Interannual variability of phytoplankton productivity and related parameters in Lake Constance: no response to decreased phosphorus loading?

In meso-eutrophic Lake Constance (Germany-Austria-Switzerland),phytoplankton bioraass, pigments and water transparency, aswell as primary productivity, have been followed between 1980and 1989. During this period, municipal phosphorus loading declinedsignificantly. Since 1981, soluble reactive phosphorus (SRP)concentrations during deep lake mixing have decreased from 3.0to currently 1 6 mmol m^–3 at a rate of 7% year^–1.Nitrate concentrations, by contrast, continued to rise. Duringthe period of maximum phosphorus loading, flushing through theoutlet and sedimentation were about equally important sinksof phosphorus from the euphotic zone. Recently, however, sedimentationand subsequent burial of P in the bottom deposits contributedabout three-quarters to the overall P-losses from the systemMain reasons for this shift are unchanged settling fluxes ofphosphorus out of the euphotic zone and decreasing concentrationsof total phosphorus in the water. Only during spring, do concentrations of soluble reactive phosphoruswithin the euphotic zone decrease in proportion to the formationof particulate organic matter. Later during the season, euphoticSRP concentrations continue to be low but are no longer matchedby high plankton biomass because phosphorus is efficiently removedby settling of particles In spite of the observed dramatic decreasein phosphorus loading since 1980, chlorophyll concentrationsand water transparency, as well as annual phytoplankton productivity(300 g C m^–2), have not shown a consistent downward trend.However, the intensity of phosphorus regeneration within theeuphoric zone, which can be used as a measure of the degreeof nutrient limitation, is likely to have increased significantlyThe most probable explanation for the insensitivity of importanttrophic state indicators to reduced nutrient loading is that,in Lake Constance, biomass accumulation to a greater extentis controlled by losses, mainly grazing by zooplankton and sedimentation,than by primary resources. This is concluded from the observationthat phytoplankton biomass always falls far short of the nutrient-dependentcarrying capacity of the system.     

Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli

Summary The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A--lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5pH6.0) and at low temperatures. These conditios were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn²⁺ ions. Offsprint requests to: G. Georgiou     

Characterization of a frequent polymorphism in the coding sequence of the Tp53 gene in colonic cancer patients and a control population

Summary We describe a simple method for characterizing a frequent polymorphism (that subsitutes an arginine for a proline) in the coding sequence of the Tp53 gene in patients with colonic cancer and in a control population. We could find no evidence that this polymorphism is associated with a marked predisposition to colorectal cancer.     

Effects of feminization of male F344 rats on induction of tumors and on nucleic acid alkylation by nitrosobis-(2-oxopropyl)amine

Groups of male and female F344 rats were treated twice weekly by gavage with 2.5 mg of nitrosobis-(2-oxopropyl)amine (BOP) for 35 weeks. Additional groups given the same treatment were male rats castrated at birth, male rats bearing an implant of a pellet containing estradiol and castrated male rats bearing an estradiol pellet. Most rats died with tumors related to the treatment; intact male rats survived the least well of the five groups. Most rats in all groups had alveolar/bronchiolar neoplasms of the lung. Many of the male rats also had follicular cell neoplasms of the thyroid and transitional cell neoplasms of the urinary bladder and kidney pelvis; there were no liver tumors in intact male rats. Almost all female rats and castrated male rats had liver neoplasms, including hepatocellular, cholangiocellular and hemangiosarcomatous neoplasms, but few neoplasms of the thyroid, kidney or bladder. The male rats feminized with estradiol, intact or castrated, had liver neoplasms, mainly cholangiocellular, and also neoplasms of the thyroid. Two rats of each of the five groups were treated at 20 weeks of age with [14C]BOP. As measured by respiration of 14CO2, metabolism of BOP was faster in the two groups of male rats with the estradiol implant than in the other groups. DNA and RNA of the liver were isolated 6 h after treatment. The extent of methylation of liver DNA as 7-methylguanine and O6-methylguanine was higher in the females and in the feminized males than in the intact male rats, but when normalized to the dose of nitrosamine per unit body weight there was little difference among the five groups.     

Secretion of lipoprotein lipid and synthesis of fatty acids, cholesterol and triacylglycerol by hepatocytes of fasted-refed rats

Synthetic rates of fatty acid, cholesterol and triacylglycerols, and contents and secretion of lipoprotein lipids, were determined in hepatocytes of rats fed ad libitum a fat-containing stock diet or of rats fasted for 48 h and then refed for 24 or 48 h with stock diet or with a glucose-rich fat-free diet. When compared with the values for the ad libitum-fed rats, fatty acid synthesis was lower in fasted rats, slightly increased in rats refed with the stock diet, but several-fold elevated after refeeding the glucose-rich fat-free diet. Cholesterol synthesis was decreased in the fasted cells, and restored to the control level upon refeeding either diet. Triacylglycerol synthesis from exogenous oleate was greatly stimulated in the cells of fasted-refed rats above the rate in cells of the ad libitum-fed rats, the increase being considerably higher after refeeding the glucose-rich fat-free diet than the stock diet. The amount of triacylglycerol secreted by the cells was also elevated by the fasting-refeeding treatment, but the difference between the two diets was much less pronounced than seen for the lipids' synthetic rates. This imbalance may underlie the huge accumulation of this lipid observed in the heptatocytes after refeeding the rats for 48 h with the glucose-rich fat-free diet.     

Chemical and physical characterization of rabbit sperm acrosome stabilizing factor

Rabbit Acrosome Stabilizing Factor (ASF) is an epididymal product that reversibly inhibits the process of sperm capacitation. The native molecular weights of the monomer and dimer ASF were determined from sedimentation and diffusion data at 129,000 and 259,000 Mr. The monomer is composed of 92,000 and 38,000 Mr subunits according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with size heterogeneity demonstrated for the latter. The stoichiometry of the subunits appears to be one-to-one by gel scanning. Amino acid and carbohydrate compositions are characteristic of a globular glycoprotein, which is high in cysteine content and is 8.3% carbohydrate by weight. The sugar composition suggests the presence of both high mannose and complex N-linked oligosaccharides with the unusual feature of appreciable amounts of glucose. The isoelectric character of ASF spans a range from 5.3 to 7.0.     

Characterization of human platelet basic protein, a precursor form of low-affinity platelet factor 4 and beta-thromboglobulin

Platelet basic protein (PBP) was purified from the supernatant of thrombin-stimulated, washed human platelets by ion-exchange, affinity, molecular sieve, and high-performance liquid chromatography (HPLC). The NH2-terminal amino acid sequence was determined by automated Edman degradation, revealing 9 unique residues followed by 10 residues of the established low-affinity platelet factor 4/beta-thromboglobulin (LA-PF4/beta TG) sequence. Among the nine were three basic residues, accounting for the high isoelectric point of PBP. Additional evidence for precursor status includes the immunological cross-reactivity of all three species and the ability of plasmin and trypsin to produce from PBP a species resembling beta TG in charge, hydrophobicity, and size. Tryptic peptide maps of PBP and LA-PF4 obtained by reverse-phase HPLC were very similar, and from each protein, a peptide was isolated which showed the amino acid composition predicted for the COOH-terminal tryptic peptide of beta TG. Normal platelets contained predominantly LA-PF4, with PBP ranging from 10% to 30% of total beta TG antigen. This was true even when fresh platelets were lysed with trichloroacetic acid in order to provide the most complete and rapid inhibition of proteolytic activity. beta TG itself was never detected in this situation or in the release supernatant of stimulated platelets, and only rarely in unprotected lysates. In agreement with earlier results, crude preparations of PBP were mitogenic for 3T3 cells, but highly purified preparations of PBP and LA-PF4 were free of this activity.     

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.