Flowering phenology and sexual allocation in single-mutation lineages of Arabidopsis thaliana

We investigated the relationship between flowering time and sexual allocation in wild-type Arabidopsis thaliana and in genetically similar lineages with single-locus mutations of floral induction genes. We examined whether the mechanisms of growth and development that govern resource investment would permit the independent evolution of reproductive phenology and sexual allocation, or whether constraints, manifested as pleiotropic effects of the single mutations, would link these two life-history traits. Flowering times differed significantly among genotypes, and, as expected, later flowering times were associated with larger vegetative size. Later flowering genotypes produced heavier floral parts (larger petals, in particular), and allocated a significantly lower proportion of biomass to androecia, especially in final allocations that included fruit biomass. At least part of this pleiotropic covariation of flowering time and sexual allocation is likely to be mediated by vegetative size and the rate of resource supply to growing reproductive tissues, because the larger fruits of late-flowering genotypes required the same time, or proportionately less time than the difference in biomass, to mature. Because fruit mass is considered an investment in female function, sexual allocation measured at the end of a growing season tends to be highly female biased in angiosperms. We consider the implications of the pleiotropic association of flowering time, vegetative size, and sexual investment for the theory of sex allocation, and suggest that the idiosyncratic phenology of sexual investment in flowering plants creates a departure from a central assumption of Fisher's seminal sex allocation argument.     

Tetrameric mouse acetylcholinesterase: continuum diffusion rate calculations by solving the steady-state Smoluchowski equation using finite element methods

The tetramer is the most important form for acetylcholinesterase in physiological conditions, i.e., in the neuromuscular junction and the nervous system. It is important to study the diffusion of acetylcholine to the active sites of the tetrameric enzyme to understand the overall signal transduction process in these cellular components. Crystallographic studies revealed two different forms of tetramers, suggesting a flexible tetramer model for acetylcholinesterase. Using a recently developed finite element solver for the steady-state Smoluchowski equation, we have calculated the reaction rate for three mouse acetylcholinesterase tetramers using these two crystal structures and an intermediate structure as templates. Our results show that the reaction rates differ for different individual active sites in the compact tetramer crystal structure, and the rates are similar for different individual active sites in the other crystal structure and the intermediate structure. In the limit of zero salt, the reaction rates per active site for the tetramers are the same as that for the monomer, whereas at higher ionic strength, the rates per active site for the tetramers are approximately 67%-75% of the rate for the monomer. By analyzing the effect of electrostatic forces on ACh diffusion, we find that electrostatic forces play an even more important role for the tetramers than for the monomer. This study also shows that the finite element solver is well suited for solving the diffusion problem within complicated geometries.     

Increased myogenic responses in uterine but not mesenteric arteries from pregnant offspring of diet-restricted rat dams

Results of epidemiological and animal studies suggest a link between poor in utero growth and cardiovascular disease in adult offspring. Few studies, however, have examined the effects of maternal undernutrition on the vasculature of pregnant female offspring, and to our knowledge, no studies have examined myogenic responses, which are essential to vascular tone development, in these animal models. Thus, myogenic responses were assessed in radial uterine arteries of pregnant female offspring to determine if diet restriction during pregnancy could contribute to transgenerational effects. These results were compared to those in mesenteric arteries, which greatly contribute to peripheral vascular resistance. Myogenic responses in the presence and absence of inhibitors for nitric oxide synthase (NOS) and prostaglandin H synthase (PGHS) were measured in arteries isolated from pregnant, 3-mo-old female offspring of control-fed (C(off)) and globally diet-restricted (DR(off)) rat dams. Although no differences were found in pregnancy weight gain, litter size, or fetal weights, placental size was significantly reduced in DR(off) compared to C(off). Enhanced myogenic reactivity was observed at the highest pressure tested (110 mm Hg) in uterine, but not in mesenteric, arteries from DR(off) compared to C(off). Inhibition of NOS, but not of PGHS, significantly increased myogenic responses in uterine arteries at pressures greater than 80 mm Hg in C(off) but, interestingly, not in DR(off) compared to untreated uterine arteries. Thus, impaired uterine vascular function in diet-restricted pregnant rat dams, which leads to similar impairment in their pregnant offspring, may be a mechanism through which transgenerational effects of unhealthy pregnancies occur.     

Structural studies on the alphabeta T-cell receptor

Alphabeta T-cell receptor (TcR) recognition of antigenic peptides bound to the major histocompatibility complex (pMHC), is integral to the cellular immune system. Crystallographic studies over the last decade have provided significant insight into this unique trimolecular recognition event. The TcR-pMHC structural information has been paralleled by biophysical studies that have further explored the emerging binding models in an attempt to answer fundamental immunological questions regarding MHC restriction, T-cell immunodominance and TcR cross-reactivity. However, despite the important data that has been generated regarding TcR-pMHC interactions, the scope of this information is still incomplete due to the limited range of TcRs that have been studied. These limitations are primarily due to difficulties in obtaining high yields of recombinant alphabeta TcR for crystallographic and biophysical analysis; here we will discuss some of the protein engineering strategies that have been employed to expand the pool of recombinant TcRs suitable for crystallographic studies and the subsequent studies that have utilized these proteins.     

Specific method for determination of gefitinib in human plasma, mouse plasma and tissues using high performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using liquid chromatography-tandem mass spectrometry (LC/MS/MS) for determination of gefitinib in human plasma and mouse plasma and tissue. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma or a 200 mg/mL tissue homogenate diluted 1/10 in human plasma with 0.3 mL acetonitrile. Separation of the compounds of interest, including the internal standard (d8)-gefitinib, was achieved on a Waters X-Terra C18 (50 mm x 2.1 mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile-water (70:30, v/v) containing 0.1% formic acid and isocratic flow at 0.15 mL/min for 3 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 1-1000 ng/mL for the human plasma samples and 5-1000 ng/mL for mouse plasma and tissue samples with values for the coefficient of determination of > 0.99. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (< 15%). This method was subsequently used to measure concentrations of gefitinib in mice following administration of a single dose of 150 mg/kg intraperitoneally and in cancer patients receiving an oral daily dose of 250 mg.     

Comparison of transient protein expression in tobacco leaves and plant suspension culture

Transient gene expression is being developed to provide a more rapid means of assessing plant tissues as a protein production platform without the labor-intensive and time-consuming process of generating stably transformed transgenic plants. Transient expression of the gus-intron reporter gene was facilitated in three different tobacco species. Two different approaches to T-DNA delivery were compared: (1) infiltration of a prototrophic strain of Agrobacterium into leaves and (2) coculture of plant cell suspension cultures with an Agrobacterium auxotroph. Wounding of plant tissues with a wire brush prior to infiltration had a large positive impact on Nicotianabenthamiana leaves but not for Nicotiana tabacum or Nicotiana glutinosa. The best expression level achieved by leaf infiltration was in N. benthamiana (0.025% total soluble protein). A cell suspension culture line of N. glutinosa achieved an expression level greater than 0.04% TSP. The tissue culture-based technique therefore provides improved levels of transient expression under aseptic conditions to facilitate improvements in expression by control of the plant cell culture and Agrobacterium coculture environments.