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PubMed2005

Amphotericin B.

Invasive fungal infections are a major cause of morbidity and mortality in immunodeficient individuals (such as AIDS patients) and in transplant recipients or tumor patients undergoing immunosuppressive chemotherapy. Amphotericin B is one of the oldest, yet most efficient antimycotic agents. However, its usefulness is limited due to dose-dependent side-effects, notably nephrotoxicity. In order to improve its safety margin, new pharmaceutical formulations of amphotericin B have been designed especially to reduce its detrimental effects on the kidneys. Since the 1980s, a wide variety of new amphotericin B formulations have been brought forward for clinical testing, many of which were approved and reached market value in the 1990s. This review describes and discusses the molecular genetics, pharmacological, toxicological, and clinical aspects of amphotericin B itself and many of its innovative formulations.

Amphotericin B↗
PubMed1982

Quantitative forward-mutation specificity of mono-functional alkylating agents, ICR-191, and aflatoxin B1 in mouse lymphoma cells.

We have analyzed forward-mutation specificity in S49 mouse T lymphoma cells. Our criteria of specificity were based upon relative mutabilities of a panel of 3 genetic markers: resistance to 6-thioguanine (6TGr), dibutyryl cAMP (bt2cAMPr), and ouabain (OUAr). We tested 2 monofunctional alkylating agents, ethyl methane-sulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and 2 heterocyclic compounds, ICR-191 and aflatoxin B1 (AFb1). AFB1 was activated with rat-liver microsomal S9 plus cofactors. Expression-time lags of each genetic marker ranged from 2 days of OUAr mutations to 6 and 8 days for bt2cAMPr and 6TGr cells, with stable induced mutant fractions thereafter. The relative activity of each agent for each marker was assessed on the basis of its mutagenic efficiency at equitoxic doses. Specificity differences between the agents were determined by taking ratios of mutagenic efficiencies (RME) for the 3 possible pairs of markers. From these quantitative correlations and other data we conclude that both MNNG and EMS induce ouabain resistance (and probably bt2cAMPr and 6TGr) by similar mechanisms, almost certainly base substitutions. In contrast, ICR-191 and AFB1 are respectively less than 2 and 3% as efficient as MNNG for OUAr mutant induction relative to the activity of each agent for 6TGr mutagenesis. We infer that ICR-191 and AFB1 very rarely cause base substitutions in S49 cells, but that their activities are consistent with production of deletions, insertions or chromosomal aberrations. S49 cells demonstrate an unusually high specificity of mutagenesis at the OUAr locus compared to several other rodent cell lines. Thus, this panel of markers in S49 cells can be used as a sensitive, reliable screening system for mutagen detection and to discriminate among major classes of mutagenic mechanisms.

Aflatoxin B1↗
PubMed2003

[Genetic features of atrial fibrillation].

Etiology of atrial fibrillation remains largely unknown. Molecular genetics is a powerful tool that may help to identify underlying mechanisms of the arrhythmia. As a matter of fact, a major step forward was the recent identification of the KCNQ1 gene coding a subunit of a potassium channel in IKS current as responsible for idiopathic atrial fibrillation in a familial autosomal dominant form. The KCNQ1 gain-of-function mutation may induce a shortening in action potential and in effective refractory periods leading to the initiation and persistence of atrial fibrillation. In the multifactorial forms of the disease, by far the most frequent, susceptibility genes and modifier genes are also currently being identified. This better understanding of the disease leads to new perspectives, particularly for therapeutists, and would enable them to work with more interest.

Atrial Fibrillation↗
PubMed1997

The ecology and biosafety of baculoviruses.

Advances in the use of molecular techniques-particularly for virus identification, the investigation of latency and the infection process, plus the development of a theoretical framework containing a higher degree of biological realism-have pushed baculovirus ecology forward in the past few years. This has created a scenario in which many hitherto intractable questions about the behaviour of natural and genetically modified baculoviruses can now be addressed.

Animals↗
PubMed2006

Sex differences after all those years? Heritability of cognitive abilities in old age.

We investigated sex differences in genetic and environmental effects on cognitive abilities among older adult twins. We drew participants from the Swedish Twin Registry; our sample included 647 twin pairs. Our cognitive measures included Synonyms, Block Design, Digit Span, Thurstone's Picture Memory, Symbol Digit, and general cognitive ability tests. Higher age was related to lower performance in all cognitive measures, except synonyms. For digit span forward, symbol digit, and general cognitive ability tasks, there was a Sex x Age interaction, with greater deficits in the performance of women compared with those of men at higher ages. We found no sex-specific genetic influences. In other words, the same genetic effects were operating for men and women. Furthermore, the magnitude of genetic effect was similar for men and women.

Adoption↗
PubMed2004

Two steps forward, one step back: the pleiotropic effects of favoured alleles.

Pleiotropy is one of the most commonly observed attributes of genes. Yet the extent and influence of pleiotropy have been underexplored in population genetics models. In this paper, I quantify the extent to which pleiotropy inhibits the spread of alleles in response to directional selection on a focal trait. Under the assumption that pleiotropic effects are extensive and deleterious, the fraction of alleles that are beneficial overall is severely limited by pleiotropy and rises nearly linearly with the strength of directional selection on the focal trait. Over a broad class of distribution of pleiotropic effects, the mean selective effect of those alleles that are beneficial overall is halved, on average, by pleiotropy. The fraction of new mutant alleles that are beneficial overall and that succeed in fixing within a population is even more severely limited when directional selection is weak, but it rises quadratically with the strength of directional selection. Finally, the mean selective effect of mutant alleles that are beneficial and succeed in fixing is reduced by one-third, on average, by pleiotropy. These results help to shape our understanding of the evolutionary inertia caused by pleiotropy.

Alleles↗
PubMed2004

Chloroplast DNA variation and postglacial recolonization of common ash (Fraxinus excelsior L.) in Europe.

We used chloroplast polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP) and chloroplast microsatellites to assess the structure of genetic variation and postglacial history across the entire natural range of the common ash (Fraxinus excelsior L.), a broad-leaved wind-pollinated and wind-dispersed European forest tree. A low level of polymorphism was observed, with only 12 haplotypes at four polymorphic microsatellites in 201 populations, and two PCR-RFLP haplotypes in a subset of 62 populations. The clear geographical pattern displayed by the five most common haplotypes was in agreement with glacial refugia for ash being located in Iberia, Italy, the eastern Alps and the Balkan Peninsula, as had been suggested from fossil pollen data. A low chloroplast DNA mutation rate, a low effective population size in glacial refugia related to ash's life history traits, as well as features of postglacial expansion were put forward to explain the low level of polymorphism. Differentiation among populations was high (GST= 0.89), reflecting poor mixing among recolonizing lineages. Therefore, the responsible factor for the highly homogeneous genetic pattern previously identified at nuclear microsatellites throughout western and central Europe (Heuertz et al. 2004) must have been efficient postglacial pollen flow. Further comparison of variation patterns at both marker systems revealed that nuclear microsatellites identified complex differentiation patterns in south-eastern Europe which remained undetected with chloroplast microsatellites. The results suggest that data from different markers should be combined in order to capture the most important genetic patterns in a species.

DNA, Chloroplast↗
PubMed1995

Construction of a phylogenetic tree for inbred strains of rat by arbitrarily primed polymerase chain reaction (AP-PCR).

We constructed a tentative genealogic tree of 13 inbred rat strains using genetic markers identified by the AP-PCR (Arbitrarily Primed Polymerase Chain Reaction) technique, which consists of PCR amplification under low stringency conditions, with only one oligonucleotide as both forward and reverse primers. We obtained discrete-state genotypes of 264 loci, presumably covering the whole rat genome. Computational analysis of this panel allowed the construction of a genealogic tree representing the relations among the 13 strains. The accordance of the present results with available data on the histories of some of these strains validates the use of the AP-PCR technique for studies on phylogeny.

Animals↗
PubMed1985

A system for detection of genetic and epigenetic alterations in Escherichia coli induced by DNA-damaging agents.

In order to compare the genetic and epigenetic effects of genotoxic agents, we have constructed Escherichia coli K12 strains that allow the detection of mutagenesis, SOS induction (epigenetic effect) and genetic recombination in the same genetic background. The epigenetic effect was detected in a similar way to any genetic alteration, i.e. by counting altered clones (colonies), using a gene fusion system that responds to a temporary epigenetic effect by a stable, heritable switch. The gene fusion consists of the E. coli gal operon and a partially deleted prophage lambda, resulting in the gal operon coming under the control of the cI and cro genes. It allows the detection of SOS induction and forward mutagenesis in the cI gene. Even a temporary inactivation of the CI repressor in this particular system leads to a stable epigenetic switch transmitted to the cellular progeny, which can be detected as Gal+ (red) colonies. The genetic (mutational inactivation of gene cI) and epigenetic (proteolytic inactivation of the product of gene cI) mechanisms leading to gal expression can be distinguished. Genetic recombination between two heteroallelic lacZ genes, one located in the bacterial chromosome, the other on an F'lac plasmid, can be detected as Lac+ colonies. Radiation and several chemical mutagens show very different capacities in generating mutants, inductants and recombinants; therefore, a dose range of any physical or chemical agent generates a set of relative values for the generation of mutants, inductants and recombinants that are characteristic of the agent.

Aflatoxin B1↗
PubMed2003

[Construction of expression vector with BG2 gene and its transformation in wheat].

beta-1,3-glucanase(BG2) is one of the pathogensis-related-proteins(PR). Study of these proteins and their related genes is one of the hot points in plant genetic engineering of disease resistance for a long time. In this research, specific primers were designed with the enzyme cleavage site of Spe I in its forward one and Not I site in the backward according to the BG2 gene sequence. Using this pair of primers, BG2 gene, which was contained in the plasmid of pRTL2, was amplified and confirmed by sequencing the amplified fragment inserted into T-easy vector. The positive clone containing BG2 gene was digested with the enzymes of Spe I/Not I and then BG2 gene was inserted into the Xba I/Not I sites of super expression binary vector pATC940. The reconstructed expression vector named as pATCBG2 was introduced into the wheat of Longfumai10 and Longfumai3 (Triticum aestivum L. em. Thell) through the particle gun transformation method. The Kanamysin resistant (Km') transformants were obtained. PCR, Dot-blotting and PCR-Southern hybridization analysis showed that the BG2 gene was integrated into the genome of wheat. Result of pathogen inoculation assay on the transgenic plants showed that the transgenic plants had a higher resistant disease score of 1-2 grade than the control.

Ascomycota↗
PubMed1979

Measurement of mutagenesis in mammalian cells.

A method using mammalian cells in vitro for detection and quantitation of mutagenic actions that appears to be useful for screening for carcinogenesis and genetic damage by environmental agents is presented. The method involves use of stable human--Chinese hamster ovary hybrid cells that have retained a single human chromosome not necessary for cell reproduction. Forward mutations are detected in genes necessary for production of specific human cell surface antigens. Such mutants form colonies in the presence of specific antisera and complement that destroy the unmutagenized cells. Use of the method is illustrated for the action of x-irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, and caffeine. The method appears to be unique in that it permits assessment of lesions that cause loss of all or most of the chromosome as well as various localized gene mutations. The former action is particularly important because of the major involvement of chromosomal lesions in an extremely important class of human genetic disease.

Amniotic Fluid↗
PubMed1997

Protein folding: optimized sequences obtained by simulated breeding in a minimalist model.

For a minimalist model of protein folding, which we introduced recently, we investigate various methods to obtain folding sequences. A detailed study of random sequences shows that, for this model, such sequences usually do not fold to their ground states during simulations. Straight-forward techniques for the construction of folding sequences, based solely on the target structure, fail. We describe in detail an optimization algorithm, based on genetic algorithms, for the "simulated breeding" of folding sequences in this model. We find that, for any target structure studied, there is not only a single folding sequence but a patch of sequences in sequence space that fold to this structure. In addition, we show that, much as in real proteins, nonhomologous sequences may fold to the same target structure.

Amino Acid Sequence↗
PubMed1997

The search for genetic clues in chronic lymphocytic leukemia.

Research on the genetic basis of CLL is progressing at a rapid pace. The development of new techniques such as FISH, comparative genomic hybridisation (CGH) and a whole range of molecular methods is being applied to identify abnormalities in this relatively common B-cell leukaemia. The abnormalities may be of a different nature. There are some which are clearly associated with particular forms of the disease and usually with aggressive characteristics. The best examples are deletions at 11q23 seen in younger patients with generalised lymphadenopathy and inferior prognosis; trisomy 12, commonly associated with an increased proportion of prolymphocytes (CLL/PL) and more progressive disease; 17p abnormalities, chiefly mutations and deletions of p53, although rare, seem to be associated with transformation such as Richter syndrome, with CLL/PL and poor response to therapy. Abnormalities at 13q, though not correlated with particular clinical syndromes, are the subject of intense interest due to the possibility that one or more tumour suppressor genes relevant to the pathogenesis of CLL may be identified. Two areas in which work is being focused are 13q14 and 13q12. Finally, the incidence of familial cases of CLL, which has been known for a number of years, will lead to an international effort to collect familial cases, which ultimately will allow a genetic linkage study to discover a CLL "susceptibility gene". The presentations at the IWCLL were up-to-date, stimulating and pointed the way forward to further rapid progress in this exciting field.

Chromosome Aberrations↗
PubMed1999

Fluorescence-based single-strand conformation polymorphism analysis of the low density lipoprotein receptor gene by capillary electrophoresis.

We describe here a new method to screen for unknown mutations in the low density lipoprotein (LDL) receptor gene by the use of capillary electrophoresis in single-strand conformation polymorphism (SSCP) analysis. To analyze the promoter and all 18 exons, 20 different amplification reactions were necessary. For each polymerase chain reaction (PCR), the forward and reverse primers were 5' fluorescent-labelled with FAM and HEX, respectively. To test the accuracy of the newly developed method, 61 genetic variants distributed in 16 exons were analyzed. Under identical electrophoresis conditions (13 kV, 30 degrees C, 30 min), 59 mutations were detected by a distinct abnormal SSCP pattern. The two remaining mutations showed only slight abnormalities, which could be amplified by increasing the electrophoresis temperature. The high accuracy, the degree of automation and the speed of analysis make fluorescence-based SSCP analysis with capillary electrophoresis ideal for rapid mutation screening and the technique is well-suited for clinical applications.

Base Sequence↗
PubMed1988

The mutY gene: a mutator locus in Escherichia coli that generates G.C----T.A transversions.

We have used a strain with an altered lacZ gene, which reverts to wild type via only certain transversions, to detect transversion-specific mutators in Escherichia coli. Detection relied on a papillation technique that uses a combination of beta-galactosides to reveal blue Lac+ papillae. One class of mutators is specific for the G.C----T.A transversion as determined by the reversion pattern of a set of lacZ mutations and by the distribution of forward nonsense mutations in the lacI gene. The locus responsible for the mutator phenotype is designated mutY and maps near 64 min on the genetic map of E. coli. The mutY locus may act in a similar but reciprocal fashion to the previously characterized mutT locus, which results in A.T----C.G transversions.

Chromosome Mapping↗
PubMedDate not supplied

Molecular events leading to fertilization--a review.

Fertilization consists of a sequence of complex events which culminates in the fusion of the genetic information provided by two parent cells, thus leading to the formation of a new individuum. The male gamete develops in the testis, acquires forward motility and fertilizing capacity during maturation in the epididymis, and after capacitation undergoes the acrosome reaction in the female reproductive tract. The female gamete matures in the cyclically developing follicles and, by the time of ovulation, reaches a quiescent stage at metaphase II of meiosis. Gamete fusion occurs in the distal tube, and involves a series of modifications on the egg surface and its investments, intended to protect the developing embryo from polyspermy.

Acrosome↗
PubMed2005

Assessment of GDNF in primate models of Parkinson's disease: comparison with human studies.

Potential future treatments for Parkinson's disease (PD) include those that not only provide symptomatic relief to patients but are also neuroprotective and/or neurorestorative. Glial cell line-derived neurotrophic factor (GDNF) may be a valuable candidate in this regard. Positive results using monkeys have encouraged the use of GDNF in human trials. These trials have unfortunately shown mixed results, illustrating the influence that various parameters of administration can have on clinical outcome. The aim of this review is to compare the findings of these clinical studies with available primate data. While bolus intraventricular injections of GDNF in primates have shown some behavioural efficacy, there was no clinical benefit in the first human trial using this method, which was most likely a result of inefficient GDNF distribution in the striatal parenchyma. In primates, however, continuous (rather than bolus) delivery of GDNF into the ventricles results in significant distribution in the striatum. While chronic delivery of GDNF into the ventricles has not been assessed in humans, intraputamenal protein delivery in two Phase I trials have demonstrated that GDNF considerably reduces PD symptoms, suggesting that the putamen is the optimal location for delivery. Primate studies have shown that vector mediated delivery of GDNF may provide a suitable means for long-term intraputamenal delivery. However, the possibility of high levels of GDNF resulting in widespread distribution of GDNF to non-targeted areas is a cause of concern, and vectors with transgene regulation are needed. The development of these vectors may be the way forward for GDNF treatment.

Animals↗
PubMed2004

[New developments in application of laser capture microdissection].

Isolation of pure targeted cells is an important and essential step for the molecular analysis of tissue lesion occurred in the progression of disease. Laser capture microdissection (LCM) is a novel technique that can be applied to obtain pure targeted cell subgroup or even a single cell quickly and precisely under the microscope, thus the problem of tissue heterogeneity in molecular analysis can be tackled successfully. In this article, the principles, advantages and disadvantages of LCM were introduced. New developments in the application of LCM were summarized in two fields (DNA analysis and gene expression analysis) respectively. Meanwhile, possible directions of the future developments of LCM were put forward.

Animals↗