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PubMed1998

Structural basis for chemical inhibition of human blood coagulation factor Xa.

Factor Xa, the converting enzyme of prothrombin to thrombin, has emerged as an alternative (to thrombin) target for drug discovery for thromboembolic diseases. An inhibitor has been synthesized and the crystal structure of the complex between Des[1-44] factor Xa and the inhibitor has been determined by crystallographic methods in two different crystal forms to 2.3- and 2.4-A resolution. The racemic mixture of inhibitor FX-2212, (2RS)-(3'-amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoic acid, inhibits factor Xa activity by 50% at 272 nM in vitro. The S-isomer of FX-2212 (FX-2212a) was found to bind to the active site of factor Xa in both crystal forms. The biphenylamidine of FX-2212a occupies the S1-pocket, and the pyridine ring makes hydrophobic interactions with the factor Xa aryl-binding site. Several water molecules meditate inhibitor binding to residues in the active site. In contrast to the earlier crystal structures of factor Xa, such as those of apo-Des[1-45] factor Xa and Des[1-44] factor Xa in complex with a naphthyl inhibitor DX-9065a, two epidermal growth factor-like domains of factor Xa are well ordered in both our crystal forms as well as the region between the two domains, which recently was found to be the binding site of the effector cell protease receptor-1. This structure provides a basis for designing next generation inhibitors of factor Xa.

Binding Sites↗

PubMed2004

Exon skipping of exonuclease 1 in MRL/MpJ mice is caused by a nucleotide substitution of the branchpoint sequence in intron eight.

In MRL/MpJ mice, there is a genetic mutation of exonuclease 1 (Exo1), in which the exon 9 is sometimes deleted. In the present study, to check the generation of the spliced exons, exon 8-intron 8-exon 9 (pCX/Ex/EIE/B and pCX/Ex/EIE/M) plasmids were temporally transfected in vitro into BALB 3T3 cells, and RT-PCR using appropriate primer pair was carried out 1 day after transfection. In these constructions, pCX/Ex/EIE/B was derived from genomic sequence of C57BL/6 mice, and pCX/Ex/EIE/M was from MRL/MpJ. A spliced band was detected in pCX/Ex/EIE/B, but was present little or very weakly in pCX/Ex/EIE/M. Next, the same spliced band was demonstrated in the pCX/Ex/EIE/M(T) plasmid, in which the branchpoint sequence (BPS) of pCX/Ex/EIE/M including the exon 9 was changed into that of pCX/Ex/EIE/B. The splicing did not occur in the dell1/B mutant, in which 1960 nucleotides of the intron 8 were deleted, whereas it was detected in the del2/B plasmid deleted 1036 nucleotides in its middle region. These results suggest that the nucleotide T to A mutation of the BPS in the intron 8 is at least a sufficient for generation of splice variants (tr-1 and tr-2 Exo1).

Amino Acid Sequence↗

PubMed2005

Combinatorial synthesis of an oligosaccharide library by using beta-bromoglycoside-mediated iterative glycosylation of selenoglycosides: rapid expansion of molecular diversity with simple building blocks.

A new method for constructing an oligosaccharide library composed of structurally defined oligosaccharides is presented based on an iterative glycosylation of selenoglycosides. Treatment of 2-acyl-protected selenoglycosides with bromine selectively generates beta-bromoglycosides, which serve as glycosyl cation equivalents in the oligosaccharide synthesis. Thus, the coupling of the bromoglycosides with another selenoglycoside affords the corresponding glycosylated selenoglycosides, which can be directly used to next glycosylation. The iteration of this sequence allows the synthesis of a variety of oligosaccharides including an elicitor active heptasaccharide. A characteristic feature of the iterative glycosylation is that glycosyl donors and acceptors with the same anomeric reactivity can be selectively coupled by activation of the glycosyl donor prior to coupling with the glycosyl acceptor. Therefore, same selenoglycosides can be used for both the glycosyl donors and the acceptors. This feature has been exemplified by a construction of an oligosaccharide library directed to elicitor-active oligosaccharides. The library composed of stereochemically defined oligoglucosides with considerable structural diversity can be constructed starting from simple selenoglycosides.

Carbohydrate Sequence↗

PubMed1997

Stochastic resonance in non-dynamical systems without response thresholds.

The addition of noise to a system can sometimes improve its ability to transfer information reliably. This phenomenon--known as stochastic resonance--was originally proposed to account for periodicity in the Earth's ice ages, but has now been shown to occur in many systems in physics and biology. Recent experimental and theoretical work has shown that the simplest system exhibiting 'stochastic resonance' consists of nothing more than signal and noise with a threshold-triggered device (when the signal plus noise exceeds the threshold, the system responds momentarily, then relaxes to equilibrium to await the next triggering event). Here we introduce a class of non-dynamical and threshold-free systems that also exhibit stochastic resonance. We present and analyse a general mathematical model for such systems, in which a sequence of pulses is generated randomly with a probability (per unit time) that depends exponentially on an input. When this input is a sine-wave masked by additive noise, we observe an increase in the output signal-to-noise ratio as the level of noise increases. This result shows that stochastic resonance can occur in a broad class of thermally driven physico-chemical systems, such as semiconductor p-n junctions, mesoscopic electronic devices and voltage-dependent ion channels, in which reaction rates are controlled by activation barriers.

Models, Theoretical↗

PubMed2001

Mismatch negativity is unaffected by top-down predictive information.

The mismatch negativity (MMN) event-related brain potential is assumed to reflect a stimulus-driven change detection process. We examined whether MMN is sensitive to volitional control by testing whether MMN is affected by the subject's foreknowledge of the sound changes. Subjects were instructed to produce a sequence of button presses by pressing one button frequently and another infrequently. In the predictable condition, the frequently pressed button triggered the standard tone, the other button the deviant tone. In the unpredictable condition, each button press triggered the next tone of a prearranged standard/deviant sequence. No difference was found in the MMN amplitude, latency, or scalp distribution between the predictable and unpredictable conditions. This suggests that there is no direct top-down control over the MMN-generating process.

Acoustic Stimulation↗

PubMed1985

The position of the epistatic S locus in the halothane linkage group in pigs.

The linkage of the Phi, Pgd, Po2, S, H and halothane sensitivity loci was followed in a Belgian Landrace family, heterozygous for these systems over 6 generations. Recombination next to the S locus occurred mainly in pigs belonging to this particular family. From this investigation the position of the S locus is proved to be outwith the Phi-Pgd region, next to Phi. Therefore the gene sequence S - Phi - Hal - H - Po2 - Pgd is proposed. Higher recombination rates were observed in the female parental line of the multiheterozygous family when compared to the male parental line. Additional data from animals, unrelated to this strain, confirm the evidence of close linkage of the S system to the nearest marker loci.

Alleles↗

PubMed2003

A genomics approach to crop pest and disease research.

Genome-wide analyses of gene function and gene expression are beginning to yield valuable information in many areas of biological research, and these genomic tools are now being applied to crop pest and disease research. DNA sequencing of cDNA libraries to generate sets of expressed sequence tags (ESTs) are allowing gene compendiums for crop diseases to be compiled. Annotation of such data collections is also providing a wealth of functional information about gene products through similarities to proteins with known function. The next phase of the functional genomics era will be to employ large-scale techniques to knock out or silence genes in order to synthesize gene-specific mutants for phenotypic analysis and to use micro-array methodology to analyze global gene expression, protein turnover and protein processing during the processes of parasitism and colonization. Application of these technologies promises to accelerate the pace that biological information relevant to crop protection accrues. The ability of researchers to assimilate this information into complex models and workable hypotheses is, thus, set to revolutionize the way we study pests and diseases of crop plants.

Crops, Agricultural↗

PubMed1997

Mapping of recombinant hemoglobin using immobilized trypsin cartridges.

A tryptic mapping procedure has been developed for a recombinant hemoglobin (rHb1.1) using an immobilized trypsin cartridge. Apohemoglobin is passed through the trypsin cartridge and the products of the digestion are captured directly onto an in-line C18 reversed-phase column. The peptides are then separated using a gradient elution. This new procedure is rapid and reproducible and can be fully automated. The total time of analysis is less than 2 h. The mapping of apohemoglobins produced an unexpected isomerization of two peptides: beta8,9 (K66-K82) and alpha8,9 (K61-K90). It appears that the isomerization may occur through transpeptidation followed by proteolysis at a newly generated site next to the site of ligation. This mapping procedure can be a useful tool for research and routine analysis of proteins.

Amino Acid Sequence↗

PubMed2005

Methods for obtaining and analyzing whole chloroplast genome sequences.

During the past decade, there has been a rapid increase in our understanding of plastid genome organization and evolution due to the availability of many new completely sequenced genomes. There are 45 complete genomes published and ongoing projects are likely to increase this sampling to nearly 200 genomes during the next 5 years. Several groups of researchers including ours have been developing new techniques for gathering and analyzing entire plastid genome sequences and details of these developments are summarized in this chapter. The most important developments that enhance our ability to generate whole chloroplast genome sequences involve the generation of pure fractions of chloroplast genomes by whole genome amplification using rolling circle amplification, cloning genomes into Fosmid or bacterial artificial chromosome (BAC) vectors, and the development of an organellar annotation program (Dual Organellar GenoMe Annotator [DOGMA]). In addition to providing details of these methods, we provide an overview of methods for analyzing complete plastid genome sequences for repeats and gene content, as well as approaches for using gene order and sequence data for phylogeny reconstruction. This explosive increase in the number of sequenced plastid genomes and improved computational tools will provide many insights into the evolution of these genomes and much new data for assessing relationships at deep nodes in plants and other photosynthetic organisms.

Amino Acid Sequence↗

PubMed1998

Learning fuzzy decision trees.

We present a recurrent neural network which learns to suggest the next move during the descent along the branches of a decision tree. More precisely, given a decision instance represented by a node in the decision tree, the network provides the degree of membership of each possible move to the fuzzy set z.Lt;good movez.Gt;. These fuzzy values constitute the core of the probability of selecting the move out of the set of the children of the current node.This results in a natural way for driving the sharp discrete-state process running along the decision tree by means of incremental methods on the continuous-valued parameters of the neural network. The bulk of the learning problem consists in stating useful links between the local decisions about the next move and the global decisions about the suitability of the final solution. The peculiarity of the learning task is that the network has to deal explicitly with the twofold charge of lighting up the best solution and generating the move sequence that leads to that solution. We tested various options for the learning procedure on the problem of disambiguating natural language sentences.

Journal Article↗

PubMed2006

Repetitive DNA elements as mediators of genomic change in response to environmental cues.

There is no logical or theoretical barrier to the proposition that organismal and cell signaling could transduce environmental signals into specific, beneficial changes in primary structure of noncoding DNA via repetitive element movement or mutation. Repetitive DNA elements, including transposons and microsatellites, are known to influence the structure and expression of protein-coding genes, and to be responsive to environmental signals in some cases. These effects may create fodder for adaptive evolution, at rates exceeding those observed for point mutations. In many cases, the changes are no doubt random, and fitness is increased through simple natural selection. However, some transposons insert at specific sites, and certain regions of the genome exhibit selectively and beneficially high mutation rates in a range of organisms. In multicellular organisms, this could benefit individuals in situations with significant potential for clonal expansion: early life stages or regenerative tissues in animals, and most plant tissues. Transmission of the change to the next generation could occur in plants and, under some circumstances, in animals.

Animals↗

PubMed1991

Motor function of the monkey globus pallidus. 2. Cognitive aspects of movement and phasic neuronal activity.

In order to study the role of the basal ganglia (BG) in cognitive aspects of movement, we recorded extracellularly from pallidal neurons in conscious monkeys while they performed a sequential wrist movement task consisting of a series of holds and ballistic jumps. The movement sequence had to be performed within specified time restraints and was predictable. We recorded the activity of 297 neurons whose discharges were related to the movement task. The movement-related response was found to be influenced by the contextual setting and by the degree of difficulty of the task in a subgroup of 82 neurons with a clear response to the first ballistic movement. Predictable and easy movements were usually represented by more prominent movement-related responses in 46% of these neurons; 35% of neurons from a different subset of 105 neurons also demonstrated a second phasic response just before the end of the final hold period of the task. This response was also found to be influenced by the predictability of the final hold period, both in its time duration and also by the direction of the following ballistic movement in double jump tasks. These findings were in keeping with a cognitive role for the BG in movement performance. In particular we suggest that the phasic neuronal activity was an internal cue generated by the BG for predictable movements of a subconscious nature which signals the end of a component of movement in a movement sequence. This cue is appropriately timed to terminate sustained neuronal activity in the SMA and to allow the next movement in the sequence to be executed.

Animals↗

PubMed2006

X-Linked inhibitor of apoptosis protein gene-based neuroprotection for the peripheral nervous system.

OBJECTIVE: The recently discovered X-linked inhibitor of apoptosis protein (XIAP) is among the most potent inhibitors of programmed cell death. In the current experiment, we examine the potential of adenoviral XIAP gene delivery to protect neurons of the peripheral nervous system using in vitro models of amyotrophic lateral sclerosis (ALS) and diabetic neuropathy. METHODS: XIAP complementary deoxyribonucleic acid was fused in frame with the green fluorescent protein sequence and cloned into a first generation adenoviral vector. The impact of XIAP gene expression on glutamate-induced apoptosis was measured in the neuronal SH-SY5Y cell line with immunohistochemistry for active caspase-3 and with cell density assays. Next, the effect of XIAP expressing neurons on the survival of uninfected neighboring neurons was measured. Finally, the impact of XIAP gene expression on glutamate-induced apoptosis was assessed in embryonic motor neuron and dorsal root ganglion cultures. RESULTS: XIAP gene expression reduced the percentage of active caspase-3 positive SH-SY5Y neurons and preserved cell density after glutamate exposure. In heterogeneously infected cultures, cells infected with XIAP were protected, but uninfected neighboring cells were not. In primary E15 models, inhibition of proapoptotic effects was demonstrated after glutamate insult in motor neurons and glucose insult in dorsal root ganglion cells. CONCLUSION: XIAP gene delivery through the neurosurgical delivery of viral vectors may provide a means for neuroprotection in ALS and diabetic neuropathy.

Amyotrophic Lateral Sclerosis↗

PubMed1987

Evolution of pseudorabies virions containing genomes with an invertible long component after repeated passage in chicken embryo fibroblasts.

The genome of pseudorabies virus consists of two components, short (S) and long (L). Only the S component is bracketed by inverted repeats, and only the S component inverts itself relative to the L component, giving rise to two isomeric forms of the genome. An attenuated vaccine strain of pseudorabies virus (Norden), however, has a genome which is found in four isomeric forms (B. Lomniczi, M. L. Blankenship, and T. Ben-Porat, J. Virol. 49:970-979, 1984). To determine the basis for the atypical structure of the genome of the Norden strain, we examined more than 40 field isolates of pseudorabies virus; all contained genomes in which the L component was fixed in only one orientation relative to the S component. Several independently generated vaccine strains which have been passaged extensively in chicken embryos and chicken embryo fibroblast (CEF) cell cultures were also analyzed; they possessed an invertible L component. Furthermore, emergence of pseudorabies virus variants with an invertible L component was observed after passage of the virus in CEF, but not in rabbit kidney or pig kidney, cells. The invertibility of the L component was associated consistently with a translocation of sequences from the left end of the genome to a position next to the inverted repeat sequence of the S component. Three observations indicate that genomes with an invertible L component (and the translocation) have a selective growth advantage over standard pseudorabies virus when grown in CEF. The proportion of virions with such genomes does not increase linearly as would be expected if the translocation events occurred repeatedly, most genomes eventually experiencing the translocation. Instead, after a lag, the proportion of such virions in the population increases relatively rapidly. The genome structures that are generated upon independent passage in CEF of each virion population were relatively homogeneous. Some heterogeneity was observed at relatively early stages of the emergence of the genomes carrying the translocation; at later stages, virions with genomes with a specific size translocation predominated in the virus population. Parallel passages in CEF of the same pseudorabies virus strain resulted in the emergence of populations of virions with genomes with different size translocations. However, in each of the passaged populations of virions the majority of virions had genomes with the same size translocation. The most likely interpretation of these results is that virions with genomes carrying the translocations that emerge upon passage of the virus in CEF have a selective advantage when grown in these cells.

Animals↗

PubMed2006

Identification and analysis of genes expressed in the adult filarial parasitic nematode Dirofilaria immitis.

The heartworm Dirofilaria immitis is a filarial parasitic nematode infecting dogs and other mammals worldwide causing fatal complications. Here, we present the first large-scale survey of the adult heartworm transcriptome by generation and analysis of 4005 expressed sequence tags, identifying about 1800 genes and expanding the available sequence information for the parasite significantly. Brugia malayi genomic data offered the most valuable information to interpret heartworm genes, with about 70% of D. immitis genes showing significant similarities to the assembly. Comparative genomic analyses revealed both genes common to metazoans or nematodes and genes specific to filarial parasites that may relate to parasitism. Characterization of abundant transcripts suggested important roles for genes involved in energy generation and antioxidant defense in adults. In particular, we proposed that adult heartworm likely adopted an anaerobic electron transfer-based energy generation system distinct from the aerobic pathway utilized by its mammalian host, making it a promising target in developing next generation macrofilaricides and other treatments. Our survey provided novel insights into the D. immitis transcriptome and laid a foundation for further comparative studies on biology, parasitism and evolution within the phylum Nematoda.

Animals↗

PubMed2001

Human immunodeficiency virus seroconversion and evolution of the hepatitis C virus quasispecies.

When chronic hepatitis C virus (HCV) infections are complicated by acquisition of human immunodeficiency virus (HIV), liver disease appears to accelerate and serum levels of HCV RNA may rise. We hypothesized that HIV might affect the HCV quasispecies by decreasing both complexity (if HIV-induced immunosuppression lessens pressure for selecting HCV substitutions) and the ratio of nonsynonymous (d(N)) to synonymous (d(S)) substitutions, because d(N) may be lower (if there is less selective pressure). To test this hypothesis, we studied the evolution of HCV sequences in 10 persons with chronic HCV infection who seroconverted to HIV and, over the next 3 years, had slow or rapid progression of HIV-associated disease. From each subject, four serum specimens were selected with reference to HIV seroconversion: (i) more than 2 years prior, (ii) less than 2 years prior, (iii) less than 2 years after, and (iv) more than 2 years after. The HCV quasispecies in these specimens was characterized by generating clones containing 1 kb of cDNA that spanned the E1 gene and the E2 hypervariable region 1 (HVR1), followed by analysis of clonal frequencies (via electrophoretic migration) and nucleotide sequences. We examined 1,320 cDNA clones (33 per time point) and 287 sequences (median of 7 per time point). We observed a trend toward lower d(N)/d(S) after HIV seroconversion in 7 of 10 subjects and lower d(N)/d(S) in those with rapid HIV disease progression. However, the magnitude of these differences was small. These results are consistent with the hypothesis that HIV infection alters the HCV quasispecies, but the number of subjects and observation time may be too low to characterize the full effect.

Adult↗

PubMed1991

A beta-galactosidase deletion mutant of Lactobacillus bulgaricus reverts to generate an active enzyme by internal DNA sequence duplication.

Several spontaneous Lac- deletion derivatives of the beta-galactosidase gene of Lactobacillus bulgaricus were analyzed for their phenotypic stability. We found that one of these mutants, lac139, carrying a deletion of 30 bp within the gene, was able to revert to a Lac+ phenotype. Genetical analysis of revertants indicated that an internal region of 72 bp was duplicated immediately next to the deletion site. The region involved in the duplication event is flanked by direct repeated sequences of 13 bp in length. Both events, the deletion and the duplication, were mediated by the presence of such short direct repeats. Enzymatic studies of the purified proteins indicated identical kinetic parameters, but showed considerable instability of the revertant protein.

Base Sequence↗

PubMed1998

Molecular coevolution within a Drosophila clock gene.

The period (per) gene in Drosophila melanogaster provides an integral component of biological rhythmicity and encodes a protein that includes a repetitive threonine-glycine (Thr-Gly) tract. Similar repeats are found in the frq and wc2 clock genes of Neurospora crassa and in the mammalian per homologues, but their circadian functions are unknown. In Drosophilids, the length of the Thr-Gly repeat varies widely between species, and sequence comparisons have suggested that the repeat length coevolves with the immediately flanking amino acids. A functional test of the coevolution hypothesis was performed by generating several hybrid per transgenes between Drosophila pseudoobscura and D. melanogaster, whose repetitive regions differ in length by about 150 amino acids. The positions of the chimeric junctions were slightly altered in each transgene. Transformants carrying per constructs in which the repeat of one species was juxtaposed next to the flanking region of the other were almost arrhythmic or showed a striking temperature sensitivity of the circadian period. In contrast, transgenes in which the repeat and flanking regions were conspecific gave wild-type levels of circadian rescue. These results support the coevolutionary interpretation of the interspecific sequence changes in this region of the PER molecule and reveal a functional dimension to this process related to the clock's temperature compensation.

Amino Acid Sequence↗