Pig complement regulator factor H: molecular cloning and functional characterization

Complement is an efficient defense mechanism of innate immunity. Factor H is the central complement regulator of the alternative pathway, acting in the fluid-phase and on self surfaces. Pigs are considered a suitable source for xenotransplantation and thus several membrane-bound pig complement regulators with importance for the acute rejection phase have been investigated. However, pig fluid-phase regulators have not been described so far. We report the cloning, expression and functional characterization of pig factor H. After constructing a pig liver cDNA library, a full-length factor H cDNA was isolated and sequenced. The predicted protein is organized in 20 short consensus repeat (SCR) domains and has an overall identity of 62% to the human protein. For functional characterization, three deletion constructs of pig factor H were expressed in insect cells. Pig factor H construct SCR 1–4 has cofactor activity for factor I-mediated cleavage of human C3b, which is similar to the human regulator. In addition, this N-terminal construct binds to human C3b, while a construct consisting of SCR 15–20 showed a weaker binding to human C3b/C3d. Pig factor H has two major binding sites for heparin, as the two constructs representing SCR 1–7 and SCR 15–20 proteins, but not the SCR 1–4 protein, bind heparin. The C-terminal construct is able to bind to human endothelial cells, as assayed by FACS. We show that pig and human factor H share functional characteristics in complement regulation and cell surface binding. Possible consequences of using pig livers for xenotransplantation are discussed.The nucleotide sequence data reported are available in the EMBL database (accession number AJ278470)     

Effects of acute and chronic protein intake on metabolism, appetite, and ghrelin during weight loss

Objective: This study evaluated the effects of acute and chronic consumption of higher dietary protein on energy expenditure, macronutrient use, appetite, and appetite‐regulating hormones during weight loss in women. Research Methods and Procedures: Thirty‐eight women chronically consuming a 750 kcal/d energy‐deficit diet with a protein content of 30% (higher protein‐chronic diet, HP‐CD, n = 21) or 18% (normal protein‐chronic diet, NP‐CD, n = 17) for 9 weeks were tested. On separate days, metabolic, appetite, and hormonal responses were measured over 4 hours when the women consumed a higher protein‐acute meal (HP‐AM) (30% of energy as protein) or a normal protein‐acute meal (NP‐AM) (18% of energy as protein). Results: With chronic diet groups combined, HP‐AM led to lower respiratory exchange ratio (0.829 ± 0.005 vs. 0.843 ± 0.008; p < 0.05), lower carbohydrate oxidation (p < 0.05), and higher fat oxidation (p < 0.05) compared with NP‐AM. HP‐AM also led to reduced self‐reported postprandial hunger (p < 0.001) and desire to eat (p < 0.001) and lower postprandial ghrelin (252 ± 16 vs. 274 ± 18 ng/mL · 240 minutes, p < 0.05) compared with NP‐AM. No differences in postprandial energy expenditure (PPEE) occurred between meals. When combining acute meals, respiratory exchange ratio was lower (p < 0.05) and protein oxidation (p < 0.001) was higher in the HP‐CD vs. NP‐CD. An acute meal‐by‐chronic diet interaction was observed with PPEE such that HP‐AM led to greater PPEE in the HP‐CD vs. NP‐CD (28.7 ± 2.7 vs. 19.9 ± 2.7 kcal/min for 195 minutes; p < 0.05). Conclusions: During weight loss, thermogenesis and protein use appear to be influenced by chronic protein intake, while appetite and ghrelin are more responsive to acute protein intake.     

Modulation of IL-4-induced human IgE production in vitro by IFN-gamma and IL-5: the role of soluble CD23 (s-CD23)

IL-4 specifically induced IgE production by peripheral blood lymphocytes or by tonsil or spleen cells from healthy donors. IL-4-induced IgE synthesis was dependent on CD4+ T cells and monocytes and was blocked by IFN-gamma, IFN-alpha, and prostaglandin E-2 (PGE-2). These substances also inhibited IL-4-induced CD23 expression and subsequent release of soluble CD23 (s-CD23). In addition, IgE production was blocked by F(ab')2 fragments of an mAb against CD23. In contrast, IL-5 enhanced IL-4-induced IgE production, provided IL-4 was added at nonsaturating concentrations. This increase in IgE production correlated quantitatively with an enhanced release of s-CD23. Collectively, these results indicate that there is a correlation between s-CD23 release and IgE production. However, s-CD23 fractionated from supernatants of the lymphoblastoid cell line RPMI-8866 was ineffective in inducing IgE production in the absence of IL-4, but acted synergistically with suboptimal concentrations of IL-4. In addition, it is demonstrated that alloreactive T-cell clones produced varying concentrations of IL-4, IL-2, or IFN-gamma upon stimulation. Only supernatants of 2/4 of these T-cell clones induced a low degree of IgE synthesis, but in the presence of anti-IFN-gamma antibodies, all four supernatants induced a strong induction of IgE production. This IgE synthesis was blocked specifically by anti-IL-4 antibodies, indicating that IL-4 is the sole inducer of IgE synthesis. Our findings demonstrate that IL-4-induced IgE production involves complex interactions of T cells, B cells, and monocytes and is positively modulated by IL-5 and s-CD23 but down-regulated by IFN-gamma, IFN-alpha, and PGE-2, respectively.     

A model for population regulation with density- and frequency-dependent selection

The life-cycle of a species with separate generations is divided into a reproduction phase and a growing-up phase. In the reproduction phase we assume random mating and selection due to genotype differences in fecundity of the parents and viability of the offspring. During the growing-up phase we assume a (deterministic) death process in continuous time with death rates for the genotypes which increase linearly with the genotype population sizes.In the absence of genotype differences the model gives logistic population regulation. With genotype differences the model generalizes the usual separate generations selection patterns. In addition to these we exhibit cases with three polymorphic equilibria or with a stable cycle.     

Speculations on carbon dioxide starvation, Late Tertiary evolution of stomatal regulation and floristic modernization

Ambient atmospheric CO₂ concentration ([CO₂]_a) has apparently declined from values above 200μmol mol⁻¹ to values below 200μmol mol⁻¹ within the last several million years. The lower end of this range is marginal for C₃ plants. I hypothesize that: (1) declining [CO₂]_a imposed a physiological strain on plants, and plant taxa evolving under declining [CO₂]_a tended to develop compensating mechanisms, including increased stomatal efficiency; (2) angiosperms were better able to adjust to declining [CO₂]_a than were gymnosperms and pteridophytes; and (3) angiosperm adjustment has been uneven. Fast-evolving taxa (e.g. grasses and herbs) have been better able to adapt to CO₂ starvation. If these propositions are true, stomatal adjustment mechanisms should show patterned variation, and a single pattern of stomatal regulation cannot be assumed.     

In vitro reconstitution of Sgk3 activation by phosphatidylinositol 3-phosphate

Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is a serine/threonine protein kinase activated by the phospholipid phosphatidylinositol 3-phosphate (PI3P) downstream of growth factor signaling via class I phosphatidylinositol 3-kinase (PI3K) signaling and by class III PI3K/Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers; however, the precise mechanism of activation of Sgk3 is unknown. Here, we use a wide range of cell biological, biochemical, and biophysical techniques, including hydrogen–deuterium exchange mass spectrometry, to investigate the mechanism of activation of Sgk3 by PI3P. We show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation. We demonstrate that binding of Sgk3 to PI3P via its regulatory phox homology (PX) domain induces large conformational changes in Sgk3 associated with its activation and that the PI3P-binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstitute Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to identifying the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.