Menthol as an alternative anaesthetic and sedative for rainbow trout,Oncorhynchus mykiss

Menthol is known for its analgesic properties, but relatively little information is available on its potential as an anaesthetic on fish. The purpose of this study was to assess anaesthetic and sedative effectiveness of menthol and its safety in rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout 180 ± 28 g (mean ± SD) within a range of 152–208 g fish^?1 were individually exposed to menthol concentrations between 10 and 150 mg l^?1 and observed for behavioural responses, induction time to anaesthesia and recovery time. Menthol concentrations of 40–150 mg l^?1 induced anaesthesia with varying exposure times. There was an exponential relationship (p < 0.001) between induction time and menthol concentration. Menthol concentrations of 80–150 mg l^?1 induced anaesthesia within three minutes of exposure and fish recovered within three minutes. Induction and recovery data showed that 80 mg l^?1 was most suitable for anaesthesia in juveniles of this species. Concentrations of 10–20 mg l^?1 had sedative effects. Menthol stock solutions prepared using ethanol and acetone and storage time of stock solutions at room temperature for up to 48 h showed no significant differences in anaesthetic efficiency. When exposure time to menthol was kept constant at three minutes, 22% of fish had temporary cessation of gill ventilation. These fish had longer recovery times than those that did not show that response. Menthol was an effective anaesthetic and could be tested as a sedative for trout.     

Trend Analysis of Biological Parameters in the Baltic (1976-1988)

Eutrophication of the nature is one of the most relevant problems for the human society today. In comparison to terrestrial and limnological ecosystems, however, the marine environment is affected with some exceptions of coastal waters in a minor degree. On the basis of data from 1976–1988 trend analysis for chlorophyll, primary production, zooplankton biomass and water transparency have been carried out for the Mecklenburg Bight and different areas of the Baltic proper. As expected from the longterm increase in the nutrient levels, also for some pelagic biological variables increasing trends could be observed. At least for chlorophyll they are significant in the 95% probability level for all investigated areas. Primary production shows also an increase, however, not significant for each subarea. For zooplankton nearly no changes could be observed. All data reflect a high interannual variability, which can partly be explained by meteorological and oceanological conditions. The results are discussed from an ecological point of view. The increase in phytoplankton variables is considered to be at least partly related to the eutrophication of the Baltic.     

Cyclin E uses Cdc6 as a chromatin-associated receptor required for DNA replication

Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specifically and saturably to chromatin in three phases. In the first phase, the origin recognition complex and Cdc6 prereplication proteins, but not the minichromosome maintenance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin E--Cdk2 to DNA. We find that cyclin E binds the NH(2)-terminal region of Cdc6 containing Cy--Arg-X-Leu (RXL) motifs. Cyclin E proteins with mutated substrate selection (Met-Arg-Ala-Ile-Leu; MRAIL) motifs fail to bind Cdc6, fail to compete with endogenous cyclin E--Cdk2 for chromatin binding, and fail to rescue replication in cyclin E--depleted extracts. Cdc6 proteins with mutations in the three consensus RXL motifs are quantitatively deficient for cyclin E binding and for rescuing replication in Cdc6-depleted extracts. Thus, the cyclin E--Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin E--Cdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin. Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro. These three phases of cyclin E association with chromatin may facilitate the diverse activities of cyclin E--Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis.     

Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers

Assimilatory nitrate reductase (NR) of higher plants is a most interesting enzyme, both from its central function in plant primary metabolism and from the complex regulation of its expression and control of catalytic activity and degradation. Here, present knowledge about the mechanism of post-translational regulation of NR is summarized and the properties of the regulatory enzymes involved (protein kinases, protein phosphatases and 14-3-3-binding proteins) are described. It is shown that light and oxygen availability are the major external triggers for the rapid and reversible modulation of NR activity, and that sugars and/or sugar phosphates are the internal signals which regulate the protein kinase(s) and phosphatase. It is also demonstrated that stress factors like nitrate deficiency and salinity have remarkably little direct influence on the NR activation state. Further, changes in NR activity measured in vitro are not always associated with changes in nitrate reduction rates in vivo, suggesting that NR can be under strong substrate limitation. The degradation and half-life of the NR protein also appear to be affected by NR phosphorylation and 14-3-3 binding, as NR activation always correlates positively with its stability. However, it is not known whether the molecular form of NR in vivo affects its susceptibility to proteolytic degradation, or whether factors that affect the NR activation state also independently affect the activity or induction of the NR protease(s). A second and potentially important function of NR, the production of nitric oxide (NO) from nitrite is briefly described, but it remains to be determined whether NR produces NO for pathogen/stress signalling in vivo.     

Localization of injected apolipophorin III in vivo - new insights into the immune activation process directed by this protein

A few years ago, it was shown that intrahemocoelic injection of the insect apolipoprotein apolipophorin III (apoLp-III) stimulates an immune response in larvae of the greater wax moth, Galleria mellonella. Since the mode of action of this activation process is unknown, we followed apoLp-III's pathway in the early phase of the immune-stimulating process, using biotin as a probe. Biotinylated apoLp-III was injected and localized using avidin-coupled horseradish peroxidase. The labeled protein was fully functional; the added amount of biotin per apoLp-III molecule used in this study only slightly decreased its ability to associate with phospholipase C-treated human low-density lipoprotein, as well as the immune-stimulating capability of apoLp-III.Gel electrophoresis with subsequent staining of biotin moieties and lipids revealed that apoLp-III undergoes lipid association in vivo within the first few minutes after injection. After two hours, no biotinylated apoLp-III was detectable in cell-free hemolymph. At this time, a subpopulation of hemocytes showed a distinct peroxidase staining. Control injections of biotinylated bovine serum albumin did not lead to similar results, giving evidence for the specificity of the phenomena observed. The results indicate that lipid association of apoLp-III occurs prior to endocytosis by immune-competent hemocytes, which is followed by the induction of a humoral immune response.     

Incorporation of beta-selenolo[3,2-b]pyrrolyl-alanine into proteins for phase determination in protein X-ray crystallography

beta-Selenolo[3,2-b]pyrrolyl-L-alanine that mimics tryptophan with the benzene ring of the indole moiety replaced by selenophene, was incorporated into human annexin V and barstar. This was achieved by fermentation and expression in a Trp-auxotrophic Escherichia coli host strain using the selective pressure incorporation method. The seleno- proteins were obtained in yields comparable to those of the wild-type proteins and exhibit full crystallographic isomorphism to the parent proteins, but expectedly show altered absorbance profiles and quenched tryptophan fluorescence. Since the occurrence of tryptophan residues in proteins is rare, incorporation of the electron-rich selenium-containing tryptophan surrogate into proteins represents a useful supplementation and even a promising novel alternative to selenomethionine for solving the phase problem in protein X-ray crystallography.     

Increased glutamine synthetase activity and changes in amino acid pools in leaves treated with 5-aminoimidazole-4-carboxiamide ribonucleoside (AICAR)

Feeding 5-aminoimidazole-4-carboxiamide ribonucleoside (AICAR) through the petiole of detached young barley leaves rapidly increased activities of NADH-nitrate reductase (NR) and glutamine synthetase (GS) in leaf extracts and at least partly prevented the usual slow decrease of these enzyme activities during prolonged illumination. Further, AICAR caused drastic changes in amino acid levels: glutamine and serine levels were increased whereas glutamate and glycine were decreased, probably indicating a higher GS activity and more rapid conversion of glycine into serine. The latter may be responsible for the higher ammonium contents found in AICAR treated leaves. We tentatively suggest that GS (located in the chloroplast) and glycine decarboxylase (located in the mitochondria) are regulated in a manner similar to NR. This is discussed in the light of recent reports that 14-3-3 isoforms exist in chloroplasts and that GS binds to 14-3-3s in vitro.     

Pro-adhesive and chemotactic activities of thrombospondin-1 for breast carcinoma cells are mediated by alpha3beta1 integrin and regulated by insulin-like growth factor-1 and CD98

Thrombospondin-1 (TSP1) is a matricellular protein that displays both pro- and anti-adhesive activities. Binding to sulfated glycoconjugates mediates most high affinity binding of soluble TSP1 to MDA-MB-435 cells, but attachment and spreading of these cells on immobilized TSP1 is primarily beta1 integrin-dependent. The integrin alpha3beta1 is the major mediator of breast carcinoma cell adhesion and chemotaxis to TSP1. This integrin is partially active in MDA-MB-435 cells but is mostly inactive in MDA-MB-231 and MCF-7 cells, which require beta1 integrin activation to induce spreading on TSP1. Integrin-mediated cell spreading on TSP1 is accompanied by extension of filopodia containing beta1 integrins. TSP1 binding activity of the alpha3beta1 integrin is not stimulated by CD47-binding peptides from TSP1 or by protein kinase C activation, which activate alphavbeta3 integrin function in the same cells. In MDA-MB-231 but not MDA-MB-435 cells, this integrin is activated by pertussis toxin, whereas serum, insulin, insulin-like growth factor-1, and ligation of CD98 increase activity of this integrin in both cell lines. Serum stimulation is accompanied by increased surface expression of CD98, whereas insulin-like growth factor-1 does not increase CD98 expression. Thus, the pro-adhesive activity of TSP1 for breast carcinoma cells is controlled by several signals that regulate activity of the alpha3beta1 integrin.     

The proto-oncogene c-myc is a direct target gene of Epstein-Barr virus nuclear antigen 2

Epstein-Barr virus (EBV) infects and transforms primary B lymphocytes in vitro. Viral infection initiates the cell cycle entry of the resting B lymphocytes. The maintenance of proliferation in the infected cells is strictly dependent on functional EBNA2. We have recently developed a conditional immortalization system for EBV by rendering the function of EBNA2, and thus proliferation of the immortalized cells, dependent on estrogen. This cellular system was used to identify early events preceding induction of proliferation. We show that LMP1 and c-myc are directly activated by EBNA2, indicating that all cellular factors essential for induction of these genes by EBNA2 are present in the resting cells. In contrast, induction of the cell cycle regulators cyclin D2 and cdk4 are secondary events, which require de novo protein synthesis.