bluuebunny/crossref_metadata_2025 huggingface dataset:
Ontogeny of the prohormone convertases PC1 and PC2 in the mouse hypophysis and their colocalization with corticotropin and alpha-melanotropin.
Authors: Marcinkiewicz M , Day R , Seidah N G , Chrétien M
Published: May 2006

In the adult pituitary, anterior lobe corticotrophs and intermediate lobe melanotrophs differentially process proopiomelanocortin (POMC). Within the corticotrophs, POMC is processed mainly to corticotropin (ACTH) and beta-lipotropin, while alpha-melanotropin (alpha MSH) and beta-endorphin are the major end products in the melanotrophs. The observed transient presence of alpha MSH-like immunoreactivity during ontogeny suggested an age-dependent variation in POMC processing in the adenohypophysis. In this tissue, cell-specific POMC products are likely the result of differential expression of the two known prohormone convertases PC1 and PC2. In the present ontogeny study done in the mouse intermediate and anterior pituitary, we examined how the expression pattern of PC1 and PC2 mRNA transcripts correlates with that of ACTH and alpha MSH-like immunoreactivities. Our data demonstrated that both PC1 and PC2 transcripts can be detected in the presumptive adenohypophysis starting on embryonic day 15 (E15). In the intermediate lobe, PC1 and PC2 mRNAs appear on E18 and E16, respectively, and their levels increased during ontogeny, reaching maximal expression in the adult. Similarly, PC1 expression in the anterior pituitary increased from E15 to adulthood. However, PC2 mRNA expression peaked between postnatal days 1 (P1) and 14 (P14) and then decreased to adult levels. The distribution of PC1 and PC2 immunoreactivity is nicely correlated with the in situ hybridization data. In the anterior lobe, during the P1-P14 postnatal period, PC2 immunoreactivity was detected within cells synthesizing an alpha MSH-like peptide(s). This observation substantiates our earlier biochemical data suggesting that PC2 is the important convertase in the processing of POMC into alpha MSH. Furthermore, the demonstrated variation in the relative ratio of PC1/PC2 expression during ontogeny rationalizes the observed plasticity of POMC processing in the adenohypophysis. It is expected that beta-endorphin processing will follow that of alpha MSH.
The abnormal phosphorylation of tau protein at Ser-202 in Alzheimer disease recapitulates phosphorylation during development.
Authors: Goedert M , Jakes R , Crowther R A , Six J , Lübke U , Vandermeeren M , Cras P , Trojanowski J Q , Lee V M
Published: May 2006

Tau is a neuronal phosphoprotein whose expression is developmentally regulated. A single tau isoform is expressed in fetal human brain but six isoforms are expressed in adult brain, with the fetal isoform corresponding to the shortest of the adult isoforms. Phosphorylation of tau is also developmentally regulated, as fetal tau is phosphorylated at more sites than adult tau. In Alzheimer disease, the six adult tau isoforms become abnormally phosphorylated and form the paired helical filament, the major fibrous component of the characteristic neurofibrillary lesions. We show here that Ser-202 (in the numbering of the longest human brain tau isoform) is a phosphorylation site that distinguishes fetal from adult tau and we identify it as one of the abnormal phosphorylation sites in Alzheimer disease. The abnormal phosphorylation of tau at Ser-202 in Alzheimer disease thus recapitulates normal phosphorylation during development.
Activation of phospholipase C beta 2 by the alpha and beta gamma subunits of trimeric GTP-binding protein.
Authors: Wu D , Katz A , Simon M I
Published: May 2006

Cotransfection assays were used to show that the members of the GTP-binding protein Gq class of alpha subunits could activate phospholipase C (PLC) beta 2. Similar experiments also demonstrated that G beta 1 gamma 1, G beta 1 gamma 5, and G beta 2 gamma 5 could activate the beta 2 isoform of PLC but not the beta 1 isoform, while G beta 2 gamma 1 did not activate PLC beta 2. To determine which portions of PLC beta 2 are required for activation by G beta gamma or G alpha, a number of PLC beta 2 deletion mutants and chimeras composed of various portions of PLC beta 1 and PLC beta 2 were prepared. We identified the N-terminal segment of PLC beta 2 with amino acid sequence extending to the end of the Y box as the region required for activation by G beta gamma and the C-terminal region as the segment containing amino acid sequences required for activation by G alpha. Furthermore, we found that coexpression of G alpha 16 and G beta 1 gamma 1 but not G beta 1 gamma 5 in COS-7 cells was able to synergistically activate recombinant PLC beta 2. We suggest that G alpha 16 may act together with free G beta 1 gamma 1 to activate PLC beta 2, while G alpha 16 may form heterotrimeric complexes with G beta 1 gamma 5 and be stabilized in an inactive form. We conclude that the regions of PLC beta 2 required for activation by G beta gamma and G alpha are physically separate and that the nature of the G beta subunit may play a role in determining the relative specificity of the G beta gamma complex for effector activation while the nature of the G gamma subunit isoform may be important for determining the affinity of the G beta gamma complex for specific G alpha proteins.
Delineation of two functional regions of transcription factor TFIIB.
Authors: Barberis A , Müller C W , Harrison S C , Ptashne M
Published: May 2006

Human transcription factor TFIIB, a protein of 316 amino acids, was subjected to limited proteolysis in order to define stable structural domains. We find that the C-terminal region of TFIIB, residues 106-316, is relatively stable, while the N-terminal region is very sensitive to proteases. Like full-length TFIIB, the stable domain, which we refer to as TFIIBc, interacts with the TATA-binding protein (TBP) on DNA. However, TFIIBc is unable to substitute for TFIIB in an in vitro transcription assay. We show by gel mobility-shift experiments that TFIIBc arrests formation of the transcription complex after binding to TBP, and we conclude that the N-terminal region of TFIIB, which is missing from TFIIBc, is responsible for the recruitment of RNA polymerase II to the promoter. We also show that TFIIBc inhibits transcription by competing with full-length TFIIB for the interaction with TBP, either in the presence or in the absence of the TBP-associated factors. The acidic transcriptional activator GAL4-VP16 does not favor the assembly of the functional transcription complex over the nonfunctional complex containing TFIIBc. Thus, if the function of GAL4-VP16 is enhancement of the interaction between TFIIB and the TFIID-DNA complex, then this function can also be exerted on the protease-resistant domain TFIIBc.
Change in the signal-response sequence responsible for asymmetric isolation between Drosophila planitibia and Drosophila silvestris.
Authors: Hoikkala A , Kaneshiro K
Published: May 2006

Drosophila planitibia and Drosophila silvestris form a species pair that is an example of species diverged through a founder event. These species exhibit asymmetric sexual isolation, courtships between D. planitibia males and D. silvestris females being more successful than courtships between D. silvestris males and D. planitibia females. When analyzing the signal-response courtship sequence in these species, we found that D. silvestris females responded to male circling by standing or preening while D. planitibia females required further signals from the male to stop walking. The main reason for the reduced mating success rate of D. silvestris males with D. planitibia females was that the females responded to male circling by walking and the males did not proceed to the head-under-wings (HUW) position of a walking female. Another critical phase in these courtships was the HUW position in D. silvestris, where males proceeded almost immediately to wing and leg vibration. The courtships between D. planitibia male and D. silvestris female proceeded in a signal-response coordination until the male went to the HUW position, where he fanned his wings for too long a period before proceeding to wing and leg vibration. Thus, it seems that the asymmetric isolation between D. planitibia (ancestral species) and D. silvestris (derived species) is mainly due to a loss of transitions in the signal-response chain of D. silvestris. A change in the behavior of the males in the HUW position has caused further isolation between the species in both directions.
A kinetic model for amyloid formation in the prion diseases: importance of seeding.
Authors: Come J H , Fraser P E , Lansbury P T
Published: May 2006

The transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by amyloid formation in the brain. The major amyloid protein is the prion protein (PrP). PrP and the beta-amyloid protein of Alzheimer disease share a similar sequence that, in both cases, may be responsible for the initiation of protein aggregation in vivo. We report here that a peptide based on this sequence in PrP (PrP96-111M) forms amyloid fibrils. The existence of a kinetic barrier to amyloid formation by this peptide was demonstrated, suggesting that formation of an ordered nucleus is the rate-determining step for aggregation. Seeding was demonstrated to occur with PrP96-111M amyloid fibrils but not with amyloid fibrils of a related peptide. This effect is consistent with the proposal that the aggregation of PrP, which characterizes TSE, involves a nucleation event analogous to the seeding of a crystallization.
Oxytricha telomere-binding protein: DNA-dependent dimerization of the alpha and beta subunits.
Authors: Fang G , Cech T R
Published: May 2006

A telomere-binding protein consisting of 56-kDa (alpha) and 41-kDa (beta) subunits binds specifically to the single-stranded T4G4T4G4 sequence at the termini of macronuclear DNA molecules in Oxytricha nova. The recent availability of separate alpha and beta polypeptides, expressed in Escherichia coli, allows investigation of the assembly of the telomeric complex ("telosome") from its individual components. By mixing wild-type subunits and electrophoretically distinct variants, we verify that the telosome contains one alpha and one beta subunit. By using telomeric DNAs of two lengths, we find that there is one DNA molecule per telosome. The DNA-protein and subunit-subunit interactions were studied by glycerol gradient sedimentation and chemical cross-linking. The formation of alpha-DNA and beta-DNA cross-links in the telomeric complex indicates that both subunits are in proximity to the DNA. When incubated together, both subunits exist predominantly as monomers in the absence of telomeric DNA. Upon binding to DNA, alpha and beta subunits directly interact with each other to form a heterodimer. We suggest that this DNA-dependent dimerization may allow each subunit to carry out distinct functions as a monomer, in addition to its participation in chromosome capping as part of the heterodimer.
Resistance of human squamous carcinoma cells to transforming growth factor beta 1 is a recessive trait.
Authors: Reiss M , Muñoz-Antonia T , Cowan J M , Wilkins P C , Zhou Z L , Vellucci V F
Published: May 2006

Because most human squamous carcinoma cell lines of the aerodigestive and genital tracts are refractory to the antiproliferative action of transforming growth factor beta 1 (TGF beta 1) in vitro, we have begun to identify the causes for resistance of squamous carcinoma cell lines to TGF beta 1 by using somatic cell genetics. Two stable hybrid cell lines (FaDu-HKc.1 and FaDu-HKc.2) were obtained by fusing a TGF beta 1-resistant human squamous carcinoma cell line, FaDu-HygR, with a human papilloma virus 16-immortalized, TGF beta 1-sensitive, human foreskin keratinocyte cell line, HKc-neoR. Whereas TGF beta 1 did not inhibit DNA synthesis in parental FaDu-HygR cells, it reduced DNA synthetic activity of HKc-neoR, FaDu-HKc.1, and FaDu-HKc.2 cells by 75-85% (IC50, 2-5 pM). Although squamous carcinoma cells express lower than normal levels of TGF beta 1 type II receptors on their cell surface, TGF beta 1 type II receptor mRNA was detected in all four cell lines. Recessive genes involved in TGF beta 1 signaling may be localized to the distal portion of chromosome 18q, as this was the sole chromosomal region of homozygous deletion in parental FaDu-HygR cells. Furthermore, our previous observation that mutant p53 decreases sensitivity of keratinocytes to TGF beta 1 was supported by the finding that the level of the mutant p53 protein expressed by the hybrid cell lines was greatly reduced. In summary, TGF beta 1 resistance of FaDu cells appears to be recessive and is presumably due to the loss of one or more post-receptor elements of the signaling pathway.
DNA target selectivity by the vitamin D3 receptor: mechanism of dimer binding to an asymmetric repeat element.
Authors: Towers T L , Luisi B F , Asianov A , Freedman L P
Published: May 2006

The 1,25-dihydroxyvitamin D3 receptor, like other members of the nuclear receptor superfamily, forms dimers in solution that are probably stabilized by a dyad symmetrical interface formed by the ligand-binding domain. This receptor, however, recognizes DNA targets that are not dyad symmetric but rather are organized as direct repeats of a hexameric sequence with a characteristic 3-bp spacing. Using molecular modeling and site-directed mutagenesis, we have identified regions within the vitamin D3 receptor zinc finger region that confer selectivity for direct repeats with appropriate spacing. Reflecting the organization of the DNA target, these regions, mapping to the tip of the first zinc finger module and the N and C termini of the second finger module, direct asymmetrical protein-protein contacts. A stereochemical model is proposed for these interactions.
Three scrapie prion isolates exhibit different accumulation patterns of the prion protein scrapie isoform.
Authors: DeArmond S J , Yang S L , Lee A , Bowler R , Taraboulos A , Groth D , Prusiner S B
Published: May 2006

To investigate the molecular basis of prion diversity, we inoculated transgenic mice expressing the Syrian hamster prion protein (PrP) with three distinct prion isolates. We compared the three isolates designated Sc237, 139H, and Me7H in Tg(SHaPrP)7 mice with clinical signs of scrapie because the incubation times with these mice are considerably shorter than the times found with hamsters. Each prion isolate produced a distinctive pattern of the scrapie isoform of PrP (PrPSc) accumulation, as determined by histoblotting, a technique developed for the regional mapping of PrPSc deposition. The PrPSc pattern with the Me7H isolate was particularly interesting because it appeared to be confined to the hypothalamus and related structures--including the interstitial nucleus of the stria terminalis, the paraventricular nucleus of the thalamus, and periaqueductal grey. Additionally, the regions of PrPSc accumulation remained highly restricted, even though the incubation time for Me7H scrapie was significantly longer than with Sc237 and 139H isolates. Neuropathological changes characterized by neuronal vacuolation and astrocytic gliosis were confined to those regions where PrPSc accumulated. These findings argue that the cell-specific propagation of prion isolates may be responsible for different properties exhibited by each of the isolates.
A cellular factor stimulates the DNA-binding activity of MyoD and E47.
Authors: Thayer M J , Weintraub H
Published: May 2006

We show that mixing purified MyoD and E47 proteins results in heterodimers that fail to bind DNA, even though MyoD and E47 homodimers can bind DNA efficiently. Addition of cell extracts or a specific fraction from a cell extract enables the heterodimer to bind DNA, but components in this fraction fail to enter the DNA complex. The activity is sensitive to heat and protease and is not ATP-dependent. The activity functions on E47 and MyoD homodimers and can stimulate DNA binding of the basic-helix-loop-helix region of MyoD. The effectiveness of the activity, for MyoD homodimers, depends on the exact DNA sequence of the binding site. Our results suggest that specific factors in the cell might control the DNA-binding properties of helix-loop-helix proteins.
A Drosophila single-strand DNA/RNA-binding factor contains a high-mobility-group box and is enriched in the nucleolus.
Authors: Hsu T , King D L , LaBonne C , Kafatos F C
Published: May 2006

We have isolated a Drosophila melanogaster cDNA encoding a high-mobility-group (HMG) box-containing protein. This protein shares 50% amino acid identity with the human putative structure-specific recognition protein, hSSRP. The gene encoding the D. melanogaster homolog, DssRP, is developmentally regulated and is expressed most abundantly in ovaries (nurse cells in particular). The protein is localized in nuclei and is particularly abundant in the nucleolus. In vitro binding studies using DssRP produced in bacteria showed that, despite expectation, the protein does not bind to structured DNA. Instead, it binds to single-stranded DNA and RNA, with highest affinity to nucleotides G and U.
Homologous recognition promoted by RecA protein via non-Watson-Crick bonds between identical DNA strands.
Authors: Rao B J , Radding C M
Published: May 2006

The RecA protein of Escherichia coli forms a nucleoprotein filament that promotes homologous recognition and subsequent strand exchange between a single strand and duplex DNA via a three-stranded intermediate. Recognition of homology within three-stranded nucleoprotein complexes, which is probably central to genetic recombination, is not well understood as compared with the mutual recognition of complementary single strands by Watson-Crick base pairing. Using oligonucleotides, we examined the determinants of homologous recognition within RecA nucleoprotein filaments. Filaments that contained a single strand of DNA recognized homology not only in a complementary oligonucleotide but also in an identical oligonucleotide, whether their respective sugar-phosphate backbones were antiparallel or parallel, and a filament that contained duplex DNA showed the same polymorphic versatility in the recognition of homology. Recognition of self by a filament that contains a single strand reveals that RecA filaments can recognize homology via non-Watson-Crick hydrogen bonds. Recognition of multiple forms of the same sequence by duplex DNA in the filament shows that it primarily senses base-sequence homology, and suggests that recognition can be accomplished prior to the establishment of new Watson-Crick base pairs in heteroduplex products. However, unlike the initial recognition of homology, strand exchange is stereospecific, requiring the proper antiparallel orientation of complementary strands.
Human immunodeficiency virus type 1 (HIV-1) strains selected for resistance against the HIV-1-specific [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro- 5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)]-beta-D-pentofurano syl (TSAO) nucleoside analogues retain sensitivity to HIV-1-specific nonnucleoside inhibitors.
Authors: Balzarini J , Karlsson A , Vandamme A M , Pérez-Pérez M J , Zhang H , Vrang L , Oberg B , Bäckbro K , Unge T , San-Félix A
Published: May 2006

We recently reported that a newly discovered class of nucleoside analogues--[2',5'-bis-O-(tert-butyldimethylsilyl)- 3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)]-beta-D - pentofuranosyl derivatives of pyrimidines and purines (designated TSAO)--are highly specific inhibitors of human immunodeficiency virus type 1 (HIV-1) and targeted at the nonsubstrate binding site of HIV-1 reverse transcriptase (RT). We now find that HIV-1 strains selected for resistance against three different TSAO nucleoside derivatives retain sensitivity to the other HIV-1-specific nonnucleoside derivatives (tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO), 1-[(2-hydroxyethoxy)methyl]-6-phenylthiothymine, nevirapine, and pyridinone L697,661, as well as to the nucleoside analogues 3'-azido-3'-deoxythymidine, ddI, ddC, and 9-(2-phosphonylmethoxyethyl)adenine. Pol gene nucleotide sequence analysis of the TSAO-resistant and -sensitive HIV-1 strains revealed a single amino acid substitution at position 138 (Glu-->Lys) in the RT of all TSAO-resistant HIV-1 strains. HIV-1 RT in which the Glu-138-->Lys substitution was introduced by site-directed mutagenesis and expressed in Escherichia coli could not be purified because of rapid degradation. However, HIV-1 RT containing the Glu-138-->Arg substitution was stable. It lost its sensitivity to the TSAO nucleosides but not to the other HIV-1-specific RT inhibitors (i.e., TIBO and pyridinone). Our findings point to a specific interaction of the 4''-amino group on the 3'-spiro-substituted ribose moiety of the TSAO nucleosides with the carboxylic acid group of glutamic acid at position 138 of HIV-1 RT.
Composite, large spirochetes from microbial mats: spirochete structure review.
Authors: Margulis L , Ashen J B , Solé M , Guerrero R
Published: May 2006

Phenomena previously unknown in free-living spirochetes are reported: large-sized cells with variable diameter (length to 100 microns, width between 0.4 and 3.0 microns), composite structure (smaller spirochetes inside larger ones), and positive phototropic behavior. These bacteria, Spirosymplokos, are compared with all other spirochete genera. The large spirochete, grown in mixed culture, was studied live and by transmission EM. The protoplasmic cylinder was replete with spherical granules 20-32 nm in diameter, and three to six periplasmic 26-nm flagella were inserted subterminally. Comparably granulated and flagellated small spirochetes were located inside the protoplasmic cylinder and in the periplasm of the large ones. When exposed to air, movement became erratic, protoplasmic cylinders retracted to lie folded inside the outer membrane, and refractile membranous structures formed. From one to four structures per still-moving spirochete were seen. Spirosymplokos was enriched from laboratory samples exposed to oxygen-rich and desiccating, but not dry, conditions for at least 4 mo after removal of microbial mat from the field.
Inhibition of T-cell reactivity to myasthenogenic epitopes of the human acetylcholine receptor by synthetic analogs.
Authors: Katz-Levy Y , Kirshner S L , Sela M , Mozes E
Published: May 2006

The synthetic peptides p195-212 and p259-271, representing amino acids 195-212 and 259-271 of the alpha subunit of the human acetylcholine receptor, preferentially stimulate T cells of patients with myasthenia gravis and are immunodominant T-cell epitopes in SJL and BALB/c mice, respectively. We designed and synthesized analogs of these peptides that contain single amino acid substitutions. An analog of peptide p195-212, no. 455 (Met-207-->Ala), was capable of inhibiting up to 100% of the proliferative responses of a p195-212-specific T-cell line originating from the high-responder strain SJL. Similarly, an analog of p259-271, no. 306 (Glu-262-->Lys), was capable of inhibiting up to 93% of the proliferative responses of the p259-271-specific T-cell line originating from high-responder BALB/c mice. Analog 306 also inhibited up to 43% of the proliferative responses of p259-271-primed lymph node cells in an in vitro proliferation assay. To test the in vivo inhibitory activity of the analogs, mice were primed with the myasthenogenic peptides in complete Freund's adjuvant concomitant with administration of the analogs in aqueous solution. Administration of analogs 455 and 306 led to decreased proliferative responses of up to 70% by peptide p199-212-primed lymph node cells and up to 85% by peptide p259-271-primed lymph node cells. Similar results were obtained whether the analogs were administered i.v. or i.p. Thus, these analogs are good candidates for specific immunomodulatory therapy for patients with myasthenia gravis.
Regression of established macroscopic liver metastases after in situ transduction of a suicide gene.
Authors: Caruso M , Panis Y , Gagandeep S , Houssin D , Salzmann J L , Klatzmann D
Published: May 2006

The herpes simplex virus type 1 thymidine kinase (HSV1-TK) converts nontoxic nucleoside analogs such as ganciclovir into phosphorylated compounds that act as chain terminators and specifically kill dividing cells. This property could be exploited for the treatment of tumors that are made up of rapidly dividing cells invading a nonproliferating tissue. For this purpose, specific expression of the suicide gene into dividing tumor cells can be further targeted by using retroviral-mediated gene transfer. We investigated whether the direct intratumoral transduction of a suicide gene might induce the elimination of malignant solid tumors. Rats with established macroscopic liver metastases were given an intratumoral injection of packaging cells producing either HSV1-TK- or lacZ-expressing recombinant retroviral particles. All rats were next treated with ganciclovir. A dramatic regression of the tumor volume was observed in the HSV1-TK-treated animals. The residual tumors were mostly made up of a massive fibrotic reaction, with the mean cancer cell mass being reduced approximately 60-fold compared to controls. In some animals, the residual tumors were devoid of cancer cells. This treatment efficacy appears in part due to a "bystander effect" in which phosphorylated ganciclovir could be transferred from cell to cell and to an active local immune reaction evidenced by massive infiltration of the tumors by macrophages and both CD4+ and CD8+ lymphocytes. This efficient therapeutic approach might be an ultimate treatment for disseminated liver metastases in humans and could also be applied to treatment of a large variety of solid tumors.
Ultraviolet-induced mutations in Cockayne syndrome cells are primarily caused by cyclobutane dimer photoproducts while repair of other photoproducts is normal.
Authors: Parris C N , Kraemer K H
Published: May 2006

We compared the contribution to mutagenesis in Cockayne syndrome (CS) cells of the major class of UV photoproducts, the cyclobutane pyrimidine dimer, to that of other DNA photoproducts by using the mutagenesis shuttle vector pZ189. Lymphoblastoid cell lines from the DNA repair-deficient disorders CS and xeroderma pigmentosum (XP) and a normal line were transfected with UV-treated pZ189. Cyclobutane dimers were selectively removed before transfection by photoreactivation (PR), leaving nondimer photoproducts intact. After UV exposure and replication in CS and XP cells, plasmid survival was abnormally reduced and mutation frequency was abnormally elevated. After PR, plasmid survival increased and mutation frequency in CS cells decreased to normal levels but remained abnormal in XP cells. Sequence analysis of > 200 mutant plasmids showed that with CS cells a major mutational hot spot was caused by unrepaired cyclobutane dimers. These data indicate that with both CS and XP cyclobutane dimers are major photoproducts generating reduced plasmid survival and increased mutation frequency. However, unlike XP, CS cells are proficient in repair of nondimer photoproducts. Since XP but not CS patients have a high frequency of UV-induced skin cancers, our data suggest that prevention of UV-induce skin cancers is associated with proficient repair of nondimer photoproducts.
Cloning a plant amino acid transporter by functional complementation of a yeast amino acid transport mutant.
Authors: Hsu L C , Chiou T J , Chen L , Bush D R
Published: May 2006

Amino acids are transported across the plasma membrane of plant cells by proton-amino acid symports. We report here the successful cloning of a neutral amino acid carrier by functional complementation. A histidine transport deletion mutant of Saccharomyces cerevisiae was transformed with an Arabidopsis thaliana cDNA library constructed in a yeast expression vector. Forty transformants, out of 10(5), allowed growth on a histidine-limiting medium. The acquired ability to grow on low histidine was shown to be strictly dependent on the protein encoded by the expression plasmid. Histidine and alanine transport activity were 10- to 20-fold greater in the transformants. The transport kinetics, inhibitor sensitivity, and substrate specificity match those of neutral system II, a neutral amino acid carrier we previously described in plasma membrane vesicles isolated from leaf tissue. The cDNA insert is 1.7 kb with an open reading frame that codes for a protein containing 486 amino acids with a calculated molecular mass of 52.9 kDa and three sites of potential N-linked glycosylation. Hydropathy analysis of the deduced amino acid sequence suggests this is an integral membrane protein with 10-12 membrane-spanning alpha-helices. Overall, the sequence of this amino acid carrier is not closely related to any other protein sequences in the GenBank data base. Interestingly, however, there are small regions of sequence that exhibit significant levels of similarity with at least seven other integral membrane proteins.
Photochemically induced cataracts in rat lenses can be prevented by AL-3823A, a glutathione peroxidase mimic.
Authors: Spector A , Wang G M , Wang R R
Published: May 2006

Oxidative stress is known to cause cataracts in lens culture systems and is believed to be an important factor contributing to human cataracts. In this communication, it is demonstrated that cataract development of cultured rat lenses produced as a result of photochemically induced oxidation in a 4% oxygen atmosphere similar to the native environment of the lens can be blocked by the transition metal complex AL-3823A. In this system, riboflavin is added to the medium as a photosensitizer. AL-3823A acts primarily as a glutathione peroxidase mimic, which catalytically metabolizes H2O2 and also has low superoxide dismutase-like activity. Measurements of H2O2, O2.-, and OH. indicate that appreciable levels of the first two of these oxidants and low levels of OH. are produced by this photochemical stressing system. The H2O2 concentrations are similar to those found in some patients with cataracts. The development of cataracts was followed over a 96-hr period. Transparency, hydration, glyceraldehyde-3-phosphate dehydrogenase activity, and protein and nonprotein thiol were monitored. All parameters show marked changes during the 96-hr period. However, in the presence of 200 microM AL-3823A, no difference between control and light-exposed lenses was observed with respect to these parameters. The results suggest that in vivo human cataract development caused by oxidative stress may be prevented by compounds of this type.
Association of the low molecular weight GTP-binding protein rap2B with the cytoskeleton during platelet aggregation.
Authors: Torti M , Ramaschi G , Sinigaglia F , Lapetina E G , Balduini C
Published: May 2006

The intracellular distribution of the low-molecular-weight GTP-binding protein rap2B was investigated in resting and agonist-activated human platelets. In both cases, platelets were lysed by Triton X-100, and cell fractions were obtained by differential centrifugations. Using a specific polyclonal antiserum, we found that rap2B in resting platelets was completely detergent-soluble. When platelets were aggregated with thrombin, the thromboxane analogue U46619, or the Ca(2+)-ATPase inhibitor thapsigargin, a significant amount of rap2B became associated with the cytoskeleton. This association was paralleled by a decrease of rap2B in the Triton X-100-soluble fraction. Translocation of rap2B to the cytoskeleton strictly depended on platelet aggregation, and maximal incorporation was found when approximately 50% aggregation was measured. Inhibition of fibrinogen binding to the glycoprotein IIb-IIIa complex completely prevented the interaction of rap2B with the cytoskeleton. These results clearly demonstrate that changes in the intracellular localization of rap2B occur during platelet activation and represent evidence that this low molecular weight GTP-binding protein may be involved in platelet function.
Amino acid residues essential for biological activity of a peptide derived from a major histocompatibility complex class I antigen.
Authors: Stagsted J , Mapelli C , Meyers C , Matthews B W , Anfinsen C B , Goldstein A , Olsson L
Published: May 2006

The stimulatory activity of peptides from the alpha 1 domain of the major histocompatibility complex (MHC) class I antigen on adipose cell glucose transport was previously shown to require a preformed, ordered conformation of the peptide. The two peptides studied previously were Dk-(61-85) (ERETQIAKGNEQSFRVDLRTLLRYY) and Dk-(69-85). We now show that systematic alanine substitution in Dk-(69-85) identifies residues that are essential for biological activity. Ordered structure of the peptides, estimated by circular dichroism, was found in all peptides with activity, but with a complex variety of spectra. Inactive peptides were in either a random coil or an ordered structure. Ordered structure, therefore, is not sufficient for activity. The peptides self-interact in the absence of cells and form aggregates that precipitate upon centrifugation. The tendency to aggregate is correlated with biological potency. Only MHC class I molecules have significant homology to the peptides studied here. The peptide self-interaction suggests that the biological effects in cells, which result from inhibition of receptor and transporter internalization, may be due to the binding (tantamount to self-interaction) of the peptide to the homologous sequences in the alpha 1 domain of the MHC class I molecule.
The Tat protein of human immunodeficiency virus type 1, a growth factor for AIDS Kaposi sarcoma and cytokine-activated vascular cells, induces adhesion of the same cell types by using integrin receptors recognizing the RGD amino acid sequence.
Authors: Barillari G , Gendelman R , Gallo R C , Ensoli B
Published: May 2006

Spindle-shaped cells of vascular origin are the probable tumor cells of Kaposi sarcoma (KS). These cells, derived from patients with KS and AIDS, proliferate in response to extracellular Tat protein of human immunodeficiency virus type 1. Normal vascular cells, believed to be the progenitors of AIDS-KS cells, acquire spindle morphology and become responsive to the mitogenic effect of Tat after culture with inflammatory cytokines. Such cytokines are increased in human immunodeficiency virus type 1-infected people, suggesting that immune stimulation (rather than immune deficiency) is a component of AIDS-KS pathogenesis. Here we show that (i) Tat promotes adhesion of AIDS-KS and normal vascular cells; (ii) adhesion of normal vascular cells to Tat is induced by exposure of the cells to the same cytokines; (iii) adhesion is associated with the amino acid sequence RGD of Tat through a specific interaction with the integrin receptors alpha 5 beta 1 and alpha v beta 3, although it is augmented by the basic region; and (iv) the expression of both integrins is increased by the same cytokines that promote these cells to acquire spindle morphology and become responsive to the adhesion and growth effects of Tat. The results also suggest that RGD-recognizing integrins mediate the vascular cell-growth-promoting effect of Tat.
Myelin/oligodendrocyte glycoprotein is a member of a subset of the immunoglobulin superfamily encoded within the major histocompatibility complex.
Authors: Pham-Dinh D , Mattei M G , Nussbaum J L , Roussel G , Pontarotti P , Roeckel N , Mather I H , Artzt K , Lindahl K F , Dautigny A
Published: May 2006

Myelin/oligodendrocyte glycoprotein (MOG) is found on the surface of myelinating oligodendrocytes and external lamellae of myelin sheaths in the central nervous system, and it is a target antigen in experimental autoimmune encephalomyelitis and multiple sclerosis. We have isolated bovine, mouse, and rat MOG cDNA clones and shown that the developmental pattern of MOG expression in the rat central nervous system coincides with the late stages of myelination. The amino-terminal, extracellular domain of MOG has characteristics of an immunoglobulin variable domain and is 46% and 41% identical with the amino terminus of bovine butyrophilin (expressed in the lactating mammary gland) and B-G antigens of the chicken major histocompatibility complex (MHC), respectively; these proteins thus form a subset of the immunoglobulin superfamily. The homology between MOG and B-G extends beyond their structure and genetic mapping to their ability to induce strong antibody responses and has implications for the role of MOG in pathological, autoimmune conditions. We colocalized the MOG and BT genes to the human MHC on chromosome 6p21.3-p22. The mouse MOG gene was mapped to the homologous band C of chromosome 17, within the M region of the mouse MHC.
Sodium azide mutagenesis: preferential generation of A.T-->G.C transitions in the barley Ant18 gene.
Authors: Olsen O , Wang X , von Wettstein D
Published: May 2006

The molecular basis for the absence of anthocyanins and proanthocyanidins in four independent sodium azide-induced ant18 mutants of barley was examined by sequencing the gene encoding dihydroflavonol 4-reductase in these mutants. Sodium azide generated 21 base substitutions, which corresponds to 0.17% of the 12,704 nucleotides sequenced. Of the substitutions, 86% were nucleotide transitions, and 14% were transversions. A.T-->G.C base pair transitions were about 3 times more frequent than G.C-->A.T transitions. No deletions or mutation hot spots were found. The absence of dihydroflavonol 4-reductase activity in ant18-159, ant18-162, and ant18-164 plants is caused by missense mutations in the respective genes. By using microprojectile bombardment, a plasmid harboring the wild-type Ant18 gene was introduced into ant18-161 mutant cells and resulted in the development of anthocyanin pigmentation, which demonstrates that the mutation is corrected by expression of the introduced gene. On the other hand, a plasmid derivative with the two ant18-161-specific base transitions at the 5' splice site of intron 3 prevented complementation. It is concluded that the absence of detectable mRNA for dihydroflavonol 4-reductase in ant18-161 cells is due to the mutations in the pre-mRNA splice donor site.
Transactivation by hepatitis B virus X protein is promiscuous and dependent on mitogen-activated cellular serine/threonine kinases.
Authors: Cross J C , Wen P , Rutter W J
Published: May 2006

The X protein of hepatitis B virus (HBV-X) can act as a transactivator of transcription but its mechanism of action remains obscure. We have analyzed HBV-X transactivation in several cell types using 13 unrelated viral and cellular promoters and found that transactivation is more or less apparent in most cell types and is promiscuous and unrelated to specific sequence motifs within the target promoters. In general, though, HBV-X appears to act on enhancer elements since HBV-X had no effect on a minimal promoter, whereas HBV-X was able to transactivate after insertion of an AP-1 minienhancer. Several lines of evidence exclude the possibility that HBV-X interacts directly with the AP-1 enhancer or its binding proteins and suggest that the proximal target of HBV-X is peripheral to the transcription complex. This hypothesis is supported by the observation that inhibition of serine/threonine kinases, which regulate AP-1 activity (phorbol ester down-regulation or staurosporine inhibition of protein kinase C and a dominant negative mutant of Raf-1), blocked the ability of HBV-X to transactivate without affecting basal promoter activity. Furthermore, basal transcription from the AP-1-dependent promoter was increased by overexpression of protein kinase C and Raf-1 but HBV-X was unable to further stimulate, indicating that these kinases act subsequently to HBV-X. These data suggest that transactivation by HBV-X is an indirect result of the activation of cellular serine/threonine kinases including protein kinase C and Raf-1. This mode of action implies that HBV-X may affect other cellular processes, besides transcription, that are regulated by these kinases.
Expression of an antisense prosystemin gene in tomato plants reduces resistance toward Manduca sexta larvae.
Authors: Orozco-Cardenas M , McGurl B , Ryan C A
Published: May 2006

The growth rates of Manduca sexta (tobacco hornworm) larvae feeding on tomato plants constitutively expressing a prosystemin antisense gene were approximately 3 times higher than growth rates of larvae feeding on nontransformed control plants. The levels of proteinase inhibitor I and inhibitor II proteins in leaves of tomato plants expressing the antisense prosystemin gene remained at undetectable levels until the sixth day of larval feeding and then increased throughout the plants to 100-125 microg/g of leaf tissue after 14 days. In control plants, levels of proteinase inhibitor I and II proteins increased rapidly from the second day of larval feeding and by the eighth day contained levels of 225 microg/g of leaf tissue and 275 microg/g of leaf tissue, respectively, and then increased slowly thereafter. Prosystemin mRNA levels in antisense and control plants after 6 days and 12 days of larval feeding correlated with levels of inhibitor I and II protein levels. These experiments demonstrate that resistance of plants toward an insect pest can be modulated by genetically engineering a gene encoding a component of the inducible systemic signaling system regulating a plant defensive response.
Cloning and tissue-specific functional characterization of the promoter of the rat diazepam binding inhibitor, a peptide with multiple biological actions.
Authors: Kolmer M , Alho H , Costa E , Pani L
Published: May 2006

Diazepam binding inhibitor (DBI) is a 10-kDa polypeptide that regulates mitochondrial steroidogenesis, glucose-induced insulin secretion, metabolism of acyl-CoA esters, and the action of gamma-aminobutyrate on GABAA receptors. To investigate the regulation of DBI gene expression, three positive clones were isolated from a rat genomic library. One of them contained a DBI genomic DNA fragment encompassing 4 kb of the 5' untranslated region, the first two exons, and part of the second intron of the DBI gene. Two other overlapping clones contained a processed DBI pseudogene. Several transcription initiation sites were detected by RNase protection and primer extension assays. Different tissues exhibited clear differences in the efficiencies of transcription startpoint usage. Transient expression experiments using DNA fragments of different length from the 5' untranslated region of the DBI gene showed that basal promoter activity required 146 bp of the proximal DBI sequence, whereas full activation was achieved with 423 bp of the 5' untranslated region. DNase I protection experiments with liver nuclear proteins demonstrated three protected regions at nt -387 to -333, -295 to -271, and -176 to -139 relative to the ATG initiation codon; in other tissues the pattern of protection was different. In gel shift assays the most proximal region (-176 to -139) was found to bind several general transcription factors as well as cell type-restricted nuclear proteins which may be related to specific regulatory patterns in different tissues. Thus, the DBI gene possesses some features of a housekeeping gene but also includes a variable regulation which appears to change with the function that it subserves in different cell types.
TATA-binding protein and the retinoblastoma gene product bind to overlapping epitopes on c-Myc and adenovirus E1A protein.
Authors: Hateboer G , Timmers H T , Rustgi A K , Billaud M , van 't Veer L J , Bernards R
Published: May 2006

Using a protein binding assay, we show that the amino-terminal 204 amino acids of the c-Myc protein interact directly with a key component of the basal transcription factor TFIID, the TATA box-binding protein (TBP). Essentially the same region of the c-Myc protein also binds the product of the retinoblastoma gene, the RB protein. c-Myc protein coimmunoprecipitates with TBP in lysates of mammalian cells, demonstrating that the proteins are also complexed in vivo. A short peptide that spans the RB binding site of the E7 protein of human papilloma virus type 16 interferes with the binding of c-Myc to TBP. The same peptide also blocks binding of adenovirus E1A protein to TBP, suggesting that c-Myc and E1A bind to RB and TBP through overlapping epitopes. Furthermore, we show that binding of RB to E1A prevents association of E1A with TBP. Our data suggest that one of the functions of RB and RB-like proteins is to prevent interaction of viral and cellular oncoproteins, such as c-Myc and E1A, with TBP.
Evidence for a chimeric nature of nuclear genomes: eubacterial origin of eukaryotic glyceraldehyde-3-phosphate dehydrogenase genes.
Authors: Martin W , Brinkmann H , Savonna C , Cerff R
Published: May 2006

Higher plants process two distinct, nuclear gene-encoded glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins, a Calvin-cycle enzyme active within chloroplasts and a glycolytic enzyme active within the cytosol. The gene for the chloroplast enzyme was previously suggested to be of endosymbiotic origin. Since the ancestors of plastids were related to cyanobacteria, we have studied GAPDH genes in the cyanobacterium Anabaena variabilis. Our results confirm that the nuclear gene for higher plant chloroplast GAPDH indeed derives from the genome of a cyanobacterium-like endosymbiont. But two additional GAPDH genes were found in the Anabaena genome and, surprisingly, one of these sequences is very similar to nuclear genes encoding the GAPDH enzyme of glycolysis in plants, animals, and fungi. Evidence that the eukaryotic nuclear genes for glycolytic GAPDH, as well as the Calvin-cycle genes, are of eubacterial origin suggests that eukaryotic genomes are more highly chimeric than previously assumed.
Modulation of early sensory processing in human auditory cortex during auditory selective attention.
Authors: Woldorff M G , Gallen C C , Hampson S A , Hillyard S A , Pantev C , Sobel D , Bloom F E
Published: May 2006

Neuromagnetic fields were recorded from human subjects as they listened selectively to sequences of rapidly presented tones in one ear while ignoring tones of a different pitch in the opposite ear. Tones in the attended ear evoked larger magnetic brain responses than did unattended tones in the latency ranges 20-50 msec and 80-130 msec poststimulus. Source localization techniques in conjunction with magnetic resonance imaging placed the neural generators of these early attention-sensitive brain responses in auditory cortex on the supratemporal plane. These data demonstrate that focused auditory attention in humans can selectively modulate sensory processing in auditory cortex beginning as early as 20 msec poststimulus, thereby providing strong evidence for an "early selection" mechanism of auditory attention that can regulate auditory input at or before the initial stages of cortical analysis.