Susceptibility of autologous target cells to lysis by lymphokine-activated effectors from interferon-α-treated chronic myelogenous leukaemia patients

Summary Chronic myelogenous leukemia (CML) patients in chronic phase display compromised lymphokine-activated killer (LAK) cell induction, which is partly restored after therapy with interferon . However, the relative resistance of the leukemic cells from these patients to autologous or allogeneic LAK lysis is not affected by this treatment. In an attempt to render CML cells more susceptible to lysis or cytostasis, they were precultured in serum-free medium with or without recombinant growth factors. In eight patients studied, interleukin-3 (IL-3) significantly enhanced the spontaneous short-term (6-day) proliferation of CML cells, with retention of ability to form colonies in methylcellulose. Culture in either medium alone or IL-3 led to a significant enrichment of CD14⁺ and CD33⁺ cells but to a reduction in CD34⁺ cells. In contrast, culture of the same cells in IL-2 (to generate autologous LAK activity) resulted in a loss of CD14⁺ and CD33⁺ as well as CD34⁺ cells but in a significant increase in CD3⁺ and CD56⁺ cells. Despite similarities in their phenotypes, IL-3 cultured cells but not those cultured in medium alone acquired susceptibility to lysis by the IL-2-cultured autologous LAK cells. These results may have significance for the design of novel combination immunotherapy in CML.This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 120)     

The effects of recombinant bovine interferon-alpha on fertility in ewes

Recombinant bovine interferon-alpha(I)1 (rBolFN-alpha) may be useful for enhancing fertility in sheep because it has extensive sequence homology with ovine trophoblast protein-1. To test the effectiveness of rBolFN-alpha, several experiments were performed in which bred females were given intramuscular injections of rBolFN-alpha around the time of maintenance of the corpus luteum. Treatment with rBolFN-alpha enhanced the fertility of ewes that were bred via natural service or embryo transfer of whole or demi-embryos. Interferon treatment was successful in enhancing lambing rate if injections were given twice daily from Days 11 to 18, 12 to 14, 12 to 15 or 12 to 16. Overall, the lambing rate for ewes bred via natural service was 94/126 (74.6%) for control ewes and 101/126 (80.2%) for rBolFN-alpha treated ewes. Litter size was not affected by treatment. Interferon treatment was not successful in increasing the lambing rate if given as a single injection on Day 12 or as a series of once-daily injections from Days 11 to 16. These results demonstrate that rBolFN-alpha can increase the lambing rate in ewes.     

A small deletion and an adjacent base exchange in a potential stem-loop region of the neurofibromatosis 1 gene

Summary A single-strand conformational polymorphism found in the DNA of a patient with neurofibromatosis 1 (NF1) was shown to be caused by a deletion of a CCACC or CACCT sequence and an adjacent transversion, located about 500 base pairs downstream from the region that codes for a functional domain of the NF1 gene product. This mutation could also be detected in the patient and in his affected daughter by heteroduplex analysis. The deletion removes the proximal half of a small potential stem-loop and interrupts the reading frame in exon 1. A severely truncated protein with a grossly altered carboxy terminus lacking one third of its sequence is expected to be formed from the mutant allele.     

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)     

Amino acid analysis by dinitrophenylation and reverse-phase high-pressure liquid chromatography

The determination of amino acids has been achieved by reverse-phase high-pressure liquid chromatography of their dinitrophenyl derivatives. The methods developed permit the quantitation of all amino acids commonly encountered in a protein hydrolysate and the effect of various parameters on this separation was systematically evaluated. The procedure eliminates the need for specialized postcolumn equipment as employed in conventional amino acid analysis and can be obtained by a simple gradient high-pressure chromatograph. The sensitivity obtained is comparable to that available by methods in common usage, being able to determine amino acids quantitatively in the low picomole range.     

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.     

Applicability of imprints to immuno-ultrastructural studies of lymphoid tissues

In order to evaluate the applicability of imprints to immuno-ultrastructural studies of lymphoid tissues, we compared distribution pattern and morphology of B cells, T cells, T-cell subsets, and follicular dendritic reticulum cells (DRC) at the light and ultrastructural level in imprints and sections of tonsils and lymph nodes. The surface antigenic profile of lymphoid cells was revealed with monoclonal antibodies in an avidin-biotin-peroxidase complex (ABC) method. Distribution of lymphoid cells in coherent areas of imprints recapitulated their disposition in sections of respective lymphoid tissues. Preservation of microanatomical relationships and ultrastructure of lymphoid cells in imprints allowed evaluation of associations and fine structural detail of lymphoid cells. Morphologic configurations of B cells in imprints, confined to round aggregates, were similar to fine structural morphology of B cells in mantle zones (MZ) and germinal centers (GC). Processes of DRCs in imprints formed conformations resembling their meshwork within follicles and mantled lymphoid cells. In imprints and sections, lymphocytes of cytotoxic/suppressor phenotype had a large amount of cytoplasm with many organelles. In contrast, cells of helper/inducer phenotype displayed a high nucleocytoplasmic (N/C) ratio and small numbers of organelles. Thus, imprints represent an easy, fast, and reliable method that lends itself to immunoultrastructural studies of lymphoid tissues.     

Effects of temperature on the development,growth, and survival of larvae and pupae of a north-temperate chrysomelid beetle

Summary Developoment, growth, and survival of larvae and pupae of the red turnip beetle, Entomoscelis americana Brown, were studied in 10 constant and four alternating temperature regimes (10 to 32.5° C), in field-cages, and in natural populations in Manitoba. This beetle has a northtemperate distribution in North America. Larval and pupal development occurs in spring and normally is completed before the end of June. Growth and development occurred at all constant temperatures tested, but survival was low at the extreme temperatures. Therefore, the threshold and upper limit were near 10 and 32.5° C. The developmental times of the sexes did not differ and decreased with temperature, except possibly at 32.5° C. The average weight of adult females increased with temperature up to 32.5° C and those of males up to 25° C. Considering developmental rate, survival, adult weight, and incidence of malformed adults, the optimum temperature was estimated to be near 27.5° C.Development was accelerated significantly (6 to 9%) in alternating regimes with temperatures differing by 10° C, but not in regimes differing by 5 and 15° C. All alternating regimes increased adult weight, 5 to 17% for females and 2 to 10% for males. Field cage studies confirmed the increase in adult weight, but not the acceleration in development.A three-parameter normal function described accurately the relationship between developmental rate and constant temperature. A computer simulation model based on this equation estimated developmental times in field cages to within one to five days. For natural populations the model overestimated the developmental times by five to 16 days. The discrepancies between model estimates and observed developmental times in natural populations apparently were due to the elevation of larval and pupal body temperatures above air temperatures by behavioral thermoregulation. The elevation of body temperature was estimated to be equivalent to the addition of 5 to 6° C to the maximum daily air temperature. The adaptations and responses of this beetle to the cool spring temperatures of the north-temperate region are discussed.Contribution No. 1164, Agriculture Canada, Research Station, Winnipeg, Manitoba, Canada     

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.