bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcellular transport of fluorescein in hepatocyte monolayers: evidence for functional polarity of cells in culture.
Authors: Barth C A , Schwarz L R
Published: May 2006

The rat liver in vivo transfers bile salts, proteins, and dyes from blood into bile. It is the purpose of this communication to demonstrate the maintenance of this transcellular transport in cultured adult rat hepatocytes. Two minutes after adding fluorescein (20 microgram/ml) to the culture medium, maximal cellular fluorescence was observed through the fluorescence microscope. Subsequently, intercellular clefts showed a steadily increasing fluorescence with a maximum between 5 and 20 min, resulting in a brightly fluorescent network of intercellular gaps. The following observations are taken as evidence that these findings reflect cellular uptake and canalicular secretion of the dye. First, the same sequence of observations was made upon addition of fluorescein diacetate (a nonfluorescent precursor of fluorescein), proving that the compound had been taken up and metabolized in the cells to fluorescein before secretion into intercellular clefts. Second, preincubation of the monolayers with the cholestatic bile salt taurolithocholate (100 mumol/liter) suppressed almost completely intercellular but not cellular fluorescence. It is concluded that hepatocytes in culture show a functional polarity permitting the transcellular transport of substances bound for biliary secretion.
Microscale nutrient patches produced by zooplankton
Authors: Lehman John T. , Scavia Donald
Published: May 2006

Both track autoradiography and grain-density autoradiography show that individual zooplankton create miniature patches of dissolved nutrients and that algae exploit those regions to absorb phosphate. The patches are short lived and can be dispersed artificially by small-scale turbulence. Our data support a simple model of encounters between algae and nutrient plumes produced by swimming zooplankton.
Formation of hybrid phycobilisomes by association of phycobiliproteins from
Nostoc
and
Fremyella
Authors: Canaani Ora , Gantt Elisabeth
Published: May 2006

Formation of phycobilisomes has been accomplished
in vitro
from isolated phycobiliprotein fractions obtained from the same blue-green alga (intrageneric) and from different blue-green algae (intergeneric). Phycobilisomes, which are supra-molecular complexes of phycobiliproteins, serve as major light-harvesting antennae for photosynthesis in blue-green and red algae. Intrageneric association into energetically functional phycobilisomes, previously reported to occur with
Nostoc
sp. allophycocyanin and phycoerythrin-phycocyanin complexes [Canaani, O., Lipschultz, C. A. & Gantt, E. (1980)
FEBS Lett.
115, 225-229], has been obtained with
Fremyella diplosiphon
. By their spectral properties (absorption, fluorescence excitation, and emission) and electron microscopic images, the native and
in vitro
-associated phycobilisomes were virtually indistinguishable. Intergeneric phycobilisomes have been produced from allophycocyanin of
Nostoc
sp. strain Mac. and phycoerythrin-phycocyanin of
F. diplosiphon
, as well as from the reverse mixtures. The yield of intergeneric phycobilisomes, favored by higher phycobiliprotein content in 0.75 M phosphate, pH 7.0/2.0 M sucrose, was 40-60%. Energy transfer to the terminal long-wavelength-emitting allophycocyanin in the phycobilisomes was evident from the 670-675 nm fluorescence emission peaks. Furthermore, excitation spectra showed the contribution of the respective phycoerythrins (
Fremyella
, λ
max
570;
Nostoc
, λ
max
573 and 553 nm), as well as that of phycocyanin and short-wavelength-absorbing allophycocyanin. Phycobilisomes of
Nostoc
and
Fremyella
, analyzed by NaDodSO
4
/polyacrylamide gel electrophoresis, possessed a number of polypeptides having similar molecular weights: the usual α- and β-phycobilin-containing polypeptides of
M
r
15,000-22,000, a faint band at
M
r
ca.
95,000, and a prominent band at
M
r
ca.
31,000. The
M
r
31,000 polypeptide is assumed to provide the recognition site for attachment of the phycoerythrin-phycocyanin complexes with the allophycocyanin core.
In vitro
association was not obtained between allophycocyanin from
Nostoc
and phycoerythrin-phycocyanin complexes from
Phormidium persicinum
or
Porphyridium sordidum
.
Circulating human mammary epithelial antigens in breast cancer.
Authors: Ceriani R L , Sasaki M , Sussman H , Wara W M , Blank E W
Published: May 2006

Heterologous specific antisera against human mammary epithelial antigens (HME-Ags), which are present in the human milk fat globule membrane and breast epithelial cells, were used in a solid-phase radioimmunoassay to determine the presence of these antigens in the sera of patients with disseminated cancer of the breast and other organs. Breast cancer patients carry high levels of HME-Ags in their circulation, while patients with disseminated nonbreast cancer, as well as normal female controls, do not. A similar release of HME-Ags in the circulation was shown by us in a model system. To further corroborate these findings, a three-step procedure for the extraction and identification of HME-Ags from the sera was devised. In this analytical procedure, circulating HME-Ags are recovered on a solid phase carrying their corresponding antibody (anti-HME) and radioiodinated in situ. Later, the labeled HME-Ags are released from the solid phase and characterized by NaDodSO4 gel electrophoresis. With this procedure, HME-Ags were isolated from sera of breast cancer patients but not from sera of nonbreast cancer patients or of normal female controls. The extracted HME-Ags had molecular masses of 150,000, 70,000, and 46,000 daltons. To further support these findings, a monoclonal antibody, BLMRL-HMFG-Mc3, directed to the 46,000-dalton HME-Ag was also used to extract its corresponding antigen from sera. Breast cancer patient sera contained such antigen while the sera of the other patients and controls did not. This highly sensitive methodology offers a specific approach to breast cancer diagnosis as well as further insight into the nature of circulating antigens with a view to increasing our understanding of breast cancer biology.
Neuropeptide Y: complete amino acid sequence of the brain peptide.
Authors: Tatemoto K
Published: May 2006

The amino acid sequence of neuropeptide Y, a 36-residue peptide recently isolated from porcine brain, has been determined by using high performance liquid chromatography for separation of its tryptic and chymotryptic fragments and subsequent sequence analysis of the isolated fragments by an improved dansyl Edman subtractive technique. The amino acid sequence of neuropeptide Y has been found to be: Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr -Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2. Neuropeptide Y has a high degree of sequence homology with peptide YY (70%), the newly isolated porcine intestinal peptide, and pancreatic polypeptide (50%). It is therefore proposed that neuropeptide Y, peptide YY, and pancreatic polypeptide are members of a newly recognized peptide family.
A
M
r
95,000 polypeptide in
Porphyridium cruentum
phycobilisomes and thylakoids: Possible function in linkage of phycobilisomes to thylakoids and in energy transfer
Authors: Redlinger Thomas , Gantt Elisabeth
Published: May 2006

Two pigmented polypeptides with the same molecular weight (
M
r
95,000) were isolated from the photosynthetic apparatus of
Porphyridium cruentum
by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A blue polypeptide from phycobilisomes had absorption and fluorescence emission spectra similar to those of allophycocyanin. A green-pigmented polypeptide from photosynthetic membranes (free of phycobilisomes) contained chlorophyll
a
. Several properties were common to the
M
r
95,000 polypeptides from both sources: (
i
) identical molecular weights, (
ii
) identical gel electrophoresis patterns after limited protease digestion, and (
iii
) immunological crossreactivity with an IgG fraction directed against the
M
r
95,000 polypeptide from phycobilisomes. On the basis of this evidence, a common polypeptide exists in phycobilisomes and thylakoids, and it probably anchors the phycobilisome to the thylakoid membrane. The fluorescence emission overlap of the blue and green polypeptides suggests that they are involved in the transfer of energy from phycobilisomes to thylakoids.
Nucleotide sequence of tobacco mosaic virus RNA.
Authors: Goelet P , Lomonossoff G P , Butler P J , Akam M E , Gait M J , Karn J
Published: May 2006

Oligonucleotide primers have been used to generate a cDNA library covering the entire tobacco mosaic virus (TMV) RNA sequence. Analysis of these clones has enabled us to complete the viral RNA sequence and to study its variability within a viral population. The positive strand coding sequence starts 69 nucleotides from the 5' end with a reading frame for a protein of Mr 125,941 and terminates with UAG. Readthrough of this terminator would give rise to a protein of Mr 183,253. Overlapping the terminal five codons of this readthrough reading frame is a second reading frame coding for a protein of Mr 29,987. This gene terminates two nucleotides before the initiator codon of the coat protein gene. Potential signal sequences responsible for the capping and synthesis of the coat protein and Mr 29,987 protein mRNAs have been identified. Similar sequences within these reading frames may be used in the expression of sets of proteins that share COOH-terminal sequences.
Chemoattractant receptor functions in human polymorphonuclear leukocytes are divergently altered by membrane fluidizers.
Authors: Yuli I , Tomonaga A , Synderman R
Published: May 2006

The chemotactic factor receptor on leukocytes initiates several cellular responses including chemotaxis, lysosomal enzyme secretion, and O2- production. The latter two responses require approximately 10-100 times more chemoattractant than is required for chemotaxis. We determined the effects of membrane fluidizers on the binding characteristics and the functional activities of the oligopeptide fMet-Leu-Phe chemotactic factor receptor on polymorphonuclear leukocytes. Fluidization was induced by aliphatic alcohols and monitored by diphenylhexatriene fluorescence polarization. Low doses of n-butanol (0.25%) and n-pentanol (0.1%) were nontoxic to the leukocytes yet reduced their diphenylhexatriene-induced polarization, indicating increased membrane fluidity. At these doses of alcohols, the affinity of the fMet-Leu-Phe receptor was enhanced from Kd = 25.5 +/- 7.6 nM to Kd = 5.2 +/- 0.9 nM and Kd = 6.0 +/- 0.9 nM, respectively. Chemotaxis was also increased, as indicated by the decrease, by a factor of approximately 1/3 in the ED50 for fMet-Leu-Phe, as well as by a 1.5-fold increase in the maximal distance of migration in the presence of 0.25% butanol or 0.1% pentanol. In contrast to chemotaxis, the alcohols depressed fMet-Leu-Phe stimulation of O2- production by 90% although they had no effect on phorbol 12-myristate 13-acetate-induced O2- production. Secretion of lysozyme was also inhibited. Thus, the affinity of the fMet-Leu-Phe receptor can be modulated by membrane fluidizers. The higher affinity state of the receptor induced by the alcohols is more efficient in transducing chemotactic signals but is deficient in mediating O2- production or secretion. Thus, the transduction mechanisms for the various biological activities of the chemotactic factor receptor are heterogeneous and can be differentially manipulated by membrane fluidizers.
Mismatch repair, gene conversion, and crossing-over in two recombination-defective mutants of Drosophila melanogaster.
Authors: Carpenter A T
Published: May 2006

Recombination-defective mutants at two loci that are known to decrease drastically the frequency of meiotic crossing-over do not decrease the frequency of gene conversion at the rosy locus. mei-9 mutant alleles produce frequent postmeiotic segregants manifested as mosaic progeny whereas controls and mei-218 mutants produce none. It is concluded that (i) recombination in Drosophila involves a biparental DNA intermediate and (ii) correction of heteroduplex DNA or recognition of biparental DNA or both is necessary, but not sufficient, for this intermediate to result in crossing-over of flanking markers. It is therefore likely, at least in Drosophila, that the isomerization step in Meselson-Radding type molecular models of recombination is under genetic control.
Stimulation of leukotriene biosynthesis in human blood leukocytes by platelet-derived 12-hydroperoxy-icosatetraenoic acid.
Authors: Maclouf J , de Laclos B F , Borgeat P
Published: May 2006

Addition of arachidonic acid to suspensions of human blood leukocytes induces the synthesis of small amounts only of the C-5 lipoxygenase products as demonstrated by HPLC. However, the coincubation of blood platelets with the leukocytes always resulted in an activation of the C-5 lipoxygenase and formation of (5S)-5-hydroxy-6,8,11,14-icosatetraenoic acid, (5S,12S)-5,12-dihydroxy-6,8,10,14-icosatetraenoic acid, and leukotriene B4 from exogenous arachidonic acid. It was found that the activation of arachidonic acid metabolism in leukocytes was caused by a labile compound because the synthesis of the C-5 lipoxygenase products did not occur when platelets were preincubated for 1 min or more with the substrate prior to the addition of the leukocytes. The use of cyclooxygenase inhibitors did not suppress the activation of the leukocytes by the platelets. However, the addition of 5,8,11,14-icosatetraynoic acid, an inhibitor of cyclooxygenase and C-12 and C-15 lipoxygenases, completely suppressed the formation of leukotrienes, although this substance is not an inhibitor of the C-5 lipoxygenase in human leukocytes. This indicated that a product of the C-12 lipoxygenase was likely the mediator of the stimulatory effect of platelets on leukocyte arachidonic acid metabolism. The finding that the direct addition of (12S)-12-hydroperoxy-5,8,10,14-icosatetraenoic acid, but not of the corresponding hydroxy derivative, could activate the leukocyte's C-5 lipoxygenase confirmed this hypothesis. These data demonstrate that an interaction between C-12 and C-5 lipoxygenases can promote the formation of leukotrienes and support the possibility of a cooperation between platelets and leukocytes in inflammation and hypersensitivity reactions. Furthermore, the finding provides a new interest for the platelet C-12 lipoxygenase.
Monoclonal antibodies recognize individual neurofilament triplet proteins.
Authors: Lee V , Wu H L , Schlaepfer W W
Published: May 2006

A series of 12 monoclonal antibodies has been prepared against crude or purified individual bovine neurofilament (NF) triplet proteins (designated as P68, P150, and P200). Six of the antibodies recognized unique determinants on individual NF subunits. The other six recognized shared determinants on at least two NF triplet proteins. Six of the antibodies recognized separated, immobilized individual NF in nitrocellulose paper. By the peroxidase-antiperoxidase technique, all of the antibodies stained neurons and their processes in a manner identical to the well-known distribution of NF in both the central and peripheral nervous system. These results strongly suggest that the NF subunits are distinct but related proteins and that the smaller NF subunits (P68 and P150) are not derived from larger NF proteins.
Are DNA precursors concentrated at replication sites?
Authors: Mathews C K , Sinha N K
Published: May 2006

We have asked whether the effective concentrations of deoxyribonucleotide 5'-triphosphates (dNTPs) at sites of DNA replication in vivo might be higher than the concentrations of dNTPs averaged over the entire cell volume. The approach involved determination of the dependence of DNA replication rate upon thymidine triphosphate concentration, both in vivo and in vitro system that closely approximates the intracellular replication apparatus. In T4 phage-infected Escherichia coli maximal rates of DNA synthesis were attained with dTTP pools of approximately 1.2 x 10(5) molecules per cell, corresponding to an average intracellular concentration of about 65 microM. When DNA synthesis was measured in the T4 purified protein system [Sinha, N. K., Morris, C. F. & Alberts, B. M. (1980) J. Biol. Chem. 255 4290--4303], maximal rates were observed at dTTP concentrations of 200--240 microM. This represents a minimal estimate, therefore, of dTTP concentration at replication sites and suggests that at least a 3- to 4-fold concentration gradient exists near these sites. We discuss why such concentration gradients might be needed and how they might be generated. We also discuss the implications of these results for understanding the relationship between intracellular dNTP pools and mutation rates. A by-product of our study was the finding that exogenous thymidine is used for T4 DNA synthesis in preference to endogenous pathways to thymidine nucleotides; at high thymidine concentrations in vivo the endogenous pathways can be completely bypassed.
Fate of receptor and ligand during endocytosis of asialoglycoproteins by isolated hepatocytes.
Authors: Bridges K , Harford J , Ashwell G , Klausner R D
Published: May 2006

The endocytosis leading to degradation of 125I-labeled asialo-orosomucoid specifically bound to the surface of freshly isolated hepatocytes was monitored as a function of time at 37 degrees C. Experimental values were determined for the rates of internalization, dissociation of the receptor-ligand complex, and degradation of the labeled ligand. Compartmental analysis and computer modeling revealed that the data were compatible with dissociation of ligand from receptor preceding ligand degradation. The rate coefficient for internalization was calculated to be an order of magnitude greater than that for receptor--ligand dissociation. Ligand internalization did not result in concomitant depletion in the total number of cell surface receptors. Our data are taken to indicate that ligand remains associated with the receptor after internalization, that the complex is dissociated prior to degradation, and that new, unoccupied receptors are promptly returned to the cell surface from an internal pool.
Selenium-containing tRNAGlu from Clostridium sticklandii: correlation of aminoacylation with selenium content.
Authors: Ching W M , Stadtman T C
Published: May 2006

Selenium-containing amino acid tRNAs are normal components of several bacterial tRNA populations. In Clostridium sticklandii seleno-nucleotides occur in at least four different tRNA species which account for 5--8% of the total tRNA population. One of these has been isolated in a highly purified form and shown to be an isoaccepting tRNAGlu. Experimental evidence indicates that the presence of the seleno-nucleotide in this tRNAGlu is essential for its acylation with glutamate.
Giant circular dichroism of high molecular weight chlorophyllide-apomyoglobin complexes
Authors: Pearlstein Robert M. , Davis Robert C. , Ditson Susan L.
Published: May 2006

Chlorophyllide
a
and the apoprotein of myoglobin (Mb) spontaneously form three types of complex. The M (
M
r
≈ 3 × 10
5
) and H (
M
r
≥ 4 × 10
6
) complexes, but not the L (
M
r
≈ 1.7 × 10
4
), display a circular dichroism (CD) spectrum that is highly red-shifted, nonconservative, and very intense—characteristics shared by the CD spectra of reaction center complexes from purple photosynthetic bacteria. At its 710-nm peak, the H complex CD spectrum has a larger magnitude, 0.06 differential absorbance per unit total absorbance, than has been reported for chlorophyll in any medium.
Aerial dispersal and drying of
Peronospora tabacina
conidia in tobacco shade tents
Authors: Aylor Donald E. , Taylor Gordon S.
Published: May 2006

Blue mold of tobacco is caused by the fungus
Peronospora tabacina
. Its spores are released in the morning when the relative humidity falls, but they require free water for germination. Traditionally, the walls of the shade tents are raised to control the pathogen by drying leaves. We show that raising the walls does not materially speed drying. It does, however, increase the wind speed at least 20 m inside the tent and spread spores farther. Because germination is not reduced, raising the walls may actually spread blue mold.
Initiation of bacteriophage lambda DNA replication in vitro with purified lambda replication proteins.
Authors: Wold M S , Mallory J B , Roberts J D , LeBowitz J H , McMacken R
Published: May 2006

We have developed a soluble enzyme system that replicates exogenously added plasmid DNA (lambda dv) bearing the replication origin of the bacteriophage lambda chromosome. The system contains pure phage lambda O and P replication proteins and a partially purified mixture of Escherichia coli replication proteins [the enzyme system of Fuller, R.S., Kaguni, J.M. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 7370-7374). The features of lambda dv replication in this system closely resemble the known characteristics of phage lambda DNA replication in vivo. The system (i) depends completely on exogenously supplied DNA, (ii) specifically replicates supercoiled plasmid DNA that contains a lambda replication origin, (iii) depends on both the lambda O protein and the lambda P protein, (iv) depends on RNA polymerase, (v) depends on host replication proteins (e.g., primase, dnaB protein, and several others that function in the priming of DNA synthesis in E. coli) as judged by antibody inhibitions, and (vi) replicates as much as 32% of added lambda dv plasmid DNA through a single complete round to generate catenated daughter molecules. Furthermore, replication of lambda dv DNA in vitro requires DNA gyrase and an ATP-regenerating system. It is notable that addition of lambda O and P proteins to the mixture of E. coli replication proteins inhibits replication of plasmids bearing the origin of the E. coli chromosome. Exploitation of this enzyme system should allow a detailed investigation of the biochemical mechanisms involved in bacteriophage lambda DNA replication and its regulation.
Intracellular K+ and the mitogenic response of 3T3 cells to peptide factors in serum-free medium.
Authors: Lopez-Rivas A , Adelberg E A , Rozengurt E
Published: May 2006

The stimulation of DNA synthesis in cultures of Swiss 3T3 cells by vasopressin, epidermal growth factor, and insulin added in serum-free medium is strikingly dependent on the intracellular K+ content or concentration. The relationship between these parameters is sigmoid; DNA synthesis commences only when the intracellular K+ increases above a certain threshold level (0.56 mumol/mg of protein; 90 mM). Addition of K+ to K+- depleted cultures reverses the block on DNA synthesis after a lag period of at least 8 hr. The sigmoid dependence of DNA synthesis on intracellular K+ is generated in early G1 phase rather than at the G1/S boundary. The effects of K+ on the G1-S transition are, at least in part, exerted through its control of protein synthesis. In serum-free medium, the K+ content is close to the threshold required for allowing a mitogenic response. The findings suggest that a small change in the intracellular K+ level can influence the ability of these cells to initiate DNA synthesis in serum-free medium.
Thyrotropin-releasing hormone mobilizes Ca2+ from endoplasmic reticulum and mitochondria of GH3 pituitary cells: characterization of cellular Ca2+ pools by a method based on digitonin permeabilization.
Authors: Ronning S A , Heatley G A , Martin T F
Published: May 2006

Treatment of 45Ca2+-loaded GH3 pituitary cells with various concentrations of digitonin revealed discrete pools (I and II) of cellular 45Ca2+ defined by differing detergent sensitivities. Markers for cytosol and intracellular organelles indicated that the two 45Ca2+ pools were correlated with the two major cellular Ca2+-sequestering organelles, endoplasmic reticulum (I) and mitochondria (II). Studies with various inhibitors were consistent with these assignments. Mitochondrial uncouplers preferentially depleted 45Ca2+ pool II while trifluoperazine selectively depleted 45Ca2+ pool I. Control experiments indicated that translocation of in situ organellar 45Ca2+ during and after permeabilization was negligible. We used the digitonin-permeabilization method to examine the effect of thyrotropin-releasing hormone (TRH) treatment on intracellular Ca2+ pools of GH3 pituitary cells. TRH was found to rapidly deplete both endoplasmic reticulum and mitochondrial exchangeable Ca2+ by 25-30%. The 45Ca2+ loss from both pools was maximal by 1 min after TRH addition and was followed by a recovery phase; mitochondrial 45Ca2+ content returned to control levels by 30 min. Previous treatment of cells with the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone blocked TRH-induced 45Ca2+ efflux from mitochondria, while previous treatment with valinomycin, an agent that depleted both 45Ca2+ pools, blocked any additional effect of TRH on these pools. We conclude that TRH rapidly promotes a net loss of exchangeable Ca2+ from GH3 cells as a result of hormone-induced mobilization of Ca2+ from endoplasmic reticulum and mitochondria.
Immunocytochemical demonstration of monoamine oxidase B in brain astrocytes and serotonergic neurons.
Authors: Levitt P , Pintar J E , Breakefield X O
Published: May 2006

An antiserum to monoamine oxidase B (MAO-B) was used to define the distribution of this metabolic enzyme in the adult rat brain immunocytochemically. MAO-B is specifically located in two major central nervous system cell classes, astrocytes and serotonin-containing neurons. Double-immunofluorescence experiments using antisera to glial fibrillary acidic protein and MAO-B showed that both protoplasmic and fibrillary astrocytes throughout the brain contain MAO-B, whereas oligodendrocytes do not contain the enzyme. Areas lacking a blood-brain barrier, such as the specialized circumventricular organs, also contain MAO-B-positive cells. A double-immunofluorescence experiment using antisera to serotonin and MAO-B enabled the positive identification of neurons containing both molecules. The catecholamine-containing neurons of the brain did not contain detectable amounts of MAO-B. The specific distribution of MAO-B in the adult central nervous system indicates that the role of MAO-B in monoamine metabolism may be more specifically defined than previously believed.
Characterization of beta-endorphin binding protein (receptor) from rat brain membranes.
Authors: Hammonds R G , Nicolas P , Li C H
Published: May 2006

Rat brain membranes (RBM) bind beta-endorphin with high affinity and specificity. We report herein the identification of a high molecular weight beta-endorphin complex (receptor) in extracts of RBM preincubated with tritiated beta-endorphin, by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Upon isoelectric focusing, this complex gives a single peak with an isoelectric point (+/- SEM) of 4.50 +/- 0.06. Sucrose density gradient experiments in H2O and 2H2O yield effective partial specific volume (v = 0.814 cm3/g and sedimentation constant s20,w = 15.6 S. Gel filtration yields an estimate of the hydrodynamic radius of 73 A. The corresponding frictional ratio of 1.12 is consistent with an elliptical spheroid with an axial ratio of 3.1-3.2. The molecular mass of the complex is estimated to be 690,000 daltons. The v of the complex is greater than that of CHAPS or most globular proteins.
Structural and functional consequences of increased tubulin glycosylation in diabetes mellitus.
Authors: Williams S K , Howarth N L , Devenny J J , Bitensky M W
Published: May 2006

The extent of in vitro nonenzymatic glycosylation of purified rat brain tubulin was dependent on time and glucose concentration. Tubulin glycosylation profoundly inhibited GTP-dependent tubulin polymerization. Electron microscopy and NaDodSO4/polyacrylamide gel electrophoresis showed that glycosylated tubulin forms high molecular weight amorphous aggregates that are not disrupted by detergents or reducing agents. The amount of covalently bound NaB3H4-reducible sugars in tubulin recovered from brain of streptozotocin-induced diabetic rats was dramatically increased as compared with tubulin recovered from normal rat brain. Moreover, tubulin recovered from diabetic rat brain exhibited less GTP-induced polymerization than tubulin from nondiabetic controls. The possible implications of these data for diabetic neuropathy are discussed.
Effects of taxol and Colcemid on myofibrillogenesis.
Authors: Toyama Y , Forry-Schaudies S , Hoffman B , Holtzer H
Published: May 2006

To determine the relationship between thin filaments, Z-bands, microtubules, intermediate filaments (IFs), T-tubules, and sarcoplasmic reticulum (SR) during myofibrillogenesis, myotubes were selectively depleted of their myofibrils with 12-tetradecanoylphorbol 13-acetate (TPA) and then were allowed to regenerate in (i) normal medium, (ii) taxol, and (iii) Colcemid. Myofibrils assembled in normal medium formed typical A-, I-, Z-, M-, and H-bands and associated IFs, T-tubules, and SR. Myofibrils assembled in taxol formed "A-bands" of aligned thick filaments interdigitating with long microtubules and "I-bands" consisting only of microtubules. These unprecedented sarcomeres lacked thin filaments, Z-bands, and associated IFs and SR. "Solitary A-bands," consisting exclusively of laterally aligned bipolar thick filaments 1.6 microM in length without either thin filaments or microtubules, were observed. Myofibrils assembled in Colcemid formed all myofibrillar components in the absence of microtubules but these did not achieve rigorous lateral alignment. Colcemid and taxol induced the formation of patchy Z-bands that invariably served as insertion sites for thin filaments, irrespective of the presence or absence of adjacent thick filaments. Z-bands may function as actin-organizing centers for each sarcomere.
Primary structure of the immunoglobulin J chain from the mouse.
Authors: Cann G M , Zaritsky A , Koshland M E
Published: May 2006

The primary structure of the murine J chain was investigated by sequence analysis of the J chain cDNA inserts from two independently cloned chimeric plasmids. The sequence data showed that (i) the two cDNA inserts accounted for all but approximately 100 5' nucleotides of the J chain mRNA and (ii) the J chain mRNA encodes a prepeptide of at least 23 amino acids, a mature protein of 137 residues, and an untranslated 3' region of 707 nucleotides exclusive of the 3' poly(A) tract. The amino acid sequence deduced for the mature mouse J chain was found to be 74% identical with that previously determined for the human J chain. By analyzing the conserved features of the sequence, a two-domain structure was generated for the J chain which correlates well with its functions in the polymerization of IgM and IgA. Moreover, by comparing the homologies of the J and heavy chains in mouse and man, evidence was obtained that the structures involved in polymerization are the most conserved elements of immunoglobulin molecules.
Cell adhesion molecules in early chicken embryogenesis.
Authors: Thiery J P , Duband J L , Rutishauser U , Edelman G M
Published: May 2006

N-CAM, the neural cell adhesion molecule, has been found at a number of regions in the early (1-5 days) chicken embryo by fluorescent antibody techniques. These regions appear to be those concerned with induction of the primary developmental axis (neural plate, neural tube, notochord, somites) or those in which later inductive events occur (neural crest cells, optic, otic, and pharyngeal placodes, cardiac mesoderm, mesonephric primordium, limb buds). The staining patterns in the latter group of regions are highly dynamic and transient and are limited to the epithelial components of the placodes and to the precursors of mesonephric tubules. In neural crest cells, N-CAM appears early, disappears during migration of the cells on fibronectin, and reappears at sites where ganglia are formed. In other regions of the nervous system, particularly those related directly to the neural tube, the N-CAM molecule is stained at all stages. The results raise the possibility that adhesion mediated by N-CAM plays a primary role in early embryogenesis as well as in later histogenesis.
Analysis of desmin and vimentin phosphopeptides in cultured avian myogenic cells and their modulation by 8-bromo-adenosine 3',5'-cyclic monophosphate.
Authors: Gard D L , Lazarides E
Published: May 2006

The intermediate filament proteins desmin and vimentin are two of the major 32P phosphate acceptors in chicken myotubes differentiating in tissue culture. Analysis of the desmin and vimentin phosphopeptides by two-dimensional tryptic peptide mapping shows that both proteins are phosphorylated at multiple sites, giving rise to 5 phosphopeptides in desmin and as many as 11 in vimentin. Addition of the cAMP analogue 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) to the culture medium of mature (8-day-old) myotubes results in a 2- to 3-fold increase in PO4 incorporation into desmin and vimentin. Two-dimensional tryptic analysis of desmin and vimentin from 8-BrcAMP-treated myotubes shows increased 32PO4 incorporation into a subset of the phosphopeptides observed in control cells. Comparison of phosphopeptides from the two proteins shows the presence of at least three comigrating peptides. All three comigrating peptides exhibit cAMP-dependent increases in 32PO4 incorporation in vimentin, while only two of the three exhibit 8-BrcAMP-dependent responses in desmin. While these peptides are the only two that are sensitive to 8-BrcAMP in desmin, vimentin contains additional peptides that exhibit increased 32PO4 incorporation in response to 8-BrcAMP. This result suggests the existence of both common and distinct phosphorylation sites between desmin and vimentin that may be differentially regulated by cAMP. Thus, desmin and vimentin, even though structurally related, may be capable of responding differently to physiological stimuli.
Electrical effects of histamine on monolayers formed in culture from enriched canine gastric chief cells.
Authors: Ayalon A , Sanders M J , Thomas L P , Amirian D A , Soll A H
Published: May 2006

To develop techniques for studying transport properties and secretory function of selected cell types in the gastric mucosa, separated fractions of dispersed canine fundic mucosal cells were placed in short-term culture to form epithelial monolayers. Cell fractions enriched in either chief, parietal, or mucous cells were prepared by using counterflow centrifugation and were plated on type I collagen. An epithelial monolayer formed by approximately equal to 36 hr. Immunofluorescence with an antipepsinogen I antibody revealed pepsinogen-containing granules in greater than 95% of the cells, regardless of whether the monolayers were formed from the mucous, chief, or parietal cell-enriched fractions. Upon achieving confluency, chief cell monolayers were mounted in Ussing chambers to study their electrical properties. Under basal conditions, monolayers (n = 6) had a spontaneous potential difference (PD) (+/- SEM) of 26 +/- 4 mV (apical surface negative), a short-circuit current (Isc) (+/- SEM) of 16 +/- 2 microA/cm2, and a transepithelial resistance (R) (+/- SEM) of 1,480 +/- 210 omega X cm2. Histamine increased the short-circuit current, an effect blocked by an H2-receptor antagonist. Seventy percent of the spontaneous PD was amiloride sensitive, suggesting sodium absorption accounted for a major component of the PD. These preparative techniques yield highly enriched chief cell monolayers, which maintain morphological and functional cellular differentiation for greater than 48 hr in culture, thus allowing study of oriented functions of a selected cell type. The present studies indicate that an H2 receptor enhances electrogenic ion transport in chief cell monolayers, indicating that histamine can act on fundic mucosal cells other than just parietal cells.
Initiation and termination of phage f1 plus-strand synthesis.
Authors: Dotto G P , Horiuchi K , Zinder N D
Published: May 2006

The origin of DNA replication of bacteriophage f1 contains a nucleotide sequence that is used both for the initiation of viral (plus) strand synthesis and for its termination. With chimeric plasmids containing two f1 functional origins in the same orientation, synthesis of chimeric plus-strand DNA is initiated, after f1 infection, at either one of the two f1 origins and is terminated at the other. Thus, the chimeric plasmids segregate into two replicons, each of them containing only one f1 origin. This system has been used to test several fragments of the f1 origin varying in size or in nucleotide sequence for their ability to function in either initiation or termination of viral strand synthesis. Our data show that the f1 origin is composed of two overlapping but distinct domains (signals), one for initiation and the other for termination of plus-strand synthesis.
Circular dichroism spectroscopy of heparin-antithrombin interactions
Authors: Stone Audrey L. , Beeler David , Oosta Gary , Rosenberg Robert D.
Published: May 2006

We have utilized circular dichroism spectroscopy to examine the interaction of antithrombin with heparin-derived oligosaccharides and mucopolysaccharides of various sizes. Our studies demonstrate that the various complexes exhibit two major types of chiral absorption spectra. The first of these patterns is seen when octasaccharide, decasaccharide, dodecasaccharide, or tetradecasaccharide fragments bind to the protease inhibitor. The circular dichroism spectra of these complexes when compared to the spectrum of free antithrombin show several distinguishing characteristics. On the one hand, there is a marked general increase in positive chiral absorption that is maximal at 296 and 288 nm and 290 and 282.5 nm. These observations indicate perturbation of “buried” and “exposed” tryptophan residues. On the other hand, a significant augmentation in circular dichroism that peaks at 269.5 and 263 nm is noted. These findings are probably due to the summed positive and negative contributions arising from tryptophan residue(s), disulfide bridge(s), and phenylalanine residue(s). Given that these heparin fragments are able to accelerate factor Xa-antithrombin interactions but not thrombin-antithrombin interactions, the above spectral transitions must be associated with either the binding of a critical domain of the oligosaccharides to the protease inhibitor or the “activation” of the protease inhibitor with respect to factor Xa neutralization. The second of these patterns is apparent when octadecasaccharide, low molecular weight heparin (6,500), and high molecular weight heparin (22,000) interact with antithrombin. The circular dichroism spectra of these complexes compared to the spectrum of free protease inhibitor are similar to the first pattern except for changes within the 292- to 282-nm and 275- to 255-nm regions. The subtraction of the first pattern from the second pattern reveals a shallow negative band between 300 and 275 nm with potential negative minima at 290 and 283 nm as well as a deep negative band between 275 and 255 nm with possible negative minima at 268 and 262 nm. This chiral absorption profile is most likely to arise from conformational changes of a disulfide bridge(s). However, we cannot completely exclude the possibility that the above circular dichroism difference curve might be explained on the basis of transitions originating from a tryptophan residue(s). Given our method for generating the above data, these spectral alterations must be associated with the binding of a second critical domain of the mucopolysaccharide to antithrombin that is required for rapid complex formation with thrombin or the activation of the protease inhibitor with respect to the neutralization of the latter enzyme.
Formation and identification of cytoskeletal components from liver cytosolic precursors.
Authors: Sahyoun N , Stenbuck P , LeVine H , Bronson D , Moncharmont B , Henderson C , Cuatrecasas P
Published: May 2006

Liver cytosol forms a macroscopic fibrillary network in the presence of low concentrations of MgCl2. This process represents the generation of 3- to 11-nm filaments from soluble precursors, involving selectively at least 12 major polypeptides. Similar polypeptides are enriched in the detergent-insoluble fraction from hepatocytes, suggesting that they may be important constituents of the native cytoskeleton. AcA 34 gel-permeation chromatography resolves the cytosol into three independently "polymerizing" peaks: A, B, and C. The formation of filaments follows biphasic kinetics in peaks B and C, whereas peak A lacks the slow phase. Filament formation in all three systems is inhibited by 1-15 mM inorganic phosphate, 10 mM NaF, or 10 mM sodium molybdate. The polymerization of peak C only is inhibited by 0.2-2 mM ATP. CaCl2 (1-100 microM) has no apparent regulatory effect. Two-dimensional polypeptide analysis and peptide mapping show that actin is a major component of peak C, while peaks A and B contain prominent polypeptides that may be related to intermediate filament subunits. In addition, all three systems contain two or three high molecular weight (greater than 170,000) polypeptides that may participate in modulating and extending the filament network. The filaments from peaks A and B are soluble in 8 M urea and reform on removal of the urea in the presence of 5 mM MgCl2. The polypeptide composition remains constant through three such cycles.
Interactions between lithium and renal transport of Krebs cycle intermediates.
Authors: Wright E M , Wright S H , Hirayama B , Kippen I
Published: May 2006

The effect of lithium on the renal transport of Krebs cycle intermediates was studied in brush border membrane vesicles isolated from the rabbit renal cortex. The di- and tricarboxylic acids are avidly transported across renal brush border membranes by a sodium cotransport system. Lithium acted as a potent, specific, competitive inhibitor (Ki = 1.2 mM) of succinate/sodium cotransport when added to the uptake medium. Similar effects were observed for citrate but not D-glucose, L-phenylalanine, L-proline, L-alanine, or L-lactate transport. Intravesicular lithium behaved as a noncompetitive inhibitor of succinate transport in the absence of sodium. These results account for the observation that therapeutic doses of lithium increase the renal excretion of Krebs cycle intermediates. The existence of a transport system for alpha-ketoglutarate in synaptosomes suggests a possible target for lithium in the central nervous system.
Collagen structural microheterogeneity and a possible role for glycosylated hydroxylysine in type I collagen
Authors: Yamauchi Mitsuo , Noyes Claudia , Kuboki Yoshinori , Mechanic Gerald L.
Published: May 2006

A three-chained peptide from type I collagen, crosslinked by hydroxyaldolhistidine, has been isolated from a tryptic digest of 5 M guanidine·HCl-insoluble bovine skin collagen (a small but as yet unknown percentage of the total collagen in whole skin). OsO
4
/NaIO
4
specifically cleaved the crosslink at its double bond into a two-chained crosslink peptide and a single peptide. The sequence of the two-chained peptide containing the bifunctional crosslink was determined after amino acid analysis of the separated peptides. The crosslink consists of an aldehyde derived from hydroxylysine-87 in the aldehyde-containing cyanogen bromide fragment α1CB5
ald
and an aldehyde derived from the lysine in the COOH-terminal nonhelical region of the α1CB6
ald
fragment. The α1CB6
ald
portion of the peptide exhibited structural microheterogeneity, containing the inverted sequence Ala-Lys-His instead of the normal sequence Lys-Ala-His. This indicates that another structural gene exists for α1(I) chain. The original three-chained peptide did not contain any glycosylated hydroxylysine or glycosylated hydroxyaldolhistidine. The lack of glycosylation of hydroxylysine-87 in α1CB5, which is usually glycosylated, allowed formation of the aldehyde, and this, coupled with the sequence inversion, may have allowed formation of the nonreducible crosslink hydroxyaldolhistidine. We suggest that the role of glycosylation, a posttranslational modification, of specific hydroxylysine residues is to prevent their oxidative deamination to aldehydes, thereby precluding formation of complex stable crosslinks. Complex crosslinks would decrease the rate of collagen turnover. The decrease, with time, would increase the population of stable crosslinked collagen molecules, which would eventually accumulate with age.
Nucleotide sequence of the gene for the
M
r
32,000 thylakoid membrane protein from
Spinacia oleracea
and
Nicotiana debneyi
predicts a totally conserved primary translation product of
M
r
38,950
Authors: Zurawski Gerard , Bohnert Hans J. , Whitfeld Paul R. , Bottomley Warwick
Published: May 2006

The gene for the so-called
M
r
32,000 rapidly labeled photosystem II thylakoid membrane protein (here designated
psbA
) of spinach (
Spinacia oleracea
) chloroplasts is located on the chloroplast DNA in the large single-copy region immediately adjacent to one of the inverted repeat sequences. In this paper we show that the size of the mRNA for this protein is ≈ 1.25 kilobases and that the direction of transcription is towards the inverted repeat unit. The nucleotide sequence of the gene and its flanking regions is presented. The only large open reading frame in the sequence codes for a protein of
M
r
38,950. The nucleotide sequence of
psbA
from
Nicotiana debneyi
also has been determined, and comparison of the sequences from the two species shows them to be highly conserved (>95% homology) throughout the entire reading frame. Conservation of the amino acid sequence is absolute, there being no changes in a total of 353 residues. This leads us to conclude that the primary translation product of
psbA
must be a protein of
M
r
38,950. The protein is characterized by the complete absence of lysine residues and is relatively rich in hydrophobic amino acids, which tend to be clustered. Transcription of spinach
psbA
starts about 86 base pairs before the first ATG codon. Immediately upstream from this point there is a sequence typical of that found in
E. coli
promoters. An almost identical sequence occurs in the equivalent region of
N. debneyi
DNA.
31P NMR studies of control of mitochondrial function in phosphofructokinase-deficient human skeletal muscle.
Authors: Chance B , Eleff S , Bank W , Leigh J S , Warnell R
Published: May 2006

Metabolic control of mitochondrial respiratory activity by Pi and ADP has been evaluated by 31P NMR measurements of the levels of Pi in normal exercising human skeletal tissues in the resting-active-resting transition and, in this contribution, in the phosphofructokinase (PFK)-deficient leg. The latter studies show near constancy of Pi in the recovery from maximal exercise of the leg, with large changes of sugar phosphate (SP) complementary to the changes of phosphocreatine (PCr). The PFK deficiency permits observation of PCr resynthesis in postexercise recovery under conditions of nearly constant Pi and ATP--a phenomenon not evident in normal exercising muscle. The constancy of free Pi is inconsistent with its role in control of mitochondrial activity, leaving ADP as a key metabolic control element. These results help clarify previous controversies on the nature of control of metabolic activity of mitochondria and extend the idea of ADP control of mitochondrial metabolic states in vivo and, in addition, provide an appropriate exercise protocol for the evaluation of a genetic deficiency affecting mitochondrial metabolism.