Use of the green fluorescent protein to rapidly assess viability of E. coli in preserved solutions

E. coli strain HB101 was genetically engineered to a fluorescent phenotype by transformation with a plasmid containing complementary DNA for a green fluorescent protein. The level of fluorescence in the transformed strain was directly proportional to the number of viable cells. There was a rapid decrease in fluorescence when transformed cells were inoculated into lamivudine solutions containing ten different preservative formulations. The decrease in fluorescence correlated to a decrease in the number of viable cells, allowing the relative antimicrobial properties of each solution to be compared. This methods provides a simple, rapid (< 2 min/assay), and accurate means of determining the effects of antimicrobial solutions on the viability of E. coli.     

Use of an autobioluminescent Salmonella hadar to monitor the effects of acid and temperature treatments on cell survival and viability on lactic acid-treated poultry carcasses

To determine the long-term effects of a lactic acid rinse on viability and recovery of pathogens, Salmonella Hadar was isolated from poultry and bioluminescent constructs obtained by transformation with the lux (CDABE) gene cassette from Photobacterium phosphoreum. Results indicated that the transformed Salmonella Hadar lux was otherwise phenotypically similar to the wild-type strain. Viability studies were performed by measuring luminescence following lactic acid treatment of turkey breast and subsequent storage at -12, 0, 5, and 10 degrees C. The ability of the S. Hadar lux strain to recover was determined by monitoring light output after incubation at 22 degrees C for 10 h. The results showed that metabolic activity was significantly (P < 0.05) affected by lactic acid and by storage temperatures of -12, 0, and 5 degrees C. The lowest recovery rate was observed after rinsing with lactic acid and storing at 5 degrees C. The study demonstrated that bacterial bioluminescence is an effective way of monitoring in "real time" the ability of bacteria to recover from stress.     

Acute effects of cell isolation on InsP profiles in adult rat cardiomyocytes

The isolation and culture of adult rat cardiomyocytes was shown to cause major changes in the contents of [3H]-labeled inositol phosphates and inositol phospholipids. Undigested heart tissue contained high levels of [3H]Ins(1,4,5)P3 (5364+/-800 ct/min/g tissue, 80+/-12 ct/min/mg protein) and mass content averaged 13.8 nmol/g tissue or 208+/-36 pmol/mg protein (mean+/-S.E.M., n=4). After collagenase digestion, [3H]Ins(1,4,5)P3 was undetectable and the mass content of Ins(1,4,5)P3 had decreased to 0.8+/-0.2 pmol/mg protein (mean+/-S.E.M., n=4, P<0.01). [3H]Ins(1,4)P2 was reduced by 80% and [3H]PtdIns(4,5)P2 by 90%. These profiles remained essentially unchanged when the isolated cells were maintained in culture for up to 24 h, even though the inositol phosphate response remained sensitive to norepinephrine. Similar to findings in intact tissue, the inositol phosphate response to norepinephrine in these cells was inhibited by neither U-73122 (5 microM) nor by neomycin (5 mM). By 48 h in culture, the relative levels of [3H]Ins(1,4,5)P3 and [3H]Ins(1,4)P2 had increased in relation to the total inositol phosphate content and responses appeared to better reflect intact tissue. However, while retaining insensitivity to neomycin, cells at 48 h were fully sensitive to U-73122 (5 microM). These data demonstrate that altered inositol phosphate responses are observed in adult cardiomyocytes from the time of isolation and that while the profiles change over time in culture, a pattern similar to that in intact heart is not re-established.     

Use of pulsed Doppler tissue imaging to assess regional left ventricular diastolic dysfunction in hypertrophic cardiomyopathy

In this study, regional diastolic patterns and their relations with transmitral Doppler inflow were investigated in hypertrophic cardiomyopathy (HC) by pulsed Doppler tissue imaging (DTI). Doppler echocardiography and DTI of basal septum and lateral wall (apical 4-chamber view) were performed in 20 patients (15 men and 5 women) with HC and in 10 healthy subjects (7 men and 3 women). Diabetes, hypertension, coronary artery and valvular disease, mitral regurgitation, New York Heart Association functional classes III to IV, sinus tachycardia, atrial fibrillation, and inadequate echocardiograms were exclusion criteria. Peak velocity and time-velocity integral of early and late waves and their ratios, and deceleration and isovolumic relaxation times were determined by standard Doppler and by DTI at the septal and lateral wall levels. The 2 groups were comparable for age, heart rate, blood pressure, and ejection fraction. Transmitral peak velocity and time-velocity integral E/A ratios were reduced (both p <0.05) and deceleration and isovolumic relaxation times prolonged (both p <0.00001) in HC. Septal DTI showed lower peak velocity and time-velocity integral e/a ratios (p <0.00001 and p <0.001, respectively) and lengthened regional deceleration (p <0.01) and isovolumic (p <0.001) relaxation times. DTI of the lateral wall showed a prolongation of deceleration and isovolumic relaxation times (both p <0.01). By dividing HC according to transmitral E/A, 8 patients with E/A <1 had lower DTI septal e/a ratio (p <0.01) and prolonged septal deceleration and isovolumic relaxation times (both p <0.01) but no changes in DTI pattern of lateral wall than 12 patients with E/A > 1. In conclusion, DTI is useful and complementary to standard Doppler imaging to characterize diastolic properties in HC, reflecting a typical pattern of intramyocardial impaired relaxation at the level of hypertrophied septum and also providing information about the degree of this regional impairment. The lateral wall presents minor changes in diastolic times, which indicate how diastolic asynchrony is not confined to the hypertrophied segment in HC.     

Use of nonlinear methods to assess effects of clonidine on blood pressure in spontaneously hypertensive rats

In spontaneously hypertensive rats (SHR), chronic infusion of clonidine failed to decrease blood pressure and blood pressure variability. We used nonlinear methods to get a deeper insight on the effects of clonidine on blood pressure dynamics. For 24 h and 4 wk, clonidine (0.1 mg . kg-1 . day-1 sc) was infused by minipumps in the conscious SHRs, and, for comparison, a vehicle was infused in SHRs and in Wistar-Kyoto rats. Blood pressure was recorded for 30 min before and after treatments. We used the Lyapunov exponent, approximated by the inverse of the lmax index derived from the recurrence plot method, to characterize nonlinear dynamics. Before treatment, lmax index of blood pressure was lower (P < 0.01) in the SHRs than in the Wistar-Kyoto rats. Clonidine significantly increased lmax (P < 0.01) to the level observed in normotensive rats, at 24 h and up to 4 wk after infusion. We conclude that clonidine has a significant chronic effect on blood pressure dynamics, as evidenced by nonlinear methods. Our study also suggests that the mechanisms governing blood pressure variations are nonlinear.     

The cellular electrophysiological effects of tedisamil in human atrial and ventricular fibers

The potential pharmacokinetic interaction between atovaquone and phenytoin was investigated in 12 healthy male volunteers. Each volunteer received a single 600 mg oral dose of phenytoin in the two treatment periods. On one occasion phenytoin was taken alone and on the other pre-treatment with 2000 mg atovaquone taken as two doses of 1000 mg as a microfluidized suspension. The mean (+/- s.d.) peak plasma concentrations (Cmax), apparent total clearance (CL/F) and terminal half-life (t1/2) for phenytoin when administered alone were 10.6(1.8) mg 1(-1), 24.3 (7.7) ml min-1 and 25(8) h, respectively. When administered together with atovaquone, phenytoin Cmax, CL/F and t1/2,z were 10.9 (2.0) mg 1(-1), 23.8 ml min-1 and 24(6) h, respectively. There were no statistically significant differences in any of these plasma pharmacokinetic parameters. There were also no statistically significant differences in the fraction of circulating drug not bound to plasma protein or urinary excretion of 5-hydroxyphenyl-phenyl-hydantoin. In conclusion, there was no effect of atovaquone on the pharmacokinetics of phenytoin or its major metabolite after a single dose.     

Patterns of cellular uptake and effects on cell survival using antimetallothionein oligodeoxyribonucleotide conjugates in vitro

Synthetic oligodeoxyribonucleotides (ODNs) offer the potential for the sequence-specific modulation of viral and cellular gene expression. However, several problems such as efficient delivery into cells, metabolic stability and delivery to specific cellular targets may limit their usefulness. Studies were designed to demonstrate that the covalent conjugation of an 18-mer ODN complementary in sequence to mRNA ODN with various polypeptide ligands, including poly(L-lysine), phosphomannan and asialo-orosomucoid, elicits a pattern of enhanced yet differential uptake into Chang and V79 cells in culture. Viability of cells exposed to conjugated ODNs was measured using a colorimetric assay (MTT). The ODNs covalently linked to poly(L-lysine) reveal an increased efficiency of antisense-directed cell killing from concentrations greater than 3 microM to less than 100 nM. Finally, poly(L-lysine) is also cytotoxic, particularly at extremes of molecular weight. Hence, these studies indicate that synthetic ODNs conjugated to peptides may offer enhanced cellular uptake leading to more efficient antisense activity. However, the cytotoxicity of ODN conjugates may limit their usefulness as research tools or therapeutic agents.     

Slow ventricular tachycardia located in the epicardium of the left ventricular base and characterized by effects of adenosine triphosphate, nicorandil and verapamil

A 31-year-old male with slow ventricular tachycardia (VT) developed a nonsustained VT with prolongation of the JT intervals after injection of contrast medium and saline into the marginal vein of the coronary sinus. The earliest activation site of the VT existed in the epicardium of the left ventricular base. Adenosine triphosphate prevented induction of VT and prolongation of JT intervals. Ventricular premature contractions showing the same morphology as the VT were also inhibited by nicorandil and verapamil. The mechanism of the VT was suggested to be abnormal automaticity due to an increase in the Ca++ current into cells after prolongation of the action potential duration induced by hypothermia.     

HMR 1883, a novel cardioselective inhibitor of the ATP-sensitive potassium channel. Part I: effects on cardiomyocytes, coronary flow and pancreatic beta-cells

The novel sulfonylthiourea HMR 1883 was investigated in in vitro systems. The rilmakalim-induced shortening of the APD90 in guinea pig right papillary muscle at pHo = 6.0 was antagonized half-maximally by glibenclamide and HMR 1883 with 0.14 microM and 0. 6 microM, respectively. Hypoxia-induced shortening of the APD90 was significantly attenuated by the sulfonylureas when applied 60 min after induction of hypoxia. In isolated guinea pig ventricular myocytes the APD90 as well as the whole-cell current was measured with the patch-clamp technique. The rilmakalim-induced shortening of the APD90 was half-maximally antagonized by glibenclamide and HMR 1883 with 10 nM and 0.4 microM, respectively (pHo = 6.5). The rilmakalim-induced whole-cell current (at 0 mV clamp-potential) was inhibited by glibenclamide and HMR 1883 half-maximally with 20 nM and 0.8 microM, respectively (pHo = 7.4). In isolated perfused guinea pig hearts, the coronary flow (CF) was increased by perfusion with hypoxic solution (20% O2). Whereas 1 microM glibenclamide completely inhibited the hypoxia-induced increase in CF, 10 microM HMR 1883 reduced it by only 18%. Pancreatic effects were investigated in rat insulinoma cells (RINm5F), which were hyperpolarized with 100 microM diazoxide. Addition of glibenclamide or HMR 1883 depolarized the cell potential half-maximally with concentrations of 9 nM and approximately 20 microM, respectively. In conclusion, the sulfonylthiourea HMR 1883 blocks KATPs in cardiac muscle cells with 10-50 fold higher potency than in pancreatic beta-cells and has little effect on the coronary vascular system. Therefore, HMR 1883 has pharmacological selectivity for cardiac myocytes and thereby may be a promising substance for the prevention of ischemia-induced ventricular fibrillation.     

Different behaviour of the non-sarcomeric cytoskeleton in neonatal and adult rat cardiomyocytes

In this study the function of transforming growth factor-beta (TGF-beta) in preimplantation mouse embryos was examined. By RT-PCR, mRNA for the signalling type I (T beta R-I) and type II (T beta R-II) receptors for TGF-beta was shown to be present in two distinct time windows: in fertilized oocytes and at the blastocyst stage. The function of TGF-beta at these times was analysed in two ways. Firstly, the TGF-beta signalling pathway was blocked by injecting a DNA construct encoding a truncated T beta R-II, that acts as a dominant-negative receptor, in fertilized oocytes, and the effect on development was determined. Secondly, inner cell masses isolated at the blastocyst stage were cultured in vitro with and without TGF-beta under conditions that favour the outgrowth of parietal endoderm. The results show that TGF-beta signalling mediated by maternally expressed receptors is important for development of preimplantation embryos beyond the two-cell stage, and suggest a regulatory role for TGF-beta in the outgrowth of parietal endoderm.     

Using analog baselines to assess the effects of naltrexone on self-injurious behavior

Healthy male and female human volunteers were exposed to 50 ppm or 100 ppm trichloroethylene (Tri) by inhalation for 4 h. Blood and urine samples were taken at various times before, during, and after the exposure period for analysis of glutathione (GSH), related thiols and disulfides, and GSH-derived metabolites of Tri. The GSH conjugate of Tri, S-(1,2-dichlorovinyl)glutathione (DCVG), was found in the blood of all subjects from 30 min after the start of the 4-h exposure to Tri to 1 to 8 h after the end of the exposure period, depending on the dose of Tri and the sex of the subject. Male subjects exposed to 100 ppm Tri exhibited a maximal content of DCVG in the blood at 2 h after the start of the exposure of 46.1 +/- 14.2 nmol/ml (n = 8), whereas female subjects exposed to 100 ppm Tri exhibited a maximal content of DCVG in the blood at 4 h after the start of the exposure of only 13.4 /- 6.6 nmol/ml (n = 8). Pharmacokinetic analysis of blood DCVG concentrations showed that the area under the curve value was 3.4-fold greater in males than in females, while the t1/2 values for systemic clearance of DCVG were similar in the two sexes. Analysis of the distribution of individual values indicated a possible sorting, irrespective of gender, into a high- and a low-activity population, which suggests the possibility of a polymorphism. The mercapturates N-acetyl-1,2-DCVC and N-acetyl-2,2-DCVC were only observed in the urine of 1 male subject exposed to 100 ppm Tri. Higher contents of glutamate were generally found in the blood of females, but no marked differences between sexes were observed in contents of cyst(e)ine or GSH or in GSH redox status in the blood. Urinary GSH output exhibited a diurnal variation with no apparent sex- or Tri exposure-dependent differences. These results provide direct, in vivo evidence of GSH conjugation of Tri in humans exposed to Tri and demonstrate markedly higher amounts of DCVG formation in males, suggesting that their potential risk to Tri-induced renal toxicity may be greater than that of females.     

Dose-dependent effects of adenosine on interstitial fluid adenosine and postischemic function in the isolated rat heart

Exogenous adenosine produces numerous beneficial effects in ischemic myocardium, but pharmacological doses of adenosine are required to exert these effects. This is thought to be due to the rapid metabolism of adenosine by coronary endothelium, although there is no direct evidence supporting this hypothesis in the ischemic/reperfused heart. The purpose of this study was to determine the relationship between vascular and interstitial fluid (ISF) adenosine levels during adenosine-induced cardioprotection. Isolated perfused rat hearts were submitted to 30-min global normothermic ischemia and 30- min reperfusion. Left ventricular developed pressure (LVDP) was measured with a fluid-filled latex balloon, and ISF adenosine was estimated with cardiac microdialysis. Control hearts were compared with hearts treated with increasing doses of adenosine (1, 10 and 100 microM) for 10 min immediately preceding ischemia. Adenosine produced dose-dependent increases in coronary effluent adenosine levels, but only 10 and 100 microM adenosine increased dialysate adenosine concentrations. All adenosine doses increased coronary flow to the same extent, but only the two higher doses decreased spontaneous heart rate. Control and 1 microM adenosine-treated hearts recovered 60 +/- 3% and 46 +/- 7% of preischemic LVDP, respectively, whereas 10 and 100 microM adenosine improved recovery to 80 +/- 5% and 90 +/- 4% of preischemic LVDP, respectively, after 30-min reperfusion. Because ISF bathes the cardiac myocytes, these results are consistent with the hypothesis that adenosine protects the ischemic rat heart via the activation of cardiac myocyte adenosine receptors.     

Use of the Personality Assessment Inventory to assess psychopathy in offender populations.

The authors investigated the validity of the Antisocial Features (ANT) scale of the Personality Assessment Inventory (PAI; L. C. Morey, 1991) with respect to assessments of psychopathy in 2 offender samples. Study 1 included 46 forensic psychiatric inpatients who were administered the Screening Version of the Hare Psychopathy Checklist (PCL:SV; S. D. Hart, D. N. Cox, and R. D. Hare, 1995). In Study 2, 55 sex offenders were administered the Hare Psychopathy Checklist—Revised (PCL—R; R. D. Hare, 1991). ANT scores correlated highly with the PCL:SV total score (r?=?.54) and moderately with the PCL—R total score (r?=?.40). ANT tapped primarily behavioral symptoms of psychopathy rather than interpersonal and affective symptoms. Also, ANT had low to moderate diagnostic efficiency regarding diagnoses of psychopathy, suggesting that it may be better used as a dimensional rather than categorical measure of this construct. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Use of D-myo inositol 1,2,6 trisphosphate to inhibit contractile activity in rat ventricular cardiomyocytes induced by neuropeptide Y and other cardioactive peptides through phospholipase C

1. D-Myo inositol 1,2,6 trisphosphate (alpha-trinositol, pp56), an isomer of the second messenger substance, inositol 1,4,5 trisphosphate, has an interesting pharmacological profile that includes antagonism of a number of neuropeptide Y (NPY)-mediated cellular processes. The ability of pp56 to inhibit selectively the myocardial contraction mediated by NPY in relation to the responses to other cardioactive peptides, including endothelin-1, calcitonin gene-related peptide (CGRP), secretin and vasoactive intestinal peptide (VIP), was assessed. In order to investigate the possible interaction of pp56 with mechanisms of inositol phosphate signalling generated in heart muscle cells by activation of the beta-isoenzyme of phospholipase C (PLC beta), noradrenaline was used as a positive control, and isoprenaline and forskolin were included as negative controls. 2. Ventricular cardiomyocytes, isolated from the hearts of adult rats, were stimulated to contract at 0.5 Hz in the presence of calcium ion (2 mM). The concentrations of agonists used were in the region of their maximally effective concentrations for myocyte contraction and the concentration of pp56 was in the range of 1-100 microM. Contractile activity was monitored by video microscopy and maximum shortening determined by image analysis. 3. In the absence of agonist, contractile amplitudes following 20 min preincubation with pp56 were not different from that observed in the absence of pp56. Pp56 (1-100 microM) inhibited significantly the positive contractile response to noradrenaline (5 microM) in the presence of propranolol (500 nM), such that the response was almost completely attenuated at the highest concentration of the inhibitor. Pp56 did not inhibit the positive contractile responses to forskolin (40 microM) or isoprenaline (100 nM). 4. NPY alone does not influence the basal level of contraction of cardiomyocytes, but can attenuate isoprenaline-stimulated contraction and can increase contractile amplitude from basal when the transient outward current is blocked with 4-aminopyridine. In the presence of isoprenaline (100 nM), the negative response to NPY (100 nM) was attenuated significantly by pp56 (1-100 microM). With 4-aminopyridine, the positive contractile response to NPY (200 nM) was decreased by pp56, although this was not statistically significant. 5. Pp56 inhibited the positive contractile responses to CGRP (1 nM) and endothelin-1 (20 nM) completely, but did not affect the responses to secretin (20 nM) or VIP (20 nM). 6. In conclusion, these data challenge the previously obtained selectivity of pp56 as an antagonist of NPY-mediated cellular processes, since responses to CGRP and endothelin-1 were at least equally sensitive. Furthermore, as pp56 discriminated clearly in its inhibition of responses to alpha-adrenoceptor by comparison with beta-adrenoceptor/adenylate cyclase stimulation, it appears that pp56 may be a useful pharmacological agent with which to distinguish between PLC beta-dependent and PLC beta-independent coupling mechanisms. On this basis, further evidence has been obtained that, in rat cardiomyocytes, the contractile responses to NPY, CGRP and endothelin-1 are attributable to the activation of PLC beta-dependent pathways, whereas the responses to secretin and VIP are mediated by PLC beta-independent pathways.     

Rapid cellular genesis and apoptosis: Effects of exercise in the adult rat.

Long-term aerobic exercise improves cognition in both human and nonhuman animals and induces plastic changes in the central nervous system (CNS), including neurogenesis and angiogenesis. However, the early and immediate effects of exercise on the CNS have not been adequately explored. There is some evidence to suggest that exercise is initially challenging to the nervous system and that the plastic changes commonly associated with chronic exercise may result as adaptations to this challenge. The current experiment assessed levels of apoptosis, angiogenesis, and neurogenesis during the first week of an exercise regimen in the adult rat. The results indicate that exercise rapidly induces these processes in the hippocampus and cerebellum. The temporal pattern of these events suggests that voluntary exercise in the adult rat rapidly and transiently induces apoptosis, followed by angiogenesis. Neurogenesis is an immediate and independent consequence of exercise in the hippocampus that may require the additional metabolic support supplied by angiogenesis. This is the first report of CNS neuronal apoptosis as a consequence of exercise in the adult rat and suggests that this process is a potential mediator of rapid exercise-induced plasticity. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Use of age and serum gamma-glutamyltransferase activity to assess passive transfer status in lambs

OBJECTIVE: To develop an algorithm for predicting passive transfer status of lambs of various ages, using the lamb's age and serum gamma-glutamyltransferase (GGT) activity. DESIGN: Prospective study. ANIMALS: 51 Suffolk, Columbia, and crossbred lambs from 1 to 16 days old. PROCEDURE: Serum was obtained from all lambs. Serum GGT activity was measured, using a commercially available kit. Serum IgG concentration was determined by use of radial immunodiffusion. Day-1 serum IgG concentration was estimated from sample IgG concentration, lamb age, and the published 14-day half-life of IgG in lambs. Stepwise multivariate regression models were developed to estimate day-1 serum IgG concentration as a function of the natural logarithm of serum GGT activity (In[GGT]) and natural logarithm of lamb age (In[age]) at the time of sampling. These regression models were then used to calculate serum GGT activities that were equivalent to various day-1 IgG concentrations in lambs of various ages. RESULTS: In(GGT) and In(age) were significantly associated with estimated day-1 IgG concentration. Day-1 serum IgG concentration could be predicted using the formula: IgG = -7,686 + 1,366(In[GGT]) + 1,199(In[age]). The model was moderately accurate in predicting serum IgG concentration (R2 = 0.52). CLINICAL IMPLICATIONS: Serum GGT activity can be used to assess passive transfer status of lambs.     

Actions of taurine on the L-type Ca2+ channel current in guinea pig ventricular cardiomyocytes

This article presents a new method for analyzing the spreading of skin erythemas. These occur as a result of the cutaneous vascular axon reflex which can be evoked by a noxious stimulation of the skin. Series of true-color images of the observed skin patch were recorded using a video camera. The images were digitized and stored on computer disk. The delineation of the reddening was segmented for every image of the sequence by a newly developed image processing method. Each image taken after the noxious stimulation was compared with the baseline before the stimulation and each image point was classified as: "unchanged" or "changed skin color." To improve the classification the CIE L*a*b* color space was used. The boundaries of the erythema were extracted from the resulting binary images. Every image of a sequence was analyzed in the same way in order to follow the time course of the flare response. The erythema reaction could be determined in an objective way using this methods. The automatically detected flare sizes were independent of human observers and had a high spatial and temporal resolution. It was used for a crossover study to assess the power of new drugs which modify the blood flow of the skin induced by an intradermal histamine application.