bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluorescent labeling of hormone receptors in viable cells: preparation and properties of highly fluorescent derivatives of epidermal growth factor and insulin.
Authors: Shechter Y , Schlessinger J , Jacobs S , Chang K J , Cuatrecasas P
Published: May 2006

Highly fluorescent analogs of insulin and epidermal growth factor were prepared by the covalent attachment of these peptides to alpha-lactalbumin molecules that were highly substituted (i.e., seven to one) with rhodamine molecules. The alpha-lactalbumin was specifically linked to the lysine residue of insulin or to the alpha-amino group of epidermal growth factor. The insulin derivative retained 1.15% of its potency in stimulating glucose oxidation in fat cells but retained about 8.3% of its binding affinity toward receptors. The epidermal growth factor derivative was completely active in binding to fibroblast receptors and 40% as potent as the native hormone in stimulating DNA synthesis. These highly fluorescent derivatives were suitable for the specific visual labeling of receptor sites in viable cells and for measuring the lateral mobilities of the receptor-hormone complexes by fluorescent photobleaching recovery techniques. By these methods it was shown that the hormone-receptor complexes can move laterally in the plane of the plasma membrane with a diffusion coefficient of (3-5) X 10(-10) cm2/sec.
Binding and kinetic data for rabbit liver fructose-1,6-bisphosphatase with Zn
2+
as cofactor
Authors: Benkovic Patricia A. , Caperelli Carol A. , Maine Margaret De , Benkovic Stephen J.
Published: May 2006

Atomic absorption determinations of zinc content were employed to demonstrate the technique to obtain zinc-free rabbit liver fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11). Reactivation of the apoenzyme by Zn
2+
is rapid (within 1 min) and restores up to 96% of the initial specific activity. Gel filtration measurements showed that the enzyme contains four binding sites for zinc per molecule, one per subunit. The dissociation constants for the initial two binding sites are less than 0.1 μM. In the presence of a substrate analog, (α + β) methyl D-fructofuranoside 1,6-bisphosphate, at a level where two analog molecules are bound per phosphatase molecule, a total of eight Zn
2+
ions bind at 8 μM Zn
2+
, revealing the presence of additional binding sites, including the catalytic one. The activity in the presence of Zn
2+
is maximal at ca. 8 μM Zn
2+
, which corresponds to saturation of the four subunit sites plus the catalytic sites in the presence of substrate. At metal ion concentrations less than 10 μM, the order of activation is Zn
2+
> Mn
2+
> Mg
2+
. In kinetic assays with two metal cofactors the effect of Zn
2+
at concentrations less than 10 μM on either the Mg
2+
or the Mn
2+
assays is inhibitory owing to the apparent formation of mixed (two different elements) metal ion-enzyme complexes possessing a catalytic activity that is measureable but lower than anticipated if the catalysis by the various metal ions is simply additive. Hence the activation by EDTA of the Mg
2+
and Mn
2+
assays is explicable in terms of Zn
2+
removal, thus eliminating mixed metal species. Collectively these observations suggest that fructose-1,6-bisphosphatase may function
in vivo
as a Zn
2+
metalloprotein.
The ovalbumin gene: structural sequences in native chicken DNA are not contiguous.
Authors: Lai E C , Woo S L , Dugaiczyk A , Catterall J F , O'Malley B W
Published: May 2006

The sequence organization of the structural ovalbumin gene and flanking sequences in native chicken DNA was studied by restriction mapping and filter hybridization using a nick-translated probe generated from pOV230, a recombinant plasmid that contains a full-length ovalbumin DNA synthesized from ovalbumin mRNA. The structural sequences of the ovalbumin gene in native chicken DNA were found to be noncontiguous because at least two restriction endonucleases that do not cut the structural sequence do cleave the natural gene into multiple fragments by cleaving within nonstructural sequences interspersed between the structural sequences. The observation that all ovalbumin DNA-containing sequences were contained within a single DNA fragment generated by BamHI digestion of total chicken DNA has allowed us to construct an inclusive restriction map of the natural ovalbumin gene which contains at least two "insert regions." These regions may be further subdivided into alternating structural and insert sequences. Both insert regions were located within the peptide-coding regions of the gene and the sizes of these insert regions were estimated to be approximately 1.0 and 1.5 kilobase pairs, respectively.
Norepinephrine, vasopressin, glucagon, and A23187 induce efflux of calcium from an exchangeable pool in isolated rat hepatocytes
Authors: Chen Jen-Ling J. , Babcock Donner F. , Lardy Henry A.
Published: May 2006

Isolated rat hepatocytes do not actively accumulate Ca
2+
during prolonged incubation
in vitro
; however, these cells do exhibit a limited exchange of intracellular with extracellular Ca
2+
. The exchangeable pool represents about 2 nmol of Ca
2+
per mg of protein. In medium containing either a low (20 μM) or high (1 mM) concentration of Ca
2+
, the divalent cation ionophore, A23187 (at concentrations of 0.03-0.1 nmol/mg of protein), causes release of
45
Ca
2+
from this exchangeable pool but does not allow net influx of extracellular Ca
2+
detectable by the use of a Ca
2+
-sensitive electrode. Like A23187, the hormones norepinephrine, vasopressin, and glucagon (at concentrations that stimulate gluconeogenesis) each induces a similar net efflux of Ca
2+
. Treatment with one hormone decreases the subsequent reponse to the others, whereas treatment with A23187 abolishes the hormonal effects upon both Ca
2+
release and gluconeogenesis. The action of norepinephrine, but not of glucagon, upon Ca
2+
efflux is prevented by the α-adrenergic antagonist, phenoxybenzamine. The action of norepinephrine is not prevented by the β-adrenergic antagonist, propranolol. Together these results indicate that the release of Ca
2+
from a common pool of exchangeable Ca
2+
is important to the action of a variety of hormones on hepatocytes. This Ca
2+
pool in the isolated hepatocyte is characterized as being similar in size and having exchange kinetics that are comparable to those reported for the major intracellular pool of Ca
2+
in the intact liver. The possibility that this pool is intramitochondrial calcium is discussed.
Carbohydrate structure and cell differentitation: unique properties of fucosyl-glycopeptides isolated from embryonal carcinoma cells.
Authors: Muramatsu T , Gachelin G , Nicolas J F , Condamine H , Jakob H , Jacob F
Published: May 2006

From embryonal carcinoma cells labeled with fucose, two main classes of glycopeptide products of Pronase digestion can be distinguished by Sephadex G-50 column chromatography: one eluted near the excluded volume and a smaller one. The large fucosyl-glycopeptides are scarcely present in differentiated cells derived from embryonal carcinoma cells (i.e., fibroblastlike cells, myoblasts, and parietal yolk-sac carcinoma). During in vitro differentiation of embryonal carcinoma cells, these large glycopeptides disappear almost completely. The small glycopeptides were analyzed by paper electrophoresis, concanavalin A-Sepharose affinity chromatography, and digestion with an endoglycosidase. The major components of these glycopeptides from embryonal carcinoma cells appear to be different from complex glycopeptides known to occur in adult cells. The glycopeptide pattern of mouse preimplantation embryos resembles that of embryonal carcinoma cells. These results suggest that the carbohydrate profile changes fundamentally during early stages of mammalian development.
Coupled cell-free synthesis, segregation, and core glycosylation of a secretory protein
Authors: Lingappa Vishwanath R. , Lingappa Jaisri R. , Prasad Rajani , Ebner Kurt E. , Blobel Günter
Published: May 2006

mRNA from rat mammary glands 13-15 days post partum was translated in a wheat germ cell-free system either in the absence or in the presence of ribosome-denuded membranes prepared from isolated rough microsomes of dog pancreas. Newly synthesized α-lactalbumin was identified by immunoprecipitation with a monospecific rabbit antiserum against rat α-lactalbumin and was characterized by partial amino-terminal sequence determination and by lectin affinity chromatography. In the absence of membranes a presumably unglycosylated form of α-lactalbumin was synthesized that bound neither to concanavalin A-Sepharose nor to
Ricinus communis
lectin-agarose and that contained an amino-terminal signal peptide region comprising 19 amino acid residues. In the presence of membranes a processed form was synthesized that lacked the signal peptide portion and that had an amino-terminal sequence identical to that of mature α-lactalbumin. Furthermore, this processed form was found to be segregated, presumably within the microsomal vesicles, because it was resistant to post-translational proteolysis. It was also found to be glycosylated, and because it bound to concanavalin A-Sepharose, from which it could be eluted specifically by α-methyl mannoside, but not to
R. communis
lectin-agarose, it was presumably core-glycosylated. Processing, segregation, and core glycosylation were observed to proceed only when membranes were present during translation and not when they were added after translation.
Homologous genes for enolase, phosphogluconate dehydrogenase, phosphoglucomutase, and adenylate kinase are syntenic on mouse chromosome 4 and human chromosome 1p
Authors: Lalley Peter A. , Francke Uta , Minna John D.
Published: May 2006

It is possible to generate interspecific somatic cell hybrids that preferentially segregate mouse chromosomes, thus making possible mapping of mouse genes. Therefore, comparison of the linkage relationships of homologous genes in man and mouse is now possible. Chinese hamster × mouse somatic cell hybrids segregating mouse chromosomes were tested for the expression of mouse enolase (ENO-1; EC 4.2.1.11, McKusick no. 17245), 6-phosphogluconate dehydrogenase [PGD; EC 1.1.1.44, McKusick no. 17220], phosphoglucomutase-2 (PGM-2; EC 2.7.5.1, McKusick no. 17190), and adenylate kinase-2 (AK-2; EC 2.7.4.3, McKusick no. 10302). In man, genes coding for the homologous forms of these enzymes have been assigned to the short arm of human chromosome 1. Analysis of 41 primary, independent, hybrid clones indicated that, in the mouse, ENO-1 and AK-2 are syntenic with PGD and PGM-2 and therefore can be assigned to mouse chromosome 4. In contrast, they were asyntenic with 21 other enzymes including mouse dipeptidase-1 (DIP-1, human PEP-C; EC 3.4.11.
*
, McKusick no. 17000) assigned to human chromosome arm 1q and mouse chromosome 1. Karyologic analysis confirmed this assignment. These data demonstrate that a large autosomal region (21 map units in the mouse and 51 map units in the human male) has been conserved in the evolution of mouse chromosome 4 and the short arm of human chromosome 1. Identification of such conserved regions will contribute to our understanding of the evolution of the mammalian genome and could suggest gene location by homology mapping.
Type C RNA virus-specific antibody in human systemic lupus erythematosus demonstrated by enzymoimmunoassay.
Authors: Mellors R C , Mellors J W
Published: May 2006

Postmortem study of proliferative glomerulonephritis associated with human systemic lumpus has previously shown that an antigen related to mammalian type C RNA viral core (p30) proteins is deposited in the renal glomerular lesions with human immunoglobulins in an immune-complex pattern. In the present work, human immunoglobulins were sequentially eluted from the lupus glomerular immune deposits and were assayed by a sensitive enzymoimmunoassay developed for the measurement of anti-p30 antibody activity against purified viral p30 proteins of mammalian type C viruses. Human immunoglobulins showing specific anti-p30 antibody activity, particularly against p30 antigen of feline endogenous virus RD-114 and to a smaller extent against p30 antigen of murine type C virus, were eluted by acid buffer from the glomerular immune deposits in two patients with lupus proliferative glomerulonephritis who have deposits of viral p30-related antigen in the same tissue lesions. This study adds support for the hypothesis that expression of type C viral antigen may be involved in the multifactorial pathogenesis of proliferative glomerulonephritis associated with human systemic lupus.
Modification of conditioned behavior of rats by neurohypophyseal hormones and analogues
Authors: Walter Roderich , Ree Jan M. Van , Wied David De
Published: May 2006

Vasopressin and other neurohypophyseal peptides affect various processes related to memory and/or learning. A single subcutaneous injection of vasopressin increases resistance to extinction of a pole-jumping avoidance response in rat. This test system has been applied in an attempt to relate structural aspects of neurohypophyseal peptides, analogues, and derivatives with truncated sequences to their effects on conditioned behavior. Thus far it can be concluded that there are more stringent requirements on certain residues in the 20-member covalent ring than in positions 8 and 9 of the linear peptide portion for neurohypophyseal hormones to be active. Critical are the contributions of residues in positions 2, 3, and 5; these results are reminiscent of those from conformation-activity correlations of the endocrine effects of neurohypophyseal hormones, in which the side chain of the residue in position 3 is critical for receptor binding and the side chains of residues in positions 2 and 5 are key for the activation of the receptor. Chemical modifications in position 4 yield analogues that are active and inactive in increasing the resistance to extinction of the avoidance response, depending on the particular structural substitution, similar to results from structure-activity studies of the endocrine activities of neurohypophyseal hormones. Because behavioral activities of vasopressin are more tolerant than endocrine activities to modifications of the hormone in positions 8 and 9, analogues with the most striking dissociation of potencies in learned behavior and endocrine responses are expected to be those with sequence alterations in the linear peptide portion. Peptides with linear part sequences of neurohypophyseal hormones showed little or no activity. The results obtained in this structure-activity study are compared with those of an earlier study in which the ability of various neurohypophyseal peptides to attenuate puromycin-induced amnesia in mice was evaluated.
Carotid body chemoreceptor function: hypothesis based on a new circuit model.
Authors: Krammer E B
Published: May 2006

Integration of our own morphological observations into recent ultrastructural, biochemical, and neuropharmacological results on the carotid body led to a new hypothesis on chemoreceptor function: (i) Glomus cells with small dense-cored vesicles (type IB cells) that store norepinephrine are chemoreceptors. (ii) Glomus cells with large dense-cored vesicles (type IA), which are postsynaptic to the other glomus cell type and presynaptic to afferent nerve endings, are dopaminergic interneurons that suppress the afferent discharge frequency during normoxia by releasing dopamine. (iii) The hypoxic stimulus causes the chemoreceptive cell to release the stored norepinephrine, which in turn brings about disinhibition of the afferent nerve endings by inhibition of the interneuron. (iv) Afferent nerve endings and interneurons interact through reciprocal synapses that form a short inhibitory feedback loop. We propose that information in the carotid body is processed in a fashion graded rather than digital, providing a fine adjusted cooperation of all elements.
Different time course of development for high-affinity choline uptake and choline acetyltransferase in the chick retina.
Authors: Bader C R , Baughman R W , Moore J L
Published: May 2006

Synthesis and storage of [3H]acetylcholine in isolated pieces of chick retina increased in two stages during embryogenesis. The first increase coincided with a 100-fold rise in the activity of choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6), but during the second increase the activity of this enzyme remained essentially constant. The second increase instead was linked to an approximately 6-fold increase in the Vmax for high-affinity uptake of choline.
Epinephrine-induced elevation of guanosine 3′:5′-cyclic monophosphate in isolated fat cells of rat
Authors: Asakawa Takeo , Ruiz Juan , Ho Ren-jye
Published: May 2006

The effects of epinephrine (as low as 0.1 μM) on guanosine 3′:5′-cyclic monophosphate (cGMP) and adenosine 3′:5′-cyclic monophosphate (cAMP) in isolated fat cells were examined. Epinephrine increased both cGMP and cAMP levels, with the elevation of cAMP preceding the rise of cGMP. Maximal elevation was obtained with 1 μM epinephrine for each nucleotide. The increase in content of cGMP and cAMP due to epinephrine was completely blocked by a β-adrenergic antagonist (5 μM propranolol). Phentolamine (10-100 μM), an α-adrenergic antagonist, enhanced the response to epinephrine resulting in elevation of cAMP levels, whereas a high concentration (100 μM) of phentolamine suppressed the elevation of cGMP. The ability of epinephrine to increase cGMP and cAMP levels was markedly diminished by “feedback regulator” partially purified from the incubation mixtures of isolated fat cells exposed to epinephrine [Ho, R.J. & Sutherland, E. W. (1971)
J. Biol. Chem.
246, 6822-6827], whereas an increase in cGMP, but not cAMP, levels was observed in isolated fat cells incubated with “feedback regulator” alone (without epinephrine). These observations suggest the possibility that the epinephrine-induced elevation of cGMP levels in isolated fat cells might be mediated by an increase in formation of intracellular “feedback regulator” due to an elevation of cAMP by epinephrine.
DNA unwinding induced by photoaddition of psoralen derivatives and determination of dark-binding equilibrium constants by gel electrophoresis
Authors: Wiesehahn Gary , Hearst John E.
Published: May 2006

Derivatives of furo[3,2-
g
]coumarin (psoralen) can bind to the DNA double helix and, in the presence of long-wavelength UV light, the bound psoralen may react covalently with pyrimidine residues on one or both strands of the helix. By using agarose gel electrophoresis, we have determined the unwinding angle associated with each of four different psoralen derivatives to be 28° ± 4°. For 4,5′,8-trimethylpsoralen (trioxsalen) the unwinding angle was found to be independent of the initial DNA superhelix density in the range that is accessible to agarose gel electrophoresis.

Also by using agarose gel electrophoresis, we have determined the unwinding angle for ethidium intercalation. This was done by the total relaxation of supercoiled DNA in the presence of a series of ethidium concentrations. By using published values for the association constant for ethidium binding to DNA and evaluating the final superhelix density (after removal of ethidium) of the DNA on gels, we calculated an unwinding angle of 29° ± 3°.

Assuming an unwinding angle of 28° for the noncovalent intercalation of psoralen derivatives, we used the same procedure to determine intercalation binding constants. The association constants for 4′-aminomethyltrioxsalen were 300-1400 M
-1
in NaCl at 0.2-0.05 M and 300-2500 M
-1
in Mg
2+
at 4-0.5 mM. The association constant for 4′-hydroxymethyltrioxsalen in 0.5 mM Mg
2+
was determined to be 70 M
-1
.
Nucleosomes associated with newly replicated DNA have an altered conformation
Authors: Seale Ronald L.
Published: May 2006

In vitro
DNA synthesis was studied in HeLa cell nuclei, with emphasis on the question of whether newly replicated DNA is associated with nucleosomes. The newly replicated DNA was twice as sensitive to digestion by micrococcal nuclease as mature chromatin DNA, reaching a limit digest at 20-25% acid-insoluble product. Examination of the intermediates of digestion by micrococcal nuclease showed the nuclease-resistant, new DNA to be complexed in nucleosomes. However, structural differences were evident at both the polynucleosomal and the core particle level. The nucleosomes on newly replicated DNA were arranged with a repeat size of 165-170 base pairs—i.e., smaller than the 185-base-pair repeat of mature chromatin. The heterogeneity of polynucleosomal multimers, evident in digests of whole chromatin, was reduced in newly replicated chromatin such that the multimers resolved as sharply defined bands. Nucleosomal core particles associated with newly replicated DNA had a different conformation from particles in mature chromatin based on the following lines of evidence: (
i
) during micrococcal nuclease digestion, the monomer nucleosomes did not accumulate but were rapidly degraded under certain conditions; (
ii
) micrococcal nuclease limit digest patterns and DNase I digestion patterns, both of which reflect internal nucleosomal protein DNA associations, differed significantly from control patterns. These findings bear directly on models postulated for nucleosome-DNA interactions during chromation replication. A possible mechanism to account for the conformational change and its role in replication are discussed.
Coordinate control of 3T3 cell proliferation by platelet-derived growth factor and plasma components.
Authors: Vogel A , Raines E , Kariya B , Rivest M J , Ross R
Published: May 2006

DNA synthesis and cell division were measured in Swiss mouse 3T3 cells cultured in different concentrations of cell-free plasma-derived serum and increasing amounts of a platelet-derived growth factor. In plasma-derived serum alone, the cells were quiescent and they were arrested in the Go/G1 phase of the cell cycle. Addition of a platelet-derived growth factor to quiescent cells maintained in plasma-derived serum stimulated both DNA synthesis and cell division. When plasma components were present at high concentration (5%, vol/vol), the amount of platelet factor added to the cultures determined the number of cell doublings. Plasma-derived molecules were required for the platelet factor to stimulate DNA synthesis and cell division in the maximal number of cells. In addition, plasma components had to be present for recently divided cells to respond to the platelet factor. When 3T3 cells were cultured in excess platelet factor and limiting amounts of plasma-derived serum (0.5%, vol/vol), the cells underwent one doubling and then ceased to proliferate. Addition of fresh plasma-derived serum to these cells induced a second round of cell division. Plasma components and the platelet-derived growth factor acted in a coordinate fashion to regulate the proliferation of Swiss 3T3 cells.
Existence of only a single functional pool of adenosine triphosphate in human erythrocytes.
Authors: Beutler E , Guinto E , Kuhl W , Matsumoto F
Published: May 2006

The question of whether separate "membrane" and "soluble" pools of ATP exist in erythrocytes has been examined. Phosphoglycerate kinase (EC 2.7.2.3)-derived ("membrane") ATP was labeled by short-term incubation with inorganic [32P]phosphate. Pyruvate kinase (EC 2.7.1.40)-derived ("soluble")ATP is not labeled under these circumstances. The specific activity of the gamma-phosphate of "soluble" ATP was then evaluated by the addition of 2-deoxyglucose and measurement of the specific activity of 2-deoxyglucose-6-[32P]phosphate formed. This specific activity was essentially the same as the overall specific activity of erythrocyte ATP gama-phosphate, indicating that no functional pools of phosphoglycerate kinase-derived and pyruvate kinase-derived ATP exist in erythrocytes.
Primary structure of murine major histocompatibility complex alloantigens: isolation, biochemical characterization, and preliminary alignment of CNBr fragments from the H-2Ib glycoprotein.
Authors: Ewenstein B M , Nisizawa T , Uehara H , Nathenson S G , Coligan J E , Kindt T J
Published: May 2006

Radiochemical methodology has been applied in studies directed toward the determination of the amino acid sequence of the murine H-2Kb gene product. Five major CNBr fragments, which comprise the NH2-terminal 80% of the intact glycoprotein and include the H-2Kb fragment isolated by papain cleavage, were isolated and characterized. By means of amino-terminal sequence analysis, homology to other products of the major histocompatibility complex, identification of partial CNBr cleavage products, and localization of the site of papain cleavage, it was possible to align the fragments obtained from the molecule in the following order: the NH2-terminal fragment, designated IIIn, contains 23 residues and is followed by fragment IIIa, which contains 29 residues. Two peptides Ib (Mr 11,500) and Ia (Mr 12,000), each of which contains a carbohydrate moiety, are followed by fragment Ic (Mr 5800), which includes the site of papain cleavage. The data indicate the presence of disulfide bonds between fragments Ib and Ia and between Ia and Ic.
Decamethonium both opens and blocks endplate channels.
Authors: Adams P R , Sakmann B
Published: May 2006

Miniature endplate currents, endplate current fluctuations ("membrane noise"), and voltage-jump current relaxations were studied in voltage-clamped frog muscle fibers during decamethonium action. All three types of experiments revealed two kinetic processes controlling the opening of endplate channels, one that reflects agonist action and another that reflects local anesthetic-like blocking activity. The kinetic constants for these two steps were evaluated from measurements of the fast and slow time constants as a function of decamethonium concentration. At -130 mV membrane potential and 13 degrees, the mean open time of decamethonium-activated channels is 2.8 msec. The forward and backward rate constants for channel blocking are 1.7 X 10(7) M-1 sec-1 and 10(3) sec-1. The voltage dependencies of the channel lifetime and of the blocking equilibrium are similar to those seen with pure agonists and local anesthetics, respectively.
Reconstitution of cholera toxin-activated adenylate cyclase.
Authors: Johnson G L , Kaslow H R , Bourne H R
Published: May 2006

Reconstitution of adenylate cyclase activity responsive to stimulation by guanylyl-5'imidodiphosphate or NaF may be achieved by mixing dilute Lubrol 12A9-solubilized extracts of wild-type S49 membranes with membranes of an adenylate cyclase-deficient variant. Experiments using N-ethylmaleimide to inactivate components of the adenylate cyclase system indicate that distinct components from both wild-type detergent extracts and adenylate cyclase-deficient membranes are essential for reconstitution. These results and conclusions confirm those of E. M. Ross and A. G. Gilman [J. Biol. Chem. (1977) 252, 6966-6969]. Detergent extracts of cholera toxin-treated wild-type membranes yield a reconstituted adenylate cyclase as responsive to GTP as to guanylyl-5'-imidodiphosphate whereas, in the absence of cholera toxin treatment, GTP has little or no effect. Cholera toxin-treated adenylate cyclase-deficient membranes and Lubrol 12A9 extracts from them, however, fail to yield a reconstituted adenylate cyclase that responds to GTP with an increase in cyclase activity. Because treatment of the adenylate cyclase-deficient variants with cholera toxin is without effect on the reconstituted cyclase, we propose that the cholera toxin substrate is absent or altered in the adenylate cyclase-deficient phenotype.
Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A.
Authors: Iglewski B H , Sadoff J , Bjorn M J , Maxwell E S
Published: May 2006

Pseudomonas aeruginosa exoenzyme S is an adenosine diphosphate ribosyltransferase distinct from Pseudomonas toxin A. Exoenzyme S catalyzes the transfer of radioactivity from all portions of radiolabeled NAD+ except nicotinamide. Digestion of the radiolabeled product(s) formed in the presence of [adenine-14C]NAD+ and exoenzyme S with snake venom phosphodiesterase yields only AMP, suggesting that ADP-ribose is present as monomers and not as poly(ADP-ribose). Exoenzyme S does not catalyze the transfer of ADP-ribose from NAD+ to elongation factor 2, as do toxin A and diphtheria toxin, but to one or more other proteins present in crude extracts of wheat germ or rabbit reticulocytes and in partially purified preparations of elongation factor I. The ADP-ribosyltransferase activity of exoenzyme S is distinct from toxin A by several tests: it is not neutralized by toxin A antibody, it is destroyed rather than potentiated by pretreatment with urea, and it is more heat stable. These latter observations and the substrate specificity suggest that exoenzyme S is different from any previously described prokaryotic ADP-ribosyltransferase.
Isolation and characterization of mutants of human diploid fibroblasts resistant to diphtheria toxin.
Authors: Gupta R S , Siminovitch L
Published: May 2006

Stable mutants highly resistant to the protein synthesis inhibitor diphtheria toxin (dipr) have been selected in human diploid fibroblast cells at a frequency of 1-8 X 10(-6). Treatment of cells with mutagens, (e.g., ethylmethanesulfonate, nitrosoguanidine, and ICR-170), increased the frequencies of dipr mutants by 50- to 500-fold in different experiments, and the optimal expression time for dipr mutation was about 5 days. All mutants examined thus far have bred true, and no effects of cell density or cross feeding have been observed on the selection. Fluctuation analysis showed that the dipr mutation occurs in these fibroblasts at the rate of 5-6 X 10(-7) mutations per cell per generation. Protein synthesis in mutant extracts was resistant to diphtheria toxin, indicating that the dipr lesion in such mutants lies in the protein synthesis machinery. The characteristics of the dipr marker should make this system particularly useful for studies of quantitative mutagenesis in human diploid cells.
Effect of myasthenic patients' immunoglobulin on acetylcholine receptor turnover: selectivity of degradation process.
Authors: Drachman D B , Angus C W , Adams R N , Kao I
Published: May 2006

Antibodies in the sera of patients with myasthenia gravis are believed to play an important role in the pathogenesis of the disorder. They have recently been shown to accelerate the degradation of acetylcholine receptors in cultured mammalian skeletal muscle and at intact neuromuscular junctions. To elucidate the mechanism of the antibody-accelerated degradation process, we have prepared cultures in which one set of acetylcholine receptors was exposed to myasthenic immunoglobulin while a second set of acetylcholine receptors, newly incorporated after exposure to the immunoglobulins, was not. The set of acetylcholine receptors with bound myasthenic immunoglobulin was degraded at 2 to 3 times the normal rate, while the second set of acetylcholine receptors without bound immunoglobulin was degraded at the control rate. This suggest that the binding of antibody from myasthenic patients alters the acetylcholine receptors in some way that causes them to be selected for preferential degradation by the muscle cells. New synthesis and incorporation of the acetyl-choline receptors into the surface membrane of cultured skeletal muscle was unaffected by exposure to myasthenic immunoglobulin.
Secretion of lipoprotein-X by perfused livers of rats with cholestasis.
Authors: Felker T E , Hamilton R L , Havel R J
Published: May 2006

The major abnormal plasma lipoprotein of cholestasis (LP-X) was isolated from blood plasma and from perfusates of isolated livers of rats with biliary obstruction. In both cases LP-X was composed mainly of about equimolar parts of phospholipids and unesterified cholesterol; the small protein component was primarily the arginine-rich apolipoprotein. By electron microscopy, LP-X appeared as a unilamellar liposome (690 A mean diameter, range 400-1000 A) with the trilaminar staining image typical of phospholipid bilayers. Extensive block staining of cholestatic livers for 48 hr with warmed uranyl acetate (37 degrees) permitted the visualization of vesicles indistinguishable from LP-X within hepatic parenchyma. These trilaminar-staining vesicles occurred predominantly within bile canaliculi. They also were seen in nearby cytoplasmic vacuoles or invaginations between hepatocytes and in the space of Disse. Similar vesicles were not seen in the endoplasmic reticulum or Golgi cisternae. These observations raise the possibility that the vesicles are formed within bile canaliculi and are transported from the canaliculi to the space of Disse within pinocytotic vacuoles.
Regulation of acetylcholine receptor levels by a cholinergic agonist in mouse muscle cell cultures
Authors: Noble Mark D. , Brown Thomas H. , Peacock John H.
Published: May 2006

The effects of continuous exposure to carbamylcholine (CbCho) on regulation and stabilization of acetylcholine receptors (AcChoR) were studied in cell cultures of G8, a continuous mouse muscle cell line. Exposure of cultures to 10-100 μM CbCho for 24-48 hr produced a 30-50% reduction in
125
I-labeled α-bungarotoxin binding. CbCho was not found to alter cell morphology, protein metabolism, or amino acid incorporation. Electrophysiological experiments demonstrated a 75% reduction in the maximum sensitivity of the myotubes to iontophoretic application of acetylcholine (AcCho). The reduction in AcCho sensitivity appeared to represent a true loss of functional receptors because there were no changes in the passive electrical properties of the cells or in the AcCho reversal potential and because receptor desensitization appeared not to be involved. Tetrodotoxin had no effect on receptor levels, either alone or in combination with CbCho. Receptor degradation in control cells could be described kinetically as a first-order process with a half-time of 19.2 hr; turnover rate in receptors remaining after prolonged exposure to CbCho was indistinguishable from that in control cells. We conclude that a receptor-active ligand can exert negative control over AcChoR levels and that prolonged exposure to an AcCho analog is not sufficient to induce a stable population of receptors in these cells.
Contraction of detergent-treated smooth muscle.
Authors: Gordon A R
Published: May 2006

After exposure of segments of rabbit taenia coli to the nonionic detergent Triton X-100, tension could be induced by increasing the [Ca2+] in the micromolar range. In the presence of a saturating [Ca2+], this preparation developed nearly 100% of the control tetanus tension recorded from the intact muscle prior to the detergent treatment. In addition, tension could be induced by increasing the [Mg2+], in the virtual absence of Ca2+. Mg2+ seems to inhibit the Ca2+-induced tension in a predominantly competitive manner.
How lac repressor recognizes lac operator.
Authors: Goeddel D V , Yansura D G , Caruthers M H
Published: May 2006

Nucleotide analogs were substituted for unmodified nucleotides at specific sites in the lac operator sequence by a combination of chemical and enzymatic procedures. The nitrocellulose filter assay was used to study the interactions of these modified operators with wild-type (SQ) and tight-binding (QX86) lac repressors. These studies implicate directly the 5 methyl of thymine and the 2 amino of guanine as important operator-repressor contact sites. Furthermore, when these findings are combined with published results from other laboratories, a model for the lac operator-lac repressor interaction can be derived. Two important postulates follow from this model. (i) The repressor interacts at specific and defined sites with the N7 of guanine, the 5 methyl of thymine, the 2 amino of guanine, and the central major groove of the operator. (ii) The repressor binds to one side of the operator.
Racemization in human lens: evidence of rapid insolubilization of specific polypeptides in cataract formation.
Authors: Garner W H , Spector A
Published: May 2006

After early life, the dry weight of normal human lenses increases at a relatively constant rate with time. Transformation from soluble to insoluble material appears to occur at a comparable rate, resulting in a constant amount of soluble material. However, in cataract the insolubilization rate is accelerated. These observations are supported by determination of D-aspartic acid/L-aspartic acid ratios. The abundance of D-aspartic acid increases with aging at a constant rate in the insoluble fraction of normal lenses but does not change in the soluble fraction. However, in cataractous lenses there is a significant decrease in the ratio in the insoluble fraction. Examination of polypeptides isolated from reduced and alkylated soluble and insoluble cataractous lens proteins as well as other data suggest the following additional conclusions: (i) the 10,000-dalton polypeptide in the insoluble fraction is derived in part from degradation of an already insoluble precursor; and (ii) the lowered abundance of D-aspartic acid in the insoluble fraction of cataractous lenses is primarily due to the rapid insolubilization of the 43,000- and 20,000-dalton range components.
Correlation of the biological activity and solution conformation of [Asp1,Ile5]- and [Phe4,Tyr8]angiotensin II.
Authors: Bleich H E , Freer R J , Stafford S S , Galardy R E
Published: May 2006

Angiotensin II is known to undergo a reversible conformational transition and a change in potency in rat uterus in vitro with pK approximately 6.5. We have shown by carbon-13 NMR that the conformational transition involves all-trans to partly cis isomerization of the His6-Pro7 peptide bond. Isomerization from all-trans at pH 6.8 to approximately 16% cis at pH 8.0 is therefore correlated with a 10-fold increase in biological activity for [Asp1,Ile5]-angiotensin II in rat uterus in vitro. Isomerization from all-trans at pH 6.8 to approximately 16% cis at pH 8.0 in the competitive inhibitor [Phe4,Tyr8]angiotensin II is correlated with exhibition of virtually no agonist activity at low pH to full agonist activity at high pH. An angiotensin II conformation with Pro7 in the cis form may therefore be the conformation with maximal binding or biological activity at the cellular receptor.
Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322.
Authors: Sutcliffe J G
Published: May 2006

I have determined the nucleotide sequence of the ampicillin resistance gene of pBR322, an Escherichia coli plasmid that encodes a penicillin beta-lactamase. This gene codes for a protein of 286 amino acid residues. The first 23 amino acids presumably form a signal for secretion, because they do not appear in the mature enzyme, whose partial amino acid sequence has been determined independently.
Kinetic analysis of receptor-controlled tracer efflux from sealed membrane fragments
Authors: Bernhardt Julius , Neumann Eberhard
Published: May 2006

A detailed kinetic analysis is presented for activator-receptor-mediated efflux of tracer substances from vesicular membrane systems in general and from sealed fragments of excitable membranes in particular. Rate constants and amplitudes, as the primary measurable quantities of the efflux kinetics, are expressed in terms of fundamental properties of vesicular membrane systems containing receptors of chemical gating systems. The experimental determination and theoretical analysis of single contributions to a complex receptor-controlled efflux has been treated for the acetylcholine receptor system; also the effect of “pharmacological densensitization” on efflux is explicitly formulated. The dependence of the measured efflux parameters on the concentration of activators can be used to derive the kinetic and thermodynamic constants for receptor activation and inactivation processes; a general kinetic scheme and two limiting cases are analyzed. The efflux of
22
Na from “excitable microsacs” of
Torpedo marmorata
is treated as an example, and the power of the rigorous analytical method is demonstrated. In particular, the analysis of efflux amplitudes from only a few data points offers an alternative to the longer lasting measurements for obtaining efflux curves when a safety factor is involved, as in the case of tracer ions like
22
Na.
Lengthening of the G1 phase is not strictly correlated with differentiation in Friend erythroleukemia cells.
Authors: Friedman E A , Schildkraut C L
Published: May 2006

Friend murine erythroleukemia cells (Friend cells) undergo erythroid differentiation in vitro with an increased probability when cells are cultured in the presence of dimethyl sulfoxide (Me2SO) or other agents. Exponentially growing Friend cells, after dilution into medium containing Me2SO, underwent a transient lengthening of the G1 phase of the cell cycle before they became committed to erythroid differentiation. For nine inducing agents, a positive correlation was found between the percentage of cells that had differentiated and synthesized heme, and the percentage of progenitor cells in which a lengthened G1 phase had previously been observed. This correlation was not found, however, with two other potent inducing agents, hypoxanthine and actinomycin D. Moreover, cells that underwent a lengthened G1 phase did not always terminally differentiate. One such example was a Me2SO-resistant, variant Friend cell line (520a) grown in the presence of Me2SO. These results imply that the prolonged G1 phase, although observed with many inducers, is not a prerequisite for erythroid differentiation with all inducers.
Effect of anti-spectrin antibody and ATP on deformability of resealed erythrocyte membranes.
Authors: Nakashima K , Beutler E
Published: May 2006

Deformability of resealed erythrocyte membranes was measured by using an ektacytometer. Divalent anti-spectrin antibody, but not monovalent anti-spectrin Fab fragments, decreased membrane deformability. Membranes resealed with MgATP were more deformable than those without MgATP. Exogenous alkaline phosphatase, which dephosphorylates spectrin, decreased membrane deformability. These results suggest that spectrin is an essential component of the system that determines erythrocyte deformability. They are consistent with the view that the role of ATP in membrane deformability is mediated through phosphorylation of the spectrin.
Neoplastic transformation of rat mammary cells exposed to 7,12-dimethylbenz[alpha]anthracene or N-nitrosomethylurea in cell culture.
Authors: Richards J , Nandi S
Published: May 2006

Primary cultures of mammary cells from virgin Lewis rats were seeded at 5 X 10(5) cells per cm2 in medium 199 supplemented with 10% fetal calf serum, insulin (5 microgram/ml), prolactin (5 microgram/ml), estradiol (5 ng/ml), progesterone (0.5 microgram/ml), and hydrocortisone (0.5 microgram/ml). On the second or third day of culture, cells were exposed to either 7,12-dimethylbenz[alpha]anthracene (0.1 microgram/ml for 24 hr) or N-nitrosomethylurea (80 microgram/ml for 2 hr). The cells were later assayed for transformation by transplanting 10(6) or 10(5) cells into gland-free mammary fat pads of 3-week-old female hosts. Untreated cells produced only normal mammary outgrowths when transplanted. Cells treated with 7,12-dimethylbenz[alpha]anthracene or N-nitrosomethylurea produced abnormal outgrowths in 11% of the transplants. These abnormal outgrowths ranged from rapidly growing adenocarcinoma to alveolar and ductal hyperplastic lesions. The results indicate that rat mammary epithelial cells can be transformed by exposure to chemical carcinogens in culture and thus represent a potential in vitro model for epithelial cell transformation.
Contranscription of genes for RNA polymerase subunits beta and beta' with genes for ribosomal proteins in Escherichia coli.
Authors: Yamamoto M , Nomura M
Published: May 2006

The lambdarifd18 transducing phage carries genes for RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) subunits beta and beta' (rpoB,C) and genes for four ribosomal proteins (rplK for L11, rplA for LI, rplJ for L10, and rplL for L7/L12). DNA segments of various sizes, which cover the rifd allele of the rpoB gene, were cloned into lambda vector phages. The hybrid phages were then analyzed for their ability to express the rpoB gene and neighboring ribosomal protein genes in ultraviolet-irradiated lambda-lysogenic and nonlysogenic bacterial hosts. The results show that the rpoB gene is cotranscribed with two neighboring ribosomal protein genes and that in the rpoB,C transcription unit is: promoter, rp1J, rp1L, rpoB, and rpoC.
Mechanisms of antibody diversity: Multiple genes encode structurally related mouse κ variable regions
Authors: McKean David J. , Bell Michael , Potter Michael
Published: May 2006

The complete amino acid sequences of the variable regions of three mouse Vκ-21 kappa chains (A22, T111, and CB101) and one partial sequence (B32) have been determined and are compared to four previously reported Vκ-21 variable regions. These eight κ variable region sequences have, with the exception of an amide difference at residue 1, identical amino-terminal 23-residue sequences, all are of the same length, and all have extensive amino acid sequence homology throughout the variable region. When these eight variable regions are grouped by sequence homology, five different groups (Vκ-21A, B, C, D, and E) are present whose members share common sets of amino acids within a group. Three groups of similar homology each contains at least two members (M63 and AB22 in Vκ-21B; M321 and T124 in Vκ-21C; and M70 and B32 in Vκ-21A). The repetition of these five characteristic subgroup sequences in this relatively small sample indicates that these subgroups are isotypes which are controlled by separate germline genes. It is unlikely that these sequences could have been randomly somatically generated in different animals from a single germ-line gene (parallel mutation). Although a limited number of comparisons are available, the sequence differences within the Vκ-21A, B, and C isotypes are limited to complementarity-determining regions and may have resulted from somatic mutations. The κ chains comprising the Vκ-21 isotypes offer a unique opportunity to compare the genetic interpretations of the primary amino acid sequence data with the nucleic acid hybridization data.
Initiation of the alternative pathway of complement: recognition of activators by bound C3b and assembly of the entire pathway from six isolated proteins.
Authors: Schreiber R D , Pangburn M K , Lesavre P H , Müller-Eberhard H J
Published: May 2006

An intact alternative pathway of complement activation was assembled from six isolated proteins present at their respective physiological concentrations (C3, 1200 microgram/ml: factor B, 200 microgram/ml; factor D, 2 microgram/ml; beta1H, 560 microgram/ml; C3b inactivator, 34 microgram/ml; and native properdin, 20 microgram/ml). Initiation of the pathway required the presence of five of these proteins not including properdin. The initial C3 convertase of the system was shown to be a fluid-phase rather than a surface-bound enzyme. The ability of the pathway to discriminate between activator and nonactivator was found to reside in the bound C3b molecule. When bound to the surface of an activator through its labile binding site, C3b interacts with surface structures of the activator through another site on the molecule. This interaction results in diminished beta1H binding to C3b and thereby allows the bound C3b molecule to escape control and participate in C3 convertase formation. Thus, initiation of the alternative pathway is a two-step process, the first being non-specific and the second being discriminatory.
A Drosophila mutant with a temperature-sensitive block in nerve conduction.
Authors: Wu C F , Ganetzky B , Jan L Y , Jan Y N
Published: May 2006

A mutant, napts (no action potential, temperature-sensitive), is described in which axonal conduction fails at high temperature. Synaptic transmission at the larval neuromuscular junction is unimpaired. Larvae and adults are rapidly paralyzed at restrictive temperatures; they recover rapidly when the temperature is decreased. The mutant gene is recessive and is located on the second chromosome at map position 56.