Intracellular metabolism of 4-hydroxynonenal in primary cultures of rabbit synovial fibroblasts.

The intracellular metabolism of 4-hydroxynonenal (HNE), a secondary product of lipid peroxidation and mediator of inflammation, which was found in the joints of patients with rheumatoid arthritis, was investigated in primary cultures of rabbit synovial fibroblasts. A consumption rate of 27.3 nmol/min x 10(6) cells was measured for the cultivated fibroblasts. It could be shown, that 4-hydroxynonenal enters the synovial fibroblasts and is metabolized mainly oxidatively to 4-hydroxynonenoic acid, intermediates of the tricarboxylic acid cycle and water and by formation of the glutathione-HNE adduct. The share of protein-bound HNE was about up to 8% of the total added HNE after 10 min of incubation. All metabolites accumulates intracellularly within the incubation time except of 4-hydroxynonenal itself. An increase of 4-hydroxynonenoic acid could be detected also extracellularly during the intracellular metabolism of 4-hydroxynonenal. Therefore, an involvement of synovial fibroblasts in the secondary antioxidant defense system of the joints during conditions of higher HNE concentrations like rheumatoid arthritis is suggested.

Inhibition of poly(ADP-ribose) formation by 4-hydroxynonenal in primary cultures of rabbit synovial fibroblasts.

The formation of poly(ADP-ribose) in primary cultures of rabbit synovial fibroblasts after treatment with active oxygen released by xanthine/xanthine oxidase is inhibited by addition of 1 and 10 microM 4-hydroxy-2,3-trans-nonenal (HNE). The endogenous formation of HNE by the xanthine/xanthine oxidase system is not responsible for the inhibitory effect of the aldehyde, owing to the low accumulation rate of the lipid peroxidation product in the system used. HNE is able to inhibit the isolated nuclear enzyme ADP-ribosyltransferase, as shown by an in vitro assay with an Ki of 4 mumol/litre. Therefore the molecular basis of HNE-mediated effects on cell proliferation, differentiation and transformation might be due to the inhibitory effect of poly(ADP-ribos)ylation.

Induction and activation of procollagenase in rabbit synovial fibroblasts after treatment with active oxygen released by xanthine/xanthine oxidase.

Treatment of rabbit synovial fibroblasts with active oxygen (AO) released by xanthine/xanthine oxidase resulted in an induction of procollagenase in these cells in concentrations ranging from 12.5 micrograms/ml xanthine plus 0.0025 U/ml xanthine oxidase to 50 micrograms/ml xanthine plus 0.01 U/ml xanthine oxidase. Preceding this there was an accumulation of poly(ADP-ribose) for the same concentration range of xanthine/xanthine oxidase. Furthermore, it was found that AO caused activation of the latent procollagenase to the active enzyme in concentrations ranging from 0.1 micrograms/ml xanthine plus 0.00002 U/ml xanthine oxidase to 1 microgram/ml xanthine plus 0.0002 U/ml xanthine oxidase. It is suggested that poly(ADP-ribosyl)ation participates in the induction of procollagenase by relaxing chromatin. Furthermore, it is proposed that AO activates latent procollagenase under physiological conditions.

Inhibition of the induction of collagenase by interleukin-1 beta in cultured rabbit synovial fibroblasts after treatment with the poly(ADP-ribose)-polymerase inhibitor 3-aminobenzamide.

3-Aminobenzamide is an inhibitor of poly-(ADP-ribosyl)ation. In concentrations from 3 to 10 mM it reduced the collagenase activity in culture supernatants of interleukin-1 beta-stimulated rabbit synovial fibroblasts. 3-Aminobenzoate, not an inhibiter of poly(ADP-ribosyl)ation, had no effect on collagenase activity at a concentration of 10 mM. We concluded that poly(ADP-ribosyl)ation plays a role in the induction of the expression of collagenase and that 3-aminobenzamide can inhibit this process.

Isolation of latent 31-kDa C-truncated stromelysin and 21-kDa stromelysin from rabbit synovial fibroblasts: an alternative activation pathway for stromelysin.

The processing of culture medium of rabbit synovial fibroblasts led to the isolation of three stromelysin-1 (MMP-3) cleavage products: A 31-kDa protein, which represents a C-truncated latent stromelysin-1, an active stromelysin-1 of 21 kDa, that originates from the 31-kDa proform by activation. A third protein had a molecular mass of 25 kDa representing the C-terminal part of prostromelysin-1 and is missing in the C-truncated latent stromelysin-1. The activation process of human prostromelysin-1 in vitro is known to lead to an active stromelysin-1 with a relative molecular mass of 45 kDa by removing the N-terminal prodomain. This active stromelysin-1 is further processed to a lower molecular mass active form of 28 kDa. Our results obtained for the highly homologous rabbit stromelysin-1 indicate that another activation pathway is possible. In a first step prostromelysin-1 is hydrolysed between Met261-Glu generating a C-truncated latent stromelysin-1, which is activated by cleavage of the Thr83-Phe bond to the 21-kDa stromelysin-1. The latent C-truncated stromelysin-1 is slowly converted even at 4 degrees C into the active form. In the presence of 50 microM ZnCl2 this activation was prevented for at least three weeks. The activation rate is largely enhanced by aminophenylmercury acetate and especially by trypsin. The differences of the 21-kDa stromelysin-1 to a 28-kDa stromelysin-1 isolated from human rheumatoid synovial fluids described earlier are discussed.

Proto-oncogene expression in cultured synovial fibroblasts of patients with rheumatoid arthritis.

Total RNA was isolated from cultured synovial fibroblasts of nine patients with rheumatoid arthritis and two controls (cruciate ligament ruptures). RNA was dot-blotted and hybridized with nine different, cloned cellular or viral oncogene probes. None of the proto-oncogenes showed a significant difference of expression in cultured fibroblasts from patients with rheumatoid arthritis when compared to the expression of control fibroblasts.

Short-lasting accumulation in osteoid bone seams of radioactive iron injected as citrate into mice.

The possible role in vivo of osseous structures in binding radioactive iron injected as a low-molecular-weight complex was studied in mice, using combined autoradiography and histomorphometry on sections of undecalcified, plastic-embedded femur epiphyses/metaphyses. A single intraperitoneal injection of 10 microCi 59Fe (1.2 micrograms Fe) per animal as citrate within 3 hours led to a preferential accumulation of this metal in the osteoid mineralized tissue interphase (osteoid seams) of bone. Within the next 2 days the labeling intensity in this localization diminished markedly to approximate levels of the bone marrow and calcified bone. The bulk of the injected radioiron was utilized according to known erythrokinetics. Findings suggest a direct entry of "free," ie, not transferrin-bound, iron into osteoid seams and its consecutive rapid removal from this site.

[Tolerability and pharmacokinetics of an intravenous immunoglobulin preparation in immunologically normal subjects and tolerability in patients with hypogammaglobulinemia resulting from chronic lymphatic leukemia]. / Verträglichkeit und Verweildauer eines intravenösen Immunglobulinpräparates bei immunologisch gesunden Personen und Verträglichkeit bei Patienten mit Hypogammaglobulinämie infolge chronischer lymphatischer Leukämie.

Tolerance, clinical effects and kinetics of an unmodified immunoglobulin preparation for intravenous use were investigated in 4 patients with advanced chronic lymphocytic leukemia. Previously, good tolerance of the preparation had been found in 49 immunologically normal patients. The four patients with secondary humoral immunodeficiency received doses of 140-360 mg IgG/kg per infusion as outpatients at monthly intervals. With one exception, no acute infections (pneumonitis), as commonly seen before, were observed during the observation time of 24 to 68 weeks, and the pre-existing chronic infections (bronchitis, sinusitis etc.) remained compensated without antibiotics. In all four patients tolerance of the preparation was good. In all cases of hypogammaglobulinemia a dose-dependent increase in the serum IgG concentration was observed immediately after the infusion. However, persistence of the serum IgG increase showed considerable interindividual differences. The half life of the tetanus and HBs antibodies (21.7 to 34.4 and 19.7 to 25.7 days respectively) found in 4 healthy volunteers is within the biological range. This indicates an unmodified structure of the antibodies of the IgG class contained in the preparation used.

Separation and characterization of two isolated lipases from Staphylococcus aureus (TEN5).

The purified lipases from Staphylococcus aureus (TEN5) showing two enzymatically active protein bands on SDS-polyacrylamide gel electrophoresis have been separated by ion-exchange chromatography. The separated proteins show some properties which are different (e.g., apparent molecular weight, charge, binding of detergent, enzymatic activity towards triolein) and some which are almost identical (spur in immunodiffusion).

Fermenter growth of Streptococcus agalactiae and large-scale production of CAMP factor.

Streptococcus agalactiae (group B) was grown in Todd-Hewitt broth (36.4 g l-1, pH 7.8) in a Braun Fermenter (type B20) to investigate the conditions of optimal bacterial growth and maximal production of CAMP factor. The influence of different gas atmospheres (air, N2, CO2, and gas mixtures) on growth, CAMP production and chain length of S. agalactiae was studied. The organisms grew best in the presence of 2% (w/v) glucose, at pH 6.2, with a constant flow of CO2. The number of diplococci and monococci under these conditions reached almost 80% of the total population.

Early and late events in streptolysin-O induced hemolysis.

Treatment of erythrocytes with activated Streptolysin (SLO) resulted in a "swelling" of the target cells. The mean cell volume (MCV) of toxin treated human - sheep - and rabbit red blood cells increased by about 15% of the original value as measured in the Coulter Counter apparatus. Release of hemoglobin was influenced by the addition of high molecular weight colloids. In contrast ATP release was independent of the osmolarity and started before an increase in MCV was observed. The experiments indicated that "colloid osmotic lysis" is involved in SLO induced hemolysis.

Amino acid sequences of antibody light chain variable regions of pedigreed rabbits: kappa light chain K49-501 (allotype b4 anti-streptococcal group A-variant polysaccharide antibody).

The amino acid sequence of positions 1--150 of a light chain, isolated from another monoclonal rabbit anti-streptococcal group A-variant polysaccharide antibody, was determined. The analysis was performed with 2 mumol of polypeptide chain, using a grossly modified Beckman 890B sequenator. This sequence stretch accounts for the whole variable region and a considerable part of the constant region at a total length of 218 amino acids. This allotype b4 light chain was isolated from a non-precipitating, end-group-specific antibody with a KD = 1.3 X 10(-5)M. This brings the present number of totally known rabbit VL sequences of antigen elicited antibodies to 21. A comparison of these 21 sequences reveals a building plan of ribbit VL homologous to that of human and murine VL regions. The observed variability does follow a pattern of linked amino acid substitutions, indicating that this information must be contained in the germ-line of the rabbit in the form of multiple VL region genes. This conclusion, however, does not rule out the occasional variant being due to somatic rearrangement. Finally, this comparison reveals that the joining peptide between positions 96--110 is also a separate entity in rabbit VL region sequences.

The VkVI subgroup of rabbit light chains: complete amino acid sequence of a third variable region (K29-213).

The amino acid sequence of the variable region of a rabbit anti-streptococcal A-variant antibody light chain was determined. By using a combination of different cleavage methods, the sequence was established. Large peptides were sequenced in an extensively modified Beckman sequentor. Light chain K29-213 belongs to a rare subgroup (kVI). Several of these light chains of antibodies with different specificities have been totally or partially sequenced. Comparison of these light chains reveals at least four germ-line-encoded variants within this subgroup.

Rabbit variable kappa light chain regions: subgroups contain polypeptides encoded by multiple genes.

Two identical light chain variable regions were identified in anti-streptococcal Group A-variant antibodies elicited in litter-mate rabbits by hyperimmunization with vaccine. In addition, one rabbit produced two additional clonally restricted antibodies to this polysaccharide antigen. The partial amino acid sequence of the light chain of one of these antibodies was identical with the dominant antibody light chain sequence, while the light chain of the other antibody, also partially established, showed significant variations in the framework-associated regions with identical CDRI and II. Since all of these light chains were from a small subset of rabbit kappa light chain pools (b4 allotype) the data suggest, together with other light chains reported in the literature, that more than one copy of variable region genes are present in the germ-line per subgroup. Furthermore, framework associated amino acid substitutions are not random; this suggests the existence of some "ordered" mechanism for linked amino acid substitutions (presumably recombination). Furthermore one light chain can pair with more than one heavy chain to yield functional antibodies.

[Lymphoproliferative diseases with small molecular (monomere) IgM paraproteinemia (proceedings)]. / Lymphoproliferative Erkrankungen mit kleinmolekularer (monomerer) 2gM-Paraproteinämie.

Phytohemagglutinin P, when used like an antiserum in immunoelectrophoresis, precipitates with pentameric IgM but not with monomeric IgM subunits. In a retrospective study this simple screening method was applied to 180 sera containing monoclonal IgM. Five cases were found to be of the monomeric type of IgM paraproteinemia. Four of these patients had lymphoproliferative diseases with lymphocytic infiltration of the bone marrow. The last case was complicated by acute myelomonocytic leukemia.

Variable-region subgroup and specificity of cold agglutinins.

The variable-region subgroup determined by amino acid sequence analysis of heavy and light chains of two monoclonal cold agglutinins with the new anti-Gd specificity is reported. Both proteins belong to the VHIII subgroup of heavy chains; one light chain falls into the V kappaI subgroup, the other has a blocked N-terminus which so far has not been observed in human kappa chains. The comparison of anti-Gd with anti-I/-i or anti-Pr cold agglutinins indicates that anti-Gd differs from other cold agglutinins with respect to variable-region subgroup. The data extend previous findings on the restriction of certain antibodies to distinct variable-region subgroups.

Cytoplasmic immunoglobulins in bone marrow cells of polyclonal and of monoclonal origin.

Cytoplasmic immunoglobulins in human bone marrow plasma cells and lymphoid cells were characterized by direct immunofluorescence with fluorochrome-labelled reagents specific for immunoglobulin heavy and light chains. The percentage distribution of cells containing IgA, IgG or IgM and kappa- or lambda-immunoglobulins was determined in bone marrow samples from 168 immunologically normal individuals, in 11 patients with polyclonal increase of bone marrow plasma cells and in 80 patients with benign or malignant monoclonal gammopathies. A clear differentiation between monoclonal and polyclonal cell populations could be obtained in all cases.