Comprehensive analysis of RHD splicing transcripts reveals the molecular basis for the weak anti-D reactivity of Del -red blood cells.

BACKGROUND: Of the Rh blood type, Del is a rare variant that elicits the weakest anti-D reactivity. In this study, we revisit the genetic changes of Del allele and characterise the RHD splicing transcripts to realise the molecular basis of Del formation in the Taiwanese population. STUDY DESIGN AND METHODS: The RHD exons from Del and D-positive individuals were amplified by polymerase chain reaction (PCR) using different primer pairs followed by sequencing analyses. In addition, full-length RHD transcripts were reversed transcribed and amplified by nested-PCR. The type and frequency of the RHD splicing transcripts were analysed after sequencing the PCR products that were subcloned into a cloning vector. RESULTS: All Del individuals had a characteristic 1227G>A mutation. No deletion of the exon sequences was found. At least nine types of RHD splicing transcripts including exons 7/8/9 deletion, 7/9 deletion, 8/9 deletion, 9 deletion, 2/3/7/9 deletion, 2/3/7/8/9 deletion, exons 7/8/9 deletion with replacement of exon 3 with RHCE exon 3, exon 9 deletion with cryptic insertion of 170 bp of intron 7 and exons 7/8/9 deletion with cryptic insertion of 117 bp of intron 3 were identified in the Del -RBC. These aberrant splicing transcripts led to production of frame shift or truncated D antigen. Notably, no full-length RHD transcript was identified in the Del -RBC. CONCLUSION: The RHD 1227G>A mutation contributes to the molecular basis of Del phenotype in the Taiwanese population. The point mutation results in aberrant frame shift or exon deletion transcripts and generates D protein with weak antigen presenting function.

Genetic and mechanistic evaluation for the weak A phenotype in Ael blood type with IVS6 + 5G>A ABO gene mutation.

BACKGROUND AND OBJECTIVES: Ael is a rare blood type that is characterized by weak agglutination of RBCs when reacts with anti-A antibody in adsorption-elution test. Although IVS6 + 5G→A mutation is known to associate with the Ael blood type, genetic and mechanistic evaluation for the weak agglutination of Ael with IVS6 + 5G→A mutation has not yet been completely addressed. MATERIALS AND METHODS: In this study, five cases of confirmed Ael individuals were analysed. The cDNAs for the A(el) alleles were obtained by cloning method for sequence analyses. The erythroleukemia K562 cells were used as the cell study model and were transfected with the A(el) expression construct. Flow cytometry analysis was then performed to determine the levels of surface antigen expression. RESULTS: The results indicated that IVS6 + 5G→A attributes to all cases of Ael . RT-PCR analyses revealed the presence of at least 10 types of aberrant A(el) splicing transcripts. Most of the transcripts caused early termination and produced non-functional protein during translation. Nevertheless, the transcript without exons 5-6 was predicted to generate functional Ael glycosyltransferase lacking 57 amino acids at the N-terminal segment. When the exons 5-6 deletion transcript was stably expressed in the K562 cells, weak agglutination of the cells can be induced by adding anti-A antibody followed by adsorption-elution test. CONCLUSION: This study demonstrates that aberrant splicing of A transcripts contributes to weak A expression and the weak agglutination of Ael -RBCs, adding to the complexity for the regulatory mechanisms of ABO gene expression.

Enhancing the stability of xylanase from Cellulomonas fimi by cell-surface display on Escherichia coli.

AIMS: The cell-surface display of Cex, which encodes xylanase and exoglucanase from Cellulomonas fimi, was constructed on Escherichia coli using PgsA as the anchor protein. Characterization of the cell-surface display of Cex was performed. METHODS AND RESULTS: PgsA was fused to the N-terminus of Cex and six histidines were utilized as spacers between the targeting and anchor proteins. Successful cell-surface display of Cex was demonstrated by Western blot and immunofluorescence analyses on E. coli C41 (DE3). According to the time-course analysis, the xylanase activity of Cex was achieved at 49Ug(-1) dry cell weight after 12 h culture at 37°C. The optimal temperature and pH ranges of the cell-surface displayed protein with whole-cell were broader than the corresponding ranges of the purified form. Further determination of thermostability indicated that the half-life of cell-surface displayed Cex was 1·6 times longer than that of purified Cex at 60°C. CONCLUSIONS: We have successfully developed the cell-surface display of xylanase on E. coli. The cell-surface display can enhance the stability of xylanase against changes in temperature and has the potential of becoming a whole-cell biocatalyst for industrial applications, such as biobleaching of paper and production of renewable energy. SIGNIFICANCE AND IMPACT OF THE STUDY: The results demonstrated that the cell-surface display of xylanase embedded in the cell membrane is more stable than that of the purified enzyme. Thus, to improve the stability of heterologous proteins production, cell-surface display using the PgsA anchor protein as a tool can be considered in E. coli.

Functional links between Disabled-2 Ser723 phosphorylation and thrombin signaling in human platelets.

Essentials Disabled-2 (Dab2) phosphorylation status in thrombin signaling of human platelet was investigated. Ser723 was the major Dab2 phosphorylation site in human platelets stimulated by thrombin. Dab2 S723 phosphorylation (pS723) caused the dissociation of Dab2-CIN85 protein complex. Dab2-pS723 regulated ADP release and integrin αIIbß3 activation in thrombin-treated platelets. SUMMARY: Background Disabled-2 (Dab2) is a platelet protein that is functionally involved in thrombin signaling in mice. It is unknown whether or not Dab2 undergoes phosphorylation during human platelet activation. Objectives To investigate the phosphorylation status of Dab2 and its functional consequences in thrombin-stimulated human platelets. Methods Dab2 was immunoprecipitated from resting and thrombin-stimulated platelet lysates for differential isotopic labeling. After enrichment of the phosphopeptides, the phosphorylation sites were analyzed by mass spectrometry. The corresponding phospho-specific antibody was generated. The protein kinases responsible for and the functional significance of Dab2 phosphorylation were defined by the use of signaling pathway inhibitors/activators, protein kinase assays, and various molecular approaches. Results Dab2 was phosphorylated at Ser227, Ser394, Ser401 and Ser723 in thrombin-stimulated platelets, with Ser723 phosphorylation being the most significantly increased by thrombin. Dab2 was phosphorylated by protein kinase C at Ser723 in a Gαq -dependent manner. ADP released from the stimulated platelets further activated the Gßγ -dependent pathway to sustain Ser723 phosphorylation. The Cbl-interacting protein of 85 kDa (CIN85) bound to Dab2 at a motif adjacent to Ser723 in resting platelets. The consequence of Ser723 phosphorylation was the dissociation of CIN85 from the Dab2-CIN85 complex. These molecular events led to increases in fibrinogen binding and platelet aggregation in thrombin-stimulated platelets by regulating αIIb ß3 activation and ADP release. Conclusions Dab2 Ser723 phosphorylation is a key molecular event in thrombin-stimulated inside-out signaling and platelet activation, contributing to a new function of Dab2 in thrombin signaling.

Transgenic mice overexpressing protein kinase Cdelta in the epidermis are resistant to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.

To determine the role of protein kinase Cdelta in mouse skin carcinogenesis, we have developed transgenic FVB/N mouse lines expressing in the epidermis an epitope-tagged protein kinase Cdelta (T7-PKCdelta) regulated by the human keratin 14 promoter. The untreated T7-PKCdelta mice displayed excessive dryness in the skin of the tail with a variable penetrance over time. Histologically, the tail skin showed hyperplasia with evidence of hyperkeratosis. The epidermis of the rest of the T7-PKCdelta mouse was unremarkable. Despite this mild phenotype, the effects of PKCdelta overexpression on mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) were dramatic. Two independent lines of T7-PKCdelta mice (16 and 37) expressing the T7-PKCdelta transgene were examined for responsiveness to skin tumor promotion by 7,12-dimethylbenz[a]anthracene and TPA. By immunoblot analysis, the T7-PKCdelta-16 and T7-PKCdelta-37 mice showed an 8- and 2-fold increase of PKCdelta protein. The T7-PKCdelta-16 mice averaged 300% more T7-PKCdelta activity than the T7-PKCdelta-37 mice did. The T7-PKCdelta-37 mice did not manifest any difference in tumor burden or incidence. However, the reduction in papilloma burden at 25 weeks of promotion for the T7-PKCdelta-16 mice relative to wild-type mice averaged 72 and 74% for males and females, respectively. The T7-PKCdelta-16 mice reached 50% papilloma incidence between 12 and 13 weeks of promotion compared with 8 weeks for wild-type mice. Furthermore, the carcinoma incidence was also reduced in T7-PKCdelta-16 mice. Carcinoma incidence at 25 weeks of promotion treatment was: wild-type females, 78%; T7-PKCdelta16 females, 37%; wild-type males, 45%; and T7- PKCdelta-16 males, 7%. Thus, PKCdelta when expressed at sufficient levels can suppress skin tumor promotion by TPA.

Loss of adenoviral receptor expression in human bladder cancer cells: a potential impact on the efficacy of gene therapy.

There is great interest in the development of gene therapeutic strategies for the treatment of benign and malignant diseases. Recombinant adenovirus has a wide spectrum of tissue specificity and is an efficient vector delivery system. Successful gene delivery, however, requires viral entry into the target cells via specific receptor-mediated uptake. Recently, a cDNA clone (the coxsackie and adenovirus receptor [CAR]) encoding a 46-kDa protein was identified as the receptor for group C adenovirus (e.g., adenovirus type 2 and 5). Currently, little is known regarding the expression of adenoviral receptor in normal tissue and cancer. In this paper, we have documented a significant difference in viral receptor levels that may be due to transcriptional regulation of the CAR gene in several human bladder cancer cell lines. The differences in viral receptor levels in these cells correlated with their sensitivity to viral infection. Transfection of receptor-negative cell line with CAR cDNA led to increased virus binding and increased susceptibility to adenovirus-mediated gene delivery. Our results demonstrate that the expression of adenoviral receptor is variable among human bladder cancer cells. This variability may have a significant impact on the outcome of adenovirus-based gene therapy.

Clonality analysis using X-chromosome inactivation patterns by HUMARA-PCR assay in female controls and patients with idiopathic thrombocytosis in Taiwan.

OBJECTIVE: Analysis of X-chromosome inactivation patterns (XCIPs) is a useful tool in the diagnosis of clonal disorders. The human androgen receptor (HUMARA) locus is especially useful for clonality study. The present study was conducted 1) to determine the heterozygosity rate for HUMARA locus in Taiwanese women, 2) to determine the frequency of excessive skewing in different cell types, and 3) to determine the utility of XCIPs in the differential diagnosis of thrombocytosis. PATIENTS AND METHODS: XCIPs by HUMARA-PCR assay were performed on purified granulocytes and T cells from 73 female patients presenting with idiopathic persistent thrombocytosis (IT), 10 patients with reactive thrombocytosis (RT), and 46 bone marrow samples from female controls. XCIPs of buccal mucosa cells were also compared with those of T cells in 57 patients with IT. The percentage of clonal granulocytes was calculated after correcting for the degree of Lyonization in T cells. RESULTS: The heterozygosity rate for the HUMARA gene was 89.1% in Taiwanese females. The median age of informative IT patients and controls was 59 (18-92) and 58 (19-89), respectively. Excessive skewing (allele ratio <0.33) was more frequent in granulocytes than in T cells in both controls (12/43 vs 9/43, p = 0.080) and IT patients (56/64 vs 25/64, p < 0.001). XCIPs were the same for both buccal mucosa and T cells in 43 patients but were different in 14 patients. Of the 43 informative controls, 31 had a polyclonal pattern; an ambiguous pattern was found in nine; and the remaining three, aged 71, 73, and 80, respectively, had a clonal pattern. A clonal pattern was found in 42 IT patients, a polyclonal pattern in 12, and an ambiguous pattern in 10 of the 64 IT patients. The frequency of clonal, polyclonal, and ambiguous patterns in the 40 IT patients with age < or = 65 was 55.0%, 30.0%, and 15.0%, respectively. None of the IT patients aged >65 had a polyclonal disease. IT patients aged >65 had a significantly higher frequency of clonal pattern (p = 0.030) and a significantly lower frequency of polyclonal pattern (p = 0.002) than those with age <65. Of the eight heterozygous patients with RT, one aged 80 exhibited a clonal pattern, and the remaining seven had a polyclonal pattern. CONCLUSIONS: The present study on Taiwanese females showed a heterozygosity rate of 89.1% for the HUMARA gene. Our results confirmed that IT is a heterogeneous disorder in terms of clonality. Twenty-three percent of IT patients exhibited a greater than 20% difference in allele expression for buccal mucosa and T cells. Presence of a clonal XCIP in young patients with IT can serve as a positive marker for the diagnosis of clonal thrombocytosis, and elderly patients with polyclonal XCIPs are unlikely to have essential thrombocythemia.

Knockdown of c-MET induced apoptosis in ABCB1-overexpressed multidrug-resistance cancer cell lines.

Inappropriate c-MET signaling in cancer can enhance tumor cell proliferation, survival, motility, and invasion. Inhibition of c-MET signaling induces apoptosis in a variety of cancers. It has also been recognized as a novel anticancer therapy approach. Furthermore, reports have also indicated that constitutive expression of P-glycoprotein (ABCB1) is involved in the HGF/c-MET-related pathway of multidrug resistance ABCB1-positive human hepatocellular carcinoma cell lines. We previously reported that elevated expression levels of PKCδ and AP-1 downstream genes, and HGF receptor (c-MET) and ABCB1, in the drug-resistant MES-SA/Dx5 cells. Moreover, leukemia cell lines overexpressing ABCB1 have also been shown to be more resistant to the tyrosine kinase inhibitor imatinib mesylate. These findings suggest that chemoresistant cancer cells may also develop a similar mechanism against chemotherapy agents. To circumvent clinical complications arising from drug resistance during cancer therapy, the present study was designed to investigate apoptosis induction in ABCB1-overexpressed cancer cells using c-MET-targeted RNA interference technology in vitro and in vivo. The results showed that cell viability decreased and apoptosis rate increased in c-MET shRNA-transfected HGF/c-MET pathway-positive MES-SA/Dx5 and MCF-7/ADR2 cell lines in a dose-dependent manner. In vivo reduction of tumor volume in mice harboring c-MET shRNA-knockdown MES-SA/Dx5 cells was clearly demonstrated. Our study demonstrated that downregulation of c-MET by shRNA-induced apoptosis in a multidrug resistance cell line.

Regulation of rat DOC-2 gene during castration-induced rat ventral prostate degeneration and its growth inhibitory function in human prostatic carcinoma cells.

Androgen is a mitogen as well as a morphogen for prostatic epithelium. However, the detailed mechanisms of these distinct androgenic actions have not yet been delineated. Therefore, we employed differential display PCR to unveil any potential genes that may be involved in these processes. In this study, we report the isolation and characterization of two alternative splicing forms (p82 and p59) of C9 complementary DNA, the rat homolog of the human deletion of ovarian carcinoma 2 (DOC-2) gene and mouse p96 phosphoprotein, from rat ventral prostate (VP). We found that C9 was up-regulated in rat VP after castration, suggesting that C9 may be regulated by androgen receptor directly or indirectly during prostate degeneration. A similar regulatory pattern was also observed in both the seminal vesicle and dorsolateral prostate, but not in the coagulating gland or other androgen-independent organs. Immunohistochemical analysis of rat VP demonstrated that C9 is detected in the basal epithelia and surrounding stromal cells after prolonged castration. Ribonuclease protection assay and Western blot analysis revealed that p59 is the predominant C9 isoform in rat VP. To unveil the function of C9 in cell growth, we transfected p59 complementary DNA into the C4-2 cells, a derivative of the LNCaP prostatic carcinoma cell line. The p59 stable transfectants exhibited a slower growth rate and an increase in the cell fraction in the G1 phase under our experimental conditions. These data indicate that C9-p59 has growth inhibitory activity for prostatic epithelial cells. Taken together, our results suggest that C9 is up-regulated during prostate degeneration process and may play an active role in the proliferation and differentiation of prostatic epithelium.

Functional expression and characterization of the mouse epitope tag-protein kinase C isoforms, alpha, beta I, beta II, gamma, delta and epsilon.

To simplify the detection of the exogenously produced protein kinase C (PKC) isoforms, we constructed the T7-Tag PKC alpha, betaI, betaII, gamma, delta and epsilon expression plasmids. T7-Tag sequences (AlaSerMetThrGlyGlyGlnGlnMetGlyArg) were inserted at the 5'-end of the translational initiation site. Transient transfection of T7-Tag PKC expression plasmids into CV-1 cells increased the levels of PKC protein, and PKC activity. T7-Tag-PKC epsilon, like native PKC epsilon, transactivated the transcription of a NF-kappa B reporter construct. These results indicate that the plasmids encoding T7-Tag PKC are functional and may be useful to define the role PKC isoforms play in cell proliferation, differentiation and tumor promotion.

Identification of a novel secretory leukocyte protease inhibitor-binding protein involved in membrane phospholipid movement.

Previous studies have suggested that human salivary secretory leukocyte protease inhibitor (SLPI) inhibits HIV-1 by binding to a host cell surface protein of unknown identity. Using the yeast two-hybrid assay, we identified a gene sequence encoding a novel SLPI-binding protein (SLPI-BP). The 1.5-kb cDNA encodes a 318-amino acid protein with a predicted transmembrane segment near the C-terminus. Sequence analysis revealed that SLPI-BP is the human scramblase protein that is involved in the movement of membrane phospholipids. Co-expression of SLPI and SLPI-BP followed by an S-protein pulldown assay confirmed the specific interaction between these two proteins. Our data represent the first report for the identity of SLPI-BP.

Long-term arsenic exposure and incidence of non-insulin-dependent diabetes mellitus: a cohort study in arseniasis-hyperendemic villages in Taiwan.

Diabetes prevalence in arseniasis-hyperendemic villages in Taiwan has been reported to be significantly higher than in the general population. The aim of this cohort study was to further evaluate the association between ingested inorganic arsenic and the incidence of non-insulin-dependent diabetes mellitus in these villages. A total of 446 nondiabetic residents in these villages were followed biannually by oral glucose tolerance test. Diabetes is defined as a fasting plasma glucose level > or = 7.8 mmol/L and/or a 2-hr post-load glucose level > or = 11.1 mmol/L. During the follow-up period of 1499.5 person-years, 41 cases developed diabetes, showing an overall incidence of 27.4/1,000 person-years. The incidence of diabetes correlated with age, body mass index, and cumulative arsenic exposure. The multivariate-adjusted relative risks were 1.6, 2.3, and 2.1 for age > or = 55 versus < 55 years, a body mass index ¿Greater/Equal to] 25 versus < 25 kg/m(2), and a cumulative arsenic exposure > or = 17 versus < 17 mg/L-years, respectively. The incidence density ratios (95% confidence intervals) between the hyperendemic villages and the two nonendemic control townships were 3.6 (3.5-3.6), 2.3 (1.1-4.9), 4.3 (2.4-7.7), and 5.5 (2.2-13.5), respectively, for the age groups of 35-44, 45-54, 55-64, and 65-74 years. The findings are consistent with our previous cross-sectional observation that ingested inorganic arsenic is diabetogenic in human beings.

Regulation of fumarase (fumB) gene expression in Escherichia coli in response to oxygen, iron and heme availability: role of the arcA, fur, and hemA gene products.

Three distinct fumarases, FumA, FumB and FumC, have been reported in Escherichia coli. While the fumA and fumC gene products are expressed under aerobic cell growth conditions, the FumB fumarase appears to be more abundant during anaerobic growth. To study the transcriptional regulation of the fumB gene, a fumB-lacZ operon fusion was constructed and analyzed in a single copy under a variety of cell culture conditions. Expression of fumB-lacZ was fourfold higher under anaerobic than aerobic growth conditions. This anaerobic response is modulated by the ArcA and Fnr proteins, which function independently as anaerobic activators of fumB gene expression. Cellular iron limitation in a fur mutant caused fumB-lacZ expression to decrease sevenfold while cellular heme limitation decreased fumB gene expression twofold. In addition, fumB-lacZ expression was shown to vary depending on the DNA superhelicity. This study further delineates the regulation of the fumB gene in cell growth.

Real-time PCR for rapid genotyping of angiotensin-converting enzyme insertion/deletion polymorphism.

OBJECTIVE: To develop a real-time PCR technique for detection of the insertion/deletion (I/D) polymorphism of angiotensin-converting enzyme (ACE) gene. DESIGN AND METHODS: Three primers were designed for performing real-time PCR in the presence of SYBR Green I as flurochrome followed by melting curve analysis. Forty human genomic DNA that have been genotyped by two-rounds of conventional PCR were used for evaluation of this technique. RESULTS: Melting curve analysis indicated the melting peak at 73.9 degrees C and 76.2 degrees C corresponding to the presence of I and D alleles, respectively. Comparable genotyping results were obtained by both conventional and real-time PCR. Besides, the mistyping of ID allele individuals by the first run of conventional PCR were accurately genotyped by single-tube real time PCR. CONCLUSIONS: The real-time PCR method presented in this study provides a rapid and sensitive way for genotyping of ACE gene that may be suitable for large-scale clinical and epidemiologic study.

The incidence of type 2 diabetes mellitus in Taiwan.

Currently, diabetes mellitus is the fifth leading cause of death in Taiwan. The trends of diabetes mortality is increasing steadily. Epidemiologic studies also showed increasing prevalence of diabetes mellitus over the past few decades. The incidence of diabetes mellitus in Taiwan has only been studied in recent 10 years. The areas that have been included as study areas for diabetes incidence are Kin-Chen (Kinmen), Chu-Dung, Pu-Tzu, Pu-Li and Pu-Tai. The reported incidence rates ranged from 1.0 to 4.0% per year for people with varying degrees of baseline plasma glucose levels not reaching the diagnosis of diabetes mellitus according to the criteria of the World Health Organization. Age, baseline glucose level, and obesity are important predictors for the development of diabetes mellitus. In the Pu-Tai study, which was aimed at following a group of people who had been living in the hyperendemic villages of blackfoot disease and had been exposed to arsenic from drinking artesian well water, the incidence of diabetes mellitus was calculated to be 27.4 per 1000 person years. The incidence of diabetes mellitus in these arseniasis-hyperendemic villages correlated with age, body mass index and cumulative arsenic exposure.

Biodegradation of hydrogen sulfide by a laboratory-scale immobilized Pseudomonas putida CH11 biofilter.

A heterotrophic Pseudomonas putida CH11 was isolated from livestock farming wastewater and applied for the treatment of H2S-containing gas. Extensive tests including removal characteristics, metabolic products, and removal efficiencies of H2S by P. putida CH11 were examined in batch and continuous systems. The optimum pH required to remove hydrogen sulfide was found in the range of 6-8. The maximum removal rate and the saturation constant were calculated to be Vm = 1.36 g S/day.kg dry bead and Ks = 45.9 ppm, respectively. The main metabolic product of H2S oxidation was determined to be elemental sulfur. When P. putida CH11 was immobilized within Ca alginate, the cells exhibited high H2S removal efficiency, in excess of 96%, at concentration of hydrogen sulfide from 10 to 150 ppm (flow rates of 36 and 72 L/h). These results suggest that P. putida CH11 immobilized within Ca alginate has the potential to be used as a H2S removal agent.

Biotreatment of ammonia from air by an immobilized Arthrobacter oxydans CH8 biofilter.

A heterotrophic Arthrobacter oxydans CH8 that was capable of removing NH3 from NH3 containing gas was isolated from livestock farming wastewater. The A. oxydans CH8 was immobilized with calcium alginate packed into filter column. Metered NH3-containing gas was partially humidified and passed through the glass column. Extensive tests including the removal characteristics, the removal efficiencies, and the metabolic products of NH3 by A. oxydans CH8 were conducted. Additionally, the operation criteria for the biofilter was also established. NH3 removel capacities were elevated by the immobilized-cell (biological conversion) method and the BDST (bed depth service time) method (physical adsorption), respectively. The optium temperature for removing NH3 was 30 degrees C, while the nitrification ability remained 80% at 40 degrees C. The high efficiency (> 97%) in the removal of NH3 was attained at 36 L/h with pH control and was not decreased because of high NH3 inlet concentration. In addition, the high maximum removal rate (1.22 g of N/day (kg of bead)) enhanced the use of the biofilter in industrial-scale NH3(g) pollution control. The ability to remove NH3 at high inlet concentration and temperature suggested that the immobilized A. oxydans CH8 biofilter has potential in processing NH3 gas.

Biological elimination of H2S and NH3 from wastegases by biofilter packed with immobilized heterotrophic bacteria.

Biotreatment of various ratios of H2S and NH3 gas mixtures was studied using the biofilters, packed with co-immobilized cells (Arthrobacter oxydans CH8 for NH3 and Pseudomonas putida CH11 for H2S). Extensive tests to determine removal characteristics, removal efficiency, removal kinetics, and pressure drops of the biofilters were performed. To estimate the largest allowable inlet concentration, a prediction model was also employed. Greater than 95%, and 90% removal efficiencies were observed for NH3 and H2S, respectively, irrespective of the ratios of H2S and NH3 gas mixtures. The results showed that H2S removal of the biofilter was significantly affected by high inlet concentrations of H2S and NH3. As high H2S concentration was an inhibitory substrate for the growth of heterotrophic sulfur-oxidizing bacteria, the activity of H2S oxidation was thus inhibited. In the case of high NH3 concentration, the poor H2S removal efficiency might be attributed to the acidification of the biofilter. The phenomenon was caused by acidic metabolite accumulation of NH3. Through kinetic analysis, the presence of NH3 did not hinder the NH3 removal, but a high H2S concentration would result in low removal efficiency. Conversely, H2S of adequate concentrations would favor the removal of incoming NH3. The results also indicated that maximum inlet concentrations (model-estimated) agreed well with the experimental values for space velocities of 50-150 h(-1). Hence, the results would be used as the guideline for the design and operation of biofilters.

Biotreatment of H2S- and NH3-containing waste gases by co-immobilized cells biofilter.

Gas mixture of H2S and NH3 in this study has been the focus in the research area concerning gases generated from the animal husbandry and the anaerobic wastewater lagoons used for their treatment. A specific microflora (mixture of Thiobacillus thioparus CH11 for H2S and Nitrosomonas europaea for NH3) was immobilized with Ca-alginate and packed inside a glass column to decompose H2S and NH3. The biofilter packed with co-immobilized cells was continuously supplied with H2S and NH3 gas mixtures of various ratios, and the removal efficiency, removal kinetics, and pressure drop in the biofilter was monitored. The results showed that the efficiency remained above 95% regardless of the ratios of H2S and NH3 used. The NH3 concentration has little effect on H2S removal efficiency, however, both high NH3 and H2S concentrations significantly suppress the NH3 removal. Through product analysis, we found that controlling the inlet ratio of the H2S/NH3 could prevent the biofilter from acidification, and, therefore, enhance the operational stability. Conclusions from bioaerosol analysis and pressure drop in the biofilter suggest that the immobilized cell technique creates less environmental impact and improves pure culture operational stability. The criteria for the biofilter operation to meet the current H2S and NH3 emission standards were also established. To reach Taiwan's current ambient air standards of H2S and NH3 (0.1 and 1 ppm, respectively), the maximum inlet concentrations should not exceed 58 ppm for H2S and 164 ppm for NH3, and the residence time be kept at 72 s.

Impaired thrombin generation in Reelin-deficient mice: a potential role of plasma Reelin in hemostasis.

BACKGROUND: Reelin is a large extracellular glycoprotein that is present in the peripheral blood. That Reelin interacts with the coagulation components and elicits a functional role in hemostasis has not yet been elucidated. OBJECTIVES: The hemostatic activity of Reelin is investigated and defined in this study. METHODS: The interplay of Reelin with coagulation components was elucidated by far-Western and liposome/platelet binding assays. In vivo and ex vivo hemostasis-related analyses of Reelin-deficient mice and plasma were also performed. RESULTS: Reelin interacted with the liposomes containing phosphatidylserine (PS) or phosphatidylcholine. Instead of interacting with known Reelin receptors (ApoE receptor 2, very low density lipoprotein receptor and integrin ß1), Reelin interacted with PS of the activated platelets. The interaction between Reelin and the coagulation factors of thrombin and FXa was also demonstrated with the Kd of 11.7 and 21.2 nm, respectively. Reelin-deficient mice displayed a prolonged bleeding time and an increase in rebleeding rate. Despite the fact that Reelin deficiency had no significant effect on the clotting time of prothrombin and activated partial thromboplastin time, the fibrin clot formation was abnormal and the fibrin clot structure was relatively loosened with reduced clot strength. Abnormal fibrinogen expression did not account for the hemostatic defects associated with Reelin deficiency. Instead, thrombin generation was impaired concomitant with an altered prothrombin cleavage pattern. CONCLUSIONS: By interacting with platelet phospholipids and the coagulation factors, thrombin and FXa, Reelin plays a selective role in coagulation activation, leading to thrombin generation and formation of a normal fibrin clot.