Search

The effects of neuropeptides on the brain are a major focus of neuroendocrine research, and little progress has been made in the identification of the target neurons for many neuropeptides. Arginine 8 -vasotocin (AVT) is aneurohypophyseal peptide present in nonmammalian vertebrates that controls many neural and behavioral functions. Here we describe synthesis and functional characterization of an AVT-Oregon green conjugate 1 (AVT-OG 1) that can be used in vivo to identify AVT target neurons. Application of AVT-OG 1 to the brainstem of an amphibian produces rapid, endosome-like internalization together with typical AVT-like neurophysiological effects. Thus, preparation of AVT-OG 1, which preserves the peptide's neurophysiological effects, is useful as a fluorescent marker for AVT target neurons. Consequently, AVT-OG 1 conjugate will have considerable utility for analyzing the neural actions of AVT in the intact brain.

Acetaldehyde, the major ethanol metabolite that is far more toxic and reactive than ethanol, has been postulated to be responsible for alcohol-induced tissue and cell injury. This study was to examine whether facilitated acetaldehyde metabolism affects acetaldehyde-induced oxidative stress and apoptosis. Transgene-encoding human aldehyde dehydrogenase-2 (ALDH2), which converts acetaldehyde into acetate, was constructed under chicken beta-actin promoter and transfected into human umbilical vein endothelial cells (HUVECs). Efficacy of ALDH2 transfection was verified using green fluorescent protein and ALDH2 enzymatic assay. Generation of reactive oxygen species (ROS) was measured using chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by 4',6'-diamidino-2'-phenylindoladihydrochloride fluorescence microscopy, quantitative DNA fragmentation, and caspase-3 assay. Acetaldehyde (0-200 microm) elicited ROS generation and apoptosis in HUVECs in a time- and concentration-dependent manner, associated with activation of the stress signal molecules ERK1/2 and p38 mitogen-activated protein (MAP) kinase. A close liner correlation was observed between the acetaldehyde-induced ROS generation and apoptosis. Interestingly, the acetaldehyde-induced ROS generation, apoptosis, activation of ERK1/2, and p38 MAP kinase were prevented by the ALDH2 transgene or antioxidant alpha-tocopherol. The involvement of ERK1/2 and p38 MAP kinase in acetaldehyde-induced apoptosis was confirmed by selective kinase inhibitors U0126, SB203580, and SB202190. Collectively, our data revealed that facilitation of acetaldehyde metabolism by ALDH2 transgene overexpression may prevent acetaldehyde-induced cell injury and activation of stress signals. These results indicated therapeutic potential of ALDH2 enzyme in the prevention and detoxification of acetaldehyde or alcohol-induced cell injury.

Polyubiquitin-encoding cDNA clones were isolated from the generative cells of lily (Lilium longiflorum) and the sperm cells of Plumbago zeylanica. The described genes encode identical amino acid sequences, with no homology outside the coding regions. This gene participates in ubiquitination of proteins, presumably enhancing protein turnover in the germline during male reproductive differentiation. In this paper we show that the gene encoding polyubiquitin is highly up-regulated in both Lilium generative cells and one of the Plumbago sperm cell types in particular.

Background: Aldosterone has been implicated in a variety of organ fibroses, but its role and mechanism in liver fibrosis remain unclear. Methods: Rat primary hepatic stellate cells (HSCs) were isolated, cultured, and characterized. HSCs were incubated with aldosterone (10-6 M) for 4 h, 8 h, 12 h, 24 h, and 48 h, after which TGF-β1 (transforming growth factor beta 1) expression was measured by real-time PCR. Rat HSCs were treated with different concentrations of aldosterone (10-6 M, 10-7 M, 10-8 M, and 10-9 M), and the expressions of PAI-1 (plasminogen activator inhibitor-1) and TGF-β1 were determined by measuring mRNA and protein. HSCs were incubated in groups containing aldosterone (10-6 M), spironolactone (10-5 M), both aldosterone and spironolactone, or neither aldosterone nor spironolactone (control), after which mRNA and protein expression of PAI-1 and TGF-β1 were measured. Collagen I expression was detected by immunohistochemical analysis of supernatants of the aldosterone (10-6 M), TGF-β1, and aldosterone plus TGF-β1 groups. SMAD expression was detected in rat HSC control, HSC plus aldosterone (10-6 M), HSC plus TGF-β1, and HSC plus aldosterone plus TGF-β1 groups. Results: HSCs were incubated with aldosterone for 4 h, 8 h, 12 h, 24 h, and 48 h after which TGF-β1 expression was measured. We found that TGF-β1 expression increased in a time dependent manner and reached a peak at 24 h. The expression of TGF-β1 in groups treated with aldosterone for 4 h, 8 h, 12 h, and 24 h was significantly different from the control group (P < 0.01). No significant difference was seen in TGF-β1 expression between the groups treated with aldosterone for 24 h and 48 h (P > 0.05). Compared with the control group, TGF-β1 expression was significantly increased after incubation with different concentrations of aldosterone (10-6 M, 10-7 M, 10-8 M, and 10-9 M) (P < 0.01). There were significant differences in the expression of TGF-β1 between 10-6 M and 10-7 M aldosterone treatment groups (P < 0.01). Compared with the control group, the expression of PAI-1 was significantly increased after incubation with different concentrations of aldosterone (10-6 M, 10-7 M, 10-8 M, and 10-9 M) (P < 0.01). PAI-1 expression was increased in the aldosterone, spironolactone, and aldosterone plus spironolactone groups. The expression of PAI-1 was significantly enhanced in the aldosterone and aldosterone plus spironolactone groups compared with the control group (P < 0.01). There was a marked enhancement of collagen I expression in the aldosterone, TGF-β1, and aldosterone plus TGF-β1 groups (P < 0.05). Collagen I expressions in the aldosterone and TGF-β1 groups were significantly different from the aldosterone plus TGF-β1 group (P < 0.01). Compared with the control group, SMAD expression was markedly elevated in the aldosterone, TGF-β1, and aldosterone plus TGF-β1 groups (P < 0.05). The expression of SMAD was significantly increased in the aldosterone plus TGF-β1 group compared with the aldosterone group (P < 0.01). Conclusion: This study demonstrated that aldosterone promoted HSC activation and the expression of TGF-β1, PAI-1, and collagen in hepatic fibrosis progression and that spironolactone administration partially reversed the effects. The aldosterone promotional effect on hepatic fibrosis was partially mediated by TGF-β1.

Phosphatidylserine decarboxylase, which is embedded in the inner mitochondrial membrane, synthesizes phosphatidylethanolamine (PE) and, in some cells, synthesizes the majority of this important phospholipid. Normal levels of PE can decline with age in the brain. Here we used yeast and worms to test the hypothesis that low levels of PE alter the homeostasis of the Parkinson disease-associated protein α-synuclein (α-syn). In yeast, low levels of PE in the phosphatidylserine decarboxylase deletion mutant ( psd1 Δ) cause decreased respiration, endoplasmic reticulum (ER) stress, a defect in the trafficking of the uracil permease, α-syn accumulation and foci, and a slow growth phenotype. Supplemental ethanolamine (ETA), which can be converted to PE via the Kennedy pathway enzymes in the ER, had no effect on respiration, whereas, in contrast, this metabolite partially eliminated ER stress, decreased α-syn foci formation, and restored growth close to that of wild-type cells. In Caenorhabditis elegans , RNAi depletion of phosphatidylserine decarboxylase in dopaminergic neurons expressing α-syn accelerates neurodegeneration, which supplemental ETA rescues. ETA fails to rescue this degeneration in worms that undergo double RNAi depletion of phosphatidylserine decarboxylase ( psd-1 ) and choline/ETA phosphotransferase ( c ept-1 ), which encodes the last enzyme in the CDP–ETA Kennedy pathway. This finding suggests that ETA exerts its protective effect by boosting PE through the Kennedy pathway. Overall, a low level of PE causes ER stress, disrupts vesicle trafficking, and causes α-syn to accumulate; such cells likely die from a combination of ER stress and excessive accumulation of α-syn.

The molecular mechanism of acetic acid tolerance in yeast remains unclear despite of its importance for efficient cellulosic ethanol production. In this study, we examined the effects of histone H3/H4 point mutations on yeast acetic acid tolerance by comprehensively screening a histone H3/H4 mutant library. A total of 24 histone H3/H4 mutants (six acetic acid resistant and 18 sensitive) were identified. Compared to the wild-type strain, the histone acetic acid-resistant mutants exhibited improved ethanol fermentation performance under acetic acid stress. Genome-wide transcriptome analysis revealed that changes in the gene expression in the acetic acid-resistant mutants H3 K37A and H4 K16Q were mainly related to energy production, antioxidative stress. Our results provide novel insights into yeast acetic acid tolerance on the basis of histone, and suggest a novel approach to improve ethanol production by altering the histone H3/H4 sequences.

In March 2013, human cases of infection with a novel A (H7N9) influenza virus emerged in China. The epidemic spread quickly and as of 6 May 2013, there were 129 confirmed cases. The purpose of this study was to analyze the epidemiology of the confirmed cases, determine the impacts of bird migration and temperature changes on the H7N9 epidemic, predict the future trends of the epidemic, explore the response patterns of the government and propose preventive suggestions.The geographic, temporal and population distribution of all cases reported up to 6 May 2013 were described from available records. Risk assessment standard was established by analysing the temperature and relative humidity records during the period of extensive outbreak in three epidemic regions in eastern China, including Shanghai, Zhejiang and Jiangsu provinces. Risk assessment maps were created by combining the bird migration routes in eastern China with the monthly average temperatures from May 1993 to December 2012 nationwide.Among the confirmed cases, there were more men than women, and 50.4% were elderly adults (age61 years). The major demographic groups were retirees and farmers. The temperature on the days of disease onset was concentrated in the range of 9°C-19°C; we defined 9°C-19°C as the high-risk temperature range, 0°C-9°C or 19°C-25°C as medium risk and0°C or25°C as low risk. The relative humidity on the days of disease onset ranged widely from 25% to 99%, but did not correlate with the incidence of infection. Based on the temperature analysis and the eastern bird migration routes, we predicted that after May, the high-risk region would move to the northeast and inland, while after September, it would move back to north China.Temperature and bird migration strongly influence the spread of the H7N9 virus. In order to control the H7N9 epidemic effectively, Chinese authorities should strengthen the surveillance of migrating birds, increase poultry and environmental sampling, improve live poultry selling and husbandry patterns and move from a "passive response pattern" to an "active response pattern" in focused preventive measures.

Objective: Hepatopulmonary syndrome (HPS) is considered as a triad of chronic liver disease, pulmonary vascular ectasia and severe hypoxemia. The study aims to investigate the pathological mechanism of intra-abdominal pressure (IAP) in HPS and establish a novel mouse model. Methods: Fifty male ICR mice were randomly divided into experimental and control group, receiving subcutaneous injection of carbon tetrachloride and water, respectively. Mice in experimental group were then divided into 4 sub-groups with the intraperitoneal injection of different volume of albumin to form different IAP (0, 5, 10 and 20 cmH2O). All the mice were then sacrificed 24 hours later and blood gas analysis was conducted. In addition, liver and lung histopathology was also examined. Results: Blood gas analysis in different IAP suggested the respiratory alkalosis. Arterial partial pressure of oxygen significantly decreased in the IAP=10 cmH2O (68.13 ± 3.56, P

Myosin associated with the male germ cells of angiosperms interacts with actin, promoting transport of the non-motile generative and later sperm cells in the pollen tube. Myosin localizing on the sperm cell plasma membrane seems negligible in Plumbago, as reflected by the absence of: (i) anti-myosin labeling using immunoelectron microscopy, (ii) sperm motility on actin matrices, and (iii) electrophoretic movement changes after addition of antibody. Sperm cells injected directly into actively streaming Nitella internodal cells, however, follow actin bundles and their movement is sensitive to ATP and Mg2+. This may be based on simple charge binding since negatively charged latex beads also migrate on actin, whereas neutral or positively-charged latex beads do not. Sperm cells are negatively charged according to capillary microelectrophoresis, whereas killed sperm cells, which are positively charged do not migrate. The sperm cell that normally fertilizes the egg has a higher calculated charge (8.277 × 103 esu/cm2) compared with the sperm cell that fuses with the central cell (6.120 × 103 esu/cm2).

A protocol is described for individually collecting two populations of sperm cells, Svn and Sua, from pollen of Plumbago zeylanica. Pollen grains were burst in 10 mM MOPS buffer containing 0.8 M mannitol (pH 4.6). Paired sperm cells released from pollen were separated using a microinjector. Svn and Sua were then collected individually with a microinjector, based upon known size differences. Collected sperm cells were washed with isolation medium and transferred to liquid nitrogen until use. Fluorochromatic reaction (FCR) test of isolated sperm cells showed a positive reaction, indicating that the isolated sperm cells are viable; most of the sperm cells retain viability for at least 2 h.

The second and last authors were incorrectly indicated as having contributed equally to the article. In addition, the first author, Jianfeng Chen, was not correctly indicated as having contributed equally to the article with the second author, Eileen Marks.

In some organs, adult stem cells are uniquely poised to serve as cancer cells of origin. It is unclear, however, whether tumorigenesis is influenced by the activation state of the adult stem cell. Hair follicle stem cells (HFSCs) act as cancer cells of origin for cutaneous squamous cell carcinoma and undergo defined cycles of quiescence and activation. The data presented here show that HFSCs are unable to initiate tumours during the quiescent phase of the hair cycle, indicating that the mechanisms that keep HFSCs dormant are dominant over the gain of oncogenes (such as Ras) or the loss of tumour suppressors (such as p53). Furthermore, Pten activity is necessary for quiescence-based tumour suppression, as its deletion alleviates tumour suppression without affecting proliferation. These data demonstrate that stem cell quiescence is a form of tumour suppression in HFSCs, and that Pten plays a role in maintaining quiescence in the presence of tumorigenic stimuli.

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine-encoding CAG expansion in the huntingtin gene. Iron accumulates in the brains of HD patients and mouse disease models. However, the cellular and subcellular sites of iron accumulation, as well as significance to disease progression are not well understood. We used independent approaches to investigate the location of brain iron accumulation. In R6/2 HD mouse brain, synchotron x-ray fluorescence analysis revealed iron accumulation as discrete puncta in the perinuclear cytoplasm of striatal neurons. Further, perfusion Turnbull’s staining for ferrous iron (II) combined with transmission electron microscope ultra-structural analysis revealed increased staining in membrane bound peri-nuclear vesicles in R6/2 HD striatal neurons. Analysis of iron homeostatic proteins in R6/2 HD mice revealed decreased levels of the iron response proteins (IRPs 1 and 2) and accordingly decreased expression of iron uptake transferrin receptor (TfR) and increased levels of neuronal iron export protein ferroportin (FPN). Finally, we show that intra-ventricular delivery of the iron chelator deferoxamine results in an improvement of the motor phenotype in R6/2 HD mice. Our data supports accumulation of redox-active ferrous iron in the endocytic / lysosomal compartment in mouse HD neurons. Expression changes of IRPs, TfR and FPN are consistent with a compensatory response to an increased intra-neuronal labile iron pool leading to increased susceptibility to iron-associated oxidative stress. These findings, together with protection by deferoxamine, support a potentiating role of neuronal iron accumulation in HD.

Next-generation sequencing (NGS) of the whole genome, whole exome, and transcriptome has enabled characterization of genetic landscapes of multiple cancers. By analyzing over 2,000 pediatric cancer patients, we have developed a comprehensive database for recurrent somatic alterations and pathogenic germline mutations as part of the St. Jude/Washington University Pediatric Cancer Genome Project. However, there is no systematic evaluation on whether NGS is able to identify germline and somatic lesions reported by existing molecular diagnostic assays and what combination of NGS platforms is best suited for clinical sequencing. Here we report the first comprehensive study that employs whole-genome sequencing at 30-45X coverage, whole-exome sequencing at 100X coverage and transcriptome sequencing using matched tumor/normal samples from cancer patients. A pilot study was carried out to perform NGS analysis on 78 children of leukemia, solid tumor or brain tumor with a total of 112 diagnostic or prognostic biomarkers previously characterized by multiple molecular diagnostic assays. We implemented an analysis pipeline that integrates the genetic lesions detected by all three NGS platforms to characterize somatic and germline single nucleotide variations (SNVs), short insertions and deletions (indels), structural variations including fusions, karyotypes, copy number alterations, loss of heterozygosity, tumor purity and tumor-in-normal contamination. The turn-around time for data analysis is 2 weeks with an overall sensitivity of 99% on detecting known biomarkers. Extensive validation of >3,000 somatic sequence mutations or structural variations from 38 cases shows that the specificity for somatic SNV, indel and structural variation is at 98%, 95% and 84% across the genome. We demonstrate that in addition to providing cross-validation, multi-platform NGS is required for detecting all genetic lesions of pathological significance including complex re-arrangements such as chromothripsis. In addition to known pathogenic or likely pathogenic mutations, our analysis has also unveiled novel pathogenic mutations (e.g. a germline deletion in TP53 in one patient with medulloblastoma) and identified multiple variants of unknown significance that may be worth further exploration (e.g. an in-frame deletion of exons 3-9 of DNMT3A in one neuroblastoma). Our study demonstrates that NGS is able to detect a wide range of genetic lesions currently characterized by multiple molecular diagnostic assays, providing critical insight into the design of clinical sequencing for ongoing studies. Citation Format: Jinghui Zhang, Michael Rusch, Joy Nakitandwe, Zhaojie Zhang, Michael N. Edmonson, Matthew Parker, Xiaotu Ma, Jared Becksfort, Andrew Thrasher, Jiali Gu, Yongjin Li, Erin Hedlund, Aman Patel, John Easton, Donald Yergeau, Bhavin Vadodaria, Xiang Chen, Tanja A. Gruber, Rose McGee, David Ellison, Sheila Shurtleff, James R. Downing. Molecular diagnosis for pediatric cancer through integrative analysis of whole-genome, whole-exome and transcriptome sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2628.

Current cancer genome data portals have focused primarily on presenting data generated from adult cancer studies. These portals typically lack features for exploring pathogenic germline mutations, somatic gene fusions, and gene expression profiling, all of which are important biomarkers for risk stratification of pediatric cancer. We have developed ProteinPaint (https://pecan.stjude.org/proteinpaint/), a web service hosting 30,000+ validated somatic SNV/indels and fusion transcripts detected in 1,654 pediatric tumor samples from 17 subtypes, 252 pathogenic or loss-of-function germline lesions detected in >1000 pediatric cancer patients of 21 subtypes, and gene expression profiles derived from RNA-Seq of 928 pediatric tumors. Cancer genomic alterations are shown on novel “disc-on-stem” skewer graphs which were designed to depict the diverse prevalence, complex allelic alteration, and temporal origin of sequence mutations and gene fusions. Adult somatic cancer mutation data from the COSMIC database can be displayed in parallel with pediatric cancer data sets for cross-study comparison. We will demonstrate examples of how ProteinPaint's integrative view of genomic alteration, gene expression and pediatric-adult data comparison has facilitated the evaluation of somatic and germline mutation pathogenicity in a clinical setting. Custom data including sequence mutations in the MAF format used by the Cancer Genome Atlas (TCGA) project, copy number alterations, and structural variations can all be imported and visualized alongside published pediatric and adult cancer data sets. Furthermore, ProteinPaint supports curation and annotation of fusion transcripts predicted from RNASeq data and analysis of tumor clonal evolution with a 2-D plot of mutation frequency of paired diagnosis and relapse samples. ProteinPaint delivers a premium user experience with animation and interactive features for visualizing large cancer mutation datasets, and can serve as a workbench to import, explore and interpret user data. Its framework continues to expand as its intuitive visualization has enabled non-bioinformatics scientists and clinicians to access and manipulate genomic data for discovery and clinical reporting. Citation Format: Xin Zhou, Michael N. Edmonson, Mark R. Wilkinson, Aman Patel, Gang Wu, Yu Liu, Yongjin Li, Zhaojie Zhang, Michael Rusch, Matthew Parker, Jared Becksfort, James R. Downing, Jinghui Zhang. Exploring genomic alterations in pediatric cancer using ProteinPaint. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2436.

The homologous genes BDF1 and BDF2 in Saccharomyces cerevisiae encode bromodomain-containing transcription factors. Although double deletion of BDF1 and BDF2 is lethal, single deletion does not affect cell viability. The bdf2∆ cells showed normal growth upon salt stress. However, the absence of Bdf1p resulted in a salt-sensitive phenotype, and the salt sensitivity was suppressed by overexpression of BDF2. In this study, we further demonstrated that BDF2 shows dosage compensation in suppressing the salt sensitivity of bdf1∆. None of the tested domains replaced the function of intact Bdf1p. The 494-626 region in Bdf1p was more important than the other domains for salt resistance. In addition, Bdf1p negatively regulated the expression of BDF2 by binding its promoter at loci -387 to -48. However, Bdf2p did not affect the expression of BDF1. In addition, Bdf1p and its defective functional domain mutants could combine with Bdf2p. This physical interaction increased the salt tolerance of bdf1∆. The mitochondrial dysfunctions caused by BDF1 deletion were restored by overexpression of BDF2 under salt stress conditions.

The Saccharomyces cerevisiae Bdf1p associates with the basal transcription complexes TFIID and acts as a transcriptional regulator. Lack of Bdf1p is salt sensitive and displays abnormal mitochondrial function. The nucleotidase Hal2p detoxifies the toxic compound 3' -phosphoadenosine-5'-phosphate (pAp), which blocks the biosynthesis of methionine. Hal2p is also a target of high concentration of Na(+). Here, we reported that HAL2 overexpression recovered the salt stress sensitivity of bdf1Δ. Further evidence demonstrated that HAL2 expression was regulated indirectly by Bdf1p. The salt stress response mechanisms mediated by Bdf1p and Hal2p were different. Unlike hal2Δ, high Na(+) or Li(+) stress did not cause pAp accumulation in bdf1Δ and methionine supplementation did not recover its salt sensitivity. HAL2 overexpression in bdf1Δ reduced ROS level and improved mitochondrial function, but not respiration. Further analyses suggested that autophagy was apparently defective in bdf1Δ, and autophagy stimulated by Hal2p may play an important role in recovering mitochondrial functions and Na(+) sensitivity of bdf1Δ. Our findings shed new light towards our understanding about the molecular mechanism of Bdf1p-involved salt stress response in budding yeast.

Cell division in yeast is a highly regulated and well studied event. Various checkpoints are placed throughout the cell cycle to ensure faithful segregation of sister chromatids. Unexpected events, such as DNA damage or oxidative stress, cause the activation of checkpoint(s) and cell cycle arrest. Malfunction of the checkpoints may induce cell death. We previously showed that under oxidative stress, the budding yeast cohesin Mcd1, a homolog of human Rad21, was cleaved by the caspase-like protease Esp1. The cleaved Mcd1 C-terminal fragment was then translocated to mitochondria, causing apoptotic cell death. In the present study, we demonstrated that Bir1 plays an important role in spindle assembly checkpoint and cell death. Similar to H(2)O(2) treatment, deletion of BIR1 using a BIR1-degron strain caused degradation of the securin Pds1, which binds and inactivates Esp1 until metaphase-anaphase transition in a normal cell cycle. BIR1 deletion caused an increase level of ROS and mis-location of Bub1, a major protein for spindle assembly checkpoint. In wild type, Bub1 was located at the kinetochores, but was primarily in the cytoplasm in bir1 deletion strain. When BIR1 was deleted, addition of nocodazole was unable to retain the Bub1 localization on kinetochores, further suggesting that Bir1 is required to activate and maintain the spindle assembly checkpoint. Our study suggests that the BIR1 function in cell cycle regulation works in concert with its anti-apoptosis function.

Sperm cells of tobacco have been intensively studied as examples of isomorphic gametes in which major cellular and organellar parameters remain statistically indistinguishable in the two sperm cells. An examination of sperm cells late in maturation, however, displays that the sperm cell associated with the vegetative nucleus becomes statistically significantly smaller than the other sperm cell in tobacco. If late divergence occurs in the two sperms of other angiosperms, sperm dimorphism may be more prevalent than has previously been assumed and dimorphism may have a major influence on the pattern of double fertilization.

Here we report that deletion of SOD1, the Cu,Zn-superoxide dismutase in Saccharomyces cerevisiae is sensitive to cell wall-perturbing agents, such as Calcofluor white and Congo red. The sensitivity was restored by retransformation with wild type SOD1 or the addition of N-acetylcysteine or reduced glutathione to the medium. Additionally, the accumulation of reactive oxygen species was observed in sod1Δ mutant in the presence of Calcofluor white or Congo red. Cell wall analysis indicated an increase of cell wall chitin and cell wall thickness in sod1Δ mutant compared to wild type. These results indicate a novel direct connection between antioxidative functions and cell wall homeostasis.

Cohesin is a protein complex that regulates sister chromatid cohesin during cell division. Malfunction in chromatid cohesin results in chromosome missegregation and aneuploidy. Here, we report that mutations of MCD1 and PDS5, two major components of cohesin in budding yeast, cause apoptotic cell death, which is characterized by externalization of phosphatidylserine at cytoplasmic membrane, chromatin condensation and fragmentation, and ROS production. Microarray analysis suggests that the cell death caused by mutation of MCD1 or PDS5 is due to the internal stress response, contrasting to the environmental or external stress response induced by external stimuli, such as hydrogen peroxide. A common feature shared by the internal stress response and external stress response is the response to stimulus, including response to DNA damage, mitochondria functions, and oxidative stress, which play an important role in yeast apoptotic cell death.

Over the last decade, yeast has been used successfully as a model system for studying the molecular mechanism of apoptotic cell death. Here, we report that Mcd1, the yeast homology of human cohesin Rad21, plays an important role in hydrogen peroxide-induced apoptosis in yeast. On induction of cell death, Mcd1 is cleaved and the C-terminal fragment is translocated from nucleus into mitochondria, causing the decrease of mitochondrial membrane potential and the amplification of cell death in a cytochrome c-dependent manner. We further demonstrate that the caspase-like protease Esp1 has dual functions and that it is responsible for the cleavage of Mcd1 during the hydrogen peroxide-induced apoptosis. When apoptosis is induced, Esp1 is released from the anaphase inhibitor Pds1. The activated Esp1 acts as caspase-like protease for the cleavage of Mcd1, which enhances the cell death via its translocation from nucleus to mitochondria.

Using a genetic screen we discovered that YGR198w (named YPP1), which is an essential Saccharomyces cerevisiae gene of unknown function, suppresses the toxicity of an alpha-synuclein (alpha-syn) mutant (A30P) that is associated with early onset Parkinson's disease. Here, we show that YPP1 suppresses lethality of A30P, but not of wild-type alpha-syn or the A53T mutant. The Ypp1 protein, when overexpressed, drives each of the three alpha-syns into vesicles that bud off the plasma membrane, but only A30P-containing vesicles traffick to and merge with the vacuole, where A30P is proteolytically degraded. We show that Ypp1p binds to A30P but not the other two alpha-syns; that YPP1 interacts with genes involved in endocytosis/actin dynamics (SLA1, SLA2, and END3), protein sorting (class E vps), and vesicle-vacuole fusion (MON1 and CCZ1) to dispose of A30P; and that YPP1 also participates in pheromone-triggered receptor-mediated endocytosis. Our data reveal that YPP1 mediates the trafficking of A30P to the vacuole via the endocytic pathway.

Budding yeast PDS5 is an essential gene in mitosis and is required for chromosome condensation and sister chromatid cohesion. Here we report that PDS also is required in meiosis. Pds5p localizes on chromosomes at all stages during meiotic cycle, except anaphase I. PDS5 plays an important role at first meiotic prophase. Failure in function of PDS5 causes premature separation of chromosomes. The loading of Pds5p onto chromosome requires the function of REC8, but the association of Rec8p with chromosome is independent of PDS5. Mutant analysis and live cell imaging indicate that PDS5 play a role in meiosis II as well.

1 Endothelin-1 (ET-1), an endothelium-derived vasoactive peptide, participates in the regulation of endothelial function through mechanisms that are not fully elucidated. This study examined the impact of ET-1 on oxidative stress, apoptosis and cell proliferation in human umbilical vein endothelial cells (HUVEC). HUVECs were challenged for 24 h with ET-1 (10 pM-10 nM) in the absence or presence of the ET(B) receptor antagonist BQ788 (1 microM) or the NADPH oxidase inhibitor apocynin (1 microM). Reactive oxygen species (ROS) were detected using chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated with 4',6'-diamidino-2'-phenylindoladihydrochloride staining and by the caspase-3 assay. Cell proliferation was measured by a colorimetric assay. Expression of NADPH oxidase, Akt, pAkt, Bcl-2, Bax, IkappaB, caveolin-1 and eNOS was evaluated by Western blot analysis. 2 ET-1 significantly enhanced ROS generation and cell proliferation following 24-h incubation, both of which were prevented by BQ788 or apocynin, consistent with the ability of ET-1 to directly upregulate NADPH oxidase. ET-1 itself did not affect apoptosis but attenuated homocysteine-induced apoptosis through an ET(B) receptor-mediated mechanism. Western blot analysis indicated that ET-1 alleviated homocysteine (Hcy)-induced apoptosis, likely acting by antagonizing the Hcy-induced decreases in Akt, pAkt, pAkt-to-Akt, Bcl-2-to-Bax ratios and increases in Bax and caveolin-1 expression. Furthermore, ET-1 downregulated expression of caveolin-1 and eNOS, which was attenuated by BQ788 or apocynin. 3 In summary, our results suggest that ET-1 affects oxidative stress, proliferation and apoptosis possibly through ET(B), NADPH oxidase, Akt, Bax and caveolin-1-mediated mechanisms.

Pds5p is a cohesin related protein. It is required for maintenance of sister chromatid cohesion in mitosis and meiosis. Here we report that pds5-1 causes cell death in yeast Saccharomyces cerevisiae during early meiosis. The pds5-1 caused cell death possesses characteristics of apoptosis and necrosis, including externalization of phosphatidylserine at cytoplasmic membrane, accumulation of DNA breaks, chromatin condensation and fragmentation, nuclei fragmentation, membrane degeneration and cell size enlargement. Our results also suggest that (1) The defect of DNA repair; (2) The production of reactive oxygen species, in pds5-1 mutant are involved in pds5-1 induced cell death.

Acetaldehyde, the major ethanol metabolite that is far more toxic and reactive than ethanol, has been postulated to be responsible for alcohol-induced tissue and cell injury. This study was to examine whether facilitated acetaldehyde metabolism affects acetaldehyde-induced oxidative stress and apoptosis. Transgene-encoding human aldehyde dehydrogenase-2 (ALDH2), which converts acetaldehyde into acetate, was constructed under chicken beta-actin promoter and transfected into human umbilical vein endothelial cells (HUVECs). Efficacy of ALDH2 transfection was verified using green fluorescent protein and ALDH2 enzymatic assay. Generation of reactive oxygen species (ROS) was measured using chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by 4',6'-diamidino-2'-phenylindoladihydrochloride fluorescence microscopy, quantitative DNA fragmentation, and caspase-3 assay. Acetaldehyde (0-200 microm) elicited ROS generation and apoptosis in HUVECs in a time- and concentration-dependent manner, associated with activation of the stress signal molecules ERK1/2 and p38 mitogen-activated protein (MAP) kinase. A close liner correlation was observed between the acetaldehyde-induced ROS generation and apoptosis. Interestingly, the acetaldehyde-induced ROS generation, apoptosis, activation of ERK1/2, and p38 MAP kinase were prevented by the ALDH2 transgene or antioxidant alpha-tocopherol. The involvement of ERK1/2 and p38 MAP kinase in acetaldehyde-induced apoptosis was confirmed by selective kinase inhibitors U0126, SB203580, and SB202190. Collectively, our data revealed that facilitation of acetaldehyde metabolism by ALDH2 transgene overexpression may prevent acetaldehyde-induced cell injury and activation of stress signals. These results indicated therapeutic potential of ALDH2 enzyme in the prevention and detoxification of acetaldehyde or alcohol-induced cell injury.

The stereo dissecting microscope is a widely used instrument for macro-structure observation and documentation. The emergence of digital imaging, along with sophisticated imaging software, makes this macro-imaging more efficient. It also makes possible certain special imaging modes that are difficult to accomplish with traditional (film) imaging, such as extended depth of focus imaging ( EDF), automatic mont aging, etc. These special imaging techniques often require dedicated hardware on the microscope, such as a motorized stage.

Small RNA cloning and sequencing is uniquely positioned as a genome-wide approach to quantify miRNAs with single-nucleotide resolution. However, significant biases introduced by RNA ligation in current protocols lead to inaccurate miRNA quantification by 1000-fold. Here we report an RNA cloning method that achieves over 95% efficiency for both 5′ and 3′ ligations. It achieves accurate quantification of synthetic miRNAs with less than two-fold deviation from the anticipated value and over a dynamic range of four orders of magnitude. Taken together, this high-efficiency RNA cloning method permits accurate genome-wide miRNA profiling from total RNAs.

Sperm cells in flowering plants are non-motile and are passive participants in their movement to the female reproductive cells. It is believed that actomyosin interaction may play a key role for sperm cell transmission in the pollen tube as well as in the embryo sac. However, indirect evidence has shown that the surface of sperm cells lacks amounts of myosin sufficient to support movement. Immunofluorescence microscopy was used in this study to further assess the presence of myosin on the surface of sperm cells ofPlumbago Zeylanica.Sperm cells ofPlumbago Zeylanicawere isolated according to published methods. Isolated sperm cells were blocked 20 min with 1% bovine serum albumin and 2% normal goat serum in phosphate buffered saline (PBS) (pH 7.3, in 15% sucrose), incubated 2 hrs in anti-myosin antibody (M-7648, Sigma Chemical Co., St. Louis, MO) diluted 1:10 with blocking solution, washed three times (5 min) with blocking solution and incubated 1 hr in 1:30 FITC-conjugated anti-rabbit IgG as secondary antibody (EY Labs, Inc., San Mateo, CA) in blocking solution. Samples were rinsed in PBS and mounted in an anti-fading solution with 1:1 PBS:glycerol with 3% n-propyl gallate.