Intracerebroventricular interleukin-6 treatment decreases body fat in rats

Recently we found that interleukin-6 (IL-6) knockout mice develop mature-onset obesity and that a single intracerebroventricular (ICV) injection of IL-6 increases energy expenditure. In the present study we investigated if chronic ICV treatment with IL-6 can suppress body fat mass. IL-6 was injected ICV daily for two weeks to rats fed a high-fat diet. IL-6 treatment but not saline treatment decreased body weight by 8.4% and decreased the relative weights of mesenteric and retroperitoneal fat pads. Consistent with this, circulating leptin levels were decreased by 40% after IL-6 treatment but not after saline treatment. Average food intake per day was decreased in the IL-6 treated group compared to the saline treated rats. IL-6 treatment did not change hepatic expression of the acute-phase protein haptoglobin, serum levels of insulin or insulin-like growth factor-I, or the weights of the heart, liver, kidneys, adrenals, and spleen. We conclude that centrally administered IL-6 can decrease body fat in rats without causing acute-phase reaction.     

The surface exposed amino acid residues of monomeric proteins determine the partitioning in aqueous two-phase systems

It is of great interest and importance how different amino acid residues contribute to and affect the properties of a protein surface. Partitioning in aqueous two-phase systems has the potential to be used as a rapid and simple method for studying the surface properties of proteins. The influence on partitioning of the surface exposed amino acid residues of eight structurally determined monomeric proteins has been studied. The proteins were characterized in terms of surface exposed residues with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP), and partitioned in two EO30PO70-dextran aqueous two-phase systems, only differing in polymer concentrations (system I: 6.8% EO30PO70, 7.1% dextran; system II: 9% EO30PO70, 9% dextran). We show for the first time that the partitioning behaviour of different monomeric proteins can be described by the differences in surface exposed amino acid residues. The contribution to the partition coefficient of the residues was found to be best characterized by peptide partitioning in the aqueous two-phase system. Compared to hydrophobicity scales available in the literature, each amino acid contribution is characterized by the slope given by the graph of log K against peptide chain length, for peptides of different length containing only one kind of residue. It was also shown that each amino acid contribution is relative to the total protein surface and the other residues on the surface. Surface hydrophobicity calculations realized for systems I and II gave respectively correlation coefficients of 0.961 and 0.949 for the linear relation between log K and calculated hydrophobicity values. To study the effect on the partition coefficient of different amino acids, they were grouped into classes according to common characteristics: the presence of an aromatic group, a long aliphatic chain or the presence of charge. Using these groups it was possible to confirm that aromatic residues have the strongest effect on the partition coefficient, giving preference to the upper EO30PO70 phase of the system; on the other hand the presence of charged amino acids on the protein surface enhances the partition of the protein to the lower dextran phase. It is also important to note that the sensitivity of the EO30PO70-dextran system for the surface exposed residues was increased by increasing the polymer concentrations. The partition coefficient of a monomeric protein can thus be predicted from its surface exposed amino acid residues and the system can also be used to characterize protein surfaces of monomeric proteins in general.     

Pleurotus and Agrocybe hemolysins,new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg(2+) but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.     

Diverse enzymatic specificities of digestive proteases, 'intestains', enable Colorado potato beetle larvae to counteract the potato defence mechanism

In response to insect attack, high levels of proteinase inhibitors are synthesised in potato leaves. This can cause inefficient protein digestion in insects, leading to reduced growth, delayed development and lower fecundity. It has been suggested that Colorado potato beetle overcomes this defence mechanism by inducing the production of a set of cysteine proteases that are resistant to potato proteinase inhibitors. Experiments with gut extracts showed that these proteases have unusual inhibition profiles as they are not inhibited by most of the cystatins but are strongly inhibited by thyropins. In this study we have isolated three cysteine proteases from adapted guts of Colorado potato beetle larvae, named intestains 1, 2 and 3, the first cysteine proteases known to be involved in extracellular protein digestion. The N-terminal sequences suggest their classification into the papain family. Intestains differ in substrate specificities and inhibitory profiles. Their substrate specificities suggest that intestains 1 and 2 are general digestive enzymes, while intestain 3 has a more specific function. The inhibitory profile of intestain 1 is similar to that of proteases of the papain family. However, the Ki values for the interaction of intestain 2 with the same set of inhibitors are several hundred fold higher, which would enable the enzyme to circumvent the potato defence mechanism characterised by high concentrations of protease inhibitors in attacked potato leaves. A further, different strategy of the Colorado potato beetle to avoid potato defence is exhibited by intestain 3, which is able to cleave off the N-terminus of model cystatin and thus inactivate the inhibitor. These results suggest that the Colorado potato beetle combines different strategies to counteract plant defence mechanisms.     

Sipaucins A-C,sesquiterpenoids from Siparuna pauciflora

The phytochemical investigation of the leaves of Siparuna pauciflora yielded three novel sesquiterpenoids: the germacrane sipaucin A, the elemane sipaucin B and sipaucin C, comprising a new type of carbon skeleton. In addition, four known aporphine alkaloids-nor-boldine, boldine, laurotetanine, and N-methyl-laurotetanine-were obtained. The evaluation of the antiplasmodial activity of the isolated compounds against two strains of Plasmodium falciparum (PoW, Dd2) showed a moderate activity of nor-boldine.     

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.     

Assignment of amide proton signals by combined evaluation of HN,NN and HNCA MAS-NMR correlation spectra

In this paper, we present a strategy for the ¹H^N resonance assignment in solid-state magic-angle spinning (MAS) NMR, using the -spectrin SH3 domain as an example. A novel 3D triple resonance experiment is presented that yields intraresidue H^N-N-C correlations, which was essential for the proton assignment. For the observable residues, 52 out of the 54 amide proton resonances were assigned from 2D (¹H-¹⁵N) and 3D (¹H-¹⁵N-¹³C) heteronuclear correlation spectra. It is demonstrated that proton-driven spin diffusion (PDSD) experiments recorded with long mixing times (4 s) are helpful for confirming the assignment of the protein backbone ¹⁵N resonances and as an aid in the amide proton assignment.