bluuebunny/crossref_metadata_2025 huggingface dataset:
Stoichiometry of GTP hydrolysis and tubulin polymerization.
Authors: Maccioni R , Seeds N W
Published: May 2006

Microtubule formation from lamb brain tubulin isolated by affinity chromatography and freed of exchangeable nucleotide requires GTP for maximal rate and extent of polymerization. The nucleotide analogs guanylylmethylenediphosphate and guanylylimidodiphosphate fail to replace GTP; in addition, neither the presence of microtubule associated proteins nor 5 M glycerol relieves the GTP requirement. The relation of GTP concentration and microtubule formation shows an association constant K = 1 X 10(4) M-1; furthermore, GDP and guanylylimidodiphosphate are competitive inhibitors of GTP for polymerization. Using a rapid filter assay for microtubule formation that allows the quantitative analysis of early polymerization kinetics and correcting for GTP hydrolysis uncoupled from tubulin polymerization, a stoichiometry of two molecules of GTP hydrolyzed per mole of tubulin dimer incorporated into microtubules has been found.
Conditionally lethal ribosomal protein mutants: characterization of a locus required for modification of 50S subunit proteins.
Authors: Kushner S R , Maples V F , Champney W S
Published: May 2006

Mutagenized P1 bacteriophage were used to transduce a marker (aroE) adjacent to the cluster or ribosomal protein genes located at 72 min on the Escherichia coli chromosome. Linked temperature-sensitive transductants were isolated and characterized. A mutant unable to grow at 44 degrees was found to be defective in protein synthesis both in vivo and in vitro. At the restrictive temperature mutant cells lost all polyribosomes. Analysis of the ribosomal proteins revealed alterations in at least four 50S subunit proteins. The mutation (called rimE1, ribosomal protein modification) mapped between rpsE and aroE. It is suggested that the rimE locus is the structural gene for an activity that modifies a selected number of ribosomal proteins.
Investigations into the relationship between structure and function of diphtheria toxin.
Authors: Everse J , Lappi D A , Beglau J M , Lee C L , Kaplan N O
Published: May 2006

Studies on the structure-function relationship of diphtheria toxin are reported. New methods are described for the preparation of pure intact ("unnicked") toxin and for the preparation of the individual A and B chains. A biological assay method for the B chain is also presented, as well as a method for the labelling of "nicked" (one peptide bond broken) diphtheria toxin with 131I such that the label is confined to only one of the two polypeptide chains. Alterations of diphtheria toxin with specific reagents reveal that modifications of the tryptophan, methionine, and arginine residues did not result in a significant loss in toxicity, whereas treatment of the toxin with omicron-phthalaldehyde or by photooxidation with rose bengal results in a complete loss of the toxic activity. Modification of tyrosine by iodination results in active toxin, whereas modification by tetranitromethane causes a loss in activity. Preliminary results also indicate that the isolated A chain is about an order of magnitude more active in incorporating adenosine diphosphoribose into translocase (elongation factor 2) than whole or nicked toxin is under identical conditions. The observed structural properties are discussed in view of the functional activity of diphtheria toxin in cell-free systems as well as in cell cultures. Evidence is presented indicating that the B chain binds to membranes: it inhibits the action of nicked toxin on HeLa cells.
Mapping of transcription sites of simian virus 40-specific late 16S and 19S mRNA by electron microscopy.
Authors: May E , Maizel J V , Salzman N P
Published: May 2006

Simian virus 40 (SV40) late 19S and 16S mRNAs were annealed to complementary regions of partially melted viral double-stranded SV40(LHpa II) DNA or SV40(LBam HI) DNA. The RNA-DNA hybrid regions within the DNA molecules were visualized as loops [with SV40(LBam HI) DNA] in the electron microscope. The data confirm the previous localizations of the 3' and 5' ends of 16S SV40 mRNA and of the 3' end of late 19S SV40 mRNA. The 5' end of the major stable SV40 late 19S mRNA has been positioned at 0.755 map unit. Thus, the sequences of viral DNA from 0.655 to 0.755 map unit, including the replication origin, are not converted into major stable species of late viral mRNA.
Charge shift electrophoresis: simple method for distinguishing between amphiphilic and hydrophilic proteins in detergent solution.
Authors: Helenius A , Simons K
Published: May 2006

Seventeen hydrophilic proteins and five amphiphilic membrane proteins were subjected to agarose gel electrophoresis in the presence of a nonionic detergent (Triton X-100), a mixture of anonionic and an anionic detergent (Triton X-100 and sodium deoxycholate), and a mixture of a nonionic and a cationic detergent (Triton X-100 and cetyltrimethylammonium bromide). The electrophoretic mobility of the hydrophilic proteins was unaffected in the three detergent mixtures. However, the mobility of the amphiphilic proteins shifted anodally in the Triton X-100-deoxycholate system and cathodally in the Triton X-100-cetyltrimethylammonium bromide system when compared to the mobility in Triton X-100 alone. The detergent-induced shift in mobility provides a simple, rapid, and sensitive method for distinguishing between hydrophilic and amphiphilic proteins.
Opposing effects of dopaminergic to cholinergic compounds on a cerebral dopamine-activated adenylate cyclase.
Authors: Tang L C , Cotzias G C
Published: May 2006

In contrast to antipsychosis drugs which inhibit the dopamine-activated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of caudate nucleus, dopaminergic drugs for treatment of Parkinson's disease stimulate this cyclase. Stimulants and inhibitors of cholinergic neurons inhibited this adenylate cyclase activity competitively and specifically. Thus, the mechanism by which dopaminergic medications ameliorate the effects of Parkinson's disease includes activation of the dopamine-sensitive adenylate cyclase. Excessive activation might be present during the psychotic episodes seen in patients with parkinsonism who are overtreated. The enzymatic effects of the drugs that affect cholinergic mechanisms seem to be generally in keeping with the pharmacological reciprocity between psychoses and extrapyramidal function, except for the anticholinergic ones which inhibited this cyclase although they can be hallucinogenic.
Ionic regulation in genetic translation systems.
Authors: Douzou P , Maurel P
Published: May 2006

The polyelectrolyte theory can provide an interpretation of the interdependence of pH, ionic strength, and polyamines one observes in the activity of ribonuclease acting on RNA. According to this theory: (i) A nucleic acid-enzyme complex and the suspending medium may be considered as two phases in equilibrium, even though within limits, the complex is soluble in water. (ii) The enzymatic catalysis is under tight control of the electrostatic potential generated by the system. Consequently, modification in electrostatic potential will induce a concomitant change in activity. (iii) The electrostatic potential can be modified through action on the system of "modulators", either "external" (ionic strength, pH, temperature, etc.) or "internal" (specific ligands, substrates, protein factors, etc.). Similarities between the reaction of ribonuclease (ribonuclease 3'-pyrimidino-oligonucleotidohydrolase; EC 3.1.4.22) and RNA and those observed with highly organized systems catalyzing DNA, RNA, and protein synthesis suggest that the electrostatic potential also provides an important regulatory mechanism in genetic translation. In this view, an essential function of nucleic acids is to provide their enzyme partners with polyanionic microenvironments within which their catalytic activities are controlled by variation in physicochemical parameters, including the proton concentration induced through "modulation" of the local electrostatic potential.
Establishment and characterization of a strain of human adrenal tumor cells that secrete estrogen.
Authors: Fang V S
Published: May 2006

We report the establishment of a new cell line, designated Fang-8, which originated from a human adrenal adenocarcinoma. It has been continuously propagated during the past 20 months. The cells exhibit an epithelioid morphology, resembling the cell structure of the zona reticularis when viewed by electron microscopy. Since passage 12, the cells showed a characteristic ring-forming property in culture. The cells produce neither testosterone, mineralocorticosteroids, nor glucocorticosteroids; instead, they possess the unique function of estrogen production. Karyotypic analysis revealed a 100% aneuploid, unstable karyotype in the hyperdiploid stem line. Identification of the Y chromosome was uncertain. In view of these characteristics, Fang-8 represents a new human cell line, unlikely to be contaminated with HeLa strains, and different from another cell line derived from human adrenal cortex andenocarcinoma, namely SW-13(ATCC CCL 105).
Cell-surface glycosyltransferases in cultured fibroblasts: increased activity and release during serum stimulation of growth.
Authors: LaMont J T , Gammon M T , Isselbacher K J
Published: May 2006

Cell-surface galactosyltransferase was studied in suspensions of intact baby hamster kidney fibroblasts with both endogenous and exogenous glycoprotein acceptors. The cell-surface location of galactosyltransferase was demonstrated in experiments with the enzyme modifier alpha-lactalbumin, which does not enter the cell. The addition of alpha-lactalbumin to the assay medium for galactosyltransferase resulted in accumulation of lactose in the medium but not in the cells. There was no detectable hydrolysis of UDP-galactose to free galactose by these cells, nor did a 100-fold molar excess of free galactose inhibit cell-surface galactosyltransferase. There was a marked increase in specific activity of cell-surface exogenous galactosyltransferase in serum-stimulated as compared to resting fibroblasts. Dividing but not resting fibroblasts released galactosyltransferase, but not sialyl- or fucosyltransferase, in soluble form into the tissue culture medium. The release of galactosyltransferase was greater from virally transformed than from nontransformed fibroblasts.
Tumorigenicity of simian virus 40-transformed human cells and mouse--human hybrids in nude mice.
Authors: Koprowski H , Croce C M
Published: May 2006

Four different human cell lines transformed by simian virus 40 (SV40) were tested for their tumorigenicity in athymic nude mice. Two of these lines, W18Va2 and GM52VA, were found to be tumorigenic when inoculated at a concentration of 1 x 10(7) cells per mouse. The other two cell lines, LN-SV and GM54VA, were found to induce very small tumors only after the injection of approximately 1 x 10(8) cells per mouse. Somatic cell hybrids between either LN-SV or GM54VA SV40-transformed human cells and normal mouse peritoneal macrophages, which have retained the human chromosomes carrying the SV40 genome, were found to be much more tumorigenic than the SV40-transformed human cell parents. These experiments suggest that the genetic background in which the human chromosomes carrying the SV40 genome are present plays a role in the modulation of the expiration of malignancy.
Immunogenicity of liposomal model membranes in mice: dependence on phospholipid composition.
Authors: Yasuda T , Dancey G F , Kinsky S C
Published: May 2006

This investigation demonstrated that antigenic expression in liposomal model membranes may be markedly influenced by phospholipid composition. Incorporation of dinitrophenylaminocaproylphosphatidylethanolamine (Dnp-Cap-PE) into egg lecithin/cholesterol/dicetylphosphate bilayers did not significantly enhance the response of AKR mice to this synthetic amphipathic antigen. In contrast, the immunogenicity of Dnp-Cap-PE was increased (as measured by either plaque-forming cell frequency of hemagglutination titer) after its insertion into liposomes prepared with beef sphingomyelin instead of egg lecithin. We also show that the response to the Dnp-Cap determinant can be stimulated by the presence of lipid A in the same bilayers without altering the relative immunogenic potency of the sphingomyelin and lecithin liposomes; similarly, incorporation of this mitogen into sphingomyelin liposomes produced a greater polyclonal (nonspecific) response. The response to Dnp-Cap-PE-sensitized liposomes (with or without lipid A) prepared with a series of synthetic phosphatidylcholines (distearoyl-, dipalmitoyl-, dimyristoyl-, dilauroyl-, dioleoyl-) suggests a direct correlation between liposomal immunogenicity and transition temperature of the phospholipid.
Quantitative correlation of dopamine-dependent adenylate cyclase with responses to levodopa in various mice.
Authors: Tang L C , Cotzias G C
Published: May 2006

We obtained 12 groups of mice with widely different neurological responses to levodopa by selecting them from different strains and submitting some of them to pretreatments. We scored the symptoms evoked by a standardized dose of levodopa in one subgroup from each group. We tested another subgroup for activation of an adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by a standardized dose of dopamine added to homogenates of the caudate nuclei of the brains of these mice. Both sets of tests were performed randomly. When the accruing two sets of data were plotted against each other there emerged a straight line which fitted the data with a coefficient of correlation of 0.97 (P less than 0.0001). The dopamine-dependent activity of the adenylate cyclase of the brain was thus shown to be a determinant of the neurological responses of intact animals to a dopaminergic drug.
Alterations of spore coat processing and protein turnover in a Bacillus cereus mutant with a defective postexponential intracellular protease.
Authors: Cheng Y S , Aronson A I
Published: May 2006

A mutant with an alteration in the major intracellular serine protease produced by postexponential Bacillus cereus was isolated by screening mutants defective in spore germination. The purified enzyme from the mutant is more labile to heat and alkaline pH than the protease from the wild type. Protease activity appears at the same time as in the wild type but only reaches 50% of the specific activity and decays more rapidly during sporulation. Coincident with the decay is a decrease in the rate of protein turnover. Generation of amino acids by turnover seems to be important for sporulation because the number of spores produced by the mutant is increased 4- to 10-fold by addition of casamino acids. As anticipated, the mutant produces spores that germinate poorly but, surprisingly, these spores are very deficient in coat protein. Coat antigen is present in cell extracts of mutant and wild type, however, both as large molecules not found on mature spores and as spore coat protein monomers. The large molecules rapidly disappear in a pulse chase experiment in the wild type with some increase in the coat monomers. In mutant extracts, however, this large coat antigen is slowly and improperly processed.
Effects of early experience upon orientation sensitivity and binocularity of neurons in visual cortex of cats.
Authors: Leventhal A G , Hirsch H V
Published: May 2006

The class of neurons within the visual cortex of normal adult cats that has the smallest receptive fields (less than or equal to 2.25 degrees2) and that responds only to low rates of stimulus motion (less than or equal to 50 degrees / sec) responds preferentially to lines oriented about either the horizontal axis (+/-22.5 degrees) or the vertical axis (+/-22.5 degrees). In animals reared without exposure to patterned visual stimulation, many of these cells display orientation preferences but are activated monocularly. In contrast, in normal animals, neurons that have larger receptive fields or that respond to higher rates of stimulus motion do not exhibit a similar bias in the distribution of their orientation preferences. Cells of this type, studied in animals reared without exposure to patterned visual stimuli, are activated binocularly but do not display orientation preferences.
A nucleotide sequence from a ribonuclease III processing site in bacteriophage T7 RNA.
Authors: Robertson H D , Dickson E , Dunn J J
Published: May 2006

Transcription of that portion of the bacteriophage T7 genome encoding early functions yields RNA molecules about 7500 nucleotides long representing this entire early region. These long transcripts can be cleaved in vitro by highly purified Escherichia coli ribonuclease III (endoribonuclease III; EC 3.1.4.24), yielding five messenger RNAs identical to those produced in vivo. During this reaction, a small RNA fragment called F5 RNA is released, which is specified by the region of the T7 genome between genes 1.1 and 1.3. The following sequence of 32P-labeled F5 RNA has been determined using standard RNA sequencing techniques: pU-A-A-G-G-U-C-G-C-U-C-U-C-U-A-G-G-A-G-U-G-G-C-C-U-U-A-G-Uoh. The relative contributions of sequence and structure to ribonuclease III processing signals are considered in light of these findings.
Structure of a large segment of the genome of simian virus 40 that does not encode known proteins.
Authors: Dhar R , Subramanian K N , Pan J , Weissman S M
Published: May 2006

The nucleotide sequence of the region of DNA of simian virus 40 extending from 0.595 to 0.790 map unit has been derived. The sequence includes the DNA complementary to the 5' end of early mRNA and to the 5' end of some of the forms of late RNA. Because there are termination codons in all three phases in early and late RNA, there is a sequence of almost 800 nucleotides of simian virus 40 DNA that probably does not code for known viral proteins. The sequence spans the 5' end of the early mRNA at 0.67 map unit and overlaps a species of late RNA whose 5' end is at 0.65 map unit and whose 3' end is at 0.77 map unit. This RNA is retained on oligo(dT)-cellulose columns in high salt concentrations. Analysis of the sequence of late strand RNA suggests that this RNA is not covalently linked to the mRNA that encodes structural proteins. There is another species of late RNA of simian virus 40 whose 5' end is at 0.775 map unit. The nucleotide sequence of this region of simian virus 40 DNA contains several examples of repeated sequences, most of which are located in DNA that does not encode known peptides. These may be analogous to the reiterated sequences that have been described in animal cell DNA.
Isolation and partial characterization of a cholesterol-requiring mutant of Chinese hamster ovary cells.
Authors: Chang T Y , Telakowski C , Heuvel W V , Alberts A W , Vagelos P R
Published: May 2006

A sterol-requiring mutant has been isolated from mutagenized Chinese hamster ovary cells. This mutant grows normally only when cholesterol is present in the medium. Cell lysis occurs within 3 days in the absence of cholesterol. The frequency of reversion of this mutant to prototrophic growth is low (less than or equal to 10(-6). Whole cell pulse experiments with [14C]acetate or [3H]mevalonate indicate that the rate of synthesis of digitonin-precipitable material is greatly diminished in the mutant cells as compared to that in normal Chinese hamster ovary cells. Enzyme assays in vitro with crude cell extracts show that the biosynthetic conversion of mevalonate to squalene and the conversion of squalene to lanosterol are not impaired in the mutant cells. Gas-liquid chromatographic analyses of radioactive sterol composition after whole cell pulse experiments with [3H]squalene and with [3H]anosterol suggest that the fundamental enzymatic defect of the mutant is at the stage of lanosterol demethylation. When cells were grown in serum-free medium, lanosterol and dihydrolanosterol accumulated intracellularly in the mutant cells before cell lysis occurred; neither of these two intermediary sterols was detected in the wild-type cells grown under the same condition.
High-resolution 31P nuclear magnetic resonance studies of metabolism in aerobic Escherichia coli cells.
Authors: Navon G , Ogawa S , Shulman R G , Yamane T
Published: May 2006

31P nuclear magnetic resonance spectra at 145.7 MHZ were obtained of concentrated suspensions of E. coli cells. The position of the Pi resonance was used to determine the pH, and in most experiments it was possible to distinguish the intracellular (pHin) and extracellular (pHex) values. During respiration pHin approached 7.55, while pHex varied from 6.0 to 8.0. With succinate as a carbon source and in a N2 environment, pHin - pHex. Upon addition of glucose, pHin greater than pHex. In the presence of an ATPase (adenosinetriphosphatase; ATP phosphohydrolase; EC 3.6.1.3) inhibitor dicyclohexylcarbodiimide, pHin remained equal to pHex even in the presence of glucose. In other experiments, oxygenation brought pHin above pHex even in the presence of dicyclohexylcarbodiimide. These experiments are consistent with Mitchell's hypothesis that, first, delta pH can be created by the reversal of the ATPase reaction and, second, that protons are pumped outward during respiration. In addition to Pi, about 10 more resonances were resolved, several of which were assigned to different phosphate metabolites.
Reconstitution of thermostable ATPase capable of energy coupling from its purified subunits.
Authors: Yoshida M , Okamoto H , Sone N , Hirata H , Kagawa Y
Published: May 2006

Purified dicyclohexylcarbodiimide-sensitive ATPase (TF0-F1) from thermophilic bacterium PS3 is composed of a water soluble part with ATP hydrolytic activity (TF1) and a water insoluble moiety (TF0). All of the five subunits (alpha, beta, gamma, delta, and epsilon) of TF1 were isolated. TF1 was reconstituted from the five subunits, which catalyzed an ATP-32Pi exchange and an ATP-driven enhancement of fluorescence of 1-anilinonaphthalene-8-sulfonate, when adsorbed on proteoliposome inlaid with TF0 (TF3-vesicles). Subunit epsilon and/or delta became firmly bound to TF0-vesicles and there was no preferential sequence in the binding. Both subunits were required for binding of the remaining subunits of TF1 to TF0-vesicles, but they did not modify the high H+ -permeability of TF0-vesicles. The addition of gamma but they did not modify the high H+-permeability of TFO-vesicles. The addition of gamma subunit together with epsilon and delta subunits caused a marked decrease of H+ -permeability of TF0-vesicles, similar to that induced by TF1. We conclude tentatively that the epsilon and delta subunits connect TF0 and the other subunits forming a part of a proton pathway, gamma is a gate of proton flow coupled to ATP hydrolysis (or synthesis), and alpha and beta subunits contain the active site for energy transformation. A possible model of subunit structure of TF1 is proposed.
Regulation of cholesterol biosynthesis by normal and leukemic (L2C) guinea pig lymphocytes.
Authors: Philippot J R , Cooper A G , Wallach D F
Published: May 2006

The cholesterol production of guinea pig leukemic (L2C) lymphocytes preceeds at greater than 30 times the rate found in normal cells. Fatty acid biosynthesis is also enhanced in L2C cells. Exposure of L2C cells to cholesterol/lecithin liposomes does not depress their sterol biosynthesis, in contrast to the behavior of normal lymphocytes [Philippot, J.R., Cooper, A.G. & Wallach, D. F. H. (1975) Biochim. Biophys. Acta 406, 161-166]. However, 25-hydroxycholesterol, an inhibitor of hydroxymethylglutaryl-CoA reductase (NADPH) [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34], the rate limiting enzyme in cholesterogenesis, and 25-hydroxycholecalciferol, a biologically potent form of vitamin D3, block sterol biosynthesis of both normal and L2C lymphocytes [Philippot, j.r., cooper, A.G. & Wallach, D.F.H. (1976) Biochem. Biophys. Res. Commun. 72, 1035-1041]. Moreover, both cell types exchange cholesterol equivalently with cholesterol/lecithin liposomes. The only difference in sterol biosynthesis observed between the two cell types is in the temperature response of the enzyme. Arrhenius plots of this enzyme activity exhibit a prominent discontinuity at about 24 degrees in the case of normal cells, but none in the case of L2C. The activation energies for L2C cells and normal cells, above the normal cell transition temperature, were not significantly different. All of the data suggest that the regulatory defect in L2C lymphocytes arises from a deficiency in these cells' internal membranes.
Translation of melittin messenger RNA in vitro yields a product terminating with glutaminylglycine rather than with glutaminamide.
Authors: Suchanek G , Kreil G
Published: May 2006

Melittin messenger RNA from queen bee venom glands has been translated in a cell-free system from wheat germ. A product larger than promelittin is formed which has the carboxy-terminal sequence-Gln-Gln-GlyCOOH. Melittin and promelittin from venom glands terminate in -Gln-GlnCONH2. The possible role of the extra glycine residue in the formation of a COOH-terminal amide via a transamidase-like reaction is discussed.
Rous sarcoma virus genome is terminally redundant: the 3' sequence.
Authors: Schwartz D E , Zamecnik P C , Weith H L
Published: May 2006

A sequence of 20 nucleotide residues immediately adjacent to the 3'-terminal poly(A) in Rous sarcoma virus (Prague strain, subgroup C) 35S RNA has been determined by extension of a riboguanylic acid-terminated oligothymidylic acid primer hybridized at the 5' end of the 3'-terminal poly(A) with purified reverse transcriptase (RNA-directed DNA polymerase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) from avian myeloblastosis virus. The sequence is 5'GCCAUUUUACCAUUCACCACpoly(A)3'. This same nucleotide sequence, excluding the poly(A) segment, has also been found at the 5' terminus of Rous sarcoma virus RNA (W. A. Haseltine, A. Maxam, and W. Gilbert, this issue pp. 989-993), and therefore the RNA genome of this virus is terminally redundant. Possible mechanisms for endogenous in vitro copying of the complete RNA genome by reverse transcriptase which involve terminally repeated nucleotide sequences are discussed.
Theorems of Barth-Lefschetz type for complex subspaces of homogeneous complex manifolds
Authors: Sommese Andrew John
Published: May 2006

Barth, Larsen, and others showed that complex submanifolds of complex projective space, C

P
N

, of small codimension strongly resemble C

P
N

both homotopically and cohomologically. These results are generalized to yield analogous results for complex subspaces of arbitrary homogeneous complex manifolds. One very special corollary that gives the flavor of the results is:

Corollary
.
Let
A
and
B
be complex submanifolds with
B
a connected submanifold of
X,
a simple Abelian variety, i.e.
, X
has no proper sub-Abelian varieties. Then:
[Formula: see text],
where basepoints are suppressed for simplicity, and
[Formula: see text].
Further, given any coherent sheaf 𝒮 on
B,
which may be assumed to be a connected local complete intersection, then:
[Formula: see text]
and given a coherent sheaf 𝒮 on
X,
then:
[Formula: see text].
Nonidentifiability in stochastic models of illness and death.
Authors: Clifford P
Published: May 2006

The interpretation of animal survival experiments in which disease incidence is determined at death or following sacrifice is shown to involve certain ambiguities. In particular, quantities of interest such as the expected duration of life for an animal contracting a specific disease at a specific age are found to be nonidentifiable. An example is constructed in which two populations of animals will appear similar to the experimenter but in which animals contracting a particular disease in one population may have double the life expectancy of similarly afflicted animals in the other population.
Chromatin subunits from baker's yeast: isolation and partial characterization.
Authors: Nelson D A , Beltz W R , Rill R L
Published: May 2006

The organization of proteins along DNA in chromatin of Saccharomyces cerevisiae (baker's yeast) was examined by analyzing the DNA and nucleoprotein products obtained after digestion of yeast nuclei with staphylococcal nuclease. Yeast DNA is digested in situ at regularly spaced cleavage sites about 160 base pairs apart. Nucleoprotein fragments were resolved and isolated by centrifugation on linear, 5-20% sucrose gradients. The predominant 11S component appears to be identical to chromatin "subunits" or "nucleosomes" isolated from higher eukaryotes, containing a 150-160 base pair length of DNA and approximately equimolar amounts of four proteins that coelectrophorese with calf histones H2A, H2B, H3, and H4, plus small amounts of three proteins that electrophorese similarly to H1 histones. Thus, the structural organization of the yeast genome is similar to that of more developed organisms, except for the smaller total repeat length. None of the yeast subunit proteins, including the possible H1 proteins, contains cysteine.
Mapping of sequences with 2-fold symmetry on the simian virus 40 genome: a photochemical crosslinking approach.
Authors: Shen C K , Hearst J E
Published: May 2006

Sequences with 2-fold axes of symmetry have been detected and mapped on the simian virus 40 (SV40) genome by their ability to form hairpin turns in single-stranded SV40 DNA. Supercoiled SV40 DNA (SV40 I) was digested with restriction enzymes EcoRI and HpaII. The resulting linear DNA molecules with lengths of the complete SV40 genome were then denatured and photochemically reacted with 4,5',8-trimethylpsoralen (trioxsalen) at 16.0 +/- 0.5 degrees and different ionic strengths. Secondary structures on the single-stranded SV40 DNA were crosslinked and their positions analyzed by electron microscopy. There were no observable hairpin turns on the denatured SV40 DNA when it was photoreacted in 1 mM Tris-HCl/0.1 mM EDTA at pH 7.0. In 20 mM NaCl, one specific hairpin turn was detected at 0.17 +/- 0.02 map units on the map of EcoRI-digested SV40 DNA, where the 3' ends of early 19S mRNA, late 19S mRNA, and 16S mRNA of SV40 have been mapped. In 30 mM NaCl there are five more major hairpin turns besides the most stable one. The centers of four of these specific hairpin turns were mapped at 0.26 +/- 0.02, 0.68 +/- 0.03, 0.84 +/- 0.02, and 0.94 +/- 0.01 units on the map of EcoRI-digested SV40. The fifth one is at or near the unique EcoRI cleavage site on SV40 DNA. The possible functions of these sequences are discussed in terms of the nature of the promoter sites, the replication origin, the processing of RNA precursors, and regulation at the translational level.
Dissociation and reassociation of immobilized porphobilinogen synthase: use of immobilized subunits for enzyme isolation.
Authors: Gurne D , Chen J , Shemin D
Published: May 2006

The dissociation and association of an immobilized preparation of the octameric enzyme porphobilinogen synthase [5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24] is described. On treatment of the immobilized preparation with 4 M urea, four subunits per octamer are removed which can be reassociated into a soluble octameric enzyme. The tetrameric bound residual protein can also be reassembled into an octameric structure, with the same initial enzyme activity, by exposing the residual bound protein to a soluble pure enzyme preparation or to a crude liver extract in the presence of urea. The dissociation of the reconstituted bound enzyme releases subunits that again can be reassembled into a soluble octameric pure protein even when the crude liver preparation is used as the donor of the subunits. Thus, a pure enzyme can be isolated in a reassociation-dissociation cycle. The use of immobilized preparations of oligomeric proteins is considered for intra- and interspecies hybridization studies and for the ready preparation of purified enzyme preparations from different species and is suggested as a model for study of the formation of an oligomeric enzyme in the presence of other polypeptides.
Evidence that dephosphorylation inactivates glucocorticoid receptors.
Authors: Nielsen C J , Sando J J , Pratt W B
Published: May 2006

Highly purified alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] from calf intestine inactivates the glucocorticoid-binding capacity of soluble preparations from mouse fibroblasts (L cells) and rat liver. The unbound receptor is sensitive to inactivation whereas the steroid-bound receptor is unaffected. The ability of the enzyme preparation to inactivate the receptor, like its ability to dephosphorylate p-nitrophenyl phosphate, is dependent on zinc and inhibited by arsenate. Both the dephosphorylating and receptor inactivating activities coelute during DEAE-cellulose purification of the enzyme. There is no detectable proteolytic activity in the purified alkaline phosphatase preparation. In a mixed system containing both glucocorticoid and estrogen receptors, the glucocorticoid receptor is selectively inactivated. Although these observations do not prove that the receptor molecule itself is the substrate, they are consistent with the proposal that the glucocorticoid receptor can be inactivated by dephosphorylation and that only the phosphorylated form of the molecule is capable of binding steroid. A phosphorylation-dephosphorylation mechanism may be responsible for determining the level of active receptor in the cell.
Regulation of cholesterol synthesis in rat adrenal gland through coordinate control of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase activities.
Authors: Balasubramaniam S , Goldstein J L , Brown M S
Published: May 2006

The activities of cytosolic 3-hydroxy-3-methylglutaryl coenzyme A synthase [3-hydroxy-3-methylglutaryl-CoA acetoacetyl-CoA-lyase (CoA-acylating), EC 4.1.3.5] and microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase[mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34], two sequential enzymes in the cholesterol biosynthetic pathway, were shown to be regulated coordinately in the adrenal gland of the rat. When the plasma cholesterol level was lowered by administration of 4-aminopyrazolopyrimidine, a treatment known to enhance cholesterol synthesis in the adrenal, synthase activity in the gland rose by 14- to 29-fold and reductase activity rose by 50- to 100-fold. The subsequent intravenous infusion of low density lipoprotein restored the plasma cholesterol level and suppressed synthase and reductase activities in parallel. The activities of adrenal 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase were also shown to exhibit a coordinate pattern of diurnal variation with peaks in both enzymes achieved at the mid-point of the dark cycle. The activity of adrenal acetoacetyl coenzyme A thiolase (acetyl CoA acetyltransferase; acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9), the enzyme preceding the synthase in the cholesterol biosynthetic pathway, and the activity of adrenal mevalonate kinase (ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36), the enzyme following the reductase, were not enhanced by cholesterol deprivation, and neither exhibited a pattern of diurnal variation. The coordinate control of 3-hydroxy-3-methylglutaryl CoA synthase and reductase in rat adrenal gland provides a model system to study the biochemical mechanism for the regulation of cholesterol synthesis in a tissue that uses cholesterol for the synthesis of steroid hormones.
Partial reaction of peptide initiation inhibited by phosphorylation of either initiation factor eIF-2 or 40S ribosomal proteins.
Authors: Kramer G , Henderson A B , Pinphanichakarn P , Wallis M H , Hardesty B
Published: May 2006

Preparations of the hemin-controlled repressor (HCR) from rabbit reticulocytes contain 3':5'-cyclic-AMP-independent protein kinase activity for the smallest subunit of the peptide initiation factor eIF-2 and for proteins of reticulocyte 40S ribosomal subunits. Binding of the ternary complex formed between Met-tRNAf, GTP, and eIF-2 to 40S ribosomal subunits is shown to be inhibited by phosphorylation of either the ribosomal subunits or eIF-2. The protein kinase activity responsible for phosphorylation of eIF-2 has been separated from the activity for phosphorylation of 40S ribosomal subunits and shown to independently block the same partial reaction of peptide initiation. It appears that different enzymes are involved, each capable of regulating peptide initiation at the same step but by a different mechanism.
Role of 3':5'-cyclic-AMP-dependent protein kinase in regulation of protein synthesis in reticulocyte lysates.
Authors: Datta A , de Haro C , Sierra J M , Ochoa S
Published: May 2006

The initiation inhibitor of reticulocyte lysates has been shown by others to be associated with a 3':5'-cyclic-AMP-independent protein kinase that catalyzes the phosphorylation of the small (38,000 daltons) subunit of the polypeptide chain initiation factor eIF-2. This factor forms a ternary complex with Met-tRNAi and GTP which, on interaction with a 40S ribosome, gives rise to a 40S complex. Ternary complex formation is inhibited by prior incubation of partially purified eIF-2 with reticulocyte inhibitor and ATP. The relation between phosphorylation and inactivation of eIF-2 is indicated by the lack of inhibition when ATP is omitted. Translation in hemin-containing reticulocyte lysates is also inhibited by cyclic-AMP-dependent protein kinases or their catalytic subunits. They act by converting proinhibitor (inactive eIF-2 kinase) present in lysates to inhibitor (active eIF-2 kinase). This reaction is analogous to the conversion of inactive phosphorylase kinase to active phosphorylase kinase.
Ribosome structure: localization of N6,N6-dimethyladenosine by electron microscopy of a ribosome-antibody complex.
Authors: Politz S M , Glitz D G
Published: May 2006

Antibodies to the minor nucleoside N6,N6-dimethyladenosine have been used to map a unique location of the nucleoside in the small subunit of the Escherichia coli ribosome. Antibodies were induced in rabbits by a nucleoside-bovine albumin conjugate and shown to be highly specific for the dimethyladenosine hapten. The antibodies were shown to interact with 30S ribosomal subunits from strain PR7, but not with subunits from its mutant strain TPR201, which is resistant to kasugamycin and lacks the two successive residues of dimethyladenosine normally found near the 3'-end of E. coli 16S ribosomal RNA. Electron micrographs of strain PR7 subunits, crosslinked by single IgG molecules, show a single binding site on the surface of the ribosome. This binding site is consistent with observations relating the 3'-end of the ribosomal RNA, binding of initiation factor IF-3 and messenger RNA, and mapping of specific ribosomal proteins.
Unexpected additional mode of energization of amino-acid transport into Ehrlich cells.
Authors: Garcia-Sancho J , Sanchez A , Handlogten M E , Christensen H N
Published: May 2006

Ehrlich cells treated with dinitrophenol and iodoacetate rapidly recover their 30-sec uptake of 2-(methyl-amino)-isobutyrate on treatment with 0.1 mM phenazine methosulfate + 20 mM sodium ascorbate before they begin to recover from the severely depressed ATP levels and alkali-ion gradients. Addition of 10 mM pyruvate also restores uptake of methylaminoisobutyrate before the alkali-ion gradients rise. This restoration is prevented by rotenone, but rotenone does not handicap restoration by phenazine methosulfate/ascorbate. Na+-independent uptake of 2-aminonorbornane-2-carboxylate by Ehrlich cells is affected the same way. Quinacrine almost completely suppresses uptake of methylaminoisobutyrate within the 30-sec uptake test, even when ATP levels are sustained by pyruvate and alkali-ion gradients are not depressed. Ouabain prevents restoration of both Na+-dependent and Na+-independent amino-acid transport by phenazine methosulfate/ascorbate or pyruvate. We interpret these results to indicate that amino-acid transport can be energized not only by known means, but also by reducing equivalents, which presumably reach the plasma membrane in the form of NADH from the mitochondria when the source of energy is pyruvate. In support of this hypothesis, the distribution of methylaminoisobutyrate between plasma membrane vesicles and their supporting media was influenced in the predictable way by NADH, quinacrine, and an uncoupling agent, proceeding on the assumption that more of the vesicles had the everted rather than the natural orientation.
Mechanism for acute control of fatty acid synthesis by glucagon and 3':5'-cyclic AMP in the liver cell.
Authors: Watkins P A , Tarlow D M , Lane M D
Published: May 2006

Labeling experiments with chicken liver cell monolayers and suspensions show that glucagon and N6, O2-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP) block fatty acid synthesis from acetate without appreciably affecting cholesterogenesis from acetate or acylglyceride synthesis from palmitate. Neither acetyl-CoA carboxylase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] activity assayed in the presence of citrate nor fatty acid synthetase activity is decreased in extracts of cells treated with glucagon. However, the cytoplasmic concentration of citrate, a required allosteric activator of acetyl-CoA carboxylase, is depressed more than 90% by glucagon or dibutyrl cyclic AMP. Pyruvate or lactate largely prevents the inhibitory action of these effectors on fatty acid synthesis by causing a large increase in cytoplasmic citrate level. Thus, it appears that glucagon, acting via cyclic AMP, inhibits fatty acid synthesis by blocking the formation of citrate, an essential activator of acetyl-CoA carboxylase.
Synthesis and glycosylation in vitro of glycoprotein of vesicular stomatitis virus.
Authors: Toneguzzo F , Ghosh H P
Published: May 2006

Coupling of ribonucleoprotein particles from L cells infected with vesicular stomatitis virus to a pre-incubated ribosomal system obtained from uninfected HeLa cells allowed synthesis of two proteins. G1 (molecular weight 63,000) and G2 (molecular weight 67,000), and all other proteins of vesicular stomatitis virus except the spike protein G (molecular weight 69,000). Analyses of the tryptic peptides showed that G1, G2, and G had identical peptide sequences. The synthesis of G2 required the presence of membranes; only G1 was synthesized in the absence of any membranes. G2 but not G1 was shown to be a glycoprotein by affinity chromatography on a concanavalin A-Sepharose column. Removal of sialic acid residues from G by neuraminidase resulted in a product having an identical mobility to G2. Digestion of G2 or G with a mixture of neuraminidase (EC 3.2.1.18), beta-galactosidase (EC 3.2.1.23), and beta-N-acetylglucosaminidase (EC 3.2.1.30), however, produced a protein of molecular weight 65,000. These data suggest that G2 is the desialated G and is formed by glycosylation of G1, which is the unglycosylated polypeptide backbone of G.
Different rate-limiting steps in excision repair of ultraviolet- and N-acetoxy-2-acetylaminofluorene-damaged DNA in normal human fibroblasts.
Authors: Ahmed F E , Setlow R B
Published: May 2006

In normal human cells the amount of excision of ultraviolet damage to DNA saturates at high doses. In these cells some chemicals mimic ultraviolet damage as far as their biological and repair characteristics are concerned. One of these chemicals is N-acetoxy-2-acetylaminofluorene. We determined whether the limited repair capacity for ultraviolet damage was affected by treatment with N-acetoxy-2-acetylaminofluorene. To measure repair we determined unscheduled DNA synthesis and the number of sites sensitive to an ultraviolet endonuclease in an assay using an extract of Micrococcus luteus. The nuclease does not act on DNA treated with the chemical. The amount of unscheduled DNA synthesis due to a combined chemical and ultraviolet treatment was the sum of those observed from the separate treatments, even at saturation doses. The combined treatment did not affect the removal of nuclease-sensitive sites. We conclude that there are different rate-limiting steps in excision repair of the ultraviolet and the chemical damage and suggest a model involving a complex of enzymes to explain the data.
Systemic alteration induced in mice by ultraviolet light irradiation and its relationship to ultraviolet carcinogenesis.
Authors: Fisher M S , Kripke M L
Published: May 2006

Chronic irradiation of mice with ultraviolet (UV) light produces a systemic alteration of an immunologic nature. This alteration is detectable in mice long before primary skin cancers induced by UV light begin to appear. The alteration results in the failure of UV-irradiated mice to reject highly antigenic, transplanted UV-induced tumors that are rejected by unirradiated syngeneic recipients. The immunologic aspect of this systemic alteration was demonstrated by transferring lymphoid cells from UV-irradiated mice to lethally x-irradiated recipients. These recipeints were unable to resist a later challenge with a syngeneic UV-induced tumor, whereas those given lymphoid cells from normal donors were resistant to tumor growth. Parabiosis of normal mice with UV-irradiated mice, followed by tumor challenge of both parabionts with a UV-induced tumor, resulted in the growth of the challenge tumors in both UV-irradiated and unirradiated mice. Splenic lymphocytes from tumor-implanted UV-treated mice were not cytotoxic in vitro against UV-induced tumors, whereas under identical conditions cells from tumor-implanted, unirradiated mice were highly cytotoxic. Our findings suggest that repeated UV irradiation can circumvent an immunologic mechanism that might otherwise destroy nascent UV-induced primary tumors that are strongly antigenic.
Unprimed spleen cell populations recognize macrophage-bound antigen with opposite net electric charge.
Authors: Teitelbaum D , Steinman L , Sela M
Published: May 2006

Previous studies have demonstrated an inverse charge relationship between the net electrical charge of antigen and the responding cells. In the present study we attempted to establish whether this phenomenon holds also for the primary recognition phase of cell-mediated immunity, when the involvement of the macrophage in presenting antigen is obligatory. Normal spleen cells were fractionated over negatively and positively charged columns. The fractionated cell populations, as well as the original cells, were sensitized in vitro on macrophage monolayers that were pulsed either with the basic encephalitogenic protein of myelin or with the acidic copolymer poly(Glu50,Tyr50). Cells eluted from glass bead columns(i.e., the more negative cells) could be sensitized only with the basic antigen, while cells eluted from poly(L-lysine)-glass bead columns (i.e., more positive cells) could be sensitized only to the acidic antigen. Thus, in delayed type hypersensitivity the inverse charge relationship prevails also for the primary immunological recognition of antigen bound to macrophages.