Chlorophyllide a Oxygenase mRNA and Protein Levels Correlate with the Chlorophyll a/b Ratio in Arabidopsis thaliana

Plants can change the size of their light harvesting complexes in response to growth at different light intensities. Although these changes are small compared to those observed in algae, their conservation in many plant species suggest they play an important role in photoacclimation. A polyclonal antibody to the C-terminus of the Arabidopsis thaliana chlorophyllide a oxygenase (CAO) protein was used to determine if CAO protein levels change under three conditions which perturb chlorophyll levels. These conditions were: (1) transfer to shaded light intensity; (2) limited chlorophyll synthesis, and (3) during photoinhibition. Transfer of wild-type plants from moderate to shaded light intensity resulted in a slight reduction in the Chl a/b ratio, and increases in both CAO and Lhcb1 mRNA levels as well as CAO protein levels. CAO protein levels were also measured in the cch1 mutant, a P642L missense mutation in the H subunit of Mg-chelatase. This mutant has reduced total Chl levels and an increased Chl a/b ratio when transferred to moderate light intensity. After transfer to moderate light intensity, CAO mRNA levels decreased in the cch1 mutant, and a concomitant decrease in CAO protein levels was also observed. Measurements of tetrapyrrole intermediates suggested that decreased Chl synthesis in the cch1 mutant was not a result of increased feedback inhibition at higher light intensity. When wild-type plants were exposed to photoinhibitory light intensity for 3 h, total Chl levels decreased and both CAO mRNA and CAO protein levels were also reduced. These results indicate that CAO protein levels correlate with CAO mRNA levels, and suggest that changes in Chl b levels in vascular plants, are regulated, in part, at the CAO mRNA level.     

Sexual Orientation Disparities in Weight Status in Adolescence: Findings From a Prospective Study

A growing number of studies among adult women have documented disparities in overweight adversely affecting lesbian and bisexual women, but few studies have examined sexual orientation–related patterns in weight status among men or adolescents. We examined sexual orientation group trends in BMI (kg/m²), BMI Z‐scores, and overweight using 56,990 observations from 13,785 adolescent females and males in the Growing Up Today Study (GUTS), a large prospective cohort of US youth. Participants provided self‐reported information from six waves of questionnaire data collection from 1998 to 2005. Gender‐stratified linear regression models were used to estimate BMI and BMI Z‐scores and modified Poisson regression models to estimate risk ratios for overweight, controlling for age and race/ethnicity, with heterosexuals as the referent group. Among females, we observed fairly consistently elevated BMI in all sexual orientation minority groups relative to heterosexual peers. In contrast, among males we documented a sexual‐orientation‐by‐age interaction indicating steeper increases in BMI with age from early‐to‐late adolescence in heterosexuals relative to sexual orientation minorities. Additional prospective research is needed to understand the determinants of observed sexual orientation disparities and to inform appropriate preventive and treatment interventions. The long‐term health consequences of overweight are well‐documented and over time are likely to exact a high toll on populations with elevated rates.     

Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes

The pathological prion protein PrP(Sc) is the only known component of the infectious prion. In cells infected with prions, PrP(Sc) is formed posttranslationally by the refolding of the benign cell surface glycoprotein PrP(C) into an aberrant conformation. The two PrP isoforms possess very different properties, as PrP(Sc) has a protease-resistant core, forms very large amyloidic aggregates in detergents, and is only weakly immunoreactive in its native form. We now show that prion-infected rodent brains and cultured cells contain previously unrecognized protease-sensitive PrP(Sc) varieties. In both ionic (Sarkosyl) and nonionic (n-octyl beta-D-glucopyranoside) detergents, the novel protease-sensitive PrP(Sc) species formed aggregates as small as 600 kDa, as measured by gel filtration. The denaturation dependence of PrP(Sc) immunoreactivity correlated with the size of the aggregate. The small PrP(Sc) aggregates described here are consistent with the previous demonstration of scrapie infectivity in brain fractions with a sedimentation coefficient as small as 40 S [Prusiner et al. (1980) J. Neurochem. 35, 574-582]. Our results demonstrate for the first time that prion-infected tissues contain protease-sensitive PrP(Sc) molecules that form low MW aggregates. Whether these new PrP(Sc) species play a role in the biogenesis or the pathogenesis of prions remains to be established.     

Studies on the mechanism of interaction of a bioresponsive endosomolytic polyamidoamine with interfaces. 1. Micelles as model surfaces

Polymers are appealing as pH-responsive elements of multicomponent systems designed to promote cytosolic delivery of macromolecular drugs (including proteins and genes), but so far the delivery efficiency achieved has been relatively modest. Therefore, the aim of this study was to apply several physicochemical techniques that are well established in the colloid field (surface tension measurements, small-angle neutron scattering (SANS), and electron paramagnetic resonance (EPR)) to probe the mechanism of endosomolytic polymer-surface interaction over the pH range 7.4 to 5.5 using the poly(amidoamine) (PAA) ISA23 x HCl and a series of "model" micelle surfaces. These micellar models were chosen to represent increasing complexity from simple, single surfactant sodium dodecylsulfate (SDS) micelles, surfactant mixtures containing bulky malono-bis-N-methylglucamide headgroups, or highly extended ethylene oxide headgroups. Spherical micelles composed of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-PC) were also used. Changes in the onset of micellization, micelle surface fluidity, and in selected cases, the overall micelle shape and size were all quantified as a function of pH in the presence and absence of ISA23 x HCl. This amphoteric PAA is negatively charged at pH 7.4 and becomes gradually more protonated on exposure to lower pH values representative of the endosomal-lysosomal pathway. As expected, the strength of polymer interaction with anionic micelles increased with a decrease in pH, while for cationic micelles the opposite was observed. Addition of bulky, nonionic surfactant headgroups led to weaker interactions. The observations from surface tension and SANS studies showed a complex pattern of interaction with both an electrostatic and hydrophobic component. Using EPR it was confirmed that ISA23 x HCl perturbed the micelle palisade layer leading to a decrease in fluidity of the interface with a lower degree of headgroup hydration, and a significant change in micelle morphology. Surprisingly, there was no interaction between ISA23 x HCl and globular micelles formed from lyso-PC (a more biologically relevant model), and this suggests that the PAA structure could be better optimized to promote rapid interaction with endosomal membranes at the physiologically relevant pH 6.5.     

Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Large-conductance Ca²⁺-activated K⁺ (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK^–/–) mice, we stimulated NADPH oxidase activity with 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O₂^– and H₂O₂ production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn²⁺, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca²⁺ and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca²⁺ by N-formyl-L-methionyl-L-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK^–/– and BK^+/+ mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity. killing assay; reactive oxygen species; BK-deficient mice; mice infection     

Temperature range of thermodynamic stability for the native state of reversible two-state proteins

The difference between the heat (T(G)) and the cold (T(G)') denaturation temperatures defines the temperature range (T(Range)) over which the native state of a reversible two-state protein is thermodynamically stable. We have performed a correlation analysis for thermodynamic parameters in a selected data set of structurally nonhomologous single-domain reversible two-state proteins. We find that the temperature range is negatively correlated with the protein size and with the heat capacity change (DeltaC(p)) but is positively correlated with the maximal protein stability [DeltaG(T(S))]. The correlation between the temperature range and maximal protein stability becomes highly significant upon normalization of the maximal protein stability with protein size. The melting temperature (T(G)) also shows a negative correlation with protein size. Consistently, T(G) and T(G)' show opposite correlations with DeltaC(p), indicating a dependence of the T(Range) on the curvature of the protein stability curve. Substitution of proteins in our data set with their homologues and arbitrary addition or removal of a protein in the data set do not affect the outcome of our analysis. Simulations of the thermodynamic data further indicate that T(Range) is more sensitive to variations in curvature than to the slope of the protein stability curve. The hydrophobic effect in single domains is the principal reason for these observations. Our results imply that larger proteins may be stable over narrower temperature ranges and that smaller proteins may have higher melting temperatures, suggesting why protein structures often differentiate into multiple substructures with different hydrophobic cores. Our results have interesting implications for protein thermostability.     

Ploidy and nuclear area as a predictive factor of histologic grade in primary breast cancer

OBJECTIVE: To compare ploidy and nuclear area with histologic grade in breast cancer using cytologic samples. STUDY DESIGN: Fine needle aspirates from 85 patients with primary breast cancer were analyzed to identify ploidy and nuclear area. The Feulgen technique was used to stain the material. We used the SAMBA 4000 image analysis system (Grenoble, France) for analyzing ploidy and nuclear area. Each patient underwent a biopsy, and the histologic grade was analyzed. RESULTS: A significant association was found between ploidy and nuclear area, between histologic grade and nuclear area, and between ploidy and histologic grade. As ploidy became aneuploid and polyploid and nuclear area became larger, histologic grade became higher. CONCLUSION: A reliable and rapid evaluation of variables for breast cancer can be achieved using cytologic preparations by measuring ploidy and nuclear area of malignant cells with an image analysis system. Ploidy and nuclear area have a significant association with histologic grade.     

Downregulation of CENPF Remodels Prostate Cancer Cells and Alters Cellular Metabolism

Metabolic alterations in prostate cancer (PC) are associated with progression and aggressiveness. However, the underlying mechanisms behind PC metabolic functions are unknown. The authors’ group recently reported on the important role of centromere protein F (CENPF), a protein associated with the centromere–kinetochore complex and chromosomal segregation during mitosis, in PC MRI visibility. This study focuses on discerning the role of CENPF in metabolic perturbation in human PC3 cells. A series of bioinformatics analyses shows that CENPF is one gene that is strongly associated with aggressive PC and that its expression is positively correlated with metastasis. By identifying and reconstructing the CENPF network, additional associations with lipid regulation are found. Further untargeted metabolomics analysis using gas chromatography‐time‐of‐flight‐mass spectrometry reveals that silencing of CENPF alters the global metabolic profiles of PC cells and inhibits cell proliferation, which suggests that CENPF may be a critical regulator of PC metabolism. These findings provide useful scientific insights that can be applied in future studies investigating potential targets for PC treatment.