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We previously showed that bovine apolipoprotein A-II (apoA-II) had antimicrobial activity against Escherichia coli and the yeast Saccharomyces cerevisiae in PBS. We have characterized here the active domain of apoA-II using synthetic peptides. A peptide corresponding to C-terminal residues Leu(49)-Thr(76) exhibited significant antimicrobial activity against E. coli in PBS, but not against S. cerevisiae. Experiments using amino-acid-substituted peptides indicated that the residues Phe(52)-Phe(53)-Lys(54)-Lys(55) are required for the activity. Peptide Leu(49)-Thr(76) induced the release of calcein trapped inside the vesicles whose lipid composition resembles that of E. coli membrane, suggesting that peptide Leu(49)-Thr(76) can destabilize the E. coli membrane. CD measurements showed that the alpha-helicity of peptide Leu(49)-Thr(76) increased from 3.5 to 36% by addition of the vesicles. When E. coli cells were incubated with peptide Leu(49)-Thr(76), some proteins were released to the external medium, probably owing to membrane destabilization caused by the peptide. In electron micrographs of E. coli cells treated with peptide Leu(49)-Thr(76), transparent nucleoids and granulated cytoplasm were observed. Amino acid substitutions, Phe(52)Phe(53)-->AlaAla (Phe(52, 53)-->Ala) in peptide Leu(49)-Thr(76) caused the loss of antimicrobial activity against E. coli, although protein-releasing activity was retained. Electron micrographs of the cells treated with peptide Leu(49)-Thr(76)(Phe(52,53)-->Ala) revealed morphological change only at the nucleoids. Therefore peptide Leu(49)-Thr(76) appears to primarily target the cytoplasm rather than the membrane of E. coli cells.

This paper presents further investigation of the properties of carbohydrate II in the cell adhesion molecule, contact site A, from Dictyostelium discoideum. A purified contact site A was digested with Achromobacter protease I to produce a 31-kDa fragment to which carbohydrate II was mainly bound and a 21-kDa fragment containing the NH2 terminus of contact site A, which was identified as Ala-Pro-Thr-Ile-Thr-Ala. The NH2 terminus of the 31-kDa fragment was Thr-Glu-Ala-Thr-Thr-Ser. It was estimated from the cDNA sequence data of contact site A that more than 20 Ser/Thr residues exist as target sites for the O-linked oligosaccharides in the 31-kDa fragment, but not for the N-linked oligosaccharides. These results suggest that carbohydrate II exists as clustered O-linked oligosaccharides in the COOH terminus of contact site A. The results of two-dimensional electrophoresis confirm that oligosaccharides of contact site A contain sialic acids. Immunoelectron microscopy was carried out to define the organelle in which O-glycosylation by carbohydrate II occurs and how carbohydrate II antigens are distributed on the cell surface. The results show that O-glycosylation can occur in the Golgi apparatus in D. discoideum as observed in other cells, although this O-glycosylation was inhibited by tunicamycin. Furthermore, gold particles were densely concentrated in cell-cell contact regions but sparsely distributed in noncontact regions.

Gypsophila elegans contains a new type 1 ribosome-inactivating protein, which we named gypsophilin. The protein was purified to apparent homogeneity by (NH4)2SO4 fractionation, ion-exchange chromatography, and adsorption chromatography. The protein was found to have a molecular mass of 28.0 kDa and a pI of about 10.1. It does not contain glycosidic linkages. The sequence of the N-terminal 22 amino acids of the protein shows a close relationship to other RIPs. The enzyme strongly inhibits protein synthesis in a rabbit reticulocyte lysate and depurinates 28S rRNA in rat liver ribosomes in a manner identical to that of ricin A-chain and other RIPs. Using a direct method for the measurement of the RNA N-glycosidase activity, the substrate specificity of gypsophilin was identified. EC50 of the protein for ribosomes of rat liver, wheat germ, and E. coli was 39.8 pM, 0.24 nM, and 0.82 microM, respectively. Gypsophilin may be one of the most active RNA N-glycosidases among the RIPs known to date. Immunoelectron microscopic localization of gypsophilin in the leaves shows that the protein is accumulated densely in the intercellular spaces and is also distributed within vacuoles in the cytoplasm.

Effect of fixative concentration, fixation time, embedding media, condition by dehydration and embedding, and incubation time with primary antibody upon the labeling intensity, was investigated by quantitative immunoelectron microscopy. Rat liver tissue and erythrocytes processed under various conditions of preparation were embedded in LR White or Lowicryl K4M. As concentration of glutaraldehyde (GA) in fixative increased, labeling density decreased. In the tissue and cells fixed with fixative containing 4% paraformaldehyde and 0.25% GA for 5-10min, cell ultrastructure and antigenicity were well preserved. As fixation was prolonged, labeling density was lowered. In the materials embedded in acrylic resins, such as LR White and Lowicryl K4M, high labeling density was obtained, compared with that obtained from embedding in epoxy resins such as Epon 812, Durcupan and Quetol 651. Temperature difference between dehydration and embedding affected strongly the preservation of antigenicity. There was no difference in labeling densities between embedding at 23°C and -20°C, when tissue slices were dehydrated at 4°C. To saturate the reaction of primary antibody with antigen on thin sections incubation should be carried out over 8hr. From these results, we recommend the following points in immunoelectron microscopy; 1) perfusion fixation with 4% paraformaldehyde+0.25% GA for 5min to 10min, 2) minimized temperature difference between dehydration and embedding of tissue, 3) use of acrylic resins for embedding, and 4) over 8 hr-incubation with primary antibody.

Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure-activity relationship of inulavosin and its benzo-derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper-binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzo-derivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo-tyrosinase that has a conformational defect.

To determine the distribution of cellular glutathione peroxidase in rat lungs, the tissues were stained immunohistochemically. Quantitative analysis was performed in certain cell types of alveolar linings, after the ultrathin sections were stained by a postembedding immunogold technique. Immunoblot analysis revealed that homogenates of rat liver, heart, and lungs all gave a single band. Under the light microscope, the following tissues were stained intensely: epithelial cells, smooth muscle cells and glands of bronchi and bronchioles, type II alveolar cells, and alveolar macrophages. Under immunoelectron microscopy, type II alveolar cells and macrophages were abundant in mitochondria. The mitochondria, nucleus, and cytoplasm of macrophages were labeled almost twice as densely as the respective compartments of type II alveolar cells. Within cell types, the mitochondria were labeled twice as densely as the nuclei. The other particles were less than half as densely labeled as the nuclei. The labeling was slightly less dense in the cytoplasm than in the nucleus. The present study revealed that glutathione peroxidase occurred predominantly in the epithelial linings and metabolically active sites in rat lungs. The tissues that were previously found to be rich in superoxide dismutases were also rich in glutathione peroxidase.

Effect of reduced OsO4 fixation on the antigenicity of rat liver catalase and erythrocyte esterase D was investigated. After fixation with mixture of 4% paraformaldehyde and 0.2% glutaraldehyde, rat liver and erythrocytes were post-fixed with reduced osmium (0.1% to 1.0%) for 1 hr. Some erythrocytes were fixed with 0.5% reduced osmium for various times (10 min to 60min). The antigenicity of both enzymes was expressed as labeling density (gold particles/μm2 of peroxisomes or erythrocytes). Fixation with reduced OsO4 greatly affected the antigenicity of both enzymes. Approximately 80% of catalase antigenicity was lost at 0.4% reduced OsO4, whereas about 80% of esterase D antigenicity was lost at 0.2%. Positive contrast of membrane structures clearly appeared at 0.6% OsO4. About 80% loss of the esterase D antigenicity was observed at 10min after fixation with 0.5% reduced OsO4. Afterwards, the antigenicity decreased very slowly. The data indicates that at the concentration of reduced OsO4 which stains the membrane structures positive contrast, 80% of the antigenicity is lost in the cases of catalase and esterase D.

In the electron micrographs of rat hepatocytes, acid phosphatase (ACPase)-positive profiles were classified as either round or elongate types by image analysis, according to a shape index. The former is typical of spherical lysosomes, and the latter are presumed to be the same structures that we have previously termed nematolysosomes. The localization of cathepsin D in these two types of ACPase-positive profiles was electron microscopically examined by a combination of enzyme cytochemistry for ACPase and postembedding immunocytochemistry for cathepsin D. Gold particles showing antigenic sites for cathepsin D were largely present in ACPase-positive structures, although the labeling intensity of gold particles varied with individual sectional profiles of these structures. Quantitative analysis of the labeling density in the two types of ACPase-positive profiles revealed that the amount of cathepsin D in the elongate-type population was smaller than that in the round-type one. This result suggests that the contents of most elongate ACPase-positive structures are different from those of spherical lysosomes and may be similar to those of endosomes. It was also frequently observed that some of the elongate ACPase-positive profiles labeled with few or no gold particles were fused with round profiles which were heavily labeled with gold particles for cathepsin D. It is possible that such fused profiles may be sites for junctions of the two different transport pathways for ACPase and cathepsin D being delivered to lysosomes. Finally, these elongate ACPase-positive structures seem to be equivalent to late endosomes or a different kind of lysosomes containing lower concentrations of hydrolases.

Administration of acetylsalicylic acid (ASA) to rats caused an alteration in intraperoxisomal localization of enzymes in liver peroxisomes as investigated by means of immunoelectron microscopy. Rats were fed on a diet containing 0.5% ASA for 2 weeks and livers were fixed by perfusion. Vibratome sections of the liver tissue were processed to routine electron microscopy (EM), alkaline 3, 3′-diaminobenzidine (DAB) reaction for catalase, and post embedding immunocytochemistry for several peroxisomal enzymes. By light microscopy (LM) of the DAB stained materials, many large spindleform peroxisomes were noted in the cytoplasm of hepatocytes, among which normal size of peroxisomes was also observed. By EM, such large peroxisomes contained fibrillar inclusions in the matrix in addition to the crystalloid core. The fibrillar inclusions were more strongly stained than the matrix after the DAB reaction for catalase. By postembedding immunoelectron microscopy, gold particles showing catalase, acyl-CoA oxidase, L-α-hydroxy-acid oxidase A and serine: pyruvate/glyoxylate aminotransferase (SPT) were closely associated with the fibrillar structures. Urate oxidase was confined exclusively to the crystalloid core, on which a few gold particles showing L-α-hydroxy-acid oxidase A was also noted. D-amino acid oxidase was associated exclusively with a clear spot located adjacent to the crystalloid core. In this clear spot, no other enzymes examined were present. The results show that the fibrillar structure induced by ASA includes at least catalase, acyl-CoA oxidase, L-α-hydroxy-acid oxidase A and SPT, but not urate oxidase and D-amino acid oxidase. D-amino acid oxidase is one of elements that constitute the clear spot. Taken together, the present results demonstrate that some subcompartments exist in the experimentally altered liver peroxisomes.

Here we describe postembedding immunoelectron microscopic technique applied to ultrathin sections of plant material. The method relies on the use of gold nanoparticles. The methods include tissue fixation, dehydration, embedding of plant specimens, and staining of semithin and ultrathin sections. The described method is suitable for localization of antigens including proteins (peroxisomal enzymes) and a low molecular substance such as ginsenoide-Rb1.

The nuage is a germ cell-specific organelle that has been studied for more than a hundred years. It was first discovered in spermatogenic cells as a perinuclear granule stained by basic dyes and visualized using a light microscope. Morphological studies using electron microscopes demonstrated that ”chromatoid body (CB)” is a nuage component in spermatids, and that “inter-mitochondrial cement (IMC)” is another nuage component found in spermatocytes. During meiosis, the IMC disappears, but the CB soon reforms in post-meiotic spermatids. Recent morphological and molecular biological studies have identified many components of nuage, and suggest that they play roles in the silencing, decay, and storage of RNA, and in the aggresome system. In this report, we summarize recent findings related to nuage and discuss their functions in spermatogenic cells.

Here we describe postembedding immunoelectron microscopic technique applied to ultrathin sections of plant material. The method relies on the use of gold nanoparticles. The methods include tissue fixation, dehydration, embedding of plant specimens, and staining of semithin and ultrathin sections. The described method is suitable for localization of antigens including proteins (peroxisomal enzymes) and a low molecular substance such as ginsenoide-Rb1.

The localization of DDX25/GRTH and gonadotropin-stimulated RNA helicase was studied in the spermatogenic cells of rat, mouse, and guinea pig by immunofluorescence and immunoelectron microscopy (IEM). Immunofluorescence studies identified four kinds of granular staining: (1) fine particles observed in meiotic cells; (2) small granules associated with a mitochondrial marker, appearing in pachytene spermatocytes after stage V; (3) short strands lacking the mitochondrial marker in late spermatocytes; and, (4) large irregularly shaped granules in round spermatids. IEM identified DDX25 signals in nine compartments: (1) fine dense particles in the meiotic cells; (2) intermitochondrial cement; (3) loose aggregates of 70–90 nm particles; (4) chromatoid bodies; (5) late chromatoid bodies; (6) satellite bodies; (7) granulated bodies; (8) mitochondria-associated granules; and, (9) reticulated bodies. Compartments (1) to (6) were previously classified into nuage while (7) to (9) were classified as nuage components by the present study. The results suggest that DDX25 functions in these nine compartments.

Ubiquitylation of membrane proteins triggers their endocytosis at the plasma membrane and subsequent lysosomal degradation through multivesicular bodies (MVBs). A dominant-negative mutant SKD1/Vps4B caused an accumulation of ubiquitylated membrane proteins in MVBs. We have identified 22 membrane proteins whose trafficking is potentially regulated by ubiquitylation. Nine of them, including transferrin receptor (TfR), are indeed ubiquitylated and/or accumulated in MVBs in the cells expressing mutant Vps4. While the recycling route and iron-regulated expression of TfR are well characterized, the mechanism by which the degradation of TfR is regulated is largely unknown. We show that an excess of iron enhances both TfR's ubiquitylation and degradation in lysosomes. Probably, the up-regulated expression of ferritin, an endogenous iron-chelating molecule, attenuated the iron-induced degradation of TfR. Exogenously introduced lysine-less TfR, compared to the wild-type one, showed resistance to the iron-induced ubiquitylation and degradation, when endogenous TfR, which most certainly heterodimerizes with exogenous ones, was depleted with siRNA. These data suggest that the iron-induced ubiquitylation and degradation of TfR along with MVB pathway physiologically plays an important role in iron homeostasis.

Several basic problems in quantitative immunoelectron microscopy were investigated. The catalase activities in the liver homogenate of guinea pig, rat, Chinese hamster and mouse were assayed biochemically, while the peroxisomal labeling density (gold particles/μm2) for this enzyme of the same animals was determined by protein A-gold technique. The species difference of the enzyme activity did not correspond to the difference of the labeling density. Furthermore, the difference of the enzyme activity did not correspond to the staining intensity of the signal developed by immunoblot for catalase. However, the labeling density largely corresponded to the immunoblot data. Peroxisomal labeling densities of the above animals were determined by protein A-gold technique using rabbit anti-rat liver catalase (RLC) or guinea pig anti-RLC. The data obtained by the rabbit antibody was not consistent with those obtained by the guinea pig antibody. Moreover, we investigated the relationship between antigen concentration and labeling density using a model system in which bovine serum albumin (BSA) or purified RLC is directly mixed with LR White resin at various concentrations. The labeling density was directly proportional to the antigen concentration within the examined range. The results clearly demonstrate that the antigen concentration is expressed by the labeling density. Such proportionality is realized only between the same species. Taken altogether, we could compare data obtained quantitative immunoelectron microscopy between the same species even if using the same lot of antibody.

In order to expand the possibility combining enzyme cytochemistry and immunocytochemistry, the post-embedding immunogold technique was applied to tissue sections stained by either the cerium-based or lead-based method for phosphatases. After enzyme cytochemical staining for alkaline phosphatase (ALPase) and acid phosphatase (ACPase), rat liver sections were dehydrated and embedded in the LR White without osmication. The ultrathin sections were labeled by post-embedding immunogold technique for anti-cathepsin D.Both the lead-based and cerium-based methods were sufficient to localize enzyme activities in the tissue before the immunoreaction. However, lead phosphate deposits, which were reaction products of phosphatases detected by the lead-based method, largely disappeared from the tissue embedded in LR White during the post-embedding immunoreaction. In contrast, cerium phosphate deposits, reaction products by the cerium-based method, were retained in the tissue even after the immunoreaction. Moreover, from the clear-cut difference in particle sizes, the cerium phosphate precipitates were readily distinguished from the immunogold particles within the same organelle. The preceding enzyme cytochemical procedures by the cerium-based method did not hinder the sensitivity and specificity of post-embedding immunogold labeling. These results indicate that the cerium-based method has the advantage of combining pre-embedding enzyme cytochemistry with post-embedding immunocytochemistry.

It is important to know the localization of medicinal substance, Rb1, of Ginseng, Panax ginseng, in this plant in order to achieve efficient extraction of Rb1 or to culture producing cells. In this report, we describe the localization of Rb1 in various parts of the plant as determined by immunofluorescence (IF) and immunoelectron microscopies (IEM). Using IF, we show that Rb1 is localized to chloroplasts, peroxisomes and cytoplasm but not to vacuoles of leaf parenchymal cells. In the leaf stem, Rb1 is localized to the vascular bundles as well as vacuoles. In the root, vacuoles of parenchymal cells are stained at various intensities. Using IEM, gold particles showing Rb1 antigenic sites are present in the compartments stained by IF technique. In addition, Rb1 is localized in the sieve elements of the phloem and degrading primary cell wall of xylem, and in the root parenchymal cells Rb1 is associated with electron dense polymorphic materials but not in starch granules. Translocation and storage of Rb1 and effective utilization of leaves are discussed.

Colloidal gold probes, including protein A-, IgG-F(ab')(2)-, and streptavidin-labeled gold particles, are useful tools for localization of antigens in cells and tissues by immunoelectron microscopy (IEM). This chapter describes different methods for the preparation of colloidal gold and conjugation of colloidal gold to protein A, IgG, and streptavidin.

The localization of vasa homolog protein in the spermatogenic cells of mice, rats, and guinea pigs was studied by immunofluorescence and electron microscopies with the antibody against mouse vasa homolog (MVH) protein. By immunofluorescence microscopy, four types of granular staining patterns were identified: (1) fine particles observed in diplotene and meiotic cells, (2) small granules associated with a mitochondrial marker and appearing in pachytene spermatocytes after stage V, (3) strands lacking the mitochondrial marker in late spermatocytes, and (4) large irregularly shaped granules in round spermatids. Immunoelectron microscopy defined the ultrastructural profiles of these MVH protein-positive granules: the first type consisted of small dense particles, the second had intermitochondrial cement (IMC), the third type, consisting of strands, had loose aggregates of either material dissociated from IMC or 70-90-nm particles, and the fourth had typical chromatoid bodies (CBs). The results suggest that MVH proteins function in these components of nuage. MVH protein-positive structures other than CBs disappeared during meiosis and CB appeared first in early spermatids. The results suggest that the formation of nuage is discontinued between spermatocytes and spermatids. The formation of nuage in spermatocytes and of CB in spermatids is discussed.

Polyamines are known to play important roles in the proliferation and differentiation of many types of cells. Although considerable amounts of polyamines are synthesized and stored in the testes, their roles remain unknown. Ornithine decarboxylase antizymes (OAZs) control the intracellular concentration of polyamines in a feedback manner. OAZ1 and OAZ2 are expressed ubiquitously, whereas OAZ-t/OAZ3 is expressed specifically in germline cells during spermiogenesis. OAZ-t reportedly binds to ornithine decarboxylase (ODC) and inactivates ODC activity. In a prior study, polyamines were capable of inducing a frameshift at the frameshift sequence of OAZ-t mRNA, resulting in the translation of OAZ-t. To investigate the physiological role of OAZ-t, we generated OAZ-t–disrupted mutant mice. Homozygous OAZ-t mutant males were infertile, although the polyamine concentrations of epididymides and testes were normal in these mice, and females were fertile. Sperm were successfully recovered from the epididymides of the mutant mice, but the heads and tails of the sperm cells were easily separated in culture medium during incubation. Results indicated that OAZ-t is essential for the formation of a rigid junction between the head and tail during spermatogenesis. The detached tails and heads were alive, and most of the headless tails showed straight forward movement. Although the tailless sperm failed to acrosome-react, the heads were capable of fertilizing eggs via intracytoplasmic sperm injection. OAZ-t likely plays a key role in haploid germ cell differentiation via the local concentration of polyamines., Author Summary Polyamines are essential for cell proliferation and differentiation, but their role in these processes is unknown. Ornithine decarboxylase antizymes (OAZs) are enzymes that control the concentration of polyamines in cells. To elucidate the role of one of these enzymes, OAZ-t, in the regulation of polyamine concentration during sperm formation, we generated mutant mice in which the OAZ-t gene was disrupted. When we observed sperm from the mice lacking a functional Oaz-t gene, we found that the sperm heads separated easily from the tails, indicating that OAZ-t is essential for the formation of a rigid junction between the head and tail during sperm development. Many of the headless tails could continue swimming, but they were unable to participate in the signaling processes required for successful fertilization. However, tailless heads could produce healthy pups when injected into unfertilized eggs. Such a phenotype has not been previously found. The mutant mice evoked rare cases of infertile human patients whose sperm behaves in a proper fashion. Our study underscores the importance of research into the processes of spermatogenesis and fertilization.

The abundance of peroxisomes within a cell is rapidly controlled depending on environmental changes and physiological conditions. It is well established that phthalate esters can cause a marked proliferation of peroxisomes (Yokota, 1986). Following induction of peroxisomes by a 2-week treatment with phthalate esters in mouse livers, peroxisomal degradation via autophagy can be induced for the subsequent week after discontinuation of the phthalate esters. Autophagic degradation of peroxisomes can be monitored by electron microscopy as well as biochemical assay for some peroxisome markers. Although most of the excess peroxisomes in the liver are selectively degraded within one week, this rapid removal is exclusively impaired in the autophagy-deficient liver.

The abundance of peroxisomes within a cell is rapidly controlled depending on environmental changes and physiological conditions. It is well established that phthalate esters can cause a marked proliferation of peroxisomes (Yokota, 1986). Following induction of peroxisomes by a 2‐week treatment with phthalate esters in mouse livers, peroxisomal degradation via autophagy can be induced for the subsequent week after discontinuation of the phthalate esters. Autophagic degradation of peroxisomes can be monitored by electron microscopy as well as biochemical assay for some peroxisome markers. Although most of the excess peroxisomes in the liver are selectively degraded within one week, this rapid removal is exclusively impaired in the autophagy‐deficient liver.

To examine the function of the amino-terminal presequence of rat peroxisomal 3-ketoacyl-CoA thiolase precursor, fusion proteins of various amino-terminal regions of the precursor with non-peroxisomal enzymes were expressed in cultured mammalian cells. On immunofluorescence microscopy, all constructs carrying the presequence part exhibited punctate patterns of distribution, identical with that of catalase, a peroxisomal marker. Proteins lacking all or a part of the prepiece were found in the cytosol. These results indicate that the presequence of the thiolase has sufficient information for peroxisomal targeting.

LETM1 is located in the chromosomal region that is deleted in patients suffering Wolf-Hirschhorn syndrome; it encodes a homolog of the yeast protein Mdm38 that is involved in mitochondrial morphology. Here, we describe the LETM1-mediated regulation of the mitochondrial volume and its interaction with the mitochondrial AAA-ATPase BCS1L that is responsible for three different human disorders. LETM1 is a mitochondrial inner-membrane protein with a large domain extruding to the matrix. The LETM1 homolog LETM2 is a mitochondrial protein that is expressed preferentially in testis and sperm. LETM1 downregulation caused mitochondrial swelling and cristae disorganization, but seemed to have little effect on membrane fusion and fission. Formation of the respiratory-chain complex was impaired by LETM1 knockdown. Cells lacking mitochondrial DNA lost active respiratory chains but maintained mitochondrial tubular networks, indicating that mitochondrial swelling caused by LETM1 knockdown is not caused by the disassembly of the respiratory chains. LETM1 was co-precipitated with BCS1L and formation of the LETM1 complex depended on BCS1L levels, suggesting that BCS1L stimulates the assembly of the LETM1 complex. BCS1L knockdown caused disassembly of the respiratory chains as well as LETM1 downregulation and induced distinct changes in mitochondrial morphology.

For the analysis of the molecular mechanism of the action of peroxisome proliferators, we attempted to establish the optimal conditions for obtaining the effects of the chemicals in vitro, employing an established cell line, Reuber rat hepatoma H4IIEC3. Histochemical analyses revealed a marked increase in the number, size, and catalase content of peroxisomes in the cells cultured on a medium containing 0.5 mM ciprofibrate, a peroxisome proliferator. The activity of acyl-CoA oxidase, the initial enzyme of the peroxisomal beta-oxidation system, was increased by more than 10-fold by the same treatment. Catalase was also induced significantly, whereas the activities of glutamate dehydrogenase and lactate dehydrogenase, mitochondrial and cytosolic marker enzymes, did not change upon the treatment. Immunoblotting and RNA-blotting analyses confirmed the increases in the amount of protein and mRNA for all the three enzymes of the peroxisomal beta-oxidation system. Cell fractionation experiments gave a partial separation of peroxisomes from other organelles for the induced culture. Thus, H4IIEC3 cells offer a good in vitro model system of the induction of peroxisomes and peroxisomal beta-oxidation enzymes by peroxisome proliferators.

We made use of autoradiographic screening to isolate two Chinese hamster ovary (CHO) cell mutants deficient in peroxisomal dihydroxyacetonephosphate acyltransferase, a key enzyme for the biosynthesis of ether glycerolipids such as plasmalogens. Morphological analysis revealed no evidence of peroxisome in these mutants. Catalase was as active as in the normal cells but was not sedimentable. Pulse-chase radiolabeling experiments and cell-free translation of RNA demonstrated that acyl-CoA oxidase, the first enzyme of the peroxisomal beta-oxidation system, was synthesized as the 75-kD form but was not converted to 53- and 22-kD mature components that were present in the wild-type CHO cells; rather, degradation was apparent. Peroxisomal thiolase was synthesized as in normal cells but remained as a larger, 44-kD precursor, whereas maturation to the 41-kD enzyme was detected in the wild-type cells. The peroxisomal 70-kD integral membrane protein was also equally synthesized, as in the wild-type cells, and was not degraded. These results suggest that assembly of the peroxisomes is defective in the mutants, whereas the synthesis of peroxisomal proteins appears to be normal. Cell-fusion studies revealed that the two mutants are recessive to the wild-type CHO cells and belong to different complementation groups. Thus, these mutants presumably contain different lesions in gene(s) encoding factor(s) required for peroxisome assembly.

Cell growth in the yeast Saccharomyces cerevisiae depends on polarization of the actin cytoskeleton. In this study, we investigated how the cell regulates the distribution of actin in response to low pH conditions, focusing on the role of mitogen-activated protein kinases, Hog1 and Slt2. Changing the extracellular pH from 6.0 to 3.0 caused a transient depolarization of the actin cytoskeleton. Actin cables were no longer visible, and actin patches appeared randomly distributed after 30 min at pH 3.0. The deletion strain hog1 Δ did not show this low-pH phenotype, suggesting that Hog1 is involved in depolarization of the actin cytoskeleton in response to low-pH stress. Yeast cells incubated at pH 3.0 also showed markedly increased endocytosis compared with the control at neutral pH, as indicated by the uptake of Lucifer Yellow (LY). Both the hog1 Δ and slt2 Δ mutants took up LY into the vacuole to a similar extent as the wild-type strain. In addition, cells grown at pH 3.0 showed a 2-fold increase in phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) levels, as did the hog1 Δ or slt2 Δ cells. Efficient uptake of LY and actin repolarization at pH 3.0 might therefore require activation of PI(4,5)P2 synthesis.

Gingipains are cysteine proteinases that are responsible for the virulence ofPorphyromonas gingivalis. Recent studies have shown thatP. gingivalisis trapped within autophagic compartments of infected cells, where it promotes survival. In this study we investigated the role of gingipains in the intracellular trafficking and survival of this bacterium in human aortic endothelial cells and any possible involvement of these enzymes in the autophagic pathway. Although autophagic events were enhanced by infection with either wild-type (WT)P. gingivalisstrains (ATCC 33277, 381, and W83) or an ATCC 33277 mutant lacking gingipains (KDP136), we have found that more than 90% of intracellular WT and KDP136 colocalized with cathepsin B, a lysosome marker, and only a few of the internalized cells colocalized with LC3, an autophagosome marker, during the 0.5- to 4-h postinfection period. This was further substantiated by immunogold electron microscopic analyses, thus implying thatP. gingivalisevades the autophagic pathway and instead directly traffics to the endocytic pathway to lysosomes. At the late stages after infection, WT strains in phagolysosomes retained their double-membrane structures. KDP136 in these compartments, however, lost its double-membrane structures, representing a characteristic feature of its vulnerability to rupture. Together with the ultrastructural observations, we found that the number of intracellular viable WT cells decreased more slowly than that of KDP136 cells, thus suggesting that gingipains contribute to bacterial survival, but not to trafficking, within the infected cells.

In the course of studying [PSI(+)], a yeast prion, we found inadvertently that Escherichia coli strain BL21 overproducing a fusion protein, in which the prion-domain of Sup35 was connected to the C terminus of glutathione S-transferase, grew normally to the stationary phase and rapidly decreased in colony-forming ability thereafter. Evidence indicated that protein polymers consisting mainly of the fusion protein GST-Sup35NM (about 70% of the mass) and its N-terminal fragments were formed in extract prepared from the cells producing GST-Sup35NM. It was further found that cells of strain BL21 accumulated the protein polymers during prolonged cultivation. Based on these results, we contend that the initially observed defect in colony forming ability is the direct or indirect consequence of intracellular formation and accumulation of the protein polymers.

Selection of good quality oocytes is important for improvement of assisted reproductive technology. Here, we studied the relationship of the mitochondrial distribution in metaphase II stage (MII) oocytes with fertility, since mitochondria in ooplasm are essential for energy production required for fertilization and embryo development. To observe mitochondria non-invasively, we used oocytes from a transgenic mouse, in which enhanced green fluorescent protein is targeted to the mitochondrial matrix and thus fluorescence is observed exclusively in the mitochondria. Control oocytes with mitochondria distributed around the nucleus showed normal embryo developmental competence, whereas oocytes with abnormal diffuse and fragmented mitochondria showed a significantly lower rate of embryo development after activation by intracytoplasmic sperm injection or strontium, which is a very effective agent for activation of mouse oocytes. Also, we showed that the reduced developmental competence of oocytes with diffuse and fragmented mitochondria caused by vitrification and thawing is similar to that of oocytes with abnormal mitochondrial foci obtained naturally. These findings suggest that abnormal mitochondrial distribution in oocytes at MII is a cause of developmental retardation and therefore normal mitochondrial distribution could be used as a criterion for selection of good oocytes.

Peroxisomes are organelles that are almost ubiquitous in eukaryotic cells. They have, however, never been described in germ cells within the testis. Since some peroxisomal diseases like Adrenoleukodystrophy are associated with reduced fertility, we have re-investigated the peroxisomal compartment of the germinal epithelium of mice using in situ hybridization, immunohistochemistry, Western blotting and immunoelectron microscopy. Within the seminiferous tubules, peroxisomes are present in Sertoli cells and in germ cells. We could show that small-sized peroxisomes of typical ultrastructure are concentrated in spermatogonia and disappear during the course of spermatogenesis. Peroxisomes of spermatogonia differ in their relative protein composition from previously described peroxisomes of interstitial cells of Leydig. Since germ cells differentiate in mouse testis in a synchronized fashion, the disappearence of peroxisomes could be a suitable model system to investigate the degradation of an organelle as part of a physiological differentiation process in higher eukaryotes.

Alpha-fetoprotein (AFP) is a major fetal serum protein, the biologic role of which has not been not fully elucidated. Recently, existence of a novel AFP mRNA isoform (del.1 AFP mRNA isoform), which is transcribed from the intron A (the intron between exons 1 and 2), has been reported in murine yolk sac and fetal liver. In the present study, we intended to identify the human homologue of the murine AFP mRNA isoform in the yolk sac tumor.To investigate the existence of the mRNA isoform (which we termed the "AFP-C mRNA isoform"), reverse transcription-polymerase chain reaction (RT-PCR) was used. Moreover, the expression analysis of the AFP-C cDNA isoform using the AFP-negative human cell line was carried out.RT-PCR revealed the existence of the AFP-C mRNA isoform in the yolk sac tumor and human hepatocellular carcinoma cells. The expression analysis clarified that the molecular size of the AFP-C was approximately 65 kd, and that the protein was not secreted, in contrast to the traditional AFP.From these results, the existence of the AFP-C mRNA isoform has been demonstrated for the first time in humans. The AFP-C located in cytoplasm possibly plays physiologic/pathogenic roles distinct from those of the traditional AFP in the yolk sac tumor and hepatocellular carcinoma.

The localization of ubiquitin (UB) signals in the acrosomes of rat spermiogenic cells was investigated by immunoelectron microscopy using two anti-UB antibodies: UB1, reacting with ubiquitinated proteins and free UB; and FK1, recognizing polyubiquitinated proteins but not monoubiquitinated proteins or free UB. Labeling of UB by UB1 (UB1 signal) was detected in the acrosomes at any stage of differentiation. In step 1 spermatids, UB1 signals were detected on the cytoplasmic surface and in the matrix of transport vesicles located between the trans-Golgi network and the acrosome. Weak signals were detected in acrosomal granules within acrosome vesicles that had not yet attached to the nucleus. In step 4–5 spermatids, the acrosome vesicles had enlarged and attached to the nucleus. Strong gold labeling was noted in a narrow space between the outer acrosomal membrane and the developing acrosomal granule, where a dense fibrous material was observed on routine electron microscopy, whereas the acrosomal granule was weakly stained by UB1 antibody. In step 6–8 spermatids, UB1 signals were detected in the fibrous material that expanded laterally to form a narrow electronless dense zone between the acrosomal granule and the outer acrosomal membrane. Labeling in the acrosomal granule increased. In step 9–11 spermatids, UB1 signals were confined to the narrow zone from the tip of the head to the periphery of the ventral fin. The matrix of the acrosome was weakly stained. In epididymal sperm, UB1 labeling in the acrosome decreased without any pretreatment, whereas staining was noted in a spot in the neck region and in the dorsal fin after trypsin digestion. On the other hand, the staining pattern with FK1 was quite different from that with UB1. The trans-Golgi network was weakly stained but the cis-Golgi network was strongly stained. The dense fibrous material just beneath the outer membrane was never stained with FK1. The results suggest that UB on the surface of transport vesicles is involved in anterograde transport from the Golgi apparatus to the acrosome. The physiological role of UB in acrosomes is not clear. Two candidates for monoubiquitinated proteins in the acrosome, which have a UB-interacting motif, were found by cyber screening.

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 (M(r)) 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 degrees C and internalized beta-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.

The distribution of lipocaline-type prostaglandin D synthase (L-PGDS) in rat brain was investigated by immunoelectron microscopy using a protein A-gold technique. In perivascular cells adjacent to the basement membrane of arterioles in the pia-arachnoid and of blood vessels in the subpial cortex, gold labeling was confined to the lumen of the dilated rough endoplasmic reticulum, and not found in the few lysosomes present in the cytoplasm. The results suggest that the perivascular cells secrete L-PGDS and seem not to degrade lipophilic molecules carried by L-PGDS. Moreover, gold particles representing the antigenic sites of L-PGDS were found in the Golgi apparatus, rough endoplasmic reticulum, vesicles, and nuclear envelope of arachnoid trabecular cells, arachnoid barrier cells, and arachnoid pia mater cells. The labeling was less detectable in the same organelles of choroid plexus epithelial cells, compared with leptomeningeal cells. In meningeal macrophages and parenchymal microglia, L-PGDS was detected in lysosomes, multivesicular bodies, and endocytic vesicles. The production of L-PGDS in perivascular cells is important to the various functions of this enzyme in brain parenchyma.

Subcellular proteomics, which includes isolation of subcellular components prior to a proteomic analysis, is advantageous not only in characterizing large macro-molecular complexes such as organelles but also in elucidating mechanisms of protein transport and organelle biosynthesis. Because of the high sensitivity achieved by the present proteomics technology, the purity of samples to be analyzed is important for the interpretation of the results obtained. In the present study, peroxisomes isolated from rat liver by usual cell fractionation were further purified by immunoisolation using a specific antibody raised against a peroxisomal membrane protein, PMP70. The isolated peroxisomes were analyzed by SDS-PAGE combined with liquid chromatography/mass spectrometry. Altogether 34 known peroxisomal proteins were identified in addition to several mitochondrial and microsomal proteins. Some of the latter may reside in the peroxisomes as well. Analysis of membrane fractions identified all known peroxins except for Pex7. Two new peroxisomal proteins of unknown function were of high abundance. One is a bi-functional protein consisting of an aminoglycoside phosphotransferase-domain and an acyl-CoA dehydrogenase domain. The other is a newly identified peroxisome-specific isoform of Lon protease, an ATP-dependent protease with chaperone-like activity. The peroxisomal localization of the protein was confirmed by immunological techniques. The peroxisome-type Lon protease, which is distinct from the mitochondrial isoform, may play an important role in the peroxisomal biogenesis.

Mitochondria are dynamic organelles that undergo frequent fission and fusion or branching. Although these morphologic changes are considered crucial for cellular functions, the underlying mechanisms remain elusive, especially in mammalian cells. We characterized two rat mitochondrial outer membrane proteins, Mfn1 and Mfn2, with distinct tissue expressions, that are homologous to Drosophila Fzo, a GTPase involved in mitochondrial fusion. Expression of the GTPase-domain mutant of Mfn2 (Mfn2 K 1 0 9 T ) in HeLa cells induced mitochondrial fragmentation in which Mfn2 K 1 0 9 T localized at the restricted domains. Immuno-electronmicroscopy revealed that Mfn2 K 1 0 9 T was concentrated at the contact domains between adjacent mitochondria, suggesting that fusion of the outer membrane was arrested at some intermediate step. Mfn1 expression induced highly connected tubular network structures depending on the functional GTPase domain. The Mfn1-induced tubular networks were suppressed by co-expression with Mfn2. In vivo depletion of either isoform by RNA interference revealed that both are required to maintain normal mitochondrial morphology. The fusion of differentially-labeled mitochondria in HeLa cells subjected to depletion of either Mfn isoform and subsequent cell fusion by hemagglutinating virus of Japan revealed that both proteins have distinct functions in mitochondrial fusion. We conclude that the two Mfn isoforms cooperate in mitochondrial fusion in mammalian cells.

NADP+-dependent isocitrate dehydrogenases (ICDHs) are enzymes that reduce NADP+ to NADPH using isocitrate as electron donor. Cytosolic and mitochondrial isoforms of ICDH have been described. Little is known on the expression of ICDHs in brain cells. We have cloned the rat mitochondrial ICDH (mICDH) in order to obtain the sequence information necessary to study the expression of ICDHs in brain cells by RT-PCR. The cDNA sequence of rat mICDH was highly homologous to that of mICDH cDNAs from other species. By RT-PCR the presence of mRNAs for both the cytosolic and the mitochondrial ICDHs was demonstrated for cultured rat neurons, astrocytes, oligodendrocytes and microglia. The expression of both ICDH isoenzymes was confirmed by western blot analysis using ICDH-isoenzyme specific antibodies as well as by determination of ICDH activities in cytosolic and mitochondrial fractions of the neural cell cultures. In astroglial and microglial cultures, the total ICDH activity was almost equally distributed between cytosolic and mitochondrial fractions. In contrast, in cultures of neurons and oligodendrocytes about 75% of total ICDH activity was present in the cytosolic fractions. Putative functions of ICDHs in cytosol and mitochondria of brain cells are discussed.

The expression of cathepsin H (CH) in differentiating rat spermatids was studied by an immunoelectron microscopic technique. Cathepsin H was detected in the acrosome throughout differentiation steps but cathepsins B, D, and L and lysosomal membrane protein (LGP107) were not. Early in the formation of the acrosome, CH signals were observed in Golgi vesicles but not in acrosomal vesicles. At steps 3–4, CH signals were associated with a fibrous material attached to the inner surface of the vesicle membrane on the Golgi side. At steps 5–6, this fibrous material accumulated to form an electron-dense sheet to which CH signals were confined. The rest of the acrosome was negative for the enzyme. At steps 11–12, the CH-positive fibrous sheet expanded from the apical to the ventral side of the sperm head. After step 16, the surface of outer dense fibers in the flagellar axoneme and reticulated bodies were stained for CH. In epididymal sperm, CH signals were detected in the acrosome as well as on the surface of the outer dense fibers running from the middle to the principal piece. By immunofluorescence staining, CH was found to be localized to the acrosome, middle piece, and principal piece.

Degradation and turnover of peroxisomes is reviewed. First, we describe the historical aspects of peroxisome degradation research and the two major concepts for breakdown of peroxisomes, i.e., autophagy and autolysis. Next, the comprehensive knowledge on autophagy of peroxisomes in mammalian and yeast cells is reviewed. It has been shown that proliferated peroxisomes are degraded by selective autophagy, and studies using yeast cells have been especially helpful in shedding light on the molecular mechanisms of this process. The degradation of extraperoxisomal urate oxidase crystalloid is noted. Overexpressed wild-type urate oxidase in cultured cells has been shown to be degraded through an unknown proteolytic pathway distinct from the lysosomal system including autophagy or the ubiquitin-proteasome system. Finally, peroxisome autolysis mediated by 15-lipoxygenase (15-LOX) is described. 15-LOX is integrated into the peroxisome membrane causing focal membrane disruptions. The content of the peroxisomes is then exposed to cytosol proteases and seems to be digested quickly. In conclusion, the number of peroxisomes appears to be regulated by two selective pathways, autophagy, including macro- and microautophagy, and 15-LOX-mediated autolysis.

Since Chinese hamster ovary (CHO) cells never express urate oxidase (UO), we tried to establish cell lines stably producing UO in order to elucidate the peroxisomal import process. The enzyme is a peroxisome targeting signal 1 (PTS1) protein harboring SKL motif at the carboxy-terminus [Biochem. Biophys. Res. Commun. 158 (1989) 991] and PEX5 protein (Pex5p) is supposed to be involved in the import process [Nat. Genet. 9 (1995) 115; J. Cell Biol. 130 (1995) 51]. We transfected a cDNA encoding rat UO into both wild type and PEX5-defective CHO cells to isolate each cell line stably producing the enzyme. While we examined the import process of UO in mutant cells, we noticed an interesting observation by using polyclonal antibody U1 or U2, which separately recognizes epitopes of UO. U1 antibody mainly interacts with epitopes in the amino-terminal region of UO. On the other hand, U2 antibody reacts with many epitopes distributed in the broad region of UO molecule. When UO produced in cultured cells was stained with U2 antibody, the enzyme was detected in peroxisomes of both wild type and PEX5-mutant cells. Whereas, U1 antibody stained the peroxisomal UO in wild type cells, but not in PEX5-mutant cells. These immunocytochemical observations suggest that the epitopes at the amino-terminal region of UO will be concealed in mutant cells. When the mutant cells were transfected with wild type PEX5 cDNA, U1 antibody came to react with UO in peroxisomes of mutant cells. The restoration indicates that the exposure of N-terminal epitopes of UO will depend upon the functional Pex5p. Immunoelectron microscopic observation showed that the peroxisomal import of UO was partially retarded in PEX5 mutant cells. The observation also supported the fact that UO was mainly localized in the peroxisomal matrix of wild type cells but in the membrane of mutant cells.

We analyzed the distribution and morphological characteristics of peroxisomes in the nematode Caenorhabditis elegans by routine electron microscopy, immunoelectron microscopy, and morphometry. Peroxisomes were mainly contained in the epithelial cells of the digestive tract and pharyngeal gland, but some were observed in other cells. Their shape varied from round to twisted. The matrix of most peroxisomes was coarse and uneven, and contained electron-dense nucleoids and frequently tubular substructures. The diameter of peroxisomes in the gut (0.185 micro m) was smaller than that in pharyngeal gland (0.262 micro m). The volume density of peroxisomes per 100 micro m(2) of cytoplasm was 1.86 in the gut and 1.75 in the pharyngeal gland. After treatment with clofibrate, the diameter of peroxisomes increased approximately 1.11-fold in the gut and 1.2-fold in the pharyngeal gland. The volume density of peroxisomes also increased by 2.2-fold in the gut and 2.6-fold in the pharyngeal gland. The labeling density for catalase-2 was almost identical between gut and pharyngeal gland peroxisomes. The results show that in C. elegans peroxisomes mainly distribute in the epithelial cells of the gut and pharyngeal gland. Peroxisomes of the pharyngeal gland are larger than those of the gut, but peroxisomes of both tissues contain catalase-2 at similar concentrations.