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Accumulation of amyloid-β peptide (Aβ) in neuronal cells and in the extracellular regions in the brain is a major cause of Alzheimer's disease (AD); therefore, inhibition of Aβ accumulation offers a promising approach for therapeutic strategies against AD. Aβ is produced by sequential proteolysis of amyloid precursor protein (APP) in late/recycling endosomes after endocytosis of APP located in the plasma membrane. Aβ is then released from cells in a free form or in an exosome-bound form. Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli. Recently, we found that one of the Stx subtypes, Stx2a, has a unique intracellular transport route after endocytosis through its receptor-binding B-subunit. A part of Stx2a can be transported to late/recycling endosomes and then degraded in a lysosomal acidic compartment, although in general Stx is transported to the Golgi and then to the endoplasmic reticulum in a retrograde manner. In this study, we found that treatment of APP-expressing cells with a mutant Stx2a (mStx2a), lacking cytotoxic activity because of mutations in the catalytic A-subunit, stimulated the transport of APP to the acidic compartment, which led to degradation of APP and a reduction in the amount of Aβ. mStx2a-treatment also inhibited the extracellular release of Aβ. Therefore, mStx2a may provide a new strategy to inhibit the production of Aβ by modulating the intracellular transport of APP.

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli, which causes fatal systemic complications. Here, we identified a tetravalent peptide that inhibited Stx by targeting its receptor-binding, B-subunit pentamer through a multivalent interaction. A monomeric peptide with the same motif, however, did not bind to the B-subunit pentamer. Instead, the monomer inhibited cytotoxicity with remarkable potency by binding to the catalytic A-subunit. An X-ray crystal structure analysis to 1.6 Å resolution revealed that the monomeric peptide fully occupied the catalytic cavity, interacting with Glu167 and Arg170, both of which are essential for catalytic activity. Thus, the peptide motif demonstrated potent inhibition of two functionally distinct subunits of Stx., Watanabe-Takahashi, Tamada, Senda et al. identify a tetravalent peptide that inhibits Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli, by targeting its receptor-binding. On the other hand, a monomeric peptide containing the same motif occupies the Stx catalytic cavity, suggesting that this peptide motif can inhibit two subunits of Stx.

The emergence of drug-resistant influenza type A viruses (IAVs) necessitates the development of novel anti-IAV agents. Here, we target the IAV hemagglutinin (HA) protein using multivalent peptide library screens and identify PVF-tet, a peptide-based HA inhibitor. PVF-tet inhibits IAV cytopathicity and propagation in cells by binding to newly synthesized HA, rather than to the HA of the parental virus, thus inducing the accumulation of HA within a unique structure, the inducible amphisome, whose production from the autophagosome is accelerated by PVF-tet. The amphisome is also produced in response to IAV infection in the absence of PVF-tet by cells overexpressing ABC transporter subfamily A3, which plays an essential role in the maturation of multivesicular endosomes into the lamellar body, a lipid-sorting organelle. Our results show that the inducible amphisomes can function as a type of organelle-based anti-viral machinery by sequestering HA. PVF-tet efficiently rescues mice from the lethality of IAV infection., The influenza virus hemagglutinin (HA) protein is a target for antivirals. Here, using a multivalent peptide library screen, the authors identify PVF-tet as an HA inhibitor and show that the peptide inhibits IAV replication by sequestering HA into amphisomes, suggesting these to represent a type of anti-viral machinery.

Shiga toxin (Stx) is a main virulence factor of Enterohemorrhagic Escherichia coli (EHEC) that causes diarrhea and hemorrhagic colitis and occasionally fatal systemic complications. Stx induces rapid apoptotic cell death in some cells, such as human myelogenous leukemia THP-1 cells expressing CD77, a receptor for Stx internalization, and the induction of apoptotic cell death is thought to be crucial for the fatal systemic complications. Therefore, in order to suppress the fatal toxicity, it is important to understand the mechanism how cells can escape from apoptotic cell death in the presence of Stx. In this study, we isolated resistant clones to Stx-induced apoptosis from highly sensitive THP-1 cells by continuous exposure with lethal dose of Stx. All of the ten resistant clones lost the expression of CD77 as a consequence of the reduction in CD77 synthase mRNA expression. These results suggest that downregulation of CD77 or CD77 synthase expression could be a novel approach to suppress the fatal toxicity of Stx in EHEC infected patient.

Highlights • Shiga toxin (Stx) rapidly reduces the level of short-lived anti-apoptotic proteins. • Stx induces activation of caspase 9 and apoptosis. • Proteasome inhibitors prevent the reduction of anti-apoptotic proteins. • Proteasome inhibitors suppress Stx-induced apoptosis. • Bortezomib prolongs the survival of mice challenged with a lethal dose of Stx., Shiga toxin (Stx) causes fatal systemic complications. Stx induces apoptosis, but the mechanism of which is unclear. We report that Stx induced rapid reduction of short-lived anti-apoptotic proteins followed by activation of caspase 9 and the progression of apoptosis. Proteasome inhibitors prevented the reduction of anti-apoptotic proteins, and inhibited caspase activation and apoptosis, suggesting that the reduction of anti-apoptotic proteins is a prerequisite for Stx-induced apoptosis. A clinically approved proteasome inhibitor, bortezomib, prolonged the survival of mice challenged by Stx. These results imply that proteasome inhibition may be a novel approach to prevent the fatal effects of Stx.

Chronic myeloid leukemia (CML) is caused by the chimeric protein p210 BCR-ABL encoded by a gene on the Philadelphia chromosome. Although the kinase domain of p210 BCR-ABL is an active driver of CML, the pathological role of its pleckstrin homology (PH) domain remains unclear. Here, we carried out phospholipid vesicle-binding assays to show that cardiolipin (CL), a characteristic mitochondrial phospholipid, is a unique ligand of the PH domain. Arg726, a basic amino acid in the ligand-binding region, was crucial for ligand recognition. A subset of wild-type p210 BCR-ABL that was transiently expressed in HEK293 cells was dramatically translocated from the cytosol to mitochondria in response to carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment, which induces mitochondrial depolarization and subsequent externalization of CL to the organelle's outer membrane, whereas an R726A mutant of the protein was not translocated. Furthermore, only wild-type p210 BCR-ABL, but not the R726A mutant, suppressed CCCP-induced mitophagy and subsequently enhanced reactive oxygen species production. Thus, p210 BCR-ABL can change its intracellular localization via interactions between the PH domain and CL to cope with mitochondrial damage. This suggests that p210 BCR-ABL could have beneficial effects for cancer proliferation, providing new insight into the PH domain's contribution to CML pathogenesis.

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), is classified into two subgroups, Stx1 and Stx2. Clinical data clearly indicate that Stx2 is associated with more severe toxicity than Stx1, but the molecular mechanism underlying this difference is not fully understood. Here, we found that after being incorporated into target cells, Stx2, can be transported by recycling endosomes, as well as via the regular retrograde transport pathway. However, transport via recycling endosome did not occur with Stx1. We also found that Stx2 is actively released from cells in a receptor-recognizing B-subunit dependent manner. Part of the released Stx2 is associated with microvesicles, including exosome markers (referred to as exo-Stx2), whose origin is in the multivesicular bodies that formed from late/recycling endosomes. Finally, intravenous administration of exo-Stx2 to mice causes more lethality and tissue damage, especially severe renal dysfunction and tubular epithelial cell damage, compared to a free form of Stx2. Thus, the formation of exo-Stx2 might contribute to the severity of Stx2 in vivo, suggesting new therapeutic strategies against EHEC infections.

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), can be classified into two subgroups, Stx1 and Stx2, each consisting of various closely related subtypes. Stx2 subtypes Stx2a and Stx2d are highly virulent and linked with serious human disorders, such as acute encephalopathy and hemolytic-uremic syndrome. Through affinity-based screening of a tetravalent peptide library, we previously developed peptide neutralizers of Stx2a in which the structure was optimized to bind to the B-subunit pentamer. In this study, we identified Stx2d-selective neutralizers by targeting Asn16 of the B subunit, an amino acid unique to Stx2d that plays an essential role in receptor binding. We synthesized a series of tetravalent peptides on a cellulose membrane in which the core structure was exactly the same as that of peptides in the tetravalent library. A total of nine candidate motifs were selected to synthesize tetravalent forms of the peptides by screening two series of the tetravalent peptides. Five of the tetravalent peptides effectively inhibited the cytotoxicity of Stx2a and Stx2d, and notably, two of the peptides selectively inhibited Stx2d. These two tetravalent peptides bound to the Stx2d B subunit with high affinity dependent on Asn16. The mechanism of binding to the Stx2d B subunit differed from that of binding to Stx2a in that the peptides covered a relatively wide region of the receptor-binding surface. Thus, this highly optimized screening technique enables the development of subtype-selective neutralizers, which may lead to more sophisticated treatments of infections by Stx-producing EHEC.

Shiga toxin (Stx) is a main virulence factor of Stx-producing Escherichia coli (STEC) that contributes to diarrhea and hemorrhagic colitis and occasionally to fatal systemic complications. Therefore, the development of an antidote to neutralize Stx toxicity is urgently needed. After internalization into cells, Stx is transferred to the Golgi apparatus via a retrograde vesicular transport system. We report here that 2-methylcoprophilinamide (M-COPA), a compound that induces disassembly of the Golgi apparatus by inactivating ADP-ribosylation factor 1 (Arf1), suppresses Stx-induced apoptosis. M-COPA inhibited transport of Stx from the plasma membrane to the Golgi apparatus and suppressed degradation of anti-apoptotic proteins and the activation of caspases. These findings suggest that inhibition of Stx retrograde transport by M-COPA could be a novel approach to suppress Stx toxicity.

Intestinal infection with Shiga toxin (Stx)-producing E.coli is a leading cause of hemolytic uremic syndrome and acute renal injury in otherwise healthy children in the US. Antibiotics are contraindicated and a therapeutic priority is agents that act intracellularly against the bacterial toxins that drive kidney injury. Our aim was to evaluate whether intravenous administration of a cell-permeable peptide (TVP) that binds to Stx2 will reduce disease severity and rescue juvenile baboons from a lethal Stx2 dose (50 ng/kg). TVP (5 mg/kg) was delivered i.v. simultaneously with toxin (prevention protocol) or at 6 or 24 h after toxin with daily 1 mg/kg supplements up to day 4 (rescue protocols). Biomarkers were monitored in blood and urine up to 28 days. TVP therapy resulted in either absence of clinical signs of acute kidney injury and normal urine output (prevention), or delayed and reduced BUN and creatinine levels (rescue) with concomitant survival. Delayed peptide administration significantly reduced thrombocytopenia, but surprisingly did not alter anemia even when monitored for 28 days in rescued survivors. This is the first successful cell-permeable therapeutic that counteracts Stx2 lethality in an animal model, which recapitulates many of the human responses to enteric infection.

Shiga toxin 2 (Stx2) is a major virulence factor in infections with Stx-producing Escherichia coli (STEC), which causes gastrointestinal diseases and sometimes fatal systemic complications. Recently, we developed an oral Stx2 inhibitor known as Ac-PPP-tet that exhibits remarkable therapeutic potency in an STEC infection model. However, the precise mechanism underlying the in vivo therapeutic effects of Ac-PPP-tet is unknown. Here, we found that Ac-PPP-tet completely inhibited fluid accumulation in the rabbit ileum caused by the direct injection of Stx2. Interestingly, Ac-PPP-tet accumulated in the ileal epithelial cells only through its formation of a complex with Stx2. The formation of Ac-PPP-tet-Stx2 complexes in cultured epithelial cells blocked the intracellular transport of Stx2 from the Golgi apparatus to the endoplasmic reticulum, a process that is essential for Stx2 cytotoxicity. Thus, Ac-PPP-tet is the first Stx neutralizer that functions in the intestine by altering the intracellular transport of Stx2 in epithelial cells.

Yip1A, a mammalian homologue of yeast Yip1p, is a multi-spanning membrane protein that is considered to be involved in transport between the endoplasmic reticulum (ER) and the Golgi. However, the precise role of Yip1A in mammalian cells remains unclear. We show here that endogenous Yip1A is localized to the ER-Golgi intermediate compartment (ERGIC). Knockdown of Yip1A by RNAi did not induce morphological changes in the Golgi, ER, or ERGIC. By analyzing a number of intracellular transport pathways, we found that Yip1A knockdown delayed the transport of Shiga toxin from the Golgi to the ER, but did not affect the anterograde transport of VSVGts045. We also found that a recombinant protein that corresponded to the N-terminal domain of Yip1A inhibited the COPI-independent retrograde transport of GFP-tagged galactosyltransferase, GT-GFP, but not the COPI-dependent retrograde transport of p58/ERGIC53. Furthermore, we found that Yip1A knockdown resulted in the dissociation of Rab6 from the membranes. These results suggested that Yip1A has a role in COPI-independent retrograde transport from the Golgi to the ER and regulates the membrane recruitment of Rab6.

LIS1, a causative gene product for type I lissencephaly, binds to and regulates the dynein motor and the centrosome. LIS1 also forms a complex with the catalytic subunits alpha1 and alpha2 of type I platelet-activating factor acetylhydrolase [PAF-AH (I)]. However, the cellular function of the catalytic subunits remains unknown. In this study, we showed that over-expression of the catalytic subunits, especially alpha2, in cultured cells induced dramatic phenotypical changes including nuclear shape change, centrosomal amplification and microtubule disorganization. We examined if these effects were due to the catalytic activity and/or binding of alpha2 to LIS1. Substitution of a single amino acid Glu39 of murine alpha1 and alpha2 by Asp (alpha2-E39D) did not affect catalytic activity but completely abolished LIS1 binding. Over-expression of either alpha2-E39D or the catalytically inactive alpha2-S48C revealed that alpha2-E39D, but not alpha2-S48C, lost its ability to induce above-mentioned phenotypic changes. Biochemical analyses showed that LIS1 present in the precipitate fraction of murine brain homogenates could be translocated to the soluble fraction by alpha2, but not by alpha2-E39D. These results suggest that over-expression of the PAF-AH (I) catalytic subunits induces centrosomal amplification and microtubule disorganization by disturbing intracellular localization of LIS1.

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli , binds to target cells through a multivalent interaction between its B-subunit pentamer and the cell surface receptor globotriaosylceramide, resulting in a remarkable increase in its binding affinity. This phenomenon is referred to as the “clustering effect.” Previously, we developed a multivalent peptide library that can exert the clustering effect and identified Stx neutralizers with tetravalent peptides by screening this library for high-affinity binding to the specific receptor-binding site of the B subunit. However, this technique yielded only a limited number of binding motifs, with some redundancy in amino acid selectivity. In this study, we established a novel technique to synthesize up to 384 divalent peptides whose structures were customized to exert the clustering effect on the B subunit on a single cellulose membrane. By targeting Stx1a, a major Stx subtype, the customized divalent peptides were screened to identify high-affinity binding motifs. The sequences of the peptides were designed based on information obtained from the multivalent peptide library technique. A total of 64 candidate motifs were successfully identified, and 11 of these were selected to synthesize tetravalent forms of the peptides. All of the synthesized tetravalent peptides bound to the B subunit with high affinities and effectively inhibited the cytotoxicity of Stx1a in Vero cells. Thus, the combination of the two techniques results in greatly improved efficiency in identifying biologically active neutralizers of Stx.

application/pdf, A two-step binding assay for globotriaosylceramide (Gb3) content was developed by histidine-tagging strategy, which is a well-established method for the purification of recombinant proteins. The complete binding of the recombinant His-tagged Shiga toxin 1B subunit (1B-His) (1 microg/ml) to the standard Gb3 adsorbed on a multi-well H type plate was observed within 30 min at 37 degrees C; and its binding could be visualized by the following applications of HisProbe-HRP (8 microg/ml) and tetramethylbenzidine (TMB) peroxidase substrate. The 1B-His binding assay was linear over the range of 1 to 100 ng of Gb3 per well. The binding of 1B-His was specific to Gb3 separated from HeLa cells, and no major cross-reactivity of other glycolipids in Folch's lower fractions extracted from HeLa cells was detected. The glycolipids in Folch's lower fractions from HeLa cells, human fibroblasts and mouse heart were suitable for this assay, but the further purification was needed for glycolipids from human plasma, thus sample preparation is critical factor for the reliable determination of Gb3 content. The 1B-His binding to Gb3 was inhibited by the addition of galactose, but not mannose. This 1B-His binding assay will be useful not only for the determination of Gb3 content, but also for screening for the compounds which inhibit the toxin-binding to Gb3. The strategy of our present method may be applicable for other binding assay, such as Cholera toxin B-subunit for ganglioside GM1., 日本薬学会 Chemical & pharmaceutical bulletin 54(4) p.522-527(2006)より転載

We previously developed linear polymers bearing clustered trisaccharides of globotriaosylceramide (Gb3) as orally applicable Shiga toxin (Stx) neutralizers. Here, using a Gb3 polymer with a short spacer tethering the trisaccharide to the core, we found that shortening the spacer length markedly reduced the binding affinity for Stx2 but not Stx1. Moreover, mutational analysis revealed that the essential binding sites of the terminal trisaccharides were completely different between Stx1 and Stx2. These results provide the molecular basis for the interaction between Stx B subunits and Gb3 polymers.

Infection with Shiga toxin (Stx)-producing Escherichia coli has been implicated to cause hemolytic uremic syndrome, which is characterized by histological abnormalities such as microvascular thrombi and tubular cell damage in the kidney. Although Stx is known to be the major virulence factor of the pathogen, it is still unclear whether Stx directly impairs renal cells in vivo to cause such histological changes and deterioration of renal function. To assess the consequence of the direct action of Stx on renal cells, left kidneys of rats were perfused with Stx1 from the renal artery through the renal vein and then revascularized. Kidneys of control animals were perfused with the vehicle alone. On day 1, apoptosis and induction of tumor necrosis factor alpha gene expression were noticed to occur in the medulla of the Stx1-perfused kidneys. On day 3, extensive tubular injuries were observed by light microscopy: aggregated platelets and monocytic infiltrates in both glomeruli and the medullary interstitium were detected by immunostaining. Tubular changes were more extensive on day 9, with areas of infarction seen in the cortex and medulla. These changes were not found to occur in the sham-operated kidneys. No obvious glomerular changes were detected by light microscopy at any time point. When nonperfused right kidneys were removed after the Stx1 perfusion of the left kidneys, the serum creatinine and blood urea nitrogen levels were increased from day 2, and acute renal failure followed on day 3. These results indicate that Stx1 caused glomerular platelet aggregation, tubular damage, and acute deterioration of renal function by acting directly on renal cells.

Shiga toxin (Stx) is a major virulence factor of Stx-producing Escherichia coli. Recently, we developed a therapeutic Stx neutralizer with 6 trisaccharides of globotriaosyl ceramide, a receptor for Stx, in its dendrimer structure (referred to as "SUPER TWIG [1]6") to function in the circulation. Here, we determined the optimal structure of SUPER TWIG for it to function in the circulation and identified a SUPER TWIG with 18 trisaccharides, SUPER TWIG (2)18, as another potent Stx neutralizer. SUPER TWIGs (1)6 and (2)18 shared a structural similarity, a dumbbell shape in which 2 clusters of trisaccharides were connected via a linkage with a hydrophobic chain. The dumbbell shape was found to be required for formation of a complex with Stx that enables efficient uptake and degradation of Stx by macrophages and, consequently, for potent Stx-neutralizing activity in the circulation. We also determined the binding site of the SUPER TWIGs on Stx.

Shiga toxin (Stx) has an A1-B5 subunit structure, and the A subunit is an RNA N-glycosidase that inhibits cellular protein synthesis. We previously reported that in Caco-2 cells Stx induced cytokines and that the RNA N-glycosidase activity was essential for the cytokine induction. It is known that the binding of the Stx-B subunit to its receptor glycolipid, Gb3, mediates an A subunit-independent signal in some types of cells, but the involvement of this signal in the cytokine induction is unclear. In this study, we investigated whether RNA N-glycosidase itself induces cytokines. IL-8 production was enhanced by Stx, ricin, and modeccin, three toxins that inhibit protein synthesis through an identical RNA N-glycosidase activity, but not by two other types of protein synthesis inhibitors, diphtheria toxin and cycloheximide. The RNA N-glycosidase-type toxins showed a similar induction pattern of cytokine mRNAs. Brefeldin A, a Golgi apparatus inhibitor, completely suppressed the cytokine induction by the toxins. Analysis by using inhibitors of toxin binding and also Stx-B subunit showed that the cytokine-inducing activity was independent of Gb3-mediated signaling. These results indicate that RNA N-glycosidase itself induces the cytokine production and that intracellular transport of toxins through the Golgi apparatus is essential for the activity.

Infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7, which causes diarrhea and hemorrhagic colitis in humans, often results in fatal systemic complications, such as neurological damage and hemolytic–uremic syndrome. Because Stx circulating in the blood is a major causative factor of these complications, the development of a Stx neutralizer that functions in the circulation holds promise as a viable therapy. Here we developed a series of carbosilane dendrimers, in which trisaccharides of globotriaosyl ceramide, a receptor for Stx, were variously oriented at their termini (referred to as SUPER TWIG), and identified a SUPER TWIG with six trisaccharides as a Stx neutralizer functioning in the circulation. This SUPER TWIG specifically bound to Stx with high affinity ( K d = 1.1 × 10 −6 M) and inhibited the incorporation of the toxin into target cells. Intravenous administration of the SUPER TWIG along with Stx to mice substantially reduced the fatal brain damage and completely suppressed the lethal effect of Stx. Moreover, the SUPER TWIG protected mice from challenge with a fatal dose of E. coli O157:H7, even when administered after the establishment of the infection. The SUPER TWIG neutralized Stx in vivo by a mechanism in which the accumulation and immediate degradation of Stx by phagocytic macrophages present in the reticuloendothelial system were induced. Taken together, our findings indicate that this SUPER TWIG is therapeutic agent against infections by Stx-producing E. coli .

Summary cally blocked signaling downstream of ZAP-70. Our studies extend the range of experimental approaches We utilized a novel peptide library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase in- for probing ZAP-70 function in vivo and introduce a technique for developing protein kinase inhibitors that volved in T cell activation. By screening more than 6 billion peptides oriented by a common Tyr residue could be useful for a wide range of protein kinases. for their ability to bind to ZAP-70, we determined a consensus optimal peptide. A Phe-for-Tyr substituted Results and Discussion version of the peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates In order to identify high-affinity inhibitors of the ZAP-70 (KI of 2 mM). The related protein Tyr kinases, Lck and protein Tyr kinase, we screened a Tyr-oriented peptide Syk, were not significantly inhibited by the peptide. library by affinity purification rather than by catalytic When introduced into intact T cells, the peptide conversion. The peptide library used contained the seblocked signaling downstream of ZAP-70, including quence Met-Ala-X-X-X-X-Tyr-X-X-X-X-Ala-Lys-Lys-Lys ZAP-70-dependent gene induction, without affecting where X indicates all amino acids except Trp, Cys, or upstream Tyr phosphorylation. Thus, screening Tyr- Tyr. The predicted degeneracy of this library is 178 < oriented peptide libraries can identify selective pep- 6.9 billion. Screening was performed in the presence of tide inhibitors of protein Tyr kinases. 100 mM ATP but in the absence of Mg 21 to prevent

Infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7 causes bloody diarrhea and hemorrhagic colitis in humans, sometimes resulting in fatal systemic complications. Among the known Stx family members, Stx2 is responsible for the most severe forms of disease. Stx2 binds to target cells via multivalent interactions between its B-subunit pentamer and globotriaosyl ceramide. After binding, it is first retrogradely transported to the Golgi and then to the endoplasmic reticulum (ER). Using a multivalent peptide library approach, we identified a tetravalent peptide that exhibits a high affinity for the Stx2 B-subunit pentamer (KD = 0.13 microM) and markedly inhibits Stx2 cytotoxicity. The tetravalent peptide exerted its inhibitory effects by inducing aberrant cellular transport of Stx2. Although the tetravalent peptide/Stx2 complex was incorporated into cells and translocated to the Golgi, this process was followed by the effective degradation of Stx2 in an acidic compartment rather than by its transfer to the ER. This peptide thoroughly protected mice from a fatal dose of E. coli O157:H7 even when administered after an established infection. Thus, the multivalent peptide library approach enabled the identification of a peptide-based Stx2 inhibitor that has remarkable therapeutic potency and appears to function by inducing aberrant cellular transport and degradation of Stx2.

Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (alpha, betaI, betaII, gamma, delta, epsilon, eta, mu, and zeta) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position +1 carboxyl-terminal of the phosphorylated Ser and basic residues at position -3. All isozymes, except PKC mu, selected peptides with basic amino acids at positions -6, -4, and -2. PKC alpha, -betaI, -betaII, -gamma, and -eta selected peptides with basic amino acid at positions +2, +3, and +4, but PKC delta, -epsilon, -zeta, and -mu preferred peptides with hydrophobic amino acid at these positions. At position -5, the selectivity was quite different among the various isozymes; PKC alpha, -gamma, and -delta selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKC mu showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position -3 to +2 for PKC alpha, -betaI, -betaII, -gamma, -delta, and -eta were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKC alpha, -betaI,-delta, -zeta, and -mu were prepared and used for the determination of Km and Vmax for these isozymes. As judged by Vmax/Km values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft.

Tricellular tight junctions (tTJs) are specialized structural variants of tight junctions within tricellular contacts of an epithelial sheet and comprise several transmembrane proteins including lipolysis-stimulated lipoprotein receptor (angulin-1/LSR) and tricellulin. To elucidate the mechanism of its formation, we carried out stepwise screening of kinase inhibitors followed by RNAi screening to identify kinases that regulate intracellular localization of angulin-1/LSR to the tTJs using a fluorescence image-based screen. We found that the activity of JNK1 and JNK2, but not JNK3, was required for the exclusive localization of angulin-1/LSR at the tTJs. Based on a bioinformatics approach, we estimated the potential phosphorylation site of angulin-1/LSR by JNK1 to be serine 288 and experimentally confirmed that JNK1 directly phosphorylates angulin-1/LSR at this site. We found that JNK2 was also involved in the phosphorylation of angulin-1/LSR. Furthermore, GFP-tagged angulin-1/LSR(S288A), in which serine 288 was substituted by alanine, was observed to be dispersed to bicellular junctions, indicating that phosphorylation of Ser288 is crucial for the exclusive localization of angulin-1/LSR and tricellulin at tTJs. Our fluorescence image-based screening for kinases inhibitor or siRNAs combined with the phosphorylation site prediction could become a versatile and useful tool to elucidate the mechanisms underlying the maintenance of tTJs regulated by kinase networks.

Understanding the substrate recognition mechanism of γ-secretase is a key step for establishing substrate-specific inhibition of amyloid β-protein (Aβ) production. However, it is widely believed that γ-secretase is a promiscuous protease and that its substrate-specific inhibition is elusive. Here we show that γ-secretase distinguishes the ectodomain length of substrates and preferentially captures and cleaves substrates containing a short ectodomain. We also show that a subset of peptides containing the CDCYCxxxxCxCxSC motif binds to the amino terminus of C99 and inhibits Aβ production in a substrate-specific manner. Interestingly, these peptides suppress β-secretase-dependent cleavage of APP, but not that of sialyltransferase 1. Most importantly, intraperitoneal administration of peptides into mice results in a significant reduction in cerebral Aβ levels. This report provides direct evidence of the substrate preference of γ-secretase and its mechanism. Our results demonstrate that the ectodomain of C99 is a potent target for substrate-specific anti-Aβ therapeutics to combat Alzheimer’s disease., γ-Secretase inhibitors are studied for their potential to treat Alzheimer’s disease, but their use is limited by side effects. Funamoto et al. show that γ-secretase preferentially cleaves substrates with short ectodomains and that inhibitors based on these ectodomains reduce disease-like pathology in mice.

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli that occasionally causes fatal systemic complications. We recently developed a tetravalent peptide (PPP-tet) that neutralizes the cytotoxicity of Stx2 using a multivalent peptide library approach. In this study, we used this technique to identify a series of tetravalent peptides that bound to Stx1, another major Stx family member, with high affinity by targeting one receptor-binding site of the B subunit. One peptide, MMA-tet, markedly inhibited Stx1 and Stx2 cytotoxicity with greater potency than PPP-tet. After forming a complex with Stx1 through its specific receptor-binding region, MMA-tet did not affect vesicular transport of the toxin to the endoplasmic reticulum but substantially rescued inhibition of the protein synthesis induced by Stx1. Oral application of MMA-tet protected mice from a fatal dose of an E. coli O157:H7 strain producing both toxins. MMA-tet may be a promising therapeutic agent against the infection.

The possible involvement of zeta isozyme of protein kinase C (PKC zeta) in phorbol ester-induced signal transduction was investigated in mouse epidermal cells. Western blot analysis of RESOURCE Q column chromatography eluates obtained from 105,000 g supernatants and particulate fractions of epidermal cells was performed using anti-PKC zeta specific antibody. Anti-PKC zeta antibody recognised proteins in low salt range corresponding to 25-125 mM NaCl (low salt-eluted PKC zeta; 1-PKC zeta) as well as high salt range corresponding to 175-300 mM NaCl (high salt-eluted PKC zeta; h-PKC zeta) in both subcellular fractions. 1-PKC zeta and h-PKC zeta were detected as a doublet protein of 79,000 and 85,000 M(r) in 105,000 g supernatants, but as a 79,000 M(r) protein in particulate fractions. Immunoprecipitated 1-PKC zeta and h-PKC zeta with anti-PKC zeta specific antibody possessed phosphatidylserine (PS)-dependent protein kinase activity, but neither 1-PKC zeta nor h-PKC zeta were further activated by 40 nM phorbol 12-myristate 13-acetate (PMA) in the presence of PS. Furthermore, 1-PKC zeta and h-PKC zeta can be autophosphorylated, indicating that both 1-PKC zeta and h-PKC zeta are PKC zeta. Treatment of intact epidermal cells with PMA or other PKC activators caused the apparent shift of 79,000 M(r) 1-PKC zeta to the 85,000 M(r) from in particulate fractions. Prolonged treatment of the cells with PMA induced the downregulation of both forms of 1-PKC zeta in particulate fractions. Under the same condition, 1-PKC zeta in 105,000 g supernatants and h-PKC zeta in both fractions did not respond to PMA. This apparent shift was reversible and the content ratio of 85,000 to 75,000 M(r) 1-PKC zeta was decreased by acid phosphatase treatment, indicating that the apparent shift results at least in part from phosphorylation of 79,000 M(r) 1-PKC zeta. Total activity of 1-PKC zeta was increased in association with the apparent shift from the 79,000 to 85,000 M(r) form in response to PMA treatment of intact epidermal cells. All of these results indicate that PKC zeta is present as multiple forms in mouse epidermal cells, and that especially 1-PKC zeta in particulate fractions play a significant role(s) in PMA-induced signal transduction in mouse epidermal cells.

Phosphatidylserine (PS) is a relatively minor constituent of biological membranes. Despite its low abundance, PS in the plasma membrane (PM) plays key roles in various phenomena such as the coagulation cascade, clearance of apoptotic cells, and recruitment of signaling molecules. PS also localizes in endocytic organelles, but how this relates to its cellular functions remains unknown. Here we report that PS is essential for retrograde membrane traffic at recycling endosomes (REs). PS was most concentrated in REs among intracellular organelles, and evectin-2 (evt-2), a protein of previously unknown function, was targeted to REs by the binding of its pleckstrin homology (PH) domain to PS. X-ray analysis supported the specificity of the binding of PS to the PH domain. Depletion of evt-2 or masking of intracellular PS suppressed membrane traffic from REs to the Golgi. These findings uncover the molecular basis that controls the RE-to-Golgi transport and identify a unique PH domain that specifically recognizes PS but not polyphosphoinositides.

Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC), including O157:H7, causes bloody diarrhea and hemorrhagic colitis in humans, occasionally resulting in fatal systemic complications, such as neurological damage and hemolytic-uremic syndrome. Because Stx is a major virulence factor of the infectious disease, a series of Shiga toxin neutralizers with various structural characteristics has been developed as promising therapeutic agents. Most of these agents function to bind to the toxin directly and inhibit the binding to its receptor present on the target cells. Other neutralizers do not inhibit receptor binding but induce aberrant intracellular transport of the toxin, resulting in effective detoxification. Such a novel type of Stx neutralizer provides a new therapeutic strategy against STEC infections. Here, recent progress of the development of Stx neutralizers is reviewed.

Globotriaosylceramide (Gb3) is a well known receptor for Shiga toxin (Stx), produced by enterohemorrhagic Escherichia coli and Shigella dysenteriae. The expression of Gb3 also affects several diseases, including cancer metastasis and Fabry disease, which prompted us to look for factors involved in its metabolism. In the present study, we isolated two cDNAs that conferred resistance to Stx-induced cell death in HeLa cells by expression cloning: ganglioside GM3 synthase and the COOH terminus region of glutamate receptor, ionotropic, N-methyl-d-asparate-associated protein 1 (GRINA), a member of the transmembrane BAX inhibitor motif containing (TMBIM) family. Overexpression of the truncated form, named GRINA-C, and some members of the full-length TMBIM family, including FAS inhibitory molecule 2 (FAIM2), reduced Gb3, and lactosylceramide was accumulated instead. The change of glycolipid composition was restored by overexpression of Gb3 synthase, suggesting that the synthase is affected by GRINA-C and FAIM2. Interestingly, the mRNA level of Gb3 synthase was unchanged. Rather, localization of the synthase as well as TGN46, a trans-Golgi network marker, was perturbed to form punctate structures, and degradation of the synthase in lysosomes was enhanced. Furthermore, GRINA-C was associated with Gb3 synthase. These observations may demonstrate a new type of posttranscriptional regulation of glycosyltransferases.

In primary cultured mouse epidermal cells, phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), induced changes in the phosphorylation levels of 10 proteins, termed KP-1 to 10, in two-dimensional PAGE. Seven of these proteins were phosphorylated and three were dephosphorylated. Similar changes were induced by other PKC activators, but not by inactive phorbol ester. Among these substrate proteins, phosphorylation of three proteins, i.e. KP-1 (pI 4.7/23,000 M r ), KP-2 (pI 4.7/20,700 M r ) and KP-10 (pI 4.7/25,000 M r was markedly enhanced by PMA and inhibited by a potent PKC inhibitor staurosporine. In vitro phosphorylation studies and phosphoamino acid analysis, using these proteins as substrate and PKC preparations obtained from epidermal cell lysate, revealed that KP-1 and -2 were directly phosphorylated by Ca 2+ -, phospholipid-dependent protein kinase (conventional-type PKC; cPKC), but not by Ca 2+ -independent, phospholipid-dependent protein kinase (novel-type PKC; nPKC). On the other hand, KP-10 was mainly phosphorylated by nPKC in intact epidermal cells. These results indicate that cPKC and nPKC in epidermal cells have different substrate specificity for endogenous proteins and may induce different signal transduction.

12-O-Tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC), induced ornithine decarboxylase (ODC) in primary cultured mouse epidermal cells. Staurosporine, a potent protein kinase C inhibitor, also induced ODC activity. Both TPA- and staurosporine-caused ODC inductions were markedly suppressed in the PKC-down-regulated cells. Another PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), inhibited both TPA- and staurosporine-caused ODC inductions. H-7 by itself never induced ODC activity. Under our experimental conditions, staurosporine induced no detectable phosphorylation of endogenous proteins. TPA induced a translocation of PKC from cytosol to membrane whereas an optimal concentration of staurosporine to induce ODC did not induce an obvious translocation of PKC. Indomethacin, a cyclooxygenase inhibitor, inhibited staurosporine-caused ODC induction, but not TPA-caused ODC induction. Staurosporine induced specific morphological changes of epidermal cells both in normal and in PKC-down-regulated cells. These results indicate that staurosporine induces ODC activity in a PKC-dependent manner and morphological changes possibly through a PKC-independent mechanism. The mechanism of ODC induction caused by staurosporine may be in some way different from that caused by TPA.

Supernatant protein factor (SPF) is a novel cholesterol biosynthesis-accelerating protein expressed in liver and small intestine. Here, we report on the physiological role of SPF by using Spf-deficient mice. Although plasma cholesterol levels were similar in chow-fed Spf-/- and wild-type (WT) mice, fasting significantly decreased plasma cholesterol levels in Spf-/- mice but not in WT mice. While fasting reduced hepatic cholesterol synthesis rate in WT mice, a more pronounced reduction was observed in Spf-/- mice. The expression of cholesterogenic enzymes was dramatically suppressed by fasting both in WT and Spf-/- mice. In contrast, hepatic SPF expression of WT mice was up-regulated by fasting in peroxisome proliferator-activated receptor alpha (PPAR-alpha)-dependent manner. These results indicate that in WT mice, the decrease of hepatic cholesterol synthesis under fasting conditions is at least in part compensated by SPF up-regulation. Fibrates, which function as a PPAR-alpha agonist and are widely used as hypotriglycemic drugs, reduced hepatic cholesterol synthesis and plasma cholesterol levels by approximately one-half in Spf-/- mice but not in WT mice. These findings suggest that co-administration of fibrates and an SPF inhibitor may reduce not only plasma triglyceride but also cholesterol levels, indicating that SPF is a promising hypocholesterolemic drug target.

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that release nucleoside 5′-monophosphate from a variety of nucleotides and nucleotide derivatives. The mammalian NPP family comprises seven members, but only three of these (NPP1–3) have been studied in some detail. Previously we showed that lysophospholipase D, which hydrolyzes lysophosphatidylcholine (LPC) to produce lysophosphatidic acid, is identical to NPP2. More recently an uncharacterized novel NPP member (NPP7) was shown to have alkaline sphingomyelinase activity. These findings raised the possibility that other members of the NPP family act on phospholipids. Here we show that the sixth member of the NPP family, NPP6, is a choline-specific glycerophosphodiester phosphodiesterase. The sequence of NPP6 encodes a transmembrane protein containing an NPP domain with significant homology to NPP4, NPP5, and NPP7/alkaline sphingomyelinase. When expressed in HeLa cells, NPP6 was detected in both the cells and the cell culture medium as judged by Western blotting and by enzymatic activity. Recombinant NPP6 efficiently hydrolyzed the classical substrate for phospholipase C, p-nitrophenyl phosphorylcholine, but not the classical nucleotide phosphodiesterase substrate, p-nitrophenyl thymidine 5′-monophosphate. In addition, NPP6 hydrolyzed LPC to form monoacylglycerol and phosphorylcholine but not lysophosphatidic acid, showing it has a lysophospholipase C activity. NPP6 showed a preference for LPC with short (12:0 and 14:0) or polyunsaturated (18:2 and 20:4) fatty acids. It also hydrolyzed glycerophosphorylcholine and sphingosylphosphorylcholine efficiently. In mice, NPP6 mRNA was predominantly detected in kidney with a lesser expression in brain and heart, and in human it was detected in kidney and brain. The present results suggest that NPP6 has a specific role through the hydrolysis of polyunsaturated LPC, glycerophosphorylcholine, or sphingosylphosphorylcholine in these organs.

In primary cultured mouse epidermal cells, protein kinase C isozyme zeta (PKCzeta) consists of multiple forms, for example, low-salt eluted PKCzeta (1-PKCzeta; 79 and 85 kDa) and high-salt eluted PKCzeta (h-PKCzeta; 79 and 85 kDa) on anion-exchange column chromatography. In this study, biochemical and biophysical differences between 1-PKCzeta and h-PKCzeta were examined by using carcinogen-initiated mouse epidermal cell-line WYF31 cells, whose growth is stimulated by tumour promoter phorbol 12-myristate 13-acetate (PMA). The binding efficiency of h-PKCzeta to anti-PKCzeta antibody-affinity column was 10 times higher than that of 1-PKCzeta. T7-tagged rat PKCzeta overexpressed in WYF31 cells was recovered only in the high-salt eluted area on the anion-exchange column. Furthermore, when rat PKCzeta was stably overexpressed in WYF31 cells, the content of h-PKCzeta increased 4 to 5 times compared to that of parental cells, but the content of 1-PKCzeta was not altered. All of these results indicate that h-PKCzeta is the product of the PKCzeta gene (referred to as PKCzeta) and that 1-PKCzeta is closely related but different from PKCzeta (referred to as PKCzeta-related kinase). Interestingly, serum starvation of WYF31 cells caused a marked increase of the content of PKCzeta-related kinase with a concomitant decrease of PKCzeta content. These changes were reversed by stimulating the cell growth with 10% foetal calf serum. Prolonged treatment of starved cells with PMA, which induces the proliferation of WYF31 cells, also caused the downregulation of PKCzeta-related kinase. These results suggest that the expression levels of PKCzeta-related kinase and PKCzeta are differently regulated, and that the increased expression of PKCzeta-related kinase might play a significant role in the growth-suppression processes of WYF31 cells.

Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

Macrophages take up and metabolize negatively charged liposomes containing free cholesterol efficiently, resulting in a massive accumulation of cholesteryl esters and triacylglycerols in their cytoplasm (Nishikawa, K., Arai, H. and Inoue, K. (1990) J. Biol. Chem. 265, 5226-5231). This system was used to assess the effects of structural variation of sterol on the intracellular transport and the metabolism of endocytosed sterols by the cells. Liposomes containing phytosterols with an extra one (campesterol) or two (beta-sitosterol, stigmasterol, fucosterol) carbons at the C-24 position of the cholesterol side-chain were endocytosed as efficiently as those containing cholesterol without exhibiting any apparent toxicity on the cells. Esterification of endocyotosed phytosterols was, however, extremely low; campesterol esterification was only 20% that of cholesterol and either beta-sitosterol or stigmasterol was not esterified appreciably. A morphological study showed that the endocytosed phytosterols were accumulated in the phagolysosomes of the cells. Blocking of esterification of endocytosed cholesterol by an acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor did not lead to cholesterol accumulation in the phagolysosomes. These data suggest that accumulation of endocytosed phytosterols in phagolysosomes is not a consequence of the inability of the cell to esterify sterols in the endoplasmic reticulum. In the light of these observations, we conclude that cultured macrophages can discriminate between sterols that differ only by a methyl or ethyl group at the C-24 position at their lysosomal compartment.

Recently this group found an endogenous substrate protein for Ca(2+)-independent novel protein kinase C (nPKC), i.e. KP-10 (pI 4.7/25,500 M(r)), in primary cultured mouse epidermal cells [Nishikawa, K. et al. (1992) Cell. Signal. 4, 757-776]. In the present study, the nPKC isozymes which phosphorylate KP-10 in these cells were determined. Western blot analysis revealed that PKC alpha, eta and zeta were present in epidermal cell 105,000 g supernatants and that the content of PKC zeta was much higher than those of PKC alpha and eta. Neither PKC beta, delta nor epsilon was detected in the 105,000 g supernatants. Phosphatidylserine and phorbol 12-myristate 13-acetate (PMA)-dependent KP-10 phosphorylating activity was immunoprecipitated by anti-PKC eta and zeta antibodies, but not by antiPKC alpha antibody. These results suggest that PKC eta and/or zeta phosphorylate KP-10 and play pivotal roles in intracellular signal pathways in intact epidermal cells.

We studied the metabolism of phospholipids exogenously added to cultures of the protozoan, Tetrahymena pyriformis. Tetrahymena cells were found to metabolize the extracellular phospholipids and the fatty acyl chains of the latter were accumulated predominantly as a form of triacylglycerol in the cells. This metabolism was considered to be initiated via endocytosis of phospholipid vesicles, as judged from the following facts: Cytochalasin B, an inhibitor of endocytosis, suppressed the metabolism almost completely. Phospholipid vesicles were incorporated into a phagosome-like structure in Tetrahymena cells, as observed under an electron microscope. When phospholipids doubly labeled with 14C and 3H at the glycerol moiety and fatty acyl chain, respectively, were incubated with Tetrahymena cells, the glycerol moiety and fatty acyl chain at the sn-2-position of the exogenous phospholipids were incorporated into the cellular triacylglycerol fraction in a 1 to 1 ratio. Monoacylglycerol acyltransferase activity was detected in the microsomal fraction of Tetrahymena cells. From these results, together with those of our previous study on lysosomal phospholipid hydrolysis in Tetrahymena (J. Biochem. 99, 125-133 (1986)), it is suggested that the extracellular phospholipids which were taken up by the cells via endocytosis were hydrolyzed through the action of lysosomal phospholipases A1 and C, and also that one of the products, sn-2-monoacylglycerol, served as an acyl acceptor for the synthesis of triacylglycerol via the microsomal "monoacylglycerol pathway."

Role of mast cells in the development of renal fibrosis: Use of mast cell–deficient rats. Background Recent clinical studies have shown that the number of interstitial mast cells increases in various types of renal disease and correlates well with the magnitude of interstitial fibrosis. The present study was conducted to assess the role of mast cells in renal fibrosis by examining an experimental glomerular disease. Methods A rat model of chronic glomerular disease, puromycin aminonucleoside-nephrosis, was induced in mast cell–deficient ( Ws/Ws ) and normal (+/+) rats. Results The area of interstitial fibrosis was widely distributed at 6weeks in both groups of rats; however, unexpectedly, the area of interstitial fibrosis was greater in Ws/Ws rats than in +/+ littermates. Biochemical analysis of the hydroxyproline content confirmed the more severe fibrosis in the Ws/Ws rats. The number of mast cells increased in both Ws/Ws and +/+ rats, concomitant with the development of interstitial fibrosis, but was confirmed to be lower in Ws/Ws than in +/+ rats. There were no differences in the numbers of interstitial macrophages and T lymphocytes between the two groups. Reverse transcription-polymerase chain reaction analysis of cytokine expression revealed that the level of mRNA for transforming growth factor-β (TGF-β), a potent profibrotic cytokine, was higher in Ws/Ws rats. In addition, heparin, one of the major components of mast cells, inhibited the expression of TGF-β mRNA in rat fibroblasts in culture. Conclusion These results suggest that mast cells do not play a major role in the pathogenesis of interstitial fibrosis in puromycin aminonucleoside nephrosis. Rather, they might be protective or ameliorative in this model through the inhibition of TGF-β production by heparin, and possibly in other models and also in humans.

Phospholipase activity in the lysosomes of the protozoan Tetrahymena pyriformis strain NT-1 was studied using phospholipids radioactively labeled in the fatty acid moieties. Lysosomal homogenates showed high phospholipase activity with an acidic pH optimum. Unlike the phospholipases in rat liver lysosomes, almost all activity was recovered from the membranous fraction of the lysosomes. The activity was partially solubilized by treatment of the membranes with a detergent or trypsin. Using specifically labeled phospholipids revealed that phospholipase. A1 and C are predominant in Tetrahymena lysosomes, no appreciable phospholipase A2 or lysophospholipase activity was detected in the fraction. There are two catabolic pathways of the hydrolysis of phospholipid: Hydrolysis is initiated by deacylation at the 1-position by phospholipase A1 and the 2-acyllysophospholipid thus formed is successively attacked by (lyso)phospholipase C; hydrolysis is initiated by cleavage of phosphodiester by phospholipase C and the diacylglycerol thus formed is attacked by lipase. Both pathways give the same end products, free fatty acid and 2-monoacylglycerol. The former pathway might be predominant in Tetrahymena lysosomes under physiological conditions since the pathway is independent of detergent. Phospholipases A1 and C activities were partially released into the medium. At least two different phospholipases C are present in the medium as judged by chromatographic behavior and their substrate specificities.