Sip1, an AP-1 Accessory Protein in Fission Yeast,Is Required for Localization of Rho3 GTPase

Rho family GTPases act as molecular switches to regulate a range of physiological functions, including the regulation of the actin-based cytoskeleton, membrane trafficking, cell morphology, nuclear gene expression, and cell growth. Rho function is regulated by its ability to bind GTP and by its localization. We previously demonstrated functional and physical interactions between Rho3 and the clathrin-associated adaptor protein-1 (AP-1) complex, which revealed a role of Rho3 in regulating Golgi/endosomal trafficking in fission yeast. Sip1, a conserved AP-1 accessory protein, recruits the AP-1 complex to the Golgi/endosomes through physical interaction. In this study, we showed that Sip1 is required for Rho3 localization. First, overexpression of rho3 ⁺ suppressed defective membrane trafficking associated with sip1-i4 mutant cells, including defects in vacuolar fusion, Golgi/endosomal trafficking and secretion. Notably, Sip1 interacted with Rho3, and GFP-Rho3, similar to Apm1-GFP, did not properly localize to the Golgi/endosomes in sip1-i4 mutant cells at 27°C. Interestingly, the C-terminal region of Sip1 is required for its localization to the Golgi/endosomes, because Sip1-i4-GFP protein failed to properly localize to Golgi/endosomes, whereas the fluorescence of Sip1ΔN mutant protein co-localized with that of FM4-64. Consistently, in the sip1-i4 mutant cells, which lack the C-terminal region of Sip1, binding between Apm1 and Rho3 was greatly impaired, presumably due to mislocalization of these proteins in the sip1-i4 mutant cells. Furthermore, the interaction between Apm1 and Rho3 as well as Rho3 localization to the Golgi/endosomes were significantly rescued in sip1-i4 mutant cells by the expression of Sip1ΔN. Taken together, these results suggest that Sip1 recruits Rho3 to the Golgi/endosomes through physical interaction and enhances the formation of the Golgi/endosome AP-1/Rho3 complex, thereby promoting crosstalk between AP-1 and Rho3 in the regulation of Golgi/endosomal trafficking in fission yeast.     

Polymyxins as Novel and Safe Mucosal Adjuvants to Induce Humoral Immune Responses in Mice

There is currently an urgent need to develop safe and effective adjuvants for enhancing vaccine-induced antigen-specific immune responses. We demonstrate here that intranasal immunization with clinically used polypeptide antibiotics, polymyxin B (PMB) and colistin (CL), along with ovalbumin (OVA), increases OVA-specific humoral immune responses in a dose-dependently manner at both mucosal and systemic compartments. Enhanced immunity by boosting was found to persist during 8 months of observation. Moreover, mice intranasally immunized with OVA plus various doses of PMB or CL showed neither inflammatory responses in the nasal cavity and olfactory bulbs nor renal damages, compared to those given OVA alone. These data suggest that polymyxins may serve as novel and safe mucosal adjuvants to induce humoral immune responses. The polymyxin adjuvanticity was found to be independent of endotoxins liberated by its bactericidal activity, as indicated by similar enhancing effects of PMB in lipopolysaccharide (LPS)-hyporesponsive and LPS-susceptible mice. However, despite the presence of preexisting anti-PMB antibodies, we observed no reduction in the adjuvant function of polymyxins when they were given intranasally. Furthermore, the titers of OVA-specific Abs in mice intranasally immunized with OVA plus PMB or CL were significantly higher than those in mice administered with polymyxin analogues, such as polymyxin B nonapeptide and colistin methanesulfonate. The levels of released β-hexosaminidase and histamine in mast cell culture supernatants stimulated by PMB or CL were also significantly higher than those stimulated by their analogues. These results suggest that both the hydrophobic carbon chain and hydrophilic cationic cyclic peptide contribute to the mucosal adjuvanticity of PMB and CL.     

Current progress in non-Edg family LPA receptor research

Lysophosphatidic acid (LPA) is the simplest phospholipid yet possesses myriad biological functions. Until 2003, the functions of LPA were thought to be elicited exclusively by three subtypes of the endothelial differentiation gene (Edg) family of G protein-coupled receptors — LPA₁, LPA₂, and LPA₃. However, several biological functions of LPA could not be assigned to any of these receptors indicating the existence of one or more additional LPA receptor(s). More recently, the discovery of a second cluster of LPA receptors which includes LPA₄, LPA₅, and LPA₆ has paved the way for new avenues of LPA research. Analyses of these non-Edg family LPA receptors have begun to fill in gaps to understand biological functions of LPA such as platelet aggregation and vascular development that could not be ascribed to classical Edg family LPA receptors and are also unveiling new biological functions. Here we review recent progress in the non-Edg family LPA receptor research, with special emphasis on the pharmacology, signaling, and physiological roles of this family of receptors. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Joint morphology in the insect leg: evolutionary history inferred from Notch loss-of-function phenotypes in Drosophila

Joints permit efficient locomotion, especially among animals with a rigid skeleton. Joint morphologies vary in the body of individual animals, and the shapes of homologous joints often differ across species. The diverse locomotive behaviors of animals are based, in part, on the developmental and evolutionary history of joint morphogenesis. We showed previously that strictly coordinated cell-differentiation and cell-movement events within the epidermis sculpt the interlocking ball-and-socket joints in the adult Drosophila tarsus (distal leg). Here, we show that the tarsal joints of various insect species can be classified into three types: ball-and-socket, side-by-side and uniform. The last two probably result from joint formation without the cell-differentiation step, the cell-movement step, or both. Similar morphological variations were observed in Drosophila legs when Notch function was temporarily blocked during joint formation, implying that the independent acquisition of cell differentiation and cell movement underlay the elaboration of tarsal joint morphologies during insect evolution. These results provide a framework for understanding how the seemingly complex morphology of the interlocking joint could have developed during evolution by the addition of simple developmental modules: cell differentiation and cell movement.     

New class azaphilone produced by a marine fish-derived Chaetomium globosum. The stereochemistry and biological activities

Four new metabolites, chaetomugilins P-R and 11-epi-chaetomugilin I, were isolated from a strain of Chaetomium globosum originally obtained from the marine fish Mugil cephalus, and their absolute stereostructures were elucidated on the basis of spectroscopic analyses, including 1D and 2D NMR techniques and various chemical transformations. Particularly, the skeleton of chaetomugilin P is different from that of other azaphilones isolated from this fungal strain to date. In addition, these compounds significantly inhibited the growth of cultured P388, HL-60, L1210 and KB cell lines.     

3-(3-Phenoxybenzyl)amino-β-carboline: a novel antitumor drug targeting α-tubulin

3-(3-Phenoxybenzyl)amino-β-carboline 2h showed extremely-high activity; the IC₅₀ value was 0.074 μM. To verify 2h-induced cell death types, we observed the chromatin condensation, the DNA fragmentation and activated caspase-3 using Hoechst 33342, agarose electrophoresis and western blot, and suggesting 2h-induced cell death type was apoptosis. Flow cytometry showed that 2h-treated cell was induced SubG1 cell population after G2/M cell cycle arrest. In addition, using affinity chromatography and peptide mass fingerprinting, we found that interacting protein with this compound was α-tubulin protein.     

Increased Selectivity, Analytical Precision, and Throughput in Targeted Proteomics

Proteomics is gradually complementing large shotgun qualitative studies with hypothesis-driven quantitative experiments. Targeted analyses performed on triple quadrupole instruments in selected reaction monitoring mode are characterized by a high degree of selectivity and low limit of detection; however, the concurrent analysis of multiple analytes occurs at the expense of sensitivity because of reduced dwell time and/or selectivity due to limitation to a few transitions. A new data acquisition paradigm is presented in which selected reaction monitoring is performed in two ways to simultaneously quantify and confirm the identity of the targeted peptides. A first set of primary transitions is continuously monitored during a predetermined elution time window to precisely quantify each peptide. In addition, a set of six to eight transitions is acquired in a data-dependent event, triggered when all the primary transitions exceed a preset threshold. These additional transitions are used to generate composite tandem mass spectra to formally confirm the identity of the targeted peptides. This technique was applied to analyze the tryptic digest of a yeast lysate to demonstrate the performance of the technique. We showed a limit of detection down to tens of attomoles injected and a throughput exceeding 6000 transitions in one 60-min experiment. The technique was integrated into a linear work flow, including experimental design, data acquisition, and data evaluation, enabling large scale proteomic studies.Proteomics is gradually complementing qualitative studies focused on protein identification relying on shotgun strategies (1, 2) with large scale quantitative experiments. This change was prompted by the growing demand for qualification and verification of putative protein biomarkers through analysis of larger cohorts of clinical samples on one hand and the need for consistent quantitative data sets to facilitate modeling in systems biology studies on the other. In either case, the number of proteins under target is quite large (tens to hundreds), and traditional immunoassay approaches are not suited for use because of the cost, time, and difficulty of developing multiplexed assays. In this context, the selected reaction monitoring (SRM)¹ technique performed on a triple quadrupole mass spectrometer is increasingly applied to quantitative proteomics because of its sensitivity, selectivity, and wide dynamic range (3–6). Mass spectrometry assays can be developed very rapidly via the use of commodity synthetic reference peptides libraries (7), and large resources of peptide MS/MS data (MRMAtlas) (8) are available to design the initial assays. However, developing a robust and precise SRM-based multiple assay remains demanding for proteomics experiments.One of the main challenges is that most proteomics samples are highly complex, and several interfering signals are detected within a given time window that require systematic verification of the target peptide identity first to ensure its accurate quantification. Using isotopically labeled counterparts of the targeted analytes is a common way to confirm the target peptide identity (9, 10). As this may not always be practical for large scale quantitative proteomics studies, an alternative way to verify the peptide identity is to use SRM-initiated full MS/MS scans (11). However, the disadvantage of this method is a lower sensitivity and selectivity compared with SRM as it uses a broader mass selection window, which results in MS/MS spectra often containing signals from multiple components co-eluting in the case of biological samples with a complex background. Furthermore, SRM-initiated MS/MS scans require much longer duty cycle times that will disrupt the predefined SRM sequence of events in the case of complex multiplexed assays even when performed on a triple quadrupole instrument equipped with a linear ion trap. Recently, instead of using MS/MS spectra, a composite MS/MS spectrum that is generated by measuring multiple fragments ions (8–10 ions) from one specific peptide has been proven to provide sufficient information for peptide identification.² The peptide is verified both by the overlay of the chromatographic elution profiles of the fragment ions and by matching the composite MS/MS spectrum that comprises multiple SRM transitions to the MS/MS spectral library entry (12, 13). Because it is based on SRM acquisition, this method provides a rapid, sensitive, and selective way to perform peptide verification, which is desirable for large scale screening experiments. The drawback of this approach is that only a limited number of compounds can be practically analyzed in one HPLC-MS run because the instrument is continuously monitoring 8–10 transitions for each peptide no matter whether the peptide is detected from the sample or not, resulting in the waste of a significant portion of the instrument time. To overcome this issue and thus increase the throughput, we propose that the instrument constantly monitor only a small subset of SRM transitions for each peptide for the actual quantification and in addition confirm the peak identity using the full set of fragment ions, which are acquired in a data-dependent mode.To provide this instrument capability, we developed an innovative instrument control software, called intelligent selected reaction monitoring (iSRM), that can use the specificity of a small subset of SRM transitions to quantify and intelligently trigger the full list for confirmation of target peptides, thereby allowing the simultaneous qualitative and quantitative analysis of up to 1000 peptides in a single LC-MS experiment. Here we describe the concept of iSRM and the work flow associated to it, and we demonstrate the increased throughput facilitating the development of SRM assays and its ability to perform large scale screens targeting a respectable number of proteins.     

The Temperature Sensitivity of a Mutation in the Essential tRNA Modification Enzyme tRNA Methyltransferase D (TrmD)

Conditional temperature-sensitive (ts) mutations are important reagents to study essential genes. Although it is commonly assumed that the ts phenotype of a specific mutation arises from thermal denaturation of the mutant enzyme, the possibility also exists that the mutation decreases the enzyme activity to a certain level at the permissive temperature and aggravates the negative effect further upon temperature upshifts. Resolving these possibilities is important for exploiting the ts mutation for studying the essential gene. The trmD gene is essential for growth in bacteria, encoding the enzyme for converting G37 to m¹G37 on the 3′ side of the tRNA anticodon. This conversion involves methyl transfer from S-adenosyl methionine and is critical to minimize tRNA frameshift errors on the ribosome. Using the ts-S88L mutation of Escherichia coli trmD as an example, we show that although the mutation confers thermal lability to the enzyme, the effect is relatively minor. In contrast, the mutation decreases the catalytic efficiency of the enzyme to 1% at the permissive temperature, and at the nonpermissive temperature, it renders further deterioration of activity to 0.1%. These changes are accompanied by losses of both the quantity and quality of tRNA methylation, leading to the potential of cellular pleiotropic effects. This work illustrates the principle that the ts phenotype of an essential gene mutation can be closely linked to the catalytic defect of the gene product and that such a mutation can provide a useful tool to study the mechanism of catalytic inactivation.     

Isolation of R-(−)-3-Hydroxy-β-ionone from Burley Tobacco

The tritium-hydrogen exchange method was used to determine the total racemization of amino acid residues of four proteins (ribonuclease A, lysozyme, soybean protein and casein) during their exposure to an alkali. Tritium was incorporated with first order kinetics into these proteins during their incubation in 0.2 n NaOH at 40°C. The tritium-hydrogen exchange increased as the temperature and the alkali concentration increased. In contrast, pepsin digestibility decreased extensively in the initial stage of the treatment. This phenomenon was verified by the subsite theory of protease, that only minor racemization renders the extended range of the peptide chain around the racemized amino acids non-susceptible to pepsin. General precautions against the racemization of food protein treated with alkalis are discussed briefly in terms of the deterioration of nutrients.     

Physiological Pineal Effects on Female Reproductive Function of Laboratory Rats: Prenatal Development of Pups,Litter Size and Estrous Cycle in Middle Age

The present study investigates whether and how the pineal or its hormone melatonin influences female reproductive functions, namely the litter size, prenatal development of offsprings, and estrous cyclicity, especially its age‐related cessation in a non‐seasonal breeder, the laboratory rat. Wistar rats were maintained under a 24 h light‐dark (12L∶12D) cycle. Female rats were divided into 3 groups: non‐operated (NO), sham‐operated (SX), and pinealectomized (PX). Surgeries were performed in 35–40 day‐old females. Starting at an age between 70 days and 7 months, female rats of all 3 groups were repeatedly mated with intact males. PX mothers more frequently delivered pups with malformations (e.g., taillessness, hydronephrosis, 7 out of 1263 pups) than control rats (0/1323; p<0.007). In the first delivery at 3 months of age, but not at later ages, PX mothers delivered more pups of lower body weight than control animals (p<0.001). Examination of vaginal smears showed that almost all female rats of the NO, SX, and PX groups had 4‐day estrous cyclicity when they were young–between 60 days and 5 months of age. At an age of 17 to 18 months, most female rats of the NO and SX groups showed irregular, continuously diestrous or pseudopregnancy‐like patterns, and 4‐day estrous cyclicity was found in only 10% of the NO or SX animals. In contrast, about 50% of the PX rats showed 4‐day estrous cyclicity at this older age (p< 0.001). Melatonin, when added to drinking water (0.4 mg/L) for 16 days during the dark phase increased the frequency of diestrous phase, except in continuously diestrous rats and very few others. This melatonin effect was strong in PX rats but relatively weak in SX rats. In conclusion, the pineal hormone appears to influence various reproductive functions and developmental processes, especially pregnancy and the timing of reproductive aging in rats. The effects of pinealectomy are more prominent at an age of 60 to 80 days (i.e., shortly after puberty) and at the beginning of the cessation of cycles in middle‐aged females.