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PubMed1988

Improved separation of multi-locus hypervariable DNA restriction fragments by field inversion gel electrophoresis and fragment detection using a biotinylated probe.

DNA probes to multiple hypervariable human genomic loci are potentially highly informative as regards individual genome identification and parentage studies. However the resultant Southern blot patterns are generally highly complex and their interpretation difficult, with one important limitation being the extent of fragment separation and resolution. The separation of large DNA restriction fragments in agarose gels may be improved, in comparison to separations obtained by conventional electrophoresis, by the use of field inversion techniques. This is demonstrated by the analysis of the complex human Satellite III related DNA polymorphism. Detection of the Satellite III related restriction fragments is achieved either by using a [35S]-labelled probe (228S) or by using the same probe in a convenient non-isotopic form constructed by the photobiotin process. In addition, the probe 228S is useful for sexing the human genome, by the identification of a Y-specific restriction fragment.

Biotin↗
PubMed1976

[FAB immunoglobulin fragments. I. The comparative characteristics of the serological and virus-neutralizing properties of a gamma globulin against tick-borne encephalitis and of the FAB fragments isolated from it].

A comparative study was made of the serological properties and virus-neutralizing activity of antiencephalitis gamma-globulin and Fab-fragments isolated from it by gel-filtration. Horse immunoglobulins against the autumno-summer tick-borne encephalitis virus could be disintegrated with the aid of papaine to monovalent Fab-fragments which (according to the complement fixation reaction, the test of suppression of the complement fixation, and the HAIT) retained the serological activity whose level was compared with that of the serological activity of gamma-globulin. Fab-fragments possessed a marked virus-neutralizing activity. The mean value of a logarithm of the neutralization index was 2.65 +/- 0.2 for Fab-fragments and 3.74 +/- 0.38 for gamma-globulin (P less than 0.01).

Animals↗
PubMed1980

Polyclonal activation of murine B lymphocytes by Fc fragments. I. The requirement for two signals in the generation of the polyclonal antibody response induced by Fc fragments.

Fc fragments derived from human IgG1 induce murine splenic B lymphocytes to undergo proliferation and differentiation to antibody-secreting cells. The polyclonal antibody response was found to require both the presence of macrophages and T cells. Spleen cell cultures from nude mice or T cell-depleted normal mice proliferate to the level of untreated control mice but do not produce polyclonal antibody unless T cells are added. Regulation of the Fc fragment induced B cell differentiation to antibody synthesis apparently occurs through two distinct signals. One signal is provided by Fc fragments for proliferation and the other by T cells for differentiation. This suggestion is supported by the observation that spleen cell preparations, devoid of T cells, are capable of proliferation to the level of normal spleen cell cultures in response to Fc fragments, but are incapable of making a polyclonal antibody response. The cell population that responds to the differentiation signal also responds to the proliferative signal. "Hot pulse" experiments demonstrated that proliferation precedes polyclonal activation.

Animals↗
PubMed1982

Cleavage of IgG by elastase-like protease (ELP) of human polymorphonuclear leukocytes (PMN): isolation and characterization of Fab and Fc fragments and low-molecular-weight peptides. Stimulation of granulocyte function by ELP-derived Fab and Fc fragments.

The elastase-like protease (ELP) from human polymorphonuclear granulocytes (PNM) is able to split human IgG into Fab and Fc-like fragments and smaller peptides. These fragments are similar but not identical to those produced by papain. They differ in their electrophoretical mobility as well as in their molecular weights. Both ELP-Fab and papain-Fab show antigen-binding capacity. In contrast to papain-derived split products of IgG, the ELP-generated Fab and Fc fragments could enhance granulocyte function. Both ELP-Fab and ELP-Fc increase the spontaneous reduction of nitroblue-tetrazolium (NBT) in granulocytes dose-dependent in a homologous as well as in an autologous system. Furthermore, the ELP-derived Fab and Fc induce an active release of ELP by PMN. It could also be demonstrated that Fab as well as Fc increased the peroxidase activity in granulocytes.

Humans↗
PubMed1975

Anaphase transport of akinetochoric fragments in tipulid spermatocytes. Electron microscopic observations on fragment-spindle interactions.

Akinetochoric chromosomal fragments in spermatocytes of mutant Pales ferruginae are transported polewards in anaphase. During migration their surfaces form radial lamellar projections between which non-kinetochoric spindle microtubules become arranged in an orderly fashion. The same morphological features had been observed earlier in intact chromosomes in late anaphase. It is assumed that the fragments are transported by some kind of poleward directed "streaming" force of the anaphase spindle, which is applied to the fragment's surface. Non-kinetochoric microtubules are thought to be engaged in the generation or, at least, in the transmission of this spindle force. Due to the morphological similarities with akinetochoric fragments, extra-kinetochoral application sites for anaphase spindle forces can be also suggested for chromosomes possessing kinetochores.

Animals↗
PubMed1981

Cloning of bacteriophage T5 DNA fragments. II. Isolation of recombinants carrying T5 PstI fragments.

The adjacent PstI-J, I and G fragments of the phage T5 DNA molecule (4.4, 4.6 and 7.2 kb, respectively) have been cloned in plasmid pBR322 and their locations verified by Southern blot analysis. The PstI I and G fragments overlap the previously cloned HindIII-P and G fragments and like those, contain no known genetic markers. In addition, one of the 12 newly isolated T5 mutants maps in this PstI-IG region. Thus, the size of the "empty" region between genes D15 and D17, which we have previously observed on the genetic map, extends to at least 11.8 kb. In contrast, the PstI-J fragment carried part of the D12 gene and the intact D14 and D15 genes. This clone is of particular interest since the D15 gene product is a nuclease and is responsible for the positive control of late gene transcription. The orientation of these genes relative to the T5 DNA molecule has been determined by a combination of restriction, deletion and complementation analyses.

Chromosome Mapping↗
PubMed1995

Perceiving exterior letters of words: differential influences of letter-fragment and non-letter-fragment masks.

Previous research shows letter-fragment masks and non-letter-fragment fields have different effects on performance with briefly presented alphabetic targets. However, popular accounts of these differences ignore mask configuration. Over a series of experiments, configurational effects of letter-fragment (LF) and non-letter-fragment (NLF) masks were compared. When the configuration of LF masks matched word boundaries, performance with exterior letter pairs from words improved, whereas performance with illegal exterior-letter pairs and single letters was unaffected. When the same changes were made to NLF masks, only an overall drop in performance occurred, with no selective effect on target type. Thus, although LF mask configuration selectively affected lexical processing, NLF mask configuration produced substantially different effects, indicating problems with contemporary accounts of masking differences that ignore influences of mask configuration.

Attention↗
PubMed1983

Generation of three different fragments of bound C3 with purified factor I or serum. II. Location of binding sites in the C3 fragments for factors B and H, complement receptors, and bovine conglutinin.

The many different recognized functions of C3 are dependent upon the ability of the activated C3 molecule both to bind covalently to protein and carbohydrate surfaces and to provide binding sites for as many as eleven different proteins. The location of the binding sites for six of these different proteins (factors B and H, complement receptors CR(1), CR(2) and CR(3) and conglutinin) was examined in the naturally occurring C3-fragments generated by C3 activation (C3b) and degradation by Factor I (iC3b, C3c, C3d,g) and trypsin (C3d). Evidence was obtained for at least four distinct binding sites in C3 for these six different C3 ligands. One binding site for B was detectable only in C3b, whereas a second binding site for H and CR(1) was detectable in both C3b and iC3b. The affinity of the binding site for H and CR(1) was charge dependent and considerably reduced in iC3b as compared to C3b. H binding to iC3b-coated sheep erythrocytes (EC3bi) was measurable only in low ionic strength buffer (4 mS). The finding that C3c-coated microspheres bound to CR(1), indicated that this second binding site was still intact in the C3c fragment. However, H binding to C3c was not examined. A third binding site in C3 for CR(2) was exposed in the d region by factor I cleavage of C3b into iC3b, and the activity of this site was unaffected by the further I cleavage of iC3b into C3d,g. Removal of the 8,000-dalton C3g fragment from C3d,g with trypsin forming C3d, resulted in reduced CR2 activity. However, because saturating amounts of monoclonal anti-C3g did not block the CR(2)-binding activity of EC3d,g, it appears unlikely that the g region of C3d,g or iC3b forms a part of the CR(2)-binding site. In addition, detergent-solubilized EC3d (C3d-OR) inhibited the CR(2)-binding activity of EC3d,g. Monocytes and neutrophils, that had been previously thought to lack CR(2) because of their inability to form EC3d rosettes, did bind EC3d,g containing greater than 5 x 10(4) C3d,g molecules per E. The finding that monocyte and neutrophil rosettes with EC3d,g were inhibited by C3d-OR, suggested that these phagocytic cells might indeed express very low numbers of CR(2), and that these CR(2) were detectable with EC3d,g and not with EC3d because C3d,g had a higher affinity for CR2 than did C3d. A fourth C3 binding site for CR(3) and conglutinin (K) was restricted to the iC3b fragment. Because of simultaneous attachment of iC3b to phagocyte CR3 and CR(3), the characteristics of iC3b binding to CR3 could only be examined with phagocytes on which the CR(1) had been blocked with anti-CR(1). Inhibition studies with EDTA and N-acetyl-D-glucosamine demonstrated a requirement for both calcium cations and carbohydrate in the binding of EC3bi to CR3 and to K. However, CR(3) differed from K in that magnesium cations were required in addition to calcium for maximum CR(3) binding activity, and NADG produced less inhibition of CR(3) activity than of K activity.

Animals↗
PubMed1991

The primary structure of skeletal muscle myosin heavy chain: III. Sequence of the 22 kDa fragment and the alignment of the 23 kDa, 50 kDa, and 22 kDa fragments.

The amino acid sequence of the 197-residue 22 kDa fragment from chicken pectoralis muscle was determined to be as follows: K-K-G-S-S-F-Q-T-V-S-A-L-F-R-E-N-L-N-K-L- M-A-N-L-R-S-T-H-P-H-F-V-R-C-I-I-P-N-E-T-K-T-P-G-A-M-E-H-E-L-V-L-H-Q-L-R- C-N-G-V- L-E-G-I-R-I-C-R-K-G-F-P-S-R-V-L-Y-A-D-F-K-Q-R-Y-R-V-L-N-A-S-A-I-P-E-G-Q- F-M-D-S- K-K-A-S-E-K-L-L-G-S-I-D-V-D-h-T-Q-Y-R-F-G-H-T-K-V-F-F-K-A-G-L-L-G-L-L-E- E-M-R-D- D-K-L-A-E-I-I-T-R-T-Q-A-R-C-R-G-F-L-M-R-V-E-Y-R-R-M-V-E-R-R-E-S-I-F-C-I- Q-Y-N-V-R-S-F-M-N-V-K-H-W-P-W-M-K-L-F-F-K, where h stands for 3-N-methylhistidine. The amino acid sequences of the 22 kDa fragment and its equivalent fragment from chicken ventricle and gizzard muscle myosins were also determined by our group. Predicted secondary structures of these 22 kDa fragment regions and of the reported chicken embryo myosin revealed some possible structural differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Facts on the fragmentation of antigens in presenting cells, on the association of antigen fragments with MHC molecules in cell-free systems, and speculation on the cell biology of antigen processing.

The processing of a protein antigen is a multi-step event taking place in antigen-presenting cells. Processing is a prerequisite for the recognition of most antigens by T lymphocytes. The antigen is ingested by endocytosis, transported to an acid cellular compartment and subjected to proteolytic fragmentation. Some of the antigen fragments bind to MHC class II molecules and are transported to the surface of the antigen-presenting cell where the actual presentation to T lymphocytes occurs. The nature of the processed antigen, how and where it is derived and subsequently becomes associated with MHC molecules are the questions discussed in this review. To us, the entire concept of processing has appeal not only because it explains some hitherto well-established, but poorly understood, phenomena such as the fact that T lymphocytes focus their attention entirely upon antigens on other cells. It has appeal also because processing allows for a thorough scrutiny of all the proteins of a cell including both the proteins which have been taken up from the environment (mostly MHC class II-restricted) and the cell's own proteins (mostly MHC class I-restricted). Through the mechanism of processing fragments of all these proteins including "internal" fragments which are not present on the globular protein's surface are brought out on the cell surface in association with MHC molecules and displayed to the T-lymphocyte system. This allows for the identification and, if necessary, the destruction of cells which in their interior harbor abnormal proteins, be they derived from virus-encoded genes or other abnormal genes.

Antigen-Antibody Complex↗
PubMed1993

Fragments of chromogranin A are present in the urine of patients with carcinoid tumours: development of a specific radioimmunoassay for chromogranin A and its fragments.

Chromogranin A is a well-known protein constituent in granules of neuroendocrine cells. It is also known that plasma levels of chromogranin A increase considerably in patients with neuroendocrine tumours and thus chromogranin A is used as a marker for these tumours. In the present study, we have shown that fragments of chromogranin A are excreted into the urine in some patients with carcinoid tumours. The chromogranin A molecule appeared in the urine N-terminally cleaved at amino acid positions 116 and 210, which are previously reported cleavage sites of the molecule. The fragments identified were mainly of about 35 kDa in size. The unprocessed chromogranin A molecule was not excreted in the urine. Five out of 40 patients excreting the fragments had slight tubular dysfunction in the kidneys. We also showed that these renally excreted split products of chromogranin A were immunogenic and could be used for production of antibodies against chromogranin A. These antibodies were used both for immunocytochemistry and for the development of a specific and sensitive radioimmunoassay for chromogranin A and its fragments. Measurements of plasma chromogranin A by radioimmunoassay appeared to be a better marker for tumour growth than were measurements of chromogranin A in the urine.

Adult↗
PubMed1991

Characterization of a chimeric plasminogen activator consisting of a single-chain Fv fragment derived from a fibrin fragment D-dimer-specific antibody and a truncated single-chain urokinase.

An Mr 57,000 single-chain chimeric plasminogen activator, K12G0S32, consisting of a variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 and of a 33-kDa (amino acids Ala132 to Leu411) recombinant single-chain urokinase-type plasminogen activator (rscu-PA-33k) was studied. K12G0S32, secreted by infected Spodoptera frugiperda insect cells at a rate of 1.5 micrograms/10(6) cells/48 h, was purified to homogeneity by ion-exchange chromatography and gel filtration. It was obtained essentially as a single-chain molecule with a Ka = 5.5 x 10(9) M-1 for immobilized fragment D-dimer, similar to that of MA-15C5. The specific activity of both its single-chain and two-chain forms on fibrin plates was 100,000 IU/mg of urokinase-type plasminogen activator (u-PA) equivalent. Activation of plasminogen by two-chain K12G0S32 obeyed Michaelis-Menten kinetics with Km = 2.9 +/- 0.6 microM and a k2 = 3.7 +/- 0.6 s-1 (mean +/- S.D.; n = 3), as compared to Km = 12 microM and k2 = 4.8 s-1 for rtcu-PA-32k (recombinant low Mr two-chain u-PA consisting of amino acids Leu144 to Leu411). Single-chain K12G0S32 induced a dose- and time-dependent lysis of a 125I-fibrin-labeled human plasma clot immersed in citrated human plasma; 50% lysis in 2 h was obtained with 0.70 +/- 0.07 micrograms/ml (mean +/- S.D.; n = 5), as compared with 8.8 +/- 0.1 micrograms/ml for rscu-PA-32k (recombinant low Mr single-chain u-PA consisting of amino acids Leu144 to Leu411) (mean +/- S.D.; n = 3). With two-chain K12G0S32, 50% clot lysis in 2 h required 0.25 +/- 0.03 micrograms/ml (mean +/- S.D.; n = 3), as compared with only 0.62 +/- 0.04 micrograms/ml (mean +/- S.D.; n = 2) for rtcu-PA-32k. These results indicate that low Mr single-chain u-PA can be targeted to a fibrin clot with a single-chain Fv fragment of a fibrin-specific antibody, resulting in a 13-fold increase of the fibrinolytic potency of the single-chain form and a 2.5-fold increase of the potency of the two-chain form.

Amides↗
PubMed1986

Comparison of tryptic fragments of von Willebrand factor involved in binding to thrombin-activated platelets with fragments involved in ristocetin-induced binding and binding to collagen.

Previously we have studied the binding domains on von Willebrand factor (vWF) involved in ristocetin-induced binding to platelets (ristocetin binding domain, RBD) and in the binding of vWF to collagen (collagen binding domain, CBD) using tryptic fragments of 125I-labelled vWF (21, 23). We have also reported on the RBD, CBD and the domain on vWF involved in the binding to thrombin activated platelets (thrombin binding domain, TBD) using vWF-fragments prepared by digestion with staphylococcal protease V8 (25). In the present study, we have digested 125I-vWF with TPCK-trypsin and we have performed at various times of digestion immuno-precipitation with Mab 9, the antibody inhibiting binding of vWF to thrombin activated platelets. The data were compared with the immunoprecipitation patterns simultaneously obtained with CLB-RAg 35 which inhibits binding of vWF in the presence of ristocetin and with CLB-RAg 201, which inhibits binding of vWF to collagen. At 90 min, Mab 9 and CLB-RAg 201 precipitated similar high molecular weight bands, whereas CLB-RAg 35 precipitated bands at 180 and 120 kDa. After 24 h, Mab 9 precipitated bands at 200, 155, 116 and 85 kDa; CLB-RAg 201 precipitated a band at 48 kDa and CLB-RAg 35 a band at 116 kDa. Two-dimensional electrophoresis demonstrated that the high molecular weight bands, precipitated by Mab 9 and CLB-RAg 201 at 90 min, were identical. The 116 kDa fragment recognized by CLB-RAg 35 had a different subunit composition than the 116 kDa fragment precipitated by Mab 9.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMedDate not supplied

[Kinetics of the reactions joining DNA fragments, catalyzed by DNA-ligase. II. Heterogeneous mixture of fragments. Optimization of the reaction conditions].

The system of kinetic equations was proposed describing the joining of heterogeneous DNA fragments by DNA-ligase. The DNA fragments were considered as flexible rods and characterized only by molecular weights and concentrations. The program for calculating the concentrations of all circular and linear DNA forms is described. The program has been written in FORTRAN IV and calculations were made with EC 10--10. For the maximal yield of the multimeric circular DNA molecules it is recommended to begin the ligation at a high DNA concentration and then after a certain fixed time to dilute the reaction mixture. The optimum time intervals and ratios of fragment concentrations were calculated. The yield of different ligation products has been determined. The calculations of optimum reaction conditions were carried out for the construction of hybrid molecules containing DNA fragments with or without selective genetic markers.

DNA↗
PubMed1980

[Fragmentation of polyploid nuclei in rat trophoblast giant cells. II. Formation of deep folds in the nuclear envelope--the beginning of fragmentation].

Using light and electron microscopy, the nuclear envelope (NE) of the trophoblast giant cells has been examined at the beginning of the spontaneous fragmentation of nuclei on the last days of pregnancy (not long before their degeneration). In the course of the polyploid nucleus division, deep and narrow invaginations appear in the NE, frequently running through the whole nucleus, from one pole towards the other, and separating it into fragments. Short finger-like outgrowings are seen extending from the long invaginations perperdicularly or at a certain angel. Both the nuclear membrances, having numerous nuclear pores, are involved in the formation of these invaginations and shorter extentions. Local enlargements are seen produced in the perinuclear space, mainly in the area of NE invaginations. Narrow folds, separating the giant nucleus into fragments, are filled with the cytoplasm rich in ribosomes. In the nuclear fragments so produced, association between the earlier dispersed chromatin and the NE becomes stronger. Large accumulations of the condensed chromatin are mainly distributed under the NE, only a few minute chromatin blocks being found in the center of the nucleus filled up with diffuse chromatin.

Animals↗
PubMed1993

13C NMR study of the mode of interaction in solution of the B fragment of staphylococcal protein A and the Fc fragments of mouse immunoglobulin G.

The mode of interaction of the B domain (FB) of staphylococcal protein A and the Fc fragments of mouse immunoglobulin G (IgG) has been investigated by 13C NMR spectroscopy. Mouse IgG1, IgG2a, and IgG2b proteins have been selectively labeled with 13C at the carbonyl carbon of His, Met, Trp or Tyr residue and used to prepare the corresponding Fc fragments by limited proteolysis. Site-specific resonance assignments have been made for each of these Fc analogues. FB was reported to form two contacts (contact 1 and contact 2) with human Fc in the crystal [Biochemistry 20 (1981) 2361-2370]. Comparisons of the chemical shift data of the Fc fragments observed in the absence and presence of FB have led us to conclude that in solution contact 1 is responsible for the formation of the Fc-FB complexes.

Animals↗
PubMed1981

Polyclonal activation of human B lymphocytes by Fc fragments. I. Characterization of the cellular requirements for Fc fragment-mediated polyclonal antibody secretion by human peripheral blood B lymphocytes.

Fc fragments derived from human immunoglobulin were found to be capable of inducing both a proliferative and polyclonal antibody response in human peripheral blood lymphocyte cultures. The cell population proliferating in response to Fc fragments belongs to the B cell lineage. Expression of polyclonal antibody formation requires the presence of both adherent monocytes and T cells. The role of the monocyte is to enzymatically cleave the Fc fragment into 19,000 mol wt Fc subfragments that are then able to induce polyclonal antibody secretion. Stimulation of polyclonal antibody production by Fc subfragments occurs in the absence of adherent monocytes but still requires the presence of T cells.

Adult↗
PubMed1991

[Study of the interaction of the monoclonal antibody to human interleukin-2 and its Fab-fragment with synthetic peptides --interleukin-2 fragments by (1)H-NMR].

Proton signals for nine synthetic peptide fragments of human interleukin-2 (region 59-78) were assigned for aqueous solutions both of pure peptides and their mixtures with LNKB-2 monoclonal antibody. The nonspecific magnetization transfer (NOE) between the antibody or its Fab-fragment and the peptides was studied upon large excess of free peptide over bound peptide. NOE spectra using modified pulse sequence, enabling to eliminate broad signals and achieve higher (peptide signal)/noise ratio were obtained. The saturation transfer experiments indicated that methyl groups of amino acid residues corresponding to Leu66,70,72, Val69 and Ala73 in interleukin-2 contact with the antibody binding site. Thus, the hydrophobic interactions are of major importance for the LNKB-2-IL-2 peptide complexes. The minimal IL-2 fragment which can still bind to LNKB-2 monoclonal antibody is -Leu70-Asn71-Leu72-.

Amino Acid Sequence↗