bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetic role of nonnative species in the folding of bovine pancreatic trypsin inhibitor.
Authors: Weissman J S , Kim P S
Published: May 2006

We have shown previously that during the oxidative folding of bovine pancreatic trypsin inhibitor only intermediates with native disulfide bonds are well populated. Nevertheless, these studies also confirmed the earlier conclusion [Creighton, T. E. (1977) J. Mol. Biol. 113, 275-293] that the rate-limiting transition in the kinetically preferred route for folding involves intramolecular disulfide bond rearrangements. Consequently, intermediates with nonnative disulfide bonds must form transiently during folding. Two specific nonnative species, denoted [30-51; 5-14] and [30-51; 5-38], in which numbers indicate residues participating in a disulfide bond, can be detected at low levels in kinetic folding experiments with bovine pancreatic trypsin inhibitor. By working with purified reversibly trapped intermediates, the role of these two nonnative species has been examined directly. These species are found to be in relatively rapid exchange with each other and with an initially formed native two-disulfide intermediate [30-51; 14-38]. Thus, the low abundance of the two nonnative species detected in kinetic folding experiments reflects primarily their low thermodynamic stability as compared to this native intermediate. To a small extent, these nonnative species form the productive native intermediate [30-51; 5-55], which is the immediate precursor to the native protein. However, an equal amount of [5-55; 14-38], a nonproductive dead-end intermediate, is also produced. Thus, the nonnative species detected during the folding of bovine pancreatic trypsin inhibitor are not committed to forming the productive native intermediate, nor do they serve to direct folding specifically toward a productive route.
Targeting of the T-cell receptor zeta-chain gene in embryonic stem cells: strategies for generating multiple mutations in a single gene.
Authors: Love P E , Tremblay M L , Westphal H
Published: May 2006

The T-cell receptor zeta chain is a member of a family of related proteins that play a critical role in coupling cell-surface receptors to intracellular signaling pathways. To study the role of zeta chain in T-cell ontogeny, we generated targeted mutations of the zeta-chain gene in murine embryonic stem cells. The mutant alleles are predicted to result either in a null phenotype or in the synthesis of a truncated protein capable of supporting T-cell-receptor surface expression but deficient in transmembrane signaling. Both of these targeting events were recovered in a single electroporation experiment with either coelectroporation or a combination deletion/truncation construct. Our results suggest that similar approaches could be used to generate multiple single mutations, modifications of more than one site within a gene, or subtle alterations that rely upon coconversion with the selectable marker gene.
Paradoxical increase in striatal neuropeptide gene expression following ischemic lesions of the cerebral cortex.
Authors: Salin P , Chesselet M F
Published: May 2006

Ischemic lesions of the cerebral cortex occur frequently in humans as a result of stroke. One major consequence of the death of cortical neurons is the loss of excitatory cortical projections to subcortical regions. Little is known, however, about the transsynaptic effect of such lesions on neurotransmitter expression in subcortical structures. We have examined the effects of ischemic cortical lesions on the peptidergic neurotransmitters enkephalin and tachykinins in the striatum, a brain region massively innervated by glutamatergic cortical inputs. The levels of enkephalin and tachykinin mRNAs increased in the striatum of adult rats after thermocoagulation of pial vessels. The effects were more pronounced in the striatal region most heavily innervated by the lesioned cortex but were also observed in other striatal regions and on the contralateral side. Increased gene expression was accompanied by increased immunoreactivity for the two peptides. Elevated levels of enkephalin mRNA were observed up to 3 months after surgery in the ipsilateral striatum. Whereas results of previous studies of acute cortical ablations suggested that excitatory corticostriatal neurons were necessary to maintain normal peptide levels in striatal efferent neurons, the present data indicate that lesions of the same corticostriatal neurons secondary to local ischemia result in a paradoxical transsynaptic activation of neuropeptide synthesis in subcortical structures. This effect may play a role in the functional consequences of cortical strokes and progressive cortical atrophy in humans and may have critical bearing for their treatment and prognosis.
Structure of inositol monophosphatase, the putative target of lithium therapy.
Authors: Bone R , Springer J P , Atack J R
Published: May 2006

Inositol monophosphatase (EC 3.1.3.25), the putative molecular site of action of lithium therapy for manic-depressive illness, plays a key role in the phosphatidylinositol signaling pathway by catalyzing the hydrolysis of inositol monophosphates. To provide a structural basis from which to design better therapeutic agents for manic-depressive illness, the structure of human inositol monophosphatase has been determined to 2.1-A resolution by using x-ray crystallography. The enzyme exists as a dimer of identical subunits, each folded into a five-layered sandwich of three pairs of alpha-helices and two beta-sheets. Sulfate and an inhibitory lanthanide cation (Gd3+) are bound at identical sites on each subunit and establish the positions of the active sites. Each site is located in a large hydrophilic cavern that is at the base of the two central helices where several segments of secondary structure intersect. Comparison of the phosphatase aligned sequences of several diverse genes with the phosphatase structure suggests that the products of these genes and the phosphatase form a structural family with a conserved metal binding site.
Methylation in the preinitiation domain suppresses gene transcription by an indirect mechanism.
Authors: Levine A , Cantoni G L , Razin A
Published: May 2006

Although the first observations of the inhibitory effect of methylation on gene activity were made almost a decade ago, the mechanism by which methyl groups affect transcription is still obscure. Here we use engineered promoters methylated in vitro in transient transfections to study the mechanism by which methylation mediates promoter repression. The results clearly show that the location of the methyl groups within the promoter region determines the extent of promoter repression. The most effective suppression was observed when methylation was in the preinitiation domain. The results also support a previous suggestion that a mediator protein is involved in the mechanism of promoter inhibition. The suppressor effect of methylation at sequences flanking the TATA box can be partially overcome in the presence of the simian virus 40 enhancer. In addition, results obtained by transient thymidine labeling of Ltk- cells that were transfected with a methylated thymidine kinase gene from herpes simplex virus, at the level of approximately one template per cell, further support the conclusion that methylation affects primarily transcription preinitiation.
Time-resolved circularly polarized protein phosphorescence.
Authors: Schauerte J A , Steel D G , Gafni A
Published: May 2006

The existence of circular polarization in room-temperature protein phosphorescence is demonstrated, and time-resolved circularly polarized phosphorescence (TR-CPP) is used to characterize unique tryptophan environments in multitryptophan proteins. Circularly polarized luminescence studies provide information regarding the excited state chirality of a lumiphore which can be used to extract sensitive structural information. It is shown by time resolving the circular polarization that it is possible to correlate the excited state chirality with unique decay components in a multiexponential phosphorescence decay profile. The present study presents a concurrent analysis of room-temperature time-resolved phosphorescence and TR-CPP of bacterial glucose-6-phosphate dehydrogenase as well as those of horse liver alcohol dehydrogenase. Only one of the two tryptophan residues per subunit of dimeric alcohol dehydrogenase is believed to phosphorescence, while the dimeric glucose-6-phosphate dehydrogenase has eight tryptophan residues per subunit and shows a corresponding complexity in its phosphorescence decay profile. The anisotropy factor [g(em) = delta I/(Itotal/2); delta I = Ileft circular-Iright circular] for alcohol dehydrogenase is time independent, suggesting a unique excited state chirality. The phosphorescence decay of glucose-6-phosphate dehydrogenase can be well fitted with four exponential terms of 4, 23, 76, and 142 msec, and the TR-CPP of this enzyme shows a strong time dependence that can be resolved into four individual time-independent anisotropy factors of -4.0, -2.1, +6.5, and +6.9 (x10(-3)), each respectively associated with one of the four lifetime components. These results demonstrate how the use of TR-CPP can facilitate the study of proteins with multiple lumiphores.
Guanine nucleotide-binding protein-coupled and -uncoupled states of opioid receptors and their relevance to the determination of subtypes.
Authors: Richardson A , Demoliou-Mason C , Barnard E A
Published: May 2006

Opioid receptors are currently classified as mu, delta, and kappa types, but various subtypes have also been proposed. We have investigated whether subtypes exist by using [3H]bremazocine. [3H]Bremazocine binds to twice as many naloxone-sensitive sites as other nonselective opioid agonists, as shown in four membrane types that have very different ratios of mu, delta, and kappa receptor types. [3H]Bremazocine binding is completely inhibited by an excess (in unlabeled form) of other opioid ligands, with Hill coefficients of 0.8-0.95. These paradoxes can be explained if there are high- and low-affinity states of the mu, delta, and kappa receptors and bremazocine binds with similar affinities to both states. We propose that these states are the guanine nucleotide-binding protein (G-protein)-coupled form and the uncoupled form of each receptor. As evidence for this proposal, the [3H]bremazocine binding suffered little or no loss with G-protein-uncoupling treatments, whereas binding of other opioid agonists was fully sensitive. We conclude that [3H]bremazocine offers a tool for the measurement of the total pools of coupled and uncoupled opioid receptors and that much of the previous characterization of opioid receptor subtypes reflects, instead, a significant pool of G-protein-uncoupled opioid receptors.
Short-chain analogs of luteinizing hormone-releasing hormone containing cytotoxic moieties.
Authors: Janáky T , Juhász A , Rékási Z , Serfözö P , Pinski J , Bokser L , Srkalovic G , Milovanovic S , Redding T W , Halmos G
Published: May 2006

Five hexapeptide and heptapeptide analogs of luteinizing hormone-releasing hormone (LH-RH) were synthesized for use as carriers for cytotoxic compounds. These short analogs were expected to enhance target selectivity of the antineoplastic agents linked to them. Native LH-RH-(3-9) and LH-RH-(4-9) containing D-lysine and D-ornithine at position 6 were amidated with ethylamine and acylated on the N terminus. The receptor-binding affinity of one hexapeptide carrier AJ-41 (Ac-Ser-Tyr-D-Lys-Leu-Arg-Pro-NH-Et) to human breast cancer cell membranes was similar to that of [D-Trp6]LH-RH. Alkylating nitrogen mustards (melphalan, Ac-melphalan), anthraquinone derivatives including anticancer antibiotic doxorubicin, antimetabolite (methotrexate), and cisplatin-like platinum complex were linked to these peptides through their omega-amino group at position 6. The hybrid molecules showed no LH-RH agonistic activity in vitro and in vivo but had nontypical antagonistic effects on pituitary cells in vitro at the doses tested. These analogs showed a wide range of receptor-binding affinities to rat pituitaries and cell membranes of human breast cancer and rat Dunning prostate cancer. Several of these conjugates exerted some cytotoxic effects on MCF-7 breast cancer cell line.
A histidine protein kinase homologue required for regulation of bacterial cell division and differentiation.
Authors: Ohta N , Lane T , Ninfa E G , Sommer J M , Newton A
Published: May 2006

Differentiation in the dimorphic bacterium Caulobacter crescentus results from a sequence of discontinuous, stage-specific events that leads to the production of a stalked cell and a new motile swarmer cell after each asymmetric cell division. As reported previously, pseudoreversion analysis of mutations in the pleiotropic developmental gene pleC identified three cell division genes: divJ, divK, and divL. We show here that one of these genes, divJ, encodes a predicted protein of 596 residues with an extensive hydrophobic N-terminal region and a C-terminal domain containing all of the invariant residues found in the family of bacterial histidine protein kinases. Our results also show that divJ is discontinuously transcribed early in the swarmer cell cycle during a period that coincides with the G1 to S transition. We propose that the DivJ protein is one member of a signal transduction pathway regulating the cell cycle and differentiation in Caulobacter and that protein modification by phosphorylation may play a central role in coupling developmental events to progress through the cell division cycle.
Cultured sympathetic neurons synthesize and release the cytokine interleukin 1 beta.
Authors: Freidin M , Bennett M V , Kessler J A
Published: May 2006

Autonomic neurons help to regulate immune responses, and there are reciprocal interactions between the nervous and immune systems. This study seeks to define some of the molecular mechanisms that may underlie such interactions. Immunoblot analysis indicated that cultured sympathetic neurons synthesize and release the cytokine interleukin 1 beta (IL-1 beta). In addition, RNA blot analysis of cultured sympathetic neurons demonstrated that the neurons contain mRNA encoding IL-1 beta. It was previously shown that explant cultures of sympathetic ganglia and dissociated cocultures of neurons with ganglionic nonneuronal cells synthesize substance P, whereas in situ levels of substance P and its mRNA are low. An antagonist at the interleukin 1 receptor markedly depressed this increase in substance P in cultures, suggesting that endogenous IL-1 beta mediates the synthetic response, at least in part. Because pure neuronal cultures do not contain substance P and neurons synthesize and release IL-1 beta, the actions of the cytokine require the presence of ganglion nonneuronal cells. These observations suggest a role for autonomic neurons in influencing immune responses by synthesizing and secreting at least two known immunoregulators, the cytokine IL-1 beta and the neuropeptide substance P.
Chimeric gag-V3 virus-like particles of human immunodeficiency virus induce virus-neutralizing antibodies.
Authors: Luo L , Li Y , Cannon P M , Kim S , Kang C Y
Published: May 2006

A 41-kDa unprocessed human immunodeficiency virus 2 (HIV-2) gag precursor protein that has a deletion of a portion of the viral protease assembles as virus-like particles by budding through the cytoplasmic membrane of recombinant baculovirus-infected insect cells. We have constructed six different combinations of chimeric genes by coupling the truncated HIV-2 gag gene to the neutralizing domain (V3) or the neutralizing and the CD4 binding domains (V3+CD4BD) of gp120 env gene sequences from HIV-1 or HIV-2. The env gene sequences were inserted either into the middle of the gag gene or at the 3' terminus of the gag gene. Virus-like particles were formed by chimeric gene products only when the env gene sequences were linked to the 3' terminus of the gag gene. Insertion of env gene sequence in the middle of the gag gene resulted in high-level chimeric gene expression but without the formation of virus-like particles. Three different chimeric genes [gag gene with HIV-1 V3 (1V3), gag gene with HIV-2 V3 (2V3), and gag gene with HIV-2 V3+CD4BD (2V3+CD4BD)] formed virus-like particles that were secreted into the cell culture medium. In contrast, the HIV-1 V3+CD4BD/HIV-2 gag construct did not form virus-like particles. The chimeric gag-env particles had spherical morphology and the size was slightly larger than that of the gag particles, but the chimeric particles were similar to the mature HIV particles. Western blot analysis showed that the gag-env chimeric proteins were recognized by antibodies in HIV-positive human serum and rabbit anti-gp120 serum. Rabbit anti-gag 1V3 and anti-gag 2V3 sera reacted with authentic gp120 of HIV-1 and HIV-2, respectively, and neutralized homologous HIV infectivity. Our results show that precursor gag protein has potential as a carrier for the presentation of foreign epitopes in good immunological context. The gag protein is highly immunogenic and has the ability to carry large foreign inserts; as such, it offers an attractive approach for HIV vaccine development.
Human herpesvirus 7 is a T-lymphotropic virus and is related to, but significantly different from, human herpesvirus 6 and human cytomegalovirus.
Authors: Berneman Z N , Ablashi D V , Li G , Eger-Fletcher M , Reitz M S , Hung C L , Brus I , Komaroff A L , Gallo R C
Published: May 2006

An independent strain (JI) of human herpesvirus 7 (HHV-7) was isolated from a patient with chronic fatigue syndrome (CFS). No significant association could be established by seroepidemiology between HHV-7 and CFS. HHV-7 is a T-lymphotropic virus, infecting CD4+ and CD8+ primary lymphocytes. HHV-7 can also infect SUP-T1, an immature T-cell line, with variable success. Southern blot analysis with DNA probes scanning 58.8% of the human herpesvirus 6 (HHV-6) genome and hybridizing to all HHV-6 strains tested so far revealed homology to HHV-7 with only 37.4% of the total probe length. HHV-7 contains the GGGTTA repetitive sequence, as do HHV-6 and Marek's disease chicken herpesvirus. DNA sequencing of a 186-base-pair fragment of HHV-7(JI) revealed an identity with HHV-6 and human cytomegalovirus of 57.5% and 36%, respectively. Oligonucleotide primers derived from this sequence (HV7/HV8, HV10/HV11) amplified HHV-7 DNA only and did not amplify DNA from other human herpesviruses, including 12 different HHV-6 strains. Southern blot analysis with the p43L3 probe containing the 186-base-pair HHV-7 DNA fragment hybridized to HHV-7 DNA only. The molecular divergence between human cytomegalovirus, on the one hand, and HHV-6 and HHV-7, on the other, is greater than between HHV-6 and HHV-7, which, in turn, is greater than the difference between HHV-6 strains. This study supports the classification of HHV-7 as an additional member of the human beta-herpesviruses.
Role of the 21-kDa protein TIMP-3 in oncogenic transformation of cultured chicken embryo fibroblasts.
Authors: Yang T T , Hawkes S P
Published: May 2006

The 21-kDa protein is an extracellular matrix (ECM) component whose synthesis is stimulated transiently during oncogenic transformation of chicken embryo fibroblasts (CEF) or after treatment of normal cells with the tumor promoter phorbol 12-myristate 13-acetate. Biochemical characterization indicates that the protein is related, but not identical, to two members of the family of tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2. The cDNA of the 21-kDa protein was recently cloned, and based upon its deduced amino acid sequence and other supporting data we propose that it is another member of this family, a TIMP-3. We now report electrophoretic purification of sufficient quantities of this protein to determine its function. The protein promotes the detachment of transforming cells from the ECM. Although its presence in the matrix may be necessary for cell release it is not the only factor involved because it does not influence the adhesive properties of nontransformed cells. It also appears to accelerate the morphological changes associated with cell transformation and stimulates the proliferation of growth-retarded, nontransformed cells maintained under low serum conditions. Based on these data we hypothesize that the 21-kDa protein promotes the development of the transformed phenotype in cultured cells.
Analysis of Escherichia coli beta-galactosidase expression in transgenic mice by flow cytometry of sperm.
Authors: Jasin M , Zalamea P
Published: May 2006

Alterations to the mammalian genome that occur during the development of germ cells, in particular during meiosis, can be introduced into the population upon fertilization. These alterations can occur through homologous recombination, genome rearrangement, or mutagenesis. Such events usually occur infrequently for any particular sequence. Because of the difficulty in analyzing a large number of offspring in a mammalian cross, we have developed a marker to detect these events in sperm, since a large number of these meiotic progeny are produced during male gametogenesis. We have expressed the Escherichia coli lacZ gene during spermatogenesis in transgenic mice and quantitated the levels of beta-galactosidase activity in single sperm with the fluorescence-activated cell sorter and a fluorogenic substrate, 5-dodecanoylaminofluorescein di-beta-D-galactopyranoside. Detection of rare positives was demonstrated in mixed sperm populations with as few as 0.01% positive sperm. Although the distribution of beta-galactosidase activity in caudal epididymal sperm populations is bimodal, it appears that beta-galactosidase, like other proteins that have been expressed postmeiotically, is distributed between transgene-positive and transgene-negative sperm.
Genomic sequences with homology to the P element of Drosophila melanogaster occur in the blowfly Lucilia cuprina.
Authors: Perkins H D , Howells A J
Published: May 2006

We have cloned two DNA elements (Lu-P1 and Lu-P2) from the Australian sheep blowfly Lucilia cuprina that are similar to the transposable P element of Drosophila melanogaster in both structure and sequence but have diverged from it and from each other considerably. Hybridization studies indicate that a third related element probably exists in another, as yet unsequenced, clone. Neither Lu-P1 nor Lu-P2 appears to be active in terms of mobility, and it is not known whether any transposition-competent copies of other related elements occur in the genome of the blowfly. However, the isolation of any P-like sequences from a species outside of the family Drosophilidae allows comparisons to be made of more widely divergent P-related elements than has been possible previously. We are unaware of any report of the presence of multiple P-like family members within a single species. The discovery of Lu-P1 and Lu-P2 in the blowfly fuels the possibility that similar elements may be widespread in insects, and perhaps in other orders of animals.
The anticodon triplet is not sufficient to confer methionine acceptance to a transfer RNA.
Authors: Senger B , Despons L , Walter P , Fasiolo F
Published: May 2006

Previous work suggested that the presence of the anticodon CAU alone was enough to confer methionine acceptance to a tRNA. Conversions of Escherichia coli nonmethionine tRNAs to a methionine-accepting species were obtained by substitutions reconstructing the whole methionine anticodon loop together with preservation (or introduction) of the acceptor stem base A73. We show here that the CAU triplet alone is unable to confer methionine acceptance when transplanted into a yeast aspartic tRNA. Both non-anticodon bases of the anticodon loop of yeast tRNA(Met) and A73 are required in addition to CAU for methionine acceptance. The importance of these non-anticodon bases in other CAU-containing tRNA frameworks was also established. These specific non-anticodon base interactions make a substantial thermodynamic contribution to the methionine acceptance of a transfer RNA.
An intrastrand d(GpG) platinum crosslink in duplex M13 DNA is refractory to repair by human cell extracts.
Authors: Szymkowski D E , Yarema K , Essigmann J M , Lippard S J , Wood R D
Published: May 2006

We have examined the ability of human cell extracts to repair the most frequent DNA adduct caused by the cancer chemotherapeutic agent cis-diamminedichloroplatinum(II). A circular DNA duplex with an intrastrand d(GpG) crosslink positioned at a specific site was synthesized. Human cell extracts were unable to induce repair synthesis in a 29-base-pair region encompassing the adduct or in adjacent regions. The same extracts could repair a single defined 2-acetylaminofluorene lesion in a similar location. When molecules containing the platinum adduct were cleaved by Escherichia coli UvrABC enzyme, human cell extracts could perform repair synthesis at the damaged site, suggesting that human enzymes fail to make incisions near the d(GpG) crosslink but can complete repair once incisions are made. This result indicates that most repair synthesis in DNA damaged with multiple cis-diamminedichloroplatinum(II) adducts takes place at lesions other than the predominant d(GpG) crosslink. These data support the idea that the clinical effectiveness of cis-diamminedichloroplatinum(II) may be explained by the inefficient repair of the major DNA adduct caused by this drug.
Nonreplicating vaccinia vector efficiently expresses recombinant genes.
Authors: Sutter G , Moss B
Published: May 2006

Modified vaccinia Ankara (MVA), a highly attenuated vaccinia virus strain that has been safety tested in humans, was evaluated for use as an expression vector. MVA has multiple genomic deletions and is severely host cell restricted: it grows well in avian cells but is unable to multiply in human and most other mammalian cells tested. Nevertheless, we found that replication of viral DNA appeared normal and that both early and late viral proteins were synthesized in human cells. Proteolytic processing of viral structural proteins was inhibited, however, and only immature virus particles were detected by electron microscopy. We constructed an insertion plasmid with the Escherichia coli lacZ gene under the control of the vaccinia virus late promoter P11, flanked by sequences of MVA DNA, to allow homologous recombination at the site of a naturally occurring 3500-base-pair deletion within the MVA genome. MVA recombinants were isolated and propagated in permissive avian cells and shown to express the enzyme beta-galactosidase upon infection of nonpermissive human cells. The amount of enzyme made was similar to that produced by a recombinant of vaccinia virus strain Western Reserve, which also had the lacZ gene under control of the P11 promoter, but multiplied to high titers. Since recombinant gene expression is unimpaired in nonpermissive human cells, MVA may serve as a highly efficient and exceptionally safe vector.
Regulation of N-region diversity in antigen receptors through thymocyte differentiation and thymus ontogeny.
Authors: Bogue M , Gilfillan S , Benoist C , Mathis D
Published: May 2006

The random addition of "N nucleotides" by terminal deoxynucleotidyltransferase (TdT) is an important component of the diversity of T-cell receptor genes. We have investigated the expression of TdT during thymocyte differentiation and thymus ontogeny. TdT gene transcripts are confined to immature thymocytes of the cortex, being down-regulated concomitantly with recombination-activating gene transcripts after positive selection of mature medullary T cells. According to in situ hybridization, TdT RNA is absent from the neonatal thymus, but it appears 3 to 5 days after birth, just before the appearance of significant N-region diversity in T-cell receptor junctional sequences but clearly after the thymus attains competence at clonal deletion.
At least three distinct proteins are necessary for the reconstitution of a specific multiprotein complex at a eukaryotic chromosomal origin of replication.
Authors: Estes H G , Robinson B S , Eisenberg S
Published: May 2006

We have reconstituted in vitro a multistage assembly of a protein complex that specifically recognizes a yeast genomic origin of replication, the autonomously replicating sequence ARS121. The first step in the assembly was the interaction of the known origin-binding factor OBF1 and another factor, OBF2, with the ARS121 origin of replication to form the OBF1-OBF2-origin complex. This complex was the substrate for the ATP-dependent binding of a third DNA-binding activity, the core binding factor, CBF. Binding of CBF to the origin, identified by the retarded mobility of the origin DNA fragment in agarose gels, required, in addition to ATP and the OBF1-OBF2-origin complex, a functional essential core nucleotide sequence. ARS121 DNA containing mutations in the core, which inactivate the origin in vivo, did not sustain stable CBF binding, whereas ARS121 DNA mutated outside the boundaries of the essential core, which has normal origin function, bound CBF as wild type. This tight, direct correlation between the ability of the origin to bind CBF and its function as an origin of replication in vivo strongly suggest that the multiprotein complex reconstituted in vitro has a key role in the initiation of DNA replication.
General splicing factors SF2 and SC35 have equivalent activities in vitro, and both affect alternative 5' and 3' splice site selection.
Authors: Fu X D , Mayeda A , Maniatis T , Krainer A R
Published: May 2006

The human pre-mRNA splicing factors SF2 and SC35 have similar electrophoretic mobilities, and both of them contain an N-terminal ribonucleoprotein (RNP)-type RNA-recognition motif and a C-terminal arginine/serine-rich domain. However, the two proteins are encoded by different genes and display only 31% amino acid sequence identity. Here we report a systematic comparison of the splicing activities of recombinant SF2 and SC35. We find that either protein can reconstitute the splicing activity of S100 extracts and of SC35-immunodepleted nuclear extracts. Previous studies revealed that SF2 influences alternative 5' splice site selection in vitro, by favoring proximal over distal 5' splice sites, and that the A1 protein of heterogeneous nuclear RNP counteracts this effect. We now show that SC35 has a similar effect on competing 5' splice sites and is also antagonized by A1 protein. In addition, we report that both SF2 and SC35 also favor the proximal site in a pre-mRNA containing duplicated 3' splice sites, but this effect is not modulated by A1. We conclude that SF2 and SC35 are distinct splicing factors, but they display indistinguishable splicing activities in vitro.
Evidence for a base-pairing interaction between U6 small nuclear RNA and 5' splice site during the splicing reaction in yeast.
Authors: Sawa H , Abelson J
Published: May 2006

U6 small nuclear RNA (snRNA) is an essential factor in mRNA splicing. On the basis of the high conservation of its sequence, it has been proposed that U6 snRNA may function catalytically during the splicing reaction. If this is the case, it is likely that U6 snRNA interacts with the splice sites in the spliceosome to catalyze the reaction. We have used UV crosslinking to analyze the interactions of U6 snRNA with the splicing substrates during the yeast splicing reaction. Crosslinked products in which the central region of U6 snRNA was joined to the 5' splice site region of mRNA precursor and lariat intermediate were identified. The crosslinking sites were precisely located in one of these products. The results suggest a possible base-pairing interaction between U6 snRNA and the 5' splice site of the mRNA precursor.
Structural analysis of the human interferon gamma receptor: a small segment of the intracellular domain is specifically required for class I major histocompatibility complex antigen induction and antiviral activity.
Authors: Cook J R , Jung V , Schwartz B , Wang P , Pestka S
Published: May 2006

Mutations of the human interferon gamma (IFN-gamma) receptor intracellular domain have permitted us to define a restricted region of that domain as necessary for both induction of class I major histocompatibility complex antigen by IFN-gamma and protection against encephalomyocarditis virus. This region consists of five amino acids (YDKPH), all of which are conserved in the human and murine receptors. Tyr-457 and His-461 are essential for activity. Approximately 80% of the amino acids of the intracellular domain of the receptor is not required for major histocompatibility complex class I antigen induction or for antiviral protection against encephalomyocarditis virus. The observation that there was no protection by IFN-gamma against vesiculostomatitis virus indicates that other factors, in addition to chromosome 21 accessory factor(s), are required to generate the full complement of transduction signals from the human IFN-gamma receptor.
pH gradients across phospholipid membranes caused by fast flip-flop of un-ionized fatty acids.
Authors: Kamp F , Hamilton J A
Published: May 2006

A central, unresolved question in cell physiology is how fatty acids move across cell membranes and whether protein(s) are required to facilitate transbilayer movement. We have developed a method for monitoring movement of fatty acids across protein-free model membranes (phospholipid bilayers). Pyranin, a water-soluble, pH-sensitive fluorescent molecule, was trapped inside well-sealed phosphatidylcholine vesicles (with or without cholesterol) in Hepes buffer (pH 7.4). Upon addition of a long-chain fatty acid (e.g., oleic acid) to the external buffer (also Hepes, pH 7.4), a decrease in fluorescence of pyranin was observed immediately (within 10 sec). This acidification of the internal volume was the result of the "flip" of un-ionized fatty acids to the inner leaflet, followed by a release of protons from approximately 50% of these fatty acid molecules (apparent pKa in the bilayer = 7.6). The proton gradient thus generated dissipated slowly because of slow cyclic proton transfer by fatty acids. Addition of bovine serum albumin to vesicles with fatty acids instantly removed the pH gradient, indicating complete removal of fatty acids, which requires rapid "flop" of fatty acids from the inner to the outer monolayer layer. Using a four-state kinetic diagram of fatty acids in membranes, we conclude that un-ionized fatty acid flip-flops rapidly (t1/2 < or = 2 sec) whereas ionized fatty acid flip-flops slowly (t1/2 of minutes). Since fatty acids move across phosphatidylcholine bilayers spontaneously and rapidly, complex mechanisms (e.g., transport proteins) may not be required for translocation of fatty acids in biological membranes. The proton movement accompanying fatty acid flip-flop is an important consideration for fatty acid metabolism in normal physiology and in disease states such as cardiac ischemia.
Results and validation of a population pharmacodynamic model for cognitive effects in Alzheimer patients treated with tacrine.
Authors: Holford N H , Peace K E
Published: May 2006

Tacrine has been studied in two clinical trials of identical design in patients with probable Alzheimer disease. One trial enrolled patients in the United States, while the other enrolled patients in France. A population pharmacodynamic model has been used to describe the cognitive component of the Alzheimer disease assessment scale (ADASC) using mixed effects nonlinear regression. The model parameters and their population variability and covariance were estimated by using NONMEM. During an observation period of up to 5 months, the rate of disease progression was 6.17 ADASC units/year. The effect of tacrine was described best by a shift in the disease progress curve (-2.99 ADASC units or 177.6 days at a dose of 80 mg/day). The placebo effects associated with tacrine and placebo treatment were similar in magnitude and time course. There was no evidence of tolerance to tacrine but tolerance to the placebo treatment developed during the study. The size of the placebo effect in the French population was 76% larger than in the United States population, and the response to placebo diminished more slowly in the French population.
Brain proteins in plants: an Arabidopsis homolog to neurotransmitter pathway activators is part of a DNA binding complex.
Authors: Lu G , DeLisle A J , de Vetten N C , Ferl R J
Published: May 2006

The G box is a well-characterized cis-acting DNA regulatory element found in the promoters of several seemingly unrelated plant genes, including the alcohol dehydrogenase (Adh) gene of Arabidopsis thaliana. Using a monoclonal antibody screening approach coupled with electrophoretic mobility shift assays, we have isolated a cDNA clone encoding a protein that is part of the in vitro protein/G box complex. The derived amino acid sequence is homologous to a class of proteins in mammalian brains described as protein kinase C inhibitors and as activators of tyrosine and tryptophan hydroxylases, the rate-limiting enzymes in the pathways leading to the catecholamines and serotonin. The fact that a homologous member of this regulatory protein family is found in plants and is associated with binding to transcriptional regulatory elements suggests a much wider role for these proteins.
Poly(L-alanine) as a universal reference material for understanding protein energies and structures.
Authors: Head-Gordon T , Stillinger F H , Wright M H , Gay D M
Published: May 2006

We present a proposition, the "poly(L-alanine) hypothesis," which asserts that the native backbone geometry for any polypeptide or protein of M residues has a closely mimicking, mechanically stable, image in poly(L-alanine) of the same number of residues. Using a molecular mechanics force field to represent the relevant potential energy hypersurfaces, we have carried out calculations over a wide range of M values to show that poly(L-alanine) possesses the structural versatility necessary to satisfy the proposition. These include poly(L-alanine) representatives of minima corresponding to secondary and supersecondary structures, as well as poly(L-alanine) images for tertiary structures of the naturally occurring proteins bovine pancreatic trypsin inhibitor, crambin, ribonuclease A, and superoxide dismutase. The successful validation of the hypothesis presented in this paper indicates that poly(L-alanine) will serve as a good reference material in thermodynamic perturbation theory and calculations aimed at evaluating relative free energies for competing candidate tertiary structures in real polypeptides and proteins.
Leukotriene C4 uses a probenecid-sensitive export carrier that does not recognize leukotriene B4.
Authors: Lam B K , Xu K , Atkins M B , Austen K F
Published: May 2006

The export of leukotriene (LT) C4 from human eosinophils, a carrier-mediated process that is temperature-dependent and saturable, was characterized further in eosinophils and in two human leukemia cell lines that do not present an intact 5-lipoxygenase pathway. In eosinophils, KG-1 cells, and dimethyl sulfoxide (DMSO)-differentiated HL-60 cells, the respective Q10 values for temperature-dependent LTC4 export were 3.7, 3.3, and 3.4 and for energy of activation were 28.2 kcal/mol, 23.0 kcal/mol, and 27.8 kcal/mol (1 kcal = 4.18 kJ). When human eosinophils, KG-1 cells, and DMSO-differentiated HL-60 cells were preloaded with defined amounts of intracellular LTC4 by incubation with LTA4 and with incremental amounts of a glutathione conjugate, S-dinitrophenyl glutathione (GS-DNP) by sequential incubation with 1-chloro-2,4-dinitrobenzene, GS-DNP inhibited the export of LTC4 in a dose-dependent manner. By plotting the ratio of total GS-DNP (cell retained plus released) to the sum of total GS-DNP plus total LTC4 against the percentage inhibition of LTC4 release, IC40 values of 0.839, 0.803, and 0.841 were obtained for eosinophils, KG-1 cells, and DMSO-differentiated HL-60 cells, respectively. When cells preloaded with LTC4 were resuspended in incremental concentrations of the organic acid transport inhibitor, probenecid, there was a dose-dependent decrease in LTC4 release; GS-DNP and probenecid inhibited LTC4 release in a cumulative fashion, whereas neither inhibited the release of LTB4 from preloaded nondifferentiated HL-60 cells. Therefore, LTC4 export from cells of bone marrow origin occurs through a probenecid-sensitive membrane carrier shared by other glutathione conjugates and distinct from the LTB4 carrier export system.
Marked increase in nitric oxide synthase mRNA in rat dorsal root ganglia after peripheral axotomy: in situ hybridization and functional studies.
Authors: Verge V M , Xu Z , Xu X J , Wiesenfeld-Hallin Z , Hökfelt T
Published: May 2006

Using in situ hybridization, we studied nitric oxide (NO) synthase (EC 1.14.23.-) mRNA in lumbar dorsal root ganglia after peripheral transection of the sciatic nerve in rats. The effect of the NO synthase inhibitor N omega-nitro-L-arginine methyl ester on the nociceptive flexor reflex was also studied in axotomized rats. Nerve section induced a dramatic increase in number of NO synthase mRNA-positive cells in the ipsilateral dorsal root ganglia. In some of these cells the peptides galanin and/or vasoactive intestinal polypeptide and/or neuropeptide Y were also strongly up-regulated. Intravenous administration of nitro-L-arginine methyl ester blocked spinal hyperexcitability at much lower dosages in axotomized than in normal animals. The results suggest involvement of NO in the function of lumbar sensory neurons, especially after axotomy, perhaps preferentially at peripheral sites.
Antiserum raised against a synthetic phosphotyrosine-containing peptide selectively recognizes p185neu/erbB-2 and the epidermal growth factor receptor.
Authors: Bangalore L , Tanner A J , Laudano A P , Stern D F
Published: May 2006

Rabbits were immunized with a synthetic phosphopeptide corresponding to a major autophosphorylation site of p185neu/erbB2 to determine the feasibility of producing tyrosine-phosphopeptide-specific antibodies. A series of adsorption and affinity chromatography steps were used to select antibodies with the desired reactivity. Immunoblot experiments showed that the resulting serum is highly specific for tyrosine-phosphorylated forms of p185 and the related epidermal growth factor receptor. The serum recognized these two receptors selectively when compared to five other receptor tyrosine kinases and several phosphorylated substrates. The serum is compatible with tissue-based assays since it detected tyrosine phosphorylation of the epidermal growth factor receptor in immunofluorescence experiments on permeabilized cells. The generality of the procedures used means that similar anti-tyrosine phosphopeptide sera can be produced that recognize other tyrosine kinases and substrates. Such sera will have numerous applications in research and clinical settings.
Secondary structure of Src homology 2 domain of c-Abl by heteronuclear NMR spectroscopy in solution.
Authors: Overduin M , Mayer B , Rios C B , Baltimore D , Cowburn D
Published: May 2006

The Src homology 2 (SH2) domain is a recognition motif thought to mediate the association of the cytoplasmic proteins involved in signal transduction by binding to phosphotyrosyl-containing sequences in proteins. Assignments of nearly all 1H and 15N resonances of the SH2 domain from the c-Abl protein-tyrosine kinase have been obtained from homonuclear and heteronuclear NMR experiments. The secondary structure has been elucidated from the pattern of nuclear Overhauser effects, from vicinal coupling constants, and from observation of slowly exchanging amino hydrogens. The secondary structure contains two alpha-helices and eight beta-strands, six of which are arranged in two contiguous, antiparallel beta-sheets. Residues believed to be involved in phosphotyrosyl ligand binding are on a face of one beta-sheet. The alignment of homologous sequences on the basis of secondary structure suggests a conserved global fold in a family of SH2 domains.
Progesterone regulates the activity of collagenase and related gelatinases A and B in human endometrial explants.
Authors: Marbaix E , Donnez J , Courtoy P J , Eeckhout Y
Published: May 2006

Explants of human endometrium were cultured to study the release of matrix metalloproteinases (MMPs). Analysis of conditioned media by zymography revealed latent and active forms of collagenase (MMP-1, EC 3.4.24.7), 72-kDa gelatinase A (MMP-2, EC 3.4.24.24), and 92-kDa gelatinase B (MMP-9, EC 3.4.24.35). These proteinases were identified by their M(r), their inhibition by tissue inhibitor of metalloproteinases, and the activation of their zymogens by trypsin or aminophenylmercuric acetate. In the absence of sex hormone, explants released large amounts of enzyme activities, as measured by densitometry of zymograms or in soluble assays. Physiological concentrations of progesterone (10-200 nM) almost totally abolished the release of collagenase, of total gelatinase activity, and of the active form of gelatinase B and largely inhibited the release of the active form of gelatinase A. These effects, which were antagonized by mifepristone (RU 38486), suggest that progesterone restrains endometrial tissue breakdown by blocking the secretion and activation of MMPs.
Characterization of the Ca(2+)-triggered conformational transition in troponin C.
Authors: Wang Z , Gergely J , Tao T
Published: May 2006

Troponin C is the Ca(2+)-binding subunit of troponin in vertebrate striated muscle. Binding of Ca2+ to troponin C is thought to induce a conformational change that triggers subsequent events in the initiation of muscle contraction. A molecular modeling study has proposed that, when Ca2+ binds to the N-terminal triggering sites, helices B and C separate from the helices D and A, thereby exposing a crucial interaction site for troponin I, the inhibitory subunit of troponin [Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644]. In the present study the question of whether this separation actually occurs is addressed directly. A mutant rabbit skeletal troponin C containing a pair of cysteines at position 12 in helix A and position 49 in the polypeptide segment linking helices B and C was created by site-directed mutagenesis. Pyrene excimer fluorescence and resonance energy transfer studies on the labeled mutant troponin C reveal a Ca(2+)-induced increase in distance between the two cysteines. Under certain assumptions, the distance increase could be estimated from the extent of energy transfer to be approximately 13 A, in good agreement with the distance increase predicted by molecular modeling. Our results provide further experimental support for the model proposed by Herzberg et al. (above).
Identification of a 45-kDa protein at the protein import site of the yeast mitochondrial inner membrane.
Authors: Scherer P E , Manning-Krieg U C , Jenö P , Schatz G , Horst M
Published: May 2006

Import of proteins into mitochondria involves the cooperation of protein translocation systems in the outer and inner membranes. We have identified a 45-kDa protein at the protein import site of the yeast mitochondrial inner membrane. This 45-kDa protein could be crosslinked to a partly translocated precursor, which cannot be imported across the inner membrane when the matrix is depleted of ATP. In addition, an antibody against this protein strongly inhibited protein import into right-side-out inner-membrane vesicles. The 45-kDa protein accounts for only 0.1% of mitochondrial protein and appears peripherally attached to the outer face of the inner membrane. The properties of this protein suggest that it is a component of the protein import system of the mitochondrial inner membrane.