Caspase-12 cleavage and increased oxidative stress during motoneuron degeneration in transgenic mouse model of ALS

Glucose-dependent insulinotropic polypeptide is an incretin hormone that stimulates insulin secretion and reduces postprandial glycaemic excursions. The glucose-dependent action of GIP on pancreatic beta-cells has attracted attention towards its exploitation as a potential drug for type 2 diabetes. Use of NMR or X-ray crystallography is vital to determine the three-dimensional structure of the peptide. Therefore, to understand the basic structural requirements for the biological activity of GIP, the solution structure of the major biologically active fragment, GIP(1-30)amide, was investigated by proton NMR spectroscopy and molecular modelling. The structure is characterised by a full length alpha-helical conformation between residues F(6) and A(28). This structural information could play an important role in the design of therapeutic agents based upon GIP receptor agonists.     

Influence of osmolytes on in vivo glucose monitoring using optical coherence tomography

Diabetes mellitus and its complications are the third leading cause of death in the world, exceeded only by cardiovascular disease and cancer. Tighter monitoring and control of blood glucose could minimize complications associated with diabetes. Recently, optical coherence tomography (OCT) for noninvasive glucose monitoring was proposed and tested in vivo. The aim of this work was to investigate the influence of changes in blood glucose concentration ([glu]) and sodium concentration ([Na+]) on the OCT signal. We also investigated the influence of other important analytes on the sensitivity of glucose monitoring with OCT. The experiments were carried out in anesthetized female pigs. The OCT images were acquired continuously from skin, while [glu] and [Na+] were experimentally varied within their physiological ranges. Correlations of the OCT signal slope with [glu] and [Na+] were studied at different tissue depths. The tissue area probed with OCT was marked and cut for histological examination. The correlation of blood [glu] and [Na+] with the OCT signal slope was observed in separate tissue layers. On average, equimolar changes in [glu] produced 2.26 +/- 1.15 greater alterations of the OCT signal slope than changes in [Na+]. Variation of concentrations of other analytes did not influence the OCT signal slope. The influence of [Na+] on relative changes in the OCT signal slope was generally less than [glu]-induced changes. OCT is a promising method for noninvasive glucose monitoring because of its ability to track the influence of changing [glu] on individual tissue layers.     

Bathy Phytochromes in Rhizobial Soil Bacteria

Phytochromes are biliprotein photoreceptors that are found in plants, bacteria, and fungi. Prototypical phytochromes have a Pr ground state that absorbs in the red spectral range and is converted by light into the Pfr form, which absorbs longer-wavelength, far-red light. Recently, some bacterial phytochromes have been described that undergo dark conversion of Pr to Pfr and thus have a Pfr ground state. We show here that such so-called bathy phytochromes are widely distributed among bacteria that belong to the order Rhizobiales. We measured in vivo spectral properties and the direction of dark conversion for species which have either one or two phytochrome genes. Agrobacterium tumefaciens C58 contains one bathy phytochrome and a second phytochrome which undergoes dark conversion of Pfr to Pr in vivo. The related species Agrobacterium vitis S4 contains also one bathy phytochrome and another phytochrome with novel spectral properties. Rhizobium leguminosarum 3841, Rhizobium etli CIAT652, and Azorhizobium caulinodans ORS571 contain a single phytochrome of the bathy type, whereas Xanthobacter autotrophicus Py2 contains a single phytochrome with dark conversion of Pfr to Pr. We propose that bathy phytochromes are adaptations to the light regime in the soil. Most bacterial phytochromes are light-regulated histidine kinases, some of which have a C-terminal response regulator subunit on the same protein. According to our phylogenetic studies, the group of phytochromes with this domain arrangement has evolved from a bathy phytochrome progenitor.Phytochromes are biological photoreceptors that were discovered in plants, where they control development throughout the life cycle in manifold ways (21, 33). Today, a large number of homologs are known also from cyanobacteria, other bacteria, and fungi, which are termed cyanobacterial phytochromes (Cphs), bacteriophytochromes (BphPs), and fungal phytochromes (Fphs), respectively (20, 24). The chromophore is autocatalytically assembled within the N-terminal part of the protein, the photosensory core module (PCM), which contains the PAS, GAF, and PHY domains (30). Typically, phytochromes are converted by light between two spectrally different forms, the red-absorbing Pr and the far-red-absorbing Pfr forms. Photoconversion is initiated by an isomerization of the covalently bound bilin chromophore (32).Plant and cyanobacterial phytochromes incorporate phytochromobilin (PΦB) and phycocyanobilin (PCB) as natural chromophores, respectively, which are covalently bound to Cys residues in the GAF domains. All characterized phytochromes that belong to these groups have a Pr ground state. Plant phytochromes can undergo dark conversion of Pfr to Pr (5), whereas the Pfr form of typical cyanobacterial phytochromes is stable in darkness (26).Bacteriophytochromes utilize biliverdin (BV) instead as a natural chromophore (1), which is covalently attached to a Cys residue in the N terminus of the PAS domain (26). Since the conjugated system of BV is longer than that of PΦB or PCB, the absorption maxima of bacteriophytochromes are found at higher wavelengths than those of cyanobacterial or plant homologs.With the discovery of a bacterial phytochrome from Bradyrhizobium sp. strain ORS278, termed BrBphP1, the first phytochrome with a Pfr ground state and dark conversion from Pr to Pfr was found (10). Thereafter, five more phytochromes with dark conversion of Pr to Pfr were described: Rhodopseudomonas palustris BphP1 (RpBphP1) from strain CEA001, RpBphP5, and RpBphP6 from strain CGA009 (11); Agrobacterium tumefaciens Agp2 (or AtBphP2) from strain C58 (18); and Pseudomonas aeruginosa BphP1 (PaBphP1) (40). These phytochromes are now termed bathy phytochromes because the absorption maxima of their ground states are bathochromically (to longer wavelengths) shifted compared to those of all other phytochromes.Moreover, some other bacterial phytochromes with unusual properties have been described. In the Ppr from Rhodospirillum centenum, a photoactive yellow protein (PYP) domain is fused to the N terminus of a phytochrome homolog. The phytochrome part of Ppr assembles with BV to form a Pr adduct. However, irradiation does not result in the formation of Pfr but in a bleaching of the Pr spectrum (23). The BV adduct of RpBphP3 from R. palustris, which has a Pr ground state, photoconverts to the so-called Pnr form with a blue-shifted absorption maximum (12). RpBphP4 from R. palustris strains Ha2 and BisB5 and Bradyrhizobium BphP3 (BrBphP3) from Bradyrhizobium BTAi1, both with a Pr ground state, photoconvert into a long-lived MetaR form (8, 42). MetaRa and MetaRc are intermediates in the photoconversion from Pr to Pfr of prototypical phytochromes (3). BphP3 from the Bradyrhizobium strain ORS 278 is an exception among bacteriophytochromes as it binds PCB as a natural chromophore. This phytochrome adopts a so-called Po (P-orange) ground state with an absorbance maximum in the orange range (11, 15). Upon irradiation, this phytochrome converts into the Pr form. RpBphP4 from R. palustris CGA009 lacks the biliverdin binding cysteine and does not bind a chromophore (42).With the rapidly growing number of bacterial genome sequences, many new bacterial phytochromes are being discovered. Thus, a large and increasing number of newly identified phytochromes remain spectroscopically uncharacterized. We established an in vivo photometry approach which allowed the rapid acquisition of spectral information about phytochromes from intact bacterial cells. In the beginning period of plant phytochrome research, in vivo photometry was extensively applied (4, 6, 29, 34). This method, in fact, allowed the identification of phytochromes for the first time in plant tissues (6), which led to the purification of phytochromes from plant extracts (37). Here, we apply in vivo photometry for the first time to organisms outside the plant kingdom. This method is especially useful for studying species with single phytochrome genes. The approach is also helpful for comparing properties of native phytochromes in vivo and of their recombinant proteins in vitro.In the present study, we concentrate on nonphotosynthetic species of the order Rhizobiales which belongs to the Alphaproteobacteria. The family Rhizobiaceae comprises plant-interacting soil bacteria. A. tumefaciens and Agrobacterium vitis can transfer genes into plants to induce plant tumors, whereas many other Rhizobiaceae can live as plant symbionts in nodules of stems or roots in which they assimilate molecular nitrogen to produce NH₄⁺, which is used by the plant for synthesis of amino acids and other nitrogen-containing molecules. A. tumefaciens C58 contains two phytochromes, termed Agp1 (or AtBphP1) and Agp2 (or AtBphP2), that have been characterized as recombinant proteins (14, 18, 26, 35) and whose spectral activities have been measured in extracts of wild-type and knockout mutants (31). A large number of phytochromes from photosynthetic Bradyrhizobium and Rhodopseudomonas species, which also belong to the order Rhizobiales, have been characterized as recombinant proteins (11), some of which have already been noted above.It turned out that most of our analyzed phytochromes undergo dark conversion of Pr to Pfr and thus belong to the group of bathy phytochromes. Such phytochromes, which absorb at around 750 nm, clearly dominate among Rhizobiales. We propose that this specific property reflects an adaptation to the light regime in the soil. Our studies also suggest that bacterial phytochromes with a C-terminal response regulator have evolved from a bathy phytochrome progenitor.     

LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9 kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co-expressed and activated LmxMPK4 in a dose-dependent manner. To our knowledge this is the first time that an in vitro activator of an essential Leishmania MAP kinase was identified and our findings form the basis for the development of drug screening assays to identify small molecule inhibitors of LmxMPK4 in the search for new therapeutic drugs against leishmaniasis.     

Short/branched-chain acyl-CoA dehydrogenase deficiency due to an IVS3+3A>G mutation that causes exon skipping

Short/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD) is an autosomal recessive disorder of l-isoleucine catabolism. Little is known about the clinical presentation associated with this enzyme defect, as it has been reported in only a limited number of patients. Because the presence of C5-carnitine in blood may indicate SBCADD, the disorder may be detected by MS/MS-based routine newborn screening. It is, therefore, important to gain more knowledge about the clinical presentation and the mutational spectrum of SBCADD. In the present study, we have studied two unrelated families with SBCADD, both with seizures and psychomotor delay as the main clinical features. One family illustrates the fact that affected individuals may also remain asymptomatic. In addition, the normal level of newborn blood spot C5-acylcarnitine in one patient underscores the fact that newborn screening by MS/MS currently lacks sensitivity in detecting SBCADD. Until now, seven mutations in the SBCAD gene have been reported, but only three have been tested experimentally. Here, we identify and characterize an IVS3+3A>G mutation (c.303+3A>G) in the SBCAD gene, and provide evidence that this mutation is disease-causing in both families. Using a minigene approach, we show that the IVS3+3A>G mutation causes exon 3 skipping, despite the fact that it does not appear to disrupt the consensus sequence of the 5′ splice site. Based on these results and numerous literature examples, we suggest that this type of mutation (IVS+3A>G) induces missplicing only when in the context of non-consensus (weak) 5′ splice sites. Statistical analysis of the sequences shows that the wild-type versions of 5′ splice sites in which +3A>G mutations cause exon skipping and disease are weaker on average than a random set of 5′ splice sites. This finding is relevant to the interpretation of the functional consequences of this type of mutation in other disease genes.     

Distribution Patterns of Isomorphic Cold-Water Dinoflagellates (Scrippsiella/Woloszynskia Complex) Causing ‘red tides’ in the Baltic Sea

During the latest years medium-sized (15–30 μm), single-celled dinoflagellates have been reported to form blooms in the northern Baltic Proper and the Gulf of Finland in winter and spring. Recent studies (Kremp et al., 2003. Proceedings of the 7th International conference of Modern and Fossil Dinoflagellates, September 21–25, Nagasaki, Japan, 66 pp.) indicate that those blooms are caused by two isomorphic species – Scrippsiella hangoei (Schiller) Larsen, and a new species, tentatively belonging to the genus Woloszynskia. Until now there has been no report on how widely distributed these phytoplankton species are in the Baltic Sea. In this study, the occurrence of Scrippsiella/Woloszynskia complex in the entire Baltic Sea was investigated, by using monitoring data from 1997 to 2003. The species occurred in a salinity range from 2 to 8 PSU. Highest concentrations were observed at salinity 4.5–6.5 PSU. Maximum cell densities of Scrippsiella/Woloszynskia complex in the water column were mainly obtained in April or in the beginning of May by the water temperature <3 °C prior to stratification was formed. In the central Gulf of Finland, the second maximum was found in 1999 and 2002 by the temperature >6 °C. Bloom formations in the Baltic Proper and in the Gulf of Finland may not only be explained by optimum temperature and salinity, but also with other factors e.g. high nutrient concentrations and good seeding conditions from the sediments.     

The effects of garlic-derived sulfur compounds on cell proliferation, caspase 3 activity, thiol levels and anaerobic sulfur metabolism in human hepatoblastoma HepG2 cells

The aim of the present studies was to determine whether the mechanism of biological action of garlic-derived sulfur compounds in human hepatoma (HepG2) cells can be dependent on the presence of labile sulfane sulfur in their molecules. We investigated the effect of allyl sulfides from garlic: monosulfide, disulfide and trisulfide on cell proliferation and viability, caspase 3 activity and hydrogen peroxide (H(2)O(2)) production in HepG2 cells. In parallel, we also examined the influence of the previously mentioned compounds on the levels of thiols, glutathione, cysteine and cysteinyl-glycine, and on the level of sulfane sulfur and the activity of its metabolic enzymes: rhodanese, 3-mercaptopyruvate sulfurtransferase and cystathionase. Among the compounds under study, diallyl trisulfide (DATS), a sulfane sulfur-containing compound, showed the highest biological activity in HepG2 cells. This compound increased the H(2)O(2) formation, lowered the thiol level and produced the strongest inhibition of cell proliferation and the greatest induction of caspase 3 activity in HepG2 cells. DATS did not affect the activity of sulfurtransferases and lowered sulfane sulfur level in HepG2 cells. It appears that sulfane sulfur containing DATS can be bioreduced in cancer cells to hydroperthiol that leads to H(2)O(2) generation, thereby influencing transmission of signals regulating cell proliferation and apoptosis.     

Human high temperature requirement serine protease A1 (HTRA1) degrades tau protein aggregates

Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed.