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PubMed2003

Polarisation of prokaryotic chromosomes.

In many prokaryotes, asymmetrical mutational or selective pressures have caused compositional skews between complementary strands of replication arms, especially sensitive in the distribution of guanine and cytosine. In Escherichia coli, most of the guanine/cytosine skew is caused by mutation rates differing on leading and lagging strands, but contribution of skewed functionally important guanine-rich motifs (Chi and Rag sites), which control chromosome repair or positioning, is noticeable. Interference between replication and gene expression plays a minor role. The situation may be different in other bacteria. Studies of chromosome processing and bacterial taxonomy might profit from consideration of chromosome polarisation.

Base Composition↗
PubMed2003

Developmental expression and gene/enzyme identifications in the alpha esterase gene cluster of Drosophila melanogaster.

Here we show how the 10 genes of the alpha esterase cluster of Drosophila melanogaster have diverged substantially in their expression profiles. Together with previously described sequence divergence this suggests substantial functional diversification. By peptide mass fingerprinting and in vitro gene expression we have also shown that two of the genes encode the isozymes EST9 (formerly ESTC) and EST23. EST9 is the major 'alpha staining' esterase in zymograms of gut tissues in feeding stages while orthologues of EST23 confer resistance to organophosphorus insecticides in other higher Diptera. The results for EST9 and EST23 concur with previous suggestions that the products of the alpha esterase cluster function in digestion and detoxification of xenobiotic esters. However, many of the other genes in the cluster show developmental or tissue-specific expression that seems inconsistent with such roles. Furthermore, there is generally poor correspondence between the mRNA expression patterns of the remaining eight genes and isozymes previously characterized by standard techniques of electrophoresis and staining, suggesting that the alpha cluster might only account for a small minority of the esterase isozyme profile.

Animals↗
PubMed1999

Evolution of the hepatitis C virus second envelope protein hypervariable region in chronically infected patients receiving alpha interferon therapy.

Sustained hepatitis C virus (HCV) RNA clearance is achieved in 8 to 12% of patients with chronic HCV infection treated with alpha interferon (IFN-alpha) at the approved dose of 3 MU three times a week for 6 months and in about 25% of those receiving this treatment for 12 months. We used single-strand conformation polymorphism analysis combined with cloning and sequencing strategies to characterize the genetic evolution of HCV second envelope gene hypervariable region 1 (HVR1) quasispecies during and after IFN therapy in patients who failed to clear HCV RNA. Sustained HCV RNA clearance was achieved in 6% of patients. Profound changes in HVR1 quasispecies major variants were estimated to occur in 70% of the patients during and after therapy. These changes were evolutionary and were characterized by shifts in the virus population, related to selection and subsequent diversification of minor pretreatment variants. The quasispecies changes appeared to be induced by changes in the host environment likely resulting from the IFN-induced enhancement and post-IFN attenuation of neutralizing and possibly cytotoxic responses against HVR1. The remaining patients had no apparent changes in HVR1 quasispecies major variants, suggesting selection of major pretreatment variants, but some changes were observed in other genomic regions. We conclude that IFN-alpha administration and withdrawal profoundly alters the nature of circulating HCV quasispecies, owing to profound changes in virus-host interactions, in patients in whom sustained HCV RNA clearance fails to occur. These changes are associated with profound alterations of the natural outcome of HCV-related liver disease, raising the hypothesis of a causal relationship.

Adolescent↗
PubMed2005

Evolutionary conservation of minor U12-type spliceosome between plants and humans.

Splicing of rare, U12-type or AT-AC introns is mediated by a distinct spliceosome that assembles from U11, U12, U4atac, U6atac, and U5 snRNPs. Although in human cells the protein composition of minor and major snRNPs is similar, differences, particularly in U11 and U12 snRNPs, have been recently described. We have identified an Arabidopsis U11 snRNP-specific 35K protein as an interacting partner of an RS-domain-containing cyclophilin. By using a transient expression system in Arabidopsis protoplasts, we show that the 35K protein incorporates into snRNP. Oligo affinity selection and glycerol gradient centrifugation revealed that the Arabidopsis 35K protein is present in monomeric U11 snRNP and in U11/U12-di snRNP. The interaction of the 35K protein with Arabidopsis SR proteins together with its strong sequence similarity to U1-70K suggests that its function in splicing of minor introns is analogous to that of U1-70K. Analysis of Arabidopsis and Oryza sativa genome sequences revealed that all U11/U12-di-snRNP-specific proteins are conserved in dicot and monocot plants. In addition, we have identified an Arabidopsis gene encoding the homolog of U4atac snRNA and a second Arabidopsis gene encoding U6atac snRNA. Secondary structure predictions indicate that the Arabidopsis U4atac is able to form dimeric complexes with both Arabidopsis U6atac snRNAs. As revealed by RNaseA/T1 protection assay, the U4atac snRNA gene is expressed as an ~160-nt RNA, whereas the second U6atac snRNA gene seems to be a pseudogene. Taken together, our data indicate that recognition and splicing of minor, AT-AC introns in plants is highly similar to that in humans.

Amino Acid Motifs↗
PubMed2002

[Modern concepts on the relationship between the agents causing plague and pseudotuberculosis].

The authors present published data and their own findings on the relationship between Yersinia pestis and Y. pseudotuberculosis and on the origination of Y. pestis from Y. pseudotuberculosis. Study of microbiological and biochemical characteristics, external membrane protein spectra, and stability of chromosomal region of pigmentation brought the authors to a hypothesis that Y. pestis minor subspecies (ssp. caucasica, altaica, hissarica, ulegeica) which are characterized by selective virulence occupy an intermediate position between Y. pseudotuberculosis and basic species of Y. pestis.

Animals↗
PubMed2006

Functional conservatism in mitochondrial evolution: insight from hybridization of arctic and brook charrs.

To assess the potential adaptive value of mtDNA, we evaluated functional properties and thermal sensitivity of key mitochondrial enzymes in two species that have originally evolved in different thermal environments (arctic charr, Salvelinus alpinus, and brook charr, S. fontinalis), as well as in their hybrids. We measured the activity of two enzymes of the electron transport system (cytochrome c oxidase and NADH-ubiquinone oxidoreductase), one enzyme of the mitochondrial matrix (citrate synthase), and one enzyme of the anaerobic glycolysis (lactate dehydrogenase) in the red muscle at three temperatures (6 degrees C, 12 degrees C and 18 degrees C). Surprisingly, the species presented no significant differences in enzyme activity, thermal sensitivity or thermostability of key metabolic enzymes even though they evolved in different thermal environments and present important differences in amino acid sequences. It seems that amino acid substitutions between those species have minor impact on the functional properties of mitochondrial enzymes studied. The thermal sensitivity results (Q(10)) obtained for inner-membrane mitochondrial enzymes differed somewhat from those of mitochondrial matrix or cytosolic enzymes. This result indicates the modulation of thermal sensitivity of all mitochondrial inner-membrane processes by a common parameter, which could be the structural and functional properties of membrane phospholipids.

Adaptation, Physiological↗
PubMed2001

Chelator-induced disappearance of carboxylate stretching vibrational modes in S2/S1 FTIR spectrum in oxygen-evolving complex of photosystem II.

Fourier transform infrared (FTIR) spectroscopy has been applied toward studies of photosynthetic oxygen evolution, especially on the effects of Ca(2+) depletion and chelating agents using S(2)/S(1) FTIR difference spectrum in the mid-IR region. Ca(2+) depletion showed little influences on the symmetric (1365/1404 cm(-1)) and the asymmetric (1587/1562 cm(-1)) stretching bands of a carboxylate, which are typical of the S(2)/S(1) vibrational features induced by the oxidation of the Mn-cluster; however, minor changes were observed in the amide regions. Addition of a chelating agent (EDTA or EGTA) to the Ca(2+)-depleted membranes resulted in the disappearance of the carboxylate bands concurrent with large modifications of the amide bands with an apparent K(d) value of approximately 0.49 mM (for EDTA). The carboxylate bands and the greater part of the amide bands were restored by the replenishment of CaCl(2), and the chelators did not affect the spectrum in the nondepleted control membranes, indicating that the effects of the chelator are reversible and manifest only in the cases in which the Ca(2+) site is unoccupied by Ca(2+). Ca(2+)-depleted membranes showed the normal S(2)Q(A)(-) thermoluminescence band, and further addition of EDTA did not show any effects on the peak temperature and peak intensity. Moreover, the Ca(2+)-depleted membranes in the presence of EDTA exhibited the S(2) multiline EPR signal with nearly the normal hyperfine splittings. These results demonstrated that the Mn-cluster is oxidized to the S(2) state with normal redox and magnetic properties in the presence of the chelator despite the loss of the carboxylate bands in the FTIR spectra. The results are interpreted as indicating that the chelator interacts with the Mn-cluster as a replacement of the native carboxylate ligand. This prevents the structural changes of the Mn-cluster and protein backbone which are induced upon the oxidation of the Mn-cluster up to the S(2) state, but preserve the redox and magnetic properties of the S(2) state Mn-cluster. The roles of Ca(2+) in the photosynthetic oxygen evolution are also discussed.

Calcium↗
PubMed2003

Binding of phage displayed Bacillus subtilis lipase A to a phosphonate suicide inhibitor.

Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants. Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties for the preparation of pharmaceutically relevant chiral compounds. The lipase gene was cloned upstream of the phage g3p encoding sequence and downstream of a modified g3p signal sequence. Consequently, the enzyme was displayed at the surface of bacteriophage fd as a fusion to its minor coat protein g3p. The phage-bound lipase was correctly folded and fully enzymatically active as determined from the hydrolysis of p-nitrophenylcaprylate with K(m)-values of 0.38 and 0.33 mM for the phage displayed and soluble lipase, respectively. Both soluble lipase and lipase expressed on bacteriophages reacted covalently with a phosphonate suicide inhibitor. The phage does not hamper lipase binding, since both soluble and phage-bound lipase have a similar half-life of inactivation of approximately 5 min. Therefore, we conclude that the Bacillus lipase can be functionally expressed on bacteriophages as a fusion to the phage coat protein g3p. The specific interaction with the suicide inhibitor offers a fast and reproducible method for the future selection of mutant enzymes with an enantioselectivity towards new substrates.

Bacillus Phages↗
PubMed2003

Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1.

The evolutionary conservation of T lymphocyte subsets bearing T-cell receptors (TCRs) using invariant alpha-chains is indicative of unique functions. CD1d-restricted natural killer T (NK-T) cells that express an invariant Valpha14 TCRalpha chain have been implicated in microbial and tumour responses, as well as in auto-immunity. Here we show that T cells that express the canonical hValpha7.2-Jalpha33 or mValpha19-Jalpha33 TCR rearrangement are preferentially located in the gut lamina propria of humans and mice, respectively, and are therefore genuine mucosal-associated invariant T (MAIT) cells. Selection and/or expansion of this population requires B lymphocytes, as MAIT cells are absent in B-cell-deficient patients and mice. In addition, we show that MAIT cells are selected and/or restricted by MR1, a monomorphic major histocompatibility complex class I-related molecule that is markedly conserved in diverse mammalian species. MAIT cells are not present in germ-free mice, indicating that commensal flora is required for their expansion in the gut lamina propria. This indicates that MAIT cells are probably involved in the host response at the site of pathogen entry, and may regulate intestinal B-cell activity.

Amino Acid Sequence↗
PubMed1999

Molecular cytogenetic analysis of the karyotype of the European Atlantic sturgeon, Acipenser sturio.

A karyotype analysis was carried out on the European Atlantic sturgeon, Acipenser sturio (2n=121 +/- 3). The telomeric sequence repeat (TTAGGG)n detected by fluorescent in situ hybridization (FISH) was mostly localized at the telomeres of all chromosomes. Ribosomal DNA (rDNA) genes were detected by silver staining techniques and by FISH with digoxigenin-labelled probe for 28S rDNA. Silver staining detected active NORs in the telomeric regions of six chromosomes, and by FISH one or two additional minor sites were detected. The 5S rDNA was found in the interstitial region of a small metacentric pair. The 5S rRNA gene was completely sequenced for the first time in a sturgeon species. The A. sturio karyotype organization is discussed in relation to phylogenesis of the species within the Acipenseridae and to polyploidization events characterizing sturgeon evolution.

Animals↗
PubMed1975

The histogenesis of mixed cervical carcinomas. The concept of endocervical columnar-cell dysplasia.

In a case of cervical adenocarcinoma in situ, accompanied by relatively minor squamous-cell dysplasia, the neoplastic glandular cells were associated with "abnormal" but not clearly cancerous surface cells of the endocervix. Measurements of the nuclear DNA of these cells revealed an aneuploid pattern similar to that of epidermoid dysplasias. It is suggested that endocervical dysplasia is a valid entity and represents a step in the evolution of cervical adenocarcinoma from reserve cells comparable to the position of epidermoid dysplasia in the genesis of epidermoid carcinoma.

Adenocarcinoma↗
PubMed2000

Resurrection of crushed magnetization and chaotic dynamics in solution NMR spectroscopy.

We show experimentally and theoretically that two readily observed effects in solution nuclear magnetic resonance (NMR)-radiation damping and the dipolar field-combine to generate bizarre spin dynamics (including chaotic evolution) even with extraordinarily simple sequences. For example, seemingly insignificant residual magnetization after a crusher gradient triggers exponential regrowth of the magnetization, followed by aperiodic turbulent spin motion. The estimated Lyapunov exponent suggests the onset of spatial-temporal chaos and the existence of chaotic attractors. This effect leads to highly irreproducible experimental decays that amplify minor nonuniformities such as temperature gradients. Imaging applications and consequences for other NMR studies are discussed.

Journal Article↗
PubMed1992

Paediatric pain--concepts for caring.

Caring for the health of children is a relatively modern concept. Furthermore, recognition of children's need for relief from pain is only just beginning to emerge and remains a subject of controversy. Sadly, in the past, children's pain was viewed as a diagnostic aid rather than an entity in its own right. This paper outlines historical perspectives which contributed, not only to the slow evolution of paediatric nursing, but in particular, to recognition of children's pain as an entity. Furthermore, it emphasizes the importance of a sound understanding of children's developmental stages as fundamental to accurate assessment of children's special needs which can only be determined through effective communication. Fundamentally, as children remain a somewhat voiceless, powerless minority group, health professionals are challenged not only to provide care, but to ensure that the care they give is indeed meeting the unique needs of each and every individual child.

Child↗
PubMed1991

Clonality and clonal evolution of hepatocellular carcinoma with multiple nodules.

To determine the clonal evolution of hepatocellular carcinoma, the integrated hepatitis B virus DNA patterns of the main tumor, satellites and/or metastatic lesions were analyzed by Southern-blot hybridization in 28 hepatocellular carcinomas, including three HBsAg-seronegative cases. Unicentric or multicentric hepatocellular carcinoma was confirmed by histopathological criteria in 89% of the cases. Among 17 unicentric hepatocellular carcinomas, minor changes of the integration pattern--including partial loss or addition of the integration sites or both--were detected in the metastatic lesions in 29% of the cases. Furthermore, none of five cases with free-form hepatitis B virus DNA in the primary tumor had detectable free hepatitis B virus DNA in the metastatic lesions. These results suggest that the alteration of integrated hepatitis B virus DNA pattern during the course of tumor growth and metastasis may occur more often than previously perceived and that the switch-off of virus replication may be related to tumor metastatic potential. In eight cases with unilateral, multicentric hepatocellular carcinoma, two clones were detected in six cases, three were seen in another and four were seen in one. One case of note was a 9-yr-old boy with two histological types and two different integration patterns, one associated with vascular invasion and lung metastasis. Three patients with bilateral hepatocellular carcinoma were confirmed to have bicentric or tricentric hepatocellular carcinoma rather than intrahepatic dissemination and had survival rates similar to those in unicentric hepatocellular carcinoma. Three invasive HBsAg-seronegative hepatocellular carcinomas were found to have hepatitis B virus DNA integration and were of unicentric origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Southern↗
PubMed1981

Lymphoid cell proliferation in non-lymphoid organs during graft-versus-host reactions (GVHR): I. Lack of correlation between mortality and grafted cell proliferation.

The course of development of graft-versus-host reactions (GVHR) following lethal irradiation was compared in two mouse donor-host combinations disparate at non H-2 loci alone or associated to H-2 complex. The pathogenesis of these reactions was very different (mortality rates faster in the second situation for example). The donor cell proliferation kinetics was followed in lymphoid (pooled lymph nodes, spleen, thymus) and in non-lymphoid organs (kidney and liver) from day 0 to day 28 post-transplantation, and showed the same profile in both GVH reactions. Our data emphasize the discordance between the clinical evolution of the GVH disease on one hand, and the stimulation of the grafted cell proliferation on the other hand, induced by a same H-2 disparity. In addition, we have demonstrated the relatively high level of the lymphocyte proliferation in non-lymphoid organs during the course of a GVHR, even in the case of minor histoincompatibility.

Animals↗
PubMed1995

Aminoglycosides--50 years on.

1. The aminoglycoside antibiotics are 50 years old. Their success and continuing use can be attributed to various factors including rapid concentration-dependent bactericidal effect, synergism with beta-lactam antibiotics, clinical effectiveness, a low rate of true resistance and low cost. 2. The aminoglycosides remain drugs of choice in many circumstances including septicaemia, other serious infections due to Gram negative bacilli, and bacterial endocarditis. 3. Nephrotoxicity and ototoxicity have been the main drawbacks clinically for the aminoglycosides. 4. There has been an evolution in dosing strategies largely aimed at reducing toxicity. Therapeutic drug monitoring has been used extensively to assist dosing, and target concentrations have been advocated, such as peak concentrations of between 6 and 10 mg l-1 and through concentrations of < 2 mg l-1 for gentamicin, tobramycin and netilmicin. 5. Recently there has been a minor revolution in the approach to aminoglycoside dosing, with a change to larger doses, given less frequently. In its most convenient form this is 'Once-daily aminoglycoside dosing'. It offers the hope of better efficacy, less toxicity, and easier administration and monitoring. 6. This article summarises the background of aminoglycoside usage, leading up to the recent changes in dosing strategy.

Aminoglycosides↗
PubMed1997

[Effect of external extra-articular ligament plasty on the results of anterior cruciate ligament reconstruction with patellar tendon, a 4 years follow-up].

PURPOSE OF THE STUDY: The purpose of this study was to compare the functional results obtained when an external extra-articular plasty was added to an anterior cruciate ligament (ACL) reconstruction using an autologous bone tendon-bone patellar tendon graft. MATERIAL AND METHODS: The authors analyzed two consecutive series of 60 and 50 patients operated by the same surgeon for a chronic rupture of the anterior cruciate ligament, one by reconstruction of the cruciate ligament with a free graft of the patellar tendon supplemented by an external extra-articular plasty made with a quadriceps tendon graft and the second with an isolated free patellar tendon graft. Anterior laxity was measured before and after surgery, by dynamic X-rays and by the Medmetric KT-1000 arthrometer. Functional results were evaluated four years after operation, with the French A.R.P.E.GE score based on sport activity level and intensity. RESULTS: Anterior laxity was not different before operation in both groups and there was no difference between males and females. Medmetric KT-1000 arthrometer showed the same negative differential laxity immediately after surgery in both groups and the same evolution during the first 4 years, without any significant difference on laxity on the middle aspect of the knee. Radiological results were different. After a 4 years follow-up, anterior laxity did not show significant difference on the medial compartment of the knee (5.3 +/- 2.3 mm and 5.5 +/- 1.7 mm), but there was a significant minor laxity in the lateral compartment for the lateral extra-articular plasty group (11.0 +/- 2.3 mm against 14.8 +/- 3.8 mm)(p = 0.002). Functional results and sport activity were similar in both groups. Examination showed 4 positive pivot shift tests (2 "sliding" and 2 positive) in the group with extra-articular plasty, even though 8 positive pivot shift tests in the isolated ACL group (5 "sliding" and 3 positive) were found. DISCUSSION: This study, as well as five others studies found in literature, was not randomized. In all these series, the surgical techniques, the rehabilitation programs and the functional score evaluation were too different to allow any pertinent comparison. Extra-articular plasty helps to control the laxity of the lateral compartment of the knee which is incompletely controlled by ACL reconstruction, particularly in chronic cases. This is proved by radiological measurements and pivot shift tests. Jensen in 1983, about 205 patients with a 4 year follow-up and Noyes, which used an allograft patellar tendon, found an advantage to do extra-articular plasty. But Strum (in 1989), as O'Brien (in 1991) and Roth (in 1987), did not found any advantage with extra-articular plasty. CONCLUSION: It is therefore obvious, after a four-year follow-up, that extra-articular supplementation presents an advantage for reconstruction of the ACL. by a free graft of the patellar tendon in chronic cases. Further randomized study will confirm that isolated ACL reconstruction is possible in some well defined categories of anterior laxity.

Adolescent↗
PubMed2001

An evolutionary-game model of tumour-cell interactions: possible relevance to gene therapy.

Evolutionary games have been applied as simple mathematical models of populations where interactions between individuals control the dynamics. Recently, it has been proposed to use this type of model to describe the evolution of tumour cell populations with interactions between cells. We extent the analysis to allow for synergistic effects between cells. A mathematical model of a tumour cell population is presented in which population-level synergy is assumed to originate through the interaction of triplets of cells. A threshold of two cooperating cells is assumed to be required to produce a proliferative advantage. The mathematical behaviour of this model is explored. Even this simple synergism (minor clustering effect) is sufficient to generate qualitatively different cell-population dynamics from the models published previously. The most notable feature of the model is the existence of an unstable internal equilibrium separating two stable equilibria. Thus, cells of a malignant phenotype can exist in a stable polymorphism, but may be driven to extinction by relatively modest perturbations of their relative frequency. The proposed model has some features that may be of interest to biological interpretations of gene therapy. Two prototypical strategies for gene therapy are suggested, both of them leading to extinction of the malignant phenotype: one approach would be to reduce the relative proportion of the cooperating malignant cell type below a certain critical value. Another approach would be to increase the critical threshold value without reducing the relative frequency of cells of the malignant phenotype.

Algorithms↗