Hygrophiloside,an iridoid glucoside from Hygrophila difformis (Acanthaceae)

A new iridoid glucoside, hygrophiloside, has been isolated from Hygrophila difformis. Hygrophiloside is apparently identical to the so-called ‘Cardanthera-Pseudoindican’. Its structure has been established by spectroscopic means and by reduction to isoaucubin.     

The structure of two distinct pancreatic amylase genes in mouse strain YBR

The amylase complex on mouse chromosome 3 encodes both salivary and pancreatic amylase. It appears that one active gene is present for salivary amylase, whereas pancreatic amylase in some strains is coded by at least 4, and perhaps by more than 10, genes. Strain YBR is different from other strains in that it produces twice as much salivary amylase. Pancreatic amylase in YBR is present as two different protein forms, A and B, the sum of which amounts to only one-third of that in, for instance, strain A/J. YBR chromosomal DNA was cloned in phage , followed by restriction and heteroduplex analysis of recombinant phages carrying amylase genes. Among 32 phage isolates, 5 carried parts of the salivary amylase sequence. The remaining phage isolates contained pancreatic amylase-like sequences and represented three nonoverlapping genomic regions, i.e., one of 34 kb containing a complete gene, PAN-II; another of 41 kb with a complete but different gene, PAN-I, plus a truncated gene, PAN-1; and finally, one of 23 kb with another truncated gene, PAN-2. Parts of the amino acid sequence of A and B have previously been determined, and we report here the sequencing of a 4-kb DNA fragment from Pan-II which establishes that this gene codes for B.This work was supported by the Danish Natural Science Research Council.     

Proenzyme to urokinase-type plasminogen activator in the mouse in vivo

We have investigated whether urokinase-type plasminogen activator (u-PA) is present in the mouse in vivo as the proenzyme or as the active enzyme. u-PA in extracts of various murine tissues was of a one-polypeptide chain form with an electrophoretic mobility indistinguishable from purified proenzyme (pro-u-PA), as demonstrated by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by immunoblotting. No 2-chain u-PA was detected in any of the extracts (detection limit 10% of that of one-chain u-PA). In bladder urine more than half of the u-PA was of the one-chain form. Together with previous immunocytochemical studies of the normal murine tissues and studies of the Lewis lung carcinoma, the present results indicate that in these tissues the one-chain proenzyme is the predominant form of u-PA in intracellular stores and for the first time demonstrates that at least in some cases the one-chain form constitutes a sizeable fraction of the u-AP in extracellular fluids in the intact organism.     

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitro-quinoxaline-2,3-dione ([3H]-CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 +/- 9.0 nM and Bmax = 3.56 +/- 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1 = 9.0 +/- 3.5 nM, Bmax = 0.15 +/- 0.05 pmol/mg protein, KD2 = 278 +/- 50 nM, and Bmax = 1.54 +/- 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L-[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.     

Coordination of growth and differentiation in the fetal lung

The male fetal lung begins to synthesize surfactant later in gestation than the female. This delay appears to be caused by androgens. We hypothesized that male fetal lung differentiation is delayed as a consequence of an extended phase of growth which is elicited by androgens. We observed that in vivo fetal lung protein synthesis relative to DNA synthesis peaked earlier in gestation in the female fetal lung and that this event was synchronous with the onset of differentiation. Pregnant rats were treated with dihydrotestosterone (DHT) during pregnancy, and fetal lung growth parameters were measured. Lung wet weight, dry weight, and DNA and protein concentrations were significantly elevated by DHT treatment. Type II cells and fibroblasts were isolated from lungs of DHT-treated fetuses. The number of total cells recovered was increased by 30%; the number of type II cells recovered was increased by 87%; and the number of fibroblasts recovered was increased by 42%. The type II cells which were recovered exhibited increased incorporation of [3H]thymidine into DNA and a reduced ratio of radiolabeled protein to radiolabeled DNA compared to that of cells from control lungs. Further studies were done in vitro with fibroblasts and type II cells isolated from untreated fetal rat lungs. Treatment of the fibroblasts with DHT during culture caused an increase in thymidine incorporation into DNA. This effect was not blocked by simultaneous treatment with cortisol, which normally causes reduced DNA synthesis and induces fibroblast differentiation. Treatment of the type II cells with DHT in culture caused a dose-dependent increase in cell number but a decrease in synthesis of disaturated phosphatidylcholine. These studies provide more direct evidence of the interrelationships between the control of growth and the control of differentiation in the fetal lung. DHT, a signal which delays the onset of expression of differentiation, also induces growth. We conclude that the controls of growth and of differentiation of the fetal lung are reciprocally linked.     

Transtubular transport of proteins in rabbit proximal tubules

The purpose of the present experiments was to study possible different pathways of intracellular transport of proteins after luminal and basolateral uptake in isolated rabbit proximal tubules. Tubules were exposed to cationized ferritin (CF) in the perfusion fluid and horseradish peroxidase (HRP) in the bath simultaneously or to HRP in the bath alone for 30 min. The peritubular fluid (bath) and perfusion fluid were then exchanged and the tubules either fixed immediately or allowed to function during chase-periods for 10, 20, 30, or 60 min before fixation to follow the migration of the proteins through the cells. The proteins were to a large extent found separated in different vacuoles and lysosomes at all time periods studied, indicating separate pathways after uptake via the luminal and basolateral membranes respectively. About 0.5% of the CF taken up by the cells was transported through the cells and became located in the intercellular spaces. HRP was transported from the peritubular fluid to the apical cytoplasm of the tubules indicated by a gradual accumulation of small HRP-containing vesicles, first in the basal part of the cells and then in the apical cytoplasm. In tubules perfused with both CF and HRP in the perfusate, the CF and HRP were found together in apical vacuoles and lysosomes. After perfusion with HRP alone, this tracer was found in similar large vacuoles and lysosomes in the apical cytoplasm, in contrast to the small HRP-filled vacuoles seen after uptake from the bath.     

The mutagenic effect of gamma rays on leaf protoplasts of haploid and dihaploid Nicotiana plumbaginifolia,estimated by valine resistance mutation frequencies

Summary Leaf protoplasts isolated from haploid and dihaploid Nicotiana plumbaginifolia plantlets were treated with different doses of gamma-rays and their survival was determined by scoring for plating efficiency at each irradiation dose. A fixed number of surviving protoplast-derived colonies was then plated in the presence of inhibitory concentrations of L-valine and incubated until growing resistant calli could be scored and mutation rates calculated. Though haploid protoplasts were found to be a little more sensitive than dihaploids to the lethal effect of radiation, the two dose-response curves of gamma-rays that induced mutagenesis were very similar. The irradiation dose capable of causing a ten-fold increase of spontaneous mutation frequencies was about 500 rads with both haploid and dihaploid protoplasts.Contribution No. 2173 of the Biology Radiation Protection and Medical Research Programme, Directorate General XII of the Commission of the European Communities     

Structural similarity between legumin and vicilin storage proteins from legumes.

The primary structures for several members of both the vicilin and legumin families of storage proteins were examined using a computer routine based on amino acid physical characteristics. The comparison algorithm revealed that sequences from the two families could be aligned and share a number of predicted secondary structural features. The COOH-terminal half of the subunits in both families displayed a highly conserved core region that was largely hydrophobic and in which a high proportion of the residues were predicted to be in beta-sheet conformations. The central region of the molecules which contained mixed areas of predicted helical and sheet conformations showed more variability in residue selection than the COOH-terminal regions. The NH2-terminal segments of subunits from the two different families could not be aligned though they characteristically had a high proportion of residues predicted to be in helical conformations. The feature which most clearly distinguished subunits between the two families was an inserted span in the legumin group with a high proportion of acidic amino acids located between the central and COOH-terminal domains. Residues in this insertion were predicted to exist mainly in helical conformation. Since considerable size variation occurs in this area amongst the legumin subunits, alterations in this region may have a minimal detrimental effect on the structure of the proteins.