Higher plant cellulose synthases

Cellulose, an aggregate of unbranched polymers of β-1,4-linked glucose residues, is the major component of wood and thus paper, and is synthesized by plants, most algae, some bacteria and fungi, and even some animals. The genes that synthesize cellulose in higher plants differ greatly from the well-characterized genes found in Acetobacter and Agrobacterium sp. More correctly designated as 'cellulose synthase catalytic subunits', plant cellulose synthase (CesA) proteins are integral membrane proteins, approximately 1,000 amino acids in length. The sequences for more than 20 full-length CesA genes are available, and they show high similarity to one another across the entire length of the encoded protein, except for two small regions of variability. There are a number of highly conserved residues, including several motifs shown to be necessary for processive glycosyltransferase activity. No crystal structure is known for cellulose synthase proteins, and the exact enzymatic mechanism is unknown. There are a number of mutations in cellulose synthase genes in the model organism Arabidopsis thaliana. Some of these mutants show altered morphology due to the lack of a properly developed primary or secondary cell wall. Others show resistance to well-characterized cellulose biosynthesis inhibitors.     

Non-synonymous single nucleotide polymorphisms in the watermelon eIF4E gene are closely associated with resistance to <Emphasis Type="Italic">Zucchini yellow mosaic virus</Emphasis>

Zucchini yellow mosaic virus (ZYMV) is one of the most economically important potyviruses infecting cucurbit crops worldwide. Using a candidate gene approach, we cloned and sequenced eIF4E and eIF(iso)4E gene segments in watermelon. Analysis of the nucleotide sequences between the ZYMV-resistant watermelon plant introduction PI 595203 (Citrullus lanatus var. lanatus) and the ZYMV-susceptible watermelon cultivar ‘New Hampshire Midget’ (‘NHM’) showed the presence of single nucleotide polymorphisms (SNPs). Initial analysis of the identified SNPs in association studies indicated that SNPs in the eIF4E, but not eIF(iso)4E, were closely associated to the phenotype of ZYMV-resistance in 70 F₂ and 114 BC_1R progenies. Subsequently, we focused our efforts in obtaining the entire genomic sequence of watermelon eIF4E. Three SNPs were identified between PI 595203 and NHM. One of the SNPs (A241C) was in exon 1 and the other two SNPs (C309A and T554G) were in the first intron of the gene. SNP241 which resulted in an amino acid substitution (proline to threonine) was shown to be located in the critical cap recognition and binding area, similar to that of several plant species resistance to potyviruses. Analysis of a cleaved amplified polymorphism sequence (CAPS) marker derived from this SNP in F₂ and BC_1R populations demonstrated a cosegregation between the CAPS-2 marker and their ZYMV resistance or susceptibility phenotype. When we investigated whether such SNP mutation in the eIF4E was also conserved in several other PIs of C. lanatus var. citroides, we identified a different SNP (A171G) resulting in another amino acid substitution (D71G) from four ZYMV-resistant C. lanatus var. citroides (PI 244018, PI 482261, PI 482299, and PI 482322). Additional CAPS markers were also identified. Availability of all these CAPS markers will enable marker-aided breeding of watermelon for ZYMV resistance.     

Cytotoxic T-Lymphocyte Epitope Immunodominance in the Control of Choroid Plexus Tumors in Simian Virus 40 Large T Antigen Transgenic Mice

The simian virus 40 (SV40) large tumor antigen (Tag) is a virus-encoded oncoprotein which is the target of a strong cytotoxic T-lymphocyte (CTL) response. Three immunodominant H-2(b)-restricted epitopes, designated epitopes I, II/III, and IV, have been defined. We investigated whether induction of CTLs directed against these Tag epitopes might control Tag-induced tumors in SV11(+) (H-2(b)) mice. SV11(+) mice develop spontaneous tumors of the choroid plexus due to expression of SV40 Tag as a transgene. We demonstrate that SV11(+) mice are functionally tolerant to the immunodominant Tag CTL epitopes. CTLs specific for the H-2Kb-restricted Tag epitope IV were induced in SV11(+) mice following adoptive transfer with unprimed C57BL/6 spleen cells and immunization with recombinant vaccinia viruses expressing either full-length Tag or the H-2Kb-restricted epitope IV as a minigene. In addition, irradiation of SV11(+) mice prior to adoptive transfer with unprimed C57BL/6 spleen cells led to the priming of epitope IV-specific CTLs by the endogenous Tag. Induction of epitope IV-specific CTLs in SV11(+) mice by either approach correlated with increased life span and control of the choroid plexus tumor progression, indicating that CTLs specific for the immunodominant Tag epitope IV control the progressive growth of spontaneous tumors induced by this DNA virus oncogene in transgenic mice.     

Unusual seasonal patterns and inferred processes of nitrogen retention in forested headwaters of the Upper Susquehanna River

Atmospheric deposition contributes a large fraction of the annual nitrogen (N) input to the basin of the Susquehanna River, a river that provides two-thirds of the annual N load to the Chesapeake Bay. Yet, there are few measurements of the retention of atmospheric N in the Upper Susquehanna’s forested headwaters. We characterized the amount, form (nitrate, ammonium, and dissolved organic nitrogen), isotopic composition (δ¹⁵N- and δ¹⁸O-nitrate), and seasonality of stream N over 2 years for 7–13 catchments. We expected high rates of N retention and seasonal nitrate patterns typical of other seasonally snow-covered catchments: dormant season maxima and growing season minima. Coarse estimates of N export indicated high rates of inorganic N retention (>95%), yet streams had unexpected seasonal nitrate patterns, with summer peaks (14–96 μmol L⁻¹), October crashes (<1 μmol L⁻¹), and modest rebounds during the dormant season (<1–20 μmol L⁻¹). Stream δ¹⁸O-nitrate values indicated microbial nitrification as the primary source of stream nitrate, although snowmelt or other atmospheric source contributed up to 47% of stream nitrate in some March samples. The autumn nitrate crash coincided with leaffall, likely due to in-stream heterotrophic uptake of N. Hypothesized sources of the summer nitrate peaks include: delayed release of nitrate previously flushed to groundwater, weathering of geologic N, and summer increases in net nitrate production. Measurements of shale δ¹⁵N and soil-, well-, and streamwater nitrate within one catchment point toward a summer increase in soil net nitrification as the driver of this pattern. Rather than seasonal plant demand, processes governing the seasonal production, retention, and transport of nitrate in soils may drive nitrate seasonality in this and many other systems.     

Variation analysis and gene annotation of eight MHC haplotypes: The MHC Haplotype Project

The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine. Horton and Gibson contributed equally to this work.     

Ancestral loss of the maxillary sinus in Old World monkeys and independent acquisition in Macaca

Cercopithecoid monkeys are unique among primates in that all species (except macaques) lack a maxillary sinus, an unusual condition among eutherian mammals. Although this uncommon distribution of cranial pneumatization was noted previously, the phylogenetic ramifications have not been investigated fully. Recently, character state optimization analysis of computed tomography (CT) data from extant Old World monkeys suggested that the loss of the sinus may have occurred at the origin of the group, unlike previous hypotheses positing only a reduction in size of the structure. To critically evaluate the "early loss" hypothesis, a recently recovered complete cranium of Victoriapithecus macinnesi from Maboko Island, Kenya, was examined by CT to determine the extent of its cranial pneumatization. This taxon is crucial for evaluating character state evolution in Old World monkeys, due to its phylogenetic position, preceding the cercopithecine/colobine split. CT analysis reveals only cancellous bone lateral of the nasal cavity, indicating that Victoriapithecus does not possess a maxillary sinus. Phylogenetic evaluation of the fossil with extant catarrhine taxa strongly supports the early loss of the sinus in cercopithecoids. The results suggest that the maxillary sinus found in the genus Macaca is not homologous with that of other eutherians, which may provide insights into the origin and function (if any) of the paranasal pneumatizations.     

Improving evolutionary models of protein interaction networks

MOTIVATION: Theoretical models of biological networks are valuable tools in evolutionary inference. Theoretical models based on gene duplication and divergence provide biologically plausible evolutionary mechanics. Similarities found between empirical networks and their theoretically generated counterpart are considered evidence of the role modeled mechanics play in biological evolution. However, the method by which these models are parameterized can lead to questions about the validity of the inferences. Selecting parameter values in order to produce a particular topological value obfuscates the possibility that the model may produce a similar topology for a large range of parameter values. Alternately, a model may produce a large range of topologies, allowing (incorrect) parameter values to produce a valid topology from an otherwise flawed model. In order to lend biological credence to the modeled evolutionary mechanics, parameter values should be derived from the empirical data. Furthermore, recent work indicates that the timing and fate of gene duplications are critical to proper derivation of these parameters. RESULTS: We present a methodology for deriving evolutionary rates from empirical data that is used to parameterize duplication and divergence models of protein interaction network evolution. Our method avoids shortcomings of previous methods, which failed to consider the effect of subsequent duplications. From our parameter values, we find that concurrent and existing existing duplication and divergence models are insufficient for modeling protein interaction network evolution. We introduce a model enhancement based on heritable interaction sites on the surface of a protein and find that it more closely reflects the high clustering found in the empirical network.     

Mechanical stiffness as an improved single-cell indicator of osteoblastic human mesenchymal stem cell differentiation

Although it has been established that cellular stiffness can change as a stem cell differentiates, the precise relationship between cell mechanics and other phenotypic properties remains unclear. Inherent cell heterogeneity and asynchronous differentiation complicate population analysis; therefore, single-cell analysis was employed to determine how changes in cell stiffness correlate with changes in molecular biomarkers during differentiation. Design of a custom gridded tissue culture dish facilitated single-cell comparisons between cell mechanics and other differentiation biomarkers by enabling sequential measurement of cell mechanics and protein biomarker expression at the single cell level. The Young’s modulus of mesenchymal stem cells was shown not only to decrease during chemically-induced osteoblast differentiation, but also to correlate more closely with the day of differentiation than did the relative expression of the traditional osteoblast differentiation markers, bone sialoprotein and osteocalcin. Therefore, cell stiffness, a measurable property of individual cells, may serve as an improved indicator of single-cell osteoblast differentiation compared to traditional biological markers. Revelation of additional osteoblast differentiation indicators, such as cell stiffness, can improve identification and collection of starting cell populations, with applications to mesenchymal stem cell therapies and stem cell-based tissue engineering.     

Organic dust augments nucleotide-binding oligomerization domain expression via an NF-{kappa}B pathway to negatively regulate inflammatory responses

Nucleotide-binding oligomerization domain 2 (NOD2) is involved in innate immune responses to peptidoglycan degradation products. Peptidoglycans are important mediators of organic dust-induced airway diseases in exposed agriculture workers; however, the role of NOD2 in response to complex organic dust is unknown. Monocytes/macrophages were exposed to swine facility organic dust extract (ODE), whereupon NOD2 expression was evaluated by real-time PCR and Western blot. ODE induced significant NOD2 mRNA and protein expression at 24 and 48 h, respectively, which was mediated via a NF-κB signaling pathway as opposed to a TNF-α autocrine/paracrine mechanism. Specifically, NF-κB translocation increased rapidly following ODE stimulation as demonstrated by EMSA, and inhibition of the NF-κB pathway significantly reduced ODE-induced NOD2 expression. However, there was no significant reduction in ODE-induced NOD2 gene expression when TNF-α was inhibited or absent. Next, it was determined whether NOD2 regulated ODE-induced inflammatory cytokine production. Knockdown of NOD2 expression by small interfering RNA resulted in increased CXCL8 and IL-6, but not TNF-α production in response to ODE. Similarly, primary lung macrophages from NOD2 knockout mice demonstrated increased IL-6, CXCL1, and CXCL1, but not TNF-α, expression. Lastly, a higher degree of airway inflammation occurred in the absence of NOD2 following acute (single) and repetitive (3 wk) ODE exposure in an established in vivo murine model. In summary, ODE-induced NOD2 expression is directly dependent on NF-κB signaling, and NOD2 is a negative regulator of complex, organic dust-induced inflammatory cytokine/chemokine production in mononuclear phagocytes.     

Combinatorial polymer electrospun matrices promote physiologically-relevant cardiomyogenic stem cell differentiation

Myocardial infarction results in extensive cardiomyocyte death which can lead to fatal arrhythmias or congestive heart failure. Delivery of stem cells to repopulate damaged cardiac tissue may be an attractive and innovative solution for repairing the damaged heart. Instructive polymer scaffolds with a wide range of properties have been used extensively to direct the differentiation of stem cells. In this study, we have optimized the chemical and mechanical properties of an electrospun polymer mesh for directed differentiation of embryonic stem cells (ESCs) towards a cardiomyogenic lineage. A combinatorial polymer library was prepared by copolymerizing three distinct subunits at varying molar ratios to tune the physicochemical properties of the resulting polymer: hydrophilic polyethylene glycol (PEG), hydrophobic poly(ε-caprolactone) (PCL), and negatively-charged, carboxylated PCL (CPCL). Murine ESCs were cultured on electrospun polymeric scaffolds and their differentiation to cardiomyocytes was assessed through measurements of viability, intracellular reactive oxygen species (ROS), α-myosin heavy chain expression (α-MHC), and intracellular Ca(2+) signaling dynamics. Interestingly, ESCs on the most compliant substrate, 4%PEG-86%PCL-10%CPCL, exhibited the highest α-MHC expression as well as the most mature Ca(2+) signaling dynamics. To investigate the role of scaffold modulus in ESC differentiation, the scaffold fiber density was reduced by altering the electrospinning parameters. The reduced modulus was found to enhance α-MHC gene expression, and promote maturation of myocyte Ca(2+) handling. These data indicate that ESC-derived cardiomyocyte differentiation and maturation can be promoted by tuning the mechanical and chemical properties of polymer scaffold via copolymerization and electrospinning techniques.