Times Higher Response Research Articles

A novel reporter gene assay for interferons based on CHO-K1 cells

Interferons (IFNs) are cytokines playing an important role in the immune response and defence against viruses. They are widely used as biopharmaceuticals. Currently, the anti-viral assay (AVA) is the most commonly used bioassay for determining interferon potency. In the search for rapid and robust but reliable methods, reporter gene assays (RGA) appear to be the most promising approach, therefore we have designed a new reporter cell line, CHO-ISRE-SEAP, suitable for determination of type I interferon potency. Chinese hamster ovary (CHO-K1) cells were stably transfected with secretory alkaline phosphatase (SEAP) gene under the control of interferon stimulated response element (ISRE) promoter. The amount of SEAP in the cell culture medium can be easily measured colorimetrically and has been found to correlate with the amount of IFN added. The new assay is widely applicable for determination of type I IFNs, such as IFN-α, IFN-β and IFN-ω, in research, development of IFN biopharmaceuticals, in batch release, etc. Interestingly, in this assay, IFN-beta shows approximately 6 times higher response than IFN-alpha, which makes it especially appropriate for measuring low levels of IFN-beta. Compared to other known RGAs, the novel CHO-ISRE-SEAP cell line-based RGA appears to have certain advantages with respect to cost and performance.

Maternal responsiveness of sows towards piglet's screams during the first 24 h postpartum

In this study we focused on sow responsiveness towards piglet screams which are proposed to have an important link to posture changes and early piglet crushing. A sow's responsiveness to screams of her piglets was investigated along the first 24 h after birth, the period of highest mortality, in 15 lactating sows housed in farrowing crates. We compared the sow's response to playbacks of screams of trapped piglets to her response to a control sound during birth, 8–12 h postpartum and 20–24 h postpartum. We did the same with playbacks of screams of fighting piglets during nursings 8–12 h postpartum and 20–24 h postpartum. The sow’ responsiveness to screams of own trapped piglets was analyzed within the whole 24 h and to screams of fighting piglets 30 min between 8 and 12 h postpartum and 30 min between 20 and 24 h postpartum. A sow was scored as responsive if she changed her posture in response to the stimuli. Sows had a four times higher response towards playbacks with screams than towards the control stimulus. The proportion of the sows’ responsiveness to screams (44%) of trapped piglets did not change significantly between birth, 8–12 h postpartum and 20–24 h postpartum. Sows responded to 28% of playbacks of fighting piglets by terminating a nursing independently from the time after birth. Playbacks and real screams of own piglets were similarly effective in evoking a response for both types of screams. There was no significant association between the sows’ responsiveness to screams of trapped piglets and piglet mortality nor between screams of fighting piglets and weight gain. In conclusion our results indicate that sows maintained their responsiveness towards piglet screams during trapping and during fighting for teats within the first 24 h. Playbacks and real screams were similarly effective in evoking a response. We discuss the importance of the sound characteristics of piglet screams related to the sows’ response.

Partial-electroded ZnO pyroelectric sensors for responsivity improvement

The design, MEMS fabrication and characterization of the ZnO pyroelectric sensor for improvement of responsivity are presented. Three groups of the sensor with various layer thickness, effective area, partially and fully covered electrode (FCE) were fabricated and investigated. The partially covered electrode (PCE) covers 50–65% area of the exposed surface for incident energy absorption. The un-covered ZnO will directly receive the incident laser energy and improve the absorption efficiency of the sensor. The measured data show that the partial-electroded sensors exhibits at least 4 times higher responsivity in the frequency range of 10–1000 Hz than the full-electroded sensors. The effects of other design parameters such as thickness of ZnO, SiO 2 and Si 3N 4 on the responsivity are also discussed.

Differences in signal transduction of two 5-HT4 receptor splice variants: compound specificity and dual coupling with Galphas- and Galphai/o-proteins.

This study documents differences in ligand binding and signal transduction properties between the human (h) 5-hydroxytryptamine (5-HT)4a and h5-HT4b receptor splice variants stably expressed in human embryonic kidney 293 cells. The fraction of the [3H]5-HT high-affinity site relative to the whole receptor population measured with [3H]GR113808 was higher for the h5-HT4a isoform (around 0.4) than for the 5-HT4b isoform (around 0.2) and was independent of the level of expression. The potency and efficacy of reference compounds tested for the cAMP response differed slightly but significantly between both variants. Most remarkably, 5-methoxytryptamine and prucalopride were found more potent on the 5-HT4b variant, whereas SDZ-HTF 919 and SB204070 were more potent on the 5-HT(4a) variant. Guanosine-5'-O-(3-[35S]thio)triphosphate binding on membranes and cAMP assays in whole cells revealed that only the h5-HT4b isoform coupled to Galphai/o-proteins in addition to its well-documented Galphas coupling. In contrast, the h5-HT4a receptor coupled only to Galphas-proteins, however, was able to trigger an increase in the intracellular calcium concentration ([Ca(2+)]i). The observed [Ca(2+)]i increase did not occur through inositol phosphate formation and was not sensitive to Bordetella pertussis toxin, forskolin, or 3-isobutyl-1-methylxanthine (pre)treatment but was due to Ca(2+) influx from the extracellular environment. Interestingly, the Ca(2+) pathway was dependent on high receptor expression levels and was compound-specific, because benzamide-like compounds triggered two to three times higher responses than indoleamines. Taken together, these data provide the first evidence for fine functional differences between C-terminal splice variants of the h5-HT4 receptor, which may contribute to a better understanding of the functional diversity of this receptor class.

Alterations of insulin response to different β cell secretagogues and pancreatic vascular resistance induced by Nω‐nitro‐L‐arginine methyl ester

1. We studied a possible interplay of pancreatic NO synthase activity on insulin secretion induced by different beta cell secretagogues and also on pancreatic vascular bed resistance. 2. This study was performed in the isolated perfused pancreas of the rat. Blockage of NO synthase was achieved with Nw-nitro-L-arginine methyl ester (L-NAME); The specificity of the antagonist was checked by using its D-enantiomer as well as by substitutive treatments with sodium nitroprusside (SNP) as a NO donor in studies of glucose-induced insulin secretion. 3. Arginine (5 mM) induced a monophasic response which was, in the presence of L-NAME at equimolar concentration, very strongly potentiated and converted into a 13 times higher biphasic one. D-NAME (5 mM) was only able to induce a 3 times higher response, but provoked a similar vasoconstrictor effect. 4. The small biphasic insulin secretion induced by L-leucine (5 mM) was also strongly enhanced, by 8 times, in the presence of L-NAME (5 mM) vs 2 times in the presence of D-NAME (5 mM). 5. beta cell responses to KCl (5 mM) and tolbutamide (0.185 mM) were only slight increased by L-NAME (5 mM) to values not far from the sum of the effects of L-NAME and of the two drugs alone. D-NAME (5 mM) was totally ineffective on the actions of both secretagogues. 6. L-NAME, infused 15 min before and during a rise in glucose concentration from 5 to 11 mM, was able in the low millimolar range (0.1-0.5 mM) to blunt the classical biphasic pattern of beta cell response to glucose and, at 5 mM, to convert it into a significantly greater monophasic one. In contrast, D-NAME (5 mM) was unable to induce similar effects. 7. SNP alone at 3 microM was ineffective but at 30 microM substantially reduced to second phase of insulin response to glucose; however, at both concentrations the NO donor partly reversed alterations in insulin secretion caused by L-NAME (5 mM) and restored a biphasic response.

Genetic control of the murine IgM plaque-forming cell response to type III pneumococcal polysaccharide.

The direct 5-day plaque-forming cell response of different inbred mouse strains to pneumococcal polysaccharide type III (SSS-III) varied from more than 10,000 per spleen, in BALB/c mice, to less than 2000 in C57BL mice. Responses of Igh congenic and recombinant inbred lines bearing different combinations of BALB/c and C57BL genes indicate that two or more gene loci are involved in controlling high or low responses. At least one is in the Igh-V region since BALB/c, BAB-14, and CB-8 KN mice (Igh-Va) had two to four times higher responses than CB-20 and CB-16 KN mice (Igh-Vb). Other gene loci must be involved, but nothing can be said about them at present.

Immunogenicity of N-(2-hydroxypropyl)-methacrylamide copolymers--potential hapten or drug carriers.

After repeated i.p. immunizations of mice with 10 micrograms of homopolymer poly (HPMA) no antibodies were detected by the ELISA test. Immunization with copolymer P-Acap-Leu-HMDA leads to a weak antibody response, while immunization with a copolymer with some side chains modified with ARS or FITC groups (P-Acap-Leu-HMDA-ARS or P-Acap-Leu-HMDA-FITC) leads to a significant antibody response detectable by PFC, ELISA and haemagglutination tests. Most of these antibodies are aimed against the modifying haptenic group, a smaller amount against side oligopeptide sequences of the carrier. Intensity of the antibody response depends on: 1) the antigen dose--the optimal dose was 10 micrograms: both the higher (100 micrograms) and the lower doses (1 and 0.1 micrograms) induced considerably lower antibody responses; 2) molar mass of the immunizing fractions--fractions of high molar mass induced up to five times higher responses than those of a low molar mass; 3) the bound haptenic group--the ARS-copolymers induced ten times lower response than the FITC-copolymers. We detected no difference between capacities of the H-2a, H-2b and H-2d haplotypes to react with anti-ARS antibodies after immunization with P-Acap-Leu-HMDA-ARS.