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PubMed1989

Assignment of the positions of chymotryptic fragments and cysteinyl groups in the primary structure of caldesmon in relation to a conformational change.

Caldesmon is universally associated with smooth muscle thin filaments, and reportedly interacts with actin, calmodulin, tropomyosin, and myosin. I attempted to determine the positions of the chymotryptic fragments which have been used to study the sites of such interactions in its primary structure. Such assignment, combined with the accumulated data of fragment studies, made it possible to clarify the functional domain organization of the caldesmon molecule. Using a specific cleavage method involving nitrothiocyanobenzoate, I also determined the number and locations of cysteinyl residues in the amino-acid sequence. Two cysteinyl groups thus determined were located close to the ends and almost at the same distance apart from the N- and C-termini of the whole molecule, respectively. Taking these locations and the extraordinary sensitivity to oxidation into consideration, I could reasonably elucidate the origin of the controversial data and the interpretation so far reported from various laboratories, concerning the length and conformation of the native molecule. Caldesmon might be folded in solution and its contour length could be almost double the accepted value, which was hydrodynamically estimated.

Amino Acid Sequence↗

PubMed2002

Initiation sites are distributed at frequent intervals in the Chinese hamster dihydrofolate reductase origin of replication but are used with very different efficiencies.

Previous radiolabeling and two-dimensional (2-D) gel studies of the dihydrofolate reductase (DHFR) domain of Chinese hamster cells have suggested that replication can initiate at any one of a very large number of inefficient sites scattered throughout the 55-kb intergenic spacer region, with two broad subregions (ori-beta and ori-gamma) preferred. However, high-resolution analysis by a PCR-based nascent strand abundance assay of the 12-kb subregion encompassing ori-beta has suggested the presence of a relatively small number of fixed, highly efficient initiation sites distributed at infrequent intervals that correspond to genetic replicators. To attempt to reconcile these observations, two different approaches were taken in the present study. In the first, neutral-neutral 2-D gel analysis was used to examine replication intermediates in 31 adjacent and overlapping restriction fragments in the spacer, ranging in size from 1.0 to 18 kb. Thirty of 31 fragments displayed the complete bubble arcs characteristic of centered origins. Taking into account overlapping fragments, these data suggest a minimum of 14 individual start sites in the spacer. In the second approach, a quantitative early labeled fragment hybridization assay was performed in which radioactive origin-containing DNA 300 to 1,000 nucleotides in length was synthesized in the first few minutes of the S period and used to probe 15 clones distributed throughout the intergenic spacer but separated on average by more than 1,000 bp. This small nascent DNA fraction hybridized to 14 of the 15 clones, ranging from just above background to a maximum at the ori-beta locus. The only silent region detected was a small fragment lying just upstream from a centered matrix attachment region--the same region that was also negative for initiation by 2-D gel analysis. Results of both approaches suggest a minimum of approximately 20 initiation sites in the spacer (two of them being ori-beta and ori-gamma), with ori-beta accounting for a maximum of approximately 20% of initiations occurring in the spacer. We believe that the results of all experimental approaches applied to this locus so far can be fitted to a model in which the DHFR origin consists of a 55-kb intergenic zone of potential sites that are used with very different efficiencies and which are separated in many cases by a few kilobases or less.

Animals↗

PubMed2003

Realization of real-time clinical data integration using advanced database technology.

As information & communication technologies have advanced, interest in mobile health care systems has grown. In order to obtain information seamlessly from distributed and fragmented clinical data from heterogeneous institutions, we need solutions that integrate data. In this article, we introduce a method for information integration based on real-time message communication using trigger and advanced database technologies. Messages were devised to conform to HL7, a standard for electronic data exchange in healthcare environments. The HL7 based system provides us with an integrated environment in which we are able to manage the complexities of medical data. We developed this message communication interface to generate and parse HL7 messages automatically from the database point of view. We discuss how easily real time data exchange is performed in the clinical information system, given the requirement for minimum loading of the database system.

Clinical Laboratory Information Systems↗

PubMed1988

Immunoglobulin synthesis in primary and myeloma amyloidosis.

Bone marrow cells from 14 patients with primary amyloidosis and two patients with myeloma amyloidosis were studied by immunofluorescence and biosynthesis experiments after incorporation of radioactive amino acids. Cells from four patients affected with non-myeloma secondary amyloidosis were also studied as controls. In primary amyloidosis, monoclonal plasma cell populations were demonstrated by immunofluorescence in virtually every case, even in patients without serum and urine monoclonal immunoglobulin and with a normal percentage of bone marrow plasma cells. Biosynthesis experiments showed the secretion of large amounts of free light chains, most often of the lambda type, in every primary or myeloma amyloidosis case and the presence of light chain fragments in almost all cases. Special features in certain patients were the synthesis of short gamma chains (two cases), assembly block at the HL half molecule level of a monoclonal IgA (one case) and secretion of decameric abnormally large kappa chains (one case). This is in contrast with non-myelomatous secondary amyloidosis where the distribution of bone marrow plasma cells was normal by immunofluorescence and where normal sized immunoglobulins were synthesized, without free light chain secretion and fragments. These data confirm that primary amyloidosis belongs to plasma cell dyscrasias and emphasize the role of free light chains and light chain fragments in the pathogenesis of amyloid deposition.

Amyloidosis↗

PubMed1998

[The costs of schizophrenia].

Schizophrenia is regarded as the most expensive mental illness because of its specific symptoms and characteristics (e.g. early onset, often chronic course, high rates of readmission to hospital treatment, high rate of disabilities and extensive rehabilitative interventions), which prove to be extremely costly. Despite this, studies on the financial aspects of schizophrenia or the provision of care for schizophrenic patients has become an issue of psychiatric research only since the beginning of the reform of mental healthcare. Early cost studies had been conducted in the United States (US) during the fifties. Since then, they have grown in number not only in the US and in Great Britain, but also on the Continent of Europe. On an international level, comprehensive literature concerning methodology has attempted to establish cost studies as an integral part of mental health services research. Germany, however, is far behind international developments. Although the fundamental lack of empirical data on costs both in psychiatry as a whole and in schizophrenia had already been ascertained in a large national survey called "Psychiatrie-Enquête" in the mid-seventies, little has changed since then. One reason for these possibly great methodological problems is associated with the assessment of cost data in fragmented community mental health care networks, which in Germany include the additional obstacle--unlike abroad--of non-availability of access to data from case registers. Psychiatric case registers are not permitted in Germany because of very strict data protection laws. Despite the problems in methodology, there is an urgent need in Germany to remedy the lack of cost data for schizophrenic-patient care. The pressure of curbing costs in health care will probably force the German mental health care services to provide detailed cost data with regular reports in the future.

Humans↗

PubMed2005

Molecular fragment replacement approach to protein structure determination by chemical shift and dipolar homology database mining.

A novel approach is described for determining backbone structures of proteins that is based on finding fragments in the protein data bank (PDB). For each fragment in the target protein, usually chosen to be 7-10 residues in length, PDB fragments are selected that best fit to experimentally determined one-bond heteronuclear dipolar couplings and that show agreement between chemical shifts predicted for the PDB fragment and experimental values for the target fragment. These fragments are subsequently refined by simulated annealing to improve agreement with the experimental data. If the lowest-energy refined fragments form a unique structural cluster, this structure is accepted and side chains are added on the basis of a conformational database potential. The sequential backbone assembly process extends the chain by translating an accepted fragment onto it. For several small proteins, with extensive sets of dipolar couplings measured in two alignment media, a unique final structure is obtained that agrees well with structures previously solved by conventional methods. With less dipolar input data, large, oriented fragments of each protein are obtained, but their relative positioning requires either a small set of translationally restraining nuclear Overhauser enhancements (NOEs) or a protocol that optimizes burial of hydrophobic groups and pairing of beta-strands.

Computer Simulation↗

PubMed1986

Overlapping deletion in two spontaneous phase variants of Coxiella burnetii.

Chromosomal DNA from the Nine Mile phase I strain of Coxiella burnetii (CB9MIC7) was cloned into the cosmid vector pHC79. The resulting gene library was probed with a radiolabelled HaeIII fragment present in the parental strain but absent from a spontaneously derived Nine Mile phase II strain (CB9MIIC4). The insert, which includes the missing HaeIII fragment, was 38.5 kb in length. When DNA from this cosmid clone was hybridized to genomic DNA of the parental CB9MIC7 and its derivative CB9MIIC4, a number of fragments were missing or altered in the latter strain. Restriction mapping localized the fragments to a contiguous portion of the chromosomal DNA fragment. The data were consistent with an 18 kb deletion in the chromosome of CB9MIIC4. Another intrastrain spontaneous derivative, CB9MI514, also lacked the sentinel HaeIII fragment and carried a deletion of approximately 29 kb within the same cloned insert. Both deletions appeared to share a common terminus, within the limits of resolution. In all other strains investigated, both phase I and phase II, the DNA represented by the insert seemed intact. The strains examined were representative of various stages of phase variation. The relationship between the observed deletions and the mechanism of phase transition in Nine Mile strains is discussed.

Chromosome Deletion↗

PubMed1980

Direct demonstration of separate receptors for growth and metabolic activities of insulin and multiplication-stimulating activity (an insulinlike growth factor) using antibodies to the insulin receptor.

Insulin and such insulinlike growth factors as multiplication stimulating activity (MSA) are related polypeptides that have common biological activities. Both insulin and MSA produce acute metabolic responses (stimulation of glucose oxidation in isolated fat cells) as well as growth effects (stimulation of [(3)H]thymidine incorporation into DNA in cultured fibroblasts). In addition, most cells have separate receptors for insulin and insulinlike growth factors, and both peptides have weaker affinity for each other's specific receptors than for their own. To determine, therefore, whether these effects are mediated by receptors for insulin, insulinlike growth factors, or both, we have selectively blocked insulin receptors with a specific antagonist, namely Fab fragments derived from naturally occurring antibodies to the insulin receptor. In rat adipocytes, 10 mug/ml of antireceptor Fab inhibited insulin binding by 90%, whereas it inhibited MSA binding <5%. The anti-insulin receptor Fab is without intrinsic biological activity, but acts as a competitive inhibitor of insulin receptors. Blockade of insulin receptors with Fab fragments produced a 30-fold rightward shift in the dose response for stimulation of glucose oxidation by both insulin and MSA. The dose-response curves for stimulation of oxidation by vitamin K(5) and spermine, agents that stimulate glucose oxidation through noninsulin receptor pathways, were not affected by the blockade of insulin receptors with Fab antibody fragments. These data suggest that this acute metabolic effect of both insulin and MSA is mediated via the insulin receptor. In cultured human fibroblasts, 10 mug/ml of Fab inhibited insulin binding by 90% and MSA binding by 15%. In fibroblasts, however, blockade of the insulin receptor did not alter the dose response for stimulation of thymidine incorporation into DNA by either insulin or MSA. Furthermore, intact antireceptor antibody immunoglobulin (Ig)G, which produces multiple other insulinlike effects, and Fab fragments of antireceptor antibody did not stimulate thymidine incorporation. These data demonstrate directly that the insulin receptor mediates the metabolic effects of insulin and MSA, whereas the growth-promoting action of both peptides is mediated by the MSA receptor or other growth factors.

Adipose Tissue↗

PubMed1987

Two distinct cell-binding domains in laminin can independently promote nonneuronal cell adhesion and spreading.

Two subfragments of laminin, E8, a major part of the long arm, and E1-4, the three short arms, promote cell adhesion and spreading. Three distinct types of adhesive behavior are seen in short term (1 h) assays, typified by secondary murine fibroblasts, adherent only on fibronectin; secondary murine myoblasts, adherent on fibronectin, laminin, and the E8 fragment; and Rugli human glioblastoma cells, adherent on fibronectin, laminin, E8, and E1-4. E8-specific polyclonal antibodies block myoblast adhesion to E8 and to laminin with identical concentration dependence; Rugli binding to E8 but not to laminin is also totally blocked by these antibodies. Heating of E8 and laminin to approximately 60 degrees C abolishes cell attachment-promoting activity for myoblasts. Adhesion of Rugli cells to E8 is also lost, but on laminin the attachment-promoting activity remains constant. This is due to an increase in the activity of E1-4 fragment as it is heated. Thus, major sites for initial cell adhesion to and spreading on laminin lie within the E8 and E1-4 fragments, but not all cells binding to laminin will bind to both fragments. These data may tentatively be explained by the existence of more than one type of receptor for laminin at the cell surface; one is needed for each fragment.

Amino Acid Sequence↗

PubMed1980

The REM cycle is a sleep-dependent rhythm.

Two studies, using data from fragmented sleep studies from three sleep laboratories, are reviewed. These studies indicate that the REM cycle is primarily governed by a sleep-dependent oscillator. These data, however, do not rule out the potential influence of endogenous or environmental variables as factors influencing the REM cycle. Further, acceptance of the REM cycle as a sleep-dependent rhythm does not lead to the denial of the basic rest-activity cycle (BRAC) proposed by Kleitman. It is questioned, however, whether the BRAC measured during waking is an extension of the REM cycle recorded during sleep.

Adolescent↗

PubMed2005

Among- and within-patch components of genetic diversity respond at different rates to habitat fragmentation: an empirical demonstration.

Habitat fragmentation is a ubiquitous by-product of human activities that can alter the genetic structure of natural populations, with potentially deleterious effects on population persistence and evolutionary potential. When habitat fragmentation results in the subdivision of a population, random genetic drift then leads to the erosion of genetic diversity from within the resulting subpopulation, random genetic drift then leads to the erosion of genetic diversity from within the resulting subpopulations and greater genetic divergence among them. Theoretical and simulation analyses predict that these two main genetic effects of fragmentation, greater differentiation among resulting subpopulation and reduced genetic diversity within them, will proceed at very different rates. Despite important implications for the interpretation of genetics data from fragmented populations, empirical evidence for this phenomenon has been lacking. In this analysis, we carry out an empirical study in population of an alpine meadow-dwelling butterfly, which have become fragmented increasing forest cover over five decades. We show that genetic differentiation among subpopulations (G(ST)) is most highly correlated with contemporary forest cover, while genetics diversity within subpopulation (expected heterozygosity) is better correlated with the spatial pattern of forest cover 40 years in the past. Thus, where habitat fragmentation has occurred in recent decades, genetic differentiation among subpopulation can be near equilibrium while contemporary measures of within subpopulation diversity may substantially overestimate the equilibrium values that will eventually be attained.

Alberta↗

PubMedDate not supplied

[Use of the antibiotics actinomycin D and distamycin A to limit the action of restriction endonucleases and to map DNA].

It is shown that distamycin A and actinomycin D protect the recognition sites of certain restriction endonucleases from the attack by these nucleases due to specific interaction of these antibiotics with double-stranded DNA. Distamycin A protects A-T containing sites and actinomycin G-C rich sites. Among Hind II recognition sites which have alternative structure (GTPyPuAC) distamycin A protects only Hpa I similar sites (GTTAAC). It is shown with several restriction endonucleases that antibiotic action depends on the nucleotide sequences in the recognition sites and in their closest environment. Proper concentrations of antibiotic give rise to larger fragments. Use of both distamycin A and actinomycin D allows to obtain a set of overlapping fragments. The data obtained with various DNAs and restriction endonucleases allow to conclude that these antibiotics may be useful for DNA mapping and for preparation of large functional fragments of DNA.

Base Sequence↗

PubMed1999

A new look at a time-worn system: oxidation of CuZn-SOD by H2O2.

This work summarizes observations from numerous investigators on the reaction of the copper-zinc superoxide dismutase with hydrogen peroxide at physiological pH in order to propose a likely sequence of events that leads to 2-oxo-histidine formation, copper loss, inactivation, and random and site-specific peptide fragmentation. New data is presented for the bovine liver enzyme that indicate copper is lost as the copper(I) form which immediately reacts with bathocuproine disulfonate to form the characteristic complex that absorbs at 485 nm. Studies in TRIS buffer ruled out the loss of copper(II) followed by reduction of the high potential copper(II)-bathocuproine disulfonate complex by buffer because TRIS is known not to reduce this complex. The rate of loss of copper(I) is not affected by the spin trap, 5,5'-dimethylpyrolline-N-oxide (DMPO), nor by replacing oxygen with argon in the reaction. In addition, changes in the native electrophoretic pattern that are correlated with copper loss and not peptide fragmentation are also unaffected by DMPO, argon, EDTA, or DTPA. These data are taken as indirect evidence that the formation of 2-oxo-histidine is the first oxidative event, unaffected by DMPO, that occurs at the bound oxidant and leads to loss of copper(I). Peptide fragmentation and the peroxidative activity of the dismutase are discussed in light of these observations.

Animals↗

PubMed1983

Immunoregulatory properties of antigenic fragments from bovine serum albumin.

Regulation of the immune response to bovine serum albumin (BSA) by defined substructures of the protein was investigated. The fragments consisted of 41 to 307 amino acids each bearing unique serologic determinants. When administered intravenously the fragments were capable of suppressing the immune response to the entire BSA molecule. The suppression was T-cell mediated and affected the determinants that were linked. The fragments also primed for a secondary anti-BSA response when given in adjuvant; however, the ability to prime was restricted to determinants on the immunizing fragment. The data demonstrate that immune responses to a large protein molecule can be regulated by a relatively small component of its structure and that the ability of a fragment to induce help is more restricted than is its suppressive potential.

Amino Acids↗

PubMed1986

[Dependence of the specific activity of antistaphylococcal immunoglobulin on the degree of its fragmentation].

In vitro experiments have revealed no perceptible decrease in the content of antitoxic antibodies to alphastaphylolysin in the preparations of antistaphylococcal immunoglobulin, containing 13-50% of Fab-fragments. These data confirm the necessity of controlling the molecular composition of the preparations of antistaphylococcal IgG by physico-chemical methods with the aim to evaluate their quality, as the fragmented preparations are rapidly eliminated from a human body.

Antibody Specificity↗

PubMed1995

Conformation of short DNA fragments by modulated fluorescence polarization anisotropy.

The technique of fluorescence polarization anisotropy (FPA) decay of intercalated ethidium has been used to study DNA conformation and dynamics, which are being recognized as primary determinants in transcription control and other cellular processes. Frequency modulated FPA when applied to two DNA molecules, a "straight" 50 base-pairs duplex fragment, and a bent fragment of similar length, has yielded different rotational diffusion coefficients for the two fragments. The data have been processed with an analytical model and with Brownian dynamics simulations, obtaining a good fit and a quantitative agreement between the two models. Both analyses have confirmed that one fragment can be described as a straight cylinder, while the other fragment is bent, with an angle estimated to be 45 degrees +/- 3 degrees. FPA has proved to be very powerful in determining simple conformations of short DNA duplexes and also particularly apt to probe the dynamical features of DNA fragments where conventional methods are either too cumbersome or fail to give quantitative results. In addition, the ligand no longer behaves ideally due to its complex structure and charge distribution. Thus for the protein the slope is no longer related simply to the net ligand charge, and the PB model gives a much larger slope than the LL model.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗

PubMed2003

Hyaluronan-oligosaccharide-induced transcription of metalloproteases.

Activated dendritic epidermal Langerhans cells and metastatic tumour cells share many properties. Both cell types can invade the surrounding tissue, enter the lymphatic system and travel to regional lymph nodes. We have recently shown that fragments of the extracellular matrix component hyaluronan, which are typically produced at sites of inflammation, can activate dendritic cells. Upon activation, dendritic cells upregulate expression of matrix metalloproteases (MMPs). These observations prompted us to investigate whether exposure to hyaluronan fragments also induces MMP expression in tumour cells. Here, we report that MMP-9, MMP-13 and urokinase plasminogen activator are upregulated in murine 3LL tumour cells after exposure to mixed-size hyaluronan. Similarly upregulated MMP-9 and MMP-13 expression was observed in primary fibroblasts. By using size-fractionated hyaluronan preparations, we show that the enhanced expression of MMP-9 and MMP-13 is only induced by small hyaluronan (HA) fragments. Although our data suggest that HA-fragment-induced MMP-9 and MMP-13 expression is receptor mediated, they rule out an involvement of the hyaluronan receptors CD44, RHAMM/IHAP and TLR-4. Finally, we show that HA fragment-induced MMP-9 transcription is mediated via NF-kappa B. Our results suggest that the metastasis-associated HA degradation in tumours might promote invasion by inducing MMP expression.

Animals↗

PubMed2000

Application of the Fragments and Structure Analysis of Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry in Peptide Sequencing.

The peptides of angiotensin II and fragment 18-39 of adrenocorticotropic hormone were determined by MALDI-TOF-MS-FAST. Main amino acid compositions and partial sequences of the peptides have been analyzed by the mass data of fragment ion peaks in the FAST spectra. The primary structures of these two peptides were identified by searching the protein databases. The research results showed that FAST is a powerful tool for determining the primary structure of peptides because of its high sensitivity, quickness and accuracy.

Journal Article↗