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PubMed2003

A novel bioartificial liver containing small tissue fragments: efficiency in the treatment of acute hepatic failure induced by carbon tetrachloride in rats.

The efficiency of a new bioartificial liver (BAL) containing small tissue fragments in the treatment of acute hepatic failure induced by carbon tetrachloride in rats was evaluated. A day after injection (i.p.) of CCl4 the animals were connected to a BAL containing liver fragments (fragment BAL) and a BAL containing no liver fragments (no-fragment BAL), and extracorporeal hemoperfusion was carried out for 4 h. The activities of alanine transaminase and lactate dehydrogenase as well as the concentrations of ammonia, glucose, urea, and amino acids in plasma were measured. A tendency to the stabilisation of ammonia, glucose, phenylalanine, tyrosine, and other amino acids was revealed at the end of hemoperfusion in poisoned rats connected to the fragment BAL. A statistically significant difference in survival between the animals connected to the fragment BAL and no-fragment BAL was found. The results obtained indicate that the bioreactor containing small liver fragments is effective in the treatment of acute hepatic failure in animals.

Amino Acids↗
PubMedDate not supplied

Human fetal cortical tissue fragments survive grafting following one week storage at +4 degrees C.

Grafting of human fetal tissue fragments has been used successfully in experimental and clinical trials. The development of techniques to store human fetal tissue fragments for longer time periods would allow to establish temporary tissue banks. We dissected several human cortical tissue fragments from one fetus and tested different storage conditions (cooling, freezing, culturing). After storage, the tissue fragments were transplanted into cavities in the cortex of host rats and the volume of the surviving grafts calculated. We report that human cortical tissue fragments grafted immediately after dissection (control group) or grafted after storage for 3 h in cryopreservation medium at room temperature survived grafting and resulted in graft sizes of 102 +/- 26 mm3 and 242 +/- 210 mm3, respectively, however, statistically not different. When the human cortical tissue fragments were slowly frozen and stored for 1 wk and/or when the fragments were cultured for 1 week in culture medium using a roller tube technique, grafts did not survive under our conditions. However, when the human cortical tissue fragments were stored for 1 week at +4 degrees C in cryopreservation medium, the graft size (48 +/- 24 mm3) was reduced but statistically not different from the control group. We conclude that human cortical tissue fragments can be stored at +4 degrees C for at least 1 wk without major loss of ability to survive grafting.

Abortion, Induced↗
PubMed2006

Quantification of fragments of human serum inter-alpha-trypsin inhibitor heavy chain 4 by a surface-enhanced laser desorption/ionization-based immunoassay.

BACKGROUND: Several proteolytically derived fragments from the proline-rich region (PRR) of human inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) have been identified by surface-enhanced or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS or MALDI-TOF-MS) as potential disease markers. METHODS: Previously, we developed a SELDI-based immunoassay that can simultaneously distinguish and quantify multiple isoforms/variants of a protein/peptide of interest. In this study, we used this high-throughput approach to quantify and characterize the extensive fragmentation within the PRR of human serum ITIH4 and determined its association with different disease conditions. The ITIH4-related fragments were first immunocaptured by use of beads coupled with peptide-specific antibodies. The eluates were then studied by SELDI-TOF-MS. In addition, freshly collected and immediately processed serum and plasma samples were used to analyze the ex vivo stability of these ITIH4 fragments. RESULTS: Human serum ITIH4 was shown to be extensively proteolytically processed within the PRR, and its fragmentation patterns were closely associated with different disease conditions. Fragmentation patterns were generally consistent with cleavages by endoprotease followed by exoprotease actions. Observed fragments changed little under different assay conditions or blood collection and processing procedures. CONCLUSIONS: The fragmentation patterns within the PRR of human serum ITIH4 are associated with different disease conditions and may hold important diagnostic information. These fragmentation patterns could be useful as potential biomarkers for detection and classification of cancer.

Adult↗
PubMed1992

Ultraviolet B-induced DNA fragmentation (apoptosis) in activated T-lymphocytes and Jurkat cells is augmented by inhibition of RNA and protein synthesis.

UVB (280-320 nm) light profoundly affects cellular immunity and immunogenicity. To further characterize the interaction of UVB light with T cells, we examined UVB-induced DNA fragmentation patterns in normal T cells and the T-cell line Jurkat. Resting or preactivated peripheral blood T cells from normal donors or Jurkat cells were exposed to various doses of UVB or gamma irradiation. Cells were sampled at 0-48 h after exposure, and DNA fragmentation was analyzed by electrophoresis in agarose gels. UVB and gamma irradiation, in a dose- and time-dependent manner, induced DNA fragmentation in Jurkat cells and in T cells preactivated with phytohemagglutinin (PHA; 10 micrograms/ml) and phorbol myristate acetate (PMA; 10 ng/ml), but not in resting T cells. The presence of RNA or protein synthesis inhibitors such as actinomycin D or cycloheximide neither inhibited nor delayed DNA fragmentation; in fact, DNA fragmentation was augmented above control values. Similarly, DNA fragmentation increased in the presence of the calcium-chelating agent ethylene-glycol-bis-tetraacetic acid (EGTA) and decreased in the presence of the calcium ionophore A23187. In the presence of ethylenediaminetetraacetic acid (EDTA), DNA fragmentation decreased. In summary, these data show that UVB-induced DNA fragmentation strongly depends upon the cell activation status, and upon the presence of divalent cations other than calcium, possibly magnesium. The data indicate furthermore that in this model, inhibition of RNA or protein synthesis can induce rather than inhibit apoptosis, suggesting that the synthesis inhibitors disrupted primarily the synthesis or action of enzymes ordinarily aimed at repairing DNA fragmentation.

Calcium↗
PubMed1992

Interactions of diphtheria toxin B-fragment with cells. Role of amino- and carboxyl-terminal regions.

The B-fragment of diphtheria toxin binds to cell surface receptors and facilitates entry of the enzymatically active A-fragment into the cytosol. The roles of the amino- and carboxyl-terminal regions of the B-fragment in interactions with the cell membrane were studied by measuring specific binding, insertion into membranes at low pH, and formation of cation-selective channels, as well as by toxicity measurements after association with active A-fragment. Deletion of the amino-terminal 12 amino acids of the B-fragment did not affect its ability to bind to receptors and to form ion channels at low pH, whereas both abilities were strongly impaired when one more amino acid (Trp206) was removed. Replacement of the amino-terminal 31 residues with an amphipathic sequence from human apolipoprotein A1 restored receptor binding but not ion channel formation. The binding to cells was virtually abolished when 9 residues were deleted from the carboxyl terminus. Deletion of only 4 residues or extension by 12 residues did not prevent specific binding, but reduced insertion, channel formation, and toxicity. Those deletions that reduced receptor binding ability increased the trypsin sensitivity of the B-fragment. The results indicate that the amino- and carboxyl-terminal regions of diphtheria toxin B-fragment are important for receptor binding, possibly because they contribute to keep the B-fragment in a binding-competent conformation. Small alterations in the carboxyl-terminal end reduced insertion, channel formation, and toxicity more than the ability of the B-fragment to bind to cells.

Amino Acid Sequence↗
PubMed2003

The role of an 80 kDa fragment of E-cadherin in the metastatic progression of prostate cancer.

PURPOSE: The purpose of this study was to evaluate an 80 kDa proteolytic fragment of E-cadherin as a potential biomarker for prostate cancer progression and to identify putative proteases that are responsible for the cleavage of E-cadherin. EXPERIMENTAL DESIGN: A wide spectrum of prostate cancer tissue and serum specimens representing different stages of prostate cancer was examined for the accumulation of the 80 kDa fragment of E-cadherin. Additionally, an expression array analysis was used to identify putative proteases that may have been involved in the cleavage of E-cadherin. RESULTS: A reproducible E-cadherin fragment was detected as a strong 80 kDa band in tissue samples. This fragment was detectable almost exclusively in metastatic sites. It was not visible in normal prostate tissue and was weak in 1 of 16 localized prostate cancers. The fragment is shed into the extracellular space and was detectable in patient serum in which the expression of the fragment showed a strong association with advanced prostate cancer. On the basis of cDNA expression analysis, several members of the metalloproteinase family could be identified as potentially responsible for the cleavage of the fragment from full-length E-cadherin. CONCLUSIONS: In this study, we present the first report of serum levels of the 80 kDa fragment of E-cadherin in prostate cancer patients. This fragment is exclusively seen in neoplastic prostate tissue and may represent a useful biomarker of prostate cancer disease progression. This study also demonstrates an association of increased levels of several metalloproteinases with metastatic prostate cancer and could provide a useful correlation between metalloproteinase expression/activity and E-cadherin cleavage and the metastatic progression of prostate cancer.

Blotting, Western↗
PubMedDate not supplied

Improved histopathological evaluation of gliomas using tissue fragments obtained by ultrasonic aspiration.

Ultrasonic aspirators are commonly used to resect brain tumors because they allow safe, rapid and accurate removal of dissected tissue. However, the tissue fragments removed by ultrasonic aspirators are used surprisingly little in daily diagnostics and in clinical research. A comparison between diagnoses made on the tissue fragments removed by the Sonocut ultrasonic aspirator and the tissue in conventional tumor biopsies was made. The correspondence between the amount of Sonocut tissue analyzed and the probability of making the most malignant diagnosis was analyzed statistically in order to limit the amount of tissue fragments, which need to be analyzed, and in order to save tissue for other purposes like molecular genetics and in vitro drug sensitivity assays. Thirty cases were included in the present study and consisted of astrocytomas, glioblastomas, oligodendrogliomas and ependymomas. The results showed that in 8 out of 30 cases only the Sonocut tissue fragments contained the tumor components that provided the most malignant diagnosis. In further 20 cases, the Sonocut tissue fragments and conventional tumor biopsies gave the same diagnoses. In the remaining 2 cases, the most malignant foci were included in the biopsy removed for peroperative frozen section investigation. When the slides with Sonocut tissue fragments were analyzed, the probability of making the most malignant diagnosis increased from 81.3% - 99.1%, when slides from 1 - 5 paraffin blocks were analyzed, respectively. When subgroups of small, medium and big tumors were analyzed, it was found that only 2 paraffin blocks from small tumors need to be prepared to reach 98.3% probability of making the most malignant diagnosis, whereas 5 paraffin blocks from big tumors need to be prepared to reach a 96.8% probability. In conclusion, the study shows that a limited amount of Sonocut ultrasonic tissue fragments improve the diagnostic evaluation of gliomas. These tissue fragments therefore must not be discarded. Only few paraffin blocks need to be prepared to reach close to 100% probability of making the most malignant diagnosis, reducing the amount of slides, which have to be analyzed and saving Sonocut tissue fragments for future use in molecular genetics and drug sensitivity assays.

Astrocytoma↗
PubMed1992

Preparation and preliminary evaluation of technetium-99m-labeled fragment E1 for thrombus imaging.

Fragment E1 labeled with 123I has been previously shown to permit imaging of thrombi in patients within as little as 20 min after injection. Because of the relatively rapid localization and blood disappearance of this protein, 99mTc would be the most clinically acceptable radionuclide for labeling Fragment E1. In this study, human fragment E1 was derivatized with a hydrazino nicotinate function to permit radiolabeling with reduced technetium. The modification reaction was carried out while the fragment E1 was protected in a complex, so that the modification occurred in nonfunctional regions of the fragment E1 molecule. After radiolabeling with 99mTc, the modified fragment E1 retained its functional activity, as judged by its binding to fragment DD in vitro. The ability of 99mTc-fragment E1 to produce images of venous thrombi was demonstrated in animal models. Images were focally positive within 20 min to 1 hr after injection. Thrombus-to-blood ratios exceeded those from 125I-fibrinogen in the same animals. This method of labeling appears to provide an alternative radiolabel to 123I without compromising the function of fragment E1.

Animals↗
PubMed1991

Induction of megabase DNA fragments by 5-fluorodeoxyuridine in human colorectal tumor (HT29) cells.

Current evidence suggests that DNA fragmentation plays an integral role in mediating cytotoxicity that results from thymidine nucleotide depletion ("thymineless death"). Recently, Ayusawa et al. [Mutat. Res. 200:221-230 (1988)] reported that dTMP starvation induces cellular processes that result in the release of 50-200-kilobase (kb) DNA fragments in FM3A cells, as detected by pulsed field gel electrophoresis (PFGE). The present study was undertaken to determine whether a similar DNA fragmentation process occurs in a human cell line in response to fluoropyrimidine treatment and, if so, to quantitate this process. When human colorectal tumor (HT29) cells were treated with 100 nm 5-fluorodeoxyuridine (FdUrd), this regimen induced the formation of high molecular weight DNA fragments, which were analyzed using three different PFGE protocols. Field inversion PFGE revealed that, in contrast to the discrete size range reported for FM3A cells, FdUrd-induced fragments in HT29 cells were broadly distributed from about 50 kb to sizes beyond the resolution of the field inversion mode (i.e., greater than 600 kb). Analysis of these same samples by contour-clamped homogeneous electric field PFGE showed that the bulk of these fragments migrated in the 1-5-megabase region. In contrast, fragments from DNA that was broken randomly by gamma-radiation appeared to be primarily in a zone corresponding to approximately 5-10 megabases. Equitoxic FdUrd and radiation treatments (100 nM FdUrd for 48 hr versus 10-Gy gamma-radiation) each increased the fraction of DNA entering the gel from about 0.07-0.09 (untreated cells) to about 0.22-0.25. To a first approximation, the time course and quantity of DNA fragmentation induced by 100 nM FdUrd appeared to correlate with the loss of clonogenicity within the 48-hr period analyzed. These findings suggest that the processes responsible for DNA fragmentation in response to a thymineless state may be different in FM3A and HT29 cells, that in both cases the breaks caused do not appear to be located randomly with respect to the entire genome, and that these processes may be related to the chain of events by which temporary dTMP starvation is made into a lethal insult.

Cell Survival↗
PubMed1991

Induction of differentiation by tumour necrosis factor in HL-60 cells is associated with the formation of large DNA fragments.

The DNA fragmentation induced by tumor necrosis factor (TNF) of differentiable human myeloid leukemic HL-60 cells has been further characterized. TNF increased the appearance of very high molecular weight DNA fragments detected by agarose gel electrophoresis. The use of pulsed-field gel electrophoresis (PFGE) revealed these fragments to be as high as 200-400 kilobase pairs. The pattern of HL-60 DNA fragmentation contrasted with that of U937 cells, which exhibited lower molecular weight, nucleosome multiple sized fragments, and greater cytotoxicity in response to TNF. The peak increase of fragments from HL-60 occurred between one and two hours of incubation, with TNF concentrations of 10 U/ml or higher, and was inhibitable by 1 mM Zn2+. Southern blotting of these fragments disclosed enrichment for c-myc related sequences compared with control probes including beta-actin and kappa and lambda light chains. Treatment of DNA with NotI or gamma-irradiation, followed by PFGE, disclosed a class of still higher molecular weight DNA, which decreased following TNF treatment, and which was apparently the precursor of the TNF-induced fragments. TNF thus rapidly increases a class of high molecular weight DNA fragments which are enriched for c-myc related sequences and may arise preferentially from higher molecular weight structures which are detectable following linearization by NotI or gamma-irradiation. Such major but non-random alterations in chromatin structure may contribute to TNF-induced monocytoid differentiation of HL-60.

Blotting, Southern↗
PubMed1990

Complementation of two overlapping fragments of SecA, a protein translocation ATPase of Escherichia coli, allows ATP binding to its amino-terminal region.

SecA is a protein translocation ATPase. The secA gene was engineered so as to code for SecA fragments of different sizes, either from the amino terminus or the carboxyl terminus. These SecA fragments, most of which formed aggregates in the cytosol, were overproduced and then purified in the presence of 6 M guanidine hydrochloride. The fragments were renatured by means of dilution and dialysis, and then examined as to their ability to interact with ATP by means of photoaffinity cross-linking with [alpha-32P]ATP. Individual SecA fragments thus renatured were inactive as to ATP binding. However, when two fragments (amino- and carboxyl-terminal ones), which structurally complemented each other and which had an overlapping region, were mixed, cross-linking was observed at the amino-terminal segments. The cross-linking was appreciably enhanced when two such fragments were first mixed together in 6 M guanidine hydrochloride and then renatured. It is concluded that SecA has an ATP-binding domain near its amino-terminal region and that the binding requires a carboxyl-terminal fragment that is large enough to cover the region deleted from the amino-terminal fragment. An amino-terminal fragment, which constituted about 92% of the entire SecA molecule, was active in not only ATP binding but also protein translocation. Based on these findings, the structure-function relationship of SecA is discussed.

Adenine Nucleotides↗
PubMed1989

Activation of a suicide process of thymocytes through DNA fragmentation by calcium ionophores and phorbol esters.

Calcium ionophore, A23187, is known to be a comitogen, but it activates a suicide process characterized by DNA fragmentation at linker regions in mouse immature thymocytes. It did not induce DNA fragmentation in T lymphocytes prepared from lymph node and spleen cells. Induction of DNA fragmentation by A23187 depends on protein phosphorylation and synthesis of mRNA and protein, because an inhibitor of protein kinase, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7), actinomycin D, and cycloheximide, respectively, inhibits the DNA fragmentation and cell death. Studies adding the inhibitors at various times show that protein phosphorylation and mRNA synthesis occur within a few hours after incubation with A23187 followed by the protein synthesis responsible for inducing DNA fragmentation. Phorbol esters, 12-O-tetradecanoyl 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBD), which are capable of activating protein kinase C, also induced similar DNA fragmentation in immature thymocytes, followed by cell death. PBD committed the suicide process after 6 h of incubation, because the DNA fragmentation above the control level was not induced when PDB was removed from the medium before 6 h of incubation. A23187 or a phorbol ester alone induced DNA fragmentation followed by cell death, whereas the addition of TPA at low concentration inhibited the DNA fragmentation induced by A23187 accompanied with an increase in DNA synthesis. The result suggests that TPA switched a suicide process induced by A23187 to an opposite process: stimulation of DNA synthesis. Physiologic factors and mechanisms which regulate cell proliferation and death in the thymus are not known at present, but the signals by protein kinases and calcium ions may regulate both cell proliferation and death, independently, synergistically or antagonistically.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Granulocytic fragments in sepsis.

We report here three patients with sepsis and one with acute pancreatitis and possible sepsis who developed granulocytic fragments on blood smears obtained prior to death. In case 1, these fragments were identified cytochemically. In case 3, granulocytic cytoplasmic projections and fragments were identified by electron microscopy of the buffy coat. All patients had leukerythroblastosis. The average corrected white blood count (WBC) was 46 X 10(9)/liter with 34 nucleated red blood cells (nRBC)/100 WBC. Patient 1 had thrombocytosis whereas patients 2, 3, and 4 were thrombocytopenic. Terminal complement levels were decreased in patients 3 and 4 as previously noted in sepsis (Sprung CL, Shultz DR, Marcial E, et al.: Complement activation in septic shock patients. Crit Care Med 14:525, 1986). A general correlation between nRBC and granulocytic fragments/100 hpf (high power field) was observed in patients 3 and 4. Granulocytic fragments were not identified on the blood smears of several patients with leukemoid reactions without erythroblastosis. Although the precise etiology of these fragments is unclear, we believe their recognition is important because all patients died within 32 hours after granulocytic fragments were identified. Furthermore, these fragments can falsely elevate the platelet count. Although myeloid fragments have previously been noted in leukemia and lymphoma, this is the first report of their association with conditions unrelated to hematologic neoplasms. These fragments can easily be recognized by careful examination of the blood smear and represent a newly recognized aspect of the septic shock syndrome.

Anemia, Myelophthisic↗
PubMed1988

[Cloning of the DNA BglII fragments of bacteriophage T4 in the vector plasmid pSCC31].

An attempt has been made to clone six BglII fragments of T4 DNA in the range of 3.3-8.1 kb in the vector plasmid pSCC31 containing a single BglII site within the gene for endonuclease EcoRI and pL promoter of phage lambda. DNA fragments were extracted from the corresponding bands of agarose gel. The following BglII fragments were cloned: the 3.3 kb fragment No. 9 containing a portion of gene 20, the gene 21 and a portion of gene 22; the 4.2 kb fragment No. 8.1 with genes 17, 18, 19 and a portion of gene 20; the 5.2 kb fragment No. 7.1 with genes 25-29 and a portion of gene 48. In the case of the fragment No. 7.1, the recombinant plasmids pRL705 and pRL707 with different orientation of phage DNA fragment were obtained. An attempt to clone the fragments No. 8.2 (4.2 kb), No. 7.2 (5.45 kb) and No. 6 (8.1 kb) was unsuccessful and this probably indicates the presence of the genes, whose products are deleterious to the growth of bacterial cell.

Bacterial Proteins↗
PubMed1988

The elevated natural killer sensitivity of targets carrying surface-attached C3 fragments require the availability of the iC3b receptor (CR3) on the effectors.

Human serum-treated Raji and Daudi cells were shown to bind C3 fragments on their surface as a consequence of their capacity to activate C via the alternative pathway. C3 molecules were detectable on the cell surfaces up to 24 h after serum exposure. The C3 fragment-coated cells showed increased sensitivity to spontaneous lymphocyte-mediated cytotoxicity. The effector lymphocytes involved in the enhanced cytotoxicity were NK cells with low buoyant density, carrying both CR3 and FcR. Blocking of the FcR and CR3 with F(ab)2 fragments from Leu-11c or Leu-15 mAb, respectively, did not influence the lysis of targets that did not carry C3 fragments. In contrast, the accessibility of CR3 on the effector lymphocytes was essential for the C3 fragment-mediated enhancement of cytotoxicity. In addition to the Leu-15 antibody, N-acetyl-D-Glucosamine, a compound known to block iC3b binding to CR3, also abrogated the C3 fragment-imposed effect. Our previous experiments showed that the C3 fragments bind to acceptor sites on target cells. The present experiments show that the C3 fragments fixed onto the target bind to CR3 on effector cells. These data substantiate the hypothesis that the bivalent C3 fragments, which are fixed on the targets, promote their interaction with lytic lymphocytes by bridging the two cells.

Binding Sites↗
PubMed1979

Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter.

Three DNA restriction fragments of established sequence containing the Escherichia coli lac genetic controlling regions were cloned. In each case a recombinant plasmid was constructed which was suitable for the subsequent large scale purification of the lac fragment. A 789-base pair HindII fragment, containing the lac operator, promoter, and cyclic AMP receptor protein binding site, was ligated into the single HindII site of the amplifiable plasmid minicolicin E1 DNA (pVH51). A 203-base pair Hae III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been "filled in" by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then constructed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hae III ends. Mapping studies on pVH51 DNA with a number of DNA restriction endonucleases, including Alu I, Taq I, and Hpa II, are described.

DNA Restriction Enzymes↗
PubMed1984

Physical characterization of a kinetoplast DNA fragment with unusual properties.

A 414-base pair fragment from a Leishmania tarentolae kinetoplast DNA minicircle has unusual physical properties. We reported previously that in comparison to phi X174 and pBR322 control fragments, the kinetoplast fragment behaves in gel electrophoresis, gel filtration, and electric dichroism experiments as if it has an unusually compact conformation. We accounted for these unusual properties by proposing that the fragment is a systematically bent helix (Marini, J.C., Levene, S.D., Crothers, D.M., and Englund, P.T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7664-7668). In this paper, we further explore the properties of the kinetoplast fragment. Because of its compact conformation, the kinetoplast fragment has difficulty in snaking through polyacrylamide gels and therefore migrates unusually slowly in electrophoresis experiments. Warming (53 degrees C) and ethanol (5-20%) partially normalize gel migration; glyoxal treatment results in denatured strands with electrophoretic mobility close to that expected for their size. In vivo modification does not appear to be responsible for the fragment's properties; its anomalous electrophoretic behavior persists after proteinase K treatment, phenol extraction, or after cloning into pBR322 and reisolation. Velocity sedimentation experiments rule out fragment aggregation. Secondary structure, such as a cruciform, is not detectable by S1 or mung bean nuclease digestion. The kinetoplast fragment has circular dichroism spectra characteristic of a B-type helix. With increasing temperature, there is an increase in the 270/280 ellipticity ratio. Circular dichroism spectra taken in the presence of ethanol show a B to A helix transition at unusually low ethanol concentrations (between 44 and 54% (w/w]. Thermal denaturation reveals a triphasic melting curve.

Animals↗
PubMed1982

Shedding of dense cell fragments into the follicular lumen early in involution of the hyperplastic thyroid gland.

Membrane-bounded fragments of thyroid epithelial cells are found in the lumen of the thyroid follicle at early stages of involution of the hyperplastic gland. To demonstrate this, thyroid glands were made hyperplastic by feeding rats a low iodine diet containing thiouracil for 3 weeks. Involution was induced by the feeding of a high iodine diet. Three types of dense cell fragments were observed in the lumen at its periphery within 5 hours after the change of diet: (1) single, relatively large fragments that contained vesicles occasionally, (2) a cluster of rounded fragments of relatively small size. Some of these had a narrow tail, and (3) elongated narrow fragments in a row parallel to the apical surface of one or more epithelial cells. The fragments are probably derived from the thyroid epithelial cells since no other cell types were generally present. By 14 hours the fragments were distributed randomly throughout the lumen. Fragments were no longer present by 12 days of involution in most follicles. Fragment formation may be a mechanism for disposal of excess plasma membrane deposited by exocytic vesicles during rapid secretion of thyroglobulin into the follicular lumen early in involution.

Animals↗