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In this paper we report that stimulation of mAChRs in PC12D cells activates Ca2+ channels that are regulated independently of intracellular Ca2+ stores. In nominally Ca2+-free medium, exposure of PC12D cells to carbachol stimulates a robust influx of Ba2+, a Ca2+ substitute. This influx is blocked by atropine, but not by inhibitors of the nicotinic acetylcholine receptor or L-, N-, or T-type voltage-regulated Ca2+ channels. By contrast, depletion of intracellular Ca2+ stores with thapsigargin only weakly stimulates Ba2+ influx. Unlike store-operated Ca2+ channels (SOCCs), which close only after intracellular Ca2+ stores refill, channels mediating carbachol-stimulated Ba2+ influx rapidly close following the inactivation of mAChRs with atropine. Ba2+ influx is inhibited by extracellular Ca2+, by the Ca2+ channel blocker SKF-96365, and by activation of protein kinase C (PKC). Exogenous expression of antisense RNA encoding the rat canonical-transient receptor potential Ca2+ channel subtype 6 (TRPC6) or the N-terminal domain of TRPC6 blocks carbachol-stimulated Ba2+ influx in PC12D cells. Expression of TRPC6 antisense RNA or the TRPC6 N-terminal domain also blocks Ba2+ influx stimulated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a diacylglycerol analog previously shown to activate exogenously expressed TRPC6 channels. These data show that mAChRs in PC12D cells activate endogenous Ca2+ channels that are regulated independently of Ca2+ stores and require the expression of TRPC6. [ABSTRACT FROM PUBLISHER]

This paper describes a method for imaging the endogenous release of glutamate from cerebral neurons. This method is based on the reactions of glutamate oxidase and peroxidase, and on the detection of hydrogen peroxide by a fluorescent substrate of peroxidase. Glutamate has been sensitively measured in vitro in the range of 20 nM to 1 µM. We used two types of Ca2+ channel inhibitors, MK-801 and ω-Conotoxin GVIA, which act to suppress Ca2+ transport at postsynaptic and presynaptic neurons, respectively. MK-801 did not inhibit the increase in glutamate release after KCl stimulation, while there was no increase in glutamate release after KCl stimulation when ω-Conotoxin GVIA was used, probably due to the inhibition of voltage-activated Ca2+ channels in the presynapse. Glutamate release and Ca2+ flow in the synaptic regions were imaged using a laser confocal fluorescence microscope. KCl-evoked glutamate release was localized around cell bodies linked to axon terminals. This procedure allows imaging that can be sensitively detected by the fluorometric enzymatic assay of endogenous glutamate release in synapses. [ABSTRACT FROM PUBLISHER]

Protein–protein interactions are essential biologic processes that occur at inter- and intracellular levels. To gain insight into the various complex cellular functions of these interactions, it is necessary to assess them under physiologic conditions. Recent advances in various proteomic technologies allow to investigate protein–protein interaction networks in living cells. The combination of proximity-dependent labelling and chemical cross-linking will greatly enhance our understanding of multi-protein complexes that are difficult to prepare, such as organelle-bound membrane proteins. In this review, we describe our current understanding of mass spectrometry-based proteomics mapping methods for elucidating organelle-bound membrane protein complexes in living cells, with a focus on protein–protein interactions in mitochondrial subcellular compartments. [ABSTRACT FROM AUTHOR]

As part of the 'Central Dogma' of molecular biology, the function of proteins and nucleic acids within a cell is determined by their primary sequence. Recent work, however, has shown that within living cells the role of many proteins and RNA molecules can be influenced by the physical state in which the molecule is found. Within living cells, both protein and RNA molecules are observed to condense into non-membrane-bound yet distinct structures such as liquid droplets, hydrogels and insoluble aggregates. These unique intracellular organizations, collectively termed biomolecular condensates, have been found to be vital in both normal and pathological conditions. Here, we review the latest studies that have developed molecular tools attempting to recreate artificial biomolecular condensates in living cells. We will describe their design principles, implementation and unique characteristics, along with limitations. We will also introduce how these tools can be used to probe and perturb normal and pathological cell functions, which will then be complemented with discussions of remaining areas for technological advance under this exciting theme. [ABSTRACT FROM AUTHOR]

Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 106 Sf9 cells in 2 ml of culture medium. [ABSTRACT FROM PUBLISHER]

As a part of the study to reveal the biological significance of de-N-glycosylation in plants, we analysed the structural features of free N-glycans (FNGs) accumulated inside cells and secreted to the extracellular space using a rice cell culture system. The structural analysis of FNGs obtained from the intracellular fraction revealed that the high-mannose type N-glycans with one GlcNAc residue (GN1-type) occurred at a concentration of ∼10 nmol/g, while the truncated complex type N-glycans with a N, N′-diacetylchitobiosyl unit (GN2-type) occurred at a concentration of ∼1 nmol/g. This result suggested that two kinds of glycoenzymes, cytosolic endo-β-N-acetylglucosaminidase (ENGase) and intracellular acidic peptide:N-glycanse (PNGase), are involved in the production of FNGs in rice cell as well as in other plant cells. On the other hand, in the culture medium, Lewis a epitope-containing complex and high-mannose type FNGs with the N, N′-diacetylchitobiosyl unit were found, suggesting extracellular acidic PNGase to be involved in the release of N-glycans from folded/processed glycoproteins in extracellular space. Furthermore, in the culture medium, we found unusual GN1-FNGs that have a biantennary complex type structure harbouring the Lewis a epitope, suggesting cytosolic ENGase and golgi N-glycan-processing enzymes to be involved in the production of these plant complex type FNGs. [ABSTRACT FROM PUBLISHER]

Activation of muscarinic acetylcholine receptors (mAChRs) causes the rapid release of Ca2+ from intracellular stores and a sustained influx of external Ca2+ in PC12D cells, a subline of the widely studied cell line PC12. Release of Ca2+ from intracellular stores and a sustained influx of Ca2+ are also observed following exposure to thapsigargin, a sesquiterpene lactone that depletes intracellular Ca2+ pools by irreversibly inhibiting the Ca2+ pump of the endoplasmic reticulum. In this study, we show that carbachol and thapsigargin empty the same intracellular Ca2+ stores, and that these stores are a subset of intracellular stores depleted by the Ca2+ ionophore ionomycin. Intracellular Ca2+ stores remain depleted during continuous stimulation of mAChR with carbachol in medium containing 2 mM extracellular Ca2+, but rapidly refill following inhibition of mAChRs with atropine. Addition of atropine to carbachol-stimulated cells causes intracellular Ca2+ levels to return to baseline levels in two steps: a rapid decrease that correlates with the reuptake of Ca2+ into internal stores and a delayed decrease that correlates with the inhibition of a Mn2+-permeable Ca2+ channel. Several lines of evidence suggest that carbachol and thapsigargin stimulate Ca2+ influx by a common mechanism: (i) pretreatment with thapsigargin occludes atropine-mediated inhibition of Ca2+ influx, (ii) carbachol and thapsigargin applied individually or together are equally efficient at stimulating the influx of Mn2+, and (iii) identical rates of Ca2+ influx are observed when Ca2+ is added to cells pretreated with carbachol, thapsigargin, or both agents in the absence of extracellular Ca2+. Taken together, these data suggest that the sustained influx of extracellular Ca2+ observed following activation of mAChRs in PC12D cells is mediated primarily by activation of a Mn2+-permeable, Ca2+ store–operated Ca2+ channel. [ABSTRACT FROM PUBLISHER]

Pepstatin A is well known to be an inhibitor of aspartic proteinases such as pepsin, cathepsins D and E. Except for its role as a proteinase inhibitor, however, the pharmacological action of pepstatin A upon cells remain unclear. In this study, we found that pepstatin A suppressed receptor activator of NF-κB ligand (RANKL)–induced osteoclast differentiation. Pepstatin A suppressed the formation of multinuclear osteoclasts dose-dependently. This inhibition of the formation only affected osteoclast cells, i.e., not osteoblast-like cells. Furthermore, pepstatin A also suppressed differentiation from pre-osteoclast cells to mononuclear osteoclast cells dose-dependently. This inhibition seems to be independent of the activities of proteinases such as cathepsin D, because the formation of osteoclasts was not suppressed with the concentration that inhibited the activity of cathepsin D. Cell signaling analysis indicated that the phosphorylation of ERK was inhibited in pepstatin A-treated cells, while the phosphorylation of IκB and Akt showed almost no change. Furthermore, pepstatin A decreased the expression of nuclear factor of activated T cells c1 (NFATc1). These results suggest that pepstatin A suppresses the differentiation of osteoclasts through the blockade of ERK signaling and the inhibition of NFATc1 expression. [ABSTRACT FROM PUBLISHER]

Stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) is activated by many types of cellular stresses and extracellular signals. Recent studies, including the analysis with knockout mice, have led to progress towards understanding the physiological roles of SAPK/JNK activation in embryonic development in addition to immune responses. SAPK/JNK activation plays essential roles in organogenesis during mouse development by regulating cell survival, apoptosis, and proliferation. Two SAPK/JNK activators, SEK1 and MKK7, are required for fetal liver formation and full activation of SAPK/JNK, which responds to various stimuli in an all-or-none manner. This article focuses on physiological roles of SAPK/JNK activation in fetal liver formation and in apoptosis regulation. [ABSTRACT FROM PUBLISHER]

Synaptotagmin IV (Syt IV) was originally described as an immediate early gene product induced by forskolin or membrane depolarization in PC12 cells; however, nothing is known about the subcellular localization and transport of the newly translated Syt IV protein in PC12 cells. In this study, we investigated the transport mechanism of Syt IV protein induced by forskolin and found that forskolin treatment dramatically increases the Syt IV protein level (approximately 10-fold, to a level comparable to that of Syt IX) and promotes the transport of Syt IV protein from the Golgi to the cell periphery by a microtubule-dependent motor(s). The expression levels and subcellular localizations of two major Syt isoforms (I and IX) in PC12 cells, on the other hand, were unaffected by such treatment. Immunoelectron microscopic analysis showed that some Syt IV signals are clearly associated with dense-core vesicles in forskolin-treated PC12 cells, although the majority of the Syt IV molecules at the cell periphery were present on clear vesicular structures other than dense-core vesicles. An N-terminal antibody-uptake experiment indicated that Syt IV–containing vesicles in forskolin-treated PC12 cells undergo Ca2+-dependent exocytosis, because uptake of the anti-Syt IV-N antibody from the culture medium was slightly, but significantly, increased after forskolin treatment. Our results indicate that forskolin (or the increased cAMP level) is important for the transport of the Syt IV protein from the Golgi to the cell periphery, but not sufficient for the sorting of all Syt IV molecules to mature dense-core vesicles. [ABSTRACT FROM PUBLISHER]

We previously noted that bovine apolipoprotein A-II (apoA-II) had a bactericidal effect causing morphological changes in the cytoplasm. To determine whether and how apoA-II and apoA-I, which have acidic isoelectric points (pIs), enter cells, we determined the rates of uptake of FITC-labeled proteins by fibroblast cells and found that they entered cells more easily at low pH than at neutral pH under conditions where endocytosis was inhibited. The enhanced uptake of proteins at low pH was also observed for other proteins examined regardless of the molecular weight (Mr) or pI in a time-dependent manner, although the efficiency of uptake varied among the proteins. Furthermore, a pH gradient was shown to be the main driving force for the translocation. As cells were viable above pH 4 for 2 h at 4°C and internalized ß-galactosidase was active under these conditions, we suggest that this procedure is applicable to the injection of proteins into cells without the use of an apparatus such as a microinjector. [ABSTRACT FROM PUBLISHER]

In mammalian cells, phosphatidylserine (PtdSer) is synthesized through the action of the endoplasmic reticulum enzymes, PtdSer synthase 1 and 2, and the decarboxylation of PtdSer accounts for the majority of phosphatidylethanolamine (PtdEtn) synthesis. PtdSer decarboxylation for PtdEtn formation occurs in the mitochondria. In addition, the transport of PtdSer from the endoplasmic reticulum to the mitochondria is probably a rate limiting step for PtdEtn synthesis through the decarboxylation pathway. Therefore, the regulation of PtdSer synthesis and its intracellular transport appear to be essential events for the maintenance of normal cellular PtdSer and PtdEtn levels. Here we describe the current understanding of the regulation of PtdSer biosynthesis and the transport of PtdSer from the ER to the mitochondria in mammalian cells. [ABSTRACT FROM PUBLISHER]

EC cells are characterized by their potent capacity to differentiate into several cell types, such as mesoderm-like cells, endoderm-like cells, or ectoderm-like cells. By subtracting the mRNAs expressed by one EC cell clone, F9 cells, with the mRNAs expressed by another EC cell clone, P19 cells, we identified six novel genes that are expressed selectively by F9 cells. One of these genes (Nucling) encodes a polypeptide of 1411 amino acids containing an ankyrin repeat, aspartyl protease motif, a leucine zipper motif, and two t-SNARE coiled-coil domains. Northern blot analyses revealed the Nucling mRNA to be detected predominantly in heart, liver, kidney and testis, but not in brain or spleen. Immunostaining analyses revealed a unique feature of Nucling that the transiently expressed protein forms aggregates exclusively around nuclear membranes. Moreover, the expression level of the Nucling gene transcript increases progressively during the early developmental stages in mice, and specifically at cardiomuscular differentiation in vitro and in vivo. These results suggest that Nucling may play some role in the gene regulation of cell differentiation during embryonal development. [ABSTRACT FROM PUBLISHER]

Macrophoma commelinae isolated from spots on leaves of Commelina communis has the ability to transform 5-acetyl-4-methoxy-6-methyl-2-pyrone (1) to 4-acetyl-3-methoxy-5-methylbenzoic acid (macrophomic acid, 2). This biotransformation includes the condensation of the 2-pyrone ring with a C3-unit precursor to form a substituted benzoic acid. We optimized conditions for induction of enzyme activity in M. commelinae, identified oxalacetate as a C3-unit precursor with cell extract, and purified the novel enzyme, macrophomate synthase. Oxalacetate inhibited the enzyme activity at a concentration higher than 5 mM, and magnesium chloride stimulated the enzyme activity. Kinetic analyses gave km of 1.7 mM for 1 at 5 mM oxalacetate, Km of 1.2 mM for oxalacetate at 5 mM 1, and kcat of 0.46 s−1 per subunit. Pyruvate was a weak substrate, with Km of 35.2 mM and kcat of 0.027 s−1 at 5 mM 1. We cloned and sequenced a cDNA encoding the macrophomate synthase. The cDNA of 1,225 bp contained an open reading frame that encoded a polypeptide of 339 amino acid residues and 36,244 Da, the sequence of which showed no significant similarity with known proteins in a homology search with BLAST programs. Transformed E. coli cells carrying the cDNA encoding the mature protein of macrophomate synthase overproduced macrophomate synthase under the control of the T7 phage promoter induced by IPTG. The purified enzyme showed the same values of Km and optimum pH as the native macrophomate synthase. [ABSTRACT FROM AUTHOR]

Fructose-1,6-bisphosphatase (FBPase) is a key gluconeogenic enzyme. The data herein show that both the enzyme activity and mRNA level of the human FBPase gene are enhanced by 9-cis retinoic acid (9cRA) and all-trans retinoic acid (atRA) as well as by 1,25-dihydroxyvitamin D3 (VD3) in human promyelocytic HL60 cells and normal monocytes in peripheral blood, which were used as an alternative source to liver for the DNA diagnosis of FBPase deficiency. To understand the molecular mechanism of this enhancing action, the 2.4 kb 5′-regulatory region of the human FBPase gene was isolated and sequenced. Using luciferase reporter gene assays, a 0.5 kb FBPase basal promoter fragment was found to confer induction by VD3, 9cRA, and atRA that was mediated by the vitamin D3 receptor (VDR), retinoid X receptor (RXR), and retinoic acid receptor (RAR). Within this region, a direct repeat sequence, 5′-TAACCTttcTGAACT-3′ (−340 to −326), which functions as a common response element for VD3, 9cRA, and atRA, was identified. The results of electrophoretic mobility shift assays indicated that VDR-RXR and RAR-RXR heterodimers bind this response element. Collectively, these observations indicate that VD3 and RA are important modulators of the expression of the human FBPase gene in monocytic cells. [ABSTRACT FROM AUTHOR]

Tom34 is a newly-found component of the mitochondrial protein import machinery in mammalian cells with no apparent counterpart in fungi. RNA blot and immunoblot analyses showed that the expression of Tom34 varies among tissues and differs from that of the core translocase component Tom20. In contrast to a previous report [Nuttal, S.D. et al. (1997) DNA Cell Biol 16, 1067–1074], the present study using a newly-prepared anti-Tom34 antibody with a high titer showed that Tom34 is present largely in the cytosolic fraction and partly in the mitochondrial and membrane fractions after fractionation of tissues and cells, and that the membrane-associated form is largely extractable with 0.1 M sodium carbonate. The in vitro import of preproteins into isolated rat mitochondria was strongly inhibited by ΔTom34 which lacks the NH2-terminal hydrophobic region of human Tom34 (hTom34). Import was also strongly inhibited by anti-hTom34. In pulse-chase experiments using COS-7 cells, pre-ornithine transcarbamylase (pOTC) was rapidly processed to the mature form. Coexpression of hTom34 resulted in a stimulation of pOTC processing, whereas the coexpression of hTom34 antisense RNA caused inhibition. The results confirm that Tom34 plays a role in mitochondrial protein import in mammals, and suggest it to be an ancillary component of the translocation machinery in mammalian cells. [ABSTRACT FROM PUBLISHER]

The human ER-60 protease cDNA was expressed in Escherichia coli BL21 (DE3) cells using the pET-20b(+) T7 promoter. The recombinant ER-60 protease was obtained in a water-soluble form and purified through four sequential chromatographies. The ER-60 protease contains two CGHC motifs. When an alanine residue was substituted for the N-terminal cysteine residue in both motifs, the protease activity was not lost. However, when the C-terminal cysteine residue in both motifs was replaced by a serine residue, the cysteine protease activity, which was inhibited by p-chloromercuribenzoic acid (pCMB) but not by diisopropyl fluorophosphate (DFP), changed to serine protease activity, which was inhibited by DFP but not by pCMB. These results indicate that the C-terminal cysteine residue(s) of the CGHC motifs may constitute the active site(s) of ER-60 protease. The ER-60 protease has a C-terminal QEDL sequence, which was proved to serve as an ER-retention signal by deletion of the QEDL sequence. However, because QEDL could not serve as the ER-retention signal for protein disulfide isomerase or ERp72, it is suggested that amino acid residue(s) of ER-60 protease, other than the QEDL sequence itself, is complimentarily responsible for the ER retention of this protein. [ABSTRACT FROM AUTHOR]

An aggregation substance of gramicidin S synthetases was found and purified by DEAE-cellulose chromatography and CM-chromatography from cell debris of Bacillus brevis Nagano. It specifically aggregated and inactivated gramicidin S synthetases 1 (GS1) and 2 (GS2). On the basis of amino acid composition analysis, reversed-phase HPLC, FAB mass spectrometry, amino acid sequence analysis, and antibacterial activity, this substance (GrS-aggregation substance) was identified as gramicidin S. A gramicidin S derivative bearing a lysine residue in place of one ornithine residue was also detected as a minor component of GrS-aggregation substance. The extent of the aggregation was dependent on the concentration and relative amount of gramicidin S. The inhibition of the enzyme activities was irreversible and the inhibition was proportional to the amount of gramicidin S, like the aggregation of the enzymes. The degree of GS2 inhibition in the amino acid-dependent ATP-PP, exchange reaction varied with the amino acids of gramicidin S and increased in order of the amino acid sequence of gramicidin S. The degree of inhibition of the overall synthesis of gramicidin S was the same as that in the leucine-dependent exchange reaction. [ABSTRACT FROM AUTHOR]

We characterized the taurine transport system in human intestinal Caco-2 cells and showed that it is subject to adaptive regulation. The activity of taurine transport in Caco-2 cells was evaluated by means of an Na+- and Cl--dependent high-affinity transport system, the characteristics of which were similar to those of the β-amino acid-specific taurine transport system previously described for various tissues. The activity of taurine transport was down-regulated on culturing in taurine-containing medium. This taurine-induced down-regulation was dependent on both the incubation time with taurine and the concentration of taurine. Hypotaurine and β-alanine were also capable of inducing this adaptive regulation, whereas α-amino acids and γ-aminoisobutyric acid were not. Kinetic analysis of control and taurine-treated cells suggested that the down-regulation was associated with a decrease in the maximal velocity of taurine transport and also with a decrease in the affinity of the taurine transporter. Cycloheximide treatment weakened the taurine-induced down-regulation. The mRNA level of the taurine transporter (HTAU type) in taurine-treated cells was markedly decreased compared with in control cells. These results indicate that a complex regulatory mechanism is involved in this down-regulation. [ABSTRACT FROM AUTHOR]

Using constructs expressing the reporter gene under the control of the entire or deleted long terminal repeats (LTRs) of 1731, a Drosophila melanogaster retrotransposable element, we show that 1731-LTR is activated by X-irradiation in a dose- and time-dependent manner, and that a sequence located in the U3 region of these LTRs is required. The cis-acting element conferring X-responsiveness shows similarities to kappaB (xB)-like binding sequence. In response to X-irradiation, S2 Drosophila cells produced an extracellular factor which activates the 1731-LTR in nonirradiated cells. This factor was detected both when transfected cells were cocultured with inducing cells and when a conditioned medium taken from irradiated cultures was added. [ABSTRACT FROM AUTHOR]

Escherichia coli strain JD518, a cydD-deficient mutant, displayed temperature-sensitive and pressure-sensitive growth. The defective cydD gene in this strain was complemented by open reading frame 3 (ORF3), previously identified in DNA from a barotolerant bacterium, strain DSS12, allowing growth of the cydD mutant under high temperature and high pressure conditions. Spectrophotometrical analysis indicated that the cytochrome bd complex which is assembled by the CydD protein was expressed in E. coli strain JD518 carrying the ORF3 gene at the same level as occurred in the wild-type strain. Our results indicate that the cydD gene functions are required for cell stability under the condition of high pressure stress in bacteria. [ABSTRACT FROM AUTHOR]

In order to elucidate possible effects of immunoglobulin on Clq metabolism at the anabolic steps, serum Clq levels and Clq mRNA of peritoneal exudate cells (PEC) and spleen cells were measured in female BALB/c mice implanted intraperitoneally with complements-fixing IgG2b- or non-C-fixing class IgG3-producing hybridomas and/or with immunoglobulin-non-productive myeloma cells (p3×63-Ag.8.653) (myeloma 653) (2 × 106/0.2 ml) or without any treatment as controls. In the IgG2b-hybridoma-treated mice, the serum C1q levels and C1q mRNA in PEC increased conspicuously as compared with those in the controls, but Clq mRNA in spleen cells was almost equal to that in the control mice. On the other hand, in the IgG3-hybridoma-treated mice, the serum Clq levels decreased significantly, but the extent of such decrease and the level of Clq mRNA in their PEC were almost equivalent to those in the myeloma 653-implanted mice. The serum Clq levels and Clq mRNA in PEC fluctuated similarly in mice injected intraperitoneally with highly purified IgG2b and/or IgG3 preparations. These results suggest some anabolic interaction, as well as catabolic interaction, between the C-fixing class of immunoglobulin and C1q. [ABSTRACT FROM AUTHOR]

Rat RBCs loaded with125 I-CA by hypotonic dialysis and isotonic resealing were evaluated as a carrier system. Loaded RBCs stored at 4°C remained unlysed (90% survival) allowing release of encapsulated126 I-CA for up to 4 days. Thereafter, cellular lysis increased significantly. IP-injected loaded RBCs reached the maximum level (50%) in circulation at 24 h post-injection. Circulating loaded RBCs showed a half-life of 8–10 days, which was advantageous for carrier function. In contrast to IP-injected free CA, which remained in circulation for only a short time, encapsulated CA showed significant levels in circulation up to 10 days post-injection. The profile of organ uptake with time is essentially not altered for loaded with respect to native cells, being higher the removal of loaded cells and mainly localized in spleen. Nevertheless, liver is the organ with highest elimination capacity for both native and loaded cells, showing its maximum at 24 h post-injection. Concomitantly, the concentration of125 I-CA in all organs studied was highest at this time. These data demonstrate that rat loaded RBCs can potentially be used as a carrier system for long-term dissemination of drug into the organism, with specially increased delivery to the spleen. They also support the use of the rat as an experimental model for biochemical and pharmacological studies in these therapeutic systems. [ABSTRACT FROM AUTHOR]

We have cloned a cDNA that codes for a putative human UDP-galactose translocator (UGT) protein. The cDNA contained an open reading frame of 1,179 base pairs encoding a novel protein of 393 amino acids. Introduction of the open reading frame sequence into a UGT-deficient mouse cell line, Had-1, complemented the genetic defect of the mutant, namely the inability to transport UDP-galactose from the cytosol to the Golgi lumen, as judged from the lectin-sensitivity spectrum of the transformant. To our knowledge, this is the first mammalian nucleotide-sugar translocator whose cDNA sequence has been described. [ABSTRACT FROM AUTHOR]

Rho, a Ras homologue of small GTPase, is present from yeast to mammals. It shuttles between the active GTP-bound form and the inactive GDP-bound form and works as a switch in stimulus-evoked cell adhesion and motility, enhancement of contractile responses, and cytokinesis. In these actions, Rho directs the reorganization of the actin cytoskeleton at a specific time and at a specific site in the cell. It also activates serum response factor possibly via a kinase cascade and mediates a growth signal to nuclei. Two signalling processes are known to lead to Rho activation: one is activation of certain types of G-protein-coupled receptors such as lysophosphatidic acid receptor, and the other is activation of other small GTPases including Ras, CDC42, and Rac. Molecules catalyzing the GDP-GTP exchange of Rho, Rho guanine nucleotide exchange factors (Rho GEF), and those catalyzing the acceleration of GTP hydrolysis, Rho GTPase activating proteins (Rho GAP), were identified as Dbl- and Bcr-containing molecules, respectively. In addition, a molecule inhibiting guanine nucleotide exchange of Rho, Rho guanine nucleotide dissociation inhibitor (Rho-GDI), was isolated and characterized. More recently, putative Rho targets possibly mediating various Rho actions have been identified by their selective interaction with GTP-bound Rho. They include lipid kinases such as phosphatidyl-inositol-5-kinase and protein serine/threonine kinases such as PKN and pl60ROCK. A model of the molecular mechanism of action of Rho constructed on the basis of these findings is presented. There are, however, still many unclarified links between cell stimulation, Rho activation and final Rho actions. [ABSTRACT FROM AUTHOR]

By means of a baculovirus expression system, a large amount of mouse midkine (MK) was produced. The protein was purified to homogeneity by heparin-Sepharose column chro-matography. The purified protein was of a mature type; the signal peptide was cleaved at the expected site. To examine the neurite-guiding activity of MK, rat embryonic brain cells (embryonic days 17–18) were cultured on plates coated with purified MK in a grid pattern. The cells attached to and extended their neurites along the substrate pattern. This interaction was strongly inhibited by heparin, but not by other glycosaminoglycans. Treatment of the cells with heparitinase was effective for inhibiting their adhesion to the substrate. These data suggest that the heparin-like domain on cell surface heparan sulfate proteoglycan is the primary site for MK binding upon interaction with nerve cells. [ABSTRACT FROM AUTHOR]

The cytocidal effect of HM-1 produced by Hansenula mrahii on yeast Saccharomyces cerevisiae; cells was studied. The HM-1 strongly inhibited the growth ofS. cerevisiae; cells at a low concentration (IC50;; 2.1 × 10−8;M) by reducing the number of viable cells. The killer action of HM-1 was most efficient when cells were actively proliferating. Cells in a resting state were resistant, but they became HM-1-sensitive after about 90 min of culturing at 30°C, concomitantly with the increment of budding index. In association with the reduction of viable cell number, ultraviolet light-absorbing cellular components were discharged from sensitive cells. HM-1 molecules appear to bind to susceptible cells rather loosely since cells incubated with HM-1 were able to proliferate after having been washed. By phase-contrast light microscopy and scanning electron microscopy, discharge of cell material was observed at the budding portions of HM-1-treated cells. Addition of sorbitol to make the culture medium isotonic partially reduced the cell death induced by HM-1. These results suggest that HM-1 acts on the budding region of proliferating yeast cells, resulting in pore formation, leakage of cell material and eventual cell death. [ABSTRACT FROM AUTHOR]