Patellin1, a novel Sec14-like protein, localizes to the cell plate and binds phosphoinositides

Membrane trafficking is central to construction of the cell plate during plant cytokinesis. Consequently, a detailed understanding of the process depends on the characterization of molecules that function in the formation, transport, targeting, and fusion of membrane vesicles to the developing plate, as well as those that participate in its consolidation and maturation into a fully functional partition. Here we report the initial biochemical and functional characterization of patellin1 (PATL1), a novel cell-plate-associated protein that is related in sequence to proteins involved in membrane trafficking in other eukaryotes. Analysis of the Arabidopsis genome indicated that PATL1 is one of a small family of Arabidopsis proteins, characterized by a variable N-terminal domain followed by two domains found in other membrane-trafficking proteins (Sec14 and Golgi dynamics domains). Results from immunolocalization and biochemical fractionation studies suggested that PATL1 is recruited from the cytoplasm to the expanding and maturing cell plate. In vesicle-binding assays, PATL1 bound to specific phosphoinositides, important regulators of membrane trafficking, with a preference for phosphatidylinositol(5)P, phosphatidylinositol(4,5)P(2), and phosphatidylinositol(3)P. Taken together, these findings suggest a role for PATL1 in membrane-trafficking events associated with cell-plate expansion or maturation and point to the involvement of phosphoinositides in cell-plate biogenesis.     

The polyprotein allergens of nematodes

Symptoms of infection with a number of parasitic nematodes may be associated with IgE antibody and associated immunopathologies. This has drawn attention to parasite allergens and provoked discussion on the wisdom of their inclusion in recombinant vaccines. Here, Larry McReynolds, Malcolm Kennedy and Murray Selkirk review work on a prominent set of allergens recently characterized in several nematode species.     

Tyzzer's disease in free-living cottontail rabbits (Sylvilagus floridanus) in Maryland

Complement-fixing (CF) antibody to Bacillus piliformis antigen was found in 9 of 14 (64%) serum samples obtained from cottontail rabbits (Sylvilagus floridanus) killed in the wild. CF antibody was not present in the serum of 8 cottontail rabbits trapped as juveniles in the same geographic areas and held in captivity for 4 years. Sero-negative cottontail rabbits died acutely with lesions typical of Tyzzer's disease following the intragastric administration of 10(3.8) ELD50 of B. piliformis spores. The possible influence of Tyzzer's disease upon the cyclic population pattern of cottontail rabbits in the wild is discussed. A hypothesis is presented that B. piliformis spores passed in the feces of diseased wild animals could contaminate pastures, hay and grain, and thereby serve as sources of infection to other animals.     

The SARS-CoV heptad repeat 2 exhibits pH-induced helix formation

The heptad repeats 1 and 2 of SARS-CoV spike, termed HR1 and HR2, play critical roles in viral entry. Moreover, HR1 and HR2 derived free peptides are inhibitors of SARS-CoV entry. In this work we used circular dichroism to show that HR2 helix formation is induced at pH 5, the pH of the endosome. In addition, we demonstrate that the HR2 helix is further stabilized at physiological ionic strengths. Together, these observations provide new insight into the mechanism of SARS-CoV entry and suggest that HR2 may be an attractive target for therapeutic intervention.     

Structure-function analysis of Methanobacterium thermoautotrophicum RNA ligase - engineering a thermostable ATP independent enzyme

ABSTRACT: BACKGROUND: RNA ligases are essential reagents for many methods in molecular biology including NextGen RNA sequencing. To prevent ligation of RNA to itself, ATP independent mutant ligases, defective in self-adenylation, are often used in combination with activated pre-adenylated linkers. It is important that these ligases not have de-adenylation activity, which can result in activation of RNA and formation of background ligation products. An additional useful feature is for the ligase to be active at elevated temperatures. This has the advantage or reducing preferences caused by structures of single-stranded substrates and linkers. RESULTS: To create an RNA ligase with these desirable properties we performed mutational analysis of the archaeal thermophilic RNA ligase from Methanobacterium thermoautotrophicum. We identified amino acids essential for ATP binding and reactivity but dispensable for phosphodiester bond formation with 5' pre-adenylated donor substrate. The motif V lysine mutant (K246A) showed reduced activity in the first two steps of ligation reaction. The mutant has full ligation activity with pre-adenylated substrates but retained the undesirable activity of deadenylation, which is the reverse of step 2 adenylation. A second mutant, an alanine substitution for the catalytic lysine in motif I (K97A) abolished activity in the first two steps of the ligation reaction, but preserved wild type ligation activity in step 3. The activity of the K97A mutant is similar with either pre-adenylated RNA or single-stranded DNA (ssDNA) as donor substrates but we observed two-fold preference for RNA as an acceptor substrate compared to ssDNA with an identical sequence. In contrast, truncated T4 RNA ligase 2, the commercial enzyme used in these applications, is significantly more active using pre-adenylated RNA as a donor compared to pre-adenylated ssDNA. However, the T4 RNA ligases are ineffective in ligating ssDNA acceptors. CONCLUSIONS: Mutational analysis of the heat stable RNA ligase from Methanobacterium thermoautotrophicum resulted in the creation of an ATP independent ligase. The K97A mutant is defective in the first two steps of ligation but retains full activity in ligation of either RNA or ssDNA to a pre-adenylated linker. The ability of the ligase to function at 65 deg C should reduce the constraints of RNA secondary structure in RNA ligation experiments.     

Non-natural glycosphingolipids and structurally simpler analogues bind HIV-1 recombinant Gp120

Interactions of recombinant gp120 (rgp120) with non-natural glycosphingolipids (GSLs) and structurally simpler analogues have been studied using a competitive adhesion assay. Conjugates of cellobiosyl ceramide and melibiosyl ceramide were synthetically prepared as water-soluble GSL analogues. These ligands were screened against a panel of biologically relevant analogues, and the results show that their interactions with rgp120 are comparable to natural cellular receptors. Glycolipid interactions with rgp120 were probed further by the synthesis and testing of structurally simpler analogues that were obtained by reductive amination of lactose, cellobiose, and melibiose with a biotinylated amino ethylene glycol moiety. RGp120 did not recognize conjugates lacking a lipid component. However, palmitoylation of the secondary amino alditols yielded compounds with comparable rgp120 affinity to the natural cellular receptor, galactosyl ceramide (GalCer). Taken together, the SAR showed that both a hydrophobic and a hydrophilic component are required for rgp120 recognition. Moreover, structural variability in the carbohydrate headgroup did not significantly alter rgp120 recognition indicating that this interaction is not highly specific.     

Characterization of the prefusion and transition states of severe acute respiratory syndrome coronavirus S2-HR2

The envelope glycoproteins of the class I family, which include human immunodeficiency virus (HIV), influenza, and severe acute respiratory syndrome coronavirus (SARS-CoV), mediate viral entry by first binding to their cellular receptors and subsequently inducing fusion of the viral and cellular membranes. In the case of SARS-CoV, heptad repeat domains of the envelope glycoprotein, termed S2-HR1 and S2-HR2, are thought to undergo structural changes from a prefusion state, in which S2-HR1 and S2-HR2 do not interact, to a postfusion state in which S2-HR1 and S2-HR2 associate to form a six-helix bundle. In the present work, the structural and dynamic properties of S2-HR2 have been characterized. Evidence is presented for an equilibrium between a structured trimer thought to represent a prefusion state and an ensemble of unstructured monomers thought to represent a novel transition state. A model for viral entry is presented in which S2-HR2 is in a dynamic equilibrium between an ensemble of unstructured monomers in the transition state and a structured trimer in the prefusion state. Conversion from the prefusion state to the postfusion state requires passage through the transition state, a state that may give insight into the design of structure-based antagonists of SARS-CoV in particular, as well as other enveloped viruses in general.     

A haplotype at chromosome Xq27.2 confers susceptibility to prostate cancer

We conducted an association study to identify risk variants for familial prostate cancer within the HPCX locus at Xq27 among Americans of Northern European descent. We investigated a total of 507 familial prostate cancer probands and 507 age-matched controls without a personal or family history of prostate cancer. The study population was subdivided into a set of training subjects to explore genetic variation of the locus potentially impacting risk of prostate cancer, and an independent set of test subjects to confirm the association and to assign significance, addressing multiple comparisons. We identified a 22.9 kb haplotype nominally associated with prostate cancer among training subjects (292 cases, 292 controls; χ² = 5.08, P = 0.020), that was confirmed among test subjects (215 cases, 215 controls; χ² = 3.73, P = 0.040). The haplotype predisposed to prostate cancer with an odds ratio of 3.41 (95% CI 1.04–11.17, P = 0.034) among test subjects. The haplotype extending from rs5907859 to rs1493189 is concordant with a prior study of the region within the Finnish founder population, and warrants further independent investigation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.