bluuebunny/crossref_metadata_2025 huggingface dataset:
Requirements of acetyl phosphate for the binding protein-dependent transport systems in Escherichia coli.
Authors: Hong J S , Hunt A G , Masters P S , Lieberman M A
Published: May 2006

In Escherichia coli, acetyl phosphate can be formed from acetyl-CoA via the phosphotransacetylase (phosphate acetyltransferase; acetyl-CoA:orthophosphate acetyltransferase, EC 2.3.1.8) reaction and from acetate (plus ATP) via the acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) reaction. By restricting acetyl phosphate formation to the phosphotransacetylase reaction alone, through the use of metabolic inhibitors, we were able to show that, with pyruvate as a source of energy, mutants defective in phosphotransacetylase are unable to transport glutamine, histidine, and methionine. However, with the same energy source, mutants defective in acetate kinase are normal in the transport of these amino acids. The inability of the phosphotransacetylase mutants to transport is due to their presumed inability to form acetyl phosphate, because pyruvate is found to be metabolized to acetyl-CoA in these mutants. Thus acetyl phosphate has been implicated in active transport. Evidence is also presented that neither the protonmotive force nor the ecf gene product is required for the shock-sensitive transport systems.
Membrane biogenesis: cotranslational integration of the bacteriophage f1 coat protein into an Escherichia coli membrane fraction.
Authors: Chang C N , Model P , Blobel G
Published: May 2006

The coat protein (CP) of bacteriophage f1 is integrated into an Escherichia coli plasma membrane fraction consisting of inverted vesicles when it is synthesized in a cell-free, coupled transcription--translation system supplemented with the inverted vesicles. By using proteolytic enzymes as probes, we found by subsequent peptide mapping and determination of the sequence of the proteolytic products that CP was inserted into the inverted vesicles in an orientation indistinguishable from that in inverted vesicles prepared from infected E. coli: only a COOH-terminal portion of approximately 10 residues was accessible to proteolysis, whereas the remainder of CP (CP') was entirely protected. Protection of CP' was dependent on the integrity of the vesicle membrane, because it was abolished when proteolysis was done in the presence of nonionic detergents. Insertion was observed when the inverted vesicles were present during translation in the cell-free system, not when they were added after translation. Thus, the asymmetric insertion of this type of integral membrane protein is strictly coupled to translation. These findings are discussed with respect to prokaryotic membrane biogenesis and are related to bacteriophage f1 assembly and infection.
Aflatoxin B1 mutagenesis, DNA binding, and adduct formation in Salmonella typhimurium.
Authors: Stark A A , Essigmann J M , Demain A L , Skopek T R , Wogan G N
Published: May 2006

Salmonella typhimurium strain TM677 was mutagenized with aflatoxin B1 (AFB1) in liquid suspension culture in the presence of a rat liver postmitochondrial supernatant. Forward mutation to 8-azaguanine resistance was measured in the treated cultures and was found to increase linearly with AFB1 concentration. DNA purified from mutagenized cells was analyzed for AFB1 adduct formation by high-pressure liquid chromatography after adduct liberation. AFB1 exposures at 0.16 and 0.32 micrometer for 35 min produced 15 and 22 AFB1--DNA adducts per genome, respectively, and induced 8-azaguanine-resistant fractions of 4.9 X 10(-4) and 9.6 X 10(-4). Approximately 70% of the AFB1 bound to DNA was chromatographically identical to 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 at the two AFB1 levels used.
Gene localization by chromosome fractionation: globin genes are on at least two chromosomes and three estrogen-inducible genes are on three chromosomes.
Authors: Hughes S H , Stubblefield E , Payvar F , Engel J D , Dodgson J B , Spector D , Cordell B , Schimke R T , Varmus H E
Published: May 2006

Chicken metaphase chromosomes were partially purified by rate zonal centrifugation, and DNA was prepared from each of the fractions of the sucrose gradient. The DNA was digested with various restriction enzymes and subjected to electrophoresis in agarose gels. The DNA was transferred to nitrocellulose filters (as described by Southern), and the filters were hybridized with cDNA probes. Four globin genes alpha A, alpha D, beta, and rho or epsilon are located on at least two chromosomes, and three of the estrogen-inducible genes of the hen oviduct--ovalbumin, ovomucoid, and transferrin--are on three different chromosomes. These experiments also confirm our earlier assignment of the endogenous viral sequence related to Rous-associated virus-0 to a separate (and larger) chromosome than the cellular sequence related to the transforming gene of avian sarcoma virus (cellular sarc), although it now appears that cellular sarc is on a small macrochromosome, rather than on a microchromosome.
Depressed cyclic AMP levels in airway smooth muscle from asthmatic dogs
Authors: Rinard Gilbert A. , Rubinfeld Abe R. , Brunton Laurence L. , Mayer Steven E.
Published: May 2006

We tested mongrel dogs by intradermal injection and tracheobronchial aerosol challenge with
Ascaris suum
antigen extract. All dogs were skin-test positive but could be segregated, on the basis of airways resistance measurements, into “asthmatic” (bronchoreactive) and “nonasthmatic” (nonbronchoreactive) groups. By using tracheal rings from these dogs, we measured the abilities of the β-adrenergic agonist, isoproterenol, to relax tracheal smooth muscle contracted by methacholine and to cause cyclic AMP (cAMP) accumulation in the presence and absence of methacholine. The magnitude of relaxation induced by any concentration of isoproterenol was always less in the smooth muscle from “asthmatic” dogs. In the same tissues the concentrations of cAMP after
in vitro
equilibration, but prior to isoproterenol addition, were significantly less in the “asthmatic” than “nonasthmatic” samples. The accumulation of cAMP due to isoproterenol was similar in both groups for every dose of isoproterenol so that the initial difference between groups in cAMP concentration was maintained in an additive fashion over the entire dose—response curve. Total protein content of trachealis muscles from both groups of dogs was the same. We conclude that β-adrenergically sensitive adenylate cyclase is not impaired in tracheal smooth muscle from “asthmatic” dogs; rather, the basal concentration of cAMP is depressed in “asthmatic” airway smooth muscle, and this difference is maintained throughout the isoproterenol dose—response curve. The depressed intracellular cAMP concentrations may be related to the decreased relaxation induced by isoproterenol in the “asthmatic” tracheal smooth muscle.
Conformation of uncomplexed natural antamanide crystallized from CH
3
CN/H
2
O
Authors: Karle Isabella L. , Wieland T. , Schermer D. , Ottenheym H. C. J.
Published: May 2006

Natural antamanide, a cyclic decapeptide from
Amanita phalloides
, has been shown to have the same conformation for the backbone and comparable side groups as the biologically active synthetic [Phe
4
, Val
6
]antamanide. Packing in the crystal is quite different for the two compounds. Large channels with a polar lining are formed in the crystals of [Phe
4
, Val
6
]antamanide, whereas in natural antamanide large channels in the crystal are completely surrounded by the lipophilic side groups of the Pro, Phe, and Ala residues. Antamanide, C
64
H
78
N
10
O
10
·8H
2
O·X, crystallizes in space group P2
1
2
1
2
1
with
a
= 15.88 Å,
b
= 34.88 Å, and
c
= 13.61 Å.
Regulation of protein synthesis in rat ventral prostate: Cell-free translation of mRNA
Authors: Parker Malcolm G. , Scrace Geoffrey T.
Published: May 2006

Androgenic steroids regulate both a class of abundant poly(A)-containing RNA and four major proteins secreted by the rat ventral prostate. That this class of RNA was mRNA coding for the major proteins was demonstrated by translating total poly(A)-containing RNA in a wheat-germ cell-free system and using the technique of hybrid arrested translation. RNA alone resulted in the synthesis of four major translation products, whereas RNA hybridized with DNA complementary to the class of abundant poly(A)-containing RNA failed to direct their cell-free synthesis. The synthesis of the polypeptides was androgen dependent, but their relative mobilities on denaturing acrylamide gels were not identical to those of the polypeptides synthesized in whole cells. Because these polypeptides are secreted to form part of the prostatic fluid, the possibility that the immediate translation products are precursors to the polypeptides was investigated. Prostatic RNA, translated in an L-cell cell-free system supplemented with microsomal membranes isolated from dog pancreas and rat prostate, caused the appearance of four translation products with relative mobilities similar to those of the androgen-dependent polypeptides synthesized in whole cells. The relationship of one of the polypeptides synthesized in minced tissue with the immediate translation products and processed polypeptides was further investigated by tryptic peptide fingerprint analysis, which revealed identical peptide maps.
Transfer of 5′-terminal cap of globin mRNA to influenza viral complementary RNA during transcription
in vitro
Authors: Plotch Stephen J. , Bouloy Michele , Krug Robert M.
Published: May 2006

We have recently demonstrated that globin mRNAs are effective primers for influenza viral RNA transcription
in vitro
catalyzed by the virion transcriptase [Bouloy, M., Plotch, S. J. & Krug, R. M. (1978)
Proc. Natl. Acad. Sci. USA
75, 4886-4890]. Here, we present direct evidence that the 5′-terminal methylated cap of the globin mRNAs is transferred to viral complementary RNA (cRNA) during transcription. Chemical (β-elimination) or enzymatic removal of the cap of globin mRNAs eliminated essentially all their priming activity. Much of this activity could be restored by recapping the β-eliminated globin mRNAs with the vaccinia virus guanylyl and methyl transferases. Globin mRNAs containing
32
P label only in the cap (m
7
G
32
pppm
6
A
m
-) were prepared by recapping β-eliminated globin mRNAs with the vaccinia virus enzymes, [α-
32
P]GTP, and unlabeled
S
-adenosylmethionine. By using this labeled globin mRNA as primer and unlabeled nucleoside triphosphates as precursors, the viral cRNA segments that were synthesized were shown to contain a
32
P-labeled 5′-terminal cap structure. Gel electrophoretic analysis indicated that the globin mRNA-primed cRNA segments were 10-15 nucleotides longer at their 5′ end than ApG-primed cRNA segments, which initiate exactly at the 3′ end of the virion RNA templates. This suggests that, in addition to the cap, about 10-15 other nucleotides are also transferred from the globin mRNA to viral cRNA. A mechanism for the priming of influenza viral cRNA synthesis by globin mRNA is proposed.
Hyperdegradation of proteins in Escherichia coli rho mutants.
Authors: Simon L D , Gottesman M , Tomczak K , Gottesman S
Published: May 2006

An Escherichia coli mutant, HDF026, defective for growth of phage T4, has been characterized biochemically and genetically. The mutant displays an elevated level of degradation of abnormal proteins, such as puromycyl polypeptides or canavanine-containing polypeptides. Genetically, HDF026 appears to be an allele of rho, which also encodes the transcription termination factor and RNA-dependent ATPase, Rho. The mutation contransduces by phage PI with ilv, weakly suppresses polar mutations in gal, and permits some growth of lambda N- phage. Temperature sensitive lambda mutants in gene O exhibit a reduced efficiency of plating at intermediate temperature on HDF026 mutants; presumably the lambda Ots protein is rapidly degraded in these strains. The ability of wild-type lambda to grow on HDF026 is also reduced, apparently the result of the lambda N product deficiency. gal escape synthesis, which reflects the level of lambda N activity, is decreased 50-66% in the HDF026 mutant. lambda r32, which requires more N function than wild-type phage, does not grow at all in HDF026. A lon mutation, which decreases protein degradation, partially reverses some of these phenotypes, suggesting that they are related to the protein hyperlability of HDF026.
Nucleosome periodicity in HeLa cell chromatin as probed by micrococcal nuclease.
Authors: Butt T R , Jump D B , Smulson M E
Published: May 2006

When HeLa cell nuclei were treated with micrococcal nuclease (nucleate 3-oligonucleotidohydrolase, EC 3.1.4.7), lysed, and centrifuged, the supernatant from early digests contained two predominant classes of polynucleosomes of repeat size 8N and 16N. With increasing digestion time, the 16 N polynucleosome appeared to be cleaved to the 8N species and finally to the basic subunit of chromatin. The size of the polynucleosomes has been determined by DNA analysis and on polyacrylamide electrophoretic gels of native chromatin particles. The 16N polynucleosome appears to be a unique higher ordered structural component of HeLa cell chromatin. Our recent report, showing that the nuclear protein-modifying enzyme poly(ADP-ribose) polymerase increases in specific activity progressively with increasing nucleosome repeat size up to 8-10N, has been extended in the present study. Activity was also elevated in the polynucleosomes of the 16N structure preferentially cleaved by micrococcal nuclease, although specific activity of the enzyme was highest in octanucleosomes. Acceptors for poly(ADP-ribose) have also been determined in these particles.
Avian acute leukemia viruses MC29 and MH2 share specific RNA sequences: Evidence for a second class of transforming genes
Authors: Duesberg Peter H. , Vogt Peter K.
Published: May 2006

The genome of the defective avian tumor virus MH2 was identified as a RNA of 5.7 kilobases by its presence in different MH2-helper virus complexes and its absence from pure helper virus, by its unique fingerprint pattern of RNase T1-resistant (T1) oligonucleotides that differed from those of two helper virus RNAs, and by its structural analogy to the RNA of MC29, another avian acute leukemia virus. Two sets of sequences were distinguished in MH2 RNA: 66% hybridized with DNA complementary to helper-independent avian tumor viruses, termed group-specific, and 34% were specific. The percentage of specific sequences is considered a minimal estimate because the MH2 RNA used was about 30% contaminated by helper virus RNA. No sequences related to the transforming
src
gene of avian sarcoma viruses were found in MH2. MH2 shared three large T1 oligonucleotides with MC29, two of which could also be isolated from a RNase A- and T1-resistant hybrid formed between MH2 RNA and MC29 specific cDNA. These oligonucleotides belong to a group of six that define the specific segment of MC29 RNA described previously. The group-specific sequences of MH2 and MC29 RNA shared only the two smallest out of about 20 T1 oligonucleotides associated with MH2 RNA. It is concluded that the specific sequences of MH2 and MC29 are related, and it is proposed that they are necessary for, or identical with, the
onc
genes of these viruses. These sequences would define a related class of transforming genes in avian tumor viruses that differs from the
src
genes of avian sarcoma viruses.
Limited action of micrococcal nuclease on trout testis nuclei generates two mononucleosome subsets enriched in transcribed DNA sequences.
Authors: Levy-Wilson B , Dixon G H
Published: May 2006

Hybridization experiments show that DNA extracted from two distinct subsets of mononucleosomes (MNI and MN2) generated by a limited action of micrococcal nuclease on trout testis nuclei is enriched approximately 7-fold in sequences that are transcribed into cytoplasmic polyadenylated RNA in trout testis cells. Both subsets of mononucleosomes contain eight core histones, but MNI also possesses one or two molecules of a small, basic, high-mobility-group (HMG) protein H6 [Levy W., B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem. 254, 609-620], bound to a DNA fragment of 140 base pairs. In contrast, MN2 contains 1 molecule of H1 but no H6, and its DNA length is somewhat longer at 140-190 base pairs. The preferential release of these two subsets of mononucleosomes is correlated with the presence of a second larger HMG protein, HMG-T, in the linker regions flanking both types of mononucleosomes. The HMG-T-containing linker regions appear to be considerably more susceptible to attack by micrococcal nuclease than H1-containing linkers. Cross-reassociation reactions between the DNA from MN1 and MN2 subsets indicate that they share a significant extent of sequence overlap but also that each subset contains specific sequences that are absent in the other subset.
In vitro regulation of DNA-dependent synthesis of Escherichia coli ribosomal protein L12.
Authors: Goldberg G , Caldwell P , Weissbach H , Brot N
Published: May 2006

The DNA of the transducing phage lambdarifd18 contains, among others, the genes for the ribosomal proteins L11, L1, L10, and L12 and the beta and beta' subunits of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). In a coupled in vitro protein-synthesis system, lambdarifd18 DNA directs the synthesis of about four to five molecules of L12 per molecule of L10. This is consistent with the finding that there are four copies of L12 per ribosome. The ratio of L12/L10 was also examined from an EcoRI fragment of lambdarifd18 that contains the L10 gene and about 50% of the L12 gene. A significantly lower ratio of truncated L12/L10 was observed compared to the intact phage. The binding of RNA polymerase to various lambdarifd18 DNA restriction fragments was used to locate possible promoter sites. These binding experiments suggest that the beta and beta' subunits of RNA polymerase are cotranscribed with at least ribosomal protein L12 and, also, that there may be an additional promoter site for the L12 gene within the structural gene for L10.
Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis
Authors: Davidson Jeffrey N. , Patterson David
Published: May 2006

A combined genetic, biochemical, and immunological approach has clarified structural relationships involving the first three enzymes of
de novo
pyrimidine biosynthesis. A procedure involving antibody and protein A-Sepharose was used to isolate the enzymes carbamoyl-phosphate synthase [ATP:carbamate phosphotransferase (dephosphorylating, amido-transferring), EC 2.7.2.9], aspartate transcarbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), and dihydro-orotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3) from Chinese hamster ovary cell CHO-K1, the uridine-requiring auxotroph Urd
-
A, and selected Urd
-
A revertants. The enzymes of Urd
-
A and the Urd
-
A revertants were significantly altered in activity, native structure, and molecular weight from those of CHO-K1. The results presented permit the conclusion that (
i
) these three enzymes reside in a single multifunctional 220,000-dalton polypeptide; (
ii
) the aspartate transcarbamoyltransferase activity is located on a portion (≈20,000 daltons) at one end of the polypeptide; (
iii
) this portion may also be required for monomers to aggregate into the multimeric from present in mammalian cells; (
iv
) the mutations in Urd
-
A and the Urd
-
A revertants lie in the structural gene for this multifunctional protein; and (
v
) increased sensitivity to proteases could account for the alterations in the structure of these enzymes in the mutants.
Release of
in vitro
-synthesized poly(A)-containing RNA from isolated rat liver nuclei: Characterization of the ribonucleoprotein particles involved
Authors: Roy Raman K. , Lau Andrew S. , Munro H. N. , Baliga B. Surendra , Sarkar Satyapriya
Published: May 2006

Nuclei isolated from rat liver were incubated under conditions in which RNA continued to be labeled with precursors for long periods. After 1 hr, during which the rate of RNA synthesis was constant, 25-30% of the newly synthesized RNA was recovered in the postnuclear supernatant. About 3-5% of this fraction was characterized as poly(A)-containing ribonucleoproteins by the following criteria: (
i
) characteristic elution profile in oligo(dT)-cellulose chromatography; (
ii
) size distribution of the molecules and their deproteinized RNAs; (
iii
) buoyant densities in CsCl gradients; (
iv
) presence of RNaseresistant fragments resembling poly(A)-protein complexes; and (
v
) identification of the protein components by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The sensitivity of labeling of the RNA synthesized and released from the nuclei to low doses of α-amanitin suggests the presence of polymerase II products in the particles. Comparison of the sizes of proteins in these particles with those of free and polysomal messenger ribonucleoproteins as well as with heterogenous nuclear ribonucleoproteins indicates that the released particles contain a protein of 78,000 daltons, which is also present in the other three classes of ribonucleoproteins, presumably at the 3′-poly(A) segments. In addition, a few other proteins, similar in size to those found in the cytoplasmic ribonucleoproteins, were also present in the released particles. It is suggested that proteins associated with heterogenous nuclear RNA are mostly exchanged before or at the time of release of mRNA from the nucleus; the remaining mRNA-associated proteins are added in the cytoplasm, possibly in relation to cytoskeleton attachment, followed by the removal of most of these proteins during polysome formation.
Tropomyosin synthesis accompanies formation of actin filaments in embryonal carcinoma cells induced to differentiate by hexamethylene bisacetamide.
Authors: Paulin D , Perreau J , Jakob H , Jacob F , Yaniv M
Published: May 2006

Hexamethylene bisacetamide (HMBA) induces in vitro the cytodifferentiation of PCC3/A/1 mouse embryonal carcinoma (EC) cells. In EC cells, actin is associated with surface structures but microfilament bundles are not seen. After 2 days of HMBA treatment, rounded EC cells are converted to flat adhesive ones with a developed cytoskeleton containing actin and tropomyosin. The ratio of actin to total proteins is constant in EC cells and their HMBA derivatives; but a striking difference is observed for one of the newly synthesized proteins (Mr 34,000) identified as tropomyosin. Synthesis of tropomyosin is followed by its association with actin microfilament bundles, as revealed by indirect immunofluorescence microscopy with specific antibodies.
Change in a secondary sexual character as evidence of incipient speciation in
Drosophila silvestris
Authors: Carson Hampton L. , Bryant Peter J.
Published: May 2006

Search for genetic changes that are pivotal in species formation has led to intraspecific studies of
Drosophila silvestris
, a giant species found only on the geologically new island of Hawaii. Males bear large, curved, modified bristles or
cilia
on the dorsal surface of the foreleg tibia and tarsus. In males from the south and west parts of the island there are two rows of cilia separated by a naked area, but in males from the north and east there is a mean of between 20 and 30 additional cilia between the two major rows on the tibia. These extra cilia are absent in closely related species of this subgroup, including the sympatric species
D. heteroneura
and three species from adjacent islands. Males use the foreleg tibiae in vibratory movements against the female's abdomen during courtship, so this character difference is likely to be important in the reproductive biology of the species. Inversion polymorphisms are similar in both northeast and southwest populations; they show large and strikingly parallel altitudinal shifts in frequency distributions involving the same inversions. Populations from various parts of the island cannot be distinguished by routine electrophoresis of soluble proteins encoded by 25 loci. Thus the “extra cilia” character is superimposed on a more ancient genetic background of similarity involving both chromosomal and electrophoretic polymorphisms. We interpret the extra cilia as a specific new embellishment of a secondary sexual character brought about by altered sexual selection occurring very recently in one part of the species range. This suggests incipient speciation.
Lack of homology between dog and human placental alkaline phosphatases
Authors: Moak Gary , Harris Harry
Published: May 2006

Alkaline phosphatases [ALPases; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] from dog and human placenta, liver, bone, kidney, and intestine were investigated by inhibition studies with L-homoarginine, L-phenylalanine, and L-phenylalanylglycyl-glycine; by thermostability studies; and by electrophoresis, both before and after treatment with neuraminidase. The inhibitions obtained for each inhibitor with dog placental ALPase closely match those obtained with dog and human liver, bone, and kidney ALPases, but are quite different from those obtained with human placental ALPase. Dog placental ALPase is thermolabile, as are dog and human liver, bone, and kidney ALPases, in marked contrast to human placental ALPase, which is very thermostable. Dog placental ALPase has the same electrophoretic mobility as dog liver, bone, and kidney ALPases after removal of sialic acid residues with neuraminidase. Desialated human placental ALPase differs electrophoretically from desialated human liver, bone, and kidney ALPases, which show the same mobilities. Dog and human intestinal ALPases are distinguished by these various criteria from the liver, bone, kidney, and placental ALPases of both species, but are similar to each other. These results suggest that the ALPase gene locus expressed in dog placenta is not homologous to that expressed in human placenta. Rather, it appears to be homologous to the ALPase locus expressed in dog and human liver and possibly also bone and kidney. Other incomplete data suggest that this may also be true for placental ALPase in other mammalian species. One possible explanation is that human placental ALPase, a relatively recent newcomer on the evolutionary scene, arose from a gene duplication that occurred subsequent to the evolutionary divergence of many other mammalian species.
Cell-mediated lysis of lipid vesicles containing eye muscle protein: Implications regarding pathogenesis of Graves ophthalmopathy
Authors: Kriss Joseph P. , Mehdi S. Qasim
Published: May 2006

We prepared artificial vesicles that are lysed upon cell-mediated immunological attack by human lymphocytes. These vesicles are made from a mixture of dimyristoyl lecithin, dipalmitoyl lecithin, and cholesterol, have eye muscle membrane protein (EMP) inserted into the bilayer wall, and contain intravesicular
99m
Tc marker. Injury to the vesicular membrane was assessed by measurement of
99m
Tc release. Thyroglobulin (Tg) and Tg-anti-Tg complex (TgA) bind to EMP-vesicles to an extent equal to or greater than to native eye muscle membranes
in vitro
; this binding requires the presence of normal human IgG. The role of Tg, TgA, IgG, and peripheral blood lymphocytes in altering membrane permeability was analyzed. Incubation of vesicles for up to 3 hr alone, with added IgG alone, or with further addition of Tg or TgA did not result in
99m
Tc release. Addition of lymphocytes from normal donors to the above four preparations showed release in the presence of TgA. Lymphocytes from each of eight patients with Graves ophthalmopathy caused release not only in the presence of TgA, but also in the presence of Tg. Separation of a patient's lymphocytes into high- and low-affinity rosette-formers (T and K cells, respectively) showed that cell-mediated vesicle lysis in the presence of TgA was greater with K cells than with T cells, while vesicle lysis in the presence of Tg was greater with T cells than with K cells. Vesicles made with inserted Tg but lacking EMP were not lysed by such T cells. Lymphocytes failed to induce permeability changes in vesicles containing other inserted proteins obtained from human nonextraocular muscle, liver, spleen, or adrenal, even if Tg or TgA were present. The results support the concept that muscle cell damage in Graves ophthalmopathy is immunological, cell-mediated, and of two types: (
i
) K lymphocytes reacting to immune complex, TgA, on the eye muscle cell surface (i.e., antibody-dependent cytotoxicity) and (
ii
) sensitized T lymphocytes reacting to Tg on the eye muscle cell surface. An antigenic role for EMP is possible, but has not been unequivocally proven.
Mechanisms and prevention of trifluoroacetylation in solid-phase peptide synthesis
Authors: Kent Stephen B. H. , Mitchell Alexander R. , Engelhard Martin , Merrifield R. B.
Published: May 2006

A novel mechanism for trifluoroacetylation in solid-phase peptide synthesis, independent of the coupling step, has been elucidated. It involves the presence of trifluoroacetoxymethyl groups on the resin support, which react with resin-bound amines by an intersite nucleophilic reaction. The trifluoroacetoxymethyl groups are generated from preexisting hydroxymethyl sites during treatment with trifluoroacetic acid in dichloromethane or by acidolysis of the benzyl ester bond between the peptide and the resin. The transfer of trifluoroacetyl from hydroxyl to amine occurs during the subsequent neutralization with tertiary amine. The mechanism was first elucidated by model studies with aminomethyl-resins. Then the expected transfer of trifluoroacetyl groups from trifluoroacetoxymethyl-resin to the α-amino group of
N
ε
-benzyloxycarbonyllysine benzyl ester in solution was demonstrated;
k
2
, 6 × 10
-4
M
-1
. Lysine-resins were used to examine the extent of trifluoroacetylation under the conditions of solid-phase peptide synthesis. After a series of acid/base cycles simulating synthetic conditions but without coupling, the poorly nucleophilic α-amino group was approximately 1-2% trifluoroacetylated per cycle when attached to resins already containing hydroxymethyl groups. Standard benzyl ester resins without preexisting hydroxymethyl groups gave comparable levels of trifluoroacetylation after the first few synthetic cycles because of gradual acid cleavage of the ester and accumulation of trifluoroacetoxymethyl sites. Peptide chain termination resulting from trifluoroacetylation by this mechanism could be prevented (<0.02% per cycle) by the use of the aminoacyl-4-(oxymethyl)-phenylacetamidomethyl-resin support, which can be synthesized free from extraneous functionalities and which is stable to trifluoroacetic acid under the conditions of solid-phase peptide synthesis.
Effect of Cibacron blue F3GA on oligonucleotide binding site of estradiol--receptor complexes of mouse uterine cytosol.
Authors: Kumar S A , Beach T A , Dickerman H W
Published: May 2006

The binding of estradiol--receptor complexes of mouse uterine cytosol to oligodeoxynucleotide celluloses is inhibited by the sulfonated polyaromatic dye Cibacron blue F3GA. The dye does not have any effect on the estradiol binding site. Additon of the dye to preformed estradiol--receptor--oligo(dT)-cellulose complex results in the release of estradiol--receptor. The inhibition of binding is competitive with respect to oligo(dT)- or oligo(dA)-Celluloses, suggesting that the effect of the dye is directly on the polynucleotide binding site of the receptor. The observed inhibitory effect of Cibacron blue is not a simple electrostatic effect of polyanions because heparin and polyglutamic acid are much less effective. The selective inhibition by Cibacron blue suggests that the polynucleotide binding domain of estradiol--receptor possesses a special "supersecondary" structure [Stellwagen, E. (1977) Acc. Chem. Res. 10, 92--98].
Darwin's finches: Population variation and sympatric speciation
Authors: Grant B. R. , Grant P. R.
Published: May 2006

The classical model of the adaptive radiation of Darwin's finches is one of repeated speciation in allopatry. Evidence presented here suggests that sympatric specification may have contributed to the radiation. On Isla Genovesa
Geospiza conirostris
displays several features that are consistent with a model of sympatric speciation. Males are polymorphic in song type. Those singing song A have significantly longer bills than those singing song B. The two groups of males forage in different ways that are functionally associated with the bill differences, particularly in the nonbreeding season when food is probably limiting. Territories of mated song A and song B males alternate in space, whereas territories of unmated males do not. This suggests that females can discriminate between males on the basis of song and position, and the pattern is consistent with a hypothesis of assortative mating within song groups. The population is therefore polymorphic; the morphs occupy different niches in which they may be separately regulated and they could be on the way to achieving full reproductive isolation through assortive mating. It is suggested that the population may oscillate between fission and fusion tendencies due to a changing selection regime in this variable and unpredictable environment. There is no evidence that one of the morphs originated allopatrically and then immigrated to Genovesa. The possibility of sympatric speciation being partly responsible for the adaptive radiation, dismissed more than 30 years ago, should be reinstated.
Detection of a transformation-related antigen in chemically induced sarcomas and other transformed cells of the mouse.
Authors: DeLeo A B , Jay G , Appella E , Dubois G C , Law L W , Old L J
Published: May 2006

Antisera prepared against BALB/c Meth A sarcoma in syngeneic or compatible F1 mice recognize a protein with an apparent molecular weight of 53,000 in extracts of [35S]methionine-labeled transformed BALB/c cells. This component, designated p53, was not detected in normal adult mouse fibroblasts, lymphoid cells, or hematopoietic cells or in mouse embryo cells or 3T3 cells. An extensive variety of antisera, including alloantisera and heterologous antisera directed against structural antigens of murine leukemia viruses, was tested for reactivity with p53; other than Meth A antisera, only comparably prepared antisera against another BALB/c sarcoma, CMS4, had anti-p53 activity. All transformed mouse cells tested were found to express p53; these tests included chemically induced sarcomas, leukemias, spontaneously transformed fibroblasts, and cells transformed by simian virus 40 and murine sarcoma virus. The presence of p53 in tumors of no known viral etiology indicates coding by resident cellular genes; this does not exclude endogenous viruses as the source of coding sequences or the possibility that transforming viruses code directly for p53.
Ca2+ and cyclic AMP regulate phosphorylation of same two membrane-associated proteins specific to nerve tissue.
Authors: Sieghart W , Forn J , Greengard P
Published: May 2006

It was shown previously that addition of cyclic AMP (cAMP) to a synaptic membrane fraction incubated with [gamma-32P]ATP stimulated the phosphorylation of two proteins, designated proteins Ia and Ib, found only in nerve tissue. Addition of Ca2+ plus veratridine to synaptosomes preincubated with 32Pi stimulated the phosphorylation of two proteins with similar apparent molecular weights. Various techniques have now been used to determine whether the two proteins phosphorylated in synaptosomes in the presence of Ca2+ plus veratridine are the same as proteins Ia and Ib phosphorylated in synaptic membranes in the presence of cAMP. The proteins phosphorylated by the two procedures were extracted under similar conditions, had similar apparent molecular weights and charges, and were digested by collagenase at similar rates and to the same radioactive intermediates and end products. Furthermore, the two sets of proteins were digested by three other proteolytic enzymes to phosphopeptides with similar molecular weights. The results indicate that Ca2+ and cAMP are each capable of regulating the phosphorylation of proteins Ia and Ib.
Phosphorylation and inactivation of glycogen synthase by phosphorylase kinase.
Authors: Soderling T R , Srivastava A K , Bass M A , Khatra B S
Published: May 2006

Skeletal muscle glycogen a4-synthase (EC 2.4.1.11) has been purified free of all synthase kinase and phosphatase activities by chromatography on a Glc-N-6-P-Sepharose affinity column and then on a phosphocellulose column. This preparation of glycogen synthase was tested as a substrate for purified skeletal muscle phosphorylase kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38). Phosphorylase kinase (1-10 microgram/ml or 0.03-0.3 microM) catalyzes rapid phosphorylation of glycogen synthase (4.5 microM) associated with conversion of the active a form to the less active b form. In the reaction, greater than 95% of the 32P incorporation from [gamma-32P]ATP goes into the synthase subunit almost exclusively in the trypsin-insensitive region which is responsible for synthase a-to-b conversion. Synthase phosphorylation or inactivations catalyzed by phosphorylase kinase is blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, is ATP dependent, is 10-fold more rapid at pH 8.6 than at pH 6.8, and is increased 10-fold by prior activation of the phosphorylase kinase with MgATP and cyclic AMP. With activated phosphorylase kinase at pH 8.2 the apparent Km and Vmax are approximately 70 microM and 4 mumol/min per mg with glycogen synthase and 70 microM and 9 mumol/min per mg with phosphorylase as substrate. It is concluded that glycogen synthase is a substrate in vitro for phosphorylase kinase, a Ca2+-dependent enzyme. The possible physiological significance of this reaction is discussed.
Polyamine-mediated phosphorylation of a nucleolar protein from
Physarum polycephalum
that stimulates rRNA synthesis
Authors: Kuehn Glenn D. , Affolter Hans-Urs , Atmar Valerie J. , Seebeck Thomas , Gubler Ueli , Braun Richard
Published: May 2006

An acidic nucleolar phosphoprotein with a subunit
M
r
of 70,000 was purified as an apparent dimer of 139,000 from isolated nuclei of the slime mold
Physarum polycephalum
. The protein was purified without the aid of strong dissociating agents after its selective phosphorylation in isolated nuclei by a polyamine-mediated reaction. Its amino acid composition resembled that of a nucleolar phosphoprotein from Novikoff hepatoma ascites cells. The phosphoprotein stimulated rRNA synthesis 5-fold by RNA polymerase I within a nucleolar, ribosomal deoxyribonucleoprotein complex isolated from nucleoli of
P. polycephalum
. It was also identified as a component of the complex. It bound with high affinity and specificity to the palindromic ribosomal DNA of 38 × 10
6
M
r
from
P. polycephalum
, which contained two coding sequences for 5.8S, 19S, and 26S rRNA. It also bound to three fragments of ribosomal DNA of
M
r
21.2 × 10
6
, 17.1 × 10
6
, and 8.1 × 10
6
, prepared by cleavage with restriction endonucleases
Hind
III,
Pst
I, and
Bam
HI, respectively. All of these fragments included the symmetry axis of the palindromic ribosomal DNA. The phosphoprotein that had been treated with alkaline phosphataseagarose to hydrolyze the phosphate groups did not stimulate transcription and did not bind to ribosomal DNA or to the restriction fragments indicated. We have thus isolated a specific phosphoprotein with the capacity to stimulate transcription of a specific set of genes in a eukaryote. These findings suggest that this phosphoprotein may specifically regulate functions of ribosomal DNA in a manner dependent on its degree of phosphorylation.
Reactions of cysteamine and other amine metabolites with glyoxylate and oxygen catalyzed by mammalian D-amino acid oxidase.
Authors: Hamilton G A , Buckthal D J , Mortensen R M , Zerby K W
Published: May 2006

Pig kidney D-amino acid oxidase [D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3] catalyzes a rapid uptake of oxygen when high concentrations (50-100 mM) of glyoxylate and the following amines are present under usual assay conditions (pH 8.3): cysteamine, 2-aminoethanol, putrescine, D,L-1-amino-2-propanol, D,L-2-amino-1-propanol, 3-amino-1-propanol, D,L-octopamine, ethylenediamine, and L-cysteine ethyl ester. Notable physiological amines that do not support a rapid O2 reaction under the above conditions include histamine, serotonin, epinephrine, norepinephrine, spermidine, spermine, and cadaverine. A more detailed kinetic investigation of the reactions involving the first four reactive amines listed above indicated that the cysteamine reaction proceeds at a rapid rate even when cysteamine and glyoxylate are present at less than millimolar concentrations, but greater than millimolar concentrations are needed in the other amine reactions in order to observe a reasonable rate. At low concentrations and pH 7.4, the cysteamine-glyoxylate substrate (presumably thiazolidine-2-carboxylic acid) reacts an order of magnitude faster than any other known D-amino acid oxidase substrate. Considerable circumstantial evidence suggests that the reaction involving cysteamine is occurring physiologically, but the reactions of other amines would be occurring in the cell at a very low rate, if at all. It is proposed that the product of the enzymic reaction may be a metabolic effector that can modify the reactivity of proteins or nucleic acids by covalent attachment.
Messenger RNA of opsin from bovine retina: Isolation and partial sequence of the
in vitro
translation product
Authors: Schechter Israel , Burstein Yigal , Zemell Ronald , Ziv Etty , Kantor Frida , Papermaster David S.
Published: May 2006

Opsin, the apoprotein of the visual pigment rhodopsin, is synthesized on membranes of the rough endoplasmic reticulum and subsequently passes through the Golgi apparatus to the rod outer segment. This pathway parallels the early stages of biosynthesis of some secretory proteins and viral membrane glycoproteins. Most of these proteins are initially synthesized as precursor molecules with a short-lived hydrophobic extra peptide segment at the NH
2
terminus. Therefore we investigated whether or not the immediate translation product of opsin mRNA contains a similar short-lived NH
2
-terminal extra peptide. The mRNA coding for opsin was isolated from bovine retina polysomes precipitated by antibodies to opsin. The mRNA directed the cell-free synthesis of a protein comparable in size to opsin that was specifically precipitated by anti-opsin antibodies. Sequence analyses of the immunoprecipitated protein labeled with six radioactive amino acids (Met, Asn, Pro, Phe, Tyr, Val) provided the following result: [Formula: see text] (X is unknown). This partial sequence of the cell-free product corresponds exactly to the published NH
2
-terminal segment of native opsin (21 residues long) and extends beyond this region. Met-1 was shown to be the initiator methionine residue, because only the initiator [
35
S]Met-tRNA
1
Met
—not the internal [
35
S]Met-tRNA
2
Met
—donated the NH
2
-terminal methionine. This finding essentially rules out the possibility that Met-1 was preceded by a peptide that was rapidly cleaved. Thus opsin, and not a precursor, is the immediate product of opsin mRNA translation.
Helical parameters of DNA do not change when DNA fibers are wetted: X-ray diffraction study.
Authors: Zimmerman S B , Pheiffer B H
Published: May 2006

We have measured the helical parameters of DNA in concentrated solutions by x-ray fiber diffraction methods. Fibers of the sodium salt of DNA were swollen with water within capillaries; the capillary served to limit water uptake, slowing dissolution. Samples containing up to 80% water gave essentially a B-form diffraction pattern and had virtually the same helical parameters [9.91 base pairs per turn (SD = 0.14); 3.34 A axial rise per residue (SD = 0.019)] as did the initial fibers [9.95 base pairs per turn (SD = 0.15); 3.33 A axial rise per residue (SD = 0.015)]. Hence, under highly solvated conditions in which the interactions between molecules should be greatly decreased, DNA maintains its classical B-form structure.
Modulation of binding and bioactivity of insulin by anti-insulin antibody: relation to possible role of receptor self-aggregation in hormone action.
Authors: Shechter Y , Chang K J , Jacobs S , Cuatrecasas P
Published: May 2006

Incubation of physiological concentrations of 125I-labeled insulin with liver membranes in the presence of anti-insulin IgG results in a 7- to 15-fold increase in the specific binding of the hormone. The low-affinity/high-capacity binding sites are replaced by an apparently homogeneous class of high-affinity sites, and the nonlinear Scatchard plots are converted to linear plots without a change in the maximum number of binding sites. Similarly, the binding of insulin to receptors in 3T3 fibroblasts is increased substantially in the presence of anti-insulin antibody, and the biological activity of subactive concentrations of insulin is enhanced by antibody in these cells. However, the affinity of 125I-labeled epidermal growth factors (EGF) in fibroblasts is not affected by anti-EGF IgG. In adipocytes anti-insulin IgG in the same concentration range only inhibits the binding of insulin and suppresses insulin-mediated glucose oxidation. Monovalent Fab' fragments from anti-insulin IgG inhibit the binding of the hormone, indicating that the enhancement of binding in liver membranes and fibroblasts requires the bivalency of the antibody.
Tactoidal state and phase transitions in systems of linear polymers of variable length
Authors: Briehl Robin W. , Herzfeld Judith
Published: May 2006

The tactoidal state in systems containing long, rod-like molecules consists of partially aligned solute molecules in equilibrium with and at a concentration not much higher than that in the conjugate isotropic phase. Under the liquid lattice model of Flory [
Proc. R. Soc. London Ser. A
, (1956) 234, 73-89], as well as under other models, tactoid formation by molecules of fixed axial ratio depends on nonideality induced by excluded volumes; the process is wholly entropy driven and requires no direct interactions between rods.

Many rod-like biological polymers exhibit reversible polymerization, so that axial ratio and length are not fixed. Polymerization and rod length will then not only induce nonideality, alignment, and phase separation, but will be affected by these. In this work these interrelations are treated under the model of Flory, modified to include a free energy of polymerization and to permit reversible changes in rod length. The primary conclusion is that, in contrast to the situation for fixed lengths, excluded volume-dependent nonideality alone does not suffice to induce a tactoidal phase separation. In the absence of attractions or repulsions between rods the anisotropic phase is highly concentrated. This phase only becomes tactoidal when a minimal level of repulsive interaction between rods is reached. Under this model, tactoid formation in systems such as deoxygenated hemoglobin S and tobacco mosaic virus depends on repulsive interactions or metastability or both. As a secondary result it is shown that rod length in the anisotropic phase is much greater than in the conjugate isotropic phase.
Nuclear gene
iojap
conditions a programmed change to ribosome-less plastids in
Zea mays
Authors: Walbot Virginia , Coe E. H.
Published: May 2006

The recessive nuclear gene
iojap
of
Zea mays
conditions a permanent, heritable deficiency in the ability of the plastid to differentiate.
iojap
-affected plastids contain a normal genome as evidenced by comparison of the restriction endonuclease digestion patterns of affected and normal plastids.
iojap
-affected plastids contain neither detectable ribosomes nor high molecular weight RNA; the affected plastids do not incorporate exogenous amino acids into protein. The lesion in plastid ribosome content occurs early in organ ontogeny because
iojap
-mediated albino stripes can occupy entire clones within affected leaves.