bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of spectrin in cell-surface receptor capping in lymphocytes.
Authors: Nelson W J , Colaço C A , Lazarides E
Published: May 2006

Human and mouse lymphocytes of T- and B-cell lineages express a protein (Mr, 240,000) that crossreacts with antibodies raised against chicken erythrocyte alpha-spectrin as judged by immunofluorescence, immunoprecipitation, and immunoautoradiography; by the same criteria, antibodies raised against chicken erythrocyte beta-spectrin do not react with any lymphocyte polypeptide. In all T and B cells analyzed, before surface-directed ligand challenge with concanavalin A and surface immunoglobulins the polypeptide antigenically related to erythrocyte alpha-spectrin is distributed diffusely at the plasma membrane. Upon challenge, the redistribution of this polypeptide is concurrent with that of the cell-surface receptors initially in patches and then in a cap. Immunoprecipitation of NaDodSO4-solubilized lymphocytes with erythrocyte alpha-spectrin antiserum shows that in all cases a polypeptide with the same apparent molecular weight as erythrocyte alpha-spectrin is precipitated. Variable amounts of another polypeptide (Mr, 235,000) are also coimmunoprecipitated. Immunoprecipitations and subsequent immunoautoradiography show that the lymphocyte polypeptide doublet has a composition similar to that of (brain) fodrin, a polypeptide doublet that previously has been found mainly in the cells of nervous tissue.
Multiple mutations in the T region of the Agrobacterium tumefaciens tumor-inducing plasmid.
Authors: Ream L W , Gordon M P , Nester E W
Published: May 2006

Three genetic loci affecting tumor morphology lie within pTiA6NC T-DNA: tms, tmr, and tml. Using deletions and multiple transposon insertions, we constructed tumor-inducing (Ti) plasmids representing every possible double and triple mutant combination. tms tmr and tms tmr tml mutants did not incite tumors on most plants and produced a very weak response on a few other hosts but tms tml and tmr tml mutants were virulent. Thus, either tms+ or tmr+ alone can promote significant tumor growth but tml+ by itself is not sufficient. On hosts where tms mutants induce tumors accompanied by shoot proliferation, addition of a tml mutation reduces or eliminates shoot proliferation, suggesting that tml+ promotes shoot development. The small calli incited by tms tmr and tms tmr tml mutants contain agropine, an indication that these plant cells incorporate T-DNA in the absence of substantial tumor growth.
Detection, rescue, and mapping of mutations in the adenovirus DNA binding protein gene
Authors: Nicolas J. C. , Young C. S. H. , Suarez F. , Girard M. , Levine A. J.
Published: May 2006

r(ts107)202 was isolated in HeLa cells as a temperature-independent revertant of H5ts107, an adenovirus mutant that maps in the structural gene of the viral DNA-binding protein. r(ts107)202 is a host-range temperature-conditional mutant: it is temperature independent for growth in HeLa cells but temperature sensitive for growth in 293 cells, a type 5 adenovirus-transformed human cell line. Marker rescue experiments using H5ts107 DNA and restriction enzyme fragments from r(ts107)202 DNA demonstrated that the mutations causing the r(ts107)202 phenotype were localized in
Hin
dIII fragment A containing the entire DNA-binding protein gene. To obtain a fine structure map of the r(ts107)202 mutations, overlap recombination between
Eco
RI fragment A (0-75.9 map units) from either Ad5wt or r(ts107)202 and
Bam
HI fragment B (59.5-100 map units) from either Ad5wt or r(ts107)202 was performed. Segregation of the H5ts107 primary-site mutation away from the accompanying reversion mutation could be demonstrated in 5 of 200 plaques when r(ts107)202
Eco
RI fragment A was crossed with the Ad5wt
Bam
HI fragment. In the reciprocal cross, none of 200 plaques contained the H5ts107 mutant. These results permitted a determination of the order of the primary-site mutation (H5ts107) and secondary-site mutation in r(ts107)202, and the frequency of recombination predicted a distance of 50-340 base pairs between these two mutations. This experimental result agrees with the nucleotide sequence of the r(ts107)202 mutant which shows that 182 base pairs separate the primary-site and secondary-site mutations in r(ts107)202.
Mechanism of four-level laser action in solution excimer and excited-state proton-transfer cases
Authors: Khan Ahsan U. , Kasha Michael
Published: May 2006

Four-level laser energy level schemes are compared from the mechanistic spectroscopic viewpoint: (
i
) noble-gas excimer, (
ii
) solution molecular excimer, (
iii
) conventional dye laser, and (
iv
) intramolecular proton transfer. The lasing action of the chlorophyll special pair is discussed as an example of a solution excimer laser, and the lasing action of 3-hydroxyflavone and other molecules is discussed as an example of an intramolecular proton-transfer laser.
Chromosome site for Epstein-Barr virus DNA in a Burkitt tumor cell line and in lymphocytes growth-transformed in vitro.
Authors: Henderson A , Ripley S , Heller M , Kieff E
Published: May 2006

The Epstein-Barr virus (EBV) genome stably persists in latently infected Burkitt tumor cells and growth-transformed B lymphocytes. These cells usually contain multiple copies of episomal viral DNA. Cytological hybridization of recombinant viral DNA fragments to metaphase chromosomes of two latently infected cell lines demonstrates that viral DNA localizes to both chromatids of one homologue of chromosome 1 in Namalwa, a Burkitt tumor cell line, and to both chromatids of one homologue of chromosome 4 in IB4, a cell line with transformed growth properties in vitro. The site of chromosome association remains stable in a clone of IB4 cells. Probes from five separate regions of the EBV genome hybridize to the same chromosome regions. A previously undescribed achromatic site is identified within the region of EBV chromosome cytological hybridization. These observations suggest that most or all of the EBV genome is integrated into the chromosomal DNA of Namalwa and IB4 cells. Although the chromosomal sites of EBV DNA association are among those regions with homology to the EBV IR3 repeated DNA sequence, EBV IR3 did not mediate recombination between EBV and chromosomal DNA.
Hybrid H-2 histocompatibility gene products assign domains recognized by alloreactive T cells.
Authors: Ozato K , Evans G A , Shykind B , Margulies D H , Seidman J G
Published: May 2006

In vitro recombination of cloned H-2 genes resulted in two allogeneically novel mouse transplantation antigens in which the C2 domains of H-2Ld and H-2Dd were exchanged. These genes were introduced into mouse L cells by DNA-mediated gene transfer. We have used these transformed fibroblast lines, expressing recombinant H-2 antigens at normal levels, as targets for alloreactive cytotoxic T lymphocytes (CTL). Transformed cell lines expressing native or hybrid H-2 molecules that share the N and C1 domains but not the C2 domain were lysed to an equivalent degree by primary and secondary anti-H-2Dd CTL. Hybrid and native H-2 antigens that have the same N and C1 domains were capable of reciprocally blocking specific lysis by the CTL. Thus, polymorphic determinants recognized by alloreactive T cells are located primarily in the N domain, the C1 domain, or both domains of the H-2 antigen. In contrast, determinants recognized by monoclonal antibodies are present throughout the H-2 protein, including the C2 domain. Antibodies that bind specifically to the C2 domain effectively inhibited CTL activity, possibly due to steric effects. The same antibodies do not competitively inhibit the binding of N/C1-specific antibodies. The predominant reactivity of CTL to the N and C1 domains suggests that humoral and cellular responses "see" a different spectrum of alloantigenic determinants.
Regulation of catabolism of microinjected ribonuclease A requires the amino-terminal 20 amino acids.
Authors: Backer J M , Bourret L , Dice J F
Published: May 2006

RNase A introduced into the cytoplasm of IMR-90 human diploid fibroblasts by erythrocyte-mediated microinjection is degraded with a half-life of approximately equal to 75 hr in the presence of fetal bovine serum. In response to serum deprivation the degradative rate of microinjected RNase A is enhanced 2-fold. RNase S protein (amino acids 21-124) is degraded with a half-life similar to that of RNase A in the presence of serum, but its catabolism is not increased during serum withdrawal. Reconstitution of RNase S protein with RNase S peptide (amino acids 1-20) restored full enzymatic activity to the S protein as well as the ability of fibroblasts to increase its catabolism during serum deprivation. Finally, RNase S peptide microinjected alone shows the full 2-fold increase in degradative rate during serum withdrawal. These results show that recognition of RNase A for enhanced breakdown during serum deprivation is based on some feature of its amino-terminal 20 amino acids. Furthermore, our results indicate that the enhanced protein catabolism during serum deprivation can be highly selective.
Quantitative correlation between proteolysis and macro- and microautophagy in mouse hepatocytes during starvation and refeeding.
Authors: Mortimore G E , Hutson N J , Surmacz C A
Published: May 2006

Cytoplasmic protein in hepatocytes is sequestered and degraded by two general classes of lysosomes, overt autophagic vacuoles (macroautophagy) and dense bodies (microautophagy). Volumes of the apparent space in each class that contain the internalized protein, together with estimates of cytoplasmic protein concentration, were used as a basis for predicting rates of protein degradation by the lysosomal system in livers of fed, 48-hr starved, and starved-refed mice. Assuming that the turnover of all sequestered protein is equal to that previously determined in overt autophagic vacuoles (0.087 min-1), we obtained close agreement between predicted and observed rates in the three conditions studied. The two autophagic components, though, exhibited different patterns of regulation. Microautophagy followed a downward course through starvation and into refeeding, a trend that explained fully the fall in absolute rates of protein degradation during starvation. By contrast, macroautophagy remained constant throughout starvation but was virtually abolished with refeeding. Whereas regulation of the latter can be explained largely by immediate responses to the supply of amino acids, present evidence together with results of others indicate that microsequestration could be linked to functional and quantitative alterations in the smooth endoplasmic reticulum. Both types of regulation contributed equally to the marked suppression of proteolysis during cytoplasmic regrowth.
Transcription termination in vitro at the tryptophan operon attenuator is controlled by secondary structures in the leader transcript.
Authors: Stroynowski I , Kuroda M , Yanofsky C
Published: May 2006

The role of alternative RNA secondary structures in regulating transcription termination at the attenuator of the tryptophan (trp) operon of Serratia marcescens was examined in vitro by transcribing mutant DNA templates having deletions of different segments of the trp leader region. Deletions that removed sequences corresponding to successive segments of postulated RNA secondary structures either increased or decreased transcription termination at the attenuator. The results obtained are consistent with the hypothesis that transcription termination results from RNA polymerase recognition of a particular RNA secondary structure, the terminator. This structure forms only in the absence of an alternative, preceding, RNA secondary structure, the antiterminator.
Autoregulatory control of translatable phytochrome mRNA levels
Authors: Colbert James T. , Hershey Howard P. , Quail Peter H.
Published: May 2006

Translatable phytochrome mRNA represents ≈5 × 10
-3
% of the total poly(A)-RNA present in etiolated
Avena
seedlings, as determined by incorporation of radioactivity into the immunoprecipitable apoprotein in a cell-free translation system. Irradiation of such seedlings with 5-
s
red light induces a decline in this mRNA that is detectable within 15-30 min, shows a 50% reduction within 50-60 min, and results in a >95% reduction within 2 hr. The effect of the red light pulse is reversed by an immediately subsequent far-red pulse to the level of the far-red-light control, indicating that phytochrome exerts autoregulatory control over its own translatable mRNA level. This result necessitates revision of existing concepts of how phytochrome concentrations are modulated
in vivo
. Red-light dose-response curves show that the response is sensitive to very low light levels. Conversion of <1% of the total cellular phytochrome to the biologically active far-red-absorbing form is sufficient to induce ≈60% of the maximal response, and 20% far-red-absorbing form saturates the response. The observed change in translatable phytochrome mRNA level is one of the most rapid phytochrome-induced alterations in any cellular mRNA yet recorded. Thus, autoregulation of phytochrome mRNA provides an attractive opportunity to examine the early sequence of events in phytochrome control of gene expression.
Nickel: A micronutrient element for hydrogen-dependent growth of
Rhizobium japonicum
and for expression of urease activity in soybean leaves
Authors: Klucas Robert V. , Hanus F. Joe , Russell Sterling A. , Evans Harold J.
Published: May 2006

Soybean plants and
Rhizobium japonicum
122 DES, a hydrogen uptake-positive strain, were cultured in media purified to remove Ni. Supplemental Ni had no significant effect on the dry matter or total N content of plants. However, the addition of Ni to both nitrate-grown and symbiotically grown plants resulted in a 7- to 10-fold increase in urease activity (urea amidohydrolase, EC 3.5.1.5) in leaves and significantly increased the hydrogenase activity (EC 1.18.3.1) in isolated nodule bacteroids. When cultured under chemolithotrophic conditions, free-living
R. japonicum
required Ni for growth and for the expression of hydrogenase activity. Hydrogenase activity was minimal or not detectable in cells incubated either without Ni or with Ni and chloramphenicol. Ni is required for derepression of hydrogenase activity and apparently protein synthesis is necessary for the participation of Ni in hydrogenase expression. The addition of Cr, V, Sn, and Pb in place of Ni failed to stimulate the activity of hydrogenase in
R. japonicum
and urease in soybean leaves. The evidence indicates that Ni is an important micronutrient element in the biology of the soybean plant and
R. japonicum
.
Immunospecific vesicle targeting facilitates microinjection into lymphocytes.
Authors: Godfrey W , Doe B , Wofsy L
Published: May 2006

Antibody-directed targeting of vesicles to cells dramatically enhances polyethylene glycol-mediated fusion and microinjection. Sealed erythrocyte ghosts, containing fluorescent bovine serum albumin, were targeted to murine spleen and thymus cells, and to lymphocyte, monocyte, and fibroblast cell lines. In all cases, targeted cell populations showed substantial levels of microinjection, whereas populations treated with the fusogen in the absence of targeting were not significantly microinjected. To achieve attachment of vesicles to selected cells, the cells were first labeled with biotin-modified antibody then treated with sealed ghosts prepared from avidin-coupled erythrocytes. This procedure should prove useful when the injection of specific cell populations is desired, or with cell types such as lymphocytes that are difficult to fuse, or when the use of limited reagents necessitates high injection efficiencies.
C3d receptors are expressed on human monocytes after in vitro cultivation.
Authors: Inada S , Brown E J , Gaither T A , Hammer C H , Takahashi T , Frank M M
Published: May 2006

Highly purified human third component of complement (C3) was used to coat sheep erythrocytes (E) that were sensitized with IgM antibody (EA), forming EAC3b over a wide range of C3 molecules per cell. EAC3b were converted to EAC3bi by incubation with purified C3b inactivator (factor I) and beta 1H globulin (factor H). EAC3bi were in turn trypsinized to produce the cellular intermediate EAC3d. Each of the cell types was carefully characterized to be certain of the type of C3 determinant expressed. These cellular complement intermediates were used to assess by rosette formation the C3 receptor activity on peripheral blood monocytes under various experimental conditions. Uncultivated monocytes from peripheral blood bound EAC3b and EAC3bi well but did not bind EAC3d significantly. However, upon cultivation on glass surfaces in the presence of fetal calf serum but not bovine serum albumin, monocytes showed a progressive increase in expression of the C3d receptor. The Fab' fragment of anti-C3c blocked binding of EAC3b completely, blocked EAC3bi partially, but failed to block binding of EAC3d to cultivated monocytes. In contrast, the Fab' fragment of anti-C3d blocked EAC3d rosette formation completely. These studies demonstrate that monocytes are capable of expressing receptor activity for a determinant on C3d but that the expression of this receptor depends on the state of activation or differentiation of the cells.
Nucleotide sequence of the structural gene (pyrB) that encodes the catalytic polypeptide of aspartate transcarbamoylase of Escherichia coli.
Authors: Hoover T A , Roof W D , Foltermann K F , O'Donovan G A , Bencini D A , Wild J R
Published: May 2006

The deoxyribonucleotide sequence of pyrB, the cistron encoding the catalytic subunit of aspartate transcarbamoylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2), has been determined. The pyrB gene encodes a polypeptide of 311 amino acid residues initiated by an NH2-terminal methionine that is not present in the catalytically active polypeptide. The DNA sequence analysis revealed the presence of an eight-amino-acid sequence beginning at Met-219 that was not detected in previous analyses of amino acid sequence. This octapeptide sequence provides an additional component of the disordered loop in the equatorial domain of the catalytic polypeptide. It had been found previously that the catalytic polypeptide is expressed from a bicistronic operon that also produces the regulatory polypeptide encoded by pyrI. A single transcriptional control region precedes the structural gene of the catalytic polypeptide and a simple 15-base-pair region separates its COOH terminus from the structural gene of the regulatory polypeptide. The chain-terminating codon of the catalytic polypeptide may contribute to the ribosomal binding site for the regulatory polypeptide and thus assist coordinate expression of the two cistrons.
Studies of osmoregulation in salt adaptation of cyanobacteria with ESR spin-probe techniques
Authors: Blumwald Eduardo , Mehlhorn Rolf J. , Packer Lester
Published: May 2006

Sucrose is accumulated in response to NaCl-induced stress in the cyanobacterium
Synechococcus
6311. Internal cell volume was measured by ESR spectra with 2,2,6,6-tetramethyl-4-oxopiperidinoxy free radical (TEMPONE) as a spin probe in order to calculate sucrose concentrations inside the cell. This method is rapid and reliable and provides an unambiguous measurement of absolute volumes in different osmotic environments. Because the osmolar concentration of sucrose does not counter-balance the osmolar concentrations of ions in the growth medium, we suggest that sucrose accumulation is one of the mechanisms involved in the process of adaptation to salt of
Synechococcus
6311. The accumulation of sucrose in non-N
2
-fixing cyanobacteria such as
Synechococcus
6311 and in N
2
-fixing cyanobacteria such as
Nostoc muscorum
suggests a common mechanism of osmoregulation of fresh water cyanobacteria in response to increasing NaCl concentrations in the growth medium.
Unusual evolutionary conservation and frequent DNA segment exchange in class I genes of the major histocompatibility complex.
Authors: Hayashida H , Miyata T
Published: May 2006

From comparisons of homologous DNA sequences for many different genes, it was shown that the silent positions of protein-encoding regions and introns evolve at high and remarkably similar rates for different genes. In addition, both silent positions and introns behave like clocks; they accumulated base substitutions at approximately constant rates with respect to geological time. The rates of evolution were estimated to be 5.5 X 10(-9), 3.7 X 10(-9), and 5.3 X 10(-9) per site per year for silent positions, short introns (less than approximately equal to 300 base pairs), and long introns (more than approximately equal to 500 base pairs), respectively. Contrary to expectation from the evolutionary clocks, DNA sequence comparison between pHLA 12.4 (a cloned HLA sequence) of man and Ld together with other H-2 genes of mouse, the class I genes of the major histocompatibility complex, revealed a surprisingly small amount of base substitution for both the introns and the silent positions; the degree of divergence is only about 60% of that of standard genes in the same species comparison. Furthermore, several segmental homologies have been observed between the class I genes of mouse, suggesting the frequent occurrence of gene conversion or double unequal crossing-over in evolution. Interrelations between the extreme polymorphism of the class I genes, the low evolutionary drift of the introns and the silent positions, and the frequent gene conversion or unequal crossing-over within the mouse genes are discussed.
Method for quantitating the molecular content of a subcellular organelle: hormone and neurophysin content of newly formed and aged neurosecretory granules.
Authors: Nordmann J J , Morris J F
Published: May 2006

A method is described for the quantitative determination of the content of subcellular organelles such as secretory granules. Purified subcellular fractions of the organelle are prepared and aliquots are assayed for hormones, for example. To determine the number of organelles per fraction, known numbers of latex particles of a size similar to the organelle are added to other aliquots of the subcellular fractions. Latex particles and organelles are then pelleted together by centrifugation. The ratio between latex particles and organelles can be determined by morphometric analysis of ultrathin sections taken through the full thickness of the pellet. The number of organelles and hence their content of the substance assayed can then be calculated. We have applied this technique to posterior pituitary neurosecretory granules, the content of which has already been estimated by a different method. Newly formed neurosecretory granules from oxen and rats were found to have a content of approximately equal to 85,000 molecules of hormone and neurophysin. Aged neurosecretory granules from the same neural lobes appeared to contain less hormone and neurophysin, but this was shown to be the result of loss of material from the granules during isolation in media of 360 mosM. Such loss could be prevented by isolation in hypertonic (660 mosM) media.
Human anti-idiotype antibodies in cancer patients: Is the modulation of the immune response beneficial for the patient?
Authors: Koprowski H , Herlyn D , Lubeck M , DeFreitas E , Sears H F
Published: May 2006

Inoculation of human subjects with mouse monoclonal antibody results in the production of anti-idiotype antibody that reacts with the binding site of the monoclonal antibody. This reaction is hapten-inhibited, suggesting that an internal image of the antigen is produced by the anti-idiotype response. The anti-idiotype antibody isolated from sera of three patients showed significant crossreactivity. Patients who developed the anti-idiotype antibody improved clinically and had long remission from their disease. The possible presence of the internal image of cancer antigen on the human immunoglobulin molecule may change the conditions under which the immune system reacts to the tumor antigen and may open new approaches to the control of tumor growth.
Cytotoxic T-cell precursors revealed in neonatally tolerant mice.
Authors: Stockinger B
Published: May 2006

Induction of neonatal tolerance leads to a profound reduction in cytotoxic T-lymphocyte precursor frequencies against the tolerated alloantigen ("tolerogen") as evaluated by limiting-dilution analysis. The curves obtained were linear within the range tested and, thus, did not yield evidence for any dissociation of cytotoxic precursors from regulatory cell populations. However, it could be shown that cytotoxic T-lymphocyte precursor frequencies against the tolerogen increased drastically if the tolerant spleen cells were adsorbed, prior to limiting-dilution culture, on monolayers of syngeneic blasts expressing receptors for the tolerogen but not if they were adsorbed on syngeneic blasts against third-party antigens. This finding implies that cytotoxic precursor cells against the tolerogen are not clonally deleted in tolerant animals but rather are suppressed by a regulatory cell population that is present at high frequency and presumably acts in an anti-idiotypic fashion.
Two antigenically related neuronal cell adhesion molecules of different specificities mediate neuron-neuron and neuron-glia adhesion.
Authors: Grumet M , Hoffman S , Edelman G M
Published: May 2006

Previous studies in this laboratory have led to the identification of the neural cell adhesion molecule, N-CAM, a homophilic ligand that mediates adhesion between neurons as well as between neurons and striated muscle precursors. By means of a similar immunological approach but with different assays, we have now identified a cell adhesion molecule on neurons (Ng-CAM) that mediates the heterotypic adhesion between neuronal membranes and glial cells. In this paper, we compare certain aspects of the structure and function of Ng-CAM and embryonic N-CAM from the chicken. Ng-CAM was localized by specific antibodies on neurons but not on glia, and double-staining methods showed that individual neurons contained both Ng-CAM and N-CAM. Embryonic Ng-CAM migrates primarily as a single component of Mr 135,000; its apparent Mr shifted to 127,000 after neuraminidase treatment. In contrast, the embryonic form of N-CAM migrates on NaDodSO4/polyacrylamide gels in the apparent Mr range of 200,000-250,000; after neuraminidase treatment, N-CAM migrates as two components of Mr 170,000 and Mr 140,000. Although both Ng-CAM and N-CAM have calcium-independent binding mechanisms, immunologically based cell adhesion assays suggested that they have different specificities in mediating cell adhesion. Whereas 0.25 micrograms of Ng-CAM partially neutralized the ability of 0.5 mg of polyspecific antineural Fab' fragments to inhibit the heterotypic binding of neuronal membrane vesicles to glial cells and larger amounts of Ng-CAM completely neutralized this inhibition, 20 micrograms of N-CAM had no neutralization activity in this assay. Reciprocally, 0.25 micrograms of N-CAM partially neutralized the ability of 0.5 mg of the same Fab' fragments to inhibit the direct homotypic aggregation of neuronal cells, but 20 micrograms of Ng-CAM had no detectable activity. Although peptide maps of the two cell adhesion molecules differed considerably and despite the differences in binding specificity of these molecules, two independently derived monoclonal antibodies were found to crossreact with both Ng-CAM and N-CAM. Therefore, these different neuronal cell adhesion molecules with distinct binding specificities share at least one antigenic determinant, raising the possibility that they arose from a common evolutionary precursor.
Role of membrane lipids in peptide hormone function: binding of enkephalins to micelles.
Authors: Deber C M , Behnam B A
Published: May 2006

In the course of their biological function, peptide hormones must be transferred from an aqueous phase to the lipid-rich environment of their membrane-bound receptor proteins. We have investigated the possible influence of phospholipids in this process, using 360-MHz 1H and 90-MHz 13C NMR spectroscopy to examine the association of the opioid peptides [Met]- and [Leu]enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) with phospholipid micelles. Binding of peptides to lipid was monitored in NMR spectra by selective chemical shift movements (e.g., the Phe aromatic ring protons) and residue-specific line broadening (e.g., of Met/Leu carbonyl- and alpha-carbon resonances). Results established that the zwitterionic hormones associate hydrophobically both with a neutral lipid (lysophosphatidylcholine) and (also electrostatically) with a negative lipid (lysophosphatidylglycerol). An association constant of Ka = 3.7 X 10(1) M-1 was calculated for the hydrophobic binding of enkephalin to lysophosphatidylcholine. NMR data suggested that enkephalin binds to the lipid with Met/Leu, Phe, and likely Tyr side-chain substituents associated with nonpolar interior regions of the micelle, whereas the COOH-terminal carboxylate moiety of the peptide is located in the surface of the lipid particle. An "attraction-interaction" model is proposed for hormone-lipid association wherein negative lipids attract the hormone electrostatically, while site-specific hydrophobic contacts facilitate its entry, concentration, and orientation into the lipid phase.
Effects of temperature on the degradation of proteins in rabbit reticulocyte lysates and after injection into HeLa cells.
Authors: Hough R , Rechsteiner M
Published: May 2006

Bovine serum albumin, pyruvate kinase, hemoglobin, and the Fc fragment of IgG were labeled and introduced into HeLa cells by erythrocyte-mediated microinjection. Degradation of the injected proteins was then measured in cells cultured at temperatures between 6 degrees C and 37 degrees C. Arrhenius plots revealed a constant Ea of 27 +/- 5 kcal/mol over this temperature interval. Similarly, the apparent Ea for the degradation of long-term endogenously labeled HeLa proteins was 22-26 kcal/mol. Both local protein unfolding and proteolysis by defined enzymes, such as trypsin or papain, proceed with EaS between 5 and 15 kcal/mol. The 2-fold higher values obtained in this study indicate that protein unfolding or simple proteolysis is not rate limiting in the degradation of injected or long-lived endogenous HeLa proteins. Moreover, the relatively uniform EaS suggest that a similar biochemical event is rate limiting in the degradation of a specific protein independent of its half-life. This event may involve a reaction in the ATP-dependent proteolytic pathway from rabbit reticulocyte lysates because we observed that EaS for ATP-dependent proteolysis in this system were also 27 +/- 5 kcal/mol.
Characterization and postulated structure of the primary emitter in the bacterial luciferase reaction
Authors: Kurfürst Manfred , Ghisla Sandro , Hastings J. Woodland
Published: May 2006

An intermediate identifiable as the emitter in bacterial bioluminescence has been demonstrated. The reaction was carried out at 1°C by mixing purified luciferase-bound FMN 4
a
-hydroperoxide with long-chain aldehyde (decanal). Simultaneous kinetic measurements of bioluminescence and absorbance showed that the decay of light emission occurred more rapidly than the appearance of the stable product, oxidized FMN, indicating the formation of a transient intermediate species subsequent to light emission. The same species was found in reaction mixtures examined immediately after light emission was completed. It has a relatively short half-life (7 min at 9°C); the chromophore is postulated to be the luciferase-bound flavin 4
a
-hydroxide and to decay to the stable product, FMN, by losing water. Both its absorption spectrum (λ
max
, 360 nm) and its fluorescence emission (λ
max
, 490 nm) are consistent with the hypothesis that this is the ground state of the primary emitter, the bioluminescent species produced in the reaction.
Transforming potential of a human protooncogene (c-fps/fes) locus.
Authors: Sodroski J G , Goh W C , Haseltine W A
Published: May 2006

The protooncogene c-fps/fes is a single vertebrate locus homologous to both the fps transforming gene of Fujinami avian sarcoma virus and the fes transforming gene of two feline sarcoma virus isolates. The human c-fps/fes locus has been previously cloned and characterized. We report that a recombinant gene in which 3' human c-fps/fes sequences replace over 80% of the feline sarcoma virus fes sequences transforms NIH 3T3 mouse fibroblasts and encodes a protein kinase. Cells transformed by this recombinant possess increased phosphotyrosine levels. These observations demonstrate that a large carboxyl portion of a human c-fps/fes protein product can functionally complement a retroviral transforming protein.
Clathrin-coated vesicles from rat liver: enzymatic profile and characterization of ATP-dependent proton transport.
Authors: Van Dyke R W , Steer C J , Scharschmidt B F
Published: May 2006

Clathrin-coated vesicles isolated from rat liver exhibited an enzymatic profile distinct from that of rat liver plasma membranes, lysosomes, microsomes, and mitochondria. The coated vesicles catalyzed ATP-dependent proton transport that acidified the vesicle interior, as measured by the fluorescence quenching of acridine orange. H+ transport by coated vesicles was not inhibited by vanadate (0.1 mM) or ouabain (2 mM) and differed from H+ transport by rat liver submitochondrial particles in its greater resistance to inhibition by oligomycin (10 pM to 10 microM) and N,N'-dicyclohexylcarbodiimide (DCCD) (0.1-100 microM) and its sensitivity to N-ethylmaleimide (0.1-2 mM). H+ transport was stimulated by valinomycin in the presence of K+, exhibited no specific cation requirement, but was dependent upon the presence of a permeant anion, with Cl- and Br- being the most effective of the anions studied. Finally, H+ transport was poorly supported by GTP, UTP, or ADP and exhibited no consistent relationship to the coated vesicle-associated ouabain-insensitive ATPase activity.
Dipyridamole inhibits reversion by thymidine of methotrexate effect and increases drug uptake in Sarcoma 180 cells.
Authors: Cabral S , Leis S , Bover L , Nembrot M , Mordoh J
Published: May 2006

The combined effect of methotrexate (MTX) with dipyridamole, an inhibitor of nucleoside transport, was studied in ascitic Sarcoma 180 cells. It was determined that 10 microM MTX inhibits by greater than 90% deoxy[3H]uridine incorporation into DNA and that this MTX concentration inhibits DNA synthesis as revealed by deoxy[3H]cytidine but not [3H]thymidine incorporation into DNA. Exogenous thymidine (greater than or equal to 1 microM) in the cell culture medium enhances DNA synthesis in nontreated cells and fully restores it in MTX-treated cells, whereas hypoxanthine has no appreciable effect on DNA synthesis. Dipyridamole inhibits deoxy[3H]cytidine and [3H]thymidine uptake by these cells (IC50 = 0.2 and 3 microM, respectively) and blocks the increase in TTP pool produced by 1 microM thymidine in MTX-treated cells (23.1 +/- 4.7 pmol per 1 X 10(6) cells vs. 80.4 +/- 18.9 pmol per 1 X 10(6) cells). Dipyridamole at 10 microM enhances [3H]MTX accumulation by Sarcoma 180 cells and diminishes the efflux of the drug in previously loaded cells. It is suggested that the combination of inhibitors of the de novo pathway for pyrimidine biosynthesis, such as MTX, with inhibitors of the salvage pathway, such as dipyridamole, may increase the cytotoxic activity of MTX alone.
Structure of the
168
Hf nucleus in high rotational states
Authors: Pauling Linus
Published: May 2006

The values of the moment of inertia of the ground-state band of
168
Hf from
J
= 0
+
to 22
+
are interpreted as involving a revolving cluster with composition increasing steadily from
p
10.5
n
14
to
p
20.5
n
24
, with radius of revolution 6.390 fm. The excited bands from 14
+
to 34
+
, 21
-
to 33
-
, and 18
-
to 30
-
have nearly the same very large moment of inertia. It is interpreted as involving two caps, each consisting of 11 tritons, on opposite sides of the nearly doubly magic sphere
p
50
n
52
, the value of the radius of revolution being 6.445 fm.
Mechanisms of cytoplasmic pH regulation in hypoxic maize root tips and its role in survival under hypoxia.
Authors: Roberts J K , Callis J , Wemmer D , Walbot V , Jardetzky O
Published: May 2006

We show that a transient lactic fermentation provides the signal triggering ethanol production in hypoxic maize root tips. The signal is cytoplasmic pH. This interaction between lactic and ethanolic fermentation permits tight cytoplasmic pH regulation during hypoxia--cytoplasmic pH remaining near neutrality for several hours. Mutant roots unable to synthesize ethanol can neither regulate cytoplasmic pH nor maintain ATP levels during extended periods of hypoxia and, like vertebrate tissues, are less tolerant of hypoxia than normal maize. This indicates that cytoplasmic pH regulation is an important factor in survival under hypoxia.
Effect of selenium deficiency on cuticle integrity in the Cladocera (Crustacea).
Authors: Keating K I , Dagbusan B C
Published: May 2006

Daphnia pulex de Geer and Daphnia magna Straus populations cannot be maintained in defined (sensu stricto) media containing less than 0.1 part per billion (ppb) of selenium. A concentration of 1 ppb is sufficient to satisfy minimal needs in otherwise sufficient media. In the first generation with no selenium added to the medium or detected in it, media deficiency is shown by a premature cuticle deterioration visually similar to senescence, by progressive loss of distal segments of second antennae (primary swimming appendages), and by a shortened lifespan. No progeny attain reproductive maturity in the second generation. Although experimental animals in prime condition exhibit a shortened lifespan in the first generation maintained at 0.5 ppb selenium, culture lines can be maintained at 0.5 ppb for indefinite numbers of generations if established as young orthoclones . Tests in organic-rich media indicate a significant sparing effect of organic additions. This selenium requirement is reminiscent of that for the stability of feathers in domestic fowl.
The rnh gene is essential for growth of Escherichia coli.
Authors: Kanaya S , Crouch R J
Published: May 2006

We have determined that a functional gene coding for ribonuclease H seems to be essential for cell growth in Escherichia coli. A strain was made with two copies of the rnh gene by lysogenizing an E. coli strain with a lambda phage bearing a copy of the rnh gene. Inactivation of one of the two copies of the rnh gene was accomplished by transformation with a linear DNA molecule that had the gene for chloramphenicol acetyltransferase inserted near the middle of the rnh gene. In recombinants that had an inactive gene replacing the normal chromosomal rnh gene, the lambda rnh prophage supplies an intact functional copy of the rnh gene. Curing the cells of the lambda rnh prophage left the cell with an inactive rnh gene and resulted in cell death. An intact functional rnh gene provided on a plasmid permits normal curing, and cured survivors were readily obtained. The technique described is probably generally applicable for assessing the requirement for other E. coli genes.
Chemically modified nucleic acids as immunodetectable probes in hybridization experiments.
Authors: Tchen P , Fuchs R P , Sage E , Leng M
Published: May 2006

Guanine residues in nucleic acids can be modified by treatment with N-acetoxy-N-2-acetylaminofluorene and its 7-iodo derivative in an in vitro nonenzymatic reaction. The modified nucleic acids (ribo or deoxyribo, single or double stranded) are recognized by specific antibodies. They can be immunoprecipitated or used as probes in hybridization experiments and detected by immunochemical techniques.
Inhibition by calmodulin of calcium/phospholipid-dependent protein phosphorylation.
Authors: Albert K A , Wu W C , Nairn A C , Greengard P
Published: May 2006

Calmodulin was previously found to inhibit the Ca2+/phospholipid-dependent phosphorylation of an endogenous substrate, called the 87-kilodalton protein, in a crude extract prepared from rat brain synaptosomal cytosol. We investigated the mechanism of this inhibition, using Ca2+/phospholipid-dependent protein kinase and the 87-kilodalton protein, both of which had been purified to homogeneity from bovine brain. Rabbit brain calmodulin and some other Ca2+-binding proteins inhibited the phosphorylation of the 87-kilodalton protein by this kinase in the purified system. Calmodulin also inhibited the Ca2+/phospholipid-dependent phosphorylation of H1 histone, synapsin I, and the delta subunit of the acetylcholine receptor, with use of purified components. These results suggest that calmodulin may be a physiological regulator of Ca2+/phospholipid-dependent protein kinase.
Monitoring fibroblast behavior in tissue culture with an applied electric field.
Authors: Giaever I , Keese C R
Published: May 2006

Mammalian fibroblasts have been cultured on evaporated gold electrodes subjected to an alternating electric field at 4000 Hz. The system consists of a large (approximately equal to 2 cm2) and a small (approximately equal to 3 X 10(-4) cm2) electrode bathed in tissue culture medium. The applied electric field produces a voltage drop at the boundary between the solution and the small electrode of a few mV at a current density of a few mA/cm2. The small population of cells that attach and spread on this electrode have a marked effect on the measured impedance and also cause it to fluctuate with time. The amplitude of these fluctuations is greatly reduced by cytochalasin B (10 microM), suggesting they are a consequence of cell movement.
Rapid mutational analysis of regulatory loci in Escherichia coli K-12 using bacteriophage M13.
Authors: Wertman K F , Little J W , Mount D W
Published: May 2006

A derivative of bacteriophage M13mp8 , designated M13mp8 /P, was prepared in which the promoter and NH2-terminal codons of bacterial genes may be fused to a portion of beta-galactosidase, resulting in an easily scorable phenotype. Because transcription from the inserted promoter remains responsive to the host regulatory system, it is simple to screen mutagenized phage for isolates with aberrant regulatory phenotypes and to determine the mutational changes by dideoxy sequence analysis. The feasibility of the method was demonstrated by isolation of a large number of mutations in the regulatory regions of two genes, lexA and recA. Base substitutions that altered the phenotype of recombinant phage were identified both in the single LexA repressor binding site of recA and in the two binding sites of lexA, as well as in other sites that likely affect translational efficiency. Our results suggest that this approach will be generally useful for mutational analysis of transcriptional and translational regulatory elements.
Cloning of the
bronze
locus in maize by a simple and generalizable procedure using the transposable controlling element
Activator
(
Ac
)
Authors: Fedoroff Nina V. , Furtek Douglas B. , Nelson Oliver E.
Published: May 2006

The
bronze
(
bz
) locus of maize has been cloned by an indirect procedure utilizing the cloned transposable controlling element
Activator
(
Ac
). Restriction endonuclease fragments of maize DNA were cloned in bacteriophage λ and recombinant phage with homology to the center of the
Ac
element were isolated. The cloned fragments were analyzed to determine which contained sequences that were structurally identical to a previously isolated
Ac
element. Two such fragments were identified. Sequences flanking the
Ac
element were subcloned and used to probe genomic DNA from plants with well-defined mutations at the
bz
locus. By this means, it was established that one of the genomic clones contained a
bz
locus sequence. The subcloned probe fragment was then used to clone a nonmutant
Bz
allele of the locus. The method described here should prove useful in cloning other loci with
Ac
insertion mutations.
Requirement for the 3' flanking region of the bovine growth hormone gene for accurate polyadenylylation.
Authors: Woychik R P , Lyons R H , Post L , Rottman F M
Published: May 2006

We examined whether the sequence extending 3' to the polyadenylylation site of the bovine growth hormone gene contains any signal that affects the polyadenylylation of the growth hormone mRNA. For this purpose, cloned copies of this gene, each containing a different length of growth hormone-specific sequence 3' to the wild-type polyadenylylation site, were used to transfect COS-1 cells. The polyadenylylation site on the mRNAs produced from the exogenously added growth hormone genes were analyzed with an S1 nuclease mapping procedure. We found that a gene containing 84 base pairs of its own 3' flanking sequence is capable of producing an accurately polyadenylylated mRNA. On the other hand, genes containing only 1, 10, or 13 base pairs of 3' flanking sequence were principally polyadenylylated at discrete sites either upstream or downstream from the wild-type position. Using a computer program, we examined whether secondary structures on the primary growth hormone transcript correlated with the site where the mRNA is polyadenylylated.
DNA sequences of the repressor gene and operator region of bacteriophage P2.
Authors: Ljungquist E , Kockum K , Bertani L E
Published: May 2006

The nucleotide sequence of the repressor gene C of the temperate phage P2 has been determined. It codes for a nonbasic polypeptide, 99 amino acids long. Twelve repressor-defective mutants have been mapped. All but one are located within the presumed coding part of the gene. There is a strong promoter sequence and an 8-base-pair inverted repeat preceding the gene. The P2 repressor protein shows structural similarity to other DNA-binding proteins. The operator region for the early replication functions was located by sequencing the DNA of three virulent mutants. The sequence indicates that there are two repressor-binding sites. In addition, one of the sites shows sequence homology with part of the operator region of the biotin operon of Escherichia coli.
Extrachromosomal copies of transposon Tc1 in the nematode Caenorhabditis elegans.
Authors: Ruan K , Emmons S W
Published: May 2006

Extrachromosomal copies of the 1.6-kilobase transposable element Tc1 are present at the level of between 0.1 and 1.0 copy per cell in Caenorhabditis elegans strain Bergerac. Extrachromosomal elements were detected and studied using Southern hybridizations employing a Tc1-specific probe. The amount of extrachromosomal Tc1 DNA was roughly constant during development in Bergerac, which has approximately 300 integrated chromosomal copies of Tc1 in its haploid genome. Extrachromosomal Tc1 DNA was not detected in strain Bristol, which has 30 chromosomal copies of Tc1. Three forms of extrachromosomal DNA were detected. The predominant form was a 1.6-kilobase linear molecule with ends corresponding to the ends of an integrated Tc1 element. The other two forms were, respectively, relaxed and supercoiled circular copies of the element. Structural assignments were based on electrophoretic mobility, the results of sedimentation velocity and equilibrium density gradient experiments, and on the sizes of the products produced by treatment of purified extrachromosomal DNA with restriction endonucleases. The suggestion is made that these extrachromosomal transposable elements are the products of excision events known to be occurring at high frequency in somatic cells in Bergerac.