Down-regulation of HSP60 expression by RNAi increases lipopolysaccharide- and cerulein-induced damages on isolated rat pancreatic tissues

The objective of this study was to investigate the function of heat shock protein 60 (HSP60) on pancreatic tissues by applying HSP60 small interfering RNA (siRNA) to reduce HSP60 expression. Rat pancreas was isolated and pancreatic tissue snips were prepared, cultured, and stimulated with low and high concentrations of cerulein (10⁻¹¹ and 10⁻⁵ mol/L) or lipopolysaccharide (LPS, 10 and 20 μg/mL). Before the stimulation and 1 and 4 h after the stimulation, the viability and the level of trypsinogen activation peptide (TAP) in the tissue fragments were determined and the levels of tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) in the culture supernatants were measured. Real-time PCR and Western blotting were used to evaluate the HSP60 mRNA and protein expression. After the administration of siRNA to inhibit HSP60 expression in the isolated tissues, these injury parameters were measured and compared. The pancreatic tissues in the control (mock-interfering) group showed a decreased viability to varying degrees after being stimulated with cerulein or LPS, and the levels of TAP, TNF-α, and IL-6 increased significantly (p < 0.05) in the tissues and/or in the culture supernatant. The expressions of HSP60 mRNA and protein were raised moderately after stimulating 1 h with low concentrations of cerulein or LPS, but decreased with high concentrations of the toxicants. In particular, the expression of HSP60 protein was reduced significantly (p < 0.05) when the tissues were stimulated by the two toxicants for 4 h. In contrast, the tissue fragments in which HSP60 siRNA was applied showed much lower tissue viability (p < 0.01) and higher levels of TNF-a, IL-6, and TAP (p < 0.01) in the tissues or culture supernatant after stimulating with the toxicants at the same dose and for the same time duration as compared with those of the control groups (p < 0.05). The results indicated that both cerulein and LPS can induce injuries on isolated pancreatic tissues, but the induction effects are dependent on the duration of the stimulation and on the concentrations of the toxicants. HSP60 siRNA reduces HSP60 expression and worsens the cerulein- or LPS-induced injuries on isolated pancreatic tissues, suggesting that HSP60 has a protective effect on pancreatic tissues against these toxicants.     

Alteration of Cpn60 expression in pancreatic tissue of rats with acute pancreatitis

The expression of heat-shock protein 60 (also known as chaperonin 60, Cpn60) in experimental acute pancreatitis (AP) is considered to play an active role in the prevention of abnormal enzyme accumulation and activation in pancreatic acinar cells. However, there are controversial results in the literature regarding the relationship between the abnormality of Cpn60 expression and AP onset and development. The purpose of this study was to investigate the alternations of Cpn60 expression and the relationship between the abnormal expression of Cpn60 and AP progression in rat severe acute pancreatitis (SAP) models. In this report, we induced SAP in Sprague–Dawley (SD) rats by reverse injection of sodium deoxycholate into the pancreatic duct, and examined the dynamic changes of Cpn60 expression in pancreatic tissues from different time points and at different levels with techniques of real-time PCR, western blotting, and immunohistochemistry. At 1 h after SAP induction, the expression of Cpn60 mRNA in the AP pancreatic tissues was higher than those in the sham-operation group and normal control group, but decreased sharply as the time period was extended, and there was a significant difference between 1 h and 10 h after SAP induction (p < 0.05). In the AP process, Cpn60 protein expression showed transient elevation as well, and the increased protein expression occurred predominantly in affected, but not totally destroyed, pancreatic acinar cells. As AP progressed, the pancreatic tissues were seriously damaged, leading to a decreased overall Cpn60 protein expression. Our results show a complex pattern of Cpn60 expression in pancreatic tissues of SAP rats, and the causality between the damage of pancreatic tissues and the decrease of Cpn60 level needs to be investigated further. Xue-Li Li and Kun Li contributed equally to this work.     

Preinduction of HSP70 promotes hypoxic tolerance and facilitates acclimatization to acute hypobaric hypoxia in mouse brain

It has been shown that induction of HSP70 by administration of geranylgeranylacetone (GGA) leads to protection against ischemia/reperfusion injury. The present study was performed to determine the effect of GGA on the survival of mice and on brain damage under acute hypobaric hypoxia. The data showed that the mice injected with GGA survived significantly longer than control animals (survival time of 9.55 ± 3.12 min, n = 16 vs. controls at 4.28 ± 4.29 min, n = 15, P < 0.005). Accordingly, the cellular necrosis or degeneration of the hippocampus and the cortex induced by sublethal hypoxia for 6 h could be attenuated by preinjection with GGA, especially in the CA2 and CA3 regions of the hippocampus. In addition, the activity of nitric oxide synthase (NOS) of the hippocampus and the cortex was increased after exposure to sublethal hypoxia for 6 h but could be inhibited by the preinjection of GGA. Furthermore, the expression of HSP70 was significantly increased at 1 h after GGA injection. These results suggest that administration of GGA improved survival rate and prevented acute hypoxic damage to the brain and that the underlying mechanism involved induction of HSP70 and inhibition of NOS activity.     

Ethanol induces secretion of oxidized proteins by pancreatic acinar cells

The pancreas is vulnerable to ethanol toxicity, but the pathogenesis of alcoholic pancreatitis is not fully defined. The intracellular oxidative balance and the characteristics of the secretion of isolated rat pancreatic acinar cells stimulated with the cholecystokinin analogue cerulein were assayed after acute oral ethanol (4 g/kg) load. Pancreatic acinar cells from ethanol-treated rats showed a significant (p < 0.02) lower content of total glutathione and protein sulfhydryls, and higher levels of oxidized glutathione (p < 0.03), malondialdehyde, and protein carbonyls (p < 0.05). Ethanol-intoxicated acinar cells showed a lower baseline amylase output compared to controls, with the difference being significantly exacerbated by cerulein stimulation. After cerulein, the release of protein carbonyls by ethanol-treated cells was significantly increased, whereas that of protein sulfhydryls was significantly decreased. In conclusion, ethanol oxidatively damages pancreatic acinar cells; cerulein stimulation is followed by a lower output of amylase and by a higher release of oxidized proteins by pancreatic acinar cells from ethanol-treated rats. These findings may account for the decreased exocrine function, intraductular plug formation, and protein precipitation in alcoholic pancreatitis.     

Rapid onset of ICAM-1 expression is a marker of effective macrophages activation during infection of <Emphasis Type="Italic">Francisella tularensis</Emphasis> LVS in vitro

Francisella tularensis is capable to modulate immunobiological activities of the host cells. We focused on the expression of ICAM-1 (CD54) on J774.2 mouse macrophage cell line infected by F. tularensis live vaccine strain (LVS) in vitro as a putative marker of subsequent elimination of infection. J774.2 cell line cells were infected by F. tularensis LVS strain (multiplicity of infection, 1:100). Cell cultures were stimulated either 3 h before infection or 3 h after infection by either lipopolysaccharide (LPS) or interferon γ (IFN-γ). The expression of ICAM-1 was determined by flow cytometry 6 h after infection. The intensity of ICAM-1 expression after 6 h of J774.2 macrophage cells infection by F. tularensis is very sensitive indicator of the effective macrophages stimulation resulting in the elimination of F. tularensis infection. The mean fluorescence intensity MFI = 49.8 is set-up by our experiments as a reliable threshold of the effective elimination of F. tularensis experimental infection with 83.3% sensitivity and 96.7% specificity, respectively. Simultaneous stimulation of J774.2 macrophage cells by LPS and IFN-γ was essential to elicit the elimination of F. tularensis infection. The ICAM-1 expression determined by flow cytometry can be considered to be highly sensitive and specific approach to predict elimination of F. tularensis infection by J774.2 macrophages.     

Freezing solution containing dimethylsulfoxide and fetal calf serum maintains survival and ultrastructure of goat preantral follicles after cryopreservation and in vitro culture of ovarian tissue

Goat ovarian cortex fragments were subjected to slow freezing in the presence of various solutions containing intracellular cryoprotectants, including 1.0 M ethylene glycol (EG), propanediol (PROH), or dimethyl sulfoxide (DMSO), with or without sucrose and/or fetal calf serum (FCS). Histological examination revealed that only the DMSO-containing solutions were able to maintain a follicular ultrastructure similar to the morphology observed in the fresh control. Therefore, fragments previously cryopreserved in DMSO solutions (with and without sucrose and/or FCS) were cultured in vitro for 48 h and then subjected to viability, histological, and ultrastructural analysis. No significant differences were observed among the percentages of morphologically normal follicles in cryopreserved ovarian tissue before in vitro culture (DMSO: 62.5%; DMSO + sucrose: 68.3%; DMSO + FCS: 60.0%; DMSO + sucrose + FCS: 60.0%) and after culture (DMSO: 60.8%; DMSO + sucrose: 64.2%; DMSO + FCS: 70.8%; DMSO + sucrose + FCS: 55.0%). Following in vitro culture, the viability analysis showed that only the freezing solution containing DMSO and FCS (75.6%) maintained a percentage of viable follicles similar to that observed after culture without cryopreservation (89.3%). As determined by ultrastructural analysis, morphologically normal preantral follicles were detected in the fresh control and in fragments cultured before and after cryopreservation with DMSO and FCS. Thus, a freezing solution containing DMSO and FCS, under the experimental conditions tested here, guaranteed the maintenance of viability and follicular ultrastructure after short-term in vitro culture.     

Regulation of auxin transport by aminopeptidases and endogenous flavonoids

 The 1-N-naphthylphthalamic acid (NPA)-binding protein is a putative negative regulator of polar auxin transport that has been shown to block auxin efflux from both whole plant tissues and microsomal membrane vesicles. We previously showed that NPA is hydrolyzed by plasma-membrane amidohydrolases that co-localize with tyrosine, proline, and tryptophan-specific aminopeptidases (APs) in the cotyledonary node, hypocotyl-root transition zone and root distal elongation zone of Arabidopsisthaliana (L.) Heynh. seedlings. Moreover, amino acyl-β-naphthylamide (aa-NA) conjugates resembling NPA in structure have NPA-like inhibitory activity on growth, suggesting a possible role of APs in NPA action. Here we report that the same aa-NA conjugates and the AP inhibitor bestatin also block auxin efflux from seedling tissue. Bestatin and, to a lesser extent, some aa-NA conjugates were more effective inhibitors of low-affinity specific [³H]NPA-binding than were the flavonoids quercetin and kaempferol but had no effect on high-affinity binding. Since the APs are inhibited by flavonoids, we compared the localization of endogenous flavonoids and APs in seedling tissue. A correlation between AP and flavonoid localization was found in 5- to 6-d-old seedlings. Evidence that these flavonoids regulate auxin accumulation in vivo was obtained using the flavonoid-deficient mutant, tt4. In whole-seedling [¹⁴C]indole-3-acetic acid transport studies, the pattern of auxin distribution in the tt4 mutant was shown to be altered. The defect appeared to be in auxin accumulation, as a considerable amount of auxin escaped from the roots. Treatment of the tt4 mutant with the missing intermediate naringenin restored normal auxin distribution and accumulation by the root. These results implicate APs and endogenous flavonoids in the regulation of auxin efflux. Received: 2 December 1999 / Accepted: 16 January 2000     

Effects of remifentanyl and fentanyl on LPS-induced cytokine release in human whole blood in vitro

Aim The present study sought insight into the effects of remifentanyl and fentanyl on LPS-induced release of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and IL-10 in human whole blood. Methods Whole blood was incubated in the presence and absence of remifentanyl and fentanyl. Effects of remifentanyl and fentanyl on spontaneous and endotoxin (lipopolysaccharide; 100 ng ml⁻¹)-stimulated cytokine release were studied in whole blood from volunteers (n = 10) cultured for 6 h. Results IL-6, TNF-α and IL-10 concentrations in groups added with LPS were significantly higher than those in control group (P < 0.01). IL-6, TNF-α and IL-10 concentrations in activation groups treated with remifentanyl or fentanyl were significantly lower than those in LPS treated group (P < 0.05). There were no significant differences on IL-6,TNF-α and IL-10 concentrations in drug-alone groups compared with control group (P > 0.05). Conclusion Remifentanyl or fentanyl alone has no effects on IL-6, TNF-α and IL-10 production, but could attenuate LPS-induced IL-6,TNF-α and IL-10 production in human whole blood. Remifentanyl and fentanyl could inhibit the expressions of IL-6, TNF-α and IL-10 induced by LPS.     

Effects of saturated fatty acids on amylase release from exocrine pancreatic segments of sheep,rats, hamsters,field voles and mice

 Stimulatory effects of saturated fatty acids consisting of 4 (butyrate), 8 (octanoate), 12 (laurate) and 16 (palmitate) carbon atoms, as well as acetylcholine on pancreatic amylase release were assessed in tissue segments isolated from sheep, rats, hamsters, field voles and mice. The amount of amylase release induced by the fatty acids (1 μmol ⋅ l^-1 to 10 mml ⋅ l^-1) and by acetylcholine (10 nmol ⋅ l^-1 to 100 μmol ⋅ l^-1) increased in a concentration-dependent manner, and the maximum response in response to the fatty acids was obtained at the maximal dose used. The maximum increase in amylase release in response to butyrate or octanoate was highly and significantly (r=0.974, P<0.001) dependent on the log value of the mean body mass in the following order: sheep>rats>hamsters>field voles>mice. On the other hand, the response to laurate and palmitate was variable among animal species. Addition of atropine (1.4 μmol ⋅ l^-1) to the medium did not reduce the responses to octanoate stimulation, but significantly reduced acetylcholineinduced responses, implying that the effects of the fatty acids were not mediated through activation of muscarinic acetylcholine receptors. Reduction of calcium ion concentration in the medium significantly inhibited the responses induced by the fatty acids and acetylcholine, suggesting that amylase release depends on extracellular calcium ions. Accepted: 14 May 1996     

Impact of seropositivity to <Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> and anti-hHSP60 on cardiovascular events in hemodialysis patients

Autoimmunity to heat shock protein 60 (HSP60) has been related to atherosclerosis. Chlamydia pneumoniae (CP), the most studied infectious agent implicated in promoting atherosclerosis, produces a form of HSP60, which can induce an autoimmune response, due to high antigenic homology with human HSP60 (hHSP60). In this study, we evaluated the correlations among anti-hHSP60 antibodies, CP infection, and cardiovascular disease (CVD) in a high-risk population, such as patients undergoing hemodialysis (HD). Thirty-two patients (67.9 ± 13.9 years; male/female, 23:9) on regular HD were enrolled. Global absolute cardiovascular risk (GCR) was assessed using the Italian CUORE Project’s risk charts, which evaluate age, gender, smoking habits, diabetes, systolic blood pressure, and serum cholesterol. The occurrence of cardiovascular events during a 24-month follow-up was recorded. Seropositivity to CP and the presence of anti-hHSP60 antibodies were tested by specific enzyme-linked immunosorbent assays. Inflammation was assessed by measurement of C-reactive protein (CRP) serum levels. Fifteen healthy sex and age-matched (61.9 ± 9.5 years; male/female, 11:4) subjects were the control group. Fifteen of 32 patients resulted seropositive for CP. CP + patients were older than CP−, while they did not differ for GCR, CRP, and dialytic parameters. CVD incidence was significantly higher in CP+ (9 CP+ vs 2 CP−, p < 0.05). Cox analysis recognized that the incidence of CVD was independently correlated with seropositivity to CP (HR, 7.59; p = 0.01; 95% CI = 1.63–35.4). On the other hand, there were no significant differences in anti-hHSP60 levels among CP+, CP− and healthy subjects: 18.11 μg/mL (14.8–47.8), 31.4 μg/mL (23.2–75.3), and 24.72 μg/mL (17.7–41.1), respectively. Anti-hHSP60 did not correlate to GCR, CRP, and incidence of CVD. In conclusion, our data suggest that anti-hHSP60 autoimmune response is not related to CP infection and CP-related CVD risk in HD patients.     

Increased serum HSP70 levels are associated with the duration of diabetes

The evolutionary conserved family of heat shock proteins (HSP) is responsible for protecting cells against different types of stress, including oxidative stress. Although the levels of HSPs can be readily measured in blood serum, the levels of HSP70 in patients with different durations of diabetes have not been studied before. We quantified serum HSP70 levels in a healthy control group (n = 36) and two groups of type 2 diabetic patients, defined as newly diagnosed diabetes (n = 36) and patients with diabetes duration of more than 5 years (n = 37). The clinical characteristics and biochemical parameters were evaluated in the studied population. We found that serum HSP70 levels were significantly higher in patients with diabetes when compared with controls (p < 0.001) and it was higher in patients with disease for more than 5 years than in newly diagnosed patients (p < 0.001). Serum HSP70 was inversely correlated with fasting blood sugar in patients with diabetes for more than 5 years (r = −0.500, p = 0.002), positively correlated with the history of hypertension in newly diagnosed patients (p < 0.001), and positively correlated with age in patients with diabetes (r = 0.531, p = 0.001). Serum level of HSP70 is significantly higher in patients with diabetes and correlates with the duration of disease. Higher HSP70 in prolonged diabetes versus newly diagnosed diabetes may be an indicator of metabolic derangement in the course of diabetes.     

Selenomethionine: an Effective Selenium Source for Sow to Improve Se Distribution,Antioxidant Status,and Growth Performance of Pig Offspring

The present study was to investigate the efficiency of maternal selenomethionine intake on growth performance, Se distribution, and antioxidant status of pig offspring by comparing with sodium selenite. A total of 12 sows (Landrace × Yorkshire) with same pregnancy were randomly divided into two groups; each group was replicated six times. These two groups received the same basal gestation and lactation diets containing 0.04 mg Se/kg, supplemented with 0.3 mg Se/kg sodium selenite and selenomethionine (i.e., seneno-dl-methylseleno), respectively. The feeding trial lasted for 60 days, with 32 and 28 days for gestation and lactation period, respectively. Compared with sodium selenite, maternal selenomethionine intake significantly (p < 0.05) increased the daily weight gain of piglet from birth to weaning. The Se concentration in the colostrum and milk and tissue Se content of piglets were significantly higher (p < 0.05) in the selenomethionine-treated group. The antioxidant status was greatly improved in piglets of selenomethionine-treated group and was illuminated by the increased total antioxidant capability, glutathione peroxidase, superoxide dismutase, and glutathione, and decreased the malondialdehyde level in the organs of piglets. The increased (p < 0.05) triiodothyronine (T₃) and decreased (p < 0.05) thyroxine (T₄) concentration indicated the improved protein synthesis and energy production in the selenomethionine-treated group. The increased (p < 0.05) pancreatic digestive enzymes of protease, amylase, and lipase activities indicated that maternal selenomethionine intake may have a positive effect on the degradation and absorption of nutrients in its piglets. In summary, we concluded that maternal selenomethionine intake increased Se deposition, antioxidant status, and nutrient use efficiency, thus providing an effective way to improve the growth performance of piglets from birth to weaning.     

Octanoate increases cytosolic Ca2+ concentration and membrane conductance in ovine pancreatic acinar cells

 In order to investigate the cellular mechanisms involved in amylase release in response to stimulation with short-chain fatty acids, changes in intracellular calcium concentration ([Ca²⁺]_i), membrane current and amylase release were measured in pancreatic acinar cells of sheep. Both octanoate and acetylcholine raised [Ca²⁺]_i in acinar cells in a concentration-dependent manner. The rise in [Ca²⁺]_i in response to the stimulation with octanoate (10 mmol ⋅ l^-1) was reduced in a medium without CaCl₂, but was markedly enhanced by reintroduction of CaCl₂ into the medium up to 2.56 mmol ⋅ l^-1. Perfusion of the cells with a medium containing octanoate (5 mmol ⋅ l^-1) or acetylcholine (0.5 μmol ⋅ l^-1) immediately raised inward current across the cell membrane at a holding-membrane potential of −30 mV. The inward current became greater as the holding potential became more negative. The equilibrium potential was 1.8 mV and 3.9 mV for octanoate and acetylcholine, respectively, being consistent with that for Cl^-. Although intracellular application of octanoate through a patch-clamp pipette also raised inward current after several minutes in some cells (4 out of 12), this possibility was significantly smaller than that for extracellular application. In other cells, even though the intracellular application of octanoate did not cause an increase in current, it always caused responses immediately after introduction of the fatty acid into the medium. Stimulation with fatty acid as well as acetylcholine raised amylase release in a concentration-dependent manner in cells dispersed from tissue segments with crude collagenase and trypsin inhibitor. Without trypsin inhibitor, crude collagenase significantly and selectively reduced the octanoate (10 mmol ⋅ l^-1)-induced amylase release. Dispersion with crude collagenase and trypsin significantly reduced both responses induced by octanoate and acetylcholine (5.5 μmol ⋅ l^-1). We conclude that fatty acids and acetylcholine increase [Ca²⁺]_i, which consequently evokes a rise in transmembrane ion (Cl^-) conductance and amylase release, and that trypsin-sensitive protein(s) in the cell membrane are involved in secretory processes activated by stimulation with fatty acids in ovine pancreatic acinar cells. Accepted: 14 May 1996     

Altered HepG2 cell models using etomoxir versus <Emphasis Type="Italic">tert</Emphasis>-butylhydroperoxide

Energetic failure which occurs in both ischemia/reperfusion and acute drug-induced hepatotoxicity is frequently associated with oxidative stress. This study displays the setting of a new cell culture model for hepatic energetic failure, i.e., HepG2 models modified by etomoxir [ETO] addition [0.1 mM to 1 mM] and compares the cell impact versus tert-butylhydroperoxide [TBOOH; 0.2 mM], an oxidative stress inducer. As it was observed with Minimum Essential Medium (MEM) without any interfering agent, decreasing temperature drastically lowered adenosine triphosphate (ATP) levels, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability test, and protein content, compared to 37°C (p = 0.02, p < 0.001 and p < 0.001, respectively), but to a larger extent in the presence of ETO or TBOOH. The alteration was generally highly dependent on the ETO concentration, time, and temperature. At 37°C 24 h after (T24h), regarding ETO concentration, R2 correlation ratio was 0.65 (p < 0.001), 0.70 (p < 0.001), and 0.89 (p < 0.001) for ATP levels, protein content, and viability, respectively. The lowest ETO concentration producing a significant effect was 0.25 mM. Concerning time dependency (i.e., T24h versus after 5 h (T5h)), at 37°C with ETO, ATP level continued to significantly decrease between T5h and T24h. In a similar way, at 37°C, the MTT viability test decrease was accelerated only between T5h and T24h for ETO concentrations higher than 0.5 mM (p = 0.016 and p = 0.0001 for 0.75 and 1 mM, respectively). On the contrary, with TBOOH, comparing T24h versus T5h, cellular indicators were improved but generally remained lower than MEM without any interfering agent at T24h, suggesting that TBOOH action was time limited probably in relation with its oxidation in cell medium. This study confirms the interest of altered ETO cell model to screen agents (or formulation) prone to prevent or treat energetic depletion in relation with oxidative stress.     

Increased heat shock protein 70 levels in induced sputum and plasma correlate with severity of asthma patients

Damage-associated molecular pattern molecules such as high-mobility group box 1 protein (HMGB1) and heat shock protein 70 (HSP70) have been implicated in the pathogenesis of asthma. The aim of our study was to examine the induced sputum and plasma concentrations of HSP70 in asthmatic patients to determine their relationship with airway obstruction. Thirty-four healthy controls and 56 patients with persistent bronchial asthma matched for gender and age were enrolled in this study. Spirometry measurements were performed before sputum induction. HSP70 levels in induced sputum and plasma were measured using the ELISA Kit. Sputum and plasma concentrations of HSP70 in asthmatics patients were significantly higher than that in control subjects (sputum, (0.88 ng/ml (0.27–1.88 ng/ml) versus 0.42 ng/ml (0.18–0.85 ng/ml), p < 0.001); plasma, (0.46 ng/ml (0.20–0.98 ng/ml) versus 0.14 ng/ml (0.11–0.37 ng/ml), p < 0.001) and were significantly negatively correlated with forced expiratory volume in 1 s (FEV1), FEV1 (percent predicted), and FEV1/FVC in all 90 participants and 56 patients with asthma. There were no significant differences in HSP70 levels between patients with eosinophilic and non-eosinophilic asthma. HSP70 levels in plasma were positively correlated with neutrophil count, and HSP70 levels in induced sputum were positively correlated with lymphocyte count. In multivariate analysis, independent predictors of sputum HSP70 were diseases and disease severity but not smoking, age, or gender, and independent predictors of plasma HSP70 were also diseases and disease severity. In conclusion, this study indicates that induced sputum and plasma HSP70 could serve as a useful marker for assessing the degree of airway obstruction in patients with asthma. However, further investigation is needed to establish the role of circulating and sputum HSP70 in the pathogenesis of asthma.     

The viability to a wall shear stress and propagation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> in the intensive membrane bioreactor

Bifidobacterium longum grew at 65 L pilot scale of the membrane bioreactor (MBR), externally fitted with ceramic membrane (0.7 m²). Cell mass at the MBR reached 22.18 g L⁻¹ as dry cell weight in 12 h, which is 8.44 times higher than cell mass attained at the vial culture. The growth rate in the vial culture was μ = 0.385 h⁻ and at the batch culture was μ = 1.13 h⁻ in the exponential period and μ = 0.31 h⁻¹ in the stationary period. In the fed-batch mode was μ = 1.102 h⁻¹ for 6 h with inoculation and declined to μ = 0.456 h⁻¹ with feeding of feed medium. The growth rate at the MBR was μ = 0.134 h⁻¹. The number of viable cells was 6.01 × 10¹² cfu L⁻¹ at the batch culture, but increased to 1.15 × 10¹⁴ cfu L⁻¹ at the MBR culture. The specific growth rate of viable cell number (colony-forming units per liter, per hour) improved by 6.01 times from the batch to the MBR culture. The wall shear stress mainly generated by the pump, and the membrane incorporated into the MBR was controlled during the cultivation at the MBR. The viability of B. longum declined to under 10% in the first 2 weeks of the 4-week stability test (40°C) as B. longum was exposed to over wall shear stress 713 Pa, but the viability improved to 30–40% in wall shear stress of 260 Pa or STR culture. The loss in the cell viability can be saved by managing with wall shear stress during the cultivation at the MBR.     

The time-profile of the PBMC HSP70 response to in vitro heat shock appears temperature-dependent

Summary. Heat shock proteins (HSPs) are synthesised by cells subsequent to a stress exposure and are known to confer protection to the cell in response to a second challenge. HSP induction and decay are correlated to thermotolerance and may therefore be used as a biomarker of thermal history. The current study tested the temperature-dependent nature of the heat shock response and characterised its time profile of induction. Whole blood from 6 healthy males (Age: 26 ± (SD) 2 yrs; Body mass 74.2 ± 3.8 kgs; VO_2max: 49.1 ± 4.0 ml·kg⁻¹·min⁻¹) were isolated and exposed to in vitro heat shock (HS) at 37, 38, 39, 40, and 41 °C for a period of 90 min. After HS the temperature was returned to 37 °C and intracellular HSP70 was quantified from the leukocytes at 0, 2, 4, and 6 h after heat treatment. The concentration of HSP70 was not different between temperatures (P > 0.05), but the time-profile of HSP70 synthesis appeared temperature-dependent. At control (37 °C) and lower temperatures (38–39 °C) the mean HSP70 concentration increased up to 4 h post HS (P < 0.05) and then returned towards baseline values by 6 h post HS. With in vitro hyperthermic conditions (40–41 °C), the time-profile was characterised by a sharp rise in HSP70 levels immediately after treatment (P < 0.05 for 40 °C at 0 h), followed by a progressive decline over time. The results suggest a temperature-dependent time-profile of HSP70 synthesis. In addition, the temperature at which HSP70 is inducted might be lower than 37 °C.     

CONTRIBUTION OF CIRCULATING LEUKOCYTES TO CYTOKINE PRODUCTION IN PANCREATIC DUCT OBSTRUCTION-INDUCED ACUTE PANCREATITIS IN RATS

Little information is available regarding the role of circulating leukocytes in the pathogenesis of acute pancreatitis (AP). Our aim was to explore the time-course of the potential role of inflammatory peripheral blood (PB) cells during AP induced in rats by pancreatic duct obstruction (PDO). Flow cytometry immunophenotyping was used to analyse the distribution of the major circulating leukocyte subsets, the activation state of circulating monocytes as reflected by both CD11b expression and TNF-α production and the relative contribution of T-cell derived pro- (TNF-α) and anti- (IL-10) inflammatory mediators at different stages of PDO-induced AP. A progressive increase in PB neutrophils and monocytes was observed up to 6 h after PDO whereas lymphocytes, as well as CD4⁺ and CD8⁺ T-cell subsets, rose as early as 1.5 h after PDO and decreased thereafter. Monocytes were activated in PB from 6 h after inducing AP as reflected by increases in both CD11b expression and spontaneous TNF-α production; nevertheless, they showed the capability of producing TNF-α at earlier AP stages by lipopolysaccharide (LPS) stimulation. In contrast, T-cells were unable to produce TNF-α during AP neither spontaneously nor after stimulation with PMA/Ionomycin. Therefore, only PB monocytes contribute to increase TNF-α levels in plasma as observed from 12 h onwards after inducing AP. Interleukin-10 was produced by T-cells 6 h after PDO only after PMA/Ionomycin stimulation. We conclude that systemic inflammatory events are triggered off at early stages of PDO-induced AP, with the activation of circulating monocytes, though not T-cells, playing a central role.     

The effects of tissue sample size and media on short-term hypothermic preservation of porcine testis tissue

The objective of this study was to develop effective strategies for hypothermic preservation of immature porcine testis tissue to maintain structural integrity and cell viability. In Experiment 1, testes from 1-week-old piglets were used to study the effects of tissue sample size (as intact testes or fragments of 100-or 30 mg) and the use of one of 9 different media on hypothermic preservation of the testis tissue for 6 days. The examined media included: Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM), Leibovitz L15 (L15), L15 with fetal bovine serum (FBS, at 10%, 20% or 50%), HypoThermosol solution-FRS (HTS), Ham’s F12, and Media 199. On days 0, 3, and 6, testis tissues were digested to compare the cell survival rates. Tissue sections were also semi-quantitatively assessed to determine the efficiency of different preservation strategies. There was no effect of testis sample size (P > 0.05), but cell survival rates of testis cells isolated from preserved testis tissues changed depending on the media and day (P < 0.05). Testis tissue within HTS did not show morphological changes after 6 days. In Experiment 2, two of the top performing media (20% FBS-L15 and HTS) were selected for immunocytochemical detection of gonocytes. Proportions of gonocytes (%) in isolated testis cells, however, did not differ between the two media on days 0, 3, or 6. These results show that testis tissue can be maintained for 3 days at 4°C with high cell survival rate, and tissue morphology can be preserved for at least 6 days in HTS.     

Properties of New,Long-Wavelength,Voltage-sensitive Dyes in the Heart

Membrane potential measurements using voltage-sensitive dyes (VSDs) have made important contributions to our understanding of electrophysiological properties of multi-cellular systems. Here, we report the development of long wavelength VSDs designed to record cardiac action potentials (APs) from deeper layers in the heart. The emission spectrum of styryl VSDs was red-shifted by incorporating a thienyl group in the polymethine bridge to lengthen and retain the rigidity of the chromophore. Seven dyes, Pittsburgh I to IV and VI to VIII (PGH I-VIII) were synthesized and characterized with respect to their spectral properties in organic solvents and heart muscles. PGH VSDs exhibited 2 absorption, 2 excitation and 2 voltage-sensitive emission peaks, with large Stokes shifts (> 100 nm). Hearts (rabbit, guinea pig and Rana pipiens) and neurohypophyses (CD-1 mice) were effectively stained by injecting a bolus (10–50 μl) of stock solution of VSD (2–5 mM) dissolved in in dimethylsulfoxide plus low molecular weight Pluronic (16% of L64). Other preparations were better stained with a bolus of VSD (2–5 mM) Tyrode’s solution at pH 6.0. Action spectra measured with a fast CCD camera showed that PGH I exhibited an increase in fractional fluorescence, ΔF/F = 17.5 % per AP at 720 nm with 550 nm excitation and ΔF/F = − 6% per AP at 830 nm with 670 nm excitation. In frog hearts, PGH1 was stable with ∼30% decrease in fluorescence and AP amplitude during 3 h of intermittent excitation or 1 h of continuous high intensity excitation (300 W Xe-Hg Arc lamp), which was attributed to a combination of dye wash out > photobleaching > dynamic damage > run down of the preparation. The long wavelengths, large Stokes shifts, high ΔF/F and low baseline fluorescence make PGH dyes a valuable tool in optical mapping and for simultaneous mapping of APs and intracellular Ca²⁺.