bluuebunny/crossref_metadata_2025 huggingface dataset:
Intradendritic recordings from hippocampal neurons.
Authors: Wong R K , Prince D A , Basbaum A I
Published: May 2006

Dendritic activity in guinea pig hippocampal CA1 and CA3 pyramidal neurons was examined by using an in vitro preparation. Histologically confirmed intradendritic recordings showed that dendrites had an average input resistance of 47.0 M omega and average membrane time constant of 33.3 msec. Active spike responses could be evoked by intracellular injection of outward current or by the activation of synaptic inputs. The predominant activity was burst firing. A typical intracellularly recorded dendritic burst consisted o spikes on a slowly increasing depolarizing potential. The spike components of the burst were of two distinct types: low threshold, fast spikes; and high threshold, slow spikes. Tetrodotoxin (1 microgram/ml) blocked the fast spikes, but slow spikes could still be evoked with direct intracellular stimulation. In contrast to dendritic responses, direct depolarization of CA1 somata did not give rise to burst generation. Orthodromic stimuli evoked large-amplitude excitatory postsynaptic potentials, followed by inhibitory postsynaptic potentials in dendrites of CA1 and CA3 neurons. In two instances, simultaneous recordings were obtained from coupled pairs of elements that were presumed to be soma and dendrite of the same CA3 pyramidal neuron. Depolarization of either element led to burst generation at that site, and the underlying slow depolarization appeared to evoke a burst at the other site. This potential postsynaptic amplifying mecahnism was not ordinarily functional because even suprathreshold orthodromic activation did not normally evoke bursting in dendrites.
Human platelet membrane receptor for bovine von Willebrand factor (platelet aggregating factor): An integral membrane glycoprotein
Authors: Cooper Herbert A. , Clemetson Kenneth J. , Lüscher Ernst F.
Published: May 2006

The platelet membrane receptor for bovine von Willebrand factor, platelet aggregating factor, has been reported to be a property of a soluble glycoprotein, glycocalicin, that is loosely attached to the platelet surface and represents one of the major glycoproteins of the platelet glycocalyx. The studies reported here, however, demonstrate that fractions from human platelets containing glycocalicin have no bovine von Willebrand factor receptor activity. Instead, only fractions containing platelet membranes have receptor activity. By using a nonionic detergent, Brij 99, active receptor can be solubilized from the membrane. Some quantitation of the intact or solubilized receptor activity is possible because the aggregation curves produced by mixtures of various dilutions of membranes and a constant concentration of standard normal bovine plasma are linear when plotted against the logarithm of the concentration of receptor. The dose—response curve obtained with Brij 99-solubilized membranes is not parallel to that obtained with intact membranes. Lectin-specificity studies of the bovine von Willebrand factor receptor, soluble in Brij 99, demonstrate binding to a wheat germ agglutinin-Sepharose 4B affinity gel but little or no binding to similar affinity gels of concanavalin A or
Lens culinaris
lectin. By using wheat germ agglutinin-Sepharose 4B as a lectin affinity column, partial purification of the receptor is possible. Stability studies of the receptor in intact membranes show essentially no loss of activity for at least 6 days when membranes are stored at 4°C in buffers containing 1 mM EDTA. One freezing and thawing cycle results in minimal loss of initial activity but the receptor activity of the thawed material is less stable over time than is fresh material. Repeated freezing and thawing destroys the activity and, once lost, it can not be recovered, even with detergents.
Isolation and characterization of purine-nucleoside phosphorylase-deficient T-lymphoma cells and secondary mutants with altered ribonucleotide reductase: Genetic model for immunodeficiency disease
Authors: Ullman Buddy , Gudas Lorraine J. , Clift Shirley M. , Martin David W.
Published: May 2006

The inherited deficiency of purine-nucleoside phosphorylase (PNPase; purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) in humans is associated with a severe deficiency of the T lymphocytes of the immune system. Because of the unsatisfactory nature of previously described model systems, we have selected, cloned, and characterized a mutant mouse T cell lymphoma (S49) completely deficient in PNPase. Of the four substrates of PNPase, only deoxyguanosine at low concentrations is toxic to the PNPase-deficient (NSU-1) cells.

In order to delineate the biochemical processes necessary for the sensitivity of the NSU-1 cells to deoxyguanosine, we have isolated a series of secondary mutants resistant to deoxyguanosine from the PNPase-deficient line. One of these mutants is defective in its ability to transport deoxyguanosine into the cell. A second type of mutant cannot phosphorylate the deoxyguanosine and is totally deficient in deoxycytidine kinase activity. A third type of mutant (NSU-1-dGuo-L) can both transport and phosphorylate deoxyguanosine and accumulates dGTP. However, unlike its parent, NSU-1-dGuo-L does not become depleted of dCTP and TTP when exposed to exogenous deoxyguanosine. This observation is accounted for by the fact that the reduction of CDP to dCDP by the ribonucleotide reductase (ribonucleoside-diphosphate reductase, 2′-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2′-oxidoreductase, EC 1.17.4.1) of NSU-1-dGuo-L cells is not normally sensitive to feedback inhibition by dGTP.

Thus, in order to exert its toxicity deoxyguanosine must be transported into the cell, be phosphorylated by deoxycytidine kinase, and be accumulated as dGTP. By inhibiting ribonucleotide reductase, dGTP depletes the cell of dCTP and to some extent TTP, thus preventing the synthesis of DNA, a process necessary for any proliferation-dependent function of T cells.
Prostaglandin synthesis in isolated rat kidney glomeruli.
Authors: Hassid A , Konieczkowski M , Dunn M J
Published: May 2006

Isolated rat kidney glomeruli converted octatritiated arachidonic acid to several prostaglandins whose production was inhibited by meclofenamate. These were, in order of decreasing abundance, prostaglandin F2 alpha, prostaglandin E2, 6-oxo-prostaglandin F1 alpha, thromboxane B2, and prostaglandin D2. These products were identified by thin-layer chromatography, before and after treatment with potassium hydroxide or sodium borohydride. Prostaglandins F2 alpha and E2 were also determined by radioimmunoassay. The major product made by glomeruli was an unidentified substance(s), whose appearance was partially inhibited by meclofenamate, and was likely to be a hydroxylated fatty acid(s). The specific activity of glomerular fatty acid cyclo-oxygenase (EC 1.14.99.1), based on radioimmunoassay for prostaglandins E2 and F2 alpha, was 10- to 40-fold higher than that of cortical tubular enzyme. These data demonstrate that glomeruli have the capability of synthesizing an array of end-products from arachidonic acid. These prostaglandins may exert important physiologic effects, because renin secretion and arteriolar resistance are regulated by the glomerulus and the afferent and efferent arterioles.
Bacteriophage f1 infection of Escherichia coli: identification and possible processing of f1-specific mRNAs in vivo.
Authors: Cashman J S , Webster R E
Published: May 2006

[3H]Uracil-pulse-labeled RNA from Escherichia coli infected with f1 bacteriophage was fractionated on polyacrylamide gels containing urea. Eight phage-specific RNA species were present with approximate lengths ranging from 2100 to 400 nucleotides. The amount of the seven largest species was increased when the infected bacteria were incubated at 41 degrees C. When the RNA was isolated and used as message in an in vitro protein-synthesizing system, most of the RNA species appeared to direct the synthesis of the phage gene VIII protein. The six largest species also directed the synthesis of the phage gene V protein. Some of the labeled smaller RNA species increased in amount after addition to rifampicin, suggesting that they may have resulted from cleavage of larger RNA species. These particular smaller RNA species also were present in infected bacteria containing a mutant RNase III. The data are discussed in terms of the regulation of synthesis of the phage-specific proteins.
Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea.
Authors: Ohlrogge J B , Kuhn D N , Stumpf P K
Published: May 2006

This communication demonstrates that all de novo fatty acid biosynthesis in spinach leaf cells requires acyl carrier protein (ACP) and occurs specifically in the chloroplasts. Antibodies raised to purified spinach ACP inhibited at least 98% of malonyl CoA-dependent fatty acid synthesis by spinach leaf homogenates. Therefore, the presence of ACP in a compartment of the spinach leaf cell would serve as a marker for de novo fatty acid biosynthesis. A radioimmunoassay capable of detecting 10(15) mol (10(-11) g) of spinach ACP was developed to measure the levels of ACP in leaf cell components isolated by sucrose gradient centrifugation of a gentle lysate of spinach leaf protoplasts. All of the ACP of the leaf cell could be attributed to the chloroplast. Less than 1% of the ACP associated with chloroplasts resulted from binding of free ACP to chloroplasts. Of interest, ACP from Escherichia coli, soybean, and sunflower showed only partial crossreactivity with spinach ACP by the radioimmunoassay. These results strongly suggest that, in the leaf cell, chloroplasts are the sole site for the de novo synthesis of C16 and C18 fatty acids. These fatty acids are then transported into the cytoplasm for further modification and are either inserted into extrachloroplastic membrane lipids or returned to the chloroplast for insertion into lamellar membrane lipids.
Replication in vitro of the 2-micrometer DNA plasmid of yeast.
Authors: Jazwinski S M , Edelman G M
Published: May 2006

Cell-free extracts prepared from growing cells of the budding yeast Saccharomyces stimulated DNA synthesis directed by the supercoiled 2-micrometer yeast DNA plasmid. The major products of the reaction were open-circular daughter molecules possessing newly synthesized full-length linear DNA strands. Some of these were ligated and supertwisted by the extracts to yield a supercoiled DNA product. Both of the complementary DNA strands of the template were replicated. In addition, the extracts induced the appearance of theta-forms of the plasmid DNA, which are presumed to be replicative intermediates. The results of experiments utilizing BrdUTP incorporation indicated that DNA repair did not contribute significantly to the overall reaction. Extracts prepared from the cell division cycle mutants cdc7 and cdc8, held in culture at the nonpermissive temperature, possessed diminished activity. Because these mutants define a dependent sequence of events leading from the start of the cell cycle through G1 to S phase, this result suggests that the activity that stimulates 2-micrometer DNA replication in vitro is subject to control in the yeast cell cycle.
Higher plant chloroplasts: Evidence that all the chlorophyll exists as chlorophyll—protein complexes
Authors: Markwell John P. , Thornber J. Philip , Boggs Russell T.
Published: May 2006

By using the polyacrylamide gel electrophoresis system described in this report, it was possible to fractionate all the photosynthetic pigments of maize (
Zea mays
L.) thylakoids into chlorophyll—protein complexes with negligible formation of free or detergent-complexed chlorophyll. Identical sodium dodecyl sulfate extracts of thylakoids have previously resulted in up to 50% of the chlorophyll migrating as free chlorophyll after electrophoresis. The major difference from previous gel electrophoresis systems is the replacement of sodium dodecyl sulfate in the electrophoresis buffer by Deriphat 160 (disodium
N
-lauryl-β-iminodipropionate), a zwitterionic detergent. The results suggest that: (
i
) no significant amount of free chlorophyll exists in the chloroplast thylakoid membranes
in vivo
, and (
ii
) most of the free pigment seen previously on gels was generated during the electrophoresis and was not a result of the solubilization technique. Additionally, the new chlorophyll-protein complexes resolved appear to have different characteristics (pigment content and size) that those observed in former systems.
Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro.
Authors: Bell E , Ivarsson B , Merrill C
Published: May 2006

Fibroblasts can condense a hydrated collagen lattice to a tissue-like structure 1/28th the area of the starting gel in 24 hr. The rate of the process can be regulated by varying the protein content of the lattice, the cell number, or the concentration of an inhibitor such as Colcemid. Fibroblasts of high population doubling level propagated in vitro, which have left the cell cycle, can carry out the contraction at least as efficiently as cycling cells. The potential uses of the system as an immunologically tolerated "tissue" for wound healing and as a model for studying fibroblast function are discussed.
Human melanoma cells have both nerve growth factor and nerve growth factor-specific receptors on their cell surfaces.
Authors: Sherwin S A , Sliski A H , Todaro G J
Published: May 2006

Human melanoma cells were examined in an indirect membrane immunofluorescence assay for surface nerve growth factor (NGF) and NGF receptors. This assay revealed that human melanoma cells have various levels of NGF and NGF receptors on the plasma membrane, whereas a variety of human sarcoma and carcinoma tumor cells and normal human fibroblasts are negative. Surface NGF could be detected on melanoma cells with a rabbit antiserum directed to NGF at titers as high as 1:64; prior adsorption of this antibody with mouse 2.5S NGF resulted in a loss of fluorescence. The melanoma cells were positive whether or not they were grown in the presence of fetal calf serum. NGF production by human melanomas is a previously unrecognized property of this differentiated cell type. Although other cells in culture have been shown to produce NGF, the association of NGF production with the presence of NGF receptors on the cell surface is rare among tumor cells, and may represent an opportunity for "autostimulation" of melanoma cells by this growth factor.
Changes in the synthesis of actin and other cell proteins after stimulation of serum-arrested cells.
Authors: Riddle V G , Dubrow R , Pardee A B
Published: May 2006

The synthesis of both cytoplasmic and nuclear proteins has been studied as quiescent, serum-deprived Swiss mouse 3T3 cells are stimulated to transit the cell cycle. In serum-arrested cells a 200,000 dalton cytoplasmic protein and a 51,000 dalton nuclear protein were found to be preferentially synthesized. In serum-stimulated cells the first major protein whose synthesis was seen to increase had a molecular mass of 42,000 daltons. This protein also showed the greatest change in synthesis during the transit from G0 to S phase. Its synthesis rose to a maximum 4--6 hr after stimulation and then declined as cells entered S phase. The protein was present in both nuclear and cytoplasmic extracts. It was identified as actin on the basis of its mobility on sodium dodecyl sulfate and isoelectric focusing polyacrylamide gels. Other proteins synthesized preferentially by stimulated cells had molecular masses of 57,000 daltons (cytoplasmic), 33,000 daltons (cytoplasmic and nuclear), and 15,000 daltons (nuclear). The synthesis of the 57,000 and 33,000 dalton proteins increased gradually after stimulation and remained high during S phase. The 15,000 dalton proteins began to be synthesized as cells entered S phase. The preferential synthesis of these proteins provides biochemical markers for the transition from quiescence to proliferation.
Analysis of the constancy of DNA sequences during development and evolution of the nematode Caenorhabditis elegans.
Authors: Emmons S W , Klass M R , Hirsh D
Published: May 2006

In order to test for the occurrence of rearrangements in DNA during development and to assess the rate of DNA divergence during evolution, we have compared restriction fragments derived from DNA from four sources: sperm cells and somatic tissues of one strain of the nematode Caenorhabditis elegans, somatic tissues of a second strain of the same species, and whole animals of a closely related species. Restriction fragments were detected by hybridizing radioactive cloned fragments to restriction digests that had been fractionated by size on agarose gels and transferred to nitrocellulose sheets. In this way, approximately 50 BamHI restriction fragments were visualized and compared. Fragments from sperm and somatic DNAs were found to be identical; 15% differed in size between the two strains. Little cross homology was found between the two species. We conclude that, if rearrangements occur in C. elegans DNA during development, they must affect fewer than a few percent of the restriction fragments or restriction sites. The difference found between the two strains and the two species is surprisingly great.
Biochemical characterization of alien H-2 antigens expressed on a methylcholanthrene-induced tumor.
Authors: Rogers M J , Appella E , Pierotti M A , Invernizzi G , Parmiani G
Published: May 2006

A methylcholanthrene-induced tumor of BALB/c (H-2d) origin had been shown to express H-2 antigens normally associated with the H-2k haplotype. The molecules expressing the alien antigens have been partially purified and shown to be distinct from molecules expressing normal H-2d antigens. Like normal H-2 antigens, the alien antigens have a molecular weight of 48,000, are glycoproteins, and are noncovalently bound to beta 2-microglobulin. The alien antigens differ from normal H-2 antigens in their susceptibility to papain digestion, behavior during gel filtration, and overall stability. Possible mechanisms accounting for the expression of the alien antigen are discussed.
Influence of major histocompatibility haplotype on autoimmune disease varies in different inbred families of chickens.
Authors: Bacon L D , Rose N R
Published: May 2006

Three partially inbred substrains of Obese strain chickens were studied for the spontaneous development of autoimmune thyroiditis. The influence of the major histocompatibility complex (B haplotype) was marked in one, less marked but still significant in a second, and barely detectable and transient in a third substrain. These differences in the effect of B haplotype may be due to the overriding action of genes other than those within the B haplotype.
Bradykinin-like immunoreactive neuronal systems localized histochemically in rat brain.
Authors: Corrêa F M , Innis R B , Uhl G R , Snyder S H
Published: May 2006

Bradykinin-like immunoreactive structures were localized in rat brain by the indirect immunofluorescence method. Specificity of staining was demonstrated by: (i) the absence of fluorescence when preimmune serum was used, (ii) the disappearance of fluorescence when sera were preadsorbed with bradykinin, and (iii) the presence of identical staining with two different antisera. Immunoreactive neuronal cells are observed only in the hypothalamus, with especially dense clusters overlying the periventricular and dorsomedial nuclei. Fibers and varicose processes are observed in the periaqueductal gray matter, hypothalamus, perirhinal and cingulate cortices, the ventral portion of caudate-putamen, and the lateral septal area.
Detection and accumulation of tetrahedral intermediates in elastase catalysis.
Authors: Fink A L , Meehan P
Published: May 2006

Tetrahedral intermediates in the reaction of elastase with specific di- and tripeptide p-nitroanilide substrates have been detected, accumulated, and stabilized at high pH by using subzero temperatures and fluid aqueous/organic cryosolvents. The tetrahedral adducts are characterized by spectra with lambda max of 359 +/- 2 nm, compared with thata of 380 nm for p-nitroaniline and 315-320 nm for the substrates. The maximal concentration of intermediate that could be accumulated varied with the different substrates from 40 to 100% of the active enzyme present. The pH dependence of the reactions indicated that formation of the tetrahedral intermediates was rate-limiting at low pH (pK* = 7.0 at -39 degrees C) and that collapse to the acylenzymes was rate-determining at high pH. When corrected for the effect of temperature and cosolvent, the rate of intermediate formation was in good agreement with that measured at 25 degrees C in aqueous solution by stopped-flow techniques.
The lambda repressor contains two domains.
Authors: Pabo C O , Sauer R T , Sturtevant J M , Ptashne M
Published: May 2006

Papain digestion of the lambda phage repressor produces two fragments that are relatively resistant to further digestion. One includes the amino terminus (residues 1-92) and the other the carboxyl terminus (residues 132-236). Calorimetry shows that the amino-terminal fragment denatures near 50 degrees C and that the carboxyl-terminal fragment denatures near 70 degrees C. Intact repressor undergoes two denaturations, one near 50 degrees C and another near 70 degrees C. These and other data show that lambda repressor consists of two domains joined by a "connector" 40 amino acids long that is sensitive to proteases. The amino-terminal domain binds DNA, and the carboxyl-terminal domain oligomerizes.
Nucleotide sequence of the ribosomal protein gene cluster adjacent to the gene for RNA polymerase subunit beta in Escherichia coli.
Authors: Post L E , Strycharz G D , Nomura M , Lewis H , Dennis P P
Published: May 2006

The 3072-nucleotide-long sequence of a segment from the 88-min region of the Escherichia coli chromosome has been determined. The sequence covers the genes for ribosomal proteins L11 (rplK), LI (rplA), L10 (rplJ), and L7/L12 ((rplL), and the 5' end of the gene for the beta subunit of RNA polymerase (rpoB), along with the presumed regulatory regions for these genes. The probable locations of the promoter for the first two genes (the L11 operon) and the promoter for the latter three genes (the proximal part of the beta operon) have been identified. We have also found that the four ribosomal protein genes preferentially use codons that are recognized efficiently by the most abundant tRNA species. These and other features of the sequence results are discussed in relation to available information obtained from both in vitro and in vivo experiments on the expression of these ribosomal and RNA polymerase subunit genes.
Purification to homogeneity of camel pituitary pro-opiocortin, the common precursor of opioid peptides and corticotropin.
Authors: Kimura S , Lewis R V , Gerber L D , Brink L , Rubinstein M , Stein S , Udenfriend S
Published: May 2006

Pro-opiocortin was purified from camel pituitaries by procedures including high-performance liquid chromatography. The precursor relationship of the pure protein to the opioid peptides and to corticotropin was confirmed. Partial chemical analysis consisting of amino acid analysis and tryptic peptide mapping was carried out with the aid of sensitive fluorescence detection.
Detection and partial sequence analysis of gastrin mRNA by using an oligodeoxynucleotide probe.
Authors: Noyes B E , Mevarech M , Stein R , Agarwal K L
Published: May 2006

We have used a specific deoxyoligonucleotide probe to detect gastrin mRNA in poly(A)-enriched RNA preparations from hog antrum. The nucleotide sequence of the oligonucleotide, d(C-T-C-C-T-C-C-A-T-C-C-A), was deduced from the unique amino acid sequence Trp-Met-Glu-Glu of gastrin. When used with hog antral RNA, the dodecanucleotide is an effective primer for the synthesis of gastrin-specific cDNA as judged by nucleotide sequence analysis of cDNA isolated by polyacrylamide gel electrophoresis. We have determined an 81-nucleotide sequence corresponding to the region of the gastrin mRNA that codes for the known amino acid sequence of the G34 progastrin intermediate species, and we have demonstrated the presence of two consecutive basic residues preceding the G34 sequence in the prohormone. Hybridization of gastrin cDNA or synthetic dodecanucleotide to hog antral RNA separated by gel electrophoresis on agarose gels in the presence of methylmercuric hydroxide indicates that the mRNA coding for gastrin is about 620 nucleotides long. These results suggest that the gastrin precursor peptide contains 110-140 amino acids. This method should be of general application for detection and characterization of mRNAs corresponding to proteins of known amino acid sequence.
Surface mapping of mouse thymocytes.
Authors: Flaherty L , Zimmerman D
Published: May 2006

The blocking method used previously for determining the relative positions of different components of the cell surface was modified by first fixing the cells with paraformaldehyde. This technique was applied to the H-2K (K), H-2D (D), TL, Lyt-1, and Lyt-2 surface components of mouse thymocytes, and the results were compared in parallel with data obtained with the original technique with unfixed cells. Previous mapping data with unfixed cells, indicating the positions of these molecules relative to one another, were confirmed with paraformaldehyde-fixed cells, with one exception. On unfixed cells, D and TL appeared sufficiently adjacent to produce mutual interference in the attachment of anti-D and anti-TL antibodies. With paraformaldehyde-fixed cells this was not so, D and TL appearing sufficiently separated from one another to obviate interference in the attachment of anti-D and anti-TL antibodies. The previously reported close association of K with Lyt-1 and of D with Lyt-2 were demonstrable equally with unfixed and paraformaldehyde-fixed thymocytes. It is suggested that activation of D sites, and alternatively of TL sites, by antibody in the present experiments brings these two molecules into apposition and that this movement may exemplify a mechanism concerned in immunological recognition and response.
Immunocytochemical localization of γ-aminobutyric acid transaminase at cellular and ultrastructural levels
Authors: Chan-Palay V. , Wu J.-Y. , Palay S. L.
Published: May 2006

γ-Aminobutyric acid transaminase (GABA-Tase; 4-aminobutyrate:2-oxaglutarate aminotransferase, EC 2.6.1.19) immunoreactivity in the rat's cerebellum was studied by light and electron microscopy with indirect immunofluorescence and peroxidase-antiperoxidase methods. Evidence is presented for neuronal and neuroglial compartments of GABA-Tase. Labeled neurons included stellate, basket, Purkinje, and Golgi cells of the cortex and a few large neurons in the deep nuclei. Labeled neuroglia included those surrounding Purkinje cells, their radial fibers in the molecular layer, and astrocytes in the granular layer and deep nuclei. No evidence for sagittal microzonation was found. At the ultrastructural level, GABA-Tase immunoreactive sites were localized to cell surface membranes, intracellular organelles, and the cytoplasmic matrix. GABA-Tase immunoreactivity at synapses could be localized precisely to pre- and postsynaptic membranes in γ-aminobutyric acid (GABA)-containing as well as non-GABA-containing neurons. Specific label was absent from tissues treated with normal rabbit preimmune sera. GABA-Tase labeling was more intense in tissues from animals anesthetized with ether than with barbiturates and after formaldehyde fixation without glutaraldehyde. Increased GABA-Tase immunoreactivity was observed on treatment with colchicine, GABA with oxamic acid, GABA, harmaline, norepinephrine and glutamate, or diazepam (in order of decreasing effectiveness). Serotonin produced no detectable change, and apomorphine and muscimol decreased the immunoreactivity.
H
+
/site, charge/site, and ATP/site ratios in mitochondrial electron transport
Authors: Pozzan T. , Virgilio F. Di , Bragadin M. , Miconi V. , Azzone G. F.
Published: May 2006

H
+
/site, charge/site, and ATP/site ratios have been determined at coupling sites I, II, and III. Three
e
-
donors have been used for coupling site III: ferrocyanide, ascorbate + tetramethyl-
p
-phenylenediamine (TMPD), and succinate + TMPD. The H
+
/site ratios are 4.0 with ferrocyanide and 6.0 with succinate + TMPD (at pH <7.0); the charge/site ratios are 6.0 with ferrocyanide and with succinate + TMPD (at pH <7.0) and 4.0 with ascorbate + TMPD; the ATP/site ratio is 1.34 with ascorbate + ferrocyanide. These ratios have been obtained in the presence of amounts of antimycin A that provide full inhibition of site II. For coupling sites I and II, ferricyanide has been used as
e
-
acceptor and succinate or NAD-linked substrates as
e
-
donors. The H
+
/site ratios are 4.0 at sites I and II; the charge/site ratios are 4.0 at site I and 2.0 at site II; the ATP/site ratios are 1.0 at site I and 0.5 at site II. Two major factors affect the stoichiometries: (
i
) dimension of [unk]

H

and (
ii
) supply of H
+
from the matrix. There is a correlation between collapse of [unk]

H

and increase of H
+
/site and charge/site ratios. This indicates that approximation of the phenomenologic stoichiometry of the H
+
pump is obtained when flow ratios are measured at level flow. That charge/site and ATP/site ratios increase when ferrocyanide is
e
-
donor and decrease when ferricyanide is
e
-
acceptor is attributed to the localization of the redox couple. This leads to separation of 1 charge/
e
-
when ferrocyanide is
e
-
donor and to consumption of 1 charge/
e
-
when ferricyanide is
e
-
acceptor. To account for an extrusion of H
+
in excess of that predicted by the loop model, it is proposed that each coupling site contains a channel acting as a H
+
pump.
Corticotropin and beta-endorphin: construction and analysis of recombinant DNA complementary to mRNA for the common precursor.
Authors: Roberts J L , Seeburg P H , Shine J , Herbert E , Baxter J D , Goodman H M
Published: May 2006

A cDNA fragment synthesized from mouse mRNA (ACTH/LPH mRNA) that codes for the precursor polypeptide containing corticotropin (ACTH), beta-lipotropin (LPH), and several other peptides has been cloned in bacteria. The mRNA was enriched for ACTH/LPH mRNA translational activity (to about 75%) prior to cDNA synthesis. It appears to contain about 1200 bases, of which approximately 450 bases are not translated. The cloned DNA fragment is complementary to the region of the mRNA coding for the protein fragment beta-LPH-(44--90); this contains all of the amino acids of [Met]-enkephalin (residues 61--65 of beta-LPH), most of the amino acids of beta-melanocyte-stimulating hormone, and all but the carboxy-terminal amino acid of beta-endorphin. Based on assignment of the amino acid sequence of mouse beta-LPH from the nucelic acid sequence, it appears that there is extensive homology of mouse beta-LPH with human and porcine beta-LPH. The data also establish the linkage between beta-melanocyte-stimulating hormone and beta-endorphin as a Lys-Arg sequence. It is hoped that this cloned DNA can be used as a probe to study the expression and structure of the ACTH/LPH gene.
Histone 1 is proximal to histone 2A and to A24.
Authors: Bonner W M , Stedman J D
Published: May 2006

Water-soluble carbodiimide crosslinks histones 1A and 1B to histone 2A and to semi-histone A24 in chromatin from mouse cells. The identities of the histone species present in the crosslinked dimers were determined by fingerprinting. The molar ratio of H1--A24 to H2A is the same as the molar ratio of A24 to H2A in these cells. The H1-H2A crosslinks form equally well in whole nuclei, lysed nuclei, and H1-containing mononucleosomes isolated from a sucrose gradient. These results suggest that there exist major H1 interactions within the nucleosome.
Inhibition of human neuroblastoma DNA polymerase activities by plant lectins and toxins.
Authors: Bhattacharya P , Simet I , Basu S
Published: May 2006

The effects of concanavalin A and ricin (RCAII, Mr 65,000) on [3H]thymidine incorporation into human neuroblastoma IMR-32 DNA showed reduction of total DNA synthesis to 50% and 70% of control, respectively. Two DNA polymerase (DNA nucleotidyltransferase, EC 2.7.7.7.) activities (alpha and beta) involved in the biosynthesis in vitro of DNA were separated by sucrose density gradient centrifugation from IMR-32 cell homogenate. The DNA polymerase alpha activity was also purified by selective precipitation with polyethylene glycol (Mr 6000) followed by agarose-concanavalin A column chromatography. The activities of both DNA polymerases were examined at various concentrations of mutagenic and nonmutagenic plant agglutinins and the toxin ricin. Concanavalin A and ricin specifically inhibited DNA polymerase alpha activity (activity reduced to 19% and 10%, respectively), whereas DNA polymerase beta activity was inhibited (reduced to 16%) by red kidney bean agglutinin (PHA-P).
Reevaluation of level of genic heterozygosity in natural population of Drosophila melanogaster by two-dimensional electrophoresis.
Authors: Brown A J , Langley C H
Published: May 2006

We have found the two-dimensional electrophoretic technique of O'Farrell to be highly efficient in the detection of charge-change substitutions in a large number of proteins. We have applied this method to determine the level of heterozygosity of the most abundant proteins in Drosophila melanogaster adults from a natural population. The estimate of per-locus heterozygosity obtained from approximately 54 loci screened was 4% with 6 loci polymorphic. This is much lower than overall estimates obtained by standard gel electrophoresis but is not different from estimates for "Group I" enzymes--i.e., those utilizing a narrow spectrum of substrates of intracellular origin. We consider these data to throw open the question of the level of genetic variability in nature.
Partial deletion of beta-globin gene DNA in certain patients with beta 0-thalassemia.
Authors: Orkin S H , Old J M , Weatherall D J , Nathan D G
Published: May 2006

We have used restriction endonuclease mapping of cell DNA to investigate the structure of the beta-globin gene in beta-thalassemias. Among 17 individuals with beta +- and beta 0-thalassemia, we observed three patients of Indian origin with beta 0-thalassemia whose DNA revealed a consistent mapping abnormality. In one beta allele in each diploid cell, 0.6 kilobase of DNA was deleted from beta-specific Pst I and Bgl II restriction fragments. This deletion involved 3' beta-globin gene sequences and eliminated the EcoRI site normally present at codons 121/122, but it did not extend to the BamHI site at codons 98--100 on the 5' side of the 0.90-kilobase intervening sequence normally present in beta-globin genes. Partial beta-globin gene deletion appears, therefore, to be a primary molecular defect seen in certain patients with beta 0-thalassemia.
Immunological tolerance: High-dose antigen-induced suppressor cells from tolerant animals inactivate antigen-presenting macrophages
Authors: Segal S. , Tzehoval E. , Feldman M.
Published: May 2006

Studies were carried out to characterize the target cell for the activity of suppressor cells induced in highzone tolerance to deaggregated human gamma globulin (HGG). We applied an
in vitro
system for the initiation of an immune response, consisting of culturing spleen lymphocytes on HGG-fed macrophages, in which initiator T cells are generated. These cells, when injected into the foot pads of syngeneic mice, recruit specific anti-HGG effector T lymphocytes. We found that HGG-fed macrophages were incapable of signaling spleen cells from HGG-tolerant animals to generate initiator cells. Spleen cells from tolerant animals, when mixed with spleen cells from normal donors, inhibited the capacity of the normal population to give rise to initiator cells after culture on HGG-fed macrophages. Thus, suppressor T cells, which inhibit education of T cells by antigen-fed macrophages, exist in the tolerant spleen. Spleen cells from HGG-tolerant animals, when seeded on macrophages fed simultaneously with HGG and keyhole limpet hemocyanin (KLH), also prevented the macrophages from signaling an anti-KLH response. Spleen cells from HGG-tolerant animals from which the suppressor cells were depleted by “affinity chromatography” on histamine columns, when seeded on macrophages fed with HGG and KLH, generated initiators to both antigens. It appears, therefore, that suppressor cells act at the level of antigen-presenting macrophages, affecting macrophages fed with the tolerogen, and therefore affecting also the immunogenic effect of other antigens presented by the same macrophages. By testing the mode of action of suppressor cells on the tolerogen-fed macrophage, we found that the suppressors manifest a cytotoxic effect on such macrophages. We propose that the suppressor cell is, in fact, an anti modified-self killer, acting on macrophages possessing surface self-antigens “modified” by the tolerogen. The similarity in cell-surface markers between suppressors and anti modified-self killers supports this concept.
Human breast carcinoma antigen is immunologically related to the polypeptide of the group-specific glycoprotein of mouse mammary tumor virus.
Authors: Ohno T , Mesa-Tejada R , Keydar I , Ramanarayanan M , Bausch J , Spiegelman S
Published: May 2006

We have shown [Mesa-Tejada, R., Keydar, I., Ramanarayanan, M., Ohno, T., Fenoglio, C. & Spiegelman, S. (1978) Proc. Natl. Acad. Sci. USA 75, 1529--1533] that an antigen immunologically related to gp52, a 52,000-dalton glycoprotein of the mouse mammary tumor virus, can be identified in sections of human breast cancer by means of an indirect immunoperoxidase technique. The specificity of the reaction was established by absorption experiments which revealed that only purified gp52, or material containing it, served to eliminate the IgG molecules responsible for the immunohistochemical reaction in the human breast tumors. We show here that the cross-reactivity between the human and murine tumor antigens is due to the polypeptide rather than the polysaccharide components of gp.52. Sugar-free gp52 prepared by deglycosylation with a mixture of glycosidases was as fully effective as the intact gp52 in removing from anti-MMTV the IgG responsible for the reaction with the human tumor antigen. In contrast, the isolated polysaccharide of gp52 was unable to exert blocking activity.
Statistics of flexible chain configurations
Authors: Wall Frederick T. , Mandel Frederic , Chin John
Published: May 2006

When Monte Carlo methods are employed to study the statistical dimensions of flexible polymer chains, it is necessary that the sampling be statistically unbiased. One Monte Carlo procedure is the so-called “slithering snake” technique, which has proved to be very useful. A question arises, however, as to how long it takes for a “slithering snake” to be completely regenerated to avoid biasing the samples around a particular configuration. It is demonstrated theoretically and verified by Monte Carlo studies that the number of iterations required to completely regenerate a sample polymer is a quadratic function of the chain length. This verification applies to chains in dilute solution but may not hold for bulk polymers.
Photoisomerization, energy storage, and charge separation: A model for light energy transduction in visual pigments and bacteriorhodopsin
Authors: Honig B. , Ebrey T. , Callender R. H. , Dinur U. , Ottolenghi M.
Published: May 2006

A simple model for the early events in visual pigments and bacteriorhodopsin is proposed. The model makes use of the likelihood that a negatively charged amino acid forms a salt bridge with the positively charged nitrogen of the retinylic chromophore. The photochemical event is a
cis-trans
isomerization in visual pigments and a
trans-cis
isomerization in bacteriorhodopsin, which in each case cleaves the salt bridge and thus separates charge in the interior of the protein. We propose that this is how the energy of a photon is transduced into chemical free energy of the primary photoproduct. The use of photoisomerization of a flexible chromophore to achieve charge separation provides a general mechanism which may be applicable to other systems. Our model explains many of the fundamental properties of visual pigments and their photoproducts. First, the extraordinarily low rate of thermally populating the ground state of the primary photoproduct, as determined from psychophysical and electrophysiological measurements, is seen as resulting from the large barrier to thermal isomerization about a double bond, perhaps enhanced by electrostatic attraction in the salt bridge. Second, the increase in energy and the spectral red shift that characterize the primary photochemical events are natural consequences of the separation of charge. Proton-dependent processes detected with picosecond techniques are proposed to be ground-state relaxation processes following the primary photochemical event. Finally, the charged groups of the salt bridge, repositioned by photoisomerization, provide a simple mechanism for vectorial proton translocation in bacteriorhodopsin.
"Alternating B-DNA" conformation for the oligo(dG-dC) duplex in high-salt solution.
Authors: Patel D J , Canuel L L , Pohl F M
Published: May 2006

The high resolution 1H and 31P NMR spectra of the (dG-dC)8 duplex have been recorded in low- and high-salt solutions in order to evaluate the structural aspects of the salt-induced transition of oligo(dG-dC) in solution [Pohl, F. M. & Jovin, T. M. (1972) J. Mol. Biol. 67, 375-396]. The NMR data require that the (dG-dC)8 duplex in 4 M NaCl adopt an "alternating B-DNA" conformation for which the symmetry unit repeats every two base pairs. By contrast, the oligomer duplex in low-salt solution is of the regular B-DNA type in solution. The chemical shift parameters for oligo(dG-dC) in high-salt solution demonstrate that every other glycosidic torsion angle and phosphodiester linkage adopts a different conformation from that observed in regular B-DNA. We demonstrate further that the generation of the "alternating B-DNA" structure is facilitated by introduction of halogen atoms at the 5 position of pyrimidine and that this probably reflects the greater overlap of this position with adjacent base pairs in high salt solution. An "alternating B-DNA" model has recently been proposed for alternating deoxy purine-deoxy pyrimidine polynucleotides based on the x-ray structure of pdA-dT-dA-dT [Klug, A., Jack, A., Viswamitra, M.A., Kennard, O., Shakked, Z. & Steitz, T.A. (1979) J. Mol. Biol., in press].
Paracatalytic modification of aldolase: A side reaction of the catalytic cycle resulting in irreversible blocking of two active-site lysyl residues
Authors: Lubini Ditmar G. E. , Christen Philipp
Published: May 2006

Paracatalytic enzyme modifications result from the oxidation of enzyme—substrate carbanions by extrinsic oxidants. During the oxidation of enzyme-activated substrates, transiently reactive intermediates are generated which, without being released from the enzyme, modify groups at the active site. For enzymes producing carbanion intermediates, the combination of the normal substrate with a suitable electron acceptor has thus been proposed as a highly specific binary system for their active site-directed modification. In this study, the structural features of paracatalytically modified fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from rabbit muscle have been elucidated. This enzyme is completely inactivated within 60 min in the presence of fructose 1,6-bisphosphate in saturating concentration and 0.5 mM hexacyanoferrate(III) (pH 7.6, 25°C). The inactivation is caused by covalent incorporation of one triosephosphate derivative per subunit. Peptide analysis showed that the triosephosphate derivative forms an
intra
chain crosslink between lysine-146 and lysine-227. According to previous independent experimental evidence, both lysyl residues are located at the active site: the ε-amino group of lysine-227 forms a Schiff base intermediate with the carbonyl group of the substrate [Lai, C. Y., Nakai, N. & Chang, D. (1974)
Science
183, 1204-1206] and alkylation of lysine-146 by the affinity labeling reagent
N
-bromoacetylethanolamine phosphate inactivates the enzyme [Hartman, F. C. & Brown, J. P. (1976)
J. Biol. Chem.
251, 3057-3062]. The present data thus establish paracatalytic modification as a mode of active site-directed enzyme modification.
Cyclic nucleotide phosphodiesterase activity in 10-nm filaments and microtubule preparations from bovine brain
Authors: Runge Marschall S. , Hewgley Paula B. , Puett David , Williams Robley C.
Published: May 2006

Cyclic nucleotide phosphodiesterase activity (3′:5′-cyclic-AMP 5′-nucleotidohydrolase, EC 3.1.4.17), which is activatable by Ca
2+
-dependent regulator protein (CDR), has been identified in cycled microtubule preparations from bovine brain. By using various methods to fractionate the microtubule preparation into subfractions (e.g., phosphocellulose chromatography to obtain purified 6S tubulin and soluble microtubule-associated proteins, and gel exclusion chromatography on Bio-Gel A-150m to obtain 10-nm filaments), we found that all the fractions exhibited some enzymic activity, but that most of the phosphodiesterase activity was localized in the 10-nm filament fraction. By using cyclic GMP as substrate, a specific activity of 921 ± 168 pmol/mg of filament protein·min was determined. Also, 10-nm filaments were prepared directly from brain homogenates by differential centrifugation and gel exclusion chromatography. This fraction also contained phosphodiesterase activity but of slightly lower specific activity (752 ± 9 pmol/mg of protein·min). The filament-associated enzymic activity was stable during storage (-70°C) and to several salt extractions at moderate ionic strength (0.5 M); the latter finding indicates that the phosphodiesterase is not adsorbed to the filaments via nonspecific electrostatic interactions. Although a chelating agent was present in the initial homogenization buffer and generally in all buffers used in preparing fractions, an activator of a smooth muscle phosphodiesterase was released upon boiling the 10-nm filaments. This activator obtained in the boiled supernatant was Ca
2+
-sensitive, trifluoperazine-sensitive, and stimulated smooth muscle phosphodiesterase to nearly the same extent as purified (exogenous) CDR; thus, it probably represents filament-associated CDR.
Synthesis and biological properties of pinane-thromboxane A2, a selective inhibitor of coronary artery constriction, platelet aggregation, and thromboxane formation.
Authors: Nicolaou K C , Magolda R L , Smith J B , Aharony D , Smith E F , Lefer A M
Published: May 2006

Pinane-thromboxane A2 (PTA2, [1alpha,2 beta(Z),-3 alpha (1E,3R*),5 alpha]-7-(3-(3-hydroxy-1-octenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-yl)-5-heptenoic acid) has been synthesized and tested for biological activity in systems responsive to thromboxane A2, stable prostaglandin endoperoxide (PGH2) analogs, and prostatacyclin (PGI2). At low concentrations, PTA2 inhibited cat coronary artery constriction induced by stable prostaglandin endoperoxide analogs, and it stabilized liver lysosomes. At slightly higher concentrations, it inhibited platelet aggregation. At still higher concentrations, PTA2 inhibited thromboxane synthetase, but it had no effect on prostacyclin synthetase. The analog also had no effect on the inhibition of platelet aggregation by PGI2 or prostaglandin D2. It is suggested that PTA2 has a suitable biochemical profile for use as an antithrombotic agent.