Gene expression profiling of ovine keratinocytes stimulated with Psoroptes ovis mite antigen--a preliminary study.

Sheep scab is caused by the noninvasive mite, Psoroptes ovis, which initiates a profound pro-inflammatory skin response leading to lesion development. To investigate these early events between the skin and the parasite, primary ovine epidermal keratinocyte cultures were generated and challenged with mite derived antigens. The kinetics of the mRNA response of these cells were monitored by microarray. The results indicated that the cells responded within 1 h of challenge, with a significant increase in the pro-inflammatory cytokine IL-8. This result was confirmed by real-time RT-PCR, and showed that IL-8 up-regulation was maximal at 1 h but declined to pre-stimulation levels at 24 and 48 h. The IL-8 mRNA response to mite wash antigens containing secretory and/or excretory proteins was also investigated and compared to the response to whole mite antigen. These studies revealed that the mite wash antigen, at a challenge dose of 10 microg/mL, was markedly more potent and induced significantly higher levels of IL-8 mRNA than the same concentration of whole mite antigen. These results are discussed in relation to mite establishment and survival on the ovine host.

Contribution of respiratory burst activity to innate immune function and the effects of disease status and agent on chemiluminescence responses by ruminant phagocytes in vitro.

The mechanisms of interaction between phagocytes and different bacteria that help resolve lung infections or contribute to lung pathology are poorly defined. Alveolar phagocytes (resident macrophages and recruited neutrophils) make a major contribution to innate immunity by mounting a respiratory burst that helps kill internalised bacteria. However, this ability may be altered during or after exposure to infection. This review considers the application and limitations of a variety of analytical methods for oxygen-dependent mechanisms of respiratory burst in phagocytes initiated by soluble and particulate activators. Particular reference is given to the study in vitro of phagocytes from healthy and diseased ruminants during either natural infection with Mycobacterium avium paratuberculosis or experimental infection with Pasteurella multocida or Mannheimia haemolytica.

Anesthetic effects on 5-hydroxytryptamine uptake by rat brain synaptosomes.

As a neurotransmitter involved in modulating central nervous system nociception and awareness, 5-hydroxytryptamine (5-HT) may play an important role in the clinical sequelae of certain anesthetic compounds. Anesthetic agents are known to affect peripheral, non-neuronal 5-HT uptake but little is known about their effects on 5-HT metabolism in the central nervous system. The effects of several anesthetic compounds on 5-HT uptake were examined in synaptosomes isolated from rat brain cortex. Inhibition of this uptake process was observed by exposure to clinically relevant concentrations of the volatile anesthetics halothane, isoflurane, and enflurane. The non-volatile agent, ketamine also inhibited uptake while the narcotic fentanyl had an effect only at the highest concentrations tested. Non-volatile agents which had neither a consistent nor significant effect on synaptosomal 5-HT uptake included pentobarbital, sufentanil, and etomidate. These alterations of 5-HT metabolism could represent a mechanism that contributes to anesthetic action.

The measurement of protein synthesis in biological systems.

Attempts to quantitate metabolism in the lung and other tissues using radioactive precursors may be subject to significant errors arising from inappropriate assumptions regarding precursor metabolism, compartmentation and specific radioactivity. This article reviews the type and magnitude of error which may complicate such measurements by presenting specific data from experiments using radioactive amino acids to estimate the rate of protein synthesis. The applicability of these observations to other metabolic systems is discussed briefly in order to develop a more general awareness of the errors which may result from incomplete validation of experimental measurements using radioisotopes.

Comparison of three salivary flow rate assessment methods in an elderly population.

Measuring salivary flow rates among the frail elderly is a challenge. The currently used spit collection method requires levels of time and cooperation that often may exclude the frail elderly who are at high risk for salivary compromise. A measurement method that is not only valid and reliable, but also feasible and acceptable is needed for use in population studies of compromised adults. This study compared two salivary flow rate assessment methods using a suction machine against the currently accepted spit collection method in an elderly population aged 75 and older. Three methods of flow rate (g/min) assessment were compared at three time periods among 16 elders (mean age 86.6 years). Flow rates using the 2-min open suction method compared well with the 10-min spit method (r=0.778) but the 2-min closed suction method did not (r=0.158). Reliability evaluation of the open suction method and the spit method was assessed using a test/retest with a 1-week interval. Both methods demonstrated good comparable reliability (spit method r=0.566, P=0.01); open suction method, (r= 0.861, P<0.01). Based on a short survey questionnaire about the three methods, 11 of the 16 elderly subjects preferred the use of the suction machine to the spit method. These results indicate that the 2-min open suction method technique is a valid and reliable means of measuring salivary flow. The lower level of patient cooperation needed, the shorter time period required, and this preliminary report of its acceptability support the use of this method in future population studies of frail elders.

Expression profiling reveals differences in immuno-inflammatory gene expression between the two disease forms of sheep paratuberculosis.

Paratuberculosis is a chronic enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP); infection of sheep results in two disease forms - paucibacillary (tuberculoid) and multibacillary (lepromatous) associated with the differential polarization of the immune response. In addition the majority of MAP-infected animals show no pathology and remain asymptomatic. Microarray and real-time RT-qPCR analyses were used to compare gene expression in ileum from sheep with the two disease forms and asymptomatic sheep, to further understand the molecular basis of the pathologies. Microarrays identified 36 genes with fold-change of >1.5 and P< or = 0.05 in at least one comparison; eight candidates were chosen for RT-qPCR validation. Sequence analysis of two candidates, CXCR4 and IGFBP6, identified three SNPs in each; five were found in all three forms of disease and showed no significant relationship to pathological type. The IGFBP6 G(3743) A SNP was not detected in asymptomatic sheep. The data show that the two forms of disease are associated with distinct molecular profiles highlighted by the differential expression of chemokine and chemokine receptor transcripts, the protein products of which might be implicated in the different cell infiltrates of the pathologies. The cells within the lesions also show evidence of abnormal activation; they express high levels of cytokine transcripts but have reduced expression levels of transcripts for T cell receptor associated molecules.

Effects of reduced pulmonary flow and hypoxia on metabolism of serotonin by rat lungs perfused in situ.

To investigate the extent to which reduced pulmonary flow may affect non-ventilatory functions of the lung, pulmonary artery pressures were altered systematically in an in vitro perfused lung preparation. Metabolic integrity of the tissue was assessed at two levels: disposition of exogenous serotonin (5-hydroxytryptamine; 5-HT) was monitored as a specific indicator of endothelial cell metabolism; and whole-tissue rates of protein synthesis and levels of ATP were evaluated as indices of general metabolic activity and energy availability. Rat lungs were perfused with recirculating cell-free buffer (37 degrees C) for 1 or 3 h at high (36) or low (3 ml.min-1.g-1) pulmonary flow; initial rates of 5-HT metabolism were measured over a subsequent 2-min interval of single-pass perfusion. Metabolism of 5-HT was inhibited and protein synthesis decreased 35% at low pulmonary flow. These changes did not appear to result directly from hypoxia, nor from the associated fall in tissue ATP. The effects of low flow were not reversed at high PO2, nor was 5-HT metabolism inhibited by restricted oxygen availability at high flow rates. After as long as 3 h exposure to a combination of low flow, ventilation (V = 0), and temperature (27 degrees C) and to the volatile anesthetic, halothane, inhibitory effects on both amine and protein metabolism were rapidly reversible. Reductions in the rate of 5-HT metabolism at reduced flow involved a decrease in the maximal velocity (Vmax: 8.0 to 2.2 nmol.min-1.g-1), without change in the apparent Km (2.6-3.2 microM) of the pathway for amine metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmalemmal metabolic activities in cultured calf pulmonary arterial endothelial cells.

We have determined kinetic characteristics of angiotensin converting enzyme, 5'-nucleotidase and transmembrane serotonin uptake and metabolism in cultured calf pulmonary arterial endothelial cells. Angiotensin converting enzyme activity was 2.8 +/- 0.03 Units/10(6) cells (N = 19; 1 Unit: amount of enzyme required to metabolize 1% of substrate, benzoyl-Phe-Ala-Pro, in 1 min under conditions of first order reaction kinetics) in confluent monolayers and 2.31 +/- 0.06 Units/10(6) cells (N = 20) in homogenates. 5'-Nucleotidase activity (substrate: 5'-AMP) was 0.25 +/- 0.01 Units/10(6) cells (N = 19) in monolayers and 0.26 +/- 0.01 Units/10(6) cells (N = 20) in homogenates. Kinetic constants for angiotensin converting enzyme were: Km = 7.6 microM, Vmax = 5.2 nmol/hour/10(6) cells and for 5'-nucleotidase: Km = 52.6 microM, Vmax = 6.3 nmol/hour/10(6) cells. These data confirm that both angiotensin converting enzyme and 5'-nucleotidase are ectoenzymes with no cytoplasmic activity. Serotonin uptake exhibited both a saturable (Km = 0.27 microM, Vmax = 17 pmol/hour/10(6) cells) and a non-saturable component.

In situ perfusion of rat lungs: stability and effects of oxygen tension.

A new method for perfusion of rat lungs in situ was developed for metabolic studies. The pulmonary circulation was cannulated without contacting the lungs, which remained in the thoracic cage. Perfusion was continued for up to 4 h with Krebs-Henseleit bicarbonate buffer, equilibrated with 95% O2- 5% CO2 and containing 4.5% bovine serum albumin, 5.6 mM glucose, and levels of amino acids normally found in rat plasma. At an arterial pressure of 20 cmH2O flow remained constant (10.9 ml/min.100 g body wt) and appeared evenly distributed among the lobes. Tidal volume was 1 ml/100 g body wt (72/min); positive end-expiratory pressure was 2 cmH2O. The preparation remained stable and metabolically active for 4 h, as evidenced by a minimal decline in dry-to-wet weight ratio, constant levels of ATP and glycogen, a high ratio of glucose uptake to lactate production, and a linear rate of incorporation of [14C]phenylalanine into protein. The lungs were unaffected when perfusate oxygen was reduced to a more physiological level (20% O2-75% N2-5% CO2). In the presence of 95% N2-5% CO2 dry-to-wet weight ratio, ATP, glycogen, and amino acid incorporation decreased, while lactate production doubled.

Effect of diabetes on metabolism of 5-hydroxytryptamine by rat lungs perfused in situ.

The effect of diabetes induced by treatment of rats with streptozotocin on metabolism of circulating 5-hydroxytryptamine (5HT) was investigated using an in situ lung perfusion preparation. Tissue uptake of 5HT and production of its metabolite, 5-hydroxyindolacetic acid, were unaffected in lungs of diabetic animals provided 2 or 20 microM exogenous 5HT. At constant perfusion pressure, pulmonary flow was not altered by substrate concentration or by streptozotocin treatment. Thus, in the experimental models of diabetes used, metabolism of circulating 5HT by the pulmonary endothelium remained unaffected.

Measurement of protein synthesis in rat lungs perfused in situ.

Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs-Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6-690mum-[U-(14)C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O(2)/CO(2) (19:1) or O(2)/N(2)/CO(2) (4:15:1)]. [U-(14)C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t((1/2)), 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [(14)C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [(14)C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.

Inhibition of synaptosomal serotonin uptake by Ketalar.

The effects of the clinical preparation of ketamine, Ketalar and its preservative, benzethonium chloride on [3H]5-hydroxytryptamine uptake were studied using rat brain synaptosomes. Ketalar caused a concentration-dependent inhibition of substrate uptake by the high affinity transport site (I50 = 20.2 +/- 2.75 microM) while benzethonium chloride had no effect. Kinetic analysis indicated the inhibition to be competitive with serotonin; the apparent km (54 nM) was increased nearly two-fold at 10 microM ketamine. This action may represent a mechanism involved in ketamine anesthesia.

Effect of age on the accumulation of lung protein following unilateral pneumonectomy in rats.

The effects of unilateral pneumonectomy (PNX) on the net synthesis of right lung protein were investigated in vivo using three groups of rats with body weights (BW) ranging from 85 to 330 g. These data were compared to those from sham-operated and normal growing control animals. After PNX, both the 2-day lag prior to the compensatory increase in right lung mass (LW) and the subsequent rate of increase in LW and LW/BW ratio were independent of two-fold differences in the basal rate of lung growth. In all PNX groups, both right LW and LW/BW reached control values for both lungs, but in the older rats the time required for complete compensation was extended from 5 days to 12 days. The rate of net accumulation of right lung protein increased two-fold in the youngest PNX rats and 6 to 8-fold in the older animals, but when these changes were normalized to the protein content of the remaining tissue, the older rats appeared to respond to PNX less efficiently. Increased tissue levels of RNA and the resulting increased capacity of the lungs for protein synthesis could account for the accelerated rate of gain in right lung protein following PNX in both adult and young animals.

Reversible inhibition of protein synthesis in lung by halothane.

Alterations in the synthesis and degradation of proteins were investigated in intact lungs exposed to the volatile anaesthetic halothane. In rat lungs perfused in situ with Krebs-Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6 mM-glucose, plasma concentrations of 19 amino acids and 690 microM-[U-14C]-phenylalanine and equilibrated with O2/N2/CO2 (4:15:1), protein synthesis, calculated based on the specific radioactivity of aminoacyl-tRNA, was inhibited by halothane. The anaesthetic did not affect degradation of lung proteins. The inhibition of protein synthesis was rapid in onset, dose-dependent, and quickly reversible. It did not appear to be associated with overall energy depletion, with non-specific changes in cellular permeability, or with decreased availability of amino acids as substrates for protein synthesis.

Lung growth in response to unilateral pneumonectomy in rapidly growing rats.

The rapidity with which lung growth is initiated and completed after pneumonectomy was examined in young rats (4 wk of age; 82 g). After left pneumonectomy, the remaining lobes of the right lung grew to equal the weight of both lungs of control animals by day 7 and within 14 days increased from 366 to 968 mg. The tissue concentrations of RNA, DNA phosphate, collagen, and noncollagen proteins did not increase during the growth response. In contrast, total amounts of these constituents increased significantly in the remaining lung of pneumonectomized animals during the 1st postoperative wk and approached levels found in both lungs of sham-operated and unoperated controls by the end of the 2nd wk after pneumonectomy. Although cell size increased in control lungs during the experimental period, there was little evidence of additional cellular hypertrophy associated with compensatory lung growth. The character of the response to pneumonectomy in these rats was similar to that observed previously in older animals (320 g). Thus in spite of the higher basal rate of lung growth in the younger rats, the pattern and rapidity of compensation after pneumonectomy was similar in both age groups.

Metabolic effects of isoflurane on rat lungs perfused in situ.

1. The current experiments studied the effects of the inhalation anesthetic, isoflurane, on 5-hydroxytryptamine (5-HT) metabolism, protein synthesis, and angiotensin-converting enzyme activity in perfused rat lungs. 2. Under first order reaction conditions, isoflurane decreased the accumulation of tissue 5-hydroxyindoleacetic acid, the principle metabolite of 5-HT in a concentration-related, competitive, and reversible manner, indicating inhibition of endothelial 5-HT uptake. 3. In apparent contrast, isoflurane appeared to stimulate uptake of 5-HT by an imipramine-sensitive process, into a cell type unable to metabolize the parent amine. 4. Isoflurane increased absolute angiotensin-converting enzyme activity only at an inspired concentration of 5%. The anesthetic did not affect lung protein synthesis.

Effects of starvation and diabetes on protein synthesis in lung.

Metabolism of lung proteins was investigated in rats starved 3 days or made diabetic with streptozotocin. Body weight was below normal in both groups, but lung weight decreased only in starved animals. Total lung protein and RNA (mg/lung) decreased during starvation and diabetes. Protein concentration (mg/g) was unchanged in either group of animals; RNA concentration decreased only during starvation. Protein synthesis, estimated in lungs perfused in situ, was reduced 22% in starvation, but remained unchanged in diabetes. Inhibition of protein synthesis was accounted for by loss of RNA. Ribosomal profiles were unchanged by starvation, suggesting an unaltered relationship between rates of peptide-chain initiation and elongation in vivo. Activity of an eIF-2-like initiation factor decreased during starvation in proportion to the loss of RNA. In diabetes, factor activity remained normal. Thus, starvation but not streptozotocin-induced diabetes, reduced the capacity of the lung to synthesize protein. No evidence for reduced efficiency of synthesis was observed.

Rapidity of compensatory lung growth following pneumonectomy in adult rats.

The rapidity with which lung growth was initiated following pneumonectomy was investigated using rats (330 g) in which lung weight-to-body weight ratio and lung cell size had stabilized. Following removal of the left lung, right lung weight increased from 823 to 1.161 mg within 7 days. Right lung weight in sham-operated animals did not change significantly. At day 7, right lung weight-to-body weight ratio in pneumonectomized rats was equal to that of both lungs in sham-operated animals; these values remained equal through day 14. Growth of individual lobes of the right lung was generally in proportion to their initial weights. Dry-to-wet weight ratio in either lung of sham-operated or pneumonectomized animals was unchanged, as compared to unoperated controls. Total right lung RNA and protein increased significantly by day 2 and reached levels equal to those in both lungs of sham-operated animals by day 7. Synthesis of lung proteins, estimated during 120 min of perfusion in situ, was elevated 25% on day 3. Incorporation of [3H]thymidine into DNA increased somewhat on day 2 and was elevated fourfold on day 3, corresponding with the initial accumulation of total DNA within the lung. These observations suggested that increased cell size may accompany early compensatory growth following pneumonectomy, but that the major portion of the response involved cellular hyperplasia.

Additive effects of pentobarbital and halothane to inhibit synthesis of lung proteins.

The effect of pentobarbital on synthesis of lung proteins was investigated, both when administered alone and in combination with halothane. When rat lungs perfused in situ with Krebs-Henseleit bicarbonate buffer containing plasma levels of 19 amino acids, 690 microM phenylalanine, 5.6 mM glucose, and 4.5 per cent fraction V bovine serum albumin were exposed to pentobarbital, a dose-related inhibition of [14C]phenylalanine incorporation into protein was observed, with a maximal inhibition (74 per cent) at a pentobarbital concentration of 324 micrograms/ml. Halothane (1-4 per cent equilibrated with O2/N2/CO2, 4:15:1) also rapidly inhibited synthesis of lung proteins in a dose-dependent manner. At the maximally effective concentration of pentobarbital, exposure of the lungs to halothane enhanced the inhibition of protein synthesis; halothane concentrations ranging from 1 to 4 per cent were equally effective. Furthermore, when lungs were exposed to a combination of pentobarbital (100 micrograms/ml) and halothane (1 per cent) at doses which had no effect when given alone, protein synthesis was inhibited 35 per cent (P less than 0.001). Thus, the metabolic effects of the anesthetics were potentiated when the drugs were administered in combination. The inhibition of protein synthesis by pentobarbital (324 micrograms/ml), with or without 4 per cent halothane, was fully reversible. A similar inhibitory effect of pentobarbital was observed in perfused rat hearts.