Biochemical and immunological characterization of pea nuclear intermediate filament proteins

In immunoblot assays, at least three putative nuclear intermediate filament (NIF) proteins were detected in nuclear envelope-matrix (NEM) and lamin (L1) fractions of nuclei from plumules of dark-grown pea (Pisum sativum L.) seedlings. These NIF proteins had apparent molecular masses of ca. 65, 60, and 54 kDa (also referred to as p65, p60, and p54), and appeared as multiple isoelectric forms, with pIs ranging from ca. 4.8 to 6.0. Polyclonal and monoclonal antibodies were raised to the 65-kDa NIF protein bands excised from gels after electrophoresis. These anti-pea antibodies were specifically cross-reactive with the pea nuclear p65, p60, and p54 proteins and also with chicken lamins. Sequence alignment of peptide fragments obtained from the 65- and 60-kDa pea NIF proteins showed similarity with animal intermediate filament proteins such as lamins and keratins and with certain plant proteins predicted to have long coiled-coil domains. These pea NIF proteins were further purified and enriched from the NEM fraction using methods similar to those used for isolating animal lamins. When negatively stained and viewed by transmission electron microscopy, the filaments in the pea lamin (L1) fraction appeared to be 6–12 nm in diameter. As assayed by immunofluorescence cytochemistry using a confocal laser-scanning microscope, fixed pea plumule cells displayed uniform as opposed to peripheral nuclear staining by several of the antibody preparations, both polyclonal and monoclonal. This report describes the biochemical and immunological properties of these pea NIF proteins.Abbreviations IF Intermediate filament - L Lamin fraction - LM Lamina-matrix fraction - MAb JLA20 Anti-chicken actin monoclonal antibody - MAb LN43 Anti-human lamin B2 monoclonal antibody - MAb PL19 Anti-pea lamin #19 monoclonal antibody - MAb TIB 131 Anti-intermediate filament monoclonal antibody - N Nuclei fraction - NEM Nuclear envelope-matrix fraction - NIF Nuclear intermediate filament - PAb PL3 Anti-pea lamin #3 polyclonal antibody     

Plasticity of size and growth in fluctuating thermal environments: comparing reaction norms and performance curves

Ectothermic animals exhibit two distinct kinds of plasticityin response to temperature: Thermal performance curves (TPCs),in which an individual's performance (e.g., growth rate) variesin response to current temperature; and developmental reactionnorms (DRNs), in which the trait value (e.g., adult body sizeor development time) of a genotype varies in response to developmentaltemperatures experienced over some time period during development.Here we explore patterns of genetic variation and selectionon TPCs and DRNs for insects in fluctuating thermal environments.First, we describe two statistical methods for partitioningtotal genetic variation into variation for overall size or performanceand variation in plasticity, and apply these methods to availabledatasets on DRNs and TPCs for insect growth and size. Our resultsindicate that for the datasets we considered, genetic variationin plasticity represents a larger proportion of the total geneticvariation in TPCs compared to DRNs, for the available datasets.Simulations suggest that estimates of the genetic variationin plasticity are strongly affected by the number and rangeof temperatures considered, and by the degree of nonlinearityin the TPC or DRN. Second, we review a recent analysis of fieldselection studies which indicates that directional selectionfavoring increased overall size is common in many systems—thatbigger is frequently fitter. Third, we use a recent theoreticalmodel to examine how selection on thermal performance curvesrelates to environmental temperatures during selection. Themodel predicts that if selection acts primarily on adult sizeor development time, then selection on thermal performance curvesfor larval growth or development rates is directly related tothe frequency distribution of temperatures experienced duringlarval development. Using data on caterpillar temperatures inthe field, we show that the strength of directional selectionon growth rate is predicted to be greater at the modal (mostfrequent) temperatures, not at the mean temperature or at temperaturesat which growth rate is maximized. Our results illustrate someof the differences in genetic architecture and patterns of selectionbetween thermal performance curves and developmental reactionnorms.     

PPAR-γ as a therapeutic target in cardiovascular disease: evidence and uncertainty

Peroxisome proliferator-activated receptor γ (PPAR-γ) is a key regulator of fatty acid metabolism, promoting its storage in adipose tissue and reducing circulating concentrations of free fatty acids. Activation of PPAR-γ has favorable effects on measures of adipocyte function, insulin sensitivity, lipoprotein metabolism, and vascular structure and function. Despite these effects, clinical trials of thiazolidinedione PPAR-γ activators have not provided conclusive evidence that they reduce cardiovascular morbidity and mortality. The apparent disparity between effects on laboratory measurements and clinical outcomes may be related to limitations of clinical trials, adverse effects of PPAR-γ activation, or off-target effects of thiazolidinedione agents. This review addresses these issues from a clinician's perspective and highlights several ongoing clinical trials that may help to clarify the therapeutic role of PPAR-γ activators in cardiovascular disease.     

Construction of a Panel of Canine–Rodent Hybrid Cell Lines for Use in Partitioning of the Canine Genome

We have constructed a collection of canine–rodent microcell hybrid cell lines by fusion of canine fibroblast microcell donors with immortalized rodent recipient cells. Characterization of the hybrid cell lines using a combination of fluorescencein situhybridization and PCR analysis of canine microsatellite repeat sequences allowed selection of a panel of hybrids in which most canine chromosomes are represented. Approximately 90% of genetic markers and genes that were tested could be assigned to 1 of 31 anonymous canine chromosome groups, based on common patterns of retention in the hybrid set. Many of these putative chromosome groups have now been validated by linkage analysis. This panel of cell lines provides a tool for development of genetic, physical, and comparative maps of the canine genome.     

Latent virus influences the generation and maintenance of CD8+ T cell memory

The influence of latent virus on CD8+ T cell memory is poorly understood. HSV type 1 specifically establishes latency in trigeminal ganglia (TG) after corneal infection of mice. In latently infected TG, IL-15 deprivation reduced the following: 1) accumulation of HSV-specific CD8+ effector T cells (HSV-CD8(eff)), 2) accumulation of CD127(+) putative HSV-CD8 memory precursors, and 3) the size and functionality of the memory (HSV-CD8(mem)) population. Although compromised in IL-15(-/-) mice, the HSV-CD8(mem) pool persisted in latently infected tissue, but not in noninfected tissue of the same mice. Anti-IL-2 treatment also dramatically reduced the size of the HSV-CD8(eff) population in the TG, but did not influence the concomitant generation of the CD127+ putative HSV-CD8(mem) precursor population or the size or functionality of the HSV-CD8(mem) pool. Thus, the size of the memory pool appears to be determined by the size of the CD127+ CD8(mem) precursor population and not by the size of the overall CD8(eff) pool. HSV-CD8(mem) showed a higher basal rate of proliferation in latently infected than noninfected tissue, which was associated with a reduced population of CD4+FoxP3+ regulatory T cells. Thus, the generation, maintenance, and function of memory CD8+ T cells is markedly influenced by latent virus.     

Escalation and extinction selectivity: morphology versus isotopic reconstruction of bivalve metabolism

Studies that have tested and failed to support the hypothesis that escalated species (e.g., those with predation-resistant adaptations) are more susceptible to elimination during mass extinctions have concentrated on the distribution and degree of morphological defenses in molluscan species. This morphological approach to determining level of escalation in bivalves may be oversimplified because it does not account for metabolic rate, which is an important measure of escalation that is less readily accessible for fossils. Shell growth rates in living bivalves are positively correlated with metabolic rate and thus are potential indicators of level of escalation. To evaluate this approach, we used oxygen isotopes to reconstruct shell growth rates for two bivalve species (Macrocallista marylandica and Glossus markoei) from Miocene-aged sediments of Maryland. Although both species are classified as non-escalated based on morphology, the isotopic data indicate that M. marylandica was a faster-growing species with a higher metabolic rate and G. markoei was a slower-growing species with a lower metabolic rate. Based on these results, we predict that some morphologically non-escalated species in previous tests of extinction selectivity should be reclassified as escalated because of their fast shell growth rates (i.e., high metabolic rates). Studies that evaluate the level of escalation of a fauna should take into account the energetic physiology of taxa to avoid misleading results.     

Interaction of the membrane-inserted diphtheria toxin T domain with peptides and its possible implications for chaperone-like T domain behavior

The T domain of diphtheria toxin is believed to aid the low-pH-triggered translocation of the partly unfolded A chain (C domain) through cell membranes. Recent experiments have suggested the possibility that the T domain aids translocation by acting as a membrane-inserted chaperone [Ren, J., et al. (1999) Science 284, 955-957]. One prediction of this model is that the membrane-inserted T domain should be able to interact with sequences that mimic unfolded proteins. To understand the basis of interaction of the membrane-inserted T domain with unfolded polypeptides, its interaction with water-soluble peptides having different sequences was studied. The membrane-inserted T domain was able to recognize helix-forming 23-residue Ala-rich peptides. In the presence of such peptides, hydrophobic helix 9 of the T domain underwent the previously characterized conformational change from a state exhibiting shallow membrane insertion to one exhibiting deep insertion. This conformational change was more readily induced by the more hydrophobic peptides that were tested. It did not occur at all in the presence a hydrophilic peptide in which alternating Ser and Gly replaced Ala or in the presence of unfolded hydrophilic peptides derived from the A chain of the toxin. Interestingly, a peptide with a complex sequence (RKE(3)KE(2)LMEW(2)KM(2)SETLNF) also interacted with the T domain very strongly. We conclude that the membrane-inserted T domain cannot recognize every unfolded amino acid sequence. However, it does not exhibit strong sequence specificity, instead having the ability to recognize and interact with a variety of amino acid sequences having moderate hydrophobicity. This recognition was not strictly correlated with the strength of peptide binding to the lipid, suggesting that more than just hydrophobicity is involved. Although it does not prove that the T domain functions as a chaperone, T domain recognition of hydrophobic sequences is consistent with it having polypeptide recognition properties that are chaperone-like.     

Type III secretion and effectors shape the survival and growth pattern of Pseudomonas syringae on leaf surfaces

The bacterium Pseudomonas syringae pv syringae B728a (PsyB728a) uses a type III secretion system (T3SS) to inject effector proteins into plant cells, a process that modulates the susceptibility of different plants to infection. Analysis of GREEN FLUORESCENT PROTEIN-expressing PsyB728a after spray inoculation without additives under moderate relative humidity conditions permitted (1) a detailed analysis of this strain's survival and growth pattern on host (Nicotiana benthamiana) and nonhost (tomato [Solanum lycopersicum]) leaf surfaces, (2) an assessment of the role of plant defenses in affecting PsyB728a leaf surface (epiphytic) growth, and (3) the contribution of the T3SS and specific effectors to PsyB728a epiphytic survival and growth. On host leaf surfaces, PsyB728a cells initially persist without growing, and show an increased population only after 48 h, unless plants are pretreated with the defense-inducing chemical benzothiazole. During the persistence period, some PsyB728a cells induce a T3SS reporter, whereas a T3SS-deficient mutant shows reduced survival. By 72 h, rare invasion by PsyB728a to the mesophyll region of host leaves occurs, but endophytic and epiphytic bacterial growths are not correlated. The effectors HopZ3 and HopAA1 delay the onset of epiphytic growth of PsyB728a on N. benthamiana, whereas they promote epiphytic survival/growth on tomato. These effectors localize to distinct sites in plant cells and likely have different mechanisms of action. HopZ3 may enzymatically modify host targets, as it requires residues important for the catalytic activity of other proteins in its family of proteases. Thus, the T3SS, HopAA1, HopZ3, and plant defenses strongly influence epiphytic survival and/or growth of PsyB728a.     

POR C of Arabidopsis thaliana: a third light- and NADPH-dependent protochlorophyllide oxidoreductase that is differentially regulated by light

During the sequencing of the genome of Arabidopsis thaliana a gene has been identified that encodes a novel NADPH-protochlorophyllide oxidoreductase (POR)-like protein (accession number AC 002560). This protein has been named POR C. We have expressed the POR C protein in Escherichia coli and have determined its in vitro activity. POR C shows the characteristics of a light-dependent and NADPH-requiring POR similar to POR A and POR B. The expression of the POR C gene differs markedly from that of the POR A and POR B genes. In contrast to the POR A and POR B mRNAs, the POR C mRNA has been shown previously to accumulate only after the beginning of illumination. In light-adapted mature plants only POR B and POR C mRNAs were detectable. The amounts of both mRNAs show pronounced diurnal rhythmic fluctuations. While the oscillations of POR B mRNA are under the control of the circadian clock, those of POR C mRNA are not. Another difference between POR B and POR C was found in seedlings that were grown under continuous white light. The concentration of POR C mRNA rapidly declined and soon dropped beyond the limit of detection, after these seedlings were transferred to the dark. On the other hand, POR B mRNA was unaffected by this light/dark shift. When seedlings were exposed to different light intensities, the amounts of POR B mRNA remained the same, while POR A and POR C mRNAs were modulated in an inverse way by these light intensity changes. POR A mRNA was still detectable in seedlings grown under low light intensities but disappeared at higher light intensities, while the mRNA concentration of POR C rose with increasing light intensities. These different responses to light suggest that the functions of the three PORs of Arabidopsis are not completely redundant, but may allow the plant to adapt its needs for chlorophyll biosynthesis more selectively by using preferentially one of the three enzymes under a given light regime.