Solid-state NMR sequential assignments of α-synuclein

Parkinson’s disease is amongst the most frequent and most devastating neurodegenerative diseases. It is tightly associated with the assembly of proteins into high-molecular weight protein species, which propagate between neurons in the central nervous system. The principal protein involved in this process is α-synuclein which is a structural component of the Lewy bodies observed in diseased brain. We here present the solid-state NMR sequential assignments of a new fibrillar form of this protein, the first one with a well-ordered and rigid N-terminal part.     

Simian immunodeficiency virus (SIV)-specific CD8+ T-cell responses in vervet African green monkeys chronically infected with SIVagm

African green monkeys (AGM) do not develop overt signs of disease following simian immunodeficiency virus (SIV) infection. While it is still unknown how natural hosts like AGM can cope with this lentivirus infection, a large number of investigations have shown that CD8(+) T-cell responses are critical for the containment of AIDS viruses in humans and Asian nonhuman primates. Here we have compared the phenotypes of T-cell subsets and magnitudes of SIV-specific CD8(+) T-cell responses in vervet AGM chronically infected with SIVagm and rhesus monkeys (RM) infected with SIVmac. In comparison to RM, vervet AGM exhibited weaker signs of immune activation and associated proliferation of CD8(+) T cells as detected by granzyme B, Ki-67, and programmed death 1 staining. By gamma interferon enzyme-linked immunospot assay and intracellular cytokine staining, SIV Gag- and Env-specific immune responses were detectable at variable but lower levels in vervet AGM than in RM. These observations demonstrate that natural hosts like SIV-infected vervet AGM develop SIV-specific T-cell responses, but the disease-free course of infection does not depend on the generation of robust CD8(+) T-cell responses.     

Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco

cDNAs encoding three proteins from barley ( Hordeum vulgare ), a class-II chitinase (CHI), a class-II β-1,3-glucanase (GLU) and a Type-I ribosome-inactivating protein (RIP) were expressed in tobacco plants under the control of the CaMV 35S-promoter. High-level expression of the transferred genes was detected in the transgenic plants by Northern and Western blot analysis. The leader peptides in CHI and GLU led to accumulation of these proteins in the intercellular space of tobacco leaves. RIP, which is naturally deposited in the cytosol of barley endosperm cells, was expressed either in its original cytosolic form or fused to a plant secretion peptide (spRIP). Fungal infection assays revealed that expression of the individual genes in each case resulted in an increased protection against the soilborne fungal pathogen Rhizoctonia solani , which infects a range of plant species including tobacco. To create a situation similar to 'multi-gene' tolerance, which traditional breeding experience has shown to provide crops with a longer-lasting protection, several of these antifungal genes were combined and protection against fungal attack resulting from their co-expression in planta was evaluated. Transgenic tobacco lines were generated with tandemly arranged genes coding for RIP and CHI as well as GLU and CHI. The performance of tobacco plants co-expressing the barley transgenes GLU/ CHI or CHI/RIP in a Rhizoctonia solani infection assay revealed significantly enhanced protection against fungal attack when compared with the protection levels obtained with corresponding isogenic lines expressing a single barley transgene to a similar level. The data indicate synergistic protective interaction of the co-expressed anti-fungal proteins in vivo .     

Molecular characterization of the diurnal/circadian expression of the chlorophyll a/b-binding proteins in leaves of tomato and other dicotyledonous and monocotyledonous plant species

Diurnal oscillations of steady-state mRNA levels encoding the chlorophyll a/b-binding proteins were monitored inLycopersicon esculentum, Glycine max, Phaseolus vulgaris, P. aureus, P. coccineus, Pisum sativum, Sinapis alba, Hordeum vulgare, Triticum aestivum andZea mays. In these plant speciescab mRNA accumulation increases and decreases periodically indicating i) that the expression of the genes for chlorophyll a/b-binding proteins (cab genes) is controlled by a circadian rhythm, and ii) that the rhythm is widely distributed among monocotyledonous and dicotyledonous plant species. A detailed characterization of the pattern ofcab mRNA expression in tomato leaves shows that the amplitude of the oscillation is dependent on i) the developmental stage of the leaves, ii) the circadian phase and duration of light and iii) the circadian phase and duration of darkness. In addition to the chlorophyll a/b-binding proteins, genes coding for other cellular functions were examined for cyclic variations of their mRNA levels. The analysis includes genes involved in i) carbon metabolism (e.g. phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, alpha amylase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase), ii) photosynthesis (large and small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, Q_B-binding protein, reaction-center protein of photosystem I) and iii) other physiological or morphological reactions (e.g. ubiquitin, actin). However, no periodic fluctuation pattern was detected for the mRNA levels of these genes in tomato and maize leaves.     

Structural and phylogenetic analyses of the GP42 transglutaminase from Phytophthora sojae reveal an evolutionary relationship between oomycetes and marine Vibrio bacteria

Transglutaminases (TGases) are ubiquitous enzymes that catalyze selective cross-linking between protein-bound glutamine and lysine residues; the resulting isopeptide bond confers high resistance to proteolysis. Phytophthora sojae, a pathogen of soybean, secretes a Ca²⁺-dependent TGase (GP42) that is activating defense responses in both host and non-host plants. A GP42 fragment of 13 amino acids, termed Pep-13, was shown to be absolutely indispensable for both TGase and elicitor activity. GP42 does not share significant primary sequence similarity with known TGases from mammals or bacteria. This suggests that GP42 has evolved novel structural and catalytic features to support enzymatic activity. We have solved the crystal structure of the catalytically inactive point mutant GP42 (C290S) at 2.95 Å resolution and identified residues involved in catalysis by mutational analysis. The protein comprises three domains that assemble into an elongated structure. Although GP42 has no structural homolog, its core region displays significant similarity to the catalytic core of the Mac-1 cysteine protease from Group A Streptococcus, a member of the papain-like superfamily of cysteine proteases. Proteins that are taxonomically related to GP42 are only present in plant pathogenic oomycetes belonging to the order of the Peronosporales (e.g. Phytophthora, Hyaloperonospora, and Pythium spp.) and in marine Vibrio bacteria. This suggests that a lateral gene transfer event may have occurred between bacteria and oomycetes. Our results offer a basis to design and use highly specific inhibitors of the GP42-like TGase family that may impair the growth of important oomycete and bacterial pathogens.     

Comparison of two expression platforms in respect to protein yield and quality: Pichia pastoris versus Pichia angusta

The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor (MTF). Both recombinant proteins were engineered to contain an N-terminal Strep- (WSHPQFEK) and a C-terminal His₆-tag. In addition, both proteins contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion. Following vector construction, transformation and zeocin amplification, the best Pichia producers were identified in a screening procedure using Western blot and a Luminex xMAP™ based high-throughput method. Recombinant NK1-fragment and MTF were purified from culture supernatants of the best producers by affinity chromatography (Ni–nitrilotriacetic acid columns). Using P. pastoris as a host for the synthesis of NK1-fragment a protein yield of 5.7 mg/l was achieved. In comparable expression experiments P. angusta yielded 1.6 mg/l of NK1-fragment. NK1-fragment apparently was not glycosylated in either system. For the production of MTF, P. pastoris was also the superior host yielding 1.2 mg/l glycosylated recombinant protein whereas P. angusta was clearly less efficient (<0.2 mg/l MTF). For both expression systems no correlation between the amount of recombinant protein and the copy number of the chromosomally integrated heterologous genes was found. In P. pastoris strains less degradation of the two model recombinant proteins was observed. Altogether, this paper provides a structured protocol for rapidly identifying productive Pichia strains for the synthesis of full-length recombinant proteins.     

Matrilin-3 is dispensable for mouse skeletal growth and development

Matrilin-3 belongs to the matrilin family of extracellular matrix (ECM) proteins and is primarily expressed in cartilage. Mutations in the gene encoding human matrilin-3 (MATN-3) lead to autosomal dominant skeletal disorders, such as multiple epiphyseal dysplasia (MED), which is characterized by short stature and early-onset osteoarthritis, and bilateral hereditary microepiphyseal dysplasia, a variant form of MED characterized by pain in the hip and knee joints. To assess the function of matrilin-3 during skeletal development, we have generated Matn-3 null mice. Homozygous mutant mice appear normal, are fertile, and show no obvious skeletal malformations. Histological and ultrastructural analyses reveal endochondral bone formation indistinguishable from that of wild-type animals. Northern blot, immunohistochemical, and biochemical analyses indicated no compensatory upregulation of any other member of the matrilin family. Altogether, our findings suggest functional redundancy among matrilins and demonstrate that the phenotypes of MED disorders are not caused by the absence of matrilin-3 in cartilage ECM.     

Retinal and optical adaptations for nocturnal vision in the halictid bee<Emphasis Type="Italic"> Megalopta genalis</Emphasis>

The apposition compound eye of a nocturnal bee, the halictid Megalopta genalis, is described for the first time. Compared to the compound eye of the worker honeybee Apis mellifera and the diurnal halictid bee Lasioglossum leucozonium, the eye of M. genalis shows specific retinal and optical adaptations for vision in dim light. The major anatomical adaptations within the eye of the nocturnal bee are (1) nearly twofold larger ommatidial facets and (2) a 4–5 times wider rhabdom diameter than found in the diurnal bees studied. Optically, the apposition eye of M. genalis is 27 times more sensitive to light than the eyes of the diurnal bees. This increased optical sensitivity represents a clear optical adaptation to low light intensities. Although this unique nocturnal apposition eye has a greatly improved ability to catch light, a 27-fold increase in sensitivity alone cannot account for nocturnal vision at light intensities that are 8 log units dimmer than during daytime. New evidence suggests that additional neuronal spatial summation within the first optic ganglion, the lamina, is involved.B.G. is thankful for travel awards from the Royal Physiographic Society, the Per Westlings Fond, the Foundation of Dagny and Eilert Ekvall and the Royal Swedish Academy of Sciences. E.J.W. is grateful for the support of a Smithsonian Short-Term Research Fellowship, the Swedish Research Council, the Crafoord Foundation, the Wenner-Gren Foundation and the Royal Physiographic Society of Lund for their ongoing support     

Structure and function of vertebrate CMP-sialic acid synthetases

Activation of sugars into nucleotide sugars is critical for their entry into biosynthetic pathways. In eukaryotic cells, the activation of the acidic nine-carbon sugar sialic acid to CMP-sialic acid takes place in the cell nucleus, whereas all other nucleotide sugars are made in the cytoplasm. Molecular cloning of vertebrate CMP-sialic acid synthetases confirmed the nuclear localization and introduced new molecular tools for directly exploring the functional mechanisms of the enzymes, as well as the physiological relevance of their nuclear transport. Although major advances have been made in understanding structure-function relationships and defining elements involved in the nuclear transport, the riddle surrounding the physiological relevance of nuclear localization awaits resolution.     

Stable haploid poplar callus lines from immature pollen culture

Embryogenesis and plant regeneration have been obtained from isolated immature pollen of two poplar hybrids ( Populus nigra L. × hybrid 'Aue1' and 'Aue2'). In total, 1487 calli or embryos, respectively, larger than 1 mm were generated in a 2-year study. By using a cytokinin containing induction medium, on average 19 calli per responsive immature catkin were formed. Additional supplementation with auxin in 2002 increased the frequency to 72 calli per catkin. Microsatellite marker analyses confirmed haploid origin in most regenerants studied. So far six out of eight obtained regenerative callus lines have maintained their haploid level up to 24 months of development. A number of haploid and doubled haploid plants of different lines are available and have been transferred to soil.