Isolation and characterization of a monoclonal antibody against pig kidney sodium- and potassium-activated ATPase

A hybridoma cell line producing mouse monoclonal antibody against pig kidney Na,K-ATPase was established. The antibody, named 38 (mAb38, IgG1), was purified from mouse ascites fluid by chromatography on a protein A-Sepharose column. Antigens immobilized on microplate wells with p-benzoquinone were used for titer assays. mAb38 cross-reacted with both dodecyloctaethyleneglycol monoether (C12E8)-solubilized enzyme and membranous sodium dodecyl sulfate (SDS)-treated enzyme from kidney with high affinity (50% binding = 0.6 nM). However, the antibody bound to neither alpha- nor beta-subunit separated by preparative SDS-polyacrylamide gel electrophoresis (PAGE). The stoichiometry of antibody binding to the purified enzyme was estimated to be about 0.86 mol of IgG per mol of alpha beta-protomer. Na,K-ATPase proteins were recovered from a column of mAb38-coupled Affi-Gel by elution with pH 3 buffer when C12E8-solubilized kidney enzyme or detergent extracts of brain microsomes were applied to it, confirming that the mAb is directed to Na,K-ATPase. mAb38 at saturation level concentrations had no effect on kidney Na,K-ATPase activity or on ouabain-sensitive Rb uptake in erythrocytes. In an immunofluorescence study, the antibody bound to intact erythrocytes much more strongly than control IgG1 (mAb50c), but the extent of the antibody binding to inside-out vesicles under hypotonic conditions was lower than that of the control. Most of the antibody binding activity remained when the kidney enzyme was treated with sialidase. These results suggest that this mAb38 was raised against an intact conformation of a cell-surface-exposed site of Na,K-ATPase.     

Changes of aldolase A and B messenger RNA levels in rat liver during azo-dye-induced hepatocarcinogenesis

The expression of aldolase A and B mRNAs during azo-dye-induced carcinogenesis in rat liver was examined. After feeding the dye for 18 weeks, the level of aldolase A mRNA increased to about 11 times that in a normal liver, with the concomitant decrease of aldolase B mRNA level to about 25% of that in a normal liver. These changes did not occur progressively during the carcinogenesis, but occurred as an additional phase after 4 week-feeding of the azo-dye. At this stage, the levels of aldolase A and B mRNAs were about 7 times and 45% of that in a normal liver, respectively. This biphasic pattern in the aldolase isozyme expression in the azo-dye-fed rat liver is discussed together with the kinetic data of the enzyme activity.     

The effect of 2-deoxy-D-glucose on insulin release by neonatal rat B cells in culture

The culture for 7 days in medium with 5.5 mM glucose and 1 mM 2-deoxy-D-glucose enhanced the glucose sensitivity of neonatal rat B cells, and even stimulated their growth in vitro. Also, 2-deoxy-D-glucose supplementation maintained insulin release evoked by leucine and 2-ketoisocaproate from B cells at day 7 at levels several times higher than at day 1. The effect of leucine was greatly augmented by glutamine, whereas that of the 2-keto acid remained almost unchanged irrespective of the presence of glutamine. These results suggest an increase in oxidative catabolism of medium nutrients in B cells grown in medium with 2-deoxy-D-glucose for 7 days, and such metabolic changes may promote the growth of B cells in vitro.     

A novel opioid peptide, leumorphin, acts as an agonist at the kappa opiate receptor

The primary structure of the common precursor of porcine beta-neo-endorphin and dynorphin (preproenkephalin B) has shown the existence of a third leucine-enkephalin (leu-enkephalin) sequence with a C-terminal extension of 24 amino acids. This nonacosapeptide, named leumorphin, was approximately 70 times more potent than leu-enkephalin in inhibiting the contraction of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. This action of leumorphin, like those of beta-neo-endorphin and dynorphin, was antagonized less effectively by naloxone than that of leu-enkephalin, but more effectively by Mr2266, an antagonist relatively specific for the kappa type opiate receptor. The inhibitory action of leumorphin or beta-neo-endorphin on the contraction of the guinea pig ileum muscle strip was reduced in a dose-dependent manner by pretreatment with dynorphin and vice versa. Leumorphin as well as beta-neo-endorphin and dynorphin inhibits the contraction of the rabbit vas deferens which is known to have only the kappa type opiate receptor. This action was also effectively antagonized by Mr2266. It is concluded that leumorphin has potent opioid activity and acts at the kappa receptor, like other opioid peptides derived from preproenkephalin B.     

Stable and unstable phase of memory in classically conditioned fly, Phormia regina: Effects of nitrogen gas anaesthesia and cycloheximide injection

This study examined the effects of nitrogen anaesthesia and cycloheximide injection on memory of the classically-conditioned fly, Phormia. 1 M NaCl solution was given to each fly as a conditioning stimulus and 0.5 M sucrose solution was the unconditioned stimulus that induced the proboscis extension response. The training period was as short as 2 min and testing was usually carried out 2 hr later. At varying times (0–60 min) between training and testing, flies were anaesthetized with nitrogen gas for 25 sec. When flies were anaesthetized immediately after training the effect of nitrogen gas was the greatest and few flies showed any conditioned response, but the sensitivity of memory to nitrogen gas declined as the interval between training and nitrogen treatment became longer, and such treatment had no effect on memory when the interval was longer than 30 min. The effect on memory of cycloheximide, an inhibitor of peptide bond synthesis, was also investigated. The injection of cycloheximide (0.37 μg) immediately after training diminished the memory, but when given 1 hr after training it had no effect on memory. These results show that the memory in Phormia has two phases, stable and an unstable phase, like long-term and short-term memory in vertebrates.     

Improvement of first-service pregnancy rate in cows with gonadotropin-releasing hormone analog

The effect of an intramuscular injection of a new analog of gonadotropin-releasing hormone (GnRH), fertirelin, on the first-service pregnancy rate in cows was investigated by a double blind experiment. A total of 1,194 cows was injected intramuscularly either with 100 mug of GnRH or placebo (physiological saline solution) at the time of first insemination postpartum. Pregnancy rate (number of cows calved/ number of cows serviced) was 57.2 % in 605 cows treated with GnRH, while the performance was 49.7 % in 589 cows of the placebo group. The difference of pregnancy rates in both groups was significant (P<0.05). GnRH injected at insemination was effective, especially in cows at the first and third lactations, cows at 101 days postpartum or later, cows with daily milk yield of 26-30 kg, and also in cows from the area where a regional average fertility was relatively low.