Antisense inhibition of the photosynthetic antenna proteins CP29 and CP26: implications for the mechanism of protective energy dissipation

The specific roles of the chlorophyll a/b binding proteins CP29 and CP26 in light harvesting and energy dissipation within the photosynthetic apparatus have been investigated. Arabidopsis was transformed with antisense constructs against the genes encoding the CP29 or CP26 apoprotein, which gave rise to several transgenic lines with remarkably low amounts of the antisense target proteins. The decrease in the level of CP24 protein in the CP29 antisense lines indicates a physical interaction between these complexes. Analysis of chlorophyll fluorescence showed that removal of the proteins affected photosystem II function, probably as a result of changes in the organization of the light-harvesting antenna. However, whole plant measurements showed that overall photosynthetic rates were similar to those in the wild type. Both antisense lines were capable of the qE type of nonphotochemical fluorescence quenching, although there were minor changes in the capacity for quenching and in its induction kinetics. High-light-induced violaxanthin deepoxidation to zeaxanthin was not affected, although the pool size of these pigments was decreased slightly. We conclude that CP29 and CP26 are unlikely to be sites for nonphotochemical quenching.     

Transgenic technology and applications in swine.

The introduction of foreign DNA into the genome of livestock and its stable integration into the germ line has been a major technical advance in agriculture. Production of transgenic livestock provides a method to rapidly introduce "new" genes into cattle, swine, sheep and goats without crossbreeding. It is a more extreme methodology, but in essence, not really different from crossbreeding or genetic selection in its result. Several recent developments will profoundly impact the use of transgenic technology in livestock production. These developments are: 1) the ability to isolate and maintain in vitro embryonic stem (ES) cells from preimplantation embryos, embryonic germ (EG) and somatic cells from fetuses; and somatic cells from adults, and 2) the ability to use these embryonic and somatic cells as nuclei donors in nuclear transfer or "cloning" strategies. Cell based (ES, EG, and somatic cells) strategies have several distinct advantages for use in the production of transgenic livestock that cannot be attained using pronuclear injection of DNA. There are many potential applications of transgenic methodology to develop new and improved strains of livestock. Practical applications of transgenesis in livestock production include enhanced prolificacy and reproductive performance, increased feed utilization and growth rate, improved carcass composition, improved milk production and/or composition and increased disease resistance. Development of transgenic farm animals will allow more flexibility in direct genetic manipulation of livestock.     

Optimizing nitrogen management in food and energy production and environmental protection: summary statement from the Second International Nitrogen Conference

Human efforts to produce food and energy are changing the nitrogen (N) cycle of the Earth. Many of these changes are highly beneficial for humans, while others are detrimental to people and the environment. These changes transcend scientific disciplines, geographical boundaries, and political structures. They challenge the creative minds of natural and social scientists, economists, engineers, business leaders, and decision makers. The Second International Nitrogen Conference was designed to facilitate communications among all stakeholders in the "nitrogen community" of the world. The Conference participants" goal in the years and decades ahead is to encourage every country to make optimal choices about N management in food production and consumption, energy production and use, and environmental protection. Scientific findings and recommendations for decision makers that emerged from the Conference are presented.     

Mechanisms of recruitment in oleic acid-injured lungs.

Lung recruitment strategies, such as the application of positive end-expiratory pressure (PEEP), are thought to protect the lungs from ventilator-associated injury by reducing the shear stress associated with the repeated opening of collapsed peripheral units. Using the parenchymal marker technique, we measured regional lung deformations in 13 oleic acid (OA)-injured dogs during mechanical ventilation in different postures. Whereas OA injury caused a marked decrease in the oscillation amplitude of dependent lung regions, even the most dependent regions maintained normal end-expiratory dimensions. This is because dependent lung is flooded as opposed to collapsed. PEEP restored oscillation amplitudes only at pressures that raised regional volumes above preinjury levels. Because the amount of PEEP necessary to promote dependent lung recruitment increased the end-expiratory dimensions of all lung regions (nondependent AND dependent ones) compared with their preinjury baseline, the "price" for recruitment is a universal increase in parenchymal stress. We conclude that the mechanics of the OA-injured lung might be more appropriately viewed as a partial liquid ventilation problem and not a shear stress and airway collapse problem and that the mechanisms of PEEP-related lung protection might have to be rethought.     

Inbreeding and experience affect response to climate change by endangered woodpeckers

In recent decades, female red-cockaded woodpeckers (Picoides borealis) have laid eggs increasingly earlier in response to a changing climate, as has been observed in several other bird species breeding at north temperate latitudes. Within each year, females that lay earlier are more productive than females that lay later. However, inexperienced females, experienced females who change mates and inbred birds have not adjusted to the changing climate by laying earlier, and have suffered reproductive costs as a result. Failure to respond to global climate change may be a further example of the reduced ability of inbred animals to respond to environmental challenges.     

Milk yield, energy balance, hormone, follicular and oocyte measures in early and mid-lactation Holstein cows

The purpose of the study was to determine the influence of energy status on metabolic and endocrine measures, follicular development, and the quality of oocytes obtained from cows during early and mid-lactation (ML). We selected Holstein cows at calving to be assigned to the early lactation (EL) group (n = 8), while we assigned cows at about day 90 postpartum to the ML group (n = 7). We obtained blood samples twice weekly from 4 weeks before aspiration to the aspiration periods for metabolite and hormone determinations. We performed ultrasound-guided transvaginal follicular aspiration (TVFA) twice weekly on all cows for a 10-week period. We obtained follicular fluid from the largest follicle > 10 mm in diameter for hormone determinations. We analyzed data by ANOVA, using the general linear model (GLM) procedures. Energy balance was positive (2.43 +/- 0.32 Mcal/kg) for ML cows and negative (-1.55 +/- 0.33 Mcal/kg) for EL cows. Serum progesterone (P4) for ML cows decreased rapidly from the first aspiration session (2.7 +/- 0.1 ng/ml) and reached a nadir at Week 8 (0.33 +/- 0.1 ng/ml), while follicular fluid P4 increased from 0.9 +/- 0.5 to 5.6 +/- 0.05 ng/ml. Serum and follicular fluid P4 remained relatively constant over the entire aspiration period for EL cows. Follicular fluid insulin-like growth factor I (TGF-I) concentrations increased linearly for EL and ML cows, but the increase was more rapid (159 +/- 36 to 200 +/- 36 ng/ml) for ML cows than for EL cows (145 +/- 36 to 164 +/- 36 ng/ml). Serum IGF-I followed the same pattern for ML cows but declined for EL cows. Early lactation cows experienced a rapid decrease in serum nonesterified fatty acids (NEFA; 0.32 +/- 0.2 to 0.22 +/- 0.2 meq/l), while serum NEFA concentrations were relatively stable (0.19 +/- 0.2 to 0.22 +/- 0.2 meq/l) for ML cows over the aspiration period. The number of follicles obtained from the twice weekly aspiration sessions increased linearly for both EL and ML cows (P < 0.05) over the 10-week period. However, the number of follicles increased from 14.2 +/- 0.5 (Day 119) to 18.1 +/- 0.5 (Day 190) in the ML cows, compared to the changes from 14.9 +/- 0.3 (Day 32) to 15.7 +/- 0.5 (Day 90) for the EL cows. These results indicate that cows are physiologically under more production stress during EL, but increasing follicular fluid and serum IGF-I throughout ML may reflect potential differences in follicle and oocyte measures, compared to cows in EL.     

The effectiveness of double-stranded short inhibitory RNAs (siRNAs) may depend on the method of transfection

RNA interference (RNAi) is a recently described powerful experimental tool that can cause sequence-specific gene silencing, thereby facilitating functional analysis of gene function. Consequently, we became interested in using RNAi to determine the function of aberrantly expressed ErbB3 in the KAS-6/1 human myeloma cell line. Despite the wealth of information available on the use of RNAi, dsRNA target design, and the transfection of dsRNA in vitro, little information is available for transfecting dsRNA into nonadherent cells from any species. In the present study, we report that gene silencing of ErbB3 was not observed in myeloma cells when dsRNA targeting ErbB3 was introduced using conventional transfection agents and protocols that have proved successful for several adherent cell lines. Silencing of ErbB3, however, was observed in T47D cells, an adherent breast carcinoma cell line, using the same transfection methods, indicating that our target sequence was functional for gene silencing of ErbB3. Interestingly, ErbB3 was silenced in myeloma cells when the dsRNA target was introduced by electroporation. Thus, our studies illustrate the striking dependence of dsRNA-mediated gene silencing in some cells on the methods of dsRNA transfection.     

Structural studies of the interaction between ubiquitin family proteins and proteasome subunit S5a

The 26S proteasome is essential for the proteolysis of proteins that have been covalently modified by the attachment of polyubiquitinated chains. Although the 20S core particle performs the degradation, the 19S regulatory cap complex is responsible for recognition of polyubiquitinated substrates. We have focused on how the S5a component of the 19S complex interacts with different ubiquitin-like (ubl) modules, to advance our understanding of how polyubiquitinated proteins are targeted to the proteasome. To achieve this, we have determined the solution structure of the ubl domain of hPLIC-2 and obtained a structural model of hHR23a by using NMR spectroscopy and homology modeling. We have also compared the S5a binding properties of ubiquitin, SUMO-1, and the ubl domains of hPLIC-2 and hHR23a and have identified the residues on their respective S5a contact surfaces. We provide evidence that the S5a-binding surface on the ubl domain of hPLIC-2 is required for its interaction with the proteasome. This study provides structural insights into protein recognition by the proteasome, and illustrates how the protein surface of a commonly utilized fold has highly evolved for various biological roles.     

Identification of mutants of Arabidopsis defective in acclimation of photosynthesis to the light environment

In common with many other higher plant species, Arabidopsis undergoes photosynthetic acclimation, altering the composition of the photosynthetic apparatus in response to fluctuations in its growth environment. The changes in photosynthetic function that result from acclimation can be detected in a noninvasive manner by monitoring chlorophyll (Chl) fluorescence. This technique has been used to develop a screen that enables the rapid identification of plants defective at ACCLIMATION OF PHOTOSYNTHESIS TO THE ENVIRONMENT (APE) loci. The application of this screen to a population of T-DNA-transformed Arabidopsis has successfully led to the identification of a number of mutant lines with altered Chl fluorescence characteristics. Analysis of photosynthesis and pigment composition in leaves from three such mutants showed that they had altered acclimation responses to the growth light environment, each having a distinct acclimation-defective phenotype, demonstrating that screening for mutants using Chl fluorescence is a viable strategy for the investigation of acclimation. Sequencing of the genomic DNA flanking the T-DNA elements showed that in the ape1 mutant, a gene was disrupted that encodes a protein of unknown function but that appears to be specific to photosynthetic organisms, whereas the ape2 mutant carries an insertion in the region of the TPT gene encoding the chloroplast inner envelope triose phosphate/phosphate translocator.