bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of tumorigenicity of rat glioblastoma directed by episome-based antisense cDNA transcription of insulin-like growth factor I.
Authors: Trojan J , Blossey B K , Johnson T R , Rudin S D , Tykocinski M , Ilan J , Ilan J
Published: May 2006

Malignant glioma is the most common brain tumor. The molecular basis of glioma tumorigenicity has not been defined. Cultured glioma cells accumulate high levels of insulin-like growth factor I (IGF-I) transcripts. We asked whether IGF-I expression is coupled to tumorigenicity, using a combined in vivo/in vitro system employing antisense RNA for IGF-I. An antisense IGF-I expression construct in an expression vector that incorporates Epstein-Barr virus replicative signals and the ZnSO4-inducible metallothionein I transcriptional promoter was assembled. Stable glioma transfectants were derived from C6 glioma cells, which constitutively express IGF-I. B-104 neuroblastoma cells, derived originally from the same tumor but not expressing IGF-I, were also transfected as controls. In the absence of ZnSO4, the C6 transfectants expressed high levels of IGF-I mRNA and protein as detected by in situ hybridization and immunocytochemistry, respectively. Addition of ZnSO4 in the culture medium resulted in high levels of antisense transcript accumulation and dramatically decreased levels of endogenous IGF-I mRNA and IGF-I protein. Subcutaneous injection of either nontransfected C6 parental cells or C6 cells transfected with vector without IGF-I sequences into rats resulted in large tumors after 2 weeks, as did transfected and nontransfected B-104 cells. However, the rats injected with transfected C6 cells yielded no tumors after 40 weeks of observation. Two weeks after injection of the transfected C6 cells a small cyst was apparent in six rats. Histologic sections revealed a few glioma cells infiltrated by a large number of mononuclear cells. No infiltration of mononuclear cells was apparent in the glioma tumors resulting from injection of parental (nontransfected) cells, suggesting that the parental cells, but not the antisense IGF-I transfectants, escape the host immune response.
Molecular cloning of an atypical voltage-gated sodium channel expressed in human heart and uterus: evidence for a distinct gene family.
Authors: George A L , Knittle T J , Tamkun M M
Published: May 2006

Previously cloned voltage-dependent sodium channels exhibit a high degree of homology to one another and appear to comprise a single multigene family. We have now isolated and characterized cDNAs from both human adult heart and fetal skeletal muscle that encode a sodium channel alpha subunit that exhibits only moderate primary structure identity with other sodium channels and is prominently expressed in both heart and uterus. The approximately 7.2-kilobase cDNA sequence, designated hNav2.1, predicts a 1682-amino acid protein that bears 52%, 49%, and 46% overall identity with sodium channels cloned from rat brain, skeletal muscle, and heart, respectively. Positively charged S4 segments are present in hNav2.1, but there are fewer basic residues in repeat domains 1, 3, and 4 than in other cloned sodium channels. The cloning of hNav2.1 provides evidence for greater evolutionary divergence among voltage-dependent sodium channels and suggests that other sodium channel gene subfamilies may exist. The unique amino acid sequences in regions known to be involved in voltage-dependent activation and inactivation suggest that hNav2.1 will have novel gating properties.
Optimal protein-folding codes from spin-glass theory.
Authors: Goldstein R A , Luthey-Schulten Z A , Wolynes P G
Published: May 2006

Protein-folding codes embodied in sequence-dependent energy functions can be optimized using spin-glass theory. Optimal folding codes for associative-memory Hamiltonians based on aligned sequences are deduced. A screening method based on these codes correctly recognizes protein structures in the "twilight zone" of sequence identity in the overwhelming majority of cases. Simulated annealing for the optimally encoded Hamiltonian generally leads to qualitatively correct structures.
Proteasomes are regulated by interferon gamma: implications for antigen processing.
Authors: Yang Y , Waters J B , Früh K , Peterson P A
Published: May 2006

Class I major histocompatibility complex (MHC) molecules present antigenic peptides of cytoplasmic origin to T cells. As the lengths of these peptides seem restricted to eight or nine amino acids, an unusual proteolytic system must play a role in antigen processing. Proteasomes, a major extralysosomal proteolytic system, are responsible for the degradation of cytoplasmic proteins. We demonstrate that several proteasomal subunits, including MHC-encoded subunits, are regulated by interferon gamma. These data and the finding that MHC-encoded and other interferon gamma-regulated proteasomal subunits are uniquely associated with proteasomes strongly suggest that the immune system has recruited proteasomes for antigen processing.
Sequence analysis of the 5' noncoding region of hepatitis C virus.
Authors: Bukh J , Purcell R H , Miller R H
Published: May 2006

We have determined the nucleotide sequence of the 5' noncoding (NC) region of the hepatitis C virus (HCV) genome in 44 isolates from around the world. We have identified several HCV isolates with significantly greater sequence heterogeneity than reported previously within the 5' NC region. The most distantly related isolates were only 90.1% identical. Nucleotide insertions were seen in three isolates. Analysis of the nucleotide sequence from 44 HCV isolates in this study combined with that of 37 isolates reported in the literature reveals that the 5' NC region of HCV consists of highly conserved domains interspersed with variable domains. The consensus sequence was identical to the prototype HCV sequence. Nucleotide variations were found in 45 (16%) of the 282 nucleotide positions analyzed and were primarily located in three domains of significant heterogeneity (positions -239 to -222, -167 to -118, and -100 to -72). Conversely, there were three highly conserved domains consisting of 18, 22, and 63 completely invariant nucleotides (positions -263 to -246, -199 to -178, and -65 to -3, respectively). Two nucleotide domains within the 5' NC region, conserved among all HCV isolates studied to date, shared statistically significant similarity with pestivirus 5' NC sequences, providing further evidence for a close evolutionary relationship between these two groups of viruses. Additional analysis revealed the presence of short open reading frames in all HCV isolates. Our sequence analysis of the 5' NC region of the HCV genome provides additional information about conserved elements within this region and suggests a possible functional role for the region in viral replication or gene expression. These data also have implications for selection of optimal primer sequences for the detection of HCV RNA by the PCR assay.
An ATP-stabilized inhibitor of the proteasome is a component of the 1500-kDa ubiquitin conjugate-degrading complex.
Authors: Driscoll J , Frydman J , Goldberg A L
Published: May 2006

Proteins conjugated to ubiquitin are degraded by a 26S (1500-kDa) proteolytic complex that, in reticulocyte extracts, can be formed by the association of three factors: CF-1, CF-2, and CF-3. One of these factors, CF-3, has been shown to be the proteasome, a 650-kDa multicatalytic protease complex. We have purified a 250-kDa inhibitor of the proteasome and shown that it corresponds to CF-2. In the presence or absence of ATP, this factor inhibited hydrolysis by the proteasome of both fluorogenic tetrapeptides and protein substrates. When the inhibitor, proteasome, and CF-1 were incubated together in the presence of ATP and Mg2+, degradation of ubiquitin-125I-lysozyme occurred. Both the inhibitory activity and the ability to reconstitute ubiquitin-125I-lysozyme degradation were very labile at 42 degrees C, but both activities were stabilized by ATP or a nonhydrolyzable ATP analog. SDS/PAGE indicated that the 250-kDa inhibitor fraction contained a major subunit of 40 kDa (plus some minor bands). The 125I-labeled inhibitor and purified proteasome formed a complex. When CF-1, ATP, and Mg2+ were also present, the 125I-labeled inhibitor along with the proteasome formed a complex of 1500 kDa. The inhibitor (CF-2) thus appears to be an ATP-binding component that regulates proteolysis within the 1500-kDa complex.
Mutations in the human CYP11B2 (aldosterone synthase) gene causing corticosterone methyloxidase II deficiency.
Authors: Pascoe L , Curnow K M , Slutsker L , Rösler A , White P C
Published: May 2006

Corticosterone methyloxidase II (CMO-II) deficiency is an autosomal recessive disorder of aldosterone biosynthesis, characterized by an elevated ratio of 18-hydroxycorticosterone to aldosterone in serum. It is genetically linked to the CYP11B1 and CYP11B2 genes that, respectively, encode two cytochrome P450 isozymes, P450XIB1 and P450XIB2. Whereas P450XIB1 only catalyzes hydroxylation at position 11 beta of 11-deoxycorticosterone and 11-deoxycortisol, P450XIB2 catalyzes the synthesis of aldosterone from deoxycorticosterone, a process that successively requires hydroxylation at positions 11 beta and 18 and oxidation at position 18. To determine the molecular genetic basis of CMO-II deficiency, seven kindreds of Iranian-Jewish origin were studied in which members suffered from CMO-II deficiency. No mutations were found in the CYP11B1 genes, but two candidate mutations, R181W and V386A, were found in the CYP11B2 genes. When these mutations were individually introduced into CYP11B2 cDNA and expressed in cultured cells, R181W reduced 18-hydroxylase and abolished 18-oxidase activities but left 11 beta-hydroxylase activity intact, whereas V386A caused a small but consistent reduction in the production of 18-hydroxycorticosterone. All individuals affected with CMO-II deficiency were homozygous for both mutations, whereas eight asymptomatic subjects were homozygous for R181W alone and three were homozygous for V386A alone. These findings confirm that P450XIB2 is the major enzyme mediating oxidation at position 18 in the adrenal and suggest that a small amount of residual activity undetectable in in vitro assays is sufficient to synthesize normal amounts of aldosterone.
Restoration of motility to an Escherichia coli fliA flagellar mutant by a Bacillus subtilis sigma factor.
Authors: Chen Y F , Helmann J D
Published: May 2006

The activation of additional promoter sites by production of an alternative sigma subunit for RNA polymerase is a common strategy for the coordinate regulation of gene expression. Many alternative sigma factors control genes for specialized, and often narrowly distributed, functions. For example, most of the alternative sigma factors in Bacillus subtilis control genes necessary for endospore formation. In contrast, the B. subtilis sigma D protein controls the expression of genes important for flagellar-based motility and chemotaxis, a form of locomotion very broadly distributed in the eubacteria. A homologous sigma factor, sigma F, controls a similar group of motility genes in the enteric bacteria. The conservation of both promoter specificity and genetic function in these two regulons allowed us to test the ability of a B. subtilis sigma factor to function within an Escherichia coli host. We demonstrate that expression of the B. subtilis sigD gene restores motility to an E. coli strain mutant in the fliA locus encoding the sigma F factor. This result suggests that the B. subtilis sigma D protein can bind to the E. coli core RNA polymerase to direct transcription initiation from at least four of the late operon promoters, thereby leading to the synthesis of flagellin, motor, and hook-associated proteins. Conversely, expression of sigma D protein in a normally chemotactic strain of E. coli (fliA+) leads to a hyperflagellated, nonchemotactic phenotype.
Adenylosuccinate lyase of Bacillus subtilis regulates the activity of the glutamyl-tRNA synthetase.
Authors: Gendron N , Breton R , Champagne N , Lapointe J
Published: May 2006

In Bacillus subtilis, the glutamyl-tRNA synthetase [L-glutamate:tRNA(Glu) ligase (AMP-forming), EC 6.1.1.17] is copurified with a polypeptide of M(r) 46,000 that influences its affinity for its substrates and increases its thermostability. The gene encoding this regulatory factor was cloned with the aid of a 41-mer oligonucleotide probe corresponding to the amino acid sequence of an NH2-terminal segment of this factor. The nucleotide sequence of this gene and the physical map of the 1475-base-pair fragment on which it was cloned are identical to those of purB, which encodes the adenylosuccinate lyase (adenylosuccinate AMP-lyase, EC 4.3.2.2), an enzyme involved in the de novo synthesis of purines. This gene complements the purB mutation of Escherichia coli JK268, and its presence on a multicopy plasmid behind the trc promoter in the purB- strain gives an adenylosuccinate lyase level comparable to that in wild-type B. subtilis. A complex between the adenylosuccinate lyase and the glutamyl-tRNA synthetase was detected by centrifugation on a density gradient. The interaction between these enzymes may play a role in the coordination of purine metabolism and protein biosynthesis.
A family of concanavalin A-binding peptides from a hexapeptide epitope library.
Authors: Scott J K , Loganathan D , Easley R B , Gong X , Goldstein I J
Published: May 2006

The lectin concanavalin A (Con A) binds methyl alpha-D-mannopyranoside (Me alpha Man) as well as alpha-D-mannosyl groups at the nonreducing terminus of oligosaccharides. Ligand peptides that mimic the binding of Me alpha Man to Con A were identified from screening an epitope library composed of filamentous phage displaying random hexapeptides. A consensus sequence was identified among affinity-purified phage; Con A binds phage bearing this sequence and is inhibited from doing so by Me alpha Man. When tested for binding against a panel of lectins, phage bearing this sequence bind only weakly to a closely related D-mannose-binding lectin, indicating that binding to Con A is highly selective. A synthetic peptide bearing the consensus sequence blocks the precipitation of Con A by dextran with an inhibition strength equivalent to that of methyl alpha-D-glucopyranoside. These results demonstrate that the specificity of Con A is not limited to carbohydrates and that highly selective sugar-mimics for lectins of plant, animal, or bacterial origin may be identified from epitope libraries.
Cloning of a rat kidney cDNA that stimulates dibasic and neutral amino acid transport and has sequence similarity to glucosidases.
Authors: Wells R G , Hediger M A
Published: May 2006

The transport of amino acids across cell membranes is believed to be mediated by integral membrane proteins with distinct substrate specificities. Using expression cloning in Xenopus oocytes and assaying for the uptake of 14C-labeled cystine, we isolated a 2.3-kilobase cDNA (D2) from a rat kidney library. D2 is expressed specifically in kidney and intestine and induces the transport of both neutral and cationic amino acids. The deduced amino acid sequence predicts a 78-kDa protein with a single transmembrane domain, a structure not typical of the known membrane transport proteins, which generally have multiple membrane-spanning regions. The putative extracellular region is highly similar to the 4F2 heavy-chain cell surface antigen and to a family of alpha-glucosidases, which raises the possibility that D2 encodes a transport activator or regulatory subunit.
A metastatic nude-mouse model of human pancreatic cancer constructed orthotopically with histologically intact patient specimens.
Authors: Fu X , Guadagni F , Hoffman R M
Published: May 2006

Pancreatic cancer is one of the most intractable and least understood of all human cancers. Pancreatic cancers is the fourth-leading cause of cancer-related mortality in the United States with less than 2% of the patients surviving for 5 yr. In an effort to help develop more effective treatment modalities for pancreatic cancer and improve detection, we report an animal model for individual human pancreatic-cancer patients. The model involves orthotopic transplantation of histologically intact pancreatic-cancer specimens to the nude-mouse pancreas, which can result in models that resemble the clinical picture including (i) extensive local tumor growth, (ii) extension of the locally growing human pancreatic cancer to the nude-mouse stomach and duodenum, (iii) metastases of the human pancreatic tumor to the nude-mouse liver and regional lymph nodes, and (iv) distant metastases of the human pancreatic tumor to the nude-mouse adrenal gland, diaphragm, and mediastinal lymph nodes. In a series of five patient cases, a 100% take rate has been demonstrated, and of 17 mice transplanted, 15 supported tumor growth. Immunohistochemical analysis of the antigenic phenotype of the transplanted human pancreatic tumors showed a similar pattern of expression of two different human tumor-associated antigens, such as tumor-associated glycoprotein 72 and carcinoembryonic antigen in the transplanted tumors when compared with the original surgical biopsy, suggesting similarity between the two. This model should, therefore, prove valuable for treatment evaluation of individual cancer patients, as well as for evaluation of experimental treatment modalities for this disease.
Stable transformation of a mosquito cell line results in extraordinarily high copy numbers of the plasmid.
Authors: Monroe T J , Muhlmann-Diaz M C , Kovach M J , Carlson J O , Bedford J S , Beaty B J
Published: May 2006

Stable incorporation of high copy numbers (greater than 10,000 per cell) of a plasmid vector containing a gene conferring resistance to the antibiotic hygromycin was achieved in a cell line derived from the Aedes albopictus mosquito. Plasmid sequences were readily observed by ethidium bromide staining of cellular DNA after restriction endonuclease digestion and agarose gel electrophoresis. The plasmid was demonstrated by in situ hybridization to be present in large arrays integrated in metaphase chromosomes and in minute and double-minute replicating elements. In one subclone, approximately 60,000 copies of the plasmid were organized in a large array that resembles a chromosome, morphologically and in the segregation of its chromatids during anaphase. The original as well as modified versions of the plasmid were rescued by transformation of Escherichia coli using total cellular DNA. Southern blot analyses of recovered plasmids indicate the presence of mosquito-derived sequences.
Clonal proliferation of murine lymphohemopoietic progenitors in culture.
Authors: Hirayama F , Shih J P , Awgulewitsch A , Warr G W , Clark S C , Ogawa M
Published: May 2006

We have used a two-step clonal culture system to unequivocally demonstrate that individual primitive lymphohemopoietic progenitor cells have the capacity for differentiation along either the myeloid or the B-lymphoid lineage. Highly enriched murine marrow cells were plated individually in culture by micromanipulation in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium, erythropoietin, steel factor (SF), and interleukin (IL) 7. Forty-five percent of the single cells formed primary colonies expressing multiple hemopoietic lineages. When aliquots from individual colonies were replated in secondary methyl cellulose culture containing SF and IL-7, 41% of the primary colonies gave rise to lymphocyte colonies. Cells of the lymphocyte colonies were blast-like and B220+, sIg-, Mac-1-, Gr-1-, Ly-1-, L3T4-, Ly-2-, and CD3-. Thirty to 70% of the cells were Thy-1+. mu-chain mRNA was detected in most of the cells by in situ hybridization with an antisense RNA probe. When lymphocyte colonies derived from a single cell were pooled and individually injected into scid mice, donor-type IgM was measurable in the serum of mice and spleens contained donor-type B cells. We then carried out initial screening of growth factors to identify growth factors that might replace pokeweed mitogen-stimulated spleen cell conditioned medium in the primary culture. Combinations of two factors that included SF plus IL-6, IL-11, or granulocyte colony-stimulating factor were all effective in the primary culture in the maintenance of the B-lymphoid potential. Interestingly, IL-3 could neither replace nor act synergistically with SF to support the lymphoid potential of the primary cultures. Our observations demonstrate that many primitive progenitors previously believed to be myeloid-committed also possess B-lymphoid potential. This culture system should prove valuable for elucidation of the mechanisms regulating early stages of lymphohemopoiesis.
Two mutations in Marfan syndrome resulting in truncated fibrillin polypeptides.
Authors: Kainulainen K , Sakai L Y , Child A , Pope F M , Puhakka L , Ryhänen L , Palotie A , Kaitila I , Peltonen L
Published: May 2006

Biochemical and molecular genetic studies have recently suggested that mutations in the gene coding for fibrillin on chromosome 15 result in Marfan syndrome. To our knowledge, only one mutation in the fibrillin gene has been published. Here we report the results of screening 20 unrelated MFS patients for mutations in fibrillin cDNA by the single-strand conformation polymorphism technique. We found two mutations, both of which appear in the heterozygote form and code for a shortened fibrillin polypeptide. The first mutation is a large in-frame deletion of 366 bases of the fibrillin mRNA, shown to result in a truncated but secreted polypeptide found in the fibroblast culture of the patient. The second mutation is a G-to-A transition resulting in the substitution of a stop codon for a tryptophan codon and thus predicting the premature termination of the polypeptide chain. We screened 60 other, unrelated MFS patients for these mutations as well as for the previously reported mutation (arginine-239 to proline) and found none of the three mutations in any of these patients. These data suggest that most MFS families carry their own distinct mutation.
Side-chain entropy opposes alpha-helix formation but rationalizes experimentally determined helix-forming propensities.
Authors: Creamer T P , Rose G D
Published: May 2006

In recent host-guest studies, the helix-forming tendencies of amino acid residues have been quantified by three groups, each obtaining similar results [Padmanabhan, S., Marqusee, S., Ridgeway, T., Laue, T. M. & Baldwin, R. L. (1990) Nature (London) 344, 268-270; O'Neil, K. T. & DeGrado, W. F. (1990) Science 250, 646-651; Lyu, P. C., Liff, M. I., Marky, L. A. & Kallenbach, N. R. (1990) Science 250, 669-673]. Here, we explore the hypothesis that these measured helix-forming propensities are due primarily to conformational restrictions imposed upon residue side chains by the helix itself. This proposition is tested by calculating the extent to which the bulky helix backbone "freezes out" available degrees of freedom in helix side chains. Specifically, for a series of apolar residues, the difference in configurational entropy, delta S, between each side chain in the unfolded state and in the alpha-helical state is obtained from a simple Monte Carlo calculation. These computed entropy differences are then compared with the experimentally determined values. Measured and calculated values are found to be in close agreement for naturally occurring amino acids and in total disagreement for non-natural amino acids. In the calculation, delta S(Ala) = 0. The rank order of entropy loss for the series of natural apolar side chains under consideration is Ala less than Leu less than Trp less than Met less than Phe less than Ile less than Tyr less than Val. Among these, none favor helix formation; Ala is neutral, and all remaining residues are unfavorable to varying degrees. Thus, applied to side chains, the term "helix preference" is a misnomer. While side chain-side chain interactions may modulate stability in some instances, our results indicate that the drive to form helices must originate in the backbone, consistent with Pauling's view of four decades ago [Pauling, L., Corey, R. B. & Branson, H. R. (1951) Proc. Natl. Acad. Sci. USA 37, 205-210].
Characterization of human androgen receptor overexpressed in the baculovirus system.
Authors: Chang C , Wang C , DeLuca H F , Ross T K , Shih C C
Published: May 2006

An essential step in the process of understanding the structure and function of the human androgen receptor (hAR) involves the production of large quantities of the hAR. For this purpose, the full-length hAR has been overproduced in insect cells by using a baculovirus genetic expression system. The recombinant hAR is produced in Sf21 insect cells at approximately 7 pmol/mg of protein (1 x 10(6) AR molecules per cell), which is 70-150 times greater than levels detected in androgen target tissues. Androgen can bind to the baculovirus-expressed hAR with high affinity (Kd = 0.46 nM), and the specificity of hormone binding in baculovirus-expressed hAR is essentially identical to that of bona fide hAR. An anti-AR monoclonal antibody can recognize the baculovirus-expressed hAR at approximately 100 kDa upon Western blot analysis. Sucrose gradient analysis shows that baculovirus-expressed hAR complexes sediment at 4 S in a high salt medium and these complexes can interact with anti-AR monoclonal antibody to form complexes that sediment at 8-10 S. Therefore, production of recombinant hAR from the baculovirus expression system will provide an alternative source of biologically active hAR for studies on the molecular mechanisms of androgen action.
The cytoplasmic component of the bacterial flagellar motor.
Authors: Khan I H , Reese T S , Khan S
Published: May 2006

We have used electron microscopy to examine freshly isolated Salmonella typhimurium and Escherichia coli basal flagellar fragments, purified without resort to extremes of pH or ionic strength. Such fragments contain the large bell-like basal structures visualized recently in freeze-substituted or fixed preparations. We have found mot (non-motile) mutants produced by lesions in fli genes (G, M, N) in which the bell structures do not coisolate with the flagellar basal body. The coisolation of the bell with the flagellar basal body was unaffected in strains lacking the genes for the motility-associated Mot proteins or for the Che family of proteins, which are necessary for chemotaxis. Proper assembly and interaction of the cytoplasmically located bell with the membrane-associated flagellar basal structures appears to be necessary for motor function. The FliG, FliM, and FliN proteins are thought to form a structural complex responsible for energization and switching of the flagellar motor. Our findings are consistent with the existence of such a complex and imply that it forms part of the flagellar bell.
Beta-amyloid precursor protein cleavage by a membrane-bound protease.
Authors: Sisodia S S
Published: May 2006

The principal component of amyloid plaques in Alzheimer disease is beta-amyloid protein, an approximately 4-kDa peptide derived from amyloid precursor proteins. Previous studies have established that amyloid precursor proteins are secreted after proteolytic cleavage within the beta-amyloid peptide. The present investigation documents that, in cultured cells, amyloid precursor protein is cleaved on the plasma membrane by a membrane-bound endoprotease and that the specificity of peptide bond hydrolysis is largely independent of the primary sequence of the precursor. The principal determinants of cleavage appear to be an alpha-helical conformation and the distance (12-13 residues) of the hydrolyzed bond from membrane.
DHR3: a Drosophila steroid receptor homolog.
Authors: Koelle M R , Segraves W A , Hogness D S
Published: May 2006

In Drosophila the steroid hormone ecdysone triggers a genetic regulatory hierarchy in which ecdysone combines with a receptor protein to form a complex that induces the transcription of a small class of "early" genes, which encode transcription factors that regulate other genes. We previously reported that one of the early genes, E75, encodes members of the steroid receptor superfamily. Using an E75 hybridization probe, we have identified two additional Drosophila genes that encode members of this superfamily. One of these is the ecdysone receptor gene, EcR, as previously reported. In this work, we examine the sequence, genomic organization, and developmental expression of the other gene, DHR3, which, like E75, encodes one of a growing number of "orphan" receptors for which ligands have not yet been identified. The structure of the DHR3 protein is strikingly similar to that of the MHR3 protein (e.g., 97% amino acid identity for the DNA binding domains), another orphan receptor encoded by an ecdysone-inducible early gene of another insect, Manduca sexta. The temporal developmental profile for DHR3 expression closely parallels that for the ecdysone titer and for the ecdysone-inducible E75 and E74 Drosophila early genes. The structural similarity to a Manduca early gene and the expression similarities to Drosophila early genes suggest that the DHR3 gene may also belong to the early gene class.
Inhibition of human immunodeficiency virus activity by phosphorodithioate oligodeoxycytidine.
Authors: Marshall W S , Beaton G , Stein C A , Matsukura M , Caruthers M H
Published: May 2006

Phosphorothioate oligodeoxynucleotides exert a sequence-independent cytoprotective effect against human immunodeficiency virus type 1 (HIV-1). We now report that phosphorodithioate-containing oligodeoxycytidines are very potent inhibitors of HIV-1 reverse transcriptase in vitro, as they exhibit an increasing inhibitory effect with length and number of phosphorodithioate internucleotide linkages. This inhibitory effect can be at least 30-fold greater with phosphorodithioate oligodeoxycytidine than for the corresponding phosphorothioate analog of similar length. In cell culture, phosphorodithioate oligodeoxycytidines are active inhibitors of syncytia formation and effectively inhibit de novo infection of target cells by HIV-1. Moreover, comparative experiments show that a deoxycytidine phosphorodithioate 14-mer is as effective an inhibitor of de novo infection as a phosphorothioate-containing 28-mer. Such potent inhibition by oligomers of relatively short length makes dithioate analogs an additional class of potential therapeutic agents against acquired immunodeficiency syndrome.
An immunophilin that binds M(r) 90,000 heat shock protein: main structural features of a mammalian p59 protein.
Authors: Callebaut I , Renoir J M , Lebeau M C , Massol N , Burny A , Baulieu E E , Mornon J P
Published: May 2006

In the rabbit, a p59 protein included in the untransformed, non-DNA binding, "8-9S," steroid receptor complexes binds heat shock protein M(r) approximately 90,000 (hsp90). Sequence data [Lebeau, M. C., Massol, N., Herrick, J., Faber, L. E., Renoir, J. M., Radanyi, C. & Baulieu, E. E. (1992) J. Biol. Chem. 267, 4281-4284] and hydrophobic cluster analysis delineate, from the N terminus, two successive domains closely related to the immunosuppressant FK506 binding immunophilin FKBP (FK506 binding protein), consistent with recent purification of the human p56 immunophilin cognate protein by FK506 affinity chromatography [Yem, A. W., Tomasselli, A. G., Heinrikson, R. L., Zurcher-Neely, H., Ruff, V. A., Johnson, R. A. & Deibel, M. R., Jr. (1992) J. Biol. Chem. 267, 2868-2871]. The first FKBP-like domain demonstrates all structural characteristics known to be necessary for immunosuppressant binding and for peptidylprolyl cis-trans isomerase (rotamase) activity. Hence, p59 is a "hsp binding immunophilin" (HBI). It is thus speculated that hsp binding immunophilin may help the assembly/disassembly mechanisms involved in steroid receptor trafficking and activity and participate in the poorly understood hsp90 function. ATP/GTP binding likely occurs within the second FKBP-like domain, near the FK506 binding site on the FKBP template. A third domain detected by the hydrophobic cluster analysis method is distantly structurally related to the two first FKBP-like domains and is followed by the C-terminal part of the protein, which contains a calmodulin binding consensus sequence. Hsp binding immunophilin may be involved in a number of immunological, endocrinological, and chaperone-mediated pathways.
Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform.
Authors: Lamas S , Marsden P A , Li G K , Tempst P , Michel T
Published: May 2006

Nitric oxide (NO) is a ubiquitous intercellular messenger molecule synthesized from the amino acid L-arginine by NO synthases in diverse cells and tissues. NO is synthesized in vascular endothelial cells and appears to play an important role in the control of blood pressure and platelet aggregation. A detailed understanding of the regulation of NO synthesis by endothelial cells has been hampered by the lack of molecular clones for endothelial NO synthase; the isolation and characterization of such clones is reported herein. The constitutive NO synthases present in endothelial cells and in brain share common biochemical and pharmacologic features. We purified NO synthase from bovine brain and determined the amino acid sequence of several tryptic peptides. The sequence of the bovine brain peptides is nearly identical to the deduced amino acid sequence previously determined for the rat brain NO synthase. These sequence data were utilized to design PCR-generated NO synthase cDNA probes, which were used to isolate clones encoding NO synthase from a bovine aortic endothelial cell (BAEC) cDNA library. A full-length NO synthase cDNA clone was isolated, representing a protein of 1205 amino acids with a molecular mass of 133 kDa; transfection of this clone in a heterologous expression system demonstrated the expected enzymatic activity. The deduced amino acid sequence of the BAEC NO synthase cDNA differs at numerous residues from the sequence determined for the purified bovine brain protein and shows 50-60% sequence identity with recently isolated molecular clones for murine macrophage and rat brain NO synthase isoforms. Bovine genomic Southern blots probed with bovine brain and BAEC NO synthase cDNA probes identify distinct bands, indicating that these cDNAs are the products of different genes. Prolonged treatment of BAECs with the cytokine tumor necrosis factor alpha, which we have previously shown to result in a marked increase in NO synthase activity, is associated with a decrease in the abundance of the 4.8-kilobase BAEC NO synthase transcript. The increase in BAEC NO synthase activity induced by tumor necrosis factor alpha is thus likely to involve posttranscriptional mechanisms or the induction of a distinct endothelial NO synthase isoform.
Germ-line mutations of the p53 tumor suppressor gene in patients with high risk for cancer inactivate the p53 protein.
Authors: Frebourg T , Kassel J , Lam K T , Gryka M A , Barbier N , Andersen T I , Børresen A L , Friend S H
Published: May 2006

Germ-line mutations in the p53 tumor suppressor gene have been observed in patients with Li-Fraumeni syndrome, brain tumors, second malignancies, and breast cancers. It is unclear whether all of these mutations have inactivated p53 and thereby provide an increased risk for cancer. Therefore, it is necessary to establish the biological significance of these germ-line mutations by the functional and structural analysis of the resulting mutant p53 proteins. We analyzed the ability of seven germ-line mutant proteins observed in patients with Li-Fraumeni syndrome, second primary neoplasms, or familial breast cancer to block the growth of malignant cells and compared the structural properties of the mutant proteins to that of the wild-type protein. Six of seven missense mutations disrupted the growth inhibitory properties and structure of the wild-type protein. One germ-line mutation retained the features of the wild-type p53. Genetic analysis of the breast cancer family in which this mutation was observed indicated that this germ-line mutation was not associated with the development of cancer. These results demonstrate that germ-line p53 mutations observed in patients with Li-Fraumeni syndrome and with second malignancies have inactivated the p53 tumor suppressor gene. The inability of the germ-line p53 mutants to block the growth of malignant cells can explain why patients with these germ-line mutations have an increased risk for cancer. The observation of a functionally silent germ-line mutation indicates that, before associating a germ-line tumor suppressor gene mutation with cancer risk, it is prudent to consider its functional significance.
Molecular basis of AMP deaminase deficiency in skeletal muscle.
Authors: Morisaki T , Gross M , Morisaki H , Pongratz D , Zöllner N , Holmes E W
Published: May 2006

AMP deaminase (AMPD; EC 3.5.4.6) is encoded by a multigene family in mammals. The AMPD1 gene is expressed at high levels in skeletal muscle, where this enzyme is thought to play an important role in energy metabolism. Deficiency of AMPD activity in skeletal muscle is associated with symptoms of a metabolic myopathy. Eleven unrelated individuals with AMPD deficiency were studied, and each was shown to be homozygous for a mutant allele characterized by a C----T transition at nucleotide 34 (codon 12 in exon 2) and at nucleotide 143 (codon 48 in exon 3). The C----T transition at codon 12 results in a nonsense mutation predicting a severely truncated AMPD peptide. Consistent with this prediction, no immunoreactive AMPD1 peptide is detectable in skeletal muscle of these patients. This mutant allele is found in 12% of Caucasians and 19% of African-Americans, whereas none of the 106 Japanese subjects surveyed has this mutant allele. We conclude from these studies that this mutant allele is present at a sufficiently high frequency to account for the 2% reported incidence of AMPD deficiency in muscle biopsies. The restricted distribution and high frequency of this doubly mutated allele suggest it arose in a remote ancestor of individuals of Western European descent.
Targeting antiretroviral nucleoside analogues in phosphorylated form to macrophages: in vitro and in vivo studies.
Authors: Magnani M , Rossi L , Brandi G , Schiavano G F , Montroni M , Piedimonte G
Published: May 2006

A number of nucleoside analogues are active against the infectivity of human immunodeficiency virus (HIV); however, their use is limited by toxic side effects and by limited phosphorylation in the infected cells. In an attempt to overcome these problems, a drug delivery system has been developed. A prototype of these drugs in a form already phosphorylated (2',3'-dideoxycytidine 5'-triphosphate; ddCTP) was encapsulated into erythrocytes. Subsequently, by the addition of Zn, an arrangement of band 3 in clusters was induced (band 3 is the major transmembrane protein in erythrocytes). The immune system recognizes these clusters as nonself, promoting autologous IgG binding and phagocytosis by cells of the monocyte-macrophage lineage. In this way, ddCTP encapsulated into erythrocytes was delivered to macrophage cells, where concentrations greater than 2 microM were found. Addition of ddCTP-loaded erythrocytes to macrophages previously infected by HIV-1 results in almost complete inhibition of HIV production over 3 weeks in culture. Administration of ddCTP-loaded erythrocytes to LP-BM5-infected mice at 10-day intervals over a period of 3 months results in reduction of lymphoadenopathy, splenomegaly, and hypergammaglobulinemia. Thus, the delivery of nucleoside analogues in phosphorylated form is feasible, and selective targeting to virus reservoirs (macrophage cells) can be accomplished by the use of autologous erythrocytes.
An infrequent point mutation of the p53 gene in human nasopharyngeal carcinoma.
Authors: Sun Y , Hegamyer G , Cheng Y J , Hildesheim A , Chen J Y , Chen I H , Cao Y , Yao K T , Colburn N H
Published: May 2006

Point mutations in the p53 gene have been detected in a variety of human cancers; the mutations are clustered in four "hot-spots" located in the coding region of exons 5, 7, and 8, which coincide with the four most highly conserved regions of the gene. We report the finding of a heterozygous G----C mutation at codon 280 (exon 8), position 2, of the p53 gene in a nasopharyngeal carcinoma (NPC) cell line, originating from Guangdong, a province in the People's Republic of China that leads the world in NPC incidence. A survey of nasopharyngeal tissues and NPC biopsies revealed that 1 out of 12 NPC samples from Hunan, another province in the People's Republic of China with high NPC incidence, had the same heterozygous mutation at codon 280 of p53, and none of 10 biopsies from Taiwan showed a mutation within exons 5-8 of the p53 gene. No other alteration of gene structure, including gross rearrangement or loss of heterozygosity or abnormality of gene expression was detected in NPC cell lines or NPC biopsies. We conclude from this study that mutational or other alterations of the p53 gene are not common in nasopharyngeal carcinogenesis and that a codon-280 mutation of p53 may be involved in less than 10% of NPC cases. This result contrasts with the relatively high frequency of p53 mutations associated with several other human carcinomas and suggests the importance of other genes in NPC genesis.
Modulation of adenylate cyclase toxin production as Bordetella pertussis enters human macrophages.
Authors: Masure H R
Published: May 2006

During the course of human infection, Bordetella pertussis colonizes sequential niches in the respiratory tract that include intracellular and extracellular environments. In vitro the expression of virulence factors such as the adenylate cyclase toxin is coordinately regulated by the bvg locus, which is an example of a two-component sensory transduction system. With this toxin as a reporter, enzyme activities were compared between a wild-type and an altered strain to determine whether bacterial entry into human macrophages affected gene expression. BPRU140, a strain containing an inducible expression vector, produced enzyme activity independent of bvg. Samples of the parent, the induced, and the uninduced BPRU140 were incubated individually with macrophages for 30 min. Extracellular bacteria were then killed by gentamicin. The number of viable intracellular bacteria and the internalized bacterial enzyme activity were measured over time. By 2.5 hr all samples reached a steady-state concentration of 10(5) bacteria per 10(6) macrophages. Following an initial peak of enzyme activity, adenylate cyclase values for the parent and the uninduced BPRU140 decreased to a basal level, while the values for the induced strain remained at least 3-fold greater. Therefore, compared with the persistence of enzyme in the induced strain BPRU140, the decrease in enzyme production by the parent and the uninduced BPRU140 upon entry into macrophages indicates in vivo down-modulation of gene expression. These observations support the hypothesis that sensory transduction contributes to adaptations for bacterial survival in the infected host.
Cooperation between the products of different nuclei in hybrid myotubes produces localized acetylcholine receptor clusters.
Authors: Gordon H , Ralston E , Hall Z W
Published: May 2006

Cultured myotubes form clusters of acetylcholine receptors (AChRs) spontaneously and at sites of nerve-muscle contact. To investigate the cellular mechanisms by which spontaneous clusters are formed, we have made hybrid myotubes between a mouse muscle cell variant, S27, that does not cluster AChRs, and one that does not make AChRs. We have also made hybrid myotubes using S27 and quail muscle cells. In both cases, clusters of AChRs were found near the non-S27 nuclei; in the case of the interspecific hybrids, mouse AChRs were associated with extracellular matrix components contributed by the quail nuclei. Our results suggest that AChRs made by one nucleus can be clustered by localized extracellular matrix produced by a different nucleus and provide an example of nuclear cooperation between the products of different nuclei within multinucleated muscle fibers.
Endosulfine, an endogenous peptidic ligand for the sulfonylurea receptor: purification and partial characterization from ovine brain.
Authors: Virsolvy-Vergine A , Leray H , Kuroki S , Lupo B , Dufour M , Bataille D
Published: May 2006

Antidiabetic sulfonylureas act through receptors coupled to ATP-dependent potassium channels. Using the binding of [3H]glibenclamide, a highly potent sulfonylurea, to rat brain membranes to follow the purification procedure, we extracted from ovine brain, purified, and partially characterized two peptides that are endogenous ligands for the central nervous system sulfonylurea receptors. These peptides, referred to as alpha and beta endosulfine, differ by their isoelectric points, the beta form being more basic. Each form of endosulfine is recognized equally by the sulfonylurea receptors from the central nervous system and from insulin-secreting beta cells. In the same concentration range that is active on the receptors, beta endosulfine releases insulin from a beta-cell line. Endosulfine is a good candidate for being implicated in the physiology of beta cells and their disorders (e.g., type II diabetes) and in certain pathologies related to modifications of ion fluxes.
A single-stranded DNA-binding protein from Crithidia fasciculata recognizes the nucleotide sequence at the origin of replication of kinetoplast DNA minicircles.
Authors: Tzfati Y , Abeliovich H , Kapeller I , Shlomai J
Published: May 2006

A sequence-specific single-stranded DNA-binding protein from the trypanosomatid protozoan Crithidia fasciculata binds to a sequence of 12 nucleotides located at the origin of replication of kinetoplast DNA minicircles. This sequence, termed the universal minicircle sequence (UMS), is conserved in the kinetoplast DNA minicircles among species of the family Trypanosomatidae. The purified protein binds specifically to the heavy strand of the DNA at this site, which consists of the sequence 5'-GGGGTTGGTGTA-3'. Binding analyses using mutated UMS dodecamers have revealed the significant contribution of each of the individual residues at the binding site, with the exception of the 3'-terminal adenine residue, to the generation of specific protein-DNA complexes. The possible role of this sequence-specific single-stranded DNA-binding protein in replication of kinetoplast DNA minicircles and the relation of the UMS to chromosomal telomeric sequences are discussed.
Meiosis-specific protein selectively associated with sex chromosomes of rat pachytene spermatocytes.
Authors: Smith A , Benavente R
Published: May 2006

During the first meiotic prophase of mammalian spermatogenesis, the sex chromosomes X and Y show a characteristic allocyclic behavior with respect to the autosomes. This is particularly evident during pachytene stage when sex chromosomes form the so-called sex vesicle. This structure is characterized by the condensed state of chromatin, transcriptional inactivity, and the limited extension of chromosome pairing, which is usually restricted to a short segment of sex chromosome axial elements. The molecular basis and functional significance of sex vesicle formation during mammalian spermatogenesis remain obscure. Here we report on the identification of a meiosis-specific sex vesicle protein we called XY40. Immunocytochemical localization on rat testis cryosections with a XY40-specific monoclonal antibody revealed that the labeling is confined to the axial elements of sex chromosomes. Biochemical characterization showed that protein XY40 (40 kDa; pI 5.7-5.8) can be extracted from rat pachytene spermatocytes and recovered in particles of 9.5 S with a native molecular mass of approximately 152 kDa. We speculate that protein XY40 may be involved in the allocyclic behavior of sex chromosomes during male meiotic prophase.