bluuebunny/crossref_metadata_2025 huggingface dataset:
Covalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases.
Authors: Shuman S , Liu Y , Schwer B
Published: May 2006

Formation of the 5' cap structure of eukaryotic mRNAs occurs via transfer of GMP from GTP to the 5' terminus of the primary transcript. RNA guanylyltransferase, the enzyme that catalyzes this reaction, has been isolated from many viral and cellular sources. Though differing in molecular weight and subunit structure, the various guanylyltransferases employ a common catalytic mechanism involving a covalent enzyme-(Lys-GMP) intermediate. Saccharomyces cerevisiae CEG1 is the sole example of a cellular capping enzyme gene. In this report, we describe the identification and characterization of the PCE1 gene encoding the capping enzyme from Schizosaccharomyces pombe. PCE1 was isolated from a cDNA library by functional complementation in Sa. cerevisiae. Induced expression of PCE1 in bacteria and in yeast confirmed that the 47-kDa Sc. pombe protein was enzymatically active. The amino acid sequence of PCE1 is 38% identical (152 of 402 residues) to the 52-kDa capping enzyme from Sa. cerevisiae. Comparison of the two cellular capping enzymes with guanylyltransferases encoded by DNA viruses revealed local sequence similarity at the enzyme's active site and at four additional collinear motifs. Mutational analysis of yeast CEG1 demonstrated that four of the five conserved motifs are essential for capping enzyme function in vivo. Remarkably, the same motifs are conserved in the polynucleotide ligase family of enzymes that employ an enzyme-(Lys-AMP) intermediate. These findings illuminate a shared structural basis for covalent catalysis in nucleotidyl transfer and suggest a common evolutionary origin for capping enzymes and ligases.
Differences in the molecular structure of c-myc-activating recombinations in murine plasmacytomas and precursor cells.
Authors: Müller J R , Potter M , Janz S
Published: May 2006

The translocation of c-myc on chromosome (chr.) 15 to an immunoglobulin heavy-chain switch region on chr. 12 is the critical oncogenic step in pristane-induced plasmacytoma (PCT) development in BALB/cAnPt mice. Applying a recently developed PCR method, we have been able to detect the most commonly occurring illegitimate recombinations between alpha-chain switch region (S alpha) and c-myc in preneoplastic B cells residing in mesenteric oil granuloma (OG) tissues 7-30 days postpristane. In this study, we compare the nucleotide sequences at the S alpha/c-myc breaksites on both the c-myc-activating chr. 12+ and the reciprocal chr. 15- from eight transplanted PCTs, seven primary PCTs, and five OGs that contained six B-cell clones. These junction sequences revealed a remarkable diversity of S alpha/c-myc recombinations. In nine cases--four PCTs and five B-cell clones--nearly precise reciprocal exchanges with a loss of only 3-35 bp in c-myc were found. Large deletions in c-myc that removed 369-878 bp were observed in seven PCTs but not in early B cells. Duplications of c-myc ranging from 103 to 229 bp were also restricted to PCTs and noticed in four cases. Clonally related but different reciprocal recombinations, 38 bp apart on chr. 12+ and 15 bp apart on chr. 15-, were isolated from two different specimens of the same OG tissue from a BALB/c mouse 30 days postpristane. A second OG from another 30-day mouse yielded four recombinational fragments--two clonally related chr. 12(+)-specific fragments and two chr. 15(-)-specific fragments--one of which carried a 143-bp insertion of a microsatellite at the breaksite. We suggest that the initial recombinational break-point regions between S alpha and c-myc in plasmacytoma precursor cells at the time of immunoglobulin heavy-chain switching are intrinsically labile and characterized by a persisting instability of c-myc, which can result in large secondary deletions of c-myc.
Three forms of gonadotropin-releasing hormone characterized from brains of one species.
Authors: Powell J F , Zohar Y , Elizur A , Park M , Fischer W H , Craig A G , Rivier J E , Lovejoy D A , Sherwood N M
Published: May 2006

Most vertebrate species have more than one form of gonadotropin-releasing hormone (GnRH) in their brains, but it is not clear whether each form has a distinct function. We report that sea bream (Sparus aurata) brains have three forms of GnRH, one of which is described herein and is called sea bream GnRH (sbGnRH). The primary structures of two forms were determined by Edman degradation and mass spectral analysis. The amino acid sequence of sbGnRH is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Ser-Pro-Gly-NH2. The second peptide is identical to a form originally isolated from chicken brains (cGnRH-II): pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2. cGnRH-II is the most ancient form of GnRH identified to date in jawed fish and the most prevalent form throughout the vertebrates. The third form of GnRH has previously been identified as salmon GnRH by cDNA studies and is confirmed here by chromatographic and immunological studies. Phylogenetic distribution of GnRH peptides suggests sbGnRH arose in the perch-like fish as a gene duplication of the existing cGnRH-II or salmon GnRH genes. All three identified GnRH peptides were synthesized and shown to release gonadotropin in vivo in the sea bream. The dominant form of GnRH stored in the pituitary was sbGnRH. Not only was the content of sbGnRH 500-fold greater than that of salmon GnRH but also cGnRH-II was not detected in the pituitary. The latter evidence suggests that sbGnRH is the endogenous releaser of gonadotropin II.
Similarity of nucleotide interactions of BiP and GTP-binding proteins.
Authors: Brot N , Redfield B , Qiu N H , Chen G J , Vidal V , Carlino A , Weissbach H
Published: May 2006

BiP is a member of the Hsp70 heat shock protein family found in the lumen of the endoplasmic reticulum, that binds to a variety of proteins destined to be secreted. Substance P (SP) has been used as a model peptide to study the interaction of BiP with protein substrates. SP stimulates BiP ATPase activity and forms a stable complex with BiP that is dissociated in the presence of levels of ATP > 50 microM. At lower concentrations of ATP, the SP remains bound to BiP, and the results are consistent with the view that a BiP-ATP complex is initially formed that reacts with SP to form a ternary complex, SP-BiP-ATP. Hydrolysis of ATP in this complex yields a SP-BiP-ADP complex. An exchange of ATP with ADP bound to BiP has also been demonstrated, and the results suggest that the interactions of BiP with ATP resemble those seen with GTP-binding proteins and GTP.
Complex correlation between excitatory amino acid-induced increase in the intracellular Ca2+ concentration and subsequent loss of neuronal function in individual neocortical neurons in culture.
Authors: Witt M R , Dekermendjian K , Frandsen A , Schousboe A , Nielsen M
Published: May 2006

Primary cultures of cerebral cortical neurons and single-cell imaging of intracellular free Ca2+ concentration ([Ca2+]i) with the ratiometric dye fura-2 were used to assess excitatory amino acid (EAA)-induced neurotoxicity; the loss of neuronal function as defined by the ability of the cells to respond to K(+)-induced depolarization by a transient increase in Ca2+ influx was measured. The responsiveness of individual neurons was measured quantitatively as the [Ca2+]i values of the second KCl (2.KCl) stimulation divided by those of the first KCl (1.KCl) stimulation, giving the value of the ratio (2.KCl/1.KCl). Exposure to EAAs led to an increase in [Ca2+]i, but no simple correlation between the increase in [Ca2+]i and neuronal responsiveness could be demonstrated. Rather, below a threshold level of [Ca2+]i (ca. 1 microM), the neuronal responsiveness was largely independent of the glutamate receptor-agonist-induced increase in [Ca2+]i. However, when [Ca2+]i increased above this threshold level, the neurons almost invariably lost the ability to respond to a K(+)-induced depolarization, particularly after exposure to glutamate. Therefore, the cortical neurons were found to be exceptionally vulnerable to the glutamate-induced loss of function when compared with the effect induced by the glutamate receptor subtype-specific agonists, N-methyl-D-aspartate, quisqualate, and 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionate. The findings suggest that the loss of neuronal membrane polarization precedes plasma membrane disruption and is a sensitive marker of EAA-induced neurodegeneration observed at the single-cell level.
DNA recognition code of transcription factors in the helix-turn-helix, probe helix, hormone receptor, and zinc finger families.
Authors: Suzuki M , Yagi N
Published: May 2006

We have previously reported that in four transcription factor families the DNA-recognition rules can be described as (i) chemical rules, which list possible pairings between the 20 amino acid residues and the four DNA bases, and (ii) stereochemical rules, which describe the base and amino acid positions in contact. We have incorporated these rules into a computer program and examined the nature of the rules. Here we conclude that the DNA recognition rules are simple, logical, and consistent. The rules are specific enough to predict DNA-binding characteristics from a protein sequence.
Short-lived complexes between myelin basic protein peptides and IAk.
Authors: Mason K , McConnell H M
Published: May 2006

Kinetic rate constants and the equilibrium dissociation constant have been determined for the reaction between an affinity-purified class II major histocompatibility complex molecule IAk and a myelin basic protein analogue peptide, fluorescein-labeled Ac(1-14)A4C15. Under the experimental conditions used, the lifetime of the peptide-free IAk molecule with respect to inactivation is 3.1 hr. The equilibrium dissociation constant, 3.3 +/- 1.7 microM, is determined from measurements of the kinetics of peptide inhibition of IAk inactivation. The measured peptide dissociation halftime is relatively short, 30 min, and the deduced association rate is 100 M-1.s-1. The rate constants and the equilibrium constant are similar to those characteristic of kinetic intermediates in reactions of peptides and class II proteins that lead to long-lived terminal complexes.
Interleukin 12 induces the differentiation of major histocompatibility complex class I-primed cytotoxic T-lymphocyte precursors into allospecific cytotoxic effectors.
Authors: Chouaib S , Chehimi J , Bani L , Genetet N , Tursz T , Gay F , Trinchieri G , Mami-Chouaib F
Published: May 2006

The production of interleukin 12 (IL-12) following allogeneic stimulation and its involvement in the differentiation of allospecific cytotoxic T lymphocytes (CTLs) have been investigated. Supernatants of mixed lymphocyte cultures had detectable levels of IL-12 p40 which were completely abrogated after depletion of responder cells from monocytes. While addition to the culture of anti-IL-12 neutralizing antibodies partially inhibited the allogeneic proliferative response and the subsequent CTL activity, addition of IL-12 stimulated both responses, suggesting that endogenously produced IL-12 plays a role in the development of alloreactivity. Furthermore, using primary mixed cultures of lymphocytes from major histocompatibility complex-recombinant siblings identical for class II antigens and displaying class I disparity, we demonstrated that addition of recombinant IL-12 at the sensitizing phase of the primary mixed lymphocyte culture induced CTL activity. Under these stimulation conditions, addition of recombinant IL-12 also triggered cell proliferation, indicating that IL-12 provides both growth and differentiation signals. The mechanism underlying this process does not appear to require IL-2, since IL-12-mediated CTL generation was not abrogated by anti-IL-2 alpha-chain antibodies. IL-12 increased granzyme B and perforin mRNA accumulation in major histocompatibility complex class I-primed lymphocytes, suggesting that this cytokine activates these two genes in CTL precursors. We conclude that IL-12 can stimulate the generation of alloreactive CTLs. We suggest that IL-12 may play a role in helper cell-independent CTL generation.
A Drosophila protein homologous to the human p70 Ku autoimmune antigen interacts with the P transposable element inverted repeats.
Authors: Beall E L , Admon A , Rio D C
Published: May 2006

P transposable elements in Drosophila are mobilized via a cut-and-paste mechanism. This mode of transposition requires repair of both a double-strand break at the donor DNA site and gapped DNA at the target site. Biochemical studies have identified a cellular non-P element-encoded DNA binding protein, termed the inverted repeat binding protein (IRBP), that specifically interacts with the outer half of the 31-bp terminal inverted repeats. Protein sequence information was used to isolate cDNA clones encoding IRBP. Sequence analysis shows that IRBP is related to the 70-kDa subunit of the human Ku autoimmune antigen. The mammalian Ku antigen binds free DNA termini and has been implicated in immunoglobulin VDJ recombination, DNA repair, and transcription. In addition, Ku is the DNA binding subunit of the double-strand DNA-dependent protein kinase. Cytogenetic mapping indicates that the IRBP gene maps to chromosomal position 86E on the right arm of the third chromosome.
Pax-3 contains domains for transcription activation and transcription inhibition.
Authors: Chalepakis G , Jones F S , Edelman G M , Gruss P
Published: May 2006

Pax-3 is a member of the Pax family of transcription factors involved in transcriptional control events during embryonic development. Here we report a functional dissection of the Pax-3 protein and describe the protein domains which are responsible for different activities. A transcription inhibition activity is located in the first 90 N-terminal amino acids and includes part of the paired domain. Furthermore, the C terminus of Pax-3 is able to confer transcriptional activation of basal promoters. Pax-3 can utilize both transcription modulating functions and activates transcription over a narrow range of protein concentration in the presence of promoter elements containing functional binding sites.
Biosynthesis of a defensive insect alkaloid: epilachnene from oleic acid and serine.
Authors: Attygalle A B , Blankespoor C L , Eisner T , Meinwald J
Published: May 2006

The biosynthesis of the azamacrolide epilachnene by the coccinellid beetle Epilachna varivestis has been studied with 2H-labeled oleic acid, 2H-labeled L-serine, and 13C,15N-labeled L-serine. The incorporation of these precursors into epilachnene defines the origin of the alkaloid's entire carbon/nitrogen skeleton. GC/MS and GC/IR studies of alkaloid produced by Epilachna fed with deuteriated oleic acid show that oleic acid loses four carbon atoms from its carboxyl end during the biosynthesis. Other details, including the mechanism of carbon-nitrogen bond formation between the fatty acid and amino acid moieties, remain to be established.
Construction of a bisaquo heme enzyme and binding by exogenous ligands.
Authors: McRee D E , Jensen G M , Fitzgerald M M , Siegel H A , Goodin D B
Published: May 2006

The crystal structure of the His-175-->Gly (H175G) mutant of cytochrome-c peroxidase (EC 1.11.1.5), missing its only heme ligand, reveals that the histidine is replaced by solvent to give a bisaquo heme protein. This protein retains some residual activity, which can be stimulated or inhibited by addition of exogenous ligands. Structural analysis confirms the binding of imidazole to the heme at the position of the wild-type histidine ligand. This imidazole complex reacts readily with hydrogen peroxide to produce a radical species with novel properties. However, reactivation in this complex is incomplete (approximately 5%), which, in view of the very similar structures of the wild-type and the H175G/imidazole forms, implies a critical role for tethering of the axial ligand in catalysis. This study demonstrates the feasibility of constructing heme enzymes with no covalent link to the protein and with unnatural ligand replacements. Such enzymes may prove useful in studies of electron transfer mechanisms and in the engineering of novel heme-based catalysts.
Interferon gamma-independent effects of interleukin 12 administered during acute or established infection due to Leishmania major.
Authors: Wang Z E , Zheng S , Corry D B , Dalton D K , Seder R A , Reiner S L , Locksley R M
Published: May 2006

Interleukin 12 (IL-12) is a powerful stimulus for the growth of activated T and natural killer cells, their generation of interferon gamma (IFN-gamma), and the differentiation of T helper type 1 (Th1) effector cells from naive precursors in vitro. These activities are consistent with the capacity of exogenous IL-12 to heal otherwise susceptible BALB/c mice infected with the intramacrophage parasite Leishmania major. Using this characterized model of CD4 cell subset differentiation, we examined the immunologic effects of IL-12 administered either at the time of infection, when naive T cells are primed, or after 14 days of infection, by which time CD4+ subset differentiation has occurred. Given with the inoculation of parasites, IL-12 induced IFN-gamma and IL-10 and markedly suppressed IL-4. Effects on IL-10 and IL-4 were comparable in mice with homozygous disruption of the IFN-gamma gene (IFN-gamma 0/0), and suppression of IL-4 was unchanged by administration of neutralizing anti-IL-10 antibody. Induction of IFN-gamma and IL-10 mRNA by IL-12 also occurred in infected SCID mice. Given after day 14 of infection, however, IL-12 not only induced IFN-gamma and IL-10 but also induced IL-4 in normal and IFN-gamma 0/0 mice. These data demonstrate direct effects of IL-12 independent of IFN-gamma, IL-10, and IL-4 and demonstrate that the ineffectiveness of IL-12 administered following infection with L. major correlates with resistance of differentiated Th2 cells to the IL-4-suppressing activity of IL-12.
Ablation of islet endocrine cells by targeted expression of hormone-promoter-driven toxigenes.
Authors: Herrera P L , Huarte J , Zufferey R , Nichols A , Mermillod B , Philippe J , Muniesa P , Sanvito F , Orci L , Vassalli J D
Published: May 2006

Ontogenic relationships between the different types of endocrine cells in the islets of Langerhans were explored by generating transgenic mouse embryos in which cells transcribing the glucagon, insulin, or pancreatic polypeptide genes were destroyed through the promoter-targeted expression of the diphtheria toxin A chain. Embryos lacking glucagon- or insulin-containing cells did not exhibit alterations in the development of the nontargeted islet cell types, whereas embryos lacking pancreatic polypeptide gene-expressing cells also lacked pancreatic insulin- and somatostatin-containing cells. These results show that neither glucagon nor insulin gene-expressing cells are essential for the differentiation of the other islet endocrine-cell types. These results also suggest that pancreatic polypeptide gene-expressing cells are indispensable for the differentiation of islet beta and delta cells because the former produce a necessary paracrine or endocrine factor and/or operate through a cell-lineage relationship.
Cholangiocytes express the aquaporin CHIP and transport water via a channel-mediated mechanism.
Authors: Roberts S K , Yano M , Ueno Y , Pham L , Alpini G , Agre P , LaRusso N F
Published: May 2006

Cholangiocytes line the intrahepatic bile ducts and regulate salt and water secretion during bile formation, but the mechanism(s) regulating ductal water movement remains obscure. A water-selective channel, the aquaporin CHIP, was recently described in several epithelia, so we tested the hypothesis that osmotic water movement by cholangiocytes is mediated by CHIP. Isolated rodent cholangiocytes showed a rapid increase in volume in the presence of hypotonic extracellular buffers; the ratio of osmotic to diffusional permeability coefficients was > 10. The osmotically induced increase in cholangiocyte volume was inversely proportional to buffer osmolality, independent of temperature, and reversibly blocked by HgCl2. Also, the luminal area of isolated, enclosed bile duct units increased after exposure to hypotonic buffer and was reversibly inhibited by HgCl2. RNase protection assays, anti-CHIP immunoblots, and immunocytochemistry confirmed that CHIP transcript and protein were present in isolated cholangiocytes but not in hepatocytes. These results demonstrate that (i) isolated cholangiocytes and intact, polarized bile duct units manifest rapid, mercury-sensitive increases in cell size and luminal area, respectively, in response to osmotic gradients and (ii) isolated cholangiocytes express aquaporin CHIP at both the mRNA and the protein level. The data implicate aquaporin water channels in the transcellular movement of water across cholangiocytes lining intrahepatic bile ducts and provide a plausible molecular explanation for ductal water secretion.
Overexpression of the gene encoding the multidrug resistance-associated protein results in increased ATP-dependent glutathione S-conjugate transport.
Authors: Müller M , Meijer C , Zaman G J , Borst P , Scheper R J , Mulder N H , de Vries E G , Jansen P L
Published: May 2006

The multidrug resistance-associated protein (MRP) is a 180- to 195-kDa glycoprotein associated with multidrug resistance of human tumor cells. MRP is mainly located in the plasma membrane and it confers resistance by exporting natural product drugs out of the cell. Here we demonstrate that overexpression of the MRP gene in human cancer cells increases the ATP-dependent glutathione S-conjugate carrier activity in plasma membrane vesicles isolated from these cells. The glutathione S-conjugate export carrier is known to mediate excretion of bivalent anionic conjugates from mammalian cells and is thought to play a role in the elimination of conjugated xenobiotics. Our results suggest that MRP can cause multidrug resistance by promoting the export of drug modification products from cells and they shed light on the reported link between drug resistance and cellular glutathione and glutathione S-transferase levels.
Hidden Markov models of biological primary sequence information.
Authors: Baldi P , Chauvin Y , Hunkapiller T , McClure M A
Published: May 2006

Hidden Markov model (HMM) techniques are used to model families of biological sequences. A smooth and convergent algorithm is introduced to iteratively adapt the transition and emission parameters of the models from the examples in a given family. The HMM approach is applied to three protein families: globins, immunoglobulins, and kinases. In all cases, the models derived capture the important statistical characteristics of the family and can be used for a number of tasks, including multiple alignments, motif detection, and classification. For K sequences of average length N, this approach yields an effective multiple-alignment algorithm which requires O(KN2) operations, linear in the number of sequences.
Enhancement in amount of P1 (hsp60) in mutants of Chinese hamster ovary (CHO-K1) cells exhibiting increases in the A system of amino acid transport.
Authors: Jones M , Gupta R S , Englesberg E
Published: May 2006

Mutants of CHO-K1 cells with varied levels of A system activity, probably the result of increases in absolute amount of the A system transporter, have corresponding increases in levels of peptides banding at 62-66 and 29 kDa. Mutant alar4-H3.9, showing the highest increase of A system activity and of 62- to 66- and 29-kDa peptides, was selected for this study. The N terminus 16-amino acid sequence of the 62- to 66-kDa peptide(s) of this mutant showed between 80% and 100% identity with the mammalian mitochondrial 60-kDa heat shock protein P1 (hsp60). Two-dimensional gel electrophoresis of the 62- to 66-kDa band showed two major, a minor, and several smaller spots (of same mass but different pI values) for both wild type (WT) and mutant, with the two major spots being of greater density in the mutant. Immunoblots with antibody to P1 identified the two major and minor peptides as P1 related. Two-dimensional gels of whole cell extracts of the WT and alar4-H3.9 confirmed these findings and indicated that the two major bands of the mutant were 2.4 times as abundant as that found for the WT. A plasma membrane fraction of the mutant, exhibiting 4.8 times more A system activity than the WT, contained 3.6 times as much P1 as the WT. Immunoblots with antibodies to P1, mitochondrial malate dehydrogenase, and to the mitochondrial F1/F0-ATPase demonstrated that the increased amount of P1 observed in the mutant was not the result of increases in amount of mitochondrial protein. Northern blot analysis demonstrated that the mutant had 2.5 times as much mRNA for P1 as the WT. The close analogy with the relationship between A system and Na+,K(+)-ATPase suggests that there is a coordinate regulation of the A system of amino acid transport, Na+,K(+)-ATPase, and P1 protein, probably as a result of mutation in a shared regulatory element. The possible role of P1 in A system function is discussed.
A proposed superfamily of transposase genes: transposon-like elements in ciliated protozoa and a common "D35E" motif.
Authors: Doak T G , Doerder F P , Jahn C L , Herrick G
Published: May 2006

The transposon-like elements TBE1, Tec1, and Tec2 of hypotrichous ciliated protozoa appear to encode a protein that belongs to the IS630-Tc1 family of transposases. The Anabaena IS895 transposase also is placed in this family. We note that most family members transpose into the dinucleotide target, TA, and that members with eukaryotic hosts have a tendency for somatic excision that is carried to an extreme by the ciliate elements. Alignments including the additional members, and also mariner elements, show that transposases of this family share strongly conserved residues in a large C-terminal portion, including a fully conserved dipeptide, Asp-Glu (DE), and a block consisting of a fully conserved Asp and highly conserved Glu, separated by 34 or 35 residues (D35E). This D35E motif likely is homologous to the previously characterized D35E motif of the family of retroviral-retrotransposon integrases and IS3-like transposases. Because it is known that the IS3-retroposon D35E region is a critical portion of a domain capable of various in vitro transposition-related reactions, the results suggest that the two families share homologous catalytic transposase domains and that members of both families may share a common transposition mechanism.
The protein phosphatase inhibitor calyculin A mimics elicitor action in plant cells and induces rapid hyperphosphorylation of specific proteins as revealed by pulse labeling with [33P]phosphate.
Authors: Felix G , Regenass M , Spanu P , Boller T
Published: May 2006

Suspension-cultured tomato cells react to microbial signals, so-called elicitors, with rapid alkalinization of the growth medium and increased biosynthesis of the stress hormone ethylene. These responses to elicitors can be blocked by staurosporine and K-252a, two specific inhibitors of protein kinases. Here we show that calyculin A, a potent inhibitor of protein phosphatases, mimics the action of elicitors and, at nanomolar concentrations, induces medium alkalinization as well as a strong increase in the activity of 1-aminocyclopropane-1-carboxylate synthase, the key enzyme of ethylene biosynthesis. Both responses were strongly inhibited by K-252a, and calyculin A induced both responses more rapidly than did a fungal elicitor, xylanase. For example, the lag phase for medium alkalinization was only 0.2-0.4 min for calyculin A, compared with 2 min for xylanase. To study changes in the dynamics of protein phosphorylation, cells were labeled with 30-sec pulses of [33P]orthophosphate. Calyculin A strongly increased phosphorylation of several polypeptide bands within 40 sec of treatment. The same phosphorylated bands also appeared in response to xylanase, but only after a lag phase of 2-3 min. These results show that the protein phosphatase inhibitor calyculin A leads to rapid hyperphosphorylation of specific proteins in cultured cells and indicate that elicitor action could be based on inhibition of a protein phosphatase as well as on activation of a protein kinase.
Humanization of murine monoclonal antibodies through variable domain resurfacing.
Authors: Roguska M A , Pedersen J T , Keddy C A , Henry A H , Searle S J , Lambert J M , Goldmacher V S , Blättler W A , Rees A R , Guild B C
Published: May 2006

Two murine monoclonal antibodies, N901 (anti-CD56) and anti-B4 (anti-CD19), were humanized by a process we call "resurfacing." A systematic analysis of known antibody structures has been used to determine the relative solvent accessibility distributions of amino acid residues in murine and human antibody variable (Fv) regions and has shown that the sequence alignment positions of surface amino acids for human and murine variable region heavy (VH) and light (VL) chains are conserved with 98% fidelity across species. While the amino acid usage at these surface positions creates surface residue patterns that are conserved within species, there are no identical patterns across species. However, surprisingly few amino acid changes need to be made to convert a murine Fv surface pattern to that characteristic of a human surface. Resurfacing was used to change the patterns of surface accessible residues in the Fv regions of the N901 and anti-B4 antibodies to resemble those found on the Fv regions of human antibody sequences. Two different procedures for selecting a human sequence were compared. For anti-B4, a data base of clonally derived human VL-VH sequence pairs was used, while for N901, sequences for VL and VH were independently selected from the Kabat et al. data base [Kabat, E. A., Wu, T. T., Reid-Miller, M., Perry, H. M. & Gottesman, K. S. (1991) Sequences of Proteins of Immunological Interest (DHHS, Washington, DC), 5th Ed.]. Resurfaced N901 and anti-B4 antibodies had apparent affinities for their cell surface ligands that were identical to those of their respective parent murine antibodies. These data provide evidence that, despite the differences in the surfaces of mouse and human Fv regions, it is possible to substitute one for the other while retaining full antigen binding affinity.
Binocular disparity discrimination in human cerebralcortex: functional anatomy by positron emission tomography.
Authors: Gulyás B , Roland P E
Published: May 2006

Neurobiological studies in higher primates
indicate that the processing of stereoscopic information takes place at early
levels in the visual cortex. To map the anatomical structures in the human brain
participating in pure stereopsis based upon binocular disparity, we measured
with positron emission tomography the changes in regional cerebral blood flow as
an indicator of metabolic activity in 10 healthy young men during visual
discrimination of binocular disparity. The data demonstrate that the
discrimination of pure stereo-optic disparity information takes place in the
polar striate cortex and the neighboring peri-striate cortices, as well as in
the parietal lobe, the prefrontal cortex, and the cerebellum. The discrimination
of stereoscopic depth is dependent on a network composed of multiple functional
fields localized in occipital- and parietal-lobe visual areas as well as in the
dorsolateral and mesial prefrontal cortex. The findings support the importance
of coactivated occipitoparietal visual areas in the processing and analysis of
binocular depth information in humans.
Evolutionary conservation of the AU-rich 3'untranslated region of messenger RNA.
Authors: Asson-Batres M A , Spurgeon S L , Diaz J , DeLoughery T G , Bagby G C
Published: May 2006

AU-rich sequence motifs (specifically sequences
containing reiterations of AUUUA) are found in the 3' untranslated region
of mammalian mRNAs encoding cytokines, adhesion molecules, and protooncogenes.
Because these AU-rich elements (3'AURE) have been observed to reduce the
stability and translational efficiency of transcripts that contain them, and
because many of these transcripts accumulate in cells exposed to inflammatory
stimuli, we reasoned that mRNAs with 3'AURE may be highly conserved and
that the AURE is a marker of mRNAs that are inducible by environmental
stressors. To test this hypothesis, we developed a polymerase chain reaction
(PCR) strategy to isolate specifically mRNAs with 3'AURE. We first
validated the effectiveness of this approach by selectively amplifying two mRNAs
containing 3'AURE from interleukin 1 (IL-1)-induced human endothelial
cells, then used the same primers in reverse transcriptase-PCR of sea urchin
RNA, and used the radiolabeled reaction products to screen a cDNA library
prepared from endotoxin-exposed sea urchin coelomocytes. We identified 124
positive clones and isolated a 1608-base-pair fragment that contains an AU-rich
consensus sequence upstream from a poly(A) tail. This sea urchin transcript
hybridizes with immobilized poly(A)(+)-selected RNA prepared from living
coelomocytes maintained in vitro for 8.5-13 h but not with RNA prepared from
freshly harvested coelomocytes. Our results provide support for the growing body
of evidence that 3' AURE are both conserved and functional and indicate
further that isolation and short-term in vitro culture of sea urchin
coelomocytes is sufficient to induce the expression of transcripts containing
3'AURE.
Formation of two hydrogen bonds from the globin to theheme-linked oxygen molecule in Ascaris hemoglobin.
Authors: De Baere I , Perutz M F , Kiger L , Marden M C , Poyart C
Published: May 2006

We have tried to find out why Ascaris hemoglobin
has such an exceptionally high oxygen affinity (P50 approximately 0.004 mmHg; 1
mmHg = 133 Pa). Following Kloek et al., we have synthesized the N-terminal
globin domain of Ascaris hemoglobin in Escherichia coli [Kloek, A. P., Yang, J.,
Mathews, F. S. & Goldberg, D. (1993) J. Biol. Chem. 268, 17669-17671]. Like
Kloek et al., we found its oxygen affinity to be as high as that of native
Ascaris hemoglobin. We thought that this high affinity might be due to the
heme-bound oxygen molecule being stabilized by two hydrogen bonds from the
globin instead of the usual one. Ascaris hemoglobin has a distal glutamine
instead of the more usual histidine as one of the potential hydrogen bond
donors. In addition, it contains a tyrosine at position 10 of B helix (B10) in
place of the leucine generally found there in vertebrate myoglobins and
hemoglobins. Following the discovery of Carver et al. that sperm whale myoglobin
with the replacement of leucine B10 by phenylalanine has a raised oxygen
affinity, we have replaced tyrosine B10 in the N-terminal domain of Ascaris
hemoglobin by either leucine or phenylalanine [Carver, T. E., Brantley, R. E.,
Jr., Singleton, E. W., Arduini, R. M., Quillin, H. L., Phillips, G. N., Jr.,
& Olson, J. S. (1992) J. Biol. Chem. 267, 14443-14450]. Either of these
replacements lowered the oxygen affinity about 100-fold, to the same level of
that of human alpha-globin chains. These results are consistent with a hydrogen
bond linking the tyrosine hydroxyl to the heme-linked oxygen, with a bond energy
of 2.7 kcal/mol.
K+ currents expressed from the guinea pig cardiac IsKprotein are enhanced by activators of protein kinase C.
Authors: Zhang Z J , Jurkiewicz N K , Folander K , Lazarides E , Salata J J , Swanson R
Published: May 2006

We have isolated cardiac cDNA and genomic clones
encoding the guinea pig IsK protein. The deduced amino acid sequence is
approximately 78% identical to the rat, mouse, and human variants of this
channel, and the structure of the gene encoding the protein is also similar to
that in other species. For example, the gene is present only once in the haploid
genome, the protein-coding sequence is present on a single uninterrupted exon,
an intron exists in the 5' untranslated domain, and multiple alternative
polyadenylation sites are used in processing the transcript. Expression of the
guinea pig protein in Xenopus oocytes results in a slowly activating,
voltage-dependent K+ current, IsK, similar to those expressed previously from
the rat, mouse, and human genes. However, in sharp contrast to the rat and mouse
currents, activation of protein kinase C with phorbol esters increases the
amplitude of the guinea pig IsK current, analogous to its effects on the
endogenous IKs current in guinea pig cardiac myocytes. Mutagenesis of the guinea
pig cDNA to alter four cytoplasmic amino acid residues alters the phenotype of
the current response to protein kinase C from enhancement to inhibition,
mimicking that of rat and mouse IsK currents. This mutation is consistent with
reports that phosphorylation of Ser-102 by protein kinase C decreases the
current amplitude. These data explain previously reported differences in the
regulatory properties between recombinant rat or mouse IsK channels and native
guinea pig IKs channels and provide further evidence that the IsK protein forms
the channels that underlie the IKs current in the heart.
The type I macrophage scavenger receptor binds togram-positive bacteria and recognizes lipoteichoic acid.
Authors: Dunne D W , Resnick D , Greenberg J , Krieger M , Joiner K A
Published: May 2006

Macrophage scavenger receptors exhibit unusually
broad binding specificity for polyanionic ligands and have been implicated in
atherosclerosis and various host defense functions. Using a radiolabeled,
secreted form of the type I bovine macrophage scavenger receptor in an in vitro
binding assay, we have found that this receptor binds to intact Gram-positive
bacteria, including Streptococcus pyogenes, Streptococcus agalactiae,
Staphylococcus aureus, Enterococcus hirae, and Listeria monocytogenes.
Competition binding studies using purified lipoteichoic acid, an anionic polymer
expressed on the surface of most Gram-positive bacteria, show that lipoteichoic
acids are scavenger receptor ligands and probably mediate binding of the
receptor to Gram-positive bacteria. Lipoteichoic acids, for which no host cell
receptors have previously been identified, are implicated in the pathogenesis of
septic shock due to Gram-positive bacteria. Scavenger receptors may participate
in host defense by clearing lipoteichoic acid and/or intact bacteria from
tissues and the circulation during Gram-positive sepsis. Since scavenger
receptors have been previously shown to bind to and facilitate bloodstream
clearance of Gram-negative bacterial endotoxin (lipopolysaccharide), these
receptors may provide a general mechanism for macrophage recognition and
internalization of pathogens and their cell surface components.
Sequence-specific "gene signatures" can beobtained by PCR with single specific primers at low stringency.
Authors: Pena S D , Barreto G , Vago A R , De Marco L , Reinach F C , Dias Neto E , Simpson A J
Published: May 2006

Low-stringency single specific primer PCR
(LSSP-PCR) is an extremely simple PCR-based technique that detects single or
multiple mutations in gene-sized DNA fragments. A purified DNA fragment is
subjected to PCR using high concentrations of a single specific oligonucleotide
primer, large amounts of Taq polymerase, and a very low annealing temperature.
Under these conditions the primer hybridizes specifically to its complementary
region and nonspecifically to multiple sites within the fragment, in a
sequence-dependent manner, producing a heterogeneous set of reaction products
resolvable by electrophoresis. The complex banding pattern obtained is
significantly altered by even a single-base change and thus constitutes a unique
"gene signature." Therefore LSSP-PCR will have almost unlimited
application in all fields of genetics and molecular medicine where rapid and
sensitive detection of mutations and sequence variations is important. The
usefulness of LSSP-PCR is illustrated by applications in the study of mutants of
smooth muscle myosin light chain, analysis of a family with X-linked nephrogenic
diabetes insipidus, and identity testing using human mitochondrial
DNA.
Vasoactive intestinal peptide increases intracellular calcium in astroglia: synergism with alpha-adrenergic receptors.
Authors: Fatatis A , Holtzclaw L A , Avidor R , Brenneman D E , Russell J T
Published: May 2006

In type I astrocytes from rat cerebral cortex, vasoactive intestinal peptide (VIP) at concentrations below 1 nM evoked an increase in intracellular calcium ion concentration. This response, however, was observed in only 18% of the astrocytes examined. alpha-Adrenergic stimulation with phenylephrine or norepinephrine also resulted in an intracellular calcium response in these cells and the threshold sensitivity of astrocytes to phenylephrine was vastly different from cell to cell. Treatment of these astrocytes with VIP (0.1 nM) together with phenylephrine at subthreshold concentrations produced large increases in intracellular Ca2+ concentration ([Ca2+]i) and oscillations. The continued occupation of the alpha-adrenergic receptor was required for sustained synergism. Both alpha-receptor stimulation and stimulation with the mixture of agonists induced the cellular calcium response by triggering release of calcium from cellular stores, since the response persisted in the absence of extracellular calcium. Furthermore, thapsigargin pretreatment, which depletes intracellular stores, abolished the agonist-induced [Ca2+]i response. VIP (0.1 nM) and phenylephrine were found to increase cellular levels of inositol phosphates; however, there was no apparent additivity in this response when the agonists were added together. These observations suggest a calcium-mediated second messenger system for the high-affinity VIP receptor in astrocytes and that alpha-adrenergic receptors act synergistically with the VIP receptor to augment an intracellular calcium signal. The synergism between diverse receptor types may constitute an important mode of cellular signaling in astroglia.
Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation.
Authors: Vane J R , Mitchell J A , Appleton I , Tomlinson A , Bishop-Bailey D , Croxtall J , Willoughby D A
Published: May 2006

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin H2, which is further metabolized to prostanoids. Two isoforms of COX exist: a constitutive (COX-1) and an inducible (COX-2) enzyme. Nitric oxide is derived from L-arginine by isoforms of nitric-oxide synthase (NOS; EC 1.14.13.39): constitutive (cNOS; calcium-dependent) and inducible (iNOS; calcium-independent). Here we have investigated inducible isoforms of COX and NOS in the acute, chronic, and resolving stages of a murine air pouch model of granulomatous inflammation. COX and NOS activities were measured in skin samples in the acute phase, up to 24 h. Activities in granulomatous tissue were measured at 3, 5, 7, 14, and 21 days for the chronic and resolving stages of inflammation. COX-1 and COX-2 proteins were assessed by Western blot. COX activity in the skin increased over the first 24 h and continued to rise up to day 14. COX-2 protein rose progressively, also peaking at day 14. COX-1 protein remained unaltered throughout. The iNOS activity increased over the first 24 h in the skin, with a further major increase in the granulomatous tissue between days 3 and 7, followed by a decrease at day 14 and a further increase at day 21. The rise in COX and NOS activities in the skin during the acute phase reinforces the proinflammatory role for prostanoids and suggests one also for nitric oxide. However, in the chronic and resolving stages, a dissociation of COX and NOS activity occurred. Thus, there may be differential regulation of these enzymes, perhaps due to the changing pattern of cytokines during the inflammatory response.
Manifold sequencing: efficient processing of large sets of sequencing reactions.
Authors: Lagerkvist A , Stewart J , Lagerström-Fermér M , Landegren U
Published: May 2006

Automated instruments for DNA sequencing greatly simplify data collection in the Sanger sequencing procedure. By contrast, the so-called front-end problems of preparing sequencing templates, performing sequencing reactions, and loading these on the instruments remain major obstacles to extensive sequencing projects. We describe here the use of a manifold support to prepare and perform sequencing reactions on large sets of templates in parallel, as well as to load the reaction products on a sequencing instrument. In this manner, all reaction steps are performed without pipetting the samples. The strategy is applied to sequencing PCR-amplified clones of the human mitochondrial D-loop and for detection of heterozygous positions in the human major histocompatibility complex class II gene HLA-DQB, amplified from genomic DNA samples. This technique will promote sequencing in a clinical context and could form the basis of more efficient genomic sequencing strategies.
Temporal phosphorylation of the Drosophila period protein.
Authors: Edery I , Zwiebel L J , Dembinska M E , Rosbash M
Published: May 2006

The period gene (per) is required for Drosophila melanogaster to manifest circadian (congruent to 24 hr) rhythms. We report here that per protein (PER) undergoes daily oscillations in apparent molecular mass as well as abundance. The mobility changes are largely or exclusively due to multiple phosphorylation events. The temporal profile of the classic short-period form of PER (PERS) is altered in a manner consistent with the mutant strain's behavioral phenotype. As changes in abundance and phosphorylation persist under constant environmental conditions, they reflect or contribute to a free-running rhythm. We suggest that the phosphorylation status of PER is an important determinant in the Drosophila clock's time-keeping mechanism.
Complete RNA editing of unspliced and dicistronic transcripts of the intron-containing reading frame IRF170 from maize chloroplasts.
Authors: Ruf S , Zeltz P , Kössel H
Published: May 2006

The maize plastome harbors within the rps4-rps14 gene cluster the reading frame IRF170, which is interrupted by two introns. Although the function of the encoded peptide of 170 amino acids is not known, the conservation of IRF170 homologs in other plastomes is a strong indication that IRF170 is a functional gene. Amplification and sequence analyses of IRF170 specific cDNAs reveals two C-to-U editing events occurring within each of the first two exons. This situation allows an analysis of the temporal order between editing and splicing of a chloroplast transcript. By using intron-specific primer combinations, cDNAs derived from partially or even unspliced IRF170 transcripts could be amplified which in all cases showed complete editing. Complete editing was also observed with a cDNA derived from a transcript in which the proximal rps4 and the 5' half of IRF170-encoded sequences were still linked. This demonstrates that editing of the IRF170 transcript is an early processing step preceding both splicing and cleavage to monocistronic mRNA.