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PubMedDate not supplied

Vibration in metal and non-metal incubators.

The purpose of this study was to determine the amount of vibration transmitted to the surface of an incubator mattress. Empty incubators with metal (n = 12) and non-metal (n = 12) bases were monitored for vibration levels when the incubators were turned "off" and when they were turned "on." High levels of low-frequency vibration were detected in both types of incubators in both conditions. The metal incubators transmitted significantly less vibration to the mattress than did the non-metal incubators at several frequencies in the "off," the "on," and the "adjusted" conditions. These results suggest that infants experience significant whole-body vibration while lying in incubators.

Acceleration↗

PubMed1988

Genetic control of prion incubation period in mice.

The prion gene complex (Prn) is located on mouse chromosome 2 between the beta-2-microglobulin (B2m) and agouti (A) genes. Within this complex are the prion protein gene (Prn-p), which encodes the only identified macromolecule (PrP) that purifies with infectious scrapie agent, and a scrapie incubation time gene (Prn-i). Using a variety of restriction endonucleases, six allelic forms of the Prn-p gene have been distinguished by their patterns of restriction fragment length polymorphisms. We had previously shown that the exceptionally long scrapie incubation period of I/LnJ mice inoculated with the Chandler isolate (over 200 days) was due to the effects of a scrapie incubation time gene tightly linked to Prn-p. So far, this long scrapie incubation time allele has been found only in those inbred mouse strains (I/LnJ, P/J and IM) that have the b allele of Prn-p. It is not known whether the incubation time gene and prion protein gene are two distinct loci or are one and the same. Putative recombinants between the incubation time phenotype and Prn-p genotype have been observed, but this could be due to effects of other genes segregating in the population. Regardless of whether or not the incubation time and PrP genes are identical, if any differences were found in the amino acid sequences of PrP encoded by the different Prn-p alleles there would be important implications for interpretation of results on 'strains' of scrapie agent. It would not be necessary to invoke nucleic acid as the informational macromolecule of the scrapie agent because differences in prion 'strains' recovered from mice with different Prn-p genotypes need not be the result of host selection but could be due to differences in host-encoded PrP.

Alleles↗

PubMed1994

In vitro incubation of golden (Syrian) hamster ovarian oocytes and human sperm with a human oviduct specific glycoprotein.

The objective of this study was to determine if human oviduct specific glycoprotein (huOGP) would associate with hamster ovarian oocytes and human sperm during in vitro incubation. The huOGP used in these studies was partially purified from human hydrosalpinx fluid. Hamster ovarian oocytes and human sperm samples were incubated in culture medium with and without huOGP. Association of huOGP was assessed by indirect immunofluorescence assay using a polyclonal antibody prepared against huOGP. Intense fluorescence of the zona pellucida, and bright but uneven fluorescence of the perivitelline space, were observed in hamster ovarian oocytes following incubation in the presence of huOGP. A similar but more uniform pattern of fluorescence was observed when hamster oviductal oocytes (positive controls) were incubated in culture medium alone. Fluorescence was absent when oocytes were assayed with preimmune serum. The association of huOGP with the zona pellucida and perivitelline space appeared to be specific since thyroglobulin, a large molecular weight glycoprotein, and human serum albumin, the major protein in oviduct fluid, did not associate with the hamster oocytes nor inhibit huOGP association when included in the culture medium. Fluorescence was absent when human sperm incubated with huOGP were assayed with antiserum to huOGP. However, human sperm fluoresced when incubated with a uterine glycoprotein, CUPED, which had previously been shown to bind to cat sperm during in vitro incubation. Sperm also fluoresced brightly when human sperm antibody was used as a positive control. Solubilization of sperm membrane proteins postincubation and analysis of these proteins by 1-D SDS-PAGE followed by immunoblotting also failed to show an association of huOGP with human sperm. Electron microscopy of sperm both pre- and postsolubilization confirmed that the sperm membranes were removed by this process. In conclusion, the association of huOGP with hamster oocytes in vitro suggests that huOGP may associate with human oocytes in vivo, whereas that may not be true for human sperm in vivo. The association of huOGP with oocytes may serve to facilitate the process of fertilization and early embryonic development within the oviduct.

Animals↗

PubMed1998

The onset of incubation in birds: can females control hatching patterns?

We investigated intraspecific variation in incubation behaviour of wild Eurasian kestrels, Falco tinnunculus, in Finland and tested whether patterns of hatching asynchrony could be predicted from patterns of incubation. The timing of the onset of incubation varied considerably for 17 female kestrels. Eggs generally hatched in the order they were laid, and both total hatching span of the clutch and the pattern of eggs hatching on certain days corresponded well with incubation behaviour. This result was consistent with the idea that females have much control over hatching patterns. In the majority (65%) of cases, the proportion of daily incubation increased monotonically with the laying sequence, a pattern described previously in other birds. Unusual patterns of incubation (35%) were most common in females with poor body condition during incubation and may be the result of energy constraints during laying.Copyright 1998 The Association for the Study of Animal Behaviour. Copyright 1998 The Association for the Study of Animal Behaviour.

Journal Article↗

PubMed1977

Adenosine triphosphate catabolism in homogenates of rat secretory enamel organs incubated in histochemical lead media.

To investigate how lead, when used as trapping agent, influences the ATP hydrolysis and to study how ATP is catalyzed in histochemical systems, homogenized secretory enamel organs were incubated in histochemical [3H]-ATP media. Aliquots from the media were taken after 3, 10, 20 and 30 min, the ATP and formed metabolites were separated by electrophoresis and radiometrically quantitated. In media lacking both lead and homogenate 2% of the ATP was spontaneously hydrolyzed during 30 min incubation at room temperature. The presence of lead caused an additional 8% hydrolysis at pH 7.2 and an additional 20% hydrolysis at pH 9.4. In the presence of homogenate, however, lead caused a net decrease of the hydrolysis of ATP as well as of ADP and AMP. This enzyme inhibition varied from around zero to some 80%, depending on pH and substrated involved. In homogenate-containing lead media, at both pH 7.2 AND 9.4, ATP was rapidly hydrolyzed primarily to ADP and subsequently to AMP and adenosine and/or inosine. After 5--10 min ADP constituted the predominant substrate at both pH:s. At pH 7.2 ADP remained so for the rest of the incubation, whereas at pH 9.4 AMP was predominant substrate at the end of the incubation. AMP was the finan catabolic product in experiments at pH 7.2, and adenosine and/or inosine at pH 9.4. Inorganic phosphate was liberated almost linearly during the whole incubation period. The results indicate that histochemical studies of substrate specific ATP-ases should be performed with short incubation times and, when high specific activities are present, in large quantities of incubation media to reduce interference by ADP and AMP hydrolyzing enzymes.

Adenosine Diphosphate↗

PubMed2000

Induction of apoptosis using 2',2' difluorodeoxycytidine (gemcitabine) in combination with antimetabolites or anthracyclines on malignant lymphatic and myeloid cells. Antagonism or synergism depends on incubation schedule and origin of neoplastic cells.

Induction of apoptosis in vitro using gemcitabine (dFdC) in combination with cladribine (2-CdA) and other cytotoxic drugs on malignant mononuclear cells (MNCs) of patients with acute myeloid leukemia (AML, n=20) and chronic lymphocytic leukemia (CLL, n =20) in myeloid (HL60, HEL) and lymphatic cell lines (HUT78, JURKAT) was investigated using different incubation conditions (simultaneous and consecutive). Furthermore, the influence of dFdC on the level of intracellular metabolites of 2-CdA was studied using high-performance liquid chromatography (HPLC). Apoptosis was evaluated using flow cytometry with 7-aminoactinomycin D. In MNCs of patients with CLL, dFdC + 2-CdA showed an antagonistic effect when applied simultaneously. This antagonism was reduced by consecutive application. The combination of dFdC with doxorubicin was synergistic, independent of incubation schedule. In blasts from newly diagnosed patients with de novo AML, all drug combinations (dFdC+2-CdA, doxorubicin, or cytosine arabinoside) were antagonistic by simultaneous incubation. Reduced antagonism or even synergism was shown (P<0.001) by consecutive incubation. The simultaneous combination of dFdC with 2-CdA in all tested cell lines resulted in a competitive inhibition on the rate of apoptosis. By changing the incubation period to a consecutive schedule, the antagonism was diminished or synergism of apoptosis was measured (P< 0.001). Using similar incubation conditions, these experiments were supported by HPLC measurement of intracellular metabolites of 2-CdA influenced by dFdC application. In conclusion, we demonstrated that the efficacy of dFdC in vitro in combination with other cytotoxic drugs depends on the incubation condition and on the origin of neoplastic cells (lymphatic vs myeloid). The data suggest that simultaneous combination therapy with purine and pyrimidine analogues may not improve the clinical efficacy of one or the other drug administered alone.

Anthracyclines↗

PubMed2000

Changes in the Ca2+-activation characteristics of demembranated rat single muscle fibres after prolonged incubation at room temperature in the presence and absence of DTT and protease inhibitors.

Prolonged incubation (24 h) of chemically skinned rat muscle preparations in rigor solutions at room temperature and in the absence of reducing agents and protease inhibitors modified the Ca2+-activation characteristics of the contractile machinery. In the absence of reducing agents and protease inhibitors, the contraction threshold for incubated fibres was shifted to lower Ca2+ concentrations and the steepness of the steady-state force-pCa (-log10)[Ca2+]) curve was decreased compared to that of control muscle fibres. Mean myosin ATPase activity under these conditions was significantly lowered by a factor of 2.7. Fibres incubated in the presence of 10 mM dithiothreitol (DTT) and protease inhibitors (100 microM pepstatin A/200 microM leupeptin) produced a maximum Ca2+-activated force per cross-sectional area that compared favourably with that of freshly dissected muscle fibres and there were no changes in the other contractile activation characteristics. Intermediate responses were obtained when fibres were incubated in the presence of either DTT or protease inhibitors. MgATPase activities of incubated preparations increased significantly following the addition of protease inhibitors and/or DTT to the incubation medium. Taken together, these results suggest that in the presence of DTT and protease inhibitors, most contractile properties are maintained at levels seen in fresh mechanically skinned fibres. The extended viability of this preparation and its closely related properties with fresh muscle fibres make it a useful model for experiments requiring longer term incubations with biological agents.

Adenosine Triphosphatases↗

PubMed2005

Post-irradiation hypoxic incubation of X-irradiated MOLT-4 cells reduces apoptotic cell death by changing the intracellular redox state and modulating SAPK/JNK pathways.

To elucidate radiobiological effects of hypoxia on X-ray-induced apoptosis, MOLT-4 cells were treated under four set of conditions: (1) both X irradiation and incubation under normoxia, (2) X irradiation under hypoxia and subsequent incubation under normoxia, (3) X irradiation under normoxia and subsequent incubation under hypoxia, and (4) both X irradiation and incubation under hypoxia, and the induction of apoptosis was examined by fluorescence microscopy. About 28-33% apoptosis was observed in cells treated under conditions 1 and 2, but this value was significantly reduced to around 18-20% in cells treated under conditions 3 and 4, suggesting that post-irradiation hypoxic incubation rather than hypoxic irradiation mainly caused the reduction of apoptosis. The activation and expression of apoptosis signal-related molecules SAPK/JNK, Fas and caspase-3 were also suppressed by hypoxic incubation. Effects of hypoxic incubation were canceled when cells were treated under conditions 3 and 4 with an oxygen-mimicking hypoxic cell radiosensitizer, whereas the addition of N-acetyl-L-cysteine again reduced the induction of apoptosis. From these results it was concluded that hypoxia reduced the induction of apoptosis by changing the intracellular redox state, followed by the regulation of apoptotic signals in X-irradiated MOLT-4 cells.

Acetylcysteine↗

PubMed1981

Interaction between macrophages and aortic smooth muscle cells. Enhancement of cholesterol esterification in smooth muscle cells by media of macrophages incubated with acetylated LDL.

Mouse peritoneal macrophages were cultured for 24 h in Dulbecco-Vogt medium containing 10% calf serum. This medium was replaced with Dulbecco-Vogt medium containing 1% bovine serum albumin to which all subsequent additions were made. Medium changes, accompanied by appropriate additions, were made every 48 or 72 h and the media were used for incubation of aortic smooth muscle cells, prelabeled with [3H]cholesterol. The amount of labeled cholesteryl ester in the smooth muscle cells incubated for 48 h with macrophage media which had been collected 48-144 h after addition of acetylated LDL was increased 3-4 times above that present prior to postincubation. A marked increment in cholesteryl ester mass occurred also after incubation of smooth muscle cells with macrophage media conditioned with acetylated LDL and this effect was shared by maleylated LDL, but not by other negatively charged compounds. The increase in labeled cholesteryl ester in smooth muscle cells was more pronounced with media collected at later time intervals of incubation with macrophages and was evident 8 hr after postincubation. Only the d less than 1.063 fraction of the medium enhanced cholesterol esterification in smooth muscle cells. The acetylated LDL reisolated from macrophage media at d less than 1.063 did not compete with native LDL for degradation by smooth muscle cells. No increase in degradation of 125I-labeled acetylated LDL preincubated with macrophages was observed above that of non-preincubated acetylated LDL. The macrophage medium conditioned with acetylated LDL depressed [14C]acetate incorporation into sterols in smooth muscle cells and this effect was abolished by extraction of the medium with diethyl ether. The ratio of free to total cholesterol in the macrophage media collected after incubation with acetylated LDL increased from 28-70%, and a decrease occurred after incubation with smooth muscle cells. The enhancement of cholesterol esterification could be abolished by addition of high density apolipoprotein/sphingomyelin mixture during incubation with macrophages, even though excretion of free cholesterol into the medium increased 3-fold. It is proposed that when smooth muscle cells are presented with a lipoprotein in which an increase in the free to esterified cholesterol ratio occurred, and which is not recognized by a specific receptor, the enhancement of cellular cholesterol esterification is due mostly to a surface transfer of lipoprotein-free cholesterol. The present results offer another view of the possible interactions between macrophages and smooth muscle cells. A modified lipoprotein, not recognized by smooth muscle cells, is ingested by macrophages, which leads to accumulation of esterified cholesterol. Part of the esterified cholesterol undergoes hydrolysis and is excreted back into the medium, leading to enrichment of the lipoproteins in the medium with free cholesterol. This enrichment with free cholesterol promotes cholesterol esterification in smooth muscle cells.

Acylation↗

PubMed1985

Reactivity of human lipoproteins with purified lecithin: cholesterol acyltransferase during incubations in vitro.

Studies have been performed to determine the proportion of the esterified cholesterol in high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) that is attributable to a direct action of lecithin: cholesterol acyltransferase on each lipoprotein fraction. Esterification of [3H]cholesterol was examined in 37 degrees C incubations of either: (a) unseparated whole plasma, (b) plasma reconstituted after prior ultracentrifugation to separate the 1.21 g/ml supernatant, (c) a mixture comprising the 1.21 g/ml supernatant of plasma and purified lecithin: cholesterol acyltransferase or (d) the same mixture as (c) after supplementation with a preparation of partially purified lipid transfer protein. Each of these incubations was performed using samples collected from four different subjects, two of whom had normal and two of whom had elevated concentrations of plasma triacylglycerol. At the completion of 3-h incubations, the lipoproteins were separated into multiple fractions by gel filtration to obtain a continuous profile of esterified [3H]cholesterol across the whole spectrum of lipoproteins. There was an appearance of esterified [3H]cholesterol in each of the major lipoprotein fractions in all incubations. In unseparated plasma, 56% of the total (mean of four experiments) was in HDL, 33% in LDL and 11% in VLDL. A comparable distribution was observed in the incubations of reconstituted plasma and in the samples to which partially purified lipid transfer protein had been added. In the absence of lipid transfer protein activity in incubations containing purified lecithin: cholesterol acyltransferase, 73% of the esterified [3H]cholesterol was in HDL, 25% in LDL and only 1% in VLDL. It has been concluded that at physiological concentrations of lipoproteins, 70-80% of the cholesterol esterifying action of lecithin: cholesterol acyltransferase is confined to the HDL fraction, with most of the remainder involving the LDL fraction. Of the newly formed esterified cholesterol incorporated into LDL during incubations of unseparated plasma, it was apparent that more than 70% was independent of activity of the lipid transfer protein. Of that incorporated into VLDL in unseparated plasma, in contrast, almost 90% was derived as a transfer from other fractions as a consequence of activity of the lipid transfer protein.

Carrier Proteins↗

PubMed1983

Sex-related difference in the metabolism of isosorbide dinitrate following incubation in human blood.

Isosorbide dinitrate (ISDN) (at a concentration of 100 ng/ml) was incubated aerobically at 37 degrees in whole blood from five male and five female normal volunteers. Following incubation of the blood samples for 0, 30, 60, 120, 240 and 360 min, the samples were centrifuged and the plasma was assayed for ISDN. A linear relationship was observed between the logarithm of the concentration of ISDN remaining and incubation time, and there was a significant difference between the T1/2 of ISDN in blood from males (90.6 min) and females (161.4 min). Very little ISDN metabolism was observed when ISDN was incubated with plasma rather than whole blood. When erythrocytes, resuspended in saline, were incubated with ISDN, there was a time-dependent loss of ISDN from the saline incubation medium. Investigation of the soluble fraction obtained after hemolysis of these erythrocytes also showed a time-dependent loss of ISDN. The saline incubation medium contained sufficient concentrations of the two major ISDN metabolites (isosorbide 2- and 5-mononitrate) to account for the observed disappearance of ISDN. The results indicate that ISDN is metabolized in the cellular compartment of blood and that the metabolic rate in males is greater than that in females.

Adult↗

PubMed1988

Acetylcholine and choline in rat adrenals and brain cortex prisms incubated at elevated concentrations of choline in the medium.

Experiments were performed with rat adrenals and brain cortex prisms incubated in vitro in order to clarify whether it is possible to increase their acetylcholine (ACh) content by adding a high concentration of choline to the medium and whether the additional ACh formed can be released by subsequent depolarization. After 60 min incubation with 0.5 mmol/l choline, the concentration of ACh in the adrenals was increased by 116% (compared to the incubation without added choline), while in cortical prisms the observed increase (by 37%) was statistically non-significant. The content of ACh in both tissues was raised by paraoxon during incubations without added choline, but paraoxon did not augment the increased concentration of ACh in tissues incubated with added 0.5 mmol/l choline. The ACh that accumulated in the adrenals during 60 min preincubations with added choline could be released during subsequent depolarizing incubations; the release was Ca2+ independent. In contrast to brain cortex prisms and to the adrenals preincubated without choline, no resynthesis of ACh occurred during the period of depolarization in the adrenals preincubated with 0.5 mmol/l choline. Large amounts of choline accumulated in both tissues during incubations with 0.5 mmol/l choline and the accumulated choline could be released by depolarization; the release of choline from the adrenals was Ca2+ independent. Free choline was produced in the adrenals (presumably from choline esters) during the periods of depolarization. The reason for differences between the effects of increased concentrations of choline on ACh in the adrenals and in brain cortex is not known.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1976

Effect of incubation in human plasma on electrophoretic mobility of brain-type creatine phosphokinase.

1. Electrophoretic mobility of brain-type creatine phosphokinase (CPK) from rabbit, rat and human decreased after incubation in human plasma for 3 h or 17 h but did not change after incubation in isotonic saline solution. The presence of the reducing agent mercaptoethanol during incubation in human plasma tended to reduce the rate of change of electrophoretic mobility of BB-CPK. 2. The activity of brain CPK was almost completely lost after incubation for 17 h in human plasma. The addition of mercaptoethanol after incubation of brain CPK in human plasma could only partially restore the enzyme activity. 3. The decrease in electrophoretic mobility of rat brain CPK occurred after incubation in an ultra-filtrate of human plasma which contained molecules whose molecular weight did not exceed 1000. 4. Gel chromatography indicated that incubation of brain CPK in human plasma did not markedly change the molecular size of the enzyme.

Animals↗

PubMed1987

External stimuli affecting incubation behavior and prolactin secretion in the duck (Anas platyrhynchos).

The importance of general environmental, including visual and tactile, stimuli on behavior and prolactin secretion during the incubation phase of reproduction in the duck (Anas platyrhynchos) was investigated. Nest occupancy rapidly increased at the end of egg laying and marked the initiation of incubation. Two recesses from the nest each day were synchronized to dawn and dusk; the median occurrence was 0.23 hr after dawn and 1.17 hr before dusk. Mean recess length was 36.1 +/- 1.9 min at dawn and 40.5 +/- 2.1 min at dusk. Plasma prolactin concentrations during incubation, 25.8 +/- 2.3 ng/ml, decreased to baseline levels, 10.8 +/- 1.9 ng/ml, within 24 hr after nestbox removal. The withdrawal of tactile, but not visual, stimuli of the clutch during incubation by either anesthesia or denervation of the incubation patch caused significant decreases in prolactin plasma concentrations within 24 hr. Prolactin plasma concentrations decreased rapidly at the end of incubation in ducks which successfully hatched young as well as in unsuccessful incubators. Temperature manipulations of the clutch, either above or below normal, caused decreases in plasma prolactin concentrations in parallel with temperature modification.

Acoustic Stimulation↗

PubMed1993

Role of antiproteolytic heparin-binding serum protein(s) in modulating the levels of sialyl- and galactosyltransferase activity released during the incubation of rat jejunal slices.

1. Sialyltransferase released into the medium during the incubation of rat jejunal slices in serum-free buffer, was susceptible to proteolytic degradation. Heat inactivated horse serum or its antiproteolytic heparin-binding fraction was found to be necessary in determining the activity of sialyltransferase released (Nadkarni et al., 1991). 2. In the present study, we have shown that heat inactivated rat serum (HRS) or its antiproteolytic heparin-binding fraction (HBF) had a role in determining the sialyltransferase activity released during jejunal slice incubations. 3. Galactosyltransferase was also released during incubations, but was not proteolytically degraded and the presence of HRS or HBF in incubations did not alter the levels of galactosyltransferase activity released. 4. Trypsin activity in serum-free incubation medium was higher compared to medium containing HRS. 5. Addition of serum-free medium obtained from 4 hr incubations of the jejunal slices, to medium obtained from parallel incubations done in the presence of HRS, caused inhibition of sialyl- but not galactosyltransferase activity. 6. In jejunal homogenates stored at -20 degrees C, sialyltransferase activity was decreased during 0-45 days of storage, whereas galactosyltransferase activity remained fairly stable for upto 56 days. 7. Inclusion of HRS or HBF in homogenates resulted in higher sialyl- but not galactosyltransferase activity compared to serum-free homogenate samples. 8. The results suggest that HRS or its antiproteolytic heparin-binding proteins have a role in determining the sialyltransferase activity released from the jejunal slices. In contrast galactosyltransferase released was not susceptible to proteolysis, and HRS or HBF was not required to express its activity.

Animals↗

PubMed1983

Changes in the density distribution of pig high density lipoproteins during incubation in vitro. Influence of esterified cholesterol transfer activity.

Pig plasma, depleted of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) by ultracentrifugation, was incubated in vitro at 37 degrees C in the presence of 0.002 M parachlormercuriphenyl sulfonate, a chemical inhibitor of lecithin: cholesterol acyltransferase. Incubation resulted in a consistent and significant shift in the density distribution of esterified cholesterol within the high density lipoprotein (HDL) fraction. After 24 h of incubation the esterified cholesterol in HDL of density greater than 1.125 g/ml was reduced by a mean of 28%, while that in HDL of density less than 1.125 g/ml was increased by a mean of 77%. Both the rate and the magnitude of this redistribution were markedly enhanced when the incubation mixture contained rabbit lipoprotein-free plasma, a rich source of the esterified cholesterol transfer protein. When pig plasma, depleted of VLDL and LDL, was subjected to rate zonal ultracentrifugation, the HDL in non-incubated samples eluted as a single symmetrical peak in a position comparable to that of human HDL3. After 24 h of incubation at 37 degrees C the density of the HDL was reduced, with a peak midway between the positions of human HDL3 and HDL2. When VLDL- and LDL-depleted pig plasma were mixed with human lipoprotein-free plasma (also a source of esterified cholesterol transfer activity) and incubated for 24 h at 37 degrees C, there was an even greater reduction in the density of pig HDL, which now eluted in a position comparable to that of human HDL2. It was concluded that the capacity of the esterified transfer protein to redistribute esterified cholesterol between different lipoprotein particles may be of importance in the process of HDL interconversion.

Animals↗

PubMed1990

A fast and efficient method for quantification of monoclonal antibodies in an ELISA using a novel incubation system.

We have developed a sensitive, reliable, optimized ELISA to measure human IgM monoclonal antibodies using a novel shaking incubator system with short incubation periods of 15 min at 37 degrees C for all stages. The shaking incubator is compared with a static incubator over a range of incubation times and temperatures. For each stage using static incubation conditions the system does not reach a saturation level and the results are inconsistent, unlike the shaking incubator. No 'edge effects' are observed in the shaking system due to even heating from beneath and across the plate. The orbital shaking ensures optimal mixing of reagents which eliminates a diffusion limited reaction rate caused by a depletion of reactants at the solid phase as observed in the static system. The optimized shaking system permits economical use of reagents since the coating antibody can be used at high dilutions.

Antibodies, Monoclonal↗

PubMed1984

NADPH-generating system: influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and mouse S9 fractions.

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroanisole O-demethylase (p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with S9 liver fractions from mice (induced with Na-phenobarbital and beta-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures. In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD). Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH. At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the D7 strain of Saccharomyces cerevisiae.

7-Alkoxycoumarin O-Dealkylase↗