GSH inhibits trypsinization of the C-terminal half of human MRP1

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance to tumor cells. The accumulated evidence has proved that GSH interacts with MRP1 and stimulates drug transport. However, the mechanism of GSH-dependent drug transport by MRP1 remains unclear. In this study, we used limited tryptic digestion of MRP1 in isolated membrane vesicles, in the presence and absence of GSH, to investigate the influence of GSH on MRP1 conformation. We found that GSH inhibited the generation of an approximately 35-kDa C-terminal tryptic fragment (including a C-terminal His tag) termed C2 from MRP1. This effect of GSH was not because of direct inhibition of trypsin activity, and agosterol A enhanced the inhibitory effect of GSH. The main cleavage site in MRP1 for the generation of the C2 fragment by trypsin resided between TMD2 and NBD2 of MRP1. Limited tryptic digestion of membrane vesicles expressing various truncated and co-expressed MRP1 fragments in the presence and absence of GSH revealed that GSH inhibited the production of the C2 fragment only in the presence of the L(0) region of MRP1. Thus the L(0) region is required for the inhibition of trypsinization of the C-terminal half of MRP1 by GSH. These findings, together with previous reports, suggest that GSH induces a conformational change at a site within the MRP1 that is indispensable for the interaction of MRP1 with its substrates.     

Neuropathological similarities and differences between schizophrenia and bipolar disorder: a flow cytometric postmortem brain study

Recent studies suggest that schizophrenia (SCH) and bipolar disorder (BPD) may share a similar etiopathology. However, their precise neuropathological natures have rarely been characterized in a comprehensive and quantitative fashion. We have recently developed a rapid, quantitative cell-counting method for frozen unfixed postmortem brains using a flow cytometer. In the present study, we not only counted stained nuclei, but also measured their sizes in the gray matter of frontopolar cortices (FPCs) and inferior temporal cortices (ITCs) from patients with SCH or BPD, as well as in that from normal controls. In terms of NeuN(+) neuronal nuclei size, particularly in the reduced densities of small NeuN(+) nuclei, we found abnormal distributions present in the ITC gray matter of both patient groups. These same abnormalities were also found in the FPCs of SCH patients, whereas in the FPCs of BPD patients, a reduction in oligodendrocyte lineage (olig2(+)) cells was much more common. Surprisingly, in the SCH FPC, normal left-greater-than-right asymmetry in neural nuclei densities was almost completely reversed. In the BPD FPC, this asymmetry, though not obvious, differed significantly from that in the SCH FPC. These findings indicate that while similar neuropathological abnormalities are shared by patients with SCH or BPD, differences also exist, mainly in the FPC, which may at least partially explain the differences observed in many aspects in these disorders.     

PHOTOAUTOTROPHIC GROWTH OF A RECENTLY ISOLATED N2-FIXING MARINE NON-HETEROCYSTOUS FILAMENTOUS CYANOBACTERIUM, SYMPLOCA SP. (CYANOBACTERIA)

Photoautotrophic growth of a marine non-heterocystous filamentous cyanobacterium, Symploca sp. strain S84, was examined under nitrate-assimilating and N₂-fixing conditions. Under continuous light, photon flux density of 55 μmol photons·m⁻² ·s⁻¹ was at a saturating level for growth, and light did not inhibit the growth rate under N₂-fixing conditions even when the photon flux density was doubled (110 μmol photons·m⁻² ·s⁻¹). Doubling times of the N₂-fixing cultures under 55 and 110 μmol photons·m⁻² ·s⁻¹ were about 30 and 31 h, respectively. Under 110 μmol photons·m⁻² ·s⁻¹ during the light phase of an alternating 12:12-h light:dark (L:D) cycle, the doubling time of the N₂-fixing culture was also about 30 h. When grown diazotrophically under a 12:12-h L:D regime, C₂H₂ reduction activity was observed mainly during darkness. In continuous light, relatively large cyclic fluctuations in C₂H₂ reduction were observed during growth. The short-term (<4 h) effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; 5 μM) indicated that C₂H₂ reduction activity was not influenced by photosynthetic O₂ evolution. Long-term (24 h) effects of DCMU indicated that photosynthesis and C₂H₂ reduction activity occur simultaneously. These results indicate that strain S84 grows well under diazotrophic conditions when saturating light is supplied either continuously or under a 12:12-h L:D diel light regime.     

Production of a recombinant full-length prion protein in a soluble form without refolding or detergents

Recombinant prion protein has been produced in insoluble form and refolded following solubilization with denaturants. It is, however, preferable to use a soluble recombinant protein prepared without artificial solubilization. In this study, a soluble recombinant prion protein was produced in Escherichia coli cells by coexpression of neuregulin I-β1 and purified to high purity.     

Development of an RNA interference method in the cladoceran crustacean Daphnia magna

Daphnids are small crustaceans ubiquitous in fresh water; they have been a subject of study in ecology, evolution, and environmental sciences for decades. To understand data accumulated in daphnid biology at the molecular level, expressed sequence tags and a genome sequence have been determined. However, these discoveries lead to the problem of how to understand the functions of newly discovered genes. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a useful tool to achieve specific gene silencing in nontransformable species. Hence, we established a technique to inject exogenous materials into ovulated eggs and developed a dsRNA-based RNAi method for Daphnia magna. Eggs were collected just after ovulation and injected with dsRNA specific to the Distal-less (Dll) gene, which functions in appendage development in invertebrates and vertebrates. We found that the dsRNA successfully triggered the degradation of Dll mRNAs, which induced the truncation of the second antenna in a dose-dependent manner. This effect was sequence specific in that: (1) an unrelated dsRNA did not induce any morphological abnormalities and (2) two non-overlapping Dll dsRNAs generated the same phenotype. This is the first report of an RNAi technique in D. magna and, together with the emerging genome sequences, will be useful for advancing knowledge of the molecular biology of daphnids.     

Semaphorin 3A induces CaV2.3 channel-dependent conversion of axons to dendrites

Polarized neurites (axons and dendrites) form the functional circuitry of the nervous system. Secreted guidance cues often control the polarity of neuron migration and neurite outgrowth by regulating ion channels. Here, we show that secreted semaphorin 3A (Sema3A) induces the neurite identity of Xenopus spinal commissural interneurons (xSCINs) by activating Ca(V)2.3 channels (Ca(V)2.3). Sema3A treatment converted the identity of axons of cultured xSCINs to that of dendrites by recruiting functional Ca(V)2.3. Inhibition of Sema3A signalling prevented both the expression of Ca(V)2.3 and acquisition of the dendrite identity, and inhibition of Ca(V)2.3 function resulted in multiple axon-like neurites of xSCINs in the spinal cord. Furthermore, Sema3A-triggered cGMP production and PKG activity induced, respectively, the expression of functional Ca(V)2.3 and the dendrite identity. These results reveal a mechanism by which a guidance cue controls the identity of neurites during nervous system development.     

Subregional 6-[<Superscript>18</Superscript>F]fluoro-ʟ-<Emphasis Type="Italic">m</Emphasis>-tyrosine Uptake in the Striatum in Parkinson's Disease

Background  

In idiopathic Parkinson's disease (PD) the clinical features are heterogeneous and include different predominant symptoms. The aim of the present study was to determine the relationship between subregional aromatic l-amino acid decarboxylase (AADC) activity in the striatum and the cardinal motor symptoms of PD using high-resolution positron emission tomography (PET) with an AADC tracer, 6-[¹⁸F]fluoro-ʟ-m-tyrosine (FMT).     

Biosynthesis of coelenterazine in the deep-sea copepod, Metridia pacifica

Coelenterazine is an imidazopyrazinone compound (3,7-dihydroimidazopyrazin-3-one structure) that is widely distributed in marine organisms and used as a luciferin for various bioluminescence reactions. We have used electrospray ionization-ion trap-mass spectrometry to investigate whether the deep-sea luminous copepod Metridia pacifica is able to synthesize coelenterazine. By feeding experiments using deuterium labeled amino acids of l-tyrosine and l-phenylalanine, we have shown that coelenterazine can be synthesized from two molecules of l-tyrosine and one molecule of l-phenylalanine in M. pacifica. This is the first demonstration that coelenterazine is biosynthesized from free l-amino acids in a marine organism.     

Inhibition of α-synuclein fibril assembly by small molecules: Analysis using epitope-specific antibodies

The conversion of soluble peptides and proteins into amyloid fibrils and/or intermediate oligomers is believed to be the central event in the pathogenesis of most human neurodegenerative diseases. Existing treatments are at best symptomatic. Accordingly, small molecule inhibitors of amyloid fibril formation and their mechanisms are of great interest. Here we report that the conformational changes undergone by α -synuclein as it assembles into amyloid fibrils can be detected by epitope-specific antibodies. We show that the conformations of polyphenol-bound α-synuclein monomers and dimers differ from those of unbound monomers and resemble amyloid fibrils. This strongly suggests that small molecule inhibitors bind and stabilize intermediates of amyloid fibril formation, consistent with the view that inhibitor-bound molecular species are on-pathway intermediates.