CART peptide: central mediator of leptin-induced adipose tissue apoptosis?

Because of connections between CART peptide containing neurons and the sympathetic nervous system (SNS) and the possible role of the SNS in leptin-induced adipose apoptosis, CART may act as a downstream effector of leptin-induced adipose apoptosis. Male Sprague-Dawley rats received continuous intracerebroventricular (i.c.v.) infusion for 4 days of either artificial cerebrospinal fluid (aCSF, 12 microl/day), leptin (15 microg/day), or CART55-102 at 2.4 microg/day (CART2.4) or 9.6 microg/day (CART9.6). Food intake (FI) was decreased 10.8% for CART2.4, 41.9% for CART9.6 and 33.4% for leptin (p<0.05). CART9.6 and leptin reduced meal size and meal number. Body weight (BW) was reduced by CART9.6 (14.6%) and leptin (11.6%) (p<0.05), but not by CART2.4. CART9.6 and CART2.4, but not leptin, caused hypothermia, and CART9.6 inhibited physical activity (p<0.05). Epididymal, inguinal and retroperitoneal fat pad weights were reduced (p<0.05) by both CART treatments and leptin; CART9.6 also reduced gastrocnemius muscle weight (18.1%, p<0.05). Leptin, but not CART, increased serum free fatty acid concentrations by 31.1% (p<0.05) and increased adipose apoptosis by 48% (p<0.05). These data show that although leptin and CART55-102 have some similar actions, CART55-102 is probably not a mediator for leptin-induced adipose apoptosis in the brain.     

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic β-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP’s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable β-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.     

Rust fungal effectors mimic host transit peptides to translocate into chloroplasts

Parasite effector proteins target various host cell compartments to alter host processes and promote infection. How effectors cross membrane‐rich interfaces to reach these compartments is a major question in effector biology. Growing evidence suggests that effectors use molecular mimicry to subvert host cell machinery for protein sorting. We recently identified chloroplast‐targeted protein 1 (CTP1), a candidate effector from the poplar leaf rust fungus Melampsora larici‐populina that carries a predicted transit peptide and accumulates in chloroplasts and mitochondria. Here, we show that the CTP1 transit peptide is necessary and sufficient for accumulation in the stroma of chloroplasts. CTP1 is part of a Melampsora‐specific family of polymorphic secreted proteins. Two members of that family, CTP2 and CTP3, also translocate in chloroplasts in an N‐terminal signal‐dependent manner. CTP1, CTP2 and CTP3 are cleaved when they accumulate in chloroplasts, while they remain intact when they do not translocate into chloroplasts. Our findings reveal that fungi have evolved effector proteins that mimic plant‐specific sorting signals to traffic within plant cells.     

Using Drugs to Probe the Variability of Trans-Epithelial Airway Resistance

Background

Precision medicine aims to combat the variability of the therapeutic response to a given medicine by delivering the right medicine to the right patient. However, the application of precision medicine is predicated on a prior quantitation of the variance of the reference range of normality. Airway pathophysiology provides a good example due to a very variable first line of defence against airborne assault. Humans differ in their susceptibility to inhaled pollutants and pathogens in part due to the magnitude of trans-epithelial resistance that determines the degree of epithelial penetration to the submucosal space. This initial ‘set-point’ may drive a sentinel event in airway disease pathogenesis. Epithelia differentiated in vitro from airway biopsies are commonly used to model trans-epithelial resistance but the ‘reference range of normality’ remains problematic. We investigated the range of electrophysiological characteristics of human airway epithelia grown at air-liquid interface in vitro from healthy volunteers focusing on the inter- and intra-subject variability both at baseline and after sequential exposure to drugs modulating ion transport.

Methodology/Principal Findings

Brushed nasal airway epithelial cells were differentiated at air-liquid interface generating 137 pseudostratified ciliated epithelia from 18 donors. A positively-skewed baseline range exists for trans-epithelial resistance (Min/Max: 309/2963 Ω·cm²), trans-epithelial voltage (-62.3/-1.8 mV) and calculated equivalent current (-125.0/-3.2 μA/cm²; all non-normal, P<0.001). A minority of healthy humans manifest a dramatic amiloride sensitivity to voltage and trans-epithelial resistance that is further discriminated by prior modulation of cAMP-stimulated chloride transport.

Conclusions/Significance

Healthy epithelia show log-order differences in their ion transport characteristics, likely reflective of their initial set-points of basal trans-epithelial resistance and sodium transport. Our data may guide the choice of the background set point in subjects with airway diseases and frame the reference range for the future delivery of precision airway medicine.     

ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding

Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.     

Age and sex differentially affect regional changes in one repetition maximum strength

To assess the influences of age and sex on regional changes in 1 repetition maximum (1RM) strength, 10 young men (20-30 years), 8 young women (20-30 years), 11 older men (65- 75 years), and 10 older women (65-75 years) were studied before and after a 24-week whole-body strength training program. Changes in 1RM strength were analyzed for each individual exercise, as well as by calculating a total body score (TBS), an upper body score (UBS), and a lower body score (LBS). The effect of age and sex on changes in 1RM strength was analyzed using a repeated measures analysis of variance. When changes in strength for individual exercises were analyzed, the chest press, lat pulldown, shoulder press, and triceps pushdown were affected by both age (p < 0.05) and sex (p < 0.05), while the biceps curls were only influenced by age (p < 0.05). For the lower body, the leg press changes in 1RM strength were influenced by age (p < 0.0001), while leg extension was influenced by sex (p < 0.05). Total body score, UBS, and LBS showed significant increases with 24 weeks of ST (p < 0.001, all). Changes in TBS and UBS were affected by age (p < 0.001, both) and sex (p < 0.05 and p < 0.001, respectively). Younger subjects showed a greater increase in strength than older subjects, and men showed a greater increase in strength compared with women. Changes in LBS were affected by age (p < 0.001), with younger subjects showing a greater increase in strength compared with the older subjects, but not by sex (p = 0.464). These data indicate that regional increases in strength are differentially affected by age and sex.     

Understory vegetation as an indicator for floodplain forest restoration in the Mississippi River Alluvial Valley,U.S.A.

In the Mississippi River Alluvial Valley (MAV), complete alteration of river‐floodplain hydrology allowed for widespread conversion of forested bottomlands to intensive agriculture, resulting in nearly 80% forest loss. Governmental programs have attempted to restore forest habitat and functions within this altered landscape by the methods of tree planting (afforestation) and local hydrologic enhancement on reclaimed croplands. Early assessments identified factors that influenced whether planting plus tree colonization could establish an overstory community similar to natural bottomland forests. The extent to which afforested sites develop typical understory vegetation has not been evaluated, yet understory composition may be indicative of restored site conditions. As part of a broad study quantifying the ecosystem services gained from restoration efforts, understory vegetation was compared between 37 afforested sites and 26 mature forest sites. Differences in vegetation attributes for species growth forms, wetland indicator classes, and native status were tested with univariate analyses; floristic composition data were analyzed by multivariate techniques. Understory vegetation of restoration sites was generally hydrophytic, but species composition differed from that of mature bottomland forest because of young successional age and differing responses of plant growth forms. Attribute and floristic variation among restoration sites was related to variation in canopy development and local wetness conditions, which in turn reflected both intrinsic site features and outcomes of restoration practices. Thus, understory vegetation is a useful indicator of functional progress in floodplain forest restoration.     

The phylogeny of the Pantropical genus Arrhipis Bonvouloir (Coleoptera, Eucnemidae)

The phylogeny of the genus Arrhipis Bonvouloir (Coleoptera, Eucnemidae) is clarified with a cladistic analysis based on five molecular markers and morphology. Sixteen species from Africa, America, Asia, and Australia are included in the analysis. Two separate Asian clades are recovered, one of them being the sister group to a clade with the American and African species. With the exception of the continental south-east Asian species, all Gondwanan regions have monophyletic faunas. According to the present data, the continental south-east Asian fauna comprises two monophyletic groups, one of which is the sister group to African and American species. Vicariance seems to be the logical explanation for the distribution of these lignicolous beetles.
© The Willi Hennig Society 2009.