l-Phenylalanyl-l-Glutamate-Stimulated, Chloride-Dependent Glutamate Binding Represents Glutamate Sequestration Mediated by an Exchange System

Stimulation of glutamate binding by the dipeptide L-phenylalanyl-L-glutamate (Phe-Glu) was inhibited by the peptidase inhibitor bestatin, suggesting that the stimulation was caused by glutamate liberated from the dipeptide and not by the dipeptide itself. It further suggests that this form of glutamate binding should be reinterpreted as glutamate sequestration and that stimulation of binding both by dipeptides and after preincubation with high concentrations of glutamate is likely to be due to counterflow accumulation. Several other criteria indicate that most of glutamate binding stimulated by chloride represents glutamate sequestration: Binding is reduced when the osmolarity of the incubation medium is increased, when membranes incubated with [3H]glutamate are lysed before filtration, and when membranes are made permeable by transient exposure to saponin. Moreover, dissociation of bound glutamate after a 100-fold dilution of the incubation medium is accelerated about 50 times by the addition of glutamate to the dilution medium. This result would be anomalous if glutamate were bound to a receptor site; it suggests instead that glutamate is transported in and out of membrane vesicles by a transport system that preferentially mediates exchange between internal and external glutamate. Glutamate binding contains a component of glutamate sequestration even when measured in the absence of chloride. Sequestration is adequately abolished only after treating membranes with detergents; even extensive lysis, sonication, and freezing/thawing may be insufficient.     

Comparative study of the composition of waxes extracted from isolated leaf cuticles and from whole leaves of Citrus: Evidence for selective extraction

The effect of different extraction methods on the composition of samples of soluble cuticular lipids (SCL) of Citrus aurantium L. was investigated. The variation of extraction yields, when whole leaves were immersed in solvent, was studied as a function of solvent type and duration of immersion. Cuticular waxes were also quantitatively extracted from isolated cuticular membranes of C. aurantium and their composition was compared to that of samples obtained by the immersion method. Significant differences were observed. Higher carbon number homologues of the aliphatic constituent classes were discriminated against when whole C. aurantium leaves were extracted by immersion. The alkyl ester fraction was almost entirely lacking in extracts from whole leaves. The dependence on carbon chain length of the saturation concentrations in chloroform of major aliphatic SCL constituents was determined. The results are discussed in terms of the major physico-chemical processes involved in the extraction of SCL.     

Relationship of hyperthermia-induced hemolysis of human erythrocytes to the thermal denaturation of membrane proteins

Hemolysis of human erythrocytes as a function of time of exposure to 47.4-54.5 degrees C was measured and correlated to thermal transitions in the membranes of intact erythrocytes as determined by differential scanning calorimetry (DSC). Curves of hemoglobin leakage (a measure of hemolysis) as a function of time have a shoulder region exhibiting no leakage, indicative of the ability to accumulate sublethal damage (i.e., damage not sufficient to cause lysis), followed by a region of leakage approximating pseudo-first-order kinetics. Inverse leakage rates (Do) of 330-21 min were obtained from 47.4-54.5 degrees C, respectively. A relatively high activation energy of 304 +/- 22 kJ/mol was obtained for leakage, eliminating the involvement of metabolic processes but implicating a transition as the rate-limiting step. Membrane protein involvement was suggested by the very low rate (10(-2) of the rate from erythrocytes) and low activation energy (50 +/- 49 kJ/mol) of hemoglobin leakage from liposomes containing no membrane protein. A model was developed that predicts a transition temperature (Tm) for the critical target (rate-limiting step) of 60 degrees C when measured at a scan rate of 1 K/min. DSC scans were obtained from intact erythrocytes and a procedure developed to fit and remove the transition for hemoglobin denaturation which dominated the scan. Three transitions remained (transitions A, B, and C) with Tm values of 50.0, 56.8, and 63.8 degrees C, respectively. These correspond to, but occur at slightly different temperatures than, the A, B, and C transitions of isolated erythrocyte membranes in the same salt solution (Tm = 49.5, 53-58, and 65.5 degrees C, respectively). In addition, the relative enthalpies of the three transitions differ between isolated membranes and erythrocytes, suggestive of membrane alterations occurring during isolation. Thus, all analyses were conducted on DSC scans of intact erythrocytes. The B transition is very broad and probably consists of several transitions. An inflection, which is seen as a distinct peak (transition B3) in fourth-derivative curves, occurs at 60.8 degrees C and correlates well with the predicted Tm of the critical target. Ethanol (2.2%) lowers the Tm of B3 by 4.0-4.5 K, close to the shift of 3.3 K predicted from its effect on hemolysis. Glycerol (10%) has very little effect on both hemolysis and the Tm of B3, but it stabilizes spectrin (delta Tm = 1.5 K) against thermal denaturation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of calcium and vanadate with fluorescein isothiocyanate labeled Ca2+-ATPase from sarcoplasmic reticulum: kinetics and equilibria

The interaction of Ca2+ and vanadate with fluorescein isothiocyanate (FITC) labeled sarcoplasmic reticulum (SR) Ca2+-ATPase has been studied by following the kinetics of changes in the reporter group fluorescence and equilibrium fluorescence levels. The vanadate species bound to the enzyme is clearly monomeric orthovanadate, probably H2VO4-. Vanadate binding is noncooperative, suggesting an absence of interactions between the Ca2+-ATPase subunits. The fluorescence experiments confirm the existence of a calcium-enzyme-vanadate complex (in the presence of magnesium). On the basis of the fluorescence properties of this complex, it is similar in its conformation to the calcium-enzyme complex, i.e., "E1-like" rather than "E2-like". However, Ca2+ binds to the enzyme-vanadate complex via sites that are only accessible from the interior of the SR vesicles. The complex Ca2E*Van, which is rapidly formed, isomerizes very slowly (t1/2 approximately 1 min) to the stable ternary complex. The mutual destabilization between bound vanadate and two bound Ca2+ ions is only 1.6 kcal/mol, much smaller than that produced by the interaction of calcium and phosphate.     

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)     

Enhanced tumor susceptibility of immunocompetent mice infected with lymphocytic choriomeningitis virus

Summary Mice infected i.v. with high doses of lymphocytic choriomeningitis virus (LCMV; 10⁵–10⁶ plaqueforming units) 8–10 days prior to challenge with the methylcholanthrene-induced fibrosarcoma tumor cell line MC57G or the melanoma cell line B16 tumor cells showed an enhanced tumor susceptibility with respect to both growth kinetics of the tumor and the minimal dose necessary for tumor take. After transient initial growth, MC57G tumor cells were all rejected by uninfected C57BL/6 mice by day 14. Mice preinfected i.v. with LCMV 3 weeks before or at the time of tumor challenge, but not those infected 2 months before or 7 days after, showed increasing tumor growth, the tumor take being 100% for 10⁶, 50% for 10⁵ and 37% for 10⁴ MC57G tumor cells injected into the footpad compared with resistance to 10⁶ cells in normal mice. B16 melanoma cells also grew more rapidly in LCMV-preinfected mice and by day 40 tumors were established with about 100 times fewer cells, i.e. about 10³ compared with 3×10⁴–3×10⁵ for uninfected mice. Analysis of the growth of tumor cells in normal and in LCMV-carrier mice revealed that the latter mice were not more susceptible to LCMV-infected than to uninfected MC57G. Since LCMV-carrier mice fail to mount LCMV-specific T cell responses, these results suggest that anti-LCMV-specific T cells may be responsible for acquired immunodeficiency hampering immune surveillance against the tumors studied.Supported by grants from the Swiss National Science Foundation 3.259–0.87 and the Kanton of Zürich     

Surface areas,lengths and volumes of Picea abies (L.) Karst. needles: determination,biological variability and effect of environmental factors

Summary A method for the rapid determination of the lengths and surface areas of very large samples of needles of Picea abies (L.) Karst. using a computer-aided image analysis system was developed. Two independent methods for measuring non-destructively the volumes of individual needles and of all needles attached to a twig were devised. The surface areas and lengths of about 38000 needles sampled from the three youngest needle age-classes (1986, 1985, 1984) of 48 trees approximately 130 years old at four sites in the Fichtelgebirge mountains (N. E. Bavaria, FRG) were measured. The frequency distributions of lengths and areas for each site and age-class are given. Variability of needle size was fairly large. Even though the sites differed in climate, soil, and air pollution levels no consistent effect of these factors on needle size could be detected. Needle lengths and surface areas did not correlate with either the total chlorophyll content of the needles or the degree of crown thinning. The needle surface area (in mm²) of fully developed P. abies needles can be estimated by the empirical equation surface area = 4.440 x needle length -24.8 (r = 0.937), and the needle volume (in mm³) by needle volume = 0.208 x projected needle area ^1.353 (r = 0.969).     

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)     

Structural analysis of the mitotic cycle in pre-gastrula Xenopus embryos

The long-known phenomenon of karyomere (chromosome vesicle) formation at early telophase of the nuclear cycle during early embryogenesis of a wide range of organisms including amphibians (Rubaschkin 1905; for review, see Richards 1917) was investigated in the early cleavage cycles of Xenopus laevis embryos before the mid blastula transition. Embryos were fixed and Epon embedded at successive time intervals and consecutive thick (3 m) and ultrathin sections cut. Using conventional light microscopy at low magnification as well as phase and/or interference contrast video microscopy at high magnification, a substantial amount of information could be obtained from the analysis of optical sections in thick-sectioned material. In addition, details of the ultrastructural organization could be analysed from corresponding ultrathin sections by electron microscopy. The light microscopic analysis of serial thick sections allowed precise determination of the arrangement and sizes of telophase karyomere structures during the embryonic nuclear division cycle. It was found that small, widely spaced 1st order karyomeres fuse to larger (2nd order) karyomeres which then progressively exhibit lateral fusion of neighbouring karyomeres. The final coalescence of adjacent karyomeres marks the onset of the reorganization of the typical interphase nuclear structure. The data are discussed with regard to the occurrence of karyomeres during the embryonic nuclear cycle of arthropods, dipteran insects, and echinoderms as well as recent progress in the use of Xenopus egg extracts for in vitro assembly of nuclear structures around protein-free DNA.