Immunocytochemical demonstration of quantitative differences in the distribution of lysozyme in human airway secretory granule phenotypes

The distribution of lysozyme in the different secretory granules (SG) of human tracheal and bronchial submucosal gland serous cells was studied by light and electron microscopy, using a post-embedding immunogold technique. SG were differentiated into 5 phenotypes according to their structure and staining electron density. All the SG-phenotypes were reactive to lysozyme. In the heterogeneous SG-phenotypes, quantitative immunocytochemistry showed that the density of lysozyme labeling was significantly higher in the electron-dense central core compared to the electron-lucent peripheral rim. At the tracheal level, the density of lysozyme did not vary significantly within the different SG-phenotypes, whereas at the bronchial level, the differences were significant. Moreover, the lysozyme labeling density was much higher in the bronchial than in the tracheal SG.     

Semi-automatic quantitation of dense markers in cytochemistry.

The quantitation of electron dense labelling is very tedious when it is done "by hand". Accordingly we developed software allowing, at electron microscopic level, a semi-automatic counting of dense markers in biological specimens. It includes the digitization of images and extraction of dense particles from the grey level of the background. The definition of the areas of interest was carried out by the observer but all quantitative calculations were done automatically. This method was applied to different biological materials (phospholipid and lysozyme labelling in secretory granules of human submucosal bronchial gland cells). The results obtained by this semi-automatic procedure were in good agreement with those obtained by manual counting of colloidal gold labelling (r = 0.97).     

中华绒螯蟹窦腺神经末梢及X-器官神经分泌细胞的类型

在电子显微镜下观察了性未成熟的中华绒螯蟹黄蟹的窦腺及X－器官的超微结构。X－器官位于眼柄神经节终髓的腹外侧,与窦腺位置斜相对,窦腺主要由神经分泌细胞的末梢和胶质细胞组成。神经末梢含有大量的膜结构包围的颗粒、线粒体、粗面内质网和许多电子透明的小泡,末梢外周有时可见指状突起。依据颗粒的大小、形状、电子致密度以及胞质特征,可区分出6种类型的窦腺神经末梢及7种X－器官神经分泌细胞。观察了末梢中神经分泌颗粒的胞吐作用方式的释放过程,并且尝试对窦腺不同末梢中的颗粒及X－器官神经分泌细胞中的颗粒作了比较,发现二者之间具有较好的对应性,即电子致密度无大的变化,形态特征相似,只是大小稍有增加。     

Subordination stress in Nile tilapia and its effect on plasma lysozyme activity

Dominant Nile tilapia had significantly higher lysozyme activity than did subordinate fishes (α=0·05). Plasma lysozyme level was not correlated with sex, parental origin, rearing length, weight, condition factor or rearing density.     

海水淹溺致大鼠急性肺损伤模型的建立

目的：建立海水淹溺肺损伤（SWD-ALl）的大鼠模型,为SWD—ALl的基础及救治研究提供平台。方法：128只健康SD大鼠随机分为4组,即对照（CG）组和海水灌注（SGI、SG2、SG4）组（分别气管内注入1、2、4mL／kg海水）。观察大鼠气管内海水灌注后症状以及各组呼吸频率、心率、动脉血气、肺湿／干重r~（W／D）,留取肺组织病理标本观察病变。结果：与对照组比较,海水灌注组大鼠心率、呼吸频率明显加快,气道有白色泡沫渗出物,口唇发绀,肺部满布湿罗音。动脉血气分析氧分压（PaO2）和二氧化碳分压（Pa—CO2）显著降低（P〈0．01）;SGl、SG2组30min测氧合指数（Pa02／FiO2）低于300mmHg,之后逐渐升高至接近正常,SG4组各时间点测PaOJFi02得均在300mmHg以下;各组间比较,SG4组肺组织湿／干重比（W／D）明显高于其他组（P〈0．01）。肺组织病理观察,光镜下肺间质水肿,肺泡隔断裂,大量的红细胞及炎性细胞浸润。结论：经气管内灌注海水4mL／kg,可成功建立海水淹溺急性肺损伤的动物模型。     

Basal secretion of lysozyme from human airways in vitro

The aim of this study was to examine the basal release of lysozyme from isolated human lung tissues. Measurements of lysozyme in the fluids derived from lung preparations were performed using a rate-of-lysis assay subsequent to acidification of the biological samples. Lysozyme released from bronchial preparations into fluids was greater than that observed for parenchymal tissues. The lysozyme quantities detected in bronchial fluids were not modified by removal of the surface epithelium. Furthermore, the quantities of lysozyme in bronchial fluids was correlated with the size of the bronchial preparations. These results suggest that the lysozyme was principally secreted by the human bronchi (submucosal layer) rather than by parenchyma tissues and that a greater release was observed in the proximal airways.     

Isoproterenol activation of prostaglandin production in guinea-pig airway muscle preparations from immature and mature animals

The aim of this study was to evaluate the capacity of isoproterenol to activate the cyclooxygenase pathway in tracheal and bronchial tissues derived from immature (198 +/- 4 g, N = 12) and mature (997 +/- 28 g, N = 12) guinea-pigs. Immunoreactive PGE2 and PGF2 alpha were measured in bath samples obtained during resting tone and when tissues had been maximally contracted or relaxed. Results from these experiments showed that histamine contractions were significantly greater in tracheal than in bronchial preparations, an effect which was independent of age. Isoproterenol and theophylline were equiactive in relaxing basal tone of tracheal and bronchial tissues when data for each tissue type was compared with results in the different age groups. This effect was also independent of age. When results were normalized for tissue wet weights, the quantities of prostaglandins produced in tissues from mature guinea-pigs were less than those generated in similar tissues derived from immature animals. These data indicate significant modifications in basal prostaglandin production in tissues from immature and mature guinea-pigs. In addition, isoproterenol stimulated prostaglandin production in airways from immature and mature animals whereas theophylline did not alter the basal production.     

Cellular compartmentation of lysozyme and alpha-amylase in the mouse salivary glands. Immunogold approaches at light and electron microscopy level

The research was planned to study the subcellular distribution of enzymatic secretory products within the secretory structures of the mouse major salivary glands at light and electron microscopy level by immunogold silver stain (IGSS) technique and double-sided post-embedding immunogold binding and silver amplification in order to speculate about their compartmentation. In particular, we experimented the above immunogold labeling approaches to localize the lysozyme and to verify its distribution patterns in relation to another secretion enzyme, alpha-amylase. Co-presence of lysozyme and alpha-amylase was observed in the convoluted granular tubule cells of the submandibular gland and in the demilunar cells of the sublingual gland as well as in the electron-dense regions of the mottled secretory granules in the parotid gland. Exclusive binding patterns of lysozyme were observed in the acinar cells of the submandibular and sublingual glands where alpha-amylase did not occur.     

黄柳与垂柳的耐热性和耐旱性比较研究

 作为一种优良的固沙植物,黄柳（Salix gordejevii）主要分布于内蒙古科尔沁沙地和浑善达克沙地,在流动沙丘边缘和半流动沙丘生长最多,属于灌木型先锋植物。实验对黄柳的耐热性和耐旱性与垂柳（Salix babylonica）进行了比较研究。两种植物的离体叶片在6个温度（即25、30、35、40、45、50 ℃）下处理1 h;在6个水势梯度（即0、-0.25、-0.5、-0.75、-1.0、-1.25 MPa）下处理24 h后,测定了其含水量、膜透性、光系统Ⅱ的最大光化学效率（由Fv/Fm计算）等参数。黄柳叶片在温度处理和干旱处理中均能保持比垂柳较高的含水量;45 ℃以下的温度对两种植物叶片的膜透性没有明显影响,但高于45 ℃的温度（本实验中为50 ℃）使黄柳和垂柳叶片膜透性剧增,且黄柳增大更多;干旱处理造成两种植物叶片膜透性增大,且垂柳的总是大于黄柳的;低于35 ℃的温度对Fv/Fm没有影响,高于40 ℃的温度使两种植物叶片的Fv/Fm明显降低,且黄柳的极显著地小于垂柳的;干旱处理总使两种植物叶片的Fv/Fm降低,当渗透势小于-0.75 MPa时,黄柳的Fv/Fm显著地大于垂柳的。这些结果说明黄柳的耐旱性比垂柳强,其耐热性比垂柳差。就光合器官而言,黄柳、垂柳耐热性和耐旱性的差别主要是由于通过光系统Ⅱ的电子传递对高温和干旱的敏感性不同造成的。     

Nonlinear mechanical property of tracheal cartilage: a theoretical and experimental study

BACKGROUND: Despite being the stiffest airway of the bronchial tree, the trachea undergoes significant deformation due to intrathoracic pressure during breathing. The mechanical properties of the trachea affect the flow in the airway and may contribute to the biological function of the lung. METHOD: A Fung-type strain energy density function was used to investigate the nonlinear mechanical behavior of tracheal cartilage. A bending test on pig tracheal cartilage was performed and a mathematical model for analyzing the deformation of tracheal cartilage was developed. The constants included in the strain energy density function were determined by fitting the experimental data. RESULT: The experimental data show that tracheal cartilage is a nonlinear material displaying higher strength in compression than in tension. When the compression forces varied from -0.02 to -0.03N and from -0.03 to -0.04N, the deformation ratios were 11.03+/-2.18% and 7.27+/-1.59%, respectively. Both were much smaller than the deformation ratios (20.01+/-4.49%) under tension forces of 0.02 to 0.01N. The Fung-type strain energy density function can capture this nonlinear behavior very well, whilst the linear stress-strain relation cannot. It underestimates the stability of trachea by exaggerating the displacement in compression. This study may improve our understanding of the nonlinear behavior of tracheal cartilage and it may be useful for the future study on tracheal collapse behavior under physiological and pathological conditions.     

Evolution of the bovine lysozyme gene family: Changes in gene expression and reversion of function

Recruitment of lysozyme to a digestive function in ruminant artiodactyls is associated with amplification of the gene. At least four of the approximately ten genes are expressed in the stomach, and several are expressed in nonstomach tissues. Characterization of additional lysozymelike sequences in the bovine genome has identified most, if not all, of the members of this gene family. There are at least six stomachlike lysozyme genes, two of which are pseudogenes. The stomach lysozyme pseudogenes show a pattern of concerted evolution similar to that of the functional stomach genes. At least four nonstomach lysozyme genes exist. The nonstomach lysozyme genes are not monophyletic. A gene encoding a tracheal lysozyme was isolated, and the stomach lysozyme of advanced ruminants was found to be more closely related to the tracheal lysozyme than to the stomach lysozyme of the camel or other nonstomach lysozyme genes of ruminants. The tracheal lysozyme shares with stomach lysozymes of advanced ruminants the deletion of amino acid 103, and several other adaptive sequence characteristics of stomach lysozymes. I suggest here that tracheal lysozyme has reverted from a functional stomach lysozyme. Tracheal lysozyme then represents a second instance of a change in lysozyme gene expression and function within ruminants. Correspondence to: D.M. Irwin     

Usefulness of the immunogold technique in quantitation of a soluble protein in ultra-thin sections

We used a model system to study whether measurements of absolute local antigen concentrations at the electron microscopic level are feasible by counting immunogold labeling density in ultra-thin sections. The model system consisted of a matrix of a variable concentration of gelatin, which was mixed with given concentrations of rat pancreas amylase and fixed according to various fixation protocols. With a relatively mild fixation, there was no clear proportionality between anti-amylase gold labeling and amylase concentration in ultra-thin cryosections. This was presumably due to uncontrolled loss of amylase from the sections. After stronger fixation with 2% glutaraldehyde for 4 hr, labeling density reflected the amylase concentration very well. We observed that matrix (gelatin) density influenced labeling density. A low gelatin concentration of 5% allowed penetration of immunoreagents into the cryosection, resulting in a high and variable labeling density. In gelatin concentrations of 10% and 20%, labeling density was lower but proportional to amylase concentration. To establish an equal (minimal) penetration of immunoreagents, we embedded model blocks with different matrix densities in polyacrylamide (PAA). In ultra-thin cryosections of these PAA-embedded blocks, anti-amylase labeling was proportional to amylase concentration even at a low (5%) gelatin concentration. Anti-amylase labeling in ultra-thin sections from Lowicryl K4M low temperature-embedded blocks was higher than in PAA sections, but the results were less consistent and depended to some extent on matrix density. These results, together with the earlier observation that acrylamide completely penetrates intracellular compartments (Slot JW, Geuze HJ: Biol Cell 44:325, 1982), demonstrate that it is possible to measure true intracellular concentrations of soluble proteins in situ using ultra-thin cryosections of PAA-embedded tissue.     

Autoradiographic mapping of pulmonary NK1 and NK2 tachykinin receptors and changes after repeated antigen challenge in guinea pigs

An autoradiographic technique was used to study the distribution of changes in pulmonary NK₁ and NK₂ receptors in guinea pig lung after repeated antigen challenge. Specific labeling of [³H] CP96345 (NK₁ receptors) and [³H] SR48968 (NK₂ receptors) was localized over the tracheal and bronchial smooth muscle; the density of binding increased towards smaller airways with a higher density for [³H] CP96345 binding. Bronchial epithelium and pulmonary blood vessels were also labeled densely with [³H] CP96345. No remarkable difference in the pattern of distribution of pulmonary NK₁ and NK₂ tachykinin receptors was observed between control, vehicle-challenged, and repeatedly antigen-challenged (weekly for three times) guinea pigs. A significant reduction in specific labeling of [³H] CP96345 (p < 0.01) and [³H] SR48968 (p < 0.05) over pulmonary structures was observed in antigen-challenged compared to control or vehicle-challenged animals. This study provides evidence that NK₁ and NK₂ tachykinin receptors are both localized to smooth muscle of all sizes in guinea pig airways and provides further evidence for a discrete distribution of NK₁ and NK₂ tachykinin receptors, consistent with their relative functional activities. In a established model of airway inflammation a decrease in the expression of NK₁ and NK₂ tachykinin receptors was evident on several different cell types within the lung, and this could influence airway and vascular reactivity.     

Semi-automatic quantitation of dense markers in cytochemistry

Summary The quantitation of electron dense labelling is very tedious when it is done by hand. Accordingly we developed software allowing, at electron microscopic level, a semi-automatic counting of dense markers in biological specimens. It includes the digitization of images and extraction of dense particles from the grey level of the background. The definition of the areas of interest was carried out by the observer but all quantitative calculations were done automatically. This method was applied to different biological materials (phospholipid and lysozyme labelling in secretory granules of human submucosal bronchial gland cells). The results obtained by this semi-automatic procedure were in good agreement with those obtained by manual counting of colloïdal gold labelling (r=0.97).     

Immunocytochemical localization of actin in the nucleolus of rat oocytes

Summary— In order to determine the localization of actin, growing and fully grown rat oocytes were immunocytochemically examined using a post-embedding ultrastructural protein-A gold technique. In quiescent oocytes, the nucleoplasm showed slightly lower levels of actin signal when compared to the surrounding cytoplasm. The highest levels of labeling were found on nucleoli showing a reticular type morphology. In oocytes at the diakinesis stage in which nucleolar compaction had occurred, the levels of labeling increased by 5–6 times those found in quiescent oocytes. Except for conspicuous accumulation of actin under the plasma membrane, compact nucleoli had significantly higher levels of labeling when compared with those found on the general cytoplasm, while the nucleoplasm with homogeneously dispersed chromatin showed significantly lower levels of associated actin signal than the general cytoplasm. In oocytes at metaphase I, the cytoplasmic region had comparable or lower levels of labeling than the cytoplasm of oocytes at diakinesis. The meiotic spindle embedded in material with medium electron density showed a similar level of labeling as the surrounding cytoplasm. On the other hand, significantly higher levels of associated actin were observed on the chromosomes of metaphase I. The actin signals were dispersed over the chromosomes and not concentrated on a specific region. These results suggest that nuclear actin may be involved in the process of chromosome construction and also the formation of the compacted structure of the nucleolus.     

Left and Right Lung Asynchrony as a Physiological Indicator for Unilateral Bronchial Obstruction in Interventional Bronchoscopy

Background

In patients with bronchial obstruction, pulmonary function tests may not change significantly after intervention. The airflow asynchrony in both lungs due to unilateral bronchial obstruction may be applicable as a physiological indicator. The airflow asynchrony is reflected by the difference in the left and right lung sound development at tidal breathing.

Objectives

To investigate the usefulness of left and right lung asynchrony due to unilateral bronchial obstruction as a physiological indicator for interventional bronchoscopy.

Methods

Fifty cases with central airway obstruction were classified into three groups: tracheal, bronchial and extensive obstruction. The gap index was defined as the absolute value of the average of gaps between the left and right lung sound intensity peaks for a 12-second duration.

Results

Before interventional bronchoscopy, the gap index was significantly higher in the bronchial (p<0.05) and extensive obstruction groups (p<0.05) than in the tracheal group. The gap index in cases with unilateral bronchial obstruction of at least 80% (0.18±0.04 seconds) was significantly higher than in cases with less than 80% obstruction (0.02±0.01 seconds, p<0.05). After intervention for bronchial obstruction, the dyspnea scale (p<0.001) and gap index significantly improved (p<0.05), although no significant improvements were found in spirometric assessments. The responder rates for dyspnea were 79.3% for gap indexes over 0.06 seconds and 55.6% for gap indexes of 0.06 seconds or under.

Conclusions

Assessment of left and right lung asynchrony in central airway obstruction with bronchial involvement may provide useful physiological information for interventional bronchoscopy.     

Immunocytochemical localization of cathepsin H in rat kidney

Summary Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For ligh microscopy, Epon sections of the kidney were stained by immunoenzyme method after removal of Epon and for electron microscopy, ultrathin sections of the Lowicryl K4M-embedded material were labeled by protein A-gold (pAg) technique. By light microscopy, fine granular staining was found in throughout the nephron, but the staining intensity considerably varied. The strongest staining was noted in the S1 segment of the proximal tubules followed by the S2 and S3 segments and the medullary collecting tubules. The glomeruli, the distal tubules, and the cortical collecting tubules were weakly stained. By electron microscopy, a gold label was found exclusively in lysosomes, which showed various sizes and labeling intensity. The results were quite consistent with the light microscopic results. The labeling intensity tended to increase as the matrix of lysosomes was condensed. Quantitative analysis of the labeling density of lysosomes demonstrated that the highest labeling density is found in the S1 segment of the proximal tubules and the labeling density of other renal segments is significantly low levels. The results indicate that a main site for cathepsin H in rat kidney is the S1 segment of the proximal tubules.     

Electromechanical effects of endothelin on ferret bronchial and tracheal smooth muscle

The effects of endothelin (ET) on transmembrane potential and isometric force were studied in ferret bronchial and tracheal smooth muscles. At rest, the muscle cells were electrically and mechanically quiescent. The mean resting potential for the bronchial cells was -70 +/- 1 mV (n = 25 cells/8 ferrets), and that of the tracheal cells was -60 +/- 1 mV (n = 7 cells/2 ferrets). ET depolarized and contracted both types of muscle cells in a concentration-dependent manner. At 1 nM ET, the bronchial muscle cells were significantly depolarized with concomitant force generation. In contrast, greater than 30 nM ET was required for the tracheal muscle cells to respond. The bronchial cells were further depolarized by 10 and 100 nM ET with electrical slow-wave activity present. The calcium channel antagonist verapamil substantially inhibited the contractions produced by 100 nM ET and abolished the slow-wave activity without affecting the base-line depolarization. Pretreatment of the bronchial muscle with 30 microM indomethacin did not affect the ET-induced contraction. These results suggest that ET modulates airway smooth muscle tone by direct activation and/or depolarization-induced activation of sarcolemmal calcium channels.     

Immune parameters in carpet shell clams naturally infected with Perkinsus atlanticus

Defence parameters of non-infected clams (Ruditapes decussatus) and clams heavily infected with Perkinsus atlanticus were assessed. Cellular (haemocyte density and phagocytic activity) and humoral (lysozyme and anti-bacterial activities, protein concentration and agglutination titre) parameters were measured in clams collected in an area enzootic for P. atlanticus. The infection intensity of each clam was assessed, and the immune parameters measured in the most infected clams were compared with those measured in the non-infected ones. Only the serum anti-bacterial activity and the agglutination titre were significantly different between infected and non-infected clams. The phagocytic rate, haemocyte density, lysozyme concentration and protein concentration were not statistically different but they showed the same trend in the two trials performed. Phagocytic rate, haemocyte concentration and anti-bacterial activity were higher in non-infected clams, while they had lower lysozyme concentration, serum protein concentration and agglutination titre than infected clams. Although infected and healthy clams were not different for every parameter measured, probably due to the high variability among individuals, P. atlanticus seems to affect the clam immune system, at least in advanced stages of the infection.     

Tracheal gland mucous cells stimulated in vitro with adrenergic and cholinergic drugs

To determine the responsiveness of tracheal mucous cells to adrenergic and cholinergic stimulation, we analyzed changes in their structure induced by neurotransmitter-like agonists. Ferret tracheal rings were exposed for 30 min in vitro to one of the following: phenylephrine, isoproterenol, or bethanechol (all at 10(-5) M), in the presence of absence of appropriate antagonists. Electron microscopy and morphometric analysis revealed that the volume density of mucous cells (Vvmc, i.e. the space occupied by mucous cells in the submucosa) significantly decreased, and the surface density of mucous cell apical membrane (Svam) increased in response to isoproterenol and bethanechol but not to phenylephrine. In metabolic labeling experiments, the morphological changes were accompanied by secretagogue-evoked release of 35S-labeled macromolecules. Taken together, these data suggest that tracheal mucous cells secrete 35S-labeled macromolecules in response to beta-adrenergic and muscarinic agonists by an exocytotic process that involves a reduction in cell size.