Sodium Deoxycholate Treatment Research Articles

GYY4137, as a slow-releasing H2S donor, ameliorates sodium deoxycholate-induced chronic intestinal barrier injury and gut microbiota dysbiosis.

Numerous studies have revealed that a long-term high-fat diet can raise intestinal deoxycholate acid concentration, which can harm intestinal mucosal barrier function in several ways. This study aims to verify the protective effect of GYY4137, as a slow-releasing H2S donor, on microbiome disturbance and the chronic injury of the intestinal mucosal barrier function caused by sodium deoxycholate. Caco-2 monolayer and mouse models were treated with a relatively high concentration of sodium deoxycholate (1.0mM and 0.2%, respectively) for longer periods (32h and 12 weeks, respectively) to understand the effects of GYY4137 on sodium deoxycholate-induced chronic intestinal barrier dysfunction and its fundamental mechanisms. A relatively long period of sodium deoxycholate treatment can remarkably increase the intestinal barrier permeability, alter the distribution and expression of tight junction proteins and generate the production of pro-inflammatory cytokines (TNF-α and IL-1β) in the Caco-2 monolayers and mouse models. Moreover, it can activate the MLCK-P-MLC2 pathway in the Caco-2 monolayers, which was further confirmed using RNA sequencing. The body weight, intestinal barrier histological score, and TUNEL index of sodium deoxycholate-treated mice worsened. In addition, an induced microbiome imbalance was observed in these mice. The above variations can be reversed with the administration of GYY4137. This study demonstrates that GYY4137 ameliorates sodium deoxycholate-induced chronic intestinal barrier injury by restricting the MLCK-P-MLC2 pathway while elevating the expression level of tight junction proteins, anti-apoptosis and maintaining the microbiome's homeostasis.

Incompletely Decellularized Tracheal Matrix Scaffold for Tissue Engineering.

Dense cartilaginous extracellular matrix makes decellularization and repopulation of tracheal cartilage difficult. However, the dense matrix isolates cartilaginous antigens from the recipient's immune system. Therefore, allorejection may be avoided by removing antigens from noncartilaginous tissues. In this study, incompletely decellularized tracheal matrix scaffolds were developed for tracheal tissue engineering. Brown Norway rat tracheae were decellularized with 4% sodium deoxycholate treatment. The cell and antigen removal efficacy, histoarchitecture, surface ultrastructure, glycosaminoglycan, collagen contents, mechanical properties, and chondrocyte viability of the scaffold were evaluated in vitro. Brown Norway rat tracheal matrix scaffolds ( n = 6) were implanted subcutaneously into Lewis rats and observed for 4 weeks. Brown Norway rat tracheae ( n = 6) and Lewis rat scaffolds ( n = 6) were implanted as controls. Histologic analysis of macrophage and lymphocyte infiltration was performed. One decellularization cycle removed all cells and antigens from noncartilaginous tissue. Incomplete decellularization preserved the structural integrity of the tracheal matrix and chondrocyte viability. Except for 31% glycosaminoglycan loss, the scaffold had comparable collagen content and tensile and compressive mechanical properties to those of the native trachea. The allogeneic scaffold showed remarkably reduced CD68 + , CD8 + , and CD4 + cell infiltration compared with the allografts and demonstrated similar cell infiltration to the syngeneic scaffold. It also maintained the three-dimensional tracheal structure and cartilage viability in vivo. Incompletely decellularized trachea did not induce immunorejection and maintained the integrity and viability of cartilage in vivo. Tracheal decellularization and repopulation can be simplified for urgent tracheal replacement. The present study describes the development of an incomplete decellularization protocol that creates a decellularized matrix scaffold for tracheal tissue engineering, aiming to provide preliminary data that this method may generate suitable tracheal scaffolds for use in tracheal replacement.

Surfactant Treatment for Efficient Gene Detection of Enteric Viruses and Indicators in Surface Water Concentrated by Ultrafiltration.

The hollow fiber ultrafiltration (HFUF)-based microbial concentration method is widely applied for monitoring pathogenic viruses and microbial indicators in environmental water samples. However, the HFUF-based method can co-concentrate substances that interfere with downstream molecular processes-nucleic acid extraction, reverse transcription (RT), and PCR. These inhibitory substances are assumed to be hydrophobic and, therefore, expected to be excluded by a simple surfactant treatment before the silica membrane-based RNA extraction process. In this study, the efficacy and limitations of the sodium deoxycholate (SD) treatment were assessed by quantifying a process control and indigenous viruses using 42 surface water samples concentrated with HFUF. With some exceptions, which tended to be seen in samples with high turbidity (> 4.0 NTU), virus recovery by the ultrafiltration method was sufficiently high (> 10%). RNA extraction-RT-quantitative PCR (RT-qPCR) efficiency of the process control was insufficient (10%) for 30 of the 42 HFUF concentrates without any pretreatments, but it was markedly improved for 21 of the 30 inhibitory concentrates by the SD treatment. Detection rates of indigenous viruses were also improved and no substantial loss of viral RNA was observed. The SD treatment was particularly effective in mitigating RT-qPCR inhibition, although it was not effective in improving RNA extraction efficiency. The methodology is simple and easily applied. These findings indicate that SD treatment can be a good alternative to sample dilution, which is widely applied to mitigate the effect of RT-qPCR inhibition, and can be compatible with other countermeasures.

Simultaneous quantitative detection of viable Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. using sodium deoxycholate-propidium monoazide with multiplex real-time PCR

Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. are common food-borne pathogens in milk that may cause serious diseases. In the present study, we established a simple, rapid, and specific method to simultaneously detect viable E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk. Three specific genes, fliC from E. coli O157:H7, cgcA from Cronobacter spp., and invA from Salmonella spp., were selected and used to design primers and probes. False-positive results were eliminated with the use of a combined sodium deoxycholate (SD) and propidium monoazide (PMA) treatment. Using the optimized parameters, this SD-PMA treatment combined with multiplex real-time PCR (SD-PMA-mRT-PCR) detected E. coli O157:H7, Cronobacter spp. and Salmonella spp. respectively, at 102 cfu/mL in pure culture or artificially spiked skim milk samples. A reasonable recovery rate (from 100 to 107%) for detection of viable bacteria using the SD-PMA-mRT-PCR assay was obtained in the presence of dead bacteria at 107 cfu/mL. The SD-PMA-mRT-PCR method developed in this study can accurately detect and monitor combined contamination with E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk and milk products.

Recombinant anthrax protective antigen: Observation of aggregation phenomena by TEM reveals specific effects of sterols

Negatively stained transmission electron microscope images are presented that depict the aggregation of recombinant anthrax protective antigen (rPA83 monomer and the PA63 prepore oligomer) under varying in vitro biochemical conditions. Heat treatment (50°C) of rPA83 produced clumped fibrils, but following heating the PA63 prepore formed disordered aggregates. Freeze-thaw treatment of the PA63 prepore generated linear flexuous aggregates of the heptameric oligomers. Aqueous suspensions of cholesterol microcrystals were shown to bind small rPA83 aggregates at the edges of the planar bilayers. With PA63 a more discrete binding of the prepores to the crystalline cholesterol bilayer edges occurs. Sodium deoxycholate (NaDOC) treatment of rPA83 produced quasi helical fibrillar aggregate, similar but not identical to that produced by heat treatment. Remarkably, NaDOC treatment of the PA63 prepores induced transformation into pores, with a characteristic extended ß-barrel. The PA63 pores aggregated as dimers, that aggregated further as angular chains and closed structures in higher NaDOC concentrations. The significance of the sterol interaction is discussed in relation to its likely importance for PA action in vivo.

A new application of a sodium deoxycholate-propidium monoazide-quantitative PCR assay for rapid and sensitive detection of viable Cronobacter sakazakii in powdered infant formula.

A rapid, reliable, and sensitive method for the detection of Cronobacter sakazakii, a common foodborne pathogen that may cause serious neonatal disease, has been developed. In this study, a rapid real-time quantitative PCR (qPCR) assay combined with sodium deoxycholate (SD) and propidium monoazide (PMA) was developed to detect C. sakazakii contamination in powdered infant formula (PIF). This method could eliminate the interference from dead or injured bacteria. Optimization studies indicated that SD and PMA at 0.08% (wt/vol) and 5µg/mL, respectively, were the most appropriate. In addition, qPCR, PMA-qPCR, SD-PMA-qPCR, and plate count assays were used to account for the number of viable bacteria in cell suspensions that were exposed to a 55°C water bath at different length of time. As a result, the viable number by PMA-qPCR showed significantly higher than of the number from SD-PMA-qPCR or plate counts. The number of viable bacteria was consistent between SD-PMA-qPCR and traditional plate counts, which indicated that SD treatment could eliminate the interference from dead or injured cells. Using the optimized parameters, the limit of detection with the SD-PMA-qPCR assay was 3.3×102 cfu/mL and 4.4×102 cfu/g in pure culture and in spiked PIF, respectively. A similar detection limit of 5.6×102 cfu/g was obtained in the presence of the Staphylococcus aureus (107 cfu/mL). The combined SD-PMA-qPCR assay holds promise for the rapid detection of viable C. sakazakii in PIF.

Rapid and accurate detection of viable Escherichia coli O157:H7 in milk using a combined IMS, sodium deoxycholate, PMA and real-time quantitative PCR process

To improve the accuracy of current methods for the detection of Escherichia coli O157:H7 foodborne disease outbreaks, two reagents, propidium monoazide (PMA) and sodium deoxycholate (SD), were utilized to eliminate the interference of dead and injured cells for qPCR that was combined with immunomagnetic separation (IMS) for cell enrichment in an IMS-SD-PMA-qPCR assay. The optimal SD concentration and the optimal incubation time with SD were recorded at 0.1% and 20 min, respectively. The number of bacteria survivors was compared using plate counts, qPCR, PMA-qPCR, and SD-PMA-qPCR assays after the cell suspensions were heat treated at 63 °C or freeze stored at −20 °C. Cell suspensions treated or not treated with PMA and treated with SD resulted in significantly lower number of bacteria survivors than those analyzed without SD treatment which indicated that dead cell DNA was eliminated with SD. More importantly, the number of bacteria survivors from those analyzed with SD-PMA-qPCR showed a direct correlation with those analyzed using the plate counts methods. In addition, in order to improve the limit of detection (LOD) and shorten the detection time, immunomagnetic separation (IMS) was adopted to capture and enrich the target bacteria. Using spiked milk as a matrix, the IMS-SD-PMA-qPCR showed a detection limit of 102 CFU/mL. Significantly, even in the presence of 106 CFU/mL of non-target bacteria, the LOD for the SD-PMA-qPCR with IMS separation for E. coli O157:H7 in spiked milk matrix was recorded at 102 CFU/mL. This combination assay holds promise for the detection of foodborne E. coli O157:H7.

Activation of NF-κB Protein Prevents the Transition from Juvenile Ovary to Testis and Promotes Ovarian Development in Zebrafish

Testis differentiation in zebrafish involves juvenile ovary to testis transformation initiated by an apoptotic wave. The molecular regulation of this transformation process is not fully understood. NF-κB is activated at an early stage of development and has been shown to interact with steroidogenic factor-1 in mammals, leading to the suppression of anti-Müllerian hormone (Amh) gene expression. Because steroidogenic factor-1 and Amh are important for proper testis development, NF-κB-mediated induction of anti-apoptotic genes could, therefore, also play a role in zebrafish gonad differentiation. The aim of this study was to examine the potential role of NF-κB in zebrafish gonad differentiation. Exposure of juvenile zebrafish to heat-killed Escherichia coli activated the NF-κB pathways and resulted in an increased ratio of females from 30 to 85%. Microarray and quantitative real-time-PCR analysis of gonads showed elevated expression of NF-κB-regulated genes. To confirm the involvement of NF-κB-induced anti-apoptotic effects, zebrafish were treated with sodium deoxycholate, a known inducer of NF-κB or NF-κB activation inhibitor (NAI). Sodium deoxycholate treatment mimicked the effect of heat-killed bacteria and resulted in an increased proportion of females from 25 to 45%, whereas the inhibition of NF-κB using NAI resulted in a decrease in females from 45 to 20%. This study provides proof for an essential role of NF-κB in gonadal differentiation of zebrafish and represents an important step toward the complete understanding of the complicated process of sex differentiation in this species and possibly other cyprinid teleosts as well.

Effect of decellularization protocol on the mechanical behavior of porcine descending aorta.

Enzymatic-detergent decellularization treatments may use a combination of chemical reagents to reduce vascular tissue to sterilized scaffolds, which may be seeded with endothelial cells and implanted with a low risk of rejection. However, these chemicals may alter the mechanical properties of the native tissue and contribute to graft compliance mismatch. Uniaxial tensile data obtained from native and decellularized longitudinal aortic tissue samples was analyzed in terms of engineering stress and fit to a modified form of the Yeoh rubber model. One decellularization protocol used SDS, while the other two used TritonX-100, RNase-A, and DNase-I in combination with EDTA or sodium-deoxycholate. Statistical significance of Yeoh model parameters was determined by paired t-test analysis. The TritonX-100/EDTA and 0.075% SDS treatments resulted in relatively variable mechanical changes and did not effectively lyse VSMCs in aortic tissue. The TritonX-100/sodium-deoxycholate treatment effectively lysed VSMCs and was characterized by less variability in mechanical behavior. The data suggests a TritonX-100/sodium-deoxycholate treatment is a more effective option than TritonX-100/EDTA and SDS treatments for the preparation of aortic xenografts and allografts because it effectively lyses VSMCs and is the least likely treatment, among those considered, to promote a decrease in mechanical compliance.

Development of a monoclonal antibody-based co-agglutination test to detect enterotoxigenic Escherichia coli isolated from diarrheic neonatal calves

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsa-stained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a co-agglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.

Presence and Elimination of the Xenoantigen Gal (α1, 3) Gal in Tissue-Engineered Heart Valves

Tissue engineering of heart valves promises to create functional autologous tissue with the potential for regeneration and growth without the limitations of current heart valve prostheses. The appropriate valve matrix is essential. Porcine heart valves are attractive because of their anatomical similarity. Decellularization is used for antigen reduction. The efficacy of published protocols varies, however. The absence of a specific immunological or unspecific inflammatory reaction is mandatory. The porcine cell-specific alpha-Gal epitope is known to be responsible for hyperacute rejection in xenotransplantation. In tissue engineering residual alpha-Gal epitope may induce severe inflammation in humans and may lead to graft failure. In this study porcine pulmonary conduits were decellularized with Triton X-100, sodium deoxycholate, Igepal CA-630, and ribonuclease treatment and were compared with specimens of the commercially available porcine decellularized SynerGraft regarding cell removal and elimination of the alpha-Gal epitope. In addition, samples of a porcine bioprosthesis were examined for the presence of the alpha-Gal epitope. In conclusion, we describe for the first time the presence of the alpha-Gal epitope in clinically used porcine bioprostheses and the first generation of a commercial tissue-engineered heart valve. In contrast, complete cell and alpha-Gal removal was achieved by a decellularization procedure developed by our group.

The involvement of smooth muscle P2X receptors in the prolonged vasorelaxation response to purine nucleotides in the rat mesenteric arterial bed.

1. ATP and adenine dinucleotides can elicit three different types of vasomotor response in the rat mesenteric arterial bed; vasocontraction, rapid relaxation (which may be masked by contraction) and slow and prolonged vasorelaxation. Contraction is mediated by smooth muscle P2X receptors and rapid relaxation by endothelial P2Y receptors. The mechanism of prolonged relaxation is, however, controversial. 2. In the present study, bolus injection of doses of alpha,beta-methylene ATP (alpha,beta-meATP; 5 pmol - 0.5 micromol; P2X receptor agonist) in methoxamine-preconstricted rat isolated mesenteric arterial beds, mimicked the action of ATP, causing contraction (R(max) 76+/-9 mmHg) followed by prolonged relaxation (78+/-11%; t(1/2) 14.6+/-1.5 min). KCl also elicited a biphasic response (R(max) contraction 73+/-8 mmHg; R(max) prolonged relaxation 70+/-6%; t(1/2) 7.7+/-1.9 min). 3. P2X receptor desensitization caused by perfusion with alpha,beta-meATP (10 microM) abolished contraction and prolonged relaxation to doses of alpha,beta-meATP (50 nmol). Rapid relaxation (32+/-7%; t(1/2) 32+/-2 s) was revealed, which was abolished by removal of the endothelium using distilled water. 4. Sodium deoxycholate treatment blocked contractile and prolonged relaxation responses to alpha,beta-meATP, ATP and KCl, whilst distilled water treatment had no significant effect on either phase of the biphasic responses. 5. These data indicate that smooth muscle P2X receptors are involved in both phases of the biphasic response (contraction followed by prolonged relaxation) to purine nucleotides in the rat isolated mesenteric arterial bed. Caution should be applied when using sodium deoxycholate to remove the endothelium because of possible damage caused by the detergent to receptors and/or the vascular smooth muscle.

Biocatalytic production of useful esters by two forms of lipase from Candida rugosa

Two distinct lipase forms were obtained from Candida rugosa lipase by Phenyl Sepharose hydrophobic interaction and DEAE Sepharose ion exchange chromatography, L1 at 45% yield and L2 at 4.7% yield. Both purified lipases were able to catalyse esterification of 1-butanol and oleic acid and trans-esterification of 2-ethyl-1-hexanol and rapeseed oil. Lipase L1 gave a 98% yield for esterification over 12 h and a 99% conversion of rapeseed oil for trans-esterification over 24 h. The minor fraction L2 gave a 97% yield for esterification over 30 h and only a 79% conversion for trans-esterification over 24 h. The superiority of fraction L1, especially in trans-esterification, could be clearly shown by reversed phased HPLC analysis. Sodium deoxycholate treatment of the purified main lipase L1 considerably improved the initial rate in both esterification and trans-esterification.

A line of rat ovarian surface epithelium provides a continuous source of complex extracellular matrix

A spontaneously immortalized, yet non-tumorigenic rat ovarian surface epithelial (ROSE 199) cell line, deposits large amounts of extracellular matrix (ECM) in response to crowding. The characteristics and components of ROSE 199-derived cell-free ECM were compared after three different preparative techniques: treatment with 20 mM ammonium hydroxide, with 1% sodium deoxycholate, or by repeated freeze-thaws. The ECMs were analyzed by histochemistry, immunofluorescence, electron microscopy, and Western immunoblotting. Components of ROSE 199 ECM included laminin, fibronectin, and collagen types I and III. Even though ROSE 199 is an epithelial cell line, striated collagen fibers formed a major part of its matrix. Thus, ROSE 199 matrix consists of both basement membrane and stromal matrix components. This matrix supported the adhesion, spreading, and growth of several cell types without altering their morphology or growth pattern, and enhanced the attachment of some cell types that spread on plastic only with difficulty. Immunofluorescence, electron microscopy, and dry weight determinations indicated that a greater proportion of matrix was retained in preparations obtained by ammonium hydroxide or freeze thaw techniques than after sodium deoxycholate treatment. Ammonium hydroxide and freeze-thaw treated matrices were also superior to sodium deoxycholate preparations as evidenced by enhanced initial cellular adhesion and spreading compared to cells plated on plastic. Residual nuclear material did not seem to affect the biological activity of this matrix. ROSE 199 extracellular matrix provides a novel, complex substratum for cell culture and for studies of matrix functions and synthesis.

Trypanosoma cruzi infection suppresses nuclear factors that bind to specific sites on the interleukin-2 enhancer.

Interleukin-2 (IL-2) gene expression, a critical early event during T lymphocyte activation, is severely suppressed in mice infected with the protozoan parasite Trypanosoma cruzi, the causative agent of human Chagas' disease. Our previous observation that reduction of IL-2 mRNA in T cells from T. cruzi-infected mice is not due to an increased degradation of the mRNA suggests a repression of the IL-2 gene at the transcriptional level. In this study, we have measured the level of nuclear factors that bind to specific sites on the IL-2 enhancer. Splenocytes and splenic T cells from acutely infected mice show a marked decrease in the level of AP-1, and a modest decrease in the level of NF-kappa B and nuclear factor of activated T cells (NF-AT). DNA-binding activity of Oct-1 was least affected in T cells from infected mice. Although the basal level of AP-1 activity is comparable in cells from uninfected and infected mice, mitogen-induced AP-1 activation is absent in the cells from T. cruzi-infected mice. Sodium deoxycholate treatment slightly enhances NF-kappa B-binding activity in splenocyte nuclear and whole-cell extracts from infected mice, suggesting that a blockage of the activation of NF-kappa B is only partially responsible for the decrease in the level of NF-kappa B in T cells from T. cruzi-infected mice. These data identify the molecular basis of IL-2 gene regulation in T. cruzi infection and suggest that T cells are anergized as a result of the infection.

Endothelium inactivation in in vitro perfused vascular beds comparison of methods

In order to choose the best procedure to inactive the endothelium from vascular beds perfused in vitro, we compared four methods: perfusion with sodium deoxycholate 0.3% for 30 sec; 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate 0.3% (CHAPS) for 2.5 min; collagenase 0.2% for 15 min, and distilled water for 10 min, using the mesenteric arterial bed (MAB) of the rat. The effectiveness of the treatments used to inactivate the endothelium was assessed functionally by using acetylcholine and sodium nitroprusside and histologically using light microscopy. Phenylephrine was used to test the contractile properties of the preparations after each treatment. After collagenase, distilled water, and CHAPS treatment, a potentiated response to phenylephrine was observed, whereas sodium deoxycholate treatment did not modify phenylephrine-induced responses. Acetylcholine-induced responses were reduced by collagenase (60% reduction), CHAPS (30% reduction), and distilled water (52% reduction) treatment, and sodium deoxycholate completely abolished acetylcholine-induced responses. Except after collagenase treatment, smooth muscle relaxant responses were not altered. Medial smooth muscle cells displayed an unchanged morphology, appearing similar to those in control mesenteric arterial beds, except for collagenase and distilled water. Despite the fact that sodium deoxycholate treatment completely abolished acetylcholine-induced response, endothelial cells were still found. No treatment totally removed endothelial cells. In conclusion, we suggest that sodium deoxycholate treatment is the best procedure to inactivate endothelial cells from vascular beds perfused in vitro since it completely abolished endothelium-dependent relaxation and did not interfere with smooth muscle vasodilating and contracting properties.

Purification of chlamydia psittaci antigen by affinity chromatography on polymyxin B agarose for use in the enzyme-linked immunosorbent assay (ELISA)

The purification of cell wall antigens of Chlamydia psittaci by affinity chromatography on polymyxin B agarose is described. Chlamydial cell wall antigens were prepared using different methods: heat treatment, ultrasonication and sodium deoxycholate treatment. The antigens were subsequently purified by gel chromatography. The highest amount of cell wall antigens was obtained by heat treatment of the chlamydiae at 90 degrees C and pH 8.5. The purified antigens showed molecular weights of 450 kDa to 700 kDa. Treatment of heat-extracted antigens with trypsin, pronase E or proteinase K did not destroy antigenic activity or alter the molecular weight. On the other hand, potassium metaperiodate treatment led to a significant decrease of both. Chlamydial cell wall antigens extracted by heat treatment and purified by affinity chromatography were used as ELISA-antigen. Using this ELISA and the complement fixation test (CFT), a total of 576 bovine sera was tested. 92 of these sera reacted positively in the ELISA and only 13 in the CFT. In addition, the prepared ELISA was compared with a commercial ELISA. 12 out of 15 sera tested were positive when using the ELISA described and 10 were positive when using the commercial ELISA.

Antigenic and immunogenic activity of flagella and fimbriae preparations from uropathogenic Proteus mirabilis

The antigenic and immunogenic activities of fimbriae and flagella from three uropathogenic strains of Proteus mirabilis were compared. Flagella were obtained by mechanical treatment and fimbriae were isolated from cells by heat shock, ammonium sulfate precipitation, sodium deoxycholate and urea treatment, and gel filtration. Both preparations inoculated to mice demonstrated high antigenicity. Titers up to 1:80,000 were determined by an enzyme-linked immunosorbent assay either against the homologous or heterologous strains. When immunized mice were challenged with homologous or heterologous hematogenous infecting doses, a good cross protection was achieved only when fimbriae were used as antigens. Cross-reactivity found between the three fimbriae antisera, and the presence of common proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of fimbriae, should validate the study of these proteins to determine the existence of a shared adhesin.

A p-nitrophenyl phosphorylcholine phosphodiesterase from mouse brain

The phosphodiesterase which catalyzes the hydrolysis of p-nitrophenyl phosphorylcholine was solubilized from mouse brain by 1% sodium deoxycholate treatment, and partially purified by HPLC gel chromatography. The enzyme, tightly bound to membranes and relatively stable, possessed apparent values of Km of 1 mM and Vmax of 150 n moles/mg. hr, and gave optimum pH of 11. Additionally, the molecular weight of the enzyme, EDTA-insensitive and divalent ions-independent, was estimated to be 150,000. Based on these results, this phosphodiesterase is supposed to be different from the phosphodiesterases reported previously.

Phospholipid requirement of alkylglycerol monooxygenase from rat liver microsomes.

The alkylglycerol monooxygenase catalyzing the cleavage of the ether bond in alkylglycerol resides in rat liver microsomes. The enzyme preparation was freed of phospholipids by sodium deoxycholate treatment followed by gel filtration in the presence of deoxycholate. The removal of phospholipids markedly decreased the alkylglycerol monooxygenase activity. The activity of the delipidated enzyme, however, could be completely restored by the addition of phospholipid vesicles without detergent. When individual phospholipids were added, anionic phospholipids such as phosphatidylglycerol and diphosphatidylglycerol were the most effective. These findings, along with our previous observation of a similar effect of liposomes on the purified enzyme, indicate that the amphipathic nature of the protein is responsible for the lipid dependence of enzymatic activity.