bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of phospholipid methylation, Ca2+ influx, and histamine release by bridging of IgE receptors on rat mast cells.
Authors: Ishizaka T , Hirata F , Ishizaka K , Axelrod J
Published: May 2006

Normal rat mast cells were stimulated by antibodies against IgE receptors (anti-RBL) or by anti-IgE, and [3H]methyl group incorporation into phospholipids, 45Ca uptake, and histamine release were examined. Anti-RBL or its F(ab')2 fragments and anti-IgE induced an increase in the incorporation of [3H]methyl into phospholipids, in 45Ca influx, and in histamine release. By contrast, Fab' monomer fragments of anti-RBL induced none of these reactions. The transient increase of [3H]methyl incorporation in lipids peaked within 15 sec after the addition of either anti-RBL or anti-IgE and fell to basal level in 30 sec. This was then followed by an influx of 45Ca that increased to a maximum in 2 min and by histamine release that reached a maximum in 3 min. Inhibition of phospholipid methylation resulted in an inhibition of 45Ca influx and histamine release. These findings demonstrate that phospholipid methylation in rat mast cells is induced by bridging of IgE receptors on the cell surface and that increased methylation of phospholipids sets the stage for an influx of Ca2+ and subsequent release of histamine.
Cyanobacterial phycobilisomes: Selective dissociation monitored by fluorescence and circular dichroism
Authors: Rigbi Meir , Rosinski Joanne , Siegelman Harold W. , Sutherland John Clark
Published: May 2006

Phycobilisomes are supramolecular assemblies of phycobiliproteins responsible for photosynthetic light collection in red algae and cyanobacteria. They can be selectively dissociated by reduction of temperature and buffer concentration. Phycobilisomes isolated from
Fremyella diplosiphon
transfer energy collected by C-phycoerythrin and C-phycocyanin to allophycocyanin. The energy transfer to allophycocyanin is nearly abolished at 2°C, as indicated by a blue shift in fluorescence emission, and is accompanied by a decrease in the circular dichroism in the region of allophycocyanin absorbance. Further dissociation of the phycobilisomes can be attained by reduction of buffer concentration and holding at 2°C. Energy transfer to C-phycocyanin is nearly abolished, and decreases occur in the circular dichroism in the region of C-phycocyanin and C-phycoerythrin absorbance. Complete dissociation of the phycobilisomes at low buffer concentration and 2°C requires extended time. Energy transfer to C-phycocyanin is further reduced and the circular dichroism maximum of C-phycoerythrin at 575 nm is lost. Circular dichroism provides information on the hexamer-monomer transitions of the phycobiliproteins, whereas fluorescence is indicative of hexamer-hexamer interactions. We consider that hydrophobic interactions are fundamental to the maintenance of the structure and function of phycobilisomes.
Amino-terminal sequence and processing of the precursor of the leucine-specific binding protein, and evidence for conformational differences between the precursor and the mature form.
Authors: Oxender D L , Anderson J J , Daniels C J , Landick R , Gunsalus R P , Zurawski G , Yanofsky C
Published: May 2006

A 2.1-kilobase Bgl II DNA fragment from Escherichia coli containing livK, the gene coding for the leucine-specific binding protein, has been cloned into the BamHI site of the plasmid vector pBR322. The DNA sequence of segments of the resulting plasmid, pOX7, established the location of the livK gene and the direction of its transcription. In vitro protein synthesis directed by pOX7DNA yielded the Mr 42,000 precursor of the leucine-specific binding protein and a small amount of the Mr 39,000 mature protein. Continued incubation of the in vitro reaction mixture after DNase and RNase treatment resulted in additional processing. The DNA sequence of the beginning of livK suggested that 23 additional amino acid residues are present as an extension of the NH2 terminus of the mature protein. Amino acid sequence analysis established that the precursor has the predicted 23-residue extension. Proteolytic digestion studies with the precursor and mature forms of the leucine-specific binding protein indicate that there are conformational differences between the two. This suggests a possible role for the signal sequence in determining the conformation of the binding protein precursor that is recognized by the membrane.
Effect of bleaching light on measurements of lateral diffusion in cell membranes by the fluorescence photobleaching recovery method.
Authors: Wolf D E , Edidin M , Dragsten P R
Published: May 2006

The Fc receptors for IgE on rat basophil leukemia 2H3 cells were labeled with either rhodamine- or fluorescein-conjugated IgE and then with Fab fragments of anti-IgE conjugated with the other dye. Fluorescence photobleaching recovery measurements of lateral diffusion were performed with one of the dyes before and after extensive bleaching of the other dye over an entire cell. Bleaching one dye did not affect subsequent measurements made with the other dye. We thus detect no evidence for photoinduced artifacts in fluorescence photobleaching recovery experiments.
Interaction of cholecystokinin with specific membrane receptors on pancreatic acinar cells
Authors: Jensen R. T. , Lemp G. F. , Gardner J. D.
Published: May 2006

We have prepared
125
I-labeled cholecystokinin and have examined the kinetics, stoichiometry, and chemical specificity with which the labeled peptide binds to dispersed acini from guinea pig pancreas. Binding of
125
I-labeled cholecystokinin was reversible, temperature-dependent, saturable, specific, and localized to the plasma membrane. Each acinar cell possessed approximately 9000 binding sites, and binding of the labeled peptide to these sites could be inhibited by cholecystokinin and structurally related peptides (e.g., gastrin and caerulein) as well as by nonpeptide competitive antagonists of the action of cholecystokinin. Binding was not inhibited by other pancreatic secretagogues such as secretin, vasoactive intestinal peptide, glucagon, physalaemin, eledoisin, kassinin, substance P, carbamoylcholine, litorin, or ranatensin or by bovine pancreatic polypeptide, atropine, neurotensin, leucineenkephalin, methionine-enkephalin, or cyclic somatostatin. With agonists as well as antagonists there was a good correlation between occupation of cholecystokinin binding sites and changes in acinar cell function. With each of six different peptide agonists maximal stimulation of enzyme secretion occurred with 40% receptor occupation and occupation of the remaining 60% caused a progressive decrease in stimulated amylase release. Agonists, but not antagonists, accelerated the dissociation of bound
125
I-labeled cholecystokinin, and these findings suggest that, in pancreatic acini, radiolabeled cholecystokinin binds to at least one class of interacting binding sites whose affinities are influenced by the extent to which these sites are occupied by agonists but not the extent to which they are occupied by antagonists.
Introduction of a viral thymidine kinase gene and the human β-globin gene into developmentally multipotential mouse teratocarcinoma cells
Authors: Pellicer Angel , Wagner Erwin F. , Kareh Adele El , Dewey Michael J. , Reuser Arnold J. , Silverstein Saul , Axel Richard , Mintz Beatrice
Published: May 2006

Teratocarcinoma (TCC) stem cells provide unique prospects for the introduction of specific genes into mice, by virtue of their dual capacity for propagation
in vitro
and for normal differentiation in embryos. In this study, we have demonstrated that foreign genes amenable to selection in culture can be transferred into the stem cells and expressed. These cells maintain expression of the gene for long periods during differentiation in tumors
in vivo
in the absence of selective pressure. The cells also integrate an unlinked nonselectable gene at high frequency.

Addition of the cloned herpes simplex virus (HSV) thymidine kinase (tk; ATP:thymidine 5′-phosphotransferase, EC 2.7.1.21) gene to cultures of tk
-
TCC cells yielded tk
+
colonies at a frequency of one colony per 4 μg of plasmid DNA. This transformation efficiency, although appreciably lower than for mouse L tk
-
cells, permits the isolation of many transformants. The HSV provenance of the transformed phenotype was verified by the characteristic electrophoretic mobility of the tk protein and by neutralization of the tk activity with specific antiserum. Moreover, blot hybridization tests revealed at least one intact copy of the viral tk gene integrated into the DNA of transformed cells. When injected into syngeneic mice, the cells formed solid tumors with various differentiating tissues. From blot hybridization comparisons with their cell lines of origin, seven of nine tumors examined had maintained the HSV tk gene without significant loss or rearrangement. Viral tk enzyme activity could also be demonstrated in at least some of the tumors. Cotransfer of the cloned human β-globin gene along with the unlinked HSV tk gene was successful in 2 of 10 tk
+
transformants. Thus, defined genes can be stably introduced into TCC cells in culture and maintained
in vivo
in a form in which they are transcribed and translated to produce a functional protein.
Bacteriophage lambda vehicle for the direct cloning of Escherichia coli promoter DNA sequences: feedback regulation of the rplJL-rpoBC operon.
Authors: Holowachuk E W , Friesen J D , Fiil N P
Published: May 2006

A derivative of bacteriophage lambda, lambda 21, has been constructed and used for the cloning of Escherichia coli DNA fragments carrying promoters. Phage lambda 21 lacks the lac promoter operator and can accept DNA fragments up to 9.8 kilobases in size at a unique HindIII restriction endonuclease site adjacent to lacZ. Recombinant phage that carry promoters are readily identified by their expression of lacZ. Lysogens of these phages in strains harboring a deletion of the chromosomal lac operon are capable of growth on lactose as sole carbon source and can be used to study some of the regulatory signals that act upon the cloned promoter. In principle, lambda 21 can be used to clone any promoter DNA sequence with HindIII termini. PJ, the primary promoter for the rplJL-rpoBC operon, and P beta, a weak promoter for rpoBC, have been cloned in lambda 21. Transcription of lacZ from PJ was found to be subjected to feedback control by ribosomal protein L10 and to a lesser extent by ribosomal protein L7/L12. This suggests a possible L10-binding site near PJ that regulates transcription from that promoter. Lysogens of the phage that carries P beta responded to two regulatory signals: a rho-sensitive termination site preceding rpoBC and induction of beta-galactosidase synthesis by rifampicin. This suggests that P beta is a bona fide promoter for rpoBC.
In vitro production of immunosuppressive factors by murine sarcoma virus-transformed mouse fibroblasts.
Authors: Mizel S B , DeLarco J E , Todaro G J , Farrar W L , Hilfiker M L
Published: May 2006

Murine sarcoma virus-transformed mouse fibroblasts produce potent immunosuppressive factors (ISF) in vitro. The partially purified ISF inhibited thymocyte proliferation induced by concanavalin A or phytohemagglutinin plus lymphocyte activating factor (Interleukin 1), lipopolysaccharide-induced spleen cell proliferation, the in vitro splenic anti-sheep erythrocyte plaque-forming cell response, and the generation of alloantigen-specific cytotoxic T cells. The effect of ISF on thymocyte proliferation was not readily reversible and required only a 4-hr exposure of the thymocytes to ISF to inhibit cell proliferation. Although ISF shares several biochemical properties with a murine sarcoma virus-transformed cell-derived sarcoma growth factor (e.g., acetic acid solubility and sensitivity to dithiothreitol), the two factors could be resolved by gel filtration on Bio-Gel P-60. Two peaks of ISF activity were found with apparent molecular weights of 12,000 and 8000. The results described here support the hypothesis that at least some of the ISF obtained from the serum of tumor-bearing hosts may be released by the tumor cells themselves. In view of the potent in vitro activity of the murine sarcoma virus-transformed fibroblast-derived ISF, it is quite possible that ISF-like molecules may play a role in subverting in vivo tumor rejection processes involving the immune system.
Suppression of tumor rejection by autologous anti-idiotypic immunity
Authors: Flood P. M. , Kripke M. L. , Rowley D. A. , Schreiber H.
Published: May 2006

Autologous anti-idiotypic responses to tumorspecific lymphocytes altered the capability of mice to reject syngeneic tumors. This was shown by using two non-crossreacting fibrosarcoma lines, 1591 and 1316, induced by ultraviolet light. Cells from these tumor lines are regularly rejected when transplanted into normal syngeneic C3H mice but grow progressively in animals immunosuppressed by irradiation with ultraviolet light or by x-irradiation and thymectomy. Immunization of normal mice with 1591-specific lymphoblasts that had been generated in mixed lymphocyte-tumor cell cultures caused a loss of resistance to 1591 tumor cells, but the animals remained resistant to 1316 tumor cells.
In vitro
, spleen cells from animals immunized with 1591-specific lymphoblasts did not generate cytolytic T cells to 1591 fibrosarcoma cells, but spleen cells from the same animals responded normally to 1316 fibrosarcoma cells. Furthermore, spleen cells from animals immunized with 1591-specific lymphoblasts contained idiotype-specific T cells that lysed 1591-specific lymphoblasts, whereas 1316-specific lymphoblasts were unaffected. Immunization of normal animals with nonresponding lymphocytes from the same mixed lymphocyte-tumor cell cultures as the 1591-specific lymphoblasts showed normal responses to both tumors
in vivo
and
in vitro
. These results suggest that changes in the balance of tumor-specific and anti-idiotypic T lymphocyte clones can influence the capability of an individual to respond effectively to tumor antigens and can determine whether a tumor grows or regresses.
Location of peptide fragments in the fibrinogen molecule by immunoelectron microscopy.
Authors: Telford J N , Nagy J A , Hatcher P A , Scheraga H A
Published: May 2006

Antibodies to the disulfide knot fragment of bovine fibrinogen have been used to locate the site of this fragment within the intact fibrinogen molecule. The antibodies were isolated from rabbit antifibrinogen antisera by affinity chromatography. Electron micrographs of reaction mixtures of bovine fibrinogen and antibodies against the disulfide knot fragment showed pairs of fibrinogen molecules crosslinked by antibody molecules as well as higher order antibody-fibrinogen complexes. From an electron microscopic investigation of the crosslinked material, we conclude that the disulfide knot lies within the central nodule of the trinodular fibrinogen molecule. Antibodies to fragment H were used in the same manner to locate this fragment within the outer nodules of the human fibrinogen molecule.
Lateral mobility of band 3 in the human erythrocyte membrane studied by fluorescence photobleaching recovery: Evidence for control by cytoskeletal interactions
Authors: Golan David E. , Veatch William
Published: May 2006

Band 3, the major intrinsic protein of the human erythrocyte membrane, was specifically labeled with the covalent fluorescent probe eosin isothiocyanate. The lateral mobility of labeled band 3 in the plane of the membrane under various conditions of ionic strength and temperature was examined by using the fluorescence photobleaching recovery technique. Low temperature (21°C) and high ionic strength (46 mM NaPO
4
) favored immobilization of band 3(10% mobile) as well as slow diffusion of the mobile fraction (diffusion coefficient
D
= 4 × 10
-11
cm
2
sec
-1
). Increasing temperature (37°C) and decreasing ionic strength (13 mM NaPO
4
) led to an increase in the fraction of mobile band 3(90% mobile) and a reversible increase in the diffusion rate of the mobile fraction (
D
= 200 × 10
-11
cm
2
sec
-1
). The increase in the fraction of mobile band 3 was markedly dissociated, however, from the increase in the diffusion rate of the mobile fraction. Thus, the fraction of mobile band 3 always increased at higher ionic strength and lower temperature than the ionic strength and temperature at which the diffusion rate increased. This dissociation was manifested kinetically on prolonged incubation of ghosts at constant ionic strength and temperature: the diffusion rate of the mobile fraction increased slowly at first and much more rapidly after the initial lag period, whereas the fraction of mobile band 3 increased almost immediately to 90% and remained maximal for the duration of the experiment. Further, changes in diffusion rate with temperature were promptly and totally reversible, whereas increases in the mobile fraction were only slowly and partially reversible. These effects were shown not to be due to complete dissociation of spectrin, the major protein of the erythrocyte cytoskeleton, from the membrane. This evidence suggests control of band 3 lateral mobility by at least two separate processes. The process that determines the diffusion coefficient of the mobile band 3 is completely reversible, and it probably involves a metastable state of cytoskeleton structure intermediate between tight binding to the membrane and complete dissociation from it.
Isovaleryl-CoA dehydrogenase: demonstration in rat liver mitochondria by ion exchange chromatography and isoelectric focusing.
Authors: Noda C , Rhead W J , Tanaka K
Published: May 2006

There has been ambiguity concerning the specificity of the enzymes that dehydrogenate short branched-chain acyl-CoAs. It previously had been assumed that isovaleryl-CoA is dehydrogenated by n-butyryl-CoA dehydrogenase [butyryl-CoA:(acceptor) oxidoreductase, EC 1.3.99.2]. To solve this problem, we fractionated five short-chain acyl-CoA dehydrogenases (isovaleryl-CoA, n-butyryl-CoA, isobutyryl-CoA, n-octanoyl-CoA, and glutaryl-CoA dehydrogenases) from rat liver mitochondria by isoelectric focusing and DEAE-cellulose column chromatography. The isovaleryl-CoA dehydrogenase [isovaleryl-CoA:(acceptor) oxidoreductase, EC 1.3.99.10] peak was almost completely separated from the peaks of n-butyryl CoA- and n-octanoyl-CoA dehydrogenases by isoelectric focusing, and it was well separated from glutaryl-CoA dehydrogenase [glutaryl-CoA:(acceptor) oxidoreductase (decarboxylating), EC 1.3.99.7] and n-octanoyl-CoA dehydrogenase by DEAE-cellulose column chromatography. The isovaleryl-CoA dehydrogenase peak partly overlapped that of n-butyryl-CoA and isobutyryl-CoA dehydrogenases in the latter procedure. These results unequivocally demonstrate that isovaleryl-CoA is oxidized by a specific isovaleryl-CoA dehydrogenase. The other dehydrogenase peaks also demonstrated activity toward a single substrate, except that isobutyryl-CoA dehydrogenase activity could not be clearly resolved from n-butyryl-CoA dehydrogenase activity.
Drosophila nucleosomes contain an unusual histone-like protein.
Authors: Palmer D , Snyder L A , Blumenfeld M
Published: May 2006

Mononucleosomes prepared from Drosophila melanogaster nuclei contain the four core histones H2A, H2B, H3, and H4 plus an additional histone-like, acid-soluble, chromosomal protein. It is probably the protein designated D2 by Alfageme et al. [Alfageme, C.R., Zweidler, A., Mahowald, A. & Cohen, L.H. (1974) J. Biol. Chem. 249, 3729-3736]. D2 elutes with histone H2A from a Bio-Gel P-100 column, but can be distinguished electrophoretically from H2A and from the other standard Drosophila core histones. The amino acid composition of D2 resembles the compositions of H2A and H2B. However, peptide mapping reveals that D2 is not a simple sequence variant of either H2A or H2B. D2 is present in nuclei from embryos and adult heads, and thus is not restricted to a narrowly defined developmental period. It is present in D. melanogaster and D. virilis, and thus appears to be conserved during the evolution of Drosophila. D2 is present in D. melanogaster chromatin with an approximate frequency of one molecule per five nucleosomes, and must therefore be associated with a subset of nucleosomes. The function of this protein is not known. Its presence in nucleosomes, evolutionary conservation, and comparatively large abundance all suggest that it is an important nucleosomal element. It will be interesting to learn whether this histone-like protein is encoded in a subset of the Drosophila histone gene cluster or is encoded separately.
Ascorbic acid inhibits replication and infectivity of avian RNA tumor virus.
Authors: Bissell M J , Hatie C , Farson D A , Schwarz R I , Soo W J
Published: May 2006

Ascorbic acid, at nontoxic concentrations, causes a substantial reduction in the ability of avian tumor viruses to replicate in both primary avian tendon cells and chicken embryo fibroblasts. The virus-infected cultures appear to be less transformed in the presence of ascorbic acid by the criteria of morphology, reduced glucose uptake, and increased collagen synthesis. The vitamin does not act by altering the susceptibility of the cells to initial infection and transformation, but instead appears to interfere with the spread of infection through a reduction in virus replication and virus infectivity. The effect is reversible and requires the continuous presence of the vitamin in the culture medium.
DNA-membrane association is necessary for initiation of chromosomal and plasmid replication in Bacillus subtilis.
Authors: Winston S , Sueoka N
Published: May 2006

We examined the effect of the inhibition of initiation of DNA replication on the membrane association of the chromosomal origin of replication of Bacillus subtilis and the Staphylococcus aureus-Bacillus pumilus chimeric plasmid pSL103, using temperature-sensitive mutants of B. subtilis that have specifically affected initiation. Inhibition of initiation of the chromosome and pSL103 in the initiation mutant dna-1 results in a decrease in the membrane association of both a marker near the chromosomal origin, purA16, and the plasmid pSL103. The membrane association of both purA16 and pSL103 can be recovered by allowing initiation to resume at the permissive temperature. In another initiation mutant, dnaB19, only the initiation and membrane association of the host chromosome are affected at the nonpermissive temperature, whereas both initiation and membrane association are not affected in the plasmid pSL103. In experiments in vitro, DNA containing the purA16 marker and pSL103 DNA molecules are both selectively released during incubation of purified DNA-membrane complexes prepared from dna-1 cells at the nonpermissive temperature. On the other hand, only purA16 DNA is released in vitro from the DNA-membrane complex prepared from dnaB19 cells. This consistent coupling between initiation and membrane association indicates that DNA-membrane association is critical for the initiation of the B. subtilis chromosome and the plasmid pSL103.
Alloantigen-induced T-cell proliferation: Lyt phenotype of responding cells and blocking of proliferation by Lyt antisera
Authors: Nakayama Eiichi , Dippold Wolfgang , Shiku Hiroshi , Oettgen Herbert F. , Old Lloyd J.
Published: May 2006

Cytotoxic T cells of the mouse express Lyt-1 as well as Lyt-2 and -3 on their surface, and T-cell cytotoxicity can be blocked by Lyt-2 and Lyt-3 (but not Lyt-1) antisera in the absence of added complement [Nakayama, E., Shiku, H., Stockert, E., Oettgen, H. F. & Old, L. J. (1979)
Proc. Natl. Acad. Sci. USA
76, 1977-1981]. This analysis has now been extended to the study of the Lyt phenotype of T cells responding to alloantigens, concanavalin A (Con A), and phytohemagglutinin (PHA) and the effect of Lyt antibody on T-cell proliferation and the generation of H-2-specific killer T cells. H-2 (D/K and I), Con A, and PHA stimulation was abolished by pretreating responding cell populations with Lyt-1 antiserum and complement. Pretreatment with Lyt-2 or -3 antiserum and complement did not decrease alloantigen or Con A stimulation but did abolish PHA stimulation. Cytotoxic cells were not generated in H-2 alloantigen-primed cultures pretreated with Lyt-1, -2, or -3 antiserum and complement. When responding cells were cultured with Lyt antiserum in the absence of added complement, Lyt-2 or -3 antiserum (but not Lyt-1 antiserum) blocked alloantigen-induced proliferation and delayed generation of killer cells. Under similar conditions, Con A and PHA stimulation was not blocked by Lyt-1,-2, or -3 antiserum. Evidence from these Lyt elimination and blocking tests and from direct Lyt phenotyping of responding cells leads to the following conclusions. Two populations of Lyt
+
cells are involved: Lyt-1
+
2
-
3
-
and Lyt-1
+
2
+
3
+
. Current evidence does not favor the existence of Lyt-1
-
2
+
3
+
cells but indicates that pre-killer and killer cells derive from the Lyt-1
+
2
+
3
+
population and have a Lyt-1
+
2
+
3
+
phenotype. H-2 (D/K and I) and PHA stimulation ordinarily activate the Lyt-1
+
2
+
3
+
population, whereas Con A and
I
region or
Mls
locus antigens activate the Lyt-1
+
2
-
3
-
population. However, when Lyt-1
+
2
+
3
+
cells are eliminated or blocked by Lyt-2 or -3 antiserum, H-2 alloantigen stimulation leads to proliferation of the Lyt-1
+
2
-
3
-
population. Blocking of H-2-induced proliferation by Lyt-2 or -3 antiserum adds further support to the possibility that molecules bearing Lyt-2 and -3 determinants are involved in T-cell recognition.
Identification and characterization of rabbit-mouse hybridomas secreting rabbit immunoglobulin chains.
Authors: Yarmush M L , Gates F T , Weisfogel D R , Kindt T J
Published: May 2006

Standard cell-fusion techniques have been used to generate hybrid cells from rabbit spleen cells and mouse myeloma cell lines. The hybrids were selected for secretion of rabbit immunoglobulin. Detailed allotype analyses were carried out for 189 cell lines found to be immunoglobulin positive: 1 produced an intact immunoglobulin molecule with antibody activity, 143 produced rabbit light (L) chains, 36 produced rabbit heavy (H) chains, and 9 cell lines gave negative results in tests for group a and b allotypes. Fusions with a nonproducing murine myeloma cell line (SP-2) yielded only L chain-secreting hybrids, whereas 27% of hybrids resulting from fusion with an L chain-producing line (PU) secreted rabbit H chains paired with mouse L chains. Several of the hybridomas have maintained the ability to produce rabbit immunoglobulin chains in culture for almost 1 year and can be propagated as ascites tumors in athymic (nude) mice. Cytogenetic analyses have detected no rabbit chromosomes in the stable hybrid cell lines.
Regulation of cyclic AMP formation in brain tissue by alpha-adrenergic receptors: requisite intermediacy of prostaglandins of the E series.
Authors: Partington C R , Edwards M W , Daly J W
Published: May 2006

The accumulations of cyclic AMP elicited by norepinephrine in slices of rat cerebral cortex or hypothalamus were markedly reduced after incubations with prostaglandin synthetase (8,11,14-eicosatrienoate, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.1) inhibitors such as indomethacin, aspirin, flufenamic acid, and acetoaminophen. Responses of cyclic AMP-generating systems to beta-adrenergic agonists or adenosine were unchanged by treatment with indomethacin and the reduction in the norepinephrine response appeared due primarily to a loss of the alpha-adrenergic component. The accumulation of cyclic AMP elicited by prostaglandin E2 [mean effective dose (EC50) 4 micro M] was increased by 2-fold by treatment with indomethacin. The alpha-adrenergic component of the norepinephrine response was fully restored by very low concentrations of prostaglandin E2 (EC50 20 nM). Prostaglandins of the F series had no effect on cyclic AMP generation under a variety of conditions. It appears that low levels of prostaglandins of the E series are required--perhaps by a calcium-dependent mechanism--for the expression of alpha-adrenergic receptor-mediated activation of cyclic AMP formation in brain tissue.
Repeated seizures induce long-term increase in hippocampal benzodiazepine receptors.
Authors: McNamara J O , Peper A M , Patrone V
Published: May 2006

Repeated seizures, whether induced by kindling or electroshock, caused a long-lasting (at least 24 hr) increase of [3H]diazepam binding in hippocampal membranes of Sprague-Dawley rats. Scatchard analyses demonstrated that increased numbers of binding sites accounted for the increase. Neither repeated hypoxia nor repeated administration of electrical current without inducing seizures caused an increase of [3H]diazepam binding. Regardless of the method used for seizure induction, the response was graded in that large numbers of seizures were required to induce significant increases, whereas fewer seizures induced only slight increases. We suggest that the receptor increases imply a heightened response to benzodiazepines and more powerful hippocampal recurrent inhibition.
Femtosecond relaxation of an iron porphyrin observed with polarization spectroscopy in a three-level system
Authors: Andrews John R. , Hochstrasser Robin M.
Published: May 2006

An experimental and theoretical study is presented of the nonlinear response of a dilute solution being pumped by two laser fields having different frequencies ω
1
and ω
2
that are both in resonance with electronic transitions of the solute. Experimental results were obtained for the dispersion of the third-order susceptibility ǀχ
(3)
(ω
2
)ǀ
2
for a dilute solution of iron(III) tetraphenylporphyrin chloride in chloroform with a fixed intense field (ω
1
) resonant with the Q
o
transition and a tunable weaker field (ω
2
) resonant with the Soret transition. The observed Lorentzian dispersion is slightly narrower than the linear absorption spectrum. The theoretical model for the third-order polarization incorporates three levels coupled to two electromagnetic fields and a population reservoir. The equations of motion for the density operator were solved in third order for the appropriate Fourier component. The resulting dispersion curve depends on both total dephasing and population decay rates, thereby demonstrating a general method for the determination of subpicosecond relaxation processes in systems to which the model is applicable. Theoretical fits to the results for iron(III) tetraphenylporphyrin chloride imply that the Soret transition is essentially homogeneously broadened with a total dephasing width of 900 ± 200 cm
-1
, and it is argued that the population decay time in this case is approximately 3.5 fsec. The broad linear absorption spectrum of the Soret band is interpreted as resulting from intramolecular perturbations between the Soret and lower energy excited states of iron(III) tetraphenylporphyrin chloride.
Semisynthesis and biological activity of porcine [LeuB24]insulin and [LeuB25]insulin.
Authors: Tager H , Thomas N , Assoian R , Rubenstein A , Saekow M , Olefsky J , Kaiser E T
Published: May 2006

Two analogs of porcine insulin with substitutions of leucine for phenylalanine in the COOH-terminal region of the insulin B chain have been prepared by a combination of solid-phase synthesis and semisynthesis. Solid-phase synthesis of the substituted octapeptides B23-B30 bearing the trifluoracetyl group on lysine-B29, enzymatic coupling of the octapeptides to bis(tertiary-butyloxycarbonyl)desoctapeptide insulin by trypsin, and deprotection of the corresponding adducts in formic acid and piperidine resulted in two insulin derivatives, one with leucine at position B24 and the other with leucine at position B25. These analogs had only about 10% and 1%, respectively, of the activity of porcine insulin in competing for the binding of [125I]iodoinsulin to both rat adipocytes and human IM-9 lymphocytes. The relative potencies of the analogs in stimulating glucose oxidation by rat adipocytes decreased in the order porcine insulin > [LeuB24]insulin > [LeuB25]insulin. However, at high concentrations both analogs had full agonists activity. Experiments in which the semisynthetic insulins were mixed with the native hormone showed that [LeuB24]insulin, but not [LeuB25]insulin, was an active antagonist of insulin action. These results suggest that the antagonistic activity of a human insulin variant having leucine at position B24 or B25 can be assigned to the molecule with the sequence Gly-Leu-Phe-Tyr (residues B23-B26) in its active site.
beta-Endorphin omission analogs: dissociation of immunoreactivity from other biological activities.
Authors: Li C H , Yamashiro D , Tseng L F , Chang W C , Ferrara P
Published: May 2006

An analog of human beta-endorphine with omission of four residues at positions 11, 14, 20, and 22 has been synthesized. This analog and other synthetic analogs with deletion of a single amino acid at position 2, 5, 6, 10, 11, 12, 13, 15, or 22 have been assayed for analgesic potency, ileal opiate activity,opiate receptor-binding activity, andimmunoreactivity. Results show that deletion of a single amino acid of the beta-endorphin molecule outside of the enkephalin segment to give des-Gln11-, des-Thr12, des-Pro13-, des-Leu14-, des-Val15-, des-Asn20-, or des-Ile22-beta-endorphin markedly reduced or abolished the immunoreactivity yet gave substantial retention of opiate potencies. Deletion of a single amino acid of beta-endorphin within the enkephalin segment (des-Gly3- or des-Met5-beta-endorphin) did not markedly affect the immunoactivity; however, the opiate activities were abolished or markedly reduced. The data indicate a clear dissociation of immunoactivity from analgesic, ileal-opiate, and opiate receptor-binding activities.
Absence of albumin mRNA in the liver of analbuminemic rats.
Authors: Esumi H , Okui M , Sato S , Sugimura T , Nagase S
Published: May 2006

Albumin synthesis in the liver of analbuminemic rats, established as a strain from a stock of Sprague-Dawley rats, was examined in vivo by labeling protein by intraperitoneal injection of L-[3H]leucine. Albumin was not synthesized in the liver of analbuminemic rats, whereas its synthesis amounted to about 14% of the total protein synthesis in the liver of normal rats. The RNA content and size distribution of the total polysomes in the liver of analbuminemic rats were not significantly different from those of normal rats. However, no functional mRNA coding for albumin was found in poly(A)-containing RNA from the liver of analbuminemic rats when tested with a cell-free translation system derived from rabbit reticulocytes. Moreover, the amount of the RNA sequence that could hybridize to purified albumin cDNA was more than 750 times greater in the liver of normal rats than in that of analbuminemic rats.
Phosphorylation and dephosphorylation of spectrin from human erythrocyte ghosts under physiological conditions: autocatalysis rather than reaction with separate kinase and phosphatase.
Authors: Imhof B A , Acha-Orbea H J , Libermann T A , Reber B F , Lanz J H , Winterhalter K H , Birchmeier W
Published: May 2006

The mechanism of phosphosylation and dephosphorylation of spectrin from human erythrocyte membranes has been examined under closely physiological conditions. The results support the hypothesis that spectrin is an autophosphorylating and dephosphorylating system. (i) Extraction from ghosts of up to 85% of the kinase (casein kinase) suggested to catalyze the reaction [see Fairbanks, G., Avruch, J., Dino, E. J. & Patel, V. P. (1978) J. Supramol. Struct. 9, 97--112] only slightly reduced spectrin component 2 phosphorylation and did not affect ATP-induced changes in the ghosts' shapes. (ii) A spectrin--actin complex isolated from endocytotic inside-out vesicles under hyperteonic conditions contained virtually no casein kinase activity and still exhibited a largely intact phosphorylation machinery. (iii) Photoaffinity labeling experiments indicated that spectrin component 2 fulfills the necessary prerequisite of the hypothesis--i.e., it contains its own ATP-binding site. (iv) Under various conditions, spectrin phosphorylation and dephospohrylation seem to be tightly coupled. The implications of these findings for the understanding of spectrin function and the maintenance of erythrocyte shape are discussed.
pH-dependent fusion between the Semliki Forest virus membrane and liposomes.
Authors: White J , Helenius A
Published: May 2006

Semliki Forest virus was mixed with liposomes containing phosphatidylcholine,phosphatidylethanolamine, sphingomyelin, and cholesterol. When the pH of the mixture was dropped to 6 or below, rapid fusion between the membranes of the virus and the liposomes occurred, resulting in the transfer of viral nucleocapsids into the liposomes. Fusion was demonstrated biochemically by trapping RNase or trypsin within the liposomes. Trapped RNase digested the viral RNA into acid-soluble form, providing a simple quantitative assay for fusion. Trapped trypsin digested the viral capsid protein. Fusion was also demonstrated by electron microscopy as the formation of large vesicles containing viral glycoproteins on the surface and nucleocapsids inside. The efficiency of fusion was 91 +/- 6%. In addition to low pH, it required that the viral glycoproteins be intact. In the target liposomes, cholesterol (but none of the individual phospholipids) was essential. Divalent cations were not required. Our previous studies with tissue culture cells indicated that the final step in the penetration of the Semliki Forest virus genome into host cells might involve a fusion event between the membrane of lysosomally trapped viruses and the lysosomal membrane [Helenius, A., Kartenbeck, J., Simons, K. & Fries, E. (1980) J. Cell Biol, 84, 404--420]. The data presented here are fully compatible with this hypothesis.
Coding capacity of a 35 percent fragment of the polyoma virus genome is sufficient to initiate and maintain cellular transformation.
Authors: Novak U , Dilworth S M , Griffin B E
Published: May 2006

Rat-1 cells were transfected with the restriction enzyme fragment of polyoma virus DNA that extends clockwise from the Bcl I site ((65.4 map units) to the EcoRI site (0/100 map units). Six transformed cell lines were obtained and one of them (BE-1) has been investigated in detail. The viral DNA that is integrated into host DNA in this line appeared to consist of two fragments arranged in a "head-to-tail" tandem with no detectable intervening host sequences. BE-1 cells contained polyoma virus small and middle tumor antigens that were indistinguishable from the corresponding tumor antigens from lytically infected cells. No large tumor antigen was detected but a "new" Mr 34,000 protein, which proved to be a truncated version of large tumor antigen, was immunoprecipitated by anti-tumor-antigen antiserum. After injection of 10(6) BE-1 cells into young syngeneic Fischer rats, tumors appeared within 3--4 weeks. Thus, the coding capacity of the Bcl I/EcoRI fragment of polyoma virus DNA is sufficient to enable the cells to produce all of small and middle tumor antigens and about a third of large tumor antigen, to transform cells stably in culture, and to produce tumors in vivo.
The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.
Authors: Ogden S , Haggerty D , Stoner C M , Kolodrubetz D , Schleif R
Published: May 2006

The locations of DNA binding by the proteins involved with positive and negative regulation of transcription initiation of the L-arabinose operon in Escherichia coli have been determined by the DNase I protection method. Two cyclic AMP receptor protein sites were found, at positions -78 to -107 and -121 to -146, an araC protein--arabinose binding site was found at position -40 to -78, and an araC protein-fucose binding site was found at position -106 to -144. These locations, combined with in vivo data on induction of the two divergently oriented arabinose promoters, suggest the following regulatory mechanism: induction of the araBAD operon occurs when cyclic AMP receptor protein, araC protein, and RNA polymerase are all present and able to bind to DNA. Negative regulation is accomplished by the repressing form of araC protein binding to a site in the regulatory region such that it stimultaneously blocks access of cyclic AMP receptor protein to two sites on the DNA, one site of which serves each of the two promoters. Thus, from a single operator site, the negative regulator represses the two outwardly oriented ara promoters. This regulatory mechanism explains the known positive and negative regulatory properties of the ara promoters.
A proton-coupled conformational switch of Escherichia coli 5S ribosomal RNA.
Authors: Kao T H , Crothers D M
Published: May 2006

We report temperature-jump kinetic studies of the early melting transition of Escherichia coli 5S rRNA. A single measurable relaxation time tau, independent of concentration, was found at 266 nm. We monitored the transition temperature tm for this process (in the range from 0 to 40 degrees C) as a function of Mg2+, Na+, K+, spermidine, and H+ concentrations. Contrary to the usual effect of salts on nucleic acid stability, addition of mono- and multivalent counterions decreases tm for the early melting transition. Also unexpectedly, we found a strong dependence of tm on pH in the physiological range of 7--8. Quantitative analysis of the data indicates that about 0.7 protons are release when the ordered (low-temperature) form melts, whereas about 2 NA+ (or K+) and 0.5 Mg2+ are taken up by the melted (high-temperature) form. We estimate the enthalpy of the transition to be 15--20 kcal/mol (63--84 kJ/mol) and also report the forward and reverse rate constants and activation energies for the transition, along with the influence of ions on the transition dynamics. Diffusion constant measurements reveal that the low-temperature form has a frictional coefficient about 10% larger than that of the high-temperature form. The data imply a low-temperature tertiary structure capable of binding a proton. Increase of pH, temperature, or counterion concentration (all at near-physiological values) causes a tertiary conformational switch to a more compact form that has greater counterion binding but less proton binding. We discuss possible physiological roles for the transition.
Sequential folding of a bifunctional allosteric protein.
Authors: Garel J R , Dautry-Varsat A
Published: May 2006

Aspartokinase I-homoserine dehydrogenase I (EC 2.7.2.4 and EC 1.1.1.3) a bifunctional and allosteric enzyme, has been renatured from its unfolded and separated polypeptide chains. Folding was measured by the reappearance of each of the two enzymatic activities, kinase and dehydrogenase, and of their allosteric inhibition by the same effector, threonine. The various observed properties yield different kinetics of folding, which shows the presence of intermediates having only some of the functional features of the native enzyme. Apparently, three successive steps can be detected during the folding of aspartokinase I-homoserine dehydrogenase I: first, a monomolecular step leads to a monomeric species with the kinase activity; then an association step leads to a dimeric species with the kinase and dehydrogenase activities, and a threonine-sensitive dehydrogenase; finally, a second association step leads to a tetrameric species with the two activities, both sensitive to threonine. The folding of this large protein appears as a sequential process during which the functional properties are regained successively, as the protein structure becomes more complex. During this process, the two regions of each polypeptide chain respectively responsible for the kinase and dehydrogenase activities seem to acquire their native conformation rather independently of each other.
Serum factors modify the cellular requirement for Ca2+, K+, Mg2+, phosphate ions, and 2-oxocarboxylic acids for multiplication of normal human fibroblasts.
Authors: McKeehan W L , McKeehan K A
Published: May 2006

The rate of multiplication of a population of cultured human lung fibroblasts is determined by the level of common nutrients and serum factors in the medium. By application of the principles of Henri--Michaelis--Menten kinetic analysis to cell growth in vitro, the relationship between the level of a macromolecular fraction of serum that contains growth factors and the concentration of individual nutrient that is required to support a half-maximal rate of cell multiplication was explored. The requirement for the majority of individual nutrients is independent of the level of serum factors in the medium. This includes all 20 amino acids, glucose, purines, pyrimidines, polyamines, choline, and inositol. In contrast, serum factors determine the cellular requirement for Ca2+, K+, Mg2+, Pi, and 2-oxocarboxylic acids. These nutrients are most likely involved in cellular processes related to the mechanism by which growth factors in serum control the cell multiplication rate.
Interaction of pergolide with central dopaminergic receptors.
Authors: Goldstein M , Lieberman A , Lew J Y , Asano T , Rosenfeld M R , Makman M H
Published: May 2006

The activity of pergolide, an N-propylergoline derivative, has been tested for stimulation of central dopaminergic receptors. Binding to dopamine receptors shows that pergolide acts as an agonist with respect to these receptors. GTP decreases the potencies of dopamine agonists and of pergolide, but not of bromocriptine, to displace [3H]spiroperidol ([3H]Spi) from striatal membrane sites. The GTP-sensitive site labeled by [3H]Spi seems to be localized on intrastriatal dopamine receptors. The potency of dopamine agonists and of pergolide to displace [3H]Spi from striatal receptor sites is reduced in membranes exposed to higher temperatures. Pergolide, but not hitherto-tested dopaminergic ergots, stimulates dopamine-sensitive adenylate cyclase in striatal homogenates. Thus, pergolide, unlike other dopaminergic ergots, acts as an agonist on GTP-sensitive components of [3H]Spi binding and stimulates dopamine receptors linked to dopamine-sensitive adenylate cyclase. The drug also induces turning behavior in rats with 6-OH-dopamine lesions and relieves tremor in monkeys with ventromedial tegmental lesions for a longer time at a lower dose than other tested dopaminergic ergots. Other studies have shown that it is effective in the treatment of patients with advanced parkinsonism.
Comparative sequence analysis of the inverted terminal repetitions from different adenoviruses.
Authors: Shinagawa M , Padmanabhan R
Published: May 2006

The comparative nucleotide sequences of the region of inverted terminal repetition from a representative member of group C (nononcogenic), group B (weakly oncogenic), and group A (highly oncogenic) adenoviruses are analyzed. Our data show that (i) the length of this unique region increases with the oncogenicity of the serotype, (ii) a unique homologous region--14 nucleotides long--is present exactly at the same distance from the terminus, and (iii) a hexanucleotide sequence, T-G-A-C-G-T, is present at the site where the terminal repetition diverges.