In situ proteolysis for protein crystallization and structure determination

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.     

Solution Structure of C. elegans UNC-6: A Nematode Paralogue of the Axon Guidance Protein Netrin-1

UNCoordinated-6 (UNC-6) was the first member of the netrin family to be discovered in Caenorhabditis elegans. With homology to human netrin-1, it is a key signaling molecule involved in directing axon migration in nematodes. Similar to netrin-1, UNC-6 interacts with multiple receptors (UNC-5 and UNC-40, specifically) to guide axon migration in development. As a result of the distinct evolutionary path of UNC-6 compared to vertebrate netrins, we decided to employ an integrated approach to study its solution behavior and compare it to the high-resolution structure we previously published on vertebrate netrins. Dynamic light scattering and analytical ultracentrifugation on UNC-6 (with and without its C-domain) solubilized in a low-ionic strength buffer suggested that UNC-6 forms high-order oligomers. An increase in the buffer ionic strength resulted in a more homogeneous preparation of UNC-6, that was used for subsequent solution x-ray scattering experiments. Our biophysical analysis of UNC-6 ΔC solubilized in a high-ionic strength buffer suggested that it maintains a similar head-to-stalk arrangement as netrins ?1 and ?4. This phenomenon is thought to play a role in the signaling behavior of UNC-6 and its ability to move throughout the extracellular matrix.     

A morphological,molecular, and histopathological redescription of Henneguya nyongensis Fomena & Bouix, 1996 (Cnidaria: Myxobolidae) infecting the gills of Peter’s elephantnose fish,Gnathonemus petersii (Günther) (Osteoglossiformes: Mormyridae), imported from Nigeria

Systematic Parasitology - A Henneguya sp., morphologically resembling Henneguya nyongensis Fomena & Bouix, 1996, was isolated from the gills of Peter’s elephantnose fish, Gnathonemus...     

Clathrin adaptors mediate two sequential pathways of intra-Golgi recycling

The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins, including some that were previously thought to pass through endosomes. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5–dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5–dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi.     

Spatial separation of ribosomes and DNA in Asgard archaeal cells

The origin of the eukaryotic cell is a major open question in biology. Asgard archaea are the closest known prokaryotic relatives of eukaryotes, and their genomes encode various eukaryotic signature proteins, indicating some elements of cellular complexity prior to the emergence of the first eukaryotic cell. Yet, microscopic evidence to demonstrate the cellular structure of uncultivated Asgard archaea in the environment is thus far lacking. We used primer-free sequencing to retrieve 715 almost full-length Loki- and Heimdallarchaeota 16S rRNA sequences and designed novel oligonucleotide probes to visualize their cells in marine sediments (Aarhus Bay, Denmark) using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Super-resolution microscopy revealed 1–2 µm large, coccoid cells, sometimes occurring as aggregates. Remarkably, the DNA staining was spatially separated from ribosome-originated FISH signals by 50–280 nm. This suggests that the genomic material is condensed and spatially distinct in a particular location and could indicate compartmentalization or membrane invagination in Asgard archaeal cells.Subject terms: Soil microbiology, Microbial ecology, Archaeal physiology     

Simultaneous nitrification and denitrification using stored substrate (PHB) as the electron donor in an SBR

The potential for PHB (poly-beta-hydroxybutyrate) to serve as the electron donor for effective simultaneous nitrification and denitrification (SND) was investigated in a 2-L sequencing batch reactor (SBR) using a mixed culture and acetate as the organic substrate. During the feast period (i.e., acetate present), heterotrophic respiration activity was high and nitrification was prevented due to the inability of nitrifying bacteria to compete with heterotrophs for oxygen. Once acetate was depleted the oxidation rate of PHB was up to 6 times slower than that of soluble acetate and nitrification could proceed due to the decreased competition for oxygen. The slow nature of PHB degradation meant that it was an effective substrate for SND, as it was oxidised at a similar rate to ammonium and was therefore available for SND throughout the entire aerobic period. The percentage of nitrogen removed via SND increased at lower DO concentrations during the famine period, with up to 78% SND achieved at a DO concentration of 0.5 mg L(-1). However, the increased percentage of SND at a low DO concentration was compromised by a 2-times slower rate of nitrogen removal. A moderate DO concentration of 1 mg L(-1) was optimal for both SND efficiency (61%) and rate (4.4 mmol N x Cmol x(-1) x h(-1)). Electron flux analysis showed that the period of highest SND activity occurred during the first hour of the aerobic famine period, when the specific oxygen uptake rate (SOUR) was highest. It is postulated that a high SOUR due to NH(4) (+) and PHB oxidation decreases oxygen penetration into the floc, creating larger zones for anoxic denitrification. The accumulation of nitrate towards the end of the SND period showed that SND was finally limited by the rate of denitrification. As PHB degradation was found to follow first-order kinetics (df(PHB)/dt = -0.19 x f(PHB)), higher PHB concentrations would be expected to drive SND faster by increasing the availability rate of reducing power and reducing penetration of oxygen into the floc, due to the corresponding increased SOUR. Process control techniques to accumulate higher internal PHB concentrations to improve PHB-driven SND are discussed.     

Synthesis of allysine ethylene acetal using phenylalanine dehydrogenase from Thermoactinomyces intermedius

Allysine ethylene acetal [(S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (2)] was prepared from the corresponding keto acid by reductive amination using phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius ATCC 33205. Glutamate, alanine, and leucine dehydrogenases, and PDH from Sporosarcina species (listed in order of increasing effectiveness) also gave the desired amino acid but were less effective. The reaction requires ammonia and NADH. NAD produced during the reaction was recyled to NADH by the oxidation of formate to CO(2) using formate dehydrogenase (FDH). PDH was produced by growth of T. intermedius ATCC 33205 or by growth of recombinant Escherichia coli or Pichia pastoris expressing the Thermoactinomyces enzyme. Using heat-dried T. intermedius as a source of PDH and heat-dried Candida boidinii SC13822 as a source of FDH,98%, but production of T. intermedius could not be scaled up. Using heat-dried recombinant E. coli as a source of PDH and heat-dried Candida boidinii 98%. In a third generation process, heat-dried methanol-grown P. pastoris expressing endogenous FDH and recombinant Thermoactinomyces98% ee.     

A familial paracentric inversion: A short review of the current status

Summary We present here a familial case of a paracentric inversion in man with a short review of the literature. A paracentric inversion of chromosome 10(q11q26) was found in the amniocytes drawn for advanced maternal age. The presence of the inversion was investigated in 35 family members in three generations. No recombinants were recognized. The significance of these data for appropriate genetic counselling and possible reproductive risks is discussed.Genetic Nurses, Department of Health and Welfare