Chemical cross-linking and mass spectrometric identification of sites of interaction for UreD, UreF, and urease

Synthesis of active Klebsiella aerogenes urease requires four accessory proteins to generate, in a GTP-dependent process, a dinuclear nickel active site with the metal ions bridged by a carbamylated lysine residue. The UreD and UreF accessory proteins form stable complexes with urease apoprotein, comprised of UreA, UreB, and UreC. The sites of protein-protein interactions were explored by using homobifunctional amino group-specific chemical cross-linkers with reactive residues being identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) of tryptic peptides. On the basis of studies of the UreABCD complex, UreD is capable of cross-linking with UreB Lys(9), UreB Lys(76), and UreC Lys(401). Furthermore UreD appears to be positioned over UreC Lys(515) according to decreased reactivity of this residue compared with its reactivity in UreD-free apoprotein. Several UreB-UreC and UreC-UreC cross-links also were observed within this complex; e.g. UreB Lys(76) with the UreC amino terminus, UreB Lys(9) with UreC Lys(20), and UreC Lys(515) with UreC Lys(89). These interactions are consistent with the proximate surface locations of these residues observed in the UreABC crystal structure. MALDI-TOF MS analyses of UreABCDF are consistent with a cross-link between the UreF amino terminus and UreB Lys(76). On the basis of an unexpected cross-link between UreB Lys(76) and UreC Lys(382) (distant from each other in the UreABC structure) along with increased side chain reactivities for UreC Lys(515) and Lys(522), UreF is proposed to induce a conformational change within urease that repositions UreB and potentially could increase the accessibility of nickel ions and CO(2) to residues that form the active site.     

Mass spectrometric analysis of Plasmodium falciparum erythrocyte membrane protein-1 variants expressed by placental malaria parasites

Surface proteins from Plasmodium falciparum are important malaria vaccine targets. However, the surface proteins previously identified are highly variant and difficult to study. We used tandem mass spectrometry to characterize the variant antigens (Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)) expressed on the surface of malaria-infected erythrocytes that bind to chondroitin sulfate A (CSA) in the placenta. Whereas PfEMP1 variants previously implicated as CSA ligands were detected, in unselected parasites four novel variants were detected in CSA-binding or placental parasites but not in unselected parasites. These novel PfEMP1 variants require further study to confirm whether they play a role in placental malaria.     

Characterization of the maize xylem sap proteome

The xylem in plants has mainly been described as a conduit for water and minerals, but emerging evidence also indicates that the xylem contains protein. To study the proteins in xylem sap, we characterized the identity and composition of the maize xylem sap proteome. The composition of the xylem sap proteome in maize revealed proteins related to different phases of xylem differentiation including cell wall metabolism, secondary cell wall synthesis, and programmed cell death. Many proteins were found to be present as multiple isoforms and some of these isoforms are glycosylated. Proteins involved in defense mechanisms were also present in xylem sap and the sap proteins were shown to have antifungal activity in bioassays.     

Inflorescence architecture: A developmental genetics approach

We are characterizing a suiteof Pisum sativum mutants that alter inflorescence architecture to construct a model for the genetic regulation of inflorescence development in a plant with a compound raceme. Such a model, when compared with those created forAntirrhinum majus andArabidopsis thaliana, both of which have simple racemes, should provide insight into the evolution of the development of inflorescence architecture. The highly conserved nature of cloned genes that regulate reproductive development in plants and the morphological similarities among our mutants and those identified inA. majus andA. thaliana enhance the probability that a developmental genetics approach will be fruitful. Here we describe sixP. sativum mutants that affect morphologically and architecturally distinct aspects of the inflorescence, and we analyze interactions among these genes. Both vegetative and inflorescence growth of the primary axis is affected byUNIFOLIA TA, which is necessary for the function ofDETERMINATE (DET).DET maintains indeterminacy in the first-order axis. In its absence, the meristem differentiates as a stub covered with epidermal hairs.DET interacts withVEGETATIVE1 (VEG1).VEG1 appears essential for second-order inflorescence (I₂) development.veg1 mutants fail to flower or differentiate the I₂ meristem into a rudimentary stub,det veg1 double mutants produce true terminal flowers with no stubs, indicating that two genes must be eliminated for terminal flower formation inP. sativum, whereas elimination of a single gene accomplishes this inA. thaliana andA. majus. NEPTUNE also affects I₂ development by limiting to two the number of flowers produced prior to stub formation. Its role is independent ofDET, as indicated by the additive nature of the double mutantdet nep. UNI, BROC, and PIM all play roles in assigning floral meristem identity to the third-order branch.pim mutants continue to produce inflorescence branches, resulting in a highly complex architecture and aberrant flowers.uni mutants initiate a whorl of sepals, but floral organogenesis is aberrant beyond that developmental point, and the double mutantuni pim lacks identifiable floral organs. A wild-type phenotype is observed inbroc plants, butbroc enhancesthe pim phenotype in the double mutant, producing inflorescences that resemble broccoli. Collectively these genes ensure that only the third-order meristem, not higher- or lower-order meristems, generates floral organs, thus precisely regulating the overall architecture of the plant. Gene symbols used in this article: For clarity a common symbolization is used for genes of all species discussed in this article. Genes are symbolized with italicized capital letters. Mutant alleles are represented by lowercase, italicized letters. In both cases, the number immediately following the gene symbol differentiates among genes with the same symbol. If there are multiple alleles, a hyphen followed by a number is used to distinguish alleles. Protein products are represented by capital letters without italics.     

Transmitted/Founder Simian Immunodeficiency Virus Envelope Sequences in Vesicular Stomatitis and Semliki Forest Virus Vector Immunized Rhesus Macaques

Identification of transmitted/founder simian immunodeficiency virus (SIV) envelope sequences responsible for infection may prove critical for understanding HIV/SIV mucosal transmission. We used single genome amplification and phylogenetic analyses to characterize transmitted/founder SIVs both in the inoculum and in immunized-infected rhesus monkeys. Single genome amplification of the SIVsmE660 inoculum revealed a maximum diversity of 1.4%. We also noted that the consensus sequence of the challenge stock differed from the vaccine construct in 10 amino acids including 3 changes in the V4 loop. Viral env was prepared from rhesus plasma in 3 groups of 6 immunized with vesicular stomatitis virus (VSV) vectors and boosted with Semliki forest virus (SFV) replicons expressing (a) SIVsmE660 gag-env (b) SIVsmE660 gag-env plus rhesus GM-CSF and (c) control influenza hemagglutinin protein. Macaques were immunized twice with VSV-vectors and once with SFV vector and challenged intrarectally with 4000 TCID₅₀. Single genome amplification characterized the infections of 2 unprotected animals in the gag-env immunized group, both of which had reduced acute plasma viral loads that ended as transient infections indicating partial immune control. Four of 6 rhesus were infected in the gag-env + GM-CSF group which demonstrated that GM-CSF abrogated protection. All 6 animals from the control group were infected having high plasma viral loads. We obtained 246 full-length envelope sequences from SIVsmE660 infected macaques at the peak of infection and determined the number of transmitted/founder variants per animal. Our analysis found that 2 of 2 gag-env vaccinated but infected macaques exhibited single but distinct virus envelope lineages whereas rhesus vaccinated with gag-env-GM-CSF or HA control exhibited both single and multiple env lineages. Because there were only 2 infected animals in the gag-env vaccinated rhesus compared to 10 infected rhesus in the other 2 groups, the significance of finding single env variants in the gag-env vaccinated group could not be established.     

Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding

A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.     

Elevated summer temperatures delay spawning and reduce redd construction for resident brook trout (Salvelinus fontinalis)

Redd (nest) surveys for resident brook trout (Salvelinus fontinalis) were conducted annually in a mountain lake in northern New York for 11 years with multiple surveys conducted during the spawning season in eight of those years. Repeated surveys throughout the spawning season allowed us to fit an individually based parametric model and estimate the day of year on which spawning was initiated, reached its midpoint, and ended during each year. Spawning phenology was then assessed relative to (1) mean of maximum daily air temperature and (2) mean of maximum daily water temperature at the lake bottom during summer in each year using a linear model. Elevated temperatures in summer were correlated with a delay in spawning and a reduction in the total number of redds constructed. Increasing the summer mean of maximum daily air temperatures by 1 °C delayed spawning by approximately 1 week and decreased the total number of redds constructed by nearly 65. Lake spawning brook trout select redd sites based on the presence of discharging groundwater that is relatively constant in temperature within and across years, leading to relatively consistent egg incubation times. Therefore, delayed spawning is likely to delay fry emergence, which could influence emergence synchrony with prey items. This work highlights non‐lethal and sub‐lethal effects of elevated summer temperatures on native resident salmonids in aquatic environments with limited thermal refugia.     

Two-step cell patterning on planar and complex curved surfaces by precision spraying of polymers

Controlling adhesion of living animal cells plays a key role in biosensor fabrication, drug-testing technologies, basic biological research, and tissue engineering applications. Current techniques for cell patterning have two primary limitations: (1) they require photolithography, and (2) they are limited to patterning of planar surfaces. Here we demonstrate a simple, precision spraying method for both positive and negative patterning of planar and curved surfaces to achieve cell patterns rapidly and reproducibly. In this method, which we call precision spraying (PS), a polymer solution is aerosolized, focused with sheath airflow through an orifice, and deposited on the substrate using a deposition head to create approximately 25 microm sized features. In positive patterning, adhesive molecules, such as laminin or polyethylenimine (PEI) were patterned on polydimethylsiloxane (PDMS) substrates in a single spraying operation. A variety of animal cell types were found to adhere to the adhesive regions, and avoid the non-adhesive (bare PDMS) regions. In negative patterning, hydrophobic materials, such as polytetrafluoroethylene (PTFE) and PDMS, were patterned on glass substrates. Cells then formed patterns on the exposed glass regions and avoided the hydrophobic regions. Cellular patterns were maintained for up to 2 weeks in the presence of serum, which normally fouls non-adhesive regions. Additionally, we found that precision spraying enabled micropatterning of complex-curved surfaces. Our results show that precision spraying followed by cell plating enables rapid and flexible cellular micropatterning in two simple steps.