Defects in protein modification precede developmental defects in l(3)c21RRW630, a temperature-sensitive Drosophila developmental mutant

The temperature-sensitive Drosophila developmental mutation, l(3)c21RRW630 (abbreviated RW630) disturbs oogenesis and has a maternal effect on embryogenesis. At restrictive temperature, RW630 alters post-translational modification of three abundant proteins. To examine the causal relationship between these biochemical defects and the developmental defects in RW630, a series of temperature-shift experiments was performed. It was found that defects in protein modification could be detected in RW630 ovaries after RW630 females had been exposed to restrictive temperature for 1 day. RW630 females treated in this fashion produce embryos which contain a low level of unmodified proteins. Nevertheless, these embryos hatch at a normal rate. Since these ovaries and these embryos are developmentally normal, but do show defects in protein modification, it is unlikely that the RW630 developmental defects cause the biochemical defects in RW630. It is more likely that accumulation of unmodified proteins after extended exposure to restrictive temperature produces the developmental defects in RW630.     

Phosphorylation of the oligosaccharide of uteroferrin by UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferases from rat liver, Acanthamoeba castellani, and Dictyostelium discoideum requires alpha 1,2-linked mannose residues

We have investigated the oligosaccharide requirements of the UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferases from rat liver, Acanthamoeba castellani, and Dictyostelium discoideum. Uteroferrin, an acid hydrolase, was phosphorylated by the three N-acetylglucosaminylphosphotransferases, and the phosphorylated oligosaccharides were isolated and analyzed by ion suppression high performance liquid chromatography. In all three cases, the phosphorylated species contained 6 or more mannose residues. Phosphorylation of the Man5GlcNAc2 oligosaccharide could not be detected even though this was the major species on the native uteroferrin. The Man5GlcNAc2 oligosaccharides lack alpha 1,2-linked mannose residues, whereas the larger oligosaccharides contain 1 or more mannose residues in this linkage. Treatment of intact uteroferrin with an alpha 1,2-specific mannosidase-generated molecules whose oligosaccharides consisted almost entirely of species with 5 mannose residues. The N-acetylglucosaminylphosphotransferases could no longer phosphorylate such molecules. These data indicate that at least 1 alpha 1,2-linked mannose residue must be present on uteroferrin's oligosaccharide for phosphorylation to occur.     

Molecular mobility and nucleocytoplasmic flux in hepatoma cells

Fluorescence microphotolysis (photobleaching) was used to measure, in single polyethylene glycol-induced polykaryons of hepatoma tissue culture cells, nucleocytoplasmic flux and intracellular mobility for a series of dextrans ranging in molecular mass from 3 to 150 kD and for bovine serum albumin. For the dextrans, the cytoplasmic and the nucleoplasmic translational diffusion coefficients amounted to approximately 9 and approximately 15%, respectively, of the value in dilute buffer. The diffusion coefficients depended inversely on molecular radius, suggesting that diffusion was dominated by viscosity effects. By application of the Stokes-Einstein equation, cytoplasmic and nucleoplasmic viscosities were derived to be 6.6 and 8.1 cP, respectively, at 23 degrees C. Between 10 and 37 degrees C nucleoplasmic diffusion coefficients increased by approximately 45-85%, whereas cytoplasmic diffusion coefficients were virtually independent of temperature. In contrast to that of the dextrans, diffusion of bovine serum albumin was more restricted. In the cytoplasm the diffusion coefficient was approximately 1.5% of the value in dilute buffer; in the nucleus albumin was largely immobile. This indicated that albumin mobility is dominated by association with immobile cellular structures. Nucleocytoplasmic flux of dextrans depended inversely on molecular mass with an exclusion limit between 17 and 41 kD. This agrees with previous measurements on primary hepatocytes (Peters, R., 1984, EMBO [Eur. Mol. Biol. Organ.] J. 3:1831-1836), suggesting that in both cell types the nuclear envelope has properties of a molecular sieve with a functional pore radius of approximately 55 A.     

Application of intracellular ATP determination in lymphocytes for HLA-typing

The amount of adenosine triphosphate (ATP) in human lymphocytes was determined using a technique based on light emission from a bioluminescent reaction with luciferin-luciferase. The amount of ATP changed when cells were incubated in the presence of specific HLA antisera and complement. For determination of intracellular ATP a modified method was applied, which was based on reduction of extracellular ATP by the addition of ATPase. The results of titration of an anti-human lymphocyte serum using the bioluminescence assay were in agreement with the results of fluorescence vitality staining. Bioluminescent HLA-determination in 57 cell samples each tested with 5 different antisera also gave good agreement (95.8%) with the conventional method. From these experimental data the calculated ATP content per lymphocyte was 0.135 ± 0.058 pg ATP.     

Selective induction of coumarin 7-hydroxylase by pyrazole in D2 mice

Pyrazole, was given to DBA/2N (D2), C57BL/6N (B6) and AKR/N mice to study its effects on hepatic drug metabolism. A decrease in the total amount of microsomal cytochrome P-450 as well as in the activities of ethylmorphine demethylase and benzo[a]pyrene hydroxylase was found. On the other hand ethoxycoumarin de-ethylase was increased 1.5-2.5-fold (depending on the strain of mouse) and coumarin 7-hydroxylase as much as sevenfold (but only in D2 mice) after pyrazole treatment. This increase was much higher than that caused by phenobarbital, the only well known inducer of coumarin 7-hydroxylase. By reconstituting the mono-oxygenase complex after purification of cytochrome P-450 we found a 40-fold increase in coumarin 7-hydroxylase and eightfold increase in ethoxycoumarin de-ethylase after pyrazole treatment. This was found only in D2 mice. An antibody previously developed against a cytochrome P-450 fraction from the the D2 strain with a high coumarin 7-hydroxylase activity inhibited the microsomal coumarin 7-hydroxylase almost 100% after pyrazole pretreatment of the animals. In the case of control or phenobarbital-treated mice the inhibition was somewhat weaker. With the reconstituted mono-oxygenase complex the inhibition of coumarin 7-hydroxylase was almost 100% both for control and pyrazole-treated D2 mice. The data indicate that pyrazole causes an induction of the microsomal monooxygenase complex different from that caused by phenobarbital or 3-methylcholanthrene and selective for coumarin 7-hydroxylation or 7-ethoxycoumarin de-ethylation. This induction was strong in D2, weak in B6 and absent in AKR/N mice.     

Eutrophication of Lakes Leman and Neuchâtel (Switzerland) indicated by oligochaete communities

In 1978–80, oligochaete communities of meso-eutrophic Lake Léman (Lake of Geneva) were compared to those of mesotrophic Lake Neuchâtel. Worm species were classified into three groups corresponding to their increasing tolerance to eutrophication: (1) oligotrophic species, mostly Peloscolex velutinus, Stylodrilus heringianus; (2) mesotrophic species, mostly Potamothrix vejdovskyi, P. bedoti; (3) eutrophic species, mostly Potamothrix hammoniensis, P. heuscheri, Tubifex tubifex. In both lakes, eutrophic species constituted the bulk of the communities in terms of absolute abundance. However, relative abundance of mesotrophic and eutrophic species was higher in Lake Léman; oligotrophic species were more important in Lake Neuchâtel. These data confirmed the trophic classification of lakes based on chemical parameters. The number of zero values, which perturbated statistical analysis, was reduced by using species groupings instead of isolated species. Thus, making the lakes more comparable even if different species were present in each one. Relative density values based on all samples were distributed among 4 density classes for the 3 species groupings. The 12 resulting frequencies described the community structure expressed in terms of eutrophication. Furthermore, these frequencies may be used for comparison of eutrophication levels in several lakes.     

Angiotensin biosynthesis and concentrations in brain of normotensive and hypertensive rats

We report here on the extraction and characterization of angiotensin I (ANG I) and angiotensin II (ANG II) from the brain of rats. High pressure liquid chromatography (HPLC) with different mobile phases combined with specific radioimmunoassays (RIA) proved to be a powerful tool for peptide characterization in biological samples; (Ile5)-ANG I, (Ile5)-ANG II and (Ile5)-ANG III could clearly be identified in cerebrospinal fluid (CSF), incubated in vivo and in vitro with renin, in total brain extracts, as well as in hypothalamus (HT), medulla oblongata (MO), cerebellum (CER) and cortex (CO). Angiotensin cleaved from CSF angiotensinogen and angiotensin extracted from brain showed retention times identical to those of plasma angiotensin and synthetic standard peptides, indicating that their amino acid sequence is probably identical. ANG I and ANG II were highest in the HT and lowest in the CO. Following bilateral nephrectomy (NX) both ANG I and ANG II persisted at control levels. Young 10 week old spontaneously hypertensive rats (SHRSP) showed significantly lower ANG I and ANG II concentrations in the HT compared with Wistar Kyoto rats (WKY). Intracerebroventricular (i.c.v.) administration of the converting enzyme inhibitor captopril caused a significant increase in ANG 1 in nephrectomized SHRSP but not in WKY. These differences were not found in 40 week old SHRSP. The data show that ANG I and ANG II are synthetized in the brain of rats. The lower concentrations and the enhanced accumulation of ANG I after converting enzyme blockade in nephrectomized young SHRSP indicate an increased turnover of angiotensin in hypertensive rats.     

Use of agar-based diet to fulfil the food and water requirements of mice

Mice were fed an agar-based diet without an additional source of water for 5 weeks. In comparison with a similar group of mice fed a commercial diet and water ad libitum, there were no significant changes in bodyweight.     

Passive Transfer of Lambert-Eaton Myasthenic Syndrome in Mice: Decreased Rates of Resting and Evoked Release of Acetylcholine from Skeletal Muscle

Mice were injected for 1-2 months daily with 10 mg immunoglobulin G (IgG) from four patients with Lambert-Eaton myasthenic syndrome (LEMS); control mice were injected with pooled human IgG from normal donors. Gastrocnemius muscles were homogenised for the assay of acetylcholine (ACh), choline acetyltransferase (ChAT), and cholinesterase (ChE). The ACh, ChAT, and ChE contents of gastrocnemius muscles from "LEMS mice" were about the same as the control values, which were 180 pmol, 40 nmol X h-1 (37 degrees C), and 15 mumol X h-1 (37 degrees C), respectively. Hemidiaphragms were treated with an irreversible ChE inhibitor (Soman) and incubated at 20 degrees C for estimation of ACh release. Resting ACh release from experimental muscles was reduced by about 25% (P2 less than 0.05) and the release evoked by 3 s-1 nervous stimulation by 50% (P2 less than 0.05). On the other hand, 50 mM KCl-induced transmitter release was not abnormal in LEMS mice. The findings indicate that IgG antibody from patients with LEMS may bind to nerve terminal determinants that are involved in quantal and nonquantal ACh release.