Postnatal deficiency of essential fatty acids in mice results in resistance to diet-induced obesity and low plasma insulin during adulthood

Our objective was to investigate the long-term metabolic effects of postnatal essential fatty acid deficiency (EFAD). Mouse dams were fed an EFAD diet or an isoenergetic control diet 4 days before delivery and throughout lactation. The pups were weaned to standard diet (STD) and were later subdivided into two groups: receiving high fat diet (HFD) or STD. Body composition, energy expenditure, food intake and leptin levels were analyzed in adult offspring. Blood glucose and plasma insulin concentrations were measured before and during a glucose tolerance test. EFAD offspring fed STD were leaner with lower plasma leptin and insulin concentrations compared to controls. EFAD offspring fed HFD were resistant to diet-induced obesity, had higher energy expenditure and lower levels of plasma leptin and insulin compared to controls. These results indicate that the fatty acid composition during lactation is important for body composition and glucose tolerance in the adult offspring.     

Radiation-sensitive Arabidopsis mutants are proficient for T-DNA transformation

Stable transformation of plants by Agrobacterium T-DNAs requires that the transgene insert into the host chromosome. Although most of the Agrobacterium Ti plasmid genes required for this process have been studied in depth, few plant-encoded factors have been identified, although such factors, presumably DNA repair proteins, are widely presumed to exist. It has previously been suggested that the UVH1 gene product is required for stable T-DNA integration in Arabidopsis. Here we present evidence suggesting that uvh1 mutants are essentially wild type for T-DNA integration following inoculation via the vacuum-infiltration procedure.     

Human cytomegalovirus UL97 Kinase is required for the normal intranuclear distribution of pp65 and virion morphogenesis

Recombinant human cytomegaloviruses that do not express UL97 kinase activity exhibit a distinctive plaque morphology characterized by the formation of highly refractile bodies late in infection. These structures were also observed in infected cells treated with the UL97 kinase inhibitor maribavir. Nuclear inclusions were purified to near homogeneity, and the constituent proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. This analysis demonstrated that the aggregates were formed principally of the tegument proteins pp65 and ppUL25 but also contained additional virion structural proteins including the major capsid protein. Immunoblotting experiments confirmed these results and identified a number of additional viral proteins present in the purified tegument aggregates. Interestingly, the formation of these structures appeared to be dependent on pp65, since it was not induced in cells infected with a recombinant virus with this open reading frame deleted. Morphologically similar aggregates could be reproduced in nuclei of uninfected cells by overexpressing pp65, and their formation was prevented by coexpressing the UL97 kinase. Inhibition of UL97 kinase activity with maribavir or mutation of an essential amino acid in the kinase abolished its ability to prevent aggregate formation. These data taken together suggest that the UL97 kinase impacts the aggregation of pp65 in the nuclei of infected cells. We propose that the kinase plays an important role in the acquisition of tegument during virion morphogenesis in the nucleus and that this activity represents an important step in the production of mature virus particles.     

Langmuir monolayer approaches to protein recognition through molecular imprinting

Surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM) have been employed to investigate ferritin adsorption to binary surfactant monolayers of cationic dioctadecyldimethylammonium bromide (DOMA) and non-ionic methyl stearate (SME). Surfactant molar ratios, miscibility, and lateral mobility were controlled to define the number, size, and distribution of "binding sites" for ferritin, which under the low ionic strength conditions investigated, adsorbed to the monolayers predominantly through electrostatic interactions. Successive adsorption/desorption cycles revealed that fluid monolayers, capable of laterally restructuring during the initial protein adsorption event, bound up to 60% more ferritin (dependent on SME:DOMA ratios) as compared to monolayers that were immobilized on a hydrophobic support during this first adsorption step. The enhanced binding of ferritin to fluid monolayers was accentuated in films having non-ionic SME as the principal component. These findings support the premise that the surfactants reorganize to form favorable interactions with an adsorbing protein, leading to protein specific charge patterns, or templates, in the films. Template assessment, however, was complicated by the presence of an irreversibly bound protein fraction, which AFM revealed to be locally ordered protein clusters.     

Shared forces of sex chromosome evolution in haploid-mating and diploid-mating organisms: Microbotryum violaceum and other model organisms

It is usually posited that the most important factors contributing to sex chromosome evolution in diploids are the suppression of meiotic recombination and the asymmetry that results from one chromosome (the Y) being permanently heterozygous and the other (the X) being homozygous in half of the individuals involved in mating. To distinguish between the roles of these two factors, it would be valuable to compare sex chromosomes in diploid-mating organisms and organisms where mating compatibility is determined in the haploid stage. In this latter group, no such asymmetry occurs because the sex chromosomes are equally heterozygous. Here we show in the fungus Microbotryum violaceum that the chromosomes carrying the mating-type locus, and thus determining haploid-mating compatibility, are rich in transposable elements, dimorphic in size, and carry unequal densities of functional genes. Through analysis of available complete genomes, we also show that M. violaceum is, remarkably, more similar to humans and mice than to yeast, nematodes, or fruit flies with regard to the differential accumulation of transposable elements in the chromosomes determining mating compatibility vs. the autosomes. We conclude that restricted recombination, rather than asymmetrical sheltering, hemizygosity, or dosage compensation, is sufficient to account for the common sex chromosome characteristics.     

Recent pulsed EPR studies of the photosystem II oxygen-evolving complex: implications as to water oxidation mechanisms

The pulsed electron paramagnetic resonance (EPR) methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the structure of the Photosystem II oxygen-evolving complex (OEC), including the paramagnetic manganese cluster and its immediate surroundings. Recent unpublished results from the pulsed EPR laboratory at UC-Davis are discussed, along with aspects of recent publications, with a focus on substrate and cofactor interactions. New data on the proximity of exchangeable deuterons around the Mn cluster poised in the S(0)-state are presented and interpreted. These pulsed EPR results are used in an evaluation of several recently proposed mechanisms for PSII water oxidation. We strongly favor mechanistic models where the substrate waters bind within the OEC early in the S-state cycle. Models in which the O-O bond is formed by a nucleophilic attack by a Ca(2+)-bound water on a strong S(4)-state electrophile provide a good match to the pulsed EPR data.     

Estrogen actions on follicle formation and early follicle development

Estradiol-17beta (E(2)) affects late follicular development, whereas primordial follicle differentiation and early activation are believed to be independent of E(2). To test this hypothesis we compared numbers of primordial and primary follicles in wild-type and E(2)-deficient, aromatase knockout (ArKO) mice, and the immunohistochemical staining or mRNA expression of Mullerian inhibiting substance (MIS), Wilms tumor 1 (WT-1), and growth differentiation factor (GDF9), which are known to effect early follicular differentiation. Proliferating cell nuclear antigen (PCNA) staining was a marker of proliferative index. The effects of E(2) replacement for 3 wk in 7-wk-old ArKO and wild-type mice on these parameters were also tested. ArKO mice had reduced numbers of primordial and primary follicles compared with wild-type mice (63%, P < 0.001 and 60%, P = 0.062, respectively). This reduction was not corrected by E(2) treatment, suggesting that E(2) affects the initial formation or activation of primordial follicles. There was a significant increase in the diameters of the oocytes in primordial follicles of ArKO mice compared with mice of the wild type. There were no differences in the immunostaining of MIS, WT-1, and PCNA in primordial and primary follicles between wild-type and ArKO mice. The only difference was as a consequence of Sertoli and Leydig cells that develop in ovaries of ArKO mice. GDF9 mRNA expression was markedly increased in ArKO ovaries. E(2) treatment restored the ovarian follicular morphology in ArKO mice, and consequently the immunostaining patterns, but had no effect on early follicle numbers. In conclusion, E(2) has a role in controlling the size of the oocyte and primordial follicle pool in mice.     

Target antigens determine graft-versus-host disease phenotype

Chronic graft-vs-host disease (cGVHD) is an increasingly frequent complication of allogeneic stem cell transplantation. Phenotypically, cGVHD differs from patient to patient; in particular, a subset of patients develops extensive cutaneous fibrosis. Similarly, graft-vs-host disease (GVHD) is distinct in inbred murine donor:recipient pairings, indicating a genetic component to disease phenotype. The B10.D2 -->BALB/c (H-2d) strain pairing uniquely recapitulates key pathologic features of fibrotic human cutaneous cGVHD. To distinguish whether this genetic component is due to differences in genes that modulate immune responses or to the specific Ags targeted, we asked whether skin-dominant cGVHD also develops in the B10 -->BALB.B (H-2b) and B10.BR -->BALB.K (H-2k) MHC-congenic pairings. Because each MHC haplotype presents different peptides and selects different T cell repertoires, GVHD in each donor:recipient pair undoubtedly targets different Ags. We found that, in contrast to BALB/c recipients, BALB.B mice never manifested skin disease while BALB.K mice developed a modified form of skin disease. Instead, BALB.B and BALB.K recipients developed systemic GVHD which was absent in BALB/c mice. Moreover, in (B10 x B10.D2)F1 -->(BALB.B x BALB/c)F1 H-2b/d transplants, recipients developed both cutaneous and systemic disease. Thus, the selection of immunodominant Ags determines the target and character of GVHD, providing insight into the genetic basis for different forms of GVHD.