Clinical importance of circulating immune complexes in human acute lymphoblastic leukemia

Summary A total of 122 sera from acute lymphoblastic leukemia (ALL) patients were analyzed for circulating immune complexes (CIC) by two methods: the ¹²⁵I-C₁q binding assay and the polyethylene glycol precipitation test (PEG). The results were correlated with induction, remission and relapse stages of the disease. Using the first method the levels of CIC in induction were 15.18±9.15, with 19/29 positive cases (65.50%), P<0.001 compared with controls. In the remission phase the levels were 9.02±5.62, 11/45 (24.49%) nonsignificant P value, and in relapse they were 16.14±11.17 28/48 (58.33%) P<0.001. The PEG precipitation test results were: 0.33±0.10, 8/22 (36.36%); 0.24±0.11, 10/48 (20.83%) and 0.28±0.10, 6/28 (21.42%), respectively. Thus the values of CIC as measured by PEG in the three clinical of phases ALL did not differ significantly from controls. This contrasts with results obtained by the radioiodinated C₁q binding assay, where the incidence of positive values was significantly higher in induction and in relapse and lower in the remission phase. These observations were extended in sequential vertical studies performed in a group of patients. These results suggest that raised CIC detected by the ¹²⁵I-C₁q method may reflect a progressive state in ALL and that quantitation of these immune complexes may provide an adequate biochemical marker for prognosis.     

Biological basis for the management of ‘lugs negra’ (Sarcothalia crispata Gigartinales,Rhodophyta) in southern Chile

The present paper describes growth dynamics in a natural bed of the resource luga negra (Sarcothalia crispata) in Guapilinao, southern Chile (41°57 S, 73°31 W). This resource is currently harvested and exported as raw material for the production of carrageenan. Seasonal variation in biomass, frond size, density and phenology was determined by periodic sampling. Natural recruitment was evaluated on different substrata added to the field; at the same time, substrata were inoculated under greenhouse conditions. Results showed that luga negra has seasonal growth: biomass increased from a minimum in spring to a maximum in mid to late summer. On the other hand, density was minimal in winter (200 ind. m^–2) and increased to 2000 ind. m^–2 in late spring. Peak abundance of mature tetrasporic fronds occurred in late summer, whereas that of cystocarpic fronds occurred in winter. Recruitment began in summer and extended into winter. Survival on different substrata were compared. Gametophytes had better survival rates on clam shells and 5 mm rope while tetrasporophytes had the best survival rate on clam shells and secondarily on boulders.     

A stochastic model for the sigmoidal behaviour of cooperative biological systems

A stochastic model for cooperative transitions in biological systems based on a Markov chain is proposed. This model requires only two parameters, the mean probability, p, and the coupling capacity, Deltap, which measure the probability of forming a new weak bond depending on the number of similar bonds already formed and it is also responsible for the transition. In this paper we show how the model works for a large number of identical molecules and how it can be useful for studying the noise around the centre of the transition where, increasing the degree of cooperativity, i.e. the number n in the well-known Hill equation, the width of the noise increases along with its fractal dimension. A simple relationship between the degree of cooperativity and the parameter Deltap is proposed, suggesting that the cooperativity of real biological transitions is related to the coupling capacity Deltap of the present model.     

Zymosan-induced luminol-amplified chemiluminescence of whole blood phagocytes in experimental and human hyperthyroidism

Luminol-amplified CL of whole blood phagocytes was studied in rats given 3 consecutive doses of 0.1 mg L-triiodothyronine T₃/kg or in hyperthyroid patients, after stimulation by zymosan. In both cases, CL was significantly increased, in effect which was produced independently of the opsonization of the zymosan particles and markedly inhibited by azide. The in vitro addition of T₃ or L-thyroxine (T₄) to whole blood phagocytes from normal rats did not modify the opsonized zymosan-dependent CL, when assayed at the concentrations found in eutrhyroid subjects or in hyperthyroid patients. Administrations of propylthiouracil (400 mg/day for 2–3 months) to hyperthyroid patients reduced the CL response observed prior to treatment, to values comparable to those found in the euthyroid group. These data indicate that hyperthyroidism elicits an enhanced respiratory burst activity of whole blood phagocytes, probably related to adaptive changes induced by thyroid hormone on the mieloperoxidase-H₂O₂ system, rather than to direct actions of the hormone molecule or changes in the opsonic capacity of plasma.     

Probing the structure and mobility of Pseudomonas aeruginosa azurin by circular dichroism and dynamic fluorescence anisotropy.

The UV dynamic fluorescence and CD of several Pseudomonas aeruginosa azurins bearing single amino acid mutation have been studied. Two classes of mutants were examined. In the first class, two hydrophobic residues in the core of the protein, Ile 7 and Phe 110, nearest to the azurin single tryptophan Trp 48, were substituted by a serine (mutants 17S and F110S). In the second class, two residues in the outer sphere of the copper ligand field were changed, obtaining the following mutants: M44K, H35F, H35L, and H35Q. All these proteins showed two fluorescence lifetimes in the copper-containing form, but only one in the copper-free form. The lifetime of the latter derivatives was different from either those of the metal-bound samples, definitely ruling out the presence of apo-like species in the holo protein. Copper-free 17S and F110S showed a more complex fluorescence decay profile requiring a distribution of lifetimes rather than a single lifetime. Holo F110S was also better fitted, in the limit of confidence, with two distributions rather than a pair of lifetimes. Time-resolved anisotropy of these two mutants as well as of wild-type (wt) protein showed two components (rotational times for wt < or = 200 ps and 7 ns, respectively). These components were not affected significantly by copper removal in the case of wt protein. Instead, the short rotational component of the mutants dropped dramatically to values near zero, indicating a much greater mobility of the tryptophanyl residue in the mutant apo azurins. These data were supported by CD measurements showing a small effect of the copper presence in the region below 250 nm, i.e., in the secondary structure, but almost a collapse of the aromatic asymmetry at 270-295 nm related to a relaxation of the structural constraint around the tryptophan. Altogether these data show that copper does not play a structural role in wt azurin, whereas it is crucial in the stabilization of 17S and F110S mutants. Furthermore, although the metal site geometry is rigidly kept in wt apo-azurin, it regains the native form only in the presence of the metal in the "core" mutants. This finding is important for the theory of entatic states in metalloproteins (Williams RJP, 1995, Eur J Biochem 234:363-381).     

Pharmacological Characterization of the Voltage-Dependent Ca2+ Channels Present in Synaptosomes from Rat and Chicken Central Nervous System

Abstract: The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (ω-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and ω-agatoxin IVA]. K⁺-induced Ca²⁺ uptake by chicken synaptosomes was blocked by ω-conotoxin GVIA (IC₅₀ = 250 nM). This toxin at 5 µM did not block Ca²⁺ entry into rat frontal cortex synaptosomes. FTX and ω-agatoxin IVA blocked Ca²⁺ uptake by rat synaptosomes (IC₅₀ = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω-agatoxin IVA affected Ca²⁺ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC₅₀ similar to the one obtained in rat preparations, whereas with ω-agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω-conotoxin GVIA was never greater than the one observed with ω-conotoxin GVIA. We also found that 60% of the Ca²⁺ uptake by rat and chicken synaptosomes was inhibited by ω-conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca²⁺ uptake in either the rat or the chicken. In conclusion, Ca²⁺ uptake by rat synaptosomes is potently inhibited by different P-type Ca²⁺ channel blockers, thus indicating that P-type channels are predominant in this preparation. In contrast, Ca²⁺ uptake by chicken synaptosomes is sensitive to ω-conotoxin GVIA, FTX, ω-agatoxin IVA, and ω-conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes.     

Mevalonate dependency of the early cell cycle mitogenic response to epidermal growth factor and prostaglandin F2α in Swiss mouse 3T3 cells

Lovastatin (LOV), a hydroxy-methylglutaryl-coenzyme A (HMGCoA) reductase competitive inhibitor, blocks epidermal growth factor (EGF)— or prostaglandin F_2α (PGF_2α)—induced mitogenesis in confluent resting Swiss 3T3 cells. This inhibition occurs even in the presence of insulin, which potentiates the action of these mitogens in such cells. LOV exerts its effect in a 2–80 μM concentration range, with both mitogens attaining 50% inhibition at 7.5 μM. LOV exerted its effect within 0–8 h following mitogenic induction. Mevanolactone (10–80 μM) in the presence of LOV could reverse LOV inhibition within a similar time period. LOV-induced blockage of PGF_2α response is reflected in a decrease in the rate of cell entry into S phase. Neither cholesterol, ubiquinone, nor dolichols of various lengths could revert LOV blockage. In EGF- or PGF_2α-stimulated cells, LOV did not inhibit [³H]leucine or [³H]mannose incorporation into proteins, while tunicamycin, an inhibitor of N′ glycosylation, prevented this last phenomenon. Thus, it appears that LOV exerts its action neither by inhibiting unspecific protein synthesis nor by impairing the N′ glycosylation process. These findings strongly suggest that either EGF or PGF_2α stimulations generate early cell cycle signals which induce mevalonate formation, N′ glycoprotein synthesis, and proliferation. The causal relationship of these events to various mechanisms controlling the onset of DNA synthesis is also discussed. © 1995 Wiley-Liss, Inc.     

Linkage disequilibrium, mutational analysis and natural selection in the repetitive region of the clock gene, period, in Drosophila melanogaster

We have used the method of disequilibrium pattern analysis to examine associations between the threonine-glycine (Thr-Gly) encoding repeat region of the clock gene period (per) of Drosophila melanogaster, and polymorphic sites both upstream and downstream of the repeat, in a number of European fly populations. The results are consistent with the view that selection may be operating on various haplotypes which share the Thr-Gly length alleles encoding 17, 20 and 23 dipeptide pairs, and that the repeat itself may be the focus for selection. These conclusions lend support to a number of other population and behavioural investigations which have provided evidence that selection is acting on the Thr-Gly region. The linkage analysis was also used to infer an approximate mutation rate (mu) for the repeat, of 10(-5) < mu < 4 x 10(-5) per gamete per generation. Direct measurements of the mutation rate using the polymerase chain reaction in a pedigree analysis of tens of thousands of individuals do not contradict this value. Consequently, the Thr-Gly repeat does not have a mutation rate that is as high as some of the non-coding minisatellites, but it is several orders of magnitude higher than the nucleotide substitution rate. The implications of this elevated mutation rate for linkage disequilibria and selection are discussed.     

Functional properties of FC-2.15, a monoclonal antibody that mediates human complement cytotoxicity against breast cancer cells

FC-2.15 is a murine IgM monoclonal antibody (mAb) that recognizes a cell-surface antigen (Ag2.15) expressed in most tumor-proliferating cells of human breast carcinomas and other neoplasias. In this study the cytotoxic ability of mAb FC-2.15, its cell-surface binding properties and endocytosis in Ag2.15-expressing (Ag2.15⁺) cells were investigated. A⁵¹Cr-release assay was used to test the FC-2.15-mediated cytotoxicity. When human serum was used as source of complement, FC-2.15 exerted a strong cytotoxic effect against human Ag2.15⁺ cells such as MCF-7 (breast cancer cell line), primary breast carcinoma cells, polymorphonuclear leukocytes and chronic myeloid leukemia cells. The mAb concentration range was 1–50 g/ml. Cytotoxicity was completely abolished when complement was inactivated. Only 3.8±2.9% of MCF-7 cells survived the treatment with FC-2.15 in the presence of human serum. A flow-cytometry assay was performed to study the Ag2.15 expression of the surviving cells and they were found to be Ag2.15^–. FC-2.15 did not mediate antibody-dependent cell cytotoxicity when different effector cells were used. Scatchard analysis with¹²⁵I-FC-2.15 on MCF-7 cells demonstrated an affinity constant of 6.9×10⁷ M^–1 and 2.8×10⁶ antigenic sites/cell.¹²⁵I-FC-2.15 was internalized to cytoplasmic vesicles reaching a maximum of 27% after 6 h incubation, followed by the release of labeled degradation products to the supernatant. FC-2.15 appears to exert its cytotoxic effect mainly in the presence of human complement, it reacts with intermediate affinity with a high-density surface antigen, and it is slowly internalized by Ag2.15⁺ cells.