bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of kidney epithelial cell growth inhibitors.
Authors: Holley R W , Böhlen P , Fava R , Baldwin J H , Kleeman G , Armour R
Published: May 2006

Two high molecular weight growth inhibitors have been isolated from the culture medium of BSC-1 cells, epithelial cells of African green monkey kidney. The purified kidney epithelial cell growth inhibitors, at ng/ml concentrations, reversibly arrest the growth of BSC-1 cells in the G1 phase of the cell cycle. Their action is selective; they are most active on BSC-1 cells, are less active as inhibitors of the growth of rat lung and human breast epithelial cells, and do not inhibit the growth of 3T3 mouse embryo fibroblasts ad human skin fibroblasts in culture. Their growth inhibitory action on BSC-1 cell cultures is counteracted by epidermal growth factor or calf serum.
Association of ribosomal genes in the fibrillar center of the nucleolus: a factor influencing translocation and nondisjunction in the human meiotic oocyte.
Authors: Mirre C , Hartung M , Stahl A
Published: May 2006

Prophase I meiosis was studied in the human oocyte obtained from 16- to 24-week-old fetuses. Electron microscopy and silver stainihg showed that, at pachytene, the ribosomal genes belonging to several chromosomes are gathered in the same nucleolar fibrillar center, where they are embedded in an argyrophilic protein. The nucleolus showed spontaneous segregation of its components due to temporary inactivation of the ribosomal genes. The fibrillar center, separated from the other nucleolar components, was penetrated as midpachytene by chromatin fibers containing rDNA emanating from one to three nucleolar bivalents. Thus, the ribosomal genes from 4-12 chromatids are temporarily juxtaposed inside the same structure. Such a structural arrangement is completely different from that observed in the pachytene-stage mouse oocyte, where two independent and active nucleoli, each displaying its own fibrillar center, were formed on the bivalents containing paired ribosomal genes. These different structural patterns are correlated with the high frequency of nondisjunction in the human oocyte and the relative infrequency of such in the mouse oocyte. The pattern observed in the human oocyte may be a cause of translocations.
Quantitation of mRNAs during mouse spermatogenesis: protamine-like histone and phosphoglycerate kinase-2 mRNAs increase after meiosis.
Authors: Erickson R P , Kramer J M , Rittenhouse J , Salkeld A
Published: May 2006

Total RNA, prepared from immature or mature mouse testes or from spermatogenic cells separated on the basis of sedimentation velocity, was translated in vitro. Mouse protamine-like histone could be identified as an in vitro translational product when [3H]arginine was used as the label. The mRNA for protamine-like histone was detected only after meiosis; the appearance of a peak of radioactivity comigrating with protamine-like histone occurred only when RNA from mature testes or late spermatid cell fractions was translated. Phosphoglycerate kinase-2 (ATP:3-phospho-D-glycerate I-phosphotransferase, EC 2.7.2.3) was identified as an in vitro translational product by affinity chromatography followed by two-dimensional gel electrophoresis or by specific immunoprecipitation when [35S]methionine was the label. The mRNA for phosphoglycerate kinase-2 was detected only in mature testes or late spermatid cell fractions. These translational assays for protamine-like histone and phosphoglycerate kinase-2 mRNAs suggest that these messages are transcribed after meiosis.
Identification and localization of glucagon-related peptides in rat brain.
Authors: Tager H , Hohenboken M , Markese J , Dinerstein R J
Published: May 2006

Immunochemical and immunocytochemical techniques have been used to identify and characterize glucagon-related peptides of the rat central nervous system. These peptides show immunoreactivity with antiglucagon sera directed towards the central portion of the hormone, but not with antisera specific for the free COOH terminus of glucagon. Highest concentrations were found in hypothalamus (6.1 +/- 1.6 ng/g wet weight) although lower amounts (approximately 2 ng/g) were found in cortex, thalamus, cerebellum, and brain stem. Gel filtration of brain extracts revealed at least two immunoreactive forms, which have molecular weights of about 12,000 and 8000. Both peptides had radioimmunoassay dilution curves parallel to the curve for glucagon and both had identical counterparts in extracts of rat intestine. Digestion of the brain and intestinal peptides with trypsin plus carboxypeptidase B released the immunoreactive COOH-terminal tryptic fragment of pancreatic glucagon from these larger forms. Immunocytochemical studies using antiglucagon serum and peroxidase-antiperoxidase staining identified glucagon-like material in neuronal cell bodie and processes in the magnocellular portion of the paraventricular nucleus, as well as in scattered cells in the supraoptic nucleus and in fibers in the median eminence. These results suggest that glucagon-containing peptides that have undergone the intestinal type of posttranslational modification are present in neuronal cells of the rat hypothalamus.
Why is there no bulk specific heat anomaly at the superconducting transition temperature of BaPb
1-x
Bi
x
O
3
?
Authors: Methfessel Christoph E. , Stewart Gregory R. , Matthias Bernd T. , Patel C. K. N.
Published: May 2006

Caloric measurements have shown that there is no bulk specific heat anomaly at the superconducting transition temperature of BaPb
0.75
Bi
0.25
O
3
.
The capsid protein of Semliki Forest virus has clusters of basic amino acids and prolines in its amino-terminal region.
Authors: Garoff H , Frischauf A M , Simons K , Lehrach H , Delius H
Published: May 2006

The amino acid sequence of the capsid (C) protein was deduced from the nucleotide sequence of the C gene. This part of the viral 42S RNA genome was transcribed into double-stranded cDNA. The cDNA was cloned in the Escherichia coli chi 1776-pBR322 host-vector system and then the base sequence was determined with the technique described by Maxam and Gilbert. The amino acid sequence of the C protein shows a clustering of basic amino acids and prolines within the first 110 amino acids.
Specificity in interaction of benzo[a]pyrene with nuclear macromolecules: implication of derivatives of two dihydrodiols in protein binding.
Authors: MacLeod M C , Kootstra A , Mansfield B K , Slaga T J , Selkirk J K
Published: May 2006

Benzo[a]pyrene (B[a]P), 7,8-dihydroxy-7,8-dihydro-B[a]P, and 9,10-dihydro-B[a]P are metabolized by hamster embryo cells to derivatives that bind to nuclear macromolecules. The selectivity for different classes of macromolecules varies depending on the compound analyzed. The ratio of DNA specific activity to protein specific activity (pmol bound/mg of macromolecules) is high (1.51) for 7,8-dihydroxy-7,8-dihydro-B[a]P, extremely low (0.03) for 9,10-dihydroxy-9,10-dihydro-B[a]P, and intermediate (0.26) for B[a]P. Histones H3 and H2A are the major targets of 7,8-dihydroxy-7,8-dihydro-B[a]P; a protein(s) with a mobility similar to that of histone H1 is heavily labeled by 9,10-dihydroxy-9,10-dihydro-B[a]P, with minor labeling of other (nonhistone) bands. The labeling pattern seen with B[a]P is a combination of the patterns seen with the two dihydrodiol metabolites studied. Analysis of the ethyl acetate-soluble metabolites suggests that hamster embryo cells produce 9,10-dihydroxy-7,8-oxy-7,8,9,10-tetrahydro-B[a]P from 9,10-dihydroxy-9,10-dihydro-B[a]P and raise the possibility that this vicinal diol epoxide is an intermediate in the binding of 9,10-dihydroxy-9,10-dihydro-B[a]P to nuclear proteins. The differences seen suggest that factors other than the intrinsic chemical reactivity of the epoxide group are extremely important in the interaction of potential ultimate carcinogens with biological systems.
Methylation of foreign DNA sequences in eukaryotic cells.
Authors: Pollack Y , Stein R , Razin A , Cedar H
Published: May 2006

The herpesvirus thymidine kinase gene has been used to introduce foreign DNA sequences into mouse L cells by DNA-mediated gene transfer. These inserted genes were then assayed for methylation at the specific sequence C-C-G-G by using the restriction enzyme isoschizomers Hpa II and Msp I. Despite the fact that 70% of the cellular C-C-G-G sites are methylated, herpesvirus sequences, plasmid DNA, and growth hormone gene DNA were found to remain unmethylated in 90% of the clones that contain these genes. DNA that had been methylated in vitro with Hpa II methylase was also inserted into L cells. The presence of this modification in the vector DNA did not, however, guarantee that these sequences remained methylated in the recipient clones. Only 10% of all transformed clones were found to contain methylated C-C-G-G sequences in the vector DNA, and these modifications were stable for 25-50 generations. Hha I and Mbo I were used to probe for methyl groups at these restriction sites, but none of the inserted sequences acquired these modifications. These results are discussed in relation to various models put forth to explain the process of methylation in eukaryotic cells.
Construction and analysis of simian virus 40 origins defective in tumor antigen binding and DNA replication.
Authors: Myers R M , Tjian R
Published: May 2006

We have inserted a 311-base pair DNA fragment containing the simian virus 40 (SV40) origin of DNA replication, the early promoter, and the tumor (T) antigen binding sites into a bacterial plasmid and cloned it. This recombinant plasmid, pSV01, binds to a purified T antigen in vitro and replicates in monkey cells when supplied with large T antigen. A series of deletion mutations was generated in the origin sequences of pSV01 DNA by mutagenesis in vitro. The replication of these mutant DNAs in monkey cells was compared with their ability to bind to purified D2 protein. Mutant DNAs deficient in binding to D2 protein also exhibit reduced levels of replication in monkey cells. These findings provide biochemical evidence that the initiation of SV40 DNA synthesis may involve a direct interaction of T antigen with sequences at the origin of replication.
Subcompartments of the G1 phase of cell cycle detected by flow cytometry.
Authors: Darzynkiewicz Z , Sharpless T , Staiano-Coico L , Melamed M R
Published: May 2006

Cellular DNA and RNA were simultaneously quantitated in individual cells of cycling and quiescent populations by flow cytometry. Based on differences in RNA content, two distinct subcompartments, G1A and G1B, were recognized in the G1 phase of exponentially growing cells. After mitosis, cells reside in the low-RNA, G1A compartment from which they cannot enter the S phase directly. An increase in RNA above a critical level is required for G1 cells to be able to initiate DNA replication; G1 cells with RNA values above this level are classified as G1B. Cell transition from G1A to G1B and from G1B to S was analyzed in a stathmokinetic experiment on L1210 cells with a doubling time of 10 hr. The half-time of cell residence in the indeterminate state of G1 was found to be 1 hr, and the duration of the deterministic portion of G1 phase was 2 hr. The indeterminate state, although not identical with G1A, is most likely located in G1A. Cell quiescence induced by serum deprivation (3T3 cells) or by addition of n-butyrate (L1210) results in cell arrest at a state which, judged by RNA content, is similar to that of G1A of exponentially growing cells. The exit from this state, however, is much slower after stimulation of these blocked cells than the transition of G1A to G1B in cells growing exponentially. Thymidine or hydroxyurea arrest cells in G1B or perhaps at the border between G1B and S. Prolonged incubation of 3T3 cells to confluence results in a marked loss of cellular RNA below the level of the G1A state. This deep quiescent state (G1Q) is distinctly different from the G1A state of cycling cells or cells blocked in G1A by serum deprivation.
Asynchronous replication of heterochromatin in maize
Authors: Pryor A. , Faulkner K. , Rhoades M. M. , Peacock W. J.
Published: May 2006

Replication patterns of four classes of heterochromatin in the chromosomes of maize were analyzed. Centromeric heterochromatin, distal heterochromatin of the B chromosomes, and knob heterochromatin all have asynchronous late replication, knob heterochromatin being the last chromosomal DNA to complete replication. Nucleolus organizer heterochromatin replicates during the period of euchromatin DNA replication. The proportion of the S period involved in asynchronous late replication is directly dependent on the amount of heterochromatin in the nucleus. Both additional knob and B-chromosome heterochromatin participate in this effect. Each additional B chromosome produces an increment in the proportion of nuclei with asynchronous replication; in nuclei with 12 B chromosomes, the proportion of the S phase showing asynchronous late replication is more than 3 times greater than that in nuclei without B chromosomes. The data are consistent with the hypothesis that delayed DNA replication of knob heterochromatin is responsible, in some genotypes, for the loss of chromosome segments and for B-chromosome nondisjunction in the second pollen grain mitosis.
Evolutionary approach to the question of immunoglobulin heavy chain switching: evidence from cloned human and mouse genes.
Authors: Ravetch J V , Kirsch I R , Leder P
Published: May 2006

We have used cloned mouse and human heavy chain genes to identify regions of conserved homology between mouse and man and between mu and alpha genes. Using heteroduplex mapping, we find that coding segments for homologous domains appear to have been well conserved between the two species, but the short intervening sequences that separate domains have diverged considerably. These studies also identify extensive regions of homology 5' to both mu and alpha constant region genes that are conserved between the two genes as well as between mouse and man. These segments encompass regions in which mu/alpha recombination occurs during the heavy chain class switch, and their extensive homology may be relevant to this process.
Expression of an X-linked gene from an inactive human X chromosome in mouse-human hybrid cells: further evidence for the noninactivation of the steroid sulfatase locus in man.
Authors: Mohandas T , Sparkes R S , Hellkuhl B , Grzeschik K H , Shapiro L J
Published: May 2006

Somatic cell hybrid clones were derived from the fusion of hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8)-deficient mouse cells and two different human fibroblast strains, each carrying an X chromosome-autosome translocation. One of these had an X/11 translocation [46,X,t(X;11)(p21;q13)] and the other had an X/19 translocation [46,X,t(X;19)(q22;q13)]. The structurally normal human X chromosome is the late-replicating (genetically inactive) chromosome in these two cell strains; the rearranged X chromosome is early replicating (genetically active). One primary hybrid clone carrying both the translocated X chromosome and the structurally normal X chromosome was isolated in hypoxanthine/aminopterin/thymidine medium from each of these two cell fusion experiments. These clones were then selected in medium containing 8-azaguanine to achieve the loss of the active human HPRT locus. Five subclones from the cell hybrid with the X/11 translocation failed to express two known human X-chromosome markers [glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) and phosphoglycerate kinase (PGK; EC 2.7.2.3)] but did express human microsomal steroid sulfatase (STS; sterol-sulfate sulfohydrolase, EC 3.1.6.2). Three of these were cytogenetically analyzed and found to contain a structurally normal human X chromosome but not the X/11 translocation. Two subclones were isolated in 8-azaguanine from the hybrid with the X/19 translocation. Cytogenetic analysis of these two clones showed the presence of a structurally normal human X chromosome; the X/19 translocation was not present. They did not express human G6PD, PGK, or HPRT but did express human STS. These results indicate that human STS is expressed from a locus on the inactive human X chromosome and support our earlier finding that the STS locus escapes X-inactivation in man.
Murine T cell Ia antigens: specificity Iat.46 maps in I-J to I-E chromosomal segment of H-2 complex.
Authors: Hayes C E , Maier J M
Published: May 2006

An alloantiserum was prepared in C57BL/10 mice hyperimmunized with concanavalin A-stimulated B10.HTG thymocytes. Absorption with separated B10.A(3R)B and T lymphocytes, followed by titration on these cell types, revealed a new I-region controlled T-cell specificity, Iat.46. The determinant is present on a subpopulation of mature peripheral T cells but not on B lymphocytes, thymocytes, or bone marrow cells. It is also observed using (A.TH X B10.HTT)F1 anti-A.TL antiserum on B10.A(3R) T lymphocytes. The k and d haplotypes are fully crossreactive with regard to Iat.46 expression. B10.HTT, C57BL/10, and BSVS do not carry the Iat.46-encoding allele. Moreover, Iat.46 is not a determinant formed by I-A--I-E gene complementation. The Iat.46-controlling gene, Ia-8, may be positioned to the left of the B10.HTT crossover and to the right of the B10.A(3R) recombinational event. Thus, this determinant may be considered either to represent complexity within the I-J subregion or to demarcate a new I subregion between I-J and I-E.
Deoxycytidine kinase-mediated toxicity of deoxyadenosine analogs toward malignant human lymphoblasts in vitro and toward murine L1210 leukemia in vivo.
Authors: Carson D A , Wasson D B , Kaye J , Ullman B , Martin D W , Robins R K , Montgomery J A
Published: May 2006

An inherited deficiency of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) produces selective lymphopenia and immunodeficiency disease in humans. Previous experiments have suggested that lymphospecific toxicity in this condition might result from the selective accumulation of toxic deoxyadenosine nucleotides by lymphocytes with high deoxycytidine kinase, levels and low deoxynucleotide dephosphorylating activity. The present experiments were designed to determine if deoxyadenosine analogs which are not substrates for adenosine deaminase might similarly be toxic toward lymphocytes and lymphoid tumors. Two such compounds, 2-chlorodeoxyadenosine and 2-fluorodeoxyadenosine, at concentrations of 3 nM and 0.15 microM, respectively, inhibited by 50% the growth of human CCRF-CEM malignant lymphoblasts in vitro. Each was phosphorylated in intact cells by deoxycytidine kinase accumulated as the nucleoside triphosphate, and inhibited DNA synthesis more than RNA synthesis. Both deoxynucleosides had significant chemotherapeutic activity against lymphoid leukemia L1210 in mice.
Single channel currents from excised patches of muscle membrane.
Authors: Horn R , Patlak J
Published: May 2006

The currents through single acetylcholine-activated channels were measured on membrane fragments that had been torn from rat muscle myotubes with patch pipettes. The membrane fragments were sealed into the pipette by using the "gigohm-seal" technique of Neher, which also permitted voltage clamp of the membrane via the patch electrode. Membrane patches were excited by sudden withdrawal of the electrode from the cell. Substitution of fluoride for chloride ions in the bathing solution could prevent or reverse the tendency for the membrane at the electrode tip to seal over into a closed vesicle. The single membrane layer at the electrode tip could remain functional for up to 30 min. The apparent single channel conductance was minimally affected by excision. The current-voltage relationships for the single channel currents show that the inside (i.e., cytoplasmic surface) of the membrane fragment was exposed to the bathing solution. In symmetric Na solutions the current-voltage curve was nearly linear and reversed at approximately 0 mV. In other bathing solutions from 40 to 500 mM NaF, the observed zero current potential was close to that predicted by the Nernst equation. We present evidence that internal Na interacts with the channel, causing both saturation of outward current and block of inward current. At + 100 mV the apparent dissociation constant for internal Na was 138 mM.
An empirical Bayes estimation problem
Authors: Robbins Herbert
Published: May 2006

Let
x
be a random variable such that, given θ,
x
is Poisson with mean θ, while θ has an unknown prior distribution
G
. In many statistical problems one wants to estimate as accurately as possible the parameter
E
(θǀ
x
=
a
) for some given
a
= 0,1,.... If one assumes that
G
is a Gamma prior with unknown parameters α and β, then the problem is straightforward, but the estimate may not be consistent if
G
is not Gamma. On the other hand, a more general empirical Bayes estimator will always be consistent but will be inefficient if in fact
G
is Gamma. It is shown that this dilemma can be more or less resolved for large samples by combining the two methods of estimation.
Monoclonal antibodies to beta-adrenergic receptors: use in purification and molecular characterization of beta receptors.
Authors: Fraser C M , Venter J C
Published: May 2006

We have developed four hybridomas that produce monoclonal antibodies to the turkey erythrocyte beta 1-adrenergic receptor and one hybridoma that produces a monoclonal antibody to the calf lung beta 2 receptor. Splenic lymphocytes from BALB/c mice immunized with partially purified turkey erythrocyte beta 1 receptors or calf lung beta 2 receptors were used with the mouse myeloma line SP2/O-Ag14 to yield hybridoma cultures producing beta receptor monoclonal antibodies of the IgG class. The anti-turkey erythrocyte beta receptor antibodies precipitated partially purified beta receptors and inhibited adrenergic ligand binding. In contrast to autoantibodies to beta 2-adrenergic receptors [Venter, J. C., Fraser, C. M. & Harrison, L. C. (1980) Science 207, 1361-1363] which do not crossreact with cardiac beta 1 receptors, monoclonal antibody FV-104 directed against the adrenergic ligand binding site of turkey erythrocyte beta receptors crossreacted equally with calf liver and lung beta 2 receptors as well as calf heart beta 1 receptors. These data suggest that some molecular homology exists between beta-adrenergic receptors of substantially diverse pharmacological classes. We utilized the monoclonal antibodies in the final stage of turkey erythrocyte beta 1 receptor purification. Turkey erythrocyte beta receptors eluted from FV-104 monoclonal antibody affinity columns with Na-DodSO4 appeared as three components of Mr 70,000, 31,000, and 22,000 on NaDodSO4/polyacrylamide gels. Iodination of material eluted from immunoaffinity columns with propranolol demonstrated the existence of only a single component (Mr, 70,000), indicating that the turkey erythrocyte beta 1 receptor can be purified to homogeneity.
In vitro transcription of normal, mutant, and truncated mouse alpha-globin genes.
Authors: Talkington C A , Nishioka Y , Leder P
Published: May 2006

We have transcribed various alpha-globin gene-containing templates by using a simplified in vitro system and have shown that the 5' end of each of the RNA polymerase II-specific transcripts corresponds to that of authentic alpha-globin mRNA. Using a truncated alpha-globin gene fragment, we then localized the promotor region of the adult alpha-globin gene to a 138-base-pair fragment of alpha-globin DNA, 141 nucleotides of which precede the initiation site. In contrast, when two naturally occurring alpha-globin gene mutants were tested as templates, no RNA polymerase II-dependent initiation was detected. Comparison of the nucleotide sequences of the normal and mutant alpha-globin genes has allowed us to focus upon nucleotide positions that might be essential for promoter activity.
Kinetic study of the interaction of oxy- and deoxyhemoglobins with the erythrocyte membrane.
Authors: Shaklai N , Sharma V S
Published: May 2006

Changes in fluorescence intensity of a membrane-embedded probe were used to study the kinetics of binding of oxy- and deoxyhemoglobin to erythrocyte membranes. For these studies, stopped-flow fluorimetric techniques were utilized. Both binding and dissociation of hemoglobin from membranes followed heterogeneous first-order kinetics. The rate constants for binding of oxyhemoglobin were about 10 times larger than those of deoxyhemoglobin; the dissociation rate constants of oxyhemoglobin were about one-quarter those of the unliganded form. The results are discussed in light of the steady-state binding constants previously derived for both oxy- and deoxyhemoglobin.
A cyclic enkephalin analog with high in vitro opiate activity.
Authors: DiMaio J , Schiller P W
Published: May 2006

A conformationally restricted analog of [Leu5]enkephalin was synthesized by cyclization of the COOH-terminal carboxyl group of leucine to the gamma-amino moiety of alpha, gamma-diaminobutyric acid (A2bu) substituted in position 2 of the peptide. Relative to [Leu5]enkephalin, the cyclic analog with D configuration in position 2, H-Tyr-cyclo(-N gamma-D-A2bu-Gly-Phe-Leu-), was 17.5 times more potent in the guinea pig ileum assay and twice as potent in the rat brain receptor binding assay, whereas its diastereomer H-Tyr-cyclo(-N gamma-L-A2bu-Gly-Phe-Leu-) showed low activity. The cyclic D isomer was also slightly more active than the open-chain reference compound [D-Nva2, Leu5]enkephalinamide in both assays, and it proved to be highly resistant to degradation by brain "enkephalinases." The steric constraints introduced in H-Tyr-cyclo(-N gamma-D-A2bu-Gly-Phe-Leu-) were shown to prevent the realization of most of the conformational features ascribed to linear enkephalin in solution or in the crystalline state and permitted an assessment of proposed models of the conformation of enkephalin when it is bound to the receptor.
A new approach to time-resolved studies of ATP-requiring biological systems; laser flash photolysis of caged ATP.
Authors: McCray J A , Herbette L , Kihara T , Trentham D R
Published: May 2006

2-Nitrobenzyl derivatives have been used for several years as photolabile protecting groups in synthetic organic chemistry. Recently, P3-1-(2-nitro phenylethyladenosine 5'-triphosphate "caged ATP" was synthesized and its photolysis was shown to generate ATP in situ. This and related reactions have great potential for structural and kinetic studies of both intact and soluble biological systems and it is thus important to define the kinetic characteristics of the photolytic reaction. Caged ATP (2.5 mM) was photolyzed at 347 nm by a single 30-nsec pulse from a frequency-doubled ruby laser of 25 mJ energy to generate 500 microM ATP. The kinetics of the overall reaction were determined by monitoring the kinetics of ATP-induced dissociation of actomyosin, a reaction of known kinetic characteristics. Release of 500 microM ATP was found to be controlled by a process having a rate constant of 2.2 X 10(9) [H+] sec-1 at 22 degrees C at pH 5.8-9.5, which corresponds to 220 sec-1 at pH 7. This process is believed to be the breakdown of an aci-nitro compound, which was identified on the basis of its spectral properties and the photochromicity of related 2-nitrobenzyl compounds.
Nucleotide sequence divergence and functional constraint in mRNA evolution.
Authors: Miyata T , Yasunaga T , Nishida T
Published: May 2006

Comparison of about 50 pairs of homologous nucleotide sequences for different genes revealed that the substitutions between synonymous codons occurred at much higher rates than did amino acid substitutions. Furthermore, five pairs of mRNA sequences for different genes were compared in species that had diverged at the same time. The evolutionary rate of synonymous substitution was estimated to be 5.1 X 10(-9) per site per year on the average and is approximately constant among different genes. It also is suggested that this property would be suitable for a molecular clock to determine the evolutionary relationships and branching order of duplicated genes. Each functional block of the noncoding region evolves with a rate that is almost constant, regardless of the types of genes. The intervening sequence and the 5' portion of the 3' noncoding region show considerable divergence, the extent of which is almost comparable to that in the synonymous codon sites, whereas the other blocks consisting of the 5' noncoding region and the 3' portion of the 3' noncoding region are strongly conserved, showing approximatley half of the divergence of the synonymous sites. This strong sequence preservation might be due to the functional requirements for transcription and modification of mRNA.
Allelic forms of beta 2-microglobulin in the mouse.
Authors: Goding J W , Walker I D
Published: May 2006

Spleen cells from BALB/c and C57BL/6 mice were cultured separately or together, and the biosynthetically labeled supernates were examined by two-dimensional polyacrylamide gel electrophoresis. Although there were no major labeled proteins in the mixed group that were not present in the separate cultures, there was a major low-molecular-weight protein that differed in charge in the two strains. This protein was identified as beta 2-microglobulin; it could be labeled with 125I on the cell surface by using the lactoperoxidase technique, was noncovalently attached to the H-2K molecule, and had the expected size and charge when compared with human beta 2-microglobulin. Both acidic and basic forms were present in (BALB/c X C57BL/6) F1 hybrids, suggesting codominant expression, although allelic exclusion was not ruled out. Either parental form could combine with one parental form of the H-2K molecule. The beta 2-microglobulin gene does not appear to be closely linked to either the H-2 or th immunoglobulin heavy-chain complexes. It is proposed that beta 2-microglobulin is an "effector subunit" of histocompatibility antigens and that its physiological role is to interact with a specific killing structure on the surface of cytolytic T lymphocytes and thereby initiate cell destruction.
Microinjection of catalytic subunit of cyclic AMP-dependent protein kinase enhances calcium action potentials of bag cell neurons in cell culture
Authors: Kaczmarek L. K. , Jennings K. R. , Strumwasser F. , Nairn A. C. , Walter U. , Wilson F. D. , Greengard P.
Published: May 2006

We have found that the calcium action potentials of bag cell neurons from the abdominal ganglion of
Aplysia
may be enhanced by intracellular microinjection of the catalytic subunit of cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). The catalytic subunit was purified from bovine heart and shown to be effective in stimulating the phosphorylation of bag cell proteins in homogenates at concentrations of 10-50 nM. Intracellular injection into isolated bag cell neurons maintained in primary culture was through pressure applied to microelectrodes filled at the tip with catalytic subunit (5-22 μM). In 11 of 16 injected cells, both the slope of the rising phase and the height of the action potentials evoked by a constant depolarizing current were markedly enhanced relative to the pre-injection control (mean increases, 73% and 35%, respectively). This effect could occur with no change in resting potential or in the latency of the action potential from the onset of the depolarizing pulse. The effect was observed with enzyme dissolved in three different salt solutions (Na phosphate, K phosphate, or KCl). In two experiments, tetrodotoxin (50 μM) added to the extracellular medium had no effect on the enhanced action potentials. Subsequent addition of the calcium antagonist Co
2+
, however, diminished or abolished the spikes. In more than half of the experiments, the injection of catalytic subunit was accompanied by an increase in the input resistance of the cells as measured by applying small hyperpolarizing current pulses. In three experiments, subthreshold oscillations in membrane potential resulted from the injections. Control injections (24 cells), carried out either with carrier medium alone or with heat-inactivated enzyme preparations, did not produce spike enhancement, increased input resistance, or oscillations. Our data suggest that the stimulation of intracellular protein phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase enhances the excitability of bag cell neurons by modifying calcium and potassium channels or currents.
Glucocorticoid hormones increase the activity of plasma membrane alkaline phosphodiesterase I in rat hepatoma cells.
Authors: Rousseau G G , Amar-Costesec A , Verhaegen M , Granner D K
Published: May 2006

In rat hepatoma cells the synthetic glucocorticoid dexamethasone causes a 3-fold increase in the activity of the plasma membrane enzyme alkaline phosphodiesterase I (oligonucleat 5'-nucleotidohydrolase, EC 3.1.4.1). The data are consistent with an induction phenomenon mediated by the glucocorticoid receptor involved in tyrosine aminotransferase induction. The effect on alkaline phosphodiesterase I is not a reflection of a general membrane effect of dexamethasone, because the activity of three other enzymes of the plasma membrane is unaffected. On the other hand, nucleoside diphosphatase (nucleoside diphosphate phosphohydrolase acting on ADP) activity is inhibited. Thus, two more enzymes sensitive to glucocorticoids have been identified in a cell line in which these hormones influence only very few gene products. This paper describes enzymatic changes in the plasma membrane of rat hepatoma cells in which glucocorticoids normalize a number of membrane-associated processes that are considered to be characteristic of transformed cells.
L-Glutamic acid as a mediator of sexual morphogenesis in
Volvox capensis
Authors: Starr Richard C. , O'Neil Rory M. , Miller Charles E.
Published: May 2006

In
Volvox capensis
the development of sexual individuals is in response to low concentrations (68 nM) of L-glutamic acid rather than to such species-specific glycoproteins as have been isolated in
Volvox carteri
or are believed to exist in a number of other species.
V. capensis
grows equally as well in light and in darkness in a medium supplemented with sodium acetate; however, L-glutamic acid is active as an inducer of the sexual form only in populations grown in the light. The site of action of L-glutamic acid and its biochemical role in the sexual response are unknown. Attempts to induce the sexual response by using the other L-amino acids, various analogs of glutamic acid, compounds of similar structure (e.g., γ-aminobutyric acid), and intermediates of biochemical pathways known to involve L-glutamic acid (e.g., α-ketoglutarate or pyroglutamic acid) have been unsuccessful. L-Glutamic acid is produced by
V. capensis
as a natural product of the digestion of the glycoproteinaceous parental matrix at the time young spheroids escape. As a population increases, so does the level of L-glutamic acid produced at each succeeding generation until the threshold of sensitivity is reached and the induction of sexual forms is effected. This serves as a mechanism for ensuring the production of sexual spheroids and their zygotes, the only phase in the life cycle resistant to drying. Thus,
Volvox
is especially adapted to an existence in ephemeral pools of water resulting from seasonal rains.
Immunohistochemical localization of cholecystokinin- and gastrin-like peptides in the brain and hypophysis of the rat
Authors: Vanderhaeghen J. J. , Lotstra F. , De Mey J. , Gilles C.
Published: May 2006

The distribution of gastrin-cholecystokinin-like peptide(s) is reported in brain and hypophysis of the rat. The unlabeled peroxidase-antiperoxidase complex immunohistochemical technique was used. Controls of specificity for various peptides were studied with solid-phase absorption. Colchicine treatment was necessary to obtain positivity in many neuronal cell bodies. In addition to their already known distribution, gastrin-cholecystokinins containing neural cell bodies and fibers were present in olfactory structures, in various preoptic and hypothalamic nuclei (except in mamillary bodies), in mesencephalic nucleus linearis rostralis, and in A-10, A-9, and A-8 regions of Dahlström and Fuxe, which include substantia nigra. From previous investigations and the present distribution study, it can be inferred that, although most of the brain material consists of cholecystokinin, gastrins may also be present in hypothalamo-posthypophyseal magnocellular cells, in nucleus tractus solitarii, and in the dorsal horn of the spinal cord. The distribution of positive cell bodies in the peripheral part of the paraventricular nucleus and in the dorsal part of the supraoptic nuclei in the hypothalamus is similar to that of oxytocin neurons. The localization of positive cell bodies in A-10, A-9, and A-8 regions of Dahlström and Fuxe is similar to that of dopaminergic neurons. The mesencephalic concentration of cell bodies and the wide distribution of fibers in striatal, hypothalamic, septal, and other hemispheric structures together with thick positive fibers in the medial forebrain bundle is consistent with the existence of ascending mesencephalic pathways, including the nigrostriate pathway.
Properties of some three-state, steady-state Ising systems, according to the Bragg-Williams approximation
Authors: Hill Terrell L. , Stein Leonard
Published: May 2006

We consider the steady-state properties of a lattice of three-state, cycling enzyme molecules, with nearest-neighbor interactions treated by the Bragg-Williams (mean field) approximation. Only a few particular cases are examined, but these illustrate the rich phase-transition possibilities of this class of systems. “Bifurcation” cases were treated in a previous paper; the present examples are of the nonbifurcation type. However, a few new theoretical properties of bifurcation cases are included.
Structural basis for apparent heterogeneity of collagens in human basement membranes: type IV procollagen contains two distinct chains.
Authors: Crouch E , Sage H , Bornstein P
Published: May 2006

Fetal cells isolated from human amniotic fluid synthesize type IV procollagen when grown in monolayer culture. The procollagen, which contains two biochemically distinct chains, was found to be structurally and immunologically related to type IV collagen chains and collagenous fragments isolated from human placenta. Limited pepsin digestion of the intact procollagen that was deposited in the cell layer during culture produced a heterogeneous population of collagenous peptides comparable to that obtained during isolation of type IV collagens from human tissues. These studies support the hypothesis that basement membranes contain at least two genetically distinct type IV procollagen chains and suggest that the heterogeneity of collagenous components obtained after pepsin digestion of tissues and isolated basement membranes can result from degradative cleavage of the procollagen at a limited number of protease-sensitive sites.
Enhancement of free radical reduction by elevated concentrations of ascorbic acid in avian dystrophic muscle.
Authors: Perkins R C , Beth A H , Wilkerson L S , Serafin W , Dalton L R , Park C R , Park J H
Published: May 2006

It has been postulated that the degenerative process in dystrophic muscle results from increased concentrations of free radicals, peroxides, or lipid hydroperoxides. Therefore, the reduction of the free radical tanol (2,2,6,6-tetramethyl-4-piperidinol-1-oxyl) by extracts of muscles of dystrophic and normal chickens was studied. Pectoral (white) and thigh (red) muscles were used. For initial rate measurements, the various muscle extracts were added to an equal volume of 0.2 mM tanol. Reaction mixtures were introduced into the EPR cavity in a standard aqueous flat cell. Rates were measured by continuously monitoring the decrease in signal amplitude of the center (MI = 0) solution tanol EPR resonance line (in-phase first harmonic absorption signal). With extracts from dystrophic white muscle, the reduction rate was 75% faster than normal, whereas in dystrophic red muscle extracts the rate was normal. This agreed with previous observations that white muscle is more severely affected than red in dystrophic chickens. The primary reductant was identified as reduced ascorbic acid, and the rate of reduction of tanol correlated directly with the concentrations of ascorbic acid in the various muscle extracts as shown by chemical analysis. The results suggest an involvement of the intracellular redox status in the pathogenesis of avian muscular dystrophy.
Preferential transcription of cloned maize chloroplast DNA sequences by maize chloroplast RNA polymerase
Authors: Jolly Setsuko O. , Bogorad Lawrence
Published: May 2006

Zea mays
chloroplast DNA-dependent RNA polymerase
in vitro
preferentially transcribes maize chloroplast DNA sequences incorporated in cloned chimeric bacterial plasmids. Preferential transcription is dependent on the presence of a 27.5-kilodalton polypeptide, the S factor, which has been purified from maize chloroplasts, and also on the template's being in the supercoiled form.
Apparent involvement of ribonuclease D in the 3' processing of tRNA precursors.
Authors: Cudny H , Deutscher M P
Published: May 2006

Escherichia coli RNase D and RNase II have been purified to homogeneity and compared for their ability to remove extra nucleotides following the -C-C-A sequence in tRNA precursors. RNase D and RNase II are single-chain proteins with molecular weights of 38,000 and 78,000, respectively. Both enzymes require a divalent cation for activity on tRNA precursors, but, in addition, RNase II is stimulated by monovalent cations. RNase D specifically removes mononucleotide residues from a mixture of tRNA precursors to generate amino acid acceptor activity for essentially all amino acids. Although RNase II can also remove precursor-specific residues, no amino acid acceptor activity is recovered. Similarly, RNase D action on the E. coli tRNATyr precursor is limited, whereas RNase II causes extensive degradation. In contrast to the processive mode of hydrolysis by RNase II, RNase D removes nucleotides randomly and slows down greatly at the -C-C-A sequence, thereby allowing the tRNA to be aminoacylated and protected from further degradation. These results suggest that RNase D is the 3'-processing nuclease in vivo and that RNase II is a nonspecific degradative enzyme. The importance of RNA conformation for correct processing is also discussed.
Two species of full-length cDNA are synthesized in high yield by melittin-treated avian retrovirus particles.
Authors: Boone L R , Skalka A
Published: May 2006

A method of activating endogenous cDNA synthesis in avian retroviruses that results in the formation of two species of full-length cDNA in high yield is described. Tests of biological activity show infectivity of at least the same order of magnitude as for full-length cDNA made by other procedures. Melittin, the major component of bee venom, is used as an alternative to nonionic detergents to make the viral envelope permeable and thus activate the endogenous RNA-dependent DNA polymerase. This compound is a toxic peptide known to interact with phospholipid membranes. It appears to be less disruptive to the viral structure than detergents, resulting in a more efficient transcription of the viral genome. Preliminary tests indicate that this method will also prove useful for studying enzymatic activities associated with other enveloped viruses.
Analysis of viral DNA sequences in hamster cells transformed by herpes simplex virus type 2.
Authors: Galloway D A , Copple C D , McDougall J K
Published: May 2006

Herpes simplex virus type 2 (HSV-2) DNA was treated with four restriction endonucleases (EcoRI, HincIII, Bgl II, and Xba I) and eight fragments were purified and labeled with 32P in vitro. The kinetics of renaturation of each of the fragments was measured in the presence of DNA extracted from 333-8-9, a hamster cell line transformed by UV light-inactivated HSV-2 strain 333, and from a series of cloned derivatives and their tumor lines. All of the lines examined contained a partial set of viral sequences present at only a few copies per cell. Passage of the cell lines in tissue culture or in animals resulted in partial loss of viral DNA. Two blocks of sequences were present in most of the lines examined; those mapping at positions 21--33 of the HSV-2 genome were detected in seven of seven cell lines tested and those at positions 60--65 were detected in six of eight. Other sequences from the L component can also be present in the DNA of HSV-2-transformed hamster cells.
Reversible dissociation of linker histone from chromatin with preservation of internucleosomal repeat.
Authors: Allan J , Staynov D Z , Gould H
Published: May 2006

Procedures are described for the dissociation of histones H1 and H5 from chicken reticulocyte chromatin without disruption of the native core histone-DNA complex. The comparative properties of native and depleted chromatin with respect to sedimentation, thermal denaturation, and sensitivity to nuclease digestion have been studied. The changes in these properties resulting from removal of the linker histones are fully reversed when histone H5 is added back to the depleted chromatin.
Camphorquinone-10-sulfonic acid and derivatives: Convenient reagents for reversible modification of arginine residues
Authors: Pande Chandra S. , Pelzig Michal , Glass John D.
Published: May 2006

Camphorquinone-10-sulfonic acid hydrate was prepared by the action of selenous acid on camphor-10-sulfonic acid. Camphorquinone-10-sulfonylnorleucine was prepared either from the sulfonic acid via the sulfonyl chloride or by selenous acid oxidation of camphor-10-sulfonylnorleucine. These reagents are useful for specific, reversible modification of the guanidino groups of arginine residues. Camphorquinonesulfonic acid is a crystalline water-soluble reagent that is especially suitable for use with small arginine-containing molecules, because the sulfonic acid group of the reagent is a convenient handle for analytical and preparative separation of products. Camphorquinonesulfonylnorleucine is more useful for work with large polypeptides and proteins, because hydrolysates of modified proteins may be analyzed for norleucine to determine the extent of arginine modification. The adducts of the camphorquinone derivatives with the guanidino group are stable to 0.5 M hydroxylamine solutions at pH 7, the recommended conditions for cleavage of the corresponding cyclohexanedione adducts. At pH 8-9 the adducts of the camphorquinone derivatives with the guanidino group are cleaved by
o
-phenylenediamine. The modification and regeneration of arginine, of the dipeptide arginylaspartic acid, of ribonuclease S-peptide, and of soybean trypsin inhibitor are presented as demonstrations of the use of the reagents. The use of camphorquinonesulfonyl chloride to prepare polymers containing arginine-specific ligands is discussed.
Arogenate (pretyrosine) is an obligatory intermediate of L-tyrosine biosynthesis: confirmation in a microbial mutant.
Authors: Fazel A M , Bowen J R , Jensen R A
Published: May 2006

Wild-type Brevibacterium flavum has been shown to possess arogenate dehydrogenase activity and to lack prephenate dehydrogenase, thereby providing presumptive evidence that arogenate (previously named "pretyrosine") is an obligatory intermediate of L-tyrosine biosynthesis. A similar enzymological pattern has been discerned in extracts made from wild-type cultures of various species of cyanobacteria. Application of rigorous molecular genetic criteria in confirmation of the exclusive role of arogenate in L-tyrosine synthesis was made possible by the isolation of an auxotrophic mutant exhibiting a nutritional requirement for L-tyrosine. The mutant was found to lack activity for arogenate dehydrogenase and to accumulate substantial amounts of arogenate behind the mutant block during starvation for L-tyrosine.