bluuebunny/crossref_metadata_2025 huggingface dataset:
The LCB2 gene of Saccharomyces and the related LCB1 gene encode subunits of serine palmitoyltransferase, the initial enzyme in sphingolipid synthesis.
Authors: Nagiec M M , Baltisberger J A , Wells G B , Lester R L , Dickson R C
Published: May 2006

The first and committed step in synthesis of the ceramide moiety of sphingolipids is catalyzed by serine palmitoyltransferase (EC 2.3.1.50), which condenses palmitoyl-CoA and serine to form 3-ketosphinganine. This step is thought to be tightly regulated to control the synthesis of sphingolipids, but data supporting this hypothesis are lacking mainly because the enzyme has resisted purification and consequent characterization. Rather than attempting to purify the enzyme from normal cells, we have taken a different tack and opted to try and overproduce the enzyme to facilitate its purification. Here we demonstrate that overproduction in Saccharomyces cerevisiae requires expression of LCB1, a previously isolated yeast gene, and LCB2, the isolation and characterization of which we describe. Several lines of evidence argue that both genes encode subunits of the enzyme; however, biochemical evidence will be needed to substantiate this hypothesis. Although overproduction was modest, 2- to 4-fold, it should now be possible to devise improved overproduction vectors for yeast or other host organisms.
Intra-host versus inter-host selection: viral strategies of immune function impairment.
Authors: Bonhoeffer S , Nowak M A
Published: May 2006

We investigate the evolution of viral strategies to counteract immunological attack. These strategies can be divided into two classes: those that impair the immune response inside or at the surface of a virus-infected cell and those that impair the immune response outside an infected cell. The former strategies confer a "selfish" individual selective advantage for intra-host competition among viruses. The latter strategies confer an "unselfish" selective advantage to the virus population as a group. A mutant, defective in the gene coding for the extracellular immune function-impairment strategy, may be protected from immune attack because the wild-type virus in the same host successfully impairs the host's immune function. Such "unselfish" defense strategies are neutral with respect to intra-host competition. We present simple models of viral intra-host and combined inter- and intra-host evolution. We show that selfish strategies can evolve by intra-host evolution. Unselfish strategies may evolve if inter-host selection pressures outweigh intra-host selection, suggesting that such strategies can only evolve in viruses with low mutation rates.
A third synaptotagmin gene, Syt3, in the mouse.
Authors: Hilbush B S , Morgan J I
Published: May 2006

The synaptotagmins are integral membrane proteins of synaptic vesicles thought to serve as Ca2+ sensors in the process of vesicular trafficking and exocytosis. Results from antibody microinjection and gene-disruption experiments have led to a controversy over whether synaptotagmins are essential for neurotransmission. However, the studies casting doubt on the role of synaptotagmins have assumed that no further isoforms of these molecules exist. Here we report the isolation of a third member of the synaptotagmin family (Syt3) from mouse brain. Although retaining the characteristic five-domain structure of the other synaptotagmins, SYT3 is considerably more divergent at the level of amino acid sequence. In the most highly conserved C2 domain, the mammalian synaptotagmins, SYT1 and SYT2, share 88% sequence identity, whereas SYT3 has only approximately 45% identity to either. Overall, SYT3 has the greatest sequence identity with rat SYT2 and marine ray p65A (both 37%), although homology to all of the known synaptotagmins is > 30%. However, SYT3 is most like p65C when comparing domain structure. Syt3 is expressed in many regions of the nervous system but is undetectable in extraneural tissues. The three murine synaptotagmins have differential expression patterns in the brain. Furthermore, in PC12 cells, Syt3 is coexpressed with Syt1 and is more abundant than the latter. This result suggests that individual neurons may have specific combinations of synaptotagmins that could provide for diversity in vesicular release.
The expression of serine carboxypeptidases during maturation and germination of the barley grain.
Authors: Dal Degan F , Rocher A , Cameron-Mills V , von Wettstein D
Published: May 2006

cDNA clones encoding three additional serine carboxypeptidases (Ser-CPs) have been isolated from a gibberellic acid-induced barley aleurone cDNA library. The three deduced Ser-CPs belong to the two-chain subfamily of Ser-CPs; they are synthesized as precursors with a putative signal peptide, propeptide, and linker peptide between the A and B chains. Their identification provides the proof for the existence of more than three Ser-CPs in cereal grains, and, based on their sequences, they may exhibit new substrate specificities. The expression of these and of the three previously isolated Ser-CPs from barley grains (CP-MI, CP-MII, and CP-MIII) has been investigated by Northern and Western analysis and RNA PCR. CP-MII is the only Ser-CP to be expressed and accumulate in the developing grain and is stored in its active form in the mature grain. All six Ser-CPs are expressed de novo in the germinating grain, in the scutellum, and/or in the aleurone. Furthermore, at least CP-MI, CP-MII, and CP-MIII are secreted into the endosperm. In addition, all Ser-CPs (except CP-MI) are also expressed in the roots and shoots of the growing seedling. This enzyme family thus appears to be ubiquitous in the barley plant, which suggests that Ser-CPs play additional roles besides their participation in the mobilization of storage proteins.
E-cadherin null mutant embryos fail to form a trophectoderm epithelium.
Authors: Larue L , Ohsugi M , Hirchenhain J , Kemler R
Published: May 2006

The cell adhesion molecule E-cadherin mediates the compaction process of mouse preimplantation embryos and is important for the maintenance and function of epithelial cell layers. To determine precisely the role of E-cadherin in epithelial biogenesis we monitored the developmental potential of embryos homozygously negative for E-cadherin that were derived from E-cadherin heterozygous transgenic mice. The homozygous negative embryos died around the time of implantation, although they did undergo compaction like their littermate controls, largely due to the presence of residual maternal E-cadherin. At the blastocyst stage, E-cadherin-negative embryos failed to form a trophectodermal epithelium or a blastocyst cavity. These results demonstrate the pivotal role of E-cadherin in one of the most basic morphogenetic events in the development of multicellular organisms, the biogenesis of an epithelium.
Intra- and extracellular amyloid fibrils are formed in cultured pancreatic islets of transgenic mice expressing human islet amyloid polypeptide.
Authors: de Koning E J , Morris E R , Hofhuis F M , Posthuma G , Höppener J W , Morris J F , Capel P J , Clark A , Verbeek J S
Published: May 2006

Islet amyloid polypeptide (IAPP) is the constituent peptide of amyloid deposits found in the islets of non-insulin-dependent diabetic patients. Formation of islet amyloid is associated with a progressive destruction of insulin-producing beta cells. Factors responsible for the conversion of IAPP into insoluble amyloid fibrils are unknown. Both the amino acid sequence of human IAPP (hIAPP) and hypersecretion of hIAPP have been implicated as factors for amyloid fibril formation in man. We have generated transgenic mice using rat insulin promoter-hIAPP or rat IAPP (rIAPP) gene constructs. No fibrillar islet amyloid was detectable in vivo in these normoglycemic mice, although small amorphous perivascular accumulations of IAPP were observed in hIAPP mice only. To determine the effects of glucose on IAPP secretion and fibrillogenesis, pancreatic islets from transgenic and control mice were examined in vitro. Islet IAPP secretion and content were increased in transgenic islets compared with control islets. IAPP-immunoreactive fibrils were formed at both intra- and extracellular sites in isolated hIAPP islets cultured with glucose at 11.1 and 28 mM for only 7 days. At 28 mM glucose, fibrils were present in deep invaginations of beta cells as observed in non-insulin-dependent diabetic patients. No fibrils were present at low glucose concentrations in hIAPP islets or at any glucose concentration in rIAPP or control islets. Thus, glucose-induced expression and secretion of hIAPP in transgenic mouse islets can lead to formation of amyloid fibrils similar to that found in non-insulin-dependent diabetes mellitus.
Membrane-targeting potentiates guanine nucleotide exchange factor CDC25 and SOS1 activation of Ras transforming activity.
Authors: Quilliam L A , Huff S Y , Rabun K M , Wei W , Park W , Broek D , Der C J
Published: May 2006

Growth factor-triggered activation of Ras proteins is believed to be mediated by guanine nucleotide exchange factors (CDC25/GRF and SOS1/2) that promote formation of the active Ras GTP-bound state. Although the mechanism(s) of guanine nucleotide exchange factor regulation is unclear, recent studies suggest that translocation of SOS1 to the plasma membrane, where Ras is located, might be responsible for Ras activation. To evaluate this model, we generated constructs that encode the catalytic domains of human CDC25 or mouse SOS1, either alone (designated cCDC25 and cSOS1, respectively) or terminating in the carboxyl-terminal CAAX membrane-targeting sequence from K-Ras4B (designated cCDC25-CAAX and cSOS1-CAAX, respectively; in CAAX, C is Cys, A is an aliphatic amino acid, and X is Ser or Met). We then compared the transforming potential of cCDC25 and cSOS1 with their membrane-targeted counterparts. We observed that addition of the Ras plasma membrane-targeting sequence to the catalytic domains of CDC25 and SOS1 greatly enhanced their focus-forming activity (10- to 50-fold) in NIH 3T3 transfection assays. Similarly, we observed that the membrane-targeted versions showed a 5- to 10-fold enhanced ability to induce transcriptional activation from the Ets/AP-1 Ras-responsive element. Furthermore, whereas cells that stably expressed cCDC25 or cSOS1 exhibited the same morphologies as untransformed NIH 3T3 cells, cells expressing cCDC25-CAAX or cSOS1-CAAX displayed transformed morphologies that were indistinguishable from the elongated and refractile morphology of oncogenic Ras-transformed cells. Thus, these results suggest that membrane translocation alone is sufficient to potentiate guanine nucleotide exchange factor activation of Ras.
Atherosclerosis and sterol 27-hydroxylase: evidence for a role of this enzyme in elimination of cholesterol from human macrophages.
Authors: Björkhem I , Andersson O , Diczfalusy U , Sevastik B , Xiu R J , Duan C , Lund E
Published: May 2006

27-Hydroxycholesterol was found in surprisingly high amounts in atherosclerotic human femoral arteries. When human macrophages were cultured in a medium containing serum, there was a significant transfer of 27-hydroxy-cholesterol and 3 beta-hydroxy-5-cholestenoic acid from the cells into the medium. Sterol 27-hydroxylase (EC 1.14.13.15) is likely to be responsible for formation of the two products as shown by use of immunoblotting, a specific inhibitor, and the 18O-labeling technique. Sterol 27-hydroxylase has the unusual ability to hydroxylate the same methyl group three times to give a carboxylic acid; thus, 3 beta-hydroxy-5-cholestenoic acid is likely to be a direct product of the enzyme. The production of these steroids increased after addition of cholesterol to the culture medium. By using deuterium-labeled cholesterol, it was ascertained that most of the oxidized products were formed from exogenous cholesterol taken up by the cells. 27-Hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid are present in the circulation and are efficiently converted into bile acids in human liver. It is suggested that conversion of cholesterol into 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid represents a general defence mechanism for macrophages and possibly also other peripheral cells exposed to cholesterol. Absence of this defence mechanism may contribute to the premature atherosclerosis known to occur in patients with sterol 27-hydroxylase deficiency (cerebrotendinous xanthomatosis).
Functional magnetic resonance imaging of human prefrontal cortex activation during a spatial working memory task.
Authors: McCarthy G , Blamire A M , Puce A , Nobre A C , Bloch G , Hyder F , Goldman-Rakic P , Shulman R G
Published: May 2006

High-speed magnetic resonance (MR) imaging was used to detect activation in the human prefrontal cortex induced by a spatial working memory task modeled on those used to elucidate neuronal circuits in nonhuman primates. Subjects were required to judge whether the location occupied by the current stimulus had been occupied previously over a sequence of 14 or 15 stimuli presented in various locations. Control tasks were similar in all essential respects, except that the subject's task was to detect when one of the stimuli presented was colored red (color detection) or when a dot briefly appeared within the stimulus (dot detection). In all tasks, two to three target events occurred randomly. The MR signal increased in an area of the middle frontal gyrus corresponding to Brodmann's area 46 in all eight subjects performing the spatial working memory task. Right hemisphere activation was greater and more consistent than left. The MR signal change occurred within 6-9 sec of task onset and declined within a similar period after task completion. An increase in MR signal was also noted in the control tasks, but the magnitude of change was less than that recorded in the working memory task. These differences were replicated when testing was repeated in five of the original subjects. The localization of spatial working memory function in humans to a circumscribed area of the middle frontal gyrus supports the compartmentalization of working memory functions in the human prefrontal cortex and the localization of spatial memory processes to comparable areas in humans and nonhuman primates.
Kinetic intermediates in the reactions between peptides and proteins of major histocompatibility complex class II.
Authors: Beeson C , McConnell H M
Published: May 2006

The kinetics of the reactions between fluorescently labeled sperm whale myoglobin-(110-121) peptide and the murine major histocompatibility complex class II protein I-Ed have been analyzed. The presence in solution of both short- and long-lived protein-peptide complexes is demonstrated by the biphasic dissociation of the myoglobin peptide from I-Ed. The formation of the long-lived terminal complex is preceded by a characteristic induction phase. It is shown that the initially formed complex of the myoglobin peptide and I-Ed is a kinetic intermediate that undergoes a unimolecular reaction to form the terminal complex. Reactions between peptides and the class II proteins thus involve an intermediate structurally distinct from the terminal complex. The terminal complex presumably has a structure that is biologically active and similar to the published class II protein-peptide crystal structure.
Interaction between the aphid transmission factor and virus particles is a part of the molecular mechanism of cauliflower mosaic virus aphid transmission.
Authors: Schmidt I , Blanc S , Esperandieu P , Kuhl G , Devauchelle G , Louis C , Cerutti M
Published: May 2006

Cauliflower mosaic virus (CaMV) aphid transmission factor (ATF or P18) is presumed to interact with both virus particles and vector mouthparts, thereby mediating virus aphid transmission. We developed a protein-protein binding assay and our results clearly show that virus particles bind strongly and specifically to P18 whether P18 was obtained from plants, a baculovirus expression system, or the pGEX-3X Escherichia coli expression system. We overproduced, using the pGEX-3X expression system, various fragments of P18 and thereby demonstrated that the C-terminal 31 amino acid residues are responsible for the interaction. Using PCR-based mutagenesis, 2 amino acid residues essential for interaction were identified. Point substitutions (amino acids 157 from Ile to Asn or 159 from Gly to Ser) were sufficient to abolish the interaction, whereas another mutation (amino acid 158 from Ile to Ser) had no effect on P18 virus binding. We evaluated whether there was a correlation between the ability of P18 to interact with CaMV particles and its biological activity. Aphid transmission assays were carried out and we demonstrated that the loss of the virus binding capacity had a dramatic effect on the ability of P18 to mediate aphid transmission. Thus, our results suggest that binding between P18 and virus particles is likely to be one of the molecular mechanisms involved in CaMV aphid transmission.
A targeted chain-termination mutation in the mouse Apc gene results in multiple intestinal tumors.
Authors: Fodde R , Edelmann W , Yang K , van Leeuwen C , Carlson C , Renault B , Breukel C , Alt E , Lipkin M , Khan P M
Published: May 2006

Germ-line mutations in the human adenomatous polyposis coli (APC) gene result in familial adenomatous polyposis, an autosomal dominant disorder characterized by the early onset of multiple adenomatous polyps in the large bowel with a high likelihood of developing colorectal carcinomas. To understand the role of APC in intestinal tumor formation, we have introduced a chain-termination mutation in the 15th exon of the mouse Apc gene and employed it to modify the endogenous gene by homologous recombination in embryonic stem cells. Mice which are heterozygous for the Apc gene modification progressively develop intestinal tumors in a manner that is similar to that observed in patients with familial adenomatous polyposis and in mice which carry a mutation called multiple intestinal neoplasia (Min). Our results indicate that the Apc gene modification is a critical event in the initiation of intestinal tumor formation and results in an autosomal dominant predisposition toward development of spontaneous colonic and intestinal tumors in mice.
Early myeloid cell-specific expression of the human cathepsin G gene in transgenic mice.
Authors: Grisolano J L , Sclar G M , Ley T J
Published: May 2006

The human cathepsin G (CG) gene is expressed only in promyelocytes and encodes a neutral serine protease that is packaged in the azurophil (primary) granules of myeloid cells. To define the cis-acting DNA elements that are responsible for promyelocyte-specific "targeting," we injected a 6-kb transgene containing the entire human CG gene, including coding sequences contained in a 2.7-kb region, approximately 2.5 kb of 5' flanking sequence, and approximately 0.8 kb of 3' flanking sequence. Seven of seven "transient transgenic" murine embryos revealed human CG expression in the fetal livers at embryonic day 15. Stable transgenic founder lines were created with the same 6-kb fragment; four of five founder lines expressed human CG in the bone marrow. The level of human CG expression was relatively low per gene copy when compared with the endogenous murine CG gene, and expression was integration-site dependent; however, the level of gene expression correlated roughly with gene copy number. The human CG transgene and the endogenous murine CG gene were coordinately expressed in the bone marrow and the spleen. Immunohistochemical analysis of transgenic bone marrow revealed that the human CG protein was expressed exclusively in myeloid cells. Expression of human CG protein was highest in myeloid precursors and declined in mature myeloid cells. These data suggest that the human CG gene was appropriately targeted and developmentally regulated, demonstrating that the cis-acting DNA sequences required for the early myeloid cell-specific expression of human CG are present in this small genomic fragment.
Evidence for multiple adaptive peaks from populations of bacteria evolving in a structured habitat.
Authors: Korona R , Nakatsu C H , Forney L J , Lenski R E
Published: May 2006

Natural selection tends to promote the divergence of populations living in different environments. Even in identical environments, however, replicate populations may diverge if they find alternative adaptive solutions. We describe the evolution of 18 bacterial populations (Comamonas sp.) founded from a single progenitor genotype and propagated separately for 1000 generations in two distinct environments, one physically unstructured (mass-action liquid) and the other structured (agar surfaces). Phenotypic diversity, as reflected in colony morphology, was greater in the structured habitat than in the unstructured habitat. More importantly, the trajectories for mean fitness, as measured by competition against the common ancestor, were more divergent for populations in the structured habitat than those in the unstructured habitat. Structured environments may accelerate evolutionary diversification by promoting genetic polymorphisms within populations, thereby increasing the complexity of genetic constraints that allow divergence among replicate populations.
Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C.
Authors: Disatnik M H , Hernandez-Sotomayor S M , Jones G , Carpenter G , Mochly-Rosen D
Published: May 2006

Phospholipase C-gamma 1 (PLC-gamma 1; EC 3.1.4.11) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate and is activated in response to growth factor stimulation and tyrosine phosphorylation. Concomitantly, the enzyme translocates from the cytosol to the particulate cell fraction. A similar process of activation-induced translocation from the cytosol to the cell particulate fraction has also been described for protein kinase C (PKC). We have previously shown that activated PKC binds to specific receptor proteins, receptors for activated C kinase, or RACKs, of approximately 30 kDa. Here, we show that PLC-gamma 1 bound to these RACKs and inhibited subsequent PKC binding to RACKs. However, unlike PKC, the binding of PLC-gamma 1 to RACKs did not require phospholipids and calcium. After epidermal growth factor treatment of intact A-431 cells, the binding of PLC-gamma 1 to RACKs increased as compared with PLC-gamma 1 from control cells. This increase in PLC-gamma 1 binding to RACKs was due to the phosphorylation of PLC-gamma 1. Additional data indicated that PLC-gamma 1 binds to RACKs in solution; epidermal growth factor receptor-dependent PLC-gamma 1 phosphorylation and activation decreased in the presence of RACKs. It is possible that, in vivo, PLC-gamma 1 associates with RACKs or with other PLC-gamma 1-specific anchoring proteins in the particulate cell fraction. Since a PKC C2 homologous region is present in PLC-gamma 1, the C2 region may mediate the activation-induced translocation of the enzyme to the cell particulate fraction and the anchoring protein-PLC-gamma 1 complex may be the active translocated form of PLC-gamma 1.
Regional, cellular, and ultrastructural distribution of N-methyl-D-aspartate receptor subunit 1 in monkey hippocampus.
Authors: Siegel S J , Brose N , Janssen W G , Gasic G P , Jahn R , Heinemann S F , Morrison J H
Published: May 2006

The regional, cellular, and subcellular distributions of N-methyl-D-aspartate (NMDA) receptor subunit 1, NMDAR-1, were investigated in monkey hippocampus by using a monoclonal antibody directed against a fusion protein corresponding to aa 660-811 of NMDAR-1. The data indicate that many neurons in each subfield of the hippocampus contain NMDAR-1 protein, although the intensity and distribution of immunoreactivity varied across regions, strata, and cellular compartments. In stratum lucidum of CA3, mossy fiber axons were immunoreactive for NMDAR-1, which may correspond to previously hypothesized presynaptic receptors. NMDAR-1-labeled postsynaptic profiles were present in stratum radiatum of CA3 but were largely absent from stratum lucidum. Such intraneuronal segregation of glutamate receptor subunits or classes may be spatially correlated with afferent systems that exhibit laminar segregation and terminate in different portions of the postsynaptic dendritic tree. For example, in CA3 pyramidal cells, NMDA receptors are postsynaptic in distal apical dendrites (stratum radiatum) where NMDA-dependent long-term potentiation in rats is mediated by associational/commissural afferents, and are absent from proximal apical dendrites (stratum lucidum), where NMDA-independent long-term potentiation is mediated by the mossy fiber input.
The B1C8 protein is in the dense assemblies of the nuclear matrix and relocates to the spindle and pericentriolar filaments at mitosis.
Authors: Wan K M , Nickerson J A , Krockmalnic G , Penman S
Published: May 2006

The B1C8 monoclonal antibody detects a 180-kDa nuclear matrix-specific protein. The protein is a component of the dense, metabolically active bodies or assemblies revealed by resinless section electron microscopy of the nuclear matrix. These assemblies are scattered through the nuclear interior, enmeshed in a complex network of 11-nm filaments. Resinless section electron microscopy of immunogold-stained nuclear matrix preparations shows B1C8 located in many but apparently not all the assemblies. In this regard, the B1C8 antigen resembles previously studied nuclear matrix proteins such as the H1B2 protein. The speckled pattern of nuclear immunofluorescence by B1C8 reflects this labeling of the dense assemblies in the nuclear matrix. Somewhat unusual is the faint staining of cytoplasmic microtubules by B1C8, which appears to be due to a weakly cross-reacting protein. During cell division, the B1C8 antigen redistributed drastically, showing the dispersion of nuclear matrix assemblies at mitosis. Speckles of B1C8 fluorescence first coalesced at prophase within the nuclear interior and then scattered into numerous cytoplasmic speckles by prometaphase. At metaphase, the B1C8 speckled cytoplasmic staining had become even more widely distributed and finely grained. Also, intense labeling appeared at the mitotic pole and on the spindle fibers themselves. The reassembly of B1C8 antigens into larger cytoplasmic speckles began at anaphase and finally, at telophase, most B1C8 labeling redistributed into speckles in the re-forming nuclei.
Mouse limb muscle is determined in the absence of the earliest myogenic factor myf-5.
Authors: Tajbakhsh S , Buckingham M E
Published: May 2006

myf-5 is the only member of the MyoD family of myogenic regulatory genes to be expressed in the mouse embryo prior to muscle cell differentiation. We have used the developing limb as a model in which to follow the formation of peripheral muscle, to address the question of whether myogenic precursor cells are already present in the limb bud before expression of myf-5. The lacZ reporter gene has been introduced into the myf-5 gene by homologous recombination so that its expression is under the control of the endogenous myf-5 locus. beta-Galactosidase (beta-gal) coloration provides a sensitive assay for myf-5+ cells. Embryos were generated from embryonic stem cells carrying this mutation, and the appearance of beta-gal+ (myf-5+) cells was followed during limb development in vivo. Limb buds, at a stage when they are beta-gal-, were cultured in vitro. After several days, beta-gal+ cells accumulated in the premuscle mass. We conclude that determined muscle precursor cells in the limb bud do not initially express any member of the MyoD family. Furthermore, myogenic precursor cells in the somite, which, according to the avian model, migrate from the ventral/lateral edge of the dermomyotome to form peripheral muscle masses, are also negative for these factors.
Minimum structural requirements for peptide presentation by major histocompatibility complex class II molecules: implications in induction of autoimmunity.
Authors: Gautam A M , Lock C B , Smilek D E , Pearson C I , Steinman L , McDevitt H O
Published: May 2006

The precise mechanisms of failure of immunological tolerance to self proteins are not known. Major histocompatibility complex (MHC) susceptibility alleles, the target peptides, and T cells with anti-self reactivity must be present to cause autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE) is a murine model of a human autoimmune disease, multiple sclerosis. In EAE, residues 1-11 of myelin basic protein (MBP) are the dominant disease-inducing determinants in PL/J and (PL/J x SJL/J)F1 mice. Here we report that a six-residue peptide (five of them native) of MBP can induce EAE. Using peptide analogues of the MBP-(1-11) peptide, we demonstrate that only four native MBP residues are required to stimulate MBP-specific T cells. Therefore, this study demonstrates lower minimum structural requirements for effective antigen presentation by MHC class II molecules. Many viral and bacterial proteins share short runs of amino acid similarity with host self proteins, a phenomenon known as molecular mimicry. Since a six-residue peptide can sensitize MBP-specific T cells to cause EAE, these results define a minimum sequence identity for molecular mimicry in autoimmunity.
Construction and characterization of a normalized cDNA library.
Authors: Soares M B , Bonaldo M F , Jelene P , Su L , Lawton L , Efstratiadis A
Published: May 2006

We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each circular template, melting and reannealing of the partial duplexes at relatively low C0t, and hydroxyapatite column chromatography, unreassociated circles were recovered from the flow through fraction and electroporated into bacteria, to propagate a normalized library without a requirement for subcloning steps. An evaluation of the extent of normalization has indicated that, from an extreme range of abundance of 4 orders of magnitude in the original library, the frequency of occurrence of any clone examined in the normalized library was brought within the narrow range of only 1 order of magnitude.
Biological responses to weak 60-Hz electric and magnetic fields must vary as the square of the field strength.
Authors: Adair R K
Published: May 2006

Under quite general conditions, the biological response j(G) to a very weak continuous 60-Hz electric or magnetic field G is shown to be proportional to the square of the field strength. This conclusion follows from the continuity of the function j(G) and the first three derivatives of j(G) with respect to G over the amplitude of G. That continuity is ensured in nominally discontinuous systems by the presence of thermal noise. I argue the validity of the conjecture that all plausible biological responses to weak 60-Hz fields vary with the square of the field strength. A specific model is used to illustrate characteristic dependencies of biological responses to exposure times.
Genetic instability in human ovarian cancer cell lines.
Authors: Orth K , Hung J , Gazdar A , Bowcock A , Mathis J M , Sambrook J
Published: May 2006

We have analyzed the stability of microsatellites in cell lines derived from human ovarian cancers and found that 5 out of 10 of the ovarian tumor cell lines are genetically unstable at the majority of the loci analyzed. In clones and subclones derived serially from one of these cell lines (2774; serous cystadenocarcinoma), a very high proportion of microsatellites distributed in many different regions of the genome change their size in a mercurial fashion. We conclude that genomic instability in ovarian tumors is a dynamic and ongoing process whose high frequency may have been previously underestimated by PCR-based allelotyping of bulk tumor tissue. We have identified the source of the genetic instability in one ovarian tumor as a point mutation (R524P) in the human mismatch-repair gene MSH2 (Salmonella MutS homologue), which has recently been shown to be involved in hereditary nonpolyposis colorectal cancer. Patient 2774 was a 38-year-old heterozygote, and her normal tissue carried both mutant and wild-type alleles of the human MSH2 gene. However the wild-type allele was lost at some point early during tumorigenesis so that DNA isolated either from the patient's ovarian tumor or from the 2774 cell line carries only the mutant allele of the human MSH2 gene. The genetic instability observed in the tumor and cell line DNA, together with the germ-line mutation in a mismatch-repair gene, suggest that the MSH2 gene is involved in the onset and/or progression in a subset of ovarian cancer.
Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.
Authors: Saouaf S J , Mahajan S , Rowley R B , Kut S A , Fargnoli J , Burkhardt A L , Tsukada S , Witte O N , Bolen J B
Published: May 2006

We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity was also transiently stimulated and was maximal at approximately 5 min after B-cell receptor surface binding. Syk activity gradually increased to a maximum at 10-30 min following receptor ligation and was found to parallel the association of Syk with the tyrosine phosphorylated Ig alpha and Ig beta subunits of the receptor. While the specific activities of the Jak1, Jak2, and Tyk2 protein tyrosine kinases were unaltered following B-cell receptor ligation, the abundance of Jak1 and Jak2 were increased 3- to 4-fold within 10 min of receptor engagement. These results demonstrate that multiple families of non-transmembrane protein tyrosine kinases are temporally regulated during the process of B-cell antigen receptor-initiated intracellular signal transduction.
A phosphatidylinositol 3-kinase is induced during soybean nodule organogenesis and is associated with membrane proliferation.
Authors: Hong Z , Verma D P
Published: May 2006

Phosphatidylinositol 3-kinase (PI3K) is an important component of various receptor tyrosine kinase complexes in mammalian cells and a key enzyme required for cell division and vacuolar protein sorting in yeast. To our knowledge, this enzyme has not been characterized in plants. We report the cloning and characterization of soybean PI3K cDNAs and present evidence for the induction of a distinctive form of this enzyme specific to nodule organogenesis. Expression of the root form of PI3K is repressed during nodule organogenesis and is reinduced in mature nodules. Primer-extension results showed that the gene encoding the nodule form of PI3K is highly expressed in young (12-15 day old) root nodules in parallel with membrane proliferation but is repressed in mature nodules. The root form of the PI3K cDNA (SPI3K-5) encodes a peptide of 814 amino acids and the nodule form (SPI3K-1) encodes a peptide of 812 amino acids. Both cDNAs share 98% sequence identity in the coding region but differ in the noncoding regions. The polypeptides encoded by soybean PI3K cDNAs show significant sequence homology (50-60% similarity and 20-40% identity) to both PI3Ks and phosphatidylinositol 4-kinases from mammalian and yeast cells. Escherichia coli expressed soybean PI3K phosphorylated phosphatidylinositol specifically at the D-3 position of the inositol ring to generate phosphatidylinositol 3-phosphate. The temporal increase of a specific PI3K activity during membrane proliferation in young nodules suggests that PI3K plays a pivotal role in development of the peribacteroid membrane forming a subcellular compartment.
Single- and multilocus DNA fingerprinting of communally breeding pukeko: do copulations or dominance ensure reproductive success?
Authors: Lambert D M , Millar C D , Jack K , Anderson S , Craig J L
Published: May 2006

In behavioral and ecological studies the "function" of dominance hierarchies is thought to be related to reproductive success. In particular, dominant males are regarded as likely to gain a reproductive advantage due to enhanced "access" to females. We compare the dominance status of adults with the frequency of copulations and the patterns of parentage in communally breeding pukeko or purple swamphen (Porphyrio porphyrio melanotus). This avian species has an unusual social system, often breeding in polygynandrous groups in which there is a strong dominance hierarchy. Typically, during the breeding season, there is considerable sexual activity, with heterosexual and homosexual copulations between adults being commonplace. Hae III-digested DNA from individuals belonging to breeding groups was hybridized to the minisatellite DNA probe YNH24, revealing putative single-locus profiles, while hybridization of the same DNA to the minisatellite probes pV47-2, 3'HVR, and per revealed typical multilocus profiles. The numbers of unattributable restriction fragments allowed the maternity and paternity of all individuals to be conclusively determined, despite high levels of band sharing among individuals within breeding groups. These close genetic similarities are a likely consequence of strong philopatry and inbreeding. We report instances of males which are high on the dominance hierarchy but have only a limited reproductive output in comparison with others and males which are subordinate but achieve a significant proportion of fertilizations. Generally these data reveal no consistent relationship between dominance, the frequency of copulations, and parentage among males. We conclude that pukeko highlight some difficulties with conventional explanations for the "function" of dominance.
Evidence for regular distribution of sterols in liquid crystalline phosphatidylcholine bilayers.
Authors: Chong P L
Published: May 2006

To investigate the lateral organization of sterols in membranes, the fluorescence intensity of dehydroergosterol at different mole fractions in liquid crystalline dimyristoyl phosphatidylcholine bilayers was examined. A number of intensity drops were observed at specific mole fractions, as predicted from a hexagonal super-lattice model. The fluorescence dips provide compelling evidence that a naturally occurring sterol is regularly distributed at fixed compositional fractions, consistent with the presence of hexagonal super-lattices in the fluid membranes. Regularly distributed regions, however, coexist with irregularly distributed regions. The extent of regular distribution varies periodically with sterol mole fraction and, consequently, similar variations take place in the membrane volume and lipid packing. This level of modulation in local membrane structure by minute changes in sterol concentration should have profound implications for the functional role of cholesterol content in cell membranes.
Transient inositol 1,4,5-trisphosphate-induced Ca2+ release: a model based on regulatory Ca(2+)-binding sites along the permeation pathway.
Authors: Swillens S , Combettes L , Champeil P
Published: May 2006

A remarkable property of Ca2+ fluxes through the inositol 1,4,5-trisphosphate (InsP3)-gated Ca2+ channel is that successive increments of InsP3 induce repeated transient release of accumulated Ca2+. The initial aim of this study was to propose a model, based on hypotheses compatible with the current description of this Ca2+ channel, which could account for such experimental observations. The key feature of the model was the assumption that the Ca(2+)-binding sites on the receptor, whose occupancy leads to immediate channel activation but to subsequent slow channel desensitization, were located somewhere along the permeation pathway and were therefore sensitive to the flux of Ca2+ rather than the cytosolic or luminal Ca2+ concentration per se. Simulation showed that, provided Ca2+ bound to both activating and inhibitory sites with adequate cooperativity, addition of submaximal concentrations InsP3 resulted in transient opening well above the stationary state. The model also rationalized the documented existence of a threshold for InsP3 action, the puzzling control of channel sensitivity to InsP3 by luminal and cytosolic Ca2+, as well as the functional heterogeneity of the Ca2+ pools.
Stromal-epithelial interaction mediates steroidal regulation of metalloproteinase expression in human endometrium.
Authors: Osteen K G , Rodgers W H , Gaire M , Hargrove J T , Gorstein F , Matrisian L M
Published: May 2006

The hallmark of the menstrual cycle is extensive steroid-dependent tissue turnover. Estrogen mediates endometrial cell growth and structural remodeling, whereas progesterone suppresses estrogen-dependent proliferation and promotes cellular differentiation. In nonfertile cycles, tissue degradation and menstruation occur as a consequence of steroidal deprivation as the ovarian corpus luteum fails. Stromal-epithelial interactions are recognized as a necessary component in mediating steroid-induced endometrial turnover. Specific mRNAs for metalloproteinases of the stromelysin family are expressed during endometrial growth and menstrual breakdown but are absent in the progestin-dominated secretory phase. This expression pattern suggests involvement of stromelysins in remodeling the extracellular matrix of the endometrium during tissue growth and breakdown and implicates progesterone in the suppression of these enzymes. We examined the regulation of endometrial stromelysins in explant cultures and found no acute effect of estradiol on their expression, whereas progesterone was a potent inhibitor of stromelysin expression. Progesterone also suppressed stromelysin expression in cultures of isolated stromal cells, but epithelial cells were progesterone insensitive. Coculture of recombined stromal and epithelial cells restored steroidal suppression of the epithelial-specific metalloproteinase. Our data confirm that progesterone inhibits endometrial stromelysins and further demonstrate the necessity for a stromal-derived factor(s) as a mediator of steroid suppression of an epithelial metalloproteinase.
Removal of transducer HtrI allows electrogenic proton translocation by sensory rhodopsin I.
Authors: Bogomolni R A , Stoeckenius W , Szundi I , Perozo E , Olson K D , Spudich J L
Published: May 2006

Sensory rhodopsin I (sR-I) is a phototaxis receptor in halobacteria, which is closely related to the light-driven proton pump bacteriorhodopsin and the chloride pump halorhodopsin found in the same organisms. The three pigments undergo similar cyclic photoreactions, in spite of their different functions. In intact cells or isolated membranes sR-I is complexed with protein HtrI, the next link in the signal transduction chain, and does not function as an electrogenic ion pump. However, illumination of sR-I in membranes lacking HtrI causes pH changes in the medium, and its photoreaction kinetics become pH-dependent. We show here that in closed vesicles, near neutral pH it functions as an electrogenic proton pump capable of generating at least -80 mV transmembrane potential. The action spectrum shows a maximum 37 nm below the 587-nm absorption maximum of the native pigment. This apparent discrepancy occurs because the 587-nm form of HtrI-free sR-I exists in a pH-dependent equilibrium with a 550-nm absorbing species generated through deprotonation of one group with a pKa of 7.2, which we have tentatively identified as Asp-76. We interpret the results in terms of a general model for ion translocation by the bacterial rhodopsins.
Fingerprinting the folding of a group I precursor RNA.
Authors: Emerick V L , Woodson S A
Published: May 2006

Evidence that folding of the Tetrahymena pre-rRNA follows a defined path and is rate-determining for splicing at physiological temperatures is presented. Structural isomers were separated by native polyacrylamide gel electrophoresis and their splicing activities were compared. GTP binding selectively shifts the active form of the pre-RNA to an electrophoretic band containing both spliced and unspliced RNA. In situ chemical modification provides evidence for base-pair rearrangements in the 5' exon and structural alterations in the intron core of partially and fully active forms. Transition to the fully active precursor requires high temperature, but the activation energy is lower than expected for opening of RNA helices. Implications for control of RNA conformation during splicing are discussed.
Iron acquisition from heme and hemoglobin by a Serratia marcescens extracellular protein.
Authors: Létoffé S , Ghigo J M , Wandersman C
Published: May 2006

Several pathogenic bacteria are able to use heme and hemoproteins as iron sources independent of siderophore production by mechanisms involving outer membrane heme-binding proteins and heme transport systems. Here we show that Serratia marcescens has such a property and we identify an extracellular heme-binding protein, HasA (for heme acquisition system), allowing the release of heme from hemoglobin. This protein is secreted by S. marcescens under conditions of iron depletion and is essential for heme acquisition.
Molecular phylogeny of the insect order Hymenoptera: apocritan relationships.
Authors: Dowton M , Austin A D
Published: May 2006

Phylogenetic relationships among the major groups of hymenopteran insects were investigated by using comparative sequence information from the mitochondrial 16S rRNA gene. The placement of the ectoparasitic Stephanidae as the sister group to the remaining Apocrita confirmed ectoparasitism as the ground plan biology for the Apocrita. Endoparasitism evolved at least eight times within the Apocrita, and the consequent association with polydnaviruses and virus-like particles evolved at least three times. The Evaniomorpha were consistently placed as basal to the remaining Apocrita but were not resolved as monophyletic. The Gasteruptiidae were resolved as the sister group to the Evaniidae, but the relationship between the Trigonalyoidea and the Evanioidea was unclear. The Proctotrupomorpha (sensu Rasnitsyn) was resolved by topology-dependent permutation tail probability (T-PTP) testing as monophyletic, with strong evidence for a sister group relationship between the Platygastroidea and the Chalcidoidea. Strong evidence was found for the monophyly of the Ichneumonomorpha (Ichneumonidae + Braconidae) and the sister-group relationship between the Aculeata (Vespomorpha) and the Ichneumonomorpha.
Transgenic copper/zinc superoxide dismutase modulates susceptibility to type I diabetes.
Authors: Kubisch H M , Wang J , Luche R , Carlson E , Bray T M , Epstein C J , Phillips J P
Published: May 2006

A growing body of evidence suggests that active oxygen is an important participant in the destruction of the pancreatic beta cell, which, in turn, leads to type I or insulin-dependent diabetes mellitus. Consequently, genetic factors predisposing susceptibility to insulin-dependent diabetes mellitus may include those that determine active oxygen metabolism. A direct test of this hypothesis is provided by a transgenic model for increased activity of Cu/Zn superoxide dismutase (EC 1.15.1.1), a principal radical scavenging enzyme. Here we demonstrate that elevated levels of this enzyme provided by a Cu/Zn superoxide dismutase transgene enhance the tolerance of pancreatic beta cells to oxidative stress-induced diabetogenesis. These results show that this transgenic approach holds promise for revealing the role of reactive oxygen in autoimmune models of diabetogenesis as well as in other models of disease pathology in which active oxygen has been implicated.