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At least 343 records · Page 19Linked to original sources
PubMed1995

Differentiation alters the expression of the two splice variants of the serotonin 5-HT3 receptor-A mRNA in NG108-15 cells.

The serotonin 5-HT3-A receptor (5-HT3R-A) mRNA has been shown recently to be expressed as two forms (5-HT3R-AL and 5-HT3R-AS) varying by the presence or the absence of a sequence of 18 bases in the region corresponding to the second cytoplasmic domain of the receptor, and generated by alternative splicing at the level of the 3' acceptor site of exon 9. As the long form of the receptor exhibits a potential phosphorylation site that is disrupted by the alternative splicing, the hypothesis of functional identity and stochastic expression of these two variants was questioned. In the present study, we used quantitative reverse transcriptase-polymerase chain reaction to examine the possible influence of culture conditions on the expression and the alternative splicing of 5-HT3R-A mRNA in NG108-15 clonal cells. Cell differentiation induced by dibutyryl cyclic AMP or theophyllin plus prostaglandin E1 in the presence of 10% serum reduced by threefold the expression of total 5-HT3R-A mRNA, and favored the short form of the message as the ratio S/L (5-HT3R-AS mRNA/5-HT3R-AL mRNA) shifted from 2.23 to 7.33 after 9 days of treatment. Culture with 0.3% serum (instead of 10%) lowered by 10-fold the level of expression of total 5-HT3R-A mRNA, but only slightly reduced the S/L ratio. However, this ratio fell to 0.06 in the presence of 0.3% serum plus 10 ng/ml basic fibroblast growth factor. These results demonstrate that external factors can influence the differential expression of the two variants of the 5-HT3R-A in NG108-15 cells. Appropriate culture conditions for the almost exclusive expression of 5-HT3R-AS mRNA or 5-HT3R-AL mRNA in NG108-15 cells should allow the identification of possible differences in the respective functional properties of each of these two forms of the native 5-HT3 receptor.

Alternative Splicing↗
PubMed2001

Design of inhibitors of Ras--Raf interaction using a computational combinatorial algorithm.

Drugs that inhibit important protein-protein interactions are hard to find either by screening or rational design, at least so far. Most drugs on the market that target proteins today are therefore aimed at well-defined binding pockets in proteins. While computer-aided design is widely used to facilitate the drug discovery process for binding pockets, its application to the design of inhibitors that target the protein surface initially seems to be limited because of the increased complexity of the task. Previously, we had started to develop a computational combinatorial design approach based on the well-known 'multiple copy simultaneous search' (MCSS) procedure to tackle this problem. In order to identify sequence patterns of potential inhibitor peptides, a three-step procedure is employed: first, using MCSS, the locations of specific functional groups on the protein surface are identified; second, after constructing the peptide main chain based on the location of favorite locations of N-methylacetamide groups, functional groups corresponding to amino acid side chains are selected and connected to the main chain C(alpha) atoms; finally, the peptides generated in the second step are aligned and probabilities of amino acids at each position are calculated from the alignment scheme. Sequence patterns of potential inhibitors are determined based on the propensities of amino acids at each C(alpha) position. Here we report the optimization of inhibitor peptides using the sequence patterns determined by our method. Several short peptides derived from our prediction inhibit the Ras--Raf association in vitro in ELISA competition assays, radioassays and biosensor-based assays, demonstrating the feasibility of our approach. Consequently, our method provides an important step towards the development of novel anti-Ras agents and the structure-based design of inhibitors of protein--protein interactions.

Algorithms↗
PubMed1999

Reversible inactivation of macaque dorsomedial frontal cortex: effects on saccades and fixations.

Neural recording and electrical stimulation results suggest that the dorsomedial frontal cortex (DMFC) of macaque is involved in oculomotor behavior. We reversibly inactivated the DMFC using lidocaine and examined how saccadic eye movements and fixations were affected. The inactivation methods and monkeys were the same as those used in a previous study of the frontal eye field (FEF), another frontal oculomotor region. In the first stage of the present study, monkeys performed tasks that required the generation of single saccades and fixations. During 15 DMFC inactivations, we found only mild, infrequent deficits. This contrasts with our prior finding that FEF inactivation causes severe, reliable deficits in performance of these tasks. In the second stage of the study, we investigated whether DMFC inactivation affected behavior when a monkey was required to make more than one saccade and fixation. We used a double-step task: two targets were flashed in rapid succession and the monkey had to make two saccades to foveate the target locations. In each of five experiments, DMFC inactivation caused a moderate, significant deficit. Both ipsi- and contraversive saccades were disrupted. In two experiments, the first saccades were made to the wrong place and had increased latencies. In one experiment, first saccades were unaffected, but second saccades were made to the wrong place and had increased latencies. In the remaining two experiments, specific reasons for the deficit were not detected. Saline infusions into DMFC had no effect. Inactivation of FEF caused a larger double-step deficit than did inactivation of DMFC. The FEF inactivation impaired contraversive first or second saccades of the sequence. In conclusion, our results suggest that the DMFC makes an important contribution to generating sequential saccades and fixations but not single saccades and fixations. Compared with the FEF, the DMFC has a weaker, less directional, more task-dependent oculomotor influence.

Anesthetics, Local↗
PubMed1990

Human epithelial tumor antigen cDNA sequences. Differential splicing may generate multiple protein forms.

The isolation and characterization of complementary DNAs (cDNAs) which code for an epithelial antigen aberrantly expressed in human breast tumor tissue are described here. The only information regarding the primary structure of this potentially important antigen has been a 20-amino-acid repeat motif. We now report the complete amino acid sequences of different forms of the human epithelial tumor antigen as deduced from the nucleotide sequence of isolated non-repeat cDNAs. The diversity of protein forms is generated by a series of alternative splicing events that occur in the regions located upstream and downstream to a central tandem repeat array. Isolated cDNAs coding for the upstream region show that differential usage of alternative splice acceptor sites may generate two protein forms containing putative signal peptides of varying hydrophobicities. The complexity of possible antigen forms is further compounded by alternative splicing events occurring in the region 3' to the repeat array. The isolated cDNAs 3' to the tandem repeats indicate that whereas one mRNA transcript is colinear with the gene, and defines an open reading frame (ORF) containing 160 amino acids downstream to the repeat array, a second cDNA correlates with a mRNA that is generated by a series of splicing events. The deduced amino acid sequence of the spliced cDNA contains an ORF that is identical for 149 amino acids downstream to the repeat array with the amino acid sequence of the unspliced cDNA. At this point it diverges and continues for an additional 179 amino acids. The sequence contains a highly hydrophobic 28-amino-acid peptide, located towards the carboxyl terminus, that may correspond to a transmembrane region. The cDNAs and deduced amino acid sequences, presented here, define the complete amino acid sequences of the epithelial tumor antigen and demonstrate the existence of multiple protein forms that probably localize to different cellular and extracellular compartments.

Amino Acid Sequence↗
PubMed1995

Genotype-dependent serologic reactivities in patients infected with hepatitis C virus in the United States.

OBJECTIVE: To evaluate the serologic reactivities in patients infected with different hepatitis C virus (HCV) genotypes to four HCV proteins that are components of the second-generation recombinant immunoblot assay. MATERIAL AND METHODS: Serum samples from 36 patients with chronic HCV infection were obtained. RNA was extracted by using chaotropic lysis and isopropanol precipitation. Reverse-transcriptase polymerase chain reaction of the NS-5 region was performed, followed by automated single-pass dideoxy sequencing of desalted amplification products. Classification of isolated HCV subtypes was based on Simmonds' system. All samples were tested for antibodies to proteins 5-1-1, C100-3, C33c, and C22-3 with the second-generation recombinant immunoblot assay. RESULTS: Reactivity to protein 5-1-1 was significantly lower for patients with genotypes 2b and 3a than for those infected with HCV types 1a or 1b (P < 0.05). Antibody reactivity to the C100-3 protein was also reduced in patients infected with HCV types 2b and 3a. CONCLUSION: These data indicate that the genotype-dependent differences in serologic reactivities are substantial among patients with chronic HCV infection.

Base Sequence↗
PubMedDate not supplied

Optimal synthesis of protein purification processes.

There has been an increasing interest in the development of systematic methods for the synthesis of purification steps for biotechnological products, which are often the most difficult and costly stages in a biochemical process. Chromatographic processes are extensively used in the purification of multicomponent biotechnological systems. One of the main challenges in the synthesis of purification processes is the appropriate selection and sequencing of chromatographic steps that are capable of producing the desired product at an acceptable cost and quality. This paper describes mathematical models and solution strategies based on mixed integer linear programming (MILP) for the synthesis of multistep purification processes. First, an optimization model is proposed that uses physicochemical data on a protein mixture, which contains the desired product, to select a sequence of operations with the minimum number of steps from a set of candidate chromatographic techniques that must achieve a specified purity level. Since several sequences that have the minimum number of steps may satisfy the purity level, it is possible to obtain the one that maximizes final purity. Then, a second model that may use the total number of steps obtained in the first model generates a solution with the maximum purity of the product. Whenever the sequence does not affect the final purity or more generally does not impact the objective function, alternative models that are of smaller size are developed for the optimal selection of steps. The models are tested in several examples, containing up to 13 contaminants and a set of 22 candidate high-resolution steps, generating sequences of six operations, and are compared to the current synthesis approaches.

Biotechnology↗
PubMed2000

Two type XVII collagen (BP180) mRNA transcripts in human keratinocytes: a long and a short form.

We have analysed BP180 mRNA expression in normal human keratinocytes. Here we report the presence in normal keratinocytes of two COL17A1 transcripts which differ by 0.6 kb in length. Both mRNAs hybridized on Northern blot with probes directed to sequences encoding intracellular and extracellular fragments of BP180. By BLAST homology search alignments we extended the 3' untranslated region (3'UTR) of the known BP180 mRNA sequence by 877 bases to completion. Three of 20 cDNAs identified by BLAST searches contained a 610 bp deletion in this new 3'UTR sequence. Northern blot analysis with a probe complementary to this deleted sequence showed binding only to the larger mRNA. The deletion of 610 nucleotides in the smaller mRNA was verified by reverse transcription-PCR and sequencing. Genomic PCR showed the new sequence to be an extension of exon 56 of the COL17A1 gene which suggests that the second mRNA is generated by differential splicing. In normal keratinocytes the level of the smaller transcript was 5-15% of that of the larger transcript whereas in a squamous cell carcinoma cell line this ratio was reversed, the smaller mRNA being three times more abundant than the larger mRNA. The biological significance of this newly identified transcript in protein synthesis and tissue expression or in cell differentiation, proliferation or adhesion is as yet unknown.

Autoantigens↗
PubMed1993

Multiple pathways of reversion in viroids for conservation of structural elements.

From site-directed mutagenesis of potato spindle tuber viroid (PSTVd) it had been concluded earlier that the formation of a thermodynamically metastable structure containing hairpin II (HP II) is critical for infectivity. In order to differentiate between structural and sequence effects, in the present work base pairs in HP II were exchanged by site-directed double mutations without significant alterations in the native rod-like structure of PSTVd. The mutants were viable and genetically stable in the first generation, but one of the two mutations reverted to the wild-type nucleotide in the second generation. Single-site mutations in the stem of HP II, which had been described as revertants to the wild-type sequence earlier, were analysed with respect to the time course of reversion and the sequence variation during reversion. All replicating sequence variants were separated by gel electrophoretic techniques and the sequences and their relative frequencies were determined. From both types of studies it can be concluded (i) that HP II is a functional element in the (-)strand replication intermediate, generated due to sequential folding during synthesis, and that it is essential for template activity of (+)strand synthesis; (ii) that G:U pairs are tolerated transiently in (-)strand HP II; the lower stability of such a HP II is compensated by additional mutations outside HP II which suppress the competition of a rod-like structure; and (iii) that the reversions are generated spontaneously during (-)strand synthesis. Furthermore, the double-stranded structure of HP II is the essential element for short term replication of PSTVd but the exact sequence of the wild-type proves to be superior with regard to fitness and replicability of PSTVd.

Base Sequence↗
PubMed1996

Clonal evolution in B-lineage acute lymphoblastic leukemia by contemporaneous VH-VH gene replacements and VH-DJH gene rearrangements.

B-cell acute lymphoblastic leukemia (B-ALL), more frequently than any other B-lineage neoplasm, exhibits oligoclonal Ig heavy chain (IgH) gene rearrangement in 15% to 43% of all cases studied. To study the molecular processes that promote multiple IgH rearrangements, a comprehensive sequence analysis of a B-ALL case was performed in which seven clonal IgH gene rearrangements were identified. The genetic profiles suggested that a single leukemic progenitor clone evolved into several subclones through dual processes of variable (VH) to preexisting diversity-joining (DJH) gene segment rearrangement and VH to VH gene replacement. Predominant IgH-V usage and the uniquely rearranged clonotype-specific VHDJH region gene sequences were identified using a novel DNA-based gene amplification strategy. Polymerase chain reaction (PCR) was directed by an IgH-J generic primer and a complement of family-specific IgH-V primers that defined the major B-cell IgH-V gene usage. Clonality of rearranged VHDJH bands was substantiated by high resolution denaturant gel electrophoretic analysis. Sequence patterns of the amplified VHDJH fragments segregated into two groups defined by common DJH sequences. Partial N region homology at the VHD junction as well as shared DJH sequences firmly established VH to VHDJH gene replacement as a mechanism generating clonal evolution in one group. In the second subset, oligoclonality was propagated by independent VH gene rearrangements to a common DJH precursor. The contributions of all clonal Ig-VHDJH repertoires for each group was approximately 50% and reflected a symmetric distribution of leukemic subclones generated by either process. Thus, oligoclonal rearrangements evolved by two independent, yet seemingly contemporaneous molecular genetic mechanisms. All seven clones displayed nonfunctional Ig-VHDJH recombinations. These observations may have relevance to the recombinatorial opportunities available during normal B-cell maturation.

Base Sequence↗
PubMed1996

Characterisation of a glutathione reductase gene and its genetic locus from pea (Pisum sativum L.).

A cDNA encoding the chloroplast/mitochondrial form of glutathione reductase (GR; EC 1.6.4.2) from pea (Pisum sativum L.) was used to map a single GR locus, named GOR1. In two domesticated genotypes of pea (cv. Birte and JI 399) it is likely that the GOR1 locus contains a single gene. However, in a semi-domesticated land race of pea (JI 281) two distinct but closely related sets of GR gene sequences were detected at the GOR1 locus. The extra GR sequences in JI 281 represent either a second intact gene or a partial or pseudogene copy. A GR gene was cloned from cv. Birte, sequenced and its structure analysed. No feature of the transcription or structure of the gene suggested a mechanism for generating any more than one form of GR. From these data plus previously published biochemical evidence we suggest that a second, distinct gene encoding for the cytosolic form of GR should be present in peas. The GOR1-encoded GR mRNA can be detected in all main organs of the plant and no alternative spliced species was present which could perhaps account for the generation of multiple isoforms of GR. The mismatch between the number of charge-separable isoforms in pea and the proposed number of genes suggests that different GR isoforms arise by some form of post-translational modification.

Amino Acid Sequence↗
PubMed1998

Preparation of functionally active cell-permeable peptides by single-step ligation of two peptide modules.

Noninvasive cellular import of synthetic peptides can be accomplished by incorporating a hydrophobic, membrane-permeable sequence (MPS). Herein, we describe a facile method that expedites synthesis of biologically active, cell-permeable peptides by site-specific ligation of two free peptide modules: one bearing a functional sequence and the second bearing a MPS. A nonpeptide thiazolidino linkage between the two modules is produced by ligation of the COOH-terminal aldehyde on the MPS and the NH2-terminal 1, 2-amino thiol moiety on the functional sequence. This thiazolidine ligation approach is performed with stoichiometric amounts of fully unprotected MPS and functional peptide in an aqueous buffered solution, eliminating the need for additional chemical manipulation and purification prior to use in bioassays. Two different MPSs were interchangeably combined with two different functional sequences to generate two sets of hybrid peptides. One set of hybrid peptides, carrying the cytoplasmic cell adhesion regulatory domain of the human integrin beta3, inhibited adhesion of human erythroleukemia cells to fibrinogen-coated surfaces. A second set of hybrid peptides, carrying the nuclear localization sequence of the transcription factor NF-kappaB, inhibited nuclear import of transcription factors NF-kappaB, activator protein 1, and nuclear factor of activated T cells in agonist-stimulated Jurkat T lymphocytes. In each assay, these nonamide bond hybrids were found to be functionally comparable to peptides prepared by the conventional method. Cumulatively, this new ligation approach provides an easy and rapid method for engineering of functional, cell-permeable peptides and demonstrates the potential for synthesis of cell-permeable peptide libraries designed to block intracellular protein-protein interactions.

Amino Acid Sequence↗
PubMed1996

The genes encoding the P39 and CG30 proteins of Bombyx mori nuclear polyhedrosis virus.

We have cloned and analysed the transcriptional properties of two closely linked genes, p39 and cg30, of Bombyx mori nuclear polyhedrosis virus (BmNPV). These genes encode a structural polypeptide and a putative transcriptional regulator of the virus, respectively. The cg30 gene is transcribed prior to and after DNA replication from a site located within the ORF of the adjacent p39 gene. Its transcription product, a 1.3 kb mRNA, is polyadenylated at a site containing consensus eukaryotic polyadenylation signals and mapping 87 bp downstream of the translation termination codon for CG30. During the later stages of infection, two additional RNAs, 2.2 and 6.5 kb, are also transcribed through the cg30 gene. The 2.2 kb RNA, representing the mRNA that encodes P39, is initiated from three relatively closely spaced sites located upstream of the P39 ORF. The 6.5 kb RNA is apparently transcribed from the promoter sequences of another gene located further upstream of the p39 gene. The 2.2 and 6.5 kb transcripts have two polyadenylation sites. The first site is the same as the one used to generate the cg30 gene-specific transcripts. The second is located 4 bp downstream of the CG30 translation termination codon. Transient expression assays show that the p39 gene sequences immediately upstream of the CG30 ORF can direct expression of a reporter gene when the latter is co-transfected with the gene encoding the early baculovirus trans-activator IE1. Thus, these sequences behave as a delayed-early baculovirus gene promoter.

Amino Acid Sequence↗
PubMed2001

Characterization of trabecular bone by dipolar demagnetizing field MRI.

A multiple spin-echo (MSE) sequence has been applied for the first time to study trabecular bone ex vivo. The second echo generated by the demagnetizing field presents discrete drops in signal intensity for certain values of the pitch of the magnetization helix created by the correlation gradient. These dips may reflect characteristic pore sizes in the trabecular bone specimens. This hypothesis is supported by similar experiments performed on a phantom with uniform pore size distribution. Trabecular bone images weighted in the MSE contrast mechanism are reported.

Animals↗
PubMed1992

Junctions between genes in the haptoglobin gene cluster of primates.

To investigate the nature of the recombination that generated the haptoglobin three-gene cluster in Old World primates, we sequenced the region between the second gene (HPR) and the third gene (HPP) in chimpanzees (15 kb), as well as the region 3' to the cluster in humans (14 kb). Comparison to the previously sequenced human haptoglobin (HP) and HPR genes showed that the junction point between HP and HPR in humans (junction 1) was not identical to the junction point between the HPR and HPP genes of the chimpanzee (junction 2). An Alu sequence was found at each junction, but both Alu sequences lacked short direct repeats of the flanking genomic DNA. The lack of direct repeats implies that both junction Alu sequences are the products of recombination between different Alu elements. In addition, other insertion and deletion events are clustered in the regions near the junction Alu sequences. The observation that Alu sequences define the junctions between genes in the haptoglobin gene cluster emphasizes the importance of Alu sequences in the evolution of multigene families.

Animals↗
PubMed2006

General strategy for the syntheses of corynanthe, tacaman, and oxindole alkaloids.

We report herein the total synthesis of the corynanthe alkaloid dihydrocorynantheol and the formal syntheses of the indole alkaloids tacamonine, rhynchophylline, and hirsutine. The strategies for assembling the corynanthe and tacaman skeletal frameworks comprised of both the classical ABD --> ABCD and ABC --> ABCD approaches wherein the variously substituted piperidinone D-rings were formed via ring-closing metathesis (RCM) followed by a 1,4-addition to introduce the requisite side chain at C(15). Since 1,4-additions to alpha,beta-unsaturated lactams represent an underdeveloped field, we conducted a series of studies with two unsaturated lactams employing organocuprates and metal enolates as the nucleophiles. These studies revealed that organocuprates derived from Grignard reagents and either stoichiometric amounts of CuCN or catalytic amounts of CuBr.DMS complex are excellent nucleophiles for such additions; TMSCl was a crucial additive for optimizing these reactions. The anion derived from ethyl 1,3-dithiolane-2-carboxylate was also an excellent nucleophile in these 1,4-additions, although the stereochemistry of such 1,4-additions to carboline-derived, unsaturated lactams was sensitive to substitution on the indole nitrogen atom. The ABD --> ABCD approach to these alkaloids featured a novel one-pot sequence of an RCM reaction and a zirconocene-catalyzed carbomagnesation followed by a second RCM to generate the D-ring.

Alkaloids↗
PubMed1996

Two Interrelated Strategies for Cephalotaxine Synthesis.

Studies of two interrelated strategies for the synthesis of members of the cephalotaxus alkaloid family have culminated in a concise route for the preparation of the parent member cephalotaxine (1). As part of efforts exploring the use of an SET-promoted photocyclization reaction of aryl-substituted silylallyliminium salts to generate the spirocyclic DE unit of the target, we noted that attempts to generate the pentacyclic amino ketone 23 by deacylation of the enol ester 20 led to production of a mixture of 23 and the macrocyclic amino enone 24. A rapid equilibrium was shown to exist between 23 and 24, favoring the latter ring-opened form. This contrasts with the behavior of desmethylcephalotaxinone (22), a key late intermediate in several earlier cephalotaxine syntheses, which is known to exist in a ring-closed form. These observations led to the design of a second generation strategy which relies on transannular cyclization of the macrocyclic amino enedione 28. In practice, the sequence following this design transforms the known iodopiperonylethanol derivative 4 to 22 in 13 steps and a 12% overall yield and, thus, corresponds to an efficient formal synthesis of cepahalotaxine.

Journal Article↗
PubMed1997

Recombinant analogues of prolactin, growth hormone, and placental lactogen: correlations between physical structure, binding characteristics, and activity.

The availability of recombinant growth hormones, prolactins, placental lactogens and a few soluble extracellular domains of their receptors have extended our ability to study the interaction of somatogenic and lactogenic hormones with their receptors. Modifications of their respective cDNAs have enabled the preparation of sufficient amounts of the corresponding proteins. The present review summarizes two aspects of these interactions: (a) the relationship between binding, the apparent ability to dimerize the receptors and biological activities in vitro and in vivo; and (b) the effect of mutations on selective changes in the ability of human growth hormone and bovine placental lactogen to interact with somatogenic and lactogenic receptors. In view of this summary, strategies for preparing a second generation of biologically relevant recombinant hormones are discussed.

Amino Acid Sequence↗