"Invisible" Detergents Enable a Reliable Determination of Solution Structures of Native Photosystems by Small-Angle Neutron Scattering.

Photosystems I (PSI) and II (PSII) are pigment-protein complexes capable of performing the light-induced charge separation necessary to convert solar energy into a biochemically storable form, an essential step in photosynthesis. Small-angle neutron scattering (SANS) is unique in providing structural information on PSI and PSII in solution under nearly physiological conditions without the need for crystallization or temperature decrease. We show that the reliability of the solution structure critically depends on proper contrast matching of the detergent belt surrounding the protein. Especially, specifically deuterated ("invisible") detergents are shown to be properly matched out in SANS experiments by a direct, quantitative comparison with conventional matching strategies. In contrast, protonated detergents necessarily exhibit incomplete matching so that related SANS results systematically overestimate the size of the membrane protein under study. While the solution structures obtained are close to corresponding high-resolution structures, we show that temperature and solution state lead to individual structural differences compared with high-resolution structures. We attribute these differences to the presence of a manifold of conformational substates accessible by protein dynamics under physiological conditions.

Mir142 loss unlocks IDH2R140-dependent leukemogenesis through antagonistic regulation of HOX genes.

AML is a genetically heterogeneous disease and understanding how different co-occurring mutations cooperate to drive leukemogenesis will be crucial for improving diagnostic and therapeutic options for patients. MIR142 mutations have been recurrently detected in IDH-mutated AML samples. Here, we have used a mouse model to investigate the interaction between these two mutations and demonstrate a striking synergy between Mir142 loss-of-function and IDH2R140Q, with only recipients of double mutant cells succumbing to leukemia. Transcriptomic analysis of the non-leukemic single and leukemic double mutant progenitors, isolated from these mice, suggested a novel mechanism of cooperation whereby Mir142 loss-of-function counteracts aberrant silencing of Hoxa cluster genes by IDH2R140Q. Our analysis suggests that IDH2R140Q is an incoherent oncogene, with both positive and negative impacts on leukemogenesis, which requires the action of cooperating mutations to alleviate repression of Hoxa genes in order to advance to leukemia. This model, therefore, provides a compelling rationale for understanding how different mutations cooperate to drive leukemogenesis and the context-dependent effects of oncogenic mutations.

Author Correction: Diversity of gut microflora is required for the generation of B cell with regulatory properties in a skin graft model.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

T-bet controls intestinal mucosa immune responses via repression of type 2 innate lymphoid cell function.

Innate lymphoid cells (ILCs) play an important role in regulating immune responses at mucosal surfaces. The transcription factor T-bet is crucial for the function of ILC1s and NCR+ ILC3s and constitutive deletion of T-bet prevents the development of these subsets. Lack of T-bet in the absence of an adaptive immune system causes microbiota-dependent colitis to occur due to aberrant ILC3 responses. Thus, T-bet expression in the innate immune system has been considered to dampen pathogenic immune responses. Here, we show that T-bet plays an unexpected role in negatively regulating innate type 2 responses, in the context of an otherwise intact immune system. Selective loss of T-bet in ILCs leads to the expansion and increased activity of ILC2s, which has a functionally important impact on mucosal immunity, including enhanced protection from Trichinella spiralis infection and inflammatory colitis. Mechanistically, we show that T-bet controls the intestinal ILC pool through regulation of IL-7 receptor signalling. These data demonstrate that T-bet expression in ILCs acts as the key transcriptional checkpoint in regulating pathogenic vs. protective mucosal immune responses, which has significant implications for the understanding of the pathogenesis of inflammatory bowel diseases and intestinal infections.

Diversity of gut microflora is required for the generation of B cell with regulatory properties in a skin graft model.

B cells have been reported to promote graft rejection through alloantibody production. However, there is growing evidence that B cells can contribute to the maintenance of tolerance. Here, we used a mouse model of MHC-class I mismatched skin transplantation to investigate the contribution of B cells to graft survival. We demonstrate that adoptive transfer of B cells prolongs skin graft survival but only when the B cells were isolated from mice housed in low sterility "conventional" (CV) facilities and not from mice housed in pathogen free facilities (SPF). However, prolongation of skin graft survival was lost when B cells were isolated from IL-10 deficient mice housed in CV facilities. The suppressive function of B cells isolated from mice housed in CV facilities correlated with an anti-inflammatory environment and with the presence of a different gut microflora compared to mice maintained in SPF facilities. Treatment of mice in the CV facility with antibiotics abrogated the regulatory capacity of B cells. Finally, we identified transitional B cells isolated from CV facilities as possessing the regulatory function. These findings demonstrate that B cells, and in particular transitional B cells, can promote prolongation of graft survival, a function dependent on licensing by gut microflora.

Comparison of population genetic patterns in two widespread freshwater mussels with contrasting life histories in western North America.

We investigate population genetic structuring in Margaritifera falcata, a freshwater mussel native to western North America, across the majority of its geographical range. We find shallow rangewide genetic structure, strong population-level structuring and very low population diversity in this species, using both mitochondrial sequence and nuclear microsatellite data. We contrast these patterns with previous findings in another freshwater mussel species group (Anodonta californiensis/A. nuttalliana) occupying the same continental region and many of the same watersheds. We conclude that differences are likely caused by contrasting life history attributes between genera, particularly host fish requirements and hermaphroditism. Further, we demonstrate the occurrence of a 'hotspot' for genetic diversity in both groups of mussels, occurring in the vicinity of the lower Columbia River drainage. We suggest that stream hierarchy may be responsible for this pattern and may produce similar patterns in other widespread freshwater species.

Leptin prevents the fall in plasma osteocalcin during starvation in male mice.

Plasma osteocalcin, a marker of osteoblastic activity, is reduced in starvation, malnutrition, and anorexia nervosa, resulting in low bone turnover osteoporosis. Contradictory findings about the role of leptin as a link between nutritional status and bone physiology have been reported. We demonstrate that leptin-deficient ob/ob and leptin-resistant db/db male mice have increased plasma osteocalcin, and that in male ob/ob mice osteocalcin is not decreased by starvation, unlike control mice. Intraperitoneal leptin administration increased plasma osteocalcin in male ob/ob mice, and prevented its fall during 24h fasting and 5 days of food restriction in normal male mice. This effect may be mediated via actions on the hypothalamic-pituitary-testicular or -growth hormone axes, or a direct action on osteoblasts. These studies support the hypothesis that the fall in leptin during starvation and weight loss is responsible for the associated reduction in osteoblast activity, and suggest a role for leptin in regulating bone turnover.

Visceral adipose tissue and metabolic complications of obesity are reduced in Prader-Willi syndrome female adults: evidence for novel influences on body fat distribution.

Visceral obesity is detrimental to health, but the mechanisms controlling body fat distribution are not fully understood. In premenopausal adult females (30 nonobese, 14 obese [body mass index >30 kg/m(2)]), variance in fasting insulin, glucose, insulin/glucose ratio, C-peptide/insulin ratio, triglycerides, and high-density lipoprotein/low-density lipoprotein-cholesterol ratio, were independently influenced by visceral but not total sc or abdominal sc adipose tissue, as measured by whole-body magnetic resonance imaging. Adult females with Prader-Willi syndrome (n = 13) had significantly reduced visceral adiposity, compared with obese controls (visceral/total sc adipose tissue ratio: 0.067 +/- 0.017 vs. 0.108 +/- 0.021), independent of their total adiposity (P < 0.001), or use of exogenous sex steroids. This is in contrast to that expected by their physical inactivity, hypogonadism, adult GH deficiency, and psychiatric problems. Females with Prader-Willi syndrome not receiving sex steroids (n = 8) had significantly reduced fasting insulin, insulin/glucose ratio, and triglycerides and increased C-peptide/insulin ratio, compared with obese controls, adjusting for total (P < 0.05) but not visceral adiposity (P = 0.3-0.6), supporting their association. The cause of the reduced visceral adiposity in Prader-Willi syndrome may reflect novel hormonal, hypothalamic, and/or genetic influences on body fat distribution.

Leptin potentiates experimental autoimmune encephalomyelitis in SJL female mice and confers susceptibility to males.

SJL (H-2s) female mice are more susceptible than males to experimental autoimmune encephalomyelitis (EAE) induced by immunization with myelin-derived peptides. The reasons for this sexual dimorphism are unclear, but may include such factors as sex-related differences in immune responsiveness, hormonal effects and sex-linked genetic factors. Recent evidence indicates that leptin modifies T cell immunity promoting T helper (Th) 1 pro-inflammatory immune responses. Circulating leptin levels show a marked sexual dimorphism, being higher in females than in males. In the present study, we investigated whether leptin treatment altered the course of relapsing-remitting EAE, induced by the proteolipid protein peptide (PLP(139-151)), in SJL susceptible females and EAE-resistant males. Administration of leptin to female SJL mice before or after disease onset significantly worsened the disease, with a concomitant increase in the PLP(139-151)-specific delayed-type hypersensitivity (DTH) reactivity and in vitro IFN-gamma secretion. Leptin treatment at priming with antigen or before disease onset rendered male SJL mice susceptible to EAE, with the appearance of PLP(139-151)-specific DTH reactivity and a switch from a Th2 to Th1 pattern of cytokine release. Our findings indicate that leptin administration to susceptible females resulted in a more severe disease, and that reduced leptin levels in male SJL mice may contribute to the gender-related differences in the induction phase of EAE.

Requirement for leptin in the induction and progression of autoimmune encephalomyelitis.

Recent evidence indicates that leptin modifies T cell immunity, and may provide a key link between nutritional deficiency and immune dysfunction. To study the influence of leptin on autoimmunity, susceptibility to experimental autoimmune encephalomyelitis induced by immunization with a myelin-derived peptide was examined in leptin-deficient, C57BL/6J-ob/ob mice, with or without leptin replacement, and in wild-type controls. Leptin replacement converted disease resistance to susceptibility in the C57BL/6J-ob/ob mice; this was accompanied by a switch from a Th2 to Th1 pattern of cytokine release and consequent reversal of Ig subclass production. Our findings suggest that leptin is required for the induction and maintenance of an effective proinflammatory immune response in the CNS.

Starvation, leptin and epithelial cell proliferation in the gastrointestinal tract of the mouse.

BACKGROUND/AIMS: Leptin, the ob/ob gene product, is a recently discovered peptide hormone, secreted by adipocytes, which can act as a satiety factor to regulate food intake. Its levels thus will be related to the presence of food in the lumen of the gut, and food intake is one of the most potent stimuli for intestinal epithelial cell proliferation. Leptin has a variety of other actions and the aim of this study was to see if one of these was to stimulate mucosal growth. METHODS: Three groups of mice were fed ad libitum, starved for 48 h or starved for 48 h and given twice-daily intraperitoneal injections of recombinant leptin (1 microg/g). RESULTS: Starvation led to a 20% decrease in body weight and a similar decrease in the weights of the intestines. Starvation also markedly inhibited intestinal epithelial cell proliferation. Leptin had little effect on the small intestine and did not stimulate proliferation. However, in the hind gut it was associated with small but significant decreases in caecal weight, distal colon mitotic counts (p = 0.036) and in colonic crypt area (approximately 20%, p<0.001). CONCLUSION: Leptin did not stimulate intestinal cell proliferation, however it did have a paradoxical inhibitory action on the caecum and colon.

Increased leptin levels in serum and peritoneal fluid of patients with pelvic endometriosis.

Pelvic endometriosis is an immune-related chronic inflammatory disease, characterized by ectopic implants of endometrium in the peritoneal cavity and associated with increased secretion of proinflammatory cytokines and neoangiogenesis. Leptin, the adipocyte-derived hormone, has been shown to have a role in food intake, basal metabolism, and reproductive function. Leptin levels are dynamically regulated, being elevated by inflammatory mediators and reduced by starvation. Leptin itself can influence the proinflammatory immune responses of CD4+ T lymphocytes, and reports have also shown this hormone to be an angiogenic factor in vitro and in vivo. We investigated whether leptin concentrations in serum and peritoneal fluid (PF) differed between 13 patients with different stages of endometriosis and 15 age- and body mass index-matched controls. We found a statistically significant (P < 0.05) increase in leptin levels in serum (30.3 +/- 14.8 ng/mL) and PF (35.9 +/- 17.4 ng/mL) of patients with endometriosis, compared with our control population (serum, 15.6 +/- 8.4; PF, 17.5 +/- 7.2 ng/mL). Regression equations, relating leptin to body mass index, were also significantly different in endometriosis patients, compared with controls. Higher levels of leptin were observed in the earlier stages of endometriosis than advanced-stage disease. These data suggest that the proinflammatory and neoangiogenic actions of leptin may contribute to the pathogenesis of endometriosis.

Leptin protects mice from starvation-induced lymphoid atrophy and increases thymic cellularity in ob/ob mice.

Thymic atrophy is a prominent feature of malnutrition. Forty-eight hours' starvation of normal mice reduced the total thymocyte count to 13% of that observed in freely fed controls, predominantly because of a diminution in the cortical CD4(+)CD8(+) thymocyte subpopulation. Prevention of the fasting-induced fall in the level of the adipocyte-derived hormone leptin by administering exogenous recombinant leptin protected mice from these starvation-induced thymic changes. The ob/ob mouse, which is unable to produce functional leptin because of a mutation in the obese gene, has impaired cellular immunity together with a marked reduction in the size and cellularity of the thymus. We found that ob/ob mice had a high level of thymocyte apoptosis resulting in a ratio of CD4(+)CD8(+) (cortical) to CD4(-)CD8(-) (precursor) thymocytes that was 4-fold lower than that observed in wild-type mice. Peripheral administration of recombinant leptin to ob/ob mice reduced thymocyte apoptosis and substantially increased both thymic cellularity and the CD4(+)CD8(+)/CD4(-)CD8(-) ratio. In contrast, a comparable weight loss in pair-fed PBS-treated ob/ob mice had no impact on thymocyte number. In vitro, leptin protected thymocytes from dexamethasone-induced apoptosis. These data indicate that reduced circulating leptin concentrations are pivotal in the pathogenesis of starvation-induced lymphoid atrophy.

Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression.

Nutritional deprivation suppresses immune function. The cloning of the obese gene and identification of its protein product leptin has provided fundamental insight into the hypothalamic regulation of body weight. Circulating levels of this adipocyte-derived hormone are proportional to fat mass but maybe lowered rapidly by fasting or increased by inflammatory mediators. The impaired T-cell immunity of mice now known to be defective in leptin (ob/ob) or its receptor (db/db), has never been explained. Impaired cell-mediated immunity and reduced levels of leptin are both features of low body weight in humans. Indeed, malnutrition predisposes to death from infectious diseases. We report here that leptin has a specific effect on T-lymphocyte responses, differentially regulating the proliferation of naive and memory T cells. Leptin increased Th1 and suppressed Th2 cytokine production. Administration of leptin to mice reversed the immunosuppressive effects of acute starvation. Our findings suggest a new role for leptin in linking nutritional status to cognate cellular immune function, and provide a molecular mechanism to account for the immune dysfunction observed in starvation.

Leptin interacts with glucagon-like peptide-1 neurons to reduce food intake and body weight in rodents.

The adipose tissue hormone, leptin, and the neuropeptide glucagon-like peptide-1 (7-36) amide (GLP-1) both reduce food intake and body weight in rodents. Using dual in situ hybridization, long isoform leptin receptor (OB-Rb) was localized to GLP-1 neurons originating in the nucleus of the solitary tract. ICV injection of the specific GLP-1 receptor antagonist, exendin(9-39), at the onset of dark phase, did not affect feeding in saline pre-treated controls, but blocked the reduction in food intake and body weight of leptin pre-treated rats. These findings suggest that GLP-1 neurons are a potential target for leptin in its control of feeding.

Plasma immunoreactive leptin concentration in end-stage renal disease.

1. Leptin is thought to be an inhibitor of appetite. As the kidney helps clear several polypeptide hormones, plasma leptin may accumulate in end-stage renal disease. 2. Plasma immunoreactive leptin was measured in four groups of subjects: haemodialysis, continuous ambulatory peritoneal dialysis and renal transplant patients and a group of healthy controls. Leptin was also measured before and after a single dialysis session. 3. There was a strong correlation between plasma immunoreactive leptin and body mass index in all groups except female haemodialysis patients. Leptin was higher in females than in males in all groups when controlled for body mass index. Mean plasma leptin [mean (SD)] was significantly higher in all renal groups [haemodialysis, 15.1 (3.6) ng/ml; continuous ambulatory peritoneal dialysis, 25.4 (4.3) ng/ml; transplants, 11.6 (2.6) ng/ml] compared with controls [5.3 (2.3) ng/ml]. There was a significant difference in the regression equations relating leptin and body mass index (dialysis > transplants > controls), even when controlled for gender. Leptin correlated modestly with serum creatinine in non-dialysis subjects. Plasma leptin immunoreactivity was slightly reduced by haemodialysis, but post-dialysis leptin was still significantly higher than that found in controls. 4. Chromatographic characterization of the high level of leptin immunoreactivity found in haemodialysis subjects showed a single elution peak corresponding to that of the highly purified human leptin standard. 5. In conclusion, leptin is higher than expected for body mass index in end-stage renal disease. Hyperleptinaemia could contribute to the anorexia and poor nutritional status commonly seen in renal failure.

Neuropeptide Y induced feeding in the rat is mediated by a novel receptor.

There are now six recognized neuropeptide Y (NPY) receptor subtypes (Y1-Y4 and two recently cloned distinct receptors labeled Y5), of which Y1 and one of the Y5's have been suggested could mediate the effect of NPY on feeding. The fragments NPY(2-36) and NPY(3-36), which bind Y1 only poorly, were injected intracerebroventricularly (icv) and found to have similar dose-response relationships to NPY in the stimulation of feeding. However NPY (13-36), which stimulates both Y2 and Y5, caused no increase in food intake, even at high doses. Maximal stimulation with the classical Y1 agonist [Pro34]-NPY produced only 50% of the maximum effect of NPY itself despite fully inhibiting adenylyl cyclase activity in vitro in a Y1 system. The novel fragment [Pro34]-NPY(3-36) is as effective at stimulating food intake as the classical Y1 analogue [Pro34]-NPY but bound to the Y1 receptor with only 1/20th of the affinity of NPY and failed to inhibit adenylyl cyclase through this receptor. [Pro34]-NPY(3-36) is therefore a relatively appetite-selective ligand. Coadministration of high dose NPY(13-36) and [Pro34]NPY did not enhance feeding compared with [Pro34]-NPY alone. In addition, the NPY Y1 receptor antagonist BIBP-3226, which does not bind Y2, Y4, or Y5 receptors, significantly reduced NPY induced feeding. These results indicate that the feeding effect of icv NPY involves a novel receptor and that it is functionally distinct from the recognized receptor subtypes.

A cardiovascular risk reduction program for the classroom.

A quasi-experimental, longitudinal study was conducted to test the effectiveness over a 1-year period of a cardiovascular risk reduction program for school-age children. The effectiveness of the program was measured with the children's knowledge of physiology of the heart and cardiovascular risk factors, their health habits, and their physical measurements. Ninety-eight children between the ages of 9 and 12 years participated in the study. The experimental group received a series of five 40-minute sessions on physiology of the heart, smoking, hypertension, diet, and physical activity. Short-term effectiveness of the program was found for the children's knowledge of physiology of the heart and smoking. Long-term effectiveness was found for running activity.