Critical Evaluation of Membrane Supports for Use in Quantitative Hybridizations

The quantification of 16S rRNA by oligonucleotide probe hybridization was investigated with MagnaGraph (Micron Separation, Inc. [MSI]), Magna Charge (MSI), Magna (MSI), Immobilon-N (Millipore Corporation), and Nytran (Schleicher & Schuell, Inc.) membranes as supports for nucleic acid immobilization. The levels of detectability provided by the Magna Charge and Immobilon-N membranes were 20 to 50 times better than those obtained with the MagnaGraph, Magna, and Nytran membranes. The variability of the signal response for individual membranes ranged from 10 to 50%, with the Magna Charge and Immobilon-N membranes demonstrating the lowest variability.     

Effect of chemical chaperone addition on production and aggregation of recombinant flag-tagged COMP-angiopoietin 1 in Chinese hamster ovary cells

To investigate the effect of chemical chaperones on the production and aggregation of flag-tagged cartilage oligomeric matrix protein-Angiopoietin1 (FCA1) in recombinant Chinese hamster ovary (CHO) cells, CHO cells were cultivated in serum-free media with various chemical chaperones, 1 mM 4-phenylbutyrate (4-PBA), 200 mM proline, 3% glycerol, 2% dimethyl sulfoxide (DMSO), and without chemical chaperone as control. The addition of chemical chaperones enhanced FCA1 production and specific FCA1 productivity, q(FCA)(1). For example, the q(FCA)(1) at 200 mM proline was fourfold higher than that at control. Unlike q(FCA)(1), the aggregation of FCA1 was strongly affected by which chemical chaperone was added. The addition of 2% DMSO and 200 mM proline significantly reduced the proportion of aggregates, but the addition of 1 mM 4-PBA and 3% glycerol was hardly effective. The proportions of aggregates were 29.5 and 55.6% at 2% DMSO and 200 mM proline, respectively, whereas it was 79.6% at control. The exact mechanism how chemical chaperones affected the aggregation of FCA1 was not investigated in this study, and therefore, more extensive works will be needed to clarify why different chemical chaperones behaved differently in reducing the aggregation of FCA1. Among chemical chaperones tested, DMSO was the most effective one in regard to enhancing the production and reducing the aggregation of FCA1 in CHO cells.     

A novel low-temperature-active β-glucosidase from symbiotic <Emphasis Type="Italic">Serratia</Emphasis> sp. TN49 reveals four essential positions for substrate accommodation

To maximize the production of flag-tagged cartilage oligomeric matrix protein angiopoietin-1 (FCA1) from Chinese hamster ovary (CHO) cells, the effects of culture pH and temperature on cell growth and FCA1 production were investigated. Cells were cultivated in a bioreactor at different culture pH (6.7, 6.9, 7.2, and 7.5) and temperatures (33 and 37 °C). Lowering the culture temperature suppressed cell growth while allowing maintenance of high cell viability for a longer culture period. The specific FCA1 productivity (q _FCA1) was increased at low culture temperature. Accordingly, the highest FCA1 concentration was obtained at pH 7.2 and 33 °C, and was approximately 4.0-fold higher than that at pH 7.2 and 37 °C. However, aggregates and a monomeric form of FCA1, which are undesirable due to reduced biological activity or immunogenicity, were significant at pH 7.2 and 33 °C. It was also found that the expression pattern of FCA1 was affected more significantly by culture pH than by the culture temperature. FCA1 aggregation dramatically decreased at culture pH 7.5 regardless of the culture temperature. Furthermore, the monomeric form of FCA1 was not observed. Taken together, optimization of culture temperature and culture pH (33 °C and pH 7.5) significantly improves the production of biologically active FCA1 with tetrameric or pentameric forms from CHO cells.     

A collaborative study to establish an International Standard Rabies immunoglobulin of human origin

Because the supply of the International Standard for Anti-rabies Serum was very low, the WHO initiated a search for a replacement product. The US Food and Drug Administration agreed to undertake a collaborative study using a human rabies immunoglobulin previously purchased for use as a US standard. The potency of this product was determined, in International Units (IU) per millilitre using the rapid fluorescent focus inhibition test for measuring rabies antibody. The mean potency value was found to be 59 IU per ampoule. In June 1984 this preparation was accepted by WHO as the International Standard for Rabies Immunoglobulin.     

Aerobic biotransformation of 4-fluorocinnamic acid to 4-fluorobenzoic acid

The biotransformation of 4-fluorocinnamic acid (FCA) using non-acclimated industrial activated sludge was investigated. FCA is a common intermediate in organic synthesis, and it is often present in aqueous waste streams. Hence, the biotransformation reactions this compound undergoes when exposed to activated sludge micro-organisms should be understood before waste streams are sent to biological wastewater treatment plants (WWTPs). FCA biotransformation was monitored using a wide range of analytical techniques. These techniques were used to monitor not only FCA disappearance, but also the formation of degradation products, in order to propose the metabolic pathway. FCA was biotransformed to 4-fluorobenzoic acid via the formation of 4-fluoroacetophenone. The removal of FCA up to 200 mg L^-1 followed first order kinetics. The half-lives for removal of FCA from the test solutions supplied with 200 mg L^-1, 100 mg L^-1, and 50 mg L^-1 were 53, 18, and 5 hours respectively.     

Characterization of serum and mucosal antibody responses in white sturgeon (Acipenser transmontanus Richardson) following immunization with WSIV and a protein hapten antigen

Serum and cutaneous mucus antibodies were monitored in white sturgeon for 15 weeks following intraperitoneal immunization. Ten fish were immunized (50 microg) with white sturgeon iridovirus (WSIV) or white sturgeon gonad (WSGO) tissue culture cells emulsified with or without FCA. An additional group was immunized with FITC:KLH+FCA. Fish were booster immunized at 6 weeks. Fish immunized with FITC:KLH+FCA produced significant serum antibodies to FITC by 6 weeks and this response peaked at 12 weeks (average titer 31,000). Mucosal antibodies to FITC were first detected at 12 weeks and significantly elevated by 15 weeks (average titer 18). Anti-WSIV antibody titers were detected in the serum by 9 weeks in fish immunized with WSIV and WSIV+FCA, but only a small number responded to immunization. At 15 weeks, four fish immunized with WSIV produced serum antibodies (average titer 838) and one fish immunized with WSIV+FCA had a serum titer of 1600. Mucosal anti-WSIV antibody titers of 8 and 16 were observed in two fish from the WSIV group at 12 weeks while four different fish from this group responded at 15 weeks (average titer 4). Western Blot using a monoclonal antibody confirmed immunoglobulin in mucus, and specificity to WSIV was further demonstrated by immunocytochemistry using serum from fish immunized with WSIV. Specific antibody was not detected in mucus of fish immunized with WSIV+FCA, WSGO, or WSGO+FCA. Collectively, these experiments demonstrate that white sturgeon can generate a specific antibody response following immunization, and is the first report showing mucosal immunoglobulin is present in this species.     

Comparison of fecal coliform agar and violet red bile lactose agar for fecal coliform enumeration in foods

A 24-h direct plating method for fecal coliform enumeration with a resuscitation step (preincubation for 2 h at 37 +/- 1 degrees C and transfer to 44 +/- 1 degrees C for 22 h) using fecal coliform agar (FCA) was compared with the 24-h standardized violet red bile lactose agar (VRBL) method. FCA and VRBL have equivalent specificities and sensitivities, except for lactose-positive non-fecal coliforms such as Hafnia alvei, which could form typical colonies on FCA and VRBL. Recovery of cold-stressed Escherichia coli in mashed potatoes on FCA was about 1 log unit lower than that with VRBL. When the FCA method was compared with standard VRBL for enumeration of fecal coliforms, based on counting carried out on 170 different food samples, results were not significantly different (P > 0.05). Based on 203 typical identified colonies selected as found on VRBL and FCA, the latter medium appears to allow the enumeration of more true fecal coliforms and has higher performance in certain ways (specificity, sensitivity, and negative and positive predictive values) than VRBL. Most colonies clearly identified on both media were E. coli and H. alvei, a non-fecal coliform. Therefore, the replacement of fecal coliform enumeration by E. coli enumeration to estimate food sanitary quality should be recommended.     

Cumulant analysis in fluorescence fluctuation spectroscopy

A novel technique for the analysis of fluorescence fluctuation experiments is introduced. Fluorescence cumulant analysis (FCA) exploits the factorial cumulants of the photon counts and resolves heterogeneous samples based on differences in brightness. A simple analytical model connects the cumulants of the photon counts with the brightness epsilon and the number of molecules N in the optical observation volume for each fluorescent species. To provide the tools for a rigorous error analysis of FCA, expressions for the variance of factorial cumulants are developed and tested. We compare theory with experiment by analyzing dye mixtures and simple fluorophore solutions with FCA. A comparison of FCA with photon-counting histogram (PCH) analysis, a related technique, shows that both methods give identical results within experimental uncertainty. Both FCA and PCH are restricted to data sampling times that are short compared to the diffusion time of molecules through the observation volume of the instrument. But FCA theory, in contrast to PCH, can be extended to treat arbitrary sampling times. Here, we derive analytical expressions for the second factorial cumulant as a function of the sampling time and demonstrate that the theory successfully models fluorescence fluctuation data.     

Characterization of new polyclonal antibodies specific for 40 and 42 amino acid-long amyloid beta peptides: their use to examine the cell biology of presenilins and the immunohistochemistry of sporadic Alzheimer's disease and cerebral amyloid angiopathy cases.

BACKGROUND: In Alzheimer's disease (AD), the main histological lesion is a proteinaceous deposit, the senile plaque, which is mainly composed of a peptide called A beta. The aggregation process is thought to occur through enhanced concentration of A beta 40 or increased production of the more readily aggregating 42 amino acid-long A beta 42 species. MATERIALS AND METHODS: Specificity of the antibodies was assessed by dot blot, Western blot, ELISA, and immunoprecipitation procedures on synthetic and endogenous A beta produced by secreted HK293 cells. A beta and p3 production by wild-type and mutated presenilin 1-expressing cells transiently transfected with beta APP751 was monitored after metabolic labeling and immunoprecipitation procedures. Immunohistochemical analysis was performed on brains of sporadic and typical cerebrovascular amyloid angiopathy (CAA) cases. RESULTS: Dot and Western blot analyses indicate that IgG-purified fractions of antisera recognize native and denaturated A beta s. FCA3340 and FCA 3542 display full specificity for A beta 40 and A beta 42, respectively. Antibodies immunoprecipitate their respective synthetic A beta species but also A beta s and their related p3 counterparts endogenously secreted by transfected human kidney 293 cells. This allowed us to show that mutations on presenilin 1 triggered similar increased ratios of A beta 42 and its p 342 counterpart over total A beta and p3. ELISA assays allow detection of about 25-50 pg/ml of A beta s and remain linear up to 750 to 1500 pg/ml without any cross-reactivity. FCA18 and FCA3542 label diffuse and mature plaques of a sporadic AD case whereas FCA3340 only reveals the mature lesions and particularly labels their central dense core. In a CAA case, FCA18 and FCA3340 reveal leptomeningeal and cortical arterioles whereas FCA3542 only faintly labels such structures. CONCLUSIONS: Polyclonal antibodies exclusively recognizing A beta 40 (FCA 3340) or A beta 42 (FCA3542) were obtained. These demonstrated that FAD-linked presenilins similarly affect both p342 and A beta 42, suggesting that these mutations misroute the beta APP to a compartment where gamma-secretase, but not alpha-secretase, cleavages are modified. Overall, these antibodies should prove useful for fundamental and diagnostic approaches, as suggested by their usefulness for biochemical, cell biological, and immunohistochemical techniques.     

Protection against toxoplasmosis in mice immunized with different antigens of Toxoplasma gondii incorporated into liposomes.

Different toxoplasma antigens were entrapped within liposomes and evaluated, in this form, for their ability to protect Swiss mice against toxoplasma infection: soluble tachyzoite antigen (L/TAg), tissue cyst (L/CAg), tachyzoite plus tissue cyst (L/TCAg) or purified antigen of tachyzoite (L/pTAg). The protein used in L/pTAg was purified from tachyzoites using a stage-specific monoclonal antibody which reacted at a molecular weight of 32 kD in SDS PAGE and silver stain using reduced condition. To compare the immuno-adjuvant action of liposomes and of Freund's Complete Adjuvant (FCA), another group of mice was immunized with soluble tachyzoite antigen (STAg) emulsified in FCA (FCA/TAg). Control groups were inoculated with (STAg) alone, phosphate-buffered saline (PBS), FCA with PBS (FCA/PBS) and empty liposomes (L/PBS). Mice were inoculated subcutaneously with these antigens six, four and two weeks before a challenge with 80 tissue cysts of the P strain of Toxoplasma gondii orally. All mice immunized with or without adjuvant showed a humoral response, as measured by Elisa. However, no correlation was found between antibody titer and protection against the challenge. All mice immunized with L/pTAg or L/TCAg survived (100), whereas 80% and 90% of mice from groups which received respectively PBS or FCA/PBS and L/PBS died. All mice immunized with antigens entrapped within liposomes (L/TAg, L/CAg, L/TCAg and L/pTAg) showed low numbers of intracerebral cysts.     

Genetic interactions of the Arabidopsis flowering time gene FCA, with genes regulating floral initiation

The genes controlling the timing of the transition from vegetative to reproductive growth are likely candidates for regulators of genes initiating floral development. We have investigated the interaction of one particular gene controlling flowering time, FCA, with the meristem identity-genes TERMINAL FLOWER 1 (TFL1), APETALA 1 (AP1) and LEAFY (LFY) and the floral repression gene EMBRYONIC FLOWER 1 (EMF1). Double mutant combinations were generated and the phenotypes characterized. The influence of strong and intermediate fca mutant alleles on the phenotype conferred by a 35S-LFY transgene was also analysed. The results support a model where FCA function promotes flowering in multiple pathways, one leading to activation of LFY and AP1, and another acting in parallel with LFY and AP1. Only the latter pathway is predicted to be non-functional in the intermediate fca-4 allele. The results are also consistent with AP1 and TFL1 negatively regulating FCA function. Combination of Columbia fca and emf1 mutant alleles confirmed that FCA is required for the early flowering of emf1. EMF1 and FCA are therefore likely to operate in different floral pathways.     

Three-Day Assay for Human Cytomegalovirus Applicable to Serum Neutralization Tests

A fluorescing cell assay (FCA) technique utilizing the indirect fluorescent-antibody method to measure human cytomegalovirus (CMV)-infected cells has been applied to the rapid determination of CMV-neutralizing antibody. Human sera with complement fixation titers to CMV of 1/32 or greater and fluorescein-conjugated rabbit anti-human globulin are the primary and secondary reagents in the fluorescent-antibody test. FCA measured in 3 days the same number of infectious units measured by plaque assay in 2 weeks. FCA and plaque assay yielded identical neutralizing antibody titers to CMV in 20 human sera.     

Humoral and peritoneal cell responses of rainbow trout (Oncorhynchus mykiss) to ovalbumin, Vibrio anguillarum and Freund's complete adjuvant following intraperitoneal and bath immunisation

ELISA methods were used to evaluate the humoral immune responses of rainbow trout (Oncorhynchus mykiss) to ovalbumin and Vibrio anguillarum. Antibody responses to ovalbumin administered intraperitoneally (i.p.) were inconsistent even when Freund's complete adjuvant (FCA) was used and the induction phase (4-6 weeks) of the response was longer compared with the response to V. anguillarum (<4 weeks). Significant elevation in antibody level was noted 3 weeks after bath vaccination with V. anguillarum but levels decreased thereafter. Humoral responses of greatest magnitude occurred where V. anguillarum was given in an emulsion with Freund's complete adjuvant (FCA). However, i.p. administration of FCA alone 3 weeks prior to i.p. immunisation with non-adjuvanted V. anguillarum resulted at 5 weeks in similar elevations in antibody to those in fish given V. anguillarum and FCA concurrently, suggesting that the effects of FCA were not limited to creation of an antigen depot. Proliferation of peritoneal inflammatory cell populations which included macrophages and plasma cells was detected histologically within 3 weeks of administration of FCA, but changes were not detected in the spleen or haematopoietic kidney.     

Experimental allergic encephalomyelitis: changes in growth and proliferation within the draining lymph node following injection of basic encephalitogenic protein in Freund's complete adjuvant

Changes in wet weight, dry mass, and DNA synthesis of draining lymph nodes from rats injected with encephalitogenic basic protein in Freund's complete adjuvant (FCA) were studied. Lymph nodes of rats injected with encephalitogenic basic protein in FCA show accelerated enlargement from the second up to the fourth day after injection, as compared to lymph nodes of rats injected with FCA alone, or with nonencephalitogenic basic protein in FCA. The greatest difference in lymph node weight was found on the fourth day. At this time cell division is higher in the group injected with encephalitogenic protein in FCA than in the group injected with FCA alone. However, the increased division of cells in situ cannot account, in and by itself, for the enlargement of the lymph node which was observed. It is concluded that migration of lymphocytes into the lymph node makes a substantial contribution to the hyperplasia of the lymph node.The results suggest that accelerated lymph node enlargement may be specific, at least in part, for induction of experimental allergic encephalomyelitis.     

Comparison of Fecal Coliform Agar and Violet Red Bile Lactose Agar for Fecal Coliform Enumeration in Foods

A 24-h direct plating method for fecal coliform enumeration with a resuscitation step (preincubation for 2 h at 37 ± 1°C and transfer to 44 ± 1°C for 22 h) using fecal coliform agar (FCA) was compared with the 24-h standardized violet red bile lactose agar (VRBL) method. FCA and VRBL have equivalent specificities and sensitivities, except for lactose-positive non-fecal coliforms such as Hafnia alvei, which could form typical colonies on FCA and VRBL. Recovery of cold-stressed Escherichia coli in mashed potatoes on FCA was about 1 log unit lower than that with VRBL. When the FCA method was compared with standard VRBL for enumeration of fecal coliforms, based on counting carried out on 170 different food samples, results were not significantly different (P > 0.05). Based on 203 typical identified colonies selected as found on VRBL and FCA, the latter medium appears to allow the enumeration of more true fecal coliforms and has higher performance in certain ways (specificity, sensitivity, and negative and positive predictive values) than VRBL. Most colonies clearly identified on both media were E. coli and H. alvei, a non-fecal coliform. Therefore, the replacement of fecal coliform enumeration by E. coli enumeration to estimate food sanitary quality should be recommended.