Activation of mouse peritoneal macrophages in vitro and in vivo by interferon-gamma

To determine the role of IFN-gamma in the activation of resident mouse peritoneal macrophages, crude macrophage-activating lymphokines were incubated with a monoclonal anti-murine IFN-gamma antibody. This treatment abolished the capacity of mitogen-induced lymphokines to enhance either H2O2 release or activity against the intracellular protozoa Toxoplasma gondii and Leishmania donovani. All macrophage-activating factor detected by these assays was also removed by passing the lymphokines over a Sepharose column to which the monoclonal anti-IFN-gamma antibody had been coupled. Therefore, pure murine rIFN-gamma was tested both in vitro and in vivo as a single activating agent. After 48 hr of pretreatment in vitro with 0.01 to 1 antiviral U/ml, macrophage H2O2-releasing capacity was enhanced an average of 6.4-fold; half-maximal stimulation was induced by 0.03 U/ml. Resident macrophages infected with T. gondii half-maximally inhibited parasite replication after 24 hr of preincubation with 0.14 U/ml of rIFN-gamma, and near complete inhibition was achieved by pretreatment with 100 U/ml. Half-maximal leishmanicidal activity was induced by 0.08 U/ml of rIFN-gamma, and 67 to 75% of intracellular L. donovani amastigotes were killed after macrophages were preincubated with 10 to 100 U/ml. Eighteen hours after parenteral injection of rIFN-gamma, peritoneal macrophages displayed a dose-dependent enhancement of H2O2-releasing capacity and antiprotozoal activity. Half-maximal enhancement required 85 to 250 U or rIFN-gamma given i.p. Peritoneal macrophages were also activated by rIFN-gamma injected i.v. and intramuscularly. These results suggest that, in the mouse model, IFN-gamma is likely to be a primary factor within mitogen-induced lymphokines responsible for activating macrophage oxidative metabolism and antiprotozoal activity, and indicate that rIFN-gamma is a potent activator of these effector functions both in vitro and in vivo. These findings provide a rationale for evaluating rIFN-gamma in the treatment of systemic intracellular infections, and indicate that murine models are appropriate for such studies.     

Organic free radical levels in seeds and pollen: The effects of hydration and aging

In view of their possible role in oxidative deterioration of seeds and pollen, organic free radicals were measured by electron spin resonance in embryonic axes and cotyledons of soybean [ Glycine max (L.) Merr], embryo and endosperm fractions of corn [ Zea mays L.] and pollen of cattail [ Typha latifolia L.]. A pronounced decline in the radical signal ensued when hydration increased above about 7% (wet weight basis) in both the seed materials and in pollen. Moderate hydration of the soybean axis followed by drying led to a small decrease in organic free radicals compared to untreated material, especially if the desiccation step was performed under nitrogen. In a comparison of soybeans of various ages under normal storage, organic free radical levels in the axis showed little or no increase with age. In marked contrast, over 5 days of accelerated aging at 40°C and near-saturating humidity, organic radical levels approximately doubled in the axis. This pronounced increase in free radical content was not associated with a decrease in the proportion of polyunsaturated fatty acids. The data suggest that hydration of seed and pollen causes a release of free radicals from the trapped state.     

Parasite specific antibodies in three strains of mice after infection with Trypanosoma cruzi

Three inbred strains of mice (BALB/cJ, C3H/HeJ and NZB/BInJ) were infected with trypomastigotes of Trypanosoma cruzi. Sera were taken at different times after infection and radioimmunoprecipitation assays were used to detect antibodies against individual T. cruzi epimastigote and trypomastigote antigens. The mouse strains differed in regard to the spectrum of antibodies and the time after infection when the various epimastigote specific antibody species appeared. NZB mice had antibodies against at least 25 polypeptides ranging in molecular weight from 20,000 to 90,000 D at 3 wk after infection, and these persisted until at least 10 wk post-infection. C3H and BALB/c had antibodies against fewer than 5 antigens at 3 wk after infection; whereas by week 10, antibodies against at least 25 polypeptides were detected. C3H mice that were most susceptible to infection (but not NZB or BALB/c mice) had antibodies against a 25,000 D molecular weight epimastigote antigen. The antibody response against trypomastigote polypeptides was more uniform. Sera from all mouse strains at 3 wk after infection precipitated the same polypeptides and the radioimmunoprecipitation patterns did not change as a function of time after infection.     

Mechanisms of dimethylbenzanthracene-induced immunotoxicity

Traditional methods for toxicological assessment have implicated the immune system as a frequent target organ of toxic insult following chronic exposure to certain environmental chemicals, radiation or therapeutic drugs (xenobiotics). Immunotoxicity is expressed as autoimmunity, chemical hypersensitivity or immunosuppression. A tiered approach for characterizing chemical and drug-induced immunomodulation has been developed and validated in laboratory animals. Polycyclic aromatic hydrocarbons (PAH) have been studied because of their ubiquitous presence in the environment and carcinogenic potential. Since immunosuppression induced by PAH carcinogens has been implicated as an epigenetic mechanism in the outgrowth of initiated cells, this tiered approach was used to characterize the mechanism of PAH immunosuppressive capacity. Previously, studies in this laboratory have demonstrated that subchronic exposure of B6C3F1 mice to PAH carcinogens suppresses both humoral immunity (HI) and cell-mediated immunity (CMI), concurrently with decreased resistance to tumor challenge. The potent carcinogenic PAH, 7,12-dimethylbenz[a]anthracene (DMBA) was subchronically administered subcutaneously at 5, 50, or 100 micrograms/g of body weight. Natural killer (NK) cell tumor cytolysis, generation of cytotoxic T-cells (CTL), and lymphoproliferation to mitogens and allogeneic splenocytes in mixed leukocyte cultures (MLC) were quantitated 3-5 days after exposure to assess CMI. Mitogen and alloantigen-induced proliferation (MLC) of splenocytes was suppressed up to 90%. CTL and NK tumor cytolysis of radiolabelled target cells were similarly depressed up to 88 and 82%, respectively. Impairment of MLC or CTL responses correlated with increased susceptibility to challenge with PYB6 sarcoma cells. HI was measured by quantitating the number of antibody (IgM) plaque-forming cells (PFC) produced in response to T-cell dependent antigen challenge (sheep erythrocytes) and was similarly suppressed up to 95%. To understand the mechanism of PAH-induced immunotoxicity, splenocytes from DMBA-exposed mice were sensitized to alloantigens in the presence of interleukin-2 (IL-2) because there were indications that T-helper cell function was suppressed. In these preliminary studies, CTL suppression could be completely restored by the addition of the T-cell growth supporting lymphokine (IL-2) during the inductive phase of CTL generation, suggesting that DMBA exposure directly or indirectly induced deficits in T-helper cell function.     

The B chain of PDGF alone is sufficient for mitogenesis.

The platelet-derived growth factor (PDGF) is a mitogen derived from human platelets consisting of two related polypeptides termed A and B chains. The entire B chain of PDGF is highly (96%) homologous to a portion of p28sis, the transforming protein of simian sarcoma virus. It has been suggested that p28sis exerts its transforming potential by mimicking the growth promoting activity of PDGF and stimulating the cell in an autocrine manner. We have directly examined the mitogenic potential of p28sis and the B chain homologous region by expressing these heterologous sequences in the yeast Saccharomyces cerevisiae. In our constructions, these proteins are encoded by portions of the v-sis gene. Expression and secretion from the yeast cell is achieved by using a yeast promoter and the alpha-factor pheromone secretory leader. The sis proteins thus expressed and secreted are immunoreactive with anti-PDGF antisera and are mitogenic for cultured fibroblasts. Furthermore, they mediate this mitogenic activity by specific binding to the PDGF cell surface receptor. Gel electrophoresis and cell binding analysis indicates that the mitogenic species is primarily a disulphide-bonded dimer. We are able to conclude that p28sis is a mitogen and that a polypeptide corresponding to the B chain alone is sufficient to account for the mitogenic activity attributed to PDGF.     

Enhanced enzymatic hydrolysis of lignocellulosic biomass pretreated by low-pressure steam autohydrolysis

Summary The use of low-pressure steam autohydrolysis in the pretreatment of corn stover and hybrid poplar has been assessed. In terms of yield of prehydrolyzed solids, minimal by-product formation and extent of subsequent enzymatic saccharification, the results of low-pressure steam pretreatment were found to be as good as or better than those reported for more severe pretreatment processes. Almost complete saccharification of the cellulose in the prehydrolyzed biomass solids was obtained within 24h with a commercial cellulase preparation — Celluclast. The presence of grinding elements (glass beads) during the enzymatic hydrolysis was found to increase the extent of saccharification by 40% to 50% over controls without any grinding elements.     

Cloning, sequencing, and functional analysis of oriL, a herpes simplex virus type 1 origin of DNA synthesis.

The herpes simplex virus type 1 genome (160 kilobases) contains three origins of DNA synthesis: two copies of oriS located within the repeated sequences flanking the short unique arm (US), and one copy of oriL located within the long unique arm (UL). Precise localization and characterization of oriL have been severely hampered by the inability to clone sequences which contain it (coordinates 0.398 to 0.413) in an undeleted form in bacteria. We report herein the successful cloning of sequences between 0.398 to 0.413 in an undeleted form, using a yeast cloning vector. Sequence analysis of a 425-base pair fragment spanning the deletion-prone region has revealed a perfect 144-base pair palindrome with striking homology to oriS. In a functional assay, the undeleted clone was amplified when functions from herpes simplex virus type 1 were supplied in trans, whereas clones with deletions of 55 base pairs or more were not amplified.     

Factors affecting tissue volume measurements in normal and edematous dog lungs

To characterize further some of the factors affecting lung tissue soluble-gas rebreathing volume (Vlt), we determined the solubility of acetylene in blood and lung tissue, the influence of the presence of pulmonary edema on tissue solubility, the effects of varying tidal volume (VT), and the tissue volume actually measured in two groups of six anesthetized paralyzed dogs: controls (C) and oleic acid-induced pulmonary edema (OA). Each animal's solubility was used to compute Vlt for comparison with gravimetric lung weight (Ql) and extravascular lung water content (Qwl). Solubility at 37.5 degrees C in blood (0.125 ml X 100 ml-1 X Torr-1) exceeded that in lung tissue (P less than 0.005): C = 0.118 and OA = 0.112 ml X 100 ml-1 X Torr-1 (NS). Vlt, expressed as %Ql, increased with increasing VT (20, 35, and 50 ml/kg) in OA (62.2, 78.9, and 94.7%, respectively, P less than 0.0001) but not in C (92.4, 94.4, and 99.3%, respectively). We conclude that solubility differs in blood and lung tissue but not in normal and edematous lungs, Vlt is not affected by VT in normal dogs but is in those with pulmonary edema, and Vlt measures Ql rather than Qwl.     

Treadmill vs. floor walking: kinematics, electromyogram, and heart rate

To identify the degree of difference between treadmill and floor walking, kinematic, electromyographic (EMG), and heart rate measurements were recorded in seven normal female subjects during walking at three speeds on the treadmill and on the floor. During treadmill walking, subjects tended to use a faster cadence and shorter stride length than during floor walking. In addition the displacements of the head, hip, and ankle in the sagittal plane showed statistically significant differences between floor and treadmill walking. Average EMG activity was usually greater on the treadmill than on the floor; however, this difference was only significant for the quadriceps. Heart rate was significantly higher during fast treadmill walking than floor walking. In general, treadmill walking was not found to differ markedly from floor walking in kinematic measurements or EMG patterns.     

Three-dimensional structure of a membrane-microtubule complex

The unicellular algae Distigma proteus contain a group of aligned microtubules associated with their cell membrane. The association is maintained in isolated membrane fragments. The membrane-microtubule complex also includes a crystalline array of membrane particles. The major peptide component of this array was identified by labeling whole cells with radioiodine. The entire complex of membrane, particles, and microtubules is sufficiently well ordered to permit reconstruction from electron micrographs by Fourier techniques. A three-dimensional model of the membrane array at a nominal resolution of 2.5 nm has been calculated. Some similarities were apparent between lattice spacings in the membrane array and in microtubules. Analysis of these lattice correlations suggests a way in which the array of membrane particles may serve as scaffolding for microtubule attachment.