Ralstonia eutropha H16 encodes two and possibly three intracellular Poly[D-(-)-3-hydroxybutyrate] depolymerase genes

Intracellular poly[D-(-)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid. Recently an intracellular PHB depolymerase gene (phaZ1) from Ralstonia eutropha was identified. We now report identification of candidate PHB depolymerase genes from R. eutropha, namely, phaZ2 and phaZ3, and their characterization in vivo. phaZ1 was used to identify two candidate depolymerase genes in the genome of Ralstonia metallidurans. phaZ1 and these genes were then used to design degenerate primers. These primers and PCR methods on the R. eutropha genome were used to identify two new candidate depolymerase genes in R. eutropha: phaZ2 and phaZ3. Inverse PCR methods were used to obtain the complete sequence of phaZ3, and library screening was used to obtain the complete sequence of phaZ2. PhaZ1, PhaZ2, and PhaZ3 share approximately 30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generating R. eutropha H16 deletion strains (Delta phaZ1, Delta phaZ2, Delta phaZ3, Delta phaZ1 Delta phaZ2, Delta phaZ1 Delta phaZ3, Delta phaZ2 Delta phaZ3, and Delta phaZ1 Delta phaZ2 Delta phaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated approximately 80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.     

Locking and unlocking of ribosomal motions

During the ribosomal translocation, the binding of elongation factor G (EF-G) to the pretranslocational ribosome leads to a ratchet-like rotation of the 30S subunit relative to the 50S subunit in the direction of the mRNA movement. By means of cryo-electron microscopy we observe that this rotation is accompanied by a 20 A movement of the L1 stalk of the 50S subunit, implying that this region is involved in the translocation of deacylated tRNAs from the P to the E site. These ribosomal motions can occur only when the P-site tRNA is deacylated. Prior to peptidyl-transfer to the A-site tRNA or peptide removal, the presence of the charged P-site tRNA locks the ribosome and prohibits both of these motions.     

The class III adenylyl cyclases: multi-purpose signalling modules

cAMP serves as a second messenger in virtually all organisms. The most wide-spread class of cAMP-generating enzymes are the class III adenylyl cyclases. Most class III adenylyl cyclases are multi-domain proteins. The catalytic domains exclusively work as dimers, catalysis proceeds at the dimer interface, so that both monomers provide catalytic residues to each catalytic center. Inspection of amino acid sequence profiles suggests a division of the class III adenylyl cyclases in to four subclasses, class IIIa–IIId. Genome projects and postgenomic analysis have provided novel aspects in terms of catalysis and regulation. Alterations in the canonical catalytic residues occur in all four subclasses suggesting a plasticity of the catalytic mechanisms. The vast variety of additional, probably regulatory modules found in class III adenylyl cyclases obviously reflects a large collection of regulatory inputs the catalytic domains have adapted to. The large versatility of class III adenylyl cyclase catalytic domains remains a major scientific challenge.     

Developmentally induced changes of the proteome in the protozoan parasite Leishmania donovani

In order to proceed through their life cycle, protozoan parasites of the genus Leishmania cycle between sandflies and mammals. This change of environment correlates with the differentiation from the promastigote stage (insect form) to the amastigote stage (intracellular mammalian form). The molecular basis underlying this major transformation is poorly understood so far; however, heat shock protein 90 (HSP90) appears to play a pivotal role. To further elucidate this process we identified proteins expressed preferentially in either of the two life cycle stages. By using two-dimensional (2-D) gel electrophoresis we observed defined changes in the protein pattern. A total of approximately 2000 protein spots were visualized. Of these, 31 proteins were present only in promastigotes. The abundance of 65 proteins increased during heat-induced in vitro amastigote differentiation, while a decreased abundance is observed for four proteins late in amastigote differentiation. Further analyses using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting 67 protein spots were identified representing 41 different proteins known from databases and eight hypothetical proteins. Further studies showed that most of the stage-specific proteins fall into five groups of functionally related proteins. These functional categories are: (i) stress response (e.g. heat, oxidative stress); (ii) cytoskeleton and cell membrane; (iii) energy metabolism and phosphorylation; (iv) cell cycle and proliferation; and (v) amino acid metabolism. Very similar changes in the 2-D protein pattern were obtained when in vitro amastigote differentiation was induced either by pharmacological inhibition of HSP90 or by a combination of heat stress and acidic pH supporting the critical role for HSP90 in life cycle control.     

Structural refinement of insecticidal plant proteinase inhibitors from Nicotiana alata

Ornamental tobacco (Nicotiana alata) produces a series of 6 kDa proteinase inhibitors belonging to the potato type II inhibitor family. These proteins inhibit trypsin and chymotrypsin, the main digestive enzymes of predatory insects, thus leading to starvation, impaired larval development or death. In this context, the three-dimensional structures of these inhibitors are important for developing novel strategies for pest control. The solution structures of C1 and T1, the two main prototypes of the N. alata inhibitors, were originally determined more than a decade ago (J. Mol. Biol. 242, 231-243 (1994) and Biochemistry 34, 14304-14311 (1995)). Since then methods for NMR structure calculations have evolved considerably. Here we report the refinement of the structures of C1 and T1 with state-of-the-art protocols for NMR structure calculations. This refinement leads to an improved quality of the structures, making them a more reliable basis for the development of novel pesticides and modeling applications.     

Colon cancer-associated DNA polymerase β variant induces genomic instability and cellular transformation

Rapidly advancing technology has resulted in the generation of the genomic sequences of several human tumors. We have identified several mutations of the DNA polymerase β (pol β) gene in human colorectal cancer. We have demonstrated that the expression of the pol β G231D variant increased chromosomal aberrations and induced cellular transformation. The transformed phenotype persisted in the cells even once the expression of G231D was extinguished, suggesting that it resulted as a consequence of genomic instability. Biochemical analysis revealed that its catalytic rate was 140-fold slower than WT pol β, and this was a result of the decreased binding affinity of nucleotides by G231D. Residue 231 of pol β lies in close proximity to the template strand of the DNA. Molecular modeling demonstrated that the change from a small and nonpolar glycine to a negatively charged aspartate resulted in a repulsion between the template and residue 231 leading to the distortion of the dNTP binding pocket. In addition, expression of G231D was insufficient to rescue pol β-deficient cells treated with chemotherapeutic agents suggesting that these agents may be effectively used to treat tumors harboring this mutation. More importantly, this suggests that the G231D variant has impaired base excision repair. Together, these data indicate that the G231D variant plays a role in driving cancer.     

Resonance Raman and FTIR spectroscopic characterization of the closed and open states of channelrhodopsin-1

Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) is a light-activated cation channel, which is a promising optogenetic tool. We show by resonance Raman spectroscopy and retinal extraction followed by high pressure liquid chromatography (HPLC) that the isomeric ratio of all-trans to 13-cis of solubilized channelrhodopsin-1 is with 70:30 identical to channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Critical frequency shifts in the retinal vibrations are identified in the Raman spectrum upon transition to the open (conductive P₂³⁸⁰) state. Fourier transform infrared spectroscopy (FTIR) spectra indicate different structures of the open states in the two channelrhodopsins as reflected by the amide I bands and the protonation pattern of acidic amino acids.     

Landscape genomics and a common garden trial reveal adaptive differentiation to temperature across Europe in the tree species Alnus glutinosa

The adaptive potential of tree species to cope with climate change has important ecological and economic implications. Many temperate tree species experience a wide range of environmental conditions, suggesting high adaptability to new environmental conditions. We investigated adaptation to regional climate in the drought‐sensitive tree species Alnus glutinosa (Black alder), using a complementary approach that integrates genomic, phenotypic and landscape data. A total of 24 European populations were studied in a common garden and through landscape genomic approaches. Genotyping‐by‐sequencing was used to identify SNPs across the genome, resulting in 1990 SNPs. Although a relatively low percentage of putative adaptive SNPs was detected (2.86% outlier SNPs), we observed clear associations among outlier allele frequencies, temperature and plant traits. In line with the typical drought avoiding nature of A. glutinosa, leaf size varied according to a temperature gradient and significant associations with multiple outlier loci were observed, corroborating the ecological relevance of the observed outlier SNPs. Moreover, the lack of isolation by distance, the very low genetic differentiation among populations and the high intrapopulation genetic variation all support the notion that high gene exchange combined with strong environmental selection promotes adaptation to environmental cues.     

Dermal application of nitric oxide releasing acidified nitrite-containing liniments significantly reduces blood pressure in humans

Vascular ischemic diseases, hypertension, and other systemic hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). NO but also its active derivates like nitrite or nitroso compounds are important effector and signal molecules with vasodilating properties. Our previous findings point to a therapeutical potential of cutaneous administration of NO in the treatment of systemic hemodynamic disorders. Unfortunately, no reliable data are available on the mechanisms, kinetics and biological responses of dermal application of nitric oxide in humans in vivo. The aim of the study was to close this gap and to explore the therapeutical potential of dermal nitric oxide application. We characterized with human skin in vitro and in vivo the capacity of NO, applied in a NO-releasing acidified form of nitrite-containing liniments, to penetrate the epidermis and to influence local as well as systemic hemodynamic parameters. We found that dermal application of NO led to a very rapid and significant transepidermal translocation of NO into the underlying tissue. Depending on the size of treated skin area, this translocation manifests itself through a significant systemic increase of the NO derivates nitrite and nitroso compounds, respectively. In parallel, this translocation was accompanied by an increased systemic vasodilatation and blood flow as well as reduced blood pressure. We here give evidence that in humans dermal application of NO has a therapeutic potential for systemic hemodynamic disorders that might arise from local or systemic insufficient availability of NO or its bio-active NO derivates, respectively.     

Distribution of neuropeptides in the primary olfactory center of the heliothine moth <Emphasis Type="Italic">Heliothis virescens</Emphasis>

Neuropeptides are a diverse widespread class of signaling substances in the nervous system. As a basis for the analysis of peptidergic neurotransmission in the insect olfactory system, we have studied the distribution of neuropeptides in the antennal lobe of the moth Heliothis virescens. Immunocytochemical experiments with antisera recognizing A-type allatostatins (AST-As), Manduca sexta allatotropin (Mas-AT), FMRFamide-related peptides (FaRPs), and tachykinin-related peptides (TKRPs) have shown that members of all four peptide families are present in local interneurons of the antennal lobe. Whereas antisera against AST-As, Mas-AT, and FaRPs give similar staining patterns characterized by dense meshworks of processes confined to the core of all antennal-lobe glomeruli, TKRPs are present only in neurons with blebby processes distributed throughout each glomerulus. In addition to local neurons, a pair of centrifugal neurons with cell bodies in the lateral subesophageal ganglion, arborizations in the antennal lobe, and projections in the inner antenno-cerebral tracts exhibits tachykinin immunostaining. Double-label immunofluorescence has detected the co-localization of AST-As, Mas-AT, and FaRPs in certain local interneurons, whereas TKRPs occurs in a distinct population. MALDI-TOF mass spectrometry has revealed nearly 50 mass peaks in the antennal lobe. Seven of these masses (four AST-As, two N-terminally extended FLRFamides, and Mas-AT) match known moth neuropeptides. The data thus show that local interneurons of the moth antennal lobe are highly differentiated with respect to their neuropeptide content. The antennal lobe therefore represents an ideal preparation for the future analysis of peptide signaling in insect brain.