Mechanical properties of the tracheal mucosal membrane in the rabbit. I. steady-state stiffness as a function of age.

Airway responsiveness is exaggerated in infancy and declines with maturation. These age-related differences (R.S. Tepper, T. Du, A. Styhler, M. Ludwig, and J.G. Martin. Am. J. Respir. Crit. Care Med. 151: 836-840, 1995; R.S. Tepper, S.J. Gunst, C.M. Doerschuk, Y. Shen, and W. Bray. J. Appl. Physiol. 78: 505-512, 1995; R.S. Tepper, J. Stevens, and H. Eigen. Am. J. Respir. Crit. Care Med. 149: 678-681, 1994) could be due to changes in the smooth muscle, the lung, and/or the airway wall. Folding of the mucosal membrane can provide an elastic load (R.K. Lambert, J. Appl. Physiol. 71: 666-673, 1991), which impedes smooth muscle shortening. We hypothesized that increased stiffness of the mucosal membrane occurs during aging, causing an increased mechanical load on airway smooth muscle and a decrease in airway responsiveness. Forty female New Zealand White rabbits between 0.75 and 35 mo of age were studied. Rectangular mucosal membrane strips oriented both longitudinally and circumferentially to the long axis of the trachea were dissected, and the stress-strain relationships of each strip were tested. The results showed that the membrane was stiffer in the longitudinal than in the circumferential direction of the airway. However, there was no significant change with age in either orientation. We conclude that the mechanical properties of the airway mucosal membrane did not change during maturation and were not likely to influence age-related changes in airway responsiveness.     

Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation.

The death receptor CD95 (APO-1/Fas), the anticancer drug etoposide, and gamma-radiation induce apoptosis in the human T cell line Jurkat. Variant clones selected for resistance to CD95-induced apoptosis proved cross-resistant to etoposide- and radiation-induced apoptosis, suggesting that the apoptosis pathways induced by these distinct stimuli have critical component(s) in common. The pathways do not converge at the level of CD95 ligation or caspase-8 signaling. Whereas caspase-8 function was required for CD95-mediated cytochrome c release, effector caspase activation, and apoptosis, these responses were unaffected in etoposide-treated and irradiated cells when caspase-8 was inhibited by FLIPL. Both effector caspase processing and cytochrome c release were inhibited in the resistant variant cells as well as in Bcl-2 transfectants, suggesting that, in Jurkat cells, the apoptosis signaling pathways activated by CD95, etoposide, and gamma-radiation are under common mitochondrial control. All three stimuli induced ceramide production in wild-type cells, but not in resistant variant cells. Exogenous ceramide bypassed apoptosis resistance in the variant cells, but not in Bcl-2-transfected cells, suggesting that apoptosis signaling induced by CD95, etoposide, and gamma-radiation is subject to common regulation at a level different from that targeted by Bcl-2.     

Mitochondrial nucleic acids as internal standards for blot hybridization analyses.

A plasmid, designated p72, constructed from human lung carcinoma DNA inserted into the promoterless herpes simplex virus thymidine kinase gene pML-TK-Bgl II vector, hybridizes strongly to human nucleic acids on Southern and Northern blots. The portion of the DNA insert responsible for the strong signal following hybridization to human DNA or RNA is a 167-bp 3' terminal portion of the mitochondrial 16S ribosomal RNA gene. The expression of this gene is constitutive in the several human cell lines that were tested and is unaffected by exposure to cytotoxic chemicals that alter the expression of nuclear genes. This plasmid offers an excellent tool for studies of perturbations of gene expression and for controlling for the variations in sample preparation, loading, and transfer in Southern or Northern analysis of nucleic acids.     

Molecular Studies on the Role of a Root Surface Agglutinin in Adherence and Colonization by Pseudomonas putida

Pseudomonas putida aggressively colonizes root surfaces and is agglutinated by a root surface glycoprotein. Mutants of P. putida derived chemically or by Tn5 insertion demonstrated enhanced or decreased agglutinability. Two nonagglutinable Tn5 mutants (Agg⁻) and two mutants with enhanced agglutinability (Agg^s) possessed Tn5 in unique restriction sites. Agg⁻ mutants colonized root surfaces of seedlings grown from inoculated seeds, but at levels lower than those observed with the Agg⁺ parent. In short-term binding studies, Agg⁻ cells adhered at levels that were 20- to 30-fold less than those for Agg⁺ parental cells. These data suggest that the agglutination interaction plays a role in the attachment of P. putida to root surfaces.     

Greater airway narrowing in immature than in mature rabbits during methacholine challenge

Shen, X., V. Bhargava, G. R. Wodicka, C. M. Doerschuk, S. J. Gunst, and R. S. Tepper. Greater airway narrowing in immature thanin mature rabbits during methacholine challenge. J. Appl. Physiol. 81(6): 2637-2643, 1996.It hasbeen demonstrated that methacholine (MCh) challenge produces a greaterincrease in lung resistance in immature than in mature rabbits (R. S. Tepper, X. Shen, E. Bakan, and S. J. Gunst.J. Appl. Physiol. 79: 1190-1198, 1995). To determine whether this maturational difference in the response to MCh was primarily related to changes in airway resistance (Raw) or changes in tissue resistance, we assessed airway narrowing in1-, 2-, and 6-mo-old rabbits during intravenous MCh challenge (0.01-5.0 mg/kg). Airway narrowing was determined frommeasurements of Raw in vivo and from morphometric measurements on lungsections obtained after rapidly freezing the lung after the MChchallenge. The fold increase in Raw was significantly greater for 1- and 2-mo-old animals than for 6-mo-old animals. Similarly, the degree of airway narrowing assessed morphometrically was significantly greaterfor 1- and 2-mo-old animals than for 6-mo-old animals. The foldincrease in Raw was highly correlated with the degree of airwaynarrowing assessed morphometrically(r² = 0.82, P < 0.001). We conclude that thematurational difference in the effect of MCh on lung resistance isprimarily caused by greater airway narrowing in the immature rabbits.

     

Effect of shoot tip and leaf removal on gravitropism in pea

Intact, light-grown pea (Pisum sativum L. cv. Alaska) seedlings were subjected to continuous horizontal gravistimulation and their growth and bending response compared with seedlings whose shoot tip and youngest leaf had been excised and with seedlings to which a counterweight to replace the mass of the decapitated tissue was added. While all seedlings achieved vertical orientation in 2 to 3 h, seedlings that were counterweighted bent upward at a significantly slower rate than the non-counterweighted, decapitated plants. In addition to this effect of mass on the rate of bending, decapitation also removed a major supply of auxin to cells in the bending zone which resulted in the slower bending of treated plants. Thus when using decapitation both the loss of mass and the time course of the response must be considered to understand its effect on gravitropism.     

Mechanisms for the mechanical response of airway smooth muscle to length oscillation

Shen, X., M. F. Wu, R. S. Tepper, and S. J. Gunst. Mechanisms for the mechanical response ofairway smooth muscle to length oscillation. J. Appl.Physiol. 83(3): 731-738, 1997.Airway smoothmuscle tone in vitro is profoundly affected by oscillations in musclelength, suggesting that the effects of lung volume changes on airwaytone result from direct effects of stretch on the airway smooth muscle.We analyzed the effect of length oscillation on active force andlength-force hysteresis in canine tracheal smooth muscle at differentoscillation rates and amplitudes during contraction with acetylcholine.During the shortening phase of the length oscillation cycle, the activeforce generated by the smooth muscle decreased markedly below theisometric force but returned to isometric force as the muscle waslengthened. Results indicate that at rates comparable to those duringtidal breathing, active shortening and yielding of contractile elementscontributes to the modulation of force during length oscillation;however, the depression of force during shortening cannot be accountedfor by cross-bridge properties, shortening-induced cross-bridgedeactivation, or active relaxation. We conclude that the depression ofcontractility may be a function of the plasticity of the cellularorganization of contractile filaments, which enables contractileelement length to be reset in relation to smooth muscle cell length asa result of smooth muscle stretch.

     

Comparative respiratory system mechanics in rodents.

Because of the wide utilization of rodents as animal models in respiratory research and the limited data on measurements of respiratory input impedance (Zrs) in small animals, we measured Zrs between 0.25 and 9.125 Hz at different levels (0-7 hPa) of positive end-expiratory pressure (PEEP) in mice, rats, guinea pigs, and rabbits using a computer-controlled small-animal ventilator (Schuessler TF and Bates JHT, IEEE Trans Biomed Eng 42: 860-866, 1995). Zrs was fitted with a model, including a Newtonian resistance (R) and inertance in series with a constant-phase tissue compartment characterized by tissue damping (Gti) and elastance (Hti) parameters. Inertance was negligible in all cases. R, Gti, and Hti were normalized to body weight, yielding normalized R, Gti, and Hti (NHti), respectively. Normalized R tended to decrease slightly with PEEP and increased with animal size. Normalized Gti had a minimal dependence on PEEP. NHti decreased with increasing PEEP, reaching a minimum at approximately 5 hPa in all species except mice. NHti was also higher in mice and rabbits compared with guinea pigs and rats at low PEEPs, which we conclude is probably due to a relatively smaller air space volume in mice and rabbits. Our data also suggest that smaller rodents have proportionately wider airways than do larger animals. We conclude that a detailed, comparative study of respiratory system mechanics shows some evidence of structural differences among the lungs of various species but that, in general, rodent lungs obey scaling laws similar to those described in other species.