bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a family of bacteriophage T4 genes encoding proteins similar to those present in group I introns of fungi and phage.
Authors: Sharma M , Ellis R L , Hinton D M
Published: May 2006

The bacteriophage T4 segA gene lies in a genetically unmapped region between the gene beta gt (beta-glucosyltransferase) and uvsX (recombination protein) and encodes a protein of 221 amino acids. We have found that the first 100 amino acids of the SegA protein are highly similar to the N termini of four other predicted T4 proteins, also of unknown function. Together these five proteins, SegA-E (similar to endonucleases of group I introns), contain regions of similarity to the endonuclease I-Tev I, which is encoded by the mobile group I intron of the T4 td gene, and to putative endonucleases of group I introns present in the mitochondria of Neurospora crassa, Podospora anserina, and Saccharomyces douglasii. Intron-encoded endonucleases are required for the movement (homing) of the intron DNA into an intronless gene, cutting at or near the site of intron insertion. Our in vitro assays indicate that SegA, like I-Tev I, is a Mg(2+)-dependent DNA endonuclease that has preferred sites for cutting. Unlike the I-Tev I gene, however, there is no evidence that segA (or the other seg genes) resides within introns. Thus, it is possible that segA encodes an endonuclease that is involved in the movement of the endonuclease-encoding DNA rather than in the homing of an intron.
Methyl jasmonate inhibition of root growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant.
Authors: Staswick P E , Su W , Howell S H
Published: May 2006

Jasmonic acid and its methyl ester, methyl jasmonate (MeJA), are plant signaling molecules that affect plant growth and gene expression. Primary root growth of wild-type Arabidopsis thaliana seedlings was inhibited 50% when seedlings were grown on agar medium containing 0.1 M MeJA. An ethyl methanesulfonate mutant (jar1) with decreased sensitivity to MeJA inhibition of root elongation was isolated and characterized. Genetic data indicated the trait was recessive and controlled by a single Mendelian factor. MeJA-induced polypeptides were detected in Arabidopsis leaves by antiserum to a MeJA-inducible vegetative storage protein from soybean. The induction of these proteins by MeJA in the mutant was at least 4-fold less in jar1 compared to wild type. In contrast, seeds of jar1 plants were more sensitive than wild type to inhibition of germination by abscisic acid. These results suggest that the defect in jar1 affects a general jasmonate response pathway, which may regulate multiple genes in different plant organs.
Cone visual pigments are present in gecko rod cells.
Authors: Kojima D , Okano T , Fukada Y , Shichida Y , Yoshizawa T , Ebrey T G
Published: May 2006

The Tokay gecko (Gekko gekko), a nocturnal lizard, has two kinds of visual pigments, P467 and P521. In spite of the pure-rod morphology of the photoreceptor cells, the biochemical properties of P521 and P467 resemble those of iodopsin (the chicken red-sensitive cone visual pigment) and rhodopsin, respectively. We have found that the amino acid sequence of P521 deduced from the cDNA was very similar to that of iodopsin. In addition, P467 has the highest homology with the chicken green-sensitive cone visual pigment, although it also has a relatively high homology with rhodopsins. These results give additional strength to the transmutation theory of Walls [Walls, G. L. (1934) Am. J. Ophthalmol. 17, 892-915], who proposed that the rod-shaped photoreceptor cells of lizards have been derived from ancestral cone-like photoreceptors. Apparently amino acid sequences of visual pigments are less changeable than the morphology of the photoreceptor cells in the course of evolution.
Elevated expression of monocyte chemoattractant protein 1 by vascular smooth muscle cells in hypercholesterolemic primates.
Authors: Yu X , Dluz S , Graves D T , Zhang L , Antoniades H N , Hollander W , Prusty S , Valente A J , Schwartz C J , Sonenshein G E
Published: May 2006

Atherosclerosis is marked by an overt inflammatory infiltrate, with enhanced recruitment of monocytes/macrophages observed in both human and experimental atherosclerosis. We previously determined that monocyte chemoattractant protein 1 (MCP-1) accounts for virtually all of the chemotactic activity produced by vascular (aortic) smooth muscle cells in culture. We now report that arteries from a primate model of atherosclerosis with dietary-induced hypercholesterolemia exhibit increased levels of MCP-1 mRNA expression in vivo, whereas their normal counterparts demonstrate minimal MCP-1 expression. Furthermore, immunohistochemistry and in situ hybridization clearly indicate that the expression of MCP-1 protein and mRNA is in the smooth muscle cells of the medial layer of the artery and in monocyte-like and smooth muscle-like cells found in the overlying intimal lesion. These studies indicate that one of the responses to dietary hypercholesterolemia is the expression of MCP-1 by vascular smooth muscle cells. This expression, when augmented with other cellular and molecular factors, could significantly contribute to the recruitment of monocytes/macrophages to the vessel wall.
Molecular basis of the membrane-anchored and two soluble isoforms of the human interleukin 5 receptor alpha subunit.
Authors: Tavernier J , Tuypens T , Plaetinck G , Verhee A , Fiers W , Devos R
Published: May 2006

By use of a 3' extension PCR strategy, cDNA clones were isolated spanning the transmembrane region and a complete cytoplasmic domain of the human interleukin 5 receptor alpha subunit (hIL5R alpha). These cDNAs differ from previously isolated clones encoding a soluble hIL5R alpha form by a sequence switch at position 1243. When expressed in COS-1 cells, only low-affinity binding of 125I-labeled human interleukin 5 was observed. Coexpression of the hIL5R beta chain led to a 2-fold increase in binding affinity. In addition, this same cloning strategy allowed us to identify a putative second soluble isoform of hIL5R alpha. Genomic data revealed that the two soluble variants arise from either a "normal" splicing event or from the absence of splicing, whereas synthesis of the membrane-anchored form requires alternative splicing.
Sexual orientation and the size of the anterior commissure in the human brain.
Authors: Allen L S , Gorski R A
Published: May 2006

The anterior commissure, a fiber tract that is larger in its midsagittal area in women than in men, was examined in 90 postmortem brains from homosexual men, heterosexual men, and heterosexual women. The midsagittal plane of the anterior commissure in homosexual men was 18% larger than in heterosexual women and 34% larger than in heterosexual men. This anatomical difference, which correlates with gender and sexual orientation, may, in part, underlie differences in cognitive function and cerebral lateralization among homosexual men, heterosexual men, and heterosexual women. Moreover, this finding of a difference in a structure not known to be related to reproductive functions supports the hypothesis that factors operating early in development differentiate sexually dimorphic structures and functions of the brain, including the anterior commissure and sexual orientation, in a global fashion.
Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs.
Authors: Ferrari S , Bannwarth W , Morley S J , Totty N F , Thomas G
Published: May 2006

Partial amino acid sequences were obtained from 22 internal tryptic peptides of rat liver p70s6k (M(r) 70,000 ribosomal protein S6 kinase), 3 of which were found to contain phosphorylated residues. To determine whether these sites were associated with p70s6k activation, the kinase was labeled to high specific activity with 32P(i) in Swiss mouse 3T3 cells. By sequential cleavage with CNBr and endoproteinase Lys-C followed by two-dimensional tryptic peptide analysis, it could be shown that all of the sites were located in a small endoproteinase Lys-C peptide of M(r) 2400. Analysis of the p70s6k protein sequence revealed a single candidate that could represent this peptide. Three tryptic peptides derived from the endoproteinase Lys-C fragment were chosen by a newly described computer program as the most likely candidates to contain the in vivo sites of phosphorylation. Synthetic peptides based on these sequences were phosphorylated either chemically or enzymatically and found to comigrate by two-dimensional thin-layer electrophoresis/chromatography with the four major in vivo labeled tryptic phosphopeptides. Three of the phosphorylation sites in these peptides were equivalent to those sequenced in the rat liver p70s6k. In addition, all four sites display the motif Ser/Thr-Pro, typical of cell cycle-regulated sites, and are clustered in a putative autoinhibitory domain of the enzyme.
The UL13 gene of herpes simplex virus 1 encodes the functions for posttranslational processing associated with phosphorylation of the regulatory protein alpha 22.
Authors: Purves F C , Roizman B
Published: May 2006

The herpes simplex virus 1 genome was shown to encode two genes, US3 and UL13, exhibiting amino acid sequence motifs common to protein kinases. Elsewhere this laboratory reported that the prominent substrate of the US3 protein kinase is the product of the UL34 gene, an essential nonglycosylated membrane protein. In the absence of the US3 kinase, the UL34 protein remains unphosphorylated but forms a complex with four proteins that become phosphorylated uniquely when UL34 is not. To investigate the role of UL13 protein in this process, recombinant viruses lacking UL13 or both UL13 and US3 were constructed. We report that UL13 is dispensable for viral replication in cell culture and is not involved in the processing of UL34 or of associated phosphoproteins. UL13 is, however, responsible for the posttranslational processing associated with phosphorylation of infected-cell protein 22, the product of the alpha 22 gene. This gene was previously reported to play a regulatory role in selected cell lines. UL13 appears to be either a protein kinase or a phosphotransferase and its major substrate is the alpha 22 protein.
Evolution of consciousness.
Authors: Eccles J C
Published: May 2006

The hypothesis of the origin of consciousness is built upon the unique properties of the mammalian neocortex. The apical dendrites of the pyramidal cells bundle together as they ascend to lamina I to form neural receptor units of approximately 100 apical dendrites plus branches receiving hundreds of thousands of excitatory synapses, the collective assemblage being called a dendron. It is proposed that the whole world of consciousness, the mental world, is microgranular, with mental units called psychons, and that in mind-brain interaction one psychon is linked to one dendron through quantum physics. The hypothesis is that in mammalian evolution dendrons evolved for more effective integration of the increased complexity of sensory inputs. These evolved dendrons had the capacity for interacting with psychons that came to exist, so forming the mental world and giving the mammal conscious experiences. In Darwinian evolution, consciousness would have occurred initially some 200 million years ago in relation to the primitive cerebral cortices of evolving mammals. It would give global experiences of a surrounding world for guiding behavior beyond what is given by the unconscious operation of sensory cortical areas per se. So conscious experiences would give mammals evolutionary advantage over the reptiles, which lack a neocortex giving consciousness. The Wulst of the avian brain needs further investigation to discover how it could give birds the consciousness that they seem to have.
Intracellular accumulation and resistance to degradation of the Alzheimer amyloid A4/beta protein.
Authors: Knauer M F , Soreghan B , Burdick D , Kosmoski J , Glabe C G
Published: May 2006

The A4 or beta protein is a peptide that constitutes the major protein component of senile plaques in Alzheimer disease. The A4/beta protein is derived from a larger, transmembrane amyloid precursor protein (APP). The putative abnormal processing events leading to amyloid accumulation are largely unknown. Here we report that a 42-residue synthetic peptide, beta 1-42, corresponding to one of the longer forms of the A4/beta protein, accumulates in cultured human skin fibroblasts and is stable for at least 3 days. The peptide appears to accumulate intracellularly, since it does not accumulate under conditions that prevent endocytosis and accumulation is correlated with the acquisition of resistance to removal by trypsin digestion. This intracellular accumulation is also correlated with the ability of the peptide to aggregate as determined by SDS/polyacrylamide gel electrophoresis. At low concentrations of the beta 1-42 peptide, which favor the nonaggregated state, no accumulation is observed. Shorter peptide analogs (28 or 39 residues) that are truncated at the C terminus, which lack the ability to aggregate in SDS gels, fail to accumulate. The accumulated intracellular beta 1-42 peptide is in an aggregated state and is contained in a dense organellar compartment that overlaps the distribution of late endosomes or secondary lysosomes. Immunofluorescence of the internalized peptide in permeabilized cells reveals that it is contained in granular deposits, consistent with localization in late endosomes or secondary lysosomes. Sequence analysis indicates that some of the internalized peptide is subject to N-terminal trimming. These results suggest that the aggregated A4/beta protein may be resistant to degradation and suggest that the A4/beta protein may arise, at least in part, by endosomal or lysosomal processing of APP. Our results also suggest that relatively nonspecific proteolysis may be sufficient to generate the A4/beta protein if this part of APP is selectively resistant to proteolysis.
Segmental determination in Drosophila conferred by hunchback (hb), a repressor of the homeotic gene Ultrabithorax (Ubx).
Authors: Zhang C C , Bienz M
Published: May 2006

The activity of homeotic genes in Drosophila cells determines segment-specific morphogenesis. Here, we provide evidence that the product of hunchback (hb), a segmentation gene, acts as a direct repressor or "silencer" of the homeotic gene Ultrabithorax (Ubx) and thus prevents ectopic activity of this gene: we show, by stable integration of reporter gene constructs, that hb protein binding sites are capable of repressing at a distance the activity of an embryonic Ubx enhancer outside the Ubx expression domain. This silencing activity is observed at advanced embryonic stages, at a time when the hb gene product is no longer detectable or required, and is dependent on the function of Polycomb (Pc). We propose a working hypothesis as to how hb protein in a "hit-and-run" fashion may effect stable and heritable silencing of the Ubx gene throughout advanced stages of development, thus mediating repression of this homeotic gene outside its realm of function.
Poly(A) RNA in Escherichia coli: nucleotide sequence at the junction of the lpp transcript and the polyadenylate moiety.
Authors: Cao G J , Sarkar N
Published: May 2006

Although it has been known for some time that bacterial mRNA molecules carry polyadenylate moieties at their 3' ends, nothing is known about the molecular structure of bacterial poly(A) RNA. To define the polyadenylylation site of a specific bacterial mRNA, we took advantage of the presence of elevated levels of poly(A) RNA in cells of Escherichia coli deficient in exoribonucleases and synthesized DNA complementary to polyadenylylated lipoprotein mRNA, encoded by the lpp gene, by using avian myeloblastosis virus reverse transcriptase and an oligo(dT)-containing primer. The 5'-terminal portion of the cDNA was amplified by the polymerase chain reaction and appropriate oligonucleotide primers, and the amplified DNA was cloned in pUC18 and subjected to nucleotide sequence analysis. Four clones were found to contain the entire 3'-terminal coding region of lpp mRNA, with poly(A) attached to either of two sites in the downstream untranslated region of the transcript. In one type of clone, the polyadenylate moiety was attached at the putative transcription termination site of lpp mRNA, whereas other clones lacked the stem-loop structure of the rho-independent transcription terminator and the polyadenylate moiety was attached to the residue just preceding the terminal stem-loop of the primary transcript. A model for the polyadenylylation of bacterial mRNA is proposed in which poly(A) polymerase and exonucleases compete for the 3' ends of mRNA molecules.
Opposite orientations of DNA bending by c-Myc and Max.
Authors: Wechsler D S , Dang C V
Published: May 2006

The control of gene transcription requires specific protein-protein and protein-DNA interactions. c-Myc, the protein product of the c-myc protooncogene, is a member of the basic helix-loop-helix leucine-zipper class of transcription factors. Although c-Myc is able to bind to a specific core hexanucleotide DNA sequence (CACGTG), its precise function in modulating transcription remains unclear. The recent discovery of Max, a basic helix-loop-helix leucine-zipper partner protein for c-Myc, suggests that the ability of c-Myc to regulate transcription is modulated by the presence of Max. By taking advantage of the altered mobility of protein-bound DNA in the mobility-shift assay, we demonstrate the homo- and heterodimeric complexes of c-Myc and Max are able to cause increased DNA flexure as measured by the circular permutation assay. Based on phasing analysis, c-Myc and Max homodimers bend DNA in opposite orientations, whereas c-Myc-Max heterodimers cause a smaller bend, in an orientation similar to that induced by Max homodimers. To address the possibility that the apparent opposite orientation of bending was the result of DNA unwinding by one of the proteins, we measured the ability of c-Myc and Max homodimers to affect DNA unwinding; we were unable to show any specific unwinding caused by c-Myc or Max. In addition to demonstrating that members of the basic helix-loop-helix leucine-zipper class of transcription factors are able to induce DNA bending, these results suggest that different transcription factor dimers are able to bind to identical DNA sequences and yet have distinct structural effects.
Interleukin 1 regulates secretion of zinc-thymulin by human thymic epithelial cells and its action on T-lymphocyte proliferation and nuclear protein kinase C.
Authors: Coto J A , Hadden E M , Sauro M , Zorn N , Hadden J W
Published: May 2006

Thymic epithelial cells (TEC) are known to secrete thymic hormones that influence maturation of T lymphocytes. One of these peptides, thymulin, requires zinc in an equimolar ratio for biological activity. A previous study [Cousins, R. J. & Leinart, A. S. (1988) FASEB J. 2, 2884-2890] showed that interleukin 1 (IL-1) in vivo stimulates zinc uptake by the thymus. Both the alpha and beta forms of IL-1, which stimulate proliferation of human TEC, also stimulate their uptake of zinc in vitro, and this latter stimulation is both dependent and independent of proliferation. Zinc induces zinc accumulation without proliferation. Two other stimulants of proliferation, bovine pituitary extract and epidermal growth factor, stimulate zinc uptake by TEC, but only in a manner dependent on proliferation. Utilizing in situ hybridization, we show that the IL-1 alpha and beta forms and zinc induce metallothionein mRNA expression TEC. Metallothionein is thought to be involved in the transfer of zinc to thymulin. IL-1 was shown to stimulate the secretion of thymulin as measured both by its ability to stimulate induction of IL-2 receptor-positive lymphocytes from human peripheral blood lymphocytes and by the azathioprine-sensitive rosette assay. In addition, the zinc-thymulin complex in the presence, but not absence, of IL-1 stimulates nuclear protein kinase C in isolated lymphocyte nuclei. IL-1 apparently regulates the synthesis or secretion and delivery of zinc-thymulin complex to the T-lymphocyte system.
Direct identification of residues of the epidermal growth factor receptor in close proximity to the amino terminus of bound epidermal growth factor.
Authors: Woltjer R L , Lukas T J , Staros J V
Published: May 2006

We have recently developed a kinetically controlled, step-wise affinity cross-linking technique for specific, high-yield, covalent linkage of murine epidermal growth factor (mEGF) via its N terminus to the EGF receptor. EGF receptor from A431 cells was cross-linked to radiolabeled mEGF (125I-mEGF) by this technique and the 125I-mEGF-receptor complex was purified and denatured. Tryptic digestion of this preparation gave rise to a unique radiolabeled peptide that did not comigrate with trypsin-treated 125I-mEGF in SDS/Tricine gels but that could be immunoprecipitated with antibodies to mEGF. The immunoprecipitated peptide was isolated by electrophoresis in SDS/Tricine gels, eluted, and sequenced. The sequence was found to correspond to that of a tryptic peptide of the EGF receptor beginning with Gly-85, which is in domain I, a region N terminal to the first cysteine-rich region of the receptor. Selective loss of signal in the 17th sequencing cycle suggests that the point of attachment of N-terminally modified 125I-mEGF to the receptor is Tyr-101. The data presented here provide identification by direct protein microsequencing of a site of interaction of EGF and the EGF receptor.
Thermodynamics of Cro protein-DNA interactions.
Authors: Takeda Y , Ross P D , Mudd C P
Published: May 2006

Using a highly sensitive pulsed-flow microcalorimeter, we have measured the changes in enthalpy and determined the thermodynamic parameters delta H, delta S degree, delta G degree, and delta C(p) for Cro protein-DNA association reactions. The reactions studied include sequence-nonspecific DNA association and sequence-specific DNA associations involving single- and multiple-base alterations and/or single-amino acid alteration mutants. (i) The association of Cro protein with nonspecific DNA at 15 degrees C is characterized by delta H = +4.4 kcal.mol-1 (1 cal = 4.18J), delta S degrees = 49 cal.mol-1.K-1, delta G degrees = -9.7 kcal.mol-1, and delta Cp congruent to 0; the association with specific high-affinity operator OR3 DNA is characterized by delta H = +0.8 kcal.mol-1, delta S degree = 59 cal.mol-1.K-1, delta G degree = -16.1 kcal.mol-1, and delta Cp = -360 cal.mol-1.K-1, respectively. Both nonspecific and specific Cro-DNA associations are entropy-driven. (ii) Plots of delta H vs. delta Cp and delta S degree vs. delta Cp for the 20 association reactions studied fall into two correlation groups with linear slopes of +9.4 K and -20.5 K and of -0.03 and -0.14, respectively. These regression lines have common intercepts, at the delta H and delta S degree values of nonspecific association (where delta Cp congruent to 0). The results suggest that there are, at least, two distinct conformational subclasses in specific Cro-DNA complexes, stabilized by different combinations of enthalpic and entropic contributions. The delta G degree and delta Cp values form an approximately single linear correlation group as a consequence of compensatory contributions from delta H and delta S degree to delta G degree and to delta Cp. Cro protein-DNA associations share some similar thermodynamic properties with protein folding, but their overall energetics are quite different. Although the nonspecific complex is stabilized predominantly by electrostatic forces, it appears that H bonds, van der Waals contacts, hydrophobic effects, and charge interactions all contribute to the stability (delta G degree and delta Cp) of the specific complex. (iii) The variations in the values of the thermodynamic parameters are in general accord with our knowledge of the structure of the Cro-DNA complex.
Purification of a murine protein-tyrosine/threonine kinase that phosphorylates and activates the Erk-1 gene product: relationship to the fission yeast byr1 gene product.
Authors: Crews C M , Erikson R L
Published: May 2006

We report the purification to near homogeneity of a 45-kDa phorbol ester-stimulated protein kinase that phosphorylates and activates the Erk-1 gene product. This kinase, which we provisionally denote MEK for MAPK/Erk kinase, phosphorylated kinase-inactive Erk-1 protein primarily on a tyrosine residue and, to a lesser extent, on a threonine. We extend our previous results and show that two forms of purified MEK activated the myelin basic protein kinase encoded by Erk-1. MEK was inactivated by the serine/threonine phosphatase 2A but not by the protein-tyrosine phosphatase 1B. Sequence analysis of peptides generated by trypsin digestion of MEK revealed similarity to the proteins encoded by the Schizosaccharomyces pombe byr1 and Saccharomyces cerevisiae STE7 genes. These data are discussed with regard to a possible signal transduction mechanism.
Glucocorticoid-suppressible hyperaldosteronism results from hybrid genes created by unequal crossovers between CYP11B1 and CYP11B2.
Authors: Pascoe L , Curnow K M , Slutsker L , Connell J M , Speiser P W , New M I , White P C
Published: May 2006

Glucocorticoid-suppressible hyperaldosteronism (GSH) is an autosomal dominant form of familial hypertension. The biochemical abnormalities seen in this disorder may be remedied by administration of dexamethasone, implying that aldosterone synthesis is being abnormally regulated by corticotropin. The final three steps of aldosterone synthesis, 11 beta- and 18-hydroxylation and 18-oxidation, are mediated by a cytochrome P450 in the zona glomerulosa of the adrenal cortex termed CYP11B2. A related isozyme in the zona fasciculata, CYP11B1, is required for cortisol synthesis; this isozyme, which is normally expressed at much higher levels than CYP11B2, only has 11 beta-hydroxylase activity. These isozymes are encoded by genes on human chromosome 8q22. We have now studied four unrelated patients with GSH. We found that each patient has one chromosome that carries three CYP11B genes instead of two. This has presumably been generated by unequal meiotic crossing-over. The extra gene is a hybrid with 5' regulatory and coding regions corresponding to CYP11B1 and 3' coding regions from CYP11B2. The breakpoint is in intron 2 in two cases, intron 3 in one, and exon 4 in one. Cells transfected with hybrid cDNAs containing up to the first three exons of CYP11B1 synthesized aldosterone at levels near that of cells carrying normal CYP11B2, but cells transfected with hybrids containing the first five or more exons of CYP11B1 could not synthesize detectable amounts of aldosterone. These data demonstrate that GSH is caused by expression of a gene that is regulated like CYP11B1 but that encodes a protein able to synthesize aldosterone.
Abundant expression of parathyroid hormone-related protein in human amnion and its association with labor.
Authors: Ferguson J E , Gorman J V , Bruns D E , Weir E C , Burtis W J , Martin T J , Bruns M E
Published: May 2006

In animal models, parathyroid hormone-related protein (PTHrP) increases placental calcium transport and inhibits contraction of uterine smooth muscle. The present studies were undertaken to characterize the expression of PTHrP in human uteroplacental tissues. PTHrP mRNA was identified by Northern analysis as a single species (approximately 1.8 kilobases) in human amnion, chorion, placenta, decidua, and myometrium. The most abundant signal was seen in amnion, where it was 10-400 times that in the other uteroplacental tissues. PTHrP mRNA abundance was decreased in amnion (but not in the other tissues) following the onset of labor (P less than 0.001). PTHrP mRNA in amnion appeared to be translated to a bioactive peptide, as PTHrP bioactivity and immunoreactive PTHrP in amnion correlated closely with PTHrP mRNA content (r = 0.86 and 0.95, respectively; P less than 0.05 and P less than 0.01). Amniotic fluid contained PTHrP, 21 +/- 6 pmol/liter (n = 10) at 16 weeks and 41 +/- 9 pmol/liter (n = 7) at 38 weeks (P = 0.05). These concentrations equaled or exceeded those found in plasma of patients with hypercalcemia secondary to PTHrP. After rupture of the fetal membranes, PTHrP mRNA in amnion was decreased by 78% (P less than 0.0001). This decrease appeared to be specific for PTHrP mRNA, as glyceraldehyde-3-phosphate dehydrogenase mRNA was unchanged following rupture of membranes. Like PTHrP mRNA, PTHrP bioactivity and immunoreactive PTHrP in amnion decreased significantly following rupture of membranes (P less than 0.03 and P less than 0.01, respectively). Since PTHrP is a potent antagonist of uterine muscle contraction, the decrease of PTHrP following rupture of the fetal membranes may play a key role in the onset of labor.
Expression of a calmodulin methylation mutant affects the growth and development of transgenic tobacco plants.
Authors: Roberts D M , Besl L , Oh S H , Masterson R V , Schell J , Stacey G
Published: May 2006

Transgenic plants were constructed that express two foreign calmodulins (VU-1 and VU-3 calmodulins) derived from a cloned synthetic calmodulin gene. VU-1 calmodulin, similar to endogenous plant calmodulin, possesses a lysine residue at position 115 and undergoes posttranslational methylation. VU-3 calmodulin is a site-directed mutant of VU-1 calmodulin that is identical in sequence except for the substitution of an arginine at position 115 and thus is incapable of methylation. Both calmodulin genes, under the control of the cauliflower mosaic virus 35S promoter, were expressed in transgenic tobacco. Foreign calmodulin protein accumulated in plant tissues to levels equivalent to that of the endogenous calmodulin. All transformed lines of VU-1 plants were indistinguishable from untransformed controls with respect to growth and development. However, all transformed lines of VU-3 plants were characterized by decreased stem internode growth, reduced seed production, and reduced seed and pollen viability. The data suggest that these phenotypes are the result of the expression of the calmodulin mutant rather than the position of transferred DNA insertion or the overall alteration of calmodulin levels. Analyses of the activity of the purified transgenic calmodulins suggest that calmodulin-dependent NAD kinase is among the potential targets that may have altered regulation in VU-3 transgenic plants.
Isoforms of the Na,K-ATPase are present in both axons and dendrites of hippocampal neurons in culture.
Authors: Pietrini G , Matteoli M , Banker G , Caplan M J
Published: May 2006

The distributions of isoforms of the Na,K-ATPase alpha subunit were determined in mature cultured hippocampal neurons and in a polarized epithelial cell line. We find that hippocampal neurons express the alpha 1 and alpha 3 isoforms in the membranes of both axons and dendrites. In contrast the alpha 1 and alpha 3 proteins are exclusively basolateral when expressed endogenously or by stable transfection in renal epithelial cells. These data suggest that epithelial cells and hippocampal neurons localize these proteins by different mechanisms. These observations contrast with those made for the vesicular stomatitis virus and the influenza glycoproteins, which are polarized in both epithelial and neuronal cells.
A factor from CD8 cells of human immunodeficiency virus-infected patients suppresses HLA self-restricted T helper cell responses.
Authors: Clerici M , Roilides E , Via C S , Pizzo P A , Shearer G M
Published: May 2006

Defective in vitro T helper cell (Th) function can occur in asymptomatic human immunodeficiency virus (HIV)-seropositive (HIV+) individuals. A characteristic, early finding is the loss of an in vitro response to recall antigens, such as influenza A virus (FLU), despite an intact Th response to alloantigen (ALLO). To determine whether suppressor cells and/or inhibitory factors could contribute to this HIV-associated Th immunodeficiency, coculture studies were performed using peripheral blood leukocytes (PBLs) from monozygotic twins, one of whom was HIV-infected (HIV+) and one of whom was uninfected (HIV-seronegative, HIV-). In vitro Th function was measured as interleukin 2 production or proliferation to FLU and ALLO. Two pairs of twins were repetitively studied. A single HIV+ individual with multiple samples of cryopreserved PBLs over 6 years (including a HIV- specimen) was also studied. PBLs from the HIV+, but not from the HIV-, individuals demonstrated defective in vitro Th function in response to FLU but not to ALLO. PBLs from HIV+ individuals could induce a similar defect in the Th function of syngeneic or autologous HIV- PBLs. This suppression was generated by CD4-depleted, but not by CD8-depleted, PBLs. A suppressive factor from CD8+ cells of HIV+ donors was generated by 24-hr unstimulated cultures of HIV+ PBLs. This factor inhibited FLU but not ALLO responses of autologous, syngeneic, or allogeneic HIV- PBLs. This suppressive effect could not be explained by HIV infection or replication during the culture period. These results demonstrate that selective abrogation of Th function to recall antigens in HIV+ individuals is associated with an inhibitory factor produced by CD8+ T cells.
Epidermal growth factor stimulates tyrosine phosphorylation in the neonatal mouse: association of a M(r) 55,000 substrate with the receptor.
Authors: Donaldson R W , Cohen S
Published: May 2006

Administration of epidermal growth factor (EGF) to neonatal mice resulted in rapid tyrosine phosphorylation of a number of specific substrates in liver, kidney, lung, bladder, skin, and brain as detected by Western blot analysis of tissue homogenates with anti-phosphotyrosine antibodies. In the liver, three prominent EGF-dependent substrates of M(r) approximately 170,000, 120,000, and 55,000 were detected. A number of less prominent EGF-dependent substrates also were noted. Maximal tyrosine phosphorylation of pp170, pp120, and pp55 occurred within 5 min of subcutaneous injection and the levels of these phosphoproteins remained elevated for at least 45 min. Direct hepatic injection of EGF resulted in the tyrosine phosphorylation of these substrates within 60 sec of treatment. Tyrosine-phosphorylated pp170 was identified as the EGF receptor (EGFR). The tyrosine-phosphorylated pp55 substrate appeared to be associated with EGFR; both pp55 and EGFR were adsorbed to EGF-Affi-Gel, wheat germ lectin-Sepharose, and anti-EGFR antibodies bound to protein A-Sepharose. pp55 was not immunoreactive with anti-EGFR antiserum by Western blot analysis, indicating that it was not a fragment of the receptor. These results were confirmed by repeating the liver experiments using 32P-labeled neonatal mice. Increased amounts of 32P-labeled pp170 and pp55 were detected in anti-EGFR immunoprecipitates from liver extracts of EGF-treated animals as compared with controls. Phospho amino acid analysis of the 32P-labeled phosphoproteins revealed that EGF stimulated both serine and tyrosine phosphorylation in pp55 as well as in EGFR. The neonatal mouse may be a useful model for the study of signal transduction mediated by a variety of growth factors.
Femtosecond spontaneous-emission studies of reaction centers from photosynthetic bacteria.
Authors: Du M , Rosenthal S J , Xie X , DiMagno T J , Schmidt M , Hanson D K , Schiffer M , Norris J R , Fleming G R
Published: May 2006

Spontaneous emission from reaction centers of photosynthetic bacteria has been recorded with a time resolution of 50 fs. Excitation was made directly into both the special-pair band (850 nm) and the Qx band of bacteriochlorophylls (608 nm). Rhodobacter sphaeroides R26, Rhodobacter capsulatus wild type, and four mutants of Rb. capsulatus were studied. In all cases the fluorescence decay was not single exponential and was well fit as a sum of two exponential decay components. The short components are in excellent agreement with the single component detected by measurements of stimulated emission. The origin of the nonexponential decay is discussed in terms of heterogeneity, the kinetic scheme, and the possibility of slow vibrational relaxation.
Molecular organization of Leishmania RNA virus 1.
Authors: Stuart K D , Weeks R , Guilbride L , Myler P J
Published: May 2006

The complete 5284-nucleotide sequence of the double-stranded RNA genome of Leishmania RNA virus 1 (LRV1) was determined and contains three open reading frames (ORFs) on the plus (+) (mRNA) strand. The predicted amino acid sequence of ORF3 has motifs characteristic of viral RNA-dependent RNA polymerases. ORF2, which may encode the major viral coat protein, overlaps ORF3 by 71 nucleotides, suggesting a +1 translational frameshift to produce a gag-pol type of fusion protein. Two alternative models for the frameshift are presented. The 5' splice leader sequence of kinetoplastid mRNAs is not in LRV1 RNA. This suggests that the 450-base region at the 5' end of the LRV1 (+)-strand, which contains ORF1 and is highly conserved among viral strains, does not encode protein but has a role in initiation of translation and/or RNA stability. The similarity of LRV1 genomic organization, replication cycle, and RNA-dependent RNA polymerase sequence to those of the yeast virus ScV L-A suggests a common ancestral origin. The possibility that LRV1 affects pathogenesis in leishmaniasis is intriguing.
Identification, characterization, and DNA sequence of a functional "double" groES-like chaperonin from chloroplasts of higher plants.
Authors: Bertsch U , Soll J , Seetharam R , Viitanen P V
Published: May 2006

Chloroplasts of higher plants contain a nuclear-encoded protein that is a functional homolog of the Escherichia coli chaperonin 10 (cpn10; also known as groES). In pea (Pisum sativum), chloroplast cpn10 was identified by its ability to (i) assist bacterial chaperonin 60 (cpn60; also known as groEL) in the ATP-dependent refolding of chemically denatured ribulose-1,5-bisphosphate carboxylase and (ii) form a stable complex with bacterial cpn60 in the presence of Mg.ATP. The subunit size of the pea protein is approximately 24 kDa--about twice the size of bacterial cpn10. A cDNA encoding a spinach (Spinacea oleracea) chloroplast cpn10 was isolated, sequenced, and expressed in vitro. The spinach protein is synthesized as a higher molecular mass precursor and has a typical chloroplast transit peptide. Surprisingly, however, attached to the transit peptide is a single protein, comprised of two distinct cpn10 molecules in tandem. Moreover, both halves of this "double" cpn10 are highly conserved at a number of residues that are present in all cpn10s that have been examined. Upon import into chloroplasts the spinach cpn10 precursor is processed to its mature form of approximately 24 kDa. N-terminal amino acid sequence analysis reveals that the mature pea and spinach cpn10 are identical at 13 of 21 residues.
In vivo requirement of integration host factor for nar (nitrate reductase) operon expression in Escherichia coli K-12.
Authors: Rabin R S , Collins L A , Stewart V
Published: May 2006

The nitrate reductase operon (narGHJI) of Escherichia coli encodes an anaerobic respiratory enzyme. Previous work has identified two cis-acting sites in the nar operon control region: a proximal site required for anaerobic induction mediated by the activator Fnr and a remote upstream site required for nitrate induction mediated by the activator NarL [Li, S. & DeMoss, J. A. (1988) J. Biol. Chem. 263, 13700-13705]. Our search for nar regulatory mutants yielded one strain with a mutation in himD, the structural gene for one of the subunits of integration host factor (IHF). Strains carrying null alleles of the IHF structural genes, himD and himA, had severe defects in nitrate induction of the nar operon but were normal for nitrate induction of the coordinately regulated fdn operon. Anaerobic expression of both operons was normal in him mutants. Gel-mobility-shift and DNase I protection experiments revealed a single IHF binding site in the nar operon control region, located midway between the upstream activation site and the promoter. We conclude that an IHF-mediated DNA bend is essential for efficient nitrate induction of the sigma 70-dependent nar operon promoter. This requirement of IHF for transcriptional activation had been noted for several sigma 54-dependent promoters.
Class-level relationships in the phylum Cnidaria: evidence from mitochondrial genome structure.
Authors: Bridge D , Cunningham C W , Schierwater B , DeSalle R , Buss L W
Published: May 2006

The phylogenetic relationships of the Recent cnidarian classes remain one of the classic problems in invertebrate zoology. We survey the structure of the mitochondrial genome in representatives of the four extant cnidarian classes and in the phylum Ctenophora. We find that all anthozoan species tested possess mtDNA in the form of circular molecules, whereas all scyphozoan, cubozoan, and hydrozoan species tested display mtDNA in the form of linear molecules. Because ctenophore and all other known metazoan mtDNA is circular, the shared occurrence of linear mtDNA in three of the four cnidarian classes suggests a basal position for the Anthozoa within the phylum.
Structure and expression of major histocompatibility complex-binding protein 2, a 275-kDa zinc finger protein that binds to an enhancer of major histocompatibility complex class I genes.
Authors: van 't Veer L J , Lutz P M , Isselbacher K J , Bernards R
Published: May 2006

We have isolated a cDNA encoding a transcription factor that binds to the enhancer of major histocompatibility complex (MHC) class I genes. MHC-binding protein 2 (MBP-2) is a 275-kDa protein, containing two sets of widely separated zinc fingers and a stretch of highly acidic amino acids, a putative transactivation domain. The two zinc finger regions, when expressed individually as bacterial fusion proteins, bind with highest affinity to the MHC class I gene enhancer. Several proteins found in mammalian nuclear extracts bind the MHC class I enhancer in an electrophoresis mobility shift assay. Only one of these, a ubiquitously expressed factor, forming a slow-migrating retarded complex, can be supershifted by a MBP-2 antiserum. The same antiserum also precipitates a protein of greater than 250 kDa from COS cells transfected with a MBP-2 expression vector. Our data indicate that MBP-2 is a transcription factor involved in the regulation of MHC class I gene expression.
A strategy for making synthetic peptide vaccines.
Authors: Ogasawara K , Naruse H , Itoh Y , Gotohda T , Arikawa J , Kida H , Good R A , Onoé K
Published: May 2006

We have determined the H-2 class II allele-specific amino acid motif of the agretope (the site of contact between the peptide antigen and the major histocompatibility complex) for a synthetic peptide composed of residues 43-58 of pigeon cytochrome c (p43-58). Residues 46 and 54 functioned as the agretope, and residues 50 and 52 functioned as the epitope (the site for contact between the peptide antigen and the T-cell antigen receptor). In general, agretopes and epitopes function independently. Thus, substitution of amino acids in the epitope does not significantly affect binding of the peptide antigen to a class II molecule. On the basis of these findings, synthetic peptide vaccines against influenza Aichi (H3N2) virus were prepared by introducing seven residues of the influenza virus hemagglutinin into the frame component residues 43-46 and 54-58 of p43-58 analogues including the agretopes for Ak or Ab previously determined on the p43-58 segment. These peptide vaccines induced both helper T-cell responses and production of antibodies that were specific for influenza Aichi hemagglutinin but not for the major histocompatibility complex binding frame in mice bearing Ak or Ab. The antibodies produced neutralize the infectivity of influenza Aichi in vitro. The present findings should provide a basis for preparing potent peptide vaccines that function without producing side effects.
Downregulation of interleukin 8 gene expression in human fibroblasts: unique mechanism of transcriptional inhibition by interferon.
Authors: Oliveira I C , Sciavolino P J , Lee T H , Vilcek J
Published: May 2006

The chemotactic cytokine interleukin 8 (IL-8) is produced upon stimulation by various agents in many cell types, including connective-tissue fibroblasts. Tumor necrosis factor (TNF) and IL-1 are potent inducers of IL-8 expression. Earlier we showed that TNF-induced stimulation of IL-8 mRNA accumulation in human FS-4 fibroblasts was inhibited by interferon beta (IFN-beta) or IFN-gamma. Here we show that this inhibition is not specific for TNF, since IFN-beta also reduced IL-8 mRNA accumulation induced by IL-1 or the double-stranded RNA poly (I-C). Treatment with IFN-beta also decreased TNF-induced IL-8 protein accumulation. Interestingly, the inhibitory effect was much less pronounced when IFN-beta was added greater than or equal to 1 hr before TNF. The inhibitory action of IFN-beta on IL-8 mRNA accumulation was undiminished in the presence of inhibitors of protein synthesis. Nuclear run-on assays demonstrated that IFN-beta caused a marked inhibition of TNF-induced IL-8 gene transcription; the transcriptional activation of several other TNF-induced genes was not inhibited by IFN-beta. The results suggest that the specific inhibition of the transcriptional activation of IL-8 by IFN is due either to a transient inactivation of a factor required for IL-8 transcription or to the activation of a selective inhibitory factor.
Expression of myosin-class II major histocompatibility complexes in the normal myocardium occurs before induction of autoimmune myocarditis.
Authors: Smith S C , Allen P M
Published: May 2006

Determining how an autoimmune response is initiated is essential to understanding the mechanisms of autoimmunity. Self-reactive T cells, self-protein, and a failure of tolerance to that self-protein are all involved in the pathogenesis of autoimmune disease; yet it is not clear how self-reactive T cells find the target self-protein to initiate an autoimmune response. Although a variety of self-proteins have been shown to be presented on both class I and class II major histocompatibility complex (MHC) molecules, the relationship of these self-proteins to autoimmune disease has not been established. To explore this further, we generated a T-cell hybridoma that recognizes mouse cardiac myosin, the self-protein that induces murine autoimmune myocarditis. Using this hybridoma as a probe to detect myosin-class II MHC complexes, we isolated a class II MHC+/CD45+ residential antigen-presenting cell (APC) population directly from the hearts of normal mice and looked for evidence of endogenous processing of cardiac myosin by these APC. In this report we show that myosin-class II MHC complexes are found on residential APC in the normal mouse heart. Induction of autoimmune myocarditis increased the expression of myosin-class II MHC in the heart and enhanced their APC functions. This result is a direct demonstration that epitopes of a self-antigen involved in initiating an autoimmune disease are endogenously processed and presented within the target organ.
AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas.
Authors: Cheng J Q , Godwin A K , Bellacosa A , Taguchi T , Franke T F , Hamilton T C , Tsichlis P N , Testa J R
Published: May 2006

We isolated cDNA clones containing the entire coding region of the putative oncogene AKT2. Sequence analysis and in vitro translation demonstrated that AKT2 encodes a 56-kDa protein with homology to serine/threonine kinases; moreover, this protein contains a Src homology 2-like domain. AKT2 was shown to be amplified and overexpressed in 2 of 8 ovarian carcinoma cell lines and 2 of 15 primary ovarian tumors. AKT2 was mapped to chromosome region 19q13.1-q13.2 by fluorescence in situ hybridization. In the two ovarian carcinoma cell lines exhibiting amplification of AKT2, the amplified sequences were localized within homogeneously staining regions. We conclude that AKT2 belongs to a distinct subfamily of protein-serine/threonine kinases containing Src homology 2-like domains and that alterations of AKT2 may contribute to the pathogenesis of ovarian carcinomas.