bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor promoter induces sister chromatid exchanges: relevance to mechanisms of carcinogenesis.
Authors: Kinsella A R , Radman M
Published: May 2006

12-O-Tetradecanoylphorbol 13-acetate (TPA), a powerful tumor promoter, is shown to induce sister chromatid exchanges (SCEs), whereas the nonpromoting derivative 4-O-methyl-TPA does not. Inhibitors of tumor promotion--antipain, leupeptin, and fluocinolone acetonide--inhibit formation of such TPA-induced SCEs. TPA is a unique agent in its induction of SCEs in the absence of DNA damage, chromosome aberrations, mutagenesis, or significant toxicity. Because TPA is known to induce several gene functions, we speculate that it might also induce enzymes involved in genetic recombination. Thus, the irreversible step in tumor promotion might be the result of an aberrant mitotic segregation event leading to the expression of carcinogen/mutagen-induced recessive genetic or epigenetic chromosomal changes.
gamma-Aminobutyric acid: a neurotransmitter candidate for cone horizontal cells of the catfish retina.
Authors: Lam D M , Lasater E M , Naka K I
Published: May 2006

In the catfish retina, horizontal cells that receive inputs exclusively from red-sensitive cones are the only neurons that accumulate exogenous gamma-aminobutyric acid under our experimental conditions. When isolated eyecups are perfused with bicuculline methochloride, an antagonist of postsynaptic gamma-aminobutyric acid receptors, responses of cone photoreceptors to a field of light (3 mm in diameter) become much slower and approach those to a small spot of light (0.3 mm). In addition, bicuculline methochloride decreases the frequency responses of cone horizontal cells to a field of light. These findings indicate that, in the catfish retina, feedback synapses from cone horizontal cells to cones are chemically mediated and may use gamma-aminobutyric acid as a neurotransmitter. Our results also confirm the hypothesis that, in the catfish retina, a function of the negative feedback is to improve the frequency responses of the system.
On the formation of protein tertiary structure on a computer.
Authors: Hagler A T , Honig B
Published: May 2006

In this paper we carry out computer simulation studies of some of the factors responsible for protein tertiary structure. We show that it is possible to obtain (fold) a compact globular conformation from a sequence of amino acids consisting of only glycines and alanines. Our results indicate that glycines play a central role in stabilizing globular structures by facilitating the formation of turns and by destabilizing helical structures. Using this simple two-amino-acid representation, which serves as a control experiment, we are able to obtain a conformation that resembles the native structure of pancreatic trypsin inhibitor, as closely as any obtained previously in folding studies. However, careful examination reveals that the true chain topology has not been reproduced here or in previous studies. We suggest that the discrepancies between calculated and observed structures are more significant than the similarities. The implications of these results for the validity of models for protein folding, the use of pancreatic trypsin inhibitor in folding studies, and the possible role of glycine in the evolution of protein structure are discussed.
Cell-free synthesis of leaf protein: Identification of an apparent precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase
Authors: Cashmore A. R. , Broadhurst M. K. , Gray R. E.
Published: May 2006

Cytoplasmic mRNA has been isolated from the leaves of pea seedlings. Translation of this RNA in the wheat germ cell-free system produces two major products, RI and RII, with molecular weights of 33,000 and 20,000, respectively. Both of these products are considerably larger than the small subunit of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], which is the major product of cytoplasmic protein synthesis
in vivo
and has a molecular weight of 14,000. Antiserum prepared against the small subunit of ribulose-1,5-bisphosphate carboxylase precipitates from the cell-free products, in 2-3% yield, three polypeptides of molecular weights 18,000, 16,000 and 14,000. The smallest of these polypeptides is indistinguishable, by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, from the small subunit of ribulose-1,5-bisphosphate carboxylase. Although the cell-free product RII is not precipitated with antiserum prepared against the small subunit of ribulose-1,5-bisphosphate carboxylase, the two polypeptides do show extensive sequence homology, as indicated by ion exchange chromatography of their tryptic peptides. The production of RII can also be achieved in a polysome-primed cell-free system, where protein synthesis is restricted to the completion of polypeptide chains that have already been initiated
in vivo
. These results indicate that RII is apparently a precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase. We suggest that the selective transport of cytoplasmically synthesized organelle proteins, like animal secretory proteins, may be achieved via the production of precursor polypeptides.
In vitro synthesis of vesicular stomatitis virus membrane glycoprotein and insertion into membranes.
Authors: Toneguzzo F , Ghosh H P
Published: May 2006

Translation in vitro of the mRNA coding for the vesicular stomatitis virus membrane glycoprotein G in a membrane-free ribosomal extract from HeLa cells allowed the synthesis of only the unglycosylated protein G1 (molecular weight, 63,000). Addition of stripped crude microsomal membranes from HeLa cells resulted in the conversion of G1 to the glycosylated protein G2 (molecular weight, 67,000). The G2 protein synthesized by the reconstructed microsomal membrane/ribosome system was found to be segregated inside the microsomal membrane vesicles and was thus protected from the proteolytic action of trypsin and chymotrypsin. Stripped membranes were required at an early stage of protein synthesis for the synthesized protein to be inserted into the membrane vesicles and to be glycosilated. The segregated protein G2, however, was not completely protected from proteolytic digestion, showing that a portion of the polypeptide chain of about 3000 daltons was present on the cytoplasmic side of the membrane vesicle. Our data thus suggest that, unlike the secretory proteins, the membrane glycoproteins are not completely discharged across the microsomal membranes.
Antibody-mediated activation of a defective beta-D-galactosidase: dimeric form of the activatable mutant enzyme.
Authors: de Macario E C , Ellis J , Guzman R , Rotman B
Published: May 2006

Sedimentation analyses of AMEF, an activatable mutant beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), and the products of its reaction with Fab fragments of activating antibody show that this enzyme exists mainly as 10S dimers. Activation of AMEF by purified antibody resulted in formation of 16S tetramers. A unifying hypothesis postulating a dimer--tetramer equilibrium accounts for this observation as the counterpart of inactivation, which was shown to involve the breakdown of tetramers into inactive subunits [Roth, R. A. & Rotman, B. (1975) Biochem. Biophys. Res. Commun. 67, 1382--1390]. Conditions are described under which AMEF loses the specific antigenic determinant(s) responsible for binding activating antibody, allowing its subsequent use as an absorption to obtain immunologically purified activating antibody,
Immunization of rats with polypeptide chains from torpedo acetylcholine receptor causes an autoimmune response to receptors in rat muscle.
Authors: Lindstrom J , Einarson B , Merlie J
Published: May 2006

Four polypeptide chains were purified from acetylcholine receptor of Torpedo californica electric organ. Their apparent molecular weights were 64,000, 57,000, 49,500, and 38,000. Rats immunized with any of the four chains produced antibodies that crossreacted with rat muscle receptors in vivo. Specificities of anti-chain sera were evaluated in vitro by reaction with native receptor solubilized from electric organs and muscles of several species and by inhibition of this reaction with the purified polypeptide chains. The chains are immunologically distinct from one another. Antigenic determinants comparable to each chain of torpedo receptor are found in receptor from both rat and human muscle. At least part of each of these determinants is exposed on the extracellular surface of the muscle membrane. The most immunogenic determinants on native receptor are lost on denaturation to polypeptide chains. Its component peptides are much less immunogenic than native receptor, and induce antibodies of different specificity. Anti-receptor antibodies of many specificities can cause experimental autoimmune myasthenia gravis.
Repair of depurinated DNA in vitro by enzymes purified from human lymphoblasts.
Authors: Bose K , Karran P , Strauss B
Published: May 2006

Alkali-labile lesions introduced into T7 DNA by treatment with methyl methanesulfonate were removed and the DNA was repaired by incubation with DNA polymerase alpha and nuclease from a human lymphoblastoid line followed by the addition of DNA ligase. The nuclease preparation contains both apurinic endonuclease and 5'-3' exonuclease activities. Dinucleotides appear to be the first product of exonuclease action. Repair of methyl methanesulfonate-induced damage can occur by the insertion of only a few nucleotides per lesion as in vivo.
Escherichia coli dnaB mutant defective in DNA initiation: isolation and properties of the dnaB protein.
Authors: Lanka E , Geschke B , Schuster H
Published: May 2006

Extracts of the DNA initiation-defective mutant Escherichia coli dnaB252 are inactive in a dnaB complementation assay but yield a ribonucleoside triphosphatase activity of native molecular weight of about 270,000 (60,000-dalton polypeptide as subunit) that can be inactivated by antibody to dnaB. On the other hand, extracts of a dnaB252(P1 bac) lysogen, in which the dnaB mutation is suppressed in vivo by the constitutive expression of the P1 dnaB analog (ban protein), are active in dnaB complementation and the activity is also sensitive to dnaB antibody. Upon further purification two proteins (with polypeptide molecular weights of 60,000 and 56,000, respectively) are found associated with each other (native molecular weight about 270,000). The larger and the smaller protein are tentatively identified as the dnaB and P1 ban protein. It is suggested that suppression of the dnaB mutation by prophage P1 bac is accomplished by a stabilization of dnaB252 by P1 ban subunit molecules in a heteromultimer.
Nascent peptide as sole attachment of polysomes to membranes in bacteria.
Authors: Smith W P , Tai P C , Davis B D
Published: May 2006

When membrane-polysome complexes from Escherichia coli were treated with puromycin, at various Mg2+ and K+ concentrations, the bulk of the ribosomes were released from the membrane. Moreover, many released ribosomes released ribosomes remained attached to mRNA (pseudopolysomes). These results suggest that ribosomes are attached to the membrane in bacteria solely by their nascent chain. Without a direct attachment the conformational changes associated with chain elongation on the ribosome cannot force the growing chain through the membrane, and so alternative sources of energy for the transfer must be considered.
Thymic hormone-like restoration by human prealbumin of azathioprine sensitivity of spleen cells from thymectomized mice.
Authors: Burton P , Iden S , Mitchell K , White A
Published: May 2006

Gel filtration of fresh human serum revealed that over 95% of the thymic-hormone-like activity was present in the fraction representing the total albumin and prealbumin. Further studies demonstrated that the activity resided in the prealbumin fraction; albumin was inactive. Prealbumin was isolated from Cohn fraction IV-1 of pooled human plasma by extraction with aqueous buffer, fractionation with (NH4)2SO4, gel filtration on Sephadex G-150, and preparative disc gel electrophoresis. The final product was homogeneous by analytical gel electrophoresis was 40,000 times more active than the starting material in an azathioprine-sensitive rosette assay. Physical and chemical characterization studies showed that the isolated product was identical to authentic human plasma prealbumin. Isolation of the prealbumin fraction from sera of adult thymectomized mice revealed that the rosette activity was substantially lower than that isolated from sera of normal mice, suggesting a thymic dependence of the prealbumin activity. In vitro and in vivo bioassays of the fraction obtained prior to the final step of the purification procedure support the conclusion that prealbumin exhibits thymic hormone-like activity.
Molecular cytochemistry: incorporation of fluorescently labeled actin into living cells.
Authors: Taylor D L , Wang Y L
Published: May 2006

Actin labeled with 5-iodoacetamidofluorescein has been incorporated into the functional pool of actin in Chaos carolinensis and Physarum polycephalum by direct microinjection. The functional activity of the labeled actin has been analyzed at three levels of organization as: (a) with the purified actin, (b) in motile extracts of cells, and (c) in living motile cells. The labeled actin exhibited normal polymerization and activated myosin ATPase to a similar extent as unlabeled controls. Labeled actin and endogenous actin were incorporated into contracted pellets to approximately the same extent in motile cell extracts. After labeled actin had been microinjected into single C. carolinensis cells, the fluorescent actin spread into both the endoplasm and etoplasm without forming distinct fibrils. In contrast, fluorescent bundles developed in the ectoplasm of P. polycephalum following microinjection of labeled actin. This experimental method in conjunction with fluorescence spectroscopic techniques could become a powerful tool for studying the intracellular distribution and structural changes of components in living cells.
Correlation between phosphorylated H1 histones and satellite DNAs in Drosophila virilis.
Authors: Blumenfeld M , Orf J W , Sina B J , Kreber R A , Callahan M A , Mullins J I , Snyder L A
Published: May 2006

Drosophila virilis DNA contains satellites I, II, and III. D. novamexicana DNA contains satellite I. D. virilis H1 histone contains subfractions a, b, c, d, and e; D. novamexicana H1 contains subfractions a, b, and c. Therefore, satellites II and III might be correlated with H1d and H1e. To test the validity of this correlation, the H1 histones of polytene nuclei, which contain less than 1% satellite DNA, were analyzed. Polytene nuclei of D. virilis contain substantially decreased levels of H1c and H1e and marginally decreased levels of H1d. Polytene nuclei of D. novamexicana contain decreased levels of H1c.H1c is correlated with satellite I (common to D. virilis and D. novamexicana); H1e is correlated with satellites II and III; H1d is not correlated with any satellite DNA, because its level is virtually unchanged in polytene cells lacking detectable amounts of satellite DNA. Alkaline phosphatase digestion of the H1 histones reveals that H1c is the phosphorylated form of H1b and H1e is the phosphorylated form of H1d. Therefore, the under-replication of satellite DNAs is correlated with the decreased phosphorylation of H1 histones. In vitro, D. virilis H1 histones preferentially bind D. virilis DNAs in the progression III greater than II greater than I greater than main band, whereas D. virilis core histones do not preferentially bind any D. virilis DNA. As an extension of these results, we suggest that phosphorylated H1 histones bind D. virilis satellite DNAs in vivo and are involved in the compaction of heterochromatin.
Incomplete dosage compensation in an evolving Drosophila sex chromosome.
Authors: Strobel E , Pelling C , Arnheim N
Published: May 2006

Cellular autoradiography was used to measure relative rates of chromosomal RNA synthesis and to examine the regulatory phenomenon of X-linked dosage compensation in Drosophila miranda, a species containing two distinct, nonhomologous X chromosomes (X1 and X2). The X1 chromosome was found to be dosage-compensated, since the rate of RNA synthesis along the single X1 chromosome in males equaled that of both X1 chromosomes in females. Unlike other sex chromosomes that have been studied, the more recently evolved X2 heterochromosome exhibited regional differences in transcriptional activity when males and females were compared. The distal 10% of the X2 was not dosage-compensated, whereas the majority of an interior segment, representing 30% of the X2 chromosome's length, was found to be dosage-compensated. Our data are consistent with the idea that the evolution of X2 dosage compensation has paralleled the differentiation of the X2 sex chromosome. In addition, gene rearrangement seems to have accompanied the acquisition of a dosage-compensory mechanism in the X2.
Control of aldosterone secretion during sodium restriction: Adrenal receptor regulation and increased adrenal sensitivity to angiotensin II
Authors: Aguilera G. , Hauger R. L. , Catt K. J.
Published: May 2006

The mechanism of increased adrenal sensitivity to angiotensin II during the aldosterone response to sodium restriction was investigated in the rat. Sodium restriction for 36 hr markedly increased the aldosterone-stimulating effect of low-dose (1 ng/min) infusion of angiotensin II and caused enhanced binding of
125
I-labeled angiotensin II to the zona glomerulosa
in vivo
. Conversely,
in vivo
binding of
125
I-labeled angiotensin II was significantly decreased after 36 hr of high-sodium intake. In isolated glomerulosa cells, the increased binding of angiotensin II after sodium restriction was shown to result from a significant increase in receptor affinity (+80%) and a smaller increase in receptor concentration (+25%). The corresponding aldosterone responses in dispersed cells showed an increase in sensitivity to angiotensin II, commensurate with the increased receptor affinity. More prolonged sodium restriction (4 days) caused a further increase in angiotensin receptor concentration (+70%) and maximal aldosterone response (+50%), whereas the binding affinity of adrenal receptors and the sensitivity of the
in vitro
aldosterone response had returned to normal. During sodium loading for 36 hr and 4 days, the converse effects on adrenal angiotensin II receptors and aldosterone production were observed. Also, in contrast to the consistent increase in angiotensin II receptors in the adrenal glands of sodium-restricted animals, the angiotensin II binding capacity of uterine smooth muscle was decreased by 40% after 7 days of sodium restriction.

The rapid regulation of receptor affinity and concentration during changes in sodium intake provides a basis for the dynamic modulation of aldosterone responses by dietary sodium content. During sodium restriction, the sequential changes in receptor affinity and concentration account for the enhanced binding and steroidogenic actions of angiotensin II
in vivo
and
in vitro
. These receptor changes, and the converse effects of sodium loading, serve as a local regulatory mechanism in the physiological control of adrenal sensitivity and aldosterone secretion. The opposite finding in smooth muscle—that sodium restriction decreases the concentration of angiotensin II receptors—is consistent with the divergent effects of changing sodium balance upon vascular and adrenal responses to angiotensin II.
Differentiation in lacunary directions
Authors: Nagel A. , Stein E. M. , Wainger S.
Published: May 2006

Let {θ

j

} be a lacunary sequence going to zero. Let [Formula: see text]. Define [Formula: see text]. We prove [Formula: see text].
Stuttering: high-level mistranslation in animal and bacterial cells.
Authors: Parker J , Pollard J W , Friesen J D , Stanners C P
Published: May 2006

In both bacterial and mammalian cells, extreme starvation for certain amino acids resulted in translational errors that could be easily detected by two-dimensional polyacrylamide gel electrophoresis. On two-dimensional gels, the faulty proteins were shown as a trail of spots with molecular weights similar to those of the authentic proteins but separated in the isoelectric focusing dimension, a phenomenon we call "stuttering." The direction of charge shift depended on the amino acid substituted and could be predicted from misreading of pyrimidines for purines at the third position of the codon. It is expected that this phenomenon will provide a rapid means of measuring the fidelity of the translational machinery from cell type to cell type.
Two chromatographically separable forms of Escherichia coli elongation factor Tu.
Authors: Geiser M , Gordon J
Published: May 2006

Two forms of the elongation factor Tu from Escherichia coli have been separated by chromatography on DEAE-Sephadex A50. Obvious chromatographic artifacts have been ruled out by investigation of the elution profile of GDP (a component of the column buffer as well as a ligand of Tu) and by rechromatography of the two components, either separately to give the component peaks or together to give a double peak. The two components have been confirmed as Tu by the poly(uridylic)-dependent polyphenylalanine synthesis and by the distribution of the Tu protein as quantitated from sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Complexes with the elongation factors Ts and G have also been ruled out by activity profiles and by quantitation of the protein distribution, again on gels. The distribution of the two forms between ribosomal and supernatant fractions has been examined: one is bound preferentially to the ribosomal fraction and the other is found in the supernatant fraction. The possible significance of this is discussed.
Promoter recognition by phage SP01-modified RNA polymerase.
Authors: Talkington C , Pero J
Published: May 2006

A modified form of Bacillus subtilis RNA polymerase containing a phage SP01-coded regulatory protein (the gene 28 product) selectively transcribes "middle" genes of the phage genome in vitro. In this paper, we identify a subset of restriction endonuclease fragments of SP01 DNA that promote specific transcription by the phage-modified polymerase. In the absence of nucleoside triphosphates, RNA polymerase containing the gene 28 protein selectively binds to these DNA fragments thereby forming stable binary complexes that can be isolated on nitrocellulose filters. In contrast, unmodified RNA polymerase containing sigma factor selectively binds to and transcribes a subset of phage DNA fragments that contain "early" sequences and that are in large part distinct from the fragments recognized by the phage-modified transcriptase. Our results strongly suggest that phage "early" and "middle" genes are transcribed from distinct promoters and that the RNA polymerase containing the gene 28 protein binds to sites that are located at or near promoters for SP01 "middle" genes.
Replica plating and in situ enzymatic assay of animal cell colonies established on filter paper.
Authors: Esko J D , Raetz C R
Published: May 2006

We have developed a simple technique for the replica plating of Chinese hamster ovary (CHO) cells. In this procedure cells are allowed to divide for 8-16 days between the plastic surface of a petri dish and a disc of Whatman no. 50 filter paper, weighted down with glass beads. The culture medium can be replaced when necessary without disturbing the growing colonies. Cells from each developing colony grow into the fibers of the paper, while others remain attached to the plate. The cell colonies transferred to the paper are viable and can be replica plated to a new petri dish with high resolution. In this way several inositol auxotrophs have been identified in a stock of mutagen-treated cells without prior enrichment. Alternatively, the cells on the paper can be rendered permeable in situ, which permits autoradiographic screening for specific biochemical defects, as reported previously for Escherichia coli [Raetz, C. R.H. (1975 Proc. Natl. Acad. Sci. USA 72, 2274-2278]. This technique is applicable to other common cell lines and is especially useful for the identification of single colonies defective in the synthesis of DNA, RNA, protein, and membrane lipids.
Two thymidylate synthetases in Bacillus subtilis.
Authors: Neuhard J , Price A R , Schack L , Thomassen E
Published: May 2006

Bacillus subtilis grown at temperatures below 37 degrees contains two thymidylate synthetases, TSaseA and TSaseB. Their presence is dependent on functional thyA and thyB genes, respectively. When cells are grown at 46 degrees they only contain TSaseA activity. This allous an easy positive selection for thyA and thyA, THYB mutants. The two TSases have been physically separated, and they show similar overall requirements for activity. However, they differ significantly in both their kinetic and their physicochemical properties.
Regulation of prolactin and somatotropin mRNAs by thyroliberin.
Authors: Evans G A , David D N , Rosenfeld M G
Published: May 2006

The tripeptide thyroliberin (thyrotropin-releasing hormone, TRH) regulates the levels of cytoplasmic prolactin and growth hormone (somatotropin) mRNAs in a line of functional rat pituitary tumor cells. Intracellular mRNA levels were determined with specific complementary DNA probes in RNA-excess hybridization reactions and independently by two-dimensional gel electrophoretic analysis of the products of mRNA-directed cell-free translation. Prolactin mRNA sequences increased from 1.1 to 4.5% of the cytoplasmic poly(A)containing mRNA within 48 hr after addition of TRH to the medium; in parallel, growth hormone mRNA sequences were decreased. Similar effects on prolactin mRNA levels were observed in cells grown in serum-containing medium or when the cells were maintained in a serum-free medium for 72 hr prior to addition of TRH, suggesting that TRH is responsible for the stimulation of prolactin mRNA levels rather than for exerting a permissive effect for another factor present in serum.
Localization of eukaryotic initiation factor 3 on native small ribosomal subunits
Authors: Emanuilov Ivan , Sabatini David D. , Lake James A. , Freienstein Christoph
Published: May 2006

The localization of eukaryotic initiation factor 3(eIF-3) on native small ribosomal subunits has been established by electron microscopy through a comparison of native small ribosomal subunits with derived subunits and with native subunits stripped of eIF-3. Small subunits derived from reticulocyte ribosomes by the puromycin/KCl method are seen in electron micrographs as elongated particles, divided by a heavily stained partition into approximately one-third and two-third domains. Most particles (60-70%) observed in electron micrographs of native small subunit preparations resemble derived small subunits, but have an additional mass attached to one side, thus producing profiles with a three-lobed appearance. The mass measures approximately 160 × 100 × 60 Å, and its particle weight is estimated to be about one-third to one-half that of a 40
S
subunit. The site of attachment of the additional mass is located on a prominence extending from the central part of the small subunit and is separated by a cleft from the smaller third of the subunit. The remaining particles in preparations of native subunits resemble the profiles seen in electron micrographs of derived subunits. After removal of eIF-3 by treatment with high concentrations of salt, profiles observed in electron micrographs of washed, native subunits were indistinguishable from those of derived subunits. Since removal of eIF-3 coincided with removal of a mass of the correct molecular weight, subunits with the three-lobed appearance are identified as native small subunits carrying eIF-3.
Species-dependent immunological differences between vertebrate brain tubulins.
Authors: Morgan J L , Holladay C R , Spooner B S
Published: May 2006

The antigenic similarities and differences between highly purified brain tubulins from lamb, mouse, and chick embryo have been examined using rabbit antisera prepared against each of these tubulins. These antisera are capable of binding 125I-labeled tubulin in homologous or heterologous combinations, demonstrating immunological similarity between the tubulins. However, there are quantitative differences in the maximum amount of binding observed. Differences between the tubulins were further resolved by radioimmunoassays, comparing the ability of each of the tubulins to inhibit the binding of each 125I-labeled tubulin to each antiserum. Competition curves generated for all possible combinations revealed quantitative immunological differences between the tubulins that imply different densities of shared antigenic determinants on all three tubulins and a unique determinant on the chick tubulin molecule.
DNA cloning in Bacillus subtilis.
Authors: Ehrlich S D
Published: May 2006

A plasmid pC194, encoding resistance to chloramphenicol, can serve as a cloning vector in Bacillus subtilis 168 for other HindIII-cleaved DNA segments. Replicons constructed by linking pC194 to several Escherichia coli plasmids can be used to introduce and compare the expression of the same genes in these two bacterial hosts.
Chromosome mapping of the CYC7 gene determining yeast iso-2-cytochrome c: structural and regulatory regions.
Authors: Sherman F , Stewart J W , Helms C , Downie J A
Published: May 2006

The primary structures of iso-1-cytochrome c and iso-2-cytochrome c in the yeast Saccharomyces cerevisiae are determined by the genes CYC1 and CYC7, respectively. The CYC1 locus was previously shown to be on the right arm of chromosome X, and the CYC7 locus is shown in this investigation to be on the left arm of chromosome V closely linked to the min1 and mak10 markers. The CYC7 locus appears to be composed of a structural region and a regulatory region. Mutations in the structural region can cause a deficiency or alteration of iso-2-cytochrome c, whereas mutations in the regulatory region can cause increases in the amount of iso-2-cytochrome c. Single-site gene conversion, occurring at a relatively high frequency of approximately 4%, caused intragenic recombination of a mutational site in the structural region and a mutational site in the regulatory region, enabling us to suggest the order of the sites in relationship to other markers on the chromosome.
Association of cyclic GMP with gene expression of polytene chromosomes of Drosophila melanogaster.
Authors: Spruill W A , Hurwitz D R , Lucchesi J C , Steiner A L
Published: May 2006

The distribution of cyclic nucleotides on polytene chromosomes isolated from Drosophilia melanogaster salivary glands was examined by using an indirect immunofluorescent technique. With a fixative that minimized the loss of chromosomal proteins, cyclic GMP, but not cyclic AMP, was observed distributed along the chromosomes. The subchromosomal distribution of cyclic GMP correlated with genetically active sites on the chromosomes. After heat-shock treatments, the intensity of cyclic GMP fluorescence was markedly enhanced at specific loci on the chromosomes, with locus 93D as the most intensely fluorescent. Autoradiographic analysis with [3H]uridine revealed that 93D was the most transcriptionally active locus within a particular nucleus. These observations suggest that cyclic GMP may participate in processes associated with transcription on polytene chromosomes. The involvement of cyclic GMP in nuclear events associated with gene expression is discussed.
Influence of dietary restriction on immunologic function and renal disease in (NZB × NZW) F
1
mice
Authors: Fernandes G. , Friend P. , Yunis E. J. , Good R. A.
Published: May 2006

In (NZB × NZW)F
1
(B/W) mice, moderate caloric intake [10 kcal (41.8 kJ) per day] from the time of weaning was associated with maintenance of lower body weight, greater capacity of spleen cells to be stimulated with T-cell mitogens, and better preserved capacity to generate cytotoxic cells in response to
in vitro
and
in vivo
stimulation with allogeneic tumor cells. Plaque-forming cell response to sheep erythrocytes was also well maintained in animals on the restricted diets when sensitization was accomplished either
in vitro
or
in vivo
. Spontaneous suppressor cell activity against plaque-forming cells that developed in controls did not appear in the mice on the restricted diet. Significantly less circulating antibody to native DNA was present in the blood of mice 10 months of age when their dietary intake had been restricted. Histological analysis revealed that the development of renal disease and the deposition of gamma globulin in the glomerular capillaries was markedly inhibited in the mice on restricted diets. Dietary restriction from the time of weaning thus appears to prolong significantly the life of autoimmunity-prone (NZB × NZW)F
1
male and female mice and to alter lymphoid cell immune function, thereby decreasing the autoimmune processes and immunological assault associated with progressive renal disease in these animals.
Alteration in suppressor cell activity in chronic active hepatitis
Authors: Hodgson Humphrey J. F. , Wands Jack R. , Isselbacher Kurt J.
Published: May 2006

We have studied peripheral blood mononuclear cells obtained from 24 patients with acute or chronic active hepatitis to determine if there was an abnormality in concanavalin A-induced suppressor cell activity compared to control subjects. Suppressor cells were generated by preincubation of the mononuclear cells with a mitogenic concentration of concanavalin A (6 μg/ml) for 48 hr followed by treatment with mitomycin C and α-methyl mannoside. Suppressor cell activity was assessed in second cultures by inhibition of concanavalin A-stimulated blast transformation of fresh allogeneic lymphocytes. Concanavalin A-stimulated suppressor activity was not elicited in mononuclear cells from the majority of patients with chronic active hepatitis in contrast to patients with acute hepatitis or acute inflammatory diseases and controls (
P
< 0.001). This finding was demonstrable in chronic active hepatitis patients in remission and relapse, both on and off prednisone therapy, and varied considerably during the course of the disease. The extent of liver injury was not related to the measured suppressor cell activity. These studies suggest that in chronic active hepatitis, a disease in which the host immune response may be involved, there appears to be a defect in concanavalin A-stimulated suppressor cells.
Positive control of D-serine deaminase synthesis in vitro.
Authors: Heincz M C , Kelker N E , McFall E
Published: May 2006

Efficient constitutive synthesis of D-serine deaminase [D-serine hydro-lyase (deaminating); EC 4.2.1.14] is obtained in vitro by using a slightly modified Zubay system programmed with dsdO6 dsdA+DNA. Synthesis from a dsdO+ dsdA+ template requires active dsdC gene product and 3':5'-cyclic AMP. In vitro synthesis of dsdC product is obtained with a dsdC+ dsdO+ dsdA+ or a dsdCc dsdO+ dsdA+ template; this synthesis is thermosensitive and can be uncoupled from D-serine deaminase synthesis by temperature shift.
Noncovalent interaction of the NH2-terminal fragment of human somatotropin with the COOH-terminal fragment of human choriomammotropin to generate growth-promoting activity.
Authors: Li C H
Published: May 2006

Complementation of the plasmin fragments of reduced-carbamoylmethylated (Cam) human somatotropin (hGH) with those of reduced-carbamoylmethylated human chorionic somatomammotropin (hCS) have been investigated. It was found that the recombinant obtained by noncovalent interaction of [Cys(Cam)53]hGH-(1-134) with [Cys(Cam)-165,182,189]hCS-(141-191) exhibits 50% growth-promoting activity and nearly full immunoreactivity. Complementation of [Cys(Cam)53]hCS-(1-133) with the COOH-terminal fragment of hGH generated lower growth-promoting and immunological activities.
Interaction of ligands with the opiate receptors of brain membranes: Regulation by ions and nucleotides
Authors: Blume Arthur J.
Published: May 2006

This study shows that nucleotides, as well as ions, regulate the opiate receptors of brain. GMP-P(NH)P and Na
+
reduce the amount of steady-state specific [
3
H]dihydromorphine binding and increase the rate of dissociation of the ligand from the opiate receptor. In contrast, Mn
2+
decreases the rate of ligand dissociation and antagonizes the ability of Na
+
to increase dissociation. The effects of GMP-P(NH)P on steady-state binding and dissociation are not reversed by washing. Only GTP, GDP, ITP, and IMP-P(NH)P, in addition to GMP-P(NH)P, increase the rate of dihydromorphine dissociation. The site of nucleotide action appears to have high affinity: <1 μM GMP-P(NH)P produces half-maximal increases in ligand dissociation. GMP-P(NH)P- and Na
+
-directed increases in dissociation have also been found for the opiate agonists [
3
H]etorphine, [
3
H]Leu-enkephalin, and [
3
H]Met-enkephalin and the opiate antagonist [
3
H]naltrexone. Mn
2+
-directed decreases in dissociation have been found for the agonist [
3
H]-etorphine and the antagonist [
3
H]naltrexone. Although the plasma membrane receptors for a number of other neuro-transmitters and hormones are also regulated by guanine nucleotides, the opiate receptors appear unique because only they show nucleotide regulation of both agonist and antagonist binding.
Kinetics of biphasic reconstitution of tobacco mosaic virus in vitro.
Authors: Fukuda M , Ohno T , Okada Y , Otsuki Y , Takebe I
Published: May 2006

The kinetics of the in vitro reconstitution of tobacco mosaic virus from its RNA and protein were studied by measuring the increase in turbidity, the development of ribonuclease-resistant infectivity, the emcapsidation of the terminal ends of the RNA, and the growth of rod length. The results showed that the reconstitution reaction consists of two processes in which the direction, timing, and rate of assembly are different. Rapid elongation of particles toward the 5' end of the RNA proceeds in the first 5-7 min to give intermediate particles of 260 nm in length in which only the 5' terminus of the RNA is encapsidated. The subsequent process requires 30-50 min, is accompanied by a slow increase in turbidity, and gives rise to rods of the full length, 300 nm. The 3' terminus becomes RNase resistant by this process with concomitant development of ribonuclease-resistant infectivity, showing that the 3'-distal portion of the RNA is encapsidated in the direction of 5' to 3'. The rate of rod elongation by the second process is less than 1/10 of that by the first process.
Differences in the isoelectric focusing patterns of gamma-glutamyl transpeptidase from normal and cancerous rat mammary tissue.
Authors: Jaken S , Mason M
Published: May 2006

Highly purified fractions of gamma-glutamyl transpeptidase [gamma-glutamyltrinsferase; (5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2] from normal and malignant rat mammary tissue were prepared. Analyses by isoelectric focusing indicate the existence of at least 12 enzymatically active species. The gamma-glutamyl transpeptidase from the tumor tissue had a greater proportion of the activity concentrated in the more negative species than the enzyme from normal tissue. Treatment of the two enzyme preparations with neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) greatly reduced this difference. When whole tissue homogenates were treated with papain to solubilize the enzyme and then focused, the same relationship held. The neuraminidase activities in the two homogenates were similar, but the sialytransferase activity (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) of the tumor homogenate was 13 times that of the normal mammary homogenate. These observations suggest that the gamma-glutamyl transpeptidase of the tumor is more heavily sialylated than that from the normal tissue, possibly reflecting the greater sialyltransferase activity of the tumor.
Functional properties of beta-galactosidase from mutant strain 13 PO of Escherichia coli.
Authors: Deschavanne P J , Viratelle O M , Yon J M
Published: May 2006

The functional properties of CZP protein, a mutant deriving from wild-type beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23) by a point mutation, were investigated. A large decrease of the specificity, as evaluated by the kcat/Km ratio, was observed, principally originated by a weaker binding of the substrates. The catalytic constants, whose values are strongly affected by the presence of divalent cations, were smaller or larger for mutant enzyme than for wild-type enzyme, depending upon the experimental conditions. Analysis of the kinetic pathway indicates, with some substrates, a change in the limiting step for the mutant enzyme compared to the wild type. Because the k'3 step is rate limiting for hydrolysis of p-nitrophenyl-beta-D-galactoside by the mutant enzyme in the absence of Mg2+ and its value is relatively small, it is possible to observe a burst of p-nitrophenol during hydrolysis. This provides conclusive evidence for the occurrence of a two-step mechanism, with a sequential release of the products.
Promotion of monolayer formation in cultured whole pancreatic islets by 3-isobutyl-1-methylxanthine.
Authors: Ohgawara H , Carroll R , Hofmann C , Takahashi C , Kikuchi M , Labrecque A , Hirata Y , Steiner D F
Published: May 2006

Normal adult rat islets usually remained intact and encapsulated, even after many days in culture. In contrast, islets cultured in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1 mM) attached more readily to the surface of plastic culture dishes and almost uniformly formed monolayers of endocrine cells. The mechanism of this effect is not known but presumably involves increases in cellular cyclic AMP content. Fibroblast growth did not appear to be stimulated by the inhibitor. These adult pancreatic endocrine monolayer cultures can be produced readily and provide useful preparations for further morphological and biochemical studies of factors affecting the differentiation, growth, and regenerative capacity of islet cells.
Mutagenesis during in vitro DNA synthesis.
Authors: Weymouth L A , Loeb L A
Published: May 2006

The error frequency of in vitro DNA synthesis using a natural DNA template has been measured with a biological assay for nucleotide substitutions. phiX174 DNA containing an amber mutation was copied in vitro by Escherichia coli DNA polymerase I, and the reversion frequency of the progeny DNA was determined by transfection of E. coli spheroplasts. E. coli polymerase I makes less than 1 mistake at the am3 locus for every 7700 nucleotides incorporated under standard reaction conditions. Substitution of Mn2+ for Mg2+ and unequal concentrations of deoxynucleoside triphosphate substrates raises this mutation frequency to greater than 1 in 1000. Thus, E. coli DNA polymerase I can copy natural DNA templates with high fidelity and its accuracy can be affected by alterations in reaction conditions.
Antigens of human trophoblasts: A working hypothesis for their role in normal and abnormal pregnancies
Authors: Faulk W. Page , Temple Anne , Lovins R. E. , Smith Nancy
Published: May 2006

This report describes the preparation and characterization of antisera to human trophoblast membranes. Rabbit antisera were raised to trophoblast microvilli prepared by differential ultracentrifugation. Antibodies to serum proteins were removed by solid-phase immunoabsorption with normal human serum, and indirect immunofluorescence experiments with cryostat sections of human placentas showed that the absorbed anti-trophoblast sera reacted with trophoblasts as well as with stromal cells and endothelium of chorionic villi. The antisera also produced membrane fluorescence when studied on viable lymphocytes and certain human cell lines. These anti-trophoblast sera were also lymphocytotoxic, and this reaction was abolished by prior absorption of the antisera with leukocytes. The leukocyte-absorbed anti-trophoblast sera retained their ability to react with trophoblasts and certain human cell lines, but no longer reacted with lymphocytes or placental stromal cells and endothelium. Two categories of trophoblast membrane antigens are thus defined: one present on trophoblasts and certain human cells lines (tentatively designated TA
1
), and the other on trophoblasts and lymphocytes, villous fibroblasts, and endothelium (tentatively designated TA
2
). A working hypothesis is proposed stating that normal pregnancy involves the generation of anti-TA
2
subsequent to blastocyst implantation and entrance of trophoblasts into the maternal circulation. This involves a mechanism similar to allogeneic cell stimulation and results in antibodies that block either the recognition or cytotoxicity of TA
1
. Failure to mount this response allows TA
1
recognition and trophoblast immunopathology. Experimental and clinical studies in support of this working hypothesis, particularly involving abortion and toxemia, are cited from published reports.
Clonal dominance: loss and restoration in adoptive transfer.
Authors: Kaplan D R , Quintans J , Köhler H
Published: May 2006

An adoptive transfer system was used to study the mechanism responsible for clonal dominance of the anti-phosphorylcholine response in BALB/c mice. The adult spleen contains phosphorylcholine-specific precursor cells that are capable of developing into antibody-producing cells after transfer into lethally irradiated animals. The neonatal liver of the BALB/c mouse lacks precursor cells specific for phosphorylcholine but contains immature cells that differentiate into specific precursors during the normal course of ontogeny. The transfer of fetal or neonatal liver cells into lethally irradiated recipients prevents the appearance of the dominant H8 clone which constitutes the majority of the clones responding to phosphorylcholine in adult BALB/c mice. However, if these cells are transferred into neonatally suppressed recipients that lack the H8 idiotype, dominance of the H8 clone can develop. The conversion of the committed immature progenitor cell into a responsive B lymphocyte precursor is a regulated event. Regulation at the level of progenitor cells determines the eventual clonal profile of the immune response to phosphorylcholine. It is suggested that selection of the dominant clone occurs at this level.