Biochemical and molecular characterization of wild-type and fused protoplasts of Beauveria bassiana and Metarhizium anisopliae.

Protoplasts were isolated from two isolates each of Beauveria bassiana and Metarhizium anisopliae using lysing enzymes. Intra- and intergeneric protoplast fusion has been carried out using 40% polyethylene glycol. The fused protoplasts of B. bassiana and M. anisopliae have been regenerated on Czapek-Dox agar media, and a total of four fusants were selected for further studies. An increase in proteinase and chitinase enzyme activity was recorded with all fusants as compared to the wild-type isolates. To understand the nature of recombination process, random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) were carried out on genomic DNA of fused and wild-type isolates. The present study demonstrates the scope and significance of the protoplast fusion technique as a rapid consistent method for identification of B. bassiana and M. anisopliae fused and wild-type isolates based on the banding pattern of RAPD and RFLP that can be reliably used ahead for further applications on these species.

Effects of ozone on airway epithelial permeability and ion transport.

Ozone is a highly reactive form of oxygen produced in the atmosphere by photochemical reactions involving substrates emitted from automobile engines. Outdoor air concentrations as high as 0.4 parts per million (ppm) occur. The respiratory tract extracts about 90% of inhaled ozone. From the chemical reactivity of ozone, it is expected to attack organic molecules located on or near the respiratory surfaces. The airways are covered with a cohesive layer of epithelial cells that forms the boundary between the external environment and the respiratory tissues. One important role of this epithelial layer is its barrier function. Airborne particles that deposit (and dissolve) in the airway surface liquid are not readily absorbed, and soluble tissue components are excluded from the surface liquid. The epithelium also controls the volume and composition of the surface liquid. One important process in this regard is the absorption and secretion of ions and water. We have studied the effects of inhalation of ozone on the barrier function (permeability to dissolved molecules) and the ion transport activity of epithelium using both in vivo and in vitro techniques. All our experiments were performed with male Hartley strain guinea pigs. Conscious, unrestrained animals were exposed to a concentration of ozone of 1 ppm for three hours in controlled environmental chambers in the Health Effects Research Laboratory, U.S. Environmental Protection Agency (EPA), Research Triangle Park, NC. Such exposures caused a marked increase in the rate of appearance in blood of various water-soluble compounds instilled onto the surface of the trachea, indicating increased permeability of the airway epithelium. This interpretation was supported by electron microscopy, which showed that the tracer molecule horseradish peroxidase was present in the intercellular spaces of tracheal epithelium from ozone-exposed, but not air-exposed (control), animals. However, when the tracheas were excised after ozone exposure and mounted in a tissue bath before measurement of permeability, no increase was found. We suggest that the effect of ozone inhalation on airway permeability requires the action of mediators that are washed out in the in vitro situation. When we exposed animals to 1 ppm for 3 hours daily, we found that the increased permeability in vivo was no longer demonstrable after the fourth exposure. The mechanisms for this type of "adaptation" are not known. The phenomenon recalls the response of human subjects to repeated daily ozone exposures. Ozone exposure caused a sharp increase in active ion transport by tracheal epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural crosslinking of lung connective tissue collagen in the blotchy mouse.

Male mice with the sex-linked mutation Blotchy (Blo) have a defect in copper metabolism which results in deficient activity of a number of copper-containing enzymes. Inbred Blo/y mice spontaneously develop lung abnormalities which resemble emphysema and often die of ruptured aortic aneurysm. Lung, tail tendon, and tibial bone collagens from inbred Blo/y and their normal (+/y) litter mates were reduced with standardized [3H]NaBH4, acid and alkaline hydrolyzed, and chromatographed in order to quantify the aldehydic crosslink precursors, and the labile reducible and nonreducible stable mature covalent intermolecular crosslinks. Reducible lung collagen crosslinks were markedly (60%) decreased in the Blo/y mice and few, if any, mature nonreducible crosslinks were present. Total aldehydes were also decreased (65%) when Blo/y was compared to +/y. In tail tendon and bone, collagen crosslinks were decreased by only 28% and 15%, respectively. Selectively severe lack of activity of the copper-dependent enzyme level oxidase in lung with only partial lack in tendon and bone could account for the results obtained. Alternatively, insufficient reducible crosslinks, coupled with increased collagen turnover in the lung could prevent formation of the more mature stable crosslinks required to provide a proper connective tissue framework for the Blo/y lung.

A quantitative study of the development of interalveolar pores in the postnatal mouse.

Quantitative analysis of regional interalveolar pore development in the lungs of C3Hf/He mice was performed using scanning electron microscopy. Lungs of male mice ages 7, 10, 14, 21, 28, and 56 days were studied following intratracheal fixation. Interalveolar pores were counted in subpleural, midzonal, and peribronchiolar regions. Interalveolar pores were rarely observed in mice ages 7, 10, and 14 days but appeared abruptly at age 21 days throughout the lung. The number of interalveolar pores increased 2-3 fold from ages 21 to 56 days. By age 28 days and thereafter interalveolar pores were more numerous in subpleural alveoli than in midzonal or peribronchiolar alveoli. We conclude that interalveolar pores appear diffusely in the lungs of mice during the third postnatal week and that regional differences in the number of interalveolar pores are established by age 28 days.

Bioelectric properties and ion flow across excised human bronchi.

Bioelectric properties and ion transport of excised human segmental/subsegmental bronchi were measured in specimens from 40 patients. Transepithelial electric potential difference (PD), short-circuit current (Isc), and conductance (G), averaged 5.8 mV (lumen negative), 51 microA X cm-2, and 9 mS X cm-2, respectively. Na+ was absorbed from lumen to interstitium under open- and short-circuit conditions. Cl- flows were symmetrical under short-circuit conditions. Isc was abolished by 10(-4) M ouabain. Amiloride inhibited Isc (the concentration necessary to achieve 50% of the maximal effect = 7 X 10(-7) M) and abolished net Na+ transport. PD and Isc were not reduced to zero by amiloride because a net Cl- secretion was induced that reflected a reduction in Cl- flow in the absorptive direction (Jm----sCl-). Acetylcholine (10(-4) M) induced an electrically silent, matched flow of Na+ (1.7 mueq X cm-1 X h-1) and Cl- (1.9 mueq X cm-12 X h-1) toward the lumen. This response was blocked by atropine. Phenylephrine (10(-5) M) did not affect bioelectric properties or unidirectional ion flows, whereas isoproterenol (10(-5) M) induced a small increase in Isc (10%) without changing net ion flows significantly. We conclude that 1) Na+ absorption is the major active ion transport across excised human bronchi, 2) Na+ absorption is both amiloride and ouabain sensitive, 3) Cl- secretion can be induced by inhibition of the entry of luminal Na+ into the epithelia, and 4) cholinergic more than adrenergic agents modulate basal ion flow, probably by affecting gland output.

Bilateral trunkal vagotomy (BTV) regarded as a pathogenic process. An experimental study.

Experimentally, bilateral trunkal vagotomy (BTV) generated some early acute modifications represented during the first 14 days by blood stasis, partial thrombosis, inflammatory infiltrates and necrobiosis of the gastric mucosa. The vascular lesions are the first to occur, the tissular ones being secondary. Later on, two and three months after surgery, chronic inflammation and metaplasia of the intestinal type have been detected as well.

Effect of allergic bronchoconstriction on airways epithelial permeability to large polar solutes in the guinea pig.

The effect of allergic bronchoconstriction on the permeability of the airway mucosa to large hydrophilic polar solutes was investigated in the guinea pig. After specific antigen (ovalbumin) challenge, there was a significant increase in the plasma levels of horseradish peroxidase (HRP) (molecular weight, approximately 40,000 daltons), 3H-dextran (approximately 10,000 daltons), and 14C-mannitol (approximately 182 daltons) compared with that in control animals aerosol-challenged with a nonspecific protein, lactoglobulin. The morphologic correlates of this enhanced transepithelial permeability after ovalbumin challenge appeared to be (1) increased HRP penetration of the epithelial tight junctions (p less than 0.001), and (2) increased mucus discharge from surface lining goblet cells. We conclude that antigen-induced bronchoconstriction leads to an increase in tracheobronchial permeability to macromolecules, and this effect is likely to be mediated by an increased paracellular as well as transcellular vesicular movement of large polar solutes across the airway epithelial barrier.

Morphologic and phenotypic analysis of an outcross line of blotchy mouse.

Blotchy is an X-linked recessive mutation at the "Mottled" locus in the mouse. The affected blotchy male (Blo/Y) mouse from an inbred genetic background demonstrates morphologic and physiologic abnormalities consistent with emphysema in adult life. Breeding of Blo/Y mice has been difficult because the inbred Blo/Y males are sterile. We report the successful development of a line of outbred Blo/Y male and Blo/Blo female nice by the controlled outcross mating of the inbred heterozygous Blo/+ female with the Argonne hybrid B6CF1 male mouse. The subsequent outcross Blo/Y progeny breed vigorously with the outcrossed Blo/+ female. The lungs of the outbred Blo/Blo female and inbred Blo/Y male mice demonstrate mild to moderate panacinar emphysema with a significant decrease in internal surface area (p less than 0.005) and an increase in mean linear intercept (p less than 0.005). In contrast, the lungs of the outbred Blo/Y is structurally normal. Despite the absence of emphysema-like changes in the outbred Blo/Y males, there were phenotypic features that suggest inherited abnormalities in connective tissue proteins including 1) high incidence of aortitis leading to premature death from aneurysmal rupture, and 2) significant decrease in the morphometrically determined parenchymal elastic fiber length in the lung (p less than 0.01). The outbred blotchy strain may be a useful experimental animal model in determining the pathogenesis of emphysema.

[Microvascularization of the liver in vagotomy]. / La microvascularisation du foie dans la vagotomie.

Sub-diaphragmatic truncal vagotomy produces early and late changes in the microvascular network of the liver. Bilateral sub-diaphragmatic truncal vagotomy was performed experimentally in the dog. After 7 days, 60 days and 2 years, the animals were subjected to laparotomy under general anaesthetic and China ink was injected into the aorta above the coeliac trunk. Samples were then taken from the hepatic parenchyma to be examined under the photomicroscope. The following early modifications were observed: absence of the injection in the central and intermediary zones of the lobe, stasis in the centro-lobular vein. These modifications are also present at 60 days. After 2 years, there is an improvement in the hepatic microcirculation, but modifications of micro-irrigation are still present.

Morphological aspects of the rat kidney preserved by cold storage. I. Glomerular morphometric changes.

The absolute density of glomeruli in the microscopic field was determined in the rat kidney preserved by cold storage for 24, 48, 72 and 96 hrs in two different media: Sacks (hyperosmolar electrolytic solution of intracellular type) and Plasmagel (gelatin solution 4%). Progressive, statistically significant (p less than 0.01) decrease of glomerular density at 24 and 48 hrs was followed by return to initial values at 96 hrs. Decrease of the glomerular density was greater with Plasmagel.

Experimental study on the effects of +Gz. Acceleration under gestational conditions. I. Ultrastructural myocardial lesions.

In the course of a complex experimental study concerning the effects of highly positive acceleration on gestation and development of the neonate, investigations were carried out of ultrastuctural myocardial lesions in the pregnant rat exposed to prolonged gravitational stress (+ 2G). The experimental animals presented various disseminated and partly irreversible myocardial lesions whose pathogeny is discussed.

The effect of pilocarpine on vesicular uptake and transport of horseradish peroxidase by the guinea pig tracheal epithelium.

The quantitative relationship between goblet cell number and airway transepithelial permeability to horseradish peroxidase (HRP) (molecular weight, approximately 40,000 daltons) was examined in the guinea pig. In animals administered 12 daily successive doses of 2.5 mg pilocarpine intraperitoneally, an increase in goblet cell population in the trachea correlated with higher plasma HRP concentrations of all 4 sampling times after intratracheal instillation of 1 mg HRP. Guinea pigs administered a single dose of pilocarpine and studied 1 h later showed a significantly higher plasma HRP concentration at 10 and at 15 min (p less than 0.025) as compared with saline control animals. The morphologic correlates of this enhanced early permeability to HRP after a single dose of pilocarpine appeared to be increased vesicular uptake and accelerated transit across the goblet cells. Our data suggest that pinocytotic vesicular uptake and transport of exogenous proteins in the secretory epithelial cells of the airways may be an important pathway for movement of large polar solutes across the epithelial barrier.

Elements of structure and ultrastructure of the blood-thymus barrier in ACTH involuted thymus.

The ACTH influence upon the thymus may be a reliable model for stress involution. In this case, the cortical lymphocytic depletion is accompanied by mast cell accumulation and increased caliber of the blood vessels. The blood-thymus barrier which has an active role in involution shows an enriched transport activity of the endothelial cells, great enlargement of the basement membrane, increased macrophage activity within the perivascular space with elevated values of acid phosphatase activity, and thickening of the fibrillar network. The epithelioreticular cells show plenty of vacuoles in their cytoplasm, the mitochondria undergo swelling processes, and their cristae are diminished. The ultrastructural data show that lymphocyte depletion is carried out by macrophage-mediated lymphocytolysis. But by counting the peripheral blood cells an earlier mechanism is revealed; i.e. migration through the enlarged but more permeable blood-thymus barrier. The epithelioreticular cells do not seem to have an active, direct implication in any of the phenomena.

The effects of small doses of oligopeptide elastase inhibitors on elastase-induced emphysema in hamsters: a dose-response study.

Our previous studies have indicated that the synthetic elastase inhibitor N-acetyl-L-alanyl-L-alanyl-L-prolyl-L-alanyl chloromethylketone (AAPACK) administered intraperitoneally in divided doses totaling 19 mg, prior to and after a single intratracheal injection of elastase, substantially inhibited the development of experimental emphysema. The present studies evaluated the effects of 1.1, 4.1 and 8.0 mg AAPACK administered in divided doses 10 min prior to and 10, 30, and 50 min after a single intratracheal dose of elastase. The development of emphysema was essentially eliminated by 4.1 and 8.0 mg of AAPACK and markedly diminished with 1.1 mg AAPACK. The AAPACK was excreted rapidly in the urine at 30 to 60 min after its administration. Elastase inhibitory capacity (EIC) in urine was elevated to 14 times that in serum at the corresponding collection times. The plasma elastase inhibitory capacity was only slightly increased after administration of AAPACK and was not a sensitive indicator of elastase inhibition in the plasma.

Effects of oligopeptide chloromethylketone administered after elastase: renal toxicity and lack of prevention of experimental emphysema.

The effectiveness of N-acetyl-L-alanyl-L-alanyl-L-prolyl-L-alanine chloromethylketone (AAPACK) in preventing the development of experimental emphysema in hamsters, when administered 60 min after exposure to elastase, was studied. When 19 mg of AAPACK was injected intraperitoneally in divided doses commencing 60 min after the intratracheal instillation of pancreatic elastase, the development of emphysema was not prevented using morphologic, morphometric, and physiologic means of evaluation. Thirty-eight per cent of hamsters given AAPACK became ill and lost weight. At autopsy, these hamsters had a renal tubular nephropathy and focal interstitial disease. The glomeruli were spared. Five of these hamsters with renal tubular lesions had azotemia. Focal necrosis was observed in the heart of 3 and in the liver of 5 animals with renal lesions. These studies indicated that AAPACK, in the protocol followed where elastase precedes administration of the inhibitor, (1) does not prevent the development of elastase-induced emphysema, and (2) does produce a unique renal tubular nephropathy.

A quantitative study of ciliary injury in the small airways of mice: the effects of nitrogen dioxide.

The frequency and types of abnormal cilia prior to and following nitrogen dioxide (NO2) exposure in the small airways of an inbred strain of mice was quantitated by transmission electron microscopy. Age-matched male and female mice were exposed to 20 ppm NO2 for 28 days. In both sexes, exposures to NO2 resulted in a significant increase (P less than 0.01) in abnormal cilia characterized by (1) decreased number of ciliary structures on cell surface, (2) focal swelling of the cytoplasmic matrix of the ciliary shaft, and (3) occasional compound cilium. The basal bodies of the ciliated cells from which atypical cilia arose appeared normal. In mice killed 30 days after termination of NO2 exposure, the ciliary morphology returned to normal and the incidence of abnormal cilia decreased to levels observed in controls. These observations indicate that subacute exposure to NO2 causes injury to the ciliary shaft; these changes are reversible within 30 days following removal from NO2.

Lung injury and repair in the blotchy mouse. Effects of nitrogen dioxide inhalation.

We studied the reparative process after inhalation exposure to 20 ppm of nitrogen dioxide (NO2) in the lungs of hemizygous blotchy male (Blo/g) and heterozygous blotchy female (Bio/+) mice. Age-matched siblings (C3Hf) without the blotchy gene at X-chromosome locus (+/y) and +/+) served as control animals. After exposure to NO2 for 28 days, there was a marked progression in the extent of emphysema in Blo/y mice associated with a significant decrease of internal surface area (p < 0.05) and an increase in the mean linear intercept (p < 0.005). In contrast, +/y, Blo/+, and +/+ mice showed mild airspace enlargement without decrease in internal surface area after similar exposures. Blo/y mice killed 1 month after cessation of NO2 exposure showed a persistent, mild chronic bronchiolitis that was more frequent and of greater severity than that present in control +/y mice. Alveolar macrophages in the Blo/y mice were larger than those in +/y, +/+, and Blo/+ mice both before and after exposure to NO2. Crystalloid inclusions were observed in the enlarged alveolar macrophages of the Blo/g mice only after exposure to NO2, but were not seen in control animals. These observations indicate that the pattern of lung injury and repair after subacute exposure to 20 ppm of NO2 in the Blo/y mouse differs from that present in age-matched siblings in that inherited abnormalities in alveolar macrophage function may exist in addition to the previously described alterations in connective tissue proteins. Both of these alterations may influence the development of emphysema in the blotchy male mouse.