Mechanisms of ammonium transport, accumulation, and retention in ooyctes and yeast cells expressing Arabidopsis AtAMT1;1

Ammonium is a primary source of N for plants, so knowing how it is transported, stored, and assimilated in plant cells is important for rational approaches to optimise N-use in agriculture. Electrophysiological studies of Arabidopsis AtAMT1;1 expressed in oocytes revealed passive, Δψ-driven transport of through this protein. Expression of AtAMT1;1 in a novel yeast mutant defective in endogenous ammonium transport and vacuolar acidification supported the above mechanism for AtAMT1;1 and revealed a central role for acid vacuoles in storage and retention of ammonia in cells. These results highlight the mechanistic differences between plant AMT proteins and related transporters in bacteria and animal cells, and suggest novel strategies to enhance nitrogen use efficiency in agriculture.     

Nonmyristoylated Matrix Protein from the Mason-Pfizer Monkey Virus Forms Oligomers

We studied the oligomeric properties of betaretroviral nonmyristoylated matrix protein (MA) and its R55F mutant from the Mason-Pfizer monkey virus in solution by means of chemical crosslinking and NMR spectroscopy. By analyzing crosslinked products and using concentration-dependent NMR chemical shift mapping, we have proven that the wild-type (WT) MA forms oligomers in solution. Conversely, no oligomerization was observed for the R55F mutant. Structural comparison of MAs explained their different behaviors in solution, concluding that the key residues involved in intermonomeric interaction are exposed in the WT MA but buried in the mutant, preventing the oligomerization of R55F. The final model of oligomerization of the WT MA was derived by concerted use of chemical shift mapping and diffusion-ordered spectroscopy measured on a set of protein samples with varying concentrations. We found that the Mason-Pfizer monkey virus WT MA exists in a monomer-dimer-trimer equilibrium in solution, with the corresponding dissociation constants of 2.3  and 0.24 mM, respectively. Structures of the oligomers calculated with HADDOCK software are closely related to the structures of other retroviral MA trimers.     

Potential bias and mitigations when using stable isotope labeled parent drug as internal standard for LC-MS/MS quantitation of metabolites

In recent years, increasing emphasis has been placed on quantitative characterization of drug metabolites for better insight into the correlation between metabolite exposure and toxicological observations or pharmacological efficacy. One common strategy for metabolite quantitation is to adopt the stable isotope labeled (STIL) parent drug as the internal standard in an isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. In the current work, we demonstrate this strategy could have a potential pitfall resulting in quantitation bias if the internal standard is subject to ion suppression from the co-eluting parent drug in the incurred samples. Propranolol and its metabolite 4-hydroxypropranolol were used as model compounds to demonstrate this phenomenon and to systematically evaluate different approaches to mitigate the issue, including atmospheric pressure chemical ionization (APCI) mode of ionization, increased internal standard concentration, quantitation without internal standard, the use of a structural analog as internal standard, and dilution of the samples. Case studies of metabolite quantitation in nonclinical and clinical studies in drug development were also included to demonstrate the importance of using an appropriate bioanalytical strategy for metabolite quantitation in the real world. We present that bias of metabolite concentrations could pose a potential for poor estimation of safety risk. A strategy for quantitation of metabolites in support of drug safety assessment is proposed.     

Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation

Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury.     

Cortical control of mastication in the cat: properties of mastication-related neurons in motor and masticatory cortices

The aim of this study is to examine mastication-specific activity of orofacial neurons in the motor and masticatory cortices of the awake cat. We examine properties of mastication-related neurons (MRNs) in masticatory (MA, the rostral region of the orbital gyrus) and motor (area P, the lateral wall of the presylvian sulcus) cortical areas that are related to mastication of cats. MRNs in MA and area P had in common mechanoreceptive fields (RFs) in perioral, mandibular and lingual regions, and many MRNs had bilateral RFs in the tongue and mandibular regions. Facial RF size was the largest in area P. Eleven percent of MRN recording sites in MA, and 43% in area P evoked various motor effects with the use of intracortical microstimulation (ICMS). MRNs of the pre-movement type showing activities prior to mastication, or masticatory or lingual EMG, were 14% in MA and 45% in area P. Based on wheat germ agglutinin–horseradish peroxidase (WGA-HRP) injection into area P and MA, cortico-cortical connections were examined. After the unilateral area P injection, were reciprocal connections between the contralateral area P and bilateral MA were demonstrated. After the unilateral MA injection, there were reciprocal connections between the contralateral MA, bilateral area P and bilateral orofacial SI (the orofacial region of the first somatosensory area). These findings suggest that accurate masticatory movements may be executed by the cortical processing in MA and area P.     

Application of Fluoresceinisothiocyanate Labeling of Protein to SDS-Electrophoresis and Subsequent Sequence Analysis

Fluoresceinisothiocyanate was applied to fluorescent labeling of protein for the determination of molecular weight by SDS Polyacrylamide gel electrophoresis. Proteins having molecular weight ranging from 15,000 to 70,000 were tested. The resolved FITC-labeled protein was eluted from the gel and was submitted to amino acid sequence analysis.     

Glycerol monomycolate, a latent tuberculosis-associated mycobacterial lipid, induces eosinophilic hypersensitivity responses in guinea pigs

Dynamic changes in the lipid composition of the cell wall occur in pathogenic mycobacteria that are often intended for adaptation to the host environment. Dormant mycobacteria should have evolved efficient maneuvers for cohabitation, allowing the microbes to persist for years within the host. Glycerol monomycolate (GroMM) has been implicated as a specific immune target in human individuals with latent, but not active, tuberculosis, but the in vivo response to GroMM and the relevance of it to latent infection remain poorly understood. Here, we immunized guinea pigs with bacillus Calmette–Guerin (BCG) expressing high levels of GroMM and then, monitored skin reactions at the site of challenge with GroMM-containing liposome. We found that BCG-immunized guinea pigs mounted enhanced skin reactions to GroMM with prominent local infiltration by eosinophils. Consistent with this, GroMM-stimulated lymph node cells upregulated the expression of T helper (Th)2-type cytokines, such as interleukin (IL)-5 and IL-10, that could potentially counteract the microbe-eliminating Th1-type cytokine response. On the basis of these observations, we predict that the host response to GroMM produced by dormant mycobacteria would contribute to their long-term survival in the host.     

The evolution of catalytic function in the HIV-1 protease

The evolution of species is a complex phenomenon based on the optimization of a multidimensional function referred to as fitness. At the level of biomolecular evolution, the fitness function can be reduced to include physiochemical properties relevant to the biological function of a particular molecule. In this work, questions involving the physical-chemical mechanisms underlying the evolution of HIV-1 protease are addressed through molecular simulation and subsequent analysis of thermodynamic properties related to the activity of the enzyme. Specifically, the impact of 40 single amino acid mutations on the binding affinity toward the matrix/capsid (MA/CA) substrate and corresponding transition state intermediate has been characterized using a molecular mechanics Poisson-Boltzmann surface area approach. We demonstrate that this approach is capable of extracting statistically significant information relevant to experimentally determined catalytic activity. Further, no correlation was observed between the effect of mutations on substrate and transition state binding, suggesting independent evolutionary pathways toward optimizing substrate specificity and catalytic activity. In addition, a detailed analysis of calculated binding affinity data suggests that ground-state destabilization (reduced binding affinity for the substrate) could be a contributing factor in the evolutionary optimization of HIV-1 protease. A numerical model is developed to demonstrate that ground-state destabilization is a valid mechanism for activity optimization given the high concentrations of substrate experienced by the functional enzyme in vivo.     

Heritability and genome-wide linkage analysis of migraine in the genetic isolate of Norfolk Island

Migraine is a common neurovascular disorder with a complex envirogenomic aetiology. In an effort to identify migraine susceptibility genes, we conducted a study of the isolated population of Norfolk Island, Australia. A large portion of the permanent inhabitants of Norfolk Island are descended from 18th Century English sailors involved in the infamous mutiny on the Bounty and their Polynesian consorts.In total, 600 subjects were recruited including a large pedigree of 377 individuals with lineage to the founders. All individuals were phenotyped for migraine using International Classification of Headache Disorders-II criterion. All subjects were genotyped for a genome-wide panel of microsatellite markers. Genotype and phenotype data for the pedigree were analysed using heritability and linkage methods implemented in the programme SOLAR. Follow-up association analysis was performed using the CLUMP programme.A total of 154 migraine cases (25%) were identified indicating the Norfolk Island population is high-risk for migraine. Heritability estimation of the 377-member pedigree indicated a significant genetic component for migraine (h² = 0.53, P = 0.016). Linkage analysis showed peaks on chromosome 13q33.1 (P = 0.003) and chromosome 9q22.32 (P = 0.008). Association analysis of the key microsatellites in the remaining 223 unrelated Norfolk Island individuals showed evidence of association, which strengthen support for the linkage findings (P ≤ 0.05).In conclusion, a genome-wide linkage analysis and follow-up association analysis of migraine in the genetic isolate of Norfolk Island provided evidence for migraine susceptibility loci on chromosomes 9q22.22 and 13q33.1.