or TMP in the presence of EGTA. The increased labelling of N1 triacylglycerols could be greatly reduced when CDP-choline was added to incubations containing EGTA and CMP. Levels of endogenous N1 diacylglycerols increased threefold following a 10-min incubation in the presence of buffer (pH 7.4) and MgCl2, when CMP and EGTA were also added. Of the major N1 phospholipids, phosphatidylcholine was most similar in fatty acid composition to the enlarged endogenous diacylglycerol pool. The rate of formation of oleoyl-CoA in fraction N1 was not significantly changed by the presence of EGTA and CMP. Rates of triacylglycerol labelling could only be modestly increased when EGTA and CMP were added to incubations containing N1 samples with artificially enlarged endogenous diacylglycerol pools (produced by phospholipase C preincubations). It is suggested that EGTA, as a Ca2+ chelator, and CMP, as a substrate, may allow an enhanced diacylglycerol production mediated by the back reaction of cholinephosphotransferase in N1. The endogenous N1 diacylglycerols produced in the absence of EGTA and CMP may come from another metabolic route.
Purification, properties, and N-terminal amino acid sequence of certain 50S ribosomal subunit proteins from the archaebacterium Halobacterium cutirubrum
Authors: Matheson Alastair T. , Yaguchi Makoto , Christensen Patricia , Rollin C. Fernand , Hasnain Sadiq
Published: December 2009

Sixteen ribosomal proteins (r-proteins) from the 50S ribosomal subunit of the archaebacterium Halobacterium cutirubrum have been purified and their amino acid composition and partial N-terminal amino acid sequence have been determined. These proteins as a group are much more acidic than the large subunit r-proteins from eubacteria or eukaryotes. Little sequence homology is evident between the 50S subunit archaebacterial r-proteins and the equivalent proteins from the eubacterium Escherichia coli.
Human placental β-galactosidase: structural and immunological observations
Authors: Jones Christopher S. , Mahuran Donald , Lowden J. Alexander , Callahan John W.
Published: December 2009

β-Galactosidase purified to apparent homogeneity from human placenta occurred in two separable fractions. A low molecular mass form (relative mass (Mr) 170 000) is composed of a single polypeptide chain (Mr 70 000). This was derived from a larger form by molecular sieve chromatography at both low (10 mM) and high (500 mM) NaCl concentration. The larger form of β-galactosidase also contains small amounts of two polypeptides with apparent Mr values of 23 000 and 35 000 daltons. Both forms of the enzyme hydrolyze synthetic aryl galactosides and natural glycolipid substrates at comparable rates. Antibodies raised in rabbits against the low Mr β-galactosidase also cross-reacts with the high Mr enzyme. The antibody preparation also cross-reacted with β-hexosaminidase even though the latter was found at very low levels in the antigen, as judged by lack of detection of representative protein bands on sodium dodecyl sulfate – polyacrylamide gel electrophoresis and enzyme activity measurements. A portion of this cross-reactivity (35%) against β-hexosaminidase could not be absorbed from the preparation without the simultaneous loss of β-galactosidase activity, suggesting that the two enzymes show a degree of antigenic identity.
Abnormal excretion of oestrogens in complicated pregnancies
Authors: Cohen Saul L. , Ho Patrick
Published: December 2009

The tremendous interpatient variations that occur for oestrogen excretion during pregnancy are largely overcome by expressing results in terms of relative concentrations (i.e., as percent of total oestrogens excreted). Specimens of urine from 28 pregnant patients with a variety of complications were assayed for their oestrogen content by an "oestrogen profile" technique and the results thus expressed were compared with those from 20 normal pregnancies. Statistically significant changes are shown in the group of patients in the "above" normal range (normal range = normal mean ± 3 × SE) for five oestrogens (oestrone, 2-hydroxyoestrone, 2-methoxyoestrone, 2-hydroxyoestradiol, and 2-hydroxyoestriol) and "below" the normal range for one oestrogen (oestriol). When the means of the oestrogen values for the different groups of patients were examined, two things became obvious; firstly, only patients with hypertension showed these changes, and secondly, two additional oestrogens (16-hydroxyoestrone and 16-epioestriol) were excreted in large amounts by the normotensive "high risk" patients. A comparison of the two methods of expressing oestrogen assay results is made quantitatively (as milligrams per day) versus relatively (as percent of total oestrogens excreted), and advantages and disadvantages of each are discussed.
Malate dehydrogenase from thermophilic Humicola lanuginosa and Mucor pusillus: purification and comparative properties of the enzymes with differing thermostabilities
Authors: Wali Anupam S. , Mattoo Autar K.
Published: December 2009

Malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) was purified from the thermophiles Humicola lanuginosa and Mucor pusillus. The H. lanuginosa enzyme was homogeneous on sodium dodecyl sulphate – polyacrylamide gels, while the M. pusillus enzyme was more than 95% pure. The two enzymes appeared to be composed of two subunits of equal size, each of 36 000 daltons (H. lanuginosa) or 33 000 daltons (M. pusillus). The native enzymes revealed molecular weights of 68 000 as determined by gel filtration. The isoelectric points of malate dehydrogenase from H. lanuginosa and M. pusillus were 3.9 and 4.2, respectively. The reduction of oxaloacetate by the H. lanuginosa enzyme was optimum at pH 8.5–9 with apparent Km's of 0.12 mM for oxaloacetate and 0.027 mM for NADH. On the other hand, M. pusillus enzyme snowed a pH optimum of 7.8–8.5 with apparent Km's of 0.075 mM for oxaloacetate and 0.1 mM for NADH. The L-malate oxidation reaction was catalyzed optimally at pH 10 by the H. lanuginosa enzyme with apparent Km's of 5.8 mM for malate and 0.1 mM for NAD, while the M. pusillus enzyme catalyzed it optimally between pH 9.5 and 10 with apparent Km's of 4.44 mM for malate and 0.16 mM for NAD. The optimum temperature for reduction of oxaloacetate was 50 °C for both the enzymes. The H. lanuginosa enzyme was resistant to heat inactivation at 40 °C, but lost 60% of its activity after 15 min at 50 °C. Mucor pusillus enzyme, on the other hand, retained 90% activity at 60 °C after 10 min. The two enzymes were protected from heat inactivation by monovalent cations (viz Na+, K+, and NH4+), as well as citrate, which may possibly involve conformational changes.
A 68 000 dalton protein genetically associated with corticotropin-sensitive adenylate cyclase activity. Purification and preliminary characterization using a specific antiserum
Authors: Schimmer Bernard P. , Robinson Ranga , Tsao Jennivine , Watt Valerie M.
Published: December 2009

Previous work from this laboratory described an association, based on genetic evidence, between a 68 000 dalton protein (p68) and corticotropin (ACTH) sensitive adenylate cyclase activity among variants of the Y1 mouse adrenocortical tumor cell line. To study the nature of this association further, we have purified p68 and raised a polyclonal anti-p68 serum in rabbits. A variant subclone of the Y1 line, in which p68 comprised approximately 10% of total soluble protein, was used as starting material. Purification of p68 was achieved by passage of a 100 000 × g supernatant fraction over DEAE-cellulose, fractionation with ammonium sulfate, and chromatography on hydroxylapatite. The purified protein had an isoelectric point of 7.3, a polarity value of 46%, and a blocked amino terminal end group. A rabbit antiserum raised against the purified p68 had a titer of 1:16 000 and specifically precipitated p68 from extracts of Y1 cells labeled with L-[35S]methionine. Using this antiserum, p68 also was detected in other cell lines including mouse erythroleukemia and Sertoli cells; rat Leydig, ovary, and glioma cells; and Chinese hamster ovary cells. The presence of p68 in a variety of cell types suggests that the function of p68 is not restricted to adrenal cells or to specific actions of ACTH.
Mechanisms of hormonal regulations in brown adipose tissue of developing rats
Authors: Skala Josef P.
Published: December 2009

The need for nonshivering heat production, a principal function of brown adipose tissue, is accentuated in neonates. Accordingly, brown fat in the rat exhibits a very pronounced process of morphological and functional maturation perinatally, reaches a peak in its differentiation and heat-generating capacity within 1–2 weeks after birth, and undergoes involutive changes later in life. The later process of dedifferentiation can be either prevented or reversed by exposing the animals to cold ambient temperature for a prolonged period of time (cold acclimatization). The regulation of both the tissue maturation processes and the superimposed acute heat production are hormone mediated. Thus, the hormone receptor system within the adipocyte membrane and the sequence of molecular events interconnecting the initial hormonal stimulus with its final intracellular effect(s) are of considerable importance. The brown adipocytes of developing rats possess adrenoreceptors that can be pharmacologically classified as β1 (linked to adenylate cyclase) and α2 (possibly linked to guanylate cyclase), multiple forms of cyclic nucleotide dependent and independent protein kinases, a protein kinase inhibitor, and at least two distinct phosphoprotein phosphatases associated with three phosphoprotein phosphatase modulators. The characteristics and developmental alterations of these regulatory components were studied in considerable detail by our group during the past decade. The results uncovered several target systems for ontogenic modifications of hormonal responses. Strong support was obtained for the hypothesis that protein phosphorylation and dephosphorylation is a major molecular mechanism involved in the regulation of both the brown adipocyte function and its proliferative activity during ontogenic development.
Interactions calmoduline–lipides : étude de l'encapsulation de la protéine en chromatographie liquide
Authors: Kercret Henri , Delaunay Thierry , Duval Jacques
Published: December 2009

The chromatographic behaviour of calmodulin (CaM) has been studied. We show two species eluted in different places on Sepharose 4B. We demonstrate that the relative amount of these two species is calcium dependent. The entrapment of calmodulin in liposomes is possible. The study of the entrapment conditions shows that only one of the two species is able to be trapped. We show that the encapsulation of calmodulin is three times higher when calcium is present.
The role of the cytoskeleton in hormone action
Authors: Hall Peter F.
Published: December 2009

The structure and functions of microfilaments and microtubules is briefly reviewed. Based on evidence from a variety of cells with various experimental approaches, it is proposed that the cytoskeleton is important in the functions of cells as follows: the cytoskeleton appears to be involved in attachment (to other cells and to substrata used in cell culture) and in movement; the cytoskeleton appears to be involved in the complex relationship between the surface and the interior of the cell. This is a two-way relationship in which the cell uses the cytoskeleton to influence the distribution of proteins within the plasma membrane and perhaps as a go-between to transmit information from the surface of the cell to the interior. Within the cell, the cytoskeleton appears to coordinate intracellular activities (e.g., between nucleus and cytoplasm) and to provide a compartmentation which restricts the movements of intracellular structures (from molecules to organelles), thereby facilitating productive encounters between such structures with an efficiency that is presumed to exceed that of random motion. The cytoskeleton may arrange the various components of the cell in space; it may direct intracellular traffic and promote intracellular movement. This loose picture of cytoskeletal activity may be more emphatic than the available data warrant. It should be regarded as informed extrapolation based upon fragments of information. When these ideas are applied to endocrine function, we can see that the secretion of hormones (exemplified by thyroid hormones and insulin) requires cytoskeletal activity to provide direction and perhaps to promote movement. In steroidogenic cells the movement of cholesterol from cytoplasm to mitochondria requires microfilaments. The antidiuretic hormone requires microfilaments and microtubules to promote transport of water through cells in response to osmotic gradients. Microfilaments adjust the compliance of the cell to provide a path for water and microtubules influence the permeability of the cell membrane to water.
The induction of α-amylase by starch in Aspergillus oryzae: evidence for controlled mRNA expression
Authors: Erratt J. A. , Douglas P. E. , Moranelli F. , Seligy V. L.
Published: December 2009

The induction of α-amylase by starch has been studied in the filamentous fungus Aspergillus oryzae. Low levels of α-amylase activity were found in both intracellular and extracellular samples from glucose-grown cultures. However, α-amylase activity increased when starch was the sole carbon source. The intracellular enzyme activity was induced by a factor of approximately 6.5, while the extracellular activity increased 20-fold over that found in the glucose-grown cultures. Regardless of the carbon source or cellular location, the molecular weight of the active protein was 52 500 ± 1800 and only this protein reacted with antibodies specific for α-amylase. A parallel study of the in vitro translated proteins directed by poly(A)+ RNA fractions indicated a presumptive α-amylase with a similar isoelectric point but with a molecular weight of approximately 54 000. This protein was most prevalent using RNA from early, exponentially growing cultures in starch medium. Immunoprecipitation data indicate that the abundance of α-amylase transcripts decreases dramatically after the first 12 h, reflecting an initial transcription control for the expression of this enzyme.
Production of monoclonal antibodies to a steroid plant growth regulator
Authors: Horgen P. A. , Nakagawa C. H. , Irvin R. T.
Published: December 2009

The active component of the brassin complex of higher plants has been shown to be a C28 steroidal lactone called brassinolide. This compound has been shown to enhance both cell division and cell growth. Monoclonal antibodies directed against a synthetic brassinosteroid have been produced in CAF1 mice. The mice were immunized with a saline solution containing the steroid hapten noncovalently bound to chloroform–methanol extracted fetal calf serum. Hybridomas were generated by fusing primed mouse spleen cells with SP2/0 myeloma cells. Sixty-one hybridoma clones were screened; four of these gave positive enzyme-linked immunosorbent assay (ELISA) values with the brassinosteroid hapten. One of these clones produced an immunoglobulin G antibody with a high specificity for the synthetic antigen. The brassinosteroid hapten which differs from the natural brassinolide by the position of a methyl group on the C24 carbon gave a considerably higher ELISA value than the natural growth regulator and several other plant steroids. The distribution of brassinolide in Brassica napus tissues was examined by ELISA with the monoclonal antibody.
The low molecular weight autoinhibitor of sexual development in Dictyostelium discoideum inhibits cell fusion and zygote differentiation
Authors: Szabo Stephen P. , O'Day Danton H.
Published: December 2009

A previous study has shown that, during the sexual cycle of Dictyostelium discoideum, zygote giant cell formation is regulated by an autoinhibitor. Experiments reported here show that the autoinhibitor inhibits two events of zygote development: cell fusion and subsequent giant cell differentiation. The autoinhibitor is heat stable and has a molecular weight around 500. Medium containing the autoinhibitor can be diluted 500-fold without loss of activity. Preliminary experiments show that, although levels of ammonia double during the 8-h period of autoinhibitor production, added ammonia does not mimic the inhibiting effect. cAMP at 1 mM inhibits both binucleate formation and differentiation, but the concentration of cyclic AMP in 28-h cultures is only 13.4 pmol, a level which does not affect zygote development. Thus, it is established that neither of these critical regulators of other developmental processes in D. discoideum is the autoinhibitor.
Carrier-mediated ion transport in lipid bilayer membranes
Authors: Laprade Raynald , Grenier François , Pagé-Dansereau Monique , Dansereau Janine
Published: December 2009

The electrical properties predicted by a widely accepted model for carrier-mediated ion transport in lipid bilayers are described. The different steps leading to ion transport and their associated rate constants are reaction at the interface between an ion in the aqueous phase and a carrier in the membrane (kRi), followed by translocation of the ion–carrier complex across the membrane interior (kis) and its dissociation at the other interface (kDi) after which the free carrier crosses back the membrane interior (ks). Results on glyceryl monooleate (GMO) membranes for a family of homologue carriers, the macrotetralide actin antibiotics (nonactin, monactin, dinactin, trinactin, and tetranactin) and a variety of ions (Na+, Cs+, Rb+, K+, NH4+, and Tl+) are presented. Internally consistent data obtained from steady-state electrical measurements (zero-current potential and conductance, current-voltage relationship) allow us to obtain the equilibrium permeability ratios for the different ions and show that for a given carrier kRi is relatively invariant from one ion to the other, except for Tl+ (larger), which implies that the ionic selectivity is controlled by the dissociation of the complex. The values of the individual rate constants obtained from current relaxation experiments are also presented and confirm the findings from steady-state measurements, as well as the isostericity concept for complexes of different ions with the same carrier (kis invariant). These also allow us to determine the aqueous phase membrane and torus membrane partition coefficients. Finally, the observed increase in kis from nonactin to tetranactin and, for all homologues, from GMO–decane to solvent-free GMO membranes, together with the concomitant decrease in kDi, can be explained in terms of modifications of electrostatic energy profiles induced by variations in carrier size and membrane thickness.
Microtubule associated proteins in microtubule preparations made with and without glycerol
Authors: Keates Robert A. B.
Published: December 2009

Preparation of microtubule protein in the presence or absence of glycerol results in differences in polymerization properties and content of microtubule associated proteins. The variation in properties appears to result from the reduced proportion of microtubule associated proteins in preparations made with glycerol. I have used the colchicine binding assay to monitor recovery of active tubulin and have found that a single factor can account for the difference. During the in vitro assembly of microtubules from the crude brain homogenate, glycerol promotes polymerization of the bulk of the tubulin, while less than half is incorporated into microtubules in the absence of glycerol. Assembly of partly purified microtubule protein is not enhanced by glycerol however. Microtubule associated proteins present in the crude homogenate are almost completely incorporated into the microtubules regardless of the presence of glycerol, and their high content in glycerol-free preparations appears to be the trivial result of low tubulin recovery. The high affinity of microtubule associated proteins for the assembled microtubules has other consequences for in vitro studies of microtubule assembly, and critical concentration plots to determine the polymerization equilibrium constant can be distorted unless the preparation used has a high content of microtubule associated proteins.
The interaction of guanine nucleotides with the adenylate cyclase complex in white adipocyte membranes of lean and obese (ob/ob) mice
Authors: Bégin-Heick Nicole
Published: December 2009

The ability of the guanine nucleotides (Gpp(NH)p and GTP-γ-S) and of fluoride to stimulate adenylate cyclase is equal in the white adipocyte of lean and ob/ob mice. This suggests that the interaction between the guanine nucleotide regulatory component (N) and the catalytic unit of adenylate cyclase is normal. In the membranes from obese mice, the addition of agonist did not diminish the concentration of guanine nucleotide required for half-maximal activation, as it did in membranes from lean mice, indicating that the interaction between the receptor and N may be altered in the obese mouse. GDP-β-S and GTP were found to be equally potent in inhibiting Gpp(NH)p-activated adenylate cyclase activity in both groups. Experiments with membranes loaded with or depleted of GDP showed that GDP did inhibit the activation of the cyclase under all conditions tested. Although depleting the membranes of obese mice of GDP improved their response to isoproterenol, it did not restore the response to levels seen in the membranes of lean mice. The data show that it is not likely the binding of GDP which limits cyclase stimulation by agonists. The defect must therefore reside in another property of N.
Partial resialylation of human asialotransferrin types 1 and 2 in the rat
Authors: Regoeczi Erwin , Chindemi Paul A. , Debanne Maria T.
Published: December 2009

125I-labeled asialotransferrin types 1 and 2 were administered in small doses to rats. The protein still in the plasma after 1–12 h was partially repurified and electrophoresed at pH 8.1, together with a transferrin standard that is composed of all six forms of the protein with respect to sialic acid content. The electrophoretic mobility of both asialotransferrins increased with time, type 2 being affected sooner than type 1. The changed mobility was due to increased electronegativity that was fully reversible by treatment of the samples with neuraminidase, thus identifying the underlying cause as partial resialylation. Asialotransferrin incubated in vitro with serum, plasma, or whole blood for 16 h exhibited no change in electrophoretic mobility. In conjunction with an earlier study on asialotransferrin type 3, it was found that the apparent speeds of resialylation of the three asialotransferrins were in the same order as their affinities for the asialoglycoprotein-binding hepatic lectin. This suggests the involvement of an endo- rather than of an ecto-transferase. Transfer of 59Fe from asialotransferrins to the liver was used to monitor the frequency of hepatocyte–asialotransferrin interactions. Iron deposition in the liver took place much more rapidly than the appearance of detectable quantities of partially resialylated asialotransferrin molecules in the circulation. It is concluded that each asialotransferrin molecule probably undergoes several passages through the hepatocyte before its glycans become modified.
Energy-dependent H+ and K+ translocation by the reconstituted yeast plasma membrane ATPase
Authors: Villalobo Antonio
Published: December 2009

A highly purified plasma membrane ATPase from the yeast Schizosaccharomyces pombe incorporated into liposomes was able to carry out translocation of H+ and K+ in the absence of the substrate ATP, when a membrane potential of appropriate polarity was applied. In the absence of ATP, the membrane potential induced K+ translocation was strongly inhibited by the ATPase inhibitor vanadate. [Formula: see text], but not [Formula: see text], stimulated the rate of ATP hydrolysis in the absence, but not in the presence, of the H+-conducting agent carbonylcyanide m-chlorophenylhydrazone. Sodium ion on either side of the membrane did not have any stimulatory effect. The potassium ion translocation driven by ATP hydrolysis appeared to have two different kinetic components. Although the ATP-dependent K+ transport strictly required the presence of a membrane potential, the rate of K+ translocation was not affected by a broad modulation of the degree of coupling (q) between ATP hydrolysis and the electrogenic H+ translocation. These experiments support the view that the yeast plasma membrane ATPase not only uses the membrane potential generated by the electrogenic H+ translocation, but also uses part of the free energy of the hydrolysis of ATP (ΔGP) to translocate potassium ion across the cytoplasmic cell membrane.
Caractéristiques fonctionnelles des activités peroxydasiques des feuilles et cals d'une plante à métabolisme acide crassulacéen, Pedilanthus tithymaloides variegatus, Euphorbiaceae
Authors: Bricage Pierre
Published: December 2009

Peroxidases (donor:hydrogen-peroxide oxidoreductase; EC 1.11.1.7) from leaf tissues and calli were extracted and compared in terms of their specific activities at different pH and temperatures, their isoenzyme patterns and physicochemical properties. Three groups of enzyme activities were detected and their purification was performed by conventional methods (ammonium sulfate fractionation, ion-exchange chromatography, gel filtration, affinity chromatography).
Changes in the cytoplasmic and nuclear activities of the ribonucleotide reductase holoenzyme and its subunits in regenerating liver cells in normal and thyroparathyroidectomized rats
Authors: Youdale T. , Frappier L. , Whitfield J. F. , Rixon R. H.
Published: December 2009

The level of the cytoplasmic ribonucleotide reductase nonheme-iron-containing L2 subunit in regenerating rat liver cells began rising about 2 h before the onset of DNA synthesis, rose sharply to a maximum level about 4 h before the DNA-synthetic activity reached its peak, and then stayed at this high level even after the cells had finished replicating their DNA. The cytoplasmic level of the CDP-specific, effector-binding L1 subunit and the holoenzyme activity began rising together about 2 h after the L2 subunit began increasing and at the same time as the DNA-synthetic activity, but subsequently rose much more slowly than the L2 subunit and continued rising even after the cells had finished making DNA. The nuclear level of the L2 subunit did not rise in the regenerating liver cells, but the nuclear level of the L1 subunit and the holoenzyme activity began rising together about the same time as the DNA-synthetic activity, peaked briefly 4–6 h before the peak DNA-synthetic activity, and dropped sharply back to the basal levels by the time the DNA-synthetic activity reached its peak, but then rose again slowly as the cells finished making DNA. Thyroparathyroidectomy 72 h before partial hepatectomy prevented the cytoplasmic and nuclear subunits and holoenzyme activity from rising and prevented most of the remaining liver cells from initiating DNA synthesis.
Phosphorylation of the transferrin receptor in isolated sheep reticulocyte plasma membranes
Authors: Johnstone Rose M. , Adam Mohammed , Turbide Claire , Larrick James
Published: December 2009

The transferrin receptor of sheep reticulocyte plasma membranes undergoes phosphorylation with [γ-32P]ATP in isolated plasma membranes. The phosphorylation is stimulated by Mn2+, Co2+, and Mg2+, but not by Ca2+, Ba2+, Zn2+, Fe2+, or Cu2+. There is no detectable effect of cyclic nucleotides on the phosphorylation of the receptor. Transferrin and a monoclonal antibody against the transferrin receptor have no apparent effect on receptor phosphorylation in intact cells or isolated membranes. Immunoprecipitates of the receptor retain ability to phosphorylate the receptor. The phosphorylation appears to be at a serine residue which turns over with a half time of 20–30 min. ATP appears to be the best, but not the only substrate for receptor phosphorylation.
Characterization and spectral properties of Proteus mirabilis PR catalase
Authors: Jouve Hélène Marie , Gaillard Jacques , Pelmont Jean
Published: December 2009

Purified catalase from a peroxide-resistant mutant (PR) of Proteus mirabilis displayed great similarities with the bovine liver catalase on the basis of its amino acid composition, content in prosthetic groups, and spectroscopic data. The bacterial enzyme was found to have 2.6 ± 0.2 mol of protoheme IX per tetramer, with an equivalent amount of titrable iron atoms. The optical absorption of P. mirabilis PR catalase in the presence of various anionic species (cyanide, azide, formate) was examined. The dissociation constant of the formate–enzyme complex was determined as 60 ± 2 mM at pH 7.5. Inhibition and spectral shifts induced by some thiol compounds were very similar to those reported with mammalian catalase. The electron paramagnetic resonance (EPR) spectra (at 9 GHz and 6 K) of bacterial catalase and its various complexes were reported. Two major different rhombic high-spin ferric signals could be seen in the g = 6 region, using either the pure enzyme or the cell crude extract. The balance between the two rhombic forms was reversibly altered by pH. Various changes in rhombicity were also observed after binding with anionic ligands. The EPR spectrum (at 40 K) of nitrosyl ferrous catalase was very similar to reported data with horse liver catalase.
Theory and practical application of coupled enzyme reactions: one and two auxiliary enzymes
Authors: Brooks S. P. J. , Espinola T. , Suelter C. H.
Published: December 2009

An extended and practical set of equations which describe coupled enzyme reactions is presented. The mathematical treatment relies on two assumptions: (a) the rate of the primary enzyme reaction is constant and (b) the reverse reactions are negligible. The treatment leads to the development of new equations which relate the time required for the concentration of a reaction intermediate to reach a defined fraction of its steady-state concentration to the kinetic parameters of the enzymes when mutarotation of one of the intermediates does not occur. The new equations reduce to those previously derived when the steady-state concentration of the intermediate is small compared with its Km value. A method for minimizing the cost of the two auxiliary enzyme system is also provided.
Theory and practical application of coupled enzyme systems: one and two coupling enzymes with mutarotation of an intermediate
Authors: Brooks S. P. J. , Espinola T. , Suelter C. H.
Published: December 2009

This paper provides equations to calculate the elasped time before the concentration of the final intermediate, in a sequence of coupled enzymatic reactions, achieves a defined fraction of its steady-state concentration when one of the intermediates undergoes mutarotation. The equations can be used to predict lag times for systems involving one coupling enzyme, as is the case when hexokinase or phosphoglucomutase activity is monitored using glucose-6-phosphate dehydrogenase as the auxiliary enzyme, or for systems of two coupling enzymes, as is the case when the activities of enzymes producing ATP (such as creatine kinase) are monitored by coupling the production of ATP to hexokinase and glucose-6-phosphate dehydrogenase. The theoretical aspects of the assay have been verified using hexokinase (as the primary enzyme) and glucose-6-phosphate dehydrogenase (as the coupling enzyme). A method of cost minimization, based on the above relationships, is also provided.