Ultrastructural localization of an extracellular protease in Pseudomonas fragi by using the peroxidase-antiperoxidase reaction

An extracellular protease, which previously has been found to correlate with the appearance of bleblike evaginations on the cell wall of Pseudomonas fragi ATCC 4973, was purified 38-fold by ammonium sulfate precipitation and Sephadex chromatography to yield a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal rabbit antiserum raised against the purified enzyme had an enzyme-linked immunosorbent assay titer of 4 X 10(7). The peroxidase antiperoxidase method was used to localize the neutral protease in P. fragi at the ultrastructural level. Electron microscopy of cell sections of this organism revealed that high concentrations of positive immunoperoxidase reaction product were located near the cell wall, whereas control sections stained with preimmune or heterologous serum did not show similar deposits to be present. These results are consistent with the hypothesis that blebs appearing on P. fragi contain high concentrations of neutral protease.     

Poly(ADP-ribose) metabolism appears normal in EM9, a mutagen-sensitive mutant of CHO cells

EM9 is a mutagen-sensitive CHO cell whose phenotype resembles that of normal CHO cells exposed to 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis. This phenotype suggested that EM9 might be defective in poly(ADP-ribose) metabolism, but we now cannot find any abnormality in the synthesis or in the degradation of poly(ADP-ribose) in permeabilized EM9 cells. Thus the effects of 3-aminobenzamide on wild-type cells may be due to the inhibition of processes other than poly(ADP-ribose) synthesis. 3-Aminobenzamide enhances the cytotoxicity of EMS toward EM9 and control cells to the same degree.     

Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.     

Energy status in the fat body of Trichoplusia ni parasitized by the insect parasite Hyposoter exiguae

The levels of adenylate nucleotides were examined in 4th-instar Trichoplusia ni larvae 3 days after parasitization by the insect parasite Hyposoter exiguae. In general, parasitization caused a decrease in the level of ATP and increased ADP and AMP levels. These changes resulted in alteration of the adenylate kinase mass-action ratio. The overall energy status of parasitized larvae, however, as indicated by energy ratios, including the “energy charge,” was affected only slightly. The result demonstrates that the host maintained an active and viable metabolic state despite extensive alterations in physiology which occur at this stage of the parasite-host association.     

Rapid recovery from K current inactivation on membrane hyperpolarization in molluscan neurons

Recovery from K current inactivation was studied in molluscan neurons using two-microelectrode and internal perfusion voltage clamps. Experiments were designed to study the voltage-dependent delayed outward current (IK) without contamination from other K currents. The amount of recovery from inactivation and the rate of recovery increase dramatically when the membrane potential is made more negative. The time course of recovery at the resting potential, -40 mV, is well fit by a single exponential with a time constant of 24.5 s (n = 7). At more negative voltages, the time course is best fit by the sum of two exponentials with time constants at -90 mV of 1.7 and 9.8 s (n = 7). In unclamped cells, a short hyperpolarization can cause rapid recovery from inactivation that results in a shortening of the action potential duration. We conclude that there are two inactivated states of the channel and that the time constants for recovery from both states are voltage dependent. The results are discussed in terms of the multistate model for K channel gating that was developed by R. N. Aldrich (1981, Biophys. J., 36:519-532).     

Interleukin 2 administered in vivo induces the growth of cultured T cells in vivo

The capacity of exogenous IL 2 to induce the growth of antigen-activated T lymphocytes in vivo was evaluated. The in vivo growth of adoptively transferred T lymphocytes that had been previously cultured long-term with IL 2 was initially examined, because in vitro such T cells are exquisitely dependent upon exogenous IL 2 for proliferation and survival. Daily administration of IL 2 in vivo, beginning on the day of cell transfer, induced these IL 2-dependent long-term cultured T lymphocytes to proliferate in vivo, and the magnitude of in vivo growth was proportional to the dose of IL 2 administered. The capacity of IL 2 to induce the in vivo growth of antigen-activated T cells not previously exposed in vitro to exogenous IL 2 was similarly studied. T lymphocytes from the spleens of immune mice, activated by 5-day culture with tumor antigen before transfer, survived poorly in vivo when injected with antigen alone, but demonstrated marked proliferation in vivo in response to antigen and exogenous IL 2. By contrast, immune spleen cells transferred with antigen, but without prior culture, proliferated without supplementary exogenous IL 2. Moreover, the growth of noncultured donor T cells was not augmented by the administration of exogenous IL 2, implying that noncultured spleen cells immune to tumor antigens can produce sufficient amounts of endogenous IL 2 in vivo to sustain maximal T cell growth over the time period examined. Importantly, the ability of exogenous IL 2 to induce donor T cell growth in vivo correlated with its ability to function in vivo to augment the anti-tumor efficacy of specifically immune donor T cells in models for the adoptive therapy of disseminated antigenic murine leukemia. Thus, the current studies highlight the potential of exogenous IL 2 to induce T cell growth in vivo and suggest that the administration of IL 2 in vivo may be useful for augmenting T cell responses that are relatively deficient in the production of endogenous IL 2.     

Recognition and response to alloantigens in vivo. I. Negative and positive selection of MLR reactivity in murine peripheral blood lymphocytes to major histocompatibility complex and Mls antigens

Specific mixed lymphocyte reaction (MLR) responsiveness to allogeneic major histocompatibility complex (MHC), or minor lymphocyte-stimulating (Mls) determinants, was depleted in the peripheral blood lymphocytes (PBL) obtained from mice 24 to 48 hr after i.v. injection of 5 to 7.5 X 10(7) MHC or Mlsa-incompatible spleen cells, respectively. Results of cell mixture experiments suggest that the generation of suppressor cells was not the explanation for this specific reduction in MLR proliferation occurring with these PBL responder cells. To gain additional insight into parameters involved in the recognition of allodeterminants in vivo, experimental manipulations of the host environment and donor cell inoculum utilized in the negative selection procedure were employed. For example, removal of the spleen in the recipient animal, an anatomic site in which injected allogeneic cells and corresponding host antigen-reactive cells (ARC) are trapped, still permitted the specific depletion in murine PBL of host ARC for donor foreign MHC antigens. This finding may implicate other sites such as the liver where unprimed host alloreactive clones are trapped. In addition, irradiation of allogeneic donor cells significantly reduced their capacity to trap alloreactive T cell clones in vivo, whereas heat treatment of the donor cells completely eliminated this ability, even though the Ia determinants were still expressed, measured by flow cytometry. After the negative selection period, kinetic analysis of proliferation showed that 3, 4, or 5 days after injection of MHC-incompatible allogeneic spleen cells, the PBL of the recipient showed specific hyperresponsiveness to the MHC-haplotype of the donor cells. Interestingly, these primed PBL responder cells had the volume distribution of small resting cells; thoracic duct lymphocytes (TDL), positively selected by adoptive transfer of T cells to irradiated semiallogeneic recipients, are reported to be mainly blast cells. In contrast to the MLR hyperresponsiveness that results from priming with MHC-incompatible splenocytes, PBL, obtained at these later time points from mice primed with Mlsa-incompatible, H-2-compatible splenocytes, showed complete unresponsiveness in MLR to these Mlsa-bearing stimulator cells, as well as some nonspecific reduction in proliferation to MHC-incompatible stimulator cells regardless of their Mls genotype.(ABSTRACT TRUNCATED AT 400 WORDS)     

An assessment of reproductive wastage in sheep

Reproductive wastage was evaluated by relating ovulation rate to lambs born or raised in range finewool ewes over a ten-year period. The results indicate that reproductive losses are of large magnitude and that these losses are concentrated in the period of ovulation to implantation and in death losses of lambs born. In this study, 33.5% of the potential lamb crop was lost in the early period. Multiple ovulating ewes showed the greatest embryonic loss. The data do not deviate from the binomial distribution, thus suggesting that these losses are largely due to chance. Further work is indicated in the first seventeen days after ovulation to examine losses due to fertilization failure, chromosomal abnormalities, and implantation failure. Abortion or absorption of the fetus was indicated in only 4% of the ewes sampled. An average of 16% of the potential lamb crop was lost from birth to weaning, with 73% of these losses occurring in the first few weeks. Management practices need to be developed to reduce these losses, and these would be expected to vary from one producer to the next depending on conditions. Increasing ovulation rate is one possible method of improving reproductive efficiency, as results of this study indicate that for each unit increase in ovulation rate, there is an increase of 0.52, 0.46, 0.33, and 0.23 in the average number of embryos surviving, lambs born, lambs marked and lambs weaned, respectively.     

Heterogeneity in the map distance between X-linked agammaglobulinemia and a map of nine RFLP loci

Summary In nine family pedigrees in which X-linked agammaglobulinemia (XLA) is segregating, a multi-point linkage analysis has been carried out. In each family, the map distance, d, between XLA and a fixed point in a known map of nine RFLP loci on the X chromosome was estimated by calculating the log likelihoods, L(d). Using a new method, the 10-point likelihood was approximated by appropriately combining three 4-point likelihoods. Homogeneity tests (admixture tests) were performed showing clear evidence for heterogeneity of XLA.