Protein digestion and proteolytic activity in the digestive tract of an omnivorous cyprinid

1. In adult and juvenile roach (Rutilus rutilus) feeding on meal worms or grass and acclimated to temperatures between 6 and 20 degrees C the following variables were determined: pH, protein and proteolytic activity of gut fluid and faeces, food consumption, duration of gut passage and efficiency of protein assimilation. 2. Proteolytic enzymes of fish are very stable against autolysis but they disappear in posterior portions of the intestine, suggesting the existence of a pinocytotic process. 3. In herbivorous roach as well as in adult carnivorous roach feeding at 20 degrees C this results in very low proteolytic activities in the faeces, whereas in juvenile fish and in adults feeding on meal worms at lower temperatures, the process of reabsorption seems to be less efficient. 4. Daily production of proteases as well as "daily proteolytic duration" are higher in herbivorous than in carnivorous roach. 5. For the same amount of protein consumed, fish feeding on grass require 10 times higher proteolytic activities than fish feeding on meal worms.     

Mitotic activity and cell elongation in geostimulated roots of Zea mays

Mitotic activity was investigated in the primary meristem of horizontally oriented excised root tips of Zea mays during the first six hours of their georeaction. The only statistically significant change that could be detected in the meristem was a decrease of the length of its upper half. No significant difference in mitotic activity was found between the upper and lower halves of roots kept continuously horizontal for 6 h. Cell proliferation thus seems relatively insensitive to changes in the redistribution of endogenous growth regulators that are believed to occur within the meristem during the onset of geotropism. In the zone of bending proximal to the meristem cell length was significantly greater in the upper half than in either the lower half or in the equivalent position in vertical control roots. Thus, cell elongation seems to be promoted in the upper half of the horizontal root. Thus, The differences in cell length were not accompanied by any change in the proportion of nuclei synthesising DNA in these elongating, non-meristematic cells.     

Primary and secondary structure specificity of the cleavage of 'single-stranded' DNA by endonuclease Hinf I.

The interaction of endonuclease Hinf I with single-stranded fd DNA was examined. The sizes of the cleavage products indicate that the enzyme cuts this substrate at the same sequences as double-stranded DNA (GANTC). To determine whether or not the recognition sites in single-stranded DNA have to be present in double-stranded form in order to be cleaved, DNA fragments containing complementary or non-complementary Hinf I sequences were prepared and treated as substrates. The results suggest that completely base-paired recognition sites are necessary for cleavage. Sequences surrounding the Hinf I pentanucleotides significantly modulate the reaction rates.     

The interactions between indomethacin and cytotoxic drugs in mice bearing B-16 melanomas

We have compared the effects of indomethacin alone (100 microgram/mouse/day) with those of indomethacin plus adriamycin, 5-FU, nitrogen mustard, thioTEPA, and vincristine on B-16 tumor cell proliferation in vivo. As we have previously described, after four days of treatment with indomethacin, subcutaneous tumors were slightly smaller and lighter in weight, but contained more melanoma cells. Addition of indomethacin to cytotoxic regimens resulted in either no change or a decrease in the effectiveness of the chemotherapy. In previous studies we demonstrated that treatment of tumor-bearing mice with a long-acting synthetic analogue of PGE2 (di-M-PGE2) stimulated the synthesis of endogenous prostaglandins. In order to evaluate if these endogenously synthesized prostaglandins were responsible for the inhibition of B-16 growth in vivo, mice were treated with di-M-PGE2 or di-M-PGE2 plus indomethacin. Addition of indomethacin did not alter the tumor inhibitory effects of di-M-PGE2.     

Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle.

In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a.     

A major lienal phosphotyrosine phosphatase is inhibited by phospholipids and inositol trisphosphate.

A major "non-receptor" phosphotyrosine-specific protein phosphatase isolated from the 30,000g pellet fraction of porcine spleen is related to the human T-cell tyrosine phosphatase (Cool et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5257-5261) and is strongly inhibited by micromolar concentrations of phosphatidyl inositol (IC50 6 microM) and phosphatidyl serine (IC50 3.7 microM). In addition, the enzyme is inhibited by myo-inositol 1,4,5-trisphosphate (IC50 ca. 2 microM) in a non-competitive manner but not by myo-inositol hexaphosphate. Since the overall cellular tyrosine phosphatase activity greatly exceeds tyrosine kinase activity, inhibition of the phosphatase may be of importance for the regulation of the extent of tyrosine phosphorylation of cellular proteins.     

Regional distribution of brain-derived neurotrophic factor mRNA in the adult mouse brain.

Brain-derived neurotrophic factor (BDNF) is a protein that allows the survival of specific neuronal populations. This study reports on the distribution of the BDNF mRNA in the adult mouse brain, where the BDNF gene is strongly expressed, using quantitative Northern blot analysis and in situ hybridization. All brain regions examined were found to contain substantial amounts of BDNF mRNA, the highest levels being found in the hippocampus followed by the cerebral cortex. In the hippocampus, which is also the site of highest nerve growth factor (NGF) gene expression in the central nervous system (CNS), there is approximately 50-fold more BDNF mRNA than NGF mRNA. In other brain regions, such as the granule cell layer of the cerebellum, the differences between the levels of BDNF and NGF mRNAs are even more pronounced. The BDNF mRNA was localized by in situ hybridization in hippocampal neurons (pyramidal and granule cells). These data suggest that BDNF may play an important role in the CNS for a wide variety of adult neurons.     

Translation of mRNA from rat-liver polysomes into tyrosine aminotransferase and tryptophan oxygenase in a protein-synthesizing system from wheat germ. Effects of cortisol on the translatable levels of mRNA for these two enzymes.

Messenger RNA was isolated from rat liver polysomes by phenol/chloroform extraction and subsequent oligo(dT)-cellulose chromatography. The mRNA was translated in a protein-synthesizing system in vitro derived from wheat germ. The system was optimized in respect to Mg2+ and K+. The presence of spermidine or spermine is necessary for the synthesis of polypeptides having molecular weights of over 20 000. In the absence of the bases only small molecular weight products are formed. The amount of protein synthesized is linearly dependent on the amount of mRNA added up to concentrations of 80 mug mRNA/ml. The synthesis of tyrosine aminotransferase and tryptophan oxygenase in the system in vitro has been demonstrated by specific immunoprecipitation and sodium-dodecylsulfate polyacrylamide gel electrophoresis of the precipitate with enzyme proteins as marker. The amount of specific product formed is linearly dependent on the amount of mRNA present. The amount of translatable tyrosine aminotransferase mRNA and tryptophan oxygenase mRNA increases after administration of hydrocortisone to adrenalectomized rats. At low doses of hormone (2 mg/100 g body weight) maximal values are observed at 4 h, control levels being reached at 6-8 h after hormone application. With higher doses of hydrocortisone (20 mg/100 g body weight) maximal values are attained at 6 h, tending to control levels 14 h after treatment. The enzyme activity curves are parallel to the mRNA curves, the peak of enzyme activity occurring 2 h after the peak of mRNA activity.     

Phosphorylation of phosphofructokinase by protein kinase C changes the allosteric properties of the enzyme

The Ca2+- and phospholipid-dependent protein kinase C from rat brain phosphorylates rabbit muscle phosphofructokinase at the same trypsin-labile site as cyclic AMP-dependent protein kinase. However, protein kinase C also effectively phosphorylates one or more separate sites. Incubation of phosphofructokinase in the presence of protein kinase C, phospholipids, Ca2+, and ATP appears to affect the allosteric properties of phosphofructokinase by shifting the fructose 6-phosphate saturation curve to lower substrate concentrations in a time-dependent manner and decreasing cooperativity of the enzyme.     

Growth and habitat separation in eight cohorts of three species of cyprinids in a subalpine lake

Synopsis Distribution and growth of the embryos, larvae and juveniles of Rutilus rutilus (roach), Scardinius erythrophthalmus (rudd) and Leuciscus cephalus (chub) from an oligotrophic subalpine lake in Tyrol, Austria, were studied during the first three to four months after hatching. R. rutilus was the first to spawn, a single cohort hatching around May 23rd. Four cohorts of S. erythrophthalmus hatched between June 19 and August 1. Three cohorts of L. cephalus hatched between July 3 and 25. The length/weight relationship of all species changed at a length of approximately 15–16 mm. R. rutilus, hatching at the lowest temperature, also showed the lowest growth rate during early life (maximum 10.4 per cent fresh body weight day^–1). In the other two species relative growth rates up to 20% day^–1) were measured. Rudd and chub remained in the shallow littoral during the whole period of observation, whereas roach left the littoral a few weeks after hatching and migrated into deeper water. A subtle shift in vertical distribution was observed for the first cohort of rudd which moved into slightly deeper water when the second cohort made its appearance.To whom correspondence should be addressed