High-resolution semi-quantitative real-time PCR without the use of a standard curve.

The repeatability and sensitivity of a simple, adaptable, semi-quantitative, real-time RT-PCR assay was investigated. The assay can be easily and rapidly applied to quantitate relative levels of any gene product without using standards, provided that amplification conditions are specific for the PCR product of interest. Using the LightCycler real-time PCR machine, a serial 10-fold dilution series (spanning four orders of magnitude) of a 379-bp cDNA template was amplified, and the PCR product was detected using SYBR Green I chemistry. The experiment was repeated on a subsequent day. The experimental design was such that the data lent itself to analysis using an appropriate method for testing repeatability. It was found that, within a single assay, for samples assayed in triplicate, a difference of 23% may be reliably detected. Furthermore, when all of the factors that contribute to variability in the assay are taken into account, such as day-to-day variation in pipetting and amplification efficiency, a 52% difference in target template can be detected using a sample size of 4. The assay was found to be linear over at least four orders of magnitude.

Differential regulation of beta3-adrenoceptors in gut and adipose tissue of genetically obese (ob/ob) C57BL/6J-mice.

1. Levels of beta3-adrenoceptor (AR) mRNA were compared using reverse transcription-polymerase chain reaction (RT PCR) in white adipose tissue (WAT), brown adipose tissue (BAT), ileum and colon from genetically obese (ob/ob) and lean (+/+) C57BL/6J mice. Functional responses to the beta3-AR agonist CL 316243 were also characterized in ileal longitudinal smooth muscle from obese and lean mice. 2. Beta3-AR mRNA levels were significantly higher in WAT (100+/-16%) and BAT (100+/-13%) from lean compared to WAT (21.0+/-0.9%; n=4; P<0.005) and BAT (14.1+/-2.2%; n=5; P<0.01) from obese mice. In contrast, beta3-mRNA levels were not significantly different in ileum (100+/-15%) and colon (100+/-22%) from lean mice, compared to ileum (78+/-13%; n=4; P= 0.31) or colon (82+/-15%; n =4; P=0.52) from obese mice. 3. Concentration-response curves to CL 316243 did not differ significantly in slope or position in ileal longitudinal smooth muscle from obese or lean mice. pEC50 (+/-s.e.mean) values were not significantly different (P= 0.59) between obese (7.90+/-0.13, n = 7) and lean (7.77+/-0.20, n = 7) mice. 4. pKB values for the beta1-AR and beta2-AR selective antagonist propranolol or the beta3-AR selective antagonist SR 58894 against relaxations to CL 316243 were similar in ileum of genetically obese (propranolol 6.31+/-0.22 and 6.13+/-0.12; SR 58894 8.22+/-0.06) and lean mice (propranolol 6.40+/-0.08 and 6.60+/-0.13; SR 58894 8.27+/-0.12) and were consistent with values previously found at beta3-AR. 5. Treatment of lean C57BL/6J mice with dexamethasone (1 mg kg(-1), i.p.) significantly reduced beta3-AR mRNA levels after 4 h in WAT (100+/-6.1 to 41.4+/-4.3; n= 16 18; P<0.0001) and BAT (100+/-8.0 to 35.1+/-5.8; n= 17; P<0.0001), but caused no change in ileum (100+/-6.1 to 101+/-17; n= 10-11; P=0.95) or colon (100+/-11 to 101+/-11; n= 11; P= 0.94). Beta3-mRNA levels in ileum and colon also did not change significantly when examined over 24 h or after the administration of a higher dose of dexamethasone (5 mg kg(-1)). 6. In summary, beta3-AR mRNA levels were considerably lower in WAT and BAT of obese compared to lean mice whereas the levels in ileum and colon were not significantly different. The similar beta3-mRNA levels in ileum of obese and lean mice were associated with indistinguishable responses of carbachol-contracted ileum to a beta3-agonist and similar affinity for beta-antagonists. Administration of glucocorticoids to lean mice reduced beta3-AR mRNA levels in WAT and BAT but not in ileum or colon. These studies show that in mice, beta3-ARs are differentially regulated in ileum and colon compared to adipose tissues.

Generic construction of single component particles that elicit humoural and cellular immune responses without the need for adjuvants.

Insoluble, pure protein particles could be advantageous as single-entity vaccines or as carriers for small peptide epitopes. Dense gas anti-solvent precipitation was employed to produce pure protein particles which were found to be insoluble in water. As particulate and multimerized antigens are more immunogenic and hence more advantageous for vaccination, particles were produced via this method using ovalbumin as a model antigen. The particles produced had a mean diameter of approximately 300nm, and remained as discrete particles at low pH. At neutral pH or in the presence of electrolyte, the particles exhibited predictable flocculation behaviour to produce aggregates 1-5microm in diameter. Immunisation of mice with these flocculates elicited specific ovalbumin antibody production, T-cell proliferation and a cytotoxic T-cell response, all in the absence of adjuvant. Thus, dense gas processing could be used as a generic method to produce pure protein particulate vaccines.

Marked difference between angiotensin-converting enzyme and neutral endopeptidase inhibition in vivo by a dual inhibitor of both enzymes.

Dual inhibition of neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE) offers the potential for improved therapy of hypertension and cardiac failure. S 21402-1 [(2S)-2-[(2S,3R)-2-thiomethyl-3-phenylbutanamido] propionic acid] is a sulfhydryl-containing potent inhibitor of both NEP (Ki = 1.7 nM) and ACE (Ki = 4.5 nM). S 21402-1 and the sulfhydryl-containing ACE inhibitor captopril were administered to rats by intraperitoneal injection (0, 0.3, 3, 30, 300 mg/kg). Urine was collected for 4 h; then plasma and kidneys were collected. The difference in NEP and ACE inhibition by S 21402-1 in vivo was greater than 1000-fold. All doses of S 21402-1 inhibited NEP, as indicated by plasma NEP activity, radioinhibitor binding to kidney sections, urinary sodium excretion and bradykinin-(1-7)/bradykinin-(1-9) ratio. However, only 300 mg/kg S 21402-1 inhibited ACE, as indicated by plasma angiotensin II/angiotensin I ratio, renin and angiotensinogen levels. Although S 21402-1 (30 and 300 mg/kg) inhibited renal NEP, as indicated by the bradykinin-(1-7)/bradykinin-(1-9) ratio in kidney, S 21402-1 had no effect on renal ACE, as indicated by the angiotensin II/angiotensin I ratio in kidney. Moreover, captopril was greater than 10-fold more potent than S 21402-1 as an ACE inhibitor in vivo. In separate experiments, the pressor response of anesthetized rats to angiotensin I showed more rapid decay in ACE inhibition by S 21402-1 than by captopril. These studies indicated that in vivo modification of S 21402-1 caused a much greater decrease in potency of ACE inhibition than NEP inhibition. Consequently, effective ACE inhibition by S 21402-1 required doses much higher than those required for NEP inhibition.

Interaction between neutral endopeptidase and angiotensin converting enzyme inhibition in rats with myocardial infarction: effects on cardiac hypertrophy and angiotensin and bradykinin peptide levels.

Combined inhibition of neutral endopeptidase 24.11 (NEP) and angiotensin converting enzyme (ACE) is a candidate therapy for hypertension and cardiac failure. Given that NEP and ACE metabolize angiotensin (Ang) and bradykinin (BK) peptides, we investigated the effects of NEP inhibition and combined NEP and ACE inhibition on Ang and BK levels in rats with myocardial infarction. We administered the NEP inhibitor ecadotril (0, 0.1, 1, 10, and 100 mg/kg/day), either alone or together with the ACE inhibitor perindopril (0.2 mg/kg/day) by 12-hourly gavage from day 2 to 28 after infarction. Ecadotril increased urine cyclic GMP and BK-(1-9) excretion. Perindopril potentiated the effect of ecadotril on urine cyclic GMP excretion. Neither perindopril nor ecadotril reduced cardiac hypertrophy when administered separately, whereas the combination of perindopril and 10 or 100 mg/kg/day ecadotril reduced heart weight/body weight ratio by 10%. Administration of ecadotril to perindopril-treated rats decreased plasma Ang-(1-7) levels, increased cardiac BK-(1-9) levels, and increased Ang II levels in plasma, kidney, aorta, and lung. These data demonstrate interactions between the effects of NEP and ACE inhibition on remodeling of the infarcted heart and on Ang and BK peptide levels. Whereas increased cardiac BK-(1-9) levels may contribute to the reduction of cardiac hypertrophy, the reduction in plasma Ang-(1-7) levels and increase in Ang II levels in plasma and tissues may compromise the therapeutic effects of combined NEP/ACE inhibition.

Effects of neutral endopeptidase inhibition and combined angiotensin converting enzyme and neutral endopeptidase inhibition on angiotensin and bradykinin peptides in rats.

The combination of neutral endopeptidase 24.11 (NEP) and angiotensin converting enzyme (ACE) inhibition is a candidate therapy for hypertension and cardiac failure. Given that NEP and ACE metabolize angiotensin (Ang) and bradykinin (BK) peptides, we investigated the effects of NEP inhibition and combined NEP and ACE inhibition on the levels of these peptides. We administered the NEP inhibitor ecadotril (0, 0.1, 1, 10, 100 mg/kg per day), either alone or together with the ACE inhibitor perindopril (0.2 mg/kg per day), to rats by 12 hourly gavage for 7 days. Ecadotril produced diuresis, natriuresis, increased urine cyclic guanosine monophosphate and BK-(1-9) levels, increased Ang II and Ang I levels in plasma, and increased Ang I levels in heart. Perindopril reduced Ang II levels in kidney, and increased BK-(1-9) levels in blood, kidney and aorta. Combined NEP/ACE inhibition produced the summation of these effects of separate NEP and ACE inhibition. In addition, perindopril potentiated the ecadotril-mediated diuresis, natriuresis and decrease in urine BK-(1-7)/BK-(1-9) ratio, which is an index of BK-(1-9) metabolism. Moreover, combined NEP/ACE inhibition increased Ang II levels in plasma and lung. These data indicate that summation of the effects of separate NEP and ACE inhibition provides the basis for the therapeutic efficacy of their combination. Whereas potentiation by perindopril of the diuretic and natriuretic effects of ecadotril may contribute to the therapeutic effects, increased Ang II levels in plasma and lung may compromise the therapeutic effects of combined NEP/ACE inhibition.