bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromophore-protein interactions and the function of the photosynthetic reaction center: a molecular dynamics study.
Authors: Treutlein H , Schulten K , Brünger A T , Karplus M , Deisenhofer J , Michel H
Published: May 2006

The coupling between electron transfer and protein structure and dynamics in the photosynthetic reaction center of Rhodopseudomonas viridis is investigated. For this purpose molecular dynamics simulations of the essential portions (a segment of 5797 atoms) of this protein complex have been carried out. Electron transfer in the primary event is modeled by altering the charge distributions of the chromophores according to quantum chemical calculations. The simulations show (i) that fluctuations of the protein matrix, which are coupled electrostatically to electron transfer, play an important role in controlling the electron transfer rates and (ii) that the protein matrix stabilizes the separated electron pair state through rapid (200 fs) and temperature-independent dielectric relaxation. The photosynthetic reaction center resembles a polar liquid in that the internal motions of the whole protein complex, rather than only those of specific side groups, contribute to i and ii. The solvent reorganization energy is about 4.5 kcal/mol. The simulations indicate that rather small structural rearrangements and changes in motional amplitudes accompany the primary electron transfer.
Endoproteolytic processing of a farnesylated peptide in vitro.
Authors: Ashby M N , King D S , Rine J
Published: May 2006

Numerous eukaryotic proteins containing a carboxyl-terminal CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid) require a three-step posttranslational processing for localization and function. The a mating factor of Saccharomyces cerevisiae is one such protein, requiring cysteine farnesylation, proteolysis of the terminal three amino acids, and carboxyl methylation for biological activity. We have used farnesylated a-factor peptides to examine the proteolytic step in the maturation of CAAX-containing proteins. Three distinct carboxyl-terminal protease activities were found in yeast cell extracts that could remove the terminal three residues of a-factor. Two of the proteolytic activities were in cytosolic fractions. One of these activities was a PEP4-dependent carboxypeptidase that was sensitive to phenylmethylsulfonyl fluoride. The other cytosolic activity was PEP4-independent, sensitive to 1,10-phenanthroline, and effectively inhibited by an unfarnesylated a-factor peptide. In contrast, a protease activity in membrane fractions was unaffected by phenylmethylsulfonyl fluoride, 1,10-phenanthroline, or unfarnesylated a-factor peptide. Incubation of membrane preparations from either yeast or rat liver with a radiolabeled farnesylated a-factor peptide released the terminal three amino acids intact as a tripeptide, indicating that this reaction occurred by an endoproteolytic mechanism and that the enzyme most likely possesses a broad substrate specificity. The yeast endoprotease was not significantly affected by a panel of protease inhibitors, suggesting that the enzyme is novel. Zinc ion was shown to inhibit the endoprotease (Ki less than 100 microM). The specific activities of the a-factor carboxyl-terminal membrane endoprotease and methyltransferase clearly indicated that the proteolytic reaction was not rate-limiting in these processing reactions in vitro.
In vitro replication of adeno-associated virus DNA.
Authors: Hong G , Ward P , Berns K I
Published: May 2006

An in vitro assay for adeno-associated virus (AAV) DNA replication has been developed. The substrate is a plasmid containing the duplex form of AAV DNA in pBR322. The AAV insert is excised or rescued from the plasmid by extracts of uninfected cells. Replication was assayed by production of full-length excised AAV DNA resistant to Dpn I digestion. The following results were obtained. (i) Only extracts of cells coinfected with AAV and adenovirus replicated the excised insert. (ii) Density label experiments showed semiconservative replication. (iii) Only the excised AAV insert was replicated; pBR322 sequences were not. (iv) Replication was dependent on the presence of the AAV terminal repeat. (v) If the terminal 55 bases were deleted from both ends of the AAV insert, no rescue took place: replication occurred and both AAV and pBR322 sequences were replicated. We conclude that the AAV terminal repeat is essential for DNA replication but that under some conditions an initiation mechanism that does not involve hairpin priming may be used.
Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.
Authors: Uzé G , Lutfalla G , Bandu M T , Proudhon D , Mogensen K E
Published: May 2006

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.
Rat p53 gene mutations in primary Zymbal gland tumors induced by 2-amino-3-methylimidazo[4,5-f]quinoline, a food mutagen.
Authors: Makino H , Ishizaka Y , Tsujimoto A , Nakamura T , Onda M , Sugimura T , Nagao M
Published: May 2006

There are reports of p53 gene mutations in various human cancers but not in rat tumor cell lines or rat primary tumor tissue. We found a p53 gene mutation in a cell line of a spontaneous squamous cell carcinoma of the rat Zymbal gland, SCC131, at codon 171 by direct sequencing of cDNA fragments amplified by PCR. We tested for p53 gene mutations in 15 primary Zymbal gland tumors induced by 2-amino-3-methylimidazo[4,5-f]quinoline by single-strand conformation polymorphism analysis of the PCR-amplified cDNA products. Samples of four tumors showed mobility shifts. Direct sequencing revealed that all these tumors had mutations in conserved regions or in scattered conserved residues. Single-strand conformation polymorphism analysis of cDNA suggested that mRNA from the wild-type allele of the p53 gene was not present in tumor cells of three of four positive cases, although genomic DNA analysis indicated that the wild-type allele was retained in all the cases. All mutations were found at a guanine base: three mutations were guanine----pyrimidine transversions and one was a deletion of a guanine base within a G+C-rich sequence. These findings indicate that 2-amino-3-methylimidazo[4,5-f]quinoline may be directly involved in induction of these mutations by forming DNA adducts at various sites in the p53 gene.
Homozygous hereditary C3 deficiency due to a partial gene deletion.
Authors: Botto M , Fong K Y , So A K , Barlow R , Routier R , Morley B J , Walport M J
Published: May 2006

The molecular mechanism of C3 deficiency in an Afrikaans patient with recurrent pyogenic infections was studied. Restriction enzyme analysis showed a gene deletion of 800 base pairs (bp) mapping to the alpha chain of C3. Amplification of genomic DNA, using the PCR, demonstrated that the deletion included exons 22 and 23 of the C3 gene. Truncated mRNA was shown in an Epstein-Barr virus-transformed B-cell line by PCR amplification of first-strand cDNA. A consequence of this deletion was that the RNA transcribed 3' to the deletion was out of frame, resulting in formation of a stop codon 19 bp downstream from the deletion. The molecular basis of the deletion was compatible with homologous recombination between two Alu sequences located in introns 21 and 23. An unrelated nonconsanguineous relative and two of a sample of 174 Afrikaans-speaking individuals were heterozygous carriers of the same gene deletion. The wide prevalence of this null allele in this population is probably due to the effects of a small founder population. The presence of this deletion in the C3 gene is not compatible with production of any functional C3, supporting the idea that study of such patients offers a valid model for understanding physiological activities of C3 in vivo in humans.
Targeted integration of the Ren-1D locus in mouse embryonic stem cells.
Authors: Miller C C , McPheat J C , Potts W J
Published: May 2006

We have introduced a Ren-1D targeting vector into embryonic stem cells containing the two highly homologous mouse renin genes Ren-1D and Ren-2. Using a polymerase chain reaction (PCR) screen designed to detect targeted integration at Ren-1D and Ren-2, we isolated 15 targeted embryonic stem cell clones, all of which had undergone a gene conversion event at the Ren-1D locus. We did not isolate any clones in which the incoming DNA had recombined with Ren-2. Over the region encompassed by our transgene, Ren-1D and Ren-2 display greater than 95% homology. Our results suggest that the machinery driving gene targeting by means of homologous recombination in mammalian cells is capable of distinguishing between these two sequences. Construction of transgenic mice with the embryonic stem cells reported here carrying a mutated renin gene will permit a greater understanding of the functions of the Ren-1D and Ren-2 gene products and their relative contribution to cardiovascular homeostasis.
Interleukin 6: insights to its function in skin by overexpression in transgenic mice.
Authors: Turksen K , Kupper T , Degenstein L , Williams I , Fuchs E
Published: May 2006

Interleukin 6 (IL-6) is a cytokine that mediates a wide range of inflammatory and immune responses. Its expression is elevated in inflammatory or immunodeficient diseases, including psoriasis, rheumatoid arthritis, and AIDS. To explore the role of IL-6 in skin, we utilized a human keratin 14 (K14) promoter to express IL-6 in the basal cells of stratified squamous epithelia of transgenic mice. Mice expressing the K14-IL-6 transgene were smaller than normal and exhibited retarded hair growth. Surprisingly, IL-6 expression did not lead to enhanced epidermal proliferation, but it did result in a thicker stratum corneum, with an otherwise seemingly normal program of differentiation. IL-6 expression did not lead to leukocytic infiltration, making it unlikely that it has direct proinflammatory activity in skin. Based on this study, one role of IL-6 relevant to host defense may be to enhance the stratum corneum, thereby providing increased protection from injurious stimuli or infection. If IL-6 plays additional roles in the skin, it is likely to act synergistically with factors that IL-6 alone cannot induce.
Genetic dissection of pancreatic trypsin inhibitor.
Authors: Goldenberg D P , Berger J M , Laheru D A , Wooden S , Zhang J X
Published: May 2006

In a previous study, a genetic screening procedure was used to identify variants of bovine pancreatic trypsin inhibitor that can fold to an active conformation but that are inactivated much more rapidly than the wild-type protein in the presence of dithiothreitol (DTT). The mechanisms by which 30 of these DTT-sensitive variants are inactivated have now been investigated. Some of the amino acid replacements cause rapid inactivation in the presence of DTT because the three disulfides of the native protein are reduced up to 300-fold faster than for the wild-type protein, leading to complete unfolding. Other substitutions, however, do not greatly increase the rate of complete reduction and unfolding but lead to accumulation of an inactive two-disulfide species. There is a striking correlation between the locations of the DTT-sensitive amino acid replacements in the three-dimensional structure of the protein and the mechanisms by which the variants are inactivated. All of the substitutions that cause rapid unfolding are clustered at one end of the folded protein, in the vicinity of the two disulfides that are reduced most slowly during unfolding of the wild-type protein, while substitutions of the other class are all located at the other end of the protein, near the trypsin binding site. These results indicate that the kinetic stability of native bovine pancreatic trypsin inhibitor and its ability to function as a protease inhibitor are largely influenced by residues in two distinguishable regions of the folded protein.
Ascorbic acid prevents oxidative stress in glutathione-deficient mice: effects on lung type 2 cell lamellar bodies, lung surfactant, and skeletal muscle.
Authors: Jain A , Mårtensson J , Mehta T , Krauss A N , Auld P A , Meister A
Published: May 2006

Glutathione deficiency in adult mice leads to lung type 2 cell lamellar body and mitochondrial damage; as reported here, these effects are associated with marked decrease of the levels of phosphatidylcholine (the main component of lung surfactant) in the lung and the bronchoalveolar lining fluid. Severe mitochondrial damage was also found in skeletal muscle. Treatment with ascorbate (1-2 mmol per kg of body weight per day), which led to greatly increased (approximately 2-fold) levels of lung and muscle mitochondrial glutathione, prevented damage to lamellar bodies and mitochondria as well as the decline of phosphatidylcholine levels in lung and alveolar lining fluid. The findings indicate that glutathione deficiency leads to depletion of lung surfactant and that this can be prevented with ascorbate. Administration of ascorbate spares glutathione and prevents cellular damage. Lamellar body degeneration in glutathione deficiency appears to be associated with oxidative damage to the perilamellar membrane, which contains the enzymes required for phosphatidylcholine synthesis. It is notable that although severe glutathione deficiency is lethal to newborn rats, which apparently do not synthesize ascorbate, adult mice are better able to survive such a deficiency because they can synthesize ascorbate. The present studies, which suggest that high doses of ascorbate may be of therapeutic value, emphasize that ascorbate and glutathione have actions in common and that they function together in a physiologically significant antioxidant system.
Diversification of the Wnt gene family on the ancestral lineage of vertebrates.
Authors: Sidow A
Published: May 2006

Diversification of the Wnt genes, a family of powerful developmental regulator molecules, is inferred by molecular evolutionary analyses. Fifty-five recently determined partial sequences from a variety of vertebrates and invertebrates, together with 17 published sequences, mostly from the mouse and Drosophila melanogaster, are analyzed. Wnt-1 through -7 originated before the last common ancestor of arthropods and deuterostomes lived. Another round of gene duplication, involving Wnt-3, -5, -7, and -10, occurred after the echinoderm lineage arose, on the ancestral lineage of jawed vertebrates. Increased constraints were imposed on the Wnt genes when jawed vertebrates originated, as indicated by an overall 4-fold lower rate of amino acid replacements in jawed vertebrates compared with invertebrates and jawless vertebrates. The Wnt genes are thus inferred to have undergone a disproportionately high amount of structural and functional evolution in the relatively short time (approximately 100 million years) between the origin of the echinoderm lineage and the first diversification of jawed vertebrates. A model is presented for the relationship of functional diversification of developmental regulators and their rates of amino acid replacement.
Opening of glibenclamide-sensitive K+ channels in follicular cells promotes Xenopus oocyte maturation.
Authors: Wibrand F , Honoré E , Lazdunski M
Published: May 2006

The vasorelaxing K+ channel opener P1060 (a pinacidil analog), gonadotropins, and cAMP were shown to activate a glibenclamide-sensitive 86Rb+ efflux from fully grown follicle-enclosed Xenopus oocytes. Glibenclamide-sensitive K+ channels are located in follicular cells. Glibenclamide (i) depressed the gonadotropin- but not the progesterone-induced maturation and (ii) did not significantly modify progesterone production in oocytes exposed to Xenopus gonadotropin. In follicle-enclosed oocytes, the opener P1060 very significantly enhanced the oocyte sensitivity to progesterone. This increased sensitivity to the hormone induced by the K+ channel opener was reversed by glibenclamide. Thus these results suggest that the opening of glibenclamide-sensitive K+ channels in follicular cells by gonadotropins (and other activators of this channel) induces a hyperpolarization in the oocyte that greatly facilitates maturation by increasing the oocyte sensitivity to progesterone.
Transduction of a foreign histocompatibility gene into the arterial wall induces vasculitis.
Authors: Nabel E G , Plautz G , Nabel G J
Published: May 2006

Autoimmune vasculitis represents a disease characterized by focal inflammation within arteries at multiple sites in the vasculature. Therapeutic interventions in this disease are empirical and often unsuccessful, and the mechanisms of immune injury are not well-defined. The direct transfer of recombinant genes and their expression in the arterial wall provides an opportunity to explore the pathogenesis and treatment of vascular disease. In this report, an animal model for vasculitis has been developed. Inflammation has been elicited by direct gene transfer of a foreign class I major histocompatibility complex gene, HLA-B7, to specific sites in porcine arteries. Transfer and expression of this recombinant gene was confirmed by a polymerase chain reaction and immunohistochemistry, and cytolytic T cells specific for HLA-B7 were detected. These findings demonstrate that expression of a recombinant gene in the vessel wall can induce a focal immune response and suggest that vessel damage induced by cell-mediated immune injury can initiate vasculitis.
Human immunodeficiency virus type 1 infection of neural xenografts.
Authors: Cvetkovich T A , Lazar E , Blumberg B M , Saito Y , Eskin T A , Reichman R , Baram D A , del Cerro C , Gendelman H E , del Cerro M
Published: May 2006

Human immunodeficiency virus type 1 (HIV-1) infection is highly specific for its human host. To study HIV-1 infection of the human nervous system, we have established a small animal model in which second-trimester (11 to 17.5 weeks) human fetal brain or neural retina is transplanted to the anterior chamber of the eye of immunosuppressed adult rats. The human xenografts vascularized, formed a blood-brain barrier, and differentiated, forming neurons and glia. The xenografts were infected with cell-free HIV-1 or with HIV-1-infected human monocytes. Analysis by polymerase chain reaction revealed HIV-1 sequences in DNA from xenograft tissue exposed to HIV-1 virions, and in situ hybridization demonstrated HIV-1 mRNA localized in macrophages and multinucleated giant cells. Pathological damage was observed only in neural xenografts containing HIV-1-infected human monocytes, supporting the hypothesis that these cells mediate neurotoxicity. This small animal model allows the study of direct and indirect effects of HIV-1 infection on developing human fetal neural tissues, and it should prove useful in evaluating antiviral therapies, which must ultimately target HIV-1 infection of the brain.
IgE-secreting cells in the thymus: correlation with induction of tolerance to IgE.
Authors: Haba S , Nisonoff A
Published: May 2006

We have shown previously that normal mice become tolerant to endogenous IgE when they are approximately 2 weeks old and that this corresponds closely with the initial appearance of IgE in serum. Tolerance evidently is restricted to T cells, since B cells responsive to IgE are present in neonatal and adult mice. The present report shows that IgE-secreting cells can be detected in the thymus between days 7 and 11 after birth and that the onset of tolerance to IgE occurs at the age of 11 days. Similar results were obtained in A/J and (BALB/c x A/J)F1 mice. This suggests that tolerance is induced in the thymus, probably by cells bearing peptide fragments of IgE. The order of appearance of IgE-secreting cells is thymus, spleen, and mesenteric lymph nodes.
The phorbol ester-dependent activator of the mitogen-activated protein kinase p42mapk is a kinase with specificity for the threonine and tyrosine regulatory sites.
Authors: Rossomando A , Wu J , Weber M J , Sturgill T W
Published: May 2006

Mitogen-activated protein kinases (MAP kinases) are activated by dual tyrosine and threonine phosphorylations in response to various stimuli, including phorbol esters. To define the mechanism of activation, recombinant wild-type 42-kDa MAP kinase (p42mapk) and a kinase-defective mutant of p42mapk (K52R) were used to assay both activator activity for p42mapk and kinase activity toward K52R in stimulated EL4.I12 mouse thymoma cells. Phorbol 12,13-dibutyrate (10 min, 650 nM) stimulated a single peak of MAP kinase activator that was coeluted from Mono Q at pH 7.5 and 8.9 with K52R kinase activity. Both activities were inactivated by the serine/threonine-specific phosphatase 2A but not by the tyrosine-specific phosphatase CD45. Phosphorylation of K52R occurred specifically on Thr-183 and Tyr-185, as determined by tryptic phosphopeptide mapping in comparison with synthetic marker phosphopeptides. These findings indicate that phorbol ester-stimulated MAP kinase kinase can activate p42mapk by threonine and tyrosine phosphorylations, and that p42mapk thus does not require an autophosphorylation reaction.
Viricidal effect of stimulated human mononuclear phagocytes on human immunodeficiency virus type 1.
Authors: Chase M J , Klebanoff S J
Published: May 2006

Human monocytes stimulated with phorbol 12-myristate 13-acetate or opsonized zymosan in vitro were viricidal to human immunodeficiency virus type 1 (HIV-1) as measured by the inability of the virus to replicate in CEM cells. Monocytes, when stimulated, release myeloperoxidase (MPO) and produce H2O2; MPO reacts with H2O2 and chloride to form hypochlorous acid, a known microbicidal agent. The viricidal activity of stimulated monocytes was inhibited by the peroxidase inhibitor azide, implicating MPO, and by catalase but not heated catalase or superoxide dismutase, implicating H2O2. Stimulated monocytes from patients with chronic granulomatous disease (CGD) or hereditary MPO deficiency were not viricidal to HIV-1 unless they were supplemented with the H2O2-generating enzyme glucose oxidase or MPO, respectively. The viricidal activity of stimulated, glucose oxidase-supplemented CGD monocytes and MPO-supplemented MPO-deficient monocytes, like that of normal stimulated monocytes, was inhibited by azide and catalase. Monocytesmaintained in culture differentiate into macrophages with loss of MPO and decreased H2O2 production. The viricidal activity of 3- to 9-day monocyte-derived macrophages was decreased unless MPO was added, whereas the loss of viricidal activity by 12-day-old monocyte-derived macrophages was not reversed by MPO unless the cells were pretreated with gamma-interferon. These findings suggest that stimulated monocytes can be viricidal to HIV-1 through the release of the MPO/H2O2/chloride system and that the decreased viricidal activity on differentiation to macrophages results initially from the loss of MPO and, with more prolonged culture, also from a decreased respiratory burst that can be overcome by gamma-interferon.
Eight base changes are sufficient to convert a leucine-inserting tRNA into a serine-inserting tRNA.
Authors: Normanly J , Ollick T , Abelson J
Published: May 2006

Each aminoacyl-tRNA synthetase must functionally distinguish its cognate tRNAs from all others. We have determined the minimum number of changes required to transform a leucine amber suppressor tRNA to serine identity. Eight changes are required. These are located in the acceptor stem and in the D stem.
Identification of Tyr-185 as the site of tyrosine autophosphorylation of recombinant mitogen-activated protein kinase p42mapk.
Authors: Rossomando A J , Wu J , Michel H , Shabanowitz J , Hunt D F , Weber M J , Sturgill T W
Published: May 2006

Tyrosine phosphorylation of 42-kDa mitogen-activated protein kinase (p42mapk) occurs during expression of the recombinant protein in Escherichia coli, as well as during in vitro phosphorylation of the protein purified from this source. Structural analyses were performed to identify the site(s) of tyrosine phosphorylation of recombinant p42mapk, both during expression of the protein in E. coli and during in vitro incubations with ATP/Mg2+/Mn2+. Mass spectrometry and phosphopeptide mapping showed that tyrosine phosphorylation of recombinant p42mapk occurs on Tyr-185, the site of regulatory tyrosine phosphorylation that occurs in mitogen-stimulated mammalian cells.
Parental bias of Ki-ras oncogenes detected in lung tumors from mouse hybrids.
Authors: You M , Wang Y , Stoner G , You L , Maronpot R , Reynolds S H , Anderson M
Published: May 2006

A mouse strain with low lung tumor susceptibility (C3H) and a strain with high lung tumor susceptibility (A/J) were reciprocally crossed to produce C3A and AC3 F1 hybrid mice. Ki-ras oncogenes were detected in spontaneous and chemically induced lung tumors obtained from the C3A and AC3 mice. To further explore the genetics of the Ki-ras gene in mouse lung tumor susceptibility, the parental origin of Ki-ras oncogenes detected in lung tumors from the F1 hybrids was determined by a strategy based on a 37-base-pair deletion in the second intron of the A/J Ki-ras allele. Ki-ras oncogenes were derived from the A/J parent in 38 of 40 tumors obtained from C3A mice and 30 of 30 tumors from AC3 mice. The observation that the activated oncogene in hybrids originates from the susceptible parent suggests that the Ki-ras gene is directly linked to mouse lung tumor susceptibility. This finding may have implications for pulmonary adenocarcinoma development in humans, since Ki-ras oncogenes are detected in 35% of this human tumor type.
Heterotrimeric G proteins in synaptoneurosome membranes are crosslinked by p-phenylenedimaleimide, yielding structures comparable in size to crosslinked tubulin and F-actin.
Authors: Coulter S , Rodbell M
Published: May 2006

We have treated rat brain synaptoneurosomes with the crosslinking agent N,N'-1,4-phenylenedimaleimide under conditions that cause extensive crosslinking of tubulin, F-actin, and the alpha and beta subunits of three major types of heterotrimeric GTP-binding regulatory proteins (G(o), Gs, Gi) present in brain membranes. The major crosslinked products are coeluted from Bio-Gel sizing columns as very large structures that do not penetrate stacking gels during SDS/PAGE. The alpha subunits but not the beta subunits of Gs, G(o) and Gi also yield crosslinked products of intermediate sizes. None of the products are as small as the heterotrimeric G proteins extracted from brain by cholate or Lubrol. However, the large and intermediate crosslinked structures are strikingly similar to the large, polydisperse structures of the alpha subunits of Gs, Gi, and G(o) extracted from synaptoneurosomes by the detergent octyl glucoside, which have sedimentation properties of multimeric proteins. Several ways in which multimeric forms of G proteins can explain the dynamic and pleiotropic actions of hormones and GTP on signal-transducing systems are discussed.
Epitope-specific suppression of antibody response in experimental autoimmune myasthenia gravis by a monomethoxypolyethylene glycol conjugate of a myasthenogenic synthetic peptide.
Authors: Atassi M Z , Ruan K H , Jinnai K , Oshima M , Ashizawa T
Published: May 2006

A synthetic peptide corresponding to a myasthenogenic region of Torpedo californica acetylcholine (AcCho) receptor (AcChoR) alpha subunit, AcChoR alpha-(125-148), was conjugated to monomethoxypolyethylene glycol (mPEG). Injection of mice with the mPEG-AcChoR alpha-(125-148) conjugate and subsequent immunization with whole Torpedo AcChoR suppressed the development of experimental autoimmune myasthenia gravis (EAMG) by electrophysiological criteria. In anti-AcChoR sera from these animals, the antibody response against unconjugated AcChoR alpha-(125-148) was decreased, while the antibody responses against whole AcChoR and other epitopes were not altered. There were no detectable changes in T-cell proliferation responses to AcChoR alpha-(125-148) or to whole AcChoR in these animals. Prior injections with a "nonsense" peptide-mPEG conjugate had no effect on responses to the subsequent immunization with whole Torpedo AcChoR. The results indicate that the mPEG-AcChoR alpha-(125-148) conjugate has epitope-specific tolerogenicity for antibody responses in EAMG and that the AcChoR alpha-subunit region comprising residues 125-148 plays an important pathophysiological role in EAMG. The epitope-directed tolerogenic conjugates may be useful for future immunotherapies of human myasthenia gravis. The strategy of specific suppression of the antibody response to a predetermined epitope by using a synthetic mPEG-peptide conjugate may prove useful in manipulation and suppression of unwanted immune responses such as autoimmunity and allergy.
Nitric oxide stimulates the ADP-ribosylation of a 41-kDa cytosolic protein in Dictyostelium discoideum.
Authors: Tao Y , Howlett A , Klein C
Published: May 2006

Nitric oxide-releasing compounds were shown to activate an ADP-ribosyltransferase activity in the cytosol of Dictyostelium discoideum. The enzyme ADP-ribosylated a cytosolic protein of approximately 41 kDa, p41. Neither cGMP nor GTP and its analogues affected this ADP-ribosylation. p41 differs from other substrates ADP-ribosylated by cholera, pertussis, or diphtheria toxins. Treatment of ADP-ribosylated p41 with snake venom phosphodiesterase released adenosine 5'-monophosphate, indicating a mono-ADP-ribose-protein linkage. This linkage was stable to neutral hydroxylamine but was sensitive to mercury ions and iodomethane, suggesting an attachment to a cysteine residue. Treatment of intact cells with nitric oxide-releasing compounds appeared to stimulate the ADP-ribosylation of p41 and this modification was reversible.
Primary structures of chicken cone visual pigments: vertebrate rhodopsins have evolved out of cone visual pigments.
Authors: Okano T , Kojima D , Fukada Y , Shichida Y , Yoshizawa T
Published: May 2006

The chicken retina contains rhodopsin (a rod visual pigment) and four kinds of cone visual pigments. The primary structures of chicken red (iodopsin) and rhodopsin have been determined previously. Here we report isolation of three cDNA clones encoding additional pigments from a chicken retinal cDNA library. Based on the partial amino acid sequences of the purified chicken visual pigments together with their biochemical and spectral properties, we have identified these clones as encoding the chicken green, blue, and violet visual pigments. Chicken violet was very similar to human blue not only in absorption maximum (chicken violet, 415 nm; human blue, 419 nm) but also in amino acid sequence (80.6% identical). Interestingly, chicken green was more similar (71-75.1%) than any other known cone pigment (42.0-53.7%) to vertebrate rhodopsins. The fourth additional cone pigment, chicken blue, had relatively low similarity (39.3-54.6%) in amino acid sequence to those of the other vertebrate visual pigments. A phylogenetic tree of vertebrate visual pigments constructed on the basis of amino acid identity indicated that an ancestral visual pigment evolved first into four groups (groups L, S, M1, and M2), each of which includes one of the chicken cone pigments, and that group Rh including vertebrate rhodopsins diverged from group M2 later. Thus, it is suggested that the gene for scotopic vision (rhodopsin) has evolved out of that for photopic vision (cone pigments). The divergence of rhodopsin from cone pigments was accompanied by an increase in negative net charge of the pigment.
Antithrombotic effects of synthetic peptides targeting various functional domains of thrombin.
Authors: Kelly A B , Maraganore J M , Bourdon P , Hanson S R , Harker L A
Published: May 2006

To determine in vivo functional roles for thrombin's structural domains, we have compared the relative antithrombotic and antihemostatic effects of (i) catalytic-site antithrombin peptide, D-Phe-Pro-Arg; (ii) exosite antithrombin peptide, the C-terminal tyrosine-sulfated dodecapeptide of hirudin; and (iii) bifunctional antithrombin peptide, a 20-mer peptide combining catalytic-site antithrombin peptide and exosite antithrombin peptide with a polyglycyl linker. All three peptides inhibited thrombin-mediated platelet aggregation and fibrin formation in vitro. In vivo thrombus formation was measured in real time as 111In-labeled platelet deposition and 125I-labeled fibrin accumulation on thrombogenic segments incorporated into chronic exteriorized arteriovenous access shunts in baboons. Under low flow conditions, the continuous infusion of peptides reduced thrombus formation onto collagen-coated tubing by half at doses (ID50) and corresponding concentrations (IC50) of 800 nmol per kg per min and 400 nmol/ml for catalytic-site antithrombin peptide, greater than 1250 nmol per kg per min and greater than 1500 mumol/ml for exosite antithrombin peptide, and 50 nmol per kg per min and 25 nmol/ml for bifunctional antithrombin peptide. Under arterial flow conditions, systemically administered bifunctional antithrombin peptide decreased thrombus formation in a dose-dependent manner for segments of collagen-coated tubing or prosthetic vascular graft ID50 and IC50 values of 120 nmol per kg per min and 15 nmol/ml; this dose also produced intermediate inhibition of hemostatic function [bleeding time, 21 +/- 3 min vs. 4.5 +/- 0.5 min (baseline values); P less than 0.001; activated partial thromboplastin time, 285 +/- 13 sec vs. 31 +/- 3 sec (baseline), P less than 0.001]. In contrast, thrombus formation onto segments of endarterectomized aorta was potently decreased by bifunctional antithrombin peptide with an ID50 value of 2.4 nmol per kg per min and an IC50 value of 0.75 nmol/ml, a systemic dose that failed to affect hemostasis. Thus, inhibiting both thrombin's catalytic and exosite domains increases antithrombotic potency by several orders of magnitude over the inhibition of either domain alone, particularly at sites of deep arterial injury.
Isolation of a gene encoding a chaperonin-like protein by complementation of yeast amino acid transport mutants with human cDNA.
Authors: Segel G B , Boal T R , Cardillo T S , Murant F G , Lichtman M A , Sherman F
Published: May 2006

A human cDNA library in lambda-yes plasmid was used to transform a strain of Saccharomyces cerevisiae with defects in histidine biosynthesis (his4-401) and histidine permease (hip1-614) and with the general amino acid permease (GAP) repressed by excess ammonium. We investigated three plasmids complementing the transport defect on a medium with a low concentration of histidine. Inserts in these plasmids hybridized with human genomic but not yeast genomic DNA, indicating their human origin. mRNA corresponding to the human DNA insert was produced by each yeast transformant. Complementation of the histidine transport defect was confirmed by direct measurement of histidine uptake, which was increased 15- to 65-fold in the transformants as compared with the parental strain. Competitive inhibition studies, measurement of citrulline uptake, and lack of complementation in gap1- strains indicated that the human cDNA genes code for proteins that prevent GAP repression by ammonium. The amino acid sequence encoded by one of the cDNA clones is related to T-complex proteins, which suggests a "chaperonin"-like function. We suggest that the human chaperonin-like protein stabilizes the NPR1 gene product and prevents inactivation of GAP.
Reconstruction of a Streptomyces linear replicon from separately cloned DNA fragments: existence of a cryptic origin of circular replication within the linear plasmid.
Authors: Shiffman D , Cohen S N
Published: May 2006

We report here the reconstruction of a functional linear replicon, the 12-kilobase Streptomyces clavuligerus plasmid pSCL, from separate DNA fragments cloned in Escherichia coli on the pUC19 plasmid. Protein-free DNA molecules containing the full-length pSCL sequence, an internally inserted thiostrepton-resistance gene, and adventitious nucleotides external to the pSCL termini were introduced into Streptomyces lividans, where the synthesis and functional attachment of replication proteins occurred and pSCL was established as an extrachromosomal linear replicon. Transformation of S. lividans with uncut supercoilded pUC19/pSCL DNA from E. coli or with a circularized 8-kilobase internal fragment of pSCL yielded circular replicons, indicating the existence of a cryptic origin of circular replication within the linear plasmid. Insertion mutations at sites that prevented the replication of pSCL linear plasmids also interfered with its replication in the circular mode.
Enhancer of splitD, a dominant mutation of Drosophila, and its use in the study of functional domains of a helix-loop-helix protein.
Authors: Tietze K , Oellers N , Knust E
Published: May 2006

Helix-loop-helix proteins play important roles in developmental processes, such as myogenesis, neurogenesis, and sex determination. The gene Enhancer of split [E(spl)] of Drosophila, a member of a gene complex that is involved in early neurogenesis, encodes a protein with a basic domain and a helix-loop-helix motif. We took advantage of a dominant mutation of this gene, E(spl)D, to define in vivo structural features of this protein for proper function. The mutation renders the otherwise recessive eye phenotype of spl dominant. By germ-line transformation of different in vitro mutagenized versions of the E(spl) gene, we could demonstrate that the basic domain of this helix-loop-helix protein is functional and necessary for expression of the dominant phenotype. These results are supported by in vitro DNA-binding assays, which showed that the basic domain is in fact necessary for DNA binding, despite the presence of a proline residue. Furthermore, we could show that the dominant enhancement of spl is caused by truncation of the E(SPL)D protein in combination with deletion of a putative regulatory element.
Distinct sequence of gonadotropin-releasing hormone (GnRH) in dogfish brain provides insight into GnRH evolution.
Authors: Lovejoy D A , Fischer W H , Ngamvongchon S , Craig A G , Nahorniak C S , Peter R E , Rivier J E , Sherwood N M
Published: May 2006

In vertebrates, gonadotropin-releasing hormone (GnRH) belongs to a family of decapeptides characterized by the conservation of residues 1, 2, 4, 9, and 10. In the jawed vertebrates only positions 5, 7, and 8 in the GnRH molecules vary. We have now purified two forms of GnRH from the brains of spiny dogfish (Squalus acanthias) by using reverse-phase high-performance liquid chromatography. The primary structures were established by automated Edman degradation and mass spectral analysis. The distinct structure of the first form (dogfish GnRH) is pGlu-His-Trp-Ser-His-Gly-Trp-Leu-Pro-Gly-NH2 (pGlu represents pyroglutamyl). The second peptide is identical to a form of GnRH originally isolated from chicken brains (chicken GnRH-II; pGlu-His-Trp-Ser-His-Gly-Trp-Tyr- Pro-Gly-NH2) and is widespread throughout the vertebrates. We are aware of no other species of cartilaginous fish in which the primary structures of two forms of GnRH have been determined. The presence of chicken GnRH-II in dogfish supports the idea that chicken GnRH-II is the oldest GnRH to evolve in jawed vertebrates. With the addition of the dogfish GnRH structure to the family, two main structural branches of GnRH can be delineated. The physiological effects of dogfish GnRH included the release of not only gonadotropin but also growth hormone from goldfish pituitary fragments.
Role of lysophosphatidylcholine in T-lymphocyte activation: involvement of phospholipase A2 in signal transduction through protein kinase C.
Authors: Asaoka Y , Oka M , Yoshida K , Sasaki Y , Nishizuka Y
Published: May 2006

2-Lysophosphatidylcholine (lysoPtdCho), a product of the hydrolysis of phosphatidylcholine catalyzed by phospholipase A2, greatly potentiates the activation of human resting T lymphocytes that is induced by a membrane-permeant diacylglycerol plus a calcium ionophore, as determined by the expression of the alpha subunit of the interleukin 2 receptor and thymidine incorporation into DNA. LysoPtdCho per se is inactive unless both diacylglycerol and a calcium ionophore are present. This effect of lysoPtdCho is also observed when diacylglycerol is replaced by a tumor-promoting phorbol ester. Other lysophosphatides including lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidic acid are inert except for lysophosphatidylethanolamine, which is far less effective than lysoPtdCho. Tracer experiments with radioactive choline indicate that, when T lymphocytes are stimulated with an antigenic signal, lysoPtdCho is indeed produced in a time-dependent fashion, although the concentration of this lysophospholipid accumulated remains to be quantitated. It suggests that phospholipase A2 is directly involved in the signal transduction pathway through protein kinase C to induce long-term cellular responses.
Directed establishment of rat brain cell lines with the phenotypic characteristics of type 1 astrocytes.
Authors: Radany E H , Brenner M , Besnard F , Bigornia V , Bishop J M , Deschepper C F
Published: May 2006

Interest in obtaining cell lines for use in studies on the development and biochemistry of the central nervous system has motivated efforts to establish cells from primary brain cultures by the use of oncogene-transfer techniques. In previous reports, cell lines derived from astrocytes in this way have had immature or abnormal phenotypes. We have explored the possibility of specifically "targeting" expression of exogenous oncogenes to differentiated astrocytes by using the promoter of the gene encoding glial fibrillary acidic protein, which is expressed almost exclusively in such cells. We report here that cell lines displaying the phenotypic characteristics of type 1 astrocytes can be established reproducibly in this manner. Given the heterogeneity of primary cultures, the availability of clonal cell lines displaying characteristics of type 1 astrocytes should greatly facilitate our understanding of the biology of these cells.
Temporal asymmetry in activation of Aplysia adenylyl cyclase by calcium and transmitter may explain temporal requirements of conditioning.
Authors: Yovell Y , Abrams T W
Published: May 2006

Cellular experiments have suggested that during classical conditioning of the gill and siphon withdrawal reflex of Aplysia, adenylyl cyclase may serve as a molecular site of convergence for Ca2+ and serotonin (5-hydroxytryptamine; 5-HT), the cellular representations of the conditioned and unconditioned stimuli (CS and US). We explored the possible molecular basis of the behavioral requirement that the CS and US be paired within a narrow time window and in the appropriate order. To examine the temporal interactions of brief pulses of Ca2+ and 5-HT in stimulating Aplysia neural cyclase, we used a perfused-membrane cyclase assay. When brief pulses of Ca2+ and 5-HT were paired, cyclase activation depended upon the sequence of the pulses: peak cyclase activation was greater when the Ca2+ pulse immediately preceded the 5-HT pulse. Examination of the rising phase of 5-HT stimulation suggested that a Ca2+ prepulse might accelerate the onset of activation by 5-HT, without affecting the final level of activation achieved with prolonged 5-HT exposure. The observed interactions between Ca2+ and transmitter in activating cyclase could contribute importantly to the temporal requirements of conditioning for CS-US pairing.
Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme.
Authors: Lowenstein C J , Glatt C S , Bredt D S , Snyder S H
Published: May 2006

Nitric oxide (NO) is a messenger molecule of macrophages, endothelial cells in blood vessels, and neurons. A neuronal form of NO synthase (NOS) has been previously cloned. We now report the molecular cloning of macrophage NOS. The macrophage enzyme displays 50% sequence identity to the neuronal enzyme. Like neuronal NOS, macrophage NOS has recognition sites for FAD, FMN, and NADPH and also has a consensus calmodulin binding site. Macrophage NOS mRNA is strikingly inducible; it is absent in quiescent macrophages or spleen but is prominent 2-6 hr after endotoxin treatment.
Binding of transition metals by apolipoprotein A-I-containing plasma lipoproteins: inhibition of oxidation of low density lipoproteins.
Authors: Kunitake S T , Jarvis M R , Hamilton R L , Kane J P
Published: May 2006

We have found transition metals tightly bound to apolipoprotein A-I-containing lipoproteins [Lp(A-I)] isolated by selected affinity immunosorption from human serum. Prominent among the metal ions detected were iron and copper. By immunoblotting the proteins of Lp(A-I), we detected both transferrin and ceruloplasmin. The transferrin-containing Lp(A-I) particles, isolated by selected affinity immunosorption against transferrin, were larger (mean diameter of 14.2 nm) and had a higher protein content than most high density lipoproteins (HDL). Ultracentrifugally isolated HDL were found to contain much less transferrin, whereas transferrin was found associated with apolipoprotein A-I from the greater than 1.21-g/ml ultracentrifugal fraction. This suggests that the complex is not recovered in the classic HDL density interval because of its very high density. HDL inhibit copper-catalyzed oxidation of low density lipoproteins (LDL) in vitro. We have found that immunoisolated Lp(A-I) are an order of magnitude more effective in inhibiting the oxidation of LDL than ultracentrifugally isolated HDL, on the basis of protein mass. When the Lp(A-I) particles containing transferrin and ceruloplasmin were removed from the bulk of Lp(A-I), inhibition of the in vitro oxidation of LDL was significantly decreased.
Phycocyanin alpha-subunit phycocyanobilin lyase.
Authors: Fairchild C D , Zhao J , Zhou J , Colson S E , Bryant D A , Glazer A N
Published: May 2006

Phycobiliproteins, unlike other light-harvesting proteins involved in photosynthesis, bear covalently attached chromophores. The bilin chromophores are attached through thioether bonds to cysteine residues. The cyanobacterium Synechococcus sp. PCC 7002 has eight distinct bilin attachment sites on seven polypeptides, all of which carry the same chromophore, phycocyanobilin. When two genes in the phycocyanin operon of this organism, cpcE and cpcF, are inactivated by insertion, together or separately, the surprising result is elimination of correct bilin attachment at only one site, that on the alpha subunit of phycocyanin. We have overproduced CpcE and CpcF in Escherichia coli. In vitro, these proteins catalyze the attachment of phycocyanobilin to the alpha subunit of apophycocyanin at the appropriate site, alpha-Cys-84, to form the correct adduct. CpcE and CpcF also efficiently catalyze the reverse reaction, in which the bilin from holo-alpha subunit is transferred either to the apo-alpha subunit of the same C-phycocyanin or to the apo-alpha subunit of a heterologous C-phycocyanin. The forward and reverse reactions each require both CpcE and CpcF and are specific for the alpha-Cys-84 position. Phycocyanobilin is the immediate precursor of the protein-bound bilin.