Band 4.1-like proteins of the bovine lens. Effects of differentiation, distribution and extraction characteristics.

Bovine lens epithelium, cortex and nucleus were screened for the presence of red-cell-membrane band 4.1-like proteins by using an immunoblot method. Lens epithelial cells were found to contain proteins of Mr 78 000 and higher (approximately 150 000) that cross-reacted with anti-(protein 4.1) sera. Fibre cells of the superficial cortex were also found to contain these two proteins, as well as an additional protein of approx. 80 000 Mr. In contrast, deep layers of the cortex and the lens nucleus contained no detectable cross-reactive protein at these Mr values. Treatment of a crude membrane fraction prepared from superficial bovine cortices with a low-ionic-strength buffer resulted in release of the high-Mr band 4.1-like protein. The 80 000- and 78 000-Mr proteins remained with the membrane fraction in low-ionic-strength buffer, but were released into solution by high-ionic-strength-buffer treatment. We have also demonstrated that the human red-blood-cell membrane, like lens epithelial cells and fibre cells, also contains a high-Mr band 4.1-like protein that is released from membranes by low-ionic-strength-buffer treatment.     

Regulatory Mechanisms of Vitamin D<Subscript>3</Subscript> on Production of Nitric Oxide and Pro-inflammatory Cytokines in Microglial BV-2 Cells

Inhibition of pro-inflammatory functions of microglia has been considered a promising strategy to prevent pathogenic events in the central nervous system under neurodegenerative conditions. Here we examined potential inhibitory effects of nuclear receptor ligands on lipopolysaccharide (LPS)-induced inflammatory responses in microglial BV-2 cells. We demonstrate that a vitamin D receptor agonist 1,25-dihydroxyvitamin D₃ (VD3) and a retinoid X receptor agonist HX630 affect LPS-induced expression of pro-inflammatory factors. Specifically, both VD3 and HX630 inhibited expression of mRNAs encoding inducible nitric oxide synthase (iNOS) and IL-6, whereas expression of IL-1β mRNA was inhibited only by VD3. The inhibitory effect of VD3 and HX630 on expression of iNOS and IL-6 mRNAs was additive. Effect of VD3 and HX630 was also observed for inhibition of iNOS protein expression and nitric oxide production. Moreover, VD3 and HX630 inhibited LPS-induced activation of extracellular signal-regulated kinase (ERK) and nuclear translocation of nuclear factor κB (NF-κB). PD98059, an inhibitor of ERK kinase, attenuated LPS-induced nuclear translocation of NF-κB and induction of mRNAs for iNOS, IL-1β and IL-6. These results indicate that VD3 can inhibit production of several pro-inflammatory molecules from microglia, and that suppression of ERK activation is at least in part involved in the anti-inflammatory effect of VD3.     

Multi‐century tree‐ring precipitation record reveals increasing frequency of extreme dry events in the upper Blue Nile River catchment

Climate‐related environmental and humanitarian crisis are important challenges in the Great Horn of Africa (GHA). In the absence of long‐term past climate records in the region, tree‐rings are valuable climate proxies, reflecting past climate variations and complementing climate records prior to the instrumental era. We established annually resolved multi‐century tree‐ring chronology from Juniperus procera trees in northern Ethiopia, the longest series yet for the GHA. The chronology correlates significantly with wet‐season (r = .64, p < .01) and annual (r = .68, p < .01) regional rainfall. Reconstructed rainfall since A.D. 1811 revealed significant interannual variations between 2.2 and 3.8 year periodicity, with significant decadal and multidecadal variations during 1855–1900 and 1960–1990. The duration of negative and positive rainfall anomalies varied between 1–7 years and 1–8 years. Approximately 78.4% (95%) of reconstructed dry (extreme dry) and 85.4% (95%) of wet (extreme wet) events lasted for 1 year only and corresponded to historical records of famine and flooding, suggesting that future climate change studies should be both trend and extreme event focused. The average return periods for dry (extreme dry) and wet (extreme wet) events were 4.1 (8.8) years and 4.1 (9.5) years. Extreme‐dry conditions during the 19th century were concurrent with drought episodes in equatorial eastern Africa that occurred at the end of the Little Ice Age. El Niño and La Niña events matched with 38.5% and 50% of extreme‐dry and extreme‐wet events. Equivalent matches for positive and negative Indian Ocean Dipole events were weaker, reaching 23.1 and 25%, respectively. Spatial correlations revealed that reconstructed rainfall represents wet‐season rainfall variations over northern Ethiopia and large parts of the Sahel belt. The data presented are useful for backcasting climate and hydrological models and for developing regional strategic plans to manage scarce and contested water resources. Historical perspectives on long‐term regional rainfall variability improve the interpretation of recent climate trends.     

Notch signaling specifies megakaryocyte development from hematopoietic stem cells

In the hematopoietic system, Notch signaling specifies T cell lineage fate, in part through negative regulation of B cell and myeloid lineage development. However, we unexpectedly observed the development of megakaryocytes when using heterotypic cocultures of hematopoietic stem cells with OP9 cells expressing Delta-like1, but not with parental OP9 cells. This effect was abrogated by inhibition of Notch signaling either with gamma-secretase inhibitors or by expression of the dominant-negative Mastermind-like1. The importance of Notch signaling for megakaryopoietic development in vivo was confirmed by using mutant alleles that either activate or inhibit Notch signaling. These findings indicate that Notch is a positive regulator of megakaryopoiesis and plays a more complex role in cell-fate decisions among myeloid progenitors than previously appreciated.     

Diffuse large B-cell lymphoma outcome prediction by gene-expression profiling and supervised machine learning.

Diffuse large B-cell lymphoma (DLBCL), the most common lymphoid malignancy in adults, is curable in less than 50% of patients. Prognostic models based on pre-treatment characteristics, such as the International Prognostic Index (IPI), are currently used to predict outcome in DLBCL. However, clinical outcome models identify neither the molecular basis of clinical heterogeneity, nor specific therapeutic targets. We analyzed the expression of 6,817 genes in diagnostic tumor specimens from DLBCL patients who received cyclophosphamide, adriamycin, vincristine and prednisone (CHOP)-based chemotherapy, and applied a supervised learning prediction method to identify cured versus fatal or refractory disease. The algorithm classified two categories of patients with very different five-year overall survival rates (70% versus 12%). The model also effectively delineated patients within specific IPI risk categories who were likely to be cured or to die of their disease. Genes implicated in DLBCL outcome included some that regulate responses to B-cell-receptor signaling, critical serine/threonine phosphorylation pathways and apoptosis. Our data indicate that supervised learning classification techniques can predict outcome in DLBCL and identify rational targets for intervention.     

On the origin of the stereoselective affinity of Nutlin-3 geometrical isomers for the MDM2 protein

The stereoselective affinity of small-molecule binding to proteins is typically broadly explained in terms of the thermodynamics of the final bound complex. Using Brownian dynamics simulations, we show that the preferential binding of the MDM2 protein to the geometrical isomers of Nutlin-3, an effective anticancer lead that works by inhibiting the interaction between the proteins p53 and MDM2, can be explained by kinetic arguments related to the formation of the MDM2:Nutlin-3 encounter complex. This is a diffusively bound state that forms prior to the final bound complex. We find that the MDM2 protein stereoselectivity for the Nutlin-3a enantiomer stems largely from the destabilization of the encounter complex of its mirror image enantiomer Nutlin-3b, by the K70 residue that is located away from the binding site. On the other hand, the trans-Nutlin-3a diastereoisomer exhibits a shorter residence time in the vicinity of MDM2 compared with Nutlin-3a due to destabilization of its encounter complex by the collective interaction of pairs of charged residues on either side of the binding site: Glu25 and Lys51 on one side, and Lys94 and Arg97 on the other side. This destabilization is largely due to the electrostatic potential of the trans-Nutlin-3a isomer being largely positive over extended continuous regions around its structure, which are otherwise well-identified into positive and negative regions in the case of the Nutlin-3a isomer. Such rich insight into the binding processes underlying biological selectivity complements the static view derived from the traditional thermodynamic analysis of the final bound complex. This approach, based on an explicit consideration of the dynamics of molecular association, suggests new avenues for kinetics-based anticancer drug development and discovery.