Agar-Gel Precipitin-Inhibition Technique for C-Reactive Protein Determinations

An application of the agargel precipitin-inhibition technique of Ray and Kadull detects the C-reactive protein present in the acute-phase human sera. The sensitivity of this procedure is compared with the capillary tube-precipitin method of Selman and Halpern.     

Agar-Gel Precipitin-Inhibition Technique for C-Reactive Protein Determinations: III. Quantitation of C-Reactive Protein in Serum Specimens

A quantitative C-reactive protein serological procedure has been developed. By use of this method, which is performed in agar-gel plates, from 2 to 654 μg of C-reactive protein per ml of titrated human serum can be detected. The method is based on the inhibition of a specific C-reactive protein antigen-antibody precipitate formed in agar-gel by the minimal reactive dilutions of each reagent in 48 hr. It is simple, sensitive, and readily reproducible.     

Tachykinins mediate bronchoconstriction elicited by isocapnic hyperpnea in guinea pigs

We tested the hypothesis that tachykinins mediate hyperpnea-induced bronchoconstriction (HIB) in 28 guinea pigs. Stimulus-response curves to increasing minute ventilation with dry gas were generated in animals depleted of tachykinins by capsaicin pretreatment and in animals pretreated with phosphoramidon, a neutral metalloendopeptidase inhibitor. Sixteen anesthetized guinea pigs received capsaicin (50 mg/kg sc) after aminophylline (10 mg/kg ip) and terbutaline (0.1 mg/kg sc). An additional 12 animals received saline (1 ml sc) instead of capsaicin. One week later, all animals were anesthetized, given propranolol (1 mg/kg iv), and mechanically ventilated (6 ml/kg, 60 breaths/min, 50% O2 in air fully water saturated). Phosphoramidon (0.5 mg iv) was administered to five of the noncapsaicin-treated guinea pigs. Eucapnic dry gas (95% O2-5% CO2) hyperpnea "challenges" were performed by increasing the tidal volume (2-6 ml) and frequency (150 breaths/min) for 5 min. Capsaicin-pretreated animals showed marked attenuation in HIB, with a rightward shift of the stimulus-response curve compared with controls; the estimated tidal volume required to elicit a twofold increase in respiratory system resistance (ES200) was 5.0 ml for capsaicin-pretreated animals vs. 3.7 ml for controls (P less than 0.03). Phosphoramidon-treated animals were more reactive to dry gas hyperpnea compared with control (ES200 = 2.6 ml; P less than 0.0001). Methacholine dose-response curves (10(-11) to 10(-7) mol iv) obtained at the conclusion of the experiments were similar among capsaicin, phosphoramidon, and control groups. These findings implicate tachykinin release as an important mechanism of HIB in guinea pigs.     

Fine structure analysis of the Chinese hamster AS gene encoding asparagine synthetase

Overlapping cDNAs for Chinese hamster ovary (CHO) asparagine synthetase (AS) were isolated from a library prepared from an AS-overproducing cell line. The sequence was determined and shown to contain an open reading frame encoding a protein of Mr 64,300. The predicted amino acid sequence for the CHO AS enzyme was compared to that of the human AS enzyme and found to be 95% homologous. A potential glutamine amide transfer domain, with sequence similarity to amidotransferases from bacteria and yeast, was identified in the N-terminal portion of the protein. The cDNAs were used to screen a library of phage containing wild type CHO DNA and the genomic AS sequences were detected on three overlapping phages. Determination of the fine structural organization showed that the CHO AS gene spanned 19 kilobases and was composed of 12 exons, three of which contained the glutamine amidotransferase domain. The 5' flanking sequences were highly G + C-rich and, like other housekeeping genes, lacked TATA and CAAT boxes.     

Mechanism of Agonist-Induced Down-Regulation and Subsequent Recovery of Muscarinic Acetylcholine Receptors in a Clonal Neuroblastoma X Glioma Hybrid Cell Line

The mechanisms of carbachol-induced muscarinic acetylcholine receptor (mAChR) down-regulation, and recovery following carbachol withdrawal, were studied in the neuroblastoma x glioma hybrid NG108-15 cell line by specific ligand binding assays. N-[3H]Methylscopolamine ([3H]NMS) and [3H]quinuclidinyl benzilate ([3H]QNB) were used as the ligands for the cell surface and total cellular mAChRs, respectively. Exposure of cells to 1 mM carbachol for 16 h decreased the specific binding of [3H]NMS and [3H]QNB by approximately 80%. Bacitracin (1-4 mg/ml) and methylamine (1-15 mM), inhibitors of transglutaminase and of endocytosis, prevented agonist-induced loss of surface mAChRs. Pretreatment of cells with the antimicrotubular agents nocodazole (0.1-10 microM) and colchicine (1-10 microM) prevented carbachol-induced loss of [3H]QNB binding, but not that of [3H]NMS binding. These results indicate that agonist-induced mAChR down-regulation occurs by endocytosis, followed by microtubular transport of receptors to their intracellular degradation sites. When carbachol was withdrawn from the culture medium following treatment of cells for 16 h, receptors recovered and were incorporated to the surface membrane. This recovery process was antagonized by monovalent ionophores monensin (0.1 microM) and nigericin (40 nM), which interfere with Golgi complex function. Receptor recovery was also prevented by the antimicrotubular agent nocodazole. Thus, recovery of receptors appears to be mediated via Golgi complex and microtubular transport to the surface membrane.