The role of myosin V in exocytosis and synaptic plasticity

In neuroscience, myosin V motor proteins have attracted attention since they are highly expressed in brain, and absence of myosin Va in man leads to a severe neurological disease called Griscelli syndrome. While in some cells myosin V is described to act as a vesicle transport motor, an additional role in exocytosis has emerged recently. In neurons, myosin V has been linked to exocytosis of secretory vesicles and recycling endosomes. Through these functions, it is implied in regulating important brain functions including the release of neuropeptides by exocytosis of large dense-core vesicles and the insertion of neurotransmitter receptors into post-synaptic membranes. This review focuses on the role of myosin V in (i) axonal transport and stimulated exocytosis of large dense-core vesicles to regulate the secretion of neuroactive substances, (ii) tethering of the endoplasmic reticulum at cerebellar synapses to permit long-term depression, (iii) recycling of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors at hippocampal synapses during long-term potentiation, and (iv) recycling of nicotinic acetylcholine receptors at the neuromuscular junction. Myosin V is thus discussed as an important modulator of synaptic plasticity.     

The neuropeptidome of Rhodnius prolixus brain

We show a sensitive and straightforward off‐line nano‐LC‐MALDI‐MS/MS workflow that allowed the first comprehensive neuropeptidomic analysis of an insect disease vector. This approach was applied to identify neuropeptides in the brain of Rhodnius prolixus, a vector of Chagas disease. This work will contribute to the annotation of genes in the ongoing R. prolixus genome sequence project. Peptides were identified by de novo sequencing and comparisons to known neuropeptides from different organisms by database search. By these means, we were able to identify 42 novel neuropeptides from R. prolixus. The peptides were classified as extended FMRF‐amide‐related peptides, sulfakinins, myosuppressins, short neuropeptide F, long neuropeptide F, SIF‐amide‐related peptides, tachykinins, orcokinins, allatostatins, allatotropins, calcitonin‐like diuretic hormones, corazonin, and pyrokinin. Some of them were detected in multiple isoforms and/or truncated fragments. Interestingly, some of the R. prolixus peptides, as myosuppressin and sulfakinins, are unique in their characteristic C‐terminal domain among insect neuropeptides identified so far.     

The molecular architecture of QdtA,a sugar 3,4‐ketoisomerase from Thermoanaerobacterium thermosaccharolyticum

Unusual di- and trideoxysugars are often found on the O-antigens of Gram-negative bacteria, on the S-layers of Gram-positive bacteria, and on various natural products. One such sugar is 3-acetamido-3,6-dideoxy-d-glucose. A key step in its biosynthesis, catalyzed by a 3,4-ketoisomerase, is the conversion of thymidine diphosphate (dTDP)−4-keto-6-deoxyglucose to dTDP-3-keto-6-deoxyglucose. Here we report an X-ray analysis of a 3,4-ketoisomerase from Thermoanaerobacterium thermosaccharolyticum. For this investigation, the wild-type enzyme, referred to as QdtA, was crystallized in the presence of dTDP and its structure solved to 2.0-Å resolution. The dimeric enzyme adopts a three-dimensional architecture that is characteristic for proteins belonging to the cupin superfamily. In order to trap the dTDP-4-keto-6-deoxyglucose substrate into the active site, a mutant protein, H51N, was subsequently constructed, and the structure of this protein in complex with the dTDP–sugar ligand was solved to 1.9-Å resolution. Taken together, the structures suggest that His 51 serves as a catalytic base, that Tyr 37 likely functions as a catalytic acid, and that His 53 provides a proton shuttle between the C-3′ hydroxyl and the C-4′ keto group of the hexose. This study reports the first three-dimensional structure of a 3,4-ketoisomerase in complex with its dTDP–sugar substrate and thus sheds new molecular insight into this fascinating class of enzymes.     

The genetic architecture of vitiligo

Vitiligo is an autoimmune disease in which destruction of skin melanocytes results in patches of white skin and hair. Genome‐wide linkage studies and genome‐wide association studies in European ancestry cases identified over 50 vitiligo susceptibility loci, defining a model of melanocyte‐directed autoimmunity. Vitiligo heritability is exceedingly high, ~2/3 coming from common and ~1/3 from rare genomic variants; ~20% of vitiligo risk is environmental. Vitiligo genetic risk is polygenic, with greater additive risk in multiplex vitiligo families than simplex cases. Vitiligo age‐of‐onset is bimodal, also involving a major genetic component; a MHC enhancer haplotype confers extreme risk for vitiligo (OR 8.1) and early disease onset, increasing expression of HLA‐DQB1 mRNA and HLA‐DQ protein and thus perhaps facilitating presentation of triggering antigens. Vitiligo triggering also involves a major environmental component; dramatic delay in vitiligo age‐of‐onset, especially from 1973 to 2004, suggests that exposure or response to a key vitiligo environmental trigger diminished during this period. Together, these findings provide deep understanding of vitiligo pathogenesis and genetic architecture, suggesting that vitiligo represents a tractable model for investigating complex disease genetic architecture and predictive aspects of personalized medicine.     

The effect of antiapoptosis genes on clarification performance

Optimal bioreactor harvest time is typically determined based on maximizing product titer without compromising product quality. We suggest that ease of downstream purification should also be considered during harvest. In this view, we studied the effect of antiapoptosis genes on downstream performance. Our hypothesis was that more robust cells would exhibit less cell lysis and thus generate lower levels of cell debris and host‐cell contaminants. We focused on the clarification unit operation, measuring postclarification turbidity and host‐cell protein (HCP) concentration as a function of bioreactor harvest time/cell viability. In order to mimic primary clarification using disk‐stack centrifugation, a scale‐down model consisting of a rotating disk (to simulate shear in the inlet feed zone of the centrifuge) and a swinging‐bucket lab centrifuge was used. Our data suggest that in the absence of shear during primary clarification (typical of depth filters), a 20–50% reduction in HCP levels and 50–65% lower postcentrifugation turbidity was observed for cells with antiapoptosis genes compared to control cells. However, on exposing the cells to shear levels typical in a disk‐stack centrifuge, the reduction in HCP was 10–15% while no difference in postcentrifugation turbidity was observed. The maximum benefit of antiapoptosis genes is, therefore, realized using clarification options that involve low shear, <1 × 10⁶ W/m³ and minimal damage to the cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:100–107, 2014     

The role of pi-acidic and pi-basic chiral stationary phases in the high-performance liquid chromatographic enantioseparation of unusual beta-amino acids

The application of 3,5-dimethylphenyl-carbamoylated-beta-cyclodextrin (Cyclobond I 2000 DMP) and 2,6-dinitro-4-trifluoromethylphenyl-ether-beta-cyclodextrin-based (Cyclobond DNP) chiral stationary phases for the high-performance liquid chromatographic enantioseparation of unusual beta-amino acids is reported. The investigated amino acids were saturated or unsaturated alicyclic beta-3-homo-amino acids and bicyclic beta-amino acids. Prior to chromatographic analyses, all amino acids were transformed to N-3,5-dinitrobenzoyl- or N-3,5-dimethylbenzoyl form to ensure a pi-acidic or pi-basic function and to enhance the pi-acidic-pi-basic interactions between analytes and chiral selectors. Chromatographic results are given as retention, separation and resolution factors. The chromatographic conditions were varied to achieve optimal separation. The sequence of elution of the enantiomers was determined in some cases.     

The visualization of natural luminescence of living cotton hairs.

Super-weak luminescence of individual living cotton cells was investigated. This phenomenon was observed under an optical microscope using a replica-reprint technique in conjunction with living cotton cells. Gelatin solution was used for visualization of luminescence in a polymer film deposited on the cotton cells. No luminescence was generated in dead and very young plant cells (1 day after flowering) but maximal luminescence was detected from apical parts of cotton hairs in the early growth stage (4-6 days after flowering). Emitted luminescence had a cone-like form for different varieties of cotton plants. This indicates a focusing of radiation connected with morphological and structural features of the apical part of cotton cells at early stages of their evolution. The electromagnetic nature of cotton cell luminescence has been shown by experiments using photomultiplier (PEM) sensing of ultraviolet radiation. Insertion of a cotton seed-bud in darkness on a PEM window increased the dark current by 6-8%. Radiation flux from developing cotton hair is actually more intense, but through the PEM window only a small part of the radiation was detected.     

The in vivo synthesis of plant sesquiterpenes by Escherichia coli.

Three plant genes encoding (+)-delta-cadinene, 5-epi-aristolochene, and vetispiradiene cyclases were expressed in Escherichia coli to evaluate the potential of this bacterium to synthesize sesquiterpenes in vivo. Various growth temperatures, carbon sources, and host strains were examined to optimize terpene production. The highest levels of sesquiterpene production occurred when the enzymes were expressed in strain DH5alpha from the trc promoter (Ptrc) of the high-copy plasmidpTrc99A in M9 medium supplemented with 0.2% (v/v) glycerol at 30 degrees C for 5-epi-aristolochene and vetispiradiene and 37 degrees C for (+)-delta-cadinene. The highest concentrations of sesquiterpenes observed were 10.3 microg of (+)-delta-cadinene, 0.24 microg of 5-epi-aristolochene (measured as (+)-delta-cadinene equivalents), and 6.4 microg of vetispiradiene (measured as (+)-delta-cadinene equivalents) per liter of culture. These sesquiterpene production levels are >500-fold lower than carotenoid production, both of which are synthesized from endogenous trans-farnesyl diphosphate (FDP) in E. coli. Based on these results, we conclude that the limiting factor for sesquiterpene synthesis in E. coli is the poor expression of the cyclase enzyme and not supply of the FDP precursor.     

The crystal structure of rabbit phosphoglucose isomerase complexed with D-sorbitol-6-phosphate, an analog of the open chain form of D-glucose-6-phosphate

Phosphoglucose isomerase (PGI) catalyzes the isomerization of D-glucose-6-phosphate (G6P) and D-fructose-6-phosphate (F6P) in glycolysis and gluconeogenesis. Analysis of previously reported X-ray crystal structures of PGI without ligand, with the cyclic form of F6P, or with inhibitors that mimic the cis-enediol intermediate led to proposed mechanisms for the ring opening and isomerization steps in the multistep catalytic mechanism. To help complete our model of the overall mechanism, information is needed about the state of PGI between the ring opening and isomerization steps, in other words, a structure of the enzyme complexed with the open form of a substrate or an analog. Here, we report the crystal structure of rabbit PGI complexed with D-sorbitol-6-phosphate (S6P), an analog of the open chain form of G6P, at 2.0 A resolution. As was seen in the PGI/F6P structure, a helix containing amino acid residues 512-520 is found in the "out" position, which provides sufficient space in the active site for a substrate in its cyclic form and which is probably the location of that helix just after ring opening (or just before ring closure). However, the S6P ligand is in an extended conformation, as was seen previously with ligands that mimic the cis-enediol intermediate. The extended conformation enables the ligand to interact with Glu357, which transfers a proton during the isomerization step. The PGI/S6P structure represents the conformation of the enzyme and substrate between the ring opening (or ring closing) step and the isomerization step and helps to complete the model for PGI's catalytic mechanism.     

The Biological Applications of Two Aggregation‐Induced Emission Luminogens

Fluorescence imaging, as a commonly used scientific tool, is widely applied in various biomedical and material structures through visualization technology. Highly selective and sensitive luminescent biological probes, as well as those with good water solubility, are urgently needed for biomedical research. In contrast to the traditional aggregation‐caused quenching of fluorescence, in the unique phenomenon of aggregation‐induced emission (AIE), the individual luminogens have extremely weak or no emissivity because they each have free intramolecular motion; however, when they form aggregates, these components immediately “light up”. Since the discovery of “turn‐on” mechanism, researchers have been studying and applying AIE in a variety of fields to develop more sensitive, selective, and efficient strategies for the AIE dyes. There are numerous advantages to the use of AIE‐based methods, including low background interference, strong contrast, high performance in intracellular imaging, and the ability for long‐term monitoring in vivo. In this review, two typical examples of AIEgens, TPE‐Cy and TPE‐Ph‐In, are described, including their structure properties and applications. Recent progress in the biological applications is mainly focused on. Undoubtedly, in the near future, an increasing number of encouraging and practical ideas will promote the development of more AIEgens for broad use in biomedical applications.     

The CatSper channel: a polymodal chemosensor in human sperm

The sperm-specific CatSper channel controls the intracellular Ca(2+) concentration ([Ca(2+)](i)) and, thereby, the swimming behaviour of sperm. In humans, CatSper is directly activated by progesterone and prostaglandins-female factors that stimulate Ca(2+) influx. Other factors including neurotransmitters, chemokines, and odorants also affect sperm function by changing [Ca(2+)](i). Several ligands, notably odorants, have been proposed to control Ca(2+) entry and motility via G protein-coupled receptors (GPCRs) and cAMP-signalling pathways. Here, we show that odorants directly activate CatSper without involving GPCRs and cAMP. Moreover, membrane-permeable analogues of cyclic nucleotides that have been frequently used to study cAMP-mediated Ca(2+) signalling also activate CatSper directly via an extracellular site. Thus, CatSper or associated protein(s) harbour promiscuous binding sites that can host various ligands. These results contest current concepts of Ca(2+) signalling by GPCR and cAMP in mammalian sperm: ligands thought to activate metabotropic pathways, in fact, act via a common ionotropic mechanism. We propose that the CatSper channel complex serves as a polymodal sensor for multiple chemical cues that assist sperm during their voyage across the female genital tract.     

The O-polysaccharide of lipopolysaccharide isolated from Vibrio fluvialis O19 is identical to that of Vibrio bioserogroup 1875 variant

A structural analysis has been carried out on the O-polysaccharide of lipopolysaccharide (LPS) isolated from Vibrio fluvialis 181-86 (Kobe) serotype O19 (O19) which has the Inaba antigen factor C of O1 V. cholerae and factors D and E in common with Vibrio bioserogroup 1875. The O-polysaccharide of O19 was characterized as an alpha (1-->2)-linked homopolymer of N-3-hydroxypropionyl-D-perosamine (4-amino-4,6-dideoxy-D-mannopyranose), which was identical to that of Vibrio bioserogroup 1875 Variant. Passive hemolysis and passive hemolysis inhibition analysis performed using anti-factor D, E and anti-factor E antisera, demonstrated that the LPS from O19 harbored O-antigenic factors identical to those of the LPS from Vibrio bioserogroup 1875 Variant.     

The therapeutic effect of rosuvastatin on cardiac remodelling from hypertrophy to fibrosis during the end-stage hypertension in rats

End-stage hypertensive heart disease is an increasing cause of cardiac mortality. Therefore, the current study focused on the cardiac remodelling from hypertrophy to fibrosis in old-aged spontaneously hypertensive rats (SHRs), and explored the therapeutic effects of Rosuvastatin and its possible mechanism(s) of action. Spontaneously hypertensive rats at age 52 weeks were randomly divided into three groups, the first two to receive Rosuvastatin at a dose of 20 mg/kg/day and 40 mg/kg/day, respectively, and the third to receive placebo, which was to be compared with Wistar-Kyoto as controls. After 2-month treatment, SBP, heart to body weight ratio (HW/BW%) and echocardiographic features were evaluated, followed by haematoxylin and eosin and Masson trichrome staining in conjunction with qPCR of foetal gene expressions. Transferase-mediated dUTP nick-end labelling assay and immunofluorescent labelling for active caspase-3 were used to detect the apoptotic cardiomyocytes. Signaling pathways involved were examined by using western blot. Old-aged SHR developed end-stage hypertensive heart disease characterized by significant enhancement of HW/BW%, LVAWd and LVPWd, and decreased LVEF and LVFS, accompanied by cardiomyocytes enlargement and fibrosis along with activation of foetal gene programme. Cardiac apoptosis increased significantly during the transition process. Rosuvastatin reduced hypertrophy significantly via AT(1) Receptor-PKCβ2/α-ERK-c-fos pathway; protected myocardium against apoptosis via Akt-FOXO1, Bcl-2 family and survivin pathways and consequently suppressed the caspase-3 activity. The present study revealed that old-aged SHRs developed cardiac remodelling from hypertrophy to fibrosis via cardiac apoptosis during the end stage of hypertensive heart disease. These pathological changes might be the consequence of activation of AT(1) Receptor-PKCβ2/α-ERK-c-fos and AKT-FOXO1/Bcl-2/survivin/Caspase3 signaling. Rosuvastatin effectively attenuated the structural changes by reversing the signaling transductions involved.     

The innate immune system in the intestine

The innate immune system provides the first line of host defense against invading pathogens. Innate immune responses are initiated by germline-encoded PRR, which recognize specific structures expressed by microorganisms. TLR are a family of PRR which sense a wide range of microorganisms, including bacteria, fungi, protozoa and viruses. TLR are also expressed in the intestine and are critical for intestinal homeostasis. Recently, cytoplasmic PRR, such as NLR and RLR, have been shown to detect pathogens that have invaded the cytosol. One of the NLR, NOD2, is thought to be involved in the pathogenesis of Crohn's disease. This review focuses on the innate immune responses triggered by PRR in the intestine.     

The genome‐centric concept: resynthesis of evolutionary theory

Modern biology has been heavily influenced by the gene‐centric concept. Paradoxically, this very concept – on which bioresearch is based – is challenged by the success of gene‐based research in terms of explaining evolutionary theory. To overcome this major roadblock, it is essential to establish new theories, to not only solve the key puzzles presented by the gene‐centric concept, but also to provide a conceptual framework that allows the field to grow. This paper discusses a number of paradoxes and illustrates how they can be addressed by the genome‐centric concept in order to further resynthesize evolutionary theory. In particular, methodological breakthroughs that analyze genome evolution are discussed. The multiple interactions among different levels of a complex system provide the key to understanding the relationship between self‐organization and natural selection. Darwinian natural selection applies to the biological level due to its unique genetic and heterogeneous features, but does not simply or directly apply to either the lower non‐living level or higher intellectual society level. At the complex bio‐system level, the genome context (the entire package of genes and their genomic physical relationship or genomic topology), not the individual genes, defines the system and serves as the principle selection platform for evolution.