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Since the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident, morphological abnormalities in lepidopteran insects, such as shrinkage and/or aberration of wings, have been reported. Butterflies experimentally exposed to radiocesium also show such abnormalities. However, because of a lack of data on absorbed dose and dose-effect relationship, it is unclear whether these abnormalities are caused directly by radiation. We conducted a low dose-rate exposure experiment in silkworms reared from egg to fully developed larvae on a

Since the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident, morphological abnormalities in lepidopteran insects, such as shrinkage and/or aberration of wings, have been reported. Butterflies experimentally exposed to radiocesium also show such abnormalities. However, because of a lack of data on absorbed dose and dose–effect relationship, it is unclear whether these abnormalities are caused directly by radiation. We conducted a low dose-rate exposure experiment in silkworms reared from egg to fully developed larvae on a 137CsCl-supplemented artificial diet and estimated the absorbed dose to evaluate morphological abnormalities in pupal wings. We used 137CsCl at 1.3 × 103 Bq/g fresh weight to simulate 137Cs contamination around the FDNPP. Absorbed doses were estimated using a glass rod dosimeter and Monte Carlo particle transport simulation code PHITS. Average external absorbed doses were approximately 0.24 (on diet) and 0.016 mGy/day (near diet); the average internal absorbed dose was approximately 0.82 mGy/day. Pupal wing structure is sensitive to radiation exposure. However, no significant differences were observed in the wing-to-whole body ratio of pupae between the 137CsCl-exposure and control groups. These results suggest that silkworms are insensitive to low dose-rate exposure due to chronic ingestion of high 137Cs at a high concentration.

lα-Aspartic acid (Asp) residues in proteins are nonenzymatically isomerized to abnormal lβ-, dα-, and dβ-Asp isomers under physiological conditions. Such an isomerization of Asp residues is considered to be a trigger of protein denaturation because it either elongates the main chain or induces a different orientation of the side chain within the protein structure or both. However, previous studies have found no direct evidence of the effects of Asp isomers on protein function. Therefore, the production of Asp-isomer-containing proteins is required to verify the effects of Asp isomerization. Here, we describe the production of an Asp-isomer-containing protein using the expressed protein ligation. As a model protein, bovine pancreatic ribonuclease A (RNase A, EC 3.1.27.5), which catalyzes the cleavage of phosphodiester bonds in RNA, was used. In this study, lα-Asp at position 121 in RNase A was replaced by lβ-, dα-, and dβ-Asp. The objective aspartic acid at position 121 is located near the active site and related to RNA cleavage. The RNase A with lα-Asp at position 121 showed a normal activity. By contrast, the catalytic activity of lβ-, dα-, and dβ-Asp-containing RNase A was markedly decreased. This study represents the first synthesis and analysis of a protein containing four different Asp isomers.

Isomerization of aspartate (Asp) is a common non-enzymatic posttranslational modification. Isomerized residues accumulate in proteins associated with age-related human disorders such as cataract and are well known to affect protein structure and function. We previously detected d -Asp-containing peptides in human serum. In this study, we investigated whether isomerized Asp residues are present in human immunoglobulin G (IgG) kappa chain by a qualitative d -amino acid analysis based on diastereomer formation and liquid chromatography tandem mass spectrometry (LC-MS/MS). We also investigated the d / l ratio of Asp residues in the IgG kappa chain in serum from donors aged 25, 37, 41, 54 and 67 years. As a result, two isomerized Asp residues, Asp151 and Asp170, were detected in the IgG kappa chain, and the d / l ratio of these residues was found to increase with aging. To assess the effects of this isomerization, we synthesized four isomeric peptides of IgG kappa chain containing l α-, l β-, d α-, or d β-Asp at position 170, and compared their secondary structures by CD spectroscopy. Peptide containing normal l α-Asp170 showed type II β-turn structure, while the other isomeric peptides showed random structure, clearly indicating that substitution of a single Asp isomer alters the secondary structure of the peptide. Because IgG is a main component of humoral immunity, Asp isomerization in IgG may reflect changes of structure and decrease in immune function. Proteome research on serum from the standpoint of racemization might enable us to develop new kinds of biomarker and new directions to study the aging process.

The formation and accumulation of D-aspartate residue (D-Asp) in proteins caused by oxidative stress leads to dysfunction and/or denaturation of proteins, and is consequently responsible for aging-related misfolding diseases such as cataracts, prion disease, and Alzheimer's disease. We sought to identify that an unknown protease selectively degrades the noxious D-Asp-containing protein, namely D-aspartyl endopeptidase (DAEP), and finally purified it from the inner mitochondrial membrane of mouse liver. In order to analyze the substrate stereoselectivity of DAEP, we synthesized a peptide corresponding to 55-65 (Thr-Val-Leu-Asp-Ser-Gly-Ile-Ser-Glu-Val-Arg) of human αA-crystallin and its corresponding diastereoisomers in which L-α-Asp was replaced with L-β-, D-α- or D-β-Asp residue at position 58. Following incubation of that peptide with purified DAEP, it was only degraded at D-α-Asp(58), independent of ATP or NAD. This result indicates that DAEP stereoselectively recognizes and degrades its substrate at the internal D-α-Asp residue. DAEP therefore seems to physiologically serve as the quality control system against the noxious D-Asp-containing protein in the long life span of mammals.

It is strongly suggested that D-aspartic acid (D-Asp)-containing proteins are spontaneously generated by oxidative stress and would cause many aging-related misfolding diseases, such as cataracts, prion disease, and Alzheimer's disease. We have identified a D-Asp-containing protein-specific protease, D-aspartyl endopeptidase (DAEP), from mammalian mitochondria, serving as a scavenger against the noxious D-Asp-containing protein. Recently, it has been shown that the activity of Lon, an ATP-dependent protease degrading oxidatively damaged proteins in mitochondria, decreases with aging by oxidative stress. However, an obvious relation between DAEP activity and oxidative stress with aging remains to be demonstrated. In the present study, we showed that there was a remarkable decrease in DAEP activity in superoxide dismutase-deficient mice, which formed excess reactive oxygen species (ROS). Our result suggests that a decrease in DAEP activity by oxidative stress may cause the accumulation of D-Asp-containing protein, leading to mitochondria-associated diseases.

D-aspartyl endopeptidase (DAEP) is a specific protease for D-aspartic acid (D-Asp)-containing protein, which has been implicated in the pathogenesis of age-related and misfolding diseases such as Alzheimer's disease. Therefore, DAEP would serve as a defensive system against the noxious D-Asp-containing protein. However, it is unclear how DAEP exerts its unique enzymatic function, since its higher-order structure remains quite unsolved. In this study, we analyzed the conformation of purified DAEP from the mitochondrial membrane of mouse by atomic force microscopy the advantage of which is its ability to study biological macromolecules and even living organisms in an ambient air environment. DAEP formed a ring-like structure with a diameter of ca. 40 nm. Our data suggest that DAEP topologically belongs to the AAA+ protease family such as proteasome, Lon, and mitochondrial membrane-bound i-/m-AAA protease.

D-Asp-containing proteins have been implicated in many aging-related diseases. To clarify the role of D-Asp-containing proteins in such diseases, we developed a screening system for these proteins using a D-aspartyl endopeptidase that specifically cleaves the proteins at the C-terminus. The digested proteins were detected by means of two-dimensional gel electrophoresis and identified using nano-liquid chromatography/tandem mass spectrometry. We were able to detect myelin basic protein, a known D-Asp-containing protein, in the brain tissues of mice; this indicates that our system is effective for screening D-Asp-containing proteins.

Lens alpha-crystallin, composed of two subunits alpha A- and alpha B-crystallin, forms large aggregates in the lens of the eye. The present study investigated the aggregate of human lens alpha-crystallin from elderly and young donors. Recombinant alpha A- and alpha B-crystallins in molar ratios of alpha A to alpha B at 1:1, corresponding to the aged sample, were also studied in detail. We found by ultra-centrifugation analysis that the alpha-crystallin aggregate from elderly donors was large and heterogeneous with an average sedimentation coefficient of 30 S and a range of 20-60 S at 37 degrees C. This was higher compared to the young samples that had an average sedimentation coefficient of 17 S. The sedimentation coefficients of recombinant alpha A- and alpha B-crystallins were approximately 12 S and 15 S, respectively. Even when recombinant alpha-crystallins were mixed in molar ratios equivalent to those found in vivo, similar S values as the native aged alpha-crystallin aggregates were not obtained. Changes in the self-association of alpha-crystallin aggregate were correlated to changes in chaperone activity. Alpha-crystallin from young donors, and recombinant alpha A- and alpha B-crystallin and their mixtures showed chaperone activity, which was markedly lost in samples from the aged alpha-crystallin aggregates.

Here we report isolation of an androgen-regulated novel gene from an androgen-dependent mouse mammary Shionogi carcinoma SC-3 cell line. Using a polymerase chain reaction-based subtraction method and Northern blotting analysis, we isolated four androgen-inducible genes from SC-3 cells. Nucleotide sequencings identified three of the genes as cyclin D1, beta-catenin, and fatty acid synthase, respectively, but the fourth, a gene tentatively named as Arg1 (androgen-regulated gene 1), remained undefined. The cloned 2.0-kb sized Arg1 cDNA encoded 414 amino acid sequences. The deduced amino acid sequences, sharing about 30% homology with cathepsin family members at a protein level, had relatively conserved residues around the three proteinase active sites reported earlier. In Northern blotting, Arg1 mRNA was found in kidney, heart, lung, and to a lesser degree, in spleen and liver. Its transcripts were also detected in male reproductive organs on RT-PCR. In addition, its expression levels in prostate were markedly reduced after castration. Unexpectedly, Arg1-expressing COS1 cells showed no significant proteinase activity to various synthesized substrates under neutral or acidic conditions in this study. This might have been due to the replacement of the cysteinyl active site for proteinase to serine residue in the Arg1 amino acid sequences. Given that Arg1 also contains a lipocaline signature known as a binding motif for small hydrophobic molecules at the center of its amino acid sequences, Arg1 is a lipocalin family gene regulated by androgens in prostate and Shionogi carcinoma cells.

Colocalization of mitochondria is the first step of intermitochondrial interaction or fusion in a cell. Here, we showed colocalization between exogenous mitochondria and endogenous ones or between exogenous proteoliposomes and endogenous mitochondria in mouse fertilized eggs by confocal laser microscopy. Isolated mitochondria from mouse liver and proteoliposomes containing mitochondrial membrane were directly labeled with red fluorescent aliphatic marker, PKH26, which is incorporated into lipid membrane, and then were microinjected into fertilized mouse eggs. Exogenous mitochondria appeared to be almost colocalized with endogenous mitochondria at the 4- and 8-cell stages, when mitochondria were stained with Rhodamine 123 (green fluorescent marker). On the contrary, when liposomes consisted of soy bean phospholipid were microinjected into the eggs as a control, their localization was different from that of endogenous mitochondria. Next, the submitochondrial particles and proteoliposomes were microinjected. Both the proteoliposomes and the submitochondrial particles appeared to colocalize with endogenous mitochondria at the 4-cell stage. These results suggest the existence of a factor that makes liposomes colocalize with mitochondria. Such a proteoliposome would be useful for the development of mitochondrial gene transfer techniques.

Thimet oligopeptidase (TOP) is a thiol- andmetallo-dependent peptidase and has been shown to beone of the β-secretase candidates. TOPexpressed in COS cells cleaved amyloid precursorprotein (APP) at the β-secretase site, and wefound a proteolytic product of APP called secretedform of APP by β-secretase (sAPPβ) in theconditioned media. Here we demonstrate thatsAPPβ was increased in conditioned media whenTOP was coexpressed in COS cells with APP and treatedwith an ADAM inhibitor SI-27. In addition, althoughTOP expressed in COS cell was localized at nuclei orGolgi apparatus, it exclusively colocalized at Golgiapparatus when APP was coexpressed with TOP.

We developed an assay method using a novel quenched fluorescent substrate (QFS) flanking the beta-cleavage site of amyloid precursor protein (APP), and purified a candidate beta-secretase from bovine brain. N-terminal amino acid analysis showed the candidate to be thimet oligopeptidase (TOP). The cDNA for human TOP was cloned from a human brain cDNA library and expressed in COS cells. The enzyme was further purified on a Ni2+-agarose column. TOP cleaved the Swedish Alzheimer's substrate (SEVNLDAEFR) as well as the normal substrate (SEVKMDAEFR). We then coexpressed TOP with APP695 in COS cells, collected transfected cells and conditioned media, and analyzed them by immunoblotting. The antibody against the specific secreted APP cleaved by beta-secretase (sAPPbeta) detected the secretion of sAPPbeta only from APP/hTOP-overexpressing cells, and not from cells overexpressing of antisense hTOP cDNA. Finally, we analyzed the immunolocalization of overexpressed hTOP in COS cells. Most hTOP was localized in the nuclei, but a small amount was localized in the Golgi or other organelles around the nuclei. These results suggest that TOP has a beta-secretase-like activity responsible for the processing of APP.

The metabolic pathway of Alzheimer's amyloid precursor protein (APP) involves restricted intracellular proteolysis by secretases, which leads to the secretion of the N-terminal soluble APP (sAPP) and the generation of a cell-associated C-terminal fragment. The precise cellular sites at which these processes occur remain unknown. In this report, we describe the route of APP sorting and the processing site using novel systems with and without sorting signals on the APP molecule. One system involves the replacement of the C-terminal ten amino acids of APP with Adenoviral E19 protein containing an endoplasmic reticulum (ER) retrieval signal (APPE19); the other involves deleting the last ten amino acids corresponding to the replaced site (APPdeltaC10). APPE19 localized mainly within the cis/medial Golgi compartment and exclusively suppresses the secretion of APP. In contrast, deletion of the C-terminal tail promotes sAPP secretion by a constitutive secretion pathway. Metabolic labeling followed by immunoprecipitation with anti-APP antibody revealed that APPE19 is rapidly degraded within 30 min and that the subsequent intracellular turnover rate is decreased with 40% of the protein retained within the cells even after a chase period a 3 h. In contrast, APPdeltaC10 is rapidly eliminated from the intracellular compartments and secreted into the culture medium. The surface internalization and recycling processes of this protein are relatively impaired compared with wild-type APP. The ratios of the levels of production to secretion of sAPP alpha, the N-terminal, soluble APP fragment released by alpha-secretase, are proportional to the secretion efficiencies among APP species, suggesting the localization of alpha-secretase within a compartment late in the constitutive secretion pathway. These secretion mutants which utilize ER targeting signals are useful tools for analyzing the location of secretases and the intracellular degradation system within a constitutive secretion pathway such as ER quality control.

Amyloid precursor protein (APP) is degraded within the amyloid β-protein (Aβ) domain and its large soluble N-terminal fragment (secreted form of APP: APP s ) is secreted into the culture media of many kinds of cells. We report here a quantitative increase in APP s secretion in the medum of human glioblastoma A172 cells grown under serum-free conditions. When A172 cells were treated with inhibitors of the arachidonate cascade, a modulation of APP s secretion was observed; the addition of small amounts of indomethacin increased secretory cleavage, but higher doses suppressed it. Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenases, also inhibited APP s secretion. These results suggest that arachidonate metabolites of the leukotriene pathway may promote APP s release upon extracellular signaling via a signal transduction pathway, while metabolites of the prostaglandin pathway inhibit APP s secretion.

UV-B irradiation is one of the risk factors in age-related diseases. We have reported that biologically uncommon D-β-Asp residues accumulate in proteins from sun-exposed elderly human skin. A previous study also reported that carboxymethyl lysine (CML; one of the advanced glycation end products (AGEs)) which is produced by the oxidation of glucose and peroxidation of lipid, also increases upon UV B irradiation. The formation of D-β-Asp and CML were reported as the alteration of proteins in UV B irradiated skin, independently. In this study, in order to clarify the relationship between the formation of D-β-Asp and CML, immunohistochemical analysis using anti-D-β-Asp containing peptide antibodies and anti-CML antibodies was performed in UV B irradiated mice. Immunohistochemical analyses clearly indicated that an anti-D-β-Asp containing peptide antibody and anti-CML antibody reacted at a common area in UV B irradiated skin. Western blot analyses of the proteins isolated from UV B irradiated skin demonstrated that proteins of 50-70 kDa were immunoreactive towards antibodies for both D-β-Asp containing peptide and CML. These proteins were identified by proteomic analysis as members of the keratin families including keratin-1, keratin-6B, keratin-10, and keratin-14.

Prior to the emergence of life, it is believed that only L-amino acids were selected for formation of proteins, and that D-amino acids were eliminated on the primitive Earth. Whilst homochirality is essential for life, recently the occurrence of proteins containing D-beta-aspartyl (Asp) residues from various tissues of elderly subjects has been reported. Here, we discuss the presence of D-beta-Asp-containing proteins in the lens, ciliary body, drusen, and sclera of the eye, skin, cardiac muscle, blood vessels of the lung, chief cells of the stomach, longitudinal and circular muscles of the stomach, and small and large intestines. Since the D-beta-Asp residue occurs through a succinimide intermediate, this isomer may potentially be generated in proteins more easily than initially thought. UV Rays and oxidative stress can accelerate the formation of the D-beta-Asp residue in proteins.

Prior to the emergence of life, it is believed that only L-amino acids were selected for formation of proteins, and that D-amino acids were eliminated on the primitive Earth. Whilst homochirality is essential for life, recently the occurrence of proteins containing D-beta-aspartyl (Asp) residues from various tissues of elderly subjects has been reported. Here, we discuss the presence of D-beta-Asp-containing proteins in the lens, ciliary body, drusen, and sclera of the eye, skin, cardiac muscle, blood vessels of the lung, chief cells of the stomach, longitudinal and circular muscles of the stomach, and small and large intestines. Since the D-beta-Asp residue occurs through a succinimide intermediate, this isomer may potentially be generated in proteins more easily than initially thought. UV Rays and oxidative stress can accelerate the formation of the D-beta-Asp residue in proteins.

Proteins have been considered to consist exclusively of l-amino acids in living tissues. However, our previous studies showed that two specific aspartyl (Asp) residues in αA- and αB-crystallins from human eye lenses invert to the d-isomers to a high degree during aging. The reaction is also accompanied by isomerization into a form containing β-Asp (isoaspartate) residues. The appearance of d- and β-Asp in a protein potentially induces large changes to the higher order structure of the protein as well as to its function. However, it remains unclear whether the formation of the Asp isomer is the direct trigger of the change to the higher order structure and function. In this study, in order to clarify the effect of the inversion to d-isomers in a protein, we synthesized peptides corresponding to the 70–88 (KFVIFLDVKHFSPEDLTVK) fragment of human αA-crystallin and its corresponding diastereoisomers in which lα-Asp was replaced with lβ-Asp, dα-Asp, and dβ-Asp at position 76 and compared their biochemical properties with that of normal peptide. The peptides containing abnormal isomers (lβ-Asp, dα-Asp, and dβ-Asp residues, respectively) were more hydrophilic than the normal peptide (containing lα-Asp), lost β-sheet structure and changed to random structures. The normal peptide promoted the aggregation of insulin while the other three isomers suppressed the aggregation of insulin. This is the first evidence that a single substitution of an Asp isomer in a peptide induces a large change to the properties of the peptide.

Asp58 and Asp151 in alpha A-crystallin of human eye lenses become highly inverted and isomerized to d-beta-Asp residues with age. Racemization was previously shown to proceed rapidly when the residue on the carboxyl side of the Asp residue is small. Asn was also demonstrated to be more susceptible to racemization than Asp in protein. In this study, the changes of rate constants for racemization at Asp58 and Asp151 and at Asn58 and Asn151 were investigated using D58N, S59T, D151N and A152V mutants obtained through site-directed mutagenesis. The rate constant of racemization at Asn151 in D151N was found to be 1.5 times more rapid than Asp151 in the wild-type. For A152V, the rate constant at Asp151 was 1/4 that of the wild-type. There were no significant differences in the rate constants of racemization for both Asp58 and Asn58 residues. The aggregate size of D58N, S59T and D151N mutants increased or increased in polydispersity and their chaperone activities decreased. The size and chaperone activity of A152V was unchanged. These results suggest that structures close to Asp58 and Asp151 residues in the protein affect the rate constant of Asp racemization and the size and chaperone function of alpha A-crystallin.

PURPOSE: The gene expression profile of human corneal endothelium (CE) was established with the gene signature system. A novel gene, GS3582, was abundantly transcribed in the CE compared with other tissues according to a human gene expression database. This protein was designated corneal endothelium-specific protein (CESP)-1. The tissue distribution and subcellular localization of CESP-1 was assessed in humans and mice, to investigate its physiological function. METHODS: Rabbit and mouse CESP-1 cDNAs were cloned, and a polyclonal anti-human CESP-1 antibody (Ab) and anti-mouse N- or C-terminal ovary-specific acidic protein (OSAP)-1 Ab were produced. CESP-1 expression was investigated in human and mouse corneas by Western blot and/or immunohistochemical analysis. The distribution of CESP-1 in human tissues was also examined by Western blot analysis. To identify the subcellular localization of CESP-1, cultured human CE was colabeled with anti-human CESP-1 Ab and anti-cytochrome c monoclonal Ab or anti-GRP78 monoclonal Ab for confocal microscopy. RESULTS: The rabbit and mouse CESP-1 cDNA sequences contained an open reading frame coding 242 and 283 amino acids, respectively. Mouse CESP-1 was entirely consistent with mouse OSAP. Western blot analysis showed that CESP-1 was expressed in the human corneal epithelium, CE, cultured CE, brain, testis, and ovary. Mouse CESP-1 was also expressed in mouse corneal epithelium and CE with anti-mouse C- but not N-terminal OSAP Ab according to immunohistochemical analysis. Subcellular localization of CESP-1 to the mitochondria was demonstrated in cultured human CE. The N-terminal of CESP-1, possessing a mitochondrial targeting sequence, may be processed after the protein is imported into the mitochondria. CONCLUSIONS: CESP-1 was distributed in the corneal epithelium, the CE and cultured human CE, as well as the brain, testis, and ovary. CESP-1 was localized in the mitochondria of cultured human CE. These findings may provide some clues about the physiological function of CESP-1.

The accumulation of D-isomers of aspartic acid (D-Asp) in proteins during aging has been implicated in the pathogenesis of Alzheimer's disease (AD), cataracts and arteriosclerosis. Here, we identified a specific lactacystin-sensitive endopeptidase that cleaves the D-Asp-containing protein and named it D-aspartyl endopeptidase (DAEP). DAEP has a multi-complex structure (MW: 600 kDa) and is localized in the inner mitochondrial membrane. However, DAEP activity was not detected in E. coli, S. cerevisiae, and C. elegans. A specific inhibitor for DAEP, i-DAEP: (benzoyl-L-Arg-L-His-[D-Asp]-CH(2)Cl; MW: 563.01), was newly synthesized and inhibited DAEP activity (IC(50), 3 microM), a factor of ten greater than lactacystin on DAEP. On the other hand, i-DAEP did not inhibit either the 20S or 26S proteasome. And we identified succinate dehydrogenase and glutamate dehydrogenase 1 as components of DAEP by affinity label using biotinylated i-DAEP. In the long life span of mammals, DAEP may serve as a scavenger against accumulation of racemized proteins in aging. Insights into DAEP will provide the foundation for developing treatments of diseases, such as AD, in which accumulation of D-Asp-containing proteins are implicated.

To describe genes expressed in human corneal endothelial cells and identify novel genes.Sixteen human donor corneas that had no history of corneal disease, infection, or intraocular surgery were used within 7 days of death. Total RNA was extracted from corneal endothelial cells with attached Descemet membranes. A 3'-directed cDNA library was constructed from mRNA by using a pUC19-based primer. These sequences were compared with each other to determine their frequency and were searched against GenBank for identification. To identify novel specific and abundant transcript genes in corneal endothelial cells, the novel genes were compared with an expressed sequence tag database, the expected sequence extended, and 5' rapid amplification of cDNA ends-polymerase chain reaction cloning performed.The human corneal endothelial cDNA library showed that the most abundant transcript was prostaglandin D2 synthase. The remaining transcript genes that were present in abundance consisted of lactate dehydrogenase-A, gene signature (GS) 3582, which is a novel gene without a known function, and matrix Gla protein. The full-length sequence of GS3582 showed similarity to genes obtained in ovary and TESTIS.A human corneal endothelial cDNA library was constructed. An expression profile of corneal endothelium provides probes to monitor physiologic and pathologic conditions of this tissue in terms of gene expression.

Ganglioside GD3 induced the release of cytochrome c from isolated rat liver mitochondria. This process was completely prevented by cyclosporin A and partially prevented by a cysteine protease inhibitor, n-acetyl-leu-leu-norleucinal. Cyclosporin A is a potent inhibitor of the permeability transition pore, whereas n-acetyl-leu-leu-norleucinal has no effect on this pore. These results indicate that the release of cytochrome c from mitochondria requires both the opening of the permeability transition pore and a cysteine protease inhibitor-sensitive mechanism. Gangliosides GD1a, GD1b, GT1b, and GQ1b along with the synthetic GD3 mimetics TMS-42 and CI-22, which are glycerophospholipids carrying a disialo residue, also induced cytochrome c release. In contrast, gangliosides GM1, GM2, and GM3 did not induce cytochrome c release. These results indicate that two sialo residues must play an important role in the induction of cytochrome c release by gangliosides.

A large soluble N-terminal fragment of Alzheimer's disease amyloid precursor protein (secreted form of APP: APPs) is produced by constitutive processing in the middle of the amyloid beta-protein portion of APP. Recent studies indicate that the activation of endogenous protein kinase C (PKC) with phorbol ester raises the rate of secretion of APPs. We constructed rat fibroblast 3Y1 cells that stably overexpress PKC isoenzymes alpha, delta, or epsilon, and analyzed the amount of APPs released from these PKC transfectants. The levels of APPs released from 3Y1 cells overexpressing PKC alpha and -epsilon were higher than those from PKC delta-transfected and control cells expressing vector only. These results suggest that specific isoforms of PKC regulate the secretion of APPs through a signaling pathway.

KINBARA, K., KITAGAKI, H., KINOUCHI, T., OKANO, M., SORIMACHI, H., ISHIURA, S. and SUZUKI, K. Processing and Secretion of Alzheimers's Disease Amyloid Precursor Protein. Tohoku J. Exp. Med., 1994, 174 (3), 209-216 - We studied in vivo expression and in vitro secretion of the Alzheimer's disease amyloid precursor protein (APP). The results indicate that secretion of APP is mediated by PKC and the initial step of the processing may occur in the acidic secretooy granules of the glial cells. Our results suggest that a metabolic switch of APP in neural cells is critical in amyloid deposition.

The major component of Alzheimer's disease amyloid is a small polypeptide referred as the amyloid beta protein, which is derived from a larger precursor, amyloid precursor protein (APP). Cell fractionation and immunological studies on the APP molecule indicate that APP is localized either in the neuron and astrocyte and that the molecule is recovered from lysosomal fraction.

Alzheimer's disease amyloid precursor protein (APP) generates a beta-amyloid protein (A beta) that is a main component of the senile plaques found in the brains of Alzheimer's disease patients. APP is thought to undergo proteolysis via two different pathways, the amyloidogenic pathway which produces A beta, and the non-amyloidogenic pathway which releases a large N-terminal fragment into the medium. The proteases that mediate these processes remain unidentified. The physiological function of APP is not clear yet. Therefore, the cytoplasmic region of APP has attracted much interest, because this region is highly conserved among species, and members of the amyloid precursor-like protein (APLP) family. Several potentially functional sequences exist in the region, including signal sequences for protein sorting and a G0-protein binding sequence. We constructed two mutants, 695 deltaNPTY and 695 deltaGYEN. They lack potential endosome/lysosome targeting signals, NPTY and GY, in the cytoplasmic domain of APP695, respectively. The mutant APPs had longer half-lives and were secreted more easily into the medium than the wild type, suggesting that these sequences are important for the secretion and metabolism of APP.