Study of fitting algorithms applied to simultaneous analysis of large numbers of peaks in gamma-ray spectra.

In this paper several nonlinear fitting algorithms without matrix inversion are proposed and investigated. The fitting algorithms represent an integral part of the analysis procedure, allowing us to simultaneously process large numbers of peaks in large blocks of data. The algorithms were applied to the analysis of both one-dimensional as well as two-fold coincidence gamma-ray spectra. The properties of the proposed methods and the suitability of employing the appropriate algorithms to different kinds of gamma-ray spectra were studied.

Equilibrium in the protein-immobilized-ligand-soluble-ligand system: estimation of dissociation constants of protein-soluble-ligand complexes from binding-inhibition data.

The equilibrium in the protein-immobilized-ligand-soluble-ligand system was examined theoretically and the equations found were used for determination of dissociation constants of protein-soluble-ligand complexes (K). These constants can be obtained from the s/b vs C plot [s/b = ratio of soluble and bound forms of the protein at equilibrium established in the presence of the soluble ligand (concn C)], which is linear if: (1) the concns of the complexes are much lower than the total concns of the immobilized and soluble ligands, and (2) if multiple interactions of an n-valent protein with the immobilized ligand essentially do not occur (i.e. the binding to the immobilized ligand is monovalent). The effect of violation of condition (1) is examined by computation simulation and is shown to be manifested as a non-linearity of the plot. Heterogeneity of the immobilized ligand (arising, for example, from the immobilization procedure) is predicted to have no effect on the K-values obtained. A more complex linear equation applicable principally for determination of K under more general conditions was also found. The conditions are defined under which the C50-values (i.e. concns of a series of ligands inhibiting the binding to an immobilized ligand by 50%) can be directly used for comparison of dissociation constants. The use of the s/b vs C plot was tested experimentally: transferrin, several glycoproteins or synthetic carbohydrate-containing copolymers were immobilized by adsorption in the wells of polystyrene microculture plates and thus served as immobilized ligands. Solutions of 125I-labelled ligand-binding proteins (lectins or monoclonal antibodies binding transferrin) were incubated in these ligand-coated wells in the presence of various amounts of soluble ligands (carbohydrates or transferrins): after equilibrium establishment the s/b values were determined and plotted against C and the values of K were obtained as the intercept of the plot with the abscissa. The method appears to be experimentally simple and the K-values of the lectin-sugar and monoclonal antibody-antigen complexes agree well with those determined by other methods.

Chromosomal aberrations and sister-chromatid exchanges (SCEs) in patients undergoing long-term Imuran therapy.

13 patients treated daily for an extended time with Imuran and prednisone and 4 patients treated in the same way with Imuran only were cytogenetically analysed for the induction of structural chromosomal aberrations and SCEs. There was an increase in the number of aberrations and SCEs in nearly all patients analysed. However, we did not find any dose-dependent cumulative effect on chromosomal damage, with the exception of 1 patient tested in a small group of 4 patients involved in a prospective cytogenetic study, who showed a significant time-dependent increase in the number of aberrations.

Rate of LCAT-mediated cholesterol esterification and serum lipids during etiroxate therapy in hyperlipoproteinemia.

Changes in the rate of the plasma cholesterol ester production mediated by lecithin: cholesterol acyltransferase (LCAT, E.C. 2.3.1.43) were examined in 15 patients suffering from types II and IV HLP who had been treated for 14 weeks with etiroxate. Whereas the plasma cholesterol concentration decreased significantly only in the initial phase of the therapy, the rate of cholesterol esterification increased gradually and attained at the end of the study a value exceeding by 50% the initial level. The final fractional turnover rate nearly equalled that characteristic for the control group of healthy subjects, in spite of the fact that the concentration of plasma cholesterol in the diseased subjects was higher by 50-100%. Triglyceride concentration decreased only transitorily in the course of the therapy with etiroxate. It is concluded that etiroxate is likely to normalize the rate of cholesterol turnover in the endogenous pool.

Complexity of the genetic control of a structural marker in the CH1 homology region of pig IgG.

The occurrence of two variant peptides P1 and P2 originating in the vicinity of the heavy-light chain disulphide bridge in the CH1 homology region was examined in IgG samples prepared from 36 randomly selected pigs. The peptides labelled with 35S at the half-cystine residues were separated on two-dimensional peptide maps and their ratio was quantitatively evaluated by a non-destructive automated method using semiconductor detectors. Fourteen individuals were found to yield only the peptide P1, the remaining 22 individuals yielded both P1 and P2 with a mean ratio of 69.4: 30.6. A model of genetic determination of the amino acid interchange, serine-leucine, distinguishing the peptides P1 and P2 was suggested. It has been assumed that the gene for one gamma-chain subclass exists only in the form coding for the peptide P1 whereas the gene for another gamma-chain subclass exists in two allelic forms coding for either P1 or P2.

Limited enzymatic cleavage of pig immunoglobulin G and of specific antibodies. I. Different resistance of various antibody types to cleavage by pepsin.

Limited proteolysis by pepsin at pH 4.5 of non-specific pig IgG and of two types of pig anti-dinitrophenyl antibodies revealed striking differences in susceptibility to proteolytic cleavage. The digests were resolved on a column of Sephacryl S-200 and the elution profiles analysed using a computer programme. The individual fragments were characterized by immunoelectrophoresis, SDS-polyacrylamide gel electrophoresis and molecular mass determination. Whereas the non-precipitating antibodies and the non-specific IgG yielded F(ab')2 and pFc' fragments in reasonable quantities, the precipitating antibody proved to be stable even at the 70th h of digestion. The precipitating antibody became susceptible to peptic cleavage only when the pH was lowered to 4.0. It was concluded that the precipitating antibody represents a subclass of pig IgG with an anomalous resistance to proteolysis at acidic pH and that this subclass constitutes only a few per cent, at most, IgG of the healthy, non-immunized animals.

On the mechanism of intercellular adhesion.

Using a 2 X 2 design, the collection rates of cells from radioactively labelled single-cell suspension on cell-coated collecting surfaces were tested for a possible H-2 effect on intercellular adhesiveness. In five donor-host combinations differing in the H-2 complex, no straight advantage of syngeneic over allogeneic relationship could unequivocally be proved. Trypsin was deliberately omitted in preparing the single-cell suspension; it was shown that the cell suspension processed in this way affected the mechanism of intercellular adhesiveness to a degree at least comparable to the role played by the properties of the collecting layer cells.

Antibody production stimulating activity of bone marrow cells from tolerant rats.

The antibody production stimulating activity of BM cells from SRBC-tolerant and normal rats were studied. Tolerance was induced by repeated injections of SRBC which were begun within 24 h after birth. BM cells obtained from tolerant animals 14-24 days after the last SRBC injection or from normal rats of the same age were used. LN cells from mice immunized with SRBC or BRBC served for the detection of BM activity. Four days after the second injection of antigen, LN cells were removed and cultivated alone or with BM cells from tolerant or control rats for 16 h. After cultivation, immune reaction was estimated by enumeration of indirect PFC. BM cells from SRBC-tolerant rats increased the number of SRBC PFC in the culture more than BM cells from control rats. On the other hand, the stimulatory activity of the BM cells from SRBC-tolerant animals for the BRBC-PFC was lower than that of normal BM cells or it was completely absent. The implications of these findings for the mechanism of the stimulating BM activity are discussed.

Chromosomal aberrations and SCE in lymphocytes of children revaccinated against smallpox.

Chromosomal changes were analysed in the peripheral lymphocytes of 14 twelve-year-old children before and 3 months and 10 months after smallpox revaccination (group A), and in peripheral lymphocytes of 8 children of the same age before and 1.5 and 8 months after revaccination (group B). A significantly increased number of aberrant cells was found after 1.5 and 3 months. In the blood samples collected 8 and 10 months after revaccination there was a decrease in the number of aberrant cells which, however, did not reach the control level. Sister chromatid exchanges (SCE) were counted in 5 children 1.5 and 8 months after revaccination, and their numbers did not differ from controls.

Dissociation of the proliferative and effector non-H-2 minor histocompatibility-alloimmune responses.

The first-set and second-set allotransplantation reactions against skin grafts and the primary and secondary proliferative graft-versus-host reactions in the popliteal lymph nodes were compared in both directions in a non-H-2 system (mouse strain combinations C57BL/10ScSnPh (further B10) and B10.C3H(40NX) further 40NX) differing at H-1 plus H-?). While 40NX recipients gave stronger reactions against B10 antigens in the allotransplantation reactions, the situation was reversed in the GVHR, B10 cells reacting more strongly against 40NX antigens. The findings of a dissociation between the mechanisms of allotransplantation reaction adn proliferative GVHR suggest that the genetic determination of the target antigens and the reacting lymphocyte populations are more complex at the minor histocompatibility systems than has been expected.

Xenogeneic bone marrow stimulating effect in vitro on antibody-producing cells.

It was described earlier that in the tissue cultures of immune mouse LN cells the number of antibody producing cells was increased 2-3-fold when syngeneic of allogeneic nonimmune BM cells were included in the cultures. In these experiments, parallel mixed cultures were set up of mouse immune LN cells with non-immune BM cells of either syngeneic or xenogeneic origin (rat, pig and chicken). Xenogeneic BM also increased the number of PFC in the mixed cultures. The intensity of the stimulation effected by xenogeneic BM of all three species tested was comparable to that induced by syngeneic BM. The number of PFC in all types of the mixed cultures was significantly higher (P less than 0.005) than in the LN cell cultures alone. The differences between the effect of syngeneic and xenogeneic BM and between the xenogeneic BM cells of different origin were not statistically significant (P less than 0.05).

[Biochemical and hematological changes in the blood of horses after the "Velká Pardubická" steeple chase]. / Biochemické a hematologické zmeny v krvi koní po dostihu "Velká pardubická".

Blood parameters were studied in two groups of horses in the "Velká Pardubická" steeple-chase in 1974, 1975 and 1976. After the race, the levels of lactate showed a manifold increase; an increase was also ascertained in the levels of glucose, sodium, potassium, haemoglobin, in the haematocrit value and in the number of erythrocytes. The following parameters significantly dropped: the levels of acid-base balance - pH, base excess, bicarbonate levels. It was proved that the values of the same parameters in horses during training were incomparably lower. It is advisable to examine horses thoroughly during training and to use the results of training for the evaluation of their condition before difficult races.

Intercellular adhesiveness of H-2 identical and H-2 disparate cells.

Using a 2 X 2 design, the collection rates of cells from radioactively labelled single-cell suspensions on cell-coated collecting surfaces were tested for a possible H-2 effect on intracellular adhesiveness. By a statistical procedure, the undesirable variability in the suspension and/or in the collecting surface could be eliminated, and the H-2 specific net effect estimated. In this way it was possible to detect (even on the background of a large non-specific variability) significant reductions of the allogeneic collection rates (i.e. between cells which differed only in their H-2 haplotypes) as compared to the syngeneic standards. It is concluded that cells of a given H-2 haplotype have preferential adhesiveness to syngeneic cells.

Cellular cooperation at the level of antibody-producing cells in chickens.

The effect of in vitro cocultivation of immune and non-immune syngeneic lymphoid cells on the antibody response was studied in chickens. Cocultivation of immune and non-immune spleen cells did not affect significantly the PFC numbers. Substantial increase in PFC was observed in mixed cultures of immune spleen cells with non-immune bone marrow cells. The existence of an enhancing effect of non-immune cells on the antibody-producing cells, which had been earlier described in mice, was observed in a phylogenetically distant species. Therefore it seems probable that it exists in most, if not all, homoiotherm animal species.

Time dependence of the effect of splenectomy on graft-versus-host reactivity of lymph node cells.

Changes in cell-mediated reactivity of lymph node cells at various intervals after splenectomy were investigated in three assays measuring the GVH reactivity of parental cells in F1 hybrids --splenomegaly test in very young recipients and popliteal lymph node enlargement assay in adults measuring the proliferative component of the reaction, and mortality assay in sublethally irradiated recipients measuring the killer activity of the cell inoculum. During the early postsplenectomy period the reactivity of the particular amounts of lymph node cells was lower than that of cells from normal donors, but at about 3 weeks after splenectomy it was higher. The increase was of short duration in the proliferation assay and at 5 weeks the reactivity declined markedly below the control values. The increase in activity persisted for 5 weeks after splenectomy in the "killer" assay. It is probable that the described changes in cell-mediated reactivity are involved in the total effect of splenectomy on the host's complex immune response, especially against normal and tumour allografts.

Attempt at "immunological castration" as an approach to the problem of the involvement of testosterone in control of expression of certain mouse antigens.

Adult male mice of the strain B10.A were immunized with a testosterone-protein conjugate, testosterone 3-(O-CARBOXYMETHYL)-oxime-bovine serum albumin which contained 27-75 steroid residues/mol BSA. Two different immunization doses of the conjugate were used, respectively, 2 x 40 mug and 2 x 200 mug in complete Freund's adjuvant or in alum adjuvant. There were two groups of control males, non-immunized and immunized with BSA in adjuvant. In the pooled immune sera, antibodies to testosterone were determined by radioimmunoassay; their titre ranged between 7 and 10. On histological sections of testes, inhibition of spermatogenesis (manifested by a sower frequency or even absence of tubules producing mature sperm, reduced frequency of tubular cells and their degenerative changes) was observed in almost all males immunized with the higher dose of the conjugate. In such animals, increased frequency of interstitial cells (except vascular elements) and enlarged nuclei of Leydig cells were found. In spite of these signs of a hyperproduction of testosterone by the Leydig cells, the product seemed to have lacked its normal biological activity as suggested not only by the low activity of spermatogenesis, but also by a significantly subnormal level of the androgen-dependent serum protein Ss.

PIP: Adult male mice of the strain B10.A were immunized with a testosterone-protein conjugate, testosterone-3-(0-carboxymethyl)-oxime-bovine serum albumin (T-BSA) which contained 27.75 steroid residues/mol BSA. 2 different immunization doses of the conjugate were used, respectively, 2 X 40 mcg and 2 X 200 mcg in complete Freund's adjuvant or in alum adjuvant. There were 2 groups of control males, nonimmunized and immunized with BSA in adjuvant. In the pooled immune sera, antibodies to T were determined by radio immunoassay: their titer ranged from 7 to 10. On histological sections of testes, inhibition of spermatogenesis (manifested by a lower frequency or even absence of tubules producing mature sperm, reduced frequency of tubular cells and their degenerative changes) was observed in almost all males immunized with the higher dose of the conjugate. In such animals, increased frequency of interstitial cells (except vascular elements) and enlarged nuclei of Leydig cells, were found. In spite of these signs of hyperproduction of T by the Leydig cells, the product seemed to lack its normal biological activity as suggested by the low activity of spermatogenesis and by significantly subnormal levels of androgen-dependent serum protein subjects.

Mutagenic effect of epichlorohydrin. I. Testing on human lymphocytes in vitro in comparison with TEPA.

The mutagenic effect of the monofunctional alkylating agent epichlorohydrin was tested on human lymphocytes in vitro and compared with the mutagenic effect of the polyfunctional alkylating agent TEPA. The same descending concentrations were used for both mutagens: 10(-4), 10(-5), 10(-6), 10(-7), 10(-8), 10(-9), 10(-10) and 10(-11) M. Similar types of chromosomal aberration were found, but the effect of ECHH was 4-5 times lower than that of TEPA. ECHH was found to be a mild mutagen. Different timing of mutagen application was used in the course of 56 h of cultivation of lymphocytes: 1 h before cultivation, one hour between the 24th and 25th h of cultivation and 24 h before the end of cultivation. From the results presented we conclude that the application of the chemical for the last 24 h of human lymphocyte cultivation should be recommended for routine mutagenicity testing.