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Scaling behavior of fragment shapes.

We present an experimental and theoretical study of the shape of fragments generated by explosive and impact loading of closed shells. Based on high speed imaging, we have determined the fragmentation mechanism of shells. Experiments have shown that the fragments vary from completely isotropic to highly anisotropic elongated shapes, depending on the microscopic cracking mechanism of the shell. Anisotropic fragments proved to have a self-affine character described by a scaling exponent. The distribution of fragment shapes exhibits a power-law decay. The robustness of the scaling laws is illustrated by a stochastic hierarchical model of fragmentation. Our results provide a possible improvement of the representation of fragment shapes in models of space debris.

Journal Article↗

Fragment complementation of calbindin D28k.

Calbindin D28k is a highly conserved Ca2+-binding protein abundant in brain and sensory neurons. The 261-residue protein contains six EF-hands packed into one globular domain. In this study, we have reconstituted calbindin D28k from two fragments containing three EF-hands each (residues 1-132 and 133-261, respectively), and from other combinations of small and large fragments. Complex formation is studied by ion-exchange and size-exclusion chromatography, electrophoresis, surface plasmon resonance, as well as circular dichroism (CD), fluorescence, and NMR spectroscopy. Similar chromatographic behavior to the native protein is observed for reconstituted complexes formed by mixing different sets of complementary fragments, produced by introducing a cut between EF-hands 1, 2, 3, or 4. The C-terminal half (residues 133-261) appears to have a lower intrinsic stability compared to the N-terminal half (residues 1-132). In the presence of Ca2+, NMR spectroscopy reveals a high degree of structural similarity between the intact protein and the protein reconstituted from the 1-132 and 133-261 fragments. The affinity between these two fragments is 2 x 10(7) M(-1), with association and dissociation rate constants of 2.7 x 10(4) M(-1) s(-1) and 1.4 x 10(-3) s(-1), respectively. The complex formed in the presence of Ca2+ is remarkably stable towards unfolding by urea and heat. Both the complex and intact protein display cold and heat denaturation, although residual alpha-helical structure is seen in the urea denatured state at high temperature. In the absence of Ca2+, the fragments do not recombine to yield a complex resembling the intact apo protein. Thus, calbindin D28k is an example of a protein that can only be reconstituted in the presence of bound ligand. The alpha-helical CD signal is increased by 26% after addition of Ca2+ to each half of the protein. This suggests that Ca2+-induced folding of the fragments is important for successful reconstitution of calbindin D28k.

Amino Acid Sequence↗

Detection of a mouse OGG1 fragment during caspase-dependent apoptosis: oxidative DNA damage and apoptosis.

We investigated the expression of mouse 8-oxoguanine DNA glycosylase 1 (mOGG1) in mouse non-parenchymal hepatocytes (NCTC) during etoposide- or mitomycin C (MMC)-induced apoptosis. We observed mOGG1 fragmentation in apoptotic cells. The apoptosis accompanying the fragmentation of mOGG1 was caspase-dependent. The mOGG1 fragment existed in both the cytoplasm and nucleus of the etoposide-treated NCTC, indicating that the mOGG1 fragment could be transferred into the nucleus. In addition, 8-hydroxyguanine (8-OH-Gua, 7,8-dihydro-8-oxoguanine) accumulated in the DNA of NCTC treated with etoposide, suggesting that the mOGG1 fragment might not function as a normal repair enzyme in etoposide-treated NCTC. Although we have not clarified in detail the mechanism and the significance of the mOGG1 fragmentation, further study of the fragmentation of DNA repair enzymes might provide insights into the relationship between oxidative DNA damage and apoptosis.

Animals↗

When log-dwellers meet loggers: impacts of forest fragmentation on two endemic log-dwelling beetles in southeastern Australia.

Anthropogenic activities continue to cause massive fragmentation and reduction of forest area worldwide. With fragmentation and reduction of habitat recognized as the greatest threats to biodiversity, the implementation of improved, informed and conservation-based forestry practices is essential, and requires a greater understanding of the responses of different organisms to forest fragmentation. While genetic techniques can add invaluable insights to fragmentation studies they have rarely been employed, particularly for multiple species. In the present study, we combined genetic information, obtained from allozyme loci and anonymous single copy nuclear DNA markers, with ecological data to investigate the impacts of forest fragmentation on two log-dwelling beetles with different life histories, in an 'islands of bush in a sea of pine' model, at Tumut in New South Wales, Australia. Both the relatively mobile (i.e. has high dispersal ability and/or broad habitat range) Adelium calosomoides and the less mobile Apasis puncticeps showed reduced mobility and gene flow in fragmented compared to continuous forest: there was significantly greater isolation by distance and stronger local structure revealed by spatial autocorrelation in fragmented forest. Analysis of patch and species characteristics revealed that genetic and demographic structure may be influenced by log degradation class for both species, and number of potential dispersal barriers, distance from continuous forest and desiccation intolerance/moisture preference for Ap. puncticeps. Thus the pine plantation matrix poses a barrier or filter for gene flow and mobility in both beetle species.

Animals↗

Binding properties of human anti-DNA antibodies to cloned human DNA fragments.

The DNA-anti-DNA antibody immune complexes were isolated from plasma of systemic lupus erythematosus (SLE) patients and DNA fragments separated from immune complexes were subjected to molecular cloning. The resulting recombinant DNA clones showed a molecular size of 37-79 base pairs, a high guanine and cytosine content, high frequencies of CpG dinucleotides, and palindromic sequences, and also clusters of G + C- and A + T-rich segments. These clones hybridized randomly to total human DNA. The reactivity of dsDNA antibodies, both monoclonal and polyclonal, from SLE was examined with a cloned SLE antigen DNA. A competitive inhibition assay showed that human monoclonal antibodies had at least one magnitude higher affinity to the cloned DNA than to the native DNA fragments. In order to characterize the factors that were recognized by antibodies, human G + C-rich and also A + T-rich 100 bp DNA fragments were cloned, and their base sequences determined. The antibody showed a higher affinity to the G + C-rich DNA fragment (71% G + C) than to the A + T-rich DNA fragment (46% G + C). When cytosines in CpG doublets in G + C-rich fragments were methylated (mCpG), the reactivity increased up to 100-fold. The native anti-DNA antibodies from SLE patients also showed preferential binding to G + C-rich fragments. These observations suggested that human anti-dsDNA antibodies may recognize some unique structures around the G + C regions or G + C clusters of DNA.

Antibodies, Antinuclear↗

R-loop mapping of calf-thymus RNA polymerase II. Transcripts in vitro on restriction fragments of adenovirus 2 DNA.

Nascent RNA, synthesized by calf thymus ENA polymerase II on the restriction endonuclease EcoRI fragment A and BamHI fragment B of adenovirus 2 DNA, was rehybridized to the template strand under conditions allowing transcription R-loop formation. Hybrids, visualized by electron microscopy, were found in looped and in branched configurations, the former being abundant, with an average loop number of roughly four per EcoRI fragment A and three per BamHI fragment B. Frequency distributions of transcription R-loops, synthesized on identical restriction fragments for different periods of time, showed similar patterns, thus pointing to a nonrandom type of transcription. A comparison of the frequency distribution of R-loops seen on the two overlapping restriction fragments reveals reasonable coincidence in the loop pattern in one out of the four possible fragment orientations, permitting correlation of the transcription R-loop pattern with the physical map of the adenovirus 2 DNA. Comparison of start sites tentatively mapped in vitro with the known promoters for RNA polymerase II in vivo suggests that a major transcription site in vitro is located in close proximity to, or identical with, two promoters in vivo at 16.1 and 16.5 map units.

Adenoviridae↗

The influence of fibrin(ogen) fragments on the kinetic parameters of the tissue-type plasminogen-activator-mediated activation of different forms of plasminogen.

In the present work we have determined Km,app and kcat,app values for tissue-type plasminogen-activator-catalyzed activation of Glu-plasminogen, Lys-plasminogen and mini-plasminogen in the absence and in the presence of fibrinogen-derived fragments. These were CNBr fragment 2, the A alpha chain remnant of CNBr fragment 2 (A alpha 148-207) and plasmin-generated fragment D-EGTA. The time course of plasmin formation from the various types of plasminogen (plg) was measured spectrophotometrically in a coupled assay system where D-valyl-L-leucyl-L-lysine p-nitroanilide served as a plasmin substrate. The kinetic constants are summarized as follows. (Values in parentheses are concentrations at which the minimum Km,app and maximum kcat,app value is reached.) (Table: see text). In conclusion our results show that CNBr fragment 2, A alpha 148-207 and to some extent D-EGTA mimic the accelerating effect of fibrin. The first two of these fragments did not accelerate activation of mini-plasminogen, lacking the kringle structures I-IV. This suggests that the stimulating effects of these two fragments were dependent on the presence of kringles I-IV of the plasminogen molecule.

Catalysis↗

Isolation of a galactose-free 20-kDa fragment exhibiting butyrylcholine esterase and aryl acylamidase activity from human serum butyrylcholine esterase by limited alpha-chymotrypsin digestion.

Purified human serum butyrylcholine esterase (approximately 90-kDa subunit), which also exhibits aryl acylamidase activity, was subjected to limited alpha-chymotrypsin digestion. Three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa were found to be produced, as observed by SDS-gel electrophoresis of the chymotryptic digest. The purified butyrylcholine esterase could fully bind to a Ricinus-communis-agglutinin-Sepharose column but after chymotryptic digestion about 15-20% of the enzyme activity remained unbound and was recovered in the run-through fractions. Sephadex G-75 chromatography of the chymotryptic digest showed an enzymatically active fragment eluted at an approximate molecular mass of 20 kDa, apart from the undigested butyrylcholine esterase eluted at the void volume. The butyrylcholine esterase fragment that did not bind to Ricinus communis agglutinin also was eluted at an approximate molecular mass of 20 kDa from a Sephadex G-75 column. This enzymatically active low-molecular-mass fragment from Sephadex G-75 chromatography showed a single protein band of approximately 20 kDa on SDS-gel electrophoresis. Neutral sugar analysis of the approximately 20 kDa fragment showed the presence of mannose only, whereas the undigested butyrylcholine esterase showed the presence of both mannose and galactose. Amino-terminal-sequence analysis of the approximately 20 kDa fragment showed the sequence Arg-Val-Gly-Ala-Leu, which agrees with amino acid residues 147-151 reported for human serum butyrylcholine esterase [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. Both cholinesterase and aryl acylamidase activities were co-eluted in all chromatographic procedures. The results suggested that limited alpha-chymotrypsin digestion of human serum butyrylcholine esterase resulted in the formation of a approximately 20-kDa enzymatically active fragment with Arg147 as its N-terminal residue and which was devoid of galactose.

Amidohydrolases↗

Plant reproductive susceptibility to habitat fragmentation: review and synthesis through a meta-analysis.

The loss and fragmentation of natural habitats by human activities are pervasive phenomena in terrestrial ecosystems across the Earth and the main driving forces behind current biodiversity loss. Animal-mediated pollination is a key process for the sexual reproduction of most extant flowering plants, and the one most consistently studied in the context of habitat fragmentation. By means of a meta-analysis we quantitatively reviewed the results from independent fragmentation studies throughout the last two decades, with the aim of testing whether pollination and reproduction of plant species may be differentially susceptible to habitat fragmentation depending on certain reproductive traits that typify the relationship with and the degree of dependence on their pollinators. We found an overall large and negative effect of fragmentation on pollination and on plant reproduction. The compatibility system of plants, which reflects the degree of dependence on pollinator mutualism, was the only reproductive trait that explained the differences among the species' effect sizes. Furthermore, a highly significant correlation between the effect sizes of fragmentation on pollination and reproductive success suggests that the most proximate cause of reproductive impairment in fragmented habitats may be pollination limitation. We discuss the conservation implications of these findings and give some suggestions for future research into this area.

Conservation of Natural Resources↗

Polymerase chain reactions with alphoid-repeat primers in combination with Alu or LINEs primers, generate chromosome-specific DNA fragments.

Y alphoid primers in combination with Alu and LINEs primers generated new DNA fragments in polymerase chain reactions (PCR) on DNA from a Y-only somatic cell hybrid but not from X-only, 3-only, or 21-only hybrids. X alphoid primers used in a similar manner generated new DNA fragments from the X-only hybrid, and 1 of the primers (X2) also generated new DNA fragments on 3-only and 21-only hybrids when used in conjunction with Alu or LINEs primers. In all but one case, consensus alphoid primers generated new chromosome-specific fragments in PCR reactions with the Alu or LINEs primers. A search for cryptic Alu- or alphoid-alone PCR products as the source for one Alu-alphoid band (chosen at random) was negative. Partial sequencing of products demonstrated that alphoid and Alu sequences were indeed contiguous in some newly synthesized DNA fragments. While Alu or LINEs primers generate smears of DNA fragments on total human DNA, the alphoid-non-alphoid repeat combinations generated electrophoretically distinguishable bands of DNA when the template was total DNA. While these were distinguishable with different chromosome-specific alphoid primers, the DNA fragments were not of the same sizes as those generated with the chromosome-only hybrids.

Animals↗

Effects of the surrounding matrix on tree recruitment in Amazonian forest fragments.

Little is known about how the surrounding modified matrix affects tree recruitment in fragmented forests. We contrasted effects of two different matrix types, Vismia- and Cecropia-dominated regrowth, on recruitment of pioneer tree species in forest fragments in central Amazonia. Our analyses were based on 22, 1-ha plots in seven experimental forest fragments ranging in size from 1 to 100 ha. By 13 to 17 years after fragmentation, the population density of pioneer trees was significantly higher in plots surrounded by Vismia regrowth than in plots surrounded by Cecropia regrowth, and the species composition and dominance of pioneers differed markedly between the two matrix types. Cecropia sciadophylla was the most abundant pioneer in fragments surrounded by Cecropia regrowth (constituting nearly 50% of all pioneer trees), whereas densities of species in Vismia-surrounded fragments were distributed more evenly. Thus the surrounding matrix had a strong influence on patterns of tree recruitment in Amazonian forest fragments.

Conservation of Natural Resources↗

Long-term landscape change and bird abundance in Amazonian rainforest fragments.

The rainforests of the Amazon basin are being cut by humans at a rate >20,000 km2/year leading to smaller and more isolated patches of forest, with remaining fragments often in the range of 1-100 ha. We analyzed samples of understory birds collected over 20 years from a standardized mist-netting program in 1- to 100-ha rainforest fragments in a dynamic Amazonian landscape near Manaus, Brazil. Across bird guilds, the condition of second growth immediately surrounding fragments was often as important as fragment size or local forest cover in explaining variation in abundance. Some fragments surrounded by 100 m of open pasture showed reductions in insectivorous bird abundance of over 95%, even in landscapes dominated by continuous forest and old second growth. These extreme reductions may be typical throughout Amazonia in small (< or =10 ha), isolated fragments of rainforest. Abundance for some guilds returned to preisolation levels in 10- and 100-ha fragments connected to continuous forest by 20-year-old second growth. Our results show that the consequences of Amazonian forest loss cannot be accurately described without explicit consideration of vegetation dynamics in matrix habitat. Any dichotomous classification of the landscape into 'forest" and "nonforest" misses essential information about the matrix.

Animals↗

Arthroscopic removal of axial osteochondral fragments of the proximoplantar aspect of the proximal phalanx using electrocautery probes in 23 standardbred racehorses.

OBJECTIVES: To describe the outcome after arthroscopic electrosurgical excision of axial osteochondral (OC) fragments of the proximal plantar aspect of the 1st phalanx (P1) in horses. STUDY DESIGN: Retrospective study. POPULATION: Twenty-three Standardbred racehorses. METHODS: Medical records of Standardbred racehorses that had arthroscopic dissection of axial OC fragments of the proximal plantar aspect of P1 with electrocautery probes were reviewed. Horses were positioned in dorsal recumbency; a 1.5% glycine solution was used to maintain joint distension. The arthroscope portal was in the proximal plantar joint pouch and OC fragments were approached using either an ipsilateral or a contralateral triangulation technique. Dissection of the fibrous attachments was performed using loop and hook electrocautery probes. Follow-up was obtained by telephone questionnaire of owners or trainers and examination of race summary records. RESULTS: Thirty-three axial OC fragments were removed from 28 metatarsophalangeal joints (left, 14; right, 14). No major operative or postoperative complications occurred. Seven of 8 (79%) of horses that raced before surgery raced after surgery. Thirty-six percent (n=4) of horses that had not raced before surgery raced after surgery. CONCLUSION: Intra-articular electrosurgery is a safe, easy alternative to conventional excision of axial OC fragments of the proximal plantar aspect of P1. CLINICAL RELEVANCE: Arthroscopic excision using electrocautery probes is a valid alternate method for removal of axial OC fragments of the proximal plantar aspect of P1. In addition, the dorsal recumbency position facilitates removal of several fragments and multiple joint surgery.

Animals↗

Localization of prophages of serological group B and F on restriction fragments defined in the restriction map of Staphylococcus aureus NCTC 8325.

Several Staphylococcus aureus strains were lysogenized by the phages of serological group B (phages phi 53, phi 85) as well as by some of serological group F (phages phi 77, phi 84) and macrorestriction fragment patterns of genomic DNA were estimated in the lysogenized, non-lysogenic and delysogenized (cured of prophages) strains. It was shown that the integration of phage DNA into chromosome of S. aureus leads to specific changes in restriction fragment pattern in all the lysogenized strains. These changes correlate well with the SmaI restriction map of S. aureus NCTC 8325 since they concern the restriction fragments defined in this map. Phages phi 53 and phi 85 integrate into SmaI fragment B. On the other hand, phages phi 77 and phi 84 integrate into SmaI fragment E of the S. aureus restriction map. The prophages of strain NCTC 8511 have their integration sites, as follows: the phage designated by us phi M integrates in fragment A, whereas the integration site for phage phi J lies in fragment E. Phage phi M was estimated to be genetically related to phages of serological group A and phage phi J to those of serological group F. Evidence was given that lysogenization of S. aureus strains by at least four prophages does not cast any doubt upon the estimation of their genetic relatedness based on their similarity in restriction pattern.

Attachment Sites, Microbiological↗

A ribosomal DNA fragment of Listeria monocytogenes and its use as a genus-specific probe in an aqueous-phase hybridization assay.

cDNAs were prepared from the total RNA of Listeria monocytogenes ATCC 19118 and used as probes to screen a genomic library of the same strain. Four clones were identified which contained ribosomal DNA fragments. Recombinant DNA from one of them was fractionated and differentially hybridized with the cDNA probes to RNA of L. monocytogenes and Kurthia zopfii. The resulting hybridization pattern revealed an HpaII fragment of 0.8 kb that was specific for the L. monocytogenes strain. The nucleotide sequence of this fragment showed 159 bases of the 3' end of the 16S rRNA gene, 243 bases of the spacer region, and 382 bases of the 5' end of the 23S rRNA gene. In dot blot hybridization assays, the 32P-labeled 784-bp fragment was specific only for Listeria species. Dot blot assays revealed that the 32P-labeled fragment can easily detect > or = 10 pg of total nucleic acids from pure cultures of L. monocytogenes, which corresponds to approximately 300 bacteria. This fragment was also used as a probe in an assay named the heteroduplex nucleic acid (HNA) enzyme-linked immunosorbent assay. In this system, the biotinylated DNA probe is hybridized in the aqueous phase with target RNA molecules and then specific HNAs are captured by HNA-specific antibodies. Captured HNA molecules are revealed with an enzyme conjugate of streptavidin. In a preliminary HNA enzyme-linked immunosorbent assay, the 784-bp fragment maintained its specificity for Listeria spp. and could detect 5 x 10(2) cells in artificially contaminated meat homogenate.

Base Sequence↗

Release of soluble peptidoglycan from growing conococci: demonstration of anhydro-muramyl-containing fragments.

Previous analysis of soluble peptidoglycan (PG) fragments released by exponentially growing gonococci implicated the combined action of both hexosaminidase and amidase activities in PG turnover. Current studies further characterized PG fragments which were labeled in the glycan with D-glucosamine and in the peptide moiety with meso-diaminopimelic acid of L- and D-alanine. Labeled PG fragments were isolated by gel filtration and characterized on the bases of (i) KD values, (ii) free amino group analysis using fluorodinitrobenzene, (iii) borohydride reduction, (iv) alkali-catalyzed beta-elimination, (v) paper chromatography in various solvents, (vi) electrophoretic mobility at various pH values, (vii) digestibility by Charonia lampas glycosidases, and (viii) content of labeled D- and L-alanine. A set of well-characterized PG fragments was used as standards. The monomer fraction (the major extracellular product) was found to contain two components. Most (about 80%) appeared to be N-acetylglucosaminyl-beta-1 leads to 4-1,6-anhydro-N-acetylmuramyl-L-ala-D-glu-meso-diaminopimelic acid; the remainder was the corresponding disaccharide tetrapeptide containing a C-terminal D-alanine. An unusual feature of these products was the presence of the anhydro-muramyl (non-reducing) ends, reflecting the activity of a gonococcal transglycosylase, and the near absence of products containing detectable reducing ends. Otherwise, the structures of the monomer fragments were typical of those expected for a gram-negative bacterium (chemotype I). The corresponding peptide-cross-linked dimer and the free disaccharide also contained nonreducing ends, exclusively. Free peptides (products of amidase activity) consisted of both tripeptide and tetrapeptide. In summary, all gonococci examined appear to possess an unusual transglycosylase activity which contributes to the release of soluble PG fragments containing nonreducing, anhydro-muramyl ends. The release of these fragments in vivo might be a unique aspect of gonococci-host interactions.

Amino Acids↗

Cutaneous inflammatory reactions to group A streptococcal cell wall fragments in Fisher and Lewis inbred rats.

Systemic administration of an aqueous suspension of group A streptococcal cell wall fragments induces severe, chronic erosive polyarthritis in LEW/N female rats, but rarely in F344/N female rats. In the present study, we attempted to exclude unresponsiveness to the cell walls as a mechanism for arthritis resistance in F344/N females. Cutaneous inflammatory reactions were assessed in both strains at various time points after direct injection of cell wall fragments of three different average molecular weights. Fragments of all sizes induced an acute inflammatory reaction, with infiltration of polymorphonuclear leukocytes and a few mononuclear cells. Small fragments (approximately 5 megadaltons) induced a transient response which resolved by day 14. Large fragments (approximately 500 megadaltons) induced severe inflammation characterized by prominent mononuclear leukocyte infiltration, whereas the intermediate-sized fragments (approximately 50 megadaltons) induced inflammation of intermediate intensity and duration. The intensity and severity of the lesions paralleled the persistence of cell wall antigens at the site of deposition. F344/N female rats responded acutely to the cell walls, with an intensity equal to or greater than that of LEW/N female rats, but the lesions tended to resolve more rapidly. These findings indicate that severity and chronicity of streptococcal cell wall-induced inflammation are dependent on the size of the fragment and provide evidence that arthritis resistance in F344/N female rats does not result from a completely unresponsive state to the proinflammatory effects of the cell walls.

Animals↗

Peptidoglycan fragments decrease food intake and body weight gain in rats.

We hypothesized that peptidoglycan (PG) fragments decrease appetite in rats. Male Lewis rats (150 g; n > or = 7) received intraperitoneal (i.p.) injections of purified soluble PG fragments that had been treated with polymyxin B-agarose to remove residual endotoxin. Food consumption and body weight gain were determined at intervals after injection. Single i.p. injections of macromolecular extensively O-acetylated PG (S-O-PG) and non-O-acetylated PG fragments (24 to 240 micrograms/kg) reduced food intake and body weight gain in a dose-dependent fashion during the first 12 h after injection. Low-molecular-weight disaccharide peptide monomers with nonreducing 1,6-anhydro-N-acetylmuramic acid ends and muramyl dipeptide (MDP; 1.6 mg/kg) were also appetite and weight gain suppressants, albeit at least 10-fold less potent than S-O-PG; however, muramidase-derived monomers and peptide cross-linked dimers with reducing muramic acid ends were inactive. Appetite suppression was not limited to the Lewis rat strain since another strain, F344, exhibited similar decreases in food intake after injection of S-O-PG or MDP. Oral administration of MDP or S-O-PG, at concentrations 3 and 20 times higher, respectively, than those that were active i.p., failed to elicit a hypophagic response. We conclude that soluble PG fragments are potent suppressants of food consumption and body weight gain in rats and that, although macromolecular PG is more potent than low-molecular-weight fragments, neither O-acetylation nor glycosidic linkage of PG fragments is required for activity. We speculate that PG fragments may contribute to loss of appetite during bacterial illness.

Acetylation↗