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PubMed2003

Contribution of the C1A and C1B domains to the membrane interaction of protein kinase C.

The hallmark for protein kinase C activation is its "translocation" to membranes following generation of lipid second messengers. This translocation is mediated by the C1 and C2 domains, two membrane-targeting modules, whose engagement on membranes provides the energy for an activating conformational change in which an autoinhibitory pseudosubstrate sequence is released from the active site. Novel and conventional protein kinase C isozymes contain a tandem repeat of C1 domains, the C1A and C1B, which each contain a binding pocket for phorbol esters/diacylglycerol. This study addresses the contribution of the C1A and C1B domains in the regulation of protein kinase C's membrane interaction using bisfunctional (dimeric) phorbol myristate acetate (PMA) molecules. We show that dimeric bisphorbols are an order of magnitude more effective at recruiting full-length PKC betaII to membranes compared with monomeric PMA and that the effectiveness of the interaction depends on the nature and length of the cross-link between the PMA moieties. Most effective were dimeric phorbol 12-acetate 13-esters linked at the 13 position with a 14 carbon spacer. The increased potency of dimeric phorbol esters is reduced if either the C1A or C1B domains are mutated so that they are unable to bind PMA, if one moiety of the dimer contains a nonfunctional phorbol, or if the binding to the isolated C1B domain is measured. Thus, the increased potency of the dimeric phorbol esters results primarily from their ability to engage, to a limited extent, both C1 modules on the same molecule. Although dimeric phorbols were more potent than monomeric phorbol esters in recruiting protein kinase C to membranes, the magnitude of the increase was still several orders of magnitude lower than what would be predicted on the basis of the reduction in dimensionality that occurs when the first C1 domain is engaged on the membrane. Thus, engaging both domains can be forced but is highly unfavored. In summary, our data reveal that both C1 domains are oriented for potential membrane interaction but only one C1 domain binds ligand in a physiological context.

Amino Acid Substitution↗

PubMed1996

Core packing defects in an engineered Cro monomer corrected by combinatorial mutagenesis.

The crystal structure of an engineered monomer of the lambda Cro repressor shows unexpected expansion of the hydrophobic core of the protein and disorder of the five C-terminal residues [Albright et al. (1996) Biochemistry 35, 735-742]. This structural information has guided the construction of a second generation of monomeric Cro proteins by combinatorial mutagenesis of selected core and C-terminal residues. Clones were identified in a library of randomized cro genes by a genetic screen for protein accumulation in Escherichia coli. Sequencing of candidate genes followed by purification and analysis of their product proteins has identified alternative arrangements of hydrophobic core residues which result in substantial increases in thermal stability. In contrast, residue replacements at the C-terminus have minor effects on stability but may increase protein expression levels.

Base Sequence↗

PubMed2004

Spongistatin synthetic studies. evolution of a scalable synthesis for the EF fragment of (+)-Spongistatin 1 exploiting a Petasis-Ferrier union/rearrangement tactic.

[structure: see text] An efficient, stereocontrolled, and scalable second-generation synthesis of (+)-3, an advanced EF subtarget for the total synthesis of (+)-spongistatin 1, has been achieved. Highlights of the strategy include preparation of the F-ring pyran via a Petasis-Ferrier union/rearrangement sequence and installation of the chlorodiene side chain employing a cyanohydrin alkylation. The longest linear sequence, 26 steps, proceeds in 8.3% overall yield.

Animals↗

PubMed1995

A second-generation YAC contig map of human chromosome 3.

A map of human chromosome 3 which integrates both physical and genetic data has been developed from the fusion of two large collections of markers and corresponding yeast artificial chromosome (YAC) clones. The map contains 972 megabase-sized YACs identified with 593 primary markers, of which 162 are highly polymorphic sequence-tagged sites (STSs) and form a closely spaced genetic linkage map; the remaining markers are hybridization-based. Chromosome 3 is now represented by 24 large YAC contigs whose order and orientation is largely known. The map generated by fusion of these hybridization- and STS-based datasets covers about 80% (over 160 megabases) of the chromosome and will provide the foundation necessary for rapid development of a detailed genetic understanding for this large autosome.

Chromosome Mapping↗

PubMed1999

An additional electrostatic interaction between adrenodoxin and P450c27 (CYP27A1) results in tighter binding than between adrenodoxin and p450scc (CYP11A1).

Mitochondrial cytochrome P450c27 (product of the CYP27A1 gene) is found to have significantly higher affinity for the common redox partner adrenodoxin than another mitochondrial P450, P450scc (product of the CYP11A1 gene). To investigate the basis of the approximately 30-fold difference in adrenodoxin binding, two sets of P450c27 mutants were generated, expressed in Escherichia coli, and purified. Mutations of one set were within the putative adrenodoxin-binding site containing conserved lysine residues also crucial in P450scc for binding adrenodoxin. The second set included mutations within a sequence aligning with the "meander region" of P450BM-3 proposed to be a site of redox-partner interactions in P450s (Hasemann, C. A., Kurumbail, R. G., Boddupalli, S. S., Peterson, J. A., and Deisenhofer, J. (1995) Structure 3, 41-62). Mutation of the P450c27 conserved lysines (K354A and K358A) led to a approximately 20-fold increase in apparent Ks for adrenodoxin, confirming that these two positively charged residues conserved in mitochondrial P450s are important for adrenodoxin binding. Mutation of Arg-418, conserved in the CYP27A1 family, to serine also decreased the affinity for adrenodoxin approximately 20-fold. This residue is predicted to be located in the meander region. A triple K354A/K358A/R418S mutation profoundly reduced adrenodoxin binding. Thus, in contrast to P450scc, where mutation of the two conserved positively charged residues results in virtually complete inhibition of adrenodoxin binding, in P450c27 there are three of such residues (Lys-354, Lys-358, and Arg-418) important for adrenodoxin interaction.

Adrenodoxin↗

PubMed2001

A novel group of phospholipase A2s preferentially expressed in type 2 helper T cells.

We report a novel phospholipase A(2) (PLA(2)), group XII (GXII) PLA(2), distinct from other cysteine-rich groups with a catalytic histidine motif, by its 20-kDa size and distribution of the 14 cysteine residues within the protein. Alternative spliced forms with distinct subcellular localization, designated GXII-1 and GXII-2, were identified by reverse transcription-polymerase chain reaction. Importantly, GXII PLA(2)s, in particular GXII-2 PLA(2), and group V PLA(2), but not group X PLA(2), were selectively expressed in murine type 2 helper T (Th2) clones and in vitro differentiated mouse CD4 Th2 cells as compared with type 1 helper T clones and cells. Stimulation with anti-CD3 appreciably up-regulated expression of GXII PLA(2)s and group V PLA(2) by steady state analysis of the Th2 cells as compared with type 1 helper T cells. These results suggest that group XII and group V PLA(2)s might participate in helper T cell immune response through release of immediate second signals and generation of downstream eicosanoids.

Alternative Splicing↗

PubMed1994

Torsion angle differences as a means of pinpointing local polypeptide chain trajectory changes for identical proteins in different conformational states.

We discuss a facile and sensitive method of determining conformational differences based on the changes in the phi and psi angle values between chemically identical proteins in different conformations. It complements the conventional r.m.s. deviation technique, but offers some advantages. Two classes of conformational difference can be distinguished by this method: (i) abrupt local trajectory deformations where the chains flanking the locus of deformation remain simultaneously superposable and (ii) localized 'hinge bending' that generates domain shifts, thereby causing only one domain to be superposable on the other at one time. In the second case, the r.m.s. deviation method requires two or more calculations of r.m.s. deviation as a function of sequence after optimal alignment of each domain to demonstrate the superposability of the shifted domains, and hence the conservation of internal domain structure. On the other hand, the method of plotting delta phi and/or delta psi as a function of sequence demonstrates in one graph the superposability of shifted domains, without the prior need to perform rotational and translational alignments whose outcomes vary with the subjective choice of alignment parameters. Also, an analysis of the r.m.s. deviation in C alpha alone will miss torsion angle rotations that happen to preserve C alpha positions. The method pinpoints residues contributing singly to localized, major movements of a conformational change; however, it is insensitive to conformational changes achieved through small, concerted movements spread over a number of residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Bacterial↗

PubMed2003

Isotropic-sequence-order learning in a closed-loop behavioural system.

The simplest form of sensor-motor control is obtained with a reflex. In this case the reflex can be interpreted as part of a closed-loop control paradigm which measures a sensor input and generates a motor reaction as soon as the sensor signal deviates from its desired (resting) state. This is a typical case of feedback control. However, reflex reactions are tardy, because they occur always only after a (for example, unpleasant) reflex-eliciting sensor event. This defines an objective problem for an organism which can only be avoided if the corresponding motor reaction is generated earlier. The goal of this study is to design a closed-loop control situation where temporal-sequence learning supersedes a tardy reflex reaction with a proactive anticipatory action. We achieve this by employing a second, earlier-occurring and causally coupled sensor event. An appropriate motor reaction to this early event prevents triggering of the original, primary reflex. Such causally coupled sensor events are common for animals, for example when smell predicts taste or when heat radiation precedes pain. We show that trying to achieve anticipatory control is a fundamentally different goal from trying to model a classical conditioning paradigm, which is an open-loop condition. To this end, we use a novel learning rule for temporal-sequence learning called isotropic-sequence-order (ISO) learning, which performs a confounded correlation between the primary sensor signal associated to the reflex and a predictive, earlier-occurring sensor input: this way the system learns the relation between the primary reflex and the earlier sensor input in order to create an earlier-occurring motor reaction. As a consequence of learning, the primary reflex will not be triggered any more, thereby permanently remaining in its desired resting state. In a robot application, we demonstrate that ISO learning can successfully solve the classical obstacle-avoidance task by learning to correlate a built-in reflex behaviour (retraction after touching) with earlier arising signals from range finders (before touching). Finally, we show that avoidance and attraction tasks can be combined in the same agent.

Adaptation, Physiological↗

PubMed1990

Sequences 5' to the polyadenylation signal mediate differential poly(A) site use in hepatitis B viruses.

Most genetic elements that employ reverse transcription generate a terminally redundant genomic RNA that serves as the template for this reaction. Because the identical polyadenylation signal is present in each terminally redundant segment, synthesis of this RNA requires that this signal be ignored on the first pass of the transcription machinery, then recognized and used on the second pass. We have studied the mechanism of this differential poly(A) site use in one family of retroid elements, the hepatitis B viruses (hepadnaviruses). Our results indicate that two features are involved: the presence of a variant poly(A) signal (TATAAA) and the participation of multiple sequences 5' to this signal that act to increase the efficiency of its use. Deletion of these upstream elements abolishes proper poly(A) site use, despite the presence of the poly(A) signal and downstream GT- and T-rich motifs known to be required for polyadenylation. Sequences from the corresponding regions of retroviral genomes can restore proper processing to these hepadnaviral deletion mutants. Thus, functionally analogous upstream elements exist in other classes of retroid elements, including those employing the canonical AATAAA hexanucleotide signal.

Animals↗

PubMed2002

Lack of self-averaging in neutral evolution of proteins.

We simulate neutral evolution of proteins imposing conservation of the thermodynamic stability of the native state in the framework of an effective model of folding thermodynamics. This procedure generates evolutionary trajectories in sequence space which share two universal features for all of the examined proteins. First, the number of neutral mutations fluctuates broadly from one sequence to another, leading to a non-Poissonian substitution process. Second, the number of neutral mutations displays strong correlations along the trajectory, thus causing the breakdown of self-averaging of the resulting evolutionary substitution process.

Cytochrome c Group↗

PubMed1997

Mitochondrial and chloroplast phage-type RNA polymerases in Arabidopsis.

In addition to the RNA polymerases (RNAPs) transcribing the nuclear genes, eukaryotic cells also require RNAPs to transcribe the genes of the mitochondrial genome and, in plants, of the chloroplast genome. The plant Arabidopsis thaliana was found to contain two nuclear genes similar to genes encoding the mitochondrial RNAP from yeast and RNAPs of bacteriophages T7, T3, and SP6. The putative transit peptides of the two polymerases were capable of targeting fusion proteins to mitochondria and chloroplasts, respectively, in vitro. The results indicate that the mitochondrial RNAP in plants is a bacteriophage-type enzyme. A gene duplication event may have generated the second RNAP, which along with the plastid-encoded eubacteria-like RNAP could transcribe the chloroplast genome.

Amino Acid Sequence↗

PubMed2001

Molecular epidemiology of Mycobacterium tuberculosis in Norway.

The incidence of tuberculosis in Norway is one of the lowest in the world, and approximately half of the cases occur in first- and second-generation immigrants. In the present study, the genetic diversity of 92% of all strains of Mycobacterium tuberculosis isolated in Norway in 1994 to 1998 was assessed using restriction fragment length polymorphism (RFLP) analysis, with the insertion sequence IS6110 and the repetitive element DR as probes, to determine the degree of active transmission between patients. The DR probe was used as a secondary molecular marker to support or rule out clustering of strains with fewer than five copies of IS6110. After exclusion of 20 cultures representing laboratory contamination, 573 different IS6110 patterns were found among the 698 strains analyzed. Of these 573 patterns, 542 were observed only once and 31 were shared by 2 to 14 isolates. Among 81 strains (11.5%) carrying fewer than five copies of IS6110, 56 RFLP patterns were found when the results of both the IS6110 and DR methods were combined. Among the 698 strains, 570 were considered to be independent cases. A total of 14.5% of the native Norwegians and 19.7% of the foreign patients were part of a cluster. Thus, the degree of recent transmission of tuberculosis in Norway is low and the great majority of the cases are due to reactivation of previous disease. Transmission between immigrants and native Norwegians is uncommon. Two outbreaks, one among native Norwegians and one mainly among immigrants, have been ongoing for several years, indicating that, even in a low-incidence country such as Norway, with a good national program for tuberculosis surveillance, certain transmission chains are difficult to break.

Adolescent↗

PubMed1989

Evolutionary pathways of the calcitonin (CALC) genes.

Recombinant DNA techniques have made it possible to establish the structure of various genes encoding polypeptide hormones. Comparison of nucleotide sequences of the calcitonin (CALC) genes in man has revealed surprising similarities and variations. These findings and the homologies among the sequences in different species offered an opportunity for speculation about relationships between these genes and about their evolutionary origin. The first gene (CALC-I) directing the synthesis of calcitonin (CT) or CT gene-related peptide (CGRP) comprises six exons and gives rise to two mRNAs by an alternative RNA-processing mechanism. The homology between CGRP and CT reflects their common origin. The human genome contains a second gene (CALC-II) that is structurally related to the CALC-I gene. The CALC-II RNA transcripts do not appear to be differentially processed, as only preproCGRP-II mRNA and not preproCT-II is detected. The first and second CT/CGRP genes probably have evolved from a common ancestor gene early in evolution. Meanwhile, a third genomic locus containing nucleotide sequences highly homologous to exons 2 and 3 of both CALC genes was detected and probably generated by duplication of a part of CALC-II. This locus is not likely to encode a CT- or CGRP-related polypeptide hormone. The CALC genes and this last (pseudo) gene are located on the short arm of chromosome 11. Recently, islet- or insulinoma-amyloid polypeptide (IAPP) was isolated as a major constituent of amyloid present in human insulinoma and in pancreatic islet amyloid in noninsulin-dependent diabetes mellitus. IAPP shows 46% amino acid sequence homology with human CGRP-II.(ABSTRACT TRUNCATED AT 250 WORDS)

Amyloid↗

PubMed1989

Molecular defects in the non-expressed H-2 E alpha genes of the f and q haplotypes.

The expression and sequences of the mouse MHC class II genes, E alpha f and E alpha q, have been studied to determine the molecular basis for their defective expression. Previous work in our laboratory showed that H-2f and H-2q mice produce no detectable E alpha protein. Northern blot analysis confirms previous results showing normal amounts of E alpha f message and a 100-fold reduction in E alpha q mRNA. Despite that observation, the rates of transcription of both defective E alpha genes, measured by nuclear run-on transcription assays, are comparable to that of the normally expressed E alpha k gene. The nucleotide sequences of the E alpha f and E alpha q genes reveal mutations generating premature translation stop codons in both genes. A single base substitution has created the stop codon TGA at amino acid -2 in the E alpha f leader sequence. A nucleotide insertion at codon 64 in the second exon of the E alpha q gene results in a frame shift and a premature stop codon at amino acid residue 69 of a mature E alpha protein. The low steady state level of E alpha q mRNA may be correlated with the unusual size distribution of the RNA, possibly due to altered RNA processing.

Animals↗

PubMed1996

The Chinese herbal remedy, T2, inhibits mitogen-induced cytokine gene transcription by T cells, but not initial signal transduction.

T2, an extract of Tripterygium wilfordii Hook F, has been reported to be effective in the treatment of a variety of autoimmune diseases, including rheumatoid arthritis. Previous studies have shown that T2 inhibited mitogen- or antigen-induced proliferation of human peripheral blood T cells and B cells, IL-2 production by T cells and Ig production by B cells. In contrast, T2 did not affect monocyte functions, such as IL-1 production and antigen presentation. The current studies sought to localize the immunosuppressive action of T2 more precisely. Results show that T2 prevented [3H]-uridine uptake by mitogen-stimulated T cells and arrested them in the early GI phase of the cell cycle. The inhibitory effects of T2 could be partially overcome by costimulating PHA activated T cells with PMA and completely nullified by costimulation with PMA plus a monoclonal antibody to CD28. Moreover, T2 had no effect on expression of IL-2R or the transferrin receptor (CD71), but inhibited production of a number of cytokines, including IL-2 and IFN-gamma by activated T cells. T2 suppressed IL-2 mRNA levels, but not IL-2R mRNA levels, in activated T cells. T2-mediated inhibition reflected suppression of IL-2 gene transcription as indicated by suppression of the expression of a reporter gene driven by the IL-2 promoter. T2 had little inhibitory effect on either IL-2 gene expression or cell cycle progression when added after initial mitogenic stimulation, indicating that an early step in the cascade of activation events was inhibited. However, initial activation events including protein tyrosine phosphorylation, the generation of diacylglycerol, IP3, and the translocation of protein kinase C were not inhibited by T2. Moreover, T2 did not inhibit the phosphatase activity of calcineurin. These results have localized the effect of T2 to a step in the T cell activation cascade after initial second messenger generation, tyrosine phosphorylation and protein kinase activation, but before IL-2 gene transcription.

Adult↗

PubMed1997

Combination chemotherapy with paclitaxel and doxorubicin for metastatic breast cancer.

The development of anthracycline/paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) combinations has gone through several phases or generations. The first dose escalation studies used prolonged infusions of doxorubicin (48 to 72 hours) and paclitaxel (24 hours). Myelosuppression, mucositis, and neutropenic fever were dose limiting, and a sequence-dependent interaction was noted. In a second phase, doxorubicin was administered by bolus, without changing the schedule of the taxane. More recently, bolus doxorubicin was combined with 3-hour paclitaxel infusions. Higher doses of both agents were administered in this latter schedule, with higher overall response rates reported. Excessive cardiac toxicity was reported with the initial trials that used this schedule. Confirmatory studies are under way pursuing several alternatives to reduce the risk of cardiac toxicity; these include limiting the cumulative dose of doxorubicin, inserting an interval between the administration of the two drugs, and adding a cardioprotective agent to the combination. The high degree of antitumor activity of this combination is quite encouraging, and additional evaluation of this regimen in controlled trials is warranted in metastatic and high-risk primary breast cancer.

Antibiotics, Antineoplastic↗

PubMed1997

Construction of a gene encoding the insect bactericidal protein attacin. Studies on its expression in Escherichia coli.

Attacin, a bactericidal small protein is produced by the giant silk moth Hyalophora cecropia. This paper deals with our efforts to clone the attacin cDNA in a bacterial vector to express it in Escherichia coli and produce the protein in sufficient amount, for further studies. We chose two inducible expression vector/bacterial cell systems: pPL-lambda/N99cI+ cells which is able to be induced by nalidixic acid, and pET3d/BL21(DE3) cells carrying a T7 RNA polymerase gene which is IPTG-inducible. After cloning in the pPL-lambda system and under no addition of the inducer, isolated transformants carried this plasmid with at least 2 concurrent deletions that drastically affected attacin expression, even though attacin gene seems to be intact as deduced by its PCR amplification. It was concluded that basal attacin expression occurred in this system and bacterial growth was limited. Plasmid deletions may have emerged by selection pressure as a way to avoid bactericidal expression and allow bacteria survival. The second cloning attempt was done in pET3d vector/BL21 cells, that should not express the cloned sequence (they lack T7 RNA polymerase gene). Transformed BL21 cells gave 3 recombinant plasmids, 2 of them presented a C deletion that generated an early stop signal in the attacin coding region. The third clone, pET-ATT18, carrying an intact gene, was transferred to BL21(DE3)-IPTG inducible cells in order to be expressed. Attacin was undetectable in stained gels or by Western blot analysis. However, expression was visualized in grown cells after 30 min of IPTG induction and 5 min of [35S]-methionine labeling, as a 22.5 kDa protein band by using gel electrophoresis and fluorography. This low level of expression drastically affected bacterial growth. Considering that attacin has no lytic activity, these results suggest that this molecule should block bacterial growth directly at the cytoplasm by an unknown mechanism, since no signal peptide coding sequence was incorporated in this gene construction, precluding periplasmic or external destination of this protein.

Anti-Bacterial Agents↗

PubMed1998

Cloning, gene structure and genomic localization of an orphan transporter from mouse kidney with six alternatively-spliced isoforms.

Two genes were identified and characterized that express cDNAs related to previously identified neurotransmitter and/or osmolyte transporters, but which are expressed specifically in the kidney. RNA transcribed from one of these two genes (XT2) was found to undergo an extensive degree of alternative splicing to generate six distinct isoforms. The intron-exon structure of the XT2 gene and the sites of alternative splicing were identified. Expression of the second gene (XT3) was found to be conserved in human kidney, and partial sequence was obtained from a human cDNA library. The expressions of both XT2 and XT3 RNAs were determined in mouse and human tissues, respectively, and the locations of the two genes within the mouse genome were identified. Screening experiments to identify the substrate(s) of these proteins failed to identify specific uptake with any of the tested compounds; however, immunofluorescent microscopy demonstrated that epitope-tagged variants of the protein products of the XT2 and XT3 cDNAs were present on the plasma membrane of transfected cells.

Alternative Splicing↗