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PubMed2005

Chromatin supraorganization, DNA fragmentation, and cell death in snake erythrocytes.

In nucleate erythrocytes of several vertebrate groups, the frequency and intensity of DNA fragmentation associated with programmed cell death vary considerably. Although hemoglobin efficiency may be related to erythrocyte life span, and hemoglobin types and erythrocyte life spans are assumed to vary in reptiles, no data on DNA fragmentation and chromatin organization as related to cell death exist for snakes. In the present study, chromatin supraorganization, DNA fragmentation, and cell death were investigated in four snake species (Crotalus durissus terrificus, Bothrops jararaca, Bothrops alternatus, and Bothrops neuwiedii), which differ in their geographical distribution and habitats, by using image analysis of Feulgen hydrolysis kinetics, the TUNEL assay, single-cell gel electrophoresis, and transmission electron microscopy. Relatively few circulating erythrocytes were found to be simultaneously committed to cell death, although there was some variation among the snake species. Conspicuous nuclear and cytoplasmic organelles suggestive of metabolic activity were seen ultrastructurally in most snake erythrocytes. The DNA of the snake erythrocyte chromatin was much more resistant to Feulgen acid hydrolysis (DNA depurination and breakdown) than that of young adult bullfrog erythrocytes, which had a high frequency and intensity of DNA fragmentation. Of the species studied, B. neuwiedii and C. d. terrificus showed the greatest resistance to Feulgen acid hydrolysis and to the DNA fragmentation, revealed by the TUNEL assay. Although B. neuwiedii also showed the lowest frequency of cells with more damaged DNA in the single-cell gel electrophoresis assay, C. d. terrificus had the highest frequency of damaged cells, possibly because of the abundance of alkaline-sensitive DNA sites. The results for DNA fragmentation and cell death in erythrocytes of B. jararaca and B. alternatus generally differed from those for C. d. terrificus and B. neuwiedii and may reflect differences in the biology of these species selected under different geographical habitats. The differences in erythrocyte cell biology reported here may be related to hemoglobin variants selected in the mentioned snake species and that would lead the cells to different resistances to unfavorable environmental conditions.

Animals↗

PubMed1998

A reexamination of the phylogenetic position of Callimico (primates) incorporating new mitochondrial DNA sequence data.

The New World monkeys are divided into two main groups, Callitrichidae and Cebidae. Callimico goeldii shares traits with both the Cebidae and the Callitrichidae. Recent morphological phyletic studies generally place Callimico as the most basal member of the Callitrichidae. In contrast, genetic studies (immunological, restriction fragment, and sequence data) have consistently placed Callimico somewhere within the Callitrichidae, not basal to this clade. A DNA sequence data set from the terminal 236 codons of the mitochondrial ND4 gene and the tRNA(His), tRNA(Ser), and tRNA(Leu) genes was generated to clarify the position of Callimico. The sequences of 887 base pairs were analyzed by maximum-parsimony, neighbor-joining, and maximum-likelihood methods. The results of these various methods are generally congruent and place Callimico within the Callitrichidae between the marmosets (Callithrix and Cebuella) and the tamarins (Saguinus and Leontopithecus). Combined analyses of all suitable nuclear and mitochondrial gene sequences confirm the position of Callimico between the marmosets and the tamarins. As available molecular evidence indicates that Callimico is more closely related to the marmosets than to the tamarins, a reconsideration of the morphological evidence in light of the consensus tree from DNA sequence analyses is warranted. The marmosets and tamarins share four morphological characters (loss of the third molar, loss of the hypocone, reduced body size, reproductive twinning). Dwarfism may have evolved repeatedly among the Callitrichidae. It is well-known that the loss of a character can occur many times independently. The reproduction of marmosets and tamarins is extremely specialized and it is difficult to imagine that this complex and unique twinning system evolved separately in marmosets and tamarins. However, it is possible that a secondary reversal to single offspring took place in Callimico.

Animals↗

PubMed1995

A method for processing diffraction data from twinned protein crystals and its application in the structure determination of an FAD/NADH-binding fragment of nitrate reductase.

A general method to deconvolute oscillation data sets from twinned protein crystals to a corresponding single-crystal data set has been developed and applied to diffraction data measured from crystals of a fragment containing the FAD- and NADH-binding domains of nitrate reductase. The procedure allows straightforward processing of diffraction data from twinned crystals. Typically, R(merge) values of reduced data sets from the nitrate reductase crystals after deconvolution are about 0.06 compared to 0.13 and higher before deconvolution. Based on these deconvoluted data sets, the structure of the FAD- and NADH-binding domains of nitrate reductase could be solved successfully. The result indicates that crystal twinning does not necessarily prevent crystallographic structure determination.

Journal Article↗

PubMed2026

Advancing cancer detection and treatment using longitudinal routine clinical data.

Cancer management remains fragmented across its continuum, from late-stage diagnosis and salvage therapies to non-personalized surveillance. Here, we present Oncoformer, a unified multimodal transformer model trained on the China Oncology Multimodal Prediction and Surveillance Study (COMPASS) cohort (3.67 million individuals, 17.7 million clinical visits) and validated on independent external cohorts, including the UK Biobank. Oncoformer integrates longitudinal electronic health records with chest X-ray imaging to address multiple clinical tasks: pan-cancer diagnosis (area under the receiver operating characteristic curve [AUROC] = 0.956), future cancer prediction up to 1 year before diagnosis (AUROC = 0.869), tumor stage inference (mean AUROC > 0.90), patient-specific treatment-response forecasting, and recurrence-free survival stratification across ten cancer types (all p < 0.01). Staging predictions were independently validated against postoperative pathological endpoints and shown to converge on core cancer genomic pathways. By translating routine clinical data into a dynamic view of cancer evolution, Oncoformer provides a framework for risk-informed cancer prediction and treatment stratification using routine clinical data.

Humans↗

PubMed1999

Frequency of spontaneous chromosome aberrations in mice: effects of age.

In this paper, we present data on chromosome aberration frequencies in mice which served as unexposed controls in a variety of radiation and chemical toxicology experiments conducted in our laboratory in recent years. All chromosome aberration data were obtained by chromosome painting. In peripheral blood lymphocytes from 102 animals, the frequencies of translocations and insertions increased significantly with age. No increase with age was seen for dicentrics or acentric fragments. When the data were analyzed by strain, the age-related increase in translocation frequencies was observed only in the 71 homozygous C57BL/6 mice and not in any of the three heterozygous strains. Very few aberrations of any type were observed in 62 bone marrow samples, and no effect of age was seen for any aberration type in this tissue. These results are similar to those observed in unexposed humans, and suggest that the increase in translocations is not the result of accumulated damage from chronic 'background' environmental exposures but instead may be due to biological processes associated with aging.

Aging↗

PubMed2004

TGF-beta2-induced cell surface tissue transglutaminase increases adhesion and migration of RPE cells on fibronectin through the gelatin-binding domain.

PURPOSE: Migration and adhesion of dislocated retinal pigment epithelial (RPE) cells to a fibronectin-rich extracellular matrix is an initial step in proliferative vitreoretinopathy (PVR). In the present study, the functional role of cell surface tissue transglutaminase (tTG) in adhesion and migration of RPE cells on fibronectin (Fn) and collagen type I (Col I) after stimulation with TGF-beta2 was investigated. METHODS: Cultured human RPE cells were treated with 1.0 ng/mL TGF-beta2 for 24 hours. Cell surface tTG expression was determined by cell fraction analysis. Attachment on Col I, full-length Fn, and its 45-kDa gelatin-binding and 110-kDa cell-binding fragment was measured with an MTT assay. Migration of RPE cells was measured by a Boyden chamber assay, and cell spreading was determined. Experiments were performed in the presence or absence of anti-tTG antibodies and anti-integrin alpha5 and beta1 antibodies. RESULTS: TGF-beta2 markedly induced expression of cell-surface tTG on RPE cells and increased attachment and migration on Fn and Col I. Blocking cell surface tTG inhibited attachment, migration, and spreading on Fn and its 45-kDa gelatin-binding fragment, whereas no effect was seen on Col I and the 110-kDa cell-binding Fn fragment. In contrast, blocking of integrin alpha5 and beta1 suppressed adhesion and migration on full-length Fn and the 110-kDa Fn fragment. CONCLUSIONS: These data demonstrate that TGF-beta2 increases expression of cell surface tTG, which in turn strengthens adhesion, migration, and spreading of RPE cells on Fn through the 45-kDa gelatin-binding Fn fragment. At the onset of PVR, this mechanism may help RPE cells to attach and migrate on Fn-containing matrices.

Adolescent↗

PubMed2005

[Linking quantitation of electrophoresis pattern and data analysis in AFLP for Oncomelania hupensis].

OBJECTIVE: To search into a method for analyzing the quantitative data in amplified fragment length polymorphism (AFLP) electrophoresis. METHODS: Oncomelania snails collected from the field were screened. Forty snails found uninfected with schistosomiasis were divided randomly into two groups and used to isolate genomic DNA. AFLP electrophoresis pattern was first transformed into quantitative data by Glyko BandScan software, and the bands were read according to different standards of band-reading to acquire the corresponding data. These data sets were analyzed by genetic statistics to get an inference set, and the analysis of this inference set was performed to reach a summary description. RESULTS: The results of genetic variation from different standards of band-reading were different With the increase of the standard value of band-reading, the indices indicating the genetic polymorphism of Oncomelania hupensis population (e.g. Shannon's information index) also increased. When the standard value reached at certain level, the values of these indices began to decrease. Compared with the above indices, the change for gene flow turned out contrary to the genetic identity. The distributions of inference results from different standards of band-reading all showed significant normal distribution. The mean value of genetic variation based on total grey was very close to that on the proportion of total grey. The average genetic identity between the "subpopulations" was 0.956 according to proportion of total grey or 0.958 from the total grey with an average genetic distance between the "subpopulations" of 0.045 and 0.043 respectively. CONCLUSION: It seems to be a reasonable and accurate method by quantifying the AFLP electrophoresis pattern followed by analyzing the data through the use of the different standards of band-reading.

Animals↗

PubMed1995

Molecular characterization of an 18 kb segment of DNA puff C4 of Bradysia hygida (Diptera, sciaridae).

The data presented here are an extension of the molecular characterization of DNA puff C4 of Bradysia hygida. A cDNA related to a gene amplified in this puff and expressed when puff C4 expands was cloned and sequenced. Analysis of the amino acid sequence deduced from the open reading frame present in the cDNA indicate that the encoded protein is secreted and comprises mostly alpha-helical coiled-coil. An 18 kb genomic segment containing the transcription unit of this gene was also cloned and the structure and expression of the 1.4 kb mRNA was determined. Quantitative slot blot hybridization of DNA complementary to the transcription unit shows that this gene is amplified about 21 times in the salivary gland, confirming data previously obtained. Fragments upstream of the 5' end, and beyond the 3' end, of the gene transcription unit were also analysed and shown to be amplified at least eight and five times, respectively. Based on these data we discuss how amplification could occur at DNA puffs.

Amino Acid Sequence↗

PubMed1991

Bone fragments a body can make.

Data obtained from various analytical techniques applied to a number of small bone fragments recovered from a crime scene were used to provide evidence for the occurrence of a fatality. Microscopic and histomorphometric analyses confirmed that the fragments were from a human skull. X-ray microanalysis of darkened areas on the bone fragments revealed a chemical signature that matched the chemical signature of a shotgun pellet recovered at the scene of the crime. The above findings supported the deoxyribonucleic acid (DNA) fingerprint evidence which, along with other evidence, was used to convict a man for the murder of his wife, even though her body was never recovered.

Bone and Bones↗

PubMed1994

Recombinant proteins L and LG. Two new tools for purification of murine antibody fragments.

Several bacterial cell wall proteins, like proteins A and G, with valuable affinity for immunoglobulins have been discovered and are currently employed in immunological techniques. In 1988, protein L, a bacterial cell wall protein with Ig-binding capacity, was isolated from the anaerobic bacterial species Peptostreptococcus magnus. Binding data with immunoglobulin fragments suggested that protein L could selectively bind the variable region of human kappa light chains. More recently a recombinant LG fusion protein was expressed in E. coli containing four repeated Ig-binding domains of protein L (fragment B1-4) and two IgG Fc-binding protein G domains (fragment CDC). Recombinant L and LG proteins were tested in the purification of murine monoclonal IgG and their fragments. After affinity-constant evaluation in different buffer systems, high-pressure affinity-chromatography columns were prepared by coupling the proteins to Affi-prep 10 resin and tested with eight different murine monoclonal antibodies and their fragments of various isotypes. Affinity-chromatography experiments confirming radioimmunoassay results showed 75% fragment-binding capacity of protein L and 100% of protein LG. These results evidenced protein LG as the most powerful Ig-binding tool so far described. Therefore, application of these proteins may be suggested in the purification of murine immunoglobulins and their fragments, including the engineered ones.

Animals↗

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↗

PubMed2003

Isolation of partial toxR gene of Vibrio harveyi and design of toxR-targeted PCR primers for species detection.

AIMS: To differentiate Vibrio harveyi from closely related Vibrio species by toxR sequence analysis and design primers for the specific detection of the shellfish pathogen. METHODS AND RESULTS: The partial toxR homologue from the shellfish pathogen V. harveyi was isolated by PCR using degenerate primers. The 578-bp toxR fragment from V. harveyi, that exhibited highest homology with partial toxR of V. parahaemolyticus (68%), is predicted to encode for a polypeptide with 192 amino acid residues. Alignment of the V. harveyi toxR nucleotide and deduced amino acid sequence with those from other Vibrio species revealed the presence of the fairly characteristic conserved transcription activation and transmembrane domain as well as the divergent membrane tether region that may be targeted for the development of species-specific oligonucleotide primers. Consequently, PCR primers that could amplify a 390-bp gene fragment in V. harveyi were designed by targeting portions of the V. harveyi toxR that display variability with toxR sequences from other Vibrio species. The 390-bp-amplicon was detected in all V. harveyi strains examined except in the nontarget bacteria and unexpectedly, in two shrimp-derived strains (VIB 391 and STD 3-101) from Thailand and Ecuador. Results show that strains exhibiting the 390-bp amplicon mostly belong to the same cluster based on previous amplified fragment length polymorphism data while strains which were previously unclustered or unclassified did not display the 390-bp PCR product. CONCLUSIONS: The toxR sequence variation could differentiate V. harveyi from closely related Vibrio species. A PCR protocol amplifying a 390-bp fragment of the V. harveyi toxR was established and could be useful in the specific and rapid detection of the species. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular approaches reported in this study could facilitate the early diagnosis and surveillance of luminous vibriosis in hatchery-reared fish and shellfish species through rapid identification and specific detection of causal agent.

Amino Acid Sequence↗

PubMed1993

Calpastatin has two distinct sites for interaction with calpain--effect of calpastatin fragments on the binding of calpain to membranes.

Calpain, a ubiquitously distributed intracellular cysteine protease in animals, is thought to be activated at the cell membrane in the presence of micromolar levels of calcium ions. Calpastatin, the sole specific proteinaceous inhibitor for calpain, inhibits not only proteolytic activity, but also the binding of calpain to cell membranes. The inhibition of calpain binding to membranes by calpastatin fragments was analyzed. A calpastatin peptide containing the inhibitory sequence did not inhibit the binding of calpain to membranes. On the other hand, binding was prevented by a fragment without the activity to inhibit proteolytic activity. These data indicate that calpain binds to cell membranes through a site (regulatory site) other than the active site and that calpastatin inhibits the binding of calpain to cell membranes via a site (regulatory inhibition site) other than the inhibitory sequence. Calpain and calpastatin can undergo independent interactions: interaction between the catalytic site of calpain and the inhibitory sequence of calpastatin, and interaction between the regulatory site of calpain and the regulatory inhibition site of calpastatin. The interaction between the regulatory site of calpain and the regulatory inhibition site of calpastatin is essential for the regulation of calpain activity.

Amino Acid Sequence↗

PubMed1993

Characterization of two major isoforms of juvenile hormone esterase from Trichoplusia ni (Lepidoptera).

The two major isoforms of juvenile hormone (JH) esterase isolated from Trichoplusia ni were fragmented by cyanogen bromide and trypsin digestion. The resulting CNBr or CNBr/trypsin fragments were characterized and compared biochemically by SDS-PAGE, isoelectric focusing, two-dimensional electrophoresis and HPLC. Similar and unique fragments were examined for sequence, antigenic determinants and carbohydrate moieties. The studies identified small regions of the proteins which possess either potentially different sequences or different post-translational modifications. The location of a glycosylated asparagine residue was determined, as well as a region containing an epitope probably composed of a linear sequence of residues. An N-terminal region was identified that contained charge variation between the two isoforms and the sequence was obtained for the only unique CNBr/trypsin fragment detected from that region. These are the first data on mapping of regions of charge variation, epitope location and glycosylation sites for this enzyme from any insect species.

Amino Acid Sequence↗

PubMed1996

Complete amino acid sequence of the A chain of mistletoe lectin I.

The complete amino acid sequence of the A chain of mistletoe lectin I was determined via Edman degradation sequencing of the N-terminus and tryptic and endoproteinase Asp-N overlapping fragments, amino acid analysis and MALDI-MS. The data obtained show a great homology with the chains of ribosome-inactivating proteins such as ricin and abrin with 111 (abrin-a) and 103 (ricin-D) amino acid residues conserved, respectively. The knowledge of the primary structure of MLA will have a fundamental impact on elucidating the biological function of medically applied mistletoe lectins on a molecular basis.

Amino Acid Sequence↗

PubMed2001

Determination of the collisionally activated dissociation of a substituted indole by orthogonal acceleration quadrupole time-of-flight mass spectrometry.

The use of orthogonal acceleration quadrupole time-of-flight (Q-TOF) mass spectrometry to determine the collisionally activated dissociation (CAD) of a test compound 1-(3-[5-[1,2,4-triazol-4-yl]-1H-indol-3-yl]propyl)-4-(2-[3-fluorophenyl]ethyl)piperazine is described. At unit-mass resolution the identity of many ions is ambiguous because of the complexity of the resulting product ion spectrum. Using the high resolution capabilities of the Q-TOF instrument, exact masses for each fragment were determined. These data were used to infer molecular formulas for each fragment through software interpretation and, by further applying chemical intuition, the majority of ions were fully assigned. Additionally, by utilizing in-source fragmentation at high cone voltage, analyses of second-generation products allowed derivation of a consistent sequential fragmentation pathway. This study clearly demonstrates the power of Q-TOF mass spectrometry to elucidate complex product ion spectra.

Indoles↗

PubMed1991

Studies on the biotin-binding site of avidin. Minimized fragments that bind biotin.

The object of this study was to define minimized biotin-binding fragments, or 'prorecognition sites', of either the egg-white glycoprotein avidin or its bacterial analogue streptavidin. Because of the extreme stability to enzymic hydrolysis, fragments of avidin were prepared by chemical means and examined for their individual biotin-binding capacity. Treatment of avidin with hydroxylamine was shown to result in new cleavage sites in addition to the known Asn-Gly cleavage site (position 88-89 in avidin). Notably, the Asn-Glu and Asp-Lys peptide bonds (positions 42-43 and 57-58 respectively) were readily cleaved; in addition, lesser levels of hydrolysis of the Gln-Pro (61-62) and Asn-Asp (12-13 and 104-105) bonds could be detected. The smallest biotin-binding peptide fragment, derived from hydroxylamine cleavage of either native or non-glycosylated avidin, was identified to comprise residues 1-42. CNBr cleavage resulted in a 78-amino acid-residue fragment (residues 19-96) that still retained activity. The data ascribe an important biotin-binding function to the overlapping region (residues 19-42) of avidin, which bears the single tyrosine moiety. This contention was corroborated by synthesizing a tridecapeptide corresponding to residues 26-38 of avidin; this peptide was shown to recognize biotin. Streptavidin was not susceptible to either enzymic or chemical cleavage methods used in this work. The approach taken in this study enabled the experimental distinction between the chemical and structural elements of the binding site. The capacity to assign biotin-binding activity to the tyrosine-containing domain of avidin underscores its primary chemical contribution to the binding of biotin by avidin.

Amino Acid Sequence↗

PubMed2001

Identification of a heparin binding domain in the N-terminal cleavage site of pro-islet amyloid polypeptide. Implications for islet amyloid formation.

Islet amyloid deposits are a characteristic pathologic lesion of the pancreas in type 2 diabetes and are composed primarily of the islet beta cell peptide islet amyloid polypeptide (IAPP or amylin) as well as the basement membrane heparan sulfate proteoglycan perlecan. Impaired processing of the IAPP precursor has been implicated in the mechanism of islet amyloid formation. The N- and C-terminal cleavage sites where pro-IAPP is processed by prohormone convertases contain a series of basic amino acid residues that we hypothesized may interact with heparan sulfate proteoglycans. This possibility was tested using affinity chromatography by applying synthetic fragments of pro-IAPP to heparin-agarose and heparan sulfate-Sepharose. An N-terminal human pro-IAPP fragment (residues 1-30) was retained by both heparin-agarose and heparan sulfate-Sepharose, eluting at 0.18 m NaCl at pH 7.5. Substitution of alanine residues for two basic residues in the N-terminal cleavage site abolished heparin and heparan sulfate binding activity. At pH 5.5, the affinity of the wild-type peptide for heparin/heparan sulfate was increased, implying a role for histidine residues at positions 6 and 28 of pro-IAPP. A C-terminal pro-IAPP fragment (residues 41-67) had no specific affinity for either heparin or heparan sulfate, and the N- or C-terminal fragments had only weak affinity for chondroitin sulfate. These data suggest that monomeric N-terminal human pro-IAPP contains a heparin binding domain that is lost during normal processing of pro-IAPP.

Amino Acid Sequence↗