32P-postlabelling of 7-methyl-dGMP ring-opened 7-methyl-dGMP and platinated dGpdG

The 32P-postlabelling technique introduced by Randerath and coworkers was used to investigate the efficiency of the phosphorylation reaction by T4 polynucleotide kinase using three synthesized adducts: 7-methyl-dGMP, ring-opened 7-methyl-dGMP and platinated dGpdG. The methylated substrates were detected at sub-fmol sensitivities. 7-Methyl-dGMP was quantitatively phosphorylated at these low concentrations. The efficiency of phosphorylation of the ring-opened product was less (about one order of magnitude) and that of Pt(dGpdG) about three orders of magnitude less. These results show that T4 polynucleotide kinase phosphorylation is an efficient reaction with 7-methyl-dGMP and with ring-opened 7-methyl-dGMP, even though in the latter case longer incubation times may have to be used to boost the reaction towards completion. By contrast, the low level of phosphorylation with Pt(dGpdG) does not appear encouraging for quantitative determination requiring a high sensitivity.     

Exogenous melatonin affects lipids and enzyme activities in mink (Mustela vison) liver

Exogenous melatonin as subcutaneous 2.7-mg implants was given to eight female and male minks in late July with an equal number of animals in the control groups. The liver enzyme activities and major lipids of liver and plasma were measured in October-November. Melatonin had very pronounced effects on the lipid and carbohydrate metabolism of the minks and there was also a clear sexual dimorphism. In the males, melatonin decreased the lipase esterase activity of the liver. In the liver of the females, however, melatonin increased the glucose-6-phosphatase activity. Due to melatonin treatment the liver triacylglycerol contents diminished in both sexes. At the same time, in the females the liver cholesterol levels were decreased. In the plasma lipids, the only change was a fall in the polar lipids of the melatonin-treated females. Melatonin seems to be responsible for the metabolic changes associated with the onset of wintering, especially for the acceleration of the deposition of subcutaneous fat reserves. The smaller females experience the effects of exogenous melatonin more rapidly than the males. Perhaps the smaller body size requires an earlier onset of metabolic preparation for the winter.     

Lignification related enzymes in Picea abies suspension cultures

Activity of a number of enzymes related to lignin formation was measured in a Picea abies (L) Karsten suspension culture that is able to produce native-like lignin into the nutrient medium. This cell culture is an attractive model for studying lignin formation, as the process takes place independently of the complex macromolecular matrix of the native apoplast. Suspension culture proteins were fractionated into soluble cellular proteins, ionically and covalently bound cell wall proteins and nutrient medium proteins. The nutrient medium contained up to 5.3% of total coniferyl alcohol peroxidase (EC 1.11.1.7) activity and a significant NADH oxidase activity that is suggested to be responsible for hydrogen peroxide (H2O2) production. There also existed some malate dehydrogenase (EC 1.1.1.37) activity in the apoplast of suspension culture cells (in ionically and covalently bound cell wall protein fractions), possibly for the regeneration of NADH that is needed for peroxidase-catalysed H2O2 production. However, there is no proof of the existence of NADH in the apoplast. Nutrient medium peroxidases could be classified into acidic, slightly basic and highly basic isoenzyme groups by isoelectric focusing. Only acidic peroxidases were found in the covalently bound cell wall protein fraction. Several peroxidase isoenzymes across the whole pI range were detected in the protein fraction ionically bound to cell walls and in the soluble cellular protein fraction. One laccase-like isoenzyme with pI of approximately 8.5 was found in the nutrient medium that was able to form dehydrogenation polymer from coniferyl alcohol in the absence of H2O2. The total activity of this oxidase towards coniferyl alcohol was, however, several orders of magnitude smaller than that of peroxidases in vitro. According to 2D 1H-13C correlation NMR spectra, most of the abundant structural units of native lignin and released suspension culture lignin are present in the oxidase produced dehydrogenation polymer but in somewhat different amounts compared to peroxidase derived synthetic lignin preparations. A coniferin beta-glucosidase (EC 3.2.1.21) was observed to be secreted into the culture medium.     

Leptin,ghrelin, and energy metabolism of the spawning burbot (Lota lota,L.)

The aim of this study was to investigate the energy metabolism of the burbot (Lota lota, n=38) before, during, and after spawning, which represents the greatest annual metabolic demand for the species. A decrease in body mass, relative weight of the livers, and glycogen concentration of the livers was observed toward the end of spawning. The prespawning period was characterized by high rates of liver glycogenolysis and lipid mobilization. Also, plasma triiodothyronine and sex steroid levels were high before reproduction. During spawning, liver lipolysis was reduced and muscle glycogenolysis stimulated. The levels of triiodothyronine and sex steroids decreased. After reproduction, liver glycogenolysis was suppressed and the rate of gluconeogenesis increased. Thyroid hormone levels were elevated after spawning. Leptin protein and a ghrelin-immunoreactive peptide were detected in burbot plasma. Their concentrations were relatively low before and during reproduction but increased after spawning. The functions of leptin and the ghrelin-immunoreactive peptide in the physiology of the burbot are not consistent with the models of their function in mammals.     

Melatonin and the wintering strategy of the tundra vole,Microtus oeconomus

Short photoperiod induces physiological changes connected to the wintering of the tundra vole, Microtus oeconomus. The aim of the present study was to investigate the effects of continuous melatonin treatment on selected hormones and enzyme activities associated with energy metabolism in the species. Liver, kidney, and muscle glycogen concentrations and glycogen phosphorylase activities, as well as liver and kidney glucose-6-phosphatase and lipase esterase activities were determined. Plasma leptin, ghrelin, thyroxine, testosterone, cortisol, and melatonin concentrations were also measured. Exogenous melatonin stimulated gluconeogenesis, increased glycogen stores, and reduced fat mobilization in kidneys. Melatonin treatment also increased the food intake of the voles. This may have been mediated via elevated ghrelin levels of the melatonin-treated animals, as ghrelin is known to increase appetite of rodents. Winter metabolism of the species does not seem to require accumulation of fat or extra stores of liver or muscle glycogen. On the contrary, successful wintering of the tundra vole presumably depends on continuous food availability.     

Seasonal weight regulation of the raccoon dog (Nyctereutes procyonoides): interactions between melatonin,leptin, ghrelin,and growth hormone

The raccoon dog (Nyctereutes procyonoides, Canidae, Carnivora) is a middle-sized omnivore with excessive autumnal fattening and winter sleep. We studied seasonal weight regulation of the species by following the plasma leptin, ghrelin, and growth hormone (GH) levels of farm-bred raccoon dogs (n = 32) for 6 months. In August, half of the raccoon dogs received continuous-release melatonin implants, and in November, half of the animals of both the sham-operated and melatonin-treated groups were fasted for 2 months. In the autumn, the plasma leptin and GH levels were low, but the ghrelin levels were relatively high and correlated positively with energy intake. This represents the period of energy storage. Leptin and GH levels peaked simultaneously in late October, and melatonin advanced the peaks by 1 week. Thereafter, the levels rapidly declined, representing the transition period from autumnal anabolism to wintertime catabolism. In the winter, the leptin and GH levels rose to high levels, but the ghrelin-leptin ratio was very low. This is the period of winter sleep, with fat accumulated in the autumn as the principal metabolic fuel. In the winter, leptin, ghrelin, and GH may work in synergy to increase lipolysis. GH may also induce winter sleep to the raccoon dog. Fasting had no effect on the hormone levels, unlike in humans and rodents. Instead of the amount of fat in the body, the main regulators of the levels of these hormones in the raccoon dog are presumably seasonal rhythms entrained by melatonin.     

Hereditary minisatellite mutations among the offspring of Estonian Chernobyl cleanup workers

A single accidental event such as the fallout released from the Chernobyl reactor in 1986 can expose millions of people to non-natural environmental radiation. Ionizing radiation increases the frequency of germline mutations in experimental studies, but the genetic effects of radiation in humans remain largely undefined. To evaluate the hereditary effects of low radiation doses, we compared the minisatellite mutation rates of 155 children born to Estonian Chernobyl cleanup workers after the accident with those of their siblings born prior to it. All together, 94 de novo paternal minisatellite mutations were found at eight tested loci (52 and 42 mutants among children born after and before the accident, respectively). The minisatellite mutation rate was nonsignificantly increased among children born after the accident (0.042 compared to 0.036, OR 1.33, 95% CI 0.80-2.20). Furthermore, there was some indication of an increased mutation rate among offspring born after the accident to workers who had received doses of 20 cSv or above compared with their siblings born before the accident (OR 3.0, 95% CI 0.97-9.30). The mutation rate was not associated with the father's age (OR 1.04, 95% CI 0.94-1.15) or the sex of the child (OR 0.95, 95% CI 0.50-1.79). Our results are consistent with both no effect of radiation on minisatellite mutations and a slight increase at dose levels exceeding 20 cSv.     

Teaching cells new tricks

The direct conversion of one differentiated cell type into another--a process referred to as transdifferentiation--would be beneficial for producing isogenic (patient's own) cells to replace sick or damaged cells or tissue. Adult stem cells display a broader differentiation potential than anticipated and might contribute to tissues other than those in which they reside. As such, they could be worthy therapeutic agents. Recent advances in transdifferentiation involve nuclear transplantation, manipulation of cell culture conditions, induction of ectopic gene expression and uptake of molecules from cellular extracts. These approaches open the doors to new avenues for engineering isogenic replacement cells. To avoid unpredictable tissue transformation, nuclear reprogramming requires controlled and heritable epigenetic modifications. Considerable efforts remain to unravel the molecular processes underlying nuclear reprogramming and evaluate stable of the changes in reprogrammed cells.     

Seasonal reproductive endocrine profile of the raccoon dog (Nyctereutes procyonoides)-effects of melatonin and food deprivation

The raccoon dog (Nyctereutes procyonoides, Canidae, Carnivora) is a middle-sized omnivore with excessive autumnal fattening and winter sleep. We studied adaptations of the species to boreal climate and photoperiod by following the plasma reproductive and thyroid hormone concentrations of farm-bred raccoon dogs (n=32) for 12 months. On August 16, 2000, and February 8, 2001, half of the raccoon dogs received continuous-release melatonin implants (the MEL group). The other half was sham-operated (the SHAM group). Between November 27, 2000, and January 25, 2001, half of the animals of both groups were fasted. The plasma testosterone concentrations of the MEL males peaked in February, a month earlier than in the SHAM males. Autumnal melatonin treatment also advanced the gestation period reflected by the plasma progesterone concentrations by seven weeks. Food deprivation in winter seems to accentuate the sex steroid response during the mating as the fasted males had higher testosterone concentrations than the fed males in February and March.     

Nitric oxide donors retard wound healing in cultured rabbit gastric epithelial cell monolayers

Effects of nitric oxide (NO) on gastric wound healing were investigated in primary rabbit gastric epithelial cell cultures. We analyzed the speed of cell migration, proliferation, and apoptosis after creating a round wound on the cell cultures. The monolayers were incubated with or without the NO donor sodium nitroprusside, oxatriazolimine 1,2,3,4-oxatriazolium, 5amino-3-(3,4-dichlorophenylchloride), or the peroxynitrite generator 3-morpholinosydnomine-N-ethylcarbamide. The possible role of cGMP as a second messenger of NO was investigated with 8-bromo-cGMP. The role of O2(-*) was evaluated using diethyldithiocarbamate and pyrogallol. The effects of superoxide dismutase and allopurinol were also investigated. NO inhibited the speed of cell migration and proliferation and induced cell apoptosis in a dose- and time-dependent manner. The effects were augmented with O2(-*) generators and ameliorated by O2-(8) scavengers, whereas cGMP had no significant effect on wound healing. NO donors retard gastric wound healing by inhibiting migration and proliferation and inducing cell apoptosis. These effects do not seem to be mediated via cGMP, but O2(-*). or peroxynitrites may be involved.