Alpha-synuclein aggregates are phosphatase resistant.

Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-min and 1-h postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.

Alpha-synuclein aggregates are phosphatase resistant.

Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-minute and 1-hour postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.

Mapping, modeling, and characterization of protein-protein interactions on a proteomic scale.

Proteins effect a number of biological functions, from cellular signaling, organization, mobility, and transport to catalyzing biochemical reactions and coordinating an immune response. These varied functions are often dependent upon macromolecular interactions, particularly with other proteins. Small-scale studies in the scientific literature report protein-protein interactions (PPIs), but slowly and with bias towards well-studied proteins. In an era where genomic sequence is readily available, deducing genotype-phenotype relationships requires an understanding of protein connectivity at proteome-scale. A proteome-scale map of the protein-protein interaction network provides a global view of cellular organization and function. Here, we discuss a summary of methods for building proteome-scale interactome maps and the current status and implications of mapping achievements. Not only do interactome maps serve as a reference, detailing global cellular function and organization patterns, but they can also reveal the mechanisms altered by disease alleles, highlight the patterns of interaction rewiring across evolution, and help pinpoint biologically and therapeutically relevant proteins. Despite the considerable strides made in proteome-wide mapping, several technical challenges persist. Therefore, future considerations that impact current mapping efforts are also discussed.

Comparison of white blood cell counts by WNR, WDF, and WPC channels in Sysmex XN hematology analyzer.

INTRODUCTION: The Sysmex XN modular system (Sysmex, Kobe, Japan) uses a novel technology for white blood cell (WBC) count and differential, using separate channels: white cell nucleated (WNR), WBC differential (WDF), and white progenitor cell (WPC) channels. We questioned how concordant WBC counts would be between them. METHODS: In a total of 6327 consecutive specimens, WBC counts were compared between WNR and WDF channels. They were also compared in three groups of WBC counts and two groups of chemotherapy status. In 508 specimens from the 4361 specimens that were run on the XN-20 module, the WPC channel was used for reflex test. Data were compared using Pearson's correlation, absolute difference, and percent difference (%D). RESULTS: WBC counts between WNR and WDF channels showed very high correlations in total specimens (r = 0.9976) and in the groups of WBC counts and chemotherapy. As WBC count increased, absolute difference increased, while %D decreased (P < 0.0001, both). Percent difference was 1.55% in total specimens and showed the highest value in the severe leukopenia group (<1.0 × 10(9)/L, 6.18%). CONCLUSIONS: This is the first large-scale study on novel channel technology for WBC counts in the Sysmex XN. WBC counts by WNR, WDF, and WPC channels are highly correlated, and they are overall interchangeable and reliable.

Evaluation of ABX Pentra DX 120 and Sysmex XE-2100 in umbilical cord blood.

INTRODUCTION: Cord blood (CB) is an important source of hematopoietic stem cells and reflects the hematologic status of neonates. ABX Pentra DX 120 (Horiba Medical, Montpellier, France) and Sysmex XE-2100 (Sysmex, Kobe, Japan) were compared in 200 CB specimens. METHODS: Complete blood count parameters including white blood cell (WBC) differential counts were compared between the two analyzers. Double differential matrix (DDX) by ABX Pentra DX 120 and hematopoietic progenitor cell (HPC) by Sysmex XE-2100 were compared with CD34(+) cells by flow cytometry. RESULTS: Most of the parameters showed acceptable correlation between the two analyzers. Although WBC differential of both analyzers showed acceptable correlation with manual counts, mononuclear cells (MNC) by ABX Pentra DX 120 better correlated with manual count than MNC by Sysmex XE-2100. NRBC by Sysmex XE-2100 better correlated with manual count than NRBC by ABX Pentra DX 120. ABX Pentra DX 120 showed better flagging performances. DDX better correlated with CD34(+) cells than HPC. CONCLUSION: Although the results from both analyzers are mostly interchangeable and reliable in CB specimens, flagging performance of ABX Pentra DX 120 seems to be superior to that of Sysmex XE-2100. DDX by ABX Pentra DX 120 would be valuable to evaluate the quality of CB for further therapeutic utilization.

Establishment of reference interval for immature platelet fraction.

INTRODUCTION: Immature platelet fraction (IPF) is a parameter for reticulated platelets. A high percentage IPF (%-IPF) is indicative of consumptive or recovering thrombocytopenic disorders in contrast to a low %-IPF seen in aplastic states. Absolute IPF (A-IPF) specifically reflects the number of immature platelets in circulation. This study aimed to establish reliable reference intervals for %-IPF and A-IPF. METHODS: Except outliers, platelet counts and IPF were determined in 2152 healthy individuals (1252 men and 900 women) and 133 umbilical cord blood from healthy full-term neonates using XE-2100 hematology analyzer (Sysmex, Kobe, Japan). The reference intervals for %-IPF and A-IPF were defined using nonparametrical percentile methods according to the Clinical and Laboratory Standard Institute (CLSI) guideline. RESULTS: Platelets,%-IPF, and A-IPF all showed nonparametrical distributions. In total individuals, the reference intervals for %-IPF and A-IPF were 0.5-3.3% (0.5-3.1% in men; 0.5-3.4% in women) and 1.25-7.02 × 10(9) /L (1.30-6.80 × 10(9) /L in men; 1.21-7.15 × 10(9) /L in women), respectively. The reference intervals for %-IPF and A-IPF in umbilical cord blood were 0.7-3.8% and 1.93-9.7 × 10(9) /L, respectively. CONCLUSIONS: This study provides the reference interval for IPF, including %-IPF and A-IPF, according to the CLSI guideline. These results could be used as fundamental data for clinical use as well as future researches.

Effects of banana, orange, and apple on oxidative stress-induced neurotoxicity in PC12 cells.

Banana, orange, and apple are the major fruits in Western and Asian diets. In order to find the effects of these fruits, neuron like PC12 cells were exposed to the extracts of these fruits before H(2)O(2) treatment. We found a significant viability of PC12 cells by the MTT reduction test, which indicated that the phenolics of banana, orange, and apple fruits prevented oxidative stress-induced neurotoxicity. Additional tests by lactate dehydrogenase and trypan blue exclusion assays showed that the extracts reduced oxidative stress-induced neuronal cell membrane damage. These results suggest that fresh apples, banana, and orange in our daily diet along with other fruits may protect neuron cells against oxidative stress-induced neurotoxicity and may play an important role in reducing the risk of neurodegenerative disorders such as Alzheimer's disease.

The hepatitis B virus encoded oncoprotein pX amplifies TGF-beta family signaling through direct interaction with Smad4: potential mechanism of hepatitis B virus-induced liver fibrosis.

Hepatitis B, one of the most common infectious diseases in the world, is closely associated with acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Many clinical investigations have revealed that hepatic fibrosis is an important component of these liver diseases caused by chronic hepatitis B. TGF-beta signaling plays an important role in the pathogenesis of fibrosis in chronic hepatitis and cirrhosis. As these diseases are associated with hepatitis B virus (HBV) infection, we examined the possibility that the HBV-encoded pX oncoprotein regulates TGF-beta signaling. We show that pX enhances transcriptional activity in response to TGF-beta, BMP-2, and activin by stabilizing the complex of Smad4 with components of the basic transcriptional machinery. Additionally, confocal microscopic studies suggest that pX facilitates and potentiates the nuclear translocation of Smads, further enhancing TGF-beta signaling. Our studies suggest a new paradigm for amplification of Smad-mediated signaling by an oncoprotein and suggest that enhanced Smad-mediated signaling may contribute to HBV-associated liver fibrosis.

Over-expression of ERT(ESX/ESE-1/ELF3), an ets-related transcription factor, induces endogenous TGF-beta type II receptor expression and restores the TGF-beta signaling pathway in Hs578t human breast cancer cells.

The epithelium-specific transcription factor, ERT/ESX/ESE-1/ELF3, binds to the TGF-beta RII promoter in a sequence specific manner and regulates its expression. In this study, we investigated whether ERT could regulate endogenous TGF-beta RII expression in Hs578t breast cancer cells. Analyses of the Hs578t parental cell line revealed low RII mRNA expression and resistance to the growth inhibitory effects of TGF-beta. Infection of this cell line with a retroviral construct expressing ERT induced higher levels of endogenous RII mRNA expression and protein expression relative to cells infected with chloramphenicol acetyltransferase (CATneo) as a control. Relative to control cells, the ERTneo-expressing Hs578t cells show approximately a 50% reduction in cell growth in the presence of exogenous TGF-beta1, as well as a fourfold higher induction of activation in transient transfection assays using the 3TP-luciferase reporter construct. When transplanted into athymic mice, ERT-expressing Hs578t cells showed decreased and delayed tumorigenicity compared with control cells. This data strongly suggests that ERT plays an important role as a transcriptional activator of TGF-beta RII expression, and that deregulated ERT expression may play a critical role in rendering Hs578t human breast cancer cells insensitive to TGF-beta's growth inhibitory effects.

A co-operative study: clinical characteristics of 334 Korean patients with moyamoya disease treated at neurosurgical institutes (1976-1994). The Korean Society for Cerebrovascular Disease.

A co-operative study was conducted to determine the clinical characteristics of patients with moyamoya disease who were diagnosed and treated at neurosurgical institutes in Korea before 1995. Twenty-six hospitals contributed 505 cases and among them, the clinical characteristics of 334 patients with definite moyamoya disease were evaluated. The number of patients began to increase from the late 1980s, and after that approximately 20 patients were treated each year. There were two age peaks: from six to 15 and from 31 to 40 years of age. Haemorrhagic manifestations occurred in approximately 43% of the patients. The major clinical manifestations were haemorrhage in adults (62.4%) and ischaemia in children (61.2%). Overall 54.5% of the patients experienced decreased consciousness levels, mainly due to intracranial haemorrhage or cerebral infarction. In the patients with ischemic manifestations, the adult patients were more likely to have cerebral infarction than the pediatric patients (80% vs. 39%) and the pediatric patients were more likely to have TIA (61% vs. 25%). Thirty eight percent of the patients underwent bypass surgery and 53% of these procedures were performed bilaterally. Treatment policies, including indications for bypass surgery and commonly used drugs, were somewhat different according to the institution. Overall favorable outcome was 73%, and the most significant factor affecting poor outcome was haemorrhagic manifestation. This article describes the characteristics of 334 patients with moyamoya disease, who were diagnosed and treated at neurosurgical institutes in Korea before 1995.

Repression of the gene encoding the TGF-beta type II receptor is a major target of the EWS-FLI1 oncoprotein.

Chromosomal translocations resulting in the expression of chimaeric transcription factors are frequently observed in tumour cells, and have been suggested to be a common mechanism in human carcinogenesis. Ewing sarcoma and related peripheral primitive neuroectodermal tumours share recurrent translocations that fuse the gene EWSR1 (formerly EWS) from 22q-12 to FLI1 and genes encoding other ETS transcription factors (which bind DNA through the conserved ETS domain). It has been shown that transduction of the gene EWSR1-FLI1 (encoding EWS-FLI1 protein) can transform NIH3T3 cells, and that mutants containing a deletion in either the EWS domain or the DNA-binding domain in FLI1 lose this ability. This indicates that the EWS-FLI1 fusion protein may act as an aberrant transcription factor, but the exact mechanism of oncogenesis remains unknown. Because ETS transcription factors regulate expression of TGFBR2 (encoding the TGF-beta type II receptor, TGF-beta RII; Refs 9,14), a putative tumour suppressor gene, we hypothesized that TGFBR2 may be a target of the EWS-FLI1 fusion protein. We show here that Ewing sarcoma [corrected] (ES) cell lines with the EWSR1-FLI1 fusion have reduced TGF-beta sensitivity, and that fusion-positive ES cells and primary tumours both express low or undetectable levels of TGFBR2 mRNA and protein product. Co-transfection of FLI1 and the TGFBR2 promoter induces promoter activity, whereas EWSR1-FLI1 leads to suppression of TGFBR2 promoter activity and FLI1-induced promoter activity. Introduction of EWSR1-FLI1 into cells lacking the EWSR1-FLI1 fusion suppresses TGF-beta RII expression, whereas antisense to EWSR1-FLI1 in ES cell lines positive for this gene fusion restores TGF-beta RII expression. Furthermore, introduction of normal TGF-beta RII into ES cell lines restores TGF-beta sensitivity and blocks tumorigenicity. Our results implicate TGF-beta RII as a direct target of EWS-FLI1.

Development of TGF-beta resistance during malignant progression.

Transforming growth factor-beta (TGF-beta) is the prototypical multifunctional cytokine, participating in the regulation of vital cellular activities such as proliferation and differentiation as well as a number of basic physiological functions. The effects of TGF-beta are critically dependent on the expression and distribution of a family of TGF-beta receptors, the TGF-beta types I, II, and III. It is now known that a wide variety of human pathology can be caused by aberrant expression and function of these receptors. The coding sequence of the type II receptor (RII) appears to render it uniquely susceptible to DNA replication errors in the course of normal cell division. By virtue of its key role in the regulation of cell proliferation, TGF-beta RII should be considered as a tumor suppressor gene. High levels of mutation in the TGF-beta RII gene have been observed in a wide range of primarily epithelial malignancies, including colon and gastric cancer. It appears likely that mutation of the TGF-beta RII gene may be a very critical step in the pathway of carcinogenesis.

Transcriptional activation of transforming growth factor beta1 and its receptors by the Kruppel-like factor Zf9/core promoter-binding protein and Sp1. Potential mechanisms for autocrine fibrogenesis in response to injury.

We have explored the regulation of transforming growth factor beta (TGF-beta) activity in tissue repair by examining the interactions of Zf9/core promoter-binding protein, a Kruppel-like zinc finger transcription factor induced early in hepatic stellate cell (HSC) activation, with promoters for TGF-beta1 and TGF-beta receptors, types I and II. Nuclear extracts from culture-activated HSCs bound avidly by electrophoretic mobility shift assay to two tandem GC boxes within the TGF-beta1 promoter but minimally to a single GC box; these results correlated with transactivation by Zf9 of TGF-beta1 promoter-reporters. Zf9 transactivated the full-length TGF-beta1 promoter in either primary HSCs, HSC-T6 cells (an SV40-immortalized rat HSC line), Hep G2 cells, or Drosophila Schneider (S2) cells. Recombinant Zf9-GST also bound to GC box sequences within the promoters for the types I and II TGF-beta receptors. Both type I and type II TGF-beta receptor promoters were also transactivated by Zf9 in mammalian cells but not in S2 cells. In contrast, Sp1 significantly transactivated both receptor promoters in S2 cells. These results suggest that (a) Zf9/core promoter-binding protein may enhance TGF-beta activity through transactivation of both the TGF-beta1 gene and its key signaling receptors, and (b) transactivating potential of Zf9 and Sp1 toward promoters for TGF-beta1 and its receptors are not identical and depend on the cellular context.

A novel ets-related transcription factor, ERT/ESX/ESE-1, regulates expression of the transforming growth factor-beta type II receptor.

A 2.5-kilobase cDNA clone that encodes a 371-amino acid novel transcription factor was isolated from a human placenta cDNA library using a yeast one-hybrid system. The novel ets-related transcription factor (ERT) showed a homology with the ETS DNA-binding domain. Using constructs of the transforming growth factor-beta (TGF-beta) type II receptor (RII) promoter linked to the luciferase gene, we have demonstrated that ERT activates transcription of the TGF-beta RII gene through the 5'-TTTCCTGTTTCC-3' response element spanning nucleotides +13 to +24 and multiple additional ETS binding sites between -1816 and -82 of the TGF-beta RII promoter. A specific interaction between ERT and the ETS binding sites was also demonstrated using an electrophoretic mobility shift assay. Deletion mapping of ERT protein suggests that the transactivation domain resides in the amino terminus while the DNA-binding domain is localized to the carboxyl-terminal region. Our results suggest that ERT might be a major transcription factor involved in the transcriptional regulation of the TGF-beta RII gene.

Unfolding properties of tryptophan-containing alpha-subunits of the Escherichia coli tryptophan synthase.

The urea-induced unfolding of the Escherichia coli tryptophan synthase alpha-subunit is examined via fluorescence measurements with tryptophan-containing alpha-subunit mutants, constructed by in vitro mutagenesis. Early unfolding studies with urea and guanidine suggested that the wild type protein unfolded in a two-step process with a stable intermediate composed of a native alpha-1 folding unit (residues 1-188) and a completely unfolded alpha-2 folding unit (residues 189-268). Recently, more detailed spectroscopic and calorimetric data from the Matthews and Yutani groups indicate that such a structure for the intermediates seems unlikely. Previously, we described the introduction of Trp residues as unfolding reporter groups separately into each of the folding domains and showed that these proteins are wild type enzymatically and in their stability to urea. The unfolding behavior of these alpha-subunits, monitored by fluorescence intensity changes at the discrete emission lambda max for each, in both equilibrium and kinetic experiments, suggest that: (a) both folding units commence unfolding simultaneously (near 2 M urea); (b) the larger alpha-1 unit unfolds in a multistep process, initially yielding a partially unfolded intermediate form which subsequently appears to unfold progressively to completion; and (c) the smaller alpha-2 unit unfolds in a single step event. These results are also clearly incompatible with the early proposals on the structure of the intermediate. It is suggested here that the intermediate is heterogeneous, consisting of a stable, partially unfolded form of alpha-1 attached to either a completely folded or completely unfolded form of alpha-2. These results are consistent with and provide an added dimension to the recent description of the proposed structure of the intermediate.

Tryptophan-containing alpha-subunits of the Escherichia coli tryptophan synthase. Enzymatic and urea stability properties.

Early studies suggested that the Escherichia coli tryptophan synthase alpha-subunit unfolded in a two-step process in which there was a stable intermediate composed of a native alpha-1 folding unit (residues 1-188) and a completely unfolded alpha-2 folding unit (residues 189-268). More recent evidence has indicated that such a structure for the intermediate seems unlikely. In this report, single Trp residues (absent in the wild-type alpha-subunit) are substituted separately for Phe residues at positions 139 (in alpha-1) and 258 (in alpha-2) to produce the F139W, F258W, and F139W/F258W mutant alpha-subunits. The UV absorbance and fluorescence properties of the F139W/F258W double mutant are identical with those of equimolar mixtures of the single mutants, suggesting that the Trp residue at each position can independently report the behavior of its respective folding unit. Each mutant alpha-subunit is wild-type enzymatically, and when UV absorbance is monitored, the urea-induced unfolding of the three tryptophan-containing alpha-subunits is virtually identical to the wild-type protein. These wild-type properties make these proteins attractive candidates for a fluorescence examination of the behavior of the individual folding units and the structure of potential intermediate(s) and as host proteins for the insertion of our existing destabilizing and/or stabilizing mutational alterations.

Effects of vasectomy on medical and psychosocial aspects.

PIP: To understand the effects of vasectomy on men, 1080 men were interviewed an average of 3.7 years after having had this operation. These men were grouped according to whether they privately paid for their operation or received it through a government program. The average age of these subjects was 37.8 years, and they had an average of 4.7 children. Reasons for vasectomy included economic considerations and the desire to limit their family. This type of operation was selected due to its permanance and reliability. Many men were motivated by family counselors or by the mass media. 68% had discussed the operation with their wives. 16% of the men had fears of impotence after the operation. Most were operated under a local anesthesia, and were not hospitalized. 57% did not bother to stay at home in recovery. Pain was encountered by 25% during the operation. Sexual activity was resumed in 3 weeks, with little change reported in eating or sleeping habits. Coital frequency decreased from 1.92-1.70 times/week. 90% of the men said they would recommend the operation to others.^ieng