Prostaglandin E2 upregulates EGF-stimulated signaling in mitogenic pathways involving Akt and ERK in hepatocytes

Prostaglandins (PGs) such as PGE2 enhance proliferation in many cells, apparently through several distinct mechanisms, including transactivation of the epidermal growth factor (EGF) receptor (EGFR) as well as EGFR-independent pathways. In this study we found that in primary cultures of rat hepatocytes PGE2 did not induce phosphorylation of the EGFR, and the EGFR tyrosine kinase blockers gefitinib and AG1478 did not affect PGE2-stimulated phosphorylation of ERK1/2. In contrast, PGE2 elicited EGFR phosphorylation and EGFR tyrosine kinase inhibitor-sensitive ERK phosphorylation in MH1C1 hepatoma cells. These findings suggest that PGE2 elicits EGFR transactivation in MH1C1 cells but not in hepatocytes. Treatment of the hepatocytes with PGE2 at 3 h after plating amplified the stimulatory effect on DNA synthesis of EGF administered at 24 h and advanced and augmented the cyclin D1 expression in response to EGF in hepatocytes. The pretreatment of the hepatocytes with PGE2 resulted in an increase in the magnitude of EGF-stimulated Akt phosphorylation and ERK1/2 phosphorylation and kinase activity, including an extended duration of the responses, particularly of ERK, to EGF in PGE2-treated cells. Pertussis toxin abolished the ability of PGE2 to enhance the Akt and ERK responses to EGF. The results suggest that in hepatocytes, unlike MH1C1 hepatoma cells, PGE2 does not transactivate the EGFR, but instead acts in synergism with EGF by modulating mitogenic mechanisms downstream of the EGFR. These effects seem to be at least in part G(i) protein-mediated and include upregulation of signaling in the PI3K/Akt and the Ras/ERK pathways.     

Understanding protein folding: small proteins in silico

Recent improvements in methodology and increased computer power now allow atomistic computer simulations of protein folding. We briefly review several advanced Monte Carlo algorithms that have contributed to this development. Details of folding simulations of three designed mini proteins are shown. Adding global translations and rotations has allowed us to handle multiple chains and to simulate the aggregation of six beta-amyloid fragments. In a different line of research we have developed several algorithms to predict local features from sequence. In an outlook we sketch how such biasing could extend the application spectrum of Monte Carlo simulations to structure prediction of larger proteins.     

Copepods act as a switch between alternative trophic cascades in marine pelagic food webs

A recent meta‐analysis indicates that trophic cascades (indirect effects of predators on plants via herbivores) are weak in marine plankton in striking contrast to freshwater plankton ( Shurin et al. 2002 , Ecol. Lett., 5, 785–791). Here we show that in a marine plankton community consisting of jellyfish, calanoid copepods and algae, jellyfish predation consistently reduced copepods but produced two distinct, opposite responses of algal biomass. Calanoid copepods act as a switch between alternative trophic cascades along food chains of different length and with counteracting effects on algal biomass. Copepods reduced large algae but simultaneously promoted small algae by feeding on ciliates. The net effect of jellyfish on total algal biomass was positive when large algae were initially abundant in the phytoplankton, negative when small algae were dominant, but zero when experiments were analysed in combination. In contrast to marine systems, major pathways of energy flow in Daphnia‐dominated freshwater systems are of similar chain length. Thus, differences in the length of alternative, parallel food chains may explain the apparent discrepancy in trophic cascade strength between freshwater and marine planktonic systems.     

Calculating reaction rate constants and estimating the efficacy of selective enzyme inhibitors

The dynamic steady state of a pair of forward and backward enzymatic reactions is dependent on the balance between the enzymes catalyzing the reactions. By selectively inhibiting one or more of the enzymes involved, this balance is shifted into a new steady state, making it possible to calculate the reaction rate constants after measurement of the reactants. Ideally, the inhibitors should completely eliminate either reaction, but this is often not the case. Here we present and discuss a method for calculating the reaction rate constants and, thus, for evaluating the efficacy of one or more inhibitors when introduced to a forward-backward pair of enzymatic reactions.     

Thermal depolymerization of alginate in the solid state

A new method of introduction carboxyl groups to chitosan sulfate by the acylation reaction between hydroxyethyl chitosan sulfates and butane dioic anhydride in homogeneous solution was used to obtain carboxybutyrylated hydroxyethyl chitosan sulfates. The structures of the derivatives were characterized by element analysis, FT-IR, ¹³C-NMR, and gel permeation chromatography. The content and position of the carboxyl groups could be controlled favorably. Their anticoagulant activity was determined for human plasma with respect to activated partial thromboplastin time (APTT), thrombin time (TT), and prothombin time (PT). The introducing of carboxyl groups to amino groups greatly prolonged the APTT and TT. The best result occurred when the degree of substitution of the carboxyl groups was about 0.4/unit that prolonged APTT and TT with about 5 and 1.5 times compared to that of the uncarboxylated hydroxyethyl chitosan sulfates; another conclusion is that introducing of carboxyl groups into N,O-position gave better results than that just into N-positions. Low S% chitosan sulfate and 6-O-desulfated chitosan sulfate showed little anticoagulant activity but their N,O-carboxybutyrylated derivatives (0.6/unit ds) showed increased APTT or TT, while their N-carboxybutyrylated derivatives (0.6/unit ds) gave no improvement. Generally, the introducing of carboxyl groups could not increase PT in spite of the position introduced.     

Improved nitrogen removal by application of new nitrogen-cycle bacteria

In order to meet increasingly stringentEuropean discharge standards, new applicationsand control strategies for the sustainableremoval of ammonia from wastewater have to beimplemented. In this paper we discuss anitrogen removal system based on the processesof partial nitrification and anoxic ammoniaoxidation (anammox). The anammox process offersgreat opportunities to remove ammonia in fullyautotrophic systems with biomass retention. Noorganic carbon is needed in such nitrogenremoval system, since ammonia is used aselectron donor for nitrite reduction. Thenitrite can be produced from ammonia inoxygen-limited biofilm systems or in continuousprocesses without biomass retention. Forsuccessful implementation of the combinedprocesses, accurate biosensors for measuringammonia and nitrite concentrations, insight inthe complex microbial communities involved, andnew control strategies have to be developed andevaluated.     

Negative and Non-Positive Epidemiological Studies

Aim. To identify and discuss validity aspects on so called negative and non-positive studies. Methods. Arguments and examples are drawn from experiences in occupational health epidemiology regarding the interpretation of more or less equivocal study results. Results and conclusions. A negative study may be defined as showing a result that goes against the investigated hypothesis of an increased (or prevented) risk. Traditionally, studies with a risk estimate (relative risk or odds ratio) above but close to unity are also referred to as negative, given a narrow confidence interval (CI) that includes unity. A risk estimate above unity with the CI including unity is non-positive, however, but an estimate below unity with upper CI bond exceeding unity might be seen as possibly negative or non-negative. A weaker “significance” than usually required should perhaps be accepted when evaluating serious hazards. In contrast to positive studies, the negative and non-positive studies tend to escape criticism in spite of questionable validity that may have obscured existing risks (or preventive effects). Even stronger arguments can be made in criticising negative and non-positive studies than positive studies, for example, regarding selection phenomena, and observational problems regarding exposure and outcome. Negative confounding should be considered although usually weak. In case-control studies, so-called over-matching may obscure an existing risk as could the “healthy worker effect” in cohort studies. Small scale non-positive studies should be made available for meta-analyses and when considering studies that do not convincingly show a risk; those who are exposed should be given the “benefit of the doubt.”     

Sequence comparison and mutational analysis of elements that may be involved in the regulation of DNA synthesis in HIV-1

The large number of sequenced clones of HIV-1 and related viruses made it possible to indicate conserved elements with potential regulatory or structural functions. Such analysis was combined with directed mutagenesis in order to investigate the importance of elements that may influence the initiation of plus-strand DNA synthesis. The main site for plus-strand initiation is a polypurine tract near the 3′ end of the viral RNA (the 3′ PPT). An exact copy of this PPT is located in the middle of the genome (the internal PPT). Upstream from the internal PPT there is an inverted repeat. Mutants designed to upset the internal PPT (i.e., purine to pyrimidine changes), as well as mutants designed to abolish the potential stem-loop formation (changes around the internal PPT or in the upstream inverted repeat) both resulted in viruses with a reduced ability to replicate. Upsetting the stem-loop formation was, however, less harmful than changing the polypurine nature of the PPT. Changing a conserved T on the 3′ side of the PPT to a C did not affect the phenotype. Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992     

Automated counting of mammalian cell colonies by means of a flat bed scanner and image processing.

BACKGROUND: Clonogenic assays are used frequently to measure the cell killing and mutagenic effects of radiation and other agents. Clonogenic assays carried out manually are tedious and time-consuming and involve a significant element of subjectivity. However, several commercial automatic colony counters are available. Based on CCD video imaging and image analysis they are relatively expensive and can analyze only one petri dish at a time. METHOD: We have developed a cheaper and more efficient device, which employs a flat bed scanner to image 12 60-mm petri dishes at a time. Two major problems in automated colony counting are the clustering of colonies and edge effects. By using standard image analysis and implementing an inflection point algorithm, these problems were greatly diminished. The resulting system was compared with two manual colony counts, as well as with automated counts with the Oxford Optronix ColCount colony counter for cell lines V79 and HaCaT. RESULTS: Comparisons assuming the manual counts to be correct showed that our automatic counter was slightly more accurate than the commercial unit. CONCLUSIONS: As a whole, our automated colony counter performed significantly better than the commercial unit with regard to processing time, cost and accuracy.