Effects of K-201 on the calcium pump and calcium release channel of rat skeletal muscle

The benzothiazepine derivative K-201 has been suggested as a potential therapeutic agent due to its antiarrhythmogenic action. To understand how the drug alters calcium release from the sarcoplasmic reticulum (SR), we investigated its effects on the SR calcium channel and calcium pump by single channel electrophysiology, whole-cell confocal microscopy, and ATPase activity measurements on control and post-myocardial infarcted (PMI) rat skeletal muscle. In bilayers, K-201 induced two subconductance states corresponding to ∼24% (S₁) and ∼13% (S₂) of the maximum conductance. Dependence of event frequency and of time spent in S₁ and S₂ on the drug concentration was biphasic both in control and in PMI rats, with a maximum at 50 μM. At this concentration, the channel spends 26 ± 4% and 24 ± 4%, respectively, of the total time in these subconductance states at positive potentials, while no subconductances are observed at negative potentials. K-201 altered the frequency of elementary calcium release events: spark frequency decreased from 0.039 ± 0.001 to 0.023 ± 0.001 s⁻¹ sarcomere⁻¹, while the frequency of embers increased from 0.011 ± 0.001 to 0.023 ± 0.001 s⁻¹ sarcomere⁻¹. Embers with different amplitude levels were observed after the addition of the drug. K-201 inhibited the Ca²⁺ ATPase characterized by IC_50,contr = 119 ± 21 μM and n _Hill,contr = 1.84 ± 0.48 for control and IC_50,PMI = 122 ± 18 μM and n _Hill,PMI = 1.97 ± 0.24 for PMI animals. These results suggest that although K-201 would increase the appearance of subconductance states, the overall calcium release is reduced by the drug. In addition, the effect of K-201 is identical on calcium release channels from control and PMI rats.     

Development and characterization of a scalable microperforated device capable of long-term zero order drug release

A drug delivery system that consists of microperforated polyimide microtubes was developed and characterized. Two groups of polyimide tubes were used. One set consisted of microtubes (I.D. = 125 μm) with 32.9 ± 1.7 μm size holes. The second set consisted of larger tubes (I.D. = 1000 μm) with 362–542 μm holes. The number of holes was varied between 1 and 3. The small tubes were loaded with crystal violet (CV) and ethinyl estradiol (EE) and the drug release studies were performed in 0.01 M phosphate buffered saline (PBS) (pH 7.1–7.4) at 37.0 ± 1.0°C for upto 4 weeks. The large tubes were loaded with CV and the drug release was studied in vitro in PBS and also ex vivo in rabbit’s vitreous humor. Linear release rates with R² > 0.9900 were obtained for all groups with CV and EE. Release rates of 7.8 ± 2.5, 16.2 ± 5.5, and 22.5 ± 6.0 ng/day for CV and 30.1 ± 5.8 ng/day for EE were obtained for small tubes. For large tubes, a release rate of 10.8 ± 4.1, 15.8 ± 4.8 and 22.1 ± 6.7 μg/day was observed in vitro in PBS and a release rate of 5.8 ± 1.8 μg/day was observed ex vivo in vitreous humor.     

A novel herbal formulation against dengue vector mosquitoes <Emphasis Type="Italic">Aedes aegypti</Emphasis> and <Emphasis Type="Italic">Aedes albopictus</Emphasis>

The objective of this study was to develop a herbal formulation to control dengue vector mosquitoes. PONNEEM, a novel herbal formulation prepared using the oils of neem (Azadirachta indica), karanj (Pongamia glabra) and their extracts, was tested for larvicidal, ovicidal and oviposition deterrent activities against Aedes aegypti and Aedes albopictus at 1, 0.5, 0.3 and 0.1 ppm concentrations. Cent percent larvicidal and ovicidal activities were observed at 0.1 ppm in the two mosquito species under laboratory and sunlight-exposed conditions up to 12 months from the date of manufacture. Oviposition deterrent activity of 69.97% and 71.05% was observed at 1 ppm concentration of PONNEEM against A. aegypti and A. albopictus, respectively. Reduction in enzyme levels for α-esterase was 0.089 ± 0.008 and 0.099 ± 0.140 μg napthol produced/min/mg larval protein; for β-esterase, it was 0.004 ± 0.009 and 0.001 ± 0.028 μg napthol produced/min/mg larval protein; for glutathione S-transferase, it was 10.4814 ± 0.23 and 11.4811 ± 0.21 μmol/min/mg larval protein and for total protein, it was 0.177 ± 0.010 and 0.008 ± 0.005 mg/individual larva in treated groups of A. aegypti and A. albopictus, respectively. The nontarget organisms such as Gambusia affinis and Diplonychus indicus were not affected. No mortality was observed in control. PONNEEM can be used effectively for the management of human vector mosquitoes.     

Nonhypotensive dose of β-adrenergic blocker ameliorates capillary deficits in the hearts of rats with moderate renal failure

Renal failure causes sympathetic overactivity and inadequate capillary growth in response to cardiomyocyte hypertrophy in experimental renal failure, as well as in uremic patients. In nonuremic animals, sympathetic overactivity was shown to suppress capillary growth. The purpose of this study was to examine whether blockade with α- and β-adrenoblockers ameliorates the capillary deficit that was documented in the hearts of rats with moderate renal failure. Male Sprague–Dawley rats, 3 days after surgical ablation [subtotal nephrectomy (SNX)] or sham operation (sham), were treated with phenoxybenzamine, metoprolol, or a combination of both: After 12 weeks, the hearts were investigated using morphometric and stereologic techniques. The length density of myocardial capillaries was lower (p<0.05) in untreated SNX than in sham (2,786±372 vs 3,397±602 mm/mm³); the decrease was abrogated by metoprolol (3,305±624 mm/mm³), but not by phenoxybenzamin (2,628±480 mm/mm³). The intercapillary distance increased (p<0.05) in SNX (20.5±1.5 μm) and tended to be lower after metoprolol treatment (19.0±1.9 μm). The media area of intramyocardial arterioles was significantly higher in untreated SNX (1,158±1,343 vs 686±771 μm² in sham). Metoprolol in nonhypotensive doses prevents the capillary deficit in the hearts of rats with moderate renal failure and presents an argument for an important role of sympathetic overactivity in the genesis of the capillary deficit in moderate chronic renal insufficiency.     

Transmural Oxygen Tension Gradients in Rat Cerebral Cortex Arterioles

Modified needle oxygen microelectrodes and vital microscopy were used to measure transmural oxygen tension gradients (PO₂) in pial arterioles with lumen diameters of 20–90  μm. A relationship between the magnitude of the transmural PO₂ gradient and arteriole wall tone was found: in control conditions, PO₂ gradients were 1.17 ± 0.06 mmHg/μm (n  = 40), while in conditions of arteriolar wall dilation the transmural PO₂ gradient decreased to 0.68 ± 0.04 mmHg/μm (p  <  0.001, n  = 38). These data provide the first measurements of transmural PO₂ gradients in pial arterioles of different calibers at different levels of vascular tone and have fundamental importance for assessing the role of arterial microvessels in tissue oxygen supply processes. The results obtained here provide evidence that oxygen consumption by the vessel wall is within the range characteristic of enveloping tissues and that oxygen consumption by the endothelial cell layer probably has no significant effect on the magnitude of the transmural PO₂ gradient. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 94, No. 4, pp. 394–405, April, 2008.     

Epac activation, altered calcium homeostasis and ventricular arrhythmogenesis in the murine heart

The recently described exchange protein directly activated by cAMP (Epac) has been implicated in distinct protein kinase A-independent cellular signalling pathways. We investigated the role of Epac activation in adrenergically mediated ventricular arrhythmogenesis. In contrast to observations in control conditions (n = 20), monophasic action potentials recorded in 2 of 10 intrinsically beating and 5 of 20 extrinsically paced Langendorff-perfused wild-type murine hearts perfused with the Epac activator 8-pCPT-2′-O-Me-cAMP (8-CPT, 1 μM) showed spontaneous triggered activity. Three of 20 such extrinsically paced hearts showed spontaneous ventricular tachycardia (VT). Programmed electrical stimulation provoked VT in 10 of 20 similarly treated hearts (P < 0.001; n = 20). However, there were no statistically significant accompanying changes (P > 0.05) in left ventricular epicardial (40.7 ± 1.2 versus 44.0 ± 1.7 ms; n = 10) or endocardial action potential durations (APD₉₀; 51.8 ± 2.3 versus 51.9 ± 2.2 ms; n = 10), transmural (ΔAPD₉₀) (11.1 ± 2.6 versus 7.9 ± 2.8 ms; n = 10) or apico-basal repolarisation gradients, ventricular effective refractory periods (29.1 ± 1.7 versus 31.2 ± 2.4 ms in control and 8-CPT-treated hearts, respectively; n = 10) and APD₉₀ restitution characteristics. Nevertheless, fluorescence imaging of cytosolic Ca²⁺ levels demonstrated abnormal Ca²⁺ homeostasis in paced and resting isolated ventricular myocytes. Epac activation using isoproterenol in the presence of H-89 was also arrhythmogenic and similarly altered cellular Ca²⁺ homeostasis. Epac-dependent effects were reduced by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibition with 1 μM KN-93. These findings associate VT in an intact cardiac preparation with altered cellular Ca²⁺ homeostasis and Epac activation for the first time, in the absence of altered repolarisation gradients previously implicated in reentrant arrhythmias through a mechanism dependent on CaMKII activity.     

Activation of big conductance Ca2+-activated K+ channels (BK) protects the heart against ischemia–reperfusion injury

Activation of the large-conductance Ca²⁺-activated K⁺ channel (BK) in the cardiac inner mitochondrial membrane has been suggested to protect the heart against ischemic injury. However, these findings are limited by the low selectivity profile and potency of the BK channel activator (NS1619) used. In the present study, we address the cardioprotective role of BK channels using a novel, potent, selective, and chemically unrelated BK channel activator, NS11021. Using electrophysiological recordings of heterologously expressed channels, NS11021 was found to activate BK α + β1 channel complexes, while producing no effect on cardiac K_ATP channels. The cardioprotective effects of NS11021-induced BK channel activation were studied in isolated, perfused rat hearts subjected to 35 min of global ischemia followed by 120 min of reperfusion. 3 μM NS11021 applied prior to ischemia or at the onset of reperfusion significantly reduced the infarct size [control: 44.6 ± 2.0%; NS11021: 11.4 ± 2.0%; NS11021 at reperfusion: 19.8 ± 3.3% (p < 0.001 for both treatments compared to control)] and promoted recovery of myocardial performance. Co-administration of the BK-channel inhibitor paxilline (3 μM) antagonized the protective effect. These findings suggest that tissue damage induced by ischemia and reperfusion can be reduced by activation of cardiac BK channels.     

Comparative Cell Shape and Diffusional Water Permeability of Red Blood Cells from Indian Elephant (Elephas maximus) and Man (Homo sapiens)

Red blood cells (RBC) from an Indian elephant (Elephas maximus) were studied by light microscopy (LM), scanning electron microscopy (SEM) and a new nuclear magnetic resonance (NMR) ‘imaging’ method based on the translational diffusion of water, NMR q-space analysis. Also, the transmembrane diffusional permeability, P _d of water in RBC was measured by using a Mn²⁺-doping NMR technique, taking human RBC as a reference. The main diameter of the elephant RBC was measured as 9.3 ± 0.7 μm by LM, 9.3 ± 0.7 μm by ‘shrinkage-corrected’ SEM, and 9.3 ± 0.4 μm by q-space anlaysis. The value is ∼1.4 μm larger than that for the human RBC. The values of P _d were, in the case of elephant RBC, 3.2 × 10⁻³ cm/s at 25 °C, 3.9 × 10⁻³ cm/s at 30 °C, 5.2 × 10⁻³ cm/s at 37 °C and 6.5 × 10⁻³ cm/s at 42 °C; all values were significantly lower than the corresponding values of P _d for human RBC, namely 4.3 × 10⁻³ cm/s at 25 °C, 5.2 × 10⁻³ cm/s at 30 °C, 6.1 × 10⁻³ cm/s at 37 °C, 7.8 × 10⁻³ cm/s at 42°C. The maximal inhibition of P _d (56%) was reached in 30 min at 37 °C with 2 mm p-chloromercuribenzene sulphonate (PCMBS) for both species of RBC. The basal permeability to water at 37 °C was estimated to be 2.3 × 10⁻³ cm/s for elephant and 2.6 × 10⁻³ cm/s for human RBC. The values of the activation energy for water permeability (E _a,d ) was significantly higher for elephant RBC (31.9 kJ/mol) than for human RBC (25.9 kJ/mol). This indicated that features other than the number of transporters per cell are likely to be important in defining the differences in water permeability in the RBC from the two species.     

Aldosterone-induced changes in the cardiac L-type Ca2+ current can be prevented by antioxidants in vitro and are absent in rats on low salt diet

Mineralocorticoid receptor (MR) activation modulates cardiac L-type Ca²⁺ current (I _CaL) and transient outward K⁺ current (I _to). The exact circumstances of MR activation, however, remain elusive. Here, we investigate the influence of corticosteroids on MR-mediated changes in cellular electrophysiology. In vitro incubation of adult rat ventricular myocytes with the MR agonist aldosterone (100 nM, 24 h) increased I _CaL density by 34% (n = 16; p < 0.01). This effect was abrogated by co-incubation with the MR antagonist spironolactone (10 μM). To investigate whether an increase in serum aldosterone concentration is sufficient for an increase in I _CaL in vivo, rats were subjected to low Na⁺ diet (LSD, 0.013% Na⁺) for 28 days. This increased serum aldosterone concentration from 0.19 ± 0.04 nM (n = 6) in control animals (0.3% Na⁺, CSD) to 16.1 ± 2.1 nM (n = 6; p < 0.0001). Strikingly, I _CaL density was similar in both CSD and LSD rats (−12.9 ± 0.9 pA pF⁻¹, n = 18 and −13.7 ± 1.1 pA pF⁻¹, n = 16, respectively), as was I _to density. In vitro, the glucocorticoid corticosterone (1 μM) also increased I _CaL and this effect was blocked by spironolactone (10 μM). Co-incubation with corticosterone (1 μM, the normal serum concentration) and aldosterone (100 nM, mimicking low Na⁺ intake) did not further increase I _CaL compared to corticosterone alone. Moreover, co-incubation of myocytes with N-acetylcysteine (10 mM) prevented the aldosterone (100 nM) or corticosterone (1 μM)-induced increase in I _CaL. In conclusion, an increase in serum aldosterone concentration in response to LSD is not sufficient for an increase in I _CaL density in cardiomyocytes in vivo. This is supported in vitro by the absence of an effect of aldosterone on I _CaL in the presence of a physiological concentration of corticosterone. Moreover, the cellular redox state may modulate MR activation. Michael Wagner and Elena Rudakova contributed equally to this work.     

Comparative Cell Shape and Diffusional Water Permeability of Red Blood Cells from Indian Elephant ( Elephas maximus ) and Man ( Homo sapiens )

Red blood cells (RBC) from an Indian elephant (Elephas maximus) were studied by light microscopy (LM), scanning electron microscopy (SEM) and a new nuclear magnetic resonance (NMR) ‘imaging’ method based on the translational diffusion of water, NMR q-space analysis. Also, the transmembrane diffusional permeability, P _d of water in RBC was measured by using a Mn²⁺-doping NMR technique, taking human RBC as a reference. The main diameter of the elephant RBC was measured as 9.3 ± 0.7 μm by LM, 9.3 ± 0.7 μm by ‘shrinkage-corrected’ SEM, and 9.3 ± 0.4 μm by q-space anlaysis. The value is ∼1.4 μm larger than that for the human RBC. The values of P _d were, in the case of elephant RBC, 3.2 × 10⁻³ cm/s at 25 °C, 3.9 × 10⁻³ cm/s at 30 °C, 5.2 × 10⁻³ cm/s at 37 °C and 6.5 × 10⁻³ cm/s at 42 °C; all values were significantly lower than the corresponding values of P _d for human RBC, namely 4.3 × 10⁻³ cm/s at 25 °C, 5.2 × 10⁻³ cm/s at 30 °C, 6.1 × 10⁻³ cm/s at 37 °C, 7.8 × 10⁻³ cm/s at 42°C. The maximal inhibition of P _d (56%) was reached in 30 min at 37 °C with 2 mm p-chloromercuribenzene sulphonate (PCMBS) for both species of RBC. The basal permeability to water at 37 °C was estimated to be 2.3 × 10⁻³ cm/s for elephant and 2.6 × 10⁻³ cm/s for human RBC. The values of the activation energy for water permeability (E _a,d ) was significantly higher for elephant RBC (31.9 kJ/mol) than for human RBC (25.9 kJ/mol). This indicated that features other than the number of transporters per cell are likely to be important in defining the differences in water permeability in the RBC from the two species.     

In vitro antifilarial activity of glutathione S-transferase inhibitors

Female adult bovine filarial worms Setaria digitata were extracted with phosphate-buffered saline (pH 7.4) and glutathione S-transferase (GST) activity and protein content were determined. The protein content, GST enzyme activity, and specific activity were 10.61 ± 3.41 mg ml⁻¹, 0.09 ± 0.019 μmol min⁻¹ ml⁻¹, and 0.009 ± 0.002 μmol min⁻¹ mg⁻¹ protein, respectively. The GST inhibition studies were performed with and without the inhibitors resulted from earlier molecular docking studies viz., ethacrynic acid, plumbagin, and curcumin for which the IC₅₀ values were 19.42, 51.41, and 114.86 μM, respectively. The in vitro macrofilaricidal activity of these molecules was studied by worm motility and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay at 24- and 48-h incubation. Plumbagin and ethacrynic acid showed 100% inhibition in worm motility at lower concentrations of 3.19 and 6.6 μM, respectively, at 48-h incubation while curcumin was effective at 54.29 μM. In MTT reduction assay, the ED₅₀ values (50% inhibition in formazan formation) for plumbagin, ethacrynic acid, and curcumin at 48-h incubation were 1.20, 2.48, and 19.86 μM, respectively. MTT reduction assay showed that plumbagin was the most effective in killing the adult S. digitata worms followed by ethacrynic acid and curcumin. In conclusion, all the three molecules selected by molecular modeling and docking studies inhibited the GST enzyme isolated from S. digitata and exhibited macrofilaricidal activity in vitro.     

Insecticidal, acaricidal and repellent effects of DEET- and IR3535-impregnated bed nets using a novel long-lasting polymer-coating technique

A novel long-lasting repellent-treated net (LLRTN) has been designed by binding the skin repellents N,N-diethyl-m-toluamide (DEET), or IR3535, onto the fibres of bed net fabric using a new polymer-coating technique. The repellent toxicological effectiveness and residual activity of a factory-based repellent-impregnated fabric has been evaluated by laboratory testing against adult Aedes aegypti mosquitoes and nymphal Ixodes ricinus ticks. By using this repellent-embedding impregnation technique, concentrations exceeding 10 g/m² could be achieved with one single polymer layer. Both DEET- and IR3535-impregnated fabrics revealed a dose-dependent insecticidal as well as acaricidal activity. One hundred percent knockdown times of DEET-treated bed nets ranged from 187.5 ± 31.8 to 27.5 ± 3.5 min against A. aegypti, and between 214 ± 47 and 22.6 ± 5 min against nymphal I. ricinus, linked to a DEET concentration of 1.08 and 10.58 g/m², respectively. With IR3535, A. aegypti produced dose-dependent 100% knockdown times varying from 87.5 ± 10.6 to 57.5 ± 3.5 min and between 131.4 ± 6.5 and 33.8 ± 5 min against nymphal I. ricinus, respectively, linked to concentrations between 1.59 and 10.02 g/m². One hundred percent repellency measured by complete landing and biting protection of impregnated fabric by using the arm-in-cage test could be achieved at DEET concentrations exceeding 3.7 to 3.9 g/m², and for IR3535 concentrations over 10 g/m². One hundred percent landing and biting protection could be preserved with DEET-treated fabrics for 29 weeks at an initial concentration of 4.66 g/m², 54 weeks at 8.8 g/m², 58 weeks at 9.96 g/m² and 61 weeks at 10.48 g/m² for DEET, and 23 weeks for IR3535-treated fabric at a concentration of 10.02 g/m². Unlike repellent-treated fabric, a brand of a commercially available long-lasting insecticide-treated net tested containing 500 mg permethrin/m² did not protect from mosquito bites. First results on bioactivity and long-lasting efficacy show that the new LLRTN technique is highly promising as a potential candidate for future malaria control strategies, especially in areas where pyrethroid resistance occurs.     

Angiogenesis in triple-negative adenoid cystic carcinomas of the breast

We compared microvascular density (MVD), lymph vessel density (LVD), and the expression of hypoxia pathway-associated proteins between primary triple-negative adenoid cystic carcinoma of the breast (TN-ACC) and grade-matched triple-negative breast carcinomas of no special type (TNBC). Twelve TN-ACC and 15 TNBC were investigated immunohistochemically for CD31, podoplanin (D2-40), von Hippel–Lindau protein (pVHL), and hypoxia-inducible factor-1alpha (HIF-1α) protein. All cases were lymph node negative (pN0). The study revealed a median MVD (CD31) of 34 vessels/mm² (mean ± SD, 41.33 ± 6.5/mm²) in the TN-ACC subgroup and a median of 55 microvessels (mean ± SD, 54.9 ± 6.3/mm²) in the TNBC subgroup. The median LVD (D2-40) was 10.5/mm² (mean ± SD, 11.9 ± 1.5/mm²) in the TN-ACC subgroup and 15.0/mm² (mean ± SD, 16.9 ± 2.5/mm²) lymph vessels in the TNBC subgroup. The differences were not statistically significant (P = 0.93, P = 0.67, respectively). pVHL was detectable in all TN-ACCs whereas two cases of TNBC had less than 5% of the positive cells. HIF-1α protein expression was significantly higher in the tumor cell population than in adjacent normal cells in both subgroups (P = 0.009 for TNBC and P = 0.028 for TN-ACC, respectively), but there was no significant difference between the two tumor groups. Up-regulation of the hypoxia-induced signaling is seen in both TN-ACC and grade-matched TNBC. Despite its perceived low malignant potential, TN-ACC of the breast does not differ in the number of blood and lymphatic vessels in comparison with the grade-matched TNBC. The reported biologic differences between TN-ACC and TNBC do not appear to result from neoangiogenesis.     

Tamoxifen treatment of myocardial infarcted female rats exacerbates scar formation

Hormonal replacement therapy in postmenopausal women was associated with an increased incidence of nonfatal myocardial infarction. Selective estrogen receptor modulators were considered an alternative pharmacological approach. However, selective estrogen receptor modulators acting via estrogen receptor-dependent and receptor-independent mechanisms may negatively influence cardiac remodeling. The present study tested the hypothesis that tamoxifen (TAM) treatment after coronary artery ligation compromised scar formation. TAM administration (10 mg kg⁻¹ day⁻¹ for 3 weeks) to postmyocardial infarcted (MI) female adult rats significantly increased scar surface area (TAM+MI = 0.67 ± 0.08 vs MI = 0.45 ± 0.06 cm²) and weight (TAM+MI = 0.071 ± 0.007 vs MI = 0.050 ± 0.006 grams). In the infarct region, a significant decrease (p < 0.05) of small calibre vessels (lumen diameter <50 μm) was observed in TAM treated post-MI rats (4.5 ± 0.8 vessels/mm²), as compared to untreated MI rats (7 ± 0.7 vessels/mm²). Consistent with the latter finding, 4-OH TAM caused a dose-dependent suppression of vascular endothelial growth factor (VEGF)-stimulated (10⁻⁹ mol/l) capillarity-like tubule formation by rat aortic endothelial cells in vitro via an estrogen receptor-independent mechanism. These data have demonstrated that TAM treatment of post-MI female rats exacerbated scar formation and may have occurred at least in part via the attenuation of new vessel formation in the infarct region.     

Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens

In the field of continuous-flow PCR, the amplification throughput in a single reaction solution is low and the single-plex PCR is often used. In this work, we reported a flow-based multiplex PCR microfluidic system capable of performing high-throughput and fast DNA amplification for detection of foodborne bacterial pathogens. As a demonstration, the mixture of DNA targets associated with three different foodborne pathogens was included in a single PCR solution. Then, the solution flowed through microchannels incorporated onto three temperature zones in an oscillatory manner. The effect factors of this oscillatory-flow multiplex PCR thermocycling have been demonstrated, including effects of polymerase concentration, cycling times, number of cycles, and DNA template concentration. The experimental results have shown that the oscillatory-flow multiplex PCR, with a volume of only 5 μl, could be completed in about 13 min after 35 cycles (25 cycles) at 100 μl/min (70 μl/min), which is about one-sixth of the time required on the conventional machine (70 min). By using the presently designed DNA sample model, the minimum target concentration that could be detected at 30 μl/min was 9.8 × 10⁻² ng/μl (278-bp, S. enterica), 11.2 × 10⁻² ng/μl (168-bp, E. coli O157: H7), and 2.88 × 10⁻² ng/μl (106-bp, L. monocytogenes), which corresponds to approximately 3.72 × 10⁴ copies/μl, 3.58 × 10⁴ copies/μl, and 1.79 × 10⁴ copies/μl, respectively. This level of speed and sensitivity is comparable to that achievable in most other continuous-flow PCR systems. In addition, the four individual channels were used to achieve multi-target PCR analysis of three different DNA samples from different food sources in parallel, thereby achieving another level of multiplexing.     

Microsporidian parasites: a danger facing marine fishes of the Red Sea

Out of 600 marine fish from the Red Sea belonging to three different species that were collected and examined for microsporidian parasites, 87 (14.5%) fish were found to be infected. The infection was recorded as cysts or xenomas embedded in the gut epithelium and the peritoneal cavity of the three fish species. The highest percent of infection with microsporidian parasites was recorded in Saurida tumbil 19.5% (39/200) followed by Pagrus pagrus 15% (45/300) and the lowest percent of infection was recorded in Epinephelus chlorostigma 3% (three out of 100). After rupture of the cysts, the spores were released and examined by light microscopy. Each spore was elongated to ellipsoidal in shape and possessed a posterior vacuole which is characteristic to phylum Microspora. They measure 1.6 ± 0.5 μm (1.5–2.4 μm) × 1.3 ± 0.1 μm (1.3–2.0 μm) in Saurida tumbil and Pagrus pagrus, respectively. The spores of Pleistophora sp recorded from E. chlorostigma were ovoid to pyriform in shape and measure 1.9 ± 0.5 μm (1.8–2.7 μm) × 1.6 ± 0.4 μm (1.5–2.4 μm).     

Trisk 32 regulates IP(3) receptors in rat skeletal myoblasts

To date, four isoforms of triadins have been identified in rat skeletal muscle. While the function of the 95-kDa isoform in excitation–contraction coupling has been studied in detail, the role of the 32-kDa isoform (Trisk 32) remains elusive. Here, Trisk 32 overexpression was carried out by stable transfection in L6.G8 myoblasts. Co-localization of Trisk 32 and IP₃ receptors (IP₃R) was demonstrated by immunocytochemistry, and their association was shown by co-immunoprecipitation. Functional effects of Trisk 32 on IP₃-mediated Ca²⁺ release were assessed by measuring changes in [Ca²⁺]_i following the stimulation by bradykinin or vasopressin. The amplitude of the Ca²⁺ transients evoked by 20 μM bradykinin was significantly higher in Trisk 32-overexpressing (p < 0.01; 426 ± 84 nM, n = 27) as compared to control cells (76 ± 12 nM, n = 23). The difference remained significant (p < 0.02; 217 ± 41 nM, n = 21, and 97 ± 29 nM, n = 31, respectively) in the absence of extracellular Ca²⁺. Similar observations were made when 0.1 μM vasopressin was used to initiate Ca²⁺ release. Possible involvement of the ryanodine receptors (RyR) in these processes was excluded, after functional and biochemical experiments. Furthermore, Trisk 32 overexpression had no effect on store-operated Ca²⁺ entry, despite a decrease in the expression of STIM1. These results suggest that neither the increased activity of RyR, nor the amplification of SOCE, is responsible for the differences observed in bradykinin- or vasopressin-evoked Ca²⁺ transients; rather, they were due to the enhanced activity of IP₃R. Thus, Trisk 32 not only co-localizes with, but directly contributes to, the regulation of Ca²⁺ release via IP₃R.     

Effect of praziquantel prolonged administration on granuloma formation around Schistosoma japonicum eggs in lung of sensitized mice

Schistosomiasis remains a major public health problem and it is an immune disease. The schistosome egg is the primary parasite factor responsible for the overt disease. The eggs release the soluble antigen, which induces intensive tissue reaction, a granulomatous reaction to the eggs. If granuloma formation could be suppressed, overt disease might not develop. Praziquantel is an effective antischistosomal drug especially for adult worms. However, whether praziquantel has a suppressing effect on granuloma formation around schistosome eggs directly remains unclear. The purpose of the present study was to investigate the effect of praziquantel, especially administered persistently, on granuloma formation around Schistosoma japonicum eggs in the lung of sensitized mice. Thirty-six mice were divided into three groups averagely. Group A was a control group. First, the mice were injected with schistosomal eggs hypodermically in abdomen, and 10 days later injected with schistosomal eggs intravenously via a tail vein. Group B was a praziquantel short administration group. In addition to the injections of schistosomal eggs as the same of Group A, the mice were administered with praziquantel in a daily dose of 300 mg/kg for 3 days, from 1 day before the intravenous injection of the eggs. Group C was a praziquantel prolonged administration group. In addition to the injections of schistosomal eggs as the same of Group A, the mice were administered with praziquantel in a daily dose of 150 mg/kg for 5 days weekly until the mice were sacrificed. Three mice of each group were sacrificed on days 7, 14, 28, and 56, respectively after the intravenous injection of the eggs, and the lung tissues were fixed with formalin and the slices were HE stained. The granulomas containing eggs in their centers were selected, and 25–30 granulomas from the animals of each group were measured at each time period. The mean areas of egg granulomas of each group were calculated, and the neutrophilic granulocytes, eosinocytes, lymphocytes, fibroblasts, and macrophages within the egg granulomas were counted. The mean numbers of them of each group were calculated. All the data of each group were analyzed and compared statistically. On day 56 after the intravenous injection of the eggs, the mean area of schistosomal egg granulomas in group B was (227.4 ± 728.0) × 10³ μm², less than that of [(297.9 ± 153.3) × 10³ μm²] in group A, and the suppression rate was 23.7% (P < 0.05). On days 7, 14, 28, and 56, the mean areas of schistosomal egg granulomas in group C were (575.8 ± 155.6) × 10³ μm², (310.5 ± 854.0) × 10³ μm², (267.7 ± 513.3) × 10³ μm², and (214.9 ± 446.4) × 10³ μm², respectively, significantly less than those of [(692.7 ± 232.6) × 10³ μm², (439.4 ± 165.0) × 10³ μm², (385.7 ± 129.3) × 10³ μm², and (297.9 ± 153.3) × 10³ μm²] in group A. The suppression rates were 16.9%, 29.3%, 30.6%, and 27.9%, respectively (P values <0.05). On day 56, the mean numbers of neutrophilic granulocytes were 11.4 ± 5.0 in group A and 5.2 ± 3.1 in group C, respectively, with the suppression rate of 54.4% in group C (P < 0.05). On day 56, the mean numbers of eosinocytes within the egg granulomas were 2.3 ± 2.0, 0.1 ± 0.3, and 0.3 ± 0.6 in groups A, B, and C, respectively, with the suppression rate of 95.7% in group B and 87.0% in group C (P values <0.05). On day 56, the mean numbers of macrophages within egg granulomas were 14.3 ± 6.9 in group C, compared with 18.6 ± 8.2 in group A, the suppression rate was 23.1% (P < 0.05). On day 56, the mean numbers of fibroblasts within the egg granulomas were 6.6 ± 4.4 and 5.8 ± 2.6 in groups B and C, respectively, and compared with 14.3 ± 7.8 in group A, the increasing extents decreased by 53.8% and 59.4%, respectively (P values <0.05). Therefore, the administration of praziquantel, especially the prolonged administration, can suppress the formation of schistosomal egg granulomas, including reduction in the areas of granulomas and suppression of the inflammatory cells and the hyperplasia of fibroblasts within granulomas.     

Afferent arteriolopathy and glomerular collapse but not segmental sclerosis induce tubular atrophy in old spontaneously hypertensive rats

In chronic renal disease, the temporal and spatial relationship between vascular, glomerular and tubular changes is still unclear. Hypertension, an important cause of chronic renal failure, leads to afferent arteriolopathy, segmental glomerulosclerosis and tubular atrophy in the juxtamedullary cortex. We investigated the pathological changes of hypertensive renal disease in aged spontaneously hypertensive rats using a large number of serial sections, where we traced and analyzed afferent arteriole, glomerulus and proximal tubule of single nephrons. Our major finding was that both afferent arteriolopathy and glomerular capillary collapse were linked to tubular atrophy. Only nephrons with glomerular collapse (n = 13) showed tubules with reduced diameter indicating atrophy [21.66 ± 2.56 μm vs. tubules in normotensive Wistar Kyoto rats (WKY) 38.56 ± 0.56 μm, p < 0.05], as well as afferent arteriolar wall hypertrophy (diameter 32.74 ± 4.72 μm vs. afferent arterioles in WKY 19.24 ± 0.98 μm, p < 0.05). Nephrons with segmental sclerosis (n = 10) did not show tubular atrophy and tubular diameters were unchanged (35.60 ± 1.43 μm). Afferent arteriolar diameter negatively correlated with glomerular capillary volume fraction (r = −0.36) and proximal tubular diameter (r = −0.46) implying reduced glomerular and tubular flow. In line with this, chronically damaged tubules showed reduced staining for the ciliary protein inversin indicating changed ciliary signalling due to reduced urinary flow. This is the first morphological study on hypertensive renal disease making correlations between vascular, glomerular and tubular components of individual nephron units. Our data suggest that afferent arteriolopathy leads to glomerular collapse and reduced urinary flow with subsequent tubular atrophy.