bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of motor neuron pools supplying identified muscles in normal and supernumerary legs of chick embryo.
Authors: Hollyday M , Hamburger V , Farris J M
Published: May 2006

Neuromuscular specificity has been investigated in chick embryos with a grafted supernumerary leg. The nerves of the lumbar plexus are divided between the two legs so that rostral nerves innervate the grafted leg and the caudal nerves supply the host's original leg. The basic topographic organization of the histologically definable motor neuron clusters of the lateral motor columns remains unchanged by the addition of a supernumerary leg. Intramuscular injections of identified leg muscles have been used to map the intraspinal location of specific motor pools in stage 38 (12-day) embryos. In the normal embryo, the gastrocnemius muscle is innervated by neurons in a central dorsal cluster of motor neurons in segments 26-29. In six experimental cases, the motor neurons supplying the gastrocnemius muscle of a rostrally placed grafted leg were consistently located in a specific medial cluster of neurons in segments 23-25. Motor neurons in this location never normally innervate a gastrocnemius muscle, even in the very young embryos during the period of naturally occurring cell death. This observation of a systematic mismatch between a particular motor cluster and an abnormally innervated muscle indicates the operation of a selective developmental process. A hierarchy of selective chemoaffinities may best explain our experimental results.
Primary structure of the membranous segment of cytochrome b5.
Authors: Ozols J , Gerard C
Published: May 2006

The primary structure of the membranous segment of porcine liver microsomal cytochrome b5 has been determined. This polypeptide is at the COOH terminus of the cytochrome molecule and consists of 43 amino acids. It is essential for the insertion of the cytochrome into the endoplasmic reticular membrane. Automated sequence analysis of tryptic and cyanogen bromide/anhydrous heptafluorobutyric acid peptides provided data from which the following unique amino acid sequence was deduced: Ile-Ala-Lys-Pro-Ser-Glu-Thr-Leu-Ile-Thr-Thr-Val-Glu-Ser-Asn-Ser-Ser-Trp-Trp-Thr-Asn-Trp-Val-Ile-Pro-Ala-Ile-Ser-Ala-Leu-Val-Val-Ser-Leu-Met-Tyr-His-Phe-Tyr-Thr-Ser-Glu-Asn. A prediction of alpha-helices, beta-structures, and beta-turns basedon the sequence of this polypeptide is also presented.
Occurrence of a major protein associated with fruiting body development in Neurospora and related Ascomycetes.
Authors: Nasrallah J B , Srb A M
Published: May 2006

Electrophoretic and immunological analysis of fruiting body (perithecial) extracts demonstrates the occurrence of a major phase-specific perithecial protein in all Neurospora species and in the closely related Gelasinospora cerealis and Sordariafimicola. The perithecial proteins from these different species fall into a number of groups with different electrophoretic mobilities. They appear to be immunologically closely related but not identical to one another even within the same genus, with only partial identity exhibited between the heterothallic and pseudohomothallic Neurospora on the one hand and the homothallic Neurospora on the other hand. In immunological analysis of fruiting body extracts of the other Ascomycetes, Podospora anserina, Cochliobolus maydis, and Aspergillus nidulans, and of ascus extracts of Saccharomyces cerevisiae, no crossreaction with the Neurospora perithecial protein was found.
Succinyl concanavalin A stimulates and antimicrotubular drugs inhibit the synthesis of a brain-specific protein in rat glial cells.
Authors: Marks A , Labourdette G
Published: May 2006

The relative synthesis of the brain-specific s100 protein increased as clonal rat glial cells, C6, progressed from logarithmic to stationary growth in monolayer culture. Drugs that disrupt microtubules, such as colchicine, vinblastine, Colcemid, and podophyllotoxin, inhibited the relative synthesis of S100 protein in stationary cultures. Colchicine (0.1 muM) caused a 50% inhibition of the relative synthesis of S100 protein whereas lumicolchicine,an isomer of colchicine that does not disrupt the microtubular system, had no effect. Succinylated concanavalin A (500 mug/ml) increased relative synthesis in logarithmic but not stationary cultures. These results suggest that signals inducing an increase in the relative synthesis of S100 protein in stationary cultures are transmitted intracellularly from the cell membrane by the microtubular network.
Morphological differentiation of neuroblastoma cells in medium supplemented with delipidated serum.
Authors: Monard D , Rentsch M , Schuerch-Rathgeb Y , Lindsay R M
Published: May 2006

Neuroblastoma cells have been found to extend neurites when grown in a medium supplemented with delipidated fetal calf serum. Fetal calf sera from different commercial sources give rise to marked differences in the degree of this spontaneous morphological differentiation. The phenomenon can be prevented by the addition of certain fatty acids; oleic acid is especially effective. The serum-free conditioned medium from glial cells can quantitatively antagonize the effect of oleic acid, suggesting that glial factor activity could be due to components [lipids and/or macromolecular factors(s)] that are able to modify the properties of the neuroblastoma cell membrane.
Variants of 3T3 cells lacking mitogenic response to epidermal growth factor.
Authors: Pruss R M , Herschman H R
Published: May 2006

We have previously demonstrated that epidermal growth factor (EGF) can serve as a potent mitogen for 3T3 cells. We have now selected variant 3T3 cell lines unable to respond to EGF, in order to define cellular events unique to the EGF response and to distinguish which of these events are necessary and which are merely correlative to mitogenesis. By simultaneously treating cells with EGF and colchicine, we eliminated those cells stimulated by EGF to enter mitosis. Of the eight clonal EGF nonresponder variants selected by this procedure, none retains a functional EGF receptor. The EGF nonresponsive va-riant lines still retain the ability to respond to other mitogens.
Role of mitotic replication genes in chromosome duplication during meiosis.
Authors: Zamb T J , Roth R
Published: May 2006

In the yeast Saccharomyces cerevisiae, DNA synthesis preceding meiosis requires the expression of replication genes used during mitosis. Diploids carrying either a temperature-sensitive lesion in cdc4 (a gene required for initiation of mitotic DNA synthesis) or a lesion in cdc8 (a gene controlling mitotic polymerization) completed premeiotic DNA synthesis at a permissive but not at a restrictive temperature. The roles of cdc4 and cdc8 were evaluated by characterizing the kinetics of premeiotic DNA synthesis after a shift to a restrictive temperature. In the cdc8 diploid, DNA synthesis was immediately inhibited, consistent with a role in polymerization. In contrast, cdc4 exhibited residual DNA synthesis characteristic of an initiation function. The cdc4 gene function was completed much earlier in the meiotic cycle than the cdc8-mediated step.
Transport of alpha-amylase across the basolateral membrane of the pancreatic acinar cell.
Authors: Isenman L D , Rothman S S
Published: May 2006

The flux of alpha-amylase (1,4-alpha-D-glucan glucanohydrolase; EC 3.2.1.1) across the basolateral membrane of the acinar cell was measured in the cell-to-bath direction using the whole rabbit pancreas in organ culture. This in vitro preparation is polarized so that apical and basolateral secretions can be collected separately. The unstimulated amylase flux from cell to bath was substantial at the initial rate (approximately three times the concurrent apical flux). With time, bath amylase approached a steady-state concentration, suggesting an equilbrating process. During the same time interval, ductal amylase secretion remained constant. At the steady state, the amylase concentration in the bath was at least an order of magnitude less than its ductal concentration. Hourly replacement of bathing medium reproduced the initial rate of amylase release into the bath for five consecutive hours. Pancreozymin (cholecystokinin), a peptide hormone, did not alter the steady-state bath amylase content, although it greatly augmented ductal amylase secretion. In contrast, a cholinergic agonist greatly increased both the flux from the cell to bath and the ductal secretion of amylase. Taken together, these results indicate a natural bidirectional permeability of the basolateral membrane to digestive enzyme and support evidence previously obtained suggesting that such a permeability might exist.
Organization and expression of early genes of simian virus 40
Authors: Crawford L. V. , Cole C. N. , Smith A. E. , Paucha E. , Tegtmeyer P. , Rundell K. , Berg P.
Published: May 2006

The early region of simian virus 40 codes for at least two immunologically related polypeptides: large-T and small-t, with apparent molecular weights of 90,000-100,000 and 15,000-20,000, respectively. Because small-t shares methionine-containing tryptic peptides with large-T, the two polypeptides are probably coded, in part, by a common nucleotide sequence. To locate the coding sequences for large-T and small-t in the DNA, the production of these proteins was examined after infection of CV-1 cells with wild-type and deletion mutants of simian virus 40. We found that a deletion at the distal portion of the early region alters the structure of large-T but not of small-t; but deletions within the region between map coordinates 0.59 and 0.55 result in an alteration or absence of small-t and a normal large-T. These findings have been rationalized by a model that proposes the existence of two early mRNAs, one coding for large-T and the other for small-t. Both mRNAs span virtually the entire early region; but the mRNA coding for large-T lacks the nucleotide sequence between map coordinates 0.59 and 0.54. We suggest that small-t is translated from the larger of the two mRNAs, beginning at or near its 5′ end and terminating at a termination codon at about map coordinate 0.54. Larger-T, on the other hand, is translated from the shorter mRNA, beginning at the same initiator codon, and, because of the deletion of the terminator codon at 0.54, translation proceeds to the terminator codon at or near map position 0.18.
In vitro inhibition of protein synthesis by purified cores from vaccinia virus.
Authors: Ben-Hamida F , Beaud G
Published: May 2006

The mechanism of the shutoff of cellular protein synthesis in vaccinia virus-infected cells has been investigated by using in vitro systems. Purified vaccinia cores cause inhibition of endogenous mRNA translation in nonpreincubated reticulocyte lysates and Ehrlich ascites tumor cell-free systems. Translation of viral mRNA from turnip yellow mosaic virus is also impaired in wheat germ cell-free extracts. The block induced by vaccinia cores in protein synthesis is not due to a decrease in the availability of mRNA but rather to an alteration of the cellular translational machinery. No nucleolytic activity able of digesting mRNA could be detected in purified vaccinia cores with three sensitive tests. There is a lack of inhibition in the poly(Phe)-poly(U) system, which bypasses the normal initiation process. An almost complete disaggregation of polyribosomes in the reticulocyte lysate appears when vaccinia cores are present. These results indicate that mRNA translation in a cell-free system is affected predominantly at the level of polypeptide chain initiation.
In vivo phosphorylation of a synthetic peptide substrate of cyclic AMP-dependent protein kinase.
Authors: Maller J L , Kemp B E , Krebs E G
Published: May 2006

A model synthetic peptide substrate of the cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase; EC 2.7.1.37), Leu-Arg-Arg-Ala-Ser-Leu-Gly, closely resembling the local phosphorylation site sequence in porcine hepatic pyruvate kinase, was shown to be phosphorylated in vivo after microinjection into Xenopus oocytes. This result demonstrates that the microinjection technique, utilizing a synthetic peptide substrate, or possibly a synthetic substrate analog inhibitor [Kemp, B. E., Benjamini, E. & Krebs, E. G. (1976) Proc. Natl. Acad. Sci. USA 73, 1038--1042], can be used to study protein phosphorylation-dephosphorylation reactions in living oocytes. This follows, since it is clear that the injected peptide was accessible to the cellular compartment containing the protein kinase.
Streptomycin causes misreading of natural messenger by interacting with ribosomes after initiation.
Authors: Tai P C , Wallace B J , Davis B D
Published: May 2006

The induction of misreading by streptomycin in vitro, previously observed with synthetic messengers, is now demonstrated with natural (endogenous or viral) messenger by the use of extracts of temperature sensitive mutants lacking Glu--tRNA or Val--tRNA synthetase. With chain-elongating but noninitiating ribosomes (i.e., purified polysomes) deprived of an aminoacyl--tRNA, streptomycin and other aminoglycosides, over a wide range of concentrations, stimulate incorporation. With ribosomes initiating in the presence of streptomycin stimulation is also observed but it is restricted, just like phenotypic suppression in cells, to very low streptomycin concentrattions which evidently allow some ribosomes to initiate and later encounter them in the course of chain elongation. The stimulation is accompanied by an increase in the size of the products; hence, it is evidently due to substitution of an incorrect aminoacyl--tRNA for a missing one. The test introduced here also has revealed a misreading effect of streptomycin on resistant ribosomes. In addition, significant intrinsic misreading was observed without streptomycin, indicating that under optimal conditions for in vitro protein synthesis an empty codon is frequently read by an incorrect aminoacyl--tRNA.
Geometric packing constraints in egg phosphatidylcholine vesicles.
Authors: Huang C , Mason J T
Published: May 2006

This investigation presents a quantitative analysis of the asymmetric transmembrane lipid packing geometry in egg phosphatidylcholine vesicles. The analysis uses hydrodynamic and nuclear magnetic resonance data obtained for homogeneous egg phosphatidylcholine vesicles and demonstrates that the average area per lipid head group and effective length of the lipid are greater on the outer monolayer than on the inner monolayer of the egg phosphatidylcholine vesicle. Results also indicate that the bilayer of the egg phosphatidylcholine vesicles has an asymmetrical interface through its center.
Cytoplasmic inheritance of rutamycin resistance in mouse fibroblasts.
Authors: Lichtor T , Getz G S
Published: May 2006

Mouse fibroblasts resistant to the drug rutamycin were isolated by selectively introducing BrdUrd into the mitochondrial genome of a line of mouse fibroblasts (clone 1 D) lacking a cytoplasmic thymidine kinase enzyme. The ATPase (ATP phosphohydrolase; EC 3.6.1.3) activity of mitochondria isolated from these cells was resistant to rutamycin. The rutamycin-resistant mutants were enucleated with cytochalasin B and fused with mouse A 9 cells resistant to 8-azaguanine and sensitive to rutamycin. Cytoplasmic hybrids, or cybrids, were selected as cells resistant to rutamycin and 8-azaguanine, and appeared at a high frequency. Other fusions between rutamycin-resistant nucleated cells and A 9 produced colonies at a much lower frequency. Finally, fusions between enucleated clone 1 D cells and A 9 cells produced no rutamycin-resistant colonies. These results indicate that rutamycin resistance is a cytoplasmically inherited characteristic in this cell line.
Diadenosine 5',5'''-P1,P4-tetraphosphate triggers initiation of in vitro DNA replication in baby hamster kidney cells.
Authors: Grummt F
Published: May 2006

Addition of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) to permeabilized G1-arrested baby hamster kidney cells resulted in the stimulation of DNA synthesis. No stimulation was observed in cells from exponentially growing cultures. The Ap4A-stimulated [3H]dTTP incorporation was inhibited by nalidixic acid, daunomycin, chloroquine diphosphate, EDTA, and N-ethylmaleimide. It was dose-dependent in regard to the amount of permeabilized cells and of Ap4A. Numerous replication eyes were formed in the DNA molecules of stimulated cells. Pulse-chase experiments showed that the synthesis of DNA was discontinuous, resulting in the appearance of approximately 4S Okazaki fragments and their ligation to high molecular weight DNA. These results strongly suggest that Ap4A stimulated the initiation of DNA synthesis in baby hamster kidney cells that had been arrested in G1 by serum deprival.
Deoxyadenosine triphosphate as a potentially toxic metabolite in adenosine deaminase deficiency.
Authors: Cohen A , Hirschhorn R , Horowitz S D , Rubinstein A , Polmar S H , Hong R , Martin D W
Published: May 2006

The inherited deficiency of adenosine deaminase (adenosine aminohydrolase; EC 3.5.4.4) activity in humans is associated with an immunodeficiency. Some of the immunodeficient and enzyme-deficient patients respond immunologically to periodic infusions of irradiated erythrocytes containing adenosine deaminase. It has been previously reported that erythrocytes and lymphocytes from immunodeficient ane enzyme-deficient children contained increased concentrations of ATP, and in the one child studied after erythrocyte infusion therapy, the intracellular level of ATP diminished. Using high-pressure liquid chromatography that resolves ATP and 2'-dATP, we have observed greater than 50-fold elevations of dATP in the erythrocytes of immunodeficient, adenosine deaminase-deficient patients but not in the erythrocytes of an immunocompetent adenosine deaminase-deficient patient. The erythrocyte dATP in two unrelated adenosine deaminase-deficient, immunodeficient patients disappeared after infusion of normal erythrocytes. We propose that deoxyadenosine, a substrate of adenosine deaminase, is the potentially toxic substrate in adenosine deaminase deficiency, and that the mediator of the toxic effect is dATP, a recognized potent inhibitor of ribonucleotide reductase.
Energy conformation study of Met-enkephalin and its D-Ala2 analogue and their resemblance to rigid opiates.
Authors: Loew G H , Burt S K
Published: May 2006

Conformational similarities of Met-enkephalin and its D-Ala2 analogue to rigid opiates were studied by both empirical and quantum mechanical methods. By both methods, conformers with maximum resemblance to rigid opiates have the highest energies. Conformers with the lowest energy had no resemblance to rigid opiates. However, several low and intermediate energy conformers were identified in which at least the NH2-terminal tyrosine residue overlaps with the phenethylamine moiety of rigid opiates and which could equally well accommodate either Gly2 or D-Ala2. The conformer among these with greatest additional resemblance to other binding sites of rigid opiates is proposed as the most likely candidate for an induced fit at the receptor site.
Protected homo-oligopeptide structure: Model for preferred conformation of a linear methionine heptapeptide in chloroform
Authors: Goodman Murray , Ribeiro Anthony A. , Naider Fred
Published: May 2006

The
1
H nuclear magnetic resonance spectra for a protected linear heptapeptide of methionine, Boc-Met
7
-OMe, were measured in dimethyl-d
6
sulfoxide and C
2
HCl
3
solutions. In the former, the peptide is disordered. In C
2
HCl
3
, preferred (short-range) interactions exist that dominate the overall secondary structure of the peptide. A model is proposed in which seven-membered ring structures are initiated at the NH
2
-terminus and propagate toward the carboxyl terminus. The first seven-membered ring contains a strong hydrogen bond between the amide NH of residue 2 and the carbonyl oxygen atom of the urethane blocking group. As one proceeds toward the carboxyl terminus the hydrogen bond strength of succeeding rings decreases, and at residue 5 the peptide chain assumes an extended structure that is stabilized by intermolecular hydrogen bonds. The model accounts for most of the results from
1
H NMR and infrared studies on the heptapeptide and is consistent with conformational energy calculations on homo-oligopeptides. It suggests that the COOH-terminal residues may serve as the nucleus for intermolecular peptide-peptide associations.
Nonexponential fluorescence decay of aqueous tryptophan and two related peptides by picosecond spectroscopy
Authors: Fleming G. R. , Morris J. M. , Robbins R. J. , Woolfe G. J. , Thistlethwaite P. J. , Robinson G. W.
Published: May 2006

Time-resolved fluorescence spectroscopy of tryptophan and two related dipeptides, tryptophylalanine and alanyltryptophan, has been carried out on the subnanosecond time scale by using picosecond exciting pulses at a wavelength of 264 nm. Detection was with an ultrafast streak camera coupled to an optical multichannel analyzer. The zwitterions of these molecules show a definite nonexponential fluorescence decay which can be analyzed in terms of two exponentials. The two decay rates increase strongly with increasing temperature, as does the weight of the faster component. In tryptophan at pH 11, where the amino group is deprotonated, there remains only a single temperature-dependent exponential. The results are interpreted in terms of two kinds of trapped conformers in the excited state that interconvert no quicker than the time scale of the fluorescence. A model is suggested in which the nonradiative processes in one conformer approximate those in the bare indole moiety. The nonradiative decay rate of the other conformer is substantially faster. It is believed that the process responsible for this fast decay is intramolecular electron transfer from the indole to the amino acid side chain. The predilection for this electron transfer depends on steric relationships as well as on the electron-attracting power of the carbonyl group. This picture is consistent with earlier fluorescence quantum yield results. In fact, a self-consistent picture emerges from the temporal and yield data that quantitatively explains most important facets of tryptophan photochemistry in aqueous solution.
Sequence of a RNA templated by the 3'-OH RNA terminus of defective interfering particles of vesicular stomatitis virus.
Authors: Semler B L , Perrault J , Abelson J , Holland J J
Published: May 2006

We have sequenced the endogenous RNA polymerase product produced by disrupted purified virions of vesicular stomatitis virus defective interfering particles by using the newer one-dimensional rapid gel sequencing techniques and confirming this with a modified two-dimensional gel vectoring technique. The sequence of this 46-nucleotide RNA is: 5'(pp)pACGAAGACCACAAAACCA-GAUAAAAAAUAAAAACCACAAGAGGG(U)COH3'. We infer that this sequence is identical to the sequence at the 5' end of infectious vesicular stomatitis virus RNA and is complementary to the sequence of the 3'-OH terminus of this defective interfering particle genome RNA.
Direct phosphorylation of brain tyrosine hydroxylase by cyclic AMP-dependent protein kinase: mechanism of enzyme activation.
Authors: Joh T H , Park D H , Reis D J
Published: May 2006

Tyrosine hydroxylase [tyrosine monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] was highly purified from rat caudate nuclei. When the pure hydroxylase was phosphorylated by incubation with cyclic AMP-dependent protein kinase and [32P]ATP, 32P and tyrosine hydroxylase activity were detected after polyacrylamide gel electrophoresis in a single protein band. After sodium dodecyl sulfate gel electrophoresis, 32P was detected only in a probably active subunit of tyrosine hydroxylase of molecular weight 62,000. Phosphorylation of the hydroxylase increased its activity by 2-fold, and was associated with an increase in Vm without any change in Km for either substrate or cofactor. We propose that the pool of native tyrosine hydroxylase is composed of a mixture of enzyme molecules in both active and probably inactive forms, that the active form is phosphorylated, and that phosphorylation produces an active form of the enzyme at the expense of an inactive one.
Gonadal luteinizing hormone receptors and adenylate cyclase: transfer of functional ovarian luteinizing hormone receptors to adrenal fasciculata cells.
Authors: Dufau M L , Hayashi K , Sala G , Baukal A , Catt K J
Published: May 2006

Luteinized rat ovaries contain a high concentration of particulate luteinizing hormone (lutropin, LH) receptors and a small quantity of lipid-associated receptors that float in the 360,000 X g supernatant fraction of ovarian homogenates. During fractionation of Lubrol-solubilized LH receptors and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from the ovary and testis, LH receptors and adenylate cyclase were coincident on gel filtration, but could be resolved during ion-exchange chromatography of soluble ovarian preparations and were completely separated by lectin-affinity chromatography on Sepharose-concanavalin A. For further analysis of receptor-adenylate cyclase coupling, the lipid-rich fraction of ovarian luteal cells was used to transfer gonadal LH receptors to isolated adrenal fasciculata cells. The lipid vesicles obtained from ovarian homogenates by flotation at 360,000 X g contained 5--10% of the ovarian LH receptors and were devoid of adenylate cyclase activity. During incubation of lipid-associated receptors with dispersed rat fasciculata cells at 16 degrees C, progressive incorporation of LH binding sites into the adrenal cells was observed. When adrenal cells bearing heterotopic LH receptors were incubated with 1 nM human choriogonadotropin, cyclic AMP production was consistently stimulated, with an accompanying increase in corticosterone production. These results indicate that LH receptors exist as separate entities from adenylate cyclase in the gonadal cell membrane and can become functionally coupled to adenylate cyclase to evoke cyclic AMP production and steroidogenesis in the host adrenal cells to which they are transferred.
Protein synthesis in rabbit reticulocytes: characteristics of a ribosomal factor that reverses inhibition of protein synthesis in heme-deficient lysates.
Authors: Ralston R O , Das A , Dasgupta A , Roy R , Palmieri S , Gupta N K
Published: May 2006

A ribosomal salt (0.5 M KCl) wash factor (RF) that reverses inhibition of protein synthesis in heme-deficient reticulocyte lysates has been resolved from the bulk of Met-tRNAfMet-binding factor (EIF-1), Co-EIF-1, and EIF-2 (ternary complex dissociation factor, TDF). The purified RF restores protein synthesis activity of heme-deficient lysates to the level observed in the presence of hemin. No direct correlation exists between amount of EIF-1 activity and ability to reverse inhibition of protein synthesis in heme-deficient lysates. Homogeneous preparations of EIF-1 are completely inactive in reversal of protein synthesis inhibition in heme-deficient lysates. These findings suggest that RF activity is not due to EIF-1 alone but may or may not require EIF-1 as a component of a complex factor.
DNA supercoiling by Xenopus laevis oocyte extracts: requirement for a nuclear factor.
Authors: Baldi M I , Mattoccia E , Tocchini-Valentini G P
Published: May 2006

A purified system is described for the introduction of negative supercoils into simian virus 40 DNA. The system consists of histones H2A, H2B, H3, and H4, DNA-relaxing enzyme, and a purified factor from Xenopus laevis stage 6 oocyte nuclei. The nuclei are prepared en masse by the technique of F. Scalenghe, M. Buscaglia, C. Steinheil, and M. Crippa [(1978) Chromosoma 60, 299-308]. The supercoiled simian virus 40 DNA prepared by this method is indistinguishable from simian virus 40 supercoiled DNA prepared from infected monkey cells.
Plasmodium knowlesi-induced antigens in membranes of parasitized rhesus monkey erythrocytes.
Authors: Schmidt-Ullrich R , Wallach D F
Published: May 2006

Highly purified Plasmodium knowlesi schizonts were used to produce a hyperimmune anti-parasite serum in a rhesus monkey. Proteins of membranes from normal and P. knowlesi-infected erythrocytes, as well as purified schizonts, were solubilized in 1% Triton X-100 and analyzed by bidimensional electrophoretic techniques. Of seven parasite-specific antigens identified in membranes of parasitized erythrocytes by crossed immune electrophoresis against monkey anti-parasite serum, only three could be detected in the purified schizonts. Bidimensional focusing-dodecyl sulfate/polyacrylamide gel electrophoresis of membranes from parasitized cells revealed three proteins, in the 55,000-90,000 molecular weight region, with isoelectric points between pH 4.5 and pH 5.2, that could not be detected in normal membranes or purified schizonts. Membranes of normal erythrocytes and uninfected erythrocytes that had been incubated with sera from monkeys with 25-50% parasitemia did not react with the monkey anti-parasite serum.
Bromodeoxyuridine mutagenesis in mammalian cells: mutagenesis is independent of the amount of bromouracil in DNA.
Authors: Kaufman E R , Davidson R L
Published: May 2006

Studies were undertaken to determine how a line of mutant Syrian hamster melanoma cells (HAB-2E) that displays unlimited growth potential when all of the thymine residues in nuclear DNA are replaced by bromouracil (BrUra) could avoid the deleterious effects of bromodeoxyuridine (BrdUrd) mutagenicity. It was found that BrdUrd could be mutagenic to these cells. However, there was a nonlinear relationship between mutagenicity and the amount of BrUra in the DNA of the HAB-2E cells. With these cells, mutagenicity apparently is determined by the concentration of BrdUrd to which the cells are exposed rather than the amount of BrUra in DNA. These results were obtained with both the induction of ouabain resistance and thioguanine resistance as markers for mutagenesis. The dependence of BrdUrd mutagenicity on BrdUrd concentration was also observed for the parental melanoma cells.
Expression of simian virus 40 early genes in transformed rat cells is correlated with maintenance of the transformed phenotype
Authors: Gaudray Patrick , Rassoulzadegan Minoo , Cuzin Francois
Published: May 2006

Early viral polypeptides synthesized in simian virus 40 rat transformants were identified by immunoprecipitation using anti-T (tumor) antigen immune serum. Four polypeptide classes could be identified, which were not detectable in extracts of nontransformed cells and were not precipitated from transformed cell extracts by nonimmune serum. Their apparent
M
r
were 92,000, 63,000, 56,000, and 19,000. A similar pattern was observed in extracts from lytically infected cells, but the relative rate of radioactive labeling of the
M
r
63,000 and 56,000 species was in this case significantly lower than in transformed cells. In
tsA30
transformants of type A, which maintain the transformed phenotype at high temperature, only minor quantitative variations of this pattern were observed when the cultures were shifted from 33° to 40.5°. In contrast, the rate of labeling of the four virus-specific polypeptides was decreased by 90% or more at high temperature in the temperature-sensitive N transformants. In all cases, a coordinated variation of the radioactivity associated with the different polypeptide classes was observed. These results suggest that the synthesis or processing, or both, of the viral early proteins may be controlled by different mechanisms in various types of simian virus 40 transformants and, furthermore, that it may be under the positive control of a virus-coded protein in transformed cells of type N.
Differences in α and β polypeptide chains of tubulin resolved by electron microscopy with image reconstruction
Authors: Crepeau Richard H. , McEwen Bruce , Edelstein Stuart J.
Published: May 2006

Electron microscopic techniques have been used to reveal two classes of subunits of tubulin in ordered arrays. Presumably the two classes correspond to the α and β polypeptide chains of tubulin that have been distinguished by chemical criteria. The two types of subunits alternate along individual protofilaments in microtubules, microtubule-precursor sheets, and extended zinc-tubulin sheets. The resolution of the two types of polypeptide chains is achieved by improved negative staining methods which produce micrographs with layer lines at 28 Å
-1
and 84 Å
-1
in optical or computed transforms, in addition to the layer lines at 21 Å
-1
and 42 Å
-1
described previously [Crepeau, R. H., McEwen, B., Dykes, G. & Edelstein, S. J. (1977)
J Mol. Biol.
116, 301-315]. In microtubules or microtubule-precursor sheets, adjacent protofilaments are staggered by about 10 Å, but parallel, in the sense that the α-β vector points in the same direction for all of the protofilaments of the microtubule. However, for the sheets assembled in the presence of zinc, adjacent protofilaments are staggered by about 21 Å and oriented in an antiparallel arrangement with alternate protofilaments related by a 2-fold screw axis. The antiparallel alignment of the protofilaments in the zinc-tubulin sheets accounts for their planarity (no tubular structures are found in the presence of moderate concentrations of zinc), since the intrinsic curvature found with parallel alignment of protofilaments in the absence of zinc would be cancelled by the antiparallel arrangement.
Natural killing and antibody-dependent cellular cytotoxicity are independent immune functions in the Minnesota miniature swine.
Authors: Koren H S , Amos D B , Kim Y B
Published: May 2006

Peripheral blood lymphocytes from Minnesota miniature pigs were tested for natural killing (NK) and antibody-dependent cellular cytotoxicity (ADCC) in a 2- to 4-hr 51Cr release assay against human myeloid and lymphoid tumor target cells. Adult specific pathogen-free and germfree animals exhibited normal levels of activity in both assays. In addition, the NK and ADCC activities of peripheral blood lymphocytes from colostrum-deprived newborn piglets were examined. These animals were obtained by hysterectomy and previously shown to be immunologically "virgin." We found that these newborn piglets exhibited normal ADCC but lacked NK activity. The differences in the ontogeny of the two activities suggest that they are distinct. Preliminary effector cell characterization studies suggest that: (i) NK and ADCC in the pig are physically not separable; (ii) the majority of the cytotoxic activity on a cell-per-cell basis is mediated by the non-T lymphocyte fraction; and (iii) the rosetted T cells, which account for about 60% of the total pig peripheral blood lymphocytes, have low but demonstrable cytotoxic activity as well.
L-aspartate: evidence for a role in cone photoreceptor synaptic transmission in the carp retina.
Authors: Wu S M , Dowling J E
Published: May 2006

A number of putative neurotransmitter substances and their antagonists were applied to the carp retina while intracellular recordings from L-type cone horizontal cells were made. Of all the substances tested, L-aspartate was found to be the most potent agent in depolarizing these horizontal cells in dark-adapted, partially light-adapted, and Co2+-treated retinas. Furthermore, DL-alpha-aminoadipate, an L-aspartate antagonist, blocked the effects of both the endogenous photoreceptor transmitter and exogenously applied L-aspartate on the horizontal cells. The results suggest that L-aspartate and the natural transmitter interact with the same population of postsynaptic receptors in the horizontal cell membrane.
Chimeric toxins: Toxic, disulfide-linked conjugate of concanavalin A with fragment A from diphtheria toxin
Authors: Gilliland D. Gray , Collier R. John , Moehring Joan M. , Moehring Thomas J.
Published: May 2006

A disulfide-linked conjugate of concanavalin A (Con A) and fragment A from diphtheria toxin has been synthesized and shown to be toxic for HeLa (human), Chinese hamster ovary (CHO), and SV3T3 (murine) cells. The conjugate was constructed by first coupling cystamine to Con A with a carbodiimide reagent and then reacting the modified Con A with reduced fragment A under conditions promoting disulfide interchange. The desired conjugate, obtained in nearly 50% yield relative to input of fragment A, was purified by affinity chromatography on Sephacryl S-200 and NAD-Sepharose; on analysis, it gave an average of 1.4 molecules of fragment A per tetrameric Con A molecule. The conjugate proved to be about equally active in inhibiting protein synthesis in HeLa, CHO, or SV3T3 cells in culture but was inactive relative to controls in a toxin-resistant strain of CHO cells containing altered elongation factor 2, the target protein of fragment A. With toxin-sensitive strains the conjugate was 100- to 1000-fold more active than controls, including fragment A, cystaminyl-Con A, and mixtures thereof, but was 1/50th to 1/500th as toxic as diphtheria toxin itself. Similar activity relative to controls was observed after intradermal inoculations in rabbits, and intravenous injections of the conjugate were lethal for mice. The activity of the conjugate in tissue culture was inhibited by Con A or α-methylmannoside but not by galactose. This and similar conjugates should be useful in studying mechanisms of entry of biologically active proteins into cells.
RNA sequencing with radioactive chain-terminating ribonucleotides.
Authors: Kramer F R , Mills D R
Published: May 2006

A rapid method for determining nucleotide sequences in RNA is described. It employs the 3'-deoxy analogues of the ribonucleoside triphosphates as specific chain terminators during RNA synthesis. For example, the inclusion of 3'-deoxyuridine 5'-triphosphate in an RNA synthesis reaction in addition to the four usual ribonucleoside triphosphate precursors results in the synthesis of a set of different-length product strands that terminate in a 3'-deoxyuridine that has been incorporated in place of uridine. To sequence an RNA, four separate reactions are run, each employing a different 3'-deoxy terminator. Parallel electrophoretic analysis of the resulting four sets of specifically terminated product chains leads to a direct reading of the nucleotide sequence. We tested this method by sequencing MDV-1 (-) RNA, a molecule that is synthesized in vitro by phage Qbeta replicase. The sequence read from the resulting gels agreed completely with the known sequence of MDV-1 (-) RNA. The bands in some regions of the sequencing gels were unusually close to one another, as has also been observed in other rapid sequencing procedures, making order assignment in these regions very difficult. Because the secondary structure of MDV-1 (-) RNA was known, it was shown that the compression of the bands is due to the persistence of secondary structures during electrophoresis. Thus, structured regions of nucleic acids may introduce difficulties for sequencing techniques that employ the currently available methods of gel electrophoresis.
In vitro RNA-RNA splicing in adenovirus 2 mRNA formation.
Authors: Blanchard J M , Weber J , Jelinek W , Darnell J E
Published: May 2006

"Splicing" of the precursor to an adenovirus mRNA was accomplished in isolated cell-free extracts. Nuclei were prepared from hypotonically swollen cells that had been labeled with [3H]uridine for 10 min prior to nuclear isolation. Addition of a "cytoplasmic" fraction was required for the splicing to occur. The nuclear precursor, a poly(A)-terminated RNA molecule approximately 5 kilobases long, contained sequences complementary to the 58.5--75.9 region of the adenovirus 2 genome, including those sequences spliced out of the mature mRNA molecule. The in vitro spliced product was a poly(A)-terminated RNA molecule identical in size to the cytoplasmic 72,000 Mr protein mRNA (2 kilobases long) in which the sequences encoded in the 70.7--75.9 region of the viral genome were spliced to those encoded at 58.7--65.6, with the sequences encoded at 66.1--70.7 deleted.
Activation energy for RNA transport from isolated rat liver nuclei.
Authors: Clawson G A , Smuckler E A
Published: May 2006

The temperature dependence of ATP-enhanced RNA delivery from rat liver nuclei to a surrogate cytoplasm was investigated. Examination of linear-rate data on Arrhenius graphs of 1/T vs. log (% RNA delivered per min) revealed an activation energy of 12.5--13 kcal/mol. When data derived from longer incubation periods was displayed on Arrhenius graphs, we observed a discontinuous graph--two distinct linear segments with slopes of differing sign which intersected near 20 degrees C. It was demonstrated that this discontinuity was not due to lipid phase transition in the nuclear membranes and that its position depended upon treatment of the nuclei and upon additives to the incubation mixtures. The decline in transport apparent in the upper-temperature domain on 20-min Arrhenius graphs was shown to be based on the diffusion of transported macromolecular RNA back into the nucleus--a process greatly amplified by the rapidity of transport in this domain. The large net inward diffusion, in concert with significantly differing activation energies for RNA transport and passive diffusion, suggests that the process of nucleocytoplasmic RNA transport is not diffusion driven. Our data have established that an integral parameter of RNA transport (namely, the activation energy) remains unchanged in various in vitro manipulations.
Reciprocal effects in human hemoglobin: direct measurement of the dimer-tetramer association constant at partial oxygen saturation.
Authors: Valdes R , Vickers L P , Halvorson H R , Ackers G K
Published: May 2006

An equilibrium gel permeation technique has been developed for determining as a function of oxygenation state the equilibrium constants for association of hemoglobin subunits. By using this method, the dimer-tetramer constant for human hemoglobin at a partial oxygenation state corresponding to 20% saturation for tetramers has been determined as 3.7 X 10(6) M-1 (dimers). Under the same conditions the corresponding constant for fully oxygenated hemoglobin is 4.1 X 10(5) M-1. These results are found to be in good agreement with the predicted behavior of the association reaction based upon oxygen binding curves measured as a function of protein concentration. Thus a high degree of consistency is found between the two independent experimental approaches to the reciprocal effects of this linkage system, lending support to the theory proposed earlier for these phenomena.
Nerve growth factor: a protease that can activate plasminogen.
Authors: Orenstein N S , Dvorak H F , Blanchard M H , Young M
Published: May 2006

The single, highly stable form of mouse submandibular gland nerve growth factor (NGF), prepared as described by Young et al. [(1978) Biochemistry 17, 1490--1498] is a protease of restricted specificity that can convert plasminogen to plasmin. In the absence of plasminogen, NGF is not fibrinolytic, nor does it hydrolyze casein at a measurable rate. Treatment of NGF with diisopropyl fluorophosphate inhibits its ability to activate plasminogen as well as its capacity to hydrolyze certain synthetic arginine esters. These results indicate that NGF is a member of the class of serine proteases. Since NGF is known to be secreted at high concentrations in mouse saliva, it may serve to activate plasminogen (with subsequent fibrinolysis) somewhere in the alimentary tract. Plasminogen activation is the only known action of NGF upon a biologically important non-neural substrate.
Temperature sensitivity of cyclic AMP production and catecholamine-induced refractoriness in a rat astrocytoma cell line
Authors: Nickols G. Allen , Brooker Gary
Published: May 2006

Intracellular cyclic AMP was increased more than 100-fold when rat C6-2B astrocytoma cells were treated with isoproterenol in the cold (4°C). When the cells were treated with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and isoproterenol, cyclic AMP levels rose more than 150-fold. The levels achieved compared favorably with the 250-fold increase in cyclin AMP produced by (-)isoproterenol at 37°C.(-)Isoproterenol at 5 nM stimulated half-maximal cyclic AMP production at 4°C and at 37°C and was blocked by (-)propranolol at both temperatures. The concentrations of cyclic AMP attained by these cells after (-)isoproterenol stimulation in the cold may be accounted for, in part, by alterations in the efflux of the nucleotide from the cells since extracellular cyclic AMP, an indicator of cyclic AMP efflux, was found to be dramatically reduced in the cold.

The cells, when exposed to (-)isoproterenol for up to 6 hr at low temperature, maintained normal responsiveness to this agent when rechallenged at 4° or 37°C. Thus, they did not display agonist-induced refractoriness during that period of exposure at 4°C, although refractoriness is always seen within 90 min at 37°C. Refractoriness, once established by (-)isoproterenol treatment at 37°C, was not reversed by exposure of the cells to cold. These data suggest that the development of catecholamine refractoriness requires a temperature-sensitive step that lies distal to the hormone-receptor interaction and cyclic AMP generaton.
Genetic alterations in potassium transport in L cells.
Authors: Gargus J J , Miller I L , Slayman C W , Adelberg E A
Published: May 2006

Starting with mutagenized cultures of the mouse fibroblastic cell line LM(TK-), we have selected mutant clones by their ability to grow at 0.2 micrometer K+, a concentration unable to support the growth of the parent cell. The mutants fall into two classes on the basis of their potassium transport properties. Both classes maintain a high intracellular K+ concentration when growing in low-potassium medium, and both are unaltered in the ouabain-sensitive Na/K pump. One class shows an increased activity of a ouabain-resistant, furosemide-sensitive K+ transport system; the other class shows a decreased activity of a specific component of K+ efflux.
Species-specific suppression of histone H1 and H2B production in human/mouse hybrids.
Authors: Ajiro K , Zweidler A , Borun T , Croce C M
Published: May 2006

Ten human/mouse hybrid cell lines that segregate either human or mouse chromosomes were examined for the expression of human- and mouse-specific histones H1 and H2B. Results of this study indicate that the human and mouse chromosomes in hybrid cells that segregate human chromosomes (M greater than H hybrids) contain only mouse histone H1 and H2B. Chromosomes in hybrid cells that segregate mouse chromosomes (H greater than M hybrids) contain only human H1 and H2B histones. Loss of the ability to produce either human or mouse histones does not seem to be due to the loss of specific human or mouse chromosomes because M greater than H hybrids retaining at least one copy of each human chromosome contain only mouse H1 and H2B and H greater than M hybrids retaining at least one copy of each mouse chromosome contain only human H1 and H2B histones. These results, together with those concerning histone H4 acetylation levels and ratios of variants of histones H3 and H2A that are like those in the dominant parent cell type, indicate that the control mechanisms affecting H1 and H2B expression in H greater than M and in M greater than H hybrid cells affect expression of histones H2A, H3, and H4 genes as well. The present data thus support the hypothesis that none of the histone genes that are active in the recessive parent cell type is expressed in hybrid lines that segregate recessive cell chromosomes.