Short communication

Application of Bradford's protein assay to adrenal gland subcellular fractions

Here, we have presented a technically simple and efficient method for preparing a continuous flow microreactor by employing immobilized β-glucosidase in a silica quartz capillary tube. Developing an immobilized enzyme layer on the inner wall of the capillary tube involved the modification of the inner wall using bifunctional crosslinking agents 3-aminopropyltriethoxysilane and glutaraldehyde before attaching β-glucosidase. The microreactor afforded unique reaction capacities compared with conventional batch operational configurations. These included enhanced pH and thermal stability during storage tests, increased conversion rates of cellobiose, and reduced product inhibition. The maximum conversion rate of soluble substrate cellobiose digestion in the microreactor was 76% at 50°C and pH 4.8 when the microreactor was operated continually over 10 h at a flow rate of 7 μL/min. This was markedly contrasting to the observed conversion rate of 56% when cellobiose was digested in a conventional batch mode under the same pH and temperature conditions. Reaction inhibition by glucose was significantly reduced in the microreactor. We postulate that the increased capacity of glucose to diffuse into the continual flowing media above the immobilized enzyme layer prevents glucose from reaching inhibitory concentrations at the substrate–enzyme interface.

The present study examined the in vivo effects of candesartan cilexetil compared with losartan on angiotensin II (Ang II) receptor binding in the rat kidney after oral administration. Male Sprague-Dawley rats (250 to 300 g) were gavaged with candesartan cilexetil or losartan in doses of 0.1, 0.3, 1, 3, 10, or 30 mg/kg, or corresponding vehicle. Rats were killed at 0, 1, 2, 8, or 24 h after drug administration, trunk blood collected, and kidneys removed. The effects of candesartan cilexetil and losartan on Ang II receptor binding were determined by quantitative in vitro autoradiography using the radioligand [¹²⁵I]-[Sar¹,Ile⁸] Ang II. Ang II receptor binding in the kidney was mainly due to AT₁ receptors with high levels of binding localized to the inner stripe of the outer medulla and glomeruli in cortical regions. Candesartan cilexetil (0.1 to 30 mg/kg) inhibited Ang II receptor binding to all anatomical sites of the kidney, in a dose-dependent manner. Losartan (0.1 to 30 mg/kg) also produced dose-dependent inhibition of Ang II receptor binding but was approximately 10- to 30-fold less potent than candesartan cilexetil. Inhibition of Ang II receptor binding was near maximal about 1 h after administration of candesartan cilexetil (10 mg/kg) or losartan (10 mg/kg), with both drugs producing persistent blockade at 24 h despite plasma renin activity and plasma drug concentrations returning to near normal levels. In vitro, candesartan, losartan, and EXP3174 (1 × 10⁻¹⁰ to 1 × 10⁻⁵ mol/L) displaced [¹²⁵I]-[Sar¹,Ile⁸] Ang II binding from AT₁ receptors in the kidney in a concentration-dependent manner with a rank order of potency of candesartan > EXP3174 > losartan. The concentration required to displace 50% of radioligand binding (IC₅₀) by candesartan, EXP3174, and losartan was 0.9 ± 0.1 nmol/L, 3.4 ± 0.4 nmol/L, and 8.9 ± 1.1 nmol/L, respectively. In conclusion, the findings of the present study suggest that candesartan cilexetil is more potent than losartan in antagonizing AT₁ receptors in the kidney in vivo. Nonetheless, both candesartan cilexetil and losartan produce rapid, complete, and sustained blockade of AT₁ receptors in the rat kidney. Tissue blockade of Ang II receptors in target organs, such as the kidney, may contribute to the beneficial effects of Ang II receptor antagonists as antihypertensive agents.

The effects of immobilization stress and/or dexamethasone (DEX) on the adrenal ornithine decarboxylase (ODC) activities of sham-operated and adrenal-medulloectomized (enucleated) male Sprague-Dawley rats were investigated. On day 11 after surgery, rats were injected with saline or DEX (1 mg/kg), 3 h before the time of sacrifice (0600 h or 1800 h). Four groups, from sham-operated and enucleated rats (ENU) treated with saline or DEX were subjected to immobilization stress for 1 h prior to sacrifice. Groups of rats from stress-sham-DEX, non stress-sham-DEX, stress-sham, non stress-sham, stress-ENU-DEX, non stress-ENU-DEX, stress-ENU, and non stress-ENU were sacrificed at 0600 h or 1800 h on day 11 after surgery. Adrenal glands were excised and later analyzed for ODC activities. Results indicated that DEX and/or immobilization stress inhibited ODC activities (p < 0.05) in normal and regenerating adrenal glands at 1800 h and ODC activity varies diurnally, the activity being greater at 1800 h than at 0600 hours (p < 0.001).

Films consisting of various numbers of alternating molecular layers of human serum albumin (HSA) and heparin were prepared on surfaces of polyethylene, polyurethane, cellulose acetate, germanium, hydrophilic and hydrophobic silica, and mica by alternating the HSA and heparin adsorption from solutions at pH 3.9. The successive adsorption of the molecular layers was observedin situby IR multiple internal reflection spectroscopy and total internal reflection fluorescence. The first HSA monolayer was irreversibly adsorbed on the substrates mainly due to the hydrophobic interaction with the surface, and the following heparin and HSA layers were successively bound due to ionic interaction between HSA and heparin. Films stable in physiological solutions were obtained by the covalent crosslinking of the final HSA–heparin assembly.