Overlapping signal sequences control nuclear localization and endoplasmic reticulum retention of GRP58

Glucose-regulated GRP58 has shown clinical applications to endoplasmic reticulum (ER) stress and cancer. GRP58 is localized in the cytosol, endoplasmic reticulum (ER) and nucleus. Twenty-four amino acids at the N-terminal hydrophobic region are known to target GRP58 to ER for synthesis at the ER membrane and translocation into the ER lumen. In addition, GRP58 contains putative nuclear localization (494KPKKKKK500) and ER retention (502QEDL505) signals. However, the role of these signals in nuclear import and ER retention of GRP58 remains unknown. Present studies investigated the signals that control nuclear localization and ER retention of GRP58. Deletion/mutation of nuclear localization signal (NLS) abrogated nuclear import of GRP58. NLS attached to EGFP localized EGFP in the nucleus. However, deletion/mutation of putative ER retention signal alone did not alter ER retention of GRP58. Interestingly, a combined deletion/mutation of NLS and ER retention signals blocked the GRP58 retention in the ER. These results concluded that overlapping NLS and ER retention signal sequences regulate nuclear localization and ER retention of GRP58.     

Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during nuclear fusion in Saccharomyces cerevisiae

During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.     

PIG-B,a membrane protein of the endoplasmic reticulum with a large lumenal domain,is involved in transferring the third mannose of the GPI anchor.

Many eukaryotic cell surface proteins are bound to the membrane via the glycosylphosphatidylinositol (GPI) anchor that is covalently linked to their carboxy-terminus. The GPI anchor precursor is synthesized in the endoplasmic reticulum (ER) and post-translationally linked to protein. We cloned a human gene termed PIG-B (phosphatidylinositol glycan of complementation class B) that is involved in transferring the third mannose. PIG-B encodes a 554 amino acid, ER transmembrane protein with an amino-terminal portion of approximately 60 amino acids on the cytoplasmic side and a large carboxy-terminal portion of 470 amino acids within the ER lumen. A mutant PIG-B lacking the cytoplasmic portion remains active, indicating that the functional site of PIG-B resides on the lumenal side of the ER membrane. The PIG-B gene was localized to chromosome 15 at q21-q22. This autosomal location would explain why PIG-B is not involved in the defective GPI anchor synthesis in paroxysmal nocturnal hemoglobinuria, which is always caused by a somatic mutation of the X-linked PIG-A gene.     

The endoplasmic reticulum (ER) translocon can differentiate between hydrophobic sequences allowing signals for glycosylphosphatidylinositol anchor addition to be fully translocated into the ER lumen

The signal sequence within polypeptide chains that designates whether a protein is to be anchored to the membrane by a glycosylphosphatidylinositol (GPI) anchor is characterized by a carboxyl-terminal hydrophobic domain preceded by a short hydrophilic spacer linked to the GPI anchor attachment (omega) site. The hydrophobic domain within the GPI anchor signal sequence is very similar to a transmembrane domain within a stop transfer sequence. To investigate whether the GPI anchor signal sequence is translocated across or integrated into the endoplasmic reticulum membrane we studied the translocation, GPI anchor addition, and glycosylation of different variants of a model GPI-anchored protein. Our results unequivocally demonstrated that the hydrophobic domain within a GPI signal cannot act as a transmembrane domain and is fully translocated even when followed by an authentic charged cytosolic tail sequence. However, a single amino acid change within the hydrophobic domain of the GPI-signal converts it into a transmembrane domain that is fully integrated into the endoplasmic reticulum membrane. These results demonstrated that the translocation machinery can recognize and differentiate subtle changes in hydrophobic sequence allowing either full translocation or membrane integration.     

A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein

When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome c1, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c1, but expressing the hybrid NLS-cytochrome c1 proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional- lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c1 to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPL1) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein. npl1-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npl1 mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in npl1/sec63 cells at the nonpermissive temperature. Thus, NPL1/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npl1 may indirectly alter assembly of proteins into the nucleus.     

Myosin‐1C uses a novel phosphoinositide‐dependent pathway for nuclear localization

Accurate control of macromolecule transport between nucleus and cytoplasm underlines several essential biological processes, including gene expression. According to the canonical model, nuclear import of soluble proteins is based on nuclear localization signals and transport factors. We challenge this view by showing that nuclear localization of the actin‐dependent motor protein Myosin‐1C (Myo1C) resembles the diffusion–retention mechanism utilized by inner nuclear membrane proteins. We show that Myo1C constantly shuttles in and out of the nucleus and that its nuclear localization does not require soluble factors, but is dependent on phosphoinositide binding. Nuclear import of Myo1C is preceded by its interaction with the endoplasmic reticulum, and phosphoinositide binding is specifically required for nuclear import, but not nuclear retention, of Myo1C. Our results therefore demonstrate, for the first time, that membrane association and binding to nuclear partners is sufficient to drive nuclear localization of also soluble proteins, opening new perspectives to evolution of cellular protein sorting mechanisms.     

The eta isoform of protein kinase C is localized on rough endoplasmic reticulum.

The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992).     

Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase

Translocation of proteins across membranes of the endoplasmic reticulum, mitochondrion, and chloroplast has been shown to be mediated by targeting signals present in the transported proteins. To test whether the transport of proteins into peroxisomes is also mediated by a peptide targeting signal, we have studied the firefly luciferase gene that encodes a protein transported to peroxisomes in both insect and mammalian cells. We have identified two regions of luciferase which are necessary for transport of this protein into peroxisomes. We demonstrate that one of these, region II, represents a peroxisomal targeting signal because it is both necessary and sufficient for directing cytosolic proteins to peroxisomes. The signal is no more than twelve amino acids long and is located at the extreme carboxy-terminus of luciferase. The location of the targeting signal for translocation across the peroxisomal membrane therefore differs from the predominantly amino-terminal location of signals responsible for transport across the membranes of the endoplasmic reticulum, chloroplast, or mitochondrion.     

Sec62 Protein Mediates Membrane Insertion and Orientation of Moderately Hydrophobic Signal Anchor Proteins in the Endoplasmic Reticulum (ER)

Nascent chains are known to be targeted to the endoplasmic reticulum membrane either by a signal recognition particle (SRP)-dependent co-translational or by an SRP-independent post-translational translocation route depending on signal sequences. Using a set of model and cellular proteins carrying an N-terminal signal anchor sequence of controlled hydrophobicity and yeast mutant strains defective in SRP or Sec62 function, the hydrophobicity-dependent targeting efficiency and targeting pathway preference were systematically evaluated. Our results suggest that an SRP-dependent co-translational and an SRP-independent post-translational translocation are not mutually exclusive for signal anchor proteins and that moderately hydrophobic ones require both SRP and Sec62 for proper targeting and translocation to the endoplasmic reticulum. Further, defect in Sec62 selectively reduced signal sequences inserted in an N_in-C_out (type II) membrane topology, implying an undiscovered role of Sec62 in regulating the orientation of the signal sequence in an early stage of translocation.     

Oligomerization Is Crucial for the Stability and Function of Heme Oxygenase-1 in the Endoplasmic Reticulum

Heme oxygenase-1 (HO-1), a stress-inducible enzyme anchored in the endoplasmic reticulum (ER) by a single transmembrane segment (TMS) located at the C terminus, interacts with NADPH cytochrome P450 reductase and biliverdin reductase to catalyze heme degradation to biliverdin and its metabolite, bilirubin. Previous studies suggested that HO-1 functions as a monomer. Using chemical cross-linking, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments, here we showed that HO-1 forms dimers/oligomers in the ER. However, oligomerization was not observed with a truncated HO-1 lacking the C-terminal TMS (amino acids 266–285), which exhibited cytosolic and nuclear localization, indicating that the TMS is essential for the self-assembly of HO-1 in the ER. To identify the interface involved in the TMS-TMS interaction, residue Trp-270, predicted by molecular modeling as a potential interfacial residue of TMS α-helices, was mutated, and the effects on protein subcellular localization and activity assessed. The results showed that the W270A mutant was present exclusively in the ER and formed oligomers with similar activity to those of the wild type HO-1. Interestingly, the W270N mutant was localized not only in the ER, but also in the cytosol and nucleus, suggesting it is susceptible to proteolytic cleavage. Moreover, the microsomal HO activity of the W270N mutant was significantly lower than that of the wild type. The W270N mutation appears to interfere with the oligomeric state, as revealed by a lower FRET efficiency. Collectively, these data suggest that oligomerization, driven by TMS-TMS interactions, is crucial for the stabilization and function of HO-1 in the ER.Heme oxygenase (HO)³ catalyzes the NADPH cytochrome P450 reductase-dependent oxidative degradation of cellular heme to biliverdin, carbon monoxide (CO), and free iron (1, 2). Biliverdin is subsequently converted to bilirubin by biliverdin reductase in the cytosol. Two HO isoforms have been identified in mammalian systems. HO-1 is a 288 amino acid protein and is expressed at high amounts in a variety of pathological conditions associated with cellular stress. There is compelling evidence that HO-1 induction represents an important cytoprotective defense mechanism against oxidative insults by virtue of the anti-oxidant properties of the bilirubin and the anti-inflammatory effect of the CO produced (2). HO-1 is anchored in the endoplasmic reticulum (ER) through a single transmembrane segment (TMS) located at the C terminus, while the rest of the molecule is cytoplasmic (3). HO-1 is sensitive to proteolytic cleavage (4), and it was recently shown that HO-1 can be proteolytically cleaved from the ER and translocated to the nucleus under certain stress conditions (5). Although the catalytic site in the cytoplasmic domain remains intact, the activity of soluble HO-1 is drastically reduced (5), indicating that ER localization is important for its full enzymatic function.Self-assembly to form dimers and higher oligomers is a common phenomenon in many membrane proteins (6, 7). Numerous studies have revealed that interactions between TMSs play an important role in the structure and function of many membrane proteins. Examples include receptors, enzymes, neurotransmitter transporters, and ion channels, in which oligomerization is crucial for their proper cellular localization and function (8). HO-1 does not contain any cysteine residues and has therefore been assumed to function as a monomer (1). To determine whether HO-1 forms oligomers in native membranes, in the present study, we performed chemical cross-linking, co-immunoprecipitation, and FRET analysis using fluorescent protein tags fused to the N terminus of HO-1. The results showed that HO-1 formed dimers/oligomers in the ER and that the TMS provided the interface for the protein-protein interactions. Interference with the TMS-TMS interaction resulted in destabilization of HO-1 and a reduction in enzymatic function.     

The nuclear reticulum in placental cells ofLilium regale is a part of the endomembrane system

Summary Placental cells line the ovarian transmitting tract inLilium regale and produce exudates for secretion. Sections through the highly lobed nuclei of these cells reveal the presence of membrane profiles which form vesicles with varying dimensions in cross section. Computer reconstruction of the nucleus reveals that the vesicle profiles form a complex reticulum of tubular cisternae, which spans the whole nucleus, enclosing a maze of continuous lumen space. Connections between the vesicles and the inner nuclear envelope are visible at various points along the nuclear envelope. This complex network of tubules which constitutes the reticulum arises from the inner nuclear membrane. The nuclear reticulum dramatically increases the inner-envelope surface area, comprising 82% of the total membrane perimeter of inner nuclear envelope and nuclear reticulum. The inner nuclear envelope invaginates into the nucleus forming the nuclear reticulum and the outer nuclear envelope evaginates into the endoplasmic reticulum (ER), indicating that there is a continuity between the lumens of the nuclear reticulum and the ER. The nuclear reticulum is labelled with zinc iodide-osmium tetroxide, a staining pattern identical to that seen in the ER. Positive reaction to the zinc iodide-osmium tetroxide indicates that the nuclear reticulum is a site for Ca²⁺ deposition. The nuclear reticulum forms an extension of the endomembrane system which reaches deep into the nucleoplasm. The lumenal continuity of this system means that there is a channel for communication from the cytoplasm into the nucleoplasm, and that this channel sequesters calcium.Abbreviations ER endoplasmic reticulum - TEM transmission electron microscope - ZIO zinc iodide-osmium tetroxide     

Intracellular processing and transport of NH2-terminally truncated forms of a hemagglutinin-neuraminidase type II glycoprotein

Six amino-terminal deletion mutants of the NH2-terminally anchored (type II orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza virus type 3 were expressed in tissue culture by recombinant SV-40 viruses. The mutations consisted of progressive deletions of the cytoplasmic domain and, in some cases, of the hydrophobic signal/anchor. Three activities were dissociated for the signal/anchor: membrane insertion, translocation, and anchoring/transport. HN protein lacking the entire cytoplasmic tail was inserted efficiently into the membrane of the endoplasmic reticulum but was translocated inefficiently into the lumen. However, the small amounts that were successfully translocated appeared to be processed subsequently in a manner indistinguishable from that of parental HN. Thus, the cytoplasmic domain was not required for maturation of this type II glycoprotein. Progressive deletions into the membrane anchor restored efficient translocation, indicating that the NH2-terminal 44 amino acids were fully dispensable for membrane insertion and translocation and that a 10-amino acid hydrophobic signal sequence was sufficient for both activities. These latter HN molecules appeared to be folded authentically as assayed by hemagglutination activity, reactivity with a conformation-specific antiserum, correct formation of intramolecular disulfide bonds, and homooligomerization. However, most (85-90%) of these molecules accumulated in the ER. This showed that folding and oligomerization into a biologically active form, which presumably represents a virion spike, occurs essentially to completion within that compartment but is not sufficient for efficient transport through the exocytotic pathway. Protein transport also appeared to depend on the structure of the membrane anchor. These latter mutants were not stably integrated in the membrane, and the small proportion (10-15%) that was processed through the exocytotic pathway was secreted. The maturation steps and some of the effects of mutations described here for a type II glycoprotein resemble previous observations for prototypic type I glycoproteins and are indicative of close similarities in these processes for proteins of both membrane orientations.     

Estradiol dependent anchoring of the goat uterine estrogen receptor activation factor (E-RAF) at the endoplasmic reticulum by a 55 kDa anchor protein (ap55)

The primary intracellular site of localization of the estrogen receptor activation factor (E-RAF) is shown here to be the endoplasmic reticulum where the protein remains anchored through an estrogen dependent mechanism. The retention of E-RAF by the endoplasmic reticulum is facilitated by two proteins: (1) a 55 kDa anchor protein (ap55) which is an integral membrane protein of the endoplasmic reticulum. ap55 is a high affinity estrogen binding protein. A conformational change induced by estrogen binding is thought to favor the anchoring process. (2) The anchoring of E-RAF by ap55 is mediated by yet another protein. This is the 66 kDa transport protein (tp66) which recognizes ap55 on the one hand and E-RAF on the other. The presence of estradiol that saturates the hormone binding sites on ap55 appears to favor the anchoring of tp66-E-RAF complex to ap55. This interaction appears to be weakened by levels of estradiol below 7 nM concentration leading to the dissociation of the tp66-E-RAF complex from ap55. The tp66-E-RAF complex moves towards the nucleus.     

Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.     

Docosahexaenoic acid inhibition of inflammation is partially via cross-talk between Nrf2/heme oxygenase 1 and IKK/NF-κB pathways

We examined the underlying mechanisms involved in n-3 docosahexaenoic acid (DHA) inhibition of inflammation in EA.hy926 cells. The present results demonstrated that pretreatment with DHA (50 and 100 μM) inhibited tumor necrosis factor-alpha (TNF-α)-induced intercellular adhesion molecule 1 (ICAM-1) protein, mRNA expression and promoter activity. In addition, TNF-α-stimulated inhibitory kappa B (IκB) kinase (IKK) phosphorylation, IκB phosphorylation and degradation, p65 nuclear translocation, and nuclear factor-κB (NF-κB) and DNA binding activity were attenuated by pretreatment with DHA. DHA triggered early-stage and transient reactive oxygen species (ROS) generation and significantly increased the protein expression of heme oxygenase 1 (HO-1), induced nuclear factor erythroid 2-related factor 2 (Nrf2) translocation to the nucleus and up-regulated antioxidant response element (ARE)-luciferase reporter activity. Moreover, DHA inhibited Nrf2 ubiquitination and proteasome activity. DHA activated Akt, p38 and ERK1/2 phosphorylation, and specific inhibitors of respective pathways attenuated DHA-induced Nrf2 nuclear translocation and HO-1 expression. Transfection with HO-1 siRNA knocked down HO-1 expression and partially reversed the DHA-mediated inhibition of TNF-α-induced p65 nuclear translocation and ICAM-1 expression. Importantly, we show for the first time that HO-1 plays a down-regulatory role in NF-κB nuclear translocation, and inhibition of Nrf2 ubiquitination and proteasome activity are involved in increased cellular Nrf2 level by DHA. In this study, we show that HO-1 plays a down-regulatory role in NF-κB nuclear translocation and that the protective effect of DHA against inflammation is partially via up-regulation of Nrf2-mediated HO-1 expression and inhibition of IKK/NF-κB signaling pathway.     

The role of topogenic sequences in the movement of proteins through membranes.

Recent advances have led to considerable convergence in ideas of the way topogenic sequences act to translocate proteins across various intracellular membranes (Table 2). Whereas co-translational translocation and processing were previously considered the norm at the endoplasmic reticulum membrane, several instances of post-translational translocation into endoplasmic reticulum microsomes in vitro have now been described. However, it must be noted that post-translational translocation in vitro is much less efficient than when endoplasmic reticulum membranes are present during translation, and it is possible that in the intact cell translocation occurs during translation. Movement of proteins into chloroplasts and mitochondria occurs after translation. When translocation is post-translational, proteins may perhaps traverse the membrane as folded domains, and the conformational effects of topogenic sequences on these domains may be as envisaged in Wickner's 'membrane-trigger hypothesis'. Both signal and transit sequences possess amphipathic structures which are capable of interacting with phospholipid bilayers, and these interactions may disturb the bilayer sufficiently to allow entry of the following domains of protein. There is increasing evidence that GTP is required to bind ribosomes and their associated nascent chains to the endoplasmic reticulum membrane. Precisely how the cell's energy is applied to achieve translocation is not clear, but one possibility at the endoplasmic reticulum is that a GTP-hydrolysing transducing mechanism may exist to couple signal sequence receptor binding to movement of the nascent chain across the membrane. Electrochemical gradients are required for protein movement to the mitochondrial inner membrane and across the bacterial inner membrane. Cytoplasmic factors such as SRP, the secA gene product or a 40 kDa protein (for mitochondrial precursors) may act by binding to topogenic sequences and preventing precursor proteins as they are translated from folding into forms which cannot be translocated. Specificity in the cell may be achieved both by targetting interactions between these cytoplasmic factors and their receptors located in target membranes, and also by specific binding of the topogenic sequences to specific proteins integrated into the target membranes. Possible candidates for the latter are the protein of microsomal membranes that reacts with a photoreactive signal peptide to give a 45 kDa complex (Fig. 1), the secY gene product of the bacterial inner membrane, and receptors on the outer membranes of chloroplasts and mitochondria. Whether these aid translocation as well as recognition is not clear.(ABSTRACT TRUNCATED AT 400 WORDS)