bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement of endothelial cell free radical generation: evidence for a central mechanism of free radical injury in postischemic tissues.
Authors: Zweier J L , Kuppusamy P , Lutty G A
Published: May 2006

Oxygen free radicals have been demonstrated to be important mediators of postischemic reperfusion injury in a broad variety of tissues; however, the cellular source of free radical generation is still unknown. In this study, electron paramagnetic resonance measurements with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrate that bovine endothelial cells subjected to anoxia and reoxygenation become potent generators of superoxide and hydroxyl free radicals. A prominent DMPO-OH signal aN = aH = 14.9 G is observed on reoxygenation after 45 min of anoxic incubation. Quantitative measurements of this free radical generation and the time course of radical generation are performed. Both superoxide dismutase and catalase totally abolish this radical signal, suggesting that O2 is sequentially reduced from O2-. to H2O2 to OH.. Addition of ethanol resulted in trapping of the ethoxy radical, further confirming the generation of OH.. Endothelial radical generation was shown to cause cell death, as evidenced by trypan blue uptake. Radical generation was partially inhibited and partially scavenged by the xanthine oxidase inhibitor allopurinol. Marked inhibition of radical generation was observed with the potent xanthine oxidase inhibitor oxypurinol. These studies demonstrate that endothelial cells subjected to anoxia and reoxygenation, conditions observed in ischemic and reperfused tissues, generate a burst of superoxide-derived hydroxyl free radicals that in turn cause cell injury and cell death. Most of this free radical generation appears to be from the enzyme xanthine oxidase. Thus, endothelial cell free radical generation may be a central mechanism of cellular injury in postischemic tissues.
Rat L (long interspersed repeated DNA) elements contain guanine-rich homopurine sequences that induce unpairing of contiguous duplex DNA.
Authors: Usdin K , Furano A V
Published: May 2006

The L family (long interspersed repeated DNA) of mobile genetic elements is a persistent feature of the mammalian genome. In rats, this family contains approximately equal to 40,000 members and accounts for approximately equal to 10% of the haploid genome. We demonstrate here that the guanine-rich homopurine stretches located at the right end of L-DNA induce oligonucleotide uptake by contiguous duplex DNA. The uptake is dependent on negative supercoiling and the length of the homopurine stretch and occurs even when the L-DNA homopurine stretches are introduced into a different DNA environment. The bound oligomer primes DNA synthesis when DNA polymerase and deoxyribonucleoside triphosphates are added, resulting in a faithful copy of the template to which the oligonucleotide had bound. The implications of this property of the L-DNA guanine-rich homopurine stretches in the amplification, recombination, and dispersal of L elements is discussed.
Human renal carcinoma expresses two messages encoding a parathyroid hormone-like peptide: evidence for the alternative splicing of a single-copy gene.
Authors: Thiede M A , Strewler G J , Nissenson R A , Rosenblatt M , Rodan G A
Published: May 2006

A peptide secreted by tumors associated with the clinical syndrome of humoral hypercalcemia of malignancy was recently purified from human renal carcinoma cell line 786-0. The N-terminal amino acid sequence of this peptide has considerable similarity with those of parathyroid hormone (PTH) and of peptides isolated from human breast and lung carcinoma (cell line BEN). In this study we obtained the nucleotide sequence of a 1595-base cDNA complementary to mRNA encoding the PTH-like peptide produced by 786-0 cells. The cDNA contains an open reading frame encoding a leader sequence of 36 amino acids and a 139-residue peptide, in which 8 of the first 13 residues are identical to the N terminus of PTH. Through the first 828 bases the sequence of this cDNA is identical with one recently isolated from a BEN cell cDNA library; however, beginning with base 829 the sequences diverge, shortening the open reading frame by 2 amino acids. Differential RNA blot analysis revealed that 786-0 cells express two major PTH-like peptide mRNAs with different 3' untranslated sequences, one of which hybridizes with the presently described sequence and the other one with that reported for the BEN cell PTH-like peptide cDNA. Primer-extension analysis of 786-0 poly(A)+ RNA together with Southern blot analysis of human DNA confirmed the presence of a single-copy gene coding for multiple mRNAs through alternate splicing. In addition, the 3' untranslated sequence of the cDNA described here has significant similarity to the c-myc protooncogene.
Sequences of the bovine and yeast ADP-ribosylation factor and comparison to other GTP-binding proteins.
Authors: Sewell J L , Kahn R A
Published: May 2006

The ADP-ribosylation factor (ARF) is a 21-kDa GTP-binding protein that serves as the cofactor in the cholera toxin-catalyzed activation of the stimulatory guanine nucleotide-binding protein of adenylate cyclase (Gs). An oligonucleotide probe based on the partial amino acid sequence was used to clone ARF from a bovine adrenal chromaffin cDNA library. The yeast (Saccharomyces cerevisiae) ARF gene was then cloned from a YCp50 genomic library by cross-species hybridization by using the coding region of the bovine gene. RNA gel blots of poly(A)+ RNA indicate that only one ARF message size (900 and 2000 base pairs) is present in yeast and cows, respectively. Comparison of the cDNA-derived amino acid sequences of ARF to other GTP-binding proteins reveals a structural relationship between ARF and the ras family of proteins. A slightly better structural relationship is detected when ARF is compared to the alpha subunits of the trimeric GTP-binding proteins, including Gs alpha. All of the biochemical characteristics of the purified ARF, including the lack of GTPase activity and the posttranslational myristoylation, are consistent with the derived sequences. Comparison of the ARF sequences to that of the chicken processed pseudogene (CPS-1), previously reported as a ras homologue, reveals that CPS-1 is actually an ARF-derived gene. These results demonstrate that ARF is a GTP-binding protein with structural features of both the ras and the trimeric GTP-binding protein families.
Chitin synthase 2 is essential for septum formation and cell division in Saccharomyces cerevisiae.
Authors: Silverman S J , Sburlati A , Slater M L , Cabib E
Published: May 2006

Previous work led to the puzzling conclusion that chitin synthase 1, the major chitin synthase activity in Saccharomyces cerevisiae, is not required for synthesis of the chitinous primary septum. The mechanism of in vivo synthesis of chitin has now been clarified by cloning the structural gene for the newly found chitin synthase 2, a relatively minor activity in yeast. Disruption of the chitin synthase 2 gene results in the loss of well-defined septa and in growth arrest, establishing that the gene product is essential for both septum formation and cell division.
Concentration-affinity equivalence in gene regulation: convergence of genetic and environmental effects.
Authors: Zuckerkandl E , Villet R
Published: May 2006

It is proposed that equivalent phenotypic effects can be obtained by either structural changes in macromolecules involved in gene regulation or changes in activity of the structurally unaltered macromolecules. This equivalence between changes in activity (concentration) and changes in structure can come into play within physiologically plausible limits and seems to represent an important interface between environment and genome--namely, between environmentally determined and genetically determined gene expression. The equivalence principle helps explain the appearance of phenocopies. It also points to a general pathway favorable to the occurrence, during evolution, of frequent episodes corresponding to Waddington's genetic assimilation and is likely to represent one component of the system responsible for the high frequency of recurrence of parallel evolution.
The copper, zinc-superoxide dismutase gene of Saccharomyces cerevisiae: cloning, sequencing, and biological activity.
Authors: Bermingham-McDonogh O , Gralla E B , Valentine J S
Published: May 2006

The gene for copper, zinc-superoxide dismutase (Cu,Zn-SOD; EC 1.15.1.1) from the yeast Saccharomyces cerevisiae has been cloned, sequenced, and shown to have physiological activity. The gene was isolated from a lambda gt11 library by using a long, unique deoxyoligonucleotide probe. The probe sequence was deduced from the known amino acid sequence by using a computer-generated yeast codon preference table. The sequence of the coding and flanking regions is reported. The cloned gene was expressed and shown to be active in vivo. A 3.2-kilobase fragment containing the coding region and 160 upstream bases, subcloned in a yeast/Escherichia coli shuttle vector, was used to transform a yeast strain lacking Cu,Zn-SOD activity. The presence of the Cu,Zn-SOD gene-containing plasmid corrected the characteristic dioxygen sensitivity of this strain. Electrophoretic transfer blots with antibody to yeast Cu,Zn-SOD showed the presence of the protein in transformants and wild-type yeast but not in the mutant. The role of Cu,Zn-SOD in defense against dioxygen toxicity is discussed in the light of these findings.
Amplified human MYC oncogenes localized to replicating submicroscopic circular DNA molecules.
Authors: Von Hoff D D , Needham-VanDevanter D R , Yucel J , Windle B E , Wahl G M
Published: May 2006

Amplification of genes can sometimes be detected by molecular hybridization but not by cytogenetic methods, suggesting that in some cases the units of amplification may be too small to be detected by light microscopy. The experiments reported here investigate whether submicroscopic amplification units are present in early passages of the human tumor cell lines HL-60 and COLO 320. The results show that such cells do contain submicroscopic, extrachromosomal, supercoiled circular molecules harboring MYC genes. The molecules in HL-60 are approximately 250 kilobase pairs (kbp), while those in COLO 320 are 120-160 kbp. The extrachromosomal molecules in HL-60 are shown to replicate semiconservatively and approximately once in one cell cycle. We propose that these submicroscopic elements are precursors of double-minute chromosomes, the usual extrachromosomal manifestation of gene amplification, since both are structurally similar and replicate autonomously.
Structural characterization of the core of the paired helical filament of Alzheimer disease.
Authors: Wischik C M , Novak M , Edwards P C , Klug A , Tichelaar W , Crowther R A
Published: May 2006

The paired helical filament, the principal constituent of the neurofibrillary tangles characteristic of Alzheimer disease, is shown to consist of two structurally distinct parts. An external fuzzy region can be removed by Pronase treatment to leave a Pronase-resistant morphologically recognizable core. Scanning transmission electron microscopy gives an estimate for the mass per unit length as 79 kDa.nm-1 before Pronase treatment and 65 kDa.nm-1 after treatment. The fuzzy region carries all the epitopes recognized by two different antisera against microtubule-associated protein tau. By contrast, a monoclonal antibody (mAb) we have raised to paired helical filament cores (mAb 423) decorates Pronase-treated filaments much more strongly than it does untreated ones. We have shown in previous papers that the epitope recognized by mAb 423 is carried by a central 9.5-kDa fragment of tau protein, which therefore forms part of the Pronase-resistant core structure. The remainder of the tau protein incorporated into the filaments must contribute part, if not all, of the fuzzy region. The mass per unit length measurements imply that the three-domain structural subunit of the core that we visualized previously by image reconstruction has a molecular mass of approximately equal to 100 kDa.
cDNA cloning of human liver monoamine oxidase A and B: molecular basis of differences in enzymatic properties.
Authors: Bach A W , Lan N C , Johnson D L , Abell C W , Bembenek M E , Kwan S W , Seeburg P H , Shih J C
Published: May 2006

The monoamine oxidases play a vital role in the metabolism of biogenic amines in the central nervous system and in peripheral tissues. Using oligonucleotide probes derived from three sequenced peptide fragments, we have isolated cDNA clones that encode the A and B forms of monoamine oxidase and have determined the nucleotide sequences of these cDNAs. Comparison of the deduced amino acid sequences shows that the A and B forms have subunit molecular weights of 59,700 and 58,800, respectively, and have 70% sequence identity. Both sequences contain the pentapeptide Ser-Gly-Gly-Cys-Tyr, in which the obligatory cofactor FAD is covalently bound to cysteine. Based on differences in primary amino acid sequences and RNA gel blot analysis of mRNAs, the A and B forms of monoamine oxidase appear to be derived from separate genes.
Properties of the ribosomal P2 protein autoantigen are similar to those of foreign protein antigens.
Authors: Elkon K , Bonfa E , Llovet R , Danho W , Weissbach H , Brot N
Published: May 2006

Approximately 15% of patients with systemic lupus erythematosus have autoantibodies that bind to a shared epitope previously shown to be located on the carboxyl-terminal 22 amino acids of three 60S ribosomal proteins, P0, P1, and P2 ("P proteins"). A hydrophilicity plot and fine epitope mapping with seven synthetic peptides revealed that the properties of the antigenic site were similar to certain properties of epitopes on foreign protein antigens--namely, the epitope was located in the most hydrophilic portion of the P2 protein and also in the terminal region of the molecule. However, this site has been highly conserved during evolution. A mouse monoclonal antibody induced by immunization with ribosomal proteins had a fine specificity similar to the lupus antibodies. This finding indicates that a highly conserved region of a lupus autoantigen may also be antigenic in some normal animals. Therefore, lupus autoantibodies may be similar in most, if not all respects, to antibodies produced by immunization.
Derivation of force fields for molecular mechanics and dynamics from
ab initio
energy surfaces
Authors: Maple Jon R. , Dinur Uri , Hagler Arnold T.
Published: May 2006

We present a technique for addressing the problem of deriving potential energy functions for the simulation of organic, polymeric, and biopolymeric systems, as well as for modeling vibrational spectroscopic properties. This method is designed to address three major objectives: deriving and comparing optimal functional forms for describing the energies of molecular deformations and interactions, developing a technique to rapidly and objectively determine reasonable force constants for intermolecular and intramolecular interactions, and determining the transferability of these potential forms and constants. The first two of these objectives are addressed in this paper, while the latter problem will be treated elsewhere. The technique uses
ab initio
molecular energy surfaces, which are described by the energy and its first and second derivatives with respect to coordinates. As an example, application to a small model compound (i.e., the formate anion) is given. A variety of analytical forms for the potential are tested against the data, to find which forms are best. The importance of anharmonicity and cross terms in accounting for structure and energy, as well as for dynamics, is demonstrated and a more accurate representation of the out-of-plane deformation for a trigonal center is derived from the energy surfaces.
Cinnamyl-alcohol dehydrogenase, a molecular marker specific for lignin synthesis: cDNA cloning and mRNA induction by fungal elicitor.
Authors: Walter M H , Grima-Pettenati J , Grand C , Boudet A M , Lamb C J
Published: May 2006

Cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyzes the final step in a branch of phenylpropanoid synthesis specific for production of lignin monomers. We have isolated a full-length cDNA clone encoding CAD, as a molecular marker specific for lignification, by immunoscreening a lambda gt11 library containing cDNAs complementary to mRNA from elicitor-treated cell cultures of bean (Phaseolus vulgaris L.). The clone comprises a single long open reading frame of 1767 base pairs, 31 base pairs of 5' leader, and 152 base pairs of 3' untranslated sequence. The deduced 65-kDa CAD polypeptide has several features that are strongly conserved in alcohol dehydrogenases. Addition of fungal elicitor to cell cultures stimulates CAD transcription, which leads to a remarkably rapid, but transient, accumulation of CAD mRNA, with no detectable lag and maximal levels after 1.5 hr. Southern blot analysis of bean genomic DNA indicates that elicitor-induced CAD is encoded by a single gene. The regulatory significance of the rapid activation of this CAD gene and the possible existence of a second, divergent CAD gene involved in lignification during xylogenesis are discussed.
Cell shape controls terminal differentiation of human epidermal keratinocytes.
Authors: Watt F M , Jordan P W , O'Neill C H
Published: May 2006

Cultures of human epidermal keratinocytes provide a useful experimental model with which to study the factors that regulate cell proliferation and terminal differentiation. One situation that is known to trigger premature terminal differentiation is suspension culture, when keratinocytes are deprived of substratum and intercellular contact. We have now investigated whether area of substratum contact, and hence cell shape, can regulate terminal differentiation. Keratinocytes were grown on circular adhesive islands that prevented cell-cell contact. By varying island area we could vary cell shape from fully spread to almost spherical. We found that when substratum contact was restricted, DNA synthesis was inhibited and expression of involucrin, a marker of terminal differentiation, was stimulated. Inhibition of proliferation was not a sufficient stimulus for involucrin synthesis in fully spread cells. When DNA synthesis and involucrin expression were plotted against contact area, classic dose-response curves were obtained. Thus cell shape acts as a signal for the terminal differentiation of keratinocytes in culture.
Expression of membrane interleukin 1 by fibroblasts transfected with murine pro-interleukin 1 alpha cDNA.
Authors: Fuhlbrigge R C , Fine S M , Unanue E R , Chaplin D D
Published: May 2006

Studies of interleukin 1 (IL-1) alpha and beta have emphasized their functional similarities. IL-1 alpha and -beta are encoded by ancestrally related genes that have diverged dramatically in primary sequence; however, only modest differences in the regulation or biological activity of IL-1 alpha and IL-1 beta have been documented. Here we show that mouse L cells transfected with murine pro-IL-1 alpha cDNA expressed biologically active, 33-kilodalton pro-IL-1 alpha, and that this pro molecule was neither processed to the 17-kilodalton mature form nor secreted. The transfected cells also expressed membrane-associated IL-1 biological activity, indicating that the pro-IL-1 alpha cDNA can direct expression of membrane-associated IL-1 and that cleavage of the pro molecule is not required for membrane presentation. In contrast, transfection of pro-IL-1 beta cDNA did not generate biologically active material in L cells. Evidence is presented that the native murine IL-1 beta precursor molecule is also biologically inactive in peritoneal exudate cells stimulated with lipopolysaccharide. These differences in distribution of the bioactive forms of IL-1 alpha and IL-1 beta may provide selective advantages for the maintenance of two gene products with similar functions.
Structure of the coding sequence and primary amino acid sequence of acetyl-coenzyme A carboxylase.
Authors: López-Casillas F , Bai D H , Luo X C , Kong I S , Hermodson M A , Kim K H
Published: May 2006

Acetyl-coenzyme A carboxylase (Ac-CoA carboxylase; EC 6.4.1.2) catalyzes the rate-limiting reaction in long-chain fatty acid biosynthesis. To investigate the mechanism of genetic control of expression of Ac-CoA carboxylase and the relationship between its structure and function, cDNA clones for Ac-CoA carboxylase were isolated. The complete coding sequence contains 7035 bases; it encodes a polypeptide chain of 2345 amino acids having a Mr of 265,220. The sequences of several CNBr peptides of Ac-CoA carboxylase were localized within the predicted protein sequence as were those peptides that contain the sites for phosphorylation. The deduced protein contains one putative site for biotinylation in the NH2-terminal half. The "conserved" biotinylation site peptide, Met-Lys-Met, is preceded by valine, whereas alanine is found in a similar position in all other known biotin-containing proteins. The primary sequences of Ac-CoA carboxylase and carbamoyl phosphate synthetase exhibit substantial identity.
CD3-associated heterodimeric polypeptides on suppressor hybridomas define biologically active inhibitory cells.
Authors: Weiner D B , Liu J , Hanna N , Bluestone J A , Coligan J E , Williams W V , Greene M I
Published: May 2006

We have investigated the relationship between CD3 expression and the suppressor T-cell function. We have isolated stable clonal cell lines of the F12.23 suppressor T-cell hybridoma that are either CD3+ or CD3-. These lines were subjected to functional assays including inhibition of in vivo hapten-specific delayed-type hypersensitivity responses, in vitro hapten-specific interleukin 2 responses, as well as hapten-specific cytotoxic T-lymphocyte assays. In all assays, the functional suppressor phenotype absolutely correlated with CD3 surface expression. Furthermore, we have immunoprecipitated heterodimeric proteins that share molecular features with some receptor polypeptides previously described. CD3 polypeptides found on the surface of suppressor T cells are phosphorylated after phorbol ester stimulation. Collectively these studies unambiguously define the suppressive supernatant function as a product of CD3+ receptor-bearing T cells.
Adenallene and cytallene: acyclic-nucleoside analogues that inhibit replication and cytopathic effect of human immunodeficiency virus in vitro.
Authors: Hayashi S , Phadtare S , Zemlicka J , Matsukura M , Mitsuya H , Broder S
Published: May 2006

Although several antiretroviral compounds are already known, almost no acyclic nucleoside derivatives lacking an oxacyclopentane have been reported to exert significant inhibition against human immunodeficiency virus type 1 (HIV-1) in vitro. We found two unsaturated acyclic nucleoside derivatives, adenallene [9-(4'-hydroxy-1',2'-butadienyl)adenine] and cytallene [1-(4'-hydroxy-1',2'-butadienyl)cytosine], that protect various CD4+ T-cell lines from the infectivity and cytopathic effect of HIV-1. These compounds inhibit the expression of HIV-1 gag-encoded protein and suppress viral DNA synthesis at concentrations that do not affect functions of normal T cells in vitro. They also inhibit the in vitro infectivity of another human retrovirus, HIV-2. Further in vitro analyses of the anti-HIV-1 activity revealed that the presence of two cumulated double bonds between the 1' and 2' carbons and between the 2' and 3' carbons confers antiretroviral activity in certain pyrimidine or purine derivatives containing a four-carbon chain. We have also found that the 4'-hydroxyl group is critical for the in vitro anti-HIV activity of adenallene. Our observations may provide structure-activity relationships for acyclic nucleoside analogues and may be of value in developing a new class of experimental drugs for the therapy of HIV-related diseases.
Dimerization of the tat protein from human immunodeficiency virus: a cysteine-rich peptide mimics the normal metal-linked dimer interface.
Authors: Frankel A D , Chen L , Cotter R J , Pabo C O
Published: May 2006

We have synthesized an 18-amino acid peptide that contains the cysteine-rich region of the tat protein from human immunodeficiency virus. Previous experiments in vitro with the intact tat protein have shown that these cysteines serve as metal ligands, causing tat to form metal-linked dimers. Ultraviolet absorption spectra show that the synthetic peptide (tat21-38) binds two Cd2+ or two Zn2+ ions per peptide monomer, and some changes in the circular dichroism spectra are seen as the metals bind. The peptide-metal complexes are completely resistant to proteolytic digestion, and mass spectrometry demonstrates that this peptide forms metal-linked dimers. The peptide can also combine with the intact tat protein to form metal-linked heterodimers. If these heterodimers are unable to trans-activate viral transcription, tat21-38 could be a lead compound for designing drugs to treat acquired immunodeficiency syndrome.
Interferon gamma induces the myristoylation of a 48-kDa protein in macrophages.
Authors: Aderem A A , Marratta D E , Cohn Z A
Published: May 2006

The lymphokine interferon gamma (IFN-gamma) induces the selective myristoylation of a macrophage protein with an apparent molecular mass of 48 kDa. The myristic acid-protein bond is resistant to treatment with hydroxylamine, suggesting that the fatty acid moiety is in an amide linkage. As little as 1 unit of IFN-gamma per ml induces the myristoylation of the 48-kDa protein, with half-maximal myristoylation being observed with 4 units/ml. The effect is observed within 1 hr after exposure to IFN-gamma and is maximal by 3-4 hr, after which it declines. IFN-alpha does not induce the myristoylation of the 48-kDa protein, and IFN-beta does so very poorly. Neither IFN-alpha nor IFN-beta has any effect on IFN-gamma-induced myristoylation of the 48-kDa protein. The 48-kDa protein is constitutively myristoylated in murine macrophages that have been activated in vivo by intraperitoneal injection of Corynebacterium parvum, suggesting that it may be an early intermediate in the activation of macrophages.
Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer.
Authors: Moscow J A , Townsend A J , Goldsmith M E , Whang-Peng J , Vickers P J , Poisson R , Legault-Poisson S , Myers C E , Cowan K H
Published: May 2006

The development of multidrug resistance in MCF7 human breast cancer cells is associated with overexpression of P-glycoprotein, changes in activities of several detoxication enzymes, and loss of hormone sensitivity and estrogen receptors (ERs). We have cloned the cDNA for one of the drug-detoxifying enzymes overexpressed in multidrug-resistant MCF7 cells (AdrR MCF7), the anionic isozyme of glutathione S-transferase (GST pi). Hybridization with this GST pi cDNA, GST pi-1, demonstrated that increased GST pi activity in AdrR MCF7 cells is associated with overexpression but not with amplification of the gene. We mapped the GST pi gene to human chromosome 11q13 by in situ hybridization. Since multidrug resistance and GST pi overexpression are associated with the loss of ERs in AdrR MCF7 cells, we examined several other breast cancer cell lines that were not selected for drug resistance. In each of these cell lines we found an inverse association between GST pi expression and ER content. We also examined RNA from 21 primary breast cancers and found a similar association between GST pi expression and ER content in vivo. GST pi mRNA content in 11 ER-positive tumors (less than or equal to 10 fmol/mg of protein) was significantly different from the GST pi content of 10 ER-negative tumors (P = 0.002; Mann-Whitney Wilcoxon test for two independent samples). The finding of similar patterns of expression of a drug-detoxifying enzyme and of ERs in vitro as well as in vivo suggests that ER-negative breast cancer cells may have greater protection against antineoplastic agents conferred by GST pi than ER-positive tumors.
Human immunodeficiency virus has an aspartic-type protease that can be inhibited by pepstatin A.
Authors: Seelmeier S , Schmidt H , Turk V , von der Helm K
Published: May 2006

The protease encoded by the human immunodeficiency virus (HIV) processes the viral gag and gag-pol protein precursor by posttranslational cleavage. In this study we have demonstrated by site-specific mutagenesis (Asp----Thr) and by pepstatin A inhibition that the recombinant HIV protease is an aspartic-type protease. Furthermore, incubation of HIV-infected H9 cells with pepstatin A inhibited part of the intracellular processing of the HIV gag protein yet had no apparent toxicity on HIV-infected cells during 48 hr of incubation.
Tryptophan repressor of Escherichia coli shows unusual thermal stability.
Authors: Bae S J , Chou W Y , Matthews K , Sturtevant J M
Published: May 2006

Differential scanning calorimetry demonstrates that the tryptophan repressor of Escherichia coli is unusually resistant to thermal denaturation. The dimeric protein undergoes reversible dissociative unfolding at pH 7.5 centered at about 90 degrees C. The thermal stability may be due in part to the unusual structure of the protein, which is composed of two identical intertwined polypeptide chains.
Dual conformations of an immunoglobulin light-chain dimer: heterogeneity of antigen specificity and idiotope profile may result from multiple variable-domain interaction mechanisms.
Authors: Stevens F J , Chang C H , Schiffer M
Published: May 2006

The structure of an immunoglobulin antigen-binding fragment (Fab) has been thought to be invariantly defined by well-conserved amino acid residues in the variable domains of the heavy and light chains. These conserved residues enable folding of the polypeptide segments into the characteristic immunoglobulin fold domains and are the major controllers of interactions between domains. However, crystallographic studies of some immunoglobulin light-chain dimers have suggested and the crystallographic structure of the Fab in an Fab-neuraminidase complex may have proven that antibodies are not restricted to a single, invariant relative positioning of the two variable domains. We propose that in some cases the detailed quaternary structural relationships between the variable domains of heavy and light chains are not restricted to those of the canonical Fab. It is unclear whether alterations of these relationships occur only after complex formation with antigen or, if in ligand-free solution, Fab conformers might coexist in relative concentrations determined by isomerization rates. In the latter case, antibody-presenting lymphocytes may be polyspecific, and the specificity of lymphocytes might be modulated by anti-idiotopic antibodies complexed to cell surface receptors. In either case, the idiotopic repertoire displayed by an antibody or lymphocyte surface receptor might be changed by the presence or absence of antigen.
Functional and structural characterization of P40, a mouse glycoprotein with T-cell growth factor activity.
Authors: Uyttenhove C , Simpson R J , Van Snick J
Published: May 2006

Antigen-independent cell lines were derived from mouse helper T-cell clones by culture in autologous supernatant obtained after stimulation with concanavalin A. A factor, termed P40, supporting the growth of these lines was purified and characterized as a basic 32- to 39-kDa single-chain glycoprotein functionally distinct from previously identified T-cell growth factors and apparently unrelated structurally to any known protein. Of a number of cell lines, only helper T cells responded to P40, and this response was not mediated by either interleukin 2 or interleukin 4.
Failure of a human immunodeficiency virus (HIV) immune globulin to protect chimpanzees against experimental challenge with HIV.
Authors: Prince A M , Horowitz B , Baker L , Shulman R W , Ralph H , Valinsky J , Cundell A , Brotman B , Boehle W , Rey F
Published: May 2006

To assess the possible efficacy of passive immunization against human immunodeficiency virus (HIV) an immune globulin was prepared from plasma of HIV-seropositive donors selected to be among those having the top 12.5% of virus-neutralizing antibody titers. The immune globulin was treated with pepsin to render it intravenously tolerable. The preparation, which we termed HIVIG, neutralized 100 tissue culture 50% infective doses (TCID50) of HIV at an average dilution of 1:1000 in neutralization tests in vitro. During preparation HIVIG was subjected to virus inactivation and removal procedures that in theory resulted in a reduction in HIV infectivity by a factor of 10(25). At a dose of 9-10 ml/kg of body weight both the virus-inactivated source plasma and the final immunoglobulin preparation were noninfective and without adverse effect in two chimpanzees. Two chimpanzees inoculated intravenously with HIVIG at 1 ml/kg and two inoculated with 10 ml/kg were challenged intravenously 1 day later with 400 TCID50 of the same strain of HIV (HTLV-IIIb) used in neutralization assays in vitro. All animals became infected. Incubation periods to virus isolation (by cocultivation with human mononuclear cells) in HIVIG recipients did not differ significantly from the incubation period seen in a control animal that received a normal anti-HIV-free immunoglobulin. These findings may have implications for understanding the failure of experimental vaccines to protect against HIV challenge in chimpanzee experiments.
An evolutionarily conserved protein binding sequence upstream of a plant light-regulated gene.
Authors: Giuliano G , Pichersky E , Malik V S , Timko M P , Scolnik P A , Cashmore A R
Published: May 2006

A protein factor, identified in nuclear extracts obtained from tomato (Lycopersicon esculentum, Solanaceae) and Arabidopsis thaliana (Brassicaceae) seedlings, specifically binds upstream sequences from the plant light-regulated gene family encoding the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RBCS). RBCS upstream sequences from tomato, pea (Pisum sativum, Leguminosae), and Arabidopsis are recognized by the factor. The factor recognition occurs via a short conserved sequence (G box) whose consensus sequence is 5'-TCTTACACGTGGCAYY-3' (where Y is pyrimidine). This sequence is distinct from the GT motif described previously in RBCS promoters. Two other conserved sequences, showing a lesser degree of evolutionary conservation, are found upstream of the G box but do not bind to the G box binding factor (GBF). Twelve nucleotides within the G box are sufficient for the formation of a stable DNA-GBF complex. GBF is found in both light-grown and dark-adapted tomato leaf extracts, but it is present in greatly reduced amounts in root extracts.
Two cis-acting elements responsible for posttranscriptional trans-regulation of gene expression of human T-cell leukemia virus type I.
Authors: Seiki M , Inoue J , Hidaka M , Yoshida M
Published: May 2006

The pX sequence of human T-cell leukemia virus type I codes for two nuclear proteins, p40tax and p27rex, and a cytoplasmic protein, p21x-III.p40tax activates transcription from the long terminal repeat (LTR), whereas p27rex modulates posttranscriptional processing to accumulate gag and env mRNAs that retain intron sequences. In this paper, we identify two cis-acting sequence elements needed for regulation by p27rex: a 5' splice signal and a specific sequence in the 3' LTR. These two sequence elements are sufficient for regulation by p27rex; expression of a cellular gene (metallothionein I) became sensitive to rex regulation when the LTR was inserted at the 3' end of this gene. The requirement for these two elements suggests an unusual regulatory mechanism of RNA processing in the nucleus.
Cloning of a Drosophila melanogaster guanine nucleotide regulatory protein beta-subunit gene and characterization of its expression during development.
Authors: Yarfitz S , Provost N M , Hurley J B
Published: May 2006

A Drosophila melanogaster gene encoding a protein with greater than 80% sequence identity to the beta subunits of mammalian guanine nucleotide-binding regulatory proteins (G proteins) has been cloned. The gene, which was mapped to 13F on the X chromosome by in situ hybridization, was cloned from a Drosophila genomic library by using a bovine transducin beta-subunit cDNA probe. Genomic DNA blot hybridization analysis indicated that there is a single Drosophila G-protein beta-subunit gene. Multiple transcripts were detected throughout development; in adult flies the mRNA is expressed at higher levels in heads than in bodies. The proposed coding region is uninterrupted by introns, but there is evidence for differential mRNA splicing in the 5' nontranslated region.
Modeling of the bryostatins to the phorbol ester pharmacophore on protein kinase C.
Authors: Wender P A , Cribbs C M , Koehler K F , Sharkey N A , Herald C L , Kamano Y , Pettit G R , Blumberg P M
Published: May 2006

The bryostatins are macrocyclic lactones that represent an additional structural class of potent activators of protein kinase C. These marine animal biosynthetic products are of unusual interest because they induce only a subset of the biological responses induced by the phorbol esters. We have now determined the binding affinities of naturally occurring and semisynthetic bryostatins for protein kinase C by competition analysis with [26-3H]bryostatin 4 as the radioactive ligand. Esterification of the hydroxyl group at C26 caused dramatic loss of activity as did inversion of the asymmetric center at this position. In contrast, neither of the ester groups at C7 and C20 had a major influence on activity. Computer modeling of the phorbol esters, related diterpenes, and indole alkaloids suggested that the C20, C9, and C4 oxygens of phorbol represented critical elements of the phorbol ester pharmacophore. The C26 oxygen of the bryostatins, together with the C1 and C19 oxygens, gave an excellent spatial correlation with this model, with a root-mean-square deviation of 0.16 A (compared to 0.10-0.35 A among phorbol-related diterpenes). The extension of the phorbol ester pharmacophore model to the bryostatins and its agreement with the structure-activity relations for the bryostatin class of compounds provide additional support for the validity of the model.
Tyrosinases of murine melanocytes with mutations at the albino locus.
Authors: Halaban R , Moellmann G , Tamura A , Kwon B S , Kuklinska E , Pomerantz S H , Lerner A B
Published: May 2006

Tyrosinase (monophenol monooxygenase; monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1) is a key enzyme in the synthesis of melanin. Reduced levels of tyrosinase play an important role in albinism. The data described here show differences in the expression and characteristics of tyrosinase in cutaneous murine melanocytes grown in culture from normal wild-type strains (C/C); from three albino locus mutants: himalayan (ch/ch), chinchilla (cch/cch), and albino (c/c); and from the double-mutant heterozygous pink-eyed chinchilla (cchp/cp). Our results suggest that the diminished pigmentation in all mutants is due to abnormal posttranslational modification of the enzyme: the levels of mRNA for tyrosinase in wild-type, himalayan, and pink-eyed chinchilla melanocytes are similar; the himalayan mutation confers a deficiency in N-linked glycosylation, which results in an extremely unstable enzyme that is also temperature sensitive; the chinchilla and albino mutations confer susceptibility to proteolytic cleavage; the pink-eye dilution confers a reduction in the levels of immunoprecipitable tyrosinase, and what little enzyme there is fails to be translocated from the trans-Golgi network to melanosomes. The kinetics of activation and inhibition of the enzyme by the cofactor dopa are unique for the mutants tested and differ from those of tyrosinase from wild-type melanocytes. The findings support the conclusion that the albino locus in mice encodes the structural gene of tyrosinase.
Evidence for regulation of reinitiation in translational control of GCN4 mRNA.
Authors: Hinnebusch A G , Jackson B M , Mueller P P
Published: May 2006

Translational control of the GCN4 gene of Saccharomyces cerevisiae is mediated by four upstream open reading frames (URFs) present in the leader of GCN4 mRNA. URFs 3 and 4 efficiently repress GCN4 expression in normal growth conditions; URFs 1 and 2 are required to overcome this repression in amino acid-starved cells. lacZ fusions to URFs 3 and 4 were used to determine the translational event that is regulated at these sequences by URFs 1 and 2. URF3-lacZ, URF4-lacZ, and GCN4-lacZ fusions are affected similarly by URFs 1 and 2 when no other URFs are present in the leader: expression from all three fusions is reduced by an amount slightly greater in repressing than in derepressing conditions. These results are inconsistent with models that postulate a differential effect of URFs 1 and 2 on initiation or elongation rates at URFs 3 and 4 versus the GCN4 coding sequences. We propose that the efficiency of reinitiation at the GCN4 AUG codon after translation of URFs 3 and 4 is the translational event that is stimulated in derepressing conditions by URFs 1 and 2.
Algorithms for the restriction-site mapping of DNA molecules.
Authors: Krawczak M
Published: May 2006

Two algorithms are described that rapidly construct restriction maps of DNA molecules from single- and double-digest data. The implementation of these algorithms into the Pascal programs AMAPK and AMARD allows such mapping to be done on microcomputers.
Gangliosides prevent glutamate and kainate neurotoxicity in primary neuronal cultures of neonatal rat cerebellum and cortex.
Authors: Favaron M , Manev H , Alho H , Bertolino M , Ferret B , Guidotti A , Costa E
Published: May 2006

Using a sensitive histofluorescence staining method that allows for a quantitation of neuronal death, we compared the protective effects of gangliosides (a group of naturally occurring glycosphingolipids), phencyclidine (PCP), and MK-801 (dibenzocyclohepteneimine) on glutamate- and kainate-induced neuronal death in primary cultures of cortical and cerebellar neurons prepared from neonatal rats. PCP and MK-801 block neurotoxicity induced by glutamate doses 50 times higher than the LD50 (LD50 in Mg2+-free medium, 10 microM) but only partially block the kainate neurotoxicity (LD50 in presence of Mg2+, 100 microM). In contrast, pretreatment with gangliosides (GT1b greater than GD1b greater than GM1) results in complete and insurmountable protection against the neurotoxicity elicited by glutamate or kainate. In primary cultures of cerebellar granule cells gangliosides, unlike PCP and MK-801, fail to block glutamate-gated cationic currents and the glutamate-evoked increase of (i) inositol phospholipid hydrolysis (ii) c-fos mRNA content, and (iii) nuclear accumulation of c-fos protein. Protection of glutamate neurotoxicity by gangliosides does not require their presence in the incubation medium; however, it is proportional to the amount of glycosphingolipid accumulated in the neuronal membranes. The ganglioside concentration (30-60 microM) that blocks glutamate-elicited neuronal death also prevents glutamate- and kainate-induced protein kinase C translocation from cytosol to neuronal membranes.
Trans-splicing in chloroplasts: the rps 12 loci of Nicotiana tabacum.
Authors: Hildebrand M , Hallick R B , Passavant C W , Bourque D P
Published: May 2006

The rps12 gene in tobacco chloroplasts consists of three exons that code for a polypeptide with homology to Escherichia coli ribosomal protein S12. C-terminal exons 2 and 3 of rps12 are located in the inverted repeat regions of the tobacco chloroplast genome. Exon 1 of rps12 is 29 kilobase pairs downstream of the nearest copy of exons 2 and 3 and 69 kilobase pairs away from the distal copy of exons 2 and 3. RNA gel blot hybridization analysis and primer extension sequencing of cDNA to rps12 encoding RNAs indicate that exon 1 and exons 2 and 3 are encoded on separate transcripts. Exon 1 and exons 2 and 3 are covalently ligated in the correct reading frame in rps12 mRNA. These results indicate that a bimolecular (trans-) splicing event occurs during the formation of mature rps12 mRNA.
Mean lifetime of microtubules attached to nucleating sites.
Authors: Rubin R J
Published: May 2006

A simple two-phase (cap; no cap) macroscopic model describing the kinetic behavior at a labile tip of a microtubule has been proposed [Hill, T. L. (1984) Proc. Natl. Acad. Sci. USA 81, 6728-6732]. In the model, a microtubule exists either in a slowly growing phase (first-order rate constant, alpha) characterized by the existence of a GTP-tubulin cap at the growing tip; or the same microtubule exists in a rapidly shrinking phase (first-order rate constant, beta), which is entered if/when the GTP-tubulin cap is lost through a fluctuation, thus exposing GDP-tubulin subunits, which constitute the body of the microtubule. Transition between the two phases--i.e., loss of a cap (first-order rate constant, k) or formation of a new cap (first-order rate constant, k') occurs very infrequently and in a stochastic manner. In vitro experiments with centrosome-nucleated microtubules by Mitchison and Kirschner and Monte Carlo kinetic simulations, based on a realistic set of microscopic rate constants that apply to the end of a microtubule, suggest this alternation between two "quasimacroscopic" phases. In this paper, I outline the calculation of the mean lifetime of a microtubule nucleated on a centrosome by using Hill's model. For a microtubule M units long in the slowly growing phase, the mean lifetime for complete depolymerization is [M(k + k') + alpha + beta](beta k - alpha k')-1, provided that beta k greater than alpha k'. If the microtubule is in the rapidly shrinking phase, then the mean lifetime is M(k + k')(beta k - alpha k')-1, provided that beta k greater than alpha k'. In case beta k less than alpha k', the microtubule grows indefinitely, and the mean lifetime is infinite.
Origin-specific DNA-binding membrane-associated protein may be involved in repression of initiation of DNA replication in Bacillus subtilis.
Authors: Laffan J J , Firshein W
Published: May 2006

Previous binding studies with labeled double-stranded Bacillus subtilis DNA fragments to a protein blot of renatured Bacillus membrane proteins showed selective binding of two adjacent origin fragments to a 64-kDa protein. The selective binding of the 64-kDa protein could be blocked by prior incubation of the blots with a specific polyclonal antibody. An in vitro replication system derived from a B. subtilis DNA-membrane complex showed initiation activity without addition of exogenous enzymes or template. When the complex was first incubated with the 64-kDa antibody or with its Fab fragments, initiation activity was enhanced. Antibodies to several other Bacillus membrane proteins as well as nonspecific antibodies did not show any significant stimulatory effect. A heavy-density-label experiment indicated that the complex initiated multiple rounds of replication in the presence of the 64-kDa antibody but not in its absence. The 64-kDa antibody plus an initiation inhibitor (streptovaricin) showed only repair and elongation activity. The 64-kDa protein may act in vivo as a repressor/regulator of initiation activity.
Modifying preselected sites on proteins: the stretch of residues 633-642 of the myosin heavy chain is part of the actin-binding site.
Authors: Chaussepied P , Morales M F
Published: May 2006

We have designed an "antipeptide" capable of firmly and specifically interacting with a preselected stretch of myosin S-1 heavy chain. Covalent attachment of this antipeptide to its target stretch, residues 633-642, does not affect the intrinsic ATPase activities of the protein but significantly reduces the actin-binding capabilities of the myosin head.