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PubMed1995

Relationship of the 37,000- and 40,000-M(r) tryptic fragments of islet antigens in insulin-dependent diabetes to the protein tyrosine phosphatase-like molecule IA-2 (ICA512).

Sera from patients with insulin-dependent diabetes immunoprecipitate 64,000-M(r) proteins, distinct from glutamate decarboxylase, that are cleaved to 37,000- and 40,000-M(r) fragments by trypsin. We investigated possible relationships between 37,000- or 40,000-M(r) fragments of antigen and the tyrosine phosphatase-like protein, IA-2 (ICA512). Antibodies from nondiabetic relatives bound differentially to 37,000- and 40,000-M(r) fragments indicating presence of distinct epitopes. Precursors of these fragments could be separated on immobilized lectins, suggesting different carbohydrate content. Levels of antibodies to 40,000-M(r) fragments were strongly associated with those to the intracellular domain of IA-2. Recombinant intracellular domain of IA-2 blocked binding of antibodies to 40,000-M(r) fragments expressed by insulinoma cells and partially blocked binding to 37,000-M(r) fragments. Furthermore, trypsinization of recombinant intracellular domain of IA-2 generated proteolytic fragments of identical M(r) to the 40,000-M(r) fragments of insulinoma antigen; 37,000-M(r) fragments were not generated. Thus, 40,000-M(r) fragments of islet autoantigen are derived from a protein similar or identical to the tyrosine phosphatase-like molecule, IA-2. The 37,000-M(r) fragments are derived from a different, although related, protein.

Animals↗
PubMed2006

Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease.

Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease (SNase110) have been studied by various biophysical and NMR methods. Samples of G-88W- and V-66W-mutant SNase110, namely G-88W110 and V-66W110, in aqueous solution and SNase110 in 2.0 M TMAO are adopted in this study. The unfolding transitions and folded conformations of the three SNase fragments were detected by far- and near-ultraviolet circular dichroism and intrinsic tryptophan fluorescence measurements. The tertiary structures and internal motions of the fragments were determined by NMR spectroscopy. Both G-88W and V-66W single mutations as well as a small organic osmolyte (Trimethylamine N-oxide, TMAO) can fold the fragment into a native-like conformation. However, the tertiary structures of the three fragments exhibit different degrees of folding stability and compactness. G-88W110 adopts a relatively rigid structure representing a most stable native-like beta-subdomain conformation of the three fragments. V-66W110- and TMAO-stabilized SNase110 produce less compact structures having a less stable "beta-barrel" structural region. The different folding status accounts for the different backbone dynamic and urea-unfolding transition features of the three fragments. The G-20I/G-29I-mutant variants of the three fragments have provided the evidence that the folding status is correlated closely to the packing of the beta-strands in the beta-barrel of the fragments. The native-like beta-barrel structural region acts as a nonlocal nucleus for folding the fragment. The tertiary folding of the three fragments is initiated by formation of the local nucleation sites at two beta-turn regions, I-18-D-21 and Y-27-Q-30, and developed by the formation of a nonlocal nucleation site at the beta-barrel region. The formation of beta-barrel and overall structure is concerted, but the level of cooperativity is different for the three 1-110 residues SNase fragments.

Amino Acid Sequence↗
PubMed2004

Fab antibody fragments: some applications in clinical toxicology.

This review provides current information on the use of antigen-binding fragments (Fab) from cleaved antibodies to treat poisoning with digoxin and other potent, low formula mass poisons, such as colchicine and tricyclic antidepressants. Anti-digoxin Fab fragments have been used successfully for many years in the management of severe poisoning with digoxin, digitoxin, and a range of other structurally related compounds, including cardiotoxins from Nerium and Thevetia sp. (oleander) and Bufo sp. (toads). However, their main use remains treating digoxin poisoning. Equimolar doses of anti-digoxin Fab fragments completely bind digoxin in vivo. The approximate dose of Fab fragments (mg) is 80 times the digoxin body burden (mg). If neither the dose ingested nor the plasma digoxin/digitoxin concentration is known, in an adult 380 mg of anti-digoxin Fab fragments should be given. The dose for elderly patients or those with renal impairment should be similar to that for those with normal renal function. Fab fragments have a plasma half-life of 12-20 hours, but this can be prolonged in patients with renal impairment. Analysis of serum ultrafiltrate using an immunoassay shown not to have matrix bias remains the most accurate approach to measuring free digoxin in the presence of anti-digoxin Fab fragments. The antibody fragments are given intravenously over 15-30 minutes after dilution to at least 250 mL with plasma protein solution, 0.9% (w/v) sodium chloride, or deionised water, except in infants where the volume infused can be reduced. Factors limiting the efficacy of Fab fragments are the dose, the duration of the infusion and any delay in administration. Guidelines for Fab fragment administration in children include (i) dilution to a final Fab concentration of 10 g/L in either 5% (w/v) dextrose or 0.9% (w/v) sodium chloride; (ii) infusion through a 0.22 microm filter; (iii) administration of the total dose over a minimum of 30 minutes; and (iv) avoiding coadministration of other drugs and/or electrolyte solutions. Fab fragments are generally well tolerated. Adverse effects attributable to Fab treatment include hypokalaemia and exacerbation of congestive cardiac failure; renal function could be impaired in some patients. Fab fragment preparations for treating acute colchicine and tricyclic antidepressant poisoning have been developed, but are not available commercially. Colchicine poisoning is rare in Western countries, and pharmacological management together with supportive care is usually effective even in severe tricyclic antidepressant overdosage. Attempts have been made to produce anti-paraquat antibodies capable of enhancing paraquat elimination from the lung, but thus far all such attempts have proved unsuccessful.

Digoxin↗
PubMed1988

Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment.

Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell-binding fragment contains a cryptic site for monocyte chemotaxis which is expressed upon enzymatic cleavage of fibronectin.

Chemotaxis, Leukocyte↗
PubMed1977

Structure of tetanus toxin. I. Breakdown of the toxin molecule and discrimination between polypeptide fragments.

Tetanus toxin was digested with papain, yielding one major polypeptide (Fragment C) with a molecular weight corresponding to 47,000 +/- 5%, thus comprising about one-third of the toxin molecule. Fragment C was antigenically active, atoxic, and stimulated the formation of antibodies neutralizing the lethal action of tetanus toxin in vivo. Furthermore, a second split product (Fragment B) was isolated from the papain digest, containing two polypeptide chains linked together via a disulfide bond. Fragment B (Mr = 95,000 +/- 5%) was atoxic and showed a reaction of nonidentity with Fragment C on immunodiffusion analysis against tetanus antitoxin. The basic two-chain structure (heavy and light chain polypeptide, cf. Matsuda, M., and Yoneda, M. (1975) Infect. Immun. 12, 1147-1153) of tetanus toxin has been confirmed and the relationship between Fragments B and C within this framework has been established. Fragment C was distinguished from the light chain by electrophoresis in sodium dodecyl sulfate and by immunodiffusion analysis, indicating that this fragment constitutes a portion of the heavy chain polypeptide. Fragment B showed a reaction of partial identity with the light as well as the heavy chain from tetanus toxin. Reduction of Fragment B with dithiothreitol followed by gel chromatography yielded a fraction which was indistinguishable from the light chain portion of the toxin molecule. It is concluded that Fragment B comprises the complementary portion of the heavy chain (remaining after scission of the polypeptide bond(s) releasing Fragment C) linked to the light chain by a disulfide bond.

Amino Acids↗
PubMed1979

Metal binding sites of a gamma-carboxyglutamic acid-rich fragment of bovine prothrombin.

The metal binding sites of a gamma-carboxyglutamic acid-rich fragment derived from bovine prothrombin were examined using paramagnetic lanthanide ions to evaluate the role of gamma-carboxyglutamic acid resideus in metal binding. A gamma-carboxyglutamic acid-rich peptide, fragment 12-44, was isolated from a tryptic digest of prothrombin. Using 153Gd(III), fragment 12-44 was found to contain one high affinity metal binding site (KD = 0.55 microM) and four to six lower affinity metal binding sites (KD approximately 4 to 8 microM). The S-carboxymethyl derivative of fragment 12-44, in which the disulfide bond in fragment 12-44 was reduced and alkylated, contained no high affinity metal binding site and four or five lower affinity sites (KD = 8 microM). The effects of paramagnetic lanthanide ions on fragment 12-44 and its S-carboxymethyl derivative were studied by natural abundance 13C NMR spectroscopy. The 13C NMR spectrum of fragment 12-44 was recorded at 67.88 MHz and the resonances were assigned by comparison to the chemical shift of carbon resonances of amino acids and peptides previously studied. The proximity between bound metal ions and carbon atoms in fragment 12-44 was estimated using Gd(III), based upon the strategy that the magnitude of the change in the transverse relaxation rate of resonances of carbon nuclei induced by bound metal ions is related in part to the interatomic distances between bound metal and carbon nuclei. Titration of fragment 12-44 with Gd(III) resulted in the selective broadening of the gamma-carboxyl carbon, C gamma, C beta, and C alpha resonances of gamma-carboxyglutamic acid, and the C epsilon of the arginines. S-Carboxymethyl fragment 12-44, which lacked the high affinity metal binding site, showed markedly decreased perturbation of the C epsilon of the arginine residues upon titration with Gd(III). These studies indicate that gamma-carboxyglutamic acid residues in prothrombin fragment 12-44 participate in metal liganding. A high affinity metal binding site in fragment 12-44 is in close proximity of Arg 16 and Arg 25 and is stabilized by the disulfide bond. On the basis of these data, a model of the metal binding sites is proposed in which the high affinity site is composed of two gamma-carboxyglutamic acid residues which participate in intramolecular metal-dependent bridging of two regions of the polypeptide chain. The lower affinity metal binding sites, formed by single or paired adjacent gamma-carboxyglutamic acid residues, then may participate in intermolecular metal-dependent protein . protein or protein . membrane complex formation.

1-Carboxyglutamic Acid↗
PubMed1976

Physiochemical characterization of proteolytic cleavage fragments of bovine colostral immunoglobulin G1 (IgG1).

Normal bovine colostral immunoglobulin G1 was subjected to enzymic digestion (pepsin, papain and trypsin) and the resulting fragments separated by a combination of molecularsieve and phosphocellulose chromatography. Fragments F(ab')2 derived from peptic digestion, fragment Fab from papain digestion and fragment Fab(t) from tryptic digestion showed complete antigenic identity with each other. Although fragment F(ab')2 (peptic digestion) had a sedimentation coefficient (S2o,w) of 5.3S, those for fragments Fab' (peptic digestion), Fab (papain digestion) and Fab(t) (tryptic digestion) were found to be 3.9S, 3.7S and 3.7S respectively. The mol.wts. calculated for the various fragments from the sedimentation equilibrium data were: F(ab')2, 104000 +/-200; Fab', 51900+/-340; Fab, 50900+/-230; Fab(t) 50900+/-300. Fragment Fc' (peptic digestion) had an S20,w of 3.2S and a mol. wt. of 42900+/-650; fragment Fc (papain digestion) had an SI0,w of 3.7S and a mol.wt. of 50800+/-300; fragment Fc(t) had an S20,w of 3.7S and a mol.wt. of 50800+/-300; fragment Fc(t) had an S20,w of 3.7S and a mol.wt. of 50800+/-450.

Agglutination↗
PubMed2000

Automated DNA fragment collection by capillary array gel electrophoresis in search of differentially expressed genes.

An automatic DNA fragment collector using capillary array gel electrophoresis has been developed. A sheath flow technique is used for not only detection but also collection of DNA fragments. In a sheath flow cell, the DNA fragments separated by 16 capillaries flow independently into corresponding sampling capillaries. The fraction collector consists of 16 sampling trays and each sampling tray is set beneath each end of the sampling capillaries to collect the flow-through DNA fragments. Certain DNA fragments are automatically sorted by controlling the movement of the sampling trays according to the signals from the system. The collector experimentally separated two mixtures of polymerase chain reaction (PCR) products: one prepared by using eight different sizes (base lengths from 161 to 562) of DNAs; and the other prepared by a differential display (DD) method with cDNA fragments. Collected DNA fragments are amplified by PCR and measured by electrophoresis. DNA fragments with base length differences of one (base lengths 363 and 364) were successfully separated. A separated DNA fragment from the DD sample was also successfully sequenced. In addition, differentially expressed DNA fragments were automatically sorted by comparative analysis, in which two similar cDNA fragment groups, labeled by two different fluorophores, respectively, were analyzed in the same gel-filled capillary. These results show that the automatic DNA fragment collector is useful for gene hunting in research fields such as drug discovery and DNA diagnostics.

Animals↗
PubMed1987

Electro-optical analysis of 'curved' DNA fragments.

The structure of some 'short' DNA fragments with 106-108 base-pairs and of a 'long' kinetoplast DNA fragment with 419 base-pairs has been analyzed by electro-optical procedures. According to their electrophoretic mobilities and circularization probabilities, it was concluded that two of our short fragments with clusters of four and five and six adenosines phased at the period of the double helix are inherently curved with an approximate curvature around 200 degrees. The dichroism decay curves of our short fragments exhibited two processes. A fast one with time constants of approx. 100 ns is attributed to bending; the bending amplitudes observed for the fragments with dA4 and dA5/6 clusters are slightly higher (23 and 29%, respectively) than those observed for control fragments (17-20%). The second process reflects the overall rotational diffusion of the whole fragments and shows some variation with the DNA sequence, but on average the rotation of fragments with dA4 and dA5/6 clusters corresponds to that observed for standard DNA. Since the rotational diffusion coefficients are very strongly dependent on the effective hydrodynamic lengths, we must conclude that the effective lengths of our fragments, including the 'curved' ones, are very similar under the conditions of our experiments. The rotation time constant for the long kinetoplast DNA is also rather close to those observed for the usual DNA fragments of corresponding length. One way to resolve the conflict of our results with conclusions obtained from other investigations would invoke the assumption that the curved fragments are not 'elastic'. According to this hypothesis, electric field pulses would stretch the curved fragments to an almost straight form and the stretched DNA would return to its equilibrium state with a time constant longer than the rotation time constant.

Base Sequence↗
PubMedDate not supplied

Shed membrane fragment modulation of CD3-zeta during pregnancy: link with induction of apoptosis.

Our objective was to identify shed placental plasma membrane fragments in the maternal circulation and determine whether these fragments are capable of down-regulating CD3-zeta chain expression and inducing apoptosis in T lymphocytes. Sera, isolated from the blood of pregnant women at 26-29 weeks gestation that subsequently had uncomplicated term deliveries, were subjected to high exclusion-limit gel chromatography to isolate placental membrane fragments. The placental origin of the fragments was confirmed by the presence of placental-type alkaline phosphates. These shed membrane fragments were further analyzed for the presence of Fas ligand (FasL) and modulation of CD3-zeta expression on cultured T-lymphocytes (Jurkat cells). The ability of the shed membrane fragments to induce apoptosis was assayed using a cell death ELISA. Components associated with Fas-dependent apoptosis (caspase-3, bcl-2 and bax) were characterized using western immunoblot following exposure to serum-derived membrane fragments. Placental membrane fragments were identified in all pregnancy sera, but not in non-pregnant controls. The 41 kDa FasL was identified in membrane fragment isolates and all samples were capable of inducing apoptosis as determined by the ELISA assay. Exposure of T lymphocytes to isolated membrane fragments suppressed the expression of CD3-zeta. The induction of apoptosis correlated with the induction and activation of caspase 3 and the induction of bax. Placenta-derived membrane fragments are detectable in the maternal circulation. These membrane fragment isolates are capable of inducing FasL-mediated apoptosis and down-regulating CD3-zeta expression, which may contribute to the immune tolerance of the fetus.

Apoptosis↗
PubMed1999

YAC fragmentation with repetitive and single-copy sequences: detailed physical mapping of the presenilin 1 gene on chromosome 14.

We constructed new LYS2 fragmentation vectors that allow direct acentric and centric fragmentation of yeast artificial chromosomes (YACs) and selection of fragmented YACs in yeast strain AB1380. The fragmentation vectors were used efficiently with repetitive (e.g., Alu), low-copy (e.g., CA-repeats) and single-copy (e.g., exons) sequences. High recombination efficiencies were obtained in fragmenting two different CEPH YACs with the Alu consensus sequence as target sequences for homologous recombination. Analysis of the acentric Alu fragmentation panel of 788H12, containing the presenilin 1 (PSEN1) gene for familial Alzheimer's disease (AD), indicated that high-resolution YAC fragmentation panels covering the entire parent YAC are obtained. Also, marker content analysis of the fragmentation panel indicated that fragmented YACs were propagated stably without rearrangements. The same fragmentation vectors were used efficiently for fragmentation of 788H12 with unique sequences, i.e., exons 3 and 12 of PSEN1 and D14S77, a polymorphic CA repeat, as target sequences. Together, our YAC fragmentation data of 788H12 provided a size estimate for the coding region of PSEN1 of 60kb and a more precise localization of D14S77 at 25kb upstream of PSEN1.

Alu Elements↗
PubMed2006

Fragment screening: an introduction.

There are clearly many different philosophies associated with adapting fragment screening into mainstream Drug Discovery Lead Generation strategies. Scientists at Astex, for instance, focus entirely on strategies involving use of X-ray crystallography and NMR. However, AstraZeneca uses a number of different fragment screening strategies. One approach is to screen a 2000 compound fragment set (with close to "lead-like" complexity) at 100 microM in parallel with every HTS such that the data are obtained on the entire screening collection at 10 microM plus the extra samples at 100 microM; this provides valuable compound potency data in a concentration range that is usually unexplored. The fragments are then screen-specific "privileged structures" that can be searched for in the rest of the HTS output and other databases as well as having synthesis follow-up. A typical workflow for a fragment screen within AstraZeneca is shown below (Figure 24) and highlights the desirability (particularly when screening >100 microM) for NMR and X-ray information to validate weak hits and give information on how to optimise them. In this chapter, we have provided an introduction to the theoretical and practical issues associated with the use of fragment methods and lead-likeness. Fragment-based approaches are still in an early stage of development and are just one of many interrelated techniques that are now used to identify novel lead compounds for drug development. Fragment based screening has some advantages, but like every other drug hunting strategy will not be universally applicable. There are in particular some practical challenges associated with fragment screening that relate to the generally lower level of potency that such compounds initially possess. Considerable synthetic effort has to be applied for post-fragment screening to build the sort of potency that would be expected to be found from a traditional HTS. However, if there are no low-hanging fruit in a screening collection to be found by HTS then the use of fragment screening can help find novelty that may lead to a target not being discarded as intractable. As such, the approach offers some significant advantages by providing less complex molecules, which may have better potential for novel drug optimisation and by enabling new chemical space to be more effectively explored. Many literature examples that cover examples of fragment screening approaches are still at the "proof of concept" stage and although delivering inhibitors or ligands, may still prove to be unsuitable when further ADMET and toxicity profiling is done. The next few years should see a maturing of the area, and as our understanding of how the concepts can be best applied, there are likely to be many more examples of attractive, small molecule hits, leads and candidate drugs derived from the approaches described.

Combinatorial Chemistry Techniques↗
PubMed1983

Analysis of HLA class I genes with restriction endonuclease fragments: implications for polymorphism of the human major histocompatibility complex.

Cellular DNA from HLA-typed individuals was digested with the restriction endonucleases HindIII, EcoRV, and EcoRI. The separated restriction endonuclease fragments were hybridized with a HLA class I cDNA probe by using the Southern transfer technique. Digestion of cellular DNA with HindIII generated 22 restriction endonuclease fragments, 11 of which showed polymorphism for presence or absence in a population sample. With EcoRV, 13 fragments were identified; 6 showed polymorphism. EcoRI generated 11 fragments, of which 1 was polymorphic. Of these 18 polymorphic fragments generated by the three restriction endonucleases, each of 5 was found to be positively associated with one allele of the HLA-A or -B allelic series (HLA-Aw24, -B8, -B15, -Bw35, and -B40). One fragment was positively associated with two HLA-A series alleles (HLA-A1 and -A11). Another fragment was positively associated with five HLA-B series alleles (HLA-B5, -B7, -B14, -Bw16, and -Bw35) and one fragment was positively associated with alleles at two loci (HLA-B14 and -Cw5). The serologically defined allele HLA-Aw24 was associated with two polymorphic fragments, one association showing a positive correlation and the other a negative correlation. Each informative family studied thus far has shown segregation of the restriction fragment with the associated serologically defined allele. The fragments associated with serologically defined alleles occurred in the population sample studied at low or moderate frequencies. The remaining polymorphic fragments occur at high frequency, suggesting that class I genes not serologically detected show less polymorphism than serologically defined class I genes.

Alleles↗
PubMed1995

Preferential isolation of DNA fragments associated with CpG islands.

We describe a procedure for preferential isolation of DNA fragments with G+C-rich portions. Such fragments occur in known genes within or adjacent to CpG islands. Since about 56% of human genes are associated with CpG islands, isolation of these fragments permits detection and probing of many genes within much larger segments of DNA, such as cosmids or yeast artificial chromosomes, which have not been sequenced. Cloned DNA fragments digested with four restriction endonucleases were subjected to denaturing gradient gel electrophoresis. Long G+C-rich sections in fragments inhibit strand dissociation after the fragments reach retardation level in the gradient; such fragments are retained in the gel after most others disappear. Nucleotide sequences of the retained fragments show that about half of these fragments appear to be derived from CpG islands. Northern analysis indicated the presence of RNA complementary to most of the retained fragments. A heuristic approach to the relation between base sequence and the kinetics of strand dissociation of partly melted molecules appears to account for retention and nonretention. The expectation that CpG island fragments will be enriched among fragments retained in a denaturing gradient is supported by rate estimates based on melting theory applied to known sequences. This method, designated SPM for segregation of partly melted molecules, is expected to provide a means for convenient and efficient isolation of genes from unsequenced DNA.

Base Sequence↗
PubMed2006

Airborne fungal fragments and allergenicity.

Exposure to fungi, particularly in water damaged indoor environments, has been thought to exacerbate a number of adverse health effects, ranging from subjective symptoms such as fatigue, cognitive difficulties or memory loss to more definable diseases such as allergy, asthma and hypersensitivity pneumonitis. Understanding the role of fungal exposure in these environments has been limited by methodological difficulties in enumerating and identifying various fungal components in environmental samples. Consequently, data on personal exposure and sensitization to fungal allergens are mainly based on the assessment of a few select and easily identifiable species. The contribution of other airborne spores, hyphae and fungal fragments to exposure and allergic sensitization are poorly characterized. There is increased interest in the role of aerosolized fungal fragments following reports that the combination of hyphal fragments and spore counts improved the association with asthma severity. These fragments are particles derived from any intracellular or extracellular fungal structure and are categorized as either submicron particles or larger fungal fragments. In vitro studies have shown that submicron particles of several fungal species are aerosolized in much higher concentrations (300-500 times) than spores, and that respiratory deposition models suggest that such fragments of Stachybotrys chartarum may be deposited in 230-250 fold higher numbers than spores. The practical implications of these models are yet to be clarified for human exposure assessments and clinical disease. We have developed innovative immunodetection techniques to determine the extent to which larger fungal fragments, including hyphae and fractured conidia, function as aeroallergen sources. These techniques were based on the Halogen Immunoassay (HIA), an immunostaining technique that detects antigens associated with individual airborne particles >1 microm, with human serum immunoglobulin E (IgE). Our studies demonstrated that the numbers of total airborne hyphae were often significantly higher in concentration than conidia of individual allergenic genera. Approximately 25% of all hyphal fragments expressed detectable allergen and the resultant localization of IgE immunostaining was heterogeneous among the hyphae. Furthermore, conidia of ten genera that were previously uncharacterized could be identified as sources of allergens. These findings highlight the contribution of larger fungal fragments as aeroallergen sources and present a new paradigm of fungal exposure. Direct evidence of the associations between fungal fragments and building-related disease is lacking and in order to gain a better understanding, it will be necessary to develop diagnostic reagents and detection methods, particularly for submicron particles. Assays using monoclonal antibodies enable the measurement of individual antigens but interpretation can be confounded by cross-reactivity between fungal species. The recent development of species-specific monoclonal antibodies, used in combination with a fluorescent-confocal HIA technique should, for the first time, enable the speciation of morphologically indiscernible fungal fragments. The application of this novel method will help to characterize the contribution of fungal fragments to adverse health effects due to fungi and provide patient-specific exposure and sensitization profiles.

Air Microbiology↗
PubMed1968

Papain digestion fragments of human IgM globulins.

Papain digestion of two Waldenström IgM globulins produced a high amount of small peptides and resulted in the formation of two end products, the Fabmicro and Fcmicro fragments. The Fcmicro fragment is characterized by a fast electrophoretic mobility, a high content in carbohydrate, and a high molecular weight. It was demonstrated that this fragment is made of heavy chain pieces belonging to several disulfide-linked monomeric subunits, presumably representing the carboxy-terminal end of the micro-chains. Fc fragments from the two macroglobulins could not be distinguished immunologically. An appreciable proportion of IgM molecules apparently underwent degradation without the formation of a stable Fc fragment. An Fc-like fragment, analogous to the reduced Fc fragment, was obtained at early stages of papain digestion of the IgM subunits. The Fabmicro fragment, with slow and individually distinct electrophoretic mobility, bears many physicochemical and immunological similarities to the Fabgamma fragment. It consists of one light chain and one Fd piece, both of which were isolated. The interaction of these two constituents was demonstrated by gel diffusion studies. Fab fragments of both IgM globulins were resolved into two subpopulations with different electric charges. In addition to these fragments, intermediary split products were observed at early stages of the degradation process, together with a high yield of small peptides mainly derived from the papain-sensitive region of the heavy chains. Immunologic data strongly suggested that this segment of micro-chains is situated between the Fd piece and the portion included in the Fc fragment. Several experiments indicated the importance of conformational antigenic specificity in both Fab and Fc regions of the IgM globulins.

Carbohydrates↗
PubMed2006

Predictors of clinical significance of residual fragments after extracorporeal shockwave lithotripsy for renal stones.

PURPOSE: To determine the predictors of the clinical outcome of residual fragments after extracorporeal shockwave lithotripsy (SWL). PATIENTS AND METHODS: During the last 10 years, residual fragments < or =5 mm were present in 99 male and 55 female patients (mean age 43.1 years) for >3 months after SWL for renal stones. Follow-up with history, urine examination, and non-contrast CT scanning was carried out every 6 months or when symptoms developed. Clinically insignificant residual fragments were diagnosed in patients who became stone free or had asymptomatic fragments of the same size during follow-up. A clinically significant outcome was considered to have occurred in patients requiring secondary intervention for regrowth of the fragments or medical treatment for frequent symptomatic episodes. Univariate (Kaplan-Meier) and multivariate (Cox-regression) analyses were performed to determine significant predictors of the outcome. The mean follow-up was 31 months (range 6-80 months). RESULTS: Stone-free status, regrowth of fragments, and persistence of fragments of the same size were present in 21 (13.6%), 52 (33.8%), and 81 (52.6%) patients, respectively. A clinically significant outcome was documented in 75 patients (48.7%) as percutaneous nephrolithotomy was needed in 2, repeat SWL in 50, and medical treatment for symptomatic episodes in 23 patients. Significant independent predictors of a clinically significant outcome were fragment size > or =4 mm and history of recurrent stone disease (P < 0.001). CONCLUSION: The term "clinically insignificant residual fragments" is not appropriate for all patients with post-SWL fragments, as 48.7% of patients had fragments that became clinically significant. Fragments of 4 to 5 mm and recurrent stone disease predicted clinical significance.

Adolescent↗
PubMed1989

Purification of limited tryptic fragments of Ca2+,Mg2+-adenosine triphosphatase of the sarcoplasmic reticulum and identification of conformation-sensitive cleavage sites.

Sarcoplasmic reticulum membranes were treated with trypsin, and samples enriched with A1a, A1b, and C fragments (Saito, K. et al. (1984) J. Biochem. 95, 1297-1304), respectively, were prepared. A1b and C fragments were purified to apparent homogeneity, and an approximately equimolar mixture of A1(Met1-Arg198), A1a, and A1b fragments free from other contaminants was also obtained through gel permeation and hydroxylapatite chromatography in the presence of sodium dodecyl sulfate. N- and C-terminal amino acid sequence analyses of these peptides were carried out in order to identify the tryptic cleavage sites responsible for the formation of these fragments. Both A1a and A1b fragments had the same C-terminal sequence as A1 fragment. Single cleavage of A1 at T3a (Lys218-Ala219) yielded A1a, while a cleavage between either Lys234-Ile235 or Arg236-Asp237 (collectively designated as T3b) resulted in A1b fragment. Thus, A1a and A1b fragments differed from A1 fragment only by their loss of short stretches corresponding to the N-terminal region of the latter. On the other hand, C fragment represented the C-terminal half of B fragment (Ala506-Gly994). It had the same C-terminal sequence as B fragment and was produced by cleavage at T4 (Lys728-Thr729). Cleavages at T3a and T3b profoundly affected the catalytic properties of SR-ATPase (Imamura, Y. and Kawakita, M. (1986) J. Biochem. 100, 133-141), and it was suggested that the segment of the ATPase molecule including the region between Ala199 and Arg236 is important in mediating the coupling between ATP splitting and Ca2+-transport.

Amino Acid Sequence↗