bluuebunny/crossref_metadata_2025 huggingface dataset:
A fission-yeast gene encoding a protein with features of protein-tyrosine-phosphatases.
Authors: Ottilie S , Chernoff J , Hannig G , Hoffman C S , Erikson R L
Published: May 2006

Degenerate oligonucleotide probes encoding sequences conserved among mammalian protein-tyrosine-phosphatases (PTPases) were used to amplify DNA fragments from a Schizosaccharomyces pombe cDNA library by polymerase chain reaction (PCR) methods. A cloned PCR product predicted peptide sequences similar to those found in PTPases but not identical to any published sequences. A S. pombe gene, designated pyp1+, was identified in a cDNA library with this PCR probe, cloned, and sequenced. The sequence of the gene predicted a 550-amino acid protein with Mr 61,586, which includes amino acid sequences that are highly conserved in mammalian PTPases. Disruption of the pyp1+ gene resulted in viable cells. Overexpression of the pyp1+ gene in S. pombe permitted detection of a protein of apparent Mr 63,000.
Calcium/calmodulin-dependent protein kinase mediates a pathway for transcriptional regulation.
Authors: Kapiloff M S , Mathis J M , Nelson C A , Lin C R , Rosenfeld M G
Published: May 2006

Calcium influx in response to extracellular signals can modulate gene transcription. A constitutive, calcium/calmodulin-independent mutant of type II calcium/calmodulin-dependent protein kinase was capable of increasing the transcription rate of specific genes independently of protein kinase C activation. This increase was mediated by transferable cis-active elements capable of binding the transcription factor CAAT/enhancer binding protein. Therefore, the activation of type II calcium/calmodulin-dependent protein kinase in response to stimuli that increase intracellular calcium is proposed to represent a distinct second messenger pathway in calcium-mediated regulation of gene transcription.
Functional analysis of a liver-specific enhancer of the hepatitis B virus.
Authors: Trujillo M A , Letovsky J , Maguire H F , Lopez-Cabrera M , Siddiqui A
Published: May 2006

The liver-specific enhancer I of the human hepatitis B virus contains several regions of DNA-protein interaction. Located within this element are also the domains of a promoter controlling the synthesis of the X open reading frame. Functional domains of the enhancer I and the X gene promoter were identified using DNase I protection analysis, deletion mutagenesis, and cell transfections. A unique liver-specific interaction was identified within this element whose binding site includes a direct sequence repeat, 5'-AGTAAACAGTA-3'. The factor(s) binding to this sequence motif was purified by oligonucleotide-affinity chromatography. Binding of this factor appears to play a key role in determining the overall enhancer function. Additionally, the interaction of several purified factors is presented. Cotransfection of liver cells with expression vectors encoding transcriptional factors resulted in trans-activation of the promoter/enhancer function. Based on the results of genetic analysis a model outlining the functional domains of the enhancer/promoter region is presented.
Severe von Willebrand disease due to a defect at the level of von Willebrand factor mRNA expression: detection by exonic PCR-restriction fragment length polymorphism analysis.
Authors: Nichols W C , Lyons S E , Harrison J S , Cody R L , Ginsburg D
Published: May 2006

von Willebrand disease (vWD), the most common inherited bleeding disorder in humans, results from abnormalities in the plasma clotting protein von Willebrand factor (vWF). Severe (type III) vWD is autosomal recessive in inheritance and is associated with extremely low or undetectable vWF levels. We report a method designed to distinguish mRNA expression from the two vWF alleles by PCR analysis of peripheral blood platelet RNA using DNA sequence polymorphisms located within exons of the vWF gene. This approach was applied to a severe-vWD pedigree in which three of eight siblings are affected and the parents and additional siblings are clinically normal. Each parent was shown to carry a vWF allele that is silent at the mRNA level. Family members inheriting both abnormal alleles are affected with severe vWD, whereas individuals with only one abnormal allele are asymptomatic. The maternal and paternal silent alleles are identical at two coding sequence polymorphisms as well as an intron 40 variable number tandem repeat, suggesting a possible common origin. Given the frequencies of the two exon polymorphisms reported here, this analysis should be applicable to approximately 70% of type I and type III vWD patients. This comparative DNA and RNA PCR-restriction fragment length polymorphism approach may also prove useful in identifying defects at the level of gene expression associated with other genetic disorders.
Immunofluorescence localization of thyroid hormone receptor protein beta 1 and variant alpha 2 in selected tissues: cerebellar Purkinje cells as a model for beta 1 receptor-mediated developmental effects of thyroid hormone in brain.
Authors: Strait K A , Schwartz H L , Seybold V S , Ling N C , Oppenheimer J H
Published: May 2006

Rat c-erbA beta 1 mRNA rises in cerebrum during the first 10 days of life, coincident with an increase in tissue triiodothyronine (T3) levels and T3-dependent brain development. These data suggest that the beta 1 receptor may mediate the T3 effect. However, in cerebellum c-erbA beta 1 mRNA levels were very low. Since cerebellar development, including dendritic arborization of Purkinje cells, is a T3-sensitive process, we assessed the levels of the beta 1 receptor protein in cerebellum during development. Antisera to unique peptide regions of beta 1 were raised. Their specificity was demonstrated by specific immunoprecipitation of the in vitro translated product, 85% immunoprecipitation of the T3 binding activity in hepatic nuclear extracts, and Western blot analysis of tissue extracts. Immunohistochemical studies using anti-beta 1 antiserum stained liver nuclei but not testis nuclei, which contain no T3 binding activity or beta 1 mRNA. In cerebellar Purkinje cells, an immunofluorescent signal, localized to the nucleus and more intense than that seen in the liver, was observed. A positive but weaker signal was also present in the granule cells. Thus, we may infer that the cerebellum contains significant concentrations of beta 1 receptor protein despite the low beta 1 mRNA content. Both the intensity of staining in Purkinje cell nuclei and immunoprecipitable beta 1 receptor binding capacity rose in the neonatal period. Antiserum to the non-T3 binding alpha 2 variant protein was also prepared and a distinctive pattern of fluorescence was observed. Strong fluorescence was seen in the nuclei of granule cells, but none was seen in Purkinje cells. The alpha 2 fluorescence in testis was high, consistent with the high levels of alpha 2 mRNA in this tissue. The fluorescent signal appeared to originate primarily in dividing spermatogonia. Our findings support the concept that the beta 1 receptor plays a central role in T3-induced brain development and strongly suggest that the Purkinje cell is a direct target for T3.
The nuclear protooncogenes c-jun and c-fos as regulators of DNA replication.
Authors: Murakami Y , Satake M , Yamaguchi-Iwai Y , Sakai M , Muramatsu M , Ito Y
Published: May 2006

Polyomavirus (Py) DNA replication is regulated by its enhancer, which contains an AP1 site, c-Jun and c-Fos, the products of nuclear protooncogenes c-jun and c-fos, form the heterodimeric transcriptional activating factor AP1. Overexpression of c-fos and c-jun genes strongly stimulated Py DNA replication from the Py origin of replication as well as transcription from the Py early promoter through the AP1 binding site. The cAMP response element (CRE)-binding protein CREB stimulated only transcription, not DNA replication, through the CRE under similar conditions. The results indicate that AP1 functions as a regulator of DNA replication and that the mechanism of activation of Py DNA replication by AP1 is distinct from that of activation of transcription from the Py early promoter.
Rhizobium meliloti produces a family of sulfated lipooligosaccharides exhibiting different degrees of plant host specificity.
Authors: Schultze M , Quiclet-Sire B , Kondorosi E , Virelizer H , Glushka J N , Endre G , Géro S D , Kondorosi A
Published: May 2006

We have shown that a Rhizobium meliloti strain overexpressing nodulation genes excreted high amounts of a family of N-acylated and 6-O-sulfated N-acetyl-beta-1,4-D-glucosamine penta-, tetra-, and trisaccharide Nod factors. Either a C(16:2) or a C(16:3) acyl chain is attached to the nonreducing end subunit, whereas the sulfate group is bound to the reducing glucosamine. One of the tetrasaccharides is identical to the previously described NodRm-1 factor. The two pentasaccharides as well as NodRm-1 were purified and tested for biological activity. In the root hair deformation assay the pentasaccharides show similar activities on the host plants Medicago sativa and Melilotus albus and on the non-host plant Vicia sativa at a dilution of up to 0.01-0.001 microM, in contrast to NodRm-1, which displays a much higher specific activity for Medicago and Melilotus than for Vicia. The active concentration range of the pentasaccharides is more narrow on Medicago than on Melilotus and Vicia. In addition to root hair deformation, the different Nod factors were shown to induce nodule formation on M. sativa. We suggest that the production of a series of active signal molecules with different degrees of specificity might be important in controlling the symbiosis of R. meliloti with several different host plants or under different environmental conditions.
A nuclear protein essential for binding of rat 1,25-dihydroxyvitamin D3 receptor to its response elements.
Authors: Ross T K , Moss V E , Prahl J M , DeLuca H F
Published: May 2006

Recombinant 1,25-dihydroxyvitamin D3 receptor from a baculovirus expression system requires a mammalian-derived nuclear accessory protein for binding to a vitamin D response element (DRE). This was established by electrophoretic mobility shift analyses using radiolabeled DNA probes consisting of DREs from two vitamin D-responsive genes. Mammalian nuclear extract was also required for the binding of wild-type porcine vitamin D receptor to a DRE. Surprisingly, the accessory factor-dependent formation of receptor-DRE complex was independent of exogenous 1,25-dihydroxyvitamin D3. A 59- to 64-kDa accessory protein from porcine intestinal nuclear extract was identified by size-exclusion chromatography. Nuclear extracts from rat liver and kidney contained accessory factor, whereas smaller amounts were detected in heart muscle. Spleen and skeletal muscle contained no detectable accessory factor.
A conserved retina-specific gene encodes a basic motif/leucine zipper domain.
Authors: Swaroop A , Xu J Z , Pawar H , Jackson A , Skolnick C , Agarwal N
Published: May 2006

Using subtraction cloning, we have isolated a human cDNA, AS321, which is expressed in retina and retinoblastoma cell lines but not in any other tissue or cell line tested. AS321 mRNA is detected in all cells of neural retina, with a high level of expression in photoreceptors. The polypeptide sequence deduced from the cDNA reveals consensus phosphorylation sites for protein kinase A and proline-directed protein kinase. Its C-terminal region contains a basic motif and a leucine zipper domain similar to the DNA binding proteins of the jun and fos oncoprotein family, and it shows a strong similarity to the product of an avian retroviral oncogene, v-maf. The gene for AS321 is conserved during evolution and is expressed in vertebrate retina. We propose to name the gene NRL (neural retina leucine zipper.
Sulfhydryl oxidation down-regulates T-cell signaling and inhibits tyrosine phosphorylation of phospholipase C gamma 1.
Authors: Kanner S B , Kavanagh T J , Grossmann A , Hu S L , Bolen J B , Rabinovitch P S , Ledbetter J A
Published: May 2006

Early events in both T-cell receptor (CD3)- and CD4-induced signal transduction pathways include tyrosine phosphorylation of protein substrates, the generation of phosphatidylinositol-phosphate breakdown products, and the mobilization of intracellular Ca2+. Oxidative stress in T cells mediated by sulfhydryl-reactive nonpolar maleimides was shown previously to down-regulate both receptor-mediated Ca2+ mobilization and interleukin 2 production. Here we show that N-ethylmaleimide suppresses both CD3- and CD4-induced Ca2+ responses in human T cells correlating with a reduction in the level of phospholipase C gamma 1 (PLC gamma 1) tyrosine phosphorylation. The inhibition of tyrosine phosphorylation of PLC gamma 1 and additional protein substrates was observed at concentrations of N-ethylmaleimide above 20 microM, whereas lower concentrations of oxidant appeared to increase tyrosine kinase activity following cell stimulation. Sulfhydryl oxidation did not directly affect the catalytic activity of PLC gamma 1, since immunopurified enzyme from N-ethylmaleimide-treated T cells was fully active. Although N-ethylmaleimide treatment of T cells did not cause a direct effect on total pp56lck kinase activity measured in vitro, the interaction between CD4 and pp56lck was oxidation-sensitive in vivo. However, CD3-induced signaling was inhibited at N-ethylmaleimide concentrations lower than that required for CD4/pp56lck dissociation, suggesting that CD3-associated tyrosine kinase activity involves acutely sensitive regulatory thiols. In addition to chemically induced sulfhydryl oxidation, naturally regulated cellular redox states appear to dictate the potential for T-cell responsiveness, since degranulating human peripheral blood neutrophils inhibited CD3-induced Ca2+ mobilization in T lymphocytes. These data indicate that signal transduction in T cells involves the activation of PLC gamma 1 by tyrosine phosphorylation through an oxidation-sensitive intermediate between surface receptors and tyrosine kinases, perhaps including the interaction between CD4 and pp56lck.
Immunoblots with rhodopsin antisera suggest that a purified mu opioid binding protein has structural characteristics of a G-protein-coupled receptor.
Authors: Gioannini T L , Weiss E R , Johnson G L , Hiller J M , Simon E J
Published: May 2006

A mu opioid binding protein (OBP), previously purified to homogeneity from bovine striatal membranes, was examined by immunoblotting with six antisera against bovine rhodopsin. An antibody against the carboxyl-terminal tail of rhodopsin and one against membrane-associated rhodopsin gave strong signals at the appropriate molecular mass (65 kDa). An antibody directed against the first cytoplasmic loop of rhodopsin was weakly reactive. Three other antibodies did not recognize OBP. This pattern of crossreactivity was identical to that previously seen with beta-adrenergic receptors. The existence of domains in the OBP, which are antigenically similar to those in two other guanine nucleotide regulatory protein-coupled receptors, supports the hypothesis that mu opioid receptors have the structure characteristic of this receptor family.
Psychophysical support for a two-dimensional view interpolation theory of object recognition.
Authors: Bülthoff H H , Edelman S
Published: May 2006

Does the human brain represent objects for recognition by storing a series of two-dimensional snapshots, or are the object models, in some sense, three-dimensional analogs of the objects they represent? One way to address this question is to explore the ability of the human visual system to generalize recognition from familiar to unfamiliar views of three-dimensional objects. Three recently proposed theories of object recognition--viewpoint normalization or alignment of three-dimensional models [Ullman, S. (1989) Cognition 32, 193-254], linear combination of two-dimensional views [Ullman, S. & Basri, R. (1990) Recognition by Linear Combinations of Models (Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge), A. I. Memo No. 1152], and view approximation [Poggio, T. & Edelman, S. (1990) Nature (London) 343, 263-266]--predict different patterns of generalization to unfamiliar views. We have exploited the conflicting predictions to test the three theories directly in a psychophysical experiment involving computer-generated three-dimensional objects. Our results suggest that the human visual system is better described as recognizing these objects by two-dimensional view interpolation than by alignment or other methods that rely on object-centered three-dimensional models.
Heat shock protein Hsp60-reactive gamma delta cells: a large, diversified T-lymphocyte subset with highly focused specificity.
Authors: O'Brien R L , Fu Y X , Cranfill R , Dallas A , Ellis C , Reardon C , Lang J , Carding S R , Kubo R , Born W
Published: May 2006

Previously, we detected a subset of gamma delta T cells in the newborn mouse thymus that responded to the mycobacterial heat shock protein Hsp60, as well as with what seemed to be a self-antigen. All of these cells expressed V gamma 1, most often in association with V delta 6+. It was not clear, however, whether similar, mature gamma delta cells with Hsp60 reactivity are common outside of the thymus, or rather, whether they are largely eliminated during development. From the data presented here, we estimate that gamma delta cells responding to Hsp60 comprise 10-20% of normal splenic and lymph node gamma delta T cells. Such cells, derived from adult spleen, always express a V gamma 1-J gamma 4-C gamma 4 gamma chain, although not all cells with this gamma chain show Hsp60 reactivity. Many of these V gamma 1+ cells also express V delta 6-J delta 1-C delta, though fewer than in V gamma 1+ cells from the newborn thymus. Extensive diversity is evident in both the gamma and delta chain junctional amino acids of the receptors of these cells, indicating that they may largely develop in the thymus of older animals or undergo peripheral expansion. Finally, we found that all such cells responding to both a putative self-antigen and to mycobacterial Hsp60 respond to a 17-amino acid synthetic peptide representing amino acids 180-196 of the Mycobacterium leprae Hsp60 sequence. This report demonstrates that a large subset of Hsp60-reactive peripheral lymphoid gamma delta T cells preexists in normal adult mice, all members of which respond to a single segment of this common heat shock protein.
Germ-line mutations of the APC gene in 53 familial adenomatous polyposis patients.
Authors: Miyoshi Y , Ando H , Nagase H , Nishisho I , Horii A , Miki Y , Mori T , Utsunomiya J , Baba S , Petersen G
Published: May 2006

We searched for germ-line mutations of the APC gene in 79 unrelated patients with familial adenomatous polyposis using a ribonuclease protection analysis coupled with polymerase chain reaction amplifications of genomic DNA. Mutations were found in 53 patients (67%); 28 of the mutations were small deletions and 2 were 1- to 2-base-pair insertions; 19 were point mutations resulting in stop codons and only 4 were missense point mutations. Thus, 92% of the mutations were predicted to result in truncations of the APC protein. More than two-thirds (68%) of the mutations were clustered in the 5' half of the last exon, and nearly two-fifths of the total mutations occurred at one of five positions. This information has significant implications for understanding the role of APC mutation in inherited forms of colorectal neoplasia and for designing effective methods for genetic counseling and presymptomatic diagnosis.
Detection of acute hepatitis C virus infection by ELISA using a synthetic peptide comprising a structural epitope.
Authors: Kotwal G J , Baroudy B M , Kuramoto I K , McDonald F F , Schiff G M , Holland P V , Zeldis J B
Published: May 2006

An enzyme-linked immunosorbent assay (ELISA) was developed by using a synthetic polypeptide (SP) whose sequence was derived from the structural region of hepatitis C virus (HCV). Results of several coded panels of sera obtained from volunteer blood donors and patients with apparent non-A, non-B hepatitis and/or hepatitis B virus used in this ELISA were compared with those of a commercially available first-generation C-100 ELISA (using nonstructural HCV antigens), an experimental second-generation C-200/C-22 ELISA (using both structural and nonstructural HCV antigens), and recombinant immunoblot assays RIBA-I and RIBA-II. In the majority of cases, the results obtained with the HCV-SP ELISA correlated well with those obtained by RIBA-II and C-200/C-22 ELISA. In contrast, many samples that were repeatedly reactive in the C-100 ELISA results were nonreactive with RIBA and HCV-SP ELISA. In addition, HCV-SP detected HCV-specific antibody that appeared within a month of infection and coincided with the earliest increase in alanine aminotransferase. In summary, we have developed an ELISA based on a structural HCV synthetic polypeptide, HCV-SP, that has high specificity and sensitivity and is capable of detecting specific antibodies in the acute phase of HCV infection.
Genetic identification of the DNA binding domain of Escherichia coli LexA protein.
Authors: Thliveris A T , Mount D W
Published: May 2006

Two genetic approaches were taken to define the DNA binding domain of LexA protein, the repressor of the Escherichia coli SOS regulon. First, several dominant negative lexA mutants defective in DNA binding were isolated. The mutations altered amino acids in a region similar to the helix-turn-helix, a DNA binding domain of other repressors and DNA binding proteins. Second, the region encoding the predicted DNA recognition helix was subjected to oligonucleotide-directed mutagenesis and mutant LexA proteins with altered or relaxed specificity for several recA operator positions were isolated. By examining the effects of a series of amino acid substitutions on repressor specificity, it was shown that a glutamic acid residue at position 45 in LexA protein is important for recognition of the first base pair (G.C) in the recA operator.
The Escherichia coli chromosome contains specific, unmethylated dam and dcm sites.
Authors: Ringquist S , Smith C L
Published: May 2006

The Escherichia coli chromosome encodes two methylases, dam and dcm, which recognize the sequences GATC and CC(A/T)GG, respectively. Specific dam and dcm sites on the E. coli chromosome were found to be unmethylated in vivo by using pulsed-filed gel electrophoresis experiments scanning megabase regions of DNA. Some sites were totally unmethylated. The dam sites display variable methylation depending on the local sequence, and, in general, their methylation shows complex modulation by growth conditions and growth rate, suggesting multiple protection mechanisms. Sites resistant to complete dam or dcm methylation appear to be distributed throughout the chromosome. These unusual sites may identify regions of the chromosome with interesting biological functions.
Integration of gene maps: chromosome 1.
Authors: Collins A , Keats B J , Dracopoli N , Shields D C , Morton N E
Published: May 2006

A composite map of 177 loci has been constructed in two steps. The first combined pairwise logarithm-of-odds scores on 127 loci into a comprehensive genetic map. Then this map was projected onto the physical map through cytogenetic assignments, and the small amount of physical data was interpolated for an additional 50 loci each of which had been assigned to an interval of less than 10 megabases. The resulting composite map is on the physical scale with a resolution of 1.5 megabases. In the future these methods may be used to incorporate locations from linkage, contigs, radiation hybrids, restriction fragments, and somatic cell maps. Dense, reliable, and well-documented maps are essential for long-range sequencing and to localize and clone disease genes.
How cytotoxic T cells manage to discriminate nonself from self at the nonapeptide level.
Authors: Ohno S
Published: May 2006

Class I major histocompatibility complex (MHC) antigens are confronted with an apparently insurmountable dilemma. Each should show a binding preference to a common enough variety of nonapeptides, so that one relevant nonapeptide can be found in at least every other viral protein to provoke a cytotoxic T-cell response. By so doing, however, the chance of that viral T epitope being self is greatly increased. Examination of human and viral nonapeptides preferred by HLA-B27 led to the following conclusions. (i) In normal cells, peptide fragments originating from 5000 or more diverse proteins vie for a finite number of class I MHC sites. Consequently, only those nonapeptides having the optimal binding affinity to a given class I MHC antigen can gain access to the plasma membrane. (ii) Tolerance is rendered only to those host nonapeptides with the optimal binding affinity. (iii) Because of the above noted tolerance, viral nonapeptides with the optimal binding affinity are invariably ignored. (iv) Viral T epitopes actually chosen are always second-echelon nonapeptides that are endowed with slightly less than the optimal binding affinity to a given class I MHC antigen. (v) Since such second-echelon nonapeptides would not gain access to the plasma membrane in normal cells, the issue of self or nonself is rendered irrelevant by this choice. (vi) Since viral T epitopes are of this type, cytotoxic T-cell responses against infected cells are expected to be effective only when a few viral proteins are made in large amounts at the expense of host proteins.
Primary structure and expression of a gamete lytic enzyme in Chlamydomonas reinhardtii: similarity of functional domains to matrix metalloproteases.
Authors: Kinoshita T , Fukuzawa H , Shimada T , Saito T , Matsuda Y
Published: May 2006

A gamete lytic enzyme (GLE) of Chlamydomonas reinhardtii is a zinc metalloprotease and mediates digestion of the cell walls of the two mating-type gametes during mating as a necessary prelude to cell fusion. The nucleotide sequence analysis of a cDNA revealed that GLE is synthesized in a preproenzyme form, a 638-amino acid polypeptide (Mr, 69,824) with a 28-amino acid signal peptide, a 155-amino acid propolypeptide, and a 455-amino acid mature polypeptide (Mr, 49,633). A potential site for autocatalytic activation was contained within the propolypeptide and a zinc binding site found within the mature polypeptide; both sites were highly homologous to those in mammalian collagenase. A putative calcium binding site was present in the near C-terminal region of the mature GLE. Both propolypeptide and mature polypeptide had potential sites for asparagine-linked glycosylation, and the Arg-(Pro)3 and Arg-(Pro)2 motifs, which are known to exist in hydroxyproline-rich glycoproteins of the Chlamydomonas cell wall. Northern blot analysis revealed that steady-state levels of the 2.4-kilobase GLE mRNA increased during growth and mitotic cell division in the vegetative cell cycle and also increased markedly during gametogenesis under nitrogen-starved conditions.
Endoplasmic reticulum resident protein of 90 kilodaltons associates with the T- and B-cell antigen receptors and major histocompatibility complex antigens during their assembly.
Authors: Hochstenbach F , David V , Watkins S , Brenner M B
Published: May 2006

In the endoplasmic reticulum (ER), newly synthesized subunits of the T-cell antigen receptor (TCR), membrane-bound immunoglobulin (mIg), and major histocompatibility complex (MHC) class I antigens must fold correctly and assemble completely into multimeric protein complexes prior to transport to the cell surface. Although folding and assembly may occur spontaneously, the concept that molecular chaperones facilitate these events is emerging. Here, an intracellular protein of 90-kDa apparent molecular mass, denoted IP90, was shown to be an ER resident protein that associated with partial complexes of the TCR, mIg, and MHC class I proteins but was absent from fully assembled complexes. We speculate that IP90 might participate in folding and assembly processes of these and other multisubunit protein complexes during their transit through the ER.
Proteins on exocytic vesicles mediate calcium-triggered fusion.
Authors: Vogel S S , Zimmerberg J
Published: May 2006

In many exocytic systems, micromolar concentrations of intracellular Ca2+ trigger fusion. We find that aggregates of secretory granules isolated from sea urchin eggs fuse together when perfused with greater than or equal to 10 microM free Ca2+. Mixing of membrane components was demonstrated by transfer of fluorescent lipophilic dye, and melding of granule contents was seen with differential interference microscopy. A technique based upon light scattering was developed to conveniently detect fusion. Two protein modifiers, trypsin and N-ethylmaleimide, inhibit granule-granule fusion at concentrations similar to those that inhibit granule-plasma membrane fusion. We suggest that molecular machinery sufficient for Ca(2+)-triggered fusion resides on secretory granules as purified and that at least some of these essential components are proteinaceous.
Molecular cloning and characterization of the human cardiac Na+/Ca2+ exchanger cDNA.
Authors: Komuro I , Wenninger K E , Philipson K D , Izumo S
Published: May 2006

The Na+/Ca2+ exchanger plays important roles in Ca2+ handling in many excitable cells. In particular, the Na+/Ca2+ exchanger is expressed at high levels in the cardiac sarcolemma and is the dominant mechanism of Ca2+ extrusion from the cells. In addition, the exchanger has been suggested to play key roles in digitalis action and in postischemic reperfusion injury of cardiac myocytes. We report here the isolation and characterization of the cDNA encoding the human cardiac Na+/Ca2+ exchanger. Twelve overlapping clones corresponding to 5.6 kilobases of the exchanger cDNA sequence were isolated from 5 x 10(5) phage plaques screened. The sequence predicted a 973-amino acid polypeptide with a putative leader peptide, 11 potential membrane-spanning regions, and one large putative cytoplasmic loop between the fifth and sixth transmembrane helices. When RNA was synthesized in vitro from the cloned cDNA and injected into Xenopus oocytes, it induced expression of Na+/Ca2+ exchange activity at high levels, confirming that this clone encodes the functional Na+/Ca2+ exchanger. Southern blot analysis indicated that the cardiac exchanger gene exists as a single copy in the human genome, although existence of other related genes cannot be ruled out. Northern blot and S1 mapping analyses revealed that the cardiac type exchanger mRNA is expressed most abundantly in the heart and next in the brain. The cardiac-type exchanger mRNA was also expressed in the retina and in skeletal and smooth muscles at very low levels. The levels of mRNA encoding the exchanger were significantly lower in fetal hearts than in adult hearts but were unchanged in the myocardium from patients with end-stage heart failure.
Constitutive synthesis of tumor necrosis factor in the thymus.
Authors: Giroir B P , Brown T , Beutler B
Published: May 2006

Although tumor necrosis factor (TNF) is a major mediator of endotoxic shock, the normal function of TNF that has preserved this protein throughout mammalian evolution remains unknown. If the protein serves a role in normal development or homeostasis, it must be produced under physiologic conditions. To determine whether TNF secretion occurs in normal animals, and to define the tissue sources of the protein, we prepared a reporter construct in which the TNF coding sequence and introns are replaced by the chloramphenicol acetyltransferase (CAT) coding sequence. This construct was inserted into the murine genome, yielding 13 transgenic founders. Macrophages harvested from 4 of the transgenic lines expressed CAT activity after stimulation with Escherichia coli lipopolysaccharide in vitro. Each of these 4 transgenic lines also constitutively expressed CAT activity in the thymus but in no other tissue examined. Cultured thymocytes secrete TNF, as demonstrated both by cytotoxicity assays and by immunoprecipitation of radiolabeled thymic culture medium. CAT activity was associated with the thymic lymphocyte population and not with thymic macrophages or dendritic cells. CAT activity was present in thymic lymphocytes irrespective of CD4 or CD8 expression; T cells from the spleen, however, had no detectable CAT activity. The biosynthesis of TNF in the thymus of normal animals implies a role for this protein in the development or regulation of the immune response.
Rat urate oxidase produced by recombinant baculovirus expression: formation of peroxisome crystalloid core-like structures.
Authors: Alvares K , Widrow R J , Abu-Jawdeh G M , Schmidt J V , Yeldandi A V , Rao M S , Reddy J K
Published: May 2006

Urate oxidase (EC 1.7.3.3), which catalyzes the oxidation of uric acid to allantoin, is present in most mammals but absent in humans and hominoid primates. In rats and most other mammals that catabolize uric acid to allantoin, this enzyme is localized within the crystalloid cores of peroxisomes present in liver parenchymal cells. To determine whether urate oxidase forms these crystalloid cores or whether core-forming protein(s) exist in association with urate oxidase, a baculovirus expression vector system was used to overproduce the full-length rat urate oxidase in Spodoptera frugiperda cells. Urate oxidase was expressed to a level of approximately 30% of the total protein in this system. Immunoblot analysis demonstrated that the baculovirus-generated protein had electrophoretic and immunologic properties similar to those of urate oxidase expressed in rat liver. Immunofluorescence and electron microscopic examination revealed that the overexpressed recombinant urate oxidase is present in both the cytoplasm and the nucleus of infected insect cells as numerous 1- to 3-microns discrete particles. These insoluble protein aggregates, which were positively stained for urate oxidase by protein A-gold immunocytochemical approach, did not appear to be delimited by a single membrane. They revealed a crystalloid structure reminiscent of rat peroxisomal core consisting of bundles of tubules with an inner diameter of approximately 50 A. The recombinant urate oxidase particles, isolated by a single-step procedure, were composed entirely of 35-kDa urate oxidase subunit. These studies indicate that rat urate oxidase is capable of forming insoluble crystalloid core-like structures.
Identification and analysis of the gene encoding human PC2, a prohormone convertase expressed in neuroendocrine tissues.
Authors: Ohagi S , LaMendola J , LeBeau M M , Espinosa R , Takeda J , Smeekens S P , Chan S J , Steiner D F
Published: May 2006

In recent studies we have identified PC2 and PC3, members of a family of serine proteases that are related structurally to subtilisin, and have provided evidence that these are involved in the tissue-specific processing of prohormones and neuropeptides. PC2 is expressed at high levels in the islets of Langerhans, where it participates in the processing of proinsulin to insulin (S.P.S. and D.F.S., unpublished data). To evaluate the regulated expression of the human PC2 (hPC2) gene we have analyzed its structure and characterized its promoter. A map of the gene was constructed by using 11 clones isolated from two human genomic DNA libraries. The gene spans greater than 130 kilobase pairs and consists of 12 exons. Comparison with the structure of the gene encoding human furin, another member of this superfamily, revealed a high degree of conservation of exon-intron junctions. The hPC2 gene was localized to chromosome 20, band p11.2. The 5' flanking region of the hPC2 gene is very G+C-rich and contains six potential Sp1 binding sites but no TATA or CAAT box. Expression of chloramphenicol acetyltransferase reporter fusions containing the putative promoter region was observed to occur in beta TC-3 mouse insulinoma cells but not in HepG2 human hepatoma cells, consistent with the known tissue-specific pattern of expression of the hPC2 gene. Analysis of the level of chloramphenicol acetyltransferase activity with several deletion mutants identified the region from -1100 to -539 from the translation start site as essential for hPC2 promoter activity.
Divergent viral superantigens delete V beta 5+ T lymphocytes.
Authors: Gollob K J , Palmer E
Published: May 2006

Several murine superantigens in association with class II major histocompatibility complex proteins have been shown to cause the deletion of T cells based on the expression of particular beta-chain variable region (V beta) gene segments. We have previously shown that mice expressing the Etc-1 superantigen, encoded by an open reading frame within the 3' long terminal repeat of the endogenous mouse mammary tumor provirus (Mtv), Mtv-9, delete T cells expressing either V beta 5 or V beta 11 gene segments. Comparison of several Mtv 3' long terminal repeat open reading frame sequences has indicated that the carboxyl terminus likely encodes the V beta specificity of these proteins. Our analysis of C57BL/6 x DBA/2 recombinant inbred strains of mouse revealed three Mtv-9-negative strains that nevertheless have a low frequency of V beta 5-expressing T cells. Here we demonstrate that a second endogenous superantigen, responsible for the deletion of V beta 5-bearing T cells, is encoded by a gene mapping to Mtv-6 on chromosome 16. Surprisingly, the carboxyl-terminal sequences of the Mtv-6 and -9 superantigens are extremely divergent, in spite of the fact that they both mediate the deletion of V beta 5+ lymphocytes.
Restriction mapping of the genome of the protozoan parasite Theileria parva.
Authors: Morzaria S P , Young J R
Published: May 2006

We have used a modified linking clone strategy and pulsed-field gel electrophoresis to derive a map of the 29 Sfi I and 4 Not I sites in the 10 million base pair genome of the protozoan parasite Theileria parva. This was achieved in the absence of classical genetic information. The map reveals four chromosomes. Several genes, including those for parasite antigens, have been located on the map, as has the single locus carrying the major repetitive sequence in this organism. The map forms the basis for a study of sexual recombination in T. parva, which will be of importance in the application of present methods of immunization. Aspects of the mapping strategy may be useful in the study of other small eukaryotic genomes.
Rapid evolution of the human gene for cytochrome c oxidase subunit IV.
Authors: Lomax M I , Hewett-Emmett D , Yang T L , Grossman L I
Published: May 2006

We have compared the DNA sequences of nine mammalian genes for cytochrome c oxidase subunit IV (COX4 genes)--four expressed genes (human, bovine, rat, and mouse) and five pseudogenes (human, chimpanzee, orangutan, squirrel monkey, and bovine)--and constructed the sequence of the ancestral mammalian COX4 gene. By analyzing these sequences to determine the pattern and rate of nucleotide substitution in each branch of the evolutionary tree, we deduced that the human gene has evolved rapidly since the origin of the primate pseudogene approximately 41 million years ago, and we discuss the suggestion that this results from coevolution of nuclear and mitochondrial genes for cytochrome c oxidase.
Parathyroid hormone-related protein in the rat urinary bladder: a smooth muscle relaxant produced locally in response to mechanical stretch.
Authors: Yamamoto M , Harm S C , Grasser W A , Thiede M A
Published: May 2006

Parathyroid hormone-related protein (PTHrP) gene expression in the pregnant rat uterus has been shown to be dependent on occupancy of the uterus by the fetus. To further test the hypothesis that the synthesis of PTHrP in smooth muscle tissue is regulated by mechanical stretch, we conducted experiments using the rat urinary bladder as a model of an expansible hollow organ. The results indicate that PTHrP mRNA levels do change in response to the stretch of the bladder wall. Under normal conditions PTHrP mRNA levels in the bladder correlated with the urine volume-namely, the extent of bladder distension. When bladders were maintained empty in vivo, PTHrP mRNA levels decreased gradually. Conversely, when bladders were distended by the accumulation of urine, levels of PTHrP mRNA increased dramatically with time. When distension was limited to one-half of the bladder, the increase in PTHrP mRNA was observed only in the distended portion. Histochemical studies performed on distended bladder tissue indicated the presence of PTHrP immunoreactivity in smooth muscle cells. Isolated organ bath studies were used to examine the possible physiological role of PTHrP in smooth muscle tonicity. In vitro responsiveness of bladder muscle strips to exogenous PTHrP was dependent on the in vivo condition of the bladder. In muscle strips obtained from bladders kept empty in vivo, PTHrP-(1-34)-NH2 relaxed carbachol-induced contraction in a dose-dependent manner but failed to relax the contraction in muscle strips from distended bladders that had high endogenous PTHrP expression. These results and the previous findings in the rat uterus suggest a physiological role of PTHrP in bladder smooth muscle function.
Encoded combinatorial chemistry.
Authors: Brenner S , Lerner R A
Published: May 2006

The diversity of chemical synthesis and the power of genetics are linked to provide a powerful, versatile method for drug screening. A process of alternating parallel combinatorial synthesis is used to encode individual members of a large library of chemicals with unique nucleotide sequences. After the chemical entity is bound to a target, the genetic tag can be amplified by replication and utilized for enrichment of the bound molecules by serial hybridization to a subset of the library. The nature of the chemical structure bound to the receptor is decoded by sequencing the nucleotide tag.
Brief visual experience induces immediate early gene expression in the cat visual cortex.
Authors: Rosen K M , McCormack M A , Villa-Komaroff L , Mower G D
Published: May 2006

Brief visual experience causes rapid physiological changes in the visual cortex during early postnatal development. A possible mediator of these effects is the immediate early genes whose protein products are involved in the rapid response of neurons to transsynaptic stimulation. Here we report evidence that the levels of immediate early gene mRNAs in the visual cortex can be altered by manipulating the visual environment. Specifically, we find that brief (1 h) visual experience in dark-reared cats causes dramatic transient inductions of egr1, c-fos, and junB mRNAs in the visual cortex but not in the frontal cortex. Levels of c-jun and c-myc mRNAs are unaffected. These results suggest that select combinatorial interactions of immediate early gene proteins are an important step in the cascade of events through which visually elicited activity controls visual cortical development.
Involvement of a chloroplast HSP70 heat shock protein in the integration of a protein (light-harvesting complex protein precursor) into the thylakoid membrane.
Authors: Yalovsky S , Paulsen H , Michaeli D , Chitnis P R , Nechushtai R
Published: May 2006

Molecular chaperones, including those belonging to the 70-kDa family of heat shock proteins (HSP70), assist both the translocation of proteins across membranes and their assembly into oligomeric complexes. We purified a chloroplast HSP70 (ct-HSP70) and demonstrated that it plays a major role in the insertion of the precursor of the major light-harvesting complex of photosystem II (pLHCP; an integral membrane protein) into the thylakoids (the inner membranes of the chloroplast). Addition of the purified ct-HSP70 is necessary for efficient insertion of pLHCP into isolated thylakoid membranes. This activity of the purified ct-HSP70 is similar to that previously reported for the total stromal extract. When the chloroplast stromal extract is depleted of HSP70, a correlative reduction in the insertion activity of pLHCP is observed. The interaction between the ct-HSP70 and pLHCP involves physical association. The purified HSP70 acts directly on the membrane protein, presumably prevents its refolding, and thereby helps to maintain its competence for insertion into membranes.
Molecular cloning and characterization of MPL, the human homolog of the v-mpl oncogene: identification of a member of the hematopoietic growth factor receptor superfamily.
Authors: Vigon I , Mornon J P , Cocault L , Mitjavila M T , Tambourin P , Gisselbrecht S , Souyri M
Published: May 2006

We have cloned the human homolog of the v-mpl oncogene transduced in the myeloproliferative leukemia retrovirus, which presents striking homologies with members of the hematopoietin receptor superfamily. We obtained two types of clones, MPLP and MPLK, which had the same 5' extremity but differed at their 3' ends. The resulting deduced polypeptides are composed of a common extracellular domain with a putative signal sequence and a common transmembrane domain, but they differ in their cytoplasmic domain after a stretch of 9 common amino acids. The extracellular domain of MPL contains the consensus sequences described for the members of the hematopoietin receptor superfamily. In addition, as for murine interleukin 3 and human and murine granulocyte-macrophage colony-stimulating factor type beta receptors, this domain can be divided into two subunits. An additional motif specific for MPL could be displayed by hydrophobic cluster analysis in the first subdomain. When RNAs from various hematopoietic cell lines were analyzed by Northern blot, MPL was detected only in the human erythroleukemia (HEL) cell line as a major 3.7-kilobase (kb) mRNA (MPLP) and a minor 2.8-kb mRNA (MPLK). However, study of MPL expression by PCR analysis indicated that MPL is expressed at a low level in a large number of cells of hematopoietic origin and that the two types of mRNAs (P and K) were always found to be coexpressed.
Archaea in coastal marine environments.
Authors: DeLong E F
Published: May 2006

Archaea (archaebacteria) are a phenotypically diverse group of microorganisms that share a common evolutionary history. There are four general phenotypic groups of archaea: the methanogens, the extreme halophiles, the sulfate-reducing archaea, and the extreme thermophiles. In the marine environment, archaeal habitats are generally limited to shallow or deep-sea anaerobic sediments (free-living and endosymbiotic methanogens), hot springs or deep-sea hydrothermal vents (methanogens, sulfate reducers, and extreme thermophiles), and highly saline land-locked seas (halophiles). This report provides evidence for the widespread occurrence of unusual archaea in oxygenated coastal surface waters of North America. Quantitative estimates indicated that up to 2% of the total ribosomal RNA extracted from coastal bacterioplankton assemblages was archaeal. Archaeal small-subunit ribosomal RNA-encoding DNAs (rDNAs) were cloned from mixed bacterioplankton populations collected at geographically distant sampling sites. Phylogenetic and nucleotide signature analyses of these cloned rDNAs revealed the presence of two lineages of archaea, each sharing the diagnostic signatures and structural features previously established for the domain Archaea. Both of these lineages were found in bacterioplankton populations collected off the east and west coasts of North America. The abundance and distribution of these archaea in oxic coastal surface waters suggests that these microorganisms represent undescribed physiological types of archaea, which reside and compete with aerobic, mesophilic eubacteria in marine coastal environments.