bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-T-cell idiotype activity in serum of mice injected with syngeneic hapten-specific T-cell lines.
Authors: Thomas W R , Mottram P L , Miller J F
Published: May 2006

T cells able to mediate specific delayed type hypersensitivity (DH) in mice in response to the haptens azobenzenearsonate (ABA), oxazolone (OX), and picryl chloride have been grown in continuous cultures. They were tested for their ability to react with the hapten in vitro and to induce anti-idiotype antibody in syngeneic mice. Incubation in vitro with OX-glycine blocked the ability of an anti-OX cell line to produce DH in vivo but had no effect on an anti-ABA cell line. However, this was inhibited by ABA conjugated to bovine serum albumin (ABA-alb) but not by irrelevant haptens such as OX-glycine or trinitrophenyl-alb. Antisera prepared by immunizing syngeneic mice with the anti-OX line blocked the inhibitory effect of OX-glycine on that line but did not reverse the effect of ABA-alb on the anti-ABA line. Neither normal mouse serum nor inappropriate anti-line antisera could influence the effect of OX-glycine on the anti-OX line. When given alone to naive mice, a specific anti-line antiserum induced DH specific for the antigen to which the line was responsive. Thus, for example, antiserum directed to an anti-OX line produced OX-specific DH. The results suggest that anti-T-cell idiotype antibodies can be induced by immunizing mice with syngeneic antigen-specific T-cell lines and that these antibodies can block the specific in vitro interaction of antigen with the corresponding antigen-specific T cells and induce specific DH in vivo.
Isolation of a
Caulobacter
gene cluster specifying flagellum production by using nonmotile Tn5 insertion mutants
Authors: Purucker Mary , Bryan Ruth , Amemiya Kei , Ely Bert , Shapiro Lucille
Published: May 2006

Caulobacter crescentus
assembles a single polar flagellum from protein components synthesized at a specific time in the cell cycle. Of the 26 genes required for flagellum production, at least 4 of them—
flaY, E, F,
and
G
—map together in a single cluster. We have isolated DNA from this region of the chromosome by using a nonmotile mutant with a Tn5 insertion into
flaE. C. crescentus
DNA carrying the Tn5-
flaE
region and adjacent sequences was cloned into pBR325 and selected by transposon-encoded kanamycin resistance. The resulting plasmid was used as a probe to isolate the
flaE
region from a wild-type gene bank and to determine the chromosomal location of several deletion and insertion mutations within the
flaY/E/F/G
cluster. At least three promotors and three major transcripts were shown to originate from the cloned gene cluster. The role of these genes in flagellar biogenesis was examined by immunoprecipitation of mutant cell extracts with antiflagellin antibody. Deletions extending rightward into this gene cluster eliminated one of the two flagellin proteins normally synthesized by
C. crescentus
. Mutations mapping to the left permitted synthesis of both normal flagellins but at significantly decreased levels. These results suggest that the leftward end of this cluster contains a region that may function in a regulatory capacity whereas the rightward end may contain sequences overlapping a flagellin structural gene.
Differential subcellular localization of in vivo-phosphorylated and nonphosphorylated middle-sized tumor antigen of polyoma virus and its relationship to middle-sized tumor antigen phosphorylating activity in vitro.
Authors: Segawa K , Ito Y
Published: May 2006

A small fraction of polyoma virus middle-sized tumor (T) antigen is phosphorylated in vivo, resulting in a small amount of phosphotyrosine and phosphothreonine and significantly larger amounts of phosphoserine. When infected cells are separated into nuclear, plasma membrane, and low-speed supernatant fractions, 80-95% of in vivo-phosphorylated middle-sized T antigen is localized to the plasma membrane fraction, while 25-50% of [35S]methionine-labeled middle-sized T antigen is found in the nuclear fraction and the same amount is found in the plasma membrane fraction. Immunoprecipitated T antigens contain a protein kinase activity that phosphorylates middle-sized T antigen at tyrosine residues. Eighty to 90% of this activity is located in the plasma membrane fraction. When immunoprecipitated T antigens are treated with alkaline phosphatase, middle-sized T antigen-phosphorylating activity decreases as 32PO4 is lost from in vivo 32P-labeled middle-sized T antigen. The possibility that in vivo-phosphorylated middle-sized T antigen located in the plasma membrane is an active tyrosine-specific kinase is discussed.
Cloning of developmentally regulated flagellin genes from Caulobacter crescentus via immunoprecipitation of polyribosomes.
Authors: Milhausen M , Gill P R , Parker G , Agabian N
Published: May 2006

Immunoprecipitation of Caulobacter crescentus polyribosomes with antiflagellin antibody provided RNA for the synthesis of cDNA probes that were used to identify three specific EcoRI restriction fragments (6.8, 10, and 22 kilobases) in genomic digests of Caulobacter DNA. The RNA was present only in polyribosomes isolated from a time interval in the Caulobacter cell cycle that was coincident with flagellin polypeptide synthesis. The structural gene for Mr 27,500 flagellin polypeptide was assigned to a region of the 10-kilobase EcoRI restriction fragment by DNA sequence analysis. Analysis of mutants defective in motility further established a correlation between the Mr 27,500 flagellin gene and the flaE gene locus [Johnson, R. C. & Ely, B. (1979) J. Bacteriol. 137, 627-634]. The other EcoRI fragments that hybridize with the immunoprecipitated polyribosome-derived cDNA probe are also temporally regulated and have features that suggest they encode other polypeptides associated with the flagellum. Modifications were required to adapt the procedure of immunoprecipitation of polyribosomes for use with Caulobacter and should be applicable to the production of specific structural gene probes from other prokaryotic systems.
Some spectrally isolated convex planar regions
Authors: Marvizi Shahla , Melrose Richard B.
Published: May 2006

The basic question raised by M. Kac as to whether a domain in Euclidean space is determined by its Dirichlet spectrum remains open. In this note, dealing only with convex planar regions, we introduce a new countable family of (generic) spectral invariants of wave type, discuss some asymptotic properties of the distribution of closed geodesics, describe a partial converse to the Poisson relation, and thereby construct a two-parameter family of spectrally isolated regions, including the circles.
Exterior gauging of an internal supersymmetry and
SU
(2/1) quantum asthenodynamics
Authors: Thierry-Mieg Jean , Ne'eman Yuval
Published: May 2006

A formally unitary Lagrangian model gauging an internal supersymmetry is proposed. The even subalgebra is gauged as a Yang-Mills theory, while the odd generators are gauged—according to Freedman's method—by skew tensor fields, equivalent dynamically to scalar Higgs fields. Chiral fermions are incorporated by following Townsend's construction and form irreducible supermultiplets graded by their helicity. The application to quantum asthenodynamics is discussed.
Changes in the topography of early region transcription during polyoma virus lytic infection.
Authors: Fenton R G , Basilico C
Published: May 2006

We have studied the pattern of transcription of the early region of polyoma virus DNA after the onset of the late phase of lytic infection of mouse cells. Following initiation of viral DNA synthesis, the early/late switch is accompanied not only by efficient production of late mRNAs but also by the appearance of previously unidentified early-strand RNAs which have certain structural features in common with the classical early mRNAs. Stable poly(A)+RNAs have been identified by blot analysis and S1 nuclease mapping that are not detected early during infection or in polyoma virus-transformed cells. One group consists of transcripts whose 5' ends map 150-200 nucleotides upstream from the major early 5' ends (at positions 148 and 153 on the polyoma virus genome) but whose splicing pattern and poly(A) addition sites are indistinguishable from those of mRNAs produced early in infection. The 5' exons of these early region transcripts contain an open translational reading frame that extends from nucleotide positions 5,255 to 124 and is capable of encoding a basic protein of 53 amino acids. Transcription of these RNAs does not appear to be negatively regulated by large tumor antigen. A transcript of 1,800 nucleotides appears to map predominantly between 93 and 26 map units and does not contain sequences present in the early mRNA 5' exons. These data suggest that, after the onset of polyoma DNA replication, the activation of new early-strand promoters leads to the expression of previously untranscribed viral DNA sequences.
Isolation of cloned cDNAs to auxin-responsive poly(A)
+
RNAs of elongating soybean hypocotyl
Authors: Walker John C. , Key Joe L.
Published: May 2006

Auxin-responsive cDNA clones have been isolated from a cDNA library prepared from elongating soybean hypocotyl poly(A)
+
RNA. The expression of two such sequences has been assessed by RNA blot hybridization analyses during normal developmental transitions in the soybean hypocotyl and during incubation of sections excised from the region of cell elongation. The concentrations of these poly(A)
+
RNAs are higher in the elongating zone than in the apical and mature zones of the hypocotyl. Both poly(A)
+
RNAs are depleted during incubation of the sections in the absence of auxin. The loss of one of these sequences (pJCW1) is prevented by the addition of auxin to the incubation medium while the other sequence (pJCW2) increases above the initial level in the presence of auxin. The addition of auxin to auxin-depleted tissue in which the sequences are depleted results in rapid accumulation of these poly(A)
+
RNAs; pJCW1 accumulates to the control level while pJCW2 increases well above the control level. These data along with others [Baulcombe, D. C. & Key, J. L. (1980)
J. Biol. Chem.
255, 8907-8913] demonstrate directly a highly selective effect of auxin on the expression of a small number of mRNAs in tissues undergoing both cell elongation and cell division in response to auxin. Although the data are suggestive of a close association betwen auxin action and altered gene expression, a causal relationship is not established. It seems highly unlikely, however, that such specific effects of auxin on gene expression are unimportant in auxin physiology.
Restriction of motion of protein side chains during the photocycle of bacteriorhodopsin.
Authors: Czégé J , Dér A , Zimányi L , Keszthelyi L
Published: May 2006

Linear dichroism was measured during the photocycle of bacteriorhodopsin. The anisotropy of the sample was produced by the photoselection method. The measurements on purple membrane fragments embedded in agar gel were performed at room temperature with 200 microseconds time resolution at several wavelengths in the 240- to 550-nm spectral region. The induced anisotropy of the retinal chromophore remained constant after the formation of the photocycle intermediate M. The anisotropy was also time independent at the characteristic peaks of the UV absorption change. These experimental data suggest that the direction of the retinal transition dipole moment remains unchanged. Moreover, the affected aromatic protein side chains also do not show any rotational motion when they are in the perturbed or ground states during the photocycle. Our data render it possible to calculate the restricted range of sudden chromophore rotations that might be coupled to the appearance and decay of the M intermediate.
Spasmogenic activity of chemotactic N-formylated oligopeptides: identity of structure--function relationships for chemotactic and spasmogenic activities.
Authors: Marasco W A , Fantone J C , Ward P A
Published: May 2006

The chemotactic N-formylated oligopeptides are potent spasmogenic agents for guinea pig ileum. Structure-activity studies with various N-formylated peptides suggest the presence of a specific receptor that resembles in specificity the formyl peptide receptor on leukocytes. A competitive antagonist of the formyl peptide receptor on leukocytes also inhibits formyl peptide-induced ileum contraction, whereas the antihistamine diphenhydramine is without effect. The contractile response caused by the synthetic N-formylated peptides differs from those induced by acetylcholine, histamine, and substance P. In particular, a latent period after treatment with the N-formyl peptides is seen before the onset of the response, and a sustained contractile response is not maintained. In addition, tachyphylaxis does occur, but complete recovery of activity is seen after a 20- to 30-min rest period. These observations suggest broad biological roles of prokaryotic signal peptides from bacteria as acute inflammatory mediators.
Tetanus toxin fragment forms channels in lipid vesicles at low pH.
Authors: Boquet P , Duflot E
Published: May 2006

Single-walled asolectin vesicles loaded with K+ at pH 7.00 released their K+ content upon incubation with tetanus toxin fragment B but only when the incubation was at pH below 5.00. Whole tetanus toxin exhibited only a weak releasing activity. Toxin light alpha chain and the carboxyl-terminal 48,000-dalton moiety of the heavy chain (fragment IIC) were unable to provoke K+ release from vesicles at any pH. K+ release from lipid vesicles could also be detected with tetanus toxin heavy beta chain at low pH. Furthermore, using a detergent binding assay ([3H]Triton X-100), we have also shown that an hydrophobic domain, localized in the 50,000-dalton terminal polypeptide of tetanus toxin heavy chain, is detectable at pH 3.60 but not at pH 5.00. These results lead us to conclude that the ability of tetanus toxin fragment B to release K+ from asolectin vesicles at low pH is due to the 50,000-dalton amino-terminal polypeptide of the heavy chain present in toxin fragment B. We propose that this phenomenon is caused by channel formation across the vesicle membrane as has been observed for the 23,000-dalton amino-terminal moiety of diphtheria toxin fragment B.
Structural diversity and differential light control of mRNAs coding for angiosperm glyceraldehyde-3-phosphate dehydrogenases
Authors: Cerff Rüdiger , Kloppstech Klaus
Published: May 2006

Subunits A and B of chloroplast glyceraldehyde-3-phosphate dehydrogenase are synthesized as higher molecular weight precursors when polyadenylylated mRNA from angiosperm seedlings is translated
in vitro
by wheat germ ribosomes. The
in vivo
levels of mRNA coding for these precursors are strongly light dependent, and the increase in translational activity stimulated by continuous white light, relative to dark-grown seedlings, is at least 5- to 10-fold for the seven plant species investigated. As opposed to this, light does not seem to change mRNA levels coding for cytosolic glyceraldehyde-3-phosphate dehydrogenase, and the polypeptides synthesized
in vitro
have the same size as the authentic subunits. In addition, precursors of the chloroplast enzyme were identified for 12 different angiosperm species and compared with their respective subunits synthesized
in vivo
. The patterns of the
in vitro
and
in vivo
products correlate in several major characteristics. They both display a remarkable interspecific heterogeneity with respect to size and number of polypeptides. The peptide extensions of the enzyme precursors calculated from these data vary between 4,000 and 12,000 daltons and seem to fall into three major size classes. The present data demonstrate that chloroplast glyceraldehyde-3-phosphate dehydrogenase, like its cytosolic counterpart, is encoded in the nucleus. Yet, the two dehydrogenases are controlled differently at both the ontogenetic and phylogenetic levels. They follow separate biosynthetic pathways with respect to light regulation, post-translational processing, and transport and also exhibit different evolutionary rates. The fast evolutionary change observed for the chloroplast enzyme contrasts sharply with the conservative structure and sequence of the cytosolic enzyme.
Replication of lambda dv plasmid in vitro promoted by purified lambda O and P proteins.
Authors: Tsurimoto T , Matsubara K
Published: May 2006

An in vitro system for replication of lambda dv plasmid DNA has been constructed. This system consists of an ammonium sulfate fraction from Escherichia coli extract, exogenously added purified lambda O and P proteins, and lambda dv DNA in closed circular form. More than 85% of the added template DNA replicated semiconservatively. In the same system, another plasmid, pBR322, also replicated, but less efficiently than lambda dv. Furthermore, its replication was independent of O and P proteins. Inhibitors of DNA gyrase entirely blocked the replication activity, whereas rifampicin, an inhibitor of RNA polymerase, showed a significant effect only when added prior to initiation of the DNA replication. DNA replication was initiated from a region near to or within the four direct repeats in lambda origin (lambda ori) and proceeded bidirectionally, as examined by DNA chain elongation termination with dideoxy CTP. A cloned DNA carrying a 350-base-pair region including the initiation site also initiated replication, dependent on O and P proteins, and its initiation occurred at the same position as with native lambda dv DNA. An A + T-rich structure neighboring the repeats was found to be essential for lambda DNA replication. Regions corresponding to ice and oop were not required for O,P-dependent initiation.
Identification of a sequence responsible for periodic synthesis of yeast histone 2A mRNA.
Authors: Osley M A , Hereford L
Published: May 2006

Sequences required for the regulated expression of a yeast histone 2A (H2A) gene have been investigated by using fusions between this gene and the Escherichia coli lacZ gene. Fusions containing the entire spacer region in which divergent transcription of the H2A and H2B genes is initiated result in low-level constitutive synthesis of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in yeast. Regulated expression (which is characterized by periodic synthesis during the S phase of the cell cycle) is restored when a 1.3-kilobase HindIII fragment containing a small region of the 3' end of the H2B gene is present in either orientation. The regulatory activity in this region appears to be coincident with a sequence that supports autonomous replication in yeast.
Semisynthesis of sperm whale myoglobin by fragment condensation.
Authors: Simmerman H K , Wang C C , Horwitz E M , Berzofsky J A , Gurd F R
Published: May 2006

Reconstruction of the sperm whale myoglobin structure was accomplished by a series of aqueous condensations of suitably protected synthetic myoglobin fragments to a large fragment prepared from the native protein. Reaction of N alpha,N epsilon 19-acetimidomyoglobin with 3-bromo-2-(2-nitrophenylsulfenyl)skatole (BNPS-skatole) yielded the fragment corresponding to residues 15-153. The covalent structure was reformed by sequential coupling of the N-hydroxysuccinimide esters of o-nitrophenylsulfenyl-L-tryptophan (residue 14) and selectively protected peptides corresponding to residues 1-5 and 6-13, which were synthesized by the solid-phase method and removed from the resin by methoxide-catalyzed methanolysis. A mixed aqueous solvent system containing methanol and N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine/trifluoroacetic acid buffer (Quadrol) solubilized the peptide and protein fragments during the condensations. Replacement of the heme moiety and immunoaffinity chromatography made possible the isolation and purification of the reconstructed native molecule. The development of this nondestructive synthetic procedure allows investigation of the structural and functional significance of individual residues by isotopic enrichment or selective amino acid substitutions.
Cytotype polymorphism of the P-M system in two wild populations of Drosophila melanogaster.
Authors: Anxolabéhère D , Nouaud D , Périquet G
Published: May 2006

In Drosophila melanogaster the interactions of the P-M system generate germ-line aberrations (e.g., sterility, mutations) found in certain interstrain hybrids. Two wild populations, from France and Tunisia, were examined in order to determine the distribution of the chromosomal P factor and the extrachromosomal cytotypes. No P factors active for potential GD sterility were found in these populations. But search for the M cytotype, which causes susceptibility to the P factors, and for the P cytotype, which causes resistance to the P factors, showed that both populations are polymorphic for the cytotypes. Such a polymorphism seems to be stable in the wild, at least over a 2-year period. Mutator activity (measured by generated mutations at the sn and ras loci) was found to be present. Some possible interactions between cytotype polymorphism, mutator activity, and the structure of Drosophila populations are discussed.
cAMP-mediated decrease in K+ conductance evoked by serotonin and dopamine in the same neuron: a biochemical and physiological single-cell study.
Authors: Deterre P , Paupardin-Tritsch D , Bockaert J , Gerschenfeld H M
Published: May 2006

The extracellular application of either serotonin or dopamine and the intracellular injection of cAMP all evoke in the same identified neurons of the snail Helix aspersa inward currents associated with a decrease in K+ conductance. The serotonin-, dopamine-, and cAMP-induced inward currents all show the same maximal amplitude. When the response to one transmitter is maximal, the response to the other is blocked. Using a single-cell microassay, we found that both serotonin and dopamine stimulate the adenylate cyclase [adenosine triphosphate pyrophosphate-lyase (cyclizing), EC 4.6.6.1] activity of the neurons giving the inward-current responses; on the other hand, the adenylate cyclase activity of a neuron that does not show the serotonin- and dopamine-induced currents was not stimulated by the transmitters. In contrast with the nonsummation of the maximal inward-current responses, the maximal stimulating effects of the transmitters on the enzyme activity are additive. The diterpene forskolin, which stimulates the adenylate cyclase activity of the single cells 9-fold, also evokes an inward current. We conclude that single snail neurons are endowed with independent serotonin and dopamine receptors linked to the adenylate cyclase. Activation of each of these receptors evokes a cAMP-mediated decrease in K+ conductance. The physiological interaction between the transmitters probably takes place at a late step in the chain of events leading from the increase in cAMP to the closing of the K+ channels.
Polar branch migration promoted by recA protein: effect of mismatched base pairs.
Authors: DasGupta C , Radding C M
Published: May 2006

Escherichia coli recA protein makes joint molecules from single-stranded circular phage DNA (viral or plus strand) and homologous linear duplex DNA by a polar reaction that displaces the 5' end of the plus strand from the duplex molecule [Kahn, R., Cunningham, R. P., DasGupta, C. & Radding, C. M. (1981) Proc. Natl. Acad. Sci. USA 78, 4786-4790]. Growth of the heteroduplex joint, which results from strand exchange or branch migration, stopped at the borders of regions of nonhomologous DNA that were variously located 145, 630, or 1202 nucleotides from the end. Accumulation of migrating branches at heterologous borders demonstrates that their migration is not the result of random diffusion but is actively driven by recA protein. Growth of the heteroduplex joint was blocked even when a heterologous insertion was located in the single-stranded DNA, a case in which the flexible single-stranded region might conceivably fold out of the way under some condition. The recA protein did not make joint molecules from phage phi X174 and G4DNAs, which are 70% homologous, but did join phage fd and M13DNAs, which are 97% homologous. In the latter case, heteroduplex joints extended through regions containing isolated mismatched base pairs but stopped in a region where the fd and M13 sequences differ by an average of 1 base pair in 10. These results suggest that in genetic recombination the discrimination of perfect or near-perfect homology from a high degree of relatedness may be attributable in part to the mechanism by which recA protein promotes strand transfer.
DNA stimulates ATP-dependent proteolysis and protein-dependent ATPase activity of protease La from Escherichia coli.
Authors: Chung C H , Goldberg A L
Published: May 2006

The product of the lon gene in Escherichia coli is an ATP-dependent protease, protease La, that also binds strongly to DNA. Addition of double-stranded or single-stranded DNA to the protease in the presence of ATP was found to stimulate the hydrolysis of casein or globin 2- to 7-fold, depending on the DNA concentration. Native DNA from several sources (plasmid pBR322, phage T7, or calf thymus) had similar effects, but after denaturation the DNA was 20-100% more effective than the native form. Although poly(rA), globin mRNA, and various tRNAs did not stimulate proteolysis, poly(rC) and poly(rU) were effective. Poly(dT) was stimulatory but (dT)10 was not. In the presence of DNA as in its absence, proteolysis required concomitant ATP hydrolysis, and the addition of DNA also enhance ATP hydrolysis by protease La 2-fold, but only in the presence of casein. At much higher concentrations, DNA inhibited proteolysis as well as ATP cleavage. Thus, association of this enzyme with DNA may regulate the degradation of cell proteins in vivo.
Microenvironmental immunoregulation: possible role of contrasuppressor cells in maintaining immune responses in gut-associated lymphoid tissues.
Authors: Green D R , Gold J , St Martin S , Gershon R , Gershon R K
Published: May 2006

The addition of Peyer's patch T cells from most strains of mice to spleen cells in primary Mishell-Dutton cultures either has no effect or augments the spleen cells' response to sheep erythrocytes. However, if the Peyer's patch T cells are treated with an anti-I-J antiserum and complement to remove contrasuppressor-inducer cells, the remaining Ly-2 cells (T cells that express Ly-2 but not Ly-1) are highly suppressive. This "latent" suppressor cell activity also can be revealed by removing contrasuppressor-acceptor (transducer) cells from the splenic assay population with either an anti-I-J or anti-Ly-2 antiserum. These findings, taken together with previous work showing that orally administered antigen leads to systemic tolerance, give experimental support to the notion that contrasuppression may be important in allowing microenvironmental immune responses (in this case the gut-associated lymphoid tissue) to take place while systemic immunity is suppressed.
Hybridization-selected translation of Bombyx mori high-cysteine chorion proteins in Xenopus laevis oocytes.
Authors: Bock S C , Tiemeier D C , Mester K , Wu M , Goldsmith M R
Published: May 2006

Xenopus laevis oocytes were injected with poly(A)+-mRNA isolated from chorionating follicular epithelium of the domesticated silk moth (Bombyx mori). On two-dimensional gel electrophoresis, the resultant translation products comigrated with authentic, secreted, chorion standards, demonstrating that the frog oocyte system synthesizes and correctly process virtually all major chorion components. A cDNA clone has been shown to contain sequences complementary to those of mRNAs encoding B mori high-cysteine (Hc) chorion proteins Hc6-Hc11. mRNAs were selected by hybridization to plasmid m5000 DNA bound to diazobenzyloxymethyl-cellulose and subsequently translated in X. laevis oocytes into forms that comigrated with authentic chorion standards. The selection of a distinct subset of Hc mRNAs under stringent hybridization conditions (70% formamide/0.2 M NaCl, 60 degrees C) suggests that they are encoded by related genes. This is consistent with the pattern obtained by hybridizing radioactive m5000 DNA to Southern blots prepared from EcoRI-cleaved B. mori chromosomal DNA.
Long-lived cytoplasmic factors that suppress adrenal steroidogenesis.
Authors: Clark M A , Shay J W
Published: May 2006

Adrenal cells secrete steroids after stimulation with corticotropin (ACTH), whereas cells reconstructed by fusing adrenal cell nuclei to fibroblast cytoplasms are temporarily (2-3 wk) unresponsive to ACTH. In this report, we characterize this inhibition by using "cybrids" (cytoplasmic hybrids) isolated by either genetic selection or a new procedure that utilizes the fluorescence-activated cell sorter and the vital mitochondrial dye rhodamine 123. Such cybrids, which contain both adrenal and fibroblast cytoplasmic components, are unable to produce steroids, suggesting the existence of cytoplasmic inhibitory factors. In order to elucidate this cytoplasmic inhibition of steroidogenesis, techniques are described that test the contribution of fibroblast mitochondria to this phenomenon. The first technique utilizes purified mitochondria, isolated from chloramphenicol (CAP)-resistant fibroblasts, to confer CAP resistance on an otherwise sensitive adrenal cell. The resulting CAPr cells, termed mitochondrial transformants, are responsive to ACTH. The second technique utilizes a procedure for isolating small fragments of cytoplasm (microcytospheres) from fibroblasts. Microcytospheres, which do not contain mitochondria, are stained with rhodamine 18, a vital membrane dye, and then fused to unstained adrenal cells. The fusion products are then isolated with the fluorescence-activated cell sorter. Approximately 30% of the fusion products are inhibited in their ability to respond to ACTH. These results suggest that the fibroblast cytoplasm contains nonmitochondrial long-lived inhibitory factors that temporarily suppress steroidogenic function in adrenal cells.
Monoclonal IgM radioimmunoassay for hepatitis B surface antigen: high binding activity in serum that is unreactive with conventional antibodies.
Authors: Wands J R , Bruns R R , Carlson R I , Ware A , Menitove J E , Isselbacher K J
Published: May 2006

Using a monoclonal IgM antibody (anti-HBs) to hepatitis B surface antigen (HBsAg) in a radioimmunoassay for hepatitis B, we have detected high binding activity in human serum that was unreactive in assays employing conventional anti-HBs reagents. The binding material was isolated from serum by affinity chromatography on monoclonal IgM anti-HBs, and comparison of the material with HBsAg (by sodium dodecyl sulfate/polyacrylamide gel electrophoresis) demonstrated that the two shared several similar polypeptides. Furthermore, comparison of the binding properties of HBsAg and concentrated monoclonal immunoreactive material with conventional and monoclonal anti-HBs reagents demonstrated some antigenic crossreactivity. The molecular weight of the monoclonal immunoreactive material was approximately 2 X 10(6). Immunoprecipitation of the material with monoclonal IgM antibodies and examination by electron microscopy revealed clumped and "spiculated" particles that resembled 22-nm hepatitis B particles coated with the same antibody. Thus, this study suggests that the high-binding-activity material, detected in serum only by the monoclonal radioimmunoassay, is not identical with HBsAg, but it shares some common properties.
Molecular basis of reovirus neurovirulence: role of the M2 gene in avirulence.
Authors: Hrdy D B , Rubin D H , Fields B N
Published: May 2006

A number of field isolates of reovirus 3 were examined to determine their relative neurovirulence after intracerebral inoculation. One isolate was found that had decreased neurovirulence. This "avirulent" strain showed the typical type 3 neural tropism but grew significantly less well in brain tissue than T3 (Dearing) and the other type 3 reoviruses. The avirulent virus was not temperature-sensitive, and its yield in mouse L cells in vitro was similar to that of the laboratory strains. To determine the reason that this clone was avirulent, we isolated a series of reassortant progeny clones from crosses between the avirulent strain and T1 (Lang) and T3 (Dearing). Using these reassortants, we showed that avirulence was a property of the M 2 gene segment. The M2 segment was also responsible for conferring greater sensitivity to chymotryptic digestion on the avirulent strain, compared to more virulent strains. Prior studies have determined that the localization of virus in different cell types in the brain (tropism) is a property of the viral hemagglutinin, the product of the S1 RNA genome segment. Our studies thus indicate that the basis for relative neurovirulence does not reside in the viral hemagglutinin and clearly illustrate the multigenic nature of neurovirulence.
Enkephalin biosynthetic pathway: a 5300-dalton adrenal polypeptide that terminates at its COOH end with the sequence [Met]enkephalin-Arg-Gly-Leu-COOH.
Authors: Jones B N , Shively J E , Kilpatrick D L , Kojima K , Udenfriend S
Published: May 2006

An enkephalin-containing polypeptide of 5300 daltons has been isolated from extracts of bovine adrenal medulla and purified to homogeneity, and its sequence has been determined by a combination of automated Edman degradation and enzymatic time-course hydrolysis. The polypeptide contains a single [Met]enkephalin sequence followed by Arg-Gly-Leu-COOH, which forms the COOH terminus of the molecule. The enkephalin sequence is preceded by a Lys-Arg linkage, typical of prohormone cleavage sites. Posttranslational processing of the polypeptide at this site would lead to release of the octapeptide [Met]enkephalin-Arg6-Gly7-Leu8.
Special phase invariant formulas of higher order: Expected values
Authors: Karle Jerome
Published: May 2006

Formulas for the cosines of the higher-order phase invariants that arise in crystal structure analysis are derived as expected values from determinantal joint probability distributions. The values of the cosines of the invariants are expressed in terms of averages over simple functions of known structure factor magnitudes. The formulas are termed “special” to distinguish them from formulas that have more general averages. The latter will be presented in a future publication. The best opportunity for obtaining useful information from these formulas is provided by embedded seminvariants formed from the invariants by use of relationships among the phases that arise from the space group symmetries.
Normal nonmetrizable Moore space from continuum hypothesis or nonexistence of inner models with measurable cardinals
Authors: Fleissner William G.
Published: May 2006

Assuming the continuum hypothesis, a normal nonmetrizable Moore space is constructed. This answers a question raised by F. B. Jones in 1931, using an axiom well known at that time. For the construction, a consequence of the continuum hypothesis that also follows from the nonexistence of an inner model with a measurable cardinal is used. Hence, it is shown that to prove the consistency of the statement that all normal Moore spaces are metrizable one must assume the consistency of the statement that measurable cardinals exist.
Accurate transcription of truncated ribosomal DNA templates in a Drosophila cell-free system.
Authors: Kohorn B D , Rae P M
Published: May 2006

An extract of Drosophila melanogaster Kc cells is shown to give specific and accurate transcription of truncated segments of cloned D. melanogaster ribosomal DNA (rDNA). When clones are digested with restriction enzymes so that the initiation site is flanked by 0.3 kilobase (kb) of nontranscribed spacer and greater than 0.4 kb of external transcribed spacer, RNA polymerase I activity in the extract parallels in vivo rRNA synthesis in selection of the coding strand of template and the site of transcription initiation. When greater than 0.3 kb of the nontranscribed spacer is contiguous with transcribed spacer, in vitro initiations evidently also occur in repeated sequences adjacent to the site of in vivo initiation; when less than or equal to 0.4 kb of the external transcribed spacer is present in a segment, expected transcripts are heterogeneous in length or not detectable. Transcription in the cell-free system requires the specific addition of D. melanogaster rDNA: neither D. virilis rDNA, vector plasmid, nor clones of D. melanogaster genes that are transcribed in vivo by RNA polymerases II and III serve as templates in the system. Drosophila rDNA units that have an interruption in the 28S rRNA coding region are not transcribed in vivo, but restriction digests of a recombinant phage DNA that contains such a unit are active as template for in vitro rDNA transcription.
Resonance Raman detection of structural dynamics at the active site in hemoglobin.
Authors: Ondrias M R , Rousseau D L , Simon S R
Published: May 2006

The iron-histidine stretching mode in deoxyhemoglobin displays a large change in frequency and width upon lowering the temperature from 300 to 10 K. The temperature dependence of the data indicates the presence of dynamic process. The dynamics of this mode in frozen hemoglobins can be qualitatively and quantitatively described as a vibrational dephasing via anharmonic coupling to other vibrations of the heme-imidazole system. the effect that occur at the melting transition in the low frequency modes cannot be quantitatively addressed at this point but may be indicative of the introduction of additional degrees of freedom predicated on protein influences that reflect differences in protein quaternary structure.
Retrovirus long terminal repeats activate expression of coding sequences for the herpes simplex virus thymidine kinase gene.
Authors: Joyner A , Yamamoto Y , Bernstein A
Published: May 2006

In this study, we demonstrate that the long terminal repeats (LTRs) of a murine retrovirus can activate expression of heterologous gene coding sequences from which a functional promoter region has been deleted. Recombinant plasmid clones were obtained that contained both cloned fragments of Friend spleen locus-forming virus (SFFV) DNA and the herpes simplex virus (HSV) thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene (tk). The effects of the LTR on tk expression were determined by constructing clones containing tk coding sequences with or without 5' sequences necessary for the initiation of transcription, inserted either 200 or 1200 base pairs downstream from the SFFV 5' LTR. The expression of the HSV TK protein by these clones was tested by gene transfer of the cloned into TK- mouse cells and assay of TK enzyme activity in TK+ transformants. These experiments demonstrate that: (i) the SFFV 5' LTR activates expression of tk coding sequences when these sequences are inserted 200 base pairs downstream from, and in the same orientation as, the LTR; (ii) tk is not activated when placed 1200 base pairs downstream from, and in the same orientation as, the LTR or when tk is inserted in either site in the opposite orientation as the LTR; (iii) the SFFV 5' LTR does not interfere with in vivo expression of tk when it is flanked by homologous 5' promoter sequences. The implication of these observations for retrovirus oncogenesis and animal cell genetics is discussed.
Properties of a Saccharomyces cerevisiae mtDNA segment conferring high-frequency yeast transformation.
Authors: Hyman B C , Cramer J H , Rownd R H
Published: May 2006

The bakers' yeast Saccharomyces cerevisiae is a facultative anaerobe, tolerant to mutations in its mitochondrial genome. Individual cytoplasmic petite mutants retain genetic information derived from any portion of the parenteral mtDNA, prompting questions concerning distribution of the DNA replication origin(s) on the yeast mitochondrial genome. The experiments described in this paper were designated to test the possibility of using high-frequency yeast transformation as a selection for yeast mtDNA sequences conferring autonomously replicating function. A complete petite mitochondrial genome was inserted into the yeast vector YIp5, and the hybrid plasmid (YRMp1) was used to transform yeast. YRMp1 promoted high-frequency transformation of both wild-type yeast cells and petite mutant hosts lacking mtDNA and was maintained in each of these strains as a high-copy-number extrachromosomal element. The stability and copy-number properties of YRMp1 are similar to those of YRp12, a recombinant plasmid containing a yeast chromosomal autonomously replicating sequence.
Natural antibodies to the structural core protein (p24) of the human T-cell leukemia (lymphoma) retrovirus found in sera of leukemia patients in Japan
Authors: Kalyanaraman V. S. , Sarngadharan M. G. , Nakao Y. , Ito Y. , Aoki T. , Gallo R. C.
Published: May 2006

In Japan, adult T-cell leukemias and lymphomas are more common than in the United States and Europe, and in the southwest part of Japan these T-cell malignancy cases appear in clusters. Therefore, we investigated the involvement in these leukemias and lymphomas of the human T-cell leukemia virus (HTLV) that was previously isolated in one of our laboratories from cultured T cells of some patients in the United States with leukemias and lymphomas involving relatively mature T cells. High titers of antibodies capable of quantitative precipitation of
125
I-labeled p24, a well characterized core protein of HTLV, were detected in 12 of 12 patients with untreated adult T-cell leukemia (ATL). (One negative was a patient on chemotherapy.) Ten of the 12 positive samples were from an area where the disease is endemic. Strong precipitating antibodies were also detected in five of seven cases of T-cell malignant lymphoma (TML) which differs from ATL by having fewer leukemic cells in the peripheral blood. High antibody titers were also observed in one of five cases of acute monoblastic leukemia and one of eight cases of chronic myelogenous leukemia in the blast phase of the disease. Low to moderate titers of antibodies were detected in several categories of leukemia (two cases of blast-phase chronic myelogenous leukemia, two cases of acute lymphoblastic leukemia of the null-cell type, and one case of acute myelogenous leukemia). Among all categories of leukemias, except ATL and TML, more cases were negative than positive for anti-p24 activity. All of 79 sera from normal Japanese, including 39 collected from the endemic ATL area of southwest Japan, were negative for antibodies to HTLV p24. All the positive reactivities observed were highly specific to HTLV. The only competition observed in the precipitation of HTLV p24 was with HTLV or with cell lines expressing HTLV and not with various animal retroviruses or a large number of human and subhuman primate cell lines, not known to be producing HTLV. The data strongly indicate an association of HTLV with the increased incidence of ATL in parts of Japan, probably with other forms of leukemias in Japan, and, less commonly, with certain T-cell malignancies in the United States.
Spontaneous vesiculation of phospholipids: a simple and quick method of forming unilamellar vesicles.
Authors: Hauser H , Gains N
Published: May 2006

Phosphatidic acid dispersions in H2O vesiculate when the pH is increased transiently (less than 2 min) from approximately 3 to 10.5-11. The same phenomenon is observed in mixed dispersions of phosphatidylcholine and phosphatidic acid in H2O when the pH is increased from approximately 3 to 7 to 8. With phosphatidic acid, this treatment produces small closed unilamellar vesicles (200-600 A) from 50-60% of the total phospholipid, with the remainder present as large unilamellar vesicles and multilamellar structures. The extent of vesiculation depends on the pH that the dispersion is exposed to. With mixed phospholipid dispersions, similar vesicles are obtained; in addition to pH, the extent of vesiculation depends on the phosphatidylcholine/phosphatidic acid (wt/wt) ratio; as this ratio increases, the percentage of small unilamellar vesicles formed decreases. The short exposure of the phospholipids to high pH does not cause lipid degradation, as assessed by thin-layer chromatography; hence, the formation of degradation products can be ruled out as being responsible for the spontaneous vesiculation. Ionization of the phosphate group, resulting in a high surface charge density, may be an important factor in the spontaneous vesiculation. The proportion of phospholipid present as small unilamellar vesicles was determined by gel filtration on Sepharose 4B and by 1H NMR. The small vesicles gave rise to a reasonably well resolved high-resolution NMR spectrum. A good correlation was found between the proportion of phospholipid giving rise to the high resolution spectrum, as derived from spectral intensity measurements, and the proportion present as small unilamellar vesicles, as derived from gel filtration.
Degradation of intra- and extrahepatic protein by livers of normal and diabetic mice: differential responses to starvation.
Authors: Hutson N J , Lloyd C E , Mortimore G E
Published: May 2006

Rates of total hepatic proteolysis were measured in normal and streptozotocin-diabetic mice during feeding and over 48 hr of starvation, during which livers in the two groups lost 37% and 54% of their protein content, respectively. Measurements were made in 15-min in situ cyclic perfusions from the linear accumulation of free valine in the presence of cycloheximide; rates were corrected for turnover of short-lived proteins because these components contribute negligibly to alterations in liver protein content. During deprivation, corrected rates, expressed on a per liver basis, remained constant in normal mice but increased markedly in the diabetic group, attaining twice prestarvation values by 48 hr. By contrast, degradation rates of long-lived intracellular proteins, calculated from the sum of their synthesis and the linear decrease in protein content, decreased predictably in both groups and in parallel with absolute rates of protein synthesis. The extra proteolysis, representing the difference between total and long-lived protein degradation, was small in fed animals but increased progressively during starvation. With diabetic mice, however, the increase was approximately 5 times that of the normal and, in absolute terms, roughly equaled the total loss of liver protein. We suggest that this fraction arose from intrahepatic breakdown of proteins that were ultimately derived from sources outside the liver. Acceleration of this novel process could play an important interim role in providing endogenous glucogenic substrate under conditions in which the demand for this substrate is high.
Transcription of adenovirus cores in vitro: major RNA products differ from those made from a DNA template.
Authors: Daniell E , Fedor M J
Published: May 2006

Intact cores of adenovirus type 5 were transcribed in vitro in extracts derived from uninfected HeLa cells, and the products were analyzed by hybridization to restricted viral DNA. Predominant transcripts from cores were distinctly different from transcripts generated from deproteinized DNA. The extracts contained RNA polymerases II and III. Transcription from cores was completely inhibited by low levels of alpha-amanitin, and there was little or no hybridization of products to the region of the genome encoding the small polymerase III-dependent virus-associated RNA that was a major transcript from the DNA template. The major transcripts from cores hybridized to the rightward-transcribed strand of the region between 75 and 89 map units, early region 3 of the adenovirus genome. There was several-fold more transcription of this region in cores than of the region downstream from the major late promoter, which predominates among polymerase II transcripts from DNA.