bluuebunny/crossref_metadata_2025 huggingface dataset:
The spfash mouse: a missense mutation in the ornithine transcarbamylase gene also causes aberrant mRNA splicing.
Authors: Hodges P E , Rosenberg L E
Published: May 2006

Ornithine transcarbamylase (ornithine carbamoyltransferase; carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) is a mitochondrial matrix enzyme of the mammalian urea cycle. The X chromosome-linked spfash mutation in the mouse causes partial ornithine transcarbamylase deficiency and has served as a model for the human disease. We show here that the spfash mutation is a guanine to adenine transition of the last nucleotide of the fourth exon of the ornithine transcarbamylase gene. This nucleotide change produces two remarkably different effects. First, this transition causes ornithine transcarbamylase mRNA deficiency because the involved exon nucleotide plays a part in the recognition of the adjacent splice donor site. As a result of the mutation, ornithine transcarbamylase pre-mRNA is spliced inefficiently both at this site and at a cryptic splice donor site 48 bases into the adjacent intron. Second, two mutant proteins are translated from these mRNAs. From the correctly spliced mRNA, the transition results in a change of amino acid 129 from arginine to histidine. This missense substitution has no discernable effect on mitochondrial import, subunit assembly, or enzyme activity. On the other hand, the elongated mRNA resulting from mis-splicing is translated into a dysfunctional ornithine transcarbamylase subunit elongated by the insertion of 16 amino acid residues.
Cloning of the major histocompatibility complex class II promoter binding protein affected in a hereditary defect in class II gene regulation.
Authors: Reith W , Barras E , Satola S , Kobr M , Reinhart D , Sanchez C H , Mach B
Published: May 2006

The regulation of major histocompatibility complex class II gene expression is directly involved in the control of normal and abnormal immune responses. In humans, HLA-DR, -DQ, and -DP class II heterodimers are encoded by a family of alpha- and beta-chain genes clustered in the major histocompatibility complex. Their expression is developmentally controlled and normally restricted to certain cell types. This control is mediated by cis-acting sequences in class II promoters and by trans-acting regulatory factors. Several nuclear proteins bind to class II promoter sequences. In a form of hereditary immunodeficiency characterized by a defect in a trans-acting regulatory factor controlling class II gene transcription, we have observed that one of these nuclear factors (RF-X) does not bind to its target sequence (the class II X box). A cDNA encoding RF-X was isolated by screening a phage expression library with an X-box binding-site probe. The recombinant protein has the binding specificity of RF-X, including a characteristic gradient of affinity for the X boxes of HLA-DR, -DP, and -DQ promoters. RF-X mRNA is present in the regulatory mutants, indicating a defect in the synthesis of a functional form of the RF-X protein.
Heightened complement sensitivity of acquired immunodeficiency syndrome lymphocytes related to diminished expression of decay-accelerating factor.
Authors: Lederman M M , Purvis S F , Walter E I , Carey J T , Medof M E
Published: May 2006

Although the human immunodeficiency virus can induce cytopathic changes in human lymphocytes in vitro, the mechanism(s) underlying progressive lymphopenia in patients with AIDS and AIDS-related complex has not been elucidated. To investigate this issue, peripheral blood lymphocytes of AIDS and AIDS-related complex patients and healthy control subjects were examined for their ability to resist homologous complement-mediated lysis. Upon sensitization with monoclonal antibodies to major histocompatibility complex class I antigen, as much as 48% lysis of patients' cells was observed in as little as a 1:32 dilution of human serum compared to 18 +/- 8% (mean +/- SD) lysis of controls' cells even in a 1:8 dilution of human serum. To investigate the mechanism of the abnormal complement sensitivity, AIDS and AIDS-related complex cells were analyzed for expression of decay-accelerating factor (DAF), a complement regulatory protein that functions intrinsically in blood cell membranes to prevent complement activation on their surfaces. Flow cytometric assays using anti-DAF monoclonal antibodies demonstrated that patients' lymphocytes and monocytes were DAF-deficient, in contrast to their polymorphonuclear leukocytes, which showed normal DAF levels. Expression of DAF was diminished on CD4+ as well as CD8+ T-lymphocyte subpopulations as opposed to expression of CD3, which was comparable in patients and controls. Incubation of normal lymphocytes with anti-DAF monoclonal antibodies or phosphatidylinositol-specific phospholipase C, an enzyme that cleaves DAF, enhanced lysis. Conversely, reconstitution of patients' cells with exogenous DAF reduced their lysis. The findings of heightened complement sensitivity and DAF deficiency of patients' lymphocytes in vitro suggest the possibility that the DAF deficit may contribute to the progressive lymphopenia of AIDS in vivo.
Synaptogenesis in visual cortex of normal and preterm monkeys: evidence for intrinsic regulation of synaptic overproduction.
Authors: Bourgeois J P , Jastreboff P J , Rakic P
Published: May 2006

We used quantitative electron microscopy to determine the effect of precocious visual experience on the time course, magnitude, and pattern of perinatal synaptic overproduction in the primary visual cortex of the rhesus monkey. Fetuses were delivered by caesarean section 3 weeks before term, exposed to normal light intensity and day/night cycles, and killed within the first postnatal month, together with age-matched controls that were delivered at term. We found that premature visual stimulation does not affect the rate of synaptic accretion and overproduction. Both of these processes proceed in relation to the time of conception rather than to the time of delivery. In contrast, the size, type, and laminar distribution of synapses were significantly different between preterm and control infants. The changes and differences in these parameters correlate with the duration of visual stimulation and become less pronounced with age. If visual experience in infancy influences the maturation of the visual cortex, it must do so predominantly by strengthening, modifying, and/or eliminating synapses that have already been formed, rather than by regulating the rate of synapse production.
cGMP is tightly bound to bovine retinal rod phosphodiesterase.
Authors: Gillespie P G , Beavo J A
Published: May 2006

Although the total concentration of cGMP in rod outer segments is thought to be substantially greater than the free concentration, no quantitatively relevant site for the bound cGMP has been described in mammalian photoreceptors. We have found that preparations of purified bovine rod photoreceptor cyclic nucleotide phosphodiesterase (PDE) contain 1.8 +/- 0.3 mol of tightly bound cGMP per mol of PDE. When subunits of the purified PDE were separated by reverse-phase HPLC in 0.1% trifluoroacetic acid and acetonitrile, a peak of material having spectral properties characteristic of a guanine ring was seen. This material was identified as cGMP by comigration with authentic cGMP on HPLC, conversion to 5-GMP by trypsin-activated rod PDE, and conversion to guanosine by a combination of trypsin-activated PDE and 5'-nucleotidase-containing snake venom. When incubated with 1 microM [3H]cGMP, only 0.1 mol of [3H]cGMP bound per mol of purified PDE, presumably because nearly all binding sites were occupied by tightly bound endogenous cGMP carried through the purification. Scatchard plots of [3H]cGMP binding have indicated that two classes of binding sites are present on the rod PDE. The off-rate of cGMP from the slowly dissociating site is extremely slow; it has a t1/2 of approximately 4 hr at 37 degrees C. At lower temperatures, very little cGMP dissociates; the amount of [3H]cGMP bound to rod PDE after 2 hr at 4 degrees C was essentially the same as at the beginning of the incubation. The observation that stoichiometric amounts of cGMP are tightly bound to PDE accounts for the inability to purify the bovine rod PDE on cGMP affinity columns or to demonstrate stoichiometric high-affinity binding sites with [3H]cGMP. More significantly, the tightly bound cGMP may resolve the apparent discrepancy between the free and total cGMP concentrations of photoreceptor outer segments.
Molecular properties of pyruvate bound to lactate dehydrogenase: a Raman spectroscopic study.
Authors: Deng H , Zheng J , Burgner J , Callender R
Published: May 2006

Lactate dehydrogenase (LDH; EC 1.1.1.27) catalyzes the addition of pyruvate to the four position of the nicotinamide ring of bound NAD+; this NAD-pyruvate adduct is bound tightly to the enzyme. We have used the adduct as a model for pyruvate in a competent ternary complex by comparing the Raman spectrum of the bound adduct with that for unliganded pyruvate. To understand the observed normal modes of pyruvate both as the bound adduct and in water, we have taken the Raman spectra of a series of 13C- and 18O-labeled pyruvates. We find that the carboxylate COO- moiety of pyruvate remains unprotonated at LDH's active site and forms an ion pair complex. The frequency of pyruvate's carbonyl C = O moiety shifts from 1710 cm-1 in water downward 34 cm-1 when pyruvate binds to LDH. This frequency shift corresponds to a ca. 34% polarization of the carbonyl bond, indicates a substantial interaction between the C = O group and enzyme, and is direct evidence for and is a measure of enzyme-induced electronic perturbation of the substrate needed for catalysis. This bond polarization is likely brought about by electrostatic interactions between the carbonyl moiety and the protonated imidazole group of His-195 and the guanidino group from Arg-109. We discuss how the data bear on the enzymatic chemistry of LDH.
Structural organization of glycophorin A and B genes: glycophorin B gene evolved by homologous recombination at Alu repeat sequences.
Authors: Kudo S , Fukuda M
Published: May 2006

Glycophorins A (GPA) and B (GPB) are two major sialoglycoproteins of the human erythrocyte membrane. Here we present a comparison of the genomic structures of GPA and GPB developed by analyzing DNA clones isolated from a K562 genomic library. Nucleotide sequences of exon-intron junctions and 5' and 3' flanking sequences revealed that the GPA and GPB genes consist of 7 and 5 exons, respectively, and both genes have greater than 95% identical sequence from the 5' flanking region to the region approximately 1 kilobase downstream from the exon encoding the transmembrane regions. In this homologous part of the genes, GPB lacks one exon due to a point mutation at the 5' splicing site of the third intron, which inactivates the 5' cleavage event of splicing and leads to ligation of the second to the fourth exon. Following these very homologous sequences, the genomic sequences for GPA and GPB diverge significantly and no homology can be detected in their 3' end sequences. The transition site from homologous to nonhomologous sequences can be localized within Alu repeat sequences. The analysis of the Alu sequences and their flanking direct repeat sequences suggest that an ancestral genomic structure has been maintained in the GPA gene, whereas the GPB gene has arisen from the acquisition of 3' sequences different from those of the GPA gene by homologous recombination at the Alu repeats during or after gene duplication.
Involvement of the TCL5 gene on human chromosome 1 in T-cell leukemia and melanoma.
Authors: Finger L R , Kagan J , Christopher G , Kurtzberg J , Hershfield M S , Nowell P C , Croce C M
Published: May 2006

We analyzed a t(1;14)(p32;q11) chromosomal translocation in a human lymphohemopoietic stem cell line derived from a patient with acute T-lymphoblastic leukemia. The chromosomal joining on the 1p+ chromosome occurred at the T-cell receptor delta diversity (D delta 2) segment, and the reciprocal chromosomal joining on the 14q- chromosome occurred at the T-cell delta diversity segment D delta 1. The involvement of delta diversity segments at the translocation junctions suggests that the translocation occurred during an attempt at D delta 1-D delta 2 joining in a stem cell. The segment of chromosome 1 at band p32, adjacent to the chromosomal breakpoint, encodes a transcriptional unit designated TCL5 (T-cell leukemia/lymphoma 5). The differential expression of the TCL5 RNA transcripts in this lymphohemopoietic stem cell line relative to several other T- and B-cell lines suggests that TCL5 gene expression is an integral event in the pathogenesis of the T-cell leukemia. Rearrangement of the TCL5 locus in a human melanoma cell line carrying a del(1p32) further implies that the TCL5 gene may play a role in malignant transformation.
Substance P receptor binding sites are expressed by glia in vivo after neuronal injury.
Authors: Mantyh P W , Johnson D J , Boehmer C G , Catton M D , Vinters H V , Maggio J E , Too H P , Vigna S R
Published: May 2006

In vitro studies have demonstrated that glia can express functional receptors for a variety of neurotransmitters. To determine whether similar neurotransmitter receptors are also expressed by glia in vivo, we examined the glial scar in the transected optic nerve of the albino rabbit by quantitative receptor autoradiography. Receptor binding sites for radiolabeled calcitonin gene-related peptide, cholecystokinin, galanin, glutamate, somatostatin, substance P, and vasoactive intestinal peptide were examined. Specific receptor binding sites for each of these neurotransmitters were identified in the rabbit forebrain but were not detected in the normal optic nerve or tract. In the transected optic nerve and tract, only receptor binding sites for substance P were expressed at detectable levels. The density of substance P receptor binding sites observed in this glial scar is among the highest observed in the rabbit forebrain. Ligand displacement and saturation experiments indicate that the substance P receptor binding site expressed by the glial scar has pharmacological characteristics similar to those of substance P receptors in the rabbit striatum, rat brain, and rat and canine gut. The present study demonstrates that glial cells in vivo express high concentrations of substance P receptor binding sites after transection of retinal ganglion cell axons. Because substance P has been shown to regulate inflammatory and immune responses in peripheral tissues, substance P may also, by analogy, be involved in regulating the glial response to injury in the central nervous system.
Cooperative activation of a eukaryotic transcription factor: interaction between Cu(I) and yeast ACE1 protein.
Authors: Fürst P , Hamer D
Published: May 2006

Cu ions activate yeast metallothionein gene transcription by altering the conformation and DNA-binding activity of the ACE1 transcription factor. We show that this conformational switch occurs in an all-or-none highly cooperative fashion (Hill coefficient = 4). Analysis of the subunit composition of ACE1 bound to DNA indicates that cooperativity results from the binding of multiple Cu(I) ions to the cysteine-rich DNA-binding domain. Surprisingly, DNA has little effect on the interaction between Cu(I) and ACE1 as assayed by partial proteolysis; this suggests that the effect of the metal on DNA binding is primarily kinetic rather than thermodynamic. Although Ag(I) also activates ACE1, it acts less cooperatively than the smaller Cu(I) ion and the resulting metalloprotein has a reduced affinity for DNA. The cooperative interaction between Cu and ACE1 allows the cell to respond to a small change in metal concentration by a large change in gene expression.
Functional inhibition of endogenously produced urokinase decreases cell proliferation in a human melanoma cell line.
Authors: Kirchheimer J C , Wojta J , Christ G , Binder B R
Published: May 2006

Binding of urokinase-type plasminogen activator (u-PA) to its receptor has been shown not only to focus proteolytic activity to the cell surface but also to exert a mitogenic effect on the human epidermal tumor cell line CCL 20.2. This report shows that u-PA is an autocrine mitogen in the human melanoma cell line GUBSB and that inhibition of receptor-bound u-PA by specific anti-u-PA antibodies causes a significant suppression of cell proliferation in this system. The GUBSB cell line secretes 70-80% of the u-PA in its active form and expresses high-affinity u-PA receptors with a Kd of 5.2 x 10(-10) M and 2.8 x 10(4) binding sites per cell. Approximately 70% of the u-PA receptors on these cells are occupied by endogenously secreted u-PA. Addition of the monoclonal anti-u-PA antibody MPW5UK (10 nM), directed against the active site of u-PA, twice daily to the cell cultures resulted in a significant decrease of [3H]thymidine incorporation by the tumor cells, whereas a 10 times higher concentration of the monoclonal antibody MPW4UK, which does not inhibit plasminogen activator activity of u-PA, was necessary to achieve the same effect. In addition, diisopropyl fluorophosphate-inactivated u-PA, in a concentration 50-fold higher than the concentration necessary to saturate the u-PA receptor (250 pM), decreased [3H]thymidine incorporation similarly to the specific antibody, proving that active u-PA is required for the mitogenic effect. Inhibition of endogenous u-PA production by cycloheximide reduced [3H]thymidine incorporation significantly; after addition of exogenous u-PA, [3H]thymidine incorporation increased again in the cycloheximide-treated cells. Therefore, inhibition of receptor-bound u-PA might represent a tool not only to inactivate cell-bound proteolytic activity, necessary for invasion, but also to exert a specific antiproliferative effect on certain tumor cells.
Analysis of mammalian cell genetic regulation in situ by using retrovirus-derived "portable exons" carrying the Escherichia coli lacZ gene.
Authors: Brenner D G , Lin-Chao S , Cohen S N
Published: May 2006

Self-inactivating derivatives of Moloney murine leukemia retrovirus containing the Escherichia coli lacZ gene were used to detect and study the regulation of transcription initiated at chromosomally located promoters in mouse fibroblasts. The introduction of splice acceptor sites in all three translational reading frames relative to lacZ and the inclusion of an in-frame ATG translation start codon in one construct allowed synthesis of beta-galactosidase fusion proteins upon insertion of retrovirus vectors containing lacZ into introns 3' to either protein-coding or noncoding exons. Selection of lacZ-expressing cells by fluorescence-activated cell sorting and the analysis of beta-galactosidase production after serum deprivation has yielded lines in which lacZ was fused to genes induced by growth arrest in the G0 state.
Immunoglobulin lambda light-chain-related genes 14.1 and 16.1 are expressed in pre-B cells and may encode the human immunoglobulin omega light-chain protein.
Authors: Hollis G F , Evans R J , Stafford-Hollis J M , Korsmeyer S J , McKearn J P
Published: May 2006

Human pre-B cells, which produce immunoglobulin heavy chain but do not produce immunoglobulin light chain, are shown to contain a 1-kilobase transcript homologous to immunoglobulin lambda light-chain genes. Detailed analysis of RNA and cDNA clones derived from these transcripts reveals that they originate from the distinct immunoglobulin lambda-like genes 14.1/16.1. Sequence analysis of these clones reveals a long open reading frame, beginning with an ATG, capable of encoding a protein of 214 amino acids with an unprocessed molecular weight of 22,944. The C-terminal half of this predicted protein is highly homologous to immunoglobulin lambda light-chain joining and constant region protein sequence, while the amino-terminal end does not share homology with variable regions. Unlike immunoglobulin genes, these genes do not undergo rearrangement prior to expression. Analysis of a panel of 26 hematopoietic cell lines reveals that expression of 14.1/16.1 is limited to pre-B cells and one B-cell line, which, like the pre-B cells, is surface immunoglobulin negative. Antisera raised against a peptide whose sequence was predicted from the 14.1 cDNA sequence identifies a 22-kDa protein in human pre-B cells. Immunoprecipitation of immunoglobulin mu-chain from these pre-B cells with anti-immunoglobulin mu antibody coprecipitates a 22-kDa protein, which is a candidate for the human immunoglobulin omega light-chain protein and may be the protein product of the 14.1/16.1 genes.
Synthesis and characterization of a series of diarylguanidines that are noncompetitive N-methyl-D-aspartate receptor antagonists with neuroprotective properties.
Authors: Keana J F , McBurney R N , Scherz M W , Fischer J B , Hamilton P N , Smith S M , Server A C , Finkbeiner S , Stevens C F , Jahr C
Published: May 2006

Four diarylguanidine derivatives were synthesized. These compounds were found to displace, at submicromolar concentrations, 3H-labeled 1-[1-(2-thienyl)cyclohexyl]piperidine and (+)-[3H]MK-801 from phencyclidine receptors in brain membrane preparations. In electrophysiological experiments the diarylguanidines blocked N-methyl-D-aspartate (NMDA)-activated ion channels. These diarylguanidines also protected rat hippocampal neurons in vitro from glutamate-induced cell death. Our results show that some diarylguanidines are noncompetitive antagonists of NMDA receptor-mediated responses and have the neuroprotective property that is commonly associated with blockers of the NMDA receptor-gated cation channel. Diarylguanidines are structurally unrelated to known blockers of NMDA channels and, therefore, represent a new compound series for the development of neuroprotective agents with therapeutic value in patients suffering from stroke, from brain or spinal cord trauma, from hypoglycemia, and possibly from brain ischemia due to heart attack.
Trapped individual ion at absolute zero temperature
Authors: Yu Nan , Dehmelt Hans , Nagourney Warren
Published: May 2006

Laser cooling and ion trapping have progressed to such an extent that one can now speak of realizing a confined atom at absolute zero temperature. In this short publication, we analyze an experiment toward such realization using a single Ba
+
ion in a miniature rf trap. The Ba
+
ion is first laser-cooled to the limit where the ion spends most of its time in the zero-point energy state. Then a test sequence allows one to verify whether or not the ion is actually in its zero-point state. The test sequence may also serve as a device for state selection of an atom at absolute zero temperature.
Association of charge clusters with functional domains of cellular transcription factors.
Authors: Brendel V , Karlin S
Published: May 2006

Using rigorous statistical methods, we have identified and evaluated unusual properties of the distribution of charged residues within the sequences of eukaryotic cellular transcription factors. Virtually all transcription factors, including GAL4, c-Jun, C/EBP, CREB, Oct-1, Oct-2, Sp1, Egr-1, CTF-1, steroid and thyroid hormone receptors, and others, carry one or more highly significant charge clusters. For the most part these clusters (conserved within families of homologous proteins) are of positive net charge but contain also substantial numbers of acidic residues. Predominantly basic charge clusters are often, but not exclusively, associated with DNA-binding domains, and vice versa. Negative charge clusters of note occur only in the yeast protein PHO4 and in the proteins encoded at the Drosophila loci zeste (zeta) and knrl. This dearth of statistically significant negative charge clusters raises questions with respect to the generality of acidic activation domains. A number of sequences (Oct-1, Oct-2, zeste, Dhr23, E75, and knrl) contain multiple charge clusters together with one or more significantly long uncharged regions. The occurrence of multiple charge clusters is a rare phenomenon (found in less than 3% of all proteins, mainly in Drosophila developmental control proteins and in transactivators of eukaryotic DNA viruses). Most of the proteins with zinc-binding "fingers" carry a mixed charge cluster centered at the zinc-finger motif preceded by a long uncharged stretch, suggestive of a modular structure for these proteins.
Predicting the orientation of eukaryotic membrane-spanning proteins.
Authors: Hartmann E , Rapoport T A , Lodish H F
Published: May 2006

We have developed a rule to predict the orientation of the first internal signal-anchor sequence in eukaryotic transmembrane proteins synthesized on the rough endoplasmic reticulum. The difference in the charges of the 15 residues flanking the first internal signal-anchor determines its orientation, with the more positive portion facing the cytosol. In proteins that span the membrane more than once, the orientation of all subsequent transmembrane segments would be determined by that of the most N-terminal one.
Rapid screening of a human genomic library in yeast artificial chromosomes for single-copy sequences.
Authors: Traver C N , Klapholz S , Hyman R W , Davis R W
Published: May 2006

A yeast artificial chromosome (YAC) library in Saccharomyces cerevisiae consisting of 30,000 clones with an average insert size of 0.1 megabase pair of human DNA has been generated from primary fibroblast DNA. A YAC vector was modified to enable the recovery of both ends of a human DNA insert in plasmids in Escherichia coli and to confer G418 resistance to mammalian cells. A rapid method for yeast colony hybridization was used that exploits the ability of yeast spheroplasts to regenerate in a thin layer of calcium alginate. This method permits direct replica plating and processing of colonies from the primary transformation plate to nitrocellulose filters. Yeast colony hybridization conditions have been established to identify, within a YAC library of human genomic DNA, artificial chromosomes with homology to human DNA probes of unique single-copy sequence. An artificial chromosome with a 0.1-megabase-pair insert from the human Xq28 region has been identified by hybridization to a DNA probe that detects a unique sequence near the 3' end of the factor VIII gene.
Molecular cloning and expression of cDNA encoding a lumenal calcium binding glycoprotein from sarcoplasmic reticulum.
Authors: Leberer E , Charuk J H , Green N M , MacLennan D H
Published: May 2006

Antibody screening was used to isolate a cDNA encoding the 160-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum. The cDNA is identical to that encoding the 53-kDa glycoprotein except that it contains an in-frame insertion of 1308 nucleotides near its 5' end, apparently resulting from alternative splicing. The protein encoded by the cDNA would contain a 19-residue NH2-terminal signal sequence and a 453-residue COOH-terminal sequence identical to the 53-kDa glycoprotein. It would also contain a 436-amino acid insert between these sequences. This insert would be highly acidic, suggesting that it might bind Ca2+. The purified 160-kDa glycoprotein and the glycoprotein expressed in COS-1 cells transfected with cDNA encoding the 160-kDa glycoprotein were shown to bind 45Ca2+ in a gel overlay assay. The protein was shown to be located in the lumen of the sarcoplasmic reticulum and to be associated through Ca2+ with the membrane. We propose that this lumenal Ca2+ binding glycoprotein of the sarcoplasmic reticulum be designated "sarcalumenin."
Actin filaments mediate Dictyostelium myosin assembly in vitro.
Authors: Mahajan R K , Vaughan K T , Johns J A , Pardee J D
Published: May 2006

Because myosin thick filaments form in the actin-rich cortex of nonmuscle cells, we have examined the role of Dictyostelium actin filaments in the assembly of Dictyostelium myosin (type II). Fluorescence energy transfer and light-scattering assembly assays indicate that self-association of Dictyostelium myosin into bipolar thick filaments is kinetically regulated by actin filament networks. Regulation is nucleotide dependent but does not require ATP hydrolysis. Myosin assembly is accelerated approximately 5-fold by actin filaments when either 1 mM ATP or 1 mM adenosine 5'-[beta,gamma-imido]triphosphate (AMP-P[NH]P) is present. However, actin filaments together with 1 mM ADP abolish myosin assembly. Accelerated assembly appears to require transient binding of myosin molecules to actin filaments before incorporation into thick filaments. Fluorescence energy-transfer assays demonstrate that myosin associates with actin filaments at a rate that is equivalent to the accelerated myosin assembly rate, evidence that myosin to actin binding is a rate-limiting step in accelerated thick filament formation. Actin filament networks are also implicated in regulation of thick filament formation, since fragmentation of F-actin networks by severin causes immediate cessation of accelerated myosin assembly. Electron microscopic studies support a model of actin filament-mediated myosin assembly. In ADP, myosin monomers rapidly decorate F-actin, preventing extensive formation of thick filaments. In AMP-P[NH]P, myosin assembles along actin filaments, forming structures that resemble primitive stress fibers. Taken together, these data suggest a model in which site-directed assembly of thick filaments in Dictyostelium is mediated by the interaction of myosin monomers with cortical actin filament networks.
A family of cyclin homologs that control the G1 phase in yeast.
Authors: Hadwiger J A , Wittenberg C , Richardson H E , de Barros Lopes M , Reed S I
Published: May 2006

Two Saccharomyces cerevisiae genes were isolated based upon their dosage-dependent rescue of a temperature-sensitive mutation of the gene CDC28, which encodes a protein kinase involved in control of cell division. CLN1 and CLN2 encode closely related proteins that also share homology with cyclins. Cyclins, characterized by a dramatic periodicity of abundance through the cell cycle, are thought to be involved in mitotic induction in animal cells. A dominant mutation in the CLN2 gene, CLN2-1, advances the G1- to S-phase transition in cycling cells and impairs the ability of cells to arrest in G1 phase in response to external signals, suggesting that the encoded protein is involved in G1 control of the cell cycle in Saccharomyces.
Altered I-A protein-mediated transmembrane signaling in B cells that express truncated I-Ak protein.
Authors: Wade W F , Chen Z Z , Maki R , McKercher S , Palmer E , Cambier J C , Freed J H
Published: May 2006

Recent evidence suggests that the major histocompatibility complex class II molecules of B lymphocytes function as signal-transducing receptors during the generation of T lymphocyte-dependent humoral immune responses. By analogy with other receptors, we postulate that perturbation of the class II molecules is coupled to the generation of intracellular second messengers through interactions involving the transmembrane and/or cytoplasmic domains of the class II molecules. We report a series of experiments that assess which amino acids of the class II molecule I-Ak are required for coupling it to the signal-transduction pathway. We prepared a series of B-lymphocyte transfectants that express I-Ak molecules with COOH-terminal truncations of either the Ak alpha or Ak beta chain or both. The ability of each transfected class II molecule to transduce a signal after being bound by monoclonal antibody was found by monitoring the translocation of protein kinase C from the cytosol to the "nuclear compartment" of the transfected B lymphocyte. Results indicate that the Ak beta chain plays the dominant role in signal transduction and that the 6 cytoplasmic amino acids of Ak beta chain most proximal to the inner plasma membrane are of greatest importance in coupling I-Ak molecules to the molecules of the signaling cascade.
Human neutrophils and mononuclear cells inhibit platelet aggregation by releasing a nitric oxide-like factor.
Authors: Salvemini D , de Nucci G , Gryglewski R J , Vane J R
Published: May 2006

Incubation of neutrophils or mononuclear cells with washed platelets (all prepared from human venous blood) resulted in an inhibition of thrombin-induced platelet aggregation that was dependent on the number of nucleated cells added. The inhibition was potentiated by superoxide dismutase and reversed by oxyhemoglobin. In the case of neutrophils the inhibition was associated with an increase in cGMP, whereas with mononuclear cells both cAMP and cGMP were increased. The inhibitory activity of neutrophils or mononuclear cells was prevented by their preincubation with NG-monomethyl-L-arginine methyl ester. L-Arginine reversed the action of NG-monomethyl-L-arginine methyl ester, whereas D-arginine was ineffective. Preincubation of the cells with catalase or mannitol did not prevent their inhibitory action on platelet aggregation. The inhibition of platelet aggregation was not due to platelet damage or to uptake of thrombin by neutrophils or mononuclear cells. It was overcome by increasing the concentration of thrombin and was absent in cell-free supernatants obtained from a suspension of neutrophils or mononuclear cells or from mixtures of platelets with neutrophils or platelets with mononuclear cells. These data provide evidence for the release of a nitric oxide-like factor from human neutrophils and mononuclear cells. In addition, evidence is provided that, as in stimulated murine macrophages and endothelial cells, the precursor of this factor is L-arginine.
Crystallographic structure of a photoreceptor protein at 2.4 A resolution.
Authors: McRee D E , Tainer J A , Meyer T E , Van Beeumen J , Cusanovich M A , Getzoff E D
Published: May 2006

The first essential step in protein photoreception is the capture and storage of energy from a photon. We have recently identified and isolated, from the purple photoautotrophic bacterium, Ectothiorhodospira halophila, a 13,000-dalton photoactive yellow protein (PYP) that has a photocycle with kinetics similar to sensory rhodopsin and a very high quantum efficiency. To study the structural chemistry of protein photoreception, we determined, refined, and analyzed the crystallographic structure of PYP at 2.4 A resolution and report here that it is composed of two perpendicular antiparallel beta-sheets that enclose the chromophore. Each of the 10 beta-strands of PYP is connected directly to its nearest neighbor with +1 topology. Globally, an asymmetric distribution of side chains places aromatic and acidic side chains in an ellipsoidal band around the chromophore with a cluster of basic side chains on one side. Locally, the electron density maps place an internal lysine and the chromophore in an apparent Schiff base linkage stabilized by a buried glutamate and a tyrosine side chain. To our knowledge, the atomic resolution structure of a protein with a reversible photoisomerization has not been reported previously. Furthermore, PYP may also represent a class of proteins that bind conjugated molecules and interact with a secondary receptor system.
Topological distribution of four-alpha-helix bundles.
Authors: Presnell S R , Cohen F E
Published: May 2006

The four-alpha-helix bundle, a common structural motif in globular proteins, provides an excellent forum for the examination of predictive constraints for protein backbone topology. An exhaustive examination of the Brookhaven Crystallographic Protein Data Bank and other literature sources has lead to the discovery of 20 putative four-alpha-helix bundles. Application of an analytical method that examines the difference between solvent-accessible surface areas in packed and partially unpacked bundles reduced the number of structures to 16. Angular requirements further reduced the list of bundles to 13. In 12 of these bundles, all pairs of neighboring helices were oriented in an anti-parallel fashion. This distribution is in accordance with structure types expected if the helix macro dipole effect makes a substantial contribution to the stability of the native structure. The characterizations and classifications made in this study prompt a reevaluation of constraints used in structure prediction efforts.
A T-cell-specific transcriptional enhancer element 3' of C alpha in the human T-cell receptor alpha locus.
Authors: Ho I C , Yang L H , Morle G , Leiden J M
Published: May 2006

A transcriptional enhancer element has been identified 4.5 kilobases 3' of C alpha (constant region alpha chain) in the human T-cell receptor (TCR) alpha-chain locus. This enhancer is active on both a TCR V alpha (variable region alpha chain) promoter and the minimal simian virus 40 promotor in TCR alpha/beta Jurkat and EL4 cells but is inactive on a V alpha promoter in human TCR gamma/delta PEER and Molt-13 cells, clone 13 B cells, and HeLa fibroblasts. The enhancer has been localized to a 116-base-pair BstXI/Dra I restriction enzyme fragment, which lacks immunoglobulin octamer and kappa B enhancer motifs but does contain a consensus cAMP-response element (CRE). DNase I footprint analyses demonstrated that the minimal enhancer contains two binding sites for Jurkat nuclear proteins. One of these sites corresponds to the CRE, while the other does not correspond to a known transcriptional enhancer motif. These data support a model in which TCR alpha gene transcription is regulated by a unique set of cis-acting sequences and trans-acting factors, which are differentially active in cells of the TCR alpha/beta lineage. In addition, the TCR alpha enhancer may play a role in activating oncogene expression in T-lymphoblastoid tumors that have previously been shown to display chromosomal translocations into the human TCR alpha locus.
Localized actions of progesterone in hypothalamus involve oxytocin.
Authors: Schumacher M , Coirini H , Frankfurt M , McEwen B S
Published: May 2006

Two ovarian hormones, estradiol and progesterone, which facilitate mating behavior in the female rat by acting on the ventromedial nuclei (VMN) of the hypothalamus, induce changes in oxytocin receptor binding in this brain region. Estradiol induced a 4-fold increase in the oxytocin receptor binding of the VMN and surrounding area and increased the number and immunostaining of oxytocin fibers in an area lateral to the ventral VMN. Progesterone, in estrogen-primed rats, caused the induced oxytocin receptors to spread over the area containing the oxytocin fibers. Infusion of oxytocin into the ventromedial hypothalamus increased the display of lordosis behavior only in females primed with both estradiol benzoate and progesterone. Thus, the sequential actions of two ovarian hormones bring a neuropeptide and its receptors into register and enable the neuropeptide to exert behavioral effects.
Activation of purified calcium channels by stoichiometric protein phosphorylation.
Authors: Nunoki K , Florio V , Catterall W A
Published: May 2006

Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of 45Ca2+ uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of 45Ca2+ uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd2+, Ni2+, and Mg2+. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels.
Structure and organization of four clustered genes that encode bombyxin, an insulin-related brain secretory peptide of the silkmoth Bombyx mori.
Authors: Kawakami A , Iwami M , Nagasawa H , Suzuki A , Ishizaki H
Published: May 2006

Four genes encoding bombyxin have been located in a 14-kilobase Bombyx genomic DNA segment. All of these genes encode preprobombyxin, the precursor molecule for bombyxin, with the domain organization of signal peptide/B chain/C peptide/A chain. Bombyxins are classified as family A or B according to their sequence homology. Two genes, each belonging to a different family, are closely apposed to form a pair with opposite orientation, presumably forming a regulatory unit for transcription. Genomic Southern blot hybridization suggested that there are many such gene pairs in the Bombyx genome. Differences between bombyxin genes and vertebrate insulin-family genes indicate that different mechanisms operate in the evolution of invertebrate and vertebrate insulin-family genes.
Site specificity of the inhibitory effects of oligo(nucleoside methylphosphonate)s complementary to the acceptor splice junction of herpes simplex virus type 1 immediate early mRNA 4.
Authors: Kulka M , Smith C C , Aurelian L , Fishelevich R , Meade K , Miller P , Ts'o P O
Published: May 2006

Oligo(nucleoside methylphosphonate)s complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5 caused specific inhibition of herpes simplex virus type 1 growth. The dodecamer d(TpTCCTCCTGCGG) (deoxynucleoside methylphosphonate residues in italic) caused 50% and 98% decreases in herpes simplex virus type 1 titers at concentrations of 15 microM and 100 microM, respectively. d(TpTCCTCCTGCGG) inhibited viral but not cellular protein synthesis and decreased splicing of immediate early pre-mRNAs 4 and 5. Inhibition was highly sequence specific. A psoralen derivative of d(TpTCCTCCTGCGG) that can covalently bind to complementary sequences after exposure to 365-nm irradiation caused 90-98% inhibition of virus growth in cells treated with oligomer (5 microM) and irradiated at 1-3 hr postinfection. The data suggest that oligo(nucleoside methylphosphonate)s of appropriate sequence and derivatization may be effective as antiviral agents.
Codon discrimination and anticodon structural context.
Authors: Lustig F , Borén T , Guindy Y S , Elias P , Samuelsson T , Gehrke C W , Kuo K C , Lagerkvist U
Published: May 2006

Site-directed mutagenesis has been used to change the nucleotide C in the wobble position of tRNA(1Gly) (CCC) to U. The mutated tRNA was tested for its ability to read glycine codons in an in vitro protein-synthesizing system programmed with the phage message MS2-RNA that had been modified by site-directed mutagenesis so as to make it possible to monitor conveniently the reading of all four glycine codons. The results showed that while the efficiency of tRNA(1Gly) (UCC) was comparable to that of mycoplasma tRNA(Gly) (UCC) in the reading of the codon GGA, the mycoplasma tRNA(Gly) was far more efficient than the tRNA(1Gly) (UCC) in the reading of the codons GGU and GGC. Thus, the anticodon UCC, when present in the structural context of the tRNA(1Gly) molecule, behaved as predicted by the wobble rules while in the structural context of the mycoplasma tRNA(Gly) it read without discrimination between the nucleotides in the third codon position, in violation of the wobble restrictions. The result with the codon GGG showed that the anticodon UCC, when present in tRNA(1Gly), was considerably less efficient in reading this codon than it was in the structural context of the mycoplasma tRNA(Gly). It would therefore seem that the anticodon UCC, when present in a certain tRNA, can be an efficient wobbler, while in the molecular environment of another tRNA it is markedly restricted in its ability to wobble.
Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase.
Authors: Rossomando A J , Payne D M , Weber M J , Sturgill T W
Published: May 2006

pp42, a low-abundance 42-kDa protein, becomes transiently phosphorylated on tyrosine after stimulation of fibroblasts by a variety of mitogens, including epidermal growth factor, platelet-derived growth factor, phorbol 12-myristate 13-acetate, thrombin, and insulin-like growth factor II. The induction of pp42 phosphorylation on tyrosine by such diverse mitogenic agents suggests an important role for pp42 in the cascade of events necessary for cell transition from G0 into the cell cycle. However, as with most proteins identified on the basis of their tyrosine phosphorylation, the function of pp42 in cellular regulation is unknown. In this manuscript we report evidence that suggests that pp42 is a serine/threonine-specific protein kinase. Stimulation of 3T3-L1 cells with insulin has been shown to activate a cytosolic serine/threonine kinase capable of phosphorylating microtubule-associated protein 2 (MAP-2) and ribosomal protein S6 kinase II. This cytosolic serine/threonine protein kinase, which itself is phosphorylated on tyrosine, has been termed "MAP kinase". We now report that pp42 phosphorylation and MAP kinase activation occur in fibroblasts in response to similar mitogens, that the two proteins comigrate on one- and two-dimensional polyacrylamide gels, and that the two proteins copurify chromatographically. The major peptides generated from purified MAP kinase by V8 protease digestion are present as a subset of the peptides in digests of pp42 excised from two-dimensional gels. Thus, the results suggest that MAP kinase is tyrosine-phosphorylated pp42.
Cytoplasmic requirement for peroxisome biogenesis in Chinese hamster ovary cells.
Authors: Allen L A , Morand O H , Raetz C R
Published: May 2006

Hybrids constructed by fusion of wild-type Chinese hamster ovary cells (CHO-K1) to peroxisome-deficient CHO mutants (ZR-78.1) contain normal peroxisomes, demonstrating that the mutation(s) are recessive. "Nuclear hybrids" prepared by fusion of CHO-K1 karyoplasts to mutant ZR-78.1 occasionally fail to regain intact peroxisomes (approximately 1/300 cells). These peroxisome-deficient nuclear hybrids closely resemble the original mutant cells by biochemical criteria, but their modal chromosome number is 36-38, the same as that of CHO hybrids generated from intact cells. When the peroxisome-deficient nuclear hybrids are fused to wild-type cytoplasts, a fraction of the fusion products (at least 70%) continue to propagate normal peroxisomes indefinitely. Peroxisome biogenesis cannot be reinitiated in cells of mutant ZR-78.1 by fusion to wild-type cytoplasts. Our results suggest that a wild-type nucleus by itself is necessary but not sufficient for restoration of normal peroxisome biogenesis and that a cytoplasmic component of wild-type cells, possibly a normal peroxisome, is also required.
Local influence of substrate molecules in determining distinctive growth patterns of identified neurons in culture.
Authors: Grumbacher-Reinert S
Published: May 2006

The growth and branching patterns of individual identified leech neurons in culture depend upon the molecular composition of the substrate. These differences in morphology have been analyzed quantitatively for nerve cells growing on the plant lectin Con A, on extracellular matrix extract (ECM) containing laminin, and on patterned substrates. The total length of neurite outgrowth was about four times greater, and the number of branching points per unit length was three times smaller on ECM laminin extract than on Con A. Single cells placed on a sharp well-defined border separating Con A and ECM-laminin extract sprouted neurites rapidly on both sides of the border without showing preference for either substrate. An individual nerve cell produced neurites with markedly different structure on the two substrates--curved and thick, with a higher branching frequency on Con A, but straight and slender with a lower branching frequency on ECM-laminin substrate. Similar changes were seen in individual neurites that crossed from one substrate to the other. These results show that local contact of the neuron with a particular substrate induces a particular pattern of local outgrowth. Hence, molecules anchored in extracellular matrix could provide information for regenerating or developing axons regarding the type of branching pattern to be produced in that region.