bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of TFIID to the CYC1 TATA boxes in yeast occurs independently of upstream activating sequences.
Authors: Chen J , Ding M , Pederson D S
Published: May 2006

Functional transcription initiation complexes can be assembled in vitro without the aid of regulatory factors that bind to upstream activating sequences. However, promoters that lack upstream activating sequences are transcribed poorly if at all in vivo, suggesting that regulatory factors are necessary for the assembly of transcription initiation complexes in cells. To test this possibility, we asked whether the general transcription factor TFIID can bind to a promoter in yeast that lacks upstream activating sequences and is transcriptionally inactive. Analysis of an inactive CYC1 core promoter by high-resolution genomic footprinting revealed efficient binding of TFIID to either of two TATA box elements. Addition of a heat shock element rendered this promoter highly responsive to induction of transcription by heat shock but did not alter the TATA box footprints in the core promoter. Inactivation of all but one TATA box by site-directed mutagenesis did not prevent TFIID from binding to the remaining wild-type TATA box independently of regulatory sequences. These results indicate that upstream regulatory factors are not required for the in vivo binding of TFIID to the CYC1 promoter and that binding of TFIID to DNA is not necessarily a rate-limiting step in the activation of transcription in cells. Differences in chromatin structure may account for why regulatory transcription factors are required for the binding of TFIID to some promoters but not to others.
Synthesis and biological activities of highly potent antagonists of growth hormone-releasing hormone.
Authors: Zarandi M , Horvath J E , Halmos G , Pinski J , Nagy A , Groot K , Rekasi Z , Schally A V
Published: May 2006

In the search for antagonists of human growth hormone-releasing hormone (hGHRH) with high activity, 22 analogs were synthesized by solid-phase methods, purified, and tested biologically. Within the N-terminal sequence of 28 or 29 amino acids of hGHRH, all the analogs contained D-Arg2, Phe(4-Cl)6 (para-chlorophenylalanine), Abu15 (alpha-aminobutyric acid), and Nle27 and most of them had Agm29 (agmatine) substituents. All the peptides, except one, were acylated at the N terminus with different hydrophobic acids--e.g., isobutyric acid (Ibu) or 1-naphthylacetic acid (Nac) in order to study the effect of N-terminal acylation on the antagonistic activity. In the superfused rat pituitary cell system, all the analogs inhibited more powerfully the GHRH-induced growth hormone (GH) release than the standard GHRH antagonist [Ac-Tyr1,D-Arg2]hGHRH-(1-29)NH2. Antagonists [Ibu0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-28) Agm (MZ-4-71), [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-28) Agm (MZ-4-243), [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-29) NH2 (MZ-4-169), [Nac0-His1,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]-hGH RH-(1-29)NH2 (MZ-4-181), and [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27,Asp28]hGH RH-(1-28)Agm (MZ-4-209) inhibited GH release at 3 x 10(-9) M. Among these peptides, MZ-4-243, MZ-4-169, and MZ-4-181 were also long acting in vitro. Antagonist MZ-4-243 inhibited GH release 100 times more powerfully than the standard antagonist and was the most potent in vitro among GHRH antagonists synthesized. Analogs with high inhibitory effects in vitro were also found to have high affinities to rat pituitary GHRH receptors. In experiments in vivo, antagonists [Ibu0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]-hGHRH-(1-28 )Agm (MZ-4-71), [Nac0,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHRH-(1-29) NH2 (MZ-4-169), and [Nac0-His1,D-Arg2,Phe(4-Cl)6,Abu15,Nle27]hGHR H-(1-29)NH2 (MZ-4-181) induced a significantly greater inhibition of GH release than the standard antagonist. In view of their high antagonistic activity and prolonged duration of action, some of these antagonists of GHRH may find clinical applications, including treatment of certain endocrine disorders and insulin-like growth factor I-dependent tumors.
Key role for pregnenolone in combination therapy that promotes recovery after spinal cord injury.
Authors: Guth L , Zhang Z , Roberts E
Published: May 2006

Controlled compressive injury to rat spinal cord was chosen to test therapies that might attenuate the progression of tissue destruction and locomotor deficits that characteristically occur after spinal injury. A highly significant reduction of damage was achieved by immediate postinjury treatment with a combination of the following: an antiinflammatory substance, indomethacin; a stimulator of cytokine secretion, bacterial lipopolysaccharide; and the parent steroid, from which all other steroids arise, pregnenolone. This treatment reduced histopathological changes, spared tissue from secondary injury, and increased restoration of motor function. Remarkably, 11 of 16 of the animals treated with the above combination were able to stand and walk at 21 days after injury, 4 of them almost normally. The results were far superior to those obtained in controls or in animals to which the substances were given separately or in combination of two. This approach may prove to be applicable to nervous system injury, in general, and to injury in other tissues.
Three chemically distinct types of oxidants formed by iron-mediated Fenton reactions in the presence of DNA.
Authors: Luo Y , Han Z , Chin S M , Linn S
Published: May 2006

Exposure of Escherichia coli to H2O2 leads to two kinetically distinguishable modes of killing: mode I killing occurs maximally near 2 mM H2O2, whereas mode II killing is essentially independent of H2O2 concentrations up to 20 mM. A major portion of H2O2 toxicity is attributed to DNA damage caused by the iron-mediated Fenton reaction. By studying DNA damage during Fenton reactions in vitro, the same complex kinetics were observed and three types of oxidants were distinguished based upon their reactivities toward H2O2 and alcohols and upon iron-chelator effects. Type I oxidants are sensitive to H2O2 but moderately resistant to ethanol; type II oxidants are resistant to both H2O2 and ethanol; type III oxidants are sensitive to H2O2, ethanol, and t-butanol. To explain these results, we hypothesize that type I oxidants are generated upon Fe2+ associated with DNA only through electrostatic interactions and cause mode I killing of E. coli; type II oxidants arise upon Fe2+, which is at least partially base-associated, and cause mode II killing; type III oxidants arise on Fe2+ free in solution and probably do not cause killing. Therefore, particular interactions of DNA with transition metals should be considered to be an integral part of the chemistry and toxicity of H2O2.
Functional three-domain single-chain T-cell receptors.
Authors: Chung S , Wucherpfennig K W , Friedman S M , Hafler D A , Strominger J L
Published: May 2006

T-cell receptors (TCRs) are membrane anchored heterodimers structurally related to antibody molecules. Single-chain antibodies can be engineered by linking the two variable domains, which fold properly by themselves. However, proper assembly of the variable domains of a human TCR (V alpha and V beta) that recognize the HLA-DR2b/myelin basic protein-(85-99) peptide complex was critically dependent on the addition of a third domain, the constant region of the TCR beta chain (C beta), to the single-chain construct. Single-chain molecules with the three-domain design, but not those with the two-domain design, expressed in a eukaryotic cell as chimeric molecules linked either to glycosyl phosphatidylinositol or to the transmembrane/cytoplasmic domains of the CD3 zeta chain were recognized by a conformation-sensitive monoclonal antibody. The chimeric three-domain single-chain TCR linked to CD3 zeta chain signaled in response to both the specific HLA-DR/peptide and the HLA-DR/superantigen staphylococcal enterotoxin B complexes. Thus, by using this three-domain design, functional single-chain TCR molecules were expressed with high efficiency. The lipid-linked single-chain TCR was solubilized by enzymatic cleavage and purified by affinity chromatography. The apparent requirement of the constant domain for cooperative folding of the two TCR variable domains may reflect significant structural differences between TCR and antibody molecules.
The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase.
Authors: Douglas C M , Foor F , Marrinan J A , Morin N , Nielsen J B , Dahl A M , Mazur P , Baginsky W , Li W , el-Sherbeini M
Published: May 2006

In Saccharomyces cerevisiae, mutations in FKS1 confer hypersensitivity to the immunosuppressants FK506 and cyclosporin A, while mutations in ETG1 confer resistance to the cell-wall-active echinocandins (inhibitors of 1,3-beta-D-glucan synthase) and, in some cases, concomitant hypersensitivity to the chitin synthase inhibitor nikkomycin Z. The FKS1 and ETG1 genes were cloned by complementation of these phenotypes and were found to be identical. Disruption of the gene results in (i) a pronounced slow-growth phenotype, (ii) hypersensitivity to FK506 and cyclosporin A, (iii) a slight increase in sensitivity to echinocandin, and (iv) a significant reduction in 1,3-beta-D-glucan synthase activity in vitro. The nucleotide sequence encodes a 215-kDa polypeptide predicted to be an integral membrane protein with 16 transmembrane helices, consistent with previous observations that the etg1-1 mutation results in echinocandin-resistant glucan synthase activity associated with the nonextractable membrane fraction of the enzyme. These results suggest that FKS1 encodes a subunit of 1,3-beta-D-glucan synthase. The residual activity present in the disruption mutant, the nonessential nature of the gene, and results of Southern blot hybridization analysis point to the existence of a glucan synthase isozyme.
Synaptic relationship between substance P and the substance P receptor: light and electron microscopic characterization of the mismatch between neuropeptides and their receptors.
Authors: Liu H , Brown J L , Jasmin L , Maggio J E , Vigna S R , Mantyh P W , Basbaum A I
Published: May 2006

Light microscopic studies have demonstrated significant mismatches in the location of neuropeptides and their respective binding sites in the central nervous system. In the present study we used an antiserum raised against a synthetic peptide corresponding to the carboxyl-terminal tail of the substance P (SP) receptor (SPR) to further explore the relationship between a neuropeptide and its receptor. Light microscopy revealed an excellent correlation between the patterns of SPR immunoreactivity and of 125I-labeled SPR-binding sites in the central nervous system. The SPR appeared to be exclusively expressed by neurons; in fact, the SPR decorates the somatic and dendritic surface of neurons, producing Golgi-like images. Electron microscopic analysis in cortex, striatum, and spinal cord revealed that approximately 70% of the surface membrane of immunoreactive neurons is SPR laden. Simultaneous electron microscopic labeling of SP and SPR demonstrated significant mismatch at the synaptic level. Although some SP terminals contacted SPR-immunoreactive membrane, no more than 15% of the SPR-laden membrane apposed synaptic terminals. These results suggest that in contrast to more "classical" central and peripheral nervous system synapses, wherein the receptor immediately apposes the site of neurotransmitter storage and release, much of the surface of SPR-expressing neurons can be targeted by SP that diffuses a considerable distance from its site of release.
Analysis of the T-cell receptor beta-chainvariable-region (V beta) repertoire in monozygotic twins discordant for humanimmunodeficiency virus: evidence for perturbations of specific V beta segmentsin CD4+ T cells of the virus-positive twins.
Authors: Rebai N , Pantaleo G , Demarest J F , Ciurli C , Soudeyns H , Adelsberger J W , Vaccarezza M , Walker R E , Sekaly R P , Fauci A S
Published: May 2006

We analyzed the T-cell receptor (TCR) V beta
repertoire in human immunodeficiency virus type 1 (HIV-1)-infected individuals
at different stages of disease. To circumvent the effect of HLA and other loci
on the expressed TCR repertoire, we compared the TCR repertoire in nine pairs of
monozygotic twins who were discordant for HIV infection. A semiquantitative
polymerase chain reaction (PCR) assay and flow cytometry enabled us to show
distinct differences in the V beta repertoire in the HIV-positive twin compared
with the HIV-negative twin. By combining PCR and cytofluorometry, these
differences were restricted to a specific set of TCR V beta segments, with
members of the V beta 13 family perturbed in six out of seven cases and those of
the V beta 21 family perturbed in four out of seven cases studied. Most of the
other V beta families remained unchanged. Our results provide direct evidence
for a skewed TCR repertoire in HIV infection.
Organization of instabilities in multispecies systems,a test of hierarchy theory.
Authors: Waltho N , Kolasa J
Published: May 2006

The hierarchy theory predicts that system
components functioning at lower levels of hierarchy operate or change at higher
rates than the components at the level(s) above. If this prediction is correct,
then interpretation of stability in complex ecological systems may be in need of
revision. We test the prediction using a model of hierarchical structure of
habitat and a coral reef fish community. We found that the variability of
ecological range and abundance increases exponentially from habitat generalists
(high in hierarchy) to specialists (low in hierarchy), as postulated by the
hierarchy theory. Our result suggests that community stability is a composite
property and should be evaluated by considering the hierarchical structure of
that community.
A multisubunit complex containing the SWI1/ADR6,SWI2/SNF2, SWI3, SNF5, and SNF6 gene products isolated fromyeast.
Authors: Cairns B R , Kim Y J , Sayre M H , Laurent B C , Kornberg R D
Published: May 2006

A complex containing the products of the
SWI1/ADR6, SWI2/SNF2, SWI3, SNF5, and SNF6 genes and four additional
polypeptides has been purified from extracts of the yeast Saccharomyces
cerevisiae. Physical association of these proteins was demonstrated by
copurification and coimmunoprecipitation. A potent DNA-dependent ATPase
copurified with the complex, and this activity was evidently associated with
SWI2/SNF2.
Enthalpy of hydrogen bond formation in aprotein-ligand binding reaction.
Authors: Connelly P R , Aldape R A , Bruzzese F J , Chambers S P , Fitzgibbon M J , Fleming M A , Itoh S , Livingston D J , Navia M A , Thomson J A
Published: May 2006

Parallel measurements of the thermodynamics
(free-energy, enthalpy, entropy and heat-capacity changes) of ligand binding to
FK506 binding protein (FKBP-12) in H2O and D2O have been performed in an effort
to probe the energetic contributions of single protein-ligand hydrogen bonds
formed in the binding reactions. Changing tyrosine-82 to phenylalanine in
FKBP-12 abolishes protein-ligand hydrogen bond interactions in the FKBP-12
complexes with tacrolimus or rapamycin and leads to a large apparent enthalpic
stabilization of binding in both H2O and D2O. High-resolution crystallographic
analysis reveals that two water molecules bound to the tyrosine-82 hydroxyl
group in unliganded FKBP-12 are displaced upon formation of the protein-ligand
complexes. A thermodynamic analysis is presented that suggests that the removal
of polar atoms from water contributes a highly unfavorable enthalpy change to
the formation of C=O...HO hydrogen bonds as they occur in the processes of
protein folding and ligand binding. Despite the less favorable enthalpy change,
the entropic advantage of displacing two water molecules upon binding leads to a
slightly more favorable free-energy change of binding in the reactions with
wild-type FKBP-12.
The kinetics of spontaneous DNA branch migration.
Authors: Panyutin I G , Hsieh P
Published: May 2006

An important step in genetic recombination is DNA branch migration, the movement of the Holliday junction or exchange point between two homologous duplex DNAs. We have determined kinetic parameters of spontaneous branch migration as a function of temperature and ionic conditions. The branch migration substrates consist of two homologous duplex DNAs each having two single-strand tails at one end that are complementary to the corresponding single-strand tails of the other duplex. Upon rapid annealing of the two duplex DNAs, a four-stranded intermediate is formed that has a Holliday junction at one end of the duplexes. Branch migration to the opposite end of the duplexes results in complete strand exchange and formation of two duplex products. The rate of branch migration is exceedingly sensitive to the type of metal ions present. In magnesium, branch migration is quite slow with a step time, tau, equal to 300 msec at 37 degrees C. Surprisingly, branch migration in the absence of magnesium was 1000 times faster. Despite this difference in rates, apparent activation energies for the branch migration step in the presence and absence of magnesium are similar. Since metal ions have a profound effect on the structure of the Holliday junction, it appears that the structure of the branch point plays a key role in determining the rate of spontaneous DNA branch migration. We discuss the role of proteins in promoting the branch migration step during homologous recombination.
Arachidonate is a potent modulator of human heat shock gene transcription.
Authors: Jurivich D A , Sistonen L , Sarge K D , Morimoto R I
Published: May 2006

Cell and tissue injury activate the inflammatory response through the action(s) of arachidonic acid and its metabolites, leading to the expression of acute-phase proteins and inflammatory cytokines. At the molecular level, little is known how arachidonic acid regulates the inflammatory response. As inflammation is also associated with local increase in tissue temperatures, we examined whether arachidonic acid was directly involved in the heat shock response. Extracellular exposure to arachidonic acid induced heat shock gene transcription in a dose-dependent manner via acquisition of DNA-binding activity and phosphorylation of heat shock factor 1 (HSF1). In addition, exposure of cells to low concentrations of arachidonic acid, which by themselves did not induce HSF1 DNA-binding activity, reduced the temperature threshold for HSF1 activation from elevated temperatures which are not physiologically relevant (> 42 degrees C) to temperatures which can be attained during the febrile response (39-40 degrees C). These results indicate that elevated heat shock gene expression is a direct consequence of an arachidonic acid-mediated cellular response.
Predictive capability of proportional hazards regression.
Authors: O'Quigley J , Flandre P
Published: May 2006

A measure of the predictive capability of a proportional hazards regression is derived. The measure is based on the residuals appropriate to proportional hazards regression. A population version is presented and can be seen not to depend on the censoring mechanism under the provision that any such censoring be independent or conditionally independent of the failure mechanism given the covariate. For the special case of a Weibull regression model, for which the covariate distribution follows binary, uniform, normal, or exponential laws, we derive analytic results. These alone give credence to the measure which can be seen to reflect strength of regression effect, as quantified by the parameter estimate, although on a scale between 0 and 1, independently of the intercept or shape parameter of the particular Weibull law and only weakly dependent on the covariate distribution. Extensions to partial and multiple measures of predictive ability are straightforward. An example is provided.
Minimal representations of E6, E7, and E8 and the generalized Capelli identity.
Authors: Brylinski R , Kostant B
Published: May 2006

We explicitly construct, in a uniform fashion, the (unique) minimal and spherical representation pi0 of the split real Lie group of exceptional type E6, E7, or E8. We obtain several algebraic and analytic results about pi0.
Truncated desmin in PtK2 cells induces desmin-vimentin-cytokeratin coprecipitation, involution of intermediate filament networks, and nuclear fragmentation: a model for many degenerative diseases.
Authors: Yu K R , Hijikata T , Lin Z X , Sweeney H L , Englander S W , Holtzer H
Published: May 2006

The earliest expression of truncated desmin in transfected PtK2 cells results in the formation of dispersed microprecipitates containing not only the truncated desmin, but also endogenous vimentin and cytokeratin proteins. Desmin microprecipitates without vimentin or vimentin microprecipitates without desmin are not observed. The microprecipitates involving cytokeratin invariably are also positive for desmin and vimentin. Over time, the precipitates enlarge into 1- to 2-microns spheroids and then fuse into amorphous chimeric juxtanuclear masses that can occupy > 30% of the cell volume. Concurrently, first the vimentin and then the cytokeratin networks are resorbed. The chimeric precipitates are not recognized or marked for degradation by the lysosomal system. Ultimately the cell nucleus fragments and the cell dies. Similar protein complexes appear in many human and animal pathologies, suggesting that a similar protein-precipitation sequence initiated by the introduction of a mutationally or environmentally altered protein molecule is at work.
Use of genetic recombination as a reporter of gene expression.
Authors: Camilli A , Beattie D T , Mekalanos J J
Published: May 2006

An understanding of the patterns of gene expression in response to specific environmental signals can yield insight into a variety of complex biological systems such as microbial-host interactions, developmental cycles, cellular differentiation, ontogeny, etc. To extend the utility of the reporter gene fusion approach to such studies, we have constructed a gene expression reporter cassette that permits the generation of transcriptional fusions to tnpR encoding resolvase, a site-specific recombinase of the transposable element gamma delta. Induction of the transcriptional fusions results in production of resolvase, which in turn, catalyzes excision of a linked tetracycline-resistance reporter gene flanked by direct repeats of res, the DNA sequences at which resolvase functions. The loss of tetracycline resistance in descendant bacteria serves as a permanent and heritable marker of prior gene expression. This gene fusion approach will allow us to assay the induction of gene expression in as few as one cell. Additionally, gene expression can be assayed at a later time and/or different place from the inducing environment facilitating the study of gene expression in complex environments such as animal tissues.
Cloning and analysis of the Saccharomyces cerevisiae MNN9 and MNN1 genes required for complex glycosylation of secreted proteins.
Authors: Yip C L , Welch S K , Klebl F , Gilbert T , Seidel P , Grant F J , O'Hara P J , MacKay V L
Published: May 2006

Proteins secreted by the yeast Saccharomyces cerevisiae are usually modified by the addition at asparagine-linked glycosylation sites of large heterogeneous mannan units that are highly immunogenic. Secreted proteins from mnn1 mnn9 mutant strains, in contrast, have homogeneous Man10GlcNAc2 oligosaccharides that lack the immunogenic alpha 1,3-mannose linkages. We have cloned and sequenced the MNN9 and MNN1 genes, both of which encode proteins with the characteristics of type II membrane proteins. Mnn9p is a membrane-associated protein with unknown function that is required for the addition of the long alpha 1,6-mannose backbone of the complex mannan, whereas Mnn1p is most likely the alpha 1,3-mannosyltransferase located in the Golgi apparatus.
Mapping of the actomyosin interfaces.
Authors: Eldin P , Le Cunff M , Vosberg H P , Mornet D , Léger J J
Published: May 2006

Recombinant DNA methods were used to obtain soluble, undenatured fragments of the heavy chain of myosin subfragment 1 (S-1). These fragments were of preselected lengths and could include protease-sensitive segments that are destroyed when other preparation methods are used. Actin binding by each of the three contiguous segments (residues 1-248, 249-524, and 518-722, essentially spanning the entire S-1 heavy chain) was demonstrated. ATP binding, comparable to that of native S-1, was obtained only with a segment consisting of residues 1-524. Competition among the various fragments for actin was also studied. The data are discussed in relation to the recently reported resolved structure of S-1 [Rayment, I., Rypnieski, R. W., Schmidt-Bäse, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G. & Holden, H. M. (1993) Science 261, 50-58].
Bone marrow transplantation corrects the enzyme defect in neurons of the central nervous system in a lysosomal storage disease.
Authors: Walkley S U , Thrall M A , Dobrenis K , Huang M , March P A , Siegel D A , Wurzelmann S
Published: May 2006

Neuronal storage disorders are fatal neurodegenerative diseases of humans and animals that are caused by inherited deficiencies of lysosomal hydrolase activity. Affected individuals often appear normal at birth but eventually develop progressive neurologic symptoms including sensory and motor deficits, mental retardation, and seizures. We have examined efficacy of bone marrow transplantation as a means of enzyme replacement, using cats with the lysosomal storage disease alpha-mannosidosis. Treated animals showed little or no progression of neurologic signs 1-2 years after transplant, whereas untreated cats became severely impaired and reached endstage disease by 6 months of age. Increased lysosomal alpha-mannosidase activity was found in brain tissue of the treated animals, and electron microscopy revealed no evidence of lysosomal storage within most neurons. Histochemical localization of acidic alpha-D-mannoside mannohydrolase (EC 3.2. 1.24), using 5-bromo-4-chloro-3-indolyl alpha-D-mannopyranoside, showed that functional enzyme was present in neurons, glial cells, and cells associated with blood vessels. This study provides direct evidence that bone marrow transplantation as treatment for a neuronal storage disease can lead to significant levels of a missing lysosomal hydrolase within neurons of the central nervous system and to compensation for the genetic metabolic defect.
Subunit interaction in the CCAAT-binding heteromeric complex is mediated by a very short alpha-helix in HAP2.
Authors: Xing Y , Zhang S , Olesen J T , Rich A , Guarente L
Published: May 2006

We dissected the domain of HAP2 that mediates subunit association in the heteromeric CCAAT-binding complex, first by genetic mutational analysis and then by structural studies. The mutational data suggest that a very short region in HAP2 mediates protein-protein association and that the structure of this domain is likely to be an alpha-helix. The CD analyses of a 15-residue synthetic oligopeptide covering this region confirm this surmise. The oligopeptide indeed formed an unusually thermal stable alpha-helix in aqueous solution. Eight amino acids that lie along one face of this helix, including three arginines, are found to be critical for protein-protein association. The partner that interacts with this helical motif is likely to be another subunit in the HAP complex, since the CCAAT-binding factor is shown to contain one molecule of HAP2. Our results suggest that very short regions in proteins can encode precise structures and mediate stable and specific protein-protein recognition and interactions.
Reading the molecular clock from the decay of internal symmetry of a gene.
Authors: Gibbs P E , Dugaiczyk A
Published: May 2006

The closely related serum albumin, alpha-fetoprotein, and vitamin D-binding proteins are derived from a common ancestor, which itself was the result of a triplication of an ancestral gene. We have aligned the sequences of these proteins against themselves to assess the degree to which the ancestral 3-fold symmetry has been retained; in a dot plot, relics of the molecular symmetry appear as a series of alignments parallel to the main diagonal. The decay of internal symmetry reflects the rate of change of a gene in a single line of evolutionary descent. We examined 11 serum albumins, 2 ceruloplasmins, 3 alpha-fetoproteins, and 3 vitamin D-binding proteins. We have found that ceruloplasmin evolves at the same rate in human and rat, whereas albumin, alpha-fetoprotein, and vitamin D-binding protein evolve at different rates. The human genes had the highest alignment scores, indicating the most preserved ancestral features. We conclude that the molecular clock may run at different rates for the same gene in different species.
Binding of histones H1 and H5 and their globular domains to four-way junction DNA.
Authors: Varga-Weisz P , Zlatanova J , Leuba S H , Schroth G P , van Holde K
Published: May 2006

We have compared chicken erythrocyte linker histones H1 and H5 binding to a synthetic four-way DNA junction. Each histone binds to form a single complex, with an affinity which permits competition against a large excess of linear duplex DNA. The affinity of H5 is higher than that of H1. The globular domain from either protein will also bind strongly, but in this case multiple binding occurs. Binding of intact H1 is inhibited by cations: Mg2+ and spermidine are very effective, Na+ much less so. This inhibition is not likely to be a general ion-competition effect, for Mg2+ is much less effective in inhibiting the binding of H1 to linear DNA. Instead, the inhibition of binding may be due to ion-dependent changes in the conformation of the four-way junction, which are known to occur under similar conditions. These results strongly suggest that the angle formed between the arms of the DNA junction could be a major determinant in the interaction of H1 with DNA crossovers.
An altered-specificity mutation in a human POU domain demonstrates functional analogy between the POU-specific subdomain and phage lambda repressor.
Authors: Jancso A , Botfield M C , Sowers L C , Weiss M A
Published: May 2006

The POU motif, conserved among a family of eukaryotic transcription factors, contains two DNA-binding domains: an N-terminal POU-specific domain (POUS) and a C-terminal homeodomain (POUHD). Surprisingly, POUS is similar in structure to the helix-turn-helix domains of bacteriophage repressor and Cro proteins. Such similarity predicts a common mechanism of DNA recognition. To test this prediction, we have studied the DNA-binding properties of the human Oct-2 POU domain by combined application of chemical synthesis and site-directed mutagenesis. The POUS footprint of DNA contacts, identified by use of modified bases, is analogous to those of bacteriophage repressor-operator complexes. Moreover, a loss-of-contact substitution in the putative POUS recognition alpha-helix leads to relaxed specificity at one position in the DNA target site. The implied side chain-base contact is identical to that of bacteriophage repressor and Cro proteins. These results establish a functional analogy between the POUS and prokaryotic helix-turn-helix elements and suggest that their DNA specificities may be governed by a shared set of rules.
Transcriptional activation by CTF proteins is mediated by a bipartite low-proline domain.
Authors: Altmann H , Wendler W , Winnacker E L
Published: May 2006

Members of the CCAAT-binding transcription factor (CTF) family of proteins stimulate the initiation of adenovirus DNA replication and act as transcriptional activators. To investigate the mechanisms underlying CTF-mediated transactivation patterns, we expressed several natural CTF variants in Saccharomyces cerevisiae and determined their transactivating activities in enzymatic assays. CTF7, which lacks the entire proline-rich region previously thought to mediate transcriptional activation by CTF proteins, enhances transcription to a greater degree than full-length CTF1, which contains the putative activation domain. CTF2, which contains a partially deleted proline-rich activation region, does not stimulate transcription at all. These findings indicate that the proline-rich region of CTF proteins is not essential for transcriptional activation in yeast. Our studies also suggest a bipartite two-domain structure of CTF-type transcriptional activation domains.
A test of lattice protein folding algorithms.
Authors: Yue K , Fiebig K M , Thomas P D , Chan H S , Shakhnovich E I , Dill K A
Published: May 2006

We report a blind test of lattice-model-based search strategies for finding global minima of model protein chains. One of us (E.I.S.) selected 10 compact conformations of 48-mer chains on the three-dimensional cubic lattice and used their inverse folding algorithm to design HP (H, hydrophobic; P, polar) sequences that should fold to those "target" structures. The sequences, but not the structures, were sent to the UCSF group (K.Y., K.M.F., P.D.T., H.S.C., and K.A.D.), who used two methods to attempt to find the globally optimal conformations: "hydrophobic zippers" and a constraint-based hydrophobic core construction (CHCC) method. The CHCC method found global minima in all cases, and the hydrophobic zippers method found global minima in some cases, in minutes to hours on workstations. In 9 out of 10 sequences, the CHCC method found lower energy conformations than the 48-mers were designed to fold to. Thus the search strategies succeed for the HP model but the design strategy does not. For every sequence the global energy minimum was found to have multiple degeneracy with 10(3) to 10(6) conformations. We discuss the implications of these results for (i) searching conformational spaces of simple models of proteins and (ii) how these simple models relate to proteins.
Role of phosphorylation in elicitation of the oxidative burst in cultured soybean cells.
Authors: Chandra S , Low P S
Published: May 2006

The oxidative burst is likely the most rapid defense response mounted by a plant under pathogen attack, and the generated oxidant species may be essential to several subsequent defense responses. In our effort to characterize the signal-transduction pathways leading to rapid H2O2/O2- biosynthesis, we have examined the role of protein phosphorylation in this resistance mechanism. K-252a and staurosporine, two protein-kinase inhibitors, were found to block the oxidative burst in a concentration-dependent manner. When added during H2O2 generation, the burst was observed to rapidly terminate, suggesting that continuous phosphorylation was essential for its maintenance. Importantly, phosphatase inhibitors (calyculin A and okadaic acid) were found to induce the oxidative burst in the absence of any additional stimulus. This may suggest that certain kinases required for the burst are constitutively active and that stabilization of the phosphorylated forms of their substrates is all that is required for burst activity. In autoradiographs of elicited and unstimulated cells equilibrated with 32PO4(3-), several phosphorylated polypeptide bands were revealed that could represent proteins essential for the burst.
Preferential isolation of DNA fragments associated with CpG islands.
Authors: Shiraishi M , Lerman L S , Sekiya T
Published: May 2006

We describe a procedure for preferential isolation of DNA fragments with G+C-rich portions. Such fragments occur in known genes within or adjacent to CpG islands. Since about 56% of human genes are associated with CpG islands, isolation of these fragments permits detection and probing of many genes within much larger segments of DNA, such as cosmids or yeast artificial chromosomes, which have not been sequenced. Cloned DNA fragments digested with four restriction endonucleases were subjected to denaturing gradient gel electrophoresis. Long G+C-rich sections in fragments inhibit strand dissociation after the fragments reach retardation level in the gradient; such fragments are retained in the gel after most others disappear. Nucleotide sequences of the retained fragments show that about half of these fragments appear to be derived from CpG islands. Northern analysis indicated the presence of RNA complementary to most of the retained fragments. A heuristic approach to the relation between base sequence and the kinetics of strand dissociation of partly melted molecules appears to account for retention and nonretention. The expectation that CpG island fragments will be enriched among fragments retained in a denaturing gradient is supported by rate estimates based on melting theory applied to known sequences. This method, designated SPM for segregation of partly melted molecules, is expected to provide a means for convenient and efficient isolation of genes from unsequenced DNA.
Gain of function mutations for paralogous Hox genes: implications for the evolution of Hox gene function.
Authors: Pollock R A , Sreenath T , Ngo L , Bieberich C J
Published: May 2006

To investigate the functions of paralogous Hox genes, we compared the phenotypic consequences of altering the embryonic patterns of expression of Hoxb-8 and Hoxc-8 in transgenic mice. A comparison of the phenotypic consequences of altered expression of the two paralogs in the axial skeletons of newborns revealed an array of common transformations as well as morphological changes unique to each gene. Divergence of function of the two paralogs was clearly evident in costal derivatives, where increased expression of the two genes affected opposite ends of the ribs. Many of the morphological consequences of expanding the mesodermal domain and magnitude of expression of either gene were atavistic, inducing the transformation of axial skeletal structures from a modern to an earlier evolutionary form. We propose that regional specialization of the vertebral column has been driven by regionalization of Hox gene function and that a major aspect of this evolutionary progression may have been restriction of Hox gene expression.
Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditioning.
Authors: Heurteaux C , Lauritzen I , Widmann C , Lazdunski M
Published: May 2006

Preconditioning with sublethal ischemia protects against neuronal damage after subsequent lethal ischemic insults in hippocampal neurons. A pharmacological approach using agonists and antagonists at the adenosine A1 receptor as well as openers and blockers of ATP-sensitive K+ channels has been combined with an analysis of neuronal death and gene expression of subunits of glutamate and gamma-aminobutyric acid receptors, HSP70, c-fos, c-jun, and growth factors. It indicates that the mechanism of ischemic tolerance involves a cascade of events including liberation of adenosine, stimulation of adenosine A1 receptors, and, via these receptors, opening of sulfonylurea-sensitive ATP-sensitive K+ channels.
Agouti regulation of intracellular calcium: role in the insulin resistance of viable yellow mice.
Authors: Zemel M B , Kim J H , Woychik R P , Michaud E J , Kadwell S H , Patel I R , Wilkison W O
Published: May 2006

Several dominant mutations at the agouti locus in the mouse cause a syndrome of marked obesity, hyperinsulinemia, and insulin resistance. Although it is known that the agouti gene is expressed in an ectopic manner in these mutants, the precise mechanism by which the agouti gene product mediates these effects is unclear. Since intracellular Ca2+ is believed to play a role in mediating insulin action and dysregulation of Ca2+ flux is observed in diabetic animals and humans, we examined the status of intracellular Ca2+ in mice carrying the dominant agouti allele, viable yellow (Avy). We show here that in mice carrying this mutation, the intracellular free calcium concentration ([Ca2+]i) is elevated in skeletal muscle, and the degree of elevation is closely correlated with the degree to which the mutant traits are expressed in individual animals. Moreover, we demonstrate that the agouti gene product is capable of inducing increased [Ca2+]i in cultured and freshly isolated skeletal muscle myocytes from wild-type mice. Based on these findings, we present a model in which we propose that the agouti polypeptide promotes insulin resistance in mutant animals through its ability to increase [Ca2+]i.
Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.
Authors: Singh K K , Samson L
Published: May 2006

Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes.
Accumulation of plasma cells in atherosclerotic lesions of Watanabe heritable hyperlipidemic rabbits.
Authors: Sohma Y , Sasano H , Shiga R , Saeki S , Suzuki T , Nagura H , Nose M , Yamamoto T
Published: May 2006

By screening a cDNA library constructed from aortic total RNA derived from Watanabe heritable hyperlipidemic (WHHL) rabbits by differential hybridization, we have obtained a cDNA encoding the kappa light chain of immunoglobulin. Northern blot analysis of total RNA prepared from aortas of WHHL and normal rabbits of various ages revealed that this light-chain mRNA accumulates gradually with age in aortas in WHHL rabbits. Northern blotting and in situ hybridization with an antisense oligonucleotide specific to rabbit immunoglobulin gamma heavy-chain mRNA also detected accumulation of this heavy-chain mRNA in advanced lesions of WHHL rabbit aortas. Moreover, immunohistochemical and electron microscopic analyses demonstrated the presence of plasma cells in the atherosclerotic lesions.
Neurosecretory habituation in PC12 cells: modulation during parallel habituation.
Authors: Martin P T , Koshland D E
Published: May 2006

PC12 cells habituate during repetitive stimulation with acetylcholine, bradykinin, or high potassium. Interspersing these stimulants did not affect the rate of habituation of the others, but it could modulate the amplitude of the norepinephrine secretion each could achieve. Stimulation with acetylcholine inhibited norepinephrine secretion caused by high potassium and bradykinin stimulation, while high potassium had no effect on acetylcholine or bradykinin, and bradykinin increased secretion caused by acetylcholine. Changes in norepinephrine secretion resulting from any of these stimulants correlated with changes in internal calcium levels. Cyclic AMP-, protein kinase C-, and calmodulin-dependent second messenger pathways all modulated norepinephrine secretion caused by acetylcholine and high potassium and showed a distinct hierarchy in their effectiveness. These data demonstrate that different receptor pathways can change the norepinephrine response of one another while not changing the levels of the molecules responsible for habituation.
Regulation of cell signaling by the cytoplasmic domains of the heat-stable enterotoxin receptor: identification of autoinhibitory and activating motifs.
Authors: Rudner X L , Mandal K K , de Sauvage F J , Kindman L A , Almenoff J S
Published: May 2006

Infection with enterotoxigenic Escherichia coli is a leading cause of traveler's diarrhea. Many enterotoxigenic E. coli strains produce heat-stable enterotoxin (ST), a peptide that binds to the intestinal receptor guanylyl cyclase C known as STaR. The toxin-receptor interaction elevates intracellular cGMP, which then activates apical chloride secretion, resulting in secretory diarrhea. In this report, we examine how the intracellular domains of STaR participate in the propagation and regulation of signaling. We show that STaR exists as an oligomer in both the presence and the absence of toxin. We also demonstrate that deletion of the intracellular kinase-homology domain produces a constitutively active mutant, suggesting that this domain subserves an autoinhibitory function. Finally, we constructed a point mutant within a highly conserved region of the cyclase domain that completely inactivates the catalytic activity of guanylyl cyclase. Cotransfection of this point mutant with wild-type receptor causes a dominant-negative effect on receptor activation. This suggests that interaction of receptor subunits is required for toxin-induced activation and that the cyclase domain is involved in this essential interaction. We propose that the binding of ST to STaR promotes a conformational change across the cell membrane. This removes the inhibitory effects of the kinase-homology domain and promotes an interaction between cyclase domains that leads to receptor activation. The data suggest a paradigm of signal transduction that may also be relevant to other members of the guanylyl cyclase receptor family.
Inflammation-induced recombinant protein expression in vivo using promoters from acute-phase protein genes.
Authors: Varley A W , Coulthard M G , Meidell R S , Gerard R D , Munford R S
Published: May 2006

We report that promoters for two murine acute-phase protein (APP) genes, complement factor 3 (C3) and serum amyloid A3 (SAA3), can increase recombinant protein expression in response to inflammatory stimuli in vivo. To deliver APP promoter-luciferase reporter gene constructs to the liver, where most endogenous APP synthesis occurs, we introduced them into a nonreplicating adenovirus vector and injected the purified viruses intravenously into mice. When compared with the low levels of basal luciferase expression observed prior to inflammatory challenge, markedly increased expression from the C3 promoter was detected in liver in response to both lipopolysaccharide (LPS) and turpentine, and lower-level inducible expression was also found in lung. In contrast, expression from the SAA3 promoter was found only in liver and was much more responsive to LPS than to turpentine. After LPS challenge, hepatic luciferase expression increased rapidly and in proportion to the LPS dose. Use of cytokine-inducible promoters in gene transfer vectors may make it possible to produce antiinflammatory proteins in vivo in direct relationship to the intensity and duration of an individual's inflammatory response. By providing endogenously controlled production of recombinant antiinflammatory proteins, this approach might limit the severity of the inflammatory response without interfering with the beneficial components of host defense and immunity.
Triplet repeat expansion in myotonic dystrophy alters the adjacent chromatin structure.
Authors: Otten A D , Tapscott S J
Published: May 2006

Myotonic dystrophy is caused by an expansion of a CTG triplet repeat sequence in the 3' noncoding region of a protein kinase gene, yet the mechanism by which the triplet repeat expansion causes disease remains unknown. This report demonstrates that a DNase I hypersensitive site is positioned 3' of the triplet repeat in the wild-type allele in both fibroblasts and skeletal muscle cells. In three unrelated individuals with myotonic dystrophy that have large expansions of the triplet repeat, the allele with the triplet repeat expansion exhibited both overall DNase I resistance and inaccessibility of nucleases to the adjacent hypersensitive site. These results indicate that the triplet repeat expansion alters the adjacent chromatin structure, establishing a region of condensed chromatin, and suggests a molecular mechanism for myotonic dystrophy.