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PubMed1984

Agonist and antagonist properties of calmodulin fragments.

Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid (EGTA) or Ca2+ was performed according to a modification of the method of Drabikowski et al. (Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124-133). The resulting peptides were purified by reverse-phase high performance liquid chromatography. Tryptic digests in EGTA yielded peptides 1-106, 1-90, and 107-148 with yields of 9, 47, and 61%, respectively. The digests performed with Ca2+ yielded peptides 1-77 and 78-148 in 35 and 45% yield. Analysis by high performance liquid chromatography indicated that the purified fragments contained less than 0.1% contamination by calmodulin, thus allowing a definitive study of the ability of these fragments to activate, or interact with, calmodulin-regulated enzymes and anti-calmodulin drugs. Each of the fragments, except 107-148, bound to a phenothiazine affinity column in a Ca2+-dependent manner. Thus, calmodulin contains two interaction sites for phenothiazines: one on the NH2-terminal half (fragment 1-77) and one on the COOH-terminal half (fragment 78-148). None of the fragments activates the protein phosphatase, calcineurin, or prevents its stimulation by calmodulin, nor does any of the fragments stimulate Ca2+-dependent cAMP phosphodiesterase. A single cleavage in the middle of the calmodulin molecule results in the rapid dissociation of the two resultant fragments and a loss of ability to activate cAMP phosphodiesterase. One fragment, 78-148, interacts with phosphodiesterase and prevents its activation by calmodulin (Ki: 1.5 +/- 0.4 X 10(-6) M). The same fragment, 78-148, can fully activate phosphorylase kinase but with a lower affinity than calmodulin (Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141-145). Thus, peptide 78-148 behaves as a calmodulin agonist or antagonist or as neither, depending on the enzyme under study.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1984

Isolation and characterization of a 33,000-dalton fragment of complement Factor B with catalytic and C3b binding activity.

Factor B is the zymogen of the catalytic site bearing subunit Bb of the C3/C5 convertase of the alternative pathway of complement. In this study, the location of the C3b binding site and the catalytic site within the Bb subunit were investigated. When human Factor B was treated with porcine elastase, fragments with respective molecular weights of 36,000, 35,000, 33,000, 31,000, and 25,000 were generated. Binding studies showed that only the 33,000-dalton fragment was capable of binding to C3b. The 33,000-dalton fragment was purified using fast protein liquid chromatography and found to be part of the Bb fragment upon testing with monoclonal antibody 15-6-19-1. Amino-terminal amino acid sequence analysis of the 33,000-dalton fragment placed it in the C-terminal half of Bb. The fragment expressed esterolytic activity as evidenced by cleavage of the synthetic substrate N alpha-acetyl-glycyl-L-lysine methyl ester and restored alternative pathway activity in Factor B-depleted serum. Its hemolytic activity was approximately 60-fold lower than that of Factor B. Comparative binding studies in the presence of metal ions using zymosan-C3b showed that the 33,000-dalton fragment bound to C3b with higher affinity than Factor B. Addition of the fragment to human serum inhibited alternative pathway activation by rabbit erythrocytes due to its high affinity for C3b and its low hemolytic activity compared to Factor B. These results show that the C-terminal 33,000-dalton portion of Bb contains not only the enzymatic site of Bb but also a C3b binding site which confers hemolytic activity upon the fragment. The observation that the fragment inhibited alternative pathway activation suggests that a synthetic peptide may be constructed that exhibits negative regulator activity in the alternative pathway.

Amino Acid Sequence↗

PubMed1984

Regeneration of native bacteriorhodopsin structure from fragments.

The previously described chymotryptic fragment of bacteriorhodopsin, C-2 (amino acids 1-71), is cleaved by 70% formic acid to two fragments, A-1 (amino acids 1-36) and A-2 (amino acids 37-71), which have been separated by high pressure liquid chromatography. The fragments A-1 and A-2, separately or together, are not able to replace C-2 in forming a stable bacteriorhodopsin-like complex with the fragment C-1 (amino acids 72-248) and all-trans-retinal. A second set of bacteriorhodopsin fragments, B-1 (amino acids 1-155) and B-2 (amino acids 156-248), have been prepared by sodium borohydride cleavage of bacteriorhodopsin. Following denaturation, fragments B-1 and B-2 reassociate in the presence of retinal to regenerate the native bacteriorhodopsin chromophore (approximately 40%). Fragment B-1 also interacts with fragment C-1 and all-trans-retinal to form a complex with spectral properties and secondary structure similar to those of bacteriorhodopsin. Vesicles prepared from the reconstituted fragment complexes (B-1 + B-2) or (B-1 + C-1), show proton pumping activities comparable to the previously described activity fragments C-1 and C-2.

Bacteriorhodopsins↗

PubMed1994

Evidence that the cation occlusion domain of Na/K-ATPase consists of a complex of membrane-spanning segments. Analysis of limit membrane-embedded tryptic fragments.

Digestion of renal Na/K-ATPase with trypsin, in the presence of rubidium and absence of calcium ions, produces so-called "19-kDa membranes," containing a C-terminal 19-kDa and smaller fragments (8-12 kDa) of the alpha chain, and a beta chain either intact or split into two fragments (Karlish, S. J. D., Goldshleger, R., and Stein, W.D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4566-4570). Cation occlusion is intact. The cation sites are thought to be located within trans-membrane segments, but the identity and number of segments involved is unknown. Analysis of Ca(2+)-induced sensitization of 19-kDa membranes to proteolysis, and characterization of the limit membrane-embedded fragments, has provided some insight into this question. Calcium ions have been shown to compete with two rubidium ions for occlusion sites on 19-kDa membranes, with a high affinity (KD approximately 2.8 microM, pH 7.5, 20 degrees C). The kinetics of displacement of rubidium by calcium ions indicate that competition is direct and is not an allosteric antagonism. At 37 degrees C, reversible displacement of rubidium ions by calcium ions is followed by an irreversible thermal inactivation of rubidium occlusion. Calcium ions partially protect rubidium occlusion sites against modification by the carboxyl reagent, N,N'-dicyclohexylcarbodiimide. We propose that calcium ions, like rubidium ions, recognize carboxyl groups at the entrance to the cation sites, but the calcium ions do not become occluded and thus fail to protect 19-kDa membranes against further proteolysis or thermal inactivation. Upon displacement of occluded rubidium, trypsin digests the Ca(2+)-bound and thermally inactivated 19-kDa membranes, and all of the membrane-embedded fragments are truncated or are split in these conditions. A related finding is that the C-terminal sequence of the 19-kDa fragment (and alpha chain), E-T-Y-Y, is digested by carboxypeptidase Y only when the rubidium occlusion is inactivated. Identification of the limit tryptic fragments indicates that polypeptide loops and the C-terminal tail of the 19-kDa fragment, N and C termini of the smaller fragments of the alpha chain, and both N and C termini of a 16-kDa fragment of the beta chain are split by proteolytic enzymes upon displacement of occluded rubidium.4+ We conclude that all fragments of 19-kDa membranes form a complex, which is stabilized and protected against proteolytic enzymes upon occlusion of rubidium ions, and which relaxes upon displacement of occluded rubidium. The cation occlusion "cage" presumably consists of litigating groups from several trans-membrane segments.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of proteolytic fragments of human factor VIIa which inhibit the tissue factor-enhanced amidolytic activity of factor VIIa.

The interaction of circulating factor VII/VIIa with tissue factor presented by cells in extravascular tissues represents the initial event in the extrinsic pathway of blood coagulation. To determine the tissue factor binding domains in human factor VIIa, we have subjected recombinant human factor VIIa to tryptic digestion and isolated two proteolytic fragments (molecular mass = 32 and 20 kDa) by a combination of immunoaffinity chromatography and reversed phase high performance liquid chromatography (HPLC) which strongly inhibits the tissue factor-enhanced amidolytic activity of factor VIIa and inhibits the activation of factor X by factor VIIa in the presence of tissue factor. The 32-kDa factor VIIa fragment consisted of residues 1-137/143 from the light chain of factor VIIa connected by a disulfide bond to residues 153-277 from the heavy chain. The 20-kDa factor VIIa fragment consisted of residues 1-137 of the light chain of factor VIIa in disulfide linkage with residues 248-266 of the heavy chain. The 32- and 20-kDa factor VIIa fragments inhibited the tissue factor apoprotein-enhanced factor VIIa amidolytic activity with Ki values of 35 and 65 nM, respectively. The Ki values for the inhibition of relipidated tissue factor apoprotein-enhanced factor VIIa amidolytic activity by the 32- and 20-kDa factor VIIa fragments were 70 and 610 nM, respectively. Factor X activation by factor VIIa-relipidated tissue factor was inhibited half-maximally by the 32- and 20-kDa factor VIIa fragments at 65 and 680 nM concentrations, respectively. Equilibrium binding studies indicated that the 32- and 20-kDa factor VIIa fragments interacted with cell surface tissue factor expressed on J82 cells in a specific and saturable manner with Kd values of 30 and 64 nM, respectively. In addition, a peptide consisting of residues 1-109 from the light chain of factor VIIa obtained by reduction and HPLC of the 20-kDa factor VIIa fragment retained inhibitory activity, but the selective removal of the gamma-carboxyglutamic acid domain from the 20-kDa factor VIIa fragment by cathepsin G cleavage resulted in the complete loss of inhibitory activity in this fragment. Our data strongly suggest that the epidermal growth factor-like domains covalently linked to the gamma-carboxyglutamic acid domain in factor VIIa constitute the high affinity tissue factor binding domain in this molecule.

Amino Acid Sequence↗

PubMed1997

[Polymorphism of B1-associated DNA fragments from normal and transform ed murine hepatocytes].

Our study of B1-associated DNA fragment polymorphism in murine hepatocytes by means of polymerase chain reaction (PCR) allowed to reveal as many as 20 DNA fragments differing in their molecular masses (m. m.) and amount of amplified products varying within the range of 100-1000 bp. Within the same inbred strain of mice (C57B1/6), spectra of B1-associated DNA fragments were similar in different periods of ontogenesis (embryos of 15 and 20 days, adult mice), in different mice of the same or different litters. A comparative analysis of the spectra of B1-associated DNA fragments from hepatocytes of two inbred strains, C57B1/6 and C3HA, has shown in general their similarities in m. m. values. But a significant distinction, that was found, involved the presence of DNA fragments with m.m. approximately 600 bp in the spectra of B1-associated DNA fragments from C3HA strain mice, that is absent in the spectra of respective fragments from hepatocytes of C57B1/6 strain mice. The spectra of B1-associated DNA fragments from transformed hepatocytes of murine hepatoma MH-22a, in general, were the same as those from hepatocytes of C3HA strain mice. At the same time, a DNA fragment with m.m. of 450 bp, not detected in normal hepatocytes, was revealed in transformed ones. Nevertheless a DNA fragment with m.m. of 600 bp, characteristic of normal hepatocytes, was not observed in the transformed hepatocytes. The B1-PCR method can be used for studying genomic polymorphism both in different populations of mice, and during malignant growth.

Animals↗

PubMed1998

Distinct steps in DNA fragmentation pathway during camptothecin-induced apoptosis involved caspase-, benzyloxycarbonyl- and N-tosyl-L-phenylalanylchloromethyl ketone-sensitive activities.

Monocytic-like leukemia U-937 cells rapidly undergo morphological changes and DNA fragmentation that is typical of apoptosis following treatment with DNA topoisomerase I inhibitor [20-S-camptothecin lactone (CPT)]. The tripeptide derivative benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone blocks Asp-Glu-Val-Asp-ase (DEVDase) activity and prevents the occurrence of high molecular weight and oligonucleosome-sized DNA fragments associated with apoptosis in CPT-treated cells. In contrast, N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) does not prevent DEVDase activity and high molecular weight DNA fragmentation but completely abrogates the appearance of oligonucleosome-sized DNA fragmentation. These results suggest that caspase 3-like activities are involved with high molecular weight DNA fragmentation pathway, whereas TPCK-sensitive activities are involved in oligonucleosome-sized DNA fragmentation pathway in CPT-treated cells. Electron micrographs reveal that caspase inhibition by benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone also abrogates the typical morphological changes associated with apoptosis, whereas TPCK does not delay these morphological changes that are typical of apoptosis. Caspase inhibition slows passage of the cells through G2 and causes a transient accumulation of these cells at the G0/G1 phase of the cell cycle following CPT treatment. In a cell-free system, when purified nuclei are incubated with apoptotic cytosolic extracts obtained from CPT-treated U-937 cells, TPCK causes a similar effect in abrogating the oligonucleosome-sized DNA fragmentation but does not affect DEVDase activity. Addition of either benzyloxycarbonyl-Val-Ala-Asp-free carboxyl group or acetyl-Asp-Glu-Val-Asp-aldehyde completely inhibits DEVDase activity in these extracts. However, acetyl-Asp-Glu-Val-Asp-aldehyde does not affect the occurrence of oligonucleosome-sized DNA fragmentation in the cell-free system, whereas the benzyloxycarbonyl derivatives benzyloxycarbonyl-Val-Ala-Asp-free carboxyl group, benzyloxycarbonyl-Val-Ala-free hydroxyl group, benzyloxycarbonyl-Val-free hydroxyl group, and benzyloxycarbonyl hydrazide abolish it markedly. Taken together, these observations show the pivotal role of DEVDase activity in triggering the apoptotic process and high molecular weight DNA fragmentation, whereas TPCK- and benzyloxycarbonyl-sensitive activities are involved in the oligonucleosome-sized DNA fragmentation pathway induced by CPT.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Strategies to reduce renal radioactivity levels of antibody fragments.

Antibody fragments such as Fab and single-chain Fv fragments possess many advantages over intact antibodies as vehicles to deliver radioactivity for diagnostic and therapeutic applications. However, radiolabled antibody fragments exhibited high and persistent localization of the radioactivity in the kidney, which compromises diagnostic accuracy and therapeutic effectiveness. Recent studies indicated that the persistent localization of renal radioactivity would be originated from the re-absorption of glomerularly-filtered radiolabeled antibody fragments, followed by the retention of the radiometabolites generated after degradation in the lysosomal compartment of the renal cells. Two major approaches have been performed to reduce the renal radioactivity levels of antibody fragments. One is to block the reabsorption of radiolabeled antibody fragments themselves at the proximal tubular cells from the luminal fluid by administration of basic amino acids such as L-lysine. The other approach is to decrease the residence time of the radiometabolites within the lysosomal compartments of the renal cells by introducing a cleavable linkage between antibody fragments and radiometabolites of rapid urinary excretion. Another approach to reduce renal radioactivity levels of antibody fragments may be to release radiolabeled compound of urinary excretion from glomerularly-filtered antibody fragments before they are reabsorbed into the renal cells by the action of brush border enzymes present on the lumen of the renal proximal cells. In this paper, recent studies of the three approaches to reduce the renal radioactivity levels of antibody fragments are briefly reviewed.

Antibodies, Monoclonal↗

PubMed2005

Non-enzymatic hinge region fragmentation of antibodies in solution.

Liquid formulations of monoclonal antibodies (MAbs) typically undergo fragmentation near the papain cleavage site in the hinge region, resulting in Fab and Fab+Fc forms. The purpose of this study was to investigate whether this fragmentation is due to proteases. Four closely-related MAbs were exchanged into a pH 5.2 acetate buffer with NaCl and stored at -20 degrees C, 5 degrees C, 30 degrees C, or 40 degrees C for 1 month. Fragmentation generated size-exclusion chromatography (SEC) peak fractions that were analyzed by electrospray mass spectrometry to identify the cleavage sites. The effects of protein inhibitors or host cell proteins on fragmentation were also studied. The extent of fragmentation was equivalent for all four antibodies, occurring in the heavy chain hinge region Ser-Cys-Asp-Lys-Thr-His-Thr sequence. The fragment due to cleavage of the Asp-Lys bond showed two forms that differ by 18 Da. A synthetic peptide with the hinge region sequence terminating with Asp did not show fragmentation or the loss of 18 Da after incubation. Protease inhibitors did not affect rates of cleavage or modify sites of fragmentation. Degradation was not affected by host cell protein content. Fragmentation appears to be a kinetic process that is not caused by low levels of host cell proteases.

Amino Acid Sequence↗

PubMed2006

Population size and density of Colobus vellerosus at the Boabeng-Fiema Monkey Sanctuary and surrounding forest fragments in Ghana.

The Boabeng-Fiema Monkey Sanctuary (BFMS) is inhabited by a growing population of Ursine colobus (Colobus vellerosus), a species that is listed as vulnerable. Smaller, degraded forest fragments that surround the BFMS also contain C. vellerosus and may provide an important habitat for the monkeys. Our objectives were to 1) determine the current population size and density of C. vellerosus at BFMS and in five surrounding fragments, 2) examine the differences in demographics between the fragments and BFMS, and 3) determine whether a relationship exists between population density and fragment size and isolation distance from BFMS. The census was a complete count and was conducted for 1 month (July 2003) by S.W. and trained research assistants. Seven census routes were walked simultaneously on 13 days. The 2003 population estimate of C. vellerosus at BFMS was 217-241 individuals (15 groups), a slight increase from the 2000 census. Numbers in the fragments (58-62, six groups) have remained stable since 1997, when the only other census of these fragments was conducted. Mean group size did not differ between the fragments and BFMS. Larger fragments had larger numbers of colobus, but there was no relationship between fragment size and colobus density. Isolation distance had no effect on population density. Our data suggest that colobus probably travel between fragments. Conservation efforts should focus on treating the small forests and their connecting areas as a single conservation unit.

Animals↗

PubMed2007

Living in forest fragments reduces group cohesion in diademed sifakas (Propithecus diadema) in eastern Madagascar by reducing food patch size.

Forest fragmentation is thought to threaten primate populations, yet the mechanisms by which this occurs remain largely unknown. However, fragmentation is known to cause dietary shifts in several primate species, and links between food resource distribution and within-group spatial dynamics are well documented. Thus, fragmentation has the potential to indirectly affect spatial dynamics, and these changes may present additional stresses to fragmented populations. I present the results from a 12-month study of Propithecus diadema at Tsinjoarivo, eastern Madagascar, including two groups in fragments and two in continuous forest. Instantaneous data on activity and spatial position were collected during all-day focal animal follows. Fragment groups had much lower cohesion, being more likely to have no neighbor within 5 and 10 m. For continuous forest groups, cohesion was highest in the rainy season (when food patches are large) and lowest in winter (when the animals rely on small-crowned mistletoes), and the chance of having no neighbor within 5 m was positively correlated with mistletoe consumption. Thus their decreased cohesion in fragment groups is inferred to result from their increased reliance on mistletoes and other small resources, which causes them to spread out among multiple patches. This scenario is consistent with the reduced body mass of subordinate individuals (males and immatures) in fragments, and suggests the occurrence of steeper within-group fitness gradients. Further research is necessary to determine whether these patterns apply to other primates; however, since fragmentation tends to cause the loss of the largest trees, many primates in fragments may lose their largest food resources and undergo similar behavioral shifts.

Analysis of Variance↗

PubMed1991

Transient electric birefringence of two small DNA restriction fragments of the same molecular weight.

The transient electric birefringence of two small DNA restriction fragments of the same molecular weight, one of which migrates anomalously slowly on polyacrylamide gels, has been investigated. Both fragments exhibit negative birefringence. The decay of the birefringence of the anomalously slowly migrating fragment is 8-9% faster than that of the normally migrating fragment. The faster birefringence decay of the anomalous fragment 12A persists under a variety of buffer conditions, suggesting that it is due primarily to static bending and/or curvature of fragment 12A. In reversing electric fields the absolute amplitude of the birefringence of fragments 12A and 12B decreased about 26% before returning to the steady state value. The minimum in the birefringence occurred faster than expected from the birefringence decay times and decreased with increasing electric field strength, suggesting that the minimum is due to a slow polarization of the ion atmosphere. For both fragments, the rise of the birefringence in the Kerr region is about 10% slower than the field-free decay. The buildup of the negative birefringence is preceded either by an interval when no birefringence is observed or by a small positively birefringent transient, suggesting that a small transverse ionic polarizability is also present. Both DNA fragments exhibit Kerr law behavior over most of the range of electric field strengths investigated. Analysis of the shapes of the saturation curves suggests that differences may exist in the polarization mechanisms of the two fragments.

Base Sequence↗

PubMed2001

Comparison by restriction fragment differential display RT-PCR of gene expression pattern in bovine oocytes matured in the presence or absence of fetal calf serum.

A novel restriction fragment differential display (RFDD) RT-PCR has been used to compare patterns of mRNA expression in bovine oocytes matured in vitro in the presence (10%) or absence of fetal calf serum (FCS). Total RNA extracted from matured and denuded oocytes was processed using display Profile kit (Display System Biotech). RFDD RT-PCR products were separated on 6% polyacrylamide gel and analyzed using a Storm 860 scanner. Selected bands representing potentially differentially expressed fragments were excised from the gel and re-amplified. Re-amplified fragments with size matched to the original fragment were cloned into the TA vector and sequenced. Initially, 10 and 15 differentially expressed fragments were isolated from oocytes matured in the presence and absence of FCS, respectively. Eight out of 10 and 10 out of 15 fragments were re-amplified successfully as evidenced by size similarity to the original fragments. Finally, the size of six inserts sequenced from each group matched the size of corresponding original as well as re-amplified fragments. Sequence comparison search revealed similarity of some isolated fragments to 18s ribosomal RNA, bovine apolipoprotein A-I, bovine mitochondrion DNA, human CGI-79 mRNA, human Ab1-interactor protein, and bovine satellite DNA. The other sequenced fragments may represent novel genes. We showed that RFDD RT-PCR can be effectively applied to contrast gene expression pattern in bovine oocytes and that presence or absence of FCS during maturation interval affects gene expression pattern in matured bovine oocytes.

Animals↗

PubMed2003

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipids: ionization and prompt fragmentation patterns.

Ionization and prompt fragmentation patterns of triacylglycerols, phospholipids (PLs) and galactolipids were investigated using matrix-assisted laser desorption/ionization (MALDI). Positive ions of non-nitrogen-containing lipids appeared only in the sodiated form, while nitrogen-containing lipids were detected as both sodiated and protonated adducts. Lipids containing acidic hydroxyls were detected as multiple sodium adducts or deprotonated ions in the positive and negative modes, respectively, with the exception of phosphatidylcholines. The positive MALDI spectra of triacylglycerols contained prompt fragments equivalent to the loss of RCOO(-) from the neutral molecules. Prompt fragment ions [PL-polar head](+) were observed in the positive MALDI spectra of all phospholipids except phosphatidylcholines. The phosphatidylcholines produced only a minor positive fragment corresponding to the head group itself (m/z 184). Galactolipids did not undergo prompt fragmentation. Post-source decay (PSD) was used to examine the source of prompt fragments. PSD fragment patterns indicated that the lipid prompt fragment ions did not originate from the observed molecular ions (sodiated or protonated), and suggested that the prompt fragmentation followed the formation of highly unstable, probably protonated, precursor ions. Pathways leading to the formation of prompt fragment ions are proposed.

Galactolipids↗

PubMed1993

In vitro study to elucidate the physical laws concerning the fragmentation of both solitary and multiple artificial stones.

These in vitro studies define the basic physical laws regarding work and energy for the successful fragmentation of human gallstones. For this purpose a standardized stone model was used consisting of plaster and glass microspheres with physical properties similar to those of human gallstones. All experiments were performed using the lithotripter model MPL9000 (Dornier). The acoustic energy passing stones of 10-30 mm ranged between 8 and 90 mJ per pulse depending on the stone size and energy setting. These results represent the basis for the three following investigations. In the first experiment the relationship between fragmentation and shock wave energy was investigated in a basket with 2 mm mesh size. Thus no layer of small fragments could shadow the acoustic energy for further fragmentation of larger fragments. A constant amount of stone material was found to be fragmented per shock-wave pulse irrespective of stone volume. A low energy threshold (2 mJ/cm3) was observed, below which fragmentation did not occur. In the second experiment, the sieve was covered with a membrane, thus simulating the in vivo situation. The presence of a layer of small fragments hindered the further disintegration of the larger fragments. The attenuation depended to a large extent on original stone volume and acoustic energy per pulse. The corresponding attenuation factor increased with the original stone volume. Thus the fragmentation of a stone with a diameter of 30 mm was attenuated twice as much as a stone of 20 mm size. The critical layer thickness at which no further disintegration took place was 2.5 mm at 18 kV, 4.2 mm at 22 kV, and 5.0 mm at 26 kV.(ABSTRACT TRUNCATED AT 250 WORDS)

Biophysical Phenomena↗

PubMed1994

1-beta-D-arabinofuranosylcytosine-, mitoxantrone-, and paclitaxel-induced apoptosis in HL-60 cells: improved method for detection of internucleosomal DNA fragmentation.

We investigated the ability of different doses and durations of exposure to the chemotherapeutic drugs 1-beta-D-arabinofuranosylcytosine (Ara-C), mitoxantrone (MTN), and paclitaxel (taxol, TXL) to induce internucleosomal DNA fragmentation and apoptosis in human acute myeloid leukemia (AML) HL-60 cells in suspension culture. At clinically achievable concentrations, all three drugs have been shown to induce apoptosis in HL-60 cells. An improved method was developed for the isolation of pure genomic DNA and the detection of drug-induced internucleosomal DNA fragmentation in < 1.0 microgram of DNA sample by agarose gel electrophoresis. Morphologic evidence for apoptosis was determined by light microscopy following Wright staining, and cell viability was assessed by trypan blue dye exclusion. Internucleosomal DNA fragmentation was observed following exposure to 1.0 microM Ara-C for 4 h, which increased with 10 and 50 microM Ara-C. Incubation with 100 microM Ara-C produced internucleosomal DNA fragmentation starting at 3 h, which increased with longer periods of exposure to Ara-C. Utilizing a schedule of 1-h exposure followed by 3-h suspension in drug-free medium, 0.25 microM MTN was found to initiate DNA fragmentation, which increased with exposure to 1.0 and 5.0 microM MTN. However, identical treatment with higher concentrations of MTN resulted in random DNA degradation. Alternatively, continuous exposure to 1.0 microM MTN for 3 h was necessary to initiate internucleosomal DNA fragmentation. This increased with exposure intervals of up to 6 h. Exposure to TXL concentrations as low as 0.01 microM for 24 h caused internucleosomal DNA fragmentation, which increased with dose escalation (0.05, 0.1, 0.5, and 1.0 microM) of TXL. Although continuous exposure to 1.0 microM TXL for a period as short as 8 h produced internucleosomal DNA fragmentation, this increased significantly with longer exposure intervals. In general there appears to be a threshold concentration and duration of exposure below which none of these three drugs activates endonucleolytic internucleosomal DNA fragmentation and apoptosis. This threshold is lower for the DNA-interactive drugs MTN and Ara-C but higher for the non-DNA-interactive drug TXL. Higher doses or prolonged treatments with the drugs produce random DNA fragmentation associated with necrotic cell death. These in vitro results may further improve our understanding of the antileukemic cytotoxic effects of these drugs, which may enable a more rational design of drug regimens for optimal treatment of AML.

Antineoplastic Combined Chemotherapy Protocols↗

PubMedDate not supplied

Angiogenic potential of microvessel fragments established in three-dimensional collagen gels.

During angiogenesis, the microvasculature displays both vessel remodeling and expansion under the control of both cellular and extracellular influences. We have evaluated the role of angiogenic and angiostatic molecules on angiogenesis in an in vitro model that more appropriately duplicates the cellular and extracellular components of this process. Freshly isolated microvessel fragments from rat adipose tissue (RFMF) were cultured within three-dimensional collagen I gels. These fragments were characterized at the time of isolation and were composed of vessel segments observed in the microvasculature of fat in situ (i.e., arterioles, venules, and capillaries). Fragments also exhibited characteristic ablumenally associated cells including smooth muscle cells and pericytes. Finally, fragments were encased in an extracellular matrix composed of collagen type IV and collagen type I/III. The elongation of microvascular elements was subsequently evaluated using morphologic and immunocytochemical techniques. The proliferation, migration, and elongation of cellular elements in microvessel fragments from rat adipose tissue was dependent on initial fragment density, matrix density, and required serum. Inclusion of endothelial cell growth factors to microvessel fragments from rat adipose tissue 3-D cultures resulted in the accelerated elongation of tube structures and the expression of von Willebrand factor in cells constituting these tubes. Molecules with reported angiostatic capacity (e.g., transforming growth factor and hydrocortisone) inhibited vessel tube elongation. In vitro methods have been developed to evaluate numerous mechanisms associated with angiogenesis, including endothelial cell proliferation, migration, and phenotypic modulation. Microvascular endothelial cell fragments described in this study represent an in vitro population of cells that accurately duplicate the in vivo microcirculatory elements of fat. The proliferation of cells and elongation of microvascular elements subsequently observed in three-dimensional cultures provides an in vitro model of angiogenesis. Microvascular formation in this system results from pre-existing microvessel fragments unlike tube formation observed when cultured endothelial cells are placed in three-dimensional gels. This form of tube formation from cultured endothelium is more characteristic of vasculogenesis. Thus, the formation of microvascular elements from microvessel fragments provides the opportunity to examine the mechanisms regulating angiogenesis in an in vitro system amenable to precise experimental manipulation.

Actins↗

PubMed2002

An immunohistochemical study of the clearance of apoptotic cellular fragments.

We investigated the distribution and fate of apoptotic bodies during human development and in the adult, using an antibody (M30) that recognizes a neo-epitope formed early in the apoptotic cascade by caspase cleavage of cytokeratin 18. In the fetus, we found extensive accumulation of M30-positive, non-phagocytosed fragments in the red pulp of the spleen, subcutaneous and submucosal vessels, the interstitium of the lung, and the glomerular mesangium of the kidneys. In the liver, M30-immunoreactive fragments were found inside macrophages in the sinusoids. The number of these fragments and the intensity of the immunostaining increased with the gestational age of the fetus. In the adult, M30-positive fragments were barely detectable in normal tissues. However, many pathological situations, including both chronic degenerative processes and metastatic cancer, were associated with accumulation of M30-positive fragments in the red pulp of the spleen. In the liver and kidney, no fragments could be detected. Remarkably, 13 of the 16 patients with metastasized cancer showed pronounced accumulation of M30-positive fragments containing hematoxylin-reactive material in the red pulp of the spleen. In the non-cancerous cases, such DNA-containing fragments were only seen in 9 of 94 cases. The results show that when apoptotic activity is high, as during development in the fetus or during metastasis and other pathological processes in the adult, the phagocytic clearance of apoptotic bodies can be overloaded. These apoptotic fragments then accumulate in the spleen. The visual detection of apoptotic fragments is concluded to reflect increased cell turnover.

Adult↗