bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of a cDNA clone encoding the leader peptide of prion protein and expression of the homologous gene in various tissues.
Authors: Robakis N K , Sawh P R , Wolfe G C , Rubenstein R , Carp R I , Innis M A
Published: May 2006

We have isolated a hamster cDNA clone representing the coding sequences for the entire precursor of prion protein (PrP) 27-30. This clone encodes a protein of 254 residues and contains an in-frame ATG codon 42 bases upstream from the one previously reported. Analysis of the predicted amino acid sequence suggests that the PrP precursor protein contains an amino-terminal signal sequence, and a membrane-spanning domain in the carboxyl terminus. Cleavage of the signal peptide would produce a mature protein of 232 amino acids. Sequences homologous to the hamster PrP cDNA were detected in hamster, mouse, sheep, human, and rabbit genomes. A related 2.5-kilobase transcript was present in the brain of normal and scrapie-infected rodents. Two homologous transcripts, 2.5 and 1.1 kilobases, were detected in the lung and heart of normal animals. No PrP mRNA was detected in spleen stroma, a tissue known to contain high titers of scrapie. Antisera raised to the 27- to 30-kDa polypeptide detected the PrP in both normal and infected brains but failed to detect this protein in either normal or infected spleens. Homologous mRNA species were detected in human, sheep, and rabbit brain, even though the latter is resistant to scrapie infection. Our data suggest that PrP is not a necessary component of the infectious agent.
Primary photochemistry of iron-depleted and zinc-reconstituted reaction centers from
Rhodopseudomonas sphaeroides
Authors: Kirmaier C. , Holten D. , Debus R. J. , Feher G. , Okamura M. Y.
Published: May 2006

The primary photochemistry of Fe-depleted and Zn-reconstituted reaction centers from
Rhodopseudomonas sphaeroides
R-26.1 was studied by transient absorption spectroscopy and compared with native, Fe
2+
-containing reaction centers. Excitation of metal-free reaction centers with 30-ps flashes produced the initial charge-separated state P
+
I
-
(P
+
BPh
-
, where P is the primary donor and BPh is bacteriopheophytin) with a yield and visible/near-infrared absorption difference spectrum indistinguishable from that observed in native reaction centers. However, the lifetime of P
+
I
-
was found to increase approximately 20-fold to 4.2 ± 0.3 ns (compared to 205 ps in native reaction centers), and the yield of formation of the subsequent state P
+
Q
A
-
(Q
A
is the primary quinone acceptor) was reduced to 47 ± 5% (compared to essentially 100% in native reaction centers). The remaining 53% of the metal-free reaction centers were found to undergo charge recombination during the P
+
I
-
lifetime to yield both the ground state (28 ± 5%) and the triplet state P
R
(25 ± 5%). Reconstitution of Fe-depleted reaction centers with Zn
2+
restored the “native” photochemistry. Possible mechanisms responsible for the reduced decay rate of P
+
I
-
in metal-free reaction centers are discussed.
Successful rescue of microsurgically produced homozygous uniparental mouse embryos via production of aggregation chimeras.
Authors: Anderegg C , Markert C L
Published: May 2006

Homozygous uniparental mouse embryos, produced by microsurgical removal of the male pronucleus from fertilized eggs and diploidization of the female pronucleus with cytochalasin, were surrounded with blastomeres from normal embryos to produce chimeric embryos. A few of these chimeras developed into viable adults, and one female has reproduced using her homozygous uniparental cells as a source of gametes. The production and use of such chimeras in breeding programs could greatly shorten the period required for producing inbred strains of mammals. The data presented demonstrate that a homozygous uniparental mammalian genome, although not lethal to all cells, is extremely deleterious to normal embryonic development. Moreover, the results support the conclusion that the genome is imprinted differently in males and females during gametogenesis so that at fertilization the genomes are not functionally equivalent, and both are required for normal development.
Human thymocytes bind to autologous and allogeneic thymic epithelial cells in vitro.
Authors: Singer K H , Wolf L S , Lobach D F , Denning S M , Tuck D T , Robertson A L , Haynes B F
Published: May 2006

The thymus plays a critical role in the generation of immunocompetent T lymphocytes. In the thymus, lymphocytes are in close contact with epithelial cells, and this contact is necessary for T-cell maturation. Using cultured human thymic epithelial (TE) cells, we have found that human thymocytes bind to human TE cells in vitro. Thymocytes bound to both allogeneic and autologous TE cells and to the epidermoid carcinoma cell line A431 but did not bind to epidermal keratinocytes or to thymic fibroblasts. Thymocyte binding to TE cells was trypsin- and cytochalasin B-sensitive. Indirect immunofluorescence assays showed that both mature (T6-, T3+) and immature (T6+, T3-) thymocytes bound TE cells. In our system, TE-thymocyte binding was not inhibited by antibodies to class I or class II major histocompatibility antigens. In vitro binding of thymocytes to TE cells may represent a correlate of in vivo TE-thymocyte interactions and provides a model system for the study of human intrathymic T-lymphocyte maturation and activation.
Effects of reagent and cell-generated hydrogen peroxide on the properties of low density lipoprotein.
Authors: Montgomery R R , Nathan C F , Cohn Z A
Published: May 2006

Low density lipoprotein (LDL) isolated from human plasma anticoagulated with EDTA (EDTA/LDL) was 4-fold more resistant to oxidation by reagent H2O2, as assayed by the thiobarbituric acid (TBA) assay, than LDL prepared from plasma anticoagulated with citrate (CDP/LDL). The LDLs required 1-3 mM H2O2 for maximal oxidation by this assay, and ED50S were 1.7 X 10(-3) M for EDTA/LDL and 4.5 X 10(-4) M for CDP/LDL. Oxidation was enhanced 2.3-fold by Cu2+ ions. Rabbit endothelial cell line monolayers released two orders of magnitude less H2O2 than was required to oxidize LDL and failed to induce TBA reactivity in either EDTA/LDL or CDP/LDL after a 24-hr coincubation. However, this LDL was subsequently degraded by mouse macrophages more rapidly than untreated LDL. Freshly isolated human monocytes (2 X 10(6) cells per ml), with or without phorbol myristate acetate (100 ng/ml) to trigger the respiratory burst, did not oxidize LDL in the TBA assay, despite producing large amounts of reactive oxygen intermediates. EDTA/LDL, CDP/LDL, and acetoacetylated LDL failed to trigger H2O2 release from human monocytes or macrophages. These results separate oxidation of LDL as measured by TBA assay from the modification of LDL by rabbit aortic endothelial cell line that leads to its subsequent enhanced degradation by macrophages.
Identification of a cyclic-AMP-responsive element within the rat somatostatin gene.
Authors: Montminy M R , Sevarino K A , Wagner J A , Mandel G , Goodman R H
Published: May 2006

We have examined the regulation of somatostatin gene expression by cAMP in PC12 rat pheochromocytoma cells transfected with the rat somatostatin gene. Forskolin at 10 microM caused a 4-fold increase in somatostatin mRNA levels within 4 hr of treatment in stably transfected cells. Chimeric genes containing the somatostatin gene promoter fused to the bacterial reporter gene encoding chloramphenicol acetyltransferase were also induced by cAMP in PC12 cells. To delineate the sequences required for response to cAMP, we constructed a series of promoter deletion mutants. Our studies defined a region between 60 and 29 base pairs upstream from the transcriptional initiation site that conferred cAMP responsiveness when placed adjacent to the simian virus 40 promoter. Within the cAMP-responsive element of the somatostatin gene, we observed an 8-base palindrome, 5'-TGACGTCA-3', which is highly conserved in many other genes whose expression is regulated by cAMP. cAMP responsiveness was greatly reduced when the somatostatin fusion genes were transfected into the mutant PC12 line A126-1B2, which is deficient in cAMP-dependent protein kinase 2. Our studies indicate that transcriptional regulation of the somatostatin gene by cAMP requires protein kinase 2 activity and may depend upon a highly conserved promoter element.
Production of immunoreactive chorionic gonadotropin during mixed lymphocyte reactions: a possible selective mechanism for genetic diversity.
Authors: Harbour-McMenamin D , Smith E M , Blalock J E
Published: May 2006

Maternal immunologic recognition of the blastocyst as well as the hormone chorionic gonadotropin (CG) has been shown to be a requirement for successful implantation of the blastocyst and maintenance of the fetal graft. On the basis of these observations, we tested whether lymphocytes could produce chorionic gonadotropic-like hormones during a mixed lymphocyte reaction (MLR). Here we described the production of immunoreactive chorionic gonadotropin (ir-CG) as a result of an allogeneic stimulus but not other mitogenic stimuli. The kinetics of production, as monitored by immunofluorescence with antibody specific to the beta chain of the human CG (hCG) molecule, paralleled the blastogenic response. Gel filtration of the de novo-synthesized ir-CG showed the molecular weight of the intact molecule to be approximately 58,000. This molecule bound to a concanavalin A affinity column and also dissociated into two subunits of approximate molecular weights 32,000 and 18,000. These molecular weights are consistent with reported molecular weights for glycosylated intact hCG, as well as the beta and alpha chains of hCG, respectively. The material from the MLR but not the control cultures elicited significant testosterone production from Leydig cells, and this activity could be blocked by antiserum to hCG. The results described here, together with the fact that the blastocyst is implanted in a lymphocyte infiltrate, lend support to our postulate that lymphocyte-derived CG enhances successful implantation of the allogeneic fetus, thus providing a mechanism for selection of genetic diversity.
Intense natural selection caused a rapid morphological transition in a living marine snail
Authors: Seeley Robin Hadlock
Published: May 2006

Shell shape and shell thickness of the intertidal snail
Littorina obtusata
changed markedly between 1871 and 1984 in northern New England. Shells collected prior to 1900 were high-spired with thin walls, whereas shells collected in 1982-84 were low-spired with thick walls. An intertidal crab (
Carcinus maenas
) which preys on
L. obtusata
expanded its range into northern New England around 1900. This suggests that the change in snail shell form was a response to predation by
Carcinus
. Field and laboratory experiments demonstrated that the high-spired form of
L. obtusata
, which can still be found in some Maine localities, is more vulnerable to predation by
Carcinus
than is the low-spired form of
L. obtusata
. Electrophoretic comparisons of high- and low-spired populations of
L. obtusata
confirmed that these populations represent different morphological forms of
L. obtusata
rather than different species [Nei's
D
(unbiased measure of genetic distance) = 0.003]. These data demonstrate that classical Darwinian selection can produce a rapid morphological transition without speciation.
Expression of the adenovirus E1A oncogene during cell transformation is sufficient to induce susceptibility to lysis by host inflammatory cells.
Authors: Cook J L , Walker T A , Lewis A M , Ruley H E , Graham F L , Pilder S H
Published: May 2006

Mammalian cells transformed by nononcogenic human adenoviruses exhibit high susceptibility to destruction by host mononuclear inflammatory cells. We have analyzed the viral gene regulation of the susceptibility of transformed cells to lysis by natural killer cells and activated macrophages. Comparisons of target cell lines transformed by overlapping segments of the adenovirus E1-transforming gene region revealed that isolated expression of a single oncogene, E1A, was sufficient to cause increased cytolytic susceptibility in the absence of detectable transformed cell-surface expression of viral transplantation antigens and irrespective of histocompatibility antigen identity between killer cells and target cells. These results suggest that oncogene functions that are not linked to the expression of previously recognized cell-surface target structures may actively induce neoplastic cell elimination by components of the host immune surveillance system.
Negative trans-regulation of T-cell antigen receptor/T3 complex mRNA expression in murine T-lymphoma somatic cell hybrids.
Authors: MacLeod C L , Minning L , Gold D P , Terhorst C , Wilkinson M
Published: May 2006

The antigen-specific T-cell receptor (TCR) is composed of variable antigen-recognition chains TCR-alpha and TCR-beta in noncovalent association with the invariant T3 multimer. The TCR-alpha and TCR-beta chains are encoded by gene segments that must be juxtaposed by rearrangement in order to be expressed. To examine whether mechanisms other than gene rearrangement might regulate TCR/T3 gene expression, somatic cell hybrids were formed among closely related murine SL12 T-lymphoma clones that differ in TCR/T3 mRNA levels. In hybrid cells formed between cell clones in which one parent is TCR-beta+ and the other is TCR-beta-, the resultant hybrid cells lack detectable TCR-beta transcripts. Since the protein synthesis inhibitor cycloheximide partially reverses TCR-beta repression in the hybrid cells, we postulate that a labile repressor protein is involved. The amount of mRNA encoding one of the T3 polypeptide chains, T3-delta, is also strongly negatively transregulated in the same hybrid cells in which TCR-beta mRNA expression is repressed. The negative trans-regulation of TCR-beta and T3-delta mRNA expression is relatively specific, since the levels of TCR-alpha mRNA and several thymocyte surface antigens are not repressed in somatic cell hybrids. Our results indicate that rearrangement of the TCR genes alone is not sufficient for TCR-beta expression and that trans-acting factors regulate the amounts of both TCR-beta and T3-delta mRNA in this system.
Selective ablation of atheromas using a flashlamp-excited dye laser at 465 nm.
Authors: Prince M R , Deutsch T F , Shapiro A H , Margolis R J , Oseroff A R , Fallon J T , Parrish J A , Anderson R R
Published: May 2006

Ablation of human atheromas with laser pulses that had only a small effect on normal artery tissue was shown in vitro in air and under saline using 1-mu sec pulses at 465 nm from a flashlamp-excited dye laser. At this wavelength, there is preferential absorption in atheromas due to carotenoids. The threshold fluence for ablation was 6.8 +/- 2.0 J/cm2 for atheromas and 15.9 +/- 2.2 J/cm2 for normal aorta tissue. At a fluence of 18 J/cm2 per pulse, the ablated mass per unit of energy ranged from 161 to 370 micrograms/J for atheromas and from 50 to 74 micrograms/J for normal aorta tissue. Ablation products consisted of cholesterol crystals, shredded collagen fibers, and small bits of calcific material. Most debris was less than 100 micron in diameter, but a few pieces were as large as 300 micron. High-speed photography of ablation in air suggested explosive ejection of debris, caused by vapor formation, at speeds on the scale of 300 m/sec. Histological analysis showed minimal thermal damage to residual tissue. These data indicate that selective laser ablation of atheromas is possible in vitro.
Structure and gene organization of bovine neuromedin K precursor.
Authors: Kotani H , Hoshimaru M , Nawa H , Nakanishi S
Published: May 2006

cDNA and genomic DNA clones for the precursor of a mammalian neuropeptide tachykinin, neuromedin K, have been isolated and characterized by molecular cloning and sequence analysis. The deduced amino acid sequence indicates that the bovine neuromedin K precursor (preprotachykinin B) consists of 126 amino acid residues including a putative signal peptide. There are two preprotachykinin B mRNAs that differ only at the 5' extremity of the untranslated regions. The major mRNA species is encoded by seven exon sequences, while the minor species includes two extra 5' exon sequences and lacks the 5' terminus of the first exon sequence for the major mRNA species. The above gene organization for the major preprotachykinin B mRNA closely resembles that for the preprotachykinin A mRNA encoding the precursor for substance P and substance K. This structural resemblance strongly suggests that the two preprotachykinin genes have evolved from a common ancestor gene. Furthermore, we have found that the preprotachykinin A and B mRNAs markedly differ in their major expression sites. The results described thus indicate that the diversity of the mammalian tachykinin system has been acquired through various cellular mechanisms including gene duplication and differential expression of duplicated genes.
Nuclease activity of 1,10-phenanthroline-copper: sequence-specific targeting.
Authors: Chen C H , Sigman D S
Published: May 2006

The nuclease activity of 1,10-phenanthroline-copper ion can be targeted to specific DNA sequences by attachment of the ligand to the 5' end of complementary deoxyoligonucleotides via a phosphoramidate linkage. To synthesize the adduct, the phosphorimidazolide of the deoxyoligonucleotide is prepared using a water-soluble carbodiimide and is then coupled to 5-glycylamido-1,10-phenanthroline. After hybridization to the target DNA, sequence-specific cleavage is observed upon the addition of cupric ion and 3-mercaptopropionic acid. Two methods of assaying the cutting of the operator sequence of the lac operon have been employed using the oligonucleotide 5'-AATTGTTATCCGCTCACAATT-3' representing sequence positions 21-1 of the template strand. In the first, the single-stranded DNA of the phage M13mp8 was the target, and cuts were detected using a primer-extension assay. In the second, the substrate was an EcoRI fragment 3' labeled in the nontemplate strand. After denaturation and reannealing to the oligonucleotide-1,10-phenanthroline adduct, cupric ion and 3-mercaptopropionic acid were added, and the products were analyzed directly on a sequencing gel. With the phenanthroline moiety attached to position 21 of the oligonucleotide carrier, cutting was observed at positions 20-25 using both assays.
Common and distinct tubulin binding sites for microtubule-associated proteins.
Authors: Littauer U Z , Giveon D , Thierauf M , Ginzburg I , Ponstingl H
Published: May 2006

A specific binding assay was developed that monitors the interaction of 125I-labeled microtubule-associated proteins (MAPs) with tubulin or its fragments bound to nitrocellulose membrane. To identify the tubulin-binding domains for MAPs we have examined the binding of rat brain 125I-labeled MAP2 or 125I-labeled tau factors to 60 peptides derived from porcine alpha- and beta-tubulin. MAP2 and tau factors specifically interacted with two peptides derived from the carboxyl-terminal region of beta-tubulin, which are located between positions 392-445 and 416-445. In addition, there is a distinct tau-binding site at the amino-terminal region of alpha-tubulin. tau factors but not MAP2 displayed strong interaction with a peptide derived from the amino-terminal domain of alpha-tubulin between positions 1 and 75. To narrow down the location of the beta-tubulin binding site that is common to MAP2 and tau factors, we have synthesized five peptides that are homologous to the corresponding sequence from the porcine or rat carboxyl-terminal region. Binding studies with the synthetic peptides suggest that amino acid residues 434-440 of beta-tubulin are crucial for the interaction of MAP2 and tau factors.
Structural evidence that endothelial cell growth factor beta is the precursor of both endothelial cell growth factor alpha and acidic fibroblast growth factor.
Authors: Burgess W H , Mehlman T , Marshak D R , Fraser B A , Maciag T
Published: May 2006

Two endothelial cell growth factors (ECGF) have been purified from bovine brain and termed alpha- and beta-ECGF [Burgess, W. H., Mehlman, T., Friesel, R., Johnson, W. V. & Maciag, T. (1985) J. Biol. Chem. 260, 11389-11392]. Amino acid sequence analysis indicates that beta-ECGF represents a 20 amino acid amino-terminal extension of alpha-ECGF and a 14 amino acid amino-terminal extension of acidic fibroblast growth factor. These data indicate that both alpha-ECGF and acidic fibroblast growth factor may be derived from beta-ECGF by posttranslational processing. Analysis of the amino-terminal 14 residues of beta-ECGF by fast-atom-bombardment mass spectrometry established the amino acid sequence of this region and the identity of the blocking group at the amino terminus (acetyl).
Monte Carlo simulations on an equilibrium globular protein folding model.
Authors: Kolinski A , Skolnick J , Yaris R
Published: May 2006

Monte Carlo simulations were performed on a diamond lattice, globular protein model in which the trans conformational state is energetically favored over the gauche states (thereby perhaps favoring a beta-sheet secondary structure) and in which nonspecific nonbonded nearest-neighbor attractive interactions are allowed. If the attractive interactions are sufficiently weak that the molecule possesses a relatively high fraction of trans states in the denatured state, then on collapse, a beta-barrel tertiary structure, highly reminiscent of the "native" structure seen in beta-proteins, spontaneously forms. If, however, the attractive interactions are dominant, a coil-to-random globule collapse transition is observed. The roles of short-, medium-, and long-range interactions and topological constraints in determining the observed tertiary structure are addressed, and the implications and limitations of the simulations for the equilibrium folding process in renal globular proteins are explored.
Regulation of melanoma by the embryonic skin.
Authors: Gerschenson M , Graves K , Carson S D , Wells R S , Pierce G B
Published: May 2006

This report focuses on the regulation of murine melanoma by the embryonic skin. A surgical technique was developed to allow injection of B16 melanoma cells into the embryo in utero. A significant decrease in incidence of tumors was noted, which correlated with the time of arrival of normally migrating premelanocytes into the skin. Media were conditioned from skin explanted at the time premelanocytes arrive in it; these media inhibited the growth of melanoma cells in vitro. Under optimal conditions the growth of melanoma cells ceased; the cells had altered morphology and failed to proliferate when placed in fresh growth media.
Identification and chemical synthesis of a ribosomal protein antigenic determinant in systemic lupus erythematosus.
Authors: Elkon K , Skelly S , Parnassa A , Moller W , Danho W , Weissbach H , Brot N
Published: May 2006

The characteristics of eukaryotic ribosomal proteins P0, P1, and P2 (P proteins) and their antigenic determinants were studied using the sera of patients with systemic lupus erythematosus (SLE). P0, P1, and P2 were isolated as a macromolecular complex by preparative isoelectric focusing and anion-exchange chromatography in the presence of 6 M urea. The apparent molecular size of the complex was 140 kDa as determined by gel filtration on a Sephadex G-200 column. P0 may, therefore, be the eukaryotic equivalent of Escherichia coli ribosomal protein L10. In addition, all three P proteins were detected in the postribosomal supernatant of HeLa cells, and P0 and P1 were found to be more acidic than their ribosome-bound counterparts. Partial proteolysis experiments revealed that SLE anti-P sera recognized one or both ends of the P2 equivalent protein from Artemia salina (eL12). Sixteen SLE sera containing antibodies to P0, P1, and P2 reacted with a carboxyl-terminal peptide 22 amino acids in length of eL12 and not with an amino-terminal peptide of 20 amino acids. Even though the carboxyl-terminal peptide completely inhibited the ability of the antiserum to react with all three proteins on an immunological blot, the same peptide produced only small decreases in binding of the SLE antibody to the native, nondenatured P proteins. These findings indicate that SLE anti-P antibodies react with a single sequential (linear) antigenic determinant on all three P proteins, but that additional antibodies recognize a conformational determinant(s).
A labile inhibitor blocks immunoglobulin kappa-light-chain-gene transcription in a pre-B leukemic cell line.
Authors: Wall R , Briskin M , Carter C , Govan H , Taylor A , Kincade P
Published: May 2006

The murine pre-B leukemic cell line 70Z/3 contains both an unrearranged immunoglobulin kappa-light-chain gene and a functionally rearranged but silent kappa-light-chain gene. Mitogenic stimulation of growing 70Z/3 cells with bacterial lipopolysaccharide (LPS) activates kappa-light-gene transcription and results in a 10- to 20-fold increase in cytoplasmic kappa-light-chain mRNA. The induction of kappa gene expression by LPS was probed by using an inhibitor of protein synthesis. Concomitant treatment of 70Z/3 cells with LPS and cycloheximide failed to block kappa-light-chain mRNA accumulation, indicating that new protein synthesis is not required for the activation of kappa-gene expression. Treatment of 70Z/3 cells with cycloheximide alone resulted in kappa-mRNA induction equivalent to those produced by LPS alone. The kappa mRNA synthesized in the presence of cycloheximide was intact and able to direct the synthesis of kappa light chains. Nuclear transcription assays revealed that cycloheximide, like LPS, activated kappa-gene transcription. These findings indicate that the trans-acting factors necessary for kappa-light-chain-gene transcription are present in pre-B cells, but their activity is blocked by short-lived inhibitory proteins.
Different types of Ca2+ channels in mammalian skeletal muscle cells in culture.
Authors: Cognard C , Lazdunski M , Romey G
Published: May 2006

This paper describes the existence of two pharmacologically distinct types of Ca2+ channels in rat skeletal muscle cells (myoballs) in culture. The first class of Ca2+ channels is insensitive to the dihydropyridine (DHP) (+)-PN 200-110; the second class of Ca2+ channels is blocked by low concentrations of (+)-PN 200-110. The two pharmacologically different Ca2+ channels are also different in their voltage and time dependence. The threshold for activation of the DHP-insensitive Ca2+ channel is near -65 mV, whereas the threshold for activation of the DHP-sensitive Ca2+ channel is near -30 mV. Current flowing through the DHP-insensitive Ca2+ channel is transient with relatively fast kinetics. Half-maximal inactivation for the DHP-insensitive Ca2+ channel is observed at a holding potential Vh0.5 = -78 mV and the channel is completely inactivated at -60 mV. Two different behaviors have been found for DHP-sensitive channels with two different kinetics of inactivation (one being about 16 times faster than the other at -2 mV) and two different voltage dependencies. These two different behaviors are often observed in the same myoball and may correspond to two different subtypes of DHP-sensitive Ca2+ channels or to two different modes of expression of one single Ca2+ channel protein.
Nuclear magnetic resonance study of the globular domain of chicken histone H5: resonance assignment and secondary structure.
Authors: Zarbock J , Clore G M , Gronenborn A M
Published: May 2006

A 1H NMR study of the 79-residue globular domain of chicken erythrocyte histone H5 (GH5) is presented. Using a combination of two-dimensional NMR techniques to demonstrate through-bond and through-space (less than 5 A) connectivities, the resonances of GH5 are assigned in a sequential manner. From a qualitative interpretation of the short-range nuclear Overhauser effects (NOEs) involving the NH and C alpha H protons, it is shown that GH5 has four alpha-helices. The approximate spatial relationship of three of these four helices relative to each other is deduced from the observation of a number of long-range NOEs. The peptide chain outside the helices appears to have little regular secondary structure and no NOEs characteristic of beta-sheets are apparent.
"Replacement" of COOH-terminal truncation of v-fms with c-fms sequences markedly reduces transformation potential.
Authors: Browning P J , Bunn H F , Cline A , Shuman M , Nienhuis A W
Published: May 2006

Protooncogenes when transduced by retroviruses may undergo structural modifications that render their gene products oncogenic. The c-fms gene encodes a transmembrane protein with tyrosine kinase activity that is very similar or identical to the receptor for the monocyte-macrophage colony-stimulating factor. Its transforming homologue (v-fms) in the Susan McDonough strain feline sarcoma virus causes fibrosarcomas in cats. Molecular cloning and sequence analysis of the cDNA that encodes the cytoplasmic domain of the human c-fms gene shows that the product of the transduced viral homologue, v-fms, is truncated at the COOH-terminal end. The COOH-terminal 40 amino acids of the c-fms gene product are replaced in the v-fms gene product by 11 amino acids encoded by the retroviral genome. Hybrid v-fms/c-fms genes, in which either the entire cytoplasmic domain or the COOH-terminal coding sequences of the v-fms gene were replaced by the corresponding segments of the c-fms gene, had a reduced ability to transform fibroblasts despite a high level of encoded protein on the cell surface. These data indicate that the COOH-terminal modifications contribute to the transforming potential of the v-fms viral oncogene product.
Functional antibody lacking a variable-region disulfide bridge.
Authors: Rudikoff S , Pumphrey J G
Published: May 2006

In 1981, Auffray et al. [Auffray, C., Sikorav, J. L., Ollo, R. & Rougeon, F. (1981) Ann. Immunol. (Inst. Pasteur) 132D, 77-88] reported a partial cDNA sequence of the heavy chain from the ABPC48 plasmacytoma whose protein product had previously been shown to bind bacterial and grass levan. In the cDNA sequence the second half-cystine of the heretofore invariant disulfide bridge had been replaced by a tyrosine. Since the presence of invariant variable-region disulfide bridges has been considered a basic structural feature of the antibody molecule necessary for proper folding and function, we have analyzed the heavy chain protein produced by ABPC48. Our results indicate that heavy chains from ABPC48 quantitatively express tyrosine in place of the normally occurring second half-cystine in the variable region. Furthermore, this antibody population is capable of both binding antigen and subsequent precipitation. Thus, the presence of a disulfide bridge in the heavy-chain variable region does not appear necessary for proper function of this antibody and may not be obligatory for antibody function in general, as has been assumed previously.
Immune responses to H-2Kd antigen expressed by recombinant vaccinia virus.
Authors: Coupar B E , Andrew M E , Boyle D B , Blanden R V
Published: May 2006

A recombinant vaccinia virus (VV-H2Kd-6) containing the coding sequence for the murine major histocompatibility complex class I antigen H-2Kd has been constructed and used to express H-2Kd on the surface of infected cells. Vaccinia expressed H-2Kd has been shown to generate an H-2Kd-specific primary cytotoxic T-cell response in mice infected with the recombinant virus and to stimulate an H-2Kd-specific cytotoxic T-cell response in vitro. Cells infected with the recombinant virus acted as targets for specific lysis by appropriate alloreactive cytotoxic T cells, albeit relatively inefficiently when compared with alloreactive recognition and specific lysis of H-2Kd-containing P815 cells. However, H-2Kd expressed by the recombinant virus was recognized efficiently as a restricting element in association with vaccinia virus antigens, while lysis of VV-H2Kd-6-infected L929 (H-2k) cells by CBA/H (H-2k) anti-C3H.OH (H-2KdDk) cytotoxic T cells was comparatively weak. These data suggest that there are quantitative or qualitative differences, or both, between H-2Kd expressed by vaccinia virus and cells of the H-2d haplotype. Qualitative differences have not been demonstrated but cannot be excluded.
Polymerization of intact beta 2-microglobulin in tissue causes amyloidosis in patients on chronic hemodialysis.
Authors: Gorevic P D , Munoz P C , Casey T T , DiRaimondo C R , Stone W J , Prelli F C , Rodrigues M M , Poulik M D , Frangione B
Published: May 2006

Systemic amyloidosis with a predilection for bone and synovium may complicate the course of patients on long-term hemodialysis. This form of amyloidosis can be typed as distinct from other amyloid diseases by using small tissue samples obtained by bone biopsy and at postmortem. Immunoblot analysis of two-dimensional gels of partially solubilized amyloid fibrils established that tissue deposits are composed of monomers, dimers, and higher polymers of beta 2-microglobulin (beta 2m) and that amyloid P component was also present. Anti-beta 2m antiserum recognized fibrils, as shown by immunoelectron microscopy. Purified monomer isolated from dissociated fibrils yielded peptides corresponding to the entire known sequence of beta 2m. Virtually all serum beta 2m, as well as that present in tissue fluid bathing amyloid fibrils, was monomeric. Hemodialysis-related amyloidosis is an example of a deposition disease occurring in hemodialysis patients. We have shown conclusively that, in this amyloid disease, polymerization of an intact normal serum protein to a fibrillar configuration may occur without proteolysis. We propose the designation A beta 2m for this form of amyloid fibril subunit protein.
Evolutionary progression at synaptic connections made by identified homologous neurones.
Authors: Shaw S R , Meinertzhagen I A
Published: May 2006

A comparative ultrastructural study of photoreceptor synapses formed upon homologous postsynaptic neurones in insects has been made by using serial-section electron microscopy in representative Diptera from a monophyletic series of 14 families. At all of the synaptic contacts there is a presynaptic dense bar, surmounted in phylogenetically more recent families by a presynaptic platform. Opposite the bar lies a pair of postsynaptic elements that invariably originate one each from two unique monopolar neurones L1 and L2. Both elements contain increasingly elaborate cisternae in more recent flies. Within the phylogenetic series, the postsynaptic ensemble itself changes from the original dyad to a tetradic configuration in more recent Muscomorpha by the addition of two new postsynaptic elements from an amacrine cell. This transition occurs once only in the series, which, gauged by the fossil record, covers divergences from the stem line extending back greater than 200 million years.
Effects of forelimb amputation and denervation on protein synthesis in spinal cord ganglia of the newt.
Authors: Bao C Y , Singer M , Ilan J
Published: May 2006

Analysis of the effects of forelimb amputation and denervation on [35S]methionine incorporation into the protein of newt brachial plexus nerve ganglia showed that amputation increases the level of protein synthesis about 4-fold as compared with that of control (nonamputated) animals. Denervation without amputation nearly doubles the level of protein synthesis as compared with controls. Analysis of labeled protein by means of two-dimensional gel electrophoresis and radioautogram revealed incorporation patterns that are similar to controls; this observation suggests that amputation affects nerve cells quantitatively rather than qualitatively, in influencing the production of putative neurotrophic agents. A group of basic proteins ranging in Mr from 15,000 to 31,000 are prominently expressed in these radioautograms from experimental ganglia and may be important in promoting forelimb regeneration in the newt.
Isolation and sequence determination of a cDNA clone related to human cytochrome P-450 nifedipine oxidase.
Authors: Beaune P H , Umbenhauer D R , Bork R W , Lloyd R S , Guengerich F P
Published: May 2006

Human liver cytochrome P-450NF is the form of cytochrome P-450 responsible for the oxidation of the calcium-channel blocker nifedipine, which has been reported to show polymorphism in clinical studies. By screening a bacteriophage lambda gt11 expression cDNA library, we isolated two clones: NF95 with an insert length of 0.8 kilobases which gave a stable fusion protein and NF25 with an insert length of 2.2 kilobases. The two clones were both sequenced and shown to be identical in their overlapping section. The sequence of NF25 is 77% similar to that reported for a rat cytochrome "P-450PCN" cDNA (PCN = pregnenolone-16 alpha-carbonitrile). The similarity decreases to 45-53% when the sequence is compared to human cytochromes P-450 belonging to other families [i.e., "pH P-450(1)," "P1-450," "P3-450," and "P-450MP." The deduced amino acid sequence is 73% similar to that of rat cytochrome P-450PCN, and the first 21 amino acids are identical to those reported for human liver cytochrome "P-450p." Sections of these clones were nick-translated and used as probes for analyses of human mRNA and genomic DNA. The number and size of bands indicate that P-450NF belongs to a multigene family, the so-called pregnenolone-16 alpha-carbonitrile-inducible family.
Type beta transforming growth factor is an inhibitor of myogenic differentiation.
Authors: Massagué J , Cheifetz S , Endo T , Nadal-Ginard B
Published: May 2006

We have investigated the effect of type beta transforming growth factor (TGF-beta) on the differentiation of skeletal muscle myoblasts. TGF-beta potently (ID50 approximately 10 pM) prevents established cell lines and primary cultures of rat and chicken embryo myoblasts from fusing into multinucleated myotubes. Inhibition of morphological differentiation by TGF-beta correlates with inhibition of the expression of muscle-specific mRNAs and proteins, strong induction of extracellular matrix type I collagen and fibronectin, and a marked tendency of the treated myoblasts to aggregate into densely multilayered arrays or clusters. Myogenic differentiation can resume after removal of TGF-beta from the medium. Examination of the time of action of TGF-beta shows that myoblasts stochastically reach a point beyond which they become insensitive to the inhibitory action of TGF-beta. This resistance of committed myoblasts to the inhibitory action of TGF-beta is not associated with any measurable change in the number or affinity of TGF-beta receptors in those cells. The results indicate that TGF-beta is a potent inhibitor of myogenesis and may regulate muscle development in vivo.
Soybean seed lectin gene and flanking nonseed protein genes are developmentally regulated in transformed tobacco plants.
Authors: Okamuro J K , Jofuku K D , Goldberg R B
Published: May 2006

We introduced a 17.1-kilobase soybean DNA fragment containing the lectin gene and at least four nonseed protein genes into the tobacco genome. As in soybean plants, lectin mRNA is present in tobacco seeds, accumulates and decays during tobacco seed development, and is translated into a protein that accumulates prior to dormancy. Each soybean nonseed protein mRNA is present in tobacco leaves, roots, stems, and seeds at levels similar to that found in soybean plants. We conclude that a differentially expressed soybean gene cluster is correctly regulated in transformed tobacco plants and that sequences controlling their expression are recognized by regulatory factors present in tobacco cells.
Analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.
Authors: Clarke L , Amstutz H , Fishel B , Carbon J
Published: May 2006

The Schizosaccharomyces pombe centromere-linked genes, LYS1 and CYH1 on chromosome I and TPS13 and RAN1 on chromosome II, have been isolated. The genetic order of these markers with respect to their centromeres was determined to establish relative directionality on the genetic and physical maps. Chromosome walking toward the centromeres reveals a group of repetitive sequences that occur only in the centromere regions of chromosomes I and II and at one other specific location in the S. pombe genome, presumably the centromere of chromosome III. The major class of large repeated sequence elements is 6.4 kilobases (kb) long (repeat K), portions of which occur at least twice on chromosome II and in several tandemly arranged intact copies at another centromeric location. Repeat K in turn contains groups of smaller repeats. Genetic recombination is strongly suppressed in the centromere II region, which contains at least 30 kb of repeated sequences. Centromeric DNA organization is much more complex in fission yeast than has been described in budding yeast (Saccharomyces cerevisiae), possibly because of the larger more condensed nature of the S. pombe chromosomes.
Mevinolin, an inhibitor of cholesterol synthesis, induces mRNA for low density lipoprotein receptor in livers of hamsters and rabbits.
Authors: Ma P T , Gil G , Südhof T C , Bilheimer D W , Goldstein J L , Brown M S
Published: May 2006

Through the use of a quantitative solution hybridization assay with 32P-labeled cDNA probes, we found that mevinolin, an inhibitor of cholesterol synthesis, elevates the level of mRNA for the low density lipoprotein receptor in livers of hamsters and rabbits. In hamsters the maximal effect (3-fold increase) occurred at 0.1% mevinolin in the diet for 10 days. The same dose produced a maximal induction (10-fold) of mRNA levels for 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme of cholesterol synthesis, and a maximal decrease (80%) in plasma cholesterol. The drug lowered the level of all cholesterol-carrying lipoproteins in plasma. In normal rabbits, mevinolin produced a 90% reduction in plasma low density lipoprotein-cholesterol levels, which was associated with a 2.5-fold increase in low density lipoprotein receptor mRNA levels. A similar induction of receptor mRNA occurred in livers of Watanabe-heritable hyperlipidemic rabbits, although the plasma cholesterol was not reduced to normal, presumably because the receptors produced by the mutant mRNA function poorly. These data are consistent with the hypothesis that mevinolin and other inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase lower plasma cholesterol levels in part by stimulating production of mRNA for the low density lipoprotein receptor in liver.
"Footprint" titrations yield valid thermodynamic isotherms.
Authors: Brenowitz M , Senear D F , Shea M A , Ackers G K
Published: May 2006

A central issue in gene regulation is the mechanism, and biological function, of the cooperative binding of regulatory protein ligands to specific sites on DNA. To elucidate the physical-chemical basis of these interactions we have developed a thermodynamically rigorous method for conducting DNase I "footprint" (protection) titration experiments. The intrinsic binding constants and also those for cooperative interactions between various sites can be resolved from the individual-site binding curves determined by this technique. Experimental studies of cI-repressor-operator binding have demonstrated that the method provides an accurate representation of the fractional saturation of a binding site. We present individual-site binding curves for a lambda operator with two competent sites that demonstrate the presence of cooperative interactions between the sites. These curves set a lower limit to the magnitude of the cooperative free energy without comparison to single-site mutant operators.
Altered effector specificities in regulators of gene expression: TOL plasmid xylS mutants and their use to engineer expansion of the range of aromatics degraded by bacteria.
Authors: Ramos J L , Stolz A , Reineke W , Timmis K N
Published: May 2006

Stimulation of transcription from positively regulated promoters involves regulatory proteins that have been activated, generally as a consequence of binding low molecular weight effector molecules. To define essential structural features of effectors for one positively acting gene regulator, the xylS-encoded protein, which activates the TOL plasmid meta-cleavage pathway operon promoters, effector activities of a wide range of benzoate derivatives have been systematically analyzed, and mutant xylS-encoded proteins exhibiting altered effector specificities have been generated and characterized. Cloned mutant xylS genes were trans dominant in partial diploids containing the wild-type xylS allele and could therefore be used to effect expansion of the range of aromatic compounds completely or partially degraded by Pseudomonas bacteria. The method developed to isolate mutant xylS-encoded proteins has general applicability and could in principle be used to isolate gene regulator specificity mutants of any inducible regulatory system.
Translation of a synthetic two-cistron mRNA in Escherichia coli.
Authors: Schoner B E , Belagaje R M , Schoner R G
Published: May 2006

A synthetic two-cistron expression system was constructed for the high-level expression of eukaryotic genes in Escherichia coli. This system was designed to overcome translational inhibition of mRNAs containing eukaryotic sequences. The first cistron in this system is a 31-base A + T-rich synthetic sequence that provides for efficient translation initiation. The second cistron contains the protein coding sequence for the eukaryotic gene. Insertion of the first cistron between the 5' untranslated region of the mRNA and the protein coding region separates the two and thereby potentially minimizes the formation of local secondary structures that might prevent ribosomes from binding and initiating translation. The 31-base cistron contains three nonsense codons (TAA), one in each of the three translational reading frames, and an 8-base Shine-Dalgarno sequence that is complementary to the 3' end of the 16S rRNA. The effects of translation of the first cistron in all three reading frames on the expression of the second cistron was examined. The most efficient expression of the second cistron seemed to occur when the stop codon that terminates translation of the first cistron is located 3' to the Shine-Dalgarno sequence and close to the AUG start codon for the second cistron. When the Shine-Dalgarno sequence was deleted from the first cistron, no detectable expression of the second cistron was observed. This two-cistron system has been used to express the gene encoding methionylalanyl bovine growth hormone with its native codons and the gene encoding methionyl human growth hormone at a level greater than 20% of total cell protein. In the case of human growth hormone, we show that the amount of gene product is not significantly diminished by placing a "functional" first cistron in front of a gene that can be expressed without a cistron.