bluuebunny/crossref_metadata_2025 huggingface dataset:
Further purification and properties of rat uterine peroxidase
Authors: Keeping Hugh S. , Kimura Shioko , Lovsted Jane , Jellinck Peter H.
Published: December 2009

Peroxidase was purified 3700-fold from homogenates of estradiol-treated rat uteri by affinity chromatography on concanavalin A (ConA) – Sepharose followed by gel filtration on Bio-Gel P-150 with high recovery of enzyme. A single protein (molecular weight (MW) 45 000) staining for heme was shown by sodium dodecyl sulfate – polyacrylamide gel electrophoresis to be present in the peak fractions of enzymic activity eluted from the ConA–Sepharose column. This protein had the same mobility as bovine lactoperoxidase (MW 78 000) in a cationic gel electrophoretic system under nondenaturing conditions. Peroxidase activity in a NaCl extract of the uterus was lower than that in a CaCl2 extract but was unaffected by prolonged storage at −20 °C. In contrast, the CaCl2-extracted enzyme lost much, of its activity under these conditions by a process which could be prevented by the addition of glycerol. The sulfhydryl reagent, N-ethylmaleimide, which caused a marked increase in the activity of uterine peroxidase, provided only partial protection against inactivation during storage of CaCl2 extracts of this enzyme at low temperature.
Inhibition of adenine and hypoxanthine uptake by guanosine in conidia of Neurospora crassa
Authors: Greer Wenda L. , Wellman Angela M.
Published: December 2009

Guanosine competitively inhibits the uptake of adenine and hypoxanthine by the general purine-base permease in conidia of Neurospora crassa. There is no reciprocal effect of adenine or hypoxanthine on guanosine uptake so it is suggested that guanosine can bind to the general purine-base permease to cause this inhibition but is not transported through this system. It is known that guanosine is transported by two. separate nucleoside transport sites. Guanosine also noncompetitively inhibits the specific adenine uptake system of germinating conidia.
Tubulin synthesis during the synchronous cell cycle of Tetrahymena
Authors: Bird R. Curtis , Zimmerman Arthur M.
Published: December 2009

The distribution of polyribosome material and the synthesis of tubulin was determined for division synchronized Tetrahymena. Cultures of Tetrahymena pyriformis GL were induced to divide synchronously by the one-heat-shock-per-generation technique. After seven heat shocks (at 34 °C for 30 min) spaced one generation time apart (157 min), the cells divided synchronously at 80 and 195 min after the end of the last heat shock (EH), Sucrose gradient analysis of polyribosomes demonstrates a progressive reduction in the relative amount of monosomes compared with the total amount of ribosomal material recovered during the period between EH and the first synchronous division. During the period between the first and second synchronous divisions the percentage of monosomes rises immediately after division and does not change after 120 min EH at which time it is indistinguishable from levels found in log growth cells. The specific activity of tubulin was determined throughout the first and second synchronous cell cycles. Tubulin synthesis was measured by labeling cells with L-[35S]methionine and determining the levels of radioactivity comigrating with purified tubulin. A comparison of the resulting electrophoretic patterns reveals a marked induction of tubulin synthesis during G2 of the first and second (free-running) synchronous cell cycle. At its peak, tubulin is synthesized to a point approximately 60% higher than levels observed during the initial part of S phase during the synchronous cell cycle. The results are discussed in reference to other morphological events that occur during the cell cycle and in reference to the regulation of tubulin synthesis.
Purification of bovine and human acrosin
Authors: Elce John S. , McIntyre Elise J.
Published: December 2009

Acrosin from human spermatozoa was required for studies of immunological interference with fertilization, but no detailed purification scheme was available for the human enzyme. Since human semen samples cannot be obtained cheaply or in large numbers and contain relatively small amounts of acrosin, development of purification procedures was carried out with bovine semen. Bovine acrosin had not previously been fully purified, and over 1 mg of pure acrosin was obtained from 100 mL of bovine semen, by a process of saline and Triton X-100 washes of the spermatozoa, 1 mM HCl extraction, gel filtration, and ion-exchange and affinity chromatography. The bovine acrosin had a molecular weight (MW) of 39 000 and a specific activity of 93 U/mg, measured with 0.5 mM benzoyl arginine ethyl ester. The same extraction procedure could be followed for human acrosin, but better yields were obtained in the purification if the ion-exchange step was omitted. The human acrosin had a MW of 49 000, and traces of a 38 000 MW component were sometimes observed. From 14 human semen samples, containing initially 7–10 U of acrosin activity, about 2.5 U (approximately 20 μg of protein) could be obtained in a pure state.
Isolation and characterization of goat serum α1-globulin protease inhibitors
Authors: Hercz Albert
Published: December 2009

α1-Globulin-type protease inhibitors were isolated from goat serum by two methods, namely preparative isoelectric focusing and preparative electrophoresis in polyacrylamide gel. The fractions obtained by the first method showed varying isoprotein compositions by analytical isoelectric focusing. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) revealed the presence of one protein in the fractions with the same velocity of migration as purified human α1-antitrypsin and a second protein with a slightly higher migration velocity. The ratios of trypsin-inhibiting to chymotrypsin-inhibiting capacities in all the fractions were the same and both inhibitors were stable upon storage. The reaction of the inhibitors with trypsin and chymotrypsin was also demonstrated by analytical isoelectric focusing.The fractions obtained by preparative gel electrophoresis (the second method) contained the same proteins but their proportions varied widely in different fractions as demonstrated by analytical electrofocusing in the presence of urea and by SDS–PAGE. The early fractions, which consisted predominantly of α1-antitrypsin, showed a high inhibiting capacity for trypsin and none or only negligible capacity for chymotrypsin. Conversely, in the late fractions, the proportions of the proteins and inhibiting capacities were reversed. At 4 °C the trypsin-inhibiting capacity was stable for weeks but the chymotrypsin-inhibiting capacity of the preparation rapidly decreased.These observations indicate that the inhibition of proteases by goat α1-globulins is due to at least two closely associated but distinguishable proteins. One of these, corresponding to human α1-antitrypsin, would have an appreciable capacity to inhibit trypsin, but unlike the latter, little or no capacity for chymotrypsin inhibition. The inhibition of chymotrypsin is due to the second, unidentified α1-globulin.
Structural studies of purified pig lymph node plasma membrane: association of cytoskeletal components with the plasma membrane and the effect of detergent solubilization
Authors: Allore Robert J. , Barber Brian H.
Published: December 2009

The reproducibility of preparation, stability at 4 °C, and detergent solubilization characteristics of plasma membrane vesicles purified from domestic pig mesenteric lymph node tissue have been examined. It was found mat 2% (w/v) Nonidet P-40 solubilized 50–60% and 2% (w/v) sodium deoxycholate solubilized 60–70% of the total membrane protein. As judged by 125I-labelled lentil lectin staining of the sodium dodecyl sulfate – polyacrylamide gel electrophoresis patterns, 2% (w/v) Nonidet P-40 solubilized approximately 73%, and 2% (w/v) sodium deoxycholate approximately 82% of the total glycoprotein. Actin and a myosin-like component were identified as major constituents of both the Nonidet P-40 and the sodium deoxycholate insoluble fractions, suggesting the possibility that the detergent-insoluble fraction may represent a membrane-associated cytoskeletal network analogous to that which has been demonstrated for the erythrocyte membrane. Consistent with such an intimate association between actin and the plasma membrane, it was found mat very little actin could be eluted from the intact membrane vesicles by dialysis against low ionic strength ATP solutions, 0.6 M KCl, or by incubation with DNase I.
The cross-linking of tubulin with imidoesters
Authors: Galella G. , Smith David B.
Published: December 2009

Tubulin was reacted with a monofunctional imidoester, ethyl acetimidate, and three bifunctional imidoesters ranging in extension from 5 to 10 Å. Extensive cross-linking was found to occur with the three bifunctional reagents resulting in the formation of dimers, trimers, tetramers, pentamers, and hexamers. In addition, a similar cross-linking pattern was observed with me monofunctional reagent. This type of cross-linking is rarely seen when proteins are reacted with imidoesters. Given that the cross-linking span of ethyl acetimidate is only 3 Å it is reasonable to infer that there are nucleophilic groups in close proximity within the tubulin dimer. Amidinated tubulin is still capable of assembling into microtubules.
Structure of the acidic capsular polysaccharide of Cryptococcus laurentii (NRRL Y-1401)
Authors: Perry Malcolm B. , Webb Ann C.
Published: December 2009

The capsular polysaccharide produced by Cryptococcus laurentii (NRRL Y-1401) is composed of D-mannose (3 mol), D-glucuronic acid (1 mol), D-xylose (1 mol), and O-acetyl (~1 mol). Methylation, periodate oxidation, partial acid hydrolysis, optical rotation, and nuclear magnetic resonance studies showed that the polysaccharide is a high molecular weight branched polymer of regular structure having a repeating pentasaccharide unit with the structure:[Formula: see text]
On the mechanism of spontaneous transfer of lipids from isolated microsomal to mitochondrial membranes
Authors: Stuhne-Sekalec L. , Stanacev N. Z.
Published: December 2009

Spontaneous (protein independent) transfer of endogenously biosynthesized radioactive lipids (phosphatidic acid, phosphatidylcholine, and diglycerides) from isolated guinea pig liver microsomal to unlabeîled mitochondrial membranes was studied as a function of addition of ATP, duration of incubation, and protein concentration of microsomal and mitochondrial membranes. It was found that transfer of phosphatidic acid, phosphatidylcholine, and diglycerides was not absolutely dependent on addition of ATP, although its presence enhanced the transfer of phosphatidylcholine. The highest amount of transferred biosynthesized phosphatidic acid, phosphatidylcholine, and diglycerides was observed after a relatively short incubation and amounted to around one-third of all biosynthesized microsomal lipids, but decreased with increasing incubation time. With increasing concentrations of proteins of mitochondria, a significant decrease of transfer of radioactive lipids from microsomal membranes was established. The same tendency, although to a lesser degree, was established with increasing concentrations of microsomal proteins.These results, together with previously published experiments from this laboratory, were taken as the evidence for the transfer of membrane-bound biosynthesized phosphatidic acid, phosphatidylcholine, and diglycerides from isolated guinea pig liver microsomal to mitochondrial membranes, by a mechanism which involves a close contact between the membrane–donor and the membrane–acceptor. Mechanisms for lipid transfer in biological membranes and in model membranes (liposomes) were compared and discussed.
The biologic activity of selenoestrogens
Authors: Longcope G. , Arunachalam T. , Caspi E.
Published: December 2009

For differential imaging of mammary tumors with estrogen receptors and without estrogen receptors we required γ-emitting estrogen analogues. In this paper we report on the binding properties of 7α-, 16α-, and 17α-methylselenoestrogens and 17α-phenylselenoestrogens relative to the binding properties of estradiol.The selenium-containing estrogens retained the ability to displace [3H]estradiol from the estrogen receptor of rabbit uterine cytosol, although in most instances the displacement was small (3–7% compared to estradiol).The most active compounds were 16α-phenylselenoestrone, 16α-methylselenoestradiol, and 17α-methylselenomethyl-estradiol which had relative binding of 23, 27, and 31%, respectively, compared with that of estradiol.16α-Methylselenoestradiol was able to translocate the estrogen cytosol receptor to the nucleus, in vitro, but was not able to increase the uterine weight when administered to mice in vivo.
Novobiocin sensitivity and chromatin conformation in wild-type and temperature-sensitive mouse L cells
Authors: Colwill R. W. , Sheinin R.
Published: December 2009

The ts A1S9 cell is a dnats mutant of mouse L cells which exhibits enhanced sensitivity to killing by the antibiotic novobiocin, as compared with the parental wild-type (WT-4) cell, another dnats mouse L cell (ts C1), and other mammalian cells. Such a response was not detected with other cytotoxic agents tested. Novobiocin inhibits DNA synthesis in ts A1S9 cells, at concentrations which have little or no effect on RNA or protein synthesis. Enhanced novobiocin sensitivity is not due to altered cellular permeability to the drug, since ts A1S9 cells do not take up more antibiotic than do WT-4 or ts+ AR mouse L cells. A ts+ revertant of ts A1S9 cells exhibits a wild-type pattern of novobiocin sensitivity, suggesting that novobiocin resistance may be encoded in the ts A1S9 locus. These findings led to a study of DNA conformation which revealed that the nuclear, chromatin-bound DNA of temperature-inactivated ts A1S9 cells sediments differently in ethidium bromide sucrose density gradients than does that of WT-4 cells grown at 38.5 °C. These observations suggest that expression of the ts A1S9 defect results in a more highly constrained conformation of the DNA, relative to that in control cells, and that in this configuration the DNA may be unable to act as template for replication.
Immunofluorescent localization of lysine-rich histones in isolated nuclei from adult and embryonic chicken erythrocytes
Authors: Mura Casilda V. , Setterfield George , Neelin James M.
Published: December 2009

Isolated nuclei from adult chicken erythrocytes were stained by indirect immunofluorescence for histones H5 and H1. Nuclei in 0.15 M NaCl stained for H5 showed internuclear variations in intensity of fluorescence from bright to dim. Most individual nuclei were homogeneously stained although some showed a bright rim around a dimmer interior. Treatment of nuclei with Tween 80 in 0.15 or 0.03 M NaCl also gave internuclear variation in intensity. Adult nuclei stained for H1 (in 0.15 or 0.03 M NaCl) showed little internuclear variation; most nuclei stained brightly with a brighter rim. Simultaneous staining of H5 and H1 in the same nuclei confirmed the variable fluorescence of H5 and consistent fluorescence of H1. Most nuclei showed the presence of both histones. Nuclei from embryonic blood cells also showed considerable internuclear variation of H5 fluorescence and less variation with H1 staining. For both histones the proportion of brightly staining nuclei increased with embryonic development. Difficulties in interpreting quantitative variations in immunofluorescence are discussed.
Injection defect in alkylated and depurinated T7 bacteriophage: analysis by DNA ejection
Authors: Mamet-Bratley M. D. , Zollinger M. , Karska-Wysocki B.
Published: December 2009

Using DNA ejection in vitro as a model, we have studied the DNA injection defect caused by alkylation and depurination of T7 bacteriophage. Phage was alkylated with 0.02 M methyl methanesulfonate for 2 h at 37 °C; alkylated phage was then incubated 24 h at 30 °C to induce depurination. These samples were treated with formamide to cause DNA ejection without dissociation of the phage capsid. After ejection, the phage preparations were analyzed by electron microscopy. DNA lengths in capsid–DNA complexes were measured; relative numbers of full, empty, and partially empty phage heads were determined. To establish the direction of DNA ejection, E. coli RNA polymerase was bound to capsid–DNA complexes. The results showed that DNA was partially ejected from both alkylated and depurinated phages. In the alkylated sample, RNA polymerase was bound to the DNA end distal to the capsid; this showed that ejection started from the genetic left end. We interpret these results to show, in confirmation of earlier results obtained by marker rescue, that alkylation causes T7 phage to partially inject its DNA, starting from the genetic left end. For depurinated phage, our results suggest that partial DNA injection is responsible, in this case as well, for the already documented injection defect.
Specific small nuclear RNAs from SV40-transformed cells stimulate transcription initiation in nontransformed isolated nuclei
Authors: Ringuette Maurice J. , Gordon Karen , Szyszko Jolanta , Krause Margarida O.
Published: December 2009

Previous studies in our laboratory have implicated small nuclear RNAs (SnRNA) in the regulation of transcription in isolated mammalian cell nuclei. The present investigation was designed to develop a transcription assay system using isolated intact nuclei with optimized RNA polymerase II activity which would be capable of reinitiation in vitro to study the mode of action of the "active" RNA.We used nuclei isolated from either human WI38 or Balb 3T3 mouse cells to test the activity of SnRNA purified from SV40-transformed WI38 or 3T3 cell lines. These systems were found to support transcription up to 60 min, 40–60% of which was polymerase II dependent. In vitro initiations were detected by [γ-32P]ATP incorporation as well as by Hg-Sepharose chromatography using (γ-S)ATP as substrate. Results supported the following conclusions: (a) SnRNA from transformed cells stimulates the transcriptional activity of nontransformed nuclei while homologous SnRNA has little or no activity; (b) the stimulation is NaOH-sensitive and is dependent on RNA polymerase II since it is eliminated by 1 μg/mL α-amanitin; (c) the active subfraction of SnRNA from mouse cells was found to be of identical size (320–350 nucleotides) to that previously identified in human and monkey cells; and (d) analysis of the transcripts obtained from control and stimulated cell nuclei revealed that SnRNA activity is due primarily to an increase in the number of initiated chains.
Isolation and characterization of histones and other acid-soluble chromosomal proteins from Physarum polycephalum
Authors: Côté Serge , Nadeau Paul , Neelin James M. , Pallotta Dominick
Published: December 2009

Chromosomal basic proteins were isolated from amoebal and plasmodial stages of the acellular slime mold Physarum polycephalum. Polyacrylamide electrophoresis on high resolution acid–urea gels separated the five histone fractions in the sequence H1, H2A, H2B, H3, andH4. Under these electrophoretic conditions Physarum histones migrated more like plant (rye) than animal (calf) histones. Furthermore, Physarum histones H1, H2A, and H2B have higher molecular weights on sodium dodecyl sulfate (SDS) gels than the corresponding calf fractions. No differences were detected between amoebal and plasmodial histones on either acid–urea or SDS–polyacrylamide gel electrophoresis. Amoebal basic proteins were fractionated by exclusion chromatography. The five histone fractions plus another major acid-soluble chromosomal protein (AS) were isolated. The Physarum core histones had amino acid compositions more closely resembling those of the calf core histones than of rye, yeast, or Dictyostelium. Although generally similar in composition to the plant and animal H1 histones, the Physarum H1 had a lower lysine content. The AS protein was extracted with 5% perchloric acid or 0.5 M NaCl, migrated between histones H3 and H4 on acid–urea polyacrylamide gels, and had an apparent molecular weight of 15 900 on SDS gels. It may be related to a protein migrating near H1. Both somewhat resembled the high mobility group proteins in amino acid composition.
On the contribution of the mitochondrial genome to the growth of Chinese hamster embryo cells in culture
Authors: Morais Réjean , Guertin Denise , Kornblatt Jack A.
Published: December 2009

The present results demonstrate that Chinese hamster embryo cell populations in culture can be adapted to grow in the presence of chloramphenicol. It is shown that tryptose phosphate broth and uridine, one of its components, prevent the growth inhibitory effect of the drug. Study of some respiratory parameters (cytochrome c oxidase, cytochrome spectra, oxygen consumption) indicated that neither the broth nor uridine prevented the inhibitory effect of chloramphenicol on mitoribosomal protein synthesis. The cells grew with mitochondria devoid of a functional respiratory chain. Auxotrophy for pyrimidines appeared to result from the absence of dihydroorotate dehydrogenase, a respiratory chain-linked enzyme that catalyzes the fourth step of de novo pyrimidine biosynthesis. These and other results suggest that the synthesis of orotic acid may be considered as one of the main contributions of mitochondria to the growth of animal cells in culture.
The effect of the chromosome banding techniques on the histone and nonhistone proteins of isolated chromatin
Authors: Burkholder Gary D. , Duczek Laurel L.
Published: December 2009

The effect of a variety of chromosome banding techniques on the histone and nonhistone proteins of isolated, fixed, air-dried chromatin has been studied. Chromatin preparations were exposed to G-banding (SSC, urea, NaCl–urea, or trypsin), R-banding (Earle's BSS), and C-banding (NaOH or Ba(OH)2) treatments, and the proteins extracted from, and those remaining in the posttreatment chromatin, were examined by SDS polyacrylamide gel electrophoresis. The results indicate that: (i) each banding treatment produces specific quantitative and qualitative changes in the proteins of chromatin; (ii) the diverse treatments producing the same type of chromosome banding have both common and unique effects on the chromosomal proteins; and (iii) the treatments producing different types of chromosome banding have substantially different effects on the chromosomal proteins. It is not known whether these protein alterations are side effects occurring coincidentally with chromosome banding, or whether they are directly involved in mechanisms of banding. Results (ii) and (iii) suggest the latter possibility is more likely. If the protein alterations occur in specific chromosome regions, they may well influence the appearance of bands on mitotic chromosomes.
Production of antibody to phosphoprotein associated with nucleosome structure
Authors: Zhao M. S. , Liew C. C.
Published: December 2009

Antibodies were produced to phosphoprotein fraction, and a phosphoprotein B2 obtained by carboxyl methyl cellulose column chromatography and sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis as described previously (Biochem. J. 183, 147 (1979)). Production of the specific antibody was confirmed by double immunodiffusion. The phosphoprotein B2 (relative mass 68 000), which was isolated from the phosphoprotein fraction by SDS–polyacrylamide gel electrophoresis, specifically reacted with the antisera, as identified by "rocket" immunoelectrophoresis. Further characterization of the antibody to the phosphoprotein was carried out by isoelectrofocusing gel electrophoresis. The phosphoprotein, previously identified in the isoelectric point (pI) region 6.2–8.5, was subsequently reacted with antisera and 125I-labelled protein A. A prominent radioactive peak was identified in the region in which the phosphoprotein was focused. The radioimmunoactivity was proportional to the amount of phosphoproteins present in the isoelectric focusing gel. The presence of phosphoprotein in two types of mononucleosomes (MN1 and MN2) was demonstrated immunologically by use of the phosphoprotein antibody. The relationship between the phosphoprotein and chromatin structure, and possible role in gene regulation is discussed.
DNA organization in nucleosomes
Authors: Bazett-Jones D. P. , Ottensmeyer F. P.
Published: December 2009

A new technique known as electron spectroscopic imaging has allowed the direct visualization of DNA within the nucleosomes of chromatin. The results presented here confirm the model which suggests that approximately two supercoil turns of DNA are wound about the nucleosome core. The structure of nucleosomes from putative transcriptionally active genes, fractionated by preferential sensitivity to DNAase II and solubility in 2 mM MgCl2, has been examined using both dark field electron microscopy and electron spectroscopic imaging. Oligomeric strands of nucleosomes in this fraction have a less distinct beaded appearance than those of bulk chromatin. The phosphorus distribution in this chromatin suggests that the DNA has a less recognizable organization, lacking a two-turn supercoil per subunit. The unique appearance of this fraction in 30 mM NaCl is reversibly changed to the classical beaded appearance when dialyzed into 0.4 M NaCl.
Differences in protein content of sister nuclei: evidence from binucleate and mononucleate cells
Authors: Armstrong S. W. , Davidson D.
Published: December 2009

DNA and protein contents of pairs of sister nuclei were determined using a combined Feulgen–dinitrofluorobenzene technique. Sister nuclei were studied in binucleate cells, induced by treatment with 0.1% caffeine, and in sister mononucleate cells of untreated roots. Excised pea roots, grown in culture, were treated with 5-aminouracil to induce mitotic synchrony and with caffeine at the time of peak mitotic index, to provide the maximum number of binucleate cells. The induced binucleate cells form a marked population which was followed through a cell cycle; sister nuclei showed a correlation of volume and protein content, r = 0.79. Protein contents of sister nuclei were rarely identical and at 1 + 2 and 1 + 6 h the difference in protein contents of sister nuclei was significant (p = 0.05). Mean nuclear protein content decreased from 1 + 2 to 1 + 6 h; then, as nuclei entered S phase, their protein content increased. From 1 + 2 to 1 + 14 h the increase in protein content, in absolute amount, was identical in both sister nuclei. This suggests that there was a biphasic pattern of protein uptake; it is differential, in sister nuclei, in the first part of G1 but is identical throughout the rest of interphase. Analysis of sister nuclei from sister mononucleate cells showed a similar pattern of change; this is further evidence, from untreated cells, of a biophasic pattern of protein uptake. Caffeine-treated nuclei had lower protein contents than untreated nuclei, yet they completed a cell cycle and entered mitosis; this suggests that nonessential proteins were no longer present. It is proposed that mitosis is asymmetrical for molecules that regulate rates of macromolecular synthesis, cell growth, and progress through a cell cycle and that once the initial asymmetry has been established, it is maintained throughout interphase, even in binucleate cells in which the two nuclei share a common cytoplasm.
Comparison of purine and pyrimidine metabolism in G1 and S phases of HeLa and Chinese hamster ovary cells
Authors: Hordern Joyce , Henderson J. Frank
Published: December 2009

Detailed quantitative studies of purine and pyrimidine metabolism in G1 and S phases of synchronized HeLa and Chinese hamster ovary cells have been carried out. Concentrations of ribonucleoside triphosphates increased approximately in proportion to the increase in cell size as cells moved from G1 to S phase. Deoxyribonucleoside triphosphate concentrations were low in G1 phase and increased 2.5- to 10-fold in S phase. Pathways and rates of metabolism of radioactive adenine, guanosine, deoxyadenosine, deoxyguanosine, uridine, cytidine, deoxyuridine, deoxycytidine, and thymidine were determined by measuring the incorporation of each precursor into individual acid-soluble nucleotides and RNA and DNA bases. Cell-cycle or size-dependent differences were detected in many of the parameters studied.
Proteins and enzymes of the brush-border membrane of mouse intestine: influence of organ culture on gel electrophoretic patterns
Authors: Berteloot A. , Hugon J. S.
Published: December 2009

Purifications of mouse intestinal brush-border membranes from control explants and scrapings of intestinal mucosa have been compared. Based on the specific activity of sucrase used as a specific marker of these membranes, higher purification factors were obtained with control explants (24.7 ± 0.9) as compared with scrapings of intestinal mucosa (14.8 ± 0.9). However, similar patterns of proteins and enzymes were obtained by sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis after membrane solubilization by 2% SDS at room temperature. After 24 h of culture, higher molecular weight species of maltase–glucoamylase–isomaltase (band 4), alkaline phosphatase (bands 9–10), and trehalase (band 17) have been observed. Enzyme species appearing in the particulate fraction of culture media were, however, identical with those found at the brush-border membrane level in control explants, except for trehalase. These results are interpreted by considering the possible adsorption of serum components to brush-border membrane proteins. It thus appears that the membrane proteins and enzymes released in the media during organ culture are identical with those synthesized in the tissue in vitro or in vivo.
Expansion of phospholipid pool size of rat intestinal villus cells during fat absorption
Authors: Shaikh N. A. , Kuksis A.
Published: December 2009

The effect of fat absorption upon the phospholipid pool size of the intestinal mucosal cells was determined in rats receiving fatty emulsions as a bolus by stomach tube or as multiple meals in the form of fat-laden laboratory chow. The phospholipid content of the mucosal scrapings and of the isolated villus cells was determined 3 to 34 h after the meals and was compared with the phospholipid content of cells from similar animals receiving water alone or 10% sucrose in water. It was shown that continuously fed animals averaged 5–10% and single meal fed animals up to 40% higher phospholipid content in their mucosal cells than the corresponding controls, when compared per milligram cell protein. The expansion of the phospholipid pool involved all phospholipid classes and correlated well with the phospholipid composition of prechylomicrons and of microsomal membranes, which undergo a significant proliferation during fat absorption. The apparent lower expansion of the phospholipid pool in the continuously fed animals correlated with the lower triacylglycerol content of the lumen and of the cells at these times.
An evaluation of mitochondrial tRNA gene evolution and its relation to the genetic code
Authors: Cedergren R. J.
Published: December 2009

Extensive sequence data on mitochondrial (mt) tRNAs give for the first time an opportunity to evaluate tRNA gene evolution in this organelle. Deductions from these gene structures relate to the evolution of tRNA genes in other cellular systems and to the origin of the genetic code. Mt tRNAs, in contrast to the prokaryotic nature of chloroplastic tRNA structure, can not at the present time be definitely related to either prokaryotic or eukaryotic tRNAs, probably because of a higher mutation rate in mitochondria.Fungal mt tRNAs having the same anticodon and function are generally similar enough to be considered homologous. Comparisons of all mt tRNA sequences contained in the same mitochondrion indicate that some tRNAs originated by duplication of a prototypic gene which, after divergence, led to tRNAs having different amino acid specificities. The deviant mt genetic code, although admittedly permitting a simpler decoding mechanism, is not useful in determining whether the origin of mitochondria had preceded or was derived from prokaryotes or eukaryotes, since the genetic code is variable even among mitochondria. Variants of the mt genetic code lead to speculation on the nature of die primordial code and its relation to the present "universal" code.
The 5S RNA – protein complex from yeast: a model for the evolution and structure of the eukaryotic ribosome
Authors: Nazar Ross N. , Yaguchi Makoto , Willick Gordon E.
Published: December 2009

The ribosomal 5S RNA – protein complex appears to be an excellent model for studies on the evolution and structure of ribosomes. In eukaryotes this complex is composed of two components, the 5S rRNA and a single ribosomal protein which in yeast has a molecular weight of about 38 000. The primary protein-binding site is located in the 3′-end region of the 5S RNA together with a small portion of the 5′ end. The primary RNA-binding site appears to be situated in the C-terminal end of the protein (YL3 in yeast) but the binding specificity requires other structural elements in the N-terminal half of the molecule. When compared with prokaryotic 5S RNA – protein complexes, various physical and chemical studies suggest that the basic structure and interactions have been conserved in the course of evolution, but that the single larger eukaryotic 5S RNA binding protein has evolved through a fusion of genes for the multiple 5S RNA binding proteins in prokaryotes.
3-Hydroxy-3-methylglutaryl-CoA reductase activity in liver, brown fat, and small intestine of developing rats
Authors: Hahn Peter , Smale Freda-Anne
Published: December 2009

3-Hydroxy-3-methylglutaryl-CoA reductase activity was found to be low in the microsomal fraction of the small intestine, liver, and brown fat in suckling rats. It was high perinatally. Treatment with phosphatase of the microsomal fraction increased activity considerably in gut, somewhat in liver, and not at all in brown fat of infant rats. Activity in the mitochondrial fraction of the small intestine snowed no developmental changes. Injections of infant rats with triiodothyronine increased and with cortisone decreased hepatic activity but had no effect on gut or brown fat. It is concluded that even though activity decreases in all three tissues after birth, it is regulated differently in liver than in gut and brown fat.
Characterization of ribosomal proteins of Physarum polycephalum: number and molecular weight
Authors: Bélanger Gilles , Lemieux Gérald
Published: December 2009

The ribosome of Physarum polycephalum was found to contain 69 different proteins, with molecular weights ranging from 10 900 to 50 000. The same result was obtained when ribosomes were prepared in presence of a protease inhibitor. A spot frequently observed in two-dimensional gels was identified as an oxidized form of S3, the major phosphoprotein.
Expression of a cloned bovine growth hormone gene in Escherichia coli minicells
Authors: Rosner Arie , Keshet Eli , Gutstein Rachel , Aviv Haim
Published: December 2009

The synthesis of polypeptides in Escherichia coli minicells, directed by a pBR322 plasmid and its derivative-carrying bovine growth hormone cDNA insert, was studied. Two polypeptides coded by the ampicillin-resistance (Apr) gene (32 000 and 28 000 daltons) and a tetracyclin-resistance (Tcr) polypeptide (36 000 daltons) were identified by insertional inactivation. Two additional polypeptides of 37 000 and 34 000 daltons of as yet unknown function were detected in all extracts regardless of the presence of the Apr or Tcr genes in the plasmid. The pBR322–BGH recombinant plasmid coded for several novel polypeptides, among them one of 46 000 daltons, presumably a fused product of the BGH and β-lactamase genes. This protein, however, was not secreted into the periplasmic space of the cells as was the β-lactamase.
Proteins synthesized by rabbit reticulocyte membrane-bound ribosomes
Authors: Lemieux Réal , Godin Claude
Published: December 2009

Rabbit reticulocyte membrane-bound ribosomes liberated by deoxycholate treatment contain degraded forms of ribosomal and messenger RNA. This degradation occurs after the liberation of the ribosomes from the membranes by the detergent because intact ribosomal and messenger RNA can be extracted from washed membranes by phenol treatment. Increasing the ionic strength of the detergent buffer prevents this RNA degradation and allows the recovery of membrane-bound ribosomes capable of protein synthesis. Comparison of the proteins synthesized in vitro by the polyribosomes shows that the main protein produced by both free and membrane-bound ribosomes is globin. However, the two types of polyribosomes could be distinguished by the nonglobin proteins they produce.
Preparation of messenger RNA from mature skeletal and cardiac muscle
Authors: Rogers Peter A. , Jones George H.
Published: December 2009

Messenger RNA (poly(A)+ RNA) has been prepared from mature skeletal muscle of adult rats by several procedures. These procedures involved oligodT-cellulose chromatography of either muscle polyribosomes (method 1), polysomal RNA isolated by CsCl gradient centrifugation of muscle polysomes (method 2), total muscle RNA isolated by CsCl gradient centrifugation (method 3), or total muscle RNA prepared by phenol extraction (method 4). Each procedure produced RNA in significant yields which sedimented in a broad band (from 4S to greater than 28S) on sodium dodecyl sulfate – sucrose gradients. In addition, poly(A)+ RNA from each procedure stimulated protein synthesis in the wheat germ cell-free system. The best combination of yield and messenger activity was obtained for poly(A)+ RNA prepared from polysomal RNA by method 2. This poly(A)+ RNA preparation stimulated the cell-free synthesis of a number of presumptive myofibrillar proteins, including myosin heavy chain and actin, in the wheat germ system. The presence of the latter protein among the cell-free products was confirmed by DNase I affinity chromatography of appropriate reaction mixtures. Poly(A)+ RNA was also isolated from rat cardiac muscle polysomal RNA by method 2. This RNA also directed the synthesis of myofibrillar proteins in the wheat germ system. The relative amounts of the proteins synthesized in the presence of skeletal and cardiac muscle poly(A)+ RNA have been compared. The data indicate that the method described is suitable for the isolation of RNA with message activity from mature mammalian muscle.
Reversible cryogenic alteration of the ultraviolet absorption spectra of the olefinic bonds in lipid membranes
Authors: Campbell Ian M. , Pawagi Asha B.
Published: December 2009

In earlier studies it was found that the association of basic polypeptides with lipid vesicles drastically altered ultraviolet absorption by the olefinic bonds of the lipid. Such an effect suggested that the polypeptide was increasing the polarity of the chromophore environment by either direct interaction with the acyl chains or by inducing their hydration. It is reported here that freeze–thaw cycling, which was expected to allow hydration of the olefinic-bond region of the membranes, caused the same spectral alteration as vesicle interaction with basic polypeptides. When these vesicles were subsequently placed under conditions that would be expected to accelerate the escape of water entrapped within the membranes (i.e., by placing them under vacuum or adding sucrose to establish a high osmotic gradient to their exterior), the absorption spectrum was rapidly restored to that for olefinic bonds in a nonpolar environment. Since placing the polylysine – dioleoylphosphatidylcholine (DOPC) vesicle interaction product under the same conditions restored the spectral intensity, at 190 nm, to between 80 and 85% of that for the lipid in a nonpolar environment, it seems that a major effect of polylysine on DOPC membranes may be through induction of hydration of their interior.
The inhibition of β-galactosidase (Escherichia coli) by amino sugars and amino alcohols
Authors: Huber R. E. , Gaunt M. T.
Published: December 2009

Kinetically determined competitive inhibitor constants, which were estimates of the binding capacity of inhibitors interacting with the free enzyme form of β-galactosidase (Escherichia coli), showed that amino sugars and alcohols inhibited the enzyme much more than did their respective parent sugars and alcohols. In most cases the inhibition was 10–30 times greater, but the inhibition by 1-aminogalactopyranose was about 300 times greater than mat of galactopyranose. When the amino groups were acetylated the inhibitory advantage was completely eliminated and partial neutralization of the amino group of an inhibitor by the presence of a group with a negative charge on the same inhibitor decreased the inhibitory advantage. Studies of binding to the galactosyl form of the enzyme (rather than to the free form) indicated that the binding at the "glucose" site was increased only slightly by the presence of the amino group. Overall, the findings suggested that β-galactosidase has a negative charge near the anomeric carbon binding position of the "galactose" site. Since negative charges are unlikely to be of any importance in binding the normally neutral β-galactosidase substrates, this charge may be important in stabilizing a positively charged reaction intermediate. Amino sugars and alcohols are thus probably inhibitors of β-galactosidase, because they are positively charged and analogous to an oxonium–carbonium ion intermediate.
Resolution of diacylglycerol moieties of natural glycerophospholipids by gas–liquid chromatography on polar capillary columns
Authors: Myher J. J. , Kuksis A.
Published: December 2009

A rapid and practical method has been developed for the gas–liquid chromatographic determination of the sn-1,2-diacylglycerol moieties of natural glycerophospholipids using polar wall-coated open tubular columns. The method gives complete resolution and quantitative estimates for all species according to molecular weight and degree of unsaturation, including stearoyl docosahexaenoylglycerol and related polyunsaturates. For this purpose the sn-1,2-diacylglycerols are obtained from the glycerophospholipids by hydrolysis with phospholipase C and are converted into the trimethylsilyl or tertiary-butyldimethylsilyl ethers. The silyl ethers are separated by gas–liquid chromatography on the capillary glass columns coated with a polar cyanopropylsiloxane polymer, in the temperature range 175–250 °C, using hydrogen as the carrier gas. Practical applications of the method are illustrated by analyses of the sn-1,2-diacylglycerol moieties of the phosphatidylcholines of soybean phosphatides, egg yolk, and rat liver. The method of analysis is applicable to other classes of glycerophospholipids and the total time requirements for the analysis of any one phospholipid class are comparable to those for a fatty acid analysis.
Biosynthesis of CDP-diacylglycerol in hog mesenteric lymph node lymphocytes
Authors: Sribney Michael , Hegadorn Carol A.
Published: December 2009

The synthesis of CDP-diacylglycerol by CTP:phosphatidate cytidylyltransferase (EC 2.7.7.41) has been studied in microsomes isolated from hog mesenteric lymph node lymphocytes. The properties of this enzyme were found to be similar in many respects to that described in rat liver microsomes; however, it is not stimulated by GTP or any other nucleotides under normal assay conditions. The enzyme requires that the phosphatidate be emulsified in a cationic detergent for optimal activity. The quaternary ammonium phospholipids lecithin and sphingomyelin were found to stimulate the formation of CDP-diacylglycerol even in the presence of optimal cationic detergent. Other phospholipids or detergents had no effect or were inhibitory to the reaction. Only in the presence of either lecithin or sphingomyelin did nucleotides such as ATP, GTP, UTP, and FTP stimulate the formation of CDP-diacylglycerol. The Km and Vmax for CTP were found to be 0.6 and 1.2 mM, respectively, while the apparent Km and Vmax for phosphatidate were 0.65 and 1.2 mM. Magnesium was found to be the only metal ion that stimulated the reaction, with an optimal concentration of 20 mM. Fluoride ions at 20 mM inhibited the reaction to the extent of 70%. The enzyme was found to be very unstable when the isolated microsomes were stored at −20 °C for 24 h, losing approximately 75% of its activity.
The biosynthesis of triterpenoid carotenoids in Streptococcus faecium UNH 564P
Authors: Davies Brian H. , Taylor Richard F.
Published: December 2009

Cell-free enzyme preparations from Streptococcus faecium UNH 564P and Phycomyces blakesleeanus strain C5-car 10(−) were used to study the incorporation of [14C]isopentenyl pyrophosphate and either all-trans-[4,8,12-3H]farnesyl pyrophosphate (FPP) or all-trans-[4,8,12,16-3H]geranylgeranyl pyrophosphate (GGPP) into squalene and the carotenoids of the organisms. It was found that the triterpenoid (C30) carotenoids of S. faecium are formed by condensation of two molecules of FPP similar to squalene biosynthesis rather than by condensation of two molecules of GGPP with subsequent degradation. Additional studies have shown that carotenoid glucoside biosynthesis in S. faecium extracts is stimulated by the addition of glucose and UDP-glucose. Such glucoside biosynthesis appears maximal in systems exposed to aeration. These results confirm that the triterpenoid carotenoids in S. faecium are symmetrical and are representatives of a unique new class of carotenoids.
Cholinephosphotransferase activities in microsomes and neuronal nuclei isolated from immature rabbit cerebral cortex: the use of endogenously generated diacylglycerols as substrate
Authors: Baker R. Roy , Chang Huu-Yi
Published: December 2009

A neuronal nuclear fraction (N1) and a microsomal fraction (P3) were isolated from homogenates of cerebral cortices of 15-day-old rabbits. A nuclear envelope fraction (E) was prepared from N1. To assay cholinephosphotransferase, diacylglycerols were first generated in the membranes of these subfractions using a phospholipase C (Bacillus cereus) preincubation. With levels of endogenous diacylglycerols producing maximal specific cholinephosphotransferase activities, an activity ratio of 1:1:5 was found for N1, P3, and E, respectively. An independent neuronal nuclear cholinephosphotransferase, concentrated in nuclear membranes, is indicated. With regard to changes in pH and concentrations of MgCl2 and CDP-choline, N1 and P3 activities responded in a similar manner. However, in contrast to P3, N1 activities were much more profoundly inhibited at low levels of Triton X-100 (0.01–0.02 w/v%) and N1 showed quite significant levels of cholinephosphotransferase activity in the absence of a phospholipase C preincubation. Choline phosphotransferase in N1 and P3 showed Km values for CDP-choline (0.028 and 0.031 mM, respectively) which were much lower than corresponding literature values determined using exogenous diacylglycerols as substrates for this enzyme. The presence of cholinephosphotransferase in neuronal nuclear membranes reflects a rather exceptional nuclear autonomy. This may be related to a need to maintain nuclear phospholipid in the absence of a well-developed endoplasmic reticulum at early stages of neuronal development or to synthesize phospholipid in response to functions unique to the nucleus.