A high-throughput mass spectrometric assay for discovery of human lipoxygenase inhibitors and allosteric effectors

Lipoxygenases (LOXs) regulate inflammation through the production of a variety of molecules whose specific downstream effects are not entirely understood due to the complexity of the inflammation pathway. The generation of these biomolecules can potentially be inhibited and/or allosterically regulated by small synthetic molecules. The current work describes the first mass spectrometric high-throughput method for identifying small molecule LOX inhibitors and LOX allosteric effectors that change the substrate preference of human lipoxygenase enzymes. Using a volatile buffer and an acid-labile detergent, enzymatic products can be directly detected using high-performance liquid chromatography–mass spectrometry (HPLC–MS) without the need for organic extraction. The method also reduces the required enzyme concentration compared with traditional ultraviolet (UV) absorbance methods by approximately 30-fold, allowing accurate binding affinity measurements for inhibitors with nanomolar affinity. The procedure was validated using known LOX inhibitors and the allosteric effector 13(S)-hydroxy-9Z,11E-octadecadienoic acid (13-HODE).     

Phenotypic plasticity and differentiation in an invasive freshwater microalga

Recent studies show that both marine and limnic microalgal species often consist of several genetically distinct populations. This is also valid for the nuisance freshwater algae Gonyostomum semen, which originates from acidic, brown water swamp lakes, but can nowadays also be found in clearer lakes with close to neutral pH. We hypothesized that the observed genetic differentiation among G. semen lake populations, reported in earlier studies, is connected to adaptation to local environmental conditions. In the present study we performed controlled laboratory experiments to test whether 12 strains originating from five lakes varied in their response to five to six different pHs, light intensities and DOC concentrations. Overall, growth (0.01–0.37 day⁻¹) was observed over a wide range of light intensities and pHs, demonstrating high potential for photoacclimation and extensive plasticity of individual strains. Moreover, we found similar growth rates and consistent growth optima for specific pHs by strains from the same lake, suggesting genetic differentiation of populations into distinct phenotypes. However, observed strain specific preferences did not always reflect environmental conditions in the lakes of origin and provided limited evidence for the hypothesized local adaptation. Instead, the observed phenotypic differentiation may indicate resilient effects of founder events. We suggest that the wide phenotypic plasticity in this species enables it to thrive in fluctuating and variable environments, and may play a role in its ability to colonize new habitats.     

Phosphorylation of elF-4E and initiation of protein synthesis in P19 embryonal carcinoma cells

Mitogenic stimulation of protein synthesis is accompanied by an increase in elF-4E phosphorylation. The effect on protein synthesis by induction of differentiation is less well known. We treated P19 embryonal carcinoma cells with the differentiating agent retinoic acid and found that protein synthesis increased during the first hour of addition. However, the phosphorylation state, as well as the turnover of phosphate on elF-4E, remained unchanged. Apparently, the change in protein synthesis after RA addition is regulated by another mechanism than elF-4E phosphorylation. By using P19 cells overexpressing the EGF receptor, we show that the signal transduction pathway that leads to phosphorylation of elF-4E is present in P19 cells; the EGF-induced change in phosphorylation of elF-4E in these cells is likely to be regulated by a change in elF-4E phosphatase activity. These results suggest that the onset of retinoic acid-induced differentiation is triggered by a signal transduction pathway which involves changes in protein synthesis, but not elF-4E phosphorylation. © 1995 Wiley-Liss, Inc.     

Collagen and non-collagenous proteins molecular crosstalk in the pathophysiology of osteoporosis

Collagenous and non-collagenous proteins (NCPs) in the extracellular matrix, as well as the coupling mechanisms between osteoclasts and osteoblasts, work together to ensure normal bone metabolism. Each protein plays one or more critical roles in bone metabolism, sometimes even contradictory, thus affecting the final mechanical, physical and chemical properties of bone tissue. Anomalies in the amount and structure of one or more of these proteins can cause abnormalities in bone formation and resorption, which consequently leads to malformations and defects, such as osteoporosis (OP). The connections between key proteins involved in matrix formation and resorption are far from being elucidated. In this review, we resume knowledge on the crosstalk between collagen type I and selected NCPs (Transforming Growth Factor-β, Insulin-like Growth Factor-1, Decorin, Osteonectin, Osteopontin, Bone Sialoprotein and Osteocalcin) of bone matrix, focusing on their possible involvement and role in OP. The different elements of this network can be pharmacologically targeted or used for the design/development of innovative regenerative strategies to modulate a feedback loop in bone remodelling.     

Physical associations of microglia and the vascular blood-brain barrier and their importance in development,health, and disease

Brain endothelial cells (BEC) of the vascular blood-brain barrier (BBB) interact with many different cell types in the brain, including microglia, the brain's resident immune cells. Physical associations of microglia with the BBB and the importance of these interactions in health and disease are an emerging area of study and likely involved in neuroimmune communication. In this mini-review, we consider how microglia and the BBB are intrinsically linked in the developing brain, discuss possible mechanisms that attract microglia to the vasculature in healthy physiological conditions, and examine the known microglial-vascular associated changes in systemic infection and various disease states. Our findings shed light on the complexities of microglial-vascular interactions and highlight the contributions of microglia to the functions of the neurovascular unit.     

Phenology of plants in relation to ambient environment in a subalpine forest of Uttarakhand,western Himalaya

Observations on phenology of some representative trees, shrubs, under-shrubs and herbs in a subalpine forest of Uttarakhand, western Himalaya were recorded. With the commencement of favorable growth season in April, occurrence of leaf fall was indicatory growth phenomenon in Quercus semecarpifolia, Q. floribunda and Abies spectabilis. However, active vegetative growth in herbaceous species starts onward April and fruit maturation and seed dehiscence are completed from mid of September to October. In general, vegetative growth and reproductive stages in majority of the studied species seems to be dependent on adequate moisture content and also flowering and fruiting in subalpine plants correlate ambient temperature.     

The role of climate change in the widespread mortality of holm oak in open woodlands of Southwestern Spain

Forest decline and increasing tree mortality are of global concern and the identification of the causes is necessary to develop preventive measures. Global warming is an emerging factor responsible for the increasing tree mortality in drought-prone ecosystems. In the southwestern Iberian Peninsula, Mediterranean holm oak open woodlands currently undergo large-scale population-level tree die-off. In this region, temperature and aridity have increased during recent decades, but the possible role of climate change in the current oak mortality has not been investigated.To assess the role of climate change in oak die-off in managed open woodlands in southwestern Spain, we analyzed climate change-related signals in century-long tree ring chronologies of dead holm oaks. We examined the high/low-frequency variability in growth and the relationship between growth and climate.Similar to other Mediterranean forests, growth was favored by precipitation from autumn of the year prior to ring formation to spring of the year of ring formation, whereas high temperatures during spring limited growth. Since the 1970s, the intensity of the high-frequency response to water availability increased simultaneously with temperature and aridity. The growth trends matched those of climatic changes. Growth suppressions occurred during droughts in the 1970s, 1980s and 1990s. Widespread stand-level, age-independent mortality occurred since 2005 and affected trees that cannot be considered old for the species standards.The close relationship between growth and climate indicate that climate change strongly controlled the growth patterns. This suggests that harsher climatic conditions, especially increased aridity, affected the tree performance and could have played a significant role in the mortality process. Climate change may have exacerbated or predisposed trees to the impact of other factors (e.g. intense management and pathogens). These observations could suggest a similar future increase in oak mortality which may occur in more northern oak open woodlands if aridity further increases.     

绿脓杆菌群集生长效应

本文通过群集的绿脓杆菌在高盐、酸性培养基上生长试验,抗金黄色葡萄球菌的拮抗试验及冷休克试验,证明了绿脓杆菌群集生长效应。主要表现为群集的绿脓杆菌可以在含10～20%NaCl 的培养基上生长;pH4环境中也可生长。同样条件下,对照几乎均为阴性。分散成单个细胞分布的绿脓杆菌对冷反应敏感,群集则有抗冷休克作用。实验条件下,密集的金黄色葡萄球菌生长对分散的绿脓杆菌有拮抗作用,群集则可抵抗这种作用而生长。讨论中初步分析了群集生长效应的机理和对细菌种群在自然生境中的稳定作用。     

Probing disorders of the nervous system using reprogramming approaches

The groundbreaking technologies of induced pluripotency and lineage conversion have generated a genuine opportunity to address fundamental aspects of the diseases that affect the nervous system. These approaches have granted us unrestricted access to the brain and spinal cord of patients and have allowed for the study of disease in the context of human cells, expressing physiological levels of proteins and under each patient's unique genetic constellation. Along with this unprecedented opportunity have come significant challenges, particularly in relation to patient variability, experimental design and data interpretation. Nevertheless, significant progress has been achieved over the past few years both in our ability to create the various neural subtypes that comprise the nervous system and in our efforts to develop cellular models of disease that recapitulate clinical findings identified in patients. In this Review, we present tables listing the various human neural cell types that can be generated and the neurological disease modeling studies that have been reported, describe the current state of the field, highlight important breakthroughs and discuss the next steps and future challenges.