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1.
Acta Chir Orthop Traumatol Cech ; 84(4): 241-246, 2017.
Article in English | MEDLINE | ID: mdl-28933326

ABSTRACT

The demographic change in industrial countries leads to an increasing population that sustains an acetabular fracture in an advanced age. Some authors predicted elderly individuals to be the most rapidly growing subgroup of patients currently sustaining acetabular fractures. Gold standard of treatment of acetabular fractures remains the open reduction and internal fixation. Relevant factors impeding surgical treatment include the significantly decreased bone stock and the incapability of the patients to partially weight bear following surgery. Therefore, special considerations should be performed when dealing with this patient group as surgical treatment is associated with several risks and often accompanied by poor outcomes. This review aims to summarize the current body of knowledge and to give a recommendation concerning a surgical treatment cascade.


Subject(s)
Acetabulum/injuries , Aging , Fracture Fixation, Internal , Hip Fractures/surgery , Fracture Fixation, Internal/methods , Guidelines as Topic , Humans , Risk Factors , Treatment Outcome
2.
Plant Sci ; 253: 194-205, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27968988

ABSTRACT

Nuclear and chloroplast markers and phenotypic characters were integrated to analyse the population genetic structure of wild cardoon, Cynara cardunculus var. sylvestris, the ancestor of cultivated globe artichoke, Cynara cardunculus var. scolymus on the island of Sardinia, Italy. The spatial scale ranged from a few metres to ∼200km. Wild cardoon appears to be genetically fragmented, with significant genetic divergence at various scales, indicating that gene flow is insufficient to counterbalance the effects of genetic drift or founder effects. Divergence between populations was higher for chloroplast (40%) than for nuclear markers (15%), suggesting that gene flow via seed was lower than via pollen. Two main genetic groups were detected; these correlated with differences in flowering time, capitula size, glossiness, and anthocyanin pigmentation. A complex population structure of wild cardoon emerged over small spatial scales, likely resulting from the interplay between gene dispersal, colonisation history and selective forces. Indeed, Sardinia appears to be a 'hybrid zone' of different gene pools. The island has unique diverse germplasm that has originated from hybridisation among different gene pools. The sampling of seeds from a few plants but from many sites is suggested as the best strategy to harvest the genetic diversity of wild cardoon.


Subject(s)
Cynara/genetics , DNA, Chloroplast/genetics , Gene Flow , Inbreeding , Italy , Microsatellite Repeats , Phenotype , Phylogeography , Polymorphism, Genetic
3.
Orthopade ; 45(1): 32-7, 2016 Jan.
Article in German | MEDLINE | ID: mdl-26614255

ABSTRACT

BACKGROUND: Fractures of the distal femur are rare injuries that are mainly treated operatively. Complication rates remain high. OBJECTIVES: This study aimed to analyse complications following the operative treatment of these fractures and to identify predictive factors that have the potential to identify patients who are at risk for a complicated course of treatment. MATERIALS AND METHODS: We retrospectively analysed all fractures of the distal femur that were treated operatively at our institution between 2005 and 2015. Besides patient and fracture-specific data, surgical details and the types of complications that occurred were recorded and analysed. RESULTS: Open soft tissue damage, the polytraumatised patient and the timing of surgery (i.e. emergency surgery) are significant risk factors for the development of a nonunion. A risk factor that predicts a postoperative infection is open soft tissue damage. Type C fractures, stabilisation as emergency surgery and an accompanying polytrauma are risk factors for a postoperative pneumonia. CONCLUSIONS: The complication rate is significantly determined by surgical factors. To reduce the rate of nonunion, infection and pneumonia, the optimisation of the patient's general condition before surgery and optimal surgical care is more important than an immediate emergency surgery.


Subject(s)
Femoral Fractures/surgery , Fracture Fixation, Internal/adverse effects , Joint Diseases/etiology , Joint Diseases/therapy , Knee Injuries/surgery , Postoperative Complications/therapy , Femoral Fractures/complications , Femoral Fractures/diagnostic imaging , Humans , Joint Diseases/diagnosis , Knee Injuries/complications , Knee Injuries/diagnostic imaging , Postoperative Complications/diagnosis , Postoperative Complications/etiology , Radiography
4.
Front Plant Sci ; 3: 312, 2012.
Article in English | MEDLINE | ID: mdl-23346091

ABSTRACT

Evolutionary studies that are aimed at defining the processes behind the present level and organization of crop genetic diversity represent the fundamental bases for biodiversity conservation and use. A Mesoamerican origin of the common bean Phaseolus vulgaris was recently suggested through analysis of nucleotide polymorphism at the nuclear level. Here, we have used chloroplast microsatellites to investigate the origin of the common bean, on the basis of the specific characteristics of these markers (no recombination, haploid genome, uniparental inheritance), to validate these recent findings. Indeed, comparisons of the results obtained through analysis of nuclear and cytoplasmic DNA should allow the resolution of some of the contrasting information available on the evolutionary processes. The main outcomes of the present study are: (i) confirmation at the chloroplast level of the results obtained through nuclear data, further supporting the Mesoamerican origin of P. vulgaris, with central Mexico representing the cradle of its diversity; (ii) identification of a putative ancestral plastidial genome, which is characteristic of a group of accessions distributed from central Mexico to Peru, but which have not been highlighted beforehand through analyses at the nuclear level. Finally, the present study suggests that when a single species is analyzed, there is the need to take into account the complexity of the relationships between P. vulgaris and its closely related and partially intercrossable species P. coccineus and P. dumosus. Thus, the present study stresses the importance for the investigation of the speciation processes of these taxa through comparisons of both plastidial and nuclear variability. This knowledge will be fundamental not only from an evolutionary point of view, but also to put P. coccineus and P. dumosus germplasm to better use as a source of useful diversity for P. vulgaris breeding.

5.
Theor Appl Genet ; 123(8): 1341-57, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21830108

ABSTRACT

Evolutionary studies in plant and animal breeding are aimed at understanding the structure and organization of genetic variations of species. We have identified and characterized a genomic sequence in Phaseolus vulgaris of 1,200 bp (PvSHP1) that is homologous to SHATTERPROOF-1 (SHP1), a gene involved in control of fruit shattering in Arabidopsis thaliana. The PvSHP1 fragment was mapped to chromosome Pv06 in P. vulgaris and is linked to the flower and seed color gene V. Amplification of the PvSHP1 sequence from the most agronomically important legume species showed a high degree of interspecies diversity in the introns within the Phaseoleae, while the coding region was conserved across distant taxa. Sequencing of the PvSHP1 sequence in a sample of 91 wild and domesticated genotypes that span the geographic distribution of this species in the centers of origin showed that PvSHP1 is highly polymorphic and, therefore, particularly useful to further investigate the origin and domestication history of P. vulgaris. Our data confirm the gene pool structure seen in P. vulgaris along with independent domestication processes in the Andes and Mesoamerica; they provide additional evidence for a single domestication event in Mesoamerica. Moreover, our results support the Mesoamerican origin of this species. Finally, we have developed three indel-spanning markers that will be very useful for bean germplasm characterization, and particularly to trace the distribution of the domesticated Andean and Mesoamerican gene pools.


Subject(s)
Crops, Agricultural/genetics , Genes, Plant/genetics , Genetic Variation , Nucleotides/genetics , Phaseolus/genetics , Base Pairing/genetics , Base Sequence , Central America , Chromosome Mapping , DNA, Intergenic/genetics , Genetic Linkage , Genetic Markers , Genetics, Population , INDEL Mutation/genetics , Molecular Sequence Data , Phylogeography , Population Dynamics , Quantitative Trait, Heritable , Recombination, Genetic/genetics , South America , Species Specificity
6.
Aktuelle Urol ; 41(6): 375-7, 2010 Nov.
Article in German | MEDLINE | ID: mdl-21082518

ABSTRACT

Neoplasms of the seminal vesicles are rare. Here we report on a patient with a low-grade phyllodes tumour of the seminal vesicle. The patient was admitted to our hospital with a tumour in the excavatio rectovesicalis diagnosed by CT scan. He had no symptoms. For further diagnosis we took transrectal ultrasound-guided biopsies, the histopathological examination showed no malignant features. One month later a follow-up CT scan demonstrated a significant enlargement of the tumour. Therefore we decided to perform a surgical exploration. During surgery we found a partially necrotic mass involving the prostate, the urinary bladder and the rectum. Both radical cystoprostatectomy with ileal conduit and anterior resection of the rectum with colostomy were necessary. Histologically the specimen showed a low-grade phyllodes tumour of the left seminal vesicle. One year after surgery the follow-up was completely normal without any residual or recurrent tumour. Frequency, histology, diagnostic investigations, therapy and prognosis of this rare tumour entity are discussed with respect to the actual literature.


Subject(s)
Genital Neoplasms, Male/diagnosis , Phyllodes Tumor/diagnosis , Seminal Vesicles , Aged , Biopsy , Endosonography , Follow-Up Studies , Genital Neoplasms, Male/pathology , Genital Neoplasms, Male/surgery , Humans , Incidental Findings , Male , Phyllodes Tumor/pathology , Phyllodes Tumor/surgery , Seminal Vesicles/pathology , Seminal Vesicles/surgery , Tomography, X-Ray Computed , Ultrasonography, Interventional
7.
Theor Appl Genet ; 121(5): 829-43, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20490446

ABSTRACT

This study focuses on the expansion of Phaseolus vulgaris in Europe. The pathways of distribution of beans into and across Europe were very complex, with several introductions from the New World that were combined with direct exchanges between European and other Mediterranean countries. We have analyzed here six chloroplast microsatellite (cpSSR) loci and two unlinked nuclear loci (for phaseolin types and Pv-shatterproof1). We have assessed the genetic structure and level of diversity of a large collection of European landraces of P. vulgaris (307) in comparison to 94 genotypes from the Americas that are representative of the Andean and Mesoamerican gene pools. First, we show that most of the European common bean landraces (67%) are of Andean origin, and that there are no strong differences across European regions for the proportions of the Andean and Mesoamerican gene pools. Moreover, cytoplasmic diversity is evenly distributed across European regions. Secondly, the cytoplasmic bottleneck that was due to the introduction of P. vulgaris into the Old World was very weak or nearly absent. This is in contrast to evidence from nuclear analyses that have suggested a bottleneck of greater intensity. Finally, we estimate that a relatively high proportion of the European bean germplasm (about 44%) was derived from hybridization between the Andean and Mesoamerican gene pools. Moreover, although hybrids are present everywhere in Europe, they show an uneven distribution, with high frequencies in central Europe, and low frequencies in Spain and Italy. On the basis of these data, we suggest that the entire European continent and not only some of the countries therein can be regarded as a secondary diversification center for P. vulgaris. Finally, we outline the relevance of these inter-gene pool hybrids for plant breeding.


Subject(s)
Gene Pool , Phaseolus/genetics , Americas , Chloroplasts/genetics , Europe , Genetic Variation , Genotype , Geography , Hybridization, Genetic , Microsatellite Repeats/genetics , Organ Size , Plant Proteins/metabolism , Principal Component Analysis , Seeds/anatomy & histology , Seeds/genetics
8.
Plant Biol (Stuttg) ; 11(4): 598-612, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19538398

ABSTRACT

Chloroplast microsatellites (cpSSRs) provide a powerful tool to study the genetic variation and evolution of plants. We have investigated the usefulness of 39 primer pairs tagging cpSSR loci on a set of eight different genera of Leguminosae (Papilionoideae subfamily) and five species belonging to the genus Phaseolus. Thirty-six 'universal' primer pairs were retrieved from the literature, one was re-designed and a further two were designed de novo. The cpSSR loci analysed were highly polymorphic across the individuals examined. Twenty-seven primer pairs were polymorphic in the overall sample, 18 within Phaseolus, and 16 in both P. vulgaris and P. coccineus. Analysis of the plastome sequences of four Leguminosae species (obtained from GenBank) showed that in the loci targeted by universal primer pairs: (i) the originally tagged cpSSRs can be lost; (ii) other cpSSRs can be present; and (iii) polymorphism arises not only from differences in the numbers of cpSSR repeats, but often from other insertion/deletion events. Multilocus linkage disequilibrium analysis suggests that homoplasy is not a major problem in our dataset, and principal component analysis indicates intelligible relationships among the species considered. Our study demonstrates that this set of chloroplast markers provides a useful tool to study the diversity and the evolution of several legumes, and particularly P. vulgaris and P. coccineus.


Subject(s)
Chloroplasts/genetics , Fabaceae/genetics , Microsatellite Repeats/genetics , Phaseolus/genetics , Base Sequence , Fabaceae/classification , Genetic Variation/genetics , Molecular Sequence Data , Phaseolus/classification
9.
Neurosci Lett ; 452(2): 214-7, 2009 Mar 13.
Article in English | MEDLINE | ID: mdl-19383442

ABSTRACT

Cerebrospinal fluid (CSF) is a promising source of biomarkers in clinically isolated syndrome (CIS), which frequently presents as a first episode of multiple sclerosis (MS). Using the two-dimensional difference in gel electrophoresis (2-D DIGE), we compared CSF samples from patients with CIS that remained CIS (CIS-CIS, n=8) over a follow-up time of 2 years and from patients with CIS that developed definite MS of the relapsing-remitting subtype (CIS-RRMS, n=8) over the same period. Protein spots that showed significant differences between patients and controls were selected for further analysis by MALDI-TOF mass spectrometry. For validation of identified spots ELISA experiments were performed. We identified one protein that was upregulated in CIS-RRMS (serin peptidase inhibitor) and eight proteins (alpha-1-B-glycoprotein, Fetuin-A, apolipoprotein A4, haptoglobin, human Zinc-alpha-2-glycoprotein (ZAG), Retinol-binding protein, superoxid dismutase 1, transferrin) that were down-regulated in CIS-RRMS vs. CIS-CIS. For Fetuin-A, our findings could be confirmed by ELISA. The pathophysiological role as well as clinical relevance of these candidate proteins in CIS remains to be further clarified by future studies.


Subject(s)
Demyelinating Diseases/cerebrospinal fluid , Demyelinating Diseases/diagnosis , Multiple Sclerosis/cerebrospinal fluid , Multiple Sclerosis/diagnosis , Proteome/analysis , Proteomics/methods , Adolescent , Adult , Biomarkers/analysis , Biomarkers/cerebrospinal fluid , Cerebrospinal Fluid Proteins/analysis , Cerebrospinal Fluid Proteins/metabolism , Disease Progression , Electrophoresis, Gel, Two-Dimensional , Enzyme-Linked Immunosorbent Assay , Female , Follow-Up Studies , Humans , Male , Middle Aged , Multiple Sclerosis, Relapsing-Remitting/cerebrospinal fluid , Multiple Sclerosis, Relapsing-Remitting/diagnosis , Proteinase Inhibitory Proteins, Secretory/analysis , Proteinase Inhibitory Proteins, Secretory/cerebrospinal fluid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Young Adult
10.
Mol Ecol ; 18(4): 603-21, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19215582

ABSTRACT

Landraces are domesticated local plant varieties that did not experience a deliberate and intensive selection during a formal breeding programme. In Europe, maize landraces are still cultivated, particularly in marginal areas where traditional farming is often practiced. Here, we have studied the evolution of flint maize landraces from central Italy over 50 years of on-farm cultivation, when dent hybrid varieties were introduced and their use was widespread. We have compared an 'old' collection, obtained during the 1950s, before the introduction of hybrids, and a recent collection of maize landraces. For comparison, a sample of maize landraces from north Italy, and of improved germplasm, including hybrids and inbred lines were also used. A total of 296 genotypes were analysed using 21 microsatellites. Our results show that the maize landraces collected in the last 5-10 years have evolved directly from the flint landrace gene pool cultivated in central Italy before the introduction of modern hybrids. The population structure, diversity and linkage disequilibrium analyses indicate a significant amount of introgression from hybrid varieties into the recent landrace populations. No evidence of genetic erosion of the maize landraces was seen, suggesting that in situ conservation of landraces is an efficient strategy for preserving genetic diversity. Finally, the level of introgression detected was very variable among recent landraces, with most of them showing a low level of introgression; this suggests that coexistence between different types of agriculture is possible, with the adoption of correct practices that are aimed at avoiding introgression from undesired genetic sources.


Subject(s)
Chimera/genetics , Crops, Agricultural/genetics , Evolution, Molecular , Genetic Variation , Zea mays/genetics , DNA, Plant/genetics , Gene Flow , Genetics, Population , Genome, Plant , Genotype , Geography , Italy , Linkage Disequilibrium , Microsatellite Repeats , Models, Statistical , Selection, Genetic
11.
Curr Genet ; 51(6): 377-92, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17426975

ABSTRACT

The main aim of this study was to test the patterns of sequence divergence and haplotype structure at the MAT locus of Pyrenophora teres, the causal agent of barley 'net blotch' disease. P. teres is a heterothallic ascomycete that co-occurs in two symptomatological forms, the net form (NF) and the spot form (SF). The mating-type genes MAT1-1-1 and MAT1-2-1 were sequenced from 22 NF isolates (12 MAT1-1-1 and 10 MAT1-2-1 sequences) and 17 SF isolates (10 MAT1-1-1 and seven MAT1-2-1 sequences) collected from Sardinian barley landrace populations and worldwide. On the basis of a parsimony network analysis, the two forms of P. teres are phylogenetically separated. More than 85% of the total nucleotide variation was found between formae speciales. The two forms do not share any polymorphisms. Six diagnostic nucleotide polymorphisms were found in the MAT1-1-1 intron (1) and in the MAT1-1-1 (3) and MAT1-2-1 (2) exons. Three diagnostic non-synonymous mutations were found, one in MAT1-1-1 and two in MAT1-2-1. For comparison with P. teres sequence data, the mating-type genes from Pyrenophora graminea were also isolated and sequenced. Divergence between P. graminea and P. teres is of a similar magnitude to that between NF and SF of P. teres. The MAT genes of P. graminea were closer to those of SF than to NF, with the MAT1-2-1 SF peptide not different from the MAT1-2-1 peptide of P. graminea. Overall, these data suggest long genetic isolation between the two forms of P. teres and that hybridization is rare or absent under field conditions, with each form having some particular niche specialization. This indicates that research on resistance to P. teres should consider the two forms separately, as different species.


Subject(s)
Ascomycota/classification , Evolution, Molecular , Genes, Mating Type, Fungal , Hordeum/microbiology , Phylogeny , Plant Diseases/microbiology , Ascomycota/genetics , Base Sequence , Chromosomes, Plant , DNA, Fungal , Genetic Variation , Polymorphism, Restriction Fragment Length
12.
Phys Rev Lett ; 90(16): 167202, 2003 Apr 25.
Article in English | MEDLINE | ID: mdl-12732000

ABSTRACT

We report the results of measurements of the dc susceptibility and the 23Na-NMR response of Na2V3O7, a recently synthesized, nonmetallic low dimensional spin system. Our results indicate that, upon reducing the temperature to below 100 K, the V4+ moments are gradually quenched, leaving only one moment out of nine active. The NMR data reveal a phase transition at very low temperatures. With decreasing applied field H, the critical temperature shifts towards T=0 K, suggesting that Na2V3O7 may be regarded as an insulator reaching a quantum critical point at H=0.

13.
Theor Appl Genet ; 106(5): 947-59, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12647071

ABSTRACT

Monoconidial cultures of Pyrenophora teres, the causal agent of barley net blotch, were isolated from leaves collected from six populations of the barley landrace "S'orgiu sardu" growing in five agro-ecological areas of Sardinia, Italy, and genotyped using AFLPs. The 150 isolates were from lesions of either the "net form" (P. teres f. sp. teres) or the "spot form" (P. teres f. sp. maculata) of the disease. Of 121 AFLP markers, 42%, were polymorphic. Cluster analysis resolved the isolates into two strongly divergent groups (F(ST) = 0.79), corresponding to the net (45% of the isolates) and the spot (55% of the isolates) forms (designated the NFR and SFR groups, respectively). The absence of intermediate genotypes and the low number of shared markers between the two groups indicated that hybridization between the two formae is rare or absent under the field condition of Sardinia. Five of the barley populations hosted both forms but in different proportions. The SFR populations were similar in overall polymorphism to the NFR populations. However, compared to the SFR form, the NFR occurred in all fields sampled and showed a higher population divergence (F(ST) = 0.43 versus F(ST) = 0.09 with all isolates; F(ST) = 0.37 versus F(ST) = 0.06 with clone corrected samples) probably due to a lower migration rate. AFLP fingerprints resolved 117 distinct genotypes among the 150 isolates sampled (78%), 87% in SFR and 68% in NFR isolates. Although the absolute numbers may be a function of the number of AFLP markers assayed, the relative difference suggests that clonality is more prevalent among the NFR isolates (with 11 of 46 haplotypes observed more than once), compared with SFR isolates (7 of 71 haplotypes). Both digenic and multilocus linkage disequilibrium analyses suggested that sexual reproduction occurs at significant levels within the NFR and SFR populations, and that the relative contribution of sexual and asexual reproduction varies among different environments.


Subject(s)
Fungi/genetics , Genetics, Population , Hordeum/microbiology , Genetic Variation , Linkage Disequilibrium , Phylogeny
14.
Prenat Diagn ; 21(12): 1049-52, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11746162

ABSTRACT

OBJECTIVES: Different aberrations in one chromosome 18 were prenatally detected during each of three different pregnancies of a healthy woman. Routine cytogenetic analysis revealed a morphologically altered maternal chromosome 18 as well. The purpose of the current study was to characterize these cytogenetic changes in detail and thus to clarify the reason for the recurrent appearance of morphologically altered chromosomes 18 in this family. METHODS: As GTG banding did not allow resolution of the kind of aberrations present in these four cases, the following molecular cytogenetic approaches were used: microdissection combined with reverse painting and multicolour banding (MCB) analysis using a chromosome 18 specific probe set. RESULTS: Molecular cytogenetic approaches revealed that fetus 1 had a derivative chromosome del(18)(q11.2q12.2), fetus 2 and the mother had the identical derivative chromosomes ins(18)(pterp11.32::q12.2q11.2::p11.32q11.2::q12.3qter) and fetus 3 had a dup(11.2q12.2). CONCLUSION: Partial monosomy in fetus 1 and partial trisomy in fetus 3 can be explained by crossing over events during maternal meiosis.


Subject(s)
Chromosomes, Human, Pair 18 , Cytogenetic Analysis , Mutagenesis, Insertional , Prenatal Diagnosis , Adult , Amniotic Fluid/cytology , Chromosome Banding , Female , Humans , In Situ Hybridization, Fluorescence , Pregnancy
15.
J Mol Biol ; 310(4): 801-16, 2001 Jul 20.
Article in English | MEDLINE | ID: mdl-11453689

ABSTRACT

We have measured the dependencies of both the dissociation rate of specifically bound EcoRI endonuclease and the ratio of non-specific and specific association constants on water activity, salt concentration, and pH in order to distinguish the contributions of these solution components to specific and non-specific binding. For proteins such as EcoRI that locate their specific recognition site efficiently by diffusing along non-specific DNA, the specific site dissociation rate can be separated into two steps: an equilibrium between non-specific and specific binding of the enzyme to DNA, and the dissociation of non-specifically bound protein. We demonstrated previously that the osmotic dependence of the dissociation rate is dominated by the equilibrium between specific and non-specific binding that is independent of the osmolyte nature. The remaining osmotic sensitivity linked to the dissociation of non-specifically bound protein depends significantly on the particular osmolyte used, indicating a change in solute-accessible surface area. In contrast, the dissociation of non-specifically bound enzyme accounts for almost all the pH and salt-dependencies. We observed virtually no pH-dependence of the equilibrium between specific and non-specific binding measured by the competition assay. The observed weak salt-sensitivity of the ratio of specific and non-specific association constants is consistent with an osmotic, rather than electrostatic, action. The seeming lack of a dependence on viscosity suggests the rate-limiting step in dissociation of non-specifically bound protein is a discrete conformational change rather than a general diffusion of the protein away from the DNA.


Subject(s)
DNA/metabolism , Deoxyribonuclease EcoRI/chemistry , Deoxyribonuclease EcoRI/metabolism , Escherichia coli/enzymology , Water/metabolism , Binding Sites , DNA/chemistry , DNA/genetics , Enzyme Stability , Hydrogen-Ion Concentration , Kinetics , Osmolar Concentration , Static Electricity , Substrate Specificity , Thermodynamics , Viscosity
16.
Cell Mol Life Sci ; 57(7): 1018-32, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10961342

ABSTRACT

Water often acts as a critical reactant in cellular reactions. Its role can be detected by modulating water activity with osmotic agents. We describe the principles behind this 'osmotic stress' strategy, and survey the ubiquity of water effects on molecular structures that have aqueous, solute-excluding regions. These effects are seen with single-functioning molecules such as membrane channels and solution enzymes, as well as in the molecular assembly of actin, the organization of DNA and the specificity of protein/DNA interactions.


Subject(s)
Intracellular Fluid/chemistry , Intracellular Fluid/metabolism , Osmotic Pressure , Water/metabolism , Actins/metabolism , DNA/chemistry , DNA/metabolism , Hemoglobins/metabolism , Hexokinase/metabolism , Humans , Ion Channels/metabolism , Protein Binding , Solutions
17.
Proc Natl Acad Sci U S A ; 97(8): 3987-92, 2000 Apr 11.
Article in English | MEDLINE | ID: mdl-10760270

ABSTRACT

There has been much confusion recently about the relative merits of different approaches, osmotic stress, preferential interaction, and crowding, to describe the indirect effect of solutes on macromolecular conformations and reactions. To strengthen all interpretations of measurements and to forestall further unnecessary conceptual or linguistic confusion, we show here how the different perspectives all can be reconciled. Our approach is through the Gibbs-Duhem relation, the universal constraint on the number of ways it is possible to change the temperature, pressure, and chemical potentials of the several components in any thermodynamically defined system. From this general Gibbs-Duhem equation, it is possible to see the equivalence of the different perspectives and even to show the precise identity of the more specialized equations that the different approaches use.


Subject(s)
Osmotic Pressure , Water/chemistry , Binding Sites , Thermodynamics
19.
J Biomol Struct Dyn ; 17(1): 19-31, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10496418

ABSTRACT

We recently showed that a nonspecific complex of the restriction nuclease EcoRI with poly (dI-dC) sequesters significantly more water at the protein-DNA interface than the complex with the specific recognition sequence. The nonspecific complex seems to retain almost a full hydration layer at the interface. We now find that at low osmotic pressures a complex of the restriction nuclease EcoRI with a DNA sequence that differs by only one base pair from the recognition site (a 'star' sequence) sequesters about 70 waters more than the specific one, a value virtually indistinguishable from nonspecific DNA. Unlike complexes with oligo (dI-dC) or with a sequence that differs by two base pairs from the recognition sequence, however, much of the water in the 'star' sequence complex is removed at high osmotic pressures. The energy of removing this water can be calculated simply from the osmotic pressure work done on the complex. The ability to measure not only the changes in water sequestered by DNA-protein complexes for different sequences, but also the work necessary to remove this water is a potentially powerful new tool for coupling inferred structural changes and thermodynamics.


Subject(s)
Deoxyribonuclease EcoRI/chemistry , Polydeoxyribonucleotides/chemistry , Water/chemistry , Betaine , Water-Electrolyte Balance
20.
Biochemistry ; 38(22): 7243-52, 1999 Jun 01.
Article in English | MEDLINE | ID: mdl-10353836

ABSTRACT

Previous electric birefringence experiments have shown that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II correlates with the ability of minifilaments to cycle between flexible and stiff conformations. The cooperative transition between conformations was shown to depend on Mg2+ concentration, on ATP binding, and on the state of phosphorylation of three serines in the C-terminal end of the heavy chains. Since the junction between the heavy meromyosin (HMM) and light meromyosin (LMM) regions is expected to disrupt the alpha-helical coiled-coil structure of the rod, this region was anticipated to be the flexible site. We have now cloned and expressed the wild-type rod (residues 849-1509 of the full-length heavy chain) and rods mutated within the junction in order to test this. The sedimentation and electric birefringence properties of minifilaments formed by rods and by native myosin II are strikingly similar. In particular, the Mg2+-dependent flexible-to-stiff transitions of native myosin II and wild-type rod minifilaments are virtually superimposable. Mutations within the junction between the HMM and LMM regions of the rod modulate the ability of Mg2+ to stabilize the stiff conformation. Less Mg2+ is required to induce minifilament stiffening if proline-1244 is replaced with alanine. Deleting the entire junction region (25 amino acids) results in a even greater decrease in the Mg2+ concentration necessary for the transition. The HMM-LMM junction does indeed seem to act as a Mg2+-dependent flexible hinge.


Subject(s)
Acanthamoeba/chemistry , Actin Cytoskeleton/chemistry , Myosin Subfragments/chemistry , Acanthamoeba/genetics , Acanthamoeba/ultrastructure , Actin Cytoskeleton/genetics , Actin Cytoskeleton/ultrastructure , Amino Acid Sequence , Animals , Birefringence , Magnesium/chemistry , Microscopy, Electron , Molecular Sequence Data , Myosin Subfragments/genetics , Myosin Subfragments/ultrastructure , Point Mutation , Protein Structure, Secondary , Structure-Activity Relationship , Ultracentrifugation
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