bluuebunny/crossref_metadata_2025 huggingface dataset:
Human cytoplasmic isoleucyl-tRNA synthetase: selective divergence of the anticodon-binding domain and acquisition of a new structural unit.
Authors: Shiba K , Suzuki N , Shigesada K , Namba Y , Schimmel P , Noda T
Published: May 2006

We show here that the class I human cytoplasmic isoleucyl-tRNA synthetase is an exceptionally large polypeptide (1266 aa) which, unlike its homologues in lower eukaryotes and prokaryotes, has a third domain of two repeats of an approximately 90-aa sequence appended to its C-terminal end. While extracts of Escherichia coli do not aminoacrylate mammalian tRNA with isoleucine, expression of the cloned human gene in E. coli results in charging of the mammalian tRNA substrate. The appended third domain is dispensable for detection of this aminoacylation activity and may be needed for assembly of a multisynthetase complex in mammalian cells. Alignment of the sequences of the remaining two domains shared by isoleucyl-tRNA synthetases from E. coli to human reveals a much greater selective pressure on the domain needed for tRNA acceptor helix interactions and catalysis than on the domain needed for interactions with the anticodon. This result may have implications for the historical development of an operational RNA code for amino acids.
Anandamide (arachidonylethanolamide), a brain cannabinoid receptor agonist, reduces sperm fertilizing capacity in sea urchins by inhibiting the acrosome reaction.
Authors: Schuel H , Goldstein E , Mechoulam R , Zimmerman A M , Zimmerman S
Published: May 2006

Anandamide (arachidonylethanolamide) is an endogenous cannabinoid receptor agonist in mammalian brain. Sea urchin sperm contain a high-affinity cannabinoid receptor similar to the cannabinoid receptor in mammalian brain. (-)-delta 9-Tetrahydrocannabinol (THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. We now report that anandamide produces effects similar to those previously obtained with THC in Strongylocentrotus purpuratus in reducing sperm fertilizing capacity and inhibiting the egg jelly-stimulated acrosome reaction. Arachidonic acid does not inhibit the acrosome reaction under similar conditions. The adverse effects of anandamide on sperm fertilizing capacity and the acrosome reaction are reversible. The receptivity of unfertilized eggs to sperm and sperm motility are not impaired by anandamide. Under conditions where anandamide completely blocks the egg jelly-stimulated acrosome reaction, it does not inhibit the acrosome reaction artificially initiated by ionomycin, which promotes Ca2+ influx, and nigericin, which activates K+ channels in sperm. These findings provide additional evidence that the cannabinoid receptor in sperm plays a role in blocking the acrosome reaction, indicate that anandamide or a related molecule may be the natural ligand for the cannabinoid receptor in sea urchin sperm, and suggest that binding of anandamide to the cannabinoid receptor modulates stimulus-secretion-coupling in sperm by affecting an event prior to ion channel opening.
Coordinate depression of bradykinin receptor recycling and microtubule-dependent transport by taxol.
Authors: Hamm-Alvarez S F , Alayof B E , Himmel H M , Kim P Y , Crews A L , Strauss H C , Sheetz M P
Published: May 2006

Significant cardiovascular side effects have limited the use of taxol as an anticancer drug. A link between decreased plasma membrane dynamics and taxol has been implied because taxol can inhibit intracellular vesicle movements. Reduced membrane recycling caused by taxol could inhibit agonist-evoked Ca2+ signaling within endothelial cells, resulting in endothelium-dependent vasodilation. Bradykinin and ATP are two agonists that evoke Ca2+ transients in endothelial cells. Since the bradykinin receptor-agonist complex is internalized and recycled whereas the ATP agonist-receptor complex is not, we expected that a taxol inhibition of recycling would decrease bradykinin but not ATP receptor activity. We found that taxol depresses (i) the frequency (to 41% of control) and velocity (to 55% of control) of microtubule-dependent vesicle transport and (ii) bradykinin-evoked cytosolic Ca2+ transients (to 76% of control) in bovine aortic endothelial cells. In studying bradykinin receptor desensitization, which reflects receptor recycling, we demonstrate that taxol inhibits bradykinin-evoked Ca2+ transients by 50%. Taxol did not significantly alter ATP-evoked Ca2+ transients in either single-exposure or desensitization experiments. We suggest that taxol's reduction of bradykinin-evoked Ca2+ transients is due to altered microtubule-dependent membrane recycling. This report describes taxol's ability to alter plasma membrane composition through effects on vesicle transport and membrane trafficking pathways. This finding provides a possible mechanism by which taxol can substantially alter cardiovascular function.
Inhibition of hematopoiesis by competitive binding of transcription factor PU.1.
Authors: Voso M T , Burn T C , Wulf G , Lim B , Leone G , Tenen D G
Published: May 2006

Transcription factors have been shown to play a role as "master switch" factors in the programming of hematopoietic cell commitment and differentiation. PU.1 is a hematopoietic-specific member of the Ets family of transcription factors. In human bone marrow CD34-enriched progenitor cells, PU.1 expression was upregulated during the early phases of granulocytic/monocytic differentiation, preceding expression of its target genes encoding CD11b and the macrophage-colony-stimulating factor receptor, whereas PU.1 was expressed at stable levels throughout erythroid differentiation. To study PU.1 function, we synthesized double-stranded phosphorothioate oligonucleotides containing a characterized PU.1 site and demonstrated their ability to specifically compete for PU.1 DNA binding. When added to CD34+ cells in vitro, wild-type PU.1-binding oligonucleotides significantly blocked hematopoietic colony formation, whereas mutated PU.1 oligonucleotides which no longer bind PU.1 had no specific inhibitory effect. These results demonstrate that PU.1 is developmentally upregulated during normal human myelopoiesis and that the function of PU.1 is critical for the development of in vitro hematopoiesis.
Insertion mutagenesis as a tool to predict the secondary structure of a muscarinic receptor domain determining specificity of G-protein coupling.
Authors: Blüml K , Mutschler E , Wess J
Published: May 2006

The N-terminal segment of the third intracellular loop (i3) of muscarinic acetylcholine receptors and other G protein-coupled receptors has been shown to largely determine the G-protein coupling selectivity displayed by a given receptor subtype. Based on secondary-structure prediction algorithms, we have tested the hypothesis that this region adopts an alpha-helical secondary structure. Using the rat m3 muscarinic receptor as a model system, a series of five mutant receptors, m3(+1A) to m3(+5A) were created in which one to five additional alanine residues were inserted between the end of the fifth transmembrane domain and the beginning of i3. We speculated that this manipulation should lead to a rotation of the N-terminal segment of the i3 domain (if it is in fact alpha-helically arranged), thus producing pronounced effects on receptor/G protein coupling. Pharmacological analysis of the various mutant receptors expressed in COS-7 cells showed that m3(+1A), m3(+3A), and m3(+4A) retained strong functional activity, whereas m3(+2A) and m3(+5A) proved to be virtually inactive. Helical wheel models show that this pattern is fully consistent with the notion that the N-terminal portion of i3 forms an amphiphilic alpha-helix and that the hydrophobic side of this helix represents the G-protein recognition surface.
Regulation in vitro of an L-CAM enhancer by homeobox genes HoxD9 and HNF-1.
Authors: Goomer R S , Holst B D , Wood I C , Jones F S , Edelman G M
Published: May 2006

Previous studies have shown that in vitro expression of the neural cell adhesion molecule (N-CAM) can be regulated by the products of homeobox genes HoxB9, -B8, and -C6. N-CAM is a Ca(2+)-independent immunoglobulin-related CAM that plays an important role in neural development. In the present study, we investigated whether the liver cell adhesion molecule (L-CAM) a member of the Ca(2+)-dependent CAM family (cadherins) is also regulated by homeobox-containing genes. In transient cotransfection experiments of NIH 3T3 cells, we observed that both HoxD9 and liver-enriched POU-homeodomain transcription factor, HNF-1, activated chloramphenicol acetyltransferase gene reporter constructs containing the L-CAM promoter and an enhancer present in the second intron of the chicken L-CAM gene. Using electrophoretic mobility-shift assays, we found that components of cell extracts from NIH 3T3 cells transfected with HoxD9 bound to a small region of the L-CAM enhancer having a consensus sequence that is a putative binding site for HNF-1. Components of extracts from the chicken hepatoma cell line LMH that had been transfected with an HNF-1 expression vector also bound to this same site. In nuclear run-on experiments with nuclei from LMH cells that were transfected with expression vectors for HoxD9 or HNF-1, L-CAM RNA levels were increased 33-fold and 4-fold respectively. Using the same run-on procedure, it was confirmed that nuclei prepared from normal embryonic chicken liver cells expressed the RNAs for HoxD9, HNF-1, and L-CAM. Taken together with previous observations, these data raise the possibility that homeobox-containing genes will have a widespread role in the place-dependent expression of CAMs belonging both to immunoglobulin-related and to cadherin families.
Quantitation of human immunodeficiency virus type 1 during pregnancy: relationship of viral titer to mother-to-child transmission and stability of viral load.
Authors: Weiser B , Nachman S , Tropper P , Viscosi K H , Grimson R , Baxter G , Fang G , Reyelt C , Hutcheon N , Burger H
Published: May 2006

To develop strategies to prevent mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1), it is important to define the factors determining it. We examined the relationship between maternal HIV-1 titer and the occurrence of mother-to-child transmission. In addition, we quantitated HIV-1 longitudinally in mothers during pregnancy, at delivery, and up to 1 year postpartum. To examine transmission, we prospectively studied 19 mother-child pairs; in 5 pairs, HIV-1 transmission occurred. We used endpoint dilution culture of peripheral blood mononuclear cells to determine maternal viral titer and found that although 4 of 6 (67%) women with viral titers of > or = 125 HIV-1 infectious units per 10(6) cells transmitted HIV-1 to their infants, only 1 of 13 (7.6%) women with lower viral titers transmitted (P = 0.01). Twelve of the 19 mothers had HIV-1 loads determined serially 3-8 times over periods ranging from 18 to 65 weeks. Viral titers varied greatly between the 12 women, but the viral load in each woman remained stable over time. In this cohort, HIV-1 viral load remained stable during pregnancy and the greater the maternal viral burden, the more likely that transmission occurred. These two related findings suggest that determination of HIV-1 titers early in pregnancy may predict which women are at high risk of transmitting to their infants and may be used to counsel HIV-1-infected women of childbearing age. These data identify maternal viral titer as a major determinant of mother-to-child HIV-1 transmission and thereby provide the scientific rationale for therapeutic strategies designed to interrupt transmission by lowering viral load.
SB 209670, a rationally designed potent nonpeptide endothelin receptor antagonist.
Authors: Ohlstein E H , Nambi P , Douglas S A , Edwards R M , Gellai M , Lago A , Leber J D , Cousins R D , Gao A , Frazee J S
Published: May 2006

An extremely potent and highly specific non-peptide, subnanomolar endothelin (ET) receptor antagonist, SB 209670, has been synthesized and characterized. SB 209670, which was rationally designed using conformational models of ET-1, selectively inhibits binding of 125I-labeled ET-1 to cloned human ET receptor subtypes ETA and ETB (Ki = 0.2 and 18 nM, respectively). SB 209670 produces concentration-dependent inhibition of ET-1-mediated vasoconstriction in isolated vascular tissues and in vivo following either intravenous or intraduodenal administration. SB 209670 produces a dose-dependent reduction in blood pressure in hypertensive rats, protects from ischemia-induced neuronal degeneration in a gerbil stroke model, and attenuates neointima formation following rat carotid artery balloon angioplasty. SB 209670 will be useful in characterizing and classifying the physiological and pathophysiological effects of ET.
Regulation of ROMK1 K+ channel activity involves phosphorylation processes.
Authors: McNicholas C M , Wang W , Ho K , Hebert S C , Giebisch G
Published: May 2006

An inwardly rectifying, ATP-regulated K+ channel with a distinctive molecular architecture, ROMK1, was recently cloned from rat kidney. Using patch clamp techniques, we have investigated the regulation of ROMK1 with particular emphasis on phosphorylation/dephosphorylation processes. Spontaneous channel rundown occurred after excision of membrane patches into ATP-free bath solutions in the presence of Mg2+. Channel rundown was almost completely abolished after excision of patches into either Mg(2+)-free bathing solutions or after preincubation with the broad-spectrum phosphatase inhibitor, orthovanadate, in the presence of Mg2+. MgATP preincubation also inhibited channel rundown in a dose-dependent manner. In addition, the effect of the specific phosphatase inhibitors okadaic acid (1 microM) and calyculin A (1 microM) was also investigated. The presence of either okadaic acid or calyculin A failed to inhibit channel rundown. Taken together, these data suggest that rundown of ROMK1 involves a Mg(2+)-dependent dephosphorylation process. Channel activity was also partially restored after the addition of MgATP to the bath solution. Addition of exogenous cAMP-dependent protein kinase A (PKA) catalytic subunit led to a further increase in channel open probability. Addition of Na2ATP, in the absence of Mg2+, was ineffective, suggesting that restoration of channel activity is a Mg(2+)-dependent process. Addition of the specific PKA inhibitor, PKI, to the bath solution led to a partial, reversible inhibition in channel activity. Thus, PKA-dependent phosphorylation processes are involved in the modulation of channel activity. This observation is consistent with the presence of potential PKA phosphorylation sites on ROMK1.
Granzyme A released upon stimulation of cytotoxic T lymphocytes activates the thrombin receptor on neuronal cells and astrocytes.
Authors: Suidan H S , Bouvier J , Schaerer E , Stone S R , Monard D , Tschopp J
Published: May 2006

Granzymes are a family of serine proteases that are harbored in cytoplasmic granules of activated T lymphocytes and are released upon target cell interaction. Immediate and complete neurite retraction was induced in a mouse neuronal cell line when total extracts of granule proteins were added. This activity was isolated and identified as granzyme A. This protease not only induced neurite retraction at nanomolar concentrations but also reversed the stellation of astrocytes. Both effects were critically dependent on the esterolytic activity of granzyme A. As neurite retraction is known to be induced by thrombin, possible cleavage and activation of the thrombin receptor were investigated. A synthetic peptide spanning the N-terminal thrombin receptor activation sequence was cleaved by granzyme A at the authentic thrombin cleavage site Leu-Asp-Pro-Arg-Ser. Antibodies to the thrombin receptor inhibited both thrombin and granzyme A-mediated neurite retraction. Thus, T-cell-released granzyme A induces cellular responses by activation of the thrombin receptor. As brain-infiltrating CD4+ lymphocytes are the effector cells in experimental allergic encephalomyelitis, granzyme A released in the brain may contribute to the etiology of autoimmune disorders in the nervous system.
Insect cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity.
Authors: Guy P M , Platko J V , Cantley L C , Cerione R A , Carraway K L
Published: May 2006

Protein kinases share a number of highly conserved or invariant amino acid residues in their catalytic domains, suggesting that these residues are necessary for kinase activity. In p180erbB3, a receptor tyrosine kinase belonging to the epidermal growth factor (EGF) receptor subfamily, three of these residues are altered, suggesting that this protein might have an impaired protein tyrosine kinase activity. To test this hypothesis, we have expressed human EGF receptor and bovine p180erbB3 in insect cells via baculovirus infection and have compared their autophosphorylation and substrate phosphorylation activities. We have found that, while the EGF receptor readily undergoes EGF-stimulated autophosphorylation and catalyzes the incorporation of phosphate into the model substrates (E4Y1)n (random 4:1 copolymer of glutamic acid and tyrosine) and GST-p85 (glutathione S-transferase fusion protein with the 85-kDa subunit of phosphatidylinositol 3-kinase), p180erbB3 autophosphorylation and substrate phosphorylation are at least 2 orders of magnitude less efficient. However, p180erbB3 is capable of binding the ATP analog 5'-p-fluorosulfonylbenzoyladenosine, indicating that the lack of observed kinase activity is probably not due to nonfunctional or denatured receptors expressed by the insect cells. On the basis of these results, we propose that p180erbB3 possesses an impaired intrinsic tyrosine kinase activity.
Chromosomal speciation and adaptive radiation of mole rats in Asia Minor correlated with increased ecological stress.
Authors: Nevo E , Filippucci M G , Redi C , Korol A , Beiles A
Published: May 2006

The evolutionary forces causing chromosomal speciation and adaptation are still enigmatic. Here we tested the Israel evolutionary model of positive association of diploid chromosome number (2n) and genetic diversity with aridity stress in subterranean mole rats, on a 30-times-larger scale in Asia Minor. We analyzed both karyotype and allozyme diversity across Turkey, based on 37 allozymic loci in 20 localities of the Spalax leucodon and 4 localities of the Spalax ehrenbergi superspecies. We found extensive chromosomal speciation in S. leucodon (2n = 38, 40, 50, 54, 60, and 62) and in S. ehrenbergi (2n = 52, 56, and 58), presumably representing from 14 to > 20 additional biological species. Genetic diversity indices were low, but, like the chromosome number (2n), positively correlated with aridity stress, increasing centripetally from the periphery toward geologically young, arid, and climatically unpredictable central Anatolia. Nei's genetic distance D across all populations averaged 0.174 (range 0.002-0.422), supporting, combined with 2n and ecogeography, the biological species status of most tested populations. Chromosome evolution is the basis of speciation and adaptation in Spalax; it provides both postmating reproductive isolation, as well as higher levels of recombination with increased 2n. A mathematical model shows that a Robertsonian fission of a single metacentric considerably increases haplotype diversity. This haplotype diversity may contribute to population adaptation to climatic stress and ecological unpredictability in space and time. The increase in diversity corroborates the nichewidth genetic-variation hypothesis.
Increasing complexity of the dystrophin-associated protein complex.
Authors: Tinsley J M , Blake D J , Zuellig R A , Davies K E
Published: May 2006

Duchenne muscular dystrophy is a severe X chromosome-linked, muscle-wasting disease caused by lack of the protein dystrophin. The exact function of dystrophin remains to be determined. However, analysis of its interaction with a large oligomeric protein complex at the sarcolemma and the identification of a structurally related protein, utrophin, is leading to the characterization of candidate genes for other neuromuscular disorders.
Monocular preferences in binocular viewing.
Authors: Purves D , White L E
Published: May 2006

Faced with an unobstructed view, both foveas can be readily aligned with a distant visual target. The minor difference in the view of the two eyes (which arises from slightly different lines of sight) presents no special problem and is, indeed, the basis of stereopsis. However, when obstructing objects are present in the foreground, the view provided by one eye becomes wholly or partially incompatible with the view of the other. We have investigated how we cope with this everyday situation by having volunteers observe distant targets through a fenestrated screen. In this circumstance, subjects naturally position themselves to view a target of interest with one eye--usually the right eye. This monocular habit in normal viewing reinforces other evidence for the unorthodox idea that visual perception arises from a union in consciousness of monocular images that are elaborated independently.
cAMP-dependent phosphorylation of Aplysia twitchin may mediate modulation of muscle contractions by neuropeptide cotransmitters.
Authors: Probst W C , Cropper E C , Heierhorst J , Hooper S L , Jaffe H , Vilim F , Beushausen S , Kupfermann I , Weiss K R
Published: May 2006

Acting through a cAMP-cAMP-dependent protein kinase (cAPK) cascade, members of two neuropeptide families, the small cardioactive peptides and myomodulins, modulate contraction amplitude and relaxation rate in the accessory radula closer (ARC) muscle of the marine mollusc Aplysia californica. An approximately 750-kDa phosphoprotein was identified in the ARC muscle as the major substrate for cAPK activated either by application of neuropeptides or by peptides released by motorneuron stimulation at physiological frequencies. Immunoblot and immunoelectron microscopy experiments revealed the widespread presence of this protein in Aplysia muscles and its colocalization with contractile filaments in the ARC muscle. Sequence analysis of proteolytic peptide fragments derived from the protein indicated that it is structurally related to the muscle protein twitchin. Finally, the level of neuropeptide-induced phosphorylation of the protein correlated well with peptidergic modulation of the relaxation rate of the muscle. We propose that twitchin in Aplysia, and perhaps in other species, may mediate the modulation of the relaxation rate of muscle contractions.
RNA-dependent RNA polymerase from plants infected with turnip crinkle virus can transcribe (+)- and (-)-strands of virus-associated RNAs.
Authors: Song C , Simon A E
Published: May 2006

RNA-dependent RNA polymerase (RdRp) was solubilized from membranes of turnip infected with turnip crinkle virus (TCV), a single-stranded, monopartite RNA virus. The RdRp activity could be separated into three peaks by Sephacryl S500HR chromatography. RdRp from peak I, which contained substantial amounts of endogenous TCV genomic RNA, and peak II were template-specific, synthesizing full-length complementary strands of exogenous TCV subviral RNAs but not control RNA templates. Peak III RdRp was nonspecific, synthesizing full-sized products for all added RNA templates. Peak II RdRp transcribed several different TCV satellite (sat) and defective interfering RNA templates in both (+)- and (-)-sense orientations but did not transcribe (+)-strands of satellite RNAs associated with unrelated viruses. Monomeric-length sat-RNA C was synthesized from a template containing as many as 220 nonsatellite bases at the 3' ends of either (+)- or (-)-strands, indicating that the RdRp was able to recognize 3'-end sequences in an internal location. Deletion of 95-242 bases from the 3' end of (+)-strand sat-RNA C abolished the synthesis of template-length product. However, transcription of template-length products was not affected by the deletion of at least 257 bases from the 3' end of (-)-strand sat-RNA C template (leaving only the 100 5'-terminal residues), implying that different mechanisms exist for synthesis of (+)-and (-)-strand satellite RNA in vitro.
Eastern Pacific molluscan provinces and latitudinal diversity gradient: no evidence for "Rapoport's rule".
Authors: Roy K , Jablonski D , Valentine J W
Published: May 2006

"Rapoport's rule," which has gained wide acceptance as a potential explanation for latitudinal and other diversity gradients, holds that mean latitudinal range of species decreases toward the equator. We analyzed latitudinal ranges of 2838 eastern Pacific marine molluscan species, a subset of which figured in the original formulation of Rapoport's rule, and failed to find the predicted trends. Instead, species diversity gradients and range magnitudes appear to vary independently, with the spatial distribution of major oceanographic barriers exerting a strong influence on latitudinal ranges. Biogeographic structure should, therefore, be an important element in the assessment of diversity patterns.
A transposable element can drive the concerted evolution of tandemly repetitious DNA.
Authors: Thompson-Stewart D , Karpen G H , Spradling A C
Published: May 2006

Recombination and conversion have been proposed to drive the concerted evolution of tandemly repeated DNA sequences. However, specific correction events within the repeated genes of multicellular organisms have not been observed directly, so their nature has remained speculative. We investigated whether the excision of transposable P elements from tandemly repeated sequences would induce unequal gene conversion. Genetically marked elements located in a subtelomeric repeat were mobilized, and the structure of the region was analyzed in progeny. We observed that the number of repeats was frequently altered. Decreases were more common than increases, and this bias probably resulted from intrinsic mechanisms governing P element-induced double-strand break repair. Our results suggest that transposable elements play an important role in the evolution of repetitious DNA.
Genomic organization and structure of Bruton agammaglobulinemia tyrosine kinase: localization of mutations associated with varied clinical presentations and course in X chromosome-linked agammaglobulinemia.
Authors: Ohta Y , Haire R N , Litman R T , Fu S M , Nelson R P , Kratz J , Kornfeld S J , de la Morena M , Good R A , Litman G W
Published: May 2006

X chromosome-linked agammaglobulinemia is a life-threatening disease that involves a failure in normal development of B lymphocytes and is associated with missense mutations in BTK, a gene encoding a cytoplasmic tyrosine kinase (Bruton agammaglobulinemia tyrosine kinase, EC 2.7.1.112), a member of the Tec family of protein-tyrosine kinases. The genomic organization has been determined by using conventional restriction fragment mapping, extended DNA sequencing, and PCR fragment-sizing approaches. The DNA sequences of the 18 coding exons composing BTK and their flanking-region sequences are reported; an additional exon(s) encodes a 5' untranslated segment. Single-base-pair substitutions and 4-nt deletions resulted in amino acid replacement, premature termination, frameshift, and exon deletion in a group of X chromosome-linked agammaglobulinemia patients exhibiting different clinical presentations and courses. The nature of the mutations is interpreted in terms of the genomic organization of the BTK gene and the disease course in individual patients. Several examples are found in which the same mutation occurs in unrelated patients, and one of these mutations occurs at the same codon that is substituted in the murine form of BTK, resulting in X chromosome-linked immunodeficiency disease. Considerable variation in presentation and disease course in X chromosome-linked agammaglobulinemia appears associated with the nature and position of different missense mutations.
Involvement of the C-terminal residues of the 20,000-dalton light chain of myosin on the regulation of smooth muscle actomyosin.
Authors: Ikebe M , Reardon S , Mitani Y , Kamisoyama H , Matsuura M , Ikebe R
Published: May 2006

The segment of smooth muscle regulatory light chain essential for the phosphorylation dependent activation of actomyosin motor activity and the binding of myosin heavy chain was identified. The C-terminal domain of the 20-kDa light chain, which is less conserved than the rest of the polypeptide among various muscle types, was mutated by either deletion or substitution of amino acid residues and the mutant light chains were then incorporated into myosin by subunit exchange. Deletion of Lys149-Ala166 markedly reduced the affinity of the light chain for the heavy chain, whereas the C-terminal five residues, Lys167-Asp171, did not contribute to the binding affinity. Deletion of Lys149-Phe158 abolished the phosphorylation-dependent activation of actomyosin ATPase activity as well as superprecipitation activity. These results suggest that the C-terminal domain of the regulatory light chain is critical for transmitting the change in the conformation of the regulatory light chain induced by phosphorylation at Ser19 to the heavy chain.
BY55 monoclonal antibody delineates within human cord blood and bone marrow lymphocytes distinct cell subsets mediating cytotoxic activity.
Authors: Bensussan A , Gluckman E , el Marsafy S , Schiavon V , Mansur I G , Dausset J , Boumsell L , Carosella E
Published: May 2006

We had previously reported, using BY55 monoclonal antibody, a cell surface 80-kDa protein restricted to human functional peripheral blood cytotoxic lymphocytes with either natural killer CD3- or cytotoxic T lymphocyte CD3+CD8+ phenotype. In the present report, we studied the cytotoxic lymphocytes in adult bone marrow and newborn cord blood as these organs are commonly used as sources of hematological stem cells for allogeneic transplantation. Our results showed that BY55 mAb labeled only 5-10% of the bone marrow lymphocytes, which included a major proportion of CD3+ CD8+ cytotoxic T lymphocytes. Interestingly, within cord blood cells, BY55+ lymphocytes represented 20-35% of the lymphocytes corresponding exclusively to a CD3- cell subset. Furthermore, we detected in cord blood no cytotoxic T lymphocyte activity but we demonstrated that the CD3-BY55+ cell subset contained the whole natural killer activity.
The phosphatidylinositol 3-kinase alpha is required for DNA synthesis induced by some, but not all, growth factors.
Authors: Roche S , Koegl M , Courtneidge S A
Published: May 2006

The phosphatidylinositol 3-kinase (PI 3-K) becomes activated when quiescent cells are stimulated with a variety of growth factors. We have microinjected antibodies specific for the p110 alpha subunit of the PI 3-K into quiescent fibroblasts and tested their effect on the ability of growth factors to stimulate exit from quiescence and entry into S phase. The antibodies inhibited platelet-derived growth factor-induced DNA synthesis, a result in keeping with previous studies using mutant platelet-derived growth factor receptors. Interestingly, functional PI 3-K was required for the first 6 hr of G1--i.e., until approximately 4 hr before the point at which the cells were committed to make DNA. A second tyrosine kinase receptor, the epidermal growth factor receptor, also required the PI 3-K for efficient signaling. However, colony-stimulating factor 1 (whose receptor is highly related to the platelet-derived growth factor receptor) could induce DNA synthesis in the absence of active PI 3-K, as could two growth factors (bombesin and lysophosphatidic acid) whose receptors are functionally coupled to G proteins. These data, therefore, demonstrate that some, but not all, growth factors require functional PI 3-K.
Detection of nondisjunction and recombination in meiotic and postmeiotic cells from XYSxr [XY,Tp(Y)1Ct] mice using multicolor fluorescence in situ hybridization.
Authors: Ashley T , Ried T , Ward D C
Published: May 2006

Current meiotic dogma holds that synapsis is required for recombination and that recombination is required for proper disjunction. The mouse chromosome aberration XYSxr [sex reversal; redesignated XY,Tp(Y)1Ct] appears to challenge this assumption, for although chromosomes X and Y often fail to synapse and recombine, there is no dramatic increase in aneuploid progeny. An explanation of this conundrum might be that X-Y univalent spermatocytes do not survive. The phenotype of sex reversal is generated by the "obligatory" crossover between the X and Y chromosomes, which always occurs proximal to a duplicated copy of the testis-determining gene Sry and transfers one copy from one chromatid of the Y chromosome to one chromatid of the X. Animals that inherit an X chromosome with the Sry gene are chromosomally female but phenotypically male. We have used fluorescence in situ hybridization (FISH) to visualize probes for the X and Y chromosomes and for the Sry sequence and chromosome 8 to track the fate of both recombinant and nonrecombinant chromosomes through metaphases I and II into spermatids and sperm. In the 219 gametes examined by multicolor FISH, the frequency of aneuploid products (XY or "O") was low (3.7%) despite a high frequency (66%) of X-Y separation at metaphase I. In balanced gametes, X and Y recombinant chromosomes slightly exceeded nonrecombinants. Both of these observations support the earlier proposal that asynapsis and nondisjunction in primary spermatocytes lead to their developmental arrest and degeneration.
Stress-inducible gene of Salmonella typhimurium identified by arbitrarily primed PCR of RNA.
Authors: Wong K K , McClelland M
Published: May 2006

Fingerprinting of RNA by arbitrarily primed PCR (RAP) can be used to identify conditionally expressed genes in prokaryotes. Differential gene expression in Salmonella typhimurium LT2 in response to peroxide treatment was examined as a system in which to demonstrate this strategy. This treatment models the induction of bacterial protective proteins that may occur when mammalian phagocytes use peroxide to fight S. typhimurium infection. To identify genes inducible by hydrogen peroxide stress, total RNA from peroxide-treated and untreated bacterial cultures were RAP fingerprinted with six different arbitrarily selected primers. A 435-base RAP product that was differentially amplified by RAP using the reverse sequencing primer was cloned and sequenced. Northern blot analysis confirmed that the RNA corresponding to this clone, RSP435, was induced when bacteria were treated with hydrogen peroxide. The RNA was not induced in an oxyR1 mutant that constitutively expressed a subset of hydrogen peroxide-inducible genes. Using pulsed-field gel electrophoresis and dot blot hybridization to an array of induced Mud-P22 integrations, the gene corresponding to RSP435 was mapped to two places, one between 19 and 21.5 min and one between 56 and 57 min. Thus, two similar or identical stress-inducible genes were found in different parts of the genome. Identification, cloning, and mapping of the conditionally expressed RSP435 cDNA were performed entirely by physical means, demonstrating that the strategy should complement genetic methods for many prokaryotic or archaebacterial systems and should be applicable to organisms in which genetic methods are difficult to perform or have not yet been developed.
The RNA of RNase MRP is required for normal processing of ribosomal RNA.
Authors: Chu S , Archer R H , Zengel J M , Lindahl L
Published: May 2006

We have isolated clones which complement the temperature sensitivity and abnormal rRNA processing pattern of the rrp2-2 mutant of Saccharomyces cerevisiae we previously described. DNA sequencing and restriction analysis demonstrated that all clones contain the NME1 gene encoding the RNA of the ribonucleprotein particle RNase MRP. Deletion analysis showed that the NME1 gene is responsible for the complementation of the rrp2-2 phenotype. A single base change was identified in the nme1 gene in the rrp2 mutant, confirming that the RRP2 and NME1 genes are identical. Our experiments therefore indicate that RNase MRP, in addition to its previously reported role in formation of RNA primers for mitochondrial DNA replication [Clayton, D. A. (1991) Trends Biochem. Sci. 16, 107-111], is involved in rRNA processing.
Beta cells arise from glucose transporter type 2 (Glut2)-expressing epithelial cells of the developing rat pancreas.
Authors: Pang K , Mukonoweshuro C , Wong G G
Published: May 2006

We observe Glut2 protein in day 11 (E11) rat embryos in an endodermal domain containing the pancreatic primordium. Glut2 expression continues as the endodermal epithelium evaginates into the surrounding mesenchyme to form the pancreatic buds. Cells of the dorsal and ventral pancreatic buds maintain Glut2 expression as the epithelium grows and branches to form ducts. As acini form at the ends of the ducts, acinar cells cease Glut2 expression. Insulin protein is first detected in small clusters at the interface between pancreatic epithelium and mesenchyme. These clusters disperse into the interstitial tissue between E13 and E17. At E17 a distinct, larger population of insulin-expressing cells arises in the Glut2-expressing ductal network. Insulin- and Glut2-coexpressing cells then appear to segregate into large aggregates to form the beta cells of the islets of Langerhans. These observations support the hypothesis that two biologically distinct populations of insulin-expressing cells arise during pancreas formation.
Genetic differences affecting the potency of stereoisomers of halothane.
Authors: Sedensky M M , Cascorbi H F , Meinwald J , Radford P , Morgan P G
Published: May 2006

The mechanism of action of volatile anesthetics is the subject of some debate. Much of the controversy has centered on whether the site of such actions is purely lipid in nature or may contain a protein target. This report studies the interaction of stereoisomers of halothane on the wild type and on a variety of genetic mutants of Caenorhabditis elegans. The mutants studied have previously been shown to have altered sensitivities to volatile anesthetics. In one mutant, fc34, (R)-halothane [the (+) isomer] was 3 times more potent than its S (-) isomer. Other mutants and wild-type animals displayed more modest differences in sensitivity to the enantiomers. The results indicate that a genetic pathway exists in C. elegans controlling sensitivity to halothane and that both lipid and protein targets may mediate halothane's effects.
Suppressors of nmtl-181, a conditional lethal allele of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene, reveal proteins involved in regulating protein N-myristoylation.
Authors: Johnson D R , Cok S J , Feldmann H , Gordon J I
Published: May 2006

Several essential Saccharomyces cerevisiae proteins require myristate to be covalently bound to their amino-terminal glycine for biological activity. Protein N-myristoylation is catalyzed by myristoyl-CoA:protein N-myristoyl-transferase, Nmt1p. nmt1-181 encodes a mutant enzyme with a Gly451-->Asp substitution. nmt181p has a reduced affinity for myristoyl-CoA and produces global defects in protein N-myristoylation at > or = 30 degrees C. nmt1-181 results in growth arrest at various stages of the cell cycle within 1 hr after cells are shifted to > or = 30 degrees C and lethality within 8 hr. The growth-arrest phenotype and loss of viability do not require components of the mating pathway and are associated with lysis sensitivity that may be related to undermyristoylation of two protein phosphatases, Ppz1p and Ppz2p. Growth can be rescued at 30 degrees C by adding myristate or sorbitol to the medium or by removing inosine. Cells can be rescued at 37 degrees C by overexpressing nmt1-181p or Nmt1p or by adding myristate to the medium. Selection of high-copy suppressors of the myristate auxotrophy and lethality observed at 37 degrees C yielded only NMT1, whereas six unlinked suppressors of the myristoylation defect (SMD1-6) were obtained when the screen was conducted at 30 degrees C. The protein products of three SMD loci were identified: (i) cdc39-delta 1.7p, which transactivates NMT1; (ii) Fas1p, the beta subunit of the fatty acid synthetase complex, activates FAS2's promoter and increases myristoylation of Gpa1p; and (iii) Pho5p, the major secreted acid phosphatase produced by this yeast. PHO5 is normally induced when yeast are grown in phosphate-depleted medium. Removal of inorganic phosphate from the medium also rescues nmt1-181 cells at 30 degrees C. PHO5's mechanism of suppression of nmt1-181 appears to involve, at least in part, activation of FAS2 transcription and a resulting effect on FAS1 expression. There is an inverse relationship between cellular N-myristoyltransferase and secreted acid phosphatase activities. These observations provide a potential mechanism for coupling phosphate metabolism with the regulation of myristoyl-CoA synthesis and protein N-myristoylation.
High-level expression of human superoxide dismutase in the cyanobacterium Anacystis nidulans 6301.
Authors: Takeshima Y , Takatsugu N , Sugiura M , Hagiwara H
Published: May 2006

A chemically synthesized gene encoding human CuZn superoxide dismutase (hSOD) was cloned into the shuttle vector pBAX18R and expressed in Anacystis nidulans 6301 (Synechococcus sp. strain PCC 6301) under the control of a ribulose-1,5-bisphosphate carboxylase/oxygenase gene (rbc) promoter derived from A. nidulans 6301. The sequences immediately upstream from the hSOD coding region and the distances between the ribosomal binding site and ATG initiation codon strongly affected the expression of the hSOD gene in A. nidulans cells. Optimal expression of hSOD was obtained with the expression vector pBAXSOD8-I, which contained a GGAGAG sequence. In defined conditions, irradiation with light increased hSOD enzyme activity in the transformants > 18-fold and the level of the hSOD protein reached a value of about 3% of the total soluble protein. The transformants that expressed hSOD acquired the ability to extenuate photooxidative damage induced by methyl viologen.
Isolation of the heme-thiolate enzyme cytochrome P-450TYR, which catalyzes the committed step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench.
Authors: Sibbesen O , Koch B , Halkier B A , Møller B L
Published: May 2006

The cytochrome P-450 enzyme (hemethiolate enzyme) that catalyzes the N-hydroxylation of L-tyrosine to N-hydroxytyrosine, the committed step in the biosynthesis of the cyanogenic glucoside dhurrin, has been isolated from microsomes prepared from etiolated seedlings of Sorghum bicolor (L.) Moench. The cytochrome P-450 enzyme was solubilized with the detergents Renex 690, reduced Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and isolated by ion-exchange (DEAE-Sepharose) and dye (Cibacron blue and reactive red 120) column chromatography. To prevent irreversible aggregation of the cytochrome P-450 enzyme, the isolation procedure was designed without any concentration step--i.e., with dilution of the ion-exchange gel with gel filtration material. The isolated enzyme, which we designate the cytochrome P-450TYR enzyme, gives rise to the specific formation of a type I substrate binding spectrum in the presence of L-tyrosine. The microsomal preparation contains 0.2 nmol of total cytochrome P-450/mg of protein. The cytochrome P-450TYR enzyme is estimated to constitute approximately 20% of the total cytochrome P-450 content of the microsomal membranes and about 0.2% of their total protein content. The apparent molecular mass of the cytochrome P-450TYR enzyme is 57 kDa, and the N-terminal amino acid sequence is ATMEVEAAAATVLAAP. A polyclonal antibody raised against the isolated cytochrome P-450TYR enzyme is specific as monitored by Western blot analysis and inhibits the in vitro conversion of L-tyrosine to p-hydroxymandelonitrile catalyzed by the microsomal system. The cytochrome P-450TYR enzyme exhibits high substrate specificity and acts as an N-hydroxylase on a single endogenous substrate. The reported isolation procedure based on dye columns constitutes a gentle isolation method for cytochrome P-450 enzymes and is of general use as indicated by its ability to separate cytochrome P-450TYR from the cytochrome P-450 enzyme catalyzing the C-hydroxylation of p-hydroxyphenylacetonitrile and from cinnamic acid 4-hydroxylase.
Ankyrin and beta-spectrin accumulate independently of alpha-spectrin in Drosophila.
Authors: Dubreuil R R , Yu J
Published: May 2006

We report the identification and initial characterization of Drosophila melanogaster ankyrin. Oligonucleotide primers based on the spectrin-binding domain of human brain ankyrin were used to amplify Drosophila genomic DNA. A cloned 184-bp PCR product was used to isolate Drosophila ankyrin cDNAs. Ankyrin cDNA probes detected a 5.5-kb transcript from embryonic poly(A)+ RNA and a single polytene chromosome locus (101F-102A). The cDNA sequence encodes a 170-kDa protein that is 53% identical to human brain ankyrin (Ank2). Antibodies directed against a recombinant fragment of Drosophila ankyrin reacted with a 170-kDa polypeptide from Drosophila embryos, larvae, S2 cells, and adult flies. The ankyrin antibody coimmunoprecipitated alpha- and beta-spectrin with ankyrin in detergent extracts of Drosophila embryo membranes. Antibodies against Drosophila ankyrin, alpha-spectrin and beta-spectrin were used to detect these proteins in wild-type and alpha-spectrin-mutant larvae. alpha-Spectrin levels were greatly diminished in mutant larvae, but levels of ankyrin and beta-spectrin were indistinguishable from wild type. The persistence of ankyrin and beta-spectrin may explain the relatively mild phenotype of alpha-spectrin mutants during early Drosophila development.
Salt-induced formation of the molten globule state of cytochrome c studied by isothermal titration calorimetry.
Authors: Hamada D , Kidokoro S , Fukada H , Takahashi K , Goto Y
Published: May 2006

Although the molten globule state has been proposed as a major intermediate of protein folding, it has proven difficult to obtain thermodynamic data characterizing this state. To explore another approach for characterizing the molten globule state, salt-induced formation of the molten globule state of horse cytochrome c at pH 1.8 was studied by isothermal titration calorimetry. By titrating the acid-unfolded cytochrome c with sodium perchlorate, an exothermic reaction was observed. The titration curve obtained from the heat was cooperative and agreed well with the conformational transition curve measured by CD at 222 nm. This result indicated that the salt-induced conformation change is well approximated by a two-state transition between the acid-unfolded and molten globule states. The heat for formation of the molten globule state estimated by isothermal titration calorimetry was consistent with the enthalpy change for unfolding of the sodium perchlorate-stabilized molten globule state at pH 1.8, which was measured by differential scanning calorimetry and CD. These results indicate that the heat of titration largely reflects the enthalpy change of the conformational transition. From these results, we consider that isothermal titration calorimetry will become a useful approach for investigating the molten globule state.