bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucocorticoid and thyroid hormones transcriptionally regulate growth hormone gene expression.
Authors: Evans R M , Birnberg N C , Rosenfeld M G
Published: May 2006

In order to define the molecular mechanisms by which glucocorticoids and thyroid hormone act to regulate growth hormone gene expression, the sites at which they exert their effects on growth hormone biosynthesis were examined in vivo and in a pituitary cell line. Glucocorticoids were shown to rapidly increase accumulation of growth hormone mRNA and nuclear RNA precursors. Glucocorticoids and thyroid hormone were shown to rapidly and independently increase growth hormone gene transcription. These events are shown to occur physiologically in animals and further establish the importance of growth hormone gene expression as a model for steroid regulation.
Host range mutants of vesicular stomatitis virus defective in in vitro RNA methylation.
Authors: Horikami S M , Moyer S A
Published: May 2006

The viral RNA polymerase of detergent-treated vesicular stomatitis virus normally synthesizes viral mRNAs in vitro that are both guanylylated and methylated to give 5'-terminal 7mGpppAm caps. We have characterized a virus host range mutant, hr 1, that is totally defective in vitro in the methylation of mRNA, although full-length polyadenylylated mRNAs with 5' termini of the form GpppA are synthesized in normal yields. A second mutant, hr 8, is partially defective in methylation and synthesizes mRNAs in vitro with primarily GpppA and some GpppAm 5' termini. When used for in vitro translation, the unmethylated hr 1 mutant mRNA shows, as expected, reduced synthesis of viral proteins. These data provide direct evidence that the vesicular stomatitis virus-associated methyltransferase activities are virus encoded.
Regulated expression of an immunoglobulin kappa gene introduced into a mouse lymphoid cell line.
Authors: Rice D , Baltimore D
Published: May 2006

We have introduced a functionally rearranged murine kappa light chain immunoglobulin (Ig) gene into an Abelson murine leukemia virus-transformed lymphoid cell line. Plasmid pSV2gpt-kappa 41, containing the kappa light chain gene from the myeloma MOPC41 and the selectable marker gene gpt, was introduced into 81A-2 cells by the calcium phosphate coprecipitation technique. Cells expressing the gpt gene were selected by growth in medium containing mycophenolic acid. One transfected cell line, kappa-2, was shown to make kappa mRNA and polypeptide chains and to assemble the kappa chain product with gamma 2b heavy chains to form an apparently complete IgG2b. When bacterial lipopolysaccharide was added to the growth medium, levels of kappa mRNA and polypeptide increased, showing regulated expression of the introduced kappa gene.
Specificity of antibodies elicited by a synthetic peptide having a sequence in common with a fragment of a virus protein, the hepatitis B surface antigen.
Authors: Neurath A R , Kent S B , Strick N
Published: May 2006

We predicted the localization of a major hepatitis B surface antigen (HBsAg) determinant within residues 135-155 [Neurath, A.R., Strick, N. & Oleszko, W.R. (1981) J. Virol. Methods 3, 115-125]. A peptide corresponding to this sequence (P135-155) was synthesized, linked to macromolecular carriers, and used to immunize rabbits. In accordance with published data on shorter peptides within the same amino acid sequence, antibodies to HBsAg were elicited. A detailed analysis of the immune response to P135-155 revealed the following: A heterogeneous population of IgG and IgM antibodies reacting with P135-155 was detected in the antisera by a variety of radioimmunoassays and enzyme-linked fluorescence immunoassays. Only a subpopulation of these antibodies reacted with HBsAg. The equilibrium constant (K) for the reaction of the antibodies with HBsAg (K = 4 X 10(5) M-1) was approximately two orders of magnitude lower than K for the reaction with P135-155 and was below K for the reaction between HBsAg and antibodies elicited by HBsAg (K greater than 10(7) M-1). Preimmunization with P135-155 did not result in an enhanced response to subsequent immunization with HBsAg. Peptides more accurately mimicking determinants on HBsAg may have to be synthesized for possible application in antiviral prophylaxis.
Alteration of epidermal growth factor-dependent phosphorylation during rat liver regeneration.
Authors: Rubin R A , O'Keefe E J , Earp H S
Published: May 2006

Epidermal growth factor (EGF) stimulates membrane protein phosphorylation in a human cell line, A-431. The known hepatic mitogenic action of EGF and the reduction in EGF receptor number that occurs during liver regeneration led us to study whether EGF-dependent protein kinase activity was present in rat liver and whether its activity was altered after partial hepatectomy. Liver membranes, preincubated with or without EGF, were phosphorylated (0 degrees C, 15 sec) and subjected to NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. In microsomal fractions, EGF at 5-2000 ng/ml produced a dose-related stimulation of 32P incorporation into a single 170,000-dalton protein (p170). In plasma membranes, a similar EGF-dependent phosphorylation was present and was substantially enriched relative to the microsomal fraction. Acid hydrolysis of labeled microsomal fraction followed by phosphoamino acid determination revealed that EGF stimulated 32P incorporation into phosphotyrosine residues. The EGF-dependent phosphorylation of p170 was compared in microsomal fractions isolated from rats 36 hr after partial hepatectomy or sham operation. In the absence of EGF, in vitro labeling of p170 was similar. EGF stimulated the labeling of p170 in both groups, but the response was clearly diminished after partial hepatectomy. In the presence of EGF, the labeling of p170 in microsomal fraction from regenerating livers was only 47 +/- 6% of that observed in membranes from sham-operated rats (p less than 0.005). Reduction of EGF-dependent phosphorylation during liver regeneration paralleled the loss of binding of 125I-labeled EGF. An increase in the EGF-independent phosphorylation of a 130,000-dalton protein was also observed after partial hepatectomy. The increase in the amount of this phosphoprotein was roughly equal to the loss of EGF-stimulated p170 phosphorylation. Several additional proteins showed increased phosphorylation in membranes from partially hepatectomized rats. These findings indicate that alterations in membrane tyrosine residue phosphorylation occur during regulated growth in vivo.
Coupling of growth arrest and differentiation at a distinct state in the G1 phase of the cell cycle: GD.
Authors: Scott R E , Florine D L , Wille J J , Yun K
Published: May 2006

The differentiation of most mammalian cells is preceded by growth arrest in the G1 phase of the cell cycle, but the characteristics of this state have not been established. We now report that the growth arrest that precedes the differentiation of BALB/c 3T3 T mouse proadipocytes must occur at a distinct state in G1 designated GD. GD-arrested cells are characterized by their ability to differentiate in the absence of DNA synthesis and by their unique sensitivity to the mitogenic effect of isobutylmethylxanthine. Proadipocytes induced to become G1 growth arrested at other states by culture in medium deficient in growth factor or nutrients, by contrast, are unable to differentiate in the absence of DNA synthesis and are not stimulated to proliferate by isobutylmethylxanthine even when they are exposed to differentiation-promoting medium prior to arrest. These data support the conclusion that, prior to the expression of a differentiated phenotype, proadipocytes must arrest their growth at a distinct state in the G1 phase of the cell cycle, GD. These data also provide the basis for the hypothesis that carcinogenesis is associated with defects in the coupling of growth arrest and differentiation at the GD state.
Regulatory regions of two transport operons under nitrogen control: nucleotide sequences.
Authors: Higgins C F , Ames G F
Published: May 2006

We have determined the nucleotide sequences of the regulatory regions from two amino acid transport operons from Salmonella typhimurium: dhuA, which regulates the histidine transport operon, and argTr, which regulates argT, the gene encoding the lysine-arginine-ornithine-binding protein, LAO. The promoter for the histidine transport operon has been identified from the sequence change in the promoter-up mutation dhuA1. Neither regulatory region has any of the features typical of the regulatory regions of the amino acid biosynthetic operons, indicating that regulation of at least these transport genes does not involve a transcription attenuation mechanism. We have identified three interesting features, present in both of these sequences, which may be of importance in the regulation of these and other operons: a "stem-loop-foot" structure, a region of specific homology, and a mirror symmetry. The region of mirror symmetry may be a protein recognition site important is regulating expression of these and other operons in response to nitrogen availability. Mirror symmetry as a structure for DNA-protein interaction sites has not been proposed previously.
Interconversion of leukotrienes catalyzed by purified gamma-glutamyl transpeptidase: concomitant formation of leukotriene D4 and gamma-glutamyl amino acids.
Authors: Anderson M E , Allison R D , Meister A
Published: May 2006

The reversible conversion of leukotriene C4 to leukotriene D4 and of the latter to leukotriene E4 were studied with highly purified homogeneous preparations of gamma-glutamyl transpeptidase, dipeptidase, and aminopeptidase M. The conversion of leukotriene C4 to leukotriene D4, catalyzed by gamma-glutamyl transpeptidase, is significantly more rapid when carried out in the presence of an amino acid mixture a closely approximating that found in blood plasma and is accompanied by gamma-glutamyl amino acid formation. Because gamma-glutamyl transpeptidase is bound to the external surface of cell membranes and thus is readily accessible to plasma amino acids, it appears that conversion of leukotriene C4 to leukotriene D4 under physiological conditions is coupled with the formation of gamma-glutamyl amino acids. The apparent Km value for leukotriene C4 in this reaction is about 6 X 10(-6) M, a value close to that found for glutathione. Conversion of leukotriene D4 to leukotriene C4 is effectively catalyzed by gamma-glutamyl transpeptidase in the presence of relatively low concentrations of glutathione. The conversion of leukotriene D4 to leukotriene E4 is catalyzed much more rapidly by renal dipeptidase than by renal aminopeptidase M. Incubation of leukotriene E4 with gamma-glutamyl transpeptidase and glutathione leads to formation of a compound with the properties of gamma-glutamyl leukotriene E4; this reaction is analogous to that shown previously in which gamma-glutamyl cystine is formed by transpeptidation between glutathione and cystine.
Spontaneous changes in nucleotide sequence in proviruses of spleen necrosis virus, an avian retrovirus.
Authors: O'Rear J J , Temin H M
Published: May 2006

We determined the nucleotide sequence of about 1 kilobase of DNA 3' to the 5' long terminal repeat of three noninfectious ad one infectious proviral DNA clones of spleen necrosis virus, an avian retrovirus, to determine if the types of nucleic acid changes involved in retrovirus mutation shed light on special features of retrovirus replication. An open reading frame was found starting 411 base pairs from the end of the long terminal repeat. It contained sequences coding for the 36 amino acids at the amino terminus of the p30 of a related reticuloendotheliosis virus [Oroszlan, S., Barbacid, M., Copeland T., Aaronson, S. A. & Gilden, R. V. (1981) J. Virol. 39, 845-854]. Therefore, the open reading frame represents the 5' end of the gag gene. A mutation in one noninfectious provirus changed the initiation codon for the gag polypeptide; a mutation in another noninfectious provirus caused premature termination of gag polypeptide synthesis; and a nontandem duplication into gag resulting from a mistake in initial (+) strand DNA synthesis changed amino acids and the reading frame in a third noninfectious provirus. These mutations appear to be responsible for the lack of infectivity of these provirus clones and indicate a higher relative frequency of mutation in this region of the genome. In addition, all four clones have multiple other mutations. These mutations are mostly base pair substitutions and many are clustered for any one clone, reflecting certain special features of retrovirus replication.
Gene dosage and gene expression in the
HLA
region: Evidence from deletion variants
Authors: Gladstone Paul , Fueresz Lucy , Pious Donald
Published: May 2006

Among variants selected in a human cell line for nonexpression of a single gene product in the
HLA
complex, most are single-gene variants, but several have been isolated that are
cis
-acting and abolish expression of a series of closely linked genes. The two most plausible mechanisms by which such variants could arise are mitotic crossing-over and chromosome deletion. In two
HLA
variants the presence of a visible chromosome deletion, a 50% reduction in activity of glyoxalase I (a closely linked marker), or both provided evidence for deletional origins. In a third variant these changes were not demonstrable. All three variants showed reductions in amount of cell surface HLA antigens: 40% for the Ia antigens (HLA-DR) and 20-25% for HLA-ABC antigens. The reductions in cell surface antigen in deletion variants have an important implication: in the case of the HLA-A, -B, and -C heterodimer, which consists of a subunit coded for within the major histocompatibility complex and another subunit (β
2
-microglobulin) coded for on a different chromosome, it is the gene of the major histocompatibility complex that is limiting. The nonmutant haplotype includes
A2
; binding of an A2 monoclonal antibody in two of the mutants was found to be approximately equal to that in the wild-type cells. Thus, loss of one copy of
HLA-ABC
genes does not lead to gene dosage compensation—i.e., increased activity by the remaining
ABC
alleles. The results with the two types of antibodies support a deletional mechanism and are inconsistent with mitotic crossing-over. Of interest with respect to the potential use of deletion variants for purposes of mapping is the fact that each of these variants has distinctive breakpoints. The absence of mitotic crossing-over in 1.2 × 10
7
cells selected suggests that the event is rare in this autosomal region, if it occurs at all.
Prostate carcinoma tumor size in rats decreases after administration of antagonists of luteinizing hormone-releasing hormone.
Authors: Redding T W , Coy D H , Schally A V
Published: May 2006

The effects of two potent antagonistic analogues of luteinizing hormone-releasing hormone (LH-RH) on the growth of two different models of rat prostate tumors have been investigated. Chronic administration of [NAc-p-F-DPhe1,p-Cl-DPhe2,DTrp3.6,D-Ala10]LH-RH (antagonist I) at 50 micrograms/day for 21 days significantly inhibited the growth of the chemically induced squamous cell carcinoma 11095 in Fisher 344 male rats. The weights of the pituitary, ventral prostate, and testes were not significantly altered. After 21 days of treatment with this analogue serum luteinizing hormone (lutropin), follicle-stimulating hormone, and testosterone levels were markedly decreased. When male Copenhagen F-1 rats bearing the Dunning 3327H prostate adenocarcinoma were injected with antagonist I at 50 micrograms/day for 6 weeks or with [NAc-p-Cl-DPhe1,2,DTrp3,DPhe6,DAla10]LH-RH (antagonist II) at 50 micrograms/day for 17 days, the percentage increase in tumor volume was decreased to half or less and the actual tumor volume was diminished 34-96% compared to controls. Tumor weight was decreased 30% and 89% after antagonist I and, respectively, compared to untreated controls. The tumor doubling time was 3- to 4-fold longer in rats receiving the inhibitory analogues than in the controls. Treatment with antagonist II decreased the weight of the whole prostate, but neither antagonist changed the weight of testes, anterior pituitary gland, or adrenals. Serum luteinizing hormone, follicle-stimulating hormone, and testosterone levels in Copenhagen F-1 rats bearing Dunning tumors were significantly decreased after treatment with the inhibitory analogues, but progesterone levels were increased. The inhibitory effects of these antagonistic analogues on rat prostate tumors suggest that these compound might be considered in the development of new types of therapy for prostate carcinoma and other endocrine-dependent neoplasias.
Regulation of serum apolipoprotein E metabolism: role of chylomicron metabolism.
Authors: DeLamatre J G , Krause B R , Roheim P S
Published: May 2006

The mechanism by which high-fat, high-cholesterol diets lower serum apolipoprotein E (apoE) concentration was studied in rats by feeding a single high-fat, high-cholesterol meal and by intravenously infusing chylomicrons containing low and high amounts of cholesterol. Serum apoE concentrations were unchanged 9 hours after an olive oil meal but were decreased by 35% after an olive oil/cholesterol-enriched meal. The decrease in serum apoE concentrations with the olive oil/cholesterol meal was accompanied by a decrease in apoE concentration in the high density lipoprotein fraction. Three hours after the intravenous infusion of cholesterol-enriched chylomicrons, serum apoE concentrations decreased 40%, whereas serum apoE concentrations decreased by only 10% when chylomicrons with low cholesterol concentrations or saline were infused. It is concluded that the metabolism of cholesterol-enriched chylomicrons results in an increased removal of serum apoE and that the cholesterol content of chylomicrons is a determinant of serum apoE removal.
Neurotypy: regional individuality in rat brain detected by immunocytochemistry with monoclonal antibodies.
Authors: Sternberger L A , Harwell L W , Sternberger N H
Published: May 2006

Hybridomas from spleen cell fusions of six BALB/c mice immunized with hypothalamus were analyzed by immunocytochemistry for antibodies reactive with paraffin sections of fixed rat brain. In a total of 135 antibody producers, 60% were brain specific. Among these, 54% reacted with glial elements, pituitary cells, or basal lamina of intracerebral capillaries, with little variation among individual hybridomas in each of these groups. Forty-six percent of brain-specific antibodies reacted with neuronal structures, localizing on nerve fibers, neurofibrils, or perikarya. Neuron-specific hybridomas could be classified into groups that localized in anatomically defineable overall patterns. Within these patterns individual hybridomas exhibited extensive qualitative localization diversity ("neurotypy"). Conceivably, the genetic message for a common "proantigen" within an overall pattern may be slightly modified during differentiation of a neuron, thus leading to minor variability in antigenic expression. During antibody formation, similar minor changes occur in the differentiation of the genetic message for the antibody variable region. Apparently, minor changes in the antibody combining site among groups of hybridomas is reflected in the detectability of minor neurotypic changes among differentiated neuronal proantigens. If neurotypy proves to be the result of single-base substitutions or of variability in alignment of peptide-coding exons, the Scharrer concept of the fundamental significance of neurosecretion could also become applicable to neuronal specialization.
Extension of the method of partial retention of diatomic differential overlap to second row atoms and transition metals
Authors: Marynick Dennis S. , Lipscomb William N.
Published: May 2006

The method of partial retention of diatomic differential overlap (PRDDO) has been extended to the elements through the first transition series. A minimum basis set of Slatertype orbitals is employed, with optional 3
d
functions on atoms in the second major row. The 3
d
shell for transition metal atoms is a fixed-contracted double-ζ combination of Slater orbitals. The method retains the basic
n
3
dependency inherent in PRDDO. Limited one-center parameterization and empirical two-center exchange integrals are included. The overall accuracy of the approach is comparable to that of the PRDDO method as previously applied to molecules containing first-row atoms only.
Comparison of acetylcholine receptor-controlled cation flux in membrane vesicles from
Torpedo californica
and
Electrophorus electricus
: Chemical kinetic measurements in the millisecond region
Authors: Hess George P. , Pasquale Elena B. , Walker Jeffrey W. , McNamee Mark G.
Published: May 2006

In earlier studies with the acetylcholine receptor (AcChoR) of
Electrophorus electricus
the rate and equilibrium constants for a model that relates the ligand binding to ion translocation were determined, and the dependence of these constants on the concentrations of carbamoylcholine and acetylcholine, over a 200- and 5000-fold range, respectively, could be predicted. AcChoR-controlled cation flux has now been measured in
Torpedo californica
vesicles by using a pulsed-quench-flow technique with a 2-msec time resolution.
Torpedo
vesicles on a weight basis may contain several hundred times more receptor sites than do
E. electricus
vesicles. Techniques have been developed to (
i
) correct for the kinetic heterogeneity of the vesicle population; (
ii
) use the inactivation of the receptor by its natural ligand to reduce influx rates at high ligand concentrations to a measurable level (this permitted
J
A
, the influx rate coefficient before the onset of inactivation, to be measured); and (
iii
) determine the rate coefficients of two processes that lead to successive inactivations (desensitization) of the receptor and occur in different time regions. An extension of a model proposed for the
E. electricus
receptor accommodates the ion translocation in
T. californica
vesicles. The features in common are: (
i
) A rapid initial flux rate [
J
A
(max) for
T. californica
is 310 sec
-1
; for
E. electricus
it is 7.5 sec
-1
]. These differences in flux rates are consistent with a difference in AcChoR density. (
ii
) A rapid inactivation process [α(max) for
T. californica
is 2 sec
-1
; for
E. electricus
it is 7 sec
-1
]. (
iii
) A slow AcChoR-controlled flux that continues after the rapid inactivation [
J
I
(max) for
T. californica
is 1.3 sec
-1
; for
E. electricus
it is 0.015 sec
-1
]. The main difference between the flux in the two types of vesicle is the existence of a second, slower, inactivation process in
T. californica
with a rate coefficient, β, of 0.12 sec
-1
. The second process leads to undetectable flux activity during the time of observation (30 sec in 10 mM carbamoylcholine). These studies are also significant because fundamental differences may exist between the mechanism of AcChoR-controlled ion flux in synaptic (
Torpedo
) and conducting (
E. electricus
) membranes.
Identification and cloning of a yeast nuclear gene (CBP1) involved in expression of mitochondrial cytochrome b.
Authors: Dieckmann C L , Pape L K , Tzagoloff A
Published: May 2006

Nuclear pet mutants of Saccharomyces cerevisiae deficient in mitochondrial respiration have been studied genetically and biochemically. Seven noncomplementing mutations leading to a deficiency of mitochondrial cytochrome b have been assigned to a single complementation group (group 60). Examination of mitochondrial RNA by blot hybridization on diazobenzyloxymethyl-paper has revealed that group 60 mutants produce a large number of novel apocytochrome b transcripts not detected in wild-type yeast. The product of the gene affected in the mutants, therefore, appears to be required either for correct transcription or for processing of apocytochrome b premessenger RNA. The gene has been designated CBP1. A representative mutant from complementation group 60 (N5-26) has been transformed to respiratory competency with a recombinant plasmid pool consisting of random fragments of wild-type yeast nuclear DNA inserted into a vector capable of replicating in yeast and Escherichia coli. The complementation of the N5-26 mutation has been shown for a number of independent transformants to be due to the presence of plasmid DNA. The plasmid pG60/T10 was further characterized to have a nuclear DNA insert of 6.7 kilobase pairs. This plasmid complements the mutations of all group 60 mutants, thus confirming that it contains the CBP1 gene.
Quantitative correspondence between the in vivo and in vitro activity of teratogenic agents.
Authors: Braun A G , Buckner C A , Emerson D J , Nichinson B B
Published: May 2006

We have tested 74 teratogenic and 28 nonteratogenic agents in a recently developed in vitro teratogen assay system. The assay identifies teratogens by their ability to inhibit attachment of ascites tumor cells to plastic surfaces coated with concanavalin A. There is a qualitative agreement between in vivo animal data and in vitro activity for 81 of the 102 agents (79%). Quantitative analysis shows a highly significant correlation coefficient of 0.69 between the inhibitory in vitro dose and the lowest reported teratogenic dose for 54 of the 60 inhibitory teratogens. The doses analyzed ranged over 5 orders of magnitude. We interpret these results to mean that attachment inhibition in concert with other, complementary, in vitro assay systems can become a useful method for the assessment of the teratogenic potential of environmental agents.
Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells.
Authors: Samulski R J , Berns K I , Tan M , Muzyczka N
Published: May 2006

We have cloned intact duplex adeno-associated virus (AAV) DNA into the bacterial plasmid pBR322. The AAV genome could be rescued from the recombinant plasmid by transfection of the plasmid DNA into human cells with adenovirus 5 as helper. The efficiency of rescue from the plasmid was sufficiently high to produce yields of AAV DNA comparable to those observed after transfection with equal amounts of purified virion DNA. Thus, the recombinant plasmid itself may be a model for studying the rescue of a latent AAV viral infection. In addition, the efficient rescue of viable AAV from the recombinant plasmid should facilitate the genetic analysis of AAV. Finally, the results of an analysis of the DNA from rescued virions indicate that an inversion of the AAV terminal sequences occurred during replication.
Regulation of protein synthesis by phosphorylation of eukaryotic initiation factor 2 alpha in intact reticulocytes and reticulocyte lysates.
Authors: Leroux A , London I M
Published: May 2006

Studies in intact rabbit reticulocytes and reticulocyte lysates provide further evidence of a functional role for the phosphorylation of eukaryotic initiation factor 2 alpha (eIF-2 alpha) in the regulation of initiation of protein synthesis in eukaryotic cells. In intact reticulocytes treated with isonicotinic acid hydrazide to inhibit heme synthesis, the phosphorylation of eIF-2 alpha was significantly greater than in control cells. In heme-deficient reticulocyte lysates and in lysates treated with double-stranded RNA, significant phosphorylation of eIF-2 alpha occurred prior to the onset of inhibition of protein synthesis; a large proportion, however, of the total eIF-2 alpha remained unphosphorylated. These findings indicate that a modest concentration of phosphorylated eIF-2 alpha can suffice to inhibit initiation, and they suggest that one of the factors with which eIF-2 must interact may be rate limiting, especially when eIF-2 alpha is phosphorylated.
Nonrandom distribution of histone mRNAs into polysomes and nonpolysomal ribonucleoprotein particles in sea urchin embryos.
Authors: Baker E J , Infante A A
Published: May 2006

In the early sea urchin embryo, newly synthesized cytoplasmic histone mRNA is found both on polysomes and free of ribosomes as nonpolysomal messenger ribonucleoprotein particles (free RNPs). The distribution of newly synthesized histone mRNAs between translating and nontranslating compartments is nonrandom and dependent on the developmental stage. Gel electrophoresis and autoradiography of polysomal and free RNP RNA from embryos at various stages show that (i) the fraction of total newly synthesized histone mRNA that is in polysomes is greater than the fraction of total newly synthesized poly(A)+mRNA that is in polysomes, at all stages examined, and (ii) among the five histone mRNAs. H1 mRNA and H4 mRNA are relatively more enriched in the free RNPs than are the mRNAs for H2A, H2B, and H3. These data suggest that histone mRNA, as a class, is more efficiently utilized as a template than the average mRNA and, of the cytoplasmic histone mRNAs, the mRNAs for histones H2A, H2B, and possibly H3 are selected more frequently for translation than those for H1 and H4. Cell-free translations of polysomal and free RNP RNAs yield different ratios of in vitro histone products, consistent with the RNA distribution data. To test the possibility that the in vivo distribution of the histone mRNAs is the consequence of different intrinsic initiation capabilities of the individual mRNAs, ribosome-binding assays were carried out and unequal binding abilities of the histone mRNAs in the reticulocyte lysate were shown. A translational level component in the regulation of histone synthesis in the sea urchin embryo is indicated.
Possible common mechanisms of morphological and growth-related alterations accompanying neoplastic transformation.
Authors: Vasiliev J M , Gelfand I M
Published: May 2006

Two main groups of phenotypic alterations usually accompany neoplastic transformations of cultured fibroblastic and epithelial cells: alterations of growth control and decreased formation of cell-substrate and cell-cell contacts. We suggest that both types of alterations are due to change of cell response to clustered membrane receptors. Clustering of certain receptors by corresponding ligands possibly induces a dual set of cellular reactions: (i) activation of cell proliferation preceded by an ordered sequence of prereplicative changes and (ii) attachment-clearing reactions that are associated with anchoring of ligand-receptor complexes by cytoskeletal components and may eventually lead to internalization of these complexes. The first stages of these two subsets of dual reactions are probably identical, but later the two chains of reactions are separated; completion of clearing reaction may stop further progress of the activation of proliferation. Attachment of the cell membrane to another surface can be regarded as a variant of unfinished clearing reaction. Transformed cells may be characterized by alterations of the dual reactions; the primary change may affect either the attachment-clearing branch or the common early stages preceding the separation of branches. These alterations may lead to facilitated activation of proliferation and also to primary or secondary decrease of the ability to perform attachment-clearing reactions in response to external ligands.
Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and in grasshopper embryos.
Authors: Jan L Y , Jan Y N
Published: May 2006

Antibodies specific for horseradish peroxidase (HRPeroxase) bind to neuronal membranes in Drosophila and serve as a specific neuronal marker. Immunocytochemical staining with these antibodies marks sensory neurons, peripheral nerves, and fiber tracks in the central nervous system of embryos, larvae, and adult flies. Similar patterns of staining also were seen in embryos of the grasshopper. It appears that an antigen associated with the nervous system and appearing early in differentiation is recognized by antibodies to HRPeroxase. Using this staining method, we followed embryogenesis of the central nervous system in Drosophila and found that the organization of central fiber tracks resembled that in the previously well-characterized grasshopper. We have used the anti-HRPeroxase antibodies to show that mutations affecting segmentation in Drosophila affect the organization of the embryonic nervous system.
Control of insulin receptor level in 3T3 cells: effect of insulin-induced down-regulation and dexamethasone-induced up-regulation on rate of receptor inactivation.
Authors: Knutson V P , Ronnett G V , Lane M D
Published: May 2006

Chronic exposure of 3T3 mouse fibroblasts to insulin or to the glucocorticoid dexamethasone induces down-regulation and up-regulation, respectively, of cell-surface and total cellular insulin binding capacity. Both processes are reversed upon withdrawal of the inducer. Scatchard analysis of insulin binding for receptors in the down- and up-regulated states indicates that the changes in binding capacity result primarily from alterations in insulin receptor level. That these alterations in total receptor level are due to changes in cell-surface receptor level is indicated by the fact that the level of trypsin-insensitive, presumably intracellular, insulin binding sites does not change appreciably upon down- and up-regulation. The effects of insulin-induced down-regulation and dexamethasone-induced up-regulation on the rates of insulin receptor synthesis and decay were assessed by the heavy-isotope density-shift technique. Cells were shifted to medium containing heavy (2H, 13C, 15N) amino acids and, at various times after the shift, light and heavy receptors solubilized from total cellular membranes were resolved by isopycnic banding on density gradients and then quantitated. It was demonstrated that the insulin- and dexamethasone-induced alterations in insulin receptor level were due entirely to changes in the rate constant for receptor inactivation. The decrease in the first-order rate constant for receptor decay caused by dexamethasone is unexpected in view of the known action of steroid hormones in the induction of the synthesis of specific proteins.
cDNA cloning and induction of the alcohol dehydrogenase gene (
Adh1
) of maize
Authors: Gerlach W. L. , Pryor A. J. , Dennis E. S. , Ferl R. J. , Sachs M. M. , Peacock W. J.
Published: May 2006

cDNA clones of
Adh1
, one of two genes encoding alcohol dehydrogenase (ADH; alcohol:NAD
+
oxidoreductase, EC 1.1.1.1) in the maize genome, have been isolated. They were derived from mRNA extracted from anaerobically treated roots of maize seedlings. Identification was initially made on the basis of molecular weight and electrophoretic properties of the
in vitro
polypeptide obtained in hybridization-release-translation experiments. The identification was confirmed by antibody precipitation and by the use of maize stocks having different genetic constitutions at the
Adh1
locus. The sequence of the longest cDNA segment, ≈900 base pairs, was determined and appears to code for 168 COOH-terminal amino acids and to have a 3′ nontranslated region of 364 base pairs. Reverse Southern hybridizations established that two different
Adh1-S
stocks produce a mRNA of 1,650 nucleotides, whereas an additional mRNA of 1,750 nucleotides is produced in three
Adh1-F
stocks. A 50-fold increase in
Adh1
mRNA level occurs during anaerobiosis, reaching a maximum at 5 hr. Return to aerobic conditions indicates a half-life of more than 18 hr for the anaerobically induced
Adh1
mRNA.
Abundant transcription of a cellular gene in T cells infected with human T-cell leukemia-lymphoma virus.
Authors: Manzari V , Gallo R C , Franchini G , Westin E , Ceccherini-Nelli L , Popovic M , Wong-Staal F
Published: May 2006

Human T-cell leukemia-lymphoma virus (HTLV) is a type C retrovirus associated with a subtype of mature T-cell malignancy in humans. HTLV also infects normal human cord blood mature T lymphocytes in vitro and induces a number of phenotypic changes in these cells, including their continuous growth and partial or complete independence of T-cell growth factor (TCGF). As part of our initial study designed to analyze gene(s) specifically activated by HTLV infection, we have isolated a recombinant DNA clone by differential screening of a cDNA library made from mRNA of a human T-cell lymphoma cell line producing HTLV. This cDNA identifies a single-copy gene in all human DNAs and a single mRNA species of 2.3 kilobases expressed at several hundred copies per cell in five HTLV-positive neoplastic T-cell lines. In addition, cord blood T lymphocytes infected with HTLV, but not the uninfected counterparts, express high levels of mRNA from this gene. A survey of different human hematopoietic cell types showed that this gene is expressed at low or undetectable levels (less than 10 copies) in human T, B, myeloid, or erythroid cell lines; in moderate amounts in lymphoid precursor (immature) cell lines; and in high amounts in lectin-activated mature T-cells, comparable to those of HTLV-infected T-cell lines. The precise function of this gene has not yet been determined.
31P NMR study of erythrocytes from a patient with hereditary pyrimidine-5'-nucleotidase deficiency.
Authors: Swanson M S , Angle C R , Stohs S J , Wu S T , Salhany J M , Eliot R S , Markin R S
Published: May 2006

The composition of phosphate metabolites and the intracellular pH in erythrocytes from a patient with hereditary pyrimidine-5'-nucleotidase deficiency were examined using 31P NMR spectroscopy. Several resonances were identified in spectra from intact cells and from extracts. The 2,3-bisphosphoglycerate line intensities were normal but the NTP resonances were about twice normal due to the presence of millimolar quantities of pyrimidine phosphates. Several intense resonances were also observed in the diphosphodiester region of the spectrum. One compound contributing to these lines has been identified as cytidine diphosphocholine. The resonances of NTPs were in a position indicating that the additional triphosphates were also bound by Mg2+. Direct measurement shows that there is a nearly proportional increase in total cell Mg2+ in the patient's cells, in agreement with the interpretation of the spectra. The intracellular pH was about 0.2 unit lower in the patient's erythrocytes. This lower pH is due to the elevation in intracellular fixed negative charges and the shift in permeable anions consequent to the Donnan equilibrium. We suggest that the lower intracellular pH may explain the lower oxygen affinity of these cells in the presence of otherwise normal 2,3-bisphosphoglycerate levels and the increased Mg2+ triphosphates level, because the Mg2+ form of NTPs is known not to alter the oxygen affinity of hemoglobin under physiologic conditions. Furthermore, the lower intracellular pH can also explain the abnormalities in glycolytic intermediates observed for these cells.
Absolute quantum yields and proof of proton and nonproton transient release and uptake in photoexcited bacteriorhodopsin.
Authors: Marinetti T , Mauzerall D
Published: May 2006

Using a sensitive differential ac conductance apparatus, we have measured transient ion movements in and the heating of bacteriorhodopsin suspensions after a light flash. The signal from the heating serves as an internal calibration of the absorbed photons and therefore the method gives the absolute quantum yield (phi) from a single measurement. At pH 4, H+ uptake precedes release, with phi = 0.4. By varying the buffer composition, we can prove that this signal is due to protons. At pH 8, however, the transient conductance increase is virtually independent of the buffer composition, showing that ions other than H+ are first released and then taken up by the purple membrane. If these ions are typical monovalent cations such as Na+ (lambda = 50 ohm-1 X cm2 X equiv-1), this process has a quantum yield of 2 or more at high salt concentrations.
Extended HLA/complement allele haplotypes: evidence for T/t-like complex in man.
Authors: Awdeh Z L , Raum D , Yunis E J , Alper C A
Published: May 2006

The chromosomal distribution of alleles for HLA-A,-B,-C, and -DR and the serum complement protein alleles of factor B and C2 and C4 was studied in normal Caucasian families. Eight combinations of HLA-B, DR, BF, C2, C4A, and C4B markers were found to occur in haplotypes at frequencies significantly higher than expected. In these combinations, which were defined as extended major histocompatibility complex haplotypes, HLA-A showed limited variation. A possible mechanism for the maintenance of extended haplotypes are human analogs of murine t mutants which are characterized by crossover suppression and male transmission bias. One human 6p haplotype, HLA-B8, DR3, SCO1, GLO 2, was found to be transmitted from males to 83% of their offspring. The same haplotype with GLO 1 had no transmission bias. It is suggested that this GLO 2-marked chromosome is a human analog of a murine t mutant.
Sequential change of carbohydrate antigen associated with differentiation of murine leukemia cells: i-I antigenic conversion and shifting of glycolipid synthesis.
Authors: Kannagi R , Levery S B , Hakomori S
Published: May 2006

Cell surface carbohydrate antigens and their metabolism were investigated during the course of differentiation of murine cultured leukemia cells (M1) into macrophage-like cells. The major glycolipids in undifferentiated M1 cells were of the ganglio series, with a small amount of lacto-series glycolipids. A novel branched structure was found as a tetraosylceramide of M1- cells. Upon differentiation, synthesis of lacto-series glycolipids was significantly enhanced and synthesis of globo-series glycolipids was newly induced but the ganglio-series synthesis was much reduced. Undifferentiated cells expressed only i antigen (i+I-Pk-); differentiated macrophage-like cells became I-antigen dominant and Pk-antigen positive (i+/-I+Pk+). The changes proceeded in two sequential steps: (i) an enhancement of lacto-series glycolipid synthesis associated with the conversion of i antigen to I antigen, and (ii) subsequent induction of globo-series glycolipid synthesis accompanied by the appearance of Pk antigen. The experimental system offers a clue for studies on the process of branching (i-to-I conversion) as well as the biological significance of three major glycolipids (globo-, lacto-, and ganglio-series) as markers of cell differentiation.
Endogenous avian retroviruses contain deficient promoter and leader sequences.
Authors: Cullen B R , Skalka A M , Ju G
Published: May 2006

A sensitive and quantitative biological assay has been utilized to measure the ability of the exogenous and endogenous avian retroviral long terminal repeats (LTR) to promote gene expression in avian cells. This assay has revealed that the exogenous virus RAV-2 LTR is approximately equal to 10-fold more active than the LTRs of endogenous viruses RAV-0, ev-1, and ev-2. The endogenous viral LTRs show approximately equal activity. Upstream flanking cellular or viral sequences have no significant modulating effect on gene expression in our assay. Unexpectedly, we have detected and localized an additional defect outside of the LTR in the 5' noncoding leader sequence of ev-1 that further decreases gene expression relative to RAV-0 by approximately equal to 10-fold.
Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells.
Authors: Loskutoff D J , van Mourik J A , Erickson L A , Lawrence D
Published: May 2006

Fibrin/agar films were prepared and used to detect plasminogen activators produced by cultured bovine aortic endothelial cells (fibrin autography). One preparation of fibrin underwent spontaneous lysis upon incubation at 37 degrees C. This lysis was prevented by antibodies to tissue-type plasminogen activator but not by antibodies to urokinase. Conditioned medium from the confluent endothelial cells was fractionated by polyacrylamide gel electrophoresis in the presence of NaDodSO4. The gels were analyzed on indicator films prepared with the spontaneously lysing fibrin (reverse fibrin autography). Unexpectedly, as the opaque fibrin film cleared, a distinct lysis-resistant zone appeared in the indicator gel at a region corresponding to Mr 55,000. Experiments were devised to determine whether the lysis-resistant zone in the indicator film reflected the presence of a cellular inhibitor in the polyacrylamide gel. The corresponding region was excised from a polyacrylamide gel, extracted with buffer, and tested directly for antifibrinolytic activity by the 125I-labeled fibrin plate method. Urokinase-mediated fibrinolytic activity was inhibited by the gel extract in a dose-dependent manner indicating the presence of such an inhibitor. Inhibitor activity was detected in Triton X-100 extracts of washed monolayers and in conditioned medium, where it accumulated with time. The endothelial cell inhibitor not only survived exposure to NaDodSO4 but also was active after incubation at pH 12 or treatment with 5% (vol/vol) 2-mercaptoethanol, 6 M urea, 4 M guanidine hydrochloride, or 1 M acetic acid. Considerable activity also remained after heating at 100 degrees C for 30 min. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a previously undetected, unusually stable fibrinolytic inhibitor of Mr 55,000. Reverse fibrin autography offers a convenient approach for studying such molecules.
Structure of the human immunoglobulin C epsilon 2 gene, a truncated pseudogene: implications for its evolutionary origin.
Authors: Hisajima H , Nishida Y , Nakai S , Takahashi N , Ueda S , Honjo T
Published: May 2006

Cloning of the overlapping DNA fragments together with Southern hybridization experiments showed the organization of the human C epsilon and C alpha gene cluster as 5'-C epsilon 2-14 kilobases-C alpha 1----C epsilon 1-13 kilobases-C alpha 2-3'. Comparison of the nucleotide sequences of the C epsilon 1 and C epsilon 2 genes revealed that four deletions have taken place in the C epsilon 2 gene and its flanking regions. The three deleted regions in the 5' side of the C epsilon 2 gene are partially filled with shorter inserted sequences. One of them has removed the CH1 and CH2 exons and a portion of the epsilon switch (S epsilon) region. The S epsilon region and the CH4 exon still retain the functional structures, whereas the CH3 exon has been inactivated by deleting its 5' intervening sequence necessary for splicing. The tetranucleotide T-G-G-G (or T-G-G-C), which is usually found in close proximity of the class-switch recombination sites in mouse myelomas, is located 5' to the three deletion sites. The results imply that the mechanism responsible for the heavy chain class-switch recombination might be relevant to the evolutionary mechanism of creation of the truncated C epsilon 2 gene. The other deletion in the 3' flanking region of the C epsilon 2 gene may be due to slipped mispairing of the short direct repeat (C-C-C-C-C) at both ends.
Electroanatomy of a unique amacrine cell in the rabbit retina.
Authors: Miller R F , Bloomfield S A
Published: May 2006

Intracellular electrophysiological recordings were obtained from a specialized class of "starburst" amacrine cells by using an isolated superperfused retina-eyecup preparation of the rabbit. These cells were injected intracellularly with horseradish peroxidase and identified with light microscopy. A computer-controlled image-processing system was used to map and display the three-dimensional dendritic organization and provide information on length and sublaminar distribution of dendritic processes. Starburst amacrines show an unusual dendritic architecture that includes thin intermediate dendritic segments. Analysis with steady-state cable equations suggests that these thin segments may provide electrical isolation of distal processes, raising the possibility that a single dendrite, which lies beyond the thin segment, may constitute a functional subunit of the cell.