bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of eIF-2 phosphatase activity in rabbit reticulocyte lysate.
Authors: Safer B , Jagus R
Published: May 2006

A sensitive assay requiring picomole amounts of [32P]eIF-2 to measure eIF-2 phosphatase activity has been developed. Dephosphorylation of [32P]eIF-2 alpha (38,000-dalton subunit) is extremely rapid (t 1/2 = 20 sec) and occurs at the same rate in both hemin-supplemented and hemin-depleted lysates. In contrast, [32P]eIF-2 beta phosphate is stable under all conditions studied. At concentrations required to produce a transient inhibition of protein synthesis, GDP prevents dephosphorylation of half of the phosphate introduced on eIF-2 alpha by the hemin-controlled repressor. Equimolar GTP is without effect. The concept that the energy charge of the guanylate pool may regulate accessibility of a phosphorylated site on eIF-2 alpha to its phosphatase and the implication of this to the mechanism of hemin-regulated translational control are discussed.
Detection of specific hybridoma clones by replica immunoadsorption of their secreted antibodies.
Authors: Sharon J , Morrison S L , Kabat E A
Published: May 2006

A sensitive and rapid method for the detection of monoclonal antibodies secreted by hybridomas is described. Mouse myeloma cells are fused with spleen cells from immunized mice and directly cloned in soft agarose containing selective medium; hybrid clones can be seen after a week. Nitro-cellulose filters that have been coated with a specific protein antigen, with antigen-coupled erythrocyte ghosts, or with other cells used as antigens are then placed on the agarose surface. After incubation to allow immunoadsorption of any secreted antibodies specific for the filter-bound antigen, the filter is removed and overlaid with a suspension of antigen-coupled erythrocytes that react with the adsorbed antibodies; after unbound erythrocytes are allowed to fall off the filter, red spots delineate the sites at which antibody-forming clones are present in the agarose. Alternatively, the filter may be treated with radiolabeled antigen followed by autoradiography. The reliability and sensitivity of the method are demonstrated with alpha-(1 leads to 3)-specific antidextran myeloma J558, and the method's applicability is established by detecting hybridomas with specificities for sheep erythrocytes and for alpha-(1 leads to 3) dextran.
Colorectal carcinoma-specific antigen: detection by means of monoclonal antibodies.
Authors: Herlyn M , Steplewski Z , Herlyn D , Koprowski H
Published: May 2006

Fusion of P3 X 63 Ag8 mouse myeloma cells with splenocytes obtained from mice immunized with cells derived from human colorectal carcinomas resulted in the production of antibody-secreting hybridomas. Two hybridomas (1083-17 and 1116-56) and their clones secreted antibodies binding specifically to human colorectal carcinoma cells either grown in culture or obtained from patients, but did not bind to normal colonic mucosa or other normal and malignant human cells. The binding specificity was consistent in three assays: radioimmunoassay, mixed hemadsorption, and immunofluorescence. Adsorption of these antibodies to colorectal carcinoma cell lines totally eliminated their specific binding.
Accelerated clearance of low-density and high-density lipoproteins and retarded clearance of E apoprotein-containing lipoproteins from the plasma of rats after modification of lysine residues
Authors: Mahley Robert W. , Weisgraber Karl H. , Innerarity Thomas L. , Windmueller Herbert G.
Published: May 2006

Selective chemical modification of lysine residues of lipoproteins by acetoacetylation dramatically altered the metabolism of the lipoproteins without significantly altering other physical or chemical properties. Modification of 30-60% of the total lysine residues of iodinated rat or human low-density lipoproteins (
125
I-LDL) resulted in a rapid removal of these acetoacetylated lipoproteins from the plasma of rats. Within minutes after intravenous injection into intact rats, greater than 80% of the total injected dose disappeared from the plasma. The rapidly cleared acetoacetylated LDL appeared in the liver, and within 6-30 min as much as 50-80% of the total injected dose of modified LDL could be accounted for in the liver. Furthermore, it was possible to demonstrate in the isolated perfused rat liver that the Kupffer cells were responsible for the lipoprotein uptake. Human high-density lipoproteins (HDL
3
) were also rapidly removed from the plasma after acetoacetylation. In striking contrast, acetoacetylation (30-60%) of two E apoprotein-containing lipoproteins (rat HDL
1
and dog HDL
c
) retarded their removal from the plasma. The accelerated removal of modified LDL and HDL
3
, in contrast to the retarded removal of modified HDL
1
and HDL
c
, suggests that the recognition and removal process is specific for a property acquired by only certain lipoproteins after acetoacetylation. Moreover, these results suggest that lysine residues of the E apoprotein may play a functional role in the recognition process for the normal clearance of HDL
1
and HDL
c
, a process that is interfered with after acetoacetylation.
Prostaglandin D2 formation by malignant melanoma cells correlates inversely with cellular metastatic potential.
Authors: Fitzpatrick F A , Stringfellow D A
Published: May 2006

B16 malignant melanoma cell lines transform arachidonic acid and its transient metabolite, prostaglandin endoperoxide H2, into prostaglandin D2. The highly metastatic line, B16 F10, forms less prostaglandin D2 compared to the moderately metastatic parent line, B16 F1. Since platelet aggregation may be one factor involved in B16 metastasis and since prostaglandin D2 inhibits platelet aggregation, this prostaglandin could affect the outcome of platelet-tumour interactions, which may contribute ultimately to metastasis. Arachidonic acid metabolism may be another one of the intrinsic biochemical properties of tumor cells that affects their metastasis. Our results suggest that quantitative release of unusual prostaglandins must be considered in this context.
Uninfected vertebrate cells contain a protein that is closely related to the product of the avian sarcoma virus transforming gene (src).
Authors: Oppermann H , Levinson A D , Varmus H E , Levintow L , Bishop J M
Published: May 2006

Neoplastic transformation of cell by avian sarcoma virus is mediated by a single viral gene (src), which encodes a phosphoprotein (pp60src) with the enzymatic activity of a protein kinase. The DNAs of vertebrate species contain a highly conserved homologue of src that is also represented in the polysomal RNA of uninfected cells and, hence, may specify a normal cellular protein. We have used antisera directed against pp60src to isolate a closely related phosphoprotein (denoted vertebrate pp60) from uninfected chicken, quail, rat, and human cells. Our data indicate that vertebrate pp60 is a homologue of pp60src, highly conserved both antigenically and chemically. Moreover, the cellular protein may possess protein kinase activity similar to that associated with pp60src. We conclude that the product of src is a slightly modified analogue of a normal cellular protein.
Migratory patterns of cloned neural crest melanocytes injected into host chicken embryos.
Authors: Bronner M E , Cohen A M
Published: May 2006

Cloned quail melanocytes grown in tissue culture for 8 days or more were injected into 2 1/2-day-old chicken embryos. The pigment cells were placed directly into the somitic lumen by means of an injection micropipette. This technique for introducing marked neural crest cells into host embryos causes far less damage than previous methods which require extirpation and replacement of the neural tube. In addition, small numbers of homogeneous cells can be implanted by this procedure. When injected into one of the posterior somites, cultured pigment cells migrated along the ventral neural crest pathway. Three days after injection the melanocytes had migrated ventral to the dorsal root ganglia and prevertebral and primary sympathetic chain ganglia and were seen associated with the adrenal gland and aortic plexi. Melanocytes were frequently found in or adjacent to the gonads and often had migrated as far as the gut.
Molecular model for the transposition and replication of bacteriophage Mu and other transposable elements.
Authors: Shapiro J A
Published: May 2006

A series of molecular events will explain how genetic elements can transpose from one DNA site to another, generate a short oligonucleotide duplication at both ends of the new insertion site, and replicate in the transposition process. These events include the formation of recombinant molecules which have been postulated to be intermediates in the transposition process. The model explains how the replication of bacteriophage Mu is obligatorily associated with movement to new genetic sites. It postulates that all transposable elements replicate in the transposition process so that they remain at their original site while moving to new sites. According to this model, the mechanism of transposition is very different from the insertion and excision of bacteriophage lambda.
Lysosomal arylsulfatase deficiencies in humans: Chromosome assignments for arylsulfatase A and B
Authors: DeLuca Chester , Brown Judith A. , Shows Thomas B.
Published: May 2006

Genetics of human lysosomal arylsulfatases A and B (aryl-sulfate sulfohydrolase, EC 3.1.6.1), associated with childhood disease, has been studied with human-rodent somatic cell hybrids. Deficiency of arylsulfatase A (ARS
A
) in humans results in a progressive neurodegenerative disease, metachromatic leukodystrophy. Deficiency of arylsulfatase B (ARS
B
) is associated with skeletal and growth malformations, termed the Maroteaux-Lamy syndrome. Simultaneous deficiency of both enzymes is associated with the multiple sulfatase deficiency disease, suggesting a common relationship for ARS
A
and ARS
B
. The genetic and structural relationships of human ARS
A
and ARS
B
have been determined by the use of human-Chinese hamster somatic cell hybrids. Independent enzyme segregation in cell hybrids demonstrated different chromosome assignments for the structural genes,
ARS
A
and
ARS
B
, coding for the two lysosomal enzymes. ARS
A
activity showed concordant segregation with mitochondrial aconitase encoded by a gene assigned to chromosome 22. ARS
B
segregated with β-hexosaminidase B encoded by a gene assigned to chromosome 5. These assignments were confirmed by chromosome analyses. The subunit structures of ARS
A
and ARS
B
were determined by their electrophoretic patterns in cell hybrids; a dimeric structure was demonstrated for ARS
A
and a monomeric structure for ARS
B
. Although the multiple sulfatase deficiency disorder suggests a shared relationship between ARS
A
and ARS
B
, independent segregation of these enzymes in cell hybrids did not support a common polypeptide subunit or structural gene assignment. The evidence demonstrates the assignment of
ARS
A
to chromosome 22 and
ARS
B
to chromosome 5. A third gene that affects ARS
A
and ARS
B
activity is suggested by the multiple sulfatase deficiency disorder.
Rapid evolution of animal mitochondrial DNA.
Authors: Brown W M , George M , Wilson A C
Published: May 2006

Mitochondrial DNA was purified from four species of higher primates (Guinea baboon, rhesus macaque, guenon, and human) and digested with 11 restriction endonucleases. A cleavage map was constructed for the mitochondrial DNA of each species. Comparison of the maps, aligned with respect to the origin and direction of DNA replication, revealed that the species differ from one another at most of the cleavage sites. The degree of divergence in nucleotide sequence at these sites was calculated from the fraction of cleavage sites shared by each pair of species. By plotting the degree of divergence in mitochondrial DNA against time of divergence, the rate of base substitution could be calculated from the initial slope of the curve. The value obtained, 0.02 substitutions per base pair per million years, was compared with the value for single-copy nuclear DNA. The rate of evolution of the mitochondrial genome appears to exceed that of the single-copy fraction of the nuclear genome by a factor of about 10. This high rate may be due, in part, to an elevated rate of mutation in mitochondrial DNA. Because of the high rate of evolution, mitochondrial DNA is likely to be an extremely useful molecule to employ for high-resolution analysis of the evolutionary process.
Neuroactive drugs inhibit trypsin and outer membrane protein processing in Escherichia coli K-12.
Authors: Gayda R C , Henderson G W , Markovitz A
Published: May 2006

Previous studies demonstrated that a cloned 2-megadalton (MDal) fragment of Escherichia coli DNA contained the structural gene for major outer membrane protein a (also known as 3b or M2 (40 kDal). The present study demonstrates that M2 is synthesized from a 42-kDal precursor that also is present in the outer membrane. The conversion of the 42-kDal precursor to M2 is inhibited by a number of different local anesthetics (procaine, piperocaine, lidocaine, cocaine), by the neuroactive drug atropine, and by the classical trypsin inhibitors N alpha-tosyllysine chloromethyl ketone (TLCK) and benzamidine. Our kinetic studies demonstrate that the amidase action of pure trypsin is inhibited competitively by the local anesthetics tested (excluding lidocaine) as well as by atropine and neostigmine. A mechanism of action for local anesthetics as well as atropine in E. coli may to be inhibit trypsinlike proteases, in a competitive manner, in the region of the outer membrane. The mechanism of action of these compounds in regulating nerve conduction in man have certain features in common with the mechanism proposed in E. coli.
Appearance in confluent vascular endothelial cell monolayers of a specific cell surface protein (CSP-60) not detected in actively growing endothelial cells or in cell types growing in multiple layers.
Authors: Vlodavsky I , Johnson L K , Gospodarowicz D
Published: May 2006

The formation of a highly organized vascular and corneal endothelial cell monolayer is associated with the appearance of a 60,000-dalton cell surface protein (CSP-60) (30,000 daltons after reduction with dithiothreitol) which is not detectable in rapidly growing endothelial cells and in subconfluent cultures that do not yet exhibit the strict morphology of a confluent monolayer. It is also absent from vascular smooth muscle cells and from endothelial cultures that are maintained in the absence of fibroblast growth factor and grow on top of each other at confluence. After disorganization of cells in a confluent endothelial monolayer by urea, EDTA, or trypsin, CPS-60 is no longer exposed on the cell surface, but it reappears as soon as the cells readopt their characteristic two-dimensional configuration. This reorganization can be achieved in the presence of cycloheximide and despite removal of fibronectin by urea, EDTA, or trypsin. Maximal amounts of fibronectin and no CSP-60 are detected in subconfluent, but not yet organized, endothelial cultures or in endothelial cells that no longer form a monolayer of nonoverlapping cells at confluence. Likewise, cultures of vascular smooth muscle cells contain fibronectin but no CSP-60. These results suggest that CSP-60, rather than fibronectin, could be involved in the adoption of a monolayer configuration by confluent endothelial cells.
Receptoral and postreceptoral visual processes in recovery from chromatic adaptation.
Authors: Jameson D , Hurvich L M , Varner F D
Published: May 2006

The time course of recovery from chromatic adaptation in human vision was tracked by determining the wavelength of light that appears uniquely yellow (neither red nor green) both before and after exposure to yellowish green and yellowish red adapting lights. Recovery is complete within 5 min after steady light exposure. After exposure to the alternating repeated sequence 10-sec light/10-sec dark, the initial magnitude of the aftereffect is reduced but recovery is retarded. The results are interpreted in terms of two processes located at different levels in the hierarchical organization of the visual system. One is a change in the balance of cone receptor sensitivities; the second is a shift in the equilibrium baseline between opposite-signed responses of the red/green channel at the opponent-process neural level. The baseline-shift mechanism is effective in the condition in which repeated input signals originating at the receptors are of sufficient strength to activate the system effectively. Hence, this process is revealed in the alternating adaptation condition when the receptors undergo partial recovery after each light exposure, but receptor adaptation during continued steady light exposure effectively protects the subsequent neural systems from continued strong activation.
Arachidonic acid metabolism in polymorphonuclear leukocytes: unstable intermediate in formation of dihydroxy acids.
Authors: Borgeat P , Samuelsson B
Published: May 2006

An unstable intermediate was detected in the transformation of arachidonic acid into 5,6-dihydroxyicosatetraenoic acids (two isomers) and 5,12-dihydroxyicosatetraenoic acids (three isomers) in rabbit peritoneal (glycogen-induced) polymorphonuclear leukocytes. Addition of 10 vol of methanol, ethanol, or ethylene glycol to short-term incubations (30-45 sec) led to the formation of the corresponding 12-O-alkyl derivatives of the 5,12-dihydroxy acids. The time for 50% disappearance of the intermediate (37 degrees C), as measured by formation of 5-hydroxy-12-O-methylicosatetraenoic acids (two isomers) upon trapping with methanol, was about 1 min in live cell preparations (pH 7.4) and about 4 min in water/acetone (1:1), pH 7.4. At pH 6.0 or below, the hydrolysis of the intermediate was too rapid to be measured by the method employed. Data supporting both enzymatic and nonenzymatic hydrolysis of the intermediate into dihydroxy acids are presented. Incubation of the cells with arachidonic acid under an atmosphere of 18O2 led to incorporation of 18O into the 5,6-dihydroxy acids and 5,12-dihydroxy acids only at C-5. The 5-hydroxyicosatetraenoic acid was also labeled at C-5. Considering the chemical reactivity of the intermediate and the structures of the derivatives obtained, it is proposed that the intermediate is 5(6)-oxido-7,9,11,14-icosatetraenoic acid.
Favin versus concanavalin A: Circularly permuted amino acid sequences
Authors: Cunningham Bruce A. , Hemperly John J. , Hopp Thomas P. , Edelman Gerald M.
Published: May 2006

We have determined the tentative amino acid sequence of the β chain (
M
r
20,000) of the lectin favin. In previous studies, we have shown that the α chain (
M
r
5600) of this lectin is homologous to a region in the middle of the concanavalin A (Con A) sequence (residues 70-119). Now we present evidence that the β chain is homologous to two discrete segments of Con A. The homology begins at residue 120 of Con A, extends to the COOH terminus (residue 237) and continues without interruption through the NH
2
-terminal 69 residues of Con A. Together, the α and β chains of favin account for a polypeptide chain equivalent in size to that of Con A. The comparison of the two proteins thus reveals a circular permutation of extensive homologous sequences. The favin molecule contains residues identical to many of the residues postulated to be involved in sugar binding by Con A, and contains all of the direct metal ligands as well as residues homologous to most of the residues that form the β-pleated sheets of Con A. These homologies suggest that the three-dimensional structures of the two lectins are likely to be very similar. Moreover, favin appears to be even more closely related in primary structure and sugar specificity to the lectins from pea and lentil, raising the possibility that all of these lectins may have structures that resemble Con A. Some of these similarities may also extend to the lectins from soybean, peanut, and red kidney bean, which have different sugar specificities but share sequence homologies with the favin β chain.
Detection of substance P in the central nervous system by a monoclonal antibody.
Authors: Cuello A C , Galfre G , Milstein C
Published: May 2006

Peptides with transmitter-like characteristics are being found in many brain areas. The application of immunocytochemical and radioimmunoassay methods has contributed much to the clarification of these neuronal systems. Here we report the development of a rat monoclonal antibody produced by a hybrid myeloma and its application to the study of one of these peptides, substance P. The hybrid clone, isolated after fusion of mouse myeloma cells with hyperimmune rat spleen cells, allowed us to obtain a standardized and permanent source of monoclonal substance P antibodies in a culture cell system. This antibody recognizes the COOH-terminal part of substance P in radioimmunoassay down to 10-20 fmol. It does not crossreact with other known mammalian brain peptides tested. By immunofluorescence the antibody was shown to bind specifically and with a remarkably low background to nerve terminals and cell bodies located in clearly defined nuclear organizations of the central nervous system.
Mechanism of toxicity of putrescine in Anacystis nidulans.
Authors: Guarino L A , Cohen S S
Published: May 2006

Putrescine is lethal to the cyanobacterium Anacystis nidulans at extracellular pH values at which significant concentrations of the nonprotonated diamine rapidly diffuse into the cell and accumulate as the charged form. Although over 98% of the accumulated putrescine is not metabolized, a small fraction is rendered trichloroacetic acid-insoluble, and about 90% of this is bound as putrescinie to proteins and cell structures. Various synthetic functions were studied in the presence of a bacteriostatic (40 microM) and a bacteriocidal (150 microM) concentration of putrescine at pH 9.5. Under lethal conditions, protein synthesis was completely inhibited after 45 min and CO2 fixation after 100 min, whereas nucleic acid synthesis was less affected. Spermidine was lost from the cell and its synthesis was arrested. These functions were much less inhibited at 40 microM putrescine. Ribosomes from putrescine-killed cells were found to be irreversibly dissociated into 30S and 50S subunits. Some putrescine (1-4 molecules) cosedimented with each subunit.
Telomere replication, kinetochore organizers, and satellite DNA evolution.
Authors: Holmquist G P , Dancis B
Published: May 2006

Robertsonian rearrangements demonstrate one-break chromosome rearrangement and the reversible appearance and disappearance of telomeres and centromeres. Such events are quite discordant with classical cytogenetic theories, which assume all chromosome rearrangements to require at least two breaks and consider centromeres and telomeres as immutable structures rather than structures determined by mutable DNA sequences. Cytogenetic data from spontaneous and induced telomere-telomere fusions in mammals support a molecular model of terminal DNA synthesis in which all telomeres are similar and recombine before replication and subsequent separation. This, along with evidence for a hypothetical DNA sequence, the kinetochore organizer, readily explains latent telomeres, latent centromeres, and reversible (one-break) Robertsonian rearrangements. A second model, involving simply recombination between like satellite DNA sequences on different chromosomes, explains not only how one satellite can simultaneously evolve on different chromosomes, but also why satellite DNA is usually located near centromeres or telomeres and why it maintains a preferred orientation with respect to the centromere.
Influenza defective interfering viral RNA is formed by internal deletion of genomic RNA.
Authors: Davis A R , Hiti A L , Nayak D P
Published: May 2006

The 3'- and 5'-terminal nucleotide sequences of the defective interfering (DI) RNAs present in a preparation of DI influenza virus were determined. It was found that all DI RNAs possessed identical terminal sequences for at least the first 13 nucleotides at the 5' end and at least the last 12 nucleotides at the 3' end. The sequence of the DI RNAs is (5')A-G-U-A-G-A-A-A-C-A-A-G-G-...-C-C-U-G-C-U-U-U-C-G-C-U-OH(3'). In addition, the same sequences were present at the 3' and 5' termini of the viral polymerase genes (P1, P2, and P3) from which these DI RNAs originate. These results indicate that DI RNAs of influenzing virus are formed by an internal deletion of the genomic RNA.
Delipidation of bacteriorhodopsin and reconstitution with exogenous phospholipid.
Authors: Huang K S , Bayley H , Khorana H G
Published: May 2006

Solubilizations of the purple membrane from Halobacterium halobium with the detergent Tritain X-100 followed by gel filtration in deoxycholate solution gave bacteriorhodopsin that was more than 99% free from endogenous lipid. The delipidated bacteriorhodopsin was reconstituted with exogenous phospholipids to form vesicles which on illumination efficiently translocated protons. The direction of proton pumping was from the outside to the interior of the vesicles, indicating that the orientation of bacteriorhodopsin in the vesicles was opposite to that in the bacterial membrane. This orientation was confirmed by cleavage of the carboxyl terminus of the protein by proteolysis from the outside of the vesicles.
An optimal multiple decision rule for signs of parameters
Authors: Bohrer Robert , Schervish Mark J.
Published: May 2006

The problem of simultaneously deciding the signs of
p
parameters based on normally distributed estimators is considered. A decision that a parameter is too close to zero to decide its sign is allowed in order that the probability of at least one incorrect decision can be kept less than a preassigned value. Concepts of upper-convexity and local optimality are defined, and for
p
= 2 or 3 the locally optimal rules in the class of upper-convex rules are found. Critical values and operating characteristics are given for the locally optimal rule and some other plausible rules when
p
= 2.
"Superkiller" mutations suppress chromosomal mutations affecting double-stranded RNA killer plasmid replication in saccharomyces cerevisiae.
Authors: Toh-E A , Wickner R B
Published: May 2006

Saccharomyces cerevisiae strains carrying a 1.5 x 10(6)-dalton double-stranded RNA genome in virus-like particles (killer plasmid) secrete a protein toxin that kills strains not carrying this plasmid. At least 28 chromosomal genes (mak genes) are required to maintain or replicate this plasmid. Recessive mutations in any of four other chromosomal genes (ski for superkiller) result in enhanced toxin production. We report that many ski- mak- double mutants are able to maintain the killer plasmid, indicating that the SKI products have an effect on plasmid replication. The ski1-1 mutation suppresses (bypasses) all mak mutations tested except mak16-1. A variant killer plasmid is described that confers the superkiller phenotype and, like chromosomal ski mutations, makes several mak genes dispensable for plasmid replication.
Expression of ribosomal proteins during Drosophila early development.
Authors: Santon J B , Pellegrini M
Published: May 2006

Newly synthesized ribosomal proteins (r-proteins) are found associated with ribosomal subunits as soon as 90 min after fertilization of Drosophila melanogaster embryos. This event substantially precedes the blastoderm stage of embryonic development, at which time the synthesis of rRNA begins. At the preblastoderm stage, embryos synthesize many and possibly all of the r-proteins, although only a small subset of these is incorporated onto ribosomes, presumably by addition or exchange onto preexisting ribosomes. After blastoderm formation, all of the newly synthesized r-proteins along with newly synthesized rRNA are incorporated into ribosomal subunits.
Evolutionary origins of vertebrate hormones: substances similar to mammalian insulins are native to unicellular eukaryotes.
Authors: Le Roith D , Shiloach J , Roth J , Lesniak M A
Published: May 2006

Tetrahymena pyriformis, Neurospora crassa, and Aspergillus fumigatus that had been grown in simple defined media were extracted with acid ethanol by a classic method for recovering insulin from pancreas. After filtration of the extracts on Sephadex G-50, distinct peaks of insulin immunoreactivity were recovered in the region typical of insulin. The gel-filtered material from the Tetrahymena had reactivity in the pork insulin radioimmunoassay about equal to its reactivity in the insulin bioassay (stimulation of lipogenesis in isolated rat adipocytes), and the gel-filtered material from neurospora had an immunoreactivity-to-bioactivity ratio of about 1:3. The material that stimulated lipogenesis could be neturalized by anti-insulin sera (i.e., 75-95% of the Tetrahymena material and 60% of the Neurospora material). Bioactive and immunoactive insulin was found in the conditioned medium equal in amount to that in the cells. The findings suggest that insulin did not arise evolutionarily in the intestinal or neural tissues of primitive vertebrates or complex invertebrates but rather has its molecular origins at least as far back as the simplest unicellular eukaryotes.
Avermectin B1a, a paralyzing anthelmintic that affects interneurons and inhibitory motoneurons in Ascaris.
Authors: Kass I S , Wang C C , Walrond J P , Stretton A O
Published: May 2006

Avermectin B1a (AVM) is an antiparasitic agent that paralyzes nematodes without causing hypercontraction or flaccid paralysis. Using selective stimulation techniques, we have shown that AVM blocks transmission between interneuron(s) and excitatory motorneurons in the ventral nerve cord of Ascaris. It also inhibits transmissin between inhibitory motoneurons and muscle but has little effect on excitatory neuromuscular transmission. Picrotoxin can reverse the AVM-induced block of interneuron-excitatory motoneuron transmission but has no effect on the inhibitory motoneuronal synapse in either the presence or absence of AVM. Our results provide an explanation of how AVM may cause paralysis of nematodes.
Synthesis and characterization of a photosensitive interface for hydrogen generation: Chemically modified p-type semiconducting silicon photocathodes
Authors: Bookbinder Dana C. , Bruce James A. , Dominey Raymond N. , Lewis Nathan S. , Wrighton Mark S.
Published: May 2006

p-Si photocathodes functionalized first with an
N,N
′-dialkyl-4,4′-bipyridinium redox reagent, (PQ
2+/+-
)
surf
, and then with a Pt precursor, PtCl
6
2-
, give significant efficiency (up to 5%) for photoelectrochemical H
2
generation with 632.8-nm light. Naked p-Si photocathodes give nearly zero efficiency, owing to poor H
2
evolution kinetics that are improved by the (PQ
2+/+-
)
surf
/Pt modification. The mechanism of H
2
evolution from p-Si/(PQ
2+/+-
)
surf
/Pt is first photoexcitation of electrons to the conduction band of Si followed by (PQ
2+
)
surf
→ (PQ
+-
)
surf
reduction. The dispersion of Pt then catalyzes H
2
O reduction to give H
2
and regeneration of (PQ
2+
)
surf
. The overall energy conversion efficiency rivals the best direct optical to chemical conversion systems reported to date.
Construction of a flash-activated cyclic electron transport system by using bacterial reaction centers and the ubiquinone-cytochrome b-c1/c segment of mitochondria.
Authors: Packham N K , Tiede D M , Mueller P , Dutton P L
Published: May 2006

Single-turnover electron transfer within the mitochondrial complex III has been studied by combining, in solution, the isolated complex from bovine heart with detergent-solubilized reaction centers of Rhodopseudomonas sphaeroides. Initiation of electron transfer by short flash activation resulted in the prompt oxidation of cytochrome c and reduction of cytochrome b. The subsequent reduction of ferricytochrome c was observed to be concomitant with the oxidation of the ferrocytochrome b, both reactions being inhibited by the addition of actimycin A. The rate of electron transfer through complex III is dependent upon the ambient redox potential poise in a way that is consistent with the presence of a redox component, presumably analogous to the photosynthetic ubiquinone Qz, which is an obligatory intermediate in electron transfer between cytochromes b and c. These results demonstrate cyclic electron transfer in a constructed assembly of mitochondrial complex III, cytochrome c, and photochemical reaction centers.
Cytoskeletal F-actin patterns quantitated with fluorescein isothiocyanate-phalloidin in normal and transformed cells.
Authors: Verderame M , Alcorta D , Egnor M , Smith K , Pollack R
Published: May 2006

Actin in cultured fibroblasts is organized into a complex set of fibers. Patterns of organization visualized with antibody to actin are similar but not identical to those visualized with fluorescein isothiocyanate-phalloidin (Fl-phalloidin), a chemical that binds to F-actin polymer with a dissociation constant of 2.7 X 10(-7) M [Wulf, E., Deboben, A., Bautz, F. A., Faulstich, H. & Wieland, T. (1979) Proc. Natl. Acad. Sci. USA 76, 4498-4502]. Fl-phalloidin reveals that transformed cells have fewer, finer, and shorter F-actin-containing structures than do normal cells. Two-color fluorescence microscopy of single cells reveals that F-actin staining by Fl-phalloidin picks out the cytoskeletal cables more sharply than does antibody to actin, due to a reduced intracellular background fluorescence. This improved resolution permits sorting of cellular Fl-phalloidin patterns into four classes ranging in organization from 90% of the cytoplasm occupied by large cables to the absence of detectable cables. Reproducible differences in pattern distributions between normal and transformed cell lines have been quantitated. Fl-phalloidin together with rhodamine-based indirect antibody to simian virus 40 tumor antigen reveals a direct relationship between the degree of pattern change and simian virus 40 nuclear antigen expression in intermediate transformed 3T3 cell lines [Risser, R. & Pollack, R. (1974) Virology 59, 477-489].
Structural effects in solvolytic reactions; carbon-13 NMR studies of carbocations
†
: Effect of increasing electron demand on the carbon-13 NMR shifts in substituted
tert
-cumyl and 1-aryl-1-cyclopentyl carbocations—correlation of the data by a new set of substituent constants, σ
C+
Authors: Brown Herbert C. , Kelly David P. , Periasamy Mariappan
Published: May 2006

The cationic carbon substituent chemical shifts (ΔδC
+
) for nine representative
meta
-substituted
tert
-cumyl carbocations are correlated satisfactorily by the σ
m
+
substituent constants (slope ρ+ = -18.18, correlation coefficient
r
= 0.990). However, the substituent chemical shifts (ΔδC
+
) for the corresponding
para
derivatives are not correlated by the σ
p
+
substituent constants. The possibility of developing a set of substituent constants capable of correlating such
13
C NMR shifts was examined. The slope of the line defined by the
meta
substituents (ρ+ = -18.18) was utilized to calculate σ
C+
constants for both
meta
and
para
substituents. The utility of these constants was then tested by their ability to correlate the
13
C NMR shifts in the cations for a different system, the 1-aryl-1-cyclopentyl cations. Indeed, these σ
C+
values correlate very well with the ΔδC
+
values, yielding ρ
C+
= -16.84,
r
= 0.999.
An atomic kinetic energy functional with full Weizsacker correction
Authors: Acharya Prabhat K. , Bartolotti Libero J. , Sears Stephen B. , Parr Robert G.
Published: May 2006

A functional is proposed for representing the electronic kinetic energy of the ground state of an
N
-electron atom or ion in terms of its electron density, [Formula: see text] Here

T
w

is the Weizsacker quantity (⅛)∫(∇ρ·∇ρ/ρ)
d
τ and
T
0
is the Thomas-Fermi quantity

C
F

∫ ρ
5 / 3
d
τ. From Hartree-Fock data on 55 neutral atoms,
C
= 1.412 ± 0.033; for 1200 atoms and ions,
C
= 1.332 ± 0.053. The proposed functional gives the derivative δ
T
/δρ its most important correct properties. The term

T
w

is shown to give the kinetic energy of the
K
shell, whereas the term (
C/N
⅓
)
T
0
gives an incorrect statistical estimate of that energy. An alternative correction -(
C/N
⅓
)
T
gives even better results.
Isolation and characterization of a Pseudomonas aeruginosa mutant producing a nontoxic, immunologically crossreactive toxin A protein.
Authors: Cryz S J , Friedman R L , Iglewski B H
Published: May 2006

Nitrosoguanidine mutagenesis of Pseudomonas aeruginosa strain PAO-1 yielded a mutant strain, PAO-PR1, which produced a protein that was immunologically indistinguishable from native toxin A and was nontoxic for cultured Chinese hamster ovary cells. In contrast to native toxin, the cell-associated and extracellular crossreactive material (CRM), designated "CRM protein," possessed no adenosine diphosphate-ribosylating activity. This CRM protein comigrated with native toxin A on sodium dodecyl sulfate/polyacrylamide gels, could be immunoprecipitated with antitoxin from culture supernatants of strain PAO-PR1, and gave a reaction of identity in immunological assays. Equivalent amounts of toxin A antigen and CRM protein antigen were produced in liquid culture by their respective strains as quantitated in a radioimmunoassay for toxin A. These data suggest that mutant strain PAO-PR1 possesses one or more missense mutations within the structural gene for toxin A that adversely affect enzymatic activity, thereby rendering the molecule nontoxic.
Nature of the open state in long polynucleotide double helices: possibility of soliton excitations.
Authors: Englander S W , Kallenbach N R , Heeger A J , Krumhansl J A , Litwin S
Published: May 2006

The existence of transiently open states in DNA and synthetic polynucleotide double helices has been demonstrated by hydrogen exchange measurements; base pairs reversibly separate and reclose, exposing nucleotide protons to exchange with solvent protons. Recently it has been possible to define the equilibrium, kinetic, and activation parameters of the major open state that determines base pair hydrogen exchange. However, there is no direct information at the moment about the conformation of the open form. Here we consider the possibility that the low energy and slow opening and closing rates observed reflect a deformation involving several adjacent base pairs. Assuming a mobile open unit capable of diffusing along the double helix, we find that available data are consistent with structures of 10 or so adjacent open pairs. It is further suggested that these structures correspond to thermally induced soliton excitations of the double helix, which retain coherence by sharing the energy of a twist deformation among several base pairs. Solitons are nonlinear excitations that can travel as coherent solitary waves, and have been recognized as an important mechanism for mediating conformational changes in polymers and condensed systems generally. Comparison of the double helix with simple mechanical analogs suggests that soliton excitations may well exist within DNA chains, and the present analysis shows that the hydrogen exchange open state is consistent with these.
Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti.
Authors: Ditta G , Stanfield S , Corbin D , Helinski D R
Published: May 2006

A broad host range cloning vehicle that can be mobilized at high frequency into Gram-negative bacteria has been constructed from the naturally occurring antibiotic resistance plasmid RK2. The vehicle is 20 kilobase pairs in size, encodes tetracycline resistance, and contains two single restriction enzyme sites suitable for cloning. Mobilization is effected by a helper plasmid consisting of the RK2 transfer genes linked to a ColE1 replicon. By use of this plasmid vehicle, a gene bank of the DNA from a wild-type strain of Rhizobium meliloti has been constructed and established in Escherichia coli. One of the hybrid plasmids in the bank contains a DNA insert of approximately 26 kilobase pairs which has homology to the nitrogenase structural gene region of Klebsiella pneumoniae.
Immunological identification and localization of the predominant nuclear protein of the amphibian oocyte nucleus
Authors: Krohne Georg , Franke Werner W.
Published: May 2006

Nuclei of vitellogenic oocytes of the frog,
Xenopus laevis
, contain a prominent protein, representing about 10% of nuclear protein, which is characterized by a polypeptide of
M
r
30,000. This protein is highly phosphorylated and acidic, displays several isoelectric variants with pI values ranging from 4.7 to 5.3, shows a high thermostability, is not stably complexed with other proteins, and is readily extracted in buffer solutions. Guinea pig antibodies against this protein have allowed its specific immunoprecipitation and localization by immunofluorescence microscopy, using both frozen tissue sections and cells grown
in vitro
. The protein is located almost exclusively in the nucleus where it appears to be spread throughout the nuclear interior. It is also a major nucleus-specific protein in vitellogenic and previtellogenic oocytes of other amphibian species as well as in other cell types, including hepatocytes, follicle epithelial cells, and cultured
Xenopus
cells, but is not detected in nuclei of transcriptionally inactive cells such as erythrocytes and spermatids. An immunologically related, if not identical, protein occurs in nuclei of higher vertebrate cells, including mammals. The properties of this abundant nuclear phosphoprotein and its possible relationship to the “nucleosome assembly factor” protein are discussed. It is suggested that this soluble protein serves a general nuclear function.
Correlation of patterns of anchorage-independent growth with in vivo behavior of cells from a murine fibrosarcoma.
Authors: Cifone M A , Fidler I J
Published: May 2006

The pattern of in vitro anchorage-independent growth of tumor cells from the murine UV-2237 fibrosarcoma correlated with their ability to produce experimental metastasis in vivo. When seeded into 0.3% Noble agar semisolid medium, cells of metastatic clones developed into larger tumor colonies at a faster rate than did cells of clones with low metastatic potential. Furthermore, when tumor cells were plated into 0.6% Noble agar, colony development by cells of low metastatic potential clones was almost completely restricted. Tumor cells from the heterogeneous parent UV-2237 fibrosarcoma were plated into dishes containing 0.6% agar semisolid medium. In separate experiments, 16 colonies were isolated 2 weeks thereafter and were established as individual cell lines in monolayer cultures. All of these cell lines produced experimental metastases as determined by in vivo lung colony assay. The data suggest that anchorage-independent growth of UV-2237 tumor cells in 0.6% Noble agar semisolid medium is selective and permits the isolation of metastatic subpopulations.
Enhancement of phospholipid transfer from Sendai virus to erythrocytes is mediated by target cell membrane.
Authors: Kuroda K , Maeda T , Ohnishi S
Published: May 2006

Transfer of phospholipid from the envelope of Sendai virus to erythrocyte membrane was measured by using spin-labeled phosphatidylcholine. The transfer was enhanced autocatalytically above a threshold dose (about five adsorbed viruses per cell). There was an inflection point in the time course of transfer, after which the transfer was greatly accelerated. The time to reach the inflection point became shorter with increased viral dose. The transfer reaction was markedly enhanced above 19 degrees C and the inflection point was observed above this temperature. There was negligible transfer from trypsin-treated virus to erythrocyte membrane, but the transfer was greatly enhanced by intact virus. The enhancement was larger with increased amount of intact virus, and the inflection point was observed in the transfer curves. All the kinetic data can be satisfactorily analyzed by a model which assumes that the virus modifies the cell membrane at the attachment site, the modification is propagated in the membrane, and transfer of phospholipid to the modified sites is greatly enhanced. The propagation rate is estimated as approximately equal to 10(-11) cm2s-1 and the activation energy as 13 kcal mol-1 (54 kJ mol-1). The viral F glycoproteins are suggested as a possible entity for the modification and its propagation: they are introduced into the target cell membrane by envelope fusion, diffuse laterally, and enhance both phospholipid exchange and envelope fusion with viruses attached to the membrane sites.
recA protein-catalyzed strand assimilation: stimulation by Escherichia coli single-stranded DNA-binding protein.
Authors: McEntee K , Weinstock G M , Lehman I R
Published: May 2006

The single-stranded DNA-binding protein of Escherichia coli significantly alters the strand assimilation reaction catalyzed by recA protein [McEntee, K., Weinstock, G. M. & Lehman, I. R. (1979) Proc. Natl. Acad. Sci. USA 76, 2615--2619]. The binding protein (i) increases the rate and extent of strand assimilation into homologous duplex DNA, (ii) enhances the formation of a complex between recA protein and duplex DNA in the presence of homologous or heterologous single-stranded DNA, (iii) reduces the rate and extent of ATP hydrolysis catalyzed by recA protein in the presence of single-stranded DNA, (iv) reduces the high concentration of recA protein required for strand assimilation, and (v) permits detection of strand assimilation in the presence of the ATP analog, adenosine 5'-O-(O-thiotriphosphate). Single-stranded DNA-binding protein purified from a binding protein mutant (lexC) is considerably less effective than wild-type binding protein in stimulating strand assimilation, a result which suggests that single-stranded DNA-binding protein participates in general recombination in vivo.
Localization of mitochondria in living cells with rhodamine 123.
Authors: Johnson L V , Walsh M L , Chen L B
Published: May 2006

The laser dye rhodamine 123 is shown to be a specific probe for the localization of mitochondria in living cells. By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment.
Enrichment of estradiol-receptor complexes in a transcriptionally active fraction of chromatin from MCF-7 cells
Authors: Scott Richard W. , Frankel Fred R.
Published: May 2006

We have examined the interaction of the estradiol receptor molecule with chromatin in MCF-7 cells, a human breast tumor cell line responsive to estradiol. Receptor was found associated with the various nucleosomal products produced by digestion with micrococcal nuclease. In order to determine whether these receptor binding sites were distributed in a random or nonrandom manner within the chromatin, we have fractionated MCF-7 cell chromatin into transcriptionally active and inactive fractions by limited micrococcal nuclease digestion followed by Mg
2+
precipitation. A comparison of the Mg
2+
-soluble and insoluble chromatin fractions showed that the Mg
2+
-soluble fraction: (
i
) was composed predominantly of mononucleosomes; (
ii
) was enriched in nonhistone proteins; (
iii
) apparently lacked histone H1; (
iv
) was enriched approximately 5-fold in transcribed sequences as measured by a cDNA probe to cytoplasmic poly(A)-RNA sequences; and (
v
) was depleted at least 5-fold of globin sequences, which is presumably a nontranscribed gene in these cells. When these cells were stimulated with β-[
3
H]estradiol, the Mg
2+
-soluble fraction showed a significant enrichment in chromatin-bound estradiol receptor: the Mg
2+
-soluble mononucleosomes showed a 3- to 4-fold enrichment and the di- and trinucleosomes, a 7- to 19-fold enrichment, when compared to the corresponding subunits in the Mg
2+
-insoluble chromatin fraction. This cofractionation of chromatin enriched in transcribed sequences and bound estradiol receptor indicated that receptor binding to MCF-7 cell chromatin was not random but, rather, occurred preferentially in specific regions of the chromatin.