bluuebunny/crossref_metadata_2025 huggingface dataset:
Human carbonic anhydrase IV: cDNA cloning, sequence comparison, and expression in COS cell membranes.
Authors: Okuyama T , Sato S , Zhu X L , Waheed A , Sly W S
Published: May 2006

We have isolated a full-length cDNA for human carbonic anhydrase IV (CA IV) from a lambda gt10 human kidney cDNA library. The 1105-base-pair (bp) cDNA contains a 47-bp 5' untranslated region, a 936-bp open reading frame, and a 122-bp 3' untranslated region. The deduced amino acid sequence is colinear with the N-terminal sequence and the sequence of several tryptic peptides of human lung CA IV. It includes an 18-amino acid signal sequence, a 260-amino acid region that shows 30-36% similarity with the 29-kDa cytoplasmic CAs (CA I, CA II, and CA III), and an additional 27-amino acid C-terminal sequence that ends in a 21-amino acid hydrophobic domain. Of the 17 "active site" residues that are highly conserved in other human CAs, 16 are also present in CA IV. Expression of the cDNA in COS cells produced a 35-kDa enzyme that was membrane associated, resistant to inactivation by SDS, contained no carbohydrate, and reacted on Western blots with antiserum to the 35-kDa CA IV from human lung. Treatment of membranes from transfected COS cells with phosphatidylinositol-specific phospholipase C released 20-30% of the expressed enzyme from membranes, indicating that at least 20-30% of the expressed enzyme was anchored to membranes by a glycosyl-phosphatidylinositol linkage.
Reversible and irreversible intermediates during photoinhibition of photosystem II: stable reduced QA species promote chlorophyll triplet formation.
Authors: Vass I , Styring S , Hundal T , Koivuniemi A , Aro E , Andersson B
Published: May 2006

Photoinhibition of photosynthesis was studied in isolated photosystem II membranes by using chlorophyll fluorescence and electron paramagnetic resonance (EPR) spectroscopy combined with protein analysis. Under anaerobic conditions four sequentially intermediate steps in the photoinhibitory process were identified and characterized. These intermediates show high dark chlorophyll fluorescence (Foi) with typical decay kinetics (fast, semistable, stable, and nondecaying). The fast-decaying state has no bound QB but possesses a single reduced QA species with a 30-s decay half-time in the dark (QB, second quinone acceptor; QA, first quinone acceptor). In the semistable state, Q-A is stabilized for 2-3 min, most likely by protonation, and gives rise to the Q-A Fe2+ EPR signal in the dark. In the stable state, QA has become double reduced and is stabilized for 0.5-2 hr by protonation and a protein conformational change. The final, nondecaying state is likely to represent centers where QA H2 has left its binding site. The first three photoinhibitory states are reversible in the dark through reestablishment of QA to QB electron transfer. Significantly, illumination at 4 K of anaerobically photoinhibited centers trapped in all but the fast state gives rise to a spinpolarized triplet EPR signal from chlorophyll P680 (primary electron donor). When oxygen is introduced during anaerobic illumination, the light-inducible chlorophyll triplet is lost concomitant with induction of D1 protein degradation. The results are integrated into a model for the photoinhibitory process involving initial loss of bound QB followed by stable reduction and subsequent loss of QA facilitating chlorophyll P680 triplet formation. This in turn mediates light-induced formation of highly reactive and damaging singlet oxygen.
Termination efficiency at rho-dependent terminators depends on kinetic coupling between RNA polymerase and rho.
Authors: Jin D J , Burgess R R , Richardson J P , Gross C A
Published: May 2006

Rho-dependent terminators constitute one of two major classes of terminators in Escherichia coli. Termination at these sites requires the concerted action of RNA polymerase and rho protein. We present evidence that the efficiency of termination at these sites is governed by kinetic coupling of the rate of transcription of RNA polymerase and the rate of action of rho protein. Termination experiments in vitro indicate that termination efficiency at a rho-dependent terminator is an inverse function of the rate of elongation of RNA polymerase, and each of the mutant phenotypes can be accounted for by the altered rate of elongation of the mutant RNA polymerase. Experiments in vivo show that fast-moving mutant RNA polymerases are termination deficient, while slow-moving mutant RNA polymerases are termination proficient and can suppress the termination deficiency of a slow-acting mutant rho protein. Because of the close coupling of rho action with RNA polymerase, small changes in the elongation rate of RNA polymerase can have very large effects on termination efficiency, providing the cell with a powerful way to modulate termination at rho-dependent terminators.
Role of steroid 11 beta-hydroxylase and steroid 18-hydroxylase in the biosynthesis of glucocorticoids and mineralocorticoids in humans.
Authors: Kawamoto T , Mitsuuchi Y , Toda K , Yokoyama Y , Miyahara K , Miura S , Ohnishi T , Ichikawa Y , Nakao K , Imura H
Published: May 2006

A gene encoding steroid 18-hydroxylase (P-450C18) was isolated from a human genomic DNA library. It was identified as CYP11B2, which was previously postulated to be a pseudogene or a less active gene closely related to CYP11B1, the gene encoding steroid 11 beta-hydroxylase (P-45011 beta) [Mornet, E., Dupont, J., Vitek, A. & White, P. C. (1989) J. Biol. Chem. 264, 20961-20967]. The nucleotide sequence of the promoter region of the P-450C18 gene is strikingly different from that of the P-45011 beta gene, although the sequences of their exons are 93% identical. The transient expression in Y-1 adrenal tumor cells of CAT constructs with a series of deletion mutants of promoter regions of both genes indicated that the two genes are regulated differently. P-450C18 as expressed in COS-7 cells exhibits steroid 18-hydroxylase activity to catalyze the synthesis of aldosterone and 18-oxocortisol and exhibits steroid 11 beta-hydroxylase activity as well. In contrast, P-45011 beta as expressed in the cultured cells exhibits steroid 11 beta-hydroxylase activity exclusively but fails to catalyze the synthesis of aldosterone and 18-oxocortisol. These results indicate that P-45011 beta and P-450C18 are products of two different genes and that the former participates in the synthesis of glucocorticoids whereas the latter participates in the synthesis of mineralocorticoids in humans.
Neurotrophin 3 is a mitogen for cultured neural crest cells.
Authors: Kalcheim C , Carmeli C , Rosenthal A
Published: May 2006

Neurotrophin 3 (NT-3) promotes the survival and induces neurite outgrowth from a subset of neural crest (NC) and placode-derived neurons. We now report that this growth factor regulates the proliferation of cultured NC progenitor cells grown in a serum-free defined medium. In cultures of somites containing NC cells at migratory stages, NT-3 promotes a 2- to 8.4-fold increase in the number of NC cells incorporating [3H]thymidine into nuclei and a 1.8- to 4.8-fold increase in NC cell number compared to controls without added factor. NT-3 also promoted, to a lesser extent, the proliferation of NC cells in homogeneous cultures established from NC clusters. In addition to its effect on NC cells, NT-3 was mitogenic to somite cells in the mixed NC/somite cultures. These data demonstrate that NT-3 can act directly on the NC cells. They also indicate that the response of NC cells to NT-3 may be modulated by the presence of somitic cells. We suggest that NT-3 may be one of the central nervous system-derived factors that mediate NC cell proliferation in vivo.
cDNA cloning and functional expression of the Schistosoma mansoni protective antigen triose-phosphate isomerase.
Authors: Shoemaker C , Gross A , Gebremichael A , Harn D
Published: May 2006

M.1 monoclonal antibody has previously been shown to passively transfer partial resistance to schistosome infection within mice and to recognize a 28-kDa antigen that has peptide sequence homology with triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1). We have now isolated the complete coding DNA for Schistosoma mansoni TPI and confirmed that this cDNA encodes the 28-kDa antigen recognized by M.1. The predicted translation product has strong homology with other TPIs, particularly from higher eukaryotes, and the sequence homology is greatest in regions known to form the active site. The complete coding DNA has been expressed within an Escherichia coli host to produce high levels of soluble, recombinant S. mansoni TPI protein. The product is recognized and purified by the M.1 antibody and is a functional TPI with an intrinsic specific activity comparable to that of rabbit and yeast TPI.
Involvement of tyrosine residues in the tanning of proteins by 3-hydroxyanthranilic acid.
Authors: Manthey M K , Pyne S G , Truscott R J
Published: May 2006

The binding of oxidized phenolic compounds to proteins is of importance in a number of biological systems, including the sclerotization of insect cuticle and the tanning of cocoons. 3-Hydroxyanthranilic acid (3HAA), an aminophenol, is a tryptophan metabolite that undergoes autoxidation readily, and proteins incubated in the presence of 3HAA and oxygen become colored and oxidized. Some moth species are thought to employ this reactivity of 3HAA with proteins for the tanning of cocoons, but the detailed mechanism of this process has not been studied previously. We show that one reaction pathway involves the covalent coupling of 3HAA with tyrosine to form a benzocoumarin derivative, a dibenzo[b,d]pyran-6-one. The stability of the benzocoumarin to conditions of acid hydrolysis normally used for protein digestion has enabled the isolation of the tyrosine adduct from bovine serum albumin that had been incubated with 3HAA. The adduct was also isolated from cocoons of Samia cynthia and Hyalophora gloveri, two species of moths reported to utilize 3HAA for cocoon tanning. These findings indicate that one mechanism of interaction of 3HAA with proteins involves a radical-radical coupling with tyrosine residues.
Identification and structural characterization of a cDNA clone encoding a membrane-bound form of the polypeptide pheromone Er-1 in the ciliate protozoan Euplotes raikovi.
Authors: Miceli C , La Terza A , Bradshaw R A , Luporini P
Published: May 2006

In the ciliate Euplotes raikovi, the same cell that secretes the pheromone Er-1, a polypeptide of 40 amino acids derived from a precursor (prepro-Er-1) of 75 amino acids, also produces a polypeptide of 130 amino acids, of which the 75 residues at the carboxyl terminus are identical to those of prepro-Er-1 and the 55 residues at the amino terminus form a new sequence. This larger Er-1 isoform is retained in membranes, where it may function as a binding site for soluble Er-1 in a mechanism of autocrine secretion. Membrane-bound and soluble Er-1 are translated from two mRNAs that apparently originate from a common micronuclear and/or macronuclear gene through alternative elimination of intervening sequences. This finding suggests that single genes responsible for the generation of isoform diversity in polypeptide hormones are present even in single-celled eukaryotes.
Interactions of liver Grp78 and Escherichia coli recombinant Grp78 with ATP: multiple species and disaggregation.
Authors: Carlino A , Toledo H , Skaleris D , DeLisio R , Weissbach H , Brot N
Published: May 2006

The hamster gene encoding the 78-kDa glucose-regulated protein (Grp78) was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. After induction with isopropyl beta-D-thiogalactopyranoside, the recombinant Grp78 was purified to homogeneity by affinity column chromatography of the fusion protein followed by thrombin cleavage. The purified recombinant protein was compared with liver Grp78 for its ability to interact with ATP. Like liver Grp78, the recombinant protein contained a weak ATPase activity and a Ca(2+)-stimulated autophosphorylation activity. However, unlike liver Grp78, in which the autophosphorylation reaction is stimulated less than 50% by CaCl2, the reaction with the recombinant Grp78 was stimulated about 15-fold in the presence of Ca2+. Although the liver protein showed at least four isoforms after two-dimensional gel electrophoresis, the recombinant Grp78 had one major species corresponding to the most basic form seen in liver. Both the liver Grp78 and the recombinant protein existed primarily as monomers and dimers. A small amount of oligomers was also present in the liver Grp78. When either protein was incubated with ATP, there was a conversion of the higher molecular weight species to the monomeric form.
Replication of patient isolates of human immunodeficiency virus type 1 in T cells: a spectrum of rates and efficiencies of entry.
Authors: Fernandez-Larsson R , Srivastava K K , Lu S , Robinson H L
Published: May 2006

Isolates of human immunodeficiency virus type 1 (HIV-1) undergo many different rates of replication, with the time course of replication being determined by the host cell and the virus. Recently, we demonstrated that the permissiveness of four CD4+ T-cell lines for the laboratory strain NL4-3 correlated with the rate and efficiency of virus entry. In this study, we have analyzed the replication of a "slow/low" isolate from the pre-AIDS period of infection and two "rapid/high" isolates from the AIDS period of infection to determine which steps in the virus life cycle determine differences in the growth characteristics of patient isolates. Differences in the growth of the patient isolates correlated with differences in entry but not postentry steps of the virus life cycle. The two rapid/high patient isolates (SF33 and SF216) underwent 50% entry in less than or equal to 0.5 hr in C8166 cells, in less than or equal to 1 hr in mitogen-stimulated peripheral blood mononuclear cells, and in greater than or equal to 2.5 hr in H9 cells. In contrast, a class 3 slow/low patient isolate required 1 hr for 50% entry into C8166 cells, 3 hr for 50% entry into peripheral blood mononuclear cells, and 5-6 hr for 50% entry into H9 cells. Entry efficiency correlated with entry rate, with the rapid/high viruses having a 2-fold higher titer and the slow/low virus having a 5-fold higher titer on C8166 than H9 cells. The laboratory strain NL4-3 displayed intermediate rates and efficiencies of entry. These data demonstrate that entry characteristics are major determinants of the pathogenic potential of patient isolates.
Neuronal lysosomal enzyme replacement using fragment C of tetanus toxin.
Authors: Dobrenis K , Joseph A , Rattazzi M C
Published: May 2006

Development of a strategy for efficient delivery of exogenous enzyme to neuronal lysosomes is essential to achieve enzyme replacement in neurodegenerative lysosomal storage diseases. We tested whether effective lysosomal targeting of the human enzyme beta-N-acetylhexosaminidase A (Hex A; beta-N-acetyl-D-hexosaminide N-acetylhexosaminohydrolase, EC 3.2.1.52) can be obtained by coupling it via disulfide linkage to the atoxic fragment C of tetanus toxin (TTC) that is bound avidly by neuronal membrane. TTC-Hex A conjugation resulted in neuronal surface binding and enhanced endocytosis of enzyme as observed in immunofluorescence studies with rat brain cultures. In immunoelectrophoretic quantitative uptake studies, rat neuronal cell cultures contained 16- and 40-fold greater amounts of enzyme after incubation with TTC-Hex A than with nonderivatized Hex A. In cerebral cortex cell cultures from a feline model of human GM2 gangliosidosis (Tay-Sachs and Sandhoff diseases), binding and uptake patterns of the enzymes were similar to those in the rat brain cell cultures. After exposure to extracellular concentrations of enzyme attainable in vivo, lysosomal storage of immunodetectable GM2 ganglioside was virtually eliminated in neurons exposed to TTC-Hex A, whereas a minimal effect was observed with Hex A. These findings demonstrate the usefulness of TTC adducts for effective neuronal lysosomal enzyme replacement.
Directed evolution of a protein: selection of potent neutrophil elastase inhibitors displayed on M13 fusion phage.
Authors: Roberts B L , Markland W , Ley A C , Kent R B , White D W , Guterman S K , Ladner R C
Published: May 2006

Inhibitors of human neutrophil elastase were engineered by designing and producing a library of phage-displayed protease inhibitory domains derived from wild-type bovine pancreatic trypsin inhibitor and fractionating the library for binding to the target protease. The affinity of one of the engineered variants for human neutrophil elastase (Kd = 1.0 pM) is 3.6 x 10(6)-fold higher than that of the parental protein and exceeds the highest affinity reported for any reversible human neutrophil elastase inhibitor by 50-fold. Thus the display phage method has allowed us to obtain protein derivatives that exhibit greatly increased affinity for a predetermined target. The technology can be applied to design high-affinity proteins for a wide variety of target molecules.
Anti-idiotype monoclonal antibody elicits broadly neutralizing anti-gp120 antibodies in monkeys.
Authors: Kang C Y , Nara P , Chamat S , Caralli V , Chen A , Nguyen M L , Yoshiyama H , Morrow W J , Ho D D , Köhler H
Published: May 2006

Murine monoclonal antibodies (mAbs) were raised against human, polyclonal, anti-gp120 antibodies (Ab1) and were selected for binding to broadly neutralizing anti-gp120 antibodies in sera positive for human immunodeficiency virus (HIV). One anti-idiotype mAb (Ab2), 3C9, was found to be specific for human anti-gp120 antibodies directed against an epitope around the conserved CD4 attachment site of gp120. The 3C9 reactive human anti-gp120 antibodies (3C9+ Ab) neutralized MN, IIIB, RF, and four primary isolates of HIV type 1 (HIV-1). Cynomolgus monkeys were immunized with 3C9 in adjuvant to test whether this anti-idiotype mAb could induce neutralizing anti-gp120 antibodies. The results show that purified anti-anti-idiotype antibodies (Ab3) from 3C9 immune sera bind to an epitope around the CD4 attachment site of gp120SF and gp120IIIB. Furthermore, purified gp120-specific Ab3 neutralize MN, IIIB, and RF isolates. These results demonstrate that primates immunized with an anti-idiotype mAb produce broadly neutralizing anti-HIV-1 antibodies. Since this anti-idiotype mAb was selected by identifying a clonotypic marker, its biological activity can be explained as the results of clonotypic B-cell stimulation.
Mobilization of dantrolene-sensitive intracellular calcium pools is involved in the cytotoxicity induced by quisqualate and N-methyl-D-aspartate but not by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate and kainate in cultured cerebral cortical neurons.
Authors: Frandsen A , Schousboe A
Published: May 2006

By using primary cultures of cerebral cortical neurons, it has been demonstrated that the antihyperthermia drug dantrolene protects against cytotoxicity induced by the excitatory amino acids quisqualate (QA) and N-methyl-D-aspartate (NMDA), whereas no effect was observed on cell damage mediated by kainate (KA) or 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA). In parallel it was shown that KA and AMPA increased the concentration of intracellular free calcium ([Ca2+]i) mainly by influx, whereas the increase in [Ca2+]i stimulated by NMDA and QA predominantly was caused by release of Ca2+ from intracellular stores, which for NMDA seemed to be mediated at least partly by Ca2+ influx. In accordance with the effects on cytotoxicity, dantrolene blocked the increase in [Ca2+]i elicited by QA and NMDA leaving the increase induced by KA and AMPA unaffected. The finding that 2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionate, which regarding toxicity is a selective KA antagonist, only reduced the KA-stimulated increase in [Ca2+]i by 30% may suggest that the elevation of [Ca2+]i is not the only element in KA-induced cytotoxicity. On the other hand, the present study underlines the importance of Ca2+ for cytotoxicity induced by some excitatory amino acids (glutamate, NMDA, and QA) and supports the current proposal that multiple mechanisms are operating, even concerning calcium homeostasis. Because excitatory amino acid-induced cytotoxicity is thought to be involved in neuropathological conditions such as ischemia, it is possible that dantrolene might be of therapeutic interest.
Interleukin 2 regulates the activity of the lyn protein-tyrosine kinase in a B-cell line.
Authors: Torigoe T , Saragovi H U , Reed J C
Published: May 2006

Recently, interleukin 2 (IL-2) has been shown to induce increased activity of the p56lck protein-tyrosine kinase (PTK) in T-cell and natural killer cell lines, and evidence for a direct interaction between the p75 subunit of the IL-2 receptor (IL-2R) and this src-family kinase has been reported. Though these findings suggest a central role for lck in IL-2 signal transduction, one problem with this idea is that not all IL-2-responsive cells express the lck gene. For this reason, we examined the effects of IL-2 on the activity of src-like kinases in a pro-B cell line, F7, that lacks p56lck but that displays high-affinity IL-2Rs and vigorously proliferates in response to this lymphokine. Of the eight known src-family PTKs, F7 cells were shown to contain only p53/56lyn, p59fyn, and a small amount of p62yes. Stimulation of resting F7 cells with IL-2 induced a rapid (detectable within 1 min and maximal at 15 min) and concentration-dependent increase in the specific activity of p53/56lyn kinase, as assessed by in vitro kinase assays. This effect of IL-2 on p53/56lyn kinase was specific, since no IL-2-inducible changes were detected in the activities of the p59fyn and p62yes kinases. Furthermore, by using a monoclonal antibody specific for the approximately 75-kDa beta subunit of the IL-2R (referred to as p75/IL-2R beta), evidence for physical association between the lyn kinase and the IL-2R complex was obtained, in that a small proportion of the p53/56lyn kinase in F7 cells, but no detectable p59fyn kinase, was coimmunoprecipitated with p75/IL-2R beta. When combined with the recent evidence that IL-2 regulates p56lck in T cells, these results indicate that some flexibility exists in the ability of various src-like PTKs to participate in IL-2 signal transduction mechanisms and raise the possibility that lineage-specific (T-versus B-cell) responses to IL-2 may be determined at least in part by the repertoire of src-like PTKs expressed in the cell.
Partial characterization of vertebrate prothrombin cDNAs: amplification and sequence analysis of the B chain of thrombin from nine different species.
Authors: Banfield D K , MacGillivray R T
Published: May 2006

The cDNA sequence of the B chain of thrombin (EC 3.4.21.5) has been determined from nine vertebrate species (rat, mouse, rabbit, chicken, gecko, newt, rainbow trout, sturgeon, and hagfish). The amino acid sequence identities vary from 96.5% (rat vs. mouse) to 62.6% (newt vs. hagfish). Of the 240 amino acids spanned in all the species compared, there is identity at 110 (45.8%) positions. When conservative changes are included, the amino acid similarity increases to 75%. The most conserved portions of the B chain are the active-site residues and adjacent amino acids, the B loop, and the primary substrate-binding region. In addition, the Arg-Gly-Asp motif is conserved in 9 of the 11 species compared, and the chemotactic/growth factor domain is well conserved in all of the 11 species compared. The least conserved regions of the B chain correspond to surface loops, including the putative thrombomodulin-binding sites and one of the hirudin-binding regions. The extent of the amino acid sequence similarity and the conservation of many of the functional/structural motifs suggests that, in addition to their role in blood coagulation, vertebrate thrombins may also play an important role in the general mechanisms of wound repair.
Single-residue changes in class I major histocompatibility complex molecules stimulate responses to self peptides.
Authors: Grandea A G , Bevan M J
Published: May 2006

Single-residue changes were introduced into the murine major histocompatibility complex class I molecule H-2Kb at positions 65 and 69, which are predicted to point up from the alpha-helix of the alpha 1 domain and not into the peptide binding groove. Mutated and wild-type genes were transfected into the murine cell line P815 (H-2d). We present evidence that the changes did not affect the binding of three foreign peptides that are recognized by cytotoxic T lymphocytes (CTL) in association with H-2Kb. Additionally, the mutants provoked strong alloreactive responses in T cells from mice expressing unmutated H-2Kb. The alloreactive CTL were specific for self peptides, which could be extracted from wild-type H-2Kb molecules, recognized in the context of the mutant class I.
Human cyclin-dependent kinase 2 is activated during the S and G2 phases of the cell cycle and associates with cyclin A.
Authors: Rosenblatt J , Gu Y , Morgan D O
Published: May 2006

We have analyzed the cell cycle regulation of human cyclin-dependent kinase 2 (CDK2), a protein closely related to the cell cycle-regulatory protein kinase CDC2. We find that CDK2 activity, like that of CDC2, oscillates during the cell cycle in cultured mammalian fibroblasts. Unlike CDC2 activity (which peaks during mitosis), CDK2 activity rises in late G1 or early S phase and declines during mitosis. Active S-phase CDK2 migrates in multiple large complexes on gel filtration, and CDK2 in one of these complexes is associated with cyclin A. These findings suggest that CDK2 and CDC2, in association with distinct cyclins, regulate separate functions in the mammalian cell cycle.
K-sam gene encodes secreted as well as transmembrane receptor tyrosine kinase.
Authors: Katoh M , Hattori Y , Sasaki H , Tanaka M , Sugano K , Yazaki Y , Sugimura T , Terada M
Published: May 2006

K-sam was first identified as a gene amplified in the stomach cancer cell line KATO-III. The size of the major transcript of the K-sam gene was 3.5 kilobases in KATO-III cells, and we have previously shown that K-sam encodes a receptor tyrosine kinase that belongs to the heparin-binding growth factor receptor, or fibroblast growth factor receptor, gene family. The K-sam gene expresses multiple sizes of mRNAs in brain tissue, the immature teratoma cell line NCC-IT, and KATO-III. RNA blot analyses with a variety of K-sam probes indicate that there are at least four classes of K-sam mRNAs. Three types of K-sam cDNAs in addition to the previously reported type of K-sam cDNA were isolated, and their nucleotide sequences encode a full-length transmembrane receptor, a secreted receptor with a tyrosine kinase domain, and a secreted receptor without a tyrosine kinase domain.
T-cell responsiveness to an oncogenic peripheral protein and spontaneous autoimmunity in transgenic mice.
Authors: Geiger T , Gooding L R , Flavell R A
Published: May 2006

Why T cells develop autoimmune reactivity to some antigens and tolerance to others is unknown. Various mechanisms can provide for T-cell tolerance. These include deletion in the thymus, exhaustive differentiation in the periphery, T-cell receptor and coreceptor downregulation, and anergy. Which mechanisms normally provide for tolerance to antigens expressed on specific tissues and why they sometimes fail is unclear. To understand this, we analyzed how a tissue-specific protein with defined timing and location of expression is recognized by T cells so as to induce tolerance or autoimmunity. We crossed mice expressing the simian virus 40 large tumor antigen on pancreatic acini beginning 4-25 days after birth with mice transgenic for a rearranged T-cell receptor that recognizes this antigen presented by the class I major histocompatibility complex molecule H-2Kk. No T-cell tolerance was found; rather, T-cell reactivity accompanied lymphocytic infiltration and pancreatic acinar destruction. This result argues that T cells may become spontaneously autoreactive to certain postnatally expressed peripheral proteins and that this reactivity may lead to autoimmune disease.
Role of neuromedin B in control of the release of thyrotropin in hypothyroid and hyperthyroid rats.
Authors: Rettori V , Pazos-Moura C C , Moura E G , Polak J , McCann S M
Published: May 2006

Neuromedin B (NB) is a recently discovered neuropeptide related to bombesin. It is localized to thyrotropes and we have previously shown that it directly inhibits thyrotropin (TSH) release from the anterior pituitary gland of euthyroid rats. In the current studies, we further evaluated the action of NB and antiserum directed against it in euthyroid rats and compared the actions with those in hypo- and hyperthyroid rats. Rats were rendered hypothyroid by treatment with propylthiouracil and hyperthyroid by treatment with thyroxine. In euthyroid rats, NB suppressed TSH release from hemipituitaries in vitro. Incubation of these pituitaries with highly specific antiserum against NB produced a stimulation of TSH release, whereas normal rabbit serum had no effect on the output of TSH. Thus, in euthyroid animals NB is a physiologically significant inhibitor of TSH release from the pituitary. In hypothyroid as in euthyroid animals, NB inhibited TSH release when microinjected into the third ventricle (3V) in the same dose (0.5 micrograms; 0.44 nmol) as in euthyroid rats. TSH release from hemipituitaries of hypothyroid animals was also suppressed by NB as in euthyroid animals. In hypothyroid animals, anti-NB antiserum was ineffective both in vivo after its microinjection into the 3V and in vitro on hemipituitaries, which suggests that the peptide has little physiologic significance in this condition, presumably because of its reduced release from the thyrotropes associated with diminished NB content in the pituitary of the hypothyroid rat. Intraventricular injection of NB failed to lower plasma TSH in hyperthyroid rats, which suggests that the action of the peptide is already maximal in hyperthyroidism. When antiserum to NB was microinjected twice into the 3V, there was a delayed increase in plasma TSH manifest 24 hr after the initial injection. TSH release from pituitaries of these animals was markedly increased in the presence of NB antiserum. Thus, NB has a physiologically significant TSH release-inhibiting action at the pituitary in the hyperthyroid as well as in the euthyroid rat. We conclude that in the euthyroid animal NB acts in an autocrine fashion to suppress TSH release from the thyrotropes directly. In hypothyroidism, NB synthesis and presumably release from the pituitary is decreased, such that there is no physiologic significance to the residual NB release, although the responsiveness to the inhibitory action of the peptide is increased, possibly via upregulation of its postulated receptors on the thyrotrope. In hyperthyroidism, the concentration of NB in thyrotropes and presumably its release is increased so that it has a physiologically significant TSH release-inhibiting action.
Alteration of a protease-sensitive region of Pseudomonas exotoxin prolongs its survival in the circulation of mice.
Authors: Brinkmann U , Pai L H , FitzGerald D J , Pastan I
Published: May 2006

Pseudomonas exotoxin A (PE) is a single-chain 66-kDa polypeptide that kills eukaryotic cells by ADP-ribosylation of translational elongation factor 2. PE is composed of three major structural domains whose functions are binding of cells (I), translocation (II), and ADP-ribosylation (III). Here we describe a protease cleavage target that is located near arginine-490 on the surface of domain III. We made several different types of mutations near arginine-490. Deletion of arginine-490 or replacement of arginine-490 and -492 with serine and lysine or with two lysines resulted in protease-resistant molecules that were fully cytotoxic and had normal ADP-ribosylation activity. However, the half-life in mouse blood of the PE delta 490 mutant was 24 min whereas that of PE was 13 min. Furthermore, two PE mutants that were protease-hypersensitive, PEGlu246,247,249 and PEGlu57,246,247,249 (in which glutamate residues replace basic residues at the indicated positions), had very short half-lives. These data indicate that protease sensitivity is an important determinant in the half-life of PE in the circulation and suggest that the half-life of other proteins may be prolonged by removal of protease sites. Deletion of arginine-492 or the replacement of amino acids 486-491 with three glycines markedly diminished ADP-ribosylation activity and cytotoxicity, indicating that this region of domain III is also important for catalytic activity.
Vesicular stomatitis virus antigenic octapeptide N52-59 is anchored into the groove of the H-2Kb molecule by the side chains of three amino acids and the main-chain atoms of the amino terminus.
Authors: Shibata K , Imarai M , van Bleek G M , Joyce S , Nathenson S G
Published: May 2006

This study describes an analysis of the interaction of individual amino acid residues of the vesicular stomatitis virus (VSV) nucleocapsid antigenic octapeptide (N52-59; Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu) with the H-2Kb molecule and T-cell receptors (TCRs). Tyr-3, Tyr-5, and Leu-8 were the positions in the peptide found to be H-2Kb contact residues by analyzing single alanine-substituted peptides in a competition assay with a Kb-restricted antigenic nonapeptide of Sendai virus. Arg-1, Gly-2, Val-4, Gln-6, and Gly-7 of the peptide were identified as putative TCR contact residues by testing the peptide analogs for their capacity to sensitize targets for VSV-specific cytolytic T-lymphocyte clones. The octamer N52-59 was the optimal length of the peptide required for binding to Kb. This peptide length requirement and the finding of an irregular interspersing of major histocompatibility complex and TCR contact residues are most consistent with the conclusion that the peptide is in an extended conformation in the antigen binding groove. Furthermore, data on binding of truncated peptides show that, although the Arg-1 side chain has been assigned as a TCR contact residue, the main-chain atoms of the N-terminal amino group are most likely involved in interacting with the major histocompatibility complex molecule. A panel of H-2Kb point mutants was constructed to explore the effect of altered amino acid residues on the binding of N52-59. Mutants with amino acid substitutions along the floor of the groove all bound the VSV peptide but modulated its interaction with Kb, apparently causing subtle changes in the spatial arrangement of some specific TCR contact residues in the peptide.
Reaction of cyanide with cytochrome ba3 from Thermus thermophilus: spectroscopic characterization of the Fe(II)a3-CN.Cu(II)B-CN complex suggests four 14N atoms are coordinated to CuB.
Authors: Surerus K K , Oertling W A , Fan C , Gurbiel R J , Einarsdóttir O , Antholine W E , Dyer R B , Hoffman B M , Woodruff W H , Fee J A
Published: May 2006

Cytochrome ba3 from Thermus thermophilus reacts slowly with excess HCN at pH 7.4 to create a form of the enzyme in which CuA, cytochrome b, and CuB remain oxidized, while cytochrome a3 is reduced by one electron, presumably with the formation of cyanogen. We have examined this form of the enzyme by UV-visible, resonance Raman, EPR, and electron nuclear double resonance spectroscopies in conjunction with permutations of 13C- and 15N-labeled cyanide. The results support a model in which one CN- binds through the carbon atom to ferrous a3, supporting a low-spin (S = 0) configuration on the Fe; bridging by this cyanide to the CuB is weak or absent. Four 14N atoms, presumably donated by histidine residues of the protein, provide a strong equatorial ligand field about CuB; a second CN- is coordinated through the carbon atom to CuB in an axial position.
Dynamics of fever and serum levels of tumor necrosis factor are closely associated during clinical paroxysms in Plasmodium vivax malaria.
Authors: Karunaweera N D , Grau G E , Gamage P , Carter R , Mendis K N
Published: May 2006

Paroxysms are sharp episodes of high fever accompanied by chills and rigors that occur periodically, once in every 48 hr in Plasmodium vivax infections. We have measured the changing levels of serum tumor necrosis factor (TNF) during paroxysms in non-immune patients infected with P. vivax malaria. The changes in TNF levels closely paralleled the rise and fall in temperature during the paroxysms but tended to precede them by 30-60 min. These observations suggest that the rise and fall in temperature during P. vivax paroxysm may be directly related to the periodic changes in TNF levels induced during these infections. The peak TNF levels reached during P. vivax infections were much higher than even those which have been recorded during severe and fatal P. falciparum infections in which TNF has been postulated to contribute to the severe manifestations of this disease.
Differential development of progenitor activity for three B-cell lineages.
Authors: Kantor A B , Stall A M , Adams S , Herzenberg L A , Herzenberg L A
Published: May 2006

Cell-transfer studies presented here distinguish three murine B cell lineages: conventional B cells, which develop late and are continually replenished from progenitors in adult bone marrow; Ly-1 B cells (B-1a), which develop early and maintain their numbers by self-replenishment; and Ly-1B "sister" (B-1b) cells, which share many of the properties of Ly-1 B cells, including self-replenishment and feedback regulation of development but can also readily develop from progenitors in adult bone marrow. The sequential emergence of these lineages, the time at which their progenitors function during ontogeny, and the distinctions among their repertoires and functions suggest that evolution has created a layered immune system in which the immune response potential of each successive lineage is adapted to its particular niche.
Assay of excised oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column.
Authors: Park E M , Shigenaga M K , Degan P , Korn T S , Kitzler J W , Wehr C M , Kolachana P , Ames B N
Published: May 2006

An immunoaffinity column is described that facilitates the analysis of oxidative damage products of DNA and RNA in urine, blood plasma, and medium isolated from cultures of Escherichia coli. In intact animals, lesions (adducts) excised from DNA are transported from the cell through the circulation and excreted in urine. In bacteria, DNA adducts are excreted directly into the medium. In either case, the adducts can be assayed as a measure of oxidative damage to DNA. A monoclonal antibody that recognizes 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo8dG;8-hydroxy-2'-deoxyguanosine), a bio-marker of oxidative damage to DNA, has been isolated, and its substrate binding properties have been characterized. The relative binding affinities of this monoclonal antibody for oxo8dG, unmodified nucleosides, or derivatives of Gua made it suitable for the preparation of immunoaffinity columns that greatly facilitate the isolation of oxo8dG, 8-oxo-7,8-dihydroguanine, and 8-oxo-7,8-dihydroguanosine from various biological fluids. Quantitative analysis of these adducts in urine of rats fed a nucleic acid-free diet and in the medium from cultures of E. coli suggests that oxo8-7,8-dihydroguanine is the principal repair product from oxo8-dG in DNA of both eukaryotes and prokaryotes. The results support our previous estimate of about 10(5) oxidative lesions to DNA being formed and excised in an average rat cell per day.
Two-subunit structure of the human thyrotropin receptor.
Authors: Loosfelt H , Pichon C , Jolivet A , Misrahi M , Caillou B , Jamous M , Vannier B , Milgrom E
Published: May 2006

The extracellular and intracellular domains of the human thyrotropin receptor were expressed in Escherichia coli and the proteins were used to produce monoclonal anti-receptor antibodies. Immunoblot studies and immunoaffinity purification showed that the receptor is composed of two subunits linked by disulfide bridges and probably derived by proteolytic cleavage of a single 90-kDa precursor. The extracellular alpha subunit (hormone binding) had an apparent molecular mass of 53 kDa (35 kDa after deglycosylation with N-glycosidase F). The membrane-spanning beta subunit seemed heterogeneous and had an apparent molecular mass of 33-42 kDa. Human thyroid membranes contained a 2.5- to 3-fold excess of beta subunits over alpha subunits. Immunocytochemistry showed the presence of both subunits in all the follicular thyroid cells, and both subunits were restricted to the basolateral region of the cell membrane.
A domain substitution procedure and its use to analyze voltage dependence of homotypic gap junctions formed by connexins 26 and 32.
Authors: Rubin J B , Verselis V K , Bennett M V , Bargiello T A
Published: May 2006

We have developed a procedure for the replacement of defined domains with specified domains from other proteins that we used to examine the molecular basis for the differences in voltage-dependent gating between connexins 26 (Cx26) and 32 (Cx32). This technique does not depend on sequence homology between the domains to be exchanged or the presence of restriction endonuclease sites. Rather, it makes use of a PCR strategy to create an adhesive "band-aid" that directs the annealing of the amplified sequence to the correct location in the recipient clone. With this technique we created a series of chimeras involving the replacement of topologically defined protein domains of Cx32 with the corresponding sequences of Cx26. We focused on domains that are predicted to line the gap junction channel as we expect that a component of the voltage-sensing mechanism resides there. Differences between Cx26 and Cx32 in the sequences of their first and second extracellular loops, the cytoplasmic loop, and the third transmembrane domain did not account for the difference in their calculated gating charges. Shifts along the voltage axis in the steady-state conductance-voltage relations of the chimeric connexins were produced by replacement of the first extracellular loop or the cytoplasmic loop and the amino-terminal half of the third transmembrane domain. These data suggest that the voltage-sensing mechanism arises from the interaction of domains lining the aqueous channel and domains deeper in the channel wall.
Cannabinoids inhibit N-type calcium channels in neuroblastoma-glioma cells.
Authors: Mackie K , Hille B
Published: May 2006

The psychoactive properties of Cannabis sativa and its major biologically active constituent, delta 9-tetrahydrocannabinol, have been known for years. The recent identification and cloning of a specific cannabinoid receptor suggest that cannabinoids mimic endogenous compounds affecting neural signals for mood, memory, movement, and pain. Using whole-cell voltage clamp and the cannabinomimetic aminoalkylindole WIN 55,212-2, we have found that cannabinoid receptor activation reduces the amplitude of voltage-gated calcium currents in the neuroblastoma-glioma cell line NG108-15. The inhibition is potent, being half-maximal at less than 10 nM, and reversible. The inactive enantiomer, WIN 55,212-3, does not reduce calcium currents even at 1 microM. Of the several types of calcium currents in NG108-15 cells, cannabinoids predominantly inhibit an omega-conotoxin-sensitive, high-voltage-activated calcium current. Inhibition was blocked by incubation with pertussis toxin but was not altered by prior treatment with hydrolysis-resistant cAMP analogues together with a phosphodiesterase inhibitor, suggesting that the transduction pathway between the cannabinoid receptor and calcium channel involves a pertussis toxin-sensitive GTP-binding protein and is independent of cAMP metabolism. However, the development of inhibition is considerably slower than a pharmacologically similar pathway used by an alpha 2-adrenergic receptor in these cells. Our results suggest that inhibition of N-type calcium channels, which could decrease excitability and neurotransmitter release, may underlie some of the psychoactive effects of cannabinoids.
Placental-specific expression from the mouse placental lactogen II gene promoter.
Authors: Shida M M , Jackson-Grusby L L , Ross S R , Linzer D I
Published: May 2006

The gene for mouse placental lactogen II (mPL-II) has been isolated and characterized. This gene contains five exons, with a transcription start site 59 nucleotides upstream of the translation initiation ATG. Introduction of a DNA construct containing 2.7 kilobases of sequence upstream of the mPL-II transcription initiation site directed the synthesis of a linked coding region for the simian virus 40 large and small tumor antigens in placental trophoblast giant cells of transgenic mice. The pattern of simian virus 40 transgene expression in the placenta was indistinguishable from that of the endogenous mPL-II gene. In contrast, the first 569 base pairs upstream of the transcription start site proved insufficient to direct placental expression. Thus, one or more elements required for placental trophoblast giant cell expression have been localized to a region between -2700 and -569 of the mPL-II gene.
Glucocorticoid receptor DNA-binding specificity is increased by the organization of DNA in nucleosomes.
Authors: Perlmann T
Published: May 2006

A DNA fragment containing glucocorticoid receptor binding sites in the mouse mammary tumor virus promoter was reconstituted in vitro with histones to form nucleosome cores, which become positioned on the DNA fragment in a sequence-specific manner. Glucocorticoid receptor binding to specific DNA sequences was analyzed by quantitative DNase I footprinting. The receptor interacted with surprisingly high affinity with one of the binding sites in the reconstituted promoter, although it was reduced by a factor of approximately 2 compared with the same site in protein-free DNA. By contrast, the affinity for random genomic nucleosomal sites was drastically reduced compared with histone-free DNA. Thus, reconstituting the promoter in vitro resulted in a 60- to 70-fold increase in binding specificity. Such an increase in selective binding may help to explain the ability of glucocorticoid receptor to effectively locate its target sites in chromatin.
LH-2: a LIM/homeodomain gene expressed in developing lymphocytes and neural cells.
Authors: Xu Y , Baldassare M , Fisher P , Rathbun G , Oltz E M , Yancopoulos G D , Jessell T M , Alt F W
Published: May 2006

A screen for early markers of B-lymphocyte differentiation has identified a homeobox gene, denoted LH-2, that has a pattern of expression distinct from that of other related genes. The LH-2 cDNA sequence encodes a polypeptide of 426 amino acids that contains a homeodomain and two repeats of a cysteine-rich domain referred to as a LIM domain. The homeodomain of the LH-2 protein is related to that of other LIM/homeodomain proteins, most strikingly with that of the Drosophila apterous protein. Expression of LH-2 was found in B- and T-lymphoid cell lines. Expression in B-cell lines was highest in lines that represent early stages of differentiation, whereas in T-cell lines there was no clear correlation with the stage of differentiation. In embryonic and adult tissues, the highest level of LH-2 expression was found in discrete regions of the developing central nervous system, primarily in diencephalic and telencephalic structures, and in a subset of lymphoid tissues. The expression pattern and structural characteristics of the LH-2 gene suggest that it encodes a transcriptional regulatory protein involved in the control of cell differentiation in developing lymphoid and neural cell types.