bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of delayed immune reactions on human epidermal keratinocytes.
Authors: Kaplan G , Witmer M D , Nath I , Steinman R M , Laal S , Prasad H K , Sarno E N , Elvers U , Cohn Z A
Published: May 2006

The epidermal changes that occur in human cutaneous immune responses have been investigated in the tuberculin reaction and in the lesions of tuberculoid and lepromatous leprosy and cutaneous leishmaniasis. In each situation, there was a dermal accumulation of monocytes and T cells, and the epidermis exhibited thickening. In the tuberculin response, the thickness of the epidermis sometimes doubled in 48-72 hr, and this was attributed to increases in both size and number of keratinocytes. In addition, the phenotype of the keratinocytes changed from Ia- to Ia+. Similar changes in keratinocyte Ia-antigen expression occurred in the epidermis overlying untreated tuberculoid leprosy and cutaneous leishmaniasis lesions, but not in lepromatous leprosy. We suggest that one or more epidermal growth factors may be generated in the course of a delayed immune reaction in the dermis.
Metabolism of lipoproteins containing apolipoprotein B-100 in blood plasma of rabbits: heterogeneity related to the presence of apolipoprotein E.
Authors: Yamada N , Shames D M , Stoudemire J B , Havel R J
Published: May 2006

Apolipoprotein B-100 is a constant component of very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL) in mammalian blood plasma. We have found that each of these classes of lipoproteins includes particles that contain apolipoprotein E (B,E particles) as well as particles that lack this protein (B particles). These two species can be separated by immunosorption on columns of anti-apolipoprotein E bound to Sepharose. We have injected radioiodinated VLDL, IDL, and LDL intravenously into recipient rabbits and have determined the concentration of radioiodine in apolipoprotein B-100 in B,E and B particles in whole-blood plasma obtained at intervals for 24 hr. We have developed a multicompartmental model that is consistent with this new information and with current concepts of lipoprotein metabolism. The model indicates that all apolipoprotein B-100 enters the blood as VLDL, of which about 90% is in B,E particles. Most VLDL B,E particles are removed rapidly from the blood, and only a small fraction is converted to IDL and eventually to LDL (overall conversion is approximately 2%). By contrast, a much smaller fraction of VLDL B particles is removed directly, and approximately 27% is converted to LDL. In addition, some B,E particles are converted to B particles as VLDL are converted to LDL, so that most LDL particles lack apolipoprotein E. Fractional rates of irreversible removal of B,E and B particles in IDL and LDL are similar. Our results indicate that the presence of apolipoprotein E is a major determinant of the metabolic fate of VLDL particles and support the hypothesis that polyvalent binding of particles containing several molecules of apolipoprotein E promotes receptor-dependent endocytosis of hepatogenous lipoproteins and limits their conversion to lipoproteins of higher density.
Isolated plasma membranes regulate neurotransmitter expression and facilitate effects of a soluble brain cholinergic factor.
Authors: Kessler J A , Conn G , Hatcher V B
Published: May 2006

The choice of which transmitter will be expressed by a neuron is influenced by diffusible differentiating factors produced by a variety of nonneuronal cells. In this study we show that human, bovine, and rat brains contain a soluble heparin-binding factor that stimulates cholinergic and peptidergic expression. We also show that neuronal contact with other cell membranes influences neurotransmitter development and acts synergistically with the effects of the soluble brain factor. Exposure of cultured sympathetic neurons to purified plasma membranes derived either from cultured Schwann cells or from cultured sympathetic neurons promoted expression of choline acetyltransferase (CAT), a cholinergic trait, and of substance P (SP). CAT activity and SP were also stimulated by a 50-kDa soluble protein that was purified 14,000-fold from human, calf, and rat brain by heparin-Sepharose affinity chromatography. CAT activity after concurrent treatment with plasma membranes and the soluble factor far exceeded the sum of the enzyme activities after the individual treatments, suggesting that membrane molecules and the factor facilitated each other. Thus, cell-surface molecules, which have been shown previously to influence neuronal morphogenesis and neurite elongation, may also help determine the transmitter phenotype of the neuron. Moreover, cell-surface molecules may modulate the effects of diffusible differentiating factors.
Sequence analysis and structure-function correlations of murine q, k, u, s, and f haplotype I-A beta cDNA clones.
Authors: Estess P , Begovich A B , Koo M , Jones P P , McDevitt H O
Published: May 2006

I-A beta-chain cDNA clones from mice of the q, k, u, s, and f haplotypes have been isolated and sequenced. Nucleotide sequence comparisons among these five A beta chains show considerable allelic variation in the region encoding the first external (beta 1) domain of the mature A beta protein. The beta 1 domain variability is clustered into three discrete regions, two of which divide the A beta chains into subgroups, suggesting an evolutionary history for the separation of alleles in inbred strains of mice. The amino acid sequences of these five chains are compared to each other and to previously published I-A beta chains. Correlations are made between the primary structural differences and the serologic and immune response characteristics mapping to the I-A subregion.
Expression of the v-mos gene alters a Mr 55,000 protein during acute infection by Moloney murine sarcoma virus.
Authors: Singh B , Sparrow J T , Hedge A M , Arlinghaus R B
Published: May 2006

Infection of the rat myoblast cell line, L6E9, with Moloney murine sarcoma virus (Mo-MuSV) clone 124, altered a cellular protein of Mr 55,000 (P55) within 2 days of infection. The alteration of P55 was observed as a reduction in its steady-state level in cell extracts. The reduction of P55 correlated with the appearance of p37mos in infected cells. Except for P55 and one other protein, no change was detected in the total protein pattern of infected cells compared to uninfected cells, as judged by either immunoblots of one-dimensional NaDodSO4 gels or direct two-dimensional gel analysis. P55 levels were unchanged when L6E9 cells were infected with Moloney murine leukemia virus or several different transforming retroviruses. To determine the specificity of this v-mos-induced effect on P55, L6E9 cells were acutely infected with a temperature-sensitive variant (ts110) of Mo-MuSV. When these cells were shifted from 39 degrees C to 33 degrees C, which activates the gag-mos gene product, the P55 level dropped by greater than 50% within 2-3 hr. Conversely, with a shift in temperature from 33 degrees C to 39 degrees C, the cells' P55 level returned to normal within 5 hr, starting at 30 min after shift. These results clearly show that v-mos expression in acutely infected L6E9 cells alters the cellular protein, P55.
NMR studies of a complex of deuterated calmodulin with melittin.
Authors: Seeholzer S H , Cohn M , Putkey J A , Means A R , Crespi H L
Published: May 2006

Completely deuterated calmodulin ([2H]CaM) has been prepared by expressing the chicken gene for CaM in Escherichia coli grown in 2H2O on a deuterated medium. The structural and dynamic properties of a 1:1 CaM/melittin (Mel) complex have been investigated by proton NMR. The spectrum of bound Mel is obtained directly from the spectrum of the [2H]CaM X Mel complex and is found to resemble strongly the spectrum of the helical species in methanol rather than that of the random coil species in water. The spectrum of bound CaM is obtained indirectly from the difference spectrum between [1H]CaM X Mel and [2H]CaM X Mel. Many changes are observed between free and bound CaM and they are distributed in both halves of the molecule, indicating that the binding of Mel affects the structure in both parts of the molecule. The rates of exchange of the amide protons of [2H]CaM with 2H2O were compared to those of [2H]CaM X Mel. The results showed that most, but not all, of the protons exchanged more slowly in the complex; after 40 hr, the residual peaks number 7 in CaM and greater than 20 in the complex. Again, changes in rates in CaM due to binding of Mel occurred in both halves of the molecule. The relative rates of amide proton exchange in CaM and its complex with Mel prove to be a sensitive criterion of differences in conformational stability and/or structure.
Regulated expression of Sindbis and vesicular stomatitis virus glycoproteins in Saccharomyces cerevisiae.
Authors: Wen D Z , Schlesinger M J
Published: May 2006

cDNAs encoding either the structural proteins (capsid and glycoproteins E1 and E2) of Sindbis virus or the glycoprotein of vesicular stomatitis virus (VSV) were fused to the Saccharomyces cerevisiae galactokinase gene (GAL1) promoter and inserted into a yeast shuttle vector. After addition of galactose to yeast transformed with this vector, 2.5-3% of total yeast protein synthesis was detected as virus proteins by specific anti-virus protein antibodies. In cells containing the Sindbis virus structural genes, the virus capsid protein was effectively released from the nascent polypeptide and two endoglycosidase H-sensitive glycoproteins were produced. One of these was identical in its gel mobility to E1 and the other appeared to be p62, a precursor to E2. A low level of E1 protein was detected on the cell's surface membranes. A single molecular weight species of glycosylated VSV glycoprotein was produced and half of the total protein could be detected at the surface membranes of yeast. Addition of long mannose chains and acylation of the virus proteins with fatty acids were not observed. Formation of virus proteins was also examined in yeast secretory mutants; one of these (sec53) failed to glycosylate the virus proteins.
Substrate free radicals are intermediates in ligninase catalysis.
Authors: Hammel K E , Kalyanaraman B , Kirk T K
Published: May 2006

The H2O2-requiring ligninase of the basidiomycete Phanerochaete chrysosporium oxidatively cleaves both lignin and lignin model compounds between C alpha and C beta (C-1 and C-2) of their aliphatic side chains. Previous work has demonstrated a reaction mechanism by which ligninase oxidizes aromatic substrates to their cation radicals, which then undergo side chain cleavage to yield carbon-centered free radicals. These carbon-centered radicals add O2 to give substrate peroxyl radicals that react further to yield the hydroxylated and carbonylated end products usually seen in experiments with ligninase. To investigate this radical mechanism, we have now designed three dimeric lignin models: 1-(3,4-dimethoxyphenyl)-2-phenylethanol (I), 1-(3,4-dimethoxyphenyl)-2-phenylpropanol (II), and 1-(3,4-dimethoxyphenyl)-2-methyl-2-phenylpropanol (III). The following results were obtained when these models were oxidized by ligninase: methyl groups at C beta of the substrate favored C alpha-C beta cleavage versus C alpha oxidation to the ketone. GC/MS and HPLC analysis showed that II gave a radical coupling dimer, 2,3-diphenylbutane, as a major (26% yield) reaction product under anaerobic conditions. The anaerobic oxidation of III yielded 2,3-dimethyl-2,3-diphenylbutane. Spin-trapping experiments with nitrosobenzene showed that model II oxidation produced alpha-methylbenzyl radicals, whereas model III oxidation gave alpha, alpha-dimethylbenzyl radicals. TLC and iodometric determinations showed that III gave cumene hydroperoxide as a major (21% yield) reaction product in air. These findings demonstrate that carbon-centered and peroxyl radicals at C beta are major products of C alpha-C beta cleavage by ligninase.
Chromatin binding of epidermal growth factor, nerve growth factor, and platelet-derived growth factor in cells bearing the appropriate surface receptors.
Authors: Rakowicz-Szulczynska E M , Rodeck U , Herlyn M , Koprowski H
Published: May 2006

We analyzed the uptake and intracellular distribution of 125I-labeled epidermal growth factor, nerve growth factor, and platelet-derived growth factor in different cell lines that express or do not express the respective surface receptors for these factors. After 1 hr of incubation, all three growth factors were detected in the cytoplasmic fraction and in the nucleus, tightly bound to chromatin. The amount of chromatin-bound growth factors continued to increase during the incubation, and analysis at 48 hr revealed each chromatin-bound labeled growth factor in a nondegraded form. After limited digestion of chromatin with DNase II (10-20% digested sequences), specific release of all three growth factors was detected only after 1 hr of incubation but not after 24 and 48 hr, suggesting that the DNA regions involved in growth factor binding became nuclease-resistant. Binding of labeled epidermal growth factor and nerve growth factor to isolated chromatin was inhibited by monoclonal antibodies specific for the respective growth factor receptor. The data suggest that chromatin binding may represent an important step in the pathway of growth factor action.
Toward computer-aided site-directed mutagenesis of enzymes.
Authors: Warshel A , Sussman F
Published: May 2006

A preliminary attempt to simulate the observed effect of a site-directed mutagenesis of rat trypsin gives encouraging results. The calculations reproduce in a semiquantitative way the observed change in the activation barrier of the rate-limiting step of amide hydrolysis. This result, which did not require any adjustable parameters, indicates that our method may provide a reliable basis for computer-aided enzyme design. In addition to the potentially practical value of the calculations, they provide important mechanistic information--that is, the change in the catalytic effect in trypsin appears to be almost exclusively due to the change in the electrostatic stabilization of the ionic configurations. This supports the view that electrostatic effects are the major factor in enzyme catalysis.
High content of creatine kinase in chicken retina: compartmentalized localization of creatine kinase isoenzymes in photoreceptor cells.
Authors: Wallimann T , Wegmann G , Moser H , Huber R , Eppenberger H M
Published: May 2006

Two isoforms of creatine kinase (CK; ATP:creatine N-phosphotransferase, E.C. 2.7.3.2), brain type (BB-CK) and mitochondrial type (MiMi-CK), but not the muscle types (MM- or hybrid MB-CK), were identified by cellulose polyacetate electrophoresis and immunoblots in retina from adult chickens. Indirect immunofluorescence labeling of cryosections of retinas revealed high concentrations of BB-CK in both rod and cone photoreceptor cells. Most of the fluorescence staining with anti-B-CK antibodies was found within the myoid and the ellipsoid portions of inner segments and the peripheral region of the outer segments. Significant staining with anti-B-CK antibodies was also found in horizontal cells and in the optical nerve fibers, with additional stratified staining in the inner plexiform layer. MiMi-CK was solely demonstrated in the ellipsoid portion of the photoreceptor cells. The presence of high concentrations of compartmentalized CK isoenzymes within photoreceptor cells (approximately equal to 30 enzyme units/mg) as well as the relatively high concentration of total creatine in these cells (approximately equal to 10-15 mM) indicates an important physiological function for CK and phosphocreatine in the energy transduction of vision.
Developmental origins of skeletal muscle fibers: clonal analysis of myogenic cell lineages based on expression of fast and slow myosin heavy chains.
Authors: Miller J B , Stockdale F E
Published: May 2006

A clonal analysis was used to show that skeletal muscle myoblasts are committed to distinct cell lineages during development. Myoblasts taken from embryonic chicken hindlimb muscles of different ages were cultured at clonal density. The content of fast and slow classes of the myosin heavy chain isoforms in the myotubes of the resulting muscle colonies was determined immunocytochemically with specific monoclonal antibodies that served as markers for the different fiber types. The muscle colonies formed by cloning myoblasts from early hindlimbs (days 4-6 in ovo) were of three types: the most numerous type, in which all myotubes in a colony contained only the fast class of myosin heavy chain; a less numerous type, in which all myotubes in a colony contained both the fast and slow classes of myosin heavy chain isoforms; and a rare type, in which all myotubes in a colony contained only the slow class of myosin heavy chain. The muscle colonies formed by cloning myoblasts from later hindlimbs (days 10-12 in ovo) were, however, all of one type, in which every myotube in a colony contained only fast myosin heavy chain. Thus, myoblasts in the early embryo (days 4-6 in ovo) were a heterogeneous population committed to three myogenic lineages: fast, mixed fast/slow, and slow, whereas myoblasts from the later embryo (days 10-12 in ovo) were only in the fast myogenic lineage. These results suggest that muscle fiber formation is rooted in two developmental phases--an early phase in which diverse fiber types are formed from intrinsically diverse populations of myoblasts and a later phase in which fibers are formed from a single population of myoblasts.
Class switch recombination is IgG1 specific on active and inactive IgH loci of IgG1-secreting B-cell blasts.
Authors: Radbruch A , Müller W , Rajewsky K
Published: May 2006

Mouse B lymphocytes can be activated polyclonally by bacterial lipopolysaccharide (LPS) to secrete Ig and perform Ig class switch. In the presence of the T-cell lymphokine B-cell differentiation factor, the frequency of IgG1-secreting cells is drastically enhanced. We show here that IgG1-secreting B cells isolated from such cultures have undergone a similar DNA rearrangement of the switch regions (S mu, S gamma 1) of the Ig heavy chain constant region genes C mu and C gamma 1 on both active and inactive IgH loci. This result argues against a stochastic model of class switch recombination and suggests programmed class-specific switch recombination in the case of the switch to IgG1. In accord with this notion, cells expressing IgM but not IgG on the surface have not deleted or rearranged C mu or S gamma 1 on either chromosome.
Sequential expression of protooncogenes during lectin-stimulated mitogenesis of normal human lymphocytes.
Authors: Reed J C , Alpers J D , Nowell P C , Hoover R G
Published: May 2006

The proliferation of non-neoplastic T lymphocytes is regulated, in part, by the coordinated expression of genes encoding T-cell growth factor (interleukin 2, IL2), IL2 receptors (IL2R), and transferrin receptors (TFR). In addition to growth factors and their receptors, protooncogenes may regulate lymphocyte proliferation. We used cloned cDNAs homologous to 21 different protooncogenes to screen for their expression at the mRNA level in human peripheral blood mononuclear cells (PBMC) stimulated with the mitogenic lectin phytohemagglutinin (PHA), and we compared the time course of accumulation of mRNAs for these protooncogenes to that of mRNAs for the IL2, IL2R, TFR, and histone H3 genes. mRNAs for c-abl, c-ets, c-yes, and N-ras were present in unstimulated PBMC. After stimulation of PBMC by PHA, we detected marked increases within 10 min in the levels of mRNA for c-fos and c-myc; within 6 hr for IL2 and IL2R mRNAs; within 14 hr for c-myb, p53, N-ras, and TFR mRNAs; and within 24-36 hr for H3 mRNA. Expression of c-abl, c-ets, and c-yes increased gradually following stimulation with PHA. None of the other protooncogenes tested was expressed in PBMC. Addition of the protein synthesis inhibitor cycloheximide, before the addition of PHA to cultures, abolished the PHA-induced accumulation of mRNAs for c-myb, N-ras, and TFR, but not of mRNAs for c-fos, c-myc, IL2, and IL2R. These data indicate that c-fos, c-myc, IL2, and IL2R belong to a group of genes expressed early, whereas c-myb, N-ras, and TFR belong to a group of genes expressed later in PHA-activated PBMC, and that the products of the c-fos and c-myc protooncogenes are not required for expression of IL2 or IL2R genes. Addition of purified IL2 augmented the expression of the later-expressed genes c-myb, p53, N-ras, and TFR in PHA-stimulated cultures of PBMC, as well as of the early genes c-myc and IL2R, but not of c-fos and IL2, thus suggesting that PHA and IL2 stimulate the expression of overlapping, but nonidentical, sets of genes in PBMC.
Transforming gene from human stomach cancers and a noncancerous portion of stomach mucosa.
Authors: Sakamoto H , Mori M , Taira M , Yoshida T , Matsukawa S , Shimizu K , Sekiguchi M , Terada M , Sugimura T
Published: May 2006

DNAs from 21 human stomach cancers, 16 metastatic stomach cancers to lymph nodes, and 21 apparently noncancerous specimens of stomach mucosae from a total of 26 patients with stomach cancer were tested for their ability to induce neoplastic transformation of NIH 3T3 cells on transfection by the calcium phosphate precipitation technique. Three samples of DNA were shown to have transforming activity; one was from a primary stomach cancer of one patient, the second was from a noncancerous portion of stomach mucosa of the same patient, and the third was from a lymph node metastasis of stomach cancer from another patient. These transformants were tumorigenic in nude mice, and DNAs from the cells could induce secondary transformants. A portion of the transforming gene from the stomach cancer of one patient, which contained the sequences expressed in the NIH 3T3 transformants, was cloned. The transforming gene did not have any homology with the transforming sequences reported previously. We have applied the term hst to this novel human transforming gene. The transforming gene, hst, was found to be present in all the primary and secondary transformants induced by the other two samples of DNA.
Human T-cell lymphotropic virus type III shares sequence homology with a family of pathogenic lentiviruses.
Authors: Gonda M A , Braun M J , Clements J E , Pyper J M , Wong-Staal F , Gallo R C , Gilden R V
Published: May 2006

The etiologic agent of the acquired immune deficiency syndrome, human T-cell lymphotropic virus type III (HTLV-III), has recently been shown to morphologically resemble and share sequence homology with visna virus, a pathogenic lentivirus. Molecular hybridization, heteroduplex mapping, and DNA sequence analyses were used to compare HTLV-III to other lentiviruses of domestic animals, including visna, caprine arthritis encephalitis, and equine infectious anemia viruses. Hybridization results showed that a substantial amount of sequence homology exists between each of these viruses and HTLV-III. In addition, a closer relationship was found between visna and caprine arthritis encephalitis viruses than for any of the other lentiviruses studied. These results, along with nucleotide and amino acid sequence comparisons, have been used in a comprehensive effort to derive a systematic relationship for lentiviruses and to provide further evidence for classifying HTLV-III with the Lentivirinae subfamily of retroviruses. This relationship predicts that similarities in biology and disease process can be expected between HTLV-III and other Lentivirinae members.
Cloning of cDNA for the contact site A glycoprotein of
Dictyostelium discoideum
Authors: Wong Lu Min , Siu Chi-Hung
Published: May 2006

A cell surface glycoprotein of
Dictyostelium discoideum
with
M
r
80,000 (gp80) has been shown to mediate the formation of the developmentally acquired EDTA-resistant cell-cell binding sites termed contact sites A. We have isolated cDNA clones encoding gp80 by immunological screening of an expression library prepared in
Escherichia coli
. Double-stranded cDNA was prepared from poly(A)
+
RNA isolated from cells at 8 hr of development and cloned into the bacteriophage expression vector λgt11. Two recombinant phages containing cDNA inserts of 1.2 and 0.8 kilobases were isolated and shown to contain sequences coding for gp80 by the immunoselect assay. Partial DNA sequence analysis also confirmed that one of these cDNA clones, λDdgp80c-19, contained the coding sequence for the amino terminus of gp80. DNA-RNA hybridization showed that the insert of λDdgp80c-19 hybridized to a single mRNA transcript of approximately 2.0 kilobases. gp80 mRNA became detectable after 6 hr of development, reached its maximum level at 9 hr, and dropped to a negligible level by 15 hr. This pattern of mRNA accumulation corresponded closely to that of gp80 synthesis in
D. discoideum
cells, suggesting that gp80 expression is regulated at the transcriptional level.
Role of conformational changes in the elution of proteins from reversed-phase HPLC columns.
Authors: Katzenstein G E , Vrona S A , Wechsler R J , Steadman B L , Lewis R V , Middaugh C R
Published: May 2006

To test the hypothesis that conformational alterations might be involved in the elution of proteins from reversed-phase HPLC columns, the conformations of proteins bound onto a C-8 alkyl-bonded silica surface have been examined in the presence of increasing concentrations of the commonly employed eluent, 1-propanol. Using a combination of photoacoustic, diffuse reflectance deconvolution Fourier transform infrared and front face fluorescence spectroscopic techniques (to minimize interference from light scattering), the existence of surface-associated protein conformational changes induced by propanol is unequivocally demonstrated. The linear relationship found between the amount of propanol needed to elute proteins from C-8 columns and the midpoint of spectrally observed structural transitions is consistent with a role for conformational changes in the elution process.
Colony stimulating factor-induced differentiation of murine M1 myeloid leukemia cells is permissive in early G1 phase.
Authors: Tsuda H , Neckers L M , Pluznik D H
Published: May 2006

Granulocyte-colony stimulating factor (G-CSF) induces differentiation of M1 murine myeloid leukemia cells into mature granulocytes/macrophages and also causes accumulation of the cells in the G1 phase of the cell cycle. We examined, therefore, whether synchronization of M1 cells in the G1 phase could affect G-CSF-induced differentiation as quantitated by expression of Fc fragment receptors (FcR) and lysozyme activity. Cells were arrested in early G1 by density inhibition in the absence of serum and in late G1 by addition of aphidicolin. Cells synchronized in early G1, when stimulated with G-CSF, showed enhanced expression of FcR and lysozyme activity. Eighty percent of the cells expressed FcR 18 hr after addition of G-CSF while, in exponentially growing cells, this percentage was reached 72 hr after addition of G-CSF. Cells synchronized in late G1 did not show enhanced expression of differentiation markers. These results imply that with respect to G-CSF-induced differentiation, the G1 phase can be separated into an early permissive and a later nonpermissive stage.
In vitro L-A double-stranded RNA synthesis in virus-like particles from Saccharomyces cerevisiae.
Authors: Fujimura T , Esteban R , Wickner R B
Published: May 2006

Most strains of Saccharomyces cerevisiae harbor L-A double-stranded RNA (dsRNA), 4.5 kilobases long, contained in virus-like particles (VLPs). These L-A VLPs can be separated by CsCl density gradient centrifugation into a main peak of particles, containing full-length L-A dsRNA, which synthesizes only plus-strand single-stranded RNA (ssRNA), and a lighter fraction of VLPs, containing plus-strand ssRNA, which has L-A dsRNA-synthesizing activity. This dsRNA-synthesizing activity was present in particles from logarithmically growing cells but not from stationary-phase cells. The newly synthesized strand of dsRNA in the lightest particles was full-length minus strand. All or almost all of the new minus strand was synthesized in vitro, and the rate of chain elongation was approximately 100 nucleotides per minute. The lightest particles synthesized plus-strand ssRNA only after completion of dsRNA synthesis, indicating that the same particle contains dsRNA- and ssRNA-synthesizing enzyme(s). We also observed dsRNA-synthesizing activity in L-BC dsRNA-containing particles similar to that in L-A VLPs.
Subgroups of variable region genes of beta chains of T-cell receptors for antigen.
Authors: Schiffer M , Wu T T , Kabat E A
Published: May 2006

The beta-chain variable regions of 6 human, 21 mouse, and 1 rabbit T-cell receptors for antigen, including two pairs of duplicates, have been classified into two subgroups. Subgroup I has an invariant Phe at residue 65 and a salt bridge can be formed between the Asp at position 86 and the 11 Arg, 2 Lys, and 1 His residues at position 64. In one instance Tyr replaces Asp at position 86 so that a salt bridge cannot be formed with the Lys at position 64. Subgroup II has an invariant Tyr at residue 65; 8 chains have Gly and 3 have Ala at position 63. These 3 have Asp at position 64. Those with Gly-63 cannot form a salt bridge in this region of the beta chain. The 3 with Asp-64 have Arg at position 86, can form a reverse salt bridge, and could constitute a third subgroup. The relation of these findings to functional parameters in beta chains of the T-cell receptors for antigen deserves further study. The use of variability plots on the assumption that the beta chains are a single population may be misleading for the variable region. Most of the variability in the expressed beta chains would be created by variable-diversity-joining assortment, junctional diversity, and the possible insertion of N sequences.
GTPase center of elongation factor Tu is activated by occupation of the second tRNA binding site.
Authors: Van Noort J M , Kraal B , Bosch L
Published: May 2006

Interaction of the elongation factor EF-Tu with the antibiotic kirromycin results in activation of the GTPase center of the factor and in induction of an additional tRNA binding site (tRNA binding site II to distinguish it from the classical tRNA binding site I). Activation of the GTPase center under these conditions is stimulated by addition of tRNA. Two-fold evidence is presented that this stimulation is due to tRNA binding to site II rather than to site I. First, a strong correlation is observed between stimulation of the GTPase activity and enhancement of the reactivity of Cys-81 of EF-Tu toward N-ethylmaleimide at various concentrations of aminoacyl-tRNA, deacylated tRNA, and N-acetylaminoacyl-tRNA. The latter effects signal tRNA binding to site II. Stimulation of the kirromycin-induced GTPase activity by tRNA binding to the factor also occurs when binding to site I is completely abolished. Such an abolishment was achieved by treating EF-Tu extensively with the thiol reagent L-1-tosylamido-2-phenylethyl chloromethyl ketone. EF-Tu X GTP thus treated has lost its ability to protect the ester bond of aminoacyl-tRNA. The relevance of these data for the sequence of events during protein synthesis and for control of translational fidelity is discussed.
Preparation of [125I-Tyr27,Leu5]beta h-endorphin and its use for crosslinking of opioid binding sites in human striatum and NG108-15 neuroblastoma-glioma cells.
Authors: Helmeste D M , Hammonds R G , Li C H
Published: May 2006

A radioligand suitable for crosslinking studies to opioid receptors has been obtained by radioiodination and purification of the monoiodotyrosine-27 derivative of the synthetic human beta-endorphin (beta h-endorphin) analogue [5-leucine]beta h-endorphin. The derivative, [27-[125I]monoiodotyrosine,5-leucine]beta h-endorphin, was crosslinked to human striatal (caudate and putamen) and NG108-15 neuroblastoma-glioma cell membranes by using disuccinimidyl suberate. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing conditions revealed four specifically labeled bands at 68, 40, 30, and 25 kDa for both human caudate and putamen, whereas NG108-15 cell membranes gave specifically labeled bands at 92, 56, 38, and 23 kDa.
Exogenous glutathione protects intestinal epithelial cells from oxidative injury.
Authors: Lash L H , Hagen T M , Jones D P
Published: May 2006

Exogenous GSH provided rat small-intestinal epithelial cells with significant protection against injury induced by t-butyl hydroperoxide or menadione. This protection was found to be dependent upon uptake of intact GSH. Uptake of GSH occurred by a Na+-dependent electrogenic system found in the basolateral membrane. Thus, rat small-intestinal epithelial cells can utilize plasma GSH to support intracellular detoxication systems that function in protection against chemically induced injury.
Evidence for soluble factors regulating cell death and cell proliferation in primary cultures of rabbit endometrial cells grown on collagen.
Authors: Lynch M P , Nawaz S , Gerschenson L E
Published: May 2006

Primary cultures of rabbit endometrial cells grown on collagen substrates exhibit cyclic changes in DNA content throughout extended periods of culture. These cycles are characterized by periods of significant increases and decreases in the DNA content of the cultures or number of cells present, yet through the entire duration of culture there is no net change in the total DNA. The rates of cell proliferation and cell death change through time in culture with the same periodicity as the changes in DNA. Neither changes in the rate of cell proliferation nor the rate of cell death alone are sufficient to account for the changes in DNA. Rather, there appears to be a feedback mechanism operating between cell proliferation and cell death such that when one increases, the other increases concomitantly in order to maintain a homeostasis in total culture mass. This homeostasis appears to be mediated by a soluble cell proliferation factor (CPF) and a cell death factor (CDF) produced by the cells. CPF and CDF may be obtained from either conditioned media or cultured cell extracts. These biological activities are heat and trypsin sensitive. The major mode of cell death in these cultures appears to be apoptosis or programmed cell death, characteristic of renewing epithelia. The data suggest that this tissue culture model system represents a renewing cell population containing stem cells and their progeny, whose total growth is strictly regulated by CPF and CDF. As such, it provides a model system in which to study homeostasis and how it may be altered in hyperplasia and neoplasia, as well as its regulation by hormones.
Abnormal recombination products result from aberrant DNA rearrangement of the human T-cell antigen receptor beta-chain gene.
Authors: Duby A D , Seidman J G
Published: May 2006

Two unusual rearrangements of the T-cell antigen receptor beta-chain gene have occurred in the human T-cell tumor line CEM. The beta chain of the T-cell antigen receptor is encoded in germ-line DNA by immunoglobulin-like gene segments that rearrange during the somatic development of T cells to form active genes. Structural analysis of rearranged immunoglobulin genes has already revealed a great deal about the mechanisms by which these genes rearrange. To further characterize the mechanism by which beta-chain genes rearrange, we have determined the organization of the rearranged beta-chain gene segments in the human T-cell tumor line CEM. Three rearranged joining (J) or diversity (D) segments of the beta-chain gene are found in CEM. One of these segments rearranged during the formation of a normal rearranged beta-chain gene that comprises a variable (V beta), D beta, and J beta gene segment associated with a constant region gene segment. Two abnormal recombination products are found at the other rearranged beta-chain locus. One product has the structure, J beta-D beta-J beta, with the J beta gene segments joined in a head-to-head fashion, while the other one consists of a V beta-D beta recombined segment not associated with a J beta gene segment. We propose that the J beta-D beta-J beta structure was formed by an inversion of 6 kilobases of DNA and subsequently, a V beta-D beta rearrangement occurred. The presence of these products in CEM has important implications for our understanding of the mechanism by which somatic rearrangements of beta-chain gene segments occur.
Mutations of the ras gene product p21 that abolish guanine nucleotide binding.
Authors: Clanton D J , Hattori S , Shih T Y
Published: May 2006

We have constructed several point mutations affecting the GTP-binding site of p21, the ras-encoded protein. Both lysine (116K) and tyrosine (116Y) mutations of asparagine-116, which, by analogy with the crystal structure of elongation factor Tu (EF-Tu), has critical interactions with the guanine base, abolish GTP binding and transforming activities of p21. These activities are retained by proteins with a mutation at position 117 or 118. Both 116K and 116Y mutant p21s, when overproduced in Escherichia coli, are apparently devoid of GTP-binding and autokinase activities. Similarly, the mutant DNAs do not transform NIH 3T3 cells in a focus-forming assay. By cotransfection with pSV-neo, cell clones resistant to the neomycin analog G418 have been isolated. Cells transfected with 116K or 116Y mutant DNA are contact inhibited. In contrast to competent clones, the defective mutants have no detectable phosphorylated p21. The present results suggest that the basic structure of the GTP-binding site is conserved between p21 and EF-Tu and that this binding site is crucial for ras gene function.
Structure of human steroid 21-hydroxylase genes.
Authors: White P C , New M I , Dupont B
Published: May 2006

We have determined the structure of cDNA and two genomic genes encoding steroid 21-hydroxylase [21-OHase; steroid 21-monooxygenase; steroid, hydrogen-donor:oxygen oxidoreductase (21-hydroxylating); EC 1.14.99.10]. If this cytochrome P-450 enzyme is defective, cortisol cannot be synthesized, resulting in congenital adrenal hyperplasia. The cDNA encoding this enzyme is 2.0 kilobases long, and the encoded protein is predicted to contain 494 amino acid residues with a molecular weight of 55,000. This enzyme is at most 28% homologous to other P-450 enzymes that have been studied. The 21-OHase genomic genes, which are located in the HLA major histocompatibility complex on chromosome 6, each contain 10 exons. This structure is distinct from other characterized P-450 genes, which contain 7 or 9 exons. Studies of individuals with homozygous deletions of the 21-OHase A or B genes suggest that only the B gene encodes an active enzyme. This is confirmed by the finding that the A gene has an 8-base deletion within codons 110-112, resulting in a frameshift that brings a stop codon into the reading frame at codon 130. A second frameshift and a nonsense mutation occur downstream. In contrast, the sequence of the exons of the B gene is identical to the cDNA sequence. The 21-OHase A gene is, therefore, a pseudogene.
Sequence homology requirements for intermolecular recombination in mammalian cells.
Authors: Ayares D , Chekuri L , Song K Y , Kucherlapati R
Published: May 2006

We have examined the homology requirements for intermolecular recombination between plasmids introduced into human, monkey, and bacterial cells. Variable-size-deletion derivatives of the prokaryotic-eukaryotic shuttle vector pSV2neo were constructed. Each of these plasmids was mixed with another pSV2neo plasmid containing a different, nonoverlapping deletion. Recombination was measured in mammalian cells and bacteria by the frequency of reconstruction of an intact neo gene. We observed that 25 base pairs of homologous sequence is sufficient to yield recombinant products, implying that synapsis and homologous pairing can occur with this level of homology. Examination of the products revealed that nonreciprocal recombination played a role in the generation of normal neo genes. In addition coconversion of linked markers was observed. Exonucleolytic action seems to play a role in gene conversion.
Induction of the Bacillus subtilis SOS-like response by Escherichia coli RecA protein.
Authors: Love P E , Yasbin R E
Published: May 2006

A plasmid that expresses the Escherichia coli RecA protein partially restored DNA repair and recombination capability and induction of the SOS-like (SOB) response in a recE4 mutant of Bacillus subtilis. In the presence of DNA-damaging agents, the E. coli RecA protein induced din operon expression, Weigle-reactivation activity, and synthesis of a B. subtilis recombination protein (Recbs) analogous to RecA but was unable to stimulate prophage induction. In addition, the RecA protein was capable of inducing the SOB response in competent recE4 strains of B. subtilis, independent of exposure to DNA-damaging agents. The results suggest that (i) the SOS response of E. coli and the SOB response of B. subtilis are strikingly similar from both a phenotypic and a regulatory standpoint and that RecA and LexA protein analogs exist in B. subtilis, (ii) the Recbs protein is capable of regulating its own production, and (iii) SOS-inducing (RecA-activating) signals are generated in B. subtilis following either DNA damage or the development of physiological competence.
Human chromosome 7 carries the beta 2 interferon gene.
Authors: Sehgal P B , Zilberstein A , Ruggieri R M , May L T , Ferguson-Smith A , Slate D L , Revel M , Ruddle F H
Published: May 2006

A cDNA clone (pAE20-4) corresponding to the 1.3-kilobase human beta 2 interferon mRNA was used as a probe in blot-hybridization experiments of DNA from a panel of human-rodent somatic cell hybrids containing overlapping subsets of human chromosomes. The DNA hybridization experiments showed that the human beta 2 interferon gene is located on human chromosome 7. This assignment is consistent with previous experimental data in which the expression of the translationally active 1.3-kilobase beta 2 interferon mRNA was assayed in various somatic cell hybrids. Blot-hybridization experiments using DNA from different human cell strains and cell lines reveal distinct EcoRI restriction fragment length polymorphisms of the human beta 2 interferon gene.
Interleukin 4 (B-cell growth factor II/eosinophil differentiation factor) is a mitogen and differentiation factor for preactivated murine B lymphocytes.
Authors: O'Garra A , Warren D J , Holman M , Popham A M , Sanderson C J , Klaus G G
Published: May 2006

Recently we described a murine T-cell hybrid that produces activities that promote the differentiation of eosinophils (eosinophil differentiation factor) and cause proliferation of the BCL1 B-cell lymphoma (B-cell growth factor II activity). Both activities appear to be associated with the same molecule, which has therefore been termed interleukin 4. The hybrid does not produce any other known lymphokines. We now find that purified interleukin 4 has no effects on small resting B cells but induces naturally occurring large B cells (which have presumably been preactivated in vivo) to synthesize DNA and to secrete IgM and low levels of IgG. B cells activated by anti-Ig antibodies apparently only become responsive to the factor once they have reached late G1 stage. All bioactivities of interleukin 4 are associated with a protein of Mr 44,000 (by NaDodSO4/PAGE). Therefore these results demonstrate that this lymphokine alone is sufficient to induce clonal expansion and maturation of activated B cells.
Molecular devices: Caroviologens as an approach to molecular wires—synthesis and incorporation into vesicle membranes
Authors: Arrhenius Thomas S. , Blanchard-Desce Mireille , Dvolaitzky Maya , Lehn Jean-Marie , Malthete Jacques
Published: May 2006

Molecular wires, which would allow electron flow to take place between different components, are important elements in the design of molecular devices. An approach to such species would be molecules possessing an electron-conducting conjugated chain, terminal electroactive polar groups, and a length sufficient to span a lipid membrane. To this end, bispyridinium polyenes of different lengths have been synthesized and their incorporation into the bilayer membrane of sodium dihexadecyl phosphate vesicles has been studied. Since they combine the features of carotenoids and of viologens, they may be termed caroviologens. Vesicles containing the caroviologen whose length approximately corresponds to the thickness of the sodium dihexadecyl phosphate bilayer display temperature-dependent changes of its absorption spectrum reflecting the gel → liquid-crystal phase transition of the membrane. The data agree with a structural model in which the caroviologens of sufficient length span the bilayer membrane, the pyridinium sites being close to the negatively charged outer and inner surfaces of the sodium dihexadecyl phosphate vesicles and the polyene chain crossing the lipidic interior of the membrane. These membranes may now be tested in processes in which the caroviologen would function as a continuous, transmembrane electron channel—i.e., as a molecular wire. Various further developments may be envisaged along these lines.
5-Azido-2'-deoxyuridine 5'-triphosphate: a photoaffinity-labeling reagent and tool for the enzymatic synthesis of photoactive DNA.
Authors: Evans R K , Johnson J D , Haley B E
Published: May 2006

We have synthesized the photoactive deoxyuridine nucleotide 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP) and used it to synthesize light-sensitive DNA by enzymatic incorporation. In the absence of ultraviolet light, 5-N3dUTP is a substrate for Escherichia coli DNA polymerase I. In in vitro DNA synthesis reactions using bacteriophage M13 single-stranded DNA as the template and 5-N3dUTP in place of dTTP, a photoactive complementary strand was synthesized by DNA polymerase I. The complementary strand was not synthesized when the 5-N3dUTP was substituted for dCTP or when it was exposed to ultraviolet light prior to the addition of DNA polymerase I. Using a synthetic lac operator template of 26 bases and a 15-base primer, we generated a photoactive 26-base-pair lac operator by enzymatically incorporating 5-N3dUMP with DNA polymerase I. Crosslinking of this photoactive DNA fragment to lac repressor was totally dependent on the presence of UV light and was reduced 78% by 150 microM isopropyl beta-D-thiogalactoside. Under the same conditions no crosslinking to lac repressor was observed using a nonphotoactive 26-base-pair lac operator. Photoactivatable deoxyuridine analogs have potential application as reagents to crosslink DNA binding proteins to 5-azidouracil-containing DNA and as active-site-directed photoaffinity labelling reagents.
Analysis of gamma delta resolvase mutants in vitro: evidence for an interaction between serine-10 of resolvase and site I of res.
Authors: Hatfull G F , Grindley N D
Published: May 2006

The resolvase encoded by the transposon gamma delta mediates a site-specific recombination between the two copies of gamma delta in a cointegrate molecule to yield the final products of transposition. Several mutants of resolvase that lack recombinational activity have been isolated previously, and one of these, which has a serine-to-leucine change at position 10, we have characterized in vitro. We also have constructed a serine-to-cysteine change at position 10 by in vitro mutagenesis and have analyzed this mutant protein in vitro. We find that the cysteine-10 mutant is defective in recombinational activity but binds to the recombinational site, res, similarly to wild-type, as assayed by gel electrophoresis of the protein-DNA complexes. In contrast, the leucine-10 mutant has a specific defect in complex formation with site I, which contains the recombinational crossover point, although it can bind and provide the ancillary functions of resolvase at sites II and III. It has been shown that a phosphoserine linkage is made to the DNA during recombination; because serine-10 is absolutely conserved amongst the family of homologous site-specific recombination proteins, it is a good candidate for the active-site serine. The properties of these resolvase mutants with substitutions at position 10 are consistent with this hypothesis and suggest that serine-10 is in close contact with the DNA at site I.