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Background/Aims: Membrane-type matrix metalloproteinases (MT-MMPs) are expressed on the cell surface and hydrolyze extracellular matrix components and signaling molecules by which they influence cancer cell migration and metastasis. Two of the six known MT-MMPs are anchored to the plasma membrane via a GPI anchor, one of which is MT4-MMP. Only little is known about MT4-MMP expression, synthesis, regulation and degradation. Methods: We analyzed several human cancer cell lines as well as tissue homogenates using Western blotting and quantitative PCR for the expression of MT4-MMP. Organelles of SK-Mel-28 cells were separated using continuous Iodixanol gradients. Glycosylation of the SK-Mel-28 protein was studied via glucosidases and site directed mutagenesis of the MT4-MMP cDNA prior to transfection. Results: We found the MT4-MMP highly expressed in human melanoma cell lines as well as skin and melanoma tissue samples. Three forms of MT4-MMP with molecular masses of 45 kDa, 58 kDa and 69 kDa were detected. Further, we demonstrate that the 58 kDa form is the mature protein in the cell membrane, while the 69 kDa form is its precursor found in intracellular compartments. The 69 kDa forms are processed by furin cleavage in the Golgi apparatus. Moreover, we identified Asn318 as the single N-glycosylation site of MT4-MMP. Conclusion: We demonstrate the novel expression of MT4-MMP in melanocytic tissues and propose a precursor/product-relationship of the different forms of MT4-MMP in melanoma cells.

Interorganelle phospholipid transfer is critical for eukaryotic membrane biogenesis. In the yeast Saccharomyces cerevisiae, phosphatidylserine (PS) synthesized by PS synthase, Pss1, in the endoplasmic reticulum (ER) is decarboxylated to phosphatidylethanolamine (PE) by PS decarboxylase, Psd1, in the ER and mitochondria or by Psd2 in the endosome, Golgi, and/or vacuole, but the mechanism of interorganelle PS transport remains to be elucidated. Here we report that Sfh1, a member of Sec14 family proteins of S. cerevisiae, possesses the ability to enhance PE production by Psd2. Overexpression of SFH1 in the strain defective in Psd1 restored its growth on non-fermentable carbon sources and increased the intracellular and mitochondrial PE levels. Sfh1 was found to bind various phospholipids, including PS, in vivo. Bacterially expressed and purified Sfh1 was suggested to have the ability to transport fluorescently labeled PS between liposomes by fluorescence dequenching assay in vitro. Biochemical subcellular fractionation suggested that a fraction of Sfh1 localizes to the endosome, Golgi, and/or vacuole. We propose a model that Sfh1 promotes PE production by Psd2 by transferring phospholipids between the ER and endosome. [ABSTRACT FROM AUTHOR]

Congenital glaucoma (CG) is a severe and inherited childhood optical neuropathy that leads to irreversible visual loss and blindness in children. CG pathogenesis remains largely unexplained in most patients. Herein we have extended our previous studies to evaluate the role of FOXC2 and PITX2 variants in CG. Variants of the proximal promoter and transcribed sequence of these two genes were analyzed by Sanger sequencing in a cohort of 133 CG families. To investigate possible oligogenic inheritance involving FOXC2 or PITX2 and CYP1B1, we also analyzed FOXC2 and PITX2 variants in a group of 25 CG cases who were known to carry CYP1B1 glaucoma-associated genotypes. The functional effect of three identified variants was assessed by transactivation luciferase reporter assays, protein stability and subcellular localization analyses. We found eight probands (6.0%) who carried four rare FOXC2 variants in the heterozygous state. In addition, we found an elevated frequency (8%) of heterozygous and rare PITX2 variants in the group of CG cases who were known to carry CYP1B1 glaucoma-associated genotypes, and one of these PITX2 variants arose de novo. To the best of our knowledge, two of the identified variants (FOXC2: c.1183C>A, p.(H395N); and PITX2: c.535C>A, p.(P179T)) have not been previously identified. Examination of the genotype-phenotype correlation in this group suggests that the presence of the infrequent PITX2 variants increase the severity of the phenotype. Transactivation reporter analyses showed partial functional alteration of three identified amino acid substitutions (FOXC2: p.(C498R) and p.(H395N); PITX2: p.(P179T)). In summary, the increased frequency in PCG patients of rare FOXC2 and PITX2 variants with mild functional alterations, suggests they play a role as putative modifier factors in this disease further supporting that CG is not a simple monogenic disease and provides novel insights into the complex pathological mechanisms that underlie CG. [ABSTRACT FROM AUTHOR]

We recently showed that folic acid (FA) could decrease the proliferation rate of colorectal cancer cells in vitro and reduce the volume of COLO-205 tumor in vivo. Since cancer cell proliferation and migration are two major events during cancer development, we further examined whether FA could also affect the migration of colorectal cancer cells. Transwell invasion assays demonstrated that FA reduced the invasion ability of colorectal cancer cell lines, COLO-205, LoVo and HT-29. Using COLO-205 as a cell model, we further delineated the molecular mechanism underlying FA-inhibited colorectal cancer cell invasion. Western blot analyses showed that FA (10 μM) activated cSrc, ERK1/2, NFκB, and p27 at serine 10 (Ser10), and up-regulated p53, p27, and KIS protein. Subcellular fractionation illustrated that FA treatment increased cytosolic translocation of p27, formation of the p27-RhoA complex, and RhoA degradation. The FA-induced migration inhibition in COLO-205 was abolished by blockade of the cSrc or ERK1/2 activity, knockdown of p27 or KIS using the siRNA technique, or over-expression of a constitutive active RhoA cDNA. Our results suggest that FA up-regulated p27 through increasing the cSrc/ERK1/2/NFκB/p53-mediated pathway. In the nucleus, FA up-regulated KIS, which in turn increased p27 phosphorylation at serine 10 (Ser10), subsequently resulting in cytosolic translocation of p27 and forming the p27-RhoA complex, thereby causing RhoA degradation, and eventually inhibited COLO-205 cell migration. Together with our previous findings suggest that FA reduced colorectal cancer development through inhibiting colorectal cancer cell proliferation and migration. [ABSTRACT FROM AUTHOR]

Rare earth elements are new and emerging contaminants in freshwater systems. Greater duckweed ( Spirodela polyrhiza L.) is a common aquatic plant widely used in phytotoxicity tests for xenobiotic substances. In this study, the cerium (Ce) accumulation potential, the distribution of Ce in bio-molecules, and ensuing biochemical responses were investigated in greater duckweed fronds when they were exposed to Ce (0, 10, 20, 40, and 60 μM). There was a concentration dependent increase in Ce accumulation, which reached a maximum of 67 mg g −1 of dry weight (DW) at 60 μM Ce after 14 d. The Ce concentrations in bio-macromolecules followed the order: cellulose and pectin > proteins > polysaccharides > lipids. In response to Ce exposure, significant chlorosis; declines in growth, photosynthetic pigment and protein contents; and cell death were noted at the highest Ce concentration. Photosystem II inhibition, degradation of the reaction center protein D1, and damage to chloroplast ultrastructure were observed in Ce treated S. polyrhiza fronds, as revealed by chlorophyll a fluorescence transients, immunoblotting, and transmission electron microscopy (TEM). O 2 .− accumulation and malondialdehyde (MDA) content in the treated fronds increased in a concentration dependent manner, which indicated that oxidative stress and unsaturated fatty acids (C18:3) were specifically affected by Ce exposure. These results suggest Ce exerts its toxic effects on photosynthesis, with a primary effect on PS II, through oxidative stress. [ABSTRACT FROM AUTHOR]

Aberrant nucleocytoplasmic localization of proteins has been implicated in many neurodegenerative diseases. Evidence suggests that cytoplasmic mislocalization of nuclear proteins such as transactive response DNA-binding protein 43 (TDP-43) and fused in sarcoma (FUS) may be associated with neurotoxicity in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. This study investigated the changes in nucleocytoplasmic distributions of the proteome and transcriptome in an in vitro model of ALS. After subcellular fractionation of motor neuron-like cell lines expressing wild-type or G93A mutant hSOD1, quantitative mass spectrometry and next-generation RNA sequencing (RNA-seq) were performed for the nuclear and cytoplasmic compartments. A subset of the results was validated via immunoblotting. A total of 1,925 proteins were identified in either the nuclear or cytoplasmic fractions, and 32% of these proteins were quantified in both fractions. The nucleocytoplasmic distribution of 37 proteins was significantly changed in mutant cells with nuclear and cytoplasmic shifts in 13 and 24 proteins, respectively (p<0.05). The proteins shifted towards the nucleus were enriched regarding pathways of RNA transport and processing (Dhx9, Fmr1, Srsf3, Srsf6, Tra2b), whereas protein folding (Cct5, Cct7, Cct8), aminoacyl-tRNA biosynthesis (Farsb, Nars, Txnrd1), synaptic vesicle cycle (Cltc, Nsf), Wnt signalling (Cltc, Plcb3, Plec, Psmd3, Ruvbl1) and Hippo signalling (Camk2d, Plcb3, Ruvbl1) pathways were over-represented in the proteins shifted to the cytoplasm. A weak correlation between the changes in protein and mRNA levels was found only in the nucleus, where mRNA was relatively abundant in mutant cells. This study provides a comprehensive dataset of the nucleocytoplasmic distribution of the proteome and transcriptome in an in vitro model of ALS. An integrated analysis of the nucleocytoplasmic distribution of the proteome and transcriptome demonstrated multiple candidate pathways including RNA processing/transport and protein synthesis and folding that may be relevant to the pathomechanism of ALS. [ABSTRACT FROM AUTHOR]

Fe65 is an adaptor protein involved in both processing and signaling of the Alzheimer-associated amyloid-β precursor protein, APP. Here, the subcellular localization was further investigated using TAP-tagged Fe65 constructs expressed in human neuroblastoma cells. Our results indicate that PTB2 rather than the WW domain is important for the nuclear localization of Fe65. Electrophoretic mobility shift of Fe65 caused by phosphorylation was not detected in the nuclear fraction, suggesting that phosphorylation could restrict nuclear localization of Fe65. Furthermore, both ADAM10 and γ-secretase inhibitors decreased nuclear Fe65 in a similar way indicating an important role also of α-secretase in regulating nuclear translocation. [ABSTRACT FROM AUTHOR]

The neuronal spine is a small, actin-rich dendritic or somatic protrusion that serves as the postsynaptic compartment of the excitatory synapse. The morphology of the spine reflects the activity of the synapse and is regulated by the dynamics of the actin cytoskeleton inside, which is controlled by actin binding proteins such as non-muscle myosin. Previously, we demonstrated that the subcellular localization and function of myosin IIb are regulated by its binding partner, filamin-A interacting protein (FILIP). However, how the subcellular distribution of myosin IIb is controlled by FILIP is not yet known. The objective of this study was to identify potential binding partners of FILIP that contribute to its regulation of non-muscle myosin IIb. Pull-down assays detected a 70-kDa protein that was identified by mass spectrometry to be the chaperone protein Hsc70. The binding of Hsc70 to FILIP was controlled by the adenosine triphosphatase (ATPase) activity of Hsc70. Further, FILIP bound to Hsc70 via a domain that was not required for binding non-muscle myosin IIb. Inhibition of ATPase activity of Hsc70 impaired the effect of FILIP on the subcellular distribution of non-muscle myosin IIb. Further, in primary cultured neurons, an inhibitor of Hsc70 impeded the morphological change in spines induced by FILIP. Collectively, these results demonstrate that Hsc70 interacts with FILIP to mediate its effects on non-muscle myosin IIb and to regulate spine morphology. [ABSTRACT FROM AUTHOR]

Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization. [ABSTRACT FROM AUTHOR]

The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene cluster, walRKJ. [ABSTRACT FROM AUTHOR]

During ER-associated degradation (ERAD), misfolded polytopic membrane proteins are ubiquitinated and retrotranslocated to the cytosol for proteasomal degradation. However, our understanding as to how polytopic membrane proteins are extracted from the ER to the cytosol remains largely unclear. To better define the localization and physical properties of ubiquitinated polytopic membrane substrates in vivo, we performed subcellular fractionation analysis of Ste6*, a twelve transmembrane protein that is ubiquitinated primarily by Doa10 E3 ligase in yeast. Consistent with previous in vitro studies, ubiquitinated Ste6* was extracted from P20 (20,000 g pellet) fraction to S20 (20,000 g supernatant) fraction in a Cdc48/p97-dependent manner. Similarly, Ubx2p, which recruits Cdc48/p97 to the ER, facilitated the extraction of Ste6*. By contrast, lipid droplet formation, which was suggested to be dispensable for the degradation of Hrd1-substrates in yeast, was not required for the degradation of Ste6*. Intriguingly, we found that ubiquitinated Ste6* in the S20 fraction could be enriched by further centrifugation at 100,000 g. Although it is currently uncertain whether ubiquitinated Ste6* in P100 fraction is completely free from any lipids, membrane flotation analysis suggested the existence of two distinct populations of ubiquitinated Ste6* with different states of membrane association. Together, these results imply that ubiquitinated Ste6* may be sequestered into a putative quality control sub-structure by Cdc48/p97. Fractionation assays developed in the present study provide a means to further dissect the ill-defined post-ubiquitination step during ERAD of polytopic membrane substrates. [ABSTRACT FROM AUTHOR]

We determined the role of N-linked glycosylation of apolipoprotein B (apoB) in the assembly and secretion of lipoproteins using transfected rat hepatoma McA-RH7777 cells expressing human apoB-17, apoB-37, and apoB-50, three apoB variants with different ability to recruit neutral lipids. Substituting Asn residue with Gln at the single glycosylation site within apoB-17 (N158) decreased its secretion efficiency to a level equivalent to that of wild-type apoB-17 treated with tunicamycin, but had little effect on its synthesis or intracellular distribution. When selective N-to-Q substitution was introduced at one or more of the five N-linked glycosylation sites within apoB-37 (N158, N956, N1341, N1350, and N1496), secretion efficiency of apoB-37 from transiently transfected cells was variably affected. When all five N-linked glycosylation sites were mutated within apoB-37, the secretion efficiency and association with lipoproteins were decreased by >50% as compared with wild-type apoB-37. Similarly, mutant apoB-50 with all of its N-linked glycosylation sites mutagenized showed decreased secretion efficiency and decreased lipoprotein association in both d < 1.02 and d > 1.02 g/ml fractions. The inability of mutant apoB-37 and apoB-50 to associate with very low-density lipoproteins was attributable to impaired assembly and was not due to the limitation of lipid availability. The decreased secretion of mutant apoB-17 and apoB-37 was not accompanied by accumulation within the cells, suggesting that the proportion of mutant apoB not secreted was rapidly degraded. However unlike apoB-17 or apoB-37, accumulation of mutant apoB-50 was observed within the endoplasmic reticulum and Golgi compartments.These data imply that the N-glycans at the amino terminus of apoB play an important role in the assembly and secretion of lipoproteins containing the carboxyl terminally truncated apoB.

The Ascomycete Cordyceps militaris, an entomopathogenic fungus, is one of the most important traditional Chinese medicines. Studies related to its pharmacological properties suggest that this mushroom can exert interesting biological activities. Aqueous (CW and HW) and alkaline (K5) extracts containing polysaccharides were prepared from this mushroom, and a β-D-glucan was purified. This polymer was analysed by GC-MS and NMR spectrometry, showing a linear chain composed of β-D-Glcp (1→3)-linked. The six main signals in the 13C-NMR spectrum were assigned by comparison to reported data. The aqueous (CW, HW) extracts stimulated the expression of IL-1β, TNF-α, and COX-2 by THP-1 macrophages, while the alkaline (K5) extract did not show any effect. However, when the extracts were added to the cells in the presence of LPS, K5 showed the highest inhibition of the pro-inflammatory genes expression. This inhibitory effect was also observed for the purified β-(1→3)-D-glucan, that seems to be the most potent anti-inflammatory compound present in the polysaccharide extracts of C. militaris. In vivo, β-(1→3)-D-glucan also inhibited significantly the inflammatory phase of formalin-induced nociceptive response, and, in addition, it reduced the migration of total leukocytes but not the neutrophils induced by LPS. In conclusion, this study clearly demonstrates the anti-inflammatory effect of β-(1→3)-D-glucan. [ABSTRACT FROM AUTHOR]

Planarians are an important model organism for regeneration and stem cell research. A complete understanding of stem cell and regeneration dynamics in these animals requires time-lapse imaging in vivo, which has been difficult to achieve due to a lack of tissue-specific markers and the strong negative phototaxis of planarians.Wehave developed the Planarian Immobilization Chip (PIC) for rapid, stable immobilization of planarians for in vivo imaging without injury or biochemical alteration. The chip is easy and inexpensive to fabricate, and worms can be mounted for and removed after imaging within minutes. We show that the PIC enables significantly higher-stability immobilization than can be achieved with standard techniques, allowing for imaging of planarians at sub-cellular resolution in vivo using brightfield and fluorescence microscopy. We validate the performance of the PIC by performing time-lapse imaging of planarian wound closure and sequential imaging over days of head regeneration. We further show that the device can be used to immobilize Hydra, another photophobic regenerative model organism. The simple fabrication, low cost, ease of use, and enhanced specimen stability of the PIC should enable its broad application to in vivo studies of stem cell and regeneration dynamics in planarians and Hydra. [ABSTRACT FROM AUTHOR]

G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system. [ABSTRACT FROM AUTHOR]

Fragile X mental Retardation Protein (FMRP) is a well-known regulator of local translation of its mRNA targets in neurons. However, despite its ubiquitous expression, the role of FMRP remains ill-defined in other cell types. In this study we investigated the subcellular distribution of FMRP and its protein complexes in HeLa cells using confocal imaging as well as detergent-free fractionation and size exclusion protocols. We found FMRP localized exclusively to solid compartments, including cytosolic heavy and light membranes, mitochondria, nuclear membrane and nucleoli. Interestingly, FMRP was associated with nucleolin in both a high molecular weight ribosomal and translation-associated complex (≥6 MDa) in the cytosol, and a low molecular weight complex (∼200 kDa) in the nucleoli. Consistently, we identified two functional nucleolar localization signals (NoLSs) in FMRP that are responsible for a strong nucleolar colocalization of the C-terminus of FMRP with nucleolin, and a direct interaction of the N-terminus of FMRP with the arginine-glycine-glycine (RGG) domain of nucleolin. Taken together, we propose a novel mechanism by which a transient nucleolar localization of FMRP underlies a strong nucleocytoplasmic translocation, most likely in a complex with nucleolin and possibly ribosomes, in order to regulate translation of its target mRNAs. [ABSTRACT FROM AUTHOR]

Abstract: During infection, hepatitis C virus (HCV) NS4B protein remodels host membranes to form HCV replication complexes (RC) which appear as foci under fluorescence microscopy (FM). To understand the role of Rab proteins in forming NS4B foci, cells expressing the HCV replicon were examined biochemically and via FM. First, we show that an isolated NS4B-bound subcellular fraction is competent for HCV RNA synthesis. Further, this fraction is differentially enriched in Rab1, 2, 5, 6 and 7. However, when examined via FM, NS4B foci appear to be selectively associated with Rab5 and Rab7 proteins. Additionally, dominant negative (DN) Rab6 expression impairs Rab5 recruitment into NS4B foci. Further, silencing of Rab5 or Rab7 resulted in a significant decrease in HCV genome replication. Finally, expression of DN Rab5 or Rab7 led to a reticular NS4B subcellular distribution, suggesting that endocytic proteins Rab5 and Rab7, but not Rab11, may facilitate NS4B foci formation. [Copyright &y& Elsevier]

The article focuses on the study which investigates the spatiotemporal dynamics of subcellular calcium (Ca) cycling during rapid pacing. One whole rat heart was made as subject to be examined using real-time confocal Ca fluorescence. The use of a set of differential equations describing Ca cycling within sarcomere was also resorted. Results reveal that dynamical factors as CaT and cycle length (CL) alternans can amplify spatial heterogeneities in the cell to yield phase-mismatched CaT alternans.

To understand bioaccumulation kinetics of metals within biota inhabiting industrially contaminated soils, toxicokinetic dynamics and subcellular fractionation were carried out with the terrestrial snail Helix aspersa in a long-term (six-month) laboratory experiment. Accumulation and elimination kinetics were determined for Cd, Pb, and Zn in both viscera and foot of snails and were described accurately by one-compartment models. The subcellular fractions were obtained by sequential centrifugations and were analyzed by isolating metal-rich granules, tissue fragments, and cytosolic fractions. Different fractions showed metal-specific binding capacities that might be useful in identifying the biological significance of accumulated metal levels in snails. Cadmium was retrieved mainly from the cytosolic fraction, where it was stored in the long term and not excreted, thus explaining the linear accumulation patterns. Most of the accumulated Pb was found in the granular fraction, and snails appeared able to excrete these concretions, leading to achievement of a steady state in internal Pb body burdens. Significant levels of Pb, however, were retrieved at the end of the depuration phase and retained in the cell debris fraction. Zinc showed affinities for both cytosolic and granular fractions, leading to intermediate uptake and excretion patterns. The dynamics of the different sequestration forms at the subcellular level support the observed kinetics of metal body burdens and, in association with the determination of uptake fluxes, allow precise assessment of metal accumulation in snails. [ABSTRACT FROM AUTHOR]

We investigated the variations of acute toxicity of mercury (Hg) in Daphnia magna under different temperatures, population origins, body sizes, and Hg pre-exposures. We measured Hg concentrations in the water and in the surviving daphnids, and used the subcellular fractionation approach to determine Hg in the metal-sensitive fraction (MSF) to predict Hg toxicity. The 24-h median lethal concentrations and 24-h lethal body burden were 12–55 μg L-1 and 10–26 mg kg-1 wet wt, respectively. High Hg tolerance accompanied by reduced Hg uptake occurred in the daphnids under extreme conditions (low temperature and high pre-exposure to Hg). Correlating Hg levels in different compartments and daphnid survival resulted in the following order of sequence: aqueous Hg > whole body Hg > Hg in the MSF. However, the threshold lethal concentration of Hg (concentration causing 1% mortality) based on the concentration of Hg in the MSF was the best indicator of Hg toxicity. Therefore, the subcellular fractionation approach is less useful in explaining acute toxicity than is sub-lethal Hg toxicity. The number of Hg binding sites in the animals varied under different conditions but the affinity of the transporter to Hg generally decreased as the animals' tolerance increased. Mercury tolerance under different conditions could be enhanced by reducing the Hg uptake, enhancing the intrinsic tolerance, and/or increasing the detoxification activity. [ABSTRACT FROM AUTHOR]

This review considers the proteins of the WASP (Wiskott-Aldrich syndrome protein) family and their role in the regulation of actin-based motility. It contains detailed classification of the WASP family proteins and data on their subcellular localization. Impairments of expression of the WASP family proteins cause certain cell pathologies. The review also deals with domain organization of these proteins and proteins interacting with various domains of the WASP proteins. Special attention is given to analysis of the role of the WASP family proteins in initiating directed actin assembly in the leading edge of the migrating cell and on the surface of some bacteria. Putative pathways of regulation of WASP proteins by various protein ligands and their links with cell signaling systems are considered. [ABSTRACT FROM AUTHOR]

Recently, the purification to homogeneity was reported of a novel broad-specificity α-mannosidase from rat liver microsomal membranes [P. Bonay and R. C. Hughes (1991) Eur. J. Biochem. 197,229– 238]. The enzyme catalyzed the ordered removal of α1 ↵2-, α1 ↵ 3- and α1 ↵6-linked mannose residues from MannGlcNAc oligosaccharide substrates where n = 4–9 . We now show by subcellular fractionation and immunocytochemistry that the novel mannosidase is present in the endoplasmic reticulum, Golgi apparatus and endosomes. Enzyme activity is enriched in a heavy Golgi membrane fraction and to lesser extent in an intermediate density Golgi membrane fraction containing GlcNAc transferase I activity and in a ‘late’ endosomal fraction. Low levels of enzyme activity were detectable in endoplasmic reticulum membranes and in ‘early’ endosomes but not in receptor-enriched and ligand-free endosomes. Assays of enzymic activity using Golgi membrane fractions in the absence and presence of Triton X-100 showed that the active site of the enzyme faces the lumen of the membrane vesicles. Antibodies directed against the purified mannosidase showed no immunological cross-reaction to known endoplasmic reticulum and Golgi mannosidases. Conversely, the purified mannosidase was not recognized by antibodies directed against endoplasmic reticulum mannosidase nor Golgi mannosidase IA. [ABSTRACT FROM AUTHOR]

Cyanophora paradoxa, a unicellular flagellate, contains cyanelles which are supposed to be cyanobacterial origin. DNA was isolated from subcellular fractions and separated according to density components in CsCl density gradients. The main DNA component, comprising more than 90% of the total DNA, has a buoyant density of 1.724g · cm&sup-3;. Several subsfractions m the range from 1.718 g · cm&sup-3; to 1,735 g · cm&sup-3; are contained in this component. This DNA of high complexity was considered to be host nuclear DNA. The DNA from the endosymbiotic cyanelles, which were isolated, treated with DNase, and purified by sucrose density gradient centrifugation exhibited a buoyant density of 1.692 g · cm&sup-3; in one strain and 1.695 g · cm&sup-3; in a second strain. Both cyanelle DNAs (cyDNA) have a complexity of approximately 126 × 10³ base pairs and comprise about 5% of the total cellular DNA content. Two additional DNA components of low complexity were isolated from crude cyanelle pellets obtained without DNase treatment. The larger of these, approximately 48 × 10³ base pairs in size, had a density of approximately 1.688 g · cm&sup-3;. The second component, about 15 × 10³ base pairs in size, banded in the density range between 1.710 g · cm&sup-3; and 1.720 g · cm&sup-3;. The latter is associated with nuclear DNA, The 48 × 10³-base-pair component was located in the cytosol and could be obtained after CsCl/ethidium bromide density gradient centrifugation at the position of covalently closed circular DNA, Both these components amounted to approximately 0.5–1% of total DNA. A further DNA component with a complexity of more than 150 × 10³ base pairs, enriched in fractions where mitochondria are expected, was not characterized further. The density was intermediate between cyDNA and nuclear DNA (1.710–1.720 g ·cm&sup-3;) and it amounted to 1–2% of the total DNA. Our results indicate that the DNA from cyanelles, believed to be endosymbiotic cyanobacteria, is not more complex than higher plant chloroplast DNAs. [ABSTRACT FROM AUTHOR]

The subcellular distribution of aryl-hydrocarbon hydroxylase, NADPH-cytochrome c reductase, NADH :cytochrome c reductase, and NADH :cytochrome b5 reductase activities in mouse liver was studied using the biochemical membrane markers microsomal glucose-6-phosphatase, mitochondrial cytochrome c oxidase, and plasma membrane 5′-nucleotidase. The rate of appearance of activity of 3-methylcholanthrene-induced aryl-hydrocarbon hydroxylase in the microsomes of C57BL/6N mice is more than twice as rapid as that in the nuclear envelope. The nuclear fraction contains less than 1% of the total cellular activities of the hydroxylase and all three reductases. All detectable basal activity of aryl-hydrocarbon hydroxylase in the nuclear fraction of control C57BL/6N and DBA′2N and 3-methylcholanthrene-treated DBA/2N mice and all detectable activities of NADPH :cytochrome c, NADH :cytochrome c, and NADH :catochrome b5 reductase in the nuclear fraction of control and 3-methylcholanthrene-treated C57BL/6N and DBA/2N mice can be completely accounted for by the degree of microsomal fragment contamination (as assessed by the microsomal marker glucose-6-phosphatase). These data raise doubts about certain previous reports of ‘nuclear’ enzyme activities in which microsomal contamination was not taken into account. However, there is more induced activity of aryl-hydrocarbon hydroxylase in the nuclear fraction of 3-methylcholanthrene-treated C57BL/6N mice than can be accounted for by the degree of microsomal membrane contribution. The routine Ah locus is known to regulate the induction by certain polycyclic aromatic chemicals of numerous drug-metabolizing enzyme activities such as aryl-hydrocarbon hydroxylase associated with cytochrome P1-450. The expression of 3-methylcholanthrene-inducible hydroxylase in nuclear membranes, like that in microsomal membranes, thus appears to be controlled by the Ah regulatory gene. [ABSTRACT FROM AUTHOR]

The acetyl-CoA carboxylase activity of all Crambe abyssinica subcellular fractions is very low as indicated by poor conversion of acetate and acetyl-CoA and a requirement for malonyl-CoA as the substrate for fatty acid synthesis. Erucic acid (C22:1) biosynthesis is most active in the oil body fraction, quite active in the denser nuclear/plastid and mitochondrial fractions and absent from the microsomal and cytoplasmic fractions. Erucic acid is formed by direct elongation of oleic acid, not by synthesis from acetate de novo. Desaturation of added stearyl-CoA was not demonstrated. Incorporation of 14C-labelled awl groups from fatty acyl-CoA thioesters into phospholipids occurred in all subcellular fractions except the supernatant from centrifugation at 105 000 × g. Triglyceride synthesis from 14C-labelled stearyl-CoA occurred in all subcellular fractions except the supernatant fraction from centrifugation at 105 000 × g. Incubations of halved seeds with labelled precursors, followed by subcellular fractionation, supported these findings except that the intact seeds had functional acid-CoA ligase and acetyl-CoA carboxylase. Incubation of halved seeds in the presence of trichloroacetic acid (2.5 mM and 12.5 mM) resulted in substantial inhibition of the biosynthesis of erucic acid but not of other fatty acids. Higher levels of trichloroacetic acid resulted in general inhibition of fatty acid and acyl lipid synthesis. [ABSTRACT FROM AUTHOR]

Ficoll-gradient fractionation of a metabolic lysate from yeast spheroplasts was used to prepare vacuole, mitochondria and cytosol fractions. Tryptophan synthase activity was exclusively found in the soluble fraction. Proteinases A, B and C as well as the tryptophan-synthase-inactivating activity were mainly found in the vacuole fraction. The heat-stable activities inhibiting proteinase and tryptophan-synthase-inactivating enzyme appeared in the soluble fraction. The subceltular separation of tryptophan synthase and the tryptophan-synthase-inactivating activities explains why in crude extracts from stationary yeast cells, high tryptophan synthase activities are observed in spite of high tryptophan-synthase-inactivating activity. The proteinases of a freshly prepared vacuole fraction from yeast spheroptasts are unable to inactivate externally added tryptophan synthase unless the organelles are disrupted by sonication or osmotic shock. [ABSTRACT FROM AUTHOR]

Lipids and acid hydrolases have been characterized in a subcellular fraction, enriched with lamellar granules (LG), derived from fetal rat epidermis. This fraction contains 23% glycosyl ceramides and ceramides, 15% free sterols, and 34% phospholipids. The lipid/protein ratio is 2.0. The sterols and sphingolipids were present in proportions similar to those previously reported in stratum corneum. These findings provide direct biochemical evidence for the widely accepted hypothesis that stratum corneum lipids are derived from exocytosis of lamellar granules into the intercellular space. The LG fraction was enriched in certain acid hydrolases including glucosidase, acid phosphatase, phospholipases A, and sphingomyelinase; other acid hydrolases, i.e., amino-glycosidases, glactosidase and aryl sulfatase (pH 5.5), and steroid sulfatase were not preferentially localized in this fraction. By modulation of phospholipids, glyco-lipids, and proteins in the membrane regions of stratum corneum, the acid hydrolases of LG may play a role relevant to the function and desquamation of stratum corneum. [ABSTRACT FROM AUTHOR]