Photosynthetic dynamics in chrysanthemum in response to single step increases and decreases in photon flux density

The time-course of CO₂ assimilation rate and stomatal conductance to step changes in photosynthetic photon flux density (PPFD) was observed in Chrysanthemum × morifolium Ramat. `Fiesta'. When PPFD was increased from 200 to 600 micromoles per square meter per second, the rate of photosynthetic CO<sub>2</sub> assimilation showed an initial rapid increase over the first minute followed by a slower increase over the next 12 to 38 minutes, with a faster response in low-light-grown plants. Leaves exposed to small step increases (100 micromoles per square meter per second) reached the new steady-state assimilation rate within a minute. Both stomatal and biochemical limitations played a role during photosynthetic induction, but carboxylation limitations seemed to predominate during the first 5 to 10 minutes. Stomatal control during the slow phase of induction was less important in low-light compared to high-light-grown plants. In response to step decreases in PPFD, photosynthetic rate decreased rapidly and a depression in CO<sub>2</sub> assimilation prior to steady-state was observed. This CO<sub>2</sub> assimilation `dip' was considerably larger for the large step (400 micromoles per square meter per second) than for the small step. The rapid photosynthetic response seems to be controlled by biochemical processes. High- and low-light-grown plants did not differ in their photosynthetic response to PPFD step decreases.     

Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

Neutering Dogs: Effects on Joint Disorders and Cancers in Golden Retrievers

In contrast to European countries, the overwhelming majority of dogs in the U.S. are neutered (including spaying), usually done before one year of age. Given the importance of gonadal hormones in growth and development, this cultural contrast invites an analysis of the multiple organ systems that may be adversely affected by neutering. Using a single breed-specific dataset, the objective was to examine the variables of gender and age at the time of neutering versus leaving dogs gonadally intact, on all diseases occurring with sufficient frequency for statistical analyses. Given its popularity and vulnerability to various cancers and joint disorders, the Golden Retriever was chosen for this study. Veterinary hospital records of 759 client-owned, intact and neutered female and male dogs, 1–8 years old, were examined for diagnoses of hip dysplasia (HD), cranial cruciate ligament tear (CCL), lymphosarcoma (LSA), hemangiosarcoma (HSA), and mast cell tumor (MCT). Patients were classified as intact, or neutered early (<12 mo) or late (≥12 mo). Statistical analyses involved survival analyses and incidence rate comparisons. Outcomes at the 5 percent level of significance are reported. Of early-neutered males, 10 percent were diagnosed with HD, double the occurrence in intact males. There were no cases of CCL diagnosed in intact males or females, but in early-neutered males and females the occurrences were 5 percent and 8 percent, respectively. Almost 10 percent of early-neutered males were diagnosed with LSA, 3 times more than intact males. The percentage of HSA cases in late-neutered females (about 8 percent) was 4 times more than intact and early-neutered females. There were no cases of MCT in intact females, but the occurrence was nearly 6 percent in late-neutered females. The results have health implications for Golden Retriever companion and service dogs, and for oncologists using dogs as models of cancers that occur in humans.     

NIM1 overexpression in Arabidopsis potentiates plant disease resistance and results in enhanced effectiveness of fungicides

The NIM1 (for noninducible immunity, also known as NPR1) gene is required for the biological and chemical activation of systemic acquired resistance (SAR) in Arabidopsis. Overexpression of NIM1 in wild-type plants (hereafter referred to as NIM1 plants or lines) results in varying degrees of resistance to different pathogens. Experiments were performed to address the basis of the enhanced disease resistance responses seen in the NIM1 plants. The increased resistance observed in the NIM1 lines correlated with increased NIM1 protein levels and rapid induction of PR1 gene expression, a marker for SAR induction in Arabidopsis, following pathogen inoculation. Levels of salicylic acid (SA), an endogenous signaling molecule required for SAR induction, were not significantly increased compared with wild-type plants. SA was required for the enhanced resistance in NIM1 plants, however, suggesting that the effect of NIM1 overexpression is that plants are more responsive to SA or a SA-dependent signal. This hypothesis is supported by the heightened responsiveness that NIM1 lines exhibited to the SAR-inducing compound benzo(1,2,3)-thiadiazole-7-car-bothioic acid S-methyl ester. Furthermore, the increased efficacy of three fungicides was observed in the NIM1 plants, suggesting that a combination of transgenic and chemical approaches may lead to effective and durable disease-control strategies.     

A Sinorhizobium meliloti lipopolysaccharide mutant altered in cell surface sulfation

The Rhizobium-legume symbiosis involves the formation of a novel plant organ, the nodule, in which intracellular bacteria reduce molecular dinitrogen in exchange for plant photosynthates. Nodule development requires a bacterial signal referred to as Nod factor, which in Sinorhizobium meliloti is a beta-(1,4)-linked tetramer of N-acetylglucosamine containing N-acyl and O-acetyl modifications at the nonreducing end and a critical 6-O-sulfate at the reducing end. This sulfate modification requires the action of three gene products: nodH, which catalyzes the sulfonyl transfer, and nodPQ, which produce the activated form of sulfate, 3'-phosphoadenosine-5'-phosphosulfate. It was previously reported that S. meliloti cell surface polysaccharides are also covalently modified by sulfate in a reaction dependent on NodPQ. We have further characterized this unique form of bacterial carbohydrate modification. Our studies have determined that one of the nodPQ mutant strains used in the initial study of sulfation of cell surface harbored a second unlinked mutation. We cloned the gene affected by this mutation (referred to as lps-212) and found it to be an allele of lpsL, a gene previously predicted to encode a UDP-glucuronic acid epimerase. We demonstrated that lpsL encoded a UDP-glucuronic acid epimerase activity that was reduced in the lps-212 mutant. The lps-212 mutation resulted in an altered lipopolysaccharide structure that was reduced in sulfate modification in vitro and in vivo. Finally, we determined that the lps-212 mutation resulted in a reduced ability to elicit the formation of plant nodules and by altered infection thread structures that aborted prematurely.     

Poly(ethylene glycol) microparticles produced by precipitation polymerization in aqueous solution

Methods were developed to perform precipitation photopolymerization of PEG-diacrylate. Previously, comonomers have been added to PEG when precipitation polymerization was desired. In the present method, the LCST of the PEG itself was lowered by the addition of the kosmotropic salt sodium sulfate to an aqueous solution. Typical of a precipitation polymerization, small microparticles or microspheres (1-5 μm) resulted with relatively low polydispersity. However, aggregate formation was often severe, presumably because of a lack of stabilization of the phase-separated colloids. Microparticles were also produced by copoymerization of PEG-diacrylate with acrylic acid or aminoethylmethacrylate. The comonomers affected the zeta potential of the formed microparticles but not the size. The carboxyl groups of acrylic-acid-containing PEG microparticles were activated, and scaffolds were formed by mixing with amine-containing PEG microparticles. Although the scaffolds were relatively weak, human hepatoma cells showed excellent viability when present during microparticle cross-linking.     

Functional analysis of the plant disease resistance gene Pto using DNA shuffling

Pto is a serine/threonine kinase that mediates resistance in tomato to strains of Pseudomonas syringae pv. tomato expressing the (a)virulence proteins AvrPto or AvrPtoB. DNA shuffling was used as a combinatorial in vitro genetic approach to dissect the functional regions of Pto. The Pto gene was shuffled with four of its paralogs from a resistant haplotype to create a library of recombinant products that was screened for interaction with AvrPto in yeast. All interacting clones and a representative sample of noninteracting clones were sequenced, and their ability to signal downstream was tested by the elicitation of a hypersensitive response in an AvrPto-dependent or -independent manner in planta. Eight candidate regions important for binding to AvrPto or for downstream signaling were identified by statistical correlations between individual amino acid positions and phenotype. A subset of the regions had previously been identified as important for recognition, confirming the validity of the shuffling approach. Three novel regions important for Pto function were validated by site-directed mutagenesis. Several chimeras and point mutants exhibited a differential interaction with (a)virulence proteins in the AvrPto and VirPphA family, demonstrating distinct binding requirements for different ligands. Additionally, the identification of chimeras that are both constitutively active as well as capable of binding AvrPto indicates that elicitation of downstream signaling does not involve a conformational change that precludes binding of AvrPto, as previously hypothesized. The correlations between phenotypes and variation generated by DNA shuffling paralleled natural variation observed between orthologs of Pto from Lycopersicon spp.     

Laminin-411 is a vascular ligand for MCAM and facilitates TH17 cell entry into the CNS

TH17 cells enter tissues to facilitate pathogenic autoimmune responses, including multiple sclerosis (MS). However, the adhesion molecules involved in the unique migratory capacity of TH17 cells, into both inflamed and uninflamed tissues remain unclear. Herein, we characterize MCAM (CD146) as an adhesion molecule that defines human TH17 cells in the circulation; following in vitro restimulation of human memory T cells, nearly all of the capacity to secrete IL-17 is contained within the population of cells expressing MCAM. Furthermore, we identify the MCAM ligand as laminin 411, an isoform of laminin expressed within the vascular endothelial basement membranes under inflammatory as well as homeotstatic conditions. Purified MCAM-Fc binds to laminin 411 with an affinity of 27 nM, and recognizes vascular basement membranes in mouse and human tissue. MCAM-Fc binding was undetectable in tissue from mice with targeted deletion of laminin 411, indicating that laminin 411 is a major tissue ligand for MCAM. An anti-MCAM monoclonal antibody, selected for inhibition of laminin binding, as well as soluble MCAM-Fc, inhibited T cell adhesion to laminin 411 in vitro. When administered in vivo, the antibody reduced TH17 cell infiltration into the CNS and ameliorated disease in an animal model of MS. Our data suggest that MCAM and laminin 411 interact to facilitate TH17 cell entry into tissues and promote inflammation.