The aspartimide problem in Fmoc-based SPPS. Part II.

The sequence dependence of base-catalysed aspartmide formation during Fmoc-based SPPS was systematically studied employing the peptide models H-Val-Lys-Asp-Xaa-Tyr-Ile-OH. The extent of formation of aspartimide and related by-products was determined by RP-HPLC. Considerable amounts of by-products were formed in the case of Xaa = Asp(OtBu), Arg(Pbf), Asn(Mtt), Cys(Acm) and unprotected Thr. Aspartimide formation could be diminished by incorporation of Asp(OMpe) or by employing milder methods for Fmoc cleavage, e.g. hexamethyleneimine/N-methylpyrrolidine/HOBt/NMP/DMSO 4:50:4:71:71 (v/v/w/v/v).     

Synthesis and application of Fmoc-His(3-Bum)-OH.

This paper presents a reevaluation of the synthesis and properties of Fmoc-His(3-Bum)-OH regarding its application in SPPS with minimal racemization of histidine residues during coupling and esterification reactions. By-product formation during the deprotection of the test peptides could be significantly reduced by scavenging the concomitantly formed HCHO, e.g. with methoxyamine.     

Molecular Evidence for the Ancient Origin of the Ribosomal Protection Protein That Mediates Tetracycline Resistance in Bacteria

The ribosomal protection proteins (RPPs) mediate the resistance to tetracycline (TC) in Gram-positive and Gram-negative bacteria. The RPPs display sequence similarity to translation elongation factors, EF-G/EF-2 and EF-Tu/EF-1α. To determine the evolutionary origin of the RPPs, we constructed a composite phylogenetic tree of the RPPs, EF-G/EF-2 and EF-Tu/EF-1α. This tree includes two universal trees for the EF-G/EF-2 and EF-Tu/EF-1α, which form clusters corresponding to the respective two groups of proteins from three superkingdoms. The cluster of RPPs was placed at a point between the EF-G/EF-2 and EF-Tu/EF-1α clusters. The branch length (substitutions/site) between the node for the RPP cluster and the primary divergence of the RPPs was statistically shorter than that between the node for this cluster and the primary divergence in the EF-G/EF-2 cluster. This indicates that the RPPs derived through duplication and divergence of the ancient GTPase before the divergence of the three superkingdoms. Furthermore, this suggests the RPPs’ extant function occurred before the streptomycetes that include the TC-producing strains. Therefore, the RPPs evolved independent of the presence of TCs and serve a function other than antibiotic resistance. The RPPs may provide ribosomal protection against other chemical substances in the environment. Reviewing Editor: Dr. Margaret Riley Takeshi Kobayashi, Lisa Nonaka have contributed significantly to the research and preparation of this article.     

Caffeic acid phenethyl ester reduces neurovascular inflammation and protects rat brain following transient focal cerebral ischemia

Ischemic stroke is a neurovascular disease treatable by thrombolytic therapy, but the therapy has to be initiated within 3 h of the incident. This therapeutic limitation stems from the secondary injury which results mainly from oxidative stress and inflammation. A potent antioxidant/anti-inflammatory agent, caffeic acid phenethyl ester (CAPE) has potential to mitigate stroke's secondary injury, and thereby widening the therapeutic window. We observed that CAPE protected the brain in a dose-dependent manner (1-10 mg/kg body weight) and showed a wide therapeutic window (about 18 h) in a rat model of transient focal cerebral ischemia and reperfusion. The treatment also increased nitric oxide and glutathione levels, decreased lipid peroxidation and nitrotyrosine levels, and enhanced cerebral blood flow. CAPE down-regulated inflammation by blocking nuclear factor kappa B activity. The affected mediators included adhesion molecules (intercellular adhesion molecule-1 and E-selectin), cytokines (tumor necrosis factor-alpha and interleukin-1beta) and inducible nitric oxide synthase. Anti-inflammatory action of CAPE was further documented through reduction of ED1 (marker of activated macrophage/microglia) expression. The treatment inhibited apoptotic cell death by down-regulating caspase 3 and up-regulating anti-apoptotic protein Bcl-xL. Conclusively, CAPE is a promising drug candidate for ischemic stroke treatment due to its inhibition of oxidative stress and inflammation, and its clinically relevant wide therapeutic window.     

Electroacupuncture regulates TRPM7 expression through the trkA/PI3K pathway after cerebral ischemia-reperfusion in rats

Recently, it was demonstrated that TRPM7 is an essential mediator of anoxia-induced neuronal death. Meanwhile, nerve growth factor (NGF) is known to have survival and neuroprotective effects by interacting with the high affinity neurotrophin receptor, tropomyosin-related kinase A (trkA). In the present study, we found that electroacupuncture (EA) treatment could up-regulate trkA expression after focal cerebral ischemia in rats. At the same time, EA therapy obviously decreased the high expression of TRPM7 induced by ischemia. Using K252a to inhibit trkA, we found that the EA-mediated down-regulation of TRPM7 was significantly suppressed in rats subjected to cerebral ischemia. TrkA can utilize two distinct signaling pathways: the phosphatidylinositol 3-kinase (PI3K) pathway and the extracellular signal-related kinase (ERK) pathway. We found that the effect of EA on TRPM7 was also inhibited by a PI3K inhibitor, while an ERK inhibitor had no effect. Taken together, our findings suggest that EA can reverse the ischemia-induced increase of TRPM7 levels through the trkA-PI3K pathway.     

Contributions of muscles to terminal-swing knee motions vary with walking speed

Many children with cerebral palsy walk with diminished knee extension during terminal swing, at speeds much slower than unimpaired children. Treatment of these gait abnormalities is challenging because the factors that extend the knee during normal walking, over a range of speeds, are not well understood. This study analyzed a series of three-dimensional, muscle-driven dynamic simulations to determine whether the relative contributions of individual muscles and other factors to angular motions of the swing-limb knee vary with walking speed. Simulations were developed that reproduced the measured gait dynamics of seven unimpaired children walking at self-selected, fast, slow, and very slow speeds (7 subjects×4 speeds=28 simulations). In mid-swing, muscles on the stance limb made the largest net contribution to extension of the swing-limb knee at all speeds examined. The stance-limb hip abductors, in particular, accelerated the pelvis upward, inducing reaction forces at the swing-limb hip that powerfully extended the knee. Velocity-related forces (i.e., Coriolis and centrifugal forces) also contributed to knee extension in mid-swing, though these contributions were diminished at slower speeds. In terminal swing, the hip flexors and other muscles on the swing-limb decelerated knee extension at the subjects’ self-selected, slow, and very slow speeds, but had only a minimal net effect on knee motions at the fastest speeds. Muscles on the stance limb helped brake knee extension at the subjects’ fastest speeds, but induced a net knee extension acceleration at the slowest speeds. These data—which show that the contributions of muscular and velocity-related forces to terminal-swing knee motions vary systematically with walking speed—emphasize the need for speed-matched control subjects when attempting to determine the causes of a patient's abnormal gait.     

Increased oxidative stress during hyperglycemic cerebral ischemia

In this review, we summarize the role of hyperglycemia during cerebral ischemia. Hyperglycemia occurring during experimental and clinical stroke has been associated with increased cerebral damage. Increased oxidative stress resulting from hyperglycemia is believed to contribute to the exacerbated damage. More specifically, superoxide, nitric oxide and peroxynitrite are believed to play an important role in cerebral damage. This also involves increased recruitment of various blood cells to the ischemic zone that contribute to inflammation. We present data from our group and others that demonstrate that free radical production is increased during hyperglycemic stroke in rodents. Recent data suggest that inflammation is an important component of ischemic damage under both normo- and hyperglycemic conditions. We summarize numerous studies that indicate that a variety of antioxidant (inhibition of free radical production, scavenging of free radicals and increasing free radical degradation) and anti-inflammatory strategies decrease cerebral infarction. Finally, we compare the success of some of these strategies in clinical trials compared to the animal models.     

Excitotoxic mechanisms in stroke: an update of concepts and treatment strategies

Cerebral damage as a consequence of glutamate-mediated excitotoxicity represents a major consequence of stroke. However, the development of effective clinical treatments for this potentially devastating condition has been largely unsuccessful to date, despite promising basic research. This review will focus on the latest advances in our understanding of the excitotoxic process including the release of glutamate as a neurotransmitter and the potential contribution of complexins, the important role of astrocytes, including its involvement in glutamate uptake, alterations in glutamate transporter levels, reversed glutamate uptake, and the vesicular release of glutamate. Recent progress in our understanding of the involvement of excitotoxicity in white matter injury following ischemic insults is also discussed, as is oxidative stress and ischemic tolerance, along with an update on the use of treatment strategies with potential therapeutic benefit including stimulation of neurogenesis. Such key issues are at the heart of future interventions directed at limiting the extent of the excitotoxic process, and remain a viable consideration for effective stroke management.     

Nature’s care: diarrhea, watershed protection, and biodiversity conservation in Flores, Indonesia

Part of the puzzle surrounding biodiversity loss lies in an incomplete understanding of how humans value the functions and services that flow from biodiversity conservation projects. This paper takes a closer look at the links between the conservation of biodiversity and the livelihoods of rural people who live on the fringes of the parks and protected areas. We revisit some of the key aspects of ecosystem valuation—purpose, methodology, and policy design and implementation—because the links between biodiversity conservation, ecosystem services, and human welfare are obscured by considerable smoke and mirrors. Using a biodiversity conservation project (Ruteng Park) on Flores Island in Indonesia as a case study, we build a concrete empirical example of ecosystem valuation. This conservation project has resulted in spatially patchy watershed protection that allows us to identify and estimate the impacts of watershed services on human health (diarrhea prevalence) in the buffer zone of the park. We conclude by offering a plan of research to improve the design of conservation interventions for protecting biodiversity and providing ecosystem services. These recommendations include developing more conceptual knowledge on the linkages between biodiversity and ecosystem services; scaling up valuation efforts of underappreciated services such as health; shifting focus from valuing services individually to valuing multiple benefits from the same area; and conducting conservation policy experiments to identify causal outcomes (including defensible estimates of ecosystem values).