bluuebunny/crossref_metadata_2025 huggingface dataset:
Adenine nucleotide binding sites on beef heart F1 ATPase: photoaffinity labeling of beta-subunit Tyr-368 at a noncatalytic site and beta Tyr-345 at a catalytic site.
Authors: Cross R L , Cunningham D , Miller C G , Xue Z X , Zhou J M , Boyer P D
Published: May 2006

2-Azidoadenine [32P]nucleotide was bound specifically at catalytic or noncatalytic nucleotide binding sites on beef heart mitochondrial F1 ATPase. In both cases, photolysis resulted in nearly exclusive labeling of the beta subunit. The modified enzyme was digested with trypsin, and labeled peptides were purified by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis of the major 32P-labeled tryptic fragments showed beta-subunit Tyr-368 to be present at noncatalytic sites and beta Tyr-345 to be present at catalytic sites. From the relationship between the degree of inhibition and extent of modification, it is estimated that one-third of the catalytic sites or two-thirds of the noncatalytic sites must be modified to give near-complete inhibition of catalytic activity.
Toward an understanding of the folding of ribonuclease A.
Authors: Scheraga H A , Konishi Y , Rothwarf D M , Mui P W
Published: May 2006

A mechanism was proposed several years ago for the regeneration of native ribonuclease A (EC 3.1.27.5) from the fully reduced form by a mixture of oxidized and reduced glutathiones. Several folding pathways, depending on the solution conditions, were deduced. It is shown here that recent criticisms of those results are due to a misinterpretation of the analysis of our data. A more detailed description of our method of analysis of our previous kinetic and energetic data is presented in order to clarify possible misconceptions.
Major histocompatibility complex gene organization in the mole rat Spalax ehrenbergi: evidence for transfer of function between class II genes.
Authors: Nizetić D , Figueroa F , Dembić Z , Nevo E , Klein J
Published: May 2006

A genomic DNA library prepared from the kidney of the mole rat Spalax ehrenbergi was screened with mouse probes representing major histocompatibility complex genes that encode alpha and beta polypeptide chains of class II molecules (alpha and beta genes). Restriction maps were constructed for the cross-hybridizing clones, and the class II genes borne by these clones were identified. By this procedure, five main regions containing class II genes were established. One region contained four genes and two gene fragments, the second region contained two genes, the third region contained one gene and one gene fragment, and the remaining two regions contained one gene each. Altogether, six beta genes, two alpha genes, and three alpha-gene fragments were identified. Two of the genes (one alpha and one beta) were established as belonging to the DQ subclass, and all other genes were found to be members of the DP subclass. (Subclass designations are based on the human HLA class II genes). No genes belonging to the DR and DO (DZ) subclasses were found in the library. The absence of DR genes in S. ehrenbergi was also indicated when other experimental methods were used. At least some of the DP loci are polymorphic and most likely also functional. Thus, in the evolution of the mole rat, the DR (and probably also the DO) loci have been deleted and their function(s) has been taken over by the DP loci, which have expanded to a great extent. These findings argue for functional interchangeability of the individual subclasses of class II loci.
Contrasting impairments in IgM and IgG responses of vitamin A-deficient mice.
Authors: Smith S M , Hayes C E
Published: May 2006

Mice fed a semipurified, vitamin A-deficient diet (A- mice) and control animals fed the same diet with added retinyl acetate (A+ mice) were used to investigate the effect of vitamin A deficiency on primary immunoglobulin responses to protein antigens. At age 6 weeks, A- mice had serum retinol concentrations that were 46% of A+ controls. When immunized with a single antigen dose, these mice produced an antigen-specific IgM response equivalent to controls, but their IgG1 and IgG3 responses were sharply diminished (less than 30% of A+ controls). At age 8 weeks, A- mice had 20% of A+ serum retinol concentrations and less than 17% of A+ liver retinyl palmitate levels. Responding to a single antigen dose, A- mice produced approximately equal to 70% as much IgM as A+ controls. Their IgG1 response was less than 30% and their IgG3 response less than 3% of A+ controls. The IgG1 response kinetics were identical in A- and A+ mice. Diminished serum antibody responses in A- mice were attributable to fewer immunoglobulin-secreting plasma cells rather than to a decline in IgM or IgG secretion rate per cell. Total serum IgG3 levels, irrespective of antigen specificity, were slightly elevated in A- mice compared to A+ controls. The inefficient clonal expansion of responding B lymphocytes and contrasting impairment of IgM and IgG responses observed in vitamin A-deficient mice are discussed with respect to a possible helper/inducer-T-lymphocyte defect.
Introduction of v-Ha-ras oncogene induces differentiation of cultured human medullary thyroid carcinoma cells.
Authors: Nakagawa T , Mabry M , de Bustros A , Ihle J N , Nelkin B D , Baylin S B
Published: May 2006

Medullary thyroid carcinoma (MTC) is an endocrine tumor of the thyroid C cells that expresses high levels of the neuroendocrine peptide hormone calcitonin. During tumor progression in the host, there is an apparent loss of differentiation in MTC cells that involves a consistent decrease in calcitonin content of the tumor cells associated with decreased expression of the calcitonin gene and/or changes in a mRNA alternative-processing pattern away from that characteristic of the parent thyroid C cell. We now report that introduction of the viral Harvey ras (v-Ha-ras) oncogene into cultured human MTC cells can reverse such changes in gene expression and can induce endocrine differentiation of the tumor cells. The expression of v-Ha-ras is associated with decreased cellular proliferation and DNA synthesis. There is a marked increase in the number of cytoplasmic secretory granules that are a classic feature of differentiated thyroid C cells. v-Ha-ras expression induces increased expression of the calcitonin gene and the processing of the primary gene transcript is shifted to favor calcitonin mRNA rather than calcitonin-gene-related peptide (CGRP) mRNA production. These studies with cultured human MTC cells provide a model system to study the role of Ha-ras and related genes in neuroendocrine differentiation. The findings suggest an important approach for identifying genes in solid tumors whose altered expression may play a role in the impaired maturational capacity characteristic of cancer cells during tumor progression.
Structure of the rat ornithine carbamoyltransferase gene, a large, X chromosome-linked gene with an atypical promoter.
Authors: Takiguchi M , Murakami T , Miura S , Mori M
Published: May 2006

Rat mitochondrial ornithine carbamoyltransferase (EC 2.1.3.3) is encoded by a gene located on the X chromosome and expressed specifically in the liver and small intestine; we have cloned this gene and determined its structure. The gene is 75 kilobases long and is split into 10 exons. The introns range in length from 85 bases to 26 kilobases. The sum of the total exons is 1.5 kilobases and occupies only 2% of the gene; this value being one of the lowest among genes heretofore reported. The first exon encodes most of the NH2-terminal presequence that functions as a mitochondrial targeting signal. Putative binding sites for the two substrates of the enzyme, carbamoyl phosphate and ornithine, are encoded by exons 3 and 9, respectively. A set of "CAAT box"- and "ATA box"-like sequences is present about 200 bases upstream from the 5' end of the mRNA. About 35 bases downstream from this set of putative promoter elements, an 11-nucleotide sequence around the 5' end of the mRNA reappears, as a direct repeat. This pair of direct repeats may play a role in pulling the cap site and the promoter elements together. Upstream and downstream from the 5' end of the mRNA there are several sequences that resemble the transcription factor Sp1 binding site, the enhancer core sequence, the consensus sequence for the glucocorticoid receptor binding sites, and the putative enhancer element of the antithrombin III gene, another gene that is expressed specifically in the liver.
Exciton interactions in reaction centers of the photosynthetic bacterium
Rhodopseudomonas viridis
probed by optical triplet-minus-singlet polarization spectroscopy at 1.2 K monitored through absorbance-detected magnetic resonance
Authors: Lous E. J. , Hoff A. J.
Published: May 2006

Linear dichroic triplet-minus-singlet [LD-(T - S)] spectra of isolated reaction centers of the photosynthetic bacterium
Rhodopseudomonas viridis
have been measured at 1.2 K with the linear dichroic absorbance-detected magnetic resonance (LD-ADMR) technique for two mutually perpendicular directions of the preferred axis. The LD-(T - S) spectra have been calibrated with respect to the corresponding (T - S) spectra as a function of applied microwave power and quantitatively interpreted using the formalism of photoselection. The transition moment of the optical transition at 1007 nm makes angles of 72° ± 5° and 15° ± 5° with the triplet
x
and
y
spin axes, respectively. The experimental spectra have been simulated employing exciton theory and using the atomic coordinates of the resolved crystal structure of the reaction center. The spectral interpretation yields the angles between the transition moments of the various absorption bands of the (T - S) spectra and the triplet axes, and between the moments themselves, with the triplet state of the primary donor
3
P localized on the P-bacteriochlorophyll
b
in the “active” (L) chain.
B-cell-stimulatory factor 1 reverses Fc receptor-mediated inhibition of B-lymphocyte activation.
Authors: O'Garra A , Rigley K P , Holman M , McLaughlin J B , Klaus G G
Published: May 2006

Intact (IgG class) rabbit anti-immunoglobulin antibodies are not mitogenic for mouse B cells but inhibit proliferation induced by F(ab')2 anti-Fab fragment antibodies (anti-Ig). In addition, cross-linkage of Fc and surface immunoglobulin receptors on B cells by intact anti-Ig inhibits inositol phospholipid breakdown (but not Ca2+ flux) resulting from ligation of antigen receptors. This system, therefore, provides a polyclonal model for B-cell inactivation by antigen-antibody complexes. T-cell-derived B-cell-stimulatory factor 1 acts synergistically with submitogenic concentrations of F(ab')2 anti-Ig to induce B-cell proliferation. We show here that B-cell-stimulatory factor 1 and intact anti-Ig also induce B cells to synthesize DNA. However, B-cell-stimulatory factor 1 does not induce inositol phospholipid breakdown, does not mobilize Ca2+ in B cells, nor does it influence the magnitude of these responses provoked by intact anti-Ig.
cDNAs for the postsynaptic 43-kDa protein of Torpedo electric organ encode two proteins with different carboxyl termini.
Authors: Frail D E , Mudd J , Shah V , Carr C , Cohen J B , Merlie J P
Published: May 2006

Postsynaptic membranes isolated from Torpedo electric organ are highly enriched in the nicotinic acetylcholine receptor and a nonreceptor protein of 43 kDa; the distribution of the 43-kDa protein and the receptor is coextensive in the electrical membrane. As a first step in understanding the regulation of 43-kDa protein expression, we have isolated and characterized 43-kDa protein cDNAs. A lambda gt11 cDNA library was constructed from Torpedo californica electric organ mRNA and screened with a pool of 26-mer oligonucleotides encoding a short tryptic fragment of the 43-kDa synaptic protein. Positive clones were purified and sequenced; the amino acid sequences were deduced, and they matched chemically determined protein sequences of the 43-kDa protein. Two distinct classes of cDNAs were obtained; one class encoded a 43-kDa protein of 389 amino acids with a calculated molecular mass of 43,988 daltons, and another class encoded a second 43-kDa protein containing 23 additional amino acids at the C terminus. Therefore, it appears that two 43-kDa proteins with different carboxyl termini are encoded by separate mRNAs. Consistent with this idea, blot hybridization analysis revealed multiple polyadenylylated 43-kDa mRNAs in electric organ. One polyadenylylated mRNA of approximately equal to 2.0 kilobases in length was apparent in both embryonic day-11 chick muscle and the mouse muscle cell line BC3H1.
Dimethyl sulfoxide-inducible cytoplasmic factor involved in erythroid differentiation in mouse erythroleukemia (Friend) cells.
Authors: Watanabe T , Oishi M
Published: May 2006

A previous report described an intracellular factor (differentiation-inducing factor I, or DIF-I) that seems to play a role in erythroid differentiation in mouse erythroleukemia (MEL) cells. We have detected another erythroid-inducing factor in cell-free extracts from dimethyl sulfoxide- or hexamethylenebis(acetamide)-treated MEL cells, which acts synergistically with DIF-I. The partially purified factor (termed DIF-II) triggered erythroid differentiation when introduced into undifferentiated MEL cells that had been potentiated by the induction of DIF-I. The activity in the extracts appeared in an inducible manner after addition of dimethyl sulfoxide or hexamethylenebis(acetamide), reached a maximum at 6 hr, and then rapidly decreased. The induction was inhibited by phorbol 12-myristate 13-acetate and also by cycloheximide. No induction was observed in a mutant MEL cell line defective in erythroid differentiation. These characteristics are consistent with the supposition that DIF-II is one of the putative dimethyl sulfoxide-inducible factors detected in previously reported cell-fusion and cytoplast-fusion experiments. The role of DIF-II in MEL-cell differentiation and in vitro differentiation in general is discussed.
Early genes that were oligomeric repeats generated a number of divergent domains on their own.
Authors: Ohno S
Published: May 2006

One of the more popular concepts to emerge in recent years is that new proteins evolved by domain exchanges between preexisting proteins. The presence of introns within eukaryotic genes is thought to enhance such exchanges. Yet domain exchanges must necessarily be the secondarily developed process in evolution, for they would have been effective only after multitudes of domains came into being. Many of the proteins with functionally divergent domains were established before the division of prokaryotes from eukaryotes; i.e., soon after the creation of life on this earth. I attribute the extreme innovativeness of early coding sequences to their construction; i.e., being repeats of oligomeric units. The rhodopsin family of proteins--with seven hydrophobic, alpha-helical transmembrane domains, four extracellular domains, and four intracytoplasmic domains--indeed arose before the division of prokaryotes from eukaryotes and later gave rise to muscarinic acetylcholine receptor and beta-adrenergic receptor among others. In this paper, I show that the entire coding sequence for porcine muscarinic acetylcholine receptor is still replete with copies of three heptameric units that are very closely related to each other. Original heptameric units are more stringently conserved in parts encoding the seven transmembrane domains, whereas new repeating units are comingled with the old in parts encoding extracellular and intracytoplasmic domains.
Induction of tumor necrosis factor expression and resistance in a human breast tumor cell line.
Authors: Spriggs D , Imamura K , Rodriguez C , Horiguchi J , Kufe D W
Published: May 2006

Tumor necrosis factor (TNF) is a polypeptide cytokine that is cytotoxic to some but not all tumor cells. The basis for resistance to the cytotoxic effects of this agent remains unclear. We have studied the development of TNF resistance in human ZR-75-1 breast carcinoma cells. ZR-75-1 cells have undetectable levels of TNF RNA and protein. However, TNF transcripts are transiently induced in these cells by exposure to recombinant human TNF. This induction of TNF RNA is associated with production of TNF-like protein in cell lysates and culture supernatants. Stable resistance to TNF-induced cytotoxicity develops when ZR-75-1 cells are exposed to increased concentrations of TNF. The TNF-resistant cells, designated ZR-75-1R, continuously express TNF transcripts and a TNF-like protein. Furthermore, ZR-75-1R cell supernatants contain cytotoxic activity that is abrogated by polyclonal antibody against TNF. The ZR-75-1R cells also possess TNF receptors that are occupied or down-regulated by the TNF-like protein. These findings thus suggest that (i) TNF induces TNF transcripts and production of a TNF-like protein in ZR-75-1 cells and (ii) resistance to TNF-induced cytotoxicity is associated with stable TNF expression.
Isolation and characterization of a T-lymphocyte somatic mutant with altered signal transduction by the antigen receptor.
Authors: Goldsmith M A , Weiss A
Published: May 2006

We have developed an approach for deriving and characterizing antigen-receptor (CD3/Ti) signal-transduction mutants. This strategy combines receptor-mediated growth inhibition and fluorescence-activated cell sorting with the Ca2+-indicator indo-1. Despite the expression of structurally normal CD3/Ti complexes, one such mutant (J.CaM1) fails to exhibit inositolphospholipid metabolism or Ca2+ mobilization in response to anti-CD3 or anti-Ti monoclonal antibodies and fails to produce lymphokines in response to these antibodies. Surprisingly, anti-Ti antibody retains its effectiveness as a stimulus for the down-regulation of CD3/Ti surface expression. These cells remain responsive to AIF-4, at least one anti-CD3 antibody, and some combinations of nonagonist anti-Ti and anti-CD3 antibodies. The mutation in J.CaM1 appears to lie in a proximal component of the signal-transduction apparatus.
Construction and expression of a recombinant antibody-targeted plasminogen activator.
Authors: Schnee J M , Runge M S , Matsueda G R , Hudson N W , Seidman J G , Haber E , Quertermous T
Published: May 2006

Covalent linkage of tissue-type plasminogen activator (t-PA) to a monoclonal antibody specific for the fibrin beta chain (anti-fibrin 59D8) results in a thrombolytic agent that is more specific and more potent than t-PA alone. To provide a ready source of this hybrid molecule and to allow tailoring of the active moieties for optimal activity, we have engineered a recombinant version of the 59D8-t-PA conjugate. The rearranged 59D8 heavy chain gene was cloned and combined in the expression vector pSV2gpt with sequence coding for a portion of the gamma 2b constant region and the catalytic beta chain of t-PA. This construct was transfected into heavy chain loss variant cells derived from the 59D8 hybridoma. Recombinant protein was purified by affinity chromatography and analyzed with electrophoretic transfer blots. These revealed a 65-kDa heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kDa disulfide-linked dimer. Chromogenic substrate assays showed the fusion protein to have 70% of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. In a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus, by recombinant techniques, we have produced a hybrid protein capable of high-affinity fibrin binding and plasminogen activation.
Structural and pathological effects of synthesis of hepatitis B virus large envelope polypeptide in transgenic mice.
Authors: Chisari F V , Filippi P , Buras J , McLachlan A , Popper H , Pinkert C A , Palmiter R D , Brinster R L
Published: May 2006

Overproduction of the hepatitis B virus (HBV) large envelope polypeptide by transgenic mice containing the entire HBV envelope coding region leads to the formation of extremely long (up to 800 nm), occasionally branching, filamentous 22-nm-diameter hepatitis B surface antigen particles that accumulate within the endoplasmic reticulum of the hepatocyte and are not efficiently secreted. As the endoplasmic reticulum expands to accommodate the increasing cellular filament stores, the hepatocytes become enlarged, hydropic, and eosinophilic and also display the characteristic features of "ground-glass" cells. As filament storage progresses, the ground-glass cells undergo coagulative necrosis and the mice develop an age-dependent lesion, whose severity is related to the intracellular concentration of envelope polypeptide, that is characterized by focal hepatocellular degeneration and necrosis, lobular macrophagic inflammation, and increased serum transaminase activity. Advanced lesions demonstrate hepatocellular hyperplasia evident as lobular architectural disarray and microscopic hepatocellular nodules, many of which no longer contain detectable HBV envelope antigens. These changes may become extreme, producing a massively enlarged liver due to multifocal nodular regenerative hyperplasia. Overproduction of the large HBV envelope polypeptide exerts major structural constraints on HBV particle formation, leading to reduced secretion and progressive intracellular accumulation of hepatitis B surface antigen, which can reach sufficiently high concentrations to be directly cytotoxic to hepatocytes in this transgenic mouse system.
Monoclonal antibody to a unique surface epitope of the human filaria Brugia malayi identifies infective larvae in mosquito vectors.
Authors: Carlow C K , Franke E D , Lowrie R C , Partono F , Philipp M
Published: May 2006

We describe properties of an IgM monoclonal antibody (NEB-D1E5) raised against the human filarial parasite Brugia malayi. The antibody reacts with a stage- and species-specific determinant located on the surface of the infective-stage larva, as determined by indirect immunofluorescence. To use this reagent in epidemiological field studies, we developed an enzyme-linked immunoassay with which B. malayi larvae can be differentiated from other filarial parasites in mosquito vectors, including the morphologically indistinguishable parasite of animals Brugia pahangi. The immunoenzyme assay was 91-94% specific and 90-97% sensitive when performed on infected mosquitoes. In the absence of mosquito tissue, the levels of specificity and sensitivity increased to 100% and 97.5-100%, respectively. Binding of antibody to the surface of living larvae was abrogated by treatment of the worms with the enzymes pronase and proteinase K and with the detergents Triton X-100, octyl beta-D-glucopyranoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS). In contrast, treatment with trypsin, endoglycosidase-F, O-Glycanase, N-Glycanase, lipase, various phospholipases, boiling, 2-mercaptoethanol at 37 degrees C, or periodate did not reduce the antigenicity of the larval surface to antibody NEB-D1E5. These results suggest that the species-specific epitope is a peptide domain attached to a hydrophobic anchoring residue.
Phosphorylation by protein kinase C of a 20-kDa cytoskeletal polypeptide enhances its susceptibility to digestion by calpain.
Authors: Pontremoli S , Melloni E , Michetti M , Sparatore B , Salamino F , Sacco O , Horecker B L
Published: May 2006

Incubation of the cytoskeletal fraction from human neutrophils with the proteolytically activated form of protein kinase C results in the phosphorylation of several components, including a 20-kDa polypeptide, probably consisting of myosin light chains. The 20-kDa polypeptide is also specifically phosphorylated by activated protein kinase C in a solubilized 20-kDa/80-kDa complex that was obtained after sonication of the insoluble cytoskeletal fraction. Phosphorylation of this polypeptide, in either the insoluble cytoskeletal fraction or the soluble 20-kDa/80-kDa complex, greatly enhances its susceptibility to digestion by the Ca2+-requiring proteinase (calpain, EC 3.4.22.17) of human neutrophils. Thus, signals that activate calpain by mobilizing intracellular calcium would lead to proteolytic activation of protein kinase C, phosphorylation of cytoskeletal proteins, and remodeling of the cytoskeleton by proteolysis of at least one cytoskeletal component.
Thymine-containing dimers as well as spore photoproducts are found in ultraviolet-irradiated Bacillus subtilis spores that lack small acid-soluble proteins.
Authors: Setlow B , Setlow P
Published: May 2006

Dormant spores of a Bacillus subtilis mutant that lacks two major small, acid-soluble spore proteins are very sensitive to UV irradiation, which in spores generates about half the amount of thymine-containing dimers formed by comparable irradiation of vegetative cells. Irradiation of mutant spores also produces spore photoproducts, but again only about one-half the amount formed in comparably irradiated wild-type spores. These findings suggest that the high UV sensitivity of the mutant spores is due to the production of pyrimidine dimers, which are not found in UV-irradiated wild-type spores, and that the high level of small, acid-soluble proteins found in wild-type spores is directly involved in spore UV resistance by facilitating a conformational change in spore DNA, preventing pyrimidine dimer formation.
Dependence of thermodynamic efficiency of proton pumps on frequency of oscillatory concentration of ATP.
Authors: Schell M , Kundu K , Ross J
Published: May 2006

In order to evaluate the utilization of variable ATP concentration produced by an oscillatory reaction (as in anaerobic glycolysis), we analyze the thermodynamic efficiency of power output of a cyclic, ATP-driven proton pump found in the plasma membrane of plant cells. The model used includes the coupling of potassium and calcium ion transport. Oscillations in the concentration of ATP can lead to either increases or decreases in efficiency compared to that at constant ATP concentration, with corresponding decreases and increases in dissipation in the irreversible processes of the proton pump, depending on the frequency of the oscillations. Variations of imposed frequencies induce, in the periodic response, variations of phase shifts between the components of the total membrane current, which consist of the pump's proton current and the currents of potassium and calcium ions. Increases in efficiency are attained when the phase shifts are such that maxima (or minima) in the proton pump current and membrane potential occur simultaneously.
Structural features of cartilage matrix protein deduced from cDNA.
Authors: Argraves W S , Deák F , Sparks K J , Kiss I , Goetinck P F
Published: May 2006

cDNAs encoding the Mr 54,000 chicken cartilage matrix protein (CMP) were selected from a cartilage cDNA expression library by immunological means. Antibodies elicited against insert-encoded protein purified from one of the clones reacted specifically with chicken CMP in immunoblots of total cartilage extract, providing positive identification of the cDNA clones isolated. The cDNAs detect a 3.4-kilobase transcript that was present in sternal cartilage and in cartilaginous but not in precartilaginous embryonic limb tissues. The cDNAs code for 416 amino acids of the chicken CMP, including its COOH terminus. There are two striking features in the deduced CMP amino acid sequence: first, it contains a region with significant homologies to repeat sequences in the precursor for epidermal growth factor; and second, it is made up of two large homologous repeat sequences. These results provide the first detailed structural information on the CMP and establish it as a developmentally regulated marker of cartilage differentiation.
Two different biosynthetic pathways for the secretion of Qa region-associated class I antigens by mouse lymphocytes.
Authors: Robinson P J
Published: May 2006

Treatment of supernates of labeled C57BL/6 mouse lymphocytes with antibodies against beta 2-microglobulin reveals the presence of two different soluble class I molecules. One molecule (Mr, 37,000) is found in supernates of both 125I surface-labeled and [35S]methionine biosynthetically labeled cells and reacts with antibodies against Qa-2 antigens. The other molecule (Mr, 42,000) is found labeled only in supernates of [35S]methionine-labeled cells and reacts with antibodies against Qb-1. Analysis of mutant and recombinant mouse strains demonstrates that both soluble class I molecules are encoded in the Qa region. Pulse-chase experiments show that the Qa-2 molecules are released more slowly than Qb-1. It is proposed that Qb-1 molecules are secreted directly, whereas Qa-2 is first expressed on the cell surface and then processed to a soluble form.
Correlations between development rates, enzyme activities, ribosomal DNA spacer-length phenotypes, and adaptation in Drosophila melanogaster.
Authors: Cluster P D , Marinković D , Allard R W , Ayala F J
Published: May 2006

Selection for "fast" preadult development rate among the progeny of flies collected in a natural population of Drosophila melanogaster produced a line that developed more rapidly than a line selected for "slow" preadult development rate. Assays for enzyme activity levels showed that the activities of alpha-glycerophosphate dehydrogenase, alcohol dehydrogenase, and malic enzyme were higher in the fast than in the slow line, but that the activity of superoxide dismutase was lower in the fast line. Differences in the frequencies of spacer-length phenotypes of X chromosome-linked rRNA genes (rDNA), which developed between the lines during the selection process, are larger than can be explained on the basis of genetic drift alone. Long rDNA spacers had high frequency in the fast line; short spacers, in the slow line. We conclude that enzyme levels affected adaptation under the selective regimes imposed and that the different X-linked rDNA spacer-length phenotypes are either adaptive in themselves or that they mark chromosomal segments carrying genes relevant to adaptation.
Structure of the gene for human beta 2-adrenergic receptor: expression and promoter characterization.
Authors: Emorine L J , Marullo S , Delavier-Klutchko C , Kaveri S V , Durieu-Trautmann O , Strosberg A D
Published: May 2006

The genomic gene coding for the human beta 2-adrenergic receptor (beta 2AR) from A431 epidermoid cells has been isolated. Transfection of the gene into eukaryotic cells restores a fully active receptor/GTP-binding protein/adenylate cyclase complex with beta 2AR properties. Southern blot analyses with beta 2AR-specific probes show that a single beta 2AR gene is common to various human tissues and that its flanking sequences are highly conserved among humans and between man and rabbit, mouse, and hamster. Functional significance of these regions is supported by the presence of a promoter region (including mRNA cap sites, two "TATA boxes," a "CAAT box," and three G + C-rich regions that resemble binding sites for transcription factor Sp1) 200-300 base pairs 5' to the translation initiation codon. In the 3' flanking region, sequences homologous to glucocorticoid-response elements might be responsible for the increased expression of the beta 2AR gene observed after treatment of the transfected cells with hydrocortisone. In addition, 5' to the promoter region, an open reading frame encodes a 251-residue polypeptide that displays striking homologies with protein kinases and other nucleotide-binding proteins.
Primary structure and subcellular localization of the knob-associated histidine-rich protein of Plasmodium falciparum.
Authors: Pologe L G , Pavlovec A , Shio H , Ravetch J V
Published: May 2006

Plasmodium falciparum-infected erythrocytes bind to venular endothelial cells by means of electron-dense deformations (knobs) on the parasitized erythrocyte surface. The primary structure of a parasite-derived histidine-rich protein associated with the knob structure was deduced from cDNA sequence analysis. The 634 amino acid sequence is rich in lysine and histidine and contains three distinct, tandemly repeated domains. Indirect immunofluorescence, using affinity-purified monospecific antibodies directed against recombinant protein synthesized in Escherichia coli, localized the knob-associated histidine-rich protein to the membrane of knobby infected erythrocytes. Immunoelectron microscopy established that the protein is clustered on the cytoplasmic side of the erythrocyte membrane and is associated with the electron-dense knobs. A role for this histidine-rich protein in knob structure and cytoadherence is suggested based upon these data.
Polymorphic expression of a human superficial bladder tumor antigen defined by mouse monoclonal antibodies.
Authors: Fradet Y , Islam N , Boucher L , Parent-Vaugeois C , Tardif M
Published: May 2006

Three mouse monoclonal antibodies (mAbs), which define a highly restricted antigen, were obtained by simultaneous immunizations with superficial papillary bladder tumor cells and mouse polyclonal serum against normal urothelium. The antigen was detected by the avidin/biotin/peroxidase method in 30/44 superficial bladder tumors (68%) but in only 4/27 infiltrating urothelial cancers (with much less intensity). No normal adult or fetal tissues tested expressed the antigen, including normal urothelium from 40 individuals, 13 of whom had a bladder tumor positive for the antigen. Only 1 of 45 nonbladder tumors showed some reactivity with one of the three mAbs. Serological tests on a large panel of human cancer cell lines and normal cultured cells were negative. The antigen is highly stable and well preserved on paraffin-embedded tissues. Electrophoretic transfer blot experiments with fresh tumor extracts showed that all three mAbs react with a determinant on a component of 300,000 Mr (pI 9.5) and 62,000 Mr (pI 6.5). The antigen shows polymorphic expression at the cellular level on tissue sections and also at a molecular level on immunoblots where the two bands are differentially detected on extracts of a series of tumors but are not visualized on normal urothelium extracts. The characteristics of this antigenic system suggest that it may provide some insights about the biology of bladder cancer. Specific detection of the antigen on 70% of superficial bladder tumors with normal cytology may be useful for their diagnosis and follow-up.
Calbindin-D in peripheral nerve cells is vitamin D and calcium dependent.
Authors: Lee Y S , Taylor A N , Reimers T J , Edelstein S , Fullmer C S , Wasserman R H
Published: May 2006

The vitamin D-induced calcium-binding protein calbindin-D (CaBP) was localized immunohistochemically in some but not all of the cell bodies and axons within the intestinalis nerve of the chicken. Unlike other nerve tissue thus far examined, the CaBP content of the intestinalis nerve was decreased in vitamin D deficiency and increased in chicken adapted to a calcium-deficient diet. These changes are qualitatively similar to the pattern of response of enterocytes. The inclusion of calcium-containing solutions within the duodenal lumen caused, directly or indirectly, a decrease in the amount of CaBP in this nerve in a dose-dependent manner. The exact role of CaBP in intestinalis nerve cells is unknown but may be in the regulation of intracellular ionic Ca2+ concentrations during excitation, although other functions of CaBP cannot be excluded.
Precise insertion of antibiotic resistance determinants into Tn21-like transposons: nucleotide sequence of the OXA-1 beta-lactamase gene.
Authors: Ouellette M , Bissonnette L , Roy P H
Published: May 2006

Several plasmid-encoded beta-lactamases are on multiresistance transposable elements. The OXA-1 beta-lactamase gene is part of Tn2603, which is borne on the R plasmid RGN238. We report here the complete nucleotide sequence of the OXA-1 beta-lactamase gene and flanking sequences. The OXA-1 gene shows a greater than 50% sequence divergence from the OXA-2 gene, yet there is significant functional similarity at the peptide level. Analysis of 5' and 3' flanking sequences shows that Tn2603 differs from its probable precursor, Tn21, by a precise 1004-base-pair insertion, containing the OXA-1 structural gene, at the target sequence AAAGTT, which is located between the Tn21 streptomycin/spectinomycin (aadA) promoter and its structural gene. A 5- for 6-base repeat of the target sequence is found at the end of the insertion. The same precise insertion and repeat of the target sequence are found for the OXA-2 gene from R46. The 5' flanking regions of two other genes, the trimethoprim-resistance gene from R388 and the gentamicin resistance (aadB) gene from pDGO100, are greater than 98% homologous to the 5' flanking sequences of the OXA-1, OXA-2, and aadA genes until they diverge at the target sequence. From the available sequence data a recombinational hot spot is defined at the nucleotide level 5' of the aadA gene of Tn21, and a second potential hot spot is proposed 3' of this gene.
Reversal of age-related effects in rat muscle phosphoglycerate kinase.
Authors: Yuh K C , Gafni A
Published: May 2006

Rat muscle phosphoglycerate kinase is one of several enzymes in which age-related effects have been identified. Thus, samples of this enzyme isolated from old rats display a greatly increased heat stability as compared with enzyme isolated from young animals. Previous studies detected no differences in the sequence of amino acids or in the net charge between the young and old forms of the enzyme and it was concluded that the age-related structural modifications are purely conformational. The present study was conducted with the aim of critically testing this hypothesis. To this end, samples of phosphoglycerate kinase purified from skeletal muscle of young and old rats were unfolded by an 18-hr incubation in a 2 M guanidine hydrochloride solution at 4 degrees C, a treatment that results in extensive loss of the three-dimensional structure of the enzyme. A complete reactivation of both enzymes was achieved by dilution of the unfolded enzyme solutions into a large excess of denaturant-free buffer followed by 4 hr of incubation at 25 degrees C. The reactivation kinetics of the unfolded young and old enzymes were practically identical and the refolded products, compared using heat-inactivation kinetics as a sensitive probe, were found to be identical. Moreover, their heat inactivation coincided with that of young untreated phosphoglycerate kinase. These results demonstrate the reversibility of age-related effects at the molecular level and provide strong support for the hypothesis that the modifications in phosphoglycerate kinase in old muscle are purely conformational and, hence, clearly postsynthetic.
The Stark effect in reaction centers from Rhodobacter sphaeroides R-26 and Rhodopseudomonas viridis.
Authors: Lösche M , Feher G , Okamura M Y
Published: May 2006

The effect of an electric field on the optical absorption (Stark effect) of reaction centers (RCs) from Rhodobacter sphaeroides and Rhodopseudomonas viridis embedded in films of poly(vinyl alcohol) was measured. The infrared bands were investigated at 295 K and 77 K. In RCs from Rp. viridis at 77 K six peaks (at 982, 849, 835, 818, 803, and 787 nm), associated with the Qy transitions of the six pigments, were resolved; in addition, a small broad band at 865 nm was resolved. In RCs from Rb. sphaeroides only five bands (at 877, 817, 802, 761, and 754 nm) assigned to the Qy transitions were resolved; in addition, two small bands at 697 and 683 nm were observed. The additional bands have been tentatively assigned to vibrational side bands, although the contribution from charge-transfer states cannot be excluded. The Stark spectra had line shapes similar to the second derivative of the absorption spectra and were interpreted in terms of the interaction between the applied electric field and the dipole moments of the ground and excited states. Analyses of the spectra yielded the apparent change in dipole moment delta mu app = f delta mu (where the factor f corrects for the difference between the local field and the applied field) and delta, the angle between delta mu---- and the transition moment mu trans. At 77 K the values of delta mu----app and delta for the peaks at 877, 802, and 761 nm in Rb. sphaeroides were 6.5 debye, 38 degrees; 2.1 debye, 23 degrees; and 3.5 debye, 8 degrees. In Rp. viridis the debye values for the peaks at 982, 835, 818, and 787 were 8.2, 40 degrees; 1.8, 50 degrees; 3.4, 14 degrees; and 2.7, 0 degrees. The large values of delta mu app associated with the long-wavelength peak of the bacteriochlorophyll dimers are consistent with a significant charge-transfer contribution to the excited state of the primary donor.
Intracellular pH in human and experimental hypertension.
Authors: Resnick L M , Gupta R K , Sosa R E , Corbett M L , Laragh J H
Published: May 2006

31P NMR spectroscopy was utilized to evaluate intracellular pH in erythrocytes from normotensive (n = 15) and from untreated (n = 16) and treated (n = 24) human essential hypertensive individuals. Intracellular erythrocyte pH was also measured in normotensive rats on different dietary calcium intakes as well as in volume-dependent deoxycorticosterone/saline and renin-dependent, 2 kidney, 1 clip (2K-1C) Goldblatt hypertensive rat models. Untreated essential hypertensives had significantly lower intracellular pH values compared with normotensive subjects [7.17 +/- 0.02 vs. 7.28 +/- 0.02 (mean +/- SEM), significance level = 0.01]. Treated hypertensives had intracellular pH values indistinguishable from normotensives [7.27 +/- 0.02 (mean +/- SEM)]. Similarly, pH values for each rat model varied inversely with blood pressure, regardless of whether increased dietary calcium intake lowered pressure (normotensive and deoxycorticosterone/saline hypertensive rats) or elevated it (2K-1C Goldblatt hypertensive rats). These results demonstrate that lower intracellular pH values are commonly observed in various hypertensive states and suggest that they may contribute to the pathophysiology of the hypertensive process. Alterations in intracellular pH may also underlie the clinically observed linkage of hypertension with other disease syndromes, such as diabetes mellitus and obesity.
An L1-like molecule, the 8D9 antigen, is a potent substrate for neurite extension.
Authors: Lagenaur C , Lemmon V
Published: May 2006

The 8D9 antigen, a cell surface protein isolated from chicken brain that is related to the L1 class of cell adhesion molecules, is shown to contain an activity that promotes the attachment of neurons and the outgrowth of neurites from chicken tecta and mouse cerebellum. When purified 8D9 antigen is attached to a nitrocellulose-coated substrate, neurons rapidly attach and extend unfasciculated neurites. Little or no attachment of astroglia, oligodendroglia, and fibroblast-like cells to the 8D9 antigen is observed. We propose that a function of the 8D9 antigen is that of a neurite extension-promoting substrate in axon fascicles and in regeneration of peripheral nerves.
Low molecular weight human pulmonary surfactant protein (SP5): isolation, characterization, and cDNA and amino acid sequences.
Authors: Warr R G , Hawgood S , Buckley D I , Crisp T M , Schilling J , Benson B J , Ballard P L , Clements J A , White R T
Published: May 2006

Pulmonary surfactant is a lipid-protein complex that promotes alveolar stability by lowering the surface tension at the air-fluid interface in the peripheral air spaces. A group of hydrophobic surfactant-associated proteins has been shown to be essential for rapid surface film formation by surfactant phospholipids. We have purified a hydrophobic surfactant protein of approximately 5 kDa that we term SP5 from bronchopulmonary lavage fluid from a patient with alveolar proteinosis and shown that it promotes rapid surface film formation by simple mixtures of phospholipids. We have derived the full amino acid sequence of human SP5 from the nucleotide sequence of cDNAs identified with oligonucleotide probes based on the NH2-terminal sequence of SP5. SP5 isolated from surfactant is a fragment of a much larger precursor protein (21 kDa). The precursor contains an extremely hydrophobic region of 34 amino acids that comprises most of the mature SP5. This hydrophobicity explains the unusual solubility characteristics of SP5 and the fact that it is lipid-associated when isolated from lung.
Lignin degradation by peroxidase in organic media: A reassessment
Authors: Lewis Norman G. , Razal Ramon A. , Yamamoto Etsuo
Published: May 2006

The effect of horseradish peroxidase/H
2
O
2
in organic medium (dioxane/aqueous buffer, pH 5, 95:5) on the depolymerization of synthetic (dehydrogenatively polymerized) lignin was reinvestigated. In contrast to previous claims [Dordick, J. S., Marletta, M. A. & Klibanov, A. M. (1986)
Proc. Natl. Acad. Sci. USA
83, 6255-6257], our results demonstrated that vigorous depolymerization of this substrate did not occur. Further, during this treatment ferulic acid was not a significant biodegradation product.
Genetic linkage map of human chromosome 7 with 63 DNA markers.
Authors: Barker D , Green P , Knowlton R , Schumm J , Lander E , Oliphant A , Willard H , Akots G , Brown V , Gravius T
Published: May 2006

High-density genetic maps of individual human chromosomes will permit accurate localization of disease-associated genes and provide signposts that will be useful in the construction of physical maps. We have constructed a genetic map of chromosome 7 with 63 polymorphic DNA markers by using segregation data from 23 three-generation families and recently developed multilocus linkage-analysis techniques. The map spans 250 centimorgans in females and 170 centimorgans in males, with much of the difference being concentrated in a few intervals. The density and informativeness of the markers are such that there is a high probability of detecting linkage to any disease gene on this chromosome for which 20 phase-known meioses are available.
T-cell gamma gene is allelically but not isotypically excluded and is not required in known functional T-cell subsets.
Authors: Heilig J S , Tonegawa S
Published: May 2006

The T-cell gamma genes, structurally related to immunoglobulin genes and the T-cell antigen-receptor alpha- and beta-chain genes, undergo somatic rearrangement in T-lineage cells. However, the role of the T-cell gamma genes has not yet been determined. To determine the potential for gamma gene expression in a set of well-characterized, cloned T-cell lines, we cloned all of the rearranged gamma genes from each cell line. The genes were sequenced to determine if the junction of the variable and joining regions maintained the proper translational reading frame. We then attempted to correlate the presence of an in-frame gamma gene with a T-cell subset. We were unable to establish such a correlation. We found evidence, however, that allelic exclusion influences the rearrangement of the gamma gene. This is consistent with the idea that the gamma gene product participates in establishing a clonally diverse population of T cells recognizing a polymorphic ligand. Isotypic exclusion does not apply to the gamma gene, however, suggesting different roles for the different gamma gene isotypes.
Spectrophotometry in vivo, a technique for local and direct enzymatic assays: application to brain acetylcholinesterase.
Authors: Testylier G , Gourmelon P
Published: May 2006

In vivo enzymology is not widely studied due to the lack of a well-adapted technology. We have developed a system that allows local and long-term spectrophotometric assays in brain tissue of live animals. It utilizes a miniaturized optical probe consisting of a multibarrel micropipette for reagent injections and optical fibers for light absorption measurements. We have applied this system to the colorimetric determination of brain acetylcholinesterase activity in rats. The reproducibility of the assay was demonstrated by repetitive assays over 24 hr, its specificity was established through the use of a highly specific organophosphorus inhibitor, and the activities measured in different brain areas agreed with the known distribution of acetylcholinesterase. No electroencephalographic abnormalities and no change in vigilance level were observed in the experimental animals. This methodology should prove to be useful for the colorimetric measurement of different enzymes or metabolites in various organs.