bluuebunny/crossref_metadata_2025 huggingface dataset:
Coupling in cytochrome
c
oxidase
Authors: Kessler R. J. , Blondin G. A. , Zande H. Vande , Haworth R. A. , Green D. E.
Published: May 2006

Cytochrome
c
oxidase (ferrocytochrome
c
: oxygen oxidoreductase; EC 1.9.3.1) can be resolved into an electron transfer complex (ETC) and an ionophore transfer complex (ITC). Coupling requires an interaction between the moving electron in the ETC and a moving, positively charged ionophore-cation adduct in the ITC. The duplex character of cytochrome oxidase facilitates this interaction. The ITC mediates cyclical cation transport. It can be replaced as the coupling partner by the combination of valinomycin and nigericin in the presence of K
+
when cytochrome oxidase is incorporated into liposomes containing acidic phospholipids or by the combination of lipid cytochrome
c
and bile acids in an ITC-resolved preparation of the ETC. Respiratory control can be induced by incorporating cytochrome oxidase into vesicles of unfractionated whole mitochondrial lipid. The activity of the ITC is suppressed by such incorporation and this suppression leads to the emergence of respiratory control. The ionophoroproteins of the ITC can be extracted into organic solvents; some 50% of the total protein of cytochrome oxidase is extractable. The release of free ionophore is achieved by tryptic digestion of the ionophoroprotein. Preliminary to this release the ionophoroprotein is degraded to an ionophoropeptide. Electrogenic ionophores, as well as uncoupler, are liberated by such proteolysis. The ITC contains a set of ionophoroproteins imbedded in a matrix of phospholipid.
Poliovirus-specific primer-dependent RNA polymerase able to copy poly(A).
Authors: Flanegan J B , Baltimore D
Published: May 2006

A template-dependent RNA polymerase has been isolated from poliovirus-infected cells by assaying for the ability of the enzyme to copy poly(A) complexed to an oligo(U) primer. The polymerase was solubilized with detergent, and RNA was removed by precipitation with 2 M LiCl. The solubilized polymerase required both poly(A) and oligo(U) for activity and was stimulated by Mg2+ but was inhibited by Mn2+. Poly(A)-oligo(U)-dependent poly(U) polymerase was not found in extracts of HeLa cells until about 2 hr after poliovirus infection, and then there was a linear increase in activity until about 5 hr. Analysis of the polymerase by glycerol gradient centrifugation showed that the majority of the activity sedimented at about 4 S, indicating that it was no longer complexed with high-molecular-weight RNA or cellular membranes. This poly(A)-oligo(U)-dependent polymerase activity could represent an important component of the poliovirus RNA-dependent RNA polymerase.
Isolation of adenylate cyclase-free, beta-adrenergic receptor from turkey erythrocyte membranes by affinity chromatography.
Authors: Vauquelin G , Geynet P , Hanoune J , Strosberg A D
Published: May 2006

The adenylate cyclase [ATP pyrophosphatelyase (cyclizing); EC 4.6.1.1] and beta-adrenergic receptor of plasma membranes of turkey erythrocytes were solubilized in an active form by treatment with either NaF or guanylylimidodiphosphate and digitonin. The solubilized enzyme was no longer stimulated by catecholamines, NaF, or guanine nucleotides. The digitonin extract was chromatographed on an alprenolol-agarose derivative. While the bulk of protein and all the adenylate cyclase activity passed unretarded through the column, the receptor was retained. It eluted free of enzyme activity with an alprenolol solution containing 1 M NaCl; the yield was 25-30%. The protein content of the alprenolol eluates was too low to be estimated by the Lowry technique and was assessed by a more sensitive fluorometric method. Under these conditions, the beta-adrenergic receptor was purified approximately 2000-fold in a single step with retention of all its pharmacological properties. These experiments establish that the beta-adrenergic receptor and the adenylate cyclase are independent entities which may be separated on a functional basis.
New detection of brain dopamine receptors with (3H)dihydroergocryptine.
Authors: Tittler M , Weinreich P , Seeman P
Published: May 2006

Because dihydroergocryptine (DHE) and closely related ergots are dopamine-mimetic agonists, we tested [3H]DHE as a possible ligand for [3H]dopamine receptors in the calf caudate. In order to avoid [3H]DHE from tagging alpha-adrenergic receptors, an excess of 500 nM phentolamine was used to block these sites, permitting the dopamine receptors to be measured separately. Specific binding of [3H]DHE was defined as total binding minus that occurring in the presence of 1 muM (+)-butaclamol. Excess phentolamine reduced the specific binding of [3H]DHE from 328 down to 138 fmol/mg, the difference presumably representing alpha-receptors. The KD for [3H]DHE was 0.55 nM (with or without phentolamine), and this high affinity site was blocked (in the presence of phentolamine) by 250nM apomorphine, 650 nM dopamine, and 1200 mM (minus)-norepinephrine, indicating that[3H]DHE was binding to dopamine receptors. All neuroleptics blocked specific [3H]DHE binding in direct relation to the clinical potency of the neuroleptic. The displacement of specific [3H]DHE binding by dopamine or by norepinephrine (in the presence of phentolamine) revealed two subsets of dopamine receptors.
Ligand-promoted weakening of intersubunit bonding domains in aspartate transcarbamoylase
Authors: Subramani Suresh , Bothwell Mark A. , Gibbons Ian , Yang Ying R. , Schachman H. K.
Published: May 2006

The cooperativity and feedback inhibition exhibited by the regulatory enzyme, aspartate transcarbamoylase (carbamoylphosphate: L-aspartate carbamoyltransferase; EC 2.1.3.2), from
Escherichia coli
are generally attributed to ligand-promoted conformational changes involving alterations in the subunit interactions. However, no quantitative estimates have been made of the effect of ligands on the strength of the intersubunit bonding domains. The native enzyme, composed of two catalytic trimers “crosslinked” by three regulatory dimers, shows little tendency to dissociate in neutral buffers at room temperature. In addition, very little exchange was observed in 2 hr between subunits within the intact enzyme and free subunits. Although exchange was enhanced in solutions of low ionic strength containing the bisubstrate analog,
N
-(phosphonacetyl)-L-aspartate, the rates of exchange were too small to permit reliable estimates of the weakening of the bonding domains caused by the ligand. Studies were conducted, therefore, on a less stable oligomeric complex which resembles the native enzyme in structure and allosteric behavior but lacks one regulatory subunit. These molecules, containing only four bonding domains between the catalytic and regulatory polypeptide chains (compared to six in the native enzyme), disproportionate to form the more stable native enzyme and free catalytic subunits. An electrophoretic technique is described for measuring the rate of disproportionation which is controlled by the rupture of the intersubunit bonding domains. This rate is enhanced about 300-fold upon the addition of the active-site ligand. Hence the ligand-promoted allosteric conversion of the enzyme-like complex from the constrained to the relaxed conformation involves a substantial weakening of the intersubunit interactions corresponding to about 1.7 kcal/mole (7.1 kJ/mole) per bonding domain between a catalytic and a regulatory chain.
A postsynthetic modification of human α-fetoprotein controls its immunosuppressive potency
Authors: Lester Eric P. , Miller J. Bruce , Yachnin Stanley
Published: May 2006

In a previous study, we demonstrated three variants of human αfetoprotein by crossed immunoelectrophoresis. In addition, we correlated the capacity of α-fetoprotein isolates from various hepatoma and fetal sources to suppress human lymphocyte transformation
in vitro
with the relative proportion of the electronegative variant, HAFP-3, present in each isolate. We have now isolated α-fetoprotein from the serum, ascitic fluid, and saline extract of tumor from a single hepatoma patient and from a homogenate of fetal livers. When tested for their capacity to inhibit human lymphocyte transformation
in vitro
, tumor and fetal liver αfetoprotein were found to be extremely potent, serum αfetoprotein had intermediate potency, and ascitic fluid α-fetoprotein was the least potent. Analysis of these isolates by crossed immunoelectrophoresis confirmed the correlation between the proportion of HAFP-3 and the immunosuppressive potency of each isolate. In addition, analysis of these isolates by isoelectric focusing in polyacrylamide gels containing 8 M urea revealed further evidence of microheterogeneity; at least six molecular variants were apparent. The proportion of one of these variants, termed HAFP-3a, in each isolate was correlated with the immunosupressive potency of the isolate. The sialic acid content of the various α-fetoprotein isolates did not vary significantly. Our data suggest that a postsynthetic modification of αfetoprotein occurs, probably after secretion, which reduces immunosuppressive potency by converting the active electronegative species to an inactive electropositive form. This modification probably involves a charged moiety other than sialic acid on the molecule.
Phosphorylation of eukaryotic protein synthesis initiation factors.
Authors: Benne R , Edman J , Traut R R , Hershey J W
Published: May 2006

Phosphorylation of eukaryotic initiation factors was examined both in intact cells and in vitro with purified components. Intact rabbit reticulocytes were incubated in a medium containing[32P]phosphate, and eight initiation factors were isolated and partially purified. The purified factors were analyzed on dodecyl sulfate/polyacrylamide gels and compared with highly purified nonradioactive factors. Significant amounts of radioactivity were found associated with initiation factors eIF-2, polypeptide 2 (molecular weight 53,000); eIF-3, polypeptides 2 and 4 (molecular weights 110,000 and 67,000); and eIF-4B. Purfied initiation factors from rabbit reticulocytes were also treated in vitro with [gamma-32P]ATP and a cyclic AMP-independent protein kinase isolated from rabbit erythrocytes. Only the factor polypeptides phosphorylated intracellularly were phosphorylated in vitro. The results suggest that the cyclic AMP-independent protein kinase is responsible for the phosphorylation of specific initiation factors in cells active in protein synthesis and that it may play a role in regulating translation.
Paired ions: Dipolar pairs as subset of diffusion pairs
Authors: Fuoss Raymond M.
Published: May 2006

Previous models for which theories of electrolytic conductance have been developed are reviewed. Discrepancies between theoretically derived values of parameters and parameters characteristic of real physical systems suggested the following revised model. Ions are counted as diffusion pairs if their center-to-center distance
r
is in the range
a
≤
r
≤
R
, in which
a
is contact distance and
R
is the diameter of the Gurney cosphere. A fraction α of these pairs diffuse to contact to form nonconducting dipolar pairs; α/(1-α) = exp(-
E
s
/
kT
), in which
E
s
is the difference in energy between a diffusion pair at
r
=
R
and a contact pair,
k
is the Boltzmann constant, and
T
is the absolute temperature. This model permits separate treatment of long-range and short-range interionic effects. The former (relaxation field and electrophoresis) depend on
R
and the values of the dielectric constant and viscosity of the pure solvent. The latter (formation of dipolar pairs) is described by
E
s
, or alternatively by
K
s
= exp(-
E
s
/
kT
) in which
K
s
is the constant describing the steady state between solvent-separated diffusion pairs and dipolar (contact) pairs. For solutions of the alkali halides, a simple empirical correlation is found between
R
and the Pauling radii of the cations, and also between
E
s
and the sum of the radii of cation and anion.
Diphtheria toxin has the properties of a lectin.
Authors: Draper R K , Chin D , Simon M I
Published: May 2006

The inhibition of protein synthesis in Chinese hamster V79 cells by diphtheria toxin is antagonized by the lectins concanavalin A, succinylated concanavalin A, and wheat germ agglutinin but not by Proteus vulgaris phytohemagglutinin or abrus agglutinin. The effects of concanavalin A and wheat germ agglutinin are reversed by methyl alpha-mannoside and N-acetylglucosamine, respectively. The inhibition of diphtheria toxin as a function of concanavalin A concentration fits a model of competitive inhibition with an apparent dissociation constant for concanavalin A of 3 X 10(-8) M. These results suggest that the diphtheria toxin receptor may be an oligosaccharide. To test this hypothesis, we screened several oligosaccharides for the ability to inhibit diphtheria toxin. The cell wall polysaccharide of Salmonella cholera suis and the ovalbumin glycopeptide were effective inhibitors. These studies suggest that diphtheria toxin may have the oligosaccharide binding properties of a lectin with specificity for N-acetylglucosamine and mannose.
Detection of prokaryotic signal peptidase in an Escherichia coli membrane fraction: endoproteolytic cleavage of nascent f1 pre-coat protein.
Authors: Chang C N , Blobel G , Model P
Published: May 2006

An inverted membrane vesicle fraction isolated from uninfected Escherichia coli and largely derived from the inner membrane has been shown to contain an endoproteolytic activity that cleaves nascent bacteriophage f1 pre-coat protein into two identifiable products. The electrophoretic mobility on sodium dodecyl sulfate/urea/polyacrylamide gels and the partial amino-terminal sequence of the larger fragment were indistinguishable from those of the mature phage coat protein. Partial amino-terminal sequence analysis showed that the smaller fragment corresponds to the amino-terminal "signal peptide" of f1 pre-coat protein. Cleavage occurred only if the membrane fraction was present during in vitro synthesis, and was not observed if it was added after completion of pre-coat protein synthesis. The cleavage reaction was strongly stimulated when the membrane fraction was present together with the nonionic detergent Nikkol. These results are consistent with and discussed in terms of the signal hyothesis.
Silkmoth chorion proteins: sequence analysis of the products of a multigene family.
Authors: Regier J C , Kafatos F C , Goodfliesh R , Hood L
Published: May 2006

Five polypeptide components have been isolated from the eggshell (chorions) of a silkmoth. Two are homogeneous on sodium dodecyl sulfate and isoelectric focusing gels, and three contain predominantly two proteins each. Amino acid analyses show that all five components are similar to each other. These proteins have been sequenced from the amino terminus. Homogeneous components yielded single sequences; heterogeneous components yielded two residues at some positions, consistent with their containing two major electrophoretic components. Striking similarities are apparent among all these sequences. These similarities can be increased dramatically by separating each of the three protein mixtures into two sequences and introducing a small number of gaps or insertions. This is due in part to bringing into register a portion that contains short repeating subunits found in all sequences. All proteins are also characterized by a region of high cysteine content near the amino terminus followed by a longer low-cysteine region. The data suggest that these proteins share a common evolutionary origin and are encoded by a multigene family.
Evidence that tumor antigens enhance tumor growth in vivo by interacting with a radiosensitive (suppressor?) cell population.
Authors: Hellström K E , Hellström I
Published: May 2006

The growth of a small number of cells from each of two chemically induced BALB/c sarcomas was enhanced when x-irradiated (15,000 rads) cells of the same sarcoma were mixed with the tumor inoculum. This enhancement did not occur if the recipients had been given a total body x-irradiation of 450 rads. Tumor neutralization (Winn) tests showed that tumor cells irradiated in vitro enhanced tumor growth only in the presence of radiosensitive cells present in the spleens of both nonimmune and tumor-bearing mice. On the basis of these findings we postulate that tumor antigen blocks effective tumor immunity by a mechanism that involves a suppressor cell population.
Characterization and mapping of RNase T1-resistant oligonucleotides derived from the genomes of Akv and MCF murine leukemia viruses.
Authors: Rommelaere J , Faller D V , Hopkins N
Published: May 2006

T1 RNA fingerprints of the genomes of Akv-1 and Akv-2 C-type viruses are indistinguishable and oligonucleotide maps of these viruses are probably the same. Akv-1 and -2 share 55--75% of their large T1-resistant oligonucleotides with four MCF viruses isolated from AKR mice or from NIH Swiss mice that inherit either the Akv-1 or Akv-2 virus-inducing locus of AKR. The majority of Akv oligonucleotides missing from T1 fingerprints of MCFs and the majority of oligonucleotides unique to MCF viruses are clustered and lie at corresponding positions in the 3' half of the oligonucleotide maps of Akv and MCF viruses. The RNA sequences present in different MCF isolates but not present in Akv-viruses are related. These results are consistent with a recombinational origin of MCF viruses, as proposed by Hartley and Rowe and their collaborators.
Structural analysis of spermine and magnesium ion binding to yeast phenylalanine transfer RNA.
Authors: Quigley G J , Teeter M M , Rich A
Published: May 2006

Refinement of the diffraction data at 2.5-A resolution from orthorhombic crystals of yeast tRNAPhe has proceeded to the point where spermine and magnesium ions can be located in the difference electron density map. Two spermine molecules are found: one is located in the major groove at one end of the anticodon stem; the other is near the variable loop and curls around phosphate 10 in a region where the polynucleotide chain takes a sharp turn. Four distinct magnesium ions have been identified: one in the anticodon loop, two in the D loop, and one coordinated with phosphates 8, 9, 11, and 12, where the polynucleotide chain is coiled. The conformation of the anticodon stem and loop is stabilized by the cations at the end of the molecule. The positions of these ions may be related to aspects of the biological activity of tRNA. The spermine and magnesium ions appear to be important in maintaining the overall folding of the tRNA molecule.
Tumor antigen(s) in cells productively infected by wild-type polyoma virus and mutant NG-18
Authors: Schaffhausen Brian S. , Silver Jonathan E. , Benjamin Thomas L.
Published: May 2006

When isolated by means of an anti-polyoma tumor (T) antiserum, the major product from mouse cells productively infected by wild-type polyoma virus is a polypeptide of 100,000 apparent molecular weight as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In cells infected by NG-18, an
hr-t
mutant carrying a deletion of about 150 base pairs in the early region of the viral DNA, a T antigen species appears that comigrates with that of the wild-type virus. Comparisons of peptides after partial proteolysis reveal no differences between mutant and wild-type products. Both wild-type and mutant 100,000 products can be labeled
in vivo
with [
32
P]orthophosphate. An independent and more reliable estimate of the molecular weight of this protein using guanidine/Sepharose chromatography yields a value of 81,000 for both mutant and wild-type species. The apparent identity of wild-type and mutant products indicates that the deletion in NG-18 lies outside of the region encoding this major T antigen species.

Immunoprecipitates from wild-type infected cells shows four bands in addition to the “100,000” band; these have apparent molecular weights of 63,000, 56,000, 36,000, and 22,000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis; the 56,000 and 36,000 species are phosphorylated. All four of these lower molecular weight bands are absent or drastically reduced in the immunoprecipitates from NG-18-infected cells.
Gamma heavy chain disease in man: translation and partial purification of mRNA coding for the deleted protein.
Authors: Alexander A , Barritault D , Buxbaum J
Published: May 2006

Lymphoid cells obtained from the peripheral blood of a patient with heavy chain disease have been established in long-term culture. They continue to produce a protein antigenically identical to the deleted gamma3 heavy chain disease protein found in the patient's serum. The availability of the cell line has made it possible to analyze the mRNA coding for this protein. The primary in vitro translation product is 1500-2000 daltons larger than the polypeptide portion of the cytoplasmic or secreted protein and has methionine at the amino terminus. The mRNA sediments at 15.5 S on sucrose gradients and therefore appears to be smaller than the 17S message coding for normal-sized mouse gamma chains. It contains a base sequence that codes for a hydrophobic amino-terminal peptide not found in the cytoplasmic protein. There was no evidence for the synthesis of translatable light chain message by these cells. The present data suggest that this protein results from a primary somatic genetic event that gave rise to a cell product bearing a normal aminoterminus sensitive to limited proteolytic digestion. The serum protein thus appears to begin in the hinge region but, in fact, contains a normal heavy chain initiation site.
Absolute configuration of the diastereomers of adenosine 5'-O-(1-thiotriphosphate): consequences for the stereochemistry of polymerization by DNA-dependent RNA polymerase from Escherichia coli.
Authors: Burgers P M , Eckstein F
Published: May 2006

The diastereomers of uridyl-(3'-5')adenyl-O,O-phosphorothioate [Up(S)A] have been separated by high-performance liquid chromatography. Their identification as RP and SP follows from the RNase A digestion of these products. It was then shown, by the same method, that the R isomer is hydrolyzed by snake venom phosphodiesterase (PDEase) approximately 500 times faster than the S isomer. Similarly, the stereoisomer of adenosine 5'-O-(1-thiotriphosphate) (ATPalphaS), until now arbitrarily designated as isomer B, is hydrolyzed ca 400 times faster by PDEase than is isomer A. From these results it is concluded that the R isomers of Up(S)A and ATPalphaS, isomers B, have the same absolute configuration. It then follows that isomer A of ATPalphaS, the preferred of the two isomers as substrate for DNA-dependent RNA polymerase, has the S configuration. The implications for the stereochemistry of action of the latter enzyme are discussed.
Purification and characterization of branched chain α-keto acid dehydrogenase complex of bovine kidney
Authors: Pettit Flora H. , Yeaman Stephen J. , Reed Lester J.
Published: May 2006

A branched chain α-keto acid dehydrogenase-dihydrolipoyl transacylase complex was purified to apparent homogeneity from bovine kidney mitochondria. As usually isolated, the complex (
s

20,
w

= 40 S) contained little, if any, dihydrolipoyl dehydrogenase. When saturated with the latter enzyme the complex had a specific activity of about 12 μmol of α-ketoisovalerate oxidized per min per mg of protein at 30° with NAD
+
as electron acceptor. In addition to α-ketoisovalerate, the complex also oxidized α-ketoisocaproate, α-keto-β-methylvalerate, α-ketobutyrate, and pyruvate. The ratios of the specific activities were 2.0:1.5:1.0:1.0:0.4, and the apparent
K
m
values were 40, 50, 37, 56, and 1000 μM. The complex was separated into its component enzymes. The branched chain α-keto acid dehydrogenase (6 S) consists of two different subunits with estimated molecular weights of 46,000 and 35,000. The dihydrolipoyl transacylase (20 S) contains apparently identical subunits of molecular weight about 52,000. In the electron microscope, the transacylase has the appearance of a cube, and the molecules of branched chain α-keto acid dehydrogenase appear to be distributed on the surface of the cube. In contrast to the pyruvate dehydrogenase complex of bovine kidney, the branched chain α-keto acid dehydrogenase complex apparently is not regulated by phosphorylation-dephosphorylation. Its activity, however, is subject to modulation by end-product inhibition.
Estrogen receptor has enhanced affinity for bromodeoxyuridine-substituted DNA.
Authors: Kallos J , Fasy T M , Hollander V P , Bick M D
Published: May 2006

The estrogen receptor (E2R) from rabbit uterus has an enhanced affinity for BrdUrd-substituted DNA compared to unsubstituted DNA. Increasing levels of BrdUrd substitution (from 0 to 100%) in a DNA sample are associated with increasingly tighter binding of E2R to that DNA as measured by equilibrium competition experiments and by rates of dissociation (or receptor transfer experiments). Although the rates of dissociation of E2R-DNA complexes vary greatly, depending on the BrdUrd-substitution level in the DNA, no differences were detected between the rates of association of E2R with unsubstituted and fully BrdUrd-substituted DNA. The E2R-DNA complex dissociates more rapidly in 150 mM KCl than in 50 mM KCl; but, at both ionic strengths, BrdUrd substitution in the DNA confers enhanced stability on the complex. The demonstration that a specific mammalian regulatory protein has an enhanced affinity for BrdUrd-substituted DNA further strengthens the possibility that BrdUrd modulates gene expression through an altered binding of regulatory proteins.
Mid-G1 marker protein(s) in 3T3 mouse fibroblast cells.
Authors: Gates B J , Friedkin M
Published: May 2006

Quiescent 3T3 mouse fibroblast cells in a state of growth arrest due to serum deprivation were exposed to [14C]isoleucine. The cell cultures were then stimulated by the addition of 10% fetal calf serum. At various times after stimulation, the 14C-labeled cells were exposed to [3H]isoleucine. Cytoplasmic extracts from the double-labeled cells were subjected to sodium dodecyl sulfate/slab gel electrophoresis. By these procedures it was found that the relative rate of synthesis of a protein species of Mr 50,000 increased after stimulation of quiescent cells, reached a maximum at 5 hr, and then decreased before the 3T3 cells began to enter the S phase. The characteristic peaking profile of mid-G1 protein synthesis exhibited by the Mr 50,000 polypeptide can serve as a useful marker for the progression of events in G1 prior to exit into S.
Sodium-dependent amino acid transport by cultured hamster cells: membrane vesicles retain transport changes due to glucose starvation and cycloheximide.
Authors: Nishino H , Christopher C W , Schiller R M , Gammon M T , Ullrey D , Isselbacher K J
Published: May 2006

Enhanced alpha-aminoisobutyric acid transport by hamster cells cultured in the absence of D-glucose has been demonstrated in isolated membrane vesicles. The observed enhancement was seen in the presence but not in the absence of Na+. Kinetic analysis of transport using both the intact cells and the membrane vesicles showed that the overall enhancement was associated with an increase in Vmax. Decreases in transport activity by intact cells resulting from extended exposure of the cells to inhibitors of protein synthesis, such as cycloheximide, were also evident in membrane vesicles. The use of metabolically inactive membrane vesicles demonstrated that amino acid uptake by intact cells is a transport property of the plasma membrane. In addition, this study shows that membrane vesicle preparations can be exploited for the purpose of studying the regulation of amino acid transport. Taken together, the data suggest that carrier turnover is involved in the regulation of amino acid transport in animal cells.
Asymmetric budding of viruses in epithelial monlayers: a model system for study of epithelial polarity.
Authors: Boulan E R , Sabatini D D
Published: May 2006

Infection of two different lines of polarized epithelial cells grown as monolayers with several types of enveloped viruses results, for each virus type, in a characteristic asymmetric budding of virions. Influenza virus (WSN strain), simian virus 5, and Sendai virus bud exclusively from the free (apical) surface of the cells, while vesicular stomatitis virus acquires its envelope only from the basolateral plasma membrane. Because different viruses select specific domains of plasma membrane in the same cell type, virus-infected epithelial monolayers can provide an excellent model system for studies of the mechanisms that generate regional differences in the distribution of plasma membrane components of epithelial cells.
Serological analysis of cell surface antigens of malignant human brain tumors
Authors: Pfreundschuh Michael , Shiku Hiroshi , Takahashi Toshitada , Ueda Ryuzo , Ransohoff Joseph , Oettgen Herbert F. , Old Lloyd J.
Published: May 2006

Sera from 30 patients with astrocytoma were tested for antibody reacting with cell surface antigens of cultured autologous astrocytoma cells. Ten percent of the patients had antibody detectable by mixed hemadsorption assays, ≈50% by immune adherence and protein A assays, and 100% by anti-C3-mixed hemadsorption assays. Absorption analysis of reactive sera with autologous, allogeneic, and xenogeneic cells permitted the definition of three classes of astrocytoma cell surface antigens. Class I antigens showed an absolute restriction to autologous astrocytoma cells. Class II antigens were shared by all astrocytomas tested and could be detected also on neuroblastoma, sarcoma, and some (but not all) melanoma cell lines; these antigens were not found on cell lines derived from carcinomas or normal tissues. Class III antigens were widely distributed on cultured normal and malignant cells of human and animal origin. In this series, sera from 2 patients recognized class I antigens, 4 patients' serum recognized class II antigens, and 13 patients' sera recognized class III antigens. Absorption tests have shown that the AJ (class II) antigen of astrocytoma is serologically related to the previously described AH (class II) antigen of melanoma; in tests of nine melanoma cell lines, there was a correspondence between the AJ and AH phenotypes. This method of autologous typing provides a way to classify the cell surface antigens of astrocytomas and to assess the clinical significance of humoral immunity to these antigens.
Direct evidence for loss of human suppressor cells during active autoimmune disease.
Authors: Strelkauskas A J , Callery R T , McDowell J , Borel Y , Schlossman S F
Published: May 2006

These studies indicate that a regulatory subset of lymphocytes is missing in patients with juvenile rheumatoid arthritis but these patients have antibodies in their serum that react with normal T cells. This regulatory subset of T cells is, however, present in patients whose serum shows little or no reactivity with normal T cells. In addition, patients who are deficient in this regulatory subset of lymphocytes significantly higher numbers of cells secreting Ig as measured by a hemolytic plaque assay. The significance of these observations is twofold: first, they represent a positive relationship among the loss of regulation overproduction of immunoglobulin, and the presence of anti-T cell antibodies and second and perhaps of equal importance, is the indication that serum from patients with autoimmune diseases may give us a readily available reagent with which to dissect further functionally distinct subsets of normal T cells in man.
Influence of hyperinsulinemia, hyperglycemia, and the route of glucose administration on splanchnic glucose exchange
Authors: DeFronzo Ralph A. , Ferrannini Eleuterio , Hendler Rosa , Wahren John , Felig Philip
Published: May 2006

The effects of hyperinsulinemia, hyperglycemia, and the route of glucose administration on total glucose utilization and on net splanchnic glucose exchange were studied in 20 normal volunteers with the hepatic venous catheter technique. Euglycemic hyperinsulinemia [induced by a priming plus continuous infusion of insulin resulting in plasma insulin levels of 400-1200 μunits (international)/ml and a variable glucose infusion] caused a 5- to 6-fold increase above basal in total glucose turnover. However, net splanchnic glucose uptake (0.5 ± 0.2 mg/kg per min) accounted for only 4-5% of total glucose utilization. When hyperglycemia (223 ± 1 mg/dl) was induced in addition to hyperinsulinemia by the intravenous infusion of glucose, splanchnic glucose uptake increased 100% to 1.0-1.1 mg/kg per min but was still responsible for only 10-14% of total glucose utilization. In other studies hyperglycemia (223 ± 2 mg/dl) was maintained constant by a variable intravenous infusion of glucose for 4 hr and oral glucose (1.2 gm/kg) was administered at 1 hr. After the oral glucose, net splanchnic glucose uptake increased to values 6-fold higher than with intravenous glucose despite unchanged plasma glucose levels and plasma insulin concentrations well below those observed in the studies with euglycemic hyperinsulinemia. The results indicate that hyperinsulinemia or hyperglycemia induced by intravenous infusion of glucose or insulin causes minimal net uptake of glucose by the splanchnic bed despite marked stimulation of total glucose turnover. In contrast, administration of glucose by the oral route has a marked stimulatory effect on net splanchnic glucose uptake. These findings suggest that orally consumed glucose causes the release of a gastrointestinal factor that enhances insulin-mediated glucose uptake by the liver.
Spectroscopic studies of oxy- and carbonmonoxyhemoglobin after pulsed optical excitation.
Authors: Greene B I , Hochstrasser R M , Weisman R B , Eaton W A
Published: May 2006

The photolysis of HbO2 and HbCO has been investigated with picosecond laser techniques. Transient absorption spectra were measured in the Soret and visible regions after excitation with 353- or 530-nm pulses. The photoproducts appeared within 8 psec and exhibited considerably broadened deoxyhemoglobin-like spectra, which persisted to 680 psec. The altered spectra are attributed to the production of deoxyheme conformational and spin states that might result from the intense excitation.
Formation of protein micelles from amphiphilic membrane proteins.
Authors: Simons K , Helenius A , Leonard K , Sarvas M , Gething M J
Published: May 2006

The membrane penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) from Bacillus licheniformis, the Semliki Forest virus spike proteins, and the Sendai virus glycoproteins have each been isolated as soluble protein aggregates that are virtually free of lipid and detergent. The sedimentation coefficients of the complexes were 18 S, 29 S, and 43 S, respectively. Mixed aggregates containing both the virus glycoproteins and the penicillinase could also be formed. Such protein micelles may serve a number of useful purposes in membrane research.
Oxidation of the methionine residues of Escherichia coli ribosomal protein L12 decreases the protein's biological activity.
Authors: Caldwell P , Luk D C , Weissbach H , Brot N
Published: May 2006

Oxidation of ribosomal protein L12 with hydrogen peroxide converts the three methionine residues to methionine sulfoxide. The oxidized protein has a decreased ability to bind to ribosomes, interact with ribosomal protein L10, be precipitated by L12 antiserum, and serve as substrate for the acetylating enzyme that converts L12 to L7. Full activity of L12 is regained when the protein is reduced with 2-mercaptoethanol. Sedimentation equilibrium analysis shows that oxidation of the methionine residues in L12 causes the conversion of the protein from the dimer to the monomer form, and the results indicate that the dimer is the active form of the protein in the above reactions.
Vitamin K-dependent carboxylase: evidence for a hydroperoxide intermediate in the reaction.
Authors: Larson A E , Suttie J W
Published: May 2006

Vitamin K is an essential cofactor for a microsomal carboxylase that converts glutamyl residues in endogenous precursor proteins to gamma-carboxyglutamyl residues in completed proteins. The same microsomal preparations convert vitamin K to its 2,3-epoxide, and it has been suggested that these two reactions (carboxylation and epoxidation) are coupled. Glutathione peroxidase, which reduces hydrogen peroxide and organic hydroperoxides, inhibits both of these reactions in a prepartion of microsomes solubilized by Triton X-100. Catalase has no effect. In the absence of vitamin K, and in the presence of NADPH, tert-butyl hydroperoxide acts as a weak vitamin K analog. At lower concentrations, tert-butyl hydroperoxide is an apparent competitive inhibitor of vitamin K for both the carboxylase and epoxidase reactions. These data are consistent with the hypothesis that both of these vitamin K-requiring reactions involve a common oxygenated intermediate, and that a hydroperoxide of the vitamin is the species involved.
Effect of transmembrane ion gradients on Raman spectra of sealed, hemoglobin-free erythrocyte membrane vesicles.
Authors: Mikkelsen R B , Verma S P , Wallach D F
Published: May 2006

Sealed hemoglobin-free erythrocyte vesicles have been isolated. Imposition of transmembrane cation gradients increases the intensity of Raman scattering in the CH3-stretching region as observed with unsealed ghosts at temperatures greater than 38 degrees C and pH less than 7.0 [Verma, S. P. & Wallach, D. F. H. (1976) Proc. Natl. Acad. Sci. USA 73, 3358--3561]. Modifications in the amide I and amide III frequencies consistent with increased helicity of membrane proteins are observed upon imposition of a cation gradient. Spectrin-free vesicles also demonstrate cation gradient-sensitive intensity changes in the CH3-stretching region. However, no evidence for cation gradient-related protein conformation changes is found with these vesicles. The transmembrane potential of these vesicles has been altered by variations in anion composition and the electrogenic activity of Na+,K+-ATPase. The membrane potential was monitored by cyanine dye fluorescence. Imposition of a membrane potential (negative inside) also increased the intensity of Raman scattering in the CH3-stretching region. These results suggest that a transmembrane potential (negative inside) and/or cation gradient can energize membranes by compression of the apolar region and transfer of protein methyl residues into polar regions.
Translation of animal virus RNA in the cytoplasm of a plant cell.
Authors: Cairns E , Gschwender H H , Primke M , Yamakawa M , Traub P , Schweiger H G
Published: May 2006

Isolated Acetabularia crenulata nuclei were injected with Mengo virus RNA solution and then implanted into anucleate posterior Acetabularia mediterranea cell fragments or fused with Acetabularia ryukyuensis cytoplasts. The injected animal virus RNA was actively translated in the plant cell cytoplasm. Mengo virus proteins were detected and localized in Acetabularia cytoplasts by use of an immunofluorescence method on the first to fifth day after injection.
Location on chromosome 1 of Rnr, a gene that regulates renin in the submaxillary gland of the mouse.
Authors: Wilson C M , Erdös E G , Wilson J D , Taylor B A
Published: May 2006

Renin activity (EC 3.4.99.19) was measured in submaxillary gland extracts from four sets of recombinant inbred mouse strains. Recombination between Rnr, a gene that mediates the susceptibility of submaxillary gland renin to induction by androgen, and Dip-1, a chromosome 1 marker, was found in only 1 of 51 recombinant inbred strains, indicating that the two genes are closely linked on chromosome 1. Renin activity in androgen-treated female mice of all recombinant inbred strains resembled that of one or the other progenitor strains, as expected if a single gene is responsible. Documentation that a single gene can have major effects on renin in the submaxillary gland of the mouse implies that single gene differences might explain known variations in renin in other species.
Differential expression of poly(A)-adjacent sequences of mammary tumor virus RNA in murine mammary cells
Authors: Dudley Jaquelin P. , Rosen Jeffrey M. , Butel Janet S.
Published: May 2006

Two DNA probes representative of either the entire mouse mammary tumor virus (MMTV) genome or the poly(A)-adjacent sequences at the 3′ end of MMTV RNA were synthesized with calf thymus DNA or oligo(dT) primers, respectively. These probes were used to study the expression of endogenous MMTV sequences in several BALB/c mammary tumor cell lines, in normal lactating BALB/c tissue, and in a cloned C3H tumor cell line. Both probes were characterized with respect to their rates of hybridization with template RNA, their size as determined by alkaline sucrose gradient centrifugation, and the thermal stability of the cDNA·MMTV RNA hybrids. In addition, the ability of the calf thymus oligodeoxy-nucleotide- or oligo(dT)-primed probes to protect
125
I-labeled MMTV RNA or
125
I-labeled poly(A)-adjacent MMTV RNA sequences from S1 nuclease digestion was determined. Hybridization analysis with these two probes indicated that (
i
) there were approximately 20-fold more oligo(dT)-primed sequences in BALB/c lactating tissue than there were sequences representing the entire genome; (
ii
) in BALB/c tumor cells, the oligo(dT):random oligonucleotide-primed cDNA sequence ratio was reduced to 4:1; and (
iii
) in virus-producer C3H tumor cells, there was only a 2-fold excess of oligo(dT)-primed sequences over that observed with a representative cDNA. These results are consistent with the presence of subgenomic viral mRNA species, integration of partial proviral copies, or altered mRNA processing.
Phosphorylation in vivo and in vitro of human histocompatibility antigens (HLA-A and HLA-B) in the carboxy-terminal intracellular domain.
Authors: Pober J S , Guild B C , Strominger J L
Published: May 2006

HLA-A and -B antigens are phosphorylated in transformed lymphoblastoid cells and peripheral blood lymphocytes, both incubated with 32Pi. The phosphate group is attached to HLA-A and -B heavy chain (p44) as identified by immunoprecipitation with anti-beta2-microglobulin IgG, sodium dodecyl sulfate/polyacrylamide gel electrophoresis, isoelectric focusing, and susceptibility to limited proteolysis by papain and trypsin. The site(s) of phosphorylation is identified as a serine residue(s) located in the hydrophilic carboxy terminus of the p44 chain. HLA antigens are also phosphorylated in isolated membranes from transformed lymphoblastoid cells that are incubated with [gamma32P]ATP. The phosphorylation of the carboxy terminus of HLA-A and -B antigens in vivo is good evidence that this portion of the molecule is intracellular. Furthermore, this modification suggests a general way in which interactions between membrane proteins and cytoskeletal elements may be regulated.
A third site of porton translocation in green plant photosynthetic electron transport
Authors: Velthuys B. R.
Published: May 2006

Prereduction of the intersystem pool in isolated spinach chloroplasts leads to the appearance of a slow phase in the field-indicating 515-nm change induced by a flash. Measurements with the pH-indicator dye cresol red show that a proton uptake is associated with this slow 515-nm change. When water is the electron donor to photosystem II, electron transfer to ferricyanide is associated with the uptake of more than one proton per electron. Tetraphenylboron upon oxidation by system II releases a proton directly into the medium; yet, flash-induced electron transport from tetraphenylboron to ferricyanide is accompanied by a net uptake of protons from the medium. The above four results demonstrate the existence of two proton translocation sites in the chain between the two photoacts; the first two observations locate the new site at the oxidizing side of the plastoquinone pool.
Systemic administration of beta-endorphin: potent hypotensive effect involving a serotonergic pathway.
Authors: Lemaire I , Tseng R , Lemaire S
Published: May 2006

In normal adult rats anesthetized with urethane, intravenous injections of beta-endorphin (30--150 micrograms kg-1) induced a transient fall of blood pressure followed by a small hypertension and a prolonged hypotension. Prior administration of naloxone completely blocked these effects, whereas naloxone, given 1 hr after beta-endorphin, did not reverse the prolonged depressor phase of the opioid peptide. The effects of beta-endorphin on the arterial blood pressure were greatly reduced in animals pretreated with p-chlorophenylalanine, a specific depletor of serotonin. Moreover, in rats pretreated with potent serotonin antagonists such as cyproheptadine, mianserin, and metergoline, beta-endorphin did not produce a significant hypotension. Furthermore, the depressor effect of beta-endorphin was potentiated by fluoxetine, a specific serotonin uptake inhibitor. These observations suggest the participation of a serotonergic pathway in the action of beta-endorphin on the arterial blood pressure.
High recovery of nitrogenase activity and of
55
Fe-labeled nitrogenase in heterocysts isolated from
Anabaena variabilis
Authors: Peterson Richard B. , Wolk C. Peter
Published: May 2006

Heterocysts were isolated from the N
2
-fixing cyanobacterium
Anabaena variabilis
after vegetative cells were disrupted by treatment with lysozyme and cavitation in a sonic cleaning bath. The acetylene-reducing (nitrogenase) activity of the isolated heterocysts,
ca
. 5.0 μmol (mg of chlorophyll
a
)
-1
min
-1
in the presence of H
2
and light, accounted for an average of 60% of the nitrogenase activity of whole filaments, and was relatively insensitive to inactivation by oxygen. Soluble extracts derived from intact filaments grown with
55
Fe, and from their heterocysts and vegetative cells, were subjected to electrophoresis. The nitrogenase and nitrogenase reductase bands (MoFe protein and Fe protein, or component 1 and component 2, respectively) were identified in these nondenaturing gels, and their radioactivities were quantitated. The isolated heterocysts accounted for an average of 91% of the nitrogenase and 69% of the nitrogenase reductase of the original filaments.
Nerve growth factor increases activity of ornithine decarboxylase in rat brain
Authors: Lewis Michael E. , Lakshmanan J. , Nagaiah K. , MacDonnell Paul C. , Guroff Gordon
Published: May 2006

Intraventricular administration of nanogram quantities of nerve growth factor to adult rats results in a marked increase in the activity of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) in the brain. The increase occurs in all major brain regions and the activity is maximal by 7.5 hr after administration. The enzyme response to nerve growth factor increases in magnitude during maturation; the relative increase in ornithine decarboxylase activity in adult animals is much greater than that in young. Neither insulin nor bovine growth hormone was able to increase ornithine decarboxylase activity to the same extent as did nerve growth factor. When brain was separated into neuronal- and glial-enriched fractions, induction of ornithine decarboxylase was found in both, but a greater increase was observed in the glial fraction.