MKK7 couples stress signalling to G2/M cell-cycle progression and cellular senescence

During the development of multicellular organisms, concerted actions of molecular signalling networks determine whether cells undergo proliferation, differentiation, death or ageing. Here we show that genetic inactivation of the stress signalling kinase, MKK7, a direct activator of JNKs in mice, results in embryonic lethality and impaired proliferation of hepatocytes. Beginning at passage 4-5, mkk7(-/-) mouse embryonic fibroblasts (MEFs) display impaired proliferation, premature senescence and G2/M cell cycle arrest. Similarly, loss of c-Jun or expression of a c-JunAA mutant in which the JNK phosphorylation sites were replaced with alanine results in a G2/M cell-cycle block. The G2/M cell-cycle kinase CDC2 was identified as a target for the MKK7-JNK-c-Jun pathway. These data show that the MKK7-JNK-c-Jun signalling pathway couples developmental and environmental cues to CDC2 expression, G2/M cell cycle progression and cellular senescence in fibroblasts.     

Influence of HSA and IgG on LDL oxidation studied by size-exclusion chromatography and phospholipid profiling using MALDI tandem-mass spectrometry

In the present study a direct detection approach combining size-exclusion chromatography (SEC) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem-mass spectrometry (MALDI-QIT-TOF-MS/MS) was applied to investigate the influence of HSA and IgG on LDL oxidation in vitro. SEC analysis showed an increase of protein aggregation during LDL-oxidation that could be essentially suppressed in the presence of HSA. In parallel, lipid peroxidation measured by TBARS assay over 24 h was inhibited by 95–100% in the presence of HSA but only 0–34% by IgG, respectively. MALDI phospholipid profiles showed considerable decrease of signals from PCs containing sn-2 PUFAs (18:2 or 20:4) accompanied by increase of sn-2 LPCs indicating for specific breakdown of PUFA-containing PLs during LDL-oxidation. These effects were nearly 100% inhibited in the presence of HSA but not by IgG, respectively. Among known pro-atherogenic PL species present in human plasma sphingomyelin (SM16:0) was bound in significant amounts to HSA but not IgG after incubation with oxLDL. Moreover, our investigation showed that LPCs containing SAFAs (16:0 or 18:0) were specifically bound to HSA, while those containing PUFAs (18:2 and 18:3) were preferentially associated with IgG. In summary, the presented methodology provides a promising platform for studying lipid–protein interactions in vivo.     

Changes in the structure-function relationship of elastin and its impact on the proximal pulmonary arterial mechanics of hypertensive calves

Extracellular matrix remodeling has been proposed as one mechanism by which proximal pulmonary arteries stiffen during pulmonary arterial hypertension (PAH). Although some attention has been paid to the role of collagen and metallomatrix proteins in affecting vascular stiffness, much less work has been performed on changes in elastin structure-function relationships in PAH. Such work is warranted, given the importance of elastin as the structural protein primarily responsible for the passive elastic behavior of these conduit arteries. Here, we study structure-function relationships of fresh arterial tissue and purified arterial elastin from the main, left, and right pulmonary artery branches of normotensive and hypoxia-induced pulmonary hypertensive neonatal calves. PAH resulted in an average 81 and 72% increase in stiffness of fresh and digested tissue, respectively. Increase in stiffness appears most attributable to elevated elastic modulus, which increased 46 and 65%, respectively, for fresh and digested tissue. Comparison between fresh and digested tissues shows that, at 35% strain, a minimum of 48% of the arterial load is carried by elastin, and a minimum of 43% of the change in stiffness of arterial tissue is due to the change in elastin stiffness. Analysis of the stress-strain behavior revealed that PAH causes an increase in the strains associated with the physiological pressure range but had no effect on the strain of transition from elastin-dominant to collagen-dominant behavior. These results indicate that mechanobiological adaptations of the continuum and geometric properties of elastin, in response to PAH, significantly elevate the circumferential stiffness of proximal pulmonary arterial tissue.     

Purification of TAXI-like endoxylanase inhibitors from wheat (Triticum aestivum L.) whole meal reveals a family of iso-forms

An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N-hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase.     

Phenotypic plasticity and precipitation response in Sonoran Desert winter annuals

Temporal environmental variation has profound influences on population dynamics and community structure. Examination of functional traits that influence resource uptake and allocation can illuminate how co-occurring species translate environmental variation into different demographic outcomes, yet few studies have considered interspecific differences in trait plasticity. We experimentally manipulated soil moisture to test the hypothesis that differences in morphological plasticity contribute to species differences in demographic response to unpredictable precipitation in Sonoran Desert winter annual plants. We compared plasticity of leaf traits and biomass allocation between Pectocarya recurvata (Boraginaceae) and Stylocline micropoides (Asteraceae), co-occurring species that differ in long-term demographic patterns. The species with highly variable population dynamics, Stylocline, had striking increases in leaf area and root biomass in response to an experimental increase in soil moisture. In contrast, the species with buffered long-term population dynamics, Pectocarya, did not differ in leaf morphology or biomass allocation between soil moisture treatments. Regardless of water treatment, Pectocarya had earlier reproductive phenology and greater fecundity than Stylocline, suggesting that differences in the timing of the phenological transitions from vegetative to reproductive growth may affect species' responses to precipitation pulses. Combining long-term observations with experimental manipulations provides a window into the functional underpinnings and demographic consequences of trait plasticity.     

Structural refinement of insecticidal plant proteinase inhibitors from Nicotiana alata

Ornamental tobacco (Nicotiana alata) produces a series of 6 kDa proteinase inhibitors belonging to the potato type II inhibitor family. These proteins inhibit trypsin and chymotrypsin, the main digestive enzymes of predatory insects, thus leading to starvation, impaired larval development or death. In this context, the three-dimensional structures of these inhibitors are important for developing novel strategies for pest control. The solution structures of C1 and T1, the two main prototypes of the N. alata inhibitors, were originally determined more than a decade ago (J. Mol. Biol. 242, 231-243 (1994) and Biochemistry 34, 14304-14311 (1995)). Since then methods for NMR structure calculations have evolved considerably. Here we report the refinement of the structures of C1 and T1 with state-of-the-art protocols for NMR structure calculations. This refinement leads to an improved quality of the structures, making them a more reliable basis for the development of novel pesticides and modeling applications.     

Optimal pennation angle of the primary ankle plantar and dorsiflexors: variations with sex, contraction intensity, and limb

Ultrasonography was used to measure the pennation angle of the human tibialis anterior (TA), lateral gastrocnemius (LG), medial gastrocnemius (MG), and soleus (Sol). The right and left legs of 8 male and 8 female subjects were tested at rest and during maximum voluntary contraction (MVC). Joint angles were chosen to control muscle tendon lengths so that the muscles were near their optimal length within the length-tension relationship. No differences in pennation angle were detected between the right and left legs. Another consistent finding was that the pennation angle at MVC was significantly greater than at rest for all muscles tested. Optimal pennation angles for the TA, MG, and Sol were significantly greater for the men than for the women. Optimal pennation angles for the TA, LG, MG, and Sol for the male subjects were 14.3 degrees, 23.7 degrees, 34.6 degrees, and 40.1 degrees respectively, whereas values of 12.1 degrees, 16.3 degrees, 27.3 degrees, and 26.3 degrees were recorded for the female subjects. The results of this study suggest the following: (1) similar values for pennation angle can be used for the right and left TA, LG, MG, and Sol; (2) pennation angle is significantly greater at MVC than at rest for all muscles tested; and (3) sex-specific values for optimal pennation angle should be used when modeling the force-generating potential of the primary muscles responsible for ankle plantar and dorsiflexion.     

Effects of Glucose, Insulin, and Supernatant from Pancreatic β-cells on Brain–Pancreas Relative Protein in Rat Hippocampus

Brain–pancreas relative protein (BPRP) is a novel protein that mainly expresses in brain and pancreas. In our previous study, we found that various stressors significantly decreased the expression of BPRP in pancreas in vivo, accompanied by changes in insulin and glucose levels, and that expression of BPRP in pancreas also decreased significantly in diabetic rats induced by Streptozocin (STZ). All these findings suggest that BPRP may be a glucose or insulin-sensitive protein. However, how the changes in insulin or glucose levels influence the expression of BPRP in hippocampus requires further study. Here, we investigated the effects of insulin or glucose on the expression of BPRP in primary cultured hippocampal neurons. We supplied hippocampal neurons with glucose, insulin, or supernatant from pancreatic β-cells, which secrete insulin into the supernatant. Our data showed that insulin had beneficial effect on the viability while no significant effect on the expression of BPRP in hippocampal neurons. On the contrary, 40 mM glucose or free glucose culture significantly decreased the expression of BPRP, while had no significant effect on the viability and apoptosis of hippocampal neurons. Further study showed that levels of insulin in the supernatant collected from pancreatic β-cells medium changed over days, and that supernatant increased the viability of hippocampal neurons, while it had no obvious effect on the expression of BPRP in hippocampal neurons. These results suggest that BPRP may be a glucose-sensitive protein.