Existence of a polar digitalis-like factor in mammalian hypothalamus

We were able to partially purify a polar digitalis-like factor from rat and bovine hypothalami based on the capacity to inhibit [3H]ouabain binding to intact human erythrocytes. This factor was characterized in reference to the digitalis-like factor that we have isolated and reported on. Hypothalamic factor shared digitalis-like activities and physicochemical properties with the one derived from human urine and mammalian plasma. These findings strongly suggest that a polar digitalis-like factor identical to the circulatory factor does exist in mammalian hypothalamus.     

Purification and characterization of membrane-bound alkaline proteases from midgut tissue of the silkworm, Bombyx mori

Membrane-bound alkaline proteases from the midgut epithelia of the silkworm, Bombyx mori, were solubilized with 1% Lubrol-WX, at pH 11.2. They were purified by gel filtration on Sepharose 6B and Ultrogel AcA-202 columns and a preparative polyacrylamide gel electrophoresis. Two proteases, caseinolytic (6B3-Tc) and benzoyl-arginine-p-nitroanilide-lytic (6B3-Tb) were obtained. Both enzymes were homogeneous as judged by polyacrylamide electrophoresis. These enzymes showed high pH optima, 11.2, and pI values, above 11, and were extremely stable over a wide range of pH. The Km values for 6B3-Tb and Tc were 0.476 mM and 2.5 mg/ml respectively. Hammarsten casein and mulberry leaf protein were rapidly hydrolyzed by Tc, whereas the hydrolytic activity of Tb for Azocoll was higher than that of Tc. The protease Tb was strongly inhibited by diisopropylfluorophosphate, p-chloromercuribenzoate, benzamidine, leupeptin, and soybean trypsin inhibitor; Tc was inhibited by diisopropylfluorophosphate, tosyl phenylalanine chloromethylketone and chymostatin, but not by tosyl lysine chloromethylketone, p-chloromercuribenzoate, or iodoacetamide. The molecular weights of the proteases were estimated to be 12,800 (Tb) and 13,300 (Tc) by Sephacryl S-300 gel filtration. The amino acid analyses showed that both proteases contain a large number of acidic amino acids but a relatively small number of basic amino acids.     

Nonresponsiveness of the thyroid gland to goitrogen in the early neonatal period in the rat: light- and electron-microscopic observations.

The thyroid response of fetal and neonatal rats to propylthiouracil (PTU) as a goitrogen was studied with observation of the thyroid glands by light and electron microscopy. On day 19 of gestation and on days 1, 3, 5 and 8 after birth, fetal and neonatal rats were given a subcutaneous injection of PTU and were autopsied 2 days later. PTU induced conspicuous goiters in fetal rats but did not in neonatal rats aged up to day 5 after birth. Beyond that age, PTU again induced goiters. Histologically, the follicular cell height in goitrous thyroid glands was significantly increased. Ultrastructurally, follicular cells in goitrous thyroid glands often had colloid droplets and lysosomes. It seems that nonresponsiveness of the thyroid glands in early neonatal rats to goitrogen is due to a temporary decline of the pituitary activity of thyrotropin secretion. About 5 days or more after birth, the pituitary-thyroid system begins to operate again in response to goitrogen.     

Comparison of stimulus-response (V-log I) functions in five types of lepidopteran compound eyes (46 species)

Summary Stimulus intensity-response relations (V-log I curves) were electrophysiologically (ERG) determined for the compound eyes of 46 lepidopteran species belonging to five different groups: butterflies (22 species), hesperids (3 species), diurnal sphingids (2 species), diurnal moths (3 species) and nocturnal moths (16 species). The V-log I curves were fitted to the Naka and Rushton equation, in whichn represents the slope of the linear part of each curve. The slopes so determined range fromn=0.35 (the shallowest slope) in nocturnal moths with the greatest dynamic range ton=0.54 (the steepest slope) in diurnal moths andn=0.53 in butterflies both of which have narrow dynamic range. Hesperids (n=0.41) and diurnal sphingids (n=0.38) have intermediate values between butterflies and nocturnal moths.The ratio of rhabdom to retinula volume is significantly higher in nocturnal moths (70–75%), however, those of butterflies and of diurnal moths are very small (2–5%), and hesperids and diurnal sphingids show intermediate ratio (ca. 25%).The slopes of V-log I curves are inversely proportional to the ratio of rhabdom to retinula volume in the various eye types. In all groups except diurnal moths, the light intensities which produce maximal and saturated responses are nearly the same, therefore the nocturnal moths which have the lowest threshold to light increase their sensitivity to dim light mainly by decreasing the slopes of V-log I curves.     

One molecule of diphtheria toxin fragment a introduced into a cell can kill the cell

Erythrocyte ghosts containing a known number of molecules of purified fragment A of diphtheria toxin with a constant amount of FITC-BSA as a fluorescence marker were prepared by dialyzing a mixture of erythrocytes and these substances against hypotonic solution. These substances were then introduced into diphtheria toxin-resistant mouse L cells by virus-mediated cell fusion of the cells with the ghosts, and mononuclear recipients that had fused with only one erythrocyte ghost were separated in a fluorescence-activated cell sorter (FACS) on the basis of their cell size and fluorescence intensity. After separation, the viability of cells containing known numbers of fragment A was examined by measuring colony-forming ability. The results demonstrated that a single molecule of fragment A was sufficient to kill a cell.This fact was confirmed by introduction into cells of fragment A from an immunologically related mutant toxin, CRM 176 (fragment A-176); this has a completely functional fragment B region, but in cell extracts, the enzymic activity of its fragment A is about 10 fold less than that of wild toxin. The cytotoxicity of CRM 176 is about two hundredths of that of the wild-type (Uchida, Pappenheimer and Greany, 1973). As expected, about 100–200 fold excess of fragment A-176 was needed to kill the cells.     

Methylamine facilitates demonstration of specific uptake of diphtheria toxin by CHO cell and toxin-resistant CHO cell mutants

We have measured the specific uptake of ¹²⁵I-labelled diphtheria toxin in the presence of methylamine by a number of cell lines with different sensitivities to diphtheria toxin. The results show a strong correlation between the toxin sensitivities of the cell lines and the amount of specific uptake. The specific association of labelled toxin with cells was clearly demonstrated even with CHO cells, a cell line with relatively low sensitivity. Thus, CHO cell mutants that are resistant to diphtheria toxin could be classified as toxin-binding or non-binding cells by this method.     

Onset of the constrictive effect of indomethacin on the ductus arteriosus in fetal rats.

The time of onset of the constrictive effect of indomethacin on the ductus arteriosus (DA) in fetal rats was assessed by measurement of the caliber of the DA after maternal treatment with indomethacin on days 19-21 of gestation. The day following overnight mating was regarded as day 0 of gestation. Observation was performed by direct exposure of the DA by hand shaving of intact frozen fetuses. On days 20 and 21, the DA was significantly constricted 3 h after maternal treatment with 1 mg/kg of indomethacin. When the DA was examined at 19 1/2 and 19 2/3 days of gestation (3 h after indomethacin exposure), it was significantly constricted at 19 2/3 days but not at 19 1/2 days. Higher doses of indomethacin (10 and 100 mg/kg) induced a significant constriction of the DA at day 19 1/2, but not at the beginning of the same day (1.00 a.m.). These results suggest that the onset of the susceptibility of the DA to the constrictive effect of indomethacin occurs in the first half of day 19 of gestation.     

Polyamine prevention of inhibition of rat liver isoleucyl-tRNA formation by poly(G), poly(I) or ribosomes.

It is shown that rat liver isoleucyl-tRNA formation in the presence of Mg²⁺ is inhibited by poly(G), poly(I) or ribosomes and that this inhibition is prevented by polyamines. The inhibition is found to be noncompetitive with respect to tRNA.