bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacteriophage lambda int protein recognizes two classes of sequence in the phage att site: characterization of arm-type sites.
Authors: Ross W , Landy A
Published: May 2006

Purified int protein from bacteriophage lambda binds to specific sites in DNA that are not part of the functional attachment sites (non-att DNA) as well as to specific sites in att DNA. Analysis of non-att sites protected from nucleases by int has permitted definition of two distinctly different consensus recognition sequences, one of which, the arm-type sequence, is characterized in this report. Both types of recognition sequence occur in attP; five copies of the arm-type consensus sequence are located distant from the crossover region in the P1, P2, and P' arm protected regions. The second type of recognition sequence occurs at the crossover region. Modification of int with N-ethylmaleimide selectively alters its interaction with arm-type sequences.
Normal serum and lipoprotein-deficient serum give different expressions of excitability, corresponding to different stages of differentiation, in chicken cardiac cells in culture.
Authors: Renaud J F , Scanu A M , Kazazoglou T , Lombet A , Romey G , Lazdunski M
Published: May 2006

Monolayers of cardiac cells from 11-day-old chicken hearts have different properties when maintained in fetal calf serum or in a lipoprotein-deficient serum (LPDS). Cells in fetal calf serum have a resting potential near -60 mV; the rate of rise of the action potential is low (less than 10 V/sec); the action potential and the contraction are essentially unaffected by tetrodotoxin (TTX); and the beating properties are unaffected by muscarinic agents. Cells in LPDS have a resting potential near -75 mV, and a fast rise of the action potential (approximately equal to 100 V/sec) that is drastically decreased by TTX with a parallel abolition of contraction, and the beat is blocked by very low concentrations of muscarinic agonists. Cells that are physiologically fully responsive to TTX and to muscarinic agents have receptors that remain stable 24 hr after protein synthesis is blocked, whereas cells that are physiologically unresponsive to TTX and muscarinic agents have receptors that are rapidly degraded with half-lives between 9 hr (TTX receptor) and 14 hr (muscarinic receptor). Differences in the physiological and biochemical properties are accompanied by changes in the cholesterol contents of the cell membranes. The properties of cardiac cells cultured in normal serum are similar to those found for cells of chicken hearts in the very early embryonic stage, whereas those of cardiac cells cultured in LPDS correspond to the late embryonic stage.
Receptor-mediated endocytosis of insulin: role of microvilli, coated pits, and coated vesicles.
Authors: Fan J Y , Carpentier J L , Gorden P , Van Obberghen E , Blackett N M , Grunfeld C , Orci L
Published: May 2006

When 125I-labeled insulin (125I-insulin) is incubated with 3T3-L1 adipocytes and cells processed for electron microscopic autoradiography, the ligand initially localizes preferentially to microvilli and coated pits. As a function of time and temperature, this initial preferential localization to microvilli is lost, and the ligand is internalized by the cell. Serial sections of apparent coated vesicles near the cell surface indicate that about half of these structures are true vesicles and, therefore, intermediates in this receptor-mediated endocytotic process. With time, 125I-insulin localizes to larger intracellular membrane-bounded structures. When cells are incubated with another ligand, cationic ferritin, that is taken up by adsorptive endocytosis, essentially the same structures are involved as for the endocytosis of 125I-insulin. The data suggest that specificity for receptor-mediated endocytosis is conferred by the specific ligand receptor and possibly by ligand-induced receptor mobility in the plane of the plasma membrane. Other structures such as coated pits, coated vesicles, larger vesicles, and secondary lysosomes are common for different ligands.
Human T cell hybridomas secreting immune interferon.
Authors: Le J , Vilcek J , Saxinger C , Prensky W
Published: May 2006

Human T-cell hybridomas were established by hybridization of concanavalin A-stimulated human peripheral blood lymphocytes with a 6-thioguanine-resistant mutant cell line, designated SH9, derived by irradiation from a cloned human cutaneous T lymphoma line, Hut102-B2. High levels of interferon (IFN) were demonstrated in the supernatants of hybridoma L265 and its subclones. Whereas no IFN was detected in SH9 cell cultures, up to 1,330 units of IFN per ml were produced spontaneously by the hybrids. On induction with 12-omicron-tetradecanoylphorbol 13-acetate, IFN synthesis in hybridoma cultures was enhanced 8- to 16-fold. Neutralization with specific antisera and determination of antiviral activities in human and bovine cells showed that the IFN secreted by the hybridomas was immune IFN (IFN-gamma). Analysis of DNA content, karyotype, and cell surface phenotype, including T cell specific antigens and receptors, confirmed the T cell hybrid nature of L265 clones. No correlation was found in the hybridomas between IFN production and the expression of HTLV, a retrovirus released by Hut102-B2 and SH9 cells.
Human alveolar macrophages produce leukotriene B4.
Authors: Fels A O , Pawlowski N A , Cramer E B , King T K , Cohn Z A , Scott W A
Published: May 2006

Human alveolar macrophages obtained by bronchoalveolar lavage were labeled overnight with [3H]arachidonic acid. The cells were stimulated with calcium ionophore A23187, and the 20:4 oxygenated metabolites released into the culture medium were identified by reverse-phase HPLC. Leukotriene B4 was the major 20:4 metabolite produced by these cultures. Leukotriene B4 was identified by its reverse-phase HPLC elution time, its UV spectrum, and its chemotactic and chemokinetic activities for neutrophils. In addition, the macrophage- and neutrophil-derived leukotriene B4 free acids and methyl esters were found to have identical HPLC retention times.
alpha-latrotoxin of black widow spider venom depolarizes the plasma membrane, induces massive calcium influx, and stimulates transmitter release in guinea pig brain synaptosomes.
Authors: Nicholls D G , Rugolo M , Scott I G , Meldolesi J
Published: May 2006

The effect of alpha-latrotoxin from black widow spider venom upon guinea pig cerebral cortical synaptosomes is described. Plasma membrane potential (delta psi p), in situ mitochondrial membrane potential (delta psi m), Ca2+ transport, gamma-amino[3H]butyrate release, [3H]noradrenaline release, and synaptosomal ATP were monitored under parallel conditions. Potentials were determined both isotopically and with a tetraphenylphosphonium-selective electrode. alpha-Latrotoxin depolarizes delta psi p selectively, both in the presence and absence of Ca2+. A slight toxin-induced depolarization of delta psi m is a consequence of a massive Ca2+ uptake across the plasma membrane. Depolarization of delta psi p is insensitive to tetrodotoxin, and Ca2+ entry is only partially inhibited by verapamil. Release of [3H]noradrenaline and gamma-amino[3H]butyrate is markedly stimulated by the toxin in the presence of Ca2+, and this effect is only slightly reduced in Ca2+-free conditions.
In vitro reconstitution of intermediate filaments form mammalian neurofilament triplet polypeptides.
Authors: Zackroff R V , Idler W W , Steinert P M , Goldman R D
Published: May 2006

Intermediate filaments (IF) were reconstituted in vitro from bovine neurofilament triplet polypeptides. Neural IF, solubilized in either low salt or 8 M urea solution, assembled into IF when returned to near-physiological solution conditions. The 68,000-dalton component of the triplet, purified to homogeneity by preparative NaDodSO4 electrophoresis, was renatured and reassembled into short (approximatley 0.05-micrometer) approximatley 10 nm-diameter filaments. These results demonstrate that the triplet polypeptides are components of neural IF and that the 68,000-dalton polypeptide is an IF structural protein.
Genetic control of heat-shock protein synthesis and its bearing on growth and thermal resistance in Escherichia coli K-12.
Authors: Yamamori T , Yura T
Published: May 2006

When Escherichia coli cells grown at 30 degrees C are transferred to 42 degrees C, synthesis of several polypeptides is markedly and transiently induced. A temperature-sensitive nonsense mutant (tsn-K165) of E. coli K-12 is found to be defective in the induction of these proteins. mRNA for one major heat-shock polypeptide (groE protein) tested is induced in the wild type but not in the mutant upon temperature shift. Hence, the mutation defines a (regulatory) gene, designated hin (heat shock induction), whose product is required for active transcription of a set of heat-inducible operons in E. coli. The results reported herein suggest that the heat-shock polypeptides controlled by the hin gene play an important role in cell growth at high temperature. The possible involvement of the hin gene product in protection against thermal killing is also discussed.
Carp horizontal cells in culture respond selectively to L-glutamate and its agonists.
Authors: Lasater E M , Dowling J E
Published: May 2006

Horizontal cells were enzymatically isolated from the carp retina and maintained in culture for 2-7 days. Cultured horizontal cells typically had resting membrane potentials of -50 to -70 mV and input resistances of 100-150 m omega. The cells were treated with a number of neurotransmitter agents and their analogues. Significant responses were evoked only by 3,4-dihydroxyphenylalanine (dopamine), L-glutamate, and certain glutamate analogues. The responses to dopamine were inconsistent; most often, the membrane hyperpolarized and input resistances increased. However, highly characteristic responses to L-glutamate and its analogues, quisqualate and kainate, were observed in virtually all of the cells tested. The responses consisted of an initial graded depolarization accompanied by a resistance increase, followed in most cases by a prolonged (1- to 2-min) regenerative depolarization. The regenerative component of the response appears to be Ca2+ dependent, while the underlying graded potential may be due to a decrease in K+ conductance of the membrane.
At least two regions of the viral genome determine the oncogenic potential of avian leukosis viruses.
Authors: Robinson H L , Blais B M , Tsichlis P N , Coffin J M
Published: May 2006

Recombinants of oncogenic and nononcogenic avian leukosis viruses were tested for their oncogenic potential in chickens. The results indicate that at least two regions of the viral genome determine the oncogenic potential of these viruses. The first region contains sequences that control viral mRNA synthesis. These sequences determine the potential of a virus to induce a low incidence of lymphomas, carcinomas, chondrosarcomas, fibrosarcomas, and osteopetrosis. The second region lies outside the sequences that control viral mRNAs synthesis. These sequences determine the ability of a virus to induce a high incidence of lymphomas or osteopetrosis.
Apamin as a selective blocker of the calcium-dependent potassium channel in neuroblastoma cells: voltage-clamp and biochemical characterization of the toxin receptor.
Authors: Hugues M , Romey G , Duval D , Vincent J P , Lazdunski M
Published: May 2006

This paper describes the interaction of apamin, a bee venom neurotoxin, with the mouse neuroblastoma cell membrane. Voltage-clamp analyses have shown that apamin at low concentrations specifically blocks the Ca2+-dependent K+ channel in differentiated neuroblastoma cells. Binding experiments with highly radiolabeled toxin indicate that the dissociation constant of the apamin-receptor complex in differentiated neuroblastoma cells is 15-22 pM and the maximal binding capacity is 12 fmol/mg of protein. The receptor is destroyed by proteases, suggesting that it is a protein. The binding capacity of neuroblastoma cells for radiolabeled apamin dramatically increases during the transition from the nondifferentiated to the differentiated state. The number of Ca2+-dependent K+ channels appears to be at most 1/5th the number of fast Na+ channels in differentiated neuroblastoma. The binding of radiolabeled apamin to its receptor is antagonized by monovalent and divalent cations. Na+ inhibition of the binding of 125I-labeled apamin is of the competitive type (Kd(Na+) = 44 mM). Guanidinium and guanidinated compounds such as amiloride or neurotensin prevent binding of 125I-labeled apamin, the best antagonist being neurotensin.
Reliability of the pair-defect-sum approximation for the strength of valence-bond orbitals
Authors: Pauling Linus , Herman Zelek S. , Kamb Barclay J.
Published: May 2006

The pair-defect-sum approximation to the bond strength of a hybrid orbital (angular wave functions only) is compared to the rigorous value as a function of bond angle for seven types of bonding situations, with between three and eight bond directions equivalent by geometrical symmetry operations and with only one independent bond angle. The approximation is seen to be an excellent one in all cases, and the results provide a rationale for the application of this approximation to a variety of problems.
Polyamines are inhibitors of gastric acid secretion.
Authors: Ray T K , Nandi J , Pidhorodeckyj N , Meng-Ai Z
Published: May 2006

The naturally occurring organic polycations such as spermine and spermidine inhibit histamine-stimulated gastric acid secretion by bullfrog gastric mucosa in vitro; spermine is much more potent than spermidine. Unlike the H2 receptor antagonists, the polyamines are completely ineffective from the nutrient side and are effective only from the secretory side of the chambered mucosa. The polyamine effects could be reversed by increasing K+ concentration in the secretory solution. Studies with isolated gastric microsomal vesicles demonstrate that the polyamines do not inhibit the gastric H+,K+-ATPase but greatly decrease the ATPase-mediated uptake of H+ under appropriate conditions. For the latter effects the presence of polyamine within the vesicle interior was found to be essential. Our data strongly suggest an uncoupling of the gastric H+,K+-ATPase system by the polyamines. The therapeutic potential of these and similar compounds in the treatment of hyperacidity and peptic ulcer is discussed.
Reversible repression and activation of measles virus infection in neural cells.
Authors: Miller C A , Carrigan D R
Published: May 2006

Conversion of acute measles virus infection to an indolent state has been achieved by treatment of infected cells of neural origin with agents that affect cyclic nucleotide metabolism. Striking results were obtained with papaverine, an inhibitor of cAMP phosphodiesterase that is capable of enhancing neural differentiation. In papaverine-treated cultures, decreased production of infectious virus was accompanied by selective disappearance of intracellular matrix proton, as detected by immunofluorescence. Viral nucleocapsid protein was enhanced in the cytoplasm while three other structural proteins--polymerase, hemagglutinin, and fusion protein--showed little change in distribution or intensity of staining. These results were specific for cells of neural origin and not observed in CV-1 or Vero cultures. cAMP, dibutyryl cAMP, 8-bromo-cAMP, and isobutylmethylxanthine all inhibited replication of virus but less so than did papaverine. Inhibition of virus replication by any of these agents was rapidly reversible, either by removal of the agent or by addition of cGMP to the culture medium and was accompanied by reappearance of the matrix protein. These results suggest that measles virus replication in neural cells depends on host factors, particularly those affecting endogenous cAMP and cGMP. Viral persistence may thus be related to the state of neural differentiation. This model system may yield information on mechanisms of recrudescence observed in some chronic diseases of the nervous system.
Isolation of functional giant smooth muscle cells from an invertebrate: structural features of relaxed and contracted fibers.
Authors: Hernandez-Nicaise M L , Bilbaut A , Malaval L , Nicaise G
Published: May 2006

The giant smooth muscle fibers of a ctenophore were isolated by enzymatic digestion. These fibers are multinucleated cells, up to 50 micrometers in diameter and 2 cm in length. Their ultrastructure and membrane electrical properties are similar to those of in situ fibers. Relaxed, coiled (partially contracted), and fully shortened states were distinguished in isolated cells and studied by scanning and transmission electron microscopy. Calcium-containing mitochondrial granules were found in the coiled cells but not in either the relaxed or the fully shortened cells. The relaxed cell is characterized in cross section by the density of myosin filaments (457 +/- 15 per micrometer2) and the thin-to-thick filament ratio (5.2 +/- 0.2). In the coiled cell, the muscle lattice does not expand uniformly, as shown by the variability of myosin spacing, and the thin-to-thick filament ratio decreases. Both clockwise and counterclockwise coiling occur along the same fiber. The implications of these findings with respect to the structure of the contractile apparatus are discussed.
Defective spectrin dimer-dimer association with hereditary elliptocytosis.
Authors: Liu S C , Palek J , Prchal J T
Published: May 2006

We examined erythrocytes from 18 patients with hereditary elliptocytosis. Spectrin from eight patients (referred to as type 1) was defective in dimer-dimer association as demonstrated in two ways. First, there was an increased amount of spectrin dimer with a concomitant decrease in tetramer as measured in erythrocyte membrane preparations extracted at 0 degrees C under low-salt conditions (the amount of spectrin dimer was 15-33% of total spectrin species compared with a normal range of 3-7%). Second, the equilibrium constants of spectrin dimer-dimer association were decreased in both solution and in situ membrane. Spectrin from the remaining 10 patients (referred to as type 2) showed normal dimer-dimer association. Membrane skeletons, produced from ghosts of both types of hereditary elliptocytosis by Triton X-100 extraction, were unstable when mechanically shaken. Because spectrin tetramers, but not dimers, can crosslink actin, we postulate that the defective spectrin dimer-dimer association in type 1 diminishes actin crosslinking and thus is responsible for membrane skeletal instability. A defective protein-protein association in type 2, however, remains to be identified.
Zn(II)-induced cooperativity of Escherichia coli ornithine transcarbamoylase.
Authors: Kuo L C , Lipscomb W N , Kantrowitz E R
Published: May 2006

The steady-state reaction of ornithine transcarbamoylase (ornithine carbamoyltransferase, carbamoyl phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) purified from the argI gene product of Escherichia coli strain K-12 exhibits Michaelis-Menten kinetics over an extended range of concentration for both L-ornithine and carbamoyl phosphate. In the presence of Zn2+, however, the saturation curve of L-ornithine becomes sigmoidal, revealing positive cooperativity for this anabolic enzyme. The kinetic data give a limiting Hill coefficient of 2.7 for this substrate at 0.3 mM Zn2+. The allosteric effect of Zn2+ on the enzyme is not altered by the concentration of carbamoyl phosphate, and the saturation curve of carbamoyl phosphate remains hyperbolic in the presence of the metal ion. At fixed substrate concentrations, initial velocity data obtained at 0.-0.3 mM Zn2+ indicate cooperative binding of the metal ion to ornithine transcarbamoylase; a Hill coefficient of 1.7 +/- 0.1 is found that is independent of the level of L-ornithine. These results suggest competitive and exclusive binding to the enzyme between L-ornithine and Zn2+ with conformational changes induced in the subunits of the enzyme only by the metal ligand. Neither Co2+ nor Cu2+ exerts an effect on the kinetic behavior of the enzyme. This finding reveals not only specific allosteric control of ornithine transcarbamoylase by Zn2+ but also the possibility of an interlocking metabolic regulation between the urea cycle and the pathway for pyrimidine biosynthesis.
Amantadine and dansylcadaverine inhibit vesicular stomatitis virus uptake and receptor-mediated endocytosis of alpha 2-macroglobulin.
Authors: Schlegel R , Dickson R B , Willingham M C , Pastan I H
Published: May 2006

The entry of many animal viruses into their host cells often proceeds via a specialized internalization pathway involving clathrin-coated regions of the plasma membrane. We have examined the effect of dansylcadaverine and amantadine on the entry of vesicular stomatitis virus (VSV) into mouse cells. Both compounds inhibit VSV entry as determined by fluorescence and electron microscopy, 3H-labeled VSV uptake, and VSV-dependent RNA synthesis assays. They also inhibit the uptake of alpha 2-macroglobulin, a protein that binds to specific membrane receptors and follows the same route of internalization. Dansylcadaverine is 20-fold more potent than amantadine in blocking virus and alpha 2-macroglobulin uptake. One cellular target for both of these amine-containing compounds appears to be the clustering of membrane-bound ligands or particles in clathrin-coated pits.
Involvement of multiple sodium ions in intestinal d-glucose transport.
Authors: Kaunitz J D , Gunther R , Wright E M
Published: May 2006

Brush border membrane vesicles isolated from rabbit small intestine were used to measure the interactions between sodium and glucose transport with a rapid uptake technique. A plot of glucose uptake rate vs. increasing sodium concentration yielded a sigmoid curve. Hill analysis revealed a coefficient of 1.9 +/- 0.02 (+/- SEM), consistent with at least two sodium ions involved in glucose transport. Transport coupling was then measured directly with double-label experiments in which the uptakes of D-glucose and sodium were determined in the presence and absence of cotransported solute. At the earliest time point, the ratio of cosubstrate-dependent sodium transport to glucose transport was 3.2 +2- 0.7 (+/- SEM). We conclude that two or more sodium ions are coupled to glucose transport across the intestinal brush border membranes.
Monoclonal antibodies identify novel neural antigens.
Authors: Hawkes R , Niday E , Matus A
Published: May 2006

Monoclonal antibodies (Mabs) were raised against synaptic plasma membranes from rat cerebellum. The hybridomas were screened with a solid-phase immunoassay, the positive lines were characterized by their immunoperoxidase staining pattern on cerebellum, and the specific polypeptide antigens were identified on protein blots. Among the Mabs described are some that stain only neurons or only glia and others that react with specific parts of cells, such as axons, dendrites, and synapses. Many Mabs reveal novel relationships between antigens and the cells in which they occur. For example, a Mab designated 7D5 reacts with a family of greater than 30 proteins but stains only glial cells. Several Mabs stain punctate sites of synaptic size and distribution in the cerebellar cortex but each reacts with a different subset of polypeptides. One of the most restricted cytological staining patterns is given by 12D5, which stains punctate sites in the granular layer of the cerebellar cortex and reacts with a single polypeptide band of apparent Mr 270,000. These results illustrate the feasibility of raising Mabs that can be used to follow the expression of specific gene products during brain development.
The complete amino acid sequence of human serum transferrin.
Authors: MacGillivray R T , Mendez E , Sinha S K , Sutton M R , Lineback-Zins J , Brew K
Published: May 2006

The complete amino acid sequence of human serum transferrin has been determined by aligning the structures of the 10 CNBr fragments. The order of these fragments in the polypeptide chain is deduced from the structures of peptides overlapping methionine residues and other evidence. Human transferrin contains 678 amino acid residues and--including the two asparagine-linked glycans--has an overall molecular weight of 79,550. The polypeptide chain contains two homologous domains consisting of residues 1-336 and 337-678, in which 40% of the residues are identical when aligned by inserting gaps at appropriate positions. Disulfide bond arrangements indicate that there are seven residues between the last half-cystine in the first domain and the first half-cystine in the second domain and therefore, a maximum of seven residues in the region of polypeptide between the two domains. Transferrin--which contains two Fe-binding sites--has clearly evolved by the contiguous duplication of the structural gene for an ancestral protein that had a single Fe-binding site and contained approximately 340 amino acid residues. The two domains show some interesting differences including the presence of both N-linked glycan moieties in the COOH-terminal domain at positions 413 and 610 and the presence of more disulfide bonds in the COOH-terminal domain (11 compared to 8). The locations of residues that may function in Fe-binding are discussed.
Phosphorylation of the regulatory subunit of yeast cAMP-dependent protein kinase.
Authors: Sy J , Roselle M
Published: May 2006

In vitro phosphorylation of the regulatory subunit of yeast cAMP-dependent protein kinase was studied. The cAMP-binding regulatory subunit (R subunit) can be multiply phosphorylated. Three distinct phosphorylation sites were inferred from the different ATP concentrations required for phosphorylation and from the presence of two discrete mobility shifts in NaDodSO4/polyacrylamide gel electrophoresis of the R subunit on phosphorylation. Limited tryptic digestion of the phosphorylated R subunit showed that a Mr 37,000 cAMP-binding peptide contained one of the phosphorylation sites and that a separate Mr 12,000 peptide contained another phosphorylation site. The yeast R subunit is therefore similar to the type II R subunit of mammalian origin, although it has a larger Mr (64,000 vs. 58,000) and is multiply phosphorylated. In vivo, both phosphorylated and unphosphorylated forms of the R subunit were found in cells grown in lactate or to stationary phase in 1.5% glucose, while cells grown in 5% glucose contained the unphosphorylated form.
Expression of hepatitis B surface antigen gene in yeast.
Authors: Miyanohara A , Toh-e A , Nozaki C , Hamada F , Ohtomo N , Matsubara K
Published: May 2006

The DNA sequence coding for hepatitis B virus surface antigen (HBsAg) was placed under control of the repressible acid phosphatase promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of autonomous replication in both yeast and Escherichia coli. Yeast transformed by this plasmid synthesized up to 5 X 10(5) molecules per cell of immunologically active HBsAg polypeptide in phosphate-free medium. The HBsAg polypeptides produced in the yeast cells were assembled into 20- to 22-nm spherical or oval particles and were immunogenic.
DNA sequence preference of the progesterone receptor.
Authors: Compton J G , Schrader W T , O'Malley B W
Published: May 2006

Highly purified hen oviduct progesterone receptor A subunit was tested for binding to several chicken gene DNAs. Sequence preference detected by nitrocellulose filter adsorption of [32P]DNA fragments obtained from recombinant plasmids revealed a marked retention of certain DNA fragments. About a 10-fold preference was seen for DNA fragments flanking the 5' end of the steroid-regulated genes ovalbumin and gene Y. No preference was seen with analogous DNA fragments from chicken beta-globin and alpha-actin genes. Restriction endonuclease mapping suggests the presence of multiple receptor interaction sites flanking the 5' terminus of the ovalbumin gene. One of these preferential binding sites was localized between -135 and -247 base pairs upstream from the start of transcription. This region contains an 18-base-pair A + T-rich sequence, a likely candidate for the binding site itself, because earlier studies had shown receptor A to have marked preference for A + T-rich DNA.
The tac promoter: a functional hybrid derived from the trp and lac promoters.
Authors: de Boer H A , Comstock L J , Vasser M
Published: May 2006

Two hybrid promoters that are functional in Escherichia coli have been constructed. These hybrid promoters, tacI and tacII, were derived from sequences of the trp and the lac UV5 promoters. In the first hybrid promoter (tacI), the DNA upstream of position -20 with respect to the transcriptional start site was derived from the trp promoter. The DNA downstream of position -20 was derived from the lac UV5 promoter. In the second hybrid promoter (tacII), the DNA upstream of position -11 at the Hpa I site within the Pribnow box was derived from the trp promoter. The DNA downstream of position -11 is a 46-base-pair synthetic DNA fragment that specifies part of the hybrid Pribnow box and the entire lac operator. It also specifies a Shine-Dalgarno sequence flanked by two unique restriction sites (portable Shine-Dalgarno sequence). The tacI and the tacII promoters respectively direct transcription approximately 11 and 7 times more efficiently than the derepressed parental lac UV5 promoter and approximately 3 and 2 times more efficiently than the trp promoter in the absence of the trp repressor. Both hybrid promoters can be repressed by the lac repressor and both can be derepressed with isopropyl beta-D-thiogalactoside. Consequently, these hybrid promoters are useful for the controlled expression of foreign genes at high levels in E. coli. In contrast to the trp and the lac UV5 promoters, the tacI promoter has not only a consensus -35 sequence but also a consensus Pribnow box sequence. This may explain the higher efficiency of this hybrid promoter with respect to either one of the parental promoters.
Induction of a B-lymphocyte receptor for a T cell-replacing factor by the crosslinking of surface IgD.
Authors: Yaffe L J , Finkelman F D
Published: May 2006

The observation that anti-immunoglobulin antibodies and T cell-replacing factor (TRF) have a synergistic effect on the stimulation of B lymphocytes to differentiate into antibody-secreting cells suggested to us the possibility that the crosslinking of B-cell surface immunoglobulin by antigen or anti-immunoglobulin antibody might induce the expression of a B-cell receptor for TRF. In order to test this possibility we studied whether spleen cells from mice injected with 400-800 micrograms of anti-IgD antibody 1-3 days before sacrifice had an enhanced capacity to adsorb TRF activity from a partially purified culture supernatant of concanavalin A-stimulated mouse spleen cells. We found that spleen cells from euthymic or congenitally athymic mice injected 24-30 hr prior to sacrifice with either affinity-purified goat anti-mouse IgD antibody or a monoclonal allospecific anti-IgD antibody had greater than 100 times the TRF adsorptive capacity of spleen cells from control mice. In contrast, spleen cells from anti-IgD treated DBA/2Ha mice, which have been shown to have an immune defect associated with the lack of a TRF receptor, were unable to adsorb TRF activity from concanavalin A-stimulated helper supernatants. This suggests that the crosslinking of B-cell surface immunoglobulin may induce B lymphocytes to express a receptor or receptors for TRF and thus enhance B-cell responsiveness to this helper factor.
Post-tetanic potentiation of acetylcholine release at the frog neuromuscular junction develops after stimulation in Ca2+-free solutions.
Authors: Misler S , Hurlbut W P
Published: May 2006

At many synapses, previous activity increases the amount of transmitter released by a single action potential. This potentiation of transmitter release is usually attributed to the local accumulation of the calcium ions that cross the axolemma during an action potential. We found that potentiated transmitter release can be observed at frog neuromuscular junctions after periods of repetitive stimulation in Ca2+-free solutions, if Ca2+ is restored after the tetanus. Potentiation is greater and more prolonged, the lower the level of extracellular K+. This component of potentiation may be due to Ca2+ that accumulates within the terminal in exchange for intracellular Na+.
Molecular cloning of human gastrin cDNA: evidence for evolution of gastrin by gene duplication.
Authors: Boel E , Vuust J , Norris F , Norris K , Wind A , Rehfeld J F , Marcker K A
Published: May 2006

An oligo(dT)-primed cDNA copy of the mRNA coding for the human gastrin precursor was constructed from poly(A)-containing RNA from a human pancreatic, gastrin-producing tumor (a gastrinoma). The cDNA was inserted into the Pst I endonuclease site of plasmid pBR322 by the use of the poly(dC) and poly(dG) tailing procedure. Clones containing gastrin sequences were selected by hybridization to a purified single-stranded 32P-labeled gastrin cDNA probe. This probe was constructed with gastrinoma mRNA as template. As primer for the cDNA synthesis, we used a synthetic oligonucleotide mixture, d(AG-A-A-AG-T-C-C-A-T-C-C-A), corresponding to the gastrin-specific amino acid sequence Trp-Met-Asp-Phe. In this way we determined the nucleotide sequence of the entire coding region (303 nucleotides), the entire 3' untranslated region (102 nucleotides), and 8 nucleotides of the 5' untranslated region. A striking homology between parts of the coding region suggests that evolution of the gastrin gene has involved a gene duplication.
Bombesin stimulation of DNA synthesis and cell division in cultures of Swiss 3T3 cells.
Authors: Rozengurt E , Sinnett-Smith J
Published: May 2006

Bombesin is shown to be a potent mitogen for Swiss 3T3 cells. At nanomolar concentrations the peptide markedly enhances the ability of fresh serum to stimulate DNA synthesis in confluent and quiescent cultures of these cells. In the presence of a low concentration (3.5%) of serum, bombesin stimulates 3T3 cell proliferation. In serum-free medium, bombesin induces DNA synthesis in the absence of any other added growth factor; half-maximal effect is obtained at 1 nM. The mitogenic effect of bombesin is dependent on dose and time, is mimicked by litorin, and is markedly potentiated by insulin, colchicine, platelet-derived growth factor, and fibroblast-derived growth factor. These mitogens increase the maximal response elicited by bombesin and decrease the bombesin concentration required to produce half-maximal effect (from 1 nM to 0.3 nM). In contrast, vasopressin, phorbol esters, or cAMP increasing agents fail to enhance the maximal level of DNA synthesis induced by bombesin. Bombesin and litorin may provide useful model peptides for studies on the mechanism(s) by which extracellular ligands control cell proliferation.
Selective phosphorylation of the IgE receptor in antigen-stimulated rat mast cells.
Authors: Hempstead B L , Parker C W , Kulczycki A
Published: May 2006

Purified rat serosal mast cells were sensitized with mouse immunoglobulin E (IgE) anti-2,4-dinitrophenyl antibody, partially depleted of phosphate, labeled with [32P]orthophosphate, and stimulated with dinitrophenylated bovine serum albumin or control protein. After 15-120 seconds at 37 degrees C, the cells were extracted with nonionic detergent. IgE receptors were purified by repetitive affinity chromatography and were analyzed by NaDodSO4/polyacrylamide gel electrophoresis and radioautography. Antigenic stimulation of intact rat mast cells produced a rapid and marked increase in the phosphorylation of the surface-exposed alpha component of the IgE receptor. However, phosphorylation of the 33,000 Mr beta component of the IgE receptor was not altered significantly by antigen stimulation. This suggests that the selective increase in phosphorylation of the IgE receptor alpha component may be part of the physiologic mediator secretion process triggered by antigen.
Model for specific rescue of normal hepatocytes during methotrexate treatment of hepatic malignancy.
Authors: Wu G Y , Wu C H , Stockert R J
Published: May 2006

We utilized the presence of hepatic receptors for galactose-terminal glycoproteins to achieve specific rescue of differentiated hepatocytes from methotrexate toxicity. This was accomplished by administration of a conjugate formed by covalent coupling of the methotrexate antagonist, folinic acid (leucovorin), to galactose-terminating (desialylated) bovine fetuin. Two cultured hepatocyte-derived cell lines were chosen to test rescue from methotrexate toxicity: Hep G2 cells are capable of receptor-mediated endocytosis of galactose-terminating glycoproteins; PLC/PRF/5 cells are not. In the presence of methotrexate alone, both cells lines were reduced in number to 20% of control populations by day 5. Growth rates of both lines returned to normal when free folinic acid was added to the medium containing methotrexate. When asialofetuin-folinic acid conjugate was given to both cell lines in the presence of methotrexate, Hep G2 cells doubled their numbers by day 4 and reached control (without methotrexate) populations by day 8. However, PLC/PRF/5 cells failed to respond to administered asialofetuin-folinic acid conjugate under identical conditions, and the growth curve was the same as that for cells which received methotrexate alone. Conjugates of asialofetuin bound to folic acid (folate derivative requiring dihydrofolate reductase activity for conversion to an active metabolite) and fetuin (non-galactose-terminal glycoprotein) linked to folinic acid also were made for control studies. These conjugates failed to rescue either cell line.
Increased rate of cell-substratum detachment of fibroblasts from patients with Duchenne muscular dystrophy.
Authors: Kent C
Published: May 2006

When skin fibroblasts from patients with Duchenne muscular dystrophy were treated with trypsin in the presence of divalent cations, they detached more rapidly from the substratum than did fibroblasts from normal individuals of similar age, sex, and passage number. This difference was observed when either the time of incubation or trypsin concentration was varied. The ease of detachment of both normal and dystrophic fibroblasts varied somewhat with culture age and plating density, although detachment was always greater for fibroblasts from dystrophic individuals. If the trypsin treatment was carried out in the absence of divalent cations, both types of fibroblasts detached rapidly from the substratum, suggesting that a divalent-cation dependent cell-substratum adhesion mechanism is altered in Duchenne muscular dystrophy fibroblasts.
Thermolysin-catalyzed peptide bond synthesis.
Authors: Wayne S I , Fruton J S
Published: May 2006

The rates of the thermolysin-catalyzed synthesis of peptides have been determined by means of HPLC. In the condensation of various N-substituted amino acids and peptides with L-leucinanilide, the enzyme exhibits preference for a hydrophobic L-amino acid as the donor of the carbonyl group of the newly formed bond. The presence of another hydrophobic amino acid residue adjacent to the carbonyl-group donor markedly enhances the rate of synthesis. In general, the effect of structural changes in both the carboxyl and amine components of the condensation reaction is in accord with the available data on the primary and secondary specificities of the thermolysin-catalyzed hydrolysis of oligopeptide substrates. A kinetic study of the condensation of benzyloxycarbonyl-L-phenylalanine with various amine components has given data on the apparent kcat and Km values for the entry of the acidic component into the condensation reaction. The results are consistent with the behavior of rapid-equilibrium random bi-reactant systems leading to ternary enzyme-substrate complexes, with a synergistic effect in the binding of the two reactants at the active site. Because the changes in the apparent kcat for the entry of the same acidic component into reaction with different amine components are greater than those in the apparent Km, it is suggested that this synergism is largely expressed at the level of the transition-state complex.
Cloning of the gene for C protein, a low molecular weight spore-specific protein from Bacillus megaterium.
Authors: Curiel-Quesada E , Setlow B , Setlow P
Published: May 2006

The structural gene for C protein, a low molecular weight spore-specific protein from Bacillus megaterium, has been cloned in Escherichia coli. Expression of the C-protein gene in E. coli requires an external transcription promoter and prevention of termination of transcription prior to transcription of all or part of the sequence coding for the C protein. The gene for the C protein is within a 5-kilobase DNA fragment, but this fragment does not code for either of the other two major low molecular weight spore proteins, suggesting that the structural genes for these proteins are not tightly linked.
Vectorial synthesis of a polysaccharide by isolated plasma membranes.
Authors: Cabib E , Bowers B , Roberts R L
Published: May 2006

To ascertain the directionality of chitin synthesis by yeast plasma membranes, the external surface of Saccharomyces cerevisiae protoplasts was labeled with ferritin--concanavalin A. After protoplast lysis, plasma membranes were isolated and treated with trypsin to activate chitin synthase (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxy-D-glucosyl-transferase, EC 2.4.1.16). The membranes were then enrobed in agar and allowed to synthesize chitin from UDP-N-acetylglucosamine. After fixation and embedding in Epon, thin sections were stained for chitin with wheat germ agglutinin--colloidal gold complexes. The chitin marker was found near the ferritin-labeled external face of the membrane--i.e., the polysaccharide was located on the outside of the membrane, as it is in the intact cell. Chitin synthase activity was not detected in intact protoplasts before or after treatment with trypsin. The enzyme became available to trypsin activation after lysis of the protoplasts. Together with similar, previously reported experiments on the inactivation of chitin synthase by glutaraldehyde, these results indicate that the enzyme faces the interior of the cell. We conclude that, both in vivo and in vitro, the synthase receives N-acetylglucosamine residues from UDP-N-acetylglucosamine at the cytoplasmic face of the membrane and transfers them vectorially to a growing chain of chitin that is concomitantly extruded to the outside.
Serotonin increases an anomalously rectifying K+ current in the Aplysia neuron R15.
Authors: Benson J A , Levitan I B
Published: May 2006

Previous work has shown that serotonin causes an increase in K+ conductance in the identified Aplysia neuron R15. This response is mediated by cAMP-dependent protein phosphorylation. The results presented here show that the K+ channel modulated by serotonin is an anomalous or inward rectifier (designated IR) that is present in R15 together with the three other distinct K+ channels previously described for this cell. Several lines of evidence indicate that this inward rectifier is partially activated in the resting cell and is further activated by serotonin. Voltage clamp analysis of resting and serotonin-evoked membrane currents at various external K+ concentrations shows that both currents have reversal potentials close to the potassium equilibrium potential, exhibit similar dependences in magnitude on external K+ concentration, and display marked anomalous rectification. The effects of particular monovalent and divalent cations are also similar on the resting and serotonin-evoked currents. Rb+, Cs+, and Ba2+ block both currents while Tl+ can substitute for K+ as a charge carrier and channel activator in both. These properties are characteristics of anomalous rectifiers in other systems. Furthermore, measurement of the voltage dependence of inactivation for the fast transient K+ current shows that this current cannot account for the anomalously rectifying K+ conductance in R15. The inward rectifier is therefore a separate current mediated by its own channels, the activity of which can be modulated by serotonin.
Regulation of human ribosomal RNA transcription.
Authors: Learned R M , Smale S T , Haltiner M M , Tjian R
Published: May 2006

We have used a cell-free polymerase I transcription system derived from HeLa cells to study the regulation of human rRNA synthesis. Analysis of deletion mutants spanning the start site of transcription at nucleotide +1 indicates that the control region affecting initiation of human rRNA synthesis is contained within sequences from nucleotides -158 to +18. This promoter region can be subdivided into (i) a central segment of approximately 40 base pair that is required for transcription and (ii) flanking sequences that influence the efficiency of transcription in vitro. We have examined the in vitro transcriptional activity of the human extract under various conditions that are thought to modulate rRNA synthesis in vivo. Cell-free extracts prepared from HeLa cells infected with adenovirus 2 synthesize human rRNA at levels greatly decreased relative to uninfected cell extracts. By contrast, in vitro transcription of human rRNA is stimulated 2- to 3-fold by the addition of purified simian virus 40 large tumor antigen to the transcription reaction. Moreover, a mutant tumor antigen known to be defective for rRNA activation in vivo is incapable of stimulating rRNA synthesis in vitro. The ability to detect these different regulatory phenomena in vitro provides us with an experimental basis for investigating the molecular mechanisms that control rRNA synthesis.
Leucinopine, a characteristic compound of some crown-gall tumors
Authors: Chang Chi-cheng , Chen Chong-maw , Adams Bruce R. , Trost Barry M.
Published: May 2006

An unusual compound has been found in crowngall tumors induced by those
Agrobacterium tumefaciens
strains that utilize neither octopine nor nopaline. The compound has been isolated and shown by proton and carbon-13 NMR spectroscopy and by synthesis to be
N
2
-(1,3-dicarboxypropyl)-L-leucine, which also exists in a cyclized (i.e., lactam) form. This compound, which we name “leucinopine,” was not detected in octopine tumors, nopaline tumors, or the tumors induced by
A. tumefaciens
strains 181 and EU6.
Host range transforming gene of polyoma virus plays a role in virus assembly.
Authors: Garcea R L , Benjamin T L
Published: May 2006

Polyoma virus host range transforming (hr-t) mutants are blocked in virion assembly. In normal 3T3 cells, a nonpermissive host, these mutants synthesize 30-40% as much viral DNA and 80-100% as much capsid proteins as does wild-type virus and yet produce only 1-2% as much infectious virus. Intermediates in virion assembly have been followed by [3H]thymidine incorporation. hr-t mutants synthesize 95S replicating minichromosomes, which accumulate as 75S forms. However, the latter fail to undergo efficient transition to 240S virion structures. This block in encapsidation is overcome in permissive hosts such as primary baby mouse kidney (BMK) epithelial cells. The block in assembly of 240S particles is accompanied by a failure to induce a series of acidic isoelectric forms of the major capsid protein, VP1. Multiple species of post-translationally modified VP1 are seen by two-dimensional gel electrophoresis in wild-type virus-infected cells. These acidic VP1 subspecies are decreased 6- to 10-fold in hr-t mutant-infected 3T3 cells but are produced in normal amounts when the same mutants infect BMK cells. When 3T3 cells are coinfected with hr-t mutant and wild-type viruses, normal amounts of the VP1 subspecies are present, and hr-t mutant viral DNA is efficiently packaged into virions. These studies demonstrate an important role of the hr-t gene of polyoma virus in virus assembly. Specifically, we propose that VP1 is a target for hr-t gene-controlled modification and that modified forms of VP1 are essential for encapsidation of viral minichromosomes.