bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of β,β-turns and unordered conformations in polypeptide chains by vacuum ultraviolet circular dichroism
Authors: Brahms S. , Brahms J. , Spach G. , Brack A.
Published: May 2006

Different conformations of polypeptides were characterized by measurements of the circular dichroism (CD) extended into the vacuum ultraviolet region. (
i
) The linear β-pleated sheet structure was characterized in a broad ultraviolet region down to 165 nm by examination of copolypeptides composed of alternating hydrophobic and hydrophilic amino-acid residues, e.g., poly(Lys-Leu-Lys-Leu). A short-wavelength intense band was found at about 169 nm, which is characteristic of β-pleated sheet conformation. (
ii
) The β-turns were experimentally measured using poly(Ala
2
-Gly
2
) in a broad spectral region down to 165 nm with accuracy. The observed CD spectrum is in excellent qualitative agreement with the theoretical curve calculated by Woody for the β-turns of type II and/or I of Venkatachalam. The similarity in shape between the theoretical curve and the observed CD spectra suggests a dominance of β-turn segments in the poly(Ala
2
-Gly
2
) structure. The presence of β-turns in poly(Ala
2
-Gly
2
) is also in agreement with the characterization of this polypeptide by solid state methods (electron microscopy and x-ray diffraction). The CD spectrum of β-turns is characterized by a very intense band at 207.5 nm and strong negative bands at 191 and 169 nm. Copolypeptides such as poly(Ala
2
-Gly
3
) and poly(Ala
3
-Gly
3
) yielded a similar type of CD spectrum, analysis of which indicates that a large fraction of their residues is contained in β-turn regions. (
iii
) The CD spectrum of the unordered chain of these alternating copolypeptides in salt-free solution is observed in the vacuum ultraviolet region.
Unequivocal demonstration of fructose-1,6-bisphosphatase in mammalian brain.
Authors: Majumder A L , Eisenberg F
Published: May 2006

Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase; EC 3.1.3.11) has been found in rat brain and identified unequivocally. The enzyme has been purified to 95% homogeneity by standard procedures, including adsorption to a phosphocellulose column followed by elution with substrate. The purified enzyme exhibits a broad optimum above pH 7.6. Both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate are substrates of this enzyme; the hydrolysis of the latter occurs at about 20% of the rate of the former, and the Km for fructose 1,6-bisphosphate is approximately 1.32 X 10(-4) M. 5'-AMP, an inhibitor of other mammalian-fructose-1,6-bisphosphatases, is without effect, and in further contrast with the other enzymes there is no metal requirement for activity. Purified brain enzyme fails to crossreact with the antibody prepared against the purified liver fructose-1,6-bisphosphatase. On the other hand, antiserum produced against the brain fructose-1,6-bisphosphatase quantitatively precipitates the enzyme activity and forms precipitin bands with preparations of brain fructose-1,6-bisphosphatase.
Chemotaxis in
Escherichia coli
: Methylation of
che
gene products
Authors: Silverman Michael , Simon Melvin
Published: May 2006

The products of three chemotaxis-specific genes in
Escherichia coli, cheM, cheD
, and
cheZ
, are methylated. The
cheZ
gene codes for the synthesis of a 24,000 molecular weight polypeptide that appears in the cytoplasm.
cheM
codes for the synthesis of a membrane-bound polypeptide with a molecular weight of 61,000.
cheD
codes for another membrane-bound polypeptide with an apparent molecular weight of 64,000. CheM
-
mutants show chemotaxis toward some attractants (Tar
-
phenotype), while CheD
-
mutants respond to other attractants (Tsr
-
phenotype). The double mutant (CheD
-
, CheM
-
) does not respond to any attractant or repellent tested. Therefore, these polypeptides play a central role in chemotaxis. They collect information from two subsets of chemoreceptors and act as the last step in the chemoreceptor pathway and the first step in the general processing of signals for transmission to the flagellar rotor. It is suggested that they may be involved in both an initial process that reflects the instantaneous state of the chemoreceptors and in an integrative, adaptive process. Two other genes,
cheX
and
cheW
, are required for the methylation of the
cheD
and
cheM
gene products.
Ultrastructural localization of cell membrane G
M1
ganglioside by cholera toxin
Authors: Hansson H.-A. , Holmgren J. , Svennerholm L.
Published: May 2006

An immunoelectron microscopic method is described for sensitive high-resolution visualization of tissuebound cholera toxin. The principle is to incubate cells or tissue sections with toxin and then to localize the bound toxin with toxin-specific peroxidase (donor:hydrogen-peroxide oxidoreductase; EC 1.11.1.7)-conjugated antibody and enzyme substrate. Thin sections are examined for electron-opaque precipitates in a transmission electron microscope. Because of the specific binding of the toxin to membrane ganglioside G
M1
, the method can be used for ultrastructural localization of this ganglioside. Semiquantitative data are obtained by titration of the limiting concentration of cholera toxin producing specific precipitates. The specificity of the method was controlled in various ways, including analyses of the correlation between the immunoelectron microscopy results and determinations of ganglioside G
M1
in tissues with different ganglioside concentrations, tissues hydrolyzed with
Vibrio cholerae
sialidase, tissues in which exogenous G
M1
has been incorporated, and lipid-extracted tissues. The immunoelectron microscopic method demonstrates that membrane G
M1
ganglioside is positioned on the external side exclusively. Cell-bound toxin remains in its original location on the plasma membrane surface of cells below 18°, but appears to be redistributed both laterally and vertically in the membrane of cells incubated at 37° for 30 min or longer. The results of this method indicate that in the central nervous system G
M1
is concentrated in the pre- and postsynaptic membranes of the synaptic terminals; a further increase in reactivity of these structures after hydrolysis of the nervous tissue with
V. cholerae
sialidase suggests that higher gangliosides of the same series are particularly increased in the pre- and postsynaptic junctions.
Enzyme regulation in neuroblastoma cells in a salts/glucose medium: Induction of ornithine decarboxylase by asparagine and glutamine
Authors: Chen Kuang Yu , Canellakis E. S.
Published: May 2006

L-Asparagine is necessary and sufficient for the maximal induction of ornithine decarboxylase (ODC) (L-ornithine carboxy-lyase, EC 4.1.1.17) activity in confluent N18 mouse neuroblastoma cells in a salts/glucose medium; L-asparagine also induces maximal ODC activity when added to a tissue culture medium. L-Glutamine is about one-half as effective as asparagine. Cholera toxin and agents that are known to raise intracellular cyclic AMP concentrations have no effect on the induction of ODC activity unless suboptimal concentrations of asparagine are present in the salts/glucose medium. Whereas actinomycin D does not inhibit induction of ODC activity by asparagine, it inhibits the induction of ODC activity in association with cyclic AMP. In the salts/glucose medium, the rate of loss of ODC activity following the inhibition of protein synthesis by cycloheximide or puromycin depends upon the presence or absence of asparagine; loss is rapid only in the absence of asparagine and does not appear to be related to the inhibition of protein synthesis. These results are discussed in the context that the overlay of the growth medium tends to mask the minimal requirements for enzyme induction, because the composition of the medium defines: (
a
) the requirements for the induction of ODC activity; (
b
) the effect, or lack of effect, of cyclic AMP (and of inducers of intracellular cyclic AMP) on the induction of ODC activity; (
c
) the effect, or lack of effect, of actinomycin D on the induction of ODC activity; and (
d
) the action of puromycin and of cycloheximide on the rate of loss of ODC activity. It will be interesting to determine whether these results are uniquely applicable to ODC, whether many of the reactions attributed to cyclic AMP in the literature may be mediated by asparagine and glutamine, and whether actinomycin D, cycloheximide, and puromycin can be relied upon to differentiate between transcriptional and post-transcriptional control.
Electron paramagnetic resonance studies of photosynthetic electron transport: Photoreduction of ferredoxin and membrane-bound iron-sulfur centers
Authors: Arnon Daniel I. , Tsujimoto Harry Y. , Hiyama Tetsuo
Published: May 2006

Electron paramagnetic resonance spectrometry was used to investigate, at physiological temperatures, light-induced electron transport from membrane-bound iron-sulfur components (bound ferredoxin) to soluble ferredoxin and NADP
+
in membrane fragments (from the blue-green alga,
Nostoc muscorum
) that had high rates of electron transport from water to NADP
+
and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH
2
) to NADP
+
. Illumination at 20° resulted in the photoreduction of membrane-bound iron-sulfur centers A and B. Photoreduction by water gave electron paramagnetic resonance signals of both centers A and B; photoreduction by DCIPH
2
was found to generate a strong electron paramagnetic signal of only center B.

When water was the reductant, the addition and photoreduction of soluble ferredoxin generated additional signals characteristics of soluble ferredoxin without causing a decrease in the amplitude of the signals due to centers A and B. The further addition of NADP
+
(and its photoreduction) greatly diminished signals due to the bound iron-sulfur centers and to soluble ferredoxin. An outflow of electrons from center B to soluble ferredoxin and NADP
+
was particularly pronounced when DCIPH
2
was the reductant. These observations provide the first evidence for a light-induced electron transport between membrane-bound iron-sulfur centers and ferredoxin-NADP
+
. The relationship of these observations to current concepts of photosynthetic electron transport is discussed.
Cell fusion induced by herpes simplex virus is promoted and suppressed by different viral glycoproteins.
Authors: Manservigi R , Spear P G , Buchan A
Published: May 2006

Some of the factors that regulate membrane fusion resulting in polykaryocyte formationhave been investigated, using the model system of human cells infected with mutants of herpes simplex virus (HSV). One of the mutant viruses used in this study (MP) failed to produce the viral glycoprotein designated C2--a nonlethal defect that has previously been correlated with the polykaryocyte-inducing phenotype of this and other mutant strains (wild-type strains of HSV usually induce the aggregation of infected cells rather than their fusion). The other mutant virus (tsB5), a temperature-sensitive conditional-lethal mutant, failed to produce glycoprotein B2 at non-permissive temperature, whereas the synthesis of all other viral products appeared to be normal. We produced and isolated seven recombinants of MP and tsB5 that expressed both of the parental alterations in glycoprotein synthesis. All of the re-combinant viruses induced the fusion of infected cells at 34 degrees (correlated with the absence of C2 expression) but were unable to cause cell fusion at 39 degrees (correlated with the absence of C2 and of B2 expression), even after infection at multiplicities high enough to ensure that all cells in the cultures synthesized viral macromolecules. These results and studies on the dominance or recessiveness of the fusion-inducing phenotype in mixed infections provide evidence that glycoprotein B2 plays a critical role in the promotion of cell fusion and that glycoprotein C2 can act to suppress fusion.
Excision of F plasmid sequences by recombination at directly repeated insertion sequence 2 elements: involvement of recA.
Authors: Deonier R C , Mirels L
Published: May 2006

The DNA of the F plasmid is joined to bacterial DNA sequences in the F' ORF203 by directly repeated insertion sequence 2 (IS2) elements. The rate of excision of the F plasmid form this F' (presumably by recombination at the directly repeated IS2s) has been estimated in both recA+ and recA- strains. Normal F is produced in the recA+ strain, but is not detected in recA-. The autonomous plasmids produced in the recA- background were F's having deletions. F excision in this particular recA+ case is specific in the sense that the directly repeated IS2s appear to be more active in recombination than similarly disposed IS3 direct repetitions in this F'.
Animal cells reversibly permeable to small molecules
Authors: Castellot John J. , Miller Michael R. , Pardee Arthur B.
Published: May 2006

A cell preparation, useful for studying the regulation of metabolism, was developed by making monolayer baby hamster kidney cells permeable. Hypertonically treated cells were permeable to nucleotides, but retained their gross cellular morphology, intact organelles, 100% of their DNA, and 91% of their total protein. The permeable cell synthesized DNA, RNA, and protein rapidly when supplied with the appropriate substrates and cofactors. They either could remain permeable or were able to “reseal” when replaced in complete medium under appropriate conditions. Optimal conditions for DNA synthesis were established for permeable cells, giving rates equal to those of intact cells. Replication rather than repair was shown by the cell-cycle dependence of DNA synthesis and its discontinuous nature. Ribonucleotide reductase was active in permeable cells, permitting equal rates of DNA synthesis when ribonucleotide diphosphates or deoxyribonucleotide triphosphates were provided. Hydroxyurea did not inhibit DNA synthesis in permeable cells supplied with deoxyribonucleotide di- or triphosphates, but drastically inhibited DNA synthesis when ribonucleotide diphosphates were supplied. Hydroxyurea is therefore primarily an inhibitor of ribonucleotide reductase.

Permeability was reversed, exposing permeable cells to [
3
H]thymidine triphosphate, which was incorporated, which labeled nuclei of cells that went on to mitosis. The reversible permeability procedure should prove especially useful in studying the functions of poorly penetrating compounds, such as drugs. Intact cells were unaffected by cytosine arabinoside triphosphate, while cells that had been made permeable and resealed were killed.
Random genetic drift in a cline
Authors: Nagylaki Thomas
Published: May 2006

A diffusion model is derived for random genetic drift in a cline. The monoecious organism occupies an unbounded linear habitat with constant, uniform population density. Migration is homogeneous, symmetric, and independent of genotype. The analysis applies to a single diallelic locus without dominance or mutation. Assuming that Hardy—Weinberg proportions obtain locally, partial differential equations are deduced for the expected gene frequency at each location and the covariance between gene frequencies at any two points of the habitat. For most selection gradients, the population density, ρ, selection intensity,
s
, and migration variance, σ
2
, appear only combined in the single dimensionless parameter, β, the ratio of the characteristic length for migration and random drift to the natural distance for migration and selection. Random drift is highly significant if β « 1; it causes only relatively small variations of order 1/β around the deterministic cline if β » 1. With β ≫ 1, the correlation between the gene frequencies at any two points is very nearly independent of β, and hence is parameter-free for particular forms of the selection gradient. For a very steep selection gradient, β = 2 [unk] 2
s
ρσ, i.e., essentially the product of the square root of the selection intensity and the neighborhood size.
Membrane particle aggregates in innervated and noninnervated cultures of Xenopus embryonic muscle cells.
Authors: Peng H B , Nakajima Y
Published: May 2006

Clusters of membrane particle aggregates were found in the cultures of Xenopus embryonic muscle cells. In innervated cultures, the aggregates were usually found in the vicinity of the nerve. In terms of particle density and morphology, they resembled the postsynaptic particle aggregates of adult skeletal muscle fibers, suggesting that they may be related to acetylcholine receptors. Similar particle aggregates were also found in noninnervated cultures. They may correspond to extrajunctional clusters of acetylcholine receptors or "hot spots."
Deuterium nuclear magnetic resonance investigation of the effects of proteins and polypeptides on hydrocarbon chain order in model membrane systems
Authors: Oldfield E. , Gilmore R. , Glaser M. , Gutowsky H. S. , Hshung J. C. , Kang S. Y. , King Tsoo E. , Meadows M. , Rice D.
Published: May 2006

Deuterium Fourier-transform nuclear magnetic resonance spectra have been obtained of 1-myristoyl 2-(14,14,14-trideutero)myristoyl phosphatidylcholine bilayers at 34.1 MHz by using the quadrupole echo pulse technique. Thereby, we have investigated the effects upon the deuterated dimyristoyl phosphatidylcholine bilayers of the following proteins and polypeptides: gramicidin A, bacteriophage f1 coat protein, beef brain myelin proteolipid apoprotein, cytochrome
b
5
, and cytochrome
c
oxidase (ferrocytochrome
c
:oxygen oxidoreductase, EC 1.9.3.1). Above

T
c

, the transition temperature between the gel and liquid crystal phases, the quadrupole splitting of the deuterium-labeled methyl group is reduced or collapsed in the presence of protein or polypeptide. No evidence has been found for ordered “boundary lipid.” Below

T
c

, the spectra show that the hydrocarbon chains are prevented from crystallizing by the protein (or polypeptide) incorporated in the membrane. Similar disordering effects above

T
c

are also seen when an unsaturated lipid, 1-(16,16,16-trideutero)palmitoyl 2-palmitoleyl phosphatidylcholine is complexed with cytochrome oxidase.
A photochemical technique for measuring lateral diffusion of spin-labeled phospholipids in membranes
Authors: Sheats James R. , McConnell Harden M.
Published: May 2006

A technique is described for the measurement of lateral diffusion of any spin-labeled molecule in planar phospholipid multilayers, using low probe concentrations and a photochemical reaction to generate the initial concentration gradient. Features of the technique include simplicity of data analysis, versatility, and applicability over a wide range of motional rates. Measurements have been made on dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine in the fluid phase (using a phospholipid with head-group spin label), and excellent agreement is found with recent nuclear magnetic resonance spin-echo measurements, as well as with other spin-label and fluorescence photobleaching results.
Biochemical studies of olfaction: binding specificity of radioactively labeled stimuli to an isolated olfactory preparation from rainbow trout (Salmo gairdneri).
Authors: Cagan R H , Zeiger W N
Published: May 2006

The extent of binding of 10 radioactively labeled odorant amino acids to a sedimentable fraction (fraction P2) derived from the olfactory rosettes of the rainbow trout Salmo gairdneri corresponded closely with their reported relative stimulatory effectiveness measured electrophysiologically. L isomers were bound to a greater extent than their respective D isomers. Binding of L-alanine was strongly and irreversibly inhibited by mercurials but was not affected by sulfhydryl-blocking reagents. Binding was saturable and reversible. Scatchard analyses gave evidence of two types of binding sites for most of the amino acids studied. The Kd values of the higher-affinity binding sites were similar among the amino acids, being in the range of 10(-6) M; differences occurred in the relative numbers of sites, n. These results, coupled with those from competition experiments, lead to the postulate that a multiplicity of types of olfactory binding sites exist in the trout: site TSA, which binds L-threonine, L-serine, and L-alanine; site L, which binds L-lysine; and site AB which binds beta-alanine. Tentative assignments are: site V, which binds L-valine; site H, which binds L-histidine; and site AD, which binds D-alanine. Site AD may be a lower affinity site for L-alanine. Binding of olfactory stimulus molecules appears to be an initial discrimination step in olfaction.
Circular dichroism analysis of mononucleosome DNA conformation.
Authors: Cowman M K , Fasman G D
Published: May 2006

Mononucleosomes were isolated from micrococcal nuclease digests of chicken erythrocyte nuclei. The circular dichroism properties of mononucleosome preparations, differing in average DNA length and in H1 and H5 content, demonstrate that the spectrum of chromatin is due only to the complete structure of its repeating subunits. The nucleoprotein spectra are all altered relative to protein-free DNA by the emergence of a single negative band at 275 nm, similar to the band observed for psi DNA. The intensity of the psi-type band depends on the proportion of DNA condensed in a specific manner. The psi-type band is proposed to be due to the compact DNA tertiary structure; i.e., the manner in which the DNA is wound around the histone core allowing interactions between adjacent turns of the superhelix. This interpretation attributes changes and variability in nucleoprotein circular dichroism spectra under different experimental conditions to alterations in DNA tertiary structure rather than secondary structure.
Single base-pair alterations in the Escherichia coli trp operon leader region that relieve transcription termination at the trp attenuator.
Authors: Stauffer G V , Zurawski G , Yanofsky C
Published: May 2006

We have isolated a set of regulatory mutants defective in transcription termination at the attenuator in the leader region of the Escherichia coli tryptophan (trp) operon. In vivo the mutants have 2- to 4-fold increased levels of expression of the trp operon above the level of the trpR parental strain. These levels are increased an additional 1.5- to 2-fold when the mutants are starved of tryptophan. Transcription termination at the trp attenuator was analyzed in vitro with DNA restriction fragments containing the termination-relief mutations. Whereas the frequency of readthrough transcription beyond the termination site is 5% with the wild-type DNA template, it is 46-76% when mutant DNAs are used as templates. The base change in the leader region of each mutant was determined by RNA and/or DNA sequencing. All the changes were between base pairs +116 and +132, in the G-C-rich segment of the leader region. The RNA residues between +114 and +134 of the leader transcript can form a stable stem and loop structure [deltaG approximately equal to -20 kcal (-84 kJ)]. All of the termination-relief mutations destabilize this structure (deltaG approximately equal to -9.0 to -10.5 kcal). These results suggest that the efficiency of transcription termination may be dependent on the integrity of the secondary structure of the above segment of the transcript of the leader region.
Globin mRNAs are primers for the transcription of influenza viral RNA
in vitro
Authors: Bouloy Michele , Plotch Stephen J. , Krug Robert M.
Published: May 2006

Because influenza viral RNA transcription
in vitro
is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription
in vivo
requires initiation by primer RNAs synthesized by the host cell, specifically by RNA polymerase II, thereby explaining the α-amanitin sensitivity of viral RNA transcription
in vivo
. Here, we identify such primer RNAs, initially in reticulocyte extracts, where they are shown to be globin mRNAs. Purified globin mRNAs very effectively stimulated viral RNA transcription
in vitro
, and the resulting transcripts directed the synthesis of all the nonglycosylated virus-specific proteins in micrococcal nuclease-treated L cell extracts. The viral RNA transcripts synthesized
in vitro
primed by ApG also directed the synthesis of the nonglycosylated virus-specific proteins, but the globin mRNA-primed transcripts were translated about 3 times more efficiently. The translation of the globin mRNA-primed, but not the ApG-primed, viral RNA transcripts was inhibited by 7-methylguanosine 5′-phosphate in the presence of
S
-adenosylhomocysteine, suggesting that the globin mRNA-primed transcripts contained a 5′-terminal methylated cap structure. We propose that this cap was transferred from the globin mRNA primer to the newly synthesized viral RNA transcripts, because no detectable
de novo
synthesis of a methylated cap occurred during globin mRNA-primed viral RNA transcription. Preliminary experiments indicate that other purified eukaryotic mRNAs also stimulate influenza viral RNA transcription
in vitro
.
Different intermediate-sized filaments distinguished by immunofluorescence microscopy.
Authors: Franke W W , Schmid E , Osborn M , Weber K
Published: May 2006

The major protein of intermediate-sized filaments in mouse 3T3 cells, for which the name vimentin is proposed, has a molecular weight of 57,000. Antibodies against vimentin and antibodies against prekeratin have been used in parallel in immunofluorescence microscopy on a variety of cultured cells as well as on frozen tissue sections. Both antibodies decorate extended wavy arrays of filaments that are different from microfilaments and microtubules. Intermediate filament bundles decorated by antibodies against prekeratin are predominant in many epithelial cells, including epithelia-derived tumor cells, and are not decorated by antibodies to vimentin. In contrast, intermediate filaments decorated by antibodies against vimentin are widespread among nonmuscle cells of mesenchymal origin, including transformed cells, and also occur in other cells. Perinuclear whorls of aggregates of intermediate filaments induced by prolonged treatment with Colcemid generally show strong decoration with antibodies against vimentin. No significant reaction with either antiserum has been observed in muscle structures or in brain nerve tissue. These observations show that intermediate filaments with similar ultrastructure and solubility characteristics can be distinguished immunologically.
Specificity and crossreactivity of idiotypes of murine antibodies induced by poly(Tyr,Glu)-poly(DLAla)-poly(Lys) and poly(Phe,Glu)-poly(DLAla)-poly(Lys)
Authors: Lifshitz Ruth , Schwartz Michal , Mozes Edna
Published: May 2006

Antibodies elicited against the two synthetic polypeptides, poly(Tyr,Glu)-poly(DLAla)-poly(Lys) [(T,G)-A-L] and poly(Phe,Glu)-poly(DLAla)-poly(Lys) [(Phe,G)-A-L], are crossreactive although the humoral responses to these immunogens are under different genetic controls. The fine specificity of the antibodies elicited by the two polypeptides was studied in the present work. Antisera against (Phe,G)-A-L bind both
125
I-labeled (T,G)-A-L and iodinated modified (Phe,G)-A-L. However, while the binding to (T,G)-A-L could be inhibited completely with the two antigens, the binding to (Phe,G)-A-L was inhibited completely with (Phe,G)-A-L and only partially with (T,G)-A-L. The binding of
125
I-labeled (T,G)-A-L to antisera against (T,G)-A-L was inhibted more efficiently by the homologous antigen than by (Phe,G)-A-L although both antigens completely inhibited the binding. (T,G)-A-L specific antibodies were purified on (T,G)-A-L immunoadsorbents from antisera of high and low responder mice to (T,G)-A-L immunized with (Phe,G)-A-L. (Phe,G)-A-L specific antibodies that did not bind (T,G)-A-L were isolated from the effluent of these columns. By use of anti-idiotypic antibodies of guinea pig against C3H.SW antibodies to (T,G)-A-L it was shown that (T,G)-A-L specific antibodies isolated from antisera against (Phe,G)-A-L of C3H.SW and C3H/DiSn mice possess part of the idiotypic determinants existing on antibodies of C3H.SW obtained by immunization with (T,G)-A-L. In contrast, antibodies to (Phe,G)-A-L that did not bind (T,G)-A-L did not share idiotypic determinants with C3H.SW antibody molecules against (T,G)-A-L. These results suggest that the B cell repertoire expressed by high and low responders to (T,G)-A-L after immunization with (Phe,G)-A-L is similar and represents only part of that of high responders immunized with (T,G)-A-L.
Evidence that catecholamine transport into chromaffin vesicles is coupled to vesicle membrane potential
Authors: Holz Ronald W.
Published: May 2006

The effects of ATP, Mg
2+
, and various agents on pH gradient, membrane potential, and catecholamine transport across membranes of intact bovine chromaffin vesicles were investigated. Methylamine and thiocyanate (SCN
-
) distributions across the vesicle membrane were used to estimate the H
+
concentration gradient and membrane potential, respectively. The H
+
concentration ratio (intravesiculanmedium) equals 16 when the medium pH is 6.9 and is unaltered by ATP and Mg
2+
. In the absence of ATP and Mg
2+
, the steady-state intravesicular S
14
CN
-
concentration is lower than the medium concentration. ATP and Mg
2+
cause an increased influx and a decreased efflux of SCN
-
that results in SCN
-
being concentrated in the vesicles 6- to 8-fold over the medium. The findings are consistent with an ATP,Mg
2+
-induced potential of approximately 50 mV (intravesicular side positive). Carbonyl cyanide
p
-trifluoromethoxyphenylhydrazone (FCCP), a H
+
translocater, and
N
-ethylmaleimide (NEM), a sulfhydryl reagent, decrease the SCN
-
ratio and, thus, the membrane potential in the presence of ATP and Mg
2+
. They have no effect on the H
+
concentration gradient. The rate of catecholamine uptake into vesicles is increased 4- to 6-fold by ATP and Mg
2+
. The ATP,Mg
2+
-stimulated uptake is inhibited by FCCP and NEM over the same concentration ranges that reduce the SCN
-
distribution (membrane potential). FCCP increases and NEM decreases vesicular membrane ATPase activity. Thus, catecholamine uptake is correlated to an inside-positive membrane potential, and not to ATPase activity. If catecholamine uptake is coupled to membrane potential, then a charged species must be involved in the transport mechanism. Reserpine and rotenone inhibit catecholamine influx but have no effect on the H
+
electrochemical gradient; they probably act at a step before coupling to the membrane potential (or the H
+
electrochemical gradient). Atractyloside, an inhibitor of nucleotide transport, has no effects on catecholamine transport or the H
+
electrochemical gradient.
Cyclic GMP injected into retinal rod outer segments increases latency and amplitude of response to illumination.
Authors: Nicol G D , Miller W H
Published: May 2006

We have injected cyclic GMP intracellularly by iontophoresis through the recording electrode into single rod outer segments of the isolated superfused retina of the toad. Bufo marinus. The two most marked effects of the injection are: (i) the latency of the hyperpolarizing membrane-potential change caused by illumination is increased from 5 to 50 times normal, the increase in latency being inversely proportional to the light stimulus intensity; and (ii) the amplitude of the hyperpolarizing receptor potential is increased. These effects are reversible. Our findings are consistent with the hypothesis that cyclic GMP is a link in the molecular chain of events that controls the inward flow of sodium ions in light and darkness. The increased latency we observe after injection of cyclic GMP may be caused by a time delay necessary for light-activated phosphodiesterase to hydrolyze the excess cyclic GMP. By this reasoning the excess cyclic GMP perpetuates the dark current long after illumination. Excitation may be controlled by cyclic GMP if, as our experiments suggest, its hydrolysis initiates the hyperpolarizing receptor potential.
Energetics of enzyme catalysis.
Authors: Warshel A
Published: May 2006

Quantitative studies of the energetics of enzymatic reactions and the corresponding reactions in aqueous solutions indicate that charge stabilization is the most important energy contribution in enzyme catalysis. Low electrostatic stabilization in aqueous solutions is shown to be consistent with surprisingly large electrostatic stabilization effects in active sites of enzymes. This is established quantitatively by comparing the relative stabilization of the transition states of the reaction of lysozyme and the corresponding reaction is aqueous solution.
Analysis of bacteriophage P1 immunity by using lambda-P1 recombinants constructed in vitro.
Authors: Sternberg N , Austin S , Hamilton D , Yarmolinsky M
Published: May 2006

We describe the dissection and reconstruction of a complex control circuit, the P1 immunity system, by a method that involves inserting EcoRI-generated fragments of P1 DNA into lambda vectors that can then be sequentially inserted into a bacterial cell. Using these techniques we have isolated lambda-P1 hybrid phages that express the products of P1 genes c1, c4, ant, and ban and, in appropriately constructed lysogens, confirmed the roles played by the first three of these products in phage immunity. In addition we have localized to particular P1 fragments the sites requisite for expression and repression of these gene products. The analysis leads to the conclusion that gpant acts in trans to antagonize repression mediated by gpc1, in support of one of two proposed models for gpant action. Moreover, two features of the immunity system are revealed: (i) a hitherto unknown component that effects gpc1 repression; and (ii) an unexpected ability of gpc4 to channel a superinfecting c1+ phage into the lysogenic state, which suggests that gpc4 activity regulates the establishment phase of lysogeny.
Stability in dynamical astronomy
Authors: Szebehely Victor
Published: May 2006

Hill's concept of stability is generalized and its relation to bifurcation theory is shown. A quantitative measure of stability is introduced that allows the comparison of the stability of different astronomical systems. Theoretical stability limits for triple stellar systems, for planetary systems, and for satellite systems are established. The measure of stability is evaluated for several known triple stellar systems as well as for the planets and for the natural satellites of the solar system. The model of the restricted problem of three bodies and values of the Jacobian constant are used to study planetary and satellite systems. The model of the general problem of three bodies is used to establish criteria for triple stellar systems.

In general, the results show a hierarchy of stability: the existing triple systems are more stable than the planetary orbits of the solar system. The satellites of the solar system are least stable; in fact, some of the satellites are close to the line of instability (the Earth's Moon) and some are actually unstable (the four outermost satellites of Jupiter).
Chemical synthesis of genes for human insulin.
Authors: Crea R , Kraszewski A , Hirose T , Itakura K
Published: May 2006

A rapid chemical procedure has been developed and used for the synthesis of 29 oligodeoxyribonucleotides to build synthetic genes for human insulin. The gene for insulin B chain, 104 base pairs, and the one for A chain, 77 base pairs, were designed from the amino acid sequence of human polypeptides. They bear single-stranded cohesive termini for the EcoRI and BamHI restriction endonucleases and are designed to be inserted separately into a pBR322 plasmid. The synthetic fragments, deca- to pentadecanucleotides, were synthesized by a block phosphotriester method with trinucleotides as building blocks. Final purification was by high-performance liquid chromatography. All 29 oligonucleotides were pure and had the correct sequences.
Localization of the site of adenylylation of glutamine synthetase by electron microscopy of an enzyme-antibody complex
Authors: Frink Raymond J. , Eisenberg David , Glitz Dohn G.
Published: May 2006

Antibodies to the nucleosidel,
N
6
-ethenoadenosine have been used to localize the site of adenylylation of the glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] of
Escherichia coli
. Antibodies were induced in rabbits by injection of a bovine albumin-ethenoadenosine conjugate. The resulting antisera strongly bound ethenoadenosine, its 5′-nucleotide, or protein conjugates of the nucleoside; little or no crossreaction was seen to adenosine, AMP, or the protein carrier. Ethenoadenylylated glutamine synthetase was prepared by modification of the enzyme by the
E. coli
adenylyltransferase, using etheno-ATP as a substrate. The ethenoadenylylated glutamine synthetase was precipitated by antibodies to ethenoadenosine in conjunction with goat anti-rabbit gamma globulin. Electron micrographs of reaction mixtures of ethenoadenylylated glutamine synthetase and anti-ethenoadenosine showed individual enzyme molecules complexed with one or more antibodies and pairs of enzyme molecules crosslinked by a single antibody. The approximate site of adenylylation was located from the apparent area of contact between enzyme and antibody. We conclude that the adenylylation sites are on the periphery of the bilayered hexagonal disc, offset by 15 ± 10° from the 2-fold axis of symmetry through a vertex of the hexagon and 20 ± 10 Å from the plane between the layers of the disc.
The duplicated human alpha globin genes lie close together in cellular DNA.
Authors: Orkin S H
Published: May 2006

The organization of alpha globin genes in normal human DNA was examined by restriction endonuclease mapping, alpha globin-specific fragments in endonuclease digests of total cell DNA were identified after electrophoresis by hybridization with [32P]cDNA following the blotting procedure of Southern [(1975) J. Mol. Biol. 98, 503--517]. The data provide direct evidence for the duplication of alpha genes and further indicate that these loci are closely linked within a single restriction fragment. The HindIII sites (codons 90/91) of these duplicated genes lie approximately 3.7 kilobases apart in the physical map proposed for this region. This organization of alpha genes can be altered in DNA of individuals with alpha-thalassemia.
Autosomal locus regulates inverse relationship between sialic acid content and capacity of mouse erythrocytes to activate human alternative complement pathway
Authors: Nydegger Urs E. , Fearon Douglas T. , Austen K. Frank
Published: May 2006

The observation that mouse erythrocytes (E
m
) from 21 inbred strains had variable capabilities to activate the human alternative complement pathway permitted the demonstration that membrane sialic acid content was inversely related to activating capacity and was regulated by codominant alleles of a single autosomal locus. Linear regression analysis also demonstrated a significant inverse correlation between the sialic acid content of E
m
from four inbred strains and the concentration of β1H required for decay-dissociation of the properdin-stabilized amplification convertase on the E
m
. E
m
from F
1
hybrids derived from strains with high and low alternative pathway activating capacities and from their backcrosses exhibited the alternative pathway activating capacities expected if the activity were regulated by alleles of a single autosomal locus. That this same locus predominantly regulated the sialic acid content of E
m
was established by the significant inverse correlation between the sialic acid content and the alternative pathway activating capacity of E
m
from mice of the F
1
and backcross generations. Although the fluid phase interaction of C3, B, and [unk]D continuously generates C3b in a reaction augmented by properdin, it is the covalent attachment of C3b to bystander surfaces deficient in sialic acid that activates the alternative complement pathway at that site because of impaired binding of β1H to C3b on such surfaces. Thus, discrimination between activating and nonactivating surfaces occurs after C3b deposition, and sialic acid deficiency represents the molecular basis for our earlier finding that activating particles circumvent the regulatory actions of the control proteins of the alternative pathway.
Normal blood cells of anemic genotype in teratocarcinoma-derived mosaic mice
Authors: Mintz Beatrice , Cronmiller Claire
Published: May 2006

In allophenic (mosaic) mice produced from blastocysts injected with teratocarcinoma stem cells of the OTT 6050 transplant line, an unexpected coat phenotype led to the discovery that the tumor-lineage cells carried the
steel
gene (

Sl
J

/+). Because
steel
also causes a macrocytic anemia, mosaics comprising both genetically anemic and normal (+/+) cells fortuitously provided a unique opportunity to examine
in vivo
the etiology of this anemia in light of previous results indicating that the lesion is extrinsic to the erythroid cells. The experiment differs from previous ones, which involved postnatal grafting, in that here hematopoietic stem cells of anemic and normal genotypes coexist throughout all developmental stages, confronted by tissues of the hematopoietic microenvironment that consist partly or solely of genetically normal cells. Therefore, the possibility exists that the anemia might be completely prevented rather than secondarily ameliorated. Moreover, variation in proportion of normal-strain cells in the hematopoietic supporting tissues could serve to “titrate” minimal requirements to promote normal erythropoiesis. Mice with mixed populations of
steel
- and normal-genotype cells in blood and other tissues were identified by means of independent markers specific for tumor vs. blastocyst strains of origin. The clinical blood picture of these mosaics proved to be indistinguishable from that of normal controls, even when only a small minority of cells in all tissues of one of the animals were genetically normal. Phenotypic blood normalcy was shown, by occurrence of the typical
steel
anemia among F
1
germ-line progeny of mosaics, not to be due to any change in the capacity of the mutant gene to elicit the anemia. The results from the mosaics thus demonstrate that the primary expression of the
steel
lesion is indeed in the hematopoietic microenvironment. However, they also reveal that a surprisingly small complement of normal cells there appears to be adequate to prevent this anemia permanently. The hypothesis is advanced that relatively short-range diffusible substances, produced by cells in the microenvironment and required for normal erythropoiesis, may account for the inductive effectiveness of small cell numbers.
Growth and maintenance of HeLa cells in serum-free medium supplemented with hormones.
Authors: Hutchings S E , Sato G H
Published: May 2006

HeLa cells grow in a nutritionally complete synthetic medium (Ham's F12) supplemented with insulin, transferrin, hydrocortisone (aldosterone), fibroblast growth factor, and epidermal growth factor. This hormone-supplemented medium supports clonal growth, long-term cultivation, and a growth rate equal to that of serum-supplemented medium. The omission of any one of the five components results in less than maximal cell growth.
Extrahypophysial distribution of corticotropin as a function of brain size.
Authors: Moldow R , Yalow R S
Published: May 2006

Determination by radioimmunoassay of corticotropin in the brains of rats, rabbits, dogs, monkeys, and human beings reveals that the dimensions within which the hormone is found is about the same for each of these species but that the anatomical regions in which the hormone is found depends on brain size. Corticotropin is widely distributed in the brain of rats but is found only in the hypothalamic region of the primate brain. The patterns of immunoreactivity observed after Sephadex gel filtration confirm that the molecular forms of corticotropin found in extrahypophysial regions are similar to those in the pituitary of each species. These findings suggest that the mammalian pituitary is the sole site of synthesis of the hormone. The observation of persistence of corticotropin in the brains of commerically hypophysectomized rats has been interpreted by others as suggesting diencephalic as well as pituitary origin for this peptide. However, our studies demonstrate that 8 weeks after hypophysectomy the rats we have received from commerical sources manifest stress-stimulated plasma corticotropin concentrations about 80% of that found in intact rats in spite of the fact that residual pituitary tissue was not found by visual inspection of the sella. Scrapings from the sella revealed a corticotropin content up to 5% that of the average rat pituitary.
Colony size distributions as a measure of in vivo and in vitro aging.
Authors: Smith J R , Pereira-Smith O M , Schneider E L
Published: May 2006

Individual human diploid cells plated at low cell density and incubated for 2 weeks develop into colonies ranging in size from one cell to several thousand cells. The resultant colony size distribution is an accurate indicator of the number of subsequent in vitro population doublings that can be attained by the parent culture. This relationship holds for both human fetal lung and adult skin fibroblast cultures. In addition, the colony size distributions obtained from fetal, young adult, and old adult human cell cultures at the same low level of in vitro passage are indicative of the in vivo age of the level of in vitro passage are indicative of the in vivo age of the cell culture donor. Cell cultures of fetal origin give rise to the highest percentage of colonies with significant proliferative abilities, whereas cultures from old adults give rise to the lowest percentage of large colonies. Therefore, colony size distributions appear to be good indicators of both in vitro and in vivo human cellular aging.
Amino- and carboxyl-terminal amino acid sequences of proteins coded by
gag
gene of murine leukemia virus
Authors: Oroszlan Stephen , Henderson Louis E. , Stephenson John R. , Copeland Terry D. , Long Cedric W. , Ihle James N. , Gilden Raymond V.
Published: May 2006

The amino- and carboxyl-terminal amino acid sequences of proteins (p10, p12, p15, and p30) coded by the
gag
gene of Rauscher and AKR murine leukemia viruses were determined. Among these proteins, p15 from both viruses appears to have a blocked amino end. Proline was found to be the common NH
2
terminus of both p30s and both p12s, and alanine of both p10s. The amino-terminal sequences of p30s are identical, as are those of p10s, while the p12 sequences are clearly distinctive but also show substantial homology. The carboxyl-terminal amino acids of both viral p30s and p12s are leucine and phenylalanine, respectively. Rauscher leukemia virus p15 has tyrosine as the carboxyl terminus while AKR virus p15 has phenylalanine in this position. The compositional and sequence data provide definite chemical criteria for the identification of analogous
gag
gene products and for the comparison of viral proteins isolated in different laboratories. On the basis of amino acid sequences and the previously proposed H-p15-p12-p30-p10-COOH peptide sequence in the precursor polyprotein, a model for cleavage sites involved in the post-translational processing of the precursor coded for by the
gag
gene is proposed.