bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular basis of aromatase deficiency in an adult female with sexual infantilism and polycystic ovaries.
Authors: Ito Y , Fisher C R , Conte F A , Grumbach M M , Simpson E R
Published: May 2006

We identified two mutations in the CYP19 gene responsible for aromatase deficiency in an 18-year-old 46,XX female with ambiguous external genitalia at birth, primary amenorrhea and sexual infantilism, and polycystic ovaries. The coding exons, namely exons II-X, of the CYP19 gene were amplified by PCR from genomic DNA and sequenced directly. Direct sequencing of the amplified DNA from the patient revealed two single-base changes, at bp 1303 (C-->T) and bp 1310 (G-->A) in exon X, which were newly found missense mutations and resulted in codon changes of R435C and C437Y, respectively. Subcloning followed by sequencing confirmed that the patient is a compound heterozygote. The results of restriction fragment length polymorphism analysis and direct sequencing of the amplified exon X DNA from the patient's mother indicate maternal inheritance of the R435C mutation. Transient expression experiments showed that the R435C mutant protein had approximately 1.1% of the activity of the wild type, whereas C437Y was totally inactive. Cysteine-437 is the conserved cysteine in the heme-binding region believed to serve as the fifth coordinating ligand of the heme iron. To our knowledge, this patient is the first adult to have described the cardinal features of a syndrome of aromatase deficiency. Recognition that such defects exist will lead to a better understanding of the role of this enzyme in human development and disease.
Pulse mass measles vaccination across age cohorts.
Authors: Agur Z , Cojocaru L , Mazor G , Anderson R M , Danon Y L
Published: May 2006

Although vaccines against measles have been routinely applied over a quarter of a century, measles is still persistent in Israel, with major epidemics roughly every 5 years. Recent serological analyses have shown that only 85% of Israelis aged 18 years have anti-measles IgG antibodies. Considering the high transmissibility of the virus and the high level of herd immunity required for disease eradication, the Israeli vaccination policy against measles is now being reevaluated. Motivated by theoretical studies of populations in perturbed environments, we examined the possibility of replacing the conventional cohort vaccination strategy by a pulse strategy--i.e., periodic vaccination of several age cohorts at the same time. Numerical studies of a deterministic age-structured model suggest that vaccination, which renders immunity to no more than 85% of the susceptible children aged 1-7 years, once every 5 years will suffice to prevent epidemics in Israel, where infection rate is highest amongst schoolchildren. The model suggests that by using such a strategy the density of susceptible individuals is always kept below the threshold above which recurrent epidemics will be maintained. Analysis of simpler, non-age-structured, models serves to clarify the basic properties of the proposed strategy. Our theoretical results indicate that the advantages and disadvantages of a pulse strategy should be seriously examined in Israel and in countries with similar patterns of measles virus transmission.
Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drug-resistance mutations in malaria.
Authors: Donald R G , Roos D S
Published: May 2006

To facilitate genetic analysis of the protozoan parasite Toxoplasma gondii, sequences derived from the parasite's fused dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene have been used to produce vectors suitable for stable molecular transformation. Mutations introduced into the DHFR coding region by analogy with pyrimethamine-resistant malaria confer drug resistance to Toxoplasma, providing useful information on the structure of fused DHFR-TS enzymes and a powerful selectable marker for molecular genetic studies. Depending on the particular drug-resistance allele employed and the conditions of selection, stable resistance can be generated either by single copy nonhomologous insertion into chromosomal DNA or by massively amplified transgenes. Frequencies of integration are independent of selection, and transgenes are stable without continued selection. Cointegration of a reporter gene adjacent to the selectable marker (under the control of an independent promoter) shows no loss of the cointegrated sequences over many parasite generations. By bringing the full power of molecular genetic analysis to bear on Toxoplasma, these studies should greatly facilitate the development of a model genetic system for Apicomplexan parasites.
Effects of central cholinergic blockade on striatal dopamine release measured with positron emission tomography in normal human subjects.
Authors: Dewey S L , Smith G S , Logan J , Brodie J D , Simkowitz P , MacGregor R R , Fowler J S , Volkow N D , Wolf A P
Published: May 2006

Previously we demonstrated that positron emission tomography (PET) can be used to measure changes in the concentrations of synaptic dopamine and acetylcholine. Whether induced directly or indirectly through interactions with other neurotransmitters, these studies support the use of PET for investigating the functional responsiveness of a specific neurotransmitter to a pharmacologic challenge. In an extension of these findings to the human brain, PET studies designed to measure the responsiveness of striatal dopamine release to central cholinergic blockade were conducted in normal male volunteers using high-resolution PET and [11C]raclopride, a D2-dopamine receptor antagonist. [11C]Raclopride scans were performed prior to and 30 min after systemic administration of the potent muscarinic cholinergic antagonist, scopolamine (0.007 mg/kg). After scopolamine administration, [11C]raclopride binding decreased in the striatum (specific binding) but not in the cerebellum (nonspecific binding) resulting in a significant decrease, exceeding the test/retest variability of this ligand (5%), in the ratio of the distribution volumes of the striatum to the cerebellum (17%). Furthermore, scopolamine administration did not alter the systemic rate of [11C]raclopride metabolism or the metabolite-corrected plasma input function. These results are consistent not only with the known inhibitory influence that acetylcholine exerts on striatal dopamine release but also with our initial 18F-labeled N-methylspiroperidol and benztropine studies. Thus these data support the use of PET for measuring the functional responsiveness of an endogenous neurotransmitter to an indirect pharmacologic challenge in the living human brain.
Overexpression, purification, and characterization of SHPTP1, a Src homology 2-containing protein-tyrosine-phosphatase.
Authors: Pei D , Neel B G , Walsh C T
Published: May 2006

A protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) containing two Src homology 2 (SH2) domains, SHPTP1, was previously identified in hematopoietic and epithelial cells. By placing the coding sequence of the PTPase behind a bacteriophage T7 promoter, we have overexpressed both the full-length enzyme and a truncated PTPase domain in Escherichia coli. In each case, the soluble enzyme was expressed at levels of 3-4% of total soluble E. coli protein. The recombinant proteins had molecular weights of 63,000 and 45,000 for the full-length protein and the truncated PTPase domain, respectively, as determined by SDS/PAGE. The recombinant enzymes dephosphorylated p-nitrophenyl phosphate, phosphotyrosine, and phosphotyrosyl peptides but not phosphoserine, phosphothreonine, or phosphoseryl peptides. The enzymes showed a strong dependence on pH and ionic strength for their activity, with pH optima of 5.5 and 6.3 for the full-length enzyme and the catalytic domain, respectively, and an optimal NaCl concentration of 250-300 mM. The recombinant PTPases had high Km values for p-nitrophenyl phosphate and exhibited non-Michaelis-Menten kinetics for phosphotyrosyl peptides.
Copper and iron are mobilized following myocardial ischemia: possible predictive criteria for tissue injury.
Authors: Chevion M , Jiang Y , Har-El R , Berenshtein E , Uretzky G , Kitrossky N
Published: May 2006

Direct evidence for substantial mobilization of copper in the coronary flow immediately following prolonged, but not short, cardiac ischemia is presented. In the first coronary flow fraction (CFF) of reperfusion (0.15 ml), after 35 min of ischemia, the level of copper (as well as of iron) was 8- to 9-fold higher than the preischemic value. The levels in subsequent CFFs decreased and reached the preischemic value, indicating that both metals appear in a burst at the resumption of coronary flow. When the first CFF was used in a reaction mixture containing ascorbate and salicylate, the latter underwent chemical hydroxylation and was converted to its dihydroxybenzoate derivatives. Likewise, this CFF promoted the ascorbate-driven DNA degradation. Subsequent 150 CFFs were serially collected and demonstrated low activities. Following 18 min of ischemia, the copper level in the first CFF of reperfusion was only 15% over the preischemic value. In contrast, the mobilization of iron into coronary flow was significant but markedly lower than after 35 min. The levels of copper and the redox activity of the first CFF correlated well with the degree of loss of cardiac function, after 18 and 35 min of ischemia, respectively. After 18 min of ischemia, cardiac function was about 50% and the damage is considered reversible, whereas after 35 min the functional loss exceeded 80% and is considered irreversible. These results are in accord with the causative role that copper and iron can play in heart injury following ischemia, by virtue of their capacity to catalyze the production of hydroxyl radicals, and could lead to the development of new modalities for intervention in tissue injury.
Stabilization of alpha-helical structures in short peptides via end capping.
Authors: Forood B , Feliciano E J , Nambiar K P
Published: May 2006

The alpha-helix-stabilizing effect of different amino acid residues at the helical termini of short peptides in aqueous solution has been determined. Several dodecapeptides containing alanine, asparagine, aspartate, glutamine, glutamate, and serine at the amino terminus and arginine, lysine, and alanine at the carboxyl terminus were synthesized, and the alpha-helical content of each peptide was measured by using circular dichroism spectroscopy. The trend in alpha-helix-inducing ability of these amino acids was found to be as follows: aspartate > asparagine > serine > glutamate > glutamine > alanine at the amino terminus and arginine > lysine > alanine at the carboxyl terminus. Our results agree with the Presta and Rose hypothesis [Presta, L. G. & Rose, G. D. (1988) Science 240, 1632-1641] on the role of end capping in helix stabilization.
Pre-mRNA splicing within an assembled yeast spliceosome requires an RNA-dependent ATPase and ATP hydrolysis.
Authors: Kim S H , Lin R J
Published: May 2006

Unlike autocatalyzed self-splicing of group I or group II introns, the removal of pre-mRNA introns in vitro occurs in the spliceosome. The spliceosome is a multicomponent complex composed of pre-mRNA, small nuclear ribonucleoprotein particles, and protein factors. ATP is required for the assembly of the spliceosome and both transesterification reactions. An RNA-dependent ATPase, the product of the yeast PRP2 gene, has been shown to be involved in the first transesterification of pre-mRNA splicing but not in spliceosome assembly. By using ATP analogs, we show that hydrolysis of ATP, mediated through a PRP2-dependent step, is required for the first catalytic event of pre-mRNA splicing. Furthermore, by using a two-step purification procedure, we have isolated a PRP2-containing spliceosome within which the first transesterification readily occurs after the addition of ATP. No additional macromolecules were required. Our results suggest that PRP2 binds to the spliceosome, interacting with an unidentified RNA species in the spliceosome, hydrolyzing ATP and allowing splicing to proceed. We postulate that PRP2 may function to induce a conformational change within the spliceosome. Alternatively, PRP2 may be involved in a proofreading step prior to splicing.
Primary structure of two-chain botrocetin, a von Willebrand factor modulator purified from the venom of Bothrops jararaca.
Authors: Usami Y , Fujimura Y , Suzuki M , Ozeki Y , Nishio K , Fukui H , Titani K
Published: May 2006

The complete amino acid sequence and location of the disulfide bonds of two-chain botrocetin, which promotes platelet agglutination in the presence of von Willebrand factor, from venom of the snake Bothrops jararaca are presented. Sequences of the alpha and beta subunits were determined by analysis of peptides generated by digestion of the S-pyridylethylated protein with Achromobacter protease I or alpha-chymotrypsin and by chemical cleavage with cyanogen bromide or 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Two-chain botrocetin is a heterodimer composed of the alpha subunit (consisting of 133 amino acid residues) and the beta subunit (consisting of 125 amino acid residues) held together by a disulfide bond. Seven disulfide bonds link half-cystine residues 2 to 13, 30 to 128, and 103 to 120 of the alpha subunit; 2 to 13, 30 to 121, and 98 to 113 of the beta subunit; and 80 of the alpha subunit to 75 of the beta subunit. In terms of amino acid sequence and disulfide bond location, two-chain botrocetin is homologous to echinoidin (a sea urchin lectin) and other C-type (Ca(2+)-dependent) lectins.
Screening for in vivo protein-protein interactions.
Authors: Germino F J , Wang Z X , Weissman S M
Published: May 2006

We describe an in vivo approach for the isolation of proteins interacting with a protein of interest. The protein of interest is "tagged" with a portion of the biotin carboxylase carrier protein (BCCP), encoded on a specially constructed plasmid, so that it becomes biotinylated in vivo. The "query" proteins (e.g., those in a cDNA library) are tagged by fusing them to the 3' end of the lacZ gene on a lambda vector in such a way that the beta-galactosidase activity is not disrupted. These phage are transfected into cells containing the plasmid encoding the BCCP-tagged protein. The infection lyses the cells and exposes the protein complexes. The BCCP-tagged protein and any associated protein(s) are "captured" by using avidin, streptavidin, or anti-biotin antibody-coated filters. The detection of bound protein is accomplished by directly assaying for beta-galactosidase activity on the filters. Positive plaques can be plaque-purified for DNA sequencing. We have tested this approach by using c-Fos and c-Jun as our model system. We show that avidin, streptavidin, or polyclonal anti-biotin (but not a monoclonal anti-biotin) antibody is capable of specifically capturing in vivo biotinylated beta-galactosidase and c-Jun and that this capture is dependent upon the presence of both avidin and the BCCP moiety. Further, complexes containing c-Jun and c-Fos can also be isolated in this manner, and the isolation of this complex is dependent on the presence of c-Fos, c-Jun, avidin, and the BCCP moiety. We discuss the possible uses and limitations of this technique for isolating proteins that interact with a known protein.
Modeling the complexation of substituted benzenes by a cyclophane host in water.
Authors: Jorgensen W L , Nguyen T B
Published: May 2006

Monte Carlo statistical mechanics simulations have been used to study the complexation of disubstituted benzenes by Diederich's octamethoxy tetraoxaparacyclophane host. Relative free energies of binding were obtained in water at 25 degrees C for benzene, p-xylene, p-cresol, p-dicyanobenzene, and hydroquinone from statistical perturbation theory. The computed results agree well with experimental data, including the binding affinity of benzene, which was determined after the calculations were completed. The computed structures for the complexes reveal details that are important for understanding the order of binding affinities. It is found that hydroquinone protrudes from one side of the complex and participates in hydrogen bonds between one hydroxyl group and two water molecules and in an intracomplex hydrogen bond between the other hydroxyl group and ether oxygens. The calculations also show a clear preference for binding p-cresol with the hydroxyl group hydrated rather than inside the host's cavity.
Autoantibodies to the GLUT-2 glucose transporter of beta cells in insulin-dependent diabetes mellitus of recent onset.
Authors: Inman L R , McAllister C T , Chen L , Hughes S , Newgard C B , Kettman J R , Unger R H , Johnson J H
Published: May 2006

Purified immunoglobulin G (IgG) from the serum of patients with insulin-dependent diabetes mellitus (IDDM) of recent onset inhibits high-Km uptake of 3-O-methyl-beta-D-glucose by rat pancreatic islets. To determine if the inhibition is the result of antibodies against GLUT-2, the high-Km glucose transporter of beta cells, we incubated IDDM sera with rat islet cells and with AtT-20ins cells transfected to express GLUT-2. IDDM sera inhibited glucose uptake in islet cells and in GLUT-2-expressing AtT-20ins cells but not in AtT-20ins cells transfected to express the low-Km isoform, GLUT-1. In 24 of 30 (77%) patients with newly diagnosed IDDM, IgG binding as measured by immunofluorescence and flow cytometry of the cells transfected to express GLUT-2 was > 2 standard deviations from the mean of the nondiabetic population; 29 of 31 (96%) of nondiabetic children were negative (P < 0.0001). Increased IgG binding could be removed by absorption with GLUT-2-expressing cells but not with GLUT-1-expressing cells. We conclude that most patients with IDDM of recent onset have autoantibodies to GLUT-2.
Assembly of avian mixed-species flocks in Amazonia.
Authors: Graves G R , Gotelli N J
Published: May 2006

Diamond's "assembly rules" model posits that competitive interactions among species govern the composition of avifaunas. Although originally applied to islands in archipelagoes, this controversial set of hypotheses is difficult to test because islands differ in habitat and resource availability, colonization history, and stochastic effects. Permanent mixed-species flocks of Amazonian birds are a model system for testing the assembly rules hypothesis because flocks occur in relatively homogeneous tracts of rain forest and because resident species are potentially interactive from minute to minute. To analyze cooccurrence patterns of species in flocks, we used null models that incorporate realistic autecological colonization parameters. Potentially competing pairs of congeneric species with similar ecologies cooccur in flocks less often than expected by chance, resulting in perfect checkerboard distributions. Interactions among more distantly related species, however, appear to have little effect on the assembly of mixed-species flocks. Checkerboard distributions enhance local species diversity within habitats by generating different combinations of species in different flocks. This process may have contributed to the immense species richness of the Amazonian avifauna.
Endogenous peptides bound to HLA-A3 possess a specific combination of anchor residues that permit identification of potential antigenic peptides.
Authors: DiBrino M , Parker K C , Shiloach J , Knierman M , Lukszo J , Turner R V , Biddison W E , Coligan J E
Published: May 2006

A motif specific to peptides that bind to the human class I major histocompatibility complex molecule HLA-A3 was identified by sequence analysis of HPLC fractions containing endogenous peptides. Twenty-six different sequences were obtained, 19 of which were nonamers. The majority of these endogenous peptide sequences contained Leu at position (P)2, while most sequences contained Tyr or Lys at P9. In addition, Phe was shared by 16 sequences at P3. Synthetic peptides corresponding to endogenous peptide sequences were shown to bind to HLA-A3. The importance of Leu at P2 and Tyr or Lys at P9 ("anchor" residues) for peptide binding to HLA-A3 was demonstrated by the following results: (i) peptides GLFGGGGGY, GLFGGGGGK, and GLGGGGFGY, but not GLFGGGGGV, specifically bound to HLA-A3 and (ii) six nonapeptides from within the influenza A nucleoprotein, matrix, and polymerase proteins, selected for synthesis based upon their possession of P2 and P9 anchor residues, were shown to bind HLA-A3. In contrast, none of a set of eight peptides that bound to HLA-A2, or six that bound to HLA-B27, bound detectably to HLA-A3. These findings provide a rationale for the design and selection of peptides that can be recognized by HLA-A3-restricted T cells.
Human immunodeficiency virus coat protein gp120 inhibits the beta-adrenergic regulation of astroglial and microglial functions.
Authors: Levi G , Patrizio M , Bernardo A , Petrucci T C , Agresti C
Published: May 2006

The goal of our study was to assess whether the human immunodeficiency virus (HIV) coat protein gp120 induces functional alterations in astrocytes and microglia, known for their reactivity and involvement in most types of brain pathology. We hypothesized that gp120-induced anomalies in glial functions, if present, might be mediated by changes in the levels of intracellular messengers important for signal transduction, such as cAMP. Acute (10 min) exposure of cultured rat cortical astrocytes or microglia to 100 pM gp120 caused only a modest (50-60%), though statistically significant, elevation in cAMP levels, which was antagonized by the beta-adrenergic receptor antagonist propranolol. More importantly, the protein substantially depressed [by 30% (astrocytes) and 50% (microglia)] the large increase in cAMP induced by the beta-adrenergic agonist isoproterenol (10 nM), without affecting that induced by direct adenylate cyclase stimulation by forskolin. Qualitatively similar results were obtained using a glial fibrillary acidic protein (GFAP)-positive human glioma cell line. The depression of the beta-adrenergic response had functional consequences in both astrocytes and microglia. In astrocytes we studied the phosphorylation of the two major cytoskeletal proteins, vimentin and GFAP, which is normally stimulated by isoproterenol, and found that gp120 partially (40-50%) prevented such stimulation. In microglial cells, which are the major producers of inflammatory cytokines within the brain, gp120 partially antagonized the negative beta-adrenergic modulation of lipopolysaccharide (10 ng/ml)-induced production of tumor necrosis factor alpha. Our results suggest that, by interfering with the beta-adrenergic regulation of astrocytes and microglia, gp120 may alter astroglial "reactivity" and upset the delicate cytokine network responsible for the defense against viral and opportunistic infections.
Three unusual modifications, a D-arabinosyl, an N-methyl, and a carbamoyl group, are present on the Nod factors of Azorhizobium caulinodans strain ORS571.
Authors: Mergaert P , Van Montagu M , Promé J C , Holsters M
Published: May 2006

Azorhizobium caulinodans strain ORS571 is a symbiont of the tropical legume Sesbania rostrata. Upon nod gene induction with naringenin, strain ORS571 secretes into the culture medium Nod factors that morphologically change the host plant--in particular, deformed root hairs (Hai/Had) and meristematic foci are formed at the basis of lateral roots. The latter infrequently develop further into nodule-like structures. The azorhizobial Nod factors are chitin tetramers or pentamers, N-acylated at the nonreducing-end glucosamine with either vaccenic acid (C18:1) or stearic acid (C18:0). They, thus, resemble the previously described Nod factors from (brady)rhizobia. The backbone lipooligosaccharide is substituted with unusual modifications, presumably involved in host-specificity determination. There is a D-arabinose branch on the reducing end and an N-methyl and O-carbamoyl substitution on the nonreducing end of the oligosaccharide chain. The previously identified nod gene nolK may be involved in the synthesis of a D-arabinose derivative. The nodS gene product is probably responsible for the N-methylation of Nod factors.
Endoplasmic reticulum-through-Golgi transport assay based on O-glycosylation of native glycophorin in permeabilized erythroleukemia cells: role for Gi3.
Authors: Wilson B S , Palade G E , Farquhar M G
Published: May 2006

An assay for endoplasmic reticulum (ER)-through-Golgi transport has been developed in streptolysin O-permeabilized murine erythroleukemia (MEL) cells. The reporter proteins are metabolically labeled native murine glycophorins, which display a distinctive shift in electrophoretic mobility after acquisition of O-linked oligosaccharides. The O-linked sugars are acquired at a site distal to a brefeldin A block, presumably in a cis Golgi compartment, and sialylation occurs in middle and/or trans Golgi compartments. In permeabilized cells supplemented with cytosolic proteins and an ATP-generating system, 20-50% of the radiolabeled precursor glycophorins can be converted to the mature, sialylated form. This maturation process is ATP- and cytosol-dependent and is blocked by guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]). Electron microscopy of permeabilized MEL cells shows retention of ER elements, stacked Golgi cisternae, free polysomes, and other subcellular components. In the presence of GTP[gamma S], dilated vesicles accumulate around the Golgi stacks. Antisera to the carboxyl terminus of the Golgi resident alpha subunit of Gi3 inhibit maturation of glycophorin. To our knowledge, a transport assay utilizing O-glycosylation of an endogenous protein as a monitor of ER-through-Golgi traffic in permeabilized cells has not been reported previously. Furthermore, the data provide evidence for heterotrimeric GTP-binding protein involvement in Golgi function.
Kindling produces long-lasting and selective changes in gene expression of hippocampal neurons.
Authors: Perlin J B , Gerwin C M , Panchision D M , Vick R S , Jakoi E R , DeLorenzo R J
Published: May 2006

To test the hypothesis that repeated subconvulsive stimulations required to induce kindling can permanently alter gene expression of hippocampal neurons, we used Northern and in situ hybridization analyses to measure steady-state mRNA levels encoding several phenotypic proteins. mRNA encoding a membrane-bound protein, ligatin, was significantly reduced in kindled brains relative to naive and sham control animals. This change in gene expression persisted for over 4 months after kindling, was associated with a decrease in ligatin protein expression, was not produced by single or multiple seizures that did not induce the kindling phenomena, and was blocked by MK801. These results provide direct evidence that kindling can cause persistent changes in the expression of specific genes in hippocampal neurons and suggest that N-methyl-D-aspartate receptor-activated changes in gene expression may be a basic molecular mechanism underlying some of the long-lasting plasticity changes seen in kindling or models of long-term memory.
Ligand-specific activation of HER4/p180erbB4, a fourth member of the epidermal growth factor receptor family.
Authors: Plowman G D , Culouscou J M , Whitney G S , Green J M , Carlton G W , Foy L , Neubauer M G , Shoyab M
Published: May 2006

This report describes the isolation and recombinant expression of a cDNA clone encoding HER4, the fourth member of the human epidermal growth factor receptor (EGFR) family. The HER4/erbB4 gene encodes a 180-kDa transmembrane tyrosine kinase (HER4/p180erbB4) whose extracellular domain is most similar to the orphan receptor HER3/p160erbB3, whereas its cytoplasmic kinase domain exhibits 79% and 77% identity with EGFR and HER2/p185erbB2, respectively. HER4 is most predominantly expressed in several breast carcinoma cell lines, and in normal skeletal muscle, heart, pituitary, brain, and cerebellum. In addition, we describe the partial purification of a heparin-binding HER4-stimulatory factor from HepG2 cells. This protein was found to specifically stimulate the intrinsic tyrosine kinase activity of HER4/p180erbB4 while having no direct effect on the phosphorylation of EGFR, HER2, or HER3. Furthermore, this heparin-binding protein induces phenotypic differentiation, and tyrosine phosphorylation, of a human mammary tumor cell line that overexpresses both HER4 and HER2. These findings suggest that this ligand-receptor interaction may play a role in the growth and differentiation of some normal and transformed cells.
The putative phosphoinositide-specific phospholipase C gene, PLC1, of the yeast Saccharomyces cerevisiae is important for cell growth.
Authors: Yoko-o T , Matsui Y , Yagisawa H , Nojima H , Uno I , Toh-e A
Published: May 2006

Using the polymerase chain reaction technique, we have isolated a gene that encodes a putative phosphoinositide-specific phospholipase C (PLC) in the yeast Saccharomyces cerevisiae. The nucleotide sequence indicates that the gene encodes a polypeptide of 869 amino acid residues with a calculated molecular mass of 101 kDa. This polypeptide has both the X and Y regions conserved among mammalian PLC-beta, -gamma, and -delta, and the structure is most similar to that of mammalian PLC-delta. This putative yeast PLC gene has been designated PLC1. Disruption of PLC1 results in slow growth or lethality for cells, depending on their genetic background and the medium, indicating that PLC1 is important for cell growth. Expression of rat PLC-delta 1 cDNA suppressed the growth defect of plc1 disruptants, strongly suggesting that PLC1 encodes PLC.
Glucokinase mutations associated with non-insulin-dependent (type 2) diabetes mellitus have decreased enzymatic activity: implications for structure/function relationships.
Authors: Gidh-Jain M , Takeda J , Xu L Z , Lange A J , Vionnet N , Stoffel M , Froguel P , Velho G , Sun F , Cohen D
Published: May 2006

The glycolytic enzyme glucokinase plays an important role in the regulation of insulin secretion and recent studies have shown that mutations in the human glucokinase gene are a common cause of an autosomal dominant form of non-insulin-dependent (type 2) diabetes mellitus (NIDDM) that has an onset often during childhood. The majority of the mutations that have been identified are missense mutations that result in the synthesis of a glucokinase molecule with an altered amino acid sequence. To characterize the effect of these mutations on the catalytic properties of human beta-cell glucokinase, we have expressed native and mutant forms of this protein in Escherichia coli. All of the missense mutations show changes in enzyme activity including a decrease in Vmax and/or increase in Km for glucose. Using a model for the three-dimensional structure of human glucokinase based on the crystal structure of the related enzyme yeast hexokinase B, the mutations map primarily to two regions of the protein. One group of mutations is located in the active site cleft separating the two domains of the enzyme as well as in surface loops leading into this cleft. These mutations usually result in large reductions in enzyme activity. The second group of mutations is located far from the active site in a region that is predicted to undergo a substrate-induced conformational change that results in closure of the active site cleft. These mutations show a small approximately 2-fold reduction in Vmax and a 5- to 10-fold increase in Km for glucose. The characterization of mutations in glucokinase that are associated with a distinct and readily recognizable form of NIDDM has led to the identification of key amino acids involved in glucokinase catalysis and localized functionally important regions of the glucokinase molecule.
Cooperativity in protein-folding kinetics.
Authors: Dill K A , Fiebig K M , Chan H S
Published: May 2006

How does a protein find its native state without a globally exhaustive search? We propose the "HZ" (hydrophobic zipper) hypothesis: hydrophobic contacts act as constraints that bring other contacts into spatial proximity, which then further constrain and zip up the next contacts, etc. In contrast to helix-coil cooperativity, HZ-heteropolymer collapse cooperativity is driven by nonlocal interactions, causes sheet and irregular conformations in addition to helices, leads to secondary structures concurrently with early hydrophobic core formation, is much more sequence dependent than helix-coil processes, and involves compact intermediate states that have much secondary--but little tertiary--structure. Hydrophobic contacts in the 1992 Protein Data Bank have the type of "topological localness" predicted by the hypothesis. The HZ paths for amino acid sequences that mimic crambin and bovine pancreatic trypsin inhibitor are quickly found by computer; the best configurations thus reached have single hydrophobic cores that are within about 3 kcal/mol of the global minimum. This hypothesis shows how proteins could find globally optimal states without exhaustive search.
An in vitro adherence assay reveals that Helicobacter pylori exhibits cell lineage-specific tropism in the human gastric epithelium.
Authors: Falk P , Roth K A , Borén T , Westblom T U , Gordon J I , Normark S
Published: May 2006

Helicobacter pylori is a microaerophilic bacterium found in the stomach of asymptomatic humans as well as patients with acid peptic disease and gastric adenocarcinoma. We have developed an in situ adherence assay to examine the cell lineage-specific nature of binding of this organism and to characterize the nature of cell surface receptors that recognize its adhesin. Fluorescein isothiocyanate-labeled H. pylori strains were bound to surface mucous cells present in the pit region of human and rat gastric units but not to mucous neck, parietal, or chief cell lineages present in the glandular domains of these units. Binding was abolished by proteinase K treatment of tissue sections and by pretreatment of the bacteria with bovine submaxillary gland mucin, a rich source of fucosylated and sialylated carbohydrates. Several lines of evidence suggest that binding to surface mucous cells is not dependent upon terminal nonsubstituted alpha 2,3- and alpha 2,6-linked sialic acids in the adhesin receptor: (i) binding was not inhibited by incubating H. pylori strains with sialylated glycoconjugates such as fetuin and free sialyllactose; (ii) immunohistochemical stainings using the sialic acid-specific Sambucus nigra and Maackia amurensis lectins and the cholera toxin B subunit did not detect any sialylated glycoconjugates in these epithelial cells; and (iii) binding was not sensitive to metaperiodate under conditions that selectively cleaved carbons 8 and 9 of terminal nonmodified sialic acids. A role for fucosylated epitopes in the glycoprotein(s) that mediate binding of H. pylori to surface mucous cells was suggested by the facts that this lineage coexpresses the adhesin receptor and major fucosylated histo-blood group antigens, that monoclonal antibodies specific for histo-blood group antigens H, B, and Leb block binding, and that the lectin Ulex europaeus type 1 agglutinin, which is specific for alpha-L-fucose, also bound to the same cells that bound the bacteria. Furthermore, human colostrum secretory IgA inhibited adhesion in a metaperiodate- and alpha-L-fucosidase-sensitive but neuraminidase-independent fashion. The in situ adherence assay should be useful in further characterizing the H. pylori adhesin and its receptor and for identifying therapeutically useful compounds that inhibit strain-specific and cell lineage-specific binding of this human pathogen.
Electrical activity-dependent regulation of the acetylcholine receptor delta-subunit gene, MyoD, and myogenin in primary myotubes.
Authors: Dutton E K , Simon A M , Burden S J
Published: May 2006

Expression of the skeletal muscle acetylcholine receptor (AChR) is regulated by nerve-evoked muscle activity. Studies using transgenic mice have shown that this regulation is controlled largely by transcriptional mechanisms because responsiveness to electrical activity can be conferred by transgenes containing cis-acting sequences from the AChR subunit genes. The lack of a convenient muscle cell culture system for studying electrical activity-dependent gene regulation, however, has made it difficult to identify the important cis-acting sequences and to characterize an electrical activity-dependent signaling pathway. We developed a muscle culture system to study the mechanisms of electrical activity-dependent gene expression. Gene fusions between the murine AChR delta-subunit gene and the human growth hormone gene were transfected into primary myoblasts, and the amount of growth hormone secreted into the culture medium from either spontaneously electrically active or inactive myotube cultures was measured. We show that 181 bp of 5'-flanking DNA from the AChR delta-subunit gene are sufficient to confer electrical activity-dependent gene expression. In addition, we show that the rate of AChR delta-subunit gene expression differs among individual nuclei in a single myotube but that highly expressing nuclei are not necessarily colocalized with AChR clusters. We also show that expression of MyoD and myogenin are regulated by electrical activity in primary myotube cultures and that all nuclei within a myotube express similar levels of MyoD and similar levels of myogenin.
Generation of beta-amyloid in the secretory pathway in neuronal and nonneuronal cells.
Authors: Busciglio J , Gabuzda D H , Matsudaira P , Yankner B A
Published: May 2006

The cellular mechanism underlying the generation of beta-amyloid in Alzheimer disease and its relationship to the normal metabolism of the amyloid precursor protein are unknown. In this report, we show that 3- and 4-kDa peptides derived from amyloid precursor protein are normally secreted. Epitope mapping and radiolabel sequence analysis suggest that the 4-kDa peptide is closely related to full-length beta-amyloid and the 3-kDa species is a heterogeneous set of peptides truncated at the beta-amyloid N terminus. The beta-amyloid peptides are secreted in parallel with amyloid precursor protein. Inhibitors of Golgi processing inhibit secretion of beta-amyloid peptides, whereas lysosomal inhibitors have no effect. The secretion of beta-amyloid-related peptides occurs in a wide variety of cell types, but which peptides are produced and their absolute levels are dependent on cell type. Human astrocytes generated higher levels of beta-amyloid than any other cell type examined. These results suggest that beta-amyloid is generated in the secretory pathway and provide evidence that glial cells are a major source of beta-amyloid production in the brain.
A method for evaluation of activity of antagonistic analogs of growth hormone-releasing hormone in a superfusion system.
Authors: Rekasi Z , Schally A V
Published: May 2006

Antagonistic analogs of growth hormone-releasing hormone (GHRH) are being synthesized in our laboratory for various clinical applications, including treatment of certain endocrine disorders and insulin-like growth factor I-dependent tumors. To evaluate the endocrine effect of these GHRH antagonists, a sensitive dynamic in vitro system has been developed. The concentration causing 50% inhibition (IC50) of the standard GHRH antagonist human [N-Ac-Tyr1,D-Arg2]GHRH-(1-29)-NH2 is 4.5 x 10(-8) M in our dispersed pituitary cell superfusion system. This value is 11 times less than that measured in earlier static pituitary cell cultures. This reliable dynamic system is simple, fast, and inexpensive and not only makes it possible to obtain quantitative data on the inhibitory capacity of the antagonists but also provides information about the intrinsic GHRH activity of the analog. The dynamic interactions of the GHRH antagonist, the GHRH receptors, and GH release can also be evaluated by this superfusion system. The pulsatile GH release induced by 10(-9) M human GHRH-(1-29)-NH2 was inhibited by two modes of application, preincubation and simultaneous administration of the GHRH antagonist (10(-9) to 10(-6) M). The reduction in GHRH-stimulated GH response was more pronounced when the cells were preincubated with the antagonist prior to GHRH infusion than for simultaneous application. The inhibitory effect of the antagonist was dose-dependent, temporary, and of the competitive type. GH release induced by nonspecific stimulus (100 mM potassium chloride) was not influenced by the GHRH antagonist. This sensitive dynamic in vitro system appears to be a suitable method for screening the biological activity of various GHRH antagonists and eliminates the drawbacks of static pituitary cell culture.
Use of a zinc-finger consensus sequence framework and specificity rules to design specific DNA binding proteins.
Authors: Desjarlais J R , Berg J M
Published: May 2006

We have designed three zinc-finger proteins with different DNA binding specificities. The design strategy combines a consensus zinc-finger framework sequence with previously characterized recognition regions such that the specificity of each protein is predictable. The first protein consists of three identical zinc fingers, each of which was expected to recognize the subsite GCG. This protein binds specifically to the sequence 5'-GCG-GCG-GCG-3' with a dissociation constant of approximately 11 microM. The second protein has three zinc fingers with different predicted preferred subsites. This protein binds to the predicted recognition site 5'-GGG-GCG-GCT-3' with a dissociation constant of 2 nM. Furthermore, selection experiments indicate that this is the optimal binding site. A permuted version of the second protein was also constructed and shown to preferentially recognize the corresponding permuted site 5'-GGG-GCT-GCG-3' over the non-permuted site. These results indicate that earlier observations on the specificity of zinc fingers can be extended to generalized zinc-finger structures and realize the use of zinc fingers for the design of site-specific DNA binding proteins. This consensus-based design system provides a useful model system with which to study details of zinc-finger-DNA specificity.
Chromostatin, a chromogranin A-derived bioactive peptide, is present in human pancreatic insulin (beta) cells.
Authors: Cetin Y , Aunis D , Bader M F , Galindo E , Jörns A , Bargsten G , Grube D
Published: May 2006

Chromogranin A (CGA) is a secretory protein present in the adrenal medulla and in a variety of endocrine organs. This protein may serve as precursor for pancreastatin (PST) and for other biologically active peptides. Recently, chromostatin (CST), a CGA derivative, has been identified that possesses high biological activity. The cellular distribution of CST in various endocrine organs is completely unknown. Using immunohistochemistry on plastic sections, we investigated the occurrence and cellular distribution of CST, PST, and CGA in human endocrine pancreas of healthy and diseased states and in the adrenal medulla. In the normal and diabetic pancreas, CST immunoreactivity was localized exclusively in beta cells, which were mostly unreactive for PST and CGA. Both latter peptides were confined mainly to glucagon (alpha) cells. Insulinoma cells displayed strong insulin, PST, and CGA immunoreactivities, but they were faintly immunoreactive for CST or unreactive. Adrenal chromaffin cells exhibited strong immunoreactivity for CGA but lacked CST and PST immunoreactivities. Based on the peculiar distributive pattern of CST, PST, and CGA, we suggest that CGA is differentially processed in chromaffin and islet tissues and in insulinoma cells. The unique cellular localization of CST in the endocrine pancreas of normal and pathological conditions may indicate that CST is involved in beta-cell function.
Male-sterility induction in transgenic tobacco plants with an unedited atp9 mitochondrial gene from wheat.
Authors: Hernould M , Suharsono S , Litvak S , Araya A , Mouras A
Published: May 2006

Cytoplasmic male sterility in plants is associated with mitochondrial dysfunction. We have proposed that a nuclear-encoded chimeric peptide formed by mitochondrial sequences when imported into the mitochondria may impair organelle function and induce male sterility in plants. A model developed to test this hypothesis is reported here. Assuming that the editing process in higher plant mitochondria reflects a requirement for producing active proteins, we have used edited and unedited coding sequences of wheat ATP synthase subunit 9 (atp9) fused to the coding sequence of a yeast coxIV transit peptide. Transgenic plants containing unedited atp9 exhibited either fertile, semifertile, or male-sterile phenotypes; controls containing edited atp9 or only the selectable marker gave fertile plants. Pollen fertility ranged from 31% to 75% in fertile plants, 10% to 20% in semifertile plants, and < 2% in male-sterile plants. Genetic and molecular data showed that the chimeric plasmid containing the transgene is inherited as a Mendelian trait. The transgenic protein is imported into the mitochondria. The production and frequency of semifertile or male-sterile transgenic plants conform to the proposed hypothesis.
Long-term retention of learning-induced receptive-field plasticity in the auditory cortex.
Authors: Weinberger N M , Javid R , Lepan B
Published: May 2006

Brief learning experience (classical conditioning) induces frequency-specific receptive-field (RF) plasticity in the auditory cortex, characterized as increased response to the frequency of the conditioned stimulus and decreased responses to most other frequencies, including the pretraining best frequency. This experiment asked (i) whether learning-induced RF plasticity, established in the waking state, can be expressed under general anesthesia and if so (ii) whether it exhibits long-term retention. Pure-tone-frequency RFs were obtained from adult guinea pigs under general anesthesia (sodium pentobarbital or ketamine) before and repeatedly after (1 hr-8 weeks) a 20- to 30-trial session of pairing a non-best-frequency tone with mild footshock. Conditioned-stimulus-specific RF plasticity was expressed under both types of anesthesia and included shifts of the pretraining best frequency toward or even to the frequency of the conditioned stimulus. Moreover, this RF plasticity exhibits long-term retention, being evident 1-8 weeks after training. This satisfies a criterion for the long-term storage of information in the auditory cortex.
Expression of the KAL gene in multiple neuronal sites during chicken development.
Authors: Legouis R , Lievre C A , Leibovici M , Lapointe F , Petit C
Published: May 2006

The human KAL gene is responsible for the X chromosome-linked Kallmann syndrome. A partial cDNA sequence from the chicken KAL homologue was determined and used to study expression of the KAL gene, by in situ hybridization, during chicken development, from day 6 of incubation. The KAL gene is mainly expressed in neurons of the central nervous system during the second half of embryonic life. High levels of transcript were detected in mitral neurons of the olfactory bulbs, in striatal neurons, in Purkinje cells of the cerebellum, in retinal neurons, and in isolated neurons of the brainstem and spinal cord. No expression was observed in glial cells. A low level of expression was observed in some mesenchymal derivatives. In the adult, expression is maintained or increased in several neuronal populations, especially in optic tectum and striatum. A possible role for the KAL protein in synaptogenesis at these stages is discussed. These results in the chicken embryo help to elucidate the mechanisms of anosmia and gonadotropin-releasing hormone deficiency, which define Kallmann syndrome. In addition, most of the occasional symptoms described in Kallmann syndrome patients, such as cerebellar ataxia, abnormal ocular movements, abnormal spatial visual attention, mirror movements, and renal aplasia, could be ascribed to malfunction of areas that, in the chicken, express the KAL gene.