Protein Content of Circulating Nucleoprotein Complexes.

In the current study we have investigated the protein content of blood plasma deoxyribonucleoprotein complexes. The complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies. Proteins were separated by SDS PAAGE and identified by MALDI-TOF mass-spectrometry. 111 and 56 proteins (excluding histones), respectively, were identified with a good score in deoxyribonucleoprotein complexes of healthy females and breast cancer patients. However, only four of these proteins were found in 30 % of all samples. Fourteen proteins previously described as tumor specific proteins were found in cancer patients whereas not one of them was found in healthy individuals. The data obtained demonstrate the involvement of different cellular and extracellular proteins in circulating cell-free DNA.

[Blood serum as a factor of chromatin condensation in trisomy-21 (Down's syndrome)]. / Syvorotka krovi kak faktor kontsentratsii khromatina pri trisomii-21 (sindrom Dauna).

The influence of blood sera from patients with Down's syndrome and healthy ones, and different serum fractions on the structure of the deoxyribonucleoprotein systems (DNP-system) has been studied. It was demonstrated that non-fractionated sera of the patients produce a condensation effect on the DNP-system in contradistinction to the sera from healthy people. The analysis of the action of single serum fractions showed that different condensation effect results from the activity of high molecular nondialysable thermosensitive components whose action disappears at gel-filtration of serum proteins. A possibility of the humoral control over chromatin structural organization in vivo is discussed in terms of the evidence on the similarity of serum proteins and chromosomal nonhistone proteins.

Immunospecificity of nonhistone chromatin proteins tightly bound to DNA from chicken thrombocytes and erythrocytes.

Erythroid cell-specific antisera capable of detecting tightly bound nonhistone chromatin protein--DNA complexes were obtained by injecting rabbits with dehistonized chicken reticulocyte chromatin. The antisera showed no crossreactivity with chromatin of thrombocytes which are regarded as cells genealogically closely related with erythrocytes. The lack of thrombocyte chromatin immunoactivity was not caused by conformational constrains. Tightly bound nonhistone protein--DNA complexes isolated from thrombocyte chromatin showed no immunological similarity with these of erythrocyte chromatin.

Nucleosome reconstitution: effect of DNA length on nuclesome structure.

Core histones (H2A, H2B, H3, and H4) are reconstituted by salt gradient dialysis with DNA molecules ranging in length from 177 bp down to 50 bp. While reconstituted particles containing 125 bp are very similar to native particles, those particles containing a single piece of shorter DNA tend to aggregate. The aggregation depends on the ionic strength and DNA length. The DNA placement on the histone core is not random as determined by pancreatic DNase I digestions of particles containing 32P 5'-end-labeled DNA. Rather, it is found that all DNA molecules, up to 161 bp in length, reassociate with core histones in such a way as to produce defined patterns of DNase I cutting with respect to the 5' ends. Particles were made that contained two pieces of 65-bp DNA. These particles are very similar to native particles under most conditions but tended to dissociation results in the production of two half-nucleosomes (hemisones).

[Cytochemical study of lymphocyte nucleoproteins in rheumatoid arthritis patients]. / Tsitokhimichno izsledvane na nukleoproteini v limfotsitite na bolni s revmatoiden artrit.

The purpose of the study is to investigate and juxtapose the cytochemical characteristics of ribonucleoproteins (RNP) and deoxyribonucleoproteins (DNP) in the lymphocytes of patients with rheumatoid arthritis (RA), of healthy lymphocytes and lymphocytes blast-transformed by phytohemagglutinins. Significant differences in the staining of lymphocytes for nucleoproteins were established in all 54 patients with RA studied. The unidirectional changes in the morphological form of the staining for RNP and DNP in blast-transformed lymphocytes and lymphocytes in RA is, most likely, in connection with the transition of the latter into active functional state and their participation in the forming of the immune inflammation. No significant difference was established in the dependence on the RA form--sero-positive or sero-negative. Regardless of the negative serological samples in the second form, there were some changes in the lymphocytes--carriers of immune reactions in organism.

Partial characterization of chromosomal proteins tightly bound to chicken erythroid DNA.

Extraction of chicken reticulocyte and erythrocyte chromatins with 2 M NaCl yields a small fraction (about 5%) of the total DNA which is very tightly bound to a class of nonhistone chromatin proteins (DNA-P). This DNA fraction has previously been shown to be significantly enriched in active gene sequences. The proteins associated with reticulocyte and erythrocyte DNA-P were analyzed by two-dimensional gel electrophoresis. Reticulocyte DNA-P yield predominantly three major proteins, designated G1, G2, and G3 with relative masses of 80 000, 50 000, and 58 000, respectively. Erythrocyte DNA-P show only two proteins which appear to be similar to the reticulocyte G1 and G2 proteins, except in much reduced quantities as revealed by two-dimensional polyacrylamide gel electrophoresis. Amino acid analysis of the three reticulocyte proteins revealed that the ratio of acidic to basic amino acid residues increased in the order G1 less than G2 less than G3, while the respective isoelectric points also increased in that order.

Core nucleosomes by digestion of reconstructed histone-DNA complexes.

Reconstructed complexes of the inner histones (H2A, H2B, H3, H4) and a variety of DNAs were digested with micrococcal nuclease to yield very homogeneous populations of core nucleosomes (nu 1). Nucleosomes containing Micrococcus luteus DNA (72% G+C); chicken DNA (43% G+C), Clostridium perfringens DNA (29% G+C); or poly(A-dT.poly(dA-dT) have been examined by circular dichroism, thermaldetenaturation, electron microscopy, and DNAse I digestion. Circular dichroism spectra of all particles show a typically suppressed ellipticity at 260--280 nm and a prominent alpha-helix signal at 222 nm. All particles show biphasic melting except nu 1 (dA-dT), which show three prominent melting transitions at ionic strength less than or equal to 1 mM. DNAse I digestion of nu 1 (dA-dT) produces a ladder of DNA fragments fiffering in lengthy by one base residue. nu 1 (dA-dT) contain 146 base pairs of DNA and exhibit an average DNA helix pitch of 10.4-10.5 bases per turn. There appear to be two regions of different DNA pitch wihtin nu 1 (dA-dT). It is suggested that the two regions of DNA pitch might correspond to the two regions of the melting profiles.

DNA on membrane receptors: a target for monoclonal anti-DNA antibody induced by a nucleoprotein shed in systemic lupus erythematosus.

Antibodies to double stranded (ds) DNA correlate with clinical evolution in systemic lupus erythematosus (SLE) although little is known about the immunogen and target for these antibodies, since ds DNA is poorly immunogenic. We now show that monoclonal anti DNA antibodies similar to those detected in human SLE can be produced by immunization of genetically non-autoimmune mice with a human circulating DNA-protein complex increased in the circulation of SLE patients. One such monoclonal antibody showed antinuclear reactivity, interacting with a 74 kd DNA-binding membrane protein, in reactions prevented by absorption with ds DNA cellulose. Our data suggests that anti ds DNA antibody reactions in SLE may be triggered by circulating nucleoproteins and directed toward membrane receptors capable of interacting with extracellular DNA.

Singlet-singlet energy transfer studies of the internal organization of nucleosomes.

We report the measurement of two specific protein to DNA distances in several conformational states of core nucleosomes by singlet-singlet energy transfer. A distance of 50-53 A separates each DNA terminus from cysteine-110 of chicken erythrocyte histone H3 in the native nucleosome. This cysteine residue must therefore be located very near the center of the nucleosome. The H3-DNA distance remained nearly constant in several unfolded forms of the core particles, as found in very low salt, in 0.6 M NaCl, and in high urea. Furthermore, it was shown that each DNA end lies within 32 A of cysteine-73 of Arbacia lixula sperm histone H4 in both the compact and the low-salt unfolded forms of the nucleosome. Because of the invariance of the two measured distances in the various conformational states of the nucleosome, we conclude that the cysteine-containing C-terminal segments of histones H3 and H4 maintain a very strong and close association with the terminal positions of the 146 base pair nucleosomal DNA. This binding may provide the primary interactions necessary for the folding of DNA into nucleosomes and for protection of 146 base pair nucleosomes from further nuclease digestion.

Reactive arthritis associated with group C and group G beta-hemolytic streptococci.

OBJECTIVE: Group A beta-hemolytic streptococci (GAS) are known to be capable of evoking sterile arthritis. Reactive arthritis (ReA) has been reported sporadically following primary infection with group C and group G beta-hemolytic streptococci (GCS, GGS). We prospectively studied 4 cases of ReA secondary to throat infection with GCS and GGS. METHODS: Four patients with arthritis secondary to throat infection were seen. Three patients were Dutch, one was Indonesian; female/male ratio was 1/3; mean age was 30 years (range 18-46). Diagnostic evaluation included culture of throat swab and serological screening. RESULTS: All patients presented with a nonmigratory asymmetrical arthritis: monoarthritis in one patient, oligoarthritis in 3. Culture of throat swab was positive in all. Antistreptolysin-O (ASO) titer rose significantly in 2 patients, and anti-DNase-B rose in 2 patients. ASO was maximal (mean 1000 U/ml; range 890-1110) and anti-DNase-B was 395 U/ml (range 290-500). Treatment consisted of feneticillin for 5 days; nonsteroidal antiinflammatory drugs were prescribed on demand. All patients recovered fully in 3 to 12 weeks. CONCLUSION: These cases provide evidence of a benign non-group A streptococcal ReA, i.e., secondary to GCS or GGS. The presence of the organism in the throat along with the elevation of antibody to streptococcal products is important for the diagnosis of GCS/GGS associated ReA. A positive throat culture is needed for differentiation from GAS associated poststreptococcal ReA, because prophylactic measures are effective only in GAS associated sequelae, but not in GCS/GGS associated ReA.