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PubMed1998

Expression of psbA genes produces prominent 5' psbA mRNA fragments in Synechococcus sp. PCC 7942.

Expression of the psbA genes, which in the cyanobacterium Synechococcus sp. PCC 7942 encode two different forms of the reaction centre D1 protein of photosystem II (D1:1 and D1:2), was studied under different light and temperature conditions. In addition to the mature 1200 nt psbA messages, three shorter mRNA fragments of 220, 320 and 900 nt were also found. All three mRNA fragments could be recognized by using different gene probes from the coding region of the psbAI gene, whereas the corresponding psbAII/III gene probes recognized only the 220 nt mRNA fragment. The 5' 320 nt mRNA fragment from the psbAI gene probably represents a degradation product, since the corresponding 3' 900 nt psbAI mRNA fragment was also detected. By contrast, the 5' 220 nt mRNA fragment of all psbA messages is suggested to be a truncated psbA transcript, since no corresponding 3' fragment was ever found. Inhibition of translation either by a protein synthesis inhibitor or by a shift of cells to lower temperature, increased the number of 1200 nt psbAII/III messages but the number of 5' 220 nt psbAII/III mRNA fragment increased even more dramatically. The first 66 bp after ATG, where the psbAI and psbAII/III genes mostly differ from each other, also appeared important in determining the amount of produced truncated psbA transcripts, as evidenced by the expression of different tac-psbA constructs in the presence of protein synthesis inhibitor. We suggest that both the psbAI and the psbAII/III genes have a latent intragenic termination site and truncated psbA transcripts are produced at high levels under stress conditions when transcription becomes uncoupled from translation. This is to prevent wasting metabolic energy in the production of unused transcripts.

Cyanobacteria↗
PubMed2006

Deconstructing fragment-based inhibitor discovery.

Fragment-based screens test multiple low-molecular weight molecules for binding to a target. Fragments often bind with low affinities but typically have better ligand efficiencies (DeltaG(bind)/heavy atom count) than traditional screening hits. This efficiency, combined with accompanying atomic-resolution structures, has made fragments popular starting points for drug discovery programs. Fragment-based design adopts a constructive strategy: affinity is enhanced either by cycles of functional-group addition or by joining two independent fragments together. The final inhibitor is expected to adopt the same geometry as the original fragment hit. Here we consider whether the inverse, deconstructive logic also applies--can one always parse a higher-affinity inhibitor into fragments that recapitulate the binding geometry of the larger molecule? Cocrystal structures of fragments deconstructed from a known beta-lactamase inhibitor suggest that this is not always the case.

Crystallography, X-Ray↗
PubMed1998

Red cell fragmentation (schistocytosis) after bone marrow transplantation.

Red cell fragmentation is often the earliest sign of thrombotic microangiopathy. Days +14, +28 and +42 blood films from 58 allograft and 32 autograft recipients were reviewed blind to determine the incidence and severity of schistocytosis (the number of fragmented red cells per 1000 red cells expressed as a percentage). Schistocytosis was graded as mild (<1%), moderate (1-1.9%) or severe (> or =2%). Schistocytes were seen in 99% of day 14 films (0.1-3.0%, median 0.4%), 97% of day 28 films (0.1-3.2%, median 0.4%), and 98% of day 42 films (0.1-4.3%, median 0.5%). Nine patients (10%) had severe fragmentation and 20 patients (22%) had moderate fragmentation at some time or the other. The difference in the extent of fragmentation between the days was not significant. Allogeneic BMT was associated with more extensive fragmentation than autologous transplantation on day 28 (P = 0.008) and day 42 (P = 0.02). Age, conditioning regimen and diagnosis had no influence. None of the patients followed-up for 6 months after transplant developed full-blown thrombotic microangiopathy. The occasional patient showing mild clinical and laboratory features of hemolysis responded well to adjustment of fluid balance and cyclosporine dose where applicable. Our data indicate that mild red cell fragmentation is a common morphologic finding after transplantation with no clinical significance in the absence of other clinical and laboratory findings suggestive of thrombotic microangiopathy, although patients with moderate or severe fragmentation should be monitored closely.

Adolescent↗
PubMed1997

Evidence that DNA fragmentation in apoptosis is initiated and propagated by single-strand breaks.

Apoptosis is characterised by the degradation of DNA into a specific pattern of high and low molecular weight fragments seen on agarose gels as a distribution of sizes between 50-300 kb and sometimes, but not always, a ladder of smaller oligonucleosomal fragments. Using a 2D pulsed field-conventional agarose gel electrophoresis technique, where the second dimension is run under either normal or denaturing conditions, we show that single-strand breaks are introduced into DNA at the initial stages of fragmentation. Using single-strand specific nuclease probes we further show that the complete fragmentation pattern, including release of small oligonucleosomal fragments can also be generated by a single-strand endonuclease. Three classes of sites where single-strand breaks accumulate were identified. The initial breaks produce a distribution of fragment sizes (50 kb to >1 Mb) similar to those generated by Topoisomerase II inhibitors suggesting that cleavage may commence at sites of attachment of DNA to the nuclear matrix. A second class of rare sites is also cut further reducing the size distribution of the fragments to 50-300 kb. Thirdly, single-strand breaks accumulate at the linker region between nucleosomes eventually causing double-strand scissions which release oligonucleosomes. These observations further define the properties of the endonuclease responsible for DNA fragmentation in apoptosis.

Journal Article↗
PubMed1967

Enzymic degradation of the Fc fragment of rabbit immunoglobulin IgG.

The digestion of the Fc fragment of rabbit immunoglobulin IgG by several proteolytic enzymes was investigated by using gel filtration and starch-gel electrophoresis in 8m-urea-formate as criteria of the extent of degradation. Though fragment Fc and mildly reduced fragment Fc proved resistant to tryptic hydrolysis, papain and pepsin cleaved the fragment at acidic pH values and appeared to give rise to a similar spectrum of products. A (limit) peptide comprising the C-terminal 113 residues of the heavy chain was isolated and identified from the pepsin-digest products of fragment Fc. The products of proteolytic digestion of fragment Fc were no longer able to inhibit passive cutaneous anaphylaxis by rabbit anti-(bovine serum albumin) or demonstrate reversed passive cutaneous anaphylaxis in the guinea pig. Nor were they able to inhibit the intestinal absorption of heterologous immunoglobulin IgG in the young mouse. These studies imply that the site or sites responsible for these biological properties of intact fragment Fc reside in the N-terminal 30-40% of the fragment.

Amino Acids↗
PubMed1968

Subfragments from the Fc fragment of human immunoglobulin G. Isolation and physicochemical charaterization.

A fragment termed fragment Fc' and a related fragment termed fragment pFc' produced by the actions of papain and pepsin respectively on human immunoglobulin G have been isolated and characterized. Amino acid analyses and experiments utilizing cyanogen bromide to cleave the methionyl bonds of the Fc' and pFc' fragments make it possible to locate both fragments within the known chain structure of the immunoglobulin G molecule. The pFc' fragment is probably a non-covalently linked dimer situated at the C-terminal end of the molecule, containing about 232 amino acid residues and having a molecular weight of 26000. The Fc' fragment is a similar dimer of about 182 residues extending from near residue 14 to near residue 105 (numbered from the C-terminal end) of the gamma-chain and has a molecular weight of 21000.

Amino Acids↗
PubMed1969

Conformation changes and dissociation of Fc fragments of rabbit immunoglobulin G as a function of pH.

1. The sedimentation coefficients of rabbit immunoglobulin G, four types of Fc fragments, univalent Fab and bivalent F(ab)(2) fragments were measured as a function of pH. 2. In conjunction with molecular-weight determinations by sedimentation equilibrium, and with the behaviour on gel filtration, this enabled the state of association of the Fc fragments to be followed. 3. The type possessing an interchain disulphide bond, 1Fc fragment, changed extensively in structure, but not in molecular weight. 4. There was good correlation between the readiness to dissociate and the chain length of the shorter Fc fragments that do not contain the interchain covalent bond. 5. The increasing resistance to dissociation as the fragments became shorter ran parallel with the ability to resist enzymic attack. 6. The site of the strong association between component chains of Fc fragment is located in the C-terminal half. 7. The gel-filtration behaviour of the Fc fragments clearly confirms that the process is governed by the Stokes radius rather than molecular weight. 8. The ultracentrifugal results were used to estimate the separations of the hydrodynamic subunits in intact immunoglobulin G, and as a basis for a schematic structure.

Animals↗
PubMed1992

Generation of forms of fragment E with differing thrombin-binding properties during digestion of fibrinogen by plasmin.

Two principal forms of fragment E are generated upon digestion of fibrinogen by plasmin, according to the concentration of enzyme used. At a high concentration of plasmin (above 10 micrograms/ml), a form lacking fibrinopeptide A (FpA) at the N-terminus of the A alpha-chain was generated. This form of fragment E caused a dose-dependent increase in thrombin clotting times but had no measurable inhibitory activity towards thrombin cleavage of D-phenylalanyl-L-pipecolyl-L-arginine p-nitroanilide. At a low concentration of plasmin (less than 1 microgram/ml), fragment E containing 35-40% of the original amount of FpA was present in the terminal digest. The FpA-containing form of fragment E inhibited thrombin cleavage of fibrinogen, inhibited amidolytic activity and bound to the enzyme with an affinity 3-fold tighter than fibrinogen itself (Kd 4.1 +/- 0.3 microM as opposed to 12.7 +/- 1.8 microM). During digestion of fibrinogen at low plasmin concentration, up to 65% of the FpA was cleaved just subsequent to the progressive release of B beta-(1-42)-peptide, and the Arg-16-Gly-17 bond of the A alpha-chain became relatively stable towards plasmin action when present in fragment E (and possibly fragment Y). It is proposed that both forms of fragment E can inhibit clotting by binding to the fibrin(ogen)-recognition site (anion-binding exosite) of thrombin. The FpA-containing form of fragment E can also inhibit binding that occurs distal to the P1 site and thereby interfere with amidolysis of the peptide substrate. Our finding of a lability of the Arg-16-Gly-17 bond in the early phase of digestion may provide an alternative explanation of the increased FpA concentrations observed during thrombolytic therapy.

Binding Sites↗
PubMed1994

Proteoglycans isolated from dissociative extracts of differently aged human articular cartilage: characterization of naturally occurring hyaluronan-binding fragments of aggrecan.

Proteoglycans extracted with 4 M guanidinium chloride from young (mean 20 years) or old (mean 79 years) macroscopically normal human articular cartilage were separated by density gradient centrifugation and Q-Sepharose chromatography and characterized by gradient gel SDS/PAGE and immunodetection before and after removal of glycosaminoglycan chains. The extracts contained two large populations of aggrecan, a population of small N-terminal aggrecan fragments, as well as decorin, biglycan and fibromodulin. The distribution of all these species in density gradient fractions has been determined. The large aggrecan populations comprised four different chondroitin sulphate-bearing core proteins while the population of smaller fragments comprised eight different components. The two smallest fragments (35 and 42 kDa), identified as the first globular domain of aggrecan (N-terminal) (G1) and containing no glycosaminoglycan, were detected only in extracts of old cartilage. A 55 and a 70 kDa fragment of G1 were present in both keratan sulphate-containing and non-keratan sulphate-containing forms. Four other fragments, each containing keratan sulphate epitopes, were identified and these contained either G1 epitopes (one 95 kDa species), or G1 and G2 epitopes (three species). These results have suggested that proteolytic processing at the N-terminus is more extensive than has previously been recognized and raises the possibility that more than one proteinase may be involved in aggrecan degradation in vivo. With the exception of the two smallest G1 fragments, the repertoire of proteoglycan fragments found in young and old human articular cartilage is essentially the same, although the relative abudnance of various species differed. The older tissue contains a larger proportion of C-terminally truncated aggrecan fragments and a significantly decreased content of decorin and biglycan.

Adolescent↗
PubMed1995

Part-word repetitions by persons who stutter: fragment types and their articulatory processes.

One of the core features of developmental stuttering is part-word repetition followed by fluent production (resolution) of the target word. The purpose of the study is to compare the spectro-temporal dimensions of fragments with the spectro-temporal dimensions of the resolution to define fragment types and develop articulatory interpretation of termination-restart cycles. From the acoustic recordings of six adult males who stutter, 142 stuttering events on words beginning with stops were isolated, excerpted, and digitized. The stuttering events occurred during either spontaneous speech or reading. The consecutive articulatory phases of fragments were classified and compared with the corresponding phases in resolutions. We found that (a) there are two basic types of fragments--those with vowels and those without, (b) the type of fragment produced is strongly influenced by the voicing status of the stop, (c) the fragments without vowels tend to have longer stop closure duration than the stops in the resolutions, (d) there are two subtypes in fragments with vowels--those with vowels shorter than and those with vowels longer than the vowels in the resolutions, (e) the shorter vowels differ spectro-temporally from the vowels in resolutions, and (f) the longer vowels differ temporally from the vowels in the resolutions. We discuss the articulatory implications of the acoustic data for each fragment type in the context of speech as an act designed to achieve contextually conditioned acoustic goals.

Adult↗
PubMed2000

The effect of sleep fragmentation on cognitive processing using computerized topographic brain mapping.

Topographic brain mapping of evoked potentials can be used to localize abnormalities of cortical function. We evaluated the effect of sleep fragmentation on brain function by measuring the visual P300 waveform using brain mapping. Eight normal subjects (Epworth Score +/- SD: 5 +/- 3) underwent tone-induced sleep fragmentation and undisturbed study nights in a randomized cross-over design. Study nights were followed by topographic brain mapping using a visual information processing test and concurrent event-related potentials. Experimental sleep fragmentation did not significantly increase objective daytime sleepiness or lower cognitive performance on a battery of cognitive function tests (all P > or = 0.1). There were no significant topographical delays in P300 latencies with sleep fragmentation (all P > 0.15). However, at sites Fz, F4, T3, C3, Cz and C4 the P300 amplitudes were reduced significantly after sleep fragmentation (all P < 0.05). A reduction in P300 amplitude has previously been interpreted as a decrease in attention. These reductions in P300 amplitudes with sleep fragmentation in frontal, central and temporal brain areas suggest that sleep fragmentation may cause a broad decrease in attention. Sleep fragmentation did not delay P300 latencies in any brain area, and so does not explain the delay in P300 latencies reported in sleep apnoeics.

Adult↗
PubMed1999

Genetic variation within a fragmented population of swietenia humilis zucc

With large tracts of once continuous forest now modified by human use to leave reduced and highly fragmented stands of trees, the determination of the genetic consequences of forest fragmentation is a priority for ascertaining the conservation value of resultant stands, and in formulating sustainable management strategies. The levels and distribution of genetic diversity over 10 microsatellite loci were investigated within a fragmented population of the neotropical tree Swietenia humilis Zucc. High levels of genetic variation, typical of a highly outcrossing species, were found in all fragments at all loci (mean HE = 0.548). The majority of the variation was within rather than between fragments (RST = 0.032), giving high indirect estimates of gene flow (Nm = 8.9), probably reflecting the genetic structure of the trees present under more continuous forest. A high proportion of loci also showed significant departures from Hardy-Weinberg equilibrium with associated significant levels of FIS. The initial effects of fragmentation were, however, seen in the fragments through the loss of low-frequency alleles present in the continuous 'control' stand. The percentage of this allelic loss increased with a decrease in fragment size.

Journal Article↗
PubMed2002

Insights into recently fragmented vole populations from combined genetic and demographic data.

We combined demographic and genetic data to evaluate the effects of habitat fragmentation on the population structure of the California red-backed vole (Clethrionomys californicus). We analysed variation in the mitochondrial DNA (mtDNA) control region and five nuclear microsatellite loci in small samples collected from two forest fragments and an unfragmented control site in 1990-91. We intensively sampled the same forest fragments and two different control sites in 1998 and 1999. Vole abundances fluctuated greatly at sizes below 50 individuals per fragment. Fragment populations had significantly lower mtDNA allelic diversity than controls, but not nuclear heterozygosity or numbers of alleles. The use of only trapping and/or mtDNA marker data would imply that fragment populations are at least partially isolated and vulnerable to inbreeding depression. In contrast, the abundance estimates combined with microsatellite data show that small fragment populations must be linked to nearby forests by high rates of migration. These results provide evidence for the usefulness of combining genetic and demographic data to understand nonequilibrium population structure in recently fragmented habitats.

Animals↗
PubMed2003

Artificial neural networks in predicting optimum renal stone fragmentation by extracorporeal shock wave lithotripsy: a preliminary study.

OBJECTIVE: To assess the ability of artificial neural networks (ANNs) to predict optimum renal stone fragmentation in patients being managed by extracorporeal shock wave lithotripsy (ESWL). PATIENTS AND METHODS: The study included 82 patients with renal stones who were being treated by ESWL. Data (input and output values) from 60 patients in whom there was optimum fragmentation of stones by ESWL were used for training the ANN. These data mainly included the 24-h urinary variables, the radiological features of the stone disease and the ESWL settings used. The predictability of the trained ANN was tested on 22 subsequent patients, by supplying the input variables of the 22 patients into the trained ANN and recording the output values (predicted values). After subjecting these patients to ESWL, the actual results (observed values) were recorded. The predicted and the observed values were then compared. RESULTS: In the 22 patients in whom predictability was tested, the trained ANN predicted optimum fragmentation at < or = 13 000 shocks/stone (as per study protocol) in 17 and optimum fragmentation at> 13 000 shocks/stone in the other five. In the 17 patients (test set) where the trained ANN had predicted optimum fragmentation at < or = 13 000 shocks/stone, the optimum fragmentation was at that value, although the predicted and observed values were not identical. The overall correlation between the predicted and the observed values was 75.5% (correlation coefficient 0.7547) in these 17 patients. Of the other five patients, none had optimum fragmentation at < 13 000 shocks/stone, as predicted by the trained ANN, giving complete accuracy for this factor. CONCLUSION: This was a pilot study, i.e. an initial attempt to use an ANN in this regard, and although there were few patients, such that it is not possible to make final recommendations, the overall predictability was approximately 75%. An encouraging outcome of the study was that the trained ANN identified patients unlikely to benefit from ESWL. Using a larger dataset and identifying more significant variables, while eliminating inputs with a negative effect, the efficiency and utility of this ANN can probably be enhanced and in future it might be possible to predict stone fragmentation with reasonable accuracy.

Humans↗
PubMedDate not supplied

A biomechanical comparison of equine third metacarpal condylar bone fragment compression and screw pushout strength between headless tapered variable pitch and AO cortical bone screws.

OBJECTIVES: To compare bone fragment compression and the mechanical pushout strength and stiffness of 6.5-mm Acutrak Plus (AP) and 4.5-mm AO cortical (AO) bone screws after stabilization of a simulated equine third metacarpal (MC3) bone complete lateral condylar fracture. STUDY DESIGN: In vitro biomechanical paired study of screw insertion variables, bone fragment compression, and screw pushout tests using a bone screw stabilized simulated lateral condylar fracture model. SAMPLE POPULATION: Six pairs of cadaveric equine MC3s. METHODS: Metacarpi were placed in a fixture and centered on a biaxial load cell in a materials testing system to measure torque, compressive force, and time for drilling, tapping, and screw insertion. Fragment compression was measured with a pressure-sensing device placed between the simulated fracture fragments during screw insertion for fragment stabilization. Subsequently, screws were pushed out of the stabilized bone fragments in a single cycle to failure. A paired t test was used to assess differences between site preparation, screw insertion, fragment compression, and screw pushout variables, with significance set at P <.05. RESULTS: Measured drilling variables were comparable for AO and AP specimens. However, the AP tap had significantly greater insertion torque and force. Mean maximum screw insertion torque was significantly greater for AO screws. For fragment compression, AP screws generated 65% and 44% of the compressive pressure and force, respectively, of AO screws. AP screws tended to have higher overall pushout strength. Pushout stiffness was similar between both screw types. CONCLUSION: The 6.5-mm tapered AP screw generated less interfragmentary compressive pressure and force but had similar pushout stiffness. Evaluation of failure patterns demonstrated that AP screws had greater pushout strength compared with 4.5-mm AO screws for fixation of a simulated complete lateral condylar fracture. CLINICAL RELEVANCE: The 6.5-mm tapered AP screw should provide ample holding strength but would provide less interfragmentary compression than 4.5-mm AO screws for repair of complete lateral condylar fractures in horses.

Animals↗
PubMed2005

Simulating the thermal behavior and fragmentation mechanisms of exohedral and substitutional silicon-doped C60.

Structures, thermal behavior, and fragmentation mechanisms of exohedral and substitutional silicon-doped C(60) containing 1-12 Si atoms are investigated by extensive molecular-dynamics simulations. A nonorthogonal tight-binding model is used to mimic the interatomic interactions in the doped fullerenes. Beginning from the minimum-energy structures, the temperature of the doped fullerenes is slowly increased until fragmentation takes place. A correlation can be established between the exohedral and substitutional structures and the corresponding fragmentation mechanisms and fragmentation temperatures. Exohedral C(60)Si(m) fullerenes fragment into two homonuclear pieces, the Si(m) cluster and the C(60) fullerene that remains intact. In contrast, the substitutional C(60-m)Si(m) heterofullerenes undergo structural transformations, including the partial unraveling of the cage, prior to fragmentation. Then, ejection of atoms or small molecules takes place from the distorted structures. The slow heating rate used, combined with long simulation runs, allows us to determine the fragmentation temperature of exohedral and substitutional Si-doped fullerenes as a function of the number of silicon atoms. Substitutional Si-doped fullerenes exhibit much higher fragmentation temperatures (1000-1500 K higher) than the exohedral fullerenes. This can be understood from the different bonding of the Si atoms in both structures.

Journal Article↗
PubMed2001

Caspase 3-cleaved N-terminal fragments of wild-type and mutant huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent proteolysis.

The Huntington's disease (HD) mutation is a polyglutamine expansion in the N-terminal region of huntingtin (N-htt). How neurons die in HD is unclear. Mutant N-htt aggregates in neurons in the HD brain; expression of mutant N-htt in vitro causes cell death. Other in vitro studies show that proteolysis by caspase 3 could be important in regulating mutant N-htt function, but there has been no direct evidence for caspase 3-cleaved N-htt fragments in brain. Here, we show that N-htt fragments consistent with the size produced by caspase 3 cleavage in vitro are resident in the cortex, striatum, and cerebellum of normal and adult onset HD brain and are similar in size to the fragments seen after exogenous expression of human huntingtin in mouse clonal striatal neurons. HD brain extracts treated with active caspase 3 had increased levels of N-htt fragments. Compared with the full-length huntingtin, the caspase 3-cleaved N-htt fragments, especially the mutant fragment, preferentially segregated with the membrane fraction. Partial proteolysis of the human caspase 3-cleaved N-htt fragment by calpain occurred in vitro and resulted in smaller N-terminal products; products of similar size appeared when mouse brain protein extracts were treated with calpain. Results support the idea that sequential proteolysis by caspase 3 and calpain may regulate huntingtin function at membranes and produce N-terminal mutant fragments that aggregate and cause cellular dysfunction in HD.

Adult↗
PubMed1975

Uptake of homologous single-stranded fragments by superhelical DNA: a possible mechanism for initiation of genetic recombination.

Superhelical [3-H]DNA (replicative form I, RFI) of bacteriophage phiX174 slowly but spontaneously took up 32-P-labeled homologous single-stranded fragments at 4 degrees. Uptake was accelerated by heating to 75 degrees. RFI did not take up single-stranded fragments derived from DNA of Escherichia coli or from separated strands of phage lambda. Uptake was inhibited by low concentrations of ethidium bromide. Relaxed circular phiX174 DNA did not take up homologous fragments. Per molecule of RFI, the complexes contained as much as 90 nucleotide residues of homologous fragment. The 32-P-lebeled fragments were largely resistant to digestion by exonuclease I, and were not displaced by heating complexes at 60 degrees for 1 min in 16 mM or 100 mM NaCl. Under comparable conditions of temperature and salt all of the fragments were displaced from complexes in which at least one phosphodiester bond was cleaved by pancreatic DNase, but a significant fraction of the fragments was retained in complexes that were relaxed by digestion with S1 nuclease. These observations are interpreted to mean that S1 nuclease digested the plus (viral) strand of the recipient RF at the site of uptake in some instances. Transfection of E. coli by heterozygous complexes produced recombinant progeny, thereby showing that genetic information can be transferred from the fragment of plus strand to progeny plus strands. We propose that both uptake of a third strand by superhelical DNA and the action of nucleases on the resulting complex may simulate early steps in genetic recombination.

Centrifugation, Density Gradient↗