Cytotoxic and noncytotoxic mechanisms involved in the in vitro anti-leukaemia effects of T cell clones established from a chronic myelogenous leukaemia patient during treatment in vivo with interferon α

Summary T cell clones derived from a chronic myelogenous leukaemia (CML) patient during interferon (IFN, Wellferon) biotherapy preferentially lysed autologous rather than allogeneic CML target cells in an apparently MHC-unrestricted fashion, but also lysed bone marrow cells from certain normal donors regardless of whether or not they shared HLA antigens with the patient. Although T cell clones inhibited both CML and normal bone marrow in the colony-forming assay, they blocked proliferation of CML cells more efficiently than bone marrow cells. This inhibitory effect was mediated at least in part by the tumour necrosis factor (TNF) and IFN secreted by the clones. Antisera to these cytokines partially prevented inhibition. Involvement of additional factors is also suggested in blocking CML cell proliferation because this was not 100% inhibited even by a combination of TNF and IFN. In addition, most clones failed strongly to block the proliferation of normal bone marrow cells, which were susceptible to inhibition by these cytokines.This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 120)     

Simultaneous binding of calcium and vanadate to the Ca2+-ATPase of sarcoplasmic reticulum

The interaction of vanadate with the Ca2+-ATPase of sarcoplasmic reticulum vesicles has been studied by making use of the ATPase activity as a measure of uncomplexed enzyme. The binding/dissociation is slow, so that initial rates can be used to study the equilibrium binding. The results indicate that in addition to a Ca2+-free complex E.Van (KV = 0.4 microM), there must also be a Ca2+-enzyme-vanadate complex (K'V = 7 microM). This observation is confirmed by the difference between the kinetics of decay of activity on vanadate addition, and on addition of ATP to enzyme preincubated with vanadate and Ca2+, which requires two enzyme-vanadate complexes. ATP increases the apparent affinity of the enzyme for vanadate by inducing calcium release. Upper limits for the kinetic parameters for vanadate binding and dissociation are estimated.     

Polymorphism of reconstituted human epidermal keratin filaments: determination of their mass-per-length and width by scanning transmission electron microscopy (STEM)

We have determined the mass-per-length (MPL) and the width of unstained freeze-dried reconstituted human epidermal keratin filaments by scanning transmission electron microscopy (STEM). Filaments were reassembled from keratins extracted from four different sources: cultured human epidermal cells (CHEC), human callus (CAL), and the living layers (LL) and stratum corneum (SC) of normal human epidermis. MPL histograms of all four keratin filament types could be fitted by a superposition of two or three Gaussians, with their respective major peaks located between 17 and 20 kDa/nm. We interpreted the multiple MPL peaks to represent different polymorphic forms of the reconstituted filaments. The number of subunits per filament cross section calculated from MPL peak positions, average subunit molecular weight, and an axial repeat of the subunits within the filament of 46.5 nm revealed an average difference between polymorphic variants of 7.5 +/- 0.9 subunits. These data suggest that reconstituted human epidermal keratin filaments are made of two to four 8-stranded "protofibrils" (i.e., made of two laterally aggregated 4-stranded protofilaments), in agreement with earlier observations. The average widths of unstained freeze-dried keratin filaments were larger than those of negatively stained filaments: 12.6 nm (9.6 nm) for CHEC, 12.3 nm (9.7 nm) for CAL, 11.6 nm (8.3 nm) for LL, and 11.3 nm (7.9 nm) for SC keratin filaments, with the values in brackets corresponding to negatively stained samples. Assuming the MPL to be proportional to the square of the filament width, there is a good correlation between the MPL and width measurements both for filaments within a given type as well as among those reconstituted from different types of keratin extracts.     

Properties of strange attractors in yeast glycolysis

The properties of periodic and aperiodic glycolytic oscillations observed in yeast extracts under sinusoidal glucose input were analyzed by the following methods. (1) Spectral analysis, rendering sharp peaks for periodic responses and enhanced broad-band noise for aperiodic oscillations. (2) Phase plane analysis, leading to closed and to open trajectories for periodic and aperiodic oscillations, respectively. (3) Rotation of a phase plane proportionally to time, revealing strange attractors associated with the aperiodic oscillations. (4) Stroboscopic plot on the phase plane, showing that the strange attractors follow a stretch-fold-press process, if the stroboscoping phase is varied. (5) Stroboscopic transfer plot, admitting a period of three transfer processes and thus implying chaos according to the Li-Yorke theorem. (6) Determination of the rate of information production by differentiation of the transfer plot, yielding approx. 0.21 bits per min for the chaotically glycolyzing yeast extract.     

Distribution of chlorpromazine and imipramine in adipose and other tissues of rats

Adipose tissue kinetics of chlorpromazine and imipramine, two drugs which are more lipophilic than thiopental, were studied in the rat. After single i.v. doses, the time-course of drug distribution was followed in adipose and various other tissues, until their concentrations in adipose tissues declined. Under these conditions the two drugs behaved almost identically. Among the tissues analyzed, the lowest concentrations were found in adipose tissue, with the exception of plasma. At its maximum concentration after about 30 minutes, total adipose tissue contained only 3 % of the dose of administered drugs. Adipose/plasma and adipose/lung concentration ratios were 2–5 and 0.05, respectively.After maximum tolerated oral doses of imipramine for 3 weeks, similar steady state concentration ratios (plasma:adipose:brain:lung 1:3:12:96) were observed. In adipose tissue the imipramine/desmethylimipramine ratio was about 1, and the desmethylimipramine steady state levels did not increase with time. Literature data indicate that many basic lipophilic drugs are not stored in adipose tissue. This is now clearly shown for chlorpromazine and imipramine, even under extreme, subchronic conditions in the case of imipramine.     

Increased lymphocyte adherence to human arterial endothelial cell monolayers in the context of allorecognition.

The interactions of alloreactive T lymphocytes with the vascular endothelium were studied in an in vitro model of lymphocyte adherence to cultured human arterial endothelial cell (HAEC) monolayers. Donor-primed lymphocytes (DPL) were shown to have significantly greater adherence to donor HAEC than were third-party primed lymphocytes. Limiting dilution analysis of adherent DPL showed an enrichment of donor-reactive lymphocytes compared with nonadherent DPL. This study examines the allospecific nature of this increased lymphocyte adherence. HAEC constitutively express class I HLA Ag and can be induced by IFN-gamma to express class II Ag. DPL adherence to class I+ HAEC was inhibited only in the presence of mAb directed against class I Ag. DPL adherence to class I+ and class II+ HAEC was inhibited in the presence of mAb directed against class I and class II Ag. Class I- and class II-specific adherence was also shown to involve CD8 and CD4 molecules, respectively, whereas lymphocyte function-associated Ag do not appear to play a major role in long term alloreactive lymphocyte adherence to HAEC. These findings suggest that alloreactive lymphocyte adherence to HAEC is mediated by two mechanisms. One is based on allorecognition, primarily of HLA Ag, and the other is related to presumably non-Ag-specific interactions between activated lymphocytes and the vascular endothelium. The studies presented provide evidence to suggest that HLA-specific lymphocyte adherence to endothelium may significantly contribute to the development of alloreactive lymphocyte infiltrates within the allograft.     

Cysteine: Depolarization-Induced Release from Rat Brain In Vitro

Compounds released on depolarization in a Ca2+-dependent manner from rat brain slices were screened to identify candidates for neuroactive substances. Lyophilized superfusates were analyzed by reversed-phase HPLC after derivatization with 9-fluorenyl N-succinimidyl carbonate. One of the compounds that showed an increase of concentration in superfusates in the presence of iodoacetamide was identified as the cysteine (Cys) derivative, S-carboxamidomethylcysteine, by fast atom bombardment mass spectrometry and other methods. This stable Cys derivative originates from endogenous, extracellular Cys. The finding led to a method for quantification of Cys in superfusates by immediate cooling of the superfusates to 0 degrees C and reaction of Cys with N-ethylmaleimide. Depolarization-induced Ca2+-dependent release of Cys was most prominent in the neocortex, followed by the mesodiencephalon, striatum, and cerebellum. This suggests that Cys is released from a neuronal compartment and might be involved in neurotransmission.     

Association of kidney and parotid Na+, K(+)-ATPase microsomes with actin and analogs of spectrin and ankyrin

Kidney Na+,K(+)-ATPase has been recently shown to bind erythroid ankyrin and to colocalize with ankyrin at the basolateral cell surface of kidney epithelial cells. These observations suggest that Na+,K(+)-ATPase is linked via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we show that Na+,K(+)-ATPase and analogs of spectrin, ankyrin and actin copurify from detergent extracts of pig kidney and parotid gland membranes. Actin, spectrin and ankyrin were extracted from purified Na+,K(+)-ATPase microsomes at virtually identical conditions as their counterparts from the erythrocyte membrane, i.e., 1 mM EDTA (spectrin, actin) and 1 M KCl (ankyrin). Visualization of the stripped proteins by rotary shadowing revealed numerous elongated spectrin-like dimers (100 nm) and tetramers (215 nm), a fraction of which (17%) was associated with globular (10 nm) ankyrin-like particles. Like erythrocyte ankyrin, kidney ankyrin was cleaved into a soluble 72 kDa fragment and a membrane-bound 90 kDa fragment. Consistent with our previous immunocytochemical findings on the pig kidney, Na+,K(+)-ATPase and ankyrin were found to be colocalized at the basolateral plasma membrane of striated ducts and acini of the pig parotid gland. The present findings confirm and extend the recently proposed concept that in polarized epithelial cells Na+,K(+)-ATPase may serve as major attachment site for the spectrin-based membrane cytoskeleton to the basolateral cell domain. Connections of integral membrane proteins to the cytoskeleton may help to place these proteins at specialized domains of the cell surface and to prevent them from endocytosis.     

Effects of abscisic acid on somatic embryo maturation of hybrid larch

Somatic embryos of hybrid larch (Larixleptoeuropaea) whichhad been matured for 4 weeks on maturation medium, developednormally on medium supplemented with 60 µM ABA, but abnormallyon medium with no ABA. A comparative structural and histochemicalinvestigation was carried out on these two types of mature embryos.At the light microscope level, differences between both treatmentswere visible only after 2–3 weeks of maturation. At aroundthis time, abnormal development becomes evident macroscopically:ABA-minus embryos remain rather stubby as opposed to the morecylindrically shaped ABA-plus embryos. Whereas somatic embryosmatured with ABA consist of densely cytoplasmic cells showinga high rate of cell division, ABA-minus embryos are largelymade up of expanded and highly vacuolate cells, indicating thatgrowth in the latter is mainly due to cell expansion and notdivision. After 4 weeks of maturation, ABA-minus embryos beginto elongate in the hypocotyl region, and precocious germinationwas observed frequently. Again, these morphogenetic events werelargely due to abnormal timing of cell expansion. Histochemically,storage proteins were found only in somatic embryos maturedfor 4 weeks with ABA. This observation is in line with resultsobtained by total protein analysis, yielding significantly lowertotal protein contents in ABA-minus embryos both on a freshweight and a per embryo basis after 4–5 weeks of maturation.Deposition of starch grains mainly in the cortex tissue of thehypocotyl region was observed within 2 weeks of maturation invarying amounts regardless of ABA supply. Polyphenols, in particularcatechins and proanthocyanidins, were present in all embryosfrom the very onset of development. They were localized preferentiallyin the proximal suspensor cells and the basal region of theembryo. However, accumulation of polyphenols was generally muchmore pronounced in embryos matured without ABA, indicating alack of biochemical regulatory competence in those embryos. Key words: Abscisic acid, embryonal development, somatic embryo, storage protein, polyphenols