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To facilitate the cytosolic delivery of larger molecules such as proteins, we developed a new cell-penetrating peptide sequence, named Pas2r12, consisting of a repeated Pas sequence (FFLIG-FFLIG) and d-dodeca-arginine (r12). This peptide significantly enhanced the cellular uptake and cytosolic release of enhanced green fluorescent protein and immunoglobulin G as cargos. We found that simply mixing Pas2r12 with cargos could generate cytosolic introducible forms. The cytosolic delivery of cargos by Pas2r12 was found to be an energy-requiring process, to rely on actin polymerization, and to be suppressed by caveolae-mediated endocytosis inhibitors (genistein and methyl-β-cyclodextrin) and small interfering RNA against caveolin-1. These results suggest that Pas2r12 enhances membrane penetration of cargos without the need for cross-linking and that caveolae-mediated endocytosis may be the route by which cytosolic delivery is enhanced.

Neural stem/progenitor cells (NSPCs) express higher levels of poly(ADP-ribose) polymerase 1 (PARP1) than mouse embryonic fibroblasts (MEFs). Inhibition of PARP induces the expression of several genes in the p53 signaling pathway, including p21, which is critical for cell cycle control at the G1/S phase, triggers apoptosis, and suppresses cell cycle progression in NSPCs. However, upon the up-regulation of p21, the cell cycle does not arrest at any specific phase. In the present study, the expression of genes specific to the G1/S and G2/M phases of the cell cycle were analyzed following treatment with PJ34 (N-[6-oxo-5,6-dihydro-phenanthridin-2-yl]-N,N-dimethylacetamide), an inhibitor of PARP. PJ34 treatment dramatically down-regulated cyclin B1 expression in NSPCs, but not in MEFs, which was confirmed by a promoter assay. Down-regulation of FoxM1 and B-MYB revealed that the down-regulation of cyclin B occurs at the transcriptional level. GADD45 was also specifically up-regulated in NSPCs. Taken together, the activation of p53 by PJ34 treatment in NSPCs induced changes in the expression of genes involved in the cell cycle. Fluorescence-activated cell sorting analysis revealed that PJ34 treatment suppressed G2/M to G1 progression in NSPCs, but not in MEFs. These data indicate that PJ34 treatment inhibits cyclin expression at the mRNA level and suppresses cell cycle progression in NSPCs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Poly(ADP-ribose) polymerase 1 (PARP-1), which catalyzes poly(ADP-ribosyl)ation of proteins by using NAD + as a substrate, plays a key role in several nuclear events, including DNA repair, replication, and transcription. Recently, PARP-1 was reported to participate in the somatic cell reprogramming process. Previously, we revealed a role for PARP-1 in the induction of neural apoptosis in a cellular model of cerebral ischemia and suggested the possible use of PARP inhibitors as a new therapeutic intervention. In the present study, we examined the effects of PARP inhibitors on neural stem/progenitor cells (NSPCs) of the mouse brain., Results: PARP-1 was more abundant and demonstrated higher activity in NSPCs than in mouse embryonic fibroblasts. Treatment with PARP inhibitors suppressed the formation of neurospheres by NSPCs through the suppression of cell cycle progression and the induction of apoptosis. In order to identify the genes responsible for these effects, we investigated gene expression profiles by microarray analyses and found that several genes in the p53 signaling pathway were upregulated, including Cdkn1a, which is critical for cell cycle control, and Fas, Pidd, Pmaip1, and Bbc3, which are principal factors in the apoptosis pathway. Inhibition of poly(ADP-ribosyl)ation increased the levels of p53 protein, but not p53 mRNA, and enhanced the phosphorylation of p53 at Ser18. Experiments with specific inhibitors and also shRNA demonstrated that PARP-1, but not PARP-2, has a role in the regulation of p53. The effects of PARP inhibitors on NSPCs were not observed in Trp53 -/- NSPCs, suggesting a key role for p53 in these events., Conclusions: On the basis of the finding that PARP inhibitors facilitated the p53 signaling pathway, we propose that poly(ADP-ribosyl)ation contributes to the proliferation and self-renewal of NSPCs through the suppression of p53 activation.

Spermatogonial stem cells (SSCs) reside in specific niches within seminiferous tubules. These niches are thought to secrete chemotactic factors for SSCs, because SSCs migrate to them upon transplantation. However, the identity of these chemotactic molecules remains unknown. Here, we established a testis feeder cell culture system and used it to identify SSC chemotactic factors. When seeded on testis cells from infertile mice, SSCs migrated beneath the Sertoli cells and formed colonies with a cobblestone appearance that were very similar to those produced by hematopoietic stem cells. Cultured cells maintained SSC activity and fertility for at least 5 months. Cobblestone colony formation depended on GDNF and CXCL12, and dominant-negative GDNF receptor transfection or CXCL12 receptor deficiency reduced SSC colonization. Moreover, GDNF upregulated CXCL12 receptor expression, and CXCL12 transfection in Sertoli cells increased homing efficiency. Overall, our findings identify GDNF and CXCL12 as SSC chemotactic factors in vitro and in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Somatic cell hybridization is widely used to study the control of gene regulation and the stability of differentiated states. In contrast, the application of this method to germ cells has been limited in part because of an inability to culture germ cells. In this study, we produced germ cell hybrids using germ-line stem (GS) cells and multipotent germ-line stem (mGS) cells. While GS cells are enriched for spermatogonial stem cell (SSC) activity, mGS cells are similar to embryonic stem (ES) cells and originally derived from GS cells. Hybrids were successfully obtained between GS cells and ES cells, between GS cells and mGS cells, and between mGS cells and thymocytes. All exhibited ES cell markers and a behavior similar to ES cells, formed teratomas, and differentiated into somatic cell tissues. However, none of the hybrid cells were able to reconstitute spermatogenesis after microinjection into seminiferous tubules. Analyses of the DNA methylation patterns of imprinted genes also showed that mGS cells do not possess a DNA demethylation ability, which was found in embryonic germ cells derived from primordial germ cells. However, mGS cells reactivated the X chromosome and induced Pou5f1 expression in female thymocytes in a manner similar to ES cells. These data show that mGS cells possess ES-like reprogramming potential, which predominates over-SSC activity.

Carbon tetrachloride (CCl(4)) is routinely used as a model compound for eliciting centrilobular hepatotoxicity. It can be bioactivated to the trichloromethyl radical, which causes extensive lipid peroxidation and ultimately cell death by necrosis. Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) can rapidly reduce the levels of β-nicotinamide adenine dinucleotide and adenosine triphosphate and ultimately promote necrosis. The aim of this study was to determine whether inhibition of PARP-1 could decrease CCl(4)-induced hepatotoxicity, as measured by degree of poly(ADP-ribosyl)ation, serum levels of lactate dehydrogenase (LDH), lipid peroxidation, and oxidative DNA damage. For this purpose, male ICR mice were administered intraperitoneally a hepatotoxic dose of CCl(4) with or without 6(5H)-phenanthridinone, a potent inhibitor of PARP-1. Animals treated with CCl(4) exhibited extensive poly(ADP-ribosyl)ation in centrilobular hepatocytes, elevated serum levels of LDH, and increased lipid peroxidation. In contrast, animals treated concomitantly with CCl(4) and 6(5H)-phenanthridinone showed significantly lower levels of poly(ADP-ribosyl)ation, serum LDH, and lipid peroxidation. No changes were observed in the levels of oxidative DNA damage regardless of treatment. These results demonstrated that the hepatotoxicity of CCl(4) is dependent on the overactivation of PARP-1 and that inhibition of this enzyme attenuates the hepatotoxicity of CCl(4).

The homing ability of spermatogonial stem cells (SSCs) allows them to migrate into niches after being transplantated into infertile testes. Transplanted SSCs attach to Sertoli cells and transmigrate through the blood-testis barrier (BTB), formed by inter-Sertoli tight junctions, toward niches on the basement membrane. The most critical step is the passage through the BTB, which limits the homing efficiency to <10%. Here we demonstrated the involvement of Rac1 in SSC transmigration. Rac1-deficient SSCs did not colonize the adult testes, but they reinitiated spermatogenesis when transplanted into pup testes without a BTB. Moreover, a dominant-negative Rac1 construct not only reduced the expression of several claudin proteins, which comprise the BTB, but also increased SSC proliferation both in vitro and in vivo. Short hairpin RNA (shRNA) -mediated suppression of claudin3, which was downregulated by Rac inhibition, reduced the SSC homing efficiency. Thus, Rac1 is a critical regulator of SSC homing and proliferation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Spermatogonial stem cells (SSCs) are the only stem cells in the body with germline potential, which makes them an attractive target for germline modification. We previously showed the feasibility of homologous recombination in mouse SSCs and produced knockout (KO) mice by exploiting germline stem (GS) cells, i.e., cultured spermatogonia with SSC activity. In this study, we report the successful homologous recombination in rat GS cells, which can be readily established by their ability to form germ cell colonies on culture plates whose surfaces are hydrophilic and neutrally charged and thus limit somatic cell binding. We established a drug selection protocol for GS cells under hypoxic conditions. The frequency of the homologous recombination of the Ocln gene was 4.2% (2 out of 48 clones). However, these GS cell lines failed to produce offspring following xenogeneic transplantation into mouse testes and microinsemination, suggesting that long-term culture and drug selection have a negative effect on GS cells. Nevertheless, our results demonstrate the feasibility of gene targeting in rat GS cells and pave the way toward the generation of KO rats.

Selenocysteine lyase (SCL) catalyzes the decomposition of L-selenocysteine to yield L-alanine and selenium by acting exclusively on l-selenocysteine. The X-ray structural analysis of rat SCL has demonstrated how SCL discriminates L-selenocysteine from L-cysteine on the molecular basis. SCL has been proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residues, but the role of SCL in selenium metabolism in vivo remains unclear. We here demonstrate that the (75)Se-labeling efficiency of selenoproteins with (75)Se-labeled selenoprotein P (Sepp1) as a selenium source was decreased in HeLa cells transfected with SCL siRNA as compared to the cells transfected with control siRNA. Immunocytochemical analyses showed high SCL expression in kidney and liver cells, where selenocysteine is recovered from selenoproteins. Mature testes of mice exhibited a specific staining pattern of SCL in spermatids that actively produce selenoproteins. However, SCL was weakly expressed in Sertoli cells, which receive Sepp1 and supply selenium to germ cells. These demonstrate that SCL occurs in the cells requiring selenoproteins, probably to recycle selenium derived from selenoproteins such as Sepp1.

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis, and are unique tissue-specific stem cells because of their ability to transmit genetic information to offspring. Generation of knockout mice using mouse SSCs became feasible after the successful establishment of protocols for the transplantation and long-term culture of these cells, called germline stem (GS) cells. Furthermore, SSCs can acquire pluripotentiality similar to that of embryonic stem (ES) cells, in addition to their highly differentiated spermatogenic potential. These ES-like cells, called multipotent GS (mGS) cells, are capable of generating knockout mice in a manner similar to that of ES cells. The use of GS and mGS cells for animal transgenesis has added a new dimension to gene-targeting technology using ES cells and somatic cell nuclear transfer, which has limited application. Furthermore, for regenerative medicine purposes, the use of mGS will settle problems such as ethics issues and immunological rejection associated with ES cells, as well as risks of insertional mutagenesis associated with integrated genes into induced pluripotent stem cells.

Background: Spermatogonial stem cells (SSCs) continuously undergo self-renewal division to support spermatogenesis. SSCs are thought to have a fixed phenotype, and development of a germ cell transplantation technique facilitated their characterization and prospective isolation in a deterministic manner; however, our in vitro SSC culture experiments indicated heterogeneity of cultured cells and suggested that they might not follow deterministic fate commitment in vitro., Methodology and Principal Findings: In this study, we report phenotypic plasticity of SSCs. Although c-kit tyrosine kinase receptor (Kit) is not expressed in SSCs in vivo, it was upregulated when SSCs were cultured on laminin in vitro. Both Kit(-) and Kit(+) cells in culture showed comparable levels of SSC activity after germ cell transplantation. Unlike differentiating spermatogonia that depend on Kit for survival and proliferation, Kit expressed on SSCs did not play any role in SSC self-renewal. Moreover, Kit expression on SSCs changed dynamically once proliferation began after germ cell transplantation in vivo., Conclusions/significance: These results indicate that SSCs can change their phenotype according to their microenvironment and stochastically express Kit. Our results also suggest that activated and non-activated SSCs show distinct phenotypes.

Although spermatogonial stem cells (SSCs) are committed to spermatogenesis, they may also convert to an embryonic stem cell-like pluripotent state at a low frequency. Because changes in DNA methylation patterns are associated with this conversion, we examined the effect of manipulating DNA methyltransferase (Dnmt) expression on the fate of cultured SSCs, germline stem (GS) cells. Dnmt1 knockdown induced apoptosis in GS cells, which was attenuated by the loss of Trp53. In contrast, GS cells proliferated normally in vitro after Dnmt3a/Dnmt3b ablation or during Dnmt3l overexpression. However, Dnmt3a/Dnmt3b double-mutant cells showed hypomethylation in the SineB1 repetitive sequence, and Dnmt3l-overexpressing cells showed hypermethylation in major and minor satellite sequences; neither cell type formed teratomas and completed spermatogenesis following transplantation into the seminiferous tubules. Although genetic manipulation did not increase the conversion of GS cells to a pluripotent state, these results underscore the important role of DNMTs in survival and spermatogenic differentiation in SSCs.

Spermatogonial stem cells (SSCs) continue to proliferate in the testis to support spermatogenesis throughout life, which makes them ideal targets for germline modification. Although recent success in the production of transgenic and knockout animals using SSCs has opened up new experimental possibilities, several problems, including the low efficiency of germ cell transplantation and poor fertility rates, remain to be resolved. In the present study, we took advantage of the xenogeneic transplantation to resolve these problems. Rat SSCs were transduced in vitro with a lentiviral vector that expressed enhanced green fluorescent protein (EGFP), and then transplanted into the testes of immunodeficient mice. The transduced rat SSCs produced EGFP-expressing spermatogenic cells, and microinsemination using these cells was used to produce transgenic rats, which stably transmitted the transgene to the next generation. Thus, xenogeneic transplantation is a powerful strategy for transgenesis, and smaller xenogeneic surrogates can be used for male germline modification using SSCs.

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis. In a manner comparable to hematopoietic stem cell transplantation, SSCs colonize the niche of recipient testes and reinitiate spermatogenesis following microinjection into the seminiferous tubules. However, little is known about the homing mechanism of SSCs. Here we examined the role of adhesion molecules in SSC homing. SSCs isolated from mice carrying loxP-tagged beta1-integrin alleles were ablated for beta1-integrin expression by in vitro adenoviral cre transduction. The beta1-integrin mutant SSCs showed significantly reduced ability to recolonize recipient testes in vivo and to attach to laminin molecules in vitro. In contrast, genetic ablation of E-cadherin did not impair homing, and E-cadherin mutant SSCs completed normal spermatogenesis. In addition, the deletion of beta1-integrin on Sertoli cells reduced SSC homing. These results identify beta1-integrin as an essential adhesion receptor for SSC homing and its association with laminin is critical in multiple steps of SSC homing.

Spermatogonial stem cells can convert into embryonic stem (ES) cell-like multipotent germline stem (mGS) cells in vitro and produce germline chimeras by blastocyst microinjection. Although homologous recombination was previously demonstrated in mGS cells, spermatogenesis was not found in chimeras, suggesting that they are not competent for germline modification. Here we conducted detailed analysis of chimeric animals to determine whether mGS cells retain germline potential after genetic manipulation. Spermatozoa that were deficient in the occludin gene could be recovered from animals that were chimeric with mGS cells that underwent homologous recombination. The phenotypes of the occludin knockout (KO) mice were similar to those reported for KO mice produced using ES cells, and the animals showed growth retardation, gastritis and male infertility. Furthermore, we found that heterozygous mGS cells acquire two copies of the G418-resistant genes and become homozygous for the targeted allele by culturing at high concentrations of G418. Cytogenetic analysis showed that the aneuploid mGS cells observed during genetic manipulation were trisomic for chromosome 8 or 11, which is a common chromosomal abnormality in ES cells. Thus, mGS cells can be used to produce KO animals, and this novel method of germline manipulation may prove useful in diverse mammalian species.

Recently Welch et al. reported that microRNA (miRNA)-34a functions as a potential tumor suppressor in neuroblastoma cells (Oncogene 26: 5017-22, 2007). Here, we conversely show that miRNA-34a supports cell proliferation in rat oxidative stress-induced renal carcinogenesis and is overexpressed in various types of human cancers. While searching for genetically unstable chromosomal areas in rat renal carcinogenesis, we found the miRNA-34 family reciprocally overexpressed in chromosomal areas with frequent allelic loss. By in situ hybridization and reverse transcription-polymerase chain reaction, cerebral neurons and Purkinje cells showed the highest expression of a major type, miRNA-34a, followed by a variety of endocrine cells and proliferating cells including germinal center lymphocytes and mouse embryonic fibroblasts and stem cells. In contrast, normal renal tubules, hepatocytes and myocardial cells showed faint expression. After 3 weeks of ferric nitrilotriacetate (Fe-NTA)-induced oxidative stress, regenerating renal proximal tubular cells showed high miRNA-34a expression. All of the Fe-NTA-induced rat renal carcinomas and an array of human cancers (151 positive cases of 177) showed high expression of miRNA-34a. Furthermore, knockdown of miRNA-34a with small interfering RNA significantly suppressed proliferation not only of renal carcinoma cells but also of HeLa and MCF7 cells. These results indicate that miRNA-34a overexpression, an acquired trait during carcinogenesis, supports cell proliferation in the majority of cancers suggesting an unexpected link in the cellular metabolism between cancer and neuronal and/or endocrine cells, which warrants further investigation.

Spermatogonial stem cells represent a self-renewing population of spermatogonia, and continuous division of these cells supports spermatogenesis throughout the life of adult male animals. Previous attempts to introduce adenovirus vectors into spermatogenic cells, including spermatogonial stem cells, have failed to yield evidence of infection, suggesting that male germ cells may be resistant to adenovirus infection. In this study we show the feasibility of transducing spermatogonial stem cells by adenovirus vectors. When testis cells from ROSA26 Cre reporter mice were incubated in vitro with a Cre-expressing adenovirus vector, Cre-mediated recombination occurred at an efficiency of 49-76%, and the infected spermatogonial stem cells could reinitiate spermatogenesis after transplantation into seminiferous tubules of infertile recipient testes. No evidence of germ-line integration of adenovirus vector could be found in offspring from infected stem cells that underwent Cre-mediated recombination, which suggests that the adenovirus vector infected the cells but did not stably integrate into the germ line. Nevertheless, these results suggest that adenovirus may inadvertently integrate into the patient's germ line and indicate that there is no barrier to adenovirus infection in spermatogonial stem cells.

Spermatogonial transplantation has demonstrated a unique opportunity for studying spermatogenesis and provided an assay for spermatogonial stem cells. However, it has remained unknown whether germ cells that matured in a xenogeneic environment are functionally normal. In this investigation, we demonstrate the successful production of xenogeneic offspring by using spermatogonial transplantation. Rat spermatogonial stem cells were collected from immature testis and transplanted into the seminiferous tubules of busulfan-treated nude mouse testis. Using rat spermatids or spermatozoa that developed in xenogeneic surrogate mice, rat offspring were born from fresh and cryopreserved donor cells after microinsemination with rat oocytes. These offspring were fertile and had a normal imprinting pattern. The xenogeneic offspring production by interspecies germ cell transplantation and in vitro microinsemination will become a powerful tool in animal transgenesis and species conservation.

Spermatogenesis originates from a small number of spermatogonial stem cells that can reinitiate spermatogenesis and produce germ cell colonies following transplantation into infertile recipient testes. Although several previous studies have suggested a single-cell origin of germ cell colonies, only indirect evidence has been presented. In this investigation, we tested the clonal origin hypothesis using a retrovirus, which could specifically mark an individual spermatogonial stem cell. Spermatogonial stem cells were infected in vitro with an enhanced green fluorescence protein-expressing retrovirus and subsequently transplanted into infertile recipient mice. Live haploid germ cells were recovered from individual colonies and were microinjected into eggs to create offspring. In total, 45 offspring were produced from five colonies, and 23 (51%) of the offspring were transgenic. Southern blot analysis indicated that the transgenic offspring from the single colony carried a common integration site, and the integration site was different among the transgenic offspring from different colonies. These results provide evidence that germ cell colonies develop from single spermatogonial stem cells.

Stem cells represent a unique population of cells with self-renewal capacity. Although they are important therapeutic targets, the genetic manipulation of tissue-specific stem cells has been limited, which complicates the study and practical application of these cells. Here, we demonstrate successful gene trapping and homologous recombination in spermatogonial stem cells. Cultured spermatogonial stem cells were transfected with gene trap or gene targeting vectors. Mutagenized stem cells were expanded clonally by drug selection. These cells underwent spermatogenesis and produced heterozygous offspring after transplantation into the seminiferous tubules of infertile mouse testes. Heterozygous mutant mice were intercrossed to produce homozygous gene knockouts. Using this strategy, the efficiency of homologous recombination for the occludin gene locus was 1.7% using a nonisogenic DNA construct. These results demonstrate the feasibility of altering genes in tissue-specific stem cells in a manner similar to embryonic stem cells and have important implications for gene therapy and animal transgenesis.

Neuronal cells injured by ischemia and reperfusion to a certain extent are committed to death in necrotic or apoptotic form. Necrosis is induced by gross ATP depletion or 'energy crisis' of the cell, whereas apoptosis is induced by a mechanism still to be defined in detail. Here, we investigated this mechanism by focusing on a DNA damage-sensor, poly(ADP-ribose) polymerase-1 (PARP-1). A 2-h oxygen and glucose deprivation (OGD) followed by reoxygenation (Reox) induced apoptosis, rather than necrosis, in rat cortical neurons. During the Reox, PARP-1 was much activated and autopoly(ADP-ribosyl)ated, consuming the substrate, NAD+. Induction of apoptosis by OGD/Reox was suppressed by overexpression of Bcl-2, indicating mitochondrial impairment in this induction process. Mitochondrial permeability transition (MPT), or membrane depolarization, and a release of proapoptotic proteins, i.e. cytochrome c, apoptosis-inducing factor and endonuclease G, from mitochondria were observed during the Reox. These apoptotic changes of mitochondria and the nucleus were attenuated by PARP-1 inhibitors, 1,5-dihydroxyisoquinoline and benzamide, and also by small interfering RNA specific for PARP-1. These results indicated that PARP-1 plays a principal role in inducing mitochondrial impairment that ultimately leads to apoptosis of neurons after cerebral ischemia.

Although stem cells are believed to divide infinitely by self-renewal division, there is little evidence that demonstrates their infinite replicative potential. Spermatogonial stem cells are the founder cell population for spermatogenesis. Recently, in vitro culture of spermatogonial stem cells was described. Spermatogonial stem cells can be expanded in vitro in the presence of glial cell line-derived neurotrophic factor (GDNF), maintaining the capacity to produce spermatogenesis after transplantation into testis. Here, we examined the stability and proliferative capacity of spermatogonial stem cells using cultured cells. Spermatogonial stem cells were cultured over 2 years and achieved approximately 10(85)-fold expansion. Unlike other germline cells that often acquire genetic and epigenetic changes in vitro, spermatogonial stem cells retained the euploid karyotype and androgenetic imprint during the 2-year experimental period, and produced normal spermatogenesis and fertile offspring. However, the telomeres in spermatogonial stem cells gradually shortened during culture, suggesting that they are not immortal. Nevertheless, the remarkable stability and proliferative potential of spermatogonial stem cells suggest that they have a unique machinery to prevent transmission of genetic and epigenetic damages to the offspring, and these characteristics make them an attractive target for germline modification.

The use of membrane-permeable peptides as carrier vectors for the intracellular delivery of various proteins and macromolecules for modifying cellular function is well documented. Arginine-rich peptides, including those derived from human immunodeficiency virus 1 Tat protein, are among the representative classes of these vectors. The internalization mechanism of these vector peptides and their protein conjugates was previously regarded as separate from endocytosis, but more recent reevaluations have concluded that endocytosis is involved in their internalization. In this report, we show that the uptake of octa-arginine (R8) peptide by HeLa cells was significantly suppressed by the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) and the F-actin polymerization inhibitor cytochalasin D, suggesting a role for macropinocytosis in the uptake of the peptide. In agreement with this we observed that treatment of the cells with R8 peptide induced significant rearrangement of the actin cytoskeleton. The internalization efficiency and contribution of macropinocytosis were also observed to have a dependency on the chain length of the oligoarginine peptides. Uptake of penetratin, another representative peptide carrier, was less sensitive to EIPA and penetratin did not have such distinct effects on actin localization. The above observations suggest that penetratin and R8 peptides have distinct internalization mechanisms.

Septin 3 is a novel member of the septin subfamily of GTPase domain proteins that was recently identified in human neuronal cells. These proteins are involved in vesicle trafficking, neurite outgrowth, and neurofibrillary tangle formation; however, the expression and functional role of septin 3 in normal neuronal tissues and as an etiological agent in neurological disorders is currently unclear. To further characterize these parameters, the present study analyzed the expression of three isoforms of septin 3 (A, B, and C) in fetal and adult human brains and polymorphism of the septin 3 exon 11 microsatellite in control, pure Alzheimer's disease (AD), Lewy body variant (LBV) of AD, and Parkinson's disease. Septin 3 mRNAs for isoforms A and B, but not C, were detected in the frontal cortex of fetus and adult human samples, as measured by reverse transcription-coupled polymerase chain reaction. Genotype analyses indicated that polymorphic septin 3 alleles were distributed in two peaks of frequency in both control and disease groups. Categorization of the alleles into short (S) and long (L) types revealed a significant difference between AD patients and controls (p = 0.034 by chi-square test). Furthermore, the S-allele homozygosity was significantly underrepresented in AD compared with control (p = 0.015 by chi-square test). These results suggest that polymorphism in exon 11 of septin 3 may have a determinative role in the pathogenesis of AD.

Septin 3 is a novel member of the septin subfamily of GTPase domain proteins. Human septin 3 was originally cloned during a screening of genes expressed in human teratocarcinoma cells induced to differentiate with retinoic acid. Alternative splicing of the septin 3 gene transcript produces two isoforms, A and B, in the human brain, though their regional expression and physiological function remain to be determined. The purpose of the present study was to identify the expression patterns of human septin 3 isoforms in normal human brain and a human neuroblastoma cell line, SH-SY5Y, after retinoic acid-induced differentiation. The expression and distribution patterns of septin 3 isoforms A and B were similar and resembled that of another septin, CDCrel-1. Septin 3A and 3B were expressed in normal human brain in a region-specific manner, with the highest level in the temporal cortex and hippocampus and the lowest level in the brainstem regions. Prominent immunoreactivity was observed diffusely in the neocortices in association with neuropils and punctate structures suggestive of synaptic junctions. Immunoprecipitation studies revealed that septin 3A, 3B, and CDCrel-1 form a complex in the frontal cortex of human brain. These findings, taken together, suggest that septin 3A and 3B, along with CDCrel-1, play some fundamental role(s) in synaptogenesis and neuronal development.

Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) in response to oxidative stress has been shown to contribute to necrotic cell death by consuming NAD+ and ATP. In the present study, PARP-1 overactivation was determined by identifying the distribution and accumulation of poly(ADP-ribose) following intraperitoneal administration of a hepatotoxic dose of carbon tetrachloride (572 mg/kg). Treated animals exhibited lipid peroxide levels 16.5-fold higher than controls. Serum activities of glutamic pyruvic transaminase and glutamic oxaloacetic transaminase were increased by 6.1-fold and 22.8-fold, respectively. Lactate dehydrogenase activity was significantly increased by 1.2-fold. Histopathological analyses revealed severe necrosis and increased poly(ADP-ribsyl)ation of cells in the centrilobular region of treated animals versus saline controls. These results demonstrate the role of PARP-1 overactivation in chemical-induced pathologies and suggest the potential role of PARP-1 inhibitors at preventing toxicity.

Clonal pheochromocytoma cell lines overexpressing cytochrome P450 2D6 (CYP2D6) were established. CYP2D6 was localized in the endoplasmic reticulum, and its enzymatic activity in the microsomal fraction was confirmed by using high performance liquid chromatography analysis with [guanidine-14C]debrisoquine as a substrate. Overexpression of CYP2D6 protected both actively dividing and differentiated cells against the toxic effects of 1-methyl-4-phenylpyridinium ion at the concentration range of 20-40 microM, as assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The production of reactive oxygen species in the mitochondria was suppressed. The cytotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was unchanged in both actively dividing and differentiated cells overexpressing CYP2D6 versus mock-transfected controls at concentrations up to 500 microM. These results suggest that the lowered enzyme activity of CYP2D6 in individuals termed "poor metabolizers" may represent a risk factor from exposure to select neurotoxicants.

The association between (GT)n dinucleotide repeats in monoamine oxidase gene loci, monoamine oxidase A (MAOA) and B (MAOB), and Parkinson's disease (PD), Alzheimer's disease (AD), and Lewy body variant (LBV) of AD were determined. MAOA-GT polymorphisms were significantly associated with pure AD and LBV. MAOA-GT allele 113 was excessively represented in pure AD and LBV compared with controls. Furthermore, the frequency of females homozygous for MAOA-GT allele 113 was higher in pure AD and LBV than controls by 2.79- and 2.77-fold, respectively. In contrast, there was no association between MAOA-GT or MAOB-GT polymorphisms and PD. These results suggest that polymorphisms within the MAOA gene may have implication in AD pathology shared by pure AD and LBV.

Non-amyloid beta (Abeta) component of Alzheimer's disease (AD) amyloid (NAC) coexists with Abeta protein in senile plaques. After exposure to NAC fibrils, cortical neurons of rat brain primary culture became apoptotic, while astrocytes were activated with extension of their processes. NAC fibrils decreased the activity of reducing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in cortical neurons more markedly (IC(50) = 5.6 microm) than in astrocytes (IC(50) approximately 50 microm). The neuron-specific toxicity of NAC fibrils was indicated also by an increased release of lactate dehydrogenase from the cells. Neuronal apoptosis was suppressed by pre-treatment with the antioxidants, propyl gallate (PG) and N-t-butyl-phenylnitrone (BPN), or overexpression of human Bcl-2. Exposure to NAC fibrils enhanced generation of reactive oxygen species (ROS) in neurons and less efficiently in astrocytes, as demonstrated by oxidation of 2',7'-dichlorofluorescin. The site of ROS generation was shown to be mitochondria by oxidation of chloromethyl-tetramethyl rosamine. Exposure to NAC fibrils increased also the nuclear translocation of nuclear factor kappa B (NF-kappaB) and enhanced its DNA-binding activity, which was inhibited by PG and BPN more efficiently in neurons than in astrocytes. These results suggest that NAC fibrils increase mitochondrial ROS generation and activate NF-kappaB, thereby causing a differential change in gene expression between neurons and astrocytes in the AD brain.